Localization of tumor necrosis factor receptors in the synovial tissue and cartilage-pannus junction in patients with rheumatoid arthritis. Implications for local actions of tumor necrosis factor α

Objective. We have previously described the location of tumor necrosis factor α (TNFα)–producing cells in synovial tissue and cartilage–pannus junction in rheumatoid arthritis (RA). To further understand the local actions of TNFα, we investigated the expression of TNF receptors (TNF-R) on cells in the same compartments in patients with RA. Methods. The expression of both p55 TNF-R and p75 TNF-R was determined using alkaline phosphatase–conjugated mouse anti–alkaline phosphatase (APAAP) and double immunofluorescence staining techniques with monoclonal antibodies. Results. In RA synovial membrane, both p55 TNF-R and p75 TNF-R were detectable in up to 90% of the cells in the lining layer, and were demonstrated on cells in deeper layers of the membrane, including vascular endothelial cells. Cells in lymphoid aggregates expressed both TNF-R, but with a predominant expression of p75 receptor. At the cartilage–pannus junction, the majority of pannus cells, especially those invading cartilage, expressed both the p55 and the p75 TNF-R. Sequential section and double immunofluorescence staining showed that the TNF-R–expressing cells were in the vicinity of TNFα-containing cells, and some TNFα-containing cells also expressed TNF-R. TNF-R–expressing cells were also detected in osteoarthritic and normal synovial tissue, but in smaller numbers and at a lower intensity. Conclusion. These results provide histologic evidence that both p55 TNF-R and p75 TNF-R are expressed by a variety of cell types in RA synovial tissue, reflecting the fact that a wide range of cells are potential targets for TNFα in this tissue. This study further supports the hypothesis that TNFα plays a major role in the pathogenesis of RA.     

Localization of tumor necrosis factor alpha in synovial tissues and at the cartilage-pannus junction in patients with rheumatoid arthritis

Using immunoaffinity-purified polyclonal anti-human recombinant tumor necrosis factor alpha (TNF alpha) F(ab')2 fragments and immunohistochemical techniques, the cells that make TNF alpha were localized in the inflamed synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Anti-TNF alpha antibody-stained cells were demonstrated in 9 of 11 RA and 2 of 4 OA but none of 5 normal synovial membranes examined. In RA, 26-64% of the lining layer cells were positive for TNF alpha. In the interaggregate area, 10-30% of the cells contained TNF alpha, often in a perivascular distribution, and up to 19% of the cells in lymphoid aggregates stained for TNF alpha. Some endothelial cells also stained with these antibodies. In OA tissues, the TNF alpha-containing cells were found predominantly in the deeper layer. Cells containing TNF alpha were also found at the cartilage-pannus junction in all 4 RA specimens examined. Double immunofluorescence analysis demonstrated that most TNF alpha-secreting cells in the RA synovial membrane expressed the monocyte/macrophage marker antigens CD11b and CD14, and a few expressed the T cell marker CD3. Our findings provide histologic evidence that TNF alpha is locally produced in the lining and deeper layers of the synovium by cells of the monocyte/macrophage lineage, supporting its role in inflammation. Further, our findings demonstrate that TNF alpha is produced by cells at the cartilage-pannus junction, which could affect chondrocyte metabolism, leading to the cartilage degradation in RA.     

Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases.

OBJECTIVE. Recently, 2 classes of cytokine inhibitors have been defined at the molecular level. The largest group comprises the extracellular domains of cell surface cytokine receptors, and includes both tumor necrosis factor receptors (TNF-R). The present study was conducted to investigate the role of TNF inhibitors in arthritis. METHODS. We measured p55 and p75 soluble TNF-R (sTNF-R) in serum and synovial fluid (SF) samples from patients with rheumatic diseases and compared their levels with levels of soluble interleukin-2 receptors (sIL-2R). Sensitive enzyme-linked immunosorbent assays (ELISA), specific for p55 and p75 sTNF-R and for sIL-2R, were used. RESULTS. Serum levels of p75 sTNF-R were 3-4-fold higher than levels of p55 sTNF-R, and both were significantly elevated in patients with osteoarthritis (OA) and rheumatoid arthritis (RA) compared with healthy controls. RA SF levels of sTNF-R were 4-5-fold higher than levels in serum, suggesting local production in the joint, and were significantly higher than levels in the SF of patients with seronegative arthropathy or OA. Furthermore, levels of p55 and p75 sTNF-R, but not sIL-2R or TNF alpha measured by ELISA, were increased in the SF of patients with clinically active RA. The soluble TNF-R in RA and OA SF were functional since they inhibited TNF activity in a cytotoxicity assay in proportion to the levels of inhibitor present. Evaluation of serially obtained serum samples suggested that sTNF-R may be a useful parameter for monitoring RA disease activity. CONCLUSION. Biologically active soluble TNF-R are up-regulated in patients with rheumatic disease and are produced locally in the joints. Measurement of serum levels of TNF-R may be useful for monitoring of disease, and determination of SF levels could be of diagnostic value.     

