Gen. Pharmac. Vol. 23, No. 3, pp. 571-574, 1992 Printed in Great Britain. All rights reserved
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
THE EFFECTS OF INDOMETHACIN ON FIBROBLAST CHEMOTAXIS GERHARD W. KALMUS, JOHN E. POULOS and CHARLESW. O'REAR Department of Biology, East Carolina University, Greenville, NC 27858-4353, U.S.A. (Received 13 M a y 199l) Abstract--1. Boyden chambers were used to investigate the effects of indomethacin on fibroblast chemotaxis to a conditioned medium. 2. It was determined that indomethacin did not inhibit, but enhanced fibroblast chemotaxis at a concentration of 10 -4 (91%)-10 6 M (79%). 3. No significant difference was found between controls and cells treated with 10-8-10-1°M indomethacin.
INTRODUCTION Chemotaxis is an i m p o r t a n t biological p h e n o m e n o n involving the directed migration o f cells along a chemical gradient. This p h e n o m e n o n is displayed by a wide variety o f cells and constitutes a major comp o n e n t o f b o t h the inflammatory response and tissue repair. The utilization o f modified Boyden chambers provides a quantitative in vitro assay for the determination o f fibroblast c h e m o a t t r a c t a n t s (Postlethwaite et al., 1976). Established fibroblast chemoattractants include conditioned medium (Mensing et al., 1983), and such connective tissue proteins as collagen and collagen derived peptides (Postlethwaite, 1978), tropoelastin and elastin filaments (Senior et al., 1982), and fibronectin (Gauss-Mueller et al., 1980). Other c h e m o a t t r a c t a n t s generated by the inflammatory response include lymphokines (Postlethwaite et al., 1976), and a derivative o f C5a (Postlethwaite et al., 1979). Fibroblast chemotaxis to the w o u n d site is followed by the synthesis o f fibronectin and collagen in an attempt to reconstruct the connective tissue matrix. Both the inflammatory response and chemotaxis involves the metabolism o f arachidonic acid into prostaglandins and leukotrienes (Schiffman et al., 1983; Mensing and Czarnetzki, 1984). Alterations in the metabolism o f arachidonic acid may affect fibroblast chemotaxis to the w o u n d site and thereby influence the reparative phase o f the host response. The purpose o f this study was to determine the effect o f an inhibitor o f arachidonic acid metabolism on fibroblast chemotaxis. MATERIALS AND METHODS Human fetal foreskin fibroblasts were grown to confluency in 25 cm2 tissue culture flasks containing Medium 199 supplemented with 8% fetal calf serum (FCS) and 1% penicillin/streptomycin at a temperature of 37.5°C in an atmosphere of 5% CO 2 and air. Cells for chemotaxis experiments were obtained between the 10th and 15th passages. Production of a conditioned medium as described by Mensing et al. (1983) involved removal of the maintainance medium followed by two washes with Hank's buffer (pH 7.4) in order to remove any trace of FCS. Cells 571
were then incubated with modified defined medium (MM) consisting of Medium 199 supplemented with thyroxin (10mg/ml), insulin (5#g/ml), transferrin (5~g/ml), selenium (95 pg/ml) and 1% penicillin/streptomycin. After 3 days, this medium, now termed conditioned medium (CM), was removed and centrifuged at 4000g for 10 min at 4°C. The resulting supernatant was stored at -40°C for use in subsequent assays. For chemotaxis assays, 200pl of CM was added to the bottom well of Boyden chambers and polycarbonate filters (8#m pore) treated with gelatin (5/zg/ml) as described by Postlethwaite et al. (1976) were placed on each well containing the CM. Fibroblasts were harvested by washing confluent T-flasks twice with Hank's buffer, followed by incubation with 1 x 10 -4 M trypsin/EDTA for 10 sec. The supernatant was discarded and the cells were allowed to incubate for 15 rain at 37°C. Detached cells were triturated and suspended in Medium 199 containing 8% FCS in order to inactivate the trypsin, centrifuged at 150 g for 3 min and resuspended in Medium 199 containing 2.5 #g/ml bovine serum albumin. Cells were then adjusted to a concentration of 3.5 × 105cells/ml. A 500/~1 aliquot of 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 CM which was in the bottom half of the chamber. Chambers were incubated for 4 hr at 37.5°C in an atmosphere of 5% CO: and air. After 4 hr of incubation, filters were fixed with 100% isopropyl alcohol, and stained with May Grunwald/Giemsa stain. The percentage cell migration through the filter depended on the number of nuclei on the bottom of the filter which was in contact with the chemoattractant. Nuclei in an area of 1 mm 2 (1/12 th of the total area of migration) were counted microscopically to quantitate results. Indomethacin was solubilized at l0 2 M in absolute alcohol. The stock solution was diluted to concentrations ranging from l0 -4 10-1°M in Medium 199. Fibroblast suspensions were exposed to concentrations ranging from 10-4-10-2M for 25 min at 37.5°C. Positive controls contained CM and no inhibitors, while negative controls contained no inhibitors with Medium 199 in the lower well. Concentrations of indomethacin utilized in this study were also examined for chemotactic activity by placing indomethacin suspended in Medium 199 in the lower half of Boyden chambers in the absence of CM. Similar experiments involved adding cells exposed to inhibitors in the upper half of Boyden chambers with lower chambers preloaded with Medium 199. Experiments were performed as described and run in duplicate. Data was subjected to a
572
GERHARD W. KALMUS et al.
