Infected Spleen Cells Research Articles

Trypanosoma cruzi-induced suppression of IL-2 production. II. Evidence for a role for suppressor cells.

Suppression of IL-2 production during experimental Chagas' disease accounts at least in part for the overall depressed state of the immune system in infected mice. The failure to produce IL-2 in response to mitogen stimulation is not the result of the lack of cells capable of producing IL-2, but appears to be due to regulation of IL-2 production by suppressor cells. This conclusion is supported by cell-mixing experiments where the ability of cells from infected mice to suppress normal spleen cell IL-2 production is evident. Although depletion of plastic and Sephadex G-10 adherent cells results in modest increases in IL-2 production by spleen cells from infected mice, even in the presence of normal adherent cells as a source of IL-1 producers, IL-2 production does not approach normal levels. Also, isolated macrophages are not by themselves suppressive for normal spleen cell IL-2 production, whereas plastic and G-10 nonadherent cells from infected mice are. Depletion of Thy-1+ and Ly-2+ cells not only completely abrogates the ability of spleen cells from infected mice to suppress normal IL-2 production, but results in a cell preparation which actually enhances IL-2 production. Anti-Ly-2 and C treatment of infected spleen cells also markedly enhances their ability to produce IL-2. These results indicate a major role for Ts cells in the regulation of IL-2 production, and a relatively minor role of macrophages as direct effector cells of suppression in this response. The ability to enhance IL-2 production in this system with PG synthesis inhibitors suggests a role for PG-producing cells such as macrophages in the suppressor mechanism, perhaps as inducers of the suppressor effector cells.

Association of reduced interleukin-2 production with genetic susceptibility to Pichinde virus in inbred strains of hamsters.

Adult inbred MHA hamsters are susceptible to lethal infections with Pichinde virus while inbred LSH hamsters resist such infections. Previous studies demonstrated higher levels of endogenous and induced natural killer (NK) activity in MHA splenocytes than in LSH splenocytes. Preferential replication of Pichinde virus in cells with NK activity was suggested by showing that the greater numbers of infected spleen cells observed in MHA hamsters could be accounted for by a cell population that cosedimented with a peak of NK activity. Increased cellularity of thymi and spleens as well as increased cells sensitive to lymphokines was also found in MHA hamsters as compared to LSH hamsters. In the present study we found that injection of anti-asialo GM 1 serum reduced NK activity but did not alter susceptibility to virus infection. However, MHA hamsters were found to be relatively deficient in the production of interleukin 2 and injection of interleukin 2 altered the mortality of hamsters infected with Pichinde virus. These findings suggest that susceptibility to lethal infection by Pichinde virus is associated with reduced ability to produce interleukin 2 in MHA hamsters.

Specific suppression of delayed hypersensitivity expression to Trypanosoma cruzi in mice.

The absence of cutaneous delayed type hypersensitivity (DTH) expression was investigated in Trypanosoma cruzi infected mice. Neither spleen cells nor peritoneal exudate cells from infected mice transferred DTH to normal recipients in local or systemic adoptive transfer experiments. Expression of DTH in T. cruzi immunized mice was suppressed specifically by Thy-1 negative spleen cells from acutely and subacutely infected animals. Suppression was observed only upon systemic transfer, but not when infected mice spleen cells were added to DTH effector cells and transferred to normal recipients. These results suggest that cutaneous DTH expression in acutely infected mice, might be blocked by mechanisms other than those described for suppression of lymphocyte proliferation and of DTH induction to T. cruzi.

Taenia taeniaeformis: Inhibition of mitogen induced proliferation and lnterleukin-2 production in rat splenocytes by larval in vitro product

Splenocytes from rats infected with Taenia taeniaeformis showed an early decreased proliferative response to the mitogen concanavalin A with larval growth. When larvae culture supernatant ( in vitro product) was added to normal rat splenocytes, there was a decrease in the proliferative response to concanavalin A and phytohemagglutinin. The ability of infected rat splenocytes to produce interleukin-2 was decreased with larval growth. Addition of in vitro product to culture medium significantly depressed interleukin-2 production by normal as well as infected rat spleen cells. Culturing normal rat splenocytes with in vitro product for 3 days induced a suppressor cell population. When these cultured cells were admixed with fresh normal rat splenocytes plus concanavalin A, the proliferative response of the fresh cells was significantly reduced. The present results suggest that in vitro product secreted by larvae in hepatic cysts causes subversion of the cellular immune response in the host. Induction of a suppressor cell population could suppress interleukin-2 production which may lead to the inhibition of differentiation and proliferation of specific cytotoxic T lymphocytes.