Preferential induction of prodestructive matrix metalloproteinase-1 and proinflammatory interleukin 6 and prostaglandin E2 in rheumatoid arthritis synovial fibroblasts via tumor necrosis factor receptor-55

OBJECTIVE: To assess expression and individual functional relevance of tumor necrosis factor receptor 55 (TNF-R55) and TNF-R75 in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFB). METHODS: Seventh to 9th passage RA SFB and OA SFB were analyzed for TNF-R expression by FACS. The SFB were then stimulated with TNF-a (1-10 ng/ml) or agonistic anti-TNF-R55 (HTR-9) and anti-TNF-R75 (UTR-1) monoclonal antibodies (1-25 micro g/ml each). Matrix metalloproteinase-1 (MMP-1), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), interleukin 6 (IL-6), and prostaglandin E(2) (PGE(2)) in culture supernatants were quantified by ELISA, and DNA fragmentation by TUNEL assay. RESULTS: RA SFB variably expressed TNF-R55 (7.2 +/- 2.2% positive cells, mean +/- SEM) and TNF-R75 (0.6 +/- 0.3%), similarly to OA SFB (6.8 +/- 2.1% and 1.6 +/- 0.8%, respectively). RA SFB constitutively secreted large amounts of TIMP-1 (1700 ng/ml), but only small amounts of MMP-1 (23.7 ng/ml), IL-6 (4.4 ng/ml), and PGE(2) (0.34 ng/ml). OA SFB secreted comparable amounts of TIMP-1 (2470 ng/ml), MMP-1 (37 ng/ml), and IL-6 (5.0 ng/ml), but significantly higher amounts of PGE(2) (0.58 ng/ml; p RA) and by separate stimulation of both TNF receptors. TNF-a-induced PGE(2) release by RA SFB (92-fold) and OA SFB (56-fold) was mediated by both TNF receptors; however, predominantly by TNF-R55. DNA fragmentation was induced exclusively by high concentrations of anti-TNF-R55 Mab and only in RA SFB. CONCLUSION: These results indicate preferential induction of prodestructive and proinflammatory mediators in RA SFB by the TNF-R55, with potential implications for understanding the pathogenesis of RA and the development of more specific therapeutic strategies.     

Increased levels of soluble tumor necrosis factor receptors in the sera and synovial fluid of patients with rheumatic diseases

Objective. Recently, 2 classes of cytokine inhibitors have been defined at the molecular level. The largest group comprises the extracellular domains of cell surface cytokine receptors, and includes both tumor necrosis factor receptors (TNF-R). The present study was conducted to investigate the role of TNF inhibitors in arthritis. Methods. We measured p55 and p75 soluble TNF-R (sTNF-R) in serum and synovial fluid (SF) samples from patients with rheumatic diseases and compared their levels with levels of soluble interleukin-2 receptors (sIL-2R). Sensitive enzyme-linked immunosorbent assays (ELISA), specific for p55 and p75 sTNF-R and for sIL-2R, were used. Results. Serum levels of p75 sTNF-R were 3–4-fold higher than levels of p55 sTNF-R, and both were significantly elevated in patients with osteoarthritis (OA) and rheumatoid arthritis (RA) compared with healthy controls. RA SF levels of sTNF-R were 4–5-fold higher than levels in serum, suggesting local production in the joint, and were significantly higher than levels in the SF of patients with seronegative arthropathy or OA. Furthermore, levels of p55 and p75 sTNF-R, but not sIL-2R or TNFα measured by ELISA, were increased in the SF of patients with clinically active RA. The soluble TNF-R in RA and OA SF were functional since they inhibited TNF activity in a cytotoxicity assay in proportion to the levels of inhibitor present. Evaluation of serially obtained serum samples suggested that sTNF-R may be a useful parameter for monitoring RA disease activity. Conclusion. Biologically active soluble TNF-R are up-regulated in patients with rheumatic disease and are produced locally in the joints. Measurement of serum levels of TNF-R may be useful for monitoring of disease, and determination of SF levels could be of diagnostic value.     

