Increased limb involvement in murine collagen-induced arthritis following treatment with anti-interferon-gamma.

We have tested the effect of administering H22, a hamster neutralizing MoAb to murine interferon-gamma (IFN-gamma) in collagen-induced arthritis. Mice were immunized with human type II collagen in adjuvant on day 1 and boosted with soluble collagen on day 21. H22 was administered (250 micrograms, intraperitoneally) either during the induction of arthritis (on days 0, 6, 13 and 20) or around the time of disease manifestation (on days 21, 28, 35 and 42). Control mice received either an isotype-matched non-neutralizing MoAb or saline. Both treatment regimes gave similar results. Treatment with H22 did not significantly affect the incidence of arthritis, time of onset, degree of oedema, histopathological severity, or level of anti-type II collagen IgG. However, a highly significant increase (P < 0.01) in the number of limbs affected by arthritis was observed in the H22-treated group, irrespective of whether the antibody was administered during the induction of arthritis, or during the time of clinical manifestation of disease. From these results it was concluded that anti-IFN-gamma treatment caused an increase in the number of arthritic lesions, but did not affect the severity of each individual lesion.     

Enhancement of DTH reaction and inhibition of the expression of class II transplantation antigens by in vivo treatment with antibodies against gamma-interferon

During the delayed type of hypersensitivity (DTH) reaction in the skin, class II transplantation antigens are expressed on the keratinocytes. This induction is attributed to the action of gamma-interferon (IFN-gamma). We have now studied the influence of antibodies against IFN-gamma on the DTH-reaction. Lewis rats were sensitized to 2,4-dinitro-1-fluorobenzene (DNFB) and challenged 5 days later with DNFB on the ears. Immediately before challenge each animal in one group (n = 16) was given 1 mg of mouse monoclonal antibodies (MoAb) against rats IFN-gamma, denoted DB-1, intraperitoneally (i.p.), another group (n = 15), 1 mg of an irrelevant MoAb and a third group (n = 11) was left untreated. The ear thickness was measured with a micrometer, before challenge and after 24, 48 and 72 h. At 72 h all rats were killed, the ears cut off, snap frozen and stained with immunoperoxidase using MoAbs OX 6 and OX 17, directed against rat class II antigens. The DB-1 treated group was found to have a larger ear swelling that was statistically significant at each time point compared with the other two groups. Furthermore, the animals given DB-1 showed class II antigen expression on Langerhans' cells, but almost none on keratinocytes. In contrast, the rats in the two other groups displayed a moderate to strong expression of class II antigens on keratinocytes as well as on Langerhans' cells. It is concluded that DB-1 can inhibit class II antigen expression on keratinocytes during the DTH- reaction and also enhance the local response.     

Prevention of adjuvant arthritis by the W3/25 anti-CD4 monoclonal antibody is associated with a decrease of blood CD4(+)CD45RC(high) T cells

Imbalance between Th1 and Th2 functions is considered to play a key role in the induction and development of several autoimmune diseases, and the correction of that imbalance has led to effective therapies of some experimental pathologies. To examine whether CD4(+)CD45RC(high) (Th1-like) and CD4(+)CD45RC(low) (Th2-like) lymphocytes play a role in the pathogenesis of adjuvant arthritis (AA) and in its prevention by anti-CD4 antibody, CD45RC expression on CD4(+) T cells was determined in arthritic rats and in animals treated with an anti-CD4 MoAb (W3/25) during the latency period of AA. The phenotype of regional lymph node lymphocytes from arthritic rats in the active phase of the disease was determined by flow cytometry. Peripheral blood lymphocytes from rats treated with W3/25 MoAb were also analysed for 2 weeks after immunotherapy finished. IgG2a and IgG1 isotypes of sera antibodies against the AA-inducing mycobacteria, considered to be associated with Th1 and Th2 responses, respectively, were also determined by ELISA techniques. Fourteen days after arthritis induction, regional lymph nodes presented an increase in CD4+CD45RC(high) T cell proportion. Preventive immunotherapy with W3/25 MoAb inhibited the external signs of arthritis and produced a specific decrease in blood CD4(+)CD45RC(high) T cells and a diminution of antibodies against mycobacteria, more marked for IgG2a than for IgG1 isotype. These results indicate a possible role of CD4(+)CD45RC(high) T lymphocytes in the pathogenesis of AA, and suggest that the success of anti-CD4 treatment is due to a specific effect on CD4(+)CD45RC(high) T subset that could be associated with a decrease in Th1 activity.     

