Depression of contact sensitivity by Pseudomonas aeruginosa-induced suppressor cells which affect the induction phase of immune response.

The cellular basis of depression of contact sensitivity to oxazolone in mice injected with Pseudomonas aeruginosa was studied. Cells from draining lymph nodes of mice sensitized with oxazolone 18 h previously were able to induce contact sensitivity to normal mice when administered in their footpads. In contrast, cells from draining lymph nodes of P. aeruginosa-injected and oxazolone-sensitized donors failed to induce contact sensitivity when injected in the footpad of normal mice and were capable of actively blocking the immunizing process brought about by lymph node cells from sensitized mice when injected together in the footpad of normal recipients. The P. aeruginosa-induced suppressor cells required antigenic stimulation, had precursors sensitive to cyclophosphamide, and did not affect the effector mechanisms of contact sensitivity. Thus, the results suggest that P. aeurginosa depresses contact sensitivity to oxazolone by enhancing the activity of suppressor cells which normally arise during the sensitization process and which affect the afferent limb of the immune response, probably by inhibiting the normal recruitment of T lymphocytes in the draining lymph nodes.     

Pseudomonas aeruginosa infection depresses contact sensitivity to oxazolone by enhancing suppressor cell activity

The depression of contact sensitivity to oxazolone in mice infected withPseudomonas aeruginosa was studied. In oxazolone-sensitized mice,P. aeruginosa infection affects cell proliferation in the lymph nodes draining the site of sensitization. This impaired cell proliferation does not seem to be due to an altered lymphocyte reactivity, since lymph node and spleen cells from infected animals show a normal mitotic responsiveness to both T and B cell mitogens. In addition, the draining lymph nodes and spleens of mice exhibiting a depressed response to oxazolone contain a cell population able actively to suppress the response to the same antigen of syngeneic recipients sensitized immediately before the cell transfer. These suppressor cells require antigenic stimulation and appear to act on the induction phase of contact sensitivity.     

Suppression of cell-mediated immunity in experimental changas' disease

Summary The effect of acute infection with the Tulahuén strain of Trypanosoma cruzi on the cellular immune response in Swiss mice was studied. Mice were immunized with either Freund's complete adjuvant or oxazolone, a skin sensitizing agent, and subsequently skin-tested with either BCG protoplasm or oxazolone to detect delayed hypersensitivity. Depression of the response to these antigens was observed in infected mice during the stage of marked parasitemia. Mice which were responsive to oxazolone before infection lost their ability to respond as the infection progressed. When immunized with live attenuated T. cruzi before infection with virulent organisms, mice developed a greater than normal sensitivity to oxazolone and survived infection. These experiments do not conclude whether immunosuppression due to infection with T. cruzi is directed toward induction or expression of the cell-mediated immune response to the antigens employed.     

Measurement of parasite-specific immune responses in vitro: evidence for suppression of the antibody response to Trypanosoma cruzi

Using the Mishell and Dutton culture system, we have developed an assay for eliciting and quantifying parasite-specific immune responses in vitro. The ability of spleen cells from noninfected and Trypanosoma cruzi-infected mice to respond to parasite-associated antigens was assessed by examining the primary plaque-forming cell response to trinitrophenylated T. cruzi (TNP-TC). The response to TNP-TC of both normal, noninfected, unprimed mice and mice infected with T. cruzi is T cell dependent and appeared to involve recognition of parasite antigens by T. cruzi-specific T cells. In most experiments, spleen cells from infected mice respond to TNP-TC at levels equal to, or below, that of spleen cells from normal mice. This near "normal" response is in apparent contrast to the suppressed response of spleen cells from infected mice to another antigen (sheep red blood cells) or TNP on a different carrier (TNP-chicken erythrocytes). Demonstration that the response of spleen cells from infected mice to TNP-TC can be potentiated by addition of interleukin 2-containing supernatants or by depletion of plastic and Sephadex G-10-adherent cells suggests that the mechanisms which control the response of infected mice to nonparasite antigens may also limit parasite-specific immune responses.     

On evasion of Trypanosoma cruzi from the host immune response. Lymphoproliferative responses to trypanosomal antigens during acute and chronic experimental Chagas' disease.

The ability of spleen cells taken from mice infected with Trypanosoma cruzi to proliferate after stimulation with specific trypanosomal antigens was investigated during the acute and chronic phases of the disease. Lymphoproliferation was minimal or undetectable during the acute period whereas the chronic phase was characterized by significant responses over a wide range of antigen concentration. Transfer of infected mouse spleen cells to cultures of splenocytes from chronically infected animals failed to modify the response of the latter to antigenic stimulation as measured by DNA synthesis. Furthermore, the responses of infected mouse spleen cells collected during the acute period and freed of Lyt 2.1-bearing lymphocytes, a subclass known to contain the suppressor T cells, did not differ significantly from those of untreated aliquots of the same cell suspensions. These results, together with the fact that the T-cell compartment of the spleen was severely depleted during the acute but not the chronic stage of the infection, suggest that the impaired immunological responsiveness of acutely infected mice may be due in part to the absence or marked reduction of responder and/or accessory T lymphocytes. An active role for suppressor T cells in the reduced response to trypanosomal antigens by lymphocytes from mice in the early, acute phase of T. cruzi infection is not supported by the present observations.     

T cell- and B cell-independent adaptive immunity mediated by natural killer cells

It is commonly believed that only T lymphocytes and B lymphocytes expressing recombination-dependent antigen-specific receptors mediate contact hypersensitivity responses to haptens. Here we found that mice devoid of T cells and B cells demonstrated substantial contact hypersensitivity responses to 2,4-dinitrofluorobenzene and oxazolone. Those responses were adaptive in nature, as they persisted for at least 4 weeks and were elicited only by haptens to which mice were previously sensitized. No contact hypersensitivity was induced in mice lacking all lymphocytes, including natural killer cells. Contact hypersensitivity responses were acquired by such mice after adoptive transfer of natural killer cells from sensitized donors. Transferable hapten-specific memory resided in a Ly49C-I(+) natural killer subpopulation localized specifically in donor livers. These observations indicate that natural killer cells can mediate long-lived, antigen-specific adaptive recall responses independent of B cells and T cells.     

Unraveling the lethal synergism between Trypanosoma cruzi infection and LPS: a role for increased macrophage reactivity

Various infections sensitize to lethal shock by promoting hyperactivation of macrophages to LPS stimulation. Although macrophages are thought to be deactivated upon contact with apoptotic cells during Trypanosoma cruzi infection, T. cruzi infection also sensitizes mice to endotoxemia. Herein, we studied the mechanisms of sensitization to endotoxemia in T. cruzi-infected mice in order to solve the paradox. Live (but not fixed) trypomastigotes from various stocks sensitized mice to endotoxemia. Mice deficient in glycolipid recognition (TLR2(-/-) and CD1d(-/-)) were sensitized by infection to challenge with LPS. Infected mice hyperproduced TNF and IL-10 upon LPS challenge. Infected TNF-R1(-/-), macrophage migration inhibitory factor (MIF)(-/-) and IFN-gamma(-/-) mice were lethally sensitized, but infected TNF-R1(-/-) mice administered anti-MIF survived shock with LPS. Macrophages from infected mice hyperproduced TNF in response to LPS stimulation and displayed increased expression of TLR4 compared to non-infected controls. Treatment with the PGE(2) synthesis inhibitor acetylsalicylic acid (AAS) in vivo reduced parasitemia and enhanced LPS-stimulated production of TNF by macrophages, but the effect was less in infected mice than in normal mice. Nevertheless, AAS treatment did not increase the susceptibility of infected mice to sublethal shock with LPS. Our results point to independent MIF and TNF/TNF-R1 lethal pathways and suggest a role for hyperactivated macrophages in T. cruzi-sensitized LPS-induced shock.     

