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Laboratory of Experimental Immunology, National Cancer Institute, Frederick, MD 21701
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, lymphotactin, macrophage-inflammatory
protein (MIP)1
, and MIP1
. This gene expression was
specific because other chemokines were not induced by anti-Ly-49D
receptors. In addition, a series of pharmacological inhibitors were
used to identify the key signaling pathways involved in the cellular
response. The primary Ly-49D signaling for IFN- production is
predominately mediated through Src kinase pathways involving membrane
proximal events, whereas MIP1 and MIP1 gene induction is more
complex and may involve multiple biochemical pathways. Thus, we
conclude that a primary role for the activating NK receptors in vivo
may be to trigger soluble factor production and regulation of the
immune response. This would place NK cells and their activating Ly-49
receptors as important initiators of microbial immunity and key
elements of the innate immune system. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inhibitory Ly-49 receptors, Ly-49A, C, G, and I, inhibit NK cell function upon binding of class I ligands on target cells (1, 2, 3). These Ly-49 inhibitory receptors, as well as inhibitory KIRs, contain cytoplasmic immune receptor tyrosine-based inhibitory motifs (ITIMs) that are phosphorylated upon stimulation, leading to the recruitment of Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1) and attenuation of intracellular signals (1, 4, 5). In contrast, the predicted amino acid sequences for the activating receptors, Ly-49D and Ly-49H, do not contain any ITIMs in their cytoplasmic domains, confirming that these are not inhibitory receptors (6, 7, 8, 9). Furthermore, it has been demonstrated that the activating receptors do not become phosphorylated after pervanadate treatment or receptor cross-linking and do not recruit SHP-1 (10). However, Ly-49D has been shown to mobilize intracellular Ca2+ and to mediate reverse Ab-dependent cellular cytotoxicity in the presence of specific mAb (10, 11).
These activating Ly-49 and KIR molecules have been shown to associate with a 12-kDa homodimeric protein, DAP12, that contains an immunoreceptor tyrosine-based activation motif that is critical for positive signaling by these receptors (10, 11). The Ly-49D and H activating receptors contain an arginine residue in their transmembrane domain that serves as a required docking site for DAP12 binding to the receptor (9, 10, 11). Association of the homodimeric DAP12 with these activating receptors is essential for phosphorylation of DAP12 following receptor triggering, leading to intracellular calcium mobilization and cytokine secretion (8, 9, 11, 12).
Most studies to date have examined the regulation of cytotoxic activity and ligand specificity of Ly-49 molecules (8, 13, 14). However, triggering of both human (6, 7) and mouse (15, 16, 17, 18) NK receptors results in both positive and negative (9, 19, 20) regulation of cytokine gene expression. Because little is known about the variety of genes that may be regulated upon triggering of the mouse Ly-49D receptor, we have examined the breadth of gene expression via microarray analysis following Ly-49D cross-linking and have provided pharmacological analysis of the biochemical pathways that are involved in Ly-49D signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Splenic NK cells were isolated from C57BL/6 mice and were grown for 7–10 days in 1000 IU/ml recombinant IL-2 (Chiron Therapeutics, Emeryville, CA) as previously described (12). Liver NK cells were isolated from IL-2-treated mice as previously described (21). Liver mononuclear cells were either used when fresh or after IL-2 expansion.
Inhibitors
Liver NK cells were incubated for 0.5 h with the concentrations of inhibitors (Calbiochem, La Jolla, CA) as indicated in the manuscript, and, at 6 h, supernatants were assayed for cytokine/chemokine production. Vehicle controls of DMSO or alcohol were used for selected inhibitors that were not soluble in water.
Antibodies
The monoclonal 4E5 (Ly-49D), 4D11 (Ly-49G2), and 24G2 (Fc receptor) were used as previously described (22). Rat IgG (BD PharMingen, San Jose, CA) was used as a control for functional studies and immunoprecipitations. Rabbit F(ab')2 anti-rat IgG was used as a cross-linking reagent. NK1.1-PE, DX-5-PE, and CD3e-PcP (BD PharMingen), as well as 4E5-FITC, were used for flow cytometric analysis.
Flow cytometry analysis
Cells were stained as previously described (12) and analyzed on a FACSort flow cytometer (BD Biosciences, San Jose, CA) and a MoFlo cytometer (Cytomation, Ft. Collins, CO). Cells were directly stained using PE- and FITC-labeled primary Abs or indirectly stained using a primary Ab followed by an isotype-specific FITC- or PE-conjugated secondary Ab or a biotinylated primary Ab followed by streptavidin PerCP (BD Biosciences).
