In vitro effects of diacerhein and rhein on interleukin 1 and tumor necrosis factor-alpha systems in human osteoarthritic synovium and chondrocytes

OBJECTIVE: To evaluate the in vitro effects of diacerhein, a new drug for the treatment of osteoarthritis (OA), and its active metabolite, rhein, on interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) synthesis and expression in human OA synovial membrane, and on the IL-1beta and TNF-alpha receptors on human OA chondrocytes. METHODS: Levels of IL-1beta and TNF-alpha were determined using specific ELISA in culture medium of human synovial membrane explants incubated in the presence of 1 microg/ml of lipopolysaccharide with or without therapeutic concentrations of diacerhein (1.4, 2.7, 5.4 x 10(-5) M) and rhein (1.7, 3.5, 7.0 x 10(-5) M). IL-1beta mRNA level was quantitated by Northern blotting. Using radioligand binding experiments, we determined the effects of these agents on the density and affinity of chondrocyte IL-1 and TNF receptors. RESULTS: IL-1beta synthesis was significantly inhibited by diacerhein and rhein, with maximum inhibition at 5.4 x 10(-5) M for diacerhein (p < 0.02) and 3.5 x 10(-5) M for rhein (p < 0.05). The effect of both agents on IL-1beta was found to be translational and/or post-translational, judging by the absence of effect on gene expression level. Both agents produced dose and time dependent decreases in the number of IL-1 receptors (IL-1R) on OA chondrocytes. This effect was mediated through a reduction in the level of the type I IL-1R as shown by experiments using a blocking monoclonal antibody against this receptor type. Both agents also markedly reduced the IL-1 induced synthesis and expression of stromelysin 1. Neither diacerhein nor rhein significantly affected the level of synthesis of TNF-alpha or the level of TNF-R. CONCLUSION: Diacerhein and rhein can effectively inhibit the synthesis of IL-1beta on human OA synovium, as well as the action of this cytokine at the cartilage level, by reducing the number of chondrocyte IL-1R. The effects of these agents seemed "selective" to the IL-1 system.     

Identification of two closely related genes, inducible and noninducible carbonyl reductases in the rat ovary

OBJECTIVE: To investigate the potential role of cytokines in psoriatic arthritis (PsA) by assessing the profiles of the proinflammatory cytokines in synovial fluid (SF) of PsA in comparison with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Levels of tumor necrosis factor-alpha (TNF-alpha), interleukin 1 (IL-1), IL-6, and IL-8 were measured in SF using ELISA. RESULTS: Levels of TNF-alpha, IL-1beta, and IL-8 were significantly higher in PsA SF than in OA SF, although lower than in RA SF. No difference was detected in the IL-6 levels between PsA and RA SF, both of which were much higher than in OA SF. CONCLUSION: The pattern of expression of proinflammatory cytokines seen in PsA is similar to that in RA. Since PsA is also a destructive arthropathy, cytokines, in particular TNF-alpha and IL-1, may be principle factors in joint destruction.     

Temporal pattern of cysteine endopeptidase (cathepsin B) expression in cartilage and synovium from rabbit knees with experimental osteoarthritis: gene expression in chondrocytes in response to interleukin-1 and matrix depletion

OBJECTIVE: To determine the temporal pattern of expression of cathepsin-B in chondrocytes and synovium in experimental osteoarthritis, and to determine possible mechanisms for upregulation and secretion of cathepsin-B from chondrocytes. METHODS: Experimental osteoarthritis was induced with partial medial meniscectomy (PM); sham operated (SH) and normal (N) rabbits were used as controls. Cathepsin-B mRNA expression was assessed with northern blotting with a 32P labelled cDNA probe. Cathepsin-B was measured in conditioned media or cell extracts using a fluorogenic substrate Z-Arg-Arg-AMC. Chondrocyte monolayers were used to determine cathepsin-B expression in response to interleukin-1 beta (IL-1 beta). Cartilage explants were used to test the effect of matrix depletion on cathepsin-B release. RESULTS: Chondrocytes obtained from experimental osteoarthritis knees did not show cathepsin-B mRNA upregulation. However, isolated chondrocytes secreted cathepsin-B into the culture medium. Enzyme release was significantly higher at 8 weeks relative to controls, but not at 12 weeks or 4 weeks. Enzyme released from synovium was significantly higher in PM group compared with SH group at 4 and 8 weeks. IL-1 beta was ineffective in upregulating steady state cathepsin-B mRNA in chondrocytes; however, it upregulated the intracellular enzyme, and this was blocked with cycloheximide. Enzymatic depletion of cartilage matrix after exposure of explants to IL-1 resulted in release of significantly higher amounts of cathepsin-B into the medium by matrix depleted chondrocytes compared with intact explants. CONCLUSIONS: In experimental osteoarthritis, cathepsin-B is upregulated in synovial tissue during the early degenerative phase. Progression of experimental osteoarthritis is accompanied by upregulation of cathepsin-B in cartilage. Cartilage and synovial cathepsin-B levels decline as experimental osteoarthritis advances to more degenerative states. IL-1 upregulates intracellular cathepsin-B by increasing cathepsin-B protein synthesis; it is not an effective stimulus for enzyme secretion. Depletion of cartilage matrix during progression of experimental osteoarthritis may contribute to secretion of cathepsin-B and perpetuation of cartilage destruction.     

Patterns of cytokine production in psoriatic synovium

OBJECTIVE: To determine patterns of cytokine production and mRNA expression in synovium from patients with psoriatic arthritis (PsA) and to compare the profile of cytokine production in PsA explants with those derived from rheumatoid (RA) and osteoarthritic (OA) synovia and psoriatic skin. METHODS: Cytokine levels were measured in supernatants from synovial and dermal explant cultures at Day 10 by ELISA. Cytokine mRNA expression in PsA whole tissue was determined by multi-gene assay. Cytokine levels in explant supernatants were compared between PsA, RA and OA, and psoriatic skin. Synovial tissues were scored histologically by a pathologist blinded to the clinical diagnosis. RESULTS: PsA explants released elevated levels of interleukin (IL)-1beta, IL-2, IL-10, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha, but not IL-4 or IL-5. A similar pattern of gene expression was detected in whole synovial tissue. These cytokine levels were greater in PsA than RA, despite higher histopathologic scores in RA explants. Production of IL-1beta, IFN-gamma, and IL-10 were strongly correlated. Levels of IFN-gamma, IL-1beta, and IL-10 were higher in psoriatic synovium than psoriatic dermal plaques. CONCLUSION: The cytokine profile in PsA is characterized by the presence of Th1 cytokines and the monokines TNF-alpha and IL-1beta and very elevated levels of IL-10. The higher levels of these cytokines in PsA compared to RA suggest the presence of different underlying mechanisms.     

Synovium as a source of increased amino-terminal parathyroid hormone-related protein expression in rheumatoid arthritis. A possible role for locally produced parathyroid hormone-related protein in the pathogenesis of rheumatoid arthritis

Proinflammatory cytokines, including tumor necrosis factor (TNF) and interleukin 1 (IL-1), mediate the joint destruction that characterizes rheumatoid arthritis (RA). Previous studies have shown that parathyroid hormone-related protein (PTHrP) is a member of the cascade of proinflammatory cytokines induced in parenchymal organs during lethal endotoxemia. To test the hypothesis that NH2-terminal PTHrP, a potent bone resorbing agent, could also be a member of the synovial cascade of tissue-destructive cytokines whose expression is induced in RA, PTHrP expression was examined in synovium and synoviocytes obtained from patients with RA and osteoarthritis (OA). PTHrP production, as determined by measurement of immunoreactive PTHrP(1-86) in tissue explant supernatants, was increased 10-fold in RA versus OA synovial tissue. Synovial lining cells and fibroblast-like cells within the pannus expressed both PTHrP and the PTH/PTHrP receptor, findings that were confirmed by in vitro studies of cultured synoviocytes. TNF-alpha and IL-1beta stimulated PTHrP expression in synoviocytes, while dexamethasone and interferon-gamma, agents with some therapeutic efficacy in the treatment of RA, inhibited PTHrP release. Treatment of synoviocytes with PTHrP(1-34) stimulated IL-6 secretion. These results suggest that proinflammatory cytokine-stimulated production of NH2-terminal PTHrP by synovial tissue directly invading cartilage and bone in RA may mediate joint destruction through direct effects on cartilage or bone, or, indirectly, via the induction of mediators of bone resorption in the tumor-like synovium.     

Making the most of aid

The synovial fluid (SF) of rheumatoid arthritis (RA) patients contains a mixture of inflammatory mediators. In order to determine whether certain cytokine patterns locally in the joint are specifically related to the chronic inflammation in RA, the concentrations of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, transforming growth factor-beta (TGF-beta), tumour necrosis factor-alpha (TNF-alpha) and IgG2b-inducing factor (IgG2bIF) were measured in SF from 22 patients with RA and 22 patients with other types of arthritic lesions. High levels of IL-10, latent and active TGF-beta and the presence of IgG2bIF are significantly correlated with RA when corrected for age. As these factors have the capacity to promote antibody production, they might contribute to the maintenance of local antibody production in RA synovial tissues. All RA-SF samples contained detectable levels of IL-10 and all except one contained IL-1beta, while concentrations in several non-RA-SF samples were below detection limits. IL-6 and TGF-beta were present in all SF samples from both RA and non-RA patients. The presence of IgG2bIF was strongly correlated with high levels of IL-10 and IL-1beta in SF. However, no distinct cytokine profile specific for the chronic inflammation characteristic of RA was found.     

Compositional mapping of mouse chromosomes and identification of the gene-rich regions

The interleukin-1 (IL-1) cytokines stimulate the synthesis of degradative enzymes in joint tissues and may play a role in the pathological joint destruction in osteoarthritis (OA). In this study, we have used immunohistochemistry and Western blot analysis to identify IL-1 in human OA cartilage. IL-1 alpha and IL-1 beta were evident in chondrocytes at the articular surface, as well as distributed throughout the cartilage. In many specimens, IL-1 beta but not IL-1 alpha was detected as a diffuse staining of the extracellular matrix especially surrounding superficial zone chondrocytes. Although chondrocyte-associated IL-1 alpha and IL-1 beta were detected in most specimens, cartilages exhibiting early osteoarthritic changes had the highest intensity of staining and the highest frequency of positive cells. Western blot analysis revealed intense immunoreactive bands corresponding to the 35 kDa precursor form of IL-1 alpha in all four chondrocyte lysates tested. The processed 18 kDa IL-1 beta species was present in only one of four chondrocyte lysates, and there was no clear evidence of precursor form within these cells. The results of this study indicate increased IL-1 alpha in cartilage showing early degenerative changes, suggesting an autocrine/paracrine role for this cytokine in OA pathogenesis.     

Pro- and anti-inflammatory cytokines in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by the accumulation of inflammatory cells into the synovium and the destruction of joints. Cytokines are important regulators of the synovial inflammation. Some cytokines, such as tumour necrosis factor (TNF)-alpha and interleukin (IL)-1, function by promoting inflammatory responses and by inducing cartilage degradation. Other cytokines, such as IL-4, IL-10 and IL-13, function mainly as anti-inflammatory molecules. Although anti-inflammatory cytokines are present in rheumatoid joints, in progressive RA their levels obviously are too low to neutralize the deleterious effects of proinflammatory cytokines. Inhibiting the action of proinflammatory cytokines by using specific cytokine inhibitors or anti-inflammatory cytokines is the basis for new therapies currently tested in patients with RA. Promising results on the use of neutralizing anti-TNF-alpha monoclonal antibodies in the treatment of RA have been reported. The results from a trial using recombinant IL-10 in the treatment of patients with RA are available in the near future and will be important in determining the therapeutic potential of this cytokine.     

Increased levels of circulating intercellular adhesion molecule 1 in the sera of patients with rheumatoid arthritis

OBJECTIVE: We sought to assess whether circulating levels of intercellular adhesion molecule 1 (ICAM-1) in patients with rheumatoid arthritis (RA) are elevated and correlate with clinical measures of disease activity and whether this ICAM-1 originates from the synovium. METHODS: Circulating ICAM-1 (cICAM-1) levels were determined by sandwich enzyme-linked immunosorbent assay of serum from 61 RA, 18 osteoarthritis (OA), and 11 inflammatory arthritis (IA) patients. In addition, paired serum and synovial fluid samples were collected from 17 RA, 9 OA, and 4 IA patients. The stability of cICAM-1 was assessed by overnight incubation at 37 degrees C. Finally, the potential degradative effects of synovial fluid proteases were assessed by incubation of recombinant soluble ICAM-1 with patient synovial fluid. RESULTS: RA sera contained significantly greater (P < 0.001) levels of cICAM-1 compared with RA synovial fluid and compared with sera or synovial fluid from the OA and IA patients. Circulating ICAM-1 levels were unaffected by overnight incubation at 37 degrees C and were unaffected by exposure to RA, OA, or IA synovial fluid. Serum levels of cICAM-1 demonstrated a weak, but significant (P < 0.05) correlation with the joint score and erythrocyte sedimentation rate in 25 RA patients treated with nonsteroidal antiinflammatory drugs. CONCLUSION: The greatest elevations of cICAM-1 were seen in RA patient sera. In both RA and OA, synovial fluid cICAM-1 levels were consistently lower than serum levels, suggesting that cICAM-1 did not originate in the synovium. Because the production of cICAM-1 can be increased by cytokines (e.g., interleukin-1, tumor necrosis factor alpha), elevated levels of circulating ICAM-1 in RA may be reflective of systemic exposure to elevated cytokine levels.     

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint

OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.     

Transforming growth factor-beta 1 stimulates articular chondrocyte proteoglycan synthesis and induces osteophyte formation in the murine knee joint

BACKGROUND: High concentrations of active transforming growth factor-beta (TGF-beta) have been found in synovial fluids from arthritic joints. TGF-beta stimulates articular cartilage proteoglycan synthesis and suppresses proteoglycan degradation in vitro. In an earlier study, we found no effect on cartilage proteoglycan metabolism shortly after a single intra-articular injection of TGF-beta 1. In the present study, we used multiple intra-articular injections and a longer time-scale. EXPERIMENTAL DESIGN: TGF-beta 1 was injected into the murine knee joint to gain insight in the consequences of its overproduction in joint diseases. This was evaluated using histologic sections of the whole knee joint and measurements of articular cartilage proteoglycan synthesis and content. RESULTS: At 6 hours after a single TGF-beta 1 injection, recruitment of polymorphonuclear leukocytes (PMNs) was observed. After 24 hours, the amount of inflammatory cells had already decreased. Multiple TGF-beta 1 injections induced synovial hyperplasia and synovitis predominantly consisting of cells of the macrophage/monocyte lineage. Both single and multiple TGF-beta 1 injections induced strong and long-lasting stimulation of articular cartilage proteoglycan synthesis. This in vivo stimulation of proteoglycan synthesis was similar in cartilage of young (3 months) and old mice (18 months). Multiple TGF-beta 1 injections resulted in an increased GAG content in patellar cartilage. After triple TGF-beta 1 injections, impressive osteophyte formation was noted at specific sites. The size and the localization of osteophytes was identical in young and old mice. Interestingly, the localization of TGF-beta 1-induced osteophytes was very similar to that of osteophytes observed in experimental arthritis and osteoarthritis models, suggesting a role for endogenous TGF-beta in osteophyte formation during joint pathology. CONCLUSIONS: Our data indicate that TGF-beta 1 injection into a normal joint induces inflammation, synovial hyperplasia, osteophyte formation, and prolonged elevation of proteoglycan synthesis and content in articular cartilage.     

Induction of release of secretory nonpancreatic phospholipase A2 from human articular chondrocytes

OBJECTIVE: Secretory nonpancreatic phospholipase A2 (sPLA2) is a known inducer/promoter of the inflammatory process in the joints. It correlates with disease activity in adult and juvenile rheumatoid arthritis. Synovial fluids contain high concentrations of sPLA2. We discovered that human articular cartilage contains large quantities of sPLA2 and that culture chondrocytes constitutively synthesize and release sPLA2. To test the mechanism controlling the release of sPLA2, we exposed cultured human articular chondrocytes to cytokines and other agents, known to induce sPLA2 in other cells. METHODS: Chondrocytes obtained from cartilage of normal appearance from rheumatoid and osteoarthritic joints, and from normal, neonatal joints were compared to rabbit articular chondrocytes. Radiolabeled Escherichia coli derived phospholipid assay and ELISA technique using monoclonal antibodies against recombinant human synovial type sPLA2 were employed. The cells were grown as monolayers as well as in alginate beads. RESULTS: Human articular chondrocytes from both arthritic and neonatal joints released sPLA2 constitutively but could not be further stimulated with interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-6, oncostatin M, lipopolysaccharide (LPS), or forskolin. Marked stimulation was observed when the cells were exposed to 8-bromo cyclic adenosine monophosphate (cAMP). Growing the cells as monolayers or in alginate beads did not change the results. In contrast to human cells, rabbit chondrocytes responded to IL-1 beta and IL-1/TNF, but not to TNF-alpha alone, with a very marked increase in extracellular sPLA2 activity. CONCLUSION: Human articular chondrocytes synthesize and constitutively release sPLA2. Such continuous release is most probably responsible for the high concentration of sPLA2 in articular cartilage and may be the source of synovial fluid sPLA2. To our knowledge, human articular chondrocytes are the only sPLA2 producing cells tested to date that do not respond to cytokine stimulation with increased sPLA2 activity; yet enhancement was seen with 8-bromo cAMP. It seems therefore that, human articular chondrocytes possess signalling mechanisms for the release of sPLA2 unlike those from other mammalian cells. The significance of this observation remains to be elucidated.     

Induction of invasive and degradative phenotype in normal synovial fibroblasts exposed to synovial fluid from patients with juvenile rheumatoid arthritis: role of mononuclear cell population

OBJECTIVE: To investigate the effect of synovial fluid (SF) from patients with juvenile rheumatoid arthritis (JRA) on proliferation and induction of degradative and invasive phenotype in normal synovial fibroblasts, and to elucidate the contribution of SF cells to this activity. METHODS: SF and/or conditioned medium (CM) from SF cells were evaluated for their ability to (1) stimulate a proliferative response, (2) induce the "activated phenotype" capable of invading cartilage matrix, and (3) promote the release of key matrix metalloproteinases (MMP) in normal synovial fibroblasts. RESULTS: Proliferation of normal synovial fibroblasts exposed to SF or CM from SF cells of patients with JRA was up to 3 times greater than untreated controls. Concomitant with induction of an activated phenotype in the treated synovial fibroblasts, the activated form exhibited up to 250% invasiveness of cartilage matrix compared to untreated synovial fibroblasts (100%), in addition to releasing increased MMP activity, not normally associated with these quiescent cells. This induction was not solely due to tumor necrosis factor-alpha, transforming growth factor-beta, interleukin 1beta (IL-1beta), and IL-6, as SF and/or CM depleted of these cytokines sustained about 40% of their invasive and inducing ability. We observed that the mononuclear cell (MNC) population that infiltrated into the joint cavity secretes this "inducing activity," which can be maintained in culture up to several weeks. CONCLUSION: Our data suggest that the cellular component of SF releases soluble factor(s) that directly or indirectly contribute to (a) proliferation of synovial fibroblasts, and (b) production and release of extracellular MMP by synovial fibroblasts, thereby inducing a degradative and invasive phenotype culminating in cartilage and bone destruction.     

Analysis of cartilage oligomeric matrix protein (COMP) in synovial fibroblasts and synovial fluids

We investigated the expression of cartilage oligomeric matrix protein (COMP) in normal and rheumatoid arthritis (RA) synovial fibroblasts. In situ hybridization (ISH) was conducted on synovial specimens from five RA patients applying specific probes for COMP or fibroblast collagen type I. ISH was combined with immunohistochemistry, applying antibodies to the macrophage marker CD68. Ribonuclease protection assay (RPA) and rapid amplification of 3'-cDNA ends (3'-RACE) were performed on total RNA from normal and RA synovial fibroblast cultures. Protein extracts from fibroblasts and culture supernatants were compared with synovial fluids and protein extracts from isolated chondrocytes by Western blot utilizing polyclonal and monoclonal antibodies (18-G3 mAb) to COMP. COMP mRNA was detected in fibroblasts of RA synovium by ISH, and in normal and RA synovial fibroblast cultures by RPA. 3'-RACE demonstrated sequence homology of chondrocyte and synovial fibroblast COMP along the coding sequence. COMP protein was detected in synovial fibroblasts and culture supernatants by immunoblot. Using polyclonal antibodies, the major portion of COMP from fibroblasts and culture supernatants was present as low-molecular-weight (LMW) bands, corresponding to those found in synovial fluids. These LMW COMP bands, however, were not detected in any of the cells or tissues tested using 18-G3 mAb. In protein extracts from chondrocytes and in COMP purified from cartilage, these LMW bands could not be detected. In conclusion, the data suggest that certain forms of COMP detected in synovial fluid are secreted from synovial fibroblasts and could be distinguished by specific mAbs from COMP secreted by chondrocytes.     

Interleukin 8 and monocyte chemoattractant protein-1 in patients with juvenile rheumatoid arthritis. Relation to onset types, disease activity, and synovial fluid leukocytes

OBJECTIVE: To measure serum and synovial fluid (SF) levels of interleukin 8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in patients with juvenile rheumatoid arthritis (JRA) and to compare them with adult rheumatoid factor-positive rheumatoid arthritis (RA). METHODS: IL-8 and MCP-1 were measured by immunoassay (1) in sera obtained from 55 children with JRA and from 16 adults with RA, and (2) in SF obtained from 30 children with JRA and 11 adults with RA. RESULTS: Patients with active systemic JRA had serum levels of IL-8 and MCP-1 higher than in controls (p<0.01) and in patients with active polyarticular or pauciarticular JRA (p<0.05). In patients with RA serum MCP-1 levels were higher than in patients with the 3 JRA onset types, while no difference was found for IL-8 levels. Patients with systemic JRA and with current systemic features had serum levels of IL-8 and MCP-1 higher (p = 0.03 and p = 0.04, respectively) than patients in which systemic features had subsided. No significant differences in SF IL-8 or MCP-1 levels were found among the 3 JRA onset types or adults with RA. In patients with JRA SF leukocyte counts were correlated with SF IL-8 levels (p = 0.002), but not with MCP-1 levels. Moreover, SF levels of both IL-8 and MCP-1 were correlated with those of IL-1beta (p<0.001) and IL-6 (p<0.01), but not with those of TNF-alpha. CONCLUSION: Elevated serum levels of IL-8 and MCP-1 in patients with systemic JRA with current systemic features at sampling suggest systemic production of the 2 chemokines during systemic phases of the disease. Similar SF levels of IL-8 and MCP-1 among the 3 JRA onset-types and RA suggest comparable local production of the 2 chemokines.     

Important immunoregulatory role of interleukin-11 in the inflammatory process in rheumatoid arthritis

OBJECTIVE: To investigate the possible immunoregulatory role of interleukin-11 (IL-11) in rheumatoid arthritis (RA). METHODS: IL-11 protein was assayed in RA tissue, and the effect of exogenous IL-11 on neutralization of endogenous IL-11 was investigated with respect to tumor necrosis factor alpha (TNFalpha), matrix metalloproteinase (MMP), and tissue inhibitor of metalloproteinases (TIMP) production. RESULTS: IL-11 was found in RA synovial membranes, synovial fluids, and blood sera. Blockade of endogenous IL-11 resulted in a 2-fold increase in TNFalpha levels, which increased to 22-fold if endogenous IL-10 was also blocked. Addition of exogenous IL-11 inhibited spontaneous TNFalpha production in RA synovium only in the presence of soluble IL-11 receptor. However, exogenous IL-11 directly inhibited spontaneous MMP-1 and MMP-3 production, and up-regulated TIMP-1 in RA synovial tissue. CONCLUSION: IL-11 has important endogenous immunoregulatory effects in RA synovium, which suggests that exogenous IL-11 may have therapeutic activity in RA.     