Anti-CD200R ameliorates collagen-induced arthritis in mice

Immunization of DBA/1 with 100 microg bovine collagen type II emulsified in Freund's adjuvant, followed by booster injection in incomplete adjuvant at 18 days, leads to development of arthritis in more than 70% of mice by 28 days postinjection. We have previously shown that the novel immunosuppressant molecule CD200Fc (linking an extracellular domain of CD200 with a murine IgG2a Fc region) can suppress induction of disease when given to mice from the time of collagen injection. This occurs in concert with a decrease in the serum levels of anti-collagen IgG ( approximately 50% reduction), with relatively more IgG2b and IgG3, decreased serum levels of TNFalpha and IFN-gamma, and decreased production of those same cytokines after restimulation of lymphocytes in vitro with collagen. Since CD200 induces suppression following engagement of a receptor (CD200R), known to be expressed on, among other cells, macrophages, we investigated whether infusion of anti-CD200R and/or CD200Fc would ameliorate established disease in DBA mice, when injections were begun following collagen immunization. Our data indicate an arrest of disease following either treatment, with modification of a number of immune parameters (serum and lymphocyte cytokine production) consistent with a general role for CD200:CD200R interactions in the regulation of induction and/or expression of autoimmune disorders. When a higher dose (250 microg/mouse) of anti-CD200R was infused into a group of overtly arthritic mice, a significant ( approximately 50%) decrease in arthritic joint score occurred over the 4-week treatment period.     

Suppression of collagen-induced arthritis by oral or nasal administration of type II collagen.

We directly compared the effects of oral and nasal administration of collagen type II (CII) on disease progression, cytokine production and T cell responses in DBA/1 mice. Lymphocytes were assayed for proliferation and cytokine production and cell lines established. T cells from fed or nasally treated groups proliferated significantly less and produced markedly less IFN-gamma than the non-fed immunized group 10 days after immunization and prior to onset of arthritis. T cell lines established from fed or nasally treated mice showed a pattern of cytokine production involving IL-4, IL-10 and TGF-beta, whereas T cell lines from the control group produced more IFN-gamma and IL-2. Suppression of clinical measures of arthritis was equivalent in the nasal and orally treated groups. Animals were then tested for IFN-gamma production 70 days after a booster immunization at a time when disease was apparent. Mucosally treated animals secreted less IFN-gamma as compared to controls, even at this late time point. Suppression of collagen induced arthritis (CIA) by nasal treatment of mice with CII was associated with diminished levels of TNF-alpha and IL-6 mRNA expression in the joints of tolerized mice, two cytokines known to be involved in the inflammatory and pathological process of CIA. These results demonstrate the induction of antigen specific Th2 and TGF-beta secreting regulatory cells following both oral and nasal treatment, which is associated with suppression of local inflammation in the joints and decreased Th1 type responses in the periphery throughout the course of the illness.     

Treatment with an anti-CD4 monoclonal antibody strongly ameliorates established rat adjuvant arthritis

Some experimental arthritic diseases can be prevented by treatment with anti-CD4 MoAbs. Trials with ongoing disease have not been successful so far. The aim of this study was to ascertain whether W3/25 could reverse adjuvant arthritis (AA), when beginning treatment on day 14, i.e. when the disease was established. Moreover, one group of animals treated with the anti-CD4 MoAb received OX8 MoAb at the same time, thus depleting CD8⁺ cells from circulation. During treatment with W3/25, a strong amelioration of inflammatory signals was observed, as assessed by means of paw volume increase and arthritic score. However, when treatment stopped, a rebound to arthritis signals occurred. The parallel depletion of CD8⁺ cells did not modify these effects, thus the combined treatment W3/25+OX8 gave the same amelioration as treatment with W3/25 alone. These findings indicate that CD4⁺ cells play an important role in perpetuating rat AA. Moreover, CD8⁺ cells do not seem to have a regulatory role in the CD4⁺ cells responsible for the inflammatory response.     

