Institute of Experimental Dermatology and
1 Institute of Physiology, University of Münster, von-Esmarch-Strasse 56, 48149 Münster, Germany
2 Institute of Physiology and Biochemistry of Nutrition, Federal Dairy Research Center, 24103 Kiel, Germany
Correspondence to: I. Eue
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Abstract |
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Keywords: 27E10, adhesion, inflammation, MRP8, MRP14, myeloid-related proteins, S100A8, S100A9
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Introduction |
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Monocytes are a heterogeneous population comprising the precursors for different tissue macrophages. It has been shown that different monocyte populations emigrate at different times in an ordered sequence into the tissue in the course of an inflammatory reaction (2). While the early-infiltrating monocytes seem to share some of the mechanisms for adhesion and transmigration with granulocytes, later-appearing monocyte subpopulations seem to utilize other mechanisms such as CD14 and RM3/1 (CD163), a newly defined member of the scavenger receptor family (4,5).
A predominant subpopulation of monocytes in the peripheral blood is characterized by the expression of myeloid related-proteins MRP8 (S100A8) and MRP14 (S100A9), two members of the S100 family of calcium binding proteins (69). These cells were shown to be among the first cells infiltrating acutely inflamed tissues in several diseases like contact dermatitis and gingivitis in humans, and irritative and allergic contact dermatitis and leishmaniasis in mice (2,6,1013). MRP8 and MRP14 are cytosolic proteins which are expressed in monocytes but are absent in mature tissue macrophages (14,15). Under inflammatory conditions and/or upon calcium mobilization, MRP8 and MRP14 are translocated to the plasma membrane and to the cytoskeleton (16,17). Both proteins form a heterodimer which is specifically recognized by the mAb 27E10 (18).
While both proteins are integrated into the plasma membrane upon calcium mobilization, they are secreted after stimulation with phorbol myristate acetate (PMA) via a novel protein kinase C-dependent pathway (19) indicating two different cellular pathways. The surface expression of the heterodimeric complex correlates with monocyte activation and can be induced in vitro with the calcium ionophore A23187, IFN- and lipopolysaccharide (16).
MRP8/14 complexes are found in high concentrations in body fluids of patients with acute and chronic diseases, and have been proposed to represent new inflammation markers (2022). The function of these proteins is obviously pleiotropic. Some of the intra- and extracellular functions are now gradually emerging. The heterodimer seems to have fungistatic and bacteriostatic properties presumably mediated by its zinc-binding properties (23,24). Recently, it was shown that the heterodimer also binds arachidonic acid with high affinity (25,26). In previous studies on cryostat sections of inflamed tissues, not only cells adhering to the endothelium but also the endothelium itself stained positive for MRP8/14 (27). This is an indication for a role of MRP8/14 in the adhesion and migration of myeloid/monocytic cells through vascular endothelium. In this context, an interesting novel function was recently described for MRP14 which was shown to regulate neutrophil adhesion to fibrinogen via ß2 integrin (CD11b/CD18, Mac-1) affinity control (28).
In order to shed more light on the cellular functions of MRP8/14 we studied adhesion and transendothelial migration of 27E10+ and 27E10 monocytes. We were able to show for the first time that 27E10+ monocytes preferentially migrate through vascular endothelium in contrast to 27E10 monocytes. By using FACS-sorted monocyte subpopulations we show a function of MRP14 and the MRP8/14 complex but not MRP8 in the regulation of the ß2 integrin Mac-1 (CD11b). In addition we found that antibodies against MRP14 can block transendothelial migration (TEM) of 27E10+ human monocytes through an EC monolayer.
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Methods |
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The human macrovascular endothelial cells (HUVEC) were prepared as previously described (3) and cultured as primary cells for up to eight passages in complete M-199 medium, supplemented with 20% FCS (PAA, Linz, Austria), 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 EC (isolated from human foreskin) which were immortalized by transfection with a pSVT plasmid containing the full coding region of the large T antigen of the simian virus 40A (33). 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% FCS at 37°C and 5% CO2. Both cell lines expressed the endothelial marker proteins PECAM (CD31) and von Willebrand factor (vWF) constitutively.
Purification of MRP8, MRP14 and the MRP8/14 heterodimer complex
MRP8 and MRP14 as well as the heterodimer MRP8/14 complex were purified from human granulocytes which were prepared from buffy coats as previously described (34).
In some experiments human recombinant MRP8 and MRP14 were used which were prepared as previously described (25). Briefly, MRP8 and MRP14 were subcloned into pQE 32, expressed and purified as follows. Bacteria were washed and lysed using lysis buffer consisting of 50 mM NaH2PO4, 20 mM TrisHCl, 8 M urea and 100 mM NaCl (Buffer 1, final pH 6.0). All chemicals were obtained from SERVA (Heidelberg, Germany). The lysate was sonified and centrifuged at 20,000 g for 30 min. The proteins were tagged with a 6xhistidine residue for purification on a TALONTM metal-affinity column (Clontech, Palo Alto, CA) using a denaturing protocol. After binding on the column the proteins were washed with Buffer 1 containing 10 mM imidazole (pH 7.0) to avoid unspecific binding. The proteins were eluted by the use of Buffer 1 containing 150 mM imidazole (pH 6.0). In order for the proteins to renature they were dialysed stepwise against decreasing urea concentrations. Subsequent PAGE revealed a protein purity of 98% (data not shown). The proteins were aliquoted and stored at 70°C or at 4°C for short-term use. All MRP preparations were checked for endotoxin content by the use of a LAL test (Biowhittaker, Walkersville, MD) and ensured to be
0.05 EU/ml.
Antibodies
The following antibodies were used in this study. Polyclonal monospecific antisera were raised in rabbits against recombinant MRP8 and MRP14. The antibodies were purified by affinity chromatography, and specificity was evaluated by Western blotting using recombinant proteins and transfected human L134 lung fibroblast cell lines as described earlier (7,17). The mAb 27E10 from our laboratory was employed to detect the MRP8/14 heterodimer complex (12 µg/ml). This antibody recognized the complex only, but not the MRP8 or MRP14 monomers (6,18). A mAb against ICAM-1 was used (84H10, 0.2 mg/ml; Immunotech, Hamburg, Germany). The CD11b (MAC-1) mAb was obtained from Dako (Hamburg, Germany; 2LPM19C) and used at a concentration of 1 µg/ml for immunostaining and 6 µg/ml for blocking experiments. Antibodies against CD62E (E-selectin) were purchased from R & D Systems (Wiesbaden, Germany) and against CD106 (VCAM-1) from DPC Biermann (Bad Nauheim, Germany). CD31 and vWF antibodies came from Monosan (Uden, Netherlands). Monoclonal mouse IgG1 (Dianova, Hamburg, Germany) and polyclonal rabbit IgG (Pharmacia, Freiburg, Germany) were used as control antibodies with irrelevant specificity. All mAb including the control antibody were used in a concentration of 1 µg/ml for immunhistochemical staining. An affinity-purified horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Dianova) was used for immunostaining.
TEM assay
Polycarbonate filters with a pore size of 5 µm (Costar, Bodenheim, Germany) were coated with fibronectin (50 µg/ml) for 30 min at room temperature. EC at 2x105 per 200 µl medium were plated per filter and grown for 1 day. For activation EC were treated with tumor necrosis factor (TNF)- (0.5 ng/ml) for 24 h. For transmigration, 600 µl of transmigration medium were added to the lower part of the chamber (complete RPMI 1640 medium, supplemented with 10% FCS and 25 mM HEPES) and 2x106 monocytes were added for TEM. The cells were allowed to migrate for the indicated time periods at 37°C. The number of migrated cells was counted using a hemocytometer or a Coulter counter after placing the plate on ice for 30 min to reverse possible monocyte attachment to the plastic. The filters were saved for later microscopic evaluation of EC density. Trypan blue exclusion was performed to analyze viability. To study the composition of monocyte subpopulations of migrated cells, cytospin slides were prepared (5x104 cells per slide), fixed with acetone for 10 min and immunostained with 27E10 or IgG1 as isotype control. At least 400 cells per slide were counted using a microscopic grid. Triplicate counts were performed in all experiments.
Immunohistochemistry
Cytospin preparations were fixed in acetone as described above, air dried for at least 1 h and submersed in PBS. Antibodies were diluted in 1% BSA in PBS. For negative controls non-specific mouse or rabbit IgG were used at concentrations corresponding to the specific test antibodies. 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 (1:40 dilution of a 12 µg/ml stock solution for 27E10, 1:100 dilution for ICAM-1). After thorough washing with 0.01% BSA, slides were treated with the 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).
CD11b ligand binding assay
Monocytes (1x106) were pretreated with 20 µg/ml of recombinant human MRP8, MRP14 or MRP8/14 complex respectively for 60 min at 37°C. After washing twice with PBS, sICAM-1 (R & D Systems) at a concentration of 10 µg/ml was added for ligand binding and incubated for 30 min at 37°C. After washing twice again, the amount of bound sICAM-1 on the cell surface (ligand binding) was estimated by immunostaining for ICAM-1 with an anti-CD54 antibody (Immunotech) in a 1:100 dilution. Immunostaining was evaluated by flow cytometry. Medium-treated monocytes served as a negative control and indicated basic sICAM-1 binding in the absence of MRP. The experiment was also controlled by native monocytes without sICAM-1 treatment. MnCl2 known to regulate ß2 integrin activity served as a positive control. To exclude LFA-1-mediated ICAM-1 binding, monocytes were pretreated with a CD11a antibody (Dianova) in control experiments. The use of sICAM-1 as a ligand for CD11b in this assay is based on observations of Rothlein et al. about the structural similarity of sICAM-1 and membrane ICAM-1 (4042). Both forms of ICAM contain five extracellular domains which are responsible for ligand binding (CD11a/b).
Flow cytometry and FACS
To minimize unspecific binding, cells were preincubated in 1% BSA/PBS for 1 h at 4°C. For each analysis, 1x106 cells were incubated with a FITC-conjugated 27E10 antibody (dilution 1:40 with HBSS) or with the isotype-matched control respectively. Staining was performed in the dark at 4°C for 60 min. Cells were stained with propidium iodide to discriminate dead cells and kept on ice until measurement.
Flow cytometry and cell sorting were performed on a Moflo high speed sorter and analyzer (Cytomation, Boulder, CO), equipped with an argon ion laser (Coherent, Palo Alto, CA) (excitation wavelength of 488 nm). Data acquisition was performed using CyCLOPS 3.4 software (Cytomation). Forward light scattering (FSC), orthogonal light scattering (SSC) and fluorescence signals (FL1 and FL3) were acquired, and stored in list mode data files. Each measurement contained a defined number of 10,000 propidium iodide-negative, vital cells. Gates of the bivariate and univariate histograms were logically connected in the following order: vital cells from gate 1 of propidium iodide/FSC and leukocytes from gate 2 of FSC/SSC to univariate FITC. The separately gated FITC signals at the mode values of 27E10 and 27E10+ cells with defined FSC, SSC, FL1 and FL3 characteristics were simultaneously isolated with a sort rate of 20,000 cells/s. Contamination with lymphocytes was avoided by careful outgating of these cells before sorting. Lymphocyte contamination was ensured to be <5% in the sorted cell populations by staining with a CD3 mAb (PharMingen, via Beckton Dickinson, Heidelburg, Germany) using either FACS analysis or cytospin slides. The separated populations were allowed to rest at least 2 h at 37°C after sorting in fresh medium before use in the assays.
MRP8/14 secretion after EC interaction
Monocytes at 2x105/well (either 27E10 or 27E10+) were added to 5x104 HUVEC which were plated in 96-well plates 2 days in advance. EC were either left untreated (medium) or activated with TNF- for 24 h. After incubating 16 h at 37°C supernatants were collected and analyzed for lactate dehydrogenase (LDH) activity as vitality and membrane integrity marker as described elsewhere (17). MRP secretion was assessed by the use of a sandwich ELISA as described earlier (17,35). EC (TNF-
treated or untreated) without monocytes served as background controls.
Respiratory burst assay
Monocytes (2x106; 200 µl) were treated with 10 µl of a 10 mM cytochrome c stock solution and the activator in an appropriate dilution. The reaction mixture was diluted to a final volume of 1 ml with HBSS containing 5 mM glucose. As a reference the same reaction mixture was prepared containing 10 µl of a 5 mg/ml superoxide dismutase stock solution in addition. After incubating for 30 min at 37°C the samples were immediately cooled down on ice, the cells were centrifuged and absorption of the supernatants was measured at 550 nm wavelength using the samples containing superoxide dismutase as reference. Molar concentration of superoxide radicals was estimated using the formula:
Measurement of intracellular [Ca2+]
Monocytes were cultured on glass coverslips which were coated with fibronectin (50 µg/ml). The cells were loaded with 5 µM Fura-2/AM in culture medium for 10 min at 37°C. Then the coverslips were mounted on a thermostated microscope tissue chamber, washed 3 times with HEPES buffer (146 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 1.4 mM Na2HPO4, 5 mM dextrose, 20 mM HEPES and 1 mM CaCl2) and incubated with 1 ml of the same buffer. Fields of ~10 cells were visualized on a Zeiss (IM35) inverted microscope and excited alternately at 340/380 nm. The emission was measured at 510 nm using a photon counting detector. The 340 and 380 nm tracings were corrected in each experiment and the autofluorescence was subtracted before calculating the 340/380 ratio (38)
Statistical analysis
The experimental results were analyzed for their statistical significance by two-tailed Student's t-test with P 0.05.
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Results |
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MRP8/14 secretion of 27E10+ and 27E10 monocytes after contact with EC
In a next set of experiments we investigated the ability of FACS-sorted 27E10+ and 27E10 cells to secrete MRP8/14 into the supernatant after contact with native and activated EC. We found a high rate of MRP8/14 secretion from 27E10+ cells (352 ± 38 ng/U) after contact with activated EC (Fig. 3a). The release of MRP8/14 from 27E10 monocytes to activated HUVEC was significantly lower. MRP secretion was also lower after contact with untreated endothelium for both 27E10+ and 27E10 monocytes. MRP release was controlled by measuring unspecific cytoplasmic protein release (LDH). We also measured only a low MRP8/14 secretion when the monocytes were incubated for the same time in cell-free conditioned medium of untreated or TNF-
-activated HUVEC (Fig. 3b
). We, therefore, conclude the requirement of direct EC to monocyte contact for MRP8/14 secretion.
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Discussion |
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In order to identify TEM mechanisms of the 27E10+ subpopulation, we utilized microvascular HMEC-1 as an EC model which differs from primary macrovascular EC (HUVEC) by its expression and inducibility of adhesion molecules. The question which of the adhesion systems are primarily utilized by 27E10+ monocytes could be answered by the identification of CD11b/CD18 (Mac-1) and ICAM-1 as the major contributors. This finding is in agreement with other reports which determine Mac-1 as one of the most important adhesion molecules for functional monocyte migration (29,36) in concert with other adhesion molecules. This is supported by our experiments (Fig. 2) using the microvascular cell line HMEC-1 which expressed ICAM-1 to a low extent in singular cells when untreated. Both ICAM-1 expression and TEM of the 27E10+ monocyte population can be induced by TNF-
in this cell line. The inhibition of 27E10+ monocyte TEM by a CD54 mAb (Fig. 2C
) further underlines the importance of this molecule for functional diapedesis of these cells. The fact that TEM of these cells is not completely suppressed by anti-CD54 points to the probability that other AM contribute to TEM of this monocyte subpopulation as well.
As several immunohistological studies on sections of acutely inflamed tissue suggest a role of MRP8 and MRP14 in adhesion and/or transmigration, we asked whether these molecules are involved as regulators in these processes. In fact, we could show that the monomer MRP14 and the heterodimer MRP8/14 are essentially involved in transmigration, because antibodies against MRP14 and MRP8/14 (27E10) but not against MRP8 could effectively block TEM of 27E10+ monocytes (Fig. 4). This raises the question whether MRP14 and MRP8/14 are able to act as adhesion molecules themselves or whether they interact with major adhesion molecules such as Mac-1 on monocytes. The fact that MRP are missing fundamental and typical structural properties of adhesion molecules such as transmembrane domains makes it less likely that MRP14 and/or MRP8/14 directly function as membrane-associated adhesion molecules. We demonstrate that MRP14 and the MRP8/14 complex, but not MRP8, differentially affect CD11b expression/activation and ICAM-1 ligand binding in monocytes. Two major conclusions can be drawn: (i) ICAM-1 binding to human monocytes is enhanced in the presence of MRP14 and the heterodimer MRP8/14 complex, but not in the presence of MRP8, and (ii) 27E10 monocytes express spontaneously less CD11b than 27E10+ cells, but CD11b expression can be stimulated by external addition of MRP14 and MRP8/14 complex. Although it has been shown that MRP14 occupies (a) binding site(s) on the surface of cells with myelo/monocytic origin there is no evidence for a direct binding of MRP to ß2 integrins so far (28,29). In neutrophils, CD11b affinity was shown to be regulated indirectly by MRP14 (28). Since we could not find evidence for a direct binding of MRP14 and CD11b in our system either, we assume that this is also true for monocytes. The pertussis toxin sensitivity of MRP14-mediated sICAM binding to CD11b points to a G protein-dependent receptor mechanism which is responsible for the up-regulation. However, the enhanced binding of sICAM-1 to monocytes in the presence of MRP14 and the MRP8/14 complex is a sign for an increased CD11b binding capacity which may be due to either (i) activation of previously expressed but non-active CD11b epitopes, (ii) increased CD11b affinity or avidity or (iii) a combination of both mechanisms. Since the ligand assay, as it was used in this study (Table 1
