Effect of Chimpi, dried citrus peel, on aquaporin-3 expression in HaCaT human epidermal keratinocytes.

BACKGROUND: Chimpi, the dried peel of Citrus unshiu or Citrus reticulata, has various pharmacological effects. Chimpi extract was recently shown to affect the skin, including its inhibitory effect against atopic dermatitis. In this study, we analyzed the effects of Chimpi extract on the functional molecule aquaporin-3 (AQP3), which is involved in water transport and cell migration in the skin. METHODS AND RESULTS: Chimpi extract was added to HaCaT human skin keratinocytes, and the AQP3 expression level was analyzed. A wound healing assay was performed to evaluate the effect of Chimpi extract on cell migration. The components of Chimpi extract and fractions obtained by liquid-liquid distribution studies were added to HaCaT cells, and AQP3 expression was analyzed. Chimpi extract significantly increased AQP3 expression in HaCaT cells at both the mRNA and protein levels. Immunocytochemical staining revealed that Chimpi extract also promoted the transfer of AQP3 to the cell membrane. Furthermore, Chimpi extract enhanced cell migration. Hesperidin, narirutin, and nobiletin did not increase AQP3 levels. Although the components contained in the fractions obtained from the chloroform, butanol, and water layer increased AQP3, the active components could not be identified. CONCLUSIONS: These results reveal that Chimpi extract may increase AQP3 levels in keratinocytes and increase the dermal water content. Therefore, Chimpi extract may be effective for the management of dry skin.

The 1st step initiation essential for allergen-specific IgE antibody production upon the 2nd step: Induction of non-specific IgE+ small B cells containing secondly-sensitized allergen-specific ones in mice firstly-sensitized with an allergen.

There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes.

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.

IL-4-dependent induction of IgE basophils in peripheral blood and IgE B cells in spleen as respective indicators of allergen sensitization and a precursor of cells secreting allergen-specific IgE antibody.

It was recently reported by us that either primary i.n. or i.p. injection of cedar pollen extract into BALB/c mice, or a second s.c. injection of the allergen into i.v. or s.c. sensitized mice, causes an IL-4-dependent increase in total IgE serum antibody to produce allergen-specific IgE antibody upon further s.c. sensitization. To determine the biology of total IgE antibody, in the present study IgE+ cells in peripheral blood or lymphoid tissues of allergen-sensitized BALB/c mice have been characterized. In peripheral blood, mice sensitized one to three times with the allergen produced a 2.5- to 4-fold increase in the number of IgE+ cells, with a time-course similar to that of the concentration of total IgE antibody in serum. These IgE+ cells were basophils. On the other hand, the number of IgE+ cells in the lymphoid tissues did not change significantly after an i.n., i.p., i.v. or s.c. injection of allergen into the mice, whereas a second s.c. injection of the allergen into the i.v.-, but not into the i.n.-, i.p.- or s.c.-, sensitized mice induced a small number of IgE+/IgM+/B220+ B cells in the spleen. In contrast, IgE+ cells were not seen in the blood or spleen of IL-4 -/- mice after sensitization with the allergen. These results suggest that IgE+ basophils in the peripheral blood, and IgE+ B cells in the spleen, might be IL-4-dependently induced as an indicator of sensitization with allergen, and a precursor of cells secreting allergen-specific IgE antibody, respectively.

Essential role of monocytes in the in vitro production of IL-4 and nonspecific IgE antibody by peripheral blood lymphocytes from mice sensitized s.c. once with cedar pollen.

To explore which cytokine or cell is essential for the production of antibodies (Abs) of the IgE class in allergic diseases, we injected cedar pollen into wild-type, interferon-gamma(-/-) (IFN-gamma(/)), or interleukin-4(-/-) (IL-4(-/-)) BALB/c mice through four (i.n., i.p., s.c., and i.v.) different routes without adjuvant. Wild-type or IFN-gamma(-/-), but not IL-4(-/-), mice sensitized once or twice showed a significant increase in total IgE Ab in their serum, revealing the essential role of IL-4 in the production of total IgE Ab. We separated peripheral blood mononuclear cells (PBMCs) from untreated or sensitized mice into monocyte-rich, lymphocyte-rich, and granulocyterich populations by Percoll density-gradient centrifugation or into specific antigen cells by flow cytometry, cultured the cells in various combinations, and examined the levels of cytokines and IgE Ab released into the medium. The PBMCs from mice sensitized s.c. once, but not those from untreated animals, produced significant amounts of IL-4 and total IgE Ab, whereas the lymphocyte-rich population alone did not. Unexpectedly, IL-4 and IgE Ab production was restored by the addition of Mac-1(+) cells in the monocyte-rich fraction to the lymphocyte-rich fraction. These results indicate the essential role of monocytes in the production of IL-4 and total IgE Ab by lymphocytes during the initial stage of sensitization.

IgE production after four routes of injections of Japanese cedar pollen allergen without adjuvant: crucial role of resident cells at intraperitoneal or intranasal injection site in the production of specific IgE toward the allergen.

The production of specific IgE antibodies directed toward cedar pollen correlates well with the onset of allergic rhinitis; but the mechanisms of allergen recognition as nonself and Ig class switch to IgE by the immune system are still not fully understood. In the present study, we injected cedar pollen into mice through 4 different routes (intranasal (i.n.), intraperitoneal (i.p.), intravenous (i.v.), and subcutaneous (s.c.)) without adjuvant 1 to 3 times, and determined time-dependent changes in the total and specific serum IgE levels compared with those in the serum levels of other isotype Igs. After an i.p. or i.n. injection of allergen into the mice, they produced a 1.5-to 1.7-fold increase in total IgE, but none in IgG, IgM, or IgA antibodies in their serum, whereas an i.v. or s.c. injection of allergen was inactive as an inducer of total IgE antibodies. Upon a 2nd (s.c.) injection of the allergen into the i.p. or i.n. sensitized mice, a large amount of allergen-specific IgE antibodies was found in the serum. In the case of i.v. or s.c. sensitized mice, however, they produced total, but not specific, IgE antibodies; and a 3rd (s.c.) injection of the allergen resulted in a large amount of specific IgE antibodies in the serum. These results imply that resident cells at the i.p. or i.n. injection site may play a crucial role in the efficient production of total and specific IgE antibodies toward the allergen.