Balance of Th1/Th2 Cytokines Associated with the Preventive Effect of Incomplete Freund's Adjuvant on the Development of Adjuvant Arthritis in LEW Rats

Complete Freund's adjuvant (CFA) could induce adjuvant arthritis (AA) in LEW rats and incomplete Freund's adjuvant (IFA) could induce oil induced arthritis (OIA) in DA but not in LEW rats. Lymph node cells (LNCs) from these AA and OIA rats showed increased mRNA expression of IFN-gamma, IL-2 and TNF-alpha but not IL-4. LNCs from IFA immunized LEW rats showed increased expression of IL-4, reduced expression of IFN-gamma and TNF-alpha and no IL-2, in contrast to IFA immunized DA rats. The pretreatment of IFA before CFA challenge could completely prevent AA in LEW rats and their LNCs showed increased expression of IL-4 and IFN-gamma but not IL-2 and TNF-alpha. In F1 (LEW x DA) rats, IFA could not induce OIA but the pretreatment of IFA before CFA challenge could induce very mild AA with 80% incidence, LNCs showing an elevated expression of all the above cytokines. These findings suggest that increased Th1 cytokine expression is associated with disease development and that increased IL-4 expression or the balance of Th2 over Th1 cytokine expression plays an important regulatory role in disease development.     

The choice of adjuvant determines the cytokine profile of T cells in proteoglycan‐induced arthritis but does not influence disease severity

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation of the synovial joints. Collagen‐induced arthritis (CIA) and proteoglycan‐induced arthritis (PGIA) are mouse models of inflammatory arthritis; CIA is a T helper type 17 (Th17) ‐dependent disease that is induced with antigen in complete Freund's adjuvant, whereas PGIA is Th1‐mediated and is induced using antigen in dimethyldioctadecyl‐ammonium bromide (DDA) as an adjuvant. To investigate whether the type of adjuvant determines the cytokine profile of the pathogenic T cells, we have compared the effect of CFA and DDA on T‐cell responses in a single arthritis model. No differences in incidence or disease severity between aggrecan‐T‐cell receptor transgenic mice immunized with aggrecan in either CFA or DDA were observed. Immunization with CFA resulted in a higher proportion of Th17 cells, whereas DDA induced more Th1 cells. However, the levels of interleukin‐17 (IL‐17) produced by T cells isolated from CFA‐immunized mice after antigen‐specific stimulation were not significantly different from those found in DDA‐immunized mice, indicating that the increased proportion of Th17 cells did not result in significantly higher ex vivo IL‐17 levels. Hence, the choice of adjuvant can affect the overall proportions of Th1 and Th17 cells, without necessarily affecting the level of cytokine production or disease incidence and severity.     

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.     

The influence of HLA-DR4 (0401) on the immune response to type II collagen and the development of collagen induced arthritis in mice

Rheumatoid arthritis (RA) is an autoimmune disease that is genetically associated with the MHC class II molecule HLA-DRbeta1*0401 (DR4). In order to determine if this MHC can influence the immune response to the candidate autoantigen type II collagen (CII), we have studied collagen induced arthritis (CIA) resistant C57BL/6 mice, made transgenic (Tg) for human DR4. These DR4 Tg mice exhibited a strong T cell proliferative response to CII and its DR4 restricted peptide p261-273 after immunization with these antigens that was not seen in the C57BL/6 wild type mice. DR4 Tg mice also exhibited an increase in IFN-gamma production in response to CII, indicating the activation of Th1 cells. While these Tg mice produced IgM anti-CII antibodies, they failed to produce a detectable level of IgG2a (Th1 type) anti-bCII antibody and did not develop CIA. This study shows that a Th1 type T cell response to CII can be established in CIA non-susceptible mice by introducing the human transgene, DR4. This T cell response, however, is not sufficient to induce an antibody isotype switch to IgG2a, nor is it sufficient for the induction of CIA. These results may help to explain why many individuals expressing HLA-DRbeta1*0401 do not develop RA.     

