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p67phoxis stably attached to the phagosome and its phagosomal accumulation correlates with ROS production. Production of ROS by the leukocyte NADPH oxidase is essential for the destruction of pathogenic bacteria inside phagosomes. The enzyme is a complex of cytosolic and membranous subunits that need to assemble upon activation. Biochemical data suggest that the complex is renewed continuously during activity. Furthermore, it is generally assumed that complex assembly and activity occur in parallel. However, information about the oxidase assembly in individual phagosomes in live cells is scarce. We studied the dynamic behavior of the crucial cytosolic NADPH oxidase component p67phoxduring phagocytosis by videomicroscopy. p67phoxis involved in the regulation of electron flow from NADPH to oxygen, leading to superoxide radical formation inside the phagosome. p67phox‐citrine, expressed in myeloid PLB‐985 cells, accumulated at the phagosomal membrane during phagocytosis of yeast particles. Using photobleaching techniques (FRAP, FLIP), we demonstrated that p67phox‐citrine diffused freely in this phagosomal membrane, but the phagosomal pool of p67phox‐citrine did not exchange with the cytosolic pool. This result suggests that once assembled in the NADPH oxidase complex, p67phoxis stable in this complex. Furthermore, the time of the presence of p67phox‐citrine at the phagosome increased substantially in the presence of complement in the opsonizing serum compared with decomplemented serum. PI(3)P also accumulated around phagosomes for twice as long in the presence of complement. The presence of p67phox‐citrine was correlated with the duration of phagosomal ROS production in different opsonization conditions. These data support the critical role of p67phoxfor ROS production on the level of individual phagosomes.

Store‐operated calcium entry (SOCE) is a key regulator in the activation of leukocytes. 3,5‐Bistrifluoromethyl pyrazole (BTP) derivatives have been identified recently as inhibitors of T lymphocyte activation. The inhibitory effect of one of these compounds, N‐(4‐[3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl]phenyl)‐4‐methyl‐1,2,3‐thiadiazole‐5‐carboxamide (BTP2), appears to be a result of inhibition of SOC influx. Polymorphonuclear neutrophils provide effective protection against bacterial infection, but they are also involved in tissue damage during chronic inflammation. As for T lymphocytes, their activation relies on SOCE. We therefore investigated the effect of BTP2 on calcium homeostasis and functional responses of human neutrophils. BTP2 significantly inhibited the calcium influx after stimulation with thapsigargin or fMLF. This inhibition was seen after 5 min of incubation with 10 μM BTP2 and after 24 h with lower concentrations. With 24 h incubation, the effect appeared irreversible, as the removal of BTP2 3 h before the experiment did not reduce this inhibition in granulocyte‐differentiated HL60 cells. In human neutrophils, BTP2 reduced superoxide anion production by 82% after 24 h of incubation. On the contrary, phagocytosis, intraphagosomal radical production, and bacterial killing by neutrophils were not reduced significantly, even after 24 h treatment with 10 μM BTP2. This work suggests that BTP2 could become an important tool to characterize calcium signaling in neutrophils. Furthermore, BTP2 or related compounds could constitute a new approach to the down‐regulation of neutrophils in chronic inflammatory disease without compromising antibacterial host defense.

The mouse has become an important model for immunological studies including innate immunity. Creating transgenic mice offers unique possibilities to study gene‐function relationships. However, relatively little is known about the physiology of neutrophils from wild‐type mice. Do they behave like human neutrophils, or are there species‐specific differences that need to be considered when extrapolating results from mice to humans? How do we isolate neutrophils from mice? For practical reasons, many studies on mouse neutrophils are done with bone marrow cells. However, human bone marrow neutrophils appear to be heterogeneous and functionally immature. We have isolated and compared neutrophils from mouse bone marrow and from peripheral blood obtained by tail bleeding. Using the same Percoll® density gradient for both preparations, we have obtained morphologically mature neutrophils from bone marrow and blood. Both cell populations responded to formylmethionyl‐leucyl‐phenylalanine (fMLF) with primary and secondary granule release and superoxide production. Quantitative analysis of our data revealed minor differences between cells from bone marrow and blood. Superoxide production and primary granule release were stimulated at lower fMLF concentrations in blood neutrophils. However, the amplitude and the kinetics of maximal responses were similar. The principal difference was the lifespan of the two cell populations. Bone marrow cells survived significantly longer in culture, which may suggest that they are receiving antiapoptic signals that are absent in the blood. Our data suggest that mice have a large reservoir of functionally competent neutrophils in their bone marrow. This reservoir may be needed to replace circulating neutrophils rapidly during infection.

Exocytosis was studied in single rat peritoneal mast cells. Granule fusion was monitored by time-resolved capacitance measurements using the patch-clamp technique. Intracellular stimulation of mast cells with 20 μM GTP-γ-S stimulates exocytosis with a calcium-dependent time course. Secretion in response to receptormediated stimulation with compound 48/80 was completely abolished by treatment with pertussis toxin (IAP) at 180 ng/ml for 4 h. The time course of exocytosis in response to GTP-γ-S remained unaffected in IAP-treated cells supporting the involvement of a second GTP-binding protein in stimulus-secretion coupling.