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The Ischemia-Responsive Protein 94 (Irp94) Activates Dendritic Cells through NK Cell Receptor Protein-2/NK Group 2 Member D (NKR-P2/NKG2D) Leading to Their Maturation¹

Centre for Cellular and Molecular Biology, Hyderabad, India

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Tumor recognition and killing, the uptake of released immunogenic substrate, and the generation of immunity are crucial aspects of dendritic cell (DC)-mediated antitumor immune response. In the context of direct tumoricidal activity, we have recently shown NK cell receptor protein-2 (NKR-P2)/NK group 2 member D (NKG2D) as a potent activation receptor on rat DCs. The activation of DCs with agonistic anti-NKR-P2 mAb, the binding of soluble NKR-P2 to the AK-5 tumor, and DC maturation with fixed AK-5 cells led us to identify a putative NKR-P2 ligand on the AK-5 cell surface. In this study we have shown that the AK-5 tumor-derived ischemia-responsive protein-94 (Irp94, a 110 kDa Hsp family member) acts as a functional ligand for NKR-P2 on DCs and enhances Irp94-NKR-P2 interaction-dependent tumor cell apoptosis via NO. Surface expression of Irp94 was also found on tumors of diverse origin in addition to AK-5. Furthermore, the Th1-polarizing cytokine IL-12, produced from Irp94-ligated BMDCs, augments NK cell cytotoxicity. Irp94-NKR-P2 interaction drives the maturation of BMDCs by up-regulating MHC class II, CD86, and CD1a and also induces autologous T cell proliferation, which displays a crucial state of DCs for adaptive antitumor immune response. These functional properties of Irp94 reside in the COOH terminus subdomain but not in the NH₂ terminus ATPase domain of Irp94. We also show the involvement of PI3K, ERK, protein kinase C, phosphatases, and NF-B translocation as downstream mediators of DCs activation upon NKR-P2 ligation with Irp94. Our studies demonstrate for the first time a novel role of a 110-kDa heat shock protein (Irp94) as a ligand for NKR-P2 on DCs, which in turn executes both innate and adaptive immunity.

	Introduction

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Dendritic cells (DCs)³are a unique subset of leukocytes, specialized for Ag processing and presentation (1). DCs are well characterized for their important role in the regulation of T cells and act as an effective arm of antitumor immunity (2, 3). Recent reports have demonstrated better prognosis and antitumor immune response upon DC infiltration in tumors (4, 5, 6). However, direct interaction between DCs and tumor cells has attained greater attention because DCs have also been shown to exert direct cytotoxic action on tumor cells (7, 8, 9).

The cytotoxic activity of human monocyte-derived DCs has been shown to be exerted through TRAIL, Fas ligand, and lymphotoxin-₁β₂ (10). Rat cytotoxic DCs express NK receptor protein 1 (NKR-P) (11) and the human and mouse NK group 2 member D (NKG2D) ortholog NKR-P2 (12, 13). It was recently reported that bone marrow DC (BMDC) cytotoxic activity requires a direct contact with target tumor cells and depends on NO production (14). NKR-P2/NKG2D is a disulfide-linked type II C-type lectin-like receptor expressed also on NK cells, CD4⁺, CD8⁺β, ⁺ T cells (15), rat CD4⁺/CD8⁺ monocytes and macrophages (16), mouse IFN-producing killer DCs (17, 18), and human myeloblastic KG1a cells (19).

NKR-P2/NKG2D interacts with multiple ligands that are usually up-regulated on stressed cells and mounts antitumor immune response. A number of NKR-P2/NKG2D ligands have been identified, including MHC class I-related chain A (MICA) and chain B and UL16-binding protein (ULBP) in humans, Rae1, H60, and murine ULBP-like transcript 1 (MULT1) in mice, and a Rae1 homologue in rat, that exhibit an enigmatic diversity of recognition components for NKG2D. NKG2D ligand-transduced tumor cells are vigorously rejected by syngeneic animals, thus displaying its effective role in antitumor immune response (20, 21, 22). NKG2D interacts with dissimilar ligands by an induced fit mechanism and displays a degenerate recognition system. Biophysical studies have explored H-bonds, hydrophobic interactions, and salt bridges as crucial components that contribute to maintain the overall shape complementarity for the induced fit recognition of NKG2D ligands. NKG2D uses a dimeric surface for ligand binding, which is a characteristic of a C-type lectin-like domain, and associates with multiple adapter units of DAP10/DAP12 to endow high sensitivity (23, 24).

The AK-5 tumor is highly immunogenic rat histiocytoma and regresses spontaneously upon s.c. transplantation in syngenic animals, whereas it kills 100% of hosts upon i.p transplantation (25). Our laboratory has recently shown enhanced cytotoxic action of DCs with an agonistic anti-NKR-P2 mAb and the binding of soluble NKR-P2 (sNKR-P2) on the AK-5 tumor cell surface in a patchy pattern (12, 13), as well as DC maturation with fixed AK-5 cells (26), which prompted us to identify a putative NKR-P2 ligand on AK-5 cell surface.

During our screening for the NKR-P2 ligand on AK-5 cells, we have identified ischemia-responsive protein 94 (Irp94) as an interacting partner for NKR-P2. Irp94 is a unique member of the 110-kDa heat shock protein (Hsp) family and a homologue of mouse APG-2 and human Hsp70RY. Irp94 is >90% identical with APG-2 and Hsp70RY, 60% homologous to other Hsp110 family members, and contains Hsp70 signatures. Irp94 up-regulation is reported in rat brain under ischemic conditions and in endoplasmic reticulum with stress-inducing drugs (27, 28, 29). A recent report has revealed the interaction of mammalian Hsp70 · peptide complex with a NKG2D receptor, which also confirms the Hsp recognition by NKR-P2/NKG2D (30).

Several studies have suggested that Hsps act as danger signals and mount immune response by activating APCs through various receptors (31). At the same time, surface expression of Hsps on tumor cells insight direct interaction between tumor and immune cells (32, 33, 34). Surface Hsp expression on tumor cells also regulates tumor growth and metastasis (35).

In the present study, we have demonstrated NKR-P2 expression on rat immature BMDCs and its up-regulation upon maturation. Irp94 surface expression was confirmed by immunostaining with anti-Irp94 mAb on AK-5 cells and on tumors of diverse origin. Irp94 specifically binds to NKR-P2-expressing DCs, NK cells, and T cells. We have studied the interaction of recombinant Irp94 with NKR-P2 on DCs and have shown that DCs stimulatory capacity resides in the COOH terminus subdomain of Irp94, whereas the NH₂ terminus ATPase subdomain is unable to stimulate DCs. Irp94 specificity to NKR-P2 was assessed as a pull-down product with the extracellular domain of NKR-P2. Specific binding of fluorescent Irp94 with NKR-P2 on DCs also corroborated its specificity and functional capability. When stimulated by an Irp94-NKR-P2 interaction, BMDCs produce NO in a Ca²⁺ dependent manner and induce apoptosis in AK-5 cells. Similarly, Irp94-NKR-P2 stimulation induces cytokine release and maturation of BMDCs, which is crucial for an adaptive immune response. Irp94-NKR-P2-induced signal transduction was investigated with the help of various pharmacological inhibitors that indicated the involvement of MAPKs in signaling pathways. NF-B translocation appears to be a crucial downstream target of Irp94-mediated activation. Thus, Irp94 acts as a novel ligand for NKR-P2 on DCs that is different from the known ligands of NKG2D.

Given the promising role of NKR-P2 in DC-mediated tumor killing, the present study was intended for a better understanding of the tumor recognition component (NKR-P2 ligand) by DCs. Our study demonstrates for the first time the role of a Hsp110 family protein as a functional ligand of a tumor recognition receptor and puts forth its significant role in a DC-mediated antitumor immune response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals and tumors

An inbred colony of Wistar rats of either sex, 5–7 wk of age, were used in this study. AK-5 tumor cells were maintained as ascites by injecting 5 x 10⁶ AK-5 cells i.p. BC-8 (in vitro adapted single cell clone of AK-5 tumor), YAC-1, MCF7, K562, A375, HeLa, J774, P19, and PCC4 tumor cells were maintained in vitro in IMDM supplemented with 10% heat-inactivated FCS. All animal experiments were done following the institutional guidelines and clearance from the animal ethics committee of the Centre for Cellular and Molecular Biology, Hyderabad, India.

