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This is to certify that the
thesis entitled
EFFECTS OF HUMAN C1- INHIBITOR ON THE CHEMOTACTIC
RESPONSIVENESS OF HUMAN NEUTROPHILIC LEUKOCYTES
presented by

David Y. Liu

has been accepted towards fulfillment
of the requirements for
PhD degree in

Microbiology and Public Health

 

Major professor

June 1 , 1976

 

LIBRARY
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ABSTRACT

EFFECTS OF HUMAN CI INHIBITOR ON THE CHEMOTACTIC
RESPONSIVENESS OF HUMAN NEUTROPHILIC LEUKOCYTES

BY

David Y. Liu

Cl inhibitor has been shown to enhance and inhibit neutrophil
chemotactic responsiveness under appropriate conditions by a direct
effect on cells. To further define the nature of this effect, the
influence of Cl inhibitor on complement and noncomplement mediated
chemotaxis was investigated.

The presence of highly purified human Cl inhibitor with either
mixed blood leukocytes suspended in 10% plasma or isolated polymorpho-
nuclear leukocytes suspended in 0.5% bovine serum albumin resulted
in significant inhibition of the chemotactic response to human C3-
derived chemotactic factors. This C3-re1ated chemotactic activity
was generated from highly purified human C3 with either trypsin or
the cellular intermediate, EAC4°xY2. A comparison of the migrating
cell distribution in micropore filters in the absence and presence
of Cl inhibitor was performed. Mixed blood leukocytes responding to
trypsin-activated C3 at 45, 90, and 135 minutes were quantitated in
10 micron increments. At each time interval CI inhibitor was associ-
ated with a greater number of cells near the top of the filter and a

shorter migration distance into the filter.

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David Y. Liu

The presence of Cl inhibitor with either mixed blood leukocytes
in 10% plasma or isolated granulocytes in 0.5% albumin resulted in
significant inhibition of the chemotactic response to CS—derived
chemotactic factors. CS-related chemotactic activity was generated
from highly purified human C5 with trypsin or isolated from
lipopolysaccharide-activated guinea pig serum as the 15,000 molecular
weight fraction from gel filtration.

The effect of Cl inhibitor on the neutrophil chemotactic
response to N-formylmethionylphenylalanine was examined. Cl-
inhibitor only when present with the neutrophils significantly
enhanced their chemotactic response to lO-GM N-formylmethionyl—
phenylalanine. An analysis of the filter distribution of migrating
neutrophils revealed a significantly higher number of cells in a zone
nearest the upper surface of the filter at 90 and 135 minutes in the
presence of Cl inhibitor and suggested an increase in the number of
cells throughout the filter in the presence of Cl inhibitor.

Another cytotaxin mediated phenomenon, chemotactic deactivation,
was examined for its possible susceptibility to the action of Cl-
inhibitor. A 10% zymosan-activated plasma filtrate was used as both
the deactivating agent and the cytotaxin. Neutrophils, which were
deactivated by incubating with zymosan-activated plasma filtrate for
30 minutes and then extensively washed, were unresponsive to subse-
quent chemotactic stimulation. The presence of Cl inhibitor during
the incubation of neutrophils with zymosan-activated plasma filtrate
resulted in partial prevention of deactivation, whereas complete
reversal of deactivation was obtained by adding Cl inhibitor to

previously deactivated and washed neutrophils.

David Y. Liu

The results of this study lend support to the conclusion that
Cl inhibitor can directly affect the chemotactic response of human
neutrophils. Well-defined conditions for the participation of Cl.
inhibitor in either enhancement or inhibition are now clearly
established so that the basic mechanism of Cl inhibitor function can
be further elucidated. Moreover, the ability of Cl inhibitor to
reverse chemotactic deactivation, which has hitherto been considered
irreversible, may be relevant to the understanding of the chemo—

tactic response of human neutrophils.

EFFECTS OF HUMAN CI INHIBITOR ON THE CHEMOTACTIC
RESPONSIVENESS OF HUMAN NEUTROPHILIC LEUKOCYTES

B ‘;
Y \o‘ 5-

was"
David Y. Liu

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1976

Dedicated to Jenny

for her endearing patience and understanding

ii

ACKNOWLEDGEMENTS

I wish to express my profound appreciation to my academic
advisor, Dr. Richard A. Patrick, for his stimulating and unselfish
guidance, confidence and encouragement throughout these studies
and during the preparation of this dissertation. His personal
kindness and consideration will always be remembered.

The advice and encouragement of my guidance committee, Drs.
Robert R. Brubaker, Harold C. Miller, C. Wayne Smith, and Jeffrey
F. Williams, are gratefully acknowledged.

I wish to express my sincere gratitude to C. Wayne Smith and
James C. Hollers, for the many valuable discussions and guidance
during my efforts to understand chemotaxis. I

The graduate assistantship and NIH traineeship awarded to me
by the Department of Microbiology and Public Health, Michigan State
University, and the excellent technical assistance offered by Ms.

Sheri Sprigg and Ms. Barbara Giese are greatly appreciated.

iii

INTRODUCTION.

TABLE OF CONTENTS

LITERATURE REVIEW . . . . . . . . . .

Biochemical Nature of CI Inhibitor .

Structure . . . . . . .
Physiologic Function. .
Phylogeny and Ontogeny.
Pathology . . . . . . .

Chemotaxis . . . . . . . . . .

Historical Perspective.
Nature 0 O O I O O O I 0
Measurement . . . . . .

Chemotactic Factors. . . . . .

REFERENCES.

Complement-Derived. . .
Non—Complement Factors.

Cellular Biochemistry of Chemotaxis . . . .

Modulators. . . . . . .
Clinical Disorders. . .

Chemotaxis in Inflammation.

ARTICLE 1 - EFFECTS OF HUMAN CI INACTIVATOR ON COMPLEMENT

AND NON-COMPLEMENT MEDIATED HUMAN NEUTROPHIL
WMAXIS O O C O O O I 0

ARTICLE 2 - EFFECTS or CT INHIBITOR ON CHEMOTACTIC

iv

DEACTIVATION OF HUMAN NEUTROPHILS

Page

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10
12

13
13
22
27
33
36
37

41

60

98

LIST OF TABLES
Table Page

1 Effects of Cl Inhibitor on Chemotactic Deactivation
of Neutrophils . . . . . . . . . . . . . . . . . . . . . 109

Flgu

ARTE

LIST OF FIGURES
Figure
ARTICLE 1

1 Effect of ClINH on the chemotactic response of
neutrophils to C3(Try) . . . . . . . . . . . . . .

2 Effect of ClINH on the chemotactic response of
neutrophils to C3(EAC4°xY2). . . . . . . . . . . .

3 Effect of C11NH on the chemotactic response of
neutrophils to human C5(Try) . . . . . . . . . . .

4 Effect of ClINH on the chemotactic response of
neutrophils to guinea pig C5a. . . . . . . . . . .

5 Distribution of leukocytes through the Millipore
f1 lter I O O O O I O O O I O O I O O O O O O O O I

6 Effect of CllNH on the chemotactic response of
neutrophils to NFMP. . . . . . . . . . . . . . . .

7 Distribution of PMN through the Millipore filter .

ARTICLE 2
1 Experimental procedure . . . . . . . . . . . . . .
2 Effect of various concentrations of ZAPF in pre-

incubation mixtures on the chemotactic response of
neutrophils O O O O O O O O O O O O O I O I O O O 0

vi

Page

68

71

73

75

78

81

83

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107

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INTRODUCTION

Approximately 10% of the total protein content in plasma consists
of a group of proteins characterized by their ability to inhibit
proteolytic enzymes. These proteinase inhibitors have a potential
pathophysiologic significance. Inherited deficiencies of several

inhibitors are associated with disease: -antitrypsin deficiency

o‘1
predisposes to emphysema and hepatic cirrhosis, antithrombin III
deficiency is characteristic of thrombo-embolic disease, and CI
inhibitor deficiency is associated with hereditary angioneurotic
edema. The proteolytic enzyme inhibitors regulate and modulate

four interrelated protease systems of the blood: coagulation,
fibrinolysis, kallikrein and complement. Several of these inhibitors
have been found to have a profound influence upon cell motility.

This new dimension of their regulatory function is currently under
intense investigation.

It is my intention to describe studies pertinent to the under-
standing of the regulation of human neutrophil Chemotaxis by CI.
inhibitor. This is done in two major sections. In the first,
research concerning the structure and function of CI inhibitor is
extensively reviewed. This involves a discussion of its control

functions and the resulting biological activities manifested in

inflammatory and hemostatic reactions. It is hoped that such a

2

discussion will provide a biochemical basis for understanding the
participation of ClINH in the chemotactic response of neutrophils.

The second part examines the studies pertaining to the evolution
of leukocyte Chemotaxis as an extremely complex phenomenon. Atten-
tion is focused on efforts to characterize the various chemotactic
stimuli. This will be supplemented by information on regulators of
the chemotactic response whether the target of their action is the
cell or the stimulus. Finally, this review will emphasize the sig-
nificance of Chemotaxis as an investigative tool which has been used
to extend our comprehension of such fundamental biological phenomena
as cell motility.

The work described in this dissertation was formulated in an
attempt to identify and characterize the nature of the effect of

CI inhibitor on neutrophil motility.

LITERATURE REVIEW

Biochemical Nature of C1 Inhibitor

 

Structure

The existence of a serum inhibitor of the first component of
complement, CI. was first demonstrated by Ratnoff and Lepow (1957).
CI inhibitor (CllNH) is an az-glycoprotein that is antigenically

identical to az-neuraminoglycoprotein (Pensky and Schwick, 1969).
Purified ClINH preparations demonstrated two bands on SDS-

acrylamide gel electrophoresis with apparent molecular weights of
105,000 and 96,000 daltons (Harpel and Cooper, 1975). Both components
are chemically, immunologically, and functional identical to ClINH.
Each of these components appeared to consist of a single polypeptide
chain since their apparent molecular weight was not altered by
disulfide bond cleavage. Haupt and associates (1970) report a
molecular weight for ClINH of 104,000 daltons as determined by
sedimentation equilibrium ultracentrifugation with a 35% carbohydrate
content. C11NH has an 820.w of 3.67 and an isoelectric point between
2.7-2.8. Analysis of ClINH isolated from the plasma of two patients
with hereditary angioneurotic edema showed an apparent molecular
weight of 109,000 daltons, an inability to form a complex with CIs

or plasmin, and a slightly different amino acid content (Harpel,

Hugli and Cooper, 1975).

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Various procedures have been reported for the purification of
ClINH. They all utilize standard biochemical techniques such as
salt fractionation, ion exchange chromatography, gel filtration,
adsorption chromatography, and preparative electrophoresis (Haupt
et a1., 1970; Schreiber, Kaplan and Austen, 1973a; Harpel and
Cooper, 1975; Nagaki, Hashimoto and Inai, 1976; Pensky and Lepow,
in press). High purity, moderate yields, long preparation times,
and low specific activities are all common characteristics of these
procedures. Three assays most commonly used for the detection and
quantification of CllNH functional activity include the inhibition
of the esterolytic activity of CI using the synthetic amino acid
esters N-acetyl-L-tyrosine ethyl ester (Levy and Lepow, 1959) and
N-a-acetyl-L-lysine methyl ester (Harpel, 1970); and the inhibition
of the activity of CI in immune hemolysis (Gigli, Ruddy and Austen,

1968).

Physiologic Function

 

CI inhibitor controls the first step in the activation of and
inhibits enzymes in all four proteolytic systems of the blood:
coagulation, fibrinolysis, kinin formation, and complement. These
enzymes include CI and its subcomponents CIs and CIr (Levy and
Lepow, 1959; Lepow and Leon, 1962; Gigli, Ruddy and Austen, 1968;
Ratnoff et a1., 1969; Nagaki, Iida and Inai, 1974), kallikrein
(Ratnoff et a1., 1969), PF/Dil (Kagen and Becker, 1963), activated
Hageman factor and plasma thromboplastin antecedent (Forbes, Pensky

and Ratnoff, 1970), activated Hageman factor fragments (Schreiber,

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Kaplan and Austen, 1973b), and plasmin (Ratnoff et a1., 1969;
Schreiber, Kaplan and Austen, 1973a).

Loss of CIs or CIr activity is associated with the formation
of a 1:1 molar complex between the inhibitor and the enzyme without
peptide bond cleavage (Nagaki, Iida and Inai, 1974; Harpel and
Cooper, 1975). Plasmin and CllNH form a 1:1 molar complex with
concomitant loss of plasmin activity and degradation of CllNH
(Harpel, 1970; Harpel and Cooper, 1975; Nagaki, Hashimoto and Inai,
1976). The reaction between kallikrein and ClINH is suggested to
follow second order kinetics leading to an inactive 1:1 molar com-
plex (Gigli et a1., 1970; Harpel, Mosesson, and Cooper, 1975).
These complexes are all resistant to denaturation by SDS or urea
(Harpel, Mosesson and Cooper, 1975). Acidic mucopolysaccharides
enhance the ability of ClINH to block the esterolytic and hemolytic
activities of CIs (Nagaki and Inai, 1976; Rent et a1., 1976). At
this time there is little known concerning the chemical mechanisms
of ClINH binding to its target protease. The necessity of peptide
bond cleavage for complex formation to be established between pro-
teolytic enzymes and their naturally occurring inhibitors continues
to be a controversial question. Nevertheless, ClINH has a broad
specificity for serine esterases. As more information is gathered
it would not be surprising if ClINH was found to express a broad

specificity similar to a -macroglobu1in for active serine-

2

proteinases and endopeptidases.

