Role of macrophages in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from SJL/J mice infected with the D variant of encephalomyocarditis virus (EMC-D virus) showed lymphocytic infiltration with moderate to severe destruction of beta cells. Immunohistochemical staining of the islet sections with several monoclonal antibodies, anti-Mac-1, anti-Mac-2, and F4/80 for macrophages, anti-L3T4 for helper/inducer T cells, and anti-Lyt2 for cytotoxic/suppressor T cells revealed that the major population of infiltrating cells at the early stage of viral infection was Mac-2-positive macrophages. In contrast, macrophages detected by anti-Mac-1 and F4/80 monoclonal antibodies were not found at the early stage of viral infection but were found at intermediate and late stages of viral infection. Helper/inducer T cells and cytotoxic/suppressor T cells also infiltrated the islets at intermediate and late stages of viral infection. Short-term treatment of mice with silica prior to viral infection resulted in an enhancement of beta-cell destruction, leading to the development of diabetes. In contrast, long-term treatment of mice with silica resulted in complete prevention of diabetes caused by a low dose of viral infection and a significant decrease in the incidence of diabetes caused by an intermediate or high dose of viral infection. Furthermore, depletion of macrophages by a specific monoclonal antibody (anti-Mac-2) resulted in a much greater decrease in the incidence of diabetes caused by an intermediate dose of viral infection. However, suppression of helper/inducer T cells and cytotoxic/suppressor T cells, by anti-L3T4 and anti-Lyt2 antibodies, respectively, did not alter the incidence of diabetes. On the basis of these data, it is concluded that macrophages, particularly Mac-2-positive macrophages, play a crucial role in the process of pancreatic beta-cell destruction at the early stage of encephalomyocarditis D virus infection in SJL/J mice.     

Possible role of macrophage-derived soluble mediators in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from DBA/2 mice infected with the D variant of encephalomyocarditis (EMC-D) virus revealed lymphocytic infiltration with moderate to severe destruction of pancreatic beta cells. Our previous studies showed that the major population of infiltrating cells at the early stages of infection is macrophages. The inactivation of macrophages prior to viral infection resulted in the prevention of diabetes, whereas activation of macrophages prior to viral infection resulted in the enhancement of beta-cell destruction. This investigation was initiated to determine whether macrophage-produced soluble mediators play a role in the destruction of pancreatic beta cells in mice infected with a low dose of EMC-D virus. When we examined the expression of the soluble mediators interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in the pancreatic islets, we found that these mediators were clearly expressed at an early stage of insulitis and that this expression was evident until the development of diabetes. We confirmed the expression of these mediators by in situ hybridization with digoxigenin-labelled RNA probes or immunohistochemistry in the pancreatic islets. Mice treated with antibody against IL-1beta or TNF-alpha or with the iNOS inhibitor aminoguanidine exhibited a significant decrease in the incidence of diabetes. Mice treated with a combination of anti-IL-1beta antibody, anti-TNF-alpha antibody, and aminoguanidine exhibited a greater decrease in the incidence of disease than did mice treated with one of the antibodies or aminoguanidine. On the basis of these observations, we conclude that macrophage-produced soluble mediators play an important role in the destruction of pancreatic beta cells, resulting in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Inhibition of virus-induced diabetes mellitus by interferon is influenced by the host strain

The diabetogenic variant of encephalomyocarditis virus (EMC-D) induces a diabetes-like syndrome in certain strains of mice. A study was done to determine if virus-induced diabetes could be prevented by interferon (IFN). It was found that the production of diabetes by EMC-D was blocked by either IFN beta or a variety of IFN-inducers in SWR/J, but not ICR Swiss mice. The replication of EMC-D in cell culture was inhibited by IFN beta. It is concluded that the response of pancreatic beta cells to the protective effect of IFN, is probably under genetic control.     

The poly(C) region affects progression of encephalomyocarditis virus infection in Langerhans' islets but not in the myocardium.

