Mesenchymal Stromal Cells Protect Endothelial Cells from Cytotoxic T Lymphocyte-Induced Lysis.

The integrity of the vasculature plays an important role in the success of allogeneic organ and haematopoietic stem cell transplantation. Endothelial cells (EC) have previously been shown to be the target of activated cytotoxic T lymphocytes (CTL) resulting in extensive cell lysis. Mesenchymal stromal cells (MSC) are multipotent cells which can be isolated from multiple sites, each demonstrating immunomodulatory capabilities. They are explored herein for their potential to protect EC from CTL-targeted lysis. CD8(+) T cells isolated from human PBMC were stimulated with mitotically inactive cells of a human microvascular endothelial cell line (CDC/EU.HMEC-1, further referred to as HMEC) for 7 days. Target HMEC were cultured in the presence or absence of MSC for 24 h before exposure to activated allogeneic CTL for 4 h. EC were then analysed for cytotoxic lysis by flow cytometry. Culture of HMEC with MSC in the efferent immune phase (24 h before the assay) led to a decrease in HMEC lysis. This lysis was determined to be MHC Class I restricted linked and further analysis suggested that MSC contact is important in abrogation of lysis, as protection is reduced where MSC are separated in transwell experiments. The efficacy of multiple sources of MSC was also confirmed, and the collaborative effect of MSC and the endothelium protective drug defibrotide were determined, with defibrotide enhancing the protection provided by MSC. These results support the use of MSC as an adjuvant cellular therapeutic in transplant medicine, alone or in conjunction with EC protective agents such as defibrotide.

The role of genetic variants of NOD2/CARD15, a receptor of the innate immune system, in GvHD and complications following related and unrelated donor haematopoietic stem cell transplantation.

Previous studies from our group indicated a role of SNPs within the innate immunity receptor NOD2/CARD15 as a risk factor for GvHD and treatment-related mortality allogeneic stem cell transplantation from HLA-identical siblings. We now extended these studies to assess the role of NOD2/CARD15 SNPs in 342 unrelated donor transplants. Overall, presence of any SNPs in patients or donor resulted in an increased risk of severe GvHD (25% in wildtype versus 38% in recipients and donors with variants, P= 0.01), which did not translate in increased mortality. When the analysis was broken down to individual SNPs, the presence of a SNP13 in the donor turned out to be the only highly significant risk factor (GvHD III/IV 22% wt, 42% SNP13 donor, P < 0.004; TRM 33% wt versus 59% SNP13 donor, P= 0.01; overall survival 49% wt versus 26% SNP13 donor, P= 0.007). This association was confirmed in multivariate analysis. Analysis of clinical risk factors suggested that this effect was most prominent in patients receiving any form of T cell depletion. Thus our observation indicates that the presence of a defect in innate immunity signalling in donor monocytes and possibly antigen presenting cells is most prominent in patients having additional T cell deficiency.

NKG2D-dependent effector function of bronchial epithelium-activated alloreactive T-cells.

Allogeneic haematopoietic stem cell transplantation (SCT) has emerged as a curative therapeutic option. However, the role of graft-versus-host disease in lung injury after SCT has yet to be determined. In the present study, primary bronchial epithelial cells and the bronchial epithelial cell line BEAS-2B were used to investigate immune responses of allogeneic CD8+ T-cells directed against respiratory epithelial cells. Following stimulation with irradiated bronchial epithelial cells, CD8+ T-cells produced significant amounts of interferon-gamma, upregulated alloantigen activation markers and proliferated highly compared with T-cells stimulated with interleukin-2 alone. Furthermore, cytotoxicity assays demonstrated that bronchial epithelial cell-specific and granzyme B-mediated cytolytic activity was induced in CD8+ T-cells. Generation of natural killer (NK) T-cells, NK-like T-cells, cytokine-induced killer cells or lymphokine-activated killer cells could be excluded by phenotyping, culture conditions and neglectable lytic activity following stimulation with interleukin-2 alone. Inhibition experiments showed that lysis of bronchial epithelial cells was not major histocompatibility complex-I restricted, but depended on NK group 2 member D signalling; a stimulatory receptor initially shown to be expressed on NK cells. The present data imply that the respiratory epithelium has an antigen presenting function and directly alloactivates cytotoxic CD8+ T-cells that show nonclassical effector function.

