Human T(H)2 cells respond to cysteinyl leukotrienes through selective expression of cysteinyl leukotriene receptor 1.

BACKGROUND: Allergic asthma is characterized by reversible airway obstruction and bronchial hyperresponsiveness associated with T(H)2 cell-mediated inflammation. Cysteinyl leukotrienes (CysLTs) are potent lipid mediators involved in bronchoconstriction, mucus secretion, and cell trafficking in asthmatic patients. Recent data have implicated CysLTs in the establishment and amplification of T(H)2 responses in murine models, although the precise mechanisms are unresolved. OBJECTIVES: Preliminary microarray studies suggested that human T(H)2 cells might selectively express cysteinyl leukotriene receptor 1 (CYSLTR1) mRNA. We sought to establish whether human T(H)2 cells are indeed a CysLT target cell type. METHODS: We examined the expression of CYSLTR1 using real-time PCR in human T(H)1 and T(H)2 cells. We functionally assessed cysteinyl leukotriene receptor 1 protein (CysLT(1)) expression using calcium flux, cyclic AMP, and chemotaxis assays. RESULTS: We show that human T(H)2 cells selectively express CYSLTR1 mRNA at high levels compared with T(H)1 cells after in vitro differentiation from naive precursors. Human T(H)2 cells are selectively responsive to CysLTs in a calcium flux assay when compared with T(H)1 cells with a rank order of potency similar to that described for CysLT(1) (leukotriene [LT] D(4) > LTC(4) > LTE(4)). We also show that LTD(4)-induced signaling in T(H)2 cells is mediated through CysLT(1) coupled to G(α)q and G(α)i proteins, and both pathways can be completely inhibited by selective CysLT(1) antagonists. LTD(4) is also found to possess potent chemotactic activity for T(H)2 cells at low nanomolar concentrations. CONCLUSIONS: These findings suggest a novel mechanism of action for CysLTs in the pathogenesis of asthma and provide a potential explanation for the anti-inflammatory effects of CysLT(1) antagonists.

Ligation of TLR9 induced on human IL-10-secreting Tregs by 1alpha,25-dihydroxyvitamin D3 abrogates regulatory function.

Signaling through the TLR family of molecular pattern recognition receptors has been implicated in the induction of innate and adaptive immune responses. A role for TLR signaling in the maintenance and/or regulation of Treg function has been proposed, however its functional relevance remains unclear. Here we have shown that TLR9 is highly expressed by human Treg secreting the antiinflammatory cytokine IL-10 induced following stimulation of blood and tissue CD3+ T cells in the presence of 1alpha,25-dihydroxyvitamin D3 (1alpha25VitD3), the active form of Vitamin D, with or without the glucocorticoid dexamethasone. By contrast, TLR9 was not highly expressed by naturally occurring CD4+CD25+ Treg or by Th1 and Th2 effector cells. Induction of TLR9, but not other TLRs, was IL-10 dependent and primarily regulated by 1alpha25VitD3 in vitro. Furthermore, ingestion of calcitriol (1alpha25VitD3) by human volunteers led to an increase of both IL-10 and TLR9 expression by CD3+CD4+ T cells analyzed directly ex vivo. Stimulation of 1alpha25VitD3-induced IL-10-secreting Treg with TLR9 agonists, CpG oligonucleotides, resulted in decreased IL-10 and IFN-gamma synthesis and a concurrent loss of regulatory function, but, unexpectedly, increased IL-4 synthesis. We therefore suggest that TLR9 could be used to monitor and potentially modulate the function of 1alpha25VitD3-induced IL-10-secreting Treg in vivo, and that this has implications in cancer therapy and vaccine design.

Fibronectin is a TH1-specific molecule in human subjects.

BACKGROUND: T(H)1 cell-mediated immunity is essential for host defense against a variety of intracellular pathogens, such as mycobacteria, salmonella, and Leishmania species. A major T(H)1-mediated effector mechanism involves the IFN-gamma-induced killing of the pathogen by infected macrophages. OBJECTIVES: The range of known T(H)1-specific effector molecules is limited, especially in human subjects. We sought to identify novel effector molecules that might be involved in T(H)1-mediated pathogen clearance. METHODS: We performed microarray-based analysis of human T(H)1 and T(H)2 cells to identify T(H)1-specific molecules. These analyses identified the extracellular matrix molecule fibronectin as a highly expressed T(H)1-specific molecule. We examined the expression of fibronectin in a variety of human cell types by using real-time RT-PCR, ELISA, and Western blotting. We also studied the role of fibronectin in modulating monocyte phenotype using in vitro culture. RESULTS: We show that human T(H)1 cells constitutively express and secrete fibronectin after in vitro differentiation from naive precursors. Furthermore, we demonstrate that ex vivo human T(H)1 cells selectively express fibronectin when compared with T(H)2 cells. The predominant isoform of fibronectin expressed by T(H)1 cells contains additional domains of the protein responsible for alpha4beta1 integrin binding and activation of Toll-like receptor 4. We show that treatment of monocytes with T(H)1 cell-derived fibronectin induces expression of the proinflammatory cytokine IL-6 while inhibiting IL-10 expression. CONCLUSIONS: Because fibronectin also plays a major role in the attachment and opsonization of numerous intracellular pathogens, we propose that it might be a critical molecule produced by T(H)1 cells involved in pathogen eradication.

Therapeutic approaches for control of transcription factors in allergic disease.

The inflammatory response observed in allergic disease involves multiple cell types but is orchestrated in part by the T(H)2 cytokines IL-4, IL-5, and IL-13. In recent years, the transcription factors that control the expression and function of these cytokines have been elucidated, including signal transducer and activator of transcription 6, GATA3, nuclear factor of activated T cells, and nuclear factor kappaB. These molecules are attractive targets for therapeutic intervention because they regulate the expression of numerous effector molecules and functions simultaneously. For instance, the immunosuppressive agents glucocorticoids and cyclosporin A both function by repressing the activity of transcription factors through a variety of mechanisms. In this review we examine the role of each transcription factor in allergic disease and discuss approaches that have been taken to therapeutically interfere with transcription factor function in allergic disease.

Human Th2 cells selectively express the orexigenic peptide, pro-melanin-concentrating hormone.

Th1 and Th2 cells represent the two main functional subsets of CD4(+) T helper cell, and are defined by their cytokine expression. Human Th1 cells express IFNgamma, whilst Th2 cells express IL-4, IL-5, and IL-13. Th1 and Th2 cells have distinct immunological functions, and can drive different immunopathologies. Here, we show that in vitro-differentiated human Th2 cells highly selectively express the gene for pro-melanin-concentrating hormone (PMCH), using real-time RT-PCR, enzyme immunoassay, and Western blot analysis. PMCH encodes the prohormone, promelanin-concentrating hormone (PMCH), which is proteolytically processed to produce several peptides, including the orexigenic hormone melanin-concentrating hormone (MCH). PMCH expression by Th2 cells was activation responsive and increased throughout the 28-day differentiation in parallel with the expression of the Th2 cytokine genes. MCH immunoreactivity was detected in the differentiated Th2 but not Th1 cell culture supernatants after activation, and contained the entire PMCH protein, in addition to several smaller peptides. Human Th1 and Th2 cells were isolated by their expression of IFNgamma and CRTH2, respectively, and the ex vivo Th2 cells expressed PMCH upon activation, in contrast to the Th1 cells. Because Th2 cells are central to the pathogenesis of allergic diseases including asthma, expression of PMCH by activated Th2 cells in vivo may directly link allergic inflammation to energy homeostasis and may contribute to the association between asthma and obesity.

Plasmacytoid dendritic cells from human lung cancer draining lymph nodes induce Tc1 responses.

Dendritic cells (DC) resident in draining lymph nodes (LN) of patients with lung cancer are proposed to have a critical role in stimulating anti-tumor immunity. CpG oligodeoxynucleotides are undergoing clinical trials in patients with lung cancer and are likely to target plasmacytoid-DC. The present study, therefore, investigated the capacity of plasmacytoid-DC from human lung cancer draining LN to respond to CpG for activation of T cell responses relevant to anti-tumor immunity. The phenotype of DC was examined by flow cytometry, and cytokine production by cytometric bead array (CBA) and ELISA. Plasmacytoid-DC, purified by cell sorting, were immature but expressed the toll-like receptor, TLR9. Plasmacytoid-DC responded to the CpG oligodeoxynucleotide, CpG 2216, by production of the proinflammatory cytokines, IFN-alpha and IL-6. DC were cocultured with normal, allogeneic T cells, and cytokine production determined by CBA and immunophenotyping. CpG 2216 enhanced IFN-gamma production and induced intracellular production of IFN-gamma by CD8(+) and CD4(+), granzyme B by CD8(+), and IL-2 by CD4(+) T cells, respectively. Ligation of CD40 on plasmacytoid-DC combined with exposure to CpG 2216 also strongly enhanced IFN-gamma production. There was no significant difference between the responses of plasmacytoid-DC from patients with lung cancer and patients with benign carcinoid tumors with no pathologic LN involvement. These results indicate that plasmacytoid DC from the draining LN of patients with lung cancer effectively induce Tc1 immunity and could, therefore, represent a novel and attractive target for immunotherapeutic intervention.

Functional plasticity of human respiratory tract dendritic cells: GM-CSF enhances T(H)2 development.

BACKGROUND: Dendritic cells within the human respiratory mucosa (RTDCs) are proposed to initiate immune responses to foreign antigens. Their capacity to polarize T-cell responses, however, has not been investigated. OBJECTIVE: To compare RTDCs with peripheral blood dendritic cells (PBDCs) with regard to phenotype, cytokine production, capacity to polarize T-cell responses, and effects of exposure to the pleiotropic cytokine, GM-CSF. METHODS: CD1a(+) RTDCs and CD1c(+) PBDCs were purified from nasal turbinates of patients with nonatopic rhinitis and peripheral blood of healthy individuals, respectively. In some experiments, matched CD1c(+) RTDCs and PBDCs from patients with rhinitis were compared. The phenotype of DC was examined by flow cytometry and cytokine production by cytometric bead array. DCs were cocultured with allogeneic naive CD4(+) T cells, and cytokine production was determined by immunophenotyping, cytometric bead array, and ELISA. RESULTS: Both RTDCs and PBDCs exhibited an immature phenotype, but RTDCs expressed lower levels of MHC class II antigen. Cross-linking of CD40 on PBDCs, but not RTDCs, induced production of IL-12p70. In mixed lymphocyte cultures, RTDCs induced a T(H)1/T(H)2 profile, whereas PBDCs induced a T(H)1 profile. Exposure of RTDCs to GM-CSF induced a T(H)2 pattern of response in the mixed lymphocyte cultures. In contrast, exposure of PBDCs to GM-CSF promoted a T(H)1 response. CONCLUSION: This report emphasizes the importance of studying tissue-derived primary DCs, demonstrates functional plasticity of RTDCs, and implicates GM-CSF in amplifying the potential of RTDCs to initiate T(H)2 responses in the airways.