The effect of lactation in the post-partum arthritis of MRL-lpr/fasmice.

OBJECTIVE: To investigate the effects of lactation on the post-partum arthritic flare in MRL-lpr/fas mice. METHODS: Three groups of mice were investigated. Group 1: females whose litters were weaned at termination of the experiment; group 2: females whose litters were weaned at parturition; group 3: females who were not bred. Clinical evaluation was carried out at 5-day intervals following parturition. Blood samples were also collected during the course of the experiment and assayed for corticosterone and prolactin. Histological evaluation of the joints was assessed at day 30. RESULTS: The incidence of swelling and erythema, the bimalleolar ankle width and the histopathology were significantly reduced by removal of the litters at parturition. This correlated well with a decrease seen in prolactin levels in these females. Corticosterone, an immunomodulatory glucocorticoid, did not play a significant role in the arthritic flare. CONCLUSION: Our findings suggest that prolactin levels contribute to the inflammation seen in MRL-lpr/fas mice following parturition.

Photodynamic therapy in immune (non-oncological) disorders: focus on benzoporphyrin derivatives.

This review examines the efficacy of photodynamic therapy in the treatment of immunological disorders. Photodynamic therapy (PDT) is a 2-step procedure. Firstly, a photosensitiser is introduced into the body, where it accumulates selectively in cells with elevated metabolism, such as cancer cells or activated cells of the immune system. Second, light is applied at a wavelength that excites the photosensitiser, producing a variety of short-lived oxygen-derived species. The effect is dependent on the doses of both photosensitiser and activating light. The mechanisms of action of PDT are multifactorial. Induction of high levels of oxidative stress results in necrotic cell death, while lower intensity oxidative stress initiates apoptosis. Sublethal doses may result in the modification of cell surface receptor expression levels and cytokine release and consequently influence cell behaviour. Immunomodulatory PDT (IPDT) utilises mainly apoptotic and sublethal doses. The studies reported here utilise verteporfin, a benzoporphyrin-derived chlorin-like photosensitiser. Veteporfin is a second generation photosensitiser, displaying rapid clearance and consequently a reduced period of skin photosensitivity compared with the first generation photosensitiser, porfimer sodium. In vivo studies showed that IPDT was effective in alleviating immunopathology in murine models of arthritis, contact hypersensitivity, experimental allergic encephalomyelitis and retention of allogeneic skin grafts. Based on these findings, early stage clinical trials with IPDT were initiated recently for the treatment of psoriasis, psoriatic arthritis and rheumatoid arthritis. While verteporfin has been the photosensitiser which pioneered IPDT, a new benzoporphyrin derivative photosensitiser, QLT0074, is under development. This has demonstrated an enhanced avidity for target cells as well as improved clearance characteristics.

Uptake of verteporfin by articular tissues following systemic and intra-articular administration.

Photodynamic therapy (PDT) using the photosensitizer BPD-Verteporfin (liposomal benzoporphyrin derivative-monoacid ring A) has been shown in previous studies to be effective in the amelioration of inflammatory arthritis in both the MRL-lpr mouse and the New Zealand White (NZW) rabbit models, and could potentially offer alleviation of certain inflammation-related symptoms of rheumatoid arthritis. Time and dose dependency of BPD-MA tissue uptake was carried out in the inflamed synovium and other articular and peri-articular tissues following intravenous and intra-articular administration in the NZW rabbit model. As some articular and peri-articular tissues are difficult to extract, this study uses a rapid fluorimetric sampling of tissues following dissolution in Soluene 350. Our results showed that i.v. injected BPD-MA preferentially distributed in the inflamed synovium, and in tissues with a high degree of vascularization. Little or no association was found with avascular tissues such as cartilage and tendons. Clearance from the synovium was rapid, supporting earlier rather than late light treatment. Much higher association of BPD-MA with the synovium was achieved using intra-articular injection, and BPD-MA concentrations were maintained at relatively steady levels for several hours. These observations support the possibility that PDT could offer a safe, highly versatile clinical option for the management of inflamed joints in autoimmune disorders.

Amelioration of antigen-induced arthritis in rabbits by induction of apoptosis of inflammatory cells with local application of transdermal photodynamic therapy.

