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Originally published online as doi:10.1189/jlb.0405190 on December 19, 2005

Published online before print December 19, 2005
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(Journal of Leukocyte Biology. 2006;79:489-498.)
© 2006 by Society for Leukocyte Biology

Cell surface expression of intermediate filament proteins vimentin and lamin B1 in human neutrophil spontaneous apoptosis

Eliane Moisan and Denis Girard1

INRS-Institut Armand-Frappier, Université du Québec, Canada

1 Correspondence: INRS-Institut Armand-Frappier, 245 boul. Hymus, Pointe-Claire (PQ), Canada, H9R 1G6. E-mail: denis.girard{at}iaf.inrs.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils represent an important source of autoantigens for antineutrophil cytoplasmic antibody associated with vasculitis. To date, two cytoskeletal proteins, vinculin and vimentin, have been reported to be expressed on the cell surfaces of activated macrophages, platelets, and apoptotic T lymphocytes. However, such cell surface expression has never been studied in human neutrophils. As we recently demonstrated that different cytoskeletal proteins were cleaved in apoptotic neutrophils, we hypothesized that some of these were expressed on the cell surface of apoptotic neutrophils. Herein, we found that among vinculin, paxillin, gelsolin, vimentin, lamin B1, {alpha}-tubulin, and ß-tubulin, only the two intermediate filament (INFIL) proteins, vimentin and lamin B1, are expressed on the cell surface of 24-h aged neutrophils [spontaneous apoptosis (SA)]. By monitoring intracellular expression of vimentin and lamin B1 during SA, we found that these two proteins were cleaved and that such cleavage was reversed by the pan caspase inhibitor N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk). When neutrophil apoptosis was delayed or suppressed by lipopolysaccharide or the cytokines granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage (GM)-CSF, or interleukin-4, the loss of intracellular expression of vimentin and lamin B1 was prevented. The INFIL proteins were absent from the cell surface when neutrophil apoptosis was delayed. Addition of z-VAD-fmk significantly decreased the cell surface expression of vimentin and lamin B1 during SA. This study provides the first evidence that apoptotic neutrophils express cytoskeletal proteins on their surface, opening the possibility that these cells may participate in the development of autoantibodies directed against cytoskeletal proteins, a condition frequently reported in several inflammatory diseases.

Key Words: cytoskeleton • microfilaments • microtubules • caspases • flow cytometry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cytoskeleton is a dynamic structure composed of numerous proteins, which form networks. Several neutrophil functions require rearrangement of the cytoskeleton, including chemotaxis, phagocytosis, degranulation, and apoptosis [1 ]. The cytoskeleton may be involved in many autoimmune disorders, as anticytoskeletal autoantibodies can be found in patients suffering from various rheumatic diseases [2 3 4 5 ]. However, the origin of these autoantibodies is still unclear. It was recently found that vinculin, a microfilament-associated protein (MFAP), was expressed on the surface of apoptotic T lymphocytes [6 ]. The intermediate filament (INFIL) protein vimentin was found on the surface of activated platelets, where it was bound to vitronectin and plasminogen activator inhibitor complexes [7 ]. Recently, Boilard et al. [8 ] showed that secreted human group IIA phospholipase A2 (PLA2) binds to vimentin on the cell surface of apoptotic T lymphocytes. The interaction between these two proteins enhanced the activity of PLA2, suggesting that vimentin may play a role in PLA2-mediated cellular arachidonic acid release. Mor-Vaknin et al. [9 ] demonstrated that stimulation of human macrophages activated phosphorylation of vimentin, leading to its expression on the cell surface and its secretion into the extracellular milieu. Furthermore, they showed that extracellular vimentin played a role during inflammation, as it acted as a protein involved in killing bacteria and in oxidative metabolite production; this activity was enhanced by proinflammatory cytokines [9 ]. In addition, a population of endothelial cells was found to express vimentin on the cell surface and to secrete it into bloodstream [10 ]. Knowing that some cytoskeletal proteins are implicated in inflammatory functions, these observations can additionally explain, in part, the presence of anticytoskeletal autoantibodies found in many autoimmune diseases.

Apoptotic cells are an important source of autoantigens [11 ]. Two studies have demonstrated that injection of apoptotic cells into normal mice resulted in production of various autoantibodies [12 , 13 ], including antivimentin autoantibodies [13 ]. Aside from erythrocytes and platelets, neutrophils are the predominant cells in the circulation, and because of this, in addition to the fact that these cells are known to undergo apoptosis spontaneously [14 , 15 ], they represent an important source of cytoskeletal autoantigens. Recently, we have demonstrated that certain cytoskeletal proteins, including vimentin, are cleaved during spontaneous or agent-induced human neutrophil apoptosis [16 17 18 19 ]. Therefore, we hypothesized that degradation of cytoskeletal proteins in neutrophils could lead to their cell surface expression.

