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ABSTRACTThe functionality of polymorphonuclear leukocytes (PMNs) once they migrate into the digestive lumen is still ill defined. More specifically, phagocytic function and bactericidal action of PMNs after transepithelial migration have not received much attention. The aim of the present study is to compare PMN behavior before and after transepithelial migration, in particular (i) phagocytosis and bactericidal activity; (ii) expression of surface molecules, particularly those involved in phagocytosis; and (iii) apoptosis. Cultured human intestinal epithelial T84 cell monolayers were used. The effect of transepithelial migration on phagocytosis was evaluated by immunofluorescence and electron microscopy and by flow cytometric assessment of the engulfment of a strain of Escherichia colitransfected with the green fluorescent protein. Superoxide production by PMNs was investigated by luminol-mediated chemiluminescence. Expression of various surface molecules on PMNs was evaluated by flow cytometry, while PMN apoptosis was assayed by morphologic changes and DNA fragmentation. E. coliphagocytosis by the PMNs was markedly increased after transepithelial migration without modification of superoxide production. CD11b/CD18 and CD47 expression was increased upon PMN transmigration, whereas CD16 expression was decreased and CD29, CD46, CD49e, CD49f, CD55, CD59, CD61, CD95 levels remained unchanged. Apoptosis in transmigrated PMNs was slightly advanced and was observed after 12 h compared to 16 h for nontransmigrated PMNs. In conclusion, the phagocytic capacity of the PMNs is augmented after transepithelial migration, with a dramatic increase in the level of CD11b/CD18 and preservation of the superoxide production. These results suggest a higher bactericidal activity of the PMNs once they have translocated into the digestive lumen.

The heterodimeric DNA fragmentation factor (DFF) is responsible for DNA degradation into nucleosomal units during apoptosis. This process needs the caspase‐dependent release of ICAD/DFF‐45, the inhibitory subunit of DFF. Here we report that triggering apoptosis via a hyperos‐motic shock in hematopoietic cells causes the appearance of mitochondrial and cytosolic alterations, activation of caspases, chromatin condensation, nuclear disruption, and DNA fragmentation. However, oligonucleosomal but not high molecular weight (50–150 kb) DNA cleavage is abolished if Cl−efflux is prevented by using NaCl to raise extracellular osmolarity or by Cl−channel blockers, even when apoptosis is initiated by other agents (staurosporine, anti‐Fas antibody). In these conditions, all the apo‐ptosis hallmarks investigated remain detectable, including the cleavage of ICAD/DFF‐45. In vitroassays with lysates of cells in which Cl−efflux is blocked confirm the lack of internucleosomal DNA degradation. These findings establish that neither caspase activation nor ICAD/DFF‐45 processing per seis sufficient to induce oligonucleosomal DNA fragmentation and that high molecular weight DNA degradation and chromatin condensation appear independently of it. Finally, they suggest that Cl−efflux is a necessary cofactor that intervenes specifically in the activation of the DFF endonuclease.—Rasola, A., Farahi Far, D., Hofman, P., Rossi, B. Lack of inter‐nucleosomal DNA fragmentation is related to Cl−efflux impairment in hematopoietic cell apoptosis. FASEB J. 13, 1711–1723 (1999)

ABSTRACTCell cycle arrest in G1in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21Cip1/Waf1/Sdi1. We provide evidence that in addition to exerting negative control of the G1/S phase transition, p21 may play a role at the onset of mitosis. In nontransformed fibroblasts, p21 transiently reaccumulates in the nucleus near the G2/M-phase boundary, concomitant with cyclin B1 nuclear translocation, and associates with a fraction of cyclin A-Cdk and cyclin B1-Cdk complexes. Premitotic nuclear accumulation of cyclin B1 is not detectable in cells with low p21 levels, such as fibroblasts expressing the viral human papillomavirus type 16 E6 oncoprotein, which functionally inactivates p53, or in tumor-derived cells. Moreover, synchronized E6-expressing fibroblasts show accelerated entry into mitosis compared to wild-type cells and exhibit higher cyclin A- and cyclin B1-associated kinase activities. Finally, primary embryonic fibroblasts derived from p21−/−mice have significantly reduced numbers of premitotic cells with nuclear cyclin B1. These data suggest that p21 promotes a transient pause late in G2that may contribute to the implementation of late cell cycle checkpoint controls.