The endothelial monocyte-activating polypeptide II (EMAP II) is a substrate for caspase-7.

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for leukocytes. The mature cytokine is formed in apoptotic cells by cleavage of the precursor proEMAP II. Here we show that caspase-7 is capable of cleaving proEMAP II in vitro. A proEMAP II mutant, in which the ASTD cleavage site was changed to the sequence ASTA, was not processed by caspase-7. The caspase-7-mediated generation and release of mature EMAP II may provide a mechanism for leukocyte recruitment to sites of programmed cell death, and thus may link apoptosis to inflammation.

Expression of EMAP II in the developing and adult mouse.

Endothelial monocyte-activating polypeptide II (EMAP II) is a chemoattractant for monocytes and granulocytes. EMAP II is translated as a precursor protein, proEMAP II, and is proteolytically cleaved to become the mature, biologically active cytokine. In this study we show that the EMAP II mRNA and the EMAP II precursor protein are constitutively expressed by all cell types analyzed in vitro, whereas the mature cytokine is only present in the supernatant of apoptotic cells. During mouse embryogenesis we found widespread expression of the EMAP II mRNA with transcripts being abundant in areas of tissue remodeling, where a large number of apoptotic cells could be detected by TUNEL staining. In the adult mouse, strong expression of the EMAP II mRNA is restricted to the brain, testis and thymus. Interestingly, prominent signals for EMAP II mRNA are found in local correlation with sites of apoptosis in thymus and testis but not in the brain. We propose that during development, the generation and release of the mature EMAP II may provide a mechanism for the recruitment of phagocytic cells to sites of programmed cell death. In the adult brain, the generation of mature EMAP II may contribute to the recruitment of monocytes and the immunosurveillance of this tissue.

Regulation of endothelial monocyte-activating polypeptide II release by apoptosis.

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

Expression of pro-inflammatory cytokines by flow-sorted alveolar macrophages in severe pneumonia.

The aim of the present study was to further characterize the role of alveolar macrophages (AM) in acute human lung inflammation by evaluating their capacity to produce pro-inflammatory cytokines such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-8. Patients with severe community-acquired pneumonia (CAP; n=12) and healthy volunteers (n=10) underwent bronchoalveolar lavage (BAL). AM were separated to high purity (>96%) using fluorescence-activated cell sorting. We determined the TNF-alpha, IL-6 and IL-8 cytokine gene expression in AM ex vivo using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Moreover, we measured in vitro unstimulated, lipopolysaccharide (LPS)- and LPS/interferon-gamma inducible TNF-alpha, IL-6 and IL-8 cytokine release and evaluated samples of BAL fluids for the same pro-inflammatory cytokines using an enzyme-linked immunosorbent assay (ELISA). We found increased TNF-alpha, IL-6 and IL-8 messenger ribonucleic acid (mRNA) levels in AM from CAP patients that were significantly elevated only for IL-8. When challenged with endotoxin in vitro, AM obtained from CAP patients showed a strongly reduced potential to release TNF-alpha and IL-6 compared to healthy controls, whereas IL-8 secretion did not differ significantly between groups. Moreover, stimulation of AM from CAP patients with LPS plus IFN-gamma augmented TNF-alpha and IL-6 cytokine release to near normal levels. Interestingly, no TNF-alpha protein was measured in BAL samples from CAP patients, whereas IL-6 and IL-8 protein levels were found to be significantly increased. Together, highly purified alveolar macrophages from community-acquired pneumonia patients show relatively low ex vivo tumour necrosis factor-alpha and interleukin-6 but not interleukin-8 messenger ribonucleic acid levels that are associated with a decreased pro-inflammatory cytokine release in vitro which, however, can be restored by concurrent interferon-gamma stimulation.

The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis.

Two distinct receptors for vascular endothelial growth factor (VEGF), the tyrosine kinase receptors Flt-1 and Flk-1/KDR, have been described. In this study we show that monocytes, in contrast to endothelium, express only the VEGF receptor Flt-1, and that this receptor specifically binds also the VEGF homolog placenta growth factor (PlGF). Both VEGF and PlGF stimulate tissue factor production and chemotaxis in monocytes at equivalent doses. In contrast, endothelial cells expressing both the Flt-1 and the Flk-1/KDR receptors produce more tissue factor upon stimulation with VEGF than after stimulation with PlGF. Neutralizing antibodies to the KDR receptor reduce the VEGF-stimulated tissue factor induction in endothelial cells to levels obtained by stimulation with PlGF alone, but do not affect PlGF-induced tissue factor induction in endothelial cells nor the VEGF-dependent tissue factor production in monocytes. These findings strongly suggest Flt-1 as a functional receptor for VEGF and PlGF in monocytes and endothelial cells and identify this receptor as a mediator of monocyte recruitment and procoagulant activity.

Ets transcription factor binding site is required for positive and TNF alpha-induced negative promoter regulation.

Thrombomodulin (TM) is expressed on vascular endothelial cells and plays an important role in the anticoagulant pathway by maintaining the thrombo-resistance of the blood vessel wall. We show that in primary human endothelial cells TM gene expression is repressed at the transcriptional level by Tumour necrosis factor (TNF alpha) through a protein kinase C independent pathway. The TM promoter is highly active in endothelial cells and is inhibited by TNF alpha. The -76/-56 region mediates both specific high basal activity and TNF alpha-repression. It binds a nuclear factor specific to endothelial cells, that appears to belong to the Ets-family by various criteria. The -76/-56 region contains three direct repeats of the ets-core sequence GGAA that are important for specific high basal activity, TNF alpha repression and trans-activation by expression of Ets-1 and 2. Although human Ets-1 (h-Ets-1) and chicken c-Ets-1 and 2 stimulate the TM promoter through the -76/-56 element, their activity is not suppressed by TNF alpha. c-Ets-1 competes and overrides TNF alpha repression in a concentration dependent manner. We propose that either a different member of the Ets domain protein family, or an Ets-associated co-factor, is the target of the TNF alpha signalling cascade in endothelial cells.