Immunoregulation in experimental schistosomiasis. II. Soluble egg antigen-induced chronic spleen cell augmentation of baseline lymphocyte reactivity

Spleen cells from mice harboring infections of Schistosoma mansoni for 20 weeks exposed to Con A or to soluble schistosome egg antigenic preparation (SEA), treated with Mitomycin C (Mc), and cocultured with spleen cells from either normal or infected mice caused an augmented baseline [3H]TdR incorporation by the otherwise unstimulated responder cells. This regulation required an in vitro induction phase. SEA-exposed, Mc-treated normal spleen cells had no effect on responder cell cultures. SEA-stimulated, Mc-treated chronic spleen cell augmentation was effective on responder cell populations from either normal mice or mice infected with S. mansoni for 8 weeks. Augmentation was most pronounced when assayed on cells from infected mice assayed over a 5-day incubation. In addition, it is demonstrated that these Con A- and SEA-elicited activities are mediated by soluble mediators which lack H-2 restriction.     

Immunoregulation in experimental schistosomiasis: in vitro induction and assay of spleen cell suppressor activity

Spleen cells from normal CBA/J mice or mice infected with Schistosoma mansoni were exposed for 48 to 72 hr to either concanavalin A (Con A), soluble egg antigen (SEA), or soluble worm antigenic preparation (SWAP), treated with mitomycin C to prevent further DNA synthesis, and admixed with either normal or sensitized syngeneic spleen cells exposed to a concentration gradient of phytohemagglutinin (PHA) or SEA, respectively. Both nonspecific (by Con A) and "antigen-specific" (by SEA and SWAP in infected mice only) induction of suppression was observed when using PHA-induced blastogenesis as the final assay. The number of mice with inducible splenic suppressive activity and the degree of PHA suppression induced by exposure to SEA appeared to decline between 8 and 20 weeks of infection. In contrast, when the response of spleen cells from mice infected for 8 weeks to SEA served as the final assay, strong suppressive activity was induced from the spleen cells of all chronically infected mice (20 weeks of infection). This model permits parallel analysis of the induction of suppressor activity by nonspecific and schistosome antigen-specific signals during the course of this chronic, immunoregulated condition, schistosomiasis mansoni.     

Recombinant granulocyte-macrophage colony-stimulating factor restores deficient immune responses in mice with chronic Trypanosoma cruzi infections

Spleen cells from mice with chronic Trypanosoma cruzi infection generate a minimal plaque-forming response to SRBC in vitro. Addition of granulocyte-macrophage (GM)-CSF to cultures of spleen cells from chronically infected mice restored the plaque-forming cells (PFC) response to normal levels. Splenic adherent cells from chronically infected mice were deficient in their ability to reconstitute the PFC response of accessory cell-depleted normal spleen cells. Preincubation of splenic adherent cells from infected mice with GM-CSF restored their ability to reconstitute the PFC response of adherent cell depleted cultures. Ia Ag expression by splenic adherent cells from chronically infected mice was significantly lower compared to Ia Ag expression of cells from normal mice. Incubation of splenic adherent cells from chronically infected mice for 48 h with GM-CSF increased levels of Ia Ag expression to approximately those of uninfected mice. Peritoneal macrophages from infected mice produced IL-1 after incubation with GM-CSF at levels equivalent to those produced by similarly treated control macrophages. Spleen cells from chronically infected mice showed significant induction of IL-2 mRNA after GM-CSF treatment, and the addition of the anti-IL-2 mAb to GM-CSF supplemented cultures of spleen cells from infected mice blocked the restoration of the anti-SRBC PFC response. Thus, the ability of GM-CSF to restore the anti-PFC response to SRBC appears to involve the up-regulation of accessory cell function that includes increased Ia Ag expression and the induction of IL-1 production. These events also involve increased IL-2 production with resultant up-regulation of the response to SRBC by spleen cells from infected mice. Finally, it was shown that treatment of infected mice with rGM-CSF completely restored their depressed PFC production in vivo.     

