POLYPEPTIDE GROWTH FACTORS AUGMENT INTERLEUKIN l-INDUCED RELEASE OF PROSTAGLANDIN E, BY RHEUMATOID ARTHRITIS SYNOVIAL CELLS IN VITRO David H. Goddard,‘,*
Scott L. Grossman,’
Robert Newton2
When stimulated with increasing amounts of interleukin l/3 (IL lfi) rheumatoid arthritis (RA), as compared with osteoarthritis (OA), synovial cells grown in RPM1 plus fetal bovine serum (FBS), released significantly more prostaglandin E, (PGE,) (p -C 0.05; paired t test, two-tailed). PGE, release by IL l&stimulated RA synovial cells grown for 14 days in serum-free RPM1 was significantly less than that released by the same cells grown in medium plus 10 % FBS (p < 0.03; two-tailed). Since these data suggest that growth factors present in FBS may augment the effects of IL lb, experiments were conducted to study the influence of four polypeptide growth factors-transforming growth factor-p (TGF-@), platelet-derived growth factor (PDGF), epiderma1 growth factor (EGF), and basic fibroblast growth factor (bFGF), on IL lb-induced release of PGE, by cultured RA synovial cells. Both EGF and bFGF significantly enhanced IL l&induced release of PGE, (p < 0.05; paired t test, one-tailed), while PDGF was synergistic with IL 18, significantly increasing release of PGE, by these cultured cells (p < 0.02; two-tailed). No such effect was seen when TGF-/3 was added to the culture medium. Taken together, these data lend support to the concept that within the synovial micro-environment small quantities of individual growth factors may potentiate the effects of IL l@ to amplify intra-articular inflammation.
o 1990 by W.B. Saunders Company.
In rheumatoid arthritis (RA) the major mediators of inflammatory destruction of cartilage and bone are the eicosanoids and metalloproteinases, released in large amounts by the cells of the hyperplastic synovial membrane. IL 1 plays a crucial role in joint destruction, by stimulating the release of prostaglandins and metalloproteinases from cells of the synovium (reviewed in reference 1). At least three polypeptide growth factorstransforming growth factor p (TGF-fl), platelet-derived
Presented in part at the Southeast Regional Meeting of the American Rheumatism Association, Tampa, FL, December 1988, and the Fifty-third Annual Meeting of the American College of Rheumatology, Cincinnati OH, June 1989. ‘The Einstein-Moss Arthritis Center, Division of Rheumatology, Department of Medicine, Albert Einstein Medical Center, Northern Division, and Temple University Medical School Philadelphia, PA. 2E.I. DuPont de Nemours and Company, Medical Products Department, Glenolden, Pennsylvania. *To whom reprint requests should be addressed at M.R.C.P., Division of Rheumatology, Albert Einstein Medical Center, Room 103, Korman Research Pavilion, 12th Street and Tabor Roads, Philadelphia, PA 19141. o 1990 by W.B. Saunders Company. 1043-4666/90/0204-0011%05.00/0 KEY WORDS: Rheumatoid arthritis/Synovial Interleukin l/Polypeptide growth factors
294
cells/Prostaglandins/
growth factor (PDGF), and epidermal growth factor (EGF)-stimulate the release of prostaglandins from normal fibroblasts in culture.2’3,3a While each of these growth factors is found in RA synovial fluids,4,5 little is known about either their individual contributions to the intraarticular inflammatory response, or about their modulating influence on IL l-induced release of prostaglandins by synovial cells. Accumulating evidence has established that polypeptide growth factors play an important role in regulating normal as well as neoplastic cell growth.6-‘2 These findings have led to the concept that cell growth may be regulated through autocrine and paracrine mechanisms.13,14While the cause of excessive growth of the rheumatoid synovial membrane is unknown, we have recently reported that RA synovial cells, in contrast to OA osteoarthritis (OA) synovial cells, grow in RPM1 1640 lacking all added growth factors-an observation consistent with the concept that RA synovial cell growth in vitro is driven by the continuous autocrine secretion of at least one polypeptide growth factor.15 Moreover, based on blocking studies using growth factor antibodies we have speculated that this growth factor is TGF-P. The finding of raised amounts of growth factors and cytokines in RA synovial fluids raises important questions concerning not only their role in regulating cell CYTOKINE,
Vol. 2, No. 4 (July),
1990: pp 294-299
PGE, release by IL l/?-stimulatedsynovialcells / 295
growth, but also in modulating inflammatory joint damage. This paper presents the results of experiments in which we have studied the effects of specific polypeptide growth factors on IL lp-induced release of PGE, by RA synovial cells in culture.
RESULTS
Influence of Fetal Bovine Serum on IL l-Induced Releaseof PGEz by Synovial Cells Table 1 shows the mean (t SEM) amounts of PGE, (pg/mL) released by three RA and three OA synovial cell cultures grown in RPM1 plus 10% fetal bovine serum (FBS), replated at 1 x IO4 cells/well, and stimulated with increasing amounts of IL lfi (0.01 to 10 ng/mL). Under basal conditions, small amounts of PGE, were released by all cultures. Following IL l@ stimulation, significantly more PGE, was released by RA, as compared with OA, synovial cells in culture (IL lp concentration range 0.1 to 10 ng/mL; p < 0.05; independent t test, two-tailed). Next, to determine whether FBS influenced the capacity of RA synovial cells to respond to stimulation with IL I@, five RA synovial cell cultures grown to confluence in either serum-free RPM1 or RPM1 plus 10% FBS and replated in 12-well plates (1 x lo4 cells/ well) were stimulated with increasing amounts of IL l/3 (concentration range 0.01 to 10 ng/mL; 6 hr at 37°C). As shown in Fig. 1, significantly less PGE, was released by RA synovial cells grown in serum-free RPM1 (concentration range 0.1 to 10 ng/mL IL l/3) as compared with the same cells grown in RPM1 plus 10% FBS (p < 0.03; paired t test, two tailed).
Influence of Polypeptide Growth Factors on IL l-Induced PGE, Releaseby RA Synovial Cells Next, we determined whether some of the polypeptide growth factors found in FBS enhanced the release of PGE, by RA synovial cells in culture following stimulation with IL lp. Six RA synovial cell lines grown to confluence in RPM1 plus 5% FBS and replated in
TABLE 1. IL lb-induced synovial cells
release of PGE, by cultured
PGE, release f SEM (pg/mL)* Dose of IL I@ (ng) 0 0.01
0.1 1.0 10.0
RA
OA
93 368 531 829 706
-e 28 + 148 f 224 * 367 + 298
153 1,054 4,448 5,496 8,150
k i f f f
75 513t 2,427t 2,598.f 1,629t
*Numbers represent mean amounts of PGE, released by RA and OA synovial cells stimulated with IL Ip determined from triplicate experiments. Ip < 0.05; paired t test, two-tailed; RA versus OA.
0.01
0.1
1.0
10
IL-1 Concentration (ng protein/ml)
Figure 1. IL 1/34nduced release of PGE2 by RA synovial cells grown in RPM1 with and without FBS. The values shown are mean t SEM PGE, release by RA synovialcells grown for 14 days in serum-freemedium (m-0, n = 5), and stimulated with increasing amounts of IL l@ (0.01 to 10 ng/mL), compared with PGE, released by IL I@-stimulated RA synovial cells (M, n = 3) grown in medium supplemented with 10% FBS. Each experiment was conducted in triplicate. Asterisks indicate significant differences (p < 0.05; two-tailed) in the amounts of PGE, released by cells grown in medium with or without FBS.
