Experimental allergic orchitis in mice. V. Resistance to actively induced disease in BALB/cJ substrain mice is mediated by CD4 + T cells

Previous studies have shown that differential susceptibility to actively induced experimental allergic orchitis (EAO) exists among various BALB/c substrains. Of 13 substrains studied, BALB/cJ mice consistently exhibit greater resistance to disease induction. Such resistance is associated with a single recessive genotypic difference in an immunoregulatory locus which is unlinked to any of the known alleles distinguishing the BALB/cJ substrain. In this study, gene complementation protocols were used to study the genetics of susceptibility and resistance to EAO. The results indicate that resistance in BALB/cJ mice is not due to a mutation in theH-2D ^d linked gene which governs the phenotypic expression of autoimmune orchitis. The mechanistic basis for disease resistance was examined using reciprocal bone marrow radiation chimeras generated between the disease-susceptible BALB/ cByJ (ByJ) substrain and BALB/cJ (Jax) mice. All constructs, including Jax - Jax and Jax - ByJ, developed severe EAO following inoculation with mouse testicular homogenate (MTH) and adjuvants whereas control chimeras immunized with adjuvants alone did not. These results suggest that an active immunoregulatory mechanism rather than a passive one, such as the lack of T cells and/or B cells with receptors for the aspermatogenic autoantigens relevant in the induction of EAO, is responsible for disease resistance in BALB/cJ mice. The role of immunoregulatory cells was examined by pretreating BALB/cJ mice with either cyclophosphamide (20 mg/kg) or low-dose whole body or total lymphoid irradiation (350 rads) 2 days prior to inoculation. BALB/cJ mice immunized with MTH plus adjuvants generate immunoregulatory spleen cells (SpCs) that, when transferred to naive BALB/cByJ recipients, significantly reduce the severity of autoimmune orchitis observed during actively induced EAO. Treatment of such cells with either cytotoxic monoclonal anti-Thy-1.2 or anti-CD4 plus C' before transfer abrogates the ability of BALB/cJ spleen cells to inhibit disease. In contrast, neither SpCs from adjuvantimmunized BALB/cJ nor MTH plus adjuvant-primed BALB/cByJ donors significantly influenced the severity of disease observed in recipients. Taken together, these results suggest that genetically controlled resistance to EAO in BALB/cJ mice is associated with a mutation in an immunoregulatory locus whose effects appear to be mediated through a cyclophosphamide and low-dose radiation-sensitive CD4⁺ T-cell population.     

Experimental allergic orchitis in mice

Inbred strains of mice were studied for their susceptibility to the induction of experimental allergic orchitis after sensitization with mouse testicular homogenate in complete Freund's adjuvant accompanied by injections of extract from Bordetella pertussis. Susceptibility to autoimmune orchitis was found to be linked to the major histocompatibility complex in BALB/c and C57BL/10 mice and mapped to genes encoded within the H-2D ^dregion. In five of six groups of bidirectional (susceptible × resistant) F₁ hybrids, H-2D ^d-linked susceptibility was inherited as a dominant autosomal trait. However, in (BALB/cByJ × DBA/2J)F₁ and (DBA/2J × BALB/cByJ)F₁ hybrids, dominant autosomal resistance to the induction of autoimmune orchitis was observed. Backcross analysis between the resistant F₁ hybrid and the susceptible BALB/cByJ parent suggests that a single independently segregating DBA/2J locus is capable of negating H-2D ^d-linked susceptibility, and controls resistance to the induction of autoimmune orchitis.Abbreviations used in this paper BP extract Bordetella pertussis extract - CFA complete Freund's adjuvant - EAO experimental allergic orchitis - Ir immune response - MHC major histocompatibility complex - MLH mouse liver homogenate - MTH mouse testis homogenate - PI pathology index     

Actively induced experimental allergic orchitis in Lewis-resistant (Le-R) rats: reversibility of disease resistance by immunization with Bordetella pertussis

Differential susceptibility to the induction of experimental allergic orchitis (EAO) was examined in Lewis/NCr and Le-R subline rats. Lewis/NCr rats were found to be fully susceptible to the induction of EAO whereas Le-R subline rats were not. Disease resistance exhibited by Le-R rats could be overcome by including Bordetella pertussis in the immunization protocol. However, reversal of resistance with B. pertussis was dependent on the dose of rat testicular homogenate in the inoculum and found to be effective only at lower doses of antigen (10 mg/rat). Disease resistance in Le-R rats as well as B. pertussis-induced reversal of resistance did not appear to be associated with either (1) a significant difference in the number of mast cells in the ductus efferentes, the anatomic location of the earliest inflammatory infiltrates, or (2) an alteration in the phenotypic expression of either innate or B. pertussis-induced sensitivity to vasoactive amines. The results are discussed in the context of the role of B. pertussis in other animal models of organ-specific autoimmune diseases. It is proposed that the phenotypic expression of resistance to EAO in Le-R rats is a result of a mutation in a common regulatory locus affecting susceptibility to multiple autoimmune diseases and whose immunoregulatory action is normally exerted during the sensitization phase of the immune response.     

