Production of gamma interferon by natural killer cells from Toxoplasma gondii-infected SCID mice: regulation by interleukin-10, interleukin-12, and tumor necrosis factor alpha.

Previous studies of mice have implicated natural killer (NK) cells as mediators of protective activity against Toxoplasma gondii through their production of gamma interferon (IFN-gamma). In the present study, we have compared NK-cell activity in infected and uninfected SCID mice. Our data reveal that infection results in increased levels of IFN-gamma in serum and elevated NK-cell activity but that these NK cells were not cytotoxic for T. gondii-infected P815 cells. Treatment with anti-IFN-gamma antibody abrogated the increase in NK-cell activity and resulted in earlier mortality of infected mice. In vivo treatment with anti-asialo GM1 antiserum reduced NK cell activity and levels of IFN-gamma in serum but did not alter time to death. Spleen cells from infected mice produced higher levels of IFN-gamma than those from uninfected mice when stimulated in vitro with live T. gondii or parasite antigen preparations. Further analysis revealed that interleukin 10 (IL-10) inhibited, whereas tumor necrosis factor alpha (TNF-alpha) and IL-12 enhanced, IFN-gamma production by spleen cells from infected or uninfected mice. The combination of IL-12 and TNF-alpha induced higher levels of IFN-gamma from whole spleen cells of infected mice than from those of uninfected mice. Depletion of the adherent cell population from the spleen cells of infected mice led to a significant reduction in the levels of IFN-gamma produced after stimulation with IL-12 plus TNF-alpha. Similar results did not occur with cells from uninfected mice. These data indicate that other cytokines produced by the adherent cell population from infected mice may be involved in maximal production of IFN-gamma by NK cells stimulated with IL-12 and TNF-alpha. To assess the importance of endogenous IL-12, a polyclonal anti-IL-12 was administered to infected SCID mice. This treatment led to earlier mortality, indicating that endogenous IL-12 mediates resistance to T. gondii.     

Drug-induced modulation of IL-2 production in experimental murine trypanosomiasis.

In this study we evaluated the effects of N-acetyl-cysteine and indomethacin in restoring IL-2 producing ability in vitro of splenocytes from mice infected with Trypanosoma equiperdum. Spleen cells from these mice were found to produce significantly lower levels of interleukin-2 (IL-2) in response to mitogen stimulation than spleen cells from uninfected control mice. This was accompanied by considerable suppression of IL-2-receptor expression, which was not attributable to the elimination of a particular T-cell subset. Impairment of IL-2 production was not due to a primary defect in L3T4+ T-cells, but rather to the presence of both adherent and non-adherent suppressor cells that apparently acted via prostaglandin-independent and dependent mechanisms. In fact, the IL-2-producing ability of lymphocytes from infected mice could be efficiently restored by in vitro exposure to N-acetyl-cysteine or indomethacin.     

Lymphokine activity production in graft-versus-host reactions across minor histocompatibility antigen barriers.

Activated T cells responding to murine minor histocompatibility antigens (HA) were characterized according to the patterns of lymphokine activity production. Although B10.D2/nSN and BALB/c are mutually non-reactive in mixed lymphocyte reaction (MLR), graft-versus-host reaction (GVHR) can be induced by the injection of a large amount of B10.D2/nSN lymphoid cells into irradiated BALB/c recipient mice. Spleen cells from such GVHR mice spontaneously produced interleukin 3 (IL-3)-dependent cell-stimulating activity in cultures, but did not produce interleukin 2 (IL-2). Normal B10.D2/nSN spleen cells also produced IL-3-like activity, but not IL-2 in MLR supernatants, in response to irradiated BALB/c splenocytes. In addition, B-cell stimulatory factor-1 (BSF-1)/interleukin 4 (IL-4) and colony-stimulating factor (CSF) activity were detected in MLR supernatants. The properties of the produced lymphokine activities were similar to those produced in syngeneic transplant mice and syngeneic MLR, but a difference in the time course of lymphokine production existed between GVHR and syngeneic transplant mice. These results indicate that T cells may be activated in vivo in allogeneic transplantation when the donor and the recipient are matched for major HA, and are non-reactive in MLR. Also, the character of lymphokine-producing T cells activated by minor HA may not be qualitatively different from those responding to irradiated syngeneic cells.     

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.     

