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Proteinase 3 Enhances Endothelial Monocyte Chemoattractant Protein-1 Production and Induces Increased Adhesion of Neutrophils to Endothelial Cells by Upregulating Intercellular Cell Adhesion Molecule-1

Department of Nephrology, Leiden University Medical Center, Leiden, The Netherlands.

Correspondence to Dr. Mohamed R. Daha, Department of Nephrology, Leiden University Medical Center, Building 1 C3-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31-0-71-5263964; Fax: 31-0-71-5248118; E-mail: M.R.Daha{at}lumc.nl

	Abstract

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Abstract. Wegener's granulomatosis is an autoimmune disease that is characterized by systemic vasculitis and granuloma formation. Early influx of polymorphonuclear neutrophils (PMN), followed at a later stage by mononuclear cells, contributes to the granulomatous inflammation. Previous studies have shown that proteinase 3 (PR3), the major autoantigen in Wegener's granulomatosis, specifically binds to endothelial cells and plays a possible role in activation of these cells by enhancing interleukin-8 production, thus providing a chemotactic and activating stimulus for PMN. The present study demonstrated that PR3 enhances the production of monocyte chemoattractant protein-1 (MCP-1) by human umbilical vein endothelial cells (HUVEC) in a dose- and time-dependent manner. The PR3-induced increase in MCP-1 production was demonstrated at both the protein and the mRNA levels and was chemotactic for monocytes. In addition, it was demonstrated that PR3 induces a dose- and time-dependent increase in the expression of intercellular adhesion molecule-1 (ICAM-1) as determined by fluorescence-activated cell sorter analysis. The PR3-induced increase in expression of ICAM-1 was also demonstrated at the mRNA level. PR3 induced a slight increase in vascular cell adhesion molecule-1 expression and had no effect on the expression of both P- and E-selectin. Incubation of HUVEC for 24 h in the presence of PR3 resulted in a significant increase in adhesion of PMN, which was reduced to baseline levels in the presence of blocking monoclonal antibody anti—ICAM-1 or anti-CD18 or a combination of both. Monocytes showed a slight but statistically not significant increase in adhesion. Incubation of HUVEC with PR3 for 4 h did not result in enhanced adhesion of either PMN or monocytes. It was hypothesized that PR3, which may be released locally at inflammatory sites after activation of cytokineprimed PMN, plays a role in endothelial cell activation by enhancing both interleukin-8 and MCP-1 production, thus providing a chemotactic and activating stimulus for both PMN and monocytes. In addition, PR3 may contribute to the ongoing inflammation by enhancing the adhesion of PMN to endothelial cells by upregulating ICAM-1 expression.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Wegener's granulomatosis (WG) is an autoimmune disease that is characterized by granulomatous inflammation of the respiratory tract, systemic necrotizing vasculitis, and crescentic glomerulonephritis (1). Early influx of neutrophils, followed at a later stage by extravasation of mononuclear cells, contributes to granuloma formation, such as seen in WG (2). Influx of leukocytes into the surrounding tissue requires upregulation of expression and/or activation of adhesion molecules, both on leukocytes and on endothelial cells. Indeed, increased expression of the ß1 (CD29) and ß2 (CD18) integrin subunits was detected on both neutrophils and monocytes, isolated from patients with WG, as compared with normal controls (3). Furthermore, increased levels of circulating intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were found in sera of patients with WG (4,5,6). Although both molecules are not expressed solely on endothelial cells, activation of these cells may contribute to the increased levels of adhesion molecules.

Proteinase 3 (PR3), a neutral serine proteinase present in the -granules, specific granules, and secretory vesicles of polymorphonuclear neutrophils (PMN) (7), is the main autoantigen to antineutrophil cytoplasmic antibodies (ANCA) in patients with WG. PR3 may be released from cytokine-primed PMN after activation by ANCA (8,9). We recently showed that PR3 binds specifically to endothelial cells, which may provide a mechanism for PR3-induced endothelial cell injury or activation (10). Indeed, incubation of endothelial cells with PR3 resulted in detachment and cytolysis (11), and recently PR3 was shown to cause apoptosis of bovine pulmonary artery endothelial cells (12) as well as human umbilical vein endothelial cells (HUVEC) (13). PR3 may also play a role in the activation of endothelial cells by enhancing interleukin-8 (IL-8) production (14), a strong chemotactic and activating factor for neutrophils.

In the present study, we investigated whether PR3, besides IL-8, also enhances the production of monocyte chemotactic protein-1 (MCP-1) by HUVEC, a chemotactic factor for monocytes. In addition, we questioned whether PR3 plays a role in leukocyte extravasation by inducing or upregulating the expression of adhesion molecules on endothelial cells, resulting in increased adhesion of leukocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation of PR3
PR3 was isolated from neutrophil azurophilic granules as described by Leid et al. (15). In brief, leukocytes were isolated from enriched buffy coats after hypotonic lysis of erythrocytes. Subsequently, leukocytes were resuspended in relaxation buffer (100 mM KCl, 35 mM MgCl₂, 10 mM HEPES [pH 7.3]) and lysed by nitrogen cavitation. Lysed cells were collected in 25 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetra-acetic acid, and the cell-free supernatant was obtained after centrifugation (900 x g, 10 min). Next, -granules were isolated from the bottom of an 84% Percoll gradient after centrifugation at 48,000 x g for 10 min. After ultracentrifugation (174,000 x g, 60 min), the green -granule—containing pellet was removed from the remaining Percoll and after centrifugation was frozen at -20°C until needed.

