The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.
SummaryCD40 is a member of the tumor necrosis factor (TNF) receptor family of cell surface proteins and was originally described as a B cell restricted antigen. Treatment of primary human monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or interferon 3' (IFN-3') resulted in the induction of CD40 mRNA and enhancement of cell surface protein expression. CD40 was found to mediate monocyte adhesion to cells expressing recombinant CD40 ligand. CD40 ligand-transfected cells provided a potent costimulus for monocyte TNF-ot and IL-6 production in the presence of GM-CSF, IL-3, or IFN-3', and enhanced IL-8 production stimulated by GM-CSF or IL-3. In addition, CD40 ligand-transfected cells acting in the absence of a costimulus induced monocytes to become tumoricidal against a human mdanoma cell target. Collectively, these data indicate that CD40 ligand is pleiotropic with potent biological activity on monocytes.C D40 is a 50-kD molecule expressed on B cells, dendritic cells, some carcinoma cell lines, and human thymic epithelium (1-3). Abs specific for CD40 exhibit stimulatory effects on IL-4 activated B cells, including the induction of B cell growth, IgE secretion, and expression of CD23 (4-7). Human and murine forms of a ligand for CD40 (CD40L) 1 were recently cloned and demonstrated to be type II integral membrane proteins expressed primarily on activated CD4 + T cells (8)(9)(10)(11). CD40L provides a strong stimulatory signal to B cells from both species (8-10), however little is known about the regulation of expression of CD40 and the effects of CD40L on other cell types.Monocytes express a variety of cell surface proteins that are thought to play an important role in antigen presentation and the cell contact-dependent interaction of monocytes with other leukocytes. The expression of many of these proteins on monocytes can be regulated by a variety of cytokines, including GM-CSF, IL-3, IL-4, IFN-3", and . In this paper, we demonstrate that GM-CSF, IL-3, and IFN-'r enhance expression of CD40 by normal human monocytes. In addition, culture of monocytes with CD40L resulted in the stimulation or costimulation of cytokine production and in the induction of monocyte tumoricidal activity. Thus, CD40L is involved in the regulation of several critical aspects 1 Abbreviation used in this paper: L, ligand. of the immune response, and may be important in the cell contact-dependent activation of both T cells and monocytes during antigen presentation. Materials and MethodsMonocyte Purification and Culture. Monocytes were purified from normal donor PBMC by countercurrent elutriation (17) and were ~95 % pure by microscopic examination of Giemsa-stained cytocentrifuge preparations. Cells were cultured in RPMI 1640 medium containing 10% low endotoxin FBS, 50 U/ml penicillin, 50/zg/ml streptomycin, and 5 x 10 -s M 2-ME. Monocytes were cultured in polypropylene tubes (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) for flow cytometry and mRNA analysis, in 24-well plates (Costar Corp., C...
SummarySignaling through the cell surface molecule, CD40, is known to play an important role in the proliferation and differentiation of B lymphocytes. Using the thymoma cell line EL4, we recently identified and cloned a cDNA encoding a routine ligand for the CD40 molecule (mCD40-L) and showed that it has biological activity in vitro. A cDNA encoding a human homologue of the mCD40-L was isolated using crosshybridization techniques from an activated peripheral blood T cell library. The predicted amino acid sequence indicates that this human ligand for CD40 (hCD40-L) is a 261 amino acid type II membrane protein that exhibits 78% amino acid identity with its murine counterpart. Northern blot and FACS | analyses suggest that the hCD40-L is restricted in its expression to T lymphocytes, and that it is most abundant on the CD4 + T cell subpopulation. Cells transfected with hCD40-L caused the proliferation of human tonsil B cells in the absence of costimuli and, in the presence of interleukin 4, induced immunoglobulin E secretion from purified human B cells. A comparison of the efficacy of the hCD40-L and mCD40-L in these assays is presented.
The vaccinia virus A39R protein is a member of the semaphorin family. A39R.Fc protein was used to affinity purify an A39R receptor from a human B cell line. Tandem mass spectrometry of receptor peptides yielded partial amino acid sequences that allowed the identification of corresponding cDNA clones. Sequence analysis of this receptor indicated that it is a novel member of the plexin family and identified a semaphorin-like domain within this family, thus suggesting an evolutionary relationship between receptor and ligand. A39R up-regulated ICAM-1 on, and induced cytokine production from, human monocytes. These data, then, describe a receptor for an immunologically active semaphorin and suggest that it may serve as a prototype for other plexin-semaphorin binding pairs.
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