Cory Teuscher

Differential susceptibility to actively induced experimental allergic encephalomyelitis and experimental allergic orchitis among BALB/c substrains

Experimental allergic orchitis (EAO) and experimental allergic encephalomyelitis (EAE) are animal models of organ-specific autoimmune disease. In this study, BALB/cByJ and BALB/cAnNCr mice were susceptible to both autoimmune diseases whereas BALB/cJ subline mice were resistant. Disease resistance in BALB/cJ mice did not appear to be a reflection of either (i) a nonspecific generalized impairment of cellular immunity or (ii) an alteration in the phenotypic expression of Bordetella pertussis-induced histamine sensitization, a phenotype which has been shown to be associated with susceptibility to both diseases. Susceptibility to both EAE and EAO was inherited as a dominant trait in F1 hybrid animals. Segregation analysis in a (BALB/cByJ X BALB/cJ) X BALB/cJ backcross population suggested that disease resistance may be associated with a single genotypic difference in a common regulatory gene affecting susceptibility to both diseases. Linkage analysis of the backcross population failed to demonstrate an association of disease resistance with the mutant raf-1b allele carried by BALB/cJ mice. The results of these studies support previous observations that multiple genotypic differences may in fact exist in mice of the BALB/cJ subline and that such differences play a significant role in the genetic control of susceptibility to EAE and EAO.

Experimental allergic orchitis in mice. II: Association of disease susceptibility with the locus controlling Bordetella pertussis-induced sensitivity to histamine

Susceptibility to the induction of murine autoimmune orchitis was found to be associated with the locus controlling Bordetella pertussis-induced sensitivity to the vasoactive amine, histamine. Only those inbred and H-2 congenic strains of mice possessing both the H-2d haplotype and the locus for susceptibility to B. pertussis-induced sensitivity to histamine developed autoimmune orchitis. In addition, segregation analysis of backcross generation mice also demonstrated a high degree of correlation between susceptibility both to disease and to histamine sensitization, which was indicative of additional multigene control. Pertussigen-histamine sensitization factor (P-HSF) was only effective in eliciting disease when it was administered on the same day, or within a period up to 6 days following sensitization with mouse testicular homogenate-emulsified in complete Freunds adjuvant. P-HSF induced sensitivity to histamine was not found to be associated with an increase in the vascular permeability of target tissue. Thus, B. pertussis-induced sensitivity to histamine appears to play a more crucial role during the sensitization phase of autoimmune orchitis induction, rather than at the inflammatory or effector phase of the disease.

An analysis of the role of tumor necrosis factor in the phenotypic expression of actively induced experimental allergic orchitis and experimental allergic encephalomyelitis

The role of tumor necrosis factor (TNF) was examined in the pathogenesis of actively induced experimental allergic orchitis (EAO) and experimental allergic encephalomyelitis (EAE) in the mouse. The ability of TNF to function as either an adjuvant or to replace pertussigen in eliciting active EAO was examined by treating groups of mice immunized for disease induction with 10 micrograms of recombinant murine TNF at various time points throughout both the induction and effector phases of the disease process. All groups of animals receiving TNF ranging from 2 days before antigen challenge to 26 days postimmunization failed to exhibit significant disease in comparison to animals treated with pertussigen, indicating that TNF can neither serve as an adjuvant nor replace pertussigen in eliciting active disease. Similarly, the role of TNF in the pathogenesis of EAO and EAE was investigated by examining the ability of a known neutralizing rabbit anti-TNF IgG antibody preparation to either inhibit the development or decrease the severity of the clinical symptoms and/or the inflammatory lesions associated with the disease processes. Groups of either B6AF1 hybrid or SJL/J mice were immunized for the induction of active EAO and EAE, respectively. They were passively immunized with either 2 mg of purified anti-TNF IgG or control anti-CFA IgG at time points ranging from 2 days before to 28 days after antigen challenge. All groups, regardless of the day of treatment with anti-TNF IgG, did not exhibit a markedly significant difference in disease outcome in comparison to either groups receiving no antibody or passively immunized with anti-CFA IgG. Taken together, these results suggest that TNF does not appear to be the principal cytokine/lymphokine involved in the pathogenesis of actively induced EAO and EAE.