Involvement of tumor necrosis factor (TNF) receptors p55 and p75 in TNF responses of acute myeloid leukemia blasts in vitro.

Tumor necrosis factor (TNF)-alpha and TNF-beta have multiple effects on human acute myeloid leukemia (AML) cells in vitro, including (1) synergistic stimulation of proliferation with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and upregulation of interleukin-3 (IL-3) and GM-CSF receptors; (2) inhibition of granulocyte-CSF (G-CSF)-induced growth and rapid downmodulation of G-CSF receptors; and (3) induction of autocrine growth. Recently, two distinct TNF receptors (TNF-Rs), TNF-R(p55) and TNF-R(p75), have been identified. In this study, we show that both receptor types may be expressed by AML blasts. It has been investigated whether the different effects of TNF on AML blasts can be explained by differential activation of the distinct TNF-R structures. For this purpose, we used the monoclonal antibodies HTR-1 and HTR-9, specifically recognizing TNF-R(p55), and UTR-1, specific for TNF-R(p75). TNF-(alpha and -beta) mediated synergistic activation with IL-3/GM-CSF, upregulation of IL-3/GM-CSF receptors, inhibition of G-CSF-induced growth, and rapid downmodulation of G-CSF receptors exclusively result from activation of TNF-R(p55). In certain cases in which TNF-alpha, rather than TNF-beta, induces AML growth through an autocrine mechanism, both TNF-R(p55) and (p75) are involved. These data indicate that the variety of TNF responses observed in AML can only be partially explained by differential activation of the TNF-R(p55) and (p75) structures, and that TNF-R(p55) on AML blasts can transduce both positive (synergism with IL-3/GM-CSF) and negative regulatory signals (inhibition of G-CSF-induced proliferation) following TNF activation.     

Serum p55 and p75 tumour necrosis factor receptors as markers of disease activity in juvenile chronic arthritis.

OBJECTIVE: To determine the expression of tumour necrosis factor alpha (TNF alpha) and its soluble receptors (p55 and p75) in the sera and synovial fluid of patients with juvenile chronic arthritis (JCA), and their correlation with disease activity parameters. METHODS: Ninety eight sera from 45 patients with JCA (14 systemic, 12 polyarticular, 19 pauciarticular), 20 sera from age matched healthy controls, and five synovial fluids from five antinuclear antibody (ANA) positive pauciarticular JCA patients were tested for the presence of TNF alpha, soluble TNF receptors p55 and p75 (sTNFRp55, sTNFRp75), and interleukin-6 (IL-6) by an enzyme amplified sensitivity immunoassay. Physician global estimate of disease activity, weekly fever score and joint score, C reactive protein (CRP), erythrocyte sedimentation rate (ESR), and haemoglobin concentration were evaluated as parameters of disease activity. The expression of p55 and p75 on peripheral mononuclear cells (MNCs) from five patients with systemic JCA and synovial MNCs from five ANA positive patients with pauciarticular JCA was evaluated by flow cytometry. RESULTS: TNF alpha serum concentrations did not differ significantly between the patients with active JCA and the control group. No correlation was found between TNF alpha and parameters of disease activity, but both p55 and p75 showed a significant positive correlation with the physician global estimate of disease activity (p < 0.001), ESR (p < 0.001), CRP (p < 0.001), and serum concentrations of IL-6 (p < 0.001). Serum concentrations of haemoglobin correlated inversely with the concentrations of p55 and p75 (p < 0.001). Synovial lymphocytes selectively expressed the p75 surface receptor. CONCLUSIONS: sTNFRp55 and sTNFRp75 each represent a sensitive marker of disease activity in JCA. Their increased expression in biological fluids may support the hypothesis that TNF alpha has a role in the pathogenesis of JCA.     

Angiopoietin-1 is expressed in the synovium of patients with rheumatoid arthritis and is induced by tumour necrosis factor alpha

OBJECTIVES: To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis. METHODS: Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha. RESULTS: Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha. CONCLUSIONS: Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.     

p55 and p75 tumor necrosis factor receptors in patients with chronic lymphocytic leukemia.

We studied the expression of the two tumor necrosis factor (TNF) receptors, p55 and p75, on B cells from patients with chronic lymphocytic leukemia (CLL), and the presence of soluble TNF receptors in serum. Expression of membrane-associated receptors was quantified by double labeling of peripheral blood mononuclear cells (PBMC) with monoclonal antibodies against CD19 and p55/p75 TNF receptors and flow cytometry. A high fraction of the CD19+ cells expressed the p55 receptor (44% +/- 34% [SD]) and p75 receptor (61% +/- 31%). In healthy controls, 0% to 1% of the CD19+ cells expressed the p55 receptor and 0% to 10% expressed the p75 receptor. Incubation of CD19+ cells with 10 ng/mL of TNF increased the incorporation of thymidine in 11 patients tested, and this was decreased to 65% (P < .05) by antibodies to the p55 receptor or the p75 receptor, and to 35% +/- 7% (P < .001) when both antibodies were combined. With an enzyme-linked immunoassay, we measured soluble TNF receptors in serum from CLL patients. The mean level of p55 receptors was increased to 12.9 +/- 8.9 ng/mL (P < .000001 v normal). The mean level of p75 receptors was increased to 13 +/- 24 ng/mL (P = .01 v normal). The membrane expression of the two receptors was positively correlated (r +/- 97, P < .01); however, there was no correlation between membrane expression and serum concentration of either receptor. Autologous serum containing high levels of soluble TNF receptors inhibited TNF-induced proliferation of CD19+ cells. In conclusion, we have demonstrated that neoplastic cells from patients with CLL have increased expression of p55 and p75 TNF receptors, and that both receptors mediate signal to proliferation. Furthermore, serum from CLL patients has elevated levels of soluble TNF receptors, which may counteract the proliferative effect of TNF.     

Expression and regulation of tumor necrosis factor, interleukin-2, and hematopoietic growth factor receptors in B-cell chronic lymphocytic leukemia

Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) express tumor necrosis factor (TNF) and interleukin-2 (IL-2) receptors, but only a low proliferative response can be elicited in vitro by TNF alpha and IL-2. To investigate the functional properties of IL-2 and TNF alpha on leukemic B cells, we evaluated (1) the regulation of expression of TNF receptors (TNF-R) and IL-2 receptors on leukemic B cells after culture with TNF alpha and IL-2; (2) the effect of the combination of TNF alpha and IL-2 in a proliferative in vitro assay; and (3) the expression and regulation by these cytokines of receptors for hematopoietic factors, including IL-3, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage colony- stimulating factor (GM-CSF). Flow cytometry analysis showed that freshly isolated leukemic cells from B-CLL patients bear the 75-kD TNF- R and the 55-kD IL-2R; TNF alpha was able to upregulate the 55-kD IL-2R but not the 75-kD TNF-R. On the other hand, IL-2 was not able to modify the expression of the above-mentioned receptors. Although each cytokine alone was unable to induce a relevant proliferation of leukemic cells, a synergistic proliferative effect was detected when these cytokines were used in combination. Leukemic B cells from B-CLL patients bear receptors for hematopoietic factors (IL-3, G-CSF, and GM-CSF) that were upregulated in vitro by IL-2 via the 55-kD IL-2R. On the contrary, TNF alpha was unable to affect the expression of the above-mentioned receptors. These results indicate (1) that IL-2 and TNF receptors are related to each other on leukemic cells in B-CLL and (2) that the IL-2R is involved in the regulation of other structures, ie, CSF receptors, thus pointing to another functional role of this receptor complex and the related cytokine in leukemic cells.     

