Cytokine Gene Expression During Infeetion of Miee Lacking CD4 and/or CD8 with Trypanosoma cruzi

The expression of lymphokine genes during infection of virulent (Tulahuén) or mild ( CA-I ) strains of T. cruzi was studied in mice lacking CD4 and/or CD8 molecules. The increased susceptibility of CD4⁻ and CD4⁻ CD8⁻ mice to infection with CA-I or Tuiahuen was parallelled by diminished IFN-γ mRNA levels. Nitric oxide release and inducible nitric oxide synthase mRNA accumulation by cells from Tulahuen infected CD4⁻ mice was also diminished. CD8⁻ (but not CD4⁻ CD8⁻ mice) showed an increased IL-4 and IL-10 mRNA accumulation upon infection with both strains of T. cruzi. A T_h2-like' response (higher IL-4 and IL-10 mRNA to IFN-γ mRNA ratio), was also observed when cells from non-infected CD8 - mice were stimulated with T cell mitogens.     

Susceptibility and Resistance to Leishmania amazonensis in H-2q Syngeneic High and Low Antibody Responder Mice (Biozzi Mice)

H-2 syngeneic H and L (Biozzi) mice provide a model to study Leishmania infections in which polar resistant and susceptible phenotypes are independent from H-2 differences. High-Ab-responder (H) and low-Ab-responder (L) mice syngeneic at the H-2 locus (H-2^q) were, respectively, susceptible and highly resistant to Leishmania amazonensis infection. L-mice resistance was associated with high IFN-γ and transient IL-4 production by lymph node (LN) cells, in contrast with sustained IL-4 and decreasing IFN-γ production by susceptible H mice. IL-12 production could be detected only in LN from resistant mice. The cytokine production pattern was consistent with preferential progression to a Th1-type response in resistant L-mice, and to a Th2-type response in susceptible H-mice. We also investigated whether this shift towards Th1- or Th2-type cytokine responses was dependent upon H or L antigen presenting cells' (APC) intrinsic ability to preferentially stimulate either T-cell subset. To this end, LN-derived T-cell lines were grown from 12-day infected mice, when both strains produced IFN-γ and IL-4. L-derived T-cell lines developed a Th2 cytokine pattern whereas H-derived T-cell lines produced IFN-γ, IL-4 and IL-10 whatever the APC origin (H or L) used for their derivation. This work constitutes the first characterization of cellular immune responses to the intracellular parasite, L. amazonensis in H-2 syngeneic mice, an infection model in which polar resistant and susceptible phenotypes are determined by non-MHC genes.     

Phenotypic and Functional Analysis of Activated B Cells of Autoimmune NZB × NZW F1 Mice

Polyclonal B-cell activation is the central theme in the production of autoantibodies and possible activation of autoreactive T cells in both human and murine lupus. The abnormal expansion of CD5⁺ B cells in murine lupus has been suggested, in particular, to be one of the most characteristic findings in these mice. Activated B cells can be separated from the B cells of resting stage by the difference in cell density. The aim of this study was to investigate the characteristics of different densities of the spleen cells separated by gradient density. Furthermore, the ability of anti-DNA antibody secretion in each percoll gradient fraction of B cells was also analysed. The results showed: a higher percentage of CD5⁺ B cells, which corresponded to the activated B-cell population, in percoll gradient 1 and 2 fractions; that splenic B cells of NZB/W F₁ mice had proliferative response to interleukin (IL)-4 or IL-5 but not to IL-10 or interferon-γ (IFN-γ); and that B cells isolated by percoll gradient produced anti-DNA antibody after stimulation with lipopolysaccharide (LPS) plus IL-5 and IFN-γ, but not IL-4 and IL-10. These data suggest that B cells at different stages of activation express differential characteristics and functions.     

Impairment of the cellular immune response in acute murine toxoplasmosis: regulation of interleukin 2 production and macrophage-mediated inhibitory effects.

Depression of the cellular immune response to Toxoplasma gondii has been reported in both mice and humans. The present study was undertaken to determine the kinetics and mechanism of the observed downregulation of interleukin 2 (IL-2) production during experimental murine toxoplasmosis. For these investigations, the cell-mediated immune response to the wild type (PTg) was compared with that to the less-virulent mutant parasite (PTgB), which is deficient in the major surface antigen, p30 (SAG-1). Spleen cells from infected A/J mice failed to proliferate in response to Toxoplasma antigens during the first week of infection. Both PTg- and PTgB-infected A/J mice exhibited a significant reduction in the concanavalin A (Con A)-induced lymphoproliferative response. Further, the response of splenocytes from mice infected with the wild-type parasite was significantly diminished compared with that of mice infected with PTgB. The lymphoproliferative response to Con A reached its nadir at day 7 and remained below control levels for at least 14 days postinfection. By day 21 postinfection, the response to Con A and to Toxoplasma antigens was restored to the level observed prior to day 7. Con A-stimulated culture supernatants of spleen cells from mice on day 7 postinfection contained significantly less IL-2 than normal mice. There was no significant difference in the numbers of binding sites or capacity of high-affinity IL-2 receptors between infected and normal mouse splenocytes as determined by Scatchard analysis. Exogenous IL-2 at different concentrations failed to restore the proliferative response of lymphocytes from infected mice to Con A. Adherent macrophages from 7-day-infected mice were able to suppress IL-2 production by normal splenocytes following stimulation with Con A. The inhibitory activity mediated by infected cells was reversed by the antibody to IL-10 but not transforming growth factor beta. There were insignificant levels of nitric oxide production in both infected and normal splenocytes. These results indicate that during acute murine toxoplasmosis, there is a well-defined period (day 7) during which both the T-cell mitogen and parasite antigen-associated lymphoproliferative response are reduced. Further, there is a reduction in the production of IL-2 and an increase in IL-10, which appear to mediate, in part, the observed downregulation of immunity to T. gondii.     

Presynaptic Membrane Receptor-Reactive T Lymphocytes in Myasthenia Gravis

The majority of patients with myasthenia gravis were shown to have T and Bcells specific for a β-bungarotoxin binding protein, presynaptic membranereceptor (PsmR). Such autoreactive T cells may be subdivided into differentsubsets according to the pattern of cytokine production. In this study theauthors examined the subpopulation of the T cells by analysing their IFN-γand/or IL-4 secretion pattern. T cell response to human muscle acetylcholinereceptor (AChR) was examined in parallel. PsmR-stimulated IFN-γ secretionwas found in 60%, and IL-4 secretion in 48% of the patients. Cells stimulated tosecrete both IFN-γ and IL-4 or IFN-γ only were the most common patterns.Treatment of the cells with a mouse anti-human HLA-DR antibody abolished thesecretion of cytokines. There was a positive correlation between the numbers ofPsmR-reactive and AChR-reactive T cells. In conclusion, the results show thatPsmR-stimulated T cells secreted IFN-γ and/or IL-4. This T cell response isMHC class II restricted. Thus, this study indicates that both Th1/Th2 or Th0subsets of the T cells are involved in the autoimmune response in thedisease.     

Interleukin-12 stimulation of lymphoproliferative responses in Trypanosoma cruzi infection.

The cytokine interleukin-12 (IL-12) is essential for resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon-gamma (IFN-gamma), a major activator of the parasiticidal effect of macrophages. A less studied property of IL-12 is its ability to amplify the proliferation of T or natural killer (NK) lymphocytes. We investigated the role of endogenously produced IL-12 in the maintenance of parasite antigen (T-Ag)-specific lymphoproliferative responses during the acute phase of T. cruzi infection. We also studied whether treatment with recombinant IL-12 (rIL-12) would stimulate T-Ag-specific or concanavalin A (Con A)-stimulated lymphoproliferation and abrogate the suppression that is characteristic of the acute phase of infection. Production of IL-12 by spleen-cell cultures during T. cruzi infection increased in the first days of infection (days 3-5) and decreased as infection progressed beyond day 7. The growth-promoting activity of endogenous IL-12 on T-Ag-specific proliferation was observed on day 5 of infection. Treatment of cultures with rIL-12 promoted a significant increase in Con A-stimulated proliferation by spleen cells from normal or infected mice. Enhanced T-Ag-specific proliferation by rIL-12 was seen in spleen cell cultures from infected mice providing that nitric oxide production was inhibited by treatment with the competitive inhibitor NG-monomethyl-L-arginine (NMMA). Enhancement of proliferation promoted by IL-12 occurred in the presence of neutralizing anti-interleukin-2 (IL-2) antibody, suggesting that this activity of IL-12 was partly independent of endogenous IL-2. Thymidine incorporation levels achieved with rIL-12 treatment of the cultures were approximately 50% of those stimulated by rIL-2 in the same cultures.     

Production of Interleukin-4, Interferon (IFN)-γ and IFN-α in Human Immunodeficiency Virus-1 Infection: An Imbalance of Type 1 and Type 2 Cytokines may Reduce the Synthesis of IFN-α

Interferon-α (IFN-α) is an important molecule in the antiviral response, but cells from HIV-1-infected individuals show a reduced ability to secrete IFN-α. We investigated an association between an imbalance of type 1/type 2 cytokines and the production of IFN-α in HIV-1 infection. We used whole blood culture to study the cytokine production profile, interferon-γ (IFN-γ) and interleukin-4 (IL-4), in response to HIV-1 antigens and to study the Sendai Virus and HSV-1-induced-production of IFN-α in seven HIV-1-infected patients. An impaired synthesis of IFN-α was obtained in patients with a predominant IL-4 production (IL-4 > IFN-γ), and we found a positive correlation between the ex vivo production of IFN-α and the IFN-γ/IL-4 ratio but not with the HIV RNA copy number in plasma. We investigated the role of T-cell-derived cytokines in the in vitro production of IFN-α by PBMC from eight healthy donors, activated with Sendai Virus or HSV-1. Whereas type 2 cytokines (IL-4, IL-13) inhibited virus-induced IFN-α synthesis, on the contrary, type 1 cytokines (IL-2, IFN-γ) enhanced it. A disarray in the T-cell-derived cytokine response may play a role in the defect of IFN-α production in HIV-1-infected individuals. Further investigations are needed to explore this hypothesis.     

The roles of interleukins in the synergistic effect of B-cell mitogens with concanavalin A on hydrocortisone-resistant thymic cell proliferation.

Investigations were made to determine the roles of interleukin (IL)-1 and IL-2 in the synergistic enhancement of DNA synthesis by concanavalin A (Con A) and bacterial lipopolysaccharide (LPS) or butanol-extracted water-soluble adjuvant (Bu-WSA) from Bacterionema matruchotii in cultures of thymic cells taken from hydrocortisone (HC)-treated C3H/HeN (LPS-responsive) and C3H/HeJ (LPS-non-responsive) mice. When the C3H/HeNCrj cells were cultured in the presence of Con A and LPS or Bu-WSA together, [3H]thymidine [( 3H]TdR) uptake of the cells was enhanced synergistically in comparison with those cultured with either one of the mitogens alone. The synergistic effect on thymic cells was dependent on Ia-positive accessory cells, since a previous treatment of the cells with anti-Iak serum and complement inhibited the response, and the inhibition could be relieved by the addition of either purified peritoneal exudate macrophages (Mø) or splenic B lymphocytes. The co-stimulation of cells with Con A and LPS or Bu-WSA also enhanced their production and release of thymic cell growth factor(s) into the culture medium. The amounts of IL-1 and IL-2 in the culture supernatants were sufficiently high to explain the activities of the growth factor(s). On the other hand, enhanced IL-2 production without significant increase in IL-1 was seen in the co-cultures of anti-Ia+ cell deprived thymic cells and purified splenic B cells prepared from C3H/HeNCrj mice in the presence of Con A and LPS or Bu-WSA, and it was seen in the cultures of C3H/HeJ thymic cells with Con A and LPS. These results suggest that the synergistic effect of Con A and LPS or Bu-WSA on the proliferative response of HC-treated thymic cells is mainly due to the enhanced production of IL-2 and its action to increase cell growth, and there are two pathways by which the enhancement of IL-2 production by Con A and LPS or Bu-WSA can occur: an IL-1-dependent pathway, or an IL-1-independent one.     

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.     

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.     

Contrary Roles of IL-4 and IL-12 on IL-10 Production and Proliferation of Human Tumour Reactive T Cells

The cytokine profile of tumour reactive T cells is likely to play a central role in their function. However, little is known about how cytokine patterns of tumour reactive T cells can be regulated. Here, the authors investigated the influence of exogenous regulatory cytokines in addition to interleukin-2 (IL-2) on cytokine patterns and the proliferation of T cells recognizing an autologous sarcoma cell line. In this system, IL-4 and IL-12 showed the most polarizing influences on tumour reactive T cells. Exogenous IL-4 induced a predominant production of IL-4 while decreasing the interferon-γ (IFN-γ) and IL-10 production by tumour reactive T cells. It also stimulated the growth of tumour reactive CD4⁺ T cell clones. In contrast, IL-12 substantially increased the production of IL-10 and IFN-γ. This was accompanied by a growth inhibition of tumour reactive T cells. The growth of CD4⁺ tumour reactive T cells was also suppressed by exogenous IL-10. This study shows that cytokine patterns and proliferation tumour reactive T cells can be significantly influenced by exogenous cytokines and confirms the hypothesis of a negative feedback loop of IL-12 by the induction of IL-10 in the context of human tumour reactive T cells.     

Interleukin-4 and Interferon-Gamma Production by Leishmania Stimulated Peripheral Blood Mononuclear Cells from Nonexposed Individuals

Interferon-gamma (IFN-γ) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-γ was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated cells were pulsed with PMA and ionomycin before IL-4 release was measured. L. donovani and L. major antigens induced IL-4 production (105–1748pg/ml) in 13 and seven cultures, and IFN-γ production (1.7- > 66IU/ml) in 14 and 11 of 20 cultures, respectively. IL-4 production rose steeply after 6 days of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-γ production was abrogated by depletion of CD2⁺ or CD4⁺ but not CD8⁺ cells. CD2⁺ or CD4⁺ but not CD8⁺ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating Leishmania reactive CD4⁺ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN-γ and/or IL-4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN-γ and IL-4 is important for the clinical outcome. Our findings call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.     

Resistant mice lacking interleukin-12 become susceptible to Trypanosoma cruzi infection but fail to mount a T helper type 2 response

Interleukin-12 (IL-12) is essential to resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon-gamma (IFN-gamma) that activates macrophages to a parasiticidal effect. Investigation of mice deprived of IL-12 genes (IL-12 knockout mice) has confirmed the important role of IL-12 and IFN-gamma in controlling parasitism in T. cruzi infection. However, it has not yet been addressed whether a shift towards a T helper type 2 (Th2) pattern of cytokine response occurred in these mice that might have contributed to the aggravation of the infection caused by IL-12 deprivation. We examined the course of T. cruzi (Y strain) infection and the regulation of cytokine responses and nitric oxide production in C57BL/6 IL-12 p40-knockout mice. The mutant mice were extremely susceptible to the infection as evidenced by increased parasitaemia, tissue parasitism and mortality in comparison with the control C57BL/6 mouse strain (wild-type) that is resistant to T. cruzi. A severe depletion of parasite-antigen-specific IFN-gamma response, without an increase in IL-4 or IL-10 production, accompanied by reduced levels of nitric oxide production was observed in IL-12 knockout mice. We found no evidence of a shift towards a Th2-type cytokine response. In IL-12 knockout mice, the residual IFN-gamma production is down-regulated by IL-10 but not by IL-4 and nitric oxide production is stimulated by tumour necrosis factor-alpha. Parasite-specific immunoglobulin G1 antibody levels were similar in IL-12 knockout and wild-type mice, whereas IL-12 knockout mice had much higher levels of immunoglobulin G2b.     

Immunobiological Studies on Experimental Visceral Leishmaniasis. III. Cytokine-Mediated Regulation of Parasite Replication

The role of T-cell-derived cytokines in the regulation of Leishmania donovani replication was studied in a murine model. It was observed that in H-2^d mice at the early and later stages of the disease IFN-γ-secreting T cells predominate, whereas in between the above stages IL-4–secreting T cells predominate. Possibly, IL-4 abrogates the protective ability of IFN-γ and thereby exponential parasite growth is ensured at the active stage of the disease. By contrast, H-2^b mice were possibly incapable of inducing IL-4-secreting T cells and therefore parasite replication remains under control at any point post infection.     

犬蛔虫（Toxocaracanis）对感染鼠的细胞免疫和IL－1、IL－2免疫调节作用的影响

应用犬蛔虫（Ｔ．ｃａｒｎｉｓ）的感染性虫卵口饲感染Ｃ３Ｈ／ＨｅＮ鼠，体外培养后观察脾细胞的免疫学变化。感染第１－４周的脾细胞用ＣｏｎＡ刺激，Ｔ细胞的增殖反应，ＩＬ－２的诱生均明显地受到抑制，而且感染鼠脾细胞抑制了正常鼠脾细胞对ＣｏｎＡ的反应。感染鼠的脾细胞经ＳｅｐｈａｄｅｘＧ－１０过柱后，Ｔ细胞则不显示被抑制作用。表明影响Ｔ细胞被抑制作用的是感染鼠的巨噬细胞。相反，感染第１－４周的鼠所产生的ＩｇＧ和ＩｇＭ等抗体的Ｂ细胞活性增强；用ＬＰＳ刺激巨噬细胞诱生的白介素（ＩＬ）ＩＬ－１也增多。     

Suppression of Tumour-Specific Cytotoxic T-Cell Responses Against the Syngeneic BALB/c Plasmacytoma ADJ-PC-5 by Tumour-Induced CD8+ Regulatory T Cells Via IFN-γ

The mechanisms of tolerance induction by tumour cells during early stages of tumourigenesis were analysed in a murine model system using the highly immunogenic BALB/c plasmacytoma ADJ-PC-5. Early stages of tumourigenesis were simulated in syngeneic BALB/c mice by repeated intraperitoneal injections with subimmunogenic doses of X-irradiated ADJ-PC-5 tumour cells. This treatment causes a state of tumour-specific tolerance in a high percentage of mice, involving a population of CD8⁺ peritoneal T cells which are able to suppress a protective tumour-specific Tc response against this tumour. Using a primary mixed lymphocyte tumour cell culture (MLTC) as an in vitro system to study suppressive mechanisms of such regulatory T cells, the role of production or consumption of a number of cytokines was analysed. The data presented here demonstrate that inhibition of a protective Tc response against ADJ-PC-5 tumour cells is due to IFN-γ production by suppressive T cells from tolerized mice, but not to IL-2 consumption. In contrast to typical CD8⁺ Tc cells, ADJ-PC-5-specific CD8⁺ Tc cells do not produce IFN-γ and are furthermore suppressed by IFN-γ. Thus, tumour-induced suppressive T cells and tumour-specific Tc cells seem to represent functionally and phenotypically different subsets of CD8⁺ T cells, possibly pointing towards a differential activation of type-1 and type-2 CD8⁺ T cells depending on the dose of tumour cells.     

Immunosuppression during acute Trypanosoma cruzi infection: involvement of Ly6G (Gr1(+))CD11b(+ )immature myeloid suppressor cells

Trypanosoma cruzi infection is associated with a severe unresponsiveness of spleen cells (SC) to antigens and mitogens. A high production of NO by concanavalin A (Con A)-stimulated SC from infected but not from control mice was observed. Neutralization of endogenous IFN-gamma production or treatment with NO synthase (NOS) inhibitor, L-N-monomethyl-arginine, blocked Con A-induced NO production and greatly restored proliferation by SC from infected mice. This was confirmed by using IFN-gammaR(-/-) and inducible NOS (iNOS)(-/- )knockout mice, since unresponsiveness to mitogens of SC from those infected mice was much less pronounced than in control littermates. Interestingly, SC unresponsiveness was associated with a huge increase in CD11b(+) cells that express Ly-6G (Gr1)(+) and other immature myeloid markers These cells were absent in infected IFN-gammaR(-/-) spleens. Purified immature Gr1(+)CD11b(+) cells produced NO and expressed iNOS upon IFN-gamma treatment, and were able to inhibit T cell proliferation. In addition, depletion of myeloid CD11b(+ )cells abrogated NO production and restored mitogen-induced proliferation, but not IL-2 synthesis, in SC from infected mice. IL-2 production and CD25 cell surface expression by mitogen-activated T cells were greatly depressed in SC from IFN-gammaR(-/-) and iNOS(-/- )mice, confirming that Gr1(+)CD11b(+) cells were not involved in their down-regulation. In contrast, IL-5, tumor necrosis factor and IFN-gamma production, and CD69 expression by T cells were not depressed in infected SC. The results indicate the existence of an immunosuppressive mechanism during T. cruzi infection, mediated through IFN-gamma-dependent NO secretion by immature Ly-6G (Gr1)(+)CD11b(+ )myeloid cells.