Neospora caninum-infected cattle develop parasite-specific CD4+ cytotoxic T lymphocytes

Cattle infected with Neospora caninum readily experience transplacental parasite transmission, presumably after maternal parasitemia, leading to abortion or birth of congenitally infected calves. Cytotoxic T lymphocytes (CTL) are important mediators of protective immunity against Toxoplasma gondii, an intracellular apicomplexan protozoan closely related to N. caninum. In this study, N. caninum-specific CTL expanded from peripheral blood mononuclear cells of two major histocompatibility complex-mismatched, experimentally infected cattle were identified by using a (51)Cr release cytotoxicity assay. Enrichment and blocking of CD4(+)- and CD8(+)-T-lymphocyte effector subsets indicated that CD4(+) CTL killed N. caninum-infected, autologous target cells and that killing was mediated through a perforin/granzyme pathway. Detection and characterization of CTL responses to N. caninum in the natural, outbred, bovine host will facilitate identification of immunogens and design of immunization strategies to induce parasite-specific CTL against transplacental N. caninum transmission in cattle.     

Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferon-secreting T lymphocytes of infected holstein cattle

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-gamma) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-gamma secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-gamma enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-gamma ELISPOT and in vitro by measuring T-lymphocyte IFN-gamma production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-gamma-secreting T lymphocytes in cattle with varied MHC genotypes.     

Toll样配体(R-848)与IL-12对人NK细胞亚群IFN-γ产生的作用

目的:研究Toll样配体(R-848)与IL-12对人NK细胞IFN-γ产生的作用和细胞亚群分析。方法:分离人外周血PBMC和纯化的NK细胞,分别与R-848、IL-12或R-848和IL-12共同培养。利用ELISA法检测培养上清中IFN-γ的水平,再利用流式检测并分析产生IFN-γ的NK细胞亚群。结果:正常人PBMC分别与不同浓度的Toll样配体R-848、LPS、CpG培养后,均以剂量依赖的方式诱导IFN-γ的产生,但以R-848的效果最佳。细胞亚群分析的结果表明,R-848对CD4 T和CD8 T细胞IFN-γ的表达无明显作用,但显著地促进CD56 细胞表达IFN-γ。同样地,在IL-12刺激之下,CD56bright和CD56dimNK细胞表达IFN-γ。当R-848和IL-12与PBMC和纯化NK细胞孵育后,对CD56bright和CD56dimNK细胞IFN-γ的表达具有协同作用。结论:Toll样配体与NK细胞Toll样受体结合后,促进CD56brightNK细胞亚群IFN-γ的产生,而且Toll样配体与IL-12具有协同作用,提示Toll样受体与细胞因子在调控NK细胞的生物活性中发挥着十分重要的作用。     

Identification and characterization of Neospora caninum cyclophilin that elicits gamma interferon production

Gamma interferon (IFN-gamma) response is essential to the development of a host protective immunity in response to infections by intracellular parasites. Neosporosis, an infection caused by the intracellular protozoan parasite Neospora caninum, is fatal when there is a complete lack of IFN-gamma in the infected host. However, the mechanism by which IFN-gamma is elicited by the invading parasite is unclear. This study has identified a microbial protein in the N. caninum tachyzoite N. caninum cyclophilin (NcCyP) as a major component of the parasite responsible for the induction of IFN-gamma production by bovine peripheral blood mononuclear cells (PBMC) and antigen-specific CD4(+) T cells. NcCyP has high sequence homology (86%) with Toxoplasma gondii 18-kDa CyP with a calculated molecular mass of 19.4 kDa. NcCyP is a secretory protein with a predicted signal peptide of 17 amino acids. Abundant NcCyP was detected in whole-cell N. caninum tachyzoite lysate antigen (NcAg) and N. caninum tachyzoite culture supernatant. In N. caninum tachyzoite culture supernatant, three NcCyP bands of 19, 22, and 24 kDa were identified. NcAg stimulated high levels of IFN-gamma production by PBMC and CD4(+) T cells. The IFN-gamma-inducing effect of NcAg was blocked by cyclosporine, a specific ligand for CyP, in a dose-dependent manner. Furthermore, cyclosporine abolished IFN-gamma production by PBMC from na?ve cows as well as PBMC and CD4(+) T cells from infected/immunized cows. These results indicate that the N. caninum tachyzoite naturally produces a potent IFN-gamma-inducing protein, NcCyP, which may be important for parasite survival as well as host protection.     

