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.     

An Inflammation Loop Orchestrated by S100A9 and Calprotectin Is Critical for Development of Arthritis

Objective

The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.

Methods

In this study, we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.

Results

Treatment with anti-S100A9 antibodies improved the clinical score by 50%, diminished immune cell infiltration, reduced inflammatory cytokines, both in serum and in the joints, and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα, IL-1β and IL-6, and of chemokines like MIP-1α and MCP-1.

Conclusion

The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively, our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore, S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed.     

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.     

Blockade of antimicrobial proteins S100A8 and S100A9 inhibits phagocyte migration to the alveoli in streptococcal pneumonia

We investigated the roles of the potent, chemotactic antimicrobial proteins S100A8, S100A9, and S100A8/A9 in leukocyte migration in a model of streptococcal pneumonia. We first observed differential secretion of S100A8, S100A9, and S100A8/A9 that preceded neutrophil recruitment. This is partially explained by the expression of S100A8 and S100A9 proteins by pneumocytes in the early phase of Streptococcus pneumoniae infection. Pretreatment of mice with anti-S100A8 and anti-S100A9 Abs, alone or in combination had no effect on bacterial load or mice survival, but caused neutrophil and macrophage recruitment to the alveoli to diminish by 70 and 80%, respectively, without modifying leukocyte blood count, transendothelial migration or neutrophil sequestration in the lung vasculature. These decreases were also associated with a 68% increase of phagocyte accumulation in lung tissue and increased expression of the chemokines CXCL1, CXCL2, and CCL2 in lung tissues and bronchoalveolar lavages. These results show that S100A8 and S100A9 play an important role in leukocyte migration and strongly suggest their involvement in the transepithelial migration of macrophages and neutrophils. They also indicate the importance of antimicrobial proteins, as opposed to classical chemotactic factors such as chemokines, in regulating innate immune responses in the lung.     

MMP-8 deficiency increases TLR/RAGE ligands S100A8 and S100A9 and exacerbates lung inflammation during endotoxemia

Matrix metalloproteinase-8, released mainly from neutrophils, is a critical regulator of the inflammatory response by its ability to cleave multiple mediators. Herein, we report the results of a model of endotoxemia after intraperitoneal LPS injection in mice lacking MMP-8 and their wildtype counterparts. Control, saline-treated animals showed no differences between genotypes. However, there was an increased lung inflammatory response, with a prominent neutrophilic infiltration in mutant animals after LPS treatment. Using a proteomic approach, we identify alarmins S100A8 and S100A9 as two of the main differences between genotypes. Mice lacking MMP-8 showed a significant increase in these two molecules in lung homogenates, but not in spleen and serum. Mice lacking MMP-8 also showed an increase in MIP-1α levels and a marked activation of the non-canonical NF-κB pathway, with no differences in CXC-chemokines such as MIP-2 or LIX. These results show that MMP-8 can modulate the levels of S100A8 and S100A9 and its absence promotes the lung inflammatory response during endotoxemia.     

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.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.     

Damage-associated molecular pattern S100A9 increases bactericidal activity of human neutrophils by enhancing phagocytosis

The damage-associated molecular-pattern S100A9 is found at inflammatory sites in infections and various autoimmune diseases. It is released at very high concentrations in the extracellular milieu by activated neutrophils and monocytes in response to various agents. This proinflammatory protein is found in infected mucosae and tissue abscesses where it acts notably as a potent neutrophil activator. In this study, we examined the role of S100A9 in the control of infections. S100A9 was found to increase human neutrophil bactericidal activity toward Escherichia coli. Although S100A9 induced the accumulation of reactive oxygen species over time through the activation of NADPH oxidase, its antimicrobial activity was mediated mainly by enhancing the efficiency of neutrophil phagocytosis. Interestingly, S100A9 did not act by increasing cell surface expression of CD16, CD32, or CD64 in neutrophils, indicating that its biological effect in FcR-mediated phagocytosis is independent of upregulation of FcγR levels. However, S100A9-induced phagocytic activity required the phosphorylation of Erk1/2, Akt, and Syk. Taken together, our results demonstrate that S100A9 stimulates neutrophil microbicidal activity by promoting phagocytosis.     

S100A8 and S100A9 in Cancer

S100A8 and S100A9 are Ca²⁺ binding proteins that belong to the S100 family. Primarily expressed in neutrophils and monocytes, S100A8 and S100A9 play critical roles in modulating various inflammatory responses and inflammation-associated diseases. Forming a common heterodimer structure S100A8/A9, S100A8 and S100A9 are widely reported to participate in multiple signaling pathways in tumor cells. Meanwhile, S100A8/A9, S100A8, and S100A9, mainly as promoters, contribute to tumor development, growth and metastasis by interfering with tumor metabolism and the microenvironment. In recent years, the potential of S100A8/A9, S100A9, and S100A8 as tumor diagnostic or prognostic biomarkers has also been demonstrated. In addition, an increasing number of potential therapies targeting S100A8/A9 and related signaling pathways have emerged. In this review, we will first expound on the characteristics of S100A8/A9, S100A9, and S100A8 in-depth, focus on their interactions with tumor cells and microenvironments, and then discuss their clinical applications as biomarkers and therapeutic targets. We also highlight current limitations and look into the future of S100A8/A9 targeted anti-cancer therapy.     

