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
Ines Eue,
Simone König1,
Jolanthe Pior2 and
Clemens Sorg3
PAN Clinic, Zeppelinstrasse 1, 50667 Köln, Germany
1 Interdisciplinary Clinical Research Center, Robert-Koch-Strasse 31, 48149 Münster, Germany
2 Clinic of Dermatology and
3 Institute of Experimental Dermatology, von-Esmarch-Strasse 56, 48149 Münster, Germany
Correspondence to:
I. Eue; E-mail: Ines.Eue{at}aol.com
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Abstract
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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
S100A9
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-
, 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,
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.
Keywords: endothelial cells,, human dermal microvascular endothelial cell line-1,, myeloid-related protein 8/14, S100A8, S100A9
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Introduction
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Members of the S100 class of calcium-binding proteins such as S100A8 and A9 [or, according to the old nomenclature, myeloid-related proteins (MRP) 8 and 14] are assumed to be crucial determinants in the initiation and development of inflammatory processes, especially regarding their role in neutrophil recruitment and cellular activation. It has been obvious both in animal models and human studies on chronic and acute inflammatory diseases such as rheumatoid arthritis, cystic fibrosis and gingivitis that one distinct monocyte subpopulation expressing the S100A8/A9 complex in a membrane-associated form belongs to the first cells which are recruited to the prone sites of inflammation (17,41). Due to increased calcium levels in mononuclear cells and neutrophils as they occur under inflammatory conditions, the cytosolic monomers of S100A8 and S100A9 are translocated to the cell membranes where they form a heterodimer in co-localization with proteins of the cytoskeleton (810,42). Activated endothelium was demonstrated to be a natural stimulator for monocytes to specifically release S100A8 and S100A9 (7,11). The secretion mechanism is known to be a protein kinase C-dependent process following a non-Golgi-mediated alternative pathway (10). According to recent mass spectrometry (MS) analysis, the most stable and very probably biological relevant form of S100A8 and A9 is a heterotetrameric complex which is formed after release (12). Whereas knowledge about the structural details of these proteins is quite extensive, little information is available at present about the role of S100A8/A9 in the regulation of cellular functions such as adhesion and transendothelial migration. The frequent appearance of S100A8/A9-expressing monocytes in inflammatory lesions as well as the high serum levels which were measured in patients with chronic inflammation suggest a key role of these cells and proteins in the modulation of the course of inflammatory processes. As recently shown, S100A9 and the S100A8/A9 complex effectively regulate CD11b expression/affinity in monocytes and neutrophils, and thus influences migration behavior (7,13).
It has been a goal of many studies to identify a natural ligand for S100 proteins. In 1992 a receptor for advanced glycation end-products was first described, which are adducts formed by glycoxidation and accumulated in disorders such as renal failure and diabetes (1417). This receptor was named receptor for advanced glycation end-products (RAGE) and represents a multiligand member of the Ig superfamily of cell surface molecules. Later, calgranulin polypetides belonging to the S100 protein class were also reported to be interaction partners of RAGE, triggering a number of downstream signals and activation parameters on endothelial, monocytic and lymphocytic cells (18). S100A8 and A9 binding to cells of the myelo/monocytic lineage was also described, although no specific binding protein or receptor was demonstrated (19). Starting from these interesting findings, the present study was designed to identify and characterize a cell membrane-associated binding protein for either the S100A8 and A9 monomers or the dimer complex on human microvascular endothelial cells (HMEC). Since the microvasculature is the preferred site for migrating inflammatory cells to pass the vessel wall during the recruitment process, dermal HMEC-1 cells were chosen as a model most closely resembling in vivo conditions. As a consequence of such an interaction of secreted S100A8/A9 complex to a cell membrane-bound ligand, a trigger of endothelial activation and signaling may promote and facilitate adhesion and migration of mononuclear cells as described in the recent literature.
