Jan 16, 2000 - purchased from Alexis Corporation and the MEK-1 inhibitor PD .... Bagrodia, S., De`rijard, B., Davis, R.J., and Cerione, R.A. (1995). Green, D.
Immunity, Vol. 12, 7–16, January, 2000, Copyright 2000 by Cell Press
RAC1/P38 MAPK Signaling Pathway Controls b1 Integrin–Induced Interleukin-8 Production in Human Natural Killer Cells Fabrizio Mainiero,*§ Alessandra Soriani,* Raffaele Strippoli,* Jordan Jacobelli,* Angela Gismondi,* Mario Piccoli,* Luigi Frati,*† and Angela Santoni*‡ * Department of Experimental Medicine and Pathology Istituto Pasteur-Fondazione Cenci Bolognetti University of Rome “La Sapienza” Rome 00161 Italy † Mediterranean Institute of Neurosciences “Neuromed” Via Atinense 18 Pozzilli 86077 Italy ‡ Laboratory of Pathophysiology Regina Elena Cancer Institute Via delle Messi d’oro 156 Rome 00158 Italy
Summary The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that b1 integrin ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the b1 integrin–mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by b1 integrins. Introduction During immune and inflammatory responses, leukocyte migration across endothelium and into the tissues is largely governed by a large number of chemoattractants and adhesion receptors. Integrins are a family of heterodimeric ab receptors that mediate cell adhesion to extracellular matrix components and cell to cell interactions (reviewed in Hynes, 1992; Giancotti and Mainiero, 1994). In recent years, a rapid accumulation of data has demonstrated that cross-linking of integrins by their ligands can lead to activation of a variety of signaling pathways (reviewed in Defilippi et al., 1997). Interestingly, coupling of integrin receptors to mitogen-activated protein kinase (MAPK) pathways has been reported (Chen et al, 1994; Wary et al., 1996; Mainiero et al., 1997, 1998). At least three distinct groups of MAPKs have been identified in mammals, including extracellular signal-regulated kinases (Erk1 and Erk2), c-Jun N-terminal kinase (Jnk), and p38 § To whom correspondence should be addressed (e-mail: fmainiero@
axrma.uniroma1.it).
(reviewed in Seger and Krebs, 1995; Su and Karin, 1996; Robinson and Cobb, 1997). Unlike Erk and Jnk, very few data are available on the activation of p38 MAPK following integrin stimulation. In mammalian cells, p38 MAPK, the Hog1p homolog, can be activated by multiple stimuli, such as proinflammatory cytokines and hemopoietic growth factors, lipopolysaccharide (LPS), and physical-chemical changes in the extracellular milieu caused by environmental stress (heat, osmotic shock, UV irradiation) (Lee et al., 1994; Su and Karin, 1996). p38 is specifically phosphorylated and activated by the MAPK kinases (MAPKK) MKK3 and MKK6 (De´rijard et al., 1995; Raingeaud et al., 1996; Enslen et al., 1998), which do not affect JNK and ERK activity. Additional components of the p38 MAPK pathway have not been fully identified yet. However, the Rho family GTPases Rac1 and Cdc42 (reviewed in Van Aelst and D’Souza-Schorey, 1997; Mackay and Hall, 1998) and the cytoplasmic kinase Pak1 (Bagrodia et al., 1995; Zhang et al., 1995) have been implicated in the control of the p38 MAP kinase signaling pathway. p38 MAPK is implicated in the regulation of inflammatory and immune responses by promoting the expression of several cytokines and controlling cell proliferation and death. To date, no evidence on the involvement of the p38 MAPK signaling pathway in the regulation of natural killer (NK) cell activation and functions is available. NK cells belong to a distinct lineage of lymphocytes that play an important role in the early phase of immune responses against certain viruses, parasites, and microbial pathogens by exhibiting cytotoxic functions and secreting a number of cytokines (reviewed in Scott and Trinchieri, 1995; Biron, 1997). NK cells circulate in the peripheral blood, are resident in the spleen, liver, lungs, and intestine, and rapidly accumulate in the parenchimas of several organs during inflammation, tumor growth, and invasion. Recent evidence indicates that NK cells can also be involved in the recruitment of various leukocyte cell types based on their capacity of synthesizing several chemokines, including IL-8 (Saito et al., 1994; Somersalo et al., 1994). IL-8 is the most throughly characterized member of the C-X-C or a-chemokine family. IL-8 is a potent polymorphonuclear cell chemoattractant, although it can affect migration of additional effector cells, such as T lymphocytes, monocytes, basophils, and eosinophils, into the target tissue. In addition to migration, IL-8 induces neutrophil adhesiveness, degranulation, and respiratory burst. Besides its central role in inflammation, IL-8 has also been implicated in other biological processes such as angiogenesis and hematopoiesis (reviewed in Baggiolini et al., 1994; Baggiolini et al., 1997; Rollins, 1997). We have previously shown that peripheral blood human NK cells express b1 integrin receptors and that their engagement transduces intracellular signals leading to activation of the Fak-related nonreceptor tyrosine kinase Pyk-2, tyrosine phosphorylation of paxillin (Gismondi et al., 1997), elevation of intracellular calcium,
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Figure 1. Ligation of b1 Integrin FN Receptors Induces IL-8 Chemokine Production in Human NK Cells (A) Human NK cells were left untreated (C) or stimulated with anti-b1 (4B4), anti-b1 (TS2/ 16) F(ab9)2 fragments, anti-CD16 (B73.1), antiCD56 (C218) mAb, FN, or BSA for 24 hr at 378C in a 5% CO2 atmosphere in duplicate wells of flat-bottom plates (10 3 106 cells/ well) in medium containing 1% BSA. After incubation, cell-free supernatants were collected, and IL-8 concentration was quantitated by ELISA. These results are representative of one out of three independent experiments. (B) Total RNA was extracted from human NK cells left untreated (-) or stimulated with antib1 (4B4) or anti-CD56 (C218) mAb for the indicated times at 378C, and RT-PCR with IL-8 or b-actin-specific primers was performed. PCR products were electrophoresed on a 1.5% agarose gel, and the gels were then stained with ethidium bromide and photographed. These results are representative of one out of three independent experiments.
