Ligation of CD40 induces the expression of vascular endothelial growth factor by endothelial cells and monocytes and promotes angiogenesis in vivo.

This study addresses a mechanism by which lymphocytes may promote vascular endothelial growth factor (VEGF) expression and angiogenesis in immune inflammation. Resting human umbilical endothelial cells (HUVECs) were found to express low levels of VEGF messenger RNA (mRNA) by reverse transcription polymerase chain reaction and ribonuclease protection assay with little or no change in expression following activation by cytokines, including tumor necrosis factor-alpha, interleukin (IL)-1, interferon gamma, or IL-4. In contrast, treatment of HUVECs and monocytes with soluble CD40 ligand (sCD40L) resulted in a marked dose-dependent induction of VEGF mRNA (approximately 4-fold), which peaked between 1 and 5 hours post-stimulation. Transient transfection of HUVECs was performed with a luciferase reporter construct under the control of the human VEGF promoter. Treatment of transfected HUVECs with sCD40L was found to enhance luciferase activity (approximately 4-fold) compared with controls, similar to the relative fold induction in mRNA expression in parallel cultures. Thus, CD40-dependent VEGF expression was a result of transcriptional control mechanisms. Treatment of HUVECs with sCD40L was also found to function in vitro to promote growth and proliferation in a VEGF-dependent manner, and CD40-dependent HUVEC growth was comparable to that found following treatment with recombinant human VEGF. Furthermore, subcutaneous injection of sCD40L in severe combined immunodeficient and nude mice induced VEGF expression and marked angiogenesis in vivo. Taken together, these findings are consistent with a function for CD40L-CD40 interactions in VEGF-induced angiogenesis and define a mechanistic link between the immune response and angiogenesis. (Blood. 2000;96:3801-3808)

Endothelial cells modify the costimulatory capacity of transmigrating leukocytes and promote CD28-mediated CD4(+) T cell alloactivation.

Activated vascular endothelial cells (ECs) express major histocompatibility complex (MHC) class II molecules in vitro and in vivo in acute and chronic allograft rejection. However, human ECs may be limited in their ability to effectively activate CD4(+) T cells, because they do not express members of the B7 family (CD80 and CD86) of costimulatory molecules. In this study, we show that ECs promote the full activation of CD4(+) T cells via trans-costimulatory interactions. By reverse transcriptase polymerase chain reaction, Western blot, and FACS((R)) analysis, we could not detect the expression of CD80 and CD86 on activated ECs and found minimal expression on purified CD4(+) T cells. In contrast, both CD80 and CD86 were expressed in allogeneic CD4(+) T cell-EC cocultures. Expression of CD86 peaked at early times between 12 and 24 h after coculture, whereas CD80 was not expressed until 72 h. Addition of anti-CD86 but not anti-CD80 monoclonal antibodies to cocultures inhibited IL-2 production and the proliferation of CD4(+) T cells to allogeneic donor human umbilical vein ECs (HUVECs), as well as to skin and lung microvascular ECs. Furthermore, we found that interferon gamma-activated ECs but not untreated ECs induced mRNA and cell surface expression of CD80 and CD86 on CD4(+) T cells, and these T cells were functional to provide a trans-costimulatory signal to autologous CD4(+) T cells. Blockade of MHC class II and lymphocyte function-associated antigen 3 but not other EC cell surface molecules on IFN-gamma-activated ECs inhibited the induction of CD86 on CD4(+) T cells. Transmigration of purified populations of monocytes across EC monolayers similarly resulted in the induction of functional CD86, but also induced the de novo expression of the cytokines interleukin (IL)-1alpha and IL-12. In addition, EC-modified monocytes supported enhanced proliferation of allogeneic and autologous CD4(+) T cells. Taken together, these data define the ability of the endothelium to modify CD4(+) T cells and monocytes for trans-costimulatory events. This unique function of the endothelium in alloimmune T cell activation has functional consequences for the direct and the indirect pathways of allorecognition.

Human endothelial cell costimulation of T cell IFN-gamma production.

In this report, we show that human endothelial cells (EC) provide costimulatory signals to mitogen-activated CD4+ T cells to augment IFN-gamma production. We also show that EC can enhance responsiveness of the T cells to IL-12. While IL-12 has no effect on IFN-gamma production by cultured CD4+ T cells in the absence of EC, addition of IL-12 to T cell-EC cocultures augments IFN-gamma production by as much as fivefold. Separation of T cells from EC by a semipermeable membrane inhibits the effect of EC and IL-12 on IFN-gamma production. Anti-LFA-3 Abs, in the absence or presence of IL-12, inhibit EC costimulation of IFN-gamma production by up to 50%, while Abs to intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), LFA-1, and very late antigen-4 (VLA-4) have relatively little effect. Pretreatment of T cells with conditioned medium from T cell-EC cocultures, or with IL-2 or IL-1 alpha similarly primes CD4+ T cells for the costimulatory effect of IL-12 on IFN-gamma production. EC costimulation of IFN-gamma production is inhibited by cyclosporine. However, in the presence of IL-12 there is a marked resistance to this inhibitory effect, suggesting that the IL-12-induced costimulatory pathway is distinct from the costimulatory pathway activated by endothelium alone. Our data are consistent with the hypothesis that, as a consequence of interactions with endothelium, T cells that migrate into an inflammatory site are primed to have enhanced responses to Ag and cytokine(s), such as IL-12, that influence T cell-cytokine production.

CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection.

BACKGROUND: CD40 is expressed by a wide variety of cells in the immune system, including endothelial cells. It binds to CD40 ligand ([CD40L] CD154), which was originally reported to be restricted in its expression to early-activated T cells. We report here the expression of CD40 and CD40L in human cardiac allografts. METHODS: A total of 123 consecutive biopsies from 11 human cardiac allograft recipients were analyzed by immunohistochemistry for the expression of CD40 and CD40L. The expression of CD40L was also examined in vitro in homogeneous cultures of umbilical vein endothelial cells by reverse transcriptase-polymerase chain reaction and by flow cytometry. RESULTS: CD40 was expressed at low levels, and CD40L was minimal or absent in histologically normal biopsies in the absence of CD3+ T-cell infiltrates. In rejection, the expression of CD40 increased on vascular endothelial cells and on graft-infiltrating leukocytes throughout biopsy specimens. Induced expression of CD40 was strongly associated with the presence of CD3+ T-cell infiltrates, acute rejection, and ischemic injury (P<0.05). CD40L was expressed in biopsies with rejection and was prominent on a subset of infiltrating leukocytes as well as on microvascular endothelial cells. In contrast to CD40, staining of endothelial CD40L was focal in most biopsies. Overall, the expression of CD40L correlated with the presence of CD3+ T-cell infiltrates and rejection (P<0.05), but not ischemic injury (P=0.9). To confirm that the endothelium can synthesize CD40L, we also evaluated the expression of endothelial CD40L in vitro. Cultured endothelial cells were found to express little constitutive CD40L that markedly increased after 24 hr of treatment with supernatants from phytohemagglutinin-activated peripheral blood mononuclear cells or by the cytokines tumor necrosis factor-alpha, interleukin-1a, interleukin-4, or interferon-gamma. CONCLUSION: Both CD40 and CD40L are expressed in vivo on infiltrating leukocytes and on microvascular endothelium in human cardiac allograft rejection. We suggest that endothelial cell CD40 and CD40L play a role in human cell-mediated immune responses such as cardiac allograft rejection.