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thesis entitled

The Effects of Cyclic Nucleotide Modulators
on Neutrophil Shape Change

presented by

Stephen L. Speilbauer

has been accepted towards fulfillment
of the requirements for

Master's degree in Clinical Laboratory
Science

 

 

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Major professor

Date May 13, 1986

 

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THE EFFECTS OF CYCLIC NUCLEOTIDE MODULATORS
ON NEUTROPHIL SHAPE CHANGE

By

Stephen L. Spielbauer

A THESIS

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

MASTER OF SCIENCE
Clinical Laboratory Sciences

Medical Technology Program

1986

fl

L/p/ ("'(‘fl/ 5/

ABSTRACT

THE EFFECTS OF CYCLIC NUCLEOTIDE MODULATORS
ON NEUTROPHIL SHAPE CHANGE

By
Stephen L. Spielbauer

Shape change is an early part of the neutrOphil
chemotactic response. Chemotaxis and shape change are
affected by cyclic nucleotide levels which are subject to
pharmacological modification. The objective of this study
was to investigate the effects of various agents associated
with altered cyclic nucleotide levels on shape change in
isolated neutrophils. N-formyl—methionyl-leucyl—
phenylalanine or arachidonic acid were used to induce shape
change. ISOproterenol and theophylline, which are agents
known to increase adenosine 3‘,5‘-mon0phosphate (cAMP) were
used to inhibit shape change. Guanosine 3‘,5‘-mon0phosphate
(COM?) and agents which increase cGMP like serotonin,
phenylephrine and carbachol were evaluated as shape change
enhancers. Shape change was evaluated by scoring wet
preparations of fixed cell suspensions for altered
neutrophil shapes. Agents which elevate CAMP inhibited shape
change. No agent tested enhanced shape change at 2 minutes.

Enhancement was not tested for at later times.

ACKNOWLEDGEMENTS

My special thanks to the members of my committee, Mrs.
Martha Thomas, Dr. Sharon Zablotney, Dr. Stuart Sleight and
Dr. Wayne Smith. I must also thank Mr. James Hollers for his
technical assistance and insite into the properties of
neutrophils. Dr. Smith and Mr. Hollers were very helpful in
designing and implementing this study. Mrs. Thomas, Dr.
Zablotney and Dr. Sleight were very helpful in preparing
this thesis.

I must acknowledge the patience and understanding

exhibited by my spouse, Kathy, throughout this time.

ii

TABLE OF CONTENTS

Page
INTRODUCTION ------------------------------------------ 1
LITERATURE REVIEW ------------------------------------- 3
Initiation of Shape Change ----------------------- 5
Calcium Flux ------------------------------------- 5
Cyclic AMP Flux ---------------------------------- 10
Cytoplasmic Movement ----------------------------- 10
METHODS AND RESULTS ----------------------------------- 15
NeutrOphil Isolation ----------------------------- 15
Serum Coated Tubes ------------------------------- 16
Assessment of Shape Change ----------------------- 16
Statistical Procedure ---------------------------- l7
FMLP Dose for Optimal Inhibition ----------------- 18

Secondary Inhibition of Shape Change Over Time --- 20
Drug Prior to FMLP Inhibition and Inhibition of
Arachidonic Acid Shape Change Over Time ---------- 20
Inhibition of Subsequent Arachidonic Acid Dose —-- 25
Serotonin Enhancement of FMLP Shape Change and
Serotonin Enhancement of Arachidonic Acid Shape

Change ------------------------------------------- 26

FMLP Shape Change Enhancement Screen ------------- 27

Enhancement of Arachidonic Acid Shape Change ----- 27

Calcium Requirement for Arachidonic Acid Shape

Change Over Time --------------------------------- 28
DISCUSSION ............................................ 29
SUMMARY AND CONCLUSIONS ............................... 32
BIBLIOGRAPHY .......................................... 33
VITAE ------------------------------------------------- 38

iii

LIST OF TABLES

Table Page
1. FMLP Dose for Optimal Inhibition ----------------- 19
2. Drug Prior to FMLP Inhibition -------------------- 22

iv

LIST OF FIGURES
Figure Page
1. Change in Shape of Neutrophils(x1600) ........... u
2. Secondary Inhibition of Shape Change Over Time --- 21

3. Inhibition of Arachidonic Acid Shape Change ------ 23

ascorbic acid

cAMP

cGMP
carbachol
EGTA

f actin
FMLP

FMP

g actin
Iso

MLCK

MT

PBS
serotonin
Theo
Untreat
urOpod
volex

W-7

LIST OF ABBREVIATIONS

L-ascorbate

adenosine 3‘,5‘-mon0phosphate
or cyclic-AMP

guanosine 3‘,5‘-mon0phosphate
carbamylcholine
ethyleneglycol—bis-glycol-tetraacetic acid
actin microfilaments
N-formly-methionly—leucyl-phenylalanine
N-formly-methionly-phenylalanine

actin monomers

isoproterenol

myosin light chain kinase

microtubules

phosphate buffered saline
5-hydroxytryptamine

theOphylline

untreated

tail like knob

6 percent hetastarch

N-(6-aminohexyl)-5-chloro-1-napthaline
sulfonamide

vi

INTRODUCTION

Neutrophil function can be tested in a variety of ways.