and costimulation of NK cytotoxic functions (Palmieri et al., 1995). More recently, we demonstrated that ligation of b1 integrins on human NK cells results in the stimulation of the Ras/ERK signaling pathway, which controls interferon (IFN)g production (Mainiero et al., 1998). Despite the increasingly prominent role of chemokines in the regulation of integrin function, relatively little is known about the ability of integrins to affect chemokine gene expression and the signaling pathways that may mediate these effects. We demonstrate here that ligation of b1 integrins, namely fibronectin (FN) receptors, on human NK cells results in the production of the proinflammatory chemokine IL-8, which requires activation of the Rac1/p38 MAPK signaling pathway. Results Ligation of b1 Integrin FN Receptors on Human NK Cells Stimulates IL-8 Production that Requires p38 MAPK We first examined if ligation of b1 integrins on human NK cells causes IL-8 production by testing the presence of this chemokine in the supernatants of human NK cells stimulated with anti-b1, anti-b1 F(ab9)2, anti-CD16, or anti-CD56 control mAb. b1 integrin cross-linking resulted in high levels of IL-8 production, comparable to those induced by engagement of CD16, one of the major NK activating receptors; by contrast, treatment of human NK cells with anti-CD56 mAb did not stimulate IL-8 production. This event did not result from the engagement of NK cell FcgRIII (CDI6) in that anti-b1 F(ab9)2 fragments were also effective (Figure 1A). We then tested whether FN, which interacts with a4b1 and a5b1 on human NK cells (Gismondi et al., 1991), may also induce IL-8 production. As shown in Figure 1A, treatment with FN, but not BSA, induced IL-8 production at significant levels. We also confirmed this result at mRNA level. RT-PCR analysis, performed on total RNA from human NK cells, showed that b1 integrin, but not CD56 antigen ligation, induced IL-8 mRNA expression that is maximal at 1.5 hr and still persistent at 4 hr after stimulation (Figure 1B). Activation of p38 MAPK has been implicated among the signaling pathways leading to regulation of cytokine
expression (Lee et al., 1994). To determine the potential role of p38 MAPK in the regulation of integrin-induced IL-8 production, we initially used SB203580, a pyridinyl imidazole drug compound that specifically binds to p38 MAPK and reversibly blocks its enzymatic activity (Lee et al., 1994). A dose-dependent inhibition of IL-8 production was obtained in b1 integrin–stimulated NK cells in the presence of different concentrations of SB203580 (Figure 2A). SB203580, at the concentration of 1 mM, also abrogated FN-induced IL-8 production (Figure 2C). By contrast, b1 integrin–induced IFNg production was not significantly affected even by the highest concentration of SB203580 (10 mM) (Figure 2B). In addition, no inhibition of IL-8 production was obtained by using the specific MEK-1 inhibitor PD98059, which completely abrogated IFNg production as previously shown (Mainiero et al., 1998) (Figures 2A and 2B). The presence of these inhibitors did not affect human NK cell viability at the time point (24 hr) when culture supernatants were harvested to determine cytokine production (data not shown). In order to provide direct evidence on the functional requirement for p38 MAPK in b1 integrin–mediated IL-8 production, we attempted to perturb p38 expression using antisense oligodeoxynucleotides (ODNs) (Nagata et al., 1998). Human NK cells were incubated with antisense (AS) and complementary sense (S) ODNs of p38 and then left untreated or stimulated with anti-b1 mAb. Western blot analysis showed that p38 was greatly diminished in cells exposed to AS-ODN when compared to cells exposed to S-ODN or to untreated control (Figure 2E). Decreased p38 expression resulted in complete inhibition of b1 integrin–induced IL-8 production, indicating that p38 MAPK is functionally involved in this event (Figure 2D).
