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The modulation of host cell apoptosis by bacterial pathogens is of critical importance for the outcome of the infection process. The capacity of Bartonella henselae and B. quintana to cause vascular tumor formation in immunocompromised patients is linked to the inhibition of vascular endothelial cell (EC) apoptosis. Here, we show that translocation of BepA, a type IV secretion (T4S) substrate, is necessary and sufficient to inhibit EC apoptosis. Ectopic expression in ECs allowed mapping of the anti-apoptotic activity of BepA to the Bep intracellular delivery domain, which, as part of the signal for T4S, is conserved in other T4S substrates. The anti-apoptotic activity appeared to be limited to BepA orthologs of B. henselae and B. quintana and correlated with (i) protein localization to the host cell plasma membrane, (ii) elevated levels of intracellular cyclic adenosine monophosphate (cAMP), and (iii) increased expression of cAMP-responsive genes. The pharmacological elevation of cAMP levels protected ECs from apoptosis, indicating that BepA mediates anti-apoptosis by heightening cAMP levels by a plasma membrane-associated mechanism. Finally, we demonstrate that BepA mediates protection of ECs against apoptosis triggered by cytotoxic T lymphocytes, suggesting a physiological context in which the anti-apoptotic activity of BepA contributes to tumor formation in the chronically infected vascular endothelium.

Cytotoxic T lymphocytes (CTLs) are important effector cells of graft-versus-host disease (GVHD) and vascular endothelial cells are target cells of allospecific CTL. A combined assessment of T-cell activation and endothelial injury should result in a specific and sensitive test for GVHD. We examined circulating T lymphocytes for effector molecules involved in CTL-mediated endothelial injury. We analyzed CD4 and CD8 T lymphocytes of 24 long-term survivors of allogeneic stem cell transplantation with or without GVHD, and nine healthy, age-matched controls for signs of CTL activation and endothelial injury. IFN-gamma transcript levels in CD8 T cells were significantly elevated in SCT recipients with GVHD compared to patients without GVHD (767 CD3epsilon units/T cell (376-2050) vs 211 CD3epsilon units/T cell (159-274), P=0.01). Fas ligand transcript levels in CD4 T cells were significantly elevated in SCT recipients without GVHD compared to patients with GVHD (20 CD3epsilon units/T cell (0-78) vs 0 CD3epsilon units/T cell (0-0), P=0.01). Von Willebrand factor plasma levels were high in patients with GVHD, but normal in patients without GVHD (209 (186-254) vs 120 (100-141), P=0.0005). This assessment of T-cell activation and endothelial injury results in a sensitive and specific test to identify patients with active chronic GVHD.

Vascular endothelial cells play a threefold role in the interaction with leukocytes. First, they are gatekeepers in leukocyte recruitment to inflammatory foci and lymphocyte homing to secondary lymphoid organs. Second, they modulate leukocyte activation. Finally, they are targets of leukocyte-derived molecules, resulting either in endothelial cell activation or death.

IL-11, a gp130-signaling cytokine, is protective in several in vivo models of immune-mediated and inflammatory injury. HUVECs express IL-11 receptor alpha-chain and gp130. Human IL-11 causes rapid (2-10 min) tyrosine phosphorylation of gp130. IL-11 at 0.1 and 10 ng/ml induces tyrosine phosphorylation of STAT3 and STAT1, respectively, although maximal responses require 50 ng/ml. Phospho-STAT3 and phospho-STAT1 levels peak rapidly (2.5 min) and disappear by 60 min. The p42 and p44 mitogen-activated protein kinases (MAPKs) are phosphorylated in response to 0.3 ng/ml IL-11 with maximal activation at 30 ng/ml IL-11. Phosphorylation of p42 and p44 MAPKs, which can be prevented by a mitogen-activated protein/extracellular signal-related kinase kinase-1 inhibitor, peaks by 15-20 min and largely disappears by 40 min. IL-11 does not activate NF-kappaB nor does it inhibit NF-kappaB activation by TNF. Similarly, IL-11 neither induces E-selectin or ICAM-1 nor blocks induction by TNF. Although IL-11 does not alter class I MHC complex molecule expression, pretreatment with 0.5 ng/ml IL-11 partially protects HUVECs against lysis by allospecific class I MHC-restricted cytolytic T lymphocytes or by anti-class I MHC Ab plus heterologous C. IL-11-induced cytoprotection is protein synthesis dependent and may depend on mitogen-activated protein/extracellular signal-related kinase kinase-1. Our results indicate that low (i.e., STAT3- and MAPK-activating) concentrations of IL-11 confer resistance to immune-mediated injury in cultured HUVECs without inhibiting proinflammatory responses.

We have shown previously that cultured HUVEC or mixtures of endothelial cells (EC) and B lymphoblastoid cells (BLC) induce the differentiation of purified CD8+ PBL into allospecific, class I MHC-restricted CTL that lyse EC, but not BLC autologous to EC. Furthermore, these EC-selective CTL lines secrete little IFN-gamma after target cell contact. In the present study, we have analyzed these polyclonal populations at a single cell level by cloning at limiting dilution and propagating the resulting CTL clones in the absence of EC. Phenotypically stable, alloreactive EC-selective CTL preferentially emerge from cocultures in which EC or EC + BLC are the initial stimulating cell types compared with cocultures stimulated by BLC alone (p = 0.005). Compared with BLC-stimulated CTL, EC-stimulated CTL clones often fail to secrete IFN-gamma after target cell contact (p = 0.0006) and constitutively express CD40 ligand (CD40L) at rest (p = 0.0006). The absence of IFN-gamma secretion does not result from a switch to IL-4 secretion. The expression of CD40L inversely correlates with the secretion of IFN-gamma after target cell contact (p = 0.0001), but correlations of CD40L expression and failure to secrete IFN-gamma with EC-selective killing did not reach statistical significance. We conclude that in a microenvironment in which allogeneic EC are in close contact with infiltrating CD8+ T cells, such as within a graft arterial intima, CTL subsets may emerge that display EC selectivity or express CD40L and secrete little IFN-gamma after Ag contact.

We compared the capacity of cultured human endothelial cells (EC) vs B lymphoblastoid cells (BLC) from the same donor to stimulate allogeneic CD8+ T cells to differentiate into CTL, assaying for allorestricted cytotoxicity, T cell growth, IFN-gamma secretion, and perforin expression. The input cell number affected specificity and potency of the resulting CTL. At low input (<10(5) cells/ well), anti-EC CTL were rarely detected. At high input (>10(6) cells/well), anti-EC CTL developed that displayed unrestricted, low-titer killing and an unstable phenotype. At intermediate input (1.0-2.5 x 10(5) cells/well), classical class I MHC-restricted, CD8+, and perforin-positive anti-EC CTL developed with reproducible frequencies. However, under all conditions EC were less efficient stimulators than BLC from the same donor. Anti-EC CTL did not kill BLC, whereas anti-BLC CTL killed BLC and EC from the same donor with comparable efficiency. When CD8+ T lymphocytes were grown in the presence of EC and BLC together, the differentiation of anti-BLC CTL was completely suppressed, while the anti-EC response was intact. The inhibition of the allogeneic anti-BLC CTL response was independent of T cell-EC contact, and proliferation of CD8+ T cells was inhibited by EC-conditioned medium. We conclude that EC are competent but less efficient activators of CTL differentiation than are BLC and that EC actively regulate differentiation and/or expansion of allospecific CTL.

Human endothelial cells cultivated on polystyrene microcarrier beads were used to study endothelial anticoagulant activity in vitro. Spontaneous whole blood coagulation was inhibited by endothelial cells on microcarriers at a surface to volume ratio of 16 cm2/ml blood. Thrombin activity generated in nonanticoagulated whole blood during 1 hour and assessed by its fibrinogen clotting effect was reduced by 87% in the presence of endothelial cells. Consistent with this observation, prothrombin fragment1+2, fibrinopeptide A, and thrombin-antithrombin III-complex release during the same period of time were inhibited by 81%, 47%, and 88%, respectively. Immunoblotting analysis of cell-free supernatants derived from the same samples demonstrated that prothrombin activation was strongly suppressed in the presence of endothelial cells. Furthermore, the incubation of nonanticoagulated whole blood with endothelialized beads for only 5 minutes after venipuncture was sufficient to prevent subsequent prothrombin activation in the cell-free supernatants of the same whole blood sample after centrifugation. These findings suggest that interruption of the coagulation cascade is probably one major mechanism of endothelial anticoagulant activity in vivo.