Constitutive neutrophil apoptosis: regulation by cell concentration via S100 A8/9 and the MEK-ERK pathway

Programmed cell death (PCD) is a fundamental mechanism in tissue and cell homeostasis. It was long suggested that apoptosis regulates the cell number in diverse cell populations; however no clear mechanism was shown. Neutrophils are the short-lived, first-line defense of innate immunity, with an estimated t = 1/2 of 8 hours and a high turnover rate. Here we first show that spontaneous neutrophil constitutive PCD is regulated by cell concentrations. Using a proteomic approach, we identified the S100 A8/9 complex, which constitutes roughly 40% of cytosolic protein in neutrophils, as mediating this effect. We further demonstrate that it regulates cell survival via a signaling mechanism involving MEK-ERK via TLR4 and CD11B/CD18. This mechanism is suggested to have a fine-tuning role in regulating the neutrophil number in bone marrow, peripheral blood, and inflammatory sites.     

Regulation of activin A release from murine bone marrow-derived neutrophil precursors by tumour necrosis factor-α and insulin

Activin A, a transforming growth factor-β family cytokine, plays a crucial role in regulating the onset and severity of many inflammatory conditions, such as acute lipopolysaccharide (LPS)-induced inflammation. Activin A is also implicated in type 2 diabetes (T2D), a disease characterised by insulin resistance, hyperglycaemia and chronic elevation of pro-inflammatory cytokines, including tumour necrosis factor (TNF-α). In the human, neutrophils contain activin A that can be released in response to TNF-α. Studies of inflammatory disease in vivo, however, generally use the mouse, so it is essential to know if murine neutrophils have similar properties. Regulation of activin A was investigated in bone marrow-derived neutrophil precursors (BMNPs) from 8 to 10 weeks old C57BL6/J male mice. The BMNPs contained 7-fold higher concentrations of activin A than bone marrow mononuclear cells. Release of activin A from isolated BMNPs was stimulated by TNF-α, but this was not due to increased activin A production. In contrast to TNF-α, LPS had no effect on isolated BMNPs, but stimulated activin A release and production in total bone marrow cell cultures. Moreover, activin A release in response to LPS, was not prevented in TNF-α null mice. Increased glucose and insulin had no effect on base-line activin A secretion by BMNPs in culture, but pre-treatment with insulin blocked the TNF-α induced release of activin A. These results indicate that murine neutrophils are a source of stored activin A, the release of which can be directly stimulated by TNF-α, although TNF-α is not the only stimulator of activin A release during inflammation. Furthermore, regulation of neutrophil activin A release by insulin may also play a role in the inflammation associated with T2D.     

S-nitrosylated S100A8: novel anti-inflammatory properties

S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-gamma, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.     

S100A8/S100A9 and their association with cartilage and bone

S100A8 and S100A9 are calcium-binding proteins expressed in myeloid cells and are markers of numerous inflammatory diseases in humans. S100A9 has been associated with dystrophic calcification in human atherosclerosis. Here we demonstrate S100A8 and S100A9 expression in murine and human bone and cartilage cells. Only S100A8 was seen in preosteogenic cells whereas osteoblasts had variable, but generally weak expression of both proteins. In keeping with their reported high-mRNA expression, S100A8 and S100A9 were prominent in osteoclasts. S100A8 was expressed in alkaline phosphatase-positive hypertrophic chondrocytes, but not in proliferating chondrocytes within the growth plate where the cartilaginous matrix was calcifying. S100A9 was only evident in the invading vascular osteogenic tissue penetrating the degenerating chondrocytic zone adjacent to the primary spongiosa, where S100A8 was also expressed. Whilst, S100A8 has been shown to be associated with osteoblast differentiation, both S100A8 and S100A9 may contribute to calcification of the cartilage matrix and its replacement with trabecular bone, and to regulation of redox in bone resorption.     

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.     

The two calcium-binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils.

Recently, we identified the two myeloid related protein-8 (MRP8) (S100A8) and MRP14 (S100A9) as fatty acid-binding proteins (Klempt, M., Melkonyan, H., Nacken, W., Wiesmann, D., Holtkemper, U., and Sorg, C. (1997) FEBS Lett. 408, 81-84). Here we present data that the S100A8/A9 protein complex represents the exclusive arachidonic acid-binding proteins in human neutrophils. Binding and competition studies revealed evidence that (i) fatty acid binding was dependent on the calcium concentration; (ii) fatty acid binding was specific for the protein complex formed by S100A8 and S100A9, whereas the individual components were unable to bind fatty acids; (iii) exclusively polyunsaturated fatty acids were bound by S100A8/A9, whereas saturated (palmitic acid, stearic acid) and monounsaturated fatty acids (oleic acid) as well as arachidonic acid-derived eicosanoids (15-hydroxyeicosatetraenoic acid, prostaglandin E(2), thromboxane B(2), leukotriene B(4)) were poor competitors. Stimulation of neutrophil-like HL-60 cells with phorbol 12-myristate 13-acetate led to the secretion of S100A8/A9 protein complex, which carried the released arachidonic acid. When elevation of intracellular calcium level was induced by A23187, release of arachidonic acid occurred without secretion of S100A8/A9. In view of the unusual abundance in neutrophilic cytosol (approximately 40% of cytosolic protein) our findings assign an important role for S100A8/A9 as mediator between calcium signaling and arachidonic acid effects. Further investigations have to explore the exact function of the S100A8/A9-arachidonic acid complex both inside and outside of neutrophils.     

Proinflammatory S100 proteins regulate the accumulation of myeloid-derived suppressor cells

Chronic inflammation is a complex process that promotes carcinogenesis and tumor progression; however, the mechanisms by which specific inflammatory mediators contribute to tumor growth remain unclear. We and others recently demonstrated that the inflammatory mediators IL-1beta, IL-6, and PGE(2) induce accumulation of myeloid-derived suppressor cells (MDSC) in tumor-bearing individuals. MDSC impair tumor immunity and thereby facilitate carcinogenesis and tumor progression by inhibiting T and NK cell activation, and by polarizing immunity toward a tumor-promoting type 2 phenotype. We now show that this population of immature myeloid cells induced by a given tumor share a common phenotype regardless of their in vivo location (bone marrow, spleen, blood, or tumor site), and that Gr1(high)CD11b(high)F4/80(-)CD80(+)IL4Ralpha(+/-)Arginase(+) MDSC are induced by the proinflammatory proteins S100A8/A9. S100A8/A9 proteins bind to carboxylated N-glycans expressed on the receptor for advanced glycation end-products and other cell surface glycoprotein receptors on MDSC, signal through the NF-kappaB pathway, and promote MDSC migration. MDSC also synthesize and secrete S100A8/A9 proteins that accumulate in the serum of tumor-bearing mice, and in vivo blocking of S100A8/A9 binding to MDSC using an anti-carboxylated glycan Ab reduces MDSC levels in blood and secondary lymphoid organs in mice with metastatic disease. Therefore, the S100 family of inflammatory mediators serves as an autocrine feedback loop that sustains accumulation of MDSC. Since S100A8/A9 activation of MDSC is through the NF-kappaB signaling pathway, drugs that target this pathway may reduce MDSC levels and be useful therapeutic agents in conjunction with active immunotherapy in cancer patients.     

S100A8/A9 activate key genes and pathways in colon tumor progression

The tumor microenvironment plays an important role in modulating tumor progression. Earlier, we showed that S100A8/A9 proteins secreted by myeloid-derived suppressor cells (MDSC) present within tumors and metastatic sites promote an autocrine pathway for accumulation of MDSC. In a mouse model of colitis-associated colon cancer, we also showed that S100A8/A9-positive cells accumulate in all regions of dysplasia and adenoma. Here we present evidence that S100A8/A9 interact with RAGE and carboxylated glycans on colon tumor cells and promote activation of MAPK and NF-κB signaling pathways. Comparison of gene expression profiles of S100A8/A9-activated colon tumor cells versus unactivated cells led us to identify a small cohort of genes upregulated in activated cells, including Cxcl1, Ccl5 and Ccl7, Slc39a10, Lcn2, Zc3h12a, Enpp2, and other genes, whose products promote leukocyte recruitment, angiogenesis, tumor migration, wound healing, and formation of premetastatic niches in distal metastatic organs. Consistent with this observation, in murine colon tumor models we found that chemokines were upregulated in tumors, and elevated in sera of tumor-bearing wild-type mice. Mice lacking S100A9 showed significantly reduced tumor incidence, growth and metastasis, reduced chemokine levels, and reduced infiltration of CD11b(+)Gr1(+) cells within tumors and premetastatic organs. Studies using bone marrow chimeric mice revealed that S100A8/A9 expression on myeloid cells is essential for development of colon tumors. Our results thus reveal a novel role for myeloid-derived S100A8/A9 in activating specific downstream genes associated with tumorigenesis and in promoting tumor growth and metastasis.