Experimental allergic orchitis in mice. VI. Recombinations within the H-2S/H-2D interval define the map position of the H-2-associated locus controlling disease susceptibility

Susceptibility to autoimmune orchitis is associated with an immune response (Ir) gene (now designated Orch-1) which was preliminarily shown to reside at or near the H-2D subregion of the major histocompatibility complex in the mouse (H-2). In this study, the role of H-2 in controlling both disease susceptibility and the phenotypic expression of infertility associated with autoimmune orchitis has been significantly extended. Of nine C57BL/10SnJ and three BALB/cAnN H-2 congenic strains, only those mice possessing the H-2d, H-2p haplotypes exhibited autoimmune orchitis accompanied by infertility. All other congenic strains, including those expressing the H-2 haplotypes v, q, b, s, r, f, and k were of the low responder phenotype. In addition, disease susceptibility was found to be inherited as a dominant trait in H-2 congenic F1 hybrid mice. In order to map the precise location of the Orch-1 locus within H-2, 32 intra-H-2 recombinant congenic strains possessing defined crossovers in various locations throughout the H-2 region were studied. The results of the analysis indicate that Orch-1 maps within the interval between the H-2S and H-2D regions. Our results also indicate that class II genes, i.e., A and E region-encoded genes, have little discernable effect in controlling disease susceptibility and resistance despite the fact that testicular lesions can be adoptively transferred with Ia-restricted CD4+ effector T cells. A comparison of the Orch-1 alleles with the genotypes of two additional markers which map within the H-2S/H-2D interval suggests the following gene order: H-2S--TNP-Ficoll--Orch-1--Tnfa--H-2D.

Actively-Induced Experimental Allergic Orchitis (EAO) in Lewis/Ncr Rats: Sequential Histo- and Immunopathologic Analysis

Active experimental allergic orchitis (EAO) was induced in Lewis/NCr rats by immunization with homologous rat testicular homogenate. Groups of animals were studied sequentially at five day intervals for histopathologic signs of disease. Inflammatory lesions were first observed in the ductus efferentes as early as 5 days following immunization. Immunohistochemical analysis of the testes, rete testis, ductus efferentes and caput, corpus and cauda epididymis of immunized rats on day five revealed that only the ductus efferentes exhibited a significant increase in the number of interstitial cells expressing Ia antigens (MRC OX-6) as well as CD4 (W3/25) positive helper/inducer T lymphocytes, CD8 (MRC OX-8) positive cytotoxic T lymphocytes and/or natural killer cells and macrophages (MRC OX-42). Increased staining for Ia antigens was also associated with both the vascular and ductal epithelial cells whereas cells within the lumen of the ducts were consistently negative for Ia antigen expression. In contrast, there was no detectable increase in the level of expression of rat MHC class I antigens (MRC OX-18) by any of the cells of the ductus efferentes. Similarly, there was no increase in the number of MAR 18.5 and/or MRC OX-12 positive B lymphocytes. By day 15, autoimmune epididymitis was observed in the cauda and corpus epididymis with the caput becoming involved by day 20. In the testes, the first histopathologic changes observed were scattered inflammatory infiltrates on day 15 and scattered foci of aspermatogenesis on day 20. Inflammatory lesions were first seen in the rete testis and the seminiferous tubules on day 25-30 with maximal involvement occurring on day 35-40. Early inflammatory lesions in the seminiferous tubules were characterized by peritubular and/or interstitial mixed cellular infiltrates. Later lesions included granuloma formation and necrosis. Autoimmune vasitis was not seen in any of the animals studied. Control rats immunized with rat liver homogenate plus adjuvants or adjuvants alone did not exhibit any of the histopathologic lesions described above. The observed results, when compared to those of previous studies examining the sequential histo- and immunopathology of active EAO in the guinea pig and mouse, support the concept that: 1) significant species specificity may exist with regard to regional differences in susceptibility to autoimmune attack within the male reproductive tract and 2) that such differences correlate with early maximal expression of Ia by cells within the male reproductive tract.

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-2Dd 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: IV. Preliminary characterization of the major murine testis specific aspermatogenic autoantigen(s).

