Activation requirements of donor T cells and host T cell recruitment in adoptive transfer of murine experimental autoimmune orchitis (EAO)

The relative roles of donor and host T lymphocytes and the T cell activation requirements in adoptive transfer of experimental autoimmune orchitis (EAO) in (C57BL/6 x A/J)F1 mice were investigated in order to gain an understanding of the pathogenesis of this disease. Depletion of T cell subsets in recipients by adult thymectomy and treatment with monoclonal antibodies against CD4 or CD8 had no effect on the incidence of EAO following adoptive transfer of activated T cells from donors immunized with testis homogenate (TH) and adjuvants. In contrast, such depletion of CD4+ T cells inhibited development of EAO in actively immunized mice. Thus, CD4+ cells are required for induction of EAO, but donor CD4+ cells are sufficient by themselves without a comparable contribution from the recipient. Adoptive transfer of EAO required that donor splenic and lymph node T cells be activated in vitro before transfer. We found that exposure to antigen (TH) for as little as 4 hr allowed EAO to occur in 25% of recipients, and by 24 hr the cells were fully competent to induce disease. Proliferation of the cells could not be measured until 2 days later. In serial double-transfer experiments, it was found that the cells must be cultured with TH before each transfer in order for the secondary recipients to develop EAO. However, it was not necessary for the transferred T cells to "see" antigen in vivo in the primary recipients, since transfer to castrated primary recipients had no effect on EAO incidence in secondary recipients. Lymphocytes isolated from diseased testes of immunized donors were competent to transfer EAO without activation in vitro, suggesting that, unlike spleen and lymph node cells, these orchitic lymphocytes were already capable of trafficking to the testis.     

Adoptive transfer of murine autoimmune orchitis to naive recipients with immune lymphocytes

A protocol was developed for reproducibly transferring experimental autoimmune orchitis (EAO) to naive recipient mice. Cell donors were (C57BL/6 x A/J)F1 mice immunized about 14 days earlier with mouse testicular homogenate with Freund's adjuvant and an extract of Bordetella pertussis. Lymphocytes from lymph nodes and spleens were equally capable of transferring disease. As few as 5 X 10(6) cells were able to transfer EAO, which began on Day 5-7 after transfer. Infiltrate of lymphocytes and macrophages in the region of the rete testis and straight tubules was the most reproducible early lesion, suggesting that this is the initial site of T cell-antigen interaction. It was not necessary to use both Mycobacteria and B. pertussis adjuvants in donor immunization to achieve transfer of EAO. Disease transfer was antigen specific since only cells from donors immunized with TH could transfer disease. In vitro stimulation of the cells with testicular antigens and/or concanavalin A was a prerequisite to successful transfer of EAO, which was dependent on the presence of L3T4+ T cells since depletion of these cells greatly diminished EAO in recipients and the lymphocyte proliferation response to testicular antigens. Disease did not depend on an antibody response by the recipients. The results imply that effector cells, once generated by immunization and fully activated or selected by in vitro stimulation, can home to specific locations in the testis, locate relevant autoantigens, and cause disease.     

Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion

Despite the immune-privileged status of the male genital tract, infection and inflammation of the male genital tract are important etiological factors in male infertility. A common observation in clinical and experimental orchitis as well as in systemic infection and inflammation are decreased levels of testosterone. Emerging data point to an immunosuppressive role of testosterone. In our study, we substituted testosterone levels in experimental autoimmune orchitis (EAO) in rat by s.c. testosterone implants. EAO development was reduced to 17% when animals were treated with low-dose testosterone implants (3 cm long, EAO+T3) and to 33% when rats were supplied with high-dose testosterone implants (24 cm, EAO+T24) compared with 80% of animals developing disease in the EAO control group. In the testis, testosterone replacement in EAO animals prevented the accumulation of macrophages and significantly reduced the number of CD4(+) T cells with a strong concomitant increase in the number of regulatory T cells (CD4(+)CD25(+)Foxp3(+)) compared with EAO control. In vitro testosterone treatment of naive T cells led to an expansion of the regulatory T cell subset with suppressive activity and ameliorated MCP-1-stimulated chemotaxis of T lymphocytes in a Transwell assay. Moreover, expression of proinflammatory mediators such as MCP-1, TNF-α, and IL-6 in the testis and secretion of Th1 cytokines such as IFN-γ and IL-2 by mononuclear cells isolated from testicular draining lymph nodes were decreased in the EAO+T3 and EAO+T24 groups. Thus, our study shows an immunomodulatory and protective effect of testosterone substitution in the pathogenesis of EAO and suggests androgens as a new factor in the differentiation of regulatory T cells.     

