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1.
J Immunol ; 179(6): 3472-9, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17804388

ABSTRACT

NK cells express different TLRs, such as TLR3, TLR7, and TLR9, but little is known about their role in NK cell stimulation. In this study, we used specific agonists (poly(I:C), loxoribine, and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12, IL-15, or IFN-alpha, and investigated the secretion of IFN-gamma, cytotoxicity, and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine, in conjunction with IL-12, but not IL-15, triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN-gamma was dependent on MEK1/ERK, p38 MAPK, p70(S6) kinase, and NF-kappaB, but not on calcineurin. IFN-alpha induced a similar effect, but promoted lesser IFN-gamma secretion. However, cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged, as well as the expression of the NKG2D receptor. Excitingly, IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12, and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12, and this effect can be further enhanced by costimulation through NKG2D. Hence, integration of the signaling cascades that involve TLR3, TLR7, IL-12, and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.


Subject(s)
Cytotoxicity, Immunologic , Interferon-gamma/metabolism , Interleukin-12/physiology , Killer Cells, Natural/immunology , Lymphocyte Activation/immunology , Receptors, Immunologic/metabolism , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 7/metabolism , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Cells, Cultured , Clone Cells , Cytotoxicity, Immunologic/drug effects , Cytotoxicity, Immunologic/immunology , Dose-Response Relationship, Immunologic , Gene Expression Regulation, Neoplastic/immunology , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/genetics , Humans , Interferon-alpha/physiology , Interleukin-12/pharmacology , Killer Cells, Natural/metabolism , Melanoma/immunology , Melanoma/pathology , Melanoma/therapy , NK Cell Lectin-Like Receptor Subfamily K , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/physiology , Receptors, Natural Killer Cell , Toll-Like Receptor 3/agonists , Toll-Like Receptor 3/immunology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/immunology , Toll-Like Receptor 9/agonists
2.
J Immunol ; 179(3): 1950-9, 2007 Aug 01.
Article in English | MEDLINE | ID: mdl-17641062

ABSTRACT

Although the development of an acidic tissue environment or acidosis is a hallmark of inflammatory processes, few studies analyze the effect of extracellular pH on immune cells. We have previously shown that exposure of murine dendritic cells (DCs) to pH 6.5 stimulates macropinocytosis and cross-presentation of extracellular Ags by MHC class I molecules. We report that the transient exposure of human DCs to pH 6.5 markedly increases the expression of HLA-DR, CD40, CD80, CD86, CD83, and CCR7 and improves the T cell priming ability of DCs. Incubation of DCs at pH 6.5 results in the activation of the PI3K/Akt and the MAPK pathways. Using specific inhibitors, we show that the maturation of DCs induced by acidosis was strictly dependent on the activation of p38 MAPK. DC exposure to pH 6.5 also induces a dramatic increase in their production of IL-12, stimulating the synthesis of IFN-gamma, but not IL-4, by Ag-specific CD4(+) T cells. Interestingly, we find that suboptimal doses of LPS abrogated the ability of pH 6.5 to induce DC maturation, suggesting a cross-talk between the activation pathways triggered by LPS and extracellular protons in DCs. We conclude that extracellular acidosis in peripheral tissues may contribute to the initiation of adaptive immune responses by DCs, favoring the development of Th1 immunity.


Subject(s)
Acidosis/metabolism , Cell Differentiation/immunology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Extracellular Fluid/metabolism , Interleukin-12/biosynthesis , Acidosis/immunology , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/enzymology , Extracellular Fluid/immunology , Humans , Hydrogen-Ion Concentration , Immunophenotyping , Interferon-gamma/biosynthesis , Interleukin-12/physiology , MAP Kinase Signaling System/immunology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Phosphatidylinositol 3-Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/physiology
3.
Parasite Immunol ; 29(5): 251-8, 2007 May.
Article in English | MEDLINE | ID: mdl-17430548

ABSTRACT

Cutaneous (CL) and mucosal leishmaniasis (ML) are characterized by a predominant type 1 immune response (IFN-gamma and TNF-alpha production) and strong inflammatory response in the lesions with few parasites. This exacerbated type 1 response is more evident in ML as compared to CL. Our main hypothesis is that a differential immune regulation of T cell activation leads to over reactive T cells in ML. In the present study, we investigated immunological factors that could explain the mechanisms behind it by comparing some immune regulatory mechanisms between ML and CL patients: frequency of cells expressing co-stimulatory molecules, apoptotic markers, T cell activation markers; and ability of neutralizing antibodies to IL-2, IL-12 and IL-15 do down-regulate IFN-gamma production in leishmania antigen-stimulated peripheral blood mononuclear cells (PBMC). Interestingly, in CL anti-IL-2 and anti-IL-15 significantly suppressed antigen-specific IFN-gamma production, while in ML only anti-IL-2 suppressed IFN-gamma production. Finally, higher frequency of CD4+ T cells expressing CD28-, CD69+ and CD62L(low) were observed in ML as compared to CL. These data indicate that an exacerbated type 1 response in ML is differentially regulated and not appropriately down modulated, with increased frequencies of activated effectors T cells, maintaining the persistent inflammatory response and tissue damage observed in ML.


