Myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis is chronic/relapsing in perforin knockout mice, but monophasic in Fas- and Fas ligand-deficient lpr and gld mice

The expression and action of Fas/Fas ligand (FasL) in multiple sclerosis has been postulated as a major pathway leading to inflammatory demyelination. To formally test this hypothesis, C57BL/6-lpr and -gld mice, which due to gene mutation express Fas and FasL in an inactive form, were immunized with myelin oligodendrocyte glycoprotein peptide(35-55). Whereas in wild-type C57BL/6 mice, experimental autoimmune encephalomyelitis (EAE), was chronic/relapsing, EAE in lpr and gld mice was characterized by a lower incidence of disease and a monophasic course. This contrasts with C57BL/6 perforin knockout mice, which showed the most severe form of EAE of all mouse strains tested, the course being chronic relapsing. The difference noted cannot be attributed to an involvement of FasL in oligodendrocyte damage since oligodendrocytes are insensitive to FasL-mediated cytotoxicity in vitro, and since in the acute phase of EAE gld mice also show CD4+ T cell infiltrates with associated demyelination in brain and spinal cord. Unlike oligodendrocytes, astrocytes were killed by FasL in vitro. It remains to be established whether this latter finding explains the different disease course of lpr and gld mice compared to wild-type and perforin knockout mice.     

Detection of immunoglobulin/c-myc recombinations in mice that are resistant to plasmacytoma induction

Interchromosomal recombinations between c-myc and immunoglobulin sequences can be found in preneoplastic lesions (oil granulomata) during pristane-induced plasmacytoma development in susceptible BALB/cAn mice. In this study we used a more sensitive approach, hybridization-enriched templates with nested PCR, to detect microclones with Ig alpha/c-myc recombinations in oil granulomata of susceptible and resistant mice. Recombinations were detected in as many as 73% (32/44) of plasmacytoma-susceptible BALB/cAn mice 30 days after an injection of pristane. Mice that are resistant to plasmacytoma induction can also harbor recombination-positive cells, but these are less frequent [2/20 DBA/2N, 8/20 (BALB/cAn x DBA/2N)F1, hereafter called CD2F1]. The clones in DBA/2N mice were small (< 400 cells), whereas in BALB/cAn, the oil granuloma harbored up to several thousand of these cells. We conclude that the molecular machinery for generating characteristic interchromosomal recombinations can be found in all strains of mice. Both the frequency of generating Ig alpha/c-myc recombinations and the expansion of recombination-positive cells are greater in susceptible mice than in resistant strains.     

Antisense knockdown of inducible nitric oxide synthase inhibits induction of experimental autoimmune encephalomyelitis in SJL/J mice

We used an antisense oligodeoxynucleotide (ODN) complementary to inducible nitric oxide synthase (iNOS) to inhibit experimental autoimmune encephalomyelitis (EAE) in female SJL/J mice, an animal model for multiple sclerosis. The antisense ODN was administered intraventricularly to mice daily for 10 days beginning at the time of adoptive transfer of myelin basic protein-specific T lymphocytes. The antisense ODN treatment significantly reduced the clinical score of EAE and blocked iNOS mRNA and protein synthesis, as well as iNOS enzyme activity within the central nervous system. The levels of nitric oxide and cyclic guanosine monophosphate were also significantly reduced by the antisense ODN treatment. Neither sense nor random ODN affected clinical EAE or iNOS expression. Moreover, the protein and enzyme activity level of constitutive neuronal nitric oxide synthase was not affected by the antisense ODN. Thus, we have shown that the iNOS antisense ODN specifically blocked iNOS expression and ameliorated the induction of EAE.     

Gelatinase B-deficient mice are resistant to experimental bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by deposition of autoantibodies at the basement membrane zone. In an experimental BP model in mice, the subepidermal blistering is mediated by antibodies directed against the hemidesmosomal protein BP180 (collagen XVII, BPAG2), and depends on complement activation and neutrophil infiltration. Gelatinase B is present in BP blister fluid and can cleave BP180. In this study we investigated the role of gelatinase B in the immunopathogenesis of experimental BP using mice containing targeted disruption of the gelatinase B (MMP-9, 92 kD gelatinase) gene. Gelatinase B-deficient mice were resistant to the blistering effect of intracutaneous anti-mBP180 antibodies, although these mice showed deposition of autoantibodies at the basement membrane zone and neutrophil recruitment to the skin comparable to that observed in the control mice. Interleukin 8 given intradermally concomitantly with pathogenic anti-mBP180 elicited a significant neutrophil recruitment into the skin in gelatinase B-deficient mice, but blistering did not occur. However, gelatinase B-deficient mice reconstituted with neutrophils from normal mice developed blistering in response to anti-mBP180 antibodies. These results implicate neutrophil-derived gelatinase B in the pathogenesis of experimental BP and might lead to novel therapeutic strategies for BP.     

IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin components including myelin oligodendrocyte glycoprotein (MOG). Myelin-specific Th1 cells enter the central nervous system (CNS) via binding of very late antigen 4 (VLA-4) to the endothelial vascular cell adhesion molecule 1 (VCAM-1). In the present study, mice with a homologous disruption of the gene encoding IL-6 are found to be resistant to MOG-induced EAE as evidenced by absence of clinical symptoms, minimal infiltration of CD3+ T cells and monocytes into the CNS and lack of demyelination. The failure to induce EAE in IL-6-/- mice is not due to the absence of priming, since lymphocytes of immunized IL-6-/- mice proliferate in response to MOG and produce pro-inflammatory cytokines including IL-2 and IFN-gamma. However, in MOG-immunized IL-6-/- mice, serum anti-MOG antibody titers were found to be drastically reduced. This observation is unlikely to be responsible for resistance to EAE, because B cell-deficient (microMT) mice proved to be fully susceptible to the disease. A striking difference between MOG-immunized wild-type (wt) and IL-6-/- mice was the expression of endothelial VCAM-1 and ICAM-1, which were dramatically up-regulated in the CNS in wt but not in IL-6-/- mice. Taking into account recent studies on the role of VCAM-1 in the entry of Th1 cells into the CNS, the absence of VCAM-1 on endothelial cells in IL-6-/- mice may explain their resistance to EAE.     

Inheritance of susceptibility to experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis is a disease of the central nervous system that can be readily induced in a variety of species by immunization with myelin proteins. It is one of the most commonly studied models of cell-mediated autoimmune disease and consequently has been important in the elucidation of many immunological functions in vivo. Nevertheless, very little is understood about the genetic and environmental factors that control the disease. Several groups have begun undertaking systematic genetic analyses in mice to identify loci that associate with increased susceptibility to disease. In this review, we will summarize the work that has been done to understand the genetic predisposition that makes certain inbred animals susceptible to disease and others resistant, and we will discuss some of the difficulties in the genetic analysis that have arisen due to the complexities of this disease model.     

CD28-deficient mice are highly resistant to collagen-induced arthritis

OBJECTIVES: The understanding of testicular histology in acquired immunodeficiency syndrome (AIDS) is essential, because the sexual route is one of the main means of transmission of the human immunodeficiency virus, which is localized primarily in the germ cells of the testes. It is important to determine whether any changes have occurred in the testicular histologic patterns in the course of the AIDS epidemic. METHODS: One hundred forty testicular specimens were available from AIDS autopsies during the AIDS epidemic (1981 to 1998). The epidemic was divided into pre-zidovudine (pre-AZT) therapy (1981 to 1987) and antiviral therapy (1988 to 1998) periods; the latter period was further subdivided on the basis of the particular treatment used. Testicular histology was evaluated and correlated with patient age, CD4 T-cell counts, and pathologic findings in other parts of the body. RESULTS: Testicular histologic findings were categorized into three groups: hypospermatogenesis (group S), spermatogenic arrest (group A), and Sertoli cell only (group O). The percentage of AIDS patients with group S histologic findings remained constant throughout the study period: 26% in the pre-AZT and 28% in the antiviral therapy periods. However, there was a reversal in the percentages of patients in groups A and O: group A decreased from 48% (pre-AZT) to 28% (antiviral) and group O increased from 26% (pre-AZT) to 44% (antiviral). There was no correlation between testicular histologic results and patient age or CD4 count. Opportunistic infections and testicular neoplasms were also identified. CONCLUSIONS: This study demonstrates that current therapy and prolongation of survival in AIDS patients are associated with a shift in the histologic findings of testes toward a more pronounced loss of germ cells. However, 28% of patients still show significant spermatogenesis at the time of death from AIDS and this subgroup cannot be identified by age or CD4 T-cell counts. The presence of large numbers of residual germ cells in these patients suggests that they may continue to be infectious throughout their disease course.     

