Enhanced autoimmune arthritis in IFN-gamma receptor-deficient mice is conditioned by mycobacteria in Freund's adjuvant and by increased expansion of Mac-1+ myeloid cells.

Induction of experimental autoimmune diseases often relies on immunization with the organ-specific autoantigens in CFA, which contains heat-killed mycobacteria. In several of these models, including collagen-induced arthritis, endogenous IFN-gamma acts as a disease-limiting factor in the pathogenesis of the disease. Here we show that in collagen-induced arthritis the protective effect of IFN-gamma depends on the presence of mycobacteria in the adjuvant. Omission of mycobacteria inverts the role of endogenous IFN-gamma to a disease-promoting factor. Thus, the mycobacterial component of CFA opens a pathway by which endogenous IFN-gamma exerts a protective effect that supersedes its otherwise disease-promoting effect. Extramedullary hemopoiesis and expansion of the Mac-1+ cell population accompanied the accelerated and more severe disease course in the IFN-gamma receptor knockout mice immunized with CFA. Treatment of such mice with Abs against the myelopoietic cytokines IL-6 or IL-12 inhibited both disease development and the expansion of the Mac-1+ population. We postulate that mycobacteria in CFA stimulate the expansion of the Mac-1+ cell population by a hemopoietic process that is restrained by endogenous IFN-gamma. These results have important implications for the validity of animal models of autoimmunity to study the pathogenesis and to evaluate cytokine-based therapy of autoimmune diseases.     

Blockade of IFN-gamma does not affect the arthritogenicity of T cells generated during the induction of adjuvant arthritis but exacerbates the polyarthritis produced by adoptive transfer of arthritogenic effector cells

IFN-gamma production is prominent in some models of autoimmune disease, including adjuvant arthritis (AA), but the role of IFN-gamma in the pathogenesis of these diseases is uncertain. Experimental manipulation (administration of cytokine, blocking cytokine action with specific antibody, disruption of genes encoding the cytokine or its receptor) has revealed both pro- and anti-inflammatory effects, depending on the nature of the manipulation and the timing of the treatment. We examined separately the effects of cytokine blockade during the afferent and efferent phases of AA in Dark Agouti rats, using an adoptive transfer system. Effects of IFN-gamma on the efferent phase were investigated by treating recipients with mAb DB-1, which blocks the activity of rat IFN-gamma. When treatment was commenced before cell transfer, the resulting polyarthritis was more severe than in controls treated with normal IgG. Commencing treatment after the adoptively transferred disease had become established caused neither amelioration nor exacerbation, but did cause some delay in resolution. In contrast, treatment of donors did not appear to affect the generation of arthritogenic cells. The main effect of IFN-gamma appears to be modulation of the arthritogenicity of the migratory effector T cells that can transfer AA.     

Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8+ T cells in vivo.

CD8+ T cells from TCR transgenic 2C mice, specific for SIYRYYGL peptide bound to H-2Kb, were adoptively transferred into C57BL/6 recipients to allow monitoring of their location, numbers, and phenotype upon peptide challenge. Recipients were primed by s.c. injection of SIYRYYGL alone or with CFA or IL-12, and the transferred cells then tracked by flow cytometry using the 1B2 mAb specific for the 2C TCR. Peptide alone induced a transient and weak expansion of 1B2+ cells in the draining lymph nodes (DLN) by day 3, but these cells were tolerant to secondary peptide challenge. In contrast, priming with CFA/peptide resulted in a large clonal expansion of 1B2+ cells in DLN by day 3, and the cells exhibited a CD25highCD44high phenotype, blast transformation, and lytic effector function. By day 5, 1B2+ cell numbers decreased in the DLN and increased in the spleen and blood. 1B2+ cells with a memory phenotype persisted through day 60 in the DLN, spleen, and blood and responded to secondary peptide challenge. Immunization with peptide, along with IL-12, mimicked the adjuvant effects of CFA with respect to phenotype, clonal expansion, effector function, and establishment of memory. IL-12 was not unique in providing this adjuvant effect however, since CFA/peptide immunization of IL-12-deficient recipient mice also resulted in 1B2+ T cell activation and clonal expansion. Thus, CFA or IL-12 can enhance Ag-specific CD8+ T cell responses to peptide, demonstrating that an inflammatory cytokine(s) can support activation and prevent tolerance induction.     

