Organ-specific distribution of CD4+ T1/ST2+ Th2 cells in Leishmania major infection

Activated CD4(+) T helper cells (Th) comprise at least two functionally distinct subsets, Th1 and Th2, which mediate different immunological effector functions. Experimental leishmaniasis is widely used to study the effector function of Th cell subsets in vivo. Healing and nonhealing Leishmania major infections have been correlated with polarized Th1 and Th2 responses, respectively. In the study presented here, a stable cell surface marker expressed on Th2 cells, T1/ST2, has been used to assess the distribution of CD4(+) T1/ST2(+) T cells in different organs of healer and nonhealer strains of mice during the course of L. major infection. The frequency of CD4(+) T cells expressing the T1/ST2 cell surface marker and Th2 cytokines in the lymphoid organs was low in both strains of infected mice; however, CD4(+) T1/ST2(+) T cells could be enriched from the lymphoid organs of infected nonhealer but not from healer strains of mice. The highest frequency of CD4(+) T1/ST2(+) T cells was detected in the footpads of mice with nonhealing disease, showing that CD4(+) T1/ST2(+) T cells home to the footpads. Since the majority of parasites persist at the local site of infection in nonhealing BALB/c mice, these results show that CD4(+) T1/ST2(+) T cells are localized at the site of active infection and inflammation in this model.     

Critical role of dendritic cells in determining the Th1/Th2 balance upon Leishmania major infection

The onset of T_h1 immunity is in part regulated by genetic background.To elucidate the cell type carrying critical factors determiningthe T_h1 response, we employed Rag-2^–/– mice on Leishmaniamajor-susceptible BALB/c and -resistant B10.D2 backgrounds.By using bone marrow (BM) chimeras generated by the transplantationof B10.D2 BM cells into BALB/c-Rag-2^–/– mice, andvice versa, it was shown that hematopoietic cells carry factorsdetermining the disease outcome and T_h1 response against L.major infection. B10.D2-Rag-2^–/– mice reconstitutedwith BALB/c CD4⁺ T cells exhibited a T_h1 response and controlledL. major infection. Wild-type BALB/c mice inoculated with L.major-parasitized B10.D2-Rag-2^–/– splenocytes alsoexhibited a T_h1 response and a mild disease outcome, whereassuch a T_h1 response was not induced when CD11c⁺ dendritic cells(DCs) were depleted from parasitized B10.D2-Rag-2^–/–splenocytes. T_h1 response was reconstituted by the additionof L. major-parasitized B10.D2 DCs but not L. major-parasitizedBALB/c DCs to DC-depleted parasitized B10.D2-Rag-2^–/–splenocytes. These results indicate that DCs determine the outcomeof the disease upon L. major infection.     

Expression of Th2 cytokines and the stable Th2 marker ST2L in the absence of IL-4 during Leishmania major infection

In this study we characterized Th2 responses in the absence of IL-4. We show that ST2L, a stable Th2 marker, is expressed at similar levels in Leishmania major-infected IL-4-deficient (IL-4(-/-)) and wild-type BALB/c (IL-4(+/+)) mice. Th2 cytokines are secreted by in vivo differentiated lymphocytes in response to specific activation in the absence of IL-4. Although IL-13 is produced, its neutralization did not alter the outcome of infection. Thus, we demonstrate that Th2 differentiation as assessed by the expression of ST2L and the production of Th2 cytokines can occur in vivo in the absence of IL-4.     

Macrophage migration inhibitory factor is essential for allergic asthma but not for Th2 differentiation

