GRP94/gp96 Elicits ERK Activation in Murine Macrophages
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m305480200
ISSN1083-351X
AutoresRobyn C. Reed, Brent Berwin, Jeffrey P. Baker, Christopher V. Nicchitta,
Tópico(s)ATP Synthase and ATPases Research
ResumoVaccination of mice with GRP94/gp96, the endoplasmic reticulum Hsp90, elicits a variety of immune responses sufficient for tumor rejection and the suppression of metastatic tumor progression. Macrophages are a prominent GRP94/gp96 target, with GRP94/gp96 reported to activate macrophage NF-κB signaling and nitric oxide production, as well as the MAP kinase p38, JNK, and ERK signaling cascades. However, recent studies report that heat shock protein elicited macrophage activation is due, in large part, to contaminating endotoxin. To examine the generality of this finding, we have investigated the role of endotoxin in GRP94/gp96-elicited macrophage activation. We report that GRP94/gp96 binds endotoxin in a high-affinity, saturable, and specific manner. Low endotoxin calreticulin and GRP94/gp96 were purified, the latter using a novel method of depyrogenation; this resulted in GRP94/gp96 and calreticulin preparations with endotoxin levels substantially lower than those of previously reported preparations. Low endotoxin GRP94/gp96 retained its native conformation, ligand binding activity, and in vitro chaperone function, yet did not activate macrophage NF-κB signaling, nitric oxide production or inducible nitric-oxide synthase production. Low endotoxin GRP94/gp96 and calreticulin did, however, elicit a marked increase in ERK phosphorylation at protein concentrations as low as 2 μg/ml. These results are discussed with respect to current understanding of the contributions of endotoxin and heat shock/chaperone proteins to the stimulation of innate immune responses. Vaccination of mice with GRP94/gp96, the endoplasmic reticulum Hsp90, elicits a variety of immune responses sufficient for tumor rejection and the suppression of metastatic tumor progression. Macrophages are a prominent GRP94/gp96 target, with GRP94/gp96 reported to activate macrophage NF-κB signaling and nitric oxide production, as well as the MAP kinase p38, JNK, and ERK signaling cascades. However, recent studies report that heat shock protein elicited macrophage activation is due, in large part, to contaminating endotoxin. To examine the generality of this finding, we have investigated the role of endotoxin in GRP94/gp96-elicited macrophage activation. We report that GRP94/gp96 binds endotoxin in a high-affinity, saturable, and specific manner. Low endotoxin calreticulin and GRP94/gp96 were purified, the latter using a novel method of depyrogenation; this resulted in GRP94/gp96 and calreticulin preparations with endotoxin levels substantially lower than those of previously reported preparations. Low endotoxin GRP94/gp96 retained its native conformation, ligand binding activity, and in vitro chaperone function, yet did not activate macrophage NF-κB signaling, nitric oxide production or inducible nitric-oxide synthase production. Low endotoxin GRP94/gp96 and calreticulin did, however, elicit a marked increase in ERK phosphorylation at protein concentrations as low as 2 μg/ml. These results are discussed with respect to current understanding of the contributions of endotoxin and heat shock/chaperone proteins to the stimulation of innate immune responses. A number of molecular chaperones, including GRP94/gp96, calreticulin (CRT), 1The abbreviations used are: CRT, calreticulin; ER, endoplasmic reticulum; APC, antigen-presenting cell; ConA, concanavalin A; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; LPS, lipopolysaccharide; IL, interleukin; DC, dendritic cell; TNF, tumor necrosis factor; JNK, Jun N-terminal kinase; NECA, N-ethylcarboxamidoadenosine.1The abbreviations used are: CRT, calreticulin; ER, endoplasmic reticulum; APC, antigen-presenting cell; ConA, concanavalin A; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; LPS, lipopolysaccharide; IL, interleukin; DC, dendritic cell; TNF, tumor necrosis factor; JNK, Jun N-terminal kinase; NECA, N-ethylcarboxamidoadenosine. and Hsp70, are capable of eliciting antitumor immune responses against their tumor of origin (1DuBois G.C. Law L.W. Appella E. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7669-7673Crossref PubMed Scopus (39) Google Scholar, 2Srivastava P.K. DeLeo A.B. Old L.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3407-3411Crossref PubMed Scopus (423) Google Scholar, 3Nair S. Wearsch P.A. Mitchell D.A. Wassenberg J.J. Gilboa E. Nicchitta C.V. J. Immunol. 1999; 162: 6426-6432PubMed Google Scholar, 4Basu S. Srivastava P.K. J. Exp. Med. 1999; 189: 797-802Crossref PubMed Scopus (210) Google Scholar). The efficacy of GRP94/gp96 in animal model studies of tumor rejection has spurred investigation into tumor-derived chaperones, principally GRP94/gp96, as immunotherapeutics for human cancers (5Janetzki S. Palla D. Rosenhauer V. Lochs H. Lewis J.J. Srivastava P.K. Int. J. Cancer. 2000; 88: 232-238Crossref PubMed Scopus (220) Google Scholar, 6Caudill M.M. Li Z. Expert Opin. Biol. Ther. 2001; 1: 539-547Crossref PubMed Scopus (34) Google Scholar, 7Belli F. Testori A. Rivoltini L. Maio M. Andreola G. Sertoli M.R. 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In this view, GRP94/gp96-peptide complexes are escorted to the MHC class I antigen presentation pathways of antigen-presenting cells (APC) to yield re-presentation of the GRP94/gp96-bound peptides on APC MHC class I molecules and activation of tumor directed CD8(+) T lymphocytes (8Srivastava P.K. Maki R.G. Curr. Top. Microbiol. Immunol. 1991; 167: 109-123Crossref PubMed Scopus (169) Google Scholar, 9Srivastava P.K. Udono H. Blachere N.E. Li Z. Immunogenetics. 1994; 39: 93-98Crossref PubMed Scopus (501) Google Scholar, 10Suto R. Srivastava P.K. Science. 1995; 269: 1585-1588Crossref PubMed Scopus (749) Google Scholar, 11Srivastava P.K. Annu. Rev. Immunol. 2002; 20: 395-425Crossref PubMed Scopus (730) Google Scholar). However, recent data demonstrate that chaperone-elicited tumor rejection stems, at least in part, from the direct interactions of these proteins with antigen-presenting cells, to yield dendritic cell (DC) maturation (12Basu S. Binder R.J. Suto R. Anderson K.M. Srivastava P.K. Int. Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1076) Google Scholar, 13Singh-Jasuja H. Scherer H.U. Hilf N. Arnold-Schild D. Rammensee H.G. Toes R.E. Schild H. Eur. J. Immunol. 2000; 30: 2211-2215Crossref PubMed Scopus (327) Google Scholar, 14Kuppner M.C. Gastpar R. Gelwer S. Nossner E. Ochmann O. Scharner A. Issels R.D. Eur. J. Immunol. 2001; 31: 1602-1609Crossref PubMed Scopus (186) Google Scholar, 15Baker-LePain J.C. Sarzotti M. Fields T.A. Li C.Y. Nicchitta C.V. J. Exp. Med. 2002; 196: 1447-1459Crossref PubMed Scopus (96) Google Scholar, 16Nicchitta C.V. Nat. Rev. Immunol. 2003; 3: 427-432Crossref PubMed Scopus (97) Google Scholar) and migration (17Binder R.J. Anderson K.M. Basu S. Srivastava P.K. J. Immunol. 2000; 165: 6029-6035Crossref PubMed Scopus (185) Google Scholar), and the release of cytokines known to exert anti-tumor effects, including TNF-α, IL-12, IL-1β, and GM-CSF (10Suto R. Srivastava P.K. Science. 1995; 269: 1585-1588Crossref PubMed Scopus (749) Google Scholar, 12Basu S. Binder R.J. Suto R. Anderson K.M. Srivastava P.K. Int. Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1076) Google Scholar, 13Singh-Jasuja H. Scherer H.U. Hilf N. Arnold-Schild D. Rammensee H.G. Toes R.E. Schild H. Eur. J. Immunol. 2000; 30: 2211-2215Crossref PubMed Scopus (327) Google Scholar, 18Chen W. Syldath U. Bellmann K. Burkart V. Kolb H. J. Immunol. 1999; 162: 3212-3219PubMed Google Scholar, 19Asea A. Kraeft S.-K. Kurt-Jones E.A. Stevenson M.A. Chen L.B. Finberg R.W. Koo G.C. Calderwood S.K. Nat. Med. 2000; 6: 435-442Crossref PubMed Scopus (1361) Google Scholar). Additionally, several chaperones, including GRP94/gp96, have been implicated in stimulating nitric oxide (NO) production by macrophages and DC (18Chen W. Syldath U. Bellmann K. Burkart V. Kolb H. J. Immunol. 1999; 162: 3212-3219PubMed Google Scholar, 20Panjwani N.N. Popova L. Srivastava P.K. J. Immunol. 2002; 168: 2997-3003Crossref PubMed Scopus (209) Google Scholar). Importantly, these peptide-independent activities are, under some circumstances, apparently sufficient to account for GRP94/gp96-elicited tumor rejection. For example, prophylactic vaccination of mice with irradiated fibroblasts, engineered to secrete GRP94/gp96 or the GRP94/gp96 N-terminal domain, yielded a marked suppression of 4T1 mammary carcinoma growth and metastasis following tumor cell challenge (15Baker-LePain J.C. Sarzotti M. Fields T.A. Li C.Y. Nicchitta C.V. J. Exp. Med. 2002; 196: 1447-1459Crossref PubMed Scopus (96) Google Scholar). It thus appears that GRP94/gp96-elicited, peptide-independent APC stimulation can play a prominent, if not primary, role in the phenomenon of chaperone-elicited tumor rejection. Several recent studies have investigated the peptide-independent mechanism(s) by which chaperones stimulate APC, implicating Toll-like receptors (TLR) and CD14 as well as a variety of downstream signaling cascades. TLR generally recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), CpG oligonucleotides, and bacterial surface proteins such as flagellin (21Janeway C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6148) Google Scholar). Hsp60, for example, elicits TNF-α and NO release from murine macrophages in a TLR4-dependent manner, suggesting that TLR4 serves as a signaling receptor for Hsp60 (22Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1364) Google Scholar). TLR2 and 4 have also been identified as Hsp70 signaling receptors, as determined through ectopic receptor expression studies (23Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15934Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar, 24Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-151112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar), and as GRP94/gp96 signaling receptors, based on similar experiments, as well as studies performed on cells derived from TLR4–/– mice (25Vabulas R.M. Braedel S. Hilf N. Singh-Jasuja H. Herter S. Ahmad-Nejad P. Kirschning C.J. da Costa C. Rammensee H.-G. Wagner H. Schild H. J. Biol. Chem. 2002; 277: 20847-20853Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). CD14 is a GPI-anchored co-receptor for LPS that may interact with signaling receptors (26Dobrovolskaia M.A. Vogel S.N. Microbes Infect. 2002; 4: 903-914Crossref PubMed Scopus (442) Google Scholar); work with Hsp70 indicates a CD14 requirement for cytokine release (19Asea A. Kraeft S.-K. Kurt-Jones E.A. Stevenson M.A. Chen L.B. Finberg R.W. Koo G.C. Calderwood S.K. Nat. Med. 2000; 6: 435-442Crossref PubMed Scopus (1361) Google Scholar). Evidence thus suggests that chaperone proteins are recognized by receptors of the innate immune system, principally those involved in the recognition of LPS and other PAMPs, to yield peptide-independent activation of APC function. At least four signaling pathways have been described in the literature for GRP94/gp96-elicited APC activation. First, GRP94/gp96 signaling by activation of NF-κB has been identified by electrophoretic mobility shift assay (12Basu S. Binder R.J. Suto R. Anderson K.M. Srivastava P.K. Int. Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1076) Google Scholar) and by the loss of IκBα by immunoblot (25Vabulas R.M. Braedel S. Hilf N. Singh-Jasuja H. Herter S. Ahmad-Nejad P. Kirschning C.J. da Costa C. Rammensee H.-G. Wagner H. Schild H. J. Biol. Chem. 2002; 277: 20847-20853Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). Additionally, MAP kinase phosphorylation has also been observed in response to GRP94/gp96; immunoblotting for phospho-p38, JNK, and ERK demonstrated increases in the phosphorylated (active) form of each, in response to GRP94/gp96 addition (25Vabulas R.M. Braedel S. Hilf N. Singh-Jasuja H. Herter S. Ahmad-Nejad P. Kirschning C.J. da Costa C. Rammensee H.-G. Wagner H. Schild H. J. Biol. Chem. 2002; 277: 20847-20853Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). It has been observed that the patterns of APC stimulation by GRP94, Hsp70, and Hsp60 bears many similarities to stimulation by LPS, in their kinetics, signaling pathways, and resulting cellular changes (12Basu S. Binder R.J. Suto R. Anderson K.M. Srivastava P.K. Int. Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1076) Google Scholar, 22Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1364) Google Scholar, 24Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-151112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar), though significant differences between LPS and GPR94/gp96, with respect to specific cellular effects and kinetics of activation, have been reported (12Basu S. Binder R.J. Suto R. Anderson K.M. Srivastava P.K. Int. Immunol. 2000; 12: 1539-1546Crossref PubMed Scopus (1076) Google Scholar). LPS, which is found on the surface of Gram-negative bacteria, is a problematic contaminant: it is very chemically stable and a potent activator of APC activation (26Dobrovolskaia M.A. Vogel S.N. Microbes Infect. 2002; 4: 903-914Crossref PubMed Scopus (442) Google Scholar), and is generally present in protein preparations unless specifically excluded. In fact, LPS levels as low as 15 pg/ml can stimulate some markers of activation in APC, including p38 phosphorylation and IL-6 release (27Bausinger H. Lipsker D. Ziylan U. Manie S. Briand J.-P. Cazenave J.-P. Muller S. Haeuw J.-F. Ravanat C. de la Salle H. Hanau D. Eur. J. Immunol. 2002; 32: 3708-3713Crossref PubMed Scopus (206) Google Scholar). Several recent reports have revived longstanding concerns about LPS contamination in purified chaperone preparations. The ability of recombinant human Hsp70 to induce TNF-α release from macrophages has recently been attributed to LPS contamination (28Gao B. Tsuan M.-F. J. Biol. Chem. 2003; 278: 174-179Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). Similarly, Bausinger et al. (27Bausinger H. Lipsker D. Ziylan U. Manie S. Briand J.-P. Cazenave J.-P. Muller S. Haeuw J.-F. Ravanat C. de la Salle H. Hanau D. Eur. J. Immunol. 2002; 32: 3708-3713Crossref PubMed Scopus (206) Google Scholar) report that recombinant human Hsp70 does not cause DC maturation or cytokine release when endotoxin levels are extremely low (less than 10 EU/mg protein). On a related note, an established GRP94/gp96 purification protocol was recently found to result in substoichiometric contamination of GRP94 by concanavalin A (29Monks S.A. Hassan-Zahraee M. Rottman J.B. Weng J. Wang Y. Sawlivich W. Simha S. Principato J. Carpenter J. Desroches B. Burke J. Truneh A. Srivastava P. Zabrecky J.R. American Association for Cancer Research Annual Meeting. 44. Cadmus Professional Communications, Toronto, Ontario, Canada2003: 2854Google Scholar), raising the possibility that other, heretofore undetected, contaminants may be present in biochemically enriched chaperone preparations. Peptide-independent stimulation of APC appears to be a central event in chaperone-mediated immune tumor rejection. Moreover, despite reports that LPS contamination accounts for some of the APC stimulation attributed to chaperones, it is evident that GRP94/gp96, at least, elicits tumor suppression in the absence of LPS. In systems in which GPR94 is secreted from cultured cells in vivo, suppression of tumor growth and metastasis is significant, peptide independent, and characterized by activation of innate immune mechanisms including DC maturation and NK cell activation (15Baker-LePain J.C. Sarzotti M. Fields T.A. Li C.Y. Nicchitta C.V. J. Exp. Med. 2002; 196: 1447-1459Crossref PubMed Scopus (96) Google Scholar, 30Strbo N. Oizumi S. Sotosek-Tokmadzic V. Podack E.R. Immunity. 2003; 18: 381-390Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It becomes paramount, then, to understand the mechanism whereby GRP94/gp96 elicits APC activation. For these reasons, we investigated whether LPS was responsible for any of the immunostimulatory activities reported for GRP94/gp96. We also tested CRT, a luminal chaperone with tumor-rejection activity similar to that of GRP94/gp96 (3Nair S. Wearsch P.A. Mitchell D.A. Wassenberg J.J. Gilboa E. Nicchitta C.V. J. Immunol. 