Azurophilic granule-containing pellets, derived from approximately 300 x 10⁹ leukocytes, were lysed by incubation in phosphate-buffered saline (PBS) containing 1% Triton X-100. PR3 and myeloperoxidase (MPO) were isolated from the lysate by cation exchange chromatography using Biorex 70 (Bio-Rad Laboratories, Richmond, CA). The -granule extract was dialyzed against phosphate-citrate buffer (80 mM Na₂HPO₄, 50 mM NaCl, adjusted to pH 7.0 with 80 mM citric acid) and applied to a column of Biorex 70, which had been equilibrated and run in the same buffer. After nonbinding proteins had washed through the column, a linear gradient up to 1 M NaCl in starting buffer was applied. Fractions from the column were analyzed for the presence of protein (BCA protein assay; Pierce Chemical Co., Rockford, IL) and PR3, elastase, and myeloperoxidase activity, as measured by cleavage of N-t-BOC-l-alanine-p-nitrophenyl ester (BOC cleavage assay), S-2484 and 2,2'-azino-bis-3-ethylbenzthioazo-line-6-sulfonic acid (ABTS), respectively.

PR3-containing fractions were concentrated by lyophilization and resuspended in PBS. For further purification, PR3 was applied to a Superdex 75 column (Pharmacia-Biotech, Uppsala, Sweden). Fractions were tested for the presence of protein and esterolytic activity (BOC-cleavage assay), and PR3-containing fractions were pooled, dialyzed against distilled water, and lyophilized. The lyophilized PR3 was dissolved in a minimal amount of distilled water and dialyzed against PBS. PR3 concentration was determined using a PR3 sandwich enzyme-linked immunosorbent assay (ELISA) and enzymatic activity by BOC-cleavage assay. The purity of the PR3 preparation was determined using analysis of enzymatic activity, ELISA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot. The PR3 preparation used for the experiments was not contaminated with MPO, neutrophil elastase, or cathepsin G. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PR3 preparation revealed a triplet of bands around 29 kD.

Isolation of Neutrophils and Monocytes
Human neutrophils and monocytes were isolated from fresh buffy coat as described before (16). Briefly, neutrophils and monocytes were isolated by differential centrifugation on Ficoll-Amidotrizoate gradients ( = 1.077 g/ml) for 20 min at 650 x g at room temperature. Purified suspensions of neutrophils were obtained from the pellet fraction of the Ficoll gradient after lysis of erythrocytes using isotonic ammonium chloride (180 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM ethylenediaminetetraacetate [pH 7.3]) for 10 min on ice. The interface fraction of the Ficoll gradient, containing the mononuclear cells, was collected and washed twice in PBS containing 0.5 U/ml heparin and 2% autologous plasma. The monocytes were purified further by centrifugal elutriation with a Beckman J2-21 M/E centrifuge using a JE-6 elutriation rotor and a standard separation chamber (Beckman Instruments Inc., Paolo Alto, CA) (17). Monocyte-enriched preparations were more than 85% pure with less than 11% lymphocytes and 4% neutrophils. Monocytes were more than 97% viable as determined by trypan blue exclusion. Cells were resuspended at a final concentration of 2 x 10⁶ cells/ml in M199/10% heat-inactivated fetal calf serum (FCS).

ELISA
PR3 Sandwich ELISA. A solid-phase sandwich ELISA, as described by Berger et al. (14), was used to measure the PR3 concentration. Briefly, 96-well microtiter plates were coated with an optimal concentration of polyclonal rabbit IgG anti-PR3 and diluted in coating buffer (74 mM NaHCO₃, 26 mM Na₂CO₃ [pH 9.6]) for 2 h at 37°C. After each incubation step, plates were washed three times with PBS/0.05% Tween 20 and all further dilutions were prepared in PBS/0.05% Tween 20/2% casein. After coating, all open binding sites on the plate were blocked using PBS/0.05% Tween 20/2% casein (30 min, 37°C). Next, twofold serial dilutions of the PR3 sample were added and tested against serial dilutions of a PR3 standard (126 µg/ml). Bound PR3 was detected using digoxigenin-conjugated rabbit IgG anti-PR3 in the first step, followed by horseradish peroxidase-conjugated sheep F(ab')₂ fragments of anti-digoxigenin. Finally, the ELISA was developed with ABTS, containing H₂O₂ (0.0005%), and the optical density was assessed at 415 nm. The standard curve started at a concentration of 630 ng/ml and was linear down to 5 ng/ml.