Subject(s)
Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Mucocutaneous/immunology , Lymphocyte Activation , T-Lymphocytes/immunology , Adolescent , Adult , Antigens, CD/pharmacology , Antigens, Differentiation/pharmacology , CTLA-4 Antigen , Child , Female , Humans , Interferon-gamma/biosynthesis , Interleukin-12/physiology , Interleukin-15/physiology , Interleukin-2/physiology , Leishmaniasis, Cutaneous/etiology , Leishmaniasis, Mucocutaneous/etiology , Male , Middle Aged
4.
Infect Immun ; 73(4): 2101-8, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15784551

ABSTRACT

Infection of C57BL/6 (B6) mice with Leishmania mexicana is associated with a minimal immune response and chronic disease. Here we show that B6 interleukin 10-/- (IL-10-/-) mice resolve their lesions and exhibit increased gamma interferon (IFN-gamma), nitric oxide production, and delayed-type hypersensitivity. This enhanced resistance was dependent upon IL-12p40, since treatment of L. mexicana-infected IL-10-/- mice with anti-IL-12p40 monoclonal antibody abrogated healing. Antibody-opsonized L. mexicana induced IL-10 production by B6 macrophages in vitro, implicating antibody binding to Fc receptors as a mechanism involved in IL-10 production in this infection. Furthermore, B6 FcRgamma-/- mice resolve L. mexicana lesions, and lymph node cells from these mice produced less IL-10 and more IFN-gamma than cells from infected wild-type mice. These data demonstrate that removal of IL-10 or FcgammaR leads to resolution of L. mexicana disease and support a model in which ligation of FcgammaR by L. mexicana-bound immunoglobulin G promotes IL-10 production, leading to chronic disease.


Subject(s)
Interleukin-10/physiology , Leishmania mexicana , Leishmaniasis, Cutaneous/immunology , Receptors, IgG/physiology , Animals , Chronic Disease , Immunoglobulin G/physiology , Interleukin-12/physiology , Interleukin-12 Subunit p40 , Mice , Nitric Oxide/biosynthesis , Protein Subunits/physiology
5.
J Immunol ; 173(1): 463-70, 2004 Jul 01.
Article in English | MEDLINE | ID: mdl-15210806

ABSTRACT

In this study, we investigated the involvement of Th1 cytokines in the expression of cell adhesion molecules (CAM) and recruitment of inflammatory cells to the heart of mice infected with Trypanosoma cruzi. Our results show that endogenously produced IFN-gamma is essential to induce optimal expression of VCAM-1 and ICAM-1 on the cardiac vascular endothelium of infected mice. Furthermore, the influx of inflammatory cells into the cardiac tissue was impaired in Th1 cytokine-deficient infected mice, paralleling the intensity of VCAM-1 and ICAM-1 expression on the vascular endothelium. Consistent with the importance of ICAM-1 in host resistance, ICAM-1 knockout (KO) mice were highly susceptible to T. cruzi infection, as assessed by mortality rate, parasitemia, and heart tissue parasitism. The enhanced parasitism was associated with a decrease in the numbers of CD4(+) and CD8(+) T lymphocytes in the heart tissue of ICAM-1 KO mice. Additionally, ICAM-1 KO mice mounted an unimpaired IFN-gamma response and IFN-gamma-dependent production of reactive nitrogen intermediates and parasite- specific IgG2a. Supporting the participation of ICAM-1 in cell migration during T. cruzi infection, the entrance of adoptively transferred PBL from T. cruzi-infected wild-type C57BL/6 mice into the cardiac tissue of ICAM-1 KO mice was significantly abrogated. Therefore, we favor the hypothesis that ICAM-1 plays a crucial role in T lymphocyte recruitment to the cardiac tissue and host susceptibility during T. cruzi infection.


Subject(s)
Chagas Disease/immunology , Intercellular Adhesion Molecule-1/physiology , T-Lymphocytes/physiology , Animals , Cell Movement , Disease Susceptibility , Female , Intercellular Adhesion Molecule-1/analysis , Interferon-gamma/physiology , Interleukin-12/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/immunology , Vascular Cell Adhesion Molecule-1/analysis
6.
Microbes Infect ; 6(6): 630-6, 2004 May.
Article in English | MEDLINE | ID: mdl-15158199

ABSTRACT

The present review illustrates the current knowledge on the autocrine effect of IL-12, and the putative contribution of IL-23, on macrophages and dendritic cells, focusing on cell activation and microbicidal activity. Here, we present convincing evidence that IL-12 is not only a connective element between accessory cells and lymphocytes, but it is also a key molecule for programming the macrophage and dendritic cell functions.


