ovalbumin and Leishmaniasis--Cutaneous

H-/K-Ras and N-Ras isoforms were proposed to lack functional specificities due to similarity in 1-165 amino acids. As recent studies implied Ras isoform-specific developmental effects, we examined their functional specificity using Leishmania major infection, anti-hapten antibody response and carrier-specific T cell response. While N-Ras overexpression increased L. major infection in resistant C57BL/6 mice, H-Ras or K-Ras overexpression reduced the infection in susceptible BALB/c mice. These Ras isoforms differentially regulated anti-TNP antibody response in TNP-Ova-primed, but not in TNP-Ficoll- or TNP-LPS-primed, BALB/c mice. Ras isoform-specific silencing selectively modulated Ova-specific T cell response. The data indicate Ras isoform-specific regulation of antigen-specific immune response.

Topics: Animals; Antibody Formation; Antigen-Antibody Reactions; Cell Line; Female; Ficoll; Haptens; Leishmania major; Leishmaniasis, Cutaneous; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Protein Isoforms; Proto-Oncogene Proteins p21(ras); RNA Interference; RNA, Small Interfering; T-Lymphocytes, Regulatory; Trinitrobenzenes

Although the strategic production of prolactin-inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4(+) T cells in peripheral lymphoid tissues and impaired CD4(+) Th1-cell differentiation in vitro. In vivo, CD4(+) T cells from OVA-immunized, PIP-deficient mice showed significantly impaired proliferation and IFN-γ production following in vitro restimulation. Furthermore, PIP-deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN-γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4(+) Th1-cell-mediated immunity.

Topics: Animals; Cell Differentiation; Cell Proliferation; Cytokines; Dendritic Cells; Disease Susceptibility; Immunity, Cellular; Interferon-gamma; Leishmania major; Leishmaniasis, Cutaneous; Lipopolysaccharides; Macrophages; Mice; Mice, Knockout; Nitric Oxide; Ovalbumin; Proteins; Th1 Cells; Th17 Cells; Th2 Cells

An effective adaptive immune response requires activation of specific CD4 T cells. The capacity of B cells to activate CD4 T cells in human cutaneous leishmaniasis caused by Leishmania (Viannia) has not been evaluated.. CD4 T cell activation by B cells of cutaneous leishmaniasis patients was evaluated by culture of PBMCs or purified B cells and CD4 T cells with Leishmania panamensis antigens. CD4 T cell and B cell activation markers were evaluated by flow cytometry and 13 cytokines were measured in supernatants with a bead-based capture assay. The effect of Leishmania antigens on BCR-mediated endocytosis of ovalbumin was evaluated in the Ramos human B cell line by targeting the antigen with anti-IgM-biotin and anti-biotin-ovalbumin-FITC.. Culture of PBMCs from cutaneous leishmaniasis patients with Leishmania antigens resulted in upregulation of the activation markers CD25 and CD69 as well as increased frequency of CD25hiCD127- cells among CD4 T cells. Concomitantly, B cells upregulated the costimulatory molecule CD86. These changes were not observed in PBMCs from healthy subjects, indicating participation of Leishmania-specific lymphocytes expanded in vivo. Purified B cells from these patients, when interacting with purified CD4 T cells and Leishmania antigens, were capable of inducing significant increases in CD25 and CD69 expression and CD25hiCD127- frequency in CD4 T cells. These changes were associated with upregulation of CD86 in B cells. Comparison of changes in CD4 T cell activation parameters between PBMC and B cell/CD4 T cell cultures showed no statistically significant differences; further, significant secretion of IFN-γ, TNF-α, IL-6 and IL-13 was induced in both types of cultures. Additionally, culture with Leishmania antigens enhanced BCR-mediated endocytosis of ovalbumin in Ramos human B cells.. The capacity of B cells specific for Leishmania antigens in peripheral blood of cutaneous leishmaniasis patients to activate CD4 T cells and induce cytokine secretion is similar to that of all cell populations present in PBMCs. This capacity implicates B cells as a plausible target for modulation of the immune response to Leishmania infection as a therapeutic strategy.

Topics: Adult; Aged; B-Lymphocytes; Biotin; CD4-Positive T-Lymphocytes; Colombia; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; Gene Expression Regulation; Humans; Immunoglobulin M; Interferon-gamma; Interleukin-6; Leishmania braziliensis; Leishmaniasis, Cutaneous; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Middle Aged; Ovalbumin; Tumor Necrosis Factor-alpha; Young Adult

Anticytokine auto-vaccination is a powerful tool for the study of cytokine functions in vivo but has remained rather esoteric as a result of numerous technical difficulties. We here describe a two-step procedure based on the use of OVA multimers purified by size exclusion chromatography after incubation with glutaraldehyde at pH 6. When such polymers are incubated with a target protein at pH 8.5 to deprotonate reactive amines, complexes are formed that confer immunogenicity to self-antigens. The chemokine GCP-2/CXCL6, the cytokines GM-CSF, IL-17F, IL-17E/IL-25, IL-27, and TGF-β1, and the MMP-9/gelatinase B are discussed as examples. mAb, derived from such immunized mice, have obvious advantages for in vivo studies of the target proteins. Using a mAb against GCP-2, obtained by the method described here, we provide the first demonstration of the major role played by this chemokine in rapid neutrophil mobilization after Leishmania major infection. Pre-activated OVA multimers reactive with amine residues thus provide an efficient carrier for auto-vaccination against 9-90 kDa autologous proteins.

Topics: Amines; Animals; Cell Movement; Chemokines; Cytokines; Glutaral; Immunization; Interleukin-17; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred C57BL; Mice, Knockout; Microtubule-Associated Proteins; Neutrophils; Ovalbumin; Protein Multimerization

Two strains of mice selected according to extreme phenotypes of susceptibility and resistance to oral tolerance (TS and TR mice, respectively) were infected with 1 x 10(7) Leishmania amazonensis promastigotes and studied comparatively. TS mice developed a minor pathology while permitting parasite growth with the presence of increased IL-4, IL-10 and IFN-gamma, and lower NO and IL-2 levels and delayed-type hypersensitivity (DTH). In contrast, in TR mice, footpad swelling was increased but parasite growth was reduced. They produced lower IL-4, IL-10 and IFN-gamma but increased NO, IL-2 levels, DTH, activated spleen macrophages and periarteriolar lymphoid sheaths. The results suggest that the tolerogenic TS mouse profile, with higher IL-10 production, impaired lesion development but also avoided macrophage leishmanicidal activity, maintaining in this manner a silent parasite load. On the other hand, the TR mouse profile contributed to lesion progression with controlled parasite load. To directly address the influence of oral tolerance on infection, mice were gavaged with OVA, and 7 days afterwards were infected and challenged to bystander suppression with OVA in the same footpad. In TR mice gavaged with 25 mg OVA the inflammatory lesion was largely enhanced, while with 5 mg OVA the lesion was diminished. In TS mice the footpad swelling was always lower. However, the bystander effect did not modify the establishment of infection; and similarly to the control non-bystander mice, parasite clearance was maintained in TR and prevented in TS mice. Therefore, a better comprehension of immunoregulation of innate and adaptive immunity in the early stages of infection is necessary for the development of protocols preventing inflammation and contributing to the elimination of parasites.

Topics: Administration, Oral; Animals; Animals, Outbred Strains; Cells, Cultured; Cytokines; Disease Models, Animal; Female; Genetic Predisposition to Disease; Immune Tolerance; Inflammation; Leishmania; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nitric Oxide; Ovalbumin; Parasite Egg Count; Phagocytosis; Selection, Genetic; Skin; Spleen

CD4(+) and CD8(+) T-cell responses have been shown to be critical for the development and maintenance of acquired resistance to infections with the protozoan parasite Leishmania major. Monitoring the development of immunodominant or clonally restricted T-cell subsets in response to infection has been difficult, however, due to the paucity of known epitopes. We have analyzed the potential of L. major transgenic parasites, expressing the model antigen ovalbumin (OVA), to be presented by antigen-presenting cells to OVA-specific OT-II CD4(+) or OT-I CD8(+) T cells. Truncated OVA was expressed in L. major as part of a secreted or nonsecreted chimeric protein with L. donovani 3' nucleotidase (NT-OVA). Dendritic cells (DC) but not macrophages infected with L. major that secreted NT-OVA could prime OT-I T cells to proliferate and release gamma interferon. A diminished T-cell response was observed when DC were infected with parasites expressing nonsecreted NT-OVA or with heat-killed parasites. Inoculation of mice with transgenic parasites elicited the proliferation of adoptively transferred OT-I T cells and their recruitment to the site of infection in the skin. Together, these results demonstrate the possibility of targeting heterologous antigens to specific cellular compartments in L. major and suggest that proteins secreted or released by L. major in infected DC are a major source of peptides for the generation of parasite-specific CD8(+) T cells. The ability of L. major transgenic parasites to activate OT-I CD8(+) T cells in vivo will permit the analysis of parasite-driven T-cell expansion, differentiation, and recruitment at the clonal level.

Topics: Animals; Antigen Presentation; Antigens, Protozoan; CD8-Positive T-Lymphocytes; Cell Proliferation; Cross-Priming; Dendritic Cells; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred C57BL; Nucleotidases; Ovalbumin; Recombinant Fusion Proteins; Transgenes

Alloreactive T lymphocytes can be primed through direct presentation of donor MHC:peptide complexes on graft cells and through indirect presentation of donor-derived determinants expressed by recipient APCs. The large numbers of determinants on an allograft and the high frequency of the alloreactive repertoire has further led to speculation that exposure to environmental Ags may prime T cells that cross-react with alloantigens. We sought to develop a model in which to test this hypothesis. We found that CD4(+) T cells obtained from C57BL/6 (B6) mice that clinically resolved Leishmania major infection exhibited statistically significant cross-reactivity toward P/J (H-2(p)) Ags compared with the response to other haplotypes. B6 animals that were previously infected with L. major specifically rejected P/J skin grafts with second set kinetics compared with naive animals. Although donor-specific transfusion combined with costimulatory blockade (anti-CD40 ligand Ab) induced prolonged graft survival in naive animals, the same treatment was ineffective in mice previously infected with L. major. The studies demonstrate that cross-reactive priming of alloreactive T cells can occur and provide direct evidence that such T cells can have a significant impact on the outcome of an allograft. The results have important implications for human transplant recipients whose immune repertoires may contain cross-reactively primed allospecific T cells.

Topics: Amino Acid Sequence; Animals; Antigens; Chickens; Cross Reactions; Disease Models, Animal; Female; Graft Rejection; Isoantigens; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Molecular Sequence Data; Muramidase; Ovalbumin; Rabbits; Skin Transplantation; T-Lymphocyte Subsets; Vaccination

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

Topics: Animals; Antigens, Protozoan; Cell Differentiation; Cells, Cultured; Dendritic Cells; Genetic Predisposition to Disease; Immunity, Innate; Interleukin-12; Interleukin-4; Leishmania major; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Ovalbumin; Protozoan Proteins; Receptors, Antigen, T-Cell, alpha-beta; Recombinant Proteins; Specific Pathogen-Free Organisms; Th1 Cells; Th2 Cells; Time Factors

The patterns of Ag-induced cytokine coexpression in normal, in vivo-primed CD4 memory T cells has remained controversial because the low frequency at which these cells occur has effectively prevented direct ex vivo measurements. We have overcome this limitation by using two-color cytokine enzyme-linked immunospot assays and computer-assisted image analysis. We found CD4 memory cells that simultaneously expressed IL-2, IL-3, IL-4, IL-5, and IFN-gamma to be rare (0-10%). This cytokine segregation was seen in adjuvant-induced type 1, type 2, and mixed immunity to OVA, in Leishmania infection regardless of the Ag dose used or how long after immunization the assay was performed. The data suggest that type 1 and type 2 immunity in vivo is not mediated by classic Th1 or Th2 cells but by single-cytokine-producing memory cells.

Topics: Animals; Antigens, Protozoan; CD4-Positive T-Lymphocytes; Cells, Cultured; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Freund's Adjuvant; Image Enhancement; Immunologic Memory; Immunophenotyping; Interferon-gamma; Interleukin-2; Interleukin-3; Interleukin-4; Interleukin-5; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Peptide Fragments; Signal Transduction; T-Lymphocyte Subsets; Th1 Cells; Th2 Cells

