Transendothelial migration of 27E10+ human monocytes

Ines Eue, Barbara Pietz, Josef Storck1, Martin Klempt2 and Clemens Sorg

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


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The myeloid-related proteins MRP8 (S100A8) and MRP14 (S100A9), two members of the S100 family of calcium-binding proteins, are co-expressed and form a cell-surface and cytoskeleton-associated heterodimer upon calcium mobilization which is recognized by the mAb 27E10. The heterodimer is abundantly expressed in the cytoplasm of granulocytes and a subpopulation of blood monocytes. Previously, we and others demonstrated endothelium-associated MRP8/14 in inflamed tissues in the vicinity of transmigrating leukocytes, suggesting a function of the proteins in this process. Here, we demonstrate that 27E10+ cells represent a fast-migrating monocyte subpopulation which preferentially utilizes an ICAM-1-dependent mechanism. The following observations imply a function of MRP8/14 in the transmigration process: (i) higher secretion of MRP8/14 from 27E10+ monocytes compared to 27E10 monocytes after interaction with activated endothelium, (ii) higher expression of CD11b on 27E10+ compared to 27E10 monocytes, (iii) up-regulation of CD11b on 27E10 monocytes in the presence of MRP14 or MRP8/14 heterodimers but not MRP8 and (iv) active participation of MRP14 but not of MRP8 in transmigration as shown by blocking with respective antibodies. We show that the interaction of 27E10+ monocytes with activated endothelium leads to MRP8/14 release which may account for the high MRP8/14 concentrations in body fluids of patients with acute or chronic inflammatory diseases. Released MRP8/14 may serve a function by enhancing CD11b expression and/or affinity in human monocytes and by participating in the transendothelial migration mechanism. Thus, MRP8/14 substantially contributes to the recruitment of monocytes to an inflammatory site.

Keywords: 27E10, adhesion, inflammation, MRP8, MRP14, myeloid-related proteins, S100A8, S100A9


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The onset of an inflammatory reaction is characterized by the recruitment of first granulocytes and later monocytes to the tissue site. The events of adhesion and transmigration of granulocytes involve a cascade of steps mediated by selectins and the leukocyte ß2 integrins (CD11/CD18) (1).

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-{gamma} 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.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells and cell culture
Human peripheral blood leukocytes were obtained from buffy coats of normal healthy donors. Monocytes were isolated in a two-step gradient centrifugation procedure as previously described (6) or by leukapheresis using a cell separator CS 3000 plus Omnix (Baxter, Unterschleissheim, Germany). Monocytes were cultured in McCoy's A5 medium (Biochrom, Berlin, Germany) and 20% (v/v) human serum in Teflon bags at 37°C and 7% CO2. For activation, monocytes were treated with 1 µM calcium ionophore A23187 (Sigma, Deissenhofen, Germany) for 1 h. The purity of the monocyte population was estimated to be >=95%. The cells were cultured in Teflon bags as previously reported (6). All media, substances and cell culture devices were free of endotoxin contamination.

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 Tris–HCl, 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)-{alpha} (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-{alpha} 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-{alpha} 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.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Migration of 27E10+ monocytes
TEM of human monocytes through macrovascular HUVEC and microvascular HMEC-1 was studied using a modified Boyden chamber assay. Monocytes were allowed to migrate for the indicated time intervals (2–30 h) and the total number of migrated cells was counted as well as the percentage of migrating monocytes staining positive for 27E10. Spontaneous monocyte TEM through HMEC-1 was found to be delayed compared to monocyte TEM through HUVEC at 4 h (Fig. 1Go). The rate of total TEM across HMEC-1 plateaued after 24 h (Fig. 1bGo), whereas a steady increase of migration was observed using HUVEC (Fig. 1aGo). Peak migration time for the 27E10 subpopulation through HUVEC was determined to be 4 h. At this early time point the majority of 27E10+ cells pass the EC layer, whereas most of the monocytes staining negative for 27E10 migrate later (6–24 h). The peak migration time for 27E10+ cells through HMEC-1 was determined to be 8 h.