Inefficient protection of human TAP-deficient fibroblasts from autologous NK cell-mediated lysis by cytokines inducing HLA class I expression

We studied HLA class I expression and susceptibility to lysis of activated autologous NK cells in normal and TAP-deficient fibroblasts. These cells were cultured in the presence or absence of cytokines known to increase the surface expression of HLA class I molecules. All the cytokines tested (IFN-alpha, IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha) increased the expression of HLA class I molecules on fibroblasts after 48-h culture, but on TAP-deficient cells this expression remained very low as compared to that of normal cells. In the presence of IFN-alpha, IFN-gamma or IFN-gamma + TNF-alpha, normal target cells became resistant to lysis by autologous NK cells, whereas this effect was much less pronounced in the case of TAP-deficient fibroblasts. Addition of an anti-HLA class I mAb to fibroblasts treated with cytokines increased lysis of normal but not of TAP-deficient cells. These results suggest that activated TAP-deficient NK cells are strongly cytotoxic to normal autologous cells and that these cells cannot be efficiently protected by cytokines inducing HLA class I expression. Thus, in human TAP deficiency, activated NK cells may contribute to the progressive lung degradation which characterizes the clinical course of these patients.     

The differential protein expression profiles and immunogenicity of tachyzoites and bradyzoites of in vitro cultured <Emphasis Type="Italic">Neospora caninum</Emphasis>

We report a study on the variations in the protein expression profiles of tachyzoites and bradyzoites of Neospora caninum. The in vitro stage conversion of N. caninum-infected Vero cells was induced by continuous treatment of infected cultures with 70 muM sodium nitroprusside (SNP) for up to 9 days. The stage conversion indicated by the expression of the bradyzoite-specific antigen BAG1 was analyzed by immunofluoresence assay. Morphological changes between tachyzoites and bradyzoites and localization of nuclei were demonstrated by transmission electron microscopy. Notably, we showed the differential protein expression profiles of tachyzoites and bradyzoites of N. caninum upon treatment with SNP. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated different protein patterns between tachyzoites and bradyzoites. Furthermore, Western blotting using rabbit polyclonal antibodies directed against tachyzoites revealed several reactive bands, one of which represented a tachyzoite-specific antigen of approximately 40 kDa remarkably expressed in the tachyzoite stage, but was absent from bradyzoites. Moreover, rabbit polyclonal serum raised against bradyzoites recognized a significant increased expression of an antigen with a MW of approximately 25 kDa in bradyzoites by Western blotting, suggesting that this protein is specifically expressed at the bradyzoite stage. Taken together, our data showed that differential protein expression profiling is a useful tool for discriminating between the two stages during tachyzoite-bradyzoite interconversion in N. caninum infections.     

Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon.

Gamma interferon (IFN-gamma) is produced in response to circulating lipopolysaccharide (LPS) and contributes to the lethality of endotoxic shock. To address the cellular source of IFN-gamma production in vivo, T cells and B cells were magnetically purified from C57BL/6 mouse spleens 5 h following endotoxin injection. IFN-gamma RNA was abundant in splenic CD4+ and CD8+ T cells and in a T- and B-cell-depleted population of splenocytes containing 34% NK1.1+ natural killer (NK) cells. Because interleukin 12 (IL-12) is a known inducer of IFN-gamma synthesis by cultured T cells and NK cells, we examined whether IL-12 might be involved in IFN-gamma release during endotoxemia. mRNA encoding the p40 subunit of IL-12 increased markedly in the spleens of C57BL/6 mice at 2 h after LPS injection, whereas p35 IL-12 mRNA was constitutively expressed at all times. Bioactive IL-12 (p70 heterodimer) was detected in mouse serum at 2 to 4 h after LPS injection. Similar results were obtained using a p40 subunit-specific enzyme-linked immunosorbent assay. Endotoxin-insensitive C3H/HeJ mice generated threefold less IL-12 p70 and IFN-gamma at these times than endotoxin-sensitive C3H/HeOuJ mice. Pretreatment of mice with polyclonal anti-mouse IL-12 antibody reduced IFN-gamma levels present at 6 h post-LPS nearly sixfold in three separate experiments. These studies support a role for IL-12 as a proximal stimulator of IFN-gamma release during endotoxemia.     

Human NK1 and NK2 subsets determined by purification of IFN-gamma-secreting and IFN-gamma-nonsecreting NK cells

The in vivo existence of human NK cell subsets similar to Th1 and Th2 cells was demonstrated in freshly isolated IFN-gamma-secreting and IFN-gamma-nonsecreting NK cells. The IFN-gamma-secreting NK subset showed a typical cytokine pattern with predominant expression of IFN-gamma, but almost no IL-4, IL-5 and IL-13. In contrast, the IFN-gamma-nonsecreting NK subset was composed of IL-4, IL-5 and IL-13-producing NK cells. Short-time stimulation or 2 weeks of in vitro differentiation of NK cells led to distinct patterns of cytokine production similar to freshly-purified IFN-gamma (+) or IFN-gamma (-) NK cell subsets. NK cells stimulated with IL-12 produced increased levels of IFN-gamma and decreased levels of IL-4. In contrast, stimulation of NK cells with IL-4 inhibited IFN-gamma, but increased IL-13 production. Freshly-purified IFN-gamma (+) and IFN-gamma (-) or in vitro differentiated NK1 and NK2 subsets showed similar cytotoxicity to K562 cells. These results demonstrate that circulating NK cells retain effector subsets in humans with distinct cytokine profiles and may display different inflammatory properties.     

Distinct gut-derived lactic acid bacteria elicit divergent dendritic cell-mediated NK cell responses

Lactic acid bacteria (LAB) are abundant in the gastrointestinal tract where they continuously regulate the immune system. NK cells are potently activated by dendritic cells (DCs) matured by inflammatory stimuli, and NK cells are present in the gut epithelium and in mesenteric lymph nodes, but it is not known how NK-DC interactions are affected by the predominantly non-pathogenic LAB. We demonstrate that human DCs exposed to different strains of gut-derived LAB consistently induce proliferation, cytotoxicity and activation markers in autologous NK cells. On the contrary, strains of LAB differ greatly in their ability to induce DC-dependent IFN-gamma production by NK cells. This suggests that DCs stimulated by gut LAB may expand the pool of NK cells and increase their cytotoxic potential. Specific LAB, inducing high levels of IL-12 in DCs, may promote amplification of a type-1 response via potent stimulation of IFN-gamma production in NK cells. Combining IFN-gamma-inducing and non-inducing LAB completely abrogates DC-mediated IFN-gamma production by NK cells, and therefore LAB modulating IFN-gamma production in NK cells may be important regulators of the immune response.     

Trogocytosis and killing of IL-4-polarized monocytes by autologous NK cells

Cross-regulations between innate immune cells have been given more and more emphasis. Here, we address the question of bidirectional interactions between activated monocytes and autologous NK cells. Classically activated monocytes (class-monocytes), obtained by priming with IFN-gamma, drive an inflammatory immune response. On the contrary, alternatively activated monocytes (alt-monocytes), obtained by stimulation with IL-4 or IL-13, engage an anti-inflammatory immune response. We show that alt-monocytes inhibit proliferation and production of IFN-gamma by autologous, IL-2-activated NK cells, whereas class-monocytes do not inhibit these NK cell functions. Reciprocally, IL-2-activated NK cells interact and undertake intensive synaptic transfer with alt-monocytes, whereas interactions with class-monocytes are weaker. This strong trogocytosis correlates with an efficient killing of alt-monocytes, mediated by natural cytotoxicity receptors and a lowered killing of class-monocytes. These results suggest that interactions between NK cells and autologous-activated monocytes modulate inflammatory responses. This might be extended further in the elimination of tumor-associated macrophages, which actively promote solid tumor progression and metastasis.     

IL-17 enhances the susceptibility of U-2 OS osteosarcoma cells to NK cell lysis

We investigated the effect of the proinflammatory cytokine interleukin 17 (IL-17) on the lysis of osteosarcoma cells by human NK cells. NK cells and U-2 OS, MG-63, HOS osteosarcoma cell lines express the IL-17 receptor, the highest amount being found on U-2 OS. Pre-incubation of NK cells with IL-17 did not affect the cytotoxicity against osteosarcomas, that was increased when U-2 OS were pre-incubated with IL-17. In IL-17 treated U-2 OS osteosarcoma cells FACS analysis demonstrated an increased expression of fibronectin among the panel of adhesion molecules assayed, and the treatment with anti-fibronectin antibodies decreased the NK cytotoxicity. The comparison between interferon gamma (IFN-gamma) treated and IFN-gamma/IL-17-treated U-2 OS showed a decreased susceptibility to NK lysis associated with a reduced expression of CD49f on U-2 OS treated with IFN-gamma/IL-17. IL-17 appears to be a modulator of NK adhesion molecules on U-2 OS cells but antagonizes with IFN-gamma on NK lysis.     

Interferon-gamma protects human thyroid epithelial cells against cell-mediated cytotoxicity

Interferons have been shown to have antagonistic effects on cell-mediated cytotoxicity. In the present study, we investigated various types of immunologically mediated cytotoxic reactions with a 51chromium release assay, using human thyroid cells as targets, which were cultured in the presence and absence of interferon-gamma (IFN-gamma). Antibody-dependent cell-mediated cytotoxicity (ADCC), natural killer (NK) cell-mediated cytotoxicity, and autologous lymphocyte-mediated cytotoxicity (ALC) were measured utilizing thyroid tissue, sera and lymphocytes from patients with autoimmune thyroid disease as well as from normal subjects. ADCC, determined with sera from patients with Hashimoto's thyroiditis, was reduced by 70% when the thyroid target cells were cultured in the presence of 1000 U human IFN-gamma/ml culture medium. Similarly, natural killer cell-mediated cytotoxicity, as well as autologous lymphocyte-mediated cytotoxicity, was significantly reduced after pretreatment of thyroid target cells with 500-1000 U IFN-gamma/ml culture medium. Although it is known that IFN-gamma renders target cells resistant to NK cell-mediated lysis, this is the first report on this effect using human epithelial cells, which may have major implications for the suggested role of IFN-gamma in the induction and perpetuation of autoimmune thyroid disease.     

Identification and characterization of Neospora caninum tachyzoite antigens useful for diagnosis of neosporosis.

The purpose of the present study was to identify antigens of the protozoan Neospora caninum that could be useful for the diagnosis of neosporosis in domestic animals. As revealed by immunoblotting, immune sera from a wide range of animal species exhibited a similar recognition pattern of four major and several minor N. caninum antigens. In contrast to preinoculation sera, all tested immune sera recognized nonreduced immunodominant 17-, 29-, 30-, and 27-kDa antigens. A 46-kDa protein which showed faint recognition by preimmune sera also exhibited a strong response by immune sera. Immunolocalization of the four immunodominant N. caninum antigens was investigated by immunogold electron microscopy using monospecific polyclonal antisera. The 17-kDa antigen appears to be associated with the body part of the rhoptries, while the 29- and 30-kDa antigens were associated with the dense granules, network, and limiting membrane of the parasitophorous vacuole. Studies were also conducted to compare antibody responses to N. caninum and the related protozoan Toxoplasma gondii. Although N. caninum and T. gondii (RH strain) tachyzoites shared a few cross-reacting antigens, the immunodominant antigens of both parasites were not recognized by heterologous sera. Also, immunogold staining with rabbit anti-Neospora hyperimmune serum exhibited almost no labeling of external membranes of Neospora tachyzoites compared with the very marked labeling seen when Toxoplasma tachyzoites (RH strain) were incubated with rabbit anti-Toxoplasma hyperimmune serum. These unique antigenic differences should be useful in developing a diagnostic assay for N. caninum.     

Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells.

We examined the ability of interleukin-12 (IL-12) and IL-18 to induce the production of gamma interferon (IFN-gamma) and nitric oxide (NO) by murine peritoneal exudate cells (PEC) and to stimulate the growth-inhibitory activity of these cells against Cryptococcus neoformans. PEC produced IFN-gamma and NO when stimulated with a combination of IL-12 and IL-18 but little or no IFN-gamma or NO when either cytokine was used alone. PEC anticryptococcal activity was mediated by IFN-gamma and NO production, since it was completely inhibited by a neutralizing anti-IFN-gamma monoclonal antibody (MAb) and N(G)-monomethyl-L-arginine, a competitive inhibitor of NO synthesis, respectively. To identify the IFN-gamma-producing cells among PEC stimulated with IL-12 and IL-18, we depleted NK cells, gammadelta T cells, or CD4+ T cells by treating PEC with specific Abs and complement. NK cell depletion strongly suppressed IFN-gamma production and almost completely inhibited NO production and anticryptococcal activity, while depletion of other cells had no such influence. Alternatively, purified NK cells by two cycles of glass adherence and magnetic separation with anti-CD3, -CD4, -CD8, and -B220 MAbs produced a greater amount of IFN-gamma by stimulation with IL-12 and IL-18 than unseparated non-glass-adherent PEC. Our results demonstrated that IL-12 and IL-18 synergistically induced NO-dependent anticryptococcal activity of PEC by stimulating NK cells to produce IFN-gamma.     

IL-12 and IFN-gamma production, and NK cell activity, in acute and chronic experimental Trypanosoma cruzi infections

Resistance to acute Trypanosoma cruzi infection is mainly associated with a Th1 immune response, characterized by gamma-interferon (IFN-gamma) production and activation of macrophages. The outcome of the Th1 response in the spleen and serum of BALB/c and C3H mice infected with T. cruzi, Tulahuén strain was studied. The levels of interleukin-12 p40 (IL-12 p40) and IFN-gamma, as well as natural killer (NK) cell cytotoxicity were determined at different time-points during the acute phase, and the production of cytokines was also studied in the chronic infection. At 2 days post-infection (pi), spleen cells from C3H mice increased their NK cell activity and the ex vivo spontaneous release of both IL-12 p40 and IFN-gamma. On the other hand, BALB/c mice reached low levels of NK cell cytotoxicity and no IFN-gamma production was detected at this time pi, but the cytokine was released at high amounts in the second week of the infection. Seric IL-12 p40 concentrations showed a 3-fold increase in both mouse strains on the second day pi and remained high throughout the acute phase. However, seric IFN-gamma levels increased during the late acute infection and were higher in BALB/c than in C3H mice. In chronically infected mice IL-12 p40 was as high as in the acute phase in the serum of both strains, but only BALB/c mice still produced IFN-gamma. To the authors' knowledge this is the first report showing the protein levels of IL-12 p40 determined in vivo in acute and chronic T. cruzi infections. The results reveal differences between both mouse strains in the mechanisms controlling the onset and fate of the Th1 response triggered by the parasite and a long lasting pro-inflammatory stimuli.     

Induction and regulation of IL-12-dependent host resistance to Toxoplasma gondii

Resistance to the intracellular protozoan parasite Toxoplasma gondii is initiated by the induction of interleukin-12 (IL-12), which stimulates interferon (IFN)-gamma synthesis by natural killer (NK) cells and T lymphocytes. This review summarizes the work of our laboratory on the mechanisms by which the parasite triggers IL-12 synthesis, and how this response is regulated to avoid the lethal effects of excessive tissue inflammation. In addition, we present an overview of our studies investigating the mechanisms by which the IFN-gamma produced as a consequence of IL-12 stimulation controls intracellular replication of the parasite in host cells.     

Interleukin-12 induces cytotoxic NK1+ alpha beta T cells in the lungs of euthymic and athymic mice.

We recently reported that interleukin-12 (IL-12) stimulated hepatic NK1.1 Ag+ alpha beta T cells with intermediate T-cell receptor (TCR; NK1+ TCRint cells) and enhanced their NK1 expression (NK1high TCRint), and that these cells acquire strong major histocompatibility complex (MHC) unrestricted cytotoxicity in C57BL/6 mice, both +/+ and nu/nu. In the present study, we find that although murine lung normally has few NK1+ TCRint cells, NK1high TCRint cells are induced in+/+ and nu/nu mice after systemic administration of IL-12; these cells exhibit strong MHC unrestricted cytotoxicity against NK-sensitive and -resistant targets. A small number of NK1high TCRint cells was also found in peripheral blood after increased amounts of IL-12 were administered. Cytotoxicity tests in vitro revealed that the cytotoxic activity of the lung mononuclear cells (MNC) of C57BL/6 mice induced by IL-12 was abrogated by the depletion of either NK1+ or CD3+ cells, but not of CD8+ cells, as reportedly was the case of hepatic MNC, suggesting that NK1high TCRint cells are an antimetastatic population not only in the liver but also in the lung of mice. IL-12 injection into mice markedly elevates serum interferon-gamma (IFN-gamma) levels. However, although IL-12-induced cytotoxicity of NK1high TCRint cells was significantly reduced by anti-IFN-gamma antibody injection (which decreased serum IFN-gamma to an undetectable level), the appearance of NK1high TCRint cells in the lung and liver was not so affected. These results suggest that IFN-gamma is an important mediator of the cytotoxicity of NK1high TCRint cells but is not an essential factor for induction of these cells. We also added data showing that IL-12 has a broad antimetastatic effect against various liver and lung metastatic tumours intravenously injected into several strains of mice, including NK-deficient bg/bg mice. It can be considered that, in addition to NK cells, CD8+ cytotoxic T cells and gamma delta T cells, NK1+ TCRint cells can be categorized as one of the cytotoxic effector populations. These novel type cells distinct from regular T cells may play an important role in monitoring intra- and perivascular areas.