Different localization of the product of the v- rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59 v- rel , the v- rel oncogene product of REV-T, we show that p59 v- rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59 v- rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59 v- rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59 v- rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59 v- rel and transformation by REV-T, and they suggest that p59 v- rel cannot transform cells in which it assumes solely a nuclear location.

Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice.

We have characterized the progress of acute murine cytomegalovirus (MCMV) infection in the spleen, liver, and salivary gland of susceptible (BALB/c) and resistant (C3H) strains of mice after intraperitoneal inoculation. Viral replication was analyzed by virus titration, infectious-center assays, and in situ cytohybridization with cloned subgenomic fragments of the MCMV genome. The most striking differences between strains were observed in the spleen. At 24 h postinfection (p.i.), both strains had a similar number of infected spleen cells. At 48 h p.i., BALB/c mice showed marked dissemination of the splenic infection which continued until 96 h p.i. In contrast, the number of infected C3H spleen cells did not increase from the 24-h level but declined later on. This early block in dissemination of MCMV infection in C3H mouse spleens was not a result of the H-2k haplotype, as BALB.K (H-2k) mice, which show an intermediate level of resistance to MCMV infection, exhibited dissemination of the infection between 24 and 48 h p.i., albeit at a reduced level. However, between 72 and 96 h p.i., we observed a decline in the number of infected spleen cells in BALB.K mice similar to that observed in C3H mice. We also demonstrated by Southern blot analysis of DNA from the infected spleen cells that the termini of the MCMV genome fuse after in vivo infection.

Different forms of membrane-associated herpes simplex virus glycoproteins induce functionally distinct subsets of herpes simplex virus-specific suppressor T cells.

Previous studies have shown that two types of virus-specific suppressor T cells (Ts) are induced in mice made tolerant with herpes simplex virus (HSV)-infected spleen cells (SC). One type of Ts blocks the afferent phase of the delayed hypersensitivity response to HSV (Ts-aff), and the other blocks the efferent or effector phase (Ts-eff). In this report we show that the induction requirements for these suppressor populations differ. Injection of SC infected for 6 h with HSV at a multiplicity of infection of 5 or less or treated with heat-inactivated virus induced only Ts-aff. Similar results were seen with SC incubated for 90 min in virus-free preparations containing only viral proteins. In contrast, the Ts-eff population was induced only by SC treated for 6 h with infectious HSV at a multiplicity of infection of 10. Collectively, these data indicate that Ts-aff are induced by adsorbed HSV antigens on SC, whereas Ts-eff are induced by nascent HSV antigens expressed on infected SC. In addition to their induction requirements, the two types of regulatory cells differ in their expression of effector function. Ts-eff but not Ts-aff require a cyclophosphamide-sensitive target cell in the immune recipient for suppressor function. The possible identity of this target cell and the significance of the different induction requirements between the two types of Ts are discussed.

Macrophage-related fibrinolysis in experimental disseminated histoplasmosis.

A model of disseminated histoplasmosis in CBA/J mice was developed. Cultures of Histoplasma capsulatum from the spleens of infected mice suggested almost complete clearance of fungi by week 3. The adherent spleen cells from infected mice showed a 2- to 20-fold increase in fibrinolysis. The increase in activity was maximal around 1 to 2 weeks and disappeared after week 3 of infection, and this paralleled the progressively decreasing number of culturable fungi from the spleen. In vitro coculture of infected spleen cells or nylon wool-purified immune T cells and proteose peptone-induced macrophages resulted in increased fibrinolysis. Peritoneal exudate cells from infected mice also showed increased fibrinolysis. The addition of soluble antigen to an in vitro culture system resulted not only in an increase in fibrinolytic activity of peritoneal exudate cells derived from infected mice but also of proteose peptone-induced macrophages. These observations suggest that spleen and peritoneal macrophages from H. capsulatum-infected mice exhibit increased fibrinolysis which in turn is indicative of macrophage activation. The mechanism of activation occurs as a result of immunologically specific T cell-macrophage interaction and by the action of histoplasma products on the macrophages. The significance of these findings and the role of the plasminogen activator assay in studies of disseminated fungal infection are discussed.

