Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex

The low molecular weight GTP binding protein Rac is essential to the activation of the NADPH oxidase complex, involved in pathogen killing during phagocytosis. In resting cells, Rac exists as a heterodimeric complex with Rho GDP dissociation inhibitor (Rho-GDI). Two types of interactions exist between Rac and Rho-GDI: a protein-lipid interaction, implicating the polyisoprene of the GTPase, as well as protein-protein interactions. Using the two-hybrid system, we show that nonprenylated Rac1 interacts very weakly with Rho-GDI, pointing to the predominant role of protein-isoprene interaction in complex formation. In the absence of this strong interaction, we demonstrate that three sites of protein-protein interaction, Arg66(Rac)-Leu67(Rac), His103(Rac), and the C-terminal polybasic region Arg183(Rac)-Lys188(Rac), are involved and cooperate in complex formation. When Rac1 mutants are prenylated by expression in insect cells, they all interact with Rho-GDI. Rho-GDI is able to exert an inhibitory effect on the GDP/GTP exchange reaction except in the complex in which Rac1 has a deletion of the polybasic region (Arg183(Rac)-Lys188(Rac)). This complex is, most likely, held together through protein-lipid interaction only. Although able to function as GTPases, the mutants of Rac1 that failed to interact with Rho-GDI also failed to activate the NADPH oxidase in a cell-free assay after loading with GTP. Mutant Leu119(Rac)Gln could both interact with Rho-GDI and activate the NADPH oxidase. The Rac1/Rho-GDI and Rac1(Leu119Gln)/Rho-GDI complexes, in which the GTPases were bound to GDP, were found to activate the oxidase efficiently. These data suggest that Rho-GDI stabilizes Rac in an active conformation, even in the GDP-bound state, and presents it to its effector, the p67phox component of the NADPH oxidase.     

Rac1、Rac2参与调节人单核细胞趋化和NADPH氧化酶活性

为了探讨小分子GTP酶蛋白Rac1和Rac2在人单核细胞中趋化迁移以及还原型辅酶II（NADPH）氧化酶活性中的作用,采用小分子干扰siRNA对人单核细胞中RAC1、RAC2分别进行特异性抑制,采用实时定量PCR技术、免疫印迹技术在RNA和蛋白质水平上确认抑制效果,使用甲酰三肽(formyl-met-leu-phe,fMLP)、人单核细胞趋化因子（monocyte chemoattractant protein-1,MCP-1）诱导单核细胞趋化;用血清调理的酵母多糖（serum opsonized zymosan,ZOP)、佛波酯（phosphomolybdic acid, PMA）激活单核细胞NADPH氧化酶活性,诱导活性氧(Reactive oxygen species, ROS)产生,以此对Rac1和Rac2作用进行研究. 结果表明,小分子干扰siRNA能够在mRNA水平和蛋白质水平分别有效抑制目的基因表达;使用Chamber assay方法发现,仅Rac1参与了fMLP、MCP-1诱导的人单核细胞趋化. Rac激活实验确证,Rac1参与MCP-1诱导的趋化;细胞色素C还原法表明,Rac1和Rac2均参与PMA和ZOP诱导人单核细胞ROS生成. 在人单核细胞中,RAC1和RAC2基因沉默模型的成功建立以及初步研究显示,Rac1和Rac2的不同作用结果将为深入研究它们在人单核细胞中的功能奠定了良好基础.     

Reactive oxygen species produced by the NADPH oxidase 2 complex in monocytes protect mice from bacterial infections

