Regulation of synovial cell growth: basic fibroblast growth factor synergizes with interleukin 1β stimulating phospholipase A2 enzyme activity, phospholipase A2 activating protein production and release of prostaglandin E2 by rheumatoid arthritis synovial cells in culture

REGULATION OF SYNOVIAL CELL GROWTH: BASIC FIBROBLAST GROWTH FACTOR SYNERGIZES WITH INTERLEUKIN lf3 STIMULATING PHOSPHOLIPASE A, ENZYME ACTIVITY 9 PHOSPHOLIPASE A, ACTIVATING PROTEIN PRODUCTION AND RELEASE OF PROSTAGLANDIN E, BY RHEUMATOID ARTHRITIS SYNOVIAL CELLS IN CULTURE1 David H. Goddard,*

Scott L. Grossman,* Robert Newton,? Mike A. Clark,$ John S. Bomalaskio

Cytokines have been implicated in the regulation of eicosanoid synthesis and synovial cell proliferation. To further define these mechanisms, we have compared the effects of basic fibroblast growth factor and platelet-derived growth factor on cell growth, prostaglandin E, (PGE,) production and phospholipase A, enzyme activity in long-term cultures of synovial cells from rheumatoid arthritis (RA) patients capable of proliferating in serum-free medium. Compared with serum-free medium alone, RA synovial cell growth was significantly enhanced by adding either basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) to the culture medium. Growing RA synovial cells for 14 days in serum-free medium plus bFGF caused them to spontaneously release significant amounts of PGE,, an effect not seen if cells were grown in serum-free medium alone, or serum-free medium plus PDGF. Enhanced release of PGE, occurred when arachidonic acid was added to bFGF but not PDGF-treated RA synovial cells, suggesting that bFGF increased cyclooxygenase enzyme activity in these cells. Moreover, phospholipase A, (PLA,) enzyme activity was found to be significantly greater in RA synovial cells grown for 14 days in serum-free medium containing bFGF alone, or bFGF plus interleukin lfi (IL-1P) compared with cells grown in either serum-free medium alone, or serum-free medium plus PDGF. Similarly, bFGF plus IL-lbstimulated release of PLA, activating protein, a novel mammalian phospholipase stimulator found in high concentrations in RA synovial fluid. These data show that bFGF functions as a mitogen for synovial cells in culture and stimulates the formation of prostaglandins by regulating at least three key steps in the eicosanoid biosynthetic pathway. Taken together, these data suggest the existence of an intimate relationship between cell growth and eicosanoid biosynthesis such that a disturbance in the normal regulation of one may lead to excessive production of the other.

From the Division of Rheumatology/Immunology/Allergy, Department of Medicine, Winthrop-University Hospital, Mineola, NY*; The DuPont Merck Pharmaceutical Company, Medical Products Department, Glendolden, PA?; Schering-Plough Corporation, Bloomfield, NJ.*; Veteran’s Affairs Medical Center, Medical College of Pennsylvania and University of Pennsylvania, Philadelphia, PAS, USA. iThese studies were supported in part by a P.H.S. grant (AR 39382) and grants from the Arthritis Foundation Eastern Pennsylvania Chapter, Arcadia Foundation, Albert Einstein Society (grant #2-0731-601-00), Veteran’s Affairs, and American Heart Grant in Aid (880936). Dr Bomalaski is

CYTOKINE,

Vol.

4, No. 5 (September),

1992: pp 377-384

an Arthritis Foundation Investigator. Address correspondence and requests for reprints to: David H. Goddard, M.D., Division of Rheumatology/Immunology/Allergy, Winthrop-University Hospital, 222 Station Plaza North, Suite 430, Mineola, NY 11501, USA. Received 6 December; revised and accepted for publication 18 March 0 1992 Academic Press Limited 1043-4666/92/050377+08 $08.00/O KEY WORDS: cytokinesieicosanoidsiarachidonic genaseiplatelet-derived growth factor

acidicyclooxy-

377

378 I Goddard

CYTOKINE,

et al.

Rheumatoid arthritis (RA) is an inflammatory disease of unknown etiology characterized by excessive growth of the synovial membrane and inflammatory destruction of cartilage and bone. Although the stimulus causing excessive synovial cell growth is unknown, cartilage resorption and bone erosion are mediated by eicosanoids and neutral proteinases, released in large quantity by cells of the hyperplastic rheumatoid synovium. At least two cytokines, interleukin 1 (IL-l) and tumor necrosis factor (Y (TNF-a) both found in raised amounts in RA synovial fluids, induce the release of these inflammatory mediators from synovial cells in culture.iJ Moreover, an increasing number of polypeptide growth factors, also found in RA synovial fluids, induce the release of prostaglandin E, (PGE,), as well as collagenase and stromelysin from normal fibroblasts in culture.%9 These findings indicate that individual growth factors and cytokines may play a central role in mediating synovial cell growth and the intra-articular inflammatory response. Based on our observations that long-term cultures of RA, but not osteoarthritis (OA) synovial cells have the capacity to proliferate in serum-free medium, and that the addition of monoclonal neutralizing antibodies to transforming growth factor p (TGF-P) cause a dose-dependent reduction in RA synovial cell growth, we have suggested that RA synovial cell growth in vitro is regulated by one of these growth factors, endogenous TGF-l3.9 We have also observed that when stimulated with IL-lp, RA, in contrast to OA, synovial cells released larger amounts of PGE,, an effect which was further enhanced by the addition of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) to the culture medium.10 Taken together, these data indicate that individual growth factors act together with IL-1S to amplify the biosynthesis and release of PGE, by RA synovial cells in culture. The rate-limiting step in eicosanoid biosynthesis is postulated to be hydrolysis of arachidonic-acid (AA) from membrane phospholipids by phospholipases, primarily phospholipase A2 (PLA,) .riJs Enhanced PLA, and phospholipase C (PLC) enzyme activity have been found in RA synovial fluids and in peripheral blood mononuclear cells from RA patients.14J5 While the steps leading to enhanced phospholipase enzyme activity have not been fully elucidated, studies in cultured fibroblasts have shown that at least two growth factors, PDGF and EGF, activate PLC, while PLA, is activated by IL-1.5,6J6J7 Furthermore, both PDGF and IL-l, but not EGF stimulate de nova synthesis and activation of cyclooxygenase, another key step in generation of PGE, from endogenous AA.16,is These findings suggest that individual cytokines regulate various aspects of the eicosanoid synthetic pathway,

Vol. 4, No. 5 (September 1992: 377-384)

and that cytokines, acting in concert, may affect multiple steps in eicosanoid generation. Recently acidic and basic forms of FGF have been identified in intact rheumatoid synovium and synovial fluid.r9,20 These pleiotropic growth factors are mitogenic for endothelial and mesenchymal cells in vitro and are powerful angiogenic factors in vivo.21 While little is presently known about the influence of these growth factors on eicosanoid biosynthesis the recent reports of enhanced PGI, release from endothelial cells and PGE, from chondrocytes stimulated with bFGF,22-24 as well as our observation of enhanced PGE, release by IL-lp stimulated RA synovial cells grown in serum-free medium plus bFGFr0 suggest FGFs may play an important role in regulating key steps in the eicosanoid biosynthetic pathway. The purpose of these studies was to determine the effects of bFGF on proliferation and the eicosanoid synthetic pathway in synovial cells. In these experiments we have compared growth, PGE, release and phospholipase and cyclooxygenase enzyme activities in RA synovial cells grown in serum-free medium supplemented with either bFGF or PDGF. We also examined the capacity of cytokines to induce release of phospholipase activating protein (PLAP), a positive regulator of PLA,.

RESULTS Influence Growth

of bFGF and PDGF on RA Synovial Cell

Figure 1 shows the significant increase in mean (+ SEM) cell number in four RA synovial cell cultures grown for 14 days in serum-free medium (day 14 = 6.93 f 0.3 x 104 vs day 1 x 2.5 + 0.25 x 104, P < 0.001; paired t test, two-tailed). As shown in Fig. 2 adding PDGF or bFGF to the culture medium resulted in a significant increase in synovial cell growth. In the case of PDGF a dose-dependent increase in cell number was observed (serum-free medium plus PDGF vs serum-free medium alone P < O.Ol), whereas in the case of bFGF the observed increase in synovial cell number was maximal between 1 ng and 5 ng of the growth factor (serum-free medium plus bFGF vs serum-free medium alone, P < 0.05). Overall, significantly greater synovial cell growth occurred in serum-free medium plus PDGF compared with bFGF (serum-free medium plus PDGF vs serum-free medium plus bFGF, P < 0.001).

bFGF

4

uay I Figure

1.

Growth

of RA synovial

Day 14

cells in serum-free

medium.

Values shown compare mean (+ SEM) numbers of RA synovial cells (N = 4 synovial cell cultures) grown for 14 days in serum-free medium (In) with synovial cell number on day 1 (0). Each growth study was performed in triplicate. By day 14, there was a significant increase in cell number compared with day 1 (day 14 = 6.9 f 0.3 x lo4 vs day 1 = 2.5 IfI 0.25 x 104, P < 0.001, paired t test, two-tailed).

Regulation

of eicosanoid

biosynthesis

in synoviocytes

/ 379

of growth factor). As shown in Fig 3B a significant and dose-dependent increase in PGE, release was observed from synovial cells grown in serum-free medium plus bFGF as compared with serum-free medium alone (P < 0.001). In contrast PGE, release by cells grown in serum-free medium plus PDGF (Fig. 3A) did not increase over baseline. Overall, significantly more PGE, was released by synovial cells grown in serum-free medium plus bFGF compared with PDGF (serum-free medium plus bFGF vs serum-free medium plus PDGF, P < 0.001). Because IL-@ stimulates the release of PGE, from synovial cells in culture, experiments next determined whether IL-1B stimulation on day 15 (1 rig/ml for 6 h at 37°C) induced the release of increased amounts of PGE, from synovial cells grown for 14

IE4 A ,E

xx

IO-O e-0 A-A

6 2 IO Log growth

factor

IO concentration

sfm +IL-I $-AA

10 (rig/ml)

I

5.0 Growth

Figure 2. cell growth.

Influence

factor

of polypeptide

concentration

growth

(rig/ml)

factors

IE4

-I

on RA synovial

Values shown are the mean (+ SEM) increase in the number of synovial cells grown in serum-free medium containing increasing concentrations of either PDGF (0) or bFGF (w). The addition of either growth factor resulted in a significant and dose-dependent increase in synovial cell growth compared with serum-free medium alone (*serum-free medium plus PDGF vs serum-free medium alone, P < 0.01; **serum-free medium plus bFGF vs serum-free medium alone, P < 0.05). Overall, significantly greater synovial cell growth occurred in serum-free medium plus PDGF compared with bFGF (serum-free medium plus PDGF vs bFGF, P < 0.001). Data shown are derived from triplicate experiments performed on each of four synovial cell cultures.

O-O O-0 A-A

2 I Log growth

factor

Figure 3. Influence of polypeptide by RA synovial cells in culture.

Influence of bFGF and PDGF Release Culture

on PGE2

Experiments next compared the amount of PGE, released by RA synovial cells grown for 14 days in serum-free medium alone, with that in serum-free medium plus bFGF or PDGF (range 0.25-20 rig/ml

concentration

growth

factors

SFM fIL-I + AA

I”00

(ng/mi)

on PGE,

release

The values shown are mean k SEM PGE, release (pgiml) by RA synovial cells grown in serum-free medium plus PDGF (A) or bFGF (B). Synovial cells were grown for 14 days in serum-free medium plus increasing concentrations of growth factors. On day 15, the medium was replaced with serum-free medium alone (48), or serum-free medium plus IL-@ (1 rig/ml;@), or serum-free medium plus AA (10pM) final concentration in ethanol; A). PGE2 release was then measured as in Materials and Methods. Under all three conditions, significantly more PGE, was released by synovial cells grown in serum-free medium plus bFGF compared with serum-free medium alone, or serum-free medium plus PDGF.

380 I Goddard

et al.

days in serum-free medium plus bFGF or PDGF (range 0.25-20 rig/ml of growth factor). Figure 3A,B shows that IL-lB-stimulated synovial cells grown in serum-free medium plus bFGF released significantly more PGE, than synovial cells grown in serum-free RPM1 alone or serum-free RPM1 plus PDGF (serumfree medium plus bFGF vs serum-free medium alone, P < 0.001; serum-free medium plus bFGF vs serumfree medium plus PDGF, P < 0.01).

Znjluence of bFGF and PZXF on Cyclooxygenase and Phospholipase Enzyme Activity bFGF Stimulates Cyclooxygenase Enzyme Activity

Since data from the preceding experiments indicate that bFGF stimulates eicosanoid biosynthesis and the ability to respond to IL-16 stimulation by increased eicosanoid production, we next undertook experiments to determine the steps regulated by this growth factor. Experiments first sought indirect evidence that bFGF modulates cyclooxygenase activity. Synovial cells were grown for 14 days in serum-free RPM1 alone, or in serum-free RPM1 plus bFGF or PDGF (final concentration 0.25-20 rig/ml). On day 15 the culture medium was removed and replaced with serum-free medium, with, and without, arachidonic acid (10 PM final concentration in ethanol). The cells were then incubated for 6 h at 37°C after which the medium was removed and assayed for PGE,. As shown in Fig. 3A,B, significantly greater amounts of PGE, were released by synovial cells grown in serum-free RPM1 plus bFGF compared with serum-free medium alone, or serum-free medium plus PDGF (serum-free medium plus bFGF vs serum-free medium alone, P < 0.0001; serum-free medium plus bFGF vs serum-free medium plus PDGF, P < 0.001).

bFGF Stimulates Pbospholipase A, Enzyme Activity

Next, experiments were undertaken to measure the effects of bFGF on PLA, enzyme activity, since this is the step proximal to the cyclooxygenase enzyme and, by increasing the formation of intracellular arachidonic acid from membrane-bound phospholipids, is the rate-limiting step in the eicosanoid synthetic pathway.rl-13 Experiments measured PLA, and PLC activity in synovial cells grown for 14 days in serum-free RPM1 alone, or serum-free RPM1 with either bFGF alone (1 rig/ml final concentration), or IL-lb alone (1 rig/ml), or bFGF and IL-@ (PDGF or bFGF, 1 rig/ml final concentration plus IL-lp 1 rig/ml final concentration). As shown in Table 1, when compared with cells grown in serum-free RPM1 alone,

CYTOKINE,

Vol.

4, No.

5 (September

1992:

377-384)

phospholipase A2 enzyme activity was significantly higher in synovial cell cultures grown in serum-free medium with bFGF or bFGF and IL-ll3 In contrast, PLC enzyme activity was not consistently altered by the addition of either bFGF or IL-lp to the growth medium. bFGF Stimulates Release of PLAP

We have recently isolated and characterized a novel mammalian PLA, activating protein termed PLAP from bovine and mammalian cell lines and from synovial fluid from patients with RA.28-32 To explore the possibility that bFGF stimulation of PLA, with resultant PGE,.production might occur through activation of this positive stimulator of PLA,, we performed ELISA tests for PLAP (Table 2). When compared with cells grown in serum-free RPM1 alone, greater amounts of PLAP were released by cells grown in serum-free medium with bFGF or bFGF and IL-@

TABLE 1. bFGF stimulates enzyme activity.” Treatment

Phospholipase

Serum-free medium bFGF IL-1 bFGF t IL-1

1.72 3.49 2,81 6.92

? k i f

pbospholipase

A,

0.26 0.26” 0.58* 0.23*‘”

A,

Phospholipase 1.88 1.52 1.37 1.83

f ++ -+

C

0.17 0.29 0.13 0.35

OExpressed as pmol substrate hydrolysedimg proteinimin. Data from a representative experiment performed in triplicate. The substrate for the phospholipase A, assay was choline-methyl-[‘HI-phosphatidylcholine, r.-a-dipalmitoyl, and arachidonyl-I-[“Cl-phosphatidylinositol. L-a-l-stearoyl2-arachidonyl for phospholipase C. UP < 0.05 by Student’s t-test compared to media alone. P < 0.01 by Student’s t-test compared to media alone.

TABLE cel1s.s Cells grown

2.

in:

SFM IL-1p FGF FGF + IL-lb SFM IL-1p bFGF bFGF + IL-lp

bFGF stimulates

Cells exposed SFM SFM SFM SFM IL-1p IL-1p IL-1p IL-l@

release of PLAP from synovial

to:

ELISA

OD,,,,,,,,

0.13 0.07* 0.16’” 0.12 0.15 0.10 0.23** O.18’L

“ELISA for PLAP expressed as optical density,,,,,,, (per 1 x IN cells). Data from a representative experiment performed in triplicate. Cells in SFM alone produced PLAP. Cells grown in the relevant cytokine at 1 nglml and then exposed to cytokine for 6 h at 1 ngiml. *P < 0.05 by Student’s t-test compared to media alone. ““P < 0.01 by Student’s I-test compared to media alone. Standard deviation of the mean for the assay was s 10%.

bFGF Regulation of eicosanoid biosynthesis in synoviocytes i 381

Influence of bFGF on the Eicosanoid Synthetic Pathway Table 3 summarizes the results of these studies which show that bFGF stimulates PGE, release and cyclooxygenase enzyme activity, as well as PLA, enzyme activity and release of PLAP, a PLA, stimulator. Taken together, these data show that bFGF positively affects multiple steps in the eicosanoid synthetic pathway.

DISCUSSION An accumulating body of data has shown that individual growth factors modulate prostaglandin release by fibroblasts and synovial cells in culture. For example, IL-l-induced release of PGE, by fibroblasts and RA synovial cells grown under serum-free conditions is enhanced by adding PDGF or bFGF to the culture medium.lcJ5 While PDGF is known to regulate several key steps in the eicosanoid biosynthetic pathway in normal fibroblasts in culture, the lack of information concerning the mechanisms of action of bFGF has led us to undertake this present study. bFGF stimulates RA synovial cell mitogenesis (Fig. 2). Using these cell cultures we have also observed that when grown in serum-free medium plus increasing concentrations of bFGF, but not PDGF, RA synovial cells spontaneously released significant amounts of PGE, (Fig. 3A,B). Furthermore, when stimulated with IL-lB, RA synovial cells grown in serum-free medium plus bFGF released more PGE, than cells grown in serum-free medium with, and without, PDGF (Fig. 3A,B). Whether long-term cultures of RA synovial cells lose the capacity to respond to PDGF is, at present, unknown. However, since the mitogenic effects of both bFGF and PDGF were similar, these findings suggest that modulation of PGE, release occurs independently of cell growth. For these studies we have chosen to use longterm cell cultures, rather than synovial cell explants or first-passage cells because these culture are not contaminated with long-lived lymphocytes or macrophages whose secretory products may be expected

TABLE 3. Influence of bFGF on the eicosanoid pathway in RA synovial cells. Eicosanoid

pathway

step

PLAP release PLA, stimulation Cyclooxygenase stimulation PGE, production

Influence

of bFGF

Increases Increases Increases Increases

synthetic

to influence synovial cell responses to added growth factors and cytokines.9 Moreover, while these cultures consist of a highly selected cell population which may not necessarily reflect that of the intact synovium, the capacity of these cells to grow in serum-free medium circumvents a significant problem inherent in conventional serum-dependent systems, namely, the extent to which mitogens present in serum supplements modify the observed interactions between growth factors and/or cytokines under study.9,26,*7 As in our earlier studylo, we could detect no discernible differences in the capacity of early, middle or late passage synovial cells to respond to stimulation with either growth factor or IL-1B (data not shown). Although the precise sequence of intracellular events leading to the formation and release of prostaglandins by cells are not fully known, generation of free intracellular AA is a key step in this pathway.lt-13 Our studies show that bFGF alone, and in combination with IL-1B, enhances PLA, enzyme activity (Table 1) and stimulates the release of PLAP from RA synovial cells in culture (Table 2). This in turn would cause a rise in the level of free intracellular AA. These findings are consistent with previous studies which have demonstrated phospholipase activities in human synovial cells and enhancement of PLA, by IL-l.rJssO Based on the results of experiments in which we observed enhanced release of PGE, by cells following the addition of exogenous AA to the culture medium, we also suggest that bFGF alone, and in combination with IL-lB, may also regulate cyclooxygenase enzyme activity, another important step in the eicosanoid synthetic pathway. With the availability of specific cDNA probes and antibodies it will clearly be important to study the effects of these growth factors and cytokines on levels of mRNAs and proteins of the appropriate enzymes. Besides modulating the activity of enzymes which regulate key steps in prostaglandin biosynthesis, bFGF could also enhance the biological effects of IL-ll3 by regulating the number of IL-l receptors expressed by cells. Support for this concept comes from studies showing that IL-la regulates expression of its own receptor on human fibroblasts through increases in the intracellular level of PGE, and PGE,? Furthermore, IL-l receptor gene expression and binding of IL-l to normal fibroblasts is enhanced by PDGF.slJ* Although there is presently no evidence to show that bFGF directly alters IL-l-receptor expression, it is nevertheless tempting to speculate that, based on this paradigm, bFGF could enhance the release of PGE, by IL-l@stimulated cells by regulating the expression of high-affinity IL-1B receptors. While these studies confirm that bFGF regulates eicosanoid biosynthesis in vitro, there is as yet no evidence to indicate how this growth factor provides

382 I Goddard

et al.

this stimulus. Unlike most secreted proteins, FGFs have no distinct signal sequence, yet many types of cells including synovial cells express high and low-affinity receptors for the growth factor.20Ji Since cells bind extracellular sources of bFGF, release of this growth factor must occur via unusual and so far uncharacterized translocation mechanisms. Recent studies have underscored the importance of matrix solubilization and degradation in angiogenesis , another key process regulated by FGFs.s3-36 Binding of FGFs to heparan sulfate found in abundance in most extracellular matrix proteins creates a pericellular reservoir of the growth factor. Several enzymes including heparinases, found in many tissues, and plasmin released by stimulated fibroblasts, release biologically active bFGF-glycosaminoglycan complexes from core proteins.32,37-39 Since basic FGF stimulates the release of plasminogen activator (PA) and metalloproteinases from endothelial cells and chondrocytes in culture,*J7-39 and since enhanced fibroblast growth and PA activity in fibroblasts is potentiated by agents such as PGE,, cholera toxin and PDGF, which enhance intracellular levels of cAMP,40 this suggests that growth factor-induced mitogenesis may be mediated in part through enhanced synthesis and release of E prostaglandins. While as yet there are no data to indicate that the mitogenic effects of bFGF occur through such mechanism, they suggest the existence of an intimate relationship between eicosanoid biosynthesis and cell growth. The clear implication of such a relationship is that disturbances in the normal regulation of one can be expected to lead to excessive features of the other; a relationship which may help to link intra-articular inflammation and excessive synovial cell growth.

MATERIALS AND METHODS Materials PDGF and bFGF were purchased from R & D Systems Minneapolis, MN. Recombinant IL-ll3 was obtained from E. I. DuPont de Nemours & Co (Wilmington, DE). Radioimmunoassay kits for PGE, and radioactive phospholipids were purchased from DuPont-NEN, Boston, MA. Cold phospholipids were purchased from Avanti, Birmingham, AL. RPM1 1640, 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 Chemical Co., bovine serum albumin (BSA) from Miles Scientific Co., Naperville, IL., fatty-acid poor bovine albumin from Calbiochem-Behring, San Diego, CA, and trypsin/EDTA from Gibco Laboratories, Grand Island, NY. All tissue culture plastics were purchased from Falcon, Oxnard, CA.

CYTOKINE, Vol. 4, No. 5 (September1992:377-384)

Synovial Cell Culture Samples of synovial membranes were obtained with informed consent from four patients with a diagnosis of defi-

nite or classical RA41 undergoing total joint replacement or other orthopedic procedures. Synovial cell cultures were established using previously described methods.9 Synovial cells were grown in RPM1 1640 supplemented with nonessential amino acids, streptomycin (100 &/ml), penicillin (100 p/ml), fungazone (25 yg/ml), and 10% FBS, and were used in passages 3-23. In the pass 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, re-suspended in RPM1 with 5% FBS, and plated in 12 well plates (1 x 104 cells/well). After overnight incubation, the cells were washed with serum-free HBSS, and grown for 14 days in serum-free RPM1 (RPM1 1640 plus 2% BSA), or serum-free RPM1 plus bFGF or PDGF (0.25-20 rig/ml final concentration). Cultures were fed twice weekly by removing half of the medium and replacing it with a similar volume of fresh medium. Similar results regarding cellular growth, PGE, and phospholipase enzyme activities were observed in all four cultures, in contrast to PLAP assays.

PGE, Release by Synovial Cells Grown in Serum-free Medium Plus Growth Factors On day 15 and other days depending on experimental protocol, cells were first washed in serum-free HBSS. and the medium replaced with RPM1 plus 2% fatty acid-poor BSA alone, or RPM1 plus fatty acid-poor BSA containing either AA (10 pM final concentration) or IL-ll3 (1 nglml final concentration). Cell cultures were then incubated for 6 hours at 37”C, after which the supernatants were collected and stored at -70°C. In order to measure cell growth, the increase in cell number over baseline were determined by Coulter counter (model ZBI, Coulter Diagnostics). Cells were detached from the plates, using trypsin/EDTA, and transferred immediately into isotonic solution (Isoton, Coulter Diagnostics) , according to manufacturer’s instructions. Triplicate counts were made on duplicate samples, and the mean value of these counts used to calculate prostaglandin E, released per 104cells.

Prostaglandin E2 Measurement PGE, levels were measured by radioimmunoassay, using commercial kits with a sensitivity to 0.5 pg/ml PGE, and low cross-reactivity with PGE, (c. 3.7%).42

PhospholipaseA2 and C Enzyme Measurement Phospholipase A, and C were assayed using the methods of Bomalaski et a1.14J5,4M7 Synovial cells were scraped off the tissue-culture plates with rubber policemen into PBS (pH 7.4) in polypropylene microfuge tubes, centrifuged for 1 min in a microfuge, and then re-suspended in 20 1.11of PBS with the following protease inhibitors: benzamidine (17 pg/ml), soybean trypsin inhibitor (10 &/ml), bacitracin (100 pglml) , and phenylmethylsulfonyl fluoride (10 @ml).

bFGF Regulation of eicosanoidbiosynthesisin synoviocytesi 383

Suspensions were then sonicated in a Branson Sonifer Cell Disrupter 200 using six on-off bursts. All assays were performed in triplicate in 1.5ml microfuge tubes. 20 ~1 of cell sonicate was used in each reaction tube, with 10 1.11 of Tris or HEPES buffer. To quantify PLC activity, the phospholipid containing labeled arachidonic acid at the R, position was used: arachidonyl-1-[i%]-phosphatidylcholine, L-a-l-palmitoyl2-arachidonyl (PC-2A; specific activity 52 mCi/mM), arachidonyl-1-[i%]-phosphatidylinositol, L-a-1-stearoyl-2arachidonyl (Pl-2A; specific activity 58 mCl/mM), all purchased from New England Nuclear, Boston. To quantify PLA, activity phospholipid was used in which the polar head group was labeled: choline-methyl-[sH]phosphatidylcholine, L-c-w-dipalmitoyl(PC-3-specific activity 497 Ci/mM) . When the [sH]-label was added, it was also necessary to add cold lipid PC, for a final concentration of 10 pM in the reaction. The organic solvent in which the lipids were dissolved was removed by blowing N, across the top of the tube. The residue was then dissolved in deoxycholate (Sigma) at 5 pg/ml, in sufficient quantity to allow 10 ul per tube. The final reaction contained a mixture which was diluted fivefold for the reaction, as 10 pl of substrate, 20 pl of the cell sonicate, 10 ~1 of buffer, and 10 ~1 final volume of 50mM NaCl and 0.4 mM CaCl, (Sigma). The reaction was started by adding the substrate, and the mixture was incubated at 37°C for 60 minutes. The reaction was stopped by adding 50 pl of CHCl,:MeOH (1:2), followed by 50 l.11of CHCl,, and then 50 ~1 of 4 M KCI. All solvents used were reagent grade. A 50 pl aliquot of the organic layer was then spotted on silica gel thin-layer chromatography plates (Analtech). Cold PL standards were also added to each lane to enhance visualization of the product. PLA, reaction products were separated using a solvent solution of CHCl,:MeOH:acetic acid:H,O (100:60:16:8). PLC reaction products were separated using a solvent solution of petroleum ether:diethyl ether:acetic acid (140:60:2). Plates were developed in iodine (Sigma) vapor. Once the iodine stains appeared, the spots were scraped from the plates in 20-ml scintillation vials; 500 ~1 of MeOH and 10 ml of Beckman HP/b scintillation fluid (Beckman Diagnostic Products) were added to each vial, and counted in a Beckman scintillation counter. Enzyme activity was expressed as pmol of substrate hydrolysed’mg of protein/min. The protein content of the cell lysates was determined by the method of Bradford.48

PLAP (Phospholipase

A, Activating

Protein) ELBA

The ELISA for PLAP was performed as described%32 using peroxidase conjugated anti-rabbit antibody and 0 phenylenediamine as substrate. The standard deviation of the mean for the assaywas less than lo%.45347

Statistical Analysis Data were analysed using the SSPSIPC + Statistical Package (SSPS Inc, Chicago, IL). One way analysis of variance was used to compare the effects of increasing doses of each growth factor on synovial cell growth, and

PGE, release. Analysis of variance (ANOVA) was used to compare the effects of the growth factors on synovial cell growth and PGE, release. Paired Student’s f-tests were used to compare the effects of bFGF, PDGF and IL-ll3 on PLA, and PLC enzyme activities and release of PLAP from synovial cells in culture. Significance was set at the 5% level.

REFERENCES 1. Dayer J-M, de Rochemonteix B, Burrus B, Demczuk S, Dinarello CA (1986) Human recombinant interleukin 1 stimulates collagenase and prostaglandin E, production by human synovial cells. Clin Invest 77:645-648. 2. Dayer J-M, Beutler B, Cerami A (1985) Cachectinitumor necrosis factor stimulates collagenase and prostaglandin E, production by human synovial cells and dermal fibroblasts. J Exp Med 162:2163. 3. 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 platelet-derived growth factor and inhibition by transforming growth factor-p. J Clin Invest 83:1267-1276. 4. Hamerman D, Taylor S, Kirschenbaum I, Klasbrun 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. 5. Habenicht AJR, Glomset JA, King WC, Mitchell CD, Ross R (1981) Early changes in phosphatidylinositol and arachidonic acid metabolism in quiescent Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem 256:12329-12335. 6. Habenicht AJR, Goerig RM, Grulich J, Rothe D, Gronwald R, Schettler G, Kommerell B (1985) The phospholipase Cidiglyceride lipase pathway contributes to arachidonic 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. 7. Diaz A, Varga J, Jiminez SA (1989) Transforming growth factor-p stimulation of lung fibroblast prostaglandin Ea. J Biol Chem 264:1154-1157. 8. Edwards AR, Murphy G, Reynolds JJ, Whitham SE, Docherty AJP, Angel P, Heath K (1987) Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 6:1899-1904. 9. Goddard DH, Grossman SL, Moore ME (1990) Autocrine regulation of rheumatoid arthritis synovial cell growth in vitro. Cytokine 2:149-155. 10. Goddard DH, Grossman SL, Newton R (1990) Polypeptide growth factors augment interleukin l-induced release of prostaglandin E, by rheumatoid arthritis synovial cells in vitro. Cytokine 2:294-299. 11. Irvine RF (1982) How is the level of free arachidonic acid controlled in mammalian cells. Biochem J 204:3-16. 12. Higgs GA, Flower RJ, Vane JR (1979) A new approach to antiinflammatory drugs. Biochem Pharmacol28: 1959-1961. 13. Flower RJ, Blackwell GJ (1976) The importance of phospholipase A, in prostaglandin biosynthesis. Biochem PharmacoI25:285-291. 14. Bomalaski JS, Clark MA, Zurier RB (1986) Enhanced phospholipase activity in mononuclear phagocytes from patients with rheumatoid arthritis. Arthritis Rheum 29:312-318.

384 I Goddard et al. 15. Pruzanski W, Vadas P, Stefanski E, Urowitz MB (1985) Phospholipase A, activity in sera and synovial fluids in rheumatoid arthritis and osteoarthritis: Its possible role as a proinflammatory enzyme. J Rheumatol12:211-216. 16. 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. 17. 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. 18. Korn JH, Downie E, Roth GJ, Ho S-Y (1989) Synergy of interleukin 1 (IL-l) with arachidonic acid and A23187 in stimulating PGE synthesis in human fibroblasts: IL-l stimulates fibroblast cyclooxygenase. Clin Immunol Immunopathol50:196204. 19. Sano H, Forough R, Maier JAM, Case JP, Jackson A, Engleka K, Maciag T, Wilder RL (1990) Detection of high levels of heparin binding growth factor-l (acidic fibroblast growth factor) in inflammatory arthritic joints. J Cell Biol 110: 1417-1426. 20. Melnyk VO, Shipley GD, Sternfeld MD, Sherman L, Rosenbaum JT (1990) Synoviocytes synthesize, bind, and respond to basic fibroblast growth factor. Arthritis Rheum 33:493-500. 21. Gospodarowicz D, Ferrara N, Schwiegerer L, Neufeld G (1987) Fibroblast growth factor: structure and biological properties. Endocr Rev 8:95-113. 22. Spencer-Green G, Caulkins K (1989) Interleukin 1 (IL-l) and fibroblast growth factor (FGF) interaction: Effects on the endothelium. Arthritis Rheum 32:S13 (abstract). 23. Chin JE, Hatfield CA, Krzesicki RF, Herblin WF (1991) Interactions between interleukin 1 and basic fibroblast growth factor on articular chondrocytes. Effects on cell growth, prostanoid production and receptor modulation. Arthritis Rheum 34:314-324. 24. Bandara G, Lin CW, Georgescu HI, Mendelow D, Evans CH (1989) Chondrocvte activation bv interleukin 1: svnereism with kbroblast growth’factor and phorbol myristate acetate. Arch Biochem Biophys 2741539-547. 25. Clark MA, Chen M-J, Crooke ST, Bomalaski JS (1988) Tumor necrosis factor (cachectin) induces phospholipase A? activity and synthesis of a phospholipase Aalactivating protein in endothelial cells. Biochem J 250:125-132. 26. Chiou WJ, Bonin PD, Singh JP (1990) Obligatory action of polypeptide growth factors for the IL-l-mediated prostaglandin E, production in fibroblasts. Potential role of growth factors in modulation of tissue response to IL-l. J Immunol145:2155-2160. 27. Akahoshi T, Oppenheim JJ, Matsushima K (1988) Interleukin 1 stimulates its own receptor expression on human fibroblasts through endogenous production of prostaglandins. J Clin Invest 82:1219-1224. 28. Baker DG, Baumgarten DF, Bomalaski JS, Zurier RB (1985) Cyclic adenosine 3’5 monophosphate stimulates prostaglandin E production in human adherent synovial cells. Prostaglandins 30:669-682. 29. Godfrey RM, Johnson WM, Newman T, Hoffstein ST (1988) Interleukin 1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA, activation and PGE, production. Prostaglandins 35:107. 30. Gilman SC, Chang J, Zeigler PR, Uhl J, Mochan E (1988) Interleukin 1 activates phospholipase A, in human synovial cells. Arthritis Rheum 31:126130. 31. Bonin PD, Singh JP (1988) Modulation of interleukin 1 receptor expression and interleukin 1 response in fibroblasts by platelet-derived growth factor. J Biol Chem 263:11052-11055. 32. Chiou WJ. Bonin PD. Harris PKW, Carter DB, Singh JP (1989) Platelet-derived growth factor induces interleukin-1 receptor gene expression in BALB/c3T3 fibroblasts. J Biol Chem 264~21442.

CYTOKINE,

Vol. 4, No. 5 (September 1992: 377-384)

33. Baird A, Ling N (1987) Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: Implications for a role of heparinase-like enzymes in the neovascular response. Biochem Biophys Res Commun 142:428-435. 34. Nakajima M, Irimura T, DiFerrante D, DiFerrante N, Nicholson GL (1982) Heparan sulfate degradation: relation of tumor invasive and metastatic properties of mouse B16 melanoma sublines. Science 213:611. 35. Bar-Ner M, Kramer MD, Schirrmacher V, Ishai-Michaeli R, Fuks Z, Vlodavasky I (1985) Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells. Int J Cancer 35:483. 36. Matzner Y, Bar-Ner MD, Yahalom J, Ishai-Michaeli R, Fuks Z, Vlodavsky I (1985) Degradation of heparan sulfate in the subendothelial extracellular matrix by readily released heparinase from human neutrophils. Possible role in invasion through basement membranes. J Clin Invest 76:1306. 37. Sakesela 0, Rifkin DB (1990) Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity. J Cell Biol 110:767-775. 38. Saksela 0, Moscatelli D, R&in DB (1987) The opposing effects of basic fibroblast growth factor and transforming growth factor beta on the regulation of plasminogen activator activity in capillary endothelial cells. J Cell Biol105:957-775. 39. Presta M, Maier JAM, Rusnati M, Moscatelli D, Ragnotti G (1988) Modulation of plasminogen activator activity in human endometrial adenocarcinoma cells by basic fibroblast growth factor and transforming growth factor-p. Cancer Res 48:6384-6389. 40. Rozengurt E, Erusalimsky J, Memhmet H, Morris C, Nanberg E, Sinnett-Smith J (1988) Signal transduction in mitogenesis: Further evidence for multiple pathways. In Molecular Biology of Signal Transduction. Cold Spring Harbor Symposia on Quantitative Biology, Vol LIII, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 945. 41. Ropes MW, Bennett GA, Cobb S (1959) Revision of diganostic criteria for rheumatoid arthritis. Arthritis Rheum 2:16-20. 42. Newton RC, Covington M (1987) The activation of human fibroblast prostaglandin E production by interleukin 1. Cell 1mmun01110:338-349. 43. Bomalaski JS, Clark MA (1987) The effect of sn-2 fatty acid substitution of phospholipase C enzyme activities. Biochem J 244~497-502. 44. Bomalaski JS, Fallon M, Turner RA, Crooke ST, Meunier PC, Clark MA (1990) Identification and isolation of a phospholipase A, activating protein (PLAP) in rheumatoid arthritis: induction of eicosanoid synthesis and an inflammatory response in joints injected in vivo. J Lab Clin Med 116:814-825. 45. Bomalaski JS, Baker DG, Brophy LM, Clark MA (1990) Monosodium urate crystals stimulate phospholipase A, enzyme activities and the synthesis of a phospholipase A, activating protein (PLAP). J Immunol145:3391-3962. 46. Bomalaski JS, Baker DG, Brophy L, Ressurection NV, Spilberg I, Munian M, Clark MA (1989) A phospholipase A,activating protein (PLAP) stimulates human neutrophil aggregation and release of lysosomal enzymes, superoxide and eicosanoids. J Immunol142:3957-3962. 47. Clark MA, Conway TA, Shorr RGL, Crooke ST (1987) Identification and isolation of a mammalian protein which is antigenitally and functionally related to phospholipase Aa stimulatory peptide melittin. J Biol Chem 262:4402-4406. 48. Bradford MM (1979) A rapid and sensitive method for quantitation of microgram amounts of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254.