Functional analysis of T lymphocyte subsets in antiviral host defense

The role of different T cell subsets in antiviral host defense was investigated by treating thymectomized C57BL/6 and CBA/J mice with monoclonal rat anti-Lyt-2 or anti-L3/T4 IgG 2b antibodies 14 and 10 days before infection. This treatment depleted the respective T cell subsets to undetectable levels in peripheral blood when assayed by immunofluorescence. In mice treated with anti-Lyt-2, induction of cytotoxic T cells was reduced to less than 1 to 2% after intravenous infection with Armstrong strain of lymphocytic choriomeningitis virus (LCMV). In addition, no primary swelling of the footpad could be detected following local inoculation of the virus. In animals treated with anti-L3/T4, antiviral cytotoxic T lymphocyte responses were reduced by a factor of 10. These L3/T4+ cell-depleted mice showed delayed footpad swelling after local injection of LCMV Armstrong. After intracerebral infection with LCMV, anti-Lyt-2-treated mice were resistant and those injected with anti-L3/T4 were totally susceptible to LCMV Armstrong-triggered immunopathologic disease. Virus could be detected in the blood of antibody-treated mice 7 days after inoculation; however, no virus could be measured in the blood of surviving anti-Lyt-2-treated animals 15 days after intracerebral infection. Serum titers of interferon-alpha,beta induced by viral infection remained unaffected by depletion of T cell subsets. Anti-L3/T4 antibody-treated C57BL/6 mice failed to generate IgG antibodies against the New Jersey strain of vesicular stomatitis virus, whereas Lyt-2+ cell-depleted mice had normal antivesicular stomatitis virus (New Jersey strain) IgG antibody titers.     

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.     

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.     

Virus-induced diabetes mellitus: mengovirus infects pancreatic beta cells in strains of mice resistant to the diabetogenic effect of encephalomyocarditis virus.

In mice, Mengovirus produces a fatal encephalitis. Plaque purification of the virus resulted in the isolation of a clone (Mengo- 2T ), which in addition to encephalitis caused diabetes. Microscopic examination of pancreases from infected mice revealed necrosis in the islets of Langerhans and infiltration of inflammatory cells. By immunofluorescence viral antigens were found in the islets, and radioimmunoassays demonstrated a substantial decrease in pancreatic immunoreactive insulin. Studies on susceptibility among inbred strains of mice showed that whereas the D variant of encephalomyocarditis virus caused diabetes only in SJL/J mice, Mengo- 2T caused diabetes in strains of mice resistant to encephalomyocarditis-induced diabetes (i.e., CBA/J, C3H/HeJ, CE/J, AKR/J, C57BL/6J). The ability of Mengo- 2T to induce diabetes in encephalomyocarditis-resistant mice was found to be due to the greater capacity of Mengo- 2T as compared to the D variant of encephalomyocarditis virus to replicate in and destroy the islets of these animals. Although Mengo- 2T and the D variant of encephalomyocarditis virus are antigenically indistinguishable by hyperimmune sera, our studies show that these viruses have different host ranges and tissue tropisms .     

Involvement of L3T4+, LYT2.2+ T cell subsets and non-T cells in the resistance of mice against Trypanosoma cruzi infection

Cellular populations involved in resistance against T. cruzi infection were characterized from mice chronically infected with the parasite. Mice transfused with spleen cells (SC), nylon-wool-non-adherent spleen cells (NWNA) or sera from mice chronically infected with T. cruzi, showed an enhanced resistance against challenge with the parasite. The protective activity of NWNA but not of SC was completely abrogated by treatment with anti-Thy1.2 monoclonal antibodies (mAb) and complement (C). Pretreatment of NWNA cells from chronically infected mice with either anti-L3T4 or anti-Lyt 2.2 mAb partially reduced the transfer of resistance. When both L3T4+ and Lyt2.2+ cells were depleted from NWNA populations, transfer of resistance was abolished. These results appear to indicate that L3T4+, Lyt2.2+ T cell subsets and non-T cells are involved in the immunity to T. cruzi.     