MCP-1 ELISA. MCP-1 production by HUVEC was quantified by sandwich ELISA as described by van den Berg et al. (18). Briefly, 96-well microtiter plates (Maxisorb F96, Nunc, Roskilde, Denmark) were coated with a monoclonal antibody (mAb) anti—MCP-1 (R&D Systems, Abington, UK) in PBS. After each incubation step, plates were washed three times with PBS/0.05% Tween 20 and all further dilutions were prepared in PBS/0.05% Tween 20/2% casein. After the wells were blocked with PBS/0.05% Tween 20/2% casein, appropriate dilutions of culture supernatants were added, followed by rabbit IgG anti—MCP-1, produced at our laboratory by immunization of rabbits with recombinant human MCP-1 (Peprotech Inc., Rocky Hill, NJ). Finally, wells were incubated with horseradish peroxidase-conjugated goat IgG anti-rabbit IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA) and plates were subsequently developed with ABTS, containing H₂O₂ (0.0005%). Optical density was measured at 415 nm, and the chemokine concentration was calculated relative to a MCP-1 standard.

Culture of Cells
HUVEC were isolated from human umbilical cord veins according to Jaffe et al. (19) and cultured as described by Ballieux et al. (11) and Miltenburg et al. (20). In brief, cells were cultured on gelatin-coated tissue culture plates or flasks in M199 medium containing Earle's salts and glutamine, supplemented with 10% FCS, penicillin-streptomycin (100 IU/ml, 100 µg/ml) (all from Life Technologies, Paisley, UK), 0.002% endothelial cell growth factor (isolated from bovine hypothalamus) (21), and 7.5 U/ml heparin (Leo Pharmaceutical Products, Weesp, The Netherlands). Cell cultures were performed at 37°C, 5% CO₂ and 95% relative humidity. Only HUVEC between passages 2 and 6 were used for experiments.

Stimulation of Cells
MCP-1 Production. To assess whether PR3 enhances the production of MCP-1 by HUVEC, we seeded cells into gelatin-coated 48-well plates (Costar, Cambridge, MA) at a concentration of 5 x 10⁴ cells/well and cultured for 24 h until confluence. Thereafter, the cells were washed with PBS and cultured for 16 h in M199 containing 2% FCS and penicillin/streptomycin (M199/2% FCS). These quiescent HUVEC were then incubated in M199/2% FCS alone or M199/2% FCS containing various concentrations of PR3. As a positive control, cells were incubated in the presence of 5 ng/ml rhIL-1 (R&D Systems). After 24 h of culture (unless stated otherwise), supernatants were harvested and assessed for the presence of MCP-1 using a sandwich ELISA.

Expression of Adhesion Molecules. To analyze the expression of adhesion molecules on HUVEC, we seeded cells at a concentration of 1 x 10⁵ cells/well in gelatin-coated six-well plates (Costar) and cultured for 24 h until confluence. Thereafter, cells were stimulated with M199/2% FCS alone or M199/2% FCS containing different concentrations of PR3, 500 U/ml recombinant human tumor necrosis factor- (rhTNF-; Peprotech Inc.), or 20 ng/ml IL-4 (R&D Systems). After stimulation of the cells for various time periods, cells were detached by trypsinization and analyzed for the expression of ICAM-1, VCAM-1, E-selectin, or P-selectin by fluorescence-activated cell sorter (FACS) analysis.

FACS Analysis
Cells were stimulated as described, detached by trypsinization, and resuspended in PBS containing 1% FCS (FACS buffer). Subsequently, cells were incubated with FACS buffer alone or buffer containing an appropriate dilution of one of the following mouse mAb: anti-ICAM-1, anti-VCAM-1 (both from Pharmingen, San Diego, CA), anti-E selectin (R&D Systems) or anti-P selectin (Serotec, Oxford, UK). Cells were washed twice in FACS buffer, and binding of mAb was detected with phycoerythrin-labeled goat F(ab')₂ anti-mouse Ig (GAM-PE; DAKO, Glostrup, Denmark). Thereafter, the cells were washed, fixed in 1% paraformaldehyde, and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Expression of adhesion molecules was assessed as median fluorescence intensity (MFI) as calculated by the Lysis II program.

Reverse Transcriptase-PCR
To assess PR3-induced mRNA expression of MCP-1, ICAM-1, VCAM-1, E-selectin, or P-selectin by HUVEC, we cultured cells until confluence and washed and incubated them with M199/2% FCS alone or M199/2% FCS containing IL-1 (5 ng/ml), TNF- (500 U/ml), IL-4 (20 ng/ml), or PR3 (10 µg/ml). Cells were stimulated for 2 h to detect E-selectin mRNA or 16 h to detect MCP-1, ICAM-1, VCAM-1, or P-selectin mRNA. HUVEC were detached by trypsinization, and total cellular RNA was isolated from 1 x 10⁶ cells using the RNAzol B method (Cinna/Biotecx, Houston, TX), according to the manufacturer's instructions (22). Fixed amounts of total cellular RNA (1 µg) were reverse-transcribed into cDNA by oligo(dT) priming, using Moloney murine leukemia virus reverse transcriptase (Life Technologies). Amplification of cDNA by PCR was performed using the primers as indicated in Table 1.

PCR reactions were performed in a semiquantitative manner using the same amount of cDNA in each mixture. Amplification of cDNA by PCR was performed under standard conditions (50 mM KCl, 10 mM Tris-HCl [pH 8.4], 20 mM MgCl₂, 0.06 mg/ml bovine serum albumin, 0.25 mM dNTP, 10 µl cDNA, 50 pmol of each primer, and 1 U of Taq DNA polymerase) for 35 cycles using 1.5 min at 95°C, 1 min at 60°C, and 3 min at 72°C (Perkin Elmer, Norwalk, CT). Equal volumes of PCR products were analyzed on 1% agarose gels containing ethidium bromide. Results were analyzed using the Eagle Eye system (Stratagene, La Jolla, CA), and for reasons of clarity, images were black/white inverted.

Chemotactic Assay
Monocyte chemotactic activity of cell-culture supernatants of HUVEC was determined essentially as described before (23) using a 48-well modified Boyden chamber (24). Briefly, HUVEC were cultured and stimulated as described for MCP-1 production. Twenty-six-µl aliquots of cell-culture supernatant of either HUVEC incubated in M199/2% FCS alone or M199/2% FCS containing PR3 (10 µg/ml) were diluted 1:1 in HEPES buffer (containing 20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1.2 mM KH₂PO₄, 1 mM MgSO₄, 5.5 mM glucose, 0.1 mM CaCl₂) and placed in triplicate wells of the lower compartments of the Boyden chamber. M199/2% FCS alone or containing rhMCP-1 (50 ng/ml) served as negative and positive controls, respectively. Controls and cell-culture supernatants were analyzed for monocyte chemotactic activity in the presence of 50 µg/ml of either control rabbit IgG or rabbit IgG anti—MCP-1. Monocytes were isolated and resuspended in M199/2% FCS diluted 1:1 in HEPES buffer, and 50 µl of monocyte suspension containing 125,000 cells was added to the upper compartments of the chamber. The compartments of the chamber were separated by a lower filter with a pore size of 0.45 µm (Millipore Products, Bedford, MA) and an upper filter with a pore size of 8 µm (Sartorius Filter, San Francisco, CA). After 2 h of incubation at 37°C, the upper filter was removed and fixed in a butanol-ethanol mixture (20/80% [vol/vol]) for 10 min and stained with Weigert solution. The filters were dehydrated with ethanol and made transparent with xylene. Migrating monocytes were counted using light microscopy; six random high-power fields (magnification, x400) were counted per well.

Adhesion Assay
HUVEC (1 x 10⁴ cells/well) were cultured in gelatin-coated 96-well plates (Costar) for 24 h until confluence. Thereafter, cells were washed twice with PBS and cultured in M199/2% FCS alone or M199/2% FCS containing either TNF- (500 U/ml) or PR3 (10 µg/ml). Cells were cultured for either 4 or 24 h and washed with warm PBS. Neutrophils or monocytes were then added to HUVEC at a concentration of 2 x 10⁵ cells/well in M199/10% FCS and allowed to adhere during 30 min at 37°C under static conditions. The wells were then washed three times with warm PBS to remove nonadherent cells. Adhesion of either neutrophils or monocytes was quantified using a modified myeloperoxidase assay as described by Bath et al. (25). Briefly, HUVEC plus adherent leukocytes were washed twice with modified PBS without Ca²⁺ and Mg²⁺ (pH 6.0), and subsequently both HUVEC plus adhering cells were permeabilized in modified PBS containing 0.5% hexadecyltrimethyl ammoniumbromide (Sigma Chemical Co., St. Louis, MO) for 30 min at room temperature. Next, a solution of O-dianisidine dihydrochloride (0.2 mg/ml in PBS [pH 6.0]) containing H₂O₂ (0.4 mM) was added and optical density was measured after 15 min of incubation at 37°C. Serial dilutions of either neutrophils or monocytes were used as a standard to calculate the number of adhering leukocytes. To study the role of the PR3-upregulated adhesion molecules on HUVEC in the adhesion of leukocytes, we washed HUVEC with PBS and incubated them with 10 µg/ml mAb anti—ICAM-1 (84H10; Beckman Coulter, Brea, CA) or anti—VCAM-1 (1G11B1; Biosource Int., Nivelles, Belgium) in M199/10% FCS for 30 min at 37°C. Alternatively, leukocytes were incubated with 10 µg/ml mAb anti-CD18 (IB4; American Type Culture Collection, Rockville, MD) or anti-CD49 d (15A8; CLB, Amsterdam, The Netherlands). Next, the adhesion assay was performed as described. The mAb remained present during the adhesion assay.

Statistical Analysis
Results were analyzed using a t test for unpaired samples.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MCP-1 Production by HUVEC
To assess whether PR3 enhances the production of MCP-1 by endothelial cells, we rendered confluent HUVEC cultures quiescent and subsequently stimulated them with different concentrations of PR3. Incubation of HUVEC in the presence of increasing concentrations of PR3 resulted in a dose-dependent increase of MCP-1 production (Figure 1A). A concentration of 10 µg/ml PR3 induced a 3.4-fold increase in MCP-1 production (25.7 ± 0.6 ng/ml) compared with basal production (7.6 ± 0.6 ng/ml). In comparison, IL-1, used as a positive control, induced an MCP-1 production of 67.1 ± 0.8 ng/ml (data not shown). The enzymatic activity of PR3 was not essential to enhance MCP-1 production, because PR3 treated with diisopropyl fluoride also resulted in enhanced MCP-1 production (data not shown).

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effect of proteinase 3 (PR3) on the production of monocyte chemoattractant protein-1 (MCP-1) by human umbilical vein endothelial cells (HUVEC). HUVEC were cultured until confluence and rendered quiescent in M199/2% heat-inactivated fetal calf serum (FCS). (A) Cells were incubated for 24 h in the presence of increasing concentrations of PR3, and MCP-1 production was measured by enzyme-linked immunosorbent assay **, P < 0.01 PR3 versus medium. (B) HUVEC were incubated in the absence () or presence ([UNK]) of PR3 (10 µg/ml) for different time periods. *, P < 0.05 PR3 versus medium. Results are expressed as mean ± SD of experiments in duplicate culture. One of three representative experiments is shown.

Incubation of HUVEC with 10 µg/ml PR3 resulted in a time-dependent increase in MCP-1 production (Figure 1B). After the cells were incubated with PR3 for 8 h, a slight increase in MCP-1 production was observed compared with cells incubated in medium alone. A significant increase in PR3-induced MCP-1 production was detected after 24 and 48 h.

The production of MCP-1 by HUVEC was confirmed at the mRNA level. HUVEC were cultured in medium alone or medium containing either PR3 (10 µg/ml) or IL-1 (5 ng/ml), and after an incubation period of 16 h, total RNA was isolated and analyzed for the expression of MCP-1 mRNA by reverse transcriptase-PCR (RT-PCR) (Figure 2). MCP-1 mRNA was present in nonstimulated cells and was upregulated in both PR3- and IL-1—stimulated cells.

View larger version (31K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Reverse transcriptase-PCR (RT-PCR) analysis to detect MCP-1 and ß-actin mRNA expression by HUVEC. Expression of MCP-1 and ß-actin mRNA was assessed by RT-PCR after stimulation of the cells for 16 h with medium alone (lane 2) or medium containing either 10 µg/ml PR3 (lane 3) or 5 ng/ml interleukin-1 (IL-1; lane 4). Lane 1 contains the negative control (no cDNA). Bands of the expected size of 257 bp for MCP-1 and 527 bp for ß-actin are shown.

To assess whether MCP-1 produced by HUVEC was functionally active, we determined monocyte chemotactic activity of cell-culture supernatants of HUVEC incubated with medium alone or medium containing PR3 (10 µg/ml). The concentration of MCP-1 in cell-culture supernatant of unstimulated or PR3-treated HUVEC was 9.8 ± 0.4 and 30.9 ± 0.7 ng/ml, respectively, as determined by ELISA. Control supernatant of unstimulated HUVEC displayed low chemotactic activity for monocytes compared with medium (196.0 ± 5.3 versus 106.7 ± 10.1 migrating cells/6 high-power fields; Table 2). Supernatant of PR3-stimulated HUVEC induced a significant increase in the number of migrating monocytes compared with supernatant of unstimulated HUVEC, which was reduced by 84% in the presence of anti—MCP-1 IgG (Table 2). As a positive control, rhMCP-1 (50 ng/ml) was used, which induced a strong increase in chemotaxis of monocytes.

Expression of Adhesion Molecules by HUVEC
To determine whether PR3 enhances the expression of the adhesion molecule ICAM-1 on HUVEC, we incubated confluent HUVEC cultures for 24 h in medium alone or medium containing PR3. Assessment of surface expression of ICAM-1 using FACS analysis showed a basal expression of ICAM-1 on HUVEC with an MFI of 365 (Figure 3A). Incubation of the cells in the presence of PR3 (10 µg/ml) resulted in a 2.9-fold increase in fluorescence intensity (MFI of 1074), indicating enhanced ICAM-1 expression. PR3 enhanced the expression of ICAM-1 in six of six primary HUVEC cultures.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Analysis of expression of adhesion molecules by fluorescence-activated cell sorter (FACS). HUVEC were cultured until confluence, washed, and incubated in medium alone to determine basal expression (---) or medium containing PR3 (—) for 24 h to assess intercellular adhesion molecule-1 (ICAM-1) (A), vascular cell adhesion molecule-1 (VCAM-1) (B), and P-selectin expression (D). Detection of E-selectin expression (C) was performed after exposure of HUVEC to PR3 for 4 h. Incubation of HUVEC with tumor necrosis factor- (TNF-; 500 U/ml; A through C) or IL-4 (20 ng/ml) (D) served as positive control (dotted gray line). After detachment of HUVEC by trypsinization, cells were incubated in the presence of monoclonal antibodies (mAb) specific for ICAM-1, VCAM-1, E-selectin, or P-selectin and binding, was detected using GAM-PE. Median fluorescence intensity (MFI) of the conjugate control was 5.2.

The effect of PR3 on the induction of VCAM-1 expression on HUVEC was less pronounced. After 24 h of incubation of HUVEC with PR3, a 1.3-fold increase in VCAM-1 expression was observed (MFI of 11.1) compared with basal expression (MFI of 8.1) (Figure 3B). TNF-, used as a positive control, enhanced both ICAM-1 and VCAM-1 expression (Figure 3, A and B). After incubation of HUVEC with PR3 for 4 h, no induction of E-selectin expression was observed, whereas TNF- induced near maximum E-selectin expression (Figure 3C). Also, incubation of HUVEC with PR3 for more than 4 h did not result in E-selectin expression. We also found no induction of P-selectin expression after incubation of HUVEC with PR3 for either 15 min or 24 h (Figure 3D). Stimulation of HUVEC with IL-4 for 24 h induced expression of P-selectin.

The effect of PR3 on the upregulation of ICAM-1 expression was studied in more detail. Incubation of HUVEC with increasing concentrations of PR3 for 24 h resulted in a dose-dependent increase in ICAM-1 expression as determined by FACS analysis (Figure 4A). A concentration of 5 µg/ml PR3 already induced a 2.4-fold increase in ICAM-1 expression, which increased to 3.9-fold at a concentration of 20 µg/ml. Higher concentrations of PR3 could not be tested because of its cytotoxic effect on HUVEC. Also, the kinetics of upregulation of ICAM-1 expression was studied using FACS analysis. TNF- induced upregulation of ICAM-1 expression, which was maximal after 24 h of stimulation and then slowly declined until 72 h of stimulation (Figure 4B). The kinetics of PR3-induced upregulation of ICAM-1 expression was found to be different from that of TNF-. A significant increase in ICAM-1 expression was observed after incubating the cells for 24 h in the presence of PR3 compared with cells incubated in medium alone. PR3-induced ICAM-1 expression continued to increase until 72 h of incubation (Figure 4B). Later time points were not measured.

View larger version (11K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effect of PR3 on ICAM-1 expression by HUVEC. (A) HUVEC were incubated with different concentrations of PR3 for 24 h, and surface expression of ICAM-1 was determined using FACS analysis. Data were expressed as MFI as determined by the Lysis II program. *, P < 0.05; **, P < 0.01 PR3 versus medium. (B) HUVEC were incubated in medium alone () or medium containing 10 µg/ml PR3 ([UNK]) or 500 U/ml TNF- () for different time periods, and the expression of ICAM-1 was assessed using FACS analysis. *, P < 0.05; **. P < 0.01; ***, P < 0.001 PR3 or TNF- versus medium. Results are expressed as mean ± SD of experiments in duplicate culture. One of three representative experiments is shown.

PR3, at a concentration of 5 µg/ml, induced a slight increase in VCAM-1 expression (1.2-fold), reaching a plateau level at 10 µg/ml of PR3 (1.3-fold). Incubation of HUVEC with 10 µg/ml PR3 reached a maximum increase in VCAM-1 expression (1.4-fold) after 48 h (data not shown).

The effect of PR3 on the expression of the above-mentioned adhesion molecules was also analyzed by RT-PCR (Figure 5). In nonstimulated cells, expression of ICAM-1 mRNA was observed, which was upregulated after stimulation of HUVEC for 16 h with either PR3 or TNF-. A very low expression of VCAM-1 mRNA was observed in nonstimulated HUVEC, which corresponded with the low basal expression observed upon FACS analysis. A slight increase in VCAM-1 mRNA expression was observed after stimulation of the cells for 16 h with PR3. TNF- induced a strong increase in VCAM-1 mRNA levels. Stimulation of HUVEC with PR3 for 2 or 16 h did not result in an increase of either E-selectin or P-selectin mRNA compared with cells cultured in medium alone (data not shown). However, cells cultured in the presence of TNF- or IL-4 clearly showed enhanced expression of either E-selectin or P-selectin mRNA, respectively.

View larger version (54K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. RT-PCR analysis to detect mRNA expression of adhesion molecules and ß-actin by HUVEC. Expression of ICAM-1, VCAM-1, and ß-actin mRNA was assessed by RT-PCR after stimulation of the cells for 16 h with medium alone (lane 2) or medium containing either 10 µg/ml PR3 (lane 3) or 5 ng/ml TNF- (lane 4). Lane 1 contains the negative control (no cDNA). Bands of the expected size of 243 bp for ICAM-1 and 523 bp for VCAM-1 are shown.

Adhesion of PMN and Monocytes to HUVEC
To assess whether PR3-induced upregulation of ICAM-1 or VCAM-1 expression indeed resulted in enhanced adhesion of either PMN or monocytes, we cultured HUVEC in the absence or presence of PR3 and washed and subsequently incubated them with PMN or monocytes. Adhesion of leukocytes was quantified using the MPO assay as described above. A linear correlation of 0.99 and 0.96 was found between the MPO content and the number of PMN and monocytes, respectively. After incubation of HUVEC with 10 µg/ml PR3 for 24 h, a 1.8-fold increase in adhesion of PMN was observed compared with medium alone (Figure 6A). However, no significant increase in adhesion of monocytes to HUVEC stimulated with PR3 for 24 h was observed compared with nonstimulated cells (Figure 6B). Stimulation of HUVEC with PR3 for 4 h did not result in enhanced adhesion of either PMN or monocytes (results not shown). TNF-, used as a positive control, induced significantly enhanced adhesion of PMN after 4 and 24 h of stimulation (39,800 ± 3615 and 16,033 ± 4761 adherent cells, respectively) compared with medium alone (7600 ± 283 and 6200 ± 1045 adherent cells, respectively). Also for monocytes, a significant increase in adhesion was observed after incubation of HUVEC with TNF- for 4 and 24 h (193,350 ± 2120 and 123,400 ± 1273 adherent cells, respectively) compared with medium alone (72,400 ± 2263 and 66,774 ± 7467 adherent cells, respectively).

View larger version (9K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Analysis of adhesion of leukocytes to HUVEC. HUVEC were cultured in 96-well plates until confluence and stimulated with either medium alone () or medium containing PR3 (10 µg/ml; ) for 24 h. HUVEC were washed with phosphate-buffered saline and polymorphonuclear neutrophils (PMN; A) or monocytes (B) (2 x 106 cells/ml) were added. After 30 min, nonadherent cells were removed and the number of adherent cells was quantified using a myeloperoxidase (MPO) assay. Serial dilutions of PMN or monocytes were used as a standard to calculate the number of adherent leukocytes. Results are expressed as number of adherent cells (mean ± SD of triplicate wells). **, P < 0.01.

Because PR3 induced a significant increase in adhesion of PMN to HUVEC, we studied the role of the PR3-upregulated adhesion molecules in this process. Treatment of unstimulated HUVEC with anti—ICAM-1 mAb or of neutrophils with anti-CD18 mAb resulted in a slight reduction of adhesion of neutrophils (10463 ± 869 and 9346 ± 1129 cells, respectively) compared with untreated HUVEC (11821 ± 1215 cells) (Figure 7). However, the combination of anti—ICAM-1 and anti-CD18 mAb induced a significant reduction in adherence of neutrophils to unstimulated HUVEC (6187 ± 1489 cells). Incubation of HUVEC with 10 µg/ml PR3 for 24 h resulted in a significant increase in adhesion of neutrophils (16549 ± 621 cells) compared with unstimulated cells (Figure 7). Treatment of HUVEC with anti—ICAM-1 mAb or of PMN with anti-CD18 mAb resulted in a significant reduction of adhesion of PMN (10591 ± 729 and 9837 ± 171 cells, respectively), whereas the combination of anti—ICAM-1 and anti-CD18 resulted in a further decrease in adhesion of neutrophils (5651 ± 1345 cells). Anti—VCAM-1 mAb had no effect on the adhesion of PMN to either unstimulated HUVEC or PR3-treated HUVEC (11976 ± 1676 and 16324 ± 1805 cells, respectively) (Figure 7). Also, pretreatment of PMN with anti-CD49d mAb did not result in a decrease in adhesion of neutrophils to HUVEC, either unstimulated or after incubation with PR3 for 24 h (data not shown).

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. The effect of mAb against different adhesion molecules on the adhesion of neutrophils to HUVEC. HUVEC were cultured in 96-well plates until confluence and exposed either to medium alone () or to medium containing PR3 (10 µg/ml) () for 24 h. HUVEC were washed and incubated with M199/10% FCS alone or containing 10 µg/ml of either mAb anti—VCAM-1 (1G11B1) or anti—ICAM-1 (LB2) (30 min, 37°C). Neutrophils (2 x 106 cells/ml), preincubated with M199/10% FCS alone or containing 10 µg/ml mAb anti-CD18 (IB4) (30 min, 37°C), were added. After 30 min, nonadherent cells were removed and the number of adherent cells was quantified using an MPO assay. Serial dilutions of PMN or monocytes were used as a standard to calculate the number of adherent leukocytes. Results are expressed as the number of adherent cells (mean ± SD of triplicate wells). One of two representative experiments is shown. **, P < 0.01; ***, P < 0.001; a, presence versus absence of mAb; b, PR3 versus medium alone.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
WG belongs to the group of ANCA-associated small-vessel vasculitides. A characteristic feature of vascular lesions in WG during the acute phase is extravasation of neutrophils, followed at a later stage by mononuclear cells (2). The pathogenesis of this disease, however, has still not been elucidated. PR3, the major autoantigen to ANCA in WG, may play a role in endothelial cell activation. Berger et al. (14) showed enhanced IL-8 production after incubation of HUVEC with either enzymatically active or inactive PR3. IL-8 is a member of the C-X-C family of chemokines and provides a chemotactic stimulus for PMN (26).

In the present article, we questioned whether PR3 would not only provide a chemotactic stimulus for neutrophils by enhancing endothelial IL-8 production but also exert a similar effect on monocytes. Indeed, incubation of HUVEC in the presence of PR3 resulted in a dose- and time-dependent upregulation of MCP-1 production by HUVEC. Enhanced MCP-1 production by PR3 was confirmed at the mRNA level. MCP-1, as a member of the C-C chemokine family, has a strong chemotactic activity on monocytes. Supernatants of PR3-stimulated endothelial cells indeed proved to be chemotactic for monocytes, and this effect was mediated predominantly via MCP-1. Other as-yet-unidentified chemotactic factors present in the supernatant of PR3-stimulated endothelial cells may be responsible for the remaining monocyte chemotactic activity. PR3, after its binding to endothelial cells and subsequent activation of these cells, thus may contribute to the extravasation of both neutrophils and monocytes by providing chemotactic and activating stimuli.

Migration of leukocytes from the blood vessel into the surrounding tissue requires closely regulated interactions between adhesion molecules, both on endothelial cells and on leukocytes. Because PR3 was found to provide a chemotactic stimulus for both neutrophils and monocytes, we also studied the effect of PR3 on the expression of adhesion molecules by endothelial cells using FACS analysis. We first studied the effect of PR3 on P-selectin expression. P-selectin is stored in the Weibel-Palade bodies of endothelial cells and is mobilized to the cell surface within minutes after stimulation with agents such as histamine or thrombin (27). Here it mediates rolling and initial adhesion of leukocytes during the acute stages of inflammation. Earlier in vitro studies demonstrated that activation of endothelial cells with, for instance IL-4, induces a prolonged increase in P-selectin expression, suggesting a role for P-selectin in chronic inflammation (28). Indeed, in sera of patients with glomerulonephritis, increased levels of soluble P-selectin were detected compared with normal control subjects, which correlated with local leukocyte accumulation (29). In the same study, persistent endothelial P-selectin expression was reported in interstitial lesions of patients with glomerulonephritis, suggesting that it may play a role in chronic inflammatory conditions. However, we found no induction of P-selectin expression after stimulation of HUVEC with PR3, for either 15 min or 24 h.

PR3 also did not induce E-selectin expression, another member of the selectin family of adhesion molecules involved in rolling and initial adhesion of leukocytes. The absence of PR3-induced E-selectin expression, as determined by FACS analysis, was confirmed at the mRNA level. Furthermore, no increase in adhesion of either PMN or monocytes was observed after stimulation of HUVEC with PR3 for 4 h, also confirming the above results. Expression of E-selectin is restricted to activated endothelial cells. In vitro studies have shown release of E-selectin from the surface of cytokine-activated endothelial cells (30,31). However, no significant increase in the level of soluble E-selectin was detected in sera of patients with active WG compared with healthy control subjects (5,6), questioning the role of E-selectin in chronic inflammatory disorders.

In sera of patients with WG, increased levels of circulating ICAM-1 and VCAM-1 were found (4,5,6). Although both molecules are not expressed solely on endothelial cells, activation of these cells may contribute to the increased levels of soluble adhesion molecules. Indeed, in vitro studies have shown release of ICAM-1 and VCAM-1 from cytokine-activated endothelial cells (30,31). Release of these adhesion molecules closely correlated with their enhanced membrane expression. In the present study, ICAM-1 expression on HUVEC was found to be upregulated clearly by PR3, as determined by FACS analysis and RT-PCR, whereas PR3 had only a marginal effect on the upregulation of VCAM-1 expression. Both molecules belong to the Ig gene superfamily of adhesion molecules, involved in firm adhesion of leukocytes. ICAM-1 has been shown to mediate adhesion of leukocytes via interaction with the ß2 integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), whereas VCAM-1 interacts with VLA-4 (CD49 d/CD29), a member of the ß1 integrin family present on monocytes and lymphocytes.

To assess whether the PR3-induced upregulation of ICAM-1 and VCAM-1 was functional, we stimulated HUVEC with PR3 and adhesion of either PMN or monocytes was quantified. After an incubation period of 24 h, PR3 induced a significant increase in adhesion of PMN to HUVEC, which was mediated via ICAM-1 as demonstrated in studies using blocking mAb. Indeed, in the presence of mAb against ICAM-1 on HUVEC or CD18 on PMN or both, adhesion of PMN to PR3-stimulated HUVEC was reduced to the levels of adhesion for unstimulated HUVEC, whereas anti—VCAM-1 or anti-CD49d mAb had no effect on adhesion of PMN to unstimulated or PR3-treated HUVEC. Because the PR3-induced expression of ICAM-1 continued to increase up to 72 h, adhesion of PMN to the endothelium may increase further in time and result in more chronic inflammation. Stimulation of HUVEC with PR3 did not result in a significant enhancement of adhesion of monocytes. Both ICAM-1 and VCAM-1 have been reported to play a role in adhesion and transmigration of monocytes to endothelial cells (32). In the present study, PR3 was found to enhance the expression of ICAM-1 and slightly upregulate VCAM-1 expression. Although we detected an increase in adhesion of monocytes, the increase was not significant, suggesting that the PR3-induced upregulation of adhesion molecules on endothelial cells was not sufficient for enhanced adhesion of monocytes.

Several hypotheses concerning the role of PR3 and ANCA in endothelial cell activation in WG have been postulated. Mayet et al. (33) suggested that activation of endothelial cells with cytokines, such as TNF- and IL-1- induced a transient membrane expression of endogenous PR3 on the cell surface. PR3 thus may become accessible to ANCA, and binding of these antibodies then will lead to endothelial cell activation (34,35,36). However, synthesis of PR3 by endothelial cells is a controversial issue. We (10) and others (37,38), using sensitive PCR-based assays, found no evidence that endothelial cells produce or express PR3.

We suggest that PR3 may be released most probably locally at inflammatory sites from cytokine-primed PMN after activation by ANCA and then bind to endothelial cells, leading to endothelial cell activation. PR3 has already been shown to enhance IL-8 production by HUVEC. In this article, we showed that PR3 also enhances MCP-1 production, thus providing chemotactic and activating stimuli for both neutrophils and monocytes. We demonstrated further that PR3 may play a direct role in the adhesion and possible transmigration of neutrophils by enhancing ICAM-1 expression and increasing adhesion of PMN to endothelial cells, thus amplifying the ongoing inflammatory cascade.

	Acknowledgments

 
The authors thank the Dutch Kidney Foundation (Grant no. C95.1458) for financial support of this research.

	References

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