ABSTRACT
John Emanuel Poulos. THE EFFECTS OF INDOMETHACIN AND DEXAMETHASONE
ON FIBROBLAST CHEMOTAXIS (Under the supervision of Dr. Gerhard Kalmus)
Department of Biology, June 1987.
Tissue damage results in an acute inflammatory response followed
by a repairative phase which is initiated by fibroblast migration
towards chemoattractants released from the wound site. Boyden
chambers were employed to investigate the effect of indomethacin and
dexamethasone on fibroblast chemotaxis towards conditioned medium. The
ability of conditioned medium to serve as a chemoattractant is possibly
due to the presence of cell secreted proteins, as well as fibronectin
and its fragments. Indomethacin is a non-steroidal anti-inflammatory
agent that inhibits prostaglandin synthase whereas, dexamethasone
inhibits phospholipase A2. It was determined that indomethacin did not
inhibit but enhanced fibroblast chemotaxis at a concentration of 1x10”^
O
to 1x10 °M. This may be due to the accumulation of arachidonic acid by
inhibition of prostaglandin synthase. This arachidonic acid is then
free to be acted upon by 5-lipoxygenase thereby producing leukotriene
B4, a potent chemoattractant for fibroblasts. Dexamethasone showed
inhibition of fibroblast chemotaxis at IxlO'^M, and enhancement of
chemotaxis at 1x10“® to lxlO“^®M. Inhibition may be due to the
induction of a chemotactic regulatory protein known as lipocortin.
Enhancement may be due to glucocorticoid stimulation of fibronectin
synthesis and secretion, accompanied with the upregulation of fibro-
nectin receptors.
THE EFFECTS OF INDOMETHACIN
AND DEXAMETHASONE ON
FIBROBLAST CHEMOTAXIS
A Thesis
Presented to
the Faculty of the Department of Biology
East Carolina University
In Partial Fulfillment
of the Requirements for the Degree
Master of Science in Biology
by
John Emanuel Poulos
June 1987
The Effects of Indomethacin and
Dexamethasone on Fibroblast Chemotaxis
By
John Emanuel Poulos
APPROVED BY:
Supervisor of Thesis :
Gerhard W. Kalmus, Ph.D
Thesis Committee:
Chairman of the Biology
Department
Dean, Graduate School
ACKNOWLEDGEMENTS
I would like to sincerely thank Dr. Gerhard Kalmus for his
immense support and supervision during both my undergraduate
and graduate career. For it was Dr. Kalmus who was instrumental
in encouraging me to pursue a scientific career. I would also
like to thank Dr. Jose Caro for giving me the opportunity to
work both, on my thesis and as a lab technician in the East
Carolina University, Department of Medicine. Extreme thanks
also goes out to Dr. Charles O'Rear, who devoted much of his
time and effort in reviewing the data presented in this thesis.
I wish to also thank Dr. James Smith for providing me with his
invaluable assistance and biochemical background.
My extreme gratitude also goes out to Mrs. Karin Kalmus for
her expertise on mammalian tissue culture and for providing me
with the technical background required for the completion of my
thesis. Thanks also goes out to Mr. Jim McLean for his
assistance during the final draft of this thesis.
I will always be indebted to the faculty of the Department of
Biology for giving me the financial support, facilities, and
education required for the attempt and completion of my Master
of Science degree in Biology.
Finally, I wish to thank my parents, Mr. and Mrs. Emanuel
Poulos who always stood behind me and offered both their love and
support in all of my endeavors.
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS i
LIST OF FIGURES AND GRAPHS iii
ABBREVIATIONS USED IN TEXT iv
INTRODUCTION 1
HISTORICAL REVIEW 2
PROPOSED RESEARCH 28
MATERIALS AND METHODS 29
RESULTS 34
DISCUSSION 47
REFERENCES 57
APPENDIX I 63
ii
LIST OF FIGURES AND GRAPHS
Page
Fig. 1 Arachidonic Acid Metabolism 5
Fig. 2 Fibronectin Molecule 10
Fig. 3 Collagen Biosynthesis . 13
Table 1 List of Cell Types and Their Attractants 15
Fig. 4 Structures of Indomethacin and Dexamethasone 25
Graph 1 Bar Graph of Fibroblast Chemotaxis Toward
Conditioned and Defined Medium 36
Fig. 5 7.5% SDS Electrophoresis of Collagen,
Modified Medium, Conditioned Medium,
and Fibronectin 37
Fig. 6 10% SDS Electrophoresis of Fibronectin,
Collagen, Conditioned Medium and
Modified Medium 38
Fig. 7 Densitometer Scan of a 12% Gel Containing
Conditioned Medium 39
Fig. 8 10% SDS Gel Utilizing Either Coomassie
Blue or Silver Nitrate Stain 40
Fig. 9 Densitometer Scan of a 10% SDS Gel Containing
Conditioned Medium and Fibronectin 41
Graph 2 Bar Graph of the Effect of Indomethacin on
Fibroblast Chemotaxis 44
Graph 3 Bar Graph of the Effect of Dexamethasone on
Fibroblast Chemotaxis 46
Table 2 Fibroblast Chemotaxis in Response to
Indomethacin and Dexamethasone 52
Abbreviations used in Text
ABP Actin Binding Protein
aa amino acids
AA Arachidonic Acid
BSA Bovine Serum Albumin
C5a Complement 5a
CM Conditioned Medium
PCS Fetal Calf Serum
Hetes Hydroxy-eicosatetraenoic acids
LTs Leukotrienes
MM Modified Medium
PA2 Phospholipase A2
PGs Prostaglandins
PGE2 Prostaglandin E2
PGG2 Prostaglandin G2
PGH2 Prostaglandin
PGI2 Prostacyclin
SDS Sodium Dodecyl Sulfate
TxA^ Thromboxane A^
1
INTRODUCTION
Chemotaxis is an important biological phenomenon
involving the directed migration of cells along a chemical
gradient. This phenomenon is displayed by a wide variety of
cells and constitutes a major component of both the
inflammatory response and tissue repair. Infection,
chemical irritation, and physical injury to tissue and
surrounding blood vessels generates localized responses by
the host. This localized response involves the release of a
wide variety of inflammatory mediators such as: histamine,
bradykinin, prostaglandins and 1 eukotrienes. These
compounds have the ability to mediate acute inflammation via
erythema, edema, platelet aggregation, along with
polymorphonuclear and monomorphonuclear cell influx.
Following this response, a reparative phase is initiated due
to fibroblast recognition of chemoattractants released from
damaged tissue and inflammatory cells. These
chemoattractants elicit the migration of fibroblasts to the
wound site, resulting in cell proliferation, collagen
synthesis, and subsequent repair of the connective tissue
matrix. Any breakdown in the reparative phase may result in
excessive fibroblast chemotaxis and extensive chronic
fibrosis. The goal of the present study was to determine if
fibroblast chemotaxis could be regulated by inhibition of
arachidonic acid metabolism.
2
HISTORICAL REVIEW
Acute Inflammation
Vasoactive peptides - Any damage to tissue, either due
to an immunological or non-immunological stimuli generates a
localized response. This response results in activation of
the coagulation and complement system. The complement
system is only activated upon immunological challenge either
from antigen stimulation or bacterial infection. Also
occurring is histamine release from mast cells, bradykinin
production, arachidonic acid (AA) metabolism, and cell
influx. The release of histamine and bradykinin initiates
constriction of the vascular endothelial cells, which
dilates terminal arterioles and increases the vascular
permeability of postcapillary venules. This
vasoconstriction followed by vasodilation results in the
dilation and loss of fluid through the microvasculature.
This is also accompanied by the exudation of plasma proteins
in inverse proportion to their molecular size (Magno
1961 ) .
