By
From the * Department of Experimental Medicine and Pathology, Istituto Pasteur-Fondazione Cenci
Bolognetti, University of Rome "La Sapienza," 00161 Rome, Italy; the Biotechnology Section,
Istituto Nazionale per lo Studio e la Cura del Tumori, 16100 Genoa, Italy; the § Mediterranean
Institute of Neurosciences "Neuromed," 86170 Pozzilli, Italy; and the
Laboratory of
Pathophysiology, Regina Elena Cancer Institute, 00100 Rome, Italy
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Abstract |
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Recent evidence indicates that integrin engagement results in the activation of biochemical
signaling events important for regulating different cell functions, such as migration, adhesion, proliferation, differentiation, apoptosis, and specific gene expression. Here, we report that 1
integrin ligation on human natural killer (NK) cells results in the activation of Ras/mitogen-activated protein kinase pathways. Formation of Shc-growth factor receptor-bound protein 2 (Grb2) and Shc-proline-rich tyrosine kinase 2-Grb2 complexes are the receptor-proximal
events accompanying the
1 integrin-mediated Ras activation. In addition, we demonstrate
that ligation of
1 integrins results in the stimulation of interferon
(IFN-
) production, which is under the control of extracellular signal-regulated kinase 2 activation. Overall, our
data indicate that
1 integrins, by delivering signals capable of triggering IFN-
production,
may function as NK-activating receptors.
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Introduction |
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Natural killer (NK) cells, a small population of circulating and tissue-resident lymphocytes, 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 such as IFN-, TNF-
, and GM-CSF (1, 2).
It is becoming increasingly clear that the final outcome
of NK cell activity results from a balance between triggering and inhibitory receptors and ligands. In the last few
years, much attention has been given to the characterization of inhibitory receptors recognizing MHC class I molecules, named killer cell-inhibitory receptors (KIRs), while
knowledge about receptors delivering positive signals is less
advanced. Activating molecules include receptors belonging to the integrin (LFA-1, 4
1), Ig (CD16, CD2,
DNAX accessory molecule 1 [DNAM-1]), and C-type lectin (NKRP-1, CD69) families (3). Recently, noninhibitory isoforms of MHC class I receptors have also been described (7), as well as the mechanisms by which they
transmit stimulatory signals (10).
Integrins are a large family of homologous cell surface receptors that mediate cell-matrix and cell-cell interactions (11, 12) and that are capable of transducing intracellular signals (13). The integrin-mediated signaling events include stimulation of phosphoinositide metabolism, elevation of intracellular pH and Ca2+ transients, activation of a number of tyrosine kinases and protein kinase C isoforms, and regulation of the small GTP-binding proteins belonging to the Ras and Rho families. In addition, coupling of integrin receptors to mitogen-activated protein kinase (MAPK)1 pathways has been reported (17), and a role for p21 Ras in the upstream events leading to extracellular signal-regulated kinase (Erk) activation has recently been demonstrated (18, 19). Integrin-induced Ras activation has been shown to involve the Shc-mediated recruitment of the Grb2-mSos complex to the plasma membrane (18, 19). Moreover, a role for p125 focal adhesion kinase (Fak) in the integrin-mediated activation of the Ras-MAPK cascade has been suggested, as formation of the Fak-Grb2-mSos complex parallels activation of Erk kinases (20).
Several findings indicate that integrins can affect expression of several genes, including those encoding cytokines.
Adhesion of monocytes to extracellular matrix components
results in activation of IL-1, TNF-
, IL-8, Gro-
, Gro-
,
and Gro-
(23). In addition,
v
3 interaction with
fibronectin (FN) and vitronectin enhances IL-4 and IL-2
production by murine
/
T cells and by a Th cell hybridoma, respectively (28, 29). In human T cells, LFA-1,
4
1,
and
5
1 provide a costimulus for the release of multiple cytokines such as IL-2, IL-4, TNF-
, GM-CSF, and IFN-
(30) and similarly, LFA-1 costimulates CD16-triggered
TNF-
production in human NK cells (35). Nonetheless,
information on the intracellular signaling events leading to
the control of gene expression by integrins has been provided so far only in epithelial and mesenchymal cells.
We have previously shown that peripheral blood human
NK cells express 4
1 and
5
1 as fibronectin receptors
(36) and
6
1 as a laminin receptor (37), and that the expression and function of
1 integrins are modulated upon
NK cell activation (37, 38). More recently, we have demonstrated that clustering of
1 integrins on human NK cells
transduces intracellular signals leading to activation of the
Fak-related nonreceptor tyrosine kinase Pyk-2, tyrosine
phosphorylation of paxillin (39), elevation of intracellular calcium, and costimulation of NK cytotoxic functions (40).
In this study, we analyzed whether
1 integrin ligation on
human NK cells results in the stimulation of the Ras/
MAPK signaling pathways and investigated the role of
these events in the production of IFN-
.
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Materials and Methods |
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Antibodies and Reagents.
The following mouse mAbs were used: anti-CD3 (Leu4), anti-CD16 (Leu11c), and anti-CD56 (Leu19) were purchased from Becton Dickinson (San Jose, CA). Anti-CD16 (B73.1) mAb was provided by Dr. G. Trinchieri (The Wistar Institute, Philadelphia, PA). Anti-CD56 (C218) mAb was provided by Dr. A. Moretta (University of Genoa, Genoa, Italy). Anti-Human NK Cell Preparation.
Cultured NK cells were obtained by incubating for 10 d nylon nonadherent PBMC (4 × 105 cells) with irradiated (3,000 rads) RPMI 8866 cells (105) as described previously (38). On day 10, contaminating T cells were eliminated by negative panning with anti-CD5 mAb, and the resulting NK cell population was ~95% CD16+CD56+CD3Cell Stimulation.
Human NK cells were resuspended in RPMI 1640 serum-free medium (50 × 106 cells/300 µl/tube) and incubated with saturating doses of the appropriate mAb for 30 min at 4°C. After washing off the unbound antibody, cells were resuspended in prewarmed RPMI 1640 medium and incubated for different time periods with polystyrene beads (2.5-µm diameter; Interfacial Dynamics Corporation, Portland, OR) coated with GAM (1.5 µg/106 cells) at 37°C (41). In some experiments, human NK cells were resuspended in RPMI 1640 serum-free medium (50 × 106/300 µl/tube) and incubated for different time periods with polystyrene beads coated with human plasma FN or its proteolytic fragments of 120 and 40 kD or with BSA. When indicated, cells were pretreated (30 min at 37°C) with MEK-1 inhibitor PD 098059 (Calbiochem-Novabiochem, La Jolla, CA).Immunoprecipitation and Immunoblot Analysis.
To estimate Ras activation, human NK cells were starved for 3 h in phosphate-free DMEM and labeled for 3 h with [32P]orthophosphate (0.5 mCi/ml, 4,500 Ci/mmol; ICN Biomedicals, Inc., Irvine, CA) in phosphate-free DMEM supplemented with 0.1% phosphate-free FCS. After stimulation, the cells were extracted and the immunoprecipitated samples subjected to Ras-GTP loading assay as described previously (19). Nucleotides bound to Ras were analyzed by TLC on polyethyleneimine-cellulose plates in 0.75 M K2HPO4, pH 3.5. Radioactivity in GDP and GTP was estimated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA). To immunoprecipitate Shc and Pyk-2, stimulated and unstimulated human NK cells were extracted in Triton lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 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 PMSF (all from Sigma Chemical Co., St. Louis, MO) for 30 min on ice. Immunoprecipitation, SDS-PAGE, and immunoblotting analysis were performed as described previously (41). Nitrocellulose-bound antibodies were detected by enhanced chemiluminescence (ECL; Nycomed Amersham plc, Little Chalfont, Bucks, UK).In Vitro Kinase Assay.
To examine Erk activity, cells were extracted with NP-40 lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) containing phosphatase and protease inhibitors for 30 min on ice. Endogenous Erk2 was immunoprecipitated with anti-Erk2 antibodies and subjected to in vitro kinase assay. The kinase reaction was initiated by adding to the immunoprecipitate 25 µl of kinase buffer (25 mM Tris, pH 7.5, 12.5 mMNorthern Blot Analysis.