Lymphocyte function in experimental African trypanosomiasis: VII. Loss of antigen-nonspecific suppressor-T-cell activity

The extent of immunosuppression occurring in mice infected with the pathogenic African trypanosomes was studied. Spleen cells from Trypanosoma rhodesiense-infected C57BL/6J mice were tested for antigen-nonspecific suppressor-T-cell (T s,) activity after concanavalin A (Con A) treatment in vitro. After exposure to Con A, control and infected mouse spleen cells were added to responder spleen cell cultures stimulated with sheep erythrocytes (SRBC). Assays for the resultant plaque-forming cell responses to SRBC revealed that antigen-nonspecific T s activity was lost during the first week of infection. Changes in infected mouse T-cell subpopulations, including a terminal loss of Lyt 2.2 + cells, accompanied but did not precede the demonstrable loss of T s, function. Splenic suppressor macrophages which arise during infections with T. rhodesiense also did not seem to be associated with the loss of antigen-nonspecific T s activity. It is concluded that the generalized immunosuppression associated with experimental African trypanosomiasis extends to the mitogen-induced T s population.

Restoration of in vitro immune responses of spleen cells from mice infected with Trypanosoma cruzi by supernatants containing interleukin 2.

During the course of Trypanosoma cruzi infection, mice become suppressed in the ability to produce IL 2. Addition of supernatants from concanavalin A-stimulated normal spleen cells or supernatants from EL 4 cells enhanced the suppressed in vitro response of infected spleen cells to sheep erythrocytes. This enhancement was demonstrable at various times after infection and was shown not to be the result of Con A stimulation alone. Spleen cells from infected mice respond to IL 2-rich supernatants in Mishell-Dutton cultures in a dose-dependent fashion. Absorption of IL 2 activity from EL-4 supernatants by CTLL-2 cells results in a decrease in the enhancing effect of the supernatants on the in vitro antibody responses of infected spleen cells. Also, the reversal of suppression occurs only if IL 2-rich supernatants are added within 24 hr of the initiation of cultures. These results indicate that defective IL 2 production may play a role in the inability of mice infected with T. cruzi to respond appropriately to neoantigens.

Further characterization of Marek's disease virus-infected lymphocytes. I. In vivo infection.

Previous reports from this laboratory identified bursa-derived lymphocytes (B cells) and non-B cells as the predominant cell types respectively involved in the early cytolytic and subsequent latent infection of chickens with Marek's disease virus (MDV). It was not known whether these differences were qualitative or quantitative or if the method for detection of latent infection (viral antigen production after 48 h of in vitro cultivation) was sensitive enough. To further define the cells involved in the various phases of MDV infection, we used monoclonal antibodies which specifically react with B cells, or T cells, or la-antigen-bearing cells. Dual fluorescence tests to detect surface markers and viral internal antigen (VIA) were conducted with infected spleen cells freshly collected from MDV-infected chickens or after in vitro cultivation of those cells. The same antibodies were also used for a rosetting procedure to yield fractions enriched or depleted of T cells, B cells or la-bearing cells. These were examined directly for viral DNA by in situ hybridization or dot blot DNA hybridization and for VIA cultivation. We learned that infected T cells also comprise part of the early cytolytic phase of MDV infection but constitute a minority population (approximately 2-3%) compared to B cells (83-92%) at 3 or 4 days post infection. Latently infected cells were definitively identified as mostly la-bearing T cells, although a few (2-4%) were B cells. Prior to in vitro cultivation, latently infected cells apparently had insufficient viral DNA for detection by in situ hybridization, but the more sensitive dot blot procedure revealed viral DNA in fractions later found positive by VIA expression after in vitro cultivation. Viral DNA replication in latently infected cells apparently had occurred after 48 h cultivation because in situ hybridization detected infected cells at that time. Treatment of cell cultures with iodo-deoxyuridine, 12-O-tetradecanoyl phorbol-13-acetate or n-butyrate failed to increase the number of spleen cells which expressed VIA.

Non-specific immunosuppression by Cryptococcus neoformans infection.

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppressive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.

