SummaryThe interaction between activated vascular endothelium and T cells has been shown to play an important role in the recruitment and activation of T cells at sites of inflammation. Here we report the expression of CD40 by vascular endothelial cells and its regulation by inflammatory agents. Using the soluble recombinant CD40 ligand, sgp39, we show that the interaction of CD40 with its ligand can lead to endothelial cell activation, which in turn leads to leukocyte adhesion. This adhesion is partly mediated by the expression of E-selectin. In addition to E-selectin expression, ~gp39 induces the expression of intercellular adhesion molecule 1 and augments the tumor necrosis factor cz-induced expression of vascular cell adhesion molecule 1. The effects of sgp39 on endothelial cells can be blocked with anti-gp39 monoclonal antibody (mAb), anti-CD40 mAb, or soluble CD40. Staining of tissues from healthy human skin using anti-CD40 mAb showed very weak expression of CD40 by the endothelium, while skin involved in inflammatory disease showed marked upregulation of CD40 expression. These studies suggest that interactions between cell surface proteins expressed by activated T cells with their receptors on vascular endothelium can stimulate the vasculature at sites of inflammation and may be involved in normal inflammatory responses and in inflammatory disease. C D40, a 50-kD glycoprotein expressed by a range of cell types of the immune system, functions as a signaling protein. It was initially described and studied on B cells, where stimulation with anti-CD40 mAb was shown to induce proliferation and isotype switching in the presence of an appropriate costimuli. For example, early studies demonstrated that anti-CD40 mAbs are synergistic with PMA, anti-CD20 mAb, or IL-4 for the induction of B cell proliferation (1-4). Further studies have shown that anti-CD40 treatment with IL-4 as the costimulator results in the secretion of IgE, IgM, IgA, and soluble CD23 (4-8). With IL-10 as the costimulator, anti-CD40 treatment results in the secretion of IgA, IgM, and IgG (9, 10). In addition, anti-CD40 presented by CD32-transfected L cells in combination with IL-10 and TGF-3 results in the production of IgA from slgD § B cells (9). CD40 is also expressed and functional on several other cell types. Monocytes respond to anti-CD40 or CD40 ligand (gp39) in conjunction with GM-CSF, IL-3, or IFN-3' by producing cytokines (11). Thymic epithelium has also been shown to express CD40, and stimulation with anti-CD40 in conjunction with IFN-3, and IL-1 leads to secretion of GM-CSF (12). CD40 is expressed by normal basal epithelium, follicular dendritic cells, and some carcinoma-and melanoma-derived cell lines (13)(14)(15)(16) been found that T cells express CD40 and respond to gp39 by the proliferation and expression of cytokines and activation markers (17), and dendritic Langerhans cells respond to ligation of CD40 by altering their morphology and surface molecule and cytokine expression (18). The molecular steps leading to the recruitment...

CD40 and CD40 ligand (gp39) mediate contact-dependent T-B cell interaction. We determined the expression of CD40 ligand by activated neonatal T cells and the response of neonatal B cells when activated through CD40. Although expression of CD40 ligand peaked simultaneously in both activated adult and neonatal cells, neonatal T cells expressed significantly less CD40 ligand surface protein and mRNA than adult T cells. Activated thymocytes also expressed far less CD40 ligand than adult T cells. Consistent with these results, activated neonatal T cells exhibited less helper function than activated adult T cells. Neonatal T cells primed and restimulated in vitro expressed CD40 ligand in amounts comparable with adult T cells and provided B cell help more effectively. This suggests that the poor expression of CD40 ligand reflects antigenic naiveté rather than an intrinsic defect of neonatal T cells. Neonatal B cells cultured with soluble CD40 ligand (sgp39) and IL-10 produced IgM in amounts comparable with adult cells, but much less IgG and IgA. Nevertheless, neonatal B cells were capable of proliferation and class switching, since sgp39 and IL-4 induced proliferation and IgE production comparable to adult B cells and production of modest amounts of IgG. Together, these results indicate that diminished CD40 ligand expression, along with decreased production of lymphokines, may be responsible, at least in part, for the transient immunodeficiency observed in human neonates.

Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhesion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I-domain of the LFA-1 alpha subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I-domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding. In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ in human and murine I-domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated. Substitution of alanine for proline at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this was due to conformational changes within the I-domain. Mutation of the aspartic acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1.

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Exposure of the Fu5 rat hepatoma cell line to glucocorticoids, such as dexamethasone and hydrocortisone, suppressed the growth rate and final density of cells grown in the presence of serum. This hormonal effect was proportional to receptor occupancy and affinity and, in addition, the glucocorticoid antagonist RU38486 prevented this response. Two classes of dexamethasone-resistant variants that failed to be growth inhibited were recovered from ethyl methylsulfonate-mutagenized populations by continuous culture in the presence of 1 microM dexamethasone. The first class, represented by the EDR3 subclone, was completely glucocorticoid unresponsive and failed to express receptor transcripts. The second class, represented by the EDR1, EDR5, and EDR7 subclones, possessed significant levels of glucocorticoid receptor but were only partially glucocorticoid responsive when stimulated with saturating levels of hormone. Introduction of functional glucocorticoid receptor genes into both classes of dexamethasone-resistant variants by a recombinant retrovirus expression vector restored glucocorticoid responsiveness and suppression of cell growth. A hypersensitive variant (BDS1), recovered by bromodeoxyuridine selection, was fully glucocorticoid responsive, and its inhibition of proliferation was more acutely regulated by dexamethasone. Taken together, our results established that the inhibition of proliferation in Fu5 rat hepatoma cells represents a new glucocorticoid response that requires the expression of a functional glucocorticoid receptor.