Student's t-test, ANOVA, and Schiff6's test. Significance was determined at a confidence level of 0.05. Conditioned medium (CM) was also subjected to 7.5% and 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as described by Laemmli (1970). Protein bands were visualized by staining with either a Coomassie Blue or silver nitrate stain. Proteins contained in the CM were compared with high and low molecular weight standards, 0.25 #g/ml type I collagen, fibronectin, and modified medium (MM). A LKB ultrascan XL laser densitometer was utilized for gel scans.
1
2
5
4
5
6
7
KO 21o 175 15o
Izo
RESULTS Indomethacin at a concentration of 1 0 - 4 M produced a 91% increase (P < 0.05) (21.79 4_- 1.07 SEM cells/mm 2) in fibroblast chemotaxis over controls (10.0+ 1). At a concentration of 10-6M, indomethacin produced a 79% increase (20.043 ___ 1.07) over controls. This effect diminished with decreasing concentrations of indomethacin. A Student's t-test (P > 0.05) revealed no significant difference between indomethacin treated cells at 10-SM (13.86 + 0.77) to 10 t°M (11.64 _+ 0.78) and positive controls containing CM. Indomethacin placed in the upper half of Boyden chambers failed to stimulate cell migration in the absence of CM. Placement of indomethacin in the lower chamber also failed to elicit chemotaxis, thus ruling out any chemoattraction by this inhibitor. A preliminary biochemical study involving characterization of the C M was also attempted. Utilizing polyacrylamide gel electrophoresis, C M which is a chemoattractant for fibroblasts, was compared to M M which elicited no chemotactic activity. A 7.5% polyacrylamide gel electrophoresis of both M M and C M revealed a 73 kDa protein (Fig. 2) with CM containing an additional 60 kDa protein. Type I collagen revealed a 210, 130, and 120 kDa protein
T 20
73 60
Fig. 2. 7.5% SDS-polyacrylamide gel electrophoresis: Lane l--collagen; Lanes 2 and 3--modified medium; Lanes 4 and 5--conditioned medium; Lanes 6 and 7--fibronectin. Bands were visualized with Coomassie Blue.
Absorbance
60 Kd
a
73 Kd ~ Kd
~
90 0Kd
IT
15
10 20 3 0 40 50 68 70 80 Y-position (ram) ......... * ...........
Cells per mm 2
~-/
T
T
90 100 110 120 130 140 TSo 160 170
T
10 60 Kd
90
i 1o 4
i lO -6'
f lO 7
J lO.8
110-tO
f 0
Indomethacin [ M ]
Fig. 1. The effect of indomethacin on fibroblast chemotaxis. Cells were obtained as described and incubated with various concentrations of indomethacin. Cells were assayed for their ability to migrate towards conditioned medium. 0 indicates positive control with no inhibitors. Vertical bar indicates SEM.
r
i
10 20 30 40 50
I
60 ¢0 80
,
i
,
i
,
i
,
I
90 tOO 110 120 130 140 150 11B0 1}'0
Fig. 3. Densitometer scan of a silver nitrate stained 10% SDS-polyacrylamide gel comparing (a) conditioned medium and (b) fibronectin.