A heritable defect in IL-12 signaling in B10.Q/J mice. I. In vitro analysis

B10.Q mice are normally susceptible to the induction of collagen-induced arthritis. We noted that one subline of B10.Q mice, B10.Q/J, was completely resistant to disease induction when immunized with collagen in CFA. B10.Q/J mice have a global defect in the generation of Th1 responses, and Ag-specific T cells derived from this strain failed to produce IFN-gamma. Because T cells from these mice could produce normal amounts of IFN-gamma when activated by IL-12/IL-18-independent stimuli, the defect appeared to be a failure to respond to IL-12. This defect extended to NK cells, which also failed to produce IFN-gamma when stimulated by IL-12. The capacity of NK cells, but not activated T cells, to produce IFN-gamma in response to IL-12 could be partially restored by IL-18. The expression of the IL-12R beta1- and beta2-chains on T cells and NK cells from B10.Q/J mice was normal. However, activated T cells from B10.Q/J mice did not signal normally through the IL-12R and manifested a defect in their capacity to phosphorylate Stat4. This defect was partial in that it could be overcome by increasing both the concentration of IL-12 and the incubation times in the Stat4 phosphorylation assays. Because Stat4 function is apparently intact in B10.Q/J mice, the defect in IL-12 signaling can be localized between the IL-12R complex and Stat4. This subtle abnormality in IL-12 responsiveness results in a profound defect in the generation of Th1 cells and the development of autoimmune disease.     

Inhibition of adjuvant arthritis by a DNA vaccine encoding human heat shock protein 60

Adjuvant arthritis (AA) is an autoimmune disease inducible in rats involving T cell reactivity to the mycobacterial 65-kDa heat shock protein (HSP65). HSP65-specific T cells cross-reactive with the mammalian 60-kDa heat shock protein (HSP60) are thought to participate in the modulation of AA. In this work we studied the effects on AA of DNA vaccination using constructs coding for HSP65 (pHSP65) or human HSP60 (pHSP60). We found that both constructs could inhibit AA, but that pHSP60 was more effective than pHSP65. The immune effects associated with specific DNA-induced suppression of AA were complex and included enhanced T cell proliferation to a variety of disease-associated Ags. Effective vaccination with HSP60 or HSP65 DNA led paradoxically to up-regulation of IFN-gamma secretion to HSP60 and, concomitantly, to down-regulation of IFN-gamma secretion to the P180-188 epitope of HSP65. There were also variable changes in the profiles of IL-10 secretion to different Ags. However, vaccination with pHSP60 or pHSP65 enhanced the production of TGFbeta1 to both HSP60 and HSP65 epitopes. Our results support a regulatory role for HSP60 autoreactivity in AA and demonstrate that this control mechanism can be activated by DNA vaccination with both HSP60 or HSP65.     

A targeted DNA vaccine augments the natural immune response to self TNF-alpha and suppresses ongoing adjuvant arthritis

Depending on the mode of immunization, a single administration of CFA may result in the development of a local inflammatory process or chronic poly adjuvant-induced arthritis (AA). Administration of naked DNA encoding TNF-alpha results in the generation of immunological memory to its gene product. Upon induction of AA, this memory effectively inhibited the development of disease. Self-specific Abs developed in DNA-vaccinated animals were neutralizing in vitro and could adoptively transfer the beneficial effect of the vaccine. Administration of CFA to induce a local delayed-type hypersensitivity response rather than AA did not lead to an elicited production of Abs to the gene product of the above vaccine. Thus, elicitation of protective immunity is dependent on the development of an autoimmune condition. Most importantly, the administration of the TNF-alpha DNA construct after the onset of disease led to a rapid, long-lasting remission. This suggests a highly effective way by which a DNA vaccine encoding an autologous proinflammatory cytokine can be used to reprogram the immune system to generate protective immunity to its own potentially harmful activities.     

The M20 IL-1 inhibitor prevents onset of adjuvant arthritis

Cytokines, specifically IL-1 and TNF, have been implicated as important mediators of joint destruction in rheumatoid arthritis (RA). Elevated levels of IL-1 in the joint fluid of patients with RA have been reported, as well as the presence of IL-1 inhibitory activity. We have reported the characterization of an inhibitor derived from a myelomonocytic cell line cloned in our laboratory which is specific for IL-1. This IL-1 inhibitor is protein in nature which specifically inhibits activityin vitro andin vivo. Previous studies showed that the inhibitor reduced acute inflammatory reactions associated with IL-1 (fever, leukocytosis, local foot pad swelling, lymph node enlargement and acute phase reactants). Thus it was of interest to study whether the M20 IL-1 inhibitor could modify adjuvant-induced chronic inflammation in rats, which is often used as a model for human RA. Administration of complete Freund's adjuvant (CFA) into Lewis rats, resulted in a severe adjuvant arthritis (AA) which reached peak severity after 14 days. Daily administration of IL-1 inhibitor, beginning after injection of CFA, abolished the appearance of AA. The parameters investigated were: joint swelling (the increase in diameter of joints), peri-articular erythema, limping of the rats and histological examination. The effect of the M20 IL-1 inhibitor was shown to be dose dependent and the IL-1 inhibitor alone had no adverse effects. These results indicate that the M20 IL-1 inhibitor may have a role in the treatment of AA and may be used to reduce pathological processes in joint inflammation.     

Activation of T cells recognizing an epitope of heat-shock protein 70 can protect against rat adjuvant arthritis.

We have previously reported that CD4+ T cells recognizing a peptide comprising residues 234-252 of the heat shock protein (HSP)70 of Mycobacterium tuberculosis (M.tb) in the context of RT1.B MHC class II molecule emerged in the peritoneal cavity during the course of Listeria monocytogenes infection in rats and suppressed the inflammatory responses against listerial infection via IL-10 production. We report in this work that pretreatment with peptide 234-252 of HSP70 derived from M.tb suppressed the development of adjuvant arthritis (AA) in Lewis rats induced using heat-killed M.tb. T cells from rats pretreated with peptide 234-252 produced a significant amount of IL-10 in response to the epitope. T cells from rats pretreated with the peptide and immunized with M.tb produced the larger amount of IL-10 in response to the peptide, but only a marginal level of IFN-gamma in response to purified protein derivative of M.tb. Administration of anti-IL-10 Ab partly inhibited the suppressive effect of pretreatment with peptide 234-252 on the development of AA. Furthermore, transfer of a T cell line specific for the epitope at the time of AA induction markedly suppressed AA. These findings suggested that T cells recognizing peptide 234-252 may play a regulatory role in inflammation during AA via the production of suppressive cytokines including IL-10.     

A novel angiogenesis inhibitor suppresses rat adjuvant arthritis

Rat adjuvant arthritis (AA) is an animal model of rheumatoid arthritis in which pannus formation and destruction of joints occur after immunization with complete Freund's adjuvant. Neovascularization is present within the synovium and may be critical for pannus growth. In this study the effects of a novel angiogenesis inhibitor, AGM-1470, on AA were evaluated. Lewis rats were immunized with CFA to induce arthritis. AGM-1470 treatment was initiated prior to arthritis onset (preventative protocol) or administered to rats with established disease (suppressive protocol). The severity of synovitis and the immunologic status of all rats were then evaluated. Using clinical and radiographic criteria, AGM-1470 significantly reduced arthritis incidence (preventative protocol) (P < 0.01) and disease severity (both protocols, P < 0.001, compared to controls) without affecting T cell function in vitro or phenotype in vivo. Additionally, histologic sections from control rats revealed marked pannus formation, destruction of bone/cartilage, and neovascularization. These findings were absent in AGM-1470-treated rats. AGM-1470 may offer a new treatment option for rheumatoid arthritis and other angiogenesis-dependent diseases.     

Exogenous nitric oxide inhibits experimental autoimmune uveoretinitis development in Lewis rats by modulation of the Th1-dependent immune response.

Experimental autoimmune uveoretinitis (EAU) is a T cell-mediated autoimmune disease of posterior uvea that closely resembles a human disease. The uveitogenic effector T cell has a Th1-like phenotype [high interferon-gamma (IFN-gamma), low interleukin-4 (IL-4)], and genetic susceptibility to EAU that is associated with an elevated Th1 response. Suppression of CD4+ Th1 cells for the treatment of autoimmune disease is an attractive potential therapeutic approach. Nitric oxide (NO) has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. In this study, we investigated the potential role of NO as an immunoregulator to alter Th1/Th2 cytokine production, as well as to inhibit the interphotoreceptor retinoid binding protein (IRBP)-induced EAU, a CD4+ Th1 cell-mediated autoimmune disease. Injection of IRBP (100 microg) into two footpads resulted in severe EAU. The beginning peak of the disease was days 12 to 15 after immunization. Oral treatment with molsidomine (MSDM), a NO donor, began 24 h before IRBP immunization to the end of the experiments, which resulted in a significant inhibition of the disease by clinical and histopathological criteria. When MSDM was administered until day 21, a complete reduction of incidence and severity of EAU was observed. To investigate the cytokine alterations from Th1 to Th2 cytokines by MSDM, the cytokines were assayed in a culture medium of IRBP-stimulated inguinal lymphocytes. IRBP-immunized rats secreted a high concentration of IFN-gamma and a low concentration of IL-10. In contrast, MSDM treatment enhanced IL-10 secretion and tended to decrease IFN-gamma secretion. In conclusion, we show that the administration of NO suppresses EAU by altering the Th1/Th2 balance of inflammatory immune responses. We suggest that NO may be useful in the therapeutic control of autoimmune uveitis.     

Kinetics of Tissue Iron in Experimental Autoimmune Encephalomyelitis in Rats

To elucidate the role of iron in the pathomechanisms of autoimmune CNS disorders, we estimated the tissue concentrations of Fe²⁺ in the brain, spinal cord, and liver in the chronic relapsing form of experimental autoimmune encephalomyelitis (EAE). The disease was induced in Dark Agouti (DA) strain of rats, by subcutaneous injection of bovine brain homogenate in complete Freund's adjuvant (CFA). Control rats consisted of unsensitized rats and of rats treated with CFA or saline. The data obtained by clinical assessment and by inductively coupled plasma spectrometry have shown that the attacks of disease (on the 12th and 22nd post-immunization day) were followed by high accumulation of iron in the liver. Additionally, during the second attack of disease, the decreased concentration of Fe²⁺ was found in cervical spinal cord. The data point to regulatory effects of iron and hepatic trace elements regulating mechanisms in the pathogenesis of EAE.     

Cytokine gene activation in synovial membrane, regional lymph nodes, and spleen during the course of rat adjuvant arthritis.

Cytokine gene activation was assessed during rat adjuvant arthritis (AA) in synovial membrane (SM), popliteal lymph node (popl-LN), and spleen, using semiquantitative, competitive RT-PCR. Changes in the popl-LN were considerably higher than in spleen or SM. In the preclinical phase (day 6), cytokine mRNA elevations occurred exclusively in the popl-LN and included IFN-gamma, IL-1beta, IL-5, IL-6, and IL-10. In the acute phase (days 13-16) all three organs became involved: (i) in the SM, significant elevations were limited to IL-1beta and IL-6, which, notably, correlated positively with the degree of arthritis; (ii) in the popl-LN, IFN-gamma, IL-1beta, IL-6, and IL-10 (but not IL-5) were still elevated, while IL-2 rose significantly; (iii) in the spleen, TNF-alpha peaked simultaneously with the arthritis score (day 16) and dramatically dropped thereafter. Upon transition into the chronic phase (day 20) the following phenomena were observed: (i) IL-1beta and IL-6 were still significantly increased in the SM; (ii) IFN-gamma, IL-1beta, IL-2, IL-6, and IL-10 were still elevated in the popl-LN; and (iii) there was a progressive rise of IL-5 mRNA in the spleen, positively correlated with the arthritis score. In conclusion, cytokines with pro- and anti-inflammatory functions overlap throughout disease, but in different organ-related patterns. Local (SM) and regional (popl-LN) IL-1beta and IL-6, elevated throughout the entire course of AA, may directly contribute to disease severity. While in AA spleen TNF-alpha appears to be a systemic marker of acute disease, spleen IL-5 may be involved in disease resolution.     

Oral administration of lipopolysaccharide exacerbates collagen-induced arthritis in mice.

We investigated whether oral administration of LPS exacerbated collagen-induced arthritis (CIA) in mice, which was an experimental model of autoimmune disease. CIA was induced by s.c. injection of type II collagen emulsified with CFA into the base of the tail (day 0) followed by a booster injection on day 21. To examine the ability of LPS to exacerbate CIA, varying doses of LPS were orally administered on day 50. The results showed that administration of LPS was followed by reactivation of CIA in a dose-related fashion. Histologically, on day 55 there were marked edema of synovium proliferated by day 50, new formation of fibrin, and intense infiltration of neutrophils accompanied with a large number of mononuclear cells. Severe destruction of cartilage and subchondral bone was also observed on day 70. The reactivation of CIA by oral administration of LPS was associated with increase in anti-type II collagen IgG and IgG2a Abs as well as varying kinds of cytokines including IL-12, IFN-gamma, IL-1beta, and TNF-alpha. Polymyxin B sulfate given either orally or i.v. suppressed the recurrence of CIA. Increased amounts of LPS were found in sera of mice given the endotoxin orally. LPS from Salmonella enteritidis, Salmonella typhimurium, and Klebsiella pneumoniae and its component, lipid A from Escherichia coli, also reactivated the disease. These findings suggest that LPS from intestinal bacteria may play a role in the exacerbation of autoimmune joint inflammation.     

Inhibitory and enhancing effects of IFN-gamma and IL-4 on SHIV(KU) replication in rhesus macaque macrophages: correlation between Th2 cytokines and productive infection in tissue macrophages during late-stage infection

HIV-1 is dual-tropic for CD4+ T lymphocytes and macrophages, but virus production in the macrophages becomes manifest only during late-stage infection, after CD4+ T cell functions are lost, and when opportunistic pathogens begin to flourish. In this study, the SHIV/macaque model of HIV pathogenesis was used to assess the role of cytokines in regulating virus replication in the two cell types. We injected complete Freund's adjuvant (CFA) intradermally into SHIV(KU)-infected macaques, and infused Schistosoma mansoni eggs into the liver and lungs of others. Tissues examined from these animals demonstrated that macrophages induced by CFA did not support viral replication while those induced by S. mansoni eggs had evidence of productive infection. RT-PCR analysis showed that both Th1 (IL-2 and IFN-gamma) and Th2 cytokines (IL-4 and IL-10) were present in the CFA lesions but only the Th2 cytokines were found in the S. mansoni lesions. Follow-up studies in macaque cell cultures showed that whereas IFN-gamma caused enhancement of virus replication in CD4+ T cells, it curtailed viral replication in infected macrophages. In contrast, IL-4 enhanced viral replication in infected macrophages. These studies strongly suggest that cytokines regulate the sequential phases of HIV replication in CD4 T cells and macrophages.     

CD4-CD8- T cells from mice with collagen arthritis display aberrant expression of type l K+ channels

Expression of voltage-gated K+ channels in mAb-defined T cell subsets from normal mice and mice with experimental autoimmune arthritis was studied with the patch-clamp whole-cell recording technique in combination with fluorescence microscopy. CD4+CD8- Th cells from DBA/1 LacJ mice with type II collagen arthritis expressed low levels of type n K+ channels, and CD4-CD8+ T cells (cytotoxic) showed small numbers of type l or n' K+ channels, like their phenotypic counterparts in normal mice. CD4-CD8-Thy-1.2+ (double negative or DN) T cells from the diseased mice, however, displayed an abundance of type l K+ channels compared to DN T cells in normal mice, or mice immunized with CFA. Furthermore, the aberrant expression of type l K+ channels correlated with the presence of active disease. DN T cells from mice with SLE, type-1 diabetes mellitus, and experimental allergic encephalomyelitis, also exhibited a high number of type l K+ channels. These results suggest that expression of numerous type l K+ channels may be a useful marker for DN T cells associated with these autoimmune disorders.     

IL-12, independently of IFN-gamma, plays a crucial role in the pathogenesis of a murine psoriasis-like skin disorder.

The onset of acute psoriasis and the exacerbation of chronic psoriasis are often associated with a history of bacterial infection. We demonstrate that while only few scid/scid mice develop disease when CD4+CD45Rbhigh T cells are transferred alone, coadministration of LPS plus IL-12 or staphylococcal enterotoxin B into scid/scid mice 1 day after CD4+CD45Rbhigh T cell transfer greatly enhances disease penetrance and severity. Most importantly, the skin lesions induced by this method exhibit many of the histologic hallmarks observed in human psoriasis. Skin infiltrating CD4+ T cells were predominantly memory/effector cells (CD45Rblow) and exhibited a highly polarized Th1 phenotype. To test whether the development of pathogenic T cells was dependent on their production of IFN-gamma, we transferred IFN-gamma-/- CD4+CD45Rbhigh T cells into scid/scid or into T, B and NK cell-deficient scid/beige mice. Surprisingly, the incidence of psoriasis was similar to scid/scid animals that received IFN-gamma+/+ T cells, although acanthosis of the skin was attenuated. In contrast, the development of psoriasis was abolished if anti-IL-12 mAb was administered on day 7 and 35 after T cell transfer. Skin-derived IFN-gamma-/- inflammatory cells, but not cells from anti-IL-12-treated animals, secreted substantial amounts of TNF-alpha, suggesting that the inflammatory effect of IFN-gamma-/- T cells may be partly exerted by TNF-alpha and that the therapeutic effect of anti-IL-12 may depend on its ability to down-regulate both TNF-alpha and IFN-gamma. Overall, these results suggest that IL-12, independently of IFN-gamma, is able to induce pathogenic, inflammatory T cells that are able to induce psoriasiform lesions in mice.     

Suppression of adjuvant arthritis in Lewis rats by oral administration of type II collagen

Adjuvant arthritis is induced by intradermal injection of Mycobacterium tuberculosis (MT) in oil. The role of immunity to type II collagen (CII) in adjuvant arthritis (AA) has not been well defined. We found that oral administration of chicken CII given 3 micrograms per feeding on days -7, -5, and -2 before disease induction consistently suppressed the development of AA. A decrease in delayed-type hypersensitivity responses to CII was also observed that correlated with suppression of AA. AA was optimally suppressed by 3 and 30 micrograms of collagen type II variably by 300 micrograms, and not by 0.3 microgram or 1 mg. Oral administration of collagen type I also suppressed AA; only minimal effects were seen with collagen type III. Suppression was Ag specific: feeding CII did not suppress experimental autoimmune encephalomyelitis; feeding myelin basic protein suppressed experimental autoimmune encephalomyelitis, but not AA. Suppression of AA could not be consistently obtained by feeding MT. Suppression of AA could be adoptively transferred by T cells from CII fed animals and could be obtained when CII was fed after disease onset. Our results suggest that autoimmunity to CII has a pathogenic role in AA and raise the possibility that cross-reactive epitopes exist between CII and MT. Alternatively, the pathogenesis of AA may be dependent on developing immunity to CII. These results further demonstrate the effectiveness of oral tolerance as a means to suppress experimental autoimmune diseases.     

Development of proteoglycan-induced arthritis is independent of IL-17

IL-17 is the hallmark cytokine for the newly identified subset of Th cells, Th17. Th17 cells are important instigators of inflammation in several models of autoimmune disease; in particular, collagen induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE), which were previously characterized as Th1-mediated diseases. Although high levels of IFN-gamma are secreted in CIA and EAE, disease is exacerbated in IFN-gamma- or IFN-gamma receptor-deficient mice due to the ability of IFN-gamma to suppress IL-17 secretion. However, in proteoglycan-induced arthritis (PGIA), severe arthritis is dependent on the production of IFN-gamma. We were therefore interested in determining the role of IL-17 in PGIA. We assessed the progression of arthritis in IL-17-deficient (IL-17-/-) mice and found the onset and severity of arthritis were equivalent in wild-type (WT) and IL-17-/- mice. Despite evidence that IL-17 is involved in neutrophil recruitment, synovial fluid from arthritic joints showed a comparable proportion of Gr1+ neutrophils in WT and IL-17-/- mice. IL-17 is also implicated in bone destruction in autoimmune arthritis, however, histological analysis of the arthritic joints from WT and IL-17-/- mice revealed a similar extent of joint cellularity, cartilage destruction, and bone erosion despite significantly reduced RANKL (receptor activator of NK-kappaB ligand) expression. There were only subtle differences between WT and IL-17-/- mice in proinflammatory cytokine expression, T cell proliferation, and autoantibody production. These data demonstrate that IL-17 is not absolutely required for autoimmune arthritis and that the production of other proinflammatory mediators is sufficient to compensate for the loss of IL-17 in PGIA.     

A single early activation of invariant NK T cells confers long-term protection against collagen-induced arthritis in a ligand-specific manner

The glycosphingolipid alpha-galactosylceramide (alpha-GalCer) has been shown to be a potent activator of invariant NKT (iNKT) cells, rapidly inducing large amounts of both Th1 and Th2 cytokines upon injection in mice. The C-glycoside analog of alpha-GalCer (alpha-C-GalCer), by contrast, results in an enhanced Th1-type response upon activation of iNKT cells. We administered a single dose of these Ags to DBA/1 mice during the early induction phase of collagen-induced arthritis and demonstrated therapeutic efficacy of alpha-GalCer when administered early rather than late during the disease. Surprisingly, the Th1-polarizing analog alpha-C-GalCer also conferred protection. Furthermore, a biphasic role of IFN-gamma in the effect of iNKT cell stimulation was observed. Whereas in vivo neutralization of IFN-gamma release induced by either alpha-GalCer or alpha-C-GalCer early during the course of disease resulted in partial improvement of clinical arthritis symptoms, blockade of IFN-gamma release later on resulted in a more rapid onset of arthritis. Although no phenotypic changes in conventional T cells, macrophages, or APCs could be detected, important functional differences in T cell cytokine production in serum were observed upon polyclonal T cell activation, 2 wk after onset of arthritis. Whereas alpha-GalCer-treated mice produced significantly higher amounts of IL-10 upon systemic anti-CD3 stimulation compared with PBS controls, T cells from alpha-C-GalCer-treated mice, by contrast, produced substantially lower levels of cytokines, suggesting the involvement of different protective mechanisms. In conclusion, these findings suggest long-term, ligand-specific, time-dependent, and partially IFN-gamma-dependent immunomodulatory effects of iNKT cells in collagen-induced arthritis.     

Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function

The effects of granulocyte-macrophage (GM)-CSF on the synthesis of MHC class II molecules and on the Ag presentation capacity by bone marrow derived macrophages (BMM phi) was investigated. BMM phi obtained by in vitro culture in the presence of macrophage-CSF were negative for synthesis of I-A molecules and induced the Ag-mediated proliferation of insulin-specific T clone cells with lower efficiency than splenic accessory cells. After pulse treatment with GM-CSF for 24 to 48 h, day 12 BMM phi exhibited highly efficient Ag presentation function which was superior to that induced by IFN-gamma. Expression of membrane-bound IL-1 was augmented significantly by GM-CSF, but not by IFN-gamma. However, the T cell clone used to probe for accessory cell function of BMM phi was not dependent on IL-1 for optimal proliferation. Concomitantly, GM-CSF induced the de novo synthesis of I-A molecules, although to a lesser extent than optimal doses of IFN-gamma. Thus GM-CSF appears to elicit properties in addition to Ia molecule synthesis and membrane IL-1 expression in BMM phi being essential for efficient accessory cell function to the T clone cells. The activation of BMM phi by GM-CSF was reversible and could be repeated. These data show that GM-CSF exerts a modulatory influence on preformed BMM phi, reversibly activating cells to Ia biosynthetic potential and pronounced accessory cell capacity, thus rendering the explanation unlikely that differentiation of precursor cells into a constitutively functional state had occurred.