The traditional chemotaxis assay simulates the i vivo

 

neutrOphil response. In this test isolated neutrophils move
through a filter towards a chemoattractant. The chemotactic
response is related to the distance the cells travel in the
filter. This process is complex which sometimes makes
interpretation of the results difficult. Simpler tests like
shape change assays, which focus on a single aspect of
neutrophil function, have been deve10ped to eliminate some
of the complexity of the chemotactic process.

Increased adherence and shape change are early
overlapping events which are part of the chemotactic
process. Under normal circumstances adherence changes begin
before the cells change shape. However, if the experiments
are conducted quickly using dilute suspensions of cells,
surface contact and adherence effects can be minimized.
Relatively low sustained concentrations of chemoattractants
transform the resting cells from a round to a long polarized
form without initiating other processes important to
chemotaxis. Shape change, which can occur and be reversed in
1 to 2 minutes, can also be tested in whole blood samples.

Although this simplifies the procedure, chemoattractant-

albumin binding and activation of platelets or complement
complicate interpretation of the results.

Cyclic nucleotides can affect monocyte or neutrOphil
chemotaxis and monocyte shape change. NeutrOphil shape
change is probably also influenced by altering cyclic
nucleotide levels. These altered levels are obtained by
treating the cells with various agents. This study was
designed to see if neutrOphil shape change would be
influenced by these agents and to compare these results with
the corresponding results obtained for monocytes by others

(8).

LITERATURE REVIEW

Shape Change

 

Shape change in neutrophils has been recognized for
some time (2). Circulating neutrophils were seen as
spherical and as these cells encountered some stimulating
factor, they adhered to the vessel walls. If the stimulant
persisted, the cells changed shape and moved to an interface
between endothelial cells lining the vessel. They then
migrated into the tissues via the process of diapedesis
(3,”).

Initial studies on neutrophil shape change were
performed on protein or gelatin coated surfaces. It was
later shown that neutrOphil shape change also occurred in
suspension, free of surface contact (5,6). Shape change in
suspension has been performed with isolated neutrophils,
whole blood neutrophils and monocytes (5,7,8). Stimulated
neutrophils changed through a continuum of shapes from round
to elongated as shown in Figure 1. These morphologic
alterations result from several overlapping mechanisms which
are divided into four tOpics (1) the initiation of shape
change, (2) calcium flux as a positive effector of shape
change, (3) adenosine 3‘,5‘-monophosphate (cyclic—AMP or
cAMP) flux as a negative effector of shape change and (4)
the resulting cytOplasmic movement. The following discussion
will cover these four topics including specific information

about neutrOphils.

 

Figure 1

Figure 1. Change in Shape of Neutrophils (x1600)

Change in shape of neutrophils (x1600) exposed to a
chemotactic factor for 2 minutes. 1a represents round cells
b is ruffled c is a bipolar cell d and e are uropod cells

l Initiation g; Shape Change

 

 

NeutrOphils have several non-hormonal receptors
including one for N—formyl-methionyl—phenylalanine (FM?)
(8). When appropriate levels of FMP were added to the cell
suspensions, receptor filling and shape change occurred in 2
minutes or less for isolated neutrophils. Whole'blood
neutrOphils by comparison, showed a slow shape change
response which could result from plasma proteins binding
FMP. In both environments the shape change response was
maintained through 45 minutes if FMP remained with the
cells. If FMP were removed from the receptors on isolated
neutrophils by dilution or addition of a competitive
inhibitor, shape change reversed in about 30 seconds (Smith,
Unpublished). N-formyl-methionyl-leucyl—phenyla1anine (FMLP)
has properties like FMP. Pure FMLP became available after
FM? and, since it is a better agonist, was used as its
replacement in later experiments (8L.Both FMLP or FMP bound
to neutrOphil receptors and induced shape change. These
filled receptors altered the neutrophil membrane and
eventually stimulated calcium influx into the cells by the
processes described below. Shape change has also been
initiated by methods which bypass the filling of membrane
receptors.

g Calcium Flux
When cells are activated calcium enters from outside

the cell or from "storage pools" within the cell.

Mitochondria, endoplasmic reticulum and the inner surface of
the cell membrane are possible storage pools (9). Calcium
bound to the inner surface of the cell membrane may be
released by chemical disturbances such as the filling of
chemotactic peptide or immune complex receptors. Extrinsic
calcium enters the cytosol with the aid of a lipid or
phospholipid ionOphore.

Two membrane processes have been investigated for their
role in the transport of calcium across cell membranes. Both
begin with receptor filling and a change in membrane
structure. Subsequent steps differ but both processes
eventually release arachidonic acid. This release is
inhibited by cAMP in platelets (10). From this point on
these membrane processes are the same. The common second
phase of these processes begins as cyclooxygenase or
lipoxygenase, in conjunction with other enzymes, convert
arachidonic acid into various prostaglandins, thromboxanes,
leukotrienes and hydrotetraenoic acids. Leukotriene Bu and
thromboxane A2 have been suggested as calcium ionophores
(11). Arachidonic acid and some of its products may simply
enhance calcium activity (12).

