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Interleukin-1β stimulates cytosolic phospholipase A2 in rheumatoid synovial fibroblasts
Vol.
184,
No.
April
30,
1992
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
712-718
Pages
INTERLEUKIN-lp STIMULATES CYTOSOLIC PHOSPHOLIPASE IN RHEUMATOID SYNOVIAL FIBROBLASTS
A,
Keren I. Hulkower, William C. Hope, Ting Chen, Celia M. Anderson, John W. Coffey and Douglas W. Morgan’ Department
Received
March
11,
of Pharmacology, Hoffmann La-Roche Inc., 340 Kingsland St. Nutley, NJ 07110
1992
SUMMARY: Phospholipase A2 (PLA,) activities in rheumatoid synovial fibroblasts (RSF) stimulated with interleukin-l/3 (IL@) were investigated. RSF incubated in the presence of IL-l@ (120 pg/ml) for 18 h secreted 35 fold more PGE, than did those incubated without IL-@. IL-@ treatment did not increase the level of secretory PLA, (sPLA,) activity or sPLA, protein in the conditioned medium or subcellular fractions of lysed RSF. In contrast, the cell-associated PLA, activity increased 3 to 4 fold in IL-la stimulated RSF when compared with the control. The IL-l/3 stimulated, cell-associated PLA, required submicromolar concentrations of calcium for activity, a characteristic consistent with the calcium sensitivity of cytosolic PLA, (cPLA,) activity reported in other cell types, such as U937 cells. These findings demonstrate that an elevation in a cytosolic PLA,, rather than a sPLA,, is associated with increased PGE, production in IL0 1992Academic Press,1°C. lp stimulated RSF.
IL-la
is a proinflammatory,
found abundantly in rheumatoid of rheumatoid
growth-promoting
cytokine (1). This cytokine is
synovial fluid (2,3) and is associated with the pathology
arthritis (4). One important biological role for IL-@ in rheumatoid
arthritis may be to increase PGE, biosynthesis (5). In rheumatoid studies have demonstrated
synoviocytes, several
that IL-lp stimulates the release of arachidonic acid, PLAz
activities, and the production of PGE, (6-8). A calcium-sensitive PLA, associated with PGE, production by IL-lp stimulated human synovial fibroblasts has been reported (8,9); ‘To whom correspondence should be addressed. FAX: 201-235-6596. AJjBREVIATIONS: AA, arachidonic acid; BSA, bovine serum albumin; D-MEW Dulbecco’s Modified Eagle Medium; D-PBS, Dulbecco’s Phosphate Buffered Saline; ELISA, enzyme linked immunosorbent assay; IL-l& recombinant human interleukin-l/3 des met; PC, phosphatidylcholine; PGE,, prostaglandin E,; PLA,, phospholipase A,; RSF, rheumatoid synovial fibroblasts. 0006-291X192 $1.50 Copyright 0 1992 by Academic Pre.r.s, Inc. Ail rights of reproduction in any form resenvd.
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however, the PLAZ activities in RSF have not been further characterized.
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Several studies
in rabbit articular chrondrocytes have shown that IL-lb induces cell-associated PLA, activity (10) and also the production of a 14 kDa sPLA, (11,12), which was thought to be associated with eicosanoid production by these cells. Recently, however, an AA-PC selective, 85 kDa cPLA,, which is distinctly different from the sPLAZ’s, was found in monocytic cell lines (13). In these cell lines, the cPLAZ is thought to regulate the release of arachidonic acid for eicosanoid biosynthesis (14). Previous studies have demonstrated that sPLA, utilizes [i4C]oleate-labelled phosphatidylcholine
E. coli membranes, but not 2-([‘4C]-arachidonyl)
in detergent-containing
mixed micelles, as substrate while the
substrate preference of the cPLA, is reversed (13,15,16 and unpublished observations). Thus, substrate preference and calcium (micromolar sPLAZ [17]) requirement
for cPLA, [13] and millimolar
for
can be used as initial criteria to distinguish these enzymes.
Based on these criteria, this report provides the first evidence that a cPLA,, and not a sPLA,, may be involved in regulation of eicosanoid biosynthesis in RSF.
MATERIALS
AND METHODS
Materials: Cell culture reagents were obtained from GIBCO (Grand Island, NY). Collagenase/dispertase from A. iophanus and B. polymyxa, bovine pancreatic DNase I and hyaluronidase from ovine testes were obtained from Boehringer-Mannheim (Indianapolis, IN). Protease inhibitors, bovine serum albumin (BSA, essentially fatty acid-free), Triton X-100 and EGTA were obtained from Sigma Chemical Co. (St. Louis, MO). Silica gel impregnated-glass fiber thin layer chromatography (TLC) sheets were from Fisher Scientific (Pittsburgh, PA). Recombinant, human interleukin-18 des met (IL-@) was prepared by Dr. S. Roy, Hoffmann-La Roche. [‘4C]Oleate-labelled E. coli was obtained from Dr. R. Franson, Medical College of Virginia, Richmond, VA. Monoclonal and polyclonal antisera to human sPLA,, recombinant human sPLAZ and enzyme linked immunosorbent assay (ELISA) reagents were provided by Dr. R. Crow1 and W. Levin, Hoffmann-La Roche. Cell Culture: Human rheumatoid synovium was obtained from the elbow or knee of patients at the time of synovectomy or total joint replacement. The tissue was washed with Dulbecco’s Phosphate Buffered Saline (D-PBS) and then minced prior to enzymatic dispersion in Dulbecco’s Modified Eagle Medium (D-MEM) containing 0.5 mg/ml collagenase, 0.25 mg/ml DNase and 0.25 mg/ml hyaluronidase for 90 min. at 37°C in 5% CO, in air. Liberated cells were collected, washed twice in D-MEM, plated and maintained in 75 or 150 cm* flasks in D-MEM containing 15% fetal bovine serum, 12.5 mM sodium HEPES buffer, pH 7.5, 2.5 pg/ml fungizone and 50 pg/ml gentamycin sulfate at 37°C in 5% CO, in air. Culture medium was changed every 4-5 days and the cells were trypsinized and passaged upon reaching confluence. Cell Activation: Confluent cultures at passage six were used for these studies. The monolayers were washed twice with Gey’s Balanced Salt Solution prior to treatment with IGlp, 120 pg/ml, in Neuman-Tytell serumless medium containing 1% penicillinstreptomycin solution for 18 h. Duplicate flasks received 1% BSA/D-PBS vehicle as controls. The conditioned media were removed from the flasks following the incubation
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period and immediately stored at -80°C until assayed for PGE, and sPLA* content. The cell sheets were rinsed twice with trypsin-EDTA and allowed to detach for lo-15 min. at 37°C. The cells were resuspended in 10 ml of buffer (20 mM sodium HEPES buffer, pH 7.3, 140 mM NaCl, 25 PM leupeptin, 10 PM pepstatin A, 1 mM phenylmethylsulfonyl fluoride and 2 mM EGTA) and counted with a hemacytometer. The cells were then pelleted at 220 x g for 5 min. and washed once prior to resuspension in 1 ml of the same buffer. The cells were stored at -80°C prior to sonication, subcellular fractionation, and assays for cPLA,, sPLA, and protein. PGE, Assay: Aliquots of conditioned medium were assayed for levels of PGE, using a commercial microtiter enzyme immunoassay kit (Advanced Magnetics, Cambridge, MA). sPLA, Determinations: Conditioned medium was assayed for sPLAz content both by measuring sPLA, activity utilizing a [‘4C]oleate-labelled E. coli substrate (18), and by immunochemically quantitating sPLA, protein via an ELISA, as described previously (19). Purified recombinant sPLA, was used as a standard for quantitation (20). Subcellular Fractionation: After thawing, 1 ml suspensions of cells (approx. 6 x lo6 cells) were pulse sonicated on ice for 30 s using a Heat Systems model W225R sonicator at a power output of 35 w with a microprobe. Sonicates were centrifuged at 178,000 x g for 10 min. at 4°C in a Beckman Airfuge ultracentrifuge using an A-95 rotor. The supernatants (i.e., the cytosolic fractions) were reserved and stored at -80°C until analysis for PLA,. The pellets were washed once before resuspending in 200 ~1 of buffer and were also kept at -80°C until further analysis. cPLA, Assay: Aliquots of each subcellular fraction (approx. 0.03 mg protein) were incubated for 1 h with shaking at 37°C in a reaction mixture (final volume 100 ~1) consisting of mixed micelles of 50 PM L-o-l-palmitoyl-2-([l-‘4C]-arachidonyl) phosphatidylcholine ([i4C]PC, from New England Nuclear, Boston, MA, adjusted to a specific activity of 10 ~CJ~rnol with non-radioactive PC from Avanti Polar Lipids, Alabaster, AL) and 100 PM Triton X-100, with 50 mM sodium HEPES buffer, pH 7.3 at 37°C 125 mM NaCl and 2 mM net Ca 2t . Total lipids were extracted from the reaction mixture by the method of Bligh and Dyer (21) and 10 pg of nonradioactive, carrier arachidonic acid was added to the lipid extract. Enzymatically released [l-14C]arachidonic acid ([l-14C]AA) was separated from unreacted [14C]PC by TLC and quantitated by liquid scintillation spectrophotometry. Protein Assay: The protein content of cell fractions was determined according to the Bensadoun and Weinstein modification (22) of the method of Lowry using BSA as a standard.
RESULTS As shown in Table 1, IL-@ treatment of rheumatoid synovial fibroblasts from two patients resulted in a 35 fold increased production of PGE,. These data are consistent with previous reports (4,8). In order to ascertain whether this dramatic increase in PGE, levels was associated with increases in PLA2 activity, the activities of both the secretory and cell-associated forms of PLAz of these cells were measured. The amount of immunoreactive
sPLA2 protein secreted by RSF was the same in the presence or absence
of IL-l/3 treatment.
Likewise, treatment with IL-@ did not increase sPLA2 activity in the
conditioned medium when the [‘4C]oleate-labelled
E. coli membrane substrate was used
to assess activity. If anything, a modest decrease in activity was seen. Additionally, 714
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Table 1 Effects of IL-lb Treatment on cPLA, Activity and the Release of sPLA, and PGE, from RSF
Patient Number
Treatment of Incubated Cells
CONTROL
IL-l/3
PGE, Release from Cells (nmol PGE, / 106 cells)
(“g spL% / 106 cells)
SPLA, Specific Activityb
CPLA, Specific ActivityC
(nmol oleic acid / h / lo6 cells)
(nmol AA / h / lo6 cells)
5.2 * 1.5 (n = 8)
4.25 f 0.18
34.1 f 0.3
0.18 f 0.02
(n = 6)
(n = 8)
(n = 6)
181 f 36
4.31 f 0.39 (n = 5)
29.3 f 0.3
0.60 t 0.01
(n = 8) [p < O.OOl]d
CONTROL
SPLA, Proteina
16.5 + 3.9
[not signif.]
(not detected)
(n = 8)
(n = 8) [not signif.]
(n = 6)
[p < O.oOl]
6.92 0.7
0.23 + 0.03
(n = 8)
(n = 4)
4.5 f 0.5
1.04 t 0.16
2 IL-l/3
555 f 128
(not detected)
(n = 8) [p < O.OOl]
(n = 8) [p < o.oq
(n = 4) [p < O.Ol]
Released sPLA, protein, measured using ELISA specific for human group II 14 kDa sPLA,. b Released sPLA, activity, measured using [‘4C]-oleate- labelled E. coli substrate. ’ cPLA, activity in 178,000 x g supernatants of lysed cells, measured using 2-([‘4C]-arachidonyl) phosphatidylcholine / Triton X-100 mixed micellar substrate. d Mean + S.E. of the number of determinations indicated. The significance of the difference from control was analyzed statistically using Student’s two-tailed t-test.
1P treatment
of RSF caused no changes in the very low levels of either sPLA, protein or
enzymatic activity, assessed with the E. coli membrane substrate, in lysates, supernatants or pellets of these cells (data not shown). In contrast, cell-associated PLAZ activity in 178,000 x g cellular supernatants of lysed IL-lp treated cells was 3.3 to 4.4 fold greater than in supernatants from control cells when micellar AA-PC was used as the substrate. Furthermore,
PLA, activity, again assessed with the micellar AA-PC substrate, in the cell
pellet was increased 1.4 to 2.2 fold by the IL-lp
treatment (data not shown).
To characterize further the PLA, that was increased in RSF following IL-@ treatment, the Ca2+ requirement
of this PLAZ was evaluated (Figure 1). Enzymatic
activity was inhibited by an excess of EGTA, and was maximal at submicromolar concentrations
of Ca’+. Maximal activity in the presence of submicromolar
characteristic of cPLA, and contrasts with sPL& which has a millimolar
Ca2+ is a
requirement
for
Ca*’ (13,17). Thus, the increased amount of PGE, produced by IL-la treated RSF is 715
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,/r-I
-8
-7
log
-6 -5 -4 [ Ca2+ 1 ( M 1
-3
-2
Enzymatic activity was measured in 178,000 x g supernatants derived from Figure 1. IL-@ treated cells (0.3 mg/ml protein), using [‘4C]AA-PC / Triton X-100 mixed micellar substrate. The reaction mixture contained 1 mM EGTA and increasing concentrations
(0 - 3 mM) of added CaCl,. The concentrations of free calcium ion ( [Ca’+rJ ) 5 1 PM were calculated using a stability constant of 1.00 x 10.’ M for the Ca*+-EGTA complex in 50 mM HEPES buffer, pH 7.3, as previously reported (29). The [Ca*‘J’s above 1 PM were estimated by subtracting 1 mM EGTA from the added [CaCl,]. Each point
represents the mean + SE. of triplicate determinations.
associated with an elevation in the activity of a cell-associated PLA2 with the characteristics of the recently described cPLA,, rather than with changes in sPLA,.
DISCUSSION
Two PLAz activities in IL-@ stimulated RSF were distinguished by substrate preference and immunoreactivity.
In addition, we demonstrated that the calcium
sensitivity of the PLA, in the 178,000 x g cellular supernatant was in the submicromolar range. The results demonstrate that neither the sPLA, protein, measured by ELISA, nor its enzymatic activity, assessed with the E. coli membrane substrate, was increased in the incubation media or in subcellular fractions after IL-@ treatment.
In contrast, the
activity of a cell-associated PLA,, with characteristics (e.g., substrate preference and calcium requirement)
similar to that of the 85 kDa cPLA, from monocytic cell lines (23),
was increased 3 to 4 fold following stimulation with IL-@. In the IL-@ treated RSF, the 3 to 4 fold increase in the activity of the cell supernatant PLAz was associated with a 35 fold increase in the production of PGE,. This may suggest the possible involvement of other phospholipases in the release of arachidonic acid that is subsequently metabolized to PGE,. Alternatively,
this apparent
discrepancy may simply indicate that the activity measured in vitro with the nonphysiologic micellar AA-PC substrate does not accurately reflect the in vivo catalytic potential of the cytosolic PLA2. Upregulation
of cyclooxygenase by IL-lp could also
contribute to the increased production of PGE, in these RSF (7,24,25). 716
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In contrast to our findings, Gilman ad. from monocyte conditioned throughout
RESEARCH COMMUNICATIONS
(8) reported that human IL-@, purified
medium, stimulated the release of PLAz and PGE,,
a 48 h time-course, from synoviocytes cultured from arthritic patients after 2-
7 passages. The secreted PLAz in that study was optimally active at 5 mM CaZt and at pH 7.4, and thus is similar to the 14 kDa sPLA2 that has been purified from human synovial fluid (17,26,27); cell-associated PLA2 was not measured. The reasons for the discrepancy in the findings between these two studies are not known; however, Ballou a al. (28) recently reported that human dermal fibroblasts do not produce sPLA, in response to recombinant
IL-@.
In addition, our findings, together with those of others investigating PLA2 and arachidonic acid metabolism in chrondrocytes (10,ll)
and synoviocytes (8), suggest
possible roles for these cell types in rheumatoid joints involving the activity of PLA,. response to IL-@ (and possibly other inflammatory
In
stimuli), chrondrocytes secrete sPLA2
indicating that these cells may be a cellular source for PLA, in synovial fluid.
While
possibly not a source of sPLA,, synoviocytes may produce eicosanoids via IL-@ stimulation of cPLA, activity. Thus, both processes may contribute to the inflammatory processes associated with rheumatoid joints. ACKNOWLEDGMENT
We are grateful to Dr. Steven Stuchin of the Hospital for Joint Disease, New York, NY, for kindly providing the rheumatoid synovia used in these studies.
REFERENCES
1. 2.
Dinarello,
C.A. (1988) FASEB J. 2, 108-115.
Wood, D.D., Ihrie, E.J., Dinarello, 26, 975-983.
C.A., and Cohen, P.L. (1983) Arthritis Rheum.
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Krane, S.M., Dayer, J-M., Simon, L.S., and Byrne, S. (1985) Collagen Relat. Res. 5, 99-117.
4. 5.
Dayer, J-M., and Demczuck, S. (1984) Springer Semin. Immunopathol. 7, 387-413. Dayer, J-M., Krane, SM., Russel, R.G.G., and Robinson, D.R. (1976) Proc. Natl. Acad. Sci. 73, 945-949. Dayer, J-M, Robinson, D.R., and Krane, S.M. (1977) J. Exp. Med. 145, 1399-1404. Case, J.P., Maier, J.A.M., Hla, T., Sano, H., Crofford, L.J., Maciag, T., and Wilder, R.L. (1990) Arthritis and Rheum. 33, S17. Gilman, S.C., Chang, J., Zeigler, P.R., Uhl, J., and Mochan, E. (1988) Arthritis and Rheum. 31, 126-130. Godfrey, R.W., Johnson, W.J., Newman, T. and Hoffstein, S.T. (1988) Prostaglandins 35, 107-114. Chang, J., Gilman, SC., and Lewis, A.J. (1986) J. Immunol. 136, 1283-1287.
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17. 18. 19. 20.
21. 22. 23. 24.
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Kerr, J.S., Stevens, T.M., Davis, G.L., McLaughlin, J.A., Harris, R.R. (1989) Biochem. Biophys. Res. Commun. 165, 1079-1084. Lyons-Giordano, B., David, G.L., Galbraith, W., Pratta, M.A., and Arner, E.C. (1989) Biochem. Biophys. Res. Commun. 164, 488-495. Clark, J.D., Milona, N., and Knopf. J.L. (1990) Proc. Natl. Acad. Sci. 87,77087712. Lin, L.-L., Lin, A.Y., and Knopf, J.L. (1991) Abstracts of the XIth Washington International Spring Symposium on Prostaglandins, Leukotrienes, Lipoxins & PAF, 90. Kramer, R.M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow, E.P., Tizard, R., and Pepinsky, R.B. (1989) J. Biol. Chem. 264, 5768-5775. Marshall, L.A., and McCarte-Roshak, A. (1991) Abstracts of the XIth Washington International Spring Symposium on Prostaglandins, Leukotrienes, Lipoxins & PAF, 154. Hara, S., Kudo, I., Chang, H. W., Matsuta, K., Miyamoto, T., and Inoue, K. (1989) J. Biochem. 105, 395-399. Hope, W.C., Patel, B.J., Fiedler-Nagy, C., and Wittreich, B.H. (1990) Inflammation 14, 543-559. Stoner, C.R., Reik, L.M., Donohue, M., Levin, W., and Crowl, R.M. (1991) J. Immunol. Methods 145, 127-136. Levin, W., Daniel, R.F., Stoner, C.R., Stoller, T.J., Swanson, J.A.W., Angellio, Y.M., Familletti, P.C. and Crowl, R.M. (1992) Protein Expression and Purification, in press. Bligh, E.G., and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241-250. Clark, J.D., Lin, L.-L., Kriz, R.W., Ramesha, C.S., Sultzman, L.A., Lin, A.Y., Milona, N., and Knopf, J.L. (1991) Cell 65, 1043-1051. O’Neill, L.A.J., Barret, M.L., and Lewis, G.P. (1987) FEBS Lett. 212, 35-39.
25.
Raz, A., Wyche, A., Siegel, N., and Needleman, P. (1988) J. Biol. Chem. 250, 3022-3028.
26.
Seilhamer, J.J., Pruzanski, W., Vadas, P., Plant, S., Miller, J.A., Kloss, J., and Johnson L.K. (1989) J. Biol. Chem. 264, 5335-5338. Lai, C.-Y., and Wada, K. (1988) Biochem. Biophys. Res. Commun. 157, 488-493. Ballou, L.R., Barker, S.C., Postlethwaite, A.E., and Kang A.H. (1990) J. Immunol. 145, 4245-4251. Tsien, R.Y. (1980) Biochemistry 19, 2396-2404.
27. 28. 29.
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184,
No.
April
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1992
2, 1992
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COMMUNICATIONS
712-718
Pages
INTERLEUKIN-lp STIMULATES CYTOSOLIC PHOSPHOLIPASE IN RHEUMATOID SYNOVIAL FIBROBLASTS
A,
Keren I. Hulkower, William C. Hope, Ting Chen, Celia M. Anderson, John W. Coffey and Douglas W. Morgan’ Department
Received
March
11,
of Pharmacology, Hoffmann La-Roche Inc., 340 Kingsland St. Nutley, NJ 07110
1992
SUMMARY: Phospholipase A2 (PLA,) activities in rheumatoid synovial fibroblasts (RSF) stimulated with interleukin-l/3 (IL@) were investigated. RSF incubated in the presence of IL-l@ (120 pg/ml) for 18 h secreted 35 fold more PGE, than did those incubated without IL-@. IL-@ treatment did not increase the level of secretory PLA, (sPLA,) activity or sPLA, protein in the conditioned medium or subcellular fractions of lysed RSF. In contrast, the cell-associated PLA, activity increased 3 to 4 fold in IL-la stimulated RSF when compared with the control. The IL-l/3 stimulated, cell-associated PLA, required submicromolar concentrations of calcium for activity, a characteristic consistent with the calcium sensitivity of cytosolic PLA, (cPLA,) activity reported in other cell types, such as U937 cells. These findings demonstrate that an elevation in a cytosolic PLA,, rather than a sPLA,, is associated with increased PGE, production in IL0 1992Academic Press,1°C. lp stimulated RSF.
IL-la
is a proinflammatory,
found abundantly in rheumatoid of rheumatoid
growth-promoting
cytokine (1). This cytokine is
synovial fluid (2,3) and is associated with the pathology
arthritis (4). One important biological role for IL-@ in rheumatoid
arthritis may be to increase PGE, biosynthesis (5). In rheumatoid studies have demonstrated
synoviocytes, several
that IL-lp stimulates the release of arachidonic acid, PLAz
activities, and the production of PGE, (6-8). A calcium-sensitive PLA, associated with PGE, production by IL-lp stimulated human synovial fibroblasts has been reported (8,9); ‘To whom correspondence should be addressed. FAX: 201-235-6596. AJjBREVIATIONS: AA, arachidonic acid; BSA, bovine serum albumin; D-MEW Dulbecco’s Modified Eagle Medium; D-PBS, Dulbecco’s Phosphate Buffered Saline; ELISA, enzyme linked immunosorbent assay; IL-l& recombinant human interleukin-l/3 des met; PC, phosphatidylcholine; PGE,, prostaglandin E,; PLA,, phospholipase A,; RSF, rheumatoid synovial fibroblasts. 0006-291X192 $1.50 Copyright 0 1992 by Academic Pre.r.s, Inc. Ail rights of reproduction in any form resenvd.
712
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1992
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however, the PLAZ activities in RSF have not been further characterized.
COMMUNICATIONS
Several studies
in rabbit articular chrondrocytes have shown that IL-lb induces cell-associated PLA, activity (10) and also the production of a 14 kDa sPLA, (11,12), which was thought to be associated with eicosanoid production by these cells. Recently, however, an AA-PC selective, 85 kDa cPLA,, which is distinctly different from the sPLAZ’s, was found in monocytic cell lines (13). In these cell lines, the cPLAZ is thought to regulate the release of arachidonic acid for eicosanoid biosynthesis (14). Previous studies have demonstrated that sPLA, utilizes [i4C]oleate-labelled phosphatidylcholine
E. coli membranes, but not 2-([‘4C]-arachidonyl)
in detergent-containing
mixed micelles, as substrate while the
substrate preference of the cPLA, is reversed (13,15,16 and unpublished observations). Thus, substrate preference and calcium (micromolar sPLAZ [17]) requirement
for cPLA, [13] and millimolar
for
can be used as initial criteria to distinguish these enzymes.
Based on these criteria, this report provides the first evidence that a cPLA,, and not a sPLA,, may be involved in regulation of eicosanoid biosynthesis in RSF.
MATERIALS
AND METHODS
Materials: Cell culture reagents were obtained from GIBCO (Grand Island, NY). Collagenase/dispertase from A. iophanus and B. polymyxa, bovine pancreatic DNase I and hyaluronidase from ovine testes were obtained from Boehringer-Mannheim (Indianapolis, IN). Protease inhibitors, bovine serum albumin (BSA, essentially fatty acid-free), Triton X-100 and EGTA were obtained from Sigma Chemical Co. (St. Louis, MO). Silica gel impregnated-glass fiber thin layer chromatography (TLC) sheets were from Fisher Scientific (Pittsburgh, PA). Recombinant, human interleukin-18 des met (IL-@) was prepared by Dr. S. Roy, Hoffmann-La Roche. [‘4C]Oleate-labelled E. coli was obtained from Dr. R. Franson, Medical College of Virginia, Richmond, VA. Monoclonal and polyclonal antisera to human sPLA,, recombinant human sPLAZ and enzyme linked immunosorbent assay (ELISA) reagents were provided by Dr. R. Crow1 and W. Levin, Hoffmann-La Roche. Cell Culture: Human rheumatoid synovium was obtained from the elbow or knee of patients at the time of synovectomy or total joint replacement. The tissue was washed with Dulbecco’s Phosphate Buffered Saline (D-PBS) and then minced prior to enzymatic dispersion in Dulbecco’s Modified Eagle Medium (D-MEM) containing 0.5 mg/ml collagenase, 0.25 mg/ml DNase and 0.25 mg/ml hyaluronidase for 90 min. at 37°C in 5% CO, in air. Liberated cells were collected, washed twice in D-MEM, plated and maintained in 75 or 150 cm* flasks in D-MEM containing 15% fetal bovine serum, 12.5 mM sodium HEPES buffer, pH 7.5, 2.5 pg/ml fungizone and 50 pg/ml gentamycin sulfate at 37°C in 5% CO, in air. Culture medium was changed every 4-5 days and the cells were trypsinized and passaged upon reaching confluence. Cell Activation: Confluent cultures at passage six were used for these studies. The monolayers were washed twice with Gey’s Balanced Salt Solution prior to treatment with IGlp, 120 pg/ml, in Neuman-Tytell serumless medium containing 1% penicillinstreptomycin solution for 18 h. Duplicate flasks received 1% BSA/D-PBS vehicle as controls. The conditioned media were removed from the flasks following the incubation
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period and immediately stored at -80°C until assayed for PGE, and sPLA* content. The cell sheets were rinsed twice with trypsin-EDTA and allowed to detach for lo-15 min. at 37°C. The cells were resuspended in 10 ml of buffer (20 mM sodium HEPES buffer, pH 7.3, 140 mM NaCl, 25 PM leupeptin, 10 PM pepstatin A, 1 mM phenylmethylsulfonyl fluoride and 2 mM EGTA) and counted with a hemacytometer. The cells were then pelleted at 220 x g for 5 min. and washed once prior to resuspension in 1 ml of the same buffer. The cells were stored at -80°C prior to sonication, subcellular fractionation, and assays for cPLA,, sPLA, and protein. PGE, Assay: Aliquots of conditioned medium were assayed for levels of PGE, using a commercial microtiter enzyme immunoassay kit (Advanced Magnetics, Cambridge, MA). sPLA, Determinations: Conditioned medium was assayed for sPLAz content both by measuring sPLA, activity utilizing a [‘4C]oleate-labelled E. coli substrate (18), and by immunochemically quantitating sPLA, protein via an ELISA, as described previously (19). Purified recombinant sPLA, was used as a standard for quantitation (20). Subcellular Fractionation: After thawing, 1 ml suspensions of cells (approx. 6 x lo6 cells) were pulse sonicated on ice for 30 s using a Heat Systems model W225R sonicator at a power output of 35 w with a microprobe. Sonicates were centrifuged at 178,000 x g for 10 min. at 4°C in a Beckman Airfuge ultracentrifuge using an A-95 rotor. The supernatants (i.e., the cytosolic fractions) were reserved and stored at -80°C until analysis for PLA,. The pellets were washed once before resuspending in 200 ~1 of buffer and were also kept at -80°C until further analysis. cPLA, Assay: Aliquots of each subcellular fraction (approx. 0.03 mg protein) were incubated for 1 h with shaking at 37°C in a reaction mixture (final volume 100 ~1) consisting of mixed micelles of 50 PM L-o-l-palmitoyl-2-([l-‘4C]-arachidonyl) phosphatidylcholine ([i4C]PC, from New England Nuclear, Boston, MA, adjusted to a specific activity of 10 ~CJ~rnol with non-radioactive PC from Avanti Polar Lipids, Alabaster, AL) and 100 PM Triton X-100, with 50 mM sodium HEPES buffer, pH 7.3 at 37°C 125 mM NaCl and 2 mM net Ca 2t . Total lipids were extracted from the reaction mixture by the method of Bligh and Dyer (21) and 10 pg of nonradioactive, carrier arachidonic acid was added to the lipid extract. Enzymatically released [l-14C]arachidonic acid ([l-14C]AA) was separated from unreacted [14C]PC by TLC and quantitated by liquid scintillation spectrophotometry. Protein Assay: The protein content of cell fractions was determined according to the Bensadoun and Weinstein modification (22) of the method of Lowry using BSA as a standard.
RESULTS As shown in Table 1, IL-@ treatment of rheumatoid synovial fibroblasts from two patients resulted in a 35 fold increased production of PGE,. These data are consistent with previous reports (4,8). In order to ascertain whether this dramatic increase in PGE, levels was associated with increases in PLA2 activity, the activities of both the secretory and cell-associated forms of PLAz of these cells were measured. The amount of immunoreactive
sPLA2 protein secreted by RSF was the same in the presence or absence
of IL-l/3 treatment.
Likewise, treatment with IL-@ did not increase sPLA2 activity in the
conditioned medium when the [‘4C]oleate-labelled
E. coli membrane substrate was used
to assess activity. If anything, a modest decrease in activity was seen. Additionally, 714
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Table 1 Effects of IL-lb Treatment on cPLA, Activity and the Release of sPLA, and PGE, from RSF
Patient Number
Treatment of Incubated Cells
CONTROL
IL-l/3
PGE, Release from Cells (nmol PGE, / 106 cells)
(“g spL% / 106 cells)
SPLA, Specific Activityb
CPLA, Specific ActivityC
(nmol oleic acid / h / lo6 cells)
(nmol AA / h / lo6 cells)
5.2 * 1.5 (n = 8)
4.25 f 0.18
34.1 f 0.3
0.18 f 0.02
(n = 6)
(n = 8)
(n = 6)
181 f 36
4.31 f 0.39 (n = 5)
29.3 f 0.3
0.60 t 0.01
(n = 8) [p < O.OOl]d
CONTROL
SPLA, Proteina
16.5 + 3.9
[not signif.]
(not detected)
(n = 8)
(n = 8) [not signif.]
(n = 6)
[p < O.oOl]
6.92 0.7
0.23 + 0.03
(n = 8)
(n = 4)
4.5 f 0.5
1.04 t 0.16
2 IL-l/3
555 f 128
(not detected)
(n = 8) [p < O.OOl]
(n = 8) [p < o.oq
(n = 4) [p < O.Ol]
Released sPLA, protein, measured using ELISA specific for human group II 14 kDa sPLA,. b Released sPLA, activity, measured using [‘4C]-oleate- labelled E. coli substrate. ’ cPLA, activity in 178,000 x g supernatants of lysed cells, measured using 2-([‘4C]-arachidonyl) phosphatidylcholine / Triton X-100 mixed micellar substrate. d Mean + S.E. of the number of determinations indicated. The significance of the difference from control was analyzed statistically using Student’s two-tailed t-test.
1P treatment
of RSF caused no changes in the very low levels of either sPLA, protein or
enzymatic activity, assessed with the E. coli membrane substrate, in lysates, supernatants or pellets of these cells (data not shown). In contrast, cell-associated PLAZ activity in 178,000 x g cellular supernatants of lysed IL-lp treated cells was 3.3 to 4.4 fold greater than in supernatants from control cells when micellar AA-PC was used as the substrate. Furthermore,
PLA, activity, again assessed with the micellar AA-PC substrate, in the cell
pellet was increased 1.4 to 2.2 fold by the IL-lp
treatment (data not shown).
To characterize further the PLA, that was increased in RSF following IL-@ treatment, the Ca2+ requirement
of this PLAZ was evaluated (Figure 1). Enzymatic
activity was inhibited by an excess of EGTA, and was maximal at submicromolar concentrations
of Ca’+. Maximal activity in the presence of submicromolar
characteristic of cPLA, and contrasts with sPL& which has a millimolar
Ca2+ is a
requirement
for
Ca*’ (13,17). Thus, the increased amount of PGE, produced by IL-la treated RSF is 715
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,/r-I
-8
-7
log
-6 -5 -4 [ Ca2+ 1 ( M 1
-3
-2
Enzymatic activity was measured in 178,000 x g supernatants derived from Figure 1. IL-@ treated cells (0.3 mg/ml protein), using [‘4C]AA-PC / Triton X-100 mixed micellar substrate. The reaction mixture contained 1 mM EGTA and increasing concentrations
(0 - 3 mM) of added CaCl,. The concentrations of free calcium ion ( [Ca’+rJ ) 5 1 PM were calculated using a stability constant of 1.00 x 10.’ M for the Ca*+-EGTA complex in 50 mM HEPES buffer, pH 7.3, as previously reported (29). The [Ca*‘J’s above 1 PM were estimated by subtracting 1 mM EGTA from the added [CaCl,]. Each point
represents the mean + SE. of triplicate determinations.
associated with an elevation in the activity of a cell-associated PLA2 with the characteristics of the recently described cPLA,, rather than with changes in sPLA,.
DISCUSSION
Two PLAz activities in IL-@ stimulated RSF were distinguished by substrate preference and immunoreactivity.
In addition, we demonstrated that the calcium
sensitivity of the PLA, in the 178,000 x g cellular supernatant was in the submicromolar range. The results demonstrate that neither the sPLA, protein, measured by ELISA, nor its enzymatic activity, assessed with the E. coli membrane substrate, was increased in the incubation media or in subcellular fractions after IL-@ treatment.
In contrast, the
activity of a cell-associated PLA,, with characteristics (e.g., substrate preference and calcium requirement)
similar to that of the 85 kDa cPLA, from monocytic cell lines (23),
was increased 3 to 4 fold following stimulation with IL-@. In the IL-@ treated RSF, the 3 to 4 fold increase in the activity of the cell supernatant PLAz was associated with a 35 fold increase in the production of PGE,. This may suggest the possible involvement of other phospholipases in the release of arachidonic acid that is subsequently metabolized to PGE,. Alternatively,
this apparent
discrepancy may simply indicate that the activity measured in vitro with the nonphysiologic micellar AA-PC substrate does not accurately reflect the in vivo catalytic potential of the cytosolic PLA2. Upregulation
of cyclooxygenase by IL-lp could also
contribute to the increased production of PGE, in these RSF (7,24,25). 716
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In contrast to our findings, Gilman ad. from monocyte conditioned throughout
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(8) reported that human IL-@, purified
medium, stimulated the release of PLAz and PGE,,
a 48 h time-course, from synoviocytes cultured from arthritic patients after 2-
7 passages. The secreted PLAz in that study was optimally active at 5 mM CaZt and at pH 7.4, and thus is similar to the 14 kDa sPLA2 that has been purified from human synovial fluid (17,26,27); cell-associated PLA2 was not measured. The reasons for the discrepancy in the findings between these two studies are not known; however, Ballou a al. (28) recently reported that human dermal fibroblasts do not produce sPLA, in response to recombinant
IL-@.
In addition, our findings, together with those of others investigating PLA2 and arachidonic acid metabolism in chrondrocytes (10,ll)
and synoviocytes (8), suggest
possible roles for these cell types in rheumatoid joints involving the activity of PLA,. response to IL-@ (and possibly other inflammatory
In
stimuli), chrondrocytes secrete sPLA2
indicating that these cells may be a cellular source for PLA, in synovial fluid.
While
possibly not a source of sPLA,, synoviocytes may produce eicosanoids via IL-@ stimulation of cPLA, activity. Thus, both processes may contribute to the inflammatory processes associated with rheumatoid joints. ACKNOWLEDGMENT
We are grateful to Dr. Steven Stuchin of the Hospital for Joint Disease, New York, NY, for kindly providing the rheumatoid synovia used in these studies.
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