Inflammation, Vol. 15, No. 2, 1991

EFFECTS OF RECOMBINANT HUMAN IL-1/3 ON PRODUCTION OF PROSTAGLANDIN E2, LEUKOTRIENE B4, NAG, AND SUPEROXIDE BY HUMAN SYNOVIAL CELLS AND CHONDROCYTES TOHRU

TAWARA,

MASASHI

MASAO

NOBUNAGA,

SHINGU, and

T A K A S H I NAONO

Department of Clinical Immunology, Medical Institute of Bioregulation Kyushu University 69 Department of Orthopedics, Beppu National Hospital Beppu, Japan 874

Abstract--The effects of recombinant human IL-1B on the production of prostaglandin Ee (PGE2), leukotriene B4 (LTB4), N-acetyl-/3-D-glucosaminidase (NAG), and superoxide by synovial cells and chondrocytes derived from osteoarthritis patients were determined. IL-I/3 markedly enhanced PGEz production in chondrocytes and, to the lesser extent, in synovial cells. Synovial cells and chondrocytes spontaneously released LTB4 into culture medium and IL-1/3 significantly inhibited LTB4 production by these cells. IL-1/3 significantly suppressed the release of NAG and superoxide by synovial cells, whereas it significantly enhanced the production of NAG and superoxide by chondrocytes. Production of intracellular superoxide dismutase by synovial cells was significantly enhanced on incubation with IL-1/3, but that of chondrocytes was not altered. IL-6, unlike IL-1/3, significantly suppressed the production of NAG and superoxide by synovial cells and chondrocytes. These results suggest that IL-1 has differing effects on the release of mediators by synovial cells and chondrocytes and that these cells also vary in their responses to IL-1/3 and IL-6.

INTRODUCTION S e v e r a l species o f h u m a n IL-1 h a v e b e e n d e s c r i b e d (1) and c o m p l e m e n t a r y D N A s for t w o distinct IL-1 species, t e r m e d IL-lc~ and IL-1/3, h a v e b e e n isolated (2). IL-1 c~ and IL-1/3 are m o n o c y t e / m a c r o p h a g e - d e r i v e d cytokines w h i c h , in addition to their effects on b l o o d and endothelial cells, are n o w r e c o g n i z e d 145 0360-399719110400-0145506.50/0 9 1991 Plenum Publishing Corporation

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as potent activators of connective tissue cells. IL-1 is thought to play an important role in chronic inflammation, particularly in rheumatoid arthritis (RA). It has been shown that IL-1 can induce cartilage destruction not only through a direct action on chondrocytes and matrix (3, 4), but also by stimulating the production of collagenase (5-8) and prostaglandin E 2 (PGEz) (5, 8-10) by synovial cells and chondrocytes; metalloproteinases by chondrocytes (11); and proteinases such as plasminogen activator (12, 13) and N-acetyl-/3-D-glucosaminidase (NAG) (14) by synovial cells. IL-1 also has been shown to promote the synthesis of extracellular matrix components such as hyaluronate (15), collagen (16), and fibronectin (16) by synovial cells. Fibronectin is a component of rheumatoid pannus (17) and may promote pannus development. It has been reported that PGE2; collagenase, a metalloproteinase; plasminogen activator, a serine proteinase; and NAG, an acid glycosidase; have important roles in tissue destruction (18-21). IL-1 has been shown to induce bone resorption (22). Thus, a number of the pathogenetic mechanisms of the joint destruction in RA may be influenced by IL-1. It has been suggested that IL-6, which is produced by various cell types, including monocytes, is a central mediator of host defense, affecting growth and differentiation of a wide spectrum of cells. It induces the final step in the maturation of B lymphocytes into immunoglobulin-secreting cells (23). Recently, it has been demonstrated that synovial cells are a potent source of IL-6, which contribute to important manifestations of inflammatory joint diseases (24). We have studied the effects of IL-1 on the production of superoxide, NAG, PGE2, leukotriene B 4 (LTB4), and superoxide dismutase (SOD) by synovial cells and chondrocytes. The effects of IL-6 on the release of NAG and superoxide from synovial cells and chondrocytes also were investigated.

M A T E R I A L S AND M E T H O D S Culture of Human Synovial Cells and Chondrocytes. Humansynovial tissue and cartilage were obtained from patients with osteoarthritis who were undergoingtotal knee replacement. These tissues were enzymaticallydissociated using 0.2% collagenase (Sigma, St. Louis, Missouri) and 0.25% trypsin (Chiba Laboratories, Chiba, Japan) and the cells subsequently cultured in RPMI 1640 medium (Gibco, Grand Island, New York) with 10% fetal calf serum (FCS) (Gibco), 100 units/ml penicillin, 100 #g/ml streptomycin, 25 ng/ml fungizone, and 2 mM L-glutamine(Gibco) according to methods previouslydescribed (8, 25). Cells were cultured in 24-well plates for three to five days until the cells reached confluency; all experiments were performed on primary and secondary cultures. The cells obtained were mostlysynovialfibroblasts. Cell numbers in each well after three to five days of culture averaged 1.8-2 • 105 cells/well in synovialcells and 1,2-1.5 x lOS/well in chondrocytes. Cytokines. Recombinanthuman IL-1/~(2 • 107 units/mg, 1 • 10 6 units/ml) was kindly

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supplied by Ohtuka Pharmaceutical Co., Ltd. (Tokushima, Japan) (26). Recombinant human IL-6 (5 • 10 6 units/rag) was a kind gift from Toshio Hirano, Osaka University, Osaka, Japan (27). Measurement of PGEz, LTB4, and NAG. In 24-well plates 0.4 ml of synovial cells and chondrocytes were plated and precultured. After the medium was replaced with 0.4 ml of fresh medium containing 10% FCS, antibiotics, and L-glutamine, various concentrations of IL-1/3 or IL-6 were added to each well and cultured for three days in a 5% CO2 incubator at 37~ The supernatants were obtained and frozen at - 8 0 ~ until measured. PGE2 and LTB4 were assayed using RIA kits (Amersham International, Amersham, UK). NAG was measured as previously described (28). Briefly, 0.2 ml of sample, diluted 1 : 1 with 0.1 ml of 0.1 M citrate buffer, pH 4.5, in glass tubes, were incubated with 3.6 mM paranitrophenyl-2-acetamide-2-deoxy-~-D-glucopymnoside (Sigma) as substrate for 4 h at 37~ The reaction was terminated with 0.3 ml of 1 M borate buffer, pH 9.8, and enzyme activity was determined colorimetrically at 405 nm as paranitrophenol released from the substrate. Values were corrected for background levels in medium. The chondrocytes in 24-well dishes were cultured with indomethacin (a kind gift from Sumitomo Pharmaceutical Co., Ltd., Osaka, Japan) or antihuman IL-1/3 antiserum (gift of Ohtuka Pharmaceutical) (29) for three days in the presence or absence of IL-1/3, and the supematants were measured for NAG activity. Superoxide Generation. Superoxide generation by synovial cells and chondrocytes was measured as previously described (30, 31). Cells were precultured in a 24-well dishes for three to five days. Medium was discarded, the cells were washed with Hanks' balanced salt solution (HBSS), and 0.4 ml of HBSS were added in each well. After the addition of varying concentrations of cytokines and ferricytochrome c (horse liver, grade III, Sigma) at a final concentration of 55 #M in a total volume of 0.5 ml, the cells were incubated for 18 h in a CO 2 incubator. The absorbance of the supernatants was measured at 550 nm. The amount of SOD-inhibitable cytochrome c reduction was calculated (32). The effects of indomethacin or antihuman IL-1/3 antibody on superoxide generation by chondrocytes were determined in the presence or absence of IL-lt3. SOD Activity. After the removal of supematants for the measurement of superoxide generation, the cells were washed once with HBSS and harvested by treatment with 0.25% trypsin (Chiba). Detached cells were washed with HBSS and the pellets then were suspended in 1 ml HBSS. The cells were sonicated using the Sonifier B-12 (Branson) at 50 W for 20 sec in an ice bath, centrifuged at 3000 rpm for 20 min, and the supematants were obtained. SOD activity of the supernatants was measured by the inhibition of ferricytochrome c reduction mediated by hypoxanthine (Sigma) (80#M) and xanthine oxidase (Sigma) (0.005-0.01 units/ml) (33).