Phylogeny and Ontogeny

Adolphs (1973) reported the presence of a specific inhibitor
of guinea pig CI in normal murine serum. Further up the phylo-
genetic tree Donaldson and Pensky (1970) found identical CllNH
antigens only in some anthropoid primates.

Synthesis of ClINH by hepatic tissue obtained from human
embryos aged only 29 days has been observed by Gitlin and Biasucci
(1969). Immunofluorescent techniques revealed that ClINH is in

5-10% of normal hepatic parenchymal cells (Johnson et a1., 1971).

Pathology

The discovery of ClINH acquired a clinical significance when
an inherited absence of the function of C1INH was diagnosed in
patients with hereditary angioneurotic edema (Landermann et a1.,
1962; Donaldson and Evans, 1963). This disease is characterized by
intermittent localized swelling of the skin and the mucous membranes
and frequent deaths from laryngeal edema.

ClINH deficiency is an autosomal dominant trait. The usual
form of the disease is characterized by extremely low levels of
C11NH. In one variant form an immunochemically normal but func-
tionally deficient inhibitor was described (Rosen et a1., 1965).
This protein exhibits genetically determined variations in electro-
phoretic mobility, binding capacity to CIs, and ability to inhibit
CIs esterolytic activity.

However, it has been suggested that many of these cases may be
artifacts induced by a spontaneous conversion of plasma prekallikrein

into kallikrein by the Hageman factor which occurs exclusively in

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the cold (Van Royen et a1., 1976). Low levels of functional ClINH
are due to the binding of ClINH by kallikrein. Laurell and
Martensson (1971) described an inactive ClINH which was found
in molecular association with albumin. This functional and anti-
genic deficiency is due to impaired synthesis. There is normal
catabolism of ClINH and immunofluorescent analysis revealed no
inhibitor antigen on liver sections from patients with this form
of edema (Johnson et a1., 1971).

In patients with ClINH deficiency, intradermal injection of
(IIs produces a wheal followed by the development of local angioedema.
Elatient plasma will spontaneously generate complement-dependent
lcinin activity. This may reflect episodic activation of Hageman
factor followed by conversion of plasminogen to plasmin. The
filasmin would then activate CI with subsequent depletion of C3 and
C55. These enzymatic activities would normally have been regulated
by ClINH. A

The pathology associated with ClINH is suggested by the dis-
covery of ClINH on the membrane of human carcinoma and blast
leukemic cells using immunofluorescence (Osther and Linnemann,
1973a,b). It was hypothesized that ClINH is a blocking factor
coating neoplastic cells resulting in inhibition of the cytotoxic

activity of complement.

Chemotaxis

 

This section of the literature review presents those aspects
of research concerned with the study of the in vitro migration of

neutrophilic polymorphonuclear leukocytes. Information concerning

8
the history of chemotaxis and the present methodological techniques
utilized in chemotaxis is provided. The structural and functional
aspects of the cytotaxins and regulators of chemotaxis are empha—
sized along with the cellular biochemistry of the chemotactic
response. Furthermore, the role of chemotaxis in inflammatory

reactions is scrutinized.

Historical Perspective

Theodor Leber (1891) performed the first definitive study of
JLeukocyte chemotaxis. Using the capillary tube technique he tested
substances for their ability to attract leukocytes into the capil-
leiry. By observing the cells continuously, Leber demonstrated the
active migration of cells into the test site. He postulated that
tflie force bringing leukocytes into an inflammatory lesion was chemo-
taxis and a foreign substance was essential to initiate cell
migration.

Metchnikoff (1893) combined these findings with others to
formulate a phagocytic theory. Metchnikoff realized that both types
Of phagocytes, macrophages and polymorphonuclear leukocytes are
probably chemotactically responsive. From an observation that the
two different types of phagocytes constituted different proportions
in different lesions with each type phagocytizing different species
of bacteria, Metchnikoff implied a cell specificity in the migra-
tional response. His speculations concerning the mechanisms of cell
motility and the influence of host substances on chemotaxis remain

relevant to this day.

 

9

The work of Von Sicherer (1899) was significant for its attempt
to make the distinction between chemotactic migration and migra-
tion resulting from vascular permeability changes. This involved
performing chemotaxis in vitro rather than introducing capillary
tubes into body cavities as had traditionally been done. Experi-
ments of Comandon (1917) introduced an in vitro technique for
rneasuring chemotaxis by directly observing cell movement between
a slide and coverslip with time-lapse cinematography. McCutcheon
eand colleagues (reviewed by McCutcheon, 1946) studied the random
axnd chemotactic migrations of leukocytes suspended in plasma while
rising the slide and coverslip method. Their investigations were

ruotable for the degree of quantitation in the experiments performed.
Tfiie skin window technique developed by Rebuck and Crowley (1955)
aallowed the in vivo examination of the migration of inflammatory
cells. In response to the stimulus, either the test substance or
‘trauma, cells migrate from the dermis to the coverslip. This
method is not quantitative unless the coverslip is replaced by a
Chamber (Perillie and Finch, 1964); however, this method finds
application in conjunction with in vitro chemotaxis assays for
clinical studies of abnormal leukocyte function.

Menkin (1938) isolated a chemotactic factor from inflammatory
exudates. This substance was active in vitro or in vivo, ninhydrin-
positive, dialyzable, heatestable, and water-soluble. Menkin named
his factor, leucotaxine. He also reported that a similar substance
could be obtained by the action of trypsin on serum proteins. Meier
and Schar (1958) reported the chemotactic activity of gramrnegative

bacterial lipopolysaccharides with no analogous activity from

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gram-positive bacteria. Meier and Schar (1955) also observed the
in vitro chemotactic activity of chicken plasma protein-anti-chicken
plasma protein precipitate. The observation that heat-inactivated
antisera reduced the chemotactic activity was not associated with

complement.

Nature

Chemotaxis is defined as the accumulation of cells at a par-
ticular locus resulting from directional migration in a chemical
ggradient of a soluble diffusible substance (Wilkinson, 1974b). In
(order to understand directional migration it is a necessity to
uunderstand the mechanisms of random migration. Robineaux (1964)

reported the anterior extension of a hyaloplasmic membrane in a
uuoving cell before the initiation of locomotion into which cellular
cxontents flowed. The migrating cell had an adhesive elongated tail
Vfliich remained adherent to the surface until broken away by the
force of forward movement.

The studies of Ramsey (1972a,b) present detailed observations
of human neutrophil movement using phase contrast cinematography.
Ramsey termed the flattened extension of a cell a lamellipodium.
The random formation of lamellipodia and the adherence of one of
these into which cytoplasm flowed are characteristics of migration.
The direction of migration is determined by which lamellipodia
received the cell contents. The remaining lamellipodia formed the
adherent tail and retraction fibers. Ramsey did not consider these
events to be characteristic of amoeboid movement. It was shown that

chemotaxis did not involve changes in rate of migration but was

ll
attributable to directional movement only. Ramsey concluded that
cell movement in the presence and absence of cytotaxin is similar
except that in the former case cell movements are directional.
However, Keller and Sorkin (1966) found that, depending on the test
conditions, the chemotactic substance enhanced random migration and
induced directional migration. By photographic tracing of cell
movement they demonstrated that cells present in a uniform concen-
tration of cytotaxin exhibited a greater degree of migration than
in the absence of cytotaxin. In the presence of a gradient of
attractant neutrophils showed strong directional migration.

The question of how a chemotactic substance can cause the
directional movement of a cell is difficult to answer. A hypothesis
that offers some explanation is that cells possess multiple surface
recognition sites which detect the stimuli. Several investigators
have provided some evidence for this. Using a micromanipulator,
Ramsey (1972a) showed that individual cells could respond repeatedly
to moving the stimulus to another location. In fact, chemotactically
active cells were observed to move towards the center of a gradient
by a convoluted path rather than by a direct route. This supports
the results of Cornely (1966) which showed the capability of leuko-
cytes to reverse the direction of migration in response to reversing
the gradient of chemotactic serum. This indicated that cells are
continuously responsive to a chemotactic gradient. Zigmond (1974),
based on time lapse observations, postulated a spatial rather than
a temporal mechanism of detecting a gradient for the cell. It is
not understood how this spatial mechanism provides the cell with

information to selectively form pseudopods. It was also gugested

12
by Keller and Sorkin (1967b) that cells recognize a chemotactic
gradient through cell surface receptors. Keller and Bessis (1975)
confirmed these studies with an analysis of the behavior of heat-
polarized leukocytes. In the absence of chemotactic stimulation,
heat-induced pseudopod formation occurs randomly in all directions.
In the presence of stimulation the proximal metapod and the distal
pseudopod migrated independently towards the chemotactic target.
This indicated that different sections of the cell are able to
recognize and respond to chemotactic stimulation independently of

each other.

Measurement

 

In 1962, Boyden introduced a revolutionary techniuqe for the
investigation of chemotaxis of macrophages and polymorphonuclear
leukocytes. In this system and in all following modifications the
principle remains the same. A micropore filter separates the cell
containing upper compartment from the lower compartment containing
the chemotactic substance. The filter membrane was of such pore
size that leukocytes could not pass through it except by active
movement towards a chemotactic stimulus. At a given time the
filters are removed and stained. The measurement of migration can
take one of two forms: (1) count the number of cells which have
migrated to a certain depth of the filter, usually the lower surface
(Boyden, 1962); or (2) measure the distance traveled by the leading
front of cells (Weksler and Hill, 1969; Zigmond and Hirsch, 1973).

Another refinement of these techniques is to study the distri-

bution of cells in the filter (Zigmond and Hirsch, 1973). This is

13

accomplished by counting cells in a series of planes at 10 micron
intervals of the filter. The distribution profile of random migra-
tion resembles one side of a normal frequency distribution curve
and significant deviations from this distribution are indications
of directional migration (Zigmond and Hirsch, 1973; Wilkinson, 1974).

Since investigators have noticed that a variable proportion of
cells on the bottom side of the filter becomes detached, several
modifications have been introduced. Keller et a1. (1972) proposed
the use of two filters, the lower being impermeable to the detached
cells. Others (Frei, Baisero and Ochsner, 1974) suggested a
direct count of cells that have fallen into the lower compartment.
In addition, a radioassay employing chromium-51 labeled cells was
described as a means to increase accuracy and efficiency (Goetzl
and Austen, 1972a; Gallin, Clark and Kimball, 1973). A complete
departure from the Boyden system was reported by Nelson, Quie and
Simmons (1975). Their system utilized agarose as a medium for
migration with separate wells for the cells and chemotactic

substance.

Chemotactic Factors

 

Complement-Derived

 

There is much evidence now that the activation of complement by
a variety of substances causes the liberation of chemotactic factors.
Boyden (1962) was the first to speculate upon the importance of
complement as a possible source of chemotactic activity in serum
activated by immune reactions. Similar results were reported by

Keller and Sorkin (1965). It was later shown that normal rabbit

14
serum but not plasma possesses chemotactic activity for rabbit
neutrophils and incubation with immune complexes increases the
chemotactic activity in plasma and serum (Borel and Sorkin, 1969).
When another inflammatory agent, endotoxin, was incubated with fresh
mammalian sera there was evidence for the generation of rabbit
neutrophil chemotactic activity by activation of the complement

system (Snyderman, Gewurz and Mergenhagen, 1968).

Trimolecular Complex C567. The initial reports of Ward, Cochrane

 

and Muller-Eberhard (1965, 1966) on the characterization of comple-
ment components involved in neutrophil chemotaxis in vitro identi-
fied C5, C6, and C7 of rabbit serum as the components of a high
molecular weight complex exhibiting chemotactic activity. It was
their belief that C7 was required for generation of the chemotactic
factor associated with human complement. Later studies demonstrated
the potential importance of other complement factors in chemotaxis.
Stecher and Sorkin (1969) generated chemotactic activity in C6-
deficient rabbit serum, as did Snyderman, Phillips and Mergenhagen
(1970). Snyderman, Gewurz and Mergenhagen (1968) could not generate
activity from CS-deficient mouse serum using endotoxin and could
only generate a low molecular weight factor in normal serum. No
complement-associated chemotactic activity was found in the high
molecular weight fraction of normal activated serum. These results
suggest that CS was essential for the generation of chemotactic
activity in whole serum. However, a more recent report by Lachmann,
Kay and Thompson (1970) confirmed the chemotactic activity of purified

human C567. It was found that the chemotactic activity generated by

15
mixing C 6 with C7 is preempted if the C36 is first available to
bind hydrophobic surfaces. This suggests that C567 may exhibit a
transient action under physiological conditions. Ward and Zvaifler
(1971) reported the presence of C867 in the synovial fluids of human
rheumatoid arthritis patients which was active for rabbit neutro-
phils. Human C867 purified by Arroyave and Muller-Eberhard (1973)

was utilized for chemotactic activity by Berenberg and Ward (1973).

CS-Derived Factors. Largely through the work of Snyderman and

 

associates (Snyderman et a1., 1969; Snyderman, Phillips and
Mergenhagen, 1970), the low molecular weight neutrophil chemotactic
factor in activated guinea pig sera was demonstrated to be a peptide
derived from C5. This chemotactic activity for rabbit neutrophils
was found in sera activated either by endotoxin or immune complexes.
Evidence for this fragment being derived from CS was as follows:
molecular weight of 15,000 on gel filtration coincidental with a
cleavage product of exogenously added 125I CS, and inhibition of
activity by anti-C5 and not anti-C3. Similar results were obtained
for rabbit serum. Other investigators have reported different
results (Clark, Frank and Kimball, 1973). Normal and C4-deficient
guinea pig sera activated by immune complexes or endotoxin when
fractionated revealed both a 17,000 and a 10,000 molecular weight
factor that were antigenically related to C5. Both fractions also
caused bluing when injected intradermally into Evan's blue treated
guinea pigs, suggesting anaphylatoxin activity.

Human serum behaves almost identically to guinea pig serum when

activated by inflammatory agents. Snyderman and Mergenhagen (1972)

l6
activated human serum with endotoxin or aggregated human gamma
globulins. A 15,000 molecular weight cytotaxin was isolated and
characterized as heat stable and antigenically related to CS.
Similar chemotactic fragments which have the characteristics of
C5a are generated by activation of the classical and alternate
pathways of complement (Gallin, Clark and Frank, 1975).

Chemotactic activity can thus be generated from C5 by activa—
tion of whole serum. CS-derived factors have also been shown to
exist by experiments utilizing purified reagents. CSa was produced
by treatment of guinea pig C5 with EACIZ§3 (Shin et a1., 1968).

This fragment possessed chemotactic activity for rabbit polymorpho-
nuclear leukocytes as well as anaphylactic activity for guinea pig
ileum. The action of EACI323 on human CS resulted in the appearance
of a chemotactic factor (12,500 daltons) for rabbit neutrophils with
no muscle-contracting activity (Ward and Newman, 1969). Trypsin
cleavage of human C5 resulted in the appearance of two chemotactic
peptides (8,500 and 12,500 daltons), neither having contractile
activity. Ward et a1. (1973) studied two bacterial proteinases
produced by Serratia marcescens and group A, B-hemolytic Strepto-
coccus. Each proteinase was able to cleave several chemotactic
factors from purified human CS and only the streptococcal proteinase
produced a CS-related factor in human serum.

CS-derived chemotactic factors can also result by the cleavage
of C5 with enzymes endogenous to cells. Lysosomal fractions from
rabbit neutrophils, peritoneal and alveolar macrophages show strong
chemotactic activity for rabbit neutrophils in the presence of fresh

normal serum (Borel, Keller and Sorkin, 1969). Lysates of lysosomal

17
granules from rabbit neutrophils contain a neutral enzyme which
cleaves human C5 into chemotactic fragments (Ward and Hill, 1970).
The enzyme cleaves C5, but not C3, into chemotactic fragments of
variable molecular weights, depending upon the conditions of inter-
action. This has been confirmed for human peripheral leukocytes
(Taubman, Goldschmidt and Lepow, 1970). Venge and Olsson (1975)
demonstrated that chymotrypsin-like cationic proteins of human
granulocytes can generate a chemotactic activity for human neutro-
phils from C5. These enzymes are confined to the azurophil (primary)
cytoplasmic granules of the cell. A CS-cleaving enzyme in lysosomes
is released from neutrophils during phagocytosis (Ward and Zvaifler,
1973). The lysosomal granules of human platelets contain a protein
fraction that acts upon C5 to liberate chemotactic activity for
human neutrophils (Weksler and Coupal, 1973) in a manner very similar
to that described for rabbit neutrophils by Ward and Hill (1970).
The expression of chemotactic activity for rabbit neutrophils is
observed if acidic proteinases from beef lung and either peritoneal
or pulmonary rabbit macrophages are incubated with guinea pig C5
(Snyderman, Shin and Dannenberg, 1972). Rabbit kidney cells infected
with herpes simplex virus release a factor which directly cleaves
guinea pig C5 in serum or in isolation to produce a neutrophil chemo-
tactic factor (Brier et a1., 1970). Similarly, fluids from chicken
embryos infected with either Newcastle disease virus or mumps virus
are associated with the elaboration of a chemotactic factor for
rabbit neutrophils from.human CS (Ward, Cohen and Flanagan, 1972).
{Theerelease of these cytotaxin generators may be secondary to virus-

induced cytotoxicity and could be lysosomal enzymes.

5”

he,
~L n

A»
.
ny.

“A
UV

5‘ _

_
u.;_

'9.

18

The use of purified enzymes and complement components has
provided meaningful information on the mechanisms of the generation
of complement—derived chemotactic factors in vitro. Nevertheless,
this does not necessarily provide information for predicting events
in vivo. The association of C5 with disease states has been well
documented. In the case of tissue injury mediated by immune
complexes, CS-derived products have been isolated. In experimental
immunologic vasculitis where deposits of immune complexes and
complement proteins develop in blood vessel walls, there exists a
correlation between the presence of chemotactic fragments of C5 and
the accumulation of neutrophils (Hill and Ward, 1972). Cleavage
products of C5 have been isolated within the first few hours after
the fixation of complement by immune complexes. Tissue extracts
from rats depleted of complement show no cytotaxins despite the
presence of immune complexes and neutrophils fail to accumulate in
the vessel walls. Two chemotactic fragments antigenically related
to C5 were found in the synovial fluids of humans with rheumatoid
arthritis (Ward and Zvaifler, 1971). In these fluids there was an
extraordinary accumulation of neutrophils. Snyderman, Phillips and
Mergenhagen (1971) demonstrated the presence of chemotactic C5
fragments in vivo after the injection of endotoxin into peritoneal
cavities of guinea pigs or mice and before the influx of leukocytes.
C5~deficient mice exhibited none of these characteristics after
injection of endotoxin. These data suggest an important function
for C5 in the early accumulation of granulocytes in inflammatory

exudates .

19

The physical or chemical nature of these CS-derived chemo-
tactic factors is not well known. The relation of these cytotaxins
to anaphylatoxin is not clear. It was recently reported that pure
human CSa anaphylatoxin isolated from serum was inactive at 10-6M
for inducing human neutrophil chemotaxis (Fernandez, Henson and
Hugli, 1975). However, Shin et al. (1968) observed both biological
activities associated with the same molecular fragment generated
from purified guinea pig C5. Furthermore, it appears that treatment
of C5 with trypsin (Ward and Newman, 1969) or lysosomal enzymes
(Ward and Hill, 1970), and the activation of guinea pig (Clark,
Frank and Kimball, 1973) and human serum (Snyderman and Mergenhagen,
1972) with endotoxin or immune complexes all result in a heterogeneous
population of chemotactically active fragments derived from C5.
Nilsson, Mandle and McConnell-Mapes (1975) alluded to this possi-
bility based on their structural investigations of CS subunit

structure and modification by trypsin and C423.

C3-Derived Factors. The paper by Bokisch, Muller-Eberhard and

 

Cochrane (1969) contributed much to an understanding of the formation
and characteristics of the biologically active C3 fragments. Treat-
ment of human C3 with trypsin, C3 convertase (C22), and the C3
inactivator complex (CoVFB) gave preparations that were chemotactic
for rabbit neutrophils. Smith et a1. (1975) confirmed these results
for human neutrophil activity generated by treatment of human C3 with
trypsin. Opferkuch, Snyderman and Borsos (1973) reported an increase
in chemotactic activity for rabbit neutrophils present in the super-

natant of mixtures of EACZE and rat serum containing EDTA as the

20
number of 22 sites increased. When streptokinase and human plasmino-
gen are added to human and rabbit serum, and purified human C3, a
chemotactic fragment of C3 is generated (Ward, 1967). It is chemo-
tactic for rabbit neutrophils in vitro, causes accumulation of rat
neutrophils in vivo, and increases rat vascular permeability. Ward
et a1. (1973) documented the ability of proteinases derived from
Serratia marcescens and groupa A, B-hemolytic Streptococcus to
generate chemotactic fragments from human C3. By antigenic analysis
of activated serum, it was discovered that only treatment with
Serratia proteinase resulted in a C3-related factor, chemotactic
for rabbit neutrophils. Allantoic fluid from chicken embryos
infected with Newcastle disease virus or mumps virus contains human
C3 cleaving activity for the production of rabbit neutrophil cyto-
taxin (Ward, Cohen and Flanagan, 1972). The studies of Hill and
Ward (1969) indicate the presence of a serine esterase in rat heart
tissue which can cleave C3, isolated or in serum, into chemotactic
fragments. Other tissue generators of C3-derived cytotaxins were
found to reside in the azurophil cytoplasmic granules of human
leukocytes (Venge and Olsson, 1975).

The in vivo significance of C3-derived chemotactic factors has
been indirectly established in two situations. In experimental
myocardial infarcts in rats, C3 cleavage products appear in soluble
extracts of infarcted tissue (Hill and Ward, 1971). If the rats are
depleted of serum C3, there is no chemotactic activity in the myo-
cardium and no intense neutrophil accumulation. The etiological
agent may be the heart tissue protease previously described by Hill

and Ward (1969). Another example is a class of human patients with

21
inflammatory nonrheumatoid arthritis featuring numerous granulocytes
in the joint fluids. A majority of these patients have C3-derived
chemotactic factors in their synovial fluids (Ward and Zvaifler,
1971). In addition, many of these fluids contain a C3-cleaving
enzyme, capable of producing chemotactic activity from C3. These
studies suggest a nonimmunologic function for C3 participation in
the acute inflammatory response to nonspecific tissue injury.

The chemical nature of C3a anaphylatoxin has been studied in
depth. However, knowledge of the relationship of structure to
chemotactic function is still inadequate. It was recently reported
that pure human C3a anaphylatoxin isolated from yeast cell activated
serum is chemotactically ineffective over a range of 10.5 to 3 x 10-7M
(Fernandez, Henson and Hugli, 1975). This was supported by Shin et
a1. (1969), who presented evidence that guinea pig C3a anaphylatoxin
isolated from lipopolysaccharide-activated serum is not chemotactic
for rabbit neutrophils. Extended treatment of C3a with trypsin
yields a fragment retaining chemotactic and losing anaphylactic

activity (Bokisch, Muller-Eberhard and Cochrane, 1969).

C35 and CoVFB. Ruddy, Austen and Goetzl (1975) consistently found

 

chemotactic activity for human neutrophils in mixtures of highly
purified CoVF, B, and D: In fact, the chemotactic activity of

CoVFB correlated with its hemolytic activity. Furthermore, mixtures
of C3b, B, and 5 could be demonstrated to have chemotactic activity.
This is evidence for the association of the appearance of chemotactic
activity with the formation of properdin pathway convertases into

necessary complexes.

22
Non-Complement Factors

Chemotactic factors not associated with the complement system
but still found in the plasma have been identified and characterized
within the last several years. A serum—derived chemotactic mediator
system, the anaphylatoxin-related binary peptide leucotactic system,
has been well described by Wissler (1972a,b) and Wissler, Stecher
and Sorkin (1972a,b). These experiments present evidence for a two
peptide system, consisting of classical anaphylatoxin (CAT) and a
co-cytotaxin (CCT). Neither of these peptides is chemotactic by
itself, but a mixture of the two causes chemotactic migration of
cells. Contact activation of normal guinea pig, rat or hog serum
with hydrophilic, insoluble substances of high molecular weight,
e.g., dextran, yeast or immune complexes, leads to the formation of
these peptides. CAT had anaphylatoxin activity in purified form.
For chemotaxis, a gradient of CAT but not of CCT was essential.
Furthermore, CCT could be replaced by various nucleotides such as
ATP, cyclic AMP or GTP. The magnitude of the chemotactic activity
was found to be strongly dependent on the absolute concentrations
and the molar ratio of the two peptide components.

Chemotactic factors can be liberated during the activation of
plasma enzyme systems other than complement. Borel and Sorkin
(1969) reported that serum clotted on glass became chemotactically
active. The chemotactic activity of kallikrein was investigated
(Kaplan, Kay and Austen, 1972; Weiss, Gallin and Kaplan, 1974).
Biological activity of kallikrein coincident with chemotactic
activity was separated from glass-clotted serum. This activity for

human neutrophils was also generated when prekallikrein was converted

23
to kallikrein by Hageman factor fragments. In support of this were
the experiments showing that reconstitution of Fletcher factor-
deficient serum with prekallikrein completely corrected the chemo-
tactic deficiency associated with kaolin-activated Fletcher factor-
deficient serum. The chemotactic defect was also corrected by
addition of intact activated Hageman factor, suggesting that the
absolute contribution of kallikrein to the chemotactic activity of
kaolin-activated normal serum is small. The coagulation and fibrino-
lytic pathways of plasma participate in the generation of chemotactic
activity.. Kay, Pepper and Ewart (1973) first observed that clot
supernatants, prepared by thrombin action on human fibrinogen, were
chemotactic for human leukocytes. Subsequently, fibrino-peptide B
was identified as a chemotactic agent in the supernatant. The
enzymatic lysis of fibrin (Stecher and Sorkin, 1972) and collagen
(Chang and Houck, 1970) can lead to the generation of chemotactic
factors in vitro.

Investigations into the discovery and characterization of chemo-
tactic factors present in inflammatory tissue have proceeded in the
last few years. Hayashi and associates (Yoshida, Yoshinaga and
Hayashi, 1968) isolated a chemotactic factor from rabbit Arthus
and burned skin lesions. Termed leucoegresin, this cytotaxin was
characterized as being antigenically and physicochemically closely
identical to rabbit IgG (Yoshinaga et a1., 1971; Yamamoto,

Yoshinaga and Hagashi, 1971). Leucoegresin was produced locally in
the skin lesion site and in vitro from rabbit and hyman 196 by the
action of inflammatory neutral SH-dependent proteases (Hayashi et a1.,

1969; Yoshinaga et a1., 1971). The chemotactic activity was also

24
produced by papain action on traditionally papain resistant sub-
classes of IgG (Yoshinaga et a1., 1972). These results suggest that
IgG is subject to structural changes due to enzymatic action leading
to the formation of neutrophil leucoegresin.

Proteins have been studied for the effect of tertiary structural
changes on their chemotactic activity. Human serum albumin, which
is not chemotactic in its native form, becomes chemotactic for human
neutrophils after denaturation by acidification, alkalation, or
reduction and alkylation, or modification of the albumin structure
by conjugation with synthetic non-polar side groups (Wilkinson and
McKay, 1971, 1972). The major conformational changes which
accompany the transition of hemoglobin to globin lead to the acqui-
sition of chemotactic activity (Wilkinson, 1973).

In view of the potent chemotactic activity of cyclic AMP for
slime molds, its potential effect on neutrophil migration has been
investigated by several laboratories. Cyclic AMP was reported to
be chemotactic for neutrophils by Leahy, McLean and Bonner (1970).
This was confirmed by Gamow and Barnes (1974), who also presented
evidence for positive and negative chemotaxis towards cyclic GMP.
Kaley and Weiner (1971) failed to find activity for cyclic AMP,
dibutyryl cyclic AMP or cyclic GMP. Tse, Phelps and Urban (1972)
actually found cyclic AMP to be inhibitory for random and chemo-
tactic migration. The evidence is confusing at best.

Kaley and Weiner (1971) and Higgs, McCall and Youlten (1975)
also reported prostaglandin E1 to be chemotactic for rabbit neutro-
phils. Turner, Campbell and Lynn (1975) did not find this to be

true, but did discover that mild aerobic oxidation of arachidonic

to

25

(catabolic precursor of prostaglandin E1) and eicosapentaenoic acids
yielded strong chemotactic activity for human granulocytes. A
component of the chemotactic oxidized arachidonic acid was identi-
fied as 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid which could
also be generated by the action of a lipoxygenase on arachidonic
acid (Turner, Tainer and Lynn, 1975). Lipoxygenase is an
aggregation-activated platelet enzyme.

Cell-associated chemotactic factors are diverse with regard
to their characteristics and origin. Eosinophil chemotactic factor
of anaphylaxis from the guinea pig lung attracted guinea pig neutro-
phils only when neutrophils comprised 90% of the total cell popula-
tion (Kay, Shin and Austen, 1973). Dialyzable transfer factor from
human leukocytes was discovered to contain a potent chemotactic
agent in vitro for human granulocytes and in vivo for monkey
granulocytes (Gallin and Kirkpatrick, 1974). The phagocytosing
neutrophil releases chemotactic factors for other neutrophils. The
appearance of chemotactic activity following human and rabbit
neutrophil phagocytosis of urate or calcium pyrophosphate crystals
has been reported (Tse and Phelps, 1970). Spilberg, Mandell and
Wbchner (1974):described this chemotactic material as lysosome-
derived and synthesized de novo. Chemotactic activity was recovered
in the supernate of leukocytes rotated in a globulin-coated tube
(Zigmond and Hirsch, 1973). Similar activity was obtained by
repeatedly freezing and thawing horse leukocytes confirming the data
of Cornerly (1966), who subjected the neutrophil to sucrose lysis
and the lysosome granule to freezing and thawing. Chick embryos

infected with either Newcastle disease virus or mumps virus and

REL}

tic

{1811

C. '1
(n
b1 2

26
monkey kidney cells infeqted with mumps virus elaborate chemotactic
activity for rabbit neutrophils. Guinea pig lymphocytes, when
stimulated in vitro by specific antigen, produce a chemotactic
stimulus for guinea pig neutrophils (Ward, Remold and David, 1970).
This may have relevance to the experiments of Ramseier (1969), who
has shown that mixed leukocyte cultures produce a factor that causes
neutrophil accumulation when injected into the skin.

"Immunechemotaxis" is a concept recently proposed from observa-
tions that immune reactions occurring on the surface of the responding
neutrophil elicit the chemotactic response of that cell (Jensen and
Esquenazi, 1975; Williams et a1., 1976).

There is still very little known about chemotactic factors
produced by bacteria. Keller and Sorkin (1967a) found culture fil-
trates from Staphylococcus albus and Escherichia coli to be chemo-
tactic for neutrophils. Ward, Lepow and Newman (1968) characterized
their bacterial factors as low molecular weight substances, the
elaboration of which was related to log phase growth. An electro-
phoretically isolated lipid moiety of E. coli chemotactic factor
was found to be chemotactic for human neutrophils (Tainer, Turner
and Lynn, 1975). The chromatographic and chemical properties of
the E. coli lipid cytotaxins were similar to those of a previously
,reported cytotaxin synthesized from arachidonic acid. The isolated
peptide components were not chemotactic. Schiffman et a1. (1975)
found chemotactic activity in the lipid material from E. coli chemo-
tactic factor in addition to low molecular weight, anionic, heat
stable peptides possessing blocked N-terminal groups. Free carboxyl

groups are required for their activity. Possibly related to these

r"

tr.

1.) '

a:

the

tiOn
the e
StEIe
Eire,“

27
findings are those of Schiffman, Corcoran and Wahl (1975) document-
ing the chemotactic activity of synthetic N-formylmethionyl

peptides.

Cellular Biochemistryiof Chemotaxis

The chemotactic response is a complex sequence of events
involving the recognition of chemotactic factors which is then
translated into directional migration. An overall coherent scenario
has yet to be established. In analyzing the mechanisms of the
chemotactic response, investigators have attempted to alter the
response through biochemical and pharmacological manipulations.
This approach yields results which must be interpreted with caution.
Many of these manipulations utilize agents which act at different
levels, some of which may be irrelevant to directional migration.

The nature of chemotactic recognition is unlikely to be as
specific as the interaction of antibody with antigen. One recogni-
tion pattern is that nonpolar groups are necessary for chemotactic
recognition. This has largely evolved from the studies of Wilkinson
(1973, 1974) and Wilkinson and McKay (1972, 1972). Cells theoreti-
cally detect a soluble protein which has many exposed non-polar
side groups and many mutually repulseive groups for the prevention
of polymerization and insolubilization. Native proteins upon denatura-
tion undergo conformational changes with non-polar groups forced to
the exterior. Cells detect these changes without a requirement for
stereospecific binding by a receptor. Interactions may take place
through hydrophobic bonding. Indeed, it was found that surface

activity and chemotactic activity of proteins are related

28
(Wilkinson, 1974a). Other mechanisms may involve interactions at
the membrane level of a different type than the one proposed by
Wilkinson. Ionic interactions between cell surface and chemo-
tactic molecules may possibly be influencing chemotaxis. Gallin,
Durocher and Kaplan (1975) demonstrated a high degree of association
between changes in cell surface charge and chemotactic activity.
Human chemotactic factors reduced the negative surface charge on
human granulocytes.

An important aspect of chemotactic recognition is that of
cell-specific chemotaxis. A mechanism for this was suggested by
Wissler, Stecher and Sorkin (1972). Their hypothesis proposes
that cell-specificity is governed by the proportions of the two
peptides CAT and CCT in the chemotactic mixture. Both rabbit neu-
trophils and guinea pig eosinophils responded to certain proportions
of CAT and CCT, while at other proportions only one of the cell
types migrated. These effects need to be confirmed in a homologous
system since CAT and CCT were generated from pig serum. Further
evidence of this type of regulation is provided by the work of Kay,
Shin and Austen (1973) which demonstrated a marked synergism between
ECF-A and C5a in their ability to stimulate eosinophil chemotaxis.
Electrophoretically isolated farctions of E. coli chemotactic factor
yielded free protein components with no chemotactic activity, a fee
lipid moiety that is chemotactic for both rabbit alveolar macrophages
and human neutrophils, and lipoprotein complexes that only stimulate
macrophage migration (Tainer, Turner and Lynn, 1975). In this case,

proteins act as modifiers of target cell specificity.

“tit

anae

mm

110?

but

hi

or

3;

ph

CE

29

Anaerobic glycolysis is probably the major energy source for
neutrophil chemotaxis. Chemotaxis is diminished by inhibitors of
anaerobic glycolysis such as iodoacetate (Carruthers, 1966) and
inhibited to a lesser degree by uncouplers of oxidative phosphoryla-
tion (ward, 1966). This suggests that oxidative metabolism is used
but is not essential for the chemotactic response. This was con-
firmed by Goetzl and Austen (1974a). They found the ability of a
chemotactic factor to stimulate glucose metabolism of human neutro-
phils through aerobic glycolysis or the hexose monophosphate shunt
is associated with a maximal chemotactic response, but alone this
stimulation is not sufficient for chemotaxis. Ouabain inhibits
chemotaxis but is reversed by raising external K+ concentration
(Ward and Becker, 1970a).

Agents, such as prostaglandins, theophylline and epinephrine,
which increase intracellular cyclic AMP of rabbit neutrophils,
decrease spontaneous motility and chemotactic responsiveness (Rivkin,
Rosenblatt and Becker, 1975). Cholera toxin increases cyclic AMP
levels and inhibits chemotaxis. Chemotactic stimuli have no sig-
nificant effect on intracellular cyclic AMP levels. The experiments
of Estensen et a1. (1973) suggested that chemotaxis is enhanced by
agents which increase cellular cyclic GMP concentration such as
phorbol myristate acetate, acetylcholine, imidazole, and 8-bromo
cyclic GMP. Until more is known about the chemotactic response,
the exact relationship between cyclic nucleotides and neutrophil
migration remains nebulous.

Some biochemical and ultrastructural information is known con-

cerning the relationship of cellular structures and chemotaxis.

30
Neutrophils exposed to CSa show a transient increase in the number
of microtubules (Goldstein et a1., 1973), and colchicine, which
prevents tubulin polymerization, inhibits chemotactic migration
(Caner, 1965). Bryant et a1. (1966) reported optimal leukocyte
migration in the presence of divalent cations. CS-derived chemo-
tactic factor has been reported to cause a redistribution of
granulocyte intracellular calcium out of the cytoplasm and into a
granule fraction in association with microtubule polymerization
(Gallin and Rosenthal, 1974). The mechanism of chemotactic factor
alteration of intracellular calcium is unknown.

Cytochalasin B is a fungal metabolite which disrupts contractile
microfilament systems. It has a complex effect on human and rabbit
neutrophils, stimulating or inhibiting it, depending on the drug
concentration (Becker et a1., 1972). However, the role of micro-
filaments in migration is confused by the findings of Zigmond and
Hirsch (1972) that cytochalasin B suppressed glycolysis by inhibiting
sugar transport into leukocytes. Cytochalasin B also causes a
decrease in the volume of rabbit neutrophils, an effect similar to
that observed with chemotactic factors (Hsu and Becker, 1975).

Drugs which inhibit RNA and protein synthesis such as puromycin
and actinomycin D were reported by Carruthers (1967) to have a slight
inhibitory effect on neutrophil random migration, but a complete
inhibitory effect on chemotactic migration.

The complementederived chemotactic factors as well as E. coli
chemotaCtic factor induce the release of lysosomal enzymes from
rabbit neutrophils without a similar effect on cytoplasmic enzymes

(Becker et al., 1974a,b). No statistical correlation was found

31
between the abilities of the chemotactic factors to stimulate migra-
tion and to induce release of lysosomal enzymes.

The chemotactic response of rabbit neutrophils to complement-
derived and E. coli chemotactic factors involves the activation of
a serine esterase (Ward and Becker, 1969; Ward and Becker, 1970b;
Becker, 1972). Activation of proesterase 1 was shown to be associated
with chemotactic activity and a correlation between the degree of
activation and level of chemotactic activity was established (Becker,
1972). The nature and mechanism of esterase 1 activity during the
chemotactic response remains to be determined. There is no informa-
tion in regard to the manner by which chemotactic factors activate
proesterase 1 and there is no evidence concerning the cell location
of proesterase l.

A phenomenon related to esterase 1 activity is deactivation
(Ward and Becker, 1968; Becker, 1972). Rabbit neutrophils incubated
with C567 or CSa are cross-deactivated to C567) Cta, C3a, or E. coli
chemotactic factor (Becker, 1972). Deactivation was also observed
with human neutrophils using kallikrein and C5a (Goetzl and Austen,
1974b), human eosinophils using histamine (Clark, Gallin and Kaplan,
1975) and ECF-A and C5a (Wasserman et a1., 1975). Kallikrein and
C5a cross-deactivated to each other (Goetzl and Austen, 1974b).
CoVFB and C33 cross-deactivated to kallikrein (Ruddy, Austen and
Goetzl, 1975), ECF-A and C5a cross-deactivated to each other
(wasserman et a1., 1975), and histamine cross-deactivated to C5a
(Clark, Gallin and Kaplan, 1975). Deactivation of human neutrophils
by kallikrein did not result in a reduction of hexose monophosphate

shunt stimulation after the introduction of kallikrein (Goetzl and

32
Austen, 1974a). Deactivation is prevented by the same phosphonates
that inhibit chemotaxis and the inhibition profiles are the same for
the two processes (Ward and Becker, 1968). This indicated that
esterase 1 is involved in both chemotaxis and deactivation.
Recently, Becker (1975) has presented preliminary evidence that
activation of esterase 1 is also involved in spontaneous motility.
Deactivated neutrophils at the present time are not well character-
ized with respect to functions.

The possibility that activation of esterase l is important for
spontaneous motility may suggest a role for serine esterases in a
phenomenon essential to motility. It has been suggested that
cell surface proteases mi ht function in cell attachment (Whur,
Payne and Koppel, 1974). Grinnell (1975) provided some recent
evidence for this possibility. He found that cell attachment to a
substratum in serum-containing medium was inhibited when protease
inhibitors were added to the medium. Furthermore, proteolytic
activation of serum-coated substrata resulted in an enhanced rate
of cell attachment.

The report by Lentnek, Schreiber and MacGregor (1976) demon-
strated that human granulocyte adherence is augmented in inflamma-
tory states and that this augmentation is mediated by inflammatory
plasma factors. The importance of granulocyte adhesiveness to the
vascular endothelium for the initiation of leukocyte migration from
venules has been shown in hamsters by Atherton and Born (1972).
Zigmond and Hirsch (1972) have suggested that a possible effect of
cytochalasin B on leukocyte motility is to cause an increase in

adhesiveness of the cells to their substrate (Carter, 1967). In

33
addition, the relationship between cell adherence and motility may
also involve surface charge. In the amoebae there is an inverse
correlation between cell adhesiveness and negativity of amoebae
surface charge (Ambrose and Forrester, 1968). Bone marrow granulo—
cyte precursor cells were described as having high negative surface
charge, poor distensibility, low adherence, and poor pseudopod
formation (Lichtman and Weed, 1972). The negative surface charge
of human granulocytes was decreased when cells were incubated with
chemotactic factors (Gallin, Durocher and Kaplan, 1975). Their
findings support the concept that an appropriate alteration in cell
adhesiveness may be critical for the chemotactic response of human

neutrophils.

Modulators

 

Agents which are known to modulate neutrophil chemotaxis exert
their activity at any of three levels: (1) formation of chemotactic
factor, (2) interaction with preformed chemotactic factors, and (3)
interference with cellular events involved in the chemotactic
response.

A recent study (Walker et a1., 1975) demonstrated that a prepa-
ration from normal human plasma reduced human leukocyte chemotaxis
only when it was added to serum before activation with zymosan.

This low molecular weight (<500) anti-inflammatory agent did not
affect cell function directly and it was concluded that the fraction
selectively inhibits the release or generation of serum chemotactic

factors in vitro.

34

Substances which interact with chemotactic factors are found
in the serum and as cellular constituents. Normal human serum was
found to contain an inactivator of chemotactic factors for rabbit
neutrophils (Berenberg and Ward, 1973). It directly and irreversibly
inactivates the chemotactic fragments of C3 and C5, C567, and E.
coli chemotactic factor. Further purification of this inactivator
revealed two inactivators, a 75 B-globulin specific for C3-derived
cytotaxin and a 45 a-globulin specific for the CS-derived cytotaxin
(Till and Ward, 1975). Both inactivators were effective against
kallikrein and E. coli chemotactic factor. Furthermore, the B-
globulin inactivator possessed the ability to inactivate C3a and
C5a anaphylatoxins, while the a-globulin inactivator could only
inhibit C3a anaphylatoxin (Ward, Data and Till, 1974). It is still
confusing as to whether or not chemotactic factor inactivator is
different from anaphylatoxin inactivator (Bokisch and Muller-
Eberhard, 1970) and the C567;INH of reactive lysis (McLeod, Baker
and Gewurz, 1975). Clinically, elevated serum levels of the chemo-
tactic factor inactivator were found in patients with Hodgkin's
disease, indicating a possible cause for deficient skin inflamma-
tory reactions in these patients (Ward and Berenberg, 1974). Sera

deficient in a -antitrypsin are also deficient in the chemotactic

1
factor inactivator (Ward and Talano, 1973). A similar acting
inactivator has been observed in tumor cells and their ascitic
fluid, normal rat tissues and serum (Brozna and Ward, 1975). Other
serum inhibitors of chemotactic factors have been associated with

chronic, hypocomplementemic glomerulonephritis (Gewurz et a1., 1967),

cirrhosis (DeMeo and Andersen, 1972), skin test anergy (Van Epps,

35
Palmer and Williams, Jr., 1974) and reticulum cell sarcoma (Ruutu
et a1., 1975).

Cell-derived products exhibit inhibitor activities towards
chemotactic factors. Incubation of neutrohil granule lysates or
post-phagocytic media with CS-related cytotaxins resulted in pro-
gressive inactivation of chemotactic activity (Wright and Gallin,
1975). The chymotrypsin-like cationic proteins of human granulo-
cytes abolish chemotactic activity upon incubation with C5 chemo-
tactic fragments (Venge and Olsson, 1975).

The serum protease inhibitors, ClINH and a -macroglobu1in

2
reduced the chemotactic activity of kallikrein and plasminogen
activator simultaneously with the suppression of kinin-generating

and plasminogen-activating activities, respectively (Goetzl and
Austen, 1974). This is presumably accomplished by interaction with

an intact serine esterase active site.

These two inhibitors in addition to ol-antitrypsin directly
affect the chemotactic response of human neutrohils. ClINH reversibly
enhances and inhibits the neutrophil chemotactic response to acti-
vated plasma and trypsin-activated C3, respectively, without
affecting spontaneous motility (Smith et a1., 1975). Goetzl (1975)
confirmed some of these results and found that al-antitrypsin, a2-
macroglobulin, and TLCK caused irreversible enhancement of random
migration and inhibition of chemotaxis. In addition, ClINH slightly
enhanced the chemotactic response to trypsin—activated C5. Clinical

studies of serum inhibitors of neutrophil chemotactic response acting

directly on the cell have been reported (Ward and Schlegel, 1969;

36
Smith et a1., 1972; Soriano et a1., 1973). These patients are
characterized by a severe susceptibility to infections.

The regulation of the chemotactic response can proceed through
factors normally present in human leukocytes. Incubation of mono-
nuclear or polymorphonuclear cells in acidic medium, with endo-
toxin or with starch particles, releases a neutrophil-immobilizing
factor (Goetzl and Austen, 1972). This factor irreversibly
suppresses chemotactic and random migration without affecting other
cell functions (Goetzl et a1., 1973). Products of human activated
lymphocytes when fractionated revealed the existence of a leukocyte
inhibitory factor which specifically inhibits human neutrophil

migration (Rocklin, 1974).

Clinical Disorders

 

The descriptions of syndromes in which isolated deficiencies of
chemotactic function are accompanied by clinical manifestations add
support to the concept of an essential role for chemotactic migra-
tion in the inflammatory response. Examples of primary defects of
chemotaxis in which the patient's serum contains inhibitors of
chemotaxis were described in the previous section. Other types of
chemotactic malfunctions are those in which there is an intrinsic
defect of neutrophil movement or the patient's serum is inadequate
as a source of chemotactic stimuli.

Intrinsic disorders of neutrophil movement and other cell
functions are reported to be associated with chronic granulomatous
disease (Steerman et a1., 1971), acquired granulomatous disease

(Singh et a1., 1972), granulocytasthenia (Higgins, Swanson and

37
Yamazaki, 1970) and pyoderma gangrenosum (Miller and Dooley, 1973).
Abnormalities of only neutrophil movement are the Chediak-Higashi
syndrome (Clark and Kimball, 1971), lazy leukocyte syndrome (Miller,
Oski and Harris, 1971), diabetes mellitus (Mowat and Baum, 1971)
and familial chemotactic defect (Miller et a1., 1973).

The second group of patients can be distinguished by the inability
of their serum to provide adequate chemotactic stimulus for their
own neutrophils or for control neutrophils. Gewurz et a1. (1967)
reported observations of failures to generate cytotaxins in C2
deficient serum. Alper et al. (1970) described a patient with
Klinefelter's syndrome, C3 deficiency and an inability to generate
chemotactic activity until C3 was added to the serum. Miller and
Nilsson (1970) found a deficiency of serum chemotactic activity
in a child with CS functional deficiency and normal serum levels of
C5. Weiss, Gallin and Kaplan (1974) discovered an abnormality of

chemotactic activity in serum from Fletcher factor deficiency cases.

Chemotaxis in Inflammation

 

It is the responsibility of investigators to demonstrate that
the in vitro observation of leukocyte directional migration using
techniques such as the Boyden chamber is a significant aspect of the
inflammatory response in vivo. However, in vivo manipulations
invariably begin a chain reaction of events that may influence cell
movement through mechanisms other than those essential for directional
migration. This makes interpretation of a direct cause-effect
relationship between eXperimental manipulation and chemotactic
migration very difficult. Nevertheless, there is strong presumptive

evidence of an in vivo correlate of chemotaxis.

38

Buckley (1963) produced tiny heat injuries in the rabbit ear
without damaging the capillaries. Granulocytes could be observed
migrating towards the site of injury. Actively motile granulocytes
moving at random were seen near the center of injury and peripheral
cells were moving directionally towards the center. Hurley (1963)
studied leukocyte emigration from rat skin using electron microscopy
and intravenous injection of visible markers. Hurley demonstrated
that leukocytes but not the injected particles or plasma protein
passed through the vascular endothelium. Histamine treatment
caused the reverse situation to occur. Therefore, Hurley could
dissociate the two phenomena of leukocyte emigration and vascular
permeability.

More recently, passive Arthus reactions in synovial tissues
were introduced in rabbits depleted of neutrophils with nitrogen
mustard (DeShazo, Henson and Cochrane, 1972). C6 deficient rabbits
exhibited protracted inflammation and neutrophil accumulation in the
synovial tissue. Arthus sites in the synovial blood vessel walls
were established by intravenous injection of antigen and injection
of antibody into the knee joint. Neutrophils injected into the
joint space migrated from the space into the synovium towards the
site of immune complex-complement reaction in the vessel walls.
There was marked inhibition of migration in C6 deficient or C3-
depleted rabbits. Furthermore, little or no vascular permeability
existed as measured by a lack of leakage of intravenously injected
125I-serum albumin out of the vessels. The review by Cochrane and

Janoff (1974) summarizes the in vivo studies which indicate the

39
importance of complement for the accumulation of neutrophils at
sites of immunological reactions.

Snyderman, Phillips and Mergenhagen (1971) investigated the
time sequence of in vivo production of chemotactic factors,
eventually identified as CS-derived fragments. They reported a
rise and fall of neutrophil chemotactic activity in the peritoneal
fluid of guinea pigs within six hours of endotoxin injection.
Neutrophils appeared during the decline of chemotactic activity
and increased in numbers during the first 24 hours. C5 deficient
mice failed to show any of these peak patterns.

Wilkinson et a1. (1973) extended the experimental procedure
of Snyderman, Phillips and Mergenhagen (1971) in order to study
the time course of appearance of eosinophil and macrophage-related
activities. The patterns for neutrophil chemotactic activity and
migration were similar to those reported previously. In addition,
peaks of macrophage and eosinophil chemotactic activities appeared
synchronously with the peak of chemotactic activity. Eosinophil
chemotactic activity declined simultaneously with neutrophil
activity but a plateau of macrophage chemotactic activity remained
for several days. Macrophages and eosinophils reached the peri-
toneal cavity slower than neutrophils, reaching a peak several days
after injection of inflammatory stimulant.

The experiments described in this section provide significant
circumstantial evidence for the probability of chemotactic migration
occurring in vivo. The experiments of Buckley (1963) demonstrated
directional migration of leukocytes. The work of Snyderman, Phillips

and Mergenhagen (1971) and Wilkinson et a1. (1973) associated the

40
formation of chemotactic activity with the appearance of the spe-
cifically responding leukocytes which suggested the importance of
a chemotactic gradient for directional migration. The data of
DeShazo, Henson and Cochrane (1972) and DeShazo et a1. (1972) offer
an isolated in vivo correlate of complement-mediated chemotaxis.
To postulate complement as a mediator of chemotactic responses in
vivo would suggest that there exists an extravascular complement
pool. Alper and Rosen (1967) proposed the possibility of 30% of
the total vascular C3 protein existing in extravascular pools.
Finally, both Hurley (1963) and DeShazo, Henson and Cochrane (1972)
dissociated leukocyte migration due to chemotactic responsiveness

from migration due to altered vascular permeability.

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Th

Tc

ARTICLE 1
EFFECTS OF HUMAN CI INACTIVATOR ON COMPLEMENT AND

1
NON-COMPLEMENT MEDIATED HUMAN NEUTROPHIL CHEMOTAXIS

DAVID Y. LIU2 AND RICHARD A. PATRICK3

DEPARTMENT OF MICROBIOLOGY AND PUBLIC HEALTH

MICHIGAN STATE UNIVERSITY, EAST LANSING, MICHIGAN 48823

This work was supported by National Institutes of Health Grants
AI 11367-03 and 5-SOl-RR-0565606-06. The complement terminology
used conforms to the recommendations of the World Health
Organization Committee on Complement Nomenclature (Immuno-
chemistry l:l37,1970).

2D. Y. Liu was supported as a Predoctoral Trainee (GM-0191108),
National Institutes of Health. Taken in part from a disser-
tation submitted by David Y. Liu to Michigan State University

in partial fulfillment of the requirements for the Ph.D.
degree.

To whom reprint requests should be addressed, Department of
Microbiology and Public Health, Michigan State University,
East Lansing, Michigan 48824.

60

61
ABSTRACT
The presence of purified human CI inactivator (ClINH) with
either mixed blood leukocytes or isolated polymorphonuclear leuko-
cytes from humans resulted in significant inhibition (44 to 94%) of
chemotaxis to human C3a and human or guinea pig C5a. C3-derived
chemotactic activity was generated from purified human C3 with either

trypsin or EAC4oxy2.

C5a was generated from purified human C5 with
trypsin or isolated from LPS-activated guinea pig serum as the
15,000 molecular weight fraction. When utilizing N-formylmethionyl-
phenylalanine as the cytotaxin, ClINH caused substantial enhancement
(183% :_46) of neutrophil chemotaxis. Well-defined conditions for
ClINH participation in either enhancement or inhibition is now

clearly established so that the basic mechanism of ClINH function

can be further elucidated.

INTRODUCTION

 

Plasma contains a class of a-globulins which have classically
been shown to contain inhibitors of proteolytic enzymes. These
protease inhibitors are believed to control the action of several
multicomponent enzyme systems that mediate blood coagulation, disso-
lution of blood clots, kinin production, and complement-associated
activities. ClINH possesses a broad inhibitory specificity for
many active enzymes functioning within these systems. These include
CI and the subcomponents CIs and CIr (1-4), kallikrein (3), PF/dil
(3), plasmin (3), Hageman Factor (5), active Hageman Factor fragments

(6), and plasma thromboplastin antecedent (5).

62

Fragments of C3 and C5 (C3a and C5a) have been reported to be
chemotactic for polymorphonuclear leukocytes (7,8). The biologically
active cleavage products are presumably generated in plasma via
activation of complement by LPS, Ag-Ab complexes, zymosan, and other
agents (9,10).

Recent reports have demonstrated that ClINH and other plasma
inhibitors affect the migration of human neutrophils (11,12) and
guinea pig macrophages (13). Smith et a1. (11) demonstrated that
human ClINH affected the migration of human polymorphonuclear
leukocytes in modified Boyden chambers. Results indicated that
human ClINH under certain circumstances increased the chemotactic
response of human peripheral blood leukocytes in vitro to activated
plasma or serum. In preliminary experiments utilizing trypsin-
activated C3 as a chemotactic source, ClINH decreased the chemo-
tactic response of human leukocytes.

‘These studies have been extended in this report to show the
effect of ClINH on human neutrophils responding to cytotaxins
generated from purified human C3 and C5, and to N-formylmethionyl-
phenylalanine. Evidence is presented to show that ClINH causes
marked inhibition of the neutrophil chemotactic response to C3a
and C5a. Moreover, these results are confirmed by the use of iso-
lated guinea pig C5a generated in whole serum. Additional experi-
ments designed to clarify the conditions of ClINH-mediated enhance-
ment of chemotaxis are also reported here. By use of the potent
cytotaxin, N-formylmethionylphenylalanine (14) we present evidence
that ClINH can cause a substantial enhancement of the chemotactic

response of migrating human neutrophils in a plasma-free system.

63

MATERIALS AND METHODS

 

Materials: All chemicals used were reagent or analytical
grade and were obtained from the following sources: ATEE4 from
Aldrich Chemical Company, Milwaukee, Wisconsin: BSA, trypsin, and
Ficoll (400,000 mol. wt.) from Sigma Chemical Co., St. Louis,
Missouri; SBTI from Calbiochem, La Jolla, California; SDS from
Pierce Chemical Co., Rockford, Illinois; Salmonella typhimurium
LPS from Difco Laboratories, Detroit, Michigan; HBSS with phenol
red from Grand Island Biological Co., Grand Island, New York;
sodium diatrizoate (Hypaque) from Winthrop Laboratories, New York,
New York; Sephadex from Pharmacia Fine Chemicals, Inc., Piscataway,
New Jersey; DEAE-cellulose from Schleicher and Schuell, Inc.,
Keene, New Hampshire; Hypatite C from Clarkson Chemical Company,
Inc., Williamsport, Pennsylvania; Dowex AG2-X10 from Bio-Rad
Laboratories, Richmond, California; and NFMP from Andrulis Chemical

Co., Bethesda, Maryland.

Methods: Human ClINH was prepared according to the method
of Pensky and Lepow (15). Briefly, this procedure involved ammonium
sulfate precipitation of fresh human serum followed by sequential
chromatography of the supernatant on Dowex AGZ-XlO, DEAE-cellulose

and Hypatite C (modified hydroxylapatite). ClINH was also purified

 

4 . .
ATEE, N-acetyl-L-terSine ethyl ester; BSA, bov1ne serum

albumin; LPS, lipopolysaccharide; Try, trypsin; SBTI, soybean trypsin
inhibitor; SDS, sodium dodecyl sulfate; HBSS, Hanks' balanced salt
solution: NFMP, N-formylmethionylphenylalanine; PMN, polymorphonuclear
neutrophils; C3(Try), trypsin-activated C3; C5(Try), trypsin-activated
C5; C3(EAC4), EAC4-treated C3: C3(EAC40XY2), EAC40XY2-activated C3;
C5a(LPS), LPS-activated guinea pig serum; hpf, high-powered field.

64
from crude ClINH generously donated by the American National Red
Cross, Bethesda, Maryland. The highly purified preparation demon-
strated a major and a minor protein band on SDS-polyacrylamide (7%
gel) electrophoresis (16) consistent with the band pattern recently
reported by Harpel and Cooper (17). Isoelectric focusing (18,19)
revealed one broad band when performed in a pH gradient 3.5-10
with an apparent pI of 4.0. The specific activity of the ClINH was
180 inhibitor ATEE units/OD280 nm (1). The protein content of the
ClINH was 330 ug/ml. In addition to the esteratic assay, ClINH was
quantitated hemolytically with human complement components according
to Gigli et a1. resulting in a specific actiVity of 133 U/ug (4).
Monospecific—goat antiserum against ClINH was a generous gift of
Dr. Chester Alper, Center for Blood Research, Harvard Medical School,
Boston, Massachusetts. This antiserum was utilized for immunochemical
quantitation by radial immunodiffusion analysis (20). Antiserum
against whole human serum revealed one precipitin arc in the a2
region in immunoelectrophoresis. Less homogeneous preparations of
ClINH (60-100 U/OD) showing three bands in polyacrylamide gel electro-
phoresis behaved identically to highly purified ClINH in chemotaxis
experiments. Unless otherwise noted, ClINH was used in chemotaxis
assays at a concentration of 7 ATEE units/ml. Human C3 and C5 were
prepared according to published procedures (21,22) and according to
Dr. B. F. Tack (personal communication). The Lowry procedure (23)
was used to determine protein content with bovine serum albumin as

the reference standard. Dilute protein solutions were quantitated

spectrophotometrically according to Murphy and Kies (24).

65

Generation of Cytotaxin: The generation of chemotactic

 

activity from serum or purified complement components was performed
in the following manner. One hundred micrograms per milliliter of
human C3 was treated with 1% trypsin (w/w) for 90 sec at 30°C,

pH 7.4, and the reaction stopped by addition of 3% SBTI. EAC4°xy2
was prepared as described by Muller-Eberhard et al. (25). A cell
button of 2 x 109 EAC4OXy2 was incubated with 1 mg human C3 (1 ml)
for 60 min at 37°C. The cells were removed by centrifugation and
the supernatants were tested for chemotactic activity. One hundred
micrograms per milliliter of human CS was treated with 1% trypsin
for 25 min at 30°C followed by addition of 3% SBTI. Guinea pig

C5a was isolated from serum according to the procedure of Snyderman
et a1. (26). Eight milliliters of guinea pig serum was incubated
with 800 ugLPS for 30 min at 37°C, followed by 30 min at 56°C and
subsequent chromatography on Sephadex G-75 (5.0 x 10 cm). Eight

milliliters of serum without LPS treatment was subjected to the

same incubation and gel filtration conditions (sham control).

Chemotaxis: Granulocyte-rich leukocyte preparations were

 

obtained by dextran sedimentation of heparinized venous blood as
previously described (27). The leukocyte-rich plasma was diluted

in HBSS and 5 m1 aliquots were layered on 4 m1 of 6.3% Ficoll -

9.9% Hypaque cushions in siliconized tubes and centrifuged at 800 x G
for 30 min at room temperature (28). Cell buttons from this centri-
fugation containing approximately 95% neutrophils were resuspended

in HBSS made 0.5-2% BSA or 10% plasma at 2.0 x 106 cells/ml for use

in the modified Boyden assay. Acrylic blind well (.2 m1) chemotaxis

66
chambers (Neuro Probe, Inc., Bethesda, Maryland) were assembled with
Millipore filters (Millipore Corporation, Bedford, Massachusetts)
of 3 um pore size. Following incubation of the chambers the filters
were fixed in methanol and stained with Mayer's hematoxylin and
eosin. The filters were dehydrated with increasing concentrations
of ethanol, cleared with xylene, and mounted on glass slides with
Permount. The chemotactic response assessed in triplicate chambers
was evaluated by the number of cells accumulating on the lower
surface on the filter after a 3 hr incubation at 37°C in an atmosphere

containing 5% CO and 95% humidity. Ten 400x fields were counted on

2
each filter and the results expressed as the percentage of positive
control. The distribution of cells throughout the filter was counted
and plotted as described by Zigmond and Hirsch (29).

The Student's t test was used with a=.05 considered significant.

Standard deviations (:.l s.d.) were based on the variability of

counting ten 400x fields.
RESULTS

Inhibition of C3a-mediated chemotaxis by ClINH: The chemo-

 

tactic response of human neutrophils to C3(Try) was markedly inhibited
when ClINH was added with the neutrophils in the upper compartment of
chemotaxis chambers (Figure 1). This inhibitory effect was observed
whether mixed blood leukocytes were suspended in 10% homologous plasma
(Figure 1A) or isolated PMN were supported in 0.5% BSA (Figure 1B).
Under these conditions and in the presence of ClINH the chemotactic
responses to C3(Try) were 7% :_8 (:_l s.d., p<.001) and 56% :_6

(p<.05) respectively when compared with the cell responses in the

67

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68

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69

absence of ClINH. That trypsin activation of C3 effectively
generated chemotactic activity was ascertained by comparison of
the PMN response to C3 and SBTI-inactivated trypsin. It should be
noted that in the plasma support system ClINH inhibition (7% :_8
of control) reduced the chemotactic response below (p<.001) that
obtained with C3 treated with SBTI-inactivated trypsin (41% :_4).

Substantial inhibition also occurred when EAC4oxy2 was used to
generate chemotactic activity from C3 (Figure 2). In this instance
ClINH decreased the cell response to 35% :_38 (p<.01) of positive
control. That EAC4oxy2 effectively generates cytotaxin is indicated
by control chambers containing EAC4-treated C3. ClINH had no sig-
nificant effect on this baseline response (p>.05).

Upon varying the concentration of ClINH present with the PMN,
maximum inhibition occurred at S and 7 U/ml and marked inhibition

was still seen at l U/ml.

Inhibition of C5a-mediated chemotaxis by ClINH: Results shown

 

in Figure 3 indicate that the chemotactic response of neutrophils to
human C5(Try) was also inhibited by ClINH. Inhibition was found
whether mixed blood leukocytes in 10% plasma (Figure 3A) or isolated
PMN in 0.5% BSA (Figure 38) were used in chemotaxin chambers. Under
these conditions and in the presence of ClINH, inhibition of C5(Try)-
mediated chemotaxis was 29% :_25 of control (p<.001) and 54% :_21
of control (p<.001) respectively. ClINH did not significantly affect
the baseline control (p>.20).

Results of confirmatory experiments with guinea pig C5a are

shown in Figure 4. The chemotactic response of purified neutrophils

70

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74

Figure 4. Effect of ClINH on the chemotactic response of
neutrophils to guinea pig C5a. Each column and bracket is the
mean :_1 s.d. for three experiments with different donors in
each experiment; Mean control (100%) chemotaxis was 23 neutrophils/
hpf; ClINH (7 units/m1), hatched columns; No ClINH, open columns;
PMN were suspended in 2% BSA; Sham represents 15,000 molecular
weight fractions from G-75 Sephadex gel filtration of guinea pig
serum previously heated at 37°C for 30 min and then 56°C for

30 min; see Materials and Methods.

75

 

 

 

 

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76

to 0.2 m1 of the C5a preparation (3 ug/ml protein) was signifi-
cantly inhibited (40% :_22 of control, p<.001) by ClINH. The 15,000
molecular weight fraction of untreated guinea pig serum (sham control)
was not significantly affected by ClINH (p>.20). Another prepara-
tion of highly purified ClINH obtained as a generous gift from Dr.
Jack Pensky (Department of Medicine, Case Western Reserve School of
Medicine) gave similar results.

The dose response of ClINH-mediated inhibition of C5(Try)
chemotaxis was similar to that found with C3(Try), i.e., maximum
inhibition (96%) at 5 and 7 U/ml with substantial inhibition (40%)

observed at l U/ml.

Cell distribution of ClINH-mediated inhibition of C3(Try)

chemotaxis: A comparison of the migrating cell distribution in

 

micropore filters in the absence and presence of ClINH was performed
(Figure 5). Mixed blood leukocytes responding to C3(Try) at 45',
90', and 135' were quantitated in 10 micron increments. The result-
ing cell distributions clearly show that at each time interval

(45', 90', or 135') ClINH was associated with a greater number of
cells near the top of the filter. In addition ClINH was associated
with shorter migration distances at all time intervals. The migra-
tion distance of the two leading neutrophils (29) at 45', 90', and
135' in the absence of ClINH was 100 pm, 120 um, and 130 pm
respectively. The penetration distance in the presence of ClINH

was diminished to 70 um, 80 um, and 90 pm respectively.

77

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79

Effect of ClINH on the chemotactic response of PMN to NFMP:
The effect of ClINH on the PMN chemotactic response to N-formyl-
methionylphenylalanine is shown in Figure 6. After establishing
that potent chemotactic activity for human neutrophils was demon-
strable with 10-6M NFMP, ClINH was placed with the PMN in chemotaxis
chambers and the response quantitated. Figure 6 is the result of
five eXperiments utilizing four cell donors. It can be seen that
ClINH caused a marked (183% i_46 of control, p<.001) enhancement
of chemotaxis (Figure 6A). Similar results were also obtained with
the preparation of ClINH donated by Dr. Jack Pensky.

The concentration of ClINH causing significant enhancement was
ascertained. ClINH at 8, 6, 4, and 2 U/ml were added with the PMN
in chambers of cells responding to 10-6M NFMP. A sharp decline in
enhancement activity to baseline levels occurred between 8 and 6
U/ml of ClINH.

In order to evaluate the reversibility of ClINH-mediated
enhancement, ClINH was incubated 30'/37°C with the PMN which were
then washed 3X with HBSS and assayed in chemotaxis chambers. The
results shown in Figure 68 indicate that a 2.7-fold enhancement
(p<.01) of chemotaxis was diminished to levels insignificantly

(p>.20) different from baseline levels due to the washing procedure.

Cell distribution of ClINH-mediated enhancement of NFMP
chemotaxis: An analysis of the distribution of migrating neutro-
phils in the absence and presence of ClINH in micropore filters is
shown in Figure 7. PMN responding to NFMP at 90', 135', and 210'

were quantitated in 10 micron increments. The distribution profiles

80

Figure 6. Effect of ClINH on the chemotactic response of
neutrophils to NFMP. A and B columns and brackets are the mean :_
l s.d. for five experiments with four different neutrophil donors;
Mean control (100%) chemotaxis for A and B was 36 and 13 neutrophils/
hpf, respectively; ClINH (7 units/m1), hatched columns; No ClINH,
open columns; PMN were suspended in 2% BSA: columns B3 represent
experiments which utilized PMN that had been preincubated 30'/37°C

with ClINH and washed 3X with HBSS.

81

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84
revealed a significantly higher number of cells in a zone nearest
the upper surface at earlier time intervals (90' and 135') when
ClINH was present with PMN. The cell distributions also suggest
that at each time interval the number of PMN'throughout the filter

was increased in the presence of ClINH.

DISCUSSION

 

Considering the data in this report and in previous reports,
the following interpretation seems plausible: human ClINH at
physiologic serum concentrations (4) alters the response of human
neutrophils to several cytotaxins by a direct effect on the cell.
The effect does not require other leukocytes since it occurs with
a purified neutrophil population and it occurs in the absence of
other plasma or serum factors. However, the nature of the effect
seems to depend on the specific cytotaxin and on the conditions
under which the cell encounters ClINH and/or cytotaxin.

ClINH and other plasma protease inhibitors have been shown to
affect leukocytes. Smith et a1. (11) reported that human ClINH
enhances the chemotactic response of human leukocytes to LPS, AgAb
complex and zymosan-activated plasma and inhibits the response to
C3(Try) but does not by itself influence spontaneous motility.
Goetzl (12) found that ClINH causes a transient, slight enhancement
of the neutrophil response to trypsin-activated C5 without any
effect on random migration. Remold and Rosenberg (13) described
an effect of ClINH on the migration of guinea pig macrophages. These
workers showed that ClINH enhances the response of macrophages to

migration inhibitory factor. Hollers et a1. (30) demonstrated that

85

ClINH prevents and reverses the ”deactivation" of human neutrophils
caused by zymosan-activated plasma filtrate. These data may ulti-
mately be related to the role of cell-associated serine esterases
in cell function. Indeed, much data support a role for esterases
in such cell functions as histamine (31), SRS-A (31) and ECF-A
(32) release from human lung fragments, serotonin release from
platelets (33), erythrophagocytosis (34), neutrophil chemotaxis
(35), lysosomal enzyme release (36), and baby hamster kidney cell
attachment to a substratum (37). Becker (35) described the activa-
tion of a cell-bound esterase essential for rabbit neutrophil chemo-
taxis as the result of a direct action of cytotaxins on the cell.
However, a direct association between the effects of the naturally
occurring plasma protease inhibitors and cell-associated serine
esterases remains to be demonstrated. Preliminary studies under-
taken to test such a possibility (Patrick and Becker, unpublished
results) failed to demonstrate any effect of ClINH on the action
of or the activation of esterase 1 on rabbit neutrophils.

The chemotactic response of human neutrophils to C3(Try) and
NFMP was not only analyzed in terms of the number of cells that had
migrated to the bottom of the filter but also in terms of the cell
distribution throughout the filter. At early time intervals (45'-
135') a significantly higher number of cells in the presence of
ClINH migrated into the filter. Furthermore, neutrophils responding
to C3(Try) exhibited a shallower penetration in the presence of
ClINH than in the absence of ClINH. PMN responding to NFMP in the
presence of ClINH demonstrated higher levels distributed throughout

the filter than in the absence of ClINH. Such an analysis (29)

86
confirmed the results seen when counting the number of neutrophils
that had migrated through the filter.

The possibility was considered that subtle variations in the
chemical structures of the cytotaxins might significantly contribute
to those conditions associated with ClINH participation in cellular
responses. There exist chemical differences within the C3a molecule
depending on the method of generation. Generation of C3a by
EAC4oxy2 cleavage of C3 is well documented (7,45,46). Budzko and
Muller-Eberhard (47) reported the close similarity in the molecular
weights and amino acid compositions of C3a(Try) and C3a(EAC4oxy2)
with a difference in their C-terminal amino acid residues. Based
on electrophoretic studies Bokisch and associates (7) concluded
that the smooth muscle contracting and chemotactic activities of
the C3a fragment are a function of different chemical groups within
the fragment. Extended treatment of C3a with trypsin resulted in
a fragment retaining chemotactic but not anaphylactic activity and
caused the originally basic fragment to become negatively charged.
In view of these differences, both trypsin and EAC4°xy2 generated
cytotaxins were examined. The negative effect of ClINH on the cell
response to C3(EAC4oxy2) was just as pronounced as that seen with
C3(Try).

Chemical differences within the C5a molecule associated with
the method of generation are not clearly understood. Human serum,
activated by LPS or immune complexes (48), contains chemotactic C5a
without anaphylactic activity. Treatment of guinea pig C5 with
EACIZES results in a chemotactic and anaphylactic fragment (49).

After prolonged trypsin treatment, human C5 retains its chemotactic

87
(8) activity but not its spasmogenic activity (50). It would seem,
therefore, that differences in experimental procedure, especially
during trypsin utilization, could result in C5a molecules with
distinct characteristics. Nilsson et a1. (46) recently reported
evidence supporting this possibility. Species differences in the
chemical nature of C5a have only been documented for anaphylactic
and not chemotactic activities. It seems feasible that trypsin
treatment of human C5 and LPS-activation of guinea pig serum could
result in two functionally similar but chemically different mole-
cules. However, the inhibitory effect of ClINH on the neutrophil
response to C5(Try) was just as pronounced as that observed with
guinea pig C5a (LPS-generated).

The chemotactic potency of a number of small N-formylmethionyl
peptides has been found to correlate with differences in chemical
structure (14). .There appeared to be requirements for N-acylation,
methionine, and minimum size, and a degree of specifiCity for hydro-
phobic residues was suggested. Future investigations may permit
inferences on the dependency of ClINH effects (enhancement or
inhibition) on such well-defined chemical differences.

Still other experimental conditions give rise to important
consequences associated with ClINH. Hollers et a1. (30), by main-
taining ClINH with the neutrophils during or after chemotactic
deactivation, achieved marked prevention or complete reversal of
this phenomenon. This clearly demonstrates the significance of
understanding the exact conditions under which ClINH and cytotaxin
encounter the cell. Goetzl (12) reported a slight enhanced neutro-

phil chemotactic responsiveness to C5(Try) at the highest concentration

88
of ClINH employed (100 ug/ml). This is not necessarily contradictory
to results reported in this study considering the significant dif-
ferences in conditions under which the cells were treated. Also, a
direct comparison of the specific activity of ClINH (12) with that
utilized in these studies cannot be made.

It is possible that C5a is involved in ClINH-mediated enhance-
ment of the neutrophil response to activated plasma. Other plasma
factors or conditions more like those utilized by Goetzl may be
required in order for enhancement to occur. It is also possible
that other naturally occurring cytotaxins participate in the enhance-
ment phenomenon. Kallikrein (51), plasminogen activator (52), the
trimolecular complex C567 (53), and C38'(54) have all been reported
to be chemotactic for neutrophils. Wissler and associates (55)
have described a chemotactic peptide system in mammalian serum that
is related to anaphylatoxin. All of these cytotaxins will be
especially prevalent in the plasma activated by LPS, Zymosan, or
immune complexes. Kallikrein may not be as relevant since ClINH
interacts directly with kallikrein to inhibit its neutrophil chemo-
tactic activity (56).

The findings presented in this report provide further evidence
to support the concept that ClINH participates in the mechanisms
of directional migration of human neutrophils without a concomitant
effect on spontaneous migration. Human ClINH at physiologic concen-
trations inhibits the chemotactic response of neutrophils to the
chemotactic fragments of C3 and C5 and enhances the response to
N-formy1methionylphenyla1anine without participation of other

plasma substances or other cell types.

89

ACKNOWLEDGEMENTS

 

The authors wish to thank Ms. Sheri Sprigg and Ms. Barbara

Giese for their excellent technical assistance.

REFERENCES

 

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serum inhibitor of C'l-esterase. Proc. Soc. Exp. Biol. Med.
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2. Lepow, I. H., and M. A. Leon. 1962. Interaction of a serum
inhibitor of Cl-esterase with intermediate complexes of the
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4. Gigli, I., S. Ruddy, and K. F. Austen. 1968. The stoichio-
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ARTICLE 2
EFFECTS OF CI INHIBITOR ON CHEMOTACTIC

DEACTIVATION OF HUMAN NEUTROPHILSl

DAVID Y. LIU AND RICHARD A. PATRICK

DEPARTMENT OF MICROBIOLOGY AND PUBLIC HEALTH

MICHIGAN STATE UNIVERSITY, EAST LANSING, MICHIGAN 48824

Condensed title: Effects of CI Inhibitor on Chemotactic
Deactivation of Neutrophils

1This work was presented in part at the annual meeting of the
Federation of the American Societies for Experimental Biology,
Anaheim, California, April 1976.

98

99
ABSTRACT

CI inhibitor has been shown to enhance and inhibit neutrophil
chemotactic responsiveness under appropriate conditions by a direct
effect on human leukocytes. To further define the nature of this
effect, we have tested the influence of CI inhibitor on another
cytotaxin mediated phenomenon, chemotactic deactivation. In the
present study, a 10% zymosan-activated plasma filtrate was used as
both the deactivating agent and the cytotaxin. The human leukocytes
used in these experiments contained greater than 93% neutrophils and
were obtained by Ficoll-Hypaque centrifugation. Neutrophils which
were deactivated by incubating with zymosan-activated plasma filtrate
for 30 min and extensively washed showed a mean chemotactic index
of 13% of control values (p<.001). The presence of CI inhibitor
during the incubation of neutrophils with zymosan-activated plasma
filtrate resulted in significant prevention of deactivation (p<.01),
increasing the mean response to 57% of control values, whereas
complete reversal of deactivation (p<.001) was obtained by adding
CI inhibitor to previously deactivated and washed neutrophils. The
results of this study lend support to the conclusion that CI inhibitor
can directly affect the chemotactic response of neutrophils. Further-
more, its ability to reverse deactivation, which has hitherto been
considered irreversible, may be relevant to the understanding of

chemotactic responsiveness.

INTRODUCTION

 

The chemotactic response of neutrophils in vitro is influenced

not only by the specific chemotactic stimulus but by other naturally

100
occurring factors which may inhibit or enhance through an influence
on the stimulus or the cell's response to the stimulus. One of
these factors is CI inhibitor (ClINH).2 This protease inhibitor
has been shown to alter the response of human neutrophils to certain
chemotactic factors (1,2). Its action seems to be directly on the
cell rather than through some plasma intermediate, and it has no
apparent effect on cells in the absence of chemotactic factors.

In an effort to characterize more completely the influence of
ClINH on the response of human neutrophils to cytotaxins, we tested
its possible influence on chemotactic deactivation. Deactivation
has been described as an alteration of the cells in a high concen-
tration of certain cytotaxins, e.g., immune complex activated serum
(3), zymosan activated serum (3), C567 (4), C5a (5,6), kallikrein
(6) or C38 (7). The altered response is seen as a decrease in the
number of cells penetrating the filter in a Boyden-type chemotaxis
chamber, and it seems to reflect a change in the cells since
repeated washing does not reverse the effect. Though the exact
(mechanism is not understood, an expending of a proenzyme (proesterase
l) necessary for migration has been proposed (4).

The results presented in this report show a profound influence

of ClINH on deactivation.

 

2 . . . . -. . .
AbbreViations used in this paper: ClINH, Cl inhibitor;
ATEE, N-acetyl-L-tyrosine ethyl ester; ZAPF, zymosan activated plasma
filtrate; HBSS, Hank's balanced salt solution; PMN, human neutrophils.

101
METHODS

Human CI inhibitor (ClINH) was prepared and evaluated for purity
and.activity as described by Pensky and Lepow (8). The specific
activities of the ClINH were 180 inhibitor ATEE-units/OD280 nm (9)
and 133 U/ug (10). The highly purified preparation upon immuno-
electrophoretic analysis revealed one precipitin arc with o2
mobility against anti-whole human serum. Isoelectric focusing
demonstrated one band, and SDS gel electrophoresis demonstrated a
major and minor band pattern consistent with the pattern recently
reported by Harpel and Cooper (11). Unless otherwise noted, ClINH
was used at 7 ATEE U/ml in all experiments.

Zymosan activated plasma filtrate (ZAPF) was prepared by
incubating zymosan (Nutritional Biochemicals Corporation, Cleveland,
Ohio) 10 mg/ml with fresh human plasma for 30 min at 37°C, diluting
this mixture 10-fold with Hank's balanced salt solution (HBSS), and
then removing the zymosan particles by centrifugation and filtra-
tion through a 0.22 um pore size filter (Millipore Corporation,
Bedford, Massachusetts).

Neutrophil enriched cell suspensions were obtained by Ficoll-
Hypaque centrifugation. Leukocytes and plasma from heparinized
dextran sedimented whole human blood were diluted with an equal
volume of HBSS and layered in 17 mm diameter siliconized tubes
above a single Ficoll-Hypaque solution consisting of 10 parts of
33.9% Hypaque (Winthrop Laboratories, New York, New York) and 24
parts of 9% Ficoll (Sigma Chemical Co., St. Louis, Missouri).

The tubes were centrifuged at 800g for 30 min at 25°C. The leuko-

cytes remaining in the cell button at the bottom of the tubes after

102
the upper layers were removed by aspiration contained greater than
98%(granulocytes of which approximately 95% were neutrophils (PMN).
Consistently less than 2% of the leukocytes were blood monocytes
or lymphocytes. No platelets could be seen in these preparations,
and the RBC to PMN ratio was approximately 5:1. The PMN viability
was greater than 98% as determined by eosin exclusion.

PMN chemotactic responsiveness was determined by a modified
Boyden technique using blind-well chemotaxis chambers (Neuroprobe,
Inc., Bethesda, Maryland). A Millipore filter of 3 pm pore size
was used, and the chambers were loaded with 2 x 104 PMN/mm2 of
exposed filter surface. The chemotactic response was evaluated
by counting the number of PMN accumulating on the lower surface
of the filter after 3 hours of incubation at 37°C in an atmosphere

containing 5% CO and 95% humidity. Ten 400x microscope fields

2
were counted on each filter and the averages of triplicate determi-
nation in each experiment were expressed as a percent of control

values. This index of chemotactic responsiveness was obtained by

the following formula:

experimental count - background count

control count - background count x 100

% of control =

where background = counts from chambers containing control cells in
the upper compartment and 10% plasma in the lower compartment, and
control = counts from chambers containing control cells in the upper
compartment and ZAPF in the lower.

Control values and background values would therefore be defined
as 100% and 0%, respectively. The comparison of experimental

results to control values in terms of percent response was necessary

103

because of the variability in cell counts due, in part, to the use
of plasma and cells from different human donors or even from the
same donor in experiments performed on different days. However,
the differences in cell counts from triplicate chambers within any
one experiment did not exceed 25%. The results in Table l were
analyzed statistically, employing the Student's t test.

PMN chemotactic deactivation was accomplished by incubating
3.0 x 107 PMN/m1 in ZAPF for 30 min at 37°C and then washing the
cells 3 times in HBSS. These cells were then adjusted to a concen-
tration of 2 x 106 PMN/m1 with 10% fresh plasma and were assayed
for chemotactic responsiveness. Cells that were incubated with 10%
plasma instead of ZAPF but were otherwise treated identically
served as controls and are referred to in the text as "control

cells."

RESULTS

Various concentrations of ZAPF and PMN were incubated together
for 30 min at 37°C to determine the experimental conditions neces-
sary for deactivation with this cytotaxin. There was no apparent
effect on the level of deactivation exerted by varying the cell
concentration from 2 x 107 to 6 x 107 PMN/ml in incubation mixtures
with ZAPF. A standard concentration for all further experiments of
3 x 107 PMN/m1 was chosen on the basis of technical convenience.
However, the efficacy of deactivation was dose-dependent on ZAPF.
The results of the experiment illustrated in Figure 2 indicate that
PMN incubated with undiluted ZAPF showed the lowest level of chemo-

tactic responsiveness as compared to control values (16%) or the

104

Figure 1. Experimental procedure.

105

PLASMA + ZYMOSAN

37° C/30’

CENTRI FUGE AND FILTER

    

 

(see Table I)

  

ZAPF + PMN

 

 

CH EMOTAXIS ASSAY
(see fig. 2) or

I 7 7 l
o . l C1INH I
37 Cl/30 (7 ATEE U/ml)

WASH

EDEACTIVATED PMN'}

 

 

 

 

Figure l

106

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DILUTIONS OF ZAPF

1:1

Figure 2

108
greatest degree of deactivation. The deactivating effects of ZAPF
were abolished at a dilution of 1:4 or higher. Undiluted ZAPF was
used in all subsequent experiments.

The effect of ClINH on the deactivation phenomenon was tested
by adding ClINH to previously deactivated cells during the chemo-
taxis assay or to the deactivating mixture (Figure 1). The results
of these experiments are summarized in Table 1. Briefly, when ClINH
was added to the PMN-ZAPF mixtures during the deactivation step and
then removed along with the ZAPF by washing the cells prior to the
chemotaxis assay, there was significant partial prevention (p<.01)
of deactivation (group 4 vs. 3). Further experiments showed no
change in the degree of prevention of deactivation by varying ClINH
concentrations between 7 and 22 U/ml; however, there was no preven-
tion of deactivation seen with ClINH at 5 U/ml. If deactivated cells
were tested for chemotactic responsiveness in the presence of ClINH,
deactivation was completely (p<.001) reversed (group 5 vs. 3),
having a chemotactic index which was not significantly (p>.4)

different from that of control cells (group 2 vs. 5).

DISCUSSION

 

Diminished chemotactic responsiveness induced by preincuba-
tion of polymorphonuclear leukocytes with chemotactic substances
is a characteristic of rabbit neutrophils (3), and human neutrophils
(6), eosinophils (10,11), and mononuclear cells (6). This altered
response has been termed "deactivation" (3). Previous results (1)
from this laboratory showed that addition of highly purified ClINH

to the media containing human neutrophils effects significant

109
Table 1

Effects of Cl Inhibitor on Chemotactic Deactivation of Neutrophils

 

 

Lower -% of Con- 5
Group Cell Compartment* Compartment trol :_s.d. n
Y

1 (PMN+P)P P o 18
2 (PMN-1P) P ZAPF 100 ’ 1 8-
3 (PMN+ZAPF) P ZAPF 13. 3:25 . 4 18
4 (PMN+ZAPF+C11NH)P ZAPF 56.6:33.5 5
5 (PMN+ZAPF) ClINH+P ZAPF 122 . 7:20 . 6 5

 

Groups 2 and 3, p<.001; 3 and 4, p<.01; 3 and 5, p<.001;
2 and 5, p<.4.

*
Cells were preincubated 30 min at 37°C with substances
in parentheses and washed 3X in Hank's solution.

+Control - group 2.

sNumber of experiments done in triplicate.

WP - 10% fresh plasma in Hank's solution.

changes (enhancement) in the chemotactic responses of the cells to
zymosan-activated plasma (ZAPF). Results reported here show that
ClINH can directly alter chemotactic deactivation of human PMN to
ZAPF.

Chemotactic deactivation of human neutrophils was partially
prevented by ClINH (Table 1, group 4). In addition, ClINH apparently
effects alterations of the "deactivated" state such that the phenomenon

becomes reversible (Table 1, group 5).

110

Phosphonate and aromatic amino acid esters have been reported
by Ward and Becker (3) to partially prevent deactivation of rabbit
neutrophils by cytotaxins derived from activated rabbit serum.

Since ClINH and phosphonate inhibitors are both known to irreversibly
alter the active site of serine esterases, it seemed plausible to
hypothesize an interaction of ClINH with the esterase 1 associated
with rabbit neutrophil chemotaxis (4). However, experiments designed
to directly test this hypothesis (Patrick and Becker, unpublished
results) failed to show that ClINH affected the action of or the
activation of esterase l on rabbit neutrophils.

Since a human corollary to rabbit esterase 1 has not been
demonstrated, no direct comparison between the effect of phosphonate
inhibitors on rabbit neutrophil deactivation and the effect of
ClINH on human neutrophil deactivation can be made at this time.

That reversal of deactivation occurs when ClINH is present with
the deactivated neutrophils during the chemotaxis assay strongly
suggests a role for ClINH in the facilitation of a chemotactically
responsive population of neutrophils. Furthermore, ClINH must be
acting at some step in the mechanisms for chemotactic responsiveness
since it does not increase spontaneous motility and since the simul-
taneous presence of cytotaxin is necessary for the effects of ClINH
to be expressed (1,2).

These data suggest an interpretation of the previous observations
that ClINH enhances the chemotactic response of neutrophils to
zymosan-activated plasma (1). If deactivation eventually occurs
during the response of cells to a chemotactic gradient and ClINH

prevents or reverses this, then the responsiveness of the cells

111

might be maintained at higher levels. This would be reflected in

a larger number of cells responding to ZAPF and migrating through

the micropore filter.

Chemotactically deactivated leukocytes are not at the present

time well characterized with regard to biochemical and cellular

functions. A recent report (12) indicated that stimulation of the

hexose monophosphate shunt by chemotactic factor was unaffected in

deactivated human neutrophils. It is hoped that the effects of

ClINH may allow for a functional characterization of chemotactic

deactivation which, in turn, may lead to a better understanding of

the chemotactic response of granulocytes.

ACKNOWLEDGEMENTS

 

The authors would like to express their appreciation to Ms.

Barbara Giese for her excellent assistance. This work was supported

by National Institutes of Health Grant AI 11367-03.

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