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.

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.

Cytotoxic T lymphocyte (CTL)3 markers (i.e., perforin and Fas ligand) are always present in biopsies of tissues undergoing rejection but are absent in those of nonrejected tissue (35). Allo-antigen-specific CTLs can be isolated from allografts being rejected but not from biopsies that show no rejection (9). The adoptive transfer of alloreactive CTLs into SCID mice can cause allograft rejection and the adoptively transferred CTLs can be recovered from grafts undergoing rejection (41). Similarly, in several well-studied viral model systems, splenic CD8 ϩ T cells with strong cytotoxic activity are readily detected 6 to 8 days after primary acute infection (16 Abstract: CD8؉ cytotoxic T lymphocytes (CTLs) generated by immunization with allogeneic cells or viral infection are able to lyse allogeneic or virally infected in vitro cells (e.g., lymphoma and mastocytoma). In contrast, it is reported that CD8 ؉ T cells are not essential for allograft rejection (e.g., heart and skin), and that clearance of influenza or the Sendai virus from virus-infected respiratory epithelium is normal or only slightly delayed after a primary viral challenge of CD8-knockout mice. To address this controversy, we gen-

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.

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