1999; 162: 6426-6432PubMed Google Scholar, 4Basu S. Srivastava P.K. J. Exp. Med. 1999; 189: 797-802Crossref PubMed Scopus (210) Google Scholar), whose ability to stimulate an innate immune response has not been characterized. Here, we report that GRP94/gp96 specifically binds LPS, and that low endotoxin preparations of GRP94/gp96 and CRT do not stimulate NF-κB activation or NO release in macrophages; both responses are, however, robustly stimulated by LPS. Low endotoxin GRP94/gp96 and CRT do induce ERK phosphorylation in macrophages, implicating the MAP kinase cascade in the phenomenon of peptide-independent GRP94/gp96- and CRT-elicted APC activation. Materials—LPS levels were determined using the QCL-1000 Limulus amoebocyte lysate kit (Cambrex, Walkersville, MD). Escherichia coli LCD25–09 LPS was labeled and purified by the method of Munford et al. (32Munford R.S. DeVaux L.C. Cronan J.E.J. Rick P.D. J. Immunol. Methods. 1992; 148: 115-120Crossref PubMed Scopus (40) Google Scholar) and was generously provided by Dr. Robert Munford (University of Texas Southwestern Medical Center, Dallas, TX). Unlabeled LPS is from E. coli strain 026:B6 and was purchased from Sigma. N-[3H]ethylcarboxamidoadenosine (NECA) was obtained from Amersham Biosciences (Piscataway, NJ). Chlorophenol red-β-d-galactopyranoside (CPRG) was obtained from Roche Applied Science (Basel, Switzerland). Phosphorothioate CpG oligonucleotide (5′ TCCATCACGTTCCTGACGTT 3′) was the generous gift of Dr. David Pisetsky (Durham VA Medical Center, Durham, NC). PD98059 was purchased from Calbiochem (La Jolla, CA). Anti-phospho-p38, JNK, and ERK antibodies were from Cell Signaling Technology (Beverly, MA). DU-120 is a rabbit polyclonal antibody directed against a domain in the GRP94 N terminus and was prepared by contract service with Cocalico Biologicals (Reamstown, PA). A rabbit antiserum against BiP was prepared by contract service with Cocalico Biologicals, using a synthetic peptide representing the 15 N-terminal amino acids of the human protein. All other reagents were purchased from Sigma. Purification of GRP94/gp96 and Calreticulin—Low endotoxin GRP94/gp96 was purified from porcine pancreas rough microsomes by a modification of the method of Wearsch and Nicchitta (33Wearsch P.A. Nicchitta C.V. Protein Expr. Purif. 1996; 7: 114-121Crossref PubMed Scopus (52) Google Scholar). In this modification, chromatography buffers were supplemented with detergents, as described below, to yield the efficient removal of LPS. All materials were decontaminated prior to use, either by soaking in 70% EtOH, 0.5 m acetic acid (34Girot P. Moroux Y. Duteil X.P. Nguyen C. Boschetti E. J. Chromatogr. 1990; 510: 213-223Crossref PubMed Scopus (35) Google Scholar), or by baking for 4 h at 200 °C. All buffers were made in pyrogen-free water. Following microsome permeabilization, luminal proteins were loaded on a Mono Q column (Amersham Biosciences) equilibrated in TTTE buffer (25 mm Tris-Cl, pH 7.8, 150 mm NaCl, 0.2% (v/v) Tween 20, 0.2% (v/v) Triton X-100, and 10 mm EDTA). The column was washed sequentially with 400 ml of TTTE, 400 ml of 25 mm Tris-Cl, pH 7.8, 150 mm NaCl, and 1% (v/v) Triton X-114, and 250 ml of 25 mm Tris-Cl, pH 7.8, 50 mm NaCl. A 50–750 mm NaCl gradient was run to elute the column, and gel filtration was performed as usual, except that the column was equilibrated and run in sterile PBS. CRT was purified from porcine pancreas rough microsomes by the method of Wearsch and Nicchitta (33Wearsch P.A. Nicchitta C.V. Protein Expr. Purif. 1996; 7: 114-121Crossref PubMed Scopus (52) Google Scholar), and subsequently decontaminated of LPS using polymyxin B-agarose beads, by the method of Wright et al. (35Wright J.R. Zlogar D.F. Taylor J.C. Zlogar T.M. Restrepo C.I. Am. J. Physiol. 1999; 276: L650-L658Crossref PubMed Google Scholar). Briefly, purified CRT was incubated overnight with polymyxin B-agarose beads in a buffer containing 25 mm K-HEPES (pH 7.4), 20 mm NaCl, 110 mm KOAc, 2 mm Mg(OAc)2, 0.1 mm CaCl2, 2 mm EDTA, and 50 mm N-octyl β-d-glucopyranoside, at 4 °C with mixing. The supernatant was dialyzed at 4 °C against several changes of PBS, to exchange buffer and remove traces of N-octyl β-d-glucopyranoside. [3H]LPS and [3H]NECA Binding Assays—[3H]LPS binding was determined by incubating 2 μg of GRP94/gp96 with various concentrations of [3H]LPS for 1 h on ice in a final volume of 250 μl of 50 mm Tris, pH 7.5. Bound versus free [3H]LPS was assayed by vacuum filtration of the binding reactions on Protran nitrocellulose membranes (Schleicher and Schuell, Keene, NH). Vacuum filtration was performed with a vacuum filtration manifold (Amersham Biosciences). Filters were rapidly washed with 3 × 4 ml of ice-cold 50 mm Tris, pH 7.5, placed in 5 ml of scintillation fluid (SafetySolve, RPI, Mt. Prospect, IL), vortexed, and counted by liquid scintillation spectrometry. All binding reactions were performed in triplicate and corrected by subtraction of background values, determined in binding reactions lacking GRP94. [3H]NECA binding assays were performed as in Rosser and Nicchitta (36Rosser M.F. Nicchitta C.V. J. Biol. Chem. 2000; 275: 22798-22805Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). GRP94/gp96 Fluorescent Labeling and Cell Binding—Low endotoxin GRP94/gp96 was labeled with fluorescein isothiocyanate (FITC) (Molecular Probes, Eugene, OR) to a ratio of ∼1 mol of FITC:mol GRP94/gp96 dimer. Labeling was performed according to the manufacturer's instructions. FITC-GRP94 binding to cells was performed as described in Wassenberg et al. (37Wassenberg J.J. Dezfulian C. Nicchitta C.V. J. Cell Sci. 1999; 112: 2167-2175Crossref PubMed Google Scholar) and evaluated by flow cytometry. Citrate Synthase Aggregation Assay—The effects of low endotoxin GRP94/gp96 on the thermal aggregation of citrate synthase were assayed essentially as described in Buchner et al. (38Buchner J. Grallert H. Jakob U. Methods Enzymol. 1998; 290: 323-338Crossref PubMed Scopus (197) Google Scholar). Binding buffer (40 mm HEPES, pH 7.5) was preheated to 43 °C. Samples containing no protein, or GRP94 (0.6 μm final), were then added to 400 μl of preheated binding buffer in a quartz microcuvette. The cuvette was placed in a spectrofluorometer (Spex Industries, Inc., Edison, NJ) thermostatted at 43 °C. Citrate synthase was then added to 0.15 μm final concentration, with stirring. Thermal aggregation of citrate synthase was recorded as light scattering, with excitation and emission wavelengths of 500 nm and a 1-nm slit width. The time course of citrate synthase aggregation was followed for 1200 s. Readings were taken every 15 s with a 5-s integration. NF-κB Activation Assay—Activation of the NF-κB signaling pathway was determined using the PathDetect NF-κB cis-reporting system (Stratagene, La Jolla, CA). RAW264.7 cells were plated to 80% confluence in 24-well plates and transfected with 0.5 μg each of pLuc-NFκB and pCMV-βGal (as a transfection efficiency control) for 5 h in the presence of LipofectAMINE (Invitrogen Life Technologies, Carlsbad, CA), as per manufacturer's protocols, then cultured in DMEM with 10% fetal bovine serum for an additional 21 h. Transfected cells were stimulated with GRP94/gp96, CRT, ConA and/or LPS for 4 h and lysed in Reporter Lysis Buffer (Promega, Madison, WI) with a freeze-thaw cycle. Luciferase levels were measured using the Promega luciferase assay system. β-Galactosidase levels were determined by CPRG assay, as in Reed et al. (39Reed R.C. Zheng T. Nicchitta C.V. J. Biol. Chem. 2002; 277: 25082-25089Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Luciferase levels were normalized to β-galactosidase levels for analysis. All reactions were performed in triplicate and corrected by subtraction of the luciferase level of transfected, untreated cells. Nitrite Release Assay—Production of nitrite, a measure of nitric oxide release, was determined with the method of Misko et al. (40Misko T.P. Schilling R.J. Salvemini D. Moore W.M. Currie M.G. Anal. Biochem. 1993; 214: 11-16Crossref PubMed Scopus (959) Google Scholar), using 2,3-diaminonaphthalene (Molecular Probes). Fluorometer (Spex Industries, Inc.) slit widths were set to 1 nm for both excitation and emission. Samples were excited at a wavelength of 365 nm and the emission at 450 nm was recorded. All reactions were performed in duplicate and corrected by subtraction of background fluorescence. Determination of MAP Kinase Activation—Peritoneal mouse macrophages were isolated from BALB/c mice by peritoneal lavage with DMEM, 10% FCS, 5–7 days after intraperitoneal injection of 1 ml 4.2% (w/v) brewer's thioglycollate broth. Macrophages were plated in 24-well plates (Corning Glass Works, Corning, NY) and selected by adherence for 1 h at 37 °C. The cells were then incubated for 7 h at 37 °C in serum-free DMEM. Cells were supplemented with the indicated concentrations of low endotoxin GRP94/gp96, low endotoxin calreticulin or CpG oligonucleotide in serum-free DMEM for the times indicated. At the end of the incubation, cells were washed in PBS, and lysed on ice in phosphoprotein lysis buffer (150 mm NaCl, 50 mm sodium phosphate (pH 7.4), 0.05% (w/v) SDS, 1% (w/v) Nonidet P-40, 2 mm EDTA, 50 mm NaF, 100 μm NaVO4, 1 mm phenylmethylsulfonyl fluoride, and 0.1 mg/ml soybean trypsin inhibitor). Cells were removed from the wells with a cell scraper, centrifuged at 15,000 rpm at 4 °C for 10 min, and the supernatants trichloroacetic acid-precipitated. After washing in acetone, protein pellets were resuspended in sample buffer, resolved by SDS-PAGE on a 10% acrylamide gels, transferred to nitrocellulose membranes and probed with anti-phosphoprotein antibodies according to the manufacturer's instructions. As an internal loading control, endogenous GRP94/gp96 levels were determined by Western blot using DU120. Quantification was performed with NIH Image 1.63 (National Institutes of Health, Bethesda, MD). GRP94/gp96 Is an LPS-binding Protein—Current evidence indicates that peptide-independent activation of APC by chaperone proteins plays a fundamental role in the phenomenon of chaperone-elicited antitumor immunity. However, LPS is a common contaminant in purified chaperone preparations, and in the case of Hsp70, recent studies have indicated that contaminating LPS is responsible for all aspects of APC activation tested (27Bausinger H. Lipsker D. Ziylan U. Manie S. Briand J.-P. Cazenave J.-P. Muller S. Haeuw J.-F. Ravanat C. de la Salle H. Hanau D. Eur. J. Immunol. 2002; 32: 3708-3713Crossref PubMed Scopus (206) Google Scholar, 28Gao B. Tsuan M.-F. J. Biol. Chem. 2003; 278: 174-179Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). We therefore sought to determine, in the case of GRP94/gp96 and CRT, which aspects of APC activation were chaperone versus LPS-mediated. To distinguish the effects of LPS from those of GRP94/gp96 and CRT on macrophage stimulation, we set out to purify very low endotoxin GRP94/gp96 and CRT. CRT can be decontaminated by incubation with polymyxin B-agarose beads (41Karplus T.E. Ulevitch R.J. Wilson C.B. J. Immunol. Methods. 1987; 105: 211-220Crossref PubMed Scopus (60) Google Scholar). However, we observed that GRP94/gp96 binds polymyxin B beads, making this purification method unsuitable (Fig. 1A). Interestingly, during the gel filtration step of our GRP94 purification (33Wearsch P.A. Nicchitta C.V. Protein Expr. Purif. 1996; 7: 114-121Crossref PubMed Scopus (52) Google Scholar), LPS present in the Mono Q pool eluted in the same fractions as GRP94/gp96 (Fig. 1B). Subsequent attempts to separate GRP94/gp96 from LPS by repeating this gel filtration step demonstrated that LPS continued to coelute with purified GRP94/gp96 (data not shown). These findings, which suggested a GRP94/gp96-LPS association, raised the question of whether GRP94/gp96 specifically binds LPS. LPS-GRP94/gp96 interactions were examined in a radioligand binding assay. In this assay, [3H]LPS and GRP94/gp96 were mixed in solution and the free versus GRP94/gp96-bound [3H]LPS resolved by vacuum filtration. [3H]LPS binding to a control protein, ovalbumin, was negligible; by contrast, we observed significant [3H]LPS binding to GRP94/gp96 (Fig. 1C). The binding was competed by an 50-fold excess of cold LPS, but was un
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