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Methods
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Reagents
EMEM, PBS and all other cell culture chemicals were purchased from Biochrome (Berlin, Germany). Human serum for cell culture was prepared from blood of healthy volunteers. FCS was purchased from PAA (Linz, Austria). FITC, phenol-extracted Salmonella lipopolysaccharide (LPS), tumor necrosis factor (TNF)-
, IL-6, orthovanadate, EDTA, Tris, Triton X-100, glycerol, PMSF, leupeptin and aprotinin were obtained from Sigma Aldrich (Diesenhofen, Germany). NHS-activated Sepharose 4 Fast Flow was purchased from Pharmacia Biotech (Uppsala, Sweden). All reagents, except for LPS, were free of endotoxin as determined by the LAL test (detection limit 0.125 ng/ml, Boehringer Mannheim, Mannheim, Germany).
Cells and cell culture
Granulocytes and lymphocytes were prepared from human buffy coats by density-gradient centrifugation as described earlier (20,21).
The human macrovascular endothelial cells (HUVEC) were prepared as previously described (22) and cultured as primary cells for up to eight passages in complete M-199 medium, supplemented with 20% FCS, 1% (v/v) L-glutamine, 1% (v/v), non-essential amino acids, and 10,000 U/ml penicillin and streptomycin.
HMEC-1 cells were derived from human dermal microvascular endothelial cells (HDME, isolated from human foreskin) which were immortalized by transfection with a pSVT plasmid containing the full coding region of the large T antigen of simian virus 40A (23). The cells were cultured in MCDB basic medium (Gibco, Karlsruhe, Germany), supplemented with epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml) and 10% human serum at 37°C and 5% CO2. Both cell lines expressed the endothelial marker proteins PECAM (CD31) and von Willebrand factor (vWF) constitutively.
The pancreatic adenocarcinoma cell lines COLO 357 FG and COLO 357 L3.3 were kindly provided by Dr I. J. Fidler (University of Texas Medical Center), and were cultured as described earlier (24).
Animals and preparation of the aorta ascendens
Apolipoprotein E (ApoE)-deficient mice were generated by gene targeting (2527), and represent a widely used and accepted animal model to study atherosclerosis in recent literature.
Mice housing and breeding was performed as described by Bourassa et al. (28). Preparation of the ascending aorta was performed as described by Paigen et al. (29). The heart tissue was shock frozen in liquid nitrogen using OCT medium. Serial sections (10 µm) were cut and prepared for immunohistochemistry.
S100A8 and S100A9 monomer, and S100A8/A9 heterodimer preparation
Murine recombinant S100A8 and S100A9 were prepared as described by Klempt et al. (30). Human S100A8 and S100A9, and the S100A8/A9 dimer complex were prepared from human granulocytes as previously described (12,31). Purity of the preparations and retained complex formation ability of the heterodimer were ascertained by SDSPAGE.
Immunohistochemistry
Preparation of frozen tissue sections of the aorta ascendens of ApoE knockout mice was performed as described in detail previously (41). Briefly, frozen sections were air dried for 30 min and fixed in acetone at room temperature for 10 min. Unspecific binding sites were blocked by overlaying the sections with 0.1% BSA for 30 min. Sections were incubated with 10 µg/ml murine recombinant S100A8 (= 1.25 µM) or S100A9 (= 0.71 µM) for 4 h and washed extensively with PBS. Subsequently, bound S100A8 or S100A9 was detected with polyclonal non-cross-reactive affinity-purified rabbit antisera and directed either against S100A8 (a-S100A8) or S100A9 (a-S100A9), respectively (1 µg/ml). The monospecificity of the antisera has been shown by the use of transfected cell lines and Western blotting as previously described (8,43). The antibodies were diluted in 1% BSA in PBS. For negative controls non-specific rabbit IgG was used (1 µg/ml). Endogenous peroxidase was blocked by 20 mM NaN3 (Merck, Darmstadt, Germany) and 0.1% H2O2 (Merck) in PBS for 5 min at room temperature. Non-specific binding was blocked by incubating the slides in 1% BSA for 1 h. The primary antibody was applied for 1 h at room temperature. After thorough washing with 0.01% BSA in PBS, slides were treated with a peroxidase-conjugated secondary antibody for 1 h at room temperature. Substrate reaction was performed with 0.01 M H2O2 containing amino-9-ethyl-carbazol (Sigma) in dimethylformamide. Slides were counterstained with Mayers' Hemalaun (Merck).
