The biological outcome of CD40 signaling is dependent on the duration of CD40 ligand expression: reciprocal regulation by interleukin (IL)-4 and IL-12

CD40 ligand (CD154) expression on activated T cells can be separated into an early TCR-dependent phase, which occurs between 0 and 24 h after activation, and a later extended phase, which occurs after 24 h and is reciprocally regulated by the cytokines IL-4 and IL-12. IL-4 represses, whereas IL-12 sustains CD154 expression. Consistent with this, Th1, but not Th2, cells express CD154 for extended periods. Differences in the duration of CD154 expression have important biological consequences because sustained, but not transient, expression of CD154 on activated T cells can prevent B cell terminal differentiation. Thus, the differential ability of Th cells to sustain CD154 expression is an important part of their helper function and should influence the activities of other CD40-expressing cell types.     

Regulation of cytokine signaling by B cell antigen receptor and CD40-controlled expression of heparan sulfate proteoglycans

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell-derived factor (SDF)-1 alpha, even though the affinities of HGF and SDF-1alpha for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation.     

A subset of CD4+ memory T cells contains preformed CD40 ligand that is rapidly but transiently expressed on their surface after activation through the T cell receptor complex

Signaling through surface CD40 is essential for selecting B cells that have mutated their immunoglobulin variable region genes in germinal centers and is an important signal in the early stages of antibody responses to T cell-dependent antigens. It is shown that a subset of CD45RO+, CD4+ T cells isolated from human tonsil contains preformed 30- 35-kD ligand for CD40. This is expressed on their surfaces within 5 min of their antigen-receptor complexes interacting with CD3 epsilon antibodies bound to ox erythrocytes. This surface expression does not require de novo protein synthesis and lasts for only 1-2 h. Preformed CD40 ligand (CD40L) was not detected in any CD4+ CD45RA+ T cells, but > 90% of all CD4+ T cells from the tonsil can be induced to express large amounts of CD40L on culture with phorbol myristate acetate and the calcium ionophore ionomycin. This expression of CD40L starts between 1 and 2 h, peaks at 6 h, and remains at a high level for > 20 h. It is totally prevented by adding a concentration of cycloheximide that inhibits CD25 synthesis by these activated cells. While CD3 epsilon antibody bound to ox red cells is a good inducer of surface expression of CD40L, it is a much less potent inducer of CD40L synthesis than phorbol myristate acetate with ionomycin. Immunohistological analysis of tonsil sections shows that cells containing CD40L are located mainly in the outer zone of germinal centers and the margins of the T zones that are rich in dendritic cells (interdigitating cells). The distribution of these cells is consistent with: (a) their interaction in T zones with B cells that have taken up and processed antigen and (b) their involvement in B cell selection in germinal centers.     

Human B cell clones can be induced to proliferate and to switch to IgE and IgG4 synthesis by interleukin 4 and a signal provided by activated CD4+ T cell clones

In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.     

Lipopolysaccharide-induced decreased protein S expression in liver cells is mediated by MEK/ERK signaling and NFκB activation: involvement of membrane-bound CD14 and toll-like receptor-4

BACKGROUND: The vitamin K-dependent protein S (PS), mainly synthesized in hepatocytes and endothelial cells, plays a critical role in the anticoagulant activity of plasma. The decreased plasma level of PS in sepsis is associated with thrombotic tendency, but the mechanism is unclear. OBJECTIVES: In the present study, we examined the effect of lipopolysaccharide (LPS) on PS expression in vivo in rat liver, and in vitro in isolated hepatocytes and sinusoidal endothelial cells (SECs) from normal rats. RESULTS: LPS induced a progressive decrease of plasma PS antigen level up to 12 h with a slight recovery at 24 h, and a transient decrease of liver PS mRNA level at 4-8 h with a complete recovery at 24 h. In the in vitro studies, LPS decreased PS antigen and mRNA levels in both hepatocytes and SECs. After LPS treatment, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) transiently increased in plasma. IL-6 increased the protein expression of PS from hepatocytes, while TNF-alpha decreased it from SECs. LPS increased CD14 in hepatocytes and decreased it in SECs, but did not affect toll-like receptor-4 (TLR-4) expression in both cells. Antirat CD14 and antirat TLR-4 antibodies inhibited LPS-induced NFkappaB activation, and a NFkappaB inhibitor suppressed LPS-induced decreased PS expression in both cells. Furthermore, MEK inhibitor blocked LPS-induced decreased PS expression in both cells. CONCLUSIONS: These findings suggest that LPS-induced decreased PS expression in hepatocytes and SECs is mediated by MEK/ERK signaling and NFkappaB activation and that membrane-bound CD14 and TLR-4 are involved in this mechanism. These findings may explain in part the decreased level of plasma PS and thrombotic tendency in sepsis.