Cytokine expression and synovial pathology in the initiation and spontaneous resolution phases of adjuvant arthritis: interleukin-17 expression is upregulated in early disease

The aim of this study was to understand the immune processes controlling the initiation and spontaneous resolution of adjuvant arthritis (AA). We investigated synovial T-cell recruitment and mRNA expression of IL-17 and other important disease related cytokines, IFN-gamma, IL-2, IL-4, TNF and TGF-beta in inguinal lymph node (ILN) and synovial membrane (SM). Arthritis severity was assessed by a numerical rating score and rats were sacrificed every 3--4 days postadjuvant induction. Further assessment involved quantitative radiology and histology of the ankle joints on each day, and the ILN and SM were removed for RNA extraction. Cytokine mRNA expression was measured using RT-PCR and densitometry. Paraffin sections of rat ankle joints were stained for T-cells (CD3) by immunohistochemistry. In the ILN, there was an increase in IL-17, TNF and IFN-gamma expression in the early stages of disease, with a secondary sustained increase in IFN-gamma expression. In the SM, there was expression of T-cell cytokines in early arthritis (day 13), and prolonged TNF and TGF-beta expression, which reflected disease progression. IL-4 mRNA expression increased in the later stages of AA. Synovial T-cell numbers transiently increased at day 6, and remained high from days 13--28. Increased pro-inflammatory cytokine expression, including IL-17, in the ILN reflects the initiating events in the early stage of disease. IL-17 may therefore play an important role in the pathogenesis of AA. The increase in IL-4 (an anti-inflammatory cytokine) in the SM in the later stages of AA suggests that IL-4 is involved in the spontaneous resolution of AA. The initial increase in IFN-gamma in the ILN may reflect a pro-inflammatory response, while the prolonged secondary increase may indicate activation of regulatory T-cells.     

Glucocorticoid inhibition of adjuvant arthritis synovial macrophage nitric oxide production: role of lipocortin 1

Nitric oxide (NO) is a mediator of inflammatory injury which is inhibited by glucocorticoids and is implicated in rheumatoid (RA) and adjuvant arthritis (AA). The glucocorticoid-induced anti-inflammatory molecule lipocortin 1 is expressed in RA synovium, but the effects of lipocortin 1 on synovial inflammation have been little studied. We investigated the effects of glucocorticoids and lipocortin 1 on inducible NO synthase (iNOS) and glucocorticoids on the induction of lipocortin 1 in AA synovial macrophages. NO production was measured by Griess assay in supernatants of day 14 AA rat synovial explants and of synovial macrophages purified from enzyme-digested synovium and treated with lipopolysaccharide (LPS) 1 μg/ml, dexamethasone (DEX) 10^?7 M , and anti-lipocortin 1 MoAb. iNOS and lipocortin 1 expression were detected by flow cytometry using specific MoAb. Cell surface lipocortin was determined by Western blot. NO was produced by all AA synovial explants and NO was released by cultured synovial macrophages (14.5 ± 2.1 μmol/24 h). iNOS was detected in synovial macrophages (ED-1⁺) by permeabilization flow cytometry. LPS increased synovial macrophage NO release (P < 0.0001) and iNOS expression (P = 0.04). DEX inhibited constitutive (P = 0.002) and LPS-induced (P < 0.001) NO release and iNOS expression (P = 0.03). DEX inhibition of synovial macrophage NO was associated with induction of cell surface and intracellular lipocortin 1. Anti-lipocortin 1 MoAb treatment reduced the inhibition of NO release by DEX (P = 0.002), but had no effect on iNOS expression. These findings demonstrate a role for lipocortin 1 in the inhibition by glucocorticoids of AA synovial macrophage iNOS activity.     

Human monoclonal rheumatoid factors augment arthritis in mice by the activation of T cells

In order to investigate the in vivo role of rheumatoid factor (RF), the effects of the administration of human monoclonal (m) IgM-RF and IgG-RF on the development of arthritis in mice were examined. The administration of human mRFs into mice immunized with type II collagen (CII) markedly enhanced the clinical score and paw swelling. The severity of arthritic joint disease with a marked infiltration of lymphoid cells, proliferation of synovial membrane, pannus formation and destruction of articular cartilage was significantly enhanced in both groups receiving RF (RF-enhanced arthritis). Skin ulcers were also observed in some of these RF-enhanced arthritis mice, whereas no such signs were observed in CII-immunized mice without mRFs. Both IgM-RF and IgG-RF increased CII-specific IgG antibodies in circulation, and the severity of arthritis correlated with the production of high titres of anti-CII antibodies. In vivo treatment of RF-enhanced arthritis mice with an anti-CD4 MoAb or an anti-CD8 MoAb inhibited the induction and progression of arthritis in these mice. Administration of RF to severe combined immunodeficient (SCID) mice with arthritis developed by the transfer of spleen cells from CII-immunized mice, prolonged the arthritis and enhanced the severity. This murine model of RF-enhanced arthritis may provide a useful tool for analysing the pathogenesis of rheumatoid arthritis in RF-positive patients.     

Inhibitory effects of incomplete Freund's adjuvant on experimental autoimmune encephalomyelitis

Freund's incomplete adjuvant (IFA), an aqueous/oil emulsion that is widely used in combination with antigenic proteins and peptides to induce tolerance, is considered to be immunologically inert. However, sporadic reports indicate that IFA may itself have inhibitory properties on induction of adjuvant induced arthritis and spontaneous diabetes. In the current study, the effects of IFA/saline were evaluated on the induction of experimental autoimmune encephalomyelitis (EAE) in three different strains of mice. IFA/saline given i.p. in two doses of > 100 microl 10 days apart were found to inhibit EAE induction to varying degrees in all three strains of mice in a dose dependent fashion. The IFA/saline injections inhibited both mitogen and antigen-induced T cell proliferation, induced elevated secretion of IFN-gamma and IL-10 by neuroantigen specific T cells, and reduced expression of cytokines, chemokines, and chemokine receptors of CNS-infiltrating mononuclear cells. These data demonstrate for the first time a direct inhibitory effect of IFA/saline on EAE, and re-emphasize the need to properly control experiments using IFA to induce antigen-specific tolerance.     

Influence of cytokines, monoclonal antibodies and chemotherapeutic drugs on epithelial cell adhesion molecule (EpCAM) and LewisY antigen expression

MoAbs against tumour-associated antigens (TAA) may be useful for the treatment of colorectal cancer. Since an increased expression of TAA may lead to enhanced antibody-dependent cellular cytotoxicity we examined whether the cytokines IL-2, IL-4, IL-6, IL-10, IL-12, interferon-alpha (IFN-alpha), IFN-gamma, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and tumour necrosis factor-alpha can influence EpCAM and LewisY expression on the surface of the colorectal carcinoma cell lines HT29, LoVo and SW480. We found that only IFN-alpha increased significantly whereas IL-4 decreased both EpCAM and LewisY expression. IFN-gamma significantly increased LewisY expression only. When tumour cells were treated with MoAb, the LewisY-specific MoAb BR55-2 down-regulated LewisY antigen expression, whereas MoAb 17-1A, which binds to EpCAM, up-regulated this TAA after 3 days of culture. The cytokines IFN-alpha or IFN-gamma combined with MoAb 17-1A enhanced further slightly the expression of EpCAM. In additional experiments with chemotherapeutic drugs commonly used for the treatment of colorectal cancer, we found that 5-fluorouracil, mitomycin-C and oxaliplatin up-regulated EpCAM and LewisY antigen expression. Raltitrexed enhanced LewisY and down-regulated EpCAM expression, whereas CPT-11 had no influence at all. The highest expression for EpCAM on HT29 cells was achieved by the combination of IFN-alpha, 5-fluorouracil and MoAb 17-1A. Our results may be useful for defining combinations of biological and chemotherapeutic drugs for the treatment of colorectal cancer. Further trials should evaluate to what extent these combinations enhance antibody-dependent cellular cytotoxicity.     

