Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression,a polarized microfilament system,and diminished responsiveness to chemoattractants in vitro

The S100A9 (MRP14) protein is abundantly expressed in myeloid cells and has been associated with various inflammatory diseases. The S100A9-deficient mice described here were viable, fertile, and generally of healthy appearance. The myelopoietic potential of the S100A9-null bone marrow was normal. S100A8, the heterodimerization partner of S100A9 was not detectable in peripheral blood cells, suggesting that even a deficiency in both S100A8 and S100A9 proteins was compatible with viable and mature neutrophils. Surprisingly, the invasion of S100A9-deficient leukocytes into the peritoneum and into the skin in vivo was indistinguishable from that in wild-type mice. However, stimulation of S100A9-deficient neutrophils with interleukin-8 in vitro failed to provoke an up-regulation of CD11b. Migration upon a chemotactic stimulus through an endothelial monolayer was markedly diminished in S100A9-deficient neutrophils. Attenuated chemokinesis of the S100A9-deficient neutrophils was observed by using a three-dimensional collagen matrix migration assay. The altered migratory behavior was associated with a microfilament system that was highly polarized in unstimulated S100A9-deficient neutrophils. Our data suggest that loss of the calcium-binding S100A9 protein reduces the responsiveness of the neutrophils upon chemoattractant stimuli at least in vitro. Alternative pathways for neutrophil emigration may be responsible for the lack of any effect in the two in vivo models we have investigated so far.     

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.     

Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide

Recently, proinflammatory activities had been described for S100A8 and S100A9, two proteins found at inflammatory sites and within the neutrophil cytoplasm. In this study, we investigated the role of these proteins in neutrophil migration in vivo in response to LPS. LPS was injected into the murine air pouch, which led to the release of S100A8, S100A9, and S100A8/A9 in the pouch exudates that preceded accumulation of neutrophils. Passive immunization against S100A8 and S100A9 led to a 52% inhibition of neutrophil migration in response to LPS at 3 h postinjection. Injection of LPS was also associated with an increase in peripheral blood neutrophils and the presence in serum of S100A9 and S100A8/A9. Intravenous injection of S100A8, S100A9, or S100A8/A9 augmented the number of circulating neutrophils and diminished the number of neutrophils in the bone marrow, demonstrating that S100A8 and S100A9 induced the mobilization of neutrophils from the bone marrow to the blood. Finally, passive immunization with anti-S100A9 inhibited the neutrophilia associated with LPS injection in the air pouch. These results suggest that S100A8 and S100A9 play a role in the inflammatory response to LPS by inducing the release of neutrophils from the bone marrow and directing their migration to the inflammatory site.     

Effect of D-alanylation of (lipo)teichoic acids of Staphylococcus aureus on host secretory phospholipase A2 action before and after phagocytosis by human neutrophils

Invading bacteria such as Staphylococcus aureus induce mobilization of professional phagocytes (e.g., neutrophils) and extracellular antibacterial proteins (e.g., group IIA phospholipase A2 (gIIA PLA2)). Accumulation of gIIA PLA2 in inflammatory fluids confers potent extracellular antistaphylococcal activity and at lower concentrations promotes bacterial phospholipid degradation during phagocytosis of S. aureus by human neutrophils. D-alanylation of (lipo) teichoic acids of S. aureus increases bacterial resistance to gIIA PLA2 approximately 100-fold, raising the possibility that the resistance of ingested S. aureus to related gV and gX secretory PLA2 present in human neutrophil granules depends on D-alanylation mediated by the dlt operon. However, we show that isogenic wild-type and dltA S. aureus are equally resistant to gV/X PLA2 during phagocytosis and when exposed to the purified enzymes. The fates of wild-type and dltA S. aureus exposed to serum and human neutrophils differed significantly only when extracellular gIIA PLA2 was also present before phagocytosis. The extreme potency of the gIIA PLA2 toward dltA S. aureus suggests that even small amounts of this extracellular enzyme mobilized early in inflammation could contribute substantially to the overall cytotoxicity of acute inflammatory exudates toward S. aureus when D-alanylation of (lipo)teichoic acids is limiting.     

House Dust Mite Allergen Regulates Constitutive Apoptosis of Normal and Asthmatic Neutrophils via Toll-Like Receptor 4

House dust mites (HDMs) induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of Dermatophagoides pteronissinus (DP) inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF) when compared to normal BALF (p<0.01), but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%). These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.     

Identification of an immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa)

Phagocytosis by neutrophils is the essential step in fighting Pseudomonas infections. The first step in neutrophil recruitment to the site infection is the interaction of P-selectin (on endothelial cells) with P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils. Pseudomonas aeruginosa secretes various proteases that degrade proteins that are essential for host defence, such as elastase and alkaline protease. Here we identify PA0572 of P. aeruginosa as an inhibitor of PSGL-1 and named this secreted hypothetical protease immunomodulating metalloprotease of P. aeruginosa or IMPa. Proteolytic activity was confirmed by cleavage of recombinant and cell-surface expressed PSGL-1. Functional inhibition was demonstrated by impaired PSGL-1-mediated rolling of IMPa-treated neutrophils under flow conditions. Next to PSGL-1, IMPa targets CD43 and CD44 that are also involved in leucocyte homing. These data indicate that IMPa prevents neutrophil extravasation and thereby protects P. aeruginosa from neutrophil attack.     

Cigarette smoke-exposed neutrophils die unconventionally but are rapidly phagocytosed by macrophages

Pulmonary accumulation of neutrophils is typical for active smokers who are also predisposed to multiple inflammatory and infectious lung diseases. We show that human neutrophil exposure to cigarette smoke extract (CSE) leads to an atypical cell death sharing features of apoptosis, autophagy and necrosis. Accumulation of tar-like substances in autophagosomes is also apparent. Before detection of established cell death markers, CSE-treated neutrophils are effectively recognized and non-phlogistically phagocytosed by monocyte-derived macrophages. Blockade of LOX-1 and scavenger receptor A, but not MARCO or CD36, as well as pre-incubation with oxLDL, inhibited phagocytosis, suggesting that oxLDL-like structures are major phagocytosis signals. Specific lipid (β-carotene and quercetin), but not aqueous, antioxidants increased the pro-phagocytic effects of CSE. In contrast to non-phlogistic phagocytosis, degranulation of secondary granules, as monitored by lactoferrin release, was apparent on CSE exposure, which is likely to promote pulmonary inflammation and tissue degradation. Furthermore, CSE-exposed neutrophils exhibited a compromised ability to ingest the respiratory pathogen, Staphylococcus aureus, which likely contributes to bacterial persistence in the lungs of smokers and is likely to promote further pulmonary recruitment of neutrophils. These data provide mechanistic insight into the lack of accumulation of apoptotic neutrophil populations in the lungs of smokers and their increased susceptibility to degradative pulmonary diseases and bacterial infections.     

Defective chemoattractant-induced calcium signalling in S100A9 null neutrophils

The S100 family member S100A9 and its heterodimeric partner, S100A8, are cytosolic Ca2+ binding proteins abundantly expressed in neutrophils. To understand the role of this EF-hand-containing complex in Ca2+ signalling, neutrophils from S100A9 null mice were investigated. There was no role for the complex in buffering acute cytosolic Ca2+ elevations. However, Ca2+ responses to inflammatory agents such as chemokines MIP-2 and KC and other agonists are altered. For S100A9 null neutrophils, signalling at the level of G proteins is normal, as is release of Ca2+ from the IP(3) receptor-gated intracellular stores. However MIP-2 and FMLP signalling in S100A9 null neutrophils was less susceptible than wildtype to PLCbeta inhibition, revealing dis-regulation of the signalling pathway at this level. Downstream of PLCbeta, there was reduced intracellular Ca2+ release induced by sub-maximal levels of chemokines. Conversely the response to FMLP was uncompromised, demonstrating different regulation compared to MIP-2 stimulation. Study of the activity of PLC product DAG revealed that chemokine-induced signalling was susceptible to inhibition by elevated DAG with S100A9 null cells showing enhanced inhibition by DAG. This study defines a lesion in S100A9 null neutrophils associated with inflammatory agonist-induced IP3-mediated Ca2+ release that is manifested at the level of PLCbeta.     

Myeloperoxidase-dependent Lipid Peroxidation Promotes the Oxidative Modification of Cytosolic Proteins in Phagocytic Neutrophils

Phagocytic neutrophils generate reactive oxygen species to kill microbes. Oxidant generation occurs within an intracellular phagosome, but diffusible species can react with the neutrophil and surrounding tissue. To investigate the extent of oxidative modification, we assessed the carbonylation of cytosolic proteins in phagocytic neutrophils. A 4-fold increase in protein carbonylation was measured within 15 min of initiating phagocytosis. Carbonylation was dependent on NADPH oxidase and myeloperoxidase activity and was inhibited by butylated hydroxytoluene and Trolox, indicating a role for myeloperoxidase-dependent lipid peroxidation. Proteomic analysis of target proteins revealed significant carbonylation of the S100A9 subunit of calprotectin, a truncated form of Hsp70, actin, and hemoglobin from contaminating erythrocytes. The addition of the reactive aldehyde 4-hydroxynonenal (HNE) caused carbonylation, and HNE-glutathione adducts were detected in the cytosol of phagocytic neutrophils. The post-translational modification of neutrophil proteins will influence the functioning and fate of these immune cells in the period following phagocytic activation, and provides a marker of neutrophil activation during infection and inflammation.     

Crosslinking of the CD69 molecule enhances S100A9 production in activated neutrophils

Expression of CD69 on neutrophils and generation of anti-CD69 autoantibodies in patients with rheumatoid arthritis (RA) have been reported. Thus natural ligands for CD69 not yet identified and/or the anti-CD69 autoantibodies possibly affect neutrophils by evoking CD69 signaling, which may further affect joint-composing cells in RA. However, the effect of the CD69 signaling in neutrophils remains largely unclear. To elucidate the issue, we tried to identify proteins affected by the crosslinking of CD69 on neutrophils using a proteomic approach. Specifically, CD69 on granulocyte-macrophage colony stimulating factor (GM-CSF)-activated neutrophils was crosslinked by anti-CD69 monoclonal antibodies, and then intracellular proteins were detected using 2-dimensional electrophoresis and further identified by mass spectrometry and subsequent protein database searching. As a result, we successfully identified multiple proteins that increased their production by the CD69 signaling. Among the proteins, we focused on one of the up-regulated proteins, S100A9 calcium binding protein (S100A9), and investigated proteome changes brought by a recombinant S100A9 in a human synovial sarcoma cell line (SW982), a human chondrosarcoma cell line (OUMS-27), and a human T leukemia cell line (Jurkat). This revealed that the recombinant S100A9 altered proteomes of SW982 and OUMS-27, and to a lesser extent, that of the Jurkat cells. Further, S100A9 induced IL-1beta production from neutrophils and the SW982 cells. These data suggest that unidentified natural ligands for CD69 and/or the anti-CD69 autoantibodies possibly affect joint-composing cell types through the increased production of S100A9 in neutrophils, providing a new insight into functions of CD69 on neutrophils in RA.     

Beneficial suicide: why neutrophils die to make NETs

Neutrophils are one of the main types of effector cell in the innate immune system and were first shown to effectively kill microorganisms by phagocytosis more than 100 years ago. Recently, however, it has been found that stimulated neutrophils can also produce extracellular structures called neutrophil extracellular traps (NETs) that capture and kill microorganisms. This Progress article gives an overview of the structure, function and generation of NETs, and their role in infections.     

Phagocytosis Is the Main CR3-Mediated Function Affected by the Lupus-Associated Variant of CD11b in Human Myeloid Cells

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis.     

A new insight into phagocytosis of apoptotic cells: proteolytic enzymes divert the recognition and clearance of polymorphonuclear leukocytes by macrophages

The recognition of phosphatidylserine (PS) on the surface of any apoptotic cell is considered to be a key event for its clearance. We challenge this concept by showing that pretreatment of neutrophils with either host or bacterial protease affects their uptake by human monocyte-derived macrophages without having an effect on cell-surface PS presentation. Specifically, whereas preincubation of apoptotic neutrophils with cathepsin G or thrombin significantly inhibited their uptake, gingipains R or clostripain enhanced phagocytosis by macrophages. Moreover, bacterial proteinases sensitized healthy neutrophils for uptake by macrophages, whereas endogenous proteinases were unable to elicit this effect. This stimulation was apparently owing to the combined effect of proteolytic cleavage of an antiphagocytic signal (CD31) and the generation of a novel 'eat-me' signal on the neutrophil surface. These results argue that neutrophil recognition and phagocytosis by macrophages is mediated by a protein ligand whose proteolytic modification could affect the local inflammatory process.     

Oxidation of calprotectin by hypochlorous acid prevents chelation of essential metal ions and allows bacterial growth: Relevance to infections in cystic fibrosis

Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both the methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis was oxidized. Oxidized calprotectin was higher in children with cystic fibrosis compared to disease controls, and further elevated in those patients with infections. Our data suggest that oxidative stress associated with inflammation in cystic fibrosis will stop metal sequestration by calprotectin. Consequently, strategies aimed at blocking extracellular myeloperoxidase activity should enable calprotectin to provide nutritional immunity within the airways.     

Examination of trafficking of phagocytosed colloid particles in neutrophils using synchrotron-based X-ray fluorescence microscopy (XFM)

Synchrotron-based X-ray fluorescence microscopy (XFM) can localise chemical elements at a subcellular level. 99mTechnetium stannous (TcSn) colloid is taken up by phagocytes via a Complement Receptor 3 mediated phagocytic process. In the current study, XFM was used to examine the intracellular trafficking of TcSn colloid in neutrophils. XFM was performed on TcSn colloid, and neutrophils labelled with TcSn colloid, in whole blood. We developed a set of pixel by pixel analysis and mapping techniques incorporating cluster analysis that allowed us to differentiate neutrophils and artefactual contaminants, and we examined the changes in element distribution that accompany neutrophil phagocytosis of TcSn colloid. Sn became associated with half the neutrophils. Within cells, Sn colocalised with iron (Fe) and sulphur (S), and was negatively associated with calcium (Ca). Despite the high sensitivity of XFM, Tc was not detected. XFM can help clarify the intracellular processes that accompany neutrophil phagocytosis. The subcellular colocalisation of Sn with Fe is consistent with fusion of the colloid-containing phagosome with neutrophil granules. The association of Sn with S suggests that proteins rich in S-containing amino acids are present in the phagosome. The negative colocalisation with Ca indicates that ongoing maturation of the TcSn colloid phagosome is no longer calcium dependent one hour after phagocytosis.     

S100A8 and S100A9 in human arterial wall. Implications for atherogenesis

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.     

Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.