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The Journal of Immunology, 2002, 168: 6084-6089.
Copyright © 2002 by The American Association of Immunologists

Apoptosis Mediated Through CD45 Is Independent of Its Phosphatase Activity and Association with Leukocyte Phosphatase-Associated Phosphoprotein1

Marylène Fortin*, Ann-Muriel Steff*, Jackie Felberg{dagger}, Isabelle Ding{ddagger}, Burkhart Schraven{ddagger}, Pauline Johnson{dagger} and Patrice Hugo2,*

* Division of Research and Development, PROCREA BioSciences, Inc., Montréal, Québec, Canada; {dagger} Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada; and {ddagger} Otto von Guericke Universität Magdeburg, Institute of Immunology, Magdeburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Besides the well-recognized role of CD45 as a major player in TCR signaling, we and others have demonstrated that cross-linking of CD45 with mAbs can induce cell death in T lymphocytes. To investigate the role of CD45 phosphatase activity in apoptosis induction, we expressed either wild-type or phosphatase-dead CD45 molecules in a CD45-deficient BW5147 T cell line. We show here that the phosphatase activity of CD45 was not required for apoptosis triggering after cross-linking of the molecule. It is noteworthy that a revertant of the CD45-negative BW5147 cell line, expressing a truncated form of CD45 lacking most of the cytoplasmic domain, was also susceptible to CD45-mediated death. Moreover, we also demonstrate that leukocyte phosphatase-associated phosphoprotein expression is totally dispensable for CD45-mediated apoptosis to occur. Taken together, these results strongly suggest a role for the extracellular and/or the transmembrane portion of CD45 in apoptosis signaling, which contrasts with the previously reported functions for CD45 in T lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD45 is a transmembrane protein tyrosine phosphatase (PTP)3 expressed on all nucleated cells in the hemopoietic system and is one of the most abundant glycoproteins at the surface of lymphoid cells (1, 2). It is now widely accepted that in lymphocytes, CD45 acts as a positive regulator of signal transduction through both T and B cell Ag receptors. Hence, mice deficient for CD45 expression demonstrate impaired T cell differentiation and activation due to defective TCR signaling as well as aberrant B cell activation (3, 4). Indeed, in CD45-deficient T lymphocytes, the protein tyrosine kinase p56lck is hyperphosphorylated (5, 6), and early TCR-mediated signaling events are altered (7, 8, 9, 10). These effects are mostly due to the lack of activation of p56lck, which is normally activated following the dephosphorylation of its C-terminal inhibitory tyrosine residue (Y505) by CD45, and by phosphorylation of its activatory tyrosine residue (Y394) after TCR engagement (11, 12). Reconstitution experiments based on the transfection of chimeric CD45 molecules in CD45-deficient cells have shown that the extracellular and transmembrane portions of CD45 are totally dispensable, but that intact phosphatase activity is both necessary and sufficient for the rescue of TCR signaling (13, 14, 15, 16). Moreover, the expression of a transgene encoding a constitutively active form of p56lck (in which the inhibitory Y505 is mutated) can restore T cell maturation in CD45-deficient mice (17). CD45-mediated activation events resulting from Ag receptor engagement are therefore clearly dependent upon its PTP activity. Furthermore, other functions of CD45 unrelated to Ag receptor signaling, such as regulation of Janus kinase-mediated cytokine signaling (18) and regulation of integrin-mediated T cell adhesion (19), have also been shown to require its PTP activity.

Previous work from our laboratory as well as others provided evidence that CD45 cross-linking with mAbs in the absence of TCR engagement induces apoptosis of murine thymocytes (20) or human mature T and B cells (21). Cell death induction, following the cross-linking of CD45 with mAbs on murine thymocytes, exhibited known hallmarks of apoptosis, such as reduction in mitochondrial membrane potential, production of reactive oxygen species, exposure of phosphatidylserine residues, and loosening of membrane phospholipids (20). However, in striking contrast to most stimuli causing thymocyte apoptosis, CD45 cross-linking with mAbs did not lead to DNA degradation into characteristic nucleosomal fragments (20, 21). Moreover, CD45-mediated killing could not be inhibited by either overexpression of the anti-apoptotic gene bcl-2 or by treatment with a broad caspase inhibitor (20). Such a caspase-independent mechanism of apoptosis induction has also been reported to occur with other stimuli (22).

