During inflammation, polymorphonuclear neutrophils (PMN) are exposed to and influenced by various cytokines, including the chemoattractant interleukin-8 (IL-8). We tested the hypothesis that IL-8 affects apoptosis in PMN. We investigated which IL-8 receptor (RI or RII) might be involved, as well as the role of Bcl-2. Human PMN were isolated and cultured up to 30 hours. Apoptosis was detected by UV and light microscopy, as well as by DNA-fragmentation assay, and quantitated by flow cytometry. Interleukin-8 significantly delayed spontaneous apoptosis at 10, 20, and 30 hours in a dose-dependent fashion. Polymorphonuclear neutrophil treatment with the highest concentration of IL-8 (100 nM) decreased the percentage of apoptotic cells from 2.1 +/- 1.5 to 0.8 +/- 0.2 after 10 hours, from 31 +/- 14 to 8 +/- 5 after 20 hours, and from 47 +/- 15 to 18 +/- 8 after 30 hours of incubation (P < 0.05 for all time points, N = 6). Interleukin-8 also inhibited TNF alpha-mediated PMN apoptosis. Incubation with 20 ng/ml TNF alpha resulted in 23 +/- 6% apoptotic cells at four hours, whereas pretreatment with IL-8 (50 nM) decreased this percentage to 11 +/- 3 (N = 5, P < 0.05). We next studied the role of both types of IL-8 receptors, RI and RII, by comparing the effect of IL-8 and the product of growth-related oncogene alpha (Gro alpha) on PMN cultured for 20 hours. Both IL-8 and Gro alpha attenuated apoptosis, although IL-8 was more effective than Gro alpha. Bcl-2 was detected by intracellular fluorescent antibody cell sorter analysis, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR). Neither resting PMN nor IL-8-treated neutrophils expressed BCL-2 protein, which was readily detected in control cells. Furthermore, we could not detect BCL-2 gene expression by RT-PCR. We conclude that IL-8 prolongs the lifespan of human neutrophils in vitro by delaying apoptosis. This effect may be important for a controlled and effective inflammatory response. The delay in apoptosis can be mediated by the IL-8 RII, while RI may provide an added effect. The actions of IL-8 on apoptosis are Bcl-2 independent.

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We have previously shown that monoclonal antibody El2 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb El2 cross-reacts with detergentsolubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of M r 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergentsolubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (M, 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PcJPNa = 20.2 in a 150/50 mM trans-bilayer NaCI gradient) whose unit conductance is 62.4 -+ 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of M r 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species.