The diabetogenic variant PV2 of encephalomyocarditis virus was cloned, and three recombinants differing in their 5' poly(C) tracts were analyzed. It is shown that the poly(C) region is not essential for infectivity in mice but does influence the virus load and degree of pathological lesions within the Langerhans' islets but not in the myocardium.     

Immunomodulation of encephalomyocarditis virus-induced disease in A/J mice.

The E variant of encephalomyocarditis (EMC) virus causes an encephalomyelitis and coagulative necrosis of the pancreas and parotid glands in some but not all strains of inbred and outbred mice. In other models of disease caused by picornaviruses, depletion of specific lymphocyte subsets abrogates the development of tissue lesions. In this study, severe encephalomyelitis and acinar pancreatitis and parotitis developed in adult male A/J mice infected with 100 PFU of EMC virus. Depletion of the CD4+ subset of T lymphocytes in vivo with a monoclonal antibody (MAb) prior to EMC virus inoculation protects mice from developing encephalomyelitis, pancreatitis, and parotitis. This effect is also seen when animals are treated with anti-CD4 and anti-CD8 in combination, but the anti-CD8 MAb alone does not ameliorate the disease. Overall, concentrations of virus in tissues from anti-CD4-treated animals are lower than in immunologically intact control mice. Small-plaque variants of virus were also recovered from the tissues in some animals in this group. CD4+ lymphocytes are involved in the expression of EMC virus-induced pancreatitis and parotitis in A/J mice. This specific subset of T cells would appear to influence EMC viral tropism or replication in various organs.     

Viruses in type 1 diabetes: brief review

Type 1 diabetes results from the progressive destruction of insulin-producing pancreatic beta cells. Although the etiology of type 1 diabetes is believed to have a major genetic component, studies on the risk of developing type 1 diabetes suggest that environmental factors, such as viruses, may be important etiological determinants. Among the viruses, the most clear and unequivocal evidence that a virus induces type 1 diabetes in animals comes from studies on the D variant of encephalomyocarditis (EMC-D) virus in mice and Kilham rat virus (KRV) in rats. A high titer of EMC-D viral infection results in the development of diabetes within 3 days, primarily due to the rapid destruction of beta cells by viral replication within the cells. A low titer of EMC-D viral infection results in the recruitment of macrophages to the islets. Soluble mediators produced by the activated macrophages such as interleukin-1Beta, tumor necrosis factor-alpha, and nitric oxide play a critical role in the destruction of residual beta cells. KRV causes autoimmune type 1 diabetes in diabetes resistant-BioBreeding rats by breakdown of immune balance, including the preferential activation of effector T cells, such as Th1-like CD45RC+CD4+ T cells and CD8+ T cells, and down-regulation of Th2-like CD45RC-CD4+ and CD4+CD25+ T cells, rather than by direct infection of pancreatic beta cells.     

Prevention of Encephalomyocarditis Virus-Induced Diabetes in Mice by Inhibition of the Tyrosine Kinase Signalling Pathway and Subsequent Suppression of Nitric Oxide Production in Macrophages

Macrophages comprise the major population of cells infiltrating pancreatic islets during the early stages of infection in DBA/2 mice by the D variant of encephalomyocarditis virus (EMC-D virus). Inactivation of macrophages prior to viral infection almost completely prevents EMC-D virus-induced diabetes. This investigation was initiated to determine whether a tyrosine kinase signalling pathway might be involved in the activation of macrophages by EMC-D virus infection and whether tyrosine kinase inhibitors might, therefore, abrogate EMC-D virus-induced diabetes in vivo. When isolated macrophages were infected with EMC-D virus, inducible nitric oxide synthase mRNA was expressed and nitric oxide was subsequently produced. Treatment of macrophages with the tyrosine kinase inhibitor tyrphostin AG126, but not tyrphostin AG556, prior to EMC-D virus infection blocked the production of nitric oxide. The infection of macrophages with EMC-D virus also resulted in the activation of the mitogen-activated protein kinases (MAPKs) p42(MAPK/ERK2)/p44(MAPK/ERK1), p38(MAPK), and p46/p54(JNK). In accord with the greater potency of AG126 than of AG556 in blocking EMC-D virus-mediated macrophage activation, the incidence of diabetes in EMC-D virus-infected mice treated with AG126 (25%) was much lower than that in AG556-treated (75%) or vehicle-treated (88%) control mice. We conclude that EMC-D virus-induced activation of macrophages resulting in macrophage-mediated beta-cell destruction can be prevented by the inhibition of a tyrosine kinase signalling pathway involved in macrophage activation.     

Role of Nitric Oxide in the Pathogenesis of Encephalomyocarditis Virus-Induced Diabetes in Mice

The D variant of encephalomyocarditis virus (EMC-D virus) causes diabetes in mice by destroying pancreatic β cells. In mice infected with a low dose of EMC-D virus, macrophages play an important role in β-cell destruction by producing soluble mediators such as interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and nitric oxide (NO). To investigate the role of NO and inducible NO synthase (iNOS) in the development of diabetes in EMC-D virus-infected mice, we infected iNOS-deficient DBA/2 mice with EMC-D virus (2 × 10² PFU/mouse). Mean blood glucose levels in EMC-D virus-infected iNOS-deficient mice and wild-type mice were 205.5 and 466.7 mg/dl, respectively. Insulitis and macrophage infiltration were reduced in islets of iNOS-deficient mice compared with wild-type mice at 3 days after EMC-D virus infection. Apoptosis of β cells was decreased in iNOS-deficient mice, as evidenced by reduced numbers of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells. There were no differences in mRNA expression of antiapoptotic molecules Bcl-2, Bcl-xL, Bcl-w, Mcl-1, cIAP-1, and cIAP-2 between wild-type and iNOS-deficient mice, whereas expression of proapoptotic Bax and Bak mRNAs was significantly decreased in iNOS-deficient mice. Expression of IL-1β and TNF-α mRNAs was significantly decreased in both islets and macrophages of iNOS-deficient mice compared with wild-type mice after EMC-D virus infection. Nuclear factor κB was less activated in macrophages of iNOS-deficient mice after virus infection. We conclude that NO plays an important role in the activation of macrophages and apoptosis of pancreatic β cells in EMC-D virus-infected mice and that deficient iNOS gene expression inhibits macrophage activation and β-cell apoptosis, contributing to prevention of EMC-D virus-induced diabetes.Type 1 diabetes results from absolute insulin deficiency caused by destruction of insulin-producing pancreatic β cells. The D variant of encephalomyocarditis virus (EMC-D virus) induces diabetes in genetically susceptible strains of mice by infecting and destroying β cells (13-18). In mice infected with a low dose (1 × 10² PFU/mouse) of EMC-D virus, macrophages play a central role in the destruction of pancreatic β cells (4, 5, 13-15), as evidenced by a significant increase in the incidence of diabetes if macrophages are activated prior to viral infection and complete prevention of EMC-D virus-induced diabetes if macrophages are inactivated prior to viral infection (4). Additional studies found that selective EMC-D viral infection of pancreatic β cells results in an initial recruitment of macrophages into the islets, followed by infiltration of other immunocytes, including T cells, natural killer cells, and B cells (5).EMC-D virus infects and activates macrophages without replication (13) and induces the production of soluble mediators such as interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS), which play important roles in the destruction of β cells (14). These infected macrophages express significantly more iNOS than either IL-1β or TNF-α (13). Treatment of EMC-D virus-infected mice with the tyrosine kinase inhibitor AG126, which inhibits nitric oxide (NO) production in EMC-D virus-infected macrophages, decreases the expression of IL-1β and TNF-α in the pancreatic islets and the incidence of diabetes and insulitis compared with those in vehicle-treated control mice (13). As well, treatment of EMC-D virus-infected mice with an iNOS inhibitor decreases the incidence of diabetes (14). These results suggest that iNOS and NO significantly contribute to the destruction of pancreatic β cells in mice infected with a low dose of EMC-D virus, although their roles are not fully understood.To directly test whether iNOS and NO play a critical role in the pathogenesis of EMC-D virus-induced diabetes in mice, we used iNOS knockout (KO) DBA/2 mice. We found that iNOS-deficient mice infected with EMC-D virus (2 × 10² PFU/mouse) showed a significantly lower incidence of diabetes. There was reduced expression of IL-1β and TNF-α in macrophages and decreased infiltration of immunocytes in the islets of iNOS-deficient mice, resulting in reduced apoptosis of β cells compared with that in EMC-D virus-infected wild-type mice. This study provides direct evidence of a role of NO in the activation of macrophages by EMC-D viral infection and in the pathogenesis of low-dose (2 × 10² PFU/mouse) EMC-D virus-induced diabetes.     

Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence

Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.     

Susceptibility of four species of small rodents to encephalomyocarditis (EMC) virus infection

The D variant of encephalomyocarditis virus (10(1)-10(5) PFU/head) was intraperitoneally inoculated into 4 species of small rodents, rats, mice, Syrian hamsters, and Mongolian gerbils, and the susceptibility of these animals to EMC virus was examined virologically and histopathologically 3 days after infection. Viral replication was detected in the brain (mice), in the heart (mice and gerbils), and in the pancreas (mice, hamsters, and gerbils). No viral replication was detected in rats. Histopathological changes were seen in the brain (mice and hamsters), in the heart (mice and gerbils), and in the pancreas (mice, hamsters, and gerbils). No histopathological changes were seen in rats. The present results suggest that it may be quite possible to produce EMC virus-induced diabetes mellitus not only in mice but also in hamsters and gerbils.     

Blockade but Not Overexpression of the Junctional Adhesion Molecule C Influences Virus-Induced Type 1 Diabetes in Mice

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing beta-cells in the pancreas. Recruitment of inflammatory cells is prerequisite to beta-cell-injury. The junctional adhesion molecule (JAM) family proteins JAM-B and JAM–C are involved in polarized leukocyte transendothelial migration and are expressed by vascular endothelial cells of peripheral tissue and high endothelial venules in lympoid organs. Blocking of JAM-C efficiently attenuated cerulean-induced pancreatitis, rheumatoid arthritis or inflammation induced by ischemia and reperfusion in mice. In order to investigate the influence of JAM-C on trafficking and transmigration of antigen-specific, autoaggressive T-cells, we used transgenic mice that express a protein of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen in the β-cells of the islets of Langerhans under the rat insulin promoter (RIP). Such RIP-LCMV mice turn diabetic after infection with LCMV. We found that upon LCMV-infection JAM-C protein was upregulated around the islets in RIP-LCMV mice. JAM-C expression correlated with islet infiltration and functional beta-cell impairment. Blockade with a neutralizing anti-JAM-C antibody reduced the T1D incidence. However, JAM-C overexpression on endothelial cells did not accelerate diabetes in the RIP-LCMV model. In summary, our data suggest that JAM-C might be involved in the final steps of trafficking and transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans.     

Viral determinants of age-dependent virulence of Sindbis virus for mice.

Many alphaviruses cause more severe disease in young animals than in older animals. The age-dependent resistance to severe disease is determined primarily by maturation of the host, but strains of virus can be selected that overcome the increased resistance of mature animals. Sindbis virus (SV) strain AR339 causes fatal encephalitis in newborn mice and nonfatal encephalitis in weanling mice, whereas NSV, a neuroadapted strain of SV, causes fatal encephalitis in weanling as well as newborn mice. We have previously shown that the E2 glycoprotein of NSV contained His-55, whereas AR339 E2 had Gln-55 (S. Lustig, A. C. Jackson, C. S. Hahn, D. E. Griffin, E. G. Strauss, and J. H. Strauss, J. Virol. 62:2329-2336, 1988) and that SV with E2 containing Gly-172 was more virulent for newborn mice than SV with E2 containing Arg-172 (P. C. Tucker and D. E. Griffin, J. Virol. 65:1551-1557, 1991). Here we tested the virulence for both newborn and older mice of SV containing a number of different amino acids at E2 position 55 (His, Gln, Lys, Arg, Glu, Gly) in combination with both Gly-172 and Arg-172. All the viruses were virulent for newborn mice, but the residues at both 55 and 172 influenced the virulence of the virus, and there were differences in virulence observed among the various viruses. However, only viruses with His-55 were fully virulent for 14-day-old mice, and this virulence was independent of the residue at position 172. Virus with Lys-55 was virulent for 7-day-old mice, although slightly attenuated relative to His-55. Viruses with His-55 grew more rapidly and to higher titer in the brains of 7- and 14-day-old mice, in N18 neuroblastoma cells, and in BHK cells. Our data suggest that His-55 is important for neurovirulence in older mice and acts by increasing the efficiency of virus replication.     

Role of Hck in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice

Soluble mediators such as interleukin-1beta, tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) produced from activated macrophages play an important role in the destruction of pancreatic beta cells in mice infected with a low dose of the D variant of encephalomyocarditis (EMC-D) virus. The tyrosine kinase signaling pathway was shown to be involved in EMC-D virus-induced activation of macrophages. This investigation was initiated to determine whether the Src family of kinases plays a role in the activation of macrophages, subsequently resulting in the destruction of beta cells, in mice infected with a low dose of EMC-D virus. We examined the activation of p59/p56(Hck), p55(Fgr), and p56/p53(Lyn) in macrophages from DBA/2 mice infected with the virus. We found that p59/p56(Hck) showed a marked increase in both autophosphorylation and kinase activity at 48 h after infection, whereas p55(Fgr) and p56/p53(Lyn) did not. The p59/p56(Hck) activity was closely correlated with the tyrosine phosphorylation level of Vav. Treatment of EMC-D virus-infected mice with the Src kinase inhibitor, PP2, resulted in the inhibition of p59/p56(Hck) activity and almost complete inhibition of the production of TNF-alpha and iNOS in macrophages and the subsequent prevention of diabetes in mice. On the basis of these observations, we conclude that the Src kinase, p59/p56(Hck), plays an important role in the activation of macrophages and the subsequent production of TNF-alpha and nitric oxide, leading to the destruction of pancreatic beta cells, which results in the development of diabetes in mice infected with a low dose of EMC-D virus.     

Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate β cell/islet dysfunction and β cell loss.     

T-helper cell and associated antibody response to synthetic peptides of the E glycoprotein of Murray Valley encephalitis virus.

A battery of 16 synthetic peptides, selected primarily by computer analysis for predicted B- and T-cell epitopes, was prepared from the deduced amino acid sequence of the envelope (E) glycoprotein of Murray Valley encephalitis (MVE) virus. We examined all of the peptides for T-helper (Th)-cell recognition and antibody induction in three strains of mice: C57BL/6, BALB/c, and C3H. Lymphoproliferative and interleukin-2 assays were performed on splenic T cells from mice inoculated with peptides in Freund's incomplete adjuvant or with MVE virus. Several peptides found to contain predicted T-cell epitopes elicited a Th-cell response in at least one strain of mice, usually with a concomitant antibody response. Peptides 145 (amino acids 145 to 169) and 17 (amino acids 356 to 376) were strongly recognized by T cells from all three inbred strains of mice. Peptide 06 (amino acids 230 to 251) primed C57BL/6 mice for Th- and B-cell reactivity with native MVE virus, and T cells from virus-immune mice were stimulated by this peptide. Peptide 06 was recognized by several Th-cell clones prepared from mice immunized with MVE, West Nile, or Kunjin virus. These results indicate that it may be feasible to design synthetic flavivirus peptides that define T-cell epitopes capable of generating a helper cell response for B-cell epitopes involved in protective immunity.     

Biological properties of strains of tick-borne encephalitis virus isolated in the natural foci of the eastern part of the Russian plain

Strains of tick-borne encephalitis virus isolated in the natural foci of infection in the eastern part of the Russian Plain (the Kirov region) were examined for their biological properties. The strains examined were 69 strains isolated from ticks Ixodes persulcatus, 62 strains obtained from patients with the clinically manifest form of tick-borne encephalitis and 56 strains isolated from the blood of patients with the inapparent form of infection. Comparative studies on laboratory animals (albino mice, golden hamsters, suckling guinea pigs and other mammals) as well as comparative serologic studies provided evidence which suggested that all virus isolates from the Kirov region were antigenically identical with the strain "Sofin" isolated in the Far East and represent thus a single causative agent of the tick-borne encephalitis virus infection. This strain of virus is supposed to exist in two variants, in dependence on ecological conditions: one of these variants is the eastern variant (strain Sofin and strains from the Kirov region) and the other one is the western variant of tick-borne encephalitis virus.     

Analysis of role of CD8+ T cells in resistance to murine AIDS in A/J mice.

The mixture of retroviruses termed LP-BM5 murine leukemia virus (MuLV) contains a replication-defective genome (BM5def), the crucial element for induction of murine AIDS (MAIDS), as well as helper B-tropic ecotropic and mink cell focus-forming MuLV. Among Fv-1b mouse strains, C57BL mice are sensitive to infection by these viruses and to development of MAIDS, but A/J mice are highly resistant to all viral components and to induction of disease. Inasmuch as previous genetic studies indicated a major role in susceptibility for the H-2D locus within the MHC, the effect of CD8+ T cells in A/J resistance to MAIDS was analyzed by depletion of this subset using mAb. A/J mice treated with anti-CD8 mAb beginning soon after inoculation with LP-BM5 MuLV developed disease within 5 wk after virus inoculation. Histopathologic and flow cytometry alteration of tissues and cells from the mAb-treated mice were identical to those seen in virus-infected MAIDS-sensitive strains, and assays for MuLV demonstrated high-level expression of ecotropic MuLV and integration of BM5def. Parallel studies of A/J mice treated with anti-CD4 mAb after infection revealed enhanced expression of ecotropic MuLV but no integration of BM5def, and no signs of MAIDS were detected. These observations indicate that CD8+ T cells are critical in the resistance of A/J mice to LP-BM5 MuLV replication and development of disease and suggest that CD4+ T cells play a role in regulation of ecotropic virus replication.     

Dysregulated B7-1 and B7-2 expression on nonobese diabetic mouse B cells is associated with increased T cell costimulation and the development of insulitis

Little is known about the pathogenic role of B cell dysfunction in T cell-mediated autoimmune disease. We previously reported that B cell hyper-responsiveness, resistance to apoptosis, and accumulation in islets occur during the onset of insulitis, but not in type 1 diabetes (T1D), in NOD mice. In this study we extended these studies to further determine how islet-infiltrated B cells contribute to this inflammatory insulitis. We demonstrate the presence of an increased percentage of B7-1(+) and a decreased percentage of B7-2(+) B cells in the spleen of autoimmune disease-prone NOD and nonobese diabetes-resistant mice compared with the spleen of nonautoimmune disease-prone C57BL/6 and BALB/c mice. An age-dependent differential expression of B7-1 and B7-2 was associated with the development of insulitis and CD4(+)CD25(+) T cell deficiency in autoimmune disease-prone mice. Whereas BCR and LPS stimulation increased B7-2 expression on B cells from autoimmune disease-prone and nonautoimmune disease-prone mice, LPS-induced B7-1 expression was higher on NOD than C57BL/6 B cells. Interestingly, increased expression of B7-1 and B7-2 was found on islet-infiltrated B cells, and this increase was associated with enhanced T cell costimulation. Islet-infiltrated B cells were shown to be a source of TNF-alpha production in islets. B7 blockade of BCR-stimulated NOD B cells by anti-B7-1 and anti-B7-2 mAbs during coadoptive transfer with diabetogenic T cells into NOD.scid mice protected these recipients from T1D. These results suggest that increased B7-1 and B7-2 expression on islet-infiltrated NOD B cells is associated with increased T cell costimulation and the development of inflammatory insulitis in NOD mice.     

Immunity to leprosy. II. Genetic control of murine T cell proliferative responses to Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured after immunization of mice at the base of the tail with antigen and challenging lymphocytes from draining lymph nodes in culture with M. leprae. This T cell response to M. leprae has been compared in 18 inbred strains of mice. C57BL/10J mice were identified as low responder mice. The congenic strains B10.M and B10.Q were found to be high responders, whereas B10.BR and B10.P were low responders. F1 (B10.M X C57BL/10J) and F1 (B10.Q X C57BL/10J) hybrid mice were found to be low responders, similar to the C57BL/10J parent, indicating that the low responsive trait is dominant. Whereas B10.BR mice were shown to be low responders to M. leprae, B10.AKM and B10.A(2R) were clearly high responders, indicating that the H-2D region influences the magnitude of the T cell proliferative response. Gene complementation within the H-2 region was evident. Genes outside the H-2 region were also shown to influence the response to M. leprae. C3H/HeN were shown to be high responder mice, whereas other H-2k strains, BALB.K, CBA/N, and B10.BR, were low responders. Gene loci that influence the T cell proliferation assay have been discussed and were compared to known background genes which may be important for the growth of intracellular parasites. Because mycobacteria are intracellular parasites for antigen-presenting cells, genes that affect bacterial growth in these cells will also influence subsequent immune responses of the host.     

Class II-restricted T cell responses in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Cross-specificity among TMEV substrains and related picornaviruses, but not myelin proteins

Following intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV), susceptible mouse strains develop a chronic demyelinating disease characterized by mononuclear cell-rich infiltrates in the central nervous system. Current evidence strongly supports an immune-mediated basis for myelin breakdown, with an effector role proposed for TMEV-specific, major histocompatibility class II-restricted delayed-type hypersensitivity, which temporally correlates with disease onset and remains chronically elevated in susceptible mice. This study examined the fine specificity of class II-restricted T cell responses in TMEV-infected mice to better define the relevant virus-encoded T cell determinant(s) responsible for triggering the demyelinating process, and to determine if class II-restricted neuroantigen-specific autoimmune responses could be detected in mice with TMEV-induced demyelination. The data clearly show that T cell responses in TMEV-infected mice are directed against determinants shared by closely related TMEV strains and are cross-reactive with related picornaviruses, such as encephalomyocarditis virus. In contrast, class II-restricted autoimmune responses against syngeneic mouse spinal cord homogenate and the two major protein components of myelin, myelin basic protein and proteolipid protein, are not demonstrable in susceptible SJL/J mice undergoing chronic TMEV-induced demyelinating disease, but are readily seen in SJL/J mice displaying chronic, relapsing experimental allergic encephalomyelitis. Cross-reactivity (or lack thereof), as determined by functional T cell analyses, was found to correlate with the extent of exact amino acid homology between the TMEV capsid proteins, the two neuroantigens, and related picornaviruses. The data thus do not support a major role for autoimmune responses against myelin proteins in TMEV-induced demyelinating disease, but are consistent with our previously proposed hypothesis that TMEV-specific T cell responses constitute a major effector mechanism of myelin breakdown.