Subject(s)
Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Dendritic Cells/immunology , Interleukin-12/physiology , Interleukins/physiology , Macrophages/immunology , Animals , Humans , Interleukin-12/immunology , Interleukin-23 , Interleukin-23 Subunit p19 , Interleukins/immunology , Macrophage Activation , Signal Transduction
7.
Infect Immun ; 71(11): 6392-401, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14573660

ABSTRACT

Herein we characterized various genetic markers and the biological behavior of a natural recombinant strain of Toxoplasma gondii (P-Br). From nine genetic markers analyzed, three (B1, ROP1, and SAG1) and three (cS10-A6, GRA6, and SAG3) markers belong to parasites from the type I and type III lineages, respectively. The SAG2 and L363 loci were shown to be type I-III chimera alleles. The cB2l-4 microsatellite marker showed a unique haplotype. The P-Br strain presented low virulence in the acute phase of infection and was cystogenic during the chronic infection. The interleukin 12/gamma interferon axis and inducible nitric oxide synthase were main determinants of resistance during the acute infection with the P-Br strain. As opposed to infection with the type II strain of T. gondii (ME-49), peroral infection with the P-Br strain led only to a light inflammatory infiltrate and no major lesions in the intestine of the C57BL/6 mice. In addition, the BALB/c (resistant to ME-49) and C57BL/6 (susceptible to ME-49) mice were shown, respectively, to be more susceptible and more resistant to cyst formation and toxoplasmic encephalitis when infected with the P-Br strain. Further, the C57BL/KsJ and DBA2/J congenic strains containing major histocompatibility complex (MHC) haplotype "d" were more resistant than the parental strains (C57BL/6 and DBA1/J), when infected with the ME-49 but not with the P-Br strain. Together, our results indicate that resistance to cyst formation and toxoplasmic encephalitis induced during infection with P-Br is not primarily controlled by the MHC haplotype d, as previously reported for type II strains of T. gondii.


Subject(s)
Cytokines/physiology , Major Histocompatibility Complex/physiology , Toxoplasmosis, Animal/immunology , Animals , Haplotypes , Interferon-gamma/physiology , Interleukin-12/physiology , Mice , Mice, Inbred Strains , Nitric Oxide Synthase/physiology , Nitric Oxide Synthase Type II , Recombination, Genetic , Toxoplasma/genetics
8.
J Immunol ; 171(7): 3711-7, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-14500670

ABSTRACT

C3H mice infected with Leishmania mexicana fail to develop a protective Th1 response, and are unable to cure. In this study, we show that L. mexicana cysteine proteases suppress the antileishmanial immune response. Previous studies demonstrated that deletion of the entire multicopy cysteine protease B (CPB) gene array in L. mexicana is associated with decreased parasite virulence, potentially attributable to factors related to parasite fitness rather than to direct effects on the host immune response. We now show that C3H mice infected with the L. mexicana deletion mutant (Deltacpb) initially develop lesions that grow at rates comparable to those of wild-type L. mexicana-infected mice. However, in contrast to controls, Deltacpb-induced lesions heal with an accompanying Th1 immune response. Lesion resolution was Th1 dependent, as Deltacpb-infected IL-12p40(-/-) and STAT4(-/-) mice developed high parasite burdens and progressive disease. Moreover, when L. major was transfected with a cosmid expressing multiple L. mexicana CPB genes, this parasite induced a significantly lower IFN-gamma response compared with wild-type L. major. These data indicate that cysteine proteases of L. mexicana are critical in suppressing protective immune responses and that inhibition of CPB may prove to be a valuable immunomodulatory strategy for chronic forms of leishmaniasis.


Subject(s)
Cathepsin B/physiology , Down-Regulation/immunology , Leishmania mexicana/enzymology , Leishmania mexicana/immunology , Leishmaniasis, Cutaneous/immunology , Th1 Cells/immunology , Th1 Cells/parasitology , Animals , Cathepsin B/deficiency , Cathepsin B/genetics , Cathepsin B/immunology , Cells, Cultured , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Down-Regulation/genetics , Gene Expression Regulation, Enzymologic/immunology , Immunity, Innate , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/biosynthesis , Interleukin-12/deficiency , Interleukin-12/genetics , Interleukin-12/physiology , Interleukin-12 Subunit p40 , Leishmania mexicana/genetics , Leishmaniasis, Cutaneous/genetics , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/prevention & control , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Protein Subunits/deficiency , Protein Subunits/genetics , Protein Subunits/physiology , Protozoan Vaccines/administration & dosage , Protozoan Vaccines/genetics , Protozoan Vaccines/immunology , STAT4 Transcription Factor , Signal Transduction/genetics , Signal Transduction/immunology , Th1 Cells/metabolism , Trans-Activators/deficiency , Trans-Activators/genetics , Trans-Activators/physiology
9.
J Immunol ; 171(2): 1001-8, 2003 Jul 15.
Article in English | MEDLINE | ID: mdl-12847273

ABSTRACT

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-gamma. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12(-/-) or IFN-gamma(-/-) mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.


Subject(s)
Anti-Allergic Agents/administration & dosage , Asthma/immunology , Asthma/prevention & control , Lipopolysaccharides/administration & dosage , Membrane Glycoproteins/physiology , Nitric Oxide Synthase/metabolism , Receptors, Cell Surface/physiology , Signal Transduction/immunology , Administration, Inhalation , Allergens/administration & dosage , Allergens/immunology , Animals , Asthma/enzymology , Asthma/genetics , Bronchi/metabolism , Bronchial Hyperreactivity/genetics , Bronchial Hyperreactivity/immunology , Bronchial Hyperreactivity/prevention & control , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Enzyme Activation/immunology , Immunity, Innate/genetics , Inflammation/embryology , Inflammation/genetics , Inflammation/immunology , Inflammation/prevention & control , Injections, Intravenous , Interferon-gamma/physiology , Interleukin-12/physiology , Lung/enzymology , Lung/immunology , Lung/pathology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mucus/metabolism , Nitric Oxide Synthase/deficiency , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/physiology , Nitric Oxide Synthase Type II , Ovalbumin/administration & dosage , Ovalbumin/immunology , Passive Cutaneous Anaphylaxis/genetics , Passive Cutaneous Anaphylaxis/immunology , Receptors, Cell Surface/deficiency , Receptors, Cell Surface/genetics , Salmonella enterica/immunology , Signal Transduction/genetics , Th2 Cells/immunology , Th2 Cells/metabolism , Toll-Like Receptor 4 , Toll-Like Receptors
10.
Infect Immun ; 70(9): 5075-80, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12183555

ABSTRACT

The role of interleukin-12 (IL-12) has been clearly established in the resistance of C57BL/6 (B6) mice to Leishmania major infection, but its involvement in the control of Leishmania mexicana infection remains to be determined. Here, we show the following. (i) L. mexicana, in contrast to L. major, induces the development of nonhealing lesions in B6 mice. (ii) Cells expressing IL-12p40, gamma interferon (IFN-gamma), NOS2, and CD40L are numerous in the footpad lesion and/or the draining popliteal lymph node of animals infected for up to 14 weeks with L. mexicana. (iii) B6 mice, either IL-12p40 deficient or treated with IL-12p40-neutralizing antibodies, display a dramatic enhancement of primary and secondary lesions leading to death 10 weeks after inoculation with L. mexicana. (iv) Splenocytes harvested 4 and 8 weeks after infection of IL-12p40(-/-) B6 mice with L. mexicana are unable to produce IFN-gamma, but secrete IL-4, IL-10, and IL-18. Thus, the early control of L. mexicana infection by B6 mice is independent of IL-12, whereas IL-12 and Th1 responses play a key role in controlling the late stages of L. mexicana infection. However, they fail to resolve lesions, in contrast to L. major infection, emphasizing the different outcomes induced by these two Leishmania species in B6 mice.


Subject(s)
Interleukin-12/physiology , Leishmania mexicana , Leishmaniasis, Cutaneous/immunology , Animals , CD40 Ligand/metabolism , Female , Interferon-gamma/metabolism , Interleukin-12/antagonists & inhibitors , Interleukin-12/genetics , Interleukin-12 Subunit p40 , Interleukin-18/metabolism , Leishmania major/pathogenicity , Leishmania mexicana/pathogenicity , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutralization Tests , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Protein Subunits , Species Specificity , Th2 Cells/immunology , Time Factors
11.
Am J Pathol ; 159(5): 1723-33, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11696433

ABSTRACT

The role of cytokines in the control of tissue parasitism and pathogenesis of experimental Chagas' disease was investigated. Wild-type and different cytokine as well as inducible nitric oxide synthase (iNOS) knockout mice were infected with the Colombian strain of Trypanosoma cruzi, and the kinetics of tissue parasitism, inflammatory reaction, parasitemia, and mortality were determined. We demonstrate the pivotal role of the interleukin (IL)-12/interferon (IFN)-gamma/iNOS axis and the antagonistic effect of IL-4 in controlling heart tissue parasitism, inflammation, and host resistance to acute infection with T. cruzi. Further, the heart and central nervous system were shown the main sites of reactivation of T. cruzi infection in mice lacking functional genes for IFN-gamma and IL-12, respectively. Our results also show that in contrast to IFN-gamma knockout (KO) mice, splenocytes from IL-12 KO mice infected with T. cruzi produced low levels of IFN-gamma upon stimulation with antigen. Consistently, high levels of anti-T. cruzi IgG2a antibodies were detected in the sera from IL-12 KO, but not from IFN-gamma KO mice, infected with the Colombian strain of T. cruzi. Thus, our results suggest that the level of IFN-gamma deficiency is a major determinant of the site of reactivation of T. cruzi infection in immunocompromised host.