Infections can influence concurrent and subsequent Th1 vs Th2 immune responses to Ags. Through pattern recognition of foreign unmethylated CpG dinucleotides, the vertebrate innate immune system can sense infectious danger and typically replies with a Th1-polarized adaptive immune response. We examined whether CpG-DNA exposure would influence subsequent responses to infection and soluble Ags. CpG-DNA injection led to local lymphadenopathy characterized by maintenance of cellular composition with some biasing toward elevated dendritic cell composition. Sustained local production of IL-12 and IFN-gamma from dendritic cells and T cells was shown. Prior injection by up to 2 wk with CpG-DNA protected BALB/c mice from Th2 driven lethal leishmaniasis. CpG-DNA injection by up to 5 wk before soluble Ag challenge resulted in the generation of Ag-specific CTL, Th1 recall responses to Ag, and Th1-polarized Ag-specific Abs. Thus, CpG-DNA instigated a local predisposition for intense CTL responses and Th1-polarized immune responses to subsequent infections or Ag challenge. The induction by the innate immune system of a locally contained hypersensitivity could represent a capacitating immune reaction yielding rapid conditioned responses to secondary infections.

Topics: Adoptive Transfer; Animals; Antigens; CD11 Antigens; CpG Islands; Cytotoxicity, Immunologic; DNA; Female; Immunity, Cellular; Immunity, Innate; Immunization; Injections, Subcutaneous; Interferon-gamma; Interleukin-12; Kinetics; Leishmania major; Leishmaniasis, Cutaneous; Lymph Nodes; Lymphatic Diseases; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Oligonucleotides; Ovalbumin; T-Lymphocytes, Cytotoxic; Th1 Cells

Gp63 is a major surface protein of Leishmania promastigotes. Its protective efficacy has been tested in several experimental models using different mouse strains, gp63 forms, adjuvants and routes of immunization, giving rise to conflicting results. This investigation was designed to determine whether these discrepancies could be ascribed to differing experimental procedures, and to compare gp63-induced protection with that achieved using live promastigotes. Preliminary experiments demonstrated that gp63 was an extremely potent immunogen compared to a standard antigen (ovalbumin). Protection against Leishmania major infection afforded by gp63 inoculation was studied in CBA mice. Injection of gp63 in saline, or of CFA, BCG, and C. parvum without antigen, induced significant protection. When gp63 and adjuvants were combined, results differed depending on the site of vaccination relative to that of the challenge infection. Vaccination with gp63 plus adjuvants in the tail (i.e. close to the site of infection) led to a stronger reduction of lesion size than the basal level of protection elicited by adjuvants alone, except in the case of CFA. Surprisingly however, when the antigen was injected at a distance from the site of infection (immunization in the hind foot pads, infection in the rump), the protective effect of gp63 was decreased by the adjuvants. Finally, vaccination at either site using live parasites (radioattenuated or virulent promastigotes) resulted in most instances in better protection than achieved by any protocol using gp63 and adjuvants. While anti-gp63 T cells proliferated in vitro in response to L. major-infected bone marrow-derived macrophages, they were unable to activate macrophages for parasite killing. This is in contrast with lymphocytes from mice immunized with live parasites, which both proliferated and stimulated significant killing of the microorganisms within 48 h.

Topics: Adjuvants, Immunologic; Animals; Disease Models, Animal; Leishmania major; Leishmaniasis, Cutaneous; Lymph Nodes; Lymphocyte Activation; Macrophages; Matched-Pair Analysis; Metalloendopeptidases; Mice; Mice, Inbred CBA; Ovalbumin; Protozoan Vaccines; T-Lymphocytes; Vaccination; Vaccines, Attenuated; Vaccines, Synthetic

Intravenous injection of antigen-coupled splenocytes has been widely used to induce specific tolerance to a variety of antigens. In this study, we investigated the effects of such a treatment on Th1 and Th2 antigen-specific immune responses. Using both well-characterized model antigens and crude homogenates from Leishmania major promastigotes, we found that intravenous injection of antigen-coupled splenocytes strongly down-regulated antigen-specific Th2 responses but had no or only moderate effects on Th1 responses. Because the susceptibility of inbred strains of mice to murine leishmaniasis has been found to be correlated with a strong Th2 response against parasite antigens, we investigated whether administration of splenocytes chemically coupled to parasite antigens could protect susceptible mice from murine leishmaniasis. We found that this was indeed the case and further demonstrated that protection was associated with a strong decrease in the number of parasite-specific Th2-like cells. Because administration of antigen-coupled splenocytes is believed to induce ligation of the T cell receptor complex without inducing a co-stimulatory signal, our results further suggest that priming of Th1 cells is less dependent on co-stimulatory signals than the priming of Th2 cells.

Topics: Adoptive Transfer; Animals; Antigens; Antigens, Protozoan; Down-Regulation; Female; Immunity, Innate; Injections, Intravenous; Leishmania major; Leishmaniasis, Cutaneous; Lymphocyte Activation; Lymphokines; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Ovalbumin; Protein Binding; Protozoan Proteins; Spleen; Th1 Cells; Th2 Cells

Continuous administration of soluble protein Ag followed by immunization with the same Ag in adjuvant results in the selective development of Ag-specific CD4+ Th2 cells in both normal and beta2-microglobulin-deficient BALB/c mice. In addition to chronic administration by mini-osmotic pump, single bolus i.p., but not i.v., injection of protein Ag induces Th2 cell expansion. Strong Th2 cell priming depends on a non-MHC-linked genetic polymorphism. It is observed in all congenic strains on BALB background tested, BALB/c, BALB/b, and BALB/k, but not in MHC-matched strains on disparate genetic background, B10.D2, C57BL/6, and C3H. DBA/2 mice appear to have an intermediate phenotype, as shown by their weaker capacity to mount Th2 responses as compared with BALB/c mice after soluble Ag administered by either mini-osmotic pumps or single bolus i.p. Conversely, induction of Th1 cell unresponsiveness by soluble protein is observed in any mouse strain tested, following any mode of Ag administration. These data demonstrate that non-MHC-linked genetic polymorphism controls the priming of Th2 but not the inhibition of Th1 cells induced by administration of soluble protein. The pattern of Th2 responses in these different strains is predictive of disease outcome following Leishmania major infection and supports the hypothesis that systemic Ag presentation in the absence of strong inflammatory signals may represent an important stimulus leading to selective Th2 cell development in susceptible mouse strains.

Topics: Animals; Antigens; beta 2-Microglobulin; Cell Differentiation; Disease Susceptibility; Dose-Response Relationship, Immunologic; Female; Immunity, Cellular; Immunization; Infusion Pumps, Implantable; Injections, Intraperitoneal; Injections, Intravenous; Leishmania major; Leishmaniasis, Cutaneous; Lymphocyte Activation; Mice; Mice, Inbred Strains; Muramidase; Ovalbumin; Phenotype; Polymorphism, Genetic; Species Specificity; Th1 Cells; Th2 Cells; Tuberculin

Leishmania-infected M phi are potential candidates for the presentation of parasite Ag to Leishmania-specific CD4+ T lymphocytes. To assess whether infected cells could function as APC, we examined the ability of bone marrow-derived M phi infected with Leishmania amazonensis amastigotes to stimulate various CD4+, l-Ad- or l-Ed-restricted T-cell hybridomas specific for the bacteriophage lambda repressor cl protein, the human chorionic gonadotropin or OVA. A reduced capacity of infected M phi to present native Ag to most T-cell hybridomas tested was noted that was probably a result of a lower expression on their plasma membrane of stimulatory [la-peptide] complexes. Neither a reduced Ag uptake nor an altered Ag processing appeared to be at the origin of the partial inability of infected M phi to present Ag. As regards the level of plasma membrane la expression, no quantitative difference could be detected between uninfected and infected M phi. Moreover, after fixation with paraformaldehyde, the ability of plasma membrane la molecules to bind immunogenic peptides was apparently not reduced in infected M phi. So, these cells most likely expressed functional la molecules on their cell surface. Interestingly, infected M phi and M phi infected then cured by a treatment with a leishmanicidal compound were similarly impaired in their capacity to present native Ag or peptides to the hybridomas, and no recovery was noted even 24 h after the leishmanicidal treatment. Furthermore, infected M phi and M phi incubated with heat-killed amastigotes or with an amastigote homogenate exhibited similar inhibitions of Ag presentation. Taken together, these results suggest that the functional failure of infected M phi to present exogenous Ag could be because either of interferences with the events leading to the meeting of la molecules with peptides derived from these exogenous Ag or to a competition for binding to la molecules between these peptides and parasite molecules.

Topics: Animals; Antigen-Presenting Cells; Antigens; CD4-Positive T-Lymphocytes; Cell Membrane; Chorionic Gonadotropin; DNA-Binding Proteins; Female; Hybridomas; Interleukin-2; Leishmania mexicana; Leishmaniasis, Cutaneous; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred BALB C; Ovalbumin; Peptides; Repressor Proteins; Viral Proteins; Viral Regulatory and Accessory Proteins