Inhibition of O2- generation by dexamethasone is mimicked by lipocortin I in alveolar macrophages.

Glucocorticoids inhibit superoxide (O2-) generation by phagocytes through a mechanism that remains unclear. We investigated this effect by using dexamethasone on guinea pig alveolar macrophages. O2- generation was induced either by the calcium ionophore A23187, a potent stimulus of phospholipase A2, or by the protein kinase C activator, phorbol myristate acetate (PMA). Dexamethasone inhibited O2- generation initiated by A23187 by 50-55%. This inhibition required: (a) more than 45 min incubation and was maximal after 2 h; (b) glucocorticoid receptor occupancy; and (c) protein synthesis. The inhibitory effect of dexamethasone could not be explained by an interaction with the respiratory burst enzyme NADPH oxidase since O2- generation was only weakly affected upon PMA stimulation. Lipocortin I, a glucocorticoid inducible and phospholipase A2 inhibitory protein, inhibited O2- generation initiated by A23187 but failed to modulate the respiratory burst activated by PMA. These results were obtained with lipocortin I purified from mouse lungs, human blood mononuclear cells, and with human recombinant lipocortin I. We propose that lipocortin I is capable of inhibiting the activation of NADPH oxidase only when membrane signal transduction involves phospholipase A2. By mimicking the effect of dexamethasone, lipocortin I may extend its potential anti-inflammatory action to the partial control of the formation of oxygen reactive species by phagocytes.

Increase in the expression of a family of small guanosine triphosphate-binding proteins, rab proteins, during induced phagocyte differentiation.

Rab is a newly identified family of small G-proteins that share 35-70% homology with the yeast Sec4p and Ypt1p involved in the regulation of the secretory pathway. Mature phagocytes display functions requiring organized intracellular traffic and, for this reason, we questioned whether phagocyte differentiation could correlate with the increased expression of rab proteins. Rabbit antisera raised against the recombinant proteins rab1Ap, 2p, 4p, and 6p were able to detect the corresponding proteins in the human monoblast leukemic cell line U937. When these cells were induced to differentiate into monocyte/macrophage-like cells displaying functional characteristics of a normal phagocyte, rab1Ap, 2p, 4p, and 6p were increased and this correlated with an increase in the rab transcripts. Using a rab5 probe, we also observed an increased expression of the rab5 gene in differentiated cells. Similarly, differentiation of the human leukemic myeloblast HL60 cell line along either monocyte or granulocyte pathways induced an increased expression of the rab proteins. Rab proteins were also detected in human neutrophils and in guinea pig alveolar macrophages. As degranulation is one of the phagocyte functions acquired in the late stage of differentiation, we investigated whether rab proteins would be involved in this process. Although rab proteins were tightly membrane bound, none of them was detected in the specific or azurophil granules purified from human neutrophils. The increased expression of rab proteins in mature phagocytes suggests that they may promote functions highly developed in these cells.

Lyn and Fgr are activated in distinct membrane fractions of human granulocytic cells.

We have previously shown that the src-family protein-tyrosine kinase Hck is localized on the azurophil granules of human granulocytes, translocates towards the phagosomes during phagocytosis of opsonized zymosan and is activated during this process. Hck is also activated upon cell stimulation with the calcium ionophore A23187, but not with PMA or the chemotactic peptide fMLP. Here, we investigated whether the src-family kinases Lyn and Fgr are activated under the same conditions. Upon stimulation of human neutrophils or retinoic acid-differentiated NB4 cells with fMLP, only Lyn is activated. Cell stimulation with opsonized zymosan or A23187 leads to simultaneous activation of Lyn and Fgr. In cell fractionation experiments with differentiated NB4 cells, the kinases show a similar subcellular localization: Both co-fractionate quantitatively with plasma-membrane marker in two fractions that sediment at 11000 g and 200000 g. Lyn is selectively activated in the 200000 g fraction, whereas Fgr is activated in the 11000 g fraction and distinct sets of tyrosine phosphorylated proteins are found in these fractions. The results suggest that these kinases exert their functional roles in distinct cellular compartments in human granulocytic cells.

Nonreceptor Protein-Tyrosine Kinases in Neutrophil Activation

Nonreceptor protein-tyrosine kinases are involved in the regulation of almost all neutrophil responses such as adhesion, chemotaxis, priming, oxidative burst, and degranulation. Here, we show that phagocytosis is also regulated by protein-tyrosine kinase activity. Using various protein-tyrosine kinase inhibitors, we further demonstrate that opsonized zymosan-induced degranulation of specific and azurophil granules is regulated by protein-tyrosine kinase activity, whereas phorbol ester-induced degranulation is not. Several of the nonreceptor protein-tyrosine kinases involving in neutrophil signal transduction are known, including Fgr, Hck, Lyn, Yes, and Syk. Among these, Hck and Fgr are localized on the azurophil and specific granules, suggesting the involvement of these two protein-tyrosine kinases in the regulation of degranulation. In this report, we characterize some of the molecular properties of Hck and Fgr. We discuss the methods generally used for the measurement of protein-tyrosine kinase activities in neutrophils highlighting precautions against proteolysis. In addition, we show that in subcellular fractions of retinoic acid-differentiated neutrophil-like NB4 cells, the 59- and 61-kDa forms of Hck are attached to the membranes of their respective compartments by different mechanisms. Finally, we discuss the functional roles of protein-tyrosine kinases in the regulation of neutrophil activation and speculate on the importance of their subcellular localization.

Expression of NADPH oxidase is induced by all-trans retinoic acid but not by phorbol myristate acetate and 1,25 dihydroxyvitamin D3 in the human promyelocytic cell line NB4.

Human promyelocytic cells, NB4, differentiate into neutrophils in response to all-trans retinoic acid (ATRA). It has recently been proposed that NB4 cells have bilineage potential because these cells are also able to differentiate into monocyte/macrophages when exposed to a combination of 1,25-dihydroxyvitamin D3 (VD3) and phorbol myristate acetate (PMA). Differentiation of myeloid cells into neutrophils or monocytes is associated with the acquisition of the O2- producing enzyme, NADPH oxidase, which plays a critical role in microbial killing. In this study, the expression of the components of the NADPH oxidase complex during the differentiation of NB4 cells into neutrophils or macrophages has been investigated. Whereas cells exposed to ATRA were able to produce O2- after 2 days of differentiation, they remain unable to generate O2- when exposed to PMA or PMA + VD3. With the exception of p21rac, none of the other oxidase components was expressed in non-differentiated cells. Addition of ATRA induced the progressive expression and accumulation of p22phox, p91phox, p47phox and p67phox. Compared to the other components, p67phox was expressed late and its expression appeared to correlate most closely with the generation of O2- in the differentiation process. In PMA or PMA + VD3-differentiated NB4 cells, expression of the NADPH oxidase components was incomplete. Therefore, ATRA induced the expression of a functional NADPH oxidase complex in neutrophil-like NB4 cells. In contrast, when NB4 cells are exposed to monocytic differentiating agents, they acquire only part of the phenotypic characteristics of monocytes and lack one of the major phagocytic functionalities, the respiratory burst oxidase.

NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils.

In addition to the extracellular production of O2- by NADPH oxidase in neutrophils stimulated by soluble stimuli, the intracellular formation of oxygen reactive species has been described. Cytochrome b559, the redox component of the NADPH oxidase complex, is mainly associated with specific granule membrane in resting neutrophils. We examined whether these granules could be a site for intracellular production of O2-. Phorbol myristate acetate (PMA)-stimulated neutrophils were fractionated by differential centrifugation, and generation of O2- was detected in both the granule and the plasma membrane-enriched fractions, but more in the granules. Translocation of p47phox and p67phox, two cytosolic components of the NADPH oxidase, was also quantitatively more important in the granules than in the plasma membrane fraction. After separation of the specific from the azurophil granules, p47phox and p67phox were found to be present only in the specific granules of PMA-activated cells. As a control, the production of O2- was studied in retinoic acid-differentiated NB4 cells that lack specific granules. During stimulation of NB4 cells with PMA, only the plasma membrane-enriched fraction was the site of O2- production. Together, these results indicate that NADPH oxidase can be functionally assembled in specific granules.

The role of the Na+/H+ antiporter in human neutrophil NADPH-oxidase activation.