), measures both increased expression and enhancement of CD11b affinity, no differentiation between the both effects can be made at this point. The fact that MRP14 and the MRP8/14 complex increase CD11b antigenic density as measured by enhancement of mean fluorescence relative to the control makes it most likely that cell adhesion strength to EC is enhanced. The situation in monocytes is apparently somewhat different from that in neutrophils (28) since we observed an up-regulation of CD11b antigenic density and induction of sICAM-1 ligand binding not only by MRP14 (like in neutrophils) but also by the MRP8/14 complex. So far, we and others could not demonstrate release of the individual proteins MRP8 or MRP14. According to our knowledge, the MRP heterodimer complex represents the only naturally occurring and biologically relevant form. As we recently showed by MALDI mass spectroscopy (37,39) the tetrameric complex seems to be the most stable complex implying that this is the biologically active form. There are only rare situations where MRP8 or MRP14 are expressed as monomers by infiltrating cells (31). Whether they are released individually as well is currently unknown. It is very unlikely that the used MRP8/14 complex preparation contained significant amounts of monomeric MRP 14 (which might have caused activation effects). We ensured equality of MRP8 and MRP14 amounts in the preparation by quantitative densitometric analysis of the gel bands after SDSPAGE (34). Since the proteins are known to form heterodimer complexes under physiological conditions we presume no monomer contamination of the MRP8/14 complex. Also, after diluting out the complex or removing it by precipitation with the only MRP8/14 complex recognizing 27E10 mAb, no remaining MRP14 monomers were found in the samples as shown by silver stained SDSPAGE gels (detection limit in the ng range).
In previous studies it had been shown that MRP are found in high concentrations in body fluids of patients with inflammatory diseases. This raises the question about the natural secretion stimulus of these proteins which are almost exclusively found as heterodimeric complexes after release. The assumption that the interaction of monocytes with activated endothelium represents the natural signal for MRP release was investigated in the present study. As we show, MRP8/14 is primarily released from activated (27E10+) monocytes after contact with TNF--activated EC. We, therefore, identified one of the natural MRP release signals from monocytes which is triggered by activated endothelium and which give an explanation for the fact that MRP plasma levels are elevated in patients with inflammatory diseases. MRP can, thus, be considered a representative early marker for inflammatory processes in human plasma. The contribution of MRP released from apoptotic cells, e.g. neutrophils, to the increase of MRP plasma levels is minor in the early events of inflammation since these cells are recognized and phagocytosed quickly (32).
Any effect of MRP on leukocytes would require binding to the cell surface and signal transduction. Even though the chemical identification of receptors for either MRP8 or MRP14 is lacking, convincing evidence for their existence has been published in myelo-monocytic cells (30). Preliminary data suggest the existence of MRP binding sites also on human EC. Taken together with our recent findings the possibility becomes likely that primarily 27E10+ monocytes release MRP8/14 after contact to EC which ligates to a EC associated counter-receptor. This could be a mechanism for the preferential adhesion and transmigration of MRP expressing monocytes. Further investigation is needed to verify this assumption.
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Acknowledgments |
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Abbreviations |
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AM adhesion molecules |
anti-MRP8 or rabbit polyclonal antibody raised against human |
anti-MRP14 recombinant MRP8 or MRP14 respectively |
EC endothelial cells |
HUVEC human macrovascular endothelial cells |
ICAM-1 intracellular adhesion molecule-1 |
LDH lactate dehydrogenase |
PECAM-1 platelet endothelial adhesion molecule-1 |
PMA phorbol myristate acetate |
TEM transendothelial migration |
TNF tumor necrosis factor |
VCAM-1 vascular endothelial adhesion molecule-1 |
vWF von Willebrand Factor |
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Notes |
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Received 10 January 2000, accepted 2 August 2000.
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References |
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