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,,
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Immunology Program and
Drug Discovery Program, and
H. Lee Moffitt Cancer Center, Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, FL 33612;
§
Department of Pathology, University of Chicago, Chicago, IL 60637;
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Department of Immunology, Mayo Clinic, Rochester, MN 55905;
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Cellular Cytotoxicity Laboratory, Austin Research Institute, Heidelberg, Victoria, Australia;
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Department of Chemistry, Yale University, New Haven, CT 06511; and
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Department of Microbiology, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ERK has largely been considered a growth- and differentiation-related
signal molecule, involved in transcriptional control of critical genes
essential for cell proliferation/maturation, but it is likely that it
plays a crucial role in other cellular events. For example, ERK has
recently been reported to activate superoxide release (11)
and modulate cell-cell or cell-matrix interactions (12, 13). Cell secretion may also be driven by ERK (14).
We therefore undertook the task of investigating whether ERK was
important in lymphocyte function against tumor cells. Target ligation
can direct two separate and distinct functions in lymphocytes, dictated
by the induction and effector phases. In the induction phase, the event
triggered by TCR coupling to its Ag on quiescent T cells leads to
signals that up-regulate genes associated with cytokine induction and
cell proliferation, resulting in clonal expansion and functional
activation (15). Upon re-encounter of the Ag, activated T
cells express lytic function in the effector phase. The signal pathways
coupled to the TCR during the initial activation phase must differ from
those during the effector phase, because different functions prevail.
Research to date has focused on the induction phase, and it is
established that the TCR is coupled to nonreceptor tyrosine kinases,
Lck and Zap70 (15). These kinases, in turn, trigger
downstream events via calcium- and Ras-dependent pathways. Zap70 can
phosphorylate a membrane-bound adaptor protein, linker for activation
of T cells (LAT), which subsequently binds phospholipase C
1
(PLC1) and growth factor receptor binding protein 2 (GRB2)
(16, 17). Activation of PLC1 leads to inositol
phosphate and intracellular calcium mobilization, while formation of
the GRB2-SOS complex recruits Ras to the membrane, turning on the
Raf/MEK/MAPK pathway (18, 19). MAPK is known to activate
several important transcription factors required for IL-2 and other
cytokine gene induction (20). The involvement of Vav and
76-kDa SH2 domain leukocyte protein in T cell signal pathways has also
been recently described, and the ability of 76-kDa SH2 domain leukocyte
protein to bind Grb2 could provide another link to the Ras-dependent
MAPK pathway (21, 22).
Studies to date have thus documented that the conserved Ras/Raf/MEK/MAPK pathway is invoked by ligands binding to their Ag receptors. This finding is expected, given that Ag recognition leads to lymphocyte activation, resulting in gene expression related to clonal expansion, a noted example of which is IL-2. However, once activated, T cells must express a separate and distinct function, i.e., the ability to lyse relevant target cells. In contrast to well-documented molecular signals that are coupled to cytokine and proliferative responses in lymphocytes, information on molecular events coupled to lytic effector function is scarce. NK cells use the same machinery as cytolytic T cells (23), and the availability of lytic NK cell lines provides a convenient, useful tool in deciphering these mechanisms. A recent interest in signal pathways associated with lytic function has led to the identification of Syk70, Vav, Rac, and Pyk2 as critical components (24, 25, 26, 27, 28, 29). We have pinpointed MAPK/ERK2 as another key signal molecule in the lytic pathway (30). We first demonstrated that target engagement rapidly activated MAPK phosphorylation and kinase function (within 5 min) in human NK cells. To link ERK2 activation to NK biological function, we recently showed that PD098059, a specific inhibitor of MEK1 that lies directly upstream of MAPK (31), could potently suppress NK lysis of live Raji tumor cells in a 5-h 51Cr release assay. More importantly, immunostaining with fluorescence-conjugated anti-perforin or granzyme B indicated that PD098059 blocked the polarization of perforin and granzyme B that occurs in NK cells upon target ligation. Transient transfection with dominant negative ERK2, but not wild-type ERK2, into NK cells effectively interfered with their lytic capacity. Thus, these results documented a pivotal role of MAPK in NK effector function and demonstrated that ERK2 may regulate the events that mobilize perforin and granzyme B to the point of contact with the engaged target cell. Two other laboratories have also recently documented the importance of MAPK in NK cells, either in direct or indirect CD16-mediated lytic function (32, 33).
In a number of models of cell growth and differentiation, the Ras-dependent MAPK pathway is inevitably involved (1, 2, 20). Whether this same pathway is called upon during the lytic process in NK cells is unknown. We thus set out to examine whether target cells triggered a Ras-dependent MAPK pathway in NK cells and compared this pathway to that stimulated by IL-2. We demonstrate here that target ligation and IL-2 activated separate signal events to lead to MAPK activation, and target cells primarily triggered a Ras-independent MAPK-dependent event in NK cells that drives lytic function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human IL-2-dependent NK92 cells, provided by Dr. H.-G. Klingeman
(Terry Fox Laboratory, Vancouver, Canada) (34), were
maintained in 
-MEM containing 100 U/ml of human rIL-2 (Chiron,
Emeryville, CA). This medium was also supplemented with 20% FCS, 2 mM
glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1%
nonessential amino acids, 1% sodium pyruvate, and 5 x
10-5 M 2-ME. All tissue culture reagents were
purchased from Life Technologies (Grand Island, NY). The NK target
cells, Raji lymphoma and K562 erythroleukemic tumor cells (American
Type Culture Collection, Manassas, VA), were cultured in RPMI 1640
containing 10% FCS with 2 mM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin (30).
Isolation of large granular lymphocytes (LGL) from peripheral blood
NK cells were derived from LGL isolated from PBMC of normal volunteers as previously described (4). After plastic adherence for 1 h at 37°C and passage through nylon wool, the nonadherent mononuclear cells were placed on a four-step discontinuous Percoll gradient. The cells recovered from 42.5–45.0% Percoll usually contained 75–90% LGL, as assessed by Giemsa-stained morphology, and were used to test NK function.
Pharmacological inhibition of MAPK and Ras
For MAPK inhibition, NK92 cells or fresh LGL (2.5 x 106 cells/ml) were incubated for 2 h at 37°C with medium or serial dilutions of PD098059 (New England Biolabs, Beverly MA) or an equal amount of DMSO to dilute the highest concentration of inhibitor (31). Ras inhibition with a farnesyl transferase inhibitor, FTI-277, also dissolved in DMSO, was performed under the same conditions, except the incubation period was extended to 24 h (35, 36).
Fixation of target cells
Raji tumor cells were washed with PBS once and incubated with 1% paraformaldehyde (methanol-free) in PBS, pH 7.4, on ice for 30 min. Then the cells were washed four times with PBS to remove all paraformaldehyde.
Preparation of GST-Raf-RBD
The GST fusion protein linked to the Raf fragment that contains the Ras-GTP binding domain, GST-Raf-RBD, was constructed using pGEX-KT. GST-Raf-RBD was recovered from Escherichia coli by sonication and lysis with 1% Triton X-100. After centrifugation of the lysate at 15,000 rpm for 15 min at 4°C, the supernatant was collected and incubated for 15 min at 4°C with glutathione agarose on a rotator. The agarose beads with the bound GST-Raf-RBD were then collected and washed three times before use.
Detection of Ras activation by GST-Raf-RBD
Active Ras was measured by its ability to bind GST-Raf-RBD. Control NK92 cells or NK92 cells treated with DMSO or FTI-277 for 24 h at 37°C were rested in IL-2-free medium for 4 h at 37°C to reduce background phosphorylation. To initiate target ligation, the NK92 cells, suspended at 1 x 107/ml, were mixed with an equal number of paraformaldehyde-fixed Raji target cells and rapidly pelleted at 1000 rpm in a microcentrifuge at 4°C, followed by incubation for 0–30 min at 37°C. For IL-2 activation, NK92 cells were incubated with 100 u/ml of IL-2 for 0–30 min at 37°C. The cell pellet was solubilized at 4°C for 30 min in lysis buffer containing 1% Nonidet P-40, 10 mM Tris, 140 mM NaCl, 0.1 mM PMSF, 10 mM iodoacetamide, 50 mM NaF, 1 mM EDTA, 0.4 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/pepstatin, and 10 µg/ml aprotinin. Cell lysates were centrifuged at 15,000 rpm in a microcentrifuge for 10 min at 4°C to remove nuclei and cell debris. To detect activated Ras, the cell lysates were incubated for 2 h at 4°C on a rotator with 15 µl of the GST-Raf-RBD/glutathione agarose bead preparation. The beads containing the bound Ras-GTP were collected by centrifugation and washed three times with washing buffer (0.1% Nonidet P-40, 10 mM Tris, 140 mM NaCl, 0.1 mM PMSF, 10 mM iodoacetamide, 50 mM sodium fluoride, 1 mM EDTA, and 0.4 mM sodium orthovanadate). Samples were then boiled for 5 min in loading buffer and separated by 10% SDS-PAGE followed by Western blotting with anti-pan- Ras (Transduction Laboratories, Lexington, KY). The proteins were detected by the enhanced chemiluminescence detection system (ECL, Amersham, Arlington Heights, IL).
Detection of active MAPK
Cell lysates prepared from IL-2-rested NK92 cells that had been exposed to fixed Raji tumor cells or IL-2 for 0–30 min at 37°C were resolved by gel electrophoresis. Active MAPK was detected by Western blotting with anti-active MAPK, which specifically recognizes the phosphorylated TEY motif in this kinase (New England Biolabs). Equal loading was assessed by reblotting with anti-pan- ERK (Transduction Laboratories).
Vaccinia viral delivery of N17Ras
Recombinant vaccinia viruses encoding wild-type H-Ras and N17-Ras were constructed using the vector, pSC11, in recombination with the WR strain of vaccinia. CD56 expressing vaccinia was used as a control. In some cases, GB-expressing vaccinia, provided by Dr. Andrew M. Scharenberg (Harvard Medical School, Boston, MA), was substituted as a control. For infection, NK92 cells were incubated with various vaccinia constructs for 1 h at 37°C in serum-free medium at a multiplicity of infection of 10. Cells were then further incubated in serum-containing medium for 5 h at 37°C.
Cytotoxicity assay
A 51Cr release assay was performed as previously described, using Raji tumor cells as targets for NK92 effector cells and K562 tumor cells for fresh LGL (30). Briefly, target tumor cells were labeled with 200 µCi of sodium [51Cr]chromate (Amersham, Arlington Heights, IL) in 0.2 ml of medium at 37°C in a 5% CO2 atmosphere for 1 h. The cells were then washed three times and added to effector cells at 5 x 103 cells/well in 96-well round-bottom microplates, resulting in E:T cell ratios ranging from 50:1 to 2.5:1 in a final volume of 0.2 ml in each well. After 5-h incubation at 37°C, 100 µl of culture supernatants were harvested and counted in a gamma counter. The percent specific 51Cr release was determined by the equation ((experimental cpm - spontaneous cpm)/total cpm incorporated) x 100. All determinations were performed in triplicate, and the SEM of all assays was calculated and was typically around 5% of the mean or less.
Immunostaining
NK92 cells, untreated or pretreated with 50 µM PD098059 for 1 h at 37°C or 15 µM FTI-277 for 24 h at 37°C, were added to Raji cells at a 1:1 ratio in a total volume of 100 µl. The cells were spun rapidly at 1000 rpm for 1 min in a cold microcentrifuge, and then incubated for 0–10 min at 37°C. DMSO-treated NK92 cells were included as a control. The cells were then centrifuged onto a microscope slide and fixed at -20°C with methanol/acetone (3/1) for 20 min (30). The slides were air-dried and rehydrated for 2 h in several changes of PBS. All procedures were performed at room temperature. Polyclonal rabbit anti-human IgM (Sigma, St. Louis, MO) was used to differentiate Raji B lymphocytic cells from NK92 cells. Monoclonal anti-human perforin (Endogen/T Cell Sciences, Woburn, MA) and anti-granzyme B (37) were used to detect these lytic components from NK92 cells. Anti-IgM together with anti-perforin (or anti-granzyme B), each diluted 1/200 with 0.1% Nonidet P-40 in 1% BSA in PBS, were applied to the slide for 1 h. After several washes with PBS for 2 h, the slides were incubated for 25 min with goat anti-rabbit IgG tetramethylrhodamine isothiocyanate (TRITC)-labeled Ab (Sigma) diluted 1/80 or goat anti-mouse Ig FITC-labeled Ab (Sigma), diluted 1/100 in 0.1% Nonidet P-40 in PBS containing 1% BSA. The slides were then washed several times with PBS and covered with coverslips in a mounting medium of antifade/DAPI. Immunofluorescence was observed with a Leitz Orthoplan 2 microscope (Rockleigh, NJ), and images were captured by a CCD camera with the Smart Capture Program (Vysis, Downers Grove, IL). On each slide 100 NK92/Raji conjugates were evaluated for perforin or granzyme B mobilization.
Several controls were performed, i.e., NK92 cells alone or Raji tumor cells alone stained only with FITC-labeled goat anti-mouse Ig or with TRITC-labeled goat anti-rabbit IgG, to check for nonspecific binding of the secondary Abs. Nonspecific binding was not detected, and the results were omitted from the figures for clarity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our earlier work had employed the NK tumor cell line, YT, to
establish the importance of MAPK (30). To carry out this
work further, we have turned to a widely used IL-2-dependent NK cell
line, NK92 (34, 38, 39). This move was taken because NK92
cells retain all the biological properties of normal NK cells and
possess a markedly higher capacity to lyse Raji tumor cells than YT
cells, ranging from about 30% at an E:T cell ratio of 5:1, while YT
effector cells only killed around 30% at 50:1. We thus needed to
re-establish the baseline that MAPK critically controls lytic function
in NK92 cells. NK92 cells were pretreated with 12.5–100 µM PD098059,
a MAPK pathway specific inhibitor, for 1 h at 37°C before
incubation with 51Cr-labeled Raji tumor cells for
5 h at 37°C to test for lysis. PD098059 pretreatment effectively
inhibited NK92 lysis of Raji tumor cells in a dose-dependent manner, as
shown in a representative experiment of four that were performed (Fig. 1
). Marked inhibition was observed with
25 µM, with complete inhibition attained at 100 µM. DMSO, tested at
the concentration used to dissolve the highest concentration of
PD098059, had little adverse effect on NK lysis.
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A). PD098059, but not DMSO,
pretreatment for 1 h was able to inhibit this event in NK cells
(Fig. 2B). Analysis of total MAPK by stripping of the
original gel and reblotting with anti-pan-ERK demonstrated that
MAPK was equally present in all lanes.
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We next undertook the task of examining whether Ras was involved.
The first attempt was to define whether Ras was activated by target
ligation in NK92 cells. Inactive Ras binds GDP, which is exchanged for
GTP upon activation, acquiring the ability to bind Raf. To pursue Ras
activation, we thus employed a GST fusion protein that is linked to the
Raf protein segment that contains the activated GST-Raf-RBD
(40). Active Ras could then be captured by binding to
GST-Raf-RBD and detected with anti-pan-Ras. NK92 cells,
IL-2-starved for 4 h, were incubated with fixed Raji tumor cells
for 0–30 min (Fig. 3). As a positive
control, a separate group of NK92 cells was stimulated with IL-2, which
is documented to activate Ras. Cell lysates were then prepared and
incubated for 2 h with GST-Raf-RBD precoupled to
glutathione-Sepharose beads. Western blot analysis of the proteins
bound to GST-Raf-RBD demonstrated that Ras was quickly activated within
2–5 min of target ligation in NK92 cells; similarly, IL-2 treatment
activated Ras, which peaked at 5–15 min and was still detectable at 30
min. Stripping and reblotting with anti-active MAPK showed that
MAPK activation followed the same rapid kinetics with target ligation
and markedly longer kinetics with IL-2 (data not shown).
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It was essential to next link Ras activation to lytic function in
NK92 cells. An effective method to interfere with Ras function is to
block its farnesylation, preventing it from localizing to the plasma
membrane and becoming functionally active. A specific farnesyl
transferase inhibitor, FTI-277, well characterized to inhibit tumor
cell growth in vitro and in vivo (35, 36), was thus
employed. NK92 cells were treated for 24 h at 37°C with 7.5–30
µM FTI-277, which represents the optimal range of concentrations
known to effectively inactivate Ras and inhibit tumor cell growth (Fig. 4A). The NK effector cells
were then tested for NK function. It was notable that FTI-277, at all
doses used, had no effect on NK lysis of
51Cr-labeled Raji tumor cells. This was
reproduced in three other experiments. We also tested FTI-277 on
freshly isolated human peripheral blood LGL and found in four
donors tested that it had no ability to interfere with NK lysis of
tumor target cells (Fig. 4B).
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C). The same aliquots of NK92 cells from Fig. 4A, pretreated for 24 h with FTI-277, were IL-2-starved
for 4 h before incubation for 0–5 min at 37°C with fixed Raji
tumor cells. Cell lysates prepared from these treatments were then
probed with anti-active MAPK. Within 5 min of incubation with Raji
tumor cells, NK92 cells, either untreated or treated with DMSO or
FTI-277, had equal ability to activate MAPK. FTI-277 therefore did not
block MAPK activation in NK92 cells Lack of effect of FTI-277 on perforin and granzyme B polarization in NK cells
Perforin and granzyme B mobilization in NK cells is a hallmark of
cytolytic events against target cells, and we have earlier demonstrated
that MAPK/ERK2 is critical for their movement (30).