Genetic analysis of the influence of pertussis toxin on experimental allergic encephalomyelitis susceptibility: an environmental agent can override genetic checkpoints

Pertussis toxin (PTX) is a potent ancillary adjuvant used to elicit several different autoimmune diseases, including experimental allergic encephalomyelitis (EAE). To delineate the genetics of PTX effect in EAE, we mapped EAE-modifying (eae-m) loci in cohorts of backcross mice immunized with and without PTX. In this study, we analyzed the genetic basis of EAE susceptibility and severity and the intermediate phenotypes of mononuclear cell infiltration, suppuration, and demyelination. In animals immunized with PTX, one major locus, eae9, controls disease susceptibility and severity. Eae9 also regulates the extent of mononuclear cell infiltration of the spinal cord in male mice. Without PTX, five eae-m loci were noted, including three new loci in intervals on chromosomes 8 (eae14), 10 (eae17), and 18 (eae18). Taken together, these results suggest that eae9 controls the effects of PTX in EAE susceptibility, and is capable of overriding the other genetic checkpoints in the pathogenesis of this disease.     

Myelin proteolipid protein-induced experimental allergic encephalomyelitis. Variations of disease expression in different strains of mice

Strains of mice with diverse genetic backgrounds were tested for susceptibility to experimental allergic encephalomyelitis (EAE) induced by myelin proteolipid protein. EAE was elicited in all strains of mice tested, but the clinical and histologic features varied. SJL (H-2s) mice had a high incidence of both clinical and histologic disease characterized by early onset of clinical signs. Inguinal lymph node T cells from diseased animals responded specifically [( 3H]thymidine incorporation) to proteolipid protein and not to myelin basic protein. In contrast, BALB/c (H-2d), DBA/1 (H-2q), C57BL/6 (H-2b), AKR (H-2k), CBA (H-2k), C3H (H-2k), B10.BR (H-2k), and C57BR (H-2k) mice showed a later onset of clinical signs and typically a lower disease incidence. However, the most marked variations in disease incidence occurred among BALB/c (H-2d) substrains in which the incidence of EAE ranged from eight of nine (BALB/cPt) to complete resistance (BALB/cWt and BALB/cORNL). Because these BALB/c substrains were initially derived from the same inbred genetic source and are serologically identical at H-2, these results suggest that expression of proteolipid protein-induced EAE in the mouse involves additional loci outside the MHC.     

Adoptive transfer of experimental allergic encephalomyelitis in mice with the aid of pertussigen from Bordetella pertussis

Adoptive transfer of experimental allergic encephalomyelitis (EAE) in (SJL X BALB/c)F1 mice was accomplished by an iv injection of 2.4 to 4.7 X 10(7) lymph node cells (LNC) from mice immunized with mouse spinal cord emulsified in complete Freund's adjuvant when both donors and recipients had been treated iv with 400 ng of pertussigen at the time of immunization for the donors and on transfer of cells for the recipients. Pertussigen was essential in both donors and recipients for development of frank EAE. Signs of EAE in recipients were delayed, appearing 21-23 days after cell transfer; the maximum response at about Day 27 is considerably delayed in comparison with other reported studies on passive transfer of EAE. Histologically, recipient mice with paralysis due to EAE had typical perivascular infiltrates of mononuclear cells in the brain and spinal cord. The mechanisms by which pertussigen promotes the development of EAE after adoptive transfer of sensitized LNC are uncertain.     

Comparison of allergic lung disease in three mouse strains after systemic or mucosal sensitization with ovalbumin antigen

Murine models of allergic lung disease have many similar traits to asthma in humans and can be used to investigate mechanisms of allergic sensitization and susceptibility factors associated with disease severity. The purpose of this study was to determine strain differences in allergic airway inflammation, immunoglobulin production, and changes in respiratory responses between systemic and mucosal sensitization routes in BALB/cJ, FVB/NJ, and C57BL/6J, and to provide correlations between immune and pathophysiological endpoints. After a single intranasal ovalbumin (OVA) challenge, all three strains of mice systemically sensitized with OVA and adjuvant exhibited higher airflow limitation than non-sensitized mice. No changes were seen in mice that were pre-sensitized via the nose with OVA. Systemic sensitization resulted in an elevated response to methacholine (MCH) in BALB/cJ and FVB/NJ mice and elevated total and OVA-specific IgE levels and pulmonary eosinophils in all three strains. The mucosal sensitization and challenge produced weaker responses in the same general pattern with the C57BL/6J strain producing less serum IgE, IL5, IL13, and eosinophils in lung fluid than the other two strains. The converse was found for IL6 where the C57BL/6J mice had more than twice the amount of this cytokine. The results show that the FVB/NJ and BALB/cJ mice are higher Th2-responders than the C57BL/6J mice and that the levels of pulmonary eosinophilia and cytokines did not fully track with MCH responsiveness. These differences illustrate the need to assess multiple endpoints to provide clearer associations between immune responses and type and severity of allergic lung disease.     

Haplotype-specific suppression of experimental allergic encephalomyelitis with anti-IA antibodies

Treatment with monoclonal antibodies directed against the IA antigens of the MHC is known to alter the course and prevent a number of experimental autoimmune diseases. To determine whether the treatment in vivo with anti-IA antibodies is haplotype-specific, we studied the development of EAE in F1 (SJL/J X BALB/c) mice following anti-IA antibody therapy. We report that treatment of animals with monoclonal antibody directed against the high responder allele product, I-As, was successful in preventing disease when therapy was begun either at the time of immunization with antigen, or following passive transfer of MBP-sensitized T cells. Therapy with antibody directed to the low responder allele product (I-Ad), while effective when used at the time of immunization with antigen, was ineffective following passive transfer of MBP-sensitized lymphocytes.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Relapsing murine experimental allergic encephalomyelitis induced by myelin basic protein

Experimental allergic encephalomyelitis (EAE), an experimental autoimmune disease of the central nervous system (CNS), is readily induced in many mammalian species by immunization with CNS tissue or myelin basic protein (MBP) purified from the CNS. EAE has been frequently used as a model for multiple sclerosis (MS). However, EAE generally presents as an acute monophasic disease in the adult animal after immunization with MBP. After recovery, the animal is resistant to rechallenge with encephalitogen (1). Two exceptions to these observations have been reported. McFarlin et al. (2) reported that a variable number of Lewis rats showed signs of a single, mild relapse about a week after recovery from MBP-induced acute EAE. Panitch and Ciccone (3) have reported induction of recurrent EAE in rats immunized with human MBP. Chronic, relapsing EAE has been induced in the mouse; however, an apparent requirement for CNS tissue had been noted (4, 5). Recently, during the course of a series of experiments on the induction of EAE in SJL/J, PL/J, and (SJL/J X PL/J)F1 (SPL F1) mice, it was observed that the F1 mice frequently had paralytic relapses after recovery from MBP-induced symptoms. Experiments were initiated to examine this phenomenon, and the findings are presented below.     

Activation of regulatory cells suppresses experimental allergic encephalomyelitis via secretion of IL-10

Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.     

In trans T cell tolerance diminishes autoantibody responses and exacerbates experimental allergic encephalomyelitis

A number of Ag-specific approaches have been developed that ameliorate experimental allergic encephalomyelitis (EAE), an animal model for the human autoimmune disease multiple sclerosis. Translation to humans, however, remains a consideration, justifying the search for more insight into the mechanism underlying restoration of self-tolerance. Ig-proteolipid protein (PLP) 1 and Ig-myelin oligodendrocyte glycoprotein (MOG) are Ig chimeras carrying the encephalitogenic PLP 139-151 and MOG 35-55 amino acid sequence, respectively. Ig-PLP1 ameliorates EAE in SJL/J (H-2(s)) mice while Ig-MOG modulates the disease in C57BL/6 (H-2(b)) animals. In this study, we asked whether the chimeras would suppress EAE in F(1) mice expressing both parental MHC alleles and representing a polymorphism with more relevance to human circumstances. The results show that Ig-MOG modulates both PLP1 and MOG peptide-induced EAE in the F(1) mice, whereas Ig-PLP1 counters PLP1 EAE but exacerbates MOG-induced disease. This in trans aggravation of MOG EAE by Ig-PLP1 operates through induction of PLP1-specific T cells producing IL-5 that sustained inhibition of MOG-specific Abs leading to exacerbation of EAE. Thus, in trans T cell tolerance, which should be operative in polymorphic systems, can aggravate rather than ameliorate autoimmunity. This phenomenon possibly takes place through interference with protective humoral immunity.     

Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-gamma

Acute experimental allergic encephalomyelitis (EAE) was induced in C57BL/6J and SJL/J mice by injection of isologous spinal cord homogenate given in conjunction with Bordetella pertussis and Freund's adjuvant. SJL/J mice showed a highly aggressive and 100% lethal form of the disease; C57BL/6J mice were much less susceptible as they had low morbidity rates (20 to 40%), low disease scores, and mostly no mortality. Treatment of these low susceptibility mice with neutralizing mAb against IFN-gamma caused an increase in morbidity rates as well as significant mortality (up to 80%). Similar antibody treatment did not affect the course of the disease in the high susceptibility SJL/J mice. However, treatment of these mice with IFN-gamma resulted in reduced morbidity and mortality. A similar but less pronounced inhibition of the disease in SJL/J mice could be obtained by administration of IFN-alpha/beta or by acute infection with lactate dehydrogenase virus. The results indicate that endogenous as well as exogenous IFN can exert a down-regulating effect on the development of EAE. They also indicate that endogenous IFN-gamma is produced during the development of EAE and plays a disease-limiting role.     

Genetic control of susceptibility to experimental allergic encephalomyelitis in mice

The genetic control of susceptibility to experimental allergic encephalomyelitis (EAE) was studied in mice. The results indicate that sensitivity to disease is not inherited in a simple Mendelian dominant way. Susceptibility to EAE is governed by genes located outside of the major histocompatibility complex and not byH-2-linkedIr genes. No correlation was observed between susceptibility to disease and the cellular immune response toward the small encephalitogenic protein.     

Central nervous system-targeted expression of the complement inhibitor sCrry prevents experimental allergic encephalomyelitis

Although generally thought of as a T cell-driven autoimmune disease, recent studies in experimental allergic encephalomyelitis (EAE), the animal model of multiple sclerosis, suggest a significant role for innate immune mechanisms. To address the possibility that the complement system plays a central role in these diseases, we developed a transgenic mouse with astrocyte-targeted production of a soluble inhibitor of complement activation, complement receptor-related protein y (sCrry). Here, we show that sCrry transgenic mice are either fully protected against EAE or develop significantly delayed clinical signs. These results indicate that complement activation may have an essential role in the pathogenesis of the disease and that complement-mediated events may occur early during the effector phase of EAE. Furthermore, this work underscores the importance of humoral immunity in amplifying a T cell-initiated pathogenic process.     

Exacerbation of experimental autoimmune encephalomyelitis in P2X7R-/- mice: evidence for loss of apoptotic activity in lymphocytes

The purinergic receptor P2X7R is a nucleotide-gated ion channel that has been proposed to function as a major regulator of inflammation. In this study we examined the role of this receptor in regulating inflammation in the CNS by determining the effects of the loss of this receptor (P2X7R-/-) on experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We show here that P2X7R-/- mice developed more severe clinical and pathological expression of EAE than wild type (WT) controls and that spleen and lymph node cells from P2X7R-/- mice proliferated more vigorously to Ag in vitro. Bone marrow (BM) radiation chimeras revealed that enhanced susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R-/- BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. Coculture of P2X7R-/- macrophages with WT lymphocytes and vice versa showed that enhanced proliferative activity resided within the P2X7R-/- lymphocyte population and correlated with reduced levels of IFN-gamma and NO and apoptosis of lymphocytes. mRNA and protein for IFN-gamma were also significantly reduced in the CNS of P2X7R-/- mice with EAE. FACS analysis of cells isolated from the CNS showed significantly fewer annexin V/propidium iodide-positive lymphocytes in the CNS of P2X7R-/- mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. From these data we conclude that enhanced susceptibility of P2X7R-/- mice to EAE reflects a loss of apoptotic activity in lymphocytes, supporting an important role for this receptor in lymphocyte homeostasis.     

Phenotypic instability between the near isogenic substrains BALB/cJ and BALB/cByJ

Closely related substrains of inbred mice often show phenotypic differences that are presumed to be caused by recent mutations. The substrains BALB/cJ and BALB/cByJ, which were separated in 1935, have been reported to show numerous highly significant behavioral and morphological differences. In an effort to identify some of the causal mutations, we phenotyped BALB/cJ and BALB/cByJ mice as well as their F1, F2, and N2 progeny for behavioral and morphological phenotypes. We also generated whole-genome sequence data for both inbred strains (~3.5× coverage) with the intention of identifying polymorphic markers to be used for linkage analysis. We observed significant differences in body weight, the weight of the heart, liver, spleen and brain, and corpus callosum length between the two substrains. We also observed that BALB/cJ animals showed greater anxiety-like behavior in the open field test, less depression-like behavior in the tail suspension test, and reduced aggression compared to BALB/cByJ mice. Some but not all of these physiological and behavioral results were inconsistent with prior publications. These inconsistencies led us to suspect that the differences were due to, or modified by, non-genetic factors. Thus, we did not perform linkage analysis. We provide a comprehensive summary of the prior literature about phenotypic differences between these substrains as well as our current findings. We conclude that many differences between these strains are unstable and therefore ill-suited to linkage analysis; the source of this instability is unclear. We discuss the broader implications of these observations for the design of future studies.