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent life-threatening bacterial and fungal infections. CGD results from defective production of reactive oxygen species by phagocytes caused by mutations in genes encoding the NADPH oxidase 2 (NOX2) complex subunits. Mice with a spontaneous mutation in Ncf1, which encodes the NCF1 (p47(phox)) subunit of NOX2, have defective phagocyte NOX2 activity. These mice occasionally develop local spontaneous infections by Staphylococcus xylosus or by the common CGD pathogen Staphylococcus aureus. Ncf1 mutant mice were more susceptible to systemic challenge with these bacteria than were wild-type mice. Transgenic Ncf1 mutant mice harboring the wild-type Ncf1 gene under the human CD68 promoter (MN(+) mice) gained the expression of NCF1 and functional NOX2 activity specifically in monocytes/macrophages, although minimal NOX2 activity was also detected in some CD11b(+)Ly6G(+) cells defined as neutrophils. MN(+) mice did not develop spontaneous infection and were more resistant to administered staphylococcal infections compared with MN(-) mice. Most strikingly, MN(+) mice survived after being administered Burkholderia cepacia, an opportunistic pathogen in CGD patients, whereas MN(-) mice died. Thus, monocyte/macrophage expression of functional NCF1 protected against spontaneous and administered bacterial infections.     

Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.     

Role of the phospholipid binding sites,PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O₂⁻), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b₅₅₈ (cyt b₅₅₈) and four cytosolic components: p40^phox, p47^phox, p67^phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b₅₅₈ to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47^phox. We also used a fused chimera consisting of p47^phox(aa 1–286), p67^phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47^phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b₅₅₈. They also have an impact on O₂.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.     

Essential role of Rac1/NADPH oxidase in nerve growth factor induction of TRPV1 expression

Nerve growth factor (NGF) regulates the nociceptive properties of a subset of small diameter sensory neurons by increasing the expression of the heat-sensing transient receptor potential (TRP) channel, TRPV1. This action involves activation of the tyrosine kinase receptor (Trk) A/p38 MAPK pathway. Recent studies indicate that activation of TrkA promotes superoxide generation via NADPH oxidase. In this study, we determined whether the NADPH oxidase pathway is involved in NGF-stimulated TRPV1 expression using a rat pheochromocytoma 12 line and rat dorsal root ganglion neurons. Treatment of these cells with NGF (100 ng/mL) increased TRPV1 protein expression (approx. twofold) but not mRNA. This increase was mimicked by H(2)O(2) and attenuated by catalase and inhibitors of NADPH oxidase. NGF stimulated NADPH oxidase activity, while 24 h exposure further increased expression of the Rac1 and gp91(phox) subunits of the holoenzyme. Inhibition of NADPH oxidase by transient transfection of a dominant negative Rac1 mutant (RacN17) plasmid blocked NGF-stimulated TRPV1 protein expression, while expression of a constitutively active Rac1 increased basal and NGF-stimulated TRPV1 levels. Inhibition of NADPH oxidase activity also attenuated NGF-dependent p38 MAPK activation. We conclude that the Rac1/NADPH oxidase pathway regulates p38 activation and TRPV1 expression which aids in the maintenance of peripheral neuron integrity and pain perception.     

Crystal structure of the Rac1-RhoGDI complex involved in nadph oxidase activation

A heterodimer of prenylated Rac1 and Rho GDP dissociation inhibitor was purified and found to be competent in NADPH oxidase activation. Small angle neutron scattering experiments confirmed a 1:1 stoichiometry. The crystal structure of the Rac1-RhoGDI complex was determined at 2.7 A resolution. In this complex in which Rac1 is bound to GDP, the switch I region of Rac1 is in the GDP conformation whereas the switch II region resembles that of a GTP-bound GTPase. Two types of interaction between RhoGTPases and RhoGDI were investigated. The lipid-protein interaction between the geranylgeranyl moiety of Rac1 and RhoGDI resulted in numerous structural changes in the core of RhoGDI. The interactions between Rac1 and RhoGDI occur through hydrogen bonds which involve a number of residues of Rac1, namely, Tyr64(Rac), Arg66(Rac), His103(Rac), and His104(Rac), conserved within the Rho family and localized in the switch II region or in its close neighborhood. Moreover, in the switch II region of Rac1, hydrophobic interactions involving Leu67(Rac) and Leu70(Rac) contribute to the stability of the Rac1-RhoGDI complex. Inhibition of the GDP-GTP exchange in Rac1 upon binding to RhoGDI partly results from interaction of Thr35(Rac) with Asp45(GDI). In the Rac1-RhoGDI complex, the accessibility of the effector loops of Rac1 probably accounts for the ability of the Rac1-RhoGDI complex to activate the NADPH oxidase.     

