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(Journal of Leukocyte Biology. 2000;68:845-853.)
© 2000 by Society for Leukocyte Biology

Activation of human neutrophils by the plant lectin Viscum album agglutinin-I: modulation of de novo protein synthesis and evidence that caspases are involved in induction of apoptosis

Anik Savoie*, Valérie Lavastre*, Martin Pelletier*, Tibor Hajto{dagger}, Katarina Hostanska{dagger} and Denis Girard*


* INRS-Institut Armand-Frappier/Santé Humaine, Université du Québec, Canada; and
{dagger} Department of Internal Medicine, University Hospital Zürich, Switzerland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The plant lectin Viscum album agglutinin-I (VAA-I) was recently found to modulate protein synthesis and to induce apoptosis in various cells of immune origin. We found that VAA-I induces de novo protein synthesis of metabolically 35S-labeled human neutrophils when used at low concentrations (<100 ng/mL) but acts as an inhibitor at higher concentrations. Using both flow cytometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining) approaches, we found that VAA-I could not modulate neutrophil apoptosis at low concentrations but could induce it in >98% of cells at 500 and 1000 ng/mL. VAA-I was also found to reverse the delaying effect of GM-CSF on neutrophil apoptosis and to inhibit GM-CSF-induced de novo protein synthesis. In contrast to GM-CSF, VAA-I does not induce tyrosine phosphorylation by itself and does not alter the GM-CSF-induced response. Among the inhibitors used, genistein, pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine HA-1077, and z-VAD-FMK, only the latter (inhibitor of caspases-1, -3, -4, and -7) was found to inhibit VAA-I-induced neutrophil apoptosis as the percentage of apoptotic cells decrease from 98 ± 1.3 to 54 ± 3.2% (n =4). Furthermore, we confirm that caspases are involved in VAA-I-induced neutrophil apoptosis as we have observed the fragmentation of the cytoskeletal gelsolin protein that is known to be caspase-3-dependent. Such degradation was reversed by the z-VAD-FMK inhibitor. We conclude that induction of neutrophil apoptosis by VAA-I is a caspase-dependent mechanism that does not involve tyrosine phosphorylation events, G-proteins, PKCs, and PLA2. In addition, we conclude that at least caspase-3 is involved. Correlation between VAA-I-induced neutrophil apoptosis and VAA-I-induced inhibition of de novo protein synthesis is discussed.

Key Words: signal transduction inhibitors • cytology • flow cytometry • gelsolin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are important cells of the immune system that are involved in host defense. Except for erythrocytes and platelets, neutrophils are the most abundant cells in the circulation and have a limited lifespan (half-life of ~12 h in the circulation). Cell turnover must therefore be under strict control. In this sense, neutrophils are known to spontaneously undergo apoptosis without addition of special reagents in the milieu. Normally, when freshly isolated human neutrophils are incubated for 24 h (37°C, 5% CO2) in RPMI-1640 supplemented with fetal calf serum or autologous serum (ranging from 5–10%), about 30–60% of cells will be in apoptosis, depending on the donor [1 2 3 4 ]. Because they have been established as terminally differentiated non-dividing cells, neutrophils were erroneously characterized as cells with little activity, namely with respect to their capacity to synthesize proteins. However, it is becoming increasingly clear that these cells are more capable of protein synthesis than previously thought [3 4 5 6 7 8 9 10 ]. Various cytokines are among proteins that are synthesized by neutrophils and this indicates that these cells have the potential to perform biological activities in both afferent and efferent arms of the immune response. Among these cytokines, some are potent modulators of neutrophil apoptosis such as interleukin-8 (IL-8) and tumor necrosis factor {alpha} (TNF-{alpha}) [5 , 6 , 9 , 11 ]. Now it is generally accepted that neutrophils play roles in host response that extend well beyond their capacities for phagocytosis and releasing cytotoxic compounds [12 ]. The importance of studying and discovering molecules that can modulate the neutrophil apoptotic rate resides in the fact that clearance of apoptotic neutrophils by cells such as macrophages leads to the resolution of inflammation [13 14 15 ]. In addition to this, deregulation of normal cell turnover via modulation of apoptosis may lead to cancer or autoimmunity.

Viscum album agglutinin-I (VAA-I) is a galactoside-specific plant lectin that possesses a molecular mass of 63 kDa [16 , 17 ]. It consists of two distinct subunits, the A chain (29 kDa) and the B chain (34 kDa). The A chain confers the protein synthesis inhibitory property to the VAA-I molecule by acting as a ribosome-inactivating agent. This is due to RNA-glycosidase activity that inhibits N-glycosilation of a single adenine within a universally conserved GAGA sequence on the 28S rRNA (at position 4324 in rat liver cells) [18 , 19 ]. The B chain allows the VAA-I molecule to bind to terminal galactoside residues on the membrane of various cells. VAA-I belongs to the family of type II ribosome-inactivating proteins including abrin, modeccin, and ricin. VAA-I was recently found to act as an immunomodulator [16 17 18 19 20 21 22 23 24 ]. This lectin is known to induce pro-inflammatory cytokine expression at both gene and protein levels in human peripheral blood mononuclear cells [17 ] and to induce apoptosis in human lymphocytes, monocytes, monocytic THP-1 cells, and murine thymocytes [16 ]. The recombinant form of the lectin (rVAA) was recently found to augment the secretion of IL-12 and to potentiate the cytokine-induced natural killer (NK) cell activation [22 ].

It was previously demonstrated, by flow cytometry, that fluorescein isothiocyanate (FITC)-conjugated VAA-I binds to monocytes and granulocytes with higher affinity than on lymphocytes [17 ]. Studies dealing with VAA-I/neutrophil interactions are limited and data reveal that VAA-I induces neutrophil aggregation, O2- and H2O2 production, and phagocytosis [25 26 27 ]. Because of the potential therapeutic use of VAA-I, at least in cancer [23 , 24 ], and its great capacity to modulate the immune response, it is important to better understand the interaction of VAA-I and human neutrophils for therapeutic purposes. Knowing that these cells are at the basis of an inflammatory reaction and that they can produce various distinct proteins, it is also important to better understand how VAA-I can alter both de novo protein synthesis and the neutrophil apoptotic rate.

In this study, we report that VAA-I can up-regulate or down-regulate de novo protein synthesis of human neutrophils depending on the concentration used and that its ability to inhibit de novo protein synthesis correlates well with its ability to induce neutrophil apoptosis. Moreover, we bring evidence that VAA-I-induced-neutrophil apoptosis is a caspase-dependent mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and agonists
Cycloheximide (CHX), genistein, pertussis toxin (50 ng/mL), staurosporine (100 nM), manoalide (1 µM), H7 (50 mM), calphostin C (1.3 µM), BPB, 4-bromophenacyl bromide (100 µM), quinacrine (200 µM), and HA-1077 (3.2 µM) were purchased from Sigma. The caspases-1, -3, -4, and -7 inhibitor carbobenzoxy-valine-alanine-aspartate-fluoromethyl ketone (z-VAD-FMK) was purchased from Calbiochem (La Jolla, CA). Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; specific activity of 9 x 106 U/mg) was a gift from the Genetics Institute, Boston, MA. This latter cytokine was used at 65 ng/mL as previously published [3 , 4 , 10 , 28 ]. Methylmercury chloride (II) was purchased from Alfa Aesar (Ward Hill, MA) and was used as an inductor of cell necrosis.

Isolation of VAA-I
The plant lectin VAA-I derived from V. album was isolated and purified as previously published, and endotoxin contamination was measured by Limulus amebocyte lysate (LAL) assay and was <5 pg/mL [21 ]. The purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; data not shown). As other agents used in the study, the lectin was present in the culture for the indicated period of incubation. Stimulation of cells was performed in 96-well plates, without agitation, at 37°C, 5%CO2, for the de novo protein synthesis assay and in 24-well plates for apoptosis [3 , 4 , 9 , 10 ]; whereas they were incubated in 5 mL polystyrene tubes in a water bath (37°C), under light agitation, for the tyrosine phosphorylation assay [9 ].

Neutrophil isolation
Cells were isolated from venous blood of healthy volunteers by centrifugation over Ficoll-Hypaque as described previously [3 , 4 , 9 , 10 , 28 ]. Blood donations were obtained from informed and consenting individuals according to institutionally approved procedures. Cell viability (>99%) was monitored by Trypan blue exclusion, and the purity (>98%) was verified by cytology from cytospin preparations followed by Diff-Quick staining [3 , 4 , 9 ]. Treatment of neutrophils with VAA-I (0.1 through 1000 ng/mL in RPMI-1640 supplemented with 10% autologous serum) did not induce cell necrosis in contrast to methylmercury after 24 h of treatment (Fig. 1 ).



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  		Figure 1. VAA-I and neutrophil cytotoxicity. Freshly isolated human cells were incubated in the presence of increasing concentrations of VAA-I (0.1–1000 ng/mL) or with methylmercury chloride (CH3HgCl), a pesticide known to induce cell necrosis, and cell viability was monitored for up to 24 h as described in Materials and Methods. (A) cells incubated with 1000 ng/mL VAA-I for 24 h; (B) cells incubated with 10-4 M CH3HgCl for 24 h. Note that CH3HgCl induces necrosis as the trypan blue dye penetrates into cells but not VAA-I. Results are representative of at least 10 experiments conducted with different blood donors.

 
Metabolic labeling and de novo protein synthesis assay
Cells (10 x 106 cells/mL in RPMI-1640 supplemented with 1% autologous serum) were metabolically labeled with 125 µCi of the Redivue Pro-Mix L-[35S] in vitro cell labeling mix (Amersham Biopharmacia) in the presence or absence of agonists, as indicated in the figure legends, for 22 h as previously published [3 , 4 , 9 , 10 ]. Cell lysates were prepared, and SDS-PAGE was performed. An equivalent of 1 x 106 cells was loaded per well (to compare appropriately de novo protein synthesis) [3 , 4 , 9 , 10 ]. After electrophoresis, gels were stained with Coomassie blue (to verify equivalent loading), dried, and exposed with Kodak film X-OMAT-RA at -80°C for 1–3 days.

Assessment of neutrophil apoptosis by cytology and by flow cytometry
Freshly isolated human neutrophils (100 µL of a 10 x 106 cells/mL suspension in RPMI-1640 supplemented with 10% autologous serum) were incubated for indicated times in the presence or absence of VAA-I or appropriate controls as indicated in the figure legends, and apoptosis was evaluated by cytology and flow cytometry. Cytocentrifuge preparations of neutrophils were performed with a Cyto-tek® centrifuge (Miles Scientific) essentially as previously described and were stained with a Diff-Quick staining kit (Baxter, FL), according to the manufacturer’s instructions [3 , 4 , 9 , 10 ]. Cells were examined by light microscopy at x400 final magnification, and apoptotic neutrophils were defined as cells containing one or more characteristic darkly stained pyknotic nuclei. An ocular containing a 10 x 10-square grill was used to count at least five different fields (>100 cells) for assessment of apoptotic cells. Results were expressed as percentage of apoptotic cells. For the flow cytometric procedure, apoptosis was investigated using FITC-Annexin-V/PI labeling as previously published [29 , 30 ]. Ten thousand cells were analyzed by FACS Calibur (Becton-Dickinson, San Jose, CA) using CellQuest program [17 ]. The number of late apoptotic and necrotic cells were subtracted in order to evaluate the percentage of apoptotic cells.

Tyrosine phosphorylation
Neutrophils (100 µL of 40 x 106 cells/mL in RPMI-1640) were incubated for 1, 5, 15, or 30 min at 37°C with buffer alone, GM-CSF (65 ng/mL), or increasing concentration of VAA-I (1–1000 ng/mL) in a final volume of 120 µL. Reactions were stopped by adding 120 µL of a 2x Laemmli sample buffer as we have detailed elsewhere [9 ]. Aliquots corresponding to 1 x 106 cells were loaded onto 10% SDS-PAGE and transferred from gel to PVDF membranes (Millipore, Bedford, MA) according to Towbin et al. [31 ]. Nonspecific sites were blocked with 2% gelatin in TBS-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) for 1 h at 37°C. The monoclonal anti-phosphotyrosine UB 05-321 (1:5000) was then incubated with membranes for 1 h at 37°C followed by washes, and incubated with a horseradish peroxidase-labeled sheep anti-mouse IgG (1:15,000, Bio/Can) for 1 h at 37°C in fresh blocking solution. Membranes were washed three times with TBS-Tween, and phosphotyrosine bands were revealed with the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham, Pharmacia Biotech). Protein loading was verified by staining the membranes with Coomassie blue at the end of the experiments.

Fragmentation of gelsolin
The degradation of gelsolin into an ~41-kDa fragment was recently found to be dependent on caspase-3 activity during neutrophil apoptosis [32 ]. We next verify whether VAA-I can induce such fragmentation in VAA-I-induced neutrophil apoptosis. Neutrophils (106 cells/mL in 24-well plate) were incubated with or without 500 ng/mL VAA-I for 24 h and then harvested for the preparation of cell lysates in Laemmli sample buffer. In other wells, cells were pre-incubated 1 h with the diluent or 50 µM z-VAD-FMK before an additional 23 h of incubation (total period of 24 h). Aliquots corresponding to 1 x 106 cells were loaded onto 10% SDS-PAGE and transferred from gel to PVDF membranes as for the tyrosine phosphorylation assay. Nonspecific sites were blocked with 1% bovine serum albumin (BSA) in TBS-Tween (25 mM Tris-HCl, pH 7.8, 190 mM NaCl, 0.15% Tween-20) for 1 h at 37°C. Membranes were incubated with monoclonal anti-human gelsolin (Sigma, clone GS-2C4) in a final dilution of 1:500 for 1 h at 37°C followed by washes, and incubated with a horseradish peroxidase-labeled sheep anti-mouse IgG (1:15,000; Bio/Can) for 1 h at 37°C in fresh blocking solution. The other steps were performed exactly as described for the tyrosine phosphorylation assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Modulation of de novo neutrophil protein synthesis by VAA-I
Results illustrated in Figure 2A demonstrate that VAA-I can induce newly synthesized polypeptides (from the intracellular fractions) at low concentrations (1 and 100 ng/mL VAA-I in lanes 1 and 2 , respectively), whereas it acts as an inhibitor at higher concentrations (1000 ng/mL, lane 3) using intracellular fractions. Figure 2B illustrates that VAA-I can also modulate newly synthesized polypeptides released in the external milieu. However, in contrast to CHX, which drastically inhibits de novo protein synthesis as seen in both extracellular (Fig. 2B) and intracellular (Fig. 2A) fractions, the highest concentration of 1000 ng/mL VAA-I does not markedly influence the pattern of the proteins detected from the extracellular fractions (Fig. 2B) . Protein lysates prepared from extracellular fractions of neutrophils treated with low concentrations of VAA-I revealed a slight increase of newly synthesized polypeptides when compared with untreated cells but not as strongly as lysates from the intracellular fraction (data not shown). Treatments of cells with GM-CSF gave the expected results as we observed a marked increase in protein synthesis, particularly near the 23-kDa region, which is probably IL-1Ra (see arrow) as previously documented [33 ].



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  		Figure 2. Modulation of de novo protein synthesis by VAA-I. Freshly isolated human neutrophils were metabolically labeled as described in Materials and Methods and incubated for 22 h in the presence or absence of stimuli. Intracellular and extracellular fractions were prepared and then run by SDS-PAGE. (A) cell lysates prepared from cells treated with 1, 100, or 1000 ng/mL VAA-I (lanes 1, 2, and 3, respectively), 65 ng/mL GM-CSF (lane 4), 10 µg/mL cycloheximide (lane 5), or buffer (lane 6). (B) samples were prepared from the external milieu of cells treated with buffer (C, control), 65 ng/mL GM-CSF (GM), 10 µg/mL cycloheximide (CHX), or 1000 ng/mL VAA-I. Results are one representative experiment out of at least 15 different experiments. Note that, in panel A, de novo synthesis of different polypeptides is increased by treatment with low concentrations of VAA-I (lanes 1 and 2) and GM-CSF (lane 4) when compared with untreated cells (small arrows in lane 6). Intensity of the bands (arrows) was quantified with the Bio-Rad Multi-AnalystTM/PC Version 1.1 program. Values are from top to bottom. Lane 1: 1.98, 2.51, 2.71, 2.71, 2.54, 3.53, 1.99. Lane 2: 1.00, 1.43, 1.48, 1.37, 1.59, 2.51, 1.46. Lane 3: 0.73, 0.83, 0.79, 0.62, 0.81, 1.30, 0.55. Lane 4: 1.02, 1.87, 1.52, 1.63, 2.09, 2.63, 0.97.

 
Modulation of neutrophil apoptotic rate by VAA-I
Staining of cells with FITC/Annexin-V and propidium iodide (PI) enabled us to separate cells in early apoptotic or late apoptotic/necrosis categories. Treatment of cells with 1, 10, or 100 ng/mL VAA-I for 24 h did not alter normal apoptotic rate (Fig. 3A ). Similarly, the number of cells categorized as late apoptotic/necrotic was quite stable. However, dramatic changes were observed when neutrophils were treated with 1000 ng/mL VAA-I. We observed that only 10 ± 4% of cells were considered as early apoptotic, whereas 90 ± 5% were in the late apoptotic/necrotic category (Fig. 3A) . When apoptotic cells were evaluated by cytology with the Diff-Quick staining procedure, we confirmed that VAA-I did not influence the apoptotic rate at low concentrations (Fig. 3B) . Incubation of cells with 65 ng/mL GM-CSF, which is known to delay neutrophil apoptosis [3 , 4 , 9 , 34 ], gave the expected results because only 9 ± 2.5% of cells were in apoptosis (data not illustrated). However, we found that virtually all cells (99±0.4%) were in apoptosis when treated with 1000 ng/mL VAA-I. Representative results are illustrated in the inset of Figure 3B . As illustrated in the bottom part of the inset, these cells were not necrotic but rather apoptotic. Necrosis is easy to evaluate by this approach because the cell permeability is altered and cells do not appeared delimited ("round"). Rather, atypical blue smears appeared after treatment with the necrotic agent methylmercury chloride (data not shown). Although the rate of apoptosis goes from minimal to 100% with high concentrations of VAA-I after 24 h of incubation at the concentrations used, we have observed in two separate experiments that about 60 and 80% of cells were apoptotic after a 12-h treatment with 175 or 250 ng/mL, respectively, whereas less than 20% of untreated cells were apoptotic.



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  		Figure 3. Induction of neutrophil apoptosis by high concentration of VAA-I. Freshly isolated human neutrophils (10 x106 cells/mL) were incubated for 22 h in the absence (0) or presence of increasing concentration of VAA-I (1–1000 ng/mL) and the number of cells in apoptosis were assessed by flow cytometry using FITC-Annexin V/PI staining (A) or by cytology from cytocentrifuge preparations (B) as described in Materials and Methods. (A) results are means ± SEM (n =3), *P < 0.05 vs. control (early apoptosis); **P < 0.05 vs. control (late apoptosis/necrotic) both by Student’s t test. Inset, about 42% of untreated cells are in early apoptosis vs. 7.8% for VAA-I-induced neutrophils, whereas it can be observed that 18.4% and 92.1% of cells are in the late apoptotic/necrotic category, respectively. (B) results are means ± SEM (n>=10), *P < 0.05 vs. control by Student’s t test. Inset, top, untreated cells after 22 h of incubation. Only one cell is in apoptosis in this field (round nucleus instead of polylobed). Inset: neutrophils treated for 22 h with 1000 ng/mL VAA-I. Note that virtually all cells are in apoptosis (pycnotic nuclei).

 
The flow cytometric assay using cells stained with FITC-Annexin-V/PI is an interesting and powerful method for measuring apoptosis, especially for early events [29 , 30 ]. However, it is sometimes difficult to distinguish between cells in late apoptosis from those considered in early necrosis by flow cytometry. In addition to the fact that neutrophils treated with up to 1000 ng/mL for 24 h still excluded trypan blue, and that it was recently documented that both late apoptotic and necrotic cells are positive for PI [30 ], we decided to perform time-course experiments and monitor apoptosis by cytology and by flow cytometry. Data from Figure 4 illustrate that no morphological changes of apoptotic cells could be detected after 4 h during all treatments studied. However, after 12 and 24 h, VAA-I was found to induce neutrophil apoptosis at 500 or 1000 ng/mL, but not at 100 ng/mL. The delaying effect of GM-CSF was also only evident after 24 h. Data illustrated in the inset of Figure 4 are those obtained using FITC-Annexin-V/PI staining and flow cytometry and represent the number of cells (mean ± SEM, n=3) categorized as early apoptosis because cells categorized as late apoptosis/necrosis were always below 9% (not shown). Unlike the cytology assay, binding of FITC-Annexin-V allowed us to readily observe the ability of 1000 ng/mL VAA-I to induce neutrophil apoptosis after 4 h (49±5.6 vs. 20.5±4.5%) and more evidently after 12 h (75.8±2.2 vs. 29.9±3.5%) of treatment. Results obtained by the two different methods correlate very well.



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  		Figure 4. Assessment of neutrophil apoptosis during time-course experiments. Cells were incubated for 4, 12, or 24 h with buffer (Ctrl), 65 ng/mL GM-CSF, or increasing concentrations of VAA-I (100, 500, or 1000 ng/mL) and apoptosis was assessed by cytology as described in Materials and Methods. Results are means ± SEM (n>=7), *P < 0.05 vs. control by Student’s t test. Inset: apoptosis was assessed by flow cytometry (FITC-Annexin V/PI staining). Results are means ± SEM (n=3) and represented mainly viable cells (early and late apoptosis) because <9% of cells were necrotic (trypan blue dye exclusion). Note that signs of apoptosis can be detected more rapidly by FITC Annexin-V binding but results are the same after 24 h; almost 100% of cells are in apoptosis with the use of 1000 ng/mL.

 
Correlation between the ability of VAA-I to inhibit de novo protein synthesis and the induction of neutrophil apoptosis
Because we found that unlike GM-CSF, VAA-I at concentrations >500 ng/mL, acts as an inhibitor of de novo protein synthesis and induces cell apoptosis, we decided to study how these two biological processes could be modulated during co-incubation. Results in Figure 5 illustrate that VAA-I can reverse the ability of GM-CSF to delay neutrophil apoptosis. The inset of Figure 5 illustrates the correlation between induction of apoptosis and inhibition of de novo protein synthesis by VAA-I (Fig. 5 , lane 4). This is unlike GM-CSF that induces de novo protein synthesis (lane 2) and delays neutrophil apoptosis.



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  		Figure 5. VAA-I reverses the GM-CSF-induced suppression of neutrophil apoptosis and the GM-CSF-induced de novo protein synthesis. Cells were incubated for 24 h in the presence of buffer (Ctrl); 65 ng/mL GM-CSF, 1000 ng/mL VAA-I; or a combination of GM-CSF (65 ng/mL) + VAA-I (1000 ng/mL) and the number of apoptotic neutrophils was evaluated by cytology as described in Materials and Methods. Results are means ± SEM (n=5). *P < 0.05 vs. control by Student’s t test. Inset: neutrophils were metabolically labeled as in legend of Figure 2 , and incubated for 24 h in the presence of buffer (lane 1), 65 ng/mL GM-CSF (lane 2), 1000 ng/mL VAA-I (lane 3), or a combination of GM-CSF and VAA-I (lane 4). Note that GM-CSF induced de novo protein synthesis (lane 2) and that VAA-I can reverse this response (lane 4).

 
VAA-I-induced neutrophil apoptosis is independent of tyrosine phosphorylation events
Because GM-CSF is known to inhibit neutrophil apoptosis via tyrosine phosphorylation [35 , 36 ] we were interested in verifying whether VAA-I-induced apoptosis also involved tyrosine phosphorylation. Results in Figure 6A indicate that VAA-I does not increase tyrosine phosphorylation by itself and that it does not alter the inducing effect of GM-CSF. Results in Figure 6B indicate that although the GM-CSF-induced suppression of neutrophil apoptosis is dependent on tyrosine kinases, the ability of VAA-I to induce cell apoptosis is independent of such a response because genistein does not reverse the effect of VAA-I. Genistein was effective based on its ability to reverse the delaying effect of GM-CSF (Fig. 6B) . The specificity of the phosphotyrosine proteins detected was confirmed because, as expected, the signal was attenuated after pretreatment with the tyrosine kinase inhibitor genistein (data not shown, n=2).



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  		Figure 6. Induction of neutrophil apoptosis by VAA-I is independent of tyrosine phosphorylation events: VAA-I does not alter GM-CSF-induced tyrosine phosphorylation. (A) cells were stimulated for 15 min with buffer (lane 1), 65 ng/mL GM-CSF (lane 2), VAA-I at 1, 10, or 1000 ng/mL (lanes 3, 4, 5, respectively), or a combination of GM-CSF + an increasing concentration of 1, 10, 1000 ng/mL VAA-I (lanes 6, 7, and 8, respectively). Immunoblotting was performed as described in Materials and Methods. Top: tyrosine phosphorylation of intracellular proteins. Bottom: Coomassie blue staining of the membrane to indicate equivalence in loading. Results shown are representative of three experiments conducted with three different blood donors. (B) Diff-quick staining of cells treated with buffer + 50 µg/mL genistein (Ctrl + genistein), 65 ng/mL GM-CSF alone (GM), GM-CSF + genistein (GM + genistein), or with 1000 ng/mL VAA-I + genistein (VAA-I + genistein). Photomicrographs are representative of three experiments conducted separately. Small arrows, apoptotic neutrophils; big arrowheads, normal neutrophils. Approximately 50, 0, 20, and 100% of cells are in apoptosis from top to bottom.

 
VAA-I-induced neutrophil apoptosis is a caspase-dependent mechanism
We tried to reverse the VAA-I-induced neutrophil apoptosis with a panel of different inhibitors and found that only the caspase inhibitor z-VAD-FMK was able to do so. Results are shown in Figure 7 , where it can be seen that the numbers of cells in apoptosis were as follows: 41.5 ± 6.3 (means ± SEM, n=4); 40.8±10.3; 28.3±5.2; 98.0±1.3; 54.5±3.2% for neutrophil treated with buffer, dimethyl sulfoxide (DMSO; 1% v/v final as used for diluting z-VAD-FMK), Z-VAD-FMK, VAA-I, and z-VAD-FMK + VAA-I, respectively. Unlike z-VAD-FMK, the use of other inhibitors such as pertussis toxin, staurosporine, H7, Calphostin C, manoalide, BpB, quinacrine, and HA-1077, were unable to reverse the VAA-I-induced neutrophil apoptosis because the number of apoptotic cells remains between 97 and 100% (n>=3). Involvement of caspases during VAA-I-induced neutrophil apoptosis was further demonstrated by the fragmentation of the cytoskeletal gelsolin protein (Fig. 8 ). Such fragmentation was largely inhibited by the z-VAD-FMK inhibitor (Fig. 8) but not the irrelevant Calphostin C inhibitor (data not shown).



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  		Figure 7. Involvement of caspases in VAA-I-induced neutrophil apoptosis. Freshly isolated human neutrophils were treated for 24 h with buffer (CTRL), diluent (1% DMSO), caspase inhibitor z-VAD-FMK (50 µM; z-VAD) alone, 500 ng/mL VAA-I alone, or 1 h with z-VAD and then VAA-I for 23 h (total of 24 h, as other conditions) and apoptotic neutrophils were quantified by cytology as described in Materials and Methods. Results are means ± SEM (n = 4). *P < 0.0005 by Student’s t test.

 


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  		Figure 8. Fragmentation of gelsolin in VAA-I-induced neutrophil apoptosis. Human neutrophils were isolated and cultured as in legend of Figure 7 . The fragmentation of the cytoskeletal gelsolin protein was visualized by immunoblotting as described in Materials and Methods. C, control cells incubated with diluent (-) or 50 µM z-VAD-FMK (+); VA, cells incubated with 500 ng/mL VAA-I with diluent (-) or 50 µM z-VAD-FMK (+). N, native gelsolin (90 kDa); p47, a 47-kDa peptide derived from a chymotryptic cleavage of gelsolin recognized by the antibody. Note the diminution of native gelsolin and an ~50-kDa band induced by VAA-I in lane 2, and the appearance of a more intense band (41 kDa) in the same lane (small arrow). The degradation of native gelsolin was inhibited by the z-VAD-FMK (compare lane 2 with lane 4). This is one representative experiment out of four. Numbers on the left are molecular mass standards.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was performed to better understand the VAA-I/neutrophil interactions. We found that VAA-I can induce protein synthesis at low concentrations, whereas it acts as an inhibitor at concentrations >500 ng/mL. Our present study is different from others not only in the cell type used but also because we have studied de novo protein synthesis from metabolically 35S-labeled cells and used SDS-PAGE to visualize them. In addition, we have monitored newly synthesized polypeptides through the use of both intracellular and extracellular fractions. Our experiments were carried out in the presence of GM-CSF, a positive control known to induce de novo protein synthesis in neutrophils [10 ] and CHX, a negative control known to inhibit protein synthesis [37 , 38 ]. Unlike CHX, which drastically inhibits de novo protein synthesis in both intracellular and extracellular fractions, high concentrations of VAA-I were found to markedly inhibit newly synthesized polypeptides only in the intracellular fraction. The pattern of newly synthesized polypeptides detected in the external milieu from VAA-I-induced cells and from control cells were similar but slightly stronger with VAA-I, whereas it was distinct from GM-CSF-induced cells. In fact, as we have highlighted in Results, we obtained the expected results with GM-CSF because this cytokine is known to induce IL-1Ra [33 ]. Our results indicate that high concentrations of 500 and 1000 ng/mL VAA-I cause inhibition of de novo protein synthesis but to a lesser extent than CHX. The fact that the number of apoptotic cells are quite similar after 22–24 h for untreated cells and neutrophils treated with low concentrations of VAA-I, ruled out the possibility that the newly synthesized polypeptides observed after the VAA-I treatments arise from cell lysis or leakage from a small number of cells. In both conditions, all cells exclude trypan blue, and the protein loading was equivalent (Coomassie blue staining).

An increasing number of products are known to modulate neutrophil apoptosis [3 , 4 , 34 , 39 , 40 ]. Identifying such agents is of biological importance in various situations such as for patients receiving drug therapy for cancer treatments [41 ], AIDS patients [42 ], or during an inflammatory state [13 14 15 ]. Herein, we report that VAA-I is a potent agent that can induce neutrophil apoptosis. Using both flow cytometry (FITC-Annexin-V/PI labeling) and cytology (Diff-Quick staining of cytocentrifuge preparations) approaches, we have demonstrated that VAA-I can induce neutrophil apoptosis at high concentrations (>500 ng/mL). In addition, our results allow us to correlate the ability of VAA-I to induce neutrophil apoptosis and concomitantly inhibit de novo protein synthesis. This is in contrast to stimuli that delay neutrophil apoptosis and induce de novo protein synthesis such as GM-CSF [3, 10, this study], IL-4 [4 ], IL-15 [3 ], sodium butyrate [37 ], and dexamethasone [38 ]. This correlation is reinforced by the following observations. (1) Low concentrations of VAA-I induced de novo protein synthesis but do not alter the neutrophil apoptotic rate. (2) We have included in all of our experiments dealing on de novo protein synthesis one of the most widely used protein synthesis inhibitors as a control, CHX, at a concentration known to induce neutrophil apoptosis [37 ] and observed that it clearly inhibits de novo protein synthesis visualized by SDS-PAGE. (3) High concentrations of VAA-I were found to markedly reverse the ability of GM-CSF to both induce de novo protein synthesis and to delay neutrophil apoptosis without inducing cell necrosis. (4) Similar properties of VAA-I were previously observed with cells other than neutrophils, namely, human lymphocytes, monocytes, monocytic THP-1 cells, and murine thymocytes [16 ]. (5) To our knowledge, there is no clear evidence in the literature that argues against the correlation of induction of neutrophil apoptosis with inhibition of de novo protein synthesis. One unique study demonstrated that typical morphological features of apoptosis, in etoposide-induced rat pheochromocytoma PC12 cells, were not associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis [43 ]. However, the concentration of CHX used in pre-incubation study or during co-incubation was low because higher concentrations were found to be toxic for these cells. In addition, unlike the present study (where proteins are visualized), these authors did not perform SDS-PAGE from metabolically labeled cells, but rather added CHX with etoposide and enumerated the number of apoptotic cells [43 ].

Results from this study allow us to establish that VAA-I does not induce tyrosine phosphorylation events in human neutrophils by itself and does not alter the GM-CSF-induced response. Involvement of tyrosine kinases to mediate biological actions was found to be of importance for several agonists [35 , 36 , 44 45 46 47 48 49 50 ]. Among them, GM-CSF and LPS were found to recruit tyrosine kinases for delaying neutrophil and eosinophil apoptosis [44 45 46 ]. Results from one recent study demonstrate that induction of human neutrophil apoptosis by the protein synthesis inhibitor CHX accelerate macrophage clearance of apoptotic neutrophils [51 ]. Whether or not VAA-I can act similarly remains unknown. In one study, it was found that treatment of cells with a specific inhibitor of Jak-2, tyrphostin B42, can reverse the ability of GM-CSF to delay eosinophil apoptosis, suggesting that this biological action of GM-CSF occurs via Jak-2 [45 ]. Herein, although pretreatments of human neutrophils by genistein can reverse the effect of GM-CSF, such treatments were ineffective in reversing the inducing effect of VAA-I. In addition, although we observed that tyrphostin B42 was able to reverse the delaying effect on neutrophil apoptosis, it was not able to reverse the VAA-I-induced neutrophil apoptosis (our unpublished observations). This suggests that VAA-I-induced neutrophil apoptosis is independent of tyrosine kinase activation and, in particular, it does not recruit Jak-2, an important molecule in neutrophil signaling [52 ]. Furthermore, in this study we bring evidence that VAA-I induces neutrophil apoptosis via a mechanism that involves caspases but not G proteins, protein kinase Cs, and phospholipase A2. Involvement of caspases was confirmed by inhibition of VAA-I-induced neutrophil apoptosis with the z-VAD-FMK inhibitor by both microscopic observations and by fragmentation of gelsolin visualized by immunoblotting. Our results are in agreement with the fact that caspases play an important role in apoptosis. Recently, it has been demonstrated that unlike anti-Fas-induced neutrophil apoptosis, specific inhibitors to caspase-3 and caspase-8 were unable to prevent spontaneous neutrophil apoptosis [53 ]. This is in agreement with our observation that the use of the z-VAD-FMK alone did not alter normal neutrophil apoptotic rate. However, it was found by others that caspase-3 is involved during spontaneous neutrophil apoptosis [54 ]. Although they speculate that the z-DEVD-FMK inhibitor is specific for caspase-3, it is specified by the manufacturer that it can inhibit as well caspases-6, -7, -8, and -10. The experimental conditions were different between the two studies, including the concentration of the inhibitor used (100 µM for the former and 20 µM in the other study). This testifies to the complexity of interpreting results. Herein, we demonstrate that among caspases-1, -3, -4, -7, the caspase-3 is at least involved. In addition, our results suggest that VAA-I can accelerate human neutrophil apoptosis without the synthesis of new factor(s). It seems that all the machinery (including the caspases) is already present in these cells and induction of apoptosis could be simply mediated by an "on/off" switch. This is in agreement with the fact that these cells are known to spontaneously undergo apoptosis without addition of external agents.

Taken together, our results demonstrate that VAA-I is a potent immunomodulator. The fact that VAA-I could act as a very potent inducer of neutrophil apoptosis offers an interesting potential therapeutic strategy in inflammatory disorders such as rheumatoid arthritis. Elaboration of a VAA-I immunotoxin that could be used to eliminate neutrophils by "killing" them by inducing apoptosis during an inflammatory response could be useful to attenuate/eliminate inflammation. This would be of great importance in general biology and medicine.


   ACKNOWLEDGEMENTS
 
This study was partly supported by Fonds de la Recherche en Santé du Québec (FRSQ) and Fonds pour la Formation de Chercheurs et l’Aide à la Recherche (FCAR). D. G. is a Scholar from FRSQ; A. S. holds a M.Sc. Natural Sciences and Engineering Research Council of Canada (NSERC) award; and M. P. holds a M.Sc. award from FCAR/FRSQ-Santé. We thank Dr. Kenneth Finnson for reading the manuscript.


   FOOTNOTES
 
Correspondance: Dr. Denis Girard, INRS/Institut Armand-Frappier/Santé Humaine, 245 boul. Hymus, Pointe-Claire, Quebec, Canada, H9R 1G6. E-mail: Denis.Girard{at}INRS-Sante.Uquebec.ca

Received December 9, 1999; revised July 1, 2000; accepted July 5, 2000.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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