Administration of recombinant human granulocyte-macrophage colony- stimulating factor after chemotherapy regulates the expression and secretion of monocyte tumor necrosis factor (TNF) and TNF receptors p55 and p75

Monocyte expression and secretion of tumor necrosis factor (TNF) and TNF receptors (TNF-R) p55 and p75 was studied in patients receiving granulocyte-macrophage colony-stimulating factor (GM-CSF) after intensive chemotherapy. TNF expression and secretion of biologically active TNF was increased at regeneration compared with that of patients who had received chemotherapy alone. This effect persisted for several weeks after cessation of growth factor therapy. GM-CSF restored the responsiveness of monocytes to bacterial lipopolysaccharide (LPS), which appeared to be diminished after chemotherapy alone. Expression and secretion of TNF-R p55 and p75 by monocytes was augmented by GM-CSF therapy in association with the increase in TNF protein. We propose that GM-CSF administration after chemotherapy restores the normal responsiveness of monocytes to a secondary stimulus such as LPS and primes monocytes to respond to LPS with increased expression and secretion of TNF and TNF-R.     

Plasma levels of tumor necrosis factor alpha and soluble tumor necrosis factor receptors in patients with narcolepsy

BACKGROUND: Narcolepsy is a disabling sleep disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and sleep paralysis. Recent studies suggest that the immune system might play a pathogenic role pointing to a possible involvement of inflammatory cytokines. METHODS: We investigated a sample of 30 patients with narcolepsy in comparison with 120 sex- and age-matched and 101 sex-, body mass index (BMI)-, and age-matched randomly selected normal controls. In these groups, plasma concentrations of tumor necrosis factor alpha (TNF-alpha) and its soluble receptors p55 and p75 (soluble TNF receptor [sTNF-R] p55 and sTNF-R p75) were measured using commercial enzyme-linked immunosorbent assays. RESULTS: The narcoleptic patients showed a significantly higher BMI compared with controls of the same age. Soluble TNF-R p75 levels were consistently elevated in the narcoleptic patients compared with their sex- and age-matched (P = .001) as well as sex-, BMI-, and age-matched counterparts (P = .003). Female narcoleptic patients exhibited higher sTNF-R p55 levels compared with their sex- and age-matched controls (P = .01), but this difference disappeared when comparing patients with sex-, BMI-, and age-matched normal controls. Tumor necrosis factor alpha levels did not differ significantly between groups. CONCLUSION: Narcoleptic patients show increased plasma levels of sTNF-R p75, suggesting a functional alteration of the TNF-alpha cytokine system, further corroborating a possible pathogenic role of the immune system in this sleep disorder.     

Isolated guinea pig gastric chief cells express tumour necrosis factor receptors coupled with the sphingomyelin pathway.

The tumour necrosis factor alpha (TNF), has been implicated in the pathogenesis of non-steroidal anti-inflammatory drug (NSAID) induced gastropathy and Helicobacter pylori induced gastritis. Both conditions are characterised by high plasma pepsinogen concentrations, which are thought to reflect an increased rate of enzyme release by the pepsinogen secreting (chief) cells. The mechanisms responsible for this cell dysfunction are unknown. This study investigates whether chief cells express TNF receptors and, if so, whether their activation results in cell death. Immunohistochemical studies conducted with monoclonal antibodies (mAbs) directed against two TNF receptor associated proteins of 55 kDa (TNF-R1) and 75 kDa (TNF-R2) showed that TNF binding sites were expressed in approximately 100% gastric chief cells. Western blot analysis of whole chief cell lysates probed with the TNF-R1 and TNF-R2 mAbs gave two distinct bands of 55 and 75 kDa in the immunoprecipitate. Incubating chief cells with TNF caused concentration and time dependent cell death, which was prevented by pretreating the cells with anti-TNF receptor mAbs. Exposing the cells to TNF reduced sphingomyelin content by 25%. Sphingomyelinase (10(-6) to 10(-2) IU/ml) mimicked the effect of TNF in that it provoked a concentration and time dependent reduction in chief cell viability and increased pepsinogen release. In conclusion, gastric chief cells express two TNF receptors partially linked to the sphingomyelin pathway. TNF induced chief cell dysfunction might be responsible for the high plasma pepsinogen concentrations seen in patients with NSAID gastropathy or H pylori induced gastritis.     

Interleukin (IL) 18 stimulates osteoclast formation through synovial T cells in rheumatoid arthritis: comparison with IL1 beta and tumour necrosis factor alpha

OBJECTIVE: To determine whether IL18 has any indirect effects on osteoclastogenesis mediated by T cells in RA synovium, and compare its effects with those of IL1 beta and TNF alpha. METHODS: Resting T cells were isolated from peripheral blood of healthy donors, and stimulated with 2 microg/ml phytohaemagglutinin (PHA) and 0.5 ng/ml IL2 for 24 hours. Synovial T cells were isolated from RA synovial tissue. The levels of soluble receptor activator of the NF-kappa B ligand (RANKL), osteoprotegerin (OPG), IFN gamma, M-CSF, and GM-CSF were determined by ELISA. Membrane bound RANKL expression was analysed by flow cytometry. Commercially available human osteoclast precursors were cocultured with T cells to induce osteoclast formation, which was determined with tartrate resistant acid phosphatase staining and pit formation assay. RESULTS: In PHA prestimulated T cells or RA synovial T cells, IL18, IL1 beta, or TNFalpha increased soluble RANKL production and membrane bound RANKL expression in a dose dependent manner. IL18, IL1 beta, and TNF alpha did not induce M-CSF, GM-CSF, IFN gamma, or OPG production in PHA prestimulated T cells or RA synovial T cells. IL18 increased the number of osteoclasts and bone resorption area on dentine slices in the coculture of human osteoclast precursors with PHA prestimulated T cells or RA synovial T cells; its ability was equivalent to that of IL1 beta, but less potent than that of TNF alpha. In the coculture system, OPG completely blocked osteoclast induction by IL18 or IL1 beta, and greatly inhibited induction by TNF alpha. CONCLUSION: IL18, IL1 beta, or TNF alpha can indirectly stimulate osteoclast formation through up regulation of RANKL production from T cells in RA synovitis; IL18 is as effective as IL1 beta, but less potent than TNF alpha.     

Vitamin D receptors in the rheumatoid lesion: expression by chondrocytes, macrophages, and synoviocytes

OBJECTIVES: The active form of vitamin D3, 1 alpha,25 dihydroxyvitamin D3 (1,25D3), through its interaction with vitamin D receptors (VDR), is reported to effect a variety of anabolic and catabolic events, especially in bone and cartilage tissues. As cartilage degradation and tissue remodelling are characteristic features of the rheumatoid lesion, the distribution and expression of VDR at sites of cartilage erosion was examined. METHODS: Immunolocalisation techniques using a rat monoclonal antibody to VDR and an alkaline phosphatase conjugated avidin/biotin detection system were used to examine VDR in 18 specimens of cartilage-pannus junction, 10 specimens of rheumatoid synovium or cartilage tissue, and four primary cultures of adherent rheumatoid synovial cells (RSC). For comparison, VDR expression was examined in 10 specimens of normal, healthy age matched articular cartilage. RESULTS: VDR was demonstrated in 15 of 18 cartilage-pannus junctions either at the interface (8 of 18), within the pannus tissue (12 of 18), and by chondrocytes often close to the erosive lesion (10 of 18). All the rheumatoid synovial tissue and 5 of 10 cartilage specimens showed cells with positive staining, but the extent of this was variable. Negligible VDR staining was observed for normal cartilage. Primary cultures of RSC also showed variability in both the numbers and proportions of macrophages or synovial fibroblasts stained for VDR (range 10-50%), this being more common in cultures with a high proportion of macrophages. CONCLUSIONS: VDR expression has been demonstrated by most specimens of cartilage-pannus junction; was associated with various cell types, including chondrocytes, but not exclusively with CD68+ macrophages. The focal nature of VDR expression within the rheumatoid lesion suggests a contributory role for 1 alpha,25D3 in the pathophysiological processes of rheumatoid arthritis.     

Chimeric tumor necrosis factor receptors with constitutive signaling activity.

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the generation of a signal. In the case of the tumor necrosis factor (TNF) receptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect. However, the quaternary structural requirements for signaling--i.e., the formation of dimers, trimers, or higher-order multimers--have remained obscure. Moreover, it has not been clear whether the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysis. We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the actual absence of the ligand. Such a molecule, inserted into an appropriate vector, could be used to project receptor-specific "TNF-like" activity to specific cells and tissues in vivo. Accordingly, we constructed sequences encoding chimeric receptors in which the extracellular domain of the mouse erythropoietin receptor (Epo-R) was fused to the "stem," transmembrane domain, and cytoplasmic domain of the two mouse TNF-Rs. Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain. These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surface. Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constitutive cytotoxic effect detected by cotransfection or clonogenic assay. Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order multimerization) was sufficient for elicitation of a biological response.