12-well tissue culture plates (1 x lo4 cells/well), were grown for 14 days in serum-free medium alone or in serum-free medium with or without IL lp (10 ng/mL), or individual growth factors (TGF-/3 at 5 ng/mL, PDGF at 5 ng/mL, EGF at 6.5 ng/mL, or bFGF at 1 ng/mL). On day 15, the cultures were washed in serum-free HBSS, and then stimulated with IL 10 (1 ng/mL) for 6 hr at 37OC. By day 14, significant increases occurred in the mean (k SEM) numbers of RA synovial cells grown in serum-free RPM1 compared with day 1 (day 14, 3.85 x lo4 + 0.43 x lo4 versus day 1,1.61 x lo4 + 0.19 x 104; p < 0.01, paired t test, two-tailed). Compared with growth in serum-free RPM1 alone, synovial cell growth was greater in serum-free RPM1 plus bFGF (serum-free RPM1 plus bFGF 4.68 x lo4 + 0.91 x lo4 versus serum-free RPM1 3.85 x lo4 * 0.44 x lo4 cells; p < 0.2, not significant) and significantly greater when either PDGF (serum-free RPM1 plus PDGF, 5.03 x lo4 + 0.38 x lo4 versus serum-free RPMI, 3.85 x lo4 * 0.44 x lo4 cells; p < 0.03; paired t test, two-tailed) or IL 10 (serum-free RPM1 plus IL-l@ 5.23 x lo4 -c 0.88 x lo4 versus serum-free RPM1 3.85 x lo4 f 0.44 x lo4 cells; p < 0.04, paired t test, two-tailed) were added to the culture medium. These increases in cell numbers were not seen when TGF-fi or EGF was added to the culture medium. As shown in figure 2A, prior to IL l@ stimulation negligible amounts of PGE, were released by synovial cells grown in serum-free RPM1 with or without added growth factors. Following IL lb stimulation, PGE, release by RA synovial cells grown in serum-free RPM1
296
/ Goddard,
Grossman,
Medium
Figure 2. serum-free
CYTOKINE,
Newton
+TGF-B
+ IL-1
+ EGF
+PDGF
Medium
+ bFGF
Influence of polypeptide growth factors on IL l-induced release RPM1 plus IL l& polypeptide growth factors, both, or neither.
of PGE,
+TGF-B
Vol. 2, No. 4 (July 1990: 294-299)
+ IL-1
by RA synovial
+ EGF
+PDGF
cells grown
in
+ bFGF
The values shown in (A) are mean f SEM PGE, release (pg/mL) by RA synovial cells with (W, n = 6) and without (0, n = 6) IL 10 stimulation (1 ng/mL for 6 hr at 37OC) in duplicate experiments. Prior to IL 18 stimulation, cells were grown for 14 days in serum-free RPM1 alone, or serum free medium plus IL 10 (10 ng/mL), or serum-free medium plus polypeptide growth factors (TGF-/3 at 5 ng/mL, PDGF at 5 ng/mL, EGF at 6.5 ng/mL, or bFGF at 1 ng/mL). On day 15, the medium was removed, cell cultures were washed with HBSS, and the medium was replaced with a similar volume of serum-free RPM1 or serum-free RPM1 plus IL 10 (1 ng/mL). PGE, release by IL l/l-stimulated cells grown in serum-free RPM1 plus PDGF, EGF, or bFGF, but not TGF-@, was significantly increased compared with release by the same cells grown in serum-free RPM1 (p < 0.05; paired t test, two-tailed). The values shown in (B) are mean f SEM PGE, release (pg/mL) by RA synovial cells with (m, n = 6) and without (0, n = 6) IL lfl stimulation (1 ng/mL for 6 hr at 37OC) in duplicate experiments. Cell cultures were grown for 14 days in serum-free RPM1 alone, or serum-free medium plus IL lfi (10 ng/mL) and individual growth factors (TGF-P b at 5 ng/mL, PDGF at 5 ng/mL, EGF at 6.5 ng/mL, or bFGF at 1 ng/mL). On day 15, the medium was removed, cell cultures were washed with HBSS and then incubated with serum-free RPM1 with or without IL lfl. Prior to IL lfl stimulation, PGE, release by RA synovial cells grown in serum-free RPM1 plus growth factors (PDGF, bFGF, or EGF) was significantly greater than by the same cells grown in serum-free RPM1 (p < 0.02; paired t test, two-tailed), serum-free RPM1 plus IL 10 alone (p < 0.05; paired t test, two-tailed), or growth factors alone (p < 0.02; paired t test, two-tailed). Following IL 10 stimulation (1 ng; 6 hr at 37OC), only RA synovial cells grown in serum-free RPMI plus IL 18 and PDGF released significantly more PGE, than did the same cells grown in serum-free RPM1 plus either IL l/3 or PDGF alone.
plus PDGF, bFGF, or EGF, but not TGF-P, increased significantly compared with cells grown in serum-free RPM1 alone (p < 0.05; paired t test, one-tailed). Next, experiments were performed to determine whether RA synovial cells grown in serum-free RPM1 with or without individual growth factors released more PGE, when IL 10 was included in the medium throughout the culture period. All six cell lines used in the preceding experiment were grown for 14 days in serumfree RPM1 with or without IL lp (10 ng/mL) and growth factors (TGF-fi 5 ng/mL, PDGF 5 ng/mL, EGF 6.5 ng/mL, or bFGF 1 ng/mL). By day 14, significant increases had occurred in the mean (t SEM) number of RA synovial cells grown in serum-free RPM1 plus IL 10 and growth factors (PDGF, bFGF, or EGF, but not TGF-0) compared with synovial cells grown in serumfree RPM1 alone (p < 0.04; paired t test, two-tailed). As shown in Fig. 2B, on day 15 and prior to IL lp stimulation, PGE, release by RA synovial cells grown in serum-free RPM1 plus IL 10 and growth factors (PDGF, EGF, or bFGF) was significantly greater than by the same cells grown in serum-free RPM1 alone (p < 0.02; paired t test, two-tailed), or serum-free RPM1 plus IL 16 alone (p < 0.05; paired t test, two-tailed), or growth factors alone (p < 0.02; paired t test, two-tailed). Follow-
ing IL 10 stimulation (1 ng, for 6 hr at 37V), only RA synovial cells grown in serum-free RPM1 plus IL l/3 and PDGF released significantly more PGE, than synovial cells grown in serum-free RPM1 plus IL l/3 alone or PDGF alone (p < 0.03; paired t test, two-tailed).
DISCUSSION It is now clear that polypeptide growth factors regulate growth of normal as well as neoplastic cells.6-12 The recent identification of several growth factors and cytokines in rheumatoid synovial effusions suggests that they may cause excessive synovial cell growth. RA and OA synovial cells grow differently in culture, with RA synovial cells displaying several characteristics that suggest transformation.4”6 We have recently reported that RA synovial cells in long-term culture have the capacity to grow in medium lacking all added growth factors and, based on the results of blocking studies with growth factor antibodies, have suggested that RA synovial cell growth in vitro is driven by endogenous TGF-P.” When we undertook a phenotypic analysis of synovial cells (RA and OA) taken from early, middle, and late passages, we observed that these cells reacted with monoclonal antibodies to vimentin but not with
PGE,releaseby IL l&stimulated synovialcells / 297 antibodies to cytokeratin, lymphocyte and monocyte/ macrophage markers, or factor VIII antigen.15 These data indicate that the cultures contain cells of mesenchyma1 lineage that are not contaminated with long-lived lymphocytes or mononuclear cells. More definitive identification is presently hampered by the lack of specific antibodies recognizing fibroblast and synoviocyte surface markers. Polypeptide growth factors stimulate cell growth through the activation of a series of discrete genes (reviewed in reference 17) and act in concert at key points in the cell cycle (reviewed in reference 18). At least two growth factors-PDGF and TGF-/3 potentiate the effects of EGF by increasing the cell expression of high-affinity receptors for EGF.19-21 These observations underscore the importance of synergy of action between individual growth factors in the regulation of cell growth; however, little information is presently available concerning the modulating influence of growth factors on IL l-Induced release of prostaglandins. Here we report the results of studies in which we have started to investigate the long-term effects of polypeptide growth factors on IL l-induced release of PGE2 by synovial cells in culture. We have chosen to study the long-term effects of these growth factors since we have observed that RA synovial cells grow slowly when cultured in serum-free medium, with an approximate doubling time of ten to twelve days. We have observed that following IL I/? stimulation, significantly more PGE2 is released by RA as compared with OA synovial cells grown in RPM1 plus FBS. This finding suggests differences in the capacity of RA and OA synovial cells in culture to respond to the same IL lb stimulus. Moreover, we observed that RA synovial cells grown in serum-free RPM1 released significantly less PGE, than the same cells grown in RPM1 plus FBS, suggesting that IL I@-induced release of PGE, may be augmented by polypeptide growth factors present in FBS. When we measured the effects of four polypeptide growth factors-TGF-P, PDGF, EGF, and bFGF-on IL l-induced release of PGE,, we found that it was enhanced by PDGF, bFGF, and EGF, but not TGF-/3. Since RA synovial cells grown in serum-free RPM1 plus growth factors release negligible amounts of PGE, and since RA synovial cell growth was enhanced by PDGF and bFGF, we think it unlikely that the differences in the amounts of PGE, released by IL l,&stimulated RA and OA synovial cells were due to the preferential survival of PGE,-releasing cells in RA synovial cell cultures. Moreover, since we could detect no discernable differences in the capacity of early, middle, or late passage synovial cell cultures to respond to IL l@ stimulation (data not shown), these findings suggest that the capacity of these cells to synthesize and release PGE, is not altered by length of time in culture.
