Endothelial cell activation and proliferation modulate NKG2D activity by regulating MICA expression and shedding.

MICA are major histocompatibility complex class I-related molecules, expressed by endothelial cells (ECs), that may be targets for alloantibodies and NKG2D-expressing natural killer (NK) and T effector cells in organ allografts. This study shows that basal levels of MICA expressed on vascular ECs is sufficient to functionally modulate the expression and activity of the immunoreceptor NKG2D in allogeneic NK cells. We found that MICA expression is differentially regulated at the EC surface in response to cytokines. TNFα upregulates MICA while IFNγ significantly decreases MICA at the EC surface. Both cytokines induce the release of soluble MICA by ECs. Modulation of NKG2D correlates with the MICA level on the EC surface. Glycosylation and metalloproteinase activities account for major post-transcriptional mechanisms controlling MICA level and the function in ECs. Our results indicate that, in addition to the NFκB pathway, the mitogen-activated protein kinase pathways JNK, ERK1/2 and p38 are key signaling pathways in the control of MICA by the cytokines. Finally, we show that EC proliferation mediated by FGF-2 or wound healing increases the MICA level. Together, our data suggest that inflammation and proliferation regulate endothelial MICA expression and shedding, enabling ECs to modulate NKG2D activity on effector NK and T cells, and provide further evidence of a role for ECs in immunoregulation.

Protective cross talk between activated protein C and TNF signaling in vascular endothelial cells: implication of EPCR, noncanonical NF-κB, and ERK1/2 MAP kinases.

Activated protein C (APC) is a natural anticoagulant protease that displays cytoprotective and antiinflammatory activities and has been demonstrated to reduce mortality of patients with severe sepsis. However, APC signaling is not fully understood. This study further investigated the antiinflammatory effects of APC in vascular endothelial cells (EC) and examined the cross talk between APC and TNF signaling. Analysis of the regulatory mechanisms mediated by APC on vascular human EC shows that APC impairs TNF signaling by triggering a preemptive activation of intracellular pathways. We found that APC signaling causes a moderate but significant induction of cell adhesion molecules (CAMs) including VCAM-1 at mRNA and protein levels. Activation of the noncanonical NF-κB and ERK1/2 are both pivotal to APC signaling leading to VCAM-1 expression. APC upregulates TNF receptor-associated factor 2 (TRAF2) and phosphorylates NF-κB p65 at Ser276 and Ser536 independently of IκB degradation. The ultimate protective antiinflammatory effect of APC in response to TNF is associated with a sustained activation of ERK1/2 and Akt while phosphorylation of NF-κB p65 is precluded. Inhibitors of ERK (PD98059 and U0126) abolish the antiinflammatory signal mediated by APC. Blocking antibodies and silencing assays also suggest that, in EC, protease-activated receptor 1 and endothelial protein C receptor (EPCR) both conduct ERK activation and VCAM-1 induction in response to APC. To conclude, APC protects EC by attenuating CAM expression during inflammation. APC engages a regulatory cross talk involving EPCR, ERK, and NF-κB that impairs TNF signaling.

Vascular endothelial cells evade apoptosis triggered by human leukocyte antigen-DR ligation mediated by allospecific antibodies.

BACKGROUND: Human leukocyte antigen (HLA)-DR ligation mediates cell death of antigen-presenting cells (APC), including mature B cells, macrophages, and dendritic cells. This study investigates the apoptotic effects of HLA class II ligation mediated by anti-HLA antibodies on activated human vascular graft endothelial cells (ECs). METHODS: HLA class II expression was examined by flow cytometry using a panel of HLA-typed vascular ECs isolated from transplant donors and compared with that of B lymphocytes. The apoptotic effects of anti-HLA-DR monoclonal antibodies (mAbs) were investigated using viability assays, DNA content analysis, and annexin-V labeling. Intracellular signaling pathways mediated by HLA-DR ligation on ECs were examined by Western blotting. RESULTS: Even with optimal stimulation, the expression of HLA-DR on interferon (IFN)-gamma-treated ECs was quantitatively lower (3-5-fold) than that on B cells. Whereas anti-HLA-DR monomorphic mAbs induced apoptosis of B cells (approximately 22%), no significant apoptosis of IFN-gamma-activated (DR-positive) ECs ( < 5%), collected from the same donor, was observed under the same conditions. Similarly, specific polymorphic anti-HLA-DR11 or -DR16 antibodies were unable to induce EC apoptosis. Nevertheless, antibody-binding to HLA-DR on ECs is sufficient to induce intracellular signaling, as evident in the modulation of tyrosine phosphorylation and protein kinase (PK)C-alpha/beta and PKB/Akt activation. Our results suggest that HLA-DR ligation induces both common and divergent signaling events in ECs and B cells. CONCLUSION: Collectively, our data suggest that, in contrast with professional APC, graft ECs evade apoptosis mediated by HLA-DR ligation, not as a result of moderate HLA-DR expression but rather as a result of a specific signaling pathway.