Arachidonic Acid - Also associated with acute
inflammation is the enhanced metabolism of AA resulting in
the synthesis of prostaglandins (PGs), leukotrienes (LTs),
and hydroxy-eicosatetraeonic acids (Hetes). Arachidonic
acid is found in abundance in the phospholipids of cell
plasma membranes and is esterified at position two of the
glycerol backbone. The availability of AA is determined by
either the cytidine diphosphate-diacyIglycerol pathway or by
3
the méthylation of phosphotidy1 ethanol amine to
phosphotidylchol ine by methy1 transferases. Inhibition of
these méthylations by agents that raise levels of S-adenosyl
homocysteine, block chemotaxis (Schiffman, 1 983).
Tissue damage, histamine, Ca+2 influx, and bradykinin,
all enhance the activity of phospholipase A2 (PA2) to
release AA and lysophosphotidy1 choline. This is the rate
limiting step and branch point for the synthesis of PGs,
LTs, and Hetes. Removal of the stimulus may result in a
rapid reincorporated of AA into the cell membrane. This is
accomplished by the action of acyl-CoA synthetase on
arachidonic acid to form arachidony1-CoA. Arachidony1-CoA
is then re-esterified by acyl transferases into the cell
membrane. However, in damaged tissues, liberated AA is
mainly metabolized in order to mediate inflammation.
and IJir.QJDj5a.x.aii.e - aa may be acted
upon by PG synthase to yield PGs, thromboxane A2 (TxA2), and
prostacyclin (Fig. 1). Prostaglandin synthase is a dimer
consisting of two similar 70 Kd subunits and catalyzes both
the bis oxygenation of AA to form prostaglandin G2 (PGG2)
(cyclo-oxygenase) and the peroxide reduction of PGG2 into
prostaglandin H2 (PGH2) (Kulmacz, 1985). Cyclo-oxygenase
incorporates molecular oxygen into the 20 carbon backbone of
AA via a dioxygenase reaction. A hydrogen is abstracted at
C-12 and peroxidation occurs at C-11. Molecular O2 is added
at C-15 and ring closure occurs between C-8 and C-12 to form
PGG2. Prostaglandin hydroperoxidase then acts on PGG2 to
4
Fig. 1 Arachidonic acid metabolism: Dex, Dexamethasone; PGS,
Prostaglandin Synthase; Indo, Indomethacin; HPETEs,
Hydroperoxyeicosatetraenoic acid; HP, Hydroperoxidase;
LS, Leukotriene Synthetase; HETEs , Hydroxyeicosatetraenoic
acid; TS, Thromboxane A2 Synthetase; EP, Endoperoxide
Isomerase; PS, Prostacyclin Synthetase. {-) indicates
inhibitor.
Ca*2 Ca+2
(Bound) -?(Free)
R1-C-O-CH^, O1 H PhosphoMpase
O Ag Arachidonic AcidCH-0-0(CH2)2 (CH2-CH:CH)^-(CH2)4-CH3
HO-P-O-CHo CHo
I I
O-CH2-CH2-N-CH3
I
CH^
COOn
OH
Cyclic Endoperoxides 5 or 15 HPETEs
Thromboxane A2 Prostaglandins Prostacyclin 5 or15-HETEs Leukotrienes
OH
COOH
6
form PGH2. Subsequent enzymatic reactions produce
prostacyclin (PGI2), prostaglandin E2 (PGE2), prostaglandin
P'2 , and TxA2 (Dawson âl-, 1 985). Prostacyclin and PGE2
are potent vasodilators and potentiate the effects of
bradykinin and histamine (Higgs si si., 1 984).
Prostacyclin has the ability to inhibit platelet aggregation
and raise c-AMP. Prostaglandin E2 also has the ability to
raise c-AMP and acts as a potent vasodilator (Higgs si sl.,
1984). Thromboxane A2 is a potent platelet aggregator and
vasoconstrictor, with the ability to mobilize Ca+2 from
intracellular stores (Gerrard, 1985). This free ionized
Ca+2 may activate PA2 or complex with calmodulin to trigger
platelet contraction.
Leukotrienes - In neutrophils AA may be oxidized by 12
or 15-1ipoxygenase to form Hetes which are potent
chemoattractants for neutrophils (Schiffman, 1983).
Arachidonic acid may also be acted upon by 5-1ipoxygenase to
yield leukotriene A^^ (LTA4). Subsequent enzymatic reactions
on LTA4 produce leukotriene Bi; (LTB4), leukotriene C4
(LTC4)j leukotriene D4 (LTD4), and leukotriene E4 (LTE4).
The enzymes responsible for the synthesis of the LTs are
found in the plasma membrane while the products are found in
the cytosol. Leukotriene B4 ¿3 3 strong chemoattractant for
fibroblasts (Mensing and Czarntzki, 1984) and neutrophils
(Ford-Hutchison si âl., 1980) and induces muscle contraction
and vascular permeability (Gerrard, 1985). Leukotriene B^
also strongly enhances plasma exudation in
combination with bradykinin and enhances Ca+2 influx into
7
cells (Eakins ^ Jl., 1980; Molsk £jt âl., 1 980); LTCi| and
L'TDii enhance the adhesiveness of neutrophils (Goetzl âl»,
1983) and promote bronchoconstriction and vascular
permeability in the skin (Gerrard, 1 985). Research has not
shown a clear role for LTE^, Further research on LTC4,
and LTE4 may indicate that these compounds have
functions similar to LTB4, p-Qr example, the ability to
elicit fibroblast migration to the wound site.
Cell Influx - Associated with acute inflammation is the
recognition of tissue damage by circulating leukocytes,
platelets, lymphocytes and macrophages. Cellular influx to
the inflammatory site involves the movement of leukocytes
and monocytes from the center of the blood vessel, followed
by adhesion to the vascular endothelium (margination).
Next, there is movement through the vascular wall and
connective tissue (diapedisis), ending in the migration by
leukocytes and monocytes in response to chemoattractants
released from the inflammatory site. These cells invade the
wound site in a defined sequence. Polymorphonuclear
leukocytes arrive first, followed by mononuclear leukocytes,
fibroblasts and finally endothelial cells (Ross, 1968). Any
antigens present at the wound site are engulfed by
macrophages and presented in a recognizable form to B and T
lymphocytes. Upon stimulation, macrophages produce
interleukin 1, which stimulates T-lymphocytes to produce
interleukin 2. Interleukin 2 stimulates lymphocytes to
differentiate into subsets of helper, suppressor, and plasma
cells. Plasma cells then migrate to the area and produce
8
antibodies that neutralize toxins, cytolyze bacterial wall
membranes (cytolytic antibodies), and elicit hypersensitive
reactions. Therefore, antigen stimulation results in
lymphocyte production of lymphokines, which are
chemoattractants for neutrophils (Altman, 1978), monocytes
(Lett-Brown, 1 976), fibroblasts (Postl ethwai te âl., 1 976)
and other lymphocytes (Ward, 1975). Neutrophils are also
attracted to the wound site where they engulf the antigen-
antibody complex into a phagosome and release hydrolytic
enzymes as well as O2-, ^^8 OH** These radicals are
generated by a respiratory burst as a result of cellular
activation. Neutrophils upon stimulation produce
chemoattractants for neutrophils and macrophages (Zigmond
and Hirsch, 1973). Neutrophils do not replicate at wound
sites, thus, the release of chemoattractants reamplifies the
signal for cellular influx. The influx by macrophages then
results in the ingestion of cellular debris and production
of a chemoattractant for fibroblasts (Wahl, I98I). At this
point, the fibroblasts initiate a reparative phase, whereby
they migrate to the wound site in an attempt to reconstruct
the connective tissue matrix.
