Phagocytic lining cells determine local expression of inflammation in type II collagen-induced arthritis.

OBJECTIVE: To investigate the in vivo role of phagocytic synovial lining cells in the local expression of inflammation in type II collagen-induced arthritis (CIA) in DBA/1J mice. METHODS: On various days before arthritis induction (day 7, 5, or 2), phagocytic lining cells were selectively depleted from the synovial layer by injecting multilamellar liposomes containing clodronate (dichloromethylene diphosphonate) directly into the knee joints. As controls, either PBS or PBS-laden liposomes were injected. CIA was induced by immunizing mice with heterologous bovine type II collagen in Freund's complete adjuvant. Arthritis onset was synchronized by a single intraperitoneal injection of lipopolysaccharide; arthritis was evaluated in hematoxylin and eosinstained knee joint sections. Chemotactic activity in synovial washout samples was detected in a Transwell chemotactic assay. Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) protein levels were measured in NOB-1 and L929 bioassays, respectively. IL-1 messenger RNA (mRNA) in synovial specimens was measured by reverse transcriptase-polymerase chain reaction. IL-1 was also detected immunohistologically in knee joint sections. RESULTS: In clodronate-laden liposome-treated, lining-depleted knee joints, there was significantly decreased inflammation compared with controls. Cell influx into the synovium was markedly decreased. Expression of IL-1 mRNA in the synovium was significantly reduced. IL-1 was detected only in some cells in the deeper synovial layer, in contrast to controls, in which large numbers of cells in the deeper synovial layer were stained. In synovial washouts from lining-depleted knee joints, biologically active IL-1 levels were reduced by 40% at 6 hours after arthritis induction. Most strikingly, chemotactic activity was highly decreased in these synovial washout samples. When IL-1 or TNF alpha was injected into the knee joints of immunized mice in which arthritis was not yet expressed, arthritis was not induced in the lining-depleted joints, whereas marked cell influx was found in control joints. CONCLUSION: Our data indicate that phagocytic lining cells play a crucial role in the local expression of inflammation in systemically induced CIA. Phagocytic lining cells probably form an important source of chemotactic factors which are set free upon activation by IL-1 or TNF alpha.

Major role for interleukin 1 but not for tumor necrosis factor in early cartilage damage in immune complex arthritis in mice.

OBJECTIVE: To determine the regulating role of interleukin-1 alpha and beta (IL-1 alpha, beta) and tumor necrosis factor alpha (TNF-alpha) on inhibition of proteoglycan synthesis and proteoglycan degradation in early immune complex arthritis (ICA) in the mouse. METHODS: In the early phases of arthritis, IL-1 and TNF were measured using cytokine specific bioassays, the NOB.1 EL-4 and L929 assay, respectively. The impact of IL-1 in proteoglycan synthesis was studied by neutralizing the formed IL-1 during early arthritis either by giving anti-IL-1 specific antibodies intravenously or IL-1 receptor antagonist (IL-1ra) intraperitoneally by osmotic pumps. TNF-alpha was neutralized by giving monoclonal antibodies directed against murine TNF-alpha. Synthesis of proteoglycans was measured ex vivo by uptake of 35S-sulfate by patellae derived from inflamed and control, noninflamed knee joints. In vivo formation of 35S-sulfate labeled proteoglycans was studied by autoradiography. Degradation of proteoglycans was measured by labeling patellae in vivo with 35S-sulfate before arthritis induction. RESULTS: High levels of IL-1 are formed during the first phase of immune complex arthritis (ICA). Neutralization of either IL-1 alpha or beta with specific polyclonal antibodies resulted only in partial blocking, whereas a combination fully blocked inhibition of proteoglycan synthesis. Full blocking was also found after systemic treatment with high amounts of IL-1 receptor antagonist (1.2 mg/day during 3 days). Influx of cells was also significantly reduced both in the anti-IL-1 as well as in the IL-1ra treated groups. Whether infiltrating cells are involved in inhibition of proteoglycan synthesis was further investigated in neutropenic mice. Significantly higher levels of IL-1 were found in arthritic joints of neutropenic compared with control mice. Suppression of proteoglycan synthesis was similar in arthritic knee joints of normal and neutropenic mice. However, only minor proteoglycan degradation was found in the latter. TNF-alpha was undetectable in the bioassay in early ICA and neutralization of TNF-alpha did not change either swelling, cell influx, proteoglycan synthesis or proteoglycan degradation. CONCLUSION: Local production of IL-1 in ICA in knee joints seems directly responsible for inhibition of proteoglycan synthesis. A direct role of IL-1 in proteoglycan loss is unlikely, but indirectly IL-1 may be involved in proteoglycan breakdown by attracting inflammatory leukocytes and activating synovial cells. TNF-alpha seemed to have no effect on either cell influx, proteoglycan synthesis or proteoglycan degradation in this model.

Early cartilage degradation in cationic immune complex arthritis in mice: relative role of interleukin 1, the polymorphonuclear cell (PMN) and PMN elastase.

OBJECTIVE: To determine the relative role of interleukin 1 (IL-1), polymorphonuclear cell (PMN) and PMN elastase in early cartilage degradation in cationic immune complex mediated arthritis (ICA) in mice. ICA is characterized by early production of IL-1, pronounced influx of PMN and marked cartilage degradation within one day. METHODS: IL-1 was neutralized by polyclonal antibodies against IL-1 alpha and IL-1 beta, given shortly before arthritis induction. The role of PMN was studied in mice made neutropenic by whole body irradiation (750 rad). The effect of elastase was examined in beige mice with PMN deficient in elastase. RESULTS: Neutralizing IL-1 during arthritis induction reduced PMN infiltration significantly and diminished cartilage degradation by 50%. In neutropenic mice, joint inflammation was virtually absent and cartilage proteoglycan loss was abolished. Finally arthritis was induced in beige mice. Although elastase appeared to be the dominant cartilage degrading factor in vitro, we found no difference in cartilage degradation in arthritic joints of beige mice and their normal littermates. CONCLUSIONS: Our data suggest that IL-1 is a crucial factor regulating PMN influx in ICA. The PMN are involved in cartilage degradation but a direct destructive role (e.g., by elastase) seems unlikely.

In vivo role of phagocytic synovial lining cells in onset of experimental arthritis.

The in vivo role of phagocytic synovial lining cells (SLC) was studied in acute experimental arthritis in the mouse. SLCs were selectively depleted by injecting liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP, Clodronate). Optimal depletion of phagocytic lining cells occurred 7 days after CL2MDP liposome injection. Eliciting an immune complex-mediated arthritis in SLC-depleted knee joints largely prevented inflammation if compared to control arthritic knee joints. Joint swelling and influx of inflammatory cells into the joint cavity was markedly diminished. Cartilage damage, in this model related to influx of inflammatory cells, was significantly decreased. Reduced influx of inflammatory cells (mainly polymorphonuclear neutrophils) was correlated to a decreased production of chemotactic factors as measured in washouts of arthritic joints in a two-compartment Transwell system. Interleukin-1-driven chemotactic factors seem to be involved. Interleukin-1 levels were significantly lowered in SLC-depleted arthritic knee joints as compared to controls. Injection of recombinant murine interleukin-1 in SLC-depleted knee joints caused less influx of inflammatory cells as compared to injection into control knee joints. A specific damage of CL2MDP liposome treatment to synovial blood vessels was excluded as intraarticular injection of human recombinant C5a in lining-depleted knee joints showed similar influx of inflammatory cells if compared to human recombinant C5a injection in control knee joints. This study indicates that in immune complex-mediated arthritis, phagocytic lining cells regulate the onset of the inflammatory response.

Cationic immune complex arthritis in mice--a new model. Synergistic effect of complement and interleukin-1.

A novel cationic immune-complex-mediated arthritis (ICA) model was developed in mice. The highly cationic protein lysozyme was coupled to poly-L-lysine (PLL) and injected intra-articularly into the knee joint of the mouse, shortly after systemic administration of specific antibodies. A vehement joint inflammation developed, characterized by severe joint swelling and the influx of predominantly polymorphonuclear (PMN) leukocyte. Unique properties were combined in this protein. First, an excellent retention of the antigen in joint structures was found, facilitating sufficient IC formation in the synovial tissue and at the cartilage surface. Secondly, PLL.lysozyme appeared to be a potent inducer of interleukin-1 (IL-1). Similar IL-1 production was measured at 6 hours, in both immune or nonimmune mice. Neutralization with antibodies against either IL-1 alpha or IL-1 beta revealed that IL-1 alpha was the dominant cytokine. Resident cells were responsible for this IL-1 production since a comparable IL-1 signal was measured after intra-articular injection of PLL.lys in neutropenic mice. We further investigated whether IL-1 and complement factors were involved in the onset of this ICA. Neutralizing the IL-1 production with antibodies directed against IL-1 alpha and beta showed a significant decrease in joint swelling. Complement depletion by cobra venom factor also prevented the onset of arthritis for the greater part. Only a minor swelling remained at 6 hours after eliciting arthritis, which was similar to the swelling after injecting the antigen alone and probably reflects IL-1 mediated inflammation. In this study, the authors show a synergistic action of IL-1 and complement in the onset of cationic ICA. Unique properties of the antigen such as excellent retention and its ability to induce IL-1 are combined within one molecule and make this antigen arthritogenic in the presence of antibodies and complement activation.

Immune complex formation between IgM rheumatoid factor and IgG generated by hyaluronic acid.

Immunoglobulin inclusions are regularly detected in granulocytes obtained from the synovial fluid of rheumatoid arthritis (RA) patients. In contrast, granulocytes isolated from the blood of the same patients usually contain no immunoglobulin inclusions. Using the indirect granulocyte phagocytosis test (IGPT), we obtained evidence that this discrepancy can be explained by the finding that hyaluronic acid (HA), a component of synovial fluid, increases the avidity of rheumatoid factor (RF), resulting in the formation of IgG-IgM-RF complexes. The addition of HA to RA sera and subsequent testing by IGPT revealed an increased uptake of the induced immune complexes by these cells, which was dependent on the HA dose. Furthermore, supplementation of normal human serum with purified IgM-RF generated a positive IGPT result in a dose-dependent manner in the presence of HA. We conclude that HA, a component of synovial fluid, might facilitate immune complex formation in the joint cavity, resulting in the inflammatory reaction in the joints of RA patients.

Circulating immune complexes and rheumatoid arthritis: the induction of immune complex formation between rheumatoid factor and IgG by polyethylene glycol.

Circulating immune complexes (CIC) as detected by the C1q binding assay (C1qBA) in sera from patients with rheumatoid arthritis (RA) were not demonstrable in these sera with the indirect granulocyte phagocytosis test (IGPT). This discrepancy could be explained by the finding that polyethylene glycol (PEG), used in the C1qBA, enhanced the binding of rheumatoid factor IgM (RFIgM) and IgG resulting in immune complex (IC) formation. The addition of PEG to RA sera and subsequent testing of these sera in the IGPT revealed increased uptake of IC by these cells, dependent on the PEG dose. Addition of purified RFIgM to a normal human serum generated a positive IGPT in a dose dependent way. We conclude that in RA sera PEG induces IC between RFIgM and IgG. Therefore, assays devised to measure CIC in RA sera which are based on PEG (like the C1qBA) overestimate the amounts of IC present in the circulation in vivo.