Recombinant S100A8 Research Articles

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation.

Abstract 5399: S100A9 drives MDSC migration and contributes to tumor progression by inhibiting the immune response

Abstract S100A9 is a pro-inflammatory molecule, secreted by myeloid cells, which has been reported to recruit leukocytes to the sites of inflammation or tumors. We have previously found that up-regulation of S100A9 expression in myeloid progenitor cells in cancer resulted in inhibition of DC differentiation and promotion of expansion of myeloid-derived suppressor cells (MDSC). It remained unclear how S100A9 could regulate MDSC expansion and function. In this study we evaluated the effects of secreted (extracellular) vs intracellular S100A9 on MDSC. Recombinant S100A9 or S100A8 proteins separately or as heterodimers did not promote MDSC expansion from hematopoietic progenitor cells (HPC), or affect their suppressive activity. However, recombinant proteins had a strong chemotactic effect on MDSC. Interestingly, this effect on immature myeloid cells isolated from naïve tumor-free mice was substantially lower. To study intracellular activity of S100A9 we used S100A9 transgenic (Tg) and knockout (KO) mice. Mice were injected with several different weakly and strongly immunogeneic tumors. As expected Tg mice had increased expansion of MDSC and KO mice had substantially reduced number of these cells. Different levels of MDSC did not affect the growth of weakly immunogeneic tumors. In contrast, overexpression of S100A9 in mice lead to significantly accelerated growth of immunogenic tumors. Lack of S100A9 resulted in fast rejection of immunogeneic tumors. Our data indicate that extracellular and intracellular S100A9 had different signalling in myeloid cells and apparently affect different aspects of their function. S100A9 over-expression, in vivo, inhibits the immune response, most likely by promoting MDSC accumulation and/or suppressive activity. Our data suggest that targeting of S100A9 in cancer can be an attractive therapeutic option. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5399. doi:1538-7445.AM2012-5399

Alarmins S100A8 and S100A9 stimulate production of pro-inflammatory cytokines in M2 macrophages without changing their M2 membrane phenotype

Background Synovial activation, which plays an important role in a subgroup of osteoarthritis (OA) patients, is regulated by M1/M2 macrophage subpopulations: pro-inflammatory M1-macrophages and regulatory M2-macrophages. Alarmins S100A8 and S100A9 are found in high concentrations in synovial fluid of rheumatoid arthritis (RA) and OA patients and are correlated to inflammation and cartilage destruction. In the current study, the authors investigated the production of S100A8 and/or S100A9 by M1- and M2-macrophages and whether S100A8 and/or S100A9 could stimulate M2-macrophages, giving them a more pro-inflammatory character. Methods Monocytes were isolated from blood of healthy volunteers by isolating peripheral blood mononuclear cells (PBMCs) using a Ficoll-gradient followed by isolating CD14+ monocytes using magnetic-activated cell sorting (MACS). CD14+ monocytes were differentiated into M1- or M2-macrophages by 6 day stimulation with either GM-CSF or M-CSF, respectively. mRNA levels of S100A8 and S100A9 in M1 and M2 macrophages were measured with RT-qPCR. Intracellular protein production was determined using FACS analysis and S100A8/A9 secretion was measured using an ELISA.M1 and M2 macrophages were stimulated with human recombinant S100A8 and S100A9. M1 and M2 markers (IL-1β, tumour necrosis factor α (TNFα), IL-6 and CD163, CD206 respectively) were measured using RT-qPCR, FACS and/or Luminex. Results S100A8 and S100A9 had a significantly higher mRNA expression (65- and 2.5-fold respectively, n=10) in human monocyte derived M1-macrophages compared to M2. Also, the levels of intracellular S100A8 and S100A9 were higher in M1 than in M2 macrophages (4.3-fold and 6.6-fold higher respectively, n=6). The secreted protein level of the S100A8/A9 heterodimer was elevated from 55.5 ng/ml in M2 to 102.8 ng/ml in M1 macrophages (n=10). Stimulation of human monocyte derived M2-macrophages for 24 h with S100A8 and S100A9 showed a marked upregulation of several pro-inflammatory markers: IL-1β expression was increased by S100A8 and S100A9 on mRNA level (33- and 16-fold, n=10). At the protein level IL-6 and TNFα were strongly upregulated by S100A8 71- and 146-fold and by S100A9 93- and 121-fold, respectively (n=6). Interestingly however, S100A8 or S100A9 stimulation did not alter the level of M2 membrane markers CD163 and CD206, both at mRNA as well as protein level. Conclusions Stimulation of M2 macrophages with S100A8 and S100A9, produced in high amounts by M1- macrophages, upregulates pro-inflammatory cytokines IL-1β, IL-6 and TNFα while M2 membrane markers CD163 and CD206 remain unchanged. S100A8 and S100A9 production during OA could therefore contribute to synovial activation by stimulating the M2 macrophage towards a more pro-inflammatory phenotype.