Regulation of granulomatous inflammation in murine schistosomiasis. IV. Antigen-induced suppressor T cells down-regulate proliferation and IL-2 production

In murine schistosomiasis mansoni the cell-mediated immune response to the deposited eggs is mediated by CD4+ delayed-type hypersensitivity effector T (TDH) cells that produce vigorous granulomatous responses in the liver and intestines of acutely infected animals. The response is significantly down-modulated in chronically infected mice by Ag-specific Ts cells. The present study was undertaken to establish an in vitro model by which TDH-Ts cell interactions could be analyzed. To this end, Ts cells were induced in vitro by preculture of chronic or acute infection spleen cells with soluble egg Ag (SEA) for 48 h. The induced cells suppressed the SEA-specific proliferation of acute infection spleen cells by 80 to 95%. The induced suppressor cells were Ag specific in both induction and elicitation of function, and were not cytotoxic to the acute infection splenic target cells. Suppression by the induced cells was manifested within the first 24 h of the SEA-induced response as IL-2 produced by acute infection spleen cells was suppressed 62%. Phenotypic analysis by flow cytometry of the induced suppressor cells showed that CD8+ cells from acute infection spleens and CD4+ and CD8+ cells from chronic infection spleens were effector Ts cells. Taken together, CD4+ and CD8+ SEA-specific Ts cells can be induced in vitro to effectively suppress the SEA-specific lymphoproliferation and IL-2 production of acute infection spleen cells. Establishment of this in vitro model will allow us to further analyze the mechanisms of Ts cell-mediated suppression of TDH cells.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

Characteristics of the splenic suppressor cell--target cell interaction in experimental African trypanosomiasis

We have examined further the relationship between immunosuppression and suppressor cell activity in experimental African trypanosomiasis. In the present study we describe the nature of the interaction between splenic suppressor macrophages from Trypanosoma rhodesiense-infected C57BL/6 mice and target effector cells in the primary in vitro PFC response to SRBC. Suppressor cell potential was expressed only when cell-cell contact of a noncytolytic nature was established between infected spleen cells and normal splenic responder cells. Isolation of suppressor cells from responder cells by a cell-impermeable membrane completely abrogated suppression. Similarly, supernatant fluids from infected spleen cell cultures could not passively transfer suppression. Suppressor cells did not act via prostaglandin synthesis in that indomethacin failed to restore responsiveness to infected spleen cells or to passively suppressed normal cultures. Inhibition of DNA synthesis by irradiation of mitomycin C treatment did not block suppressor cell function, but suppressor cell effects were inhibited by exposure of infected spleen cells to silica particles or to heat treatment. We conclude that suppressor cell effects in experimental African trypanosomiasis are consistent with a suppressor macrophage acting via a noncytolytic cell-cell interaction with responder target cells.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Cellular interactions in lymphocyte proliferation: effect of syngeneic and xenogeneic macrophages.

Secondary cell-mediated responses to ectromelia virus infection were studied using an in vitro system. Lymphoid “responder” cells from mice which had recovered from intravenous primary infection at various times prior to sacrifice, were cultured with syngeneic, virus-infected macrophages or spleen cells as “stimulator” cells at 39 °C, a temperature which prevented the virus from exerting cytopathic effects against responder cells. This restrictive temperature and medium with 2-mercaptoethanol at 10^?4M often gave viable cell yields of more than 100% of the original responder cells over 4 days of culture. Preliminary experiments showed that spleen cells from primed mice, cultured with syngeneic, infected spleen cells from normal mice gave the most powerful secondary cytotoxic cell responses as measured by ⁵¹Cr release from virusinfected H-2-compatible target cells. The cytotoxic cells were sensitive to anti-θ and complement treatment and lysed H-2-compatible, virus-infected target cells much more efficiently than infected, allogeneic target cells, thus indicating that they were T cells. Some activity against uninfected H-2-compatible target cells was also generated, but this was largely independent of the presence of virus-induced antigen, (i.e. infected stimulator cells were unnecessary) and therefore seemed to be a consequence of the cultural conditions. Cold target competition showed that this activity was the responsibility of a T cell subset separate from the virus-specific cytotoxic T cells. The peak of cytotoxic activity against virus-infected targets occurred at 4 days of culture and DNA synthesis was maximal on day 3. The concentration of cytotoxic T cells at the peak was eight-fold higher than at the peak of the splenic primary response in vivo, Memory T cells (precursors of secondary cytotoxic T cells) appeared in spleen within 12–14 days of primary infection in vivo, reached a plateau at 5–6 weeks and persisted for at least 16 months. Spleen cells appeared partly refractory to secondary stimulation in vitro at 8–10 days post-priming. This did not seem to be due to cellular migration from spleen to lymph nodes or peritoneal cavity, but its cause was not determined. Primary responses in vitro were not detectable under conditions optimal for secondary responses, thus suggesting a major quantitative, or qualitative difference between virgin and memory T cells.     