Subject(s)
Central Nervous System Diseases/pathology , Central Nervous System Diseases/parasitology , Chagas Cardiomyopathy/pathology , Chagas Cardiomyopathy/parasitology , Chagas Disease/pathology , Chagas Disease/parasitology , Interferon-gamma/physiology , Interleukin-12/physiology , Animals , Central Nervous System/parasitology , Central Nervous System/pathology , Disease Susceptibility , Female , Heart/parasitology , Interferon-gamma/deficiency , Interferon-gamma/genetics , Interleukin-12/deficiency , Interleukin-12/genetics , Interleukin-4/deficiency , Interleukin-4/genetics , Interleukin-4/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout/genetics , Myocarditis/parasitology , Myocarditis/pathology , Myocardium/pathology , Nitric Oxide Synthase/deficiency , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/physiology , Nitric Oxide Synthase Type II , Recurrence
12.
Arch Med Res ; 32(2): 108-12, 2001.
Article in English | MEDLINE | ID: mdl-11343806

ABSTRACT

BACKGROUND: Activation and clonal expansion of T cells require not only the recognition of processed antigen on the surface of the antigen-presenting cell (APC) by T-cell receptor (TCR), but also involve co-stimulatory signals that are provided by the simultaneous engagement of cell surface molecules expressed by both the APC and the T cell. Interaction between CD40 and its ligand (CD40L) is known to mediate host immune response and T-cell-mediated effector functions in mycobacterial infections in mice. In this work, we investigated the capacity of Mycobacterium bovis (M. bovis) BCG to induce the expression of CD40L on human T cells. METHODS: Human cells were obtained from healthy adults by centrifugation using Ficoll/Hypaque. Cells (1 x 10(6)) were incubated in RPMI medium with BCG. After incubation at 37 degrees C in 5% CO(2) atmosphere for 40 h, cells were collected and double-stained with anti-CD40L-PE and anti-CD4-FITC or anti-CD8-FITC mAb. The quantification of positively stained population was based on samples stained with isotype control antibodies analyzed on a FACScan. RESULTS: M. bovis BCG stimulation induced a significant amount of CD40L expression on CD4+ T cells, while CD40L was not significantly detected on human CD8+ T cells. The highest CD40L expression on BCG-activated T cells in synergism with interleukin-12 endogenous occurred after a 40-h cell culture with a dose of 10 microg/mL of BCG (84.86 +/- 11.77; mean +/- standard deviation [SD]). This CD40L expression on BCG-activated human T cells was significantly inhibited by anti-IL-12 mAb (5.08 +/- 1.7; mean +/- SD). In contrast, anti-IFN-gamma and anti-IL-2 mAb did not have an important role in this expression. CONCLUSIONS: These results indicate that the capacity of BCG to induce CD40L expression on human cells represents a novel mechanism underlying the regulation of cellular responses against tuberculosis. Furthermore, the results showed a direct role of IL-12 to enhance the expression of CD40L on BCG-activated human cells.


Subject(s)
CD40 Ligand/metabolism , Interleukin-12/physiology , Mycobacterium bovis/metabolism , Adult , Humans , In Vitro Techniques
13.
Immunol Lett ; 76(1): 55-62, 2001 Feb 01.
Article in English | MEDLINE | ID: mdl-11222914

ABSTRACT

Peripheral blood mononuclear cells from leprosy patients and normal individuals were analysed for their ability to lyse autologous macrophages pulsed with the Mycobacterium leprae 10 kDa heat shock protein (hsp10), an antigen considered to have an important role in the protective responses in leprosy. Strong cytotoxic responses, with an involvement of gammadelta T and class-I and class-II restricted alphabeta T cells and/or CD16+56+ cells, were observed in normal individuals, paucibacillary (PB) and those multibacillary (MB) patients with undetectable bacillary load. On the contrary, only a weak class-II restricted cytotoxic response was observed in those MB patients with positive bacillary load (MB(+)). Simultaneous addition of IFNgamma plus TNFalpha and IL-12 during hsp10 stimulation could partially upregulate the low cytotoxic response observed in MB(+) by enhancing class-II restricted T cell activity and by development of gammadelta T and/or CD16+56+ cell activity. Our results suggest that the ability to mount an effective cytotoxic response against hsp10-pulsed macrophages in leprosy patients is closely related to the patient's bacterial load and not to the clinical form of the disease.


Subject(s)
Chaperonin 10/immunology , Cytotoxicity Tests, Immunologic , Leprosy/immunology , Leprosy/microbiology , Macrophages/immunology , Mycobacterium leprae/growth & development , Mycobacterium leprae/immunology , Adult , Aged , CD56 Antigen/biosynthesis , Cell Differentiation/immunology , Cells, Cultured , Chaperonin 10/metabolism , Female , Humans , Interferon-gamma/physiology , Interleukin-12/physiology , Killer Cells, Natural/immunology , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Male , Middle Aged , Receptors, Antigen, T-Cell, alpha-beta/biosynthesis , Receptors, Antigen, T-Cell, gamma-delta/biosynthesis , Receptors, IgG/biosynthesis , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/microbiology , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/microbiology , Tumor Necrosis Factor-alpha/physiology
14.
Scand J Immunol ; 51(3): 262-70, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10736095

ABSTRACT

Interleukin-12 (IL-12) is a major immunomodulatory cytokine that represents a functional bridge between the early resistance and the subsequent antigen specific adaptive immunity. TNF-alpha and IFN-gamma have an important role in the generation of hsp65 specific cytotoxic T lymphocytes (CTL) that lyse hsp65-pulsed autologous macrophages (hsp65 CTL). Since a positive feedback mechanism between TNF-alpha, IFN-gamma and IL-12 has been described, we undertook to evaluate the role of IL-12 on the hsp65 CTL generation in leprosy patients. Our results show that the presence of IL-12 during the first 24 h of the in vitro antigen stimulation amplifies the hsp65 cytotoxic response whenever both IFN-gamma and TNF-alpha are present. The addition of these three cytokines (CKs) was able to abrogate the inhibitory effect of IL-10 on hsp65 CTL in cells from paucibacillary patients (PB) but not that of IL-4 in PB and normal controls (N). Both IL-12 or anti IL-4 enhanced the cytotoxic activity in cells from multibacillary patients (MB). Anti IL-4 upregulated the binding of IFN-gamma and did not modify that of TNF-alpha so the low CTL activity could be as a result of IL-4 by a decrease of the IFN-gamma binding on MB cells. Cells from those MB patients taking thalidomide (MB-T) did neither bind IFN-gamma nor TNF-alpha even when antigen or anti-IL-4 were added, demonstrating that thalidomide inhibits either the in vitro binding or receptor expression of both TNF-alpha and IFN-gamma. Development of CD56 effector cells during the hsp65 stimulation was observed in PB and N by the addition of IL-12 plus TNF-alpha and IFN-gamma, while in MB and MB-T anti IL-4 was also required. So, the inhibitory effect of IL-4 on either production of IFN-gamma, TNF-alpha and/or IL-12 or their receptors could be the mechanism underlying the lack of the hsp65 CTL generation in cells from MB.


Subject(s)
Bacterial Proteins , Chaperonins/immunology , Cytotoxicity, Immunologic/immunology , Interferon-gamma/biosynthesis , Interleukin-12/physiology , Interleukin-4/physiology , Mycobacterium leprae/immunology , T-Lymphocytes, Cytotoxic/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Adult , Aged , CD56 Antigen/biosynthesis , Cells, Cultured , Chaperonin 60 , Down-Regulation/immunology , Drug Synergism , Female , Humans , Immune Sera/pharmacology , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/metabolism , Interferon-gamma/physiology , Interleukin-10/physiology , Interleukin-12/immunology , Interleukin-12/metabolism , Interphase/immunology , Leprosy/immunology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lymphocyte Activation/immunology , Major Histocompatibility Complex/immunology , Male , Middle Aged , Protein Binding/immunology , T-Lymphocytes, Cytotoxic/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/physiology
15.
J Pediatr ; 136(2): 176-80, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10657822

ABSTRACT

OBJECTIVE: Patients with the hyperimmunoglobulinemia E (hyper-IgE) syndrome are reported to have defective production of interferon gamma (IFN-gamma). Because IFN-gamma is a major activator of polymorphonuclear leukocytes (PMNs), this could result in defective PMN chemotaxis and markedly elevated IgE levels because of the unopposed action of interleukin (IL)-4. IL-12, an important enhancer of IFN-gamma production, also suppresses IgE production. This study assessed the IL-12/IFN-gamma pathway in patients with hyper-IgE syndrome. METHODS: Production of IL-12 and IFN-gamma by mononuclear cells from 10 patients with hyper-IgE syndrome in response to a number of stimuli was determined, as well as the effect of IL-12 on IFN-gamma release and cell proliferation. RESULTS: IL-12 and IFN-gamma production by the patients' cells was similar to that of control subjects independent of the stimulus used, except for Staphylococcus aureus, with which cells of patients with hyper-IgE syndrome released markedly less IFN-gamma (19.8%; P <.002). The ability of recombinant IL-12 to enhance IFN-gamma release from patients' cells in response to all stimuli was, however, significantly lower than with control cells (12% to 51%; P <.03). CONCLUSION: The lymphocytes of patients with hyper-IgE syndrome have an impaired response to IL-12, resulting in decreased IFN-gamma production, which may be of key importance in the pathogenesis of the immune abnormalities of hyper-IgE syndrome.


Subject(s)
Interferon-gamma/physiology , Interleukin-12/physiology , Job Syndrome/immunology , Adolescent , Adult , Candida albicans/immunology , Cells, Cultured , Child , Female , Humans , Immunoglobulin E/metabolism , In Vitro Techniques , Interferon-gamma/biosynthesis , Interleukin-12/biosynthesis , Job Syndrome/genetics , Leukocytes, Mononuclear/immunology , Male , Staphylococcus aureus/immunology
16.
Clin Exp Immunol ; 119(2): 299-304, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10632666

ABSTRACT

In this study we have compared the immune response of normal human cells cultured in vitro to two virulent strains of Leishmania major (CC1 and LV39), and to an avirulent vaccine strain (dhfr-ts-) made by targeted deletion of the essential gene DHFR-TS. We utilized an in vitro system in which naive T cells from normal human donors were primed with autologous Leishmania-infected macrophages. All three parasites infected macrophages and transformed into amastigotes within the cells. However, whereas LV39 and CC1 replicated in macrophages, dhfr-ts- did not. When peripheral blood lymphocytes (PBL) were stimulated with autologous macrophages infected with any of the three parasites, the lymphocytes produced a type-1-biased cytokine response. Finally, addition of IL-12 during the first stimulation period increased the production of interferon-gamma but decreased IL-5 secretion. On the other hand, anti-IL-12 resulted in the opposite effect.


Subject(s)
Cytokines/biosynthesis , Leishmania major/immunology , Leishmania major/pathogenicity , Th1 Cells/immunology , Animals , Cells, Cultured , Humans , Interferon-gamma/biosynthesis , Interleukin-12/physiology , Interleukin-5/biosynthesis , Leishmania major/enzymology , Leishmania major/genetics , Tetrahydrofolate Dehydrogenase/deficiency , Tetrahydrofolate Dehydrogenase/genetics , Th1 Cells/metabolism , Th1 Cells/parasitology , Th2 Cells/immunology , Th2 Cells/metabolism , Th2 Cells/parasitology , Thymidylate Synthase/deficiency , Thymidylate Synthase/genetics , Virulence
17.
Medicina (B Aires) ; 59 Suppl 2: 84-90, 1999.
Article in Spanish | MEDLINE | ID: mdl-10668248

ABSTRACT

Specific antibodies and the activation of phagocytic cells by IFN-gamma are the key elements of the immune response involved in protection of the T. cruzi infected host. The central role of the IFN-gamma in vivo seems to be the activation of the inducible nitric oxide synthetase of macrophages (iNOS) and the production of nitric oxide (NO degree) for the intracellular destruction of the parasite. Interleukin 12 (IL-12), the cytokine that stimulates NK cells for IFN-gamma production, seems to trigger the TH1 response in the acute phase. Other cell types, such as lymphocytes Thy-1+CD4-CD8-, CD4+ and CD8+, are also involved in IFN-gamma production. The down regulation of the TH1 response could in part depend on the decrease in the macrophage activation, as a result of the controlled parasite burden, and on the production of IL-10 and transforming growth factor beta (TGF-beta). The protective TH1 immune response seems to be also related to both the tissue damage and the alterations of the immune response observed during the infection. We studied the kinetics of both NK cell activity, and the production of IL-12 and/IFN-gamma by spleen cells, as well as the seric levels of these cytokines, in BALB/c and C3H mice infected with T. cruzi, Tulahuén strain. In the spleen, we found that the production of IL-12 and the NK cell activity increased in the very early acute infection, and that in C3H the effect was higher than in BALB/c mice. IFN-gamma increased in C3H at the same time, but in the BALB/c strain it increased later in the acute phase. The infection induced a very early increase in the seric levels of IL-12, that remained high throughout the acute phase, in both mouse strains. However, the levels of IFN-gamma in the serum increased a few days before the peak of parasitemia, reaching higher values, and earlier, in BALB/c than in C3H mice. Surprisingly, in the chronic infection IL-12 production remained high in both mouse strains, but IFN-gamma production was only observed in BALB/c mice. The immune response was predominantly TH1 in both mouse strains, in spite of the higher susceptibility of BALB/c compared to C3H. The early control of the parasite burden could be evaluated as the expression of the TH1 response in spleen cells, while the seric levels of IL-12 and IFN-gamma would be related to the induction of tissue damage. Our data indicate that the protective TH1 immune response has a different expression according to the host-parasite relationship, and that the factors controlling the response are of primary importance to determine the quali- and quantitative expression of IL-12/NK/IFN-gamma as well as their involvement in resistance and tissue damage.


Subject(s)
Chagas Disease/immunology , Interferon-gamma/physiology , Interleukin-12/physiology , Killer Cells, Natural/immunology , Nitric Oxide/blood , Th1 Cells/immunology , Trypanosoma cruzi/immunology , Animals , Chronic Disease , Immunity, Cellular , Interferon-gamma/blood , Interleukin-12/blood , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Species Specificity , Spleen/cytology , Spleen/immunology , Th2 Cells/immunology , Trypanosoma cruzi/pathogenicity
18.
Braz J Med Biol Res ; 31(1): 111-5, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9686186

