S100A9 deficiency alters adenosine-5'-triphosphate induced calcium signalling but does not generally interfere with calcium and zinc homeostasis in murine neutrophils

The two calcium- and zinc-binding proteins, S100A9 and S100 A8, abundant in myeloid cells are considered to play important roles in both calcium signalling and zinc homeostasis. Polymorphonuclear neutrophils from S100A9 ko mice are also devoid of S100A8. Therefore, S100A9-deficient neutrophils were used as a model to study the role of the two S100 proteins in the neutrophils's calcium and zinc metabolism. Analysis of the intracellular zinc level upon pyrithione and (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1) treatment revealed no differences between S100A9-deficient and wildtype neutrophils. Similar, the calcium signals were not distinguishable from S100A9-deficient and wildtype neutrophils upon stimulation with platelet activating factor (PAF), thapsigargin or macrophage inflammatory protein 1 alpha (MIP-1 alpha), indicating despite their massive expression S100A8/A9 do neither serve as calcium nor as zinc buffering proteins in granulocytes. In contrast, stimulation with adenosine-5'-triphosphate (ATP) induces a significant stronger increase of the intracellular free calcium level in S100A9-deficient cells compared to wildtype cells. Moreover, the ATP-induced calcium signal was still different when the cells were incubated in calcium free buffer suggesting that pirinergic receptors of the P(2Y) class could be involved in this signalling pathway.     

Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations

S100A8 and S100A9 are two proinflammatory molecules belonging to the S100 family of calcium-binding proteins. Common to all S100 proteins S100A8 and S100A9 form non-covalently associated complexes which have been shown to exhibit different functional properties. Besides dimerization, recent research is focused on the importance of higher oligomeric structures of S100 proteins induced by bivalent cations. While S100A8/S100A9-heterodimers are formed in the absence of calcium, tetramerization is strictly calcium-dependent. Heterodimer formation is not a simple process and our biophysical analyses (FRET, ESI-MS) demonstrate that simply mixing both subunits is not sufficient to induce complex formation. Steps of denaturation/renaturation are necessary for the recombinant complex to show identical biophysical properties as S100A8/S100A9 obtained from granulocytes. In addition to calcium both proteins are able to bind zinc with high affinity. Here we demonstrate for the first time by different biophysical methods (MALDI-MS, ESI-MS, fluorescence spectroscopy) that zinc-binding, like calcium, induces (S100A8/S100A9)(2)-tetramers. Using mass spectrometric investigations we demonstrate that zinc triggers the formation of (S100A8/S100A9)(2)-tetramers by zinc-specific binding sites rather than by interactions with calcium-specific EF-hands. The zinc-induced tetramer is structurally very similar to the calcium-induced tetramer. Thus, like calcium, zinc acts as a regulatory factor in S100A8/S100A9-dependent signaling pathways.     

Crystal structure of human S100A8 in complex with zinc and calcium

Background

S100 proteins are a large family of calcium binding proteins present only in vertebrates. They function intra- and extracellularly both as regulators of homeostatic processes and as potent effectors during inflammation. Among these, S100A8 and S100A9 are two major constituents of neutrophils that can assemble into homodimers, heterodimers and higher oligomeric species, including fibrillary structures found in the ageing prostate. Each of these forms assumes specific functions and their formation is dependent on divalent cations, notably calcium and zinc. In particular, zinc appears as a major regulator of S100 protein function in a disease context. Despite this central role, no structural information on how zinc bind to S100A8/S100A9 and regulates their quaternary structure is yet available.

Results

Here we report two crystallographic structures of calcium and zinc-loaded human S100A8. S100A8 binds two zinc ions per homodimer, through two symmetrical, all-His tetracoordination sites, revealing a classical His-Zn binding mode for the protein. Furthermore, the presence of a (Zn)₂-cacodylate complex in our second crystal form induces ligand swapping within the canonical His₄ zinc binding motif, thereby creating two new Zn-sites, one of which involves residues from symmetry-related molecules. Finally, we describe the calcium-induced S100A8 tetramer and reveal how zinc stabilizes this tetramer by tightening the dimer-dimer interface.

