Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to G(i). In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.

To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D, Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 ,uM H202 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H202. H202 at 40 MuM or higher inhibited DNA synthesis, and induced SSB within less than 30 s.These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis.

H202, in concentrations achieved in the proximity of stimulated leukocytes, induces injury and lysis oftarget cells. This may be an important aspect of inflammatory iD'jury of tissues. Cell lysis in two target cells, the murine macrophagelike tumor cell line P388D1 and human peripheral lymphocytes, was found to be associated with activation of poly(ADP-ribose) polymerase (EC 2.4.2.30), a nuclear enzyme. This enzyme is activated under various conditions of DNA damage. Poly(ADPribose) polymerase utilizes nicotinamide adenine dinucleotide (NAD) as substrate and has been previously shown to consume NAD during exposure of cells to oxidants that was associated with inhibition of glycolysis, a decrease in cellular ATP, and cell death. In the current studies, inhibition of poly(ADPribose) polymerase by 3-aminobenzamide, nicotinamide, or theophylline in cells exposed to lethal concentrations of H202 prevented the sequence of events that eventually led to cell lysis-i.e., the decrease in NAD, followed by depletion of ATP, influx of extracellular Ca2+, actin polymerization and, finally, cell death. DNA damage, the initial stimulus for poly(ADPribose) polymerase activation, occurred despite the inhibition of this enzyme. Cells exposed to oxidant in the presence of the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide failed to demonstrate repair of DNA strand breaks.Oxidants induce cell injury and lysis in various target cells both in vivo and in vitro (1)(2)(3)(4) ], a nuclear enzyme using NAD as its substrate and forming nicotinamide and poly(ADP-ribose) polymers, which are ester-bound to various nuclear proteins. Poly-(ADP-ribose) polymerase activation has been associated with conditions that cause DNA damage (9-12) and results in depletion of NAD in irradiated cells (9) and cells exposed to alkylating agents (10, 11). Poly(ADP-ribose) polymerase can be inhibited by 3-aminobenzamide (which is considered to be specific), nicotinamide, theophylline, and high doses of thymidine (13).The effect of inhibition of poly(ADP-ribose) polymerase on H202-induced cellular injury is not known. Since this enzyme appears to exert a central function in the biochemical chain of events following exposure of cells to the oxidant H202, it is important to know whether its action is essential to the development of injury to the target cell. Therefore, NAD, ATP, cellular Ca2+, actin polymerization, and cell lysis as parameters of oxidant-induced cell injury were determined in the presence or absence of inhibitors of poly(ADP-ribose) polymerase. MATERIALS AND METHODSCell Culture. P388D1 cells were cultured in RPMI 1640 medium (Irvine Scientific) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 ,ug of gentamycin sulfate (M. A. Bioproducts, Walkersville, MD) per ml.

In leukocytes, C3a and C5a cause chemotaxis in a Gi-dependent, pertussis toxin (PT)-sensitive fashion. Because we found that HUVECs and immortalized human dermal microvascular endothelial cells express small numbers of C3aRs and C5aRs, we asked what the function of these receptors was on these cells. Activation of the C3aR caused transient formation of actin stress fibers, which was not PT-sensitive, but depended on rho activation implying coupling to Gα12 or Gα13. Activation of the C5aR caused a delayed and sustained cytoskeletal response, which was blocked by PT, and resulted in cell retraction, increased paracellular permeability, and facilitated eosinophil transmigration. C5a, but not C3a, was chemotactic for human immortalized dermal microvascular endothelial cells. The response to C5a was blocked by inhibitors of phosphatidylinositol-3-kinase, src kinase, and of the epidermal growth factor (EGF) receptor (EGFR) as well as by neutralizing Abs against the EGFR and heparin-binding EGF-like factor. Furthermore, immune precipitations showed that the EGFR was phosphorylated following stimulation with C5a. The C5aR in endothelial cells thus uses a signaling cascade–transactivation of the EGFR–that does not exist in leukocytes, while the C3aR couples to a different G protein, presumably Gα12/13.