Role of CD4+ and CD8+ T cells in murine resistance to street rabies virus.

Mice of the SJL/J and BALB/cByJ inbred strains are naturally resistant to street rabies virus (SRV) injected via the intraperitoneal route. To determine the cellular mechanism of resistance, monoclonal antibodies specific for CD4+ or CD8+ subsets of T cells were used to deplete the respective cell population in SRV-infected animals. Elimination of CD4+ T-helper cells abrogated the production of immunoglobulin G (IgG) neutralizing antibodies in response to rabies virus infection and reversed the resistant status of SJL/J and BALB/cByJ mice. In contrast, in vivo depletion of CD8+ cytotoxic T cells had no measurable effect on host resistance to SRV. These results indicate that serum neutralizing antibodies of the IgG class are a primary immunological mechanism of defense against rabies virus infection in this murine model of disease. CD8+ cytotoxic T lymphocytes, which have been shown to transfer protection in other rabies virus systems, appear to have no role in protecting mice against intraperitoneally injected SRV.     

Modulation of lymphocyte subsets in Peyer's patches of mice treated with monoclonal antibody against helper T-cells

Treatment of mice with anti-L3T4, a monoclonal antibody directed against helper T-cells, impairs clearance of intestinal Giardia muris infection. The present study examined the effect of anti-L3T4 treatment on mouse Peyer's patch cytoarchitecture and on the distribution of T-cell subsets within microenvironments of the follicle. Female BALB/c mice, aged 8 weeks, were given 4-7 weekly injections of either anti-L3T4 (1 mg/wk) or PBS (control group), and Peyer's patches were examined by immunohistochemistry or flow cytometry. In anti-L3T4-treated mice, Peyer's patch follicles (B-cell areas) were about two thirds the size of follicles in controls, and virtually all the size difference occurred in germinal centers. Peyer's patches were depleted of L3T4+ cells, yet the proportion of Thy-1.2+ (all T) cells was not decreased correspondingly, and the distribution of Thy-1.2+ cells in the patches was similar to that in control mice. In anti-L3T4-treated mice, Thy-1.2+ cells consisted of (a) Ly-2+ (cytotoxic/suppressor T) cells, and (b) a population of Thy-1.2+ cells that were neither L3T4+ nor Ly-2+. After treatment, Ly-2+ cells accounted for most of the T-cells in interfollicular areas and were also scattered in follicles, in germinal centers, and below the dome epithelium--in areas where L3T4+ cells predominated in control mice. Thy-1.2+ cells that were L3T4- and Ly-2- were mainly localized below the dome epithelium. These shifts indicate complex interrelationships among different lymphocyte subsets in Peyer's patches.     

T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T-cell and antibody responses.

This study documents the differential requirements of T4+ T helper cells in the induction of virus-specific cytotoxic T-lymphocyte (CTL) and antibody responses during acute lymphocytic choriomeningitis virus infection. Two monoclonal antibodies (GK1.5 and RL172.4) directed against the L3T4 (T4) molecule were used for depleting T helper cells from mice. Depletion of T4+ cells caused a pronounced suppression of antiviral antibody response (20-fold decrease) but had minimal effect on virus-specific CTL response (less than 2-fold reduction). Despite the elimination of greater than 90% of T helper cells, anti-L3T4-treated mice were able to generate a CTL response of sufficient magnitude to control the viral infection. In contrast, depletion of Lyt2+ T cells abrogated the CTL response and the ability to eliminate virus. Thus, our results underscore the importance of the Lyt2+ T-cell subset in controlling infection with this virus and show that a deficiency of T4+ T cells is likely to have a more severe effect on antibody production than on CTL responses.     

