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Am. J. Respir. Cell Mol. Biol., Volume 18, Number 3, March 1998 315-322

Human Eosinophils Express Bcl-2 Family Proteins: Modulation of Mcl-1 Expression by IFN-gamma

Anne Druilhe, Michel Arock, Liliane Le Goff, and Marina Pretolani

Unité de Pharmacologie Cellulaire, Institut Pasteur; and Centre National de la Recherche Scientifique Unité de Recherche Associéc No. 625, Centre Hospitalier Universitaire Pitié-Salpétrière, Paris, France


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The expression of the Bcl-2 family proteins Bax, Mcl-1, Bcl-2, and Bcl-xL, was examined in human peripheral blood eosinophils or in umbilical-cord-blood-derived eosinophils. Immunoblot analysis disclosed high amounts of the proapoptotic factor Bax in freshly purified eosinophils of both types. Although cord-blood-derived eosinophils expressed easily detectable levels of Mcl-1, Bcl-2, and Bcl-xL, only traces or no expression of these three antiapoptotic proteins were found in peripheral blood eosinophils. Incubation of both eosinophil types for 1 to 3 days in a cytokine-deprived medium led to apoptosis, without changes in the expression of Bax, Mcl-1, Bcl-2, or Bcl-xL. Although addition of interleukin-5 or interferon-gamma (IFN-gamma ) to the culture medium increased the survival of both eosinophil types, a rise in the levels of Mcl-1 was observed only in IFN-gamma -treated cord-blood eosinophils. Together, these results indicate that human eosinophils have a specific profile of Bcl-2-family protein expression that depends on their maturation status and may be modulated by stimuli that influence their survival.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Increased numbers of eosinophils are frequently observed in blood and/or tissues in different diseases, including asthma, allergic rhinitis, atopic dermatitis, and the hypereosinophilic syndrome (1). Release by eosinophils of cytotoxic basic proteins, extracellular-matrix-degradation enzymes, lipid mediators, oxygen radicals, and inflammatory cytokines may contribute to the pathogenesis of these diseases (2). As recently proposed (3), blood and tissue eosinophilia may result at least in part from a defect in eosinophil apoptosis. This process has been extensively studied in a variety of cell types, leading to the discovery of different factors, such as the proapoptotic antigen Fas and its natural ligand, Fas-ligand (Fas-L) (4). Recent studies have shown that human blood eosinophils constitutively express a functional Fas antigen on their surface, since its stimulation induces their apoptotic death (5). The expression of Fas-L, however, was undetectable (8), suggesting that mechanisms unrelated to the Fas/Fas-L pathway may be involved in apoptosis observed upon the in vitro culture of human eosinophils in the absence of cytokines (5, 9). We therefore hypothezised that other factors, such as the pro- and antiapoptotic members of the Bcl-2 family, may play a role in this process.

The bcl-2 oncogene was isolated from the t(14;18) chromosomal breakpoint found in most cases of non-Hodgkin's B-cell lymphoma (10, 11), and was shown to encode an intracellular protein that resides in the mitochondrial, nuclear, and endoplasmic reticulum membranes (12, 13). The first evidence for an effect of the Bcl-2 protein on cellular viability came from a study demonstrating that its overexpression promotes survival in cytokine-deprived lymphoid and myeloid cell lines (14). In addition, Bcl-2 was shown to display inhibitory functions on cell death induced by chemotherapeutic drugs; c-Myc overexpression; formation of reactive oxygen species; lipid peroxidation; heat shock; UV- or gamma -radiation; some viruses; and calcium, azide, and glucose deprivation (15).

Recently, a family of genes with sequence similarities to bcl-2 has emerged (15). The bcl-x gene, isolated by low-stringency hybridization to bcl-2, encodes two distinct proteins: Bcl-xL, which increases cell viability as Bcl-2, and Bcl-xS, which shows opposite effects (16, 17). Bax protein was isolated by coimmunoprecipitation with Bcl-2 from a human B-cell line, and accelerates the death of cells in this line (18). Additionally, the mcl-1 gene was cloned in differentiated myeloid cells and shown to encode an antiapoptotic factor (19, 20).

The expression of these proteins depends on cell type, as shown for Bcl-2, which is undetectable in neutrophils but is present in T- and B-lymphocytes (21). Differentiation status may also influence the levels of Bcl-2, Bcl-xL or Mcl-1 (16, 19, 21). Moreover, various stimuli that act on apoptotic cell death, such as sphingosine; Ras activation; adhesion; cytokines including interleukin (IL)-2, IL-3, IL-10, and tumor necrosis factor-alpha (TNF-alpha ); and cluster of differentiation (CD)3, CD28 or CD40 activation, modulate the expression of these proteins (21, 24).

In the present study, the expression of Bcl-2, Bcl-xL, Bcl-xS, Bax, and Mcl-1 was examined in terminally differentiated human peripheral blood eosinophils and in umbilical cord blood eosinophils, which contain high proportions of immature cells (32). Both eosinophil types were cultured in cytokine-deprived medium, a condition leading to their apoptosis (5, 9), or in the presence of IL-5 or interferon-gamma (IFN-gamma ), two cytokines known to prolong eosinophil survival and/or prevent eosinophil apoptosis (9, 33).

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents

Percoll and Ficoll-Paque were obtained from Pharmacia (Orsay, France); Diff-Quik dye from Baxter Dade AG (Duedingen, Switzerland); RPMI 1640 and fetal calf serum (FCS) from Gibco BRL (Cergy Pontoise, France); antibiotic-antimycotic solution and L-glutamine from Boehringer Mannheim (Meylan, France); prostaglandin E2 (PGE2), 2-mercaptoethanol, propidium iodide, naphthol AS-XM phosphate, levamisole, Fast red salt TR, mouse anti-beta actin monoclonal antibodies (mAb) (clone AC-74) from Sigma (St. Quentin Fallavier, France); recombinant human IL-5 (rhIL-5) from Immugenex (Los Angeles, CA); mouse antihuman Bcl-2 mAb (clone 124), goat antimouse Ig, alkaline phosphatase antialkaline phosphatase (APAAP), murine IgG1 isotype control, and fluorescein isothiocyanate (FITC)- conjugated F(ab')2 fragment of goat antimouse Ig from Dako (Trappes, France); rabbit antihuman Mcl-1 antibody (Ab) from Pharmingen (San Diego, CA); peroxidase-conjugated sheep antimouse Ig, donkey antirabbit Ig, rainbow-protein markers, and ECL western blotting detection system from Amersham (Les Ulis, France); and BioRad protein assay reagent from BioRad (Munich, Germany). Rabbit antihuman and antimouse Bax (N-20), and rabbit antihuman and antimouse Bcl-xL/S (S-18) Abs were supplied by Tebu (Le Perray en Yvelines, France) for Santa Cruz Biotechnology (Santa Cruz, CA). rhIL-3 was a kind gift of Dr. U. K. Brown (Sandoz Biotechnology, Basel, Switzerland), and rhIFN-gamma was provided by Dr. P. Devillier, Roussel Uclaf (Romainville, France).

Eosinophil and Peripheral Blood Mononuclear Cell Preparations

Human peripheral venous blood from healthy donors (eosinophil counts ranging from 49 × 109/liter to 493 × 109/liter) was obtained from the Centre de Transfusion Sanguine (Paris, France) and used to purify eosinophils and peripheral blood mononuclear cells (PBMC). When indicated, eosinophils were isolated from individuals with blood hypereosinophilia (eosinophil counts ranging from 1,046 × 109/liter to 10,848 × 109/liter) of various etiologies (two with leukemia, one with a parasitic infection, and one with hypereosinophilic syndrome). These patients were taking no medication at the time of blood sampling. Informed consent was obtained from these donors.