C3-Activating proteases on human lymphoblastoid cells superinfected with epstein-barr virus

The role played by macrophages in feedback inhibition of the immune response during murine brucellosis, allowing establishment of chronic infection, was investigated using a number of approaches. First, it was shown that the degree of splenomegaly (a measure of macrophage influx) following infection with Brucella abortus strain 19 did not correlate with the course of bacterial numbers in the spleen of CBA, BALB/c, and C57B1/10 mice. Second, it was shown that a rough, mucoid mutant, B. abortus strain 19R, although causing a chronic infection, did not induce splenomegaly. Nor could “suppressor macrophages” be demonstrated in these spleens. Delayed-type hypersensitivity during this infection was lower than with B. abortus strain 19. Third, no nonspecific suppression of unrelated immune responses in CBA mice infected with B. abortus strain 19 could be demonstrated, despite the very large numbers of macrophages in the spleen. The responses tested included delayed-type hypersensitivity and cell-mediated resistance to Listeria monocytogenes, antibody response to sheep erythrocytes (both serum antibody and plaque-forming cells in the spleen) and skin-graft rejection.     

Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

Activation of lymphoid cells by BCG in vitro

Bacillus Calmette-Guerin (BCG) stimulated splenic and thymic lymphocytes in vitro as measured by uptake of ³H-thymidine. This activation of lymphocytes by BCG required the presence of a critical concentration of macrophages. Thymus cells containing no more than 0.25% macrophages were stimulated by BCG, but reduction of macrophages below this level by adherence to plastic abolished the response. Reconstitution with purified macrophages completely restored the response. A high concentration of adherent cells (“macrophages”) depressed the response of splenic lymphocytes, as judged by the improvement in DNA synthesis after reduction of the proportion of adherent cells in the spleen cell population.Bacillus Calmette-Guerin augmented the production of lymphocyte-activating factor (LAF) from purified splenic adherent cells, but the presence of lymphocytes made that augmentation considerably greater.These data reaffirm the bidirectional nature of the relationship between lymphocytes and macrophages. They further show that BCG can create highly activated populations of each type of cell, in part by enhancing their interaction.     

Mechanisms of killing of measles virus-infected cells by human lymphocytes: interferon associated and unassociated cell-mediated cytotoxicity

The recent interest in natural killer (NK) cells in immunosurveillance and the ability of infection with certain organisms to modulate NK activity led us to examine the influence of Toxoplasma gondii infection on mouse NK cells. Infection of BALB/c mice with 5 × 10³ virulent Toxoplasma intraperitoneally (ip) resulted in significantly enhanced NK activity in peritoneal exudate cells (PC) and in spleen cells (SC). Intravenous (iv) and subcutaneous (sc) challenge of BALB/c mice with Toxoplasma also resulted in enhanced natural killer (NK) activity in PC and SC. In BALB/c mice, as well as in other strains (A/J, C57BL/6, C3H/HeJ, CeH/HeN, [A/J × C3H]F₁), peak augmentation of PC and SC NK activity was observed 3 days following ip Toxoplasma challenge. Administration of silica to mice abolished Toxoplasma-induced NK cytotoxicity. BALB/c mice chronically infected with Toxoplasma had significantly higher endogenous NK activity than did controls in PC but not in SC. Chronically infected BALB/c mice boosted with virulent Toxoplasma ip exhibited significantly enhanced NK activity in PC but not in SC. Thus, acute and chronic infection with Toxoplasma modulates NK activity in addition to macrophage activation and thereby provides a system that should facilitate study of the relative contribution of NK cells and activated macrophages in resistance to tumor growth and spread.     

Changing patterns of lymphocyte proliferation, IL-2 production and utilization, and IL-2 receptor expression in mice infected with Schistosoma mansoni

In murine schistosomiasis mansoni the soluble egg Ag (SEA)-induced L3T4+ T cell-mediated circumoval granulomatous response peaks at the acute stage (8w) and undergoes Lyt-1+, Lyt-2+ suppressor cell mediated down-modulation at the chronic stage (20w) of the infection. In the present study lymphoproliferation, IL-2 production, utilization, and IL-2R display were examined in splenic lymphocytes of infected mice. The L3T4+ subset was the major SEA-specific proliferative population at both stages of the infection. Chronic infection spleen cells or the L3T4+ subset proliferated less compared with their acute infection counterparts. Elimination of the Lyt-2+ subset did not improve diminished proliferation. Chronic infection cells and the Lyt-2+ subset stimulated with SEA and exogenous rIL-2 regained their proliferative ability to a level comparable with acute infection cells. Ag-stimulated acute infection T cells produced 40 to 50 chronic infection cells less than 5 U/ml IL-2. Elimination of L3T4+ T cells diminished, and the Lyt-2+ T cells left unchanged IL-2 production in the acute infection cells. Elimination of Lyt-2+ subset from the chronic infection population did not enhance IL-2 production. Exogenously added rIL-2 was equally utilized by acute or chronic infection T cells. Scatchard plots of [125I]IL-2 binding showed similar numbers of high affinity receptors on acute (189) and chronic infection cells (118), but the number of low affinity receptors was higher on the acute (2229) vs the chronic infection lymphocytes (578). Analysis of IL-2R expression by two-color fluorescence and flow cytometry revealed that 30 to 40% of the acute, chronic infection L3T4+ cells displayed receptor. Receptor expression increased by added SEA, or SEA + rIL-2. R display among Lyt-2+ cells was only 12 to 18%. SEA stimulation enhanced receptor display more among the acute, SEA + rIL-2 stimuli raised receptor expression only among chronic infection lymphocytes. These data do not show inherent defect in IL-2R display, or utilization in chronic infection T cells. Diminished IL-2 production appears to be the cause of decreased T cell responsiveness.     

Macrophages as effector cells of protective immunity in murine schistosomiasis: IV. Coincident induction of macrophage activation for extracellular killing of schistosomula and tumor cells

Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ($\text{1}\text{3}$ to $\text{1}\text{5}$ those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.     

Suppression of antitumor immunity by macrophages in spleens of mice bearing a large MOPC-315 tumor

Summary We had shown previously that progression of MOPC-315 plasmacytoma growth is associated with an increase in the percentage of macrophages in the spleen as well as a decrease in the ability of tumor-bearer spleen cells to mount an antitumor cytotoxic response upon in vitro immunization. Here we provide evidence that macrophages in the MOPC-315 tumor-bearer spleen are responsible at least in part for the suppression of the generation of antitumor cytotoxicity. Accordingly, removal of most macrophages by depletion of phagocytic cells or Sephadex G-10-adherent cells from spleens of mice bearing a large tumor resulted in augmented antitumor immune potential. Also, Sephadex G-10-adherent spleen cells from tumor-bearing (but not normal) mice drastically suppressed the in vitro generation of antitumor cytotoxicity by normal spleen cells. The suppressive activity of these adherent cells did not reside in contaminating suppressor T cells, since it was not reduced by treatment with monoclonal anti-Thy 1.2 antibody plus complement. The Sephadex G-10-adherent cell population from the tumor-bearer spleen suppressed the in vitro generation of antitumor cytotoxicity against autochthonous tumor cells but not against allogeneic EL4 tumor cells, and hence the suppression was apparently specific. The suppressive activity of the Sephadex G-10-adherent cell population from tumor-bearer spleens was overcome by treatment of the tumor-bearing mice with a low curative dose of cyclophosphamide. This immunomodulatory effect of a low dose of the drug in overcoming the suppression mediated by the Sephadex G-10-adherent cell population enables the effector arm of the immune system of tumor-bearing mice to cooperate effectively with the drug's tumoricidal activity in tumor eradication.This paper was presented in part at the annual meeting of the American Association of Immunologists, Chicago, Illinois, 10–15 April 1983     

The requirement of Ia-positive accessory cells for the induction of tumor-specific cytotoxic T lymphocytes in vitro

The mechanism for the induction of cytotoxic T cells specific for tumor-associated antigens was studied by using fractionated responder T cells, tumor cells, and accessory cells in vitro. The tumor-specific cytotoxic T cells were induced by culturing immunized spleen cells with the tumor cells in vitro for 5 days. Nylon-column-purified T cells alone did not induce cytotoxic T cells upon culture with tumor cells, but the addition of normal spleen cells as accessory cells did successfully induce the cytotoxic T cells, suggesting that the presence of accessory cells is required for the activation of tumor-specific cytotoxic T cells in vitro. The accessory function was associated with spleen cell populations adhering to a plastic dish, a Sephadex G-10 column or a nylon wool column, and was sensitive to anti-Ia serum and C treatment, but was resistant to anti-Ig serum or anti-Thy 1 serum and C treatment, suggesting that the accessory cells are Ia-positive macrophages. Not only syngeneic but also allogeneic macrophages had the accessory function and the allogeneic macrophages were also Ia positive. These results suggest that Ia-positive macrophages play a crucial role in the induction of tumor-specific cytotoxic T cells in vitro. The possible role of Ia-positive accessory cells in the induction of tumor-specific cytotoxic T cells is discussed from the standpoint of cellular interactions.     

Balancing immunity and pathology in visceral leishmaniasis

Experimental visceral leishmaniasis (VL) caused by infection with Leishmania donovani results in the development of organ-specific immunity in the two main target tissues of infection, the spleen and the liver. The liver is the site of an acute resolving infection associated with the development of inflammatory granulomas around infected Kupffer cells, and resistance to reinfection. Paradoxically, the spleen is an initial site for the generation of cell-mediated immune responses, but ultimately becomes a site of parasite persistence with associated immunopathological changes. These include splenomegaly and a breakdown in tissue architecture that is postulated to contribute to the immunocompromized status of the host. The progressive development of splenic pathology is largely associated with high levels of TNF and interleukin (IL)-10. Follicular dendritic cell (DC) networks are lost, whereas TNF mediates the destruction of marginal zone macrophages and gp38(+) stromal cells, and IL-10 promotes impaired DC migration into T-cell areas with consequent ineffective T-cell priming. Splenic stromal cell function is also altered, promoting the selective development of IL-10-producing DC with immunoregulatory properties. Ultimately, a fine immunological balance determines responses that effectively promote parasite clearance in the liver and those that promote pathology in the spleen, and future investigation aims to separate these responses to offer further means of parasite control in chronically infected VL patients.     

Mechanisms of depression of splenic natural killer cell function in C57BL/6 mice infected with Leishmania donovani

C57BL/6 mice chronically infected with the protozoan parasite Leishmania donovani exhibit profoundly depressed splenic natural killer (NK) cell activity as measured by in vitro cytolysis of lymphoma target cells. Injection of infected mice with an interferon (IFN) inducer or in vitro treatment of infected splenocytes with IFN, a phorbol ester, or indomethacin failed to restore their NK activity to the degree shown by age-matched, uninfected mice. Fractionation of infected splenocytes by nylon wool, Sephadex G-10, or carbonyl iron and magnetism treatments was also unable to effect an increase in NK activity. Addition of infected splenocytes to uninfected ones in in vitro NK assays suppressed the NK activity of the latter, and the suppression could be partially or wholly abrogated by prior fractionation of infected splenocytes by the methods noted above. In vitro treatment of infected splenocytes with concanavalin A revealed the presence of NK activity in these cell populations. The results indicate that splenocytes in L. donovani-infected mice become insensitive to IFN stimulation; and the impairment of another, possibly IFN-independent pathway of NK-cell activation may also contribute to the observed L. donovani-induced depression in splenic NK activity in C57BL/6 mice.     

Enhancing and suppressive effects of macrophages on T-lymphocyte stimulation in vitro.

The immunoregulatory effect of peritoneal and splenic macrophages on Con A-stimulated mouse splenic T lymphocytes was investigated in vitro using [¹²⁵I]UdR incorporation as a measure of lymphocyte proliferation. [¹²⁵I]UdR incorporation was enhanced by the addition of increasing numbers of splenic or low doses of peritoneal adherent cells to macrophagedepleted splenic lymphocytes. The addition of increasing numbers of peritoneal macrophages beyond 5–10%, however, proportionally suppressed T-cell proliferation. Activated splenic macrophages obtained from mice 6 days after infection with Listeria monocytogenes were suppressive, whereas macrophages obtained from immune donors 9–10 days after infection were not, so that a chronological association appeared to exist between macrophage activation and immunosuppression. The addition of 2-mercaptoethanol to the cell cultures increased [¹²⁵I]UdR incorporation without affecting the stimulatory and suppressive effects of splenic and peritoneal macrophages, respectively. Heat-killed and freeze-thawed macrophages lost their capacity to enhance or inhibit lymphocyte transformation. Macrophages treated with mitomycin C to inhibit DNA synthesis retained their regulatory functions. These studies suggest differential regulatory roles for spleen versus peritoneal macrophages on T-lymphocyte responses to Con A stimulation in vitro.