In the present study, we investigated potential cell surface expression of different cytoskeletal proteins on the surface of apoptotic human neutrophils using flow cytometry. In parallel, we studied the intracellular expression of cytoskeletal proteins. Our results indicate that the two INFIL proteins vimentin and lamin B1, but not the MFAP paxillin, gelsolin, and vinculin nor the microtubule proteins {alpha}- and ß-tubulin, are expressed on the cell surface of human apoptotic neutrophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals, agonists, and antibodies
RPMI 1640, HEPES, penicillin/streptomycin (P/S), bovine serum albumin (BSA), Viscum album agglutinin-1 (VAA-I), cycloheximide (CHX), lipopolysaccharide (LPS), mouse monoclonal antivinculin antibody (clone hVIN-1), mouse monoclonal antipaxillin antibody (clone PXC-10), mouse monoclonal antigelsolin antibody (clone GS-2C4), mouse monoclonal anti-{alpha}-tubulin (clone B-5-1-2), mouse monoclonal anti-ß-tubulin (clone 2-28-33), and polyclonal goat anti-human vimentin (clone V4630) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Mouse monoclonal antivimentin (clone V9), rabbit polyclonal antivimentin (clone H84), and goat polyclonal antilamin B1 (C-20) antibodies were purchased from Santa Cruz Biotechnology (CA). Vimentin antibody (clone 3B4) was obtained from Chemicon (Temecula, CA). Horseradish peroxidase (HRP)-conjugated antibodies, phycoerythrin (PE)-conjugated AffiniPure F(ab')2 fragments, goat anti-mouse immunoglobulin G (IgG; Fab')2 fragment-specific PE-conjugated AffiniPure F(ab')2 fragments and donkey anti-goat IgG (H+L) were purchased from Jackson Immunoresearch (West Grove, PA). Control isotypic antibodies Ms IgG1 and Ms IgG2a were obtained from PharMingen (Mississauga, Ontario, Canada), and rabbit IgG and goat IgG were from R&D Systems (Hornby, Ontario, Canada). N-benzyloxy-carbonyl-V-A-D-O-methylfluoromethyl ketone (z-VAD-fmk) was purchased from Calbiochem (Pasadena, CA). Granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, and interleukin-4 (IL-4) were purchased from PeproTech Inc. (Rocky Hill, NJ).

Human neutrophils isolation
Neutrophils were isolated from the venous blood of healthy volunteers by dextran sedimentation followed by centrifugation over Ficoll-Paque (Amersham Pharmacia Biotech Inc., Baie d’Urfé, Québec, Canada), as described previously [16 , 18 ]. Blood donations were obtained from informed and consenting individuals according to our institutionally approved procedures. Cell viability (>98%) was monitored by trypan blue exclusion, and the purity (>98%) was verified by cytology from cytocentrifuged preparations colored with the Hema 3 stain set (Biochemical Sciences Inc., Swedesboro, NJ).

Assessment of neutrophil apoptosis
Freshly isolated human neutrophils (10x106 cells/mL in RPMI 1640-HEPES-P/S, supplemented with 10% autologous serum) were incubated for 24 h in the presence or absence of neutrophil agonists. Cytocentrifuged samples of neutrophils were prepared using a Cyto-tek® centrifuge (Miles Scientific, Naperville, IL) and processed as documented previously [16 , 18 , 20 ]. Cells were examined by light microscopy at 400x final magnification, and apoptotic neutrophils were defined as cells containing one or more characteristic, darkly stained pyknotic nuclei.

Apoptosis was also assessed by flow cytometry following staining with fluorecein isothiocyanate (FITC)-annexin-V, as described previously [19 , 21 ]. Briefly, cells were washed in phosphate-buffered saline (PBS) and resuspended in 100 µl 1x binding buffer (10 mM HEPES/NaOH, pH 7.2, 140 mM NaCl, and 2.5 mM CaCl2), mixed with 2 µl FITC-conjugated annexin-V (Biosource, Montréal, Canada). Cells were gently vortexed and incubated for 15 min at 4°C in the dark. A volume of 400 µl binding buffer was added, and incubation was continued for an additional 15 min in the dark before fluorescein-activated cell sorter analysis (10,000 events) using a FACScan (Becton Dickinson, San Jose, CA).

Cell surface expression of cytoskeletal proteins by flow cytometry
Neutrophils (10x106 cells/mL RPMI-HEPES-P/S) were incubated at 37°C, 5% CO2, in the presence or absence of the indicated neutrophil agonists for 24 h. Cells were harvested in cold PBS and blocked with PBS containing 20% autologous serum for 30 min on ice. Cells were washed in PBS and incubated for 30 min on ice with 2 µg/ml mouse monoclonal anticytoskeletal antibodies (antipaxillin, antigelsolin, antivinculin, anti-{alpha}-tubulin, anti-ß-tubulin, antilamin B1, or the four antivimentin antibodies). Appropriate isotypic control antibodies were used to compare with the proteins of interest. Cells were washed in PBS and incubated for 30 min on ice with FITC-conjugated goat anti-mouse, FITC-conjugated rabbit anti-goat, or FITC-conjugated goat anti-rabbit antibodies. Cell surface expression was analyzed using a FACScan. Results are expressed as a Gmean fluorescence obtained by subtracting the GMean value of the isotypic control from the GMean value obtained with the anticytoskeletal antibody directed against the protein of interest, as the Gmean of the isotypic control varied between the fresh and apoptotic conditions. In other experiments, dual labeling was performed using FITC-annexin-V and antivimentin (V9) or antilamin B1, followed by their corresponding PE-conjugated antibodies described above.

In some experiments, as apoptotic cells lost CD16 expression, we sorted them from normal apoptotic neutrophils by negative immunomagnetic selection using anti-human CD16-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) as we have published previously [22 ].

Degradation of cytoskeletal proteins
Neutrophils (10x106 cells/mL) were incubated in the presence or absence of agonists for the indicated periods of time, and the expression of cytoskeletal proteins was performed by Western blot as published previously [16 , 18 ]. Briefly, cells were harvested for the preparation of cell lysates in 2x Laemmli’s sample buffer. Aliquots corresponding to 250,000 cells were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred from gel to polyvinylidene difluoride membranes. Nonspecific sites were blocked with 1–5% nonfat dry milk or 1% BSA (vinculin and lamin B1) in Tris-buffered saline-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) for 1 h at room temperature. Membranes were washed and incubated with anti-human cytoskeletal antibodies [mouse monoclonal antivimentin (1:2000 for clones H84, V4630, and V9 or 1:200 for clone 3B4); goat polyclonal antilamin B1 (1:500); or mouse monoclonal antivinculin (1:150)] overnight at 4°C. After several washes, membranes were incubated with HRP-labeled goat anti-mouse IgG or rabbit anti-goat antibodies (1:20,000 for vimentin and lamin B1; 1:50,000 for vinculin) for 1 h at room temperature in fresh blocking solution. Bands were revealed with the enhanced chemiluminescence-Western blotting detection system (Amersham Pharmacia Biotech Inc.).

Statistics
Statistical analysis was performed with SigmaStat for Windows Version 2.03 with a one-way ANOVA. Statistical significance was established at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intermediate filaments are expressed on the surface of apoptotic neutrophils
Knowing that some immune cells express cytoskeletal proteins on their cell surface [6 7 8 9 10 ], we investigated the possibility that apoptotic neutrophils express some cytoskeletal proteins on their cell surface. As illustrated (Fig. 1A ) among the different cytoskeletal proteins tested, the three MFAP paxillin, gelsolin, and vinculin, as well as the two major components of microtubules, {alpha}-tubulin and ß-tubulin, were not expressed on the cell surface of freshly isolated neutrophils or in spontaneous apoptosis (SA). In contrast, we found that the two INFIL vimentin and lamin B1 proteins were expressed on the cell surface of apoptotic neutrophils. As expected [16 , 18 , 20 ], the percentage of neutrophils undergoing SA ranged between 35% and 45% (Fig. 1B) . Cell surface expression of vimentin and lamin B1 on apoptotic cells was reproducible (Fig. 1C) .


Figure 1
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  		Figure 1. Cell surface expression of vimentin (Vim) and lamin B1 (Lam B1) on apoptotic neutrophils. Freshly isolated human neutrophils (10x106 cells/ml) were aged for 24 h or not (Fresh), and cell surface expression of the cytoskeletal proteins (A) was assessed by flow cytometry using the corresponding isotypic control antibody (dark cytograph) as described in Materials and Methods. Results are from one representative experiment of at least five. Arrows indicate cell surface expression (white area). Pax, Paxillin; Gel, gelsolin; Vinc, vinculin; {alpha}-Tub, {alpha}-tubulin; ß-Tub, ß-tubulin. The apoptotic rate (B) was assessed by cytology as described in Materials and Methods. (C) A compilation of the Gmeans (mean±SEM, n=5) obtained for the cell surface expression of vimentin (using the V9 clone) and lamin B1 in fresh and 24-h aged neutrophils. *, P < 0.05, by ANOVA. ND, Not detected.