Trypanosoma cruzi-induced immunosuppression: B cells undergo spontaneous apoptosis and lipopolysaccharide (LPS) arrests their proliferation during acute infection

Acute infection with Trypanosoma cruzi is characterized by multiple manifestations of immunosuppression of both cellular and humoral responses. B cells isolated at the acute stage of infection have shown marked impairment in their response to polyclonal activators in vitro. The present work aims at studying the B cell compartment in the context of acute T. cruzi infection to provide evidence for B cell activation, spontaneous apoptosis and arrest of the cell cycle upon mitogenic stimulation as a mechanism underlying B cell hyporesponse. We found that B cells from acutely infected mice, which fail to respond to the mitogen LPS, showed spontaneous proliferation and production of IgM, indicating a high level of B cell activation. Furthermore, these activated B cells also exhibited an increase in Fas expression and apoptosis in cultures without an exogenous stimulus. On the other hand, B cells from early acute and chronic infected mice did not present activation or apoptosis, and were able to respond properly to the mitogen. Upon in vitro stimulation with LPS, B cells from hyporesponder mice failed to progress through the cell cycle (G0/G1 arrest), nor did they increase the levels of apoptosis. These results indicate that B cell apoptosis and cell cycle arrest could be the mechanisms that control intense B cell expansion, but at the same time could be delaying the emergence of a specific immune response against the parasite.     

Impairment of contrasuppressor activity in mice infected with the paramyxovirus of Newcastle disease.

Spleen cells from mice infected with the virus of Newcastle disease (NVD) fail to mediate the passive transfer of contact sensitivity to simple chemical haptens such as picryl chloride (Pcl) and oxazolone (Ox). The inhibitory effect of NDV can be bypassed by treating recipient mice with low doses of cyclophosphamide (Cy), suggesting that the T-effector cell which mediates the passive transfer of contact sensitivity is not affected by NDV infection. Vicia Villosa-adherent cells from immune mice display contrasuppressor activity and restore the ability of cells from NDV-infected mice to transfer contact sensitivity to naive recipients. In contrast, Vicia Villosa-adherent cells from NDV-infected mice fail to exert any contrasuppressor activity. Furthermore, contrasuppressor activity can also be detected in the culture supernatants of Vicia Villosa-adherent cells from uninfected, sensitized mice, but not in culture supernatants of Vicia Villosa-adherent cells from NDV-infected mice. The present results suggest that a Vicia Villosa-adherent contrasuppressor cell population is impaired by NDV infection.     

RIL—2对急性克氏锥虫感染小鼠的抗SRBC直接溶血空斑反应及抗虫保护性免疫影响的初步研究

为研究ＩＬ—２在克氏锥虫感染过程中出现的免疫抑制机制中的作用，８只克氏锥虫感染的Ｃ３Ｈ／ＨｅＪｎ小鼠每日皮下注射两次，每日总量５０００μ重组人ｒＩＬ－２；对照组相同数量小鼠皮下注射等量的无钙、镁离子的ＰＢＳ。实验结果表明ｒＩＬ－２注射组小鼠脾细胞对羊红细胞的反应能力由ＰＢＳ注射鼠的１５。２％提高到９３。５％（Ｐ＜０．０５）。相当接近于正常的未感染小鼠的空斑形成细胞数。ｒＩＬ－２注射组小鼠仅表现轻度降低的寄生虫血症，其动态变化在两组间未显示出差异。除ＰＢＳ组的１只小鼠在感染后第３６ｄ死亡外，ｒＩＬ－２以及ＰＢＳ注射组的其它动物都在克氏锥虫急性感染期存活。尽管ｒＩＬ－２注射并未明显地提高克氏锥虫急性感染期Ｃ３Ｈ／ＨｅＪｎ小鼠的抗虫保护性免疫，本实验提示ＩＬ－２不足是克氏锥虫急性感染期Ｃ３Ｈ／ＨｅＪｎ小鼠非特异性免疫抑制的原因之一。     

The role of T cells in the passive transfer of contact sensitivity and their occurrence in the bone marrow

Contact sensitivity to 4-ethoxymethylene-2-phenyl oxazolone can be transferred to irradiated recipients by lymph node, spleen and bone marrow cells from sensitized donors. The transfer by lymph node, spleen and bone marrow cells was abolished by anti-θ serum whose specificity was confirmed by absorption with CBA brain. Transfer by peritoneal exudate cells was relatively resistent to treatment with anti-θ serum. Mice deprived of T cells did not develop contact sensitivity following immunization, and their bone marrow and peritoneal exudate cells failed to transfer contact sensitivity. It was concluded that T cells were required both for the induction and for the passive transfer of contact sensitivity.     