Transient killer cells mediating antibody-dependent cell cytotoxicity to chicken erythrocytes in mouse spleen during post natal development

Anti CRBC-ADCC increased in spleens of C3H/He mice from 2 weeks after birth to a peak value at 7-9 weeks of age and then declined to at least 17 weeks. Spleen cells from 8 week-old mice were fractionated by Percoll discontinuous density gradients and Sephadex G-10 columns. At least two cell fractions mediating ADCC to CRBC targets have been identified. Percoll gradient separation revealed one cell fraction with monocyte and another with lymphocyte morphology. The spleen cells adherent to Sephadex G-10 columns were responsible for most ADCC and this fraction accounted for the majority of killer cells at 8 weeks of age. These adherent cells were responsible for most superoxide anion production when stimulated with opsonised zymosan. The ontogenetic pattern of Sephadex-adherent killer cells closely resembles that for natural killer (NK) cells. This is discussed with regard to the probable influx of monocyte-like cells in the spleen during the postnatal development of mice.     

Production of tumor necrosis factor alpha, interleukin-1 alpha, and interleukin-6 during murine coccidioidomycosis.

The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin-6 (IL-6) were induced in mice infected with Coccidioides immitis. Analyses of the cytokine profiles of two inbred mouse strains which differ in their susceptibility to pulmonary challenge with C. immitis revealed higher levels of IL-6 in lungs from DBA/2 mice (resistant strain) than in those from BALB/c mice (susceptible strain) beginning at day 6 and continuing through day 15 postinfection. Spleen cells from both mouse strains secreted TNF-alpha, IL-1 alpha, and IL-6 in vitro in response to stimulation with killed spherules but differed in that spleen cells from the resistant strain produced increased levels of these cytokines earlier after pulmonary challenge and at increased levels throughout the course of the disease.     

Effect of interleukin 12 neutralization on host resistance and gamma interferon production in mouse typhoid.

Innately resistant (Ityr) A/J mice infected with the virulent Salmonella typhimurium C5 strain suppress the early exponential bacterial growth in the reticuloendothelial system toward the end of the first week of infection, with spleen and liver bacterial counts reaching a plateau phase. In vivo administration of neutralizing anti-interleukin-12 (IL-12) antibodies did not affect early bacterial growth in the tissues (days 1 to 3) but impaired the establishment of the plateau, with higher spleen and liver counts by day 7 of the infection in anti-IL-12 treated mice than in untreated controls. Gamma interferon (IFN-gamma) was detectable in the sera and spleen homogenates of both control and anti-IL-12-treated mice on days 3 and 7 of the infection. Noticeably, IFN-gamma levels were significantly lower in anti-IL-12 treated mice than in control animals. Splenocytes from uninfected A/J mice released IFN-gamma in response to concanavalin A (ConA) or to S. typhimurium C5. In vitro IL-12 neutralization dramatically impaired the IFN-gamma response to S. typhimurium but not to ConA. Splenocytes harvested from infected anti-IL-12 treated mice on day 7 of the infection produced significantly lower amounts of IFN-gamma upon in vitro stimulation with ConA and with a Salmonella protein-rich extract than did cells from similarly infected untreated control animals. Spleen cells from infected mice showed lower proliferative (mitogenic) responses to ConA and to a Salmonella soluble extract than did cells from uninfected mice. In vivo anti-IL-12 treatment significantly restored the ability of splenocytes from infected mice to proliferate in response to the antigens and ConA. In vivo neutralization of IL-12n in innately susceptible BALB/c mice ((ItyS)) immunized with a live attenuated aromatic-dependent Salmonella vaccine reduced host resistance to virulent oral challenge with S. typhimurium C5. Thus, in primary Salmonella infections, IL-12 mediates the suppression of growth of virulent salmonellae in the reticuloendothelial system, positively modulates IFN-gamma production, and is involved in the immunosuppression which accompanies the acute stages of the disease. IL-12 also contributes to host resistance to virulent organisms in secondary infections.     

Novel rosette formation of murine spleen cells with autologous red blood cells

Spleen cells of normal BALB/c mice formed rosettes with autologous red blood cells, and the formation was calcium ion dependent. Peritoneal exudate cells, bone marrow cells and thymocytes did not form such rosettes. Spleen cells were passed over a Sephadex G-10 column or incubated on a plastic surface in order to eliminate adherent cells from them. Cells obtained by both these methods were unable to form rosettes. B cell-, T cell- and natural killer cell-enriched fractions in spleen cells were unable to form rosettes either. Some of the mouse IgG subclasses suppressed rosette formation when added to its site. These are monoclonal antibodies whose specificities are directed against Aspergillus niger glucose oxidase. Moreover, Aspergillus niger glucose oxidase suppressed the rosette formation when spleen cells had been treated with it as well as when it had been added to the site of rosette formation. These findings suggest that some murine spleen cells have receptors to a structure on autologous red blood cells, which is recognized by an anti-Aspergillus niger glucose oxidase monoclonal antibody.     