In the first of these membrane processes a chemical
cycle begins as phosphatidylinositol, incorporated in the
membrane, is converted to phosphatidylinositol 4,5-
diphosphate. This is Split into diacylglycerol and inositol

trisphosphate. Phosphatidic acid, a product of

diacylglycerol, combines with inositol-l-phosphate from
several sources to produce phosphatidylinositol in the
membrane and complete the cycle. Phospholipase A2 and
possibly phospholipase C are able to convert diacylglycerol,
an intermediates from this cycle, to arachidonic acid (13).
The common second phase, discussed above, leads to ionOphore
production. Inositol triphosphate and phosphatidic acid, may
also be ionophores (14,15).

The second membrane process suggests that membrane
stimulation induces multiple methylations of
phosphatidylethanolamine in the inner membrane and a
concomitant transfer to the outside of the product,
phosphatidylcholine. This "transmethylation" results in
local changes in membrane viscosity and permeability.
Phospholipase A2, which is calcium dependent, is then
eXposed to increased calcium in these local fluid areas.
This eXposure produces arachidonic acid from
phosphatidylcholine. As before, the common second phase
produces ionophores from arachidonic acid production
(16,17). It has been reported that both phosphatidylinositol
and transmethylation pathways are functional in human
neutrOphils but that transmethylation is not the major
source of arachidonic acid (15,13).

Calcium flux can be regulated in ways that bypass the
initial membrane changes discussed above by directly

controlling phospholipase A2 activity. Mellitin and phorbol

esters are phospholipase A2 activators, while mepacrine and
tetracaine are inhibitors (16). In rat leukocytes and rabbit
neutrOphils, an endogenous inhibitor of phospholipase A2 is
produced by the glucocorticoids hydrocortisone, prednisolone
and dexamethasone. This endogenous inhibitor is transcribed
and translated from the cell‘s DNA which eXplains the time
delay associated with the action of these glucocorticoids
(18). This is a common mode of action for steroid hormomes
(19). In the presence of resting levels of calcium
diacylglycerol, a precursor to arachidonic acid, activates
protein kinase C. This kinase mimics the calcium-calmodulin
complex and also induces the cell to change shape (14,12).
Granulocytes have an active transport system for removing
calcium from the cytosol (20).

The release of storage pool calcium is important to
neutrOphil shape change because extracellular calcium is not
needed for neutrOphil shape change in response to FMP or
FMLP (5)(Hollers unpublished data). This could mean that the
increase in intracellular calcium needed for shape change is
small and may not require ionOphore production. Membrane
changes that release calcium from their inner surface could
be sufficient.

Calmodulin

Many processes are regulated by changing calcium

concentration. Calmodulin promotes the activity of calcium

by acting as an intracellular receptor for calcium. For

example, calcium or calmodulin alone had little effect on
lfl.!i££9 microtubule assembly. However, when calcium and
calmodulin were combined, microtubule assembly was prevented
(21). The cell‘s calmodulin content remains relatively
constant. Changing calcium concentration and binding
regulates these cellular mechanisms (22). Calmodulin is
often associated with membranes and contractile filaments.
It binds 11 calcium ions per molecule and is a common
"conserved" protein found in plants and animals. As binding
between calcium and calmodulin occurs there is a
conformational change and the active site of the molecule is
exposed. Phenothiazine, an antipsychotic, and the N-(6—
aminohexy)-5-chloro—1-napthaline sulfonamide (W-7) group of
compounds are selective inhibitors of calmodulin that
compete for the active site (23). They are inhibitors of the
respiratory burst and degranulation in neutrOphils
(23,2u,25,26). Calcium-calmodulin is involved in many
cellular mechanisms such as prostaglandin metabolism,
mitosis, guanosine 3‘,5‘-mon0phoshpate (cGMP) flux, cAMP
flux, actin-myosin interaction, actin and microtubule
structure and calcium flux across the cell membrane
(19,27,28,29). The calcium-calmodulin complex regulates
several of the enzymes involved in controlling cAMP and cGMP
levels and serves as part of the link between calcium and

the cyclic nucleotides.

10

Changing calcium concentrations from 10"7 to 10'” M in
neutrophils induces many of the responses associated with
chemotaxis and shape change. Calmodulin, as a receptor, is
responsible for making calcium so versatile.

; Cyclic AMP Flux

It has been shown that increased levels of cAMP have an
inhibitory effect on neutrophils, platelets and monocytes
(1,19,29,30,31). Cyclic AMP has a high energy bond and is
stable in the cytosol. Adenylate cyclase produces cAMP while
phosphodiesterases hydrolyze cAMP to AMP. The neutrophil
adenylate cyclase complex is activated by beta agonists like
iSOproterenol. Phosphodiesterase activity is blocked by
theOphylline or caffeine resulting in elevated cAMP
(29,30,31,32,33,34). Phosphodiesterase activity is promoted
by calcium—calmodulin resulting in lower cAMP levels (10).

fl_ Cytoplasmic Movement

The three structural proteins involved in physical
neutrOphil shape change are actin, myosin and tubulin. These
monomers assemble to form large "active" structures. Actin
forms crosslinked microfilaments, which are responsible for
transient bulging structures near the cell surface. Myosin
forms bundles which apply traction to microfilaments.
Tubulin forms microtubules, which give structure to the cell
core and may transport lysosomes toward the outside. The
monomers are in equilibrium with the respective large

"active" structures. These events will be discussed.