Interleukin-6 functions in autoimmune encephalomyelitis: a study in gene-targeted mice

The encephalitogenic peptide pMOG 35-55 from the myelin oligodendrocyte glycoprotein was used to induce experimental autoimmune encephalomyelitis (EAE) in H-2b mice with the interleukin-6 (IL-6) gene intact or disrupted. The IL-6+/+ mice developed a chronic form of EAE ascending paralysis, whereas the IL-6-/- mice were resistant to the disease. Injections of recombinant IL-6 following pMOG immunization induced severe disease in the IL-6-/- mice. Histological examination of brain and spinal cord sections showed that the perivascular infiltration of inflammatory cells evident in IL-6+/+ mice was absent in the IL-6-/- animals and could be restored by exogenous IL-6 administration. Anti-MOG antibody levels were much lower in the IL-6-/- mice, but were not restored to high levels by IL-6 injections which elicited the development of pMOG 35-55-induced EAE. T lymphocytes reactive to the pMOG antigen were recovered from lymph nodes of both types of mice and Tcell lines could be established from both. Adoptive transfer of Tcell lines from IL-6+/+ mice induced EAE in the mice with the intact IL-6 gene but less in the IL-6-deficient mice, indicating that the resistant phenotype cannot be explained solely by lack of encephalitogenic Tcells. The absence of cell infiltrates in the brain and spinal cords of IL-6-/- mice upon adoptive transfer of the pathogenic Tcells from IL-6+/+ mice is consistent with a function of IL-6 in the local perivascular inflammatory process.     

Experimental autoimmune myasthenia gravis induction in B cell-deficient mice

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG). Autoantibody-induced functional loss of nicotinic acetylcholine receptor (AChR) at the postsynaptic membrane is an important pathogenic feature of both MG and EAMG. To evaluate the extent at which the humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B cell knockout (muMT) and wild-type C57BL/6 mice with AChR and complete Freund's adjuvant. The ability of AChR-primed lymph node cells to proliferate and secrete IFN-gamma in response to AChR and its dominant peptide alpha146-162 were intact in muMT mice as in wild-type mice. Similar amounts of mRNA for IFN-gamma, IL-4 and IL-10 in AChR-reactive lymph node cells were detected in muMT and wild-type mice. However, muMT mice had no detectable anti-AChR antibodies and remained completely free from clinical EAMG. We conclude that B cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T cell priming.     

Neutralizing antibodies to IFN-gamma-inducing factor prevent experimental autoimmune encephalomyelitis

Specific oligonucleotide primers were used to identify and isolate IFN-gamma-inducing factor (IGIF) from the brain of rats with developing experimental autoimmune encephalomyelitis (EAE), a T cell-mediated autoimmune disease of the central nervous system that serves as a model for multiple sclerosis. IGIF was highly transcribed in the brain at the onset and during the course of active EAE. PCR products encoding rat IGIF were used to generate the recombinant protein that was used to induce anti-IGIF neutralizing Abs. These Abs significantly reduced the production of IFN-gamma by primed T cells proliferating in response to their target myelin basic protein epitope and by Con A-activated T cells from naive donors. When administered to rats during the development of either active or transferred EAE, these Abs significantly blocked the development of disease. Splenic T cells from protected rats were cultured with the encephalitogenic myelin basic protein epitope and evaluated for production of IL-4 and IFN-gamma. These cells, which proliferated, exhibited a profound increase in IL-4 production that was accompanied by a significant decrease in IFN-gamma and TNF-alpha production. Thus, we suggest that perturbation of the Th1/Th2 balance toward Th2 cells is the mechanism underlying EAE blockade by anti-IGIF immunotherapy.     

Lewis rats given antibodies against denatured acetylcholine receptor become resistant to induction of experimental autoimmune myasthenia gravis

The embryotoxic and dysmorphogenic effects of mercuric chloride (HgCl2) have been studied in mouse embryos cultured in vitro. In addition, the alterations induced in the developing branchial nerves and ganglia were analyzed. Mouse embryos with 6-8 pairs of somites were exposed for 26 hr to increasing concentrations (0, 12.5, 25, 50 microM) of HgCl2. After this period, a first set of embryos was removed and a second set of embryos transferred to culture medium without HgCl2 and remained in culture for an additional 22 hr. Both sets of embryos were examined for (1) survival, (2) presence of external dysmorphogenesis, (3) growth, and (4) differentiation. Dose-related alterations of these parameters were observed. The main target was the cephalic neural tube (mainly the forebrain), but several other systems were also affected (e.g., the turning of the embryos, the optic system). The 48-hr cultured embryos were immunostained using a monoclonal antineurofilament antibody to visualize defects in the development of branchial nerves and ganglia. HgCl2 induced a pronounced retardation in the differentiation of ganglion/nerve V and a slight retardation in the differentiation of ganglia/nerves VII and IX. The ganglia/nerves VIII and X were not retarded. In addition, hight percentages of abnormalities of ganglion/nerve V and fusions between ganglia/nerves IX and X were observed in these embryos. Disorganized fibers between ganglia/nerves VII-VIII and IX and between ganglia/nerves IX and X were also more frequently observed. At the highest concentration, asymmetric defects were induced by HgCl2 with a more pronounced effect observed on the right side of the embryos. These results demonstrate the usefulness of this approach in evaluating the susceptibility of the developing branchial nerves to the adverse effects of developmental toxicants.     

Challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient, mice

Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTalpha-/- mice, TNF-/- mice, and a new LTalpha-/- line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTalpha gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTalpha with soluble TNF receptor in unmanipulated wild-type or TNF-/- mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.     

A new strategy for treatment of autoimmune diseases in chimeric resistant MRL/lpr mice

A new strategy for the treatment of autoimmune diseases in chimeric resistant MRL/lpr mice is established. The strategy includes injection of cyclophosphamide (CY), fractionated irradiation (5 Gy x 2), bone grafts (to recruit stromal cells), and two transplantations of whole bone marrow cells (WBMCs) from allogeneic normal C57BL/6 mice (CY/2X/Bone/2BMT). MRL/lpr mice, thus treated, survived more than 40 weeks (1 mouse survived for >40 weeks, 7 for >50 weeks, and 4 for >60 weeks after these treatments). Immunohistological studies showed that the mice were completely free from both lymphadenopathy and autoimmune diseases such as systemic lupus erythematosis and rheumatoid arthritis. The levels of autoantibodies (IgM/IgG rheumatoid factors and IgM/IgG anti-ssDNA antibodies [Abs]) in the treated mice decreased to those in the normal mice. In addition, successful cooperation among T cells, B cells, and antigen-presenting cells (APCs) was observed. Abnormal T cells with immunophenotypes of B220+/Thy-1+/CD3+/CD4-/CD8- present in untreated MRL/lpr mice disappeared, and the hematolymphoid cells of the treated mice were of donor origin. However, the mice that had been irradiated with 8.5 Gy and then reconstituted with T-cell-depleted BMCs plus bone grafts died within 2 weeks due to the side effect of irradiation. The depletion of CD8+ cells (not CD4+ cells) from WBMCs resulted in graft failure; 60% of the recipient mice, thus treated, died within 2 weeks, and all recipients died by 15 weeks. Furthermore, limiting dilution assays showed that approximately more than 0.5% of T cells contained in the BMCs are necessary not only for engraftment of BMCs but also for long-term disease-free survival of the recipients. In contrast, recipients that had received CD4-depleted BMCs with CY plus fractionated irradiation (5Gy x 2) survived for more than 40 weeks without showing graft-versus-host reaction (GVHR). This indicates that CD8(+)cells in the BMCs are essential for the successful engraftment of the donor-type hematolymphoid cells.     

Non-obese diabetic (NOD) mice are genetically susceptible to experimental autoimmune prostatitis (EAP)

Rodents develop inflammatory, non-infectious, prostatitis upon autoimmuniz-ation with male accessory gland (MAG) extracts in complete Freund's adjuvant (CFA). Although there appears to be differences among strains, with respect to susceptibility to induction, specific details are not known about the genetic bases of such differences. Because NOD mice have inherited a genetic predisposition to autoimmune lesions affecting, apart from the islets of Langerhans, a large array of secretory glands such as salivary glands, thyroid, parathyroids and adrenal cortex, we selected this strain to assess the influence of inherited genes upon experimentally-induced autoimmune prostatitis (EAP). Indeed, MAG extracts injected into young NOD males in association with CFA cause a severe inflammatory reaction in the prostate, accompanied by a humoral and T cell-mediated response. NOD mice develop a more aggressive form of EAP than Wistar rats, the strain of reference used to establish the model. In NOD mice, disease begins earlier, affects 100% of the animals, does not require boosting and leads to florid infiltrates circumscribed to lateral and dorsal prostatic lobes. Immune mice develop a T cell-mediated response to MAG assessed by in vitro proliferation and accompanied by the release of IFN-gamma, whereas IL-4 is not detectable in the same culture super-natants. To assess the influence of the NOD background genes upon EAP susceptibility, we tested C57BL/6.H2(g7) mice in parallel. NOD mice are considerably more susceptible to EAP induction than congenic C57BL/6.H2(g7) mice. Both strains demonstrate a detectable humoral and cell-mediated response against MAG, but the histopathological manifestations are considerably more dramatic in NOD than in the C57BL/6.H2(g7) strain. Our results thus support the notion that NOD mice have background genes which favour severe autoimmune manifestations, irrespective of the target tissue.     

Humane endpoints are an objective measure of morbidity in Venezuelan encephalomyelitis virus infection of mice

The clinical signs are described of Venezuelan encephalomyelitis virus (VEEV) infection in mice after both airborne and subcutaneous (s.c.) challenge. Group clinical scores reflected the known pathogenesis of infection by both s.c. and airborne challenge, and with epizootic and enzootic strains of VEEV. This observation confirms the specific relationship of the observed clinical signs to VEEV infection. Within an experiment, those who are assessing the animals for clinical signs must have a common understanding of their appearance, including severity, and should be unaware of the allocation of treatments. If these conditions are met, the progress of clinical signs may be used to determine objectively the time of culling for humane endpoints.     

E- and P-selectin are not involved in the recruitment of inflammatory cells across the blood-brain barrier in experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE) inflammatory cells cross the endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). Here we show that E- and P-selectin are not involved in the recruitment of inflammatory cells across the BBB. Neither expression of E- nor P-selectin is induced in BBB-forming endothelium at any time after initiation of EAE. Some of the inflammatory cells present in the CNS during EAE express ligands for E- or P-selectin. However, anti-E- and P-selectin antibodies influence neither immigration of inflammatory cells across the BBB nor the development of EAE. In general, suppression of E- and P-selectin expression on BBB endothelium is dependent on factors derived from the CNS microenvironment, eg, astrocytes. Our results suggest that during EAE suppression of E- and P-selectin expression on the BBB provides a CNS-specific mechanism to reduce leukocyte recruitment into the CNS.     

Suppression of experimental autoimmune encephalomyelitis by tyrphostin AG-556

Tyrosine kinase blockers from the AG 126/AG-556 tyrphostin family are shown to inhibit the lipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha (TNFalpha), nitric oxide (NO), and prostaglandin E2 (PGE2) in primary rat astrocytes cultures. The tyrphostin AG-556 which was previously shown to be effective against sepsis in mice and dogs also show excellent efficacy in inhibiting experimental autoimmune encephalomyelitis (EAE) in mice. AG-556 does not block the activation of JNK/SAPK and of p38/HOG and therefore seems to act at a target down stream to these kinases which is activated in stress or at a target on an obligatory parallel pathway. These findings together with previous results showing inhibition of sepsis in mice and dogs suggest that protein tyrosine kinase (PTK) blockers of the AG-556 family may be considered in the management of human autoimmune disorders such as multiple sclerosis (MS).