RESULTS

Effects o f lL-1t3 on Production o f PGE2 and LTB 4. IL-1/3 at c o n c e n t r a t i o n s r a n g i n g f r o m 3 3 . 3 to 1200 u n i t s / m l s i g n i f i c a n t l y e n h a n c e d P G E 2 p r o d u c tion by synovial cells and chondrocytes. Mean percent increase of PGE 2 release b y c h o n d r o c y t e s f o l l o w i n g i n c u b a t i o n w i t h IL-1 at c o n c e n t r a t i o n s o f 3 3 . 3 , 2 0 0 , a n d 1200 u n i t s / m l w e r e 8 0 7 % , 7 7 0 % , a n d 6 5 1 % , r e s p e c t i v e l y , a n d t h o s e b y synovial cells were 116%, 49%, and 52%, respectively. The differences b e t w e e n c h o n d r o c y t e s a n d s y n o v i a l ceils at e a c h IL-1 c o n c e n t r a t i o n w e r e statistically s i g n i f i c a n t ( P < 0 . 0 0 1 ) ( d a t a n o t s h o w n ) . S y n o v i a l cells a n d c h o n d r o c y t e s w i t h o u t IL-1/~ p r o d u c e d 3 . 0 +_ 0 . 8 a n d 3 . 4 _+ 0 . 9 p g / t u b e ( m e a n a n d SD o f six e x p e r i m e n t s ) o f L T B 4 i n t o t h e c u l t u r e m e d i u m , r e s p e c t i v e l y . LTB4

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production was significantly suppressed by 1200 units/ml IL-1B in the case of synovial cells and by 33.3 and 200 units/ml IL-1/3 in the case of chondrocytes (Table 1). There was no significant detachment of synovial cells and chondrocytes when the cells were observed after three days of culture in the presence of IL-1/5 or IL-6 at any concentrations used, suggesting that neither IL-1/3 nor IL-6 were cytotoxic to the cells. There were no changes of cell numbers during three days of culture in synovial cells and chondrocytes in the presence or absence of IL-1/3. Effects of lL-113 or IL-6 on NAG Release. NAG activity released by synovial cells in the presence of 33.3 and 200 units/ml IL-1B was not significantly different from control level, whereas it was significantly inhibited by 1200 units/ ml IL-1B. NAG release by chondrocytes, on the contrary, was significantly enhanced by 200 and 1200 units/ml IL-1/3. IL-6 significantly suppressed NAG release by synovial cells at 16.7 units/ml but did not show any effects on NAG release by synovial cells at concentrations of 2.8 and 100 units/ml. IL-6, on the other hand, did not influence NAG release by chondrocytes at concentrations of 2.8, 16.7, and 100 units/ml (Table 2). Effects of lL-113 or IL-6 on Superoxide Release. Synovial cells and chondrocytes spontaneously produced 1.74 + 0.49 and 3.47 ___ 0.28 nmol (mean and SD of six experiments) of superoxide respectively into the medium during 18 h of culture. IL-1B at concentrations of 33.3 and 200 units/ml significantly suppressed superoxide generation by synovial cells, while there were no suppressive effects at 1200 units/ml. IL-1/3 at 200 and 1200 units/ml significantly stimulated chondrocyte superoxide release, whereas 33.3 units/ml IL-1B did not influence such release. IL-6, on the other hand, significantly inhibited superoxide production by synovial cells and chondrocytes at concentrations of 16.7 and 100 units/ml; 2.8 units/ml IL-6 did not show any effect on superoxide generation by these cell types (Table 3).

Table 1. Effects of IL-1B on Production of PGE2 and LTB4 by Synovial Cells and Chondrocytes ~ PGE2 (pg/tube)

LTB4 (pg/tube)

m-l~3 (units/ml) 0 33.3 200 1200

Synovial ceils 44.2 95.3 66.0 67.3

+ 45: 4-

5.0 23.4**** 10.7"** 10.4"**

Chondrocytes 35.3 320.0 307.5 265.0

4- 7.3 + 25.3**** + 46.7**** 4- 36.2****

Synovial cells 3.0 2.7 2.5 1.7

+ 0.8 5:1.4 5:0.8 4- 0.1"

Chondrocytes 3.4 2.2 2.1 2.8

4- 0.9 4- 0.6* 4- 0.5*** 4- 0.6

aSynovial cells or chondrocytes in 24-well dishes were cultured with or without IL-1/3 for three days, and the supematants were measured for PGE2 and LTB4. Each datum is the mean and SD of six separate experiments. Statistical significance calculated by Student's t test; *P < 0.05, ***P < 0.01, ****P < 0.001 (vs. control).

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Table 2. Effect of IL-1/3 or IL-6 on NAG release by Synovial Cells and Chondrocytes ~ NAG(~mol/h) IL-1/~ or IL-6

(units/ml)

Synovial cells

Chondrocytes

IL-IB

0 (control) 33.3 200 1200

86.3 107.6 79.0 69.7

+ 18.6 + 14.6 ___31.4 + 10.3"

73.0 71.4 '~5.2 177.5

_ 11.6 _+ 18.4 _ 8.9*** +_ 23.2****

IL-6

0 (control) 2.8 16.7 100

66.6 54.6 48.9 67.2

+ 14.1 + 9.1 + 8.4* _+ 8.0

115.7 99.0 101.3 118.8

+ 19.0 _+ 14.0 + 15.0 ___ 13.7

Synovial cells or chondrocytes in 24-well dishes were cultured with or without IL-1/3 or IL-6 for three days, and the supematants were measured for NAG activity. Each datum is the mean and SD of six separate experiments. Statistical significance calculated by Student's t test; *P < 0.05, ***P < 0.01, ****P < 0.001 (vs. control).

Table 3. Effect of IL-I~3 or IL-6 on Superoxide Release by Synovial Cells and Chondrocytes" Superoxide (nmol/18 h) IL-1/3 or IL-6

(units/ml)

Synovial cells

Chondrocytes

IL-1/~

0 (control) 33.3 200 1200

1.76 1.37 1.02 1.67

+ 0.33 + 0.22*** +__0.24*** + 0.55

3.47 3.42 6.90 8.37

+ + + +

IL-6

0 (control) 2.8 16.7 100

1.74 1.38 1.03 1.04

+ 0.49 + 0.13 + 0.34* ___0.38*

3.47 3.27 2.87 2.93

+ 0.28 + 0.17 +_ 0.40* + 0.43*

0.51 0.56 2.24* 1.99"**

"Synovial cells or chondrocytes in 24-well dishes were incubated with or without IL-1/3 or IL-6 plus ferricytochrome c for 18 h and superoxide generated was measured. Each datum is the mean and SD of six separate experiments. Statistical significance calculated by Student's t test; *P < 0.05, ***P < 0.01 (vs. control).

Effects of lL-1t3 on Intracellular SOD Activity. S y n o v i a l cells a n d c h o n d r o c y t e s w e r e c u l t u r e d w i t h o r w i t h o u t IL-1/3 f o r 18 h, a n d t h e cells w e r e coll e c t e d , s o n i c a t e d , a n d c e n t r i f u g e d . T h e S O D activity o f the s u p e r n a t a n t s w a s m e a s u r e d . I n t r a c e l l u l a r S O D activity o f s y n o v i a l c e l l s c u l t u r e d w i t h 200 and 1200 u n i t s / m l IL-1/3 w a s s i g n i f i c a n t l y g r e a t e r t h a n that o f s y n o v i a l cells c u l t u r e d w i t h o u t IL-1/3. H o w e v e r , c h o n d r o c y t e s c u l t u r e d w i t h IL-1/3 at 3 3 . 3 , 2 0 0 , a n d

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1200 units/ml did not express increased SOD activity as compared to control (Table 4). Effects of lndomethacin or Anti-IL-l f3 Antibody on Superoxide Generation and NAG Release by Chondrocytes. As shown in Table 5, indomethacin or anti-IL-l~ antiserum, either alone or in combination with IL-1/3, significantly inhibited superoxide generation and NAG release by chondrocytes. Indomethacin or anti-IL-1/3 antiserum, at the concentrations used, did not show any ferTable 4. Effects of IL-1B on Intracellular SOD Activity of Synovial Ceils and Chondrocytes~ Intmcellular SOD activity (units) IL-I~ (units/ml)

Synovial cells

0 (control) 33.3 200 1200

0.21 4- 0.06 not done 0.42 ___ 0.05* 0.46 4- 0.04***

Chondrocytes 0.75 0.79 0.79 0.79

+ + + 4-

0.01 0.04 0.06 0.08

~Synovial cells or chondrocytes in 24-well dishes were cultured with or without IL-1/3 for 18 h. The cells were washed with HBSS, trypsinized, and collected into polystyrene tubes. The ceils in HBSS were sonicated and centrifuged. The supernatants were measured for SOD activity. Each datum is the mean and SD of six separate experiments. Statistical significance calculated by Student's t test; *P < 0.05, ***P < 0.01 (vs. control).

Table 5. Effects of Indomethacin or Antihuman IL-1/3 Antiserum in Presence or Absence of IL-ll8 on Production of Superoxide and NAG by Human Chondrocytesa

Chondrocytes cultured with (1) (2) (3) (4) (5) (6) (7) (8)

HBSS (control) Indomethacin (4 r Indomethacin (8 #M) Antihuman IL-I~ (3200• Antihuman IL-l~8 (800• IL-1B (600 units/ml) (6) + Indomethacin (8/~M) (6) + Antihuman IL-1/~ (800x)

Superoxide (nmol/18 h) 1.74 1.3t 1.04 1.41 1.08 2.52 1.12 1.17

+ 0.5 + 0.3 4- 0.21"* +_ 0.37 _+ 0.26* + 0.31"* 4- 0.21" 4- 0.2*

NAG (/,M/h) 99,0 91.6 83.7 86.0 80.0 149.2 85.1 85.2

_+ 6.4 4- 11.8 + 8.2*** 4- 5.7*** 4- 3.8**** 4- 12.8"*** _+ 7.6** 4- 4.2***

aCells were incubated in 24-well dishes with reagents for 18 h in the presence or absence of ferricytochrome c, and the superoxide generated was measured. Cells also were cultured in RPMI 1640 with 10% FCS in the presence of reagents for three days, and NAG release was measured. Data are the mean and SD of six repeated experiments. Statistical significance calculated by Student's t test; *P < 0.05, **P < 0.02, ***P < 0.01, ****P < 0.001 (vs. control).

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ricytochrome c reducing or any SOD-like activity, when assessed by incubation with ferricytochrome c for 18 h and with hypoxanthine-xanthine oxidase plus ferricytochrome c for the periods up to 20 min, respectively (Table 5).

DISCUSSION One of the most notable features of rheumatoid joints is the proliferation of synovial membrane. The growth of synovial cells is often associated with the growth of the pannus, a vascular and fibrous extension of the perichondral portion of the synovial membrane, which grows over the cartilage and invades the cartilage matrix. The articular cartilage in RA is frequently overlaid by a cellular pannus that actively invades the cartilage (34). The cartilage pannus junction is infiltrated by mononuclear cells that appear to degrade the surrounding cartilage matrix (35). Factors released from these mononuclear cells have been identified as important mediators of joint destruction. One such mediator is IL-1. Therefore, interaction of synovial cells and chondrocytes with IL-1 may trigger joint tissue destruction in RA. In the present study, we investigated how IL-1 influenced the production of mediators by synovial cells and chondrocytes. PGE 2 production by synovial cells and chondrocytes was significantly stimulated by incubation with IL-1, but IL-1 stimulation of PGE 2 was significantly greater in chondrocytes than in synovial ceils. Dayer et al. first reported that synovial cells produced collagenase and PGE2 (7) and that mononuclear cell factor, believed to be identical to IL-1, stimulated collagenase and PGE 2 production by synovial cells (36). It also has been suggested that production of collagenase (9) and PGE2 (9, 10) by chondrocytes is stimulated by IL-1. Since the production of cyclooxygenase products is dependent on prior release of arachidonic acid by a phospholipase enzyme, the observations that IL-1 can activate phospholipase A 2 (37, 38) and cyclooxygenase (39) in synovial cells and chondrocytes provide the mechanism by which IL-1 stimulates PGE 2 production. The reason why there is a difference in the PGE2 response to IL-1 between chondrocytes and synovial cells is not clear, but it is possible that IL-1 binding affinity for the cell membrane receptors of the two cell types is distinct. Both synovial cells and chondrocytes spontaneously produced LTB4, although the amounts produced were minimal. LTB4 production by these cell types was, in contrast to PGE 2, significantly inhibited by IL-1. Since LTB4 is produced by the oxygenation of arachidonic acid by lipoxygenase, the substrate of this reaction is the same as that for PGEE production mediated by cyclooxygenase. Increased consumption of arachidonic acid by cyclooxygenase may be one of the mechanism by which IL-1 inhibits LTB4 production. Such production by synovial cells and chondrocytes has not been reported previously. Since LT]~4

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is a potent inflammatory mediator that acts as chemotactic, degranulating, and vascular permeability-inducing factor, IL-1 inhibition of LTB4 production seems to be paradoxical. On examining the effect of IL-1/3 on the enzyme activity of synovial cells and chondrocytes, as indicated by levels of activity and release of acid glycosidase NAG, we found that IL-1/3 enhanced NAG release by chondrocytes. In the case of synovial cells, 1200 units/ml 1L-l/3 significantly suppressed NAG release. The reason for this difference in response to IL-1/3 between synovial ceils and chondrocytes is not clear. Clarris et al. (14) have shown that IL-1/~ stimulates NAG release by synovial ceils. They have used a single dose oflL-1. Muirden (21) found that rheumatoid synovial tissue contained increased levels of acid hydrolases. At pH values low enough to promote significant activity of lysosomal enzymes, such as might occur in joint tissues, NAG may have an important role in the destruction of joint tissues or activation of latent neutral proteases such as plasminogen activator (40). It has been shown that mononuclear cell factor or IL-1/3 induces neutral protease plasminogen activator in synovial cells (13). Synovial cells and chondrocytes spontaneously released superoxide into extracellular fluid during an 18-h incubation. IL-1/3 at concentrations of 200 and 1200 units/ml markedly enhanced superoxide generation by chondrocytes in a dose-dependent fashion, whereas IL-I/3 at concentrations of 33.3 and 200 units/ml significantly suppressed superoxide generation by synovial cells, suggesting that the superoxide response to IL-1/~ is different in chondrocytes and synovial cells. Since SOD scavenges superoxide, we have measured intracellular SOD activity of these cells to clarify the mechanism of the difference between the two cell types. The results obtained suggest that SOD activity in synovial cells is significantly enhanced by 1L-l/3, but SOD activity of chondrocytes is not enhanced by IL-1/3, resulting in decreased superoxide generation in synovial cells and increased superoxide generation in chondrocytes following IL-1/3 stimulation. It has been shown that superoxide release by synovial cells and chondrocytes in response to tumor necrosis factor is different between the two cell types (41). Shingu et al. (42) reported that cultured human fibroblasts derived from normal skin generate superoxide that is markedly inhibited by IL-1/3, suggesting that superoxide generation of fibroblastic cells is inhibited by IL-1/3. Yaron et al. (43) have shown that IL-1/3 stimulates glycosaminoglycan synthesis in human synovial fibroblasts but inhibits production of glycosaminoglycan in human cartilage cultures. In the present study, we have used chondrocytes isolated from cartilage tissue by enzyme digestion. Thus, IL-1/3 causes opposite effects on the production of various mediators and matrix components in different types of joint tissue cells. Why the same cytokine induces an enhancement of superoxide generation in chondrocytes and an inhibition of superoxide production in synovial cells, or induces different response in SOD

Effects of IL-I[~

153

activity or NAG production in chondrocytes and synovial cells is not understood. However, these differences may be related to differences inherent in the different cell types or in membrane receptor affinity for IL-1/3. It has been reported that RA synovial cells possess I L - l a and/3 receptors that have sufficiently high affinity to mediate the biological properties of these two classes of IL-1 (44). The characteristics of these receptors on RA synovial ceils are very similar to those of normal human embryonic lung fibroblasts (45). There are no reports directly comparing the characteristics of I L - l a and/3 receptors on synovial cells and chondrocytes. The effects of intrinsic IL-1 or PGE2 on the production of NAG and superoxide were investigated using anti-human IL-1/3 antiserum and indomethacin. Anti-IL-1/~ antiserum significantly inhibited superoxide and NAG released by chondrocytes in the presence or absence of exogenous IL-1/~, suggesting that IL-1 regulates an autocrine activation of cellular metabolism. Therefore, it is difficult to be certain whether IL-1/3 effects on superoxide and NAG are specific for IL-1/3 or not, because anti-IL-1/3 antiserum itself inhibits them in the absence of exogenous IL-I~. Similarly, indomethacin significantly suppressed the production of superoxide and NAG, suggesting that intrinsic PGE2 mediated the release of superoxide and NAG in chondrocytes. It has been shown that spontaneous production of plasminogen activator by synovial cells is potentiated by exogenously added prostaglandins (46), but IL-I~ induction of NAG activity in synovial cells is not mediated by PGE 2 (14). The latter observation is contradictory to the present findings that IL-1 induction of superoxide and NAG in chondmcytes is mediated by PGE 2. It has been shown that collagenase stimulation by IL-1 is not associated with increased PGE2 accumulation (47). IL-1 stimulation of glycosaminoglycan production by synovial cells has been reported to be completely (48) or partially (47) mediated by PGE 2 or not associated with PGE2 (49). These observations, together with the present observations that spontaneous production of superoxide and NAG is closely associated with intrinsic PGE2, indicate that available data are in conflict as to whether prostaglandin E2 mediates IL-1 induction of various mediators by joint tissue cells. It has been shown that IL-1 activity is present in cartilage-conditioned medium as well as synovial-conditioned medium that is active in stimulating the synthesis of collagenase activator protein, suggesting that autocrine synthesis of IL-1 regulates cartilage destruction (50). The results obtained in our experiments using anti-human IL-1/3 imply that autocrine synthesis of IL-1/3 also modifies the production of superoxide and NAG. There are recent data suggesting the presence of IL-lc~ and fl mRNA in human chondrocytes with greater than 90 % homology to human monocyte IL-1 mRNA. IL-6 has been characterized recently as a mediator of multiple inflammatory responses. IL-6 is a pleiotropic lymphokine active on T and B lymphocytes and on hepatocytes, which are stimulated by IL-6 to produce acute-phase reac-

154

Tawara et al.

tants (51). It has been shown that human endothelial cells produce IL-6 and IL-1 stimulates IL-6 production in this cell type (52). Human synovial cells have been shown to produce IL-6, which was increased by IL-1 and tumor necrosis factor (24). Therefore, it is important to determine whether IL-6, whose production is enhanced by IL-1, has any effect on joint tissue cells and can thus contribute to IL-1-mediated tissue destruction. Unlike IL-1, IL-6 did not enhance but rather suppressed the production of superoxide and NAG by both chondrocytes and synovial cells. Sironi et al. (52) also have found that, unlike IL-1, IL-6 did not stimulate endothelial cell neutrophil adhesion and the production of prostacyclin by endothelial cells and that IL-6 was not mitogenic for endothelial cells. These results, which are in accord with our observations, suggest that IL-1 does not modulate the functions of these cells via IL-6 and that the proinflammatory role of IL-6 is fundamentally distinct from that of IL-1, as far as available data are concerned. IL-6 may be a central mediator of host defense responses rather than an inflammatory mediator of tissue destruction. Finally, our findings indicate that IL-1/~ released by infiltrated mononuclear cells (53) or joint tissue cells during an inflammatory reaction influences the production or synthesis of superoxide, NAG, PGF~, and LTB4 by synovial cells and chondrocytes in culture. Our observation that the biologic effects obtained with the same cytokine were different in synovial ceils and chondrocytes was of special interest. Since the target cells and cytokine used in this study were of human origin, the present findings suggest that cytokine-joint tissue cell interaction has relevance to the connective tissue alterations frequently observed in RA patients. Acknowledgments--We are grateful to Dr. Morris Ziff for helpful discussions. We are thankful to Dr. John Hamilton for critical review of the manuscript. We thank Mrs. N. Ham~imatu and Miss E. Kohno for excellent technical assistance. We are grateful to the Ministry of Welfare for support.

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