Here we sought to gain insights into the molecular mechanism by which CD45 cross-linking with mAbs induces apoptosis in murine T cells and, more specifically, the relative importance of CD45 PTP activity in this process. We have used BW5147 thymoma T cell lines expressing various mutants of the CD45 molecule to directly show that neither CD45 PTP activity nor most of the CD45 intracytoplasmic domain is required for apoptosis signaling. Moreover, we demonstrate that CD45-mediated apoptosis does not rely on the presence of the CD45-associated protein leukocyte phosphatase-associated phosphoprotein (LPAP). These results are in sharp contrast to all other activities previously attributed to CD45 in leukocytes, which were demonstrated to rely on the PTP activity of the molecule.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and mice

The thymoma T cell line BW5147.G1.4 (ATCC TIB-48, American Type Culture Collection, Manassas, VA), hereafter named BW5147, and its CD45-negative variant T200-a (ATCC TIB-233, American Type Culture Collection), hereafter named T200-, have been described previously (23, 24). A revertant of the T200- cell line (hereafter named REV) was also used in this study (provided by Dr. R. Hyman, Department of Cancer Biology, The Salk Institute, San Diego, CA). These cells express a truncated form of CD45 lacking most of its cytoplasmic domain (25). Thymocytes were obtained from 4- to 8-wk-old C57BL/6 mice (Charles River, St. Constant, Canada) or LPAP-deficient mice (26).

Generation of T200- cell lines expressing wild-type or PTP-dead CD45 molecules

The bicistronic retroviral vector LZRS-pBMN-IRES-enhanced green fluorescent protein (provided by Dr. H. Spits, Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands) was used to generate two retroviral constructs, comprising the murine cDNAs for either the wild-type CD45 RABC molecule (27) or the PTP-dead version, which is identical with the wild-type (RABC) molecule except that it contains a C817S mutation in the proximal PTP domain (28). The retroviral constructs were transfected with the Lipofectamine reagent (Life Technologies, Burlington, Canada) into the BOSC23 packaging cell line (provided by Dr. D. Baltimore, Rockefeller University, New York, NY) (29). After puromycin selection, the supernatants from confluent BOSC23 cultures were harvested and used to infect T200- cells. One week after infection, stable expression of either wild-type or PTP-dead CD45 was monitored using biotinylated anti-CD45 mAb (ALI-4A2) (30) and PE-conjugated streptavidin (BD Biosciences, San Jose, CA), and cells expressing both enhanced green fluorescent protein and CD45 were sorted on a FACStar cytometer (BD Biosciences). To confirm that the transduced cells did indeed express the correct CD45 molecules, RNA was isolated from each cell type, and RT-PCR fragments containing the catalytic cysteine (C817) were subcloned into a plasmid vector and subjected to restriction analysis, which allowed distinction between wild-type and PTP-dead molecules (data not shown).

Apoptosis induction through CD45 cross-linking using Ab-coated beads

Latex microbeads (Interfacial Dynamics Corp., Portland, OR), 6 µm in diameter, were coated with either pan-specific anti-CD45 mAb (ALI-4A2) (30) or isotype-matched control mAb (H8H9D1) (31) as described previously (32). Briefly, 20 x 106 microbeads were incubated overnight at room temperature with 100 µg mAb in 1 ml PBS with gentle agitation. The beads were then blocked with FCS and washed twice in PBS. For apoptosis induction experiments, thymocytes from C56BL/6 or LPAP knockout (KO) mice (5 x 105 cells in 125 µl RPMI/10% FCS) or BW cell lines (2 x 105 cells in 200 µl RPMI/5% FCS) were added to 96-well plates, and a 10 µl stock suspension of mAb-coated beads was added to reach various bead-to-cell ratios (ranging from 0.5:1 to 8:1). After a 3-h incubation at 37°C in 5% CO2, the samples were harvested, washed in PBS, and stained for apoptotic markers. When indicated, the cells were treated with 10-6 M dexamethasone (dex; Sigma Aldrich, Oakville, Canada) and/or 50 µM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk; Kamiya Biomedical, Seattle, WA).

Apoptosis detection by flow cytometry

Loosening of membrane phospholipids and exposure of phosphatidylserine residues were detected using merocyanin-540 (MC540; Molecular Probes, Eugene, OR) or FITC-conjugated annexin V (BD Biosciences), respectively. The staining procedures were described previously (20). Samples were run on a Coulter EPICS XL flow cytometer (Coulter Electronics, Ville St. Laurent, Canada) and analyzed using Win MDI software (http://facs.scripps.edu). For determination of the percentage of apoptotic cells, electronic gating was performed to exclude beads from the analysis.