Although it has been previously reported3a that TGF-6 induces the release of prostaglandins by normal fibroblasts in culture, we found a lack of such an effect on RA synovial cells in culture. We believe that our finding is consistent with one of the known biologic properties of this growth factor,22 namely enhancement of extracellular matrix deposition through downregulation of the synthesis and release of mediators known to degrade extracellular matrix proteins. When we measured PGE, release by RA synovial cells grown in medium containing IL l/3 and individual growth factors (PDGF, bFGF, or EGF, but not TGF-0) we found it to be significantly greater than release by cells grown in medium plus either IL l/3 or growth factors alone. Moreover, further stimulation of synovial cells on day 15 with IL lp caused a significant increase in the amounts of PGE, released by cells grown in serum-free medium plus IL lp and PDGF. Taken together, these data indicate that at least three polypeptide growth factors-PDGF, bFGF, and EGF-act in synergy with IL l/3 to enhance the release of PGE, by RA synovial cells in culture, findings that are consistent with those recently reported by Kumkumian et a1.23 Polypeptide growth factors could enhance IL l-induced release of PGE, by synovial cells by three separate but interrelated mechanisms: first, by increasing the numbers of IL l/3 receptors expressed on cells; second, by increasing the size of the intracellular arachidonic acid (AA) pool; and third, by increasing intracellular levels of cyclooxygenase, causing the enhanced release of PGE, from intracellular arachidonic acid. Recently, IL lcr was shown to stimulate expression of its own receptor on human fibroblasts by increasing endogenous levels of PGE, and PGE2.24 This effect is thought to be mediated through activation of the prostaglandin-adenylate cyclase pathway. Polypeptide growth factors could increase IL 1 receptor expression indirectly by increasing endogenous levels of these prostaglandins. While there is presently no evidence to show that polypeptide growth factors directly regulate IL 1 receptor expression, PDGF and TGF-/3 both regulate the expression of high-affinity EGF receptors.20v21Based on this paradigm, it is tempting to speculate on a role for PDGF in the regulation of IL 1 receptor expression on cells. While the sequence of intracellular events resulting in the generation of prostaglandins and thromboxanes has not yet been fully elucidated, it is generally believed that the rate-limiting step in prostaglandin synthesis is the generation of intracellular AA.25 In mammalian cells the intracellular AA pool is low. When required AA is generated from membrane-bound phospholipids by phospholipase A, and phospholipase C. In mammalian cells, PDGF and EGF preferentially activate the phospholipase C/diglyceride lipase pathway stimulating AA release from phosphatidylinositol.2’3’26 IL 1 in-
298
/ Goddard,
Grossman,
Newton
creases intracellular AA by activating phospholipase A,.27 Both PDGF and IL 1, but not EGF, stimulate de novo synthesis and activation of cyclooxygenase.28 Our finding that PDGF and IL lp act in synergy to enhance release of PGE, from RA synovial cells in culture is entirely consistent with these previous observations. Of further interest is the observation that bFGF augments IL l-induced release of PGE, by RA synovial cells in culture. Although the mechanisms by which this effect is mediated are unknown, the recent reports that bFGF augments IL l-induced release of PGI, by endothelial cells29 and PGE, by chondrocytes,30 indicates that the effects of this growth factor are not unique to synovial cells. Moreover, these observations suggest that this growth factor may play an important role in the inflammatory response. From the foregoing, it is clear that polypeptide growth factors and cytokines have a complex effect on eicosanoid biosynthesis. Our studies in vitro, have shown that at least three growth factors augment IL l-induced release of PGE,. These data suggest that within the synovial micro-environment small quantities of individual growth factors and cytokines may act together to amplify the ongoing intra-articular inflammatory response and consequent destruction of cartilage and bone.
MATERIALS
AND
METHODS
Materials TGF-P, PDGF, and bFGF were purchased from R & D Systems, Minneapolis, MN. EGF was purchased from Sigma Chemical Company, St. Louis, MO. Recombinant IL lp (endotoxin levels tl ng/mg) was obtained from E.I. DuPont de Nemours & Co (Wilmington, DE). Radioimmunoassay kits for PGE, were purchased from DuPont-New England Nuclear, Boston, MA. RPM1 1640, Hanks’ balanced salt solution (HBSS), non-essential amino-acids, streptomycin, penicillin, gentamicin, and fungazone were all purchased from Flow Labs, McLean, VA. Fetal bovine serum was purchased from Hyclone, Logan, UT, crude collagenase from Sigma, bovine serum albumin (BSA) from Miles Scientific Co., Naperville, IL, fatty-acid poor bovine albumin from Calbiothem-Behring, San Diego, CA, and trypsin/EDTA from Gibco Laboratories, Grand Island, NY. All tissue culture plastics were purchased from Falcon, Oxnard, CA. Samples of synovial membranes were obtained with informed consent from patients undergoing total joint replacement or other orthopedic procedures. Synovial samples were obtained from sevenpatients with a diagnosis of definite RA3’ and five patients with clinical and radiologic features of OA.
Synovial Cell Culture Synovial cell cultures were established using previously described methods.‘5,32Synovial samples cut into small pieces (~5 mm*), were incubated overnight in RPM1 1640 supplemented with non-essential amino-acids, streptomycin (100 pg/mL), penicillin (100 U/mL), fungazone (25 Hg/mL), 10%
CYTOKINE, Vol. 2, No. 4 (July 1990:294-299) FBS, and crude collagenase(200 U/mL) at 37OCin 10% CO, in air. Dispersed cells were then collected by low-speed centrifugation, washed twice in HBSS and once in RPM1 plus 10% FBS, resuspendedin 8 mL of RPM1 plus 10% FBS, and seededinto two tissue culture flasks (25 cm*, Falcon, Oxnard, CA). At confluence, primary cultures were split weekly, and used in passages3-23.
PGE, Releaseby IL I@-Stimulated Synovial Cells In experiments that measured PGE, release by RA and OA synovial cells stimulated with IL 1, synovial cell cultures were grown to confluence in RPM1 plus 10% FBS. Synovial cells from each of the RA and OA synovial cell cultures tested were detached from the culture flasks by trypsin/EDTA, resuspendedin RPM1 with 10% FBS, and replated in 12-well tissue culture plates (1 x lo4 cells per well). After overnight incubation, cells were washed in supplemented HBSS (HBSS plus 10% FBS, 2 mM L-glutamine, and gentamicin [50 pg/mL]), and stimulated with IL 10 (100 ~1 final volume in RPM1 plus 2% fatty acid-poor BSA; final concentration depending on experimental protocol-range 0.01 to 10 ng/ mL), for 6 hr at 37OC. The cell supernatants were collected and stored in polypropylene tubes (Sarstedt) at -7OOC.
Influence of Polypeptide Growth Factors on IL l-Induced Releaseof PGEz by RA Synovial Cells To study the influence of growth factors on IL l-induced releaseof PGE,, the methods were modified as follows. In the passage preceding the start of the study, RA synovial cells were grown to confluence in RPM1 plus 5% FBS. Cells were detached by adding trypsin/EDTA, resuspended in RPM1 with 5% FBS, and plated in 12-well plates (1 x lo4 cells/well). After overnight incubation, the cells were washed with serumfree HBSS, and grown for 14 days in serum-free RPM1 (RPM1 1640 plus 2% BSA), or serum-free RPM1 plus IL lb (10 ng/mL), or growth factors (TGF-@ at 5 ng/mL, PDGF at 5 ng/mL, bFGF at 1 ng/mL, or EGF at 6.5 ng/mL), or IL lp plus individual growth factors. Cultures were fed twice weekly by removing half of the medium and, depending on experimental protocol replacing the medium with a similar volume of fresh medium containing IL 10, growth factors, or IL lp and growth factors. On day 15, the cultures were washed in serum-free HBSS, and stimulated with 100 ul of IL lb (1 ng/mL in RPM1 with 2% BSA) for 6 hr at 37OC. The supernatants were then collected and stored at -7OOC.
