Immunity, Vol. 12, 7–16, January, 2000, Copyright 2000 by Cell Press

RAC1/P38 MAPK Signaling Pathway Controlsb1 Integrin–Induced Interleukin-8 Productionin Human Natural Killer Cells

(reviewed in Seger and Krebs, 1995; Su and Karin, 1996;Robinson and Cobb, 1997). Unlike Erk and Jnk, veryfew data are available on the activation of p38 MAPKfollowing integrin stimulation.

In mammalian cells, p38 MAPK, the Hog1p homolog,

Fabrizio Mainiero,*§ Alessandra Soriani,*Raffaele Strippoli,* Jordan Jacobelli,*Angela Gismondi,* Mario Piccoli,*Luigi Frati,*† and Angela Santoni*‡

*Department of Experimental Medicine and Pathologycan be activated by multiple stimuli, such as proinflam-Istituto Pasteur-Fondazione Cenci Bolognettimatory cytokines and hemopoietic growth factors, lipo-University of Rome “La Sapienza”polysaccharide (LPS), and physical-chemical changesRome 00161in the extracellular milieu caused by environmentalItalystress (heat, osmotic shock, UV irradiation) (Lee et al.,†Mediterranean Institute of Neurosciences “Neuromed”1994; Su and Karin, 1996). p38 is specifically phosphory-Via Atinense 18lated and activated by the MAPK kinases (MAPKK)Pozzilli 86077MKK3 and MKK6 (Derijard et al., 1995; Raingeaud et al.,Italy1996; Enslen et al., 1998), which do not affect JNK and‡Laboratory of PathophysiologyERK activity. Additional components of the p38 MAPKRegina Elena Cancer Institutepathway have not been fully identified yet. However, theVia delle Messi d’oro 156Rho family GTPases Rac1 and Cdc42 (reviewed in VanRome 00158Aelst and D’Souza-Schorey, 1997; Mackay and Hall,Italy1998) and the cytoplasmic kinase Pak1 (Bagrodia et al.,1995; Zhang et al., 1995) have been implicated in thecontrol of the p38 MAP kinase signaling pathway.Summary

p38 MAPK is implicated in the regulation of inflamma-tory and immune responses by promoting the expres-The MAP kinase (MAPK) p38 plays a key role in regulat-sion of several cytokines and controlling cell prolifera-ing inflammatory responses. Here, we demonstratetion and death. To date, no evidence on the involvementthat b1 integrin ligation on human NK cells results inof the p38 MAPK signaling pathway in the regulation ofthe activation of the p38 MAPK signaling pathway,natural killer (NK) cell activation and functions iswhich is required for integrin-triggered IL-8 produc-available.tion. In addition, we identified some of the upstream

NK cells belong to a distinct lineage of lymphocytesevents accompanying the b1 integrin–mediated p38that play an important role in the early phase of immuneMAPK activation, namely, the activation of the Racresponses against certain viruses, parasites, and micro-guanine nucleotide exchange factor (GEF) p95 Vav,bial pathogens by exhibiting cytotoxic functions andthe small G protein Rac1, and the cytoplasmic kinasessecreting a number of cytokines (reviewed in Scott andPak1 and MKK3. Finally, we provide direct evidenceTrinchieri, 1995; Biron, 1997). NK cells circulate in thethat p95 Vav and Rac control the activation of p38peripheral blood, are resident in the spleen, liver, lungs,MAPK triggered by b1 integrins.and intestine, and rapidly accumulate in the parenchi-mas of several organs during inflammation, tumor

Introduction growth, and invasion.Recent evidence indicates that NK cells can also be

During immune and inflammatory responses, leukocyte involved in the recruitment of various leukocyte cellmigration across endothelium and into the tissues is types based on their capacity of synthesizing severallargely governed by a large number of chemoattractants chemokines, including IL-8 (Saito et al., 1994; Somersaloand adhesion receptors. et al., 1994). IL-8 is the most throughly characterized

Integrins are a family of heterodimeric ab receptors member of the C-X-C or a-chemokine family. IL-8 is athat mediate cell adhesion to extracellular matrix com- potent polymorphonuclear cell chemoattractant, al-ponents and cell to cell interactions (reviewed in Hynes, though it can affect migration of additional effector cells,1992; Giancotti and Mainiero, 1994). In recent years, a such as T lymphocytes, monocytes, basophils, and eo-rapid accumulation of data has demonstrated that sinophils, into the target tissue. In addition to migration,cross-linking of integrins by their ligands can lead to IL-8 induces neutrophil adhesiveness, degranulation,activation of a variety of signaling pathways (reviewed and respiratory burst. Besides its central role in inflam-in Defilippi et al., 1997). Interestingly, coupling of integrin mation, IL-8 has also been implicated in other biologicalreceptors to mitogen-activated protein kinase (MAPK) processes such as angiogenesis and hematopoiesis (re-pathways has been reported (Chen et al, 1994; Wary et viewed in Baggiolini et al., 1994; Baggiolini et al., 1997;al., 1996; Mainiero et al., 1997, 1998). At least three Rollins, 1997).distinct groups of MAPKs have been identified in mam- We have previously shown that peripheral blood hu-mals, including extracellular signal-regulated kinases man NK cells express b1 integrin receptors and that their(Erk1 and Erk2), c-Jun N-terminal kinase (Jnk), and p38 engagement transduces intracellular signals leading to

activation of the Fak-related nonreceptor tyrosine ki-nase Pyk-2, tyrosine phosphorylation of paxillin (Gis-§ To whom correspondence should be addressed (e-mail: fmainiero@

axrma.uniroma1.it). mondi et al., 1997), elevation of intracellular calcium,

Immunity8

Figure 1. Ligation of b1 Integrin FN Recep-tors Induces IL-8 Chemokine Production inHuman NK Cells

(A) Human NK cells were left untreated (C) orstimulated with anti-b1 (4B4), anti-b1 (TS2/16) F(ab9)2 fragments, anti-CD16 (B73.1), anti-CD56 (C218) mAb, FN, or BSA for 24 hr at378C in a 5% CO2 atmosphere in duplicatewells of flat-bottom plates (10 3 106 cells/well) in medium containing 1% BSA. After in-cubation, cell-free supernatants were col-lected, and IL-8 concentration was quantitatedby ELISA. These results are representative ofone out of three independent experiments.(B) Total RNA was extracted from human NKcells left untreated (-) or stimulated with anti-b1 (4B4) or anti-CD56 (C218) mAb for the indi-cated 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 stainedwith ethidium bromide and photographed. These results are representative of one out of three independent experiments.

and costimulation of NK cytotoxic functions (Palmieri et expression (Lee et al., 1994). To determine the potentialrole of p38 MAPK in the regulation of integrin-inducedal., 1995). More recently, we demonstrated that ligation

of b1 integrins on human NK cells results in the stimula- IL-8 production, we initially used SB203580, a pyridinylimidazole drug compound that specifically binds to p38tion of the Ras/ERK signaling pathway, which controls

interferon (IFN)g production (Mainiero et al., 1998). MAPK and reversibly blocks its enzymatic activity (Leeet al., 1994). A dose-dependent inhibition of IL-8 produc-Despite the increasingly prominent role of chemo-

kines in the regulation of integrin function, relatively little tion was obtained in b1 integrin–stimulated NK cells inthe presence of different concentrations of SB203580is known about the ability of integrins to affect chemo-

kine gene expression and the signaling pathways that (Figure 2A). SB203580, at the concentration of 1 mM,also abrogated FN-induced IL-8 production (Figure 2C).may mediate these effects.

We demonstrate here that ligation of b1 integrins, By contrast, b1 integrin–induced IFNg production wasnot significantly affected even by the highest concentra-namely fibronectin (FN) receptors, on human NK cells

results in the production of the proinflammatory chemo- tion of SB203580 (10 mM) (Figure 2B). In addition, noinhibition of IL-8 production was obtained by using thekine IL-8, which requires activation of the Rac1/p38

MAPK signaling pathway. specific MEK-1 inhibitor PD98059, which completely ab-rogated IFNg production as previously shown (Mainieroet al., 1998) (Figures 2A and 2B). The presence of theseResultsinhibitors did not affect human NK cell viability at thetime point (24 hr) when culture supernatants were har-Ligation of b1 Integrin FN Receptors on Human NK

Cells Stimulates IL-8 Production that Requires vested to determine cytokine production (data notshown).p38 MAPK

We first examined if ligation of b1 integrins on human In order to provide direct evidence on the functionalrequirement for p38 MAPK in b1 integrin–mediated IL-8NK cells causes IL-8 production by testing the presence

of this chemokine in the supernatants of human NK cells production, we attempted to perturb p38 expressionusing antisense oligodeoxynucleotides (ODNs) (Nagatastimulated with anti-b1, anti-b1 F(ab9)2, anti-CD16, or

anti-CD56 control mAb. b1 integrin cross-linking re- et al., 1998). Human NK cells were incubated with anti-sense (AS) and complementary sense (S) ODNs of p38sulted in high levels of IL-8 production, comparable to

those induced by engagement of CD16, one of the major and then left untreated or stimulated with anti-b1 mAb.Western blot analysis showed that p38 was greatly di-NK activating receptors; by contrast, treatment of hu-

man NK cells with anti-CD56 mAb did not stimulate IL-8 minished in cells exposed to AS-ODN when comparedto cells exposed to S-ODN or to untreated control (Figureproduction. This event did not result from the engage-

ment of NK cell FcgRIII (CDI6) in that anti-b1 F(ab9)2 2E). Decreased p38 expression resulted in completeinhibition of b1 integrin–induced IL-8 production, indi-fragments were also effective (Figure 1A).

We then tested whether FN, which interacts with a4b1 cating that p38 MAPK is functionally involved in thisevent (Figure 2D).and a5b1 on human NK cells (Gismondi et al., 1991),

may also induce IL-8 production. As shown in Figure1A, treatment with FN, but not BSA, induced IL-8 pro-duction at significant levels. We also confirmed this re- The p38 MAPK Is Activated by Ligation of b1

Integrin FN Receptors on Human NK Cellssult at mRNA level. RT-PCR analysis, performed on totalRNA from human NK cells, showed that b1 integrin, but We directly tested if ligation of b1 integrins on human

NK cells could result in p38 MAPK activation. Humannot CD56 antigen ligation, induced IL-8 mRNA expres-sion that is maximal at 1.5 hr and still persistent at 4 hr NK cells were stimulated with anti-b1, anti-b1 F(ab9)2,

anti-CD16, or anti-CD56 mAb, and phosphorylation ofafter stimulation (Figure 1B).Activation of p38 MAPK has been implicated among p38 MAPK was examined using an antibody against the

active, phosphorylated form of p38 MAPK. Western blotthe signaling pathways leading to regulation of cytokine

Integrins Control IL-8 via RAC1/P38 in NK9

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 withanti-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 for24 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. Afterincubation, IL-8 concentration was quantitated by ELISA in the cell-free supernatants (D), and the amount of p38 was examined on total celllysates by Western blot analysis; as loading controls, the amounts of p44/42 MAPK proteins are shown on the bottom (E). These results arerepresentative of one out of three independent experiments.

analysis showed that b1 integrin stimulation causes sig- at 15 min after stimulation. No changes in Pak1 activitywere observed in untreated or anti-CD56 mAb-treatednificant activation of p38 MAPK at levels comparable to

those induced by engagement of CD16. By contrast, NK cells used as control (Figure 4A).We also investigated if b1 integrin cross-linking onanti-CD56 control mAb treatment did not result in any

significant activation of p38 MAPK. In addition, treat- human NK cells could induce activation of MKK3and/or MKK6. Western blot experiments showed thatment with FN, but not BSA, induced activation of p38

MAPK (Figure 3A). The analysis of the p38 MAPK activa- b1 integrin cross-linking causes significant levels of acti-vation of MKK3. The time course reveals that it is rapid,tion time course shows that it is rapid, is still persistent

at 30 min, and returns to basal levels at 90 min after peaks at 5 min, is still persistent at 30 min, and returnsto basal levels after 90 min. In contrast, anti-CD56 con-stimulation (Figure 3B). Similar results were obtained by

analyzing p38 MAPK activation by in vitro kinase assay trol mAb treatment did not result in any significant acti-vation of MKK3 (Figure 4B). The levels of b1 integrin–on p38 immunoprecipitates, using GST-ATF-2 as sub-

strate (Figure 3C). The presence of SB203580, at the triggered MKK3 phosphorylation were comparable tothose obtained with anisomycin or sorbitol (data notsame concentrations that abolished b1 integrin–induced

IL-8 production, completely inhibited p38 but not Erk shown). By contrast, no activation of MKK6 was ob-served in b1 integrin–stimulated human NK cells, al-and Jnk MAPK activation (Figure 3D).though MKK6 stimulation was readily induced by aniso-mycin and sorbitol (Figure 4C).Pak1 and MKK3 Are Activated by b1 Integrin

Cross-Linking on Human NK Cells These data indicate that activation of p38 MAPKthrough ligation of b1 integrins on human NK cells isThe mammalian protein-serine kinase Pak1, MKK3, and

MKK6 have been implicated in the control of the p38 associated with significant and persistent activation ofPak1 and MKK3, but not MKK6, strongly suggestingMAPK activity (Bagrodia et al., 1995; Derijard et al., 1995;

Zhang et al., 1995; Raingeaud et al., 1996; Enslen et al., their involvement in this event.1998). We examined if b1 integrin cross-linking inducesPak1 activation in human NK cells. An in vitro kinase Cross-Linking of b1 Integrins on Human NK Cells

Activates Rac1, which Controls p38assay performed on anti-Pak1 immune complexes iso-lated from NK cells stimulated with anti-b1 or anti-CD56 MAPK Activation

It is well known that the serine/threonine kinase Pak1Mab showed that Pak1 activity was significantly in-creased following b1 integrin ligation, as visualized by binds to GTP-bound Rac1 (Martin et al., 1995). Despite

a large amount of evidence implicating Rac1 in integrin-phosphorylation of the exogenous substrate MBP. Pak1activation was already maximal at 5 min and declined mediated functional responses (D’Souza-Schorey et al.,

Immunity10

Figure 3. Activation of p38 MAPK by b1 Inte-grin Cross-Linking on Human NK Cells

(A) Human NK cells were left untreated (-) orstimulated with anti-b1 (4B4), anti-b1 (TS2/16) F(ab9)2 fragments, anti-CD16 (B73.1), anti-CD56 (C218) Mab, FN, or BSA for the 15 minat 378C, and p38 MAPK activation was exam-ined by Western blot analysis performed ontotal cell lysates. As loading controls, theamounts of p38 are shown on the bottom.(B and C) Human NK cells were left untreated(-) or stimulated with anti-b1 (4B4) mAb forthe indicated times at 378C, and p38 MAPKactivation was examined as above (B) or onp38 immunoprecipitates by in vitro kinaseassay using GST-ATF-2 as substrate (C). Asloading controls, the amounts of p38 areshown on the bottom.(D) Human NK cells were incubated for 30min at 378C with different concentrations ofSB203580 and then stimulated with anti-b1(4B4) or anti-CD56 (C218) mAb for 10 min at378C, and p38, Erk, and Jnk MAPK activationwere examined by Western blot analysis per-formed on total cell lysates. As loading con-trols, the amounts of p38, Erk, and Jnk MAPKproteins are shown. These results are repre-sentative of one out of three independent ex-periments.

1998; Price et al., 1998), no direct demonstration of Rac1 Cross-Linking of b1 Integrins on Human NK CellsInduces Vav Tyrosine Phosphorylation, which Controlsactivation upon integrin receptor triggering has been

provided so far. Thus, we performed GTP-loading exper- p38 MAPK Activation and IL-8 ProductionThe protooncogene p95 Vav, selectively expressed iniments to examine if ligation of b1 integrins on human

NK cells results in the activation of Rac1. After in vivo hematopoietic cells, acts as GEF for the low molecularweight Rho-family GTPase Rac1, and its GEF activity islabeling with 32P-orthophosphate, human NK cells were

stimulated with anti-b1 or anti-CD56 mAb. Chromato- regulated by PTK-dependent tyrosine phosphorylation(Crespo et al., 1997). To investigate the possible involve-graphic analysis of nucleotides bound to Rac1 indicated

that b1 integrin stimulation results in a 2-fold increase ment of Vav in b1 integrin–induced Rac1 activation, hu-man NK cells were stimulated with anti-b1 or anti-CD56in the proportion of GTP-bound Rac1 (from 27% to 55%).

Anti-CD56 mAb-treated control sample showed a p21 mAb, and anti-Vav immunoprecipitates were examinedby immunoblotting with anti-pTyr mAb. Marked tyrosineRac1 GTP/GDP1GTP ratio comparable to that of the

untreated sample (Figure 5A). phosphorylation of Vav was detected after b1 integrincross-linking; Vav phosphorylation was rapid, peakedTo directly assess the role of Rac1 in b1 integrin–

induced activation of p38 MAPK, human NK cells were at 5 min, and declined at 15 min. No changes in thephosphorylation status of Vav were observed in anti-infected with recombinant vaccinia virus encoding wild-

type (WT) Rac1 or dominant-negative N17-Rac1. In- CD56 mAb-treated NK cells used as control (Figure 6A).These results indicate that Vav undergoes tyrosinefected human NK cells were then left untreated or stimu-

lated with anti-b1 mAb, and phosphorylation of p38 phosphorylation in b1 integrin–stimulated NK cells andstrongly suggest that its GEF activity can be involvedMAPK was examined as above. Western blot analysis

showed that dominant-negative N17-Rac1 completely in- in b1 integrin–mediated p21 Rac1 and p38 MAP kinaseactivation.hibits b1 integrin–induced p38 MAPK activation, whereas

wild-type Rac1 overexpression causes a slight increase In order to provide direct evidence on the functionalrequirement for Vav in b1 integrin–mediated p38 MAPKin the b1 integrin–induced p38 MAPK activation (Figure

5B). In addition, infection with wild-type virus alone (WR) activation and IL-8 production, we attempted to perturbVav expression using ODNs (Galandrini et al., 1999).did not affect p38 activation.

These data indicate that the small G protein Rac1 Human NK cells were incubated with Vav AS- or S-ODNsand then left untreated or stimulated with anti-b1 mAb.controls b1 integrin–induced p38 MAP kinase activation

on human NK cells. Western blot analysis showed that immunodetectable

Integrins Control IL-8 via RAC1/P38 in NK11

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 Rac1control the activation of p38 MAPK triggered by b1 inte-grins.

p38 MAPKs were initially characterized as enzymesactivated in response to stresses such as heat, cold, UVirradiation, osmolar shock, and to the proinflammatorycytokines, tumor necrosis factor (TNF)a, and interleu-kin-1 (IL-1) (Lee et al., 1994; Su and Karin, 1996). Cross-linking of the T cell receptor (TCR), CD28, or Fas onproliferating T cells, or cross-linking of the B cell recep-tor (BCR) or CD40 on freshly isolated or LPS-activatedsplenic B cells (Salmon et al., 1997; Craxton et al., 1998;Zhang et al., 1999) also results in rapid activation ofp38 MAPK, and this event has been implicated in theregulation of cytokine production and apoptosis.

No evidence on the role of p38 MAPK activation inthe regulation of NK cell functions has been providedso far. Moreover, although several studies have de-scribed 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 alsoinitiate p38 MAPK cascade. The results of this studydemonstrate that ligation of b1 integrin FN receptors onhuman NK cells stimulates the phosphorylation and theenzymatic activity of p38 MAPK to levels comparableto those induced by CD16 engagement.

Control of p38 MAPK activation has been shown toinvolve Rac and Cdc42, members of the Rho-relatedsmall GTPase family that are implicated in a wide spec-trum of cellular processes, namely cytoskeletal or-ganization, membrane trafficking, transcription factor

Figure 4. Activation of Pak1 and MKK3, but Not MKK6, by b1 Inte- regulation, cell cycle progression, and cellular transfor-grin Cross-Linking on Human NK Cells

mation (reviewed in Van Aelst and D’Souza-Schorey,(A) Human NK cells were left untreated (-) or stimulated with anti- 1997; Mackay and Hall, 1998). The role of Rac1 in regu-b1 (4B4) or anti-CD56 (C218) mAb for the indicated times at 378C.

lating transcription factor activity through its ability toOn anti-Pak1 immunoprecipitates, an in vitro kinase assay was per-activate MAPK cascades has been more extensivelyformed using MBP as substrate. As loading controls, the amountsstudied on the Jnk pathway (Coso et al., 1995; Mindenof Pak1 immunoprecipitated are shown on the bottom. These results

are representative of one out of three independent experiments. et al., 1995), whereas relatively little is known about Rac-(B and C) Human NK cells were left untreated (-) or stimulated with mediated regulation of the p38 MAPK pathway. Rac1anti-b1 (4B4) or anti-CD56 (C218) mAb for the indicated times or has been found to be coupled to and regulate the activitywith anisomycin ([AN];10 mg/ml, 45 min) or sorbitol ([Srb]; 0.5 M, 30 of p38 MAPK in response to inflammatory cytokinesmin) at 378C, and activation of MKK3 or MKK6 was examined by

such as IL-1 (Bagrodia et al., 1995). Moreover, recentWestern blot analysis performed on total cell lysates. As loadingevidence shows impaired p38 MAPK phosphorylationcontrols, the amounts of MKK3 or MKK6 are shown on the bottom.induced by fLMP stimulation in Rac-2-deficient neutro-These results are representative of one out of three independent

experiments. phils (Roberts et al., 1999). Consistent with these stud-ies, by the use of recombinant vaccinia virus encodingthe dominant-negative N17-Rac1 mutant, we show that

Vav was greatly diminished in cells exposed to AS-ODN Rac1 controls b1 integrin–mediated activation of p38when compared to cells exposed to S-ODN or to un- MAPK. Moreover, we provide direct demonstration oftreated control. AS-ODN but not S-ODN treatment mark- the ability of integrins to induce GDP/GTP exchangeedly reduced b1 integrin–induced p38 MAPK activation activity on endogenous Rac1, although several studiesas well as IL-8 production, indicating that Vav activity using transient or inducible expression of either acti-is functionally involved in these events (Figures 6B and 6C). vated or dominant-negative Rac1 mutants have sug-

gested a role for Rac in the formation of membraneDiscussion ruffles and lamellipodia, as well as in the integrin-medi-

ated cell spreading on FN (D’Souza-Schorey et al., 1998;Here, we demonstrate that b1 integrin ligation on human Price et al., 1998).peripheral blood NK cells results in the activation of the We have also shown the potential upstream signalingRac/p38 MAPK signaling pathway that is required for events leading to b1 integrin–mediated Rac1/p38 MAPKintegrin-triggered IL-8 production. In addition, we identi- activation. Rac1 activation paralleled the tyrosine phos-fied some of the upstream events accompanying the phorylation of GEF for Rac1, Vav (Crespo et al., 1997).

Like dominant-negative N17-Rac1 infected human NKb1 integrin–mediated p38 MAPK activation, namely the

Immunity12

Figure 5. Cross-Linking of b1 Integrins onHuman NK Cells Activates Rac1, which Con-trols p38 MAP Kinase Activation

(A) TLC of the nucleotides eluted from Rac1immunoprecipitates of (32P)orthophosphate-labeled NK cells. Cells were stimulated for 5min with appropriate doses of anti-b1 (4B4)or anti-CD56 (C218) mAb at 378C before lysis.The position at which GTP and GTP stan-dards run is indicated. Numbers indicate themolar ratio of GTP over total nucleotides fromquantitation by direct scanning for b radiationof the same experiment. These results arerepresentative of one out of three indepen-dent experiments.(B) Human NK cells were infected with recom-binant vaccinia virus encoding wild-type (WT)Rac1, dominant-negative N17-Rac1, or wild-

type vaccinia virus alone (WR). Infected human NK cells were left untreated (-) or stimulated with anti-b1 (4B4) mAb for 59 at 378C, and p38activation was examined by Western blot analysis performed on total cell lysates. As loading controls, the amounts of p38 protein are shownon the center panel. As overexpression control, the amounts of Rac1 protein are shown on the bottom. These results are representative ofone out of three independent experiments.

cells, no b1 integrin–induced p38 MAPK activation was NK cells following interaction with sensitive targets (Ga-landrini et al., 1999). On the other hand, a role for theobserved in Vav AS-ODN treated cells, further support-

ing a role for Vav in Rac1 activation. Vav tyrosine phos- Fak family kinase Pyk-2 may also be envisaged, as wehave recently demonstrated the ability of b1 integrinsphorylation was previously observed upon aIIbb3 cell

adhesion on fibrinogen or Ab-mediated b1 and b2 inte- to stimulate tyrosine phosphorylation of Pyk-2 in humanNK cells (Gismondi et al., 1997), and Pyk-2 activationgrin ligation in myeloid cells (Cichowski et al., 1996;

Gotoh et al., 1997). At present, no knowledge of the has been reported as a receptor proximal event control-ling Rac and p38 MAPK activation in response to stressprotein tyrosine kinases (PTKs) responsible for b1 inte-

grin–induced Vav phosphorylation is available. Much ev- stimuli (Tokiwa et al., 1996; Pandey et al., 1999).Our data also indicate that activation of p38 MAPKidence indicates that PTKs belonging to the Src and the

Syk/Zap-70 families may be implicated in this event. through ligation of b1 integrins on human NK cells isassociated with a significant and persistent activationaIIbb3 stimulates Vav1 tyrosine phosphorylation in a

Syk-dependent manner (Miranti et al., 1998), and associ- of Pak1 and MKK3, strongly suggesting that they arepotential constituents of the p38 MAPK signalingation between tyrosine-phosphorylated Syk and tyro-

sine-phosphorylated Vav has been reported in human pathway.

Figure 6. Ligation of b1 Integrins on NK CellsInduces Tyrosine Phoshorylation Vav, whichControls p38 MAPK Activation and IL-8 Pro-duction

(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-VavmAb. Resulting protein complexes were re-solved by 7% SDS-PAGE and immunoblottedwith anti-pTyr. As loading controls, theamounts of Vav immunoprecipitated areshown on the bottom. These results are rep-resentative of one out of three independentexperiments.(B) Human NK cells were exposed to Vav-ASor -S ODNs for 56 hr and then left untreatedor stimulated with anti-b1 (4B4) mAb. Vav ex-pression and p38 activation were examinedby Western blot analysis performed on totalcell lysates. As loading controls, the amountsof Syk and p38 MAPK proteins are shown.(C) Human NK cells were incubated with VavODNs and stimulated as above for 24 hr at378C in a 5% CO2 atmosphere in duplicatewells of flat-bottom plates (10 3 106 cells/well) in medium containing 1% BSA. After in-cubation, IL-8 concentration was quantitatedby ELISA in the cell-free supernatants.

Integrins Control IL-8 via RAC1/P38 in NK13

strongly suggest that MKK3 is responsible for the inte-grin-triggered p38 activation. The MAPK kinase kinases(MKKKs) involved in the activation of MKK3 throughintegrins are presently unknown, and further studies arerequired to define their function and specificity.

Finally, our results demonstrate that the ligation of b1integrins on human NK cells results in the stimulationof IL-8 production that is under the control of p38 MAPKactivation, as shown by the use of the specific syntheticinhibitor SB203580 and p38 AS-ODN. Moreover, consis-tent with the finding that Vav controls p38 MAPK activa-tion, we demonstrated a role for Vav in b1 integrin–induced IL-8 production using Vav AS-ODN. As in ourstudy, activation of p38 MAPK was found indispensablefor the neutrophil IL-8 production stimulated by granulo-cyte-macrophage colony stimulating factor (GM-CSF),LPS, as well as TNFa, but not for that induced by phorbolmyristate acetate (PMA) or ionomycin (Zu et al., 1998).

The mechanisms involved in the p38 MAPK-mediatedIL-8 production by integrins are unknown. It is conceiv-able, however, that they may be related to the ability ofp38 MAPK to regulate the activity of several transcrip-tion factors involved in the control of the IL-8 gene pro-moter including Elk-1, NF-kB, ATF-2, and CREB (Priceet al., 1996; Su and Karin, 1996; Tan et al., 1996; Craxtonet al, 1998).

In sum, the results of this study suggest a model thatFigure 7. The RAC1/P38 MAPK Signaling Pathway Controls b1 Inte-

depicts a Rac1-dependent pathway generated by thegrin–Induced IL-8 Production in Human NK Cellsligation of b1 integrins on human NK cells leading to

Our model depicts a Rac1-dependent pathway generated by thep38 MAPK activation that controls the production of theligation of b1 integrins on human NK cells leading to p38 MAPKproinflammatory chemokine IL-8 (Figure 7). The identityactivation that controls the production of the proinflammatory che-

mokine IL-8. b1 integrin ligation induces tyrosine phosphorylation of PTKs involved in Vav tyrosine phosphorylation andof the protooncogene p95 Vav, which may catalyze Rac1 activation. of the MKKK(s) that cause MKK3 activation is underRac1 activation is associated with b1 integrin–induced activation investigation.of Pak1 and MKK3 and controls p38 MAPK activation leading to What is the pathophysiological relevance of IL-8 pro-the production of IL-8.

duced 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 IL-Pak 1 belongs to a family of closely related serine/18 (Puren et al., 1998) and upon stimulation throughthreonine kinases that mediate several of the down-CD16, and IL-8 secreted by activated NK cells has beenstream effects of Rac and Cdc42, including activationshown to support a4b1 and a5b1-mediated migrationof Jnk and p38 MAPKs, and reorganization of the actinof T cells (Somersalo et al., 1994). Moreover, decidualcytoskeleton (Bagrodia et al., 1995; Zhang et al., 1995;NK cells as well as NK cells generated from humanSells and Chernoff, 1997). Rapid activation of Pak1 cata-duodenal mucosa express IL-8 mRNA and produce IL-8lytic activity is induced by a large number of extracellular(Saito et al., 1994).ligands including growth factors and inflammatory and

b1 integrins have been shown to control NK cell adhe-chemotactic cytokines and is initiated by the binding ofsion to endothelial cells and migration of NK cells intoGTP-Rac1 or GTP-Cdc42, which promote its autophos-the normal and neoplastic tissues (Allavena et al., 1991;phorylation (Sells and Chernoff, 1997). Recent evidenceSomersalo and Saksela, 1991; Fogler et al., 1996). More-indicates that cross-linking of CD3/TCR complex or Fasover, tissue uterine NK cells migrate over a dense hor-on T cells also results in rapid activation of Pak1, andmonally regulated ECM rich in FN and laminin.this event is involved in the regulation of gene expres-

Based on our data, it can be hypothesized that NKsion and apoptosis (Rudel et al., 1998).cells that enter early into the sites of inflammation, byThe MAPK kinases MKK3 and MKK6, which sharesecreting IL-8 upon interaction with activated endothe-80% amino acid identity, are considered upstream acti-lium or ECM components, may participate in the recruit-vators of the p38 MAPK signaling pathway. Recent datament of additional effector cells, such as neutrophilsindicate that the dominant-negative mutant of MKK3 butand T lymphocytes into the target tissue, thus affectingnot of MKK6 inhibits p38 activity in response to hyperos-the progression and evolution of the inflammatory re-molarity or methylmetane sulfonate (MMS) (Pandey etsponse. In the decidua, where NK cells are the predomi-al., 1999), and TNF-induced cytokine and IL-12 is im-nant population during the early stages of gestation, NKpaired in MKK3-deficient mice production (Lu et al, 1999;cell–derived IL-8 might contribute to regulation of theWysk et al., 1999). In accordance with these reports,selective leukocyte access at the maternal/fetal in-our evidence that b1 integrin stimulation on NK cells

causes significant activation of MKK3, but not MKK6, terface.

Immunity14

Experimental Procedures In Vitro Kinase AssayTo analyze Pak1 activation, cells were extracted with Triton lysisbuffer as above. Endogenous Pak1 was immunoprecipitated withAntibodies and Reagents

Anti-CD16 (B73.1) mAb was kindly provided by Dr. G. Trinchieri anti-Pak1 antibodies and subjected to in vitro kinase assay. Thekinase reaction was initiated by adding to the immunoprecipitate(Wistar Institute, Philadelphia, PA). Anti-CD56 (C218) mAb was gen-

erously provided by Dr. A. Moretta (University of Genoa, Genoa, 25 ml of kinase buffer (25 mM Tris [pH 7.5], 12.5 mM b-glycerophos-phate, 7.5 mM MgCl2, 20 mM cold ATP, and 0.5 mM sodium orthovan-Italy). Anti-b1 (4B4) was purchased from Coulter Immunology. Anti-

b1 (TS2/16) F(ab9)2 fragments were prepared by proteolytic digestion adate) containing 5 mCi of g[32P]-ATP (4,500 Ci/mmol, ICN) and 2.5mg of myelin basic protein (MBP) (Sigma). After 30 min of incubationas described previously (Gismondi et al., 1995). The anti-phospho-

tyrosine (anti-pTyr) 4G10 and the anti-Vav mAb were purchased at 308C, the samples were boiled in sample buffer and separatedby SDS-PAGE. The gels were dried, and the 32P-labeled proteinsfrom UBI. The affinity-purified rabbit antisera against Rac1, Pak1,

MKK3, Erk, Jnk, and p38 MAPKs, the goat antiserum against MKK6, were made visible by autoradiography.To examine p38 MAPK activity, cells were extracted for 30 minand the anti-Syk mAb were obtained from Santa Cruz Biotechnol-

ogy. The affinity-purified rabbit antiserum against mouse Ig was on ice with modified Triton lysis buffer (25 mM Hepes [pH 7.5], 300mM NaCl, 0.1% Triton X-100, 0.2 mM EDTA, 20 mM b-glycerophos-purchased from Zymed Laboratories. Affinity-purified (Fab9)2 frag-

ments of goat anti-mouse Ig (GAM) were purchased from ICN-Cap- phate, 1.5 mM MgCl2, and 0.5 mM DTT) containing phosphataseand protease inhibitors. Endogenous p38 MAPK was precipitatedpel. GST-ATF-2 fusion protein was a kind gift of Dr. C. J. Der (Univer-

sity of North Carolina at Chapel Hill). Human plasma FN was with anti-p38 antibodies and subjected to in vitro kinase assay. Afterwashing, the beads were incubated with 25 ml of kinase buffer aspurchased from GIBCO. The p38 MAPK inhibitor SB203580 was

purchased from Alexis Corporation and the MEK-1 inhibitor PD above containing 1 mg of GST-ATF-2. After 30 min of incubation at308C, the samples were boiled in sample buffer and separated by098059 from Calbiochem-Novabiochem. Anisomycin and sorbitol

were purchased from Sigma. SDS-PAGE. The gels were dried, and the 32P-labeled proteins weremade visible by autoradiography.

Human NK Cell Preparation and StimulationHighly purified (97%) cultured NK cells were obtained by incubating RT-PCR

Total RNA was extracted using RNAfast (Molecular System). For thefor 8 days nylon nonadherent peripheral blood mononuclear cells(PBMC) (4 3 105 cells) with irradiated (3000 rads) RPMI 8866 cells 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,(1 3 105) as previously described (Mainiero et al., 1994).Human NK cells were stimulated as previously described (Mai- 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 volumeniero et al., 1998). When indicated, cells were pretreated (30 min at378C) with the p38 MAPK inhibitor SB203580 or MEK-1 inhibitor PD 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 each098059 before stimulation. In some experiments, before stimulation,NK cells were infected with recombinant vaccinia virus encoding 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,wild-type (WT) Rac1, dominant-negative N17-Rac1, or wild-typevaccinia virus alone (WR), kindly provided by Dr. Leibson (Mayo 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), synthesisClinic and Fundation, Rochester, Minnesota) (Billadeau et al., 1998).In brief, semipurified recombinant vaccinia virus was used to infect for 1 min at 728C, and a final elongation at 728C for 5 min. Primer

sequences used for IL-8 were sense, (59-TCTCAGCCCTCTThuman NK cells for 1 hr in serum-free medium at a multiplicity ofinfection of 20:1. The remainder of the infection (4 hr) was carried CAAAAACTTCT-39) and antisense, (59-ATGACTTCCAAGCTGGCC

GTGCT-39). Primer sequences used for b-actin were sense, (59-GGGout in RPMI 1640 containing 10% FCS. In other experiments, beforestimulation, NK cells were treated with antisense (AS) and com- TCAGAAGGATTCCTATG-39) and antisense, (59-GGTCTCAAACAT

GATCTGGG-39). PCR products were electrophoresed on a 1.5%plementary sense (S) oligodeoxynucleotides (ODNs) for Vav (Galan-drini et al., 1999) or p38 (Nagata et al., 1998). The sequences were agarose gel in Tris-borate-EDTA buffer, and the gels were then

stained with ethidium bromide and photographed.as follows: AS-Vav, (59-CATTGGCGCCACAGCTCCAT-39); S-Vav,(59-ATGGAGCTGTGGCGCCAATG-39); AS-p38, (59-GGCCTCTCCTGCGACATCTT-39); and S-p38, (59-AAGATGTCGCAGGAGAGGCC-39). IL-8 and IFNg Production AssayHuman NK cells (2.5 3 105/ml) were exposed to ODNs (100 mg/ml) Untreated or treated NK cells were seeded in duplicate wells of flat-in heat-inactivated (658C for 30’) culture medium for 56 or 72 hr. bottom 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.Immunoprecipitation and Immunoblot Analysis

To estimate Rac1 activation, human NK cells were starved for 3 hr IL-8 concentration was quantitated with an ELISA kit (BioSource).IFNg concentration was quantitated with an ELISA kit (EuroClone,in phosphate-free RPMI and labeled for 3 hr with [32P]-orthophos-

phate (0.5 mCi/ml) (4,500 Ci/mmol, ICN, Biomedicals) in phosphate- Torquay, UK).free RPMI supplemented with 0.1% phosphate-free fetal calf serum.After stimulation, the cells were extracted and the immunoprecipi- Acknowledgmentstated samples subjected to Rac-GTP loading assay as previouslydescribed for Ras (Mainiero et al., 1997). We thank Dina Milana, Anna Maria Bressan, Alessandro Procaccini,

To immunoprecipitate Vav, stimulated and unstimulated human Antonio Sabatucci, and Patrizia Birarelli for expert technical assis-NK cells were extracted in Triton lysis buffer (50 mM Hepes [pH tance and Sandro Valia for photographic assistance. We also thank7.5], 150 mM NaCl, and 1% Triton X-100) containing 1 mM sodium Paul J. Leibson, Alessandro Moretta, and Giorgio Trinchieri for re-orthovanadate, 2.5 mM sodium pyrophosphate, 25 mM sodium fluo- agents. This work was partially supported by grants from the Italianride, 0.01% aprotinin, 4 mg/ml pepstatin A, 10 mg/ml leupeptin, and Association for Cancer Research (AIRC), Istituto Superiore di Sanita1 mM phenylmethanesulfonyl fluoride (PMSF) (all from Sigma) for 30 Italy-USA “Therapy of Tumors” Program, Ministero dell’Universitamin on ice. Immunoprecipitation, SDS-PAGE, and immunoblotting e della Ricerca Scientifica e Tecnologica (MURST), Ministero dellaanalysis were performed as previously described (Mainiero et al., Sanita, and CNR special project on Biotechnologies.1995). Nitrocellulose-bound antibodies were detected by chemilu-minescence with ECL (Amersham Life Sciences, Little Chalfont, UK).

Received July 8, 1999; revised December 1, 1999.Phoshorylated forms of Erk, Jnk, and p38 MAPKs, MKK3, andMKK6 were detected by Western blot analysis by using Phospho-Plus p44/42 Erk, SAPK/Jnk, and p38 MAPK and MKK3/MKK6 Ab ReferencesKit (New England Biolabs, Hitchin, UK) following protocols providedby the manufacturer. The identity of MKK3 and MKK6 was estab- Allavena, P., Paganin, I., Martin-Padura, I., Peri, G., Gaboli, M., De-

jana, E., Marchisio, P.C., and Mantovani, A. (1991). Molecules andlished by Western blot analysis using specific antibodies.

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