S100A8/A9 binding assay
Endothelial cells were grown to ~75% confluence in tissue culture T25 flasks, activated with either 100 U/ml TNF-
, 10 µg/ ml LPS or 5000 U/ml IL-6, and trypsinized with 0.05% trypsin and 0.02% EDTA. After washing the cells were rested for 3 h in medium at 37°C in order for the S100A8/A9-binding protein to regenerate. Then, 1x106 cells/well were seeded in round-well plates and blocked in 0.1% BSA in PBS, which also served as negative controls for unspecific binding. Aliquots of 10 µg/ml S100A8 (= 1.25 µM), S100A9 (= 0.71 µM) or S100A8/A9 (= 0.45 µM) were added for binding and incubated for 2 h at 37°C. After washing 3 times with PBS/FACS staining was performed using either a-S100A8, a-S100A9 or 27E10 (1 µg/ml) for the detection of endothelial cell-bound S100A8 or S100A9 monomer or S100A8/A9 complex. 27E10 is a mAb recognizing the heterodimer only, but not the S100A8, or A9 monomer (42). Rabbit or mouse IgG1 was used to control specificity of antibody binding. Goat anti-rabbit or anti-mouse FITC-conjugated secondary antibodies (Dianova, Hamburg, Germany; 1:100 diluted) were used. Stained cells were analyzed using a FACSscan flow cytometer (Becton Dickinson, Heidelberg, Germany). Then, 10,000 propidium iodide-negative cells were counted, and mean fluorescence intensity (MFI) was monitored and calculated in comparison to the control as well as percentage of positive cells. For statistical evaluation, three independent experiments were performed and the data were expressed as mean ± SD. Two-tailed Student's t-test was performed to calculate statistical significance (P
0.01). In experiments measuring calcium dependence one set of cells was incubated in medium without Ca2+ and one set of cells in the presence of 1 mM Ca2+. To test the response of S100A8/A9 binding to cytokines, the cells were activated in the indicated concentrations in the cell culture flasks and trypsinized afterwards. To study specificity of binding increasing amounts of unlabeled S100A8/A9 complex were added to the cells. After washing, the cells were labeled with 0.9 nM S100A8/A9FITC, washed and analyzed by flow cytometry.
FITC labeling of S100A8/A9
FITC labeling of the S100A8/A9 complex was performed according to a standard protocol of Goding (44). Unbound FITC was removed by washing with PBS using pre-equilibrated PD 10 gel filtration columns (Pharmacia Biotech, Uppsala, Sweden)
Purification of the S100A8/A9-binding protein
Large-scale preparation of the S100A8/A9-binding protein was performed following an affinity chromatography protocol and a cell lysate of TNF-
-activated HMEC-1 cells. A lysis buffer described by Eue et al. (32) was used. HMEC-1 cells were plated in Petri dishes and grown to 75% confluence. Before lysis the monolayer was washed 5 times with cold PBS containing 1 mM orthovanadate and 5 mM EDTA. Cells were scraped into the lysis buffer using a rubber policeman and stored on ice for 20 min. The cell lysate was centrifuged for 15 min at 14,000 g at 4°C. The supernatants were collected, aliquoted and stored until use at 20°C. For final binding to the S100A8/A9-coupled affinity matrix, the endothelial cell lysate was dialyzed against PBS overnight at 4°C. The average protein content of the lysate was 1 mg/ml as measured by the Bradford protein assay.
