Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury.

Acute lung injury (ALI) is an inflammatory disorder associated with recruitment and activation of neutrophils in lungs. Rac2, a member of the Rho GTPase subfamily, is an essential regulator of neutrophil degranulation, superoxide release, and chemotaxis. Here, we hypothesized that Rac2 is important in mediating lung injury. Using a model of IgG immune complex-mediated ALI, we showed that injury was attenuated in rac2(-/-) mice compared with wild-type (WT) mice undergoing ALI, with significant decreases in alveolar leukocyte numbers, vascular leakage, and the inflammatory mediators, myeloperoxidase (MPO) and matrix metalloproteinases (MMPs). Reduced injury in rac2(-/-) mice was not associated with diminished cytokine and chemokine production, since bronchoalveolar lavage (BAL) levels of IL-17, TNF, CCL3, CXCL1, and CXCL2 were similarly increased in WT and rac2(-/-) mice with ALI compared with sham-treated mice (no ALI). BAL levels of MMP-2 and MMP-9 were significantly decreased in the airways of rac2(-/-) mice with ALI. Immunohistochemical analysis revealed that MMP-2 and MMP-9 expression was evident in alveolar macrophages and interstitial neutrophils in WT ALI. In contrast, MMP-positive cells were less prominent in rac2(-/-) mice with ALI. Chimeric mice showed that Rac2-mediated lung injury was dependent on hematopoietic cells derived from bone marrow. We propose that lung injury in response to immune complex deposition is dependent on Rac2 in alveolar macrophages and neutrophils.

Neutrophil primary granule release and maximal superoxide generation depend on Rac2 in a common signalling pathway.

Neutrophils play an integral role in innate immunity by undergoing degranulation and respiratory burst in response to inflammatory stimuli. Rac2, a monomeric GTP-binding protein, has been shown to be involved in several neutrophil functions, including primary granule release and superoxide (O(2)(-.) generation. We hypothesized that Rac2 is a common signalling molecule required for primary granule translocation and maximal O(2)(-.) production. Using bone marrow neutrophils from Rac2 knockout (KO) mice and wild type C57Bl/6 mice, we found that primary granule elastase and myeloperoxi dase release were absent in Rac2 KO neutrophils upon chemoattractant stimulation. Rac2 KO neutrophils also failed to produce maximal levels of extracellular O(2(-.) generation in response to phorbol myristate acetate (PMA). Although PMA was ineffective at eliciting primary granule mediator release, it induced secondary granule exocytosis in both WT and Rac2 KO neutrophils. Thus, the signalling pathway leading to primary granule release utilized Rac2, which was also necessary for full activation of O(2)(-.) generation in stimulated neutrophils. These findings indicate that O(2)(-.) release and secondary granule secretion may use protein kinase C (PKC) - dependent pathways, whereas primary granule exocytosis appears to rely on PKC-independent signalling events. These findings shed light on possible signalling mechanisms involved in granule secretion from activated neutrophils responding to different stimuli.

Anaplasma phagocytophilum utilizes multiple host evasion mechanisms to thwart NADPH oxidase-mediated killing during neutrophil infection.

Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O(2)(-)) and initiate oxidative killing of microorganisms. A. phagocytophilum's unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects of A. phagocytophilum infection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. Following neutrophil binding, Anaplasma invasion requires at least 240 min. During its prolonged association with the neutrophil plasma membrane, A. phagocytophilum stimulates NADPH oxidase assembly, as indicated by increased cytochrome b(558) mobilization to the membrane, as well as colocalization of Rac and p22(phox). This initial stimulation taxes the host neutrophil's finite oxidase reserves, as demonstrated by time- and bacterial-dose-dependent decreases in secondary activation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). This stimulation is modest, however, and does not diminish oxidase stores to nearly the extent that Escherichia coli, serum-opsonized zymosan, FMLP, or PMA do. Despite the apparent activation of NADPH oxidase, no change in ROS-dependent chemiluminescence is observed upon the addition of A. phagocytophilum to neutrophils, indicating that the bacterium may scavenge exogenous O(2)(-). Indeed, A. phagocytophilum rapidly detoxifies O(2)(-) in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22(phox) and gp91(phox). Thus, A. phagocytophilum employs at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing.

Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects.

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O(2)(-)-mediated cytotoxicity. PMA-induced O(2)(-) release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O(2)(-) release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400-800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22(phox)-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22(phox). Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O(2)(-). Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.

Rac2 is critical for neutrophil primary granule exocytosis.

Neutrophil degranulation is important in many inflammatory disorders, although the intracellular mechanisms underlying this process remain poorly understood. The Rho GTPase, Rac2, has been implicated in control of degranulation in earlier studies. We hypothesized that Rac2 selectively regulates neutrophil primary granule release. Using bone marrow and peritoneal exudate neutrophils from rac2(-/-) mice in comparison with similar cells from wild-type C57Bl/6 mice, we found that primary granule myeloperoxidase and elastase release was absent in Rac2(-/-) neutrophils in response to chemoattractant stimulation, cytochalasin B/f-Met-Leu-Phe (CB/fMLP), and CB/leukotriene B4. Rac2(-/-) neutrophils also failed to exhibit mobilization of the primary granule marker CD63+ during CB/fMLP stimulation as determined by confocal microscopy. Priming of Rac2(-/-) neutrophils with tumor necrosis factor (TNF) or by peritoneal elicitation did not rescue the defect in primary granule release. However, phosphorylation of p38 mitogen-activated protein (MAP) kinase in Rac2(-/-) neutrophils was evident in response to CB/fMLP and/or TNF. Primary granule density and morphology were normal in Rac2(-/-) neutrophils. Secondary specific and tertiary granule release, measured by lactoferrin immunoassay and zymography, was normal in response to CB/fMLP and adhesion to fibronectin. These findings suggest an obligatory role for Rac2 in regulation of primary granule release by neutrophils.