Monosodium urate and calcium pyrophosphate crystals differentially activate the excitation-response coupling sequence of human neutrophils

The activation patterns of human neutrophils elicited by unopsonized monosodium urate and calcium pyrophosphate dihydrate crystals were investigated. The parameters chosen, the mobilization of calcium and the synthesis of leukotrienes, are generally accepted to be relevant to the activation of the cells and their pathophysiological roles. Both particles were found to elicit increases in cytoplasmic free calcium and leukotriene synthesis. However, the rank order of potency of these two stimuli was found to be sharply dependent on the test chosen. Monosodium urate crystals were significantly more effective than calcium pyrophosphate dihydrate crystals in terms of calcium mobilization, while the latter are more potent at inducing leukotriene synthesis. These results demonstrate that these two phagocytic particles which are related to separate inflammatory joint diseases differentially activate the excitation-response coupling sequence of human neutrophils.     

Pertussis toxin as a probe of neutrophil activation

In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed.     

Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.     

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.     

Development of calcium and secretory responses in the human promyelocytic leukemia cell line HL60

We have begun to characterize the development of the excitation-response coupling sequence in the human promyelocytic leukemia cell line HL60. Using the recently developed fluorescent calcium probe quin-2, it was found that DMSO induced myeloid differentiation of the HL60 cells is accompanied by the development of a calcium response to the addition of the chemotactic factors fMet-Leu-Phe and leukotriene B4. The characteristics (time course, concentration dependence, stereospecificity, and metabolic dependence) of the calcium response are extremely similar to those previously described in human neutrophils. These results imply that functional receptors for leukotriene B4 appear in HL60 cells upon the induction of differentiation and also lend strong support to the use of these HL60 cells as a model of human myeloid differentiation. We have also characterized the emergence of a secretory response to fMet-Leu-Phe and leukotriene B4 in cytochalasin B treated HL60 cells. In addition, it is found that differentiation was required for the calcium ionophore A23187 to express its secretory activity toward the HL60 cells. This last set of results implies that differentiation is accompanied by the coordinated appearance of surface receptors and cytoplasmic factors required for the expression of cellular responsiveness.     

Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.     

Guanine nucleotide-induced polymerization of actin in electropermeabilized human neutrophils

The effects of exogenous guanine nucleotides on the polymerization of actin in human neutrophils were tested in an electropermeabilized cell preparation. Close to 40% permeabilization was achieved with a single electric discharge as measured by nucleic acid staining with ethidium bromide or propidium iodide with minimal (less than 2%) release of the cytoplasmic marker lactate dehydrogenase. In addition, electropermeabilized neutrophils retained their capacity to produce superoxide anions and to sustain a polymerization of actin in response to surface-receptor dependent stimuli such as chemotactic factors. Electropermeabilization produced a rapid and transient permeabilization that allowed the entry of guanine nucleotides into the cells. GTP and, to a larger extent, its nonhydrolyzable analog guanosine 5'-O-2-thiotriphosphate (GTP[S]), induced a time- and concentration-dependent polymerization of actin, as determined by increased staining with 7-nitrobenz-2-oxa-1,3-diazolylphallacidin. The effects of the aforementioned guanine nucleotides were antagonized by GDP[S], but were insensitive to pertussis toxin. Cholera toxin potentiated to a small degree the amount of actin polymerization induced by GTP[S]. These results provided direct evidence for the involvement of GTP-binding proteins in the regulation of the organization of the cytoskeleton of neutrophils, an event that is of crucial importance to the performance of the defense-oriented functions of these cells.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

[Fréquence des maladies transmises sexuellement chez des étudiants universitaires.]

OBJECTIVE: To estimate the incidence rate of sexually transmitted diseases (STDs) among university students and evaluate the associated sociodemographic factors. DESIGN: Mail survey in April 1990. Included in the questionnaire were questions about the subjects'' STD experience since their admission to the university and the type and date of the infection. SUBJECTS: Of the 19,682 undergraduate students 2920 subjects, in 10 groups of 292, were randomly selected. A total of 1731 (59.4%) completed the questionnaire. MAIN OUTCOME MEASURES: Estimated annualized incidence rates of genital human papillomavirus infection and Chlamydia infection. RESULTS: The estimated annualized incidence rates of genital human papillomavirus and Chlamydia infections were 2.2% and 1.5% respectively. Among the students who indicated being infected with genital human papillomavirus 59% were 18 to 21 years old (p < 0.05), 76% were women (p < 0.01) and 69% had more than one sexual partner (p < 0.01). No statistically significant associations were observed between age, sex and Chlamydia infection. On the other hand, 95% of the cases of Chlamydia infection were found among those who had more than one sexual partner (p < 0.01). CONCLUSION: University students continue to have sexual activities at risk for STDs and should be specifically targetted by general practitioners and health services in an effort to slow the spread of STDs.