Differentiation of human tumour-associated dendritic cells into endothelial-like cells: an alternative pathway of tumour angiogenesis.

Until recently, the only accepted mechanism of tumour vascularization was the sprouting of endothelial cells (EC) from pre-existing vessels, while recent studies suggest the contribution of stem cell-derived endothelial progenitors as well as cells from the myeloid lineage. Here, we show a new way of endothelial differentiation that involves the specific modulation of monocytes by the tumour environment. The tumour milieu is characterized by the presence of cytokines and lactate which induce the differentiation of tumour-invading monocytes into tumour-associated dendritic cells (DC). Additional incubation of tumour-associated DC with pro-angiogenic factors, such as vascular endothelial growth factor and oncostatin M, led to transdifferentiation into endothelial-like cells. The cells showed strong expression of von Willebrand factor and VE-Cadherin, both classical EC markers, while leukocytic markers were reduced. In addition, they were able to form network-like structures on matrigel, which could be blocked by the DNA-based drug Defibrotide. This finding may be of great therapeutic relevance for tumour therapy.

Functional implication of BMP4 expression on angiogenesis in malignant melanoma.

Analyses of malignant melanomas revealed a strong expression of bone morphogenic proteins (BMPs) and their autocrine effect in promoting cell invasion and migration. Here, we report a paracrine effect of BMPs on the vascular network. Both BMP2 and BMP4 induced tube formation as well as the migratory efficiency of microvascular endothelial cells. Melanoma cells with reduced BMP activity attracted less endothelial cells in invasion assays than control cells. Furthermore, reduction of BMPs in melanoma cells had a strong effect on vasculogenic mimicry. Tube formation on matrigel was analysed for melanoma cells as well as in co-cultures of endothelial and melanoma cells. Melanoma cells with reduced BMP activity were not capable of forming cord-like structures by themselves and additionally inhibited tube formation of the endothelial cells. Genes involved in angiogenesis turned out to be strongly downregulated in these cell clones. Tumors derived from cells with impaired BMP activity showed reduced tumor growth or large necrotic areas owing to lack of angiogenesis in in vivo analyses.

Hep27, a member of the short-chain dehydrogenase/reductase family, is an NADPH-dependent dicarbonyl reductase expressed in vascular endothelial tissue.

Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His(6) fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components.

Expression of heme oxygenase-1 protects endothelial cells from irradiation-induced apoptosis.

A common side effect of therapeutic use of ionizing irradiation is endothelial cell damage resulting in a variety of clinical complications. Thus, preservation of endothelial function after irradiation could potentially limit toxicity. Using the murine endothelioma cell line bEnd2 we show here that induction of heme oxygenase-1 (HO-1) by cobalt protoporphyrine IX (CoPP) inhibits irradiation-induced apoptosis. In contrast, HO-1 induction by tin protoporphyrine IX (SnPP), a HO-1 inhibitor, does not affect survival after irradiation. The protective effects of CoPP could be partially reversed by blockade of HO-1 function with SnPP after induction by CoPP. Vice versa, blockade of HO-1 function by SnPP was reversible using CoPP. Treatment with CoPP inhibited cytochrome c release induced by irradiation. These results demonstrate that HO-1 induction and activation prior to irradiation inhibits endothelial apoptosis and might be used for possible cell protection in vivo.

Interferon (IFN)-gamma is a main mediator of keratinocyte (HaCaT) apoptosis and contributes to autocrine IFN-gamma and tumour necrosis factor-alpha production.

BACKGROUND: Apoptosis of keratinocytes or intestinal epithelial cells is an important pathophysiological mechanism of organ damage during acute graft-versus-host disease. OBJECTIVES: To analyse in detail the mediators and their mutual interaction leading to keratinocyte apoptosis. METHODS: Experiments were performed using a keratinocyte cell line (HaCaT) and human skin explant cultures. RESULTS: Supernatants (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLCs) induced apoptosis in HaCaT cells and also in keratinocytes from skin biopsies. Although both interferon (IFN)-gamma and Fas ligand (FasL) were detected in MLC-SN by enzyme-linked immunosorbent assay, the apoptosis-inducing capacity could be fully abrogated by neutralization of IFN-gamma, but not by neutralization of FasL. Recombinant (r) IFN-gamma induced HaCaT keratinocyte apoptosis in a dose- and time-dependent manner. Induction of HaCaT apoptosis by rFasL alone was induced only at higher doses than present in MLC-SN, but apoptosis was dramatically enhanced in the presence of rIFN-gamma. Further synergistic effects with IFN-gamma in the induction of apoptosis were also observed with agonistic antitumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor 2 antibody, soluble TRAIL and TNF-alpha. However, in contrast to FasL and TRAIL, TNF-alpha alone did not induce HaCaT apoptosis. Interleukin-1beta and lipopolysaccharide did not enhance the apoptosis-inducing effect of IFN-gamma. Beside its apoptosis-inducing capacity in HaCaT cells, rIFN-gamma also induced autocrine IFN-gamma production, and combined treatment with IFN-gamma and TNF-alpha induced autocrine TNF-alpha production. Neutralization of autocrine IFN-gamma protected HaCaT cells from apoptosis. CONCLUSIONS: Taken together, our data suggest a central role for IFN-gamma in HaCaT keratinocyte apoptosis but also show the importance of co-acting mediators such as TNF-alpha, TRAIL and FasL, which potentiate the effect of paracrine and autocrine IFN-gamma and TNF-alpha release.

Oligotide, a defibrotide derivative, protects human microvascular endothelial cells against fludarabine-induced activation, damage and allogenicity.

Fludarabine is a nonmyeloablative immunosuppressant increasingly used as a component of alternative reduced-intensity conditioning regimens prior to allogeneic stem cell transplantation (SCT). However, we have previously shown that 2-fluoroadenine 9-beta-D-arabinofuranoside (F-Ara) as the active metabolized form of fludarabine induces damage, activation and allogenicity in human microvascular endothelial cells (HMEC). We had also identified the pharmaceutic compound Defibrotide (DF), originally used in the treatment of veno-occlusive disease and thrombotic microangiopathy, as being protective against F-Ara-induced dysfunction of HMEC, importantly, without affecting the antileukemic effect of F-Ara. In the present report, we show that a recently developed derivative of DF, Oligotide, similarly downregulates F-Ara-induced activation and damage of HMEC as well as their antigenicity for allogeneic CD8+ T cells. In addition, Oligotide could also block F-Ara-mediated transendothelial migration of peripheral blood cells across the HMEC barrier. Taken together, these observations argue for a potential clinical use of both DF and Oligotide in pre transplant conditioning.

Differences in LPS-induced activation of bronchial epithelial cells (BEAS-2B) and type II-like pneumocytes (A-549).

Lipopolysaccharide (LPS) as a major component of the outer membrane of gram-negative bacteria stimulates various cells to initiate a signalling cascade which ultimately leads to cell activation and expression of immunoregulatory or inflammatory cytokines. The human respiratory epithelium is an important environmental interface, but differences in LPS-induced cell activation between bronchial and alveolar epithelial cells have not yet been investigated in detail. First, the expression of Toll-like receptors (TLRs), as pattern-recognition receptors, was investigated for the bronchial epithelial cells and type II-like pneumocytes, demonstrating that they fulfil the prerequisites for LPS signalling. Thereafter, the effects of LPS, soluble CD14 (sCD14) and LPS-binding protein (LBP) on the release of interleukin-6 (IL-6) and IL-8 were studied. In the presence of LPS, sCD14 induced a significant and concentration-dependent cytokine release in type II-like pneumocytes, whereas the response of bronchial epithelial cells to sCD14 stimulation was low, implicating sCD14-independent activation mechanisms. Furthermore, LBP revealed inhibitory effects on the activation of alveolar epithelial cells, which may represent a novel local defence mechanism during gram-negative infection. We conclude that distinct pathways exist for LPS-induced activation of bronchial and alveolar epithelial cells.

The detection of apoptosis in a human in vitro skin explant assay for graft versus host reactions.

AIMS: Keratinocyte apoptosis is a major pathogenic mechanism in dermal complications, such as graft versus host disease (GVHD), after allogeneic bone marrow transplantation. However, the mechanisms by which recipient target cells undergo apoptosis in GVHD are still unclear, but may result from DNA damage caused by chemotherapeutic agents and/or by direct cytokine action. The basis of this investigation was to correlate keratinocyte apoptosis with (1) the severity of graft versus host reactions (GVHR) in vitro and (2) the clinical grade (0--III) of GVHD. METHODS: Skin sections generated from an in vitro skin explant model for detecting experimental or clinically relevant GVHR were investigated for the detection of apoptotic nuclei using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique. This investigation also aimed to establish whether the TUNEL assay could be used as an additional, predictive method for the severity of GVHD before transplantation in potential patient/donor pairs given standard GVHD prophylaxis (cyclosporin A and methotrexate). RESULTS: By comparing mean values of apoptosis for each GVHR grade in a cohort of 83 retrospective skin sections it was shown that as the severity of GVHR increased there was a parallel increase in the percentage of apoptotic cells (p < 0.0001). However, the correlation between clinical GVHD grade II--III and overall keratinocyte apoptosis (> 2.6%) did not reach this degree of significance (chi(2): 4.2; degrees of freedom, 1; p = 0.04; Fisher's exact test: p = 0.06). CONCLUSIONS: The detection of apoptosis correlated with degree of GVHR using an in vitro assay and a higher degree of apoptosis tended to correlate with more severe GVHD. Further studies in a larger cohort of patients, using other methods to detect apoptosis in conjunction with the TUNEL assay, may give additional insight into the complex immunopathophysiology of GVHD.

Histological correlation between different centers using the skin explant model to predict graft-versus-host disease following bone marrow transplantation.

Acute graft-versus-host disease (GVHD) remains the major complication of allogeneic stem cell transplantation (SCT) with an incidence of 40-60% and a mortality of up to 50%. Several assays have been developed to try to predict the development of GVHD including the mixed lymphocyte culture reaction, cytotoxic and helper T lymphocyte precursor frequency assays. In the Northern region of England we have used an in vitro skin explant model for predicting GVHD in MHC compatible bone marrow transplant recipients since 1988. The aims of the present study was to test the reproducibility of the model in two other bone marrow transplant centers in Europe. The assay consists of incubating patient skin explants with effector cells from mixed donor versus recipient lymphocyte cultures and the subsequent detection of graft-versus-host reactions by histopathological grading (0-IV) of the skin explants. 503 slides from 134 patients were evaluated. All were graded for negative GVHR grade 0-I or positive grade II-IV. Results from control and test slides significantly correlated between centers to the p value of 0.0001 by Fisher's exact probability test. These results show that the skin explant assay is reproducible between centers and supports the continued use of the assay to predict GVHD in allogeneic stem cell transplant recipients.

Antagonistic effects of c-myc and Epstein-Barr virus latent genes on the phenotype of human B cells.

Epstein-Barr virus (EBV) immortalized cells and Burkitt lymphoma cells have a completely different growth pattern and phenotype. EBV immortalized cells express a set of 11 viral genes to accommodate B cell activation and proliferation, whereas EBV-positive Burkitt lymphoma cells highly express the c-myc oncogene that is activated through translocation into 1 of the immunoglobulin loci and EBNA1 as the only viral protein. We have developed a primary human B cell line conditionally immortalized by Epstein-Barr virus in which the viral gene program responsible for the induction of proliferation can be switched on and off by the addition or withdrawal of estrogen (cell line EREB2-5). Starting from this cell line we have generated 2 daughter cell lines that proliferate in a c-myc dependent fashion, 1 with a highly active exogenous c-myc gene (cell line A1) and 1 with a regulatable c-myc gene that can be switched on by withdrawal and switched off by addition of tetracycline (cell line P493-6). The comparison of the 3 cell lines has allowed us to dissect the contribution of c-myc and EBV genes to the regulation of the growth pattern and expression of cell surface molecules. We show that MYC and EBNA2 (and their respective target genes) have opposing effects on the expression of several surface markers involved in B cell activation. We show that MYC contributes to the phenotype of Burkitt lymphoma cells by upregulating CD10 and CD38 and downregulating activation markers. The phenotype of the cells is determined on one hand by the absence of the viral gene products EBNA2 and LMP1 that mediate the phenotype of activated lymphoblasts and to a lesser extent by an active contribution of the c-myc gene.

In vitro-generated stem cell leukaemia showing altered cell cycle progression with distinct signalling of the tyrosine-phosphorylated rasGAP-associated p62(dok) protein.

In an attempt to gain more insight into the events of leukaemic transformation, a cell line overexpressing MHC class II (DR) was generated by transfecting an early CD34-negative haematopoietic progenitor stem cell line with the appropriate constructs. The stable transfection with genes for DR antigens leads to cellular transformation. The DR(+) transformed cell clones express a tyrosine-phosphorylated DR heterodimer and show a significantly different morphology. DR(+) clones present the morphology of an immature myeloid neoplasia expressing alpha-naphthyl-acetate-esterase (ANAE), but neither myeloperoxidase nor CD34. While D064 cells predominately grow adherent as fibroblast-like cells, the DR(+) clones display a decrease in adherent growth. Although both cell lines express similar amounts of the interleukin-6 (IL-6) signal transducer gp130, the DR-transfected cells still show activation of STAT factors by IL-6, whereas D064 cells do not. Although the transformed clones present acceleration of cell-cycle transition and growth, the G(0)/G(1) progression inhibitor p27(kip-1) is up-regulated, while the expression of proteins involved in the S/G(2) phase transition, such as cyclin B and cdc2 (p34), is suppressed. Instead cyclin D3, one of the G(0)/G(1) progression factors, is up-regulated, as well as tyrosine-phosphorylated p62(dok), suggesting dysregulation of cell cycle-controlling proteins. In addition, DR(+) leukaemia-like cells also overexpress Bcl-2, while bax expression is suppressed, compared with the wild-type (wt) parental haematopoietic stem cell line.

Endothelial apoptosis is induced by serum of patients after cardiopulmonary bypass.

OBJECTIVE: Increased serum levels of a multitude of mediators like interleukins, tumor necrosis factor, elastase, adhesion molecules, and endotoxin have been described following cardiopulmonary bypass (CPB). The biological consequences of this complex response are unclear. METHODS: Serum samples of nine patients scheduled for elective coronary artery bypass grafting were obtained preoperatively and 1, 6, and 12 h after weaning from CPB. Additional serum samples were obtained perioperatively from four patients undergoing major lung resection and from four healthy volunteers. The apoptosis-inducing activity of serum samples on endothelial cells was examined using a tissue culture assay system. Endothelial cells were derived from human umbilical cords and incubated for 48 h with serum samples in various dilutions during their second passage. The culture plates were fixed with methanol/acetone and stained with the DNA dye diamidinophenylindole. Apoptotic and normal cells were identified and counted using phase contrast and fluorescence microscopy. RESULTS: The proportion of apoptotic endothelial cells was 5.6-fold higher in culture plates incubated with diluted (30%) serum samples obtained at 6 h after weaning from CPB when compared to plates incubated with preoperative samples (P=0.0077). A smaller effect occurred already at 1 h in some patients, whereas at 12 h after weaning from CPB no increased endothelial apoptosis was observed. No proapoptotic activity was found in preoperative as well as in control samples from patients undergoing lung resection or from healthy volunteers. CONCLUSIONS: Serum of patients after CPB exerts a strong apoptosis inducing activity on human endothelial cells. Apoptotic death of endothelial cells following CPB may be responsible for postoperative vascular and bypass dysfunction including phenomena like increased capillary permeability.

Quiescence of CD34-negative haematopoietic stem cells is mediated by downregulation of Cyclin B and no stat activation.

The CD34-negative, adherent growing, fibroblast-like canine haematopoietic stem cell line D064 was recently identified as the earliest progenitor population in the bone marrow. D064 cells are predominately quiescent. Quiescence is mediated by the accumulation of the cyclin-dependent kinase inhibitor p27(kip-1)and in parallel, by the downregulation of Cyclin B, leading to an accumulation of quiescent cells in the G(0)/G(1)-phase of the cell cycle. Stem cell factor (SCF), the ligand for the tyrosine kinase receptor c-kit, usually induces differentiation of the CD34-negative stem cells into CD34-positive haematopoietic precursors. SCF also suppresses the expression of c-myc-dependent Cyclin E, which is not transcribed initially, but expression occurs later on. Interleukin 6 (IL-6) instead rather promotes proliferation, but fails to induce proliferation in the majority of CD34-negative stem cells due to no STAT activation in quiescent cells. Nevertheless, the potential of quiescent D064 cells to proliferate eventually, becomes apparent by the low-level expression of IL-6 dependent STAT factors. D064 cells also spontaneously start to express Bax, while Bcl-2 is downregulated in parallel. In summary, CD34-negative haematopoietic stem cells dwell in the marrow or other niches as quiescent cells, until they can respond to autocrine or paracrine growth factor-mediated signals.

Reverse signaling through transmembrane TNF confers resistance to lipopolysaccharide in human monocytes and macrophages.

We have previously reported that the CD14+ monocytic subpopulation of human PBMC induces programmed cell death (apoptosis) in cocultured endothelial cells (EC) when stimulated by bacterial endotoxin (LPS). Apoptosis is mediated by two routes, first via transmembrane TNF-alpha (mTNF) expressed on PBMC and, in addition, by TNF-independent soluble factors that trigger apoptosis in EC. Neutralizing anti-TNF mAb completely blocked coculture-mediated apoptosis, despite the fact that there should have been additional soluble cell death factors. This led to the hypothesis that a reverse signal is transmitted from the TNF receptor on EC to monocytes (MO) via mTNF that prevents the production of soluble apoptotic factors. Here we have tested this hypothesis. The results support the idea of a bidirectional cross-talk between MO and EC. Peripheral blood MO, MO-derived macrophages (MPhi), or the monocytic cell line Mono Mac 6 were preincubated with human microvascular EC that constitutively express TNF receptor type I (TNF-R1) and subsequently stimulated with LPS. Cell-free supernatants of these preparations no longer induced EC apoptosis. The preincubation of MO/MPhi with TNF-reactive agents, such as mAb and soluble receptors, also blocked the production of death factors, providing further evidence for reverse signaling via mTNF. Finally, we show that reverse signaling through mTNF mediated LPS resistance in MO/MPhi as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.

Naive monocytes can trigger transendothelial migration of peripheral blood cells through the induction of endothelial tumour necrosis factor-alpha.

In this manuscript we describe a potentially new mechanism by which unstimulated human monocytes activate endothelial cells (EC) through the secondary induction of endothelial tumour necrosis factor alpha (TNF-alpha). Serum free supernatants (SN) of peripheral blood mononuclear cells (PBMC) strongly induce the expression of intercellular adhesion molecule 1 (ICAM-1, CD54), vascular cell adhesion molecule 1 (VCAM-1, CD106), and endothelial-leukocyte adhesion molecule 1 (ELAM-1, CD62E) on human EC 24 and 4 h post treatment, respectively. Further characterization of the responsible subpopulation revealed the CD14+ monocytes and a monocytic cell line (MM6) to produce an endothelial activating factor (EAF). The EAF also triggers an adhesion and a transendothelial migration (TEM) of peripheral blood cells. Using neutralization with an anti TNF-alpha MoAb MAK195, EAF is not identical with TNF-alpha, but induces the expression of endothelial TNF-alpha, since MAK195 blocked TEM only when coincubated with EC, not with monocytes. Furthermore, intracellular TNF-alpha was significantly upregulated in EC after treatment with SN-MM6. Another evidence for a secondary autocrine mechanism was provided by culturing the EC with a conditioned medium of SN-MM6 treated EC. This conditioned medium induces an adhesion molecule expression and TEM in a similar way to SN-MM6 and can completely be inactivated by anti TNF-alpha. Taken together, these data may have an impact for, e.g. transplantational settings that donor monocytes may trigger an inflammatory response in the absence of further activation signals by eliciting an endogenous TNF-alpha response in the host.