Determination of Synovial Cell Number Immediately following IL lb stimulation, synovial cells were detached from the plates by trypsin/EDTA, transferred into isotonic solution (Isoton, Coulter Diagnostics, Hialeah, FL), and counted on a Coulter counter (model ZBI, Coulter Diagnostics). Triplicate counts were made on duplicate samples, and the mean value of the counts were used to calculate PGE, releasedper lo4 synovialcells.
PGEz Measurement PGE, levels were measured by radioimmunoassay, using commercially available kits that have been previously vali-
PGE, release by IL lb-stimulated dated and found to show low cross-reactivity (approximately 3.7%).33
Statistical
with
PGE,
Analysis
Data was analyzed using paired and unpaired Student’s t tests as appropriate. Significance was set at the 5% level.
REFERENCES 1. Dinarello CA (1988) Interleukin-1. Dig Dis Sci (Suppl) 33:258-35s. 2. Habenicht AJR, Glomset JA, King WC, Mitchell CD, Ross R (198 1) Early changes in phosphatidylinositol and arachadonic acid metabolism in quiescent Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem 25612329-12335. 3. Habenicht AJR, Goerig RM, Grulich J, Rothe D, Gronwald R, Schettler G, Kommerell B (1985) The phospholipase C/diglyceride lipase pathway contributes to arachadonic acid (AA) release and prostaglandin (PG) E, formation in platelet-derived growth factor (PDGF) stimulated Swiss 3T3 cells. Adv Prostaglandin Thromboxane Leukotriene Res 13:37-39. 3a. Diaz A, Varga J, Jiminez SA (1989) Transforming growth factor-p stimulation of lung fibroblast prostaglandin Ez production. J Biol Chem 264:1154-l 157. 4. Lafyatis R, Remmers EF, Roberts A, Yocum DE, Sporn MB, Wilder RL (1989) Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous plateletderived growth factor and inhibition by transforming growth factor-@. J Clin Invest 83:1267-1276. 5. Hamerman D, Taylor S, Kirschenbaum I, Klagsbrun M, Raines EW, Ross R, Thomas KA (1987) Growth factors with heparin binding affinity in human synovial fluid. Proc Sot Exp Biol Med 186:384-389. 6. Sporn MB, Roberts AB (1985) Autocrine growth factors and cancer. Nature 3 13:745-747. 7. James R, Bradshaw RA (1984) Polypeptide growth factors. Annu Rev Biochem 53:259-292. 8. Ross R, Glomset J, Kariya B, Harker LA (1984) A plateletdependent serum factor that stimulates the proliferation of arterial smooth muscle in vitro. Proc Nat1 Acad Sci USA 71:1207-1210. 9. Gajdusek C, DiCorleto P, Ross R, Shwartz SM (1980) An endothelial cell-derived growth factor. J Cell Biol85:467-472. 10. Rizzino A, Bowen-Pope DF (1985) Production of PDGFlike factors by embryonal carcinoma cells and response to PDGF by endoderm-like cells. Dev Biol 110:15-22. 11. Berk BC, Alexander RW, Brock TA, Gimbrone MA, Webb CR (1986) Vasoconstriction: A new activity for platelet-derived growth factor. Science 232:87-90. 12. Waterfield MD, Scrace GT, Whittle N, Stroobant P, Johnson A, Warteson A, Westermark B, Heldin CH, Huang JS, Deuel TF (1983) Platelet-derived growth factor is structurally related to the putative transforming protein ~28”‘~ of the simian sarcoma virus. Nature 304:35-39. 13. Sporn MB, Todaro GJ (1980) Autocrine secretion and malignant transformation of cells. N Engl J Med 303:878-880. 14. Williams LT, Escobedo JA, Keating MT, Coughlin SR (1988) Signal transduction by the platelet-derived growth factor receptor. Cold Spring Harbor Symp Quant Biol53:455-465. 15. Goddard DH, Grossman SL, Moore ME (1990) Autocrine regulation of rheumatoid arthritis synovial cell growth in vitro. Cytokine2:149-155. 16. Anastassiades TP, Ley L, Wood A, Irwin D (1978) The
synovial cells / 299
growth kinetics of synovial fibroblasts cells from inflammatory and noninflammatory arthropathies. Arthritis Rheum 21:461-466. 17. Rollins BJ, Oquendo P, Stiles CD (1988) Structure and function of platelet-derived growth factor inducible gene products. In Beach D, Basilic0 C, Newport J (eds) Cell Cycle Control in EukaryotesCurrent Communications in Molecular Biology. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, pp 9-14. 18. Bravo R (1988) Complexity of the early genomic response to growth factors in mouse fibroblasts. In Beach D, Basilic0 C, Newport J (eds): Cell Cycle Control in Eukaryotes-Current Communications in Molecular Biology. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, pp 15-21. 19. Armelin HA, Armelin MCS (1987) The interaction of peptide growth factors and oncogenes. In Guroff G (ed) Oncogenes, Genes, and Growth Factors, New York, Wiley Interscience pp 361-363. 20. Davis RJ, Czech MP (1987) Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by plateletderived growth factor in protein kinase-C deficient human fibroblasts. J Biol Chem 262:6832-6841. 2 1. Assoian RK (1985) Biphasic effects of type @-transforming growth factor on epidermal growth factor receptors in NRK fibroblasts. Functional consequences for epidermal growth faotorstimulated mitosis. J Biol Chem 260:9613-9617. 22. Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B (1987) Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 105:1039-1045. 23. Kumkumian GK, Lafyatis R, Remmers EF, Case JP, Kim S-J, Wilder RL (1989) Platelet-derived growth factor and IL-1 interactions in rheumatoid arthritis: Regulation of synoviocyte proliferation, prostaglandin production, and collagenase transcription. J Immunol 143:833-837. 24. Akahoshi T, Oppenheim J, Matsushima K (1987) Induction of Il- 1 receptor expression on fibroblasts by glucocorticoid hormone, prostaglandins, and interleukin-1 (IL-l) (abstract). J Leukocyte Biol 42~579. 25. Irvine RF (1982) How is the level of free arachadonic acid controlled in mammalian cells. Biochem J 204:3-16. 26. Habenicht AJR, Goerig M, Grulich J, Rothe D, Gronwald R, Loth U, Schettler G, Kommerell B, Ross R (1985) Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase. J Clin Invest 75:1381-1387. 27. Burch RM, Connor JR, Axelrod J (1988) Interleukin-1 amplifies receptor-mediated activation of phospholipase A, in 3T3 fibroblasts. Proc Nat1 Acad Sci USA 85:6306-6309. 28. Baird A, Mormede P, Bohlen P (1985) Immunoreactive fibroblast growth factor in cells of peritoneal exudate suggests its identity with macrophage growth factor. Biochem Biophys Res Comm 126:358-364. 29. Spencer-Green G, Caulkins K (1989) Interleukin-I (IL-l) and fibroblast growth factor (FGF) interaction: Effects on the endothelium (abstract). Arthritis Rheum 32:S13. 30. Bandara G, Lin CW, Georgescu HI, Mendelow D, Evans CH (1989) Chondrocyte activation by interleukin-1: Analysis of the synergistic properties of fibroblast growth factor and phorbol myristate acetate. Arch Biochem Biophys 274:539-547. 31. Ropes MW, Bennett GA, Cobb S (1959) 1958 revision of diagnostic criteria for rheumatoid arthritis. Arthritis Rheum 2:16-20. 32. Dayer J-M, Krane SM, Ruse11RGG, Robinson DR (1976) Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. Proc Nat1 Acad Sci USA 73:945-949. 33. Newton RC, Covington M (1987) The activation of human fibroblast prostaglandin E production by interleukin-1. Cellular Immuno1 110:338-349.
Scott L. Grossman,’
Robert Newton2
When stimulated with increasing amounts of interleukin l/3 (IL lfi) rheumatoid arthritis (RA), as compared with osteoarthritis (OA), synovial cells grown in RPM1 plus fetal bovine serum (FBS), released significantly more prostaglandin E, (PGE,) (p -C 0.05; paired t test, two-tailed). PGE, release by IL l&stimulated RA synovial cells grown for 14 days in serum-free RPM1 was significantly less than that released by the same cells grown in medium plus 10 % FBS (p < 0.03; two-tailed). Since these data suggest that growth factors present in FBS may augment the effects of IL lb, experiments were conducted to study the influence of four polypeptide growth factors-transforming growth factor-p (TGF-@), platelet-derived growth factor (PDGF), epiderma1 growth factor (EGF), and basic fibroblast growth factor (bFGF), on IL lb-induced release of PGE, by cultured RA synovial cells. Both EGF and bFGF significantly enhanced IL l&induced release of PGE, (p < 0.05; paired t test, one-tailed), while PDGF was synergistic with IL 18, significantly increasing release of PGE, by these cultured cells (p < 0.02; two-tailed). No such effect was seen when TGF-/3 was added to the culture medium. Taken together, these data lend support to the concept that within the synovial micro-environment small quantities of individual growth factors may potentiate the effects of IL l@ to amplify intra-articular inflammation.
o 1990 by W.B. Saunders Company.
In rheumatoid arthritis (RA) the major mediators of inflammatory destruction of cartilage and bone are the eicosanoids and metalloproteinases, released in large amounts by the cells of the hyperplastic synovial membrane. IL 1 plays a crucial role in joint destruction, by stimulating the release of prostaglandins and metalloproteinases from cells of the synovium (reviewed in reference 1). At least three polypeptide growth factorstransforming growth factor p (TGF-fl), platelet-derived
Presented in part at the Southeast Regional Meeting of the American Rheumatism Association, Tampa, FL, December 1988, and the Fifty-third Annual Meeting of the American College of Rheumatology, Cincinnati OH, June 1989. ‘The Einstein-Moss Arthritis Center, Division of Rheumatology, Department of Medicine, Albert Einstein Medical Center, Northern Division, and Temple University Medical School Philadelphia, PA. 2E.I. DuPont de Nemours and Company, Medical Products Department, Glenolden, Pennsylvania. *To whom reprint requests should be addressed at M.R.C.P., Division of Rheumatology, Albert Einstein Medical Center, Room 103, Korman Research Pavilion, 12th Street and Tabor Roads, Philadelphia, PA 19141. o 1990 by W.B. Saunders Company. 1043-4666/90/0204-0011%05.00/0 KEY WORDS: Rheumatoid arthritis/Synovial Interleukin l/Polypeptide growth factors
294
cells/Prostaglandins/
growth factor (PDGF), and epidermal growth factor (EGF)-stimulate the release of prostaglandins from normal fibroblasts in culture.2’3,3a While each of these growth factors is found in RA synovial fluids,4,5 little is known about either their individual contributions to the intraarticular inflammatory response, or about their modulating influence on IL l-induced release of prostaglandins by synovial cells. Accumulating evidence has established that polypeptide growth factors play an important role in regulating normal as well as neoplastic cell growth.6-‘2 These findings have led to the concept that cell growth may be regulated through autocrine and paracrine mechanisms.13,14While the cause of excessive growth of the rheumatoid synovial membrane is unknown, we have recently reported that RA synovial cells, in contrast to OA osteoarthritis (OA) synovial cells, grow in RPM1 1640 lacking all added growth factors-an observation consistent with the concept that RA synovial cell growth in vitro is driven by the continuous autocrine secretion of at least one polypeptide growth factor.15 Moreover, based on blocking studies using growth factor antibodies we have speculated that this growth factor is TGF-P. The finding of raised amounts of growth factors and cytokines in RA synovial fluids raises important questions concerning not only their role in regulating cell CYTOKINE,
Vol. 2, No. 4 (July),
1990: pp 294-299
PGE, release by IL l/?-stimulatedsynovialcells / 295
growth, but also in modulating inflammatory joint damage. This paper presents the results of experiments in which we have studied the effects of specific polypeptide growth factors on IL lp-induced release of PGE, by RA synovial cells in culture.
RESULTS
Influence of Fetal Bovine Serum on IL l-Induced Releaseof PGEz by Synovial Cells Table 1 shows the mean (t SEM) amounts of PGE, (pg/mL) released by three RA and three OA synovial cell cultures grown in RPM1 plus 10% fetal bovine serum (FBS), replated at 1 x IO4 cells/well, and stimulated with increasing amounts of IL lfi (0.01 to 10 ng/mL). Under basal conditions, small amounts of PGE, were released by all cultures. Following IL l@ stimulation, significantly more PGE, was released by RA, as compared with OA, synovial cells in culture (IL lp concentration range 0.1 to 10 ng/mL; p < 0.05; independent t test, two-tailed). Next, to determine whether FBS influenced the capacity of RA synovial cells to respond to stimulation with IL I@, five RA synovial cell cultures grown to confluence in either serum-free RPM1 or RPM1 plus 10% FBS and replated in 12-well plates (1 x lo4 cells/ well) were stimulated with increasing amounts of IL l/3 (concentration range 0.01 to 10 ng/mL; 6 hr at 37°C). As shown in Fig. 1, significantly less PGE, was released by RA synovial cells grown in serum-free RPM1 (concentration range 0.1 to 10 ng/mL IL l/3) as compared with the same cells grown in RPM1 plus 10% FBS (p < 0.03; paired t test, two tailed).
Influence of Polypeptide Growth Factors on IL l-Induced PGE, Releaseby RA Synovial Cells Next, we determined whether some of the polypeptide growth factors found in FBS enhanced the release of PGE, by RA synovial cells in culture following stimulation with IL lp. Six RA synovial cell lines grown to confluence in RPM1 plus 5% FBS and replated in
TABLE 1. IL lb-induced synovial cells
release of PGE, by cultured
PGE, release f SEM (pg/mL)* Dose of IL I@ (ng) 0 0.01
0.1 1.0 10.0
RA
OA
93 368 531 829 706
-e 28 + 148 f 224 * 367 + 298
153 1,054 4,448 5,496 8,150
k i f f f
75 513t 2,427t 2,598.f 1,629t
*Numbers represent mean amounts of PGE, released by RA and OA synovial cells stimulated with IL Ip determined from triplicate experiments. Ip < 0.05; paired t test, two-tailed; RA versus OA.
0.01
0.1
1.0
10
IL-1 Concentration (ng protein/ml)
Figure 1. IL 1/34nduced release of PGE2 by RA synovial cells grown in RPM1 with and without FBS. The values shown are mean t SEM PGE, release by RA synovialcells grown for 14 days in serum-freemedium (m-0, n = 5), and stimulated with increasing amounts of IL l@ (0.01 to 10 ng/mL), compared with PGE, released by IL I@-stimulated RA synovial cells (M, n = 3) grown in medium supplemented with 10% FBS. Each experiment was conducted in triplicate. Asterisks indicate significant differences (p < 0.05; two-tailed) in the amounts of PGE, released by cells grown in medium with or without FBS.
