Lucy A. Gildea

Interleukin‐4‐ and interleukin‐13‐mediated host protection against intestinal nematode parasites

Summary:  Intestinal worm infections characteristically induce T‐helper 2 cell (Th2) cytokine production. We reviewed studies performed with mice infected with either of two intestinal nematode parasites, Nippostrongylus brasiliensis or Trichinella spiralis, that evaluate the importance of the Th2 cytokine interleukin‐4 (IL‐4) and IL‐13 in protection against these parasites. These studies demonstrate that while IL‐4/IL‐13 protect against both parasites by activating signal transducer and activator of transcription 6 (Stat6) through IL‐4 receptor α (IL‐4Rα) ligation, Stat6 activation protects against these parasites through different mechanisms. Stat6‐dependent gene transcription promotes expulsion of N. brasiliensis solely through effects on non‐bone marrow‐derived cells that may include enhancement of intestinal smooth muscle contractility, changes in intestinal epithelial cell function, and increased intestinal mucus secretion. In contrast, Stat6 signaling promotes immunity to T. spiralis both through effects on bone marrow‐derived cells that can be reproduced by treating mice with IL‐4 or IL‐13 and through effects on non‐bone marrow‐derived cells. The former effects appear to include T‐cell‐dependent induction of intestinal mastocytosis, while the latter sensitize non‐bone marrow‐derived cells to mast cell‐produced mediators. We argue that a limited ability of the host immune system to distinguish among different nematode parasites has led to the evolution of a stereotyped Th2 response that activates a set of effector mechanisms that protects against most intestinal nematode parasites.

Selective stimulation of IL-4 receptor on smooth muscle induces airway hyperresponsiveness in mice

IL-4Rα expression on airway smooth muscle cells is sufficient for the development of airway hyperresponsiveness.

Human dendritic cell activity against Histoplasma capsulatum is mediated via phagolysosomal fusion.

ABSTRACT Histoplasma capsulatum is a fungal pathogen that requires the induction of cell-mediated immunity (CMI) for host survival. We have demonstrated that human dendritic cells (DC) phagocytose H. capsulatum yeasts and, unlike human macrophages (Mø) that are permissive for intracellular growth, DC killed and degraded the fungus. In the present study, we sought to determine whether the mechanism(s) by which DC kill Histoplasma is via lysosomal hydrolases, via the production of toxic oxygen metabolites, or both. Phagosome-lysosome fusion (PL-fusion) was quantified by using fluorescein isothiocyanate-dextran and phase and fluorescence microscopy and by electron microscopy with horseradish peroxidase colloidal gold to label lysosomes. Unlike Mφ, Histoplasma-infected DC exhibited marked PL-fusion. The addition of suramin to Histoplasma-infected DC inhibited PL-fusion and DC fungicidal activity. Incubation of Histoplasma-infected DC at 18°C also concomitantly reduced PL-fusion and decreased the capacity of DC to kill and degrade H. capsulatum yeasts. Further, culture of Histoplasma-infected DC in the presence of bafilomycin, an inhibitor of the vacuolar ATPase, did not block DC anti-Histoplasma activity, indicating that phagosome acidification was not required for lysosome enzyme activity. In contrast, culture of Histoplasma-infected DC in the presence of inhibitors of the respiratory burst or inhibitors of NO synthase had little to no effect on DC fungicidal activity. These data suggest that the major mechanism by which human DC mediate anti-Histoplasma activity is through the exposure of yeasts to DC lysosomal hydrolases. Thus, DC can override one of the strategies used by H. capsulatum yeasts to survive intracellularly within Mø.

Overexpression of Interleukin-4 in Lungs of Mice Impairs Elimination of Histoplasma capsulatum

ABSTRACT Protection against the pathogenic fungus Histoplasma capsulatum requires Th1 cytokines. Since interleukin-4 (IL-4) can inhibit both Th1 cytokine production and activity, we examined the effects of overproduction of IL-4 in the lung on the course of pulmonary histoplasmosis. IL-4 lung transgenic mice manifested a higher fungal burden in their lungs, but not spleens, compared to wild-type infected controls. Despite the higher burden, the transgenic animals were ultimately capable of controlling infection. The adverse effects of IL-4 on H. capsulatum elimination were not observed during the early phase of infection (days 1 to 3) but were maximal at day 7 postinfection, prior to the induction of cell-mediated immunity. Analysis of total body and lung cytokine levels revealed that gamma interferon and tumor necrosis factor alpha production were not inhibited in the presence of excess IL-4. Our results with transgenic mice were supported by additional in vivo studies in which allergen induction of pulmonary IL-4 was associated with delayed clearance of H. capsulatum yeast and increased fungal burden. These findings demonstrate that excess production of endogenous IL-4 modulates protective immunity to H. capsulatum by delaying clearance of the organism but does not prevent the generation of a Th1 response that ultimately controls infection.

Examination of Phenotypic Changes in Peripheral Blood‐Derived Dendritic Cells Following Exposure to a Contact Allergen: Cell Surface Marker and Gene Expression

The evaluation of the potential to induce allergic contact dermatitis is an important component of the overall safety assessment process of a new chemical entity that may be encountered through the skin. Currently, the murine local lymph node assay (LLNA) is the method of choice for assessing skin sensitization potential. However, despite the success of the LLNA as an alternative test method that results in a reduction in and refinement of animal usage, it does require the continued use of animals. Therefore, the development of an in vitro predictive test method for skin sensitization is still necessary. A number of different approaches have been taken to develop such a method including the examination of changes in cell surface marker expression in cultured human dendritic cells (DC). The purpose of this current study was to examine the effect of allergen exposure on peripheral blood mononuclear cell (PBMC)‐derived DC using a panel of cell surface markers known to be important in the development of allergic contact dermatitis; CD86, CD80, CD83, CD54, CD40, and CD1a. Changes in the expression of these markers were measured at the level of transcription and cell surface expression. The DC derived from the adherent cell fraction of human PBMC were exposed to either 1 mM or 5 mM dinitrobenzene sulfonic acid (DNBS), the water‐soluble analog of the strong contact allergen 2,4‐dinitrochlorobenzene for 24 hr. After treatment, surface markers expression was measured by flow cytometry, and total RNA was obtained for microarray analysis using Affymetrix U95Av2 Genechips®. Increases in both the cell surface expression and transcript levels of CD86 were observed for PBMC‐DC exposed to DNBS, with 5 mM DNBS inducing the greater change. Cell surface expression of CD80 and CD54 was similar for 1 mM DNBS treated and control cells, and no significant changes were seen at the transcript level. Treatment with 5 mM DNBS resulted in a decrease in cell surface expression for both of these markers. Although a significant allergen‐induced decrease in the transcript level for CD54 was noted, CD80 transcripts were increased. No marked effects on CD83 cell surface or gene expression were observed for either the 1 mM or 5 mM DNBS‐treated cells. Cell surface expression of CD40 and CD1a was somewhat variable in DC preparations treated with 1 mM DNBS, and no significant changes in transcript levels were induced. However, treatment with 5 mM DNBS induced a more marked decrease in cell surface expression of CD40 and CD1a and a significant decrease in the transcript level for CD1a. In summary, we found that changes in cell surface expression in PBMC‐DC following 24‐hr exposure to a contact allergen were reflective of the transcript levels for each of the markers examined with the exception of CD80 in cells treated with 5 mM DNBS.

Transcript Profiling of T Lymphocytes and Dendritic Cells in a Co–culture System Using Anti‐CD3 and Allergen Activation

The interaction of antigen‐specific T lymphocytes with hapten‐bearing dendritic cells (DC) and the subsequent activation and clonal expansion of specific T lymphocyte populations are critical steps in the induction of skin sensitization. Therefore, we have sought to characterize changes in gene expression in T lymphocytes stimulated by incubation with allergen‐treated DC compared with anti‐CD3‐treated T cell‐DC cocultures as a method to identify potential markers of skin sensitization. Human T cells and autologous, mature peripheral blood‐derived DC were co–cultured in the presence or absence of anti‐CD3 monoclonal antibody (mAb) for 6 hours at a 10:1 responder:stimulator ratio. In a separate experiment, autologous DC and T cells from a donor sensitized to the potent contact allergen dinitrochlorobenzene (DNCB) were isolated. T cells were cultured for 6 hours at a responder to stimulator ratio of 20:1 with mature DC that had been treated with either 1 mM 2,4‐dinitrobenzenesulfonic acid (DNBS; the water soluble analog of DNCB), or media alone for 15 minutes. Total RNA was prepared and changes in gene expression were analyzed using Affymetrix U95Av2 GeneChips®. Comparative analysis of Affymetrix mean signal values from triplicate control cultures with those from anti‐CD3‐treated samples revealed highly significant (p ≤ 0.001) changes in the expression of 344 transcripts of the total of approximately 12,000 represented on the chip. However, mean signal values for T cells cocultured with allergen‐treated DC compared to vehicle‐treated DC‐T cell co–cultures identified only 17 significant gene changes (p ≤ 0.001), 11 of which were also identified as having changed significantly in response to stimulation with anti‐CD3. In parallel assays, antigen‐specific T cell proliferative responses were assessed as a function of tritiated thymidine incorporation. Increased T cell proliferative responses were observed in the cultures that contained both DNBS‐treated DC and T cells as well as the anti‐CD3 treated cultures compared with their respective controls. These data suggest that this approach can be used to identify genes that might serve as indicators of contact allergy and may be used in an in vitro predictive assay for skin sensitization.