Distribution of histopathology and Ia positive cells in actively induced and passively transferred experimental autoimmune orchitis

Histopathology in testes from mice with actively induced experimental orchitis (EAO) (active EAO) and those from recipients of testis-sensitized lymphocytes (passive EAO) had different distributions. In passive EAO, maximum orchitis existed in the straight tubules, rete testis, and ductus efferentes, obstruction of which led to extreme dilatation of seminiferous tubules. Unusual intralymphatic granulomata also resulted in dilated testicular lymphatics. In active EAO, maximum orchitis affected seminiferous tubules under the testicular capsule, away from the rete testes. Vasitis was common and occurred in both active and passive EAO. In normal testes, IA+ F4/80+ cells were sparse but formed a cuff around the straight tubules. After immunization with testis in adjuvant or with adjuvant alone, the number, size, and staining intensity of IA+ cells increased dramatically beginning on day 5, 7 days before disease onset. Simultaneously, epithelial cells confined to the ductus efferentes became Ia+. Although recipients of sensitized lymphocytes also developed epithelial Ia in the ductus efferentes, they did not show changes in testicular interstitial Ia+ cells. Our findings indicate that testicular autoantigens are not completely sequestered, but are accessible to and can react with passively transferred immune lymphocytes in well-defined regions of the germ cell compartment. These regions coincided to a large extent with maximum expression of periductal or epithelial Ia. Changes in Ia+ cells in the testis, which are inducible by adjuvants and precede orchitis, may account in part for the different distribution of histopathology of active EAO.     

Inhibition by serum of autoimmune aspermatogenic orchitis.

Inhibition by serum of experimental autoimmune orchitis (EAO) was studied in guinea-pigs. It was found that the capacity of an autoantigen, antigen P, purified from guinea-pigs' spermatozoa, to produce lesions of EAO could be inhibited by mixing antigen P with a small amount of normal human serum before injection into guinea-pigs. In protected animals, cell-mediated immunity and humoral antibodies to antigen P were also significantly suppressed.     

Antigen presentation in human autoimmune thyroid disease

Monocyte/macrophage function in patients with autoimmune thyroiditis was investigated by their presentation of two distinct antigens; sheep red blood cells (SRBC) and human thyroglobulin (hTg) using in vitro systems designed for antibody induction. Purified peripheral blood monocyte/macrophages were primed by prefeeding with antigen for 60 min at 37 degrees C, washed, and co-cultured with autologous lymphocytes under a variety of incubation conditions. The most successful system employed 5% monocyte/macrophages with autologous T-B cells in the presence of the mitogen Staphylococcus aureus and B-cell differentiating factors. Under such conditions the anti-SRBC plaque-forming cell (PFC) response was amplified equally (approximately 10-fold) by SRBC-fed monocyte/macrophages in normal controls and patients with autoimmune thyroiditis rendered euthyroid with thyroxine replacement. hTg-fed monocyte/macrophages induced a 4-fold increase in anti-hTg PFC in selected patients with autoimmune thyroiditis examined under similar conditions (mean 36 +/- 3 PFC per 10(6) T-B cells). These data indicated that antigen processing by monocyte/macrophages was normal in patients with autoimmune thyroid disease.     

Involvement of tumor necrosis factor-alpha in the pathogenesis of autoimmune orchitis in rats

We studied the testicular macrophages of rats with experimental autoimmune orchitis (EAO) and analyzed whether the tumor necrosis factor-alpha (TNFalpha) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNFalpha concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNFalpha on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNFalpha-positive testicular macrophages, the TNFalpha concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNFalpha could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNFalpha could trigger germ cell apoptosis. We also demonstrated that TNFalpha inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.     

Identification of a dendritic cell population in normal testis and in chronically inflamed testis of rats with autoimmune orchitis

Experimental autoimmune orchitis (EAO) in the rat is the primary chronic animal model for the investigation of one of the main causes of male infertility, viz., testicular inflammation. Dendritic cells (DC) are potent antigen-presenting cells that play a fundamental role in autoimmune disease. We investigated the number of DC in normal testis and examined whether DC infiltrated the testis during the development of EAO. EAO was induced by active immunization with testis homogenate and adjuvants in two strains of rat (Wistar and Sprague Dawley). The presence of DC in testis was determined, 50 and 80 days after the first immunization, by immunohistochemical staining with specific antibodies (OX-62 and CD11c), and then the total number of DC was measured by stereological analysis. Labeled cells were found only in the interstitial compartment and within granulomas of EAO animals. The number of DC in EAO testes increased compared with control rats in both strains, whereas the number of OX-62+ and CD11c+ cells in adjuvant controls remained unchanged compared with untreated rats. Interspecies variations in the quantity of DC were found, with the total number of DC per testis in untreated and adjuvant control Sprague-Dawley rats being about three times higher than that seen in Wistar rats. Moreover, the increase in DC numbers at 80 days was less prominent in EAO testes of Sprague-Dawley rats than in the Wistar strain in which EAO was more severe and showed a higher number of granulomae. Thus, we have identified the DC population in normal and chronically inflamed testis. The increase in DC observed in EAO suggests that, under inflammatory conditions, the modified action(s) of these cells is a factor in the induction of the autoimmune response in testis.This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Germany (Me 1323/4–1 &4–2) and grants from Universidad de Buenos Aires (UBA) and Consejo Nacional de Investigaciones Científícas y Técnicas (CONICET, Argentina). The authors are Research Members (L.L.) or Fellows (C.R.) of CONICET and the UBA (V.A.G.).     

Immune ablation and stem-cell therapy in autoimmune disease. Experimental basis for autologous stem-cell transplantation

Treatment of rats suffering from florid chronic progressive systemic arthritis or from chronic remitting/relapsing encephalomyelitis with autologous bone marrow transplantation (BMT) is highly effective. This finding was unexpected as the genotype of the bone marrow largely determines the susceptibility of both spontaneous and induced autoimmune diseases in rodents. The success of autologous stem-cell transplantation depends on the completeness of eradication of the effectors of autoimmune disease, in other words activated and memory T lymphocytes. The reviewed experimental data, when translated to the clinic, indicate that the patients should be subjected to a conditioning regimen that induces maximal lymphoablation and that the autologous transplant has to be T-cell depleted.     

Immune ablation and stem-cell therapy in autoimmune disease: Experimental basis for autologous stem-cell transplantation

Treatment of rats suffering from florid chronic progressive systemic arthritis or from chronic remitting/relapsing encephalomyelitis with autologous bone marrow transplantation (BMT) is highly effective. This finding was unexpected as the genotype of the bone marrow largely determines the susceptibility of both spontaneous and induced autoimmune diseases in rodents. The success of autologous stem-cell transplantation depends on the completeness of eradication of the effectors of autoimmune disease, in other words activated and memory T lymphocytes. The reviewed experimental data, when translated to the clinic, indicate that the patients should be subjected to a conditioning regimen that induces maximal lymphoablation and that the autologous transplant has to be T-cell depleted.     

Pathogenesis of experimental allergic orchitis. III. T lymphocyte requirement in local adoptive transfer by peritoneal exudate cells.

