Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by del Pozo, V.
Right arrow Articles by Lahoz, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by del Pozo, V.
Right arrow Articles by Lahoz, C.
(Journal of Leukocyte Biology. 2000;68:31-37.)
© 2000 by Society for Leukocyte Biology

Expression on human eosinophils of CD148: a membrane tyrosine phosphatase. Implications in the effector function of eosinophils

Victoria del Pozo*, Fiorella Pirotto{dagger}, Blanca Cárdaba*, Isabel Cortegano*, Soledad Gallardo*, Marta Rojo*, Ignacio Arrieta*, Esther Aceituno*, Pilar Palomino*, Antoni Gaya{dagger} and Carlos Lahoz*

* Immunology Department, Fundación Jiménez Díaz, Madrid, Spain; and
{dagger} Servei d’Immunologia, Hospital Clinic, Barcelona, Spain

Correspondence: Dr. Carlos Lahoz, Immunology Department, Fundación Jiménez Díaz, Av. Reyes Catolicos 2, 28040 Madrid, Spain. E-mail: clahoz{at}fjd.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The role of protein tyrosine phosphatases (PTP) is crucial in regulating the phosphorylation status of cells. CD148 is a recently described membrane-type PTP. In this study, we have demonstrated that this molecule is expressed on human eosinophils and eosinophilic cell line EoL-3. Interestingly, our data also showed that this molecule acts as a transduction molecule on these cells. Thus, the crosslinking of CD148 was able to induce the degranulation and the induction of superoxide anion generation. By using specific inhibitor and by western blotting, we have shown that tyrosine kinase activation is involved in this transduction pathway. In addition, we have shown the presence of a serine/threonin kinase activity associated with CD148. In conclusion, the activation capacity of CD148 on eosinophils suggests a potential role of this molecule on inflammatory diseases, such as allergic and parasitic diseases, associated with eosinophilia.

Key Words: eosinophils • protein kinases/phosphatases • inflammatory mediators • superoxide anion • allergic reactions


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils are specialized blood cells that can carry out a wide range of functions in allergic, parasitic, and chronic inflammatory disease [1 ]. Some of their functions are mediated by the secretion of cytokines [2 3 4 5 ]; presentation of Ag to T lymphocytes [6 ]; secretion of cytotoxic products after receptor activation, such as nitric oxide, eosinophils cationic protein, major basic protein, and eosinophil-derived neurotoxin [7 8 9 ]; and generation of toxic-free radicals [10 11 12 ]. These eosinophil products are potentially important in the pathophysiology of allergic diseases.

The phosphorylation of proteins on tyrosine residues is a crucial event in the regulation of cellular processes, because the total level of protein phosphorylation is dependent on the enzymes, protein tyrosine phosphatases (PTP) and protein tyrosine kinases (PTK). Thus, the activation and inactivation of both enzymes determine the state of cell activation [13 , 14 ]. An increasing number of intracellular and transmembrane tyrosine phosphatases have been described during past years. Among the hematopoietic cells, CD45 is the most relevant membrane tyrosine phosphatase [15 16 17 ].

Recently, a new PTP (CD148) has been identified on the membrane of hematopoietic cells [18 ]. We have described the presence of this molecule on the membrane of human lymphocytes, monocytes, and, with higher intensity, granulocytes [19 ] and its identity with a previously described membrane PTP, HPTP{eta}/DEP-1 [20 , 21 ]. We have described recently the signaling capabilities of CD148 on human T lymphocytes. Our results showed that the crosslinking of CD148 is able to induce an increase in [Ca2+]i and tyrosine phosphorylation of several substrates [22 ]. In neutrophils, it has been described that cocrosslinking of CD148 with Fc{gamma}RIIa inhibits O2- production but does not inhibit [Ca2+]i rise, in contrast with CD45, which inhibits [Ca2+]i and O2- generation [23 ]. In addition, it has been shown that CD45 is able to modulate the activation of the inducible respiratory burst in eosinophils [24 ].

The aim of this study was to analyze the expression of CD148 on human eosinophils and its potential role as a signaling molecule on reactions mediated by eosinophils. Our results show the presence of CD148 on the membrane of human eosinophils and the human eosinophilic leukemia cell line EoL-3. The crosslinking of this molecule is able to induce the degranulation of eosinophils, measured by the release of eosinophil cationic protein (ECP) and eosinophil protein X (EPX), as well as the induction of superoxide anion generation. In addition, we have shown that CD148 is associated with a serine/threonin kinase that could be important in mediating these signaling mechanisms. The release of these mediators after activation with CD148 indicates the potential role of this molecule on inflammatory diseases associated with eosinophilia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Polinic patients were selected by the Allergy Department from Fundación Jiménez Díaz (Madrid, Spain) during pollen season, and they had an eosinophil count higher than 600 mm-3. Nonallergic persons were used as healthy donors. Heparinized peripheral blood was obtained after informed consent and approval by the Hospital Ethical Committee. Eosinophils were purified after a negative immunoselection technique using magnetic beads [9 , 25 ]. This procedure yielded >98% pure eosinophils detected using toluidine blue dye and eosin dye with no detectable lymphocyte or monocyte contamination. The absence of monocyte/macrophage was demonstrated in purified preparations of eosinophils by flow cytometry using CD64 (Landerdiagnostico, S.A., Madrid, Spain) as a marker of monocyte/macrophages and CD32 for eosinophil stain. EoL-3 was a generous gift from Dr. R. G. Lynch (University Iowa, Iowa City).

Cells were cultured in RPMI-1640 medium (Life Technologies, Refrenwshire, Scotland), supplemented with sodium pyruvate (5 mM), L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 µg/ml) (Flow Laboratories, Irvine, Scotland), and 10% heat-inactivated fetal bovine serum (FBS; Life Technologies).