Macrophage migration inhibitory factor (MIF) is increased in asthmatic patients and plays a critical role in the pathogenesis of asthma. We show here that mice lacking MIF failed to develop airway hyper-responsiveness (AHR), tissue eosinophilia, and mucus metaplasia. Analysis of the bronchoalveolar fluids revealed a substantial reduction of IL-13, eotaxin and cysteinyl-leukotrienes. The lack of these cardinal features of asthma in MIF(-/-) mice occurs regardless of high concentrations of IL-4 in the lung and OVA-specific IgE in the serum. Antigen-specific lymphocyte proliferation and IL-13 production were similarly increased in the draining lymph nodes of OVA-immunized and challenged MIF(-/-) mice compared to WT, but were reduced in the spleen of MIF(-/-), thus indicating differential roles of MIF in these compartments. Stimulation of naive CD4(+) cells with anti-CD3 antibody demonstrated that MIF(-/-) cells produced increased amounts of IFN-gamma and IL-4 compared to WT CD4(+) cells. Finally, treatment of sensitized BALB/c mice with neutralizing anti-MIF antibody abrogated the development of ARH and airway inflammation without affecting the production of Th2 cytokines or IgE. The present study demonstrates that MIF is required for allergic inflammation, adding important elements to our knowledge of asthma pathogenesis and suggesting that neutralization of MIF might be of therapeutic value in asthma.     

Beta1 integrin is not essential for hematopoiesis but is necessary for the T cell-dependent IgM antibody response

Several experimental evidences suggested that beta1 integrin-mediated adhesion of hematopoietic stem cells (HSC) is important for their function in the bone marrow (BM). Using induced deletion of the beta1 integrin gene restricted to the hematopoietic system, we show that beta1 integrin is not essential for HSC retention in the BM, hematopoiesis, and trafficking of lymphocytes. However, immunization with a T cell-dependent antigen resulted in virtually no IgM production and an increased secretion of IgG in mutant mice, while the response to a T cell-independent type 2 antigen showed decreases in both IgM and IgG. These data suggest that beta1 integrins are necessary for the primary IgM antibody response.     

CXCR3-/- mice mount an efficient Th1 response but fail to control Leishmania major infection

Chemokines play a critical role in recruitment of leukocytes to the site of infection, which is essential for host defense. We analyzed the role of CXC chemokine receptor 3 (CXCR3) in the control of cutaneous leishmaniasis using CXCR3-/- C57BL/6 mice. We found that Leishmania major-infected CXCR3-/- mice mount an efficient Th1 response as evident by markedly increased serum levels of Th1-associated IgG2a and significant production of IFN-gamma and IL-12 by the draining lymph node cells, restrict systemic spread of infection, but fail to control parasite replication at the site of infection and develop chronic non-healing lesions. Furthermore, the inability of CXCR3-/- mice to control cutaneous L. major growth was associated with fewer CD4+ and CD8+ T cells and significantly lower levels of IFN-gamma in their lesions as compared to CXCR3+/+ mice. These results demonstrate that CXCR3 plays a critical role in the host defense against cutaneous leishmaniasis caused by L. major. Furthermore, they also suggest that the susceptibility of CXCR3-/- mice to L. major is due to impaired CD4+ and CD8+ T cell trafficking and decreased production of IFN-gamma at the site of infection rather than to their inability to mount a parasite-specific Th1 response.     

Dendritic cell subsets and the regulation of Th1/Th2 responses

Cells of the dendritic family are suited to perform two distinct functions at two discrete locations. In the peripheral tissues, dendritic cells (DC) act as sentinels for "dangerous" antigens. They then migrate into the lymphoid organ, where they initiate activation of T lymphocytes which are specific for these antigens. During their migration, DC shift from an antigen-capturing mode to a T cell sensitizing mode. In addition to switching on the immune response, subtypes of DC appear to influence the character of T cell differentiation, i.e. the Th1/Th2 balance. We will review the cellular and molecular bases of Th1-Th2 development by DC subsets, and will focus primarily, although not exclusively, on mouse DC.     

Costimulatory molecule OX40L is critical for both Th1 and Th2 responses in allergic inflammation

T cell activation and cytokine secretion are important mediators of inflammation in allergic asthma. The costimulatory pathway CD28/CD80/CD86 has been shown to play an important role in T cell activation in allergic asthma, but less is known about the effect of other costimulatory molecules in allergy. The costimulatory molecule OX40 ligand (OX40L), a member of the tumor necrosis factor superfamily, has been shown to be important in T cell priming and cytokine production. We investigated the role of OX40L in a murine model of allergic inflammation using OX40L(-/-) mice. In this model, following OVA sensitization and challenge, mice develop features of allergic inflammation including elevated levels of total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation. In the absence of OX40L, total serum IgE, pulmonary eosinophils, cytokines, and pulmonary inflammation were all significantly reduced compared to wild-type controls. Levels of eotaxin mRNA, an eosinophil-specific chemoattractant, were also markedly reduced, paralleling the significant reduction in pulmonary eosinophils. Levels of allergen-induced Th1 as well as Th2 cytokines were also significantly reduced. Together, the data support a critical role for OX40L signals in allergic responses.     

The IL-1 receptor 1 is critical for Th2 cell type airway immune responses in a mild but not in a more severe asthma model

IL-1 alpha and IL-1 beta are potent pro-inflammatory cytokines that regulate many physiological systems by binding and signaling to the same receptor termed IL-1 receptor type 1 (IL-1R1). We have investigated the role of IL-1 for pulmonary immune responses in models of allergic asthma using IL-1R1-deficient (IL-1R1(-/-)) mice. In a model of mild asthma, based on repeated sensitization of mice with low doses of ovalbumin in the absence of any adjuvant and multiple intranasal challenges, the pulmonary eosinophilic inflammation and goblet cell hyperplasia were strongly reduced in IL-1R1(-/-) as compared to control BALB/c mice. Moreover, priming of CD4(+) T cells in bronchial lymph nodes and their recruitment to the lung was affected in IL-1R1(-/-) mice associated with impaired antibody responses including IgG, IgE, and IgA. In contrast, sensitization of mice in the presence of alum adjuvant, a more severe asthma model, rendered the IL-1 pathway dispensable for the development of pulmonary allergic Th2 responses, as eosinophilic inflammation, antibody responses, and CD4(+) T cell priming in lymph nodes were comparable between IL-1R1(-/-) and wild-type mice. These results suggest a critical role of IL-1/IL-1R1 for development of allergic Th2 responses, but its requirement can be overcome by using alum as adjuvant for sensitization.     

Signaling lymphocytic activation molecule (SLAM) is differentially expressed in human Th1 and Th2 cells

We have used a real-time quantitative RT-PCR technique (TaqMan, PE Biosystems) to identify genes that are differentially expressed by human polarised CD4(+) T cell subsets (Th1 or Th2). The goal was to test the feasibility of the detection method in profiling the expression of a set of marker genes important for Th1 and Th2 differentiation. We demonstrate that in polarised human Th1 cells signaling lymphocytic activation molecule (SLAM), a member of the immunoglobulin superfamily, is expressed at 7-25-fold higher levels than in Th2 cells. Along with SLAM, expression of the IL-12 receptor chain beta 2 (IL-12R beta 2) and the IFN-gamma receptor chain beta (IFN-gamma R beta) proved to be useful molecular markers indicating the state of T cell polarisation, as previously reported. Treatment with IL-12 increased SLAM mRNA expression in T cells by 3-4-fold, whereas a number of other cytokines including PDGF-BB, IFN-alpha A, IFN-alpha A/D, IFN-beta, IFN-gamma or IL-9 had no effect. Stimulating T cells by co-ligating CD3 and CD28 increased SLAM protein surface expression in both Th1 and Th2 cells. In conclusion, real-time RT-PCR detection was found to be an accurate, sensitive and highly reproducible method for fast profiling of mRNA expression in Th1 and Th2 cell subsets.     

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice

Background and aim: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice.

Material and methods: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-γ as well as Th2 cytokines such as IL-4 and IL-5 were measured.

Results: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-γ. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells.

Conclusion: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.     

Interleukin-12/T cell stimulating factor,a cytokine with multiple effects on T helper type 1 (Th1) but not on Th2 cells

At least two subsets of CD4⁺ T helper cell lymphocytes termed T_h1 and T _h, 2 exist in the mouse and probably in humans. They are characterized by the secretion of different lymphokines and by their functional behavior. Dysregulated expansion of one or the other subset may be one reason for the development of certain diseases. Thus, it is of importance to define the signals involved in the differentiation and activation of the two T_h cell subsets. It is known and has been confirmed in this report that the cytokine interleukin (IL)-1 acts onT_h2 cells but not on T_h1 cells. We now report that a previously identified cytokine which was provisionally termed T cell stimulating factor is identical with IL-12 and exhibits a reciprocal behaviour to IL-1. IL-12 has several effects on T_h1 cells. It can induce the proliferation of certain T_h1 cells in combination with IL-2. Synthesis of interferon (IFN)-γ by T_h1 cells can be triggered by IL-2 plus IL-12. In contrast to the IFN-γ production observed after T cell receptor (TcR) CD3 stimulation of T_h1 cells with lectin Concanavalin A the IFN-γ production induced by IL-12+IL-2 is insensitive to the immunosuppressive drug cyclosporin A. Furthermore, IL-12 enhances the TcR/CD3-induced synthesis of IFN-γ of several T_h1 clones. Finally, IL-12 (+ IL-2) induces homotypic cell aggregation of T_h1 clones. This type of cell aggregation depends on the participation of LFA-1 and ICAM-1 molecules. In all activation systems with T_h1 cells no effect of IL-1 was demonstrable. In contrast, only IL-1 but not IL-12 served as a co-stimulatory signal for several T_h2 cell lines activated via the TcR/CD3 complex.     

RIP2 contributes to Nod signaling but is not essential for T cell proliferation, T helper differentiation or TLR responses

Receptor-interacting protein 2 (RIP2), also known as CARDIAK and RICK, has been reported to play a role in both adaptive T cell responses and innate immunity as a mediator in TLR signaling and nucleotide-binding oligomerization domain (Nod) signaling. Because initial reports remain controversial, we have further examined both innate and adaptive immune responses in RIP2-deficient mice on the C57BL/6 background. Despite the up-regulation of RIP2 after T cell activation, we could not detect any defect in T cell proliferation or Th1/Th2 responses in RIP2-KO mice. Furthermore, we found that TLR responses in RIP2-deficient macrophages were normal. However, our analysis showed that Nod signaling was impaired in macrophages from RIP2-deficient mice. In conclusion, our data demonstrate a critical role for RIP2 in Nod signaling, while T cell proliferation, T helper differentiation and TLR responses were unaffected by the absence of RIP2.     

CD40-CD40 ligand costimulation is not required for initiation and maintenance of a Th1-type response to Leishmania major infection

Although previous studies demonstrated a requirement for CD40-CD40 ligand (CD40L) interaction in the development of resistance to Leishmania infection, we recently showed that mice lacking the gene for CD40L (CD40L(-/-) mice) can control Leishmania major infection when they are infected with reduced numbers of parasites. In this study, we examine the cytokine pattern in healing versus nonhealing CD40L(-/-) mice and investigated whether CD40 activation is required for resistance to reinfection. We observed that CD4(+) cells in healed CD40L(-/-) mice produce high levels of gamma interferon compared to cells from nonhealing, high-dose-inoculated mice. In addition, we observed a higher frequency of interleukin-12 (IL-12)- producing cells and a reduced number of IL-4-producing cells in mice infected with reduced numbers of parasites. Importantly, we found that healed CD40L(-/-) mice are highly resistant to reinfection with a large parasite inoculum. In addition, by comparing the cytokine patterns at an early and late stage of infection in nonhealing CD40L(-/-) mice, we demonstrated that nonhealing CD40L(-/-) mice produce a weak Th1-type response during the early stage of infection, but this response wanes as a Th2-type response emerges during late stages of infection. Anti-IL-4 antibody treatment, starting either at the beginning of infection or at week 4 postinfection enabled CD40L(-/-) mice to control a high-dose infection. Together, these results show that CD40-CD40L interaction, although important for IL-12 production in high-dose infections, is not required for either the development or maintenance of resistance in mice infected with reduced numbers of parasites.     

Delta-like 1 is necessary for the generation of marginal zone B cells but not T cells in vivo

Notch receptors and their ligands contribute to many developmental systems, but it is not apparent how they function after birth, as their null mutants develop severe defects during embryogenesis. Here we used the Cre-loxP system to delete the Delta-like 1 gene (Dll1) after birth and demonstrated the complete disappearance of splenic marginal zone B cells in Dll1-null mice. In contrast, T cell development was unaffected. These results demonstrated that Dll1 was dispensable as a ligand for Notch1 at the branch point of T cell-B cell development but was essential for the generation of marginal zone B cells. Thus, Notch signaling is essential for lymphocyte development in vivo, but there is a redundancy of Notch-Notch ligand signaling that can drive T cell development within the thymus.     

Suppression of Th1 and Th17, but not Th2, responses in a CD8+ T cell-mediated model of oral tolerance

The role of CD8⁺ T cells in oral tolerance remains unclear. To address this, we developed a model to induce CD8⁺ Tregs by feeding the major histocompatibility complex class I immunodominant epitope of OVA, OVA_(257–264). OVA_(257–264) feeding induced tolerance similar to that observed in OVA protein-fed mice, capable of suppressing the production of Th1 and Th17 cytokines and inhibiting a Th1-driven delayed-type hypersensitivity response following immunization with whole OVA (wOVA) protein. OVA_(257–264) peptide-induced suppression could be transferred to naive mice with CD8⁺ cells, but not CD8-depleted cells, isolated from mesenteric lymph nodes of peptide-fed mice. Interestingly, while capable of inhibiting Th1 and Th17 responses, OVA_(257–264) feeding could not suppress any feature of a Th2 inflammatory response, though OVA protein feeding could, suggesting that these cells function through a different mechanism than their CD4⁺ counterparts generated in response to feeding with wOVA. Thus, CD8⁺ T cells are functionally capable of mediating tolerance to Th1 and Th17 responses.     

WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection.

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors. The physiological role of WSX-1, which is expressed mainly in T cells, was investigated in gene-targeted WSX-1-deficient mice. IFN-gamma production was reduced in isolated WSX-1(-/-) T cells subjected to primary stimulation in vitro to induce Th1 differentiation but was normal in fully differentiated and activated WSX-1(-/-) Th1 cells that had received secondary stimulation. WSX-1(-/-) mice were remarkably susceptible to Leishmania major infection, showing impaired IFN-gamma production early in the infection. However, IFN-gamma production during the later phases of the infection was not impaired in the knockout. WSX-1(-/-) mice also showed poorly differentiated granulomas with dispersed accumulations of mononuclear cells when infected with bacillus Calmette-Guerin (BCG). Thus, WSX-1 is essential for the initial mounting of Th1 responses but dispensable for their maintenance.     

Vimentin is required for dengue virus serotype 2 infection but microtubules are not necessary for this process

The present study investigated the effect of microtubules (MTs) and vimentin during dengue virus serotype 2 (DV2) infection. Immunostaining showed that DV2 infection induced MT and vimentin reorganization. Colocalization of DV2 antigens with MTs or vimentin were often observed in ECV304 cells. MT-disrupting agents could enhance DV2 release but did not affect other steps of virus replication. In contrast, disruption of vimentin inhibited DV2 infection. Our results suggest that an MT-dependent mechanism may not be necessary for DV2 infection, and MT disruption may promote DV2 release. However, vimentin is required for DV2 infection.     

Expression of human pim family genes is selectively up-regulated by cytokines promoting T helper type 1, but not T helper type 2, cell differentiation

Cytokines are the most important inducers of T helper (Th) cell differentiation. Interleukin-12 (IL-12) and interferon-alpha (IFN-alpha) are responsible for human Th1-cell differentiation, while IL-4 is the critical cytokine promoting Th2-cell development. These two subsets of cells co-ordinate immunological responses to pathogens as well as autoimmune or allergic reactions. The pim family of proto-oncogenes encodes serine/threonine-specific kinases involved in cytokine-mediated signalling pathways in haematopoietic cells. Here we demonstrate that expression of pim-1 and pim-2 mRNAs is selectively up- or down-regulated in human cord-blood-derived CD4+ cells freshly induced to polarize towards Th1 or Th2 cells, respectively, whereas their expression is inhibited in both cell types by the immunosuppressive transforming growth factor beta (TGF-beta). Moreover, the Th1-specific cytokines IL-12 and IFN-alpha, but not the Th2-specific cytokine IL-4, transiently up-regulate pim-1 and pim-2 mRNA expression in human peripheral blood T cells and natural killer cells. In addition, the Pim-1 protein levels are strongly up-regulated by Th1-specific cytokines in all of these cell types. Taken together, our results suggest that pim genes and their protein products are involved in the early differentiation process of T helper cells.