The NHS-activated Sepharose 4 Fast Flow was washed and equilibrated according to the manufacturer's instructions. NHS-Sepharose was equilibrated in cold coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3). Either pure S100A9 monomer or S100A8/A9 complex was dialyzed against coupling buffer overnight. Coupling was performed overnight at 4°C under overhead rotation in a protein to NHS-Sepharose ratio of 5 mg/1 ml NHS-matrix. After coupling protein concentration of the supernatant was controlled by measuring the OD280 using a Kontron UV spectrometer (adsorption coefficient
= 7.5 ml/mg, d = 1 cm), and comparing the supernatant samples before and after NHS adsorption by SDSPAGE (+ ß2-mercaptoethanol). The matrix was washed extensively with coupling buffer and unspecific binding sites were blocked by a 2 h incubation of the coupled NHS matrix in 0.1 M Tris, 0.2 M glycine, pH 8. Subsequently, the coupled and blocked Sepharose was washed alternating with an alkaline buffer (0.1 M NaCO3, 0.5 M NaCl, pH 8,3) and an acidic buffer (0.1 M sodium acetate, 0.5 M NaCl, pH 4) to remove residues of unbound MRP protein. The coupled Sepharose was finally stored in PBS at 4°C for further use.
HMECTNF lysate and MRP-coupled NHS-Sepharose were co-incubated at room temperature for 6 h on an overhead rotator at 3 mg lysate protein/ml coupled NHS-Sepharose. Afterwards the Sepharose was washed 3 times with large amounts of cold PBS. Supernatants were monitored for protein content by UV spectrometry. After the final washing step the Sepharose was resuspended in a minimum volume of SDSPAGE sample buffer and boiled in a water bath for 5 min. The supernatant was subjected to SDSPAGE.
Matrix-assisted laser desorption ionization (MALDI)-MS and database query
After SDSPAGE the Coomassie-stained band of purified S100A8/A9-binding protein was prepared for MS in a procedure adapted from Shevchenko et al. and Zhang et al. (45,46). The gel slice was excised and destained in 25 mM NH4HCO3 containing 50% methanol. The gel was washed in acetic acid/methanol/water 10/45/45 v/v/v for 30 min and washed in water for 30 min. It was shrunk in acetonitrile and dried. The gel slice was reswollen in 20 µl of 50 mM NH4HCO3 containing 400 ng trypsin. Extra trypsin solution was removed and enough buffer was added to cover the gel slice. Digestion was carried out at 37°C overnight. Digestion was stopped by adding 510 µl of 100% acetic acid and the supernatant was removed to a clean Eppendorf tube. The gel slice was extracted twice with 70 µl acetonitrile, and supernatants were pooled and lyophilized. The dried extract was redissolved in 7 µl 0.1% aqueous TFA and purified using ZipTips (Millipore, Bedford, MA). Peptides were eluted with 7 µl 70% acetonitrile. Samples of 10 mg
-cyano were washed with acetone and dissolved in 1 ml of 50/50 v/v acetonitrile/ethanol containing 1% of 0.1% aqueous TFA. Then, 0.7 µl of this matrix preparation was spotted onto the target followed by the same amount of sample. Both solutions were mixed directly on the target. MALDI-MS was carried out using a TofSpec-2E instrument (Micromass, Manchester, UK). Digests were run in positive ion reflection mode using a matrix suppression of 500. Masses were externally calibrated and internally corrected using either the lock mass option of the instrument or trypsin autolysis peaks providing m/z values >50 p.p.m. up to m/z 3000. Data were searched against publicly available protein databases such as Swissprot and NCBI.
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Results
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S100A8/A9 in vivo binding
ApoE-deficient mice are characterized by pathological endothelial changes resulting from hypercholesteremia. The animals develop advanced atherosclerotic lesions at an age of 56 months under a normal chow diet. This model was used to demonstrate differential in situ binding of murine recombinant S100A8 and A9 between normal and pathologically changed endothelium of the aorta ascendens. As Fig. 1
indicates, S100A9 binds to the endothelium of ApoE null mice (Fig. 1C
), whereas no binding was observed in wild-type mice aorta (Fig. 1E
) and in the isotype control (Fig. 1D
). S100A8 binding was negligible in both ApoE null (Fig. 1B
) and wild-type mice. Binding of S100A8 and A9 was detected by the use of polyclonal non-cross-reactive rabbit antisera, produced against the recombinant murine proteins (a-mS100A8 and a-mS100A9 respectively). An antibody directed against vWF, a constitutive endothelial cell marker, was used as a positive control (Fig. 1A
).