Antibodies
Unconjugated horseradish peroxidase (HPRO) and a fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragment of goat anti-mouse (GAM) immunoglobulin (IgG) were purchased from Caltag Laboratories (Burlingame, CA). Isotype control was from Becton Dickinson (San Jose, CA), PE-very late antigen-4 and anti-DR2 were purchased from Pharmingen (San Diego, CA), and anti-Fc{gamma}RII (CD32) monoclonal antibody (mAb) IV.3 was provided by Landerdiagnostico S.A. CD148 mAb 143-41 has been described previously [18 ]. The two different polyclonal antisera we have used, 42 and 70, were generated and characterized by Jallal et al. [26 ] and kindly provided by Dr. Holsinger, Stanford University Medical School, Stanford, CA. Antiphosphotyrosine biotin-conjugated mAb (PY99) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunofluorescence assay
Cells were washed with phosphate-buffered saline (PBS) and incubated with specific monoclonal or isotype-matched control mAb for 30 min on ice. For two-color analysis, the simultaneous combination of anti-CD148 (FITC)-conjugated mAb and PE-conjugated VLA-4 was used. Where applicable, granulocytes were identified based on light-scatter characteristics.

Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from 2 x 106 purified eosinophils by the guanidine-thiocyanate method previously described [27 ], and 1 µg of RNA was converted to cDNA by the RT enzyme reaction (AMV transcriptase-reverse, Promega, Madison, WI) in a total volume of 20 µl.

PCR was performed in a final volume of 50 µl containing 2–4 µl of RT reaction product. The primers used for amplification of CD148 were 5' 148U1964 and 3' 148L2310, and amplification product was 346 bp [20 ]. A 15-µl aliquot from each PCR reaction was electrophoresed in a 2% agarose gel containing 0.5% ethidium bromide. The gel was then photographed under UV transillumination and submitted to southern blot hybridization.

Southern blot hybridization
One-third of the PCR products was fractionated on a 1.5% agarose gel and blotted onto nylon Zeta-Probe membranes (Bio-Rad Laboratories, Hercules, CA) using 0.4 N NaOH as transfer medium. Membranes washed were prehybridized in 6x saline-sodium phosphate ethylene diaminetetraacetate (EDTA; SSPE), 0.1% sodium dodecyl sulfate (SDS), 10x Denhart’s solution (0.2% Ficoll, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin (BSA); Pentax fraction V, Sigma Chemical Co., St. Louis, MO), and 0.1 mg/ml herring sperm DNA for 1 h at Tm-5 for each case.

Oligonucleotide probe (5' TGG ACT GGA AGA GCC CTG ACG GTG C 3'; 150 ng), specific for an internal sequence of the primers used in the amplification, was used as described [9 ].

EPX, ECP, and superoxide anion (O2-) generation
Eosinophils, 2 x 106/ml, were resuspended in RPMI with 20 mM HEPES and 5% fetal calf serum (FCS) and were incubated with isotype control, CD32 (5 µg/ml), CD148 (5 µg/ml), or oxophorbol 12,13-dibutyrate (Pbu2; 10 ng/ml) in microplates containing 100 µM cytochrome c (Type VI, Sigma). After addition of 5 µg/ml F(ab')2 GAM for 40 min at 37°C, plates were cooled and centrifuged at 1100 g for 10 min at 4°C.

Quantitative measurement of EPX was performed by Pharmacia EPX RIA (Pharmacia LKB Biotechnology, Upsala, Sweden). Tryptase and ECP production was measured by fluoroimmunoassay (Unicap Pharmacia). Superoxide production was quantified by measuring the reduction of cytochrome c at 550 nm. Data were calculated by substracting from samples the O.D. values obtained in the presence of 0.05 mg/ml superoxide dismutase (SOD; Sigma) and converted to nmoles of O2- release by using an extinction coefficient of 29.5 mm/cm.

In some experiments, different doses of inhibitors—genistein as a tyrosin kinase inhibitor and sodium pervanadate as a tyrosin phosphatase inhibitor—were added during the culture. These agents did not affect cell viability. Cells were treated with 500 µl of a solution containing 100 µg/ml of propidium iodide, 0.05% Nonidet P-40, and 0.02% RNase to assay apoptotic cells. After 1 h of incubation, the stained DNA was analyzed by flow cytometry.

Phosphotyrosine immunoblotting
Cells (2 x 106) were resuspended in 50 µl of RPMI 1640 medium containing 25 mM HEPES (pH 7.2) and were stimulated with 5 µg/ml of anti-CD148 mAb and 5 µg/ml of GAM for different periods of time. When the effects of genistein and pervanadate were studied, cells were preincubated for 1 h and 10 min in the presence or absence of each compound, respectively, before stimulation. After stimulation, cells were lysed by the addition of 80 µl of ice-cold lysis buffer (20mM Tris-HCl, 30mM Na4P2O7, 50 mM NaF, 40 mM NaCl, 5 mM EDTA, pH 7.4) containing 1% Nonidet P-40, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM Na3 VO4, and 0.5% deoxycholic acid. After 10 min on ice, the samples were centrifuged (5000 g, 1 min) to remove nuclear and cellular debris. The supernatants (70 µl) were mixed with 4 x SDS sample buffer (250 mM Tris-HCl, pH 6.8, 9.2% SDS, 40% glycerol, 20% 2-mercaptoethanol (2-ME), 0.004% bromophenol blue), boiled for 15 min, and loaded on 8,75% SDS-polyacrylamide gel electrophoresis (PAGE). The separated proteins were transferred to a nitrocellulose. The blocked membrane was incubated overnight with 0.1 µg/ml of the antiphosphotyrosine biotin-conjugated mAb (Santa Cruz Biotechnology) and, after washing, was incubated for 2 h with streptavidin-HRP (Pharmingen). Reactive proteins were detected with the enhanced chemiluminescence (ECL) system (Amersham Life Sciences, Arlington Heights, IL).

