Heidrun Lindner

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-α (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 (MΦ), 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/MΦ 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/MΦ as indicated by the down-regulation of LPS-induced soluble TNF and IL-6 as well as IL-1 and IL-10.

Influence of bacterial endotoxin on radiation-induced activation of human endothelial cells in vitro and in vivo: protective role of IL-10.

Previous work from our group has contributed to demonstrate the role of conditioning related release of proinflammatory cytokines in induction of acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT). In the present report we show that ionizing radiation (IR) in a clinical relevant dose upregulates intercellular adhesion molecule 1 (ICAM-1) on cultured human microvascular endothelial cells (HMEC). Bacterial endotoxin (lipopolysaccharide, LPS) in a concentration corresponding to serum levels seen during clinical endotoxemia, is capable of further enhancing ICAM-1 expression on irradiated cells. Adhesion assays with freshly isolated peripheral blood mononuclear cells (PBMC) revealed that increased ICAM-1 on IR-treated endothelial cells led to an increased adhesion of PBMC. Again, this effect could be superinduced by LPS. Recombinant human interleukin 10 (IL-10), an antagonistic cytokine known to function as an LPS antagonist, was able to counteract the LPS-mediated enhancement of IR-triggered ICAM-1 induction and PBMC adhesion. In contrast, IL-10 could not inhibit irradiation caused effects. IL-10 seemed to interfere with the translocation of preformed intracellular ICAM-1 to the cell membrane. To investigate whether this superinductive function of IR and LPS on endothelial cells is of clinical relevance, mice were treated with total body irradiation (TBI) and inoculated with a single dose of LPS. Immunohistochemical analyses of murine tissues demonstrated that LPS superinduces IR-triggered ICAM-1 also in vivo. These findings may be of clinical importance as they suggest that the endothelium is activated after radiotherapy or TBI used for conditioning in bone marrow transplantation. The activated endothelium in turn may facilitate the accumulation of effector cells at sites of inflammation.

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‐α). 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‐α MoAb MAK195, EAF is not identical with TNF‐α, but induces the expression of endothelial TNF‐α, since MAK195 blocked TEM only when coincubated with EC, not with monocytes. Furthermore, intracellular TNF‐α 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‐α. 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‐α response in the host.

Differential modulation of IL-1-induced endothelial adhesion molecules and transendothelial migration of granulocytes by G-CSF

Granulocyte colony stimulating factor (G‐CSF) is widely used for mobilization of haemopoietic stem cells into the peripheral blood. However, little is known about the mechanisms involved in mobilization and the immune modulatory effects of this growth factor. In this report we show that G‐CSF down‐regulated intercellular adhesion molecule 1 (ICAM‐1) induced by Interleukin‐1 (IL‐1) on human endothelial cells. Interestingly, the G‐CSF‐mediated down‐modulation of IL‐1‐induced ICAM‐1 appeared to be biphasic. In pharmacological concentrations (>300 ng/ml), and in dose ranges of plasma G‐CSF levels above that of non‐febrile healthy individuals (30 pg/ml), a significant decrease in surface ICAM‐1 could be observed. This could be explained, at least in part, by an increased autocrine G‐CSF production by endothelial cells in response to IL‐1 and exogenous G‐CSF. In contrast to ICAM‐1, IL‐1‐triggered VCAM‐1 expression was superinduced by G‐CSF with the optimal concentration of 30 pg/ml. To evaluate the functional significance of these findings, 51Cr adhesion assays with peripheral blood mononuclear cells (PBMC) or granulocytes known to lack the VCAM‐1 counter‐receptor very late antigen 4 (VLA‐4) and IL‐1‐stimulated endothelial cells, in the presence or absence of G‐CSF, were performed. G‐CSF could not inhibit the IL‐1‐induced adhesion of PBMC to endothelial cells, which may be due to the differential adhesion molecule modulation. In contrast, granulocyte adhesion induced by IL‐1 could effectively be blocked by co‐incubation with G‐CSF. Finally, G‐CSF also inhibited transendothelial migration of granulocytes through IL‐1‐activated endothelial cells in a concentration‐dependent manner.