Whether Ras is required for this event thus needs to be evaluated. NK92
cells, pretreated 24 h at 37°C with 15 µM FTI-277 or DMSO at
the concentration used to dissolve the Ras inhibitor, were incubated
with Raji tumor cells for 0–10 min at 37°C. NK92 cells were also
pretreated for 1 h at 37°C with 50 µM PD098059 to inhibit MAPK
activation before target ligation. The cells were then cytocentrifuged
onto slides for assessment of perforin and granzyme B polarization.
Perforin or granzyme B, contained only in NK92 cells and not Raji tumor
cells, was detected by FITC staining with specific mAbs. Raji tumor
cells could be differentiated from NK92 cells by TRITC staining with
anti-human IgM. Immunostaining demonstrated that tumor engagement
for only 10 min mobilized perforin and granzyme B unidirectionally
toward the target cell, and a representative NK92/Raji conjugate is
shown under each treatment in Fig. 5. At
0 min of target binding, FITC-labeled perforin was evenly distributed
in the cytoplasm of NK92 cells (Fig. 5A, upper
panel). Upon 10-min incubation at 37°C, the DMSO-treated NK92
cell that had bound TRITC-labeled IgM+ Raji
target cells now showed almost complete polarization of FITC-labeled
perforin at the site of tumor contact (Fig. 5B, upper
panel). However, FTI-277 had no ability to interfere with perforin
polarization in NK92 cells (Fig. 5C, upper
panel). PD098059, which specifically inhibited MAPK activation,
clearly interrupted this process (Fig. 5D, upper
panel). Similar observations were made with granzyme B (Fig. 5, lower panel). Enumeration of NK/Raji conjugates showed that
the percentage of conjugates with polarized perforin rose from 4.8 to
32% with 10 min of binding. PD098059 decreased the percentage of
polarized conjugates to 16.9%, but FTI-277-treated conjugates remained
at 34%. Similar percentages were obtained with granzyme B. Confocal
microscopy showed that all perforin was located at the contact point by
10 min (data not shown). Thus, Ras does not appear to be essential for
granule movement in NK92 cells, but MAPK is.
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It is critical to ensure, however, that FTI-277 is able to block
Ras activation in our experiments. We thus treated NK92 cells with or
without FTI-277 for 24 h at 37°C, rested the NK92 cells in
IL-2-starved medium for 4 h at 37°C, and then activated them for
5 min at 37°C with either fixed Raji tumor cells or 500 U/ml of IL-2.
IL-2 was included as a positive control. Cell lysates were prepared and
incubated for 2 h with GST-Raf-RBD precoupled to
glutathione-Sepharose beads, and the adsorbed complexes were resolved
by gel electrophoresis. Detection of bound Ras-GTP by Western blot
analysis with anti-pan-Ras indicated that both IL-2 and Raji tumor
cells readily activated Ras (Fig. 6A). FTI-277 was able to
inhibit this activation in a dose-dependent manner. To show specificity
and effectiveness of FTI-277 in inhibiting only farnesyl transferase,
but not geranyl-geranyl transferase, we next performed Western blot
analysis of NK92 cells with anti-H-Ras and anti-Rap1A after
FTI-277 treatment. FTI-277 at 15 µg/ml specifically inhibited Ras
farnesylation in NK92 cells (detected by a slower migrating
unprenylated band in Western blots using anti-H-Ras), but did not
interfere with Rap1A geranylation in NK92 cells (Fig. 6B).
Thus, our results indicated that FTI-277 effectively inhibited Ras
farnesylation and activation in NK cells, but could not interfere with
MAPK activation or tumor cell lysis.
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To definitely determine whether MAPK activation and lytic function
were independent of Ras in NK cells, we resorted to a molecular
approach. We delivered dominant negative N17 Ras into NK92 effector
cells via a vaccinia virus vector and assessed, first, Ras activity in
the infected cells before analysis of MAPK activity and tumoricidal
function. Infection was conducted for 6 h, which provided
sufficient time for ample production of proteins from viral delivery
without loss of cell viability. After viral infection, NK92 cells were
then IL-2-starved 4 h before activation for 0–5 min with either
IL-2 or fixed Raji tumor cells. Wild-type Ras and an irrelevant gene,
CD56, were introduced into separate NK92 cell pools as controls.
Expression of the irrelevant gene, CD56, had no effect on Ras
activation in NK92 cells, as measured by Ras-GTP binding to
GST-Raf-RBD, and the levels of Ras activation by IL-2 and Raji tumor
cells were similar to those by mock-infected control NK92 (Fig. 7). Expression of wild-type Ras caused a
significant production of activated Ras even without IL-2 or Raji
treatment. N17 Ras expression, in contrast, caused loss of detection of
activated Ras in both IL-2- and Raji-stimulated NK92 cells. Thus, we
have a working system to assess what fate MAPK will have in a
Ras-defective NK92 cell.
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Assessment of active MAPK in N17 Ras-expressing NK92 cells was
next performed (Fig. 8A). NK92
cells, mock-infected or carrying the irrelevant gene, GB, showed
similar enhanced levels of MAPK activated by Raji tumor cells. NK92
expressing wild-type Ras had active MAPK even before Raji stimulation.
Most importantly, N17 Ras expressing NK92 cells showed equal ability as
normal NK92 cells to activate MAPK upon target ligation. Thus, MAPK
activation triggered by target ligation is via a Ras-independent
mechanism. Using the same pool of cells, we tested their tumorical
capacity (Fig. 8B). We found that N17 Ras expressing NK92
cells had almost equal ability to lyse
51Cr-labeled tumor cells as mock-infected and
GB-infected NK92 cells. We also made the observation that
overexpression of wild-type Ras consistently caused a moderate decrease
in NK lytic function. These results were reproduced in two other
experiments. It has been reported that MAPK activation by IL-2 in T
cells such as Jurkat is a Ras-dependent MAPK event. To ensure that our
system was working appropriately, we also examined this process in NK92
cells. As in T cells, expression of N17 Ras completely abolished MAPK
activation in IL-2-stimulated NK cells (Fig. 9). Unlike target cell ligation, IL-2
caused a Ras-dependent MAPK event. Thus, various pathways exist to
activate MAPK in NK cells, and we have evidence to indicate that NK
lytic function, activated by target engagement, is controlled by a
Ras-independent MAPK signal pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pharmacological inhibition of Ras is one means to analyze Ras participation in NK function. To definitely identify the role of Ras in MAPK activation and lytic function, we undertook the genetic approach of introducing dominant negative N17-Ras into NK92 cells via vaccinia delivery. Viral delivery of N17-Ras abolished Ras activation in NK92 cells triggered by either IL-2 or Raji tumor cells. Irrelevant CD56 gene delivery, used as a control, had no adverse effect on Ras activation. Of key importance was that although N17-Ras was able to suppress Ras, it could not interfere with MAPK activation in NK92 cells triggered by Raji tumor cells. To ensure that N17-Ras could block a Ras-dependent MAPK pathway, we also evaluated its effect on IL-2 activation in NK92 cells. IL-2 activation of MAPK was completely dependent on intact Ras. These results suggest that the tumoricidal process triggered in NK cells by target ligation is controlled via a Ras-independent MAPK-dependent signal event. It is thus important to note that lymphocytes may actually use separate signal pathways in the initial activation phase and the later effector phase. Initially, Ag ligation may drive a Ras-dependent MAPK mechanism that results in gene expression that is required for cell proliferation and differentiation (1, 2, 20). Later Ag ligation in the effector lymphocyte calls upon a Ras-independent MAPK mechanism, which now rapidly mobilizes lytic components, such as perforin and granzyme B, without the need for new gene expression. Another key difference is that IL-2-driven Ras/MAPK activation persists for up to 30 min, while target-induced MAPK activation is very transient. This suggests that a longer period of MAPK activation is required for the induction of cell cycle control-related gene expression in response to IL-2, as opposed to a short burst of MAPK activation for rapid granule mobilization, which is accomplished within 5 min upon contact with target tumor cells.
Several candidates could serve in place of Ras to activate MAPK in the lytic process. Phosphoinositol 3-kinase (PI 3-kinase) interfaces with Ras in insulin and G protein-coupled receptor signaling (43, 44), but in integrin- or IL-2-mediated signaling, PI 3-kinase can act downstream of Ras to regulate MAPK (45, 46). Syk70 overexpression in JCAM1 Jurkat T cells can enable TCR cross-linking to activate MAPK/ERK2, but two distinct pathways can emerge (47, 48). For MAPK activation, Lck-negative Jurkat T cells require Ras, but normal Jurkat cells do not. Thus, Syk70 can potentially bypass Ras to activate MAPK. Because PI 3-kinase can be triggered by either CD94 or FcR cross-linking in NK cells (49, 50), and Syk70 is critical for signaling NK activation (24, 25, 26), it is possible that either of these molecules could control MAPK in NK cells during the lytic process.
Other potential pathways include protein kinase C and protein kinase A. Ca-dependent isoforms of protein kinase C control MAPK by bypassing Ras to activate Raf-1 (51, 52, 53), while protein kinase A uses Rap1 (instead of Ras) to activate Raf, resulting in MEK activation and subsequent MAPK activation (54, 55). Work is being planned to determine whether there is a specific upstream pathway in NK cells that is critical for MAPK activation to attain functional lysis of tumor cells.
In terms of the clinical significance of our study, the finding that NK cells use a Ras-independent pathway to kill tumor cells might be of therapeutic advantage. A major recent focus in pharmaceuticals is in the targeting of oncogenes that are mutated in cancer. Ras has been a prime target for the development of new therapeutic agents, because of the high frequency of Ras mutations in human tumors (36). If Ras is not involved in NK lytic function, a rational drug design targeted specifically against Ras could generate new therapeutic agents that might spare a key immune system in cancer patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Julie Y. Djeu, Immunology Program, H. Lee Moffitt Cancer Center, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612.
3 Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; FTI, farnesyl transferase inhibitor; ERK, extracellular regulated kinase 2; LGL, large granular lymphocyte; PLC, phospholipase C; RBD, Ras-GTP binding domain; PI 3-kinase, phosphoinositide 3-kinase; MEK-1, MAPK/ERK kinase 1; TRITC, tetramethylrhodamine isothiocyanate; RK, MAPKAP kinase 2 reactivity kinase; GRB2, growth factor receptor binding protein 2.
Received for publication May 30, 2000. Accepted for publication July 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 associate with a 36- to 38-kilodalton phosphotyrosine protein after T-cell receptor stimulation. Mol. Cell. Biol. 14:4435.. Science 275:394.RIIIA-ligand interaction. J. Exp. Med. 179:551. activates RAF-1 by direct phosphorylation. Nature 364:249.[Medline]
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 M M Whalen and S O Odman-Ghazi Effects of adenylyl cyclase and protein kinase A inhibition on signaling enzymes in natural killer cells: comparison to tributyltin Human and Experimental Toxicology, June 1, 2006; 25(6): 333 - 340. [Abstract] [PDF]
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 D. G. T. Hesslein, R. Takaki, M. L. Hermiston, A. Weiss, and L. L. Lanier Dysregulation of signaling pathways in CD45-deficient NK cells leads to differentially regulated cytotoxicity and cytokine production PNAS, May 2, 2006; 103(18): 7012 - 7017. [Abstract] [Full Text] [PDF]
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 N. J. Waterhouse, V. R. Sutton, K. A Sedelies, A. Ciccone, M. Jenkins, S. J. Turner, P. I. Bird, and J. A. Trapani Cytotoxic T lymphocyte-induced killing in the absence of granzymes A and B is unique and distinct from both apoptosis and perforin-dependent lysis. J. Cell Biol., April 10, 2006; 173(1): 133 - 144. [Abstract] [Full Text] [PDF]
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F. Guo, C. Sigua, P. Bali, P. George, W. Fiskus, A. Scuto, S. Annavarapu, A. Mouttaki, G. Sondarva, S. Wei, et al. Mechanistic role of heat shock protein 70 in Bcr-Abl-mediated resistance to apoptosis in human acute leukemia cells Blood, February 1, 2005; 105(3): 1246 - 1255. [Abstract] [Full Text] [PDF]
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J. A. Yoder, R. T. Litman, M. G. Mueller, S. Desai, K. P. Dobrinski, J. S. Montgomery, M. P. Buzzeo, T. Ota, C. T. Amemiya, N. S. Trede, et al. Resolution of the novel immune-type receptor gene cluster in zebrafish PNAS, November 2, 2004; 101(44): 15706 - 15711. [Abstract] [Full Text] [PDF]
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D. F. Barber, M. Faure, and E. O. Long LFA-1 Contributes an Early Signal for NK Cell Cytotoxicity J. Immunol., September 15, 2004; 173(6): 3653 - 3659. [Abstract] [Full Text] [PDF]
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O. D. Perez, D. Mitchell, G. C. Jager, and G. P. Nolan LFA-1 signaling through p44/42 is coupled to perforin degranulation in CD56+CD8+ natural killer cells Blood, August 15, 2004; 104(4): 1083 - 1093. [Abstract] [Full Text] [PDF]
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 P. Garcia, A. B. de Heredia, T. Bellon, E. Carpio, M. Llano, E. Caparros, P. Aparicio, and M. Lopez-Botet Signalling via CD70, a member of the TNF family, regulates T cell functions J. Leukoc. Biol., July 1, 2004; 76(1): 263 - 270. [Abstract] [Full Text] [PDF]
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P. K. Epling-Burnette, J. S. Painter, P. Chaurasia, F. Bai, S. Wei, J. Y. Djeu, and T. P. Loughran Jr Dysregulated NK receptor expression in patients with lymphoproliferative disease of granular lymphocytes Blood, May 1, 2004; 103(9): 3431 - 3439. [Abstract] [Full Text] [PDF]
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 H. Xu, H. An, Y. Yu, M. Zhang, R. Qi, and X. Cao Ras Participates in CpG Oligodeoxynucleotide Signaling through Association with Toll-like Receptor 9 and Promotion of Interleukin-1 Receptor-associated Kinase/Tumor Necrosis Factor Receptor-associated Factor 6 Complex Formation in Macrophages J. Biol. Chem., September 19, 2003; 278(38): 36334 - 36340. [Abstract] [Full Text] [PDF]
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B. Zhong, K. Jiang, D. L. Gilvary, P. K. Epling-Burnette, C. Ritchey, J. Liu, R. J. Jackson, E. Hong-Geller, and S. Wei Human neutrophils utilize a Rac/Cdc42-dependent MAPK pathway to direct intracellular granule mobilization toward ingested microbial pathogens Blood, April 15, 2003; 101(8): 3240 - 3248. [Abstract] [Full Text] [PDF]
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D. Khurana and P. J. Leibson Regulation of lymphocyte-mediated killing by GTP-binding proteins J. Leukoc. Biol., March 1, 2003; 73(3): 333 - 338. [Abstract] [Full Text] [PDF]
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K. Jiang, B. Zhong, D. L. Gilvary, B. C. Corliss, E. Vivier, E. Hong-Geller, S. Wei, and J. Y. Djeu Syk Regulation of Phosphoinositide 3-Kinase-Dependent NK Cell Function J. Immunol., April 1, 2002; 168(7): 3155 - 3164. [Abstract] [Full Text] [PDF]
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J. M. Cunnick, S. Meng, Y. Ren, C. Desponts, H.-G. Wang, J. Y. Djeu, and J. Wu Regulation of the Mitogen-activated Protein Kinase Signaling Pathway by SHP2 J. Biol. Chem., March 8, 2002; 277(11): 9498 - 9504. [Abstract] [Full Text] [PDF]
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 J. Y. Djeu, K. Jiang, and S. Wei A View to a Kill: Signals Triggering Cytotoxicity Clin. Cancer Res., March 1, 2002; 8(3): 636 - 640. [Abstract] [Full Text] [PDF]
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S. S. Chuang, P. R. Kumaresan, and P. A. Mathew 2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN-{gamma} Release in Human NK Cells Involves Distinct Pathways J. Immunol., December 1, 2001; 167(11): 6210 - 6216. [Abstract] [Full Text] [PDF]
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Y. M. Vyas, K. M. Mehta, M. Morgan, H. Maniar, L. Butros, S. Jung, J. K. Burkhardt, and B. Dupont Spatial Organization of Signal Transduction Molecules in the NK Cell Immune Synapses During MHC Class I-Regulated Noncytolytic and Cytolytic Interactions J. Immunol., October 15, 2001; 167(8): 4358 - 4367. [Abstract] [Full Text] [PDF]
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J. A. Yoder, M. G. Mueller, S. Wei, B. C. Corliss, D. M. Prather, T. Willis, R. T. Litman, J. Y. Djeu, and G. W. Litman Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster PNAS, June 5, 2001; 98(12): 6771 - 6776. [Abstract] [Full Text] [PDF]
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