Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles

The Rho family GTPase Rac is a crucial participant in numerous cellular functions and acts as a molecular switch for signal transduction. Mice deficient in hemopoietic-specific Rac2 exhibited agonist-specific defects in neutrophil functions including chemoattractant-stimulated filamentous actin polymerization and chemotaxis, and superoxide production elicited by phorbol ester, fMLP, or IgG-coated particles, despite expression of the highly homologous Rac1 isoform. In this study, functional responses of Rac2-null murine macrophages were characterized to examine whether Rac2 also has nonredundant functions in this phagocytic lineage. In contrast to murine neutrophils, in which Rac1 and Rac2 are present in similar amounts, Rac1 was approximately 4-fold more abundant than Rac2 in both bone marrow-derived and peritoneal exudate macrophages, and macrophage Rac1 levels were unchanged by the absence of Rac2. Accumulation of exudate macrophages during peritoneal inflammation was reduced in rac2(-/-) mice. FcgammaR-mediated phagocytosis of IgG-coated SRBC was also significantly decreased in Rac2-null macrophages, as was NADPH oxidase activity in response to phorbol ester or FcgammaR stimulation. However, phagocytosis and oxidant production stimulated by serum-opsonized zymosan was normal in rac2(-/-) macrophages. Macrophage morphology was also similar in wild-type and Rac2-null cells, as was actin polymerization induced by FcgammaR-mediated phagocytosis or M-CSF. Hence, Rac2-null macrophages have selective defects paralleling many of the observed functional defects in Rac2-null neutrophils. These results provide genetic evidence that although Rac2 is a relatively minor isoform in murine macrophages, it plays a nonoverlapping role with Rac1 to regulate host defense functions in this phagocyte lineage.     

MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis

Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immune responses to microbial infection. Recent studies have shown that Toll-like receptors （TLRs） play an important role in promoting the clearance of bacteria by up-regulating the phagocytic activity of macrophages. However, information regarding the signaling mechanism of TLR-mediated phagocytosis is still limited. Here, we provide evidence that the stimulation of TLR4 with LPS leads to activation of multiple signaling pathways including MAP kinases, phosphatidylinositide 3-kinase （PI3K）, and small GTPases in the murine macrophage-like cell line RAW264.7. Specific inhibition of Cdc42/Rac or p38 MAP kinase, but not PI3K, reduced TLR4-induced phagocytosis of bacteria. Moreover, we have found that either inhibition of actin polymerization by cytochalasin D or the knockdown of actin by RNAi markedly reduced the activation of Cdc42 and Rac by LPS. TLR4-induced activation of Cdc42 and Rac appears to be independent of MyD88. Taken together, our results described a novel actin-Cdc42/Rac pathway through which TLRs can specifically provoke phagocytosis.     

Nonphlogistic clearance of late apoptotic neutrophils by macrophages: efficient phagocytosis independent of beta 2 integrins

Neutrophils undergo constitutive death by apoptosis, leading to safe nonphlogistic phagocytosis and clearance by macrophages. Recent work has shown that before secondary necrosis, neutrophils exhibiting classical features of apoptosis can progress to a morphologically defined late apoptotic state. However, whether such neutrophils could be safely cleared was unknown. We now report that human late apoptotic neutrophils could be purified from cultured neutrophil populations undergoing constitutive death and were subsequently ingested by human monocyte-derived macrophages by serum-independent mechanisms that did not trigger the release of IL-8 or TNF-alpha. Such ingestion was specifically inhibited by Abs to thrombospondin-1 and the alpha(v)beta(3) vitronectin receptor. Murine bone marrow-derived macrophage phagocytosis of late and early apoptotic neutrophils occurred by similar mechanisms, proceeding with the same efficiency as that observed for wild-type controls when macrophages from [alpha(m)](-/-) or [beta(2)](-/-) mice were used. We conclude that specific nonphlogistic, beta(2) integrin-independent mechanisms involving thrombospondin-1 and alpha(v)beta(3) allow macrophages to ingest late apoptotic neutrophils without eliciting inflammatory cytokine secretion.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcgamma receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.     

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5′ adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.     

Vav proteins in neutrophils are required for FcgammaR-mediated signaling to Rac GTPases and nicotinamide adenine dinucleotide phosphate oxidase component p40(phox)

Phagocytes generate reactive oxygen species, the regulation of which is important in eliminating ingested microbes while limiting tissue damage. Clustering of FcgammaRs results in the activation of Vav proteins, Rho/Rac guanine nucleotide exchange factors, and results in robust superoxide generation through the NADPH oxidase. In this study, studies in neutrophils isolated from mice deficient in Vav or Rac isoforms demonstrate a critical role for Vav3 in Rac2-dependent activation of the NADPH oxidase following FcgammaR clustering. However, studies in cytokine-primed cells revealed a strict requirement for Vav1 and Vav3 and Rac1 and Rac2 in the FcgammaR-mediated oxidative burst. In comparison, Vav was not essential for PMA or G protein-coupled receptor-mediated superoxide generation. The FcgammaR-mediated oxidative burst defect in Vav-deficient cells was linked to aberrant Rac activation as well as Rac- and actin-polymerization-independent, but PI3K-dependent, phosphorylation of the NADPH oxidase component p40(phox). In macrophages, Vav regulation of Rac GTPases was required specifically in FcgammaR-mediated activation of the oxidative burst, but not in phagocytosis. Thus, Vav proteins specifically couple FcgammaR signaling to NADPH oxidase function through a Rac-dependent as well as an unexpected Rac-independent signal that is proximal to NADPH oxidase activation and does not require actin polymerization.     

Role of CrkII in Fcgamma receptor-mediated phagocytosis

Phagocytosis of IgG-opsonized pathogens by Fcgamma receptors requires extensive remodeling of the actin cytoskeleton, a process regulated by the small GTPase Rac. Vav was thought to be the guanine nucleotide exchange factor responsible for the activation of Rac, but recent evidence indicates that Fcgamma receptor-mediated phagocytosis is unaffected in macrophages lacking all three isoforms of Vav. We therefore tested whether another GEF, DOCK180, participates in Fcgamma receptor-initiated phagocytosis. DOCK180 associates with the adaptor protein Crk, which mediates recruitment of the GEF to sites of tyrosine phosphorylation. CrkII and DOCK180 were found to accumulate at the phagocytic cup. Knockdown of Crk or DOCK180 in murine macrophages using small interfering RNA inhibited phagocytosis of IgG-opsonized particles. Moreover, transfection of dominant negative CrkII prevented both recruitment of DOCK180 and the activation of Rac at the phagocytic cup. This is the first report of a role for either Crk or DOCK180 in Fcgamma receptor-mediated phagocytosis. The Crk-DOCK180 complex is involved in the clearance of apoptotic cells, which unlike the ingestion of IgG-opsonized particles, is an anti-inflammatory process. The finding that CrkII-DOCK180 is also responsible, at least in part, for the effects of Fcgamma receptors implies that additional, parallel pathways must account for the associated pro-inflammatory effect.     

Mitochondria and AMP-activated Protein Kinase-dependent Mechanism of Efferocytosis

Defective clearance of apoptotic cells is frequently associated with perpetuation of inflammatory conditions. Our results show a rapid activation of AMP-activated kinase (AMPK) in macrophages upon exposure to apoptotic cells or lysophosphatidylcholine, a specific phospholipid that is produced and released from dying cells. AMPK activation resulted from inhibition of mitochondrial oxygen consumption and ATP production and further depended on Ca²⁺ mobilization and mitochondrial reactive oxygen species generation. Once activated, AMPK increased microtubule synthesis and chemokinesis and provided adaptation to energy demand during tracking and engulfment. Uptake of apoptotic cells was increased in lungs of mice that received lysophosphatidylcholine. Furthermore, inhibition of AMPK diminished clearance of apoptotic thymocytes in vitro and in dexamethasone-treated mice. Taken together, we conclude that the mitochondrial AMPK axis is a sensor and enhancer of tracking and removal of apoptotic cell, processes crucial to resolution of inflammatory conditions and a return to tissue homeostasis.     

Murine low-density lipoprotein receptor-related protein 1 (LRP) is required for phagocytosis of targets bearing LRP ligands but is not required for C1q-triggered enhancement of phagocytosis

C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared with LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep RBC suboptimally opsonized with IgG or complement, compared with cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared with normal mouse serum, and the addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.     

Effects of fibronectin on actin organization and respiratory burst activity in neutrophils,monocytes, and macrophages

Previous studies have shown that fibronectin (Fn) enhances phagocytosis and killing of antibody-coated bacteria by neutrophils and macrophages. In an attempt to understand the mechanism of this enhancement, we have investigated the effects of Fn on phagocytosis-related actin organization as well as respiratory burst activity in neutrophils, monocytes and culture-derived macrophages. Employing an NBD-phallacidin flow cytometric analysis of filamentous actin formation, we found that Fn promotes rapid actin polymerization within 30 seconds in neutrophils, monocytes, and macrophages, but not lymphocytes. Enhancement of actin polymerization by Fn was concentration-dependent and mediated by a pertussis toxin- but not cholera toxin- sensitive G protein. Inhibition of protein kinase C by sphingosine (20 μM), calcium influx by verapamil (0.1 mM), or intracellular calcium mobilization by 8-(N, N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate HCI (TMB-8; 0.1 mM) did not block Fn-enhanced actin polymerization in phagocytes. Incubation of neutrophils and macrophages on microtiter plates precoated with Fn suppressed superoxide (O₂^?) production induced by IgG- and IgA- opsonized group B streptococci. In contrast, Fn significantly enhanced IgA- and IgG-mediated O₂^? production by freshly isolated monocytes. These data suggest that Fn enhances phagocytosis, presumably through G protein-coupled cytoskeleton reorganization and augments O₂^? production by circulating monocytes. In contrast, it appears to suppress O₂^? production by the active phagocytic cells, neutrophils and macrophages. This may result in enhanced phagocytosis and intracellular killing of microorganisms without damaging interstitial tissues. © 1994 Wiley-Liss, Inc.     

