Emmanuelle Rollet-Labelle

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

Significance On activation, blood platelets package components from their cytoplasm into microparticles (MPs), tiny vesicles released by cytoplasmic membrane budding and shedding. Given that MPs can impact other cellular lineages on internalization, we aimed to decipher the mechanisms promoting MP internalization by cellular recipients. We modeled MP internalization by neutrophils and identified a predominant lipid, 12(S)-hydroxyeicosatetranoic acid, as a mediator critical for the promotion of MP internalization. MPs were found inside neutrophils from individuals with rheumatoid arthritis, and their presence in neutrophils in the joints of mice treated with arthritogenic serum is dependent on the expression of enzymes implicated in the generation of 12(S)-hydroxyeicosatetranoic acid. These findings reveal a unique molecular mechanism implicated in MP internalization relevant to inflammatory processes. Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Class IA Phosphatidylinositide 3-Kinases, rather than p110γ, Regulate Formyl-Methionyl-Leucyl-Phenylalanine-Stimulated Chemotaxis and Superoxide Production in Differentiated Neutrophil-Like PLB-985 Cells

Class I PI3Ks, through the formation of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3), are thought of as essential elements of the neutrophil response to chemotactic factors. Moreover, the recent development of PI3K-deficient mice and isoform-specific inhibitors enabled examinations of the contribution of the distinct PI3K isoforms in neutrophil activation. However, the results of these various studies are conflicting, and the exact role of the different PI3K isoforms is not yet clearly established, particularly in human cells. In the present study, we used a different approach to assess the role of the distinct PI3K isoforms in response to the chemotactic agent fMLP. We inhibited PI3K activities by the transient expression following nucleofection of dominant negative mutants of either p85α or p110γ in the human myeloid cell line PLB-985, which can be induced to express a neutrophil-like phenotype. The data obtained with this approach showed that the production of PI(3,4,5)P3 triggered by fMLP is biphasic, with a peak of production observed in a short time period that entirely depends on p110γ activity, and a delayed phase that is mediated by class IA PI3K. We also provide evidence that the PI3K-dependent functional responses (i.e., superoxide production and chemotaxis) induced by the chemotactic factor mainly involve PI3K IA and, by implication, the delayed phase of PI(3,4,5)P3 production, whereas p110γ and the early peak of PI(3,4,5)P3 do not play major roles in the initiation or the control of these responses.

Signaling through CD16b in human neutrophils involves the Tec family of tyrosine kinases

Tec kinases belong to the second largest family of nonreceptor tyrosine kinases. Although these kinases are expressed in myeloid cells, little is known about their implication in neutrophil function. We recently reported the participation of Tec kinases in the responses of human neutrophils to the bacterial peptide N‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine via G‐coupled protein receptors. In this study, we extended our investigations of Tec kinases to the signaling of the glycosylphosphatidylinositol‐linked receptor CD16b, which is highly and specifically expressed in neutrophils. The results obtained indicate that Tec is translocated to the plasma membrane, phosphorylated, and activated upon CD16b cross‐linking and that the activation of Tec is inhibited by Src‐specific inhibitors as well as by the phosphatidylinositol‐3 kinase inhibitor, wortmannin. As no specific inhibitor of Tec exists, the role of Tec kinases was further investigated using a‐Cyano‐b‐hydroxy‐b‐methyl‐N‐(2,5‐dibromophenyl)propenamide (LFM‐A13), a compound known to inhibit Bruton’s tyrosine kinase. We show that this compound also inhibits the kinase activity of Tec and provide evidence that the mobilization of intracellular calcium and the tyrosine phosphorylation of phospholipase Cγ2 (PLCγ2) induced upon CD16b engagement are inhibited by LFM‐A13. We also show that Tec kinases are important for CD16b‐dependent degranulation of neutrophils. In summary, we provide direct evidence for the implication of Tec in CD16b signaling and suggest that Tec kinases are involved in the phosphorylation and activation of PLCγ2 and subsequently, in the mobilization of calcium in human neutrophils.

Crystal-induced neutrophil activation. IX. Syk-dependent activation of class Ia phosphatidylinositol 3-kinase

The deposition of monosodium urate (MSU) crystals in the joints of humans leads to an extremely acute, inflammatory reaction, commonly known as gout, characterized by a massive infiltration of neutrophils. Direct interactions of MSU crystals with human neutrophils and inflammatory mediators are crucial to the induction and perpetuation of gout attacks. The intracellular signaling events initiated by the physical interaction between MSU crystals and neutrophils depend on the activation of specific tyrosine kinases (Src and Syk, in particular). In addition, PI‐3Ks may be involved. The present study investigates the involvement of the PI‐3K family in the mediation of the responses of human neutrophils to MSU crystals. The results obtained indicate that the interaction of MSU crystals with human neutrophils leads to the stimulation of class Ia PI‐3Ks by a mechanism that is dependent on the tyrosine kinase Syk. We also found an increase in the amount of p85 associated with the Nonidet P‐40‐insoluble fraction derived from MSU crystal‐stimulated human neutrophils. Furthermore, MSU crystals induce the formation of a complex containing p85 and Syk, which is mediated by the Src family kinases. Finally, evidence is also obtained indicating that the activation of PI‐3Ks by MSU crystals is a critical element regulating phospholipase D activation and degranulation of human neutrophils. The latter response is likely to be involved in the joint and tissue damage that occurs in gouty patients.

Recruitment of the cross-linked opsonic receptor CD32A (FcγRIIA) to high-density detergent-resistant membrane domains in human neutrophils

We have previously shown that CD32A (or FcgammaRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-beta-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-beta-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.

Early Events in the Activation of FcγRIIA in Human Neutrophils: Stimulated Insolubilization, Translocation to Detergent-Resistant Domains, and Degradation of FcγRIIA

The signal transduction mechanisms associated with the ligation of FcγRIIA in human neutrophils are as yet only incompletely characterized. In the present study, we have investigated the distribution and fate of FcγRIIA following its cross-linking. The results obtained indicate that cross-linking of FcγRIIA led, within a few seconds, to its translocation into a nonionic detergent-insoluble fraction. This was followed, within a couple of minutes, by a substantial loss of immunoreactive FcγRIIA in the cells. The stimulated degradation of FcγRIIA was blocked by the Src kinase inhibitor PP1 but not by wortmannin, ST-638, piceatannol, or cytochalasin B. Cross-linked FcγRIIA could be solubilized by saponin (in the presence of Nonidet P-40) and by β-octylglucoside. Sucrose gradient analysis of the distribution of FcγRIIA revealed that its cross-linking led to its translocation into the pellets and not the light buoyant density fractions classically associated with lipid rafts. Disruption of cholesterol-containing membrane microdomains with filipin prevented the degradation of FcγRIIA but did not inhibit the stimulation of the pattern of tyrosine phosphorylation or the mobilization of calcium that followed FcγRIIA cross-linking. These data suggest that both cholesterol-rich domains and Src kinases are required for the degradation of the activated FcγRIIA and provide new insights into the early events following FcγRIIA cross-linking.

Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling

In stimulated neutrophils, the majority of tyrosine-phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells that are lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserve and solubilize tyrosine-phosphorylated proteins but also retain their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and hypertonic buffers containing non-ionic detergents resulted in the solubilization of a significant fraction of tyrosine-phosphorylated proteins. Furthermore, we observed in neutrophils in which CD32 was cross-linked that the tyrosine kinase activity of Lyn was enhanced in the soluble fraction recovered from the hypertonic lysis but not in that derived from the first hypotonic lysis. Furthermore, we detected tyrosine kinase activity and the presence of the tyrosine kinase Syk in association with CD32 in the soluble hypertonic lysis fraction. This fraction also contained most of the tyrosine-phosphorylated proteins including Cbl, Syk and CD32 itself. The results of this study provide a new experimental procedure for the investigation of tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types.

Crystal-Induced Neutrophil Activation: XI. Implication and Novel Roles of Classical Protein Kinase C

Monosodium urate (MSU) crystals are among the most potent proinflammatory stimuli, and an innate immune inflammatory response to the crystal surface is involved in the pathology of gouty arthritis. Furthermore, MSU crystals have recently been identified as danger signals able to induce the maturation of dendritic cells. Release of the crystals into the joint cavity promotes an acute inflammation characterized by a massive infiltration of neutrophils that leads to tissue damage. Protein kinase C (PKC) represents a family of serine/threonine kinases that play central signaling roles in multiple cellular responses. This family of kinases is divided into three subfamilies based on second messenger requirements: conventional (or classical), novel, and atypical. Despite their role in signal transduction, very little is known about the involvement of the PKC family in the inflammatory reaction induced by MSU crystals. In the present study, we show that MSU crystals activate conventional PKC isoforms, and that this activation is necessary for the MSU crystal-induced degranulation and generation of a chemotactic activity in the supernatants of MSU crystal-stimulated human neutrophils. Evidence is also obtained that the tyrosine kinase Syk is a substrate of PKC and that the PKC-mediated serine phosphorylation of Syk is necessary to its interaction with the regulatory subunit of PI3K kinases (p85) and thus to the subsequent activation of these lipid kinases. These results suggest novel means of modulating neutrophil responses (through the specific regulation of PKC) during the acute phase of MSU crystal-induced inflammation.

CD16b associates with high-density, detergent-resistant membranes in human neutrophils

CD16b is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI (glycosylphosphatidylinositol) anchor. GPI-anchored proteins often preferentially localize to DRMs (detergent-resistant membranes) that are rich in sphingolipids and cholesterol and play an important role in signal transduction. Even though the responses to CD16b engagement have been intensively investigated, the importance of DRM integrity for CD16b signalling has not been characterized in human neutrophils. We provide direct evidence that CD16b constitutively partitions with both low- and high-density DRMs. Moreover, upon CD16b engagement, a significant increase in the amount of the receptor is observed in high-density DRMs. Similarly to CD16b, CD11b also resides in low- and high-density DRMs. In contrast with CD16b, the partitioning of CD11b in DRMs does not change in response to CD16b engagement. We also provide evidence for the implication of Syk in CD16b signalling and its partitioning to DRMs in resting and activated PMNs (polymorphonuclear neutrophils). Additionally, DRM-disrupting agents, such as nystatin and methyl-beta-cyclodextrin, alter cellular responses to CD16b receptor ligation. Notably, a significant increase in the mobilization of intracellular Ca2+ and in tyrosine phosphorylation of intracellular substrates after CD16b engagement is observed. Altogether, the results of this study provide evidence that high-density DRMs play a role in CD16b signalling in human neutrophils.

Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates. II. A sequential lysis protocol for the analysis of tyrosine phosphorylation-dependent signalling

In stimulated neutrophils, the majority of tyrosine phosphorylated proteins are concentrated in Triton X-100 or NP-40 insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine phosphorylation are, however, performed using the supernatants of cells lysed in non-ionic detergent-containing buffers (RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserved and solubilized tyrosine phosphorylated proteins but also retained their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and hypertonic buffers containing non-ionic detergents resulted in the solubilisation of a significant fraction of tyrosine phosphorylated proteins. Furthermore, we observed that in monosodium urate crystals-stimulated neutrophils, Lyn activity was enhanced in the soluble fraction recovered from the hypertonic fraction, but not from that of the first hypotonic lysis. The distribution of tyrosine phosphorylated proteins between the NP-40 soluble and insoluble fractions was both substrate- and agonist-dependent. In neutrophils stimulated with fMet-Leu-Phe, MSU crystals or by CD32 ligation, the tyrosine phosphorylated proteins were mostly insoluble. On the other hand, in GM-CSF-treated cells, the phosphoproteins were more equally distributed between the two fractions. The results of this study provide a new experimental procedure for the investigation of tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types.