Generation and isolation of DCs and NK cells

Bone marrow cells were obtained from the femur of rats. After RBC lysis (hypotonic shock with 0.84% NH₄Cl), bone marrow cells were plated in culture medium (IMDM, 10% heat inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml kanamycin) for 1 h for the removal of resident macrophages. The nonadherent cells were collected and replated in complete medium supplemented with GM-CSF (obtained from GM-CSF-secreting Chinese hamster ovary (CHO) cells; GM-CSF content > 10 ng/ml) and mouse rIL-4 (5 ng/ml; R & D Systems) for 6 days to generate immature DCs. The thus obtained BMDCs were OX-62, CD11c, MHC II, and CD1a positive. Fewer than 2–3% of the attached cells were positive for OX-41 (the signal inhibitory regulatory protein SIRP).

Spleens were removed aseptically from experimental animals and teased in cold PBS. Total splenocytes were obtained by Ficoll-Hypaque density gradient (at 28°C for 20 min), washed, plated for 30 min for macrophage (attached cell) elimination, and the nonadherent cells were collected and the splenic DCs (SDCs) isolated by using magnetic beads (Dynal) coated with OX-62 mAb as per manufacturer’s description; detachment of DCs was performed by a Detach-a-Bead mixture (Dynal). The isolated cells were plated in IMDM-FCS overnight at 37°C in a CO₂ incubator. Using the above-mentioned protocol, we obtained 95% pure DCs as assessed by morphology and phenotypic markers (OX-62, MHC II, and CD86).

Splenocytes from normal rats were used for NK cell isolation with the aid of magnetic beads coated with 3.2.3 mAb as described earlier (22). Beads were washed extensively and the nonadherent NK cells were collected after Detach-a-Bead separation. Splenocytes from normal rats were used for CD8⁺T cell isolation. CD8⁺ T cells were isolated with anti-CD3 followed by anti-CD8 Ab (Serotec) by using Dynal bead-coated Abs. The purity of CD8⁺ T cells was >90% as assessed by anti-CD3 Abs. All of the treatments were performed on day 6 immature adherent BMDCs. For maturation, BMDCs were incubated with LPS (1 µg/ml) for 24 h. Rat PBMCs were isolated from blood (heparinized) by Ficoll density centrifugation.

Reagents and Abs

Hybridoma OX-62 ( integrin) was kindly provided by Dr. M.J. Puklavec (University of Oxford, Oxford, U.K.) and GL-1 (CD86) by Dr. V. Kuchroo (Harvard Medical School, Boston, MA). Anti-mouse IL-12 (clone C17.15; p40 subunit) was kindly provided by Dr. G. Trinchieri (Schering-Plough, Dardilly, France). mAbs 3.2.3 (NKR-P1) and OX-41 (the signal inhibitory regulatory protein SIRP) were from Serotec. Anti-mouse Hsp70 Ab (StressGen) was used to confirm the presence of ectopically expressed Hsp70 on CHO cells. MHC II (OX-6), CD1a (FITC tagged), and mouse inducible NO synthase (iNOS) mAb were from BD Bioscience. A hybridoma-secreting mAb (clone 1A6) against NKR-P2 was raised in our laboratory in BALB/c mice (12). Rabbit anti-mouse NF-B (p65 subunit), polyclonal anti-NKG2Dab, and IB were from Santa Cruz Biotechnology. Anti-rat/-mouse/-rabbit IgG, either Alexa Fluor 488 or Alexa Fluor 594, were used as secondary Abs in accordance with primary Abs. Alexa Abs were purchased from Molecular Probes. W1400 (iNOS inhibitor) and PD98059 were from Calbiochem. LPS (Escherichia coli serotype 0127:B8), wortmannin, Ly294002, okadaic acid, polymyxin B (PMB)-coated agarose beads, and anti-GST Ab were purchased from Sigma-Aldrich. The NF-B specific inhibitory peptides SN50 and SN50M were purchased from BIOMOL International.

cDNA expression library construction and screening

Poly(A)⁺ RNA was isolated from AK-5 tumor cells using TRIzol reagent (Invitrogen Life Technologies). The cDNA expression library from AK-5 cells was constructed using the ZAP-cDNA synthesis kit (Stratagene) as per manufacturer’s protocol. The library was titrated and the appropriate dilution was plated on a Luria-Bertani plate along with the host strain XL1 Blue MRF to obtain well-separated plaques. The plaques were immobilized onto nitrocellulose membrane on prewet with isopropyl β-D-thiogalactoside (Sigma-Aldrich). Replica blots were obtained to maintain negative controls during screening. The screening protocol was based on the far-Western principle wherein the blots were incubated with soluble recombinant NKR-P2-GST fusion protein or with GST alone overnight at 4°C, followed by washing and incubation with anti-GST Ab and the appropriate secondary Ab conjugated with alkaline phosphatase. The signal was developed using a chromogenic substrate, namely NBT/5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich). The blots were aligned with plaques and positive plaques were cored out and used for subsequent secondary and tertiary screening. Finally, individual positive plaques from enriched plates were processed for obtaining the insert encoding the interacting protein. The insert was sequenced from multiple independent plaques and the sequence identity was obtained using BLAST (basic local alignment search tool) software (National Center for Biotechnology Information).

Generation of recombinant Irp94 proteins, anti-Irp94 Abs, and FITC conjugation of Irp94

The recombinant Irp constructs Irp94 full length (1–840), Irp94 COOH terminus (493–840), and Irp94 NH₂ terminus (1–175) were cloned in pET series vectors. The recombinant proteins, namely the COOH terminus subdomain cIrp94 (aa 493–840) and the NH₂ terminus ATPase nIrp94 (aa 1–175) were overexpressed in E. coli BL21 DE3. Full-length Irp94 (aa 1–840) was expressed in Rosetta DE3. Cloned inserts were verified by DNA sequencing. Ni-NTA-agarose was used as the affinity matrix, The cell lysate was prepared in 10 ml of lysis buffer (50 mM Tris-Cl (pH 8), 300 mM NaCl, 10 mM imidazole, 1 µg/ml leupeptin, aprotinin, and pepstatin and 0.15 mM PMSF) and the washing was conducted with 50 mM Tris-Cl (pH 8), 300 mM NaCl, and 20 mM imidazole. Bound proteins were eluted with 50 mM Tris-Cl (pH 8), 300 mM NaCl, and 259 mM imidazole. All the three proteins were expressed as His-tagged fusion proteins and purified by using a Ni-NTA agarose column, and the identity of each protein was confirmed by MALDI-TOF analysis. Extensive washing before protein elution was adopted as an essential practice to diminish the chances of LPS contamination during purification. The protein preparations were treated with PMB-coated agarose beads (Sigma-Aldrich) to remove LPS. cIrp94 was injected s.c. in mice and antisera (polyclonal anti-Irp94) were collected and used for immunostaining. The mAb 2F4 against Irp94 was generated using the recombinant cIrp94 protein. The specificity of mAb 2F4 was checked by the immunostaining of AK-5 cells and with full-length Irp94 (FlIrp94) cDNA-transfected CHO cells. Nontrypsinized, fixed, FlIrp94-transfected and Hsp70 (Hsp70 · C)-transfected (Lipofectamine; Invitrogen Life Technologies) CHO and parental control cells were stained with a NKR-P2-GST fusion protein followed by detection with anti-GST and anti-rat Ig-FITC Abs and the surface staining was analyzed by FACS. Similarly, nontrypsinized full-length NKR-P2 cDNA transfected-CHO cells and parental CHO cells were stained with FITC-cIrp and analyzed by FACS.

cIrp94 was conjugated to FITC as described (36). Briefly, for each milliliter of protein solution (2 mg/ml) 50 µl of FITC dye was added. After 8 h of incubation in dark at 4°C, unbound dye from the conjugate complex was removed by gel filtration (Sephadex G-25). FITC-cIrp94 protein integrity was further checked by SDS-PAGE and was found to be unaffected by FITC-coupling protocol. The molar ratio of absorbance for FITC-cIrp94 was 0.7 (optimal range 0.3–1.4).

GST pull-down assay

The soluble NKR-P2-GST fusion protein was prepared as described earlier (12, 22). The soluble recombinant NKR-P2-GST fusion protein and GST alone were immobilized on glutathione-agarose beads. Soluble recombinant Irp94 protein was incubated with Ni-NTA-agarose in buffer containing 50 mM Tris-Cl (pH 8), 300 mM NaCl, and 20 mM imidazole at 4°C for 4 h. To this complex a NKR-P2-GST fusion protein or GST alone was added in an equimolar ration and incubated for 4 h at 4°C. At the end of incubation, the beads were washed five times with PBS and the bound proteins were released in Laemmli sample buffer (2x), boiled for 5 min, and resolved by electrophoresis on 10% SDS acrylamide gels. The gels were silver-stained to see the pull-down products.

Tumor cell apoptosis assay, immunofluorescence, flow cytometry, and confocal microscopy

Day 6 adherent immature BMDCs were activated with cIrp94 for 6 h in the presence or absence of inhibitors (W1400 or EGTA) and preneutralized cIrp94 (cIrp94:mAb2F4; 2 h at 4°C) followed by washing and coculture with nonadherent tumor targets (BC-8 cells) at a 5:1 ratio. Induction of apoptosis in tumor cells was assessed by flow cytometry after propidium iodide staining of fixed cells Briefly, harvested tumor cells were fixed in 80% methanol for 15 min and washed with PBS. The cells were stained with propidium iodide (0.05 mg/ml propidium iodide in 0.1% sodium citrate, 0.3% Nonidet P-40, and 0.02 mg/ml RNase for 30 min). The viability of effector cells (BMDCs) remained unaffected after coculture as judged by MTT assay.

Cells were fixed in 2% paraformaldehyde for surface staining or with methanol:acetone (1:1) for intracellular staining for 20 min at 4°C followed by incubation with the specific mAbs iNOS, IL-12p40, MHC II, B7-2, and FITC-cIrp94 for 90 min and then washed and treated with the appropriate secondary Ab for 45 min (Alexa). The CD1a mAb was FITC-conjugated. Stained cells were analyzed on a FACS (BD Biosciences). BMDCs were cultured and stimulated on coverslips. After fixation with 2% paraformaldehyde for surface staining and methanol:acetone for intracellular staining, cells were stained with a primary mAb for 90 min followed by an appropriate fluorochrome-conjugated secondary Ab for 45 min. FITC-cIrp94 (1 mg/ml) was incubated with DCs for 90 min followed by washing. Immunostained cells were observed using a confocal laser scanning microscope for mAb1A6, NF-B, and FITC-cIrp94 staining (Carl Zeiss). AK-5 tumor cells were fixed in 2% paraformaldehyde (5 min) for surface staining or with 80% methanol for intracellular staining and probed with mAb2F4 (90 min) and the secondary Ab (30 min) and scanned on a confocal microscope.

Estimation of NO

BMDCs were incubated at 2 x 10⁵ cells/well in 96-well plates along with the indicated quantities of the proteins nIrp94, cIrp94, and FlIrp94 or controls in IMDM (Invitrogen Life Technologies) supplemented with 10% heat inactivated FCS for the indicated times. Cell-free culture supernatants were aspirated after incubation and NO content was measured with Griess reagent. The absorbance at 540 nm was measured using an ELISA reader (SpectraMax 190; Molecular Devices). NO content (micromoles per 2 x 10⁵ cells) was quantified from a standard curve generated using sodium nitrate.

Cytotoxicity assay

The cytotoxicity assay was performed by a 4-h ⁵¹Cr-release assay (as described previously in Ref. 20). DC-activated NK cells were incubated with ⁵¹Cr-labeled YAC-1 cells (E:T = 25:1) for 4 h. The target cells were labeled with 250 µCi of Na₂Cr⁵¹O₄ at 37°C for 45 min with shaking. The cells were washed thrice with PBS to remove the free radioactive label. ⁵¹Cr released in the medium was counted in a Packard gamma counter and the percentage of cytotoxicity was calculated as follows: percentage of cytotoxicity = 100 x (experimental cpm – spontaneous cpm/total cpm – spontaneous cpm).

RT-PCR and Western blotting

Total cellular RNA was isolated using the TRIzol reagent. cDNA was prepared using oligo(dT) 12–18 primer and avian myeloblastosis virus reverse transcriptase (Promega). Semiquantitative RT-PCR was conducted using specific primers for NKR-P2 and iNOS genes along with GAPDH primers. The NKR-2 and iNOS PCR products were analyzed in 1.5% agarose gel and the PCR product was confirmed by Southern hybridization with the corresponding cDNA probes.

BMDCs were stimulated with the cIrp94 for the indicated times and lysed in Laemelli buffer. Lysates were resolved by electrophoresis on 10% SDS-polyacrylamide gels and transferred to nitrocellulose paper by an electroblotting unit. Membranes were blocked with 3% BSA in TBST buffer for 3 h at room temperature. Membrane were incubated with anti-mouse iNOS and IB mAb for 2 h, washed five times, and incubated with the appropriate secondary Ab conjugated to alkaline phosphatase; afterward, the signal was developed by NBT/5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich). The Ponceau S-stained blots were used to ascertain equal loading in all lanes.

Enzyme-linked immunofiltration assay (ELIFA; a cytokine assay)

The IL-12p40 levels in culture supernatants were measured by ELIFA (Pierce). Briefly, a nitrocellulose membrane was cut off and placed in an ELIFA unit where the Ag solution (culture supernatants) was filtered through the nitrocellulose membrane, which trapped the protein specifically, followed by blocking with BSA (3%); the primary Ab (anti-IL-12p40 mAb) was then passed through the membrane. The membrane was washed and incubated with a HRP-tagged secondary Ab. Afterward, the signal was developed using the chromogenic substrate o-phenylenediamine (2 mg/ml in PBS). Afterward, the reaction was stopped by adding 50 µl of 2 N sulfuric acid. The signal was read at 490 nm along with positive control.

MLR assay

For the MLR assay, T lymphocytes were enriched by nylon wool followed by anti-CD3 mAb-coated beads (Dynal). The purity of T cells was >95% as assessed by FACS using anti-CD3 mAb. After 24 h of stimulation with nIrp94, cIrp94, and FlIrp94 proteins, attached BMDCs were washed and cultured at the appropriate ratio with autologous T lymphocytes for 3 days in conjunction with fractions of the proteins that had been used for DC treatment. Proliferation was measured after 3 days. At 18 h before termination, 0.5 µCi of [³H]thymidine was added per well. Cells were harvested and incorporated radioactivity was measured in a beta counter.

Statistical analysis

Student’s t test was performed to analyze the differences between control and experimental groups. Differences were assumed to be significant at p < 0.01.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Expression of Irp94 and its interaction with NKR-P2

To identify the interacting ligand(s) for NKR-P2, the AK-5 cDNA expression library was screened and Irp94 (a 110-kDa Hsp family) was found to be positive. The interaction of Irp94 with a soluble recombinant NKR-P2-GST fusion protein was confirmed in multiple rounds of secondary and tertiary screening with GST alone as negative control. To find out the interacting segment in FlIrp94 (full length: aa 1–840), we generated two deletion constructs, namely cIrp94 (the COOH-terminal subdomain; aa 493–840) and nIrp94 (the NH₂-terminal ATPase domain; aa 1–175) (Fig. 1A). The interaction between Irp94 and NKR-P2 (extracellular domain) was confirmed in an in vitro pull-down assay. An equimolar ratio of soluble recombinant NKR-P2-GST or GST alone was immobilized on glutathione-agarose beads and the interaction with a pure soluble recombinant Irp94-His tag protein was studied. The assay demonstrated specific interaction of NKR-P2 and Irp94 by pulling down Irp94 along with the recombinant extracellular domain of NKR-P2 over the GST and bead alone as negative controls (Fig. 1B). FlIrp94 and cIrp94 stimulated BMDCs, whereas nIrp94 neither interacted nor stimulated BMDCs. The specificity of Irp94 to NKR-P2 was confirmed by staining with sNKR-P2 to ectopically expressed surface Irp94, whereas Hsp70 · C showed little binding (Fig. 1C), and by the binding of FITC-cIrp94 to ectopically expressed surface NKR-P2 (Fig. 1D). Either a patchy localized or a patchy nonuniform staining of Irp94 was observed on the plasma membrane of the AK-5 cell surface with the 2F4 mAb against Irp94 (Fig. 1E). The specificity of the 2F4 mAb was established by the competitive binding of 2F4 mAb on AK-5 cells, which show significantly less binding (78% reduction in mean fluorescence intensity (MFI)) upon preneutralization with recombinant cIrp94 (Fig. 1F). Surface expression of Irp94 was confirmed on AK-5 cells after immunostaining with polyclonal anti-Irp94 Ab and with mAb 2F4, both of which were generated against cIrp94 (Fig. 1G). Surface expression of Irp94 increases significantly upon sublethal heat shock, suggesting a heat-inducible translocation of Irp94 on the plasma membrane (Fig. 1H). The affinity and specificity of recombinant Irp94 for NKR-P2 prompted us to scan a panel of tumor cell lines for the expression of Irp94. Irp94 expression was detected in several tumor cell lines of murine and human origin as seen by the semiquantitative RT-PCR (Fig. 1I). In addition to AK-5 cells, surface expression of Irp94 was also analyzed on a panel of tumor cell lines by live cell staining with mAb 2F4, and most of the tumor cell lines were found to express Irp94; low levels of expression were observed on rat peritoneal macrophages, the rat fibroblast cell line F111, and rat PBMCs, but not on splenocytes and bone marrow cells (Fig. 1 J).

View larger version (46K): [in this window] [in a new window]   FIGURE 1. Expression and interaction of Irp94 with NKR-P2. A, Domain configuration of the expressed cDNA deletion constructs of Irp94. The COOH terminus (C-terminus) subdomain (cIrp94), the NH2 terminus (N-terminus) ATPase domain (nIrp94), and full-length Irp94 (FlIrp94) were used as constructs. cIrp94 and FlIrp94 stimulate DCs, whereas nIrp94 fails to do so. B, Irp94 specifically interacts with the sNKR-P2-GST pull-down assay product of NKR-P2 (total extracellular domain), where Irp94 is pulled down along with NKR-P2 as shown in lane 2. Lanes 1 and 3 represent GST and immobilized agarose beads, respectively. M, Marker. C, Binding of sNKR-P2 to ectopically expressed Irp94. CHO cells (panel 1), CHO transfected with Hsp70 · C (panel 2), or FlIrp94 cDNA (3 ) were stained with sNKR-P2-GST followed by detection with rat anti-GST Ab and FITC-conjugated anti-rat Ig. Filled histogram, secondary Ab; open histogram with dotted line, anti GST Ab; open histogram with bold line, sNKR-P2. Binding intensity (MFI) is represented by numbers along with lines pointing to the relevant histograms. Values from one representative experiment of the two experiments are shown. D, FACS analysis of FITC-cIrp94 binding on ectopically expressed NKR-P2. Live CHO cells (dotted line) or CHO transfected with full length NKR-P2 cDNA (bold line) were incubated with FITC-cIrp94 and analyzed by FACS. Binding intensity (MFI) is represented in numbers along with lines pointing to the relevant histograms. The filled histogram denotes autofluorescence. One representative experiment of the two experiments is shown. E, Surface staining of Irp94 on AK-5 cells. AK-5 plasma membrane was stained with mAb 2F4. Upper left panel, localized surface staining; lower left panel, nonunifrom patchy staining. The nucleus (N) was stained with propidium iodide. The respective transmission pictures are also shown (right panels). F, Specificity of mAb 2F4. Fixed AK-5 cells were incubated with mAb 2F4 (bold line) or with 2F4 preincubated with recombinant cIrp94 (broken line) (50 µg/ml for 2 h at 4°C). Cells were stained with anti-mouse Alexa Fluor 488 and analyzed by FACS. MFI is represented by the numbers along with lines pointing to the histograms. The filled histogram shows the secondary Ab control. G, Expression of Irp94 on AK-5 cells. AK-5 cells were stained with mouse polyclonal and mAb 2F4 against Irp94. Panel 1, live AK-5 cells; panel 2, methanol-fixed AK-5 cells; filled histogram, secondary Ab control; open histogram with dotted line, polyclonal anti-Irp94; open histogram with bold line, mAb 2F4. One representative experiment of three experiments is shown. H, Enhanced surface expression of Irp94 upon sublethal heat shock. BC8 cells were subjected to heat shock (4°C, 41°C, 42°C, and 43°C for 45 min; recovery time, 6 h) and stained with mAb2F4. Filled histogram, secondary Ab control; open histogram, mAb 2F4. Binding intensity (MFI) is represented in numbers along with lines pointing to the relevant histograms. I, Semiquantitative RT-PCR analysis showing expression of Irp94 on AK-5 (rat histiocytoma), A498 (human renal cell carcinoma), A375 (human melanoma), BT549, Colo205 (human adenocarcinoma), Colo357 (human pancreatic tumor cell), DU145 (human prostrate carcinoma), HL60 (human myeloid tumor), LSTRA (murine lymphoma), and OVCAR (human ovarian carcinoma). GAPDH control is also shown. J, Surface expression analysis of Irp94 with mAb 2F4 on cells from various lines: F111 (normal rat fibroblast), peritoneal macrophage, rat splenocytes, rat bone marrow cells, rat PBMCs, BC-8 (single cell clone of AK-5), J774 (murine macrophage tumor cell line), A498 (human renal cell carcinoma), MCF-7 (human breast cancer cell), A375 (human melanoma), YAC-1 (murine lymphoma), K562 (human erythromyeloid leukemic cell line), P19 and PCC4 (mouse embryonal carcinoma), and HeLa (human cervical carcinoma). Cells were stained with mAb 2F4 (open histogram) in the background of secondary Ab staining (filled histogram). One representative experiment of the two experiments is shown.

The isolated pure BMDCs were immature as assessed by dendritic morphology and the quantitation of phenotypic markers like MHC II, CD1a, B7-2, integrin, and CD11c. Upon LPS treatment, significant up-regulation of MHCII, CD86, and CD1a takes place whereas integrin and CD11c expression remain unchanged as assessed by flow cytometry (Fig. 2A). In continuation with our earlier observations, where a concomitant increase in NKR-P2 levels was seen in DCs activated in vitro after coculture with fixed AK-5 tumor cells as well as in DCs (both splenic and axillary lymph nodes) obtained from animals bearing AK-5 tumors (12). We further analyzed NKR-P2 expression on immature and LPS matured BMDCs, and NKR-P2 was found to be up-regulated on LPS-matured BMDCs (Fig. 2B). RT-PCR analysis also correlated well with the increase in NKR-P2 levels upon cIrp94 and LPS treatment on both BMDCs and SDCs (Fig. 2C). This clearly suggests that NKR-P2 is up-regulated upon stimulus with cIrp94 on both splenic and bone marrow derived DCs. However, nIrp94 did not up-regulate NKR-P2 expression on either splenic or bone marrow DCs (data not shown).

View larger version (36K): [in this window] [in a new window]   FIGURE 2. Up-regulation of NKR-P2 on DCs. A, Quantitation of the phenotypic markers MHC II, CD86, CD1a, OX-62 ( integrin), and CD11c. Significant up-regulation of maturation markers (MHC II, CD86, and CD1a) was observed upon LPS treatment on day 6 BMDCs in comparison to untreated cells as detected by FACS analysis. Results shown are representative of more than five similar experiments with different sets of in vitro generated BMDCs. B, Day 6 immature and LPS-matured BMDCs were harvested and the surface expression of NKR-P2 was analyzed on a FACS with mAb 1A6. Shown are secondary Ab control (left panel), day 6 immature (middle panel) and, LPS-matured (right panel) BMDCs. Results shown are representative of three similar experiments with different sets of in vitro generated BMDCs. C, RT-PCR analysis of NKR-P2 expression. NKR-P2 expression increases with the activation signals, viz cIrp94 and LPS. BMDCs are shown in the two upper panels and SDCs in the two lower panels. RNA was isolated after 24 h of stimulation with either cIrp94 or LPS, and amplification of the NKR-P2 transcript was further confirmed by Southern hybridization using NKR-P2 specific probe (data not shown). The GAPDH control is also shown.

To study NKR-P2-Irp94 interaction at the cellular level, cIrp94 was labeled with FITC and the binding of FITC-cIrp94 on DCs was analyzed. FITC-cIrp94 binding was specific to that of NKR-P2 as judged by its binding pattern on immune SDCs (DCs obtained from an AK-5 tumor-bearing rat) where NKR-P2 is expressed as a raft-like clustered pattern, in contrast to SDCs from normal rats where NKR-P2 is expressed all along the cell membrane. This distinct atypical pattern of colocalization of FITC-cIrp94 with NKR-P2 provides an insight into its binding specificity with NKR-P2 on DCs (Fig. 3A). Interaction between surface Irp94 on AK-5 tumor cells and NKR-P2 on DCs was also observed in DCs-AK-5 conjugate staining (data not shown).

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Irp94 binding to NKR-P2 on DCs. A, Confocal analysis of NKR-P2 distribution on naive and immune SDCs and the corresponding binding of FITC-cIrp94 to NKR-P2. FITC-cIrp94 and NKR-P2 colocalizes all along the cell periphery on naive SDCs in contrast to the clustered colocalization on immune SDCs. The upper panels (original magnification x40) show multiple cells and the lower panels show a single cell at a higher original magnification (x100). B, Competitive binding of FITC-cIrp94 to BMDCs. Panels 1 and 3 represent positive controls of FITC-cIrp94-stained BMDCs. Panel 2 represents cells pretreated with mAb 1A6 to block NKR-P2 followed by staining with FITC-cIrp94. Similarly, panel 4 shows cells preblocked with unlabeled recombinant cIrp94 before staining with FITC-cIrp94. In both the cases, significant reduction in FITC-cIrp94 binding is observed. C, Specific binding of FITC-cIrp94 to NK cells and resting CD8+ T cells. Purified NK cells (panel 1) and T cells (panel 2) were stained with FITC-cIrp94 (open histogram with bold line) or preblocked with mAb1A6 and stained with FITC-cIrp94 (open histogram with dotted line). Binding intensity (MFI) is represented in numbers along with lines pointing to the relevant histogram. Filled histogram denotes autofluorescence. One representative experiment of two experiments is shown.

Activation of DCs upon NKR-P2-Irp94 interaction

Because activated BMDCs are known to produce significantly higher amounts of NO as compared with resting DCs, we measured NO production as a measure of iNOS activity to assess DCs activation. Day 6 immature DCs were incubated with increasing concentrations of cIrp94 or with LPS as positive control, which led to NO secretion in a dose-dependent manner as a response to cIrp94-NKR-P2 interaction. The production of NO induced by NKR-P2 ligation or LPS stimulation was significantly inhibited by the reversible inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 200 µM), which acts as a competitive substrate of iNOS (Fig. 4A). Time-dependent NO secretion was measured in response to cIrp94 interaction, and a significant increase was seen at 24 h. After 24 h, the decreased levels of NO could be the result of unstable nitrite due to acidification of the medium (Fig. 4B). However, NO production was close to that of unstimulated BMDCs with BSA at similar protein concentrations.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Binding of Irp94 to DCs leads to NO secretion. A, Dose-dependent production of NO with cIrp94 (50–200 µg/ml) and LPS (0.5–5 µg/ml). Production of NO was significantly reduced in the presence of L-NAME (200 µM) as the competitive substrate of iNOS for both cIrp94 and LPS. LPS-mediated activation and its inhibition with L-NAME were used as experimental positive control. Results shown are representative of three similar experiments. B, BMDCs were incubated with 100 µg/ml BSA, cIrp94, and 1 µg/ml LPS for different time periods and NO content was measured. Maximum NO production was achieved at 24 h of incubation. BSA had minimal effect on NO production. C, NO production with different domains of Irp94 (100 µg/ml). nIrp94, cIrp94, and FlIrp94 recombinant proteins were incubated with BMDCs and the NO content was measured after 24 h. The nIrp94 protein failed to activate BMDCs, whereas significant release of NO was observed with cIrp94 and FlIrp94. Results shown are representative of three similar experiments. D, NO release in BMDCs cocultured with FlIrp94-transfected CHO cells. BMDCs were cocultured with paraformaldehyde-fixed, FlIrp94-transfected and parental CHO cells for 24 h at a 25:1 ratio. NO content was measured in the cell-free culture supernatants. E, Irp94 activates BMDCs via NKR-P2/NKG2D. Control BMDCs or NKR-P2/NKG2D preblocked BMDCs (with anti-NKG2D Ab) were incubated with cIrp94 for 24 h along with the relevant control and NO content was measured in the culture supernatant. The stimulatory action (agonistic nature) of anti-NKR-P2 mAb1A6 is also shown in the split bar. The data are representative of three similar experiments. F, RT-PCR analysis of the iNOS transcript upon BMDCs treatment with cIrp94 or LPS. Up-regulation of iNOS transcript was observed with cIrp94 and LPS-stimulated DCs. GAPDH bands represent loading control (Con). G, cIrp94 induced expression of iNOS protein in BMDCs after 24 h treatment with either cIrp94 or LPS. iNOS expression was studied by immunoblotting using anti-mouse iNOS mAb. H, Intracellular iNOS levels were analyzed after 24 h treatment with cIrp94 or LPS on day 6 BMDCs, followed by analysis on FACS.

To corroborate the specific activating nature of Irp94 during cellular contact, we transfected Fl-Irp94 in CHO cells and confirmed the surface expression of Irp94 with mAb 2F4 staining (data not shown). Furthermore we cocultured fixed FlIrp94-transfected CHO cells (CHO:FlIrp94) and untransfected CHO cells as control (CHO/CHO:Irp94:BMDCs) with BMDCs at a 25:1 ratio. As a result of cellular contact, a 3-fold increase in the released NO was found in a cell-free supernatant in comparison to parental CHO-induced BMDCs. A slight increase in NO levels in control CHO coculture was also seen that may be due to some unknown interactions between the two distinct cell types (Fig. 4D). To explore the stimulatory specificity of Irp94 through NKR-P2/NKG2D, we preblocked NKR-P2/NKG2D with an anti-NKG2D polyclonal Ab and measured NO content in the culture supernatant. The stimulatory capacity of cIrp94 was significantly inhibited upon NKG2D preblocking (100 µg/ml) as measured by NO content after 24 h. However, the agonistic anti-NKR-P2 mAb could not be used as preblocking reagent for NKR-P2 at similar concentration (Fig. 4E). iNOS status in Irp94-stimulated BMDCs was also checked by looking at iNOS transcript levels by RT-PCR (Fig. 4F) and iNOS protein by immunoblotting and FACS (Fig. 4, G and H). The iNOS mRNA and protein levels were increased significantly upon cIrp94 interaction. Additionally, NO secretion was also observed upon the coculture of Irp94-positive fixed tumor cells with BMDCs, and there was an increase in the production of TNF- as well by cIrp94-activated BMDCs as assessed by L929 bioassay (data not shown).

To exclude endotoxin contamination as a cause of responsiveness to cIrp94 and other proteins, we performed a Limulus amebocyte lysate (BioWhittaker) assay and the detectable endotoxin content was found to be <0.006 U/mg of Irp94 preparations, which is within the sensitivity limits of the assay (0.125 endotoxin units). PMB is a peptide antibiotic that neutralizes LPS action by chemical coupling to lipid A (37). Heat denaturation abolished the cIrp94-activating ability of BMDCs, whereas it had negligible effect on PMB neutralization, which was used to ensure inhibition of the lipid A-mediated action of LPS. In parallel assays, LPS activity was completely abolished with PMB neutralization, whereas LPS activity was consistent upon heat denaturation (Fig. 5). These observations, when put together, completely rule out the possibility of LPS contamination in our experiments.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. NO production by BMDCs stimulated with Irp94 is devoid of LPS contamination. NO production of BMDCs stimulated with cIrp94 is shown. BMDCs were stimulated with cIrp94, BSA, and LPS in different forms. Native recombinant cIrp94, cIrp94 in the presence of 50 µg/ml PMB, and heat-denatured cIrp94 (100°C for 10 min) were the three separate forms. Heat-denatured cIrp94 failed to activate BMDCs whereas PMB-coupled cIrp94 retained its functional properties while heat inactivation did not harm LPS effectiveness, which was significantly brought down with PMB-neutralized LPS. BSA treatment for 6 h did not show any activation. Represented histograms are indicative of >10 separate experiments, with different batches of purified Irp94 throughout the study.

Our earlier observations on DC activation with the agonistic anti-NKR-P2 mAb 1A6 and with fixed AK-5 cells (12, 26) prompted us to evaluate the Irp94-induced cytotoxic action of BMDCs against tumor. Immature BMDCs induced apoptosis in BC-8 tumor targets when cocultured for 24 h. Pretreatment of immature BMDCs with cIrp94 enhanced the killing of tumor cells, which strengthens our earlier observation that NKR-P2 acts as an activation receptor and that the identified Irp94 is its ligand present on the tumor cell surface, which mediates a basal level of tumor cell killing even with uninduced BMDCs. However, under similar conditions nIrp94 could not augment tumor cell killing (data not shown). We have measured the induction of apoptosis in BC8 cells under different conditions. cIrp94 treatment of BMDCs caused a 3-fold increase in apoptosis, while cIrp94 treatment in conjunction with W1400 (iNOS specific inhibitor; 5 µM) or EGTA (Ca²⁺ chelator; 2 mM) resulted in a significant reduction of apoptosis. Preneutralization of cIrp94 with mAb 2F4 also resulted in the inhibition of apoptosis (Fig. 6A), whereas mAb 2F4 itself did not induce apoptosis in BC8 cells. A parallel and comparable effect of these inhibitors, viz W1400, EGTA, and mAb 2F4, was visible as an inhibition of NO release in the case of cIrp94-activated BMDCs (Fig. 6B). These observations are highly suggestive of the fact that Ca²⁺ mobilization modulates NO secretion upon NKR-P2-Irp94 interaction and, as a consequence, regulates the effector function of BMDCs.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. cIrp94-activated BMDCs induce apoptosis in BC8 cells. A, BMDCs were activated with cIrp94 for 6 h and cocultured with BC-8 cells at a 5:1 ratio (E:T) for 24 h. In a parallel experiment, BMDCs were pretreated with W1400 (5 µM for 1 h), EGTA (2 mM for 30 min), or mAb 2F4 neutralized cIrp94 (200 µg for 2 h at 4°C) and cocultured with BC-8 tumor targets. The percentages of apoptotic cells were scored by flow cytometry after staining with propidium iodide. B, BMDCs were stimulated with cIrp94 in conjunction with W1400 (5 µM for 1 h), EGTA (2 mM for 30 min), or mAb 2F4-neutralized cIrp94 mAb 2F4 (200 µg for 2 h at 4°C) and NO content was estimated in the cell-free culture supernatants after 24 h. The data are representative of three similar experiments.

We first investigated whether BMDCs produce IL-12 upon stimulation with cIrp94. Intracellular staining for IL-12p40 showed increased levels of the cytokine in cIrp94-treated BMDCs, which was also observed with LPS (Fig. 7A). A much more significant increase (5-fold) in IL-12p40 levels was detected in the supernatants of cIrp94- and FlIrp94-stimulated BMDCs, whereas BMDCs treated with nIrp94 did not show an increase in IL-12p40 production, thereby supporting our conclusions regarding the nonfunctional nature of nIrp94 (Fig. 7B). To check whether IL-12 produced by activated BMDCs could augment the cytotoxic potential of NK cells, we performed a 4-h ⁵¹Cr-release assay with NK cells cocultured with cIrp94-pulsed DCs against YAC-1 cells at a 20:1 ratio. In the presence of cIrp94-activated DC, NK cell cytotoxicity against YAC-1 showed 4-fold increase that was inhibited by neutralizing with an anti-IL-12 mAb. However, neither untreated nor cIrp94-induced BMDCs showed any significant cytotoxicity against YAC-1 in a 4-h ⁵¹Cr release assay (Fig. 7C).

View larger version (27K): [in this window] [in a new window]   FIGURE 7. Activation of naive NK cells by cIrp94-treated BMDCs is mediated through IL-12. A, Intracellular IL-12p40 staining of cIrp94- and LPS-activated BMDCs after 24 h. Enhanced IL-12p40 levels are seen in cIrp94- and LPS-treated BMDCs. B, IL-12 levels in the control-, nIrp94-, cIrp94-, FlIrp94-, and LPS-treated BMDC culture supernatants were estimated by ELIFA. C, Role of IL-12 secreted by cIrp94-stimulated BMDCs on NK cell activation. IL-12 produced by cIrp94-stimulated BMDCs was neutralized by anti-IL12 mAb and its effect on NK cell cytotoxicity against YAC-1 was estimated in a 4-h 51Cr-release assay. The data are representative of three similar experiments.

The effect of cIrp94 on the expression of BMDC maturation markers like MHCII, B7-2, and CD1a was examined, as these are important for Ag presentation by DCs. After 24 h of induction with cIrp94, the surface expression of the maturation markers increased significantly in comparison to LPS, which was used as a positive control (Fig. 8A). However, BSA and nIrp94 did not influence the maturation status of DCs under similar conditions (data not shown). The functional significance of Irp94-mediated DC maturation was assayed in a MLR assay. When compared with medium and nIrp94 as control, BMDCs pulsed with cIrp94 and FlIrp94 for 3 days induced significant autologous T cell proliferation (Fig. 8B). The stimulatory capacity of cIrp94 was significantly blocked by preneutralization with anti-Irp94mAb 2F4 (Fig. 8C).

View larger version (40K): [in this window] [in a new window]   FIGURE 8. Maturation of BMDCs after treatment with Irp94. A, DCs maturation markers like MHC II, CD86, and CD1a were analyzed after 24 h of culture in presence of cIrp94 (100 µg/ml) and LPS (1 µg/ml) using specific Abs. Positive cells were analyzed by flow cytometry. B, DCs were incubated with different parts of Irp94 for 24 h. Cells were incubated with autologous T cells at different (T cell:BMDC) ratios for 3 days and in conjunction with Irp94 fractions for another 3 days. Eighteen hours before harvesting, [3H]thymidine (1 µCi/well) was added. Cells were harvested and washed and the incorporated radioactivity was counted. C, cIrp94 and preneutralized cIrp94 (cIrp94:mAb 2F4) were used as stimulators in the MLR assay described above and cell proliferation was measured. The data shown is representative of three similar experiments.

Irp-94/NKR-P2 mediated translocation of NF-B to nucleus and regulation of signaling

To investigate the signaling mechanism triggered by cIrp94 ligation on BMDCs, we used several inhibitors of signaling cascade. BMDCs were pretreated with the inhibitors before cIrp94 treatment and the resulting effect on DCs was read out as their NO-secreting capacity. The results obtained show involvement of PI3K (wortmannin and Ly294002), ERK (PD98059), serine/threonine phosphatase (okadaic acid), protein kinase C (H7), and protein tyrosine kinase (genistein) as the specific mediators of cIrp94-mediated BMDC activation (Fig. 9A). NF-B is activated in the early stages of iNOS induction and the Ag-presentation function of DCs. iNOS, being a well-known target of NF-B, prompted us to check the activation status of NF-B upon cIrp94-NKR-P2 interaction. The NF p65 molecule translocated to the nucleus within 2 h after the activation of BMDCs with cIrp94 and LPS, as recorded by confocal microscopy (Fig. 9B). The NF-B inhibitory peptide SN50 significantly reduced the Irp94-induced NO production by BMDCs, which suggests the involvement of the NF-Bp50 subunit in the stimulation (Fig. 9C). Degradation of IB was also observed in a time-dependent manner with cIrp94 stimulation, thereby confirming the involvement of NF-B in Irp94-NKR-P2 interaction-dependent BMDC activation (Fig. 9D).

View larger version (33K): [in this window] [in a new window]   FIGURE 9. NKR-P2 mediated regulation of signal transduction. A, Effect of protein kinase and phosphatase inhibitors on NO production by cIrp94-treated BMDCs. BMDCs were incubated for 24 h with 10 µM H7, 2 µM wortmannin (Worta.), 25 µM PD98059, 10 µM genistein, 10 µM okadaic acid (O.acid), or 20 µM Ly294002 in the presence or absence of 100 µg/ml cIrp94. NO content was measured in culture supernatants. Results shown are representative of three similar experiments. B, cIrp94 induced translocation of NF-B to the nucleus. DCs were induced with cIrp94 on coverslips for 2 h, fixed, and permeabilized with methanol:acetone (1:1) for 30 min. Washed cells were then incubated with anti-NF-B Ab. Cells were stained with a FITC-conjugated secondary Ab. The nucleus was located by staining with propidium iodide. Translocation of NF-B to nucleus was evident in the nuclei of stimulated cells, suggesting the translocation of NF-B to the nucleus (original magnification 60x). C, Inhibition of Irp94-induced NO production by SN50 (NF-B inhibitory peptide). BMDCs were preincubated with SN50 (active) and SN50M (inactive control) for 2 h, followed by treatment with cIrp94 for 24 h. NO content in the supernatant was measured by Griess reagent. D, Degradation of IB in BMDCs treated with cIrp94. BMDCs (106cells/well) were induced with cIrp94 for the indicated time periods. Cells were lysed and immunoblotted with a rabbit anti-mouse IB Ab. The Ponceau S blot is also shown for equal loading of proteins.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The NKG2D-ligand system has been assessed as a multifunctional skill to activate the innate immune system in response to tumor- and virus-infected cells. NKG2D ligand nonredundancy has been observed in intraspecies conditions such as MICA/B, which was initially identified for human NKG2D and has striking dissimilarity with mouse Rae1β, H60, and MULT1. Among themselves, Rae1β, H60, and MULT1 show only 30% similarity in amino acid sequence but are completely different from MICA/B. However, the human UL16 binding protein ULBP, which was identified using a different approach, has very little similarity with mouse NKG2D ligands (20). NKR-P2/NKG2D is a unique member of the C- type lectin-like receptor family and binds its ligands with variable affinities by dimeric surface. The dimeric surface is rich in basic amino acids and contributes to the recognition of dissimilar ligands. However, overall shape complementarity is an imperative factor for ligand recognition (23). Thus, NKG2D-ligand system works in a broad range of diversity but mounts an efficient immune response. Recently the instrumental role of the Hsp70 · peptide complex in NKG2D-mediated IFN- secretion and its active role in the induction of tolerance against experimental autoimmune encephalomyelitis was demonstrated (41). Our observations about a 110-kDa Hsp working as a NKR-P2 ligand on rat DCs provides the evidence of additional diversity for an efficient tumor recognition system that is also supported by the binding of the Hsp70 · peptide complex to NKG2D. A significant binding of NKG2D to a fluorescent Hsp70 · peptide complex has been found that contained HSPA1a, Hsp70 protein 8, Grp78, and other Hsp70 members (30). We have also observed a low binding of sNKR-P2 to a membrane Hsp70 · C-transfected CHO cell. Taken together, these observations confer a broad range of Hsp recognition by NKR-P2/NKG2D. Although a wide range of "MHC-I-like" ligands to NKG2D is known (23), further structural studies are needed to reveal the exact mode of Hsp binding to NKG2D/NKRP2. Heat shock transcription elements regulate expression of Hsp; however, MICA/B up-regulation in many tumors is also thought to be the consequence of activation of a heat shock transcription element in the promoters of corresponding genes, an event known to accompany transformation (42). Thus, the regulation of human NKG2D and rat NKR-P2 ligands with heat shock transcription elements supports a common regulatory system.

Hsps perform essential functions in the cellular physiology and are usually up-regulated under stressed conditions. Although Hsps do not possess a membrane anchorage domain and their membrane translocation process is not very well understood, many of the Hsps have been demonstrated to be present on the tumor cell surface.

A recent study provides insight into the involvement of endolysosome in Hsp70 membrane transport (43); however, we could not find the increased lysosomal-associated membrane protein 1 (LAMP-1) expression on the AK-5 tumor surface, suggesting a distinct mechanism for its transport to the membrane. Expression of surface gp96 has been found to be evolutionarily conserved on various tumors, and highly immunogenic tumors express more of gp96 on cell surface (44). Hsp72 surface expression on stressed tumor cells also augments the cytotoxicity of NK cells (45). Recent reports provide evidence of the surface expression of Hsp90 on B cell lymphomas upon EBV infection (46), the expression of Hsp60 on stressed endothelial cells (47), and the expression of Hsp72 on neutrophils by LPS (48), suggesting the surface expression of Hsps under stressed conditions. Hsps can elicit immune responses if expressed on plasma membrane (35). In the transgenic model, enforced surface Hsp expression leads to autoimmune disorder (49). The surface expression of Irp94 by many tumor cell lines, but not by normal cells, also suggests that cellular stress induces up-regulation of Irp94. These findings support our observations regarding surface expression of Irp94 on tumor cells, which increases upon heat shock on AK-5 cells. During necrotic and apoptotic tumor cell death, released exogenous Hsps and Hsp-immunogenic peptide complexes stimulate APCs through the CD91 receptor and initiate an antitumor immune response (50). Similarly, TLR2, TLR4 (51), CD40 (52), CCR5 (53), and CD316 (54) act as receptors for exogenous Hsps and function as Th1-polarizing agents. However, Hsp binding and stimulation through NKG2D/NKRP2 display a novel mode of action in contrast to that of distinct pattern recognition receptors. Higher Hsp110 has been shown to act as a "danger signal" to DCs (55), and Hsp105/Hsp110-pulsed BMDCs induce the regression of intestinal adenomas in vivo (56). Recently, it was reported that some member of the "Hsp70-superfamily" binds to scavenger receptors extracellularly (57). However, scavenger receptors negatively regulate antitumor immunity (58).

Our data suggest that the stimulatory capacity of Irp94 resides in the COOH-terminal subdomain and that the ATPase domain is not necessary for Irp94 function. Similarly, the stimulating epitope of microbial Hsp70 resides in the base of the COOH terminus domain and activates DCs through the CD40 receptor and the adjuvant function of Hsp70 on human monocyte-derived DCs (59, 60). Irp94 contains the Hsp70 signature, which is broadly conserved for various Hsp110 and Hsp70 family members (27). We have previously shown AK-5 cells to be deficient in mounting a heat shock response and therefore they undergo apoptosis (61). Moreover, AK-5 cells do not express Hsp70 on the cell surface (data not shown). Hence, in AK-5 Irp94 functions exclusively as the interacting partner for NKR-P2.

The evidence that cIrp94 is a specific binding partner of NKR-P2 comes from four different sets of observations. First, the GST pull-down assay clearly shows the binding of NKR-P2 with Irp94, where Irp94 is pulled down along with extracellular domain of NKR-P2. Second, there is specific binding of sNKR-P2 to ectopically expressed surface Irp94 and binding of FITC-cIrp94 to ectopically expressed NKR-P2. Third, FITC-conjugated cIrp94 colocalizes with NKR-P2, displaying a similar expression pattern as that of NKR-P2 on immune SDCs, where both are seen to be concentrated at same point(s) on the membrane as compared with naive splenic DC, where colocalization was observed all along the periphery. Fourth, in a competitive binding assay a marked inhibition of FITC-cIrp94 binding was observed upon the preblocking of NKR-P2 with mAb1A6 and untagged Irp94 on BMDCs. A similar inhibition of FITC-cIrp94 binding was also observed on NK and resting T cells upon NKR-P2 preblocking. These independent observations clearly prove the binding specificity of Irp94 for NKR-P2.

We have demonstrated the interaction of Irp94 with NKR-P2 on DCs, which activates them to secrete L-arginine and Ca²⁺-dependent NO that in turn induces apoptosis in tumor targets. NO is a well-known tumoricidal molecule that executes tumor cell killing by the down-regulation of cyclin D1, the inhibition of vital enzymes essential for tumor growth, and the activation of caspases (62). In vitro, iNOS induction in DCs has been shown with Hsps, IFN-, and endotoxins and upon CD40 ligation (63, 64). Enhanced NO production is also reported in mouse thymic DCs in response to self-antigens and alloantigens (65). NO production has also been documented under in vivo conditions (66, 67, 68), whereas a subset of DCs encounters bacterial infections in murine spleen through NO (69). Mouse BMDCs have also been shown to perform tumoricidal action through NO, but no specific surface interaction was defined in these studies (70). The production of NO by DCs after interaction with ectopically expressed Irp94 on CHO cells again confirms its efficiency during cellular contact, because Irp94-NKR-P2-mediated DC activation involves the interaction of surface proteins and killing by soluble NO. To rule out the susceptibility of DCs during coculture, we performed an MTT assay on effector BMDCs, which showed a consistent viability of DCs during coculture (data not shown).

DCs have been shown to produce the Th1-polarizing cytokine IL-12 in addition to IL-1β, IFN-, and IL-18, whereas IL-10 and IL-4 production are documented during tolerogenic stages (71). We also observed increased IL-12 levels intracellularly and in culture supernatants from cIpr94-activated BMDCs. In an AK-5 tumor regression model, IL-12-activated NK cells have been identified as the major effector cells (72). Hence, we checked whether cIrp94-activated BMDCs could augment the cytotoxic action of naive NK cells. We observed DC-mediated activation of NK cells through IL-12, thereby supporting our earlier findings of DC activation with fixed AK-5 cells in vitro and in vivo (26) and the anti-tumor cross-talk between DCs and NK cells (73). This crucial activity of Irp94 suggests that it may enhance innate immune response or break immune tolerance status in tumors that regress spontaneously.

In various studies, tumor-infiltrating mature DCs have been found to be associated with the regression of the tumor (4). We also observed up-regulation of MHC-II, B7-2, and CD1a as phenotypic maturation markers upon treatment of DCs with Irp94, which correlates very well with our earlier findings of DC maturation with fixed AK-5 cells (26) and with the surrogate ligand mAb1A6 (data not shown). In autologous MLR-assays, Irp94-treated BMDCs exhibit enhanced T cell proliferative capacity. This phenotypic and functional maturation through NKR-P2 displays the crucial adaptive immune response of BMDCs. Earlier, DC-mediated priming of the tumor-specific T cell immune response has been shown with complex assortments of tumor Ags; however, a recent approach of targeting the NKG2D ligand (MICA) for generating T cell-specific immune responses through DCs supports our findings and demonstrates the importance of the NKG2D ligand in DC-mediated immunotherapy (74). Hsps have been shown to induce NO production and DC maturation in vitro (75), and recently iNOS has been found to regulate the DC maturation process by inhibiting the caspase-like activity of immature DCs (76). Hence, Irp94-mediated iNOS induction and the maturation of BMDCs appear to be a concerted action and are assumed to govern both innate and adaptive antitumor immune responses.

Because NO is a crucial effector of DC-mediated cytotoxicity, the signal transduction mechanism of Irp94-mediated NO production was investigated. The results from the studies with pharmacological inhibitors demonstrated that NO production with Irp94-NKR-P2 interaction is regulated by PI3K, tyrosine kinase, ERK kinase, serine threonine protein phosphatase, and protein kinase C. These observations suggest the involvement of a MAPK pathway in NKR-P2-mediated DC activation. Similar pathways seem to operate in DCs for iNOS induction (77) and with Hsp60 interaction in DCs (78). However, it will be interesting to investigate the accurate signal transduction cascade upon NKR-P2 ligation with Irp94.

NF-B transcription factor (p50-p65) is the prototype of a family of homodimeric and heterodimeric protein complexes comprised of subunits related to the c-rel protooncogene. NF-B activation plays an important role in innate and adaptive immunity (79) and has been found to execute iNOS induction with Hsps (80, 81). Cytotoxic NO production after Irp94 treatment represents the strong induction of iNOS, which is reported to be regulated by NF-B activation. Furthermore, NF-B acts as a potential transcription factor in the initiation of MHC-II, CD86, and CD80 up-regulation, as well as in IL-12 and TNF- production (82). We have observed the translocation of NF-B to the nucleus after 2 h of treatment with Irp94. cIrp94-treated BMDCs show a strong translocation as compared with LPS in the nucleus, which suggests the early activation of NF-B with NKR-P2 ligation. NF-B activation takes place due to the phosphorylation and degradation of IB in the cytoplasm (77); data on cIrp94-mediated degradation of IB also support the involvement of NF-B activation during Irp94-NKR-P2 interaction. Thus, our data suggest NF-B to be a key executioner of Irp94-mediated activation of BMDCs. Earlier NF-B activation and maturation are reported for exogenously released Hsps from necrotic tumor cells (75). In the present study, we show the efficiency of surface Hsp to induce similar cascade in BMDCs.

Our results demonstrates the role of a surface Hsp in eliciting DC-mediated immune responses through NKR-P2/NKG2D. Surface Hsp uses multiple rationales to activate immune cells and also induces activation of DCs (35, 83). A strong correlation between Hsp surface expression and tumor immunogenicity has been reported (84). Moreover surface Hsps also regulate the immunogenicity of cancer cell death (85).

Thus, our findings have important implications in host resistance against tumors. Our data highlight the novel expression of Irp94 on the tumor cell surface and elucidate the mechanism of Hsp-mediated tumor recognition through DCs. Also, our data demonstrate the maturation of DCs through the interaction between Hsp and NKR-P2, which leads to the generation of adaptive immune response in DCs. Although many cytotoxic subsets of DCs have been described in the literature, our findings suggest the common cytotoxic action of DCs through a tumor recognition receptor, because bone marrow precursors generate both lymphoid and myeloid subsets of DCs. NKR-P2 function on DCs establishes a significant link between adaptive and innate immunity through the tumor recognition receptor. However, the detailed operating signal cascade, which governs the activation and maturation status of DCs after NKR-P2 ligation, needs to be worked out.

	Acknowledgments

 
We thank Drs. M. J. Puklavec and Vijay Kuchroo for providing the hybridomas OX-62 and B7.2, respectively. We also thank Dr. Eric Dissen and Dr. Y. Yagita for providing the NKR-P2 and Irp94 cDNA probes, respectively. We thank B. Savithri for the initial study of the work. We also thank Dr. Cecilla Melani for providing the pSignalHsp70 · C construct. We thank A. Leela Kumari, A. Mubarak Ali, B. V. V. Pardhasaradhi, N. Rangaraj, and N. Dwarakanath for the technical help. We thank T. Hemalatha for secretarial help.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by a grant from Department of Biotechnology, Government of India. R.M.S. is a recipient of Council for Scientific and Industrial Research fellowships.

² Address correspondence and reprint requests to Dr. Ashok Khar, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, AP 500007, India. E-mail address: khar{at}ccmb.res.in

³ Abbreviations used in this paper: DC, dendritic cell; BMDC, bone marrow-derived DC; CHO, Chinese hamster ovary; cIrp94, COOH terminus subdomain Irp94; ELIFA, enzyme-linked immunofiltration assay; FlIrp94, full-length Irp94; Hsp, heat shock protein; iNOS, inducible NO synthase; Irp94, ischemia-responsive protein 94; L-NAME, N^G-nitro-L-arginine methyl ester; MFI, mean fluorescence intensity; MICA/B, MHC class I-related chain A or chain B; MULT1, murine UL16-binding protein-like transcript 1; nIrp94, NH₂ terminus ATPase Irp94; NKG2D, NK group 2 member D; PMB, polymyxin B; sNKR-P2, soluble NKR-P2; NKR-P1 or -2, NK cell receptor protein-1 or -2; SDC, splenic DC.

Received for publication February 8, 2007. Accepted for publication November 8, 2007.

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