OBJECTIVE: To study the efficacy and mechanism of local transdermal photodynamic therapy (tPDT) in rabbits with antigen-induced arthritis (AIA). METHODS: AIA in rabbits on day 14 postinduction was treated with an intravenous injection of benzoporphyrin-derivative monoacid ring A (BPD; Verteporfin) and subsequent transdermal exposure of the knee joint to light. BPD uptake and PDT-induced apoptosis of the synovium was studied applying fluorescence confocal microscopy and immunohistochemistry. The (histo)pathology of the joints was assessed at day 28. RESULTS: Treatment with tPDT resulted in significant amelioration of synovial inflammation and an almost complete prevention of pannus formation and bone and cartilage destruction. BPD uptake was detectable in activated T cells and macrophages, and there was significant PDT-induced increase in the number of apoptotic cells in the synovium. CONCLUSION: Because photodynamic therapy is both specific and noninvasive, our findings suggest that it could be used for treating arthritic joints in humans.

An antagonist of monocyte chemoattractant protein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model.

An antagonist of human monocyte chemoattractant protein (MCP)-1, which consists of MCP-1(9-76), had previously been characterized and shown to inhibit MCP-1 activity in vitro. To test the hypothesis that, by inhibiting endogenous MCP-1, the antagonist has antiinflammatory activity in vivo, we examined its effect in the MRL-lpr mouse model of arthritis. This strain spontaneously develops a chronic inflammatory arthritis that is similar to human rheumatoid arthritis. Daily injection of the antagonist, MCP-1(9-76), prevented the onset of arthritis as monitored by measuring joint swelling and by histopathological evaluation of the joints. In contrast, controls treated with native MCP-1 had enhanced arthritis symptoms, indicating that the inhibitory effect is specific to the antagonist. In experiments where the antagonist was given only after the disease had already developed, there was a marked reduction in symptoms and histopathology, although individuals varied in the magnitude of the response. The mechanism of inhibition of disease is not known, although the results suggest that it could be more complex than the competitive inhibition of ligand binding that is observed in vitro. The demonstration of the beneficial effects of an MCP-1 antagonist in arthritis suggests that chemokine receptor antagonists could have therapeutic application in inflammatory diseases.

Prolonged skin allograft survival after photodynamic therapy associated with modification of donor skin antigenicity.

BACKGROUND: The ability to prolong graft survival, in some cases by depleting donor antigen-presenting cells (APCs), and the subsequent demonstration that lymphocytes stimulated by non-APCs become anergic, suggested that graft survival and tolerance induction might be achieved by manipulating donor APCs to render them incompetent. This possibility was tested in histoincompatible murine skin allograft with photodynamic therapy (PDT). METHODS: Skin sections (C57BL/6) were exposed in vitro to low doses of benzoporphyrin derivative monoacid ring A (BPD) (verteporfin) and light (A=690+/-10 nm; low-dose PDT) before implantation on recipients (BALB/c). Furthermore, the effect of the treatment on the surface molecules of donor-derived Langerhans cells (LC) was evaluated by fluorescence-activated cell sorter analysis; the effect of treatment on the LC alloreactivity in the mixed epidermal cell lymphocyte reaction was also evaluated. RESULTS: Pretreating skin to be grafted with low-dose PDT can significantly prolong the survival of allografts from 9.3+/-2.2 (n=42) days (control group) to 16.9+/-1.7 days (n=20; treated group). Moreover, low-dose PDT significantly down-regulated the major histocompatibility complex and costimulatory (B7) molecules (60-90% reduction) on LC, but not LC-specific endocytic receptor (DEC-205), CD45, intercellulr adhesion molecule 1, LC viabilities, and ectophosphatase activity on LC. Additionally, this treatment significantly suppressed the ability of LC to stimulate alloreactive T cells to proliferate. CONCLUSIONS: Since engaging T cell receptors in the absence of costimulation results in suboptimal activation of T cells and ultimately anergy, it appears that the immunomodulatory effects of low-dose PDT associated with extended engraftment may depend upon decreased LC expression of major histocompatibility complex and costimulatory molecules.

Post-partum flare in MRL-lpr/lpr mice is associated with a parallel increase of N-acetylglucosamine on serum IgG.

Our objective was to investigate IgG glycosylation and disease activity post partum in the MRL-lpr/lpr mouse. Disease activity was monitored using bimalleolar ankle swelling. Levels of galactose and N-acetylglucosamine (GlcNAc) on IgG isolated from serum were measured using biotinylated lectins. Our results show that disease severity increased post partum. This post-partum flare correlated significantly with an increase in IgG GlcNAc expression. The disease severity increased post partum in the MRL-lpr/lpr mouse model similar to the changes seen in rheumatoid arthritis (RA).

Stimulation of enzyme and cytokine production by methyl mercaptan in human gingival fibroblast and monocyte cell cultures.

The volatile sulphur compound methyl mercaptan (CH3SH) is a by-product of protein metabolism and a principal component of oral malodour. This investigation examines the effect of CH3SH on the enzymatic activities of cathepsins B and G and elastase, and on the production by human gingival fibroblasts of two key factors, prostaglandin E (PGE) and cAMP, of the PGE2-cAMP-dependent pathway, which may contribute to the increased production of collagenase and tissue destruction in human periodontal disease. The results demonstrate that CH3SH alone, or in combination with interleukin-1 (IL-1) or lipopolysaccharide, can significantly enhance the secretion of PGE2, cAMP and procollagenase by human gingival fibroblasts. CH3SH also stimulated mononuclear cells to produce IL-1, which can increase cAMP production, and act in synergism with the direct effect of CH3SH on cAMP. CH3SH also significantly enhanced the activity of cathepsin B, moderately suppressed that of cathepsin G, but did not significantly affect elastase. These results provide evidence that CH3SH could be a contributing factor in the enzymatic and immunological cascade of events leading to tissue degradation in periodontal diseases.

Evaluation of a model for post-partum arthritis and the role of oestrogen in prevention of MRL-lpr associated rheumatic conditions.

Sixty-eight percent of female MRL-lpr mice developed a post-partum exacerbation of their mild spontaneous arthritis within 30 days of parturition. The flare became evident between 5 and 15 days after delivery. Histologically it was characterized by a significant increase of subsynovial inflammation and synovial hyperplasia without changes in the level of cartilage and bone erosion. Immunohistologically, marked subsynovial and frequent synovial staining of MHC class II bearing cells was noted, along with the sporadic presence of CD3, CD4, and CD43 receptor-bearing cells in the subsynovium. Injection of physiological levels (0.08 mg/kg) of estradiol on days 2, 3, 9, 15 and 20 post-partum delayed and reduced the flare to 23% of the animals. Administration of pharmacological amounts (0.4 mg/kg per day for 2 weeks following Freund's complete adjuvant injection) prevented adjuvant-enhanced arthritis, reducing the incidence from 67% to the baseline 21% level. Deleterious changes in the underlying systemic lupus erythematosus (SLE), as demonstrated by proteinuria and mortality rate increases, were elicited only by the employed pharmacological amounts of estradiol. These results indicate that the MRL-lpr mice might serve as a model for post-partum flare of arthritis in SLE and rheumatoid arthritis by providing an approach to study the complexity of the effects of pregnancy on autoimmune diseases, and to obtain further evidence for the involvement of oestrogen in arthritis.

Lpr and MRL background gene involvement in the control of adjuvant enhanced arthritis in MRL-lpr mice.

The MRL-lpr mouse strain develops a mild spontaneous arthritis which can be enhanced by the intradermal injection of complete Freund's adjuvant (CFA). In this study we examined the requirement of the lpr gene and background MRL genes on CFA-enhanced murine arthritis. MRL+, MRL-lpr, AKR/J, and B6-lpr mice (experimental) and B6 mice (control) were injected intradermally with CFA containing M. tuberculosis H37 RA. The development of swelling and erythema was monitored for 1 month after the injection, when the histopathology of the joints was investigated. It was found that while 74% of both 7-month-old MRL + and 3-month-old MRL-lpr mice and 11% of AKR/J mice displayed clinically visible arthritis, B6, B6-lpr, and 3-month-old MRL+ did not develop the condition after CFA treatment. In accordance with the clinical observations, the histopathological changes were manifested only in older MRL+, AKR/J and 3-month-old MRL-lpr mice. One month after the CFA injection, milder changes were observed in the MRL+ than in the MRL-lpr mice, with the MRL+ mice developing a disease of similar severity to uninjected MRL-lpr mice. The AKR/J mice demonstrated the least severe histopathological changes. In the long term (150 days) more severe destructive changes could be demonstrated in the cartilage and bone of the MRL+ mice although the average histological scores did not show statistically significant differences from those found in the MRL+ 30 days after injection. The serological evaluation of the adjuvant-injected mice demonstrated significantly enhanced antibody production to type II collagen and M. tuberculosis, but did not correlate with the disease activity. These observations suggest that while the lpr gene causes a more severe early effect, background genes other than the lpr are more involved in the adjuvant-enhanced arthritis-afflicted mice.

The use of transcutaneous photodynamic therapy in the prevention of adjuvant-enhanced arthritis in MRL/lpr mice.

The use of transcutaneous photodynamic therapy (PDT) has been investigated in the prevention of adjuvant enhanced arthritis in MRL/lpr mice. Mice receiving adjuvant were treated with PDT at 10-day intervals starting on the day of adjuvant administration. PDT was carried out by intravenous injection of the photosensitizer, benzoporphyrin derivative-monoacid ring A, followed by its transcutaneous activation with light. Adjuvant-injected animals displayed a delayed onset and reduced incidence and severity of arthritis when compared to untreated animals. Most importantly, inflammatory structural damage to cartilage and bone tissues was prevented by PDT. PDT was found to have no adverse effects on animals as assessed by mitogen responses, hematopoiesis, and serum enzyme levels. As mitogen-activated MRL/lpr splenocytes were shown to be more susceptible to in vitro photodynamic treatment, it is postulated that the observed effects were the result of selective destruction of adjuvant-activated lymphocytes in the circulation and/or joints.

Photodynamic therapy; a comparison with other immunomodulatory treatments of adjuvant-enhanced arthritis in MRL-lpr mice.

Although numerous experimental immunomodulatory regimens have been reported to be effective in the treatment of rheumatoid arthritis, they also produce undesirable side effects. An alternative specific modality of localized treatment is photodynamic therapy (PDT). In this study we treated 13-week-old MRL-lpr mice whose spontaneous arthritis was enhanced by intradermal injection of Freund's complete adjuvant (FCA). One group received transcutaneous photodynamic therapy at days 0, 10, and 20, following the FCA injection. The other groups were injected with 1 mg/kg per day indomethacin, 40 mg/kg per day cyclosporin A (CsA), or treated with 3 Gy sublethal whole body irradiation (WBI). The development of swelling was monitored for 1 month, at which time proteinuria, lymphadenopathy and the histopathology of the joints and kidneys were assessed. The results demonstrated that PDT and the conventional treatments significantly ameliorated swelling of the hindlimbs from 70% in the untreated FCA-injected animals to below the 19% level characteristic of the unmanipulated control. Histological examination showed a reduction in pannus formation, and cartilage and bone destruction, the characteristics of adjuvant-enhanced arthritis. PDT did not affect the survival rate, lymphoproliferation, or proteinuria of the treated animals. However, indomethacin increased proteinuria, and was less effective in preventing cartilage and bone destruction. Furthermore, lower doses of CsA and WBI exacerbated arthritis activity. These results indicate that photodynamic therapy can inhibit the development of adjuvant-enhanced arthritis in MRL-lpr mice with similar effectiveness to the conventional treatments, but without their negative side effects.

Complete Freund's adjuvant induces an earlier and more severe arthritis in MRL-lpr mice.

A study was performed on the effect of CFA on the spontaneous arthritis of MRL-lpr mice. The development of swelling and erythema was monitored for 1 mo after injecting 13- to 14-wk-old mice intradermally with CFA, at which time the histopathology of the joints and serologic responses to extracellular matrix proteins were investigated. In a series of six experiments, 67 to 82% of mice showed early clinical evidence of arthritis in contrast to the low percentage observed in control animals. Similarly, the histopathologic analyses on the CFA-injected mice indicated a significantly higher frequency of advanced histopathologic alterations, characterized by cartilage erosion and pannus formation. The serologic evaluation of the adjuvant-injected mice demonstrated a significant enhanced antibody production to type I and type II collagens, DNA, and the Mycobacterium tuberculosis-positive control. This reproducible adjuvant-enhanced model of murine arthritis will be extremely useful in evaluating experimental therapeutic regimes as the arthritis is initiated earlier and exhibits an enhanced frequency and severity compared with the spontaneous arthritis seen in MRL-lpr mice.

Antibodies to extracellular matrix proteins in the sera of MRL-lpr mice.

Autoimmune MRL-lpr mice develop a spontaneous arthritis displaying similar articular and extra-articular features to rheumatoid arthritis in humans. In this study we used an ELISA assay to evaluate the serological responses of MRL-lpr mice to select extracellular matrix proteins associated with the joint. Significant levels of antibodies to collagens types I, II, III, IV, and V were demonstrated starting between 17 and 20 weeks of age. Moreover, the sera contained a strong reactivity to fibronectin. Responses to proteoglycans and laminin were weaker but still detectable. Specificity studies on pooled sera from MRL-lpr mice suggest that the autoantibodies produced are highly cross-reactive. The results indicate that the MRL-lpr mouse strain exhibits similar anti-extracellular matrix antibody profiles to those seen in varying frequencies in the sera and synovial fluid of patients with rheumatoid arthritis.