 
The tail domain of vimentin is recognized on the surface of apoptotic neutrophils
Vimentin is composed of three domains: the amino-terminal domain (head domain), the central core (rod domain), and the carboxy-terminal domain (tail domain). This is illustrated in Figure 2A . Several antivimentin antibodies are known to target specific parts of the protein [8 , 23 ]. Taking advantage of the availability of antivimentin antibodies directed against the different domains, we next investigated which domain of the molecule was detected on apoptotic neutrophils, using four different antibodies. Using three antivimentin antibodies, H84, 3B4, and V9, which are directed against the head, rod, and tail domains of the molecule, respectively, and V4630, an antibody directed against a region overlapping between the rod and tail domains, we found that only V9 stained vimentin on the cell surface of apoptotic neutrophils, as assessed by flow cytometry (Fig. 2B) .


Figure 2
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  		Figure 2. Cell surface expression of vimentin is only detected with an antibody directed against the tail domain. The specificity of the different antivimentin antibodies used in this study is indicated on the illustrated structure of vimentin (A). (B) Freshly isolated neutrophils (10x106 cells/ml) were aged for 24 h (SA) or not (Fresh), and surface expression of vimentin was assessed as described in Materials and Methods. Note that staining was observed only with the V9 clone antivimentin antibody directed against the tail domain of the molecule (arrow). Results are from one representative experiment out of at least five.

 
Dual labeling of vimentin and lamin B1 on the cell surface of human neutrophils undergoing SA
To further support that apoptotic neutrophils express intermediate filament proteins on the cell surface, dual labeling was performed using FITC-annexin-V and antivimentin (V9; Fig. 3B ) or antilamin B1 (Fig. 3D) , followed by their corresponding PE-conjugated antibodies. As illustrated in Figure 3 , apoptotic cells, which are annexin-V-positive, express vimentin (Fig. 3B) and lamin B1 (Fig. 3D , upper/right quadrants). The basic levels of fluorescence are also illustrated in Figure 3 , A and C, for corresponding isotypic controls. Apoptotic cells are illustrated in the upper/right quadrants (FITC-annexin-V and cytoskeletal antigen-positive cells). To support that apoptotic neutrophils express intermediate filament proteins on their surface, we sorted apoptotic cells by negative immunomagnetic selection using anti-human CD16-coated magnetic beads, based on the fact that apoptotic neutrophils lose CD16 cell surface expression [24 ], and we stained them with the antilamin B1 anibody. As illustrated in the inset of Figure 3 , CD16– neutrophils express a high level of lamin B1 when compared with CD16+ cells.


Figure 3
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  		Figure 3. Dual labeling of vimentin and lamin B1 on the cell surface of human neutrophils undergoing SA. Freshly isolated human neutrophils (Fresh) were aged for 22–24 h (SA), and dual labeling was performed using FITC-annexin-V and antivimentin (V9; B) or antilamin B1 (D), followed by their corresponding PE-conjugated antibodies, described above as in Materials and Methods. The corresponding basic level of fluorescence (isotypic controls) is illustrated (A, for vimentin; C, for lamin B1). Numbers in quadrants are the percent of cells from the total population. Apoptotic cells are illustrated in the upper/right quadrants (FITC-annexin and antibody-positive cells). (Inset) Apoptotic cells were sorted by negative immunomagnetic selection using anti-human CD16-coated magnetic beads as described in Materials and Methods. Cell surface expression of lamin B1 was measured by flow cytometry, and the results are expressed as Gmean of fluorescence (numbers in brackets). Results are from one representative experiment out of three.

 
Degradation of vimentin in apoptotic neutrophils
As only the V9 antivimentin antibody among the four that were tested detected vimentin on the cell surface of apoptotic neutrophils, we then investigated the intracellular degradation pattern of vimentin obtained with V9 antibody in comparison with the three other antivimentin antibodies. We previously demonstrated that the plant lectin VAA-I was a potent inducer of neutrophil apoptosis, which induced degradation of vimentin via caspases [16 , 18 ]. We therefore studied the degradation of vimentin in SA and in VAA-I-induced apoptosis in parallel. As illustrated in Figure 4 , all antibodies recognized the native form of vimentin, as well as different fragments known to appear over time when cells undergo apoptosis [19 ]. The fragments are not related to the sizes of head, rod, or tail fragments of vimentin. It is interesting that addition of the pan caspase inhibitor z-VAD-fmk reversed such degradation, whether or not neutrophils underwent SA or if apoptosis was accelerated by VAA-I. It is clear that the caspase inhibitor partially inhibits the cleavage, suggesting that proteases other than caspases are involved. As others [9 ], we observed some fragments of vimentin in fresh cells, suggesting that proteolysis occurred during cell isolation.


