Total Serum IgE and Latex Specific IgG
There was a significant increase in total serum IgE levels in animals exposed to latex antigens as compared to controls (Fig. 1A). This increase was arrested in mice challenged with latex and treated with curcumin. However, the difference between curcumin treated and untreated mice was not statistically significant.
Latex specific IgG1, IgG2a, IgG2b, and IgG3 was readily detected in the serum of latex-sensitized mice (Fig. 1C). This increase in specific antibody was several fold greater than the baseline antibody levels detected in unexposed controls. The control mice treated with curcumin only (no latex sensitization) showed only baseline values.
Peripheral Blood Eosinophils
The numbers of eosinophils in the peripheral blood of latex-sensitized mice were markedly elevated as compared to PBS controls (Fig. 1B). Notably, curcumin treatment significantly decreased the numbers of eosinophils in latex sensitized mice (p
Antigen Induced Lymphocyte Stimulation
Latex allergens (MNA) were unable to induce the proliferation of spleen cells from latex sensitized mice (data not shown), possibly due to toxicity of the latex extract preparation [19]. In contrast, the recombinant latex allergen, Hev b 6 was used to stimulate spleen cells, enhanced proliferation was detected in cells from latex antigen exposed mice (data not shown). Curcumin treatment of latex sensitized mice only marginally affected Hev b 6 induced lymphocyte proliferation (data not shown). Concanavalin-A induced stimulation of lymphocytes from latex challenged mice showed reduced proliferation as compared to controls, and this decreased proliferation was partially restored in latex challenged mice treated with curcumin (results not shown).
Cytokine Production by Spleen Cells
Cytokines were not detected in the culture supernatants of cultured spleen cells from curcumin treated or PBS treated control mice (Fig. 1D &1E). Spleen cells stimulated with Hev b 6 also failed to produce detectable levels of cytokines. Varying levels of cytokines were detected in the culture supernatants of antigen stimulated cells and from latex sensitized mice treated with curcumin. Reduced levels of IFN-γ were detected from cells of latex sensitized mice compared to cells from normal mice, while increased amounts of IFN-γ were produced from the cells of mice challenged with latex and treated with curcumin (Fig. 1D). Although, IL-4 production was only slightly reduced in culture supernatants from latex sensitized mice treated with curcumin (Fig. 1E), overall the cytokine profiles indicated that curcumin shifted the latex-induced Th2 response towards a Th1 type of response. No major differences were detected for IL-5, IL-10, and IL-13 (results not shown).
Analysis of Lung Tissue
Messenger RNA was isolated from the lung tissue and studied for Ly75 (CD205), MMP9, OAT and TSLP expression. The results indicated that there were diverse responses in the different groups of mice (Fig. 2). Expression of OAT, MMP9, and Ly75 (CD205) were increased in latex sensitized mice as compared to untreated PBS controls, while TSLP levels were similar. There was a marked reduction in the expression of all four genes in latex sensitized mice treated with curcumin.
Flow Cytometric Analysis of Lung Cells
Expression of costimulatory molecules on lung cells from experimental mice were examined by flow cytometry, including CD28, OX40, and CTLA-4 on T-cells, and CD80, CD86, and OX40L on B cells and macrophages. A representative histogram shown in panel A1 of Figure 3 depicts CD80 expression on lung B cells from the different treatment groups. Increased percentages of B cells expressing CD80 were detected in the lungs of latex-sensitized mice as compared to PBS controls (Fig. 3A2). Curcumin treatment reduced the expression of CD80 on lung B cells of latex-sensitized mice (Fig. 3A1), and the reduced expression was reflected by decreased percentages of lung B cells expressing CD80 (Fig. 3A2) and decreased CD80 median fluorescence values (Fig. 3A3). Lung B cells from mice sensitized with latex and treated with curcumin also showed reduced CD86 and OX40L expression as compared to lung B cells from latex-sensitized mice (Fig. 3B and 3C, respectively), and the expression of CD80, CD86, OX40L on lung macrophages exhibited a similar pattern (Fig. 3D–F). Finally, percentages of lung CD4+ T cells expressing OX40 and OX40 median fluorescence values were decreased in latex-sensitized/curcumin treated mice as compared to latex-sensitized mice (Fig. 3G). Curcumin treatment also resulted in slightly reduced percentages of lung CD4+ T cells expressing CTLA-4 (21.6% vs. 29.3% in latex-sensitized mice as compared to 7.6% in control mice), while no differences were observed in regard to CD28 expression (data not shown).
Lung Histology
Mice sensitized with latex antigen showed significant interstitial inflammation with peribronchiolar and perivascular infiltrates (Fig. 4). The inflammatory cells primarily consisted of small lymphocytes with plasma cells and epitheloid histiocytes. In PAS stained sections, increased numbers of bronchial epithelial cells, particularly PAS positive goblet cells were discernable (Fig. 4B, E, F &4I). Bronchial epithelial cell hyperplasia was predominant in latex challenged mice (Fig. 4C). A marked increase in eosinophils was evident in latex challenged mice (Fig. 4C &4D), but was considerably reduced in latex challenged mice treated with curcumin (Fig. 4G &4H). In the curcumin treated, latex sensitized mice there was only minimal perivascular edema and moderate perivascular cuffing with infiltration of neutrophils and mononuclear cells. These mice also had fewer lesions consistently devoid of eosinophils, although some neutrophils and mononuclear cells were discernible (Fig. 4G &4H). There was also less bronchial epithelial hyperplasia and no goblet cells (Fig. 4G, H &4I). Control mice treated with PBS and curcumin showed normal lung pathology (Fig. 4A &4B).
Immunohistochemistry
Lung tissue sections were stained for IL-4, IL-5, IL-10, IL-13 and IFN-γ. Cytokine producing cells in the lungs of PBS-treated control mice could only be detected in low numbers for IL-10 and IFN-γ (Fig. 5D). There was a marked increase in lung cells secreting IL-4, IL-5, IL-13, and IFN-γ in the latex-challenged mice (Fig. 5A, B, C, D). Curcumin treatment of latex sensitized mice resulted in a marked decrease in IL-4, IL-5, and IL-13 expressing cells (Fig. 5A, B &5C).
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