Experimental autoimmune encephalomyelitis can be prevented and cured by infection with Trypanosoma cruzi

Trypanosoma cruzi is an intracellular parasite that induces a strong Th1-type response and immunosuppression during the acute phase of infection. To study how the infection with T. cruzi would modulate the development of an autoimmune disease, we immunized C57BL/6 mice and IL-10 or iNOS knock-out mice of the same background with the encephalitogenic MOG 35-55 peptide and infected them with T. cruzi. Our results demonstrate that infection with T. cruzi completely prevents EAE development and furthermore induces complete and lasting remission in mice that were infected with this parasite after they had developed clinical EAE. Nitric oxide and IL-10 participate in triggering the mechanisms associated with EAE suppression by the infection. Decreased lymphoproliferation and increased frequencies of Annexin-positive cells and of T cells bearing CD95, CD95L or CTLA-4 were observed in the spleen from immunized/infected mice, as well as lower IL-2 and increased TGF-beta production in comparison with only immunized mice. Our results indicate that several effector and regulatory mechanisms of the immune response that arise during the acute phase of T. cruzi infection lastingly affect the expansion and/or effector functions of encephalitogenic cells, preventing the onset or inducing complete remission of EAE.     

Gamma interferon suppresses acute and chronic Trypanosoma cruzi infection in cyclosporin-treated mice.

To determine if exogenous gamma interferon is effective in immunosuppressed mice infected with Trypanosoma cruzi, recombinant murine gamma interferon was administered to cyclosporin-treated mice with either acute or chronic T. cruzi infection. Gamma interferon significantly decreased parasitemia and prevented death in acutely infected mice. Parasitemias and mortality of mice treated with both gamma interferon and cyclosporin were similar to those of immunocompetent controls. In chronically infected mice, cyclosporin treatment produced significantly more organ explant cultures positive for T. cruzi. Fewer positive cultures, particularly for spleen and heart, were obtained from cyclosporin-treated mice when they also received gamma interferon. Ketoconazole treatment of mice resulted in no positive cultures. Cyclosporin treatment did not prevent activation of peritoneal macrophages by parenteral gamma interferon, nor did it have a consistent effect on serum titers of alpha/beta or gamma interferon in response to a second challenge inoculum of T. cruzi. These data indicate that exogenous gamma interferon suppresses acute and chronic T. cruzi infection in cyclosporin-treated mice but that gamma interferon is not as effective as the relatively specific antimicrobial ketoconazole. Gamma interferon activates macrophages despite cyclosporin treatment, and its effects appear to be tissue specific.     

The migratory behavior of T blasts to contact sensitivity reactions in activelyand passively sensitized mice.

The arrival of cells from lymph nodes immunized with the contact sensitizing agents oxazolone and picryl chloride at ears challenged with these antigens was studied inmice using the technique of labelling with -51Cr. An apparent specificity of arrival was seen because the immune cells transfered contact sensitivity passively, giving rise to an inflammatory response in the ear, to which a subpopulation of cells (T blasts) was non-specifically attracted. It was also shown that there are at least two distinct populations of cells with the ability to move to inflammatory sites: the first, found in immunized lymph nodes, moves to contact sensitivity reactions in both actively and passively sensitized mice; the second, found in bone marrow and oil-induced peritoneal exudates, moves to contact sensitivity reactions in actively sensitized mice, whereas in passively sensitized mice, the arrival of these cells at contact sensitivity reaction is poor. It is suggested that the ability of T blasts to move to sites of inflammation my be useful as an assay technique for contact sensitivity reactions.     

Suppression of contact hypersensitivity in mice by ultraviolet irradiation is associated with defective antigen presentation.