The Na+/H+ antiporter has been shown to regulate activation of the human neutrophil. To delineate the role of the antiporter in the stimulation cascade, superoxide [O2-) generation was observed in PMA-stimulated neutrophils in which intracellular pH (pHi) was artificially manipulated by the use of nigericin, a K+/H+ ionophore, with and without use of the Na+/H+ antiporter inhibitor, dimethylamiloride (DA). Decreased O2- generation was observed in a Na+-free, 140 mM K+ buffer, but addition of nigericin restored this parameter of neutrophil activation to levels found in physiologic Na+ media. Further, the inhibitory effects of DA on O2- generation by cells incubated in a 10 mM Na+, 130 mM K+ buffer were totally reversed by a similar concentration of nigericin. O2- generated by a membrane preparation of the NADPH-oxidase, made from cells incubated under the same experimental conditions, paralleled whole cell studies, but the activity of the oxidase did not vary when suspended in the various reaction mixtures. These experiments support the role of the Na+/H+ antiporter in neutrophil activation as a metabolic regulator of pHi that influences receptor-coupled reactions proximal to expression of the NADPH-oxidase itself.

Regulation of production of soluble Fc gamma receptors type III in normal and pathological conditions.

CD16 (Fc gamma R type III), a low affinity IgG Fc receptor, is found in two forms, a transmembrane Fc gamma RIIIa expressed by NK cells and monocytes and a phosphatidylinositol-linked Fc gamma RIIIb present on neutrophils. Exposure of neutrophils to inflammatory signals induces a rapid loss of CD16 expression and release of a soluble form of CD16 (sCD16). Soluble CD16 circulates in plasma, levels being reduced in sera from patients with multiple myeloma. In the present manuscript the authors summarize work that aimed to better understand: (i) the role of proteinases in sCD16 production and CD16 membrane shedding; and (ii) the regulation of sCD16 levels in multiple myeloma patients and the possible biological consequences of its decrease in this disease. Soluble CD16 was purified from human serum. Its N-terminal sequencing demonstrated that it originates from neutrophil CD16 and its C-terminal sequencing showed that the cleavage site was between Val 196 and Ser 197, close to the membrane anchor. Analysis of the effect of protease inhibitors revealed that the cleavage leading to sCD16 production by PMA-activated neutrophils was metalloproteinase-dependent. In addition, membrane and sCD16 were sensitive to serine proteinases released by azurophil granules or added under purified form. The reduction of sCD16 levels that occurs in patients with multiple myeloma was associated with a slight decrease in circulating neutrophils, but not with a significant defect in sCD16 production by neutrophils, as detected in vitro. Moreover, addition of a recombinant sCD16 to plasmocytoma lines did not significantly modify their proliferation and Ig secretion.

Effect of intracellular oxygen-free radicals on the formation of lipid derived mediators in rat renomedullary interstitial cells.

An intracellular generation of oxygen free radicals was induced by phenazine methosulfate (PMS) in rat renomedullary interstitial cells (RMIC) in culture. This response was associated with an increase in PGE2 and 15 HETE production. The synthesis of cyclooxygenase and lipoxygenase derivatives in PMS-treated cells was inhibited by indomethacin and NDGA respectively. Inhibitors of PLA2 such as mepacrine and dexamethasone were able to inhibit partially the PGE2 synthesis induced by PMS. The formation of lyso-platelet activating factor, a product of membrane-bound phospholipid, by a PLA2 catalyzed reaction was also stimulated in PMS-treated cells. Superoxide dismutase added to the incubation medium enhanced the PMS-dependent PGE2 synthesis whereas catalase decreased it, suggesting the involvement of H2O2 in this process. In addition, a depletion of soluble thiol groups was observed in PMS-treated cells. Treatment of RMIC by the thiol oxidative agent, diamide, mimicked the effect of PMS on PGE2 synthesis, whereas diamide did not increase the formation of lyso-PAF indicating its inability to stimulate PLA2. These results suggest that cyclooxygenase may be involved in this process, indeed added arachidonate, bypassing PLA2, enhanced PGE2 synthesis in PMS-treated cells further supporting the involvement of cyclooxygenase. In conclusion, generation of oxygen free radicals by PMS in RMIC enhanced the synthesis of lipid derived mediators. A decrease in the cellular thiol content is partially involved in cyclooxygenase activation but does not appear to be involved in PLA2 activation.

Neutrophil-associated inflammatory responses in rats are inhibited by phenylarsine oxide.

NADPH oxidase is a phagocyte-specific enzyme which produces O2- and so initiates a cascade of reactive oxygen species formation. Inflammatory diseases involve overproduction of reactive oxygen species which induce tissue damage. Phenylarsine oxide has been described previously as a complete and direct inhibitor of NADPH oxidase in vitro that acts by covalently binding to vicinal thiol groups of a membrane-associated component of the enzyme. In the present work, the potential anti-inflammatory effect of phenylarsine oxide was tested on two experimental models in rats, carrageenan-induced paw oedema and lipopolysaccharide-mediated lung inflammation. Intraperitoneal injection of phenylarsine oxide reduced (i) reactive oxygen species production by rat phagocytes, (ii) neutrophil infiltration into the lung after inhalation of lipopolysaccharide and (iii) neutrophil-dependent oedema induced by carrageenan in hindpaws. We conclude that phenylarsine oxide has anti-inflammatory properties which are probably exerted by its ability to inhibit neutrophil NADPH oxidase-dependent reactive oxygen species production. The present work provides the basis for the development of new anti-inflammatory, arsenic-free agents reacting at the phenylarsine oxide site, which seems to be the Achilles' heel of NADPH oxidase.

Interference of BN 52021 (ginkgolide B) with the bronchopulmonary effects of PAF-acether in the guinea-pig.

The interaction between the ginkgolide BN 52021 and the effects of PAF-acether on the bronchopulmonary system of the guinea-pig was studied. In pentobarbitone or ethyl carbamate-anaesthetized animals, BN 52021 (1 mg/kg i.v. or 10 mg/kg p.o.) inhibited bronchoconstriction, the hematocrit increase and the accompanying thrombopenia and leukopenia induced by PAF-acether (33-100 ng/kg) and failed to block the bronchoconstriction produced by collagen, arachidonic acid and the tripeptide formyl-Met-Leu-Phe (FMLP). BN 52021, 3 mg/kg, reduced the bronchoconstriction induced by aerosolized PAF-acether. BN 52021, 300 microM, also inhibited the superoxide production by PAF-acether-stimulated alveolar macrophages and failed to reduce the same effects when triggered by FMLP (0.01-1 microM). BN 52021 blocked the formation of thromboxane-triggered by PAF-acether (100 ng) injected into perfused lung, under conditions where the effects of arachidonic acid where not modified. Finally, pretreatment of parenchyma lung strips with BN 52021 (100 microM) partially inhibited the contraction induced by PAF-acether (0.1 microM) and suppressed the accompanying release of thromboxane. BN 52021 is a selective antagonist of the effects of PAF-acether on the bronchopulmonary system and on circulating blood cells of the guinea-pig.

Pharmacological profile of 48740 R.P., a PAF-acether antagonist.

The pyrrolo-thiazole derivative 48740 R.P. inhibited the platelet-activating factor (PAF-acether)-induced aggregation of human and rabbit platelets and was poorly effective against ADP- and arachidonic acid-induced platelet aggregation. 48740 R.P. prevented the activation of guinea-pig alveolar macrophages by PAF-acether, and the PAF-acether-induced thromboxane B2 production from guinea-pig lungs. 48740 R.P. (3 mg/kg i.v.) antagonized selectively in anaesthetized guinea-pigs the bronchoconstriction due to PAF-acether without affecting that due to acetylcholine, histamine, serotonin, thromboxane A2 analogue U-46,619 and arachidonic acid. A higher dose of 48740 R.P. (10 mg/kg i.v.) was required to block the thrombocytopenia and the leucopenia induced by PAF-acether in the propranolol-treated guinea-pigs. 48740 R.P. (30 mg/kg i.v.) antagonized the PAF-acether effects when bronchoconstriction was induced by aerosolized PAF-acether. 48740 R.P. is a selective antagonist of PAF-acether under in vitro and in vivo conditions.

Cytotoxicity of phenazine methosulfate in isolated rat hepatocytes is associated with superoxide anion production, thiol oxidation and alterations in intracellular calcium ion homeostasis.

The metabolism of phenazine methosulfate (PMS) by isolated rat hepatocytes is associated with superoxide anion production, and with a substantial decrease in intracellular levels of reduced glutathione, most of which is oxidized to GSSG. A marked loss of protein-free sulfhydryl groups also occurs when intracellular glutathione is depleted, and cytotoxicity follows. These effects are associated with the inhibition of the plasma membrane Ca2+-ATPase and with intracellular accumulation of calcium ion which is preferentially sequestered in mitochondria. Maintenance of protein sulfhydryl groups in the reduced state by dithiothreitol (DTT) prevents the alterations in intracellular calcium homeostasis and protects against toxicity.

Differential induction of stress proteins and functional effects of heat shock in human phagocytes.

Induction of specific heat shock (HS) proteins (HSP) has been described as a response of human monocytes to phagocytosis, and HSP may play protective roles in infection and immunity. Here we compared the stress response in monocytes and polymorphonuclear neutrophils during exposure to the classical inducers of HSP, i.e., HS and cadmium. We also investigated the stress response in these two phagocytic cells after particulate (phagocytosis) and nonparticulate [f-Met-Leu-Phe (FMLP)] activation of the respiratory burst enzyme NADPH oxidase. HS and cadmium induced stress protein synthesis in both cell types. In contrast, phagocytosis induced HSP in monocytes only, while FMLP did so in neutrophils only. This differential regulation of stress proteins might relate to physiological and functional differences between monocytes and neutrophils. With respect to functional effects of HS, we examined, in human monocytes and in neutrophils, the effect of HS on NADPH oxidase-mediated O2- generation as well as on phagocytosis, bacterial killing, and superoxide dismutase (SOD) activity. In monocytes, as in neutrophils, NADPH oxidase activity was inhibited by HS, while thermotolerance prevented this inhibition. Phagocytosis and bacterial killing were unaltered by HS. SOD activity transiently increased in monocytes but decreased in neutrophils upon exposure to HS. These observations indicate differential induction of HSP in human phagocytes and differential regulation of phagocytes' functions by HS.

[Effect of trimetazidine on membrane changes induced by oxygen free radicals in human red cells]. / Effet de la trimétazidine sur les altérations membranaires induites par les radicaux libres oxygénés dans les globules rouges humains.

It is well known that oxygen free radicals are generated in different ischaemic tissues and can mediate cell injury. Furthermore, a loss of intracellular potassium is involved in heart ischemia. We previously developed a method to study a possible connection between these two phenomena in human erythrocytes treated in vitro with an oxygen free radical generator: phenazine methosulfate. We showed that transport systems specific to potassium are poorly inhibited (sodium-potassium pump; sodium-potassium cotransport) or unaffected (Gardos effect, chlorine-dependent potassium transport); that the erythrocyte potassium loss is essentially due to an increase in passive potassium permeability resulting from lipid peroxidation and that alterations of transport pathways are enhanced by diethyldithiocarbamate, a superoxide dismutase inhibitor. On the other hand, a cardioprotective drug, trimetazidine, indicated in ischaemic heart disease, which has no calcium antagonist action or coronary vasodilator effect, was studied as a possible candidate for preventing such damage by free radicals. Red cells collected from healthy donors previously treated with trimetazidine were incubated in vitro with phenazine methosulfate and diethyldithiocarbamate. We observed significant decreases in oxygen free radical-dependent passive potassium permeability and lipid peroxidation. This strongly suggests that trimetazidine possesses a free radical scavenger activity which may explain its cardioprotective role.

Activation of NADPH-oxidase by arachidonic acid involves phospholipase A2 in intact human neutrophils but not in the cell-free system.

The mechanism involved in the stimulation of NADPH-oxidase by arachidonic acid (AA) in intact human neutrophils was studied and compared with that involved in a cell-free system. [3H]-AA was released from pre-labeled cells upon AA stimulation, and phospholipase A2 inhibitors reduced in parallel the release of [3H]-AA and superoxide. Cyclooxygenase, lipoxygenase or protein kinase inhibitors failed to affect either response. In a cell-free system, no release of [3H]-AA was observed after AA addition, whereas NADPH-oxidase was activated; the generation of superoxide was not inhibited by phospholipase inhibitors and was not initiated by adding phospholipase A2 to the preparation. Thus AA stimulates NADPH-oxidase through a phospholipase A2 mediated pathway in intact cells, but activates the oxidase independent of phospholipase A2 in a broken cell system, suggesting distinctive mechanisms of activation for each system.

Effect of trimetazidine on membrane damage induced by oxygen free radicals in human red cells.

The effect of trimetazidine, 1-(2, 3, 4 trimethoxybenzyl)piperazine di-hydrochloride, on membrane damage induced by oxygen free radicals in red cells was studied in seven healthy volunteers after oral administration. Red cells collected prior to and after a 7 day treatment period with trimetazidine were incubated in the presence of phenazine methosulphate (an intracellular oxygen free radical generator) and diethyldithiocarbamate (a Cu-Zn superoxide dismutase inhibitor). The loss of intracellular K+ induced by oxygen free radicals and the membrane content of peroxidated lipids were significantly reduced in red cells collected after the period of treatment. These results indicate a potent antioxidant activity of trimetazidine which could explain its cardioprotective role during ischaemic and reperfusion phases in which oxygen free radicals are generated and probably implicated in the genesis of cardiac cell injury.

Hck is activated by opsonized zymosan and A23187 in distinct subcellular fractions of human granulocytes.

Regulation of neutrophil responses is known to involve tyrosine phosphorylation. Hck, a major neutrophil protein-tyrosine kinase, becomes expressed during differentiation of human promyelocytic NB4 cells into neutrophil-like cells. Hck is mainly localized in a secretory granule-enriched cell fraction, but it is also present in a granule-free membrane fraction and the cytosol. Hck is rapidly and transiently activated upon stimulation of differentiated NB4 cells or human neutrophils with serum-opsonized zymosan or the calcium ionophore A23187, but not by phorbol 12-myristate 13-acetate. In NB4 cells, Hck is also weakly activated by fMet-Leu-Phe. Cell fractionation showed that opsonized zymosan and A23187 induce Hck activation in distinct subcellular fractions. Both stimuli activate Hck in the secretory granule-enriched fraction, but only A23187 activates the kinase in the granule-free membrane fraction. Our results suggest that Hck might regulate early signal transduction events induced by opsonized zymosan and A23187, and that the different subcellular fractions of Hck might serve discrete functions, one of which could be regulation of the degranulation response.

Annexin 3 is associated with cytoplasmic granules in neutrophils and monocytes and translocates to the plasma membrane in activated cells.

Annexins are soluble proteins capable of binding to phospholipid membranes in a calcium-dependent manner. Annexin 3, a 33 kDa protein mainly expressed in neutrophils, aggregates granules in cell-free assays, and a 36 kDa variant of this protein, specifically expressed in monocytes, has recently been identified. To obtain further information on these proteins, we defined their subcellular localization in resting and activated cells by immunofluorescence microscopy. Both proteins were associated with cytoplasmic granules in resting cells. We obtained evidence to indicate that, in neutrophils which possess a heterogenous granule population, annexin 3 was more likely to be associated with the specific granules. In cells activated with phorbol 12-myristate 13-acetate or opsonized zymosan, the 33 kDa and 36 kDa proteins translocated to the plasma or the phagosome membrane. Upon stimulation with A23187, annexin 3 translocated to the plasma membrane only in neutrophils. We also report that while annexin 3 was associated with restricted membranes in intact cells, it binds indiscriminately to every membrane fraction in cell-free assay. In conclusion, association of both forms of annexin 3 with granules suggests that these proteins could be implicated in processes of granule fusion.

Association of rap1 and rap2 proteins with the specific granules of human neutrophils. Translocation to the plasma membrane during cell activation.

Activation of human neutrophils involves the degranulation of specific and azurophil granules. This process is GTP-dependent and the presence of small GTP-binding proteins (SGBPs) has been detected in the two granule populations. At present, none of these SGBPs has been definitely identified. In order to characterize some of these proteins and obtain further insights as to their potential role in degranulation processes, we have used specific antibodies directed against the ras-related rap1 and rap2 proteins. By immunoblot analysis, we observed that rap2p is predominantly located in specific granules, whereas rap1p is detected both in specific granules and a fraction enriched in plasma membranes. Neither rap1p nor rap2p was found in the cytosol or in azurophil granules. Similarly, by indirect immunofluorescence, we observed that cytoplasmic granules were stained with anti-rap1p antibodies and anti-rap2p antibodies, and the plasma membrane was labeled with both antibodies but more distinctly with anti-rap1p than with anti-rap2p antibodies. rap1p and rap2p are tightly bound to the membrane of specific granules since they cannot be extracted by high salt or alkaline buffers. In addition, treatment of intact specific granules with pronase induced the degradation of rap proteins suggesting that they are exposed to the cytoplasmic face of the granules. Degranulation of neutrophils consists of the translocation and subsequent fusion of granules with the plasma membrane. Activation of this process induced the accumulation of rap proteins in the plasma membrane as observed by subcellular fractionation and indirect immunofluorescence experiments; this was not associated with the appearance of a soluble form of these proteins, showing that they remain membrane-bound during this process. The identification and subcellular localization of rap1p and rap2p at the surface of specific granules and the observation that they translocate to the plasma membrane upon cell stimulation without appearance of soluble forms constitute an important step toward the understanding of their physiological functions in human neutrophils.