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Fig. 1. Binding of murine recombinant S100A9 (C) but not S100A8 (D) (10 µg/ml) to the endothelium of the aorta ascendens in the heart of mice deficient in ApoE production. The expression of vWF was monitored as a positive control for endothelial cell identification (A) and background staining was excluded using rabbit IgG (B). Aorta endothelium from wild-type mice did not bind S100A9 (E). Magnification: x100 (A and B) and x400 (C and E).
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S100A8 and A9 monomer and complex binding to HMEC-1
S100A8 only slightly binds to activated HMEC-1 cells (Fig. 2A
), whereas S100A9 monomer binding is more effective (Fig. 2B
). The S100A8/A9 complex most efficiently binds HMEC-1 as detected by the 27E10 mAb (Fig. 2C
).

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Fig. 2. Binding of human S100A8 (A), S100A9 (B) and the S100A8/A9 complex (C) to LPS-activated HMEC-1 cells. Binding of S100A8 and A9 was measured by the use of the appropriate homologous polyclonal rabbit antibodies as described in Methods; binding of the S100A8/A9 complex was detected using the 27E10 mAb. S100A8, A9 and A8/A9 were used at concentrations as indicated in Methods.
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The binding of S100A8/A9 to activated HMEC-1 cells is trypsin sensitive as indicated in Fig. 3
. If the cells were used in the binding assay directly after trypsinization, almost no binding was observed (Fig. 3A
). Increasing with time of rest after trypsinization (1 and 3 h, Fig. 3B and C
), S100A8/A9 binding also increased pointing to the protein character of the binding site. Furthermore, S100A8/A9 complex binding to endothelial cells was demonstrated to depend on calcium ions since the binding to activated HMEC-1 cells was significantly reduced in the absence of calcium (Fig. 3D
). No significant difference in binding of the monomers depending on calcium was obvious.
Both monomers and the complex also bind constitutively to primary macrovascular endothelial cells (HUVEC) (Fig. 4
). Whereas binding of the S100A8/A9 complex to HMEC-1 cells could be stimulated by LPS and TNF-
as compared to medium-treated cells, no such induction was observed in HUVEC cells (Fig. 4
). Specificity of S100A8/A9 binding to activated HMEC-1 cells was demonstrated (i) by a dose-responding binding curve (Fig. 5A
) and (ii) by the ability of unlabeled S100A8/A9 complex to compete for the binding sites with FITC-labeled complex (Fig. 5B
). S100A8/A9 binding was saturated at a concentration of ~5 µM. Increasing concentrations of unlabeled S100A8/A9 (ranging from 0.45 to 4500 µm) inhibited binding of FITC-labeled S100A8/A9 (9 µM).

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Fig. 5. Doseresponse curve of S100A8/A9FITC binding to LPS-activated HMEC-1 cells after 2 h of incubation, which is saturated at 5 µM S100A8/A9FITC (A). S100A8/A9 binding (9 µM) is inhibited by increasing doses of unlabeled S100A8/A9 (B).
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Neither monomer nor heterodimer complex binding could be observed on lymphocytes prepared from human peripheral blood (Table 1
) or on fibroblasts (data not shown). Interestingly, considerably effective binding of both S100A9 and S100A8/A9 was found on cancer cells, whereas binding, mainly of the complex but also of the S100A9, was greatly increased when the highly metastatic pancreatic adenocarcinoma cell line (clone L 3.3 ) was used (Table 1
).