Conclusions

Our structures of Zn²⁺/Ca²⁺-bound hS100A8 demonstrate that S100A8 is a genuine His-Zn S100 protein. Furthermore, they show how zinc stabilizes S100A8 tetramerization and potentially mediates the formation of novel interdimer interactions. We propose that these zinc-mediated interactions may serve as a basis for the generation of larger oligomers in vivo.

     

S100A8/9 induces cell death via a novel, RAGE-independent pathway that involves selective release of Smac/DIABLO and Omi/HtrA2

A complex of two S100 EF-hand calcium-binding proteins S100A8/A9 induces apoptosis in various cells, especially tumor cells. Using several cell lines, we have shown that S100A8/A9-induced cell death is not mediated by the receptor for advanced glycation endproducts (RAGE), a receptor previously demonstrated to engage S100 proteins. Investigation of cell lines either deficient in, or over-expressing components of the death signaling machinery provided insight into the S100A8/A9-mediated cell death pathway. Treatment of cells with S100A8/A9 caused a rapid decrease in the mitochondrial membrane potential (DeltaPsi(m)) and activated Bak, but did not cause release of apoptosis-inducing factor (AIF), endonuclease G (Endo G) or cytochrome c. However, both Smac/DIABLO and Omi/HtrA2 were selectively released into the cytoplasm concomitantly with a decrease in Drp1 expression, which inhibits mitochondrial fission machinery. S100A8/A9 treatment also resulted in decreased expression of the anti-apoptotic proteins Bcl2 and Bcl-X(L), whereas expression of the pro-apoptotic proteins Bax, Bad and BNIP3 was not altered. Over-expression of Bcl2 partially reversed the cytotoxicity of S100A8/A9. Together, these data indicate that S100A8/A9-induced cell death involves Bak, selective release of Smac/DIABLO and Omi/HtrA2 from mitochondria, and modulation of the balance between pro- and anti-apoptotic proteins.     

Implication of extracellular zinc exclusion by recombinant human calprotectin (MRP8 and MRP14) from target cells in its apoptosis-inducing activity

BACKGROUND: Calprotectin is a calcium-binding and zinc-binding protein complex that is abundant in the cytosol of neutrophils. This factor is composed of 8 and 14 kDa subunits, which have also been termed migration inhibitory factor-related proteins MRP8 and MRP14. We previously reported that rat calprotectin purified from inflammatory neutrophils induces apoptosis of various tumor cells or normal fibroblasts in a zinc-reversible manner. AIM: The present study was undertaken to elucidate which subunit is responsible for the apoptosis-inducing activity, and to explore the mechanism of zinc-reversible apoptosis induction. METHODS: The apoptosis-inducing activity of recombinant human MRP8 (rhMRP8) and recombinant human MRP14 (rhMRP14) was examined against EL-4 lymphoma cells in vitro. To determine whether zinc deprivation by calprotectin was essential for the cytotoxicity, the activity of calprotectin was tested under conditions where physical contact between the factor and the cells was precluded by a low molecular weight cut-off dialysis membrane. RESULTS: The cytotoxicity of rhMRP14 against EL-4 cells was first detected at 10 microM in a standard medium, whereas rhMRP8 caused only marginal cytotoxicity at 40 microM. A mixture of both proteins showed higher specific activity (onset of cytotoxicity at 5 microM). When the cells were cultured in divalent cation-depleted medium, each dose-response curve was shifted to about a four-fold lower concentration range. Calprotectin was found to induce cell death even when the complex and the target cells were separated by dialysis membrane. A membrane-impermeable zinc chelator, diethylenetriamine pentaacetic acid (DTPA), also induced target cell apoptosis in a similar time-course as calprotectin. Moreover, the activities of calprotectin and DTPA were completely inhibited by the presence of zinc ions. CONCLUSION: These data indicate that calprotectin has higher specific activity to induce apoptosis than the Individual subunits, and that the mechanism is exclusion of zinc from target cells.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

Calcium-dependent tetramer formation of S100A8 and S100A9 is essential for biological activity