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.     

Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5'-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or hydrogen peroxide (H(2)O(2)) to activate AMPK. Although ratios of AMP to adenosine 5'-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.     

Enteropathogenic Escherichia coli activates the RhoA signaling pathway via the stimulation of GEF-H1

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF-H1.     

Photo-enhancement of macrophage phagocytic activity via Rac1-mediated signaling pathway: Implications for bacterial infection

Phagocytosis and the subsequent destruction of invading pathogens by macrophages are indispensable steps in host immune responses to microbial infections. Low-power laser irradiation (LPLI) has been found to exert photobiological effects on immune responses, but the signaling mechanisms underlying this photobiomodulation of phagocytosis remains largely unknown. Here, we demonstrated for the first time that LPLI enhanced the phagocytic activity of macrophages by stimulating the activation of Rac1. The overexpression of constitutively activated Rac1 clearly enhanced LPLI-induced phagocytosis, whereas the overexpression of dominant negative Rac1 exerted the opposite effect. The phosphorylation of cofilin was involved in the effects of LPLI on phagocytosis, which was regulated by the membrane translocation and activation of Rac1. Furthermore, the photoactivation of Rac1 was dependent on the Src/PI3K/Vav1 pathway. The inhibition of the Src/PI3K pathway significantly suppressed LPLI-induced actin polymerization and phagocytosis enhancement. Additionally, LPLI-treated mice exhibited increased survival and a decreased organ bacterial load when challenged with Listeria monocytogenes, indicating that LPLI enhanced macrophage phagocytosis in vivo. These findings highlight the important roles of the Src/PI3K/Vav1/Rac1/cofilin pathway in regulating macrophage phagocytosis and provide a potential strategy for treating phagocytic deficiency via LPLI.     

Characterization of the effects of cross-linking of macrophage CD44 associated with increased phagocytosis of apoptotic PMN

Control of macrophage capacity for apoptotic cell clearance by soluble mediators such as cytokines, prostaglandins and lipoxins, serum proteins, and glucocorticoids may critically determine the rate at which inflammation resolves. Previous studies suggested that macrophage capacity for clearance of apoptotic neutrophils was profoundly altered following binding of CD44 antibodies. We have used a number of different approaches to further define the mechanism by which CD44 rapidly and specifically augment phagocytosis of apoptotic neutrophils. Use of Fab' fragments unequivocally demonstrated a requirement for cross-linking of macrophage surface CD44. The molecular mechanism of CD44-augmented phagocytosis was shown to be opsonin-independent and to be distinct from the Mer/protein S pathway induced by glucocorticoids and was not functional for clearance of apoptotic eosinophils. CD44-cross-linking also altered macrophage migration and induced cytoskeletal re-organisation together with phosphorylation of paxillin and activation of Rac2. Investigation of signal transduction pathways that might be critical for CD44 augmentation of phagocytosis revealed that Ca(2+) signalling, PI-3 kinase pathways and altered cAMP signalling were not involved, but did implicate a key role for tyrosine phosphorylation events. Finally, although CD44 antibodies were able to augment phagocytosis of apoptotic neutrophils by murine peritoneal and bone marrow-derived macrophages, we did not observe a difference in the clearance of neutrophils following induction of peritonitis with thioglycollate in CD44-deficient animals. Together, these data demonstrate that CD44 cross-linking induces a serum opsonin-independent mechanism of macrophage phagocytosis of apoptotic neutrophils that is associated with reduced macrophage migration and cytoskeletal reorganisation.     

Apoptotic Neutrophils Augment the Inflammatory Response to Mycobacterium tuberculosis Infection in Human Macrophages

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.     

在LPS诱导的炎症模型中低功率激光照射对小胶质细胞吞噬功能的影响

小胶质细胞的激活在神经退行性疾病的病理发生过程中发挥了重要的作用.一旦被激活,他们便具有类似巨噬细胞的吞噬功能以及释放炎症因子的能力,前者有利于保护中枢神经系统的功能,而后者则会加重神经元的死亡.然而,在神经退行性疾病的发生过程中,脑内的小胶质细胞却不能有效地对死亡细胞甚至Aβ进行吞噬.因此,调控小胶质细胞的吞噬功能被认为是寻求神经保护治疗手段的一个有效策略.在本研究中,我们的实验结果表明了20 J/cm2的LPLI能够增强LPS激活的小胶质细胞的吞噬功能.我们发现LPLI介导的小胶质细胞的吞噬功能增强是一个基于actin聚合的Rac1依赖的过程,持续激活的Rac1（Rac1Q61L）相比野生型Rac1可以诱导更多的actin聚合,而显性负效应的Rac1 （Rac1T17N）却显著抑制了actin的聚合.另外,我们运用一个基于荧光能量共振转移的Raichu-Rac1质粒也进一步证实了在LPLI下Rac1的激活,并且这一激活过程是由PI3K/Akt通路所介导的.我们的研究为控制神经退行性疾病的进程提供了一个可行的的治疗策略.     

Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases

A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versus RhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60 cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities.