Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1202624 on July 15, 2003

Published online before print July 15, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1202624v1
74/4/551    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by David, A.
Right arrow Articles by Aviram, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by David, A.
Right arrow Articles by Aviram, I.
(Journal of Leukocyte Biology. 2003;74:551-557.)
© 2003 by Society for Leukocyte Biology

Interaction of proteinase 3 with CD11b/CD18 (ß2integrin) on the cell membrane of human neutrophils

A. David*, Y. Kacher*, U. Specks{dagger} and I. Aviram*,1

* Department Biochemistry, Tel Aviv University, Israel; and
{dagger} Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, Minnesota

1 Correspondence: Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: avirama{at}post.tau.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener’s granulomatosis, is a serine proteinase stored in granules of human neutrophils. As previously shown, PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. The current study demonstrates that membrane-bound PR3 colocalizes with the adhesion molecule CD11b/CD18 (ß2 integrin). Immunoprecipitation experiments using plasma membranes of phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils revealed coimmunoprecipitation of PR3 with CD11b/CD18, indicating their location in the same complex. PR3 was also detected in TritonX-100-insoluble cytoskeleton of plasma membranes isolated from unactivated and activated neutrophils. Release of cytoskeletal PR3 by salt treatment implied electrostatic interaction with the enzyme. The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) augmented membrane expression of PR3 in unactivated and PMA-stimulated neutrophils. PMSF significantly reduced adhesion of neutrophils to fibrinogen-coated plates and their NADPH oxidase activity. Moreover, the addition of exogenous PR3 (1–5 µg/ml) augmented the CD11b/CD18-dependent adhesion of neutrophils. Taken together, these results implicate the ß2 integrin of neutrophils in their membrane association with PR3 and suggest a role of PR3 in the modulation of cell adhesion.

Key Words: adhesion • NADPH oxidase • serine protease • PMSF • cytoskeleton


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase 3 (PR3; reviewed in ref. [1 ]) previously named myeloblastin, belongs to the family of serine proteases of neutrophil azurophilic granules, also comprising neutrophil elastase and cathepsin G [2 , 3 ]. Earlier studies localized PR3 to azurophilic granules [2 ]; later publications documented its presence in secretory vesicles and on the plasma membrane of freshly isolated human neutrophils [4 5 6 7 ]. Priming and appropriate stimulation increased the membrane expression of PR3 [5 , 6 ], which was identified as the main target autoantigen for cytoplasmic-staining antineutrophil cytoplasmic antibodies (c-ANCA) in the autoimmune small vessel vasculitis, Wegener’s granulomatosis [4 , 8 9 10 ]. Binding of c-ANCA to membrane-associated PR3 induces exocytosis of granule proteases and release of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-generated superoxide, which may contribute to the tissue necrosis and vasculitis characteristic of Wegener granulomatosis [11 ]. Recent characterization of PR3 as a multifunctional protein is based on the evidence that in addition to its proteolytic activity, essential for killing invading microorganisms, PR3 participates in myeloid differentiation [12 ] and independently of its enzymatic activity, exhibits antimicrobial properties [13 ] and regulates activity of the NADPH oxidase [14 ].

The molecular mechanisms of the association of PR3 with the plasma membrane and the functional significance of this association are still under investigation. In view of the recently presented evidence for binding of the PR3 homologs, elastase and cathepsin G, to CD11b/CD18 [15 , 16 ], we addressed the possibility that neutrophil membrane-bound PR3 interacts with integrins, which are heterodimeric membrane receptors that participate in intercellular adhesion and in adhesion of cells to the extracellular matrix [17 18 19 ]. The main adhesion receptor of neutrophils is the ß2 integrin CD11b/CD18 (Mac-1) [18 19 20 21 ]. In primed or activated cells, CD11b/CD18 mediates binding of immobilized ligands, e.g., intercellular adhesion molecule-1 (ICAM-1), fibrinogen, and complement fragment C3bi [22 ]. Our data, presented below, indicate colocalization of CD11b/CD18 with membrane-bound PR3 and suggest a possible, functional involvement of the enzyme in cell adhesion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Tumor necrosis factor {alpha} (TNF-{alpha}) was obtained from PeproTech Inc. (Rocky Hill, NJ); dextran and Ficol-Hypaque were from Pharmacia (Uppsala, Sweden). PR3 was from Wieslab AB (Lund, Sweden).

Antibodies
Goat antisera to gp91phox, p22phox, p47phox, and p67phox were a generous gift of Dr. Thomas L. Leto (National Institutes of Health, Bethesda, MD). Monoclonal anti-PR3 (6A6, 4A5) and rabbit anti-PR3 were purchased from Wieslab AB. The polyclonal goat anti-CD11b was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies (mAb), anti-CD18 mAb (CBL 514), anti-CD11b mAb (ICRF44), and anti-CD44 (MEM-85) were from IQ Products (Groningen, The Netherlands). Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG), phycoerythrin (PE)-labeled donkey anti-mouse F(ab')2, and cyanine (Cy3)-labeled goat anti-rabbit IgG were obtained from Jackson ImmunoResearch (West Grove, PA). Goat IgG was from Sigma Chemical Co.

Preparation of neutrophils
Human polymorphonuclear leukocytes (PMNs) were isolated from fresh buffy coats by standard procedures of dextran sedimentation, hypotonic lysis of erythrocytes, and Ficol-Hypaque density gradient centrifugation [14 ]. Cells were resuspended in Krebs-Ringer phosphate (KRP)-buffered solution (131 mM NaCl, 15.7 mM NaPi, pH 7.4, 5.2 mM KCl, 2 mM glucose, 1.3 mM MgSO4, 0.9 mM CaCl2).

Flow cytometry
Unactivated neutrophils (defined as isolated cells that were not intentionally primed or stimulated) or cells prestimulated with phorbol 12-myristate 13-acetate (PMA; 100 ng/ml; 107/ml in RPMI 1640/5% fetal calf serum/0.01% azide) were incubated with a primary antibody for 45 min on ice, washed, and further incubated in the dark (45 min) with appropriately labeled secondary antibodies. Washed cells were fixed with 1% paraformaldehyde at room temperature and analyzed for fluorescence in a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Immunofluorescence confocal microscopy
Cell suspensions were treated as for flow cytometry. For double-labeling experiments, the two kinds of primary antibodies or the two conjugated secondary antibodies were simultaneously incubated with the cells at 4°C. Fixation was performed as described above. In some experiments, fixed cells were permeabilized before incubation with antibodies with 0.1% Triton X-100 for 10 min at room temperature. Fixed cells were transferred to glass slides, coated with mounting medium (Vector Laboratories, Burlingame, CA), and visualized by confocal microscopy (Zeiss microscope LSM 510, Carl Zeiss, Thornwood, NY).

Adhesion and measurement of O2- production
Neutrophil adhesion was measured following the protocol described by Bellavite et al. [23 ]. The 96-well plates were precoated with fibrinogen (0.05 mg/ml, 100 µl/well) in 0.1% bovine serum albumin (BSA) containing Tris-buffered saline (20 mM Tris, pH 7, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2) for 1 h at 37°C and were washed twice with the same buffer (200 µl/well). Cells (2x105/well in KRP) were transferred onto the coated plates, preincubated for 10 min at 37°C, and stimulated with TNF-{alpha} (20 ng/ml), PMA (100 ng/ml), or formyl-Met-Leu-Phe (fMLP; 10-7 M) in a total volume of 100 µl in duplicates. After incubation and three washes, adherent cells were quantitated by measuring their acid phosphatase content [23 ]. For this purpose, 10 mM p-nitrophenyl phosphate in 0.15 M acetate buffer, pH 5.3, 0.2% Triton X-100, was added to the adherent cells (75 µl/well) and incubated for 10 min at 37°C. The reaction was terminated by the addition 100 µl/well of 1 M NaOH, and the color was determined in a microtiter plate reader (Thermomax, Molecular Devices, Sunnyvale, CA) at 405 nm. In adhesion plots, adherent cells are shown as % of total.

O2- production was measured as superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (0.8 mg/ml) with NADPH as substrate [14 ]. Reduced cytochrome c was estimated at 550 nm.

Neutrophil fractionation
Cells (2x108/ml) in 10 mM potassium phosphate-buffered saline (pH 7), supplemented with 1 mM EGTA, 3.5 mM phenylmethylsulfonyl fluoride (PMSF), 15 µg/ml leupeptin, were sonicated on ice (three 20-s pulses of a Microson 25-W microtip) [14 , 24 ]; unbroken cells were removed by low-speed (250 g) centrifugation. Granules were sedimented by centrifugation at 15,000 rpm for 15 min at 4°C (Eppendorf centrifuge 5403). Plasma membranes and cytosol were separated by ultracentrifugation at 48,000 rpm for 45 min at 4°C (SORVAL Ti-50).

Immunoprecipitation and Western blot
Plasma membranes were isolated from the cells as described above and were solubilized in immunoprecipitation buffer (150 mM NaCl, 0.5% Nonidet P-40, 0.5 mM EDTA, 1 mM PMSF, 1 mM EGTA, 10 mM Tris-HCl, 15 µg/ml leupeptin). Lysates were precleared with IgG bound to protein A-Sepharose (Calbiochem, San Diego, CA). Primary antibodies (polyclonal anti-PR3 or anti-CD11b) were incubated overnight with the supernatants followed by the addition of protein A-Sepharose for 3 h at 4°C and were pelleted. Immunoprecipitates were washed four times with immunoprecipitation buffer containing 1% BSA and were suspended in Laemmli sample buffer [25 ]. The beads were boiled for 5 min and sedimented, and the supernatants were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to nitrocellulose and incubated with anti-PR3 (6A6 or 4A5) or anti-CD11b (ICRF44), followed by horseradish peroxidase-conjugated antibodies. Labeled proteins were detected using the luminol-p-coumarine enhanced chemiluminescence technique.

Preparation of membrane cytoskeleton
Following procedures described by El Benna et al. [26 ], membrane aliquots from unactivated and PMA-activated cells (5x107), isolated as described above, were incubated in 1 ml membrane-cytoskeleton buffer (25 mM Hepes, pH 7.4, 60 mM Pipes, 2 mM MgCl2, 0.75% Triton X-100,10 mM EGTA, 8 µg/ml aprotinin, 156 µg/ml benzamidine, 20 µg/ml leupeptin, 1 mM PMSF) for 20 min on ice. Lysates were layered on top of 4 ml extraction buffer (6% sucrose, 1% Triton X-100, 100 mM Tris, pH 7, plus protease inhibitors listed above), and the Triton-soluble and insoluble fractions were separated by ultracentrifugation at 48,000 rpm for 120 min at 4°C (SORVAL Ti-50). Triton-insoluble fractions were washed with extraction buffer. Proteins in supernatants were precipitated by cold methanol (overnight at –20°C) followed by centrifugation.

Statistical analysis
Data are presented as mean values ± SEM and are analyzed by a paired one-tailed Student’s t-test; P values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Colocalization of ß2 integrin and membrane-bound PR3 on the plasma membrane of unactivated and activated neutrophils
Immunofluorescence staining and confocal microscopy were used to examine the localization of membrane-bound PR3 (red) with respect to ß2 integrin CD11b/CD18 (green). The yellow staining in these dual-labeling experiments indicated overlapping areas of green and red fluorescent labels and suggested colocalization of the {alpha} (CD11b; Fig. 1A ) or ß2 (CD18) subunits with PR3 on the plasma membrane of PMA-stimulated cells (Fig. 1B) . Colocalization was also observed in plasma membranes of unactivated cells. In control experiments, neutrophils were labeled for CD44 and PR3 (Fig. 1C) . Separate green and red patches were observed, incompatible with colocalization. Secondary antibodies used as controls yielded no staining (data not shown).



View larger version (76K):
[in this window]
[in a new window]
  		Figure 1. Distribution of PR3 and CD11b (A) or CD18 (B) or CD44 (C) in the plasma membranes of human neutrophils stimulated with PMA (100 ng/ml) for 15 min at 37°C. The activated neutrophils were treated on ice with the primary antibodies (rabbit anti-PR3 and manti-CD11b or manti-CD18) followed by secondary antibodies, goat anti-rabbit IgG–Cy3 (red), and goat anti-mouse IgG–FITC (green). Colocalization of the two proteins results in a yellow staining. Shown is a representative of three experiments performed with cells of different donors.

 
The effect of incubation of unactivated or PMA-prestimulated PMN (15 min, 37°C, followed by washing) with goat polyclonal anti-CD11b (45 min on ice) on membrane-bound PR3 was evaluated by flow cytometry. Surface-expressed PR3 was detected using mAb 6A6 and PE-labeled donkey anti-mouse IgG F(ab')2 as the secondary antibody. Unexpectedly, we observed that the treatment with anti-CD11b significantly augmented the amount of PR3 measured in the plasma membrane of PMA-preactivated neutrophils (154%±8.7% of untreated stimulated controls; P<0.05). Anti-CD11b treatment did not affect PR3 detected in unactivated neutrophils (98.7%±12.3%). These results are compatible with proximity of PR3 to ß2 integrin in the plasma membrane of PMA-activated neutrophils.

Coimmunoprecipitation of CD11b with PR3
To confirm the conclusion described above, we immunoprecipitated membrane-bound PR3 from plasma membranes of PMA-activated neutrophils using polyclonal rabbit anti-PR3. Immununoprecipitated proteins were resolved by SDS-PAGE and subjected to immunoblotting with monoclonal anti-CD11b (Fig. 2 ). Detection of the CD11b subunit of the ß2 integrin in lane 1 of the immunoblot indicates that CD11b coimmunoprecipitated with PR3 (Fig. 2A) . The CD11b subunit was not detected when rabbit IgG was used as a control (lane 2). When immunoprecipitation was performed with goat anti-CD11b, a 29-kDa band of PR3 was detected on the Western blot (Fig. 2B) . The results corroborate the colocalization of PR3 and CD11b/CD18 in a membrane-bound complex suggested by confocal imaging.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 2. Immunoblot analysis of immunoprecipitates from PMA-activated plasma membranes of neutrophils (2x107 cells equivalents) with: (A) lane 1, rabbit anti-PR3, and lane 2, rabbit IgG; (B) lane 1, goat anti-CD11b, and lane 2, goat serum. Immunoprecipitates were run on SDS-PAGE, 7.5% and 12%, respectively. Blots were probed with (A) manti-CD11b (ICRF44) and (B) manti-PR3 (6A6).

 
Association of PR3 with the neutrophil cytoskeleton
Neutrophil solubilization in Triton X-100 preserves actin filaments and yields two fractions upon centrifugation of the lysates: a Triton-insoluble fraction operationally defined as a cytoskeleton and a Triton-soluble fraction containing noncytoskeletal proteins. A similar detergent treatment of washed neutrophil plasma membranes (the 100,000 g pellet), isolated from unactivated or PMA-stimulated neutrophils, produced Triton X-100-insoluble membrane cytoskeletons [26 ], resolved by SDS-PAGE, as shown in Figure 3 . PR3 was detected in insoluble fractions of unactivated and activated Triton X-100-insoluble pellets (lanes 3 and 4), whereas the detergent-soluble residues were free of PR3 (Fig. 3 , lanes 1 and 2). The presence of PR3 in the Triton-insoluble fraction reflects association of PR3 with the cytoskeleton of unactivated as well as PMA-activated neutrophils. Ionic strength dependence of the association was tested by incubation of the isolated membrane cytoskeletons (1 h, 4°C) with 1 M NaCl. Release of PR3 by the salt treatment indicated involvement of electrostatic interactions of PR3 with cytoskeletal elements.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 3. Distribution of PR3 in Triton-soluble and insoluble fractions of plasma membranes of unactivated and PMA-activated human neutrophils (A) and effect of treatment with NaCl on the PR3 content in the Triton-insoluble fraction (B). (A) Lanes 1 and 2, Triton-soluble fractions (sol); lanes 3 and 4, Triton-insoluble fractions (csk) of unactivated (–) and PMA-activated (+) cells, respectively. (B) Lanes 1 and 2, Membrane cytoskeleton of unactivated and PMA-activated cells; lanes 3 and 4, same after treatment with 1 M NaCl at 4°C, 1 h. Cell equivalents (5x107) of each fraction were separated in 12% SDS-PAGE gels, and the blots were probed with manti-PR3 (4A5). The results are representative of three separate experiments.

 
Effect of exogenous PR3 on CD11b/CD18-dependent neutrophil adhesion to fibrinogen-coated plates
Priming of neutrophils with TNF-{alpha} and activation with fMLP or PMA increase density of CD11b/CD18 molecules on the cell membrane and their avidity for ligands, resulting in cell adhesion to immobilized fibrinogen [22 ]. Consistent with dependence of adhesion on CD11b/CD18, pretreatment of a suspension of PMN with polyclonal goat anti-CD11b (60 min, 4°C, followed by short centrifugation) but not with an irrelevant goat IgG inhibited subsequent PMA-induced adhesion (Fig. 4A ). Exogenous PR3, present during the preincubation with anti-CD11b, prevented this anti-CD11b-mediated inhibition of adhesion. The abolition of the anti-CD11b effect was exerted by enzymatically active as well as by PMSF-inactivated PR3. The effect of PR3 was dose-dependent; substitution of PR3 with identical concentrations of BSA had no effect on the inhibition by the antibody, suggesting specificity of the PR3 effect (Fig. 4B) . In the absence of anti-CD11b, preincubation with exogenous PR3 enhanced weakly adhesion of PMA-stimulated cells from 36.2% ± 8.4 to 42.5% ± 2.45 (inactive PR3) or to 42.8% ± 2.13 (active PR3; n=4; data not shown). These findings may suggest that exogenous PR3 modulates adhesion of PMA-stimulated neutrophils to fibrinogen by interacting with CD11b/CD18. This effect, independent of PR3 proteolytic activity, requires further investigation.



View larger version (43K):
[in this window]
[in a new window]
  		Figure 4. The effect of goat anti-CD11b (1 µg/ml) and exogenous PR3 on the adhesion of PMA-stimulated neutrophils to fibrinogen-coated plates. (A) The effect of anti-CD11b and 4 µg/ml exogenous PR3 (PMSF-inactivated PR3(i), n=8, or active PR3, n=4) on adhesion (means±SEM). Neutrophils were preincubated (1 h, on a shaker, 4°C) without (control) or with polyclonal goat anti-CD11b and exogenous PR3, pelleted before transfer to fibrinogen-coated plates, and stimulated with PMA. (B) Dose-dependent effect of PR3 (inactivated with PMSF) preincubated with neutrophils in the presence of goat anti-CD11b. The results are representative of four experiments.

 
PMSF blocks CD11b/CD18-mediated adhesion of PMN to fibrinogen
To examine possible effects of serine proteases on adhesion, neutrophils were activated in the presence of the irreversible serine protease inhibitor PMSF. The inhibitor added to the cells concomitantly with the stimulant significantly reduced adhesion of PMA-, fMLP-, or TNF-{alpha}-treated neutrophils to fibrinogen-coated plates (Fig. 5A ). The extent of inhibition by PMSF depended on its concentration (data not shown) and on the time of its addition. The most pronounced effect was observed on concurrent addition of the inhibitor and the stimulant (Fig. 5C) . PMSF added during preincubation or 20 min after PMA exerted significantly smaller effects. Notably in neutrophils pretreated with PMSF in suspension (20 min), washed, aliquoted on plates, and stimulated, no residual effect of PMSF on stimulation-induced adhesion was observed (data not shown). However, the PMSF-treated neutrophils lost their esteratic activity on N-t-Boc-L-alanine p-nitro phenyl ester, a substrate cleavable by PR3 and neutrophil elastase [3 ] (data not shown). Furthermore, the adhesion of these neutrophils was suppressed on stimulation in the presence of re-added PMSF. This finding implied that PMSF was effective only when present during cell stimulation and that inhibition of activity and reduction of adhesion were not mediated by the same cellular component. It is of note that the presence of other inhibitors of serine proteases, {alpha}-1 antitrypsin (0.5 mg/ml), aprotinin (0.5–4 µg/well), or 4-(2-aminoethyl)-benzenesulfonyl fluoride (1 mM), during stimulation had no effect on the adhesion (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 5. The effect of PMSF (1 mM) on (A) the adhesion to fibrinogen-coated plates and on (B) NADPH-oxidase activity of neutrophils (2x105) activated with: PMA [100 ng/ml in dimethyl sulfoxide (DMSO)], fMLP (10-7 M), or TNF-{alpha} (20 ng/ml). PMSF was added with the stimulants; adhesion and cytochrome c reduction were determined separately after 30 min of incubation at 37°C (means±SEM, n=5). Basal adhesion of unstimulated cells was 11.2% ± 3.0 SEM (n=7). (C) Effect of PMSF added at various time points relatively to stimulants on PMA- and fMLP-elicited adhesion. The results are representative of three experiments.

 
Effect of PMSF on the activity of neutrophil NADPH oxidase
To further characterize the effect of PMSF on neutrophil activation in the context of adhesion to fibrinogen, superoxide released by neutrophils in microplates was monitored as a SOD-inhibitable reduction of cytochrome c. PMA-stimulated neutrophils allowed to adhere to fibrinogen-coated plates in the presence of PMSF released superoxide ions at a lower rate than PMSF-untreated controls (Fig. 5B) , suggesting inhibition of the NADPH oxidase.

Influence of PMSF on the expression of membrane-bound PR3
The effect of PMSF was further tested by flow cytometry estimating the expression of membrane-bound PR3 and CD11b/CD18 in unactivated and PMA-activated neutrophils. Pretreatment of neutrophils with 1 mM PMSF (20 min, room temperature), followed by washing and subsequent stimulation, progressively up-regulated the amount of membrane-bound PR3 detected in unactivated and PMA-activated cells (Fig. 6 ). Replacement of PMSF by {alpha}1-antitrypsin had no effect on the membrane-bound PR3 (data not shown). This finding may suggest that the observed effect of PMSF is independent of its protease inhibitor property.



View larger version (30K):
[in this window]
[in a new window]
  		Figure 6. Fluorescence-activated cell sorter (FACS) analysis of the effect of PMSF (1 mM) on time-dependent expression of PR3 in plasma membranes of unactivated and PMA-activated (100 ng/ml) neutrophils. PMSF was preincubated (20 min, room temperature) with the cells and was washed out before activation (means±SEM, n=3; *, P<0.05). MFI, Mean fluorescence intensity.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Detection of PR3 on the plasma membrane of neutrophils isolated from healthy donors and its increase by stimulation are in keeping with results reported by others [4 5 6 7 ]. Immunofluorescence confocal microscopy analysis of membrane-bound PR3 and CD11b/CD18 presented in this communication demonstrates colocalization of the two proteins in plasma membranes of unactivated and PMA-activated human cells (Fig. 1A and 1B) . The notion that both proteins reside within the same complex was further supported by coimmunoprecipitation of CD11b/CD18 and PR3 from the plasma membranes of PMA-preactivated neutrophils (Fig. 2A and 2B) and by identification of PR3 in detergent-insoluble cytoskeleton of plasma membranes (Fig. 3A) . Colocalization and coimmunoprecipitation, however, do not imply that the interaction between PR3 and CD11b/CD18 is direct. The apparent association of PR3 with CD11b/CD18 might be mediated by additional components clustering in cytoskeletal domains of plasma membranes and/or in detergent-insoluble membrane microdomains (rafts) [27 ].

PMNs are known to constitutively express an inactive form of integrins, which are converted into the active, high-affinity, and avidity form on arrival of an appropriate signal. Cellular activation is also associated with an increased density of CD11b/CD18 on the cell membrane, reflecting translocation of integrin molecules from subcellular storage granules to the cell surface [17 18 19 20 21 22 ]. Active CD11b/CD18 molecules facing the medium participate in adhesion of neutrophils to endothelial cells, fibrinogen, gelatin, serum, or ICAM-1-coated plates [19 , 20 ]. Association of CD11b/CD18 with neutrophil elastase [15 ] and other ligands has also been documented [22 ], and involvement of CD11b/CD18 in the binding of a soluble endothelial protein C receptor to PR3 on activated neutrophils has recently been described [28 ]. Although it is tempting to conclude that ß2 integrin functions as a membrane receptor or one of the receptors for PR3 released from the cell, additional evidence for this conclusion is required.

The unexpected finding that treatment (in cold) of preactivated neutrophils with anti-CD11b augmented the amount of membrane-bound PR3 detected by FACS also supported interaction of PR3 with CD11b/CD18. We can only speculate that this augmentation might be a result of the unmasking of previously buried antigenic epitopes on membrane-bound PR3 caused by the association of ß2 integrin with its cognate antibodies. Further evidence for an interaction between PR3 and CD11b/CD18 is provided by our observation that pre-exposure of cells to exogenous PR3 in the presence of anti-CD11b prevents the antiadhesive effect of the antibody on PMA-stimulated neutrophils in a manner that is dose-dependent but independent of the proteolytic activity of PR3 (Fig. 4A) . It is of note that the concentrations of PR3 used in these experiments (1–5 µg/ml) are compatible with the amount of the enzyme that may be discharged into inflammatory tissues [7 ].

Previous studies reported modulation of CD11b/CD18-mediated adhesion of neutrophils by the homologous serine proteases elastase and cathepsin G [15 , 16 ]. In keeping with this modulation, we showed that the treatment of neutrophils with the covalent, unspecific serine protease inhibitor PMSF reduced CD11b/CD18-mediated adhesion to fibrinogen-coated plates, implicating a serine protease activity in the ß2 integrin-dependent adhesion (Fig. 5A) . However, neutrophils pretreated with PMSF and washed free of the reagent exhibited normal adhesion upon stimulation. Lysates of PMSF-pretreated cells lost their N-t-Boc-L-alanine p-nitro phenyl ester-hydrolyzing activity, indicating inactivation of cellular serine proteases known to cleave this substrate, including elastase and PR3 [3 ] (data not shown). Taken together, these data suggest that PMSF attenuates adhesion, not through a serine protease-inhibiting action but rather by another modification that requires cell activation. Binding of PMSF to proteins at noncatalytic sites has previously been documented [29 , 30 ]. It is noteworthy that the activation-coupled effect of PMSF also reduced superoxide release by activated NADPH oxidase (Fig. 5B) . Earlier communications reported a reduction of NADPH oxidase activity by PMSF and other serine protease inhibitors [30 , 32 ].

Detection of membrane-bound PR3 in PMSF-pretreated neutrophils by FACS implies that inhibition of the enzymatic activity of PR3 by PMSF did not disrupt the association of the enzyme with the plasma membrane. On the contrary, the amount of membrane-bound PR3 was enhanced by the pretreatment with the inhibitor (Fig. 6) . This finding may be ascribed to susceptibility of membrane-bound PR3 or of its putative neutrophil membrane receptor to an autoproteolytic or proteolytic cleavage by a serine protease that impedes its subsequent detection. As PMSF treatment of cells is not specific for PR3, it will be of interest whether similar augmentation will be observed in the case of elastase or cathepsin G.

Taken together, our studies provide evidence for interaction of PR3 with activated CD11b/CD18 on the plasma membrane of human neutrophils. This interaction seems to affect the adhesive properties of neutrophils and may thus lead to functional alterations of the cells in an inflammatory environment.


   ACKNOWLEDGEMENTS
 
This work was supported in part by The United States–Israel Binational Science Foundation, Grant 1999012. A. D. and Y. K. were equally contributing authors.

Received December 25, 2002; revised May 23, 2003; accepted May 27, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Van der Geld, Y. M., Limburg, P. C., Kallenberg, C. G. (2001) Proteinase 3, Wegener’s autoantigen: from gene to antigen J. Leukoc. Biol. 69,177-190[Abstract/Free Full Text]
  2. Dewald, B., Rindler-Ludwig, R., Bretz, U., Baggiolini, M. (1975) Subcellular localization and heterogeneity of neutral proteases in neutrophilic polymorphonuclear leukocytes J. Exp. Med. 141,709-723[Abstract]
  3. Rao, N. V., Wehner, N. G., Marshall, B. C., Gray, W. R., Gray, B. H., Hoidal, J. R. (1991) Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties J. Biol. Chem. 266,9540-9548[Abstract/Free Full Text]
  4. Csernok, E., Ernst, M., Schmit, W., Bainton, D. F., Gross, L. (1994) Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo Clin. Exp. Immunol. 95,244-250[Medline]
  5. Halbwachs-Mecarelli, L., Bessou, G., Lesavre, P., Lopez, S., Witko-Sarsat, V. (1995) Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool FEBS Lett. 374,29-33[CrossRef][Medline]
  6. Witko-Sarsat, V., Cramer, E. M., Hieblot, C., Guichard, J., Nusbaum, P., Lopez, S., Lesavre, P., Halbwachs-Mecarelli, L. (1999) Presence of proteinase 3 in secretory vesicles: evidence for a novel, highly mobilizable pool distinct from azurophilic granules Blood 94,2487-2496[Abstract/Free Full Text]
  7. Campbell, E. J., Campbell, M. A., Owen, C. (2000) Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity and susceptibility to inhibition J. Immunol. 165,3366-3374[Abstract/Free Full Text]
  8. Goldschmeding, R., van der Schoot, C. E., ten Bokkel Huinink, D., Hack, C. E., van den Ende, M. E., Kallenberg, C. G., von dem Borne, A. E. (1989) Wegener’s granulomatosis autoantibodies identify a novel diisopropylfluorophosphate-binding protein in the lysosomes of normal human neutrophils J. Clin. Invest. 84,1577-1587
  9. Niles, J. L., McCluskey, R. T., Ahmad, M. F., Arnaout, M. A. (1989) Wegener’s granulomatosis autoantigen is a novel neutrophil serine proteinase Blood 74,1888-1893[Abstract/Free Full Text]
  10. Jennette, J. C., Hoidal, J. R., Falk, R. J. (1990) Specificity of anti-neutrophil cytoplasmic autoantibodies for proteinase 3 Blood 75,2263-2264[Free Full Text]
  11. Falk, R. J., Terrel, R. S., Charles, L. A., Jennette, J. C. (1990) Anti-neutrophil cytoplasmic autoantibodies induce neutrophil to degranulate and produce oxygen radicals in vitro Proc. Natl. Acad. Sci. USA 87,4115-4119[Abstract/Free Full Text]
  12. Bories, D., Raynal, M. C., Solomon, D. H., Darzynkiewicz, Z., Cayre, Y. E. (1989) Down-regulation of a serine protease, myeloblastin, causes growth arrest and differentiation of promyelocytic leukemia cells Cell 59,959-968[CrossRef][Medline]
  13. Campanelli, D., Detmers, P. A., Nathan, C. F., Gabay, J. E. (1990) Azurocidin and a homologous serine protease from neutrophils. Differential antimicrobial and proteolytic properties J. Clin. Invest. 85,904-915
  14. Tal, T., Sharabani, M., Aviram, I. (1998) Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation J. Leukoc. Biol. 63,305-311[Abstract]
  15. Cai, T. Q., Wright, S. D. (1996) Human leukocyte elastase is an endogenous ligand for the integrin CR3 (CD11b/CD18, Mac-1, {alpha}mß2) and modulates polymorphonuclear leukocyte adhesion J. Exp. Med. 184,1213-1223[Abstract/Free Full Text]
  16. Renesto, P., Halbwachs-Mecarelli, L., Bessou, G., Balloy, V., Chignard, M. (1996) Inhibition of neutrophil-endothelial cell adhesion by a neutrophil product, cathepsin G J. Leukoc. Biol. 59,855-863[Abstract]
  17. Williams, M. A., Solomkin, J. S. (1999) Integrin-mediated signaling in human neutrophil functioning J. Leukoc. Biol. 65,725-736[Abstract]
  18. Harris, E. S., McIntyre, T. M., Prescot, S. M., Zimmerman, G. A. (2000) The leukocyte integrins J. Biol. Chem. 275,23409-23412[Free Full Text]
  19. Berton, G., Lowell, C. A. (1999) Integrin signaling in neutrophils and macrophages Cell. Signal. 11,621-635[CrossRef][Medline]
  20. Arnaout, M. A. (1990) Structure and function of the leukocyte adhesion molecules CD11/CD18 Blood 75,1037-1050[Free Full Text]
  21. Nathan, C., Srimal, S., Farber, C., Sanchez, E., Kabbash, L., Asch, A., Gailit, J., Wright, S. D. (1989) Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins J. Cell Biol. 109,1341-1349[Abstract/Free Full Text]
  22. Jones, S. L., Knaus, U. G., Bokoch, G. M., Brown, E. J. (1998) Two signaling mechanisms for activation of alphaM beta2 avidity in polymorphonuclear neutrophils J. Biol. Chem. 273,10556-10566[Abstract/Free Full Text]
  23. Bellavite, P., Chirumbolo, S., Mansoldo, C., Gandini, G., Dri, P. (1992) Simultaneous assay for oxidative metabolism and adhesion of human neutrophils: evidence for correlation and dissociations of the two responses J. Leukoc. Biol. 51,329-335[Abstract]
  24. Klinger, E., Aviram, I. (1992) Involvement of GTP in cell-free activation of neutrophil NADPH oxidase: studies with GTP analogs Biochem. J. 285,635-639
  25. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227,680-685[CrossRef][Medline]
  26. El Benna, J., Ruedi, J. M., Babior, B. M. (1994) Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation J. Biol. Chem. 269,6729-6734[Abstract/Free Full Text]
  27. Nebl, T., Pestonjamasp, K. N., Leszyk, J. D., Crowley, J. L., Oh, S. W., Luna, E. J. (2002) Proteomic analysis of a detergent-resistant membrane skeleton from neutrophil plasma membranes J. Biol. Chem. 277,43399-43409[Abstract/Free Full Text]
  28. Kurosawa, S., Esmon, C. T., Stearns-Kurosawa, D. J. (2000) The soluble endothelial protein C receptor binds to activated neutrophils: involvement of proteinase-3 and CD11b/CD18 J. Immunol. 165,4697-4703[Abstract/Free Full Text]
  29. Mark, D. E., Lazzari, K. G., Simons, E. R. (1988) Are serine proteases involved in immune complex activation of neutrophils? J. Leukoc. Biol. 44,441-447[Abstract]
  30. Rao, K. M., Castranova, V. (1988) Phenylmethylsulfonyl fluoride inhibits chemotactic peptide-induced actin polymerization and oxidative burst activity in human neutrophils by an effect unrelated to its anti-proteinase activity Biochim. Biophys. Acta 969,131-138[Medline]
  31. Gomez-Cambronero, J., Mege, J-L., Molski, T. F. P., Naccache, P. H., Becker, E. L., Sha’afi, R. I. (1989) Actions of the protease inhibitor phenylmethylsulfonyl fluoride on neutrophil granule enzyme secretion and superoxide production induced by fMet-Leu-Phe and phorbol 12- myristate-13-acetate Int. Arch. Allergy Appl. Immunol. 89,362-368[Medline]
  32. Lundqvist, H., Dahlgren, C. (1995) The serine protease inhibitor diisopropylfluoro-phosphate inhibits neutrophil NADPH-oxidase activity induced by the calcium ionophore ionomycin and serum opsonized yeast particles Inflamm. Res. 44,510-517[CrossRef][Medline]



This article has been cited by other articles:


Home page
 BloodHome page
S. von Vietinghoff, G. Tunnemann, C. Eulenberg, M. Wellner, M. Cristina Cardoso, F. C. Luft, and R. Kettritz
NB1 mediates surface expression of the ANCA antigen proteinase 3 on human neutrophils
Blood, May 15, 2007; 109(10): 4487 - 4493.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
J. M. Williams, T. R. Pettitt, W. Powell, J. Grove, C. O.S. Savage, and M. J.O. Wakelam
Antineutrophil Cytoplasm Antibody-Stimulated Neutrophil Adhesion Depends on Diacylglycerol Kinase-Catalyzed Phosphatidic Acid Formation
J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1112 - 1120.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
S. Bauer, M. Abdgawad, L. Gunnarsson, M. Segelmark, H. Tapper, and T. Hellmark
Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils
J. Leukoc. Biol., February 1, 2007; 81(2): 458 - 464.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
D. Reumaux, P. L. Hordijk, P. Duthilleul, and D. Roos
Priming by tumor necrosis factor-{alpha} of human neutrophil NADPH-oxidase activity induced by anti-proteinase-3 or anti-myeloperoxidase antibodies
J. Leukoc. Biol., December 1, 2006; 80(6): 1424 - 1433.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Stefanidakis and E. Koivunen
Cell-surface association between matrix metalloproteinases and integrins: role of the complexes in leukocyte migration and cancer progression
Blood, September 1, 2006; 108(5): 1441 - 1450.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1202624v1
74/4/551    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by David, A.
Right arrow Articles by Aviram, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by David, A.
Right arrow Articles by Aviram, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS