Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protectiveysuppressive effects

Protectiveysuppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)–Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protectiveysuppressive, are expressed at a high level on almost all CD11b1 BMDMs for 5–8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigenpresenting cells. The results indicate that macrophages of the protectiveysuppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice. After the recognition of HLA DR2 as a haplotype negatively associated with type I diabetis and, therefore, protective against the disease (1), a number of protective major histocompatibility complex (MHC) class II genes have been identified in humans (2) and mice. In the mouse, I-E alleles have often been found to suppress antibody responses and are known to exert an ameliorating effect in collagen-induced arthritis (CIA), a disease that has an obligatory initial T helper type 2 (Th2) phase (3–5). Recently, the I-Ab allele has been found to be suppressive in CIA (5) and several antibody responses (6) while a suppressive effect of the H-2b haplotype on IgG responses has been noted (7, 8) and may be part of the same phenomenon. In contrast, the I-Ad and I-Ak alleles are neutral, i.e., have not been found to exert a suppressive effect on antibody responses, whereas I-Aq is a positive response gene in CIA and has not otherwise been tested for suppressive activity. All of these effects are dominant, in that the presence of a single copy of the gene is sufficient to obtain the suppressiveyprotective effect (4, 9). They represent no more than a bias, because each of these genes can also serve as a positive response gene for other antigens. Differential expression of protective MHC class II molecules in the various antigen-presenting cells (APCs) is one hypothesis that has been proposed to explain the protective effect (2, 5, 10, 11), although other possibilities remain open (3, 12). The findings leading to the present study are as follows. An A 3 G substitution in the X box of the I-Ab gene promoter (compared with I-Ad, I-Ak, and I-Aq) is responsible for heightened signaling in a macrophage cell line that is reversible by site-specific mutagenesis (13). Early interleukin (IL) 4 transcription is strongly down-regulated in the presence of a single I-Ab gene (9), and anti-IL-4 mAb given early in the response mimics the protectiveysuppressive effect of the gene (4). Thus, these findings suggest that the presence of an I-Ab gene results in the release of a cytokine counter to IL-4, presumably IL-12. Meanwhile others have shown that ligation of class II molecules on macrophages elicits prompt release of IL-12, mainly via facilitated ligation of CD40 (14); the same effect can be obtained with monocytes (15) and dendritic cells (16, 17), although there the IL-12 release may occur too late to account for the early suppression of IL-4 production. It therefore seemed likely that the level of expression of an MHC class II molecule might gate this back signal from a T cell to its APC so that heightened or more extensive expression would increase the IL-12 signal and thus exert a protectiveysuppressive effect via Th1 bias (18). To test this hypothesis, the expression of class II molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was measured on fresh splenic macrophages, on activated macrophages represented by bone marrowderived macrophages (BMDMs), on fresh splenic B cells, and on lipopolysaccharide (LPS)-activated splenic B cells. The latter cell type, known to show relatively high expression (19) and not expected to release an IL-4 counter cytokine (20), served as a control. In addition, the cytokine responses of T cells restricted by protective, neutral, or disease-associated haplotypes were investigated. The data presented herein suggest that the differential MHC class II expression observed on BMDMs causes immune deviation among the responding T cells. MATERIALS AND METHODS Mice. The strains used for measuring the time course of MHC II expression on BMDMs were B10.A(5R) (I-Ab), The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y956936-5$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: MHC, major histocompatibility complex; Th, T helper; BMDM, bone marrow-derived macrophage; APC, antigen-presenting cell; CIA, collagen-induced arthritis; IL, interleukin; LPS, lipopolysaccharide; FITC, fluorescein isothiocyanate; FACS, flow cytometry; ABC, antibody-binding capacity; KLH, keyhole limpet hemocyanin; cIg, chicken Ig; mfi, mean fluorescence intensity. *To whom reprint requests should be addressed. e-mail: mueller@ drfz.de. †Bessis, N., Boissier, M.-C., Caput, D., Fradelizi, D. & Fournier, C. Ninth International Congress of Immunology, July 23–29, 1995, p. 898, abstr. 5327.