Galectin-3 is critical for the development of the allergic inflammatory response in a mouse model of atopic dermatitis

Abstract

Galectin-3 belongs to a family of beta-galactoside-binding animal lectins expressed in several cell types, including epithelial and immune cells. To establish the role of galectin-3 in the development of allergic skin inflammation, we compared inflammatory skin responses of galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice to epicutaneous sensitization with ovalbumin (OVA). OVA-treated gal3(-/-) mice exhibited markedly reduced epidermal thickening, lower eosinophil infiltration, and lower serum IgE levels compared with gal3(+/+) mice. The former evoked lower interleukin-4, but higher interferon-gamma, mRNA expression at OVA-treated skin sites. Moreover, gal3(-/-) splenocytes from OVA-sensitized mice secreted more interleukin-12 compared with gal3(+/+) splenocytes. In addition, antigen presentation by gal3(-/-) dendritic cells to T cells in vitro were T helper cell (Th1)-polarized relative to presentation by gal3(+/+) dendritic cells. When exposed to OVA, recipients engrafted with T cells from gal3(-/-) OVA-specific T cell receptor transgenic mice developed significantly reduced dermatitis and a markedly lower Th2 response compared with recipients of comparable gal3(+/+) T cells. We conclude that galectin-3 is critical for the development of inflammatory Th2 responses to epicutaneously administered antigens; in its absence, mice develop a Th1-polarized response. This regulatory effect of galectin-3 on Th development is exerted at both the dendritic cell and T cell levels. Our studies suggest that galectin-3 may play an important role in the acute phase of human atopic dermatitis.

Figure 1

Galectin-3 expression is up-regulated in inflammatory cells in the skin in atopic dermatitis. A: a) Immunohistochemical staining for galectin-3 in normal skin, ×60 objective. (b, c) Immunohistochemical staining for galectin-3 and CD4 in lesional skin from AD patients. Presence of galectin-3+ lymphocytes (arrows, b) in the same vicinity as CD4⁺ lymphocytes (c) in serial sections was noted. Counterstained with hematoxylin. Magnification = ×10. Magnification inset ×60 objective. B: Double immunohistochemical staining for galectin-3 and CD4 in nonlesional (a) and lesional (b) skin from the same AD patient. Scanty CD4⁺ lymphocytes (red) were present in nonlesional skin (a) that were galectin-3-negative (arrow). In contrast, large numbers of CD4⁺ cells were found in lesional skin (arrows, b), and many cells express galectin-3 (brown). Cells co-expressing CD4 and galectin-3 appear reddish-brown (arrows); ×60 objective. Results are representative of five AD patients. C: Immunohistochemical staining for galectin-3 in skin from PBS controls (a) and OVA-treated (b) mice. Significantly larger numbers of galectin-3+ cells (arrows, brown) were present at the OVA-sensitized skin sites compared with PBS-treated sites. Counterstained with hematoxylin, ×10 objective. D: Double immunofluorescence staining for galectin-3 and CD4⁺ in mouse skin treated with OVA. Increased numbers of infiltrating CD4⁺ (green) lymphocytes were present in the dermis. Cells co-expressing galectin-3 (red) and CD4 appear partially yellow (arrows). An expanded region of the boxed area in the left panel is shown in the right panel. Nuclei visualized with Hoechst dye 33342 appear blue, ×20 objective.

Figure 2

OVA-sensitized skin sites of gal3^−−/−- mice show significantly reduced inflammation. A: Measurements of epidermal thickness in H&E-stained tissues. Quantification of the skin cellular infiltrates after Giemsa staining for (B) eosinophils and (C) mononuclear cells. Bars represent mean cell numbers ± SEM (n = 6 gal3^+/+, n = 5 gal3^−/−). *P < 0.05.

Figure 3

Gal3^−/− mice develop a lower cytokine Th2 response, but a higher Th1 response. A: Total IgE and OVA-specific IgE in sera were determined by ELISA. B: The OVA-specific IgG₁ and IgG_2a were determined by ELISA, and ratios of IgG_2a to IgG₁ were calculated. C: Real-time PCR was performed to quantify mRNA. The results were normalized to glyceraldehyde-3-phosphate dehydrogenase. Bars represent mean ± SEM (n = 6 gal3^+/+, n = 5 gal3^−/−). *P < 0.05; **P < 0.01.

Figure 4

Gal3^−/− APCs exhibit a Th1-polarized response. A: Splenocytes from gal3^+/+ and gal3^−/− mice sensitized with OVA as described in Figure 2 were cultured in the presence of OVA (50 μg/ml) for 72 hours. The concentration of IL-12 in the supernatants was determined by ELISA (n = 6 gal3^+/+, n = 5 gal3^−/−). B: CD4⁺ T cells from OVA-TCR tg mice were co-cultured with bone marrow-derived DCs from gal3^+/+ and gal3^−/− mice in the presence of OVA_323–339 peptide for 3 days. The same number of T cells (5 × 10⁵) from the two groups was further stimulated with anti-CD3 and anti-CD28 Ab for 48 hours. The concentrations of cytokines were measured by ELISA. Results are representative of three experiments. Bars represent mean ± SEM. *P < 0.05; **P < 0.01.

Figure 5

A 1-week OVA sensitization induces significant thickening of epidermis in recipients of T cells from OVA-TCR tg mice. OVA-specific gal3^+/+ and gal3^−/− T cell-enriched populations generated in vitro were adoptively transferred into wild-type mice (5 × 10⁶ cells/mouse) and recipient mice were exposed to OVA for 1 week. A: H&E staining of sensitized skin sites (magnification = original ×200). B: Epidermal thickness measurements. Bars represent mean ± SEM (n = 5 animals per group). *P < 0.05. Similar results were obtained in a separate experiment.

Figure 6

Skin sites of recipients of gal3^−/− T cells have markedly decreased eosinophils in the dermis. In vitro-generated OVA-specific gal3^+/+ and gal3^−/− T cell enriched populations (A and B) or purified CD4⁺ T cells (C) were adoptively transferred into wild-type mice (5 × 10⁶ cells/mouse) and recipient mice were exposed to OVA for 1 week. A: Giemsa staining of sensitized skin sites (magnification = original ×400). Arrows indicate eosinophils. B and C: Number of infiltrating cells in sensitized skin sites. Bars represent mean ± SEM (n = 5 animals per group). **P < 0.01; ***P < 0.001. Similar results were obtained in a separate experiment.

Figure 7

Recipients of gal3^−/− T cells exhibit lower IgE levels but a higher IgG_2a/IgG₁ ratio in the serum. Adoptive transfer was conducted as in Figure 5. A: Total IgE levels in sera were determined by ELISA. B: The OVA-specific IgG₁ and IgG_2a were determined by ELISA, and ratios of OVA-specific IgG_2a to IgG₁ were calculated. Bars represent mean ± SEM (n = 5 animals per group). *P < 0.05. Similar results were obtained in a separate experiment.