PTP assays and Western blots

BW cell lines were lysed in 0.5% Brij-97 TNE (10 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.2 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin). Anti-murine CD45 mAb (I3/2), recognizing a common extracellular determinant of CD45 (33), was conjugated to cyanogen bromide-activated Sepharose beads (4 mg/ml) and used to immunoprecipitate CD45. CD45 was immunoprecipitated using 10 µl I3/2-conjugated Sepharose beads per 2 x 106 cells per 200 µl lysis buffer, and the immunoprecipitate was washed with 0.2% Brij-97 TNE. The CD45 immunoprecipitate was tested for phosphatase activity using the malachite green assay. Substrate for the PTP assay was a phosphorylated 9-mer peptide corresponding to the autophosphorylation site of Lck, EDNEpYTARE, used at a final concentration of 2 mM. Details of the CD45 immunoprecipitate and malachite green phosphatase assay were described previously (34). R01.1, a rabbit antisera against the cytoplasmic domain of CD45 (35) and 131, a rabbit antisera against a peptide derived from the common region of the extracellular domain (36), were used in Western blots to detect the intracellular and extracellular regions of CD45, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Apoptosis induction following CD45 cross-linking with mAb-coated beads in the BW5147 cell line is caspase independent and does not involve bystander effect

We have previously reported that CD45 cross-linking with immobilized mAbs on murine thymocytes resulted in apoptotic cell death by virtue of a caspase-independent pathway (20). To better define the domains of the CD45 molecule responsible for cell death induction, we used the CD45-negative variant of the BW5147 cell line (T200-) reconstituted with either wild-type or mutant CD45 molecules. We first wanted to ascertain that apoptosis induction following CD45 cross-linking on the BW5147 cell line proceeded through the same pathway as for thymocytes. For this purpose we directly compared apoptosis induction in thymocytes and BW5147 cells, using pan-specific anti-CD45 mAbs coated on microbeads, allowing very high levels of CD45-mediated apoptosis (Fig. 1GoA). Importantly, neither thymocytes nor BW5147 cell death was inhibited by the addition of the broad caspase inhibitor z-VAD-fmk, while apoptosis in thymocytes treated with dex was readily blocked by this drug (Fig. 1GoA). Moreover, z-VAD-fmk was inefficient at inhibiting CD45-mediated apoptosis in BW5147 cells in a dose-response experiment, where z-VAD-fmk was used at concentrations up to 200 µM (data not shown). These data strongly support the idea that a similar caspase-independent apoptotic pathway is triggered in BW5147 cells and thymocytes in response to CD45 engagement.



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  		FIGURE 1. Apoptosis induction following CD45 cross-linking with mAb-coated beads in the BW5147 cell line is caspase independent and does not involve a bystander effect. A, BW5147 cells or thymocytes from C57BL/6 mice were preincubated for 15 min in the absence or the presence of 50 µM z-VAD-fmk. The cells were then incubated with either isotype control or anti-CD45-coated beads at a 4:1 or 2:1 bead-to-cell ratio for BW5147 or thymocytes, respectively. Thymocytes were also treated with dex at 10-6 M. After a 3-h incubation, the samples were harvested, stained with FITC-conjugated annexin V, and analyzed by flow cytometry. Results are expressed as the mean percentage (±SD) of triplicate determinations of annexin-positive cells (over background death obtained with isotype control beads) and are representative of three independent experiments. B, BW1547 cells (CD45-positive) and T200- cells (CD45-negative) were mixed in equal proportions and incubated with isotype control or anti-CD45 mAb-coated beads at a 4:1 bead-to-cell ratio. After a 3-h incubation, the samples were harvested and stained with MC540 and an FITC-conjugated pan-specific anti-CD45 mAb (M1/89). Electronic gating was used to determine the percentages of apoptotic cells within the CD45-positive or CD45-negative cell populations. Results are expressed as the mean percentage (±SD) of triplicate determinations of MC540-positive cells (over background death obtained with isotype control beads).

 
We then verified that apoptosis directly ensues from CD45 cross-linking and not from a bystander effect, which we define as indirect killing of the cells mediated through the release into the medium of a soluble pro-apoptotic molecule. To this end, BW1547 (CD45-positive) and T200- (CD45-negative) cells were mixed in equal proportions and incubated with anti-CD45-coated beads. Fig. 1GoB shows that apoptotic cells were exclusively found within the CD45-positive population, thus excluding the possibility of a nonspecific bystander effect in this system. In addition, we asked whether aggregation of the cells, possibly resulting from the use of mAb-coated beads as opposed to mAb-coated plates, could be responsible for nonspecific cell death induction. We incubated BW5147 cells with mAb-coated beads directed against another abundant cell surface molecule (anti-LFA-1-coated beads) and found that there was no apoptosis induction (data not shown). This suggests that the sole aggregation of cells by mAb-coated beads does not explain apoptosis observed using anti-CD45-coated beads. Taken together, these results provide evidence that anti-CD45-coated beads and the BW5147 cell line can be used as a model system to study CD45-mediated apoptosis in T cells.

C817S-mutated, PTP-dead CD45 molecules can restore sensitivity toward CD45-mediated apoptosis in CD45-deficient cells

Mutation of the critical cysteine residue (C817), located in the membrane proximal phosphatase (D1) domain of CD45, was previously shown to totally abrogate PTP activity of the molecule when tested in vitro against a variety of phosphorylated substrates or in cells in response to TCR engagement (15, 28). To directly test the requirement for PTP activity in CD45-mediated apoptosis, we reconstituted the T200- cell line (CD45 deficient) with either wild-type (CD45 RABC) or mutated, catalytically dead molecules (CD45 C817S). These two cell lines were sorted by FACS to obtain similar levels of CD45 expression at their surface (Fig. 2GoA). As expected, re-introduction of wild-type CD45 molecules in T200- cells restored the sensitivity toward CD45-mediated apoptosis in a dose-dependent manner (Fig. 2GoB). Importantly, expression of a PTP-dead version of CD45 also allowed the T200- cells to become sensitive to apoptosis induction following CD45 cross-linking (Fig. 2GoB). Of note, CD45-mediated apoptosis in the RABC cells or C817S cells could not be inhibited by incubation with z-VAD-fmk (data not shown). These results thus strongly suggest that CD45-induced death can occur without any requirement for PTP activity.



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  		FIGURE 2. PTP activity is not required for CD45-mediated apoptosis. A, CD45 expression levels, as determined with biotinylated anti-CD45 mAb (ALI-4A2) and PE-conjugated streptavidin, are shown for T200- (dashed line), RABC (thick line), or C817S (solid fill) cells, expressing none, wild-type, or PTP-dead CD45 molecules, respectively. B, T200-, RABC, or C817S cells were treated for 3 h with increasing amounts of either isotype-control or anti-CD45 beads to obtain various bead-to-cell ratios and stained for apoptosis induction using MC540. Results are expressed as the mean percentage (±SD) of triplicate determinations of MC540-positive cells (above control) and are representative of three independent experiments.

 
Most of the cytoplasmic domain of CD45 is dispensable for apoptosis induction

Given the lack of requirement for PTP activity for efficient cell death induction, we speculated that one or both of the PTP domains of CD45 might be required for interaction with downstream signaling molecules involved in the apoptotic pathway. To verify this hypothesis, we took advantage of a revertant of the T200- cell line (REV) characterized by the expression of a CD45 molecule lacking virtually all the cytoplasmic domain, including both PTP domains (25). We sorted both the REV and the BW5147 cell lines either to increase or decrease CD45 expression, respectively, which resulted in cells displaying similar level of expression (Fig. 3GoA). As illustrated in Fig. 3GoB, REV cells were killed by anti-CD45-coated beads, with a similar dose-response curve as for BW5147 cells. In addition, z-VAD-fmk could not inhibit CD45-mediated apoptosis in REV cells (data not shown). These results indicate that none of the PTP domains of CD45 is involved in apoptosis induction. However, given that the precise amino acid sequence of mutant CD45 expressed by REV cells is not known, we cannot exclude the possibility, although highly unlikely, that a short intracytoplasmic segment (25) mediates interactions with downstream protein partners.



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  		FIGURE 3. Most of the CD45 cytoplasmic domain is dispensable for apoptosis induction. A, CD45 expression levels, as determined with biotinylated anti-CD45 mAb (ALI-4A2) and PE-conjugated streptavidin, are shown for T200- (dashed line), BW5147 (thick line), or REV (solid fill) cells, expressing none, wild-type, or truncated CD45 molecules, respectively. B, T200-, BW5147, or REV cells were treated for 3 h with increasing amounts of either isotype control or anti-CD45 beads to obtain various bead-to-cell ratios and stained for apoptosis induction using MC540. Results are expressed as the mean percentage (±SD) of triplicate determinations of MC540-positive cells (above control) and are representative of three independent experiments.