Participation of Rac GTPase activating proteins in the deactivation of the phagocytic NADPH oxidase

The aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase.     

Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells

Neurodegenerative diseases are attributed to impairment of the ubiquitin–proteasome system (UPS). Oxidative stress has been considered a contributing factor in the pathology of impaired UPS by promoting protein misfolding and subsequent protein aggregate formation. Increasing evidence suggests that NADPH oxidase is a likely source of excessive oxidative stress in neurodegenerative disorders. However, the mechanism of activation and its role in impaired UPS is not understood. We show that activation of NADPH oxidase in a neuroblastoma cell line (SHSY-5Y) resulted in increased oxidative and nitrosative stress, elevated cytosolic calcium, ER-stress, impaired UPS, and apoptosis. Rac1 inhibition mitigated the oxidative/nitrosative stress, prevented calcium-dependent ER-stress, and partially rescued UPS function. These findings demonstrate that Rac1 and NADPH oxidase play an important role in rotenone neurotoxicity.     

Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly

The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome b(559). This process can be reproduced in an in vitro system consisting of phagocyte membranes, p47(phox), p67(phox), and Rac, activated by an anionic amphiphile. We now show that post-translationally processed (prenylated) Rac1 initiates NADPH oxidase assembly, expressed in O(2) production, in a cell-free system containing phagocyte membrane vesicles and p67(phox), in the absence of an activating amphiphile and of p47(phox). Prenylated Cdc42Hs, a GTPase closely related to Rac, is inactive under the same conditions. Results obtained with phagocyte membrane vesicles can be reproduced fully by replacing these with partially purified cytochrome b(559), incorporated in phosphatidylcholine vesicles. Prenylated, but not nonprenylated, Rac1 binds spontaneously to phagocyte membrane vesicles and also to artificial, protein-free, phosphatidylcholine vesicles, a process counteracted by GDP dissociation inhibitor for Rho. Binding of prenylated Rac1 to membrane vesicles is accompanied by the recruitment of p67(phox) to the same location and the formation of an assembled NADPH oxidase complex, producing O(2) upon the addition of NADPH. Amphiphile and p47(phox)-independent NADPH oxidase activation by prenylated Rac1 is inhibited by Rho GDP dissociation inhibitor and by phosphatidylcholine vesicles, both competing with membrane for prenylated Rac1. We conclude that, in vitro, targeting of Rac to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly, suggesting that the principal or, possibly, the only role of Rac is to recruit cytosolic p67(phox) to the membrane environment, to be followed by the interaction of p67(phox) with cytochrome b(559).     

Continuous translocation of Rac2 and the NADPH oxidase component p67(phox) during phagocytosis

In this study, the translocation of the NADPH oxidase components p67(phox) and Rac2 was studied during phagocytosis in living cells. For this purpose, green fluorescent protein (GFP)-tagged versions of these proteins were expressed in the myeloid cell line PLB-985. First, the correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis of serum-treated zymosan by wild-type PLB-985 cells and PLB-985 X-CGD (chronic granulomatous disease) cells, which lack expression of flavocytochrome b(558). Subsequently, these constructs were used for fluorescence recovery after photobleaching studies to elucidate the turnover of these proteins on the phagosomal membrane. The turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actin cytoskeleton for correct localization of these proteins was investigated by disrupting the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatment of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 once phagocytosis was initiated. In addition, the continuous exchange of flavocytochrome b(558)-bound p67GFP and GFP-Rac2 for cytosolic p67GFP and GFP-Rac2 was still intact in cytochalasin B-treated cells, indicating that the translocation of these proteins does not depend on a rearrangement of the actin cytoskeleton.     

Apocynin prevents cyclooxygenase 2 expression in human monocytes through NADPH oxidase and glutathione redox-dependent mechanisms

In the present study we report the preventive effect of apocynin, an active constituent of the Himalayan herb Picrorhiza kurrooa, on cyclooxygenase-2 (Cox-2) synthesis and activity in human adherent monocytes exposed to serum treated zymosan (STZ) and phorbol myristate acetate (PMA). Apocynin markedly decreases the intracellular reduced/oxidized glutathione ratio (GSH/GSSG) and prevents nuclear factor-kappaB (NF-kappaB) activation in stimulated monocytes. Moreover, it reduces intracellular reactive oxygen species (ROS) generation, NADPH oxidase activity in monocyte homogenates and translocation of p47phox subunit in monocyte membranes. p47phox levels are also reduced in lysates of apocynin-treated monocytes. The inhibition of Cox-2 by apocynin is completely abrogated by GSH provision. Results from this study indicate that apocynin inhibits Cox-2 synthesis and activity induced in monocytes by an increased oxidative tone and provide an explanation for the protective effect exerted by this compound in numerous cell and animal models of inflammation. Attenuation of NADPH oxidase derived ROS coupled with GSH/GSSG reduction and suppression of NF-kappaB activation are highlighted as the molecular mechanisms responsible for Cox-2 inhibition.     

HaRas activates the NADPH oxidase complex in human neuroblastoma cells via extracellular signal-regulated kinase 1/2 pathway

In this study we have investigated the effects of the small GTP-binding-protein Ras on the redox signalling of the human neuroblastoma cell line, SK-N-BE stably transfected with HaRas(Val12). The levels of reactive oxygen species (ROS) and superoxide anions were significantly higher in HaRas(Val12) expressing (SK-HaRas) cells than in control cells. The treatment of cells with 4-(2-aminoethyl) benzenesulfonylfluoride, a specific inhibitor of the membrane superoxide generating system NADPH oxidase, suppressed the rise in ROS and significantly reduced superoxide levels produced by SK-HaRas cells. Moreover, HaRas(Val12) induced the translocation of the cytosolic components of the NADPH oxidase complex p67(phox) and Rac to the plasma membrane. These effects depended on the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK1/2) pathway, as the specific MEK inhibitor, PD98059, prevented HaRas-mediated increase in ROS and superoxide anions. In contrast, the specific phosphoinositide 3-kinase (PI3K) inhibitors LY294002 and wortmannin were unable to reverse the effects of HaRas(Val12). Moreover, cholinergic stimulation of neuroblastoma cells by carbachol, which activated endogenous Ras/ERK1/2, induced a significant increase in ROS levels and elicited membrane translocation of p67(phox) and Rac. ROS generation induced by carbachol required the activation of ERK1/2 and PI3K. Hence, these data indicate that HaRas-induced ERK1/2 signalling selectively activates NADPH oxidase system in neuroblastoma cells.     

NADPH oxidase activation is required for migration by LIGHT in human monocytes

Homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus (HSV) glycoprotein D (gD) for herpes virus entry mediator (HVEM; TR2) (LIGHT), a ligand of herpes virus entry mediator (HVEM), increased reactive oxygen species (ROS) and enhanced the destruction of bacteria in human monocytes. In this study, rhLIGHT was found to increase the expression of the chemokine receptors, chemokine receptor 1 (CCR1) and CCR2, as well as to accelerate the migration activity of human monocytes. Additionally, rhLIGHT was found to increase ROS via NADPH oxidase p47^phox phosphorylation, which was found to be required for LIGHT-induced NF-κB activation, CCR1 and CCR2 expression, migration and IL-8 and TNF-α production. Taken together, these results indicate that NADPH oxidase activation is required for rhLIGHT-induced migration in human monocytes.     

Role of Rac GTPase activating proteins in regulation of NADPH oxidase in human neutrophils

Precise spatiotemporal regulation of O₂⁻-generating NADPH oxidases (Nox) is a vital requirement. In the case of Nox1–3, which depend on the small GTPase Rac, acceleration of GTP hydrolysis by GTPase activating protein (GAP) could represent a feasible temporal control mechanism. Our goal was to investigate the molecular interactions between RacGAPs and phagocytic Nox2 in neutrophilic granulocytes. In structural studies we revealed that simultaneous interaction of Rac with its effector protein p67^phox and regulatory protein RacGAP was sterically possible. The effect of RacGAPs was experimentally investigated in a cell-free O₂⁻-generating system consisting of isolated membranes and recombinant p47^phox and p67^phox proteins. Addition of soluble RacGAPs decreased O₂⁻ production and there was no difference in the effect of four RacGAPs previously identified in neutrophils. Depletion of membrane-associated RacGAPs had a selective effect: a decrease in ARHGAP1 or ARHGAP25 level increased O₂⁻ production but a depletion of ARHGAP35 had no effect. Only membrane-localized RacGAPs seem to be able to interact with Rac when it is assembled in the Nox2 complex. Thus, in neutrophils multiple RacGAPs are involved in the control of O₂⁻ production by Nox2, allowing selective regulation via different signaling pathways.     

Co-regulation of constitutive nitric oxide synthases and NADPH oxidase by the small GTPase Rac

Nitric oxide (NO), generated by NO synthases (NOSs), has multifarious roles in signal transduction. Reactive oxygen species (ROS), generated by ubiquitous NADPH oxidases (NOXs), also participate in cellular signaling. However, the coordination of signals conveyed by NO and ROS is poorly understood. We show that the small GTPase Rac, a component of some NOXs, also interacts with and regulates the constitutively-expressed NOSs. Cellular NO and O(2)(-) production increase or decrease together following activation or inhibition of Rac, and Rac inhibition reveals transduction mechanisms that depend upon NO (vasodilation), ROS (actin polymerization) or both (cytoskeletal organization). Thus, signaling by NO and ROS may be coordinated through a common control element.     

Mechanism of homocysteine-induced Rac1/NADPH oxidase activation in mesangial cells: role of guanine nucleotide exchange factor Vav2.

We have demonstrated that homocysteine (Hcys) stimulates de novo ceramide synthesis and thereby induces NADPH oxidase activation by increase of Rac GTPase activity in rat mesangial cells (RMCs). However, which isofrom of Rac GTPases is involved in Hcys-induced NADPH oxidase activity and what mechanism mediates Hcys-induced Rac GTPase activation remain unknown. The present study first addressed the role of Rac1 and then determined the contribution of a subfamily of Guanine Nucleotide Exchange Factors (GEFs), Vav, to the action of Hcys on Rac and NADPH oxidase activities in RMCs. By small interfering RNA (siRNA), it was found that Rac1-siRNA attenuated Hcys-induced superoxide (O(2)(-)) production. To explore the mechanism activating Rac by Hcys, GEF-Vav was examined. Vav2 was found to be a predominant isoform among Vav family in RMCs. In Vav2-siRNA transfected RMCs, Hcys-induced Rac activity was blocked, which was accompanied by significant reduction of Hcys-induced O(2)(-). production. This Vav2-siRNA also blocked Rac activation induced by C16-Ceramide (C16-Cer), an intermediate lipid product stimulated by Hcys. Furthermore, we found that Hcys induced Vav2 phosphorylation in a time-dependent manner, which could be induced by C16-Cer and blocked by inhibition of de novo ceramide synthesis. These results suggest that Vav2 importantly contributes to Hcys-induced increase in Rac1 activity and consequent activation of NADPH oxidase in RMCs via ceramide-associated tyrosine phosphorylation.     

HIV-1 Nef associates with p22-phox, a component of the NADPH oxidase protein complex

Altered neutrophil function may contribute to the development of AIDS during the course of HIV infection. It has been described that Nef, a regulatory protein from HIV, can modulate superoxide production in other cells, therefore altered superoxide production in neutrophils from HIV infected patients, could be secondary to a direct effect of Nef on components of the NADPH oxidase complex. In this work, we describe that Nef, was capable of increasing superoxide production in human neutrophils. Furthermore, a specific association between Nef and p22-phox, a membrane component of the NADPH oxidase complex, was found. We propose that this association may reflect a capability of Nef to modulate by direct association, the enzymatic complex responsible for one of the most efficient innate defense mechanisms in phagocytes, contributing to the pathogenesis of the disease.