CD40 Ligand Binds to α5β1 Integrin and Triggers Cell Signaling

Walid Mourad,Wen Guo,John A. Wilkins,Rejean Lapointe,Yahye Merhi,Youssef El-Fakhry,Marlène Bouillon,Ehssan Sharif-Askari,Claire Lé;veillé;,Julie Bolduc,Carlos Reyes-Moreno

doi:10.1074/jbc.m608342200

Abstract

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L(-/-) and CD40(-/-) mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an alphaIIbbeta3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40- and alphaIIbbeta3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-alpha5beta1 monoclonal antibody P1D6, and soluble alpha5beta1. The direct binding of sCD40L to purified alpha5beta1 was confirmed in a solid phase binding assay. Binding of sCD40L to alpha5beta1 was modulated by the form of alpha5beta1 expressed on the cell surface as the activation of alpha5beta1 by Mn(2+) or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of alpha5beta1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an alpha5beta1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble alpha5beta1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, alpha5beta1, and alphaIIbbeta3) for CD40L.

Highlights

The CD40 ligand (CD40L),4 known as CD154 or gp39, is a type II transmembrane protein that belongs to the tumor necrosis factor (TNF) superfamily and is expressed on a variety of hematopoietic and non-hematopoietic cells
Our results show that soluble form of biologically active CD40L trimer (sCD40L) binds to CD40-negative human monocytic U937 cells, an interaction that is inhibited by anti-␣5␤1 integrin (VLA-5) mAb as well as by soluble ␣5␤1 integrin (s␣5␤1)
The binding of sCD40L to immobilized purified ␣5␤1 confirms the direct interaction of sCD40L with this integrin. sCD40L induces the translocation of ␣5␤1 to the Triton X-100-insoluble fraction, the rapid activation of MAPKs ERK1/2 in U937 cells, and IL-8 gene expression, confirming the existence of a third functional receptor for CD40L on ␣5␤1

Summary

EXPERIMENTAL PROCEDURES

Cells—The myelomonocytic cell line U937 (ATCC, Manassas, VA) and the B cell lymphoma cell line BJAB Cell Binding Assay—The wells of microtiter plates (Nunc Maxisorp, VWR International Ltd., Mississauga, Ontario, Canada) were coated with gelatin (30 mg/ml in PBS) for 2 h at 37 °C. Solid Phase Binding Assay—The wells of microtiter plates (Nunc Maxisorb) were coated with 4 ␮g/ml of purified s␣5␤1, ␣V␤3, ␣V␤5, or soluble recombinant CD40-Fc in PBS (pH 7.5, 50 ␮l/well) overnight at room temperature. After three washes with PBS-T, rsCD40L was added to the wells at the indicated concentration, and the plate was incubated for 3 h at room temperature. Receptor Translocation to the Cytoskeleton—U937 and BJAB cells were stimulated with rsCD40L (250 ng/5 ϫ 105 cells) for 30 min at 37 °C in binding assay medium and washed three times in PBS. The ratio was blotted as -fold increase of IL-8 mRNA after rsCD40L treatment versus time of the treatment

RESULTS

DISCUSSION

Wilkins and Walid Mourad