S100 proteins comprise the largest family of calcium-binding proteins. Members of this family usually form homo- or heterodimers, which may associate to higher-order oligomers in a calcium-dependent manner. The heterodimers of S100A8 and S100A9 represent the major calcium-binding proteins in phagocytes. Both proteins regulate migration of these cells via modulation of tubulin polymerization. Calcium binding induces formation of (S100A8/S100A9)2 tetramers. The functional relevance of these higher-order oligomers of S100 proteins, however, is not yet clear. To investigate the importance of higher-order oligomerization for S100 proteins, we created a set of mutations within S100A9 (N69A, E78A, N69A+E78A) destroying the high-affinity C-terminal calcium-binding site (EF-hand II). Mutations in EF-hand II did not interfere with formation of the S100A8/S100A9 heterodimer as demonstrated by yeast two-hybrid experiments and pull-down assays. In contrast, mass spectrometric analysis and density gradient centrifugation revealed that calcium-induced association of (S100A8/S100A9)2 tetramers was strictly dependent on a functional EF-hand II in S100A9. Failure of tetramer formation was associated with a lack of functional activity of S100A8/S100A9 complexes in promoting the formation of microtubules. Thus, our data demonstrate that calcium-dependent formation of (S100A8/S100A9)2 tetramers is an essential prerequisite for biological function. This is the first report showing a functional relevance of calcium-induced higher-order oligomerization in the S100 family.     

Role of reactive oxygen species in zinc deficiency-induced hepatic stellate cell activation

We previously reported that zinc deficiency caused a reduction in intracellular glutathione at 8 h after the addition of zinc chelator, diethylenetriamine pentaacetic acid (DTPA), compared with control levels in rat hepatic stellate cells. In this study, we investigated the role of reactive oxygen species and glutathione on the mechanism of zinc deficiency-induced hepatic stellate cell activation, via assessing collagen synthesis. Isolated hepatic stellate cells were incubated with or without DTPA. Type I collagen expression in hepatic stellate cells was detected by immunohistochemistry, and then quantification of the intensity of type I collagen expression was analyzed using a computer with NIH image. Intracellular glutathione was measured using HPLC. H(2)O(2) release from hepatic stellate cells into the overlying medium was assayed using a fluorimetric method. H(2)O(2) release by DTPA-treated hepatic stellate cells significantly increased from 4 h, but returned to control levels after zinc supplementation. When catalase was added to the culture at 6 h after the addition of DTPA, the staining for type I collagen was as weak as at control levels. Diphenyliodonium chloride, the inhibitor of NADPH oxidase, produced a marked reduction in zinc deficiency-induced H(2)O(2) release. The results of this study show that the depletion of intracellular glutathione levels triggers a progression of collagen synthesis in zinc deficient-hepatic stellate cells and this depletion may be induced by the stimulation of cellular production of H(2)O(2).     

Interaction between S100A8/A9 and annexin A6 is involved in the calcium-induced cell surface exposition of S100A8/A9

The calcium binding S100A8/A9 complex (MRP8/14; calgranulin) is considered as an important proinflammatory mediator in acute and chronic inflammation and has recently gained attention as a molecular marker up-regulated in various human cancers. Here, we report that S100A8/A9 is expressed in breast cancer cell lines and is up-regulated by interleukin-1beta and tumor necrosis factor-alpha in SKBR3 and MCF-7 cells. We identified the phospholipid-binding protein annexin A6 as a potential S100A8/A9 binding protein by affinity chromatography. This finding was verified by Southwestern overlay experiments and by coimmunoprecipitation with the S100A8/A9-specific monoclonal antibody 27E10. Immunocytochemical experiments demonstrated that S100A8/A9 and annexin A6 colocalize in SKBR3 breast cancer cells predominantly in membranous structures. Upon calcium influx both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells. Subcellular fractionation studies suggested that after A23187 stimulation membrane association of S100A8/A9 is not enhanced. However, both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells upon calcium influx. Experiments with artificial liposomes indicated that S100A8/A9 is able to associate with membranes independently of both annexin A6 and independently of calcium. Finally, cell surface expression of S100A8/A9 could not be observed in A23187-treated A431 and HaCaT cells. Both cell lines are known to be devoid of annexin A6. Repression of annexin A6 expression by small interfering RNA in SKBR3 cells abolishes the cell surface exposition of S100A8/A9 upon calcium influx, suggesting that annexin A6 contributes to the calcium-dependent cell surface exposition of the membrane associated-S100A8/A9 complex.     

Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

Introduction  

Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9.     

Human S100A9 Protein Is Stabilized by Inflammatory Stimuli via the Formation of Proteolytically-Resistant Homodimers

S100A8 and S100A9 are Ca²⁺-binding proteins that are associated with acute and chronic inflammation and cancer. They form predominantly heterodimers even if there are data supporting homodimer formation. We investigated the stability of the heterodimer in myeloid and S100A8/S100A9 over-expressing COS cells. In both cases, S100A8 and S100A9 proteins were not completely degraded even 48 hrs after blocking protein synthesis. In contrast, in single transfected cells, S100A8 protein was completely degraded after 24 h, while S100A9 was completely unstable. However, S100A9 protein expression was rescued upon S100A8 co-expression or inhibition of proteasomal activity. Furthermore, S100A9, but not S100A8, could be stabilized by LPS, IL-1β and TNFα treatment. Interestingly, stimulation of S100A9-transfected COS cells with proteasomal inhibitor or IL-1β lead to the formation of protease resistant S100A9 homodimers. In summary, our data indicated that S100A9 protein is extremely unstable but can be rescued upon co-expression with S100A8 protein or inflammatory stimuli, via proteolytically resistant homodimer formation. The formation of S100A9 homodimers by this mechanism may constitute an amplification step during an inflammatory reaction.     

S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells

S100 proteins, a multigenic family of calcium-binding proteins, have been linked to human pathologies in recent years. Deregulated expression of S100 proteins, including S100A8 and S100A9, was reported in association with neoplastic disorders. In a previous study, we identified enhanced expression of S100A8 and S100A9 in human prostate cancer. To investigate potential functional implications of S100A8 and S100A9 in prostate cancer, we examined the influence of over-expressed and of purified recombinant S100A8 and S100A9 proteins in different prostate epithelial cell lines. S100A8 and S100A9 were secreted by prostate cancer cells, a finding which prompted us to analyze a possible function as extracellular ligands. S100A8/A9 induced the activation of NF-kappaB and an increased phosphorylation of p38 and p44/42 MAP kinases. In addition, extracellular S100A8/A9 stimulated migration of benign prostatic cells in vitro. Furthermore, in immunofluorescence experiments, we found a strong speckled co-localization of intracellular S100A8/A9 with RAGE after stimulating cells with recombinant S100A8/A9 protein or by increasing cytosolic Ca2+ levels. In summary, our findings show that S100A8 and S100A9 are linked to the activation of important features of prostate cancer cells.     

S100A14 stimulates cell proliferation and induces cell apoptosis at different concentrations via receptor for advanced glycation end products (RAGE)

S100A14 is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effects on different types of cells. However, exact extracellular roles of S100A14 have not been clarified yet. Here we investigated the effects of S100A14 on esophageal squamous cell carcinoma (ESCC) cell lines. Results demonstrated that low doses of extracellular S100A14 stimulate cell proliferation and promote survival in KYSE180 cells through activating ERK1/2 MAPK and NF-κB signaling pathways. Immunoprecipitation assay showed that S100A14 binds to receptor for advanced glycation end products (RAGE) in KYSE180 cells. Inhibition of RAGE signaling by different approaches including siRNA for RAGE, overexpression of a dominant-negative RAGE construct or a RAGE antagonist peptide (AmphP) significantly blocked S100A14-induced effects, suggesting that S100A14 acts via RAGE ligation. Furthermore, mutation of the N-EF hand of S100A14 (E39A, E45A) virtually reduced 10 μg/ml S100A14-induced cell proliferation and ERK1/2 activation. However, high dose (80 μg/ml) of S100A14 causes apoptosis via the mitochondrial pathway with activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase. High dose S100A14 induces cell apoptosis is partially in a RAGE-dependent manner. This is the first study to demonstrate that S100A14 binds to RAGE and stimulates RAGE-dependent signaling cascades, promoting cell proliferation or triggering cell apoptosis at different doses.     

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.     

Amyloid Formation by the Pro-Inflammatory S100A8/A9 Proteins in the Ageing Prostate

Background

The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer''s disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon − prostate tissue remodelling in middle-aged and elderly men.