Natural Killer Cell Activity during Murine Schistosomiasis mansoni

Natural killer cell (NK) activity of spleen cells from CBA/J and C57BL/6 mice infected with Schistosoma mansoni was assayed using 51Cr-labeled YAC-1 cells as target cells. No significant difference in NK activity was detected between infected and age-matched control spleen cells from 1 to 4 wk after infection. The NK-mediated cytotoxic abilities of both infected and age-matched control mice fell in parallel, as expected, on an age-related basis. However, on a cell-to-cell basis the splenic NK activity of mice infected from 8 to 18 wk was significantly less than that of age-matched control mice. Meanwhile, because of the splenomegaly associated with patent infection, the total number of nucleated splenocytes in chronically infected mice increased to fourfold that of age-matched control mice. Therefore, the total available NK activity per spleen was increased by chronic infection. The NK activities of spleen cells from age-matched control mice and those infected for 18 wk could be augmented by in vivo administration of polyinosinic-polycytidylic acid, indicating the continued responsiveness of NK cells from chronically infected mice to activation. Spleen cell NK activity in age-matched control and infected C57BL/6 mice exhibited a pattern similar to that of CBA/J mice.

In vitro and in vivo histamine-producing cell-stimulating factor (or IL3) production during Nippostrongylus brasiliensis infection: coincidence with self-cure phenomenon.

Spleen cells from Nippostrongylus brasiliensis-infected mice produce large amounts of histamine in response to adult worm antigen. This phenomenon results from the production of HCSF (histamine-producing cell-stimulating factor, probably related to IL3) by sensitized lymphocytes. This factor acts on its target cells (presumably mast cell precursors) by inducing a rapid increase in histamine synthesis. Similarly, parasite infection generates enhanced histamine production by spleen cells in response to concanavalin A (Con A). This results from increases in both HCSF production and the HCSF sensitivity of its target cells. In all cases, maximal histamine and HCSF productions are obtained on day 8 after infection and coincide with parasite rejection. Methyl prednisolone suppresses HCSF production by infected mouse spleen cells in response to worm antigen or Con A. HCSF activity is found in vivo on day 8 in the sera of infected mice, 4 h after they are challenged with an i.v. injection of adult worm antigen. No activity is detected in the sera of normal mice with or without antigen injection. Sera from infected mice that did not receive the antigen exhibit a slight HCSF activity on day 8. Our data bring the first evidence of the existence of an in vivo production of HCSF.

The decline of immunological resistance of aging mice to Trypanosoma musculi

Aged mice of several strains studied developed much more severe infections of Trypanosoma musculi (mouse-specific parasite) than did young adults. Reduced resistance of the aged mice, assessed from the resistance conferred on irradiated recipients by transfer of normal and infected donor spleen cells, resulted from a much slower development of immunity to the parasite, reflecting depleted immune competence, and from the expression in aged mice of an altered internal milieu unfavorable for normal function of lymphoid cells. An analysis of antibody-dependent cytotoxic reactions against T. musculi revealed no differences; therefore, intrinsic changes in antibody-dependent cytotoxic effector cells are not responsible for the decline in resistance to trypanosomes. The possibility that quantitative or qualitative changes in humoral antibody production might account for defective resistance of aged mice was minimized by demonstrating that antibodies play a minor role in the recovery from infection with T. musculi. Evidence of a significant role (possibly indirect) of macrophages in resistance to T. musculi was obtained. Altogether, our data raise the possibility that the decline in natural killer activity that is susceptible to amplification by macrophage-derived interferon may account for the defective resistance of aged mice to trypanosomes.

Immunologic deficiency during experimental Chagas' disease (Trypanosoma cruzi infection): role of adherent, nonspecific esterase-positive splenic cells.

The involvement of adherent splenic cells in the production of deficient lymphocyte responses during the acute phase of experimental Chagas' disease was investigated. When cultured together, purified adherent splenocytes from mice acutely infected with Trypanosoma cruzi caused a significant reduction in the responses of normal mouse spleen cells to T and B cell-specific mitogens. Similar observations were made when infected mouse adherent splenocytes were co-cultured with normal mouse nonadherent cells. Exchange of adherent cells in infected mouse spleen cells suspensions for adherent cells from uninfected mice resulted in increased responses to stimulation with the T and B cell mitogens tested. Treatment of infected mouse cell suspensions with indomethacin improved the responsiveness of these cells to the mitogens. These results support the concept that the immunosuppression that is characteristic of experimental acute Chagas' disease is at least in part mediated by an adherent cell population and is dependent on a prostaglandin-mediated mechanism.