11

Actin Microfilaments

Actin monomers (g-actin) form actin microfilaments (f-
actin). If these microfilaments become long and abundant,
they will be crosslinked by actin-binding protein. This
crosslinked mesh forms a solid bulge. The transition from g-
actin and short microfilaments to long microfilaments is
controlled by acumentin, profilin and gelsolin, the last of
which is calcium dependent. Acumentin, independent of
calcium, blocks the slow growing end of a microfilament and
gelsolin blocks the fast growing end but only if the calcium
concentration is greater than 10"7 M. Profilin sequesters g-
actin. If calcium is less than 10"7 M, gelsolin frees the
fast growing end of the microfilaments which lengthen and
are crosslinked (14). Mesh formation depends on low calcium
levels. Other factors may be involved but are not
understood.

The crosslinked mesh forms near the plasma membrane of
the neutrOphil and excludes cell organelles (35). As
neutrophils change shape, the uniform layer of actin becomes
structured in 2 areas inducing a tail-like knob (urOpod) and
a broad ruffled structure, see Figure 1 (e). If local
membrane calcium pumps become activated, intracellular
calcium levels drop and actin mesh formation is enhanced
(20). It is the actin mesh that is primarily responsible for

shape change.

12

Myosin

Myosin is the energy transducing part of the system. It
compresses and moves parts of the mesh along by pulling on
actin microfilaments. Some myosin is trapped in the gel at
formation and contributes to its compression when myosin is
activated (36). Myosin bundles draw actin filaments from
liquid areas of the cell to areas where the crosslinked mesh
is forming (1A).

The calcium-calmodulin complex promotes contraction of
myosin by indirectly phosphorylating it with an active
myosin light chain kinase (MLCK). In the presence of 10'7 to
10'5 M calcium, the calcium—calmodulin complex binds and
activates MLCK. Active MLCK induces myosin contraction and
movement. Cyclic AMP—dependent protein kinase and cAMP
inactivate MLCK through phosphorylation. In this state MLCK
cannot complex with calcium-calmodulin. These events are in
equilibrium. If high calcium levels exist, myosin
contraction will occur. However, cAMP can reverse the
contraction in the presence of high calcium levels by
shifting the equilibrium (37). Details of this relationship
have been worked out in platelets (38,39).

Recent information indicates other factors released
directly by membrane activation are involved here» Protein
kinase C, activated by diacylglycerol in the presence of
less than 10"7 M calcium, can also phosphorylate the light

chain of myosin. Arachidonic acid and other related lipids

13

are able to enhance the binding of calcium-calmodulin to
MLCK. Some of the suspected calcium ionOphores may be simply
enhancers of calcium activity (12).

Calcium—calmodulin and cAMP are involved in control of
neutrOphil movement. In neutrophils ingesting zymosan
particles, cAMP and a cAMP-dependent protein kinase are
present in the area of the pseudOpods (MD). This area was
observed by others to contain actin mesh and myosin (36). In
resting neutrOphils, no concentrated areas of cAMP or cAMP-
dependent kinase were seen (NO). This indicates that in
neutrOphils with active pseudOpods, cAMP and the kinase are
exerting some control over myosin activation. Myosin bundles
could be collecting actin filaments from areas of higher
calcium to the base of the low calcium actin bulge where
they will be crosslinked into the bulge structure (20). The
degree of myosin activation involved in shape change is
unknown.

Microtubules

Microtubules (MT) are a structural component of
neutrophils and play some role in shape change, secretion of
granules and phagolysosome formation. In resting neutrophils
fewer MT are seen than in neutrOphils that have changed
shape. Microtubules assemble at one end and are disassembled
at the other. The rate of the turnover increases with
increased calcium. The core of an activated neutrOphil

contains MT while the outer actin bulges do not (41,u2).

114

This segregation of assembled components could be due to
differing calcium concentrations.

Colchicine, a plant alkaloid, blocks the assembly of
MT. If colchicine is added to round neutrophils they change
shape as areas of actin mesh appear and the MT are reduced.
Treatment with colchicine also inhibits granule secretion
(“2). The details of MT function are not clear.

Microtubules, crosslinked microfilaments and myosin
each play some role in the process of cytOplasmic movement
that results in shape change. Microfilaments seem to be the
most visible component in the early stages of this process.
Many questions remain unanswered.

In summary, neutrOphil shape change is generally
initiated by filling some surface receptor which induces
calcium concentrations to increase to 10'5M. Calcium binds
to calmodulin which allows actin, myosin and tubulin to
assemble more completely and change the shape of the
neutrOphil. This process can be altered at several points by

beta agonists, lipids, drugs, ions and other agents.