The Repairative Phase
The final phase of the inflammatory response involves
the migration of the fibroblasts to the wound site and
subsequent synthesis of glycosaminoglycans, proteoglycans,
fibronectin, and collagen, resulting in the appearance of
scar tissue. Attachment to a substratum is an important
9
step in this response and fibroblasts require fibronectin
for both chemotaxis and adherence (Gauss-Muel 1 er ^
1980). Fibronectin is a glycoprotein consisting of two
similar polypeptide chains which are linked at their carboxy
(COOH) terminus by disulfide linkages (Fig. 2). These
chains are designated alpha and beta, with the alpha chain
being slightly larger. The higher molecular weight of the
alpha chain is due to the presence of additional amino acids
within the heparin/fibrin COOH terminal region (Sekiguchi ^
âl., 1985). Differences in the molecular weights of
fibronectin have been reported and may be due to different
degrees of glycosylation (Albini ^ âl., 1983» Sekiguchi Mi
âl.» 1985). The fibronectin chains may further be
subdivided into 3 types of regions due to either their
cysteine content, protein folding, or fragmentation patterns
(Erickson, 1985). Fibronectin has the ability to bind to
such proteins as heparin, fibrin, gelatin, collagen, and
actin, as well as cell surfaces (Albini mí âl., 1 983;
Pierschbacher MÍ âl.» 1985; and, Sekiguchi mí Ml-, 1 985).
Scatchard analysis reveals that fibroblasts contain 5.3 x
10+8 binding sites per cell for fibronectin. The
linearality of the Scatchard plot indicates a single class
of receptors with a Kd of 8 x 10-7m (Akiyama and Yamada,
1985). Adherence mediated by fibronectin may be required
for the proper attachment and detachment in the
microtubule/microfilament based motility apparatus.
Fibronectin is secreted by fibroblasts and binds collagen at
the collagen binding site located near the fibronectin NH2
10
Fig. 2 Fibronectin molecule containing binding regions and the three types
of sequence homologies. (Taken from Erickson, 1985).
terminus (Gauss-Muel1 er ^ âl-, 1 980). Binding to collagen
converts fibronectin into a more extended form, thus
enhancing fibronectin binding to other molecules.
Fibroblasts then use gangliosides, located in their cell
membrane to bind to the fibronectin/col 1 agen complex. The
fibroblasts use the carbohydrate moiety of gangliosides to
bind to the complex at the cell attachment site of
fibronectin, located near the COOH terminus. This cell
attachment region may be responsible for 30/& of the
fibronectin molecule containing a folded B structure
(Alexander, 1 979). Thus fibronectin, or its fragments
generated by tissue damage, may serve in facilitating
fibroblast migration as a prerequisite to connective tissue
reconstruction.
Initial development and regeneration of connective
tissue shows an increase in hyaluronate. This latter
disappears with fibroblast proliferation and enhanced
collagen synthesis. Collagen is the primary structural
element in connective tissue and is made up of three
polypeptide alpha-chains wound in a regular helix. The
collagen polypeptide chains are synthesized on ribosomes and
injected into the lumen of the endoplasmic reticulum as pro
alpha-chains, with extension peptides at both the NH2 and
COOH terminus (Fig. 3). In the endoplasmic reticulum
each pro alpha-chain combines with two others to form a
hydrogen bonded triple stranded helical molecule. The
extension peptides aid in guiding this triple helix
formation. Along with the assembly of the triple helix is
12
Fig. 3 Collagen biosynthesis by fibroblasts.
(Taken from Alberts et , 1983).
PROCESSES
OCCURRING WITHIN
INTRACELLULAR
MEMBRANES
ER, GOLGI,
SECRETORY VESICLES]
procollagen
molecule
un ' OH ^
CLEAVAGE OF -
EXTENSION PEPTIDES 9”
collagen molecule
OH* * OH \
\ ASSEMBLY INTO
\ MICROFIBRIL
microfibril
ASSEMBLY INTO
MATURE
COLLAGEN FIBRIL
14
the hydroxylation of proline and lysine and post-
translational glycosylation of hydroxylysine residues.
During secretion, the extension peptides of the procollagen
molecule are cleaved off, thus forming tropocol1 agen. These
terminal extension peptides have the ability to regulate
collagen deposition by inhibition of collagen synthesis at
the level of m-RNA translation. Once formed, tropocol1 agen
associates into microfibrils and microfibrils associate into
collagen fibrils. Subsequent crosslinking by extracellular
lysyl oxidase forms collagen fibers. Ten to forty percent
of all newly synthesized collagen can be eliminated by
intracellular degradation via lysosomal enzymes, thus
allowing the cell to dispose of imperfect chains. After
collagen synthesis there is a remodeling of the connective
tissue matrix by collagenase, gelatinase , elastase, and
other hydrolytic enzymes. Remodeling is controlled by
factors that cause the release, activation, and inhibition
of proteolytic enzymes balanced by the synthesis and
deposition of matrix proteins and collagen (Kang and
Mainardi, 1 985).
Fibroblast Chemoattractants
The recognition of damage to the host and subsequent
migration to the site of inflammation by the fibroblast is
believed to be elicited by the release of two classes of
chemoattractants (Table 1). Compounds included in the first
class of fibroblast chemoattractants include connective
tissue fragments such as collagen and collagen derived
Table 1
List of cell types and their attractants; + (attractant), ? (possible attractant),
- (inhibitor). Revised from Schiffman et al., 1983.
FIBROBLASTS NEUTROPHILS MONOCYTES LYMPHOCYTES PLATELETS
PDGF ? ? +
Plasminogen ? + +
activators
HETEs + +
Lymphokines + + + +
LTB4 + +
TxA2 ? +
Fibrin ? +
Tropoelastin/ +
elastin
Collagen + +
Fibronectin +
Complement C5a + +
Colchicine
Cytochalasin B
16
peptides (Postlethwaite, 1978), tropoelastin and elastin
filaments (Senior ^ âi.j 1982), and fibronectin (Gauss-
Mueller, 1980). The second class of chemoattractants
includes proteins generated by the inflammatory response
such as lymphokines (Postlethwaite ^ âl., 1 976), and
complement 5a [C5a] (Postlethwaite ^ âl., 1 979).
Complement 5a is a product of the 5th component of
complement. Complement 5a contains 74 amino acid (aa)
residues with the first 69 aa serving as the recognition
site and the last 5 aa at the COOH terminus serving as the
active site (Synderman slÍ âJL., 1981). This glycoprotein is
cleaved from the NH2 terminus of the oc chain of C5 during
complement activation. Chemotaxis to C5a is diminished by
the loss of the terminal arginine by the action of serum
carboxypeptidase (Synderman, I98I). Activated platelets,
lymphocytes, and neutrophils present at acute inflammatory
sites also release such potent fibroblast chemoattractants
as platelet derived growth factor [PDGF] (Seppa ^ b1.,
1982), lymphokines (Postl ethwai te si si., 1 976), and
leukotriene (Mensing and Czarnetzki, 1984). All these
compounds serve as chemical messengers in eliciting
fibroblast chemotaxis to the wound site for initiation of
the reparative phase.
Mechanisms of Chemotaxis
Morphological Events - Migration by cells may occur by
either random migration, haptotaxis, chemokinesis, or
chemotaxis. Random migration is migration in the absence of
17
any known stimuli, whereas haptotaxis is movement up a
gradient due to adhesiveness. Chemokinesis is chemically
stimulated, nondirectional motility in the absence of a
chemotactic gradient, whereas, chemotaxis is directional
motility in the presence of a concentration gradient.
Chemotaxis is the most investigated type of motility due to
its implication in the inflammatory response. Chemotaxis
involves mechanisms of either temporal or spatial sensing
(Synderman and Goetzl, I98I; Schiffman, 1983). Temporal
sensing involves the perception of the chemoattractant with
linear movement for a fixed period of time. This is
associated with lag periods where the cell stops, and
perceives a new concentration before moving again. Bacteria
are prime examples of this mechanism, using one
chemoreceptor and memory. Spatial sensing is associated
with eukaryotic cells and exhibits alteration of cell shape
and morphological orientation toward the concentration
gradient. Migration is in a curvilinear fashion without
pauses, and with detection of the gradient across the length
of the cell via multiple receptors. This type of sensing
allows differentiation of concentrations as little as 0.1%
across the cell surface (Synderman and Goetzl, I98I). This
is accomplished by altering the distribution, number, and
affinity of chemotactic receptors. Exposure to a
chemoattractant results in polarized elongation of the cell
with the appearance of a broad 1amel1ipodium anteriorly.
