Sex-dependent, early cytokine production by NK-like spleen cells following infection with the D variant of encephalomyocarditis virus (EMCV-D)

Spleen cell cultures from diabetes-resistant ICR Swiss females exhibited an increase in expression of Ia antigens 24 hours post-infection (PI) with EMCV-D while comparable spleen cell cultures from diabetes-susceptible males of this strain did not exhibit this increase in Ia antigens expression. A monoclonal antibody specific for mouse interferon-gamma (IFN gamma) eliminated this increase in Ia antigens expression. Interferon-gamma (IFN gamma) and interleukin 2 (IL-2) production by EMCV-D-infected spleen cell cultures were monitored at 4-hour intervals for 24 hours. Female spleen cells produced IFN gamma earlier (less than 16 hours PI) and in greater amounts than did comparably treated male spleen cells. Addition of a monoclonal rat anti-mouse IL-2 to virus-infected cultures did not significantly affect the early (less than 16 hours PI) production of IFN gamma by spleen cells of females. Treatment of the spleen cell donors with rabbit anti-asialo GM1 (AAGM1) abolished early production of IFN gamma in virus-infected female spleen cell cultures and reduced the early IL-2 production by infected male and female cells. These results suggest that an NK-like cell is responsible for the early female IFN gamma production; this may be a factor in the resistance of female ICR Swiss mice to EMCV-D-induced diabetes.     

Tropism and histopathology of the D, B, K, and MM variants of encephalomyocarditis virus

Variants of encephalomyocarditis virus (EMCV), which are immunologically indistinguishable by hyperimmune serum, produce different disease syndromes in mice. For instance, in ICR Swiss male mice, EMCV-B produces no overt illness, EMCV-MM produces severe neurological signs followed by death, EMCV-D destroys pancreatic beta cells producing a disease syndrome resembling insulin-dependent diabetes mellitus, and EMCV-K is lethal but produces no overt signs of infection. The present study was done to determine the tissue tropism and histopathology of each of these EMCV variants in the ICR Swiss mouse model. The data show the highest concentrations in the following organs: EMCV-D in the pancreas, EMCV-B in the pancreas, EMCV-MM in the cerebrum, and EMCV-K in the medulla/brainstem. They also show that the pathological lesions produced by each variant correlate well with viral titers.     

Role of macrophages,interferon gamma and procoagulant activity in the resistance of genetic heterogeneous mouse populations to mouse hepatitis virus infection

Summary Genetic heterogeneous mouse populations selected for high (H_III) and low (L_III) antibody response were used to study some aspects of mouse hepatitis virus 3 (MHV3) infection, such as the resistance pattern, virus replication in the liver and peritoneal exudate or in cultured peritoneal macrophages, the interferon (IFN) synthesis in the serum and peritoneal exudate and the procoagulant activity (PCA) of the peritoneal exudate (PEC) and spleen cells (SC). The H_III mice, when compared to their L_III mice counterparts, were susceptible to MHV3 infection showing higher virus titres in the liver and peritoneal exudate, comparable IFN alpha/beta or IFN gamma titres in the peritoneal exudate or in the serum, and higher levels of PCA of PEC and SC. A higher virus titre was detected in the supernatants of H_III mouse macrophages infected with MHV3. The activation of H_III mouse macrophages with LPS, IFN alpha/beta or IFN gamma, in contrast to that of L_III mouse macrophages, did not induce an antiviral effect with partial restriction of the MHV3 replication. The LPS antiviral activity was shown to be partially exerted by IFN alpha/beta synthesis. The IFN gamma was shown to be more effective in inducing an antiviral state in L_III macrophages, when compared to IFN alpha/beta. The data obtained are consistent with the notion that the resistance mechanisms to the MHV3 infection involve the PCA and the sensitivity of macrophages to IFN.     

Production of lymphokines and interferon by immune cells involved in recovery of mice from herpes simplex virus type 2 hepatitis

Adoptive transfer of spleen cells from mice 4 days after infection with herpes simplex virus type 2 (HSV-2) reduced the virus titer in the liver of recipient mice infected 24 h before transfer. Macrophage chemotactic factor (CF) and macrophage migration inhibition factor (MIF) were produced by day 3 of infection in spleen cell cultures stimulated with HSV-2, but not with control antigen, i.e. 1 day before the cells are active in adoptive transfer. Interferon was produced in cultures established throughout the infection but not in normal spleen cells. From days 1 to 5 of infection interferon was produced irrespective of in vitro restimulation, although the highest amounts were always produced after stimulation with the specific antigen. Spleen cells from mice infected for 6 days produced interferon only when stimulated with HSV-2. The cells from 6-day-immune mice active in adoptive transfer and CF and MIF production were found to be Thy 1+, Ig- and Lyt2-. Both Thy 1+ and plastic adherent cells were necessary for interferon production, whereas Ig+ and Lyt2+ cells did not produce interferon. The interferon was acid stable and neutralized by antiserum against alpha/beta-interferon and thus has the characteristics of alpha-interferon. The data indicate that a delayed type hypersensitivity reaction with lymphokine-induced macrophage recruitment into infectious foci may be a central feature of the recovery process in HSV-2-induced hepatitis. A possible role of interferon produced by the accumulated cells needs further investigation.     

Role of interferon in streptococcal infection in the mouse

In previous studies, we have shown the rapid in vitro induction of IFN gamma from human T cells by highly purified peptic extracts of M proteins from Streptococcus pyogenes. The present report extends these in vitro studies and shows that a mixture of both alpha/beta and gamma IFN were present in spleen cell homogenates after in vivo treatment with M protein wild-type (M+) or mutant (M-) S. pyogenes strains. The levels of bacterial-induced IFN were found to be greater in M+ treated animals. Additional studies in vivo showed that pretreatment of mice with heat-killed M+ S. pyogenes organisms significantly protected mice to pneumococcal infection compared to similarly treated M- or control animals (P less than 0.001). Further, antibodies to mouse IFN alpha/beta and antibodies specific to a synthetic N-terminal peptide of mouse IFN gamma enhanced the death of animals due to pneumococcal infection and blocked the protection observed in animals previously treated with heat-killed M+ organisms. Most importantly, treatment of mice with either type of IFN alone enhanced the survival of mice to levels similar to that observed by treatment with M+ organisms (P less than 0.05). The results strongly suggest that IFN can play a crucial role, directly or indirectly, in controlling infection by Streptococcus pneumoniae and perhaps other streptococci.     

D variant of encephalomyocarditis virus (EMCV-D)-induced diabetes following natural killer cell depletion in diabetes-resistant male C57BL/6J mice

The involvement of natural killer (NK) cells in the development of diabetes in the normally resistant 9-10 week old C57BL/6J male mice by the D variant of encephalomyocarditis virus (EMCV-D) was examined. Inoculation of purified EMCV-D induced maximum NK cell activity in splenic cell populations on day 4 post-inoculation as determined by lysis of YAC-1 target cells in a standard 51chromium release microcytotoxicity assay. Selective depletion of NK cells by the administration of rabbit anti-asialo GM1 sera prior to challenging the C57BL/6J mice with EMCV-D, resulted in diminished splenic NK cell activity, increased EMCV-D viral titers in the pancreas, spleen, heart and brain, and the induction of diabetes in 60-80% of the mice. The data suggest that NK cells play a role in host protection against the diabetogenic EMCV-D.     

Different roles are played by alpha beta and gamma delta T cells in acquired immunity to Chlamydia trachomatis pulmonary infection.

Using gene knockout and wild-type C57BL/6 mice, we examined the role of alpha beta and gamma delta T cells in the resolution of Chlamydia trachomatis mouse pneumonitis (MoPn) biovar pulmonary infection. The results show that alpha beta T-cell-deficient (alpha-/-) mice, when compared with wild-type control mice, have dramatically increased mortality rate and greater in vivo growth of MoPn. The alpha beta T-cell-deficient mice were as susceptible to MoPn infection as T- and B-lymphocyte-deficient (RAG-1-/-) mice. Moreover, both alpha beta T-cell-deficient and RAG-1 mutant mice failed to mount delayed-type hypersensitivity (DTH) responses to organism-specific challenge and showed undetectable interferon-gamma (IFN-gamma) production by spleen cells upon in vitro organism-specific restimulation. In contrast, gamma delta T-cell-deficient mice exhibited intact DTH responses and their mortality rate and in vivo chlamydial growth were comparable to those in wild-type controls. More interestingly, gamma delta T-cell-deficient mice showed significantly higher levels of IFN-gamma production than did wild-type mice. The data indicate that the alpha beta T cell is the major T-cell population for acquired immunity to chlamydial infection and that gamma delta T cells may play an ancillary role in regulating the magnitude of alpha beta T-cell responses.     

Interferon treatment inhibits early events in vaccinia virus gene expression in infected mice.

We have analyzed the role of exogenous administration of mouse interferon (IFN alpha + beta) on the replication of vaccinia virus in peritoneal cells and in the spleen of Balb/c mice. Mice were pretreated for 16 hr with IFN and then infected with a vaccinia virus recombinant expressing luciferase under an early or late virus promoter, and the enzyme activity was measured in the course of virus infection. A dose of IFN as low as 10(3) units/mouse abolished the appearance of luciferase activity in cells of the peritoneal cavity and in spleen cells. The IFN-mediated inhibition of luciferase activity was observed even when mice were infected 4 days after the administration of IFN. The IFN-treated animals were considered free of virus since neither luciferase nor viral proteins were detected in target cells several days after virus infection. Despite a severe IFN-mediated inhibition of luciferase activity, the appearance of luciferase on mRNA levels was not inhibited 6 hr after virus infection. Our finding revealed that replication of vaccinia virus in Balb/c mice is exquisitively sensitive to inhibition by IFN and that this effect occurs at early times postinfection, most likely as a result of a translational block.     

Investigation on extracellular slime from Pseudomonas aeruginosa as interferon inducer in mice

The investigation concerns interferon (IFN) production in the sera and spleens of 129/Ao/Boy mice induced with Pseudomonas aeruginosa slime extract. Interferon was present in the serum as early as 2 h after i.v. and i.p. injection of the immunogenic dose of the slime - 100 micrograms/mouse. Likewise, in the spleen the same dose induced interferon production at the second hour after its administration. In the spleen interferon was synthetized longer, even up to 7 days. On the other hand, it disappeared from the serum after 24 h. In vitro investigations on interferon induction in peritoneal cells and spleen revealed that after slime extract stimulation, only non-adherent cells are capable of IFN production; while adherent cells are not. Interferon synthesis in peritoneal cells in vitro was much enchanced if for the experiments, cells isolated 2 - 4 h after i.v. administration of mice with Ps. aeruginosa slime extract, were used. Besides, the stimulatory effect of the extract on interferon production was well marked in the Newcastle virus-induced peritoneal cells. For comparison, interferon obtained after induction with slime extract in vivo (in the serum) and in vitro (in peritoneal cells) was tested for its properties. The interferons although both acidstable, displayed significant differences. IFN obtained in vitro from peritoneal cells culture appeared thermolabile and susceptible for neutralization with gamma-globulin of rabbit serum against interferon from Newcastle virus-induced L929 cells (anti-MuIFN alpha/beta). IFN from serum was thermostabile, undergoing only slight neutralization with anti-MuIFN alpha/beta globulin.     

Effect of mosquito salivary gland treatment on vesicular stomatitis New Jersey virus replication and interferon alpha/beta expression in vitro

The sensitivity of vesicular stomatitis (VS) viruses to interferon (IFN)-mediated antiviral effects has been well documented. Previous studies in our laboratory have shown the ability of mosquito saliva to enhance vesicular stomatitis New Jersey (VSNJ) virus infection in mice. To investigate the effect of mosquito saliva on virus replication and IFN alpha/beta expression, virus titers were analyzed at various time points after infection in cells that were treated with mosquito salivary gland homogenate (SGH). Salivary gland treatment of mouse fibroblast cells (L929) resulted in a significant increase in virus growth kinetics compared with untreated controls. In contrast, Vero cells, which are deficient in the IFN alpha/beta response, did not yield increased viral titers in the time points examined. Treatment of L929 cells with an IFN alpha/beta neutralizing antibody also slightly increased virus yield. Ribonuclease protection assays revealed that induction of IFN alpha2 expression was reduced in L929 cells treated with SGH. Modulation of IFN alpha/beta by mosquito saliva may be a critical determinant of the transmission and pathogenesis of VSNJ virus.     

Liver regeneration and immune system--morphological and cytochemical studies of activated spleen cells during the liver regeneration after partial hepatectomy

Morphological and cytochemical changes were investigated in spleen cells after 70% hepatectomy in mice. During liver regeneration after the hepatectomy, the spleen weight gradually rose to a peak at 6 day. In the spleen, the number of POD-positive myelocytic cells and NCAE-positive granulocytic cells also reached a peak 4 days after the operation and then decreased. On the other hand, ANBE-positive monocytic cells gradually increased up to 9 day and didn't decrease for this period. On day 9 of the culture, the boundary between the red and the white pulps of the spleen became unclear. In this spleen, the clusters of blast cells were sporadically observed. Splenic T cells cocultured with nonparenchymal adherent normal liver cell or nonparenchymal adherent normal liver cell supernatant developed into granulocyte colonies in earlier periods and monocyte colonies in later periods. These findings suggest that the factors released from liver cells may regulate strictly spleen cell activation. Blast cell formation in the culture with nonparenchymal adherent normal liver cell supernatant was amplified by anti interferon (alpha + beta) antibody. These facts indicate that the functional network of cytokines is formed by the interaction of various cytokines (IL-1, IL-6, CSF, IFN, etc.) in nonparenchymal adherent normal liver cell supernatant.     

Production of interferon alpha and interferon gamma by peripheral blood leukocytes from patients with chronic hepatitis B virus infection

Peripheral blood leukocytes from patients with chronic hepatitis B virus (HBV) infection were studied for their capacity to produce interferon (IFN) alpha or IFN gamma. Yields of IFN alpha in leukocyte cultures stimulated with influenza A virus or human leukemic cells were significantly lower than those obtained from healthy controls. Production of IFN gamma in response to induction with protein A of Staphylococcus aureus was also significantly diminished. Defects of IFN production in leukocyte cultures showed no correlation with active viral replication or the degree of severity of HBV-associated liver disease. The demonstration of partial defects of endogenous IFN production provides a rationale for using IFN replacement therapy in patients with chronic HBV infection.     

The effect of age of cattle on the in vitro production of interferon by peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) from normal cattle of different ages and from specific pathogen-free (SPF) calves, 2 to 4 weeks old, were cultured with bovine herpes virus type 1 (BHV1), parainfluenza-3 virus (PI3) and phytohaemagglutinin (PHA). The interferon (IFN) produced was characterized by acid stability and neutralizing antisera to recombinant bovine interferons. The virus preparations were presented either live or inactivated and as cell-bound virus or free virions. PBMC from cattle of all ages produced IFN-alpha when stimulated with live BHV1 and PI3 viruses. IFN-alpha was also produced with inactivated BHV1, even in cell cultures from SPF calves. However, inactivated PI3 virus failed to induce IFN in PBMC cultures from normal cattle, but approximately half of the animals, mostly calves, produced IFN-gamma spontaneously in 48 h cultures in the absence of added antigen. PHA induced IFN-gamma at an optimal concentration of 20 micrograms per ml after 3 days in culture. An age-related maturation of the IFN response was observed as PBMC from calves less than 2 weeks old produced little or no IFN when induced with either PHA or inactivated BHV1, although some IFN-alpha was produced in cultures containing live virus. Both adherent and non-adherent cells from adults and calves over 2 weeks old produced IFN on induction with inactivated BHV1 but only the non-adherent cell population produced IFN spontaneously or in response to inactivated PI3.     

Concanavalin A-induced interferon gamma production by murine spleen cells and T cell lines. Lack of correlation with Lyt 1,2 phenotype

Concanavalin A(Con A)-induced interferon-gamma (IFN gamma) production by resting or preactivated murine spleen cells negatively selected with monoclonal antibodies specific for Lyt 1,2 antigens plus complement (C) and by interleukin 2(IL-2)-dependent T cell lines of different Lyt phenotype was studied. The data show that most of the IFN-gamma produced upon stimulation of resting spleen cells was a product of Lyt 1+2+ cells. Lyt 1-2+ cells were negative for IFN gamma production. When spleen cells that had been preactivated for 3 days with Con A were restimulated with Con A, Lyt 1+2+, Lyt 1+2- as well as Lyt 1-2+ cells produced IFN gamma in a relationship of approximately 5:3:1. In both cases the picture remained unaltered independently when the supernatants were harvested after 1, 3 or 5 days. Furthermore, two IL-2 dependent T cell lines were studied in regard to Con A-induced IFN gamma production. Line 1.3 was Thy 1+, Lyt 1-2+, whereas line 20.9. was Thy 1+, Lyt 1+2-. Both lines produced initially high titers of IFN gamma upon stimulation with Con A. After prolonged passage in vitro, however, they progressively lost the capacity to produce IFN gamma.     

Paradoxical lessening of autoimmune processes in non-obese diabetic mice after infection with the diabetogenic variant of encephalomyocarditis virus

In order to gain insight into the interaction between autoimmunity and viral infection in the onset of insulin-dependent diabetes, non-obese diabetic (NOD) mice which spontaneously develop autoimmune diabetes were inoculated with the diabetogenic variant of the encephalomyocarditis virus (EMCV-D) before the onset of the disease. The pre-diabetic period was divided into two phases: the early phase (days 88 to 116) during which development of spontaneous diabetes is rare and the late phase (day 123 to 200) during which the incidence of spontaneous diabetes is high. As controls ICR mice of common ancestry were also inoculated. During the early phase diabetes was observed in 4/10 inoculated, 0/13 control NOD and 7/13 inoculated ICR males vs. 6/12 inoculated, 1/11 control NOD and 0/15 inoculated ICR females. However, in NOD female, virus-induced diabetes prevalence was variable from one experiment to another. In parallel the flow cytometric analysis showed a high percentage of L3T4+ T lymphocytes in the pancreas of inoculated female NOD mice 10 days after the infection. At this time a large proportion of both L3T4+ and Ly-2+ cells expressed the interleukin 2 receptor. During the late phase no new case of diabetes occurred in inoculated NOD mice but one case was observed in control NOD males and five in control NOD females. This prevention of autoimmune diabetes was constantly found in other experiments. Insulitis was milder in inoculated NOD mice of both sexes than in control NOD. Adoptive transfer of diabetes into irradiated 8-week-old males by splenocytes from 28-week-old females was successful in five out seven attempts with control splenocytes and in zero out of six attempts with splenocytes from inoculated mice. This immunosuppression was specific as the ability of lymphocytes to respond to soluble or allogeneic antigens was preserved. In the early phase EMCV-D precipitated the onset of diabetes in females NOD mice by amplifying L3T4+ T lymphocyte-mediated immune mechanisms. During the late phase viral infection had lessened immune processes in animals which had resisted or recovered from virus-induced diabetes.     

Nondetectable levels of interferon gamma is a critical host defense during the first day of herpes simplex virus infection

Previous studies have demonstrated the importance of IFN alpha/beta in resistance to primary viral infections. However, the role of IFN gamma in primary infections is unclear. The present studies were undertaken to determine whether IFN gamma induction was an important early host defense against primary HSV infection. The approach was to block the IFN gamma response with antibodies to IFN gamma prior to infection and at various times post-infection (p.i.). The data indicates that treatment of mice with anti-IFN gamma prior to infection enhanced mortality (89% vs 37%). Anti-IFNs given at various times post HSV challenge proved most effective within the first 24 h of infection. The above results suggest for the first time that IFN gamma mediates important host defense(s) early during primary HSV infection. Similar results were obtained using antibody to IFN alpha/beta.     

Pulmonary epithelial T cells induced by viral infection express T cell receptors alpha/beta.

Cells were recovered from the lungs of mice by bronchoalveolar lavage before and after infection with respiratory syncytial (RS) virus. Uninfected mice yielded mostly macrophages, but after RS virus infection lymphocytes also appeared. Recovered cells were stained for CD4, CD8' and either CD3, T cell receptor (TcR) alpha/beta or TcR gamma/delta and analyzed by 3-color flow cytometry. Both the CD4+CD8- and CD4-CD8+ subpopulations stained uniformly for CD3 and TcR alpha/beta, while none stained for TcR gamma/delta. Of the CD4-CD8- cells, about 5% stained for CD3 and TcR alpha/beta during the first week after infection, although this figure increased during the second week. A small and variable fraction of this subset stained for TcR gamma/delta. These results oppose the view that lymphocytes expressing TcR gamma/delta predominate in initial epithelial immune responses to viral infections.     

Some properties of mycoplasma virus Br1

Morphologically mycoplasma virus Br 1 is a typical contractile-tailed bacteriophage with a head 77 nm in diameter and a tail 104 nm long. Its type of nucleic acid was not determined. Br 1 was closely associated with its host cell and assays reflected infectious centres. During growth of Br 1 in mycoplasma cultures at multiplicities of infection (MOI) greater than 0.001, there was a lag period: this was up to 23 hours at an MOI of 35. The mean generation time of a mycoplasma culture infected at MOI up to 235 was 2 hours, compared with 1 hour for an uninfected culture. However in these infected cultures there were viable mycoplasmas all of which appeared to be fully susceptible to Br 1 infection and did not seem to be carrying the virus. Br 1 formed plaques on M. bovirhinis but failed to produce plaques on strains of 8 Mycoplasma, 2 Acholeplasma and 4 bacterial species.     

Membranes of activated CD4+ T cells expressing T cell receptor (TcR) alpha beta or TcR gamma delta induce IgE synthesis by human B cells in the presence of interleukin-4.

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.