Effects of indomethacin on fibroblast chemotaxis corresponding to the one ~2- and two ~ - polypeptide chains comprising this glycoprotein. Fibronectin also contained a 175, 150, 137 and 60kDa protein. Samples of CM were then subjected to electrophoresis utilizing higher concentrations of acrylamide, in order to resolve lower molecular weight proteins. A silver stain of the 10% gel was subsequently employed to enhance faint staining bands in both fibronectin and CM. Densiphotometric analysis of this gel revealed co-migrating proteins of 70, 73, 90 and 150 kDa (Fig. 3). DISCUSSION This study attempted to determine how an inhibitor of a specifc branchpoint in arachidonic acid metabolism would affect the chemotactic response of fibroblasts. Indomethacin, a nonsteroidal antiinflammatory agent, was utilized as a representative inhibitor of arachidonic acid metabolism. Indomethacin is a competitive inhibitor of prostaglandin synthase, thus inhibiting the formation of prostaglandins, prostacyclin, and thromboxane A2. This nonsteroidal anti-inflammatory agent is employed during the acute inflammatory response in an attempt to limit the pain and swelling resulting from prostaglandin production. Earlier studies involving the treatment of leukocytes with indomethacin revealed a decrease in both prostaglandin levels and leukocyte accumulations (Higgs et al., 1980; Salmon et al., 1983). Our study is the first to show that enhancement of fibroblast migration may occur with treatment of indomethacin. The possibility exists of a mechanism whereby inhibition of prostaglandin production in fibroblasts leads to a substrate diversion of arachidonic acid into the 5-1ipoxygenase pathway to produce leukotriene B4, a potent chemoattractant for fibroblasts (Mensing and Czarnetzki, 1984). The drug concentrations utilized in these experiments may not reflect physiological concentrations, however they approximate therapeutic concentrations employed to limit acute inflammation at intraocular or intra-articular sites. Further studies utilizing RIAs should attempt to quantitate changes in levels of leukotriene U 4 and/or arachidonic acid metabolites in fibroblasts during utilization of anti-inflammatory agents. This should resolve either similarities or differences between agents modulating arachidonic acid metabolism in both fibroblasts and leukocytes. Our experiments utilized a medium conditioned by human fibroblasts for chemotactic assays. The potency of CM in serving as a chemoattractant has been previously reported (Mensing et al., 1983), and may be attributed to the presence of fibronectin (Albini et al., 1983), and to a lesser degree to collagen (Postlethwaite, 1978) and/or their fragments. Electrophoresis of CM revealed many proteins which were not present in MM. This led to the conclusion that the chemotactic activity may reside in the presence of these proteins. Comparison of CM with fibronectin revealed co-migrating proteins with molecular weights of 180, 90, 73, 70, and 60 kDa. These protein bands correlated well with fragments obtained in studies characterizing the cell binding and ligand binding domains of fibronectin (Albini et al., 1983;
573
Hiyashi and Yamada, 1983; Sekinguichi et al., 1985). The CM utilized in our study may contain either intact or proteolytic fragments of collagen, elastin, fibronectin or other growth factors that elicit fibroblast chemotaxis. Although the biochemical characterization of CM is far from complete, further studies will attempt to isolate and characterize the chemoattractants present within the media. This should involve monoclonal antibodies directed against either fibronectin, collagen, or other isolated proteins, in order to determine if the removal of these proteins by immunoprecipitation or addition of these antibodies to the CM removes the chemotactic activity. The emphasis of this paper was not in characterization of the CM but in the ability of indomethacin to modulate fibroblast chemotaxis. The ability of indomethacin in inhibiting prostaglandin production and the influx of inflammatory cells during acute inflammation does not necessarily indicate that this agent could be utilized as an effective inhibitor of fibroblast stimulation and migration. Conditions may exist whereby nonsteroidal anti-inflammatory agents enhance fibroblast influx into the wound site, resulting in such pathological conditions as the formation of keloids, joint contractures, proliferative retinopathy and corneal deposits. Future characterization of the receptors involved or mechanisms unique to fibroblast chemotaxis may elucidate strategies in controlling fibroproliferative diseases. REFERENCES
Atbini A., Richter H. and Pontz B. F. (1983) Localization of the chemotactic domain in fibronectin. FEBS 156, 222-226. Gauss-Mueller V., Kleinman H. K., Martin G. R. and Schiffman E. (1980) Role of attachment factors and attractants in fibroblast chemotaxis. J. Lab. Clin. Med. 96, 1071 1080. Higgs G., Eakins K. E., Mugridge K. G., Moncada S. and Vane J. R. (1980) The effects of non-steroidal anti-flammatory drugs on leukocyte migration in carrageein induced inflammation. Eur. J. Pharm. 66, 81-86. Hiyashi M. and Yamada K. (1983) Domain structure of the carboxyl-terminal half of human plasma fibronectin. J. Biol. Chem. 258, 3332-3340. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680~585. Mensing H. and Czarnetzki B. M. (1984) Leucotriene B4 induces in vitro fibroblast chemotaxis. J. Ira,est. Derrn. 82, 9-12. Mensing H., Pontz B. F., Mueller P. K. and Gauss-Mueller V. (1983) A study of fibroblasts and conditioned medium as chemoattractants. Eur. J. Cell Biol. 29, 268-273. Postlethwaite A. E. (1978) Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen derived peptides. Proc. Natn. Acad. Sci., U.S.A. 75, 871-875. Postlethwaite A. E., Synderman R. and Kang A. H. (1976) The chemotactic attraction of human fibroblasts to a lymphocyte derived growth factor. J. Exp. Med. 144, 1188-1203.
Postlethwaite A. E., Synderman R. and Kang A. H. (1979) Generation of a fibroblast chemoattractant in serum by activation of complement. J. Clin. Invest. 64, 1379-1385. Salmon J., Simmons P. M. and Moncada S. (1983) The effects of BW 755C and other anti-inflammatory drugs on eicosanoid concentrations and leukocyte accumulations
574
GERHARD W. KALMUSel al.
in experimentally induced acute inflammation. J. Pharm. Pharmac. 35, 808-813. Schiffman E., Geetha V., Pencev D., Warabi H. and Mato J. (1983) Agents and Supplements, Vol. 12, pp. 106-120. Birkhaus, Basel. Sekinguichi K., Siri A., Zardi L. and Hakomori S. (1985) Differences in domain structure between human
fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts. J. Biol. Chem. 260, 5105-5114. Senior R. M., Griffin G. L. and Mecham R. (1982) Chemotactic response of fibroblasts to tropoelastin and elastin derived peptides. J. Clin. Invest. 70, 6144518.
0306-3623/92 $5.00 + 0.00 Copyright © 1992 Pergamon Press Ltd
THE EFFECTS OF INDOMETHACIN ON FIBROBLAST CHEMOTAXIS GERHARD W. KALMUS, JOHN E. POULOS and CHARLESW. O'REAR Department of Biology, East Carolina University, Greenville, NC 27858-4353, U.S.A. (Received 13 M a y 199l) Abstract--1. Boyden chambers were used to investigate the effects of indomethacin on fibroblast chemotaxis to a conditioned medium. 2. It was determined that indomethacin did not inhibit, but enhanced fibroblast chemotaxis at a concentration of 10 -4 (91%)-10 6 M (79%). 3. No significant difference was found between controls and cells treated with 10-8-10-1°M indomethacin.
INTRODUCTION Chemotaxis is an i m p o r t a n t biological p h e n o m e n o n involving the directed migration o f cells along a chemical gradient. This p h e n o m e n o n is displayed by a wide variety o f cells and constitutes a major comp o n e n t o f b o t h the inflammatory response and tissue repair. The utilization o f modified Boyden chambers provides a quantitative in vitro assay for the determination o f fibroblast c h e m o a t t r a c t a n t s (Postlethwaite et al., 1976). Established fibroblast chemoattractants include conditioned medium (Mensing et al., 1983), and such connective tissue proteins as collagen and collagen derived peptides (Postlethwaite, 1978), tropoelastin and elastin filaments (Senior et al., 1982), and fibronectin (Gauss-Mueller et al., 1980). Other c h e m o a t t r a c t a n t s generated by the inflammatory response include lymphokines (Postlethwaite et al., 1976), and a derivative o f C5a (Postlethwaite et al., 1979). Fibroblast chemotaxis to the w o u n d site is followed by the synthesis o f fibronectin and collagen in an attempt to reconstruct the connective tissue matrix. Both the inflammatory response and chemotaxis involves the metabolism o f arachidonic acid into prostaglandins and leukotrienes (Schiffman et al., 1983; Mensing and Czarnetzki, 1984). Alterations in the metabolism o f arachidonic acid may affect fibroblast chemotaxis to the w o u n d site and thereby influence the reparative phase o f the host response. The purpose o f this study was to determine the effect o f an inhibitor o f arachidonic acid metabolism on fibroblast chemotaxis. MATERIALS AND METHODS Human fetal foreskin fibroblasts were grown to confluency in 25 cm2 tissue culture flasks containing Medium 199 supplemented with 8% fetal calf serum (FCS) and 1% penicillin/streptomycin at a temperature of 37.5°C in an atmosphere of 5% CO 2 and air. Cells for chemotaxis experiments were obtained between the 10th and 15th passages. Production of a conditioned medium as described by Mensing et al. (1983) involved removal of the maintainance medium followed by two washes with Hank's buffer (pH 7.4) in order to remove any trace of FCS. Cells 571
were then incubated with modified defined medium (MM) consisting of Medium 199 supplemented with thyroxin (10mg/ml), insulin (5#g/ml), transferrin (5~g/ml), selenium (95 pg/ml) and 1% penicillin/streptomycin. After 3 days, this medium, now termed conditioned medium (CM), was removed and centrifuged at 4000g for 10 min at 4°C. The resulting supernatant was stored at -40°C for use in subsequent assays. For chemotaxis assays, 200pl of CM was added to the bottom well of Boyden chambers and polycarbonate filters (8#m pore) treated with gelatin (5/zg/ml) as described by Postlethwaite et al. (1976) were placed on each well containing the CM. Fibroblasts were harvested by washing confluent T-flasks twice with Hank's buffer, followed by incubation with 1 x 10 -4 M trypsin/EDTA for 10 sec. The supernatant was discarded and the cells were allowed to incubate for 15 rain at 37°C. Detached cells were triturated and suspended in Medium 199 containing 8% FCS in order to inactivate the trypsin, centrifuged at 150 g for 3 min and resuspended in Medium 199 containing 2.5 #g/ml bovine serum albumin. Cells were then adjusted to a concentration of 3.5 × 105cells/ml. A 500/~1 aliquot of 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 CM which was in the bottom half of the chamber. Chambers were incubated for 4 hr at 37.5°C in an atmosphere of 5% CO: and air. After 4 hr of incubation, filters were fixed with 100% isopropyl alcohol, and stained with May Grunwald/Giemsa stain. The percentage cell migration through the filter depended on the number of nuclei on the bottom of the filter which was in contact with the chemoattractant. Nuclei in an area of 1 mm 2 (1/12 th of the total area of migration) were counted microscopically to quantitate results. Indomethacin was solubilized at l0 2 M in absolute alcohol. The stock solution was diluted to concentrations ranging from l0 -4 10-1°M in Medium 199. Fibroblast suspensions were exposed to concentrations ranging from 10-4-10-2M for 25 min at 37.5°C. Positive controls contained CM and no inhibitors, while negative controls contained no inhibitors with Medium 199 in the lower well. Concentrations of indomethacin utilized in this study were also examined for chemotactic activity by placing indomethacin suspended in Medium 199 in the lower half of Boyden chambers in the absence of CM. Similar experiments involved adding cells exposed to inhibitors in the upper half of Boyden chambers with lower chambers preloaded with Medium 199. Experiments were performed as described and run in duplicate. Data was subjected to a
572
GERHARD W. KALMUS et al.
Student's t-test, ANOVA, and Schiff6's test. Significance was determined at a confidence level of 0.05. Conditioned medium (CM) was also subjected to 7.5% and 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as described by Laemmli (1970). Protein bands were visualized by staining with either a Coomassie Blue or silver nitrate stain. Proteins contained in the CM were compared with high and low molecular weight standards, 0.25 #g/ml type I collagen, fibronectin, and modified medium (MM). A LKB ultrascan XL laser densitometer was utilized for gel scans.
