Signaling Through CD43 Induces Natural Killer Cell Activation, Chemokine Release, and PYK-2 Activation

IL-2–cultured NK cells were obtained essentially as described.41 In brief, peripheral blood T lymphocytes (PBL) were cultured with irradiated (5 Gy) RPMI 8866 lymphoblastoid cells for 6 to 9 days in RPMI 1640 supplemented with 10% fetal calf serum (FCS; complete medium), followed by a negative selection step using an anti-CD3 MoAb plus rabbit complement (Behring, Marburg, Germany). The CD3⁻ cells (<5% CD3⁺) were cultured with 50 IU/mL of recombinant human IL-2 (rhIL-2) until use. These cell populations are hereafter referred to as NK cells. After each purification process, the resulting population was characterized by flow cytometry analysis. We routinely obtained a cell population with a proportion of CD56⁺ and CD16⁺cells greater than 95% and with less than 5% of CD3⁺, CD19⁺, or CD14⁺ contaminating cells.

The anti-CD43 MoAb TP1/36 (IgG1), HP2/21 (IgM), the nonactivatory anti-CD44 MoAb HP2/9 (IgG1), and the anti-CCR5 MoAb CCR5-01(IgM) have been previously described.42,43 The anti-CD56 K218 (IgG1) was kindly provided by Dr A. Moretta (Istituto Nazionale per la Ricerca sul Cancro e Centro Biotecnologie Avanzate, University of Genova, Genova, Italy). F(ab′)₂ fragments were obtained by pepsin digestion of purified antibody.41 PYK-2 antipeptide polyclonal antibodies C-19 and N-19 were from Santa Cruz Biotechnology Inc (Santa Cruz, CA). The anti-Tyr(P) PY20 MoAb was from Transduction Laboratories (Lexington, KY). [γ³²P]ATP (4,000 Ci/mmol) was from ICN (Irvine, CA). Protein A-agarose and protein G-agarose were from Boehringer Mannheim (Mannheim, Germany). ECL reagents were from Amersham (Buckinghamshire, UK). Tyrosine kinase inhibitor genistein was from Calbiochem-Novabiochem Ltd (Nottingham, UK). All other reagents used were of the purest grade available.

Plates were precoated overnight at 4°C with 2.5 μg/mL of antibody in adhesion buffer (20 mmol/L Tris/HCl, 150 mmol/L NaCl, pH 8.2), blocked with 1% bovine serum albumin (BSA) in adhesion buffer for 1 hour at room temperature, and then washed twice with phosphate-buffered saline (PBS).

NK cells (10⁶) were incubated in polypropylene tubes, to prevent signaling through integrins, in a final volume of 1 mL complete medium in the presence of 5 μg/mL of different MoAbs for different times at 37°C, 5% CO₂. Cells were then centrifuged and the chemokines present in the supernatant were quantified. Human RANTES was measured using the Cytoscreen immunoassay kit (Biosource International, Inc, Camarillo, CA), and human macrophage inflammatory protein (MIP)-1α and MIP-1β were measured using the Quantikine immunoassay kit (R&D Systems, Minneapolis, MN), following the manufacturer’s instructions.

Redirected lysis assays were performed as described.41 NK cells were in triplicate tested in a 4-hour ⁵¹Cr release assay against the murine P815 (FcγR⁺) mastocytoma cell line at different E/T ratios. MoAb were preincubated for 15 minutes with the target cells before NK cell addition. The percentage of specific lysis was calculated as described previously.41Data are expressed as the arithmetic mean of triplicates. In each case, spontaneous release was always less than 10% of the maximum lysis.

IL-2–activated NK cells were washed twice with RPMI. Experiments were initiated by adding the cells (10 × 10⁶ cells, unless otherwise stated) to 60-mm cultured dishes precoated with the relevant antibodies. Cells were incubated in ice for 15 minutes and then were incubated at 37°C for the indicated times. The incubation was stopped by solubilizing the cells in 1 mL of ice-cold lysis buffer (10 mmol/L Tris/HCl, pH 7.65, 5 mmol/L EDTA, 150 mmol/L NaCl, 30 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 2 mmol/L sodium orthovanadate, 1% Triton X-100, 50 μg/mL aprotinin, 50 μg/mL leupeptin, 5 μg/mL pepstatin, and 1 mmol/L phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 14,000 rpm for 10 minutes and the pellets were discarded. After centrifugation, supernatants were transferred to fresh tubes and proteins were immunoprecipitated at 4°C overnight with protein G-agarose–linked MoAbs directed against Tyr(P) proteins (PY20 MoAb) or protein G-agarose–linked goat polyclonal anti–Pyk-2 (C-19) antibody. Immunoprecipitates were washed 3 times with lysis buffer and either used for in vitro kinase reaction (see below) or extracted in 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (200 mmol/L Tris/HCl, pH 6.8, 0.1 mmol/L sodium orthovanadate, 1 mmol/L EDTA, 6% SDS, 2 mmol/L EDTA, 4% 2-mercaptoethanol, and 10% glycerol) by boiling 5 minutes, were fractionated by SDS-PAGE (7.5%), and were further analyzed.

Reactions were performed as described.44 Briefly, immunoprecipitates were washed and pelleted (2,500 rpm 10 minutes at 4°C) 3 times in lysis buffer and twice with kinase buffer (20 mmol/L HEPES, 3 mmol/L MnCl₂, pH 7.35). Pellets were dissolved in 40 μL of kinase buffer and reactions were initiated by adding 10 μCi of [γ³²P] ATP. The reactions were performed at 30°C for 15 minutes and were stopped on ice by adding 10 mmol/L EDTA. After the in vitro kinase reactions, the pellets were washed in lysis buffer containing 10 mmol/L EDTA, extracted for 5 minutes at 95°C in 2× SDS-PAGE sample buffer, and analyzed by SDS-PAGE. After fixing and drying of the gels, autoradiography was performed at −80°C. Autoradiograms were analyzed using an AGFA Studio ScanIIsi scanner (Montiel, Belgium) and bands were quantified using the Bio-Rad Molecular Analyst Software (Bio-Rad, Hercules, CA).

Cell lysis and immunoprecipitations were performed as described above. After SDS-PAGE, proteins were transferred to Immobilon membranes using a Bio-Rad SD Transblot. Membranes were blocked using 3% nonfat dried milk in PBS, pH 7.2, and incubated for 2 hours at 22°C with the polyclonal antibody anti–Pyk-2 (C-19 or N-19) or with the anti-Tyr (P) PY20 MoAb, all diluted 1:500 in PBS containing 3% nonfat dried milk. After incubating membranes with horseradish peroxidase-conjugated secondary antibodies, immunoreactive bands were visualized using ECL reagents.

Upon activation, NK cells are able to secrete cytokines that modulate NK cytotoxic response and other immune events.45,46Different chemokines, including RANTES, MIP-1α, MIP-1β, and lymphotactin, are secreted by NK cells.43,47-49 These chemokines trigger the directional migration or chemotaxis of NK cells, regulate cell polarization and redistribution of adhesion molecules, stimulate intracellular Ca²⁺ mobilization and cytolytic granule release, and augment NK-mediated cytotoxicity.50-53To investigate the possible role of CD43 as a triggering molecule in NK cells, we assayed the effect on chemokine secretion by NK cells of anti-CD43 MoAbs that have previously been described to stimulate integrin-mediated adhesion, cell polarization, and homotypic aggregation in T lymphocytes.28,54 IL-2–activated NK cells constitutively secrete MIP-1α, MIP-1β, and RANTES (Fig 1A through C). Engagement of CD43 with TP1/36 MoAb significantly increases the production of these chemokines by NK cells. The increased release of chemokines in NK cell supernatants was clearly detectable after 4 hours of incubation with the anti-CD43 TP1/36 and persisted after 8 hours of incubation (Fig 1A). Treatment of NK cells with cycloheximide abolished the secretion of chemokines induced by anti-CD43, thus indicating the requirement of protein synthesis for this process (data not shown). Engagement of CD43 with the anti-CD43 MoAb (HP2/21) of a different isotype also increased chemokine production by NK cells (Fig 1B). Furthermore, the incubation of NK cells on TP1/36 Fab′₂ fragment-coated dishes also stimulated the secretion of chemokines (Fig 1C). In contrast, control isotype-matched MoAbs against the highly expressed membrane adhesion molecule CD44 or the chemokine receptor anti-CCR5 did not affect cytokine production, ruling out the possibility of an Fc-receptor mediated effect (Fig 1B and C).

We next assessed the possible effect of the anti-CD43 MoAb TP1/36 on the cytotoxic activity of NK cells. IL-2–activated NK cells treated with the anti-CD43 MoAb showed a significant increase of cytotoxic activity. Similarly to the chemokine production response, the increase in NK cell-mediated cytotoxicity triggered by anti-CD43 varied among donors (Fig 2). Control isotype-matched MoAb did not modify the cytotoxic activity of effector NK cells (Fig2). These data further indicate that CD43 functions as a stimulatory molecule in NK cells.

Positive regulation of NK cell activity involves protein kinase activation.2-4 To examine the possible involvement of protein tyrosine kinases in the signals triggered by CD43, NK cells were pretreated with the tyrosine kinase inhibitor genistein55 before stimulation with anti-CD43 MoAb, and then chemokines released were quantitated in cell supernatants. As shown in Fig 3, CD43-induced production of RANTES and MIP-1β was abolished by genistein, suggesting that genistein-sensitive tyrosine kinases are required for signaling through CD43 in NK cells.

We next examined the effect of anti-CD43 TP1/36 MoAb on the tyrosine kinase activities present in NK cells. Cells plated on dishes coated with the anti-CD43 MoAb TP1/36 were allowed to attach for different times and then lysed. Tyrosine-phosphorylated proteins were immunoprecipitated from the cell lysates with the MoAb PY20, and the resulting immunocomplexes were incubated with [³²P]-γ-ATP and then analyzed by SDS-PAGE. As shown in Fig 4A, we observed a time-dependent phosphorylation of proteins in the molecular weight (Mr) of 110 to 80 kD and in the 48 to 83 kD range (Fig 4A, left panel).

PYK-2 has been recently identified as a 115-kD tyrosine kinase homologous to FAK that is expressed in different cell types, including NK cells.36-38,56 A protein band displaying an Mr equal to that of PYK-2 was among the phosphorylated proteins present in the lysates stimulated with the anti-CD43 TP1/36 MoAb. To determine whether PYK-2 was a component of the Mr 110- to 180-kD bands in the PY20 immune complexes, parallel cultures of NK cells were treated with the anti-CD43 MoAb for 45 minutes, in vitro kinase analysis was performed, and [³²P]-labeled phosphotyrosinated proteins were eluted from the complexes by denaturation and then reimmunoprecipitated with the anti–PYK-2 polyclonal antibody. The results shown in Fig 4A (right panel) demonstrate that PYK-2 is a constituent of the Mr 110- to 180-kD Tyr (P) bands induced after engagement of CD43. We confirmed by Western blotting that CD43 induces tyrosine phosphorylation of PYK-2. Lysates obtained from cells that were plated on dishes coated with the anti-CD43 TP1/36 MoAb for different times were immunoprecipitated with the C-19 anti–PYK-2 antibody and tyrosine phosphorylation of PYK-2 was determined by immunoblotting using the MoAb PY20. As shown in Fig 4B, tyrosine phosphorylation of PYK-2 increased gradually, reaching a maximum after 60 minutes of CD43 stimulation.

To confirm directly that CD43 stimulates PYK-2 activity, NK cells were incubated for 60 minutes on dishes coated with the anti-CD43 MoAb TP1/36. Cells were then lysed and immunoprecipitated with the C-19 anti–PYK-2 polyclonal antibody. PYK-2 immunoprecipitates were incubated with [³²P]-γ-ATP and analyzed by SDS-PAGE. As shown in Fig 5A, CD43 induced an increase in the PYK-2 autokinase activity. F(ab′)₂ of anti-CD43 TP1/36 MoAb also induced a similar activation PYK-2 (Fig 5C). The increase in PYK-2 phosphorylation of activity was not observed when NK cells were plated on dishes precoated with the nonstimulating isotype control anti-CD44 MoAb HP2/9 or when NK cells were incubated with conditioned medium obtained from cells plated on TP1/36 MoAb-coated dishes for 60 minutes, ruling out an autocrine loop in the activation of PYK-2 (Fig 5A and data not shown). Immunoblotting with anti–PYK-2 C-19 antibody immunoprecipitates performed in parallel verified that similar amounts of PYK-2 were recovered after stimulation with anti-CD43 MoAbs or its F(ab′)₂ fragments (Fig 5A and C; IP: C-19; WB: C-19). Densitometric scanning showed that anti-CD43 MoAb TP1/36 induced a 4- ± 0.5-fold increase (n = 5) in the phosphorylation level of PYK-2 (Fig 5B).

Kinetic studies of CD43-induced activation of PYK-2 showed that the increase in the autokinase activity of PYK-2 reached a maximum after 45 to 60 minutes, returning to basal levels after 180 minutes. When soluble anti-CD43 MoAb was added directly to the cell suspension, it was also able to induce activation of PYK-2 (Fig 6A and B). Taken together, these results show that engagement of CD43 increases the phosphotyrosine kinase activity of PYK-2.

We provide evidence here suggesting that CD43 positively regulates NK cell activity through phosphorylation signals. In addition, we have investigated the involvement of the protein tyrosine kinase PYK-2 in this signaling pathway. Despite the fact that a definitively confirmed ligand for CD43 is still lacking and its controversial function as an adhesion molecule, our data further support the role of this receptor as an accessory molecule in lymphocytes. In this regard, we have explored different aspects of the activation events triggered by CD43 in NK cells. Our results show that engagement of CD43 induces the augmented secretion of chemokines. This effect appeared to be independent of homotypic cell aggregation triggered by the anti-CD43 MoAb,28 as demonstrated by the results obtained with antibody-coated dishes, in which cells adhere and do not aggregate. This fact allows us to rule out the possibility of an activation effect as a consequence of cell-cell interactions.

Chemokines have been shown to be major factors regulating the directed migration of leukocytes, including NK cells, in inflammation and immunity.57-59 In addition, it has been described that chemokines stimulate Ca²⁺ mobilization and cytolytic granule release, promote cytotoxic activity, and regulate the adhesiveness of NK cells.51,52 We have previously reported that chemokine secretion by NK cells is dramatically increased by contact with target cells and that the release of these soluble factors induces the chemotaxis of additional NK cells. This suggests that chemokines may function by amplifying the immune response at sites of NK cell activation.43 It is therefore conceivable that the chemokines RANTES, MIP1-α, and MIP-1β, released upon activation through CD43, may increase the cytotoxic activity of NK cells at the targeted tissue and may direct the migration of other neighboring NK cells towards the target cells. In addition, these chemokines may contribute to modulate the function of other leukocytes. In this regard, it is worth mentioning that, in addition to its cytotoxic activity, growing evidence points to the role of NK cells as regulators of the immune response.46 

The role of CD43 as a positive modulator of NK cell function was further reinforced by our finding that engagement of this receptor increases the cytotoxic activity of NK cells. Furthermore, CD43 induced phosphorylation and activation of tyrosine kinase proteins that are thought, in general, to transduce the positive signals that trigger the cytotoxic process. We have analyzed in detail some of the molecular components of the phosphorylation pathways triggered through CD43. PYK-2 was among the tyrosine kinases that became activated. PYK-2 is a member of the FAK nonreceptor tyrosine kinase family expressed by different cell types, including brain cells, platelets, and other hematopoietic cells. The cascade of signaling events that trigger PYK-2 activation includes various stimuli that elevate intracellular calcium or induce PKC activation.36,37 Hence, a possible linkage between CD43 engagement and PYK-2 activation could be established, because it has been reported that ligation of CD43 induces PKC activation.33 Nevertheless, it should be taken into account that PYK-2 tyrosine phosphorylation can be mediated via different pathways in various cell sytems. The Src tyrosine kinase Fyn has been reported to selectively phosphorylate PYK-2 in T cells. This phosphorylation does not depend on intracellular Ca²⁺.60 Fyn, which associates with the cytoplasmic tail of CD43,34 could be another candidate for the molecule responsible for the phosphorylation and activation of PYK-2 triggered through CD43.

The precise role of PYK-2 in the activation of NK cells through CD43 currently remains unknown. The homology sequence between FAK and PYK-2 seems to correspond to the functions that these two PTKs perform in the cell, although the exact role of PYK-2 may differ depending on the cell type.38 In this regard, studies using FAK⁻ mutant mice have shown that expression of PYK-2 is induced in fibroblasts from these mice and that this expression counterbalances FAK signaling functions triggered by β1 integrins.61 This suggested that the expression of both proteins is regulated in a mutually exclusive fashion and that they might undertake similar functions in the cell. Nevertheless, as occurs with FAK, PYK-2 is best known for its functional association to β1, β2, and β3 integrins and its activation upon interaction of these adhesion receptors with their ligands.56,62,63 Recently, it has been described that NK cells express PYK-2, but not FAK, and that outside-in signaling triggered by β1 integrin receptors stimulates PYK-2 activation.56 Moreover, the constitutive association of PYK-2 to paxillin, a cytoskeletal protein that is also associated to pp125^FAK, and to other proteins present in focal adhesions such as vinculin has been recently reported.56,64 Integrins are two-way signaling receptors, and it is therefore feasible that PYK-2, as occurs with different second-messengers and mediators involved in the outside-in integrin signaling, may also participate in transducing signals from inside the cell to the exterior. The possibility that CD43-mediated activation of PYK-2 may regulate integrin affinity must be taken into account, because CD43 has been reported to regulate β1 and β2 integrin-mediated lymphocyte adhesion,28 a phenomenon that may lead to an increase in the cytotoxic activity of NK cells.

Nevertheless, it is also feasible that PYK-2 may function independently of integrin receptors and participates in other different signaling events during CD43-induced activation. Hence, PYK-2 phosphorylation has been involved in other processes, such as the signal events triggered by the interaction of CCR5 with either the chemokine RANTES or the HIV envelope protein gp120, the stress signals, or cytokine signaling.65-67 Interestingly, different reports have shown that engagement of the T-cell receptor triggers PYK-2 activation, which is selectively phosphorylated by Fyn, and the association of PYK-2 to Lck.60,68,69 

Our results add novel elements to the range of protein kinases that participate in the positive regulation of NK cell activity. It is clear that the function of NK cells is modulated by the interplay between negative and positive signals within the cell. Membrane receptors as well as the signaling pathways involved in the activation of NK cells have not been studied to the same extent as the inhibitory receptors that provide specific recognition.2-4 The CD16 molecule functions as a prototype activating receptor in NK cells, and the signaling through this molecule has been extensively investigated (reviewed in Lanier2 and Yokoyama4). Engagement of CD16 triggers recruitment of tyrosine kinase of the src-family and tyrosine phosphorylation of residues contained within the immunoreceptor tyrosine-based motifs (ITAMs) in the cytoplasmic domains of CD16-associated zeta and gamma chains, followed by activation of ZAP-70, activation of phospholipase C, and the MAP kinase activation pathway. As occurs with CD43, ligation of CD16 stimulates cytokine production.70 These findings, together with the fact that CD43 has been reported to be associated to the Src family tyrosine kinase Fyn,34 suggest that the activation pathway triggered by these two receptors, as well as by other positive modulators of NK cell activity, may share more common elements. Therefore, it is likely that CD16 receptor triggers PYK-2 phosphorylation. However, this issue requires further investigation. It is therefore feasible that signals triggered by positive regulators of NK cell-mediated cytotoxicity may converge in a common signaling pathway. This activatory cascade of signals may be induced by different receptors depending on the type of NK cell cytotoxicity (ie, antibody-dependent cellular cytotoxicity or MHC-recognition regulated killing). Triggering of this cascade by CD43, as well as by other costimulatory receptors, such as CD16, may serve to augment the primary activation of NK cells through specific MHC receptors.

The authors thank Dr Miguel López-Botet for critical reading of the manuscript.