ABSTRACT
Interleukin-12 (IL-12) is a cytokine that promotes cell-mediated immunity to intracellular pathogens by inducing type 1 helper T cell (TH1) responses and interferon-gamma (IFN-gamma) production. IL-12 binds to high-affinity beta1/beta2 heterodimeric IL-12 receptor (IL-12R) complexes on T cell and natural killer cells. Three unrelated individuals with severe, idiopathic mycobacterial and Salmonella infections were found to lack IL-12Rbeta1 chain expression. Their cells were deficient in IL-12R signaling and IFN-gamma production, and their remaining T cell responses were independent of endogenous IL-12. IL-12Rbeta1 sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain. The lack of IL-12Rbeta1 expression results in a human immunodeficiency and shows the essential role of IL-12 in resistance to infections due to intracellular bacteria.
Subject(s)
Interleukin-12/immunology , Mycobacterium avium-intracellulare Infection/immunology , Mycobacterium bovis , Receptors, Interleukin/genetics , Salmonella Infections/immunology , Tuberculosis/immunology , Adult , Child, Preschool , Codon, Terminator , Disease Susceptibility , Female , Frameshift Mutation , Genes, Recessive , Humans , Interferon-gamma/biosynthesis , Interleukin-12/metabolism , Lymphocyte Activation , Mutation , Receptors, Interferon/metabolism , Receptors, Interleukin/deficiency , Receptors, Interleukin/metabolism , Receptors, Interleukin-12 , Sequence Deletion , T-Lymphocytes/immunology , Interferon gamma ReceptorABSTRACT
The CD28-CD80/CD86 costimulatory pathway provides a critical signal for T cell activation. Only recently rat CD80 and CD86 have been cloned and monoclonal antibodies have been generated. In this study we examined the expression of these molecules in lymphoid tissue and on purified subsets of antigen-presenting cells (APC). The target tissue of cyclosporin A-induced autoimmunity, i.e. the skin and tongue, were also examined for expression of CD80 and CD86. Whereas CD80 was hardly detected in the lymphoid tissues, CD86 was clearly expressed by non-lymphoid cells in the thymus, as well as in the secondary lymphoid organs. With respect to lymphoid cells, only germinal center B cells exhibited clear CD86 expression. Phenotypic analysis by flow cytometry revealed that only dendritic cells, both of thymic and splenic origin, expressed the full array of stimulatory molecules required for the proper activation of naive T cells. On development of cyclosporin A-induced autoimmunity, non-professional APC, i.e. epithelial cells, started to express MHC class II, but not the costimulatory ligands CD80 and CD86. However, CD86 staining was observed in the target tissue and was associated with Langerhans cells as well as infiltrating leukocytes. Altogether, our results show that also in the rat strong stimulatory capacity for primary immune responses is associated with the expression of the costimulatory ligands CD80 and CD86. As concluded from the in situ expression CD86 may be the predominant costimulatory ligand early in immune responses.
Subject(s)
Antigen-Presenting Cells/metabolism , Antigens, CD/biosynthesis , B7-1 Antigen/biosynthesis , Lymphoid Tissue/immunology , Membrane Glycoproteins/biosynthesis , Animals , Autoimmunity/drug effects , B7-2 Antigen , Cells, Cultured , Cyclosporine/administration & dosage , Histocompatibility Antigens Class II/biosynthesis , Injections, Subcutaneous , Lymphoid Tissue/drug effects , Organ Specificity/drug effects , Organ Specificity/immunology , Rats , Rats, Inbred LewABSTRACT
The pathogenesis of scleroderma is still elusive, although autoimmune mechanism involvement has been suggested. The present study was designed to investigate whether or not rat scleroderma would appear as one of the symptoms in a recently described model for autoimmune disease. The model is based on manipulation or reconstitution of the immune system after lethal irradiation and syngeneic bone marrow transplantation by temporary administration of the immunosuppressive drug cyclosporine-A. Withdrawal of cyclosporine-A 6-12 wk after bone marrow transplantation gives rise to autoimmune reactions causing clinical pathology similar to graft-versus-host disease. We discuss the chronic phase of this disease, approximately 30 wk after cyclosporine-A was withheld at that time-about one-third of the rats had developed histologic skin lesions comparable to those seen in patients with scleroderma. Therefore, we propose this model as a new, experimental autoimmune model for scleroderma in humans.
Subject(s)
Autoimmune Diseases/chemically induced , Cyclosporins , Scleroderma, Systemic/immunology , Acute Disease , Alopecia/etiology , Animals , Autoimmune Diseases/pathology , Bone Marrow Transplantation/immunology , Chronic Disease , Disease Models, Animal , Graft vs Host Disease/immunology , Graft vs Host Disease/pathology , Rats , Rats, Inbred Strains , Scleroderma, Systemic/pathology , Skin/pathology , X-RaysABSTRACT
A panel of monoclonal antibodies (mAbs) against human thyroglobulin (hTg) was obtained by somatic fusion of the nonsecreting myeloma cell line P3X66 Ag8/0 and spleen cells of Balb/c mice immunized with purified hTg. Antibody secreting clones were selected by solid phase enzyme immunoassay and analyzed for cross-reaction with Tg from several animal species. Twelve out of 15 mAbs cross-reacted with both rat and mouse Tg and 11 Mabs cross-reacted with bovine Tg. The cross-reaction with mouse Tg paralleled that of rat Tg, whereas discrete differences between the cross-reactivity patterns with bovine Tg were observed. Two clones secreted mAbs specific for hTg. We further characterized the mAbs and found that three mAbs recognized T4-containing determinants and one mAb reacted with both T4 and T3-containing determinants on the Tg molecule. The binding of the mAbs to hormonogenic determinants depended upon the thyroid hormone content of the molecule and the integrity of the three dimensional structure of Tg. One other mAb reacted with four peptides of CNBr-cleaved hTg, indicating the recognition of a repetitive determinant in the hTg molecule distinct from the hormonogenic regions.
Subject(s)
Antibodies, Monoclonal , Thyroglobulin/immunology , Animals , Cell Line , Epitopes/analysis , Humans , Immunoenzyme Techniques , Mice , Mice, Inbred BALB C , Plasmacytoma , Thyroglobulin/analysis , Thyroid Gland/analysis , Thyroid Gland/cytologyABSTRACT
The effect of target organ manipulation by means of denervation and treatment with anabolic steroids on the severity of disease in EAMG was assessed in inbred rats. Unilateral limb denervation, a procedure known to increase the AChR content of muscle, 'protected' the denervated leg against antibody-mediated AChr loss in acute EAMG induced by passive transfer of mAb 35 directed against the main immunogenic region. Also in chronic EAMG, brought about by immunizing rats with AChR in complete Freund's adjuvant, the AChR loss of the denervated leg was about one fourth (13.5 vs. 53%) of the control leg. In both acute and chronic EAMG the amount of AChR complexed with antibody was lower in the denervated leg. This lower AChR occupancy with antibody in the denervated leg occurred also in conditions of marked antibody excess and was therefore due to enhanced AChR synthesis. Next the effect of treatment with a weakly virilizing anabolic steroid nandrolone in chronic and acute EAMG was examined in order to examine whether a hypothesized enhanced synthesis of AChR would protect animals from disease. In the absence of an immunosuppressive effect, in terms of concentration of antibodies to AChR, nandrolone treatment protected the rats from severe disease in the chronic EAMG model as shown by the fact that of the 9 rats 6 showed mild (1+) disease and 3 no disease at all; conversely 6 out of 9 control rats showed severe (3+) disease. Rats treated with nandrolone showed a 48 +/- 1.7% loss of AChR compared to a loss of 58 +/- 3.6% in the control rats, suggesting enhanced AChR synthesis. When nandrolone-pretreated rats were given acute EAMG by passive transfer of mAb 35 a paradoxical effect was seen. In contrast to the controls all of the rats pretreated with nandrolone showed severe signs of EAMG; this was associated with a higher loss of AChR and increased consumption of complement C4 as suggested by decreased concentrations of C4 in the serum. Results show increased AChR synthesis to protect against chronic EAMG both in terms of clinical disease (nandrolone) as well as AChR loss (nandrolone, denervation). In addition it was shown that nandrolone increases serum C4 consumption which in the complement-dependent acute EAMG model causes enhancement of the severity of clinical disease and increased AChR loss.
Subject(s)
Myasthenia Gravis/metabolism , Receptors, Cholinergic/biosynthesis , Acute Disease , Animals , Antibodies, Monoclonal/administration & dosage , Chronic Disease , Complement C3/metabolism , Complement C4/metabolism , Disease Models, Animal , Female , Immunization, Passive , Muscle Denervation , Myasthenia Gravis/etiology , Myasthenia Gravis/immunology , Nandrolone/pharmacology , Rats , Rats, Inbred Lew , Receptors, Cholinergic/drug effectsABSTRACT
Myasthenia gravis (MG) is an organ-specific autoimmune disease in which autoantibodies against nicotinic acetylcholine receptors (AChR) at the postsynaptic membrane cause loss of functional AChR and disturbed neuromuscular transmission. The immunopathogenic mechanisms responsible for loss of functional AChR include antigenic modulation by anti-AChR antibodies, complement-mediated focal lysis of the postsynaptic membrane, and direct interference with binding of acetylcholine to the AChR or with ion channel function. The loss of AChR and subsequent defective neuromuscular transmission is accompanied by increased expression of the different AChR subunit genes, suggesting a role for the target organ itself in determining susceptibility and severity of disease. Experimental autoimmune myasthenia gravis (EAMG) is an animal model for the disease MG, and is very suitable to study the immunopathogenic mechanisms leading to AChR loss and the response of the AChR to this attack. In this article the current concepts of the structure and function of the AChR and the immunopathological mechanisms in MG and EAMG are reviewed.
Subject(s)
Autoimmune Diseases/immunology , Myasthenia Gravis/immunology , Animals , Autoantibodies/immunology , Disease Models, Animal , Humans , Mice , Myasthenia Gravis/physiopathology , Rabbits , Receptors, Cholinergic/immunologyABSTRACT
The polymerase chain reaction (PCR) is a sensitive method for the analysis of cytokine mRNA expression. The amount of specific mRNA in tissues involved in an inflammatory immune response can be low and therefore requires highly sensitive detection of the PCR products. In our study we have compared different detection techniques in order to replace the commonly used detection by means of radiolabeled probes. Besides the detection of DNA in agarose gels by ethidium bromide (EB), we used detection by digoxigenin (DIG)-labeled probes, as well as the direct incorporation of DIG-labeled nucleotides in the PCR, in comparison to detection by means of 32P-labeled probes. In vitro activated rat lymph node cells, lymph node tissue, and acutely or chronically rejected rat heart allografts were examined for expression of mRNA of the cytokines IL-2 and IFNgamma. The directly DIG-labeled PCR appeared to be the best alternative for detection of PCR products by means of radiolabeled probes. While IL-2 mRNA was not detected by means of EB and IFNgamma mRNA was only detected at the highest PCR cycle numbers in acutely and chronically rejected rat heart allografts, both cytokine mRNA's were readily detected by directly DIG-labeled PCR.
Subject(s)
Graft vs Host Disease/diagnosis , Heart Transplantation/immunology , Interferon-gamma/biosynthesis , Interleukin-2/biosynthesis , Myocardium/chemistry , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Blotting, Southern , Digoxigenin , Graft vs Host Disease/immunology , Interferon-gamma/genetics , Interleukin-2/genetics , Lymph Nodes/chemistry , Rats , Rats, Inbred BN , Rats, Inbred Lew , Sensitivity and Specificity , Specific Pathogen-Free Organisms , Transplantation, HomologousABSTRACT
Several studies have now confirmed our original observation that transfusions of large doses of donor-specific allogeneic erythrocytes (E) induce humoral unresponsiveness to E-associated antigens (including major histocompatibility complex (MHC) class I antigens) in rats, which is associated with prolonged survival of subsequently inserted renal grafts. To examine whether this tolerance was due to induction of suppressor (T) cells or to anergy or deletion of antigen-specific B cells, an in vitro system was developed that allowed generation of primary immune responses of rat splenocytes to allogeneic erythrocytes. Cocultivation of B and non-B cells from tolerant and normal rats in this system showed that E transfusions had induced B cell tolerance in the recipients; no evidence was obtained that suppressor T cells were involved in the maintenance of unresponsiveness. On the contrary, non-B cells from tolerant rats could "help" normal B cells for antibody formation to allogeneic E. In vivo, tolerance to allogeneic E could be broken in one rat strain combination by skin allografts. Therefore, the state of B cell tolerance induced by E transfusions may be referred to as "reversible clonal anergy".
Subject(s)
B-Lymphocytes/immunology , Blood Transfusion , Erythrocyte Transfusion , Immune Tolerance , Animals , Graft Survival , Rats , Rats, Inbred BN/immunology , Rats, Inbred Lew/immunology , Rats, Inbred Strains/immunology , Skin Transplantation , Transplantation, HomologousABSTRACT
Transfusions of highly purified LEW erythrocytes (E) administered to BN recipients prior to insertion of LEW kidneys markedly prolonged the survival of these allografts (greater than 35 days). Administration of E from syngeneic (BN), third-party (PVG), and MHC-congenic LEW.1N or BN.1L rats did not improve LEW kidney graft survival to the same extent (less than 14 days). BN.1L E were shown to carry at least the same quantity of LEW MHC antigens on their surface as LEW E, thus the failure to prolong LEW kidney graft survival is due to the absence of LEW non-MHC antigens from BN.1L E. Attempts to substitute for this deficiency by mixing LEW.1N E to BN.1L E prior to transfusion failed to restore the beneficial effect, demonstrating that donor E-mediated prolonged renal allograft survival requires the presence of both MHC and non-MHC alloantigens on the same E.
Subject(s)
Erythrocytes/immunology , Graft Survival , Histocompatibility Antigens/immunology , Kidney Transplantation , Animals , Antigen-Antibody Reactions , Blood Transfusion , Erythrocyte Transfusion , Major Histocompatibility Complex , Male , Rats , Rats, Inbred BN/immunology , Rats, Inbred Lew/immunology , Transplantation, HomologousABSTRACT
To study immune reactive and thrombotic mechanisms involved in chronic renal allograft rejection, Lewis rat kidneys were transplanted into bilaterally nephrectomized Brown Norway recipients tolerant of LEW erythrocyte antigens. Such BN rats fail to produce anti class I MHC alloantibodies after insertion of a LEW kidney. The LEW renal allografts experience a transient rejection episode without proteinuria followed by the development of chronic rejection, clinically characterized by glomerular proteinuria in the presence of stable renal function. Immunohistological studies of such chronically rejected LEW renal allografts showed the occurrence of glomerular and interstitial infiltration of predominantly monocytes and T cells. CD4-positive T cells dominated over CD8-positive T cells in the chronically rejected LEW renal grafts. IgG deposition was found deposited throughout the renal vasculature--this in contrast to IgM, which was observed only in the glomerular vasculature. Glomerular antibodies were not directed to endothelial class II MHC antigens, and showed only weak complement fixation as demonstrated by C3 staining. Selective glomerular IgM deposition was associated with vascular (platelet-containing) thrombi, and focal and segmental fibrinoid necrosis. In contrast, acutely rejected LEW renal grafts in unmodified BN recipients showed IgM deposition as well as thrombus formation throughout the entire renal vasculature. The results demonstrate that the antibody response to endothelial--and, in particular, glomerular endothelial non-MHC antigens--may bring about chronic vascular renal allograft rejection. How the formation of glomerular thrombotic lesions may be assisted by endothelial reactivity to cytokines from local immune reactive cells is discussed.
Subject(s)
Graft Rejection/immunology , Kidney Glomerulus/blood supply , Kidney Transplantation/immunology , Animals , Antibody Formation/immunology , Blood Coagulation , Endothelium/cytology , Endothelium/immunology , Immunity, Cellular/immunology , Immunohistochemistry , Kidney/blood supply , Kidney/metabolism , Kidney Glomerulus/physiology , Rats , Rats, Inbred BN , Rats, Inbred Lew , Thrombosis/etiologyABSTRACT
BN rats were immunized with one or three doses of 1 X 10(8) highly purified LEW erythrocytes (LEW-E) yielding IgM antibody (IgM-BN rats) and IgG antibody (IgG-BN rats) to LEW class I antigens, respectively. LEW kidneys transplanted into IgM-BN rats elicited cytotoxic T cell responses and lymphocytotoxic antibody responses comparable to those elicited by LEW renal grafts in unmodified BN rats. However, LEW kidneys were rejected by IgM-BN hosts in a slightly delayed fashion compared with controls (mean rejection times (MRTs), 9.4 versus 7.1 days); delayed rejection was associated with the absence of anti-LEW IgG hemagglutinins from the recipients' blood and the absence of vasculonecrotic lesions from rejected renal grafts. LEW kidneys inserted into IgG-BN rats were rejected in a slightly accelerated fashion compared with controls (MRT, 6.6 days). Lymphocytotoxins developed in IgG-BN recipients of LEW kidneys in a fashion similar to that of controls, but cytotoxic T cell responses were delayed up to the 6th day after transplantation. These observations confirm our previous finding that cytotoxic T cells do not play a decisive role in acute rejection in this model. The association observed between delayed or accelerated rejection of LEW kidneys by BN rats sensitized with LEW-E and the absence or presence of anti-donor IgG hemagglutinins in the blood of these recipients after transplantation suggests an important role for IgG anti-donor class I antibodies in the rejection of LEW renal allografts by BN rats.
Subject(s)
Antilymphocyte Serum/immunology , Graft Enhancement, Immunologic , Graft Rejection , Kidney Transplantation , T-Lymphocytes/immunology , Animals , Erythrocytes/immunology , Histocompatibility Antigens/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Male , Rats , Rats, Inbred BN , Rats, Inbred Lew , Time Factors , Transplantation, HomologousABSTRACT
First set rat renal allografts transplanted over the strong Ag-B histocompatibility locus elicit antibodies of the gammaM class demonstrable at the time of graft rejection. These early gammaM alloantibodies with guinea pig complement are cytotoxic in vitro to high antigen density target cells like lymph node cells and splenocytes but not to low antigen density target cells like thymocytes and bone marrow cells. With rat complement, gammaM alloantibodies are required in far greater amounts to kill some high density target cells. This in vitro discrepancy between rat and guinea pig complement is not caused by the presence of natural antibody in guinea pig serum nor by a deficiency of complement components in rat serum, but is dependent on the antigen antibody interaction studied. In vivo studies show early gammaM alloantibodies to be cytotoxic to donor lymphoid cells but to enhance renal allografts. These cytotoxic and enhancing qualities reside in the same preparation of immunoglobulin and are influenced by antigen density. These studies suggest that the failure to damage donor kidneys by early, probably low avidity, antibody is caused by a low concentration of antigen on the endothelial cells within the renal graft, and or an inability of this antibody antigen interaction to activate syngeneic complement.
Subject(s)
Immunoglobulin M , Isoantibodies/biosynthesis , Kidney Transplantation , Animals , Antibody Specificity , Cell Movement , Complement System Proteins , Cytotoxicity Tests, Immunologic , Graft Rejection , Guinea Pigs , Immunization, Passive , Lymph Nodes/cytology , Rats , Rats, Inbred BN , Rats, Inbred Lew , Time Factors , Transplantation, HomologousABSTRACT
It is demonstrated that passenger lymphocytes migrate out of rat renal allografts into host spleens in a radioresistant fashion. These mobile passenger lymphocytes within BN kidney and heart transplants are immunocompetent, since they elicit a graft-versus-host (GVH) reaction in the spleens of (LEW x BN)F2 hybrid hosts. The greater GVH reaction in (LEW x BN)F1 recipients of BN kidneys reflects the greater number of mobile passenger lymphocytes in the kidney when compared to the heart. The mobile passenger lymphocytes within BN renal allografts also cause a proliferative response in the spleens of LEW hosts as well as an accelerated rejection of BN renal allografts when compared to BN cardiac allografts, for the differences between BN kidney and heart, both in terms of splenomegaly elicited in LEW as well as tempo of rejection, are abolished by total body X-irradiation of the donor with 900 rad. Results indicate that a mobile passenger lymphocyte mediated GVH reaction in the central lymphoid organs of the host augments the host response to allogeneic kidneys and contributes materially to first-set renal allograft rejection; this GVH reaction on the other hand is not conspicuously present in LEW recipients of BN cardiac allografts and has therefore little effect on first-set cardiac allograft rejection.
Subject(s)
Graft Rejection , Heart Transplantation , Kidney Transplantation , Lymphocytes/immunology , Animals , Cell Division , Cell Movement/radiation effects , Chromium Radioisotopes , Graft vs Host Reaction , Kidney/cytology , Male , Myocardium/cytology , Organ Size , Rats , Rats, Inbred BN , Rats, Inbred Lew , Spleen/anatomy & histology , Spleen/cytology , Time Factors , Transplantation, Homologous , X-RaysABSTRACT
Passive transfer of latent rat cytomegalovirus (R-CMV) infection by means of vascularized organ transplants was examined in inbred rat strains. LEWIS (LEW) rats 4-5 weeks old were infected with RCMV and used as donors at 5 months of age when the infection had become latent. Well-perfused LEW hearts and kidneys were transplanted into unmodified or 500-rad x-irradiated syngeneic or allogeneic Brown Norway (BN) recipients; recipients were sacrificed 3 weeks after transplantation, and RCMV virus from various organs was quantitated by means of a plaque assay. Passive transfer of latent infection could be accomplished with renal allografts (60%) and renal isografts (40%). When BN hosts were x-irradiated LEW renal allografts invariably transferred the latent infection (100%); cardiac allografts rarely did so (8%). X-irradiation of syngeneic hosts did not enhance the capacity of LEW kidneys to transfer the latent infection. The latent infection could not be transferred with thoracic duct lymphocytes. Results show the passive transfer of latent infection with well-perfused vascularized organ allografts to be a relative organ-specific phenomenon.
Subject(s)
Cytomegalovirus Infections/transmission , Heart Transplantation , Immunization, Passive , Kidney Transplantation , Animals , Cytomegalovirus/immunology , Cytomegalovirus Infections/etiology , Cytomegalovirus Infections/immunology , Disease Models, Animal , Immunosuppression Therapy , Rats , Rats, Inbred BN , Rats, Inbred Lew , Salivary Glands/microbiology , Whole-Body IrradiationABSTRACT
Cyclosporine (CsA) and FK506 are structurally unrelated immunosuppressants, but function in similar ways. FK506 and rapamycin (RAPA), on the other hand, have structural similarities, but act by different mechanisms to yield immunosuppression. Besides their immunosuppressive action, CsA and FK506 are known to interfere with T-cell development. CsA treatment after lethal X-irradiation and syngeneic bone marrow transplantation results in autoimmune disease, which is referred to as CsA-induced autoimmunity. In this study, we examined the effect of RAPA on T-cell development by flow cytometry and immunohistochemistry in female Lewis and Brown Norway rats. Irradiation and syngeneic bone marrow transplantation were performed before a 4-week course of RAPA administration to determine de novo T-cell development in relation to possible autoimmune phenomena. RAPA interfered with the maturation of thymocytes to the CD4+CD8+ DP stage, which resulted in a relative increase in TCRalphabeta(-) immature thymocytes, localized in a rim along the outer cortex. The thymus of RAPA-treated animals had a thinner cortex, leading to stronger thymic atrophy. In the periphery, only a few T cells were observed at the end of RAPA treatment. In the Lewis rat, a normal CD4/CD8 T-cell ratio and an increased Th1/Th2 ratio was observed within the T-cell population. Six weeks after cessation of RAPA therapy, the T-cell compartment was restored to normal, with respect to number and phenotype. In Brown Norway rats, however, T-cell areas were barely detectable at the end of RAPA treatment. The CD4/CD8 T-cell ratio was decreased as a result of a lower number of CD4 T cells; the Th1/Th2 ratio was increased but Th2 remained higher. Similar to Lewis rats, the situation was almost normalized 6 weeks after cessation of RAPA administration. However, Brown Norway rats, in contrast to Lewis rats, showed T-cell infiltration and concomitant induction of MHC class II in the submandibular salivary gland, as well as insulitis, in the pancreas. Possible relationships to Sjogren's disease and diabetes remain to be established.
Subject(s)
Autoimmune Diseases/chemically induced , Autoimmune Diseases/immunology , Immunosuppressive Agents/pharmacology , Polyenes/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , Animals , Autoimmune Diseases/pathology , Autoimmunity/drug effects , Disease Susceptibility , Female , Flow Cytometry , Phenotype , Rats , Rats, Inbred BN , Rats, Inbred Lew , Sirolimus , T-Lymphocyte Subsets/drug effects , T-Lymphocytes/immunology , Th1 Cells/drug effects , Th1 Cells/immunology , Th2 Cells/drug effects , Th2 Cells/immunology , Thymus Gland/cytologyABSTRACT
BACKGROUND: To gain insight in the pathogenesis of vascular lesions in heart allograft rejection, we investigated effects of allosera reactive with major histocompatibility complex (MHC) or non-MHC alloantigens on graft endothelial cells (EC) in a rat transplantation model. METHODS: Anti-MHC and anti-non-MHC allosera were obtained from Brown Norway (RT.1(n)) recipients of a Lewis (RT.1(1)) or congenic LEW.1N (RT.1(n)) heart allograft respectively. Reactivity with endothelial alloantigens was studied in vitro using a series of three rat heart endothelial cell (RHEC) lines of Lewis origin. Phenotypic studies of MHC and non-MHC alloantigen expression, and adhesion molecule induction on EC were performed by immunostaining and fluorescence-activated cell sorting analysis. Complement-mediated cytotoxicity of allosera was studied using a 51Cr release assay. RESULTS: Both anti-MHC allosera and anti-non-MHC allosera showed reactivity with all three RHEC lines. EC stimulation with tumor necrosis factor-alpha and interferon-y resulted in increased reactivity of anti-MHC but not of anti-non-MHC allosera. Anti-MHC allosera showed complement-mediated cytotoxicity for EC, which was strongly increased when cytokine-stimulated EC were used. With anti-non-MHC allosera, only minor cytotoxicity was measured, irrespective of the activation of EC. Anti-MHC and anti-non-MHC allosera from the day of rejection (days 7-8 and days 29-35, respectively) had similar subclass profiles of allospecific IgG, except for allospecific IgM, which was only detected in anti-MHC allosera. Complement-mediated cytotoxicity of anti-MHC allosera from the day of rejection was effected mainly by IgM alloantibodies, whereas, in allosera taken 4 days after rejection, a predominance of cytotoxic alloantibodies of the IgG class was observed. No indications were found that either alloantibody reactivity alone or in combination with complement activation led to EC activation processes relevant to intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 induction. CONCLUSIONS: Our data show that, in heart allograft rejection, MHC but also non-MHC alloantigens on EC are target structures in the alloantibody response. Alloantibodies reactive with endothelial MHC, but not those reactive with non-MHC alloantigens, may significantly contribute to vasculopathy by complement-mediated cytotoxicity. Although no evidence was found that alloantibodies reactive with graft EC induce adhesion molecule expression, they may trigger other EC mechanisms relevant to graft vasculopathy.
Subject(s)
Endothelium, Vascular/immunology , Graft Rejection , Heart Transplantation/immunology , Histocompatibility Antigens/immunology , Isoantibodies/blood , Isoantigens/immunology , Animals , Cytotoxicity, Immunologic , Endothelium, Vascular/cytology , Immunoglobulin G/blood , Immunoglobulin M/blood , Intercellular Adhesion Molecule-1/biosynthesis , Male , Rats , Rats, Inbred Lew , Transplantation, Homologous , Vascular Cell Adhesion Molecule-1/biosynthesisABSTRACT
Lethally irradiated Lewis rats reconstituted with syngeneic bone marrow and given cyclosporine for 4 weeks develop a graft-versus-host-like disease upon withdrawal of CsA. Autoreactive T cells inducing this thymus-dependent autoimmune disease, termed CsA-AI, are demonstrable by adoptive transfer, provided regulatory cells in recipient rats are eliminated. Earlier studies have not unequivocally defined the effector T cells responsible for development of CsA-AI. Some of these studies suggest that both CD4 and CD8 T cells are required, while other studies indicate disease transfer by CD4 or CD8 T cells only. To further clarify this issue, it was necessary to study putative effector T cells in a well-defined setting. Hence, adoptive transfer studies were designed wherein the effect of the T cells of interest could be studied without being influenced by T cells of unwanted origin. Accordingly, recipient rats were thymectomized prior to irradiation, lymph node cells (LNC) from diseased donor rats were depleted of CD4 or CD8 cells before adoptive transfer, and recipients were treated in vivo with CD4- or CD8-depleting mAb. The results showed that CsA-AI developed after adoptive transfer with LNC depleted of either CD4 or CD8 cells. Analysis of PBL and of histologic specimens confirmed the absence of the depleted subset. In both instances, the typical MHC class II expression on keratinocytes and the presence of ED1+ macrophages were identical to the lesions in the primary donors, where both CD4 and CD8 T cells were present. Analysis of the T cell Receptor beta-chain variable region repertoires revealed that their expression patterns in LNC of diseased donors or recipients was comparable to that in normal thymus or LNC--hence, there was no restricted BV repertoire. Taken in toto, our observations indicate that CsA-AI involves both CD4 and CD8 T cells, and that these subsets can generate identical macroscopic and microscopic signs of disease.
Subject(s)
Autoimmune Diseases/chemically induced , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/physiology , Cyclosporine/adverse effects , Adoptive Transfer , Animals , Cell Separation , Female , Flow Cytometry , Graft vs Host Disease/chemically induced , Histocompatibility Antigens Class II/physiology , Immunoglobulin Variable Region/genetics , Keratinocytes/immunology , Lymphocyte Subsets/cytology , Phenotype , Rats , Rats, Inbred Lew , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
The thymus-dependent model of cyclosporine-induced autoimmunity (CsA-AI) in the Lewis rat requires a lethal total body X-irradiation and rescue with syngeneic or autologous bone marrow and cyclosporine (CsA) administration for at least 4 weeks; two to three weeks after cessation of CsA, the animals develop a graft-versus-host-like disease. The obligatory role of the thymus in the etiology of CsA-AI has been established unequivocally, but the way in which disease is thymus dependent is a topic of debate. In the present study we demonstrate that the model of CsA-AI requires the presence of a thymus for at least 2 weeks after total body irradiation and CsA administration, but that X-irradiation of the thymus itself is not necessary to bring about disease. Transplantation of neonatal thymus shows in addition that in the absence of X-irradiation of the thymus, CsA therapy is required to generate autoreactive cells, but that disease occurs only if peripheral autoregulatory cells are eliminated by X-irradiation.
Subject(s)
Autoimmunity/drug effects , Cyclosporine/adverse effects , Thymus Gland/radiation effects , Animals , Female , Rats , Rats, Inbred Lew , Thymectomy , Time Factors , Whole-Body IrradiationABSTRACT
Three biweekly infusions of a x 10(10) highly purified LEW (RT1l) erythrocytes (LEW-E) administered to BN (RT1n) rats commencing at 1 month of age failed to elicit alloantibody or cell-mediated cytotoxicity against LEW target cells. The magnitude of the proliferative response of lymphocytes taken from LEW-E-infused BN rats in unilateral mixed lymphocyte culture against LEW stimulator cells was identical to that of control BN lymphocytes. LEW renal grafts inserted in LEW-E-infused BN rats showed a markedly prolonged survival which was specific since Wistar (RT1u) renal allografts were acutely rejected. LEW kidneys grafted to unmodified and LEW-E-infused BN recipients elicited cytotoxic antibody responses to Ia-like antigens and cellular immune responses of identical magnitude and specificity. On the other hand, LEW renal grafts evoked hemagglutinating alloantibody in control BN recipients but failed to do so in LEW-E-infused BN recipients. This unresponsiveness to LEW-E-associated antigens was specific since Wistar renal grafts elicited anti-Wistar hemagglutinin responses in LEW-E-infused BN recipients. These results suggest that antibodies to LEW-E-associated alloantigens play an essential role in the acute rejection of LEW renal grafts by BN recipients.
Subject(s)
Antibodies , Erythrocytes/immunology , Graft Rejection , Kidney Transplantation , Animals , Antibody Formation , Antigens , Antilymphocyte Serum , Graft Survival , Immunity, Cellular , Male , Rats , Rats, Inbred BN , Rats, Inbred Lew , Transplantation, HomologousABSTRACT
In experimental autoimmune myasthenia gravis anti-rat nicotinic acetylcholine receptor (AChR) antibody titers correlated significantly with the AChR-antibody complexes found in muscle. It was shown that at least a large part of the AChR-antibody complexes are formed in vitro, which can be prevented by washing of the muscle homogenate. Using a modified assay, no differences in AChR-antibody complexes could be detected between rats with and without symptoms of experimental autoimmune myasthenia gravis. Also no difference in AChR loss nor in inhibition of alpha-bungarotoxin binding to AChR was found between these groups of rats. However, a significant difference in the reduction of AChR function was found, using an assay measuring agonist-induced 22Na+ flux into the TE671 cell line.