Interferon-Mediated Enhancement of Thyroid Major Histocompatibility Complex Antigen Expression

Epithelial expression of class II antigens encoded by the major histocompatibility complex (MHC) has been proposed as a means by which autoimmune thyroid disease may be initiated and maintained. We studied a rat thyroid epithelial cell line (FRTL-5), which constitutively expresses class I (OX18) but not class II (OX6 or OX17) determinants to quantify in vitro MHC antigen induction using flow cytometry. Recombinant rat gamma interferon (rIFN-gamma) induced dose-dependent expression of OX6 (I-A) antigen at greater than 48 h (maximum 80-90% of cells in culture at 100 U/ml), which was abrogated by DB-1, a monoclonal antibody to rat IFN-gamma. OX17 antigen (I-E) was also induced (86%) and OX18 (class I) markedly increased under these conditions. Other thyroid-active agents including the calcium ionophore A23187, dibutyryl cyclic AMP, thyroid-stimulating autoantibodies from Graves' disease patients (LATS), and TSH, caused no I-A induction. Supernatants from spleen cells stimulated with plant lectins (concanavalin A or phytohaemagglutinin), but not lectin alone, evoked substantial class II induction, which was inhibited by DB-1. These findings suggest that IFN-gamma is the central mediator of thyroid epithelial class II expression. FRTL-5 provides a powerful model for the analysis of thyroid MHC class II dynamics and a potential means of analysing the role of epithelial class II in autoimmune pathogenesis.     

Effect of cyclophosphamide and its analogs on collagen arthritis in rats

The effects of cyclophosphamide (CY) and its structurally related analogs, ifosfamide (Ifo), sufosfamide (Sufo), and mafosfamide (Mafo), on collagen arthritis in Sprague-Dawley rats were examined. Prophylactic treatment with 7.5-10 mg/kg/day of CY. 15 mg/kg/day of Ifo, and 10-15 mg/kg/day of Sufo for the first 10 days starting on the same day as the type II collagen immunization suppressed arthritis induction as well as humoral immune response to type II collagen. Prophylactic treatment with Mafo at doses ranging from 10 to 40 mg/kg/day for 10 days was ineffective in suppressing the disease development. When drug treatment was started only during the immediate preclinical phase of arthritis, the development of arthritis was suppressed in the animals treated with 10 mg/kg/day of CY and 15 mg/kg/day of Ifo from Day 5 to Day 14. Additional studies demonstrated that treatment with 10 mg/kg/day of CY and 15 mg/kg/day of Ifo started at the time of disease onset significantly suppressed the severity of arthritis compared with the control group. These results show the effectiveness of Ifo and CY on this animal model of polyarthritis and suggest the possibility of clinical use of Ifo for the treatment of human arthritides similar to CY.     

CD8+ cells suppress oil-induced arthritis

Oil-induced arthritis is a genetically restricted polyarthritis that develops in the DA rat after injection of the mineral oil Freund's incomplete adjuvant. Here, we investigated the role of the potentially disease-limiting cell populations CD8+ T cells, gammadelta T cells, natural killer (NK) cells and NK T cells in inguinal lymph nodes for the development of this adjuvant-induced arthritis. Flow cytometry analysis before and at disease onset revealed a higher proportion of lymph node T cells expressing NKR-P1 in the disease-resistant LEW.1AV1 compared with the disease-susceptible DA strain, suggesting that NK T cells might be disease protective. However, prophylactic in vivo administration of an anti-NKR-P1 MoAb (clone 10/78) did not consistently affect the disease course. The proportion of CD8+ T cells and the ratio CD4+/CD8+ T cells in inguinal lymph nodes did not differ significantly between DA and LEW.1AV1 rats before or at disease onset. Nevertheless, prophylactic in vivo depletion of CD8+ cells by the OX8 MoAb in the DA strain resulted in an earlier disease onset compared with the control group, demonstrating that CD8+ cells regulate arthritis development. In vivo depletion of gammadelta T cells by the V65 MoAb did not alter the disease course, indicating that the disease-suppressive CD8+ cells are alphabeta T cells or NK cells.     

Effect of in vivo administration of anti-CTLA-4 monoclonal antibody and IL-12 on the induction of low-dose oral tolerance

Oral tolerance has been characterized as an immunological hyporesponsiveness to fed antigen. Previous studies have suggested that high-dose oral tolerance involves the preferential interaction of B7 with CTLA-4 on the T cell. To determine whether similar mechanisms are involved in the induction of low-dose oral tolerance, mice were treated with anti-CTLA-4 monoclonal antibody (MoAb), with or without IL-12, at the time of feeding. Results showed that anti-CTLA-4 MoAb alone failed to restore cellular proliferation, antibody titres and IFN-gamma levels; however, IL-4 cytokine levels in OVA-fed mice were partially restored. In contrast, administration of IL-12 along with anti-CTLA-4 MoAb to mice during feeding completely prevented the suppression of Th1 immune responses, as shown by increased serum IgG2a titres, IFN-gamma production and cell proliferation. These results suggest that blocking B7-CTLA-4 interactions in the presence of IL-12 prevents the induction of low-dose oral tolerance at the Th1 cell level.     

Interaction with Complement Receptor 1 (CD35) Leads to Amelioration of Sepsis-Triggered Mortality but Aggravation of Arthritis During Staphylococcus aureus Infection

The aim of this study was to assess the importance of complement receptor 1 (CR1, CD35) in Staphylococcus aureus arthritis and sepsis. The murine model of haematogenously acquired septic arthritis was used, injecting toxic shock syndrome toxin 1 (TSST-1)-producing S. aureus LS-1 intravenously. CR1 was blocked using immunoglobulin G (IgG) rat antimouse CR1 monoclonal antibody (MoAb) (8C12). Evaluation of arthritis was performed clinically and histopathologically. In addition, the effect of blocking CR1 was assessed on the phagocytic activity of leucocytes and on T-cell dependent and independent inflammation. Seven days after inoculation with bacteria, 96% of CR1 MoAb-treated mice had clinical symptoms of arthritis compared with 58% of the control animals (P < 0.01). The severity of arthritis, expressed as mean arthritic index, was 2.9 +/- 0.5 and 1.4 +/- 0.5, respectively (P = 0.004). Fifteen days after bacterial inoculation, all CR1 MoAb-treated mice had severe arthritis (mean arthritic index 6.3 +/- 0.6), while only 77% of controls were affected (mean arthritic index 2.9 +/- 0.6; P = 0.002). The potential explanation of these findings is that treatment with CR1 MoAb significantly increases the polymorphonuclear cell-dependent inflammatory response as a result of enhanced vasodilatation in treated animals. We conclude that treatment with CR1 MoAb leads to amelioration of sepsis-induced mortality during S. aureus infection, possibly as a result of the increased phagocytic activity of peripheral phagocytes.     

Anti-CD2 (OX34) MoAb treatment of adjuvant arthritic rats: attenuation of established arthritis, selective depletion of CD4+ T cells, and CD2 down-modulation

Anti-CD2 MoAbs have previously been shown to induce tolerance and to block B cell differentiation, T cell and monocyte activation. Since these immune functions are important in joint inflammation, we asked whether administration of the anti-CD2 MoAb OX34 has a beneficial effect on established rat adjuvant arthritis, a model of human rheumatoid arthritis, and how it affects CD2-bearing leucocyte subsets. Female Lewis rats with established adjuvant arthritis received a total of 5 mg OX34 or isotype-matched control MoAb starting on day 15 after adjuvant injection. Weight and arthritis score (AS) were measured in a blinded fashion. Peripheral blood cells were analysed for numbers of leucocyte subsets at various time points. Animals were killed on day 30 and lymphatic organs were processed for immunohistology. Clinically, OX34 treatment led to increased body weight and reduced AS. Although OX34 binds to CD4⁺ and CD8⁺ T cells in a comparable fashion, OX34 treatment reduced CD4⁺ T cells, but not CD8⁺ T cells. Among CD4⁺ T cells CD45RC⁺ (‘naive’) T cells virtually disappeared; CD45RC⁻ (‘recently activated’) T cells were slightly reduced. A reduction of CD4⁺ T cells was also found in the lung, liver, bone marrow, spleen and lymph nodes. Down-modulation of the CD2 molecule by OX34, again, affected CD4⁺ T cells, suggesting a specific signal for CD4⁺ but not CD8⁺ T cells. In conclusion, the anti-CD2 MoAb OX34 attenuates established rat adjuvant arthritis. In spite of similar binding to CD4⁺ and CD8⁺ T cells, OX34 depletes only CD4⁺ T cells and down-modulates the CD2 molecule on these cells. These results suggest a therapeutic benefit from CD2-directed therapy for chronic types of arthritis.     

Immunization with Alum–Collagen II Complex Suppresses the Development of Collagen-Induced Arthritis in Rats by Deviating the Immune Response

Collagen-induced arthritis (CIA) is an experimental rat model sharing a number of features with human rheumatoid arthritis (RA). The model is associated with a proinflammatory (TH1) type of immune response and treatments with cytokines associated with TH2 immune responses are beneficial. Since agents with TH1-inducing properties, such as Freund's incomplete adjuvant (FIA), are necessary for disease induction, it is of interest to investigate whether an adjuvant with TH2-inducing properties affects CIA in a different way than does FIA. The authors studied arthritis development in DA rats after immunization with the TH2 stimulatory adjuvant alum adsorbed to rat collagen type II (CII) or collagen II fragments. Such treatments suppressed disease development both prophylactically and therapeutically. This beneficial effect of alum–CII immunization was associated with an increase in the IgG1 anti-CII antibody response as compared to untreated rats or rats pretreated with alum alone. Treatment with alum without the addition of collagen did not have any clinical effect. In addition, alum–CII treated rats had a significantly higher expression of IL-4 mRNA than untreated rats in the lymph nodes, 7 days after CIA induction. The authors suggest that alum–CII induces a TH2 immune response against rat CII which counteracts the development of CIA.     

Amelioration of rat adjuvant arthritis by therapeutic treatment with bindarit, an inhibitor of MCP-1 and TNF-α production

OBJECTIVE: This study was designed to evaluate therapeutic effects of bindarit, an indazolic derivative able to inhibit monocyte chemoattractant protein-1 (MCP-1) production, in adjuvant induced arthritis in rats. MATERIALS AND METHODS: Arthritis was induced by Freund's complete adjuvant injection. Bindarit was given as a 0.5% medicated diet starting on day 11 after adjuvant injection. The course of arthritis was monitored by sequential paw volume measurement and by radiologic and histologic evaluations. Human osteoblast cell line Saos-2 stimulated with Interleukin-1 (IL-1) was used to assess in vitro bindarit effect on MCP-1 release. In addition, in vivo effects of bindarit on cytokine production were studied in mice injected with lipopolysaccharide (LPS). Immune function studies were performed in mice by evaluating ex vivo antibody response to ovalbumin and splenocytes proliferation to Concanavalin A (Con A). RESULTS: In adjuvant-induced arthritis in rats, bindarit possessed therapeutic activity resulting in a significant inhibition of paw inflammation. Evidence for a disease-modifying activity was also indicated by amelioration of radiologic alterations and by histological evaluation of joints. Additional evidence for beneficial effects in osseous inflammation was provided by an in vitro assay in which bindarit inhibited the release of MCP-1 from IL-1 stimulated osteoblast cells. Moreover, in a murine model of LPS-induced cytokine production bindarit reduced MCP-1 and tumor necrosis factor (TNF)-alpha increase without affecting IL-1 and IL-6 levels. Finally, the drug, given as a 0.5% medicated diet for 14 days, did not affect either anti-ovalbumin serum antibody production or splenocytes proliferative response in mice. CONCLUSIONS: Results obtained indicate that bindarit beneficial effects in experimental arthritis are correlated to MCP-1 and TNF-alpha inhibition and suggest that the control of cytokines and chemokines production can have considerable relevance as regards strategies for the treatment of chronic inflammatory diseases.     

Vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type II collagen-induced arthritis in the same rat strain are different diseases.

The DA rat is highly susceptible to induction of arthritis after immunization with homologous type II collagen (CII) emulsified in Freund's incomplete adjuvant (FIA), resulting in collagen-induced arthritis (CIA). The DA rat also develops arthritis after injection of FIA alone (oil-induced arthritis (OIA)). This finding allows a direct comparison of two different models for rheumatoid arthritis; one induced with a defined auto-immunogen and one with a pure adjuvant. Both CIA and OIA develop approximately 2 weeks after induction but OIA is a self-limited acute disease whereas CIA induced with homologous CII follows a chronic disease course. Immunization with CII leads to a strong autoantibody response to CII while injection of FIA leads to no or very limited anti-CII antibody response. The Lewis rat develops neither CIA nor OIA while F1 (DA x Lewis) rats develop CIA but not OIA. Olive oil or CII emulsified in olive oil does not induce arthritis in DA rats. Pretreatment with CII in olive oil vaccinates against CIA but not OIA whereas pretreatment with FIA vaccinates against OIA but not CIA. These findings demonstrate that inclusion of CII in the adjuvant leads to a disease distinct from OIA which is characterized by a CII autoimmune response and chronicity of the disease course.