12-well tissue culture plates (1 x lo4 cells/well), were grown for 14 days in serum-free medium alone or in serum-free medium with or without IL lp (10 ng/mL), or individual growth factors (TGF-/3 at 5 ng/mL, PDGF at 5 ng/mL, EGF at 6.5 ng/mL, or bFGF at 1 ng/mL). On day 15, the cultures were washed in serum-free HBSS, and then stimulated with IL 10 (1 ng/mL) for 6 hr at 37OC. By day 14, significant increases occurred in the mean (k SEM) numbers of RA synovial cells grown in serum-free RPM1 compared with day 1 (day 14, 3.85 x lo4 + 0.43 x lo4 versus day 1,1.61 x lo4 + 0.19 x 104; p < 0.01, paired t test, two-tailed). Compared with growth in serum-free RPM1 alone, synovial cell growth was greater in serum-free RPM1 plus bFGF (serum-free RPM1 plus bFGF 4.68 x lo4 + 0.91 x lo4 versus serum-free RPM1 3.85 x lo4 * 0.44 x lo4 cells; p < 0.2, not significant) and significantly greater when either PDGF (serum-free RPM1 plus PDGF, 5.03 x lo4 + 0.38 x lo4 versus serum-free RPMI, 3.85 x lo4 * 0.44 x lo4 cells; p < 0.03; paired t test, two-tailed) or IL 10 (serum-free RPM1 plus IL-l@ 5.23 x lo4 -c 0.88 x lo4 versus serum-free RPM1 3.85 x lo4 f 0.44 x lo4 cells; p < 0.04, paired t test, two-tailed) were added to the culture medium. These increases in cell numbers were not seen when TGF-fi or EGF was added to the culture medium. As shown in figure 2A, prior to IL l@ stimulation negligible amounts of PGE, were released by synovial cells grown in serum-free RPM1 with or without added growth factors. Following IL lb stimulation, PGE, release by RA synovial cells grown in serum-free RPM1
296
/ Goddard,
Grossman,
Medium
Figure 2. serum-free
CYTOKINE,
Newton
+TGF-B
+ IL-1
+ EGF
+PDGF
Medium
+ bFGF
Influence of polypeptide growth factors on IL l-induced release RPM1 plus IL l& polypeptide growth factors, both, or neither.
of PGE,
+TGF-B
Vol. 2, No. 4 (July 1990: 294-299)
+ IL-1
by RA synovial
+ EGF
+PDGF
cells grown
in
+ bFGF
The values shown in (A) are mean f SEM PGE, release (pg/mL) by RA synovial cells with (W, n = 6) and without (0, n = 6) IL 10 stimulation (1 ng/mL for 6 hr at 37OC) in duplicate experiments. Prior to IL 18 stimulation, cells were grown for 14 days in serum-free RPM1 alone, or serum free medium plus IL 10 (10 ng/mL), or serum-free medium plus polypeptide growth factors (TGF-/3 at 5 ng/mL, PDGF at 5 ng/mL, EGF at 6.5 ng/mL, or bFGF at 1 ng/mL). On day 15, the medium was removed, cell cultures were washed with HBSS, and the medium was replaced with a similar volume of serum-free RPM1 or serum-free RPM1 plus IL 10 (1 ng/mL). PGE, release by IL l/l-stimulated cells grown in serum-free RPM1 plus PDGF, EGF, or bFGF, but not TGF-@, was significantly increased compared with release by the same cells grown in serum-free RPM1 (p < 0.05; paired t test, two-tailed). The values shown in (B) are mean f SEM PGE, release (pg/mL) by RA synovial cells with (m, n = 6) and without (0, n = 6) IL lfl stimulation (1 ng/mL for 6 hr at 37OC) in duplicate experiments. Cell cultures were grown for 14 days in serum-free RPM1 alone, or serum-free medium plus IL lfi (10 ng/mL) and individual growth factors (TGF-P b at 5 ng/mL, PDGF at 5 ng/mL, EGF at 6.5 ng/mL, or bFGF at 1 ng/mL). On day 15, the medium was removed, cell cultures were washed with HBSS and then incubated with serum-free RPM1 with or without IL lfl. Prior to IL lfl stimulation, PGE, release by RA synovial cells grown in serum-free RPM1 plus growth factors (PDGF, bFGF, or EGF) was significantly greater than by the same cells grown in serum-free RPM1 (p < 0.02; paired t test, two-tailed), serum-free RPM1 plus IL 10 alone (p < 0.05; paired t test, two-tailed), or growth factors alone (p < 0.02; paired t test, two-tailed). Following IL 10 stimulation (1 ng; 6 hr at 37OC), only RA synovial cells grown in serum-free RPMI plus IL 18 and PDGF released significantly more PGE, than did the same cells grown in serum-free RPM1 plus either IL l/3 or PDGF alone.
plus PDGF, bFGF, or EGF, but not TGF-P, increased significantly compared with cells grown in serum-free RPM1 alone (p < 0.05; paired t test, one-tailed). Next, experiments were performed to determine whether RA synovial cells grown in serum-free RPM1 with or without individual growth factors released more PGE, when IL 10 was included in the medium throughout the culture period. All six cell lines used in the preceding experiment were grown for 14 days in serumfree RPM1 with or without IL lp (10 ng/mL) and growth factors (TGF-fi 5 ng/mL, PDGF 5 ng/mL, EGF 6.5 ng/mL, or bFGF 1 ng/mL). By day 14, significant increases had occurred in the mean (t SEM) number of RA synovial cells grown in serum-free RPM1 plus IL 10 and growth factors (PDGF, bFGF, or EGF, but not TGF-0) compared with synovial cells grown in serumfree RPM1 alone (p < 0.04; paired t test, two-tailed). As shown in Fig. 2B, on day 15 and prior to IL lp stimulation, PGE, release by RA synovial cells grown in serum-free RPM1 plus IL 10 and growth factors (PDGF, EGF, or bFGF) was significantly greater than by the same cells grown in serum-free RPM1 alone (p < 0.02; paired t test, two-tailed), or serum-free RPM1 plus IL 16 alone (p < 0.05; paired t test, two-tailed), or growth factors alone (p < 0.02; paired t test, two-tailed). Follow-
ing IL 10 stimulation (1 ng, for 6 hr at 37V), only RA synovial cells grown in serum-free RPM1 plus IL l/3 and PDGF released significantly more PGE, than synovial cells grown in serum-free RPM1 plus IL l/3 alone or PDGF alone (p < 0.03; paired t test, two-tailed).
DISCUSSION It is now clear that polypeptide growth factors regulate growth of normal as well as neoplastic cells.6-12 The recent identification of several growth factors and cytokines in rheumatoid synovial effusions suggests that they may cause excessive synovial cell growth. RA and OA synovial cells grow differently in culture, with RA synovial cells displaying several characteristics that suggest transformation.4”6 We have recently reported that RA synovial cells in long-term culture have the capacity to grow in medium lacking all added growth factors and, based on the results of blocking studies with growth factor antibodies, have suggested that RA synovial cell growth in vitro is driven by endogenous TGF-P.” When we undertook a phenotypic analysis of synovial cells (RA and OA) taken from early, middle, and late passages, we observed that these cells reacted with monoclonal antibodies to vimentin but not with
PGE,releaseby IL l&stimulated synovialcells / 297 antibodies to cytokeratin, lymphocyte and monocyte/ macrophage markers, or factor VIII antigen.15 These data indicate that the cultures contain cells of mesenchyma1 lineage that are not contaminated with long-lived lymphocytes or mononuclear cells. More definitive identification is presently hampered by the lack of specific antibodies recognizing fibroblast and synoviocyte surface markers. Polypeptide growth factors stimulate cell growth through the activation of a series of discrete genes (reviewed in reference 17) and act in concert at key points in the cell cycle (reviewed in reference 18). At least two growth factors-PDGF and TGF-/3 potentiate the effects of EGF by increasing the cell expression of high-affinity receptors for EGF.19-21 These observations underscore the importance of synergy of action between individual growth factors in the regulation of cell growth; however, little information is presently available concerning the modulating influence of growth factors on IL l-Induced release of prostaglandins. Here we report the results of studies in which we have started to investigate the long-term effects of polypeptide growth factors on IL l-induced release of PGE2 by synovial cells in culture. We have chosen to study the long-term effects of these growth factors since we have observed that RA synovial cells grow slowly when cultured in serum-free medium, with an approximate doubling time of ten to twelve days. We have observed that following IL I/? stimulation, significantly more PGE2 is released by RA as compared with OA synovial cells grown in RPM1 plus FBS. This finding suggests differences in the capacity of RA and OA synovial cells in culture to respond to the same IL lb stimulus. Moreover, we observed that RA synovial cells grown in serum-free RPM1 released significantly less PGE, than the same cells grown in RPM1 plus FBS, suggesting that IL I@-induced release of PGE, may be augmented by polypeptide growth factors present in FBS. When we measured the effects of four polypeptide growth factors-TGF-P, PDGF, EGF, and bFGF-on IL l-induced release of PGE,, we found that it was enhanced by PDGF, bFGF, and EGF, but not TGF-/3. Since RA synovial cells grown in serum-free RPM1 plus growth factors release negligible amounts of PGE, and since RA synovial cell growth was enhanced by PDGF and bFGF, we think it unlikely that the differences in the amounts of PGE, released by IL l,&stimulated RA and OA synovial cells were due to the preferential survival of PGE,-releasing cells in RA synovial cell cultures. Moreover, since we could detect no discernable differences in the capacity of early, middle, or late passage synovial cell cultures to respond to IL l@ stimulation (data not shown), these findings suggest that the capacity of these cells to synthesize and release PGE, is not altered by length of time in culture.
