Recombinant human interleukin-1 β–induced increase in levels of proteoglycans,stromelysin, and leukocytes in rabbit synovial fluid

Objective. To evaluate the effects of intraarticular injection of recombinant human interleukin-1β (IL-1β) on levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid (SF), and to determine the effects of leukocyte depletion on SF proteoglycan and stromelysin levels. Methods. Levels of leukocytes and of proteoglycans, stromelysin, and collagenase were evaluated 12 hours after the intraarticular injection of various doses of IL-1, and over a 24-hour period after injection at a single dose level. We used a monoclonal antibody (MAb) against leukocyte integrins, which markedly depressed leukocyte accumulation in SF, to evaluate the role of synovial leukocytes on IL-1–induced increases in SF proteoglycan and stromelysin levels. Results. Levels of both proteoglycans and stromelysin increased in the IL-1–injected joints between 4 hours and 24 hours after the injection of a single 200-ng dose of IL-1. The highest levels of stromelysin and proteoglycans were achieved with IL-1 doses ≥ 100 ng. Infiltration of polymorphonuclear cells (PMN) into the joint fluid of the IL-1–injected rabbits also increased, in a dose-dependent manner. Treatment of rabbits with MAb 1B4 markedly reduced infiltration of PMN into the joint, without affecting either stromelysin or proteoglycan levels. Conclusion. Taken together, the data suggest that there is a coordinate increase in SF stromelysin and proteoglycan levels in rabbits injected with IL-1, and that leukocytes play a minimal role in the accumulation of proteoglycans and stromelysin in the SF.     

Human recombinant interleukin-1β stimulates glycosaminoglycan production in human synovial fibroblast cultures

Human recombinant interleukin-1β (rIL-1β) stimulated glycosaminoglycan (GAG) production in human synovial fibroblast cultures. A dose-dependent increase in GAG production was found, to a maximum of 500%. Increase was detected at doses as low as 1 pg/ml of rIL-1β, reached a maximum at 10-100 pg/ml, and was apparent 10 hours after addition of rIL-1β. Stimulation of GAG was always accompanied by increased accumulation of prostaglandin E (PGE) in culture media and by increased collagenase production in approximately one-half the experiments. Indomethacin (5 m̈/ml) completely inhibited PGE stimulation by rIL-1β, but only partially inhibited that of GAG overproduction and had no effect on collagenase production. Hydrocortisone (2 m̈/ml) inhibited stimulation of all 3 parameters. Stimulation of hyaluronate in synovial cultures prevailed over that of sulfated GAG, which occurred to à lesser extent. Our results support earlier suggestions that interleukin-1 is a major active mononuclear cell factor that is capable of inducing profound changes in connective tissue cell function.     

Recombinant human interleukin-1 beta-induced increase in levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid.

OBJECTIVE. To evaluate the effects of intraarticular injection of recombinant human interleukin-1 beta (IL-1 beta) on levels of proteoglycans, stromelysin, and leukocytes in rabbit synovial fluid (SF), and to determine the effects of leukocyte depletion on SF proteoglycan and stromelysin levels. METHODS. Levels of leukocytes and of proteoglycans, stromelysin, and collagenase were evaluated 12 hours after the intraarticular injection of various doses of IL-1, and over a 24-hour period after injection at a single dose level. We used a monoclonal antibody (MAb) against leukocyte integrins, which markedly depressed leukocyte accumulation in SF, to evaluate the role of synovial leukocytes on IL-1-induced increases in SF proteoglycan and stromelysin levels. RESULTS. Levels of both proteoglycans and stromelysin increased in the IL-1-injected joints between 4 hours and 24 hours after the injection of a single 200-ng dose of IL-1. The highest levels of stromelysin and proteoglycans were achieved with IL-1 doses greater than or equal to 100 ng. Infiltration of polymorphonuclear cells (PMN) into the joint fluid of the IL-1-injected rabbits also increased, in a dose-dependent manner. Treatment of rabbits with MAb 1B4 markedly reduced infiltration of PMN into the joint, without affecting either stromelysin or proteoglycan levels. CONCLUSION. Taken together, the data suggest that there is a coordinate increase in SF stromelysin and proteoglycan levels in rabbits injected with IL-1, and that leukocytes play a minimal role in the accumulation of proteoglycans and stromelysin in the SF.     

Effects of recombinant human interleukin-1β on rabbit articular chondrocytes

Using an in vitro model, we characterized the production of prostaglandin E₂, 6-keto-prostaglandin F_1α, and thromboxane B₂ by normal rabbit articular chondrocytes stimulated by interleukin-1β. The prostanoids were produced in a dose- and time-dependent manner, which was sensitive to actinomycin D and cycloheximide. Interleukin-1 also had a pronounced, but reversible, cytostatic effect on chondrocytes, which was not attributable to prostanoid or polyamine synthesis.     

In vivo expression of stromelysin in synovium and cartilage of rabbits injected intraarticularly with interleukin-1β

Objective. To examine the in vivo expression of the matrix metalloproteinase stromelysin in the synovium and articular cartilage of rabbits injected intraarticularly with recombinant human interleukin-1β (IL-1). Methods. The direct isolation of messenger RNA (mRNA) from articular cartilage without the prior isolation of chondrocytes is described. The in vivo expression of stromelysin was examined at the mRNA level by Northern blot analysis, and at the protein level by in situ immunolocalization and by enzyme-linked immunosorbent assay. Results. In the synovium of IL-1–injected joints, stromelysin mRNA levels were highest at 4 hours and declined to background levels within 24 hours. In the cartilage of IL-1–injected joints, stromelysin mRNA was elevated at 4 hours and continued to increase until 8 hours, before declining. Stromelysin mRNA expression preceded a similar increase in stromelysin protein levels in both synovium and cartilage. Conclusion. Intraarticular injection of IL-1 induced the endogenous expression of stromelysin mRNA and protein in both synovium and cartilage. The kinetics of stromelysin expression correlated well with the accumulation of stromelysin and proteoglycan in synovial fluids. Therefore, the de novo synthesis of stromelysin in cartilage may have contributed to the loss of proteoglycan from that tissue.     

Stimulation of the hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β

Monocyte/macrophage polypeptides (monokines) alter the properties of synovial cells. This interaction could explain some of the properties of the inflamed synovium in rheumatic disease. Only recently has it been possible to test the action of purified monokines on the target synovial cells. We report here that recombinant human tumor necrosis factor α, tumor necrosis factor β (lymphotoxin), interleukin-1α, and interleukin-1β stimulate the hyaluronic acid (HA) levels of human synovial fibroblast-like cells. The effect of monokines was generally inhibited by indomethacin, suggesting the involvement of an endogenous cyclooxygenase product in the stimulation, and by the glucocorticoid, dexamethasone. In contrast, all-trans-retinoic acid stimulated synovial cell plasminogen activator activity but did not increase the HA levels. These findings could help to explain the raised HA levels found in the joint fluids and in the circulation of patients with rheumatic disease.     

Transforming growth factor β exerts opposite effects from interleukin-1β on cultured rabbit articular chondrocytes through reduction of interleukin-1 receptor expression

Objective. The aim of the study was to determine whether transforming growth factor β (TGFβ) modulates the effects and the receptor expression of interleukin-1 (IL-1) in rabbit articular chondrocytes (RAC). Methods. Collagen, glycosaminoglycan, and collagenase production, together with ¹²⁵I-labeled IL-1 binding, were analyzed in RAC cultures. Results. TGFβ reduced both IL-1 effects on matrix metabolism and IL-1 receptor expression. Conclusion. TGF/3 acts as an antagonist of the effects of IL-1 through down-regulation of its receptor expression.     

Melatonin attenuates TNF‐α and IL‐1β expression in synovial fibroblasts and diminishes cartilage degradation: Implications for the treatment of rheumatoid arthritis

The hormone melatonin has many properties, including antioxidant, anti‐inflammatory, and immunomodulatory effects. Melatonin has been demonstrated to be beneficial in several inflammatory autoimmune diseases, but its effects in rheumatoid arthritis (RA) remain controversial. We sought to determine how melatonin regulates inflammation in RA. We found that melatonin dose‐dependently inhibits tumor necrosis factor‐α (TNF‐α) and interleukin (IL)‐1β expression through the PI3K/AKT, ERK, and NF‐κB signaling pathways. We also identified that melatonin inhibits TNF‐α and IL‐1β production by upregulating miR‐3150a‐3p expression. Synovial tissue specimens from RA patients and culture of human rheumatoid fibroblast‐like synoviocytes confirmed that the MT1 receptor is needed for the anti‐inflammatory activities of melatonin. Importantly, melatonin also significantly reduced paw swelling, cartilage degradation, and bone erosion in the collagen‐induced arthritis mouse model. Our results indicate that melatonin ameliorates RA by inhibiting TNF‐α and IL‐1β production through downregulation of the PI3K/AKT, ERK, NF‐κB signaling pathways, as well as miR‐3150a‐3p overexpression. The role of melatonin as an adjuvant treatment in patients with RA deserves further clinical studies.     

Interleukin-1β induces cytosolic phospholipase a2 and prostaglandin h synthase in rheumatoid synovial fibroblasts

Objective. In order to investigate potential regulatory mechanisms for the increased production of prostaglandin E₂ (PGE₂) in interleukin-1β (IL-1β)–stimulated rheumatoid synovial fibroblasts (RSF), this study examined the induction of phospholipase A₂ (PLA₂) and prostaglandin H synthase (PGHS) enzymes and the correlation of these events with PGE₂ production in IL-1β–stimulated RSF. Methods. Protein and messenger RNA (mRNA) levels of cytosolic PLA₂ (cPLA₂) and PGHS-2 enzymes in IL-1β–stimulated RSF were measured by Western and Northern blotting, respectively, using specific antisera and complementary DNA probes. Enzymatic activity of cPLA₂ was determined in cell-free reaction mixtures utilizing mixed micelles of ¹⁴C-phosphatidylcholine and Triton X-100 as the substrate. PGE₂ levels were quantitated using a commercial enzyme immunoassay kit. Results. Incubation of RSF with IL-1β increased the mRNA and protein levels for the high molecular weight cPLA₂ as well as for the mitogen/growth factor–responsive PGHS (PGHS-2). The IL-1 receptor antagonist completely abolished the induction of these two enzymes and the stimulation of PGE₂ production by IL-1β in RSF. In contrast, levels of the other known forms of these enzymes, i.e., the 14-kd secretory group II PLA₂ (sPLA₂) and the constitutive form of PGHS (PGHS-1), were unaffected by IL-1β treatment. Conclusion. These are the first data to demonstrate the coordinate induction by IL-1 of cPLA₂ and PGHS-2 in RSF. The time-course for the induction of these enzymes suggests that their increase contributes to the increased production of PGE₂ in IL-1–treated RSF, and may help explain the capacity of RSF to produce large amounts of PGE₂.     

Reduction of leukocyte and interleukin-1β concentrations in the synovial fluid of rheumatoid arthritis patients treated with methotrexate

Objective. To examine the effect of methotrexate (MTX) on the numbers of leukocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with active rheumatoid arthritis (RA). Methods. Twelve patients were treated with MTX; 5 patients not taking MTX served as controls. Samples of PB and SF were collected at 0, 1, 4, and 8 weeks of the study. Disease activity was scored, and total leukocytes, neutrophils, lymphocytes, and CD4+, CD8+, DR+, and CD25+ lymphocyte subsets were analyzed in PB and SF. Interleukin-1β (IL-1β) concentrations in SF were determined. Results. Patients treated with MTX showed significant clinical improvement. No change in PB leukocytes or lymphocyte subsets was observed in either patient group over the 8-week study period. In contrast, the number of leukocytes, the number and proportion of neutrophils, and the concentration of IL-1β in the SF of patients treated with MTX were reduced. In addition, in MTX-treated patients, there was an appreciable decrease in SF CD8+ lymphocytes, but not CD4+, DR+, or CD25+ lymphocytes. Conclusion. These findings suggest that in RA, MTX acts, at least in part, by reducing the migration of leukocytes into the inflamed synovium. Local reduction of IL-1β secretion may contribute to this effect.     

A case of spontaneous femoral neck fracture associated with multicentric reticulohistiocytosis. Oversecretion of interleukin-1β, interleukin-6, and tumor necrosis factor α by affected synovial cells

We describe a case of left femoral neck fracture associated with multicentric reticulohistiocytosis (MR). Biopsy specimens from a skin nodule and from synovial tissue showed histiocytic multinucleated giant cells (MR cells) that are characteristic of MR. A surgical specimen from the resected femoral head revealed that multinucleated giant cells and mononuclear cells invaded the marginal subchondral bone, without evident pannus. These cells also infiltrated into the fracture site, with bone resorption by activated osteoclasts. Immunohistochemical studies of synovium from the left hip joint showed positive staining for interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α, and abundant cytokine production by cultured synovial cells was demonstrated. These findings suggest that the subchondral invasion and intramedullary infiltration by MR cells caused articular destruction and/or fracture as a result of oversecretion of the cytokines.     

Effect of interferon α, γ, and tumor necrosis factor α on the HLA-A,B, C expression of cell lines derived from human liver

ABSTRACT— Our study was undertaken to determine whether human recombinant interferon α(rIFNα), γ(rIFNγ), and tumor necrosis factor α(rTNFα) exert an effect on the HLA-A, B, C expression of human liver cell lines. The HLA-A, B, C expression was assayed by immunoperoxidase staining and enzyme-linked immunosorbent assay. rIFNα and γ enhanced the HLA-A, B, C expression of the three cell lines tested, Chang cells, SK-Hep-1, and PLC/PRF/5. The activity of rIFNγ proved more than 8000 times more potent than that of rIFNα in Chang cells, 30 times in SK-Hep-1, and 20 times in PLC/PRF/5, respectively. rTNFα also enhanced the HLA-A, B, C expression of the three cell lines. The enhancement of HLA-A, B, C expression by rIFNα and γ reached a peak on day 3, and that by rTNFα on day 5. These findings suggest that IFNα, IFNγ, and TNFα may play similar roles in enhancement of HLA-A, B, C expression of hepatocytes in hepatitis and hepatoma cells.     

Relationship between α1-antitrypsin inactivation and tumor necrosis factor α concentration in the synovial fluid of patients with rheumatoid arthritis

Objective. To examine the relationship between α₁-antitrypsin (α₁AT) specific activity and tumor necrosis factor α (TNFα) concentration in synovial fluid from 48 patients with rheumatoid arthritis. Methods. The specific activity of α₁AT was calculated from the measurement of α₁AT concentration (by rocket immunoelectrophoresis) and elastase inhibitory capacity. TNFα was detected by enzyme-linked immuno-sorbent assay. Results. TNFα concentrations correlated with the extent of α₁AT inactivation. Conclusion. Our findings are consistent with a role of elastase in TNFα release within the inflamed joint.