Purification of a HMEC-1-associated S100A9- and S100A8/A9-binding protein
Following the protocol described in Methods, binding proteins for the S100A9 monomer and the S100A8/A9 complex were isolated from a lysate of activated HMEC-1 cells using a non-ionic detergent for membrane solubilization. In one set of experiments S100A9 monomer was coupled to NHS-Sepharose and in another set S100A8/A9 complex was used in order to elucidate whether or not monomer interaction to activated endothelium differs from that mediated by the complex. The binding strength of the target protein to both S100A9 and S100A8/A9 was very high, so that common methods of ligand elution from the column turned out to be inefficient (high ion strength, increased pH). Only after boiling the matrix in SDSPAGE sample buffer was a single protein eluted from the column. The Mr of the protein isolated from the NHS-S100A9 column was almost identical to that isolated from the NHS-S100A8/A9 complex column (~160x103, Fig. 6A
). Control experiments, in which HMEC-1 lysate was incubated with `empty' matrix (uncoupled NHS-Sepharose), revealed no unspecific elution of any protein. The protein bands were cut out from the gel and processed for MALDI-MS as described in Methods. To demonstrate S100A9 and S100A8/A9 interaction to the HMEC-1-associated ligand protein, a modified Western blot procedure was performed. Lysates of activated HMEC-1 cells (protein concentration 1 mg/ml) were separated in a SDSPAGE gel, transferred to nitrocellulose, blocked and incubated with either S100A9 or S100A8/A9 (10 µg/ml) for 2 h at room temperature using agitation. After extensive washing, protein binding was detected using an a-S100A9 antibody in a standard Western blot protocol. In both cases proteins with a Mr similar to that obtained in the Coomassie-stained gel were identified (Fig. 6B and C
).

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Fig. 6. Coomassie-stained SDSPAGE gel of S100A8/A9-binding protein which was affinity chromatography purified from activated HMEC-1 cell lysate as indicated in Methods (A). The band was cut out and subjected to MALDI-MS. The HMEC-1TNF lysate was separated by SDSPAGE, transferred to nitrocellulose and subsequently incubated with S100A9 (B) or S100A8/A9 (C). The bound protein was detected employing a standard Western blotting procedure using a-S100A9 (1 µg/ml) and alkaline phosphatase-labeled goat anti-rabbit secondary antibody (Dianova; 1:2000 dilution). In both cases a protein of ~160 kDa was identified.
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MALDI-MS analysis
After tryptic in-gel digests of the isolated proteins MALDI-MS was performed, and two different peptide maps of the S100A9- and S100A8/A9-binding proteins were obtained as demonstrated in Fig. 7
for the S100A9 ligand protein.
Surprisingly, identity of the isolated proteins as suspected on the basis of a similarity in Mr could not be confirmed. Very clearly, the S100A9-binding protein was identified to be the
2-macroglobulin precursor (
2-MG; SwissProt accession no. P01023). Such a clear identification of the protein was not possible with the binding protein eluted from the S100A8/A9 complex column.
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Discussion
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We describe for the first time binding proteins on HMEC for members of the S100 class of calcium-binding proteinsS100A9 monomer and S100A8/A9 dimer. This is of importance since the natural ligands for these proteins on endothelial cells may serve as the long searched for interpretation for the fact that S100A8/A9-positive monocytes belong to the primarily infiltrating cell population in acute inflammation processes. There have been early reports in the literature about S100A8 and A9 co-localized to vascular endothelium at places where leukocytes migrate into inflamed tissue areas, suggesting a role of these proteins in leukocyte trafficking (33). The idea of a receptor protein for S100A8 and A9 was further followed by Koike et al. (19), who described binding of these proteins to certain myelo-monocytic cells. As we demonstrate here, S100A8 and A9 binding is not restricted to monocytic cells. Human micro- and macrovascular endothelial cells also specifically bind S100A9 and the S100A8/A9 complex, whereas binding is inducible by inflammatory cytokines only in microvascular endothelial cells. This seems to be in agreement with the biological situation: transmigration of trafficking leukocytes into tissue mostly happens through the walls of smaller vessels. Blood stream and shear forces are too high in macrovascular vessels, and therefore prevent leukocyte attachment, adhesion and migration.
In order to confirm our results from in vitro binding studies in an in vivo situation we used ApoE knockout mice as a model. These animals have been well characterized with a proven pathology of the vessel wall of the aorta ascendens and defined disturbance of endothelial functions due to atherosclerosis. As we demonstrate, S100A9 but not S100A8 specifically binds to the endothelium (unspecific binding is excluded by preadsorption with BSA) also in this tissue-associated ex vivo condition, whereas significant lower binding was observed in healthy mice of the same genetic background (wild-type ApoE+/+). These results further support the idea of S100A9 endothelial cell binding as a pathologically relevant process which is promoted by endothelial activation as it occurs during inflammation or atherosclerosis. As we recently showed, arachidonic acid is a specific inhibitor of S100A8/A9 binding to HMEC-1 cells (47). Since it has been known for some time that the S100A8/A9 specifically binds to arachidonic acid (30), we assume an interference or competition of this fatty acid with the endothelial S100A8/A9 binding site. The exact functional mechanism and physiological importance of this process remains open at this point to further studies.
As we show, endothelial cells are not the only cell types expressing a binding protein for S100A9 and the S100A8/A9 heterodimer. Human pancreatic adenocarcinoma cells, especially a clone with high metastatic potential, effectively bind S100A9 and the S100A8/A9 complex. S100A8 and A9 have earlier been described to act cytotoxically toward tumor cells. This mechanism is obviously mediated by initial binding of the proteins to a membrane-associated binding protein as we show here. It is our intention to study the downstream signaling effects in tumor cells after S100A8/A9 binding in a future project. The fact that lymphocytes and fibroblasts do not express binding sites for S100A8/A9 underlines the specificity of the binding effect. It is very obvious that S100A8 neither bound in one of the systems which were used in this study nor was it possible to show any other regulatory effect of this protein in bioassays used so far in our and other laboratories, e.g. regarding regulation of CD11b expression/affinity, transmigration and adhesion (7,13). This lack of biological activity is drastically contrasted by the embryo resorption described to take place in S100A8 knockout mice that suggests a crucial role of this protein during early developmental processes (34).
The fact that
2-MG was identified as an interaction protein in endothelial cells for monomer S100A9 was initially surprising given the nature and occurrence of this protein.
2-MG is a physiologic serum protein which occurs in high concentrations (24 mg/ml) in the blood. Biochemically, it is a proteinase inhibitor which inhibits all classes of proteinases by a unique `trapping' mechanism. Prerequisite for S100A9 binding to
2-MG would be the physiologic occurrence of this protein or a receptor for it on endothelial cells. Since HMEC-1 cells are cultured in human serum-containing medium, the possibility of
2-MG binding to the cells would theoretically exist, even though the cells were extensively washed before preparation of the lysate. This question is controversially discussed in the recent literature. Whereas some studies report the absence of
2-MG receptor (which is identical to the low-density lipoprotein receptor-related protein) on endothelial cells, Becker and Harpel detected it on human endothelium (35). Other reports describe the induction of a
2-MG receptor on arterial endothelium under pathological conditions such as inflammation or atherosclerosis (36). Unfortunately, nothing is known, yet, about possible differential expression of
2-MG or its receptor in the endothelium of ApoE/ versus wild-type mice. It is currently under investigation whether ApoE/ mice express higher levels of
2-MG or
2-MG receptor due to pathological endothelial conditions, which would explain extensive binding of S100A9 but not A8.
Since we used activated endothelial cells in our in vitro study as well, the possibility of an induced
2-MG receptor could be likely. Another hypothesis is that
2-MG binds to endothelial cells via growth factors or cytokines which are known to interact with
2-MG. Transforming growth factor-ß and vascular endothelial growth factor are known interaction partners of
2-MG (3739). Receptors for both factors are expressed on microvascular endothelial cells (39,40). It is, therefore, not unlikely to find
2-MG on the surface of HMEC-1 cells. Nevertheless, the biological relevance of the S100A9
2-MG interaction needs to be discussed critically since so far no evidence is available that S100A9 is released as a singular protein from monocytes or granulocytes. As recently shown, the stability of S100A8 and A9 proteins is greatest when the proteins occur as a heterodimer or even as a heterotetramer (12), implying it to be the biologically relevant form.
The data which were received by MALDI-MS clearly demonstrate that the S100A8/A9 heterodimerization is accompanied by conformational changes within the protein complex and distinct changes of binding sites for its interaction partners as compared to the monomers S100A8 and A9.
Although there was not sufficient evidence for identification of the endothelial cell-associated target protein of the S100A8/A9 complex, we can clearly state that it is definitely different from the isolated binding partner of the monomer S100A9,
2-MG. Our results leave open the possibility that the interaction protein of the S100A8/A9 heterodimer in activated HMEC-1 cells is a new, unidentified molecule. Future studies are planned using isolated cell membranes for lysate preparation.
The hypothesis of
2-MG being a ligand for S100A9 was supported by inhibition studies using an antibody directed against
2-MG. Preliminary data show that S100A9 binding to activated HMEC-1 cells in the presence of the antibody is clearly reduced compared to untreated or control antibody-treated cells. Future experiments are needed to confirm these results, and show a possible co-precipitation of S100A9 and its ligand.
The formerly assumed existence of only one unique receptor or binding protein for the whole class of S100 proteins does not seem to be acceptable. The structural similarity among the proteins of this class, including the EF hand motif as responsible binding element, has been used as an intriguing argument in the past to suggest, for example, RAGE as a unique target molecule for S100 proteins. This controversy cannot be finally solved by the present study since too little comparative information about the two described receptor/binding proteins is available, mainly due to the lack of appropriate antibodies or probes. Nevertheless, some data make it less likely that the HMEC-1-associated S100A9-binding protein described here is identical to RAGE: (i) different Mr, (ii) no indication for peptide map similarity or amino acid sequence homology and (iii) a different spectrum of effects to endothelial cells resulting from proteinreceptor ligation. EN-RAGERAGE ligation was reported to cause enhanced expression of the adhesion molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, which could not be proven for the binding of S100A8/A9 to its counter-receptor on endothelial cells. The observed down-regulation of E-selectin was shown for S100A8/A9 after binding to endothelial cells was, vice versa, not described for the EN-RAGERAGE interaction. This may be either due to the differential methodology and use of different endothelial cells or it may, indeed, point to a different receptor protein for S100A8/A9. Future comparative studies are needed to answer these questions.
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Acknowledgments
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The ApoE null mice were received from Dr A. v. Eckardstein, Institute of Atherosclerois Research, Münster. We thank him and Dr C. Langer for their kind support and helpful discussions. We are also grateful to Dr G. v. Dehn who taught us the technique of preparing the aorta ascendens. The project was supported by a grant from the Federal Ministry for Education, Science, Research and Technology (BMBF) #IZKF.C5. We thank H. Hater and G. Gehlmann for excellent technical assistance. S. K. acknowledges a research grant from the Department of Research and Technology in Germany (01KX9820/B) and the use of the mass spectrometer of the Protein Sequencing Laboratory of IZKF (Medical School, University of Münster).
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Abbreviations
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2-MG 2-macroglobulin |
ApoE apolipoprotein E |
HMEC human dermal microvascular endothelial cell line |
HUVEC human macrovascular endothelial cell |
LPS lipopolysaccharide |
MALDI matrix-assisted laser desorption ionization |
MRP myeloid-related protein |
MS mass spectroscopy |
RAGE receptor for advanced glycation end-products |
vWF von Willebrand factor |
TNF tumor necrosis factor |
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Notes
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Transmitting editor: T. Hunig
Received 5 March 2001,
accepted 6 December 2001.
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