METHODS AND RESULTS

NeutrOphil Isolation

 

Sterile Dulbecco‘s phosphate buffered saline (PBS),
containing 0.1 % glucose, was prepared in the laboratory.
Ficoll and hypaque, obtained from Sterling-Winthrop Labs,
New York, NY 10016, were used to prepare a ficoll-hypaque
cushion (43). Six percent hetastarch (volex) was used to
enhance sedimentation, McGaw Laboratories, Ann Arbor, MI
A8108. All reagents used were brought to room temperature.
Five milliliters of citrated blood, diluted with 5 ml of PBS
in disposable glass tubes, were layered over A ml of Ficoll-
Hypaque and centrifuged in a 20 C centrifuge at ”00 G for 20
minutes. The cells, suspended in Ficoll—Hypaque, were
transferred to a serum protein-coated tube and diluted to
twice the original volume with PBS. A total of 1.7 ml of
volex was added. The tubes were undisturbed for 30 minutes
to permit cells to sediment. The supernatant was removed to
another serum-coated tube and washed with large amounts of
PBS using a 15 minute 200 G spin for each wash. The cells
were suspended in 1 m1 of PBS and counted. The concentration
of neutrophils was adjusted to 107 per ml with PBS and the
cells stored at 5 C for up to 5 hours. This preparation
resulted in a mixture of 5 % platelets, 62 1 red blood cells
and 33 % neutrOphils. Cell viability was assessed during the

experiments.

15

16

Serum Coated Tubes
Pre-cleaned disposable glass tubes were selected to
avoid chemical contamination. Tubes were coated with a 1:1
dilution of human serum to prevent attachment of the cells
to the glass surface and were kept moist until use.

Assessment of Shape Change

 

 

Suspensions of 5X105 to 1X106 neutrOphils per ml were
used in eXperiments to assess shape change. Shape change was
preserved to allow measurement by cell fixation with 1 1
glutaraldehyde. This was done by diluting the tube contents
with cold 2 % glutaraldehyde and holding at 5 C for 30
minutes. One hundred neutrOphils from each suspension were
classified according to cell shape by observing fixed wet
mounts at a magnification of 1000X using a phase contrast
micrOSCOpe. For analysis the shape change spectrum was
divided into 4 cell shapes labeled round, ruffled, bipolar
and urOpod (Figure 1). The percentage of neutrophils in the
bipolar and urOpod categories were added together. This
result was labeled % shape change.

For example:
round ruffled bipolar urOpod % Shape Change
5% 23% ”2% + 30% = 72%
Unpublished data showed that if neutrophils in suspension
and in a non-round shape were killed before fixation, they
would quickly become round. All neutrOphils classed as

ruffled, bipolar and uropod were viable during the test;

17

hence, in the above example, 95 1 were viable. To induce
shape change means to produce or bring forth some 1 shape
change, for example 72 1. To enhance shape change means to
add to some baseline 1 shape change, for example > 72 1. To
inhibit shape change means to restrict the baseline 1 shape
change to some lower value, for example < 72 1.

N-formyl-methionyl-leucyl-phenylalanine (FMLP) or
arachidonic acid (Sigma Chemical Company, St. Louis, MO
63178) were used to induce shape change. TheOphylline (ICN
Biochemicals Cleveland, OH 48812) and iSOproterenol were
used to inhibit shape change. Phenylephrine, 5-
hydroxytryptamine (serotonin), cGMP, L—ascorbate (ascorbic
acid), carbamycholine (carbachol), acetylcholine, succinyl
choline and D-tubocuraine (Sigma Chemical) were agents
thought to enhance shape change.

Statistical Procedure

 

Most of the eXperiments used a randomized block design
with 2 or 4 blocks (r=number of blocks). The first and
second eXperiments were factorial in design which allows
testing for an interaction between factors. The factors were
theophylline and isoproterenol. In the other experiments,
treatments consisting of inhibitors or enhancers of shape
change were evaluated. Analysis of variance testing for
significant differences was done by using the F-test at the
95 1 level of significance. In some instances where the null

hypothesis was rejected, Tukey‘s method for multiple

18

comparisons was used to determine which means were different
from the others. The original data, being eXpressed as a
percentage, were put through an arcsin square root of the
percentage transformation to eliminate skewed distributions
near 0 or 100 1. The eXperiments in which serotonin was the
enhancer were analyzed using multivariate analysis of
variance from the statistical package for the social
sciences. Other analysis of variance was calculated using
the Apple version of VisiCalc (NA,H5,46,M7).

FMLP Dose For Optimal Inhibition

 

—H

Neutrophils were treated with isoproterenol (1X10 M),

theOphylline (1x10'3 M), the combination of theOphylline at

-11 M (T+I) or were

1x10'3 M plus iSOproterenol at 1x10
untreated for 2 minutes. The neutrOphils were then combined
with FMLP concentrations ranging from 11(10'1O M to 5X10"9 M
in the presence of isoproterenol, theOphylline, (T+I) or PBS
(untreated) for 5 minutes. Percent shape change was measured
and recorded. See Table 1. Instances where neutrophils were
combined with 1X10"9 M FMLP have rzfl. Other instances have
r=2. All following experiments have r=4 and were performed
at 37 C unless otherwise indicated.
Results

Isoproterenol caused no significant inhibition.

Significant inhibition of shape change by theophylline

occurred at several FMLP concentrations including the

optimal concentration of 1x10'9M FMLP.

19

Table 1 FMLP Dose for Optimal Inhibitiona

(FMLP) 0 1x10‘10 3x10'10 6X1O'1O 1x10‘9 5x10‘9
---------------- 1 Shape Change------—---—-----
(Untreat) N 9 22 55 77 96
b o
(Theo) 3 2 5 31 5A 9“
C C C
(T+I) 2 2 1M 34 an 88
(Iso) 4 8 25 52 78 98

 

aN-formyl-methionyl-leucyl-phenyalanine (FMLP) Dose for
Maximum Inhibition Different doses of FMLP are used to
induce the 1 shape change seen in the table. Theophylline
and ISOproterenol were tested as inhibitors of 1 shape
change at different doses of FMLP.

(FMLP) N-formyl—methionyl-leucyl-phenyalanine at
various molar concentrations

(Untreat) Untreated with an inhibitor
(Theo) Theophylline at 1x10'3 M

(T+I) The combination of theOphylline plus
iSOproterenol at the concentrations used when these
inhibitors were used alone.

-u M

(150) Isoproterenol at 1X10

bIn these instances the 1 shape change was
significantly different from the corresponding untreated 1
shape change. Significant differences indicate differences
at the 95 1 level of significance. 1

0In these instances the 1 shape change was
significantly different from the corresponding untreated 1
shape change. ISOproterenol did not interact with
theOphylline.

20

Secondary Inhibition of Shape Change Over Time

 

Neutrophil percent shape change levels were known to be
relatively stable for about 30 minutes or more for FMLP and
FMP (Hollers, unpublished data). In this eXperiment
neutrophils were combined with 1X10"9 M FMLP for 1 minute
and 50 seconds and then treated with isoproterenol (1X10'u
M), theophylline (1X10"3 M), (T+I) or were untreated.
Portions for 1 shape change evaluation were taken at 0, 1,
2, 3, LI, 5, 7, 10, 15 and 20 minutes. See Figure 2.

Results
TheOphylline was able to significantly inhibit the
shape change induced by FMLP from 3 through 20 minutes.
ISOproterenol caused a significant interaction with
theOphylline at A minutes and caused significant inhibition
when present alone at 4 minutes.
Drug Prior to FMLP Inhibition

and
Inhibition of Arachidonic Acid Shape Change Over Time

 

 

Time studies were done on neutrOphils which were
treated with theOphylline (11(10'3 M), (T+I) or were
untreated for 2 minutes before combination with FMLP (1X10"9
M) or arachidonic acid (5X10"7 M) at 0 time. Portions were
taken at -10 seconds, 1, 2, 3, 11 and 5 minutes. This was
done to compare arachidonic acid with FMLP as a shape change

inducing agent. See Table 2 and Figure 3.

21

 

1(30

9C)-

70 4

60 d

 

 

 

5O -

4o—

2 Shape Change

30 —

20 -

10 -

 

 

 

I I I I I I I I I I I I I I

T I
O 2 4 6 8 1O 12 14 16 18 20

flinut-a
fl Outta-tad + Thunphgllino 0 1+1 A Inoproturonni

Figure 2

Figure 2. Secondary Inhibition of Shape Change Over Time.

Shape change caused by 1X10”9 M N-formyl-methiony—
leucyl-phenyalanine (FMLP) was tested for inhibition by
theophylline, isoproterenol, and theophylline plus
isOproterenol (T+I) 2 minutes after shape change began. The
average of 11 values as 1 shape change were plotted against
time through 20 minutes.

TheOphylline significantly inhibited the shape change
induced by FMLP from 3 through 20 minutes. Isoproterenol
caused a significant interaction with theOphylline at 4
minutes and caused significant inhibition when present alone
at 4 minutes. Significant differences indicate differences
at the 95 1 level of significance.

22

Table 2 Drug Prior to FMLP Inhibitiona

(Time) 0 1 2 3 A 5
----------------- 1 Shape Change---—-—-------—--
(Untreat) 3 72 83 74 66 67
b b
(Theo) 3 59 71 59 55 M8
0 c,d c,d c,d d c,d
(T+I) 3 5” 57 51 N7 42

 

aDrug Prior to N—formyl—methionyl-leucyl-phenyalanine
(FMLP) Inhibition The inhibitors theOphylline or
theOphylline plus iSOproterenol were introduced prior to
induction of shape change by FMLP. Percent shape change was
recorded through 5 minutes after the addition of FMLP.

(Time) Time in minutes
(Untreat) Untreated with an inhibitor
(Theo) Theophylline at 1X10'3 M

(T+I) The combination of isoproterenol at 1X10'u M plus
theophylline at the concentration used above.

bIn these instances the 1 shape change was not
significantly different from the corresponding untreated 1
shape change. Significant differences indicate differences
at the 95 1 level of significance.

°In these instances the 1 shape change was n__o__t
significantly different from the corresponding theophylline
1 shape change.

dIn these instances the 1 shape change was
significantly different from the corresponding untreated 1
shape change.

23

Figure 3. Inhibition of Arachidonic Acid Shape Change.

The first 5 minutes

NeutrOphils were eXposed to 5X10"7 M arachidonic acid 2
minutes after treatment with the inhibitors theOphylline or
theOphylline plus iSOproterenol (T+I). The untreated
neutrophils were not combined with an inhibitor before
eXposure to arachidonic acid. The average of 4 values as 1
shape change were plotted against time through 5 minutes.

TheOphylline caused significant inhibition only at 3
minutes. The combination (T+I) caused significant inhibition
from 1 to 9 minutes. TheOphylline was significantly
different from (T+I) at 2 minutes. Untreated shape change
and inhibition were maximal at 2 to 3 minutes and decreased
by 5 minutes. Significant differences indicate differences
at the 95 1 level of significance.

The second 5 minutes

At 5 minutes a second dose of arachidonic acid was
added to neutrophils inhibited with theOphylline or (T+I). A
portion of the untreated neutrOphils were eXposed to a
second dose of arachidonic acid at 5 minutes and are labeled
double untreated. The remaining untreated neutrOphils were
not altered by a second eXposure to arachidonic acid and are
labeled untreated. The average of 4 values as 1 shape change
were plotted against time from 5 through 10 minutes.

At 6, 7 and 8 minutes, double untreated neutrOphils
were significantly different from untreated neutrophils.
Treated neutrOphils did not differ significantly from the
untreated neutrOphils. The inhibiting drugs were more
effective during the second dose of arachidonic acid. The
double untreated 1 shape change response was transient, like
the untreated response in the first 5 minutes.

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25

Results

When shape change was induced by FMLP, both
theophylline and (T+I) generally caused significant
inhibition at all times. When arachidonic acid induced shape
change, theOphylline caused significant inhibition only at 3
minutes. The combination of theophylline plus iSOproterenol
caused significant inhibition from 1 to 4 minutes.
Theophylline inhibition was significantly different from
(T+I) inhibition at 2 minutes. When arachidonic acid induced
shape change, both induction and inhibition were maximal at
2 to 3 minutes and decreased by 5 minutes.

Inhibition of Subsequent Arachidonic Acid Dose

 

 

This eXperiment was designed to determine whether
neutrOphils combined with arachidonic acid for 5 minutes, as
described above, were completely refractory to a second
treatment with arachidonic acid. This was done by adding
more arachidonic acid at the 5 minute time to neutrophils
that had encountered arachidonic acid in the previous
eXperiment. The concentration of arachidonic acid was 5X10"7
M at 0 time and was increased to 1.9)(10"6 M at 5 minutes.
This was based on the assumption that the arachidonic acid
concentration did not change from 0 to 5 minutes. No attempt
was made to quantitate arachidonic acid. Untreated
neutrophils that had been exposed to one dose of arachidonic
acid in the first 5 minutes were divided into 2 untreated

groups during the second 5 minutes. One group (double

26

untreated) was given a second dose of arachidonic acid. The
other group (untreated) was not given a second arachidonic
acid dose and served as a baseline for comparison. Groups
treated with inhibiting drugs also received a second dose of
arachidonic acid at 5 minutes. Portions taken in the second
5 minutes were labeled with respect to 0 time and were taken
at 6, 7, 8, 9 and 10 minutes. See Figure 3.
Results

At 6, 7 and 8 minutes, the double untreated neutrophils
were significantly different from the untreated neutrophils.
The treated neutrophils did not differ significantly from
the untreated neutrophils. When treated with inhibiting
drugs, the neutrophils failed to change shape when combined
with the second dose of arachidonic acid. The second shape
change response induced by arachidonic acid was transient.

Serotonin Enhancement gf FMLP Shape Change

and
Serotonin Enhancement gf Arachidonic Acid Shape Change

 

 

Neutrophils were treated with PBS (untreated) or with
serotonin at a final concentration of 1X10”5 M for 2
minutes. Concentrations of FMLP ranging from 0 to 5x10’9 M
were then combined with these neutrOphils for 2 minutes
before measurements were taken. This was repeated

'6 M for

substituting arachidonic acid ranging from O to 1X10
1 minute before measurement, in place of the range of FMLP

concentrations.

27

Results
These data were submitted to a multivariate anaylsis of
variance which indicated that serotonin treated and
untreated neutrOphils were not significantly different.

FMLP Shape Change Enhancement Screen

 

A standard level of 1 shape change was established by
combining neutrOphils with 5X10"1O M FMLP for 2 minutes
before measurement. Neutrophils were treated with a drug for
3 minutes before FMLP was added. Each drug was tested at 3
different concentrations within a 100-fold range. The drugs
used were cGMP, succinyl choline, ascorbic acid,
phenylephrine, acetylcholine, D—tubocuraine, carbachol and
the combination D-tubocuraine plus acetylcholine. Some of
these drugs were dissolved in DMSO. Dimethyl—sulfoxide
controls were run with the tests. r=1 for this eXperiment.

Results

A baseline level of 1 shape change was established and
1 shape change resulting from enhancing drugs was compared
to the baseline level. This was a one-time eXperiment in
which FMLP was used to induce shape change. None of the drug
treatments appeared to enhance shape change, but ascorbic
acid did seem to inhibit its These results were used to
screen drugs for inclusion in the next eXperiment.

Enhancement of Arachidonic Acid Shape Change

 

 

The previous format was used to test cGMP,

phenylephrine and carbachol as enhancers of 1 shape change

28

induced by 5X10"8 M arachidonic acid. Each drug was tested
at N concentrations within a 1000-fold range. A baseline
level of 1 shape change was established by combining
neutrophils with arachidonic acid in the absence of
enhancing drugs. The enhancing drug results were compared to
the baseline. r=9 in this eXperiment.
Results

None of the drugs tested gave results that were
significantly different from the baseline 1 shape change
which was 60 1.

Calcium Requirement for Arachidonic
Acid Shape Change Over Time

 

 

All previous eXperiments included calcium in the PBS.
Some calcium-free PBS containing 1X10"6 M ethyleneglycol-
bis-glycol-tetraacetic acid (EGTA) was prepared. The EGTA
was added to bind small amounts of calcium that might be
contaminating the calcium-free PBS. After the normal
isolation process neutrOphils were washed with and stored
for 20 minutes in calcium-free (EGTA) PBS. Arachidonic acid
dissolved in calcium—free (EGTA) PBS at a final
concentration of 1X10"7 M was combined with neutrophils.
Untreated portions used regular PBS. Portions were taken at
0,1, 2, 3,11and 51ninutes.r=1 for this eXperiment.

Results

The results suggest that arachidonic acid induced shape

change does not require calcium, but further testing is

needed to confirm this.

DISCUSSION

Each cell preparation had to meet certain shape change
guidelines before data could be accepted. First, the
unstimulated 1 shape change had to be 8 1 or less. Second, a
maximum 1 shape change of greater than 80 1 had to be
obtained to insure viability of the cells. Several cell
preparations failed to meet an acceptable maximum or minimum
1 shape change and were rejected.

In the first two experiments iSOproterenol alone was an
effective inhibitor at only one time. Theophylline was an
effective inhibitor and showed an interaction with
isoproterenol. The inhibition does not require preincubation
with the inhibitor before FMLP is added. These results are
consistent with the results from similar chemotactic and
shape change studies of monocytes except that monocytes are
more responsive to iSOproterenol than neutrOphils (1).

Arachidonic acid-induced shape change is brief compared
to that induced by FMLP. Surface receptors and multiple
activation pathways could eXplain the persistence of FMLP-
induced shape change. TheOphylline and (T+I) are effective
inhibitors of arachidonic acid-induced shape change. The
shape change burst can be repeated by increasing the
concentration of arachidonic acid. It seems that, if the
shape change mechanism and the inhibition mechanism have
been primed by one pass, the inhibition is stronger during

the second shape change burst. This may be due to a build-up

29

30

of cAMP. It appears that, as with FMLP, extracellular
calcium is not needed (5). However, both arachidonic acid
and FMLP-induced shape change may be inhibited by depleting
the intracellular calcium pools (9). Monocyte shape change
induced by FMLP is delayed by a calcium-free medium (1).
This does not appear to be true for neutrOphils.

Monocyte shape change can be induced by a variety of
agents that are not effective for neutrOphils under similar
conditions. These agents are thought to increase calcium and
cGMP in the cells. They will also enhance monocyte shape
change or chemotaxis induced by FMLP (1). In neutrOphils,
under similar conditions, shape change enhancement was not
seen; however, enhancement of neutrOphil chemotaxis was
seen. It is assumed that the agents used are able to alter
the levels of the cyclic nucleotides as the literature
indicates (29,30,31,N8). In these eXperiments, that ability
seems to be limited for neutrOphils.

Monocytes, activated to similar levels of shape
change, are more responsive then neutrOphils to agents that
alter cyclic nucleotides (1). Neutrophils did not respond to
drugs that should increase the shape change level at 2
minutes. However these drugs are effective in neutrOphil
chemotaxis eXperiments which can last up to an hour
(29,30,31,48). It is not clear why neutrOphil shape change
was not enhanced by serotonin, carbachol and phenylephrine.

These results suggest that neutrophils are less responsive

31

than monocytes to agents that enhance or inhibit the i3
vitro cell response. It may be possible to alter the time of
arrival and relative number of the different cell types at

an inflammatory site by using these enhancers or inhibitors.

SUMMARY AND CONCLUSIONS

This study focused on neutrOphil shape change and how
it is affected by altering the levels of intracellular cAMP
and cGMP. Theophylline increased cAMP and inhibited shape
change induced by arachidonic acid or FMLP. Within certain
time limits iSOproterenol, when combined with theOphylline,
increased the inhibition. Serotonin, phenylephrine and
carbachol have been shown to enhance neutrophil and monocyte
chemotaxis and monocyte shape change (1,30,31,48). These
enhancers are thought to increase cGMP. Serotonin,
phenylephrine and carbachol did not enhance neutrophil shape
change as eXpected. These results indicate that neutrOphils
are less sensitive to agents that elevate cGMP and cAMP than
monocytes. It may be possible to use this difference in

sensitivity to manipulate the inflammatory response.,

32

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VITAE

VITAE

The author was born in Dubuque, Iowa, on May 20, 1952.
He lived on the family farm near Volga, Iowa until he
started college at Upper Iowa University, Fayette, Iowa in
1970. After receiving a B.A. in chemistry he entered the Air
Force to become a medical technologist. The first 2 years
were spent training in Texas. The last 2 years he worked in
central Louisiana. On July 23, 1977 the author married Kathy»
Gaunt in Wyoming, Michigan. In the fall of 1978 they left
the Air Force to attend Michigan State University. In 1980
the author started a Master‘s degree program in Clinical
Laboratory Science. He also taught in the Clinical

Laboratory Sciences Program at Ferris State College.

38