Role of Protein Tyrosine Phosphatase Non-Receptor Type 7 in the Regulation of TNF-α Production in RAW 264.7 Macrophages

Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.     

Surfactant protein A activation of atypical protein kinase C zeta in IkappaB-alpha-dependent anti-inflammatory immune regulation

The pulmonary collectin surfactant protein (SP)-A has a pivotal role in anti-inflammatory modulation of lung immunity. The mechanisms underlying SP-A-mediated inhibition of LPS-induced NF-kappaB activation in vivo and in vitro are only partially understood. We previously demonstrated that SP-A stabilizes IkappaB-alpha, the primary regulator of NF-kappaB, in alveolar macrophages (AM) both constitutively and in the presence of LPS. In this study, we show that in AM and PBMC from IkappaB-alpha knockout/IkappaB-beta knockin mice, SP-A fails to inhibit LPS-induced TNF-alpha production and p65 nuclear translocation, confirming a critical role for IkappaB-alpha in SP-A-mediated LPS inhibition. We identify atypical (a) protein kinase C (PKC) zeta as a pivotal upstream regulator of SP-A-mediated IkappaB-alpha/NF-kappaB pathway modulation deduced from blocking experiments and confirmed by using AM from PKCzeta-/- mice. SP-A transiently triggers aPKCThr(410/403) phosphorylation, aPKC kinase activity, and translocation in primary rat AM. Coimmunoprecipitation experiments reveal that SP-A induces aPKC/p65 binding under constitutive conditions. Together the data indicate that anti-inflammatory macrophage activation via IkappaB-alpha by SP-A critically depends on PKCzeta activity, and thus attribute a novel, stimulus-specific signaling function to PKCzeta in SP-A-modulated pulmonary immune response.     

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.     

Surfactant Protein-A Modulates LPS-Induced TLR4 Localization and Signaling via β-Arrestin 2

The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in β-arrestin 2^−/− AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2^−/− mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A^−/− mice is significantly reduced compared to SP-A^+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A^−/− mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.     

PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow-mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow-mediated peroxynitrite (ONOO(-)) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53-Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO(-) increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301-410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ-PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.     

Role of protein kinase C-alpha in the control of infection by intracellular pathogens in macrophages.

The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In this study, we investigated the role of macrophage PKC-alpha in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections. To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-alpha. While phagocytosis of L. donovani promastigotes was not affected by DN PKC-alpha overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection. Intracellular survival of a L. donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection. However, IFN-gamma-induced leishmanicidal activity was not affected by DN PKC-alpha overexpression. Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila. In contrast, DN PKC-alpha-overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L. pneumophila. Permissiveness to L. pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC-alpha-overexpressing RAW 264.7 clones. Collectively, these results support a role for PKC-alpha in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.     

The role of Rab6 GTPase in the maturation of phagosome against Staphylococcus aureus

Phagocytosis, an evolutionarily conserved process in animals, plays a central role in host defense against pathogens. As reported, Rab6 GTPase was involved in the regulation of hemocytic phagocytosis in invertebrates. However, the role of Rab6 GTPase in mammalian phagocytosis remains to be addressed. In this study, the results showed that Rab6 GTPase took great effects on phagocytosis of mouse leukemic monocyte macrophages (RAW 264.7 cells). It was revealed that Rab6 GTPase was required during the phagosome maturation by its interaction with bicaudal-D1 (BICD1) protein. Further data presented that the Rab6 GTPase-regulated phagocytosis could influence the proliferation of Staphylococcus aureus in macrophages. Therefore, our study demonstrated a novel insight into the mechanism of regulation of mammalian phagocytosis by Rab6 GTPase and a novel strategy for the control of Staphylococcus aureus.     

Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that can replicate within infected macrophages. The ability of M. tb to arrest phagosome maturation is believed to facilitate its intracellular multiplication. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tb modulates their localization during inhibiting phagolysosome biogenesis remain elusive. We compared the localization of 42 distinct Rab GTPases to phagosomes containing either Staphylococcus aureus or M. tb. The phagosomes containing S. aureus were associated with 22 Rab GTPases, but only 5 of these showed similar localization kinetics as the phagosomes containing M. tb. The Rab GTPases responsible for phagosome maturation, phagosomal acidification and recruitment of cathepsin D were examined in macrophages expressing the dominant-negative form of each Rab GTPase. LysoTracker staining and immunofluorescence microscopy revealed that Rab7, Rab20 and Rab39 regulated phagosomal acidification and Rab7, Rab20, Rab22b, Rab32, Rab34, Rab38 and Rab43 controlled the recruitment of cathepsin D to the phagosome. These results suggest that phagosome maturation is achieved by a series of interactions between Rab GTPases and phagosomes and that differential recruitment of these Rab GTPases, except for Rab22b and Rab43, to M. tb-containing phagosomes is involved in arresting phagosome maturation and inhibiting phagolysosome biogenesis.     

The antimicrobial peptide, tilapia hepcidin 2-3, and PMA differentially regulate the protein kinase C isoforms, TNF-α and COX-2, in mouse RAW264.7 macrophages

The antimicrobial and immunomodulatory functions of the antimicrobial peptide, tilapia hepcidin (TH)2-3, were previously studied. Herein, we report the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 macrophages. Treatment with TH2-3 at 40 or 80 μg/ml did not affect the cell morphology, but TH2-3 at 120 μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. The coexistence of the PKC inhibitor, Ro-31-8220, prevented morphological changes induced by either PMA or 120 μg/ml TH2-3 in RAW264.7 cells. Since PMA is known to induce expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, activation of the TNF-α promoter in response to TH2-3 and PMA treatments in lipopolysaccharide (LPS)-stimulated cells was compared. In LPS-stimulated RAW264.7 cells, TNF-α promoter activity was significantly suppressed by TH2-3, but not by PMA. In addition, PMA activated prostaglandin synthase-associated cyclooxygenase (COX)-2 proteins on the cell surface, while the presence of TH2-3 inhibited its expression. Western blotting demonstrated that the expressions of PKC-μ, phosphorylated (p)-PKCμ at serine (S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells.     

Up-regulation of ATP binding cassette transporter A1 expression by very low density lipoprotein receptor and apolipoprotein E receptor 2

Activation of very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (apoER2) results in either pro- or anti-atherogenic effects depending on the ligand. Using reelin and apoE as ligands, we studied the impact of VLDLR- and apoER2-mediated signaling on the expression of ATP binding cassette transporter A1 (ABCA1) and cholesterol efflux using RAW264.7 cells. Treatment of these mouse macrophages with reelin or human apoE3 significantly increased ABCA1 mRNA and protein levels, and apoAI-mediated cholesterol efflux. In addition, both reelin and apoE3 significantly increased phosphorylated disabled-1 (Dab1), phosphatidylinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ), and specificity protein 1 (Sp1). This reelin- or apoER2-mediated up-regulation of ABCA1 expression was suppressed by 1) knockdown of Dab1, VLDLR, and apoER2 with small interfering RNAs (siRNAs), 2) inhibition of PI3K and PKC with kinase inhibitors, 3) overexpression of kinase-dead PKCζ, and 4) inhibition of Sp1 DNA binding with mithramycin A. Activation of the Dab1-PI3K signaling pathway has been implicated in VLDLR- and apoER2-mediated cellular functions, whereas the PI3K-PKCζ-Sp1 signaling cascade has been implicated in the regulation of ABCA1 expression induced by apoE/apoB-carrying lipoproteins. Taken together, these data support a model in which activation of VLDLR and apoER2 by reelin and apoE induces ABCA1 expression and cholesterol efflux via a Dab1-PI3K-PKCζ-Sp1 signaling cascade.     

Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages

The oxidized phospholipids (oxPl) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are cytotoxic components of oxidized LDL (oxLDL). Sustained exposure to oxLDL or isolated oxPl induces apoptotic signaling in vascular cells, which is a hallmark of the late phase of atherosclerosis. Activation of sphingomyelinase, the coordinate formation of ceramide and activation of caspase 3/7 as well as the activation of stress-associated kinases are causally involved in this process. Here, we provide evidence for a role of PKCδ in oxPl cytotoxicity. Silencing of the enzyme by siRNA significantly reduced caspase 3/7 activation in RAW 264.7 macrophages under the influence of oxPl. Concomitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluorescence microscopy provided direct evidence for oxPl-protein interaction. Both oxPl recruited an RFP-tagged PKCδ to the plasma membrane in a concentration-dependent manner. In addition, two color cross-correlation number and brightness (ccN&B) analysis of the molecular motions revealed that fluorescently labeled PGPC or POVPC analogs co-diffuse and are associated with the fluorescent protein kinase in live cells. The underlying lipid-protein interactions may be due to chemical bonding (imine formation between the phospholipid aldehyde POVPC with protein amino groups) and physical association (with POVPC or PGPC). In summary, our data supports the assumption that PKCδ acts as a proapototic kinase in oxPl-included apoptosis of RAW 264.7 macrophages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling.     

Mycobacterial Nucleoside Diphosphate Kinase Blocks Phagosome Maturation in Murine Raw 264.7 Macrophages

Background

Microorganisms capable of surviving within macrophages are rare, but represent very successful pathogens. One of them is Mycobacterium tuberculosis (Mtb) whose resistance to early mechanisms of macrophage killing and failure of its phagosomes to fuse with lysosomes causes tuberculosis (TB) disease in humans. Thus, defining the mechanisms of phagosome maturation arrest and identifying mycobacterial factors responsible for it are key to rational design of novel drugs for the treatment of TB. Previous studies have shown that Mtb and the related vaccine strain, M. bovis bacille Calmette-Guérin (BCG), disrupt the normal function of host Rab5 and Rab7, two small GTPases that are instrumental in the control of phagosome fusion with early endosomes and late endosomes/lysosomes respectively.

Methodology/Principal Findings

Here we show that recombinant Mtb nucleoside diphosphate kinase (Ndk) exhibits GTPase activating protein (GAP) activity towards Rab5 and Rab7. Then, using a model of latex bead phagosomes, we demonstrated that Ndk inhibits phagosome maturation and fusion with lysosomes in murine RAW 264.7 macrophages. Maturation arrest of phagosomes containing Ndk-beads was associated with the inactivation of both Rab5 and Rab7 as evidenced by the lack of recruitment of their respective effectors EEA1 (early endosome antigen 1) and RILP (Rab7-interacting lysosomal protein). Consistent with these findings, macrophage infection with an Ndk knocked-down BCG strain resulted in increased fusion of its phagosome with lysosomes along with decreased survival of the mutant.

Conclusion

Our findings provide evidence in support of the hypothesis that mycobacterial Ndk is a putative virulence factor that inhibits phagosome maturation and promotes survival of mycobacteria within the macrophage.     

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.     

Daxx通过上调caveolin-1的表达介导Ox-LDL诱导巨噬细胞胆固醇蓄积和凋亡

为探讨Daxx对氧化型低密度脂蛋白(oxidized low-density lipoprotein,Ox-LDL)诱导巨噬细胞胆固醇蓄积和凋亡的介导作用及其可能的分子机制,用高效液相色谱法检测细胞内胆固醇含量,油红O染色观察细胞内脂滴的形成情况,流式细胞术和吖啶橙/溴化乙锭(AO/EB)染色法研究Ox-LDL对细胞凋亡的影响,Real time RT-PCR检测细胞内Daxx mRNA的表达水平,Western blot检测caveolin-1蛋白的表达,用特异性siRNA沉默Daxx在RAW264.7 细胞中的表达．Ox-LDL上调Daxx mRNA和caveolin-1的表达、增加细胞内胆固醇含量、促使RAW264.7细胞凋亡,用特异性siRNA干扰Daxx在RAW264.7细胞中的表达能降低caveolin-1的表达、减少细胞内胆固醇含量、以及抑制细胞凋亡．上述结果表明,Daxx对Ox-LDL诱导RAW264.7巨噬细胞胆固醇蓄积和凋亡具有介导作用,这一作用可能与Daxx上调caveolin -1的表达有关．     

Vasoactive hormone adrenomedullin and its binding protein: anti-inflammatory effects by up-regulating peroxisome proliferator-activated receptor-gamma

Sepsis is a critical inflammatory condition from which numerous patients die due to multiple organ failure and septic shock. The vasoactive hormone adrenomedullin (AM) and its binding protein (AMBP-1) are beneficial in sepsis by abrogating the progression to irreversible shock and decreasing proinflammatory cytokine release. To investigate the anti-inflammatory mechanism, we studied to determine the effect of the AM/AMBP-1 complex on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-gamma expression in vivo and in vitro and was associated with increased TNF-alpha production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-gamma levels in both models, resulting in TNF-alpha suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-alpha production in the absence of PPAR-gamma. LPS caused the suppression of intracellular cyclic AMP (cAMP), which was prevented by simultaneous AM/AMBP-1 treatment. Although incubation with dibutyryl cAMP significantly decreased LPS-induced TauNuF-alpha release, it did not alter PPAR-gamma expression. Through inhibition studies using genistein and PD98059 we found that the Pyk-2 tyrosine kinase-ERK1/2 pathway is in part responsible for the AM/AMBP-1-mediated induction of PPAR-gamma and the anti-inflammatory effect. We conclude that AM/AMBP-1 is protective in sepsis due to its vasoactive properties and direct anti-inflammatory effects mediated through both the cAMP-dependent pathway and Pyk-2-ERK1/2-dependent induction of PPAR-gamma.     

AS-703026 Inhibits LPS-Induced TNFα Production through MEK/ERK Dependent and Independent Mechanisms

Chronic obstructive pulmonary disease (COPD) is characterized by intense lung infiltrations of immune cells (macrophages and monocytes). Lipopolysaccharide (LPS) activates macrophages/monocytes, leading to production of tumor necrosis factor α (TNFα) and other cytokines, which cause subsequent lung damages. In the current study, our results demonstrated that AS-703026, a novel MEK/ERK inhibitor, suppressed LPS-induced TNFα mRNA expression and protein secretion in RAW 264.7 murine macrophages, and in murine bone marrow-derived macrophages (BMDMs). Meanwhile, TNFα production in LPS-stimulated COPD patents’ peripheral blood mononuclear cells (PBMCs) was also repressed by AS-703026. At the molecular level, we showed that AS-703026 blocked LPS-induced MEK/ERK activation in above macrophages/monocytes. However, restoring ERK activation in AS-703026-treated RAW 264.7 cells by introducing a constitutive-actively (CA)-ERK1 only partially reinstated LPS-mediated TNFα production. Meanwhile, AS-703026 could still inhibit TNFα response in ERK1/2-depleted (by shRNA) RAW 264.7 cells. Significantly, we found that AS-703026 inhibited LPS-induced nuclear factor κB (NFκB) activation in above macrophages and COPD patients’ PBMCs. In vivo, oral administration of AS-703026 inhibited LPS-induced TNFα production and endotoxin shock in BALB/c mice. Together, we show that AS-703026 in vitro inhibits LPS-induced TNFα production in macrophages/monocytes, and in vivo protects mice from LPS-induced endotoxin shock. Thus, it could be further studied as a useful anti-inflammatory therapy for COPD patients.     

RILP interacts with the VPS22 component of the ESCRT-II complex

The Rab-interacting lysosomal protein (RILP) has been identified as an effector for the small GTPases Rab7 and Rab34. It has been demonstrated that Rab7 and RILP are key proteins for the biogenesis of lysosomes and phagolysosomes. Indeed, expression of dominant negative mutants of Rab7 or of the C-terminal half of RILP impairs biogenesis and function of these organelles. In this study we have isolated, using the yeast two-hybrid system, the EAP30/SNF8/VPS22 subunit of the ESCRT-II complex as a RILP interacting protein. We demonstrated that VPS22 interacts with the N-terminal half of RILP. The interaction data obtained with the two-hybrid system were confirmed by co-immunoprecipitation. In addition, confocal immunofluorescence revealed colocalization of GFP-RILP and HA-VPS22. These data suggest that RILP could have a role in the biogenesis of multivesicular bodies.     

Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A

Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.     

Direct binding of Toll-like receptor 2 to zymosan,and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.     

Surfactant protein A enhances mycobacterial killing by rat macrophages through a nitric oxide-dependent pathway

Surfactant-associated protein A (SP-A) is involved in surfactant homeostasis and host defense in the lung. We have previously demonstrated that SP-A specifically binds to and enhances the ingestion of bacillus Calmette-Guerin (BCG) organisms by macrophages. In the current study, we investigated the effect of SP-A on the generation of inflammatory mediators induced by BCG and the subsequent fate of ingested BCG organisms. Rat macrophages were incubated with BCG in the presence and absence of SP-A. Noningested BCG organisms were removed, and the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide were measured at varying times. TNF-alpha and nitric oxide production induced by BCG were enhanced by SP-A. In addition, SP-A enhanced the BCG-induced increase in the level of inducible nitric oxide synthase protein. Addition of antibodies directed against SPR210, a specific macrophage SP-A receptor, inhibited the SP-A-enhanced mediator production. BCG in the absence of SP-A showed increased growth over a 5-day period, whereas inclusion of SP-A dramatically inhibited BCG growth. Inhibition of nitric oxide production blocked BCG killing in the presence and absence of SP-A. These results demonstrate that ingestion of SP-A-BCG complexes by rat macrophages leads to production of inflammatory mediators and increased mycobacterial killing.