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dissertation entitled

HUMAN NEUTROPHIL INTERACTION WITH IMMUNE COMPLEXES:
EFFECTS OF IMMUNE COMPLEXES ON THE EXPRESSION OF
C3b (CRI), C3bi (CRIII), AND Fc RECEPTORS, AND ON
THE SYNTHESIS OF LEUKOTRIENE-BA BY NEUTROPHILS.

presented by

ELAHE TORABI

has been accepted towards fulfillment
of the requirements for

Ph. D . degree in Anatomy

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HUMAN NEOTROPEIL INTERACTION MITE IMMUNE COMPLEXES:
EFFECTS OF IMMUNE COMPLEXES ON THE EXPRESSION
OF C3b (CR1), C3bi (CRIII), AND Pc
RECEPTORS, AND ON THE SYNTHESIS or
LEUKOTRIENE—B4 BY NEOTROPEILS

BY
Elahe Torabi

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Anatomy

1987

Copyright by
ELAHE TORABI
1987

EUMAN NEUTROPEIL INTERACTION MITE IMMUNE COMPLEXES:
EEPECTS or IMMUNE COMPLEXES ON THE EXPRESSION
or C3!) (CR1), C3bi (CRIII), AND Pc
RECEPTORS, AND ON TEE SYNTHESIS or
LBUKO'I'RIm-B4 BY NBUTROPBILS

BY
Elahe Torabi

AN ABSTRACT OF A DISSERTATION

Submitted to
Michigan State University
in partial fulfilment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Anatomy

1987

ABSTRNUT
HUMAN MEDTROPHIL INTERACTION WITH IMMUNE COMPLEXES:
EFFECTS OF IMMUNE COMPLEXES ON THE EXPRESSION
OF C3b (CR1), C3b1 (CRIII), AND PC

RECEPTORS, AND ON THE SYNTHESIS OF
LBUKOTRIEMB-B4 BY NBUTROPHILS

BY

Elahe Torabi

Human peripheral blood neutrophils obtained from
healthy adults were examined _i_n 11312. The expression of
C3b, C3bi and Fc receptors on the surfaces of isolated
neutrophils, as well as neutrophils in whole blood was
determined using monoclonal antibodies and immunofluorescent
techniques. :hn addition, the effects of chemotactic
dipeptide N-formyl-L-methionyl-L-phenylalanine (f-Met-Phe),
leukotriene B4 (LTB4), a temperature transition (i.e., 4°C-
---> 37°C), and immune complexes (ICs) on expression of
neutrophils receptors were examined. Furthermore, the
release of LTB4 by neutrophils after interaction with ICS
was studied.

It was shown that unactivated neutrophils in whole
blood exhibit a minimal number of C3b, C3bi receptors, while
expressing a large number of receptors for PC on their
surface. Each of the chemotactic factors, f-Met-Phe, LTB4,
and the temperature transition significantly enhanced the

expression of C3b, C3bi and Fc receptors on the plasma

membranes of neutrophils in whole blood. When neutrophils
were isolated by the standard isolation procedure, the
expression of C3b and C3bi receptors were only enhanced
significantly on cell surfaces upon stimulation with f-Met-
Phe or LTB4. No Significant increased expression of Fc
receptors was observed on the isolated neutrophil surface.

The interaction of ICS with neutrophils was studied.
Neutrophils in whole blood avidly bound and ingested the
insoluble ICS. However, the uptake of soluble ICS by
neutrophils was 1-5% compared to the insoluble ICS uptake.
The interaction of ICS with neutrophils depressed the
expression of C3b and Fc receptors, in contrast to the
expression of the C3bi receptor which was significantLy
enhanced. In addition, soluble and insoluble ICS induced
the synthesis of LTB4 from the endogenous arachidonic acid
via the S-lipoxygenase pathway. The interaction between the
Fc receptor and the Fc portion of the antibody molecule in
ICS was required for the release of LTB4 by neutrophils.

The results suggest that ICS modulate the surface
receptors associated with the immune adherence, and may be
responsible for the depressed locomotion and phagocytic
activity of neutrophils observed in some patients with
inflammatory diseases. The LTB4 released by neutrophils

upon the interaction with ICs could potentiate the

inflammatory reaction by increasing leukocytic infiltration.

Dedicated to my husband, Gregory C. Bader, my son,
Alexander, my daughter, Sarah, and my parents, Zinat Amel
and Ali Torabi, whose moral support, understanding, and

encouragement made this work possible.

ACKNOWLEDGEMENTS

I wish to express my appreciation to all of those who
helped with the completion of this study. I would
especially like to thank my major Professors:

C. Wayne Smith, M.D., Professor, Departments of
Pediatrics and Cell Biology, Clinical Care Center, Houston,
TX., for his advise and academic counseling. His support
and encouragement over the past several years will always be
remembered.

Ckflui R. Kateley, Ph.D., Director of Immunology/Tissue
Bank, Edward W. Sparrow Hospital, for his continuimg
guidance, support, patience, and for the many opportunities
he has provided for me.

I would also like to extend my sincere appreciation to
the members of my committee: Dr. Ronald Patterson, Dr.
Robert Echt, and Dr. Lawrence M. Ross for their constructive
critical review of my research and dissertation, and for
their guidance.

It is with sincere appreciation that I also thank
Wilford E. Maldonado, M.D., Director of Laboratories, Edward
W. Sparrow Hospital, for the use of reagents, equipment, and
facilities.

I also greatly appreciate the assistance, support, and
encouragement given by my colleagues: Massoom Ahmadizadeh,
Ph.D.; Joann Barry, B.S.: Chris Brown, 8.5.: Sally Chirio,
B.S.; Sue Codere, M.S.; Debbie Eagen, B.S.; Judie Federico,
B.S.; Barb Forney, M.S.: Karen Hess, M.S.; Chris Jordan:
Martin Oaks, Ph.D.: Dan O'Malley, 3.8.: Debbie Pavlak, B.S.:
Allyson Penwell, B.S.; Melani Roberts; Dace Valduss, M.S.;
Peggy Weber, 8.8.; and Dan Zelinski, 8.8.

Last, but not the least, I wish to express my gratitude
to my husband, Gregory C. Bader, for his support,
encouragement, and many hours of editing, to my four year
old son, Alexander T. Bader, for his enduring patience and
love, and to my parents, Ali-Asghar Torabi and Zinat Amel-
Boshrooyeh, for their continuing support and encouragement.

vi

TABLE OF CONTENTS

LIfl m TAKES O O O O O O O O O O O O O O ..... O O O O O 0

LIST OF FIGURE 0 O O O O O O O O O O O O O O O O O O O O O O

Mlm O O O O O O O O O O O O O O O O O ..... O

RWIm OE. lem 0 O O O O O O O O O O O I O O O O O O O O O O

I.

II.

III.

NEU'I'ROPHIIS: Composition of Cytoplasmic Granules, Plasma
Meubrane Receptors and Qtoskeletal Proteins . . . . . .

A. CytoplasmicGranules.................
B. CytoskeletalProteins ................
C. PlasmMenbraneReoeptors
C.1. IFA-l,p150-95 and m1(C3bi)glyooproteins . . .
C.2.C3breceptor(CRI) .............
C.3.C3dgreceptor
C.4.Lamininreceptor
C.S.Fcreceptor...................

PUMICNS CF W118: Signal Mechanisms for Neutrophils

ChemtacticFactorsandOpsonms
A. ActivatingFactorsforNeutrophils. ..

A.1. Couplement oonponents . . . .

A.2.Bacterialproducts................
A.3.Cellularproducts................

A.3.1. Platelet activating factor (PAF) . . .

A.3.2. Ieukotriene B4 (LTB4) . . . . . . . . .
A4.(psonins.
A.S. IOTIOphores: Calcium ionophore A23187 . . . . . .
A.6.Teuperaturetransition

W WISMS OF W113 - THE MEMBRANE RESPONSE
MEX: mrphology of Incarnation, Adherence, Ingestion
am mantllatim O O O I O O I I I 0 O O O O O O O O O O O

A. Morphology .

B. mm 0 O O O O O O O O O O O O O O O O O O O O O
C. Migratim O O O O O O O O O O I O O O O ..... O O
D. PhagocytosisandDegranulatim...........

vii

11
13

13
18
23

27

31
33
34
34
35
35
36

37
38

41

42
45

52

IV. NEUTROPHIISINIDMJNITYANDINFLAMJATION.......... 53

A. IrmnmeCOtnplexes: Background 55

B. Inmune Complexes: Clearance Versus Vascular
localization.....................57

C. Neutrophils Interaction with Inmune Corrplexes . . . . . 59

C.1. Extracellular granule release . . . . . . . . . . . 60
C.2. Production of toxic metabolites of oxygen

(O',H202)andphagocytosis............65
C.3.NeutrophllMigration...............72
C.4. Release of Platelet Activating Factor (PAP) . . . . 73
C.5.Releaseofleukotrienes..............81

C.5.1.Lipoxygenasepathway...........83

C.5.2. leukotriene B4 biosynthesis by
neutrophilsandother cell types . . . . . 86
C.5.3. Effects of LTB4 on neutrophil functions . . 91

C.5.3.l. Mechanism(s) of LTB4'
neutrole activation . . . . . . 9S

SECTIQJ I
THE EFFECTS OF CHEMOTACTIC PEPTIDE, IHJKOI‘RIENE B4, AND TEMPERATURE
TRANSITICN (N THE EXPRESSICN OF C3b, C3bi AND Fc RECEPTOIB (1)] HUMAN
NEUTIDPHIIS

MATERIALS W W O O O O O O O O O O O O O O O O O O O I O O O 99
1. Preparation of Chemotactic Solutions . . . . . . . . . . . . 99

A. Chfl'fOtflCtiCpeptidesooo...ooo....oo...99
Bo Ie‘JkOt-riene34eooo0.00000000000000099

II.MonoclonalAntibodieS.....................99
III.Blood‘Collection......................100
IV. IsolationofHumanNeutrophils .............100
V. NeltrophilPretreatment...................102
VI.InuunOfluorescenceStudies.................102
VII.StatisticalE\raluation 104
RESULTS..............................105
I. C3b, C3bi, and Fc Receptor Expression by Human Neutrophils
inWholeBlood.......................105

viii

II . Effects of Tenperature Transition on Membrane Expression of
C3b,C3bi,anchReceptors.................108

III. Effects of LTB4 on Membrane Ebcpression of C3b, C3bi, and Fc
Rxeptors O I O O O O O O O O O I O O O O I O O O O O O O 112

IV. Effects of the Isolation Procedure on Receptors Expression . 116

DIMSSIW O O O O O O O O O I O O O O O O O O 0 O O O O O O O O O O 119

SECTION] II

MWOFWWINWIWUNEWCNMWSIW
OFC3b, C3bi, ANDFC REEEPIORSCNHUMANNEUI‘KDPHIIS INWI‘DLEBIIDD

MATERIAISANDMETIDDS.......................129

1. Preparation of Chemotactic Solutions . . . . . . . . . . . .129

A. Chemotacticpeptide ..... .............129

II. MonoclonalAntibodies 129

III.InmuneComplexes......................129
A. Preparation of monomer albumin by high

performanceliquidchromatography...........129

B. Preparation of immune conplexes ............ 131

IV. BloodCollection......................140

V. Uptake of Imuune Couplexes by Human Neutrophils in Whole
Bl“ O O O O O O O O O O O O O O O O O O O O O O O O O I O O 140

V1. Uptake of Soluble Innune Couple-res by Human Erythrocytes . . 141
VILNeutrophilPretreatnent..................142
VIII.StatisticalEvaluation 143
RESULTS ..............................l44

I. Uptake of Imnme Complexes by Hanan Neutrophils in Whole
81w 0 O O O O O O O O O O O I O O I O O 0 O O O O O 0 O O O 144

A. Uptake of insoluble inmme conplexes in neutrophils . . 144
B. Uptake of soluble immne couplexes by human neutrophils. 153

II. Uptake of Soluble Immne Couplexes by Hurrah Erythrocytes . . 155
III. Modulation of C3b, C3bi, and PC Receptors on human

ix

Neutrophils in Whole Blood by Inmune Conplexes . . . . . . . 155

DISCUSSIW O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 170

SECTICN III

MEFFECPSOFSOHJBIEANDNSOLUBIEIMMECGIPIECESWTIESYNTIESISOF
IEUKOI'RIENEB4BYHUMANNEUI‘ROPHIIS

MATERIALS AND W I O O O O O O O O O O O O O O ........ 182
I. PreparationofReagents ....... ......182
A. Calcium ionophore A23187 .............. . 182

B. Aracrlidomc ch-d (M) O O O I O O O O O O O O O O O O O 182

C. CytochalasinB(CB)......... ...... ....182

D. Nordihydroguaiaretic Acid (NDGA) . . . . . . . . . . . . 183
II. MonoclonalAntibodies........ ...... ......183
III. Preparationof InmmeConplexes . . . . . . . . . . . . . . . 183
IV. Blood Collection and Isolation of Hmnan Neutrophils . . . . . 183
V. Incubation of Human Neutrophils with Inmme Couplexes . . . . 183

VI. Pretreatment of Human Neutrophils with C3b, C3bi and EC
MomclonalAntibodies ...................184

VII. Analysis of L184 by Radioinmunoassay (RIA) ........ . 185
REULTS. O O O O O O O O O O O O O O O O O O O O O O O I O O O O l O 187
I. Stinulation of LTB4 Release by calcium Ionophore A2318? . . . 187

11. Release of LTB4 from Human Neutrophils Induced by Hmlan
Cmplems O O O O O O O O O O O O O O O O O O O O O O O O O O 187

III. Dose-Related Release of LTB4 From Neutrophils Induced by Immne
Mlms O O O O O O O O O O O O O O O O O O O O O O O O O O 194

IV. Effects of Cytochalasin B (CB) and Nordihydroguaiaretic Acid
(NDGA) onICs—InducedLTB4Release . . . . . . . . . . . . . 198

V. Role of C3b, C3bi and Fc Receptors on L184 Release From
Human Neutrophils Stimulated with INsoluble-Inmune
cmlms O O O O O O O O O O O 0 O O O O O O O O O O O O O O 198
DIWSSIm O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 203
SW. 0 O O O I O O O O 0 O O O O O O O O O O O O O O O O O O 0 O 212

LIST CE W O O O O O ..... O O O O O O O O O O O O O O 216

1.1

1.2

1.3

2.1

2.2

2.3
2.4

2.5

2.6

3.1

3.2

3.3

LISTG'm

Neutrophilgranuleccnponents.............

Expression of receptors on neutrophils induced by
f-Met-Phe and by temperature transition . . . . . . . .

Percentage of positive neutrophils with various
the8 0 O O O O O O O O O O I I O O O O O O O O 0

Expression of C3b, C3bi, and Fc receptors on neutrophils
inwholebloodorafter isolation . . . . . . . . . . .

Retention time for different protein molecules using a
Waters PROI'EIN-PAKcolmmandaBDI-IPICsupport . . . .

Uptake of inmune canplexes by human nwtrophils in whole
blm O O O O O O O O O O O O O O O O O O O O O O O O O

Uptake of immeccnplexesbyneutrophils . . . . . . .

Effects of immune complexes on C3bi receptor expression
onhmnanneutrophilsinwholeblood . . . . . . . . . .

Effect of imnune couplexes m Fc receptor expression on
hmnanneutrophilsinwholeblood . . . . . . . . . . .

Effect of immme couplexes on C3b receptor expression on
hmanneutrophilsinwholeblood . . . . . . . . . . .

Release of mm; from human neutrophils stinulated with
calcj-m imre O O O O O O O O O O O O O O O O O O 0

Effect of insoluble inmune complexes on the metabolism
of arachidonic acid in human neutrophils . . . . . . .

Release of LTB4 from human neutrophils incubated with
various reagents in the presence or absence of human
sermorbcvinesermnalbumin.............

PM;

10

110

113

118

133

148
154

160

164

166

188

191

193

LevelofLTB4inhumanserumandinthesupernatantsof
stinulatedneutrophils 196

Effect of C3b, C bi and Fc receptors blocked by
monoclonal antibodies on immune complexes-induced LTB4
releasefromhmianneutrophils . . . . . . . . . . . . 202

Hm

R.1.

R.2.
R.3
R.4.
1.1

1.2.

1.3

1.4

2.1
2.2

2.3

2.4

2.5

2.6

2.7

2.8

WC! Elm

Changeinshapeof neutrophilsexposedto
chemotacticfactors................

General structureofaphospholipid. . . . . . . .
Metabolism of arachidonic acid . . . . . . . . . .

Metabolismofleukotrienes

Cytogram of cells within the whole blood preparations

Cytof luorograph of C 3b, C 3bi and Fc receptors on
resting neutrophils inwhole blood . . . . . . . .

Ecpression of receptors on neutrophils in whole blood

Effect of LTB4 on the expression of receptors on
neutrophilsinwholeblood

Analysis of a protein mixture using a 3D HPIC support

Electrophoresis of protein molecules in agarose gel

Chromatogram of human serum albumin using a 30
m swrt O O O O O O O O I I O O O O O O O O O

Histogramoftheimnunoprecipitin. . . . . . . . .

The standard curve of different molecular weight
proteins 0 O O O O O O O O O O O O O O O O O O O O

Dose-response curves for the uptake of immne

couplexes by stimulated and non-stimulated human
W18 1.“ mule b1“ 0 O O O O O O O O O O 0

Fluorescence photomicrograph of neutrophils bearing
FI'IC-immmecarplexes...............

Fluorescence photomicrograph of neutrophils bearing
SOIUDIEFI'IC'WCGVPIEXGS o o o o o o o o o o o

xiii

P13

43
82
84
87
106

107

111

115
134
136

. 137

138

139

146

151

156

PI“

2.9

2.10

2.11

2.12

3.1

3.2

Uptake of soluble FITC-immme complexes by human
mm 0 O O O O O O I O I O O O O O O O O O

Dose-response curves for soluble and insoluble
complexes-decreased PC receptor expression on
hmnanneutrophilsinwholeblood . . . . . . . . .

Dose-response curves for insoluble and soluble-
complexes-decreased C3b receptor expression on
humanneutrophilsinwholeblood . . . . . . . . .

Dose-response curves for soluble and insoluble-
ccmplexes—increased C3bi receptor expression on
hmtenneutrophilsinwholeblood . . . . . . . . .

Dose-response curves for imuune conplexes-induced
release Of LTB4 from human neutrophils . . . . . .

Release of LTB4 from human neutrophils induced with
insoluble immne complexes: Effects of CB and NDGA

157

167

168

169

197

199

INTRODUCTION

Human neutrophils are highly specialized for several
biological functions. One of their primary biological roles
includes ingestion, neutralization, and digestion of
microorganisms. inn addition, neutrophils have also been
implicated iJiicauSing damage to extracellular tissues
Observed in various types of inflammatory tissue injury
(Issekutz, 1984; Issekutz et al., 1983; Ward et al., 1983).
This tissue damage is thought to result from the release of
mediators such as, lysosomal enzymes, Platelet - activating
factor (PAP), leukotriene-B4 (LTB4), and perhaps others by
neutrophils (Fanton and Ward, 1982; Williams, 1981).

Activation of neutrophils involved in these processes
appears to result from the interaction of a stimulant
(ligand) with a cell surface receptor (Becker, 1986:
Cochrane, 1984). Different activating factors such as
chemotactic peptides, leukotriene-B4, calcium ionophore
A23187, and temperature transition have been reported to
activate neutrophils (Fearon and Collins, 1983; Ford—
Hutchinson, 1981: Hoffstein and Weissman; Painter at al.,
1984; Schiffmann and Wahl, 1975). Neutrophil activation can
be accompanied by cellular chemical changes (e.g.,
consumption of oxygen, oxidative metabolism of glucose,

1

2
generation of superoxide anion, production of metabolites of
arachidonic acid, release of lysosomal enzymes, etc.) and/or
cellular physical changes (e.g., changes in cellular shape,
expression of membrane components, enhanced adhesiveness,
directional ndgration, and phagocytosis, etc.)(Becker, E.,
1986; Fearon and Collins, 1983; Ford-Hutchinson, 1983; Smith
et al., 1979: Wright and Gallin, 1977: Zigmond, S.H., 1978).

While the membrane and cytoplasmic events that control
neutrophil functions have been poorly understood, recent
studies have provided some new insights in this area. It
has been Shown that the plasma membrane of neutrophils
contain receptors for the Fc portion of immunoglobulin
G(IgG), formylated peptides, leukotriene B4, and the
complement components (i.e., C3 and C5)(Fearon, 1980: Fleit
et al., 1982: Goetzl and Hoe, 1979). These receptors are
important in the performance of many of the functions of
these cells.

At least two distinct receptors for the third component
of complement (i.e., C3) have been demonstrated on the
neutrophil surface: one receptor recognizing C3b, the
product of C3 activation, and has been termed the C3b
receptor, or CR1 (i.e., C3b/C4b receptor) (Fearon, 1984:
Wong, et al., 1983), while the second receptor recognizing
C3bi, the product of C3b cleavage by the serum proteins
factors H and I, and has been termed CRIII, or the C3bi

receptor (Ross & Lembris, 1982; Yoon & Fearon, 1985). The

3

C3bi, a cell surface glycoprotein, is present on human
neutrophils, monocytes, and null cells (Todd, Nadler &
Schlossman, 1981) and appears to be involved in some of the
neutrophil adherence reactions (e.g., adherence to a
substratum, aggregation, orientation, motility, phagocytosis
and cytotoxicity) (Arnaout et al., 1983; Beller et al.,
1982: Dana et al., 1984). A cooperative interaction between
Fc and CR1 receptors involving the endocytic process of
neutrophils has been suggested in studies by Jack and Fearon
(1984).

The capacity of neutrophils to respond to
microorganisms or other particles appears dependent on the
number of receptors expressed on their plasma membrane.
Defects in neutrophil functions, associated with the
deficiency or abnormal expression of complement receptors,
has been described in several patients and neonates
(Anderson et al., 1984: Arnaout et al., 1984; Crowely et
al., 1980; Thompson et al., 1984) Studies have shown an
increased expression of complement receptors on the
neutrophil surface upon activation (Berger et al., 1984,
1985; Pearon and Collins, 1983). An enhanced expression of

neutrophil C3b receptors has been reported to occur in vivo

 

with patients experiencing intravascular complement
activation secondary to hemodialysis or thermal injury (Lee

et al., 1984; Moore et al., 1986). Therefore, it appears

4
that these receptors play an important role in neutrophil
functions.

It is believed that much of the tissue injury seen in
inflammatory diseases (e.g. rheumatoid arthritis,
vasculitis, systemic lupus erythematosus, chronic
inflammatory bowel disease, and certain forms of
glomerulonephritis) is mediated by the formation and
deposition of immune complex in tissues. The interaction of
immune complexes, complement, and neutrophils is believed to
be important in the pathogenesis of immune complex diseases
(Starkebaum et al., 1982). The interaction between immune
complexes and neutrophils proceeds in several steps; 1)
adherence, 2) phagocytosis, 3) cellular metabolic
alteration, and 4) release of mediators from neutrOphilS
(Goetzlet al., 1984: Lucisano and Mantovani, 1984). The
phlogestic potential of immune complexes relates to several
factors: the ability of the immune complexes to, a) activate
the complement system, b) stimulate neutrophils to release
granule contents, platelet-activating factor, superoxide
anion, and hydrogen peroxide, and c) induce the release of
leukotrienes from monocytes/macrophages (Camussi et al.,
1981: Gimbron et al., 1984; Johnson and Ward, 1981: Ringertz
et al., 1982: Rouzer et al., 1980).

Leukotrienes are a group of mediators of inflammation

(Samuelsson, 1983). These are formed within neutrophils

5

after stimulation with an activating agent, formyl-
methionyl-1eucyl-phenylalanine (fMLP) or the calcium
ionophore via the lipoxygenase pathway (Jubiz et al., 1982:
Stenson and Parker, 1979). Leukotriene-B4 has been
identified as a potent mediator of leukocyte function.
Leukotriene-B4 is a potent chemokinetic and aggregating
agent for neutrophils and has been shown to cause
degranulation, superoxide generation, receptor expression,
and mobilization of membrane-associated calcium (Lew et al.,
1984: Naccache et al., 1981: Shaafi et al., 1982; and Volpi
et al., 1984).

The study presented herein in this thesis investigated,
1) the effects of the chemotactic factor N-formyl-L-
methionyl-phenylalanine (f-Met-Phe), leukotriene-B4, and
temperature transition on the expression of C3b (CR1), C3bi
(CRIII or M01), and Fc receptor on human neutr0phils in
whole blood as well as isolated neutrophils, 2) the
modulation of these receptors on human neutrophil surfaces
upon interaction with soluble and insoluble immune complexes
and 3) the release of leukotriene-B4 by human neutrophils
after interaction with immune complexes. A whole blood
assay was developed, and the expression of surface receptors
was examined using monoclonal antibodies, immunofluorescent
techniques, and flow cytofluorography. The immune complexes
were composed of human serum albumin and an IgG fraction of

rabbit anti-human albumin. A radioimmunoassay (RIA) was

6
employed In) measure the amount of leukotriene-B4 released
into the extracellular media by human neutrophils following
exposure to immune complexes.

The results from this study Showed that the interaction
of human neutrophils with immune complexes induced the
synthesis of leukotriene-B4 from endogenous arachidonic
acid. In addition, this interaction enhanced the expression
Of C3bi (CRIII) receptors and depressed the expression of Fc
and C3b (CR1) receptors on neutrophil surfaces in whole
blood. The interaction between the Fc receptor on the
neutrophil surface and the Fc portion of the antibody in the
immune complex was required, but not sufficient for the
release of LTB4 from neutrophils. The mechanism is
currently unknown, but the presence of serum was required.
LeukotrienevB4, as well as f-Met-Phe and temperature
transition (i.e., 4°C --> 37°C) induced a significant
expression of C3b, C3bi and Fc receptors on neutrophils in
whole blood. These results suggest that the interaction of
human neutrophils with immune complexes may potentiate the
inflammatory reaction by the release of leukotriene—B4 and
may contribute to the depressed locomotion and phagocytic

activities seen in some patients with inflammatory diseases.

REVIEW OF LITERATURE

The purposes of this review are to 1) provide
information concerning morphology, structure and physiology
(3f human neutrophils, 2) introduce information regarding
the activating agents for neutrophils, 3) discuss events of
activation which occur following neutrophil activation, and
4) present information to facilitate an understanding of
the physiopathology of immune complexes and neutrOphilS,

along with their involvements in inflammatory reactions.

I. NEUTROPHILS: Composition of cytoplasmic granules,

plasma membrane receptors and cytoskeletal proteins

Neutrophils are polymorphonuclear cells which are
produced in the bone marrow, mature there into cells
containing a variety of enzymes and potent mediators
sequestered in granules, and then emigrate and circulate in
the blood. Release of neutrophils from the marrow into
blood sinuses is generally related to their maturation
(Weiss, 1970). However, other factors such as, blood flow
(Dornfest et al., 1862), number of neutrophils in blood,
ibacterial products (Bishop et al., 1968), and “colony
stimulating factors" (Athens et al., 1961: Parker and
Metcalk, 1974), also influence neutrophil release from the
marrow.

In the peripheral blood, neutrophils constitute
approximately 60-70% of the blood leukocytes. The
neutrophil is an end cell, incapable of cell division. The
most prominent feature of a mature neutrophil is the highly
lobulated nucleus. The cytoplasm contains numerous
membrane-bound granules, few ribosomes and mitochondria,
scant rough endoplasmic reticulum, diminished Golgi complex,
and is rich in glycogen. The paucity of mitochondria and
the abundance of glycogen in neutrophils reflect the

predominance of the anaerobic mode of metabolism, whnfli

9
permits neutrophils to function in the poorly oxygenated

environment of damaged tissues.

A. Cytoplasmic granules

 

Based on morphological and histochemical examination of
the mature neutrophil, at least three discrete types of
granules (i.e., primary, secondary and tertiary) have been
identified. The azurophilic "primary" granules are the first
to appear in the developing neutrophils (i.e., promyelocyte
stage) (Klebanoff, 1978; Silber and Moldow, 1983). Mature
primary granules contain acid hydrolases, neutral proteases,
myeloperoxidase,lysozyme, and acid mucopolysaccharide (Table
R.l) (Baggiolini et al., 1985: Bainton and Farquhar, 1968;
Pember et.et1., 1983). Myeloperoxidase is present
exclusively in the azurophil granules of the resting
neutrophil.

The secondary "specific" granules appear during the
myelocyte stage of neutrophil development (Bainton et al.,
1971; Capone et al., 1964: Scott and Horn, 1970). These
granules are smaller (0.5 pm) and less electron-dense than
are the primary granules. Secondary granules contain
lysozyme, alkaline phosphatase, the neutral protease
collangenase, vitamin BIZ-binding proteins, and the
glycoprotein lactoferrin (Table R.1) (Baggiolinin et al.,
1985: Bainton et al., 1971; Masson et al., 1985). Secondary

granules contain approximately two thirds of the

10

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11

neutrophil's total lysozyme. Recently, Cramer and Breton-
Gorius (1987), demonstrated ultrastructural localization of
lysozyme in both the primary and secondary granules of blood
and bone marrow neutrophils utilizing immunoelectron
microscopy.

The third type of granules in mature neutrophils are
.referred to as Chparticles or tertiary granules. The
tertiary granules are relatively small (approximately 0.3
pm), contain a number of acid hydrolases also found in
primary granules (Table R.1), the neutral protease
gelatinase and the plasminogen activator (Baggiollini et
al.,):1985; Murata and Spicer, 1973; Sopata and Dancewicz,
1974; Wetzel et al., 1967). Recent evidence suggests that a
proton pump ATPase, responsible in part for the
acidification of phagolysosomes following neutrophil
phagocytosis, is associated with tertiary granules

(Mollinedo and Schneider, 1984; Mollinedo et al., 1986).

B. Cytoskeletal proteins

 

The cytoskeleton of neutrophil is formed by micro-
tubules and microfilaments (Stossel et al., 1984). Micro—
tubules are composed primarily of tubulin. Tubulin is a
complex dimeric protein which can polymerize into micro-
tubular structures both in XEXS and in vitgg, in the
presence of Ca++ and GTP (Borisy et al., 1974).

Microtubules form a cytoskeleton which is utilized in the

12
movement of some interacellular organelle. Colchicine is an
alkaloid which binds with tubulin and causes the rapid and
complete disassembly of cytoplasmic microtubules in all
mammalian cells (Oliver, 1978). The contracting
microfilaments consist of actin and myosin. Microfilaments
appear to function in the maintenance of cell shape,
motility and in the intracellular movement of organelles
(Howell and Tyhurst, 1982: Neiderman, et al., 1983; Oliver,
1978). Cytochalasin B (CB), a metabolite of the mold
Elmintho-Sporium Dematioideum, causes a disruption of
microfilaments and an associated loss of the cellular
functions dependent on them (Oliver, 1978). In addition, CB
inhibits the active transport of sugar across the cell
membrane at lower concentration than those required to
affect microfilament organization (Enstensen and Plagemann,
1972). In neutrophils, CB inhibits Chemotaxis and
phagocytosis and enhances secretion to the outside of the
cell, (Becker, et al., 1972; Becker, 1976: Malech, et al.,
1977). It has been presumed that they do this by preventing
the gelation of actin with actin binding proteins, which
would allow granules to gain access to the membrane, a
process otherwise prevented by subplasmalemmal

microfilamentous structures (Hartwig and Stossel, 1976).

13

C. Plasma membrane receptors

 

Neutrophils have different receptors on their plasma
membrane, including receptors for the Fc portion of IgG,
formylated peptides, and the complement components (i.e.,
C3b, C3bi, and C5) (Fearon, 1980; Fleit, et al., 1982:
Goetzl and Hoe, 1979; Sanchez-Madrid et al., 1983). The
presence of B-adrenergic receptors and insulin-binding sites
have also been demonstrated, even though these receptors

have not been purified (Silber and Moldow, 1983).

c.1. LEA-1, p150-95, and M01 (C3bi) glycoproteins

 

The use of monoclonal antibodies to cell surface
structures has identified surface glycoproteins (GPS) (i.e.,
MOl, LFA-l, and p150, 95) which functionally appear to be
involved in some cellular adhesiveness reactions (Anderson
et al., 1985, 1986: Arnaout et al., 1983; Beller et al.,
1982; Dana et al., 1984; Springer et al., 1984, 1986).
Structurally these GPS consist of two noncovalently linked
glycoproteins of an alpha subunit and a beta subunit
(Arnaout et al., 1983; Davignon et al., 1981: Sanchez-Madrid
et al., 1983). The beta subunits (94 kilodalton) of these
GPS are highly homologous or identical, and the alpha chain
contain the antigenic epitopes.

LEA-1, an antigen associated with lymphocyte functions,
is a heterodimer (alpha subunit, 177 KD; beta subunit, 94

KD) and is present.cn1!r and B cells, monocytes, and

14

neutrophils (Davignon et al., 1981; Sanchez-Madrid et al.,
1983). Studies have shown the involvement of LEA-l in
neutrophil-mediated antibody-dependent cellular cytotoxicity
(ADCC) and lymphocyte-mediated natural killer cytotoxicity
(NKD) (Kohl et al., 1984), homotypic adherence of B
lymphocytes and monocytes (Mentzer et al., 1985, 1986), T-
cell dependent antibody production (Fisher et al., 1986;
Howard et al., 1986) and the interaction between activated
macrophages and tumor cells (Strassman et al., 1986).

The p150, 95 GP has been found on neutrophil and
monocyte membranes and it appears to be involved in
adherence functions of these cells (Anderson et al., 1986:
Gallilh, 1985; Harlan et al., 1985; Sanchez-Madrid et al.,
1983; Springer et al., 1986). Diene et al., (1985) have
shown that both Gp-lSO deficient and monoclonal antibody-
treated normal neutrophils failed to adhere to cultured
human umbilical vein endothelial cell monolayers when
activated by phorbol myristate acetate (PMA).

M01, a structure closely associated or identical with
the human complement C3bi receptor, consists of two
noncovalently associated alpha chain of approximately 155 KD
and beta chain of 94 KD. It is present on human
neutrophils, monocytes, natural killer cells, null cells,
and erythrocytes (Arnaout et al., 1983; Sanches-Madrid et
al., 1983). M01, is also known as OKMl, Mac-l and p150, 95

(Berger et al., 1984: Dana et al., 1984; Springer et al.,

15

1986). M01 recognizes the complement protein fragment C3bi,
the product of C3b cleavage by the serum proteins factor H
and I, and is termed CR3 (CRIII), or the C3bi receptor (Ross
and Lambris, 1982; Yoon and Fearon, 1985). Wright et al.,
(1987) have shown that CRIII recognizes a region of C3 that
contains the sequence Arg-Gly-Asp (RGD), and appears to be a
member of a larger family of adhesion-promoting receptors
(i.e., fibronectin receptor, vitronectin receptor, platelet
glycoprotein IIb/III,and CSAT antigen). It is believed that
the residues beyond the RGD triplet participate in binding.
The exposure of normal neutrophils to anti-M01 or

anti-LFA-l monoclonal antibodies produces functional defects
of neutrophils which are linked to cell adhesive properties
(e.g., adherence to a substratum, aggregation, orientation,
motility, phagocytosis and cytotoxicity) (Anderson, et al.,
1984; Arnaout et al., 1983: Beller et al., 1982: Kohl et
al., 1984). Arnaout et al.,(l983) reported that monoclonal
antibodies to M01 (C3bi) receptors specifically inhibited
rosetting between neutrophils and sheep erythrocytes coated
with C3bi. In addition, anti-MOI blocked phagocytosis by
human neutrophils of C3-or IgG-Opsonized (C3-coated)
particles as well as lysosomal enzyme release and superoxide
generation by neutrophils in response to Opsonized zymosan
(coated with C3bi). However, the anti-M01 antibody did not
inhibit IgG Fc, C3b or C3d receptor-mediated binding of

erythrocytes coated with the respective proteins.

16

Impaired phagocytosis, Opsonized zymosan-induced
degranulation and superoxide production have previously been
noted in neutrophils from a patient with recurrent bacterial
infections (Arnaout et al., 1982). Defects of neutrophil
functions associated with the deficiency or absence of the
surface glyCOprotein antigens (e.g., M01 and LFA—l) have
been described in several patients (Anderson et al., 1984;
Arnaout et al., 1984; Crowely et al., 1980; Dana et al.,
1984; Kohl et al., 1984; Thompson et al., 1984). Their
clinical symptoms are characterized by recurrent bacterial
or fungal infection, progresssive periodontitis, persistent
leukocytosis, and/or delayed umbilical cord separation
(Bowen et al., 1982). The patients' neutrophils have shown
severely depressed adherence, Chemotaxis and phagocytic
function.

Depressed neutrophil adherence-dependent functions have
also been reported in human neonatal neutrophils (Anderson
et al., 1981 and 1984: Boner et al., 1932; Klein et al.,
1977). Becker-Freement et al., (1984) have shown that
neonatal neutrophils exhibit depressed baseline levels of
M01 receptors on their plasma membranes when compared to
adult neutrophils. Neonatal neutrophils demonstrated a
dose-dependant increased expression of M01 receptor
following activation, but the degree of expression was
significantly less than those expressed by activated adult

neutrophils. It has also been reported that the levels of

17

lactoferrin (i.e., secondary granule enzyme) in lysates
prepared from neonatal neutrophils were approximately 50% of
those found in adult lysate. However, the amounts of
lysozyme (i.e., secondary granule enzyme) and
myeloperoxidase euui beta-glucuronidase (i.e., primary
granule enzyme) were comparable to those found in adult
neutrophils (Ambruso et al., 1984; Becker-Freement et al.,
1984). Upon stimulation, neonatal neutrophils secreted
equivalent amounts of myeloperoxidase and lysozyme as adult
neutrophils, but the level of lactoferrin was 50% of that
secreted by adult cells. Becker-Freeman et al., (1984) have
suggested that the low levels of M01 receptor expression and
lactoferrin content in neonatal neutrophils may contribute
to the various depressed functions observed in neonates and
their increased susceptibility to bacterial infections.

Neutrophil activation promotes an increased expression
of M01 receptors on the cell membrane (Arnaout et al., 1984;
Berger et al., 1984). Few studies favor the secondary
granules as tuna interacellular pool for M01 antigens
(Arnaout et al., 1984; Todd et al., 1984; O'Shea et al.,
1985). Todd et al., (1984) examined neutrophils and their
subcellular components by applying immunological and
electrophoretic techniques. They demonstrated that the
major intracellular pool for M01 antigens was located
primarily in the specific (secondary) granules. Arnaout et

al., (1984) and Todd et al., (1984) have sUggested that

18

expression of M01 antigens on neutrophil membrane is
probably due to degranulation and fusion of the specifflc
granule fractions with the plasma membrane. O'Shea et al.,
(1985) presented evidence showing that neutrophils from a
patient with a secondary granule deficiency were unable to
exhibit an enhanced expression of M01 receptor upon
activation. Further evidence for the association of the M01
antigens with the secondary (specific) granules was
demonstrated by the myeloid lines KGl and THP-l. These
cells lack secondary granules and do not express M01
receptors on their plasma membrane (Arnaout et al., 1984).
However; Petrequin and coworkers (1985) have presented
evidence suggesting that the M01 antigens may be more
closely associated with tertiary than secondary granules.
Under controlled conditions, activated neutrophils expressed
a 3-fold increase in cell surface MOI antigens and
concomitant extracellular gelatinase (tertiary granule
enzyme) release without significant release of secondary
granule vitamin BlZ-binding protein or primary granule

myeloperoxidase.

C.2. C3b receptor (CR1)

 

Neutrophils exhibit on their plasma membrane receptor
which recognizes C3b, the major Cleavage fragment of C3, and
is termed the C3b receptor, or CRI (i.e., C3b/C4b receptor)

(Fearon, 1980, 1984, 1986). C3b receptor recognizes the C3c

19

region of C3, has greatest affinity for C3b, and also binds
iC3b and C4b. C3b (CR1) receptor is composed of a single
polypeptide chain that exhibits genetically determined
differences in size ranging from 160,000 to 260,000 Daltons
that are not the result of differences in carbohydrate (Wong
et al., 1983). Recently, Holers and associates (1987),
reported that the molecular weight polymorphism is deter-
mined at the genomic level and may have been generated by
unequal crossing-over. C3b (CR1) receptor is also present
on human erythrocytes, eosinophils, monocytes, macrOphages,
B lympho-cytes, a subpopulation of T lymphocytes, and
glomerular podocytes. C3b receptor has been purified and
specific monoclonal or polyclonal antibodies have been
prepared which have provided a useful tool in studying the
function and modulation of this receptor in human cells.

Human erythrocytes express an average of 500-600 C3b
receptors per cell, and a wide range of 100-1000 receptor
sites per cell has been found among different normal
individuals (Arnaout, et al., 1981: Fearon, 1980). The
function of C3b receptors on erythrocytes may be to pro-mote
the clearance of immune complexes from the circulatnnn
Several studies have reported a relative deficiency of C3b
receptors (n1 erythrocytes in patients with systemic lupus
erythematosus (Dykman, et al., 1984: Fearon, 1986; Minota,
et al., 1984; Wilson et al., 1982). Studies from Boston and

Japan have implicated the role of genetic factors in the

20

occurrence of the deficiency (although, other reports have
suggested that the deficiency may also be secondarily
acquired in some individuals).

Circulating neutrophils express only a limited number
(i.e., average of 5,000) of C3b sites per cell, but
activation of these cells induces a rapid up-regulation
(i.e., increased expression) of these receptors (i.e., as
many as 50,000 per cell) available for binding of ligands
(Berger et al., 1984: Pearon and Collins, 1983: Torabi et
al., 1986). Up-regulation of neutrophil-C3b receptors has
been reported to occur i_n 2.519. with patients experiencing
intravascular complement activation secondary to
hemodialysis or thermal injury (Lee et al., 1984; Moore et
al.,..l986). Increased expression of C3b receptors on
neutrophil membrane is a temperature dependent process and
does not require the presence of extracellular Ca‘”.
Although, the release of intracellular Ca++ is necessary and
appears to be sufficient for increased C3b receptor
expression in response to different stimuli (Berger et al.,
1985). The latent receptors are presumed to be
intracellular, although the precise Site has not been
identified. O'Shea and his associates (1985a), examined
membrane- and granule-enriched fractions of neutrophils for
the presence of C3b receptor antigens using RIA and

immunofluorescent techniques. They found that the C3b

21

receptor was present only in the plasma membrane-enriched
fraction.

The primary function of C3b receptor on neutrophils is
to mediate or enhance the endocytosis of soluble complexes
and particles to which C3b or C4b have attached during
activation of complement (Fearon, 1984: Mantovani et al.,
1972). Earlier studies indicated that the phagocytosis of
particles was mediated through EC receptor interaction, and
could not occur via C3b receptor alone. It had been
suggested that C3 fragments facilitate recognition of
particle-cell attachment without leading to ingestion
(Newman, 1979; Scribner and Fahrney, 1976: Taylor et al.,
1983). It was considered that C3b receptor has relatively a
passive role in phagocytosis, and is involved only in the
adherence In: the phagocytes of particles bearing C3b.
However, recent studies indicate an active role of C3b
receptor in cellular reactions. Neutrophils activated with
phorbol esters ingested particles bearing only C3b, and
neutrophils internalized C3b receptors that had been cross-
linked with F(ab')2 anti-C3b receptor or with multimeric C3b
(Chagelian et al., 1985; Fearon et al., 1981; Jack et al.,
1986: Wright and Silverstein, 1982). Further evidence for
the active role of C3b receptor is provided by the
association of the C3b receptor with the cytoskeleton of
neutrophils (Jack and Fearon, 1983, 1984). A rapid capping

of the C3b receptors on the plasma membrane of neutrophils

22

occurred after binding of F(ab')2 anti-C3b receptor, but not
after uptake of Fab' anti-C3b receptor (Jack and Fearon,
1984). Capping on neutrophils did not represent a passive
aggregation by the anti-receptor antibody of laterally
diffusing receptors. In contrast, the reaction was
accompanied by the subplasmalemmal accumulation of actin
network at the Site of caps. The distribution of the
receptor was inhibited by the presence of chlorpromazine,
which may interfere with intracellular Ca‘H-dependent
reactions by binding to calmodulin (Salisbury et al., 1981)
or of cytochalasin D, which prevents actin elongation (Lin
et al., 1980). Jack et al.,,(1986) studied the nature of
C3b receptors in human erythrocytes. They observed no
association between cross-linking C3b receptors on
erythrocytes and the cytoskeleton, and indicated that
erythrocyte C3b receptors may differ structurally from
neutrophil C3b receptors in it's cytoplasmic extension.
These investigators strengthen their suggestion by the
recent finding that activation of protein kinase C with
phorbol myristate acetate causes the phosphorylation of C3b
receptor in neutrOphils but not in erythrocytes (Changelion
and Fearong, 1986). O'Shea and his coworkers (1985b), and
Changelion and his associates (1985), studied internali-
zation of the C3b receptors by tumor-promoting phorbol
myristate acetate (PMA). Phorbol myristate acetate had a

biphasic effect on the plasma membrane expression of C3b

23

receptors by neutrophils. Treatment of neutrophils with PMA
first led to translocation of latent C3b receptors to the
plasma membrane and this reaction was followed within
minutes by the internalization of the C3b receptors.
Internalization of C3b receptors by PMA, occurred in the
absence of ligand, suggesting an effect on the C3b receptor.
This process was temperature-dependent and required
functional microfilaments. Despite the decreased expresion
of C3b receptors on neutrophils membrane after treatment
with PMA, C3b-dependent phagocytosis was markedly enhanced.
The mechanism by which PMA altered the phagocytic function
<xf C3b receptor has not been interpreted. O'Shea and his
associates (1985b), suggested the association of C3b
receptors with the cytoskeleton may be impor-tant to the

process of ”activation" of C3b phagocytosis.

C.3. C3dg receptor

 

The existence of a fourth type of C3 receptor on human
neutrophil membranes has been demonstrated by Vik and Fearon
(1985). 'This receptor binds the C3d region of iC3b, and
C3dg and is distinct from CRI (C3b), CRII (C3d), and CRIII
(C3bi). A lO-fold variation in receptors for C3dg was
Observed among normal individuals which was not caused by
variable expression of latent receptors, as has been

observed for C3b and C3bi receptors. Since pretreatment of

24
neutrophils with the chemotactic peptide, N-formyl-L-
methionyl-L-leucyl-L-phenylalanine (FMLP), did not greatly
augment the subsequent uptake Of C3dg. Binding of 1251-
labelled C3dg to neutrophils was saturable, and an average
of 12,400 sites/cell among normal individuals was reported.
Specific binding of C3dg was cation independent and was
competitively inhibited by iC3b and C3d, whereas C3b was
less able to compete with C3dg for binding to these receptor
sites. On the other hand, anti-Mac-l and OKMlO, monoclonal
antibodies reported to be directed to the iC3b binding of
CRIII (C3bi), did not inhibit the cellular uptake of C3dg.
Vick and Fearon (1985), suggested that, two iC3b-binding
proteins are present on neutrophils, with CRIII (C3bi)
perhaps having more critical functions in the adherence of
iC3b-coated particles and the C3dg-binding protein having a
role in the cellular uptake of soluble complexes bearing

iC3b or C3dg.

C.4. Laminin receptor

Recently, Bryant et al., (1987) and Yoon et al.,
(1987), demonstrated that neutrophils possess a receptor for
laminin. Laminin (MW 800x103 - lOOOxlO3 daltons) is a large
and abundant basement membrane-specific glycoprotein
(Foidart et al., 1980; Timple et al., 1979). It contains
structurally and functionally distinct domains for attach-

ment to the extracellular matrix components such as type IV

25

collagen, to heparan sulfate proteoglycan, as well as to
cell surface receptors (Kennedy et al., 1983: Liotta, 1984;
Rao, et al., 1982: Terranova et al., 1980 and 1982: Von der
Mark and Kuhl, 1985). Laminin has been shown to interact
with a variety of cell types including human breast carci-
noma (Hand et al., 1985; Terranova et al., 1983), murine
fibrosarcoma (Malinoff and Wicha, 1983), mouse melanoma (Rao
et al., 1983), and human melanoma (Turpeenniemi-Hujanen et
al., 1986) by binding to specific cell surface receptors (MW
68,000 to 72,000 daltons). In addition, existance of a
50,000 daltons bacterial laminin has been reported (Lopes et
al., 1985). Furthermore, laminin promotes haptotactic
migration of murine 816 melanoma cells (McCarthy and Furcht,
1984).

Terranova and his associates (1986), studied the
effects of laminin on rabbit neutrophil motility and attach-
ment. Laminin was shown to be chemotactic for neutrophils.
In the Boyden chamber and human amnion system, laminin was
shown to stimulate migration of neutrophils. Pretreatment
of neutrophils with antibody to laminin blocked neutrophil
migration. In an attachment assay system, laminin was found
In: increase protease-treated neutrophil attachment to type
IV (basement membrane) collagen-coated plastic and to a
plastic substrate itself; In addition, laminin caused
Significant release of the specific granule constituent

vitahdJI BlZ-binding protein. However, laminin had no

26

detectable effect on the release of lysozyme (found in
specific and primary azurophil granules), of beta
glucuronidase (a primary granule marker), or of the
cytoplasmic enzyme, lactic dehydrogenase. These investi-
gators suggested that neutrophils bind laminin on their
plasma membrane, use laminin to attach to basement (type IV)
membrane collagen, and migrate toward a gradient of laminin.
These properties may be important for the migration of
neutrophils from circulation to sites of infection.

Bryant and his coworkers (1987), reported about a
Single class Of saturable high affinity binding Sites (kd =
6.15 nM/L) for laminin on rabbit neutrophils and 3.6x104
sites per cells. Human granulocytes were shown to possess a
high affinity laminin receptor, Kd = 1.2 nM with 2.18x104
Sites per cell using 125I-labelled laminin. The number of
binding sites on myeloid cells from chronic myelogenous
leukemic patients and those from chronic lymphocytic
leukemic patients was reported to be at least lO-fold leSs.

Yoon and his associated (1987), examined the effect of
stimulation on the expression of laminin receptors on human
neutrophils. Unstimulated cells exhibited very low levels
of binding for 125I-laminin. However, upon treatment of
neutrophils with PMA, an eighteen-fold to twenty-fold
increase of laminin binding to cells was Observed. The

chemotactic peptide, FMLP, also induced an enhanced

27

expression of laminin receptors. At 10‘6M of FMLP a five-
fold to ten-fold increase was observed. Cytochalasin-B
pretreatment enhanced sensitivity to FMLP. In the presence
of cytochalasin 8(5 ug/ml), 10’7M of FMLP, stimulated
Specific laminin binding, with Kd = 3.9 nM and 64Jhu04
binding sites/cell. In addition, treatment of neutrophils
with A2318? (10‘6M), an effective secretagogue, resulted in
an increased expression of laminin binding sites. Yoon and
his coworkers (1987), have implicated the secondary/tertiary
granule release with increased surface expression of the
laminin receptor. Electroblot transfer and autoradiography
of subcellular fractions from unstimulated neutrophils
exhibited the presence of a 68,000 dalton laminin-binding
component in the secondary/tertiary (beta) fraction, which
these investigators presumed to be intracellular laminin

receptor pool.

C.5. Fc receptors

 

Human neutrophils, as well as monocytes, macrophages
and lymphocytes possess Fc receptors (FcR) on their mem-
branes that bind the Fc domain of IgG and may, as a conse-
quence, initiate the adherence and destruction of IgG-
Coated targets (Dickler, 1976: Fleit and Unkeless, 1982).
This IgG-dependent, leukocyte-mediated destruction may be
important in host defense against infectious microorganisms,

in various types of inflammatory diseases, in the

28

destruction of autologous cells in immune hemolytic anemia,
and perhaps in other immunologic disorders. These FCR each
have a characteristic binding affinity and IgG subclass
specificity. For example, evidence indicates the presence
of two distinct Fc receptors on mouse macrophages. The
trypsin-resistance Fc receptors for binding to 1962b and
IgG1 and the trypsin-sensitive Fc receptors which bind IgG2a
(Unkeless, 1977). Mellman and Unkeless (1980) purified a
monoclonal rat anti-mouse Fc antibody (i.e., 2.462 IgG) that
was specifically directed against the trypsin-resistance
IgGZb.

Fleit and his associates (1982), analyzed the struc-
ture, distribution, and heterogeneity of human neutrophil Fc
receptors using monoclonal antibodies (mAb). They prepared
two monoclonal antibodies, 3G8 and 4F7, which Fab fragments
of both inhibited binding to neutrophils of soluble rabbit
IgG complexes and sheep erythrocytes coated with rabbit IgG.
Approximately 135,000 Fc receptor sites per neutrophil were
shown using the 368 antibody. The neutrophil FcR
immunoprecipi-tated with 3G8 or 4F? Fab-Sepharose exhibited
a broad band ranging from 51,000 to 73,000 daltons, when
examined by sodium dodecyl sulfate/polyacrylamide gel
electrophoresis. Ln many cases, this broad band was
resolved into two poorly separated components, centered at
66,000 and 53,000 daltons. These investigators found the

3G8 antigen to be present on all neutrophils, mature chronic

29

myelogenous leukemia cells, eosinophils, 10-20% of $19-
bearing lymphocytes, and 6% of erythrocyte-rosetting T
lymphoCytes. No binding of 3G8 Fab was observed on the FcR-
bearing cell lines, the human promyelocyte cell line HL-60
and macrophage-like line U937., Raji, Daudi, and K562 or
blood monocytes. On the other hand, these cell lines and
monocytes bound human IgG1 with high avidity, while the
human neutrophils bound IgGl dimer with a low avidity (i.e.,
ka 1/100th of that of monocytes). Neutrophils did not bind
monomeric IgGl. Kurlander and Batker (1982), reported that
monocytes and neutrophils differ strikingly in their avidity
for IgGl polymers. In contrast to the avid binding of IgGl
to monocytes, IgGl bound to neutrophils very loosely at
37°C, and even at 4°C, where binding is more stable. IgGl
polymers bound to neutrophils 100-1000-fold less avidly than
they bound to monocytes at 37°C. Both neutrophils and
monocytes bound IgG1 and IgG3 with roughly comparable
avidity, and bound IgG4 and 192 much less avidly. Fleit and
his coworkers (1982), have suggested that the human
neutrophil FcR for 196 is different from that on monocytes.
With respect to both antigenic composition and binding of
monomeric IgGl.

A second class of 196 Fc receptors on human neutrophils
has been identified by using the monoclonal antibody 1V3
(mAb-IV3) (Looney et al., 1986). The second class of FcR

has a 40,000 daltons M.W. and is distinct from the FcR

30

(51,000 - 73,000 daltons) on neutrOphils recognized by mAb-
368. This 40,000 daltons FcR is also present on human
monocytes, eosinophils, platelets, U937, and K562 cells.
Fab fragments of mAb-IV3 or the intact molecule (i.e., IgG
mAb) were capable of completely and selectively inhibiting
immune complex-mediated generation of superoxide by human
neutrophils; superoxide generation by other stimulants
(i.e., PMA and FMLP) was not prevented by mAb-IV3. Looney
et al., (1986), indicated the 40,000 daltons FcR recognized
by mAb-IV3 is the human homologue of a class of murine
receptors recognized by monoclonal antibody 2.462, and which
on macrophages is called FcRII.

Perussia and Trinchieri (1984), have described a mAb-
B73.1 which reacts with a discrete population of human
lymphocytes, natural killer (NK) and antibody-dependent
killer (K) cells. Monoclonal antibody 873.1 and mAb-368
reacted with the same human lymphocyte subset containing
virtually all NK and K cytotoxic activity. These antibodies
recognize two distinct epitopes on the same FcR on the
membranes of these cells.

Walsh and Kay (1986), examined the binding of
immunoglobulin classes and subclasses to human neutrophils
and eosinophils using human 196-1, 196-2, 196-3, IgG-4, IgA-
1, IgA-Z, IgM, 19D and IgE myeloma proteins. Sheep
erythrocytes (E) were coated with different myeloma proteins

and the rossette formation by neutrophils and eosinophils

31

was analyzed. The rossette assay in which human myeloma
proteins are covalently linked to red cells, allows the
identification of human Ig(Fc) receptors using a homologous
system. .Lt was shown that human neutrophils bound
homologous IgG subclass of myeloma protein, and readiLy
expressed demonstrable receptors for IgA-l and IgA-Z.
Neutrophils did not express receptors for IgE, IgM or IgD.
On the other hand, eosinophils formed rosettes with E-IgE,
but not with E-IgA-l or E-IgA-Z. Similar to neutrophils,

eosinophils expressed receptors for IgG, and lack receptors

for IgM or 190 myeloma proteins.

11. FUNCTIONS OF NEUTROPHILS: Signal mechanism for

 

neutrophils - Chemotactic factors and opsonins

The primary function of the neutrophil is to protect
the host against pyogenic infection. It's function is
closely integrated with that of macrophages and lymphocytes,
cells also involved in response to infection. Polymorpho-
nuclear neutrophils (PMN) in the peripheral blood are
attracted to sites of infection by Chemotactic factors,
which are produced by the interaction of plasma proteins
with antigens or pathogens. The diffusion of these
chemotactdx:.factors creates a chemical gradient which
influences the direction of neutrophils migration. Plasma,

in addition to generating chemical attractants, provides

32
Specific proteins (e.g. immunoglobulins and/or complement)
that coat foreign materials, rendering them capable of
ingestion by neutrophils. This process of immunoglobulin
and complement coating is referred to as "opsonization". It
can enhance recognition of foreign particles by phagocytic
cells through binding to specific receptors on the surface
of phagocytes (e.g. Fc receptor, C3b receptor) (Fanton and
Ward, 1982; Mantovani, 1975). Neutrophils ingest the
Opsonized particles by surrounding them with moving
pseudopodia. These pseudopodia fuse to enclose the particle
within a vesicle called the phagosome. The cytoplasmic
granules of the neutrophil fuse with the phagosome and
release their contents into it, a process called
"degranulation". The neutrophil reduces oxygen
enzymatically to generate reactive metabolites such as
superoxide anion (0'2), hydrogen peroxide (H202) and
hydroxyl radical (011'). Various oxygen radicals with
materials released into the phagosome by the cytoplasmic
granules contribute to the anti-bacterial defense mechanism
of the neutrophils. Some of the granule contents and oxygen
metabolites may leak from the neutrophils into extracellular
environment, where they can injure the surrounding tissue
elements, as well as the foreign particles. This effect can
be counteracted and prevented to some extent by some of the
plasma inhibitors. The leakage results from secretion and

from incompletely fused phago-somes. In addition, under

33
some circumstances, activation of neutrophils initiates
synthesis of arachidonic acid (AA) which is normally stored
in biological membrane structures. Leukotreines,
metabolites of AA are important mediators in inflammatory
reactions, which can modulate neutrophil func tion. This
side effect of neutrophil response against antigens or
pathogens may be an important cause of tissue inflammation

and in certain locations may be harmful to the host.

A. Activating factors for neutrophils

 

In general the circulating neutrophils may respond to a
focus of inflammation (whether on going or potentLal) by
vascular margination, emigration and Chemotaxis, adherence
to particles or surfaces, phagocytosis, degranulation, and
secretion of granule constituents. The first essential step
for these processes is a signal mechanism(s) to prompt the
neutrophils. Activating factors (i.e., chemotactic factors,
opsonins, aggregated IgG, bacterial endotoxin, cellular
enzymes, and etc.) play an important role for the signal
mechanism(s). Bacterial invasion or tissue damage initiates
the elaboration of chemoattractants. The activation of
protein cascade systems (e.g. complement system, clotting
system) in plasma can generate activating factors (AF).
Activating factors also can be derived from lymphocytes
(e.g. lymphokines), monocytes, and neutrophils (O'Flaherty

and Ward, 1979).

34

A.1. Complement components

Complement components are important biological
substrates for the formation of activating factors. A small
molecular weight, heat stable fragment of the fifth
component of complement (e.g. C5a) generated during the
activation of complement by either the classical or
alternate pathway exerts chemotactic activity (Chenoweth and
Inuni, 1980; Jose et al., 1981). The native molecule (C5a)
possesses classical anaphylatoxic properties, i.e., it
causes smooth muscle contraction, tdstamine release from
mast cells and enhanced vascular permeability. A
carboxypeptidase B-type enzyme present in plasma converts
C5a to C5a—desArg, leading to a loss of its biological
activity. However, the chemotactic activity can be largely
restored by the addition of an unidentified "helper factor"
which is also present in plasma (Chenoweth and Hugli, 1980).
Various components such as aggregated IgG, antigen-antibody
complexes, bacterial cadetoxin, microbial cell walls,
damaged tissue and cellular enzymes can generate complement-

related chemotactic factors.

A.2. Bacterial products

Products of bacterial growth are chemotactic, which
appear to be related to two groups of compounds; N-Formyl-
methionyl-oligopeptides and oxidized lipids (Sahu and Lynn,

197?; Schiffmann and Wahl, 1975: Schiffmann et al., 1975).

35
The formylated peptides can be synthesized in large
quantities in pure form, and can be used as probes for

studying neutrophil responses to chemotactic peptides.

A.3. Cellular products

 

A.3.l Platelet activating factor (PAF)

 

Platelet activating factor is a low molecular weight
lipid molecule (1-lyso-glycerophosphocholine) which is
released from sensitized leukocytes after challenge with
specific antigen (O'Flaherty and Wykle, 1983). Studies
indicate that basophils, mast cells, macrophages and
neutrophils are capable of releasing PAF (Betz et al., 1980:
Lotner et al., 1980). £2 gitgg, neutrophils have released
PAF in response to different stimuli such as PMA, calcium
ionophore A23187, serum-Opsonized zymosan, aggregated IgG,
and surface-bound immune complexes (Betz and Henson, 1980;
Lotner et al., 1980; Virella et al., 1982). Platelet
activating factor is a potent chemotactic substance for
human neutrophils and modulates other leukocyte functions
(Goetzl et al., 1980: Shaw et al., 1981). In addition, PAF
has aggregating and degranulating effects on rabbit, guinea-
pig, dog, and human platelets (Lynch et al., 1979;
O'Flaherty and Wykle et al., 1983). Furthermore, it has
been suggested that PAF may mediate human anaphylaxis.

Platelet activating factor enhances capillary permeability

36
in humans and causes anaphylactic symptoms in subhuman

primates (Benveniste, 1974; Pinckard et al., 1980).

A.3.2 Leukotriene-B4

 

Leukotriene-B4 (LTB4), an arachidonic acid metabolite,
can be synthesized through the lipoxygenase pathway by
different types of cells, including human neutrophils.
Leukotriene-B4 was first identified as a potent mediator of
leukocyte function by Ford-Hutchinson et al., (1980), who
indicated that it was a potent chemokinetic and aggregating
agent for neutrophils. In addition, it hs been demonstrated
that LTB4 causes degranulation, superoxide generation,
receptor expression, and mobilization of membrane-associated
calcium (Lew et al., 1984; Naccache et al., 1981; Ringertz
«et al., 1982; Shaafi et al., 1981: Showell et al., 1982;

Volpi et al., 1984).

A.4. Opsonins

The complement mediated reactions which generate
chemotactic factors in plasma also elaborate by-products
which coat foreign particles and opsonize them. Immunoglo-
bulin (Ig) and fragments of complement (e.g., C3, C3b) are
considered opsonins (Opsonins; Greek, "to prepare food”)
(Goldstein et al., 1976). The opsonic fragments (C3b, 1g)
bind with extreme tenacity to the surface of a particle by

both hydrophobic and covalent interactions. This can

37
enhance recognition of foreign particles by neutrOphils
through binding to Specific receptors (e.g. C3b and Fc
receptors) on the plasma membranes of cells. It is
generally accepted that binding via C3b receptor serves only
to provide more intimate contact between the neutrophils
surface and the ligand (e.g. 196) which serve as a trigger
for time internalization phase of phagocytosis (Mantovani,

1975: Scribner and Fahrney: 1976).

A.5. Ionophores; Calcium ionophore A23187

 

Ionophores are small hydrophobic molecules that
dissolve lJl lipid bilayers of cell membranes and increase
the ion permeability of the bilayer (Alberts et al., 1983).
Most are synthesized by microorganisms (presumably as
biological weapons to weaken their competitors), and some
have been used as antibiotics. Ionophores have been widely
used to increase membrane permeability to specific ions in
studies on synthetic bilayers, cell organelles, and intact
cells. The ionophore A2318? is a mobile ion carrier, which
transports divalent cations such as Ca++ and Mg+*. When
living cells are exposed to A23187, Ca++ rushes into the
cytosol down a concentration gradient. A2318? is widely
used in cell biology to increase the concentration of free
Ca++ in the cytosol.

Increased cytoplasmic free Ca++ occur rapidly following

the binding of ligands to their appropriate receptors on

38

neutrophils (Becker and Stossel, 1980; Korchak et al.,
1984). In addition, ligand binding to surface receptor
initiates a graded displacement of intracellular bound Ca++
from membranous stores into the neutrophil cytoplasm.
Further-more, ligand-receptor binding enhances membrane
permeability to calcium. It is believed, these events
(i.e., increased interacellular Ca++ and membrane
permeability) may serve as a second messenger to mediate the
subsequent physiological responses.

Calcium ionophore A23187, mimics the effect of ligand-
receptor occupancy on neutrophils without binding to
receptors.

Treatment of neutrophils with the calcium ionophore
A23187 have been shown to initiate the responses of 0'2
generation (Korchak et al., 1984; Smolen et al., 1981),
degranulation (Hoffstein and Weissman, 1978; Smolen et al.,
1981), up-regulation of the C3b and C3bi receptors (Berger
et al., 1985), and synthesis of LTB4 (Borgeat and Samuels-

son, 1979; Seeger et al., 1986; and personal observation).

A.6. Temperature transition
Accumulating data indicates that temperature transition
of isolated neutrophils can induce cellular activation.
Temperature mediated enhanced receptor expression have
been observed (Berger et al., 1984; Fearon and Collins,

1983; personal observation). Fearon and Collins (1983),

39

reported up-regulation Of C3b on neutrophils and monocytes
that had been subjected to mechanical force during centrifu-
gation and resuspension and had been subsequently incubated
at temperatures greater than 20°C. Berger et al., (1984),
also observed a spontaneous increase in expression of C3b
and C3bi receptors on isolated neutrophils upon warming to
37°C. Charo et al., (1985), reported that temperature
significantly influenced adherence of neutrophils to cul-
tured endothelial cells or albumin-coated plastic. Neutro-
phils incubated with endothelial cells at 37°C for 10
minutes became tightly adherent, and resisted dislogment
forces. In contrast, almost all of the neutrophihs
incubated with endothelial cells at 4°C attached very
loosely and were easily dislodged.

Actin polymerization is an event which occurs following
neutrophil activation (Anderson et al., 1982; Oliver and
Berlin, 1982). Howard and Oresajo (1985), reported that the
temperature transition from 4°C to 37°C caused a four-fold
increase in neutrophils actin contents, as compared to actin
contents of cells held at 4°C.

Spontaneous temperature-mediated extracellular granule
release by neutrophils has been reported. Heerdt (1986),
presented evidence suggesting existence of a population of
peroxidase negative lactoferrin-containing granules in human
neutrophils which are selectively released early in response

to cellular activation or in response to a temperature

4O
transition from 4°C to 37°C. She detected Significant
quantities of lactoferrin in supernatant within one minute
after neutrophils isolated and maintained at 4°C were warmed
to 37°C. The extracellular lactoferrin release was not due
to aging of cells or cell death, but required that the cells
be isolated in the cold. Dewald et al., (1982), observed
that incubation of aged neutrophils (i.e., neutrophils
isolated from buffy coat cells held at 4°C for 24 hours) at
37°C for 5 minutes, in the absence of any stimulus was
sufficient to induce a rapid and massive release of
gelatinase (tertiary granule) which was accompanied by some
release of vitamin BIZ-binding protein (secondary granule).
However, incubation of neutrophils isolated from freshly
drawn blood did not induce the release of these substances.
Similar release to those of aged neutrophils was observed
only when freshly isolated neutrophils were incubated with
FMLP or ionophore A23187. In the other hand, Corcin et al.,
(1970), have detected vitamin BIZ-binding protein in
supernatants from freshly isolated neutrophils incubated for
five minutes at 37°C. No measurable amount of vitamin BIZ-
binding protein was present in supernatants from neutrophils
incubated up to five hours at 4°C. Other investigators have
reported presence of lysozyme in the supernatant of
neutrophils incubated for 30 minutes at 37°C (Goldstein et

al., 1974; Wright and Gallin, 1979).

41

III. SENSORY MECHANISMS OF NEUTROPHILS - THE MEMBRANE

 

RESPONSE COMPLEX: Morphology of Locomotion, Adherence,

 

Ingestion and Degranulation

The exposure of neutrophils to chemotactic factors
initiates a series of coordinated biochemical and biological
events including alteration of ion fluxes and membrane
permeability, morphological polarization, locomotion,
adhesiveness, release of granule contents, superoxide anion
generation, and release of arachidonic acid metabolites.
These responses are initiated by the generation of signal
mechanism (S) as a result of chemotactic factors binding to
their specific receptors on neutrophils.

Neutrophils lJl‘the peripheral blood are attracted to

Sites of infection by chemoattractants generation i_n vivo

 

through bacterial invasion, tissue degredation or activation
of the complement system. The initial neutrophil response
is margination and adherence to endothelial cells of
vasculature. The marginated neutrophils then migrate out of
the microcirculation into specific tissue compartments,
where they phagcytose the infectious agents, and eventually
will burst and die. Neutrophils have never been observed
re-entering circulation and are true end cells with half
lives of minutes to hours. The leukocyte margination and

migration through the blood vessels to the inflammed tissue

42
'was first noted by Dutrochet in 1824, who studied the

vasculature of the tadpole's tail during inflammation.

A. Morphology

 

Morphologically, freshly isolated, nonactivated,
neutrophils are basically rounded, as seen by light
microscopy. By scanning electron microscopy, the cells are
seen to have relatively smooth surfaces (Figure R.1a).
Within seconds of stimulation, cells begin to exhibit small
ruffles and other surface irregularities (Figure R.1b),
'which are randomly extended over all regions of the cell
surface. 'The ruffles become fewer and longer and
predominately at one pole of the cells which is referred to
as lamellipodium (pseudopodium). The remainder of the cell
is rounded with some small irregularities (Figure R.1c).
With continued exposure to chemotactic factors, the cell
becomes elongated and forms a knoblike tail called the
"uropod". The neutrophil has now a bipolar configuration
(Figure R.l. d,e,f), with the lamellipodium in the front
region, the uropod at the rear region, and a mid-region
which is relatively smooth with small surface ridges.

Bipolar formation can also occur rapidly (i.e., within
2 minutes after the exposure to Chemotactic factor) in
suspension, in the absence of chemotactic gradient or
attachment of cells to a surface, indicating that

neutrophils have an interinsic polarity (Smith et al.,

43

 

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44
1979). Shape change responses require intact functional
microfilaments, glucose metabolism, and are temperature
dependent (Heerdt et al., 1984; Smith et al., 1979). Shape
change response is reversible and cells become spherical
upon removal of the chemotactic factor by washing.

The Change in cell shape from spherical to ruffled
occurs within seconds, and it has been suggested that
membrane is added to the cell surface from an intracellular
store (Heerdt, 1986; Hoffstein et al., 1982). It has been
demonstrated that the increased cell surface membrane was
mediated by specific granule membrane merger, was dose-
dependent, and kinetically a transient event (Heerdt, 1986;
Hoffstein et al., 1982).

During locomotion toward a chemotactic source,
neutrophils acquire a bipolar configuration. The anterior
lamellipodium ruffles as the neutrophil moves, at a rate of
up to 50 pm/minute. Within the cell, bundles of
microfilaments form along the inner surface of the plasma
membrane at the advancing front (Oliver et al., 1978). The
centromere moves to a position in front of the nucleus and
microtubules form around the centromere, radiating
peripherally toward, but not contacting, the advancing
surface of the cell along with intermediate-sized filaments
(Hoffstein et al., 1977; Malech et al., 1977). The mean
number of microtubules in stimulated neutrophils

significantly increase, implying the involvement of

45
microtubules in stimulated shape change. Microtubule
assembly appears to be transient and the number of
microtubules returns to baseline levels a few minutes after
activation (Hoffstein et al., 197?). Anderson et al.,
(1982) demonstrated that microtubules lengthen along the
axis of cellular polarity and shorten perpendicular to the
polar axis. As the cell moves, the cytoplasmic contents
behind the anterior lamellipodium streams forward into the
front part of the cell, almost obliterating the
lamellipodiunu, The uropod is organelle-free, composed of
microfilaments and frequently a core of intermediate
filaments (Oliver and Berlin, 1982). At this point, some
granules appear to contact the cell periphery, and the
release of lysosomal contents occurs at this time (Stossel
and Boxer, 1983). It has been observed that neutrophils
moving through the vasculature also formed 'lamellipoida"
between endothelial cells and that the cell contents
streamed from the tail to the lamellipodium (Marchesi and

Florey, 1960).

B. Adherence

 

Adherence is a cell property by which non-adhesive
circulating neutrophils stick to endothelial cells of the
vessel wall and emigrate into the inflammed tissues. A

degree of neutrophil adherence appears essential for

46
margination, locomotion, and phagocytosis, all pre-
requisites for normal neutrophil function in host defense.

Most of the data used to evaluate neutrOphil
adhesiveness has been obtained using in yitrg assays. These
techniques allow for neutrophil attachment to an artificial
substrate (i.e., plastic or glass coated with different
types of substances) or to cultured endothelial cells.
Despite the diversity of the assay systems employed, the
results obtained for neutrophil function in adherence are
generally in agreement. However, the importance of these
results derived from in yitrg techniques may not apply to
neutrophil adhesiveness in giyg since the substrates used,
either glass or endothelial monolayers, are foreign to the
neutrophil and the testing is done under artificial
conditions.

Studies have shown that neutrophil adherence is
glucose, temperature and extracellular Mg** dependent, but
is independent of microfilaments, cell polarity and
extracellular Ca++ (Charo et al., 1985; Kvarstein, 1969;
Smith et al., 1979). Upon activation, neutrophils become
more adhesive which is probably due to an enhanced
expression of adhesion sites on the plasma membrane (Berger
et al., 1984; Smith et al., 1979). In a non-activating
condition, neutrophils express minimal numbers of adhesion
sites (receptors) on their plasma membrane, activation of

cells with an optimal dose of stimulating factors enhances

47

the availability of the adhesion Sites on neutrophil
surfaces up to 10 to 12-fold (Berger et al., 1984; Torabi et
al., 1986). Studies have documented that the latent
adhesion Sites (receptors) are stored in specific granules
of neutrophils, which will be incorporated into the cell
surface, possibly by the fusion of specific granules with
the plasma membrane of stimulated neutrophils (Todd et al.,
1984). Smith and his coworkers (1979), reported that
chemoattractant mediated-enhanced adherence is not
reversible by washing the cells free of the Chemotactic
factor.

Since neutrophil adherence is implicated in cell
motility, numerous investigators have focused on studies of
the relationship of adherence and motility. Smith and
Hollers (1981), studied the attachment and crawling of
neutrophils on an albummin-coated glass. The regions of
adherence to the substrate were under the lamellipodia, cell
body, tail and tips of retraction fibers. It was observed
that the detachment of the adherent neutrophils from the
substratum occured first at the region of the lamellipodia
with the result that cells hung by their uropods, and
eventually dropped off the substratum. In another assay
system, these investigators studied the attachment and
movement of albumin-coated beads on neutrophil surfaces in
suspension upon activation with chemotactic factors. It was

shown that exposure of neutrophils to low concentrations of

48

chemotactic factor causes the appearance of adhesion sites
for albumin coated glass on the neutrophils surface. A
second exposure of these neutrophils to a high concentration
of chemotactic factor initiates the movement of these
adherence Sites Ix) the uropod with the appearance of new
adhesion sites at the front (lamellipodium) of the cell. It
was hypothesized that the continued appearance of adhesion
sites and the movement of these adherence Sites to the tail
upon sensing a concentration gradient of chemotactic factor
provides the frictional forces required for locomotion. It
has also been suggested that the surface proteins in moving
cells continously circulate within the nembrane from the
front of the cell to its trailing end, then become
internalized. After a hypothetical passage through the
cytoplasm, these proteins become incorporated again into the
front section of the cell (Harris, 1976).

The use of monoclonal antibodies to cell surface
structures has identified surface glycoprotein molecules,
M01 (C3bi) and LFA-l, which appear to be involved in some of
the adherence reactions (Arnaout et al., 1983; Beller et
al.,. 1982). M01 is a structure Closely associated or
identical with the human complement C3bi receptor. The
adherence associated glycoproteins, M01 and LFA-l are
required for normal adhesion-dependant neutrophil functions.
Defects of neutrophil function associated with the

deficiency or absence of the surface glycoprotein antigens

49
(e.g. M01 and LFAl) have been described in several patients
(Anderson et al., 1984; Arnaout et al., 1984; Crowely et
al., 11980). Their clinical symptoms are characterized by
recurrent bacterial (n: fungal infection, progressive
periodontitis, persistent leukocytosis, and/or delayed
unbilical cord separation (Arnaout et al., 1982; Bowen et
al., 1982; Thompson et al., 1984). The patients neutrophils
have shown severly depressed adherence, Chemotaxis and
phagocytic function. Depressed neutrophil adherence-
dependent functions have also been reported in human
neonatal neutrophils (Anderson et al., 1984; Klein et al.,
1977). In contrast to the normal adult cells, neonatal
neutrophils exhibit only slight elevations in adherence upon
stimulation with chemotactic factors. In addition, these
cells do not demonstrate normal redistribution of adhesion
sites (Anderson et al., 1981; Smith and Hollers, 1980). The
abnormal adherence function of neonatal neutrophils may be
due to a deficiency of glyCOproteins (M01 and LFA-l) which
are present on mature neutrophils. Further evidence
supporting the involvement of these glycoproteins in
adherence and adherence-associated functions is provided by
utilizing monoclonal antibody against the adherence
associated glyCOproteins. Arnaout and his coworkers (1983),
demonstrated that pretreatment of neutrophils with
monoclonal anti-MOI antibody inhibited rosetting between

neutrophils and erythrocytes coated with C3bi. It also

50
inhibited ingestion of IgG or C3-coated particles by

neutrophils.

C. Migration

 

Chemotaxis, the unidirectional migration of cells in
response tx: a gradient of Specific chemical substances is
believed to be an important process by which leukocytes
migrate from the blood to inflammatory sites in the tissues.
One of the processes during neutrophil migration is
attachment to the endothelial basement membrane. It has
been suggested that the attachment might occur through
binding tn: the basement membrane glycoprotein laminin
(Terranova et al., 1986; Yoone et al., 198?). Human
neutrophils possess on their plasma membrane receptors to
bind laminin (Bryant et al., 1987). Laminin stimulates
neutrophil adherence, and migration, as well as the release
of granule contents (Terranova et al., 1986). On the other
hand, activating agents (i.e., FMLP, PMA, and etc.), enhance
the expression of laminin receptors on human neutrophils
(Yoon et al., 1987). It has been speculated that
neutrophils could respond chemotactically to laminin, and
could preferentially use laminin as an attachment factor to
type IV collagen during their emigration from the
vasculature (Terranova et al., 1986).

Attempts have been made to develop in yitgg techniques

which simulate -i_n_ vivo conditions, to study cellular

51

motility. Since most leukocyte chemotactic factors increase
the rate of cell locomotion at moderate concentrations and
inhibit the rate at high concentrations, careful
consideration of assay systems is essential. Two basic
types of systems exist to detect and measure Chemotaxis. In
the first, one measures the migration and distribution of a
sample of a cell population. In the second, one measures
the locomotion of individual cells (Wilkonson, 1974; Zigmond
and Hirsch, 1973). Widely used techniques evaluate
leukocyte migration in response to chemoattractant on
plastic (Zigmond, 1977), under agarose, through micropore
filters (Gallin and Quie, 1978), and through cultured
endothelial cells (Beesley et al., 1979). Few significant
in yi_vo techniques have been developed. The skin window
technique which was developed by Rebuck and Crowley (1955)
is the most common one. A circular abrasion is made on the
human forearm and test agents are added to the abrasion.
The site is then covered with a sterile glass coverslip and
the accumulation of leukocytes in response to the trauma or
test agent is observed. The skin window is still a popular
technique which is used by a number of groups in clinical
studies.

Studies indicate that neutrophil chemotactic responses
require establishment of a chemotactic factor gradient,

presence of albumin, extracellular Ca++ and Mg‘I, glucose,

52
microfilament and microtubules function (Wilkinson, 1974;

Zigmond, 1978).

D. Phagocytosis and Degranulation

The formation of pseudopodium is also essential for
ingestion. As the neutrophil meets a particle, the
pseudopodium flows around the particle, its extensions fuse,
and it thereby encompasses the particle. The particle thus
becomes enclosed within a phagosome into which cytoplasmic
granules are rapidly releasing their contents. Locomotion
is not prerequisite for ingestion. If neutrophils come in
contact with a particle not generating a chemotactic
substance, pseudopodia form abruptly at the contact point
and envelope the particle. Pseudopodia form whether
neutrophils are suspended in a liquid medium or are adhered
to a surface. The neutrophil membrane adheres firmly to the
particles it ingests, probably to provide the frictional
force needed to move pseudopodia around the particles
(Hoffstein et al., 1982; Stossel and Boxer, 1983).

Neutrophils become more adhesive during Chemotaxis or
phagocytosis. It has been suggested that this may be due to
secretion of fibronectin by neutrophils (Stossel and Boxer,
1983). Ln addition, the release Of specific granule
products and binding to the plasma membrane of "sticky”
proteins from these granules, such as acidic proteins and

lacatoferrin increase neutrophil adhesiveness (Stossel and

53

Boxer, 1983). Recently, it has been reported that specific
granules might be the major intracellular source for M01 and
laminin glycoproteins which are believed to have an
important role in neutrophil adherence. (Todd et al., 1984;
Yoon et al.,.il987). Furthermore, the granules of
neutrophils contain substances which are capable of
splitting a chemotactic factor from C5 directly or by
activating complements (Wright and Gallin, 1975, 1977).
Moreover, release of platelet activating factor and
leukotriene 84 by neutrophils, which have chemotactic
activity for neutrophils may contribute to enhanced
neutrophil adhesiveness (Berger et al., 1985; Camussi et
al., 1981; Ford-Hutchinson, 1981).

The recognition of signals, the formation of
pseudopodia, membrane fusion, and membrane adhesiveness are
all characteristics associated with the functional responses

of neutrophils.

IV. NEUTROPHILS IN IMMUNITY AND INFLAMMATION

Acute inflammatory reactions may be initiated in
tissues by a wide variety of stimuli such as microorganisms,
immune complexes, or direct physical and chemical trauma.
During acute inflammation, vascularized tissues develop
well-known Clinical signs of erythema and warmth due to
vasodilation and swelling due to the exudation of fluid and

protien from the local microvasculature.

54

Experimental models of acute inflammation have
suggested that neutrophils play a central role in the degree
of vasodilation and of increase in permeability leading to
protein and fluid exudation (Issekutz, 1984; Issekutz et
al., 1983). Studies indicate that platelet-activating
factor (PAF) may be responsible for the associated protein
and fluid exudation in inflammatory reaction. Platelet-
activating factor can be produced in 22259 and in 2139 by
neutrophils following neutrophil activation by a variety of
stimuli (Camussi et al., 1981; Lotner et al., 1980; Virella
et al.,, 1982). Platelet-activating factor can induce
prolonged vascular permeability and aggregation of
platelets. Therefore, PAF, once generated in sufficient
quantities by chemotactic factor, could be responsible for
the transient platelet accumulation in the microvasculature
of accutely inflamed tissues, an event that coincides with
and is also dependent on neutrophils margination and
migration (Issekutz, 1984; Issekutz et al., 1983). In
addition, metabolites of arachidonic acid (i.e.,
prostaglandins, thromboxanes and leukotrienes) participate
in inflammatoy reaction by direct or indirect action
(Samuelsson, 1983). Leukotrienes C, D, and E (Cysteinyl-
containing leukotrienes), the constituents of the Slow
reacting substance of anaphylaxis (SRS-A) exert potent
effects on smooth muscle, increase vascular permeability in

postcapillary venules, and stimulate mucus secreation

55
(Goetzl et al., 1984; Samuelasson, 1983). Leukotriene-B4
has potent effects on eosinophils and neutrophils that is
related to their adhesion to postcapillary venules and
extravasation as well as migration to areas of inflammation
and degranulation. Leukotriene-B4 enhances the
proliferation of supressor T lymphocytes and inhibits that
of helper T lymphocytes (Goetzl et al., 1984). Studies have
shown that stimulation of neutrophils with the activating
agents FMLP, Ca A23187, and zymosan-coated serum induced
release of LTB4 by neutrophils (Borgeat et al., 1979;
Calesson et al .,1981; Jubiz et al.,1982). Elavated levels
of leukotrienes have been detected in tissues or exudates
from patients with some spontaneous or elicited
hypersensitivity and inflammatory states (Goetzl et
al.,1984; Barnes and Costello, 1984). Therefore, the
activation of neutrophils may lead to the production of
vasopermeability mediators (possibly PAP and leukotrienes).
Which account for the protein exudation and swelling.
Alternatively, the seperation of endothelial gap by
neutrophils during migration may be directly responsible for
the protein and fluid exudation in the absence of a specific

vasopermeability meditor (Issekutz, 1984).

A. Immune Complexes: Background

 

It is generally recognized that inflammatory injury in

tissues may result from the deposition of immune complexes

56
(IC) in tissues (Fish et al., 1966). This has been
demonstrated not only during the formation of antigen-
antibody complexes in 2139 but also after the injection of
immune complexes that have been performed in 21552 (Johnson
et al., 1979 and 1981; Scherzer and Ward, 1978; Von Pirquet
and Schick, 1951).

The concept that circulating immune complexes (antigen-
antibody) could play a role in human disease was first
suggested by Clemens Von Pirquet and Schick in 1905. Their
studies on the efficiency of horse anti-diphtheria toxin led
them to suggest that the reaction between a foreign protein
and the host antibody could have a harmful, as well as a
beneficial effect on the host. They postulated that the
variation in onset and clinical signs of rash, fever,
lymphadenopathy, joint involvement, and, rarely,
glomerulonephritis after therapeutic horse serum
administration was initiated by the interaction of the
foreign antigen and the host antibody. 0n the basis of
these observations Clemens Von Pirquet coined the term
allergy in 1906 to define the host's altered immunologic
reactivity (hypersensitivity), which develops during initial
exposure to antigen or in an accelerated tempo upon
reexposure. Despite this early recognition of possible
pathogenic role of circulating immune complexes, it was only
recently that the role of immune complexes in human disease

has been actively studied. In 1953 Germuth made the

57
observatixni that immune complexes were present in the
circulation at the time of the tissue injury. Later, these
initial observations were extended and it was possible to
correlate the levels of circulating soluble immune complexes
with their deposition in glomeruli and vessel walls and the
subsequent development of tissue injury. However, in some
cases, the detectable immune complexes may not correlate

with disease activity (Johnson and Ward, 1982; Jones, 1983).

B. Immune Complexes: Clearance Versus Vascular

Localization

 

Physiochemical characteristics of immune complexes have
a role in tissue injury. The most pathogenic complexes are
those that are insoluble, either at antigen equivalence or
slight antigen excess. These same complexes are also more
effective in activation of the complement system (Johnson
and ward, 1982; Scherzer and Ward, 1978). The quality and
immunoglobulin class of the antibody involved in immune
complexes appears to be important. Immune complexes
composed of IgM antibodies are larger and more rapidly
cleared than are those formed from 19A or IgG antibodies.
One of the most important features of immune complexes is
related to their complement fixing activity. Therefore,
immune complexes composed of more than one antigen molecule
and cross-linked latice-wise by several bivalent IgG

molecules are often capable of an effective complement

58

activation. This is also true for immune complexes that
consist of IgM antibodies, which in general more effectively
activate complememnt. In contrast, IgA immune complex
deposits within tissues clinically are not associated with
intense complement cascade activation or the other
amplifying inflammatory effects associated with such
sequelae (Ghebrehiwet, 1983; Williams, 1981).

The clearance of circulating immune complexes by the
mononuclear phagocyte system (MPS), formerly called the
reticulo-endothelial system (RES) takes place primarily in
the liver and spleen. The former mediated predominantly by
C3b receptors, the latter by Fc receptors. Studies have
shown that the MPS can be saturated with large doses of
immune complexes. With a subsequent persistence of immune
complexes in the circulation resulting in increased
nonhepatic tissue deposition which may play a role in the
pathogenesis of lesions in immune complex diseases
(Haakenstad and Mannik, 1974). On the other hand,
activation of the system enhances the number and function of
PC and C3b receptors, increases uptake of circulating immune
complexes, and decreases vascular circulating complex
localization (Barcelli et al., 1981). The concentration of
immune complexes in the circulation depends on the rate of
immune complex formation and on the rate of immune complex
removal. The rate of immune complex formation in turn

depends on the rate of specific antigen availability and on

59
the rate of antibody synthesis. The removal of circulating
immune complexes by the RES depends on the physiochemical
nature of the antigen, characteristics of the antibody
moiety' Li.e., affinity and immunoglobulin class), the
lattice of immune complexes, and the status of the RES.

In addition to the role of the reticuloendothelial
system in the clearance of circulating immune complexes, the
presence of Fc receptors on a number of circulating blood
celljs such as monocytes, macrophages, lymphocytes,
polymorphonuclear cells, and platelets is of great

importance (Williams, 1981).

C. Neutrophils Interaction with Immune Complexes

 

The interaction of immune complexes, complement, and
neutrophils is thought to be important in the pathogenesis
of immune complex diseases (Starkebaum et al., 1982). The
interaction between immune complexes (IC) and neutrophils
proceeds in several steps; a) adherence, b) phagocytosis,
cu cellular metabolic alteration, and d) release of
mediators from neutrophils (Goetzl et al., 1984; Lucisano
and Mantovani, 1984).

Studies have shown that the phlogestic potential of
immune complexes relates to several factors; a) the ability
of the immune complexes to activate the complement system
resulting in the generation of chemotactic factors which are

ultimately responsible for the attraction of neutrophils, b)

60
immune complexes can stimulate neutrophils to release
mediators (i.e., lysosomal proteases), which mediate tissue
damage directly by hydrolyzing the susceptible substates
such as basement membrane, collagen, elastin, etc., or
indirectly by enhancing the inflammatory reaction via the
generation of chemotactic peptide from C5, c) immune
complexes can induce neutrophils to generate superoxide
anion and hydrogen peroxide which may play an important role
in subsequent tissue damage, d) interaction of immune
complex with neutrophils can result in release of platelet-
activating factor which can induce neutrophil platelet
aggregation, and e) immune complexes can induce the release
of leukotrienes from monocytes/macrophages, and neutrophils
(personal observation), which leukotrienes, in turn, can
modulate cellular functions of neutrophils and other cell
types (Camussi et al., 1981; Gimbrone et al., 1984; Johnson

and Ward, 1981; Ringertz et al., 1982; Rouzer et al., 1980).

C.1. Extracellular Granule Release

 

Neutrophils contain a number of different granules
which can release their lysosomal constitutents (e.g. acid
proteases, cationic proteins and etc.) upon a proper
stimulation. The involvement of these mediators in'tissue
injury in experimental animals has been well established.
Several groups of investigators have shown that immune

complexes can induce the release of lysosomal constituents

61

from neutrophils (Hawkins and Peeters, 1971; Henson and
Oades, 1975). As was mentioned earlier, neutrophils possess
surface membrane "receptors" for a fragment of the third
component of complement (i.e., C3b) and for PC regions of
certain immunoglobulin classes which have undergone a
conformational change either as a result of union with an
antigen or as a result of aggregation. Recognition at the
cell surface and binding of such "altered" immunoglobulin
mediate adherence and ingestion of immunoglobulin coated-
particles or aggregated immunoglobulins by neutrophils
(Goldstein et al., 1976; Mantovani, 1975).

In 1971, Hawkins and Peters studied the response of
rabbit peripheral blood neutrophils in gitgg to immune
complexes (IC) in both percipitate and soluble form. They
observed that normal rabbit neutrophils, when incubated with
immune complexes, released into the external-medium a number
of intracellular enzymes (e.g. P-Glucoroniades and Cathepsin
E). Homologous antigen-antibody complexes in precipitate
form and soluble antigen-antibody complexes made in antigen
excess of 20 times equivalence proved equally effective as
releasing agents. Highly soluble immune complexes made at
100 times equivalence antigen excess were totally
ineffectiwe. However, the greatest release of enzymes was
observed from those neutrophils exposed to immune complexes

in precipitate fornu .Release of enzymes appeared to

62

correlate with uptake of immune complexes from the external
medium, and the release occurred from intact viable cells.

The effect of immune complexes in both phagocytosable
and nonphagocytosable forms on rabbit neutrophils was
studied by Peter Henson (1971a, 1971b). Bovin Serum Albumin
- rabbit anti BSA was used as the immune complex. In each
case, release of constituents from the neutrophil granules
was observed. the noted that smaller quantities of
immunoglobulins were required to induce release if bound to
time nonphagocytosable surface than if phagocytosed by the
neutrophils in suspension. Similar observations have been
reported using aggregated immunoglobulin as the stimulus. In
1975, Henson and Oades studied the effect of soluble and
insoluble aggregated human gamma-globulin on the hexose
monophosphate pathway (HMP) and exocytosis of granules on
human neutrophils. They reported that insoluble aggregated
gamma-globulin in suspension stimulated HMP and secretion of
enzymes, while soluble aggregates stimulated HMP only and
had no effect upon secretion. However, soluble aggregates
bound.tx>(a surface readily induced exocytosis of granules

from the adherent neutrophils. This process may mimic

IS'

vivo situations where neutrophils adhere to immune

 

complexes (n1 surfaces such as the glomerular basement
membrane and release their granule enzymes, which may then

cause tissue injury.

63

The Fc receptors on the neutrophil surface have
different specificities for particular classes and
subclassees of immunoglobulins. Henson and Spiegelbeng
(1972), studied the effect of different classes and
subclasses of immunoglobulin from normal human and myeloma
serum. They reported that aggregated human myeloma proteins
of subclasses, IgGl, 1962, 1963, I964, IgAl and IgA2 and
normal 196 induce the release of lysosomal enzyme beta-
glucuronidase from human neutrophils. In contrast, IgD, IgE
and IgM macroglobulins did not stimulate this release.
Enzyme secretion occurred with the insoluble form of
aggregates. The soluble aggregates did not induce
liberation of enzymes when incubated with neutrophils in
suspension. Later, Lucisano and Mantovani (1984),
investigated the response of rabbit neutrophils to immune
complexes (ovalbumin-Rabbit Immunoglobulin) of IgM and of
1961. They observed that insoluble IgM immune complexes
were able to stimulate the release of lysosomal enzymes
beta-glucuronidase, alkaline, and acid phosphatase, in
contrast to heat-aggregated IgM, which had only a small
effect. The reaction required the presence of extracellular
Ca++. Insoluble IgG immune complexes from the equivalence
region were the most effective, those at antibody excess
have a smaller but comparable capacity, whereas the immune
complexes at antigen excess were the least effective. They

noted that the enzyme release induced by IgM immune

64

complexes was not inhibited by competition with free
immunoglobulin in the medium, either IgM or IgG
inapproximate physiologic concentrations; contrarily, with
IgG IC, free IgG (but not IgM) could completely block the
reaction. They suggested that ”this difference may have a
bearing in gizg; IgG present in the plasma or interstitial
fluid would competitively block the interaction between the
tissue-deposited IgG immune complexes and neutrophils,
inhibiting the lysosomal enzyme release, whereas no such
interference would exist with the IgM immune complexes. It
is possible that for IgG immune complexes the presence of
complement could be an important factor in the mechanism of
tissue injury, because interaction could be made through C3
receptors,thus bypassing the competitive inhibition".

Johnson and Ward (1981), suggested that the leukocytic
neutral proteases released from lysosomal granules have the
potential to amplify the acute inflammatory response. With
human neutrophils, the major neutral proteases, elastase and
chymotrypsin, have the ability, given the proper conditions,
to generate leukotactic peptides from C3 and C5. They
suggested that perhaps, leukocytic proteases act mainly in
the amplification of acute cellular inflammatory reactions

rather than as primary effectors of tissue damage.

65

C.2 Production of Toxic Metabolites of Oxygen (O‘2L§292L_

 

and Phagocytosis.

 

In Neutrophils, binding of ligands to their appropriate
receptors (Fc, concanavalin A, chemotactic peptide and
phorbol myristate acetate receptors) and perturbation of the
membrane with the calcium ionophore A23187, have been shown
in: initiate several responses including the generation of
toxic metabolites of oxygen (0'2,H202) (Becker et al., 1979;
Korchak et al., 1984; Smolen et al., 1980). A lag period
(within seconds) exists between receptor-ligand binding and
the onset of oxygen metabolites generation, and the length
of this lag period is specific to the particular stimulus.

Korchak et al., have reported that the chemotactic
peptide N-formyl-methionyl-leucylphenyl-alanin (f-Met-leu-
Phe) exhibited the shortest lag period of approximately 16
seconds, the length of the lag period being independent of
the f-Met-Leu-Phe concentration. In the presence of immune
complexes (150 ug/ml), a lag period of 45 seconds was
observed. The length of this lag period, unlike f-Met-Leu-
Phe, was dependent on the concentration of stimulus.

Studies have shown that products of oxygen metabolism
are able to inflict damage on isolated cells. Johnson and
Ward (1981), using an animal model have shown that
production of H202 and/or it's metabolic products play a key
role in the acute lung injury associated with deposition of

immune complexes within the tissue of the lung. ward at

66

al., (1983), have also shown that rat alveolar macrophages

can be activated by 1 vivo exposure to immune complexes

 

resulting in the production of oxygen metabolites. The
production of oxygen metabolites was a linear function of
cell number, the duration of incubation, and the amount of
immune complex employed. In the case of neutrophils, there
was a direct relationship between the amounts of immune
complex internalized, secretory release of lysosomal
enzymes, and production of 0‘2 and H202. With both
neutrophils as well as alveolar nacrophages, maximal
production of 0‘2 occurred with the largest complexes formed
under conditions of antigen equivalence.

A sensitive indicator of increased neutrophils
oxidative metabolism, as well as of production of oxygen
radicals such as superoxide anion and hydroxyl radical, is
the emission of light, termed "chemiluminescence". A
portion of these oxidative reactions yield electronically
excited products relaxing to ground state by photon
emission. The resulting light emission, or chemiluminescence
can be measured using a scintillation spectrometer modified
for single photon counting. In this manner, CL has been
applied to the study of phagocyte physiology and
pathophysiology as well as humoral opsonic capacity (Doll
and Salvaggio, 1982).

Starkebaum et al., (1981), examined the ability of

soluble and insoluble immune complexes, IgG aggregates and

67

anti-neutrophil antibody to stimulate human neutrophils' CL.
The stimulation of neutrophils' CL and uptake of immune
complexes by neutrophils were both closely correlated with
the percent precipitation of immune complexes. Nevertheless,
increased levels of neutrophils' CL were observed with
soluble immune complexes that were poorly ingested by the
neutrophils. A similar dependence of peak CL on immune
complex size in guinea-pig peritoneal macrophages was also
observed. Soluble aggregated IgG also enhanced the
neutrophils' CL as compared to equal amounts of monomeric
IgG. Inn the presence of fresh, normal, human serum (NBS),
IgG aggregates induced significantly higher levels of
neutrophils' CL than did equal amounts of IgG aggregates in
phosphate buffer. Heat-inactivated normal serum didn't have
a significant effect on neutrophils' CL, which a role of
complement in amplication of this response has been
suggested. Rabbit antiserum to neutrophils also was found to
stimulate neutrophils' CL.

Doll and Salvaggio (1982), observed that incubating
neutrophils with either aggregated immunoglobulin or immune
complexes reduced or inhibited the CL response of the cells
to subsequent challenge with a particulate stimulant. A
direct correlation between percent inhibition in CL and the
amount of complex exposed to neutrophils was observed. This
observatitni is consistent with other reports that the

microbicidal activity of neutrophils is depressed following

68
immune complex exposure (Matheisz and Allen, 1979). It has
been suggested that the inhibition observed in neutrophils
CL after preincubation with immune complexes and the
subsequent challenge with a secondary phagocytic stimulus

may serve as a diagnostic assay for the detection of in vivo

 

immune complexes (Doll et al., 1980).

The interaction of lymphocyte surface-bound immune
complexes and neutrophilswas studied by Archibald et al.,
(1983). It was found that CL could be induced by
stimulation of neutrophils with surface-bound aggregated
human gamma-globulin (AHG) or bovine serum albumin-immune
complexes (BSA-1C). Opsonization of the ABC or bovine serum
albumin - immune complexes (BSA-1C) with NHS enhanced the CL
that was produced. Moreover, B lymphocyte-enriched cell
preparations with surface-bound AHG stimulate the production
of neutrophils CL to a much greater extent than T
lymphocyte-enriched cell preparation.

The production of oxident metabolites, although not
enough to cause a significant decrease in lymphocyte
viability can cause impaired lymphocyte function (i.e.,
antibody-dependent and non antibody-mediated cytotoxicity,
attachment to sheep red blood cells, concanavalin A cap
formation, and etc.). Physiologically, this may relate in
the long term to immunologic malfunction observed in
patients with high levels of circulating immune complexes

(Archibald et al., 1983).

69

Studies have shown neither phagocytosis nor lysosomal
degranulation are prerequisites for enhanced 0'2 production
(Goldstein et al., 1975). The 0‘2 generating system is very
likely associated with the external plasma membrane of
neutrophils. Study employing distrupted neutrophils was
shown that 072 generating activity was associated with
membrane fraction.

Weiss and Ward (1982), studied the human neutrophil
responses 11) antigen-antibody complex (Ag-Ab) prepared at
different ratios (i.e.: 1:8, 1:4, 1:2, 1:1 and 2:1 ratio of
Ag:Ab) . The Ag-Ab complexes were made using hyperimmune
rabbit IgG rich in antibody to bovine serum albumin (BSA).
They found striking differences between; 1) the ability of
immune complexes to fix Complement (C'); 2) the ability of
immune complexes to be internalized by neutrophils, 3) the
ability of the complexes to mediate lysosomal enzyme
release, and 4) the ability of complexes to stimulate 0'2
and H202 generation from human neutrophils. There was no
correlation between cell uptake of the immune complexes,
enzyme release, and production of 0'2 and H202.

At an antigen:antibody weight ratio of 1:8 (molar ratio
of 1:2.7), both the ingestion of complexes and lysosomal
enzyme release were maximal, whereas 0'2 and H202 production
were best achieved by complexes containing an Ag:Ab ratio of
1:2 (molar ratio of 1.5:1). Indeed, at an Ag:Ab ratio of

1:1, the ingestion of complexes was almost undetectable,

70
enzyme release was supressed by more than 50%, and the
amount of 0‘2 and H202 produced were near maximal levels.

The greatest amount of C' fixation resulted when the
Ag:Ab ratio was 1:8, somewhat less fixation resulted with
the ratio was 1:4, and no significant amount of C' fixation
resulted when the ratio of Ag:Ab was increased.

They found that the Fc portion of the IgG molecule
interacts with Fc receptors on the neutrophil surface to
initiate cell activation, since immune complexes containing
pepsin-degraded IgG in the form of F(ab')2 with an
antigen:antibody ratio of 1:2 were unable to stimulate 0'2
or H202 production by the neutrophils. They suggested that
surface activation of the neutrophil with immune complexes
via the Fc receptor can lead to quantitatively independent
changes in internalization, enzyme release, and oxygen
metabolite generation.

These investigators found that small immune complexes
that are formed at antigen exess, do not effectively fix C',
and are not effectively internalized can maximally stimulate
the generation of 0‘2 and H202 from neutrophils. They
suggested that the fact that non-C'-fixing immune complexes
are optimal in the stimulation of neutrophils to generate O'
2 and H202 could be taken to indicate that soluble complexes
in relative antigen excess have considerable phlogistic
potential, although it has been believed for some time that

complexes of this type do not have a role in the

71

pathogenesis of immune complex-induced tissue damage.
Therefore, the larger complexes (containing less antigen)
fix:(2' and initiate the inflammatory reaction via the
production of C5a. Even though the same complexes can also
stimulate neutrophils, resulting in genereation of 0‘2 and
H202 with the induction of tissue damage, these complexes
will be rapidly ingested, terminating the inflammatory
reaction. .Lf small immune complexes (containing relative
antigen excess) are present, those may lead to a more
intense generation of 0'2 and H202 as well as a more
sustained production of the toxic metabolites, since the
complexes are not rapidly ingested by neutrophils.

Starkebaum et al., (1982), examined the effect of
immune complexes on human neutrophil phagocytic function.
They employed human serum albumin (HSA) as an antigen and
rabbit immunoglobulin rich in IgG antibodies to HSA as the
antibody. Direct uptake of immune complexes by neutrophils
was closely related to the percent of precipitation of the
complexes. Less than 1% of soluble complexes prepared at 3-
fold antigen excess were internalized by neutrophils
compared to 65% internalization of insoluble complexes made
at equivalence. Preincubation of neutrophils with immune
complexes depressed the subsequent ingestion of insoluble
immune complexes which correlated with the percentage of
precipitation of the complexes utilized for preincubation.

Furthermore, preincubation of neutrophils in normal serum

72
containing soluble aggregates of IgG decreased the
internalization of insoluble immune complexes by neutrophils
in a dose-dependent fashion. Maximum binding of soluble
immune complexes or IgG aggregates to neutrophils occurred
after 5 in: 15 minutes, however, inhibition of neutrophils
phagocytosis was seen only after 90 minutes of preincubation
with the complexes or with IgG aggregates. They showed that
the decreased phagocytic activity induced by soluble
complexes was not due to capping of Fc receptors of
neutrophil surfaces. They suggested that prolonged contact
of neutrophils with soluble immune complexes or IgG
aggregates results in decreased phagocytic activityof the‘
cells via a process that involves metabolic activation of
the cells and may be accompanied by the loss or denaturation
of surface (196) Fc receptors. This reaction may have an
important role 2". vi_vg, where the interaction of soluble
immune complexes with circulating neutrophils could lead to

abnormalities in phagocyte function.

C.3. Neutrophils Migration

 

Evidence indicates that the exposure of neutrophils to
immune complexes depresses neutrophils migration. Deahlgren
and Elwing (1983), examined locomotion of neutrophils on
solid surfaces with bound antigen-antibody complexes.
Locomotion of neutrophil was inhibited on surfaces coated

with bilayers of human serum albumin and the corresponding

73
antibody. Once immobilized on an antigen antibody coated
surface, neutrophils did not move chemotactically in
response to formylmethionyl-leucyl-phenylalanine (FMLP).
However, the receptor for FMLP appeared to be intact since
the cells responded metabolically to FMLP as judged from the
chemiluminescence response. Furthermore, locomotion
inhibition was observed also in the present of super oxide
dismutase (SOD) and/or catalase (scavengers of 0'2 and
H202), thus, autooxidation induced by surface bound Ag-Ab
complexes was ruled out as the basis for locomothmi

inhibition.

C.4. Release of Platelet Activating Factor

It is likely that platelets play a role in inflammatory
reactions. These cells contain and can release a wide
variety of mediators including vesoactive amines and
peptide, proteases, as well as chemotactic factor for
neutrophils, and some other nmdiators. Issekutz et al.,
(1984), and Lundberg, et al., (1984), observed that during
the acute inflammatory reaction induced in the dermis of
rabbits by killed or live E. Coli, maximal pdatelet
deposition occurred early, during the first 1 to 2 hours
prior tuaiany histologic evidence of hemorrhage or
thrombosis. They investigated the relationship between the
deposition of platelets and the infiltration of neutrophils

into the inflamed tissue since neutrophils infiltration is

74

also an early event. A temporal association between
leukocyte accumulation and platelet deposition in the
lesions was observed. Platelet deposition in response to
inflammatory stimuli did not occur in neutrophil-depleted
but platelet-sufficient rabbits. Platelet responses to
inflammatory stimuli were normal when neutropenia was
prevented. They suggested that platelets selectively
deposit in acutely inflamed tissues primarily during
neutrophil margination in and emigration across, the
microvasculature. It is not clear which mediators may be
involved in this neutrophil platelet intereaction.
Platelet-activating factor (PAF) is the most likely mediator
which has been suggested.

Platelet activating factor has been reportely released
from a variety of cell types, in several species, and by
different stimuli (O'Flaherty et al., 1983: Pinckard et al.,
1979). Evidence has implicated the basophil as the cell of
origin. However, recent studies have shown that other cell
types such as mast cells, macrophages and neutrophils are
capable of releasing PAF (Betz et al., 1980; Lotner et al.,
1980).

PAF is a l-lyso-glycerophosphocholine, which has
aggregating and degranulating effects on rabbit, guinea-
pig, dog, and human platelets (Lynch et al., 1979;
O'Flaherty and Wykie, 1983). In addition, PAF has the

ability to aggregate and degranulate rabbit and human

75

neutrophils in nonomolar and lower concentration. With
similar potency it stimulates human neutrophils Chemotaxis
and oxidative metabolism, hnuman monocyte aggregation,
infiltratdxni of neutrophils into rabbit skin, and various
other actions. Platelet activating factor does not activate
rats platelet (Camussi et al., 1982; O'Flaherty and Wykie,
1983; Shaw et al., 1981).

In 1981, Shaw et al., studied the effect of AGEPC on
human neutrophil exocytosis, migration, superoxide
production and aggregation over a concentration range of
10'10 and 1075M. AGEPC is a class of lipid mediator
documented to be functionally and structurally identical to
rabbit-derived platelet-activating factor. In this study,
AGEPC was prepared by partial chemical synthesis from beef
heart choline plasmalogen. AGEPC-induced exocytosis of
azurophilic (myeloperoxidase and beta-glucuronidase) and
specific (lactoferrin and lysozyme) lysosomal granules was
rapid (Tl/2 = 20 seconds). Similar to C5a, secretion caused
by PAF was minimally affected by the absence of
extracellular Ca*+, but was significantly inhibited by
replacing glucose in the media by 2-deoxyglucose. The
enzyme release was markedly temperature dependent, and did
not occur at 4°C. The process was dependent on the presence
of cytochalasin B, but was unassociated with release of
cytoplasmic IJHL. PAF caused neutrophil migration into

cellulose filters over a concentration range of 10"10 to

76

10'5M. A gradient analysis of this migration showed that
AGEPC induced migration was primarily chemotactic in nature,
with little stimulation of random migration (chemokinetic
stimulation). An unusual characteristic for both enzyme
release and migration was a decrease in response between
10'6M and 10'5M AGEPC. The authors suggested that this
decreased responsiveness might be due to rapid neutrophil
desensitization occurring at high AGEPC concentration,
limiting the overall cellular response. The degree of
desensitization for lysozyme and MP0 secretion was dependent
(M1 the concentration of AGEPC during the initial exposure
and desensitization was much more complete for the
azurophilic granule marker, MPO, than for the specific
granule enzyme lysozyme.

The PAF-induced desensitization for secretion appeared
to be stimulus specific, in that neutrophils desensitized
for subsequent challenge with PAF, responded normally to
C5a. Moreover, neutrophils preincubated with C5a prior to
the addition of CB, responded normally to AGEPC, whereas
they were fully desensitized for lysozyme and MP0 release by
subsequent challenge with C5a.

Neutrophil aggregation and superoxide production
occurred upon exposure to AGEPC. However, in comparison
with secretion and Chemotaxis, superoxide production and

aggregation failed to show a peak or plateau of the response

77
relationship, possibly indicating different control
mechanisms for limiting these processes.

Studies have documented that neutrophils activated with
different stimuli are able to release PAF (Camussi et al.,
1977 8 1980; Virella et al., 1982 & 1983).

131.1977 Camussi et. al. studied the effect of immune
complexes, complement and neutrophils on human and rabbit
mastocytes and basophils. They showed that: 1) in the
presence CHE immune complexes, with or without the
complement, neutrophils released not only lysosomal enzymes
but also neutrophil cationic proteins (CP) which were
capable of degranulating human basophils and human and
rabbit mastocytes; 2) C3a and C5a anaphylatoxins, generated
during complement activation by immune complexes, had an
effect on basophils/mastocytes similar to that of CP; and 3)
histamine and PAF released upon stimulation of basophils and
mastocytes by CP and anaphylatoxins. Aggregation of
platelets and release of their vasoactive amine content
occurred following the exposure of platelets to PAF. The
release of PAF by anaphylatoxins and CP was an active
process, which was suppressed in the absence of Ca++ and at
low temperature. The effect of immune complexes on
neutrophils was enhanced when neutrophils were incubated
with immune complexes in Ab excess in the presence of serum.
These investigators suggested that basophils, mastocytes and

platelets become involved in inflammatory reaction not only

78
by way of the IgE-mediated mechanism but also as a result of
the interaction of immune complexes with complement and
immune complexes with neutrophils.

The intravascular release of PAF was demonstrated after
the intravenous injection of immune complexes which
temporarily correlated with the development of neutropenia.
Furthermore, Camussi et. al.(1982) showed that in gixg
injection of purified PAF into rabbits lead both to
formation of intravascular neutrophil aggregates and to
development of acute neutrOpenia , which had the same
features as those observed after challenge with immune
complexes, C5a and CP. They suggested that immune complex-
induced neutropenia is either due to CSa production or to
the interaction between immune complexes and neutrophil
surface receptors, resulting in phagocytosis of immune
complexes and release of neutrophil CP. Both C5a, CP and
phagocytosis are effective stimuli for the release of PAF
from neutrophils. Platelet activating factor in turn

aggregates neutrophils which i vivo could embolize to

 

microvascular sites, thus possibly playing a pathogenic role
in several human immunopathologic states (Issekutz, 1984).
Virella et a1. (1982) investigated the interactions of
immune complexes and human neutrophils. They found that
human neutrophils can be stimulated by large aggregated
complexes (heat-aggregated IgG, chemically polymerized IgG,

or heavily aggregated human complexes) and by surface-bound

79

immune complexes to release enzymes (lysozyme, beta
glucuronidase) and PAF which are able to induce platelet
aggregation and ATP release from the platelets. Surface-
bound immune complexes were most effective in stimulating
the release of PAF. They used several substrates for their
preparation: plastic-absorbed antigen, sepharose-coupled
antigen and polymerized antigen.

In another study Virella et al. (1983) reported that
incubation of human neutrophils with homologous red blood
cells (RBC) preincubated with soluble immune complexes
resulted in the activation of neutrophils. The
corresponding neutrophil supernatants were able to induce
platelet aggregation. The stimulation by RBC was also
complement-independent and the immune complexes prepared in
antigen excess were usually more effective in stimulating
neutrophils. They observed that significant immunoglobulin
binding only occur when the RBC were incubated with antigen
antibody complexes, and not with antibody alone, the
incubation) of immune complex-coated RBC was an effective
stimulus for the release of PAF by neutrophils.

Lotner et al. (1980) reported the release of PAF from
both human and rabbit neutrophils after phagocytic stimulus.
Human neutrophils from nonatopic individuals were isolated
and incubated with opsonized zymosan. They found that the
resultant supernatant had the ability to react with washed

rabbit platelets and initiate aggregation and release of

80

previously incorporated tritiated serotonin, and with human
platelets to induce aggregation.

Betz and Henson (1980) studied the generation of human
PAF using neutrophils from normal and Chronic granulomatous
disease (CDG) donors. The release of PAF occurred in
response to different neutrophils stimuli, indicating PMA
and the calcium ionophore A23187, which initiate the
relatively selective release of specific granule consti-
tuents. The release of PAF was dissociable from neutrophils
degranulation and was dependant on the presence of extra-
cellular Ca++. Furthermore, cytochalasin B was required for
either enzyme or PAF release by soluble stimuli; however, it
had little or no effect on ZC-induced (serum-opsonized
zymosan) PAF production at concentrations that consistently
enhanced secretion. They suggested that, cytochalasin B may
not be required for PAF production, but only for release and
that once the signal for production has occured, release
will follow as a natural consequence in the presence of
cytochalasin B. The release of PAF was also normal in
neutrophils from patients with chronic granulomatous
disease, indicating that the formation and release of PAF
was not dependent upon an intact superoxide generating
pathway.

With the use of appropriate inhibitors, it has been
documented that the release of PAF is independant of both

adenosine-diphosphate (ADP) and arachidonic acid (AA)

81
pathways. Albumin and calcium are required for PAF acti-
vity: It appears that albumin binds to PAF and stabilizes

its activity (Camussi et al.,1981; Lotner et al. 1980).

C.5. Release of leukotreines

Phospholipids are the major constituent of cell
membranes and are subjected to degradation by phospho-
lipases. Phospholipids contain a glyceral backbone which is
joined to two fatty acid chains and a phosphate group, which
is iJI turn attached to another small hydrophilic compound
such as ethanolamine, choline, inositol, or serine (Figure
R.2) (Alberts et al., 1983; Nalbandian and Henry, 1978).
Each phospholipid molecule has a hydrophobic tail, composed
of two fatty acid chains, and a hydrophilic polar head
group, where the phosphate is located.

Phospholipase A2 cleaves unsaturated fatty acids from
the phospholipid, leaving a by-product of lysophospholipid.
Other phospholipases such as A1, C, D or B can cleave bonds
at additional sites of the phospholipid. If the fatty acid
released by phospholipase A2 contains 20 carbons and four
double bonds it is called an eicosatetraenoic acid. The
prefix eicos indicates 20 carbons, and tetra-enoic indicates
four double bonds; 20:4 is the symbolic representation of

that combination.

82

 

P139 Aimed (GHIVHHIVSNHD

 

 

P199 43 19.5 (GHLVHHLVSD

 

 

   

 

G L Y C E'R O L

PHOSPHATE

—'

   

 

 

 

 

 

'

(TEASE

 

 

 

 

 

Figure R.2. General Structure of a Phospholipid

83

Arachidonic acid is 5, 8, 11, 14 eicosatetraenoic acid.
The numbers designate the location of it's double bonds.
Once freed from the phospholipid, arachidonate can be
metabolized by at least two distinct pathways: (1) the
lipoxygenase pathway, whose products are the S-hydroxyei-
cosatetraenoic acids (S-HETE) and leukotrienes, and (2) the
cyclooxygenase pathway, whose products are prostaglandins,
prostacyclin (PGIZ), and thromboxane (Figure R.3).
Leukotrienes and prostaglandins are formed in many tissues

of the body.

C.5.1 Lipoxygenase Pathway

The lipoxygenase pathway has been described in
relatively few cell types: platelets, neutrophils,
lymphocytes, mast cells, macrophages, testis, a line of rat
basophilic leukemia cells (RBL-l), and skin (Borgeat and
Sameulsson, 1979; Stenson and Parker, 1979). The products
of the lipoxygenase pathway are mono-, di-, and trihydroxy
fatty acids. They are formed by the addition of molecular
oxygen to arachidonic acid to form unstable hydroperoxy
fatty acid intermediates. These peroxide intermediates are
then reduced, either enzymatically by a peroxidase or
spontaneously, to more stable hydroxy fatty acids. There
are a number of different hydroxy fatty acids formed through
the lipoxygenase pathway by different cell types.

Neutrophils make predominately S-hydroxy-é, 8, 11, 14-

MEMBRANE

84

figfifigfigu’g‘amffiigfié §§§§£§§
WE'VE?“ '2 = WW?)

Arachidonic acid

 

CY CLOOXYGEINASE POXY GENASE
PATH/M . PA’I‘I-lWAY.

5- -Lipoxygenase

  
    

 

 
 

 

Prostaglandins 5—HPEI'E
P602 TXAZ Dehydrase
PGE ' S-HEI'E
PGFZa - leukotriene A4 (LTA4)
, GZutathione—
PGIZ S— trans ferase
H dro lase Leukotriene C4 (L'IC4)
Glutamyl
I transpeptidase
leukotriene B4 ‘ -
(L134) LTD4
Non-enzymatic ‘
, L'IE4
_ H 0 _ K-Glutamyl
5,6 DHEI‘E...Z__ 55,12 mm , ”-———— transpeptidase
LTF4

12-o-Methy1 derivative SRS-A; LTC4. LTD4, & LTE4

Non enzymatic degradation products

 

 

 

Figure R.3 Metabolism of arachidonic acid ( AA ).

85
eicosatetraenoic acid (S-HETE), and 5, 12-dihydroxy-6, 8,
10, 14-eicosatetraenoic acid (5, 12-diHETE, leukotriene B).
In addition, neutrophils also make 8-HETE, 9-HETE, and 11-
HETE in small quantities. It is not clear if these are all
products of the same enzyme or if there is a series of
distinct but related lipoxygenases.

In neutrophils arachidonic acid is converted to an
unstable peroxide, S-HPETE (5-hydroperoxyeicosatetraenoic
acid) by the S-lipoxygenase (Figure R.3). S-HPETE can be
reduced to S-HETE and also can be converted to the unstable
expoxide leukotriene A4 by leukotriene A synthetase.
Leukotriene A4 (LTA4) is converted by enzymatic hydration
(i.e., leukotriene A hydrolase) to leukotriene B4 (LTB4) and
by tflua addition of 6-S-gluthathione to leukotriene C4
(LTC4). Alternatively, LTA4 can undergo nonenzymatic
degradation. The nonenzymatic degredation products have
much less biologic activity than LTB4 (Figure R.3).
Leukotriene C4 is metabolized to leukotriene D4 by
elimination of glutamyl residue. Further transformation of
LTD4 by elimination of glycine yields leukotriene E4.
Together LTC4, LTD4 and LTE4 form what has been known for
many years as the slow-reacting substance of anaphylaxis
(SRS-A). LTC4, LTD4 and LTE4 (cysteine-containing
leukotrienes) are released from leukocytes and have been
found in lung tissue after antigenic challenge. LTC4, LTD4

and LTE4 are bronchoconstrictors and increase the

86
permeability of microvasculature, while LTB4 is chemotactic
and chemokinetic for human leukocytes (Goetzl, 1983;
Samuelsson, 1983; Stenson et al., 1984).

In addition, further metabolism of leukotrienes may
convert the primary principles to uediators of different
activities or to inactive products (Figure R.4). The
conversion of LTB4 to 20-hydroxyl-LTB4 and 20-carboxy1-LTB4
depresses the neutrophil chemotactic potency and the smooth
muscle contractile activity in several in 23532 systems
(Ford-Hutchinson et al., 1983; Goetzl, 1983; Hansson et al.,
1981). The term "leukotriene” was chosen because the
compounds were discovered in leukocytes and the common

structure feature is a conjugated triene.

C.5.2 Leukotriene B4 Biosynthesis by Neutrophils and Other

 

Cell Types

 

Studies have shown that exposure of neutrophils to
different stimuli results in the release of the LTB4.
Leukotriene B4 is produced by other leukocyte preparations
including human eosinophils (Goetzl, et al., 1980),
monocytes (Ferreri et al., 1986), and both resting and
elicited rat peritoneal macrophages (Doig and Ford-
Hutchinson, 1980). It has also been detected in perfusates
from isolated guinea-pig lungs challenged with antigen
(Morris et al., 1979). In 2319, LTB4 has been shown to be

present in amniotic fluid embolism (Azegami and Mori, 1986),

87

MEMBRANE PHCSPHCHIPIDS

Phospholipase A2

(Arachidonic acid

S-HPETE

 

 

S-HETE
l‘r/////EIAQ

LTBl) LTCl-l LTD!)

LIE“

20-0H-LTB
4
LTC4-SULFOXIDES
LTC4-SULFONE
20-COOH-LTB4 1

6-TRANS ISOMERS
OF LTB4

Figure 3. 4 Fatabolism of leukotrienes .

88
in synovial fluid from patients with either non-inflammatory
arthropathies or rheumatoid arthritis (Klickstein et al.,
1980). Leukotriene B4 has also been found in rat peritoneal
fluid five minutes after the anaphylactic challenge (Bray et
al., 1981) and is present in the sputum of patients with
cystic fibrosis (Cromwell et al., 1981).

In 1979, Borgeat and Samuelsson studied the effects of
ionophore A23187 and the exogenous arachidonic acid (AA) on
LTB4 synthesis by human neutrophils. Addition of AA to the
suspension of neutrophils led to variable results when cells
from several subjects were compared. Some cell preparations
showed little activity with respect to the conversion of
added AA to LTB4. However, addition of A23187 together with
AA caused a strong conversion of the fatty acid into LTB4.
Ionophore A23187 alone also had strong stimulatory effects
on the synthesis of LTB4 from endogenous substrates. These
investigators suggested that, ionophore A23187 not only
activates Ca‘H-dependent phospholipase, it might also
directly activate the enzymatic system (or unmask it's
activity) involved in the formation of LTB4.

Jubiz et al., (1982), studied the LTB4 synthesis by
stimulating neutrophils with FMLP. Human neutrophil
suspension (10 ml, 100x106 cells/ml) was incubated with FMLP
(final concentration ranged from 10"5 M to 10'8M) for 10
minutes. ‘After extraction and purification, HPLC was

performed and the amount of LTB4 metabolites were estimated

89

from the peak area. The predominant product was 20-
carboxyl-LTB4 (20-COOH-LTB4), which was derived from LTB4 by
lo-oxidation. A dose-response relationship between FMLP
concentration and the amount of 20-carboxyl—LTB4 released
was observed. The amount of 20-carboxyl-LTB4 recovered was
290110 ng at 10'5M FMLP, 160:5 ng at 10'7M FMLP, 100_+_60 at
10'8 FMLP and 10:10 ng at zero concentration of FMLP. In
addition to the dicarboxylic acid (20-COOH-LTB4), two other
compounds were isolated for the FMLP incubation, including
the 20-hydroxyl-LTB4 (20-OH-LTB4) compound. The two
metabolites of LTB4 (i.e., 20-carboxyl-LTB4 and 20-
hydroxyl-LTB4) exhibited chemotactic properties for human
neutrophils but were less active in this respect than the
parent compount, LTB4.

Phagocytosis of serum-coated particles (i.e., zymosan
and staphylococcus aureus) by human neutrophils have shown
to induce the release of LTB4. Henricks et al., (1985),
reported that human neutrophils challenged with serum-coated
staphylococcus aureus in the presence of exogenous AA
released LTB4. The release of LTB4 was dependent on the
number of bacteria ingested by the neutrophils. In a
similar study, Claesson et al., (1981), demonstrated that
ingestion of serum-treated zymosan particles by human
neutrophils resulted in the release of LTB4 from the
endogenous substrate. Increased levels of cyclic AMP led to

complete inhibition of LTB4 synthesis. Since cyclic AMP is

90
an important mediator of many processes in leukocytes, these
investigators suggested that elevated cyclic AMP levels
might decrease Chemotaxis by inhibiting the fOrmation and
liberation of LTB4.

Release of LTB4 and LTC4 from human monocytes
stimulated with aggregated immunoglobulins (Ig) was examined
by Ferreri and his coworkers (1986). They found that
aggregated IgG, IgA and IgE, but not IgM or monomeric IgG,
human myeloma proteins stimulate peripheral blood monocytes
to release LTC4 and LTB4. Release of leukotrienes was Ca++
dependent. Phagocytosis of Ig aggregates was not required
for the leukotrienes release, since cytochalasin B did not
inhibit the release. In fact, in the case of IgG, release
(If the leukotrienes was actually potentiated several-fold.
Similar effects of cytochalasin B on the release of LTB4
from monocytes has been reported by Williams et al.,(l986).
They reported that FMLP only initiated the generation of
LTB4 from monolayers of human monocytes pretreated with
cytochalasin B. Ferreri and his coworkers suggested that
cross-linking of Fc receptors by aggregated Ig induced
synthesis of LTB4 and LTC4 by human monocytes.

The role of functional microtubules for the formation
of LTB4 by neutrophils was investigated by Reibman and her
associates (1986). Colchicine or vinblastine (i.e.,
microtubular-distruptive agents) decreased the formation of

LTB4 and S-HETE from human neutrophils stimulated with

91
A23187 in the absence of exogenous AA even more than in its
presence. It was pointed out that colchicine did not act by
inhibiting the uptake and utilization of exogenous
arachidonate, In“: either decreased the release of
arachidonate from membrane phospholipids, or altered the
interaction of arachidonate with the lipoxgenases. In
addition, colchicine might have modulated the synthesis of
lipoxgenase products by an effect on cyclic AMP.
Distruption of microtubules by colchicine has been shown to
increase the levels of cyclic AMP in the neutrophil (Keller
et al., 1984; Rudolph et al., 1977). Increased levels of
cyclic AMP has also been shown to inhibit LTB4 synthesis by
neutrophils stimulated with serum-treated zymosan (Claesson
et al., 1981). The distruption of microtubules by
colchicine might have led to an increase in cyclic AMP
levels, which might then have inhibited the formation of the
LTB4 and S-lipoxygenase products. Therefore, it appears
that intact functional microtubules and the presence of Ca++
are essential for optimal activity of the lipoxygenase

pathway by human neutrophils.

C.5.3 Effects of LTB4 on Neutrophil Functions
Saturable, sterospecific receptors (a mean of 4400
high-affinity and 270,000 low-affinity) for the potent

chemotactic factor of LTB4, on human neutrophils have been

92
identified (Kreisle and Parker, 1983). The binding of LTB4
to its receptor initiates activation of the neutrophil.
Leukotriene B4 enhances the neutrophils adhesiveness

which may play an important pathophysiologic role in vivo.

 

Ringertz and his associates (1982), studied the effect of
LTB4 and its metabolites on human neutrophil aggregation.
The peak response occurred for LTB4 at 10'7M and for FMLP at
10'5M, but, while the lowest concentration of FMLP that
caused aggregation was 10'9M, LTB4 had some effect even at
10'10M. The 20-hydroxy1-LTB4 was more active than 20-
carboxyl-LTB4, and almost as active as LTB4, but did not
initiate any aggregation at 10'10M.

Gimbrone et al., (1984), examined the role of
leukotrienes in the regulation of neutrophil adhesion to
cultured endothelial cells. They observed that LTB4 could
effectively enhance neutrophil adherence to endothelial cell
surfaces in contrast the LTC4, LTD4 and LTE4 that had little
or no effect on human neutrophill adhesiveness. The
response was dose related and sensitive to the protein
(i.e., Albumin) content of the incubation medium. At near
physiologic albumin concentration (40 mg/ml), activation was
noticeable at >10‘8M of LTB4, whereas at lower albumin
concentration, (1 mg/ml), the threshold was reduced to
(10'11M. At 10‘6M of LTB4, the magnitude of adhesiveness
was enhanced several fold. These investigators also studied

the effect of the S-HETE (i.e., leukocyte-S-lipoxygenase)

93

and the 12-HETE (i.e., platelet 12-lipoxygenase) that has
been reported to have chemoattractant activity. Both S-HETE
and 12-HETE enhanced neutrophil adherence to serum- or
fibronectin-coated coverslips, but neither compound had a
significant effect on neutrophil adherence to endothelial
monolayers. In addition, LTC4 and LTD4 that have been shown
to increase neutrophil adherence to Sephadex G-25, were
ineffective in stimulating neutrophil adherence to
endothelial monolayers. These investigators suggested that
the mechanism of neutrophil adherence to endothelial cell
surfaces may be qualitatively different than that involved
in neutrophil interactions with artificial surfaces.
Leukotriene B4 may modulate leukocyte-vessel wall inter-
actions.

A similar study has been done by Palmblad and her
associates (1981), who investigated the effects of LTs on
neutrOphil adherence to nylon fibers and migration under the
agarose gel. They found that LTB4 stimulated the migration
of neutrophils under the agarose gel with an optimum
concentration at 10'5M. Whereas the 20-carboxyl-LTB4
induced this response at 10‘5M. The LTC4 and the 5-HETE did
not influence neutrophil migration. At the same optimum
concentration LTB4 and 20-carboxyl-LTB4 enhanced neutrophil
adherence to nylon fibers. However, neither LTs nor S-HETE
stimulated tflue spontaneous or phagocytosis-associated

chemiluminescence, nor the bactericidal capacity. Migration

94
of neutrophils through human endothelial monolayers has also
been stimulated by LTB4. Whereas, LTC4 and LTD4 were
inactive as chemoattractants for neutrophil migration
(Hopkins et al., 1984).

The release of lysosomal enzymes from neutrophils by
leukotrienes have been studied (Hafstrom et al., 1982;
Showel et al., 1982). LTB4 was found to induce a signifi-
cant release of lysozyme and beta-glucuronidase from
cytochalasin B-treated human neutrophils at 10'5M, but not
at lower concentrations. Neutrophils not treated with
cytochalasin B also released small amounts of lysozyme but
not of beta-glucuronidase once stimulated with 10'5M of
LTB4. However, 20-carboxy1-LTB4, LTC4 or S-HETE did not
stimulate secretion of any of the enzymes. The release of
both enzymes from cytochalasin B-treated human neutrophils
by LTB4 at 10'5M was approximately half of that detected
after stimulation with FMLP at the same concentration
(Hafstrom et al., 1981). Showell and his associates (1982),
examined the secretory activity of LTB4 toward rabbit
neutrophils. They observed that LTB4 (10'7M), induced a
substantial release of lysozyme into the extracellular
medium, which was absolutely dependent on the presence of
cytochalasin B, and was enhanced by extracellular calcium.
This study indicated that rabbit neutrophils responded to
exogenous LTB4 in a significantly different manner than

human cells.

95

C.5.3.l Mechanism (S) of LTB4-Neutrophil Activation

Based on the similarities between the effects of LTB4
and chemotactic peptide (f-Met-Leu-Phe, FMLP) on
neutrophils, it has been suggested that a common mechanism
is responsible which leads to neutrophil activation (Molski
et al., 1981; Shaafi et al., 1981). The activation of
neutrophils by chemotactic factors and other stimuli is
believed to be mediated by a rise in the level of
intracellular calcium and/or activation of the protein
kinase C system (Lew et al., 1984; Shaafi et al., 1981;
Volip et al., 1984). Protein kinase C is widely distributed
in tissues and organs (Nishizuka, 1984 and 1986). The
activation of protein kinase depends onCa++ as well as
phospholipid, particularly phosphatidylserine. Chemotactic
peptide (i.e., f-Met-Leu-Phe) and LTB4, activate several of
the neutrophil responses by increasing the concentration of
intracellular free calcium by mobilization of the same pool
(Goldman et al., 1985; Molski et al., 1981; Volpi et al.,
1984). Although similar in many ways, the activation of the
neutrophil by these two stimuli exhibits significant and
important differences. For example, leukotriene B4, like f-
Met-Leu-Phe, mobilizes calcium and is a potent chemotactic
agent. On the other hand, LTB4 is less potent for cell
degranulation and a very poor stimulus for the metabolic
burst. In addition, contrary to f-Met-Leu-Phe, it does not

stimulate tflua production of leukotrienes, or S-HETE, from

96
endogenous arachidonate, nor does it stimulate the release
of platelet-activating factor (Hafstrom et al., 1981;

Palmblad et al., 1981; Prescott et al., 1984).

97

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

IC NEUTROPHILS C'
'T' '1- ‘T‘
Activation .‘
Activation C b
" C —-- C3a ——- C3ai
Activation by C5 CSa CSal
chemotactic ’ Helper factor
factors. Activation
FI: .-
ANAPHYLATOXINS ; Cause;
v 1- Smooth muscle contraction.
2- Histamine release from mast
cells.
3- Enhanced vascular permeability.
# causes anaphylactic symptoms in
_47 subhuman primates.
p’
l- Lysosomal release, CP. /
2_ 02 ml 1 lites. l- Stimulates neutrophils and
eosznophils.
3- LTB4.-= _, ’- 2— Enhances proliferation of
4_ PAF t J suppressor T-lymphocytes.
K .: ' 3- Inhibits proliferation of
* * helper T-lymphocytes.
. . 4— Suppresses proliferation of
Actiration “' IgG-producing B-lymphocytes
. (mouse).
Release ‘MAST ',
4
BASOPHILS.
(Causes; 1- Enhanced vascular permeability,
2- Platelet aggregation. ‘———}
L... Serotonin release,
Prostaglandins,
Arachidonate, ADP, and etc.
Acts as chemotactic factor.-.————L'IB4
‘\ .
L‘ :‘ I

 

 

 

 

 

NEUTROPHIL ACETVATION.

 

Overview.

SECTION I

THE EFFECTS OF N-PORNYL-L-NETEIONYL-L—PEENYLALANINE,
LEUKOTRIENE-El, AND TEMPERATURE TRANSITION ON THE
EXPRESSION OP C3b, C3bi, AND PC RECEPTORS ON HUMAN

NEUTROPHILS IN WHOLE BLOOD AND ISOLATED NEUTROPHILS

98

MATERIALS AND METHODS

I. PREPARATION OF CHEMOTACTIC SOLUTIONS

A. Chemotactic Peptides

The synthetic chemoattractant, N-formyl-L-methionyl-
phenylalanine (f—Met-Phe), was obtained from Sigma Chemical
Company, St. Louis, MO., and stored at -70°C as a 10‘3M
stock solution in 0.1% dimethyl sulfoxide. As needed, the
10'3M f-Met-Phe solution was thawed and diluted to working
concentrations.

B. Leukotriene B4

 

Leukotriene B4 (LTB4) (MW. 336) was obtained from
Calbiochem, Behring Diagnostics, La Jolla, CA, 92037. An
aqueous stock solution of 10'4M was prepared and stored
frozen under liquid nitrogen. As needed, an aliquot was
diluted further with Hanks' buffer (HBSS) to the desired

concentration.

II. MONOCLONAL ANTIBODIES
Monoclonal antibody to the human neutrophil Fc receptor
was kindly provided by Dr. J.C. Unkeless, Mt. Sinai
Hospital, School of Medicine, New York. The anti-human Fc
receptor was a mouse immunoglobulin of the IgG1 class (3G8).
The Fc receptor antibody was diluted ten-fold with PBS
99

100
containing 5.0% bovine serum albumin (BSA) and stored at-
7OQC. Monoclonal antibody to the human C3b receptor was
obtained from Dako Corp., Santa-Barbara, CA. The anti-C3b
receptor was a mouse immunoglobulin of the IgG1 class, and
stored in the dark at 4-6°C as recommended. Monoclonal anti-
human C3bi receptor (i.e., M01) was a mouse IgM which was
purchased from Coulter Immunology, Hialeah, Fdorida. A
mouse monoclonal antibody against human-T4 antigen which was
IgG1 and a purified mouse IgM were also obtained from
Coulter Immunology. Fluorescein isothiocyanate (FITC)-
labeled goat F(abr)2 anti-mouse immunoglobulins (FITC-GAM)
was purchased from Cappel, Cooper Biomedical, West Chester,

PA.

III. BLOOD COLLECTION

 

Peripheral blood was collected in acid citrate dextrose
(ACD) solution (Bectin-Dickinson, Rutherford, New Jersey) by
the standard venipuncture technique from healthy adult
volunteers, who had not taken non-steroidal anti-inflam-
matory drugs during the preceeding 10 days. All the
experiments were immediately performed after the blood was

drawn.

IV. ISOLATION OF HUMAN NEUTROPHILS
Five milliliters of citrated blood was overlaid on 4-5

ml of Ficoll-Hypaque (2.16 grams Ficoll, 3.396 grams

101
hypaque; q.s. 34 ml distilled water) (Sigma Chemical
Company, St. Louis, MO), and centrifuged at 800 G for 30
minutes at 4°C. Ficoll-Hypaque solution provided a density
gradient to separate granulocytes and erythrocytes from
platelets and other leukocytes in the peripheral blood.
After centri- fugation, the supernatant was removed by
suction and the cell button, consisting of granulocytes and
erythrocytes, was resuspended in 9.0 m1 of CA++-free HBSS.
A 1.0 ml of a 6% (W/V) solution of dextrafi (70,000 MW; Sigma
Chemical Company) in Ca++-free.HBSS was mixed with the 9.0
m1 cell suspension to enhance erythrocyte sedimentation.
The mixture was incubated for 45—60 minutes at 20°C. The
granulocyte rich supernatant was removed and washed with an
equal vodume of HBSS. The cells were then resuspended in
HBSS and counted by an electronic cell counter (Coulter
Counter, Model 231). This cell suspension contained greater
than 90% granulocytes, of which approximately 95% were
neutrophils. Rare platelets or mononuclear cells were
observed in the suspension and the neutrophil-to-erythrocyte
ratio was consistently greater than 5:1. The neutrophil
viability was greater than 98% as determined by trypan blue
(0.04% W/V) exclusion. The neutrophils were resuspended at

107/ml in HBSS and were used within one hour of isolation.

102

V. NEUTROPHIL PRETREATMENT
Neutrophil suspensions (106 cells/0.1 ml HBSS) or whole
blood (0.1.lnl) were incubated with 0.1 m1 of a specified
concentration of f-Met-Phe, LTB4, or HBSS for a selected
time interval and temperature. The treated cells while in
the pretreatment solution, were then brought to 4°C and
immunofluorescence studies were performed on the cells. All
cell pretreatments were carried out in polystyrene micro-

centrifuge tubes (1.2 ml).

VI. IMMUNOFLUORESCENCE STUDIES

An indirect immunofluorescent procedure was employed
using the different monoclonal antibodies and an FITC-
conjugated goat anti-mouse IgG. The experiments were
carried out at 4°C. For the procedure, 0.1 ml of whole
blood was added to a 1 ml polystyrene microcentrifuge tube
containing 0.1 ml PBS, LTB4, or f-Met-Phe as described in
Neutrophil Pretreatment. The contents of the tubes were
gently mixed using a Vortex mixer and incubated at the
desired temperature for 10 minutes. After incubation, the
different monoclonal antibodies, Fc, M01, C3b, T4 and mouse
IgM were added in excess, as determined in preliminary
saturation experiments, and the mixtures were incubated at
4°C for 10 minutes. The blood was washed by centrifugation
three times using HBSS or 10’6M f-Met-Phe and then the

fluorescein-conjugated goat anti-mouse IgG solution was

103

added and incubated with the cells for an.additional 10
minutes at 4°C. The blood was washed an additional three
times and the erythrocytes were removed using 1 ml of lysing
reagent prepared by adding Coulter Immunolyse concentrate to
HBSS at a 1:25 dilution. After incubation in the lysing
reagent for 20 seconds, 250 pl of Coulter fixative was added
to each tube and the cells were washed 2-3 times until the
residual erythrocytes were removed. All washes were
performed by centrifugation at 600 g for one minute in a
microcentrifuge (Model 59A, Fisher) using 1 m1 HBSS or 10'5M
f-Met-Phe. When isolated neutrophils in suspension were
used, the lysing step for erythrocytes was omitted.

The cell-surface fluorescence was analyzed using an
EPICS-V flow cytofluorograph (Coulter Electronics, Inc.,
Hialeah, FL.) set at 488 nm. Forward light scatter, right
angle light scatter (i.e., 90°LS), and green fluorescence
*were measured as each cell passed through the argon laser
beam. Fluorescence emission was logarithmically amplified
and displayed as a fluorescence profile histogram.
Fluorescence intensity of neutrophils was reported as the
mean fluorescent channel, average intensity of fluorescence
emitted by at least 10,000 cells measured. The T4 mono-
clonal antibody and a Mouse IgM were used to determine the

non-specific binding to neutrophil surface.

104

VII. STATISTICAL EVALUATION

 

The data are expressed in terms of a mean +_ standard
error of the mean; n representing the number of separate
experiments and in all cases, the number of separate donors.
The analysis of variance (ANOVA) and the least significant
difference (lsd) tests were used to assess significance

(Steel & Torrie, 1985).

RESULTS

I. 93b, C3bi, AND FC RECEPTOR EXPRESSION BY HUMAN

 

NEUTROPHILS IN WHOLE BLOOD

 

The assay for assessing the expression of C3b, C3bi and
Fc receptor antigens on neutrophils was performed by
staining the cells in whole blood with different monoclonal
antibodies at 4°C. After staining the cells were analyzed
using a flow cytometer that simultaneously measured the
forward angle light scatter (FALS), right angle light
scatter (90°LS), and fluorescence of each cell as it passed
through an argon laser beam. Polymorphonuclear leukocytes,
monocytes, and lymphocytes have different light-scattering
properties, and are represented by three distinct clusters
When 90°LS and FALS signals are displayed as a cytogram.
Each cluster can be gated and the fluorescence of cells
comprising the cluster can be measured. Figure 1.1 shows
the cytograms of peripheral blood leukocytes.
Immunofluorescent analysis showed a distinct difference in
neutrophil expression of C3b, C3bi and Fc receptors. C3b
and C3bi receptors were minimally expressed on the plasma
membrane of resting neutrophils, while Fc receptors were
expressed in greater amounts as indicated by the difference
in the mean fluorescent channel distribution (Figure 1.2).

105

Figure 1.1

106

200

150

100

50

(channel number)

10

 

FORWARD ANGLE LIGHT SCATTER (FALS)

1 50 100 150 200 250

RIGHT-ANGLE LIGHT SCATTER (90 L3)
(channel number)

Cytogram produced by the forward light scatter (Y
axis) and right-angle light scatter (X axis) of
cells within the whole blood preparations after
the erythrocytes being lysed. Signals for
neutrophils, monocytes, lymphocytes, erythrocytes
and cell debris have been recorded at different
regions of the cytogram according to their light-
scattering properties. The dotted lines that form
a box in the cytogram delineate the neutrophil
clusters and select these cells for analysis of~
immunofluorescence.

Figure 1 . 2

107

 

Cbntnfl. CIbi

400
((EUII)

L

1

300

 

200

100

 

 

400

300

200

NIMBER OF WPHIIS/ CHANNEL

100

 

 

 

 

 

 

Quantitation by flow cytofluorography of C3b, C3bi
and Fc receptors on resting neutrophils in whole
blood. Samples (0.1 m1) of freshly drawn
anticoagulated peripheral blood were immediately
placed at 4°C. NeutrOphils were stained for the
antigens using monoclonal antibodies and FITC-
conjugated F(ab')2 fragments of goat anti mouse
immunoglobulins as described in Methods. A mouse
IgG1 anti-T4 and a purified mouse IgM were used to
determine the non-specific binding to neutrophil
surface and served as controls. Relative
immunofluorescence of neutrophils was determined
using the Coulter EPICS-V. Ten thousand cells were
analyzed. The cytograms depict the number of
neutrophils (Y axis) having variable amounts of
fluorescence (X axis).

108
II. EFFECTS OF TEMPERATURE TRANSITION ON MEMBRANE

EXPRESSION OF C3b,§3§_j;' AND FC RECEPTORS

 

 

To determine the possible effect of temperature
irransition on C3b, C3bi and Fc receptor expression, freshly
drawn blood was anticoagulated, then immediately divided
:into four samples designated as A, B, C, and D. All samples
were incubated at different temperatures as following. A
éand B samples were placed at 4°C, and C and D samples were
Theld at 37°C. To D sample, the chemotactic factor f-Met-Phe
(final concentration 10’6M), was added. All the samples (A,
B, C, and D) were incubated for 10 minutes. After the first
incubation, sample B that was held at 4°C was transferred
into 37° CL (All blood samples were then incubated for an
additional 10 minutes. At the end of the second incubation,
all the samples (A, B, C, and D) were brought to 4°C and the
neutrophils were stained for the receptor antigens and
assessed for fluorescence. Neutrophils in the whole blood
samples (i.e., A and B) maintained at 4°C or 37°C had a low
mean fluorescence. However, neutrophils in whole blood
samples (i.e., C) that had been incubated at 4°C for the
first incubation and then transferred into 37°C for the
second incubation had significantly increased the expression
of their receptors relative to the cells held at 4°C (Table
1.1). Addition of f-Met-Phe to the whole blood also caused
a significant increase in the expression of C3b, C3bi and Fc

receptors on neutrophil surfaces (Figure 1.3).

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110

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 1.3

111

 

 

 

         

 

 

 

 

3
co : (M01)
. g C3bi
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‘ 50 m '50 m 50 m ‘50 am

Flue re scence channel number

Expression of receptors on neutrOphil in
whole blood. The broken lines represent
neutrophils in whole blood stained for the
receptors, and incubated at 4°C for 10
minutes, as described in Methods. The dotted
lines represent neutrophils in whole blood
incubated in 10‘6M f-Met-Phe for 10 minutes
at 37°C, and stained for the antigens using
monoclonal antibodies as described. T4
monoclonal antibody represents the
nonspecific binding to neutrophil surface.
Relative immunofluorescence of cells was
determined using the Coulter EPICS-V. The
number of neutrophils (10,000 analyzed) is

plotted against log green fluorescence
(Channels 1-255).

112

The percentage of positive neutrophils labeled with
C3b, C3bi and EC receptor antibodies were analyzed. The
results showed a significant effect of f-Met-Phe stimulation
on the percentage of neutrophils expressing C3b and C3bi
receptors, while no effect was observed on the percentage of
neutrophils expressing Fc receptors (Table 1.2). More than
94% of neutrophils expressed Fc receptor on their surfaces
before and after incubation with f-Met-Phe. Less than 10%
of neutrophils exhibited the C3b receptor before incubation
with f-Met-Phe; while 94% expressed this receptor following
a short incubation with chemotactic peptide. For the C3bi
receptor, 45% of neutrophils expressed this receptor before

incubation with f-Met-Phe and 98% did after incubation.

III. EFFECTS OF LTB4 ON MEMBRANE EXPRESSION OF C3b, C3bi,

AND FC RECEPTORS

To assess the influence of LTB4 on neutrophil receptor
expression, 0.1.rm1 of fresh blood was incubated with
different concentrations of LTB4 (10x10'12M to 5x10'8M) at
37°C. After 10 minutes, the different monoclonal antibodies
and FITC-F(ab')2 goat anti-mouse immunoglobulins were added
as described in methods. The expression of C3b, C3bi and Fc
receptors (n1.neutrophils were all increased significantly
after incubation with LTB4 at 10'10M to 5 x 10'3M
Concentrations. When compared to neutrophils in whole blood

incubated with mass (Figure 1.4).

113

Table 1.2 Percentage of positive neutrophils with various

 

 

antibodies.
Receptors % Fluorescence*
on N P**
Neutrophils PBS f-Met-Phe
(10‘6M
C3b 6 6 94 SD
C3bi (M01) 6 45 98 SD
Fe 6 94 95 NS

 

Stimulated and non-stimulated neutrophils in whole blood
were stained for C3b, C3bi and Fc receptors at 4°C.
Relative fluoresence of cells was measured using the Coulter
EPICS V.

(*)

(**)

Results are expressed as the percentage of positive
neutrophils expressing various receptors on their
plasma membranes. These were calculated by subtraction
of background fluoresence after labeling with control
antibodyu 4A Coulter MDADS using the IMMUNO software
program was applied for calculation of data. Ten
thousand cells were analyzed.

Statistical analysis was done using the T-test variance
by transformation of the percentage data into arcsine
value at the P-value of 0.05.

(N) Represents the number of experiments.

114

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115

 

 

 

 

 

 

 

 

 

 

 

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Figure 1.4 Effect of LTB4 on the expression of C3b, C3b' , and Fc receptors on neutrophils in whole blood.

116
Under this condition, LTB4 at lO‘llM and 10'12M did not
significantly effect neutrophil receptor expression for C3b,

C3bi or Fc.

IV. EFFECTS OF THE ISOLATION PROCEDURE ON RECEPTOR

EXPRESSION

 

To determine the possible effects of the isolation
procedure on receptor expression, anticoagulated blood from
three donors was divided into paired aliquots and
neutrophils were isolated from one aliquot by the standard
Ficoll-Hypaque centrifugation as described in Methods. The
isolated neutrophils were then used for the experiment and
stained with monoclonal antibodies for different antigens.
The other blood aliquot was used as whole blood and
immediately stained for the receptors. Neutrophils in whole
blood had low mean fluorescence for C3b and C3bi receptors
(i.e., '7 and 25) compared with the fluorescence levels of
these receptors on the isolated neutrophils (i.e., 46 and
82) (Table 1.3). Addition of f-Met-Phe or LTB4 to PMNs in
whole blood or neutrophils in suspension induced a
significant increase in C3b and C3bi receptor expression on
neutrophils in whole blood and on the isolated neutrophils.
However, there was no significant effect of stimulation of
Fc receptor expression on isolated neutrophils. The
increased Fc receptor expression was only significant on

neutrOphils in whole blood.

117

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118

 

 

 

 

 

 

 

 

 

 

 

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DISCUSSION

The purposes of the study in this section (I), are to
l) investigate the expression of C3b, C3bi and Fc receptors
on human neutrophils in whole blood, 2) evaluate the effect
of chemotactic factors (i.e., f-Met-Phe and LTB4) and
temperature transition on the expression of these receptors
in neutrophils, and 3) to examine the differences, which
might exist, between the isolated neutrophil and the
neutrophil in whole blood as they relate to surface receptor
expressions.

In this study, monoclonal antibodies and
cytofluorography were used to assess the expression of
receptors for C3b, C3bi and Fe on the surface of human
neutrophils.

The results indicate that resting neutrophil in whole
blood expressed relatively few receptors for C3b and C3bi,
but expressed greater numbers of surface receptors for Fc
(Figure 1.2). With neutrophils isolated using Ficoll-
Hypaque gradients and maintained at 4°C, the results were
different for C3b and C3bi receptor expressions than those
<xf neutrophils in whole blood. As the data demonstrate in
Table 1.3, the isolation procedure significantly increased
the expression of C3b and C3bi, but not the Fc receptor. A

119

120

6.5-fold increase for C3bi and a 3.2-fold increase in C3bi
receptor expression was observed upon isolation. Part of
these results are consistent with the observations of Fearon
and Collins (1983) who reported a spontaneous increase in
the expression of C3b receptors on human neutrophils due to
the isolation procedure. However, contrary to the results
presented here, Berger et al., (1984), reported the
existence of no significant difference in the expression of
C3b and C3bi receptors between the isolated neutrophils and
the neutrophils in whole blood. This difference could be
due to the assay system. In the whole blood assay used by
Berger et al., (1984), after addition of monoclonal anti-
bodies, the whole blood was subjected to a rotator for 30
minutes at 4°C. The buffy coat cells were then obtained by
centrifugation, and erythrocytes were lysed by a hypotonic
procedure. The leukocytes were washed again and then
stained with FITC-conjugate. Therefore, it is possible that
the rotatory action and the isolation of the buffy coat
cells before the completion of immunofluorescent staining
have activated neutrophils. As a result, a higher baseline
level for C3b and C3bi receptor expression on neutrophils
might have been obtained. In the whole blood assay applied
in this study, neither a rotator was used nor were the buffy
coat cells isolated prior to the completion of the staining.

Activation of the isolated neutrophils or neutrophils

in whole blood with either of the chemotactic factors (i.e.,

121

f-Met-Phe or LTB4) induced a significant increase in C3b and
C3bi receptor expression on neutrophils (Table 1.3). The
magnitude of stimulation for both the isolated neutrophils
and the neutrophils in whole blood was similar. However, f-
Met-Phe or LTB4 stimulation did not significantly enhance
the up-regulation of Fc receptors on the isolated
neutrophils when compared with isolated cells in the buffer.
Stimulation with chemotactic factors induced only a
significant increase in the expression of the Fc receptors
on neutrophils in whole blood. Of particular interest is
the presence of a significant difference between the
stimulated-isolated neutrophils and the stimulated
neutrophils in whole blood in regards to their Fc receptor
expressions. As the data demonstrate in Table 1.3, isolated
neutrophils stimulated with chemotactic factors expressed
significantly less Fc receptors than the stimulated
neutrophils in whole blood. Further studies are necessary
to clarify the possible mechanism(S) involved in the
alteration of Fc receptor expression apparent in the
isolated neutrophils.

The results in this study clearly indicate that LTB4, a
major product of 5-lipoxygenase pathway in activated
neutrophils, can stimulate the expression of C3b, C3bi and
Fc receptors on the neutrophil surface. A noticeable
enhanced expression of these receptors (i.e., C3b, C3bi and

PC) was observed at 10’10M concentration of LTB4. The

122

up-regulatixni of surface receptors reached its maximum at
approximately 5 x 10'9 to 10 ’8M concentration of LTB4 (5 x
10'3M was the hughest LTB4 concentration used in this
study). The results may suggest that modulation of these
receptors by LTB4 could partly account for the activation of
neutrophils seen in gitgg and in gizg. Previous in gitgg
experiments have shown that LTB4 stimulated the adhesion of
neutrophils to endothelial cell surfaces, and enhanced the
migration of neutrophils through endothelial monolayers
(HOpkins et al., 1984; Gimbron et al., 1984). In 2129
studies have demonstrated neutrophil accumulations following
the injection of LTB4 into the rabbit eye and guinea-pig
peritoneum. Neutrophil accumulation in humans has also been
observed following application of skin chambers containing a
solution of LTB4 over skin abrasions on the forearm.
Additionally, in the skin of the rabbit, in the presence of
vasodilator (i.e., PGEZ) and neutrophils, the leukotrienes
produced a marked increase in vascular permeability, which
appeared to result from the interaction of neutrophils with
vascular endothelial cells (wedmore and Williams, 1981).
Therefore, LTB4 may have an intracellular role as a
modulator of neutrophil functions partly through its action
upon these receptors.

Neutrophils in whole blood exposed to a temperature
transition also exhibited an enhanced expression of C3b,

C3bi and Fc receptors on their surfaces. The purpose of this

123

study was to find out if a temperature transition could
contribute to the up-regulation of receptors. Several
studies have reported that a temperature transition (i.e.,
40C --> 37°C) can stimulate neutrophils adhesiveness and the
release of granule contents from these cells (Charo et al.,
1985; Goldstein et al., 1974; Heerdt, 1986). Additionally,
different laboratories have reported data regarding the
expression of surface receptors which contradict each other.
As a result, uninterpretable information has been reported,
and in some cases, technical procedures have been suggested
to be implicated in these discrepancies.

In the most common standard technique for isolation of
neutrophils, whole blood (with 37°C body temperature) is
subjected to erythrocyte sedimentation at room temperature
(RT) (i.e., approximately 18-22°C) or at 37°C for 45-90
minutes. Then the leukocyte rich plasma will be washed, and
leukocytes will be fractionated by passage through a
specific gradient using centrifugal force. This step will
usually take place at 4°C or RT. After isolating the
neutrophils, the cell suspension will be refrigerated until
the performance of the experiments which generally are
carried out at 37°C. Therefore, it is obvious that neutro-
phils will be exposed to different temperatures during the
isolation.

Berkow and associates (1983), reported that, compared

to neutrophils isolated with ficoll/hypaque gradients, cells

124

obtained by the elutriation technique generated signifi-
cantly more superoxide anion and released significantly more
vitamin B12 binding protein, lysozyme and beta-glucuronidase
when stimulated with FMLP in the presence of cytochalasin B.
In contrast to this report is the study by Heerdt (1986) who
studied the effect of temperature transition and technical
procedures on the release of lactoferrin (specific granules)
from neutrophils. She applied two different techniques, the
ficoll/hypaque gradient and the elutriation technniques for
the isolation of neutrophils. She reported that the
technique played no apparent role on the extracellular
granule release. Temperature transition (i.e., 37°C, body
temperature --> 4°C, isolation temperature --> 37°C,
experimental temperature) played the key role in the release
of granule contents.

In this study, in order to control the temperature
transitions and prevent the effect of other factors (e.g.,
centrifugal force, washing, and etc.) during neutrophil
preparations, the experiment was designed so that the
isolation procedure was omitted. A whole blood assay was
developed and the effect of temperature on the expression of
neutrophils was studied. All the necessary reagents and
tubes were prepared and left at the desired temperature
prior to the blood drawing. The experiment was carried out
immediately after the blood was drawn. Blood samples were

incubated at the desired temperature for 10 minutes, then

125

subjected to a temperature transition and incubated for
another 10 minutes. The expression of the receptors was
evaluated immediately by staining the neutrophil receptors
in the whole blood at 4°C as described in Methods. As the
data demonstrates in Table 1.1, when blood was kept at 4°C
for ten minutes and then warmed to 37°c, neutrophils
expressed ea significantly increased up-regulation of C3b,
C3bi and Fc receptors. In contrast, neutrophils in whole
blood kept at either 4°C or 37°C for the entire incubation
period and not subjected to a temperature transition
exhibited no significant differences in their receptor
expression. The data suggests that the up-regulation of
these receptors is through a metabolic process(es) which can
be initiated by a temperature transition or a chemotactic
stimuli. This metabolic activity appears to occur at a much
slower rate by temperature manipulation, since the up-
regulation of these receptors did not reach maximal
expression, similar to those induced by chemotactic factors.
However, it should be considered that, a longer incubation
time might be essential for the maximum receptor expression
to occur by temperature transition. Further investigation is
required to clarify this matter.

In considering if the C3bi receptor is partially
related to neutrophils adhesion to albumin-coated surfaces
or cultured endothelial cells, the results presented here

for the expression of C3bi are consistent with the

126
observations of Charo et al., (1985). These investigators
reported that a temperature increase from 4°C to 37°C
enhanced neutrophil adhesion to albumin coated plastic and
cultured endothelial cells.

The data presented above appear to suggest that
temperature transition has a significant role in the
discrepancy among data reported by different laboratories.
However, centrifugal force, the action of breaking the cell
pellet, and other cell manipulation during the isolation
procedure should not be neglected. The whole blood assay
developed in this study appears to eliminate many of the
problems (i.e., cell loss, damage, activation, etc.) which
are associated with neutrophil isolation. It provides an
assay system with less uncertainty about different factors
which might interfere with the overall interpretations. In
addition, the expression of receptors is examined in whole
blood milieu, which represents a more relevant physiologic
condition.

In conclusion, the results reported here indicate that
1) unactivated neutrophils in whole blood exhibit a minimal
number of C3b and C3bi receptors, while expressing a much
greater number of receptors for Fc on their surfaces, 2)
activation of neutrophils in whole blood by f-Met-Phe, LTB4,
or temperature transition (ite., 4°C --> 37°C) induced a
significant up-regulation of C3b, C3bi, and Fc receptors on

their plasma membranes, 3) although the up-regulation of

127

C3b, C3bi and Fc receptors by temperature transition was
significantly enhanced on neutrophils in whole blood, it was
not near the maximal expression of these receptors when
induced by the chemotactic peptide, f-Met-Phe, 4) isolated
neutrophils iJ1(a non-stimulatory medium had significantly
more receptors for C3b and C3bi on their surfaces than
neutrophils in whole blood, isolation had only a moderate
effect on the up-regulation of the Fc receptor, and 5)
activation of the isolated neutrophils with f-Met-Phe or
LTB4 induced only a significant expression of C3b and C3bi
receptors on their surfaces.

The results reported here raise the possibility that;
1) LTB4 which has been considered a potent mediator of
hypersensitivity and inflammation, may modulate neutrophil
functions by it's effect on the expression of C3b, C3bi and
Fc receptors, becoming involved in the regulation of
leukocyte-endothelial cell adhesion. 2) The discrepancy
existing among different reports regarding the up-regulation
of neutrophil receptors might be due to the temperature
transitions which occur during the isolation of neutrophils.
One particular issue is Fc receptor expression, where up-
regulation may be a physiologic process obscured by cell
isolation procudures. in The whole blood assay developed
in this study may facilitate assessment of neutrophil
responsiveness in clinical and research laboratories, by
eliminating many of the difficulties associated with

neutrophil isolation.

SECTION II

THE EFFECTS OF SOLUBLE AND INSOLUBLE IMMUNE COMPLEXES

ON THE EXPRESSION OF C3b, C3bi, AND Fc RECEPTORS

ON HUMAN NEUTROPHILS IN WHOLE BLOOD

128

MATERIALS AND METHODS

I. PREPARATION OF CHEMACTIC SOLUTION

 

A. Chemotactic Peptides
The synthetic chemoattractant, N-formyl-L-methionyl-L-
phenylalanine (f-Met-Phe), was prepared as described in

Section I.

II. MONOCLONAL ANTIBODIES

 

Monoclonal antibody to human Fc, C3b, C3bi and T4
antigens were all prepared as described in Methods of

Section I.

III. IMMUNE COMPLEXES

 

Immune Complexes (ICs) were prepared with normal human
serum albumin (HSA) (Sigma Chemical Company) and rabbit IgG
antibodies (Cappel, Cooper Biomedical Inc., Malvern, PA) to
HSA.

A. Preparation of Monomer Albumin by High-Performance

Liquid Chromatography

Human Albumin was gel filtered on Waters PROTEIN-
PAKBOOSW column (7.5 mm (ID) X 300. mm) (Waters, Millipore
Corporation, Milford, Mass) using high-performance liquid

chromatography (HPLC). PROTEIN-PAK3oosw column is packed

129

130

with a rigid, hydrophilic porous silica gel, separates
proteins ranging in molecular weight from 10,000 to 500,000
Daltons (native globular). The column provides rapid
separation, purification and characterization of proteins.
PROTEIN-PAR column accomadates flow rates of up to two
ml/min., providing high efficiency separations in minutes
compared to hours or days required to accomplish separations
with conventional soft gel techniques. This column can be
used to isolate up to 50 mg of protein per separation. The
Perklin-Elmer TriDetTM Detector/30TM HPLC (Norwalk, Conn.)
support was used. The 3D HPLC system consists of three
units, TriDet Detector, Series 100 Pump, and the R50
Recorder. The series 100 Pump is for the accurate delivery
of mobile phase. The sample to be analyzed is loaded into
the injector. The sample flows from the injector through a
packed column (in this case, Waters PROTEIN-PAK3OOSW) and
IJHH) the unique trifunctional detector flowcell. The
flowcell is capable of simultaneously monitoring three
physical properties of the sample (ultraviolet absorbance,
fluorescence and conductivity). The R50 recorder receives
the detector output from only one channel and displays it in
chart form (chromatogram).

The column was equilibrated with 0.1 M Potassium
Phosphate dibasic, pH 7.0, at a flow rate of 1. ml per
minute. ‘The column was calibrated with the following
substances: Blue dextram (2000 X 103 M.W.), catalase (210 X

103 M.W.), aldolase (158 x 103 M.W.), human albumin

131

(66 X 103 M.W.) ovalbumin (43 X 103 M.W.), chymotrypsinogen
A (25 X 103 M.W.) and ribonuclease A (13.7 X 103 M.W.).
Concentrations of the standard solutions were at 10 mg/0.1
ml buffer. A 0.05 ml of each standard solution was
separately loaded into the injector, and the protein
molecules were scanned using an ultraviolet (UV) mode. The
retention time for each protein standard was recorded and a
calibration (standard) curve was prepared (Table 2.1 and
Figure 2.5). ;A four-protein mixture of blue dextran,
catalase, human albumin and ribonuclease A was used to
observe tine resolution, and each protein fraction was
collected at their main peak. Each protein fraction was
identified by it's specific retention time using the
standard curve, and was verified by electrophoresis (Figure
2.1 and 2.2). A solution of human serum albumin (HSA) was
prepared at 250 ug/ml buffer, clarified by centrifugation if
necessary. 0.1 ml of HSA solution was loaded into the
injector, the monomer albumin molecules were identified by
their specific retention time using the standard curve, and
were collected (Figure 2.3). The protein content of the
monomer albumin solution was determined using a modified
Folin method (Appendix A).
B. Preparation of Immune Complexes

Immune complexes were prepared by adding a constant
amount of anti-HSA antibody (i.e., 0.2 ml antibody which
contained 1000 ug antibody protein) to various amounts of

antigen HSA (i.e., 5 to 1000 pg). After incubation for one

132

lhour at 37°C and overnight at 5°C, the complexes were
centrifuged at 2000Xg for 15 minutes at 5°C. The complexes
were then washed twice with cold PBS and resuspended in 1 ml
of PBS. The total protein in the precipitates was measured
by a modified Folin method (Appendix A) with human IgG
(Accurate Chemical and Scientific Corp., Westbury, New York,
11590) used as the standard. By quantitative precipitin
analysis equivalence was found to be 10 pg HSA per 100 pg
antibody (Figure 2.4). For these studies, insoluble immune
complexes were prepared at equivalence, and soluble
complexes were made in antigen excess as indicated in
Results. For some experiments, labeled immune complexes
were used in which a fluorescein isothiocyanate-(FITC)
labeled rabbit (IgG) anti-human albumin (Cappel, Cooper
Biomedical) was used as the antibody. Also, immune
complexes which lack the Fc portion of their antibody were
prepared. These complexes were made of rabbit F(abr)2 anti-
human albumin and monomer HSA. In addition, a different
type of immune complex was made. In this complex, a ggat
anti-human albumin (Cappel, Cooper Biomedical) was used as
the antibody source.

All the products were sterilized as provided by the
companies and the reagents were examined for endotoxin
contamination using a Limulus Amebocyte Lysate (LAL) test

(Appendix B).

133

 

Protein Molecular Retention Time
Weight (Minutes)
Blue Dextran 2000 4.2
Catalase 210 X 103 6.6
Aldolase 158 x 103 6.9
Albumin (Human) 66 x 103 7.3
Ovalbumin 43 x 103 8.1
Chymotrypsinogen A 25 x 103 9.2
Ribonuclease A 13.7 X 103 9.4

 

Table 2.1 Retention time for different protein molecules
using a Waters PROTEIN-PAK3OOSW Column and a 3D
HPLC support. 2-5 mg of different protein
molecules loaded into a 3D HPLC support. Column:
7.5 mm X 300 mm Waters PROTEIN-PAK3OOSW; mobile
phase; 0.1 M potassium phosphate dibasic, pH 7.0;
flow rate: 1 ml/minute.

134

Peak # Protein MW
1) Blue Dextran 2000 X 103
2) Catalase 210 X 103
3) Human Albumin 66 X 103
4) Ribonuclease A 13.7 x 103

(1)

 

<2)
(3) (4)

Absorbance (UV)

I NJ ECT

 

 

 

 

 

 

 

 

 

TIME (minute)

Figure 2.1 Analysis of a protein mixture using a 3D HPIC support.
Colulm: 7.5 m X 300 m Waters Pml'EIN-PAK3OOSW;
Mobile phase: 0.1 M phosphate dibasic, pH 7.0;
Flowimue: :lnfl/mnnue.

 

135

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136

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Figure 2.3

Absorbance (UV)

INJECT

 

137
(2)

Mobile phase:

0.1M potassium phosphate
dibasic

pH=7.0

Flow rate;

1 mLmun.

(1)

 

 

 

I I A L A l A A A A

1 2 3 4 5 6 7 8 9 10 11 12

TIME (minute)

Chromatogram of human serum albumin using a
Waters PROTEIN-PAK3OOSW Column and a 3D HPLC
support. 0.1 ml of HSA solution (250 pg/ml)
loaded into the injector, and ultraviolet
(UV) mode was selected to scan the albumin
molecules, and the Chromatogram was recorded.
By applying the calibration curve and the
retention time, it is apparent that peak #1
is correspondent to polymers (retention time
= 6.2 minutes; MW > 200 X 103) and peak #2 is
correspondent to monomers (retention time =
7.3 minutes; MW = 66 X 103).

my Jul "I01 I up‘f) 1

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138

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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150 200‘"J :500

I
-11. ----.. .. __1 -_ 1., -1

20 30 4O 50 100
ANTIGEN CONCENTRATION ( pg protein )

Histogram of the immunoprecipitin occuring
between anti-HSA (Rabbit) and monomer HSA.
Varying concentrations of monomer HSA (S to
1000 pg protein) were added to a constant
concentration of antibody (i.e., 1000 pg
antibody protein). After incubation for 1
hour at 37°C and overnight at 5°C, the
complexes were centrifuged and washed 2 times
with coLd PBS. The total protein in the
precipitates was measured. The various
concentrations of antigen (X-axis) is plotted
against the total protein in the
precipitates. The data demonstrates the
point of equivalence to be 100. pg of HSA per
1000 pg of antibody protein (i.e., 1:10 Ag/Ab
ratio).

 

 

139

 

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_._.__.————’

utmfififlzn.2 3 4 5 6 7 8 9 10, 11 12 13 14
RETENTION TIME (minute)
Figure 2.5 The standard curve of different molecular weight

proteins using a 3D-HPLC system.

140
IV. BLOOD COLLECTION
Peripheral blood was collected from healthy adult

volunteers as described in Methods, Section I.

V. UPTAKE OF IMMUNE COMPLEXES BY HUMAN NEUTROPHILS IN

WHOLE BLOOD

 

0.1 ml of whole blood was added to a 1 ml polystyrene
microcentrifuge tube containing 0.1 ml PBS or f-Met-Phe
(final concentration = 10’5M). The contents of the tubes
were gently mixed using a Vortex mixer and incubated for 10
minutes at 37°C. The treated cells while in the pretreatment
solution, were then incubated with different types of FITC-
immune complexes for 15 minutes at 37°C. The samples were
then washed three times with magnesium-free HBSS and the
erythrocytes were removed using 1 ml of a lysing reagent
prepared by adding Coulter Immunolyse concentrate to Mg++-
free HBSS at a 1:25 dilution. The contents of the tubes
were immediately mixed, then after 20 seconds 0.25 ml of
Coulter fixative was added to each tube and mixed well using
a pipet. The cells were then washed 2-3 times with HBSS
until the residual erythrocytes were removed. All washes
were performed by centrifugation at 600 g for one minute in
a microcentrifuge (Model 59A, Fisher) using 1..m1 Mg++-free
HBSS. Florescence of neutrophils was analyzed using an
EPICS-V flow cytofluorograph (Coulter Electronics, Inc.,

Hialeah, FL) set at 488 nm. Forward angle light scatter

141

(FALS), right angle light scatter (i.e., 90°LS), and green
fluorescence were measured as each cell passed through the
argon laser beam. Fluorescence emission was logarithmically
amplified and displayed as a fluorescence profile histogram.
Fluorescence intensity of neutrophils was reported as the
mean fluorescent channel, average intensity of fluorescence
emitted by at least 10,000 cells measured. In addition,
neutrophils were mounted on glass slides, coverslipped and
examined for immunofluorescence using a Zeiss microscope
with UV illumination.

In a few experiments, neutrophils in whole blood were
exposed to immune complexes which were not conjugated with
FITC. The presence of the immune complexes on the surface
of neutrophil was demonstrated by staining the complexes
with the FITC-antibody against them. In duplicate samples,
neutrophils in whole blood (0.1 ml) were pretreated with
cytochalasin-B (10 pM) for five minutes at 37°C. The
treated cells were then incubated with immunce complexes
while in the pretreatment solution as described at the

above.

1H5 UPTAKE OF SOLUBLE IMMUNE COMPLEXES BY HUMAN

ERYTHROCYTES

 

1 ml of citrated blood was placed into a FALCON
polystyrene conical tube (17 X 120 mm, Becton Dickinson,

Lincoln Park, New Jersey), and washed three times with

142
saline. Then, a 20% solution of erythrocytes was prepared
by resuspending the cell pellet in Mg++-free HBSS. 0.1 ml
of erythrocytes was added to a 1..m1 polytyrene micro-
centrifuge tube containing different concentrations of FITC-
labeled soluble immune complexes or HBSS. The contents of
the tubes were gently mixed and incubated at 37°C. After 15
minutes, erythrocytes were washed two times with MgII-free
HBSS, and the cells were then fixed with 1% paraformal-
dehyde. To examine the presence of FITC-labeled soluble
immune complexes on erythroc tes, the cells were mounted on
glass slides, coverslipped, and examined using a Zeiss

fluorescence microscope.

VII. NEUTROPHIL PRETREATMENT

 

Neutrophils in whole blood (0.1 ml) were incubated with
0.1 ml of a specific concentration of f-Met-Phe, soluble
immune complexes, insoluble immune complexes, or MgH-free
HBSS for 15 minutes at 37°C. The treated cells in whole
blood were washed twice with Mg++-free HBSS and divided into
two groups for different experiments. For the first group,
the treated cells were brought to 4°C and stained for C3b,
C3bi and Fe antigens by an indirect immunofluorescent
technique using monoclonal antibodies as described in
Methods, Section I. For the second group, the treated cells
were exposed to f-Met-Phe (final concentration 10'5M) for

ten minutes at 37°C. The cells were then stained for C3b,

143

C3bi and Fc antigens at 4°C by an indirect immunofluorescent

technique.

VIII. STATISTICAL EVALUATION

The data are expressed in terms of a mean 1 standard
error of the mean; n representing the number of separate
experiments and in all cases, the number of separate donors.
The analysis of variance (ANOVA) and the least significant

difference (lsd) tests were used to assess significance.

RESULTS

I. UPTAKE OF IMMUNE COMPLEXES BY HUMAN NEUTROPHILS IN

WHOLE BLOOD

 

A. Uptake Of Insoluble Immune Complexes By Neutrophils

These experiments were performed with insoluble
(precipitated) immune complexes composed of human monomer
albumin and fluorescein conjucated IgG fraction of anti-
human albumin at equivalence as described in Methods.
Samples (0.1 ml) of freshly drawn anticoagulated whole blood
were exposed to different concentrations of insoluble
fluoresceinated-immune complexes (FL-ICs). After 15
minutes incubation at 37°C, the blood samples were washed
twice with magnesium-free HBSS, and the leukocytes fixed
after lysing tflua erythrocytes. The uptake of immune
complexes by neutrophils was quantitated by measuring the
relative fluorescence of neutrophils using the Coulter
EPICS-V. The results showed that the uptake of immune
complexes by neutrophils was in a dose-dependent manner
(Figure 2.6). Saturation of uptake by neutrophils was
achieved at a dose of 60—100 ug of antibody protein in the
immune complexes used (Table 2.2).

An immunofluorescence microscopic examination was also
performed, and neutrophils exhibited diffusely distributed

144

Figure 2.6

145

Dose-response curves for the uptake of immune
complexes by stimulated and non-stimulated
human neutrophils. Stimulated and non-
stimulated neutrophils in whole blood (0.1
ml) were incubated with FITC-immune complexes
(ICs) containing different concentrations of
antibodies to human serum albumin for 15
minutes at 37°C. Relative fluorescence of
cells was measured using the Coulter EPICS-V.
Solid lines represent neutrophils which were
exposed tn) insoluble ICs. The broken lines
represent neutrophils which were exposed to
soluble ICs prepared at 10 times antigen
excess (IC-lOXAg). (0), non-stimulated
neutrophils in whole blood incubated with
ICs. (+), neutrophils in whole blood were
preincubated with f-Met-Phe (10'5M) for 15
minutes at 37°C, and then were exposed to
ICs.

Results are expressed as the mean fluorescent
channel (Channels 1-255) which is the average
fluorescence of each population. 10,000
cells were analyzed. The concentration of ICs
is plotted against the log green fluorescence
(Channel 1-255). Representative figure from
three experiments.

 

146

     

 

200 ,
Insoluble ICS

180 b
160 (
140 ,
g 120 .
E 100 t
E 80 .
g 60 t
40 .
20 .

.— __ f- I. Soluble ICs

l
5 10 20 40 60 80 100
yg'nnmme(xnphmes
Figure 2.6 Dose-response curves for the uptake of immune

complexes by stimulated and non-stimulated
human neturophils in whole blood.

(0 ) = Non-stimulated neutrophils

(+ ) = f-Met-Phe stimulated neutrophils

147

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148

 

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149

clusters of immune complexes (Figure 2.7.a, and 2.7.b). On
rare occasions, small aggregates of neutrophils (2-3 cells)
were noticed when cells were exposed to a lower concen-
tration of immune complexes (i.e., 2.5 to 20 pg antibodies
protein) (Figure 2.7.c). As the concentration of immune
complexes immune complexes increased, over 20 pg antibody
protein, a greater aggregation of neutrophils were observed.
There was a moderate aggregation of neutrophils when the
cells were incubated with 80-100 pg of immune complexes
(Figure 2.7.d). All the experiments were performed in a
magnesium (Mg++) free system, since the presence of the Mg++
caused a strong aggregation of cells after exposure to
immune complexes.

The effects of the chemotactic factor upon the uptake
of immune complexes by human neutrophils were examined.
Neutrophils in whole blood were pre-incubated with f-Met-Phe
(10‘5M) for 10 minutes at 37°C prior to incubation with
immune complexes. The stimulated neutrophils in whole blood
were then exposed to different concentrations of immune
complexes, and the uptake was measured by flow cytometry.
Stimulated neutrophils had only a slight increase in the
uptake of the immune complexes compared to those neutrophils
which had not been stimulated with f-Met-Phe (Figure 2.6).

Insoluble immune complexes which lack the Fc portion of

the antibody were formed at equivalence. The complexes were

Figure 2.7

150

Fluorescence photomicrographs of neutrophils
bearing FITC-immune complexes. Neutrophils
in whole blood (0.1ml) were incubated with
insoluble Immune Complexes (5 g) at 37°C.
After a 15 minute incubation, cells were
washed twice with Mg**-free HBSS and the
erythrocytes were lysed. The leukocytes were
then fixed with 1% paraformaldehyde. (a and
b) Neutrophils having diffusly distributed
clusters of ICs. (c) Neutrophils were
exposed to a low concentration of ICs (i.e.,
5 pg antibody protein). (d) Neutrophils were
exposed to a high concentration of ICs (i.e.,
80 pg antibody protein). All
photomicrographs were taken using a Zeiss
microscopewith a UV light source (i.e.,
Mercury Lamps HBO). Magnification
approximately 1000 X for figure 2.7.b, and
250 X for figures 2.7.c and 2.7.d.

 

 

 

 

 

(b)

Figure 2.7 Fluorescence photomicrographs of neutrophils
bearing FITC—immune complexes.

 

(d)

Figure 2.7 Fluorescence photomicrographs of neutrophils
bearing FITC-immune complexes.

153
composed of human serum albumin (HSA) and fluorescein
conjugated F(abr)2 fragments of anti-human albumin. These
insoluble immune complexes served as negative controls.
There was only a minor uptake of these complexes by
neutrophils in whole blood.

To assess whether insoluble immune complexes were
internalized by the neutrophils in whole blood, cells were
pretreated with cytochalasin B (CB) before exposure to
immune complexes. Pretreatment of neutrophils with CB
inhibited internalization of immune complexes. Insoluble
immune complexes were rapidly ingested by the untreated
neutrophils, and were not present on the surfaces of the
cells to bind to the FTTC-antibody against them. However,
when phagocytosis of immune complexes was prevented by CB
treatment, complexes were left on the cell surfaces and

bound to the FITC-antibody against them (Table 2.3).

B. Uptake Of Soluble Immune Complexes By Human Neutrophils

In Whole Blood

 

These experiments were similar to those which were
performed with insoluble immune complexes except that
soluble immune complexes were used instead. The soluble
immune complexes were made at 10 times antigen excess as
described in Methods. The results showed that the uptake of
soluble immune complexes by neutrophils was also in a dose-

dependent manner; however, the uptake was very poor compared

154
to those of insoluble immune complexes (Table 2.2). In
immunofluorescence microscopy, neutrophils exhibited only a
very weak fluorescence with high concentrations of insoluble
immune complexes (i.e., 80 - 100 pg), and there was no

apparent clumping of cells (Figure 2.8).

 

 

Table 2.3: Uptake of Insoluble Immune Complexes* by
Neutrophils
*Relative
Source Fluorescence N
HBSS-treated neutrophils 12 i 7 2
CB-treated neutrophils 85 1 12 2

 

Neutrophils in whole blood were pre-incubated with
Hank's buffer (HBSS) or with cytochalasin B (CB) for five
minutes at 37°C. The treated cells while in the
pretreatement solution, were then exposed to insoluble
immune complexes (10 pg) for 15 minutes at 37°C. After two
washes, the cells were exposed to F(ab')2 fragments of the
antibody against the immune complexes. Results are
expressed as the mean fluorescence 1 SEM for 10,000
neutrophils.

(*) Immune complexes composed of human serum
albumin and IgG fraction of anti-human
albumin.

(N) Represents the number of experiments

155

II. UPTAKE OF SOLUBLE IMMUNE COMPLEXES BY HUMAN

 

ERYTHROCYTES

 

Since all the experiments for the uptake of soluble
immune complexes were performed on human neutrophils in
whole blood, the possible uptake of these complexes by
erthyrocytes was examined. A 20% suspension of human
erythrocytes was prepared as described in Methods. Samples
(i.e., 0.1 ml) of the erythrocyte suspension were exposed to
different concentrations of soluble immune complexes (i.e.,
5, 7.5, 10, 20, 40, 80 and 100 pg) for 15 minutes at 37°C.
Under these conditions, immunofluorescent microscopic
examination showed no apparent FITC-soluble immune complexes
on erythrocytes. Occasional fluoresceinated leukocytes

were observed (Figure 2.9).

III. MODULATION OF C3b, C3bi, AND FC RECEPTORS ON HUMAN

 

NEUTROPHILS IN WHOLE BLOOD BY IMMUNE COMPLEXES

 

These studies were performed to clarify the mechanism
whereby preincubating neutrophils with immune complexes led
to reduced phagocytic activity and decreased migration of
the cells. Neutrophils in whole blood were pre-incubated
with 5 pg of insoluble or soluble immune complexes at 37°C.
After 15 minutes, the cells were washed twice with Mg++-free
HBSS, and the cells were then stained for different antigen

receptors. In order to stimulate the maximum receptor

Figure 2.8

156

 

Fluorescence photomicrograph of neutrophils bearing
soluble FITC-immune complexes. Neutrophils in whole
blood (0.1 ml) were incubated with 80 g of soluble-
Immune Complexes for 15 minutes at 37 C. Cells were
washed twice with Mg++-free HBSS and the
erythrocytes were lysed, then the leukocytes were
fixed. Neutrophils exhibited only a very weak
fluorescence. Photomicrograph was obtained using a
Zeiss microscope with a UV source. Magnification
approximately 250 times.

Figure 2.9

157

 

Uptake of soluble FITC-immune complexes (FITC-ICs)
by human erythrocytes. 0.1 ml of a 20% suspension
of human erythrocytes incubated with 80 pg of
soluble immune complexes prepared at 10 X antigen
excess. After a 15 minute incubation at 37°c,
erythrocytes were washed twice with HBSS, and the
cells then fixed with 10% paraformaldehyde.
Erythrocytes were examined using a Zeiss
microscope with a UV and white light sources.
Note that there is no evidence of soluble FITC-
ICs binding to erythrocytes. The arrow shows a
leukocyte bearing soluble FITC-ICs. Magnification
approximately 400 times.

158

expression after the exposute of the cells to the immune
complexes, duplicate samples of whole blood were treated as
follows: As the neutrophils in whole blood (0.1 ml) were
pre-incubated with 5 pg of soluble or insoluble immune
complexes for 15 minutes at 37°C and washed twice, the cells
were then exposed to f-Met-Phe (10'5M) for 10 minutes at
37°Ch Neutrophils were then stained for C3b, C3bi and PC
antigens at 4°C.

The results showed that 5 pg of insoluble or soluble
immune complexes caused a significant increase in C3bi
receptor expression on the human neutrophil plasma membrane.
The results also indicated that this increase was near the
maximum expression of C3bi receptors as caused by f-Met-Phe
(10’5M) alone. However, when insoluble immune complexes
which lack the Fc portion on their antibodies were used, no
increase in the expression of C3bi receptors were observed.
This C3bi receptor expression was similar to that expressed
by neutrophils which were incubated in HBSS alone (Table
2.4).

Immune complexes had different effects on EC receptor
expression on the plasma membrane of neutrophils. Pre-
incubation of neutrophils in whole blood with 5 pg of
insoluble immune complexes resulted in a significant

decrease of the expression of Fc receptors when compared to

 

159

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Acme

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.ecoHe mmmm cu oeuecsocu mHHcmouucec How eecHe> ecu cuus oeuemeou Act.

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comfl.mo eoceomeuosHm emeue>m ecu mu cOucz AmmNIH mHeccchv
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¢.N manna

160

 

In; m. oH oHooHom

 

on H u omH m Izo-oH. ono-uoz-o
on m H.OHH m one: Add m. oH oHosHom
on H u omH m 12o-OH. ond-oox-u Hod m. oH oHooHomoH
om m u «HH m mmm: Add m. oH oHnsHonoH
I _ucesmmuw m..ce.mH
mz m + no m name Add m. oH oHooHonoH
on o u omH m Azo-oHt occ-uox-o Azo-oH. ocd-uox-e
N “.mo m mmmm mmmc

mcoHuHooc ecoHuuooc

..d oocoonouooHn z Aooam on .oHe oH. .oomm on .oHa mH.
ae>HueHem coHumccocH ocooem coHueccocH umuwm

 

oooHc eHoc3 cw mHHcmouucec cmecc co cOuwmeumxe
.Houmeoeu Hcmu co meermeoo ecDEEH mo uoemmm v.m eHcea

161

those of neutrophils which were pre-incubated in HBSS alone
(Table 2.5). In parallel, replicate samples of blood were
restimulated with f-Met-Phe (10‘5M), after cells were pre-
incubated with 5 pg insoluble immune complexes. This
experiment was performed to see if f-Met-Phe stimulation
would up-regulate more Fc receptors on the plasma membrane
of neutrophils. No significant up-regulation of Fc
receptors was observed on the neutrophil surface. The
relative fluorescence of Fc receptors on neutrophils
incubated with insoluble immune complexes was 99, and for
neutrophils pre-incubated with insoluble immune complexes
and then exposed to f-Met-Phe being 100 (Table 2.5).

Soluble immune complexes at 5 pg antibody protein did
not reduce the number of Fc receptors on human neutrophils
surface (Table 2.5). However, soluble immune complexes at
higher concentrations decreased the expression of Fc
receptors (n1 plasma membranes, the effect being dose-
dependent. Figure 2.10 shows the dose-response curves for
soluble and insoluble immune complexes. Statistical
analysis showed that there was an inverse relationship
between the expression of Fc receptors on neutrophil
surfaces and the concentration of immune complexes to which
cells were exposed to.

Under these circumstances, the effect of immune

complexes (n1 C3b receptor expression of human neutrophils

162
was similar to those of Fc receptors (Table 2.6). A
decreased expression of C3b receptors was observed and the
effect was dose-dependent (Figure 2.11). Soluble complexes
at low concentrations did not reduce the expression of C3b
on neutrophil surfaces. However, an inhibitory effect was
observed when higher concentrations of soluble Complexes

were used (Figure 2.11).

 

163

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.eoceuemmuo ucMOumucmue

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eoHe>Im ecu um umeu AomHv eoceuemuuo ucMOHMHcmHm umeeH ecu ocums
oeeuomuem we: comHummeoo eHmuuHDE m cecu .umeu Ae>occ. eocmfiue>
mo mumxumce ecu mcflmeae xc ecoo mm3 muthmce Heouuwuumum Mira

.ecoHe ecm
IueZIm cu oeumcoocu mHHcmouusec ecu pow mech> ecu cuu3 oeuemeou

.ecoHe
mmmm cH oeumccocu mHHcmouucec ecu pom meoHe> ecu cufi3 oeuemeou

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2mm + Heccmco uceoEeuosHm cmee ecu mm oemmeucxe eum muHsmem

65
com.

Az.

.mv

Aces.

Acev

Ate

.>-monm HeuHsou ecu ecumc pecueueueo
mm: eoceomeuoon e>HumHem .moocuez cu oecuuomeo we cmu ou hoocuuce
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(w Ho mmmc cH oeocemmcweu eHeB mHHeo ecu .mecmms ozu ueumc .oeuMOuocu
we mucemmeu mooHHm> cu oeumcsoculeum eue3 oooHc eHocB cu mHHcmouucez

m.~ eacee

 

164

 

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om H H mm H mmmm Add m. 0H oHosHonoH
l .uceemeum NA.cm.mH
mz H + HHH H mum: Add m. 0H oHosHoncH
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v H OHH H mmmm mmmm
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«e>HueHem coHumcsocH ocooem couuecnocH umuum

 

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.eoceuemuuo ucmoHMHcmHm

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erHOLHec mes cOmHHemeoo eHQHuHDE m cecu .umeu Am>occv eoceuue>
mo mumemcm ecu mcHmeme ac ecoo mez mumxHecm HmoHuwHumum ece

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)uezlm cH oeumccocH mHHcmoHucec ecu you mecHe> ecu cufiz oeummeou

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comwluo eoceomeuocHu emmue>m ecu mH coHcs AmmmIH mHeccch.
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166

 

Ami m. 0H oHooHom

 

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167

akz= feMet-Phe

160 E] = HBSS

140 H\
[3 ' =Ekflubkaoammmes;
120 "““‘-~..' b2= -o.25
r = 0.98
r = -O . 99

100

on
o
O

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RELATIVE FLUORESCENCE

b“: -o.41
A r“= 0.65
50 r = -0.81

 

S 20 40 60 80 100 120

IMMUNE COMPLEXES (‘pg )

Figure 2.10 Dose-response curves for soluble and insoluble
complexes - decreased Fc receptor expression on
human neutrophils in whole blood. Neutrophils
in whole blood preincubated with various
concentrations of immune complexes as indicated
for 15 minutes at 37°C. After two washes,
cells were stained with a monoclonal antibody
to PC antigens. Fluorescence was determined
using the Coulter EPICS V. Results are
expressed as the mean relative fluorescence for
10,000 cells. Mean fluorescence for
neutrophils stimulated with f-Met-Phe is
indicated by an asterisk (*). Each point
represents the mean for 3 determinations.

60
g 5 so
3 4.
H
3E 30
id
52 E 20
10

Figure 2.11

168

—)k = f-Met- Phe ( 10'6M )

L D=HBBS

Sohfifle<xmpham5;

V
1‘
fl

b2= -0.65

‘ r = 0.98

I- = ”0.99
’ . = Ifisohiflecxnphaes;

b = -0.36

U r2= 0.54

' r = -0.74

 

 

l l I I I I I I | I I | I
s 20 4o 80 160
MINE W ( pg)

Dose-response curves for insoluble and soluble
complexes - decreased C3b receptor expression
on human neutrophils in whole blood.
Neutrophils in whole blood preincubated with
various concentrations of immune complexes as
indicated, for 15 minutes at 37°c, After two
washes, cells were stained with a monoclonal
antibody to C3b antigens. Fluorescence was
determined using the Coulter EPICS V. Results
are expressed as the mean relative
fluorescence for 10,000 cells. Mean
fluorescence for neutrophils stimulated with f-
Met-Phe is indicated by an asterisk (*). Each
point represents the mean for 3 determinations.

120

100

80

60

40

UkundqgnznxzpuMfl
FELHEREIFDWXESCBII:

20

Figure 2.12

169

f-Met-Phe ( 10-6M )

lflES

SflxbhaICs

Ov-- C] 3F’

Ihmohflfleiflb

 

LJIIILiJJiLL

5 20 4o 80
mmm(pg)

Dose-response curves for insoluble and soluble
complexes-increased C3bi receptor expression on
human neutrophils in whole blood. Neutrophils
in whole blood pre-incubated with various
concentrations of immune complexes as indicated
for 15 minutes at 37°C. After two washes,
cells were stained with a monoclonal antibody
(i.e., M01) to C3bi antigens. Fluorescence was
determined using the Coulter EPICS V. Results
are expressed as the mean relative fluorescence
for 10,000 cells. Mean fluorescence for
neutrophils stimulated with f-Met-Phe is
indicated by an asterisk (*). Each point
represents the mean for 3 determinations.

DISCUSSION

The purpose of the study in section II was to examine
the effects of soluble and insoluble immune complexes (ICs)
on the expression of C3b, C3bi and Fc receptors on human
neutrophils. Previous studies have documented that exposure
of neutrophils to immune complexes depresses the locamotion
and phagocytic activity of these cells. C3b, C3bi and Fc
receptors are implicated in immune adherence and adhesion-
associated functions (i.e., locomotion, phagocytosis,
adhesion, etc.) of neutrophils. Thus, it was of interest to
evaluate the fate of these receptors on neutrophils which
have been exposed to immune complexes. The experiment was
designed so that the interaction between the neutrophil and
immune complexes occured in a whole blood udlieu, whnfli
represents a more relevant physiological condition.

Insoluble-immune complexes were avidly bound by human
neutrophils in whole blood. Neutrophils rapidly ingested
the insoluble-immune complexes and this process was
inhibited by the pretreatment of neutrophils in whole blood
with 5 PM cytochalasin B. However, neutrophils poorly bound
the soluble-immune complexes. The uptake of soluble-immune
complexes was approximately 1-5% of the insoluble-immune
complexes uptake by neutrophils depending on the
concentration of the complexes used. These results are

170

171
consistent with those of Starkebaum et al., (1982), who

studied the uptake of immune complexes labeled with 125

I by
isolated neutrophils. These investigators reported that
less than 1% of the soluble complexes prepared at a 3-fold
antigen excess were taken up by the neutrophils compared to
a 65% uptake of insoluble complexes made at equivalence.

The presence of a Mg++ ion in buffer solutions caused a
strong aggregation of leukocytes which occurred during
washing of the cells. Therefore a Mg++-free buffer was used
for the assay. Under the condition used in this study
(i.e., absence of Mg++ and presence of Ca++ in the buffer)
no aggregation of neutrophils was observed upon the
interaction of immune complexes and neutrophils, except when
a higher concentration of complexes (i.e., 40-100 F9) was
used in which slight aggregations were observed (Figure
2.7.d).

The uptake of immune complexes by neutrophils in whole
blood was dependent of the concentration of immune complexes
present in the media. Increasing the dose of insoluble-
immune complexes (i.e., 2.5, 5, 7.5, 10, 20, 40, 60, 80, and
100 pg) in the incubation media with neutrophils increased
the uptake of the complexes by neutrophils which reached a
plateau at concentrations higher than 20 pg immune complexes
(Table 2.2 and Figure 2.6). When insoluble-complexes which
lack tuna Fc portions on the antibody molecules were used,

only a minor uptake was observed. These results suggest

172

that the uptake of immune complexes by neutrophils in whole
blood possibly occured via Fc receptors which are saturable.

The binding of soluble-complexes (formed by complement)
tn: human erythrocytes (i.e., RBC) via C3b (CRI) receptors
have been previously reported (Schifferli et al., 1986.,
Schifferli anui Peters, 1983; Sherwood and Virella, 1986).
It was of concern in this study to find out if the soluble-
complexes bound to RBC and thus were not available for
binding to the neutrophils. Under the condition of the
assay system used in this study no apparent binding of the
insoluble-complexes to RBC was observed (Figure 2.9).
Explanation for this discrepancy is out of the scope of this
study. Since the purpose of this experiment was to find out
if under the experimental condition used in this study the
soluble-complexes will bind to RBC, not to provide an assay
system for the evaluation of immune complex binding to RBC.
However, a few points have to be mentioned. A different
assay system was used in this study. The sensitivity of the
assay used here is for neutrophils which express
approximately 135,000 Fc receptors per cell. Erythrocytes
express an average of 500-600 C3b receptors per cell. In
addition, the characteristics of antigen and antibody, the
ratio of antigen to antibody, and the physical properties of
the reaction medium (i.e., plasma, extracellular fluid,
lymph, and so forth) are very important factors which

influence immune adherence.

173

Neutrophils preincubated with insoluble-complexes
exhibited significantly reduced Fc receptors on their
surfaces. The expression of Fc receptors by neutrophils
correlated inversely with the concentrations of complexes
present during the incubation. At lower concentrations of
insoluble-complexes (i.e., S, 10 pg) a 30-40% reduction at
the higher concentrations of insoluble-complexes (i.e., 20,
40, 80 pg) :3 50-55% reduction in Fc receptors on the
neutrophil surface were observed. Soluble-complexes at
lower concentration (i.e., 5, 10 pg) slightly decreased the
expression of Fc receptors, but at higher concentrations
(i.e., 100 pg) the reduction was more noticable. A 20%
reduction in Fc receptor expression on neutrophils was
observed when cells were preincubated with 80 pg of soluble-
complexes. These results are in agreement with those of
Starkebaum et al., (1982) who utilized a different
experimental assay system to examine the effect of immune
complexes (”1 human neutrophils phagocytic function. They
observed that preincubating isolated neutrophils with
insoluble complexes caused significantly greater inhibition
of the uptake of additional insoluble complexes than did
preincubating neutrophils with an equal amount of soluble
complexes. They reported that maximum inhibition of uptake
of insoluble complexes occured only after 90 ndnutes of
preincubation of the neutrophils with immune complexes or

aggregated IgG.

174

Down-regulation of Fc receptors on the neutrophil
surface reached its maximum plateau at 40 pg concentration
of insoluble-complexes in the incubating media (Figure
2.10). It is interesting to notice that the maximum uptake
of insoluble-immune complexes by neutrophils also reached
its plateau at approximately this concentration, indicating
saturation of the receptors associated with insoluble-
complexes binding at this concentration (Figure 2.6). In
the presence of saturating levels of insoluble-complexes
approximately forty percent of the total Fc receptors on the
neutrophil were free of ligands and were available for
binding to anti-Fc antibody (i.e., 3G8). On the other hand,
pretreatment of neutrophils with anti-Fc antibody completely
inhibited the uptake of insoluble-complexes, suggesting that
Fc receptors play the main role in the banding of these
complexes to the cells. A question might be raised of, how
the anti-Fc antibody could completely inhibit the uptake of
insoluble-complexes, while the exposure of neutrophils to
saturating levels of insoluble-complexes did not block all
the available Fc receptors on the cell surface. This
question could be explained by the assumption that some Fc
receptors are present on the surfaces of neutrophils which
do not bind to the Fc portion of IgG molecules in immune
complexes, but will bind to the anti-Fc antibody. Further
study is required to find an apprOpriate answer to the above

question.

175

Immune complexes also decreased the expression of C3b
(CRI) receptors on human neutrophils in whole blood. Like
the Fc receptor, the down-regulation of C3b receptors on
neutrophils is inversely correlated with the concentration
of complexes present during incubation (Figure 2.11).
Insoluble-immune complexes at lower concentration (i.e., 5,
10 pg) induced a 30-40% reduction in the expression of C3b
receptors, while the soluble-complexes used 15-20%
reduction. A maximum reduction (i.e., 50%) of the C3b
receptor expression was observed when neutrophils were
incubated with 20, 40, (n: 80 pg of insoluble-immune
complexes. With soluble-immune complexes a 50% reduction
was observed when 80 pg of complexes were present in the
incubating media. It is interesting to notice that soluble-
immune complexes at 80 pg depressed the expression of C3b
receptors as much as the insoluble—complexes did at the same
concentration (Figure 2.11). A higher concentration of
soluble-immune complexes was not tested in this study to see
if this was the maximum reduction or not. These results
clearly indicate that C3b receptors are also associated with
the uptake of immune complexes by neutrophils.

Results of the effect of soluble-immune complexes on
C3b and Fc receptors show that the soluble-complexes induced
a greater inhibition of C3b receptors (i.e., at 80 pg IC -->
50% reduction) than of Fc receptors (i.e., at 80 pg IC -->

20% reduction). These results may suggest that C3b

176

receptors are associated with the uptake of soluble-immune
complexes more than the Fc receptors are. It is suggested
that the primary function of the C3b (CRI) receptor on
neutrophils and monocytes is to mediate or enhance the
endocytosis of soluble complexes and particles to which C3b
has bound covalently (Abrahamon and Fearon, 1983; Fearon,
1986). In addition, in vitrg study has shown that binding
of soluble antibody/d3 DNA-ICS to neutrophils occurs
principally via the CRI receptor (Taylor, et. al., 1983).

It was of interest to see if the reduction of the C3b
receptors on the neutrophil was due to the fact that they
luui not been fully up-regulated on the cell surface.
Therefore, after the neutrophils were incubated with immune
complexes and washed twice, they were exposed to the
chemotactic factor, f-Met-Phe. No up-regulation of the C3b
receptors on neutrophils preincubated with 5 pg of
insoluble-immune complexes was observed upon restimulation
wild: f-Met-Phe (Table 2.5). In fact, there was a
significant down-regulation of C3b receptors on these
neutrophils (i.e., preincubated with 5 pg of insoluble
complexes) restimulated with f-Met-Phe when compared with
the untreated neutrophils (i.e., preincubated in buffer
only) stimulated with f-Met-Phe. On the other hand,
soluble-immune complexes at a low concentration (i.e., 5 pg)
which did not induce a great inhibition of C3b receptors,

significantly enhanced the up-regulation of C3b receptors on

177
neutrophil surfaces (Table 2.6). The results indicate that
a low concentration of soluble-immune complexes enhances the
up-regulation of C3b receptors and a higher concentration of
soluble complexes enhances the down-regulation of these
receptors.

‘When neutrophils were treated with soluble-immune
complexes lacking the Fc portions on their antibody
molecules, no changes were observed in the expression of C3b
receptors. The level of C3b receptor expression was equal
to that of cells in the control buffer solutions. These
results may indicate that the attachment of immune complexes
to neutrophils is initiated via the interaction between the
Fc portion of IgG and the Fc receptors of the cell.

So far, the results demonstrated that interaction of
immune complexes with neutrophils depressed the expression
of C3b and Fc receptors on the cell surface. The decreased

neutrophil phagocytic activity which have been shown in vivo

 

upon exposure to immune complexes could be due to down-
regulation of C3b and Fc receptors. In addition the data
may suggest that the decreased expression of these receptors
could be responsible for the impaired phagocytic functions
found with neutrophils from some patients with rheumatoid
arthritis and systemic lupus erythematosus.

The effect of immune complexes on the expression of
C3bi (CRIII or M01) was different from those of C3b and Fc

receptors on neutrophils in whole blood. Soluble and

178

insoluble-immune complexes induced a significant up-
regulation of C3bi receptors at different concentrations of
immune complexes (i.e., 5,20, 40, and 80 pg) (Figure 2.12).
In fact, there was no apparent difference in the magnitude
of the enhanced receptor expression of 5 pg of soluble and
insoluble-immune complexes (Table 2A). Insoluble-immune
complexes lacking Fc portions on their antibody molecules
did not enhance the expression of C3bi (CRIII) on
neutrophils. Restimulation of immune complexes-pretreated
neutrophils with f-Met-Phe slightly increased the expression
of C3bi receptors above the level of expression observed
vdJfli untreated neutrophils activated with f-Met-Phe. The
data presented here may suggest that the inhibition of
neutrophil migration induced by immune complexes in vitro,
might be due to a significant increase in the number of C3bi
receptors on neutrophil surfaces. The C3bi (CRIII) receptor
is associated with the adherence property of the neutrophil.
Upon exposure of neutrophils to immune complexes, they
become very adherent to the substratum, resulting in their
depressed locomotion.

In conclusion, the results presented in this section
(II) indicated that: 1) neutrophils in whole blood avidly
bound and ingested the insoluble-immune complexes, the
uptake of complexes was directly correlated with the
concentration of complexes pmesent in the blood, and the

uptake was saturable, 2) the uptake of soluble-immune

179

complexes by neutrophils in whole blood was 1-5% of the
insoluble-immune complexes. Similiar to those of insoluble
immune complexes, the uptake of soluble-immune complexes was
directly related to the concentration of complexes present
in the blood. 3) Insoluble-immune complexes induced a
significant down-regulation of Fc receptors on the
neutrophil surface. Soluble-immune complexes at higher
concentrations had a similar effect on Fc receptors though
of'ai lesser magnitude. 4) Insoluble-immune complexes
depressed the C3b receptor expression on the neutrophil
surface. Although, the soluble-immune complexes at very low
concentrations (i.e., 5 pg), enhanced the expression of C3bi
(CRIII), at a higher concentration (i.e., 80 pg) depressed
the expression of C3b receptors to the same magnitude as the
insoluble-immune complexes did. 5) Insoluble and soluble-
immune complexes at low or high concentrations,
significantly enhanced the up-regulation of C3bi (CRIII)
receptors on neutrophils in whole blood. 6) None of the
results summarized so far, were observed when immune
complexes lacking the Fc portion on their antibody molecules
were applied in the experiment.

This study suggests that uptake of immune complexes is
primarily initiated by the interaction of the Fc portion of
the antibody molecule in immune complexes and the Fc
receptor on the neutrophil surface. The presence of the Fc

receptor is required for this interaction. Once the

180
interaction occurs, C3b receptors become involved in the
attachment of immune complexes with the neutrophil. Upon
this attachment on the neutrophil surface a decrease in the
number of C3b and Fc receptors occurs concomitant with an
increase in the C3bi (CRIII) receptors, which together
impair the normal functions of the neutrophil (i.e.,
adherence, locomotion, phagocytosis, etc.). The abnormal
migratory auul phagocytic activities of neutrophils in
response to foreign particles and infectious microorganisms

results in a prolonged infection and inflammatory reactions.

SECTION III
TEE EFFECTS OF SOLUBLE AND INSOLUBLE IHHUNE COMPLEXES ON

THE SYNTHESIS OF LEUKOTRIENE-34 BY HUMAN NEUTROPHILS

181

MATERIALS AND METHODS

I. PREPARATION OF REAGENTS

 

A. Calcium Ionophore A23187

 

Calcium ionophor (M.W. 523.6), was obtained from
Calbiochem, Behring Diagnostic, La Jolla, CA, and stored at
-70°C as a: 10‘3 stock solution in 0.1% dimethylsulfoxide.
As needed, the 1.:ma calcium ionophore solution was diluted
to working solution.

B. Arachidonic Acid (AA)

 

The arachidonic acid (M.W. 304.) procine liver, (cis,
cis, cis, - 5, 8, ll, lA-eicosatetraenoic, 20:4),
(Calbiochem, La Jolla, CA) was provided in glass ampule as a
liquid. The purity of this substance was greater than 99%
by GC and TLC. A.stock solution of 0.1 M was prepared and
stored at -70°C. The stock solution was diluted further
with Ca++ and Mg++-free HBSS to the desired concentrations.

C. Cytochalasin B

 

Cytochalasin B (CB) (M.W. 479.6) was purchased from
Sigma Chem. Co., St. Louis, MO. A stock solution of 10"3 M

was stored at -70°C, and diluted further with Ca++ and Mg++-

free HBSS to the desired concentrations.

182

183

D. Nordihydroguaiaretic Acid

 

Nordihydroguaiaretic acid (NDGA), (M.W. 302.4)., (4,
4'-(2, 3-Dimethyl-1, 4-butanediyl) - bis [1,2-benzenediol]),
was obtained from Sigma Chem. Co., St. Louis, MO, and stored
at 4°C as a 10"3 stock solution in 0.1% ethanol. As needed,

the 1 mM NDGA was diluted to working concentrations. Fresh

stock solution was prepared weekly.

II. MONOCLONAL ANTIBODIES
Monoclonal antibody to human Fc, C3b, C3bi and T4

receptors were all prepared as described in Methods of

Section I.

III. PREPARATION OF IMMUNE COMPLEXES

Immune complexes were prepared as described in Methods

of Section II.

IV. BLOOD COLLECTION AND ISOLATION OF HUMAN NEUTROPHILS
Human venous blood from healthy donors was collected,

and neutrophils were isolated as described in Methods of

Section I.

V. INCUBATION OF HUMAN NEUTROPHILS WITH IMMUNE COMPLEXES
Neutrophils (5 X 106 cells/0.1 ml HBSS) were incubated
with 0.9 m1 of a specified concentration of calcium

ionophore and immune complexes (ICs) in polystyrene tubes,

184

for 15 minutes at 370C. At the end of the incubation, tubes
were placed in ice water for 5 minutes to stop the reaction,
and were then centrifuged for 10 minutes at 2000 G at 4°C.
The supernatants were immediately analyzed for their LTB4
contents as described below. In a few experiments,
neutrophils were pretreated prior to their incubation with
immune complexes or calcium ionophore. Briefly, neutrophils
(5 X 106 cells) were preincubated with specified concen-
tration of arachidonic acid, CB, NDGA, or HBSS for a
selected time interval and temperature. The treated cells
‘were then incubated with immune complexes or calcium
ionophore while in the pretreatment solutions.

For the experiments in which human serum (HS) was used,
a desired concentration of HS was added to immune complexes
prior tn: their incubation with neutrophils. Heat
inactivated HS was prepared by incubating the sera for 30

minutes at 56°C.

VI. PRETREATMENT OF HUMAN NEUTROPHILS WITH C3b, C3bi AND PC

 

MONOCLONAL ANTIBODIES

 

5 l! 106 neutrophils in 0.1 m1 HBSS were preincubated
for 15 minutes at 37°C with saturating amounts of monoclonal
antibodies against C3b, C3bi or Fc receptors. The treated
cells were then incubated with the desired concentrations of
immune complexes or HBSS for 15 minutes further at 37°C, in

the presence of monoclonal antibodies. At the end of the

185
incubation, the cell mixtures were chilled in ice water and
centrifuged (2000 G, 10 minutes) at 4°C. The supernatants

were then assayed for their LTB4 contents.

VII. ANALYSIS OF LTB4 BY RADIOIMMUNOASSAY (RIA)
Immunoreactive LTB4 was measured by RIA, using the
Amersham (Arlington Heights, IL) TRK.840 kit. Briefly, 0.1
ml of cells supernatant with fixed amounts of antibody and
tritiated leukotrien B4 was added into glass tubes (12 mm X
75 mm). After a 15 to 18 hour incubation at 40c, tubes were
transferred into an ice water bath and allowed to equili-
brate for 10 minutes. Then a 0.2 ml of dextran-coated
charcoal suspension was added to the tubes, mixed well, and
incubated in ice water for 10 minutes. Charcoal separates
the bound LTB4 from the unbound LTB4. At the end of the
incubations, the tubes were centrifuged at 2000 G for 15
minutes at 4°C. Immediately after centrifugation, the
supernatants were decanted gently into the scintillation
vials containing 15-18 ml of scintillation cocktail. The
amount of 3H-LTB4 in the supernatant was determined by
counting for four minutes in a beta scintillation counter
(Searle Analytic Inc., ISOCAp/300, Model 6868). A standard
curve was prepared by plotting bound cpm against pg of LTB4
per assay tube on a semi-log graph paper. The concentration
of LTB4 in each sample was then calculatd from the standard

curve. 'This assay was sensitive for the determination of

186
LTB4 in zitrg over the range 12.5 to 400 pg per assay tube.
According to the manufacturer, the anti-sera to LTB4 cross-
reacted 0.03% with LTC4 and LTD4, (0.03% with PGEZ, and

other prostaglandins and arachidonic acid, and 0.14% with

5(8), 12(S)-di HETE.

RESULTS

I. STIMULATION OF LTB4 RELEASE BY CALCIUM IONOPHORE A23187.

An initial assessment of the enzymatic capacity of
human neutrophils to metabolize endogenous arachidonic ascid
via the C5-lipoxygenase pathway was accomplished by using
the calcium ionophore A23187. Isolated neutrophils (5 X
105/ml) were incubated with 5 pM of calcium ionophore for 15
minutes at 37°C, after which their supernatants were
collected and subjected to LTB4 analysis by RIA. Ionophore

induced release of LTB4 by human neutrophils (Table 3.1).

II. RELEASE OF LTB4 FROM HUMAN NEUTROPHILS INDUCED BY IMMUNE
COMPLEX

Human neutrophils (5 X 105/1 ml) were incubated with 15
ug of insoluble immune complexes for 15 minutes at 37°C, and
the release of LTB4 was determined by RIA. Under this
condition, insoluble complexes did not induce release of any
measurable amount of LTB4 by neutrophils using the RIA
assay.

In a few experiments, the release of LTB4 by
neutrophils was measured in the presence of arachidonic acid
(AAA. In a preliminary series of experiments, an AA
concentration of 50 pM was the lowest concentration yielding

187

188

Table 3.1 Release of LTB4 from human neutrophils stimulated
with Calcium ionophore A23187.

 

Nanograms per 106 cells*

 

 

Stimulus N
LTB4

None (HBSS) <0.01** 7

A23187 6.4 i 2 7

 

Neutrophils (5 x 105/1 ml) were incubated with A23187
(5 pM) or HBSS for 15 minutes at 37°C, after which the
supernatants were collected and analyzed by RIA.

(*) Nanograms of LTB4 released from neutrophils are

expressed per 106 cells. Values represent mean 1
SEM.

(**) Minimum sensitivity level of the RIA assay for
LTB4 was 0.012. Any value smaller than the
sensitivity level (i.e., >0<0.012) is shown as
(0.01.

(N) represents the number of experiments.

189

optimal release of LTB4 by neutrophils. Therefore,
thisconcentration was applied in this part of the study.
Human neutrophils (5 X 105) were pre-incubated with 50 pM AA
for 2 minutes, and then exposed to insoluble immune
complexes for 15 minutes at 37°C. The results showed that
insoluble ICs induced the release of LTB4 by neutrophils in
the presence of exogenous AA (Table 3.2).

The effects of human serum (HS) or bovine serum albumin
(BSA) on the release of LTB4 by the human neutrophils upon
the stimulation with insoluble complexes was assessed. In a
preliminary series of experiments, a 10% HS and a 10% BSA
solution were used. The insoluble-immune complexes (15 pg)
were exposed to a 10% HS or a 10% BS solution and then the
neutrophils (5 X 106) were incubated with these complexes
for 15 minutes at 37C. The LTB4 released by neutrophils
into the supernatants were measured by RIA. The results
showed that the presence of 10% HSA with the insoluble-
complexes induced the release of LTB4 by neutrophils.
However, when the serum was inactivated by incubating it for
30 minutes at 56°C, the effect of insoluble-complexes on the
release of LTB4 was abolished. In addition, no measurable
amount of LTB4 was detected in the supernatant when a 10%
BSA solution was added to the insoluble-complexes (Table
3.3).

Since the presence of serum was required for the

release of LTB4 by neutrophils, different concentrations of

190

Table 3.2 Neutrophils (5 x 105/1 ml) in the presence or
absence of 50 pM arachidonic acid were exposed to
immune complexes and various reagents as
designated, for 15 minutes at 37°C. The
supernatants were then collected and analyzed for
LTB4. Abbreviations: Hank's buffer (HBSS),
immune complexes (IC), and antibody (AB).

(*) Nanograms of ImB4 released from neutrophils
are expressed per 106 cells. Values
represent mean 1 SEM.

(**) Minimum sensitivity level of the RIA assay
for LTB4 was 0.012. Any value smaller than
the minimum sensitivity level (i.e.,
>0<0.012) is shown as (0.01.

(***) Antibody was the IgG fraction of rabbit anti-
human albumin which was used for the
formation of Immune Complexes.

(N) represents the number of experiments.

191

 

 

 

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m moo. + Ho.o Issuesv magnumznw mmmm
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m . Ho.ov Amamsv moH mansflomcH mwmm
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m saao.ov mmmm mmmm
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z cofiumnsocw scaumnsoca
«mHHwo boa Mom msmumocmz pcooom umuwm

 

.maflnmouusmc amass cw cwom
oacoownomum mo EwflHonmumE mnu so wmxmamsoo mazesfl mansmomca mo uommmm m.m magma

192

Table 3.3 Neutrophils (5 x 105) were incubated with various
reagents as indicated, in the presence or absence
of HS and BSA for 15 minutes at 37°C. (AA+),
neutrophils were exposed to 50 pM arachidonic acid
before incubation with different reagents. (AA-),
neutrophils were not exposed to arachidonic acid
before incubation.

Abbreviations of reagents used are bovine serum
albumin (BSA), heat inactivated human serum (1-
HS), Human serum (HS), Hank's buffer (HBSS),
calcium ionophore A23187 (A23187), antibody (Ab),
and immune complexes (ICS).

(*) Nanograms of LTB4 released from neutrophils
are expressed per 1X106 cells. Values
represent mean 1 SEM.

(**) Minimum sensitivity level of this RIA assay
for LTB4 was 0.012. Any value smaller than
the minimum sensitivity level (i.e.,
>0<0.012) is shown as <0.01.

(***) Maximum sensitivity level of the RIA assay
was 0.5 ng/tube. The values higher than this
level (i.e., >0.5ng) is shown as >0.5, since
no dilution was made at the time the
experiments were performed.

(****) Antibody was the IgG fraction of rabbit anti-
human albumin which was used for the
formation of Immune Complex.

(N) represents the number of experiments.

193

 

 

N m.o A sasm.0A hmamm<
m Hoo.o m Ho.o ~oo.o_n ~o.o «mm was + A01 ms. muHumHnssomaH
m moo.o n Ho.o I Ho.ov mmus was + Am: ms. moHumHnsHomaH
N moo.o.n os.o Ho.o + os.o mm was + As; ms. mosumsnsHomcm
m «H.o + H~.o Ho.ov 161 ms. moHumHnsflomcu
m ~oo.o m Ho.o ~oo.o m ~o.o «mm «as + is; ma.....n¢
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N moo.o m Ho.o moo.o u Ho.o «mm was + Ammmm. 6:02
m soo.o n Ho.o I Ho.ov mans mos + Immune mcoz
m soo.o n mo.o moo.o + mo.o mm mos + Amman. mcoz
m mo.o + as.o .«Hc.ov Amman. waoz
+<< uac

 

«waamu boa mom mamumocmz msasewum

«mag

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Ca mucmmmmu mDOwum> nufiz omumnsocw waficmouusma amen: Eoum vmeq mo mmmoflmm m.m manna

194
serum solutions were used. The LTB4 in the supernatant of
the stimulated neutrophils and in the serum solutions were
determined. Small amounts of LTB4 were detectable in a 10%
solutdxni of the serum from some individual subjects. The
variation among individual donors was great. However, in
lower concentrations of serum (i.e., 2.5% and 5.%) no
detectable levels of LTB4 were observed (Table 3.4). The
release of LTB4 by insoluble-immune complexes was higher

when a lower concentration of serum was applied (Table 3.4).

III. DOSE-RELATED RELEASE OF LTB4 FROM NEUTROPHILS INDUCED

 

BY IMMUNE COMPLEXES

 

Neutrophils were incubated with various concentrations
of soluble and insoluble-immune complexes for 15 minutes at
37°C, and the release of LTB4 was determined by RIA. The
threshhold dose for activation of lipoxygenase enzyme system
by insoluble-immune complexes was 5 pg for S X 106 cells/1
ml (Figure 3.1). Soluble-immune complexes with an excess of
antigen concentration at 10 times that of equivalence were
also able to stimulate the LTB4 release, but this capacity
was two to fourfold less than those of insoluble-immune
complexes. Saturation for immune complexes-induced release
was achieved at a dose of 30-50 pg for both complexes. The
variatdtni among individual donors was great, which is
typical of arachidonic acid nmtabolism. In a few cases,
when 50 pg insoluble-complexes were used, no detectable
level of LTB4 was measured in the supernatant of

neutrophils.

195

Table 3.4 Human neutrophils (5 x 105/1 ml) were incubated
with insoluble-immune complexes (10 pg) and
calcium ionophor A23187 (5 pH) in the presence of
serum for 15 minutes at 37°C. The LTB4 present in
the serum solutions and supernatants were measured
by RIA.

Abbreviations of reagents used are Hank's buffer
(HBSS), human serum (HS), and insoluble immune
complexes (ICs).

(*) Nanogram of LTB4 present in 1 ml solution of
serum, or released into 1 ml of supernatant
by 5 x 106 neutrophils. Values represent mean
+ SEM.

(**) Minimum sensitivity level of the RIA assay
for LTB4 was 0.012. Any value smaller than
this value (i.e., >0<0.012) is shown as
(0.01.

(N) represents the number of experiments.

196

 

 

 

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N mHHmo mosxm\mc NN.o m Nv.o .moae mm + moH + mssmo
N mHHmo sosxm\mc No.o n e¢.o Lam.N. mm + mos + mHHmo
N mHHmo soaxm\oc oN.o n om.o .a.m. mm + mom + mHHmo
N mHHmo coaxm\mc «N.o + Nm.o Awm.Nv mm + moH + mHHmo
m Hs\ma No.o .Nos. mm
N Hs\ma No.ov .wm.N. mm
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N Hs\mc .«No.ov .mm.N. mm
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z Nomad mousom
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L'IB4 ( ng / 5X106 cells )

Figure 3.1

0.5

0.4

0.3

0.2

0.1

0.0

197

. thohifleifls

' EkflpbhaICs

 

 

 

 

2.5 5 10 20 40 60 80 100

meme<kmphac(lxgoflxxmehl)

Dose-response curves for immune complexes-
induced release of LTB4 from human
neutrophils. Neutrophils were incubated for
15 minutes at 37°C with soluble or insoluble
immune complexes, and the amount of LTB4 was
determined by RIA. Representative figure
from four experiments. Values represent mean
+ SD.

198

IV. EFFECTS OF CYTOCHALASIN-B AND NORDIHYDROGUAIARETIC

ACID (NDGA) ON IMMUNE COMPLEXES-INDUCED LTB4 RELEASE

To assess whether phagocytosis of immune complexes by
neutrophils was required for initiation of LTB4 synthesis,
cells were pretreated with cytochalasin B (CB) (10 pM)
before exposure to immune complexes. Pretreatment of
neutrophils with CB did not inhibit immune complexes-induced
LTB4 release. In fact, CB enhanced immune complexes-induced
LTB4 synthesis by 1.5-fold to 3-fold (Figure 3.2).
Cytochalasin B alone did not have any effect on the release
of LTB4 from neutrophils.

When neutrophils were pre-incubated with the
lipoxygenase inhibitor NDGA (10 pM), LTB4 release induced by

immune complexes was completely inhibited (Figure 3.2).

V. ROLE OF C3b, C3bi AND FC RECEPTORS ON LTB4 RELEASE FROM

 

HUMAN NEUTROPHILS STIMULATED WITH INSOLUBLE-IMMUNE

 

COMPLEXES

 

The role of these receptors in initiation of LTB4
synthesis by neutrophils, upon stimulation with immune
complexes was assessed using monoclonal antibodies.
Neutrophils (5 X 106) were preincubated with monoclonal
antibodies against C3b, C3bi or Fc receptors for 15 minutes
at 37°C. The cells were then exposed to insoluble-immune
complexes (10 pg) and incubated for 15 minutes further at
37°C. The supernatants were collected and analyzed for the

LTB4 by RIA. The results showed that anti-Fc completely

Figure 3.2

LTBtHng/5X106 Cell >

199

 

A B C
ICS CB-ICS MBA-ICS

Release of LTB4 from human neutrophils
induced with insoluble immune complexes
(ICs): Effects of CB and NDGA. The results of
three experimental conditions are given; (A),
neutrophils (5 it 105) suspended in HBSS
exposed to insoluble—1C3 (10 pg) for 15
minutes at 37°C; (B), neutrophils (5 x 105)
preincubated with CB (10 pM) and then exposed
to insoluble-ICS (10 pg) for 15 minutes at
37°C; (C), neutrophils (5 x 105) preincubated
with NDGA (10 pM) and then exposed to
insoluble-ICS (10 pg) for 15 minutes at 37°C.
The amounts of LTB4 were then determined by
RIA in the supernatants.

Abbreviations: Hank's buffer (HBSS),
Cytochalasin B (CB), nordihydroguaiaretic
acid (NDGA), and immune complexes (ICs).
Each point represents the mean :_SD for three
determinations.

200
inhibited the release of LTB4 by neutrophils (Table 3.5).
Human neutrophils (5 )I 106) incubated with insoluble
complexes in the presence of 5% serum released 0.106 1 0.037
ng of LTB4, while, cells preincubated with anti-Fc and then
exposed to insoluble complexes released (0.001 mg of LTB4.
However, pre-incubation of neutrophils with antibodies to
C3b, C3bi receptors, or with both antibodies simultaneously,
did not inhibit immune complexes-induced LTB4 release. In
fact, C3b, or C3bi receptor blocked with the monoclonal
antibodies potentiated immune complexes-induced LTB4 release
(Table 3.5). No effect was observed with a control mouse
monoclonal IgG directed against human T4 receptor or with a

purified mouse IgM.

201

Table 3.5 Neutrophils (5 x 105) preincubated with Hank's
buffer (HBSS) or different monoclonal antibodies
for 15 minutes at 37°C. The cells were then
exposed to Hank's buffer or insoluble immune
complexes (ICs) (10 pg) in the presence of 5%
serum for an additional 15 minutes at 37°c, The
supernatants were collected and analyzed for LTB4
by RIA.

(*) Nanograms of LTB4 released from neutrophils
are expressed per 5 x 106 cells. Values
represent mean i SEM.

(**) Minimum sensitivity level of the RIA assay
for LTB4 was 0.012 ng. Ahy value smaller
than this level (i.e., >0<0.012) is shown as
(0.001.

(***) Insoluble immune complexes composed of human
serum albumin and IgG fraction of Goat anti—
human albumin.

(****) Insoluble immune complexes composed of human
serum albumin and IgG fraction of Rabbit
anti-human albumin.

 

 

 

 

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N (Noo.ov mom: NnNouNuaa
N oo.o + moN.o Ame muHumesflomcH NnNouNucN
N Noo.ov mmmm omusuac
m“ N Noo.ov Am. moHannsNomcH omnwucm
2
N No.o + ooH.o ....Am. moHannoHomcH moms
m abo.ov «film: mUHIOHQSHOmGH mmmm
N o.o + .NN NNHNNN mmmm
m «aaoo.ov mmmm mmm:
maoflusooa mooNuNocN
z . ommu .ooNN o .ch NHL .ooNN o .css NH.
mHHmU moaxm Mom msmumocmz coflumnsocH ocoomm nowumnnocH umuflm
.mHanmouusm: amen: scum mmmoamu omen coupon“ moxoflmsoo
m::EEH :o mmfioonwucm HmcoHoocoe an omeONQ muoumooou on can flnmu .Qmu mo uoommm m.m wanna

DISCUSSION

The central role of arachidonic acid (AA) metabolites
in hypersensitivity reactions and inflammation has led to an
explosive growth of interest and research activity regarding
these substances. Leukotriene B4, one of the AA
metabolites, is chemotactic for granulocytes and promotes
neutrophil aggregation as well as the expression of
receptors (i.e., C3b, C3bi, and PC) (discussed in Section
I). Human neutrophils can metabolize AA to several
important types of eicosanoids, including LTB4. On the
other hand, neutrophils are implicated in inflammatory
reactions. Of particular interest was whether the
interaction of immune complexes and neutrophils can initiate
the synthesis of LTB4 by these cells.

Human neutrophils were isolated as described in
Methods. Immune complexes were prepared from human serum
albumin (HSA) and the IgG fraction of rabbit antLHummn
albumin. The amounts of LTB4 released by neutrophils into
the extracellular media was measured by RIA.

The data presented in this study demonstrate that
immune complexes stimulate peripheral blood neutrophils to
release LTB4 from the endogenous AA. Insoluble-immune
complexes were the most potent stimulators when compared

203

204

with soluble complexes. Immune complexes stimulated
neutrophils via interaction with Fc receptors, since the
release of LTB4 was completely inhibited by the exposure of
neutrophils to anti-Fc antibody (i.e., 368). In the
absence of exogenous AA, the release of LTB4 was dependent
on the presence of a small fraction (i.e., 5%) of human
serum. The role of serum in the synthesis of LTB4 by human'
neutrophils is not clear and further studies are necessary.
Cytochalasin B (CB), a potent inhibitor of phagocytosis, did
not prevent the synthesis of LTB4, indicating that cross-
linking of Fc receptors on neutrophils in the absence of
phagocytosis activated the C5-lipoxygenase pathway of AA
metabolism. To my knowledge, this is the first observation
describing release of LTB4 by soluble and insoluble immune
complexes in the absence of exogenous AA from human neutro-
phils. This study indicates that not only insoluble-immune
complexes but also soluble—complexes, which has been
believed for some time to be a "dead end" and not have a
role in the pathogenesis of immune complexes induced tissue
damage, can contribute to inflammatory reactions by
stimulating neutrophils to produce LTB4.

The result obtained for the abscence of LTB4 released
from neutrophils stimulated with immune complexes in the
absence of human serum (i.e., 5%) is in agreement with the
observations of Smith et al., (1986). These investigators

reported that aggregated IgG (i.e., 400 pg/ml) failed to

205

stimulate neutrophils (5 J: 106) to release LTB4. Hewever,
they showed that in the presence of exogenous AA (i.e.,
30pM), neutrophils stimulated with aggregated IgG released
LTB4. Smith et al., (1986) did not examine the effect of
aggregated 196 on LTB4 synthesis by neutrophils in the
presence of human serum. In the study presented here,
immune complexes, in the presence of AA also stimulated the
release of LTB4 by neutrophils (Table 3.2). However, the
addition of AA to suspensions of human neutrophils by itself
also led to the release of LTB4 as has previously been
reported by Borgeat and Samuelsson (1979). The level of the
LTB4 release was two to three-fold higher from neutrophils
activated with the stimuli (i.e., ICs or f-Met-Phe) in the
presence of AA than the level of LTB4 released by
unstimulated neutrophils in the presence of AA alone (Table
3.2). Never the less, this study concentrated only on
endogenous AA metabolism, since the use of an exogenous
substrate may not reflect the fate of AA released from
endogenous cellular phospholipids after receptor stimu-
lation.

In the absence of exogenous AA, the presence of human
serum (HS) was required for the production of LTB4 by
neutrophils stimulated with immune complexes. Substitution
of serum with a bovine serum albumin solution or heat-
inactivated serum did not promote LTB4 release. The

presence of serum in the incubating media also enhanced the

206

production of LTB4 by neutrophils stimulated with ionOphore
A23187. The data suggest that the presence of serum was not
required for the Opsonization of immune complexes, and the
heat labile factor present in human serum might somehow have
enhanced the reaction which is involved in LTB4 synthesis by
neutrophils, since it also increased the production of LTB4
by the soluble stimulus A23187.

Inhibition of immune complexes-induced LTB4 release
with the lipoxygenase inhibitor NDGA suggests that LTB4 was
formed by enzymatic transformation of fatty acid in the
plasma membranes of neutrophils.

Both insoluble and soluble-complexes were capable of
stimulating the release of LTB4 by neutrophils. However,
the quantities of LTB4 released by soluble-complexes were
two to four-fold less than those of insoluble-complexes.
Although the amounts of LTB4 released after immune complexes
stimulation are relatively low compared with A23187 stimu-
lation, they are significant. As it is presented in section

I of this study, LTB4 at 10-10

M (i.e., approximately equals
to 0.03 pg/ml) could induce a significant up-regulation of
C3b, C3bi and FC receptors on the neutrophil surface. The
amount of immune complexes tested in this study were within
the probable concentration range found in the circulation of
some patients with rheumatoid arthritis or systemic lupus

erythematosus. Therefore, it is likely that interaction of

neutrophils with immune complexes will result in the

207

production of quantities of LTB4 that can influence local
inflammatory reactions. In addition, peripheral monocytes
can synthesize LTB4 as well as LTC4 and PGE2 upon
interaction with aggregated IgG, IgA and IgE (Ferreri et
al., 1986). Therefore leukocytes in peripheral blood upon
stimulation with immune complexes can release a more
substantial amount of leukotrienes. This suggestion could
be supported further by a recent report by Gresele and
associates (1986) who reported that higher amounts of LTB4
are produced by whole blood than those produced by isolated
neutrophils.

Phagocytosis of insoluble particulate substances such
as opsonized zymosan (Claesson et al., 1981) and
Staphylococcus aureus (Henricks et al., 1985) have been
shown to initiate the release of LTB4 from human
neutrophils. However, the data presented in this study show
that human neutrophils can also release LTB4 in the absence
of a phagocytic stimulus. Inhibition of immune complexes
phagocytosis by CB treatment did not inhibit LTB4 pro-
duction. IUl fact, the release of LTB4 was actually poten-
tiated. An enhanced production of LTB4 by CB-treated
monocytes after stimulation with aggregated immunoglobulin
has also been reported (Ferreri et al., 1986). This
enhancement is not understood. Cytochalasin B also
increases the magnitude of the Quin—2 fluorescence response

elicited by fMLP (Korchak et al., 1984) and LTB4 (Goldman,

208

unpublished data),(Quin-2 measures the cytoplasmic concen-
tration of interacellular calcium). It is believed that an
increase in intracellular Ca++ may serve to emhance the
physiological responses of the cell. Perhaps the increments
in cytosolic free calcium induced by CB, somehow contributes
to the enhancement of LTB4 production. In addition, CB
stimulates the release of neutrophil granules contents. The
fusion of granule membranes with the plasma membrane may
provide additional phospholipids and AA on the cell surface,
which upon stimulation could be metabolized further to LTB4.
Furthermore, the release of granule contents into the
extracellular medium may somehow contribute to the release
of LTB4 by neutrophils. Extensive research and study is
required to clarify these speculations.

It was of interest to discover how the metabolism of
AA by immune complexes is related to Fc, C3b (CRI), and/or
the C3bi (CRIII) receptors on human neutrophils. To fulfill
this part of the study, neutrophils were preincubated with
different monoclonal antibodies against Fc, C3b, or C3bi
receptors. Cells in the presence of the antibody were then
exposed to immune complexes and the amount of LTB4 released
into the extracellular medium was measured. When Fc
receptors on the neutrophil surface were blocked with anti-
Fc antibody (i.e., 368), the release of LTB4 was completely
inhibited. On the other hand, pretreatment of neutrophils

with either C3b or C3bi antibodies potentiated the release

209

of LTB4 upon exposure to insoluble complexes. A much higher
enhancement of LTB4 release was observed when both
receptors, C3b and C3bi on the neutrophil surface were
simultaneously blocked by their respective antibodies. The
treatment of neutrophils by antibodies alone did not induce
the release of LTB4. Complete inhibition of LTB4 release by
anti-Fc antibody indicates that the interaction between the
neutrophil Fc receptor and IgG molecules of immune complexes
is necessary for the initiation of AA metabolism by these
cells. This suggestion is further supported by the finding
that insoluble-immune complexes containing an IgG fraction
of goat anti—human albumin did not induce the release of
LTB4 by neutrophils (Table 3.5). As previously reported,
human neutrophils failed to bind particles coated with goat
immunoglobulins, apparently because of a difference between
the Fc component of goat immunoglobulins and other animal
immunoglobulins (Newman and Johnston, 1979; York, 1983).

The enhancement of LTB4 release from immune compleXes-
stimulated neutrophils by blocking the C3b and C3bi
receptors with their respective antibodies is not understood
at present. To my knowledge, this is the first observation
regarding this issue, and since the study of the
mechanism(s) involved in this reaction(s) was not within the
scope of this research investigation, it will remain unknown

until some future study can clarify it.

210

The results may suggest that the release of LTB4 by
neutrophils upon exposure to soluble-complexes is initiated,
somewhat, through the Fc receptor interaction and is further
enhanced by binding to C3b receptors. Since, it appears
that soluble-complexes bind more to C3b receptors (as
discussed in Section II). If this hypothesis is true, then
higher quantities of LTB4 are expected to be released upon
interaction of neutrophils with higher concentrations of
complexes. However, in this study a decline of the LTB4
released by neutrophils was cmmerved when the cells were
exposed to higher concentrations of both soluble and
insoluble-complexes (Figure 3.1). The following assumptions
may offer a solution to this puzzle. The occupancy of C3b
receptors by immune complexes may be different from that of
the anti-C3b antibody therefore causing different physio-
logical responses to occur, as can be observed from the
previous study. Abrahamson and Fearon (1983) reported that
F(ab')2 antibodies (but not Fab') to the C3b receptor were
rapidly internalized by neutrophils at 37°C. They suggested
that soluble-immune complexes bearing C3b may be similarly
internalized by neutrophils. Contrary to this suggestion,
Taylor et al., (1983) observed no significant intern-
alization of soluble antibodies/dsDNA immune complexes after
they were bound by neutrophils. Another assumption is that,
the decline in LTB4 might not be due to the presence of a

high concentration of immune complexes, resulting instead as

211
a further metabolism of LTB4 to w-oxidation products, as
previously reported in neutrophils stimulated with FMLP
(Jubiz et al., 1982; Goetzl, 1983).

In conclusion, the results reported here indicate that:
l) soluble and insoluble immune complexes induced the
synthesis of LTB4 from endogenous arachidonic acid through
the 5-lipoxygenase pathway in human neutrophils, 2) the
transformation of arachidonic acid to LTB4 initiated by the
interaction of Fc receptors with IgG molecules of immune
complexes, 3) the synthesis of LTB4 was not only due to the
Fc receptor occupancy by the ligand, since anti-Fc antibody
alone did not induce the release of LTB4, and 4) the release
of LTB4 was potentiated upon preincubation of neutrophils
with cytochalasin B (CB), and antibodies against C3b (CRI)
and C3bi (CRIII) receptors.

This study suggests that the interaction of neutrophils
with immobilized immune complexes (such as those deposited
in blood vessel walls or glomerular basement membranes), and
with soluble circulating immune complexes (which have been
believed to be a ”dead end"), could initiate metabolism of
arachidonic acid. Such a mechanism could contribute to
inflammatory reactions characterized by the infiltration of

leukocytes.

SUMMARY

In this study it was shown that unactivated neutrophils
in whole blood exhibit a minimal number of C3b (CRI), C3bi
(CRIIlJ :receptors, while expressing a large number of
receptors for EC on their surface. Each of the chemotactic
factors, f-Met-Phe, LTB4, and a temperature transition
(i.e., 4°C --> 37°C) significantly enhanced the up-
regulation of C3b, C3bi and Fc receptors on the plasma
membranes of neutrophils in whole blood. When neutrophils'
were isolated by the standard isolation procedure (i.e.,
dextran sedimentation and ficoll/hypaque gradients), the
expression of C3b and C3bi receptors were only enhanced
significantly on cell surfaces upon stimulation with f-Met-
Phe or LTB4. No significant increased expression of Fc
receptors was observed on the isolated neutrophil surface.

The interaction of immune complexes with neutrophils
was studied. Neutrophils in whole blood avidly bound and
ingested the insoluble immune complexes. However, the
uptake of soluble immune complexes by neutrophils was 1-5%
compared to the insoluble immune complex uptake. The
interaction of immune complexes with neutrophils depressed
the expression of C3b and Fc receptors, in contrast to the
expression of the C3bi receptor which was significanthy

212

213

enhanced. In additdxni, soluble and insoluble immune
complexes induced the synthesis of leukotriene B4 from the
endogenous arachidonic acid via the 5-lipoxygenase pathway.
The interaction between the Fc receptor and the Fc portion
of the antibody molecule in immune complex was required but
not sufficient for the release of LTB4 by neutrophils (the
mechanism is unknown, however, the presence of a small
fraction of human serum was required). Pre-incubation of
neutrophils with antibodies against C3b, C3bi receptors or
cytochalasin B prior to the interaction with immune
complexes potentiated the release of LTB4 from these cells.

The results suggest that immune complexes modulate the
surface receptors associated with the immune adherence, and
may be responsible for the depressed locomotion and
phagocytic activity of neutrophils observed in some patients
with inflammatory diseases (Brandt and Hedberg, 1969;
Corberand et al., 1977). The leukotriene B4 released by
neutrophils upon the interaction with immune complexes could
potentiate the inflammatory reaction by increasing

leukocytic infiltration.

APP‘DICES

APPENDIX A

 

This modified “FOLIN” assay was used for the
measurement of protein in Immune Complexes. The assay is
sensitive for the determination of protein in vitro over the
range 5-100 ug per assay tube.

REAGENTS

1)

2% Na2Co3 in 0.1 N NaOH

20. gm Na Co (Sodium Carbonate)

5.56 ml 0 1 N NaOh or 4 gm of NaOH pellets
Up to 1000. ml with distilled water

 

2) 1% CuSO4 (W/V)
3) 2% Natartrate (Na2C4H405,2H20)
Fresh solution was prepared, since it precipitates
with standing.
4) Phenol reagent of Folin-Ciocalteau
Diluted 1:2 with distilled H20.
(the light sensitive phenol reagent was stored in
the dark).
PROCEDURE
1) Measured out an aliquot 0.05 ml of sample.
2) Added 2 ml of Na C03 mixture.
Mixed in the folIowing order:
1 ml of 2% Natartrate
1 ml of 1% CuSo4
100 ml of 2% Na2C03 in 0.1 N NaOH.
3) Added 0.2 ml of phenol reagent and mixed
immediately.
4) Read at OD 700 after 30 minutes incubation at RT.

214

 

APPENDIX B

 

LIMULUS AMEBOCYTE LYSATE

The Limulus Amebocyte Lysate (LAL) test is a
quantitative test for gram negative bacterial endotoxin. The
test kit was purchased from Whittaker M.A. Bioproducts,
Walkersville, Maryland. The use of LAL for the detection of
endotoxin evolved from the observation by Bang that a gram-
negative infection of Limulus polyphemus, the horseshoe
crab, resulted in fatal intravascular coagulation. Levin
and Bang later demonstrated that this clotting was a result
of the reaction between endoxtin and a clottable protein in
the circulating amebocytes of Limulus. Levin and Bang
prepared a lysate from washed amebocytes which was an
extremely sensitive indicator of the presence of endotoxin.
They have purified and characterized the clottable protein
from LAL and have shown the reaction with endotoxin to be
enzymatitu The lysate prepared from the circulating
amebocytes of the horse-shoe crab Limulus polyphemus
standardized to detect at least 1.25 EU/ml of FDA Reference
endotoxin.

215

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