A single dose of radiation from FS40 sunlamps results in systemic depression of delayed-type hypersensitivity (DTH) to 2-chloro-1,3,5-trinitrobenzene (TNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB). Immunosuppression is proportional to the log10 dose of radiation and exhibits a delayed time course. Animals sensitized one day after ultraviolet (u.v.) treatment respond normally, but sensitization 3-15 days after treatment results in about 70% suppression of the DTH response. The dose response of DTH in normal and u.v.-treated animals to 1,3,5-trinitrophenyl (TNP) conjugated adherent splenocytes from normal or u.v.-treated donors was investigated. When normal mice were immunized with TNP-conjugated adherent splenocytes from normal or u.v.-treated donors, a DTH response could be elicited in these animals by injection of TNP-conjugated splenocytes into the ear. However, u.v.-irradiated recipients could not be sensitized by TNP-conjugated adherent cells from u.v.-treated mice but were sensitized by such cells from normal mice. Lysed, TNP-conjugated, normal adherent splenocytes did not immunize u.v.-irradiated recipients, but did immunize normal recipients. These results confirmed that antigen presentation is deficient in u.v.-treated mice. The time of appearance of the antigen-presenting defect in the spleen cells of u.v.-treated mice was the same as for the depression of contact sensitivity, strengthening the evidence for a causal relationship between defective antigen presentation and depression of contact sensitivity.     

Trypanosoma cruzi-induced immunosuppression: blockade of costimulatory T-cell responses in infected hosts due to defective T-cell receptor-CD3 functioning.

A model of experimental Trypanosoma cruzi murine infection with chemically induced metacyclic forms (opossum clone Dm28c) showed a marked state of T-cell unresponsiveness during acute phase, but lacked evidence of suppressor cell activity. Spleen cells from infected mice were suppressed in vitro in responses to T-cell activators concanavalin A, anti-Thy1 monoclonal antibody (MAb), and anti-CD3 MAb compared with spleen cells from control littermates. Activation with accessory cell-independent stimulus provided by immobilized anti-CD3 was defective in splenic CD4-positive T cells from infected mice, but not in such cells from control mice. No evidence of splenic suppressor cell activity was found in cell-mixing experiments using nylon-passed T cells from control and infected donors. Kinetic experiments showed that there was a discrete stage in infection when T cells were already suppressed in response to anti-CD3 but still responded to anti-CD69 MAb. In these T cells, immobilized anti-CD3 failed to enhance simultaneous CD69 responses, although anti-CD3 enhanced CD69 responses in control T cells from uninfected donors. These results demonstrate an intrinsic defect in T-cell receptor-mediated T-cell activation, which could be a mechanism generating T-cell suppression during infection by T. cruzi.     

Antibody responses to contact sensitizing agents. Effect of sensitized cells.

Lymphocytes from mice immunized by painting with the contact sensitizing agent picryl chloride have been shown to produce regulatory effects on the DNA synthesis and contact sensitivity responses of normal mice painted with picryl chloride. This report describes the effect of these cells on antibody responses of normal mice to picryl chloride. Lymph node cells taken 5--7 days after painting increased early IgM antibody responses of normal mice to picryl chloride. Spleen cells were not effective. The increase was mediated by T cells as judged by anti-theta treatment and nylon wool filtration and could not be produced by killed, irradiated or allogeneic cells. A similar activity could be demonstrated in cells from mice painted with another contact sensitizing agent, oxazolone. The effect was strictly specific and cells from mice painted with picryl chloride or oxazolone would only increase responses to picryl chloride or oxazolone, respectively, even when presented with a mixture of the two antigens. The cel-s increasing antibody production were different from T cells previously shown to mediate contact sensitivity and T cells shown to regulate DNA synthesis in that they could not be generated in adult-thymectomized mice.     