Cell lysis and immunoprecipitation
Cells (50x106) of EoL-3 were washed with ice-cold PBS, pH 8.0, and surface-biotinylated by incubating with biotinamidocaproic-acid-3-sulfo N-hydroxysuccinimide ester (1 mg/50x106; Sigma) during 30 min at room temperature. After washing with culture media with 10% of FCS, the cells were scraped into lysis buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM MnCl2, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 mM PMSF, 50 mM sodium fluoride). After removal of cell debris by centrifugation (14,000 g, 20 min), lysates were incubated with 10 µg of anti-CD148 polyclonal antiserum, and 42 and 30 µl of protein A-Sepharose for 3 h at 4°C with gentle agitation.

Immunoprecipitates were washed six times with buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM orthovanadate, 10 mM PMSF) and heated in SDS sample buffer for 5 min (90°C). Proteins were resolved by 8% SDS-PAGE and transferred to nitrocellulose. The blots were incubated with streptavidin-peroxidase conjugate (Boehringer Mannheim, Mannheim, Germany). After three washes, the filter was developed by a chemoluminescence substrate ECL (Amersham, Buckinghamshire, UK).

In vitro kinase assay
For in vitro kinase assays, cells were incubated in serum-free medium with 1 mM orthovanadate for 90 min. Cells were then washed with ice-cold PBS, pH 7.4, and scraped into lysis buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mM sodium pyrophosphate, 1 mM orthovanadate, 1 mM MnCl2, 10 µg/ml leupeptin, 2 mM PMSF, 10 µg/ml aprotinin) for 15 min on ice. After removal of cell debris by centrifugation (14,000 g, 20 min), lysates were incubated with 5 µl polyclonal antiserum, and 42 and 30 µl protein A-Sepharose beads (Pharmacia) for 3 h at 4°C with gentle agitation. Precipitates were washed with 4 x 1 ml HNTG buffer without EDTA and incubated with kinase assay buffer (100 mM NaCl, 20 mM HEPES, 5 mM MnCl2, 5 mM MgCl2, 1 µM adenosine 5'-triphosphate (ATP), 1 mM orthovanadate, 1 mM NaF, 5 µCi [{gamma}-32P] ATP (7000 Ci/mmol; Nuclear Iberica S.A., Spain) for 15 min at 4°C. The samples were diluted with HNTG with 20 mM EDTA, washed twice, and then mixed with SDS sample buffer. The samples were resolved on eight, 5% SDS-PAGE, and phosphorylated proteins were visualized by autoradiography.

Phosphoaminoacid analysis
After electrophoresis of the immunocomplex eluates from the kinase assay in eight, 5% SDS-PAGE gels, proteins were electrotransferred to membranes of nitrocellulose. These filters were exposed to detect the phosphorylated proteins. A 150-kDa protein was excised, extensively washed with distilled water to remove glycine and sodium chloride from the membrane, and incubated with 200 µl 6 N HCl at 110°C for 2 h [28 ]. After hydrolysis, the reaction tube was centrifuged at 14,000 g for 5 min, and the acid was transferred to a new tube, dried, resuspended in 200 µl water, and dried again. The residue was resuspended in 10 µl electrophoresis buffer, pH 3.5 (pyridine/acetic acid/water; 5:50:945), containing 1 µl of a phosphoaminoacid standard solution (phosphoserine, phosphothreonin, and phosphotyrosine, 5 mg/ml each). Samples of 2–10 µl were spotted on cellulose precoated plates (Merck, Rahway, NJ) and analyzed by 1-D thin-layer chromatography (TLC; 1000 V, 45 min). Nonradioactive standards were detected with 0.25% nihydrin in ethanol and radiolabeled phosphorylated amino acid by autoradiography.

Statistical analysis
Experiments were performed at least three times in duplicate or triplicate using different cell preparations. Data are expressed as the mean ± SD. Significance was determined by Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CD148 on eosinophils
To assay the expression of CD148 on human eosinophils, an immunofluorescence analysis on granulocytes from allergic patients, selected on the basis of light-scatter characteristics, was performed. Thus, granulocytes were double-immunostained by using a combination of antibodies anti-PE-VLA-4 and FITC-CD148. The representative results are shown in Figure 1A . As can be observed, all the granulocytes express CD148. Among them, the eosinophils (VLA-4+ cells) showed a high level of expression of CD148. When granulocytes from normal donors were analyzed in the same way, positivity was found in the few cells labeled with VLA-4. EoL-3, a leukemic eosinophilic cell line, was also tested for the expression of CD148. The immunofluorescence analysis of EoL-3 cells showed a clear reaction of 143-41 mAb with this cell line (Fig. 1A) , however Jurkatt, a T-cell line, does not express CD148 on its membrane.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 1. A, Flow cytometric analysis of eosinophils. Whole blood was stained with anti-CD148-FITC and anti-VLA-4-PE, as described in Materials and Methods. Granulocytes were selected on the basis of cell scatter and forward size, and VLA-4 expression was used to differentiate eosinophils (E) from neutrophils (N) (upper histograms). Also, expression of CD148 on EoL-3 and Jurkatt cells was assayed (middle and lower histograms). As negative controls, cells were stained with a control isotype-matched antibody (left histograms). B, Immunochemical characterization of CD148 molecule. EoL-3 cells were surface-biotinylated, and the CD148 molecule was immunoprecipitated by using polyclonal antibody anti-CD148 (lane 2) or preimmune serum (lane 1), as described in Materials and Methods. Samples were analyzed on 8% SDS-PAGE under reducing conditions. After blotting to nitrocellulose membranes and incubation with avidin-peroxidase, western blots were developed using ECL. C, RT-PCR specific to human CD148 of different purified eosinophil preparations. Upper row, Lanes E1–E4 are CD148 amplification products of eosinophils, from normal individual (E1) and allergic individuals (E2–E4); the amplified PCR fragment is 346 bp, and southern blot hybridization was performed with a 32P-labeled specific CD148 internal probe. Lane N, Neutrophils used as positive control. As CD148 negative cells, we used T- and B-cell lines from mouse. C is a negative control to which no RT has been added. Lower row, ß-Actin PCR was performed to assay loading differences.