Gut-associated immune effector responses in immunocompetent and immunocomprimised mice with Giardia lamblia

Abstract Significantly higher Giardia lamblia trophozoites load in the intestine of infected mice accompanied pronounced influx of suppressor/cytotoxic T cells (Lyt 2.2⁺), T cells (Thy 1.2⁺) and significant reduction in IgA-containing cells in the gut during the establishment and peak phases of infection. The induction of helper/inducer T cells (Lyt 1.1⁺) and significant enhancement of IgA-containing cells in gut resulted in the decline of the trophozoite loads. However, the prior treatment of animals with dexamethasone alone resulted in significant reduction in helper/inducer T cells (Lyt 1.1⁺) and the IgA-containing cells in the gut; the percents of suppressor/cytotoxic T cells (Lyt 2.2⁺) and IgM-containing cells remained unaltered. Although the G. lamblia infection in such animals further significantly increased the influx of suppressor/cytotoxic T cells, the late response of helper/inducer T cells and IgA-containing cells was abrogated during the decline phase of infection. The significant reduction in the trophozoite load — despite immuno-suppressive therapy — appeared to be due to unaltered IgM response in such animals which probably took over the function of IgA in defense against G. lamblia . The data of the investigation thus suggested a role of helper/inducer T cells and antibodies producing cells in gut as important effector cells resulting in the termination of primary G. lamblia infection.     

Enumeration of T lymphocyte subsets by monoclonal antibodies in young and aged humans

The percentage and absolute number of peripheral blood mononuclear cells reactive with monoclonal antibodies identifying mature thymocytes and T cells (T3+), helper/inducer cells (T4+), and suppressor/cytotoxic cells (T8+) was determined in 19 young (mean age 35 yr) and 31 elderly (mean age 72 yr) individuals. The percent representation but not the absolute number of T cells (T3+) declined significantly (p less than 0.001) in the elderly, and the decline was attributable to both an absolute and relative decrease in the representation of the subpopulation of cytotoxic/suppressor (T8+) cells. The percentage and number of helper/inducer (T4+) T cells was comparable in both age groups.     

The role of CD4(+) T cells in biphasic hind limb paralysis induced by the D variant of encephalomyocarditis virus (EMC-D) in DBA/2 mice.

DBA/2 CrSlc mice infected with the D variant of encephalomyocarditis virus (EMC-D) (10 PFU/head) developed biphasic hind limb paralysis due to spinal cord lesion. The early phase lesion was characterized by demyelination with infiltration of macrophages in the funiculus lateraris and the late phase lesion by degeneration of motor neurons with infiltration of CD4(+) T cells in the cornu ventrale. In the present study, treatment with anti-Mac1 monoclonal antibody (MAb) or anti-CD4 MAb prior to virus infection (-3 to -1 days) reduced the early phase lesion and the incidence of the first paralysis. Signals of viral RNAs were observed only in a few oligodendrocytes in the funiculus lateraris. Treatment with anti-CD4 MAb from 31 to 33 days post infection when mice showed recovery from the first paralysis reduced the late phase lesion and prevented the second paralysis. Signals of viral RNAs were still detected in a few degenerated neurons in the cornu ventrale. These results indicate that while macrophages and CD4(+) T cells participate in the early phase lesion and paralysis and only CD4(+) T cells in the late phase lesion and paralysis.     

Interaction of reovirus with cell surface receptors. IV. The reovirus type 3 receptor is expressed predominantly on murine Lyt-2,3+ and human T8+ cells

Reovirus type 3 binds to approximately 20% of murine and human T cells via the viral hemagglutinin, a small outer capsid polypeptide. By using purified viral particles as a ligand in a standard plate separation technique, we have been able to enrich human peripheral blood and murine splenic T cells for reovirus receptor-positive cells (reovirus 3+) to levels of 88 to 92%. Analysis of reovirus 3+ T cells with monoclonal antibodies that identify inducer and suppressor/cytotoxic cells demonstrated that in the mouse, 68% of reovirus 3+ cells were Lyt-2+, and in the human, 60% were T8+. In reciprocal experiments, when subpopulations of murine and human T cells were prepared with the use of monoclonal anti-T cell reagents, 16% of Lyt-1+ and 81% of Lyt-2+ cells bound reovirus, whereas 30% of T4+ and 65% of T8+ cells bound reovirus. To determine whether reovirus type 3 identified a functional as well as a phenotypic category of cells, an antigen-specific cytotoxic T cell assay was employed. There was complete loss of cytotoxic activity in the reovirus 3+ cell population and slight enhancement of cytotoxic activity in the cell population from which reovirus 3+ cells were removed. This suggested that reovirus was binding to functionally active suppressor cells. Furthermore, adoptive transfer of antigen-specific T cells that were enriched for reovirus 3+ cells demonstrated suppression of cytoxic T cell activity. These results suggest that reovirus type 3 may identify a structure common to a subclass of murine and human T cells and that by using the virus as a natural biologic probe for cell surface receptors, one may be able to functionally segregate murine cytotoxic from suppressor T cells.     

Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1+ 2- T lymphocytes

Optimal conditions were established for the adoptive transfer of experimental allergic encephalomyelitis (EAE) in SJL/J mice. Lymph node cells from SJL/J mice primed in vivo with myelin basic protein (BP) were incubated in vitro with BP. These cells proliferated specifically to BP and when transferred at the optimal conditions into syngeneic mice induced EAE in 100% of the recipients. The in vitro proliferative response to BP was dependent on the presence of Lyt 1+ 2- T lymphocytes. Furthermore, when the activated LNC were treated before transfer with anti-Thy 1 or anti-Lyt 1 antibody and C, neither clinical nor histologic signs were observed in the recipients, whereas treatment with anti-Lyt 2 antibody and C had no effect. These results indicated that Lyt 1+ 2- T cells are responsible for the transfer of EAE.     

T cell synergy in the primary MLR: proliferative kinetics, effector cell generation, and IL 2 production

A primary rat MLR was initiated, and on each of 8 consecutive days during the evolving culture, an aliquot of cells was separated into its constitutive helper/inducer (W3/25+) and suppressor/cytotoxic (OX8+) T cell subsets by a monoclonal antibody, Degalan-bead immunoadsorbent column technique. This allowed a detailed kinetic analysis of T cell proliferation, the generation of effector cells, and the production of IL 2 by each subset relative to net whole culture supernatant IL 2 activity. The primary MLR demonstrates an early period of helper/inducer cell proliferation, IL 2 production and accumulation, followed by a period of suppressor/cytotoxic cell (OX8+) proliferation and IL 2 consumption during which there are distinct waves of allospecific suppressor, followed by cytotoxic activity. If fresh T cells of the helper/inducer or suppressor/cytotoxic phenotype were preseparated and then cultured alone with irradiated allogeneic stimulator cells, proliferation was noted in both subsets despite no demonstrable IL 2 activity in cultures of the suppressor/cytotoxic cells. Finally, a suppressed primary MLR exhibited proliferative inhibition of both T cell subsets.     

Abnormalities of the in vitro cellular and humoral responses to tetanus and influenza antigens with concomitant numerical alterations in lymphocyte subsets in Down syndrome (trisomy 21)

Peripheral blood leukocytes (PBL) from noninstitutionalized individuals with trisomy 21, paired with closely age-matched and/or family members as controls, were analyzed for different aspects of their cellular and humoral immune responses, and were phenotypically characterized by means of various monoclonal antibodies. Both the in vitro PBL proliferative and antibody responses to a bacterial antigen (tetanus toxoid) and to viral antigens (influenza A/Bangkok and B/Singapore) were significantly decreased in trisomy 21. In addition, bacterial and viral antigen-induced in vitro interleukin 2 (IL 2) production was markedly reduced, although mitogen (PHA)-stimulated IL 2 production was not impaired. The functional abnormalities observed in trisomy 21 PBL occur concomitantly with numerical alterations in circulating lymphocyte subsets in these same individuals. Although no difference was observed between the trisomic and control groups in the percentage of total T and B lymphocytes, a decreased level of Leu-3a + 3b-positive cells (T helper/inducer cells) and an increased level of Leu-2a-positive cells (T suppressor/cytotoxic cells) that co-expressed Leu-15 (suppressor alone) were noted.     

Evaluation of the circulating and splenic lymphocyte subpopulations in patients with non-Hodgkin lymphomas and Hodgkin's disease using monoclonal antibodies

The number of B lymphocytes, T lymphocytes and their helper/inducer, cytotoxic/suppressor and NK/K subpopulations was measured in peripheral blood and spleen cell suspensions from patients with Hodgkin's disease (HD) in the active stage of the disease and in remission status, as well as in Non-Hodgkin lymphomas (NHL) in active stage of the disease. B lymphocytes were determined by direct immunofluorescence and T lymphocytes with the E rosette technique. Helper/inducer, cytotoxic/suppressor, and NK/K T lymphocytes were determined by indirect immunofluorescence with the monoclonal antibodies OKT4, OKT8 and Leu 7 (HNK1). In the same way, Lyt3 was used for determination of the total T lymphocytes. Whereas in peripheral blood of the NHL group an increase of B lymphocytes and a slight reduction of T lymphocytes could be observed, with normal distribution of the subpopulations, in patients with active HD as well as in those in remission, a marked absolute and relative decrease of T helper/inducer cells was found with normal cytotoxic/suppressor and NK/K proportion. In contrast to this, a significant increase of helper/inducer T lymphocytes with decreased cytotoxic/suppressor T proportion was found in spleen cell suspensions of patients with HD.     

Two-dimensional flow cytometric analysis of T cell subsets maintained in IL-2 medium after autologous mixed leukocyte reaction activation

Human T cells are stimulated with an autologous mixed leukocyte reaction (AMLR) and can be propagated in interleukin-2. Staining of the cultured cells with the combination of two monoclonal antibodies was evaluated by two-dimensional flow cytometry at weekly intervals. AMLR activation resulted in an initial preservation of the CD4+ (helper/inducer T) subset predominance over the CD8+ (suppressor/cytotoxic T) cells, noted on normal circulating blood lymphocytes. However, during culture in interleukin-2, there was a progressive increase in the percentages of CD8+ Leu 15- cytotoxic T, CD4+ Leu 8- helper T, and CD3+ HLA-DR+ activated T cells, and a concomitant decrease in those of CD4+ Leu 8+ suppressor inducer T and CD8+ Leu 15+ suppressor T cells if the responder sheep red blood cell (SRBC)-rosetting T cells were made up by tris ammonium chloride, but not by hypotonic shock treatment to lyse SRBC. The significant difference between hypotonic shock-T cells and ammonium chloride-T cells in the phenotypic changes of T cell subsets after long-term culture in an interleukin-2 medium may suggest a regulatory role of the ammonium chloride-sensitive T cells in the AMLR.     

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.     

Influence of stress on the maturity of T-cells

Stress is known to influence the immune function via an effect on the central nervous system. We previously presented data showing that stress alters the population of T-cell subsets in mice. The variations of T-cell subsets in the thymus, peripheral blood, and spleen in mice similarly stressed by immobilization or by unavoidable and opioid-dependent stress were measured by flow cytometry using the monoclonal antibodies anti-L3T4, anti-Lyt 1, anti-Lyt 2 and anti-Thy 1, 2. Immobilization stress was applied for three days and T-cell subsets were measured on the days 1, 2 and 3, as well as on day 7 after release from immobilization. Lyt 2-positive cells in the thymus were the most sensitive to stress, showing significant variations. The proportion of immature T-cells increased in the thymus, blood and spleen of the stressed mice. When diazepam or naloxone were administered 30 min before the initiation of stress, these variations tended to decrease. Thus, the ratio of T-cell subsets varied with the duration of immobilization stress. This appeared to be partly mediated by the opioid system and the central nervous system.