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Release in Human NK Cells Involves Distinct Pathways1
Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and matrix
metalloproteinases, and NK cell invasiveness. Although there has been
several molecules shown to interact with 2B4, the signaling mechanism
of 2B4-mediated activation of NK cells is still unknown. In this study,
we found cross-linking of 2B4 on YT cells, a human NK cell line,
results in the increased DNA binding activity of activator protein-1
(AP-1), an important regulator of nuclear gene expression in
leukocytes. We investigated the possible role of various signaling
molecules that may be involved in the activation of lytic function of
YT cells via 2B4. Treatment of YT cells with various specific
inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and
Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple
MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of
transcription and p38 inhibited 2B4-mediated IFN- release indicating
distinct pathways are involved in cytotoxicity and cytokine release. In
this study we also show that 2B4 constitutively associates with the
linker for activation of T cells (LAT) and that 2B4 may mediate NK cell
activation via a LAT-dependent signaling pathway. These results
indicate that 2B4-mediated activation of NK cells involves complex
interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic
function and cytokine production may be regulated by distinct
pathways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TGF
, IL-1, IL-10 and GM-CSF as well as matrix
metalloproteinases (MMPs) (6, 7, 8, 9). The mechanisms that
control NK cell activation and cytotoxicity are believed to be
determined by a delicate balance between stimulatory and inhibitory
signals received from surface receptors (10, 11, 12). NK cells
also mediate the rejection of MHC mismatched bone marrow stem cells
(13). Engagement of cytolytic activity can be inhibited by
MHC class I molecules on target cells interacting with MHC class I
receptors expressed on the surface of NK cells. NK cell inhibitory
receptors belong to the Ig and lectin gene superfamilies. One common
feature of the killer cell inhibitory receptors is the presence of
immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their
cytoplasmic domains (3, 10). The ITIM motifs become
phosphorylated upon receptor binding which results in the recruitment
of protein SH2 domain bearing tyrosine phosphatase
(SHP)3 1 and/or SHP-2.
These phosphatases can dephosphorylate several substrates within the
activating signaling cascade or a distal activating receptor and
inhibit NK cell activation (14, 15). Although there has been great progress in understanding the inhibitory mechanisms that regulate NK cell function, our knowledge of the activating signaling pathways is slowly emerging. Several surface molecules have been identified that can activate natural cytotoxicity. Activating receptors such as 2B4, CD2, CD16, KIR3DS1-5, NKp30, NKp44, and NKp46 are members of the Ig superfamily (16, 17, 18). These molecules bear high homology on their extracellular domains while lacking the immunoreceptor tyrosine-based inhibitory motifs on their cytoplasmic tails. There are also members of the lectin gene superfamily that can transduce an activating signal. Members of the lectin superfamily, such as NKR-P1A and NKR-P1C, NKG2 family, and the LY49 family can form homo- and heterodimer complexes that can be inhibitory or stimulatory. Although many of the activating surface molecules have been identified, information on the signaling cascade from the cell surface to within is fragmentary.
2B4 was originally identified on mouse NK cells and the subset of T cells that mediate non-MHC-restricted killing (18, 19, 20, 21). It is a novel member of the CD2 subfamily which includes signaling lymphocyte-activation molecule, CD48, CD58, CD84, CS1, and Ly9, and is expressed on all NK cells, a subset of CD8+ T cells, basophils, and monophils (22, 23, 24). The cytoplasmic domain of 2B4 contains novel tyrosine motifs (TxYxxV/I) that associate with signaling adaptor molecule, signaling lymphocyte-activation molecule-associated protein (SAP) whose defect forms the basis for X-linked lymphoproliferative syndrome (XLP) (25, 26, 27, 28). It is thought that NK cells in XLP patients are defective in their activation through 2B4 signaling (29, 30, 31). In addition to modulating cytolytic function, 2B4 activation of NK cells induces cytokine production as well as invasiveness (9, 19, 32). CD48 was recently identified as the high-affinity counterreceptor of 2B4 in both mice and humans (33, 34). CD48–2B4 interactions are physiologically important since they enhance the lytic function of human NK cells (23). It has been reported that 2B4 may function as a coreceptor in human NK cell activation (35).
In this paper, we investigated whether the AP-1 pathway is activated in
human NK cells upon 2B4 stimulation. AP-1 is an important regulator of
nuclear gene expression in leukocytes, having been found responsive to
a wide range of stimuli and regulating a large number of genes
(36, 37). We observed that AP-1 DNA-binding activity
increased in response to 2B4 stimulation, in particular AP-1 complexes
containing JunB. To dissect the mechanisms involved in
2B4-mediated cytotoxicity, we set out to examine whether 2B4
stimulation of YT cells utilized the Ras-dependent mitogen activated
protein kinase (MAPK) pathway in natural cytotoxicity and Ab-dependant
cellular cytotoxicity (ADCC). Our results show that 2B4-induced NK cell
cytotoxicity is dependent on the extracellular signal-regulated kinase
(ERK) and p38 MAPK pathways and can be inhibited by inhibitors of Ras
and Raf. Examination of 2B4-stimulated release of IFN- revealed the
role of the p38 MAPK but not the ERK1/2 pathway in IFN- production.
