L. van den Bersselaar

IL-1-independent role of IL-17 in synovial inflammation and joint destruction during collagen-induced arthritis.

T cell IL-17 displays proinflammatory properties and is expressed in the synovium of patients with rheumatoid arthritis. Its contribution to the arthritic process has not been identified. Here, we show that blocking of endogenous IL-17 in the autoimmune collagen-induced arthritis model results in suppression of arthritis. Also, joint damage was significantly reduced. In contrast, overexpression of IL-17 enhanced collagen arthritis. Moreover, adenoviral IL-17 injected in the knee joint of type II collagen-immunized mice accelerated the onset and aggravated the synovial inflammation at the site. Radiographic and histologic analysis showed markedly increased joint destruction. Elevated levels of IL-1β protein were found in synovial tissue. Intriguingly, blocking of IL-1αβ with neutralizing Abs had no effect on the IL-17-induced inflammation and joint damage in the knee joint, implying an IL-1 independent pathway. This direct potency of IL-17 was underscored in the unabated IL-17-induced exaggeration of bacterial cell wall-induced arthritis in IL-1β−/− mice. In conclusion, this data shows that IL-17 contributes to joint destruction and identifies an IL-1-independent role of IL-17. These findings suggest IL-17 to be a novel target for the treatment of destructive arthritis and may have implications for tissue destruction in other autoimmune diseases.

Intra-articular IL-10 gene transfer regulates the expression of collagen-induced arthritis (CIA) in the knee and ipsilateral paw

We studied the effects of local IL‐10 application, introduced by a recombinant human type 5 adenovirus vector, in the mouse knee joint during the early phase of CIA. One intra‐articular injection with the IL‐10‐expressing virus (Ad5E1mIL‐10) caused substantial over‐expression of IL‐10 in the mouse knee joint, using virus dosages which did not induce distracting inflammation. High expression of IL‐10 was noted for a few days, being maximal at day 1. One intra‐articular injection of Ad5E1mIL‐10 in the knee joints of collagen type II (CII)‐immunized mice, before onset of CIA was noted, reduced the incidence of collagen arthritis in that knee. Of high interest, the protective effect of local IL‐10 expression by Ad5E1mIL‐10 was not restricted to the knee joint alone. The arthritis incidence in the ipsilateral paw was highly suppressed. In contrast, local IL‐10 over‐expression was not effective when treatment was started after onset of CIA. Further analysis in the acute streptococcal cell wall‐induced arthritis model revealed that local IL‐10 over‐expression markedly suppressed the production of tumour necrosis factor‐alpha (TNF‐α) and IL‐1α, but had no significant effect on IL‐1β and IL‐12 production in the inflamed synovium. These data indicate that local over‐expression of IL‐10 in the knee joint of mice regulates the expression of collagen arthritis, probably through down‐regulation of TNF‐α.

Cationization of catalase, peroxidase, and superoxide dismutase. Effect of improved intraarticular retention on experimental arthritis in mice.

Several enzymes and other proteins were made cationic either by coupling to polylysine or by shielding of anionic sites. These cationic proteins, all having an isoelectric point greater than 8.5 exhibited excellent retention in articular structures when injected in mouse knee joints. Autoradiography and histochemistry showed that cationic forms of catalase, superoxide dismutase, and horseradish peroxidase were firmly retained by synovial and cartilaginous tissues. The half-life of these enzymes in the joint is thus significantly extended compared with native enzymes. The native enzymes and their cationic derivatives were tested for antiinflammatory properties in mice, using antigen-induced arthritis and zymosan-induced arthritis. It was found that injection of cationic catalase or peroxidase induced a marked suppression of some parameters of the inflammatory response in both types of arthritis, as measured by 99m technetium pertechnetate uptake and leakage of 125I-labeled albumin. Native catalase and peroxidase were less, or not at all effective. Cationic superoxide dismutase or cationic nonenzyme proteins did not suppress inflammation. The observed suppression of two different types of inflammation (an immune and a nonimmune arthritis) by catalase and peroxidase suggests that elimination of peroxides contributes to the suppression of an inflammatory response. We would hypothesize that cationic enzymes offer the possibility for investigating the mechanisms of inflammation and, in addition, might be interesting from a therapeutical point of view.

Reversible depletion of synovial lining cells after intra-articular treatment with liposome-encapsulated dichloromethylene diphosphonate

SummaryWe studied the depletion and repopulation of synovial lining cells in mice. A single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP) in the mouse knee joint caused selective elimination of synovial lining cells. Depletion of cells occurred within a few days as evidenced by light microscopic, electronmicroscopic and immunohistochemical studies. Maximal depletion was seen on day 7. Repopulation was observed in the following weeks, starting at the bone side of the joint. Until day 30, full recovery (60% recovery) was not observed in the lining lying adjacent to the dermis. Side effects on cartilage metabolism, such as inhibition of proteoglycan synthesis or degradation of proteoglycans from the matrix was minor but significant, 1 and 2 days after liposome treatment but thereafter full recovery was observed. Selective elimination of lining cells from the joint enabled us to study the in vivo role of these cells in the onset and subsequent pathology of experimental arthritis. An immune-complex-mediated experimental arthritis elicited in lining cell depleted joints that had received CL2MDP-liposomes 7 days earlier prevented inflammation as compared to controls.

Role of macrophage-like synovial lining cells in localization and expression of experimental arthritis.

(1995). Role of Macrophage-Like Synovial Lining Cells in Localization and Expression of Experimental Arthritis. Scandinavian Journal of Rheumatology: Vol. 24, No. sup101, pp. 83-89.

Phagocytic synovial lining cells in experimentally induced chronic arthritis: down-regulation of synovitis by CL2MDP-liposomes

SummaryChronic inflammation of the joint is characterized by the long-term presence of macrophage-like cells in the multilayered synovium. We examined whether synovial phagocytic cells which have settled in the inflamed lining layer play a role in perpetuating synovitis by selectively eliminating them from chronically arthritic murine knee joints. For this purpose we used liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP, Clodronate). Injection of CL2MDP-liposomes into acutely inflamed knee joints (6h, 1 and 3 days) had no significant effect on late chronic synovitis (14 and 21 days after arthritis induction) as observed in haematoxylin and eosin-stained total knee joint sections. Liposomes did not reach the lining layer, as seen with fluorescent liposomes. Additional in vitro studies revealed that activated polymorphs were not affected by CL2MDP-liposomes within 16 h of incubation. Liposomes formed clusters, however, in the presence of intact polymorphs or extracts of polymorphs. In contrast, a significant down-regulation of late synovitis was observed if CL2MDP-liposomes were given during the chronic phase (day 7). Phosphate-buffered saline (PBS) alone or PBS-liposomes had no effect on synovitis. A single injection of CL2MDP-liposomes eliminated many of the phagocytic lining cells and deeper lying inflammatory cells for at least 4 weeks. Free CL2MDP had a minor but significant effect. This study indicates that phagocytic synovial lining cells play an important role in propagating chronic synovitis. To eliminate them from inflamed knee joints, CL2MDP-liposomes should be injected in the chronic and not in the early arthritic phase.

Role of phagocytic synovial lining cells in experimental arthritis

We investigated thein vivo role of phagocytic synovial lining cells (SLC) in the onset of experimental arthritis by depleting phagocytic SLC prior to arthritis induction. Phagocytic SLC were depleted by a single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP). Seven days after injection optimal depletion was observed and this time point was chosen for induction of arthritis in SLC depleted joints. Joint swelling was highly reduced after elicitation with either zymosan, immune complexes or antigen, as compared to observations in normal non-depleted joints. In addition cellular infiltration was markedly reduced. Further study in the immune complex mediated arthritis revealed that reduced cell influx in SLC depleted knee joints was correlated to lowered chemotactic activity and IL-1 levels as measured in washouts of joint tissues. This indicates that IL-1 driven chemotactic factors might be involved. Furthermore reduced cell influx was also correlated to significantly diminished loss of35S-prelabeled PG from the cartilage. Out data indicate that SLC are directly involved in the onset of joint inflammation.

Electrical charge of a protein determines penetration and localization in hyaline articular cartilage

SummarySufficient antigen retention in joint structures is a prerequisite for sustained antigen-induced arthritis. In this in vitro study we investigated the retention of native and charge-modified bovine serum albumin (BSA) with patellar cartilage of different species (mouse, rat, rabbit, and man). Association of BSA with cartilage due to charge of the protein with that due to immune complex formation was compared. Using radiolabeled proteins we showed quantitatively that the retention of highly positively charged BSA is considerably higher (200–500 times) than retention of the anionic BSA in all cartilage species examined. Mouse, rat, and human cartilage bind more protein per mg dry weight, compared to rabbit cartilage. No clear-cut relation was found with the glycosaminoglycans (GAG) contents. Retention of native BSA by anti-BSA antibodies was low in the dense hyaline patellar cartilage in all species. Enhanced immune-complex formation was found in marginal regions of rabbit patellar cartilage, consisting of fibrous-like cartilage. Localization studies by autoradiography showed that cationic BSA penetrates deeply into the cartilage matrix. Even in a thick cartilage specimen, such as rabbit cartilage, very deep penetration into the calcified zone was demonstrated. This study indicates that binding and deep penetration of cationized protein in cartilage is not restricted to mouse specimen, as had been found previously, but is a general phenomenon.