Alarmins S100A8 and S100A9 stimulate production of pro-inflammatory cytokines in M2 macrophages without changing their M2 membrane phenotype

BackgroundSynovial activation, which plays an important role in a subgroup of osteoarthritis (OA) patients, is regulated by M1/M2 macrophage subpopulations: pro-inflammatory M1-macrophages and regulatory M2-macrophages. Alarmins S100A8 and S100A9 are...

Abstract 3648: S100A9 play a role in MDSC expansion and migration but not in their activation

Abstract S100A9 is a pro-inflammatory molecule, secreted by myeloid cells, which has been reported to recruit leukocytes to the sites of inflammation or tumors. Up-regulation of S100A9 expression in myeloid progenitor cells was implicated in inhibition of DC differentiation and promotion of expansion of myeloid-derived suppressor cells (MDSC). These cells have been well described as a major component used by the tumor to be protected from the immune system. In order to determine if extracellular and intracellular S100A9 has similar effects on myeloid cell differentiation and function, we evaluated the effect of recombinant S100A9. Our results demonstrated that in contrast to overexpression of S100A9 in hematopoietic progenitor cell (HPC), the treatment of HPC with recombinant S100A9 did not affect their differentiation to mature macrophages, or dendritic cells. The same treatment does not affect the suppressive activity of MDSC purified from naïve or tumor-bearing mice either. However, recombinant S100A9 had strong chemotactic effect on MDSC. Interestingly, its effect on immature myeloid cells isolated from naïve tumor-free mice was substantially lower. Recently, it has been suggested that MDSC need two different signals: one – required for their expansion and inhibited differentiation of mature myeloid cells and the other one – for activation. Our data suggest that S100A9 may mediate first signals but not the second one. Intracellular up-regulation of S100A9 promotes expansion of MDSC and contributes to block of their differentiation, whereas extracellular S100A9 via cell receptors induce MDSC migration. A better understanding of how MDSC migrate into the tumor and how S100A9 is involved in this process could lead to the generation of new treatment aiming to block the migration of MDSC inside the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3648. doi:10.1158/1538-7445.AM2011-3648

Dynamic Mobility of Immunological Cells Expressing S100A8 and S100A9 in vivo: A Variety of Functional Roles of the two Proteins as Regulators in Acute Inflammatory Reaction