Immunoprecipitations revealed the constitutive association of linker
for activation of T cells (LAT) with 2B4.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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YT (human NK cell line), K562 (human erythroleukemia cell line), and P815 (mouse lymphoma cell line) were cultured in culture medium (RPMI 1640 supplemented with 10% FBS (HyClone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, 10 mM HEPES, and 10 mM nonessential amino acids). Cells were maintained at 37°C in a humidified 5% CO2/95% air incubator. Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD) unless otherwise noted. C1.7 Ab, which recognizes human 2B4 (25), was purchased from Coulter (Orlando, FL). Mouse Abs against c-Jun (catalog no. SC-822X), JunB (catalog no. SC-8051X), anti-phospho-p38 (catalog no. SC-7973), anti-phospho-ERK (catalog no. SC-7383), polyclonal goat Abs anti-p38 (catalog no. SC-535), and anti-ERK (catalog no. SC-94) and protein A/G PLUS-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LAT rabbit polyclonal Ab was purchased from Upstate Biotechnology (Lake Placid, NY). All enzymes were purchased from New England Biolabs (Beverly, MA) unless otherwise stated. Poly(dI-dC) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). All custom-synthesized oligonucleotides used in this study were supplied by Integrated DNA Technologies (Coralville, IA). All inhibitors used in this study were purchased from Calbiochem (San Diego, CA).
Nuclear extraction and EMSA
Nuclear extracts were isolated from YT cells and NK-92 cells and
YT cells incubated with mAb C1.7 (200 ng/ml concentration)
(38) and protein DNA-binding reactions were conducted in
EMSAs (39). One million YT cells were incubated with C1.7
mAb for 2, 4, 6, and 8 h in 12-well plates at 37°C in a
humidified 5% CO2/95% air incubator. After the
stimulation period, the cells were collected, washed in ice-cold PBS,
and lysed as described elsewhwere (38). A typical binding
reaction mixture contained 2 µg of nuclear protein, 1 µg of
poly(dI-dC), and radiolabeled oligonucleotide (20,000 cpm, 
0.2 ng)
in 10 µl of reaction volume. The double-stranded radiolabeled
oligonucleotide probe codes for the human 2B4 (h2B4) promoter
sequence lies (-111 to -89) relative to the start of transcription
and contains a functional AP-1 site (40). The
mixture was incubated on ice for 30 min and then electrophoresed
through a 4% polyacrylamide gel under nondenaturing conditions in
0.25x Tris-borate-EDTA at 200 V for 70 min. The gel was dried and then
exposed to film. The bands were visualized by autoradiography and
quantified using the image quantitation program AlphaEase (Alpha
Innotech, San Leandro, CA).
51Cr release cytotoxicity assay
K562 cells and P815 cells, where indicated, were used as target cells and labeled by incubating 1 x 106 cells with 2 MBq of Na251CrO4 (NEN Research Products, Boston, MA) for 90 min at 37°C under 5% CO2 in air. The target cells were then washed three times in culture medium. Ten thousand labeled target cells (100 µl) were incubated with the effector YT cell suspension (100 µl), with and without mAb C1.7 (200 ng/ml). Effector YT cells were resuspended and added at 1, 2, 5, 10, and 20 times the number of labeled target cells. After incubation for 4 h at 37°C under 5% CO2 in air, the cells were pelleted at 250 x g for 5 min, 100 µl of the supernatants was removed, and their radioactivity was measured. The percentage of specific lysis was calculated by the following equation: (a - b/c - b) x 100, where a is the radioactivity of the supernatant of target cells mixed with effector cells, b is that in the supernatant of target cells incubated alone, and c is that in the supernatant after lysis of target cells with 1% Nonidet P-40. All data points in each graph represent the average of four independent trials with similar results. Determination of statistical significance was determined on each data point representing 2B4-mediated cytotoxicity assays performed with inhibitor-treated effector cells or target cells compared with assays conducted with nontreated effector and target cells with Student’s t test. Data groups were considered significantly different when p < 0.05.
Inhibitor treatment of cells
In assays using inhibitors, both effector YT cells and target K562 and P815 cells were subjected to treatment in culture medium as indicated. Cells were incubated in actinomycin D (20 µg/ml) for 2 h to inhibit RNA polymerase. To inhibit p38, cells were incubated in SB203580 at 10 and 50 µM concentrations for 1 h. Cells were incubated in PD089059 (100 µM) for 1 h to inhibit MAPK kinase 1 (MEK1). Cells were incubated in FTI-277 at 7.5 and 30 µM for 16 h to inhibit H-Ras processing. All incubations with inhibitors were conducted at 37°C under 5% CO2 in air. At the end of the incubation period, the cells were used in 51Cr release cytotoxicity assays.
Immunoblot analysis
YT cells were incubated (1 x 107/100 µl; 37°C) for the indicated times with the different stimuli. These were C1.7 mAb (10 µg/ml) and PMA (50 ng/ml). After stimulation, the cells were lysed with 900 µl of lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 mM HEPES (pH 7.5), 0.15 M NaCl, 10% glycerol, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, 1 mM EDTA, and 10 µg/ml each of aprotinin and leupeptin). Forty micrograms of protein lysate was analyzed in 8% SDS-PAGE (reducing conditions). Western blots were performed according to the manufacturer’s chemiluminescence detection system instructions (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Western blots were hybridized with anti-phospho-p38 and anti-phospho-ERK mAbs to detect phosphorylated forms of p38 and ERK, respectively. The Western blots were then stripped and reprobed with anti-p38 and anti-ERK Abs to detect total amounts of p38 and ERK, respectively.
IFN- release assay
Inhibitor-treated or untreated YT cells (500,000) were
stimulated or unstimulated with C1.7 mAb (400 ng/ml) in flat-bottom
24-well plates for 1 h at 37°C under 5%
CO2 in air. Target K562 cells (50,000) were then
added. After incubation for 16 h at 37°C under 5%
CO2 in air, 100 µl of cell-free supernatant was
collected. IFN- concentration was then quantitated with an ELISA kit
according to the manufacturer’s instructions (CLB, Amsterdam, The
Netherlands). Each condition was tested in at least four independent
trials.
Immunoprecipitations
Approximately 1 x 108 YT cells were either left untreated or treated with 1 µg/ml C1.7 mAb for 2 h at 37°C. Cells were then lysed with 1% digitonin, 10 mM Tris (pH 7.4), 150 mM NaCl, 100 µg/ml PMSF, and protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO) for 1 h on ice. The lysates were precleared with the addition of control mouse IgG Ab (Upstate Biotechnology) for 3 h followed by protein G/A plus agarose and rotated for 3 h at 4°C. The precleared supernatant were used to immunoprecipitate 2B4 with C1.7 mAb (5 µg/ml) overnight with slow shaking followed by protein G/A plus agarose mix for 3 h at 4°C. Isotype control IgG (22b5) was used as negative control immunoprecipitations. Immunoprecipitates were separated by 10% SDS-PAGE and detected as described in Immunoblot analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies on the transcriptional regulation of the mouse and
human 2B4 gene have revealed the presence of
functional AP-1 sites in both promoters (40, 41). The AP-1
site that resides -106 to -100 relative to the start of transcription
is necessary for human 2B4 gene expression (40). Recently,
it has been found that engagement of NK cell cytotoxicity regulates
AP-1 expression (42). To determine whether 2B4 stimulation
of YT cells, a human NK cell line, results in activation of AP-1, we
isolated nuclear protein after 2B4 stimulation. We then performed EMSAs
with double-stranded radiolabeled probes coding for the promoter
sequence (-111 to -89) of the human 2B4 promoter that contains a
functional AP-1 site (Fig. 1
).
Examination of the levels of AP-1 DNA-binding activity through
densitometric analysis revealed that AP-1 DNA-binding activity
increased in response to 2B4 cell stimulation, reaching maximal
stimulation 6 h after 2B4 stimulation of the cells (Fig. 1, B and C). Supershift analysis using Ab specific
for c-Jun and JunB found AP-1 factors that contained either protein
component increased (Fig. 1, D–F). However, the presence of
the supershifted band of JunB increased proportionately as shown in
Fig. 1B, indicating that 2B4 stimulation of NK cells results
in selective activation of AP-1 heterodimers involving JunB.
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Previously, we and others have established that 2B4 stimulation of
NK cell lines results in an increase in spontaneous cytotoxic and
redirected ADCC (rADCC) activity (9, 22, 23, 35). AP-1
induction and other critical transcriptional events may be involved in
2B4 signaling in NK cells. To determine whether 2B4-mediated
cytotoxicity is transcription dependent, 51Cr
release cytotoxicity assays were performed using effector and target
cells pretreated for 2 h with 20 µg/ml actinomycin D.
Actinomycin D inhibits RNA polymerases by complexing with the DNA
preventing transcription. Pretreatment of YT cells with actinomycin D
also inhibited natural cytotoxicity against target K562 cells (data not
shown). Pretreatment of YT effector cells resulted in significantly
lower levels of 2B4-mediated cytotoxicity of K562 cells
(p < 0.05) and rADCC
(p < 0.02) against P815 cells than those
mediated by control nontreated effector cells (Fig. 2). However, pretreatment of target cells
with actinomycin D did not alter their susceptibility to NK cell
cytotoxicity. Thus, these results show that critical transcriptional
events are involved in 2B4-mediated cytotoxicity.
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Several transcription factors including AP-1, NF-
B, and Ets can
be activated by the MAPK signaling pathways. We undertook the task of
examining the roles of the p38 and MEK1/ERK signaling pathways in the
2B4 signal cascade. To accomplish this, we used the selective inhibitor
of p38, SB203580, in pretreatments of YT cells and target K562 cells
before their use in 51Cr release cytotoxicity
assays. YT cells pretreated with increasing concentrations of SB203580
and then stimulated with anti-2B4 mAb resulted in progressively
lower spontaneous cytotoxicity against K562 cells
(p < 0.05; Fig. 3A). SB203580-pretreated NK
cells’ ability for rADCC against P815 cells was also diminished.
Pretreatment of either target cell failed to inhibit 2B4-stimulated
cytotoxicity significantly. These results show that p38 plays a role in
2B4-stimulated YT cells in ADCC as well as spontaneous
cytotoxicity.
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B). This indicates the that MEK1/ERK signaling pathway
regulates 2B4 activation of the lytic function of NK cells. It appears
that 2B4 signaling utilizes multiple MAPK pathways in activating
cytolytic function. We have examined the effect of pretreatment with
PD089059 and SB203580 on YT cells. We found that inhibition with either
MEK1 or p38 decreased the natural cytotoxicity of YT cells against
target K562 cells (data not shown). Previous studies have found that
the MEK1/ERK pathway is required for NK cells to acquire
lymphokine-activated killer activity. However, once the NK cells
were engaged, treatment with the inhibitor PD089059 had little effect
on NK activity (44). The MEK1/ERK signaling pathway can
activate AP-1, members of the Ets-1 family, whereas activation of the
p38 MAPK pathway leads to NF-B activation (37, 45, 46, 47).
Activation of these pathways can lead to important transcriptional
events, such as AP-1 activation, that are essential for the engagement
of NK cell cytotoxicity.
Signal transduction through the p38 and ERK MAPK pathways requires the
phosphorylation of p38 and ERK, respectively. To determine whether 2B4
ligation results in the alteration of phosphorylation states of p38 and
ERK, YT cells were stimulated with C1.7 mAb and then lysed and analyzed
by Western blot. 2B4 engagement by anti-2B4 mAb resulted in
tyrosine phosphorylation of both p38 and ERK (Fig. 4). However, PMA stimulation of YT cells
resulted in phosphorylated forms of ERK only.
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In general, the ERK pathway can be activated by signaling through
Ras-Raf (36, 37). It has been recently reported that Ras
becomes activated after target ligation or IL-2 induction
(48). To determine whether 2B4 activates NK cell function
in a Ras-dependent manner, we performed 51Cr
release assays with FTI-277-treated cells. FTI-277 is a farnesyl
transferase inhibitor that prevents H-Ras from localizing to the plasma
membrane and becoming functionally active both in vivo and in vitro.
This induces accumulation of nonfarnesylated cytoplasmic H-Ras, which
bind Raf protein to form inactive Ras-Raf complexes (49).
YT cells were treated for 16 h at 37°C with 7.5 and 30 µM
FTI-277, known levels that inhibit Ras activation (48).
Pretreatment of YT cells with FTI-277 markedly diminished
2B4-stimulated NK cell cytotoxicity against K562 and P815 target cells
(Fig. 5). However, FTI-277 treatment of
target cells had no effect on target lysis in the same assay (Fig. 5).
These 51Cr release cytotoxicity assays were also
conducted using ZM 336372, an inhibitor of Raf. ZM336372 treatment of
YT effector cells also significantly inhibited 2B4-mediated cytolytic
activity (data not shown). Thus, it appears Ras and Raf are essential
for 2B4-stimulated NK cell cytotoxicity in YT cells.
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release is dependent on the p38 pathway
IFN- production by NK cells is a major function of NK cells in
response to NK cell-target cell contact (1). Our studies
have revealed that binding of 2B4 by C1.7 mAb results in the increase
in cytolytic activity of YT cells and the activation of Ras, Raf, and
the ERK and p38 MAPK pathways. Using similar inhibitor treatment
protocols, we investigated whether 2B4-induced IFN- production was
under the control of the same pathways. Pretreatment of YT cells with
actinomycin D and SB203580 inhibited IFN- production (Fig. 6), whereas inhibitors of Ras, Raf, and
the ERK MAPK pathway did not. The induction of IFN- production
required the activity of p38 as shown by the ability of SB203580 to
completely inhibit 2B4-induced cytokine production.
