The superficial layer of human articular cartilage is more susceptible to interleukin-1induced damage than the deeper layers.
код для вставкиСкачатьARTHRITIS & RHEUMATISM Vol. 39, No. 3, March 1996, pp 478-488 0 19%, American College of Rheumatology 478 THE SUPERFICIAL LAYER OF HUMAN ARTICULAR CARTILAGE IS MORE SUSCEPTIBLE TO INTERLEUKIN-1-INDUCED DAMAGE THAN THE DEEPER LAYERS H. J. HAUSELMANN, J. FLECHTENMACHER, L. MICHAL, E. J.-M. A. THONAR, M. SHINMEI, K. E. KUElTNER, and M. B. AYDELOTTE Objective. To compare the responses of chondrocytes from superficial and deep layers of normal human articular cartilage to interleukin-1 (IL-1) and IL-1 receptor antagonist protein (IRAP), and to evaluate the binding sites for IL-1 on these cells. Methods. Cartilage and chondrocytes from superficial and deeper layers of human femoral condyles were cultured with and without IL-1 in the presence and absence of IRAP. The effect of these agents on %proteoglycan synthesis and catabolism and production of stromelysin and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by biochemical and immunologic assays. Receptor binding was evaluated using ‘251-labeledIL-1. Results. IL-1 induced more severe inhibition of proteoglycan synthesis and a lower ratio of secreted TIMP-kstromelysin in chondrocytes from superficial cartilage than those from deeper cartilage. IRAP blocked responses to IL-1 more effectively in chondroDr. Hauselmann’s work was supported by Swiss National Science Foundation grant 3 1-33791.92, a Swiss Fellowship from the Bernardi Foundation, University of Bern, and by the Hemnann Foundation, Vaduz, Liechtenstein. Dr. Thonar’s work was supported by NIH grant AG-04736. Dr. Kuettner’s and Dr. Aydelotte’s work was supported by NIH grant AR-39239 and by an Arthritis Research Grant from Hoechst A-G, Werk Kalle-Albert, Germany. H. J. Hauselmann, MD: Rush Medical College at RushPresbyterian-St. Luke’s Medical Center, Chicago, Illinois, the University Hospital, Zurich, Switzerland, and the University of Berne, Berne, Switzerland; J. Flechtenmacher, MD, Rush Medical College at Rush-Presbyterian-St. Luke’s Medical Center, and the University of Ulm, Ulm, Germany; L. Michal, BS, E. J-M. A. Thonar, PhD, K. E. Kuettner, PhD, M. B. Aydelotte, PhD: Rush Medical College at Rush-Presbyterian-St Luke’s Medical Center; M. Shinmei, MD, PhD: National Defense Medical College, SaitamaKen, Japan. Address reprint requests to H. J. Hauselmann, MD, Department of Rheumatology, University Hospital, 8091 Zdrich, Switzerland. Submitted for publication April 10, 1995; accepted in revised form September 22, 1995. . cytes from deep cartilage than those from superficial cartilage. Chondrocytes from the articular surface showed approximately twice the number of h i g h - m i t y binding sites for IL-1 as did cells from deep cartilage. Conclusion. Chondmytes from the surface of articular cartilage show a greater vulnerability to the harmful effects of IL-1 and are less responsive to the potential therapeutic effects of IRAP than cells in the deeper layers of the tissue. Treatment of cartilage tissue in vitro with IL-1 (1,2) or injection of IL-1 into the synovial cavity (3,4) results in inhibition of synthesis of proteoglycans (PGs) (5-9), enhanced production of prostaglandin E2 (PGE,) (10) and metalloproteinases (1 l), and excessive catabolism of PGs. Together these conditions contribute to loss of cartilage matrix and inadequate tissue repair. Based on experimental results and on the finding of IL-1, PG fragments, and proteolytic enzymes in inflamed joints (12-14), it is widely accepted that IL-1 plays an important role in cartilage erosion in inflammatory joint diseases. Because PG synthesis is usually upregulated during the early stages of osteoarthritis (15), IL-1 has been given little attention as a potential mediator of early metabolic changes in articular cartilage. In patients, on the other hand, especially during the inflammatory episodes which almost always develop at late stages of the disease, IL-1 appears to play a significant role in suppressing the synthesis and repair mechanism (16). IL-1 a and IL-Ip, the 2 agonist isoforms of 1L-1 (17-21), bind to specific cell-membrane receptors and set in motion complex signal transduction pathways which are not yet fully understood (22). The human recombinant form of IL-1 receptor antagonist protein also binds to the same receptors, but since it fails to initiate signal transduction or elicit biological responses, it blocks cellular ARTICULAR SURFACE SUSCEPTIBILITY TO IL-1 responses to IL-1 (23) and acts as a pure antagonist (24-28). Two structurally related cell membrane receptors for IL-1 have been identified, the 80-kd type I receptor and the 68-kd type I1 receptor (29-32). The type I receptor occurs on all cells that are responsive to IL-I, including human chondrocytes (16,33), while the type I1 receptor occurs on B