Martin S. Kluger

Human Endothelial Cell Presentation of Antigen and the Homing of Memory/Effector T Cells to Skin

Abstract: Dermal microvascular endothelial cells (ECs) form a continuous lining that normally bars blood‐borne T lymphocytes from entering the skin, but as part of the response to foreign antigen, dermal ECs undergo alterations in their surface proteins so as to provide signals to circulating T cells that lead to their activation and recruitment. Several observations suggest that human dermal microvascular ECs may help initiate cutaneous immune reactions by presentation of cognate antigens to circulating T memory cells: (1) antigen‐specific inflammatory responses in the skin, as in other organs, involve accumulation of memory and effector T cell populations that are enriched in cells specific for the eliciting antigen; (2) recall responses to intradermal protein antigens in the skin start very rapidly within two hours of challenge; (3) dermal microvascular ECs in humans and other large mammals basally display high levels of class I and class II MHC molecules, the only known purpose of which is to present antigenic peptides to lymphocytes; (4) the lumen of dermal capillaries are narrower than the diameter of circulating T cells, ensuring surface contact; and (5) cultured human ECs effectively present antigens to resting memory T cells isolated from the circulation. Upon contact with activated T cells or their secreted products (cytokines), dermal ECs themselves become activated, increasing their capacity to recruit memory and effector T cell populations in an antigen‐independent manner. Specifically, activated ECs express inducible leukocyte adhesion molecules such as E‐selectin, ICAM‐1, and VCAM‐1; and several lines of evidence, including neutralizing antibody experiments and gene knockouts, have supported a role of these molecules in T cell recruitment. Dermal ECs have unique expression patterns of adhesion molecules that can determine the subsets of memory T cells that are recruited into the skin. For example, slow internalization of E‐selectin allows more persistent expression of this protein on the surface of dermal ECs, favoring interactions with CLA‐1+ T cells. VCAM‐1 expression, normally confined to venular EC may extend to capillaries within the dermal papillae and contribute to epidermal inflammation, recruiting α4β7 integrin‐expressing T cells that also express the cadherin‐binding integrin αEβ7. New models involving transplantation of normal and genetically modified human dermal ECs into immunodeficient mice may be used to further explore these properties.

Cytoprotection of Human Umbilical Vein Endothelial Cells Against Apoptosis and CTL-Mediated Lysis Provided by Caspase-Resistant Bcl-2 Without Alterations in Growth or Activation Responses

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-κB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Interferon-γ Augments CD95(APO-1/Fas) and Pro-Caspase-8 Expression and Sensitizes Human Vascular Endothelial Cells to CD95-Mediated Apoptosis

We have examined the effects of interferon (IFN)-gamma on expression and function of CD95 (APO-1/Fas) and associated proteins in cultured human umbilical vein and dermal microvascular endothelial cells (HUVEC and HDMEC, respectively). Unstimulated cells express only low levels of CD95; IFN-gamma produces a time- and concentration-dependent increase of CD95 in both cell types at the mRNA and cell surface protein levels. IFN-gamma also produces an increase in expression of pro-caspase-8 (FLICE/MACH) but does not significantly change expression of either Fas-associated death domain (FADD) protein or cellular FLICE inhibitory protein (cFLIP), other proteins associated with the CD95 death-inducing signaling complex (DISC). Neither resting nor IFN-gamma-treated EC express detectable CD95L mRNA or protein. Untreated HUVEC and HDMEC show minimal apoptosis when transduced to express CD95L. Treatment of CD95L-transduced cells with IFN-gamma causes apoptosis within 24 to 36 hours that can be blocked by antagonistic anti-CD95 antibody or by the caspase-inhibitory peptide zVAD-FMK. The extent of apoptosis is increased by co-treatment with either the protein synthesis inhibitor cycloheximide or the phosphatidylinositol 3-kinase inhibitor LY294002. Untransduced HUVEC treated with IFN-gamma also undergo CD95-initiated apoptosis when mixed with CD95L-transduced HUVEC or when incubated with pharmacologically activated cytolytic T lymphocytes. Overexpression of CD95 in HUVEC confers sensitivity to CD95L in the absence of IFN-gamma-treatment. We conclude that IFN-gamma induces sensitivity of endothelium to CD95L-mediated apoptosis, and that this response may result from increased expression of CD95 and/or pro-caspase-8.

Heparin Displaces Interferon-γ–Inducible Chemokines (IP-10, I-TAC, and Mig) Sequestered in the Vasculature and Inhibits the Transendothelial Migration and Arterial Recruitment of T Cells

Background— Heparin, used clinically as an anticoagulant, also has antiinflammatory properties and has been described to inhibit interferon (IFN)-&ggr; responses in endothelial cells. We investigated the effects of heparin on the IFN-&ggr;–inducible chemokines IP-10/CXCL10, I-TAC/CXCL11, and Mig/CXCL9, which play important roles in the vascular recruitment of IFN-&ggr;–producing Th1 cells through interactions with their cognate receptor, CXCR3. Methods and Results— Patients undergoing coronary artery bypass grafting were studied because coronary atherosclerosis is recognized as a Th1-type inflammatory disease and the subjects required systemic heparinization. Plasma levels of IP-10, I-TAC, and Mig increased immediately after heparin administration and diminished promptly after heparin antagonism with protamine. These effects were independent of detectable circulating IFN-&ggr; or the IFN-&ggr; inducer interleukin-12. We confirmed previous reports that heparin inhibits the IFN-&ggr;–dependent production of CXCR3 chemokine ligands using atherosclerotic coronary arteries in organ culture. In addition to prolonged treatment decreasing chemokine secretion, heparin rapidly displaced membrane-associated IP-10 from cultured endothelial cells that did not express CXCR3 and reduced the IP-10–dependent transendothelial migration of T helper cells under conditions of venular shear stress. Finally, heparin administration to immunodeficient mouse hosts decreased both the recruitment and accumulation of memory T cells within allogeneic human coronary arteries. Conclusions— Besides inhibiting IFN-&ggr; responses, heparin has further immunomodulatory effects by competing for binding with IP-10, I-TAC, and Mig on endothelial cells. Disruption of CXCR3+ Th1 cell trafficking to arteriosclerotic arteries may contribute to the therapeutic efficacy of heparin in inflammatory arterial diseases, and nonanticoagulant heparin derivatives may represent a novel antiinflammatory strategy.

Lymphotoxin-α1β2 and LIGHT Induce Classical and Noncanonical NF-κB-Dependent Proinflammatory Gene Expression in Vascular Endothelial Cells

Activation of the classical and noncanonical NF-κB pathways by ligation of the lymphotoxin (LT)-β receptor (LTβR) plays a crucial role in lymphoid organogenesis and in the generation of ectopic lymphoid tissue at sites of chronic inflammation. Within these microenvironments, LTβR signaling regulates the phenotype of the specialized high endothelial cells. However, the direct effects of LTβR ligation on endothelial cells remain unclear. We therefore questioned whether LTβR ligation could directly activate endothelial cells and regulate classical and noncanonical NF-κB-dependent gene expression. We demonstrate that the LTβR ligands LIGHT and LTα1β2 activate both NF-κB pathways in HUVECs and human dermal microvascular endothelial cells (HDMEC). Classical pathway activation was less robust than TNF-induced signaling; however, only LIGHT and LTα1β2 and not TNF activated the noncanonical pathway. LIGHT and LTα1β2 induced the expression of classical NF-κB-dependent genes in HUVEC, including those encoding the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Consistent with this stimulation, LTβR ligation up-regulated T cell adhesion to HUVEC. Furthermore, the homeostatic chemokine CXCL12 was up-regulated by LIGHT and LTα1β2 but not TNF in both HUVEC and HDMEC. Using HUVEC retrovirally transduced with dominant negative IκB kinase α, we demonstrate that CXCL12 expression is regulated by the noncanonical pathway in endothelial cells. Our findings therefore demonstrate that LTβR ligation regulates gene expression in endothelial cells via both NF-κB pathways and we identify CXCL12 as a bona fide noncanonical NF-κB-regulated gene in these cells.

Knockdown of TNFR1 by the sense strand of an ICAM-1 siRNA: dissection of an off-target effect

Tumor necrosis factor (TNF) initiates local inflammation by triggering endothelial cells (EC) to express adhesion molecules for leukocytes such as intercellular adhesion molecule-1 (ICAM-1 or CD54). A prior study identified siRNA molecules that reduce ICAM-1 expression in cultured human umbilical vein EC (HUVEC). One of these, ISIS 121736, unexpectedly inhibits TNF-mediated up-regulation of additional molecules on EC, including E-selectin (CD62E), VCAM-1 (CD106) and HLA-A,B,C. 736 siRNA transfection was not toxic for EC nor was there any evidence of an interferon response. 736 Transfection of EC blocked multiple early TNF-related signaling events, including activation of NF-κB. IL-1 activation of these same pathways was not inhibited. A unifying explanation is that 736 siRNA specifically reduced expression of mRNA encoding tumor necrosis factor receptor 1 (TNFR1) as well as TNFR1 surface expression. A sequence with high identity to the 736 antisense strand (17 of 19 bases) is present within the 3′UTR of human TNFR1 mRNA. An EGFP construct incorporating the 3′UTR of TNFR1 was silenced by 736 siRNA and this effect was lost by mutagenesis of this complementary sequence. Chemical modification and mismatches within the sense strand of 736 also inhibited silencing activity. In summary, an siRNA molecule selected to target ICAM-1 through its antisense strand exhibited broad anti-TNF activities. We show that this off-target effect is mediated by siRNA knockdown of TNFR1 via its sense strand. This may be the first example in which the off-target effect of an siRNA is actually responsible for the anticipated effect by acting to reduce expression of a protein (TNFR1) that normally regulates expression of the intended target (ICAM-1).

MEK5 is Activated by Shear Stress, Activates ERK5 and Induces KLF4 to Modulate TNF Responses in Human Dermal Microvascular Endothelial Cells

Please cite this paper as: Clark, Jensen, Kluger, Morelock, Hanidu, Qi, Tatake, Pober (2011). MEK5 is Activated by Shear Stress, Activates ERK5 and Induces KLF4 to Modulate TNF Responses in Human Dermal Microvascular Endothelial Cells. Microcirculation18(2), 102–117.

Claudin-5 Controls Intercellular Barriers of Human Dermal Microvascular but Not Human Umbilical Vein Endothelial Cells

Objective—To assess the role claudin-5, an endothelial cell (EC) tight junction protein, plays in establishing basal permeability levels in humans by comparing claudin-5 expression levels in situ and analyzing junctional organization and function in 2 widely used models of cultured ECs, namely human dermal microvascular (HDM)ECs and human umbilical vein (HUV)ECs. Methods and Results—By immunofluorescence microscopy, ECs more highly express claudin-5 (but equivalently express vascular endothelial-cadherin) in human dermal capillaries versus postcapillary venules and in umbilical and coronary arteries versus veins, correlating with known segmental differences in tight junction frequencies and permeability barriers. Postconfluent cultured HDMECs express more claudin-5 (but equivalent vascular endothelial-cadherin) and show higher transendothelial electric resistance and lower macromolecular flux than similarly cultured HUVECs. HDMEC junctions are more complex by transmission electron microscopy and show more continuous claudin-5 immunofluorescence than HUVEC junctions. Calcium chelation or dominant negative vascular endothelial-cadherin overexpression decreases transendothelial electric resistance and disrupts junctions in HUVECs, but not in HDMECs. Claudin-5 overexpression in HUVECs fails to increase transendothelial electric resistance or claudin-5 continuity, whereas claudin-5 knockdown in HDMECs, but not in HUVECs, reduces transendothelial electric resistance and increases antibody accessibility to junctional proteins. Conclusion—Claudin-5 expression and junctional organization control HDMEC and arteriolar-capillary paracellular barriers, whereas HUVEC and venular junctions use vascular endothelial-cadherin.

IL-10 inhibits endothelium-dependent T cell costimulation by up-regulation of ILT3/4 in human vascular endothelial cells

Effects of IL‐10 on endothelium‐dependent T cell activation have not been investigated in detail. We confirm expression of the IL‐10 receptor and effective signaling via STAT‐3 in human umbilical vein endothelial cells (HUVEC). In CD4 T cell cocultures with HUVEC, pretreatment of endothelial cells with IL‐10 resulted in significant dose‐dependent inhibition of CD4 T cell proliferation, which also occurred when IL‐10 was removed after pretreatment before starting cocultures. Th1/Th2 polarization of proliferated T cells, endothelial nitric oxide (NO), or IL‐12 production were unchanged. However, IL‐10 stimulation resulted in up‐regulation of SOCS‐3, a negative regulator of cytokine secretion, and induction of the inhibitory surface molecules immunoglobulin‐like transcript 3 and 4 (ILT3/ILT4) in EC, potentially involving glucocorticoid‐induced leucine zipper (GILZ). Addition of blocking antibodies against ILT3/ILT4 to EC/T cell cocultures resulted in nearly complete reestablishment of T cell proliferation. In contrast, addition of soluble ILT3 or overexpression of ILT3 in cocultures significantly reduced T cell proliferation. No induction of foxp3+ regulatory T cells was seen. In conclusion, the T cell costimulatory potential of human EC is markedly suppressed by IL‐10 due to up‐regulation of ILT3/ILT4, obviously not involving generation of Treg. This identifies a novel action of IL‐10 in EC and a potential therapeutical target for local immunomodulation.

Regulation of arterial-venous differences in tumor necrosis factor responsiveness of endothelial cells by anatomic context

We analyzed tumor necrosis factor (TNF) responses of human umbilical artery and vein endothelial cells (HUAECs and HUVECs) in organ and cell culture. In organ culture, TNF induced expression of E-selectin, VCAM-1, and ICAM-1 on HUVECs but only ICAM-1 on HUAECs. Activation of nuclear factor-kappaB, c-jun, and ATF2 by TNF was comparable in HUAECs and HUVECs, whereas binding of transcription factors and p300 co-activator to the E-selectin enhancer was lower in HUAECs compared to HUVECs. In cell culture, HUAECs rapidly acquired inducible E-selectin and VCAM-1 whereas ICAM-1 inducibility decreased. Culture of HUVECs rapidly decreased TNF responses of all three genes. By 72 hours in cell culture, TNF-treated HUVECs and HUAECs showed comparable adhesion molecule induction and transcription factor binding to the E-selectin enhancer. Freshly isolated HUAECs expressed higher levels of Kruppel-like factor 2 (KLF2) than HUVECs, consistent with greater KLF2 induction by arterial levels of shear stress in vitro. KLF2 expression decreased rapidly in both cell types during culture. Transduction of HUVECs with KLF2 reduced TNF-mediated induction of E-selectin and VCAM-1 while increasing ICAM-1 induction and reduced transcription factor/co-activator binding to the E-selectin enhancer. In conclusion, the differential responses of HUAECs and HUVECs to TNF in organ culture correlate with transcription factor/co-activator binding to DNA and converge during cell culture. Flow-induced expression of KLF2 contributes to the in situ responses of HUAECs but not of HUVECs.