Differential expression of two major cytokines produced by neutrophils, interleukin-8 and the interleukin-1 receptor antagonist, in neutrophils isolated from the synovial fluid and peripheral blood of patients with rheumatoid arthritis

OBJECTIVE: Neutrophils have been shown to produce interleukin-8 (IL-8) and interleukin-1 receptor antagonist (IL-1ra) in large amounts compared with other cytokines. Since IL-8 has a proinflammatory action whereas IL-1ra is antiinflammatory, our objective was to examine the relative levels of production of these cytokines by synovial fluid (SF) neutrophils isolated from patients with rheumatoid arthritis (RA). METHODS: We measured cytokine production using an enzyme-linked immunosorbent assay and analyzed messenger RNA accumulation in cells by Northern blot. RESULTS: SF neutrophils produced significantly more IL-8 and IL-1 beta, but significantly less IL-1ra, than peripheral blood neutrophils. CONCLUSION: These observations provide new information on the production of pro- and antiinflammatory molecules by neutrophils in the SF environment, and their possible role in RA.     

RANTES expression and contribution to monocyte chemotaxis in arthritis

Rheumatoid arthritis (RA) is characterized by recruitment of leukocytes from the vasculature into inflamed synovial tissue (ST) and synovial fluid (SF), which depends, in part, upon the continued maintenance of chemotactic stimuli. RANTES is a potent chemoattractant for leukocytes including monocytes and CD45RO+ memory T lymphocytes. The aim of this study was to determine the production, the source, and the function of antigenic RANTES in arthritis. We detected antigenic RANTES in SFs from RA and OA patients (100 +/- 22.7 and 72 +/- 30.7 pg/ml, respectively). CM from RA ST fibroblasts stimulated with interleukin-1beta or tumor necrosis factor-alpha contained significantly more antigenic RANTES than unstimulated CM (452 +/- 181.6 and 581 +/- 200.2 pg/ml, respectively, versus 12 +/- 4.4 pg/ml, P < 0.05). PHA-stimulated RA SF mononuclear cells secreted 5- to 15-fold more antigenic RANTES than did nonstimulated mononuclear cells, while LPS induced secretion up to 4-fold. We immunolocalized antigenic RANTES to sublining macrophages (28 +/- 3.7 and 8 +/- 2.0% immunopositive cells), perivascular macrophages (56 +/- 6.9 and 19 +/- 3.4%), and synovial lining cells (37 +/- 5.8 and 60 +/- 10.4%) in RA and OA tissue, respectively. Anti-RANTES neutralized 20.2 +/- 1.3% of the RA SF chemotactic activity for normal peripheral blood monocytes (P < 0.05). These results demonstrate antigenic RANTES in RA and OA ST and SF and identify RANTES as a chemoattractant for monocytes in the RA joint.     

Liquid ventilation for treatment of meconium aspiration syndrome in a piglet model

OBJECTIVES: Osteoarthritis (OA) is a common joint deterioration initiated by multiple factors. To better understand related factors in the development of this disease, we focused on the mechanical stress loaded on articular cartilage. MATERIALS AND METHODS: The anterior cruciate ligaments of rabbit knee joints were transected, and expression of protein kinase C (PKC) examined immunohistochemically. The PKC activator 12-o-tetradecanoyl-phorbol-13-acetate (TPA) was then administered intraarticularly. To determine the involvement of gas mediators, a cartilage defect was made on the medical femoral condyle of rabbit knee joints. Hydrostatic pressure was loaded on the cartilage taken from the surrounding defects, and levels of superoxide anion and nitric oxide (NO) were measured. Bovine chondrocytes were subjected to cyclic mechanical stretch using a Flexercell Strain Instrument. Proteoglycan synthesis and PKC activity were measured. Expression of matrix metalloproteinase (MMP)-3 and tissue inhibitor of metalloproteinase (TIMP)-1 in articular cartilages obtained from OA patients were examined using Northern blots. RESULTS: Chondrocytes from experimentally induced OA were stained positively with anti-alpha-PKC antibody. Intraarticular administration of TPA prevented the development of OA changes. Cyclic tensile stretch loaded on chondrocytes decreased proteoglycan synthesis and PKC activity. Thus, PKC is involved in the stress-mediated degradation of articular cartilage. Cartilage defects led to degradation of surrounding cartilage and to enhanced superoxide anion and NO synthesis. We also noted increased and decreased expressions of MMP-3 and TIMP-1 mRNA in human OA cartilage, respectively. CONCLUSION: PKC, gas mediators (superoxide anion, NO), and proteinases are all involved in OA.