cells, monocytes, neutrophils, and bone marrow cells (34). However, some cells can express both types of receptor, a feature which may increase the range of responses elicited by IL-1 (34,35). The coexpression of type I and type I1 receptors for IL-1 has also recently been reported in human synoviocytes (36). The smaller type I1 receptor has a shorter cytoplasmic domain (31,34,37,38). Apparently, it is not a signalling receptor, but acts as a “decoy target” at the cell surface or extracellularly in soluble form, trapping IL-I and thereby diminishing the effective signalling of IL-1 via its type I receptor (39). Although IL-la and p bind to the same receptors, IL-1a seems to bind preferentially to the 80-kd type I IL-1R (40) and IL-Ip binds best to the 68-kd type I1 IL-1R (41). IRAP reportedly binds well to the membrane-bound type I IL-1R and soluble murine type I1 IL-lR, but only poorly to membranebound type I1 IL-1R of murine p r e B cells (42). IRAP can inhibit the binding of both IL-la and p to the type I IL-1R on T cells and to the type I1 IL-1R on polymorphonuclear leukocytes and Raji human B lymphoma cells (26,43). Earlier studies of cartilage organ cultures and chondrocyte cultures were valuable for evaluating the direct effects and mechanisms of action of modulators such as IL-1 (5-9). In those studies, the effects of IL-1 were examined on the entire thickness of cartilage (or chondrocytes therefrom), without consideration of the heterogeneity of articular cartilage. However, articular cartilage is a highly complex, stratified tissue with depth-related differences in biochemical composition, macromolecular organization, and biomechanical properties, which result ultimately from metabolic specialization among the resident chondrocytes (for review, see ref. 44). In the arthritides, the distribution of lesions and the rate of their progression are related to this heterogeneity within the tissue. For example, in osteoarthritis, the earliest damage is detected in the most highly cellular, thin superficial zone (45). In addition, even in healthy joints, the progressive thinning of articular cartilage with age appears to be mainly at the expense of the deeper layers. We have previously demonstrated that in cul- 479 ture, tissue slices and subpopulations of articular chondrocytes isolated from dfierent layers of cartilage continue to express metabolic features characteristic of their specific anatomical origins (46,47). Preliminary experiments showed that in culture, both human and bovine chondrocytes in specific layers of articular cartilage respond in distinctly different, but characteristic, ways to treatment with IL-la, IL-lp, and IRAP (48-50). Results of more detailed studies are reported here, and show that in comparison with cells from deep layers of human articular cartilage, the chondrocytes closest to the synovial cavity are more responsive to IL-1, are less readily protected by the naturally occurring inhibitor, IRAP, and possess more receptors for 1L-1. In addition, we present evidence that there are clear differences between the effectiveness of the 2 isoforms of IL-1 in inhibiting PG synthesis by human chondrocytes. To our knowledge, this is the first report of affinity studies between ligand and receptors performed in chondrocytes isolated from different layers of cartilage and examined in suspension culture, under conditions in which the normal cellular phenotype is conserved (51). SUBJECTS AND METHODS Chondrocyte and cartilage cultures. Tissues were obtained from 30 femoral condyles (15 donors), 4 ankles (2 donors), and 1 shoulder (1 donor) from donors who had no history of joint disease and had macroscopically normal joint cartilage. Tissue was obtained through the Regional Organ Bank of Illinois and through the Examiners Office of the University of Bern, according to the institutions’ protocols and with institutional approvals. Sixteen of the donors were male (ages 14, 17, 22, 24, 29, 32, 37, 42 [2 donors], 50, 51,55 [2 donors], 57, and 63 [2 donors]) and 2 were female (ages 13 and 43). For some experiments, articular cartilage representing the entire thickness of uncalcified tissue was cut from the weight-bearing surfaces of the medial and lateral femoral condyles. For most experiments, in order to obtain tissue representing different layers of articular cartilage, very thin superficial slices were collected separately from tissue that was subsequently harvested from the combined middle and deep layers of articular cartilage of the medial and lateral femoral condyles. These 2 pools of tissue were rinsed, blotted, and weighed to determine the ratio of wet weights. For most of the experiments, the chondrocytes were isolated by protease digestion and cultured in agarose gels (46). Results were confirmed by culturing intact cartilage slices. For binding studies, to evaluate IL-lR, chondrocytes were cultured in alginate, another supporting gel in which chondrocytes show normal phenotype expression (52), but from which the cells can be released readily with chelating agents and recovered for measurement of cell-bound radioactivity (52). Cultures were fed daily with Ham’s F-12 HAUSELMANN ET AL 480 medium (or with F-12DME for alginate cultures) supplemented with 10% pooled adult human serum and 25 &ml ascorbate, and incubated at 37°C in a humidified atmosphere of 5% CO, in air. Treatment with cytokines. After maintenance for 4-6 days in control medium, triplicate or quadruplicate groups of cultures were treated with IL-1a or IL-1p (human recombinant form; Genzyme, Boston, MA) at concentrations of 0.001-2.0 ng/ml. Other cultures were treated simultaneously with IL-1 and IRAP (1-500 ng/ml) (human recombinant form of IRAP kindly donated by Dr. Robert Smith, The Upjohn Company, Kalamazoo, MI). Cytokines were added with the medium each day to maintain the desired concentrations. Measurement of the synthesis and catabolism of PG. To assess PG synthesis, cultures were treated with cytokines for 3 days, and then incubated for 4 hours with 20 &i/ml of "S-sulfate (specific activity 2540 Ci/mg; Amersham, Arlington Heights, IL). PGs were extracted from the tissue slices and agarose gel cultures under dissociative conditions in 4M guanidinium chloride containing protease inhibitors, as described previously (47). Samples were stored at -70°C until analyzed. '%-labeled PGs in the media and dissociative extracts were quantified by liquid scintillation spectroscopy after chromatography on Sephadex G-25M in PD-10 columns, as described previously (47). Parallel cultures were digested overnight at 60°C with papain, and the DNA content was measured by fluorescence using Hoechst dye 33258 (53). To measure the effect of IL-1 on PG catabolism, chondrocytes in the agarose cultures were labeled overnight in the absence of IL-I, then rinsed thoroughly and chased for 7 days with and without IL-I a. The rate of loss of '%-labeled PGs from the agarose gel was calculated daily by measuring the 3SS-labeledPGs appearing in the medium, as described previously (4732). Binding studies to evaluate IL-1 receptors. Receptors for IL-I in human chondrocytes from superficial and deep layers of articular cartilage were evaluated in direct-binding assays according to published methods (5435). The following modification was adopted: before and during the binding study, the cells were maintained in a 3-dimensional culture system within alginate gels (52). After 4 days of culture in control medium, alginate beads containing 4 0 , ~ 5 0 , 0 0 0 chondrocytes were distributed into %-well plates (1 bead well) and incubated for 4 hours at 22°C in binding buffer, in the presence of '251-labeledIL-la or IL-1p (New England Nuclear, Boston, MA) up to a concentration of 36 ng/ml, with triplicate samples for each concentration of IL-1. The beads were washed extensively, and the cells were released from alginate with 5 mM EDTA, recovered by centrifugation or filtration, and then bound radioactivity was measured in a gamma counter. Specific binding was calculated as the difference in binding of '251-labeledIL-I a or p alone and in the presence of a 250-fold concentration of unlabeled IL-1a or p (54). The molar ratios of '"I to IL-la and to IL-1p were the same. To correct for differences in cell numbers, parallel cultures of alginate beads incubated with unlabeled IL-la or p were analyzed for DNA content (53). Results were calculated manually and controlled with an appropriate computer program (Scatchard analysis; Biosoft, Cambridge, UK). v 0,001 0,Ol 0,1 1 hrlL-la (ng/ml) Figure 1. Synthesis of 35S-proteoglycans (35S-PGs) by cultured articular chondrocytes. Chondrocytes from full-thickness cartilage of 2 femoral condyles, 1 from a 22-year-old male (0)and 1 from a 63-year-old male (0). were cultured in agarose gel and treated for 3 days with human recombinant interleukin-l a (hrIL- I a) at the concentrations shown. Note the dose-related inhibition of PG synthesis in response to treatment with IL-la. Results were corrected for DNA content and are expressed as percentages of "S-PG synthesis in untreated control cultures. Bars show the SD of the mean (n = 4). Similar results from additional experiments are shown in Table 1. Assays for stromelysin, tissue inhibitor of metalloproteinases 1 (TIMP-I), and PGE,. Medium was harvested after each day of treatment of cartilage slices; TIMP-1 and stromelysin were measured by enzyme-linked immunosorbent assay (56,57). The assay system for stromelysin (MMP-3) measures not only proMMP-3, but also the active form of MMP-3, as well as MMP-3 complexed with TIMP-I or TIMP-2. The assay system for TIMP-I is capable of measuring both free TMP-1 and TIMP-I complexed with MMP-I (collagenase). PGE, was measured by a specific, commercially available radioimmunoassay (Cayman, Ann Arbor, MI). Results