Methodology/Principal Findings

By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions.

Conclusions/Significance

These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.     

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.     

The crystal structure of the human (S100A8/S100A9)2 heterotetramer, calprotectin, illustrates how conformational changes of interacting alpha-helices can determine specific association of two EF-hand proteins

The EF-hand proteins S100A8 and S100A9 are important calcium signalling proteins that are involved in wound healing and provide clinically relevant markers of inflammatory processes, such as rheumatoid arthritis and inflammatory bowel disease. Both can form homodimers via distinct modes of association, probably of lesser stability in the case of S100A9, whereas in the presence of calcium S100A8 and S100A9 associate to calprotectin, the physiologically active heterooligomer. Here we describe the crystal structure of the (S100A8/S100A9)(2) heterotetramer at 1.8 A resolution. Its quaternary structure illustrates how specific heteroassociation is energetically driven by a more extensive burial of solvent accessible surface areas in both proteins, most pronounced for S100A9, thus leading to a dimer of heterodimers. A major contribution to tetramer association is made by the canonical calcium binding loops in the C-terminal halves of the two proteins. The mode of heterodimerisation in calprotectin more closely resembles the subunit association previously observed in the S100A8 homodimer and provides trans stabilisation for S100A9, which manifests itself in a significantly elongated C-terminal alpha-helix in the latter. As a consequence, two different putative zinc binding sites emerge at the S100A8/S100A9 subunit interface. One of these corresponds to a high affinity arrangement of three His residues and one Asp side-chain, which is unique to the heterotetramer. This structural feature explains the well known Zn(2+) binding activity of calprotectin, whose overexpression can cause strong dysregulation of zinc homeostasis with severe clinical symptoms.     

High Levels of S100A8/A9 Proteins Aggravate Ventilator-Induced Lung Injury via TLR4 Signaling

Background

Bacterial products add to mechanical ventilation in enhancing lung injury. The role of endogenous triggers of innate immunity herein is less well understood. S100A8/A9 proteins are released by phagocytes during inflammation. The present study investigates the role of S100A8/A9 proteins in ventilator-induced lung injury.

Methods

Pulmonary S100A8/A9 levels were measured in samples obtained from patients with and without lung injury. Furthermore, wild-type and S100A9 knock-out mice, naive and with lipopolysaccharide-induced injured lungs, were randomized to 5 hours of spontaneously breathing or mechanical ventilation with low or high tidal volume (V_T). In addition, healthy spontaneously breathing and high V_T ventilated mice received S100A8/A9, S100A8 or vehicle intratracheal. Furthermore, the role of Toll-like receptor 4 herein was investigated.

Results

S100A8/A9 protein levels were elevated in patients and mice with lung injury. S100A8/A9 levels synergistically increased upon the lipopolysaccharide/high V_T MV double hit. Markers of alveolar barrier dysfunction, cytokine and chemokine levels, and histology scores were attenuated in S100A9 knockout mice undergoing the double-hit. Exogenous S100A8/A9 and S100A8 induced neutrophil influx in spontaneously breathing mice. In ventilated mice, these proteins clearly amplified inflammation: neutrophil influx, cytokine, and chemokine levels were increased compared to ventilated vehicle-treated mice. In contrast, administration of S100A8/A9 to ventilated Toll-like receptor 4 mutant mice did not augment inflammation.

Conclusion

S100A8/A9 proteins increase during lung injury and contribute to inflammation induced by HV_T MV combined with lipopolysaccharide. In the absence of lipopolysaccharide, high levels of extracellular S100A8/A9 still amplify ventilator-induced lung injury via Toll-like receptor 4.     

FGF-2, IL-1beta and TGF-beta regulate fibroblast expression of S100A8

Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+-binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon gamma (IFNgamma), tumour-necrosis factor (TNF) and TGF-beta did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged by heparin. Interleukin-1beta (IL-1beta) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-2 and IL-1beta was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-2-responsive elements were distinct from the IL-1beta-responsive elements in the S100A8 gene promoter. FGF-2-/heparin-induced, but not IL-1beta-induced responses were significantly suppressed by TGF-beta, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-2/IL-1beta, down-regulation by TGF-beta, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.