Cytokine measurement
Cytokines were measured using IFN- and chemokine ELISA kits
(R&D Systems, Minneapolis, MN). Cell stimulations were performed at
cell concentrations of 1–5 x 106/ml. Abs
were added at a concentration of 1 µg/106 cells
for 30 min at 4°C. Cells were then washed and plated on 24-well
Costar (Corning, Corning, NY) plates that were precoated with 2
µg/well rabbit F(ab')2 anti-rat IgG and
blocked with medium containing 10% FCS. Unless stated otherwise,
samples were collected after 5–6 h incubation (37°C, 5%
CO2) and were measured in duplicate against the
standard curve of the assay and reported as pg/ml. In all assays, the
SD of the cytokine measurement was <5 pg/ml.
RNA preparation, microarray, and data analysis
Approximately 200 x 106 C57BL/6
splenic NK cells expanded with 1000 U/ml IL-2 were stimulated with
anti-Ly-49D (4E5) or control IgG for 6 h. These expanded NK
cells were 85%
CD3-NK1.1+. In one
experiment, primary liver NK cells were isolated from animals treated
for 3 days with IL-2, as previously described (21).
Poly(A)+ RNA was isolated according to the
manufacturer’s protocol using a FastTrack 2.0 mRNA isolation kit
(Invitrogen, Carlsbad, CA). cDNA generation, hybridization, and data
collection were performed by Incyte Genomics. In brief, alterations in
gene expression were evaluated by reverse transcription of
poly(A)+ RNAs in the presence of Cy3 or Cy5
fluorescent labeling dyes followed by hybridization to a mouse GEM 2
microarray chip. Each chip displays a total of 8734 elements of which
7854 are unique genes/clusters. These unique gene/clusters can be
further defined as 3205 annotated and 4649 unannotated sequences.
Subsets of genes were selected for further study based on differential
Cy3/Cy5 expression ratios that were 
2 in response to Ab-cross-linking
treatment. Differential expression of representative selected genes was
confirmed by RNase protection assay (RPA). Definition of terms for the
gene chip can be found at
http://reagents.incyte.com/support/ gem/unigem_leg.html.
RNase protection assay
The multiprobe RPA was performed using the mCK-1 or mCK-5 probe set (BD PharMingen) or a custom multiprobe set (Torrey Pines Biolabs, La Jolla, CA). Total cellular RNA was extracted using TRIzol (Life Technologies, Gaithersburg, MD), and 5 µg of the total was hybridized with a [33P]UTP-labeled RNA probe (1–1.5 x 106 cpm/sample) prepared according to the manufacturers directions (BD PharMingen) using the PharMingen RiboQuant in vitro transcription kit. Following hybridization, the samples were treated with RNase A and T1 according to the procedure provided by BD PharMingen. The RNase was inactivated and precipitated using a master mixture containing 200 µl RNase inactivation reagent (Ambion, Austin, TX), 50 µl ethanol, 5 µg yeast tRNA, and 1 µl GycoBlue coprecipitate (Ambion) per RNA sample. The samples were mixed well, incubated at -70°C for 30 min, and centrifuged at 14,000 rpm for 15 min at room temperature. The pellets were suspended in 3 µl of BD PharMingen sample buffer and subjected to PAGE as recommended by the manufacturer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine NK cells express Ly-49 receptors that can initiate either inhibitory or activating signals to regulate lytic function. Ly-49A, C, and G2 have been shown to inhibit NK cell function upon recognition of class I ligands on target cells (265, 152, 229, 258, 231, 262). These inhibitory receptors contain ITIMs in their cytoplasmic domains that are phosphorylated upon stimulation leading to the recruitment of SHP-1 and attenuation of intracellular signals (5, 6). However, receptors exist in both systems that can activate NK cells. Murine Ly-49D activates NK cells by inducing reverse Ab-dependent cellular cytotoxicity of FcR+ targets. Upon activation, this molecule associates with and stimulates the rapid phosphorylation of a specific protein, DAP12. This associated phosphoprotein exists as a disulfide-linked dimer that becomes highly tyrosine-phosphorylated upon specific receptor cross-linking. The in vivo function of these receptors is not known, but the activating NK receptors have been shown to be functional in activation of cytotoxicity (5, 6) and cytokine production (20). In addition, Ly-49D was only found on CD3-NK1.1+ cells. Extensive analysis of T cells, including NK T cells, has indicated that the activating Ly-49D is not present on cells other than NK cells (8, 10, 13).
In the present study, we sought to characterize the signals that
are induced upon Ly-49D receptor cross-linking. For our initial assay,
we examined both freshly isolated and IL-2-expanded NK cells (data not
shown) (20) for the kinetics of anti-Ly-49D (4E5)
receptor-induced IFN- gene transcription. In IL-2-expanded NK cells,
IFN- gene expression could be observed by 3 h and became
maximal by 6 h, whereas the kinetics of gene induction in primary
cells was faster, as maximal mRNA expression was observed between 1 and
3 h (data not shown). IL-2-expanded NK cells activated for 6
h were used due to the inability to obtain sufficient poly(A) RNA from
fresh cells. In addition, controls for nonspecific activation of NK
cells by FcR and or other surface Ly-49, as well as specific activation
of cells by F(ab')2 anti-Ly-49D (4E5), were
previously reported (12). In addition, anti-FcR Ab
(24G2) activated when fresh or IL-2-expanded NK cells did not produce
IFN- or chemokines during the 6 h of treatment (Table I
). Although CD16 cross-linking has been
shown to induce cytokines, the contribution of CD16 to the Ly-49D
induction was not evident under the conditions examined. Based on these
results, IL-2-expanded activated NK cells (85%
CD3-NK1.1+) were used to
generate poly(A) mRNA for microarray analysis using the mouse GEM 2
microarray. Results of this experiment, highlighting genes that were
decreased or increased by >2-fold or greater, are shown in Table II. As can be seen in Table II, only a
few genes were significantly decreased (>2 balanced differential
expression). In contrast, as shown in Table I, Ly-49D activation
induced a number of genes that fell into two major categories, e.g.,
cytokine or chemokine genes and apoptosis-related genes. The latter is
not unexpected, because our previous studies have shown that activating
NK receptors can induce apoptosis-mediated events in both human and
mouse NK cells (8, 23). The other genes that were
activated were either regulatory proteins or secretory proteins.
Interestingly, in addition to lymphotactin, four of the six genes most
strongly induced were chemokine genes: MIP1, MIP1, MIP1-C10, and
single C motif (SCM)1. It has been shown previously that NK
cells both respond to and make chemokines, but the induction of
chemokines via activating NK receptors suggests an important role for
NK cells in attracting other immune effector cells upon interaction of
this receptor with its target ligand.
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is the result of a single
analysis. Due to the amount of poly(A) RNA required for analysis and
the cost, it was not possible to perform multiple experiments. However,
verification of gene chip results was performed by subsequent
measurement of mRNA by RPA and cytokine secretion. To verify the
results of the microarray analysis, NK cells from two different
experiments were stimulated with either IgG or 4E5 and plated on plates
coated with anti-rat IgG. These cells were then evaluated for mRNA
expression by RPA, and typical results are shown in Fig. 1. Consistent with the microarray
analysis, MIP1 and MIP1 mRNA but not RANTES and monocyte
chemoattractant protein (MCP)-1 mRNA were induced by activating the
Ly-49D NK receptor. In addition, an increase of TNF- and inducible
protein-10 mRNAs was observed with RPA analysis. As indicated
above, these treatments also induced a strong increase in IFN-
mRNA (data not shown). To further verify that activating NK
receptors could stimulate chemokine expression, a series of experiments
were performed in which fresh NK cells treated with IgG or 4E5 were
placed on plates coated with anti-rat IgG, supernatants collected
after 6 h, and cytokine or chemokine protein production evaluated
using specific capture ELISAs. The summary of four experiments is shown
in Fig. 2. Parallel to that which was
observed in the mRNA analysis, NK cells made significant quantities of
IFN-, MIP1, and MIP1 protein after anti-Ly-49D (4E5)
cross-linking. Chemokines KC and MCP-1 were not produced, whereas low
levels of MIP-2 were made spontaneously but not altered by NK receptor
cross-linking. We were not able to analyze lymphotactin (SCM1) protein
because an ELISA is not commercially available. Thus, in addition to
mRNA, cytokine and chemokine protein expression was strongly increased
upon Ly-49D receptor cross-linking.
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To date, detailed analysis of the biochemical signaling
pathways downstream of Ly-49 activating receptors has not been
reported. Previous studies have demonstrated that Syk kinases
(24) are involved in downstream phosphorylation events
that are mediated through mouse and human activating receptors.
Therefore, we analyzed the effects of a series of pharmacological
inhibitors to identify possible pathways activated by these receptors.
These inhibitors were tested for their effects on cytokine/chemokine
protein expression by inclusion into assays in which fresh NK cells
were cross-linked with anti-Ly-49D (4E5) for 6 h. Inhibitors
were tested at a two-log range above and below the published
IC50. Results are shown in Table III for IFN-, MIP1, and MIP1 and
are derived from three experiments. Values represent the observed
IC50 determined by regression analysis. It is
compared with the published IC50 from a chemical
source. IFN- expression was very sensitive to Src family kinase and
calcium-dependent phosphatase inhibitors as well as
phosphatidylinositol (PI)-3 kinase inhibitors. These results are
all consistent with the tyrosine phosphorylation of the DAP12 molecule
upon Ly-49D cross-linking and subsequent signaling through Syk in a
calcium-dependent pathway. Close analysis of the
IC50s for the different compounds indicated that
the PI-3 kinase inhibitor Ly204002 and Src family inhibitor
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine
(PP2) required significantly higher doses (20- to 100-fold) to
inhibit MIP1 and MIP1 (Table III) than IFN-. These doses were
considered nonspecific, and these results suggest that the pathway(s)
involved in the activation of these genes may be distinct from that of
IFN-. Examination of p38 kinase (SB 203580) and mitogen-activated
protein kinase kinase (PD 98059) inhibitors demonstrated
mitogen-activated protein kinase kinase involvement in Ly-49D
signaling, but not p38 kinase.
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was compared with MIP1
and MIP1 (data not shown), both IFN and MIP were rapidly induced in
the first 3 h. Although this data is consistent with rapid
induction of new mRNA, we wanted to determine whether some of the gene
expression might have been due to the presence of preexisting protein
stored in granules that was then released upon Ly-49D activation. In an
attempt to evaluate this possibility, we inhibited new mRNA synthesis
by pretreatment of the NK cells with actinomycin D and then evaluated
both MIP1 and MIP1 and IFN- production. Both IFN- and
MIP1 production (data not shown) were completely blocked by
actinomycin D, indicating that virtually all gene expression is the
result of new mRNA expression upon receptor cross-linking. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The majority of the mRNAs that were strongly induced as
analyzed by microarray were chemokine genes. RPAs and chemokine protein
production analysis validated the microarray results, as cross-linking
the Ly-49D mouse NK receptor induced high levels of IFN-,
lymphotactin, MIP1, and MIP1. This gene induction was specific
because other chemokines such as KC and MIP-2 were unaffected. Although
NK cells have previously responded to chemokines (25, 26),
our data demonstrates that these cells can be strong producers of these
inflammatory mediators. Although the results presented here indicate
that Ly-49D is a potent inducer of IFN- and chemokines, our findings
do not rule out the possibility that activation through other NK
receptors (e.g. NKG2, FcR, etc.) also results in the induction of these
potent immune modulators.
To gain more in insight into the activation pathways of both cytokine
(IFN-) and chemokine (MIP1, MIP1) gene expression, a series of
pharmacological inhibitors was used to identify the key signaling
pathways involved in the cellular response. Previous studies with
Ly-49D have indicated that membrane proximal tyrosine phosphorylation
of DAP12 was a tysosine kinase-mediated event that leads to calcium
mobilization (20, 24) and was proposed to be mediated by
Src family kinases. This hypothesis was confirmed, as complete
inhibition of IFN- expression was observed with the p72 Syk and Src
kinase family inhibitors, Picannetol and PP2, respectively.
Interestingly, the Src kinase inhibitor PP2 did not completely block
MIP1 and MIP1 production at the reported
IC50 levels. In contrast, protein kinase C,
Ca2+ phosphatase, and calmodulin phosphatase
inhibitors completely blocked both MIP1 and MIP1 as well as
IFN- production at IC50 levels. These data
would suggest that the primary Ly-49D signaling for IFN- production
is predominately mediated through Src kinase pathways, whereas MIP1
and MIP1 gene induction is more complex and may involve multiple
biochemical pathways.
Regardless, these results collectively suggest the primary role for the
activating NK receptors is cytokine and chemokine production. Previous
studies have implicated Ly-49D as a positive regulator of cytotoxicity
of Dd-expressing targets (13, 14).
Although these results are clear, they are difficult to demonstrate
unless high-sorted subsets of NK cells (>95%
Ly-49D+) (13) or transfected
effectors (14) are used. Demonstration of a direct
cytotoxic role for Ly-49D against H-2Dd targets
using unseparated NK cells has been very difficult, and considerable
cytotoxic activity can be demonstrated in Ly-49D-negative NK cells. In
contrast, strong IFN- production has been shown upon target
interaction with unseparated NK cells as well as by receptor
cross-linking in this report and previous (18, 20, 27)
studies. Our current study extends our findings with regard to
chemokine genes (lymphotactin, MIP1, MIP1) and suggests that a
primary function of these activating NK receptors in immune regulation
is one of immunomodulatory factor production. Recent reports of the
phenotype of activating receptor DAP12-knockout mice (28)
and receptor signaling disruption (29) demonstrated that
the cytotoxic phenotype of NK cells was essentially intact. The
DAP12-knockout mice (28) lacked or demonstrated a
significant reduction of Ly-49D expression but exhibited little or no
difference in direct cytotoxicity against a variety of NK targets.
These mice lacking this signaling moiety did demonstrate a lack of
experimental allergic encephalomyelitis (EAE). Several studies have
indicated that chemokines and cytokines can dramatically alter the
extent and character of EAE (30, 31). IFN- has been
implicated as a direct regulator in EAE by acting on T cell
proliferation and directing chemokine production, with profound effects
on the onset and progression of the disease (30). Other
studies (31) have suggested that cytokine and chemokine
expression correlates with the Th1/Th2 paradigm contributing to the
genetic basis of the EAE immune response. Thus, NK cells from the the
DAP12-knockout mice should lose their ability to express IFN- and
chemokines through activating Ly-49. The NK cells from the
DAP12-disruption (29) mice also demonstrated minor effects
on myeloid target killing, and there were profound defects on dendritic
cell migration. These DAP12-disruption mice exhibited a dramatic
accumulation of dendritic cells in mucocutaneous epithelia, associated
with an impaired hapten-specific contact sensitivity. These data
strongly suggest a unique role for DAP12 in innate immunity through the
expression of secreted factors that regulate other hematopoietic cells.
A defect in the initial production of cytokines and chemokines by NK
cells might explain the defect in dendritic cell localization, as it is
well established that macrophage and dendritic cells’ migration is
controlled by chemokine production. DAP12 was shown (32)
to be present in nonlymphoid cells including dendritic cells and
monocytes. More recently (33, 34), DAP12 has been
demonstrated as a signal-regulatory molecule with signal-regulatory
protein 1 and MDL-1, receptors that are commonly found on
either monocytes and/or dendritic cells. These reports suggest that
DAP12 transduce immunoreceptor tyrosine-based activation motif-mediated
activation signals that will regulate monocyte and dendritic cell
functions. Chemokines (35) and chemokine receptors
(36, 37, 38) have been shown to regulate dendritic cell
localization in tissues and to regulate trafficking via the lymph or
blood to lymphoid organs. Gangur and Oppenheim predict
(38) that suppression of chemokines would interrupt the
sequence of signals that culminate in an allergic response.
In addition, in vivo transplantation studies in perforin-null mice have shown that NK-mediated hybrid resistance is intact (39). This result suggests a critical role for the expression of soluble factors by NK cells, as opposed to direct cytotoxicty. Thus, one might conclude that activating NK receptors are more involved in soluble immune regulation than in cytotoxic regulation. Although the inhibitory NK receptors have been shown to strongly regulate both cytotoxic and secretory functions in vitro, their in vivo role has not been thoroughly defined. Thus, we conclude that a primary role for the activating NK receptors in vivo is to trigger soluble factor production and regulation of the immune response at the site of receptor activation. This would place NK cells and their activating Ly-49 receptors as important initiators of microbial immunity and key elements of the innate immune system.
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Footnotes |
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2 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; ITIM, immune receptor tyrosine-based inhibitory motif; SHP-1, Src homology 2 domain-containing protein tyrosine phosphatase; MIP, macrophage-inflammatory protein; RPA, RNase protection assay; EAE, experimental allergic encephalomyelitis; PI, phosphatidylinositol; MCP, monocyte chemoattractant protein; SCM, single C motif.
Received for publication September 28, 2000. Accepted for publication February 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion. J. Immunol. 164:603. and chemokines induce NK cell migration and enhance NK-mediated cytolysis. J. Immunol. 155:3877.[Abstract]
chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production. J. Leukocyte Biol. 59:81.[Abstract]
shapes immune invasion of the central nervous system via regulation of chemokines. J. Immunol. 164:2759.1 is a DAP12-associated activating receptor expressed in myeloid cells. J. Immunol. 164:9.This article has been cited by other articles:
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B. G. Dorner, A. Scheffold, M. S. Rolph, M. B. Huser, S. H. E. Kaufmann, A. Radbruch, I. E. A. Flesch, and R. A. Kroczek MIP-1alpha , MIP-1beta , RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines PNAS, April 30, 2002; 99(9): 6181 - 6186. [Abstract] [Full Text] [PDF]
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