Although it has been previously reported3a that TGF-6 induces the release of prostaglandins by normal fibroblasts in culture, we found a lack of such an effect on RA synovial cells in culture. We believe that our finding is consistent with one of the known biologic properties of this growth factor,22 namely enhancement of extracellular matrix deposition through downregulation of the synthesis and release of mediators known to degrade extracellular matrix proteins. When we measured PGE, release by RA synovial cells grown in medium containing IL l/3 and individual growth factors (PDGF, bFGF, or EGF, but not TGF-0) we found it to be significantly greater than release by cells grown in medium plus either IL l/3 or growth factors alone. Moreover, further stimulation of synovial cells on day 15 with IL lp caused a significant increase in the amounts of PGE, released by cells grown in serum-free medium plus IL lp and PDGF. Taken together, these data indicate that at least three polypeptide growth factors-PDGF, bFGF, and EGF-act in synergy with IL l/3 to enhance the release of PGE, by RA synovial cells in culture, findings that are consistent with those recently reported by Kumkumian et a1.23 Polypeptide growth factors could enhance IL l-induced release of PGE, by synovial cells by three separate but interrelated mechanisms: first, by increasing the numbers of IL l/3 receptors expressed on cells; second, by increasing the size of the intracellular arachidonic acid (AA) pool; and third, by increasing intracellular levels of cyclooxygenase, causing the enhanced release of PGE, from intracellular arachidonic acid. Recently, IL lcr was shown to stimulate expression of its own receptor on human fibroblasts by increasing endogenous levels of PGE, and PGE2.24 This effect is thought to be mediated through activation of the prostaglandin-adenylate cyclase pathway. Polypeptide growth factors could increase IL 1 receptor expression indirectly by increasing endogenous levels of these prostaglandins. While there is presently no evidence to show that polypeptide growth factors directly regulate IL 1 receptor expression, PDGF and TGF-/3 both regulate the expression of high-affinity EGF receptors.20v21Based on this paradigm, it is tempting to speculate on a role for PDGF in the regulation of IL 1 receptor expression on cells. While the sequence of intracellular events resulting in the generation of prostaglandins and thromboxanes has not yet been fully elucidated, it is generally believed that the rate-limiting step in prostaglandin synthesis is the generation of intracellular AA.25 In mammalian cells the intracellular AA pool is low. When required AA is generated from membrane-bound phospholipids by phospholipase A, and phospholipase C. In mammalian cells, PDGF and EGF preferentially activate the phospholipase C/diglyceride lipase pathway stimulating AA release from phosphatidylinositol.2’3’26 IL 1 in-
298
/ Goddard,
Grossman,
Newton
creases intracellular AA by activating phospholipase A,.27 Both PDGF and IL 1, but not EGF, stimulate de novo synthesis and activation of cyclooxygenase.28 Our finding that PDGF and IL lp act in synergy to enhance release of PGE, from RA synovial cells in culture is entirely consistent with these previous observations. Of further interest is the observation that bFGF augments IL l-induced release of PGE, by RA synovial cells in culture. Although the mechanisms by which this effect is mediated are unknown, the recent reports that bFGF augments IL l-induced release of PGI, by endothelial cells29 and PGE, by chondrocytes,30 indicates that the effects of this growth factor are not unique to synovial cells. Moreover, these observations suggest that this growth factor may play an important role in the inflammatory response. From the foregoing, it is clear that polypeptide growth factors and cytokines have a complex effect on eicosanoid biosynthesis. Our studies in vitro, have shown that at least three growth factors augment IL l-induced release of PGE,. These data suggest that within the synovial micro-environment small quantities of individual growth factors and cytokines may act together to amplify the ongoing intra-articular inflammatory response and consequent destruction of cartilage and bone.
MATERIALS
AND
METHODS
Materials TGF-P, PDGF, and bFGF were purchased from R & D Systems, Minneapolis, MN. EGF was purchased from Sigma Chemical Company, St. Louis, MO. Recombinant IL lp (endotoxin levels tl ng/mg) was obtained from E.I. DuPont de Nemours & Co (Wilmington, DE). Radioimmunoassay kits for PGE, were purchased from DuPont-New England Nuclear, Boston, MA. RPM1 1640, Hanks’ balanced salt solution (HBSS), non-essential amino-acids, streptomycin, penicillin, gentamicin, and fungazone were all purchased from Flow Labs, McLean, VA. Fetal bovine serum was purchased from Hyclone, Logan, UT, crude collagenase from Sigma, bovine serum albumin (BSA) from Miles Scientific Co., Naperville, IL, fatty-acid poor bovine albumin from Calbiothem-Behring, San Diego, CA, and trypsin/EDTA from Gibco Laboratories, Grand Island, NY. All tissue culture plastics were purchased from Falcon, Oxnard, CA. Samples of synovial membranes were obtained with informed consent from patients undergoing total joint replacement or other orthopedic procedures. Synovial samples were obtained from sevenpatients with a diagnosis of definite RA3’ and five patients with clinical and radiologic features of OA.
Synovial Cell Culture Synovial cell cultures were established using previously described methods.‘5,32Synovial samples cut into small pieces (~5 mm*), were incubated overnight in RPM1 1640 supplemented with non-essential amino-acids, streptomycin (100 pg/mL), penicillin (100 U/mL), fungazone (25 Hg/mL), 10%
CYTOKINE, Vol. 2, No. 4 (July 1990:294-299) FBS, and crude collagenase(200 U/mL) at 37OCin 10% CO, in air. Dispersed cells were then collected by low-speed centrifugation, washed twice in HBSS and once in RPM1 plus 10% FBS, resuspendedin 8 mL of RPM1 plus 10% FBS, and seededinto two tissue culture flasks (25 cm*, Falcon, Oxnard, CA). At confluence, primary cultures were split weekly, and used in passages3-23.
PGE, Releaseby IL I@-Stimulated Synovial Cells In experiments that measured PGE, release by RA and OA synovial cells stimulated with IL 1, synovial cell cultures were grown to confluence in RPM1 plus 10% FBS. Synovial cells from each of the RA and OA synovial cell cultures tested were detached from the culture flasks by trypsin/EDTA, resuspendedin RPM1 with 10% FBS, and replated in 12-well tissue culture plates (1 x lo4 cells per well). After overnight incubation, cells were washed in supplemented HBSS (HBSS plus 10% FBS, 2 mM L-glutamine, and gentamicin [50 pg/mL]), and stimulated with IL 10 (100 ~1 final volume in RPM1 plus 2% fatty acid-poor BSA; final concentration depending on experimental protocol-range 0.01 to 10 ng/ mL), for 6 hr at 37OC. The cell supernatants were collected and stored in polypropylene tubes (Sarstedt) at -7OOC.
Influence of Polypeptide Growth Factors on IL l-Induced Releaseof PGEz by RA Synovial Cells To study the influence of growth factors on IL l-induced releaseof PGE,, the methods were modified as follows. In the passage preceding the start of the study, RA synovial cells were grown to confluence in RPM1 plus 5% FBS. Cells were detached by adding trypsin/EDTA, resuspended in RPM1 with 5% FBS, and plated in 12-well plates (1 x lo4 cells/well). After overnight incubation, the cells were washed with serumfree HBSS, and grown for 14 days in serum-free RPM1 (RPM1 1640 plus 2% BSA), or serum-free RPM1 plus IL lb (10 ng/mL), or growth factors (TGF-@ at 5 ng/mL, PDGF at 5 ng/mL, bFGF at 1 ng/mL, or EGF at 6.5 ng/mL), or IL lp plus individual growth factors. Cultures were fed twice weekly by removing half of the medium and, depending on experimental protocol replacing the medium with a similar volume of fresh medium containing IL 10, growth factors, or IL lp and growth factors. On day 15, the cultures were washed in serum-free HBSS, and stimulated with 100 ul of IL lb (1 ng/mL in RPM1 with 2% BSA) for 6 hr at 37OC. The supernatants were then collected and stored at -7OOC.
Determination of Synovial Cell Number Immediately following IL lb stimulation, synovial cells were detached from the plates by trypsin/EDTA, transferred into isotonic solution (Isoton, Coulter Diagnostics, Hialeah, FL), and counted on a Coulter counter (model ZBI, Coulter Diagnostics). Triplicate counts were made on duplicate samples, and the mean value of the counts were used to calculate PGE, releasedper lo4 synovialcells.
PGEz Measurement PGE, levels were measured by radioimmunoassay, using commercially available kits that have been previously vali-
PGE, release by IL lb-stimulated dated and found to show low cross-reactivity (approximately 3.7%).33
Statistical
with
PGE,
Analysis
Data was analyzed using paired and unpaired Student’s t tests as appropriate. Significance was set at the 5% level.
REFERENCES 1. Dinarello CA (1988) Interleukin-1. Dig Dis Sci (Suppl) 33:258-35s. 2. Habenicht AJR, Glomset JA, King WC, Mitchell CD, Ross R (198 1) Early changes in phosphatidylinositol and arachadonic acid metabolism in quiescent Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem 25612329-12335. 3. Habenicht AJR, Goerig RM, Grulich J, Rothe D, Gronwald R, Schettler G, Kommerell B (1985) The phospholipase C/diglyceride lipase pathway contributes to arachadonic acid (AA) release and prostaglandin (PG) E, formation in platelet-derived growth factor (PDGF) stimulated Swiss 3T3 cells. Adv Prostaglandin Thromboxane Leukotriene Res 13:37-39. 3a. Diaz A, Varga J, Jiminez SA (1989) Transforming growth factor-p stimulation of lung fibroblast prostaglandin Ez production. J Biol Chem 264:1154-l 157. 4. Lafyatis R, Remmers EF, Roberts A, Yocum DE, Sporn MB, Wilder RL (1989) Anchorage-independent growth of synoviocytes from arthritic and normal joints. Stimulation by exogenous plateletderived growth factor and inhibition by transforming growth factor-@. J Clin Invest 83:1267-1276. 5. Hamerman D, Taylor S, Kirschenbaum I, Klagsbrun M, Raines EW, Ross R, Thomas KA (1987) Growth factors with heparin binding affinity in human synovial fluid. Proc Sot Exp Biol Med 186:384-389. 6. Sporn MB, Roberts AB (1985) Autocrine growth factors and cancer. Nature 3 13:745-747. 7. James R, Bradshaw RA (1984) Polypeptide growth factors. Annu Rev Biochem 53:259-292. 8. Ross R, Glomset J, Kariya B, Harker LA (1984) A plateletdependent serum factor that stimulates the proliferation of arterial smooth muscle in vitro. Proc Nat1 Acad Sci USA 71:1207-1210. 9. Gajdusek C, DiCorleto P, Ross R, Shwartz SM (1980) An endothelial cell-derived growth factor. J Cell Biol85:467-472. 10. Rizzino A, Bowen-Pope DF (1985) Production of PDGFlike factors by embryonal carcinoma cells and response to PDGF by endoderm-like cells. Dev Biol 110:15-22. 11. Berk BC, Alexander RW, Brock TA, Gimbrone MA, Webb CR (1986) Vasoconstriction: A new activity for platelet-derived growth factor. Science 232:87-90. 12. Waterfield MD, Scrace GT, Whittle N, Stroobant P, Johnson A, Warteson A, Westermark B, Heldin CH, Huang JS, Deuel TF (1983) Platelet-derived growth factor is structurally related to the putative transforming protein ~28”‘~ of the simian sarcoma virus. Nature 304:35-39. 13. Sporn MB, Todaro GJ (1980) Autocrine secretion and malignant transformation of cells. N Engl J Med 303:878-880. 14. Williams LT, Escobedo JA, Keating MT, Coughlin SR (1988) Signal transduction by the platelet-derived growth factor receptor. Cold Spring Harbor Symp Quant Biol53:455-465. 15. Goddard DH, Grossman SL, Moore ME (1990) Autocrine regulation of rheumatoid arthritis synovial cell growth in vitro. Cytokine2:149-155. 16. Anastassiades TP, Ley L, Wood A, Irwin D (1978) The
synovial cells / 299
growth kinetics of synovial fibroblasts cells from inflammatory and noninflammatory arthropathies. Arthritis Rheum 21:461-466. 17. Rollins BJ, Oquendo P, Stiles CD (1988) Structure and function of platelet-derived growth factor inducible gene products. In Beach D, Basilic0 C, Newport J (eds) Cell Cycle Control in EukaryotesCurrent Communications in Molecular Biology. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, pp 9-14. 18. Bravo R (1988) Complexity of the early genomic response to growth factors in mouse fibroblasts. In Beach D, Basilic0 C, Newport J (eds): Cell Cycle Control in Eukaryotes-Current Communications in Molecular Biology. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, pp 15-21. 19. Armelin HA, Armelin MCS (1987) The interaction of peptide growth factors and oncogenes. In Guroff G (ed) Oncogenes, Genes, and Growth Factors, New York, Wiley Interscience pp 361-363. 20. Davis RJ, Czech MP (1987) Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by plateletderived growth factor in protein kinase-C deficient human fibroblasts. J Biol Chem 262:6832-6841. 2 1. Assoian RK (1985) Biphasic effects of type @-transforming growth factor on epidermal growth factor receptors in NRK fibroblasts. Functional consequences for epidermal growth faotorstimulated mitosis. J Biol Chem 260:9613-9617. 22. Sporn MB, Roberts AB, Wakefield LM, de Crombrugghe B (1987) Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 105:1039-1045. 23. Kumkumian GK, Lafyatis R, Remmers EF, Case JP, Kim S-J, Wilder RL (1989) Platelet-derived growth factor and IL-1 interactions in rheumatoid arthritis: Regulation of synoviocyte proliferation, prostaglandin production, and collagenase transcription. J Immunol 143:833-837. 24. Akahoshi T, Oppenheim J, Matsushima K (1987) Induction of Il- 1 receptor expression on fibroblasts by glucocorticoid hormone, prostaglandins, and interleukin-1 (IL-l) (abstract). J Leukocyte Biol 42~579. 25. Irvine RF (1982) How is the level of free arachadonic acid controlled in mammalian cells. Biochem J 204:3-16. 26. Habenicht AJR, Goerig M, Grulich J, Rothe D, Gronwald R, Loth U, Schettler G, Kommerell B, Ross R (1985) Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase. J Clin Invest 75:1381-1387. 27. Burch RM, Connor JR, Axelrod J (1988) Interleukin-1 amplifies receptor-mediated activation of phospholipase A, in 3T3 fibroblasts. Proc Nat1 Acad Sci USA 85:6306-6309. 28. Baird A, Mormede P, Bohlen P (1985) Immunoreactive fibroblast growth factor in cells of peritoneal exudate suggests its identity with macrophage growth factor. Biochem Biophys Res Comm 126:358-364. 29. Spencer-Green G, Caulkins K (1989) Interleukin-I (IL-l) and fibroblast growth factor (FGF) interaction: Effects on the endothelium (abstract). Arthritis Rheum 32:S13. 30. Bandara G, Lin CW, Georgescu HI, Mendelow D, Evans CH (1989) Chondrocyte activation by interleukin-1: Analysis of the synergistic properties of fibroblast growth factor and phorbol myristate acetate. Arch Biochem Biophys 274:539-547. 31. Ropes MW, Bennett GA, Cobb S (1959) 1958 revision of diagnostic criteria for rheumatoid arthritis. Arthritis Rheum 2:16-20. 32. Dayer J-M, Krane SM, Ruse11RGG, Robinson DR (1976) Production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. Proc Nat1 Acad Sci USA 73:945-949. 33. Newton RC, Covington M (1987) The activation of human fibroblast prostaglandin E production by interleukin-1. Cellular Immuno1 110:338-349.