Active experimental allergic orchitis (EAO), characterized by inflammation of the testes (autoimmune orchitis), aspermatogenesis, epididymitis and vasitis was induced in mice using a panel of tissue antigens as immunogens. Immunization with allogeneic murine tissue homogenates emulsified in complete Freunds adjuvant (CFA) accompanied by the injection of pertussigen revealed that only adult murine testicular and epididymal homogenates are capable of eliciting murine EAO. All other tissue antigens studied including prepubertal mouse and epididymal homogenates failed to elicit significant disease. Immunization with xenogeneic testicular antigens also failed to elicit significant disease indicating that the major murine aspermatogenic autoantigen(s) is also highly species specific. Sensitization with allogeneic mouse testicular homogenates (MTH) from different disease resistant strains was for the most part no less potent in inducing significant disease than was immunization with mouse testicular homogenates from disease susceptible strains. However, testicular homogenates from NZB/B1NJUnm and MRL/MpJ-/+Unm mice were significantly less potent at inducing autoimmune epididymitis as compared to other strains, indicating possible interstrain differences in the immunogenicity of the aspermatogenic autoantigen(s) relevant to eliciting epididymitis. Attempts at solubilization and purification of the major murine aspermatogenic autoantigen(s) utilizing techniques employed for the purification of aspermatogenic autoantigens such as AP3 from guinea pig (GP) testes were unsuccessful. Additional extraction procedures resulted in solubilization of the relevant autoantigen(s) only after reduction in the presence of 6 M guanidine hydrochloride. These data suggest that: (1) there may be a much more limited number of aspermatogenic autoantigens in murine testes as compared to GP testes; (2) the disease inducing determinant(s) may be expressed as either a sequential antigenic determinant(s) or as an antigenic determinant(s) in the carbohydrate portion of a glycoprotein or glycopeptide; and (3) the disease inducing autoantigen(s) may be present in situ in a highly insoluble form requiring active processing within the target organ in order to generate soluble antigen capable of being seen by immune reactants.

BALB/c Substrain Differences in Susceptibility to Experimental Allergic Encephalomyelitis (EAE)a

Experimental allergic encephalomyelitis (EAE) is currently the best animal model for multiple sclerosis (MS). As an experimental system, murine EAE promises to be a fertile area for study since its natural course after either active or passive induction and its demyelinative histopathology closely mimic MS.’ In addition, the mouse immune system is the most completely understood of all mammalian systems; thus, basic immunological, immunogenetic, and molecular biological analyses should be able to progress rapidly in the rnurine EAE model. However, one block to progress has been the lack of a homozygous, inbred strain of mouse that offers features relative to EAE that would permit dissection of the system. With hope of identifying such a strain, we undertook the present study. Various substrains of BALB/c mice were obtained from a number of independent sources (TABLE 1). The mice were injected in each rear footpad with 0.05 ml of the encephalitogenic emulsion. Controls received the same amount of a saline complete Freund’s adjuvant (CFA) emulsion. On the day of injection, or the third day after injection, the mice were inoculated with 0.1 ml of crude pertussigen’ intravenously and 0.05 ml intraperitoneally. The mice were then observed for the appearance of clinical signs of EAE for a period up to thirty days. At the termination of the observation period, the mice were sacrificed and their central nervous systems were studied histologically for EAE infiltrates. Encephalitogenic emulsion: a 0.9 N solution containing 40 mg/ml of homogenized, lyophilized mouse (SWR) C N S tissue was prepared.’ Incomplete Freund’s adjuvant (DIFCO, Detroit, Michigan) was supplemented with 4.0 mg/ml of killed mycobacterium (H37Ra, DIFCO). Equal volumes of these two fluids were mixed through a fine bore needle to form a nonseparating emulsion. Thus, each mouse received 2.0 mg of C N S tissue in adjuvant containing 0.2 mg of mycobacterium. The available results of this ongoing study are shown in TABLE 1 and the apparent relative substrain susceptibility is shown in TABLE 2. Of interest is the apparent intermediate degree of susceptibility exhibited by some of the substrains; however, the small numbers in certain groups do not permit an exact assignment of relative susceptibility. No control animals showed signs of EAE and a smaller percentage of controls died within seven days of pertussigen administration. Our study produces some interesting points. The BALB/c mouse has been

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.

Serum Testosterone (T) Levels in Balb/CBYJ and Balb/CJ Substrain Mice: Potential Relationship with Differential Susceptibility to Experimental Allergic Orchitis (EAO)

BALB/cByJ substrain mice are highly susceptible to the induction of EAO whereas BALB/cJ mice are resistant. BALB/cJ mice have been reported to have elevated levels of serum T, which may account for disease resistance in light of its purported immunosuppressive effects. Serum T levels were determined by radioimmunoassay on 40 BALB/cByJ and 41 BALB/cJ age matched male mice. Our results indicate that the two substrains do not differ significantly from each other in their mean serum T levels (BALB/cByJ-4.50 ng/ml and BALB/cJ-3.16 ng/ml; p = 0.601) suggesting that the EAO resistant phenotype exhibited by BALB/cJ mice is not a result of the immunosuppressive effects of T.