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Fig. 1. Total TEM of human monocytes ({blacktriangleup}, {square}) and 27E10+ monocytes (bar graphs) through macrovascular HUVEC (a) and microvascular HMEC-1 (b) within 2–30 h. EC were plated on fibronectin-coated polycarbonate filters and monocytes (44.8 ± 6.3% 27E10+) were added to the EC monolayer for TEM. Total cell number of migrated monocytes was counted at the indicated time points and the percentage of 27E10+ monocytes was determined by immunhistochemical staining. The data shown are mean ± SD of four independent experiments.

 
ICAM-1-dependent transmigration of 27E10+ monocytes
In order to identify mechanisms by which 27E10+ monocytes migrate through EC we compared the expression and inducibility of a panel of adhesion molecules on both HUVEC and HMEC-1 before and after stimulation with TNF-{alpha}, and correlated it with the TEM of 27E10+ monocytes. Corresponding with previously published findings, HUVEC cells were shown to constitutively express ICAM-1 (42.9 ± 9.3% positive cells). In contrast, ICAM-1 expression on untreated HMEC-1 was negligible in single cells (Fig. 2aGo). After activation with TNF-{alpha} both cell types expressed high amounts of ICAM-1 (82.9 ± 3.2 and 84.9 ± 3.5% positive cells respectively). Both cell types stained positive for the two EC markers CD31 and vWF, and could not be up-regulated by TNF-{alpha} treatment (Fig. 2aGo). The expression of E-selectin and VCAM-1 on HUVEC was induced after 4 h treatment with TNF-{alpha} from a basic level of ~5 to 20 or 40% positive cells respectively, whereas the expression of these adhesion molecules on HMEC-1 was much lower and only slightly inducible by TNF-{alpha} (Fig. 2aGo).



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Fig. 2. Differential expression of adhesion molecules ICAM-1, E-selectin, VCAM-1, PECAM-1 and vWF (FVIII) in HUVEC and HMEC-1 cells under native ({square}) and inflammatory ({blacksquare}) conditions (a) and their correlation with TEM of the 27E10+ subpopulation (b). EC were treated with TNF-{alpha} for 4 h (0.5 ng/ml) or medium as control. The expression of the single adhesion molecules was evaluated by immunhistochemical staining as indicated in Methods. (b) Time-dependent TEM of 27E10+ monocytes across HUVEC and HMEC-1 before (•) and after ({blacksquare}) TNF-{alpha} treatment. While no difference in TEM was observed using HUVEC, a peak shift in migration time from 8 to 4 h after TNF treatment was apparent with HMEC-1 cells. Comparing these data with the adhesion molecule expression under the same conditions a clear correlation appeared for ICAM-1 (prominent ICAM-1 induction after TNF treatment in HMEC-1 cells, but low induction in HUVEC cells). (c) Additionally, TEM of 27E10+ monocytes was inhibited by saturating ICAM-1 binding sites on monocytes with an anti-CD54 mAb (0.2 mg/ml). The data shown are from one experiment representative of three.

 
Spontaneous TEM of 27E10+ monocytes through ICAM-1-deficient HMEC-1 was clearly delayed compared to TEM across HUVEC after 4 h (peak migration time 8 versus 4 h for HUVEC, Fig. 2bGo). If both EC lines were treated with TNF-{alpha} for 4 h, TEM of 27E10+ monocytes was demonstrated to be equal across both cell types. This was also the case for ICAM-1-expression on the EC. If 27E10+ TEM before and after stimulation with TNF-{alpha} and the expression of five different adhesion molecules on untreated and stimulated HUVEC and HMEC-1 was compared, a clear correlation became apparent only for ICAM-1 (Fig. 2a and bGo) but not for the other adhesion molecules tested. In addition, we found a mAb against ICAM-1 to efficiently but not completely inhibit TEM of 27E10+ monocytes through HUVEC (Fig. 2cGo). The experiment was controlled by an antibody against an irrelevant epitope (anti-IgG1). These data indicate the involvement of ICAM-1 as a key molecule for TEM of 27E10+ monocytes.

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. 3aGo). 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-{alpha}-activated HUVEC (Fig. 3bGo). We, therefore, conclude the requirement of direct EC to monocyte contact for MRP8/14 secretion.



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Fig. 3. Differential release of MRP8/14 from 27E10+ ({blacksquare}) and 27E10 ({square}) monocytes after interaction with medium or TNF-{alpha}-treated HUVEC cells (a). Endothelial cells were plated in 96-well plates and 1x105 sorted monocytes (27E10+ or 27E10) were added per well. The supernatants were analyzed for MRP8/14 content. LDH was measured as vitality marker and indicator of membrane stability. Endothelial cells without monocytes served as background control. (b) Requirement of cell–cell contact for the enhancement of MRP8/14 release caused by activated EC. The addition of cell-free conditioned TNF-{alpha}-treated HUVEC medium did not lead to an increase of MRP8/14 secretion. The data represent mean ± SD of three independent experiments, each carried out in quadruplicate. *P <= 0.05.

 
Functional importance of MRP14 and the MRP8/14 complex but not of MRP8 for TEM of 27E10+ monocytes
Using monospecific polyclonal antibodies directed against either MRP8 or MRP14 and a mAb against the MRP8/14 heterodimer (27E10) we investigated which of the proteins are functionally essential for successful TEM of the 27E10+ monocyte subpopulation. Monocytes staining surface-positive for 27E10 to 50.4% were preincubated with the respective blocking antibodies and added to activated HUVEC for TEM. Anti-MRP14 was found to effectively inhibit TEM of 27E10+ monocytes (41.4 ± 2.1% inhibition), whereas anti-MRP8 did not show an inhibitory effect (Fig. 4Go). The monoclonal 27E10 antibody inhibited TEM by 14.6 ± 2.7%. The experiment was controlled by a monoclonal mouse IgG and a polyclonal rabbit IgG antibody. Equal antibody binding to the monocyte surface was ensured by FACS staining (Fig. 4aGo–c).



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Fig. 4. Inhibition of TEM of 27E10+ monocytes through TNF-{alpha}-activated HUVEC by anti-MRP14 and 27E10 but not anti-MRP8. Monocytes (50.4% 27E10+ cells) preincubated with antibodies directed either against MRP8, MRP14 or the MRP8/14 complex (27E10) were used in the TEM assay. A monoclonal mouse IgG antibody and a polyclonal rabbit IgG served as isotype controls. All antisera were used at a concentration of 6 µg/ml. * indicates significant inhibition as compared to the control with P <= 0.05. Equal antibody binding to monocytes was ensured by flow cytometry with anti-MRP8 (a), anti-MRP14 (b) and 27E10 (c).

 
MRP14 and MRP8/14 induced regulation of CD11b expression/activation
The obvious participation of MRP14 in monocyte TEM raised the question whether MRP14 or MRP8/14 function as adhesion-like molecules themselves or whether known adhesion mechanisms are controlled by MRP. CD11b (Mac-1) as a key molecule being responsible for firm leukocyte adhesion to the endothelium via ICAM-1- and fibrinogen-ligand binding was studied under the influence of MRP8, MRP14 and the MRP8/14 heterodimer. We tested binding of sICAM-1 to its counter-receptor CD11b by flow cytometry and found it significantly increased in the presence of MRP14 (84.6% cells staining positive for CD54 versus 36.2% in control cells) and MRP8/14 complex (79.6% positive cells versus 36.2% in medium treated cells), whereas MRP8 had no effect (Table 1Go). MnCl2, a known regulator of CD11b affinity, was used as positive control. Since ICAM-1 serves not only as a ligand for CD11b but also for LFA-1 (CD11a), we performed a control experiment in which the cells were pretreated with an anti-LFA-1 antibody in order to saturate CD11a binding sites. Similar results were obtained after subsequent incubation with MRP8, MRP14, MRP8/14 and the sICAM-1 ligand (see Table 1Go, insets).


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Table 1. MRP14 and MRP8/14 induced enhancement of sICAM-1-ligand binding to CD 11b on human monocytes.
 
Since we were not able to find evidence for a direct binding of MRP14 or MRP8/14 to CD11b (immunoprecipitation experiments, data not shown), an indirect activation mechanism, presumably via another so far not identified receptor, is supposed. In a next experiment the sensitivity of this putative receptor/binding protein to Bordetella pertussis toxin was studied by investigating MRP14-mediated sICAM binding (Fig. 5Go). Ligand binding to CD11b, up-regulated by MRP14 but not influenced by MRP8, was dose-dependently inhibited in the presence of 0–1 µg/ml pertussis toxin, suggesting a G-protein-coupled activation mechanism.



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Fig. 5. Pertussis toxin sensitivity of MRP14-mediated sICAM-1 binding. Monocytes were treated with different concentrations of B. pertussis toxin for 1 h. Subsequently, MRP8 or MRP14 (10 µg/ml) were added and the cells were washed. The sICAM-1 ligand binding assay was performed as indicated in Methods. (a) sICAM-1 binding to CD11b on monocytes was not significantly enhanced in the presence of MRP8 (*). (+) represents IgG control, (#) represents medium treated cells. (b) Enhanced sICAM-1 binding on monocytes after treatment with MRP14 (*) compared to medium-treated cells (#) and the IgG control (+). (c) Dose-dependent reduction of MRP14-mediated sICAM-1 binding in the presence of B. pertussis toxin 0 (#); 0.5 (Ø) and 1 µg/ml ({lozenge}).

 
To substantiate the idea that MRP14 and the heterodimer complex are involved in the regulation of CD11b expression and/or activation, we analyzed 27E10+ and 27E10 monocytes (isolated by FACS sorting) with regard to their CD11b expression. As we found by flow cytometry, 27E10+ monocytes stain highly positive for CD11b, whereas mean fluorescence was lower in 27E10 monocytes (Fig. 6bGo). When 27E10 monocytes were incubated with MRP8 (20 µg/ml for 4 h) CD11b mean fluorescence was either slightly down-regulated or not affected (Fig. 6bGo). In contrast, MRP14 and MRP8/14 up-regulated CD11b to an extent similar to 27E10+ cells (Fig. 6bGo).



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Fig. 6. Differential CD11b expression in 27E10+ and 27E10 monocytes as assessed by flow cytometry. (a) Monocytes were separated by FACS sorting using a directly FITC-labeled 27E10 antibody into a positive and a negative subpopulation. (b) CD11b expression in 27E10 monocytes after external treatment with either medium, MRP8, MRP14 or MRP8/14 complex (10 µg/ml, 4 h at 37°C) and in 27E10+ monocytes.

 
During the process of TEM, monocytes pass different stages of intracellular activation and regulation of cell surface molecules. This process includes enhancement of adhesion molecule expression, production of cytokines and superoxide radical formation. In order to analyze whether MRP14 and MRP8/14 complex mediated up-regulation is a specific effect we studied the influence of these proteins on other monocyte activation parameter as well. As expected, oxidative burst was clearly increased in supernatants of cells treated with PMA (10 µg/ml), whereas none of the MRP-stimulated superoxide radical production in the cells (Fig. 7aGo). Increase of intracellular calcium is a characteristic signal during cellular activation by various stimulants. As indicated in Fig. 7Go(b), 50 µM histamine caused a clear increase in intracellular Ca2+ concentration within 60 s after addition, whereas MRP8 and MRP14 failed to induce such a Ca2+ flux even after up to 10 min after addition of the substances. Cells only slightly responded to stimulation with MRP8/14 complex over a period of 10 min.



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Fig. 7. MRP8, MRP14 and MRP 8/14 do not induce respiratory burst nor calcium flux in human monocytes. (a) Respiratory burst in monocytes in response to stimulation with either MRP8, MRP14 and MRP8/14 complex (10 µg/ml), or PMA (10 µg/ml) as a positive control. (b) Calcium flux in monocytes after treatment with either MRP8, MRP14 and MRP8/14 complex, or histamine as positive control. Arrows indicate addition of the appropriate substances.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In search of functions for MRP8, MRP14 and their heterodimeric complex, we investigated their possible role in the adhesion and TEM of human peripheral blood monocytes. Consistent with earlier in vivo reports (11,34), we could demonstrate MRP8/14 expressing monocytes (27E20+) to represent an early-migrating cell population across the endothelial barrier also in vitro.

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. 2Go) 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-{alpha} in this cell line. The inhibition of 27E10+ monocyte TEM by a CD54 mAb (Fig. 2CGo) 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. 4Go). 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 1Go), 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 SDS–PAGE (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 SDS–PAGE 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-{alpha}-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.


    Acknowledgments
 
We appreciate the expert technical assistance of Heike Hater, Karin Fischer and Ruth Goez. We also thank Frank Schönlau and Claus Kerkhoff for critical reading of the manuscript. This work was supported by the DFG So 87/11 Fund.


    Abbreviations
 
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

    Notes
 
Transmitting editor: A. Radbruch

Received 10 January 2000, accepted 2 August 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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