Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0303095 on September 12, 2003

Published online before print September 12, 2003
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(Journal of Leukocyte Biology. 2003;74:1083-1093.)
© 2003 by Society for Leukocyte Biology

T cell aggregation induced through CD43: intracellular signals and inhibition by the immunomodulatory drug leflunomide

Esther Layseca-Espinosa*,{dagger}, Gustavo Pedraza-Alva*, José Luis Montiel*, Roxana del Río*, Nora A. Fierro*, Roberto González-Amaro{dagger} and Yvonne Rosenstein*,1

* Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos; and
{dagger} Department of Immunology, Facultad de Medicina, Universidad Autónoma de San Luis Potosí, México

1 Correspondence: Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 62250, Mexico. E-mail: yvonne{at}ibt.unam.mx


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CD43 coreceptor molecule has been shown to participate in lymphocyte adhesion and activation. Leukocyte homotypic aggregation results from a cascade of intracellular signals delivered to the cells upon engagement of different cell-surface molecules with their natural ligands. This phenomenon requires an active metabolism, reorganization of the cytoskeleton, and relocalization of cell-surface molecules. The aim of this study was to identify some of the key members of the signaling cascade leading to T lymphocyte homotypic aggregation following CD43 engagement. CD43-mediated homotypic aggregation of T lymphocytes required the participation of Src kinases, phospholipase C-{gamma}2, protein kinase C, phosphatidylinositol-3 kinase, as well as extracellular-regulated kinase 1/2 and p38. Data shown here suggest that these signaling molecules play a central role in regulating actin cytoskeleton remodeling after CD43 ligation. We also evaluated the ability of immunomodulatory drugs such as leflunomide to block the CD43-mediated homotypic aggregation. Leflunomide blocked the recruitment of targets of the Src family kinases as well as actin polymerization, diminishing the ability of T lymphocytes to aggregate in response to CD43-specific signals, suggesting that this drug might control the migration and recruitment of lymphoid cells to inflamed tissues.

Key Words: adhesion • leukosialin • cytoskeleton • lymphocyte • signaling pathways


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intercellular adhesion phenomena play an important role in normal and pathological processes, such as immune response, thrombosis, and metastasis of tumor cells. Cell–cell adhesion is essential for the appropriate migration, differentiation, and activation of lymphoid cells [1 ]. In addition, leukocyte–leukocyte interactions are thought to constitute a mechanism to recruit leukocytes to inflamed tissues. Thus, understanding the molecular interactions that regulate cell aggregation is a first step for the generation of immunomodulatory drugs targeted to reduce recruitment of lymphoid cells to inflammatory foci.

Leukocyte homotypic aggregation results from a cascade of intracellular signals delivered to the cells following engagement of different cell-surface molecules with their natural ligands or with specific antibodies. Molecules such as protein tyrosine kinases (PTKs), phosphatidylinositol-3 kinase (PI-3K), phospholipase C (PLC)-{gamma}, Vav, and mitogen-activated protein kinase (MAPK) participate in cytoskeleton reorganization and relocalization of membrane receptors, leading to cell-adhesion phenomena [2 3 4 5 ]. Syk couples activated immunoreceptors to downstream signaling events that mediate diverse cellular responses including adhesion, proliferation, differentiation, and phagocytosis [6 7 8 9 ]. PLC-{gamma} has been involved in multiple cellular processes such as cytoskeletal assembly, mitogenesis, chemotaxis, and secretion. Activation of PLC-{gamma} requires phosphorylation of key tyrosine residues by kinases such as Syk as well as its targeting to the plasma membrane through the association of its PH domain with Pi 3,4,5-triphosphate (PIP3), a product of PI-3K [10 11 12 ]. Two isoforms of PLC-{gamma} have been identified in mammals. PLC-{gamma}1 is a ubiquitously expressed protein, and PLC-{gamma}2 is restricted to hematopoietic lineages. PLC-{gamma}1 is required for T cell and natural killer (NK) function, whereas PLC-{gamma}2 has been found to be more important in mast cells, NK cells, B cells, and platelets, yet little is known about PLC-{gamma}2 function in T lymphocytes [13 ].

The CD43 coreceptor is an abundant glycoprotein (~1.5x105 molecules/cell) expressed on the membrane of all hematopoietic cells except erythrocytes and resting B cells [14 , 15 ]. CD43 has a 235-amino acid (aa) extracellular domain, a 23-aa transmembrane domain, and a 123-aa cytoplasmic domain, all encoded by a single exon [16 ]. The intracytoplasmic region of the protein is necessary to transduce signals; it is rich in potentially phosphorylable threonines and serines [17 , 18 ] but lacks tyrosine residues as well as catalytic activity. CD43 engagement on human peripheral blood T cells and monocytes leads to cell activation and proliferation through the generation of second messengers such as diacylglycerol and inositol phosphates, protein kinase C (PKC) activation and Ca2+ mobilization [19 , 20 ]. In addition, CD43 ligation on human T cells induces the association of CD43 with Src family kinases, presumably through the interaction of their Src homology 3 domain with a proline-rich region of the CD43 intracytoplasmic tail [21 , 22 ]. Following Fyn and Lck recruitment, CD43-mediated signals result in the formation of macromolecular complexes, which comprise the molecular adapters Shc, Grb2, and SLP-76, as well as the guanine exchange factor Vav, ultimately resulting in the activation of the MAPK pathway and regulation of gene expression [23 , 24 ]. Recently, CD43 was found to interact with members of the ezrin-radixin-moesin (ERM) family of proteins [25 , 26 ], a group of molecules that link membrane receptors to the actin cytoskeleton and play a critical role in processes such as cytokinesis and cell adhesion.

The real function of CD43 remains elusive. This molecule has been implicated in T cell activation, enhancing T cell response to allogeneic or mitogenic stimulation [27 , 28 ] and CD43-specific signals have been reported to be sufficient to activate T cells in the absence of T cell receptor (TCR) engagement [20 21 22 23 24 ]. Based on its abundance and on its elongated and heavily sialylated structure, this molecule has been considered a barrier to cellular interactions, functioning as an antiadhesive molecule. However, CD43 has been shown to participate in diverse homotypic and heterotypic adhesion phenomena [29 , 30 ], whereby it may be regulating lymphoid cell migration toward antigen-presentation sites [31 , 32 ]. By mimicking the interaction between CD43 and one of its putative ligands {intercellular adhesion molecule (ICAM)-1, major histocompatibility complex (MHC)-I, human serum albumin; [33 34 35 ]}, different anti-CD43 monoclonal antibodies (mAb) induce homotypic aggregation of monocytes, neutrophils, and T lymphocytes [29 , 36 , 37 ]. The aim of this study was to identify some of the key members of the signaling cascade leading to T lymphocyte homotypic aggregation following CD43 engagement. CD43-dependent signals mediated the homotypic adhesion of T lymphocytes through the participation of Src kinases, PLC-{gamma}2, PKC, PI-3K, extracellular-regulated kinase (ERK), and p38. Interestingly, the MAPKs ERK and p38 seemed to be recruited through independent pathways. Furthermore, our data suggest that Src kinases, ERK, p38, and PI-3K play a central role in regulating the actin cytoskeleton remodeling, resulting in CD43 engagement. We also found that the immunomodulatory drug leflunomide inhibited the CD43-mediated signaling pathway leading to cytoskeleton remodeling, decreasing the CD43-mediated lymphocyte aggregation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Jurkat cells and the Lck-deficient JCaM.1 cells were cultured in RPMI 1640 (Hyclone, Logan, UT), supplemented with 5% fetal calf serum (FCS), 5% bovine calf serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, and 50 µM ß-mercaptoethanol. Peripheral blood mononuclear cells were isolated from healthy adult donors by Ficoll-Hypaque gradient centrifugation as described previously [21 ]. Prior to stimulation, T cells were arrested for 24 h at 37°C in RMPI–2% FCS.

Antibodies
L10, an immunoglobulin G (IgG)1 mAb that recognizes CD43 [14 ], was used pure or from ascites and cross-linked with rabbit anti-mouse IgG (RaMIG). The anti-PLC-{gamma}2, anti-Syk, anti-pERK, anti-ERK, anti-p-p38, anti-p38, and anti-Vav antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The antiphosphotyrosine 4G10 mAb was from Upstate Biotechnology (Lake Placid, NY).

Chemicals
Phorbol 12-myristate 13-acetate (PMA), cytochalasin B, colchicine, and 1-butanol were from Sigma Chemical Co. (St. Louis, MO). U73122, Ro318220, LY294002, PD98059, PD169316, SB202190, SB202474, PP2, PP3, herbimycin A, cyclosporin A, and pertussis toxin were from Calbiochem (San Diego, CA). All inhibitors were prepared in dimethyl sulfoxide (DMSO).

Homotypic aggregation assays
T cells (1x106) in supplemented RPMI were incubated into 48-well plates in the presence or absence of each inhibitor at 37°C for 1 h. Thereafter, anti-CD43 mAb L10 (1 µl ascites) and RaMIG (1 µg/ml) were added, and plates were incubated at 37°C for an additional 2 h. Finally, cells were fixed by adding 2% p-formaldehyde, and aggregates were evaluated by microscopy. The degree of cell aggregation was scored as follows: 0, the majority of cells were nonaggregated, as observed when cells were left unstimulated or incubated with cytochalasin B; +, 30% of cells were forming aggregates; ++, 60% of cells were forming aggregates; and +++, >90% of cells were forming aggregates as observed with L10 + RaMIG.

Confocal microscopy analysis
T lymphocytes were incubated in the presence of the L10 mAb plus RaMIG–fluorescein isothiocyanate (FITC) for 1 h at 37°C in 5% CO2. Then, cells were washed with phosphate-buffered saline to remove excess antibody and were fixed with 1% p-formaldehyde at room temperature. CD43 localization was evaluated with a MRC-600 confocal scanning system equipped with a krypton/argon laser (Bio-Rad, Hercules, CA) coupled to an Axioscope microscope (Zeiss, Germany) with a PlanNeofluar 63X W Korr (aperture 1.2) objective.

T cell activation, immunoprecipitation, and immunoblot
Purified T cells (2x107) were incubated in 500 µl cold RPMI with L10 (4 µg/ml) mAb for 15 min at 4°C. Thereafter, RaMIG (4 µg/ml) was added to each tube, and the cells were further incubated for 15 min at 4°C. When cells were activated with PMA, the chemical was added to the cells just before activation. Following preincubation, cells were activated at 37°C for different periods of time and lysed in 100 µl lysis buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5% Triton X-100, 0.5 mM dithiothreitol, 10 mM ß-glycerophosphate, 1 mM Na3VO4, 5 mM NaF, 4 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin) for 45 min a 4°C. Lysates were spun (14,000 rpm for 10 min at 4°C), and cellular equivalents were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, or precleared supernatants were incubated overnight with the indicated antibody at 4°C. Immune complexes were collected with protein A-Sepharose 4B for 1 h at 4°C and were washed twice with cold TNE-T (150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA, 1% Triton X-100), twice with TNE (150 mM NaCl, 50 mM Tris, pH 7.5, 5 mM EDTA), and once with dH2O. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in TBS-T (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) or with 3% bovine serum albumin in TBS-T for 2 h at room temperature and incubated with the indicated antibody. After three washes with TBS-T, the appropriate horseradish peroxidase-conjugated secondary antibody (Biomeda, Foster City, CA) was added to the membranes, and proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, UK). To probe with another antibody, membranes were stripped, washed, and then blotted.

Actin polymerization assays
Purified human T lymphocytes (1x106) were preincubated in the presence or absence of each inhibitor for 2 h at 37°C. Subsequently, cells were incubated with the anti-CD43 mAb L10 and RaMIG or were left untreated and then activated at 37°C for 10 min. Thereafter, changes in actin polymerization were evaluated as described [38 ]. Briefly, T lymphocytes were fixed, permeabilized, stained with BODIPY® FL phallacidin (Molecular Probes, Eugene, OR), and analyzed by flow cytometry on a FACSort. Results are displayed as the percentage of increase in the mean fluorescence intensity (MFI) relative to unstimulated cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD43 localizes at the site of cell–cell contact
The CD43-specific signals have been shown to induce homotypic aggregation of lymphoid cells [29 , 36 , 37 ]. To further assess the role of this molecule in the CD43-dependent cell aggregation, we evaluated its membrane distribution by confocal microscopy. It is interesting that we found that CD43 concentrated at cell–cell contact areas in CD43-induced T lymphocyte aggregates (Fig. 1 ), whereas when T cells were induced to aggregate with PMA, CD43 was evenly distributed on the cell surface (data not shown). These data suggest that under our experimental conditions, CD43 is an important element for the formation of supramolecular complexes necessary for intercellular adhesion of T cells induced through this receptor.



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  		Figure 1. CD43 is concentrated at the cell–cell contact area in T lymphocyte aggregates resulting from CD43 engagement. Normal peripheral blood T lymphocytes were incubated with the L10 mAb + RaMIG–FITC for 1 h at 37ºC before fixation with 1% p-formaldehyde and observation by confocal microscopy (100x). (A) Localization of CD43 is shown in a typical cell aggregate; (B) differential interference contrast image of the same aggregate; (C) merge.

 
Signaling pathway involved in the CD43-dependent homotypic aggregation of T lymphocytes
To characterize the pathway involved in the homotypic adhesion of T lymphocytes resulting from CD43-specific signals, we evaluated the effect of different pharmacological inhibitors. At the concentration and length of incubation each inhibitor was used, cell viability was always greater than 90%, and none of the inhibitors altered the CD43 expression level on the surface of T lymphocytes, as determined by flow cytometry analysis (data not shown). As shown in Table 1 and Figure 2 , cells incubated with the L10 mAb for 2 h formed large aggregates (Fig. 2B) , whereas those treated with cytochalasin B did not (Fig. 2C) , indicating that remodeling of the actin cytoskeleton is required for the CD43-dependent, homotypic aggregation. In addition, we confirmed that homotypic aggregation was temperature-dependent [39 ], as we observed aggregate formation at 37°C but not at 4°C (data not shown). The Src tyrosine kinases, PLC-{gamma}-, PKC-, and PI-3K-specific inhibitors (PP2, U73122, Ro318220, and LY294002, respectively) completely abrogated the CD43-mediated, homotypic adhesion of T lymphocytes (Fig. 2D 2E 2F , and Table 1 ). The MAPK inhibitors PD98059 (MEK1/2; Fig. 2G ), PD169316 (p38), and SB202190 (p38) and the general inhibitor of PTK herbimycin A induced only a partial blockade (Table 1) . As expected, PP3 (Fig. 2H) and SB202474, the negative controls for PP2 and SB202190, or DMSO had no effect. Finally, molecules, such as calcineurin (Fig. 2I) , PLD, the {alpha} subunit of heterotrimeric G proteins, or the microtubule machinery, were not found to participate in the CD43-mediated adhesion. Altogether, these data suggest that Src family tyrosine kinases, PLC-{gamma}, PKC, and PI-3K, together with the MAPKs ERK1/2 and p38, are important players in the CD43-dependent pathway leading to homotypic aggregation of T lymphocytes.


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  		Table 1. Effect of Different Inhibitors on T Lymphocyte Aggregation Mediated by CD43

 


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  		Figure 2. Signaling pathway involved in the CD43-mediated T lymphocyte aggregation. T lymphocytes were preincubated without inhibitor (A and B) or with cytochalasin B (C), PP2 (D), U73122 (E), LY294002 (F), PD98059 (G), PP3 (H), or cyclosporine A (I) for 1 h at 37°C and subsequently, were activated with the anti-CD43 mAb L10 and RaMIG (B–I) or left untreated (A). After 2 h of incubation at 37°C, cells were fixed in 2% p-formaldehyde, and aggregation was evaluated by microscopy. Identical data were obtained in five separate experiments.

 
Src kinases, MAPKs, and PI-3K participate in CD43-mediated actin polymerization
To assess the participation of all these molecules in cytoskeleton rearrangements, we evaluated the binding of phalloidin as a measure of actin cytoskeleton remodeling in response to CD43 signals in the presence or absence of different pharmacological inhibitors. As shown in Figure 3 , CD43 ligation resulted in enhanced actin polymerization, and the PI-3K and Src-kinase inhibitors LY294002 and PP2 as well as the MAPK inhibitors PD98059 (MEK1/2) and SB202190 (p38) all significantly reduced the CD43-mediated enhancement in F-actin levels, suggesting that these molecules participate in the signaling pathway leading to the CD43-dependent actin remodeling required for homotypic aggregation.



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  		Figure 3. Src kinases, MAPKs, and PI-3K participate in the CD43-mediated actin polymerization. (A) Purified human T lymphocytes (1x106) were preincubated in the presence or absence of each inhibitor for 2 h at 37°C. Subsequently, cells were incubated with the anti-CD43 mAb L10 and RaMIG or were left untreated and then activated at 37°C for 10 min. Thereafter, T lymphocytes were fixed, permeabilized, and stained with BODIPY® FL phallacidin and analyzed by flow cytometry. Results are displayed as the percentage of increase in MFI relative to unstimulated cells. Data shown are representative of three independent experiments (LY29, LY294002; PD98, PD98059; SB24, SB202474; SB21, SB202190). (B) Representative histogram from an experiment with the LY294002 inhibitor. The bold line corresponds to unstimulated cells (MFI, 113); the thin line, to CD43-activated T lymphocytes that were not pretreated with inhibitor (MFI, 143); and the dotted line, to CD43-activated T cells pretreated with LY294002 (MFI, 111).

 
Src kinases are upstream of Syk and PLC-{gamma}2 in the CD43-mediated signaling pathway
Next, we assessed the recruitment of the signaling molecules involved in the CD43-dependent homotypic aggregation of T cells. Activation of PLC-{gamma} requires the phosphorylation of tyrosine residues [13 ]. Although PLC-{gamma}1 predominates in T cells, we could not detect tyrosine phosphorylated PLC-{gamma}1 upon CD43 engagement (data not shown) but found that the basal level of tyrosine phosphorylation of PLC-{gamma}2 increased in a time-dependent manner, up until 2 h after activation, the time at which aggregate formation was evaluated (data not shown and Fig. 4A , compare lanes 3, 5, and 8 vs. 1). The antiphosphotyrosine blot revealed the presence of a tyrosine-phosphorylated 72-kDa protein that paralleled the phosphorylation of PLC-{gamma}2 (data not shown). The fact that Syk has been found to associate with and to phosphorylate PLC-{gamma}2 [40 , 41 ] prompted us to investigate whether this protein was Syk. The anti-Syk blot of the PLC-{gamma}2 immunoprecipitates showed that there is a constitutive association of Syk with PLC-{gamma}2 (data not shown). However, Syk tyrosine phosphorylation increased in a time-dependent manner in response to CD43-mediated signals and was still detectable 2 h after activation (Fig. 4B , compare lanes 3, 5, and 8 vs. 1).



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  		Figure 4. Src kinases are upstream of Syk, PLC-{gamma}2, and Vav in CD43-activated T lymphocytes. Purified T lymphocytes were preincubated in the presence of PP2 or PP3 or without inhibitor for 1 h at 37°C. Subsequently, cells were incubated with the anti-CD43 mAb L10 and RaMIG or were left untreated and activated at 37°C for different periods of time. Cell lysates (1x106 cells) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblotting with the antiphosphotyrosine mAb 4G10 (A–C, upper panels). Membranes were stripped and reprobed with anti-PLC-{gamma}2 (A, lower panel), anti-Syk (B, lower panel), or anti-Vav (C, lower panel) polyclonal Ab. Identical results were obtained in four similar experiments.

 
Src kinases are thought to phosphorylate and activate Syk [8 , 42 ]. As the Src-kinase inhibitor PP2 blocked the CD43-dependent homotypic cell adhesion (Fig. 2D and Table 1 ), we evaluated whether the CD43-induced phosphorylation of PLC-{gamma}2 and Syk was dependent on Src-kinase activity. Tyrosine phosphorylation of PLC-{gamma}2 was abolished by PP2 (Fig. 4A , compare lanes 4, 6, and 9 with lanes 3, 5, and 8, respectively). In addition, Syk tyrosine phosphorylation was inhibited when T lymphocytes were preincubated with PP2 (Fig. 4B , lanes 4, 6, and 9 vs. lanes 3, 5, and 8, respectively). As expected, PP3 did not affect tyrosine phosphorylation of Syk or PLC-{gamma}2. Furthermore, the tyrosine phosphorylation of Vav, a downstream substrate for Syk implicated in cytoskeleton rearrangement [5 ], was inhibited by PP2 but not PP3 (Fig. 4C) . Altogether, these results suggest that activation of Src kinases is necessary to recruit Syk, PLC-{gamma}2, and Vav, ultimately promoting cell adhesion in response to CD43 ligation.

Lck is necessary for PLC-{gamma}2 recruitment in response to CD43 signals
Fyn and Lck have been reported to be activated in response to CD43-specific signals [21 , 22 ]. To investigate the specific role of Lck or Fyn in the CD43-dependent homotypic aggregation, we evaluated the participation of Lck and Fyn on PLC-{gamma}2 phosphorylation in response to CD43 engagement in Jurkat cells and the Lck-deficient cell line JCaM.1. In Jurkat cells, as in resting normal T lymphocytes, PLC-{gamma}2 was constitutively phosphorylated (Fig. 5 , lane 1), and following CD43 ligation, its phosphorylation levels increased, reaching a maximum at 2 min and decreasing below basal levels thereafter (Fig. 5 , lanes 2–6). In contrast, in JCaM.1 cells, PLC-{gamma}2 tyrosine phosphorylation was difficult to detect throughout the experiment (Fig. 5 , lanes 7–12), suggesting that Lck is necessary for PLC-{gamma}2 tyrosine phosphorylation and that Fyn is not sufficient.



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  		Figure 5. Lck recruitment following CD43 ligation is necessary for PLC-{gamma}2 tyrosine phosphorylation. J22 or JCaM.1 cells were stimulated with the anti-CD43 mAb L10 and RaMIG or were left untreated for the indicated times. Cell lysates (1x106) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblot with the antiphosphotyrosine mAb 4G10 (upper panels). Membranes were stripped and reblotted with an anti-PLC-{gamma}2 polyclonal Ab (lower panels). Similar results were obtained in three separate experiments.

 
PI-3K is upstream of Syk and PLC-{gamma}2 in the CD43-mediated signaling pathway
PIP3 promotes the activation and recruitment of PLC-{gamma} to the membrane through its N-terminal PH domain [11 , 12 ]. Based on the fact that PI-3K inhibitors blocked CD43-mediated aggregation and actin remodeling, we investigated whether there was a cross-talk between PI-3K and the Syk and PLC-{gamma}2 molecules in the pathway leading to homotypic adhesion following CD43 engagement. The PI-3K inhibitor LY294002 reduced the tyrosine phosphorylation of PLC-{gamma}2 throughout the duration of the experiment (Fig. 6A , lanes 4, 6, 8, and 10 vs. 3, 5, 7, and 9, respectively). In contrast, Syk tyrosine phosphorylation was only inhibited at longer times of stimulation (30 and 60 min; Fig. 6B , lanes 8 and 10 vs. 7 and 9, respectively) and at early times, was not affected. These data suggest that PI-3K regulates PLC-{gamma}2 recruitment and that at longer times, it modulates Syk activation.



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  		Figure 6. PI-3K is upstream of Syk and PLC-{gamma}2 in T lymphocytes stimulated through CD43. Purified T lymphocytes were preincubated in the presence of LY294002 or with DMSO for 1 h at 37°C. Subsequently, cells were activated at 37°C with the anti-CD43 mAb L10 and RaMIG for different periods of time. Cell lysates (1x106 cells) were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to immunoblotting with the antiphosphotyrosine mAb 4G10 (A and B, upper panels). Membranes were stripped and reprobed with anti-PLC-{gamma}2 (A) or anti-Syk (B) polyclonal Ab. Graphs represent tyrosine phosphorylation fold increase corrected for the amount of protein loaded in each lane. Similar data were obtained in two additional experiments.

 
ERK and p38 are recruited through independent pathways following CD43 ligation
Src kinases activation results in tyrosine phosphorylation of substrates such as PLC-{gamma} and Vav as well as activation of several signaling pathways including the MAPK pathway. Our data with pharmacological inhibitors led us to evaluate whether ERK activation was dependent on Src-kinase activity in the CD43-specific signaling cascade. We found that ERK phosphorylation increased within 2 min of activation with L10 and remained so up until 2 h following activation (Fig. 7A , lanes 3, 6, and 8 vs. 1). ERK2 was predominantly activated in response to CD43 stimulation, whereas ERK1 was phosphorylated to a lesser extent at all time points. ERK phosphorylation was significantly reduced by PP2 (Fig. 7A , lanes 4, 7, and 9 vs. lanes 3, 6, and 8, respectively) but not PP3 (lane 5), suggesting that Src kinases are upstream of ERK in this signaling pathway.



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  		Figure 7. Cross-linking CD43 on the surface of T lymphocytes induces Src kinases and PLC-{gamma}-, PI-3K-, and PKC-dependent phosphorylation of ERK. (A) T lymphocytes preincubated in the presence of PP2 or PP3 or without inhibitor were further incubated with the anti-CD43 mAb L10 and RaMIG and activated at 37°C for different periods of time. (B) T lymphocytes pretreated with U73122, Ro318220, or DMSO were activated at 37°C following stimulation with the anti-CD43 mAb L10 and RaMIG or PMA. (C) T lymphocytes were preincubated in the presence of LY294002 or DMSO for 1 h at 37°C. Subsequently, cells were stimulated with the anti-CD43 mAb L10 and RaMIG and activated at 37°C for different periods of time. Cell lysates (1x106 cells) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with an anti-pERK mAb. Membranes were stripped and reprobed with an anti-ERK2 polyclonal Ab. The graph represents the phosphorylation fold increase corrected by the amount of ERK present in each lane. Data shown were reproduced in two separate experiments.

 
PKC plays an important role in ERK activation in response to TCR and CD43 engagement [43 ]. As the role of PLC-{gamma} in the activation of PKC is well established, we decided to explore the involvement of PLC-{gamma}2 and PKC in the activation of the MAPK pathway induced through CD43. The PKC inhibitor Ro318220 abrogated the CD43-mediated ERK phosphorylation (Fig. 7B , compare lanes 6, 9, and 12 vs. lanes 4, 7, and 10, respectively), whereas it was only partially reduced by the PLC-{gamma}2 inhibitor U73122 (Fig. 7B , compare lanes 5, 8, and 11 vs. lanes 4, 7, and 10, respectively). As expected, Ro318220 blocked PMA-induced ERK phosphorylation, whereas U73122 had no effect. Finally, the PI-3K inhibitor LY294002 also diminished ERK phosphorylation (Fig. 7C , lanes 4, 6, and 8 vs. 3, 5, and 7, respectively). Overall, these data suggest that in the signaling pathway leading to the CD43-mediated, intercellular adhesion of T lymphocytes, Src kinases, PLC-{gamma}, PKC, and PI-3K participate in ERK recruitment.

p38 has been implicated in actin-filament dynamics in mammalian cells [44 , 45 ]. Data from Table 1 and the actin-remodeling assays (Fig. 3) suggested that p38 also participated in the CD43-mediated signaling pathway leading to adhesion. It is interesting that the CD43-induced p38 phosphorylation was enhanced by the PKC inhibitor Ro318220 (Fig. 8A , lanes 4, 6, and 8 vs. 3, 5, and 7, respectively) and the PI-3K inhibitor LY294002 (Fig. 8B , lanes 4, 6, and 8 vs. 3, 5, and 7, respectively), indicating that ERK and p38 are likely activated through different pathways in response to CD43 signals.



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  		Figure 8. CD43-induced activation of p38 occurs independently of PKC and PI-3K. Purified T lymphocytes were preincubated in the presence of Ro318220 (A), LY294002 (B), or with DMSO for 1 h at 37°C. Subsequently, cells were incubated with the anti-CD43 mAb L10 and RaMIG or were left untreated and activated at 37°C for different periods of time. Cell lysates (1x106 cells) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with an anti-p-p38 mAb. Membranes were stripped and reprobed with an anti-p38 polyclonal Ab. Data shown were reproduced in two separate experiments.

 
Leflunomide inhibits CD43 signals leading to homotypic aggregation of T lymphocytes
We evaluated the effect of two drugs known to regulate T lymphocyte adhesion on the CD43-dependent T lymphocyte aggregation. Leflunomide is an immunomodulatory drug that reduces spontaneous clustering of mononuclear cells in rheumatoid arthritis patients [46 ]. Pentoxifylline is a nonselective phosphodiesterase inhibitor with immunoregulatory and anti-inflammatory effects [47 ]. A771726 (200 µM), the active metabolite of leflunomide, induced a significant reduction in the CD43-mediated homotypic aggregation (Table 1 and Fig. 9A ), whereas pentoxifylline (1 mM) had no effect (Table 1) . Furthermore, A771726 partially inhibited actin polymerization (Fig. 9B) . Consistent with previous reports, where A771726 was found to inhibit Fyn and Lck kinase activity [48 ], we found that this compound was able to inhibit PLC-{gamma}2 (Fig. 9C) and ERK phosphorylation (Fig. 9D) . In contrast, A771726 had no effect on CD69 expression by T lymphocytes activated through CD43 (Fig. 9E) . These data suggest that leflunomide has a selective, inhibitory effect on CD43-mediated homotypic adhesion of T lymphocytes by affecting Src-tyrosine kinase activation and cytoskeleton rearrangements.



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  		Figure 9. Leflunomide inhibits homotypic aggregation but not activation of T cells. (A, B) T lymphocytes were preincubated in the presence of A771726 or DMSO for 1 h at 37°C. Subsequently, cells were activated or not with the anti-CD43 mAb L10 and RaMIG, and after 2 h of incubation at 37°C, cells were fixed, and lymphocyte aggregation (A) and actin polymerization (B) were assessed as described in Materials and Methods. (C, D) Cell lysates (1x106) of T lymphocytes preactivated at 37°C for 5 min with the L10 anti-CD43 mAb were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (E) T lymphocytes preincubated or not with A771726 were stimulated or not with the activating L10 anti-CD43 mAb and analyzed for CD69 expression by flow cytometry. The thin line corresponds to unstimulated cells; the bold line, to CD43-activated T lymphocytes pretreated with A771726; and the dotted line, to CD43-activated T cells that were not pretreated with A771726. Similar data were obtained in two separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A better knowledge of the molecules that regulate cell aggregation as well as of the signaling pathways that participate in these phenomena is necessary to have a deeper understanding of the molecular mechanisms involved in extravasation and migration of leukocytes toward inflammatory foci. Anti-CD43 mAb, such as L11, have been reported to inhibit T lymphocyte migration toward lymphoid tissues as well as sites of inflammation [31 , 32 ]. In addition, several anti-CD43 mAb have been found to induce lymphocyte aggregation [29 , 39 ]. Although several molecules have been identified in the signaling cascade initiated in response to CD43 engagement, the specific pathways involved in T lymphocyte adhesion through this receptor have not been elucidated yet. Using the homotypic aggregation of T lymphocytes as a model to study the intercellular adhesion phenomena triggered through CD43 [49 ], we identified specific molecules required for the homotypic aggregation of T lymphocytes following CD43 ligation with the L10 mAb. Although the use of natural ligands is the most physiological way to study the role of a cell-surface molecule, we mimicked the interaction between CD43 and its potential ligands with an anti-CD43 mAb instead of the natural ligands, as these molecules (e.g., MHC-I molecules or ICAM-1) interact with other important receptors on the T cell. The specific mAb we used has been shown by us and others to recruit multiple signaling molecules [21 , 23 , 24 , 50 ].

Our data indicate that CD43 engagement promotes cell–cell interactions. It is feasible that CD43 induces homotypic aggregation of T lymphocytes by two distinct mechanisms. CD43 might trigger a signaling cascade that results in cell aggregation through activation of adhesion molecules such as integrins. Alternatively, CD43 might interact with its putative ligand(s) [33 34 35 ], directly mediating cell–cell interactions. The fact that anti-CD43 mAb promote homotypic adhesion of leukocytes through lymphocyte function-associated antigen-1-dependent and -independent pathways [29 ] supports either possibility. Data reported here also provide evidence that CD43-mediated signals are sufficient to induce cytoskeleton rearrangements as well as CD43 relocalization to the cell–cell contact area in resting, normal human T lymphocytes. Furthermore, the fact that in all experiments reported here, anti-CD43 mAb were added in solution suggests that in contrast to what was previously reported [49 ], a contact area with a substratum is not required for the CD43-mediated cell adhesion and CD43 relocalization.

Results from aggregation assays and biochemical data reported here allowed us to identify Lck, Syk, PLC-{gamma}2, PKC, PI-3K, Vav, ERK, and p38 as key components of the CD43-mediated reorganization of actin cytoskeleton and homotypic adhesion of human, normal peripheral blood T lymphocytes. The CD43-induced signaling pathway leading to homotypic aggregation of T cells is proposed in Figure 10 . CD43 engagement results in Fyn/Lck tyrosine phosphorylation [21 , 22 ], facilitating the activation of PLC-{gamma}2, Syk, and Vav. Recruitment of PLC-{gamma}2 and PLC-{gamma}-dependent PKC isoforms would lead to ERK phosphorylation. Conversely, PI-3K could participate in Syk and PLC-{gamma}2 activation as well as in the recruitment of novel isoforms of PKC and through them, of ERK [51 ]. Altogether, these molecules induce rearrangements of the actin cytoskeleton and contribute to the formation of cell aggregates.



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  		Figure 10. Signaling pathway leading to CD43-mediated, homotypic aggregation of human T lymphocytes. CD43 engagement results in Fyn/Lck tyrosine phosphorylation, facilitating the activation of PLC-{gamma}2, Syk, and Vav. Recruitment of PLC-{gamma}2 and PLC-{gamma}-dependent PKC isoforms would lead to ERK phosphorylation. Conversely, PI-3K could participate in Syk and PLC-{gamma}2 activation as well as in the recruitment of novel isoforms of PKC and through them, of ERK. Altogether, these molecules induce rearrangements of the actin cytoskeleton and contribute to the formation of cell aggregates. Continuous lines correspond to data reported in this work and dotted lines, to data previously published.

 
The fact that the PLC-{gamma}-specific inhibitor U73122 abrogated the CD43-mediated homotypic aggregation of T lymphocytes pointed at this enzyme as an important player in the CD43 signaling pathway leading to cell adhesion. Consistent with data published by Anzai et al. [52 ], we found that CD43 engagement resulted in tyrosine phosphorylation of PLC-{gamma}2 but not PLC-{gamma}1 (data not shown). Furthermore, we show that following CD43 ligation, Src kinases, particularly Lck, participate in PLC-{gamma}2 recruitment, as this molecule was not tyrosine-phosphorylated in the Lck-deficient but Fyn-positive JCaM.1 cells (Figs. 4 and 5) . CD43 engagement also resulted in large aggregates of Jurkat but not of JCaM.1 cells (data not shown), suggesting that Lck is necessary for PLC-{gamma}2 recruitment and further downstream events leading to CD43-mediated cytoskeleton remodeling and cell–cell aggregation. Our data also suggest that PLC-{gamma}2 is downstream of PI-3K in the CD43-mediated aggregation of T lymphocytes. In contrast with our results, Anzai and colleagues [52 ] did not observe an active role for PI-3K in the CD43-mediated adhesion. This discrepancy may arise from different experimental systems: They investigated the signals, delivered by a mAb (DF-T1) that recognizes a CD43 neuraminidase-sensitive epitope in the myeloid cell line MO7e, adhered to fibronectin, whereas we analyzed fresh, normal peripheral blood T lymphocytes stimulated by a mAb (L10), which recognizes a neuraminidase-resistant epitope.

PLC-{gamma}1 and PLC-{gamma}2 are expressed in hematopoietic cells. PLC-{gamma}1 is the predominant isoform activated in T cells downstream of the TCR, and PLC-{gamma}2 has been reported to be critical for mast cell degranulation, NK cell activation, platelet aggregation induced by collagen, and B cell activation through the B cell receptor [53 , 54 ]. However, little is known about the role of PLC-{gamma}2 in T cells. The fact that this particular isoform is recruited in T lymphocytes in response to CD43-specific signals may help to elucidate the factors that determine the selective activation of PLC-{gamma} isoforms in T lymphocytes and other cells as well as the functional consequences of this selection in intercellular adhesion phenomena of T cells.

It has been shown that members of the MAPK family take part in cell adhesion-related events and that specific isoforms of PKC are able to recruit ERK to focal adhesions [55 , 56 ]. In addition, ERK participates in the actin reorganization required for the establishment of integrin-induced adhesions [57 ]. Our experiments using the MEK1/2 inhibitor PD98059 suggest that ERK is involved in the CD43-mediated cell adhesion. The fact that PI-3K and PKC inhibitors abrogated the CD43-dependent phosphorylation of ERK, whereas the PLC-{gamma} inhibitor only had a partial effect (Fig. 7) suggests that in addition to the PLC-{gamma}2-dependent PKC isoforms, other isoforms, recruited through PI-3K but in a PLC-{gamma}2-independent manner, lead to the CD43-specific activation of ERK.

p38 MAPK has been implicated in cell adhesion as well as in monocyte chemotaxis [58 , 59 ]. We found that the p38 inhibitors PD169316 and SB202190 partially blocked the CD43-mediated homotypic adhesion of human T lymphocytes (Table 1) and actin cytoskeleton remodeling (Fig. 3) , suggesting that p38 also participates in these phenomena. It is interesting that opposed to what we found for ERK, PKC and PI-3K seem to play a negative, regulatory role on p38 recruitment, as the CD43-induced phosphorylation of p38 was augmented in the presence of PKC and PI-3K inhibitors (Fig. 8) . PD169316 had no effect on the CD43-dependent ERK phosphorylation (data not shown), ruling out the possibility that p38 inhibitors might affect the CD43-mediated activation of ERK and as a consequence, block lymphocyte adhesion. Overall, our data suggest that p38 and ERK are recruited through different pathways in response to CD43 signals, consistent with reports describing that distinct members of the MAPK family have differential activation requirements [4 , 60 ]. Additional investigation will provide information about the specific pathways involved in these phenomena.

Intercellular adhesion is essential throughout the different facets of the immune response. It has been postulated that CD43 is involved in T cell adhesiveness, traffic, homing, and activation. Certain immunomodulatory drugs exert their action, at least partially, by regulating the adhesion process of lymphoid cells. In patients with rheumatoid arthritis, a decrease of the spontaneous clustering of mononuclear cells is achieved after leflunomide therapy [46 ]. The plasma level of leflunomide in patients taking 25 mg/day is 190 µM [61 ]. At this concentration, leflunomide has been reported to inhibit protein tyrosine kinases of the Src family [48 ]. Data we report here show that this drug significantly reduces the CD43-mediated aggregation of T lymphocytes, the actin polymerization, and tyrosine phosphorylation of PLC-{gamma}2 and ERK (Fig. 9) , suggesting that by blocking Fyn and/or Lck, leflunomide affects the CD43-signaling pathway leading to cell aggregation. The fact that leflunomide did not inhibit CD69 expression on T lymphocytes suggests that this drug has a selective effect on adhesion/motility, and it does not seem to affect other facets of T cell activation. It has been described that expression of CD69 requires activation of members of the PKC family [62 ]. In our system, specific PKC isoforms activated by CD43 through a Src-independent pathway could mediate the expression of CD69.

Recently, the interaction of CD43 with the ERM molecules, a family of proteins linking membrane receptors to the actin cytoskeleton and participating in cell-shape remodeling, was found to be essential for CD43 relocalization to the uropod and formation of the immunological synapse following TCR engagement [27 , 28 , 49 , 63 ]. Syk has also been shown to participate in actin cytoskeleton reorganization through the phosphorylation of Vav [5 ] and association with ezrin and moesin [64 ]. In addition, recent data from our laboratory suggest that {zeta}-associated protein-70 (ZAP-70) is also recruited in response to CD43 [65 ]. Whether the CD43-induced movement of CD43 toward the cell–cell contact area is also dependent on ERM function and interaction with Syk or ZAP-70 is under present investigation.

Our data suggest that CD43 signals participate in T cell homotypic aggregation through a signaling machinery that involves Src kinases, PI-3K, PKC, PLC-{gamma}2, ERK, as well as p38, ultimately leading to cytoskeleton remodeling and relocalization of CD43 toward the site of cell–cell contact. A better knowledge of the molecular mechanisms underlying lymphocyte traffic and activation will lead to the identification of targets for new, therapeutic agents aimed to treat diverse inflammatory disorders.


   ACKNOWLEDGEMENTS
 
This work was supported by Grant IN209400 (Y. R.) from Dirección General de Apoyo al Personal Académico, Universidad Nacional Autónoma de México, and Grants 25307-M (Y. R.) and G35943-M (R. G-A. and Y.R) from Consejo Nacional de Ciencia y Tecnología, México. E. L-E. was the recipient of a doctoral fellowship from CONACyT. We thank Dr. Eduardo Huerta for leukocyte concentrates, Drs. Angélica Santana and Carlos Rosales for critical reading of the manuscript, and Erika Melchy and Xóchitl Alvarado for technical help.

Received March 9, 2003; revised July 8, 2003; accepted July 28, 2003.


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 RESULTS
 DISCUSSION
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