1
2
5
4
5
6
7
KO 21o 175 15o
Izo
RESULTS Indomethacin at a concentration of 1 0 - 4 M produced a 91% increase (P < 0.05) (21.79 4_- 1.07 SEM cells/mm 2) in fibroblast chemotaxis over controls (10.0+ 1). At a concentration of 10-6M, indomethacin produced a 79% increase (20.043 ___ 1.07) over controls. This effect diminished with decreasing concentrations of indomethacin. A Student's t-test (P > 0.05) revealed no significant difference between indomethacin treated cells at 10-SM (13.86 + 0.77) to 10 t°M (11.64 _+ 0.78) and positive controls containing CM. Indomethacin placed in the upper half of Boyden chambers failed to stimulate cell migration in the absence of CM. Placement of indomethacin in the lower chamber also failed to elicit chemotaxis, thus ruling out any chemoattraction by this inhibitor. A preliminary biochemical study involving characterization of the C M was also attempted. Utilizing polyacrylamide gel electrophoresis, C M which is a chemoattractant for fibroblasts, was compared to M M which elicited no chemotactic activity. A 7.5% polyacrylamide gel electrophoresis of both M M and C M revealed a 73 kDa protein (Fig. 2) with CM containing an additional 60 kDa protein. Type I collagen revealed a 210, 130, and 120 kDa protein
T 20
73 60
Fig. 2. 7.5% SDS-polyacrylamide gel electrophoresis: Lane l--collagen; Lanes 2 and 3--modified medium; Lanes 4 and 5--conditioned medium; Lanes 6 and 7--fibronectin. Bands were visualized with Coomassie Blue.
Absorbance
60 Kd
a
73 Kd ~ Kd
~
90 0Kd
IT
15
10 20 3 0 40 50 68 70 80 Y-position (ram) ......... * ...........
Cells per mm 2
~-/
T
T
90 100 110 120 130 140 TSo 160 170
T
10 60 Kd
90
i 1o 4
i lO -6'
f lO 7
J lO.8
110-tO
f 0
Indomethacin [ M ]
Fig. 1. The effect of indomethacin on fibroblast chemotaxis. Cells were obtained as described and incubated with various concentrations of indomethacin. Cells were assayed for their ability to migrate towards conditioned medium. 0 indicates positive control with no inhibitors. Vertical bar indicates SEM.
r
i
10 20 30 40 50
I
60 ¢0 80
,
i
,
i
,
i
,
I
90 tOO 110 120 130 140 150 11B0 1}'0
Fig. 3. Densitometer scan of a silver nitrate stained 10% SDS-polyacrylamide gel comparing (a) conditioned medium and (b) fibronectin.
Effects of indomethacin on fibroblast chemotaxis corresponding to the one ~2- and two ~ - polypeptide chains comprising this glycoprotein. Fibronectin also contained a 175, 150, 137 and 60kDa protein. Samples of CM were then subjected to electrophoresis utilizing higher concentrations of acrylamide, in order to resolve lower molecular weight proteins. A silver stain of the 10% gel was subsequently employed to enhance faint staining bands in both fibronectin and CM. Densiphotometric analysis of this gel revealed co-migrating proteins of 70, 73, 90 and 150 kDa (Fig. 3). DISCUSSION This study attempted to determine how an inhibitor of a specifc branchpoint in arachidonic acid metabolism would affect the chemotactic response of fibroblasts. Indomethacin, a nonsteroidal antiinflammatory agent, was utilized as a representative inhibitor of arachidonic acid metabolism. Indomethacin is a competitive inhibitor of prostaglandin synthase, thus inhibiting the formation of prostaglandins, prostacyclin, and thromboxane A2. This nonsteroidal anti-inflammatory agent is employed during the acute inflammatory response in an attempt to limit the pain and swelling resulting from prostaglandin production. Earlier studies involving the treatment of leukocytes with indomethacin revealed a decrease in both prostaglandin levels and leukocyte accumulations (Higgs et al., 1980; Salmon et al., 1983). Our study is the first to show that enhancement of fibroblast migration may occur with treatment of indomethacin. The possibility exists of a mechanism whereby inhibition of prostaglandin production in fibroblasts leads to a substrate diversion of arachidonic acid into the 5-1ipoxygenase pathway to produce leukotriene B4, a potent chemoattractant for fibroblasts (Mensing and Czarnetzki, 1984). The drug concentrations utilized in these experiments may not reflect physiological concentrations, however they approximate therapeutic concentrations employed to limit acute inflammation at intraocular or intra-articular sites. Further studies utilizing RIAs should attempt to quantitate changes in levels of leukotriene U 4 and/or arachidonic acid metabolites in fibroblasts during utilization of anti-inflammatory agents. This should resolve either similarities or differences between agents modulating arachidonic acid metabolism in both fibroblasts