are shown for media samples on the second day of treatment. RESULTS Effects of IL-1 on the entire mixed population of human chondrocytes. Human chondrocytes isolated from healthy knee cartilage and cultured in agarose gels showed a dose-dependent inhibition of PG synthesis in response to IL-la (Figure 1). Interestingly, the age of the donor appeared to play an important role in the metabolic response; chondrocytes and cartilage slices from the younger donors (ages 22 and 29) showed similar inhibition of PG synthesis with lower concentrations of 1L-1a (Table 1 and Figure 1) than did cells from the older donors (ages 42, 55, and 63). Because of the small numbers of young donors, a statistical analysis between the average concentrations of IL-1 a required to inhibit 35Sincorporation into PGs ARTICULAR SURFACE SUSCEPTIBILITY TO IL-1 Table 1. Mean concentrations of interleukin-la (IL-la) required to inhibit incorporation of 35S into proteoglycans by 50% (I(&) in cultured slices of human articular catilage from the knees of 2 young and 4 older donors* Donor age 42 55 63 63 Mean 2 SD age 56 f 10 IC, IL- I a (pdml) Donor age IC, IL- 1 a (Pdml) 7.5 9.0 9.0 15 22 29 2.5 3.0 10.1 2 3.3t 26 2.75 120 glx 100 $ 80 0 0 g 60 s 40 4 20 " 0 v) * Results corrected for cell number (DNA content). t Mean 48 1 * SD. by 50% in young and old donors was not performed (Table 1). The results were corrected for the cell number (DNA content) and, in Figure 1, are expressed as a percentage of the incorporation in untreated control cultures of isolated chondrocytes. Very little catabolic response was observed in human chondrocytes; over a period of 7 days, IL-la at 0.2 ng/ml and 2 ng/ml stimulated the loss of PGs by only -10% (data not shown). Differential responses to IL-1 by subpopulations of chondrocytes. When populations of chondrocytes from different layers of human cartilage were compared, it was evident that chondrocytes from the superficial layer were much more responsive to both isoforms of IL-1 at low concentrations than were those from deeper layers of articular cartilage (Figure 2). Interestingly, however, in both cell populations, PG synthesis was inhibited to a greater extent by IL-1p than by IL-la (Figure 2). Similar results with respect to differences between isolated superficial and deep chondrocytes in response to IL-la and p were observed in 2 other experiments using knee joint cartilage from 2 additional human organ donors ages 24 and 51 (data not shown). In addition, in slices of human articular cartilage (2 knee joints, 1 ankle joint) from 3 different donors ages 13,37, and 51 years, similar differences in inhibition of PG synthesis were observed in response to treatment with IL-la and p (Table 2). The results in both cell and cartilage culture were comparable, and illustrate the pronounced sensitivity of the superficial cartilage to IL-I, and the greater responsiveness of human chondrocytes to IL-I p than to the same concentration of IL-1a.Human chondrocytes from either superficial or deep layers of cartilage showed little catabolic response to IL-I (data not shown). 0,Ol 0,1 h rlL-1a/p (ng/ml) 1 Figure 2. Comparisons of the inhibitory effects of treatment for 3 days with human recombinant interleukin-1a and p (hrIL-1alp; opedsolid symbols) on the synthesis of 35S-proteoglycans(35S-PGs) by articular chondrocytes derived from superficial (circles) and deep (squares) zones of the femoral condyles of a 13-year-old female. Knee chondrocytes from the articular surface showed relatively greater inhibition than those from deep cartilage, and PG synthesis was suppressed more by treatment with IL-Ip than with IL-la. Results were corrected for DNA content, and are expressed as percentages of the radioactivity in untreated control cultures. Bars show the SD of the mean (n = 3). These results are representative of 3 experiments on cartilage from 3 different donors (see Table 2). Responses to IL-1 in the presence of I M P . Chondrocytes derived from different zones of human cartilage also responded differently to simultaneous treatment with IL-I a and IRAP. In chondrocytes from the deeper layers of cartilage, the IL-1 *induced inhiTable 2. Effect of interleukin-1 a (IL-1a)and IL-Ip (0.5 n d d ) on the incorporation of 35S into proteoglycans in cultured slices of cartilage from 3 donors of different ages* Superficial cartilage Donor age 13 37 51 Mean f SD age 39.8 2 14.8 Deep cartilage Donor age 13 37 51 Mean 2 SD age 39.8 2 14.8 IL- 1a IL-IP 11.4 18.8 11.35 4.2 9.0 13.6 ? 3.8ti 5.21 6.5 ? 2.2t§ 55.8 90.0 55.85 14 40.0 14.0 67.2 2 19.7t 22.7 f IS.Ot§ * Results corrected for cell number (DNA content). Values are percentages of the incorporation in untreated control slices. t Mean -e SD. t P 5 0.05 versus deep cartilage. 5 P I0.05 versus IL-I a. HAUSELMANN ET AL 482 120 I 120 100 80 60 40 20 IL-I(nghl) 0 0 0.5 0.5 0.5 0.5 IRAP(nglm1) 0 500 0 5 50 500 I- Control IL-la IL-10 IRAP IL-la IL-10 + I M P +IMP B A F m 3. A, Chondrocytes from the superficial and deeper layers of femoral condyles (from a 14-year-old male) were cultured in agarose gel and treated for 3 days with interleukin-la (IL-la) and IL-I receptor antagonist protein (IRAP) at the concentrations shown. Synthesis of 3SS-proteoglycans(3sS-PGs),corrected for DNA content, was calculated as a percentage of that in untreated control cultures. Hatched bars show superficial cells (from 10% of cartilage wet weight); open bars show the remaining deeper cells (from 90% of cartilage wet weight). Values are the mean and SD (n = 3). Note that at its highest concentration, IRAP restored F G synthesis to the control value in cells from deep cartilage, but failed to reverse the inhibitory effect of IL-1a in cells from the articular surface. These results are representative of 2 experiments on knee cartilage from 2 different male donors (ages 14 and 55). B, Cartilage slices from the superficial and deeper layers of femoral condyles (from a 32-year-old and a 51-year-old male) were cultured with and without IL-la,IL-Ip, I M P , and with both an active cytokine and IRAP. Concentrations of stromelysin and tissue inhibitor of metalloproteinases 1 (TIMP-I) in samples of media were measured by enzyme-linked immunosorbent assay on the second day of treatment. Results shown for each treatment group are the TIMP-1:stromelysin ratio. Note that in both deep and superficial cartilage, treatment with IL-la or p caused a profound lowering of the ratio, but this was more marked in the superficial than in the deep cartilage. In deep cartilage treated with IL-laor B, the antagonist, I M P , was much more effective in restoring the TIMP-1:stromelysin ratio toward control values than in tissue from the surface of the joint. bition of PG synthesis could be overcome almost completely with an 1RAP:IL-la molar ratio of 1,000 (Figure 3A). In contrast, in cultures of chondrocytes from the superficial layer of cartilage, IRAP only partially blocked IL-1*induced inhibition, with PG synthesis being restored to -30% of that in control cultures (Figure 3A). Similar results were obtained using human articular chondrocytes from the superficial and deeper layers of knee cartilage (femoral condyles) of a 55-year-old donor (data not shown). Chondrocytes from the different layers were cultured in agarose and treated under identical conditions as the cultured chondrocytes shown in Figure 3A (femoral condyles from a 14-year-old). In another experiment, when the molar ratio of IRAPAL-1a was increased to lO,OOO, the response of the superficial cells to IL-la could be blocked almost completely (data not shown). The concentrations of stromelysin, TIMP-1, and PGE, in harvested culture media were measured following treatment with IL-la and p; the results are shown in Table 3. This preliminary examination of conditioned medium showed that in the absence of IL- 1 treatment, superficial cartilage slices secreted significantly more PGE, than did deeper tissues (Table 3). From these results, we calculated the TIMP-1: stromelysin ratios in culture media from superficial and deep cartilage slices (obtained from 4 knees of 2 organ donors ages 32 and 51) that had been treated with both active isoforms of IL-1; the results are shown in Figure 3B. After treatment with IL-1a and p, the TIMP-1:stromelysin ratio in the culture medium decreased 26-fold in superficial slices and 9-fold in deep slices, compared with control slices (Table 3). Consistent with the results described above for PG metabolism, IRAP was more effective in blocking IL-1induced responses in deeper cartilage than in superficial cartilage. When cultures were treated simultaneously with IL-laor pand IRAP,the TIMP-1:stromelysin ratio reached control levels in cultures of deep cartilage, but remained <50% of the control value for cartilage from the surface of the joint (Figure 3B). Receptors for IL-1. Specific binding experiments with 12sI-labeled IL-1a were performed on chondrocytes isolated from the full thickness of femoral cartilage after 5 days of culture in alginate beads (52). The results showed 1,OOO copies of the receptor - ARTICULAR SURFACE SUSCEPTIBILITY TO IL-I 483 Table 3. Effect of interleukin-la (IL-la) or IL-lp (0.5 ng/ml) and their inhibitor 1L-l receptor antagonist protein (IRAP) (500 ng/ml) on the secretion of TIMP-1, stromelysin, and PGE, in media from cultured superficial and deep cartilage slices from human knee joints* Superficial TIMP-I Stromelysin SEZ Deep TIMP-I Stromelysin PGE, + IRAP IL-Ip + IRAP IL-1 a IL- I p IRAP 5.2 0.14 f 0.015 1.2 2 2.1 0.71 2 0.5 4.2 t 7.6 0.5 t 1.2 0.69 t 0.25 110.9 t 83.7 4.5 t 2.4 0.27 2 0.19 0.19 2 0.31 3 f 1.4 0.19 f 0.11 0.012 f 0.01 5.4 2 2.8 0.28 2 0.2 0.015 2 0.01 1.2 1.9 2 1.0 0.5 t 0.3 2.7 2 3.2 1.4 2 1.3 0.5 t 0.35 27.4 2 44.5 2.3 2 1.3 0.08 2 0.09 0.01 f 0.01 2.5 2 1.3 0.09 f 0.07 0.24 f 0.4 2.6 f 1.3 0.11 2 0.08 ND Control 6.3 0.14 0.017 2 2 3.5 f 0.1 t 0.05 0.01 2 0.01 IL-la * The concentrations in media harvested on the second day of treatment with cytokines are expressed as follows: for tissue inhibitor of metalloproteinases 1 (TIMP-I) ng/ml; for stromelysin &d; and for prostaglandin E2(PGE,) pg/mY106 cells/24 hours. See also Figure 3B. ND = not done. Values are the mean f SD (n = 4). per cell, corresponding to an affinity of 0.9 X 10'oM-' in a 1 binding-site model (Figure 4A). Similar results with respect to the number of binding sites per cell and the affinity of IL-1a to its receptor were obtained in a second experiment on chondrocytes isolated from the knee joints of a 29-year-old donor. At IL-la concentrations that inhibited PG synthesis by 50% (0.0030.015 ng/ml), fewer than 1% of the receptors were occupied in these experiments for specific binding of '251-labeled IL-1a. However, when the specific binding of 1251-labeledIL-1/3 was examined selectively on 1.oo cells from the superficial and deep layers of human articular cartilage obtained from the normal femoral condyles of 2 male donors and 1 female donor, there was a >Zfold difference in the number of maximal binding sites of the low-to-intermediate affinity type between these 2 cell populations (Table 4 and Figure 4B). Chondrocytes from the deep zone had only - 1,800 copies of the intermediate-affinity type, while chondrocytesfrom the superficial cartilage showed 4,760 ? 2,280 (mean 2 SD) copies of the low-affinity type. In contrast, we could not detect any significant 0 h k 0.75 f 0.50 8 > 0 a, v D 50 0.25 m i 0.0 0.5 1 0 B (molec./cell*) 0.00 0.0 'xl03 0.5 1.5 1.0 2.0 Free 1251-IL-la (nM) A 2.5 3.0 0.0 * x i 0-3 0.5 1.5 1.0 2.0 2.5 3.0 Free 1251-IL-1 p (nM) B Figure 4. A, Specific binding curve of 'zsI-labeled interleukin-I a (IL-la) for chondrocytes cultured in alginate, derived from the full-thickness articular cartilage from the normal femoral condyles of a 42-year-old male. Inset, Scatchard plot derived from binding study based on 1 binding-site model. Ka = 0.92 x lO'OM-', and 960 2 52 siteskell (n = 3). Similar results were obtained in a second experiment using chondrocytes derived from full-thickness articular cartilage of the normal femoral condyles of a 29-year-old male (K, = 10"M-' and 1,300 siteskell). B, Specific binding curve of 'z51-labeledIL-1p for superficial and deep chondrocytes cultured in alginate, derived from the normal femoral condyles of a 17-year-old male. 0 = cells from the superficial layer (10% wet weight of cartilage); 0 = cells from the remaining, deeper layers (WO wet weight of cartilage). Inset, Scatchard plot. In both populations, binding data are compatible with binding sites of 2 affinities: for superficialchondrocytes K, 1 = 1.25 t 0.04 x IOI'M-' with 1,OOO t 27 siteskell and K, 2 = 2.7 2 0.03 x 10"M-' with 3,500 t 42 siteskell; for deep chondrocytes K, 1 = 1.11 2 0.1 x 10"M-' with 525 f 54 siteskell and K, 2 = 3 2 0.2 x IO'OM-' with 1,800 2 127 siteskell (n = 3). Similar results were obtained using superficial and deep chondrocytes derived from the normal femoral condyles of 2 other donors (ages 13 and 24) and cultured in alginate (see Table 4). 484 HAUSELMANN ET AL Table 4. Specific binding sites and ailhities of IZSI-labeledIL-1s for superficial and deep chondrocytes derived from normal femoral condyles of 2 male donors and 1 female donor and cultured in alginate* ~ Superficial Superficial B, Donor age 17 24 13 Mean f SD age, 18 f 5 Deep Bmax 2 Bmax 1 1,cm 360 3,500 7,400 3,400 525 850 1,033 680 4,760 f 2,280t 803 f 257t 1 - Deep Ka 1 (XIO") Ka 2 (XI04 Ka 1 (X10") 1,800 - I .25 1.33 - 0.27 1.5 0.63 1.11 3.0 0.43 1,800 1.29 0.8 f 0.63t B, 2 - 1.51 f 1.33t Ka 2 ( x 10'9 3.0 - 3.0 * Maximal binding sites (Bmx 1 and B,,, 2) and attinities (Ka1 and K, 2, in M-')of subsets of superficial and deep chondrocytes are shown. Specific binding of "'I-labeled interIeukin-l@(IL-1s) was determined in the presence of a 250-fold concentration of unlabeled IL-Ip. Binding data from the chondrocytes of the 17-year-old donor are compatible with binding sites of 2 affinities in both populations of chondrocytes. Binding data from the chondrocytes of the 24-year-old donor are compatible with 2 binding sites only in the superficial layer, whereas binding data from the chondrocytes of both layers from the 13-year-old donor are compatible with binding sites of 1 affinity. t Mean f SD. difference between the number of the high-affinity binding sites in these 2 cell populations (803 ? 257 binding sites in deep chondrocytes versus 680 in superficial chondrocytes). Results of the Scatchard plot indicated binding sites of lower and higher affinities in the superficial cells from 2 of 3 donors, and in the deep chondrocytes from 1 of 3 donors. Importantly, the difference in affinity of IL-1p for the 2 classes of binding sites was almost 2 orders of magnitude (62-fold) in superficial cells, but only 1 order of magnitude (20-fold)in deep cells (Table 4). In both cell populations, the binding constant of the sites with high affinity for IL-1p were almost 10 times higher than that for sites binding IL-1a (Figures 4A and B and Table 4). DISCUSSION The results reported here demonstrate striking metabolic differences in the responsiveness to IL- 1 between chondrocytes derived from superficial and deep layers of normal human articular cartilage, and show the value of analyzing separately these diverse chondrocyte populations. The specific results discussed below are from experiments on normal human cartilage and chondrocytes from 18 donors, as described in Subjects and Methods. Because of the fundamental difficulty of obtaining sufficient samples of normal human articular cartilage, it was not always possible to repeat each experiment numerous times. This problem was compounded when the cartilage was subdivided into zones to compare the responses of different populations of chondrocytes, since yields of cartilage and cells were lower. However, results of experiments with similar samples were consistent, and supported the results shown here. As indicated above, most experiments were done 3 or 4 times, and a minimum of 2 times, with similar results. Cells and tissue slices from the articular surface showed more severe suppression of PG synthesis in response to IL-1 treatment than did cells and cartilage from deeper layers. In comparison with chondrocytes from the articular surface, at least a 10-fold greater concentration of IL- 1(Y was required for similar inhibition of PG synthesis in cells from the deep layer. While the superficial cartilage is more cellular than the deeper tissue, our results for both cell and cartilage slice cultures were corrected for content of DNA; therefore, the difference in cellularity of the native tissue cannot account for the observed variations in responsiveness. The results also demonstrate that human chondrocytes showed a consistent and significantly greater suppression of PG synthesis when treated with IL-lp than with IL-la; this was the case irrespective of the layer of the cartilage, as has been recently reported elsewhere (58). These results were supported by our findings that IL-Ip stimulated secretion of PGE, significantly moreso than did IL-la (10-fold in chondrocytes from the deep zone and 25-fold in those from the superficial zone) (Table 3). These results are also consistent with a recent report that IL-la and p have different effects on the arachidonic acid cascade in cultured human articular chondrocytes (59), and they demonstrate a strong correlation between the effectiveness of the 2 isoforms of IL-1 to stimulate secretion of PGE, and inhibit PG synthesis in chondrocytes. ARTICULAR SURFACE SUSCEPTIBILITY TO IL-1 Human articular chondrocytes from a 50-yearold donor responded to both isoforms of IL-1 with only a weak catabolic response, and in this respect, they differed significantly from the results of studies utilizing mostly young bovine cartilage and chondrocytes (8,9). Other investigators have also observed a weak catabolic response to 1L-1 in most samples of human cartilage, but results vary with the donor, and possibly also with the anatomical site (60) and age (Bayliss MT: personal communication). The reason for the common resistance of human cartilage to showing a catabolic response to IL-1 is not understood, but the results suggest that apart from the reasons discussed above, there may be different regulatory pathways for anabolic and catabolic events in normal human articular chondrocytes. In addition, other mediators may be required to permit the action of IL-1 above the threshold response. Ours were the first direct binding studies in a 3-dimensional culture system to show 2 classes of binding sites with almost 2 orders of magnitude (62fold) difference in affinity for IL- 1p in human articular chondrocytes of the superficial zone but only 1 order of magnitude (20-fold) difference in deep zone cells. This could be seen in 2 of 3 donors for superficial cells and in 1 of 3 donors for deep cells. In contrast, chondrocytes from at least 2 different donors appeared to express only 1 class of binding sites of intermediate affinity for IL-1a. This suggests that after stimulation with IL-1p, both populations of human articular chondrocytes expressed 2 different classes of IL- 1 binding sites, at least in 2 of 3 donors with respect to superficial chondrocytes and 1 of 3 donors with respect to deeper chondrocytes. The reason for this variability is not presently known. In earlier studies on Raji B lymphoma and other cells (29,30,33),there was a 10-100-fold stronger binding of IL-lp to type I1 IL-lR, in comparison with 1L-la and IRAP. For those experiments, equivalent concentrations of recombinant IL-1a and p from different species, including human recombinant forms, were used (31). In addition, our results revealed important differences in the number of receptors in subpopulations of human chondrocytes. Chondrocytes from the surface of the cartilage had approximately twice the number of receptors for IL-1 with respect to the low-affinity binding class in comparison with cells from the deep cartilage of the same joint (50). So far, published reports on IL-1 receptors of chondrocytes cultured in monolayer have indicated a single class of IL-1 receptor (61), but some differences 485 in affinity were reported. For example, rabbit chondrocytes, cultured in monolayers, contained approximately 1,620 receptors per cell, with high affinity for IL-lp ( K , of 10'3M-'), corresponding to the 80-kd or type I receptor (5435). While results of our binding studies with IL-la and p (Figures 4A and B) support these earlier findings of high-affinity IL-1 receptors on articular chondrocytes (54), they also clearly show, for the first time, that the IL-1 receptors present on human articular chondrocytes from superficial and deep zones possess different binding properties for IL-1p. Whether only the type I receptor is present in chondrocytes or whether treatment with IL-1 causes an upregulation of the type I1 receptor on certain cells remains to be established. However, preliminary evidence from this laboratory points to the additional presence of the 68-kd receptor (type I1 IL-1R) in chondrocytes from both superficial and deep layers of human articular cartilage following treatment with IL-1 (Hauselmann et al: unpublished observations). It has recently been reported that human synoviocytes, cells which bear a close developmental and anatomical relationship to articular chondrocytes, express both type I and type I1 receptors for IL-1 (36). Subpopulations of human chondrocytes from superficial and deep layers of articular cartilage showed significant differences not only in their responsiveness to IL-1 a and p, but also in their responsiveness to IL-1 in the presence of I M P . IL-14nduced inhibition of PG synthesis was completely blocked by IRAP in chondrocytes from the deep cartilage, but only partially blocked in cells from the surface of the cartilage. Chondrocytes from deep layers of cartilage assemble much more extracellular matrix than do cells from the cartilage surface. However, it is unlikely that variations in the accumulation of matrix around the chondrocytes could account for the observed differences in effective blocking of the IL-1 response by IRAP; rather, the antagonist may have more ready access to cartilage surface chondrocytes that have sparse matrix. This difference in the effectiveness of IRAP may instead result from a difference in binding affinities of IRAP for the 2 isoforms of IL-1, together with differences in the number and affinity of receptors for IL-1 in the 2 cell populations. In the joint, the synthesis of stromelysin and TIMP are known to be influenced by the presence of 1L-1 (3,62,63). After treatment with IL-la and p, the TIMP- 1:stromelysin concentration ratio in culture medium decreased 26-fold in superficial slices and 9-fold in deep slices (compared with control slices), demon- 486 strating a significant increase in stromelysin and a decrease in TIMP-1 concentration after treatment with these cytokines. This change in the TIMP-1: stromelysin ratio is very similar to the reported 20-fold decrease of the TIMP-1:stromelysin ratio in synovial fluid from patients with joint trauma compared with synovial fluid from normal controls (64).In our cultures, the effectiveness of IRAP in blocking the IL-linduced decrease in the ratio of TIMP-1:stromelysin varied according to the cell population and the active isoform of IL-1. In this regard, IRAP was, again, less effective as an antagonist in chondrocytes from the articular surface than in those from deeper cartilage (Table 3 and Figure 3B). The data presented here emphasize the significant variations in the effectiveness of IRAP in blocking specific IL-1 effects in these subpopulations of chondrocytes. Based on these results, we propose first, that among the more abundant IL-1 receptors in chondrocytes at the surface of the articular cartilage, there is a class which has a higher binding afhity to IL-lp and can be less readily inhibited by IRAP. These cellular differences in types and affinities of IL-1 binding sites may, in part, explain the observed variations between populations of chondrocytes. Second, the higher number of binding sites for IL-1p in the superficial layers, compared with the deeper layers, of cartilage may explain to some extent why IRAP can only partially inhibit the effects of IL-la or p in most of our experiments on mixed populations of human chondrocytes derived from full-thickness cartilage (data not shown). 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