Higher efficacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis

Th17 cells and their cytokines are linked to the pathogenesis of rheumatoid arthritis, a chronic autoimmune disease characterized by joint inflammation. Th17 development is initiated by combined signaling of TGF-β and IL-6 or IL-21, and can be reduced in the absence of either IL-6 or IL-21. The aim of this study was to assess whether combinatorial IL-6/IL-21 blockade would more potently inhibit Th17 development, and be more efficacious in treating arthritis than targeting either cytokine. We assessed in vitro Th17 differentiation efficacy in the absence of IL-6 and/or IL-21. To investigate in vivo effects of IL-6/IL-21 blockade on Th17 and arthritis development, antigen-induced arthritis (AIA) was induced in IL-6-/- x IL-21R-/- mice. The therapeutic potential of this combined blocking strategy was assessed by treating mice with collagen-induced arthritis (CIA) with anti-IL-6R antibodies and soluble (s)IL-21R.Fc. We demonstrated that combined IL-6/IL-21 blocking synergistically reduced in vitro Th17 differentiation. In mice with AIA, absence of IL-6 and IL-21 signaling more strongly reduced Th17 levels and resulted in stronger suppression of arthritis than the absence of either cytokine. Additionally, anti-IL-6/anti-IL-21 treatment of CIA mice during the arthritis induction phase reduced disease development more potent than IL-6 or IL-21 inhibition alone, as effective as anti-TNF treatment. Collectively, these results suggest dual IL-6/IL-21 inhibition may be a more efficacious therapeutic strategy compared to single cytokine blockade to suppress arthritis development.

FRI0005 Combination Blocking of IL-22 and IL-17 During Experimental Arthritis Potently Reduces TH17-Driven Disease Progression

Background Rheumatoid arthritis (RA) patients show elevated levels of IL-22 and IL-22-producing T helper cells that correlate to erosive disease, suggesting a role for this cytokine in the pathogenesis of RA. Interestingly, IL-22 is a dual cytokine with pro- and anti-inflammatory properties, and its effects might be regulated by cooperation and crosstalk with IL-17. Objectives The purpose of this study was to elucidate the role of IL-22 in the development of a spontaneous model of experimental arthritis by using IL-1Ra knockout mice. Additionally, we aimed to investigate the therapeutic potential of combined IL-22/IL-17 blocking during experimental arthritis. Methods IL-1Ra-deficient mice develop spontaneous arthritis due to excess IL-1 signaling, and we previously demonstrated the importance of IL-17 and Th17 cells in this model1. To investigate the role of IL-22 in this arthritis model, we compared IL-1Ra-/- x IL-22+/+ mice to IL-1Ra-/- mice lacking IL-22 expression. Paw joint swelling was scored weekly, and mice were sacrificed at the age of fifteen weeks. In addition, arthritic IL-1Ra-/- x IL-22-/- mice were treated with anti-IL-17 antibodies to determine the therapeutic potency of this combined blocking strategy during experimental arthritis. Results IL-1Ra-/- mice that also lack IL-22 showed strongly reduced arthritis development, reaching a disease incidence of only 54% at the age of 15 weeks compared to 93% in IL-1Ra-/- x IL-22+/+ mice. In addition, arthritis severity of the mice that did develop arthritis was significantly reduced by 30.6% in the absence of IL-22. Interestingly, combined blocking of IL-22 and IL-17 using IL-1Ra-/- x IL-22-/- mice treated with neutralizing anti-IL-17 antibodies after the onset of arthritis demonstrated clear additive effects compared to blocking these single cytokines alone, thereby potently reducing progression of this Th17-driven arthritis model. Conclusions These findings suggest that IL-22 plays an important role both in the initiation and augmentation of Th17-dependent experimental arthritis, and that targeting IL-22, especially in combination with IL-17 therefore seems an interesting, potent strategy in RA treatment. References Koenders MI, Devesa I, Marijnissen RJ, Abdollahi-Roodsaz S, Boots AM, Walgreen B, di Padova FE, Nicklin MJ, Joosten LA, van den Berg WB. Interleukin-1 drives pathogenic Th17 cells during spontaneous arthritis in interleuking-1 receptor antagonist-deficient mice. Arthritis Rheum. 58(11): 3461-70, 2008. Disclosure of Interest None declared