In experimental allergic orchitis (EAO), a lesion characterized by mononuclear invasion of seminiferous tubules can be adoptively transferred within 1 to 4 days by testicular injection of peritoneal exudate cells (PEC) from syngeneic strain 13 guinea pigs (GP) immunized with homologous testicular antigens in complete Freund's adjuvant (CFA). This study examined the role of T lymphocytes, macrophages, and polymorphonuclear neutrophils (PMN) in the adoptive transfer. Guinea pig PEC contained 7% T lymphocytes, rare B lymphocytes, and over 90% of macrophages and PMN. After T lymphocytes were depleted by rabbit erythrocyte (E) rosette and Hypaque-Ficoll gradient centrifugation, cell preparations that contained 73% of original macrophages and 15% original T lymphocytes were obtained, and these cells did not transfer EAO (0 of 18 testes). In contrast, cell preparations enriched in T lymphocytes by nylon wool column or E rosette contained 1.5% of the original macrophages and 59% of the original T lymphocytes transferred EAO to 70% of the testes, starting at 1.5 x 10(6) T lymphocytes per testis. The number of T lymphocytes correlated with the incidence of adoptive transfer; the correlation existed regardless of the number of macrophages or PMN present. Finally, EAO was adoptively transferred to recipients that had total-body irradiation. The results indicate that (a) T lymphocytes are capable of transferring lesions of EAO, (b) in the transfer, the T lymphocytes did not function as helper T cells, since the transfer need not involve participation of host lymphoid cells, and (c) by inference, testis antigen-reactive T lymphocytes exist.     

Proinflammatory effects of exogenously administered IL-10 in experimental autoimmune orchitis

We studied the effects of exogenously administered recombinant murine interleukin (IL)-10 on the development of experimental autoimmune orchitis (EAO) in C3H/He mice. IL-10 significantly augments histological signs of EAO when administered for 6 consecutive days from days 15 to 20 after primary immunisations with testicular germ cells. These data demonstrate that IL-10, in addition to its well-known antiinflammatory property, also has proinflammatory functions capable of up-regulating testicular immunoinflammatory processes in vivo.     

Lymphocytes binding polyriboadenylic acid and synthesizing antibodies to nucleic acids in autoimmune and normal mice.

Splenic lymphocytes from both normal and autoimmune mice bind significant quantities of polyriboadenylic acid (poly rA) when incubated with radiolabeled poly rA for 40 min at 37 degrees C. This poly rA binding is specifically inhibited by an excess of nonradioactive poly rA and by anti-mouse immunoglobulin. Poly rA binding is decreased by exposing spleen cells to Pronase and is restored by subsequent culture for 18 to 72 hr. Poly rA-binding activity is associated more with bone marrow-derived than with thymus-derived lymphocytes. These results suggest the presence of autoantigen-binding lymphocytes in normal as well as autoimmune mice. Furthermore, spleen cells from normal and autoimmune mice cultured for 72 hr synthesize and secrete antibodies to poly rA and DNA. These antibodies can be recovered from the culture supernatants by a solid immunoadsorbent technique and antibody immunoprecipitation. The synthesis of antibodies to nucleic acids by normal spleen cells suggests that autoreactive lymphocytes may be released from normal immunoregulatory control during in vitro culture conditions.     

Effect of alpha-melanotropin hormone on serum levels of luteinizing hormone and progesterone in experimental rat autoimmune oophoritis

We studied the effect of alpha-melanotropin hormone (alpha-MSH) on experimental autoimmune oophoritis (EAO), an inflammatory process induced in female rats. During proestrus, serum levels of LH and progesterone in rats with EAO were higher than those of control rats. However, administration of alpha-MSH to these rats decreased the levels of LH. Similarly, in the following diestrus, rats with EAO had high levels of LH but treatment with alpha-MSH decreased the levels to diestrus 2 control values. Treatment with alpha-MSH also reduced the LH levels of control rats in diestrus 2 compared to untreated controls. However, alpha-MSH treatment had no effect on progesterone levels of either control or rats with EAO. Thus, although alpha-MSH induced notable changes in levels of LH, this decrease was unable to block the illness.     

Thyroglobulin peptides associate in vivo to HLA-DR in autoimmune thyroid glands

Endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express HLA class II (HLA-II) molecules that are presumably involved in the maintenance and regulation of the in situ autoimmune response. HLA-II molecules thus expressed by thyroid cells have the "compact" conformation and are therefore expected to stably bind autologous peptides. Using a new approach to study in situ T cell responses without the characterization of self-reactive T cells and their specificity, we have identified natural HLA-DR-associated peptides in autoimmune organs that will allow finding peptide-specific T cells in situ. This study reports a first analysis of HLA-DR natural ligands from ex vivo Graves' disease-affected thyroid tissue. Using mass spectrometry, we identified 162 autologous peptides from HLA-DR-expressing cells, including thyroid follicular cells, with some corresponding to predominant molecules of the thyroid colloid. Most interestingly, eight of the peptides were derived from a major autoantigen, thyroglobulin. In vitro binding identified HLA-DR3 as the allele to which one of these peptides likely associates in vivo. Computer modeling and bioinformatics analysis suggested other HLA-DR alleles for binding of other thyroglobulin peptides. Our data demonstrate that although the HLA-DR-associated peptide pool in autoimmune tissue mostly belongs to abundant ubiquitous proteins, peptides from autoantigens are also associated to HLA-DR in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response.     

Experimental autoimmune thyroiditis in mice chronically treated from birth with anti-IgM antibodies

The role of T lymphocytes in the pathogenesis of experimental autoimmune thyroiditis in mice is well established while the role of B lymphocytes is unclear. Mice with thyroid lesions have thyroglobulin antibodies whereas these antibodies can occur in mice immunized with Tg that do not develop thyroid lesions. To determine whether thyroglobulin antibodies are necessary for the development of the thyroid infiltrates with mononuclear cells, which are characteristic for experimental autoimmune thyroiditis, AKR mice chronically treated from birth with goat anti-mouse IgM antibodies were immunized with mouse thyroglobulin in Freund's complete adjuvant when they were 7 weeks old. Control mice, similarly immunized, were chronically injected from birth with normal goat gamma-globulin. Three weeks after immunization, all mice were sacrificed, thyroglobulin antibodies in the serum were measured by hemagglutination assay and enzyme-linked immunosorbent assay, and thyroid pathology was assessed. The serum concentration of IgG and IgM, the percentage of B and T lymphocytes in the spleen (flow cytometry), and the in vitro proliferative response of spleen lymphocytes to stimulation by PHA, LPS, and Tg were also measured. All mice treated with anti-IgM antibodies did not have detectable thyroglobulin antibodies but 63% of these mice and 88% of control mice (all of which had thyroglobulin antibodies) had thyroid lesions. Mice treated with anti-IgM antibodies that did not have thyroid lesions had a more pronounced depression of B lymphocytes than similarly treated mice that had thyroid lesions. These experiments suggest that thyroglobulin antibodies are not necessary for the development of thyroid infiltrates with mononuclear cells. B lymphocytes could still participate in the production of experimental autoimmune thyroiditis by presenting thyroglobulin to helper T lymphocytes.     

The use of patient-specific stem cells in different autoimmune diseases

Autoimmune diseases are developed when the immune system mistakenly attacks the body’s cells. These inflammatory disorders can be inherited or triggered by external forces, such as type 1 diabetes, which is caused by the immune system's destruction of pancreatic beta cells. So far, stem cells such as hESC and iPSC have been used to treat autoimmune disorders such as type 1 diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE), although these procedures have certain ethical concerns. On the other hand, bone marrow-derived mesenchymal stem cells (BM-MSC) are thought to be the best source of stem cells. Later, it was shown that mesenchymal stem cells produced from autologous adipose tissues have a great potential for producing huge volumes of stem cells. In-vitro and in-vivo investigations using autologous hematopoietic stem cells and autologous mesenchymal stem cells have been carried out on various rodent and human models, while clinical trials for inflammatory diseases such as multiple sclerosis and diabetes mellitus have yielded promising results. We attempted to summarise the usage of diverse stem cells in the therapy of various autoimmune disorders in this review. Shortly, we expect that the use of autologous stem cells will provide a new perspective on the treatment of autoimmune disorders.     

Prevention of experimental autoimmune encephalomyelitis and induction of tolerance with acute immunosuppression followed by syngeneic bone marrow transplantation.

Experimental autoimmune encephalomyelitis (EAE) is an inducible autoimmune disease widely used as a model of the acute/relapsing stage of multiple sclerosis. In the present study we examined the effect of acute immunosuppression induced by total body irradiation (TBI) (900 to 1100 centigray (cGy)) or by a single high dose of cyclophosphamide (CY) (300 mg/kg), followed by syngeneic bone marrow transplantation (SBMT), on the development of EAE in SJL/J mice. EAE was induced in SJL/J mice by immunization with spinal cord homogenate in adjuvant. Treatment with TBI (900 cGy) and SBMT on day 6 postimmunization caused a delayed onset and a marked reduction in the incidence and severity of EAE. A higher dose of irradiation (1100 cGy) or the administration of CY followed by SBMT completely abrogated the development of paralysis. None of the 21 mice treated with CY and SBMT, and only 1 of 7 mice treated with TBI (1100 cGy) and SBMT developed clinical signs of EAE during a period of 3 months. Furthermore, mice treated with CY and SBMT became resistant to rechallenge with the same encephalitogenic inoculum. In addition, the lymphocytes obtained from these mice did not proliferate in vitro in response to myelin basic protein or tuberculin-purified protein derivative, unlike lymphocytes from immunized but untreated animals. This absence of reactivity was not associated with alterations in the proportion of the L3T4 and Lyt-2 T-cell subsets nor with a loss in T cell competence as evidenced by the full response of lymphocytes to the T cell mitogen Con A and to a nonrelevant Ag (OVA). Our results indicate that the elimination of effector lymphocytes either by myeloablative doses of CY or ionizing irradiation followed by rescue with SBMT inhibits the development of the autoimmune process in EAE and leads to induction of tolerance to the immunizing Ag by newly developing lymphocytes. This approach of combining immunoablation and reconstitution with autologous bone marrow transplantation may be applicable in the treatment of life-threatening neurologic autoimmune diseases.     

Autoimmune orchitis can be induced by thymic lymphocytes autosensitized against syngeneic sertoli cells in vitro

Thymic lymphocytes from normal inbred Lewis/Wistar rats were cocultured with syngeneic Sertoli cell-peritubular cell preparations in the presence of heterologous or allogeneic serum. Thymic cells cultured in this manner bound to Sertoli cells, became autosensitized , and markedly altered syngeneic Sertoli cell surface properties and remodeling functions in vitro. In contrast, control thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not become sensitized. Coculture of autosensitized thymic cells with syngeneic seminiferous tubule segments, or local transfer of such lymphocytes into syngeneic rat testes, resulted in intratubular infiltration by "light cells." Intratesticular injection of autosensitized thymic cells was followed by derangement of the seminiferous epithelium, and by morphologic changes characteristic of experimental autoimmune orchitis. Thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not elicit these changes. Thymic cells incubated with peritubular cells in heterologous or autologous serum behaved like control thymocytes, and were not sensitized. Data presented indicate that thymic cells are potentially capable of recognizing syngeneic Sertoli cell self-antigens. We speculate that factors normally present in serum may inhibit the recognition by thymic lymphocytes of antigenic determinants present on Sertoli cells. We discuss the possibility that the modulation of interactions between immature thymic lymphocytes and Sertoli cells is implicated in the prevention of autoimmune reactions against the testis.