and leukocytes. Our experiments utilized a medium conditioned by human fibroblasts for chemotactic assays. The potency of CM in serving as a chemoattractant has been previously reported (Mensing et al., 1983), and may be attributed to the presence of fibronectin (Albini et al., 1983), and to a lesser degree to collagen (Postlethwaite, 1978) and/or their fragments. Electrophoresis of CM revealed many proteins which were not present in MM. This led to the conclusion that the chemotactic activity may reside in the presence of these proteins. Comparison of CM with fibronectin revealed co-migrating proteins with molecular weights of 180, 90, 73, 70, and 60 kDa. These protein bands correlated well with fragments obtained in studies characterizing the cell binding and ligand binding domains of fibronectin (Albini et al., 1983;
573
Hiyashi and Yamada, 1983; Sekinguichi et al., 1985). The CM utilized in our study may contain either intact or proteolytic fragments of collagen, elastin, fibronectin or other growth factors that elicit fibroblast chemotaxis. Although the biochemical characterization of CM is far from complete, further studies will attempt to isolate and characterize the chemoattractants present within the media. This should involve monoclonal antibodies directed against either fibronectin, collagen, or other isolated proteins, in order to determine if the removal of these proteins by immunoprecipitation or addition of these antibodies to the CM removes the chemotactic activity. The emphasis of this paper was not in characterization of the CM but in the ability of indomethacin to modulate fibroblast chemotaxis. The ability of indomethacin in inhibiting prostaglandin production and the influx of inflammatory cells during acute inflammation does not necessarily indicate that this agent could be utilized as an effective inhibitor of fibroblast stimulation and migration. Conditions may exist whereby nonsteroidal anti-inflammatory agents enhance fibroblast influx into the wound site, resulting in such pathological conditions as the formation of keloids, joint contractures, proliferative retinopathy and corneal deposits. Future characterization of the receptors involved or mechanisms unique to fibroblast chemotaxis may elucidate strategies in controlling fibroproliferative diseases. REFERENCES
Atbini A., Richter H. and Pontz B. F. (1983) Localization of the chemotactic domain in fibronectin. FEBS 156, 222-226. Gauss-Mueller V., Kleinman H. K., Martin G. R. and Schiffman E. (1980) Role of attachment factors and attractants in fibroblast chemotaxis. J. Lab. Clin. Med. 96, 1071 1080. Higgs G., Eakins K. E., Mugridge K. G., Moncada S. and Vane J. R. (1980) The effects of non-steroidal anti-flammatory drugs on leukocyte migration in carrageein induced inflammation. Eur. J. Pharm. 66, 81-86. Hiyashi M. and Yamada K. (1983) Domain structure of the carboxyl-terminal half of human plasma fibronectin. J. Biol. Chem. 258, 3332-3340. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680~585. Mensing H. and Czarnetzki B. M. (1984) Leucotriene B4 induces in vitro fibroblast chemotaxis. J. Ira,est. Derrn. 82, 9-12. Mensing H., Pontz B. F., Mueller P. K. and Gauss-Mueller V. (1983) A study of fibroblasts and conditioned medium as chemoattractants. Eur. J. Cell Biol. 29, 268-273. Postlethwaite A. E. (1978) Chemotactic attraction of human fibroblasts to type I, II, and III collagens and collagen derived peptides. Proc. Natn. Acad. Sci., U.S.A. 75, 871-875. Postlethwaite A. E., Synderman R. and Kang A. H. (1976) The chemotactic attraction of human fibroblasts to a lymphocyte derived growth factor. J. Exp. Med. 144, 1188-1203.
Postlethwaite A. E., Synderman R. and Kang A. H. (1979) Generation of a fibroblast chemoattractant in serum by activation of complement. J. Clin. Invest. 64, 1379-1385. Salmon J., Simmons P. M. and Moncada S. (1983) The effects of BW 755C and other anti-inflammatory drugs on eicosanoid concentrations and leukocyte accumulations
574
GERHARD W. KALMUSel al.
in experimentally induced acute inflammation. J. Pharm. Pharmac. 35, 808-813. Schiffman E., Geetha V., Pencev D., Warabi H. and Mato J. (1983) Agents and Supplements, Vol. 12, pp. 106-120. Birkhaus, Basel. Sekinguichi K., Siri A., Zardi L. and Hakomori S. (1985) Differences in domain structure between human
fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts. J. Biol. Chem. 260, 5105-5114. Senior R. M., Griffin G. L. and Mecham R. (1982) Chemotactic response of fibroblasts to tropoelastin and elastin derived peptides. J. Clin. Invest. 70, 6144518.