After removal of the mononuclear cells, normodense eosinophils from healthy individuals were isolated with a 1.082 g/ml Percoll gradient, hypotonic lysis of erythrocytes, and depletion of neutrophils by the immunomagnetic method, as previously described (5). In the case of hypereosinophilic donors, a 1.077 g/ml Percoll gradient was used to isolate hypodense eosinophils. Viability (98.5 ± 0.3% and 97.6 ± 1.9% viable cells, n = 20 and n = 4, for normal and hypereosinophilic donors, respectively) and purity (98.6 ± 0.3% and 91.0 ± 6.1% normodense and hypodense eosinophils, n = 20 and n = 4, respectively) of the final cell suspension were evaluated by the trypan blue exclusion method and after staining of cytospin preparations with Diff-Quik dye, respectively.

To purify PBMC, blood was mixed with 3% dextran and allowed to settle at 20°C for 30 min. Buffy coat was next layered onto a Ficoll-Paque gradient. Expression of Bcl-2-family proteins was evaluated in freshly purified PBMC, which constitutively express high levels of Bcl-2, Bcl-xL, Bax, and Mcl-1 (21).

Eosinophils differentiated from umbilical cord blood cells were obtained as described (32). Briefly, umbilical cord blood was layered over Ficoll-Paque gradient. The mononuclear cell fraction was then cultured in RPMI 1640 medium supplemented with 0.3 µM PGE2, 50 µM 2-mercaptoethanol, and 10% FCS. Eosinophil differentiation was obtained by enriching the culture medium with 2 ng/ml rhIL-3 and 5 ng/ml rhIL-5. Washed cells were used after 3 wk of culture, the medium being replaced every week. Viability and purity of the final cell suspension were of 95.6 ± 1.1% viable cells and 89.1 ± 1.4% eosinophils (n = 16), respectively. Contaminant cells were mostly basophils (12.4 ± 2.8%, n = 16), as identified by toluidine blue staining.

Peripheral or umbilical cord blood eosinophils (1.5 × 106/ml) were resuspended in culture medium consisting of RPMI 1640 supplemented with 1% antibiotic-antimycotic solution, 10% FCS, and 2 mM L-glutamine, and were cultured in flat-bottom 12- or 96-well culture plates at 37°C in a 5% CO2 atmosphere for 1 and 3 d, in the absence or presence of 5 ng/ml rhIL-5 or 1,000 U/ml rhIFN-gamma .

Determination of Eosinophil Apoptosis by Flow Cytometry

Eosinophils (0.3 × 106) were resuspended in a solution containing 0.1% Triton X100, 0.1% sodium citrate, and 50 µg/ml propidium iodide, as described (34). Cells were applied to a FACScan flow cytometer (Becton Dickinson, Le Pont de Claix, France), and a total of 5,000 ungated cells were analyzed, using LYSIS software (Becton Dickinson). The percent of weakly propidium iodide-stained apoptotic nuclei was determined.

Evaluation of Bcl-2-family Protein Expression by Immunoblot Analysis

PBMC, peripheral blood eosinophils, or cord blood eosinophils were incubated for 10 min at 4°C with the lysis buffer containing 0.2% NP-40, 50 mM NaCl, 10 mM 4-(2-hydroxyethyl)-1-piperazine-N'-2-ethanesulfonic acid (pH 8.0), 0.5 M sucrose, 1 mM EDTA disodium salt, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 7 mM 2-mercaptoethanol, and were subsequently centrifuged for 15 min at 12,000 × g. Supernatant was mixed with glycerol and the protein content was determined with the BioRad protein assay. Aliquots containing 25 µg or 100 µg of total proteins, or of rainbow-protein markers (14.3-200 kD) were mixed with a 3× loading buffer consisting of 150 mM Tris (pH 6.8), 300 mM dithiothreitol (DTT), 6% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue, and 30% glycerol solution. Samples were boiled for 5 min and subjected to SDS-10% polyacrylamide gel electrophoresis (PAGE), except for Mcl-1 detection, for which SDS-8.5% PAGE was used. After migration in a 26 mM Tris (pH 8.3), 192 mM glycine and 0.1% SDS buffer, size-fractionated proteins were electroblotted onto nitrocellulose filters in a 26 mM Tris (pH 8.3), 192 mM glycine, and 20% methanol buffer. Blots were immediately incubated once at 20°C for 10 min, with a reaction buffer consisting of 20 mM Tris (pH 7.6), 140 mM NaCl, 0.1% Tween-20, and 5% nonfat evaporated milk. The membrane was next reacted for 1 h at 20°C with the anti-Bcl-2, anti-Bax, anti-Bcl-xL/S, or anti-Mcl-1 Abs at 1:250, 1:500, 1:100 and 1:1,000 dilutions, respectively. After two washes lasting 10 min at 20°C each, peroxidase-conjugated antimouse or antirabbit Ig Abs, both at a 1:1,000 dilution, were applied to the blot for 1 h at 20°C. Immunoblots were again washed three times and were then incubated for 1 min with the ECL western blotting detection system. Films were exposed for 1 min or 40 min before being developed. beta -Actin detection was next performed on each blot. In this case, the first and second Abs were applied to the membrane for 10 min at 1:4,000 and 1:2,000 dilutions, respectively, and the film was exposed for 15 to 30 s.

The anti-Bcl-xL/S Ab reacts with an identical epitope present in both Bcl-xL and Bcl-xS and thus allows distinction of these proteins through the relative sizes of their bands (~ 30 kD for Bcl-xL and ~ 21 kD for Bcl-xS; [17]).

The intensities of expression of Bcl-2, Bax, Bcl-xL/S, Mcl-1, and beta -actin were quantified using a Masterscan densitometer and ZeroDscan software (both from Scanalytics, Billerica, MA). Results are expressed as a ratio of the OD values of Bcl-2, Bax, Bcl-xL/S or Mcl-1 bands to the OD values of the beta -actin bands.

Immunocytochemical and Flow-cytometric Detection of Bcl-2

Peripheral or cord-blood-derived eosinophils and PBMC were cytocentrifuged (Hettich, Tuttlingen, Germany) on glass slides, fixed in acetone for 10 min at 20°C, wrapped in a plastic film, and kept at -20°C until use. Antibodies were diluted in a buffer (50 mM Tris, 150 mM NaCl, pH 7.6) supplemented with 1% bovine serum albumin (BSA). Cells were incubated for 1 h at 20°C with 6 µg/ml anti-Bcl-2 mAb or murine IgG1 isotype control, followed by goat antimouse Ig and APAAP, both at a 1:25 dilution. Substrate solution, consisting of 0.48 mM naphthol AS-XM phosphate, 2% dimethylformamide, 100 mM Tris (pH 8.2), 1.3 mM levamisole, and 194 mM Fast red salt TR, was applied for 30 min at 20°C. A light hematoxylin counterstaining was next performed. Slides were washed twice for 10 min between each step. The proportion of Bcl-2-positive cells was calculated after counting approximately 200 cells in randomly selected fields.

For flow-cytometric detection of Bcl-2, peripheral or cord-blood-derived eosinophils and PBMC (0.4 × 106) were centrifuged and resuspended in 200 µl of phosphate buffered saline (PBS) (NaCl 140 mM, Na2HPO4 9.2 mM, NaH2PO4 1.3 mM, pH 7.4) containing 0.25% formaldehyde. After 30 min incubation at room temperature, 1 ml of PBS supplemented with 10% FCS was added. Cells were next centrifuged and the pellets were resuspended in 200 µl of PBS containing 10% FCS and 0.025% saponin. Antibodies were diluted in this buffer. After 10 min incubation at 4°C, cells were centrifuged and next incubated for 1 h at 4°C with 6 µg/ml anti-Bcl-2 mAb or murine IgG1 isotype control. Cells were washed twice in the PBS-FCS-saponin buffer and incubated for 1 h at 4°C with 20 µg/ml FITC-conjugated F(ab')2 fragment of goat antimouse Ig. Cells were then washed once in PBS-FCS buffer, centrifuged, resuspended in the same buffer, and immediately subjected to flow cytometry. A total of 5,000 ungated cells were analyzed using the LYSIS software.

Statistical Analysis

Results are expressed as mean ± SEM of the indicated number of experiments. One-way analysis of variance (ANOVA) was used to determine significance among the groups. If a significant variance was found, a Student's t test for unpaired or paired values was used to assess comparability between the means. A value of P < 0.05 was considered significant.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Detection of Bcl-2-family Proteins in Freshly Purified Peripheral Blood Eosinophils

The expression of Bax, Bcl-2, Mcl-1, and BclxL/S by freshly purified peripheral blood normodense eosinophils from healthy donors was first examined through immunoblotting, and compared with that of PBMC, included as a positive control. Using similar experimental conditions (25 µg proteins, 1 min film exposure time), we found high levels of Bax in eosinophils and in unstimulated PBMC (Figure 1). By densitometry, we established that the intensity of Bax expression was similar in both cell types, since OD Bax/ OD beta -actin values for eosinophils and PBMC were 1.4 ± 0.4 and 0.7 ± 0.2, respectively (n = 4 to 7, differences not statistically significant). Unstimulated PBMC also contained Bcl-xL and Mcl-1, but not Bcl-xS, even though amounts of proteins (100 µg) and film exposure time (40 min) were greater than those used for Bax detection (Figure 1). Under the same conditions, eosinophils failed to express Bcl-xL and Bcl-xS, and only traces of Mcl-1 were detected in half of the eosinophil preparations (Figure 1). Similarly, although Bcl-2 was easily detectable in PBMC, only traces of this protein were observed in four of eight eosinophil samples, although high quantities (100 µg) of proteins were loaded onto gels and exposure times lasted up to 40 min (Figure 1).


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  		Figure 1.   Immunoblot analysis of Bcl-2-family proteins in normodense eosinophils from peripheral blood of healthy donors and in freshly purified PBMC. Bcl-2-family protein expression was examined in freshly purified eosinophils and PBMC, or in eosinophils cultured for 1 or 3 d in the absence of cytokines (N) or in the presence of 5 ng/ml rhIL-5 (IL) or 1,000 U/ml rhIFN-gamma (IF). Blots were reacted with anti-Mcl-1, anti-BclxL/S, anti-Bcl-2, anti-Bax, and anti-beta -actin Abs. Positions of molecular-weight markers are indicated in kD. Each blot is representative of three to eight experiments.

The expression of Bcl-2 was next analyzed with immunocytochemistry and flow cytometry. Only 1.0 ± 0.7% (n = 5) peripheral blood eosinophils expressed Bcl-2, as determined through immunocytochemistry (Figure 2), whereas practically all lymphocytes in PBMC preparations stained positively for this antigen (data not shown). No staining was shown in isotype control-treated cells (data not shown). Flow-cytometric analysis also failed to demonstrate the presence of Bcl-2 in human blood eosinophils, in contrast to PBMC, which showed a marked expression of this protein (Figure 3).


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  		Figure 2.   Identification of Bcl-2-positive cells on cytospin preparations from normodense peripheral blood (a) or umbilical-cord-blood-derived (b, and insert) eosinophils through immunocytochemistry and light hematoxylin counterstaining. Positive cells show a broad range of red stain intensities in the cytoplasm. Scale bars = 39 µm (a and b) and 13 µm (insert).


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  		Figure 3.   Representative flow-cytometric profiles, showing the absence of Bcl-2 in peripheral blood eosinophils (a), and its presence in cord-blood-derived eosinophils (b) and unstimulated lymphocytes (c). Cells were incubated with the control isotype (full lines) or with the anti-Bcl-2 Ab (dashed lines). The expression of Bcl-2 in lymphocyte populations was obtained after gating these cells from total PBMC.

The expression of Bcl-2 was further examined in freshly purified hypodense eosinophils obtained from hypereosinophilic donors. As with normodense blood eosinophils from healthy individuals, these cells failed to express Bcl-2, as determined by immunoblot and immunocytochemical analysis (data not shown).

Detection of Bcl-2-family Proteins in Umbilical-Cord-Blood-derived Eosinophils

We next analyzed the expression of Bcl-2-family proteins in eosinophils obtained by culture of umbilical-cord-blood-derived mononuclear cells for 3 wk in the presence of 2 ng/ml rhIL-3 and 5 ng/ml rhIL-5. As shown for peripheral blood eosinophils, these cells did not contain Bcl-xS, and expressed high levels of Bax (Figure 4). However, cord blood-derived cells expressed amounts of Bcl-2 and Bcl-xL similar to those of PBMC (Figure 4). Indeed, OD Bcl-2/OD beta -actin values for cord blood-derived eosinophils and PBMC were 1.0 ± 0.3 and 2.1 ± 1.0, respectively, and OD Bcl-xL/OD beta -actin values were 2.2 ± 0.5 and 3.4 ± 1.7, respectively (n = 4 to 6). Mcl-1 was also detectable in cord-blood-derived eosinophils, although wide variability in the intensities of its basal expression was observed in the different cell preparations (OD Mcl-1/OD beta -actin produced values of 0.0 to 17.3, n = 6). This phenomenon was unrelated to the purity of the cell samples (percent eosinophils versus OD Mcl-1/OD beta -actin values: r = 0.645, P > 0.1, n = 6; percent basophils versus OD Mcl-1/OD beta -actin: r = 0.336, P > 0.1, n = 6).


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  		Figure 4.   Time- and cytokine-dependent modulation of the expressions of Bcl-2-family proteins in umbilical-cord-blood-derived eosinophils. The expression of the Bcl-2-family proteins was analyzed in umbilical-cord-blood eosinophils freshly purified or cultured for 1 or 3 d in the absence (N) or in the presence of 5 ng/ml rhIL-5 (IL) or 1,000 U/ml rhIFN-gamma (IF). Blots were reacted with the anti-Mcl-1, anti-Bcl-xL/S, anti-Bcl-2, or anti-Bax Ab. Each membrane was next reacted with an anti-beta -actin mAb. Positions of molecular-weight markers are indicated in kD. Each blot was performed on four to seven different preparations.

Immunocytochemical analysis showed that 34.6 ± 4.6% of umbilical-cord-blood-derived cells were Bcl-2 positive (n = 9; Figure 2), a finding confirmed by flow cytometry (Figure 3).

Time- and Cytokine-dependent Modulation of Apoptosis and Expression of Bcl-2-family Proteins

Normodense peripheral or umbilical-cord-blood-derived eosinophils were cultured for 1 or 3 d in the absence or in the presence of 5 ng/ml rhIL-5 or 1,000 U/ml IFN-gamma , and the number of apoptotic nuclei was assessed through flow cytometry.

Incubation of normodense peripheral or umbilical-cord-blood eosinophils for 1 or 3 days in a cytokine-deprived medium resulted in an increase in the number of apoptotic nuclei (Table 1). This phenomenon was unaccompanied by changes in the expression of Bax, Bcl-2, Bcl-xL/S, Mcl-1, or beta -actin (Figures 1, 4, and 5). However, a slight decrease in the expression of Bcl-xL was observed in cord-blood- derived eosinophils at Day 1, since the OD Bcl-xL/OD beta -actin values at Days 0 and 1 were 2.2 ± 0.5 and 1.3 ± 0.1, respectively (n = 6, differences not statistically significant).

                              
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TABLE 1
Modulation by cytokines of apoptosis of normodense peripheral or umbilical-cord-blood-derived eosinophils


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  		Figure 5.   Time- and cytokine-dependent changes in the intensity of Mcl-1 expression in cord-blood-derived eosinophils and in unstimulated PBMC. Cord-blood-derived eosinophils were incubated for 0, 1, or 3 d in the absence (None) or in the presence of 5 ng/ml rhIL-5 or 1,000 U/ml rhIFN-gamma . Blots were incubated with the anti-Mcl-1 and anti-beta -actin Abs. The intensity of Mcl-1 expression was defined as the ratio of OD Mcl-1/OD beta -actin values, as determined by densitometry. Results are expressed as mean ± SEM of six experiments. *P < 0.05 as compared with untreated or IL-5-treated eosinophils.

When rhIL-5 or rhIFN-gamma was added to the culture medium, a reduction in apoptotic death of peripheral blood eosinophils was observed, the effect of both cytokines being less pronounced in cord-blood-derived cells (Table ). These phenomena were unaccompanied by changes in the expression of Bax, Bcl-2, Bcl-xL/S, or beta -actin in either eosinophil type (Figure 1). In contrast, a clear increase in Mcl-1 expression was observed at Days 1 and 3 in rhIFN-gamma -treated cord-blood eosinophils exclusively (Figures 1, 3, and 5). No modifications in the expression of this protein were found in rhIL-5-treated cells from either origin (Figures 1, 3, and 5).

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we examined the presence of Bax, Bcl-2, Bcl-xL and Bcl-xS, and Mcl-1 in human eosinophils. We showed that normodense eosinophils isolated from the blood of healthy individuals express constitutively high levels of the proapoptotic factor Bax, and practically no or only traces of the cell-death repressors Bcl-2, Bcl-xL, and Mcl-1. Bcl-xS, which positively regulates programmed cell death, was also undetectable. The pattern of Bcl-2-family protein expression that we found resembles that reported for neutrophils, but was very different from that observed in PBMC, since these cells constitutively express Bcl-2, Bcl-xL, Mcl-1, and Bax, but not Bcl-xS (21). The tendency of mature eosinophils, like neutrophils, to express high quantities of the proapoptotic factor Bax and low amounts of the anti-cell-death proteins Bcl-2, Bcl-xL, and Mcl-1 may reflect their short life span in vitro (35). These results, however, are not compatible with the slower rate of in vitro eosinophil as opposed to neutrophil apoptosis (35). This suggests that other factors belonging to the Bcl-2 family and with anti-cell-death properties may be expressed by eosinophils and may be involved in their spontaneous apoptosis. Accordingly, the A1, bfl-1, and bcl-w genes have been shown to encode proteins with sequence homologies to Bcl-2 (36- 38). Overexpression of the A1 protein delays apoptosis after cytokine deprivation in a murine IL-3-dependent cell line (39), and its gene was recently cloned in cultured human endothelial cells (36). Enforced expression of bcl-w, a gene highly conserved between the mouse and human, renders murine lymphoid and myeloid cells refractory to several cytotoxic treatments (37), whereas bfl-1 suppresses p53-induced apoptosis in rodent epithelial cells (38, 40). Lack of specific reagents for detecting A1, Bfl-1, or Bcl-w proteins prevented the analysis of their expression in this study.

As discussed earlier, previous studies failed to detect Bcl-2 in mature neutrophils, whereas its presence at high levels was reported in their bone-marrow precursors (22). Since a decrease in Bcl-2, Mcl-1 and Bcl-xL expression has also been described during T-cell maturation in vivo (16, 21, 23), we postulated that the differentiation status of eosinophils might also influence the expression of Bcl-2-family proteins. We therefore examined the level of these proteins in umbilical-cord-blood-derived eosinophils, which have been previously defined as immature cells (32). Interestingly, these eosinophils exhibit a pattern of Bcl-2-family protein expression similar to that found in unstimulated PBMC, since they contain constitutively high amounts of Bcl-2, Bcl-xL, and Bax, and detectable levels of Mcl-1, but not of Bcl-xS. However, since immature eosinophils were obtained after 3 wk of culture of hematopoietic precursors in the presence of IL-3 and IL-5, the hypothesis may be raised that the difference in the profile of expression of the Bcl-2-family proteins would be linked to their activation by long-term exposure to these cytokines. Our observations that: (1) neither IL-5 nor IFN-gamma modified the levels of Bcl-2, Bcl-xL, or Mcl-1 in peripheral blood eosinophils from healthy donors, and that: (2) hypodense eosinophils from hypereosinophilic individuals, considered to be in vivo-activated cells (2), failed to express Bcl-2 in a manner similar to that of normodense blood eosinophils, may exclude this possibility.

Culture of peripheral or cord-blood eosinophils in the absence of cytokines induced a time-dependent increase in their apoptosis, confirming previous reports (5, 9). Spontaneous apoptotic death in both eosinophil types was unaccompanied by changes in the levels of Bcl-2, Mcl-1, Bcl-xL, or Bax, indicating that these events might be unrelated. Although the constitutive pattern of Bcl-2-family protein expression was different for peripheral and umbilical-cord-blood-derived eosinophils, the kinetics and the degree of their apoptosis were similar. These findings suggest that the discrepancy in the profiles of Bcl-2-family protein expression by these two eosinophil types is not predictive of the evolution of cell survival, but may reflect a potential difference in the sensitivity to apoptotic stimuli, as shown for other cell types (15). Alternatively, additional mechanisms may be implicated in eosinophil death, including the expression of other pro- (41) or antiapoptotic (36) members of the Bcl-2 family, and/or of factors unrelated to this family. Among these molecules, the intracellular IL-1-converting enzyme-like proteases are claimed to play a critical role in controlling apoptotic death in different cell types (44). Whether these molecules also participate actively in the regulation of eosinophil apoptosis is an area for further investigation.

Our findings also extend those in previous studies, showing that IL-5 and IFN-gamma prolonged the survival of peripheral and umbilical-cord-blood eosinophils (9, 33). Previous studies have shown that inhibition of eosinophil apoptosis by IL-5 depends on the synthesis of new proteins (9). The members of the Bcl-2 family may be good candidates for such proteins, since induction of Bcl-2 expression has been observed in an erythroleukemic cell line upon culture with IL-3 (27). However, no modifications in the expression of the Bcl-2-family proteins were found when both eosinophil types were incubated with IL-5. In contrast, IFN-gamma may modulate cord, but not peripheral, blood eosinophil apoptosis by augmenting the levels of Mcl-1.

In conclusion, our findings demonstrate that Bcl-2, Bcl-xL, Mcl-1, and Bax are expressed constitutively by umbilical-cord-blood eosinophils, and that the expression of some of these proteins probably decreases during cell maturation, as suggested for neutrophils and T- and B-lymphocytes (16, 21). The expression of Mcl-1 is augmented by IFN-gamma , indicating that this member of the Bcl-2 family may represent a novel target for other stimuli that influence eosinophil survival.

    Footnotes

Abbreviations: antibody, Ab; alkaline phosphatase-antialkaline phosphatase, APAAP; cluster of differentiation, CD; Fas-ligand, Fas-L; peripheral blood mononuclear cells, PBMC.

(Received in original form May 8, 1997).

Acknowledgments: This work was supported by Grant 1388 from the Association pour la Recherche sur le Cancer, Paris, France. The authors are grateful to Dr. Nicole Israël and Ms. Fabienne Aillet (Unité de Biologie des Rétrovirus, Institut Pasteur, Paris, France) for help in performing initial immunoblot assays, and to the nurses of the Maternité du Centre Hospitalier Universitaire Pitié-Salpétrière (Paris, France) for providing umbilical cord blood.
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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