 
Immunoprecipitated CD45 molecules from the T200- revertant or the C817S transfectant do not have detectable PTP activity

The demonstration that both C817S and truncated CD45 molecules could transmit an apoptotic signal, and the fact that the exact nature of the truncation in the REV cells is not known, prompted us to confirm the lack of in vitro phosphatase activity of these CD45 variants. To this end, immunoprecipitated CD45 molecules from the T200- transfectants and revertant were tested for PTP activity against a phosphopeptide corresponding to the autophosphorylation site of Lck. While CD45 molecules from RABC-transduced T200- cells could efficiently dephosphorylate the substrate (Fig. 4GoA), CD45 immunoprecipitated from REV or C817S cells were completely devoid of phosphatase activity (Fig. 4GoA) even when 10 times more cell equivalents were used (data not shown). These results are consistent with previous reports (6, 28) and totally rule out the possibility that residual PTP activity in the C817S or the REV CD45 molecules could account for apoptosis signaling.



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  		FIGURE 4. Immunoprecipitated CD45 molecules from the T200- revertant or the C817S transfectant do not have detectable PTP activity. A, Phosphatase activity of CD45 immunoprecipitated from 5 x 104 cell equivalents from T200- cells, T200- revertant (REV), or T200- cells transfected with the catalytically dead mutant C817S or with the wild-type CD45 RABC. The rate of phosphatase activity was determined by measuring the amount of phosphate released over time using the malachite green reagent as described previously (34 ). B, Western blots of CD45 immunoprecipitated from 8 x 105 cell equivalents using 131, an antiserum raised against a peptide derived from the extracellular region of CD45 (left panel), or R01.1, an antisera raised against the cytoplasmic domain of CD45 (right panel). The positions of the molecular mass markers are indicated on the left in kilodaltons. Results are representative of three independent experiments.

 
CD45 molecules expressed by the RABC or C817S transfectants could be readily detected by Western blot, using antisera directed against either the extracellular (Fig. 4GoB, left panel) or cytoplasmic (Fig. 4GoB, right panel) domain. In addition, truncated CD45 molecules expressed in the REV cells could be detected by the extracellular anti-CD45 antiserum, yet with reduced molecular mass (~100 kDa), as previously reported (25) (Fig. 4GoB, left panel). In contrast, the REV molecule could not be detected by an antiserum directed against the cytoplasmic domain of CD45 (Fig. 4GoB, right panel). This antiserum can detect the membrane-proximal region of CD45 (i.e., the first 67 aa of the cytoplasmic portion) expressed as a fusion protein with maltose-binding protein as well as both recombinant D1 and D2 PTP domains of CD45 (J. Felberg and P. Johnson, unpublished observations). This suggests that the truncated CD45 expressed in REV cells is indeed missing most of the cytoplasmic domain, including both phosphatase domains. Therefore, none of the PTP domains of CD45 is involved in apoptosis signaling upon cross-linking of the molecule.

Apoptosis induction through CD45 does not depend on its association with LPAP

Given that most of the intracellular part of CD45 is dispensable for apoptosis signaling, we hypothesized that molecules interacting with CD45 through its transmembrane portion could be involved in cell death induction. One candidate for such a function would be LPAP (also termed CD45-AP). Indeed, it has been shown that LPAP and CD45 interact through their respective transmembrane domains (37, 38). LPAP is a 32-kDa phosphoprotein of as yet unknown function that is specifically expressed in T and B cells (37, 39, 40, 41). Although the biological function of LPAP is not well defined, one interesting observation is that the lymph nodes from LPAP-deficient mice show an increase in cellularity compared with those from wild-type mice, which could be indicative of a role for LPAP in the regulation of CD45-mediated apoptosis (26). We therefore tested the sensitivity of LPAP KO thymocytes to CD45-mediated killing. As depicted in Fig. 5Go, LPAP KO thymocytes were as susceptible as wild-type thymocytes to anti-CD45 bead treatment, notwithstanding the fact that LPAP KO thymocytes exhibited a slight decrease in CD45 expression (26). These data undoubtedly show that LPAP deficiency does not impinge on CD45-mediated killing.



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  		FIGURE 5. Apoptosis induction through CD45 does not rely on its association with LPAP. Thymocytes from C57BL/6 (control) or LPAP KO mice were incubated with either isotype control or anti-CD45-coated beads at a 2:1 bead-to-cell ratio. After a 3-h incubation, the samples were harvested and stained with MC540. Results are expressed as the mean percentage (±SD) of triplicate determinations of MC540-positive cells (over background death obtained with isotype control beads) and are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented above definitely demonstrate that anti-CD45 mAb-mediated apoptosis in murine T cells is independent of CD45 PTP activity and of most of its intracytoplasmic domain. Given that p56lck is a major substrate for CD45 in T cells, these results are supported by our previous observation that CD45 cross-linking could efficiently induce apoptosis in thymocytes from p56lck-deficient mice (20). Our results, however, could be in apparent discrepancy from those previously reported by Klaus et al. (21) for CD45-mediated apoptosis in human T and B lymphocytes, which was proposed to rely on CD45 PTP activity. This might be explained by the fact that these authors based this assumption on the indirect observation that CD45-mediated apoptosis could be blocked by broad PTP inhibitors, which may have targeted protein tyrosine phosphatases other than CD45. We have also shown that apoptosis induction through CD45 cross-linking can occur in thymocytes from LPAP KO mice. This may suggest either that heretofore unknown protein partners interacting with CD45 through the transmembrane portion could be implicated or that the transmembrane domain of CD45 is completely dispensable for apoptosis signaling. In that sense it would be of interest to verify whether glycosylphosphatidylinositol-linked forms of CD45 would still be able to trigger cell death. Likewise, CD45 is known to coassociate in cis via its extracellular domain with other molecules on the T cell surface, including TCR and CD4 (42), Thy-1 (43), CD2 (44), and CD26 (45). It is therefore possible that one or more of these protein partners is required for transmitting an apoptotic signal. We can already exclude an essential role for CD4 or TCR, as we have shown that thymocytes from CD4 KO or TCR{alpha} KO mice (20) are sensitive to CD45-mediated death.

In vivo, CD45 cross-linking could be achieved through binding to one of its putative physiologic ligands, such as galectin-1, a member of the {beta}-galactoside binding protein family, expressed in thymus, spleen, lymph nodes, and bone marrow (46). Indeed, in vitro CD45 cross-linking using recombinant galectin-1 was shown to induce apoptosis in human thymocytes (47) and in activated human T cells and T leukemic cell lines (48). However, in contrast to anti-CD45 mAb-mediated apoptosis (20, 21), galectin-1-mediated death is associated with DNA degradation (47, 48), suggesting that other molecules are involved in galectin-1-induced apoptosis. In fact, the oligosaccharide ligand recognized by galectin-1 is ubiquitously distributed, and many T cell surface glycoprotein receptors were shown to bind to galectin-1 (46), among which CD43 and CD7 also appeared to participate in galectin-1-induced apoptosis (49, 50). Therefore, it is clear that although nonphysiologic, specific mAbs to CD45 constitute a better alternative to study apoptosis induction through CD45, as galectin-1 acts through several receptors simultaneously. Moreover, it is possible that the concentration of CD45 molecules in defined membrane domains (e.g., rafts) through specific contacts with cis-interacting partners, rather than interaction with a particular ligand, would be sufficient for the transmission of an apoptotic signal under given conditions.

Taken together, our data all converge toward a role for the extracellular and/or transmembrane domain of CD45 in apoptosis signaling. Indeed, our results provide compelling evidence that neither CD45 PTP activity, most of the CD45 intracellular domain, nor LPAP expression is required to trigger apoptosis following anti-CD45 mAb cross-linking in T cells. To our knowledge, this is the first function reported for CD45 in leukocytes that does not rely on its PTP activity.


   Acknowledgments
 
We thank Dr. H. Spits for the kind gift of the bicistronic retroviral vector, Dr. R. Hyman for the gift of the REV cell line, Dr. D. Baltimore for the BOSC23 packaging cell line, C. Arguin for expert technical assistance, and the Flow Cytometry Unit at the Clinical Research Institute of Montréal for cell sorting.


   Footnotes
 
1 This work was supported by PROCREA BioSciences, Inc. Back

2 Address correspondence and reprint requests to Dr. Patrice Hugo, PROCREA BioSciences, Inc., Division of Research and Development, 6100 Royalmount, Montréal, Québec, Canada H4P 2R2. E-mail address: phugo{at}procrea.qc.ca Back

3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; dex, dexamethasone; KO, knockout; LPAP, leukocyte phosphatase-associated phosphoprotein; MC540, merocyanin 540; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Back

Received for publication March 9, 2001. Accepted for publication April 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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