In vitro culture of coxsackievirus group B, type 3 immune spleen cells on infected endothelial cells and biological activity of the cultured cells in vivo.

Spleen cells from male BALB/c mice infected 7 days earlier by an intraperitoneal injection of 3 X 10(4) PFU of a myocarditic strain of coxsackievirus B-3 lysed virus-infected endothelial cells in a 51Cr release assay. Cytotoxic activity in the in vivo sensitized spleen cell population could be further increased by culturing the immune spleen cells from infected mice on virus-infected or uninfected endothelial cells for 6 to 7 days in vitro. Cytotoxicity of in vitro cultured spleen cells to infected targets was mediated by T lymphocytes since reactivity was abolished by treatment of the spleen cells with anti-thy 1.2 serum and complement. Reciprocal assays with BALB/c and C57BL cells indicated that maximum cytotoxicity occurred when spleen cells were sensitized on syngeneic endothelial cells. Other experiments showed that spleen cells sensitized to coxsackievirus B-3 or encephalomyocarditis virus were selectively cytolytic to targets infected with the homologous virus. Adoptive transfer of T cells cultured in vitro on infected endothelial cells retained their ability to induce myocarditis in T-lymphocyte-deficient mice.     

Direct evidence for granuloma-inducing activity of interleukin-1. Induction of experimental pulmonary granuloma formation in mice by interleukin-1-coupled beads.

Pulmonary granulomas were induced in BALB/c mice by the intratracheal injection of Sephadex G-50 and latex beads. Very large granulomas developed around Sephadex G-50 beads. Minimal inflammation was produced in mice given latex beads. Aqueous extracts prepared from pulmonary granuloma lesions induced in mice by Sephadex G-50 beads contained high levels of interleukin-1 (IL-1) activity but not interleukin-2 (IL-2) activity. IL-1 activity in the extracts correlated with granuloma size. In a subsequent step, large granulomas were induced by the intratracheal injection of Sepharose 4B beads coupled to fractions of the extracts containing IL-1 activity (ie, granuloma-derived IL-1) prepared from Sephadex G-50-induced granulomatous lungs. In addition, large granulomas were induced by the intratracheal injection of recombinant IL-1-coated Sepharose 4B beads. In contrast, very small granulomas were seen when IL-2-coated or plain Sepharose 4B beads were injected into mice. These results indicate that IL-1 participates in the induction and/or expression of granulomas.     

Early IFN-gamma production is related to the presence of interleukin (IL)-18 and the absence of IL-13 in experimental Trypanosoma cruzi infections

Gamma-interferon (IFN-gamma) production, the hallmark of the Th1 immune response, has been shown to play a central role in the resistance to Trypanosoma cruzi infections, in particular when produced in the very early acute infection. BALB/c mice infected with T. cruzi, Tulahuén strain, reach high parasitemias during the acute phase, and their spleen cells release IFN-gamma in the second week of the infection, while those of the resistant C3H strain produce the cytokine earlier, at 2 days post-infection (pi). We studied in the spleen cells supernatants of infected BALB/c and C3H mice, the spontaneous production of cytokines involved in the induction, interleukin (IL)-18 and IL-12 p70, as well as in the downregulation, IL-13 and IL-10, of the Th1 immune response. We found that, at 2 days pi, only C3H mice produced IL-18, while IL-12 p70 was detected in both mouse strains. Moreover, at this time pi splenocytes from BALB/c mice spontaneously produced high amounts of IL-13. At 14 days pi, despite the increased levels of IL-13 and IL-10 detected in C3H mice, they still showed high concentrations of IL-18 and IL-12 p70. In contrast, spleen cells from BALB/c mice did not secrete IL-18, IL-12 p70 and IL-13 at this time pi, but produced higher amounts of IL-10 than C3H mice. Non of these cytokines was found increased in the cell supernatants of chronically infected mice. The addition of lipopolysaccharide (LPS) or Concanavalin A (Con A) to the cell cultures did not enhance the production of IL-18 and IL-12 at the time points tested. On the other hand, at 21 days pi, when parasitemia peaked, an inhibition of both the LPS induced IL-10 release and the IL-13 production upon Con A stimulation was observed in C3H, but not in BALB/c mice. We did not find an increase of IL-18, IL-10, or IL-12 p70 in the serum of the infected mice, despite the high seric IL-12 p40 concentrations reached during the infection. The data show that the different kinetics of the production of these cytokines in the spleen of both mouse strains could have a key role in the in vivo regulation of IFN-gamma production. In these experimental models, early IFN-gamma release and thus resistance to T. cruzi infection, could be related to the combined effect of both IL-18 and IL-12p70 in the absence of IL-13.     

Cellular basis of decreased immune responses to pneumococcal vaccines in aged mice.

Previously, model systems were developed in our laboratory to study murine immune responses to the 23-valent pneumococcal polysaccharide vaccine Pnu-Imune, both in vivo and in vitro (M. Garg and B. Subbarao, Infect. Immun. 60:2329-2336, 1992; M. Garg, A. M. Kaplan, and S. Bondada, J. Immunol. 152: 1589-1596, 1994). Using these systems, we found that aged mice did not respond to the vaccine in vivo or in vitro. Cell separation studies showed that the unresponsiveness of the aged spleen cells to the vaccine was not due to an intrinsic B-cell defect or to T-cell-mediated immunosuppression but resulted from an accessory cell deficiency. Irradiated spleen cells from young mice enabled the old mouse spleen cells to respond to the vaccine. Interestingly, irradiated spleen cells from old mice also restored the vaccine responsiveness in old mice but were required in greater numbers than the young mouse spleen cells to induce similar levels of response. The accessory cell was an adherent cell that could be removed by passage through Sephadex G-10 and thus may be a macrophage. Accessory function could also be provided by the cytokine interleukin-1 (IL-1), IL-4, or IL-5 but not IL-2 or IL-6. Thus, one reason for the deficient immune response to pneumococcal vaccine in aged mice is a quantitative defect in adherent accessory cells.     

Permissiveness of athymic ("nude") mice towards congenic memory cells

Spleen cells from BALB/c or BALB-Igb mice immunized against the determinant oligo-D-alanine were transferred to the following recipients: normal BALB/c; lethally irradiated BALB/c; and congenitally athymic BALB/c-nu. Irradiated as well as nude recipients permitted the development of a strong adoptive antibody response, while the response in normal BALB/c recipients was very low ("isogeneic barrier"). Using allotypically marked spleen cells from BALB-Igb donors it was shown that the antibodies in irradiated as well as in nude recipients were produced by donor cells. The same conclusion was drawn by assessing isoelectric focusing spectra, which in each transfer displayed the individually characteristic pattern of the donor. In addition to specific antibodies, the donor cells produced considerable levels of IgG, as characterized by allotype, but again only in nude and in irradiated recipients. The ready permissiveness of nude recipients towards congenic memory cells could be abolished when prospective recipients were restored, some time prior to transfer, by BALB/c thymus or BALB/c spleen cells. The results are interpreted to suggest that the isogeneic barrier in normal recipients is due to a thymus-dependent suppression or rejection of memory cells by the recipients.     

Interleukin 2 augmentation of the defective natural killer cell activity in patients with primary Sjögren''s syndrome.

BALB/c mice are highly susceptible to Leishmania major infection and develop a disseminated lethal disease. Previous experiments indicate that during infection the spleen is heavily populated with large mononuclear cells containing amastigotes. Morphologically these cells resemble undifferentiated monocytes and granulocytes. In this study we examined myelopoiesis in BALB/c and C57BL/6 (resistant) mice during infection with L. major. The number of macrophage-granulocyte precursors in the spleen of infected BALB/c mice, determined by colony forming units in soft-agar cultures (cfu-c), increased steadily to a level of about 60 times that of normal sex- and age-matched controls. In C57BL/6 mice, spleen cfu-c peaked at about 1 month post-infection (four times that of normal controls) and declined thereafter to about two times normal levels. The number of cfu-c in the bone marrow did not change significantly in either strain during the infection. Colony stimulating activity (CSA) was found in supernates of cultures of adherent cells from the spleen of infected BALB/c mice. Under the same conditions, CSA was non-detectable in supernates of nonadherent spleen cells of infected mice, and those of adherent or nonadherent spleen cells of control animals. A possible role of undifferentiated macrophage-granulocytes in the exquisite susceptibility of BALB/c mice to L. major infection is discussed.     

Spleen cell cytokine secretion in Mycobacterium bovis BCG-infected mice.

Three susceptible mouse strains, i.e., BALB/c (H-2d), C57BL/6 (H-2b), and major histocompatibility complex-congenic BALB.B10 (H-2b), were infected intravenously with 4 x 10(6) CFU of live Mycobacterium bovis BCG and analyzed 4 weeks later for in vitro spleen cell cytokine secretion in response to purified protein derivative (PPD), BCG culture filtrate (CF), BCG cellular extract, total BCG, the purified extracellular 30-32-kDa antigen (the fibronectin-binding antigen 85), or the intracellular 65-kDa heat shock protein. C57BL/6 and BALB.B10 mice produced 5- to 10-fold more gamma interferon and interleukin-2 (IL-2) when stimulated with CF, PPD, and antigen 85 than BALB/c mice did. When stimulated with BCG extract and whole BCG, gamma interferon and IL-2 levels were generally lower and comparable in the three strains. IL-4 was detected in spleen cell culture supernatants from infected BALB/c mice but not from C57BL/6 or BALB.B10 mice. IL-5 could not be detected. C57BL/6 and BALB.B10 spleen cells also produced more tumor necrosis factor alpha and IL-6 after stimulation with PPD and CF than BALB/c cells did. Finally, BCG vaccination generated efficient protective immunity in C57BL/6 and BALB.B10 mice but not in BALB/c mice. These data suggest that secreted mycobacterial CF antigens selectively induce a strong TH1 response in BCG-infected C57BL/6 and BALB.B10 mice, whereas in BALB/c mice this response is partly counterbalanced by TH2 cells.     

Lysis of infected myofibers by coxsackievirus B-3-immune T lymphocytes.

Spleen cells from male adult BALB/c mice given intraperitoneal injections of purified coxsackievirus B-3 were examined for the ability to lyse syngeneic neonatal myofibers in culture. Cytotoxicity against infected and uninfected targets was measured with the use of an in vitro 51Cr release assay. Immune spleen cells obtained 4--7 days after infection were cytotoxic for viral-infected myofibers. Peak reactivity was observed 5 days after infection. At this time immune spleen cells showed significantly less reactivity against uninfected myofibers. Cytotoxicity against infected targets was mediated by T lymphocytes, since reactivity was abolished by treatment with anti-thy 1.2 and complement. Treatment with anti-Ig and complement caused no loss of activity. Reciprocal assays performed with BALB/c and CBA cells showed that maximal cytotoxicity occurred against infected syngeneic myofibers, providing further evidence that viral-specific effector cells were T lymphocytes. In addition, hyperimmune rabbit anti-coxsackievirus B-3 antiserum could not block immune spleen cell lysis of infected targets, suggesting that coxsackievirus-infected myofibers expressed surface membrane antigens not recognized by specific neutralizing antibody.     

T cells and macrophages in Trypanosoma brucei-related glomerulopathy.

In a previous study, susceptibility for Trypanosoma brucei-related glomerulopathy in mice was shown to be dependent on non-major histocompatibility complex genes. Glomerular disease in this model could not be explained by the production of autoantibodies alone. In order to analyze which part of the defense system, in addition to the B-cell compartment, is involved in the development of this infection-related glomerular disease, groups of athymic (BALB/c rnu/rnu), splenectomized, or macrophage-depleted BALB/c mice were inoculated with T. brucei parasites. Polyclonal B-cell activation, invariably observed in infected BALB/c mice, was absent in BALB/c rnu/rnu mice. Glomerular disease in athymic mice, however, as defined by albuminuria and deposition of immune complexes, was not different from that seen in euthymic infected BALB/c mice. Splenectomy prior to inoculation of parasites led to a decreased incidence of albuminuria in 40% of the animals, whereas splenectomy 21 days after inoculation reduced albuminuria significantly, suggesting a role for spleen cells in the induction of glomerular disease. After macrophage depletion with liposome-encapsulated dichlorodimethylene-diphosphonate, infected BALB/c mice developed significantly higher albuminuria levels for a period up to 2 weeks after depletion. Therefore, it was concluded that the development of T. brucei-related glomerular disease is independent of thymus-matured T cells, while the involvement of macrophages in the development of proteinuria is inhibitory rather than disease inducing. Spleen cells other than thymus-dependent T cells, B cells, and macrophages should be investigated for their role in the pathogenesis of this glomerulopathy.     

Suppressor cells induced by influenza virus inhibit interleukin-2 production in mice.

PR8 virus depressed interleukin-2 (IL-2) and natural killer (NK) cell activity in BALB/c infected mice. IL-2 production was not dependent on (i) a decreased number of T cells or (ii) a primary defect in IL-1 production, but on a T-suppressor cell subpopulation. In fact, when T suppressor cells were removed from infected spleen cells, we observed normal levels of IL-2 activity.