Augmented followed by suppressed levels of natural cell-mediated cytotoxicity in mice infected with Toxoplasma gondii.

The cytotoxic activity of effector cells from mice infected with Toxoplasma gondii was tested in a 4- to 5-hr (51)Cr release assay, using RL [vertical male] 1 and YAC-1 target cells. They showed enhanced cytotoxicity with a peak on the 3rd day postinfection followed by suppression with a peak on the 12th day. The cytotoxicity seemed to be exhibited by natural killer (NK) cells because: (i) pretreatment of the effector cells with antiasialo GM(1) or antiasialo GM(2) plus complement abolished the cytotoxic activity; (ii) the altered cytotoxicity levels were also induced in nude mice; and (iii) the activity was elicited by nonadherent-nonphagocytic cells. The alteration occurred simultaneously in various lymphoid organs with a similar profile. Neither spleen nor bone marrow cells of 12-day-postinfected mice inhibited NK activity of uninfected mice. Culture fluids of the infected mouse spleen and bone marrow cells did not affect the normal mouse NK activity. The proportion of effector cells capable of binding to target cells was constant during the infection. There was no positive correlation between NK activity and serum interferon level; i.e., interferon was detected in the serum of 12-day-postinfected mice but not in that of 3-day-postinfected or uninfected mice. Passively administered interferon or polyinosinic-polycytidylic acid could not restore the suppressed NK activity of 12-day-postinfected mice. Moreover, in vitro treatment of spleen cells from 12-day-postinfected mice with interferon failed to restore the suppressed NK activity. These results suggested that after toxoplasma infection, defective sensitivity to interferon was induced in NK precursor cells, and differentiation to functionally active NK cells might be blocked.

Suppression of responses to cryptococcal antigen in murine cryptococcosis.

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.

Delayed hypersensitivity granuloma formation and modulation around Schistosoma mansoni eggs in vitro. II. Regulatory T cell subsets.

Schistosoma mansoni granuloma modulation was first described in an in vivo murine model of schistosomiasis and was characterized as a diminished cellular responsiveness to newly formed eggs in chronic infections. This phenomenon of modulation was studied using an in vitro model of granuloma formation. Negative selection procedures using anti-Lyt-1.2, 2.2, and Qa-1 sera and complement on spleen cell populations from normal or S. mansoni-infected C57BL/6 or (C57BL/6 X A/J)F1 mice were examined for their effect on in vitro granuloma formation or modulation. These data suggest that there is a Lyt-1+2-, Qa-1+ T cell present in chronically infected spleen cells that is capable of inducing feedback suppression. In addition, selective removal of a population of Lyt-1-2+ T cells from chronically infected spleen cells augmented the ability of that population to form granulomas in vitro. The selective removal of Lyt-1+2- cells ablated the ability of chronically infected spleen cells to form in vitro granulomas. Granuloma formation is primarily dependent on a population of Lyt-1+, Qa-1+ cells, and modulation is dependent on 2 populations, 1 acting directly (Lyt-1-2+) and the other through a (Lyt-1+2-, Qa-1+) feedback mechanism of suppression.

Use of in vivo infected spleen cells to study Fv-2 r-mediated resistance of mice to friend spleen focus-forming virus

In vivo infection of DBA/2 and D2. Fv-2 r mice with 10 5 FFU of FV resulted in a rapid adsorption of virus onto the spleen cells. Thus, these cells could be used in the analysis of Fv-2 expression on FV target cells without any difficulty in avoiding extracellular FV particles. Higher frequencies of cells capable of forming IC could be detected in the spleen than in the bone marrow at 2 and 48 h post infection and levels were very low in D2. Fv-2 r mice. When recently infected cells were transferred to secondary Fv-2 s recipients shortly after infection had been established, the virus-replicating cells released SFFV equally well regardless of whether they originated from Fv-2 s or Fv-2 r mice. This suggests that suppression of SFFV replication may not take place in Fv-2 r cells at an early stage of infection.