Gamma Interferon Modulates CD95 (Fas) and CD95 Ligand (Fas-L) Expression and Nitric Oxide-Induced Apoptosis during the Acute Phase of Trypanosoma cruzi Infection: a Possible Role in Immune Response Control

We have previously shown that splenocytes from mice acutely infected with Trypanosoma cruzi exhibit high levels of nitric oxide (NO)-mediated apoptosis. In the present study, we used the gamma interferon (IFN-gamma)-knockout (IFN-gamma(-/-)) mice to investigate the role of IFN-gamma in modulating apoptosis induction and host protection during T. cruzi infection in mice. IFN-gamma(-/-) mice were highly susceptible to infection and exhibited significant reduction of NO production and apoptosis levels in splenocytes but normal lymphoproliferative response compared to the infected wild-type (WT) mice. Furthermore, IFN-gamma modulates an enhancement of Fas and Fas-L expression after infection, since the infected IFN-gamma(-/-) mice showed significantly lower levels of Fas and Fas-L expression. The addition of recombinant murine IFN-gamma to spleen cells cultures from infected IFN-gamma(-/-) mice increased apoptosis levels, Fas expression, and NO production. In the presence of IFN-gamma and absence of NO, although Fas expression was maintained, apoptosis levels were significantly reduced but still higher than those found in splenocytes from uninfected mice, suggesting that Fas-Fas-L interaction could also play a role in apoptosis induction in T. cruzi-infected mice. Moreover, in vivo, the treatment of infected WT mice with the inducible nitric oxide synthase inhibitor aminoguanidine also led to decreased NO and apoptosis levels but not Fas expression, suggesting that IFN-gamma modulates apoptosis induction by two independent and distinct mechanisms: induction of NO production and of Fas and Fas-L expression. We suggest that besides being of crucial importance in mediating resistance to experimental T. cruzi infection, IFN-gamma could participate in the immune response control through apoptosis modulation.     

Inhibition of Trypanosoma cruzi-specific immune responses by a protein produced by T. cruzi in the course of Chagas' disease.

Immunosuppression is readily demonstrable in the acute phase of Trypanosoma cruzi infection but subsides during the chronic phase. In vitro, living T. cruzi induces important alterations in mitogen-activated human T and B lymphocytes and inhibits their capacity to proliferate. These effects are reproduced by a protein spontaneously released by this parasite, termed trypanosomal immunosuppressive factor (TIF). In this study we asked whether TIF would also inhibit a T. cruzi-specific immune response and if it is produced in a mammalian host during infection. A significant reduction in the level of [3H]thymidine incorporation by spleen cells from chronically infected mice stimulated with a T. cruzi antigen preparation ensued when TIF was added to the cultures. Production of TIF in T. cruzi-infected individuals was denoted by the ability of serum IgG from either chronically infected patients or mice to abolish, in a concentration-dependent manner, the capacity of TIF to suppress interleukin-2 receptor expression by phytohaemagglutinin-stimulated human lymphocytes. This neutralizing activity was absent in the IgG fractions prepared from sera of healthy volunteers, noninfected mice or mice killed at different times during acute T. cruzi infection. Circulating anti-TIF antibodies represent indirect evidence of TIF production in vivo which, together with TIF-mediated inhibition of T. cruzi-specific lymphoproliferation, raise the possibility that TIF controls anti-parasite immune responses in vivo. The presence of TIF-neutralizing antibodies during chronic but not acute T. cruzi infection may be one of the reasons why immunosuppression is confined to the acute stage.     

Depression of contact sensitivity by enhancement of suppressor cell activity in Pseudomonas aeruginosa-injected mice.

Heat-killed Pseudomonas aeruginosa depresses contact sensitivity to oxazolone in C56BL/6 mice. The draining lymph nodes and spleens of mice exhibiting an impaired reactivity to oxazolone contain a cell population capable of depressing the response to oxazolone of recipients sensitized immediately before cell transfer. The suppressive activity of these cells appears to be antigen specific, since they do not affect the response to picryl chloride and because they do not arise in P. aeruginosa-injected but not oxazolone-sensitized mice. These suppressor cells occur in the draining lymph nodes and spleen at 3 and 4 days after sensitization, respectively, and have precursors sensitive to cyclophosphamide. It is concluded that P. aeruginosa depresses contact sensitivity to oxazolone by enhancing the suppressor cell activity of the regulatory cells which arise during conventional sensitization.