 
To confirm the expression of CD148 on the EoL-3 cell line, we decided to immunoprecipitate the molecule from the cell surface. Thus, cells were biotinylated, and CD148 was immunoprecipitated by using an affinity-purified anti-DEP-1 antiserum. As can be observed in Figure 1B 1a clear band of ~240 kDa was observed, which is similar to that obtained by immunoprecipitating peripheral blood leukocytes (PBLs) [19 ]. A similar result was obtained by using 143-41 mAb (data not shown).

CD148 mRNA expression on eosinophils from allergic individuals and one healthy donor was studied by RT-PCR. RNA from purified eosinophils was obtained and reverse transcribed before PCR analysis. A primer pair amplifying a 346-bp segment of CD148 was used. As can be observed in Figure 1C , CD148 transcripts were present on four different preparations of pure human eosinophils. The specificity of PCR product was confirmed by southern blot, followed by hybridization with an internal probe (upper row). RNA from granulocyte preparation was used as positive control, because CD148 in neutrophils has been previously described [23 ]. RNA from T and B murine cell lines was included as negative controls, because the primers used are species-specific. In addition, it was included as a negative control to which RT was not added.

Eosinophil activation after CD148 crosslinking
One of the immediate consequences of eosinophil activation is the release of ECP and EPX. Thus, to test the activation capability of CD148 on eosinophils, we decided to measure the level of this protein on supernatants of human eosinophils purified from allergic patients after crosslinking with anti-CD148. As can be seen in Figure 2 , when freshly isolated eosinophils were stimulated via CD148 with 143-41 mAb, a statistically significant production of EPX and ECP was observed with respect to isotype control (P<0.05). The levels obtained were very similar to those obtained after activation via Fc{gamma}RII with anti-CD32 mAb. The use of an irrelevant mAb of the same isotype of anti-CD32 and anti-CD148 alone, or a control antibody reactive with eosinophils such as anti-DR2, induces a minimum production of ECP or EPX. In addition, none of these mAbs alone without the crosslinking agent (GAM) generated ECP or EPX production (data not shown). A nonrelated mediator such as tryptase, was used as negative control.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 2. Production of toxic mediators by eosinophils. Human-purified eosinophils were incubated with 5 µg/ml of anti-CD148, anti-CD32, anti-DR2, and control isotype, and after crosslinking with GAM F(ab')2, ECP, EPX, and tryptase, production was determined. Data are presented as mean ± SD of four independent human eosinophil preparations. *P < 0.05; significant augmentation of mediators by crosslinking CD148 with GAM. Pbu2 was added to 10 ng/ml as positive control.

 
To address the question of whether human eosinophils can be functionally activated by crosslinking of CD148, purified human eosinophils were incubated with 143-41 mAb (anti-CD148), and after crosslinking with GAM antibody, the generation of O2- was assessed. We used aggregation of CD32 (Fc{gamma}RII) as positive control. A weak basal level was detected on isotype-treated eosinophils. However, after CD148 crosslinking, purified eosinophils produced a significant quantity of superoxide anion comparable with those produced by engagement of CD32. Table 1 shows the mean ± SEM of nmoles of O2- produced in four independent experiments. Also, in this case, the crosslinking with GAM was necessary, because these mAbs alone generated little O2-. When control anti-DR2 was added, O2- production was not significant.


View this table:
[in this window]
[in a new window]
  		Table 1. Production of Anion Superoxide by Human Eosinophils

 
Crosslinking of CD148 in eosinophils produces tyrosine phosphorylation
We have shown that the crosslinking of CD148 is able to induce tyrosine phosphorylation on T lymphocytes [22 ]. To analyze whether a similar situation was also occurring on eosinophils, Eol-3 cells were stimulated with CD148 and crosslinked with F(ab')2 GAM. After immunoblotting, a tyrosine phosphorylation of cellular proteins with approximate molecular masses of 113, 105, 73, 38, and 32 kDa was found (Fig. 3A ). Tyrosine phosphorylation peaked at 15 min after crosslinking with GAM IgG and began to disminish to 30 min.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 3. A, Kinetics of tyrosine phosphorylation in Eol-3 cells stimulated by CD148. EoL-3 cells (2x106 cells/sample) were stimulated with anti-CD148 mAb (143-41) for the indicated period of time. The EoL-3 cells were lysed with lysis buffer, as described in Materials and Methods, and cell lysates were resolved on 8.75% SDS-PAGE and immunoblotted with biotinylated antiphosphotyrosine mAb (PY99). The immunoreactive proteins were detected with an ECL system. The arrows indicate 32, 38, 73, 105, and 113 kDa proteins. The figure is representative of two independent experiments showing similar results. B, Effects of genistein and pervanadate on CD148-induced tyrosine phosphorylation. Eol-3 cells were preincubated in the presence of 150 µM of genistein or 10 µM of pervanadate and were stimulated for 15 min with CD148 mAb. Tyrosine-phosphorylated proteins were detected by immunoblot, as described above. The arrows indicate 32, 38, 73, and 105 kDa proteins from bottom to top.

 
These results led us to investigate the effect of extracts of genistein, a well-characterized tyrosine kinase inhibitor in CD148-induced protein tyrosine phosphorylation, from drug-treated CD148-stimulated cells immunoblotted with antiphosphotyrosine Ab. To do this, Eol-3 cells were treated for 1 h before stimulation with 150 µM of genistein. As shown in Figure 3B , under these conditions, genistein inhibited the tyrosine phosphorylation of at least two proteins. However, exposured of Eol-3 to 10 µM of pervanadate for 10 min resulted in tyrosine phosphorylation of many intracellular proteins (Fig. 3B) .

We studied the effect of genistein and pervanadate on the O2- production induced by CD148 crosslinking on human purified eosinophils. As can be observed in Figure 4 , the O2- production after CD148 crosslinking had a dose-dependent inhibition in the presence of genistein. This inhibition was significant from 100 to 200 µM (P<0.05). In presence of pervanadate, an inhibitor of protein phosphatases, a dose-dependent inhibition of the O2- production was observed. Doses from 10 to 200 µM produce a complete inhibition (P<0.001).



View larger version (9K):
[in this window]
[in a new window]
  		Figure 4. Dose-response curves of different inhibitors on superoxide anion release. Different doses of genistein and sodium pervanadate were added to the human eosinophil preparations, and its effect on the O2- production induced by CD148 crosslinking was evaluated. Data represent mean ± SD, n = 4. The anion superoxide inhibition induced by inhibitors was significant with respect to that obtained after crosslinking with CD148, with doses above 150 µM of genistein and 10 µM of pervanadate, P < 0.05.

 
In all cases, it was confirmed that the inhibitions were not a result of an increased mortality, because the cell viability was not affected by the treatment with the different inhibitors (data not shown).

In vitro kinase assay
The inhibition observed in the presence of genistein implies the activation of tyrosine kinase. Taking into account that this enzymatic activity could not be developed directly by CD148, we considered it plausible to assume the existence of some kinase closely associated to CD148. To analyze the potential interaction of CD148 with a tyrosine or serine/threonin kinase, we decided to immunoprecipitate CD148 from the eosinophilic cell line Eol-3. The immunoprecipitates were subjected to in vitro kinase assays as described in Materials and Methods. The results, displayed in Figure 5 , clearly show that the incubation of CD148 immunocomplexes with P32-{gamma}-ATP resulted in the phosphorylation of several substrates. The most prominent bands detected correspond to proteins of 150, 135, 75, 67, and 50 kDa (Fig. 5A , lane 1). An additional band with a molecular weight (MW) higher than 240 kDa was also consistently observed. According to its mobility on SDS-PAGE, this band could correspond to CD148 itself. To confirm this hypothesis, EoL-3 cells were biotinylated before immunoprecipitation with an anti-CD148 polyclonal antibody, the membrane was incubated with streptavidin-peroxidase, and the proteins immunoprecipitated with the anti-CD148 antiserum could be visualized (Fig. 5A , lane 2). As can be observed in Figure 5A , the phosphorylated protein shows a MW higher than those corresponding to CD148 (arrow).



View larger version (36K):
[in this window]
[in a new window]
  		Figure 5. A, Western analysis of the in vitro kinase assay. EoL-3 cells were surface-biotinylated previously to the in vitro kinase assay, as described in Materials and Methods. After transferring to the nitrocellulose membrane, the phosphorylated bands were visualized by autoradiography (lane 1), and simultaneously the biotinylated proteins were evidenced by incubating with avidin peroxidase and revealed by chemiluminescence (lane 2). Lane 3 represents MW markers. B, Phosphoaminoacid analysis of in vitro-phosphorylated, 150-kDa protein. The band corresponding to 150 kDa was excised and treated, as described in Materials and Methods for phosphoaminoacid analysis. The relative positions of the ninhydrin-stained phosphoaminoacid markers are indicated. *150-kDa protein used for phosphoaminoacid analysis.

 
Next, we were interested in determining the amino acid specificity of the protein kinase activity present in CD148 immune complexes. Western blot analysis with antiphosphotyrosine mAb failed to detected phosphorylation of tyrosine residues in CD148 immunoprecipitates after in vitro kinase assays (data not shown). Thus, we decided to perform a phosphoaminoacid analysis of the 150-kDa phosphorylated band obtained after in vitro kinase assay (Fig. 5A , lane 1). The result of the analysis (Fig. 5B) established that the phosphorylated amino acids correspond to serine and threonin, thus suggesting the existence of a serine/threonin kinase associated to CD148.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated that purified human eosinophils and the eosinophilic leukemia cell line, Eol-3, express CD148 on its membrane, a recently identified receptor-type PTP. We have presented several pieces of evidence showing that CD148 is expressed on eosinophil membranes. First of all, we have identified CD148 on membrane eosinophils by flow cytometry. Secondly, CD148 was immunoprecipitated from an eosinophilic cell line with mAb (143-41) and polyclonal (antiserum 42) antibody. In both cases, a unique band around 240 kDa was obtained (Fig. 1B) . In Eol-3, the MW of the immunoprecipitated band does not seem to differ from that observed by us on PBLs [19 ]. Thirdly, CD148 gene expression was also confirmed by RT-PCR in purified eosinophils.

More interestingly, this molecule seems to be able to modulate eosinophil activation, measured by superoxide anion production and the release of ECP and EPX.

Previous studies about signal transduction in eosinophils have implicated PKC and Ca2+ mobilization [29 , 30 ], as well as tyrosine kinases [31 32 33 ], as a PTK-dependent signaling pathway that has been described to play an important role in triggering the eosinophil degranulation. Preliminary results from our laboratory point to calcium mobilization in eosinophils after CD148 cosslinkinking (data not shown).

Tyrosine phosphorylation is also involved in the superoxide anion production induced by vascular cell adhesion molecule 1 (VCAM-1) [34 ]. Thus, taking into account our previous results, we decided to analyze the potential signaling capabilities of CD148 on human eosinophils. Our results clearly showed that crosslinking CD148 was sufficient for inducing the respiratory burst and releasing specific toxic proteins from eosinophil granules, such as ECP and EPX, similarly to the stimulation with anti-CD32 or Pbu2. The importance of the release of these substances is based on the fact that these mediators are toxic to the tissues, playing a determinant role in the pathogenesis of asthma [35 , 36 ].

The involvement of PTKs was demonstrated by anti-pTyr western blot analysis of cell lysates and was confirmed with the use of several specific inhibitors. A dose-dependent inhibition was observed when a tyrosine kinase inhibitor, genistein, was used in the generation of oxigen metabolites. In fact, there are several reports supporting the implication of tyrosine kinases on superoxide anion production by eosinophils [37 , 38 ]. Kato et al. [31 ] demonstrated that pervanadate without stimulus induces eosinophil degranulation. In our case, the stimulation with CD148, which induces anion superoxide release, is inhibited by pervanadate (Fig. 4) . Pervanadate is a phosphotyrosine phosphatase inhibitor and has been known to act as a powerful stimulus for the accumulation of tyrosine phosphorylated proteins. Thus, the result obtained on phosphotyrosine immunoblot is not surprising; phosphorylation is similar to CD148, a phosphotyrosine phosphatase.

It could be considered contradictory that a tyrosine phosphatase could be able to induce tyrosine phosphorylation. However, a similar situation has been described for CD45, where the association of this molecule with p56lck, a scr-family tyrosine kinase, results in an increased activity of the PTK after CD45 crosslinking [39 ]. However, the results we have obtained in in vitro kinase assays do not show the existence of a PTK directly associated with CD148, at least in our experimental design. Instead, our result with the in vitro kinase assays showed the existence of a serine/threonin kinase activity present in the immunoprecipitates of CD148. Nevertheless, other kinases may act, because we only analyzed the p150 band, as a result of scarcity of the material. In fact, it has been identified as a constitutively associated 64-kDa serine/threonin kinase associated with DEP-1 in some epithelial tumor cell lines [26 ]. In addition, these authors have observed in anti-DEP-1 immunoprecipitates of pervanadate-treated cells the presence of a 62-kDa protein highly phosphorylated on tyrosine residues and have suggested that both proteins could be the same. If this were the case, it would be possible that the dephosphorylation of this kinase by CD148 would activate this enzyme, thus initiating a signaling pathway. One example of overlapping activities is when Bcr is phosphorylated on tyrosine, which greatly inhibits its serine/threonin kinase activity [40 ]. Conversely, the intervention of raf-1 kinase, identified as an important intermediate in signal transduction pathways, has been described recently as essential for eosinophil activation and degranulation and has a serine/threonin kinase activity [41 ].

Another possible explanation for our data is that after CD148 crosslinking, an inactivation of these receptors occurs, and tyrosine phosphatase is produced. Thus, CD148 is normally active playing an inhibitory role, but when it is crosslinked, it is inactivated, so tyrosine phosphorylation and superoxide production are enhanced. According to these data, it has been published that crosslinking or dimerization of tyrosine phosphatase receptors produces the inactivation of these proteins [42 , 43 ].

After in vitro kinase assay, several bands appeared phosphorylated. We have demonstrated that the phosphorylated protein shows a higher MW on SDS-PAGE and does not seem to correspond to CD148. Although in the cytoplasmic domain of CD148, there are several potential serine and threonin phosphorylation sites, and we have evidence that CD148 becomes phosphorylated after Pbu2 treatment (unpublished results), CD148 does not seem to be a substrate of the kinase that coimmunoprecipitates. Initial attempts to precipitate the kinase activity associated with CD148 using a glutathione S-transferase fusion protein of the intracellular domain of CD148 had not been successful. Although it should be tested in several other conditions, this result could reflect an indirect association of both molecules.

It should be noted that, only by using tyrosine phosphatase inhibitors, it has been possible to totally inhibit the signal transduction through CD148. Thus, it could be possible to establish the hypothesis that the dephosphorylation of some tyrosine residue is the first step in the activation pathway. The inhibitory effect observed after incubating with tyrosine kinase inhibitor, however, suggested that a more complex signaling pathway is involved.

In conclusion, this study demonstrates that the recently described protein tyrosine phosphatase, CD148, is present on eosinophil membranes and could play an important role in the process of eosinophil activation.


   ACKNOWLEDGEMENTS
 
This work has been supported in part by grant SAF96-0298 from the Spanish Ministerio de Educación y Cultura and grant 96-0788 from Fondo de Investigaciones Sanitarias.

Received December 16, 1999; revised February 17, 2000; accepted February 18, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sedgwick, J. B., Calhoum, F., Busse, W. W. (1994) Eosinophils and asthma Gleich, G. J. Kay, A. B. eds. Eosinophils in Allergy and Inflammation ,443-454 Marcel Dekker New York.
  2. Del Pozo, V., De Andrés, B., Martin, E., Maruri, N., Zubeldia, J. M., Palomino, P., Lahoz, C. (1990) Murine eosinophils and IL-1:{alpha}IL-1 mRNA detection by in situ hybridization J. Immunol. 144,3117-3122[Abstract]
  3. Kita, H., Ohnishi, T., Okubo, Y., Weiler, D., Abrams, J. S., Gleich, J. G. (1991) Granulocyte/macrophage colony-stimulating factor and interleukin 3 release from human peripheral blood eosinophils and neutrophils J. Exp. Med. 174,745-748[Abstract/Free Full Text]
  4. Hamid, Q., Barkans, J., Meng, Q., Ying, S., Abrams, J. S., Kay, A. B., Moqbel, R. (1992) Human eosinophils synthesize and secrete interleukin 6, in vitro Blood 80,1496-1501[Abstract/Free Full Text]
  5. Ying, S., Humbert, M., Barkans, J., Corrigan, C. G., Pfister, R., Menz, G., Larche, M., Robinson, D. S., Durham, S. R., Kay, A. B. (1997) Expression of IL-4 and IL-5 mRNA and protein product by CD4+ and CD8+ T cells, eosinophils, and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrisic) asthmatics J. Immunol. 158,3539-3544[Abstract]
  6. Del Pozo, V., De Andres, B., Martin, E., Cárdaba, B., Fernandez, J. C., Gallardo, S., Tramón, P., Leyva-Cobian, F., Palomino, P., Lahoz, C. (1992) Eosinophil as antigen-presenting cell: activation of T cell clones and T cell hybridization by eosinophils after antigen processing Eur. J. Immunol. 22,1919-1925[Medline]
  7. Frigas, E. (1982) The eosinophil and tissue damage Folia Allergol. Immunol. Clin. 29,51-55
  8. Desreumaux, P., Capron, M. (1996) Eosinophils in allergic reactions Curr. Opin. Immunol. 8,790-795[Medline]
  9. Del Pozo, V., de Arruda-Chaves, E., de Andrés, B., Cárdaba, B., López-Farre, A., Gallardo, S., Cortegano, I., Vidarte, L., Jurado, A., Sastre, J., Palomino, P., Lahoz, C. (1997) Eosinophils tanscribe and translate messenger RNA for inducible nitric oxide synthase J. Immunol. 158,859-864[Abstract]
  10. De Andres, B., Del Pozo, V., Martin, E., Palomino, P., Lahoz, C. (1990) Release of O2- and LTC4 by murine eosinophils: role of intra- and extracellular calcium Immunology 69,271-276[Medline]
  11. De Andrés, B., Raskasz, E., Hagen, M., McCormik, M. K., Mueller, A. L., Metwali, A., Sandor, M., Britigan, B. E., Weinstock, J. V., Lynch, R. G. (1997) Lack of Fc-{varepsilon} receptors on murine eosinophils: implications for the functional significance of elevated IgE and eosinophils in parasitic infections Blood 89,8826-8836
  12. Vadas, M. A., Lucas, C. M., Gamble, J. R., Lopez, A. F., Skinner, M. P., Berndt, M. C. (1994) Regulation of eosinophils functions by P-selectin Gleich, G. J. Kay, A. B. eds. Eosinophils in Allergy and Inflammation ,443-454 Marcel Dekker New York.
  13. Sun, H., Touks, N. K. (1994) The co-ordinated action of protein tyrosine phosphatases and kinases in cell signaling Trends Biochem. Sci. 19,480-485[Medline]
  14. Hunter, T. (1995) Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling Cell 80,225-236[Medline]
  15. Clark, E. A., Ledbetter, A. A. (1989) Leukocite cell surface enzymology: CD45 (LCA, T200) is a protein tyrosine phosphatase Immunol. Today 10,225-228[Medline]
  16. Martorell, J., Borche, L., Rojo, I., Vives, J. (1987) A second signal for T cell mitogenesis provided by monoclonal antibodies CD45 (T200) Eur. J. Immunol. 17,1447-1451[Medline]
  17. Alsinet, E., Inglés-Esteve, J., Vilella, R., Lozano, F., Milá, J., Rojo, I., Martorrell, J., Vives, J., Gayá, A. (1990) Differential effect of anti-CD45 monoclonal antibody on human B cell proliferation: a monoclonal antibody recognizing a neuroaminidase-sensitive epitope of the T-200 molecule enhances anti-immunoglobulin-induced proliferation Eur. J. Immunol. 20,2801-2804[Medline]
  18. Schraven, B., Hegen, M., Autschbactch, F., Gayá, A., Schwartz, C., Meuer, S. (1999) Cluster report CD148 Kishimoto, T. eds. Leukocyte Typing VI Garland New York.
  19. De la Fuente-Garcia, M. A., Nicolás, J. M., Freed, J. H., Palou, E., Thomas, A. P., Vilella, R., Vives, J., Gayá, A. (1998) CD148 is a membrane protein tyrosine phosphatase present in all hematopoietic lineages and is involved in signal transduction on lymphocytes Blood 91,2800-2809[Abstract/Free Full Text]
  20. Honda, H., Inazawa, J., Nishida, J., Yazaki, Y., Hiras, H. (1994) Molecular cloning, characterization, and chromosomal localization of a novel protein-tyrosine phosphatase, HPTP{eta} Blood 84,4186-4194[Abstract/Free Full Text]
  21. Östman, A., Yang, Q., Tonks, N. K. (1994) Expression of DEP-1, a receptor-like protein tyrosine-phosphatase, is enhanced with increasing cell density Proc. Natl. Acad. Sci. USA 91,9680-9684[Abstract/Free Full Text]
  22. Palou, E., de la Fuente, M. A., Nicolas, J. M., Vilardell, C., Vives, J., Gaya, A. (1997) CD148, a membrane protein tyrosine phosphatase, is able to induce tyrosine phosphorylation on human lymphocytes Immunol. Lett. 57,101-103[Medline]
  23. Hundt, M., Schmidt, R. E. (1997) Functional characterization of receptor-type protein tyrosine phosphatase CD148 (HPTP{eta}/DEP-1) in Fc{gamma} receptor IIa signal transduction of human neutrophils Eur. J. Immunol. 27,3532-3535[Medline]
  24. Liles, W. C., Ledbetter, J. A., Waltersdorph, A. W., Klebanoff, S. J. (1995) Cross-linking of CD45 enhances activation of the respiratory burst in response to specific stimuli in human phagocytes J. Immunol. 155,2175-2184[Abstract]
  25. Hansel, T. T., Pound, J. D., Pilling, D., Kitas, G. D., Salmon, M., Gentle, T. A., Lee, S. A., Thompson, R. A. (1989) Purification of human blood eosinophil by negative selection using immunomagnetic beads J. Immunol. Methods 122,97-103[Medline]
  26. Jallal, B., Mossie, K., Vasiloudis, G., Knyazev, P., Zachwieja, P., Clairvoyant, F., Schilling, J., Ullrich, A. (1997) The receptor-like protein-tyrosine phosphatase DEP-1 is constitutively associated with a 64-kDa protein serine/threonin kinase J. Biol. Chem. 272,12158-12163[Abstract/Free Full Text]
  27. Chomczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction Anal. Biochem. 162,156-159[Medline]
  28. Kamps, M. P., Sefton, B. M. (1989) Acid and base hydrolysis of phosphoproteins bond to immobilon facilitates analysis of phosphoaminoacids in gel fractionated proteins Anal. Biochem. 176,22-27[Medline]
  29. Dent, G., Muñoz, N. M., Rühlmann, E., Zhu, X., Leff, A. R., Magnussen, H., Rabe, K. F. (1998) Protein kinase inhibition enhances platelet-activating factor-induced eicosanoid production in human eosinophils Am. J. Respir. Cell. Mol. Biol. 18,136-144[Abstract/Free Full Text]
  30. Lindsay, M. A., Perkins, R. S., Barnes, P. J., Giembycz, M. A. (1998) Leukotriene B4 activates the NADPH oxidase in eosinophils by a pertussis toxin-sensitive mechanism that is largely independent of arachidonic acid mobilization J Immunol 160,4526-4534[Abstract/Free Full Text]
  31. Kato, M., Abraham, R. T., Kita, H. (1995) Tyrosine phosphorylation is required for eosinophil degranulation induced by immobilized immunoglobulins J. Immunol. 155,357-366[Abstract]
  32. Coffer, P. J., Schweizer, R. C., Dubois, G. R., Maikoe, T., Lammers, J-W. J., Koenderman, L. (1998) Analysis of signal transduction pathways in human eosinophils activated by chemoattractants and the T-helper 2-derived cytokines interleukin-4 and interleukin-5 Blood 91,2547-2557[Abstract/Free Full Text]
  33. Pazdrak, K., Adachi, T., Alam, R. (1997) Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival J. Exp. Med. 186,561-568[Abstract/Free Full Text]
  34. Nagata, M., Sedgwick, J. B., Bates, N. E., Kita, H., Busse, W. W. (1995) Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation J. Immunol. 155,2194-2202[Abstract]
  35. DeChatelet, L. R., Shirley, P. S., McPhail, L. C., Huntley, C. C., Muss, H. B., Bass, D. A. (1977) Oxidative metabolism of the human eosinophil Blood 50,525-535[Abstract/Free Full Text]
  36. Fujisawa, T., Terada, A., Atsuta, J., Iguchi, K., Kamiya, H., Sakurai, M. (1998) Clinical utility of serum levels of eosinophil cationic protein (ECP) for monitoring and predicting clinical course in childhood asthma Clin. Exp. Allergy 28,19-25[Medline]
  37. Nagata, M., Sedgwick, J. B., Busse, . W. (1997) Synergistic activation of eosinophil superoxide anion generation by VCAM-1 and GM-CSF Int. Arch. Allergy Immunol. 114(Suppl. 1),78-80
  38. Elsner, J., Höchtetter, R., Kimmig, D., Kapp, A. (1996) Human eotaxin represents a potent activator of the respiratory burst of human eosinophils Eur. J. Immunol. 26,1919-1925[Medline]
  39. Marie-Cardine, M., Maridonneau-Parini, Y., Fisher, S. (1994) Activation and internalization of p56lck upon CD45 triggering of Jurkat cells Eur. J. Immunol. 24,1255-1261[Medline]
  40. Liu, J., Wu, Y., Ma, G. Z., Lu, D., Haataja, L., Heisterkamp, N., Groffen, J., Arlinghaus, R. B. (1996) Inhibition of Bcr serine kinase by phosphorylation Mol. Cell. Biol. 16,998-1005[Abstract]
  41. Pazdrak, K., Olszewska-Pazdrak, B., Stafford, S., Garofalo, R. P., Alam, R. (1998) Lyn, jak2, and raf-1 kinases are critical for the antiapoptotic effect of interleukin 5, whereas only raf-1 kinase is essential for eosinophil activation and degranulation J. Exp. Med. 188,421-429[Abstract/Free Full Text]
  42. Bilwes, A. M., den Hertog, J., Hunter, T., Noel, J. P. (1996) Structural basis for inhibition of receptor protein-tyrosine phosphatase-{alpha} by dimerization Nature 382,555-559[Medline]
  43. den Hertog, J. G., Noel, S. J., Sap, J., Hunter, T. (1999) Dimerization inhibits the activity of receptor-like protein-tyrosine phosphatase-alpha Nature 401,606-610[Medline]



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
S. Kellie, G. Craggs, I. N. Bird, and G. E. Jones
The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation
J. Cell Sci., February 1, 2004; 117(4): 609 - 618.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by del Pozo, V.
Right arrow Articles by Lahoz, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by del Pozo, V.
Right arrow Articles by Lahoz, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS