The role of T lymphocytes in the pathogenesis of Coxsackie virus B3 heart disease

Myocarditis in athymic BALB/c-nu/nu (nu/nu) mice infected with Coxsackie virus B3 was studied to determine whether inflammatory mononuclear cell infiltration was produced by transfer of spleen cells ob BALB/c-nu/+ (nu/+) mice infected with the same virus. In addition, spleen cells of uninfected nu/+mice were transferred into athymic nu/nu mice infected with Coxsackie virus B3. Athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of nu/+ mice infected with the same virus developed marked to moderate myocarditis with inflammatory mononuclear cell infiltration. However, athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of uninfected nu/+ mice developed moderate to mild degeneration or necrosis of the muscle fibres, although mild mononuclear cell infiltration was found in only one mouse. The incidence of myocardial lesions of athymic nu/nu mice infected with Coxsackie virus B3 after transfer of infected-spleen cells of nu/+ mice was about the same is that in infected nu/+ mice after transfer of spleen cells of uninfected nu/+ mice. However, degrees in the intensity of the muscular changes and inflammatory mononuclear cell infiltration in the former was significantly greater than that in the latter. The results show, therefore, that Coxsackie virus B3 infection stimulated production of cytotoxic activity in the spleen which was evident on Day 6. From the present results, it is confirmed that myocarditis in mice infected with Coxsackie virus B3 is thymus-dependent, supporting previous investigations.     

The role of T lymphocytes in the pathogenesis of Coxsackie virus B3 heart disease.

Myocarditis in athymic BALB/c-nu/nu (nu/nu) mice infected with Coxsackie virus B3 was studied to determine whether inflammatory mononuclear cell infiltration was produced by transfer of spleen cells ob BALB/c-nu/+ (nu/+) mice infected with the same virus. In addition, spleen cells of uninfected nu/+mice were transferred into athymic nu/nu mice infected with Coxsackie virus B3. Athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of nu/+ mice infected with the same virus developed marked to moderate myocarditis with inflammatory mononuclear cell infiltration. However, athymic nu/nu mice infected with Coxsackie virus B3 after transfer of spleen cells of uninfected nu/+ mice developed moderate to mild degeneration or necrosis of the muscle fibres, although mild mononuclear cell infiltration was found in only one mouse. The incidence of myocardial lesions of athymic nu/nu mice infected with Coxsackie virus B3 after transfer of infected-spleen cells of nu/+ mice was about the same is that in infected nu/+ mice after transfer of spleen cells of uninfected nu/+ mice. However, degrees in the intensity of the muscular changes and inflammatory mononuclear cell infiltration in the former was significantly greater than that in the latter. The results show, therefore, that Coxsackie virus B3 infection stimulated production of cytotoxic activity in the spleen which was evident on Day 6. From the present results, it is confirmed that myocarditis in mice infected with Coxsackie virus B3 is thymus-dependent, supporting previous investigations.     

Relationship between colony-stimulating activity and interferon production during infection.

Normal mouse spleen, when cultured in vitro for 3 days in the presence of 10(8) heat-killed Listeria monocytogenes organisms, produced colony-stimulating factors (CSF) that were capable of supporting the production of haemopoietic colonies by bone marrow cells in semi-solid agar, or supporting bone marrow proliferation in liquid medium. In contrast, when the spleen cells were prepared from mice that had been infected with Listeria monocytogenes, colony-stimulating activity (CSA) was no longer detectable over a period from Day 3 to Day 17 post-infection. Suppression of CSA was imposed on normal spleen cells when nylon-wool filtered, T-cell enriched spleen cells from infected mice were co-cultured with normal spleen cells. Suppression largely coincided with the production of interferon by whole spleen from infected mice, and when interferon-gamma (IFN-gamma) was neutralized by antibody CSA was again detected. An early IFN-gamma-independent decrease in CSA production was also detected 2-3 days post-infection. The relevance of this system to the control of CSF production in vivo is discussed.     

Mechanisms of eosinophilia in BALB/c-nu/+ and congenitally athymic BALB/c-nu/nu mice infected with Toxocara canis.

We studied the mechanism of eosinophilia in BALB/c-nu/+ (nu/+) and BALB/c-nu/nu (nu/nu) mice infected with Toxocara canis. Eosinophilia with two peaks on days 11 and 21 of infection was observed in infected nu/+ mice, and with a peak on day 11 in nu/nu mice. Interleukin-5 (IL-5) mRNA was expressed on day 5 of infection in the lung and spleen of nu/+ mice and in the lung of nu/nu mice, but not in the spleen of nu/nu mice. Large numbers of eosinophils and lymphocytes infiltrated the lung of both mice 1 week after infection. The number of larvae in the lung was the largest on day 5. Anti-IL-5 monoclonal antibody (mAb) treatment completely inhibited eosinophilia of both mice, with no change of larval distribution. Administration of anti-CD4 or anti-CD3 mAb markedly reduced the second peak of eosinophilia on day 21 of infection in nu/+ mice, and slightly reduced the first peak of eosinophilia on day 11 in both mice. Anti-CD8 mAb had no effect on the eosinophilia. These results suggest that eosinophilia in both mice is caused by IL-5, and that IL-5 is produced by cells other than CD4+ T cells, in addition to CD4+ T cells.     

Immunoglobulin synthesis in nude (nu/nu), nu/+ and reconstituted nu/nu mice infected with a demyelinating strain of Semliki Forest virus

Infections with the avirulent (A7/74) strain of Semliki Forest virus which causes primary demyelination of the central nervous system in mice have been studied further in nude athymic (nu/nu) mice and their immunocompetent (nu/+) litter mates to measure the production of immunoglobulins. This has been done by radial immunodiffusion and enzyme-linked immunosorbent assays. Half the nude mice examined were able to synthesize specific IgG but at levels 1,000-fold lower than their nu/+ littermates. The majority of nude mice reconstituted with spleen cells from nu/+ mice 1 day before infection with virus were able to synthesize specific IgG nearly as well as the nu/+ animals.     

Chlamydia trachomatis pneumonia in the immune,athymic and normal BALB mouse.

This paper compares the histopathology of pneumonia due to murine Chlamydia trachomatis (MoPn, mouse pneumonitis agent) in susceptible athymic nude mice (nu/nu), resistant heterozygous littermates (nu/+) and very resistant immunized nu/+ mice. While all groups had an early heterophil response, successful host defence correlated with the presence of large numbers of plasma cells, lymphocytes, monocytes, and lipid laden macrophages. Reticulate bodies were seen in all groups, predominantly in type I alveolar epithelial cells. By 24 h in the immune nu/+ group, no intact organisms were visible. Optimal control of infection was thus rapid and not clearly related to heterophils. These studies show that the histopathology of chlamydial infection may be quite atypical in the immunocompromised host, mononuclear cells seem critical in host defence, and B cell activation with plasma cell infiltration is dependent on intact T cell function in this model.     

Chlamydia trachomatis pneumonia in the immune, athymic and normal BALB mouse

This paper compares the histopathology of pneumonia due to murine Chlamydia trachomatis (MoPn, mouse pneumonitis agent) in susceptible athymic nude mice (nu/nu), resistant heterozygous littermates (nu/+) and very resistant immunized nu/+ mice. While all groups had an early heterophil response, successful host defence correlated with the presence of large numbers of plasma cells, lymphocytes, monocytes, and lipid laden macrophages. Reticulate bodies were seen in all groups, predominantly in type I alveolar epithelial cells. By 24 h in the immune nu/+ group, no intact organisms were visible. Optimal control of infection was thus rapid and not clearly related to heterophils. These studies show that the histopathology of chlamydial infection may be quite atypical in the immunocompromised host, mononuclear cells seem critical in host defence, and B cell activation with plasma cell infiltration is dependent on intact T cell function in this model.     

The importance of interferon-gamma in an early infection of Chlamydia psittaci in mice.

Athymic mice (nu/nu) and their hairy littermates (nu/+) were infected experimentally with Chlamydia psittaci and the role of endogenous interferon-gamma (IFN-gamma) on the resolution of the infection was studied. The pathological changes produced in the spleen, liver and lung were exacerbated by administration of a monoclonal antibody (mAb) to IFN-gamma and an increased number of viable chlamydiae were recovered from the tissues of both nu/+ and nu/nu mice treated in this way.     

Etiology of the liver granulomatous response in Schistosoma mansoni-infected athymic nude mice.

The effect of schistosome infection on the presence and maturation of splenic T lymphocytes in C3H/HeN nu/nu and nu/+ mice was examined. Spleens of uninfected nu/nu mice contained very low numbers (u to 2%) of T lymphocytes. This percentage did not increase throughout the 10 weeks of the infection. Spleens of uninfected nu/+ littermates contained 28.8% T cells, which decreased to 15.0% by week 10 of the infection. Similarly, whereas spleen cells of normal or infected nu/nu mice were nonresponsive to concanavalin A, the initial high response of nu/+ mice gradually diminished. Both nu/nu and nu/+ spleen cells responded well to lipopolysaccharide initially, but by 10 weeks their responsiveness declined. Sera of five infected nu/nu mice contained no antibodies to egg antigens, and one had a low titer (log2 5.0). In contrast, the mean titer of sera from six nu/+ mice was log2 10.7 Nu/+ mice had typical florid lesions, but nu/nu mice mounted sparse granulomatous reactions around eggs in the liver without evidence for hepatocellular damage. Dispersed liver granulomas of nu/nu mice contained 1.2% T and 20.3% B lymphocytes. Lesions of nu/+ mice contained 12.9% T and 18.4% B cells. Eighty percent of the macrophages from nu/nu and nu/+ granulomas displayed high density/avidity Fc receptors. Production of migration inhibition factor-active lymphokine by liver granulomas and spleens of schistosome-infected nu/nu mice is suggestive of the immune role of B cells in the granulomatous inflammation.     

Impaired protective immunity and T helper 2 responses in alymphoplasia (aly) mutant mice infected with Trichinella spiralis

The alymphoplasia (aly) mutation of mice prevents the development of systemic lymph nodes and Peyer's patches. The mutant homozygotes (aly/aly) are partially deficient in both humoral and cell-mediated immune functions. In the present study, we show that adult worm expulsion was slightly delayed and that T helper 2 (Th2)-type responses were partially defective in aly/aly mice after infection with Trichinella spiralis. Male aly/aly and aly/+ mice (8-weeks old) were infected with 400 muscle larvae. There was no difference in worm recovery between the two groups on day 5. However, worm recovery in aly/aly mice was significantly higher than that in aly/+ mice on day 14. Mucosal mast cells increased in number and peaked 14 days after infection in aly/+ mice. aly/aly mice were deficient in their mucosal mast cell response through out the primary infection. To examine the existence of mast cell precursors, aly/aly mice were treated with recombinant interleukin-3 (rIL-3) before infection. The mast cell response was poorly induced in aly/aly mice treated with rIL-3. An immunoglobulin E (IgE) response was not detected in aly/aly mice during the course of infection. Serum IgG1 levels in aly/aly mice were significantly lower than that of aly/+. The serum IgG2a levels increased in both strains of mice. However, IgG2a production in aly/aly mice on day 14 was half as much as that in aly/+mice. Stimulation of splenic T cells in vitro with anti-CD3 monoclonal antibody (mAb) showed that spleen cells from aly/+ mice on day 5 produced more IL-4 than spleen cells from aly/aly mice. IL-4 production from aly/aly mice on day 14 was half that from aly/+ mice. Interferon-gamma (IFN-gamma) was produced in both aly/aly and aly/+ mice on day 14. Proliferation assay showed that T cells of aly/aly mice responded poorly when cultured with antigen-presenting cells. These results suggest that aly gene is needed for the induction of protective immunity and Th2 responses in mice infected with T. spiralis.     

Mycobacterium lepraemurium infection of nude athymic (nu/nu) mice.

Nude athymic (nu/nu) mice on a BALB/c background and their heterozygous euthymic litter mates (nu/+) were infected with either 10(8) or 10(6) Mycobacterium lepraemurium organisms intravenously or in the left hind footpad (LHF). After LHF infection with 10(8) M. lepraemurium organisms, nu/+ mice slowly developed a response that consisted of LHF swelling and local resistance to Listeria monocytogenes. The lower inoculum induced a proportionately lower response in nu/+ mice, but the nu/nu mice developed neither LHF swelling nor resistance to L. monocytogenes in response to either dose of M. lepraemurium. Counts of M. lepraemurium in the LHF revealed no difference between the nu/+ mice and nu/nu mice. After intravenous infection the nu/+ mice developed splenomegaly, but did not otherwise differ from nu/nu mice with respect to resistance to intravenous challenge with L. monocytogenes or growth of M. lepraemurium in the spleen. In light of the poor responsiveness of nu/+ mice in this experiment, they were then compared with CB6 and B6D2 mice, which are genetically susceptible and resistant to M. lepraemurium, respectively. These mice were infected with either 10(8) or 10(6) M. lepraemurium cells or 10(6) Mycobacterium bovis BCG cells in the LHF. Once again the nu/+ mice responded poorly to M. lepraemurium, the CB6 mice responded very strongly, and the B6D2 mice gave an intermediate response with respect to LHF swelling and resistance to L. monocytogenes. However, M. lepraemurium grew to higher numbers in the LHF of nu/+ and CB6 mice than in B6D2 mice, revealing, in CB6 mice, a dissociation between resistance to L. monocytogenes and M. lepraemurium. All three mouse strains responded strongly to M. bovis BCG, but there was a suggestion that nu/+ mice might be more susceptible to this agent than the other two strains. I concluded that the failure of nu/+ mice to restrict the growth of M. lepraemurium more than nu/nu mice was due to the intrinsic genetic susceptibility of both types of mice. In effect, the nu/+ mice behaved like nu/nu mice, as if they too were deficient in T lymphocytes that were responsive to M. lepraemurium.     

T-cell-independent resistance to infection and generation of immunity to Francisella tularensis.

The intraperitoneal 50% lethal dose (LD50) for Francisella tularensis LVS in both normal control heterozygote BALB/c nu/+ mice and BALB/c nu/nu mice was 2 x 10(0). Both nu/+ and nu/nu mice given 10(7) LVS bacteria or more intradermally (i.d.) died, with a mean time to death of about 7 to 8 days. On the other hand, nu/+ mice given 10(6) LVS bacteria or less survived for more than 60 days and cleared systemic bacteria, while nu/nu mice given 10(6) LVS bacteria or less survived for more than 10 days but died between days 25 and 30. Thus, the short-term (i.e., < 10-day) i.d. LD50 of both nu/nu and nu/+ mice was 3 x 10(6), but the long-term (i.e., > 10-day) i.d. LD50 of nu/nu mice was less than 7 x 10(0). The short-term survival of i.d. infection was dependent on tumor necrosis factor and gamma interferon: treatment of nu/nu mice with anti-tumor necrosis factor or anti-gamma interferon at the time of i.d. infection resulted in death from infection 7 to 8 days later, whereas control infected nu/nu mice survived for 26 days. nu/nu mice infected with LVS i.d. generated LVS-specific serum antibodies, which were predominantly immunoglobulin M: titers peaked 7 days after i.d. infection but declined sharply by day 21, after which mice died. Surprisingly, nu/nu mice given 10(3) LVS bacteria i.d. became resistant to a lethal challenge (5,000 LD50s) of LVS intraperitoneally within 2 days after i.d. infection; nu/nu mice similarly infected with LVS i.d. and challenged with Salmonella typhimurium (10 LD50s) were not protected. nu/nu mice given nu/+ spleen cells intravenously as a source of mature T cells survived i.d. infection for more than 60 days and cleared bacteria. Taken together, these studies demonstrate that i.d. infection of nu/nu mice with LVS rapidly generates T-cell-independent, short-term, specific protective immunity against lethal challenge, but T lymphocytes are essential for long-term survival.     

Pathogenesis of Cryptococcus neoformans in congenitally immunodeficient beige athymic mice.

Mortality after intravenous challenge with 10(4) Cryptococcus neoformans demonstrated that doubly immunodeficient beige athymic (bg/bg nu/nu) mice were more susceptible to systemic cryptococcosis than either bg/bg or nu/nu mice. Infected bg/bg nu/nu mice also had a shortened lifespan compared with their bg/bg nu/+ littermates. Beige athymic (bg/bg nu/nu) but not bg/bg nu/+mice developed cryptococcal lesions in the skin, demonstrating that C. neoformans is dermatotropic in a T-cell-deficient host. Higher numbers of C. neoformans were isolated from the lungs and spleen of infected bg/bg nu/nu than bg/bg nu/+ mice as early as day 3 after challenge, indicating that in lymphoid-rich organs, T cells can alter the course of systemic cryptococcosis early in the infection. Despite extensive abscess formation in the brains of bg/bg nu/+ mice, dissemination and growth rate of C. neoformans in the brain was similar in both genotypes. The primary histopathological feature in tissues from bg/bg nu/nu mice infected with C. neoformans consisted of foci of encapsulated yeast cells with minimal to no inflammatory response. In contrast to bg/bg nu/nu mice, bg/bg nu/+ mice mounted a vigorous inflammatory response to C. neoformans that progressed from acute to chronic inflammation. Beige athymic mice are a new animal model that will be useful in clarifying the innate and acquired immune factors important in resistance to cryptococcosis.     

Differential susceptibility and resistance of immunocompetent and immunodeficient mice to fatal Hantaan virus infection

Summary Susceptibility and resistance to fatal Hantaan virus meningoencephalitis were studied in immunocompetent (nu/+) and congenitally T-cell deficient (nu/nu) CD-1 mice of different ages. Susceptibility of nu/+ mice to fatal infection was age-dependent, as evidenced by 100 percent mortality in mice inoculated intracerebrally with Hantaan virus (strain 76–118) during the first week of life, 60 percent mortality in mice inoculated at 9 days of age, and no disease or death in mice inoculated at 14 to 42 days of age. Deaths occurred significantly earlier in nu/+ mice inoculated at 5 and 7 days of age than in nu/+ mice less than 24 hours old. Nu/nu littermates of the same age did not exhibit a similar inverse relationship between age and survival times. Moreover, nu/nu mice 14 days or older remained susceptible, albeit with delayed onset of illness and time of death. Virus titers in brain tissues of nu/+ mice inoculated at 7 days and of nu/nu nice inoculated at 7, 9 and 14 days of age were nearly identical. In older nu/+ mice, peak virus titers were comparatively lower, but they did not seem to be influenced by the magnitude of the neutralizing antibody response. The more rapidly fatal course in nu/+ mice inoculated at a week of age than at birth, and the differential resistance between weanling nu/+ and nu/nu mice to Hantaan virus meningoencephalitis suggest that cell-mediated immunity may be responsible for both enhancement of disease and recovery from infection.With 5 Figures     

A role in vivo for tumor necrosis factor alpha in host defense against Chlamydia trachomatis.

In a mouse model of pneumonia caused by murine Chlamydia trachomatis (mouse pneumonitis agent [MoPn]), tumor necrosis factor alpha (TNF-alpha) antigen and bioactivity were demonstrated in vivo in the lung during MoPn infection in both athymic (nude) and heterozygous (nu/+) mice. Antibody to TNF-alpha that was exogenously given neutralized the TNF-alpha in the lung, significantly accelerated mortality, and caused a borderline increase in MoPn counts in the lung by culture in nu/+ mice. Lipopolysaccharide-induced TNF-alpha activity or injections of recombinant murine TNF-alpha significantly but modestly protected nu/+ mice against MoPn-induced mortality. TNF-alpha is produced in vivo during C. trachomatis infection and plays a role in host defense.     

Bacterial translocation from the gastrointestinal tract of athymic (nu/nu) mice.

The immune system may be one host defense mechanism preventing viable indigenous bacteria from translocating from the mouse gastrointestinal lumen to the mesenteric lymph nodes, spleen, liver, or kidney. The role of T-cell-dependent immunity in preventing bacteria from translocating from the gastrointestinal tract was tested with congenitally athymic nude (nu/nu) mice, heterozygous (nu/+) mice, and thymus-grafted nude (nu/nu) mice. Viable bacteria were cultured from 50% of the mesenteric lymph nodes, spleens, livers, and kidneys of athymic (nu/nu) mice, whereas heterozygous (nu/+) mice exhibited viable bacteria in only 5.2% of these organs. Both aerobic and strictly anaerobic bacteria were cultured from these organs with Escherichia coli and Lactobacillus predominanting. Grafting thymuses to the athymic (nu/nu) mice restored their immunological responses to sheep erythrocyte antigens. The incidence of bacterial translocation from the gastrointestinal tract was reduced from 50% in the athymic (nu/nu) mice to 7.8% in the thymus-grafted (nu/nu) mice. Thus, T-cell-dependent immunity restored by thymic grafts inhibited the translocation of certain indigenous bacteria from the gastrointestinal tract to the spleen, liver, and kidney in nu/nu mice.     

T-independent macrophage changes in murine malaria

A study to investigate the participation of T cells in macrophage-mediated responses during malaria was performed in nude (nu/nu) and littermate (nu/+) mice infected with Plasmodium berghei (PB). We found that in both groups of mice spleen cells suppressed the mitogenic response to LPS. Both nu/+ and nu/nu infected mice also showed liver macrophage activation, reflected by increased plasminogen activator release. These findings suggest that at least some of the macrophage changes during malaria infection are T-independent.     

Tumor necrosis factor alpha is a cytotoxin induced by murine Chlamydia trachomatis infection.

A mouse model of pneumonia caused by murine Chlamydia trachomatis (mouse pneumonitis agent) was used to demonstrate that whole spleen cells from both nude athymic mice (nu/nu) and heterozygous mice (nu/+) produced tumor necrosis factor alpha in vitro in response to mouse pneumonitis agent antigen. The tumor necrosis factor alpha measured in these supernatants by immunoassay was shown to have bioactivity in a cytotoxic assay in which uninfected target cells were used. This cytotoxicity was distinct from the gamma interferon-related cytotoxicity against C. trachomatis-infected targets that we described previously.     

Studies on immune responses to parasite antigens in mice. V. Different susceptibilities of hypothymic and intact mice to Babesia rodhaini.

Hypothymic BALB/c.nu/nu mice are more resistant than intact BALB/c.nu/+ mice to Babesia rodhaini and a proportion survive a dose of infected blood which is uniformly lethal to nu/+ mice. This proportion of nu/nu survivors is not affected by administration of an anti-Babesial drug, whereas the majority of nu/ + mice develop a long-lasting resistance to infection. The data suggest that T cell dependent activities are involved both in the acceleration of parasitaemia and in the development of drug-assisted resistance.     

Mitogen- and antigen-specific proliferation of T cells in murine toxoplasmosis is inhibited by reactive nitrogen intermediates.

Acute and chronic infections with Toxoplasma gondii result in a nonspecific suppression of immunologic function in mice and humans. Proliferation of spleen cells in response to concanavalin A (ConA) and toxoplasma lysate antigen (TLA) was studied during the course of infection in mice susceptible (CBA/Ca) and resistant (BALB/c) to development of toxoplasmic encephalitis to determine if reactive nitrogen intermediates (RNI) are involved in the suppression of the proliferative responses. Maximal suppression of proliferation of spleen cells in response to ConA and TLA was observed on days 7 and 14 after infection and correlated with elevated levels of nitrite in spleen cell culture supernatants. By day 68 postinfection in BALB/c mice, proliferative responses returned to normal levels, whereas in CBA/Ca mice, they remained suppressed. The addition of an inhibitor of production of RNI (NG-monomethyl-L-arginine) increased proliferation of spleen cells in response to both ConA and TLA at days 7, 14, and 21 after infection. Depletion of adherent cells from spleen cell preparations obtained from acutely infected mice followed by their repletion with adherent spleen cells from uninfected mice resulted in increased proliferation of spleen cells from infected mice and a significant decrease in nitrite in the cultures. These results indicate that production of RNI by macrophages contributes significantly to the suppression of the spleen cell proliferation observed in the acute stage of toxoplasmosis.