ABSTRACT

Host resistance to Trypanosoma cruzi infection is dependent on both natural and acquired immune responses. During the early acute phase of infection in mice, natural killer (NK) cell-derived IFN-gamma is involved in controlling intracellular parasite replication, mainly through the induction of nitric oxide biosynthesis by activated macrophages. We have shown that IL-12, a powerful inducer of IFN-gamma production by NK cells, is synthesized soon after trypomastigote-macrophage interaction. The role of IL-12 in the control of T. cruzi infection in vivo was determined by treating infected mice with anti-IL-12 monoclonal antibody (mAb) and analyzing both parasitemia and mortality during the acute phase of infection. The anti-IL-12 mAb-treated mice had higher levels of parasitemia and mortality compared to control mice. Also, treatment of infected mice with mAb specific for IFN-gamma or TNF-alpha inhibited the protective effect of exogenous IL-12. On the other hand, TGF-beta and IL-10 produced by infected macrophages inhibited the induction and effects of IL-12. Therefore, while IL-12, TNF-alpha and IFN-gamma correlate with resistance to T. cruzi infection, TGF-beta and IL-10 promote susceptibility. These results provide support for a role of innate immunity in the control of T. cruzi infection. In addition to its protective role, IL-12 may also be involved in the modulation of T. cruzi-induced myocarditis, since treatment of infected mice with IL-12 or anti-IL-12 mAb leads to an enhanced or decreased inflammatory infiltrate in the heart, respectively. Understanding the role of the cytokines produced during the acute phase of T. cruzi infection and their involvement in protection and pathogenesis would be essential to devise new vaccines or therapies.


Subject(s)
Disease Models, Animal , Interleukin-12/physiology , Trypanosoma cruzi/pathogenicity , Trypanosomiasis/immunology , Animals , Immunity, Innate , Mice
19.
Braz J Med Biol Res ; 31(1): 163-9, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9686195

ABSTRACT

The attenuated vaccine against Schistosoma mansoni induces Th1-mediated protective immunity and we have sought to identify a role for IL-12 in this model. Elevated levels of IL-12 (p40 mRNA) were detected in the lymph nodes (LN) and the lungs of vaccinated mice, whilst treatment of vaccinated mice with anti-IL-12 antibodies decreased the ratio of IFN gamma:IL-4 secreted by in vitro-cultured LN cells. However, there was only marginal abrogation of the level of resistance in these mice. Soluble antigens from the lung-stage of the parasite (SLAP) appeared to be efficient stimulators of IFN gamma and IL-12 secretion. These antigens when used to immunise mice in conjunction with IL-12 as an adjuvant, elicited a polarised Th1 response with abundant IFN gamma secretion but no IL-4. This immunisation regime also induced significant protection against reinfection, whereas inoculation of mice with SLAP alone did not. The induction of a dominant Th1 response using SLAP + IL-12 probably operates via IFN gamma production by natural killer (NK) cells stimulated by IL-12, since in vivo ablation of NK cells using anti-NK1.1 antibody reduced CD4(+)-dependent IFN gamma production from cultured LN cells by over 97%. Nevertheless, in mice with a genetic disruption of the IFN gamma receptor, administration of SLAP + IL-12 induced levels of IFN gamma equal to those in wild-type mice, thus showing that in this model IL-12 can directly prime T cells independent of IFN gamma. Clearly, IL-12 has a critical role in protective immunity to schistosomes and it may aid the development of an effective vaccine against this disease.


Subject(s)
Interleukin-12/physiology , Schistosoma mansoni/pathogenicity , Schistosomiasis mansoni/immunology , Schistosomiasis mansoni/prevention & control , Vaccines , Animals , Mice , Th1 Cells
20.
Rev. bras. pesqui. méd. biol ; Braz. j. med. biol. res;31(1): 111-5, Jan. 1998.
Article in English | LILACS | ID: lil-212545

ABSTRACT

Host resistance to Trypanosoma cruzi infection in dependent on both natural and acquired immune responses. During the early acute phase of infection in mice, natural killer (NK) cell-derived IFN-gamma is involved in controlling intracellular parasite replication, mainly through the induction of nitric oxide biosynthesis by activate macrophages. We have shown that IL-12, a powerful inducer of IFN-gamma production by NK cells, is synthesized soon after trypomastigote-macrophage interaction. The role of IL-12 in the control of T. cruzi infection in vivo was determined by treating infected mice with anti-IL-12 monoclonal antibody (mAb) and analyzing both parasitemia and mortality during the acute phase of infection. The anti-IL-12 mAb-treated mice had higher levels of parasitemia and mortality compared to control mice. Also, treatment of infected mice with mAb spectific for IFN-gamma or TNF-alpha inhibited the protective effect of exogenous IL-12. On the other hand, TGF-beta and IL-10 produced by infected macrophages inhibited the induction and effects of IL-12. Therefore, while IL-12, TNF-alpha and IFN-gamma correlate with resistance to T. cruzi infection, TGF-beta and IL-10 promote susceptibility. These results provide support for a role of innate immunity in the control of T. cruzi infection. In addition to its protective role, IL-12 may also be involved in the modulation of T. cruzi-induced myocarditis, since treatment of infected mice with IL-12 or anti-IL-12 mAb leads to an enhanced or decreased inflammatory infiltrate in the heart, respectively. Understanding the role of the cytokine produced during the acute phase of T. cruzi infection and their involvement in protection and pathogenesis would be essential to devise new vaccines or therapies.


Subject(s)
Mice , Animals , Disease Models, Animal , Interleukin-12/physiology , Trypanosoma cruzi/pathogenicity , Trypanosomiasis/immunology , Trypanosomiasis/physiopathology , Immunity, Innate
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