Total RNA was extracted using RNAfast (Molecular Systems, San Diego, CA), size-fractionated in 1% agarose/formaldehyde gels, transferred by capillarity onto nitrocellulose filters, baked at 80°C for 2 h, and hybridized to cDNA probes specific for human IFN-IFN- Production Assay.
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Results |
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GTP-loading experiments were performed
to examine if ligation of 1 integrins on human NK cells
results in activation of Ras. After in vivo labeling with
[32P]orthophosphate, human NK cells were incubated with
saturating concentrations of anti-
1 or anti-CD56 control
mAb and stimulated for the indicated times at 37°C with
polystyrene beads coated with GAM F(ab')2 fragments. As
shown in Fig. 1, chromatographic analysis of nucleotides
bound to Ras indicated that
1 integrin stimulation results
in a threefold increase in the proportion of GTP-bound
Ras (from 8 to 21%). Ras activation was rapid, peaking at
5 min and returning to basal levels after 40 min. Anti-CD56 mAb-treated control samples showed a p21 Ras GTP to
GDP plus GTP ratio comparable to that of the untreated
sample (not shown).
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These results indicate that cross-linking of 1 integrins
on human NK cells causes activation of Ras.
Integrin-induced Ras activation involves tyrosine phosphorylation
of the adaptor protein Shc and Shc-mediated recruitment of the adaptor protein Grb2 (18, 19). Since Grb2 is stably associated with the Ras-GTP exchange factor mSos, the association of Grb2 with Shc is likely to bring mSos in close
proximity to its target Ras (42). To investigate whether ligation of 1 integrins on NK cells results in tyrosine phosphorylation of Shc, human NK cells were stimulated with
anti-
1 or anti-CD56 mAb as above. Anti-Shc immunoprecipitates were examined by immunoblotting with anti-pTyr mAb. Marked tyrosine phosphorylation of both the
46- and 52-kD isoforms of Shc was detected after
1 integrin cross-linking (Fig. 2).
1 integrin-mediated Shc phosphorylation was rapid and persistent, in that it peaked at 5 min and declined at 40 min. No changes in the phosphorylation status of Shc were observed in untreated or anti-CD56 mAb-treated NK cells used as control. Interestingly,
an additional tyrosine-phosphorylated protein of ~145 kD
was consistently coimmunoprecipitated with Shc from
1 integrin-stimulated NK cell lysates; tyrosine phosphorylation of
Shc and p145 occurred with the same kinetics (Fig. 2). The nature of this protein has not been investigated, but it is likely to correspond to one of the isoforms of the lipid phosphatase Ship, which combines with the Shc phosphotyrosine-binding (PTB) domain upon tyrosine phosphorylation (43). To
examine the possibility that tyrosine-phosphorylated Shc
associates with the SH2 domain-containing adaptor protein
Grb2, Shc immunoprecipitates were immunoblotted with an
anti-Grb2 antibody. The results indicate that Grb2 forms a
complex with Shc in
1 integrin-stimulated NK cells. No
Shc-Grb2 association was observed in NK cells untreated or
treated with anti-CD56 mAb used as control (Fig. 2).
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Similar data were obtained upon cross-linking of 4
1 and
5
1 FN receptors with polystyrene beads coated with FN
or its 120- and 40-kD proteolytic fragments recognized by
5
1 and
4
1, respectively (Fig. 3). These results indicate
that cross-linking of
1 integrin FN receptors on human NK
cells induces Shc tyrosine phosphorylation and its association
with Grb2. The time course of Shc phosphorylation and
Grb2 association strictly parallels the
1 integrin-mediated p21 Ras activation, suggesting that the formation of the
Shc-Grb2 complex is important for Ras activation.
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The ability of the Fak
family members to combine with Grb2 upon integrin
engagement has been shown, and a role for p125Fak in the
integrin-mediated activation of the Ras-MAPK cascade has been suggested (20, 21). We reported recently that human
peripheral blood NK cells express Pyk-2 and not p125Fak,
and that ligation of 1 FN receptors results in Pyk-2 activation (39). To investigate whether Pyk-2 can associate
with Shc and Grb2 upon
1 integrin cross-linking, human
NK cells were stimulated with anti-
1 or anti-CD56 mAb
as above. Pyk-2 immunoprecipitates were then analyzed for the presence of Shc and Grb2 by immunoblotting with
specific mAbs. As shown in Fig. 4, the 46-kD isoform of
Shc and the adaptor protein Grb2 were detected in the
Pyk-2 immunoprecipitates after
1 integrin cross-linking.
Shc and Grb2 association to Pyk-2 was observed at 5 min
and was still evident at 15 min; formation of this complex
parallels Pyk-2 tyrosine phosphorylation. No association of
Shc and Grb2 with Pyk-2 was observed in untreated or
control mAb-treated NK cells.
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These results suggest that tyrosine-phosphorylated Pyk-2
forms a complex with Shc and Grb2 upon stimulation of
NK cells through 1 integrins, suggesting that Pyk-2 may
cooperate with the Shc-Grb2 complex to fully activate Ras
in response to integrin ligation.
We next examined if ligation of 1 integrins on human NK cells results in activation of Erk and Jnk. Human NK cells were stimulated with
anti-
1 or anti-CD56 mAb and subjected to Erk2 and Jnk
kinase assay. As shown in Fig. 5,
1 integrin stimulation
causes a significant activation of Erk2 (A) and Jnk (B). This
activation is already evident at 1 min and still persistent at
20 min. In contrast, anti-CD56 control mAb treatment did
not result in any significant activation of Erk2 or Jnk.
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These data indicate that ligation of 1 integrins on human NK cells causes a significant and persistent activation
of Erk and Jnk.
We first examined if ligation of 1 integrins on human NK cells can
affect IFN-
mRNA expression. Northern blot analysis
was performed by using total RNA from human NK cells
stimulated with anti-
1 or anti-CD56 mAb. Fig. 6 shows
that
1 integrin ligation induces IFN-
mRNA expression,
which was maximal at 30 min and declined at 3 h after
stimulation. Treatment of human NK cells with anti-CD56
mAb used as control did not influence IFN-
mRNA expression (Fig. 6 A).
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To investigate whether 1 integrin-induced IFN-
mRNA expression is under the control of Erk activation,
we used the synthetic inhibitor PD 098059, which specifically prevents activation of the Erk-activating kinase MEK-1
(44, 45). PD 098059, at the 50 µM concentration that
completely inhibits
1 integrin-induced Erk2 activation
(not shown), abrogated the stimulation of IFN-
mRNA
expression. The same concentration of PD 098059 did not
affect IFN-
mRNA levels in human NK cells treated with
anti-CD56 control mAb (Fig. 6 B).
Finally, we evaluated if ligation of 1 integrins on human NK cells stimulates IFN-
production by testing the
presence of this cytokine in the supernatants of NK cells,
either untreated or treated for 24 h at 37°C with anti-
1 or
anti-CD56 control mAb. As shown in Fig. 7,
1 integrin
cross-linking resulted in induction of IFN-
production at
levels comparable to those induced by CD16 engagement
(not shown). In contrast, treatment of human NK cells
with anti-CD56 mAb used as control did not stimulate IFN-
production.
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The induction of IFN- synthesis required the activity
of Erk2 as shown by the ability of PD 098059 to completely inhibit
1 integrin-induced cytokine production
(Fig. 7).
These findings indicate that ligation of 1 integrins on
human NK cells stimulates IFN-
production, and that this
event requires Erk2 activation.
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Discussion |
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The ability of integrins to cause activation of Ras has
been previously described in several adherent primary cells
and cell lines (18, 19). In regard to the immune system,
there is only one report showing that 2
1 triggers Ras activation in Jurkat T leukemia cells (46). Here, we demonstrate that
1 integrin ligation on human peripheral blood
NK cells results in activation of the Ras/MAPK pathway,
which is involved in integrin-triggered IFN-
production.
In addition, we provide information on the receptor-proximal events accompanying the
1 integrin-mediated Ras
activation, namely tyrosine phosphorylation of Shc and its
association with Grb2, and the association of tyrosine-phosphorylated Pyk-2 with Grb2 and Shc.
Tyrosine phosphorylation of both the 46- and 52-kD
isoforms of Shc was observed after mAb-mediated cross-linking of 1 integrins, as well as upon interaction of NK
cells with FN and its proteolytic 120- and 40-kD fragments, which are recognized by
5
1 and
4
1, respectively. The time course of
1 integrin-triggered Shc phosphorylation strictly paralleled Ras activation, suggesting
that the formation of the Shc-Grb2 complex is important
in this event.
These results are in accordance with previous studies indicating the involvement of the Shc-Grb2 complex in Ras
activation upon engagement of v
3,
4, or several
1 integrins on various adherent cell types (18, 19, 41).
Integrin-mediated activation of Ras has also been suggested to involve the nonreceptor tyrosine kinase p125Fak,
and the formation of the Fak-Grb2-mSos complex upon
cell adhesion to FN has been described (20, 22). No data
are available yet on the involvement of the recently discovered member of the Fak family, Pyk-2, in the activation of
Ras through integrins, although the association of Pyk-2
with Grb2 has been observed in FN-activated COS cells (47). However, recent observations indicate that Pyk-2
regulates Ras in response to neurotransmitters, inflammatory cytokines, and cellular stresses by recruiting the Grb2-
mSos complex to the plasma membrane (48). We have recently demonstrated that human peripheral blood NK cells
express Pyk-2 but not p125Fak, and that tyrosine phosphorylation of Pyk-2 occurs rapidly after ligation of 1 integrins by natural ligands or specific mAbs (39). Here we
show that integrin ligation induces association of tyrosine-phosphorylated Pyk-2 with Grb2 and Shc. Interestingly,
only the 46-kD isoform of Shc was detected in Pyk-2 immunoprecipitates, suggesting that the p46 and p52 Shc may
have different functional properties. In line with this hypothesis, it has been reported that p66 Shc, which is mainly
expressed in epithelial cells, mediates opposite effects on
the epidermal growth factor (EGF) receptor/MAPK/Fos signaling pathways with respect to the other splicing isoforms (49).
Overall, our results suggest that the formation of the
Shc-Grb2 and Shc-Pyk-2-Grb2 complexes may couple 1
integrins to the Ras pathway in human NK cells. The relative contribution of Shc-Grb2 and Shc-Pyk-2-Grb2 complexes in the signaling events leading to integrin-mediated
activation of Ras is currently unknown. Upon integrin engagement, both tyrosine-phosphorylated Shc and Pyk-2 may bind to the SH2 domain of Grb2 (22, 48, 50);
whether Pyk-2 interacts directly with Shc has not yet been
defined. Knowledge of the molecular basis regulating the
association of Shc with Grb2-associated Pyk-2 is necessary
for a better understanding of whether Shc and Pyk-2 represent additive complementary pathways required to fully activate Ras in response to integrin ligation, or if one of these
pathways is dominant and the other redundant.
The results of this study also indicate that ligation of 1
integrins on human NK cells stimulates the enzymatic activities of both Erk2 and Jnk, the last kinases of MAPK cascades controlled by Ras activation.
Activation of Erk and Jnk in response to integrin stimulation has been reported previously (17, 51). However,
the majority of these studies were performed in adherent
cell types, including primary human keratinocytes, endothelial cells, and 3T3 fibroblasts. In the immune system, it
has been reported that 4
1 costimulates (pp45) MAPK
activation in resting T cells (52), but no evidence is available on the ability of integrins to activate Jnk.
Control of Erk activation has been shown to involve Shc
and Ras as indicated by the ability of their dominant negative forms to prevent Erk2 activation in response to cell adhesion to fibronectin or laminin (18, 19, 53). However,
Ras-independent signaling events have also been implicated in control of MAPK activation (54). Jnk activation
upon integrin ligation involves multiple pathways, since it
is prevented by either Ras and Rac dominant negative
forms or the phosphatidylinositol 3-kinase inhibitor, wortmannin (19). Overall, it is conceivable that Ras activation may be a crucial event controlling 1 integrin-triggered
MAPK cascades in NK cells.
Finally, we provide evidence that ligation of 1 integrins
on human NK cells results in the stimulation of IFN-
mRNA expression and production, which are under the
control of Erk2 activation as shown by the use of the specific MEK-1 inhibitor, PD 98059. Similarly, a role for
Erk2 activation in the control of IFN-
synthesis in T cells
triggered through the TCR complex has been recently
demonstrated (55). The involvement of Erk in the regulation of IFN-
production may be related to its ability to
activate c-Fos transcription and to regulate the formation of
the activating protein (AP)-1 heterodimer (56), which is
involved in the control of the IFN-
gene promoter (57,
58). In support of this hypothesis, several studies indicate
that integrins activate several transcriptional factors (25, 31,
59, 60); furthermore, recent studies in
4
1-stimulated
monocytes have demonstrated an association between Erk
activation and nuclear factor (NF)-
B translocation, which
leads to the expression of the tissue factor gene (61).
Our data indicate that 1 integrins, by delivering signals
capable of triggering IFN-
production, may function as
NK-activating receptors. In a previous study, we have
shown that cross-linking of
4
1 and
5
1 by specific
mAbs or by the natural ligands vascular cell adhesion molecule 1 (VCAM-1) and FN costimulates but does not trigger
natural cytotoxicity (40). Taken together, these results suggest that different receptor signaling thresholds and/or different signaling events are required to elicit distinct NK cell
functions: it is possible that
1 integrin-mediated signaling does not reach the threshold for induction of NK cytotoxicity, or that
1 integrins are unable to transmit the signal(s)
required for the activation of the NK lytic program. In this
regard, ligation of
1 integrins on NK cells does not stimulate phospholipase C
(PLC-
) activation and inositol-1,4,5-triphosphate (PtdIns(1,4,5)P3 generation [Milella, M.,
personal communication]), a signaling pathway triggered
by the NK receptors capable of activating the cytotoxic
function. Furthermore, in accordance with this evidence, certain target cell lines resistant to NK cell lysis and unable to stimulate detectable phosphoinositide turnover were found
to be capable of inducing IFN-
production, although the
basis of this phenomenon remains unclear (Perussia, B.,
personal communication).
What is the pathophysiological relevance of IFN- produced by NK cells in response to
1 integrin stimulation?
1 integrins have been shown to control NK cell adhesion
to endothelial cells and migration of NK cells into the normal and neoplastic tissues (62). Based on our data, it can
be hypothesized that IFN-
produced by NK cells upon
interaction with activated endothelium or extracellular matrix components may contribute to affect the local inflammatory reaction by regulating several functions of endothelial cells, including expression of ICAM-1 and VCAM-1
adhesion receptors, cytokine release, and nitric oxide production (65, 66). Finally, IFN-
production by NK cells in
response to
1 integrins may be relevant for the antiviral
activity exerted by the NK cells at the tissue level, in particular in the liver. It has been recently reported (67) that
NK cells control murine CMV infection in the liver by secreting IFN-
, which exerts its antiviral action through the
induction of nitric oxide synthesis; it has also been suggested that unlike in the spleen, NK cell-dependent resistance to murine CMV infection in this organ does not require NK cell contact with virus-modified target cells.
Thus, integrins may be indicated as one of the receptors
triggering IFN-
synthesis by liver NK cells which closely
interact with the endothelial cells in the hepatic sinusoids.
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Footnotes |
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Address correspondence to Fabrizio Mainiero, Department of Experimental Medicine and Pathology, University of Rome "La Sapienza," viale Regina Elena, 324, 00161 Rome, Italy. Phone: 39-6-4468448; Fax: 39-6-4468448; E-mail: asantoni{at}axcasp.caspur.it
Received for publication 28 May 1998.
This work was partially supported by grants from the Italian Association for Cancer Research (AIRC), Istituto Superiore di Sanità Italy-USA "Therapy of Tumors" Program, Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST) 40% and 60%, Ministero della Sanità, and by a Consiglio Nazionale delle Ricerche special project on Biotechnologies.We thank Dina Milana, Anna Maria Bressan, Alessandro Procaccini, Antonio Sabatucci, and Patrizia Birarelli for expert technical assistance, and Ilio Piras for photographic assistance. We also thank Dr. F.G. Giancotti for carefully reviewing the manuscript.
Abbreviations used in this paper Erk, extracellular signal-regulated kinase; FN, fibronectin; Fak, focal adhesion kinase; GAM, goat anti-mouse IgG F(ab')2; Grb2, growth factor-bound protein 2; GST, glutathione S-transferase; Jnk, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MEK, MAPK kinase; Pyk-2, proline-rich tyrosine kinase 2.
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