Increased Escherichia coli phagocytosis in neutrophils that have transmigrated across a cultured intestinal epithelium.

The 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 coli transfected 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. coli phagocytosis 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.

Impairment of HIV polymorphonuclear leukocyte transmigration across T84 cell monolayers: an alternative mechanisms for increased intestinal bacterial infections in AIDS?

Our objective was to study the influence of HIV infection of polymorphonuclear leukocytes (PMN) on transepithelial migration. To date, reports of functional PMN chemotaxis in AIDS are contradictory. This is the first attempt to assess this function via an in vitro model allowing transmigration of neutrophils through an intestinal epithelial barrier. PMN were isolated from 45 HIV-infected patients and 45 healthy volunteers. PMN transmigration across T84 epithelial cells was initiated by applying either various concentrations of formyl-met-leu-phe peptide (f-MLP) or interleukin-8 and assayed by quantification of myeloperoxidase activity. CD11b, CD18, and CD47 expression on PMN was compared before and after transepithelial migration by flow cytometry analysis. CD11b expression was studied by electron microscopy. Apoptosis of transmigrated HIV PMN and control PMN was investigated by morphology and DNA fragmentation characterization. Compared to control PMN, HIV PMN exhibited a decrease in transepithelial migration that directly correlated with CD4+ counts. Basal and transepithelial migration-mediated expression of CD11b, CD18, and CD47 were unmodified in HIV PMN compared to control PMN. Electron microscopy labeling confirmed no difference in CD11b expression on HIV and control PMN. The index of apoptosis in transmigrated HIV PMN and control PMN was identical. These data provide evidence of a defect in the f-MLP-induced chemotaxis of PMN from HIV-infected patients across an intestinal epithelial barrier. This defective migration is not due to a quantitative modification of CD11b, CD18 and CD47 on HIV PMN suggesting a more subtle alteration. The impairment in the transmigration function may contribute in vivo to an increased susceptibility to intestinal bacterial infection in HIV-infected patients.

Neutrophil F-actin and myosin but not microtubules functionally regulate transepithelial migration induced by interleukin 8 across a cultured intestinal epithelial monolayer.

The role of the polymorphonuclear leukocyte (PMN) cytoskeleton during the transmigration across colonic epithelial cells is not very well understood. In order to study the role of different components of the PMN cytoskeleton during transepithelial migration across a colonic epithelial cell monolayer (T84), PMN were preincubated with drugs affecting either the actin cytoskeleton (cytochalasin B, iota toxin of Clostridium perfringens, and phalloidin) or the microtubules (colchicine and taxol). The role of PMN myosin during transepithelial migration was investigated using the inhibitor 2,3-butanedione monoxime (BDM) and DC3B toxin. PMN intracellular Ca2+, during neutrophil adhesion and translocation across the epithelium, was assessed by the Ca2+ chelator 1, 2bis-(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM). Transmigration of PMN was initiated by applying either interleukin-8 or formyl-met-leu-phe (fMLP). While colchicine and taxol preexposure did not influence PMN transepithelial migration, treatment with cytochalasin B, iota toxin, phalloidin, BDM, DC3B toxin and BAPTA-AM greatly diminished migration of PMN across T84 monolayers. Similarly, cell-cell contacts established between PMN and epithelial cells during the transmigration were diminished after treatment of PMN with iota toxin or cytochalasin B. These data show that the neutrophil actin cytokeleton and myosin, but not the microtubules, evoke a Ca2+ -dependent motility that facilitates migration across the colonic epithelial barrier.

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest.

p27[KIP1] (p27) is a cyclin dependent kinase inhibitor, involved in the negative regulation of G1 progression in response to a number of anti-proliferative signals. In this study we show, in growing mouse hybridoma (7TD1) and human myeloma (U266) cell lines, that p27 is highly expressed but slightly upregulated when cells are arrested, regardless to the phases of the cell cycle. In contrast, the specific blockade of these cells in early G1 phase reveals the induction of a protein of 23 kDa (p23) specifically recognized by polyclonal anti-p27 antibodies raised against the NH2 terminal part of p27 but not by anti-p21[CIP1] antibodies. Experiments using caspase inhibitors strongly suggest that p23 results from the proteolysis of p27 by a 'caspase-3-like' protease. This cleavage leads to the cytosolic sequestration of p23 but does not alter its binding properties to CDK2 and CDK4 kinases. Indeed, p23 associated in vivo with high molecular weight complexes and coprecipitated with CDK2 and CDK4. We demonstrate by transfection experiments in SaOS-2 cells that p23 induces a G1 phase growth arrest by inhibition of cyclin/CDK2 activity. In summary we describe here a caspase-cleaved form of p27, induced in absence of detectable apoptosis and likely involved in cell cycle regulation.

Nuclear accumulation of p21Cip1 at the onset of mitosis: a role at the G2/M-phase transition.

Cell cycle arrest in G1 in response to ionizing radiation or senescence is believed to be provoked by inactivation of G1 cyclin-cyclin-dependent kinases (Cdks) by the Cdk inhibitor p21(Cip1/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 G2 that may contribute to the implementation of late cell cycle checkpoint controls.

Interleukin 6 stimulates a tyrosine kinase activity potentially involved in mouse hybridoma cell growth.

In this report the authors describe the characterization of a cytosolic tyrosine kinase activity (IL-6PTK) stimulated by interleukin 6 (IL-6). IL-6PTK appears 6 h after IL-6 addition and is inhibited by tyrphostin but not genistein. It is active under its phosphorylated form although it is not immunoprecipitated by antiphosphotyrosine antibodies, suggesting that autophosphorylation occurs on residues other than tyrosine. Using the ATP-binding site covalent label, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), two phosphoproteins have been identified of 52 and 59 kDa respectively, that could potentially harbour IL-6PTK activity. The intracellular elevation of cAMP, which inhibits 7TD1 cell proliferation, decreases as the same time IL-6PTK activity suggesting that the cAMP-dependent kinase could act as a negative regulator of this tyrosine kinase species. Taken together the results strongly suggest that a tyrosine kinase (IL-6PTK) might be involved in the cascade of events leading to the proliferation of 7TD1 cells under IL-6 stimulation.

Cell cycle expression of p53 protein, c-Myc gene product and tyrosine-phosphorylation level determined by image analysis in human breast cancer cells.

OBJECTIVE: To investigate the cell cycle expression of p53 protein, c-myc gene product and tyrosine phosphorylation level in human breast cancer cells. STUDY DESIGN: Using a multifluorescence imaging procedure, the concentration per cell in different phases of the cell cycle can be evaluated by analyzing the bivariate contour plot of DNA content versus antigen concentration. RESULTS: Low fluorescence intensity was observed in the G0/G1 phase for the three markers. The analysis of individual cells demonstrated that approximately 10% of cells were negative. During the G1/S transition, the fluorescence intensity of the three antigens increased rapidly. However, after the mild S-phase, the increase of c-myc was more marked than the tyrosine phosphorylation level, whereas p53 protein remained stable, with a slight tendency to decrease. CONCLUSION: This study confirmed that the p53 protein and c-myc gene product could perform a regulatory function in G1/S transition and, consequently, may play an important role in malignant transformation. Like-wise, the variations of tyrosine kinase activity were linked to cellular progression throughout the cell cycle and could be a useful marker of alteration in the growth-factor signaling pathway. Thus, the multifluorescence imaging procedure may provide useful information on the mechanisms of the cell cycle and on malignant transformation.

Thrombin and trypsin-induced Ca(2+) mobilization in human T cell lines through interaction with different protease-activated receptors.

This study was conducted to determine whether serine proteinases may induce [Ca(2+)]i mobilization in different hematopoietic cell lines and to analyze their mechanisms of action. We show that in addition to thrombin and thrombin receptor agonist peptide (TRP, SFLLRN), trypsin induced [Ca(2+)]i mobilization in a highly thrombin-sensitive Jurkat T cell clone. Thrombin, TRP, and trypsin were found to induce [Ca(2+)]i release in three different Jurkat T cell clones differing in the level of T cell receptor expression. Similar results were obtained with a prothymocytic leukemic cell line, HPB.ALL, although these cells were much more responsive to trypsin than to thrombin and TRP. Other cell types such as THP1, a myelomonocytic cell line, or CEM, a CD4(+) positive leukemic cell line, were unresponsive to thrombin, TRP, and trypsin. The effect of trypsin was mimicked by SLIGRL, a peptide corresponding to the cleaved amino-terminal sequence of the recently characterized murine trypsin-activated receptor (PAR2). At suboptimal concentrations, the effects of SFLLRN and SLIGRL were additive, whereas saturating doses of peptides did not further increase [Ca(2+)]i mobilization in Jurkat T cells, indicating that both peptides were able to mobilize the same pool of calcium. Northern blot analysis of mRNAs from different leukemic cell lines indicated a remarkable correlation between PAR2 expression in different cell lines and SLIGRL or trypsin responses in the same cells. The expression of the "trypsin receptor" was also confirmed by polymerase chain reaction analysis. Moreover, a 24 h treatment of Jurkat cells by an anti-CD3 monoclonal antibody, a condition known to down-regulate thrombin receptor expression, induced loss of thrombin and TRP responses but only partially affected trypsin stimulation of [Ca(2+)]i release. Finally, after a first stimulation with either thrombin or trypsin, Jurkat cells were still able to respond to trypsin or thrombin, respectively, demonstrating that thrombin and trypsin essentially activated their own receptors. Our data provided evidence that 1) the human T leukemic cell line Jurkat and other T cell lines express at least two different functional protease-activated receptors, the thrombin receptor and a highly sensitive trypsin receptor, likely the human counterpart of the murine PAR2, and 2) at variance with the commonly accepted model, trypsin exerts most of its effect in T leukemic cell lines by thrombin receptor-independent mechanisms.

Cyclic AMP stimulates a JunD/Fra-2 AP-1 complex and inhibits the proliferation of interleukin-6-dependent cell lines.

Interleukin-6 (IL-6) is a proinflammatory cytokine which also acts as a growth factor for some murine hybridomas (7TD1) or human myelomas (U266). We demonstrate that elevation of cAMP cellular content inhibits IL-6-stimulated cell growth, by blocking cells mainly in G1 phase. This inhibition is associated with increased expression of the Fos family protein Fra-2. Treatment of cells with 8Br-cAMP results in increased DNA-binding activity of two distinct AP-1 complexes; JunD/Fra-2 and JunB/Fra-2, and also in elevated AP-1 transactivation. When 8Br-cAMP is withdrawn from the medium, cells enter S phase and Fra-2 protein levels and AP-1 DNA-binding activity decrease to their basal value indicating that a temporally correlation exists between the 8Br-cAMP-mediated induction of JunD/Fra-2 AP-1 complex and the 7TD1 and U266 cell growth inhibition.

Immunofluorescent quantification of tyrosine phosphorylation of cellular proteins in whole cells by flow cytometry.

Tyrosine phosphorylation of proteins, a major event in the transduction of mitogenic signals, was analysed by flow cytometry with a fluorescent antiphosphotyrosine monoclonal antibody, on formaldehyde-fixed, permeabilized cells. We have used this method (PY-Facs) to study activation of normal human T lymphocytes and cells of a leukemic T-cell line: Jurkat. In contrast to normal T cells, Jurkat cells as well as three other leukemic cell lines display a higher constitutive level of tyrosine phosphorylation. This level of tyrosine phosphorylation results from an equilibrium that can be up-regulated by the tyrosine phosphatase inhibitor, vanadate peroxide, and down-regulated by the tyrosine kinase inhibitors, genistein and staurosporine. We have also observed an increased tyrosine phosphorylation of proteins after mitogenic stimulation of Jurkat cells via T-cell receptor triggering. In addition, the entry of normal purified T cells from G0 phase into the cell cycle after co-stimulation with a phorbol ester and an anti-receptor antibody is correlated with a pronounced increase in tyrosine phosphorylation. We thus confirmed that this biochemical event was tightly associated with the activation status of the cells. The rapidity and sensitivity of the method we describe here make it particularly convenient for routine use and processing of a large number of samples, e.g., during analysis of human tumors. Moreover, because it retains sufficiently the integrity of treated cells and does not alter expression of membrane antigens, this method is suitable for multiparametric analysis, particularly for simultaneous studies associating the measure of tyrosine phosphorylation levels with possible modifications of membrane or intracellular structures as well as with cell cycle status.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin and thrombin receptor agonist peptide induce early events of T cell activation and synergize with TCR cross-linking for CD69 expression and interleukin 2 production.

Thrombin stimulation of the T leukemic cell line Jurkat induced a transient increase in [Ca2+]i. Proteolytic activity of the enzyme was required for this effect since diisopropyl fluorophosphate-thrombin failed to increase [Ca2+]i. Furthermore, hirudin and anti-thrombin III inhibited the thrombin-induced [Ca2+]i rise in Jurkat T cells. A synthetic thrombin receptor agonist peptide (TRP) of 7 residues (SFLLRNP) was found to be as effective as thrombin for [Ca2+]i mobilization, and both agonists induced Ca2+ release exclusively from internal stores. Thrombin stimulated tyrosine phosphorylation of several proteins of molecular mass 40, 42, 70, 120, and 130 kDa. There was a good correlation between thrombin-induced tyrosine phosphorylation of the latter three proteins and Ca2+ mobilization. Thrombin and TRP also caused translocation of protein kinase C from the cytosol to the plasma membrane. As a likely consequence of these events, thrombin activated the nuclear factor NF-kB. Several cell lines of hematopoietic origin including the leukemic T cell line HPB.ALL and the erythroleukemic cell line K562 were responsive to thrombin, whereas others such as THP1, a myelomonocytic cell line, and BL2, a Burkitt lymphoma were refractory to thrombin or TRP stimulation. The magnitude of the thrombin response in the different cell types paralleled the expression of the thrombin receptor mRNA. We found that activation of Jurkat T cells by a combination of phytohemagglutinin and phorbol 12-myristate 13-acetate led to a dramatic inhibition of thrombin receptor mRNA expression and to a concomitant loss of the thrombin response. Finally, we demonstrate that thrombin and TRP enhanced CD69 expression and interleukin 2 production induced by T cell receptor cross-linking in both Jurkat T cells and peripheral blood lymphocytes. These findings highlight the role of thrombin as a potential regulator of T lymphocyte activation.

Inhibition of prostacyclin-induced Ca2+ mobilization by phorbol esters in Ob1771 preadipocytes.

In addition to cAMP production, a transient elevation of intracellular free Ca2+ has been shown to take place in preadipose cells upon stimulation by carbaprostacyclin (cPGI2), both messengers acting in synergy to initiate adipose cell differentiation (Vassaux, G., Gaillard, D., Ailhaud, G., and Négrel, R. (1992) J. Biol. Chem.267, 11092-11097). Further studies reported herein show that this Ca2+ transient is i) elicited by the natural prostaglandin PGI2, ii) independent of the presence of extracellular Ca2+, suggesting a mobilization of Ca2+ from intracellular pools and ii) unaffected by cAMP elevating agents. Moreover, and in contrast to the InsP3-dependent Ca2+ signal evoked by PGF2 alpha, that induced by PGI2 is fully abolished by pretreatment with phorbol esters (EC50: 1-5 nM). Furthermore, experiments designed to empty the Ca2+ pools, using PGI2 or PGF2 alpha as Ca2+ mobilizing agents as well as pretreatments with drugs, allow to conclude that PGI2 mobilizes Ca2+ from an InsP3 sensitive, ryanodine insensitive intracellular pool. Altogether, these results strongly suggest that PGI2 mobilizes Ca2+ from an intracellular store common to that affected by InsP3, by means of a mechanism which remains to be elucidated.