The p38 MAPK Is Activated by Ligation of b1 Integrin FN Receptors on Human NK Cells We directly tested if ligation of b1 integrins on human NK cells could result in p38 MAPK activation. Human NK cells were stimulated with anti-b1, anti-b1 F(ab9)2, anti-CD16, or anti-CD56 mAb, and phosphorylation of p38 MAPK was examined using an antibody against the active, phosphorylated form of p38 MAPK. Western blot
Integrins Control IL-8 via RAC1/P38 in NK 9
Figure 2. b1 Integrin–Induced IL-8 Chemokine Production in Human NK Cells Requires p38 MAPK (A–C) Human NK cells were incubated for 30 min at 378C with different concentrations of SB203580 or PD098059 and then stimulated with anti-b1 (4B4), anti-CD56 (C218) mAb, FN, or BSA for 24 hr at 378C in a 5% CO2 atmosphere in duplicate wells of flat-bottom plates (10 3 106 cells/well) in medium containing 1% BSA. After incubation, IL-8 or IFNg concentrations were quantitated by ELISA in the cell-free supernatants. (D and E) Human NK cells were incubated with p38 AS or S-ODNs for 72 hr and then left untreated or stimulated with anti-b1 (4B4) mAb for 24 hr at 378C in a 5% CO2 atmosphere in duplicate wells of flat-bottom plates (10 3 106 cells/well) in medium containing 1% BSA. After incubation, IL-8 concentration was quantitated by ELISA in the cell-free supernatants (D), and the amount of p38 was examined on total cell lysates by Western blot analysis; as loading controls, the amounts of p44/42 MAPK proteins are shown on the bottom (E). These results are representative of one out of three independent experiments.
analysis showed that b1 integrin stimulation causes significant activation of p38 MAPK at levels comparable to those induced by engagement of CD16. By contrast, anti-CD56 control mAb treatment did not result in any significant activation of p38 MAPK. In addition, treatment with FN, but not BSA, induced activation of p38 MAPK (Figure 3A). The analysis of the p38 MAPK activation time course shows that it is rapid, is still persistent at 30 min, and returns to basal levels at 90 min after stimulation (Figure 3B). Similar results were obtained by analyzing p38 MAPK activation by in vitro kinase assay on p38 immunoprecipitates, using GST-ATF-2 as substrate (Figure 3C). The presence of SB203580, at the same concentrations that abolished b1 integrin–induced IL-8 production, completely inhibited p38 but not Erk and Jnk MAPK activation (Figure 3D). Pak1 and MKK3 Are Activated by b1 Integrin Cross-Linking on Human NK Cells The mammalian protein-serine kinase Pak1, MKK3, and MKK6 have been implicated in the control of the p38 MAPK activity (Bagrodia et al., 1995; De´rijard et al., 1995; Zhang et al., 1995; Raingeaud et al., 1996; Enslen et al., 1998). We examined if b1 integrin cross-linking induces Pak1 activation in human NK cells. An in vitro kinase assay performed on anti-Pak1 immune complexes isolated from NK cells stimulated with anti-b1 or anti-CD56 Mab showed that Pak1 activity was significantly increased following b1 integrin ligation, as visualized by phosphorylation of the exogenous substrate MBP. Pak1 activation was already maximal at 5 min and declined
at 15 min after stimulation. No changes in Pak1 activity were observed in untreated or anti-CD56 mAb-treated NK cells used as control (Figure 4A). We also investigated if b1 integrin cross-linking on human NK cells could induce activation of MKK3 and/or MKK6. Western blot experiments showed that b1 integrin cross-linking causes significant levels of activation of MKK3. The time course reveals that it is rapid, peaks at 5 min, is still persistent at 30 min, and returns to basal levels after 90 min. In contrast, anti-CD56 control mAb treatment did not result in any significant activation of MKK3 (Figure 4B). The levels of b1 integrin– triggered MKK3 phosphorylation were comparable to those obtained with anisomycin or sorbitol (data not shown). By contrast, no activation of MKK6 was observed in b1 integrin–stimulated human NK cells, although MKK6 stimulation was readily induced by anisomycin and sorbitol (Figure 4C). These data indicate that activation of p38 MAPK through ligation of b1 integrins on human NK cells is associated with significant and persistent activation of Pak1 and MKK3, but not MKK6, strongly suggesting their involvement in this event. Cross-Linking of b1 Integrins on Human NK Cells Activates Rac1, which Controls p38 MAPK Activation It is well known that the serine/threonine kinase Pak1 binds to GTP-bound Rac1 (Martin et al., 1995). Despite a large amount of evidence implicating Rac1 in integrinmediated functional responses (D’Souza-Schorey et al.,
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Figure 3. Activation of p38 MAPK by b1 Integrin Cross-Linking on Human NK Cells (A) Human NK cells were left untreated (-) or stimulated with anti-b1 (4B4), anti-b1 (TS2/ 16) F(ab9)2 fragments, anti-CD16 (B73.1), antiCD56 (C218) Mab, FN, or BSA for the 15 min at 378C, and p38 MAPK activation was examined by Western blot analysis performed on total cell lysates. As loading controls, the amounts of p38 are shown on the bottom. (B and C) Human NK cells were left untreated (-) or stimulated with anti-b1 (4B4) mAb for the indicated times at 378C, and p38 MAPK activation was examined as above (B) or on p38 immunoprecipitates by in vitro kinase assay using GST-ATF-2 as substrate (C). As loading controls, the amounts of p38 are shown on the bottom. (D) Human NK cells were incubated for 30 min at 378C with different concentrations of SB203580 and then stimulated with anti-b1 (4B4) or anti-CD56 (C218) mAb for 10 min at 378C, and p38, Erk, and Jnk MAPK activation were examined by Western blot analysis performed on total cell lysates. As loading controls, the amounts of p38, Erk, and Jnk MAPK proteins are shown. These results are representative of one out of three independent experiments.
1998; Price et al., 1998), no direct demonstration of Rac1 activation upon integrin receptor triggering has been provided so far. Thus, we performed GTP-loading experiments to examine if ligation of b1 integrins on human NK cells results in the activation of Rac1. After in vivo labeling with 32P-orthophosphate, human NK cells were stimulated with anti-b1 or anti-CD56 mAb. Chromatographic analysis of nucleotides bound to Rac1 indicated that b1 integrin stimulation results in a 2-fold increase in the proportion of GTP-bound Rac1 (from 27% to 55%). Anti-CD56 mAb-treated control sample showed a p21 Rac1 GTP/GDP1GTP ratio comparable to that of the untreated sample (Figure 5A). To directly assess the role of Rac1 in b1 integrin– induced activation of p38 MAPK, human NK cells were infected with recombinant vaccinia virus encoding wildtype (WT) Rac1 or dominant-negative N17-Rac1. Infected human NK cells were then left untreated or stimulated with anti-b1 mAb, and phosphorylation of p38 MAPK was examined as above. Western blot analysis showed that dominant-negative N17-Rac1 completely inhibits b1 integrin–induced p38 MAPK activation, whereas wild-type Rac1 overexpression causes a slight increase in the b1 integrin–induced p38 MAPK activation (Figure 5B). In addition, infection with wild-type virus alone (WR) did not affect p38 activation. These data indicate that the small G protein Rac1 controls b1 integrin–induced p38 MAP kinase activation on human NK cells.
Cross-Linking of b1 Integrins on Human NK Cells Induces Vav Tyrosine Phosphorylation, which Controls p38 MAPK Activation and IL-8 Production The protooncogene p95 Vav, selectively expressed in hematopoietic cells, acts as GEF for the low molecular weight Rho-family GTPase Rac1, and its GEF activity is regulated by PTK-dependent tyrosine phosphorylation (Crespo et al., 1997). To investigate the possible involvement of Vav in b1 integrin–induced Rac1 activation, human NK cells were stimulated with anti-b1 or anti-CD56 mAb, and anti-Vav immunoprecipitates were examined by immunoblotting with anti-pTyr mAb. Marked tyrosine phosphorylation of Vav was detected after b1 integrin cross-linking; Vav phosphorylation was rapid, peaked at 5 min, and declined at 15 min. No changes in the phosphorylation status of Vav were observed in antiCD56 mAb-treated NK cells used as control (Figure 6A). These results indicate that Vav undergoes tyrosine phosphorylation in b1 integrin–stimulated NK cells and strongly suggest that its GEF activity can be involved in b1 integrin–mediated p21 Rac1 and p38 MAP kinase activation. In order to provide direct evidence on the functional requirement for Vav in b1 integrin–mediated p38 MAPK activation and IL-8 production, we attempted to perturb Vav expression using ODNs (Galandrini et al., 1999). Human NK cells were incubated with Vav AS- or S-ODNs and then left untreated or stimulated with anti-b1 mAb. Western blot analysis showed that immunodetectable
Integrins Control IL-8 via RAC1/P38 in NK 11
Figure 4. Activation of Pak1 and MKK3, but Not MKK6, by b1 Integrin Cross-Linking on Human NK Cells (A) Human NK cells were left untreated (-) or stimulated with antib1 (4B4) or anti-CD56 (C218) mAb for the indicated times at 378C. On anti-Pak1 immunoprecipitates, an in vitro kinase assay was performed using MBP as substrate. As loading controls, the amounts of Pak1 immunoprecipitated are shown on the bottom. These results are representative of one out of three independent experiments. (B and C) Human NK cells were left untreated (-) or stimulated with anti-b1 (4B4) or anti-CD56 (C218) mAb for the indicated times or with anisomycin ([AN];10 mg/ml, 45 min) or sorbitol ([Srb]; 0.5 M, 30 min) at 378C, and activation of MKK3 or MKK6 was examined by Western blot analysis performed on total cell lysates. As loading controls, the amounts of MKK3 or MKK6 are shown on the bottom. These results are representative of one out of three independent experiments.
Vav was greatly diminished in cells exposed to AS-ODN when compared to cells exposed to S-ODN or to untreated control. AS-ODN but not S-ODN treatment markedly reduced b1 integrin–induced p38 MAPK activation as well as IL-8 production, indicating that Vav activity is functionally involved in these events (Figures 6B and 6C). Discussion Here, we demonstrate that b1 integrin ligation on human peripheral blood NK cells results in the activation of the Rac/p38 MAPK signaling pathway that is required for integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the b1 integrin–mediated p38 MAPK activation, namely the
activation of Rac GEF p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, our data directly demonstrate that p95 Vav and Rac1 control the activation of p38 MAPK triggered by b1 integrins. p38 MAPKs were initially characterized as enzymes activated in response to stresses such as heat, cold, UV irradiation, osmolar shock, and to the proinflammatory cytokines, tumor necrosis factor (TNF)a, and interleukin-1 (IL-1) (Lee et al., 1994; Su and Karin, 1996). Crosslinking of the T cell receptor (TCR), CD28, or Fas on proliferating T cells, or cross-linking of the B cell receptor (BCR) or CD40 on freshly isolated or LPS-activated splenic B cells (Salmon et al., 1997; Craxton et al., 1998; Zhang et al., 1999) also results in rapid activation of p38 MAPK, and this event has been implicated in the regulation of cytokine production and apoptosis. No evidence on the role of p38 MAPK activation in the regulation of NK cell functions has been provided so far. Moreover, although several studies have described the ability of integrins to activate ERK and JNK (Chen et al, 1994; Wary et al., 1996; Mainiero et al., 1997, 1998), it is unknown whether these receptors may also initiate p38 MAPK cascade. The results of this study demonstrate that ligation of b1 integrin FN receptors on human NK cells stimulates the phosphorylation and the enzymatic activity of p38 MAPK to levels comparable to those induced by CD16 engagement. Control of p38 MAPK activation has been shown to involve Rac and Cdc42, members of the Rho-related small GTPase family that are implicated in a wide spectrum of cellular processes, namely cytoskeletal organization, membrane trafficking, transcription factor regulation, cell cycle progression, and cellular transformation (reviewed in Van Aelst and D’Souza-Schorey, 1997; Mackay and Hall, 1998). The role of Rac1 in regulating transcription factor activity through its ability to activate MAPK cascades has been more extensively studied on the Jnk pathway (Coso et al., 1995; Minden et al., 1995), whereas relatively little is known about Racmediated regulation of the p38 MAPK pathway. Rac1 has been found to be coupled to and regulate the activity of p38 MAPK in response to inflammatory cytokines such as IL-1 (Bagrodia et al., 1995). Moreover, recent evidence shows impaired p38 MAPK phosphorylation induced by fLMP stimulation in Rac-2-deficient neutrophils (Roberts et al., 1999). Consistent with these studies, by the use of recombinant vaccinia virus encoding the dominant-negative N17-Rac1 mutant, we show that Rac1 controls b1 integrin–mediated activation of p38 MAPK. Moreover, we provide direct demonstration of the ability of integrins to induce GDP/GTP exchange activity on endogenous Rac1, although several studies using transient or inducible expression of either activated or dominant-negative Rac1 mutants have suggested a role for Rac in the formation of membrane ruffles and lamellipodia, as well as in the integrin-mediated cell spreading on FN (D’Souza-Schorey et al., 1998; Price et al., 1998). We have also shown the potential upstream signaling events leading to b1 integrin–mediated Rac1/p38 MAPK activation. Rac1 activation paralleled the tyrosine phosphorylation of GEF for Rac1, Vav (Crespo et al., 1997). Like dominant-negative N17-Rac1 infected human NK
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Figure 5. Cross-Linking of b1 Integrins on Human NK Cells Activates Rac1, which Controls p38 MAP Kinase Activation (A) TLC of the nucleotides eluted from Rac1 immunoprecipitates of (32P)orthophosphatelabeled NK cells. Cells were stimulated for 5 min with appropriate doses of anti-b1 (4B4) or anti-CD56 (C218) mAb at 378C before lysis. The position at which GTP and GTP standards run is indicated. Numbers indicate the molar ratio of GTP over total nucleotides from quantitation by direct scanning for b radiation of the same experiment. These results are representative of one out of three independent experiments. (B) Human NK cells were infected with recombinant vaccinia virus encoding wild-type (WT) Rac1, dominant-negative N17-Rac1, or wildtype vaccinia virus alone (WR). Infected human NK cells were left untreated (-) or stimulated with anti-b1 (4B4) mAb for 59 at 378C, and p38 activation was examined by Western blot analysis performed on total cell lysates. As loading controls, the amounts of p38 protein are shown on the center panel. As overexpression control, the amounts of Rac1 protein are shown on the bottom. These results are representative of one out of three independent experiments.
cells, no b1 integrin–induced p38 MAPK activation was observed in Vav AS-ODN treated cells, further supporting a role for Vav in Rac1 activation. Vav tyrosine phosphorylation was previously observed upon aIIbb3 cell adhesion on fibrinogen or Ab-mediated b1 and b2 integrin ligation in myeloid cells (Cichowski et al., 1996; Gotoh et al., 1997). At present, no knowledge of the protein tyrosine kinases (PTKs) responsible for b1 integrin–induced Vav phosphorylation is available. Much evidence indicates that PTKs belonging to the Src and the Syk/Zap-70 families may be implicated in this event. aIIbb3 stimulates Vav1 tyrosine phosphorylation in a Syk-dependent manner (Miranti et al., 1998), and association between tyrosine-phosphorylated Syk and tyrosine-phosphorylated Vav has been reported in human
NK cells following interaction with sensitive targets (Galandrini et al., 1999). On the other hand, a role for the Fak family kinase Pyk-2 may also be envisaged, as we have recently demonstrated the ability of b1 integrins to stimulate tyrosine phosphorylation of Pyk-2 in human NK cells (Gismondi et al., 1997), and Pyk-2 activation has been reported as a receptor proximal event controlling Rac and p38 MAPK activation in response to stress stimuli (Tokiwa et al., 1996; Pandey et al., 1999). Our data also indicate that activation of p38 MAPK through ligation of b1 integrins on human NK cells is associated with a significant and persistent activation of Pak1 and MKK3, strongly suggesting that they are potential constituents of the p38 MAPK signaling pathway.
Figure 6. Ligation of b1 Integrins on NK Cells Induces Tyrosine Phoshorylation Vav, which Controls p38 MAPK Activation and IL-8 Production (A) Human NK cells were left untreated (-) or stimulated with anti-b1 (4B4) or anti-CD56 (C218) mAb for the indicated times at 378C. Cells were immunoprecipitated with anti-Vav mAb. Resulting protein complexes were resolved by 7% SDS-PAGE and immunoblotted with anti-pTyr. As loading controls, the amounts of Vav immunoprecipitated are shown on the bottom. These results are representative of one out of three independent experiments. (B) Human NK cells were exposed to Vav-AS or -S ODNs for 56 hr and then left untreated or stimulated with anti-b1 (4B4) mAb. Vav expression and p38 activation were examined by Western blot analysis performed on total cell lysates. As loading controls, the amounts of Syk and p38 MAPK proteins are shown. (C) Human NK cells were incubated with Vav ODNs and stimulated as above for 24 hr at 378C in a 5% CO2 atmosphere in duplicate wells of flat-bottom plates (10 3 106 cells/ well) in medium containing 1% BSA. After incubation, IL-8 concentration was quantitated by ELISA in the cell-free supernatants.
Integrins Control IL-8 via RAC1/P38 in NK 13
Figure 7. The RAC1/P38 MAPK Signaling Pathway Controls b1 Integrin–Induced IL-8 Production in Human NK Cells Our model depicts a Rac1-dependent pathway generated by the ligation of b1 integrins on human NK cells leading to p38 MAPK activation that controls the production of the proinflammatory chemokine IL-8. b1 integrin ligation induces tyrosine phosphorylation of the protooncogene p95 Vav, which may catalyze Rac1 activation. Rac1 activation is associated with b1 integrin–induced activation of Pak1 and MKK3 and controls p38 MAPK activation leading to the production of IL-8.
Pak 1 belongs to a family of closely related serine/ threonine kinases that mediate several of the downstream effects of Rac and Cdc42, including activation of Jnk and p38 MAPKs, and reorganization of the actin cytoskeleton (Bagrodia et al., 1995; Zhang et al., 1995; Sells and Chernoff, 1997). Rapid activation of Pak1 catalytic activity is induced by a large number of extracellular ligands including growth factors and inflammatory and chemotactic cytokines and is initiated by the binding of GTP-Rac1 or GTP-Cdc42, which promote its autophosphorylation (Sells and Chernoff, 1997). Recent evidence indicates that cross-linking of CD3/TCR complex or Fas on T cells also results in rapid activation of Pak1, and this event is involved in the regulation of gene expression and apoptosis (Rudel et al., 1998). The MAPK kinases MKK3 and MKK6, which share 80% amino acid identity, are considered upstream activators of the p38 MAPK signaling pathway. Recent data indicate that the dominant-negative mutant of MKK3 but not of MKK6 inhibits p38 activity in response to hyperosmolarity or methylmetane sulfonate (MMS) (Pandey et al., 1999), and TNF-induced cytokine and IL-12 is impaired in MKK3-deficient mice production (Lu et al, 1999; Wysk et al., 1999). In accordance with these reports, our evidence that b1 integrin stimulation on NK cells causes significant activation of MKK3, but not MKK6,
strongly suggest that MKK3 is responsible for the integrin-triggered p38 activation. The MAPK kinase kinases (MKKKs) involved in the activation of MKK3 through integrins are presently unknown, and further studies are required to define their function and specificity. Finally, our results demonstrate that the ligation of b1 integrins on human NK cells results in the stimulation of IL-8 production that is under the control of p38 MAPK activation, as shown by the use of the specific synthetic inhibitor SB203580 and p38 AS-ODN. Moreover, consistent with the finding that Vav controls p38 MAPK activation, we demonstrated a role for Vav in b1 integrin– induced IL-8 production using Vav AS-ODN. As in our study, activation of p38 MAPK was found indispensable for the neutrophil IL-8 production stimulated by granulocyte-macrophage colony stimulating factor (GM-CSF), LPS, as well as TNFa, but not for that induced by phorbol myristate acetate (PMA) or ionomycin (Zu et al., 1998). The mechanisms involved in the p38 MAPK-mediated IL-8 production by integrins are unknown. It is conceivable, however, that they may be related to the ability of p38 MAPK to regulate the activity of several transcription factors involved in the control of the IL-8 gene promoter including Elk-1, NF-kB, ATF-2, and CREB (Price et al., 1996; Su and Karin, 1996; Tan et al., 1996; Craxton et al, 1998). In sum, the results of this study suggest a model that depicts a Rac1-dependent pathway generated by the ligation of b1 integrins on human NK cells leading to p38 MAPK activation that controls the production of the proinflammatory chemokine IL-8 (Figure 7). The identity of PTKs involved in Vav tyrosine phosphorylation and of the MKKK(s) that cause MKK3 activation is under investigation. What is the pathophysiological relevance of IL-8 produced by NK cells in response to b1 integrin stimulation? Production of IL-8 by NK cells has been previously reported in response to cytokines such as IL-2 and IL18 (Puren et al., 1998) and upon stimulation through CD16, and IL-8 secreted by activated NK cells has been shown to support a4b1 and a5b1-mediated migration of T cells (Somersalo et al., 1994). Moreover, decidual NK cells as well as NK cells generated from human duodenal mucosa express IL-8 mRNA and produce IL-8 (Saito et al., 1994). b1 integrins have been shown to control NK cell adhesion to endothelial cells and migration of NK cells into the normal and neoplastic tissues (Allavena et al., 1991; Somersalo and Saksela, 1991; Fogler et al., 1996). Moreover, tissue uterine NK cells migrate over a dense hormonally regulated ECM rich in FN and laminin. Based on our data, it can be hypothesized that NK cells that enter early into the sites of inflammation, by secreting IL-8 upon interaction with activated endothelium or ECM components, may participate in the recruitment of additional effector cells, such as neutrophils and T lymphocytes into the target tissue, thus affecting the progression and evolution of the inflammatory response. In the decidua, where NK cells are the predominant population during the early stages of gestation, NK cell–derived IL-8 might contribute to regulation of the selective leukocyte access at the maternal/fetal interface.
Immunity 14
Experimental Procedures Antibodies and Reagents Anti-CD16 (B73.1) mAb was kindly provided by Dr. G. Trinchieri (Wistar Institute, Philadelphia, PA). Anti-CD56 (C218) mAb was generously provided by Dr. A. Moretta (University of Genoa, Genoa, Italy). Anti-b1 (4B4) was purchased from Coulter Immunology. Antib1 (TS2/16) F(ab9)2 fragments were prepared by proteolytic digestion as described previously (Gismondi et al., 1995). The anti-phosphotyrosine (anti-pTyr) 4G10 and the anti-Vav mAb were purchased from UBI. The affinity-purified rabbit antisera against Rac1, Pak1, MKK3, Erk, Jnk, and p38 MAPKs, the goat antiserum against MKK6, and the anti-Syk mAb were obtained from Santa Cruz Biotechnology. The affinity-purified rabbit antiserum against mouse Ig was purchased from Zymed Laboratories. Affinity-purified (Fab9)2 fragments of goat anti-mouse Ig (GAM) were purchased from ICN-Cappel. GST-ATF-2 fusion protein was a kind gift of Dr. C. J. Der (University of North Carolina at Chapel Hill). Human plasma FN was purchased from GIBCO. The p38 MAPK inhibitor SB203580 was purchased from Alexis Corporation and the MEK-1 inhibitor PD 098059 from Calbiochem-Novabiochem. Anisomycin and sorbitol were purchased from Sigma. Human NK Cell Preparation and Stimulation Highly purified (97%) cultured NK cells were obtained by incubating for 8 days nylon nonadherent peripheral blood mononuclear cells (PBMC) (4 3 105 cells) with irradiated (3000 rads) RPMI 8866 cells (1 3 105) as previously described (Mainiero et al., 1994). Human NK cells were stimulated as previously described (Mainiero et al., 1998). When indicated, cells were pretreated (30 min at 378C) with the p38 MAPK inhibitor SB203580 or MEK-1 inhibitor PD 098059 before stimulation. In some experiments, before stimulation, NK cells were infected with recombinant vaccinia virus encoding wild-type (WT) Rac1, dominant-negative N17-Rac1, or wild-type vaccinia virus alone (WR), kindly provided by Dr. Leibson (Mayo Clinic and Fundation, Rochester, Minnesota) (Billadeau et al., 1998). In brief, semipurified recombinant vaccinia virus was used to infect human NK cells for 1 hr in serum-free medium at a multiplicity of infection of 20:1. The remainder of the infection (4 hr) was carried out in RPMI 1640 containing 10% FCS. In other experiments, before stimulation, NK cells were treated with antisense (AS) and complementary sense (S) oligodeoxynucleotides (ODNs) for Vav (Galandrini et al., 1999) or p38 (Nagata et al., 1998). The sequences were as follows: AS-Vav, (59-CATTGGCGCCACAGCTCCAT-39); S-Vav, (59-ATGGAGCTGTGGCGCCAATG-39); AS-p38, (59-GGCCTCTCCTG CGACATCTT-39); and S-p38, (59-AAGATGTCGCAGGAGAGGCC-39). Human NK cells (2.5 3 105/ml) were exposed to ODNs (100 mg/ml) in heat-inactivated (658C for 30’) culture medium for 56 or 72 hr. Immunoprecipitation and Immunoblot Analysis To estimate Rac1 activation, human NK cells were starved for 3 hr in phosphate-free RPMI and labeled for 3 hr with [32P]-orthophosphate (0.5 mCi/ml) (4,500 Ci/mmol, ICN, Biomedicals) in phosphatefree RPMI supplemented with 0.1% phosphate-free fetal calf serum. After stimulation, the cells were extracted and the immunoprecipitated samples subjected to Rac-GTP loading assay as previously described for Ras (Mainiero et al., 1997). To immunoprecipitate Vav, stimulated and unstimulated human NK cells were extracted in Triton lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, and 1% Triton X-100) containing 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 25 mM sodium fluoride, 0.01% aprotinin, 4 mg/ml pepstatin A, 10 mg/ml leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF) (all from Sigma) for 30 min on ice. Immunoprecipitation, SDS-PAGE, and immunoblotting analysis were performed as previously described (Mainiero et al., 1995). Nitrocellulose-bound antibodies were detected by chemiluminescence with ECL (Amersham Life Sciences, Little Chalfont, UK). Phoshorylated forms of Erk, Jnk, and p38 MAPKs, MKK3, and MKK6 were detected by Western blot analysis by using PhosphoPlus p44/42 Erk, SAPK/Jnk, and p38 MAPK and MKK3/MKK6 Ab Kit (New England Biolabs, Hitchin, UK) following protocols provided by the manufacturer. The identity of MKK3 and MKK6 was established by Western blot analysis using specific antibodies.
In Vitro Kinase Assay To analyze Pak1 activation, cells were extracted with Triton lysis buffer as above. Endogenous Pak1 was immunoprecipitated with anti-Pak1 antibodies and subjected to in vitro kinase assay. The kinase reaction was initiated by adding to the immunoprecipitate 25 ml of kinase buffer (25 mM Tris [pH 7.5], 12.5 mM b-glycerophosphate, 7.5 mM MgCl2, 20 mM cold ATP, and 0.5 mM sodium orthovanadate) containing 5 mCi of g[32P]-ATP (4,500 Ci/mmol, ICN) and 2.5 mg of myelin basic protein (MBP) (Sigma). After 30 min of incubation at 308C, the samples were boiled in sample buffer and separated by SDS-PAGE. The gels were dried, and the 32P-labeled proteins were made visible by autoradiography. To examine p38 MAPK activity, cells were extracted for 30 min on ice with modified Triton lysis buffer (25 mM Hepes [pH 7.5], 300 mM NaCl, 0.1% Triton X-100, 0.2 mM EDTA, 20 mM b-glycerophosphate, 1.5 mM MgCl2, and 0.5 mM DTT) containing phosphatase and protease inhibitors. Endogenous p38 MAPK was precipitated with anti-p38 antibodies and subjected to in vitro kinase assay. After washing, the beads were incubated with 25 ml of kinase buffer as above containing 1 mg of GST-ATF-2. After 30 min of incubation at 308C, the samples were boiled in sample buffer and separated by SDS-PAGE. The gels were dried, and the 32P-labeled proteins were made visible by autoradiography. RT-PCR Total RNA was extracted using RNAfast (Molecular System). For the preparation of cDNA, 1 mg of total RNA was incubated at 428C with 2 ml 103 reaction buffer, 4 ml of 25 mM MgCl2, 2 ml of 10 mM dNTPs, 2 ml of 0.8 mg/ml oligo-p(dT) primer, 50 U RNase inhibitor, and 20 U AMV reverse transcriptase (Boehringer Mannheim) in a total volume of 20 ml. A 50 ml solution, containing 5 ml of PCR reaction buffer 103, 3 ml of 25 mM MgCl2, 1 ml of 10 mM dNTPs, 2.5 ml of each primer at a concentration of 20 mM, 0.3 ml of 5 U/ml Taq DNA polymerase (Perkin-Elmer), and 1 ml of the cDNA reverse mixture, was subjected to 30 cycles of PCR, consisting of 1 min denaturation at 948C, 1 min annealing at 638C (IL-8) or 558C (b-actin), synthesis for 1 min at 728C, and a final elongation at 728C for 5 min. Primer sequences used for IL-8 were sense, (59-TCTCAGCCCTCTT CAAAAACTTCT-39) and antisense, (59-ATGACTTCCAAGCTGGCC GTGCT-39). Primer sequences used for b-actin were sense, (59-GGG TCAGAAGGATTCCTATG-39) and antisense, (59-GGTCTCAAACAT GATCTGGG-39). PCR products were electrophoresed on a 1.5% agarose gel in Tris-borate-EDTA buffer, and the gels were then stained with ethidium bromide and photographed. IL-8 and IFNg Production Assay Untreated or treated NK cells were seeded in duplicate wells of flatbottom plates (Costar)(10 3 106 cells/well) in medium containing 1% BSA. After incubation (24 hr, 378C in a 5% CO2 atmosphere), cell-free supernatants were collected. IL-8 concentration was quantitated with an ELISA kit (BioSource). IFNg concentration was quantitated with an ELISA kit (EuroClone, Torquay, UK). Acknowledgments We thank Dina Milana, Anna Maria Bressan, Alessandro Procaccini, Antonio Sabatucci, and Patrizia Birarelli for expert technical assistance and Sandro Valia for photographic assistance. We also thank Paul J. Leibson, Alessandro Moretta, and Giorgio Trinchieri for reagents. This work was partially supported by grants from the Italian Association for Cancer Research (AIRC), Istituto Superiore di Sanita` Italy-USA “Therapy of Tumors” Program, Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST), Ministero della Sanita`, and CNR special project on Biotechnologies. Received July 8, 1999; revised December 1, 1999. References Allavena, P., Paganin, I., Martin-Padura, I., Peri, G., Gaboli, M., Dejana, E., Marchisio, P.C., and Mantovani, A. (1991). Molecules and
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