Figure 4
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  		Figure 4. Comparison of the fragments of vimentin detected by the different antivimentin antibodies and role of caspases in the degradation of intracellular vimentin in spontaneous or VAA-I-induced neutrophil apoptosis. Neutrophils (10x106 cells/ml) were incubated with or without (SA) 1000 ng/ml VAA-I for 24 h in the presence (+) or absence (–) of 50 µM pan-caspase inhibitor (Z-VAD). The antivimentin clones are illustrated under each blot. Results are from one representative experiment out of at least three. Membranes were stained by Coomassie blue at the end of the experiments to illustrate equal loading. F, Fresh.

 
Role of caspases in the cell surface expression of vimentin and lamin B1
As degradation of vimentin occurred during SA, addition of caspase inhibitors was previously found to inhibit SA [16 , 18 ], and degradation of this INFIL protein occurred in human neutrophils, we next investigated the potential role(s) of caspases in the cell surface expression of vimentin and lamin B1, another INFIL protein. As illustrated in Figure 5 , addition of the pan-caspase inhibitor z-VAD-fmk significantly decreased the cell surface expression of both proteins. The apoptotic rate (means±SEM, n=3) diminished from 56.3 ± 2.7% to 30.3 ± 9% when z-VAD-fmk was added.


Figure 5
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  		Figure 5. Potential role of caspases for cell surface expression of vimentin and lamin B1 in SA. Neutrophils (10x106 cells/ml) were incubated for 24 h in the presence (+) or absence (–) of 50 µM pan-caspase inhibitor (z-VAD), and cell surface expression of vimentin (A) and lamin B1 (B) was assessed in parallel with the same blood donor as described in Materials and Methods. The corresponding apoptotic rate is illustrated (C), as assessed by measuring the number of FITC-annexin-V-positive cells. Results (GMean) are means ± SEM (n=3). *, P < 0.05, by ANOVA.

 
Cell surface expression of vimentin and lamin B1 when neutrophil apoptosis is delayed or suppressed
To further associate cell surface expression of vimentin and lamin B1 with a signal that induced or accelerated neutrophil apoptosis, we decided to investigate intracellular degradation and cell surface expression of these two proteins when apoptosis was delayed or suppressed by different neutrophil agonists. As illustrated in Figure 6 , cell surface expression of vimentin and lamin B1 was only observed in SA and not (or rarely, barely detectable) when apoptosis was delayed by G-CSG, GM-CSF, LPS, or IL-4. The apoptotic rate was monitored by cytology and/or flow cytometry after staining with FITC-annexin V and was <45% for SA when the antiapoptotic agents were added to the culture.


Figure 6
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  		Figure 6. Cell surface expression of vimentin and lamin B1 when neutrophil apoptosis is delayed. Neutrophils (10x106 cells/ml) were incubated in the presence of buffer (SA), GM-CSF (65 ng/ml), G-CSF (50 ng/ml), LPS (1 µg/ml), or IL-4 (100 ng/ml) for 24 h. Cell surface expression was assessed by flow cytometry as described in Materials and Methods. Results are from one representative experiment out of at least four. Arrows, Cell surface expression of vimentin and lamin B1 in SA. Note the striking decrease in protein expression when apoptosis is delayed with the antiapoptotic agents.

 
Intracellular expression of vimentin and lamin B1 proteins during neutrophil apoptosis
Next, we decided to study the expression of vimentin and lamin B1 when neutrophil apoptosis was delayed, as compared with SA, and to verify the role of caspases in the intracellular expression of these two proteins. As illustrated in Figure 7 , expression of the native forms of vimentin and lamin B1 was down-regulated drastically in SA when compared with delayed apoptosis. As expected [18 ], expression of the MFAP vinculin remained relatively stable in the presence or absence (SA) of the antiapoptotic molecules. Knowing that degradation of vimentin occurs by a caspase-dependent mechanism in neutrophils [18 ], we investigated the role of caspases in the degradation of lamin B1, in parallel with vimentin in SA and in VAA-I-induced apoptosis. As illustrated in Figure 7 , addition of the pan-caspase inhibitor (z-VAD) reversed the cleavage of vimentin and lamin B1 in SA and in VAA-I-induced apoptosis. The level of expression of vimentin and lamin B1 in the presence of z-VAD in SA or in VAA-induced apoptosis was similar. However, the levels of vimentin and lamin B1 were, under all conditions, weaker than the basal levels observed in fresh cells, suggesting that G-CSF, GM-CSF, LPS, and IL-4 did not induce de novo protein synthesis of these proteins but rather prevented their loss of expression. To investigate the potential role of general protein synthesis in the process, we next performed experiments with CHX. As illustrated in Figure 8 , addition of CHX to the culture during SA or when cells were incubated with the antiapoptotic agents G-CSF, GM-CSF, LPS, or IL-4 was found to reduce the levels of expression of vimentin and lamin B1, suggesting that protein synthesis is involved in the prevention of degradation of vimentin and lamin B1.


Figure 7
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  		Figure 7. Intracellular expression of intermediate filaments when neutrophil apoptosis is delayed or accelerated. Neutrophils (10x106 cells/ml) were incubated in the presence of buffer (SA) or the antiapoptotic agents G-CSF (50 ng/ml), GM-CSF (65 ng/ml), LPS (1 µg/ml), or IL-4 (100 ng/ml) or with the proapoptotic agent VAA-I (1000 ng/ml) in the presence (+) or absence (–) of the pan-caspase inhibitor z-VAD-fmk. Cell lysates were prepared, and Western blot experiments were performed as described in Materials and Methods using the V9 antivimentin, the antilamin B1, or the antivinculin antibodies. Results are from one representative experiment out of at least three. Note that cleavage of vimentin and lamin B1 is partially reversed by z-VAD-fmk but that the level of protein expression did not return to the basal levels observed in fresh cells. Vinculin was used to illustrate equal loading, as this cytoskeletal protein is not cleaved during SA or VAA-I-induced neutrophil apoptosis [18 ].

 

Figure 8
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  		Figure 8. The loss of intracellular intermediate filament expression is prevented by a de novo protein synthesis-dependent mechanism. Neutrophils (10x106 cells/ml) were incubated for 24 h with buffer (SA) or the antiapoptotic agents in the presence (+) or absence (–) of CHX (2 µg/ml). Western blot experiments were performed as described in Materials and Methods. Results are from one representative experiment out of at least three. Note that for all antiapoptotic agents, the levels of expression of vimentin and lamin B1 are decreased in the presence of CHX. The corresponding apoptotic rates, as assessed by cytology, are illustrated in the table on the right.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study demonstrating that human neutrophils can express cytoskeletal proteins on their cell surface. It is interesting that among the seven different proteins we tested, namely, paxillin, gelsolin, vinculin, {alpha}-tubulin, ß-tubulin, vimentin, and lamin B1, only the two INFIL proteins, vimentin and lamin B1, are expressed on the cell surface, suggesting that this is a specific mechanism. Moreover, these proteins are expressed when neutrophils are in apoptosis.

As demonstrated in the present study, vinculin is not expressed on the surface of apoptotic neutrophils. This is in agreement with our previous work, indicating that vinculin is not cleaved during induction of neutrophil apoptosis by VAA-I [18 ], tributyltin [17 ], or methylmercury [19 ]. However, Melendez et al. [23 ] have shown that vinculin expression was decreased (a decrease of 33%) during related adhesion focal tyrosine kinase/pyk2-induced apoptosis in cardiomyocytes. In contrast to this latter study, Harrington et al. [25 ] demonstrated that vinculin was not cleaved during epithelial cell apoptosis induced by adenosine. In another study [26 ], expression of vinculin was not modulated by interferon-{gamma} (IFN-{gamma})-induced apoptosis in the human oropharyngeal epidermoid carcinoma KB cell line. These studies concur with our results, indicating that vinculin remains stable, whether apoptosis is induced or delayed [17 18 19 ]. This further demonstrates that cytoskeletal proteins are not all processed in the same way during the apoptotic program. Vinculin is a structural component of focal adhesions, and the reason why it is not cleaved during neutrophil apoptosis is unclear but may be related to the fact that potential cleavage sites are protected by an unknown mechanism. Similar reasoning may also be applied to {alpha}- and ß-tubulin, as these proteins are not cleaved [18 ] and are not expressed on the surface of apoptotic neutrophils (this report). However, cleavage of a particular cytoskeletal protein does not necessarily result in its expression on the apoptotic cell surface; this is the case for paxillin and gelsolin, which are cleaved by caspases [16 , 18 ] but are not detected on the surface of apoptotic neutrophils (this report).

INFIL are known to be cleaved into many fragments by caspases during apoptosis. It is not clear if cell surface expression of vimentin and lamin B1 originates from particular fragmentation products by caspases (or other proteases) or if another mechanism is involved. In various cell types, vimentin is cleaved by caspase-3, -6, -8, and -9 [27 28 29 30 ], whereas lamin B1 has been found to be cleaved by at least caspase-3 and -6 [31 , 32 ]. The role of caspases in the intracellular degradation of vimentin and lamin B1 in neutrophils is clear, as treatment with the pan-caspase inhibitor (z-VAD-fmk) partially reversed their cleavage. However, although we demonstrated that addition of z-VAD-fmk reversed the cell surface expression of these two proteins, a direct link with caspase activity in this process is less evident, as addition of this inhibitor also reversed apoptosis, suggesting that cell surface expression of INFIL is also diminished. What is clear is that when apoptosis is suppressed or delayed by G-CSF, GM-CSF, LPS, or IL-4, there are no (or few) INFIL proteins on the neutrophil cell surface. It is of note that even in the presence of the antiapoptotic agents, cleaved vimentin and lamin B1 are evident, and in some cases, the levels of these cleaved products are as high as in the SA samples. This would argue that the antiapoptotic agents induce synthesis of these proteins, but cleavage still occurs. It is of interest that all of these antiapoptotic agents are known to, themselves, induce de novo protein synthesis in neutrophils [33 34 35 ]. In addition, the role of protein synthesis in delaying neutrophil apoptosis has been demonstrated for G-CSF and GM-CSF [33 ] and for LPS [34 ]. This is not clear for IL-4 [35 ]. The cytokine IL-15, like IL-4, is a CD132-dependent cytokine and inhibits the activity of caspase-3 and -8, resulting in a decreased ability to cleave vimentin [36 ]. Whether this occurs with IL-4 remains to be determined. This raises the possibility that G-CSF, GM-CSF, and LPS prevent the loss of vimentin and lamin B1 expression by inactivating some caspases. It is likely that some proteins requiring continuous synthesis are those that govern caspase activity by an as-yet unknown mechanism. It is noteworthy that the role of protein synthesis in suppression of neutrophil apoptosis is not limited to LPS or cytokines, as dexamethasone also reportedly inhibited apoptosis via continuous protein synthesis [37 ]. The fact that G-CSF and GM-CSF inhibited neutrophil apoptosis via a protein synthesis-dependent as well as a protein synthesis-independent mechanism indicates the complex mode of action of a given molecule inhibiting neutrophil apoptosis [33 ].

Antivimentin and antilamin B1 surface staining is not the result of secondary necrosis, as all cytoskeletal proteins tested (other than INFIL) are not detected on the neutrophil cell surface by flow cytometry. Moreover, among four different antivimentin antibodies, only one detected vimentin on the cell surface of apoptotic neutrophils. In addition, cell viability of neutrophils was systematically monitored by trypan blue exclusion. Cell surface expression of vimentin and lamin B1 appeared to be a relatively late event during neutrophil apoptosis, as 14-h aged cells did not significantly express the protein on their surface (data not shown). Paradoxically, it is known that INFIL are cleaved relatively early during the apoptotic process (few hours), as compared with other cytoskeletal proteins [38 ]. To ensure that cytoskeletal proteins other than INFIL are not expressed on the neutrophil surface after 24 h, we verified cell surface expression of paxillin, gelsolin, and vinculin on 44-h aged neutrophils and did not observe any of these MFAP, whereas vimentin and lamin B1 were still detected, and cells were all negative for trypan blue staining (data not shown).

Recently, it has been demonstrated that vimentin is citrullinated during macrophage cell death, suggesting that some of its arginine residues are deiminated to citrulline residues [39 ]. A few studies have demonstrated that citrullinated vimentin is found in the serum of rheumatoid arthritis patients [3 , 40 ]. Peptidylarginine deiminase (PADI) is an enzyme involved in post-translational modification of peptidylarginine to citrulline in the presence of calcium ions and can change conformation and properties of proteins after their citrullination. Expression of PADI was observed in neutrophils from the synovia of rheumatoid arthritis patients [41 , 42 ]. In the future, it will be interesting to answer whether vimentin (and lamin B1), detected on the surface of apoptotic neutrophils, is citrullinated.

Neutrophils can generate tyrosyl radicals after activation with phorbol 12-myristate 13-acetate (PMA), IFN-{gamma}, or tumor necrosis factor {alpha} [43 ]. Those radicals act in an autocrine manner by cross-linking to endogenous proteins exposed to the medium. As shown by confocal microscopy, tyrosylated proteins were initially located in patches on the cell surface, internalized, and subsequently degraded. It is interesting that many tyrosylated proteins in neutrophils, including vimentin, have been identified. It was demonstrated that upon cell activation, vimentin was tyrosylated slightly, and the carboxy-terminal part of the protein was phosphorylated at Thr-425. The authors suggested that vimentin phosphorylation was a requirement for its translocation to the plasma membrane. However, they did not demonstrate the presence of vimentin on the neutrophil cell surface.

Using protein kinase C modulators such as PMA and okadaic acid, it was shown that secretion and surface expression of vimentin were regulated by phosphorylation events in macrophages and in a population of endothelial cells [9 , 10 ]. The implication of vimentin phosphorylation for its cell surface expression on apoptotic neutrophils remains to be investigated. However, activation of neutrophils by the antiapoptotic agents GM-CSF, G-CSF, LPS, and IL-4, which are known to induce phosphorylation events in neutrophils, maintained intracellular INFIL expression, probably via de novo protein synthesis, but did not lead to vimentin and lamin B1 cell surface expression. As assessed by immunoblotting, we failed to detect the presence of vimentin and lamin B1 in the extracellular milieu after neutrophil activation with these agents (data not shown).

Antilamin B autoantibodies are detected in many autoimmune diseases, including autoimmune liver diseases, rheumatoid arthritis, and systemic lupus erythematosus (SLE) [44 ]. Dieude et al. [5 ] have demonstrated that antilamin B autoantibodies from SLE patients do not bind to the surface of apoptotic blebs in Jurkat T cells and human umbilical vein endothelial cells. Using confocal microscopy, they demonstrated that lamin B was not present on the surface of these blebs but rather remains buried within the blebs, rendering them inaccessible to external antilamin B antibodies. However, unlike these cells, neutrophils do not form typical blebs when they undergo apoptosis.

Boilard et al. [8 ] determined the specificity of various antivimentin antibodies. In their study, they determined that the goat polyclonal V4630 antivimentin antibody recognized the rod and tail domain of the protein. They found that rod and tail domains of vimentin were exposed on the cell surface of human apoptotic T lymphocytes, as the V9 and V4630 antibodies recognized the protein on the cell surface. Herein, we demonstrated that only the antibody directed against the tail domain of vimentin-stained apoptotic neutrophils, suggesting that unlike T lymphocytes, apoptotic neutrophils exposed only a part of the tail domain of vimentin on the surface.

Although we only detected the presence of INFIL on the surface of apoptotic neutrophils, we cannot rule out the possibility that some of the proteins we tested (as well as other cytoskeletal proteins) are expressed on the cell surface, as it is possible that the antibodies we used in this study may not recognize particular epitopes on the neutrophil surface. Although the antibodies used to detect gelsolin, paxillin, vimentin (clone V9), and lamin B1 are all directed against the carboxy-terminal end of proteins, only those directed against vimentin and lamin B1 stained apoptotic neutrophils.

Xu et al. [10 ] suggested that secreted vimentin could play a role in mediating the movement of circulating blood cells across the endothelium, a process in which activated macrophages and activated platelets participate. Knowing that cell surface and secreted vimentin are involved in various biological functions, it is tempting to speculate that the protein exposed on the surface of apoptotic neutrophils could serve as an "eat me" signal, helping phagocytes to recognize and ingest apoptotic neutrophils, but this remains to be demonstrated. In addition, lamin B1 could also be involved in this process. As previously mentioned, the antilamin B1 antibody we used is also directed against the carboxy-terminal part of the protein. However, in contrast to vimentin, antibodies directed against different parts of lamin B1 are not well characterized. In addition to the potential role in apoptotic neutrophil elimination, neutrophils may represent an important source of cytoskeletal autoantigens for the development of autoantibodies directed against cytoskeletal proteins, normally sequestered inside the cell, at least for the two INFIL proteins vimentin and lamin B1.

The results of this study establish for the first time that apoptotic neutrophils express some cytoskeletal proteins on their surface. Among different proteins that we have tested, including members of the three classes of cytoskeletal filaments, namely, vinculin, paxillin, and gelsolin (microfilaments), vimentin and lamin B1 (INFIL), and {alpha}- and ß-tubulin (microtubules), only those of the INFIL are detected on apoptotic neutrophils.


   ACKNOWLEDGEMENTS
 
This study was supported by Natural Sciences and Engineering Research Council of Canada (NSERC). E. M. holds a Ph.D. award from Fonds de la Recherche en santé du Québec (FRSQ), and D. G. is a scholar from FRSQ. We thank Mary Gregory for reading this manuscript.

Received April 12, 2005; revised October 26, 2005; accepted November 1, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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