The preventive effects of incomplete Freund’s adjuvant and other vehicles on the development of adjuvant-induced arthritis in Lewis rats

The present study showed a novel finding that the development of adjuvant-induced arthritis (AA) in Lewis rats was completely prevented by incomplete Freund's adjuvant (IFA) injected 21 or 28 days before complete Freund's adjuvant (CFA) challenge. Hexadecane also completely prevented AA and squalane, methyl oleate and pristane moderately prevented AA, though pristane by itself induced mild arthritis in two out of five rats. Concanavalin A-stimulated lymph node cells (LNCs) isolated from AA rats were able to adoptively transfer the severe polyarthritis to all the naive recipients or even to the IFA pretreated recipients with earlier onset and more rapid progression than those of AA. The LNCs from the donors who had been pretreated with IFA and subsequently challenged with CFA could induce mild arthritis in only two out of eight naive recipients, whereas all the recipients who were challenged with CFA immediately after intravenous injection of these LNCs developed significantly less severe arthritis. However, the LNCs from IFA-pretreated donors failed to prevent AA. According to the T helper type 1 (Th1)/Th2 paradigm, it was suggested that the adjuvant-active vehicles such as IFA, hexadecane, squalane, methyl oleate and pristane, can affect and deviate the Th1/Th2 balance of immune responses in host. CFA could promote the propagation of Th2 cells rather than Th1 cells in these vehicle-pretreated rats through as yet undetermined mechanisms, eventually resulting in the prevention of AA. Finally, we discussed a regulatory role of adjuvant vehicles for induction and suppression of AA.     

Effect of rolipram, a phosphodiesterase IV inhibitor, on allergic footpad swelling using various adjuvants in mice

We studied the effect of rolipram, a phosphodiesterase (PDE) IV inhibitor, on allergic footpad swelling in mice. For this study, varying adjuvants including complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and Imject Alum (Alum) were used because the extent of antigen-specifically induced T helper type 1 (Th1) and Th2 responses had been shown to depend on adjuvants used. To induce allergic footpad swelling, we immunized mice with ovalbumin (OVA) emulsified in either CFA or IFA, dissolved in Alum or in phosphate-buffered saline (PBS) as a control (day 0), followed by subcutaneous injection of the antigen into footpads on day 21. Rolipram was given orally to the animals daily from days 0-20. Results showed that treatment with rolipram was followed by an increase in early swelling at 0.5 h and a decrease in late swelling at 6 and 24 h in the CFA group. In the IFA group, rolipram significantly enhanced swelling at, but not after, 30 min. In the Alum and the PBS groups, the PDE inhibitor failed to affect the OVA-specific footpad reaction at all times examined. Treatment of the CFA and IFA groups with rolipram significantly inhibited the production of the Th1 antibody anti-OVA immunoglobulin G2a (IgG2a), and the drug enhanced Th2 cell-dependent anti-OVA IgE production. In both groups, rolipram also enhanced the secretion of Th2 cytokines including interleukin-4 (IL-4) and IL-10. These findings suggest that rolipram may facilitate early allergic footpad swelling mediated by Th2 immune responses, while the late phase of swelling associated with Th1 responses may be attenuated by the PDE IV inhibitor.     

T cell cytokine polarity as a determinant of immunoglobulin A (IgA) glycosylation and the severity of experimental IgA nephropathy

Immunoglobulin A (IgA) glycosylation, recognized as an important pathogenic factor in IgA nephropathy (IgAN), is apparently controlled by the polarity of T helper (Th) cytokine responses. To examine the role of cytokine polarity in IgAN, inbred mice were immunized by intraperitoneal priming with inactivated Sendai virus (SeV) emulsified in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), which promote Th1- or Th2-immune response, respectively, and then boosted identically twice orally with aqueous suspensions of inactivated virus. Next, some mice were challenged intranasally with infectious SeV. Mice primed with CFA or IFA had equal reductions in nasal viral titre relative to non-immune controls, and equally increased serum levels of SeV-specific IgA antibody. Mice primed with CFA showed higher SeV-specific IgG than those with IFA. Splenocytes from mice primed with IFA produced copious amounts of interleukin (IL)-4 and IL-5, but little interferon-gamma and IL-2; those primed with CFA had reciprocal cytokine recall responses. Total serum IgA and especially SeV-specific IgA from mice primed with IFA showed a selective defect in sialylation and galactosylation. Although the frequency and intensity of glomerular deposits and haematuria did not differ, glomerulonephritis in mice primed with IFA and challenged with infectious virus was more severe than in those given CFA, as judged by serum creatinine level. We conclude that the polarity of T cell cytokines controls the pattern of IgA glycosylation and exerts direct or indirect effects on functional glomerular responses to immune complex deposition.     

Single immunization of newborn mice with heterologous type-II collagen induces arthritic disease

Collagen-induced arthritis (CIA) is a widely accepted model of autoimmune disease with significant similarities to rheumatoid arthritis in humans. CIA is provoked in susceptible strains upon immunization of adult mice with native type-II collagen in complete Freund's adjuvant (CFA). Neonatal exposure to antigen is supposed to result in T cell clone deletion and induction of tolerance. Here we report that the neonatal injection of bovine type-II collagen (bCII) to ICR (CD-2) mice triggers the development of autoimmune chronic joint inflammation. Compared with standard CIA significant joint swelling was not observed and anti-collagen antibodies were not detected if the second challenge with the antigen was not supplied. Histopathologic examination of the joints showed cell infiltration, synovial hyperplasia and at the later period bone destruction. Mice immunized as neonates expressed Ag-specific proliferative response and delayed type hypersensitivity (DTH) reaction to bCII.     

Pulmonary inflammation in mice with collagen‐induced arthritis is conditioned by complete Freund's adjuvant and regulated by endogenous IFN‐γ

Following immunization with collagen II (CII) in complete Freund's adjuvant (CFA), DBA/1 mice develop arthritis of major joints. This collagen‐induced arthritis (CIA) is used as a model for rheumatoid arthritis (RA) in man. Inflammatory changes in lung tissue commonly occur in RA. However, evidence for pulmonary inflammation in CIA is scarce and ambiguous. Here, we demonstrate pulmonary inflammation accompanying CIA in wild‐type DBA/1 mice. In IFN‐γ receptor‐deficient (IFN‐γR KO) mice, inflammation was more frequent and more severe. Injection of CFA only (without CII) proved to be as efficient in eliciting pulmonary inflammation as immunization with CFA + CII, though being less effective in causing arthritis. Significant correlation in severity between joint and pulmonary involvement could not be demonstrated. Macroscopic, microscopic, and functional characteristics of pulmonary inflammation in the mice resembled those seen in human RA. Increased inflammation in IFN‐γR KO mice was accompanied by augmented expression of various cytokines and chemokines, as measured by RT‐PCR on affected tissue. Treatment with a TNF‐α inhibitor ameliorated lung pathology. We conclude that CIA in DBA/1 mice is accompanied by pulmonary inflammation. Although both disease processes are kept in check by endogenous IFN‐γ, lack of strict parallelism indicates that overlap in their pathogeneses is partial.     

In vivo activation of myelin oligodendrocyte glycoprotein-specific T cells in healthy control subjects

Collagen-induced arthritis (CIA) is a murine model of autoimmune-mediated polyarthritis. CIA can be prevented by the administration (intravenously) of CII, inducing regulatory CD4+ T cells which produce Th2 cytokines. However, the relative importance of IL-4 in suppressing arthritis remains unclear. To address this question, a neutralizing monoclonal antibody to IL-4 was given to mice treated with tolerized, CII-specific cells. The antibody significantly reversed the expected suppression of arthritis. Moreover, CII administered intravenously to DBA/1 IL4-/- mice (developed by backcrossing C57B1/6 IL4-/- to wild-type DBA/1 mice) was completely ineffective in suppressing disease. These data support the importance of IL-4 in the regulation of autoimmune arthritis. Compensatory increases in mRNA message for other Th2 cytokines were observed, but they did not restore suppression of arthritis. Antibodies to CII, mostly IgG2a, were increased in IL4-/- mice. These studies represent a unique opportunity to analyze the role of IL-4 and its absence on an autoimmune murine model of arthritis.     

Delayed type hypersensitivity (DTH) to type II collagen (CII) in DBA-1 mice.

Sensitization of DBA-1 mice with Type II collagen (CII) in complete Freund's adjuvant can cause polyarthritis. A possible link between CII-induced arthritis and delayed type hypersensitivity (DTH) has been suggested, so we decided to investigate the susceptibility of DBA-1 mice to CII induced DTH reactions. The mice were primed with a dose of 10 micrograms CII i.p. 4 days before challenging with 40 micrograms CII in the ear. Swelling was measured 48 h later and was found to be reproducible. Responsiveness to CII could be transferred with whole spleen cell populations from primed animals or with enriched spleen T cells, thus confirming the cellular nature of the reaction. Lymph node cells from CII/CFA footpad immunized animals were restimulated with CII in vitro. These cells were able to passively transfer DTH sensitivity in vivo and exhibited specificity for this antigen in vitro in proliferation assays.     

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.     

Antibody responses to a cytochrome c peptide do not correlate with lymphokine production patterns from helper T-cell subsets.

This paper examines helper T-cell responses and antibody titres and isotypes following immunization with a peptide antigen in association with three different adjuvants. B10.A mice were primed with pigeon cytochrome c fragment 81-104 in association with the adjuvants complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum. Strong antibody responses, dominated by IgG1, were observed upon priming with CFA and IFA. In contrast, priming with alum induced a weak antibody response with little or no detectable antigen-specific IgG1. These differences did not correlate with differences in T-cell priming, as immunization with peptide in association with all three adjuvants induced comparable T-cell proliferative responses and frequencies of antigen-specific cells. In addition, no significant differences in interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-4 production could be found, suggesting that the adjuvants did not differentially affect Th1 and Th2 cells.     

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.     

Intravenous tolerization with type II collagen induces interleukin-4-and interleukin-10-producing CD4+ T cells

Intravenous (i.v.) administration of type II collagen (CII) is an effective way to induce tolerance and suppress disease in the collagen-induced arthritis (CIA) model. In this study, we demonstrated that a single i.v. dose of CII (as low as 0.1 mg/mouse) completely prevented the development of CIA. This suppression was accompanied by decreases in levels of antibody specific for the immunogen, bovine CII and autoantigen, mouse CII. Splenocytes obtained from CII-tolerized mice and stimulated with CII in vitro produced predominantly the T helper 2 (Th2)-type cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10). In contrast, cells obtained from mice immunized with CII produced predominantly interferon-gamma (IFN-gamma). Two-colour flow cytometric analysis of cytokine expression and T-cell phenotype demonstrated that CD4+ cells and not CD8+ or gammadelta+ cells were the predominant regulatory cells producing IL-4 and IL-10. Transgenic mice bearing a T-cell receptor (TCR) specific for CII had a greater increase in the number of IL-4-secreting CD4+ cells, as well as a marked increase of IL-4 in culture supernatants. This cytokine was produced by transgene-bearing T cells. Elucidation of mechanisms for the induction of tolerance in mature T cells is an important line of study in autoimmune models because of the potential application for treating organ-specific autoimmune disease.     

Methotrexate,combined with cyclophosphamide attenuates murine collagen induced arthritis by modulating the expression level of Breg and DCs

To explore the mechanism of methotrexate (MTX) and its combination with cyclophosphamide (CTX) in collagen-induced arthritis (CIA), we investigated the levels of several immune cells and cytokines in mice with different treatments. CIA was induced in DBA/1 mice at the age of 7 weeks by primary immunization with 100 μl emulsion containing 2 mg/ml bovine type II collagen which was mixed with complete Freund's adjuvant (CFA). The booster immunization was performed with 50–100 μl emulsion containing 2 mg/ml bovine type II collagen (CII) mixed with incomplete Freund's adjuvant (IFA). MTX, CTX or both were administrated after the booster immunization. Therapeutic effect was evaluated by arthritic scores, X-rays and assessment of histopathological joint destruction. The expression of TNF-α, IL-6, IL-23, IL-10 were also measured. The frequencies of different immune cell subsets in the lymph node, spleen and bone marrow were determined by flow cytometry analysis. Our results showed that CTX and MTX treatment attenuated the severity of arthritis of CIA mice and reduced the levels of several cytokines. CTX and MTX treated mice showed a lower frequency of B cells in bone marrow. Also, when treated the CIA mice with MTX, alone or together with CTX, the lymph nodes and spleen exhibited a decrease in regulatory B cells (Breg) and dendritic cells (DCs). Notably, the combination of MTX and CTX had a more pronounced effect. By measuring the levels of different immune cells those participated in the development of rheumatoid arthritis (RA), our experiment may help to evaluate the therapeutic effects and prognosis of arthritic diseases.     

Effect of Candida albicans cell wall glucan as adjuvant for induction of autoimmune arthritis in mice

Collagen-induced arthritis (CIA) is an experimental model of rheumatoid arthritis (RA) and has aided research into the pathogenesis of inflammatory joint disease. Typically, Type II collagen (CII) emulsified with Freund's complete adjuvant (FCA) is injected into DBA/1 mice. After a booster injection, the mice develop inflammation of the paws. But the fact that the immunization of CII alone does not induce arthritis suggests that activation of the immune system by an adjuvant is necessary for induction of the arthritis. In the present study, we investigated the ability of beta-glucans derived from Candida albicans to act as an adjuvant to induce autoimmune arthritis. DBA/1 mice were injected with CII emulsified with FCA or particulate beta-glucan, OX-CA, on day 0 and given a booster at day 21. Mice immunized with CII plus OX-CA developed arthritis at around 7-10 days after the booster injection. Similarly, mice administered CII emulsified with FCA developed arthritis with the same time course. The mice immunized with CII and OX-CA had a more severe arthritis than those immunized with CII and FCA. Histological changes and production of anti-CII antibody were observed regardless of the type of injection. In addition, components of C. albicans were also tested for their ability to induce arthritis as an adjuvant. The results showed that CSBG, which is a soluble beta-glucan, acted as an adjuvant for CIA but CAWS, which is a mannoprotein-beta-glucan complex, did not. In conclusion, beta-glucan derived from C. albicans acted as an adjuvant and the injection with CII resulted in arthritis with the production of anti-CII autoantibody. The results strongly suggested that fungal metabolites such as beta-glucans have the capacity to induce and exacerbate autoimmune diseases such as RA.     

CD4+ T cells from collagen-induced arthritic mice are essential to transfer arthritis into severe combined immunodeficient mice.

The role of T lymphocytes in the adoptive transfer of collagen-induced arthritis (CIA) in DBA/1J mice to severe combined immunodeficient (SCID) mice was investigated. Spleen cells from non-immunized, type I collagen (CI) or type II collagen (CII)-immunized DBA/1J mice were injected into SCID mice which lack functional T and B cells. Specific antigenic stimulation of arthritogenic cells was required since only lymphocytes from arthritic CIA mice plus simultaneous administration of CII transferred arthritis to 11 of 12 SCID mice with a marked increase in CII antibody titre. However, CI-immunized or non-immunized DBA/1J mice cells did not induce arthritis in SCID mice. SCID recipients of pre-arthritic CIA lymphocytes presented increase in CII antibody, but showed no clinical signs of arthritis, suggesting that antibodies to CII alone can not induce CIA. Depletion of CD4+ T cells inhibited the transfer of arthritis to SCID mice, with a decrease in CII antibody titre in chimaeras. In contrast, depletion of CD8+ T cells enhanced the onset of arthritis in SCID mice. The results imply that CD4+ T cells are required for the induction of CIA. In addition, CD8+ T cells might have a suppressive role in the etiology of this disease. It is probable that memory CD4+ T cells stimulate production of antibodies to CII and subsequent arthritis. This study clarifies the role of T lymphocytes in the transfer of CIA to SCID mice.