The immunological properties of rat S100A8 (r-S100A8) and S100A9 (r-S100A9) in immune cells are poorly understood. Enzyme-linked immunosorbent assay (ELISA) for r-S100A9 enabled us to discuss the differential functional roles of the two proteins, and their localization in the cells was observed microscopically. Recombinant human S100A8 (rh-S100A8) or S100A9 (rh-S100A9) were intravenously administrated into rats with LPS-induced liver damage. ELISA was used to measure the serum concentration of S100A9 in the rats. Western blotting and a preparative ELISA were used to prove specificity and avidity of monoclonal antibodies for r-S100A8 and r-S100A9. Immunohistochemical staining was carried out to visualize intracellular localization of the two proteins in the immune cells using the antibodies. When rh-S100A8 was intravenously injected in the rats (B group), the serum concentration of r-S100A9 apparently decreased as compared with that of the positive control rats (A group). The activities of AST, ALT, and LD in the rat sera (B group) also significantly went down in comparison with those of the rats (A group). Although both the S100A8 and S100A9 were abundantly expressed in activated immune cells, quite difference of not only their intracellular localization but also distribution of the cells expressing the two proteins was microscopically observed. In the rats (B group), less number of the immune cells or less amount of r-S100A8 and r-S100A9 in the cells than those of the rats (A group) was also seen. The r-S100A8 could serve as a regulator of acute inflammatory reaction in the rats with LPS-induced damage.

S100A8 enhances osteoclast-mediated bone resorption in experimental antigen-induced arthritis through activation of toll-like receptor 4

ObjectiveRheumatoid arthritis (RA) is characterised by severe bone erosions caused by enhanced osteoclast formation and activity. Significantly elevated S100A8 and S100A9 levels in serum and synovial fluid of RA patients...

S100A8 and S100A9 induce a catabolic shift in chondrocytes from human osteoarthritis patients

Background and objectiveS100A8 and S100A9 are damage associated molecular patterns that are associated with inflammation and cartilage and bone erosion during human rheumatoid arthritis. They are produced in large amounts...

S100A8 causes a shift toward expression of activatory Fcγ receptors on macrophages via toll‐like receptor 4 and regulates Fcγ receptor expression in synovium during chronic experimental arthritis

The levels of both Fcγ receptor (FcγR) and the alarmins S100A8 and S100A9 are correlated with the development and progression of cartilage destruction during antigen-induced arthritis (AIA). This study was undertaken to study the active involvement of S100A8, S100A9, and S100A8/S100A9 in FcγR regulation in murine macrophages and synovium during AIA. Recombinant murine S100A8 (rS100A8) was injected into normal mouse knee joints, and the synovium was isolated for analysis of FcγR messenger RNA (mRNA) expression by reverse transcription-polymerase chain reaction (RT-PCR). Macrophages, including bone marrow macrophages derived from Toll-like receptor 4-deficient (TLR-4(-/-)) mice, and polymorphonuclear cells (PMNs) were stimulated with S100 proteins, and levels of FcγR mRNA and protein were measured using RT-PCR and fluorescence-activated cell sorting analyses. AIA was induced in the knee joints of S100A9-deficient (S100A9(-/-)) mice, compared with wild-type (WT) controls, and the extent of cartilage destruction was determined using immunohistochemical analysis. Intraarticular injection of rS100A8 into the knee joints of normal mice caused a strong up-regulation of mRNA levels of activating FcγRI (64-fold increase) and FcγRIV (256-fold increase) in the synovium. Stimulation of macrophages with rS100A8 led to significant up-regulation of mRNA and protein levels of FcγRI and FcγRIV, but not FcγRIII, while the effects of S100A9 or S100A8/S100A9 complexes were less potent. Stimulation of PMNs (32Dcl3 cell line) with S100 proteins had no effect on FcγR expression. Up-regulation of FcγRI and FcγRIV was abrogated in rS100A8-stimulated macrophages from TLR-4(-/-) mice, indicating that the induction of FcγR expression by S100A8 is mediated by TLR-4. FcγR expression in the inflamed synovium of S100A9(-/-) mice was significantly lower on day 14 after arthritis induction when compared with WT controls, and these findings correlated with reduced severity of matrix metalloproteinase-mediated cartilage destruction. S100A8 is a strong promoter of activating FcγRI and FcγRIV in macrophages through the activation of TLR-4, and acts as a regulator of FcγR expression in inflamed synovium in chronic experimental arthritis.

Identification of serum proteins that bind with S100A8, S100A9 and S100A8/A9: Clinical significance of using proteins for monitoring the postoperative condition of liver recipients

Serum proteins that non-specifically bind with human S100A8/A9 (h-S100A8/A9) have been proposed. Our aim was to isolate and identify these proteins, and verify their clinical significance for monitoring the postoperative condition of liver recipients, and further to discuss the transportation of human fibronectin (h-FN) with h-S100A8/A9 and its functional role in vivo. To isolate the serum proteins, recombinant human S100A8, S100A9 and S100A8/A9 affinity columns were used. Proteins were identified by mass spectrometry. Two enzyme-linked immunosorbent assays (ELISA) were used to measure h-S100A8/A9 and h-FN in the sera of liver recipients. Flow cytometry was employed to detect h-S100A8/A9 and h-FN on immunological cells. Western blotting was used to confirm serum constituents using antibodies specific to each constituent. One of the proteins was identified with h-FN, and its fluctuation pattern in the serum of the recipient was in contrast to that of CRP. Flow cytometry showed a positive reaction for h-S100A8/A9 and h-FN on neutrophils and monocytes, indicating that both proteins exist on these cells. The h-FN could be transported with S100A8/A9 in blood and/or on immunological cells, and effectively prevent further attack by various internal oxidants or repair damaged liver tissue in vivo.

Effects of S100A9 in a rat model of asthma and in isolated tracheal spirals

S100A9 is a member of the S100 family of proteins that contain two EF-hand calcium-binding motifs. We previously reported that S100A9 was differentially expressed during the early airway response phase of asthma and can be regulated by acupuncture. To understand the possible role of S100A9 in asthma, the effects of the S100A9 were investigated in a rat model of asthma and in isolated tracheal spirals. The pulmonary function and isometric tension were measured after the administration of purified recombinant S100A9. The results of in vivo experiments showed that S100A9 (0.1 μg/kg) significantly decreased the pulmonary resistance and increased the dynamic compliance. The in vitro experimental results showed that S100A9 (100, 200, 400, or 800 ng/ml, final concentrations) significantly reduced the isometric tension of isolated tracheal spirals. These results suggest that S100A9 elicits dose-dependent anti-asthmatic effects and may provide further insight into the treatment of asthma.

Effect of human immunodeficiency virus infection on S100A8/A9 inhibition of peripheral neutrophils oxidative metabolism

Neutrophils are endowed with a highly active oxidative metabolism that is crucial for their antimicrobial functions but can produce oxidative conditions disruptive to the host. Opportunistic infections associated with HIV disease and ex vivo studies of neutrophils from HIV patients suggest that neutrophil dysfunctions significantly contribute to HIV disease. The calcium-binding proteins S100A8 and S100A9 are abundant cytosolic constituents of human neutrophils. Our previous work has shown that S100A8 and S100A9 inhibit neutrophil oxidative metabolism. In this study, we tested the hypothesis that neutrophils from HIV infected subjects respond differently to S100A8 and S100A9 when compared to neutrophils isolated from control HIV naive subjects. Neutrophils, freshly isolated from whole blood, were tested in a 96-well plate assay for their ability to oxidize the DCFH-DA probe. The neutrophils from HIV+ and HIV− subjects were stimulated with LPS and inhibited with recombinant S100A8 and S100A9. Our data indicate that when compared to neutrophils isolated from HIV− subjects, neutrophils from HIV+ subjects display an exaggerated response to LPS and a diminished response to S100A8 and S100A9 inhibition. Our data support our hypothesis and signify that, in HIV disease, dysregulated neutrophil responses to endotoxins stimulation and S100A8/A9 inhibition may contribute to a higher risk for oxidative stress associated ailments. The mechanism for the observed differences in neutrophil response and their biological significance in the course of HIV disease should be addressed in further studies.

S100A8/A9 Induces Apoptosis and Inhibits Metastasis of CasKi Human Cervical Cancer Cells

S100 proteins, a family of Ca(2+)-binding proteins, have been linked to several human diseases in recent years. Deregulated expression of S100 proteins, including S100A9 and its partner S100A8, was reported to be associated with neoplastic disorders. In our previous study using serial analysis of gene expression, we identified decreased expressions of S100A9 and S100A8 in human cervical squamous cell carcinoma. To investigate the functions of S100A8 and S100A9 in cervical cancer, we purified recombinant S100A8 and S100A9 proteins and treated CaSki human cervical cancer cells with these proteins. We found that S100A8/A9 induced apoptosis and inhibited migration of CaSki cells; S100A8/A9 also reduced the expression of matrix metalloproteinase (MMP)-2 in CaSki cells. In summary, this study suggests that S100A8 and S100A9 have inhibitory effects on the proliferation of CaSki carcinoma cells by inducing cell apoptosis and on the invasiveness of CaSki cells.

Stimulation of chondrocyte‐mediated cartilage destruction by S100A8 in experimental murine arthritis

To investigate whether S100A8 is actively involved in matrix metalloproteinase (MMP)-mediated chondrocyte activation. S100A8 and S100A9 proteins were detected in inflamed knee joints from mice with various forms of murine arthritis, using immunolocalization. Murine chondrocyte cell line H4 was stimulated with proinflammatory cytokines or recombinant S100A8. Messenger RNA (mRNA) and protein levels were measured using reverse transcriptase-polymerase chain reaction and intracellular fluorescence-activated cell sorting (FACS). Breakdown of aggrecan on the pericellular surface of the chondrocytes was measured using VDIPEN and NITEGE antibodies and FACS, and breakdown in patellar cartilage was measured by immunolocalization. S100A8 and S100A9 proteins were abundantly expressed in and around chondrocytes in inflamed knee joints after induction of antigen-induced arthritis or onset of spontaneous arthritis in interleukin-1 (IL-1) receptor antagonist-knockout mice. Stimulation of chondrocytes by the proinflammatory cytokines tumor necrosis factor alpha, IL-1beta, IL-17, and interferon-gamma caused strong up-regulation of S100A8 mRNA and protein levels and up-regulation to a lesser extent of S100A9 levels. Stimulation of chondrocytes with S100A8 induced significant up-regulation of MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 mRNA levels (up-regulated 4, 4, 3, 16, 8, and 4 times, respectively). VDIPEN and NITEGE neoepitopes were significantly elevated in a concentration-dependent manner in chondrocytes treated with 0.2, 1, or 5 microg/ml of S100A8. (VDIPEN levels were elevated 17%, 67%, and 108%, respectively, and NITEGE levels were elevated 8%, 33%, and 67%, respectively.) S100A8 significantly increased the effect of IL-1beta on MMP-3, MMP-13, and ADAMTS-5. Mouse patellae incubated with both IL-1beta and S100A8 had elevated levels of NITEGE within the cartilage matrix when compared with patellae incubated with IL-1beta or S100A8 alone. These findings indicate that S100A8 and S100A9 are found in and around chondrocytes in experimental arthritis. S100A8 up-regulates and activates MMPs and aggrecanase-mediated pericellular matrix degradation.

S100A8 AND S100A9 INCREASE PANCREATIC AND COLORECTAL CANCER CELL MOTILITY AND PROLIFERATION

Introduction: We previously reported that Smad4 negative pancreatic tumours have fewer S100A8, but not S100A9-expressing stromal monocytes than Smad4 positive tumours. We have since extended this finding to colorectal tumours. Here we investigate the effects of these proteins on pancreatic and colorectal cancer cell motility and proliferation. Methods: Recombinant GST-tagged S100A8, S100A9 and control GST proteins were generated and purified from E. coli. The effects of these proteins on the motility and proliferation of pancreatic and colorectal cancer cell lines, including lines where Smad4 has been re-expressed or depleted, were assessed using Boyden Chamber and MTT assays respectively. Results: Incubation with GST-S100A8 and GST-S100A9 singly or in combination significantly increased pancreatic and colorectal cancer cell motility by 2-10 and 6-15 fold respectively compared to GST. Colorectal cancer cell proliferation was significantly increased in a dose-dependent manner after 24 h or 36 h of incubation with recombinant S100 proteins. Pancreatic cancer cells showed an increase in proliferation when incubated with a combination of S100A8 and S100A9. The effects of Smad4 loss or re-expression on the response to these proteins will be presented. Conclusion: S100A8 and S100A9 significantly increase pancreatic and colorectal cancer cell motility and proliferation. Our data indicate that these proteins may play an important role in cancer growth and spread.

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.

S100A8 and S100A9 activate MAP kinase and NF-κB 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.

Fibroblast growth-stimulating activity of S100A9 (MRP-14).

Fibroblasts play a critical role in chronic inflammation and wound healing. In this study, a fibroblast growth-stimulating factor was purified from the exudate of carrageenan-induced inflammation in rats. The purified protein was a disulfide-linked homodimer. Amino acid sequence analysis of the peptides generated by cleavage with cyanogen bromide and proteinase V8 resulted in identification of the protein as S100A9. Recombinant S100A9 as well as its disulfide-linked homodimer stimulated the proliferation of fibroblasts at a similar concentration of the purified protein. The concentration of S100A9 in the exudate was determined by immunoblot analysis. The total protein concentration in the exudate reached a maximum 4 days after carrageenan injection and then slightly decreased, whereas the concentration of S100A9 reached a maximum at day 3 and then decreased rapidly. These studies show that S100A9 is present at a high concentration in the exudate of carrageenan-induced inflammation in rats, and that S100A9 stimulates proliferation of fibroblasts, suggesting that it plays a role in chronic inflammation.

S100A8, S100A9 and the S100A8/A9 heterodimer complex specifically bind to human endothelial cells: identification and characterization of ligands for the myeloid-related proteins S100A9 and S100A8/A9 on human dermal microvascular endothelial cell line-1 cells.

The natural ligands of the S100 EF hand proteins S100A8 and A9 [myeloid-related proteins 8 and 14] have long been searched for in order to further the understanding of the role of the S100A8/A9-expressing monocyte subpopulation in progressing inflammatory processes. We demonstrate that S100A8, S100A9 and the S100A8/A9 heterodimeric complex bind to human dermal microvascular endothelial cell line (HMEC)-1 with an increasing binding capacity progressing from S100A8 < or = S100A9 < or = S100A8/A9. Similar results were obtained in the apolipoprotein E knockout mouse model, where preferably recombinant S100A9 but no S100A8 bound to the endothelium of the aorta ascendens. The binding of the S100A8/A9 heterodimer complex to activated HMEC-1 is specific as demonstrated by a dose-responding and satiable binding curve and the competition of FITC-labeled versus unlabeled protein. The protein character of the binding site was proven by treatment with trypsin. S100A8/A9 binding to HMEC-1 is inducible by lipopolysaccharide and tumor necrosis factor-alpha, and in the presence of calcium. A 163-kDa protein was isolated from a cell lysate of activated HMEC-1 cells using an affinity-chromatography protocol. The endothelial cell-associated ligand proteins isolated by the use of the S100A9 monomer and the S100A8/A9 dimer were subjected to mass spectrometry for protein identification. Clearly, alpha(2)-macroglobulin was identified as a binding partner for the S100A9 monomer, whereas no protein could be identified from the database for the ligand of the S100A8/A9 dimer.

Oxidation Regulates the Inflammatory Properties of the Murine S100 Protein S100A8

The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites. S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.