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Both 2B4 and LAT are localized in the glycolipid-enriched
microdomains (GEM) fractions of the cell membrane (50). To
determine whether LAT associates with 2B4 in NK cells, 2B4 was
immunoprecipitated from the YT cells, which was either unstimulated or
stimulated with anti-2B4 mAb. Immunoprecipitates were analyzed by
Western blot using anti-2B4 mAb and subsequently after stripping
the blot was reprobed with anti-LAT polyclonal Ab. As shown in Fig. 7, LAT is associated with 2B4
consitutively. Densitometric analysis indicates that the level of
association between 2B4 and LAT remains unchanged after 2B4
stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MAPK signaling pathways activates AP-1 and other transcription factors
including NF-B and Ets proteins. IL-2 activation of human NK cells
results in an increase in ERK activity but not p38 activity, and
IL-2-activated NK functions are dependent on the ERK pathway
(44). FcR-triggered TNF-
secretion and
integrin-mediated IFN- production also are dependent on the ERK
pathway (52, 53). In our study, YT cytotoxicity in
spontaneous and rADCC against target K562 and P815 cells was increased
by 2B4 ligation with Ag-specific mAb. Pretreatment of YT cells with
various cell-permeable specific inhibitors revealed the activation of
the Ras/Raf/ERK and p38 MAPK pathways during 2B4-mediated signaling.
Previously, it has been found that p38 and MEK1/ERK signaling are
involved in direct tumor cell lysis by NK cells while the c-Jun
N-terminal kinase MAPK pathway does not play a significant role
(44, 48, 52). The p38 MAPK pathway’s role in other NK
cell functions include FcRIIIA-induced granule exocytosis and target
cell-induced IFN- mRNA accumulation (52). Although some
studies have shown that activation of natural cytotoxicity can be Ras
independent, IL-2 activation of the MAPK pathway was completely
dependent on intact Ras (44, 48). It appears that the role
of these proteins in natural cytotoxicity is complex and dependent on
the activation state of the NK cells.
2B4 stimulation of NK cells may utilize distinct but overlapping
pathways from those engaged by other receptors and these pathways
regulate different aspects of NK cell function. Ras inhibitor, FTI-277,
could not interfere with perforin and granzyme B polarization toward
the contact point with a target cell (48). 2B4 stimulation
of NK cells revealed the activation of the ERK and p38 pathway as well
as an integral role for Ras (
Figs. 3–5). However, pretreatment of YT
cells with an inhibitor of Ras did not inhibit 2B4/target
cell-stimulated production of IFN- (Fig. 6). Alternatively,
treatment of YT cells using actinomycin D and the p38 inhibitor
SB203580 did inhibit IFN- production. Additionally, SB202190, a p38
inhibitor, inhibited accumulation of target (K562)-induced IFN- mRNA
in NK cells (52). This suggests that 2B4-mediated IFN-
production is Ras/Raf independent, unlike 1
integrin- and IL-2-activated secretion of IFN- (44, 53).
Our study demonstrated that 2B4 associates constitutively with LAT
(Fig. 7). Bottino et al. (54) have also shown that 2B4 is
constitutively associated with LAT and Ab ligation of 2B4 leads to
phosphorylation of 2B4 and LAT. Furthermore, phosporylated LAT recruits
other signaling molecules, phospholipase C (PLC) and Grb2
(54). The role of PLC1 or PLC2 in natural
cytotoxicity has not been clearly defined. Both are found in NK cells
and become tyrosine phosphorylated upon FcR activation
(55). In other signaling pathways, LAT is required for
FcR-dependent phosphorylation of PLC. LAT has been found to
associate with Grb2, Gads, and PLC1 in T cell Ag receptor-mediated
signaling (56, 57). In T cells, the activation pathways
through LAT may be determined by the signaling molecules that it
associates with (58, 59, 60). The Ras pathway is blocked in
LAT-deficient cells, resulting in inhibition of MAPK activation and
transcriptional activity of AP-1 (61, 62). On the other
hand, overexpression of LAT in NK cells leads to increased ADCC and
spontaneous cytotoxicity (56). LAT has emerged as a major
transmembrane adaptor protein in T cell activation after TCR engagement
by Ag-MHC (58, 59, 60, 63). TCR engagement results in the
phosphorylation of LAT which then associates with other adaptor
proteins including PLC, Grb-2, Grap, phosphatidylinositol 3-kinase,
and Gads which can then form multimeric signaling complexes by the
recruitment of other adaptor proteins including Vav, SLP-76 Sos, Shc,
Pyk-2, and Graf (58, 59, 60). Many of these adaptor proteins
are also expressed in NK cells and have been found to play various
roles in the signaling cascade in natural cytotoxicity and ADCC.
Grb2-Sos complex associates with LAT and activates the Ras-dependent
MEK/ERK pathway (36, 43, 64). Many other kinases have also
been implicated in NK cell activation including phosphatidylinositol
3-kinase (65), Syk (66), and Pyk2
(67, 68, 69). Pyk2 is recruited from the cytoplasm and
associates with paxillin and microtubule organizing center and
transported to the GEM fraction where it associates with Grb2, Shc, and
possibly Vav (53, 67, 68, 70). Pyk2 can activate the
Ras/Raf kinase pathway as well as the Rac-1 pathway (71, 72). Overexpression of Pyk2 leads to the activation of p38 MAPK
(73). Thus, it appears there are complex interactions that
occur at the GEM where 2B4 signaling may be controlled by LAT and the
adaptor molecules it associates with. It has been suggested that 2B4
functions as a coreceptor in human NK cell activation and requires
coengagement of other activating receptors (35). It is
possible that NK cell activation is dependent on the recruitment of key
adaptor molecules to the GEM fractions before engagement of cell
cytotoxicity can occur.
It has been found that NK inhibitory receptors can inhibit
phosphorylation of 2B4, thus blocking NK cell activation
(15). A possible mechanism may be dependent on SHP-1
recruitment by inhibitory killer Ig-related receptors that could
dephosphorylate 2B4 directly or other downstream signaling molecules
including LAT, SLP-76, Pyk2, and PLC (15, 74, 75, 76). It
has been shown that the 2B4 cytoplasmic tail can associate many adaptor
molecules including SHP-2 and SAP (25). It has also been
found that 2B4 can associate with SHP-2 in murine NK cells and SHP-1 in
primary NK cell isolates (31, 77). Several studies
indicate defective 2B4 signaling in NK cells from XLP patients
(26, 27, 29, 30, 31). Mutations in SAP results in defective NK
cell activation through 2B4, possibly due to loss of inhibition of
SHP-1–2B4 interactions (25, 30). It is conceivable that
SAP regulates 2B4 function by preventing SHP-1 from associating and
dephosphorylating the 2B4 cytoplasmic tail as well as other adaptor
molecules that are part of the signaling complex.
Activation of NK cells via h2B4 triggers many events that may be
controlled through transcription including an increase in transcription
of IFN- and MMP-2 (9, 78). Although we have found 2B4
signaling results in the activation of both the ERK and p38 MAPK
pathway, surprisingly, only inhibitors of the p38 pathway resulted in
the inhibition of 2B4-mediated IFN- secretion. This indicates that
the MAPK pathways regulate different aspects of NK cell activation.
Examination of the role of another NK cell surface receptor KIR2DL4
(CD158d) also supports the concept of functional distinction between
cytotoxicity and cytokine production (79). KIR2DL4
induction of resting NK cells induced IFN- production but not
cytotoxicity. We have observed that the LLT-1 receptor
(80), another NK cell molecule, induces IFN- secretion
while failing to increase cytolytic activity (H. K. Pham,
S. S. Chuang, and P. A. Mathew, unpublished observation).
Cumulatively, these data show that NK cell functions may be
differentially controlled and stimulated as determined by the signals
of individual receptors.
In summary, our study demonstrates that activation of NK cells through surface 2B4 is mediated via pathways that may involve adaptor molecules including LAT, Ras, and Raf, resulting in the activation of the p38 and MEK1/ERK MAPK pathways. This data along with other recent findings reveal the complexity of 2B4 signaling and its role in NK cell activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Porunelloor A. Mathew, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699. E-mail address: pmathew{at}hsc.unt.edu
3 Abbreviations used in this paper: SHP, SH2 domain bearing tyrosine phosphatase; SAP, signaling lympocyte-activation molecule-associated protein; XLP, X-linked lymphoproliferative syndrome; MAPK, mitogen-activated protein kinase; ADCC, Ab-dependent cellular cytotoxicity; ERK, extracellular signal-regulated kinase; LAT, linker for activation of T cells; h2B4, human 2B4; MEK1, MAPK kinase 1; rADCC, redirected ADCC; GEM, glycolipid-enriched microdomains; MMP, metalloproteinase; PLC, phospholipase C.
Received for publication March 28, 2001. Accepted for publication September 26, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation by MAP kinase cascades. Immunobiology 198:35.[Medline]
receptor activation induces the tyrosine phosphorylation of both phospholipase C (PLC)-1 and PLC-2 in natural killer cells. J. Exp. Med. 176:1751.1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling. J. Biol. Chem. 275:23355.1 and the Ras pathway. Immunity 9:617.[Medline]
signaling complex in human natural killer (NK) cells. J. Exp. Med. 184:2243. production following 2B4 activation in human NK cells. In Vivo 14:625.[Medline]
production, but not cytotoxicity, by the killer cell Ig-like receptor KIR2DL4 (CD 158d) in resting NK cells. J. Immunol. 167:1877.This article has been cited by other articles:
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 E. Morel and T. Bellon HLA Class I Molecules Regulate IFN-{gamma} Production Induced in NK Cells by Target Cells, Viral Products, or Immature Dendritic Cells through the Inhibitory Receptor ILT2/CD85j J. Immunol., August 15, 2008; 181(4): 2368 - 2381. [Abstract] [Full Text] [PDF]
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S. Malarkannan, J. Regunathan, H. Chu, S. Kutlesa, Y. Chen, H. Zeng, R. Wen, and D. Wang Bcl10 Plays a Divergent Role in NK Cell-Mediated Cytotoxicity and Cytokine Generation J. Immunol., September 15, 2007; 179(6): 3752 - 3762. [Abstract] [Full Text] [PDF]
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J. C. D. Wiseman, L. L. Ma, K. J. Marr, G. J. Jones, and C. H. Mody Perforin-Dependent Cryptococcal Microbicidal Activity in NK Cells Requires PI3K-Dependent ERK1/2 Signaling J. Immunol., May 15, 2007; 178(10): 6456 - 6464. [Abstract] [Full Text] [PDF]
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P. Eissmann and C. Watzl Molecular Analysis of NTB-A Signaling: A Role for EAT-2 in NTB-A-Mediated Activation of Human NK Cells. J. Immunol., September 1, 2006; 177(5): 3170 - 3177. [Abstract] [Full Text] [PDF]
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 H. Sasanuma, A. Tatsuno, S. Hidano, K. Ohshima, Y. Matsuzaki, K. Hayashi, C. A. Lowell, D. Kitamura, and R. Goitsuka Dual function for the adaptor MIST in IFN-{gamma} production by NK and CD4+NKT cells regulated by the Src kinase Fgr Blood, May 1, 2006; 107(9): 3647 - 3655. [Abstract] [Full Text] [PDF]
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 X. Jiang, B. A. Orr, D. M. Kranz, and D. J. Shapiro Estrogen Induction of the Granzyme B Inhibitor, Proteinase Inhibitor 9, Protects Cells against Apoptosis Mediated by Cytotoxic T Lymphocytes and Natural Killer Cells Endocrinology, March 1, 2006; 147(3): 1419 - 1426. [Abstract] [Full Text] [PDF]
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I. Saborit-Villarroya, J. M. Del Valle, X. Romero, E. Esplugues, P. Lauzurica, P. Engel, and M. Martin The Adaptor Protein 3BP2 Binds Human CD244 and Links this Receptor to Vav Signaling, ERK Activation, and NK Cell Killing J. Immunol., October 1, 2005; 175(7): 4226 - 4235. [Abstract] [Full Text] [PDF]
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B. Morandi, R. Costa, M. Falco, S. Parolini, A. De Maria, G. Ratto, M. C. Mingari, G. Melioli, A. Moretta, and G. Ferlazzo Distinctive Lack of CD48 Expression in Subsets of Human Dendritic Cells Tunes NK Cell Activation J. Immunol., September 15, 2005; 175(6): 3690 - 3697. [Abstract] [Full Text] [PDF]
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 A. A. Maghazachi Insights into Seven and Single Transmembrane-Spanning Domain Receptors and Their Signaling Pathways in Human Natural Killer Cells Pharmacol. Rev., September 1, 2005; 57(3): 339 - 357. [Abstract] [Full Text] [PDF]
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M. Martin, J. M. Del Valle, I. Saborit, and P. Engel Identification of Grb2 As a Novel Binding Partner of the Signaling Lymphocytic Activation Molecule-Associated Protein Binding Receptor CD229 J. Immunol., May 15, 2005; 174(10): 5977 - 5986. [Abstract] [Full Text] [PDF]
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A. Aoukaty and R. Tan Role for Glycogen Synthase Kinase-3 in NK Cell Cytotoxicity and X-Linked Lymphoproliferative Disease J. Immunol., April 15, 2005; 174(8): 4551 - 4558. [Abstract] [Full Text] [PDF]
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S. V. Vaidya, S. E. Stepp, M. E. McNerney, J.-K. Lee, M. Bennett, K.-M. Lee, C. L. Stewart, V. Kumar, and P. A. Mathew Targeted Disruption of the 2B4 Gene in Mice Reveals an In Vivo Role of 2B4 (CD244) in the Rejection of B16 Melanoma Cells J. Immunol., January 15, 2005; 174(2): 800 - 807. [Abstract] [Full Text] [PDF]
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A. Munitz, I. Bachelet, S. Fraenkel, G. Katz, O. Mandelboim, H.-U. Simon, L. Moretta, M. Colonna, and F. Levi-Schaffer 2B4 (CD244) Is Expressed and Functional on Human Eosinophils J. Immunol., January 1, 2005; 174(1): 110 - 118. [Abstract] [Full Text] [PDF]
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A. Mavropoulos, G. Sully, A. P. Cope, and A. R. Clark Stabilization of IFN-{gamma} mRNA by MAPK p38 in IL-12- and IL-18-stimulated human NK cells Blood, January 1, 2005; 105(1): 282 - 288. [Abstract] [Full Text] [PDF]
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P. Roda-Navarro, M. Mittelbrunn, M. Ortega, D. Howie, C. Terhorst, F. Sanchez-Madrid, and E. Fernandez-Ruiz Dynamic Redistribution of the Activating 2B4/SAP Complex at the Cytotoxic NK Cell Immune Synapse J. Immunol., September 15, 2004; 173(6): 3640 - 3646. [Abstract] [Full Text] [PDF]
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 K.-M. Lee, M. E. McNerney, S. E. Stepp, P. A. Mathew, J. D. Schatzle, M. Bennett, and V. Kumar 2B4 Acts As a Non-Major Histocompatibility Complex Binding Inhibitory Receptor on Mouse Natural Killer Cells J. Exp. Med., May 3, 2004; 199(9): 1245 - 1254. [Abstract] [Full Text] [PDF]
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R. Vankayalapati, P. Klucar, B. Wizel, S. E. Weis, B. Samten, H. Safi, H. Shams, and P. F. Barnes NK Cells Regulate CD8+ T Cell Effector Function in Response to an Intracellular Pathogen J. Immunol., January 1, 2004; 172(1): 130 - 137. [Abstract] [Full Text] [PDF]
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C. N. Baxevanis, A. D. Gritzapis, and M. Papamichail In Vivo Antitumor Activity of NKT Cells Activated by the Combination of IL-12 and IL-18 J. Immunol., September 15, 2003; 171(6): 2953 - 2959. [Abstract] [Full Text] [PDF]
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S. G. Tangye, K. E. Nichols, N. J. Hare, and B. C. M. van de Weerdt Functional Requirements for Interactions Between CD84 and Src Homology 2 Domain-Containing Proteins and Their Contribution to Human T Cell Activation J. Immunol., September 1, 2003; 171(5): 2485 - 2495. [Abstract] [Full Text] [PDF]
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X. Gu, A. Laouar, J. Wan, M. Daheshia, J. Lieberman, W. M. Yokoyama, H. R. Katz, and N. Manjunath The gp49B1 Inhibitory Receptor Regulates the IFN-{gamma} Responses of T Cells and NK Cells J. Immunol., April 15, 2003; 170(8): 4095 - 4101. [Abstract] [Full Text] [PDF]
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C. Watzl and E. O. Long Natural Killer Cell Inhibitory Receptors Block Actin Cytoskeleton-dependent Recruitment of 2B4 (CD244) to Lipid Rafts J. Exp. Med., January 6, 2003; 197(1): 77 - 85. [Abstract] [Full Text] [PDF]
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J. Klem, P. C. Verrett, V. Kumar, and J. D. Schatzle 2B4 Is Constitutively Associated with Linker for the Activation of T Cells in Glycolipid-Enriched Microdomains: Properties Required for 2B4 Lytic Function J. Immunol., July 1, 2002; 169(1): 55 - 62. [Abstract] [Full Text] [PDF]
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S.-i. Yusa, T. L. Catina, and K. S. Campbell SHP-1- and Phosphotyrosine-Independent Inhibitory Signaling by a Killer Cell Ig-Like Receptor Cytoplasmic Domain in Human NK Cells J. Immunol., May 15, 2002; 168(10): 5047 - 5057. [Abstract] [Full Text] [PDF]
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) and JunB (
) activation observed in C and
D, respectively. Arrow, AP-1-DNA complex. Bracket, Ab
supershifted DNA-protein complex. N.S. are nonspecific
bands. Free represents unbound probe.
) or
with C1.7 mAb at 200 ng/ml concentrations (
). Pretreated effector
cells (x) were then incubated (1 h, 37°C) with C1.7 mAb (200 ng/ml).
Pretreated target cells (
) were incubated with 2B4-stimulated
effector cells. All data points in each graph represents the average of
four independent trials with similar results.
) concentrations of inhibitors were then incubated with
2B4-stimulated effector cells All data points in each graph represent
the average of four independent trials with similar results.