Located at the posterior end of the cell is a thin uropod
with terminal arborations. Located beneath the plasma
18
membrane and within the cell are the cytoskeletal processes.
Throughout the cell, are microtubule organizing centers that
function as the point of origin for microtubules.
Concentrated just beneath the plasma membrane is the actin
microfilament network. There is a localization of receptors
in the 1 ammel 1 ipodia, with vesicle concentration in the
uropod. Chemoattractant binding results in the ligand-
receptor complex being picked up at the leading edge,
followed by transport over the dorsal surface of the cell.
Old cell mass is endocytosed at the rear with new cell mass
and receptors arising from the Golgi complexes to be
reincorporated into the new cell membrane (Nemere ^ âl-,
1984). Both Golgi complexes and microtubules are organized
in the direction of migration. Cytochalasin B blocks actin
filament rearrangement and pseudopod retraction without the
loss of orientation. This indicates that microfilaments are
critical for the contractile events of directed locomotion
and morphological changes. Colchicine inhibits microtubules
without altering the formation of pseudopodia, thus
indicating their importance in the initiation and
stabilization of cellular orientation toward a chemotactic
gradient (Synderman and Goetzl, 1981; Schiffman, 1982).
Biochemical Events - Chemoattractant binding also stimulate
Ca+2 influx both extracel 1 ul arly and from membrane bound
compartments, thereby, increasing the concentration of
ionized intracellular Ca+2 (Naccache ¿í ¿1., 1977). Calcium
release is accomplished by an increase in permeability to
Na+ through membrane potential insensitive channels.
19
resulting in H+ leaving the cell and subsequent membrane
polarization. A pH change also arises and Ca+2 is released.
These events elicit activation of a membrane bound Na+-
K+ ATPase. A calcium influx activates PA2 resulting in
the production of PGs, LTs, and Hetes, each with the ability
to reamplify the response in other cells. Increases in free
ionized Ca+2 may activate a tyrosine specific kinase, a c-
AMP kinase or calmodulin. A specific kinase may be needed
to phosphory1 ate a chemotactic regulatory protein known as
lipocortin. Recent research indicates that lipocortin acts
as a regulatory protein in chemotaxis (Hirata, 1980). This
is due to the fact that the phosphory 1 ated form of
lipocortin has the ability to inhibit chemotaxis by binding
to PA2 (Blackwell 52., 1980; Hirata, 198O). Associated
with chemotaxis, is a brief increase in c-AMP. However high
levels of c-AMP inhibit chemotaxis, while high levels of c-
GMP enhance chemotaxis (Tse &i, 52-, 1972; Rivkin £2 52-,
1974). This may be explained by the following mechanism.
Attractant binding followed by Ca+2 influx and c-AMP
production may stimulate a c-AMP dependant kinase and
calmodulin. The c-AMP dependant kinase may serve to
phosphorylate lipocortin at tyrosine residues. Binding of
Ca+2 by calmodulin then results in a conformational change
and enzyme activation. Once activated, calmodulin may
accelerate phosphodiesterase activity resulting in
termination of the c-AMP response and elevation of guanosine
triphosphate levels. Guanosine triphosphate may then induce
conformational changes in the 6S tubulin dimers resulting in
20
polymerization of microtubules from microtubule organizing
centers. Motility is then generated by the polymerization-
depolymerization of microtubules or by a sliding mechanism
through ATP hydrolysis by dynein arms. Free ionized Ca+2
also stimulates microfilament mediation of chemotaxis. The
mechanisms of motility may be very similar to the mechanisms
of sliding actin/myosin filaments in muscle. However, in
migrating cells the actin/myosin complex is three
dimensional, and regulated by an actin binding protein (ABP)
and gelsolin (Stendahl ¿i âl., 1980). Increases in actin
network structure by ABP enhances the contractility of actin
by myosin. The ABP crosslinks actin to form an actin
lattice with interspersed myosin filaments, thus providing a
contracting mass. Gelsolin divides actin filaments in the
presence of high Ca+2 by shorting and severing actin
filaments between points of crosslinking. The actin lattice
moves from high concentrations of Ca+2 to low concentrations
of Ca+2. Free actin filaments interacting with myosin move
away from domains of decreased crosslinking because of the
increased efficiency of contraction in the more crosslinked
domains. Therefore, any part of the cell attached to the
lattice would move accordingly. Calmodulin would then play
a role in the events leading up to the phosphorylation of
myosin light chains, and subsequent motility. The ATPase
activity of the myosin head group would then generate the
energy for the sliding mechanism between the actin and
myosin filaments.
21
Fibrotic Diseases and their Relationship with the
Leukotrienes and Thromboxane A2
The function of the acute inflammatory response is to
complete healing with restoration of normal tissue
architecture and function. Any breakdown in this process
may result in little to no attempt at wound healing with
resulting fibrosis and excessive connective tissue
formation. This chronic inflammatory response may be due to
the continuation of the initial inflammatory stimulus. This
continuation leads to enhanced tissue destruction by
proteases, as well as elevated radical formation, AA
metabolism, and cell influx. Released proteases have the
ability to modify proteins at the inflammatory site with
subsequent antibody formation against this "self antigen".
Enhanced AA metabolism and leucocyte influx may generate
such oxidative radicals as OH*, H2O2, and O2-. These
radicals are generated either by endoperoxide formation or
by respiratory bursts in activated leukocytes. Thus
excessive AA metabolism may generate radicals that attack
surrounding tissue, or act as chemoattractants in
stimulating cellular influx. These events may result in the
appearance of many pathological disorders including
rheumatoid arthritis, breast cancer, and many types of
pulmonary fibrotic diseases. The pathogenesis of these
diseases seems to indicate that the generation of
chemoattractants such as the LTs and TXA2 serve to elicit
fibroblast migration to the afflicted site resulting in
22
excessive fibrotic buildups. Examination of a few of the
diseases associated with excessive fibroses follows.
Rheumatoid Arthritis - Rheumatoid arthritis is
characterized by high percentages of neutrophils and
elevated levels of LTs within synovial fluid. As the
disease progresses, fibrotic involvement is encountered. It
is interesting to note that patients with severe arthritis
have been shown to contain antibodies for the protein
lipocortin (Hirata, 1981). Lipocortin inhibits
phospholipase activity in vitro. Studies postulate that a
c-AMP dependent kinase phosphorylâtes the lipocortin
inhibitor (Hirata ^ ^1., 1981), resulting in the
inactivation of lipocortin and initiation of AA metabolism.
Thus, prolonged inactivation of lipocortin may result in
excessive chemotaxis by neutrophils and fibroblasts and an
overproduction of LTs. The released LTs may possibly elicit
the attraction of more neutrophils and fibroblasts to the
afflicted area.
Breast Cancer - Diffuse fibrotic buildup is also
associated with many forms of breast cancer. It has been
shown that tumor cells obtained from patients with breast
cancer secrete a potent chemoattractant for fibroblasts
(Gleiber and Schiffman, 1984). Research has also shown that
breast tumor cells treated with ionophore A-23187 and L-
cysteine produce LTs (Nelson âl., 1 982). Thus,
fibroblasts may migrate to tumor cells due to a
chemoattraction for both the breast tumor cells and possibly
23
the LTs. This migration to the tumor cells is followed by
fibroblast proliferation and subsequent formation of dense
fibrotic masses.
Progressive .§y¿,tjemig Sclerosis and Q.ther Fib£.p.ti.s
Diseases - Progressive systemic sclerosis is another example
of a disease characterized by diffuse fibrosis. The primary
event in this disease is postulated to be endothelial cell
injury in blood vessels (Kang and Mainardi, 1985). The
increased cell damage produces interstitial edema,
fibroblast stimulation, and fibrosis. Two more examples of
fibrotic diseases encountered by clinicians is diffuse
interstitial fibrosis and idiopathic fibrosis. It is
believed that the local production of specific chemotactic
factors recruits inflammatory and immune effector cells into
the alveolar interstitium (Kang and Mainardi, 1985). These
chemotactic factors may prove to be LTs released from
leukocytes and/or thromboxane A2 released from platelets.
As a consequence, this inflammatory response results in
extensive damage to lung connective tissue, and endothelial
membranes. Following this damage, extensive fibroses may be
encountered.
Despite the high occurrence of these diseases, medical
therapy is not curative and often unsatisfactory and
ineffective. Thus, our understanding of their pathogenesis
is incomplete. Excessive fibrosis is often associated with
an overproduction of AA metabolites, therefore inhibitors of
their synthesis or recognition may prove to be of
therapeutic value in the treatment of many fibrotic
24
diseases. One mechanism of treatment may be through the
inhibition of AA metabolism by glucocorticoids or non-
steroidal anti-inflammatory agents.
Anti-Inflammatory Agents
Indomethacin - Indomethacin (Fig.4) is a non-steroidal
anti-inflammatory agent with the ability to inhibit
granuloma formation, edema, pain, and PG synthesis.
Indomethacin inhibits PA2 at very high doses and cyclo-
oxygenase at a concentration of 14.3 ng/ml. Indomethacin is
classified as a substrate competitive irreversible inhibitor
(Kulmancz, 1983). This compound competes with AA for cyclo-
oxygenase but does not covalently bind to the enzyme. The
active site of indomethacin contains an -methyl acetic
acid side chain, which binds to contours on cyclo-oxygenase
at sites which accommodate large groups of aryl acetic acids
(Shen and Winter, 1977)* Indomethacin has also been shown
to suppress leukocyte influx (Blackham 1979) as well as PG
formation. Therefore, indomethacin may serve as an
important mediator of both the inflammatory response and
cell migration.
- Dexamethasone (Fig. 4) is an
optically active glucocorticoid with the ability to induce
hyperglycemia followed by secondary hyperinsulinimia.
Glucocorticoids increase the degradation and decrease the
synthesis of fat, proteins, DNA, and RNA. Glucocorticoids
also have the ability to inhibit lymphokine production and
(Hirata, 1983; Munck 1984). Recently it has been found
25
a. Indomethacin b. Dexamethasone
Fig. 4 Structures of a.) indomethacin and b.) dexamethasone
26
that glucocorticoids inhibit both the expression of Fc
receptors and tumoricidal activity of macrophages (Munck
âl», 1984). Dexamethasone rapidly penetrates the cell due
to its hydrophobic nature and binds reversibly to its
cytosolic receptor to form a non-covalent complex of high
affinity.
Receptor - The glucocorticoid receptor
is a single polypeptide with 5,000 to 50,000 sites per cell.
It has a molecular weight of 85-95 Kd containing three
distinct domains; a steroid binding domain, a DNA binding
domain, and an immunoactive domain (Rousseua, 1984).
Steroid binding increases the affinity of the receptor for
sites in the cell nucleus. Binding of the receptor to the
steroid is second order with disassociation of the complex
being first order (Baxter, 1976). The steroid binding
domain is rich in lysine and arginine residues which bind
the steroid. This binding is inhibited by proteases and
sulfhydryl reagents. The steroid binds the receptor when
the receptor is phosphory1ated and reduced, thus requiring
the availability of thiol groups. A thioredoxin associated
factor prevents the exposure of basic aa located on the DNA
binding subunit. This prevents binding to DNA by the
inactivated receptor. Molybdate protects reduced thiol
groups on the receptor which are essential for steroid
binding. Steroid binding initiates dephosphorylation and
release of the thioredoxin associated factor with
dissociation of the receptor into monomeric units in order
to allow DNA binding. Binding of the complex to nuclear
27
receptors initiates transcription and translation with
subsequent protein synthesis. The receptor is then recycled
in the cytoplasm, reduced and rephosphory1ated in
preparation for subsequent binding.
Lipocortin - Lipocortin (M.W. 40 Kd) is found in both
untreated and dexamethasone treated cells. Treatment of
cells with dexamethasone rapidly increases the supply of
this protein in the cell membrane. Lipocortin reduces the
Vmax but not the {Cm of PA2, thus forming stoichiometric
complexes (Hirata, 1983)* Lipocortin inhibits this enzyme
by binding to the hydrophobic PA2 Ca+2 binding site. This is
substantiated by the ability of lipocortin to bind four
moles of Ca+2 (Hirata, 1983 )• Phosphorylation of
lipocortin results in a loss in the ability to inhibit
phospholipase A2 and chemotaxis (Hirata, I98O, 1981).
Studies indicate that chemoattractant binding induces a Ca+2
influx which activates PA2 and the phosphorylation of
lipocortin. Further research needs to be conducted to
determine if phosphorylation is by a Ca+2 kinase, tyrosine
dependant kinase, or cAMP dependant kinase
V
28
PROPOSED RESEARCH
The purpose of this study was to determine the effect
of inhibitors of AA metabolism on fibroblast migration.
Inhibitors were selected for their ability to inhibit PA2
activity and cyclo-oxygenase. Inhibitors of AA metabolism
may be grouped into two classes; nonsteroidal anti-
inflammatory agents and glucocorticoids. A representative
of each class was examined to determine its effect on
fibroblast chemotaxis. Indomethacin represented the
nonsteroidal anti-inflammatory agents, while dexamethasone
represented the glucocorticoids. Indomethacin directly
inhibits cyclo-oxygenase activity. Whereas, glucocorticoids
inhibit PA2 through the induction of lipocortin ( Blackwell
et âl., I98O; Hirata, 1981). This study examined the
effects of these anti-inflammatory compounds on fibroblast
chemotaxis towards conditioned medium.
29
MATERIALS AND METHODS
Cell Culture Techniques - Human fetal foreskin
fibroblasts (Earl Clay Co., Norcross, GA) were grown to
confluency in 25 cm2 tissue culture T-flasks containing
medium 199 (Sigma, St. Louis, MO) supplemented with 8% fetal
calf serum [FCS] (Sigma, St. Louis, MO), and 1%
penicillin/streptomycin. Propagation of the cells consisted
of removal of the maintenance media followed by three washes
with Hank's buffer (pH 1.2) consisting of 0.14M NaCl, 5mM
KCL, 1.5mM KH2PO4, 15mM NaHC03, and 0.7mM Na2HP04-7H20.
Fibroblasts were harvested by a five minute trypsinization
procedure utilizing a solution of 0.016% trypsin (Diffco,
Detroit, MI.) and 0.09% EDTA in Hank's buffer.
Trypsinization was then terminated by the addition of
maintenance medium to the T-flasks. Cells were seeded at a
concentration of 1x10+5 cells/ml and maintained at a
temperature of 37*5 °C with 95% O2 and 5% CO2. Cells from
the 10th to 15th passages were used for this study.
Isolation of Conditioned Medium - Fibroblasts were
harvested as described, adjusted to a concentration of 3*0 x
10+6 cells/ml in Medium 199 with 8% FCS and grown to
confluency. Attempts at growing cells to confluency
utilizing a modified defined medium (see below) revealed
poor cell viability. The maintenance medium was decanted
and the cells were washed three times to remove any trace of
FCS. Cells were then incubated with a modified defined
medium consisting of Medium 199 supplemented with thyroxine
30
(10 mg/ral), insulin (5 ug/ml), transferrin (5ug/ml),
selenium (5 ug/ml) [Collaborative Res. Inc., Lexington, MA],
and 1% penicillin/streptomycin. After 3 days, the
conditioned medium was removed and centrifuged at 4,000g for
10 min. at 40c. The resulting supernatant was removed and
stored at -40OC for subsequent assays.
Partial Characterization of the Conditioned Medium. -
Conditioned media was subjected to sodium dodecyl sulfate
(SDS) 7.5, 10, and 12% polyacrylamide gel electrophoresis as
described by Laemmli (1970). A one hundred microliter
quantity of sample was suspended in a 100 ul cocktail
containing a 1.4% Tris stacking buffer with 2% SDS, 10%
glycerol and 5% bromophenol blue. Samples were then loaded
into respective gels, and electrophoresed at 12.5 mA per gel
at a constant current. Protein bands were visualized by
staining with either a Coomassie Blue or Silver Nitrate
stain. For Coomassie Blue staining, gels were fixed in 50%
methanol and 7*5% acetic acid for 4 to 6 hours. Gels were
then stained with Coomaasie Blue (0.25%) for 15 min.
Destaining consisted of repeated immersions of the gels in
15% methanol and 7.5% acetic acid. Silver Nitrate staining
of gels consisted of a 45 min. fix in 6% glutaraldehyde.
Gels were then stained with 0.9% AgNO^, and developed in a
solution containing 0.005% citric acid and 0.02%
formaldehyde. Proteins contained in the conditioned media
were compared with high molecular weight standards (BioRad,
Richmond, CA), 0.250 ug/ml Type I Collagen (Flow Labs,
McLean, VA), fibronectin (Sigma, St. Louis, MO,
31
Collaborative Res. Inc., Lexington, MA), and defined medium.
A LKB ultrascan XL laser densitometer was utilized for gel
scans.
Chemotactic Assays - Polycarbonate 15mm filters
(Nucleopore Corp., Pleasanton, CA) were placed in chemotaxis
filter baskets (East Carolina University Physics Shop,
Greenville, NC) and washed with 0.5% acetic acid for 20 min.
at 60 oc. Filters were then rinsed with distilled H20 and
immersed in a solution containing 0.0025% gelatin (Sigma,
St. Louis, MO) for 60 min at 100 oc. Filters were dried at
37.5 °C overnight. Two hundred microliters of conditioned
medium was added to the bottom well of modified Boyden
chambers. Gelatin coated filters were placed on each well
containing the test solution. Initial cell harvesting
procedures were preformed as described by Gleiber (1985).
In initial indomethacin studies, T-flasks containing
confluent fibroblasts were washed twice with Hank's buffer
and incubated with 0.025% trypsin/EDTA for 10 min. at 37*5
°C. Modification of this procedure for dexamethasone
studies involved washing the cells twice for 1 min. in
Hank's buffer. Cells were then washed with 0.025%
trypsin/EDTA for 10 seconds. The supernatant was discarded
and the cells were allowed to incubate in the trypsin
backwash for 15 min. at 37.5 oC. In all experiments. Medium
199 containing 8% FCS was added to the T-flasks in order to
inactivate the trypsin. Cells treated by the backwash
method displayed a two fold increase in cell response to all
treatments. Therefore, later studies utilized this method
32
for cell isolation. After inactivation by trypsin, the cell
suspension was centrifuged at 150g for three min. The
supernatant was removed and cells were resuspended in Medium
199 containing 2.5 ug/ml bovine serum albumin (BSA). Cells
were then adjusted to a concentration of 3.5 x 10+5
cells/ml. A 500 microliter aliquot of the cell suspension
was added to the upper half of the Boyden chamber. Thus,
the filter separated the cell suspension which was in the
top from attractant which was in the bottom half of the
chamber. Chambers were incubated for 4 hours at 37.5 °C in
an atmosphere of 5% CO2 and 95% O2. After 4 hours of
incubation, the filters were removed, fixed in 100%
isopropyl alcohol, and stained with a May Greenwald/Giemsa
stain. The percent cell migration through the filter
depended on the number of nuclei on the bottom of the filter
which was in contact with the chemoattractant ( see Appendix
I). Nuclei in an area of Imm^ (1/12th of the total area of
migration) were microscopically counted to quantitate
results. Experiments were run in duplicate and data was
subjected to a Student's t-test, analysis of variance, and
Schiffe's test. Significance was determined at a confidence
level of 0.05.
A preliminary study was conducted in order to determine
the chemotactic activity of conditioned medium . The cell
suspensions were obtained as described and added to the
upper half of Boyden chambers. The lower wells of the
chambers contained either: Medium 199, Medium 199
supplemented with 8% PCS, Modified Medium 199» or
33
conditioned medium.
Subsequent inhibition studies utilized conditioned
medium (CM) as a chemoattractant. Indomethacin (Sigma,
St.Louis, MO) and dexamethasone (Sigma, St. Louis, MO) were
solubilized at 1 x 10-2m in 100% ETOH. These stock
solutions were diluted to concentrations ranging from 1 x
10-^ to 1 X 10-10m in Medium 199. Fibroblast suspensions
were exposed to concentrations of inhibitors ranging from 1
X 10-4 to 1 X 10-IOm for 25 min. at 37*5 °C. Positive
controls contained conditioned medium and no inhibitors,
while negative controls contained no inhibitors with Media
199 in the lower well. Indomethacin (10-4m) and
dexamethasone (lO-l^M) were also examined for chemotactic
ability. Inhibitors were adjusted to their respective
concentrations in Medium 199 and added to the lower half of
Boyden chambers. Similar experiments involved adding cells
exposed to inhibitors to the upper half of the Boyden
chambers with lower chambers preloaded with Medium 199.
Experiments were then performed as described.
34
RESULTS
Conditioned Medium - Medium 199 elicited no fibroblast
chemotaxis (Graph 1). Supplementing this medium (modified
media, MM) with thyroxine, insulin, selenium and transferrin
resulted in the appearance of chemotactic activity (6.3 2.6
cells/mm2). Addition of 8% FCS to Medium 199 also resulted
in enhanced chemotaxis (12.25 1 cells/mm2). The highest
chemotactic activity resided in the MM which had been
exposed to fibroblasts for three days (19.5 2.5 cells/ml).
This CM was then utilized for all subsequent assays.
A 7.5% polyacrylamide gel electrophoresis of both MM
and CM revealed a 73 Kd protein (Fig. 5), with CM containing
an additional 60 Kd protein. Type I collagen revealed a
210, 130, and 120 Kd protein corresponding to the one alpha
2 and two alpha 1 polypeptide chains comprising this
glycoprotein. Fibronectin obtained from Sigma revealed two
faint 230 and 220 Kd proteins corresponding to the alpha and
beta chains of fibronectin. Fibronectin from Sigma also
contained a 175, 150, 137 and 60 Kd protein. Identical
proteins with weights of 230, 220, 175, and 150kd were
obtained with fibronectin from Collaborative Research.
Samples were then subjected to electrophoresis utilizing
higher concentrations of acrylamide, in order to determine
lower molecular weight proteins. A 10% (Fig. 6) and 12%
(Fig. 7) acrylamide gel revealed the appearance of an
additional 70 Kd and 28 Kd protein in the CM. A silver
stain of the 10% acrylamide gel (Fig. 8) was subsequently
employed to enhance faint staining bands in both fibronectin
35
P
Graph 1.- Total number of fibroblasts migrating per Imm*^
in response to various types of media. Cells
were prepared as described in materials and
methods.
Fibroblast Chemotaxis Toward Conditioned and Defined Media
22
20 I n = 2
18
16
14
12
10
8
I
6
4
2
0 CHo
Conditioned Modified Media 199 Media 199
Media Media 199 8% FCS No FCS
37
1 2 3 4 5 6 7
210 Kd
_ ^
175
150
120
73
60
Fig. 5 7.5% SDS electrophoresis: Lane 1, Collagen: Lane 2 and
3, MM: Lane 4 and 5, CM: Lane 6 and 7, Fibronectin.
38
Fig, 6 10% SOS electrophoresis: Lane 1, Fibronectin: Lane 2,
Collagen: Lane 3, CM: Lane 4, MM.
39
Absorbance
Fig. 7 A Densitometer scan of a ^2% gel containing CM.
40
1 2 3 4
Fig. 8 10% SOS electrophoresis utilizing either a Coomassie
Blue or AgN03 stain: Lane 1, Coomassie Blue stain of
Fibronectin; Lane 2, AgN03 stain of Fibronectin; Lane
3, Coomassie stain of CM; Lane 4, AgN03 stain of CM.
Mwrtáicf
41
Fig. 9 A Densitometer scan of a 10% SDS silver nitrate stained gel
containing a.) CM and b.) Fibronectin
42
and CM. Thi s method rev ea 1 ed an addi ti ona 1 137 and 90 Kd
proteins in CM, and 90, 80 and 60 Kd proteins in
fibronectin.
Inhibition Studies - Fibroblasts treated with Ix10-4M
indomethacin revealed a 91Í increase in migration over
controls (Graph 2). There was also a 79% increase in
chemotaxis in cells treated with 1 x 10-6m indomethacin.
This effect diminished with decreasing concentrations of
indomethacin. A Student's t-test (a =0.05) revealed no
significant difference between indomethacin treated cells at
1 X 10-8m to 1 X IO-TOm and positive controls containing
conditioned medium. Indomethacin placed in the bottom of
Boyden chambers failed to stimulate cell migration.
Indomethacin placed in the upper halves of Boyden chambers
failed to stimulate cell migration in the absence of
conditioned medium.
Dexamethasone at a concentration of 1 x 10-4m inhibited
fibroblast chemotaxis by 14% (Graph 3). Cell migration in
response to 1 x 10-6m was not statistically different from
positive controls. This indicated no cellular inhibition or
enhancement. Enhancement of fibroblast chemotaxis by dexa-
methasone was observed at 1 x 10-8m (29%), and
1 X 10“'*0m (33%)* Boyden chambers were also prepared
contain-ing Medium 199 in the lower chamber. Upper chambers
containing cells treated with 1 x 10-10m dexamethasone
failed to stimulate chemotaxis in the absence of conditioned
medium. Untreated cells also failed to migrate to dexa-
methasone, thus ruling out any chemoattraction by inhibitors.
43
Graph 2 - Fibroblasts were obtained as described in materials and
methods and incubated with various concentrations of
indomethacin. Cells were then assayed for their ability
to migrate towards conditioned medium.
44
The Effect of Indomethacin on Fibroblast Chemotaxis
Indomethacin [M]
45
Graph 3 - Fibroblasts were obtained as described and subjected to
various concentrations of dexamethasone. Chemotaxis
assays then determined the effect of dexamethasone on
fibroblast chemotaxis towards conditioned medium.
46
The Effect of Dexamethasone on Fibroblast Chemotaxis
Cells
per
1mm
Dexamethasone [M]
47
DISCUSSION
An initial aspect of this study was to determine and
obtain in high yield an active chemoattractant for
fibroblasts. Work prior to this study revealed that
fibroblasts migrated to maintenance medium which had been
exposed to confluent layers of fibroblasts. This medium
contained 8% FCS, thus implicating the role of growth
factors or hormones associated with FCS in stimulating
chemotaxis. With this in mind the possibility of other
modified media in stimulating chemotaxis was investigated.
A modified medium supplemented with thyroxine, insulin,
transferrin and selenium was incubated for three days with
confluent layers of fibroblasts, and elicited the highest
chemotactic activity. This medium was then utilized for all
subsequent assays. This supports work by Mensing (1983),
who found that medium conditioned by human fibroblasts
elicited chemotactic activity. Thus, the release of
chemotactic factors into their medium may serve to regulate
such cell specific activities as adherence, migration,
replication, and maintenance of the extracellular matrix.
Many of the chemotactic factors contained in conditioned
medium include: fibronectin (Albini, 1983), collagen
(Postl ethwai te si âJ.., 1979), and/or their fragments as well
as a nonalbumin 60 Kd protein yet to be identified (Mensing
S.Í ál., 1 983)
Electrophoresis of CM revealed many proteins which were
not present in MM. This lead to the conclusion that the
chemotactic activity of CM resided in the presence of these
48
proteins. A large majority of the chemotactic activity of
CM is due to the secretion of fibronectin, or its fragments,
by fibroblasts. Therefore, comparisons were made between co-
migrating proteins in samples of CM and fibronectin. The
cell attachment segment of fibronectin is located within the
central region of both the alpha and beta chains of
fibronectin (Sekiguchi and Hakomori, 1 983; Ingham ^ al.,
1984; Hiyashi and Yamada, 1984; Sekiguchi ^ 5l., 1 985).
The chemotactic activity associated with this region is due
to a chemotactic sequence localized within the COOH terminal
end of the cell attachment region (Albini ^ âl., 1 983)»
Fragmentation of the fibronectin chains has determined that
this chemotactic region is located 90 Kd from the COOH
terminal end of fibronectin. A trypsin (Hiyashi and Yamada,
1 983; Sekiguchi âJ.., 1983), elastase (Ingham ^ 3^1-,
1984) and cathepsin D (Albini âl., 1983) digest of the
fibronectin molecule releases the cell attachment region as
a 140-155 Kd polypeptide fragment. A subsequent plasmin
digest of this fragment releases a 90 Kd polypeptide which
retains chemotactic activity (Albini ¿i âl., 1983)- A SDS
electrophoresis of CM and fibronectin revealed the presence
of both the 150 and 90 Kd polypeptides. Both samples also
contained a 60 Kd protein which may represent the terminal
heparin/fibrin binding regions of fibronectin (Hiyashi and
Yamada, 1983)* A 28 Kd protein was also contained within CM
and may actually be a fibronectin fragment with heparin
binding ability. This is based upon the generation of a
heparin binding 30 Kd peptide by thermolysin fragmentation
49
of the cell attachment region. Therefore, the 150 Kd band
within CM may represent the cell attachment region of
fibronectin, with the 90 Kd band representing a subfragment
of the cell attachment region. The chemotactic activity
within CM may reside in the presence of chemotactic amino
acid sequences within fibronectin and its fragments.
Elimination of fibronectin from conditioned medium results
in a 50-60% reduction in its chemotactic activity (Mensing
âJ.., 1 983). Remaining activity is accounted for by the
presence of a protein in the size range of albumin (Mensing
et 1983). Therefore, the 60 Kd protein observed in the
CM may be either a 60 Kd fibronectin fragment containing the
chemotactic sequence or an unidentified chemotactic protein.
Further work involving antibodies raised against fibronectin
fragments should resolve this discrepancy.
Damage to tissue results in the release of a wide
variety of inflammatory mediators. These include AA
metabolites, as well as C5a, PDGF, lymphokines, collagen,and
tropoelastin/el astin with their fragments. These compounds
serve as chemoattractants in eliciting cellular influx into
the damaged tissue sites. Of primary importance in the
acute inflammatory response is the metabolism of AA, which
generates a wide variety of compounds that mediate the
inflammatory response. Of importance are the LTs and Hetes,
which elicit cellular influx into the wound site.
Inflammatory cells migrating to the wound site also release
chemoattractants, thereby enhancing the signal for cellular
influx. Thus, chemoattractants serve as cell to cell
50
signals in eliciting cell migration, and ultimate tissue
reconstruction, in response to tissue damage. Arachidonic
acid metabolism not only generates chemoattractants but
serves as the principal biochemical mechanism in chemotaxis.
The Ca+2 influx associated with this pathway may activate a
wide variety of kinases, or serve to initiate the
polymerization of microtubules and microfilaments.
Therefore this study attempted to determine how inhibition
of specific branchpoints in AA metabolism would affect the
chemotactic response of fibroblasts.
Indomethacin, a nonsteroidal anti-inflammatory agent
and dexamethasone, a steroidal glucocorticoid, were utilized
as inhibitors of AA metabolism. Indomethacin (10-6m) is a
irreversible competitive inhibitor of cyclo-oxygenase, thus
inhibiting the formation of PGs, PGI, and TxA2 from AA. A
reduction in prostaglandin levels would result in a decrease
in the redness, swelling and pain at the wound site. The
potency of this drug in inhibiting cyclo-oxygenase resides
in the presence of the N-benzyl and -methyl acetic acid
side chains which are attached to the indole ring of
indomethacin (Lombardino, 1 985). Inhibition of PG synthase
by high concentrations of indomethacin (10-4_io-8m) resulted
in an enhanced chemotactic response by the fibroblasts
(Table 2). This response decreased with lower
concentrations of indomethacin, and cells treated with
10-10m indomethacin were not significantly different from
controls. A chemotactic enhancement due to inhibition of
prostaglandin production has been reported previously in
51
Table 2 - A Summary of the effects of Indomethacin and Dexamethasone
on Fibroblast Chemotaxis.
Fibroblast Chemotaxís in Response to Anti-inflammatory Agents
indomethacin (n = 7) Dexamethasone (n = 6)
Concentration Cell % (-) Inhibition Cells % (-) Inhibition^
per per
1mm (-) Enhancement 1mm (+) Enhancement
10""^ 21.79Î1.07 +91 % 21.42 iO.76 -14%
10'® 20.43 Í 1.07 +79% 24.92 iO.84 -0.3%
10'^ 14.57Î0.64 +27%
10-® 13.86^0.77 +21% 32.33 Í0.82 +29%
10"^° 11.64Î0.78 + 1.8% 33.17Í 1.12 +33%
53
neutrophils (Higgs éí âl., 1980; Goetzl, 1980; Malmsten ^
1980; Palmblad âl.> 1980). This chemotactic
enhancement may be the result of a substrate diversion by
AA. This involves the inhibition of PG synthase, resulting
in an excess concentration of free AA. This free AA may
then be acted upon by 5-1ipoxygenase, thereby, increasing
the production of LTB^ (Salmon âJ.., 1 983). Leukotriene B4
is a potent chemoattractant for fibroblasts and its
production could result in an enhancement of chemotaxis.
Enhancement of chemotaxis was also observed with cells
treated with 10-8-10-IOm dexamethasone (Table 2). This may
be due to the ability of low concentrations of
glucocorticoids to stimulate the production of fibronectin
(Marceau ^ âJ..| 1 980, Oliver âJL.» 1 983; Lien ^i..,
1984). Oliver and co-workers have proposed that two
mechanisms govern fibronectin synthesis. One is responsible
for basal rates of fibronectin synthesis, which is
glucocorticoid independent; and the other is responsible for
induced rates of fibronectin synthesis, which is regulated
by glucocorticoid receptors. Dexamethasone treatment of
fibroblasts results in a two fold increase in the rate of
fibronectin biosynthesis in normal cells, and a ten fold
increase in fibrosarcoma cells (Oliver ^ âl-, 1983)-
Treatment of fibroblasts with hydrocortisone results in a
13-19% increase in fibronectin levels, with a more dramatic
effect in the accumulation of fibronectin in the medium
(Lien £_fc âl., 1 984). Thus, the enhancement observed with
10*8 to 10-10m dexamethasone treated cells may be due to an
54
enhanced production or presence of fibronectin within the
upper well of the Boyden chamber. This could then result in
an up-regulation of fibronectin receptors. Detection of a
higher chemotactic gradient towards CM would then result in
chemotaxis. An inhibition of chemotaxis was observed with
10-4m dexamethasone treated cells. This may be attributed
to the induction of a chemotactic regulatory protein known
as lipocortin (Blackwell jgi âl., 1 980; Hirata ^ âl-, 1 980).
Lipocortin is found within a wide variety of cells and
phosphorylation of this protein results in the loss in its
ability to inhibit PA2 and chemotaxis (Hirata âl.> 1 980).
Chemoattractant binding results in a Ca+2 influx which
activates PA2 and either a Ca’’’^ dependant kinase or o-AMP
dependant kinase. These kinases would then phosphory1 ate
lipocortin and render it inactive. Treatment of cells with
glucocorticoids results in a rapid release of existing pools
of lipocortin from cells, followed by a slower release of
inhibitor due to subsequent protein synthesis and release
(Walker et al.,1986). Following a two hour treatment with
glucocorticoids, there is a six fold increase in lipocortin-
specific mRNA’s (Walker ^ âJL., 1 986). Therefore, the
induction of lipocortin by 10“4m dexamethasone results in
the inhibition of PA2 which prevents AA metabolism and
fibroblast chemotaxis.
Glucocorticoids have been classically described as
having the ability to enhance gluconeogenesis, inhibit
glucose and amino acid uptake, and catecholamine 0-methyl
transferase. They are further implicated in the stress
55
response involving a synergistic enhancement and
prolongation of hyperglycemia, initially induced by
epinephrine. Current evidence points to a more direct role
for glucocorticoids, i.e., terminators of the anti-
inflammatory response. This is due to their ability to
inhibit AA metabolism, chemotaxis, lymphokine and interferon
production, and Fc expression (Munck âi., 1984). Thus,
the release of glucocorticoids following an inflammatory
response may serve as a terminating signal in this response,
thereby protecting the host from excessive cellular influx,
hydrolytic enzyme release and prolonged immunological
stimuli. Decreases in circulating glucocorticoid levels
would then result in a lower concentration of
glucocorticoids in peripheral tissues. These lower levels
of glucocorticoids would then induce fibronectin synthesis
in preparation of connective tissue repair. Therefore,
glucocorticoids serve as a signal to end the acute
inflammatory response and initiate a reparative phase
whereby, fibroblasts reconstruct the connective tissue
matrix.
Dexamethasone and indomethacin are termed anti-
inflammatory agents due to their ability to inhibit
prostaglandin production. However both compounds under
certain conditions may stimulate chemotaxis. Excessive or
prolonged use may alleviate pain and swelling, but may also
enhance cellular influx into the wound site. This
chemotactic enhancement would result in excessive cell
proliferation, enzyme release, and generation of radicals,
56
and tissue destruction. Subsequent research should involve
the utilization of dual inhibitors that selectively prevent
both PG and LT production. Prostaglandin synthase requires
a hydroperoxide activator and a heme group for the catalysis
of AA to PGH2 (Kulmacz, 1986). Thus, the synthesis and
availability of inhibitors of peroxide incorporation or
peroxide scavengers may result in the availability of a
nontoxic class of inhibitors of both chemotaxis and AA
metabolism. Other inhibitors may involve radical scavengers,
or substrate analogues such as the omega 3 fatty acids.
Research is also required in the determination of the
effects of anti-inflammatory agents on cell proliferation,
collagen synthesis, and col 1agenase/elastase activity.
57
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63
APPENDIX I
Morphological Studies of Fibroblast Chemotaxis
64
Light micrographs of both activated and quiescent fibroblasts
on a nucleopore filter; A) lOOX, B) 400X,
65
Thru focus series of fibroblast migration in response to a
chemoattractant; (C) top of filter, (D) middle of filter,
(E) bottom of filter, (p) pore. 1,000X.
66
ê
67
Scanning electron micrograph of; (F) a fibroblast responding
to a chemoattractant located beneath the filter, note the
filopodia which serve in chemoreception and motility, and
(G) a fibroblast oriented on top of the pore in response
to a chemoattractant. 5,000X.
68
Posterior view of fibroblast within the pore. 10,000X.
0>
(O