Key points are not available for this paper at this time.
Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor α (TNFα) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70–1) with a LPS content of 1.4 pg/μg was unable to induce TNFα release by RAW264.7 murine macrophages at concentrations up to 5 μg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70–2) at 1 μg/ml with a LPS content of 0.2 ng/μg was able to induce TNFα release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70–1 to induce TNFα release was not because of defective physical properties since rhHsp70–1 and rhHsp70–2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70–2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFα-inducing activity of rhHsp70–2. The addition of LPS at the concentration found in rhHsp70–2 to rhHsp70–1 resulted in the same TNFα-inducing activity as observed with rhHsp70–2. The TNFα-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70–1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFα release from murine macrophages and that the observed TNFα-inducing activity in the rhHsp70–2 preparation is entirely due to the contaminating LPS. Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor α (TNFα) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70–1) with a LPS content of 1.4 pg/μg was unable to induce TNFα release by RAW264.7 murine macrophages at concentrations up to 5 μg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70–2) at 1 μg/ml with a LPS content of 0.2 ng/μg was able to induce TNFα release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70–1 to induce TNFα release was not because of defective physical properties since rhHsp70–1 and rhHsp70–2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70–2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFα-inducing activity of rhHsp70–2. The addition of LPS at the concentration found in rhHsp70–2 to rhHsp70–1 resulted in the same TNFα-inducing activity as observed with rhHsp70–2. The TNFα-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70–1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFα release from murine macrophages and that the observed TNFα-inducing activity in the rhHsp70–2 preparation is entirely due to the contaminating LPS. heat shock protein 70 recombinant human Hsp70 tumor necrosis factor lipopolysaccharide endotoxin units Limulus amebocyte lysate The 70-kDa heat shock proteins (Hsp70s)1 are highly conserved proteins expressed both constitutively (Hsc70) and under stressful conditions (Hsp70) in all prokaryotes and eukaryotes (1Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1467) Google Scholar, 2Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2426) Google Scholar). Members of the Hsp70 protein family play essential roles as molecular chaperones in the cytosol, mitochondria, and endoplasmic reticulum. Hsp70s are required for nascent or misfolded protein folding (3Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2786) Google Scholar, 4Hendrick J.P. Hartl F.U. FASEB J. 1995; 9: 1559-1569Crossref PubMed Scopus (205) Google Scholar), protein translocation into endoplasmic reticulum and mitochondria (5Ng D.T. Walter P. Curr. Opin. Cell Biol. 1994; 6: 510-516Crossref PubMed Scopus (27) Google Scholar,6Matouschek A. Pfanner N. Voos W. EMBO Rep. 2000; 1: 404-410Crossref PubMed Scopus (129) Google Scholar), and uncoating of clathrin-coated vesicles (7Greene L.A. Eisenberg E. J. Biol. Chem. 1990; 265: 6682-6687Abstract Full Text PDF PubMed Google Scholar, 8Gao B. Biosca J. Craig E.A. Greene L.A. Eisenberg E. J. Biol. Chem. 1991; 266: 19565-19571Abstract Full Text PDF PubMed Google Scholar, 9Jiang R. Gao B. Prasad K. Greene L.E. Eisenberg E. J. Biol. Chem. 2000; 275: 8439-8447Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These molecular chaperone functions of Hsp70s require the enzymatic hydrolysis of ATP and cycles of bound nucleotide exchange. Recently, Hsp70s were found to be present in circulation, and their levels were increased in a number of pathological conditions (10Pockley A.G. Shepherd J. Corton J.M. Immunol. Invest. 1998; 27: 367-377Crossref PubMed Scopus (253) Google Scholar, 11Rea I.M. McNerlan S. Pockley A.G. Exp. Gerontol. 2001; 36: 341-352Crossref PubMed Scopus (139) Google Scholar, 12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar). Studies also show that Hsp70 is a potent activator of the innate immune system (13Srivastava P. Nat. Rev. Immunol. 2002; 2: 185-194Crossref PubMed Scopus (860) Google Scholar, 14Wallin R.P. Lundqvist A. More S.H. von Bonin A Kiessling R. Ljunggren H.G. Trends Immunol. 2002; 23: 130-135Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). Using recombinant human Hsp70 (rhHsp70), it has been demonstrated that Hsp70 induces the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) and interleukin-6 via the CD14 and Toll-like receptor (TLR)-mediated signal transduction pathway (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar, 15Asea 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, 16Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 17Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar). Thus, it has been suggested that Hsp70, through its cytokine function, serves as a danger signal and that Hsp70 could be the endogenous ligand for the TLRs, both TLR-2 and TLR-4 (16Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar,17Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar). The CD14 and TLR complexes are pattern recognition receptors involved in the innate immunity for the pathogen recognition and host defense (18Medzhitov R. Nat. Rev. Immunol. 2001; 1: 135-145Crossref PubMed Scopus (3246) Google Scholar, 19Antal-Szalmas P. Eur. J. Clin. Invest. 2000; 30: 167-179Crossref PubMed Scopus (125) Google Scholar). CD14, the endotoxin (lipopolysaccharide (LPS)) receptor, is a glycophosphatidylinositol-anchored membrane protein lacking transmembrane and intracellular domains (20Stelter F. Chem. Immunol. 2000; 74: 25-41Crossref PubMed Google Scholar, 21Tapping R.I. Tobias P.S. Chem. Immunol. 2000; 74: 108-121Crossref PubMed Google Scholar). TLRs are Type I transmembrane proteins with an extracellular domain containing a leucine-rich repeat and a cytoplasmic domain analogous to that of the interleukin-1 receptor family (18Medzhitov R. Nat. Rev. Immunol. 2001; 1: 135-145Crossref PubMed Scopus (3246) Google Scholar, 22Jones B.W. Heldwein K.A. Means T.K. Saukkonen J.J. Fenton M.J. Ann. Rheum. Dis. 2001; 60: iii6-iii12PubMed Google Scholar). Together with CD14, TLR4 initiates signaling cascades in response to LPS, an abundant glycolipid of the outer membrane of the Gram-negative bacterial cell wall, whereas TLR2 initiates the signal cascades in response to bacterial lipoproteins, Gram-positive bacteria, mycoplasma, yeast, and spirochetes (18Medzhitov R. Nat. Rev. Immunol. 2001; 1: 135-145Crossref PubMed Scopus (3246) Google Scholar, 22Jones B.W. Heldwein K.A. Means T.K. Saukkonen J.J. Fenton M.J. Ann. Rheum. Dis. 2001; 60: iii6-iii12PubMed Google Scholar, 23Hirschfeld M., Ma, Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (969) Google Scholar, 24Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar). Because rhHsp70 is produced by Escherichia coli expressing human Hsp70 cDNA, the final preparation may be contaminated with bacterial products such as LPS and lipoproteins. Contamination of rhHsp70 with LPS and/or lipoproteins could be responsible for the reported cytokine functions of Hsp70. In the current study, we demonstrated that the ability of commercially available rhHsp70 preparations to induce TNFα production by murine macrophages is solely due to the contaminating LPS. Recombinant human Hsp70 proteins were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Recombinant Hsp70 was cloned from a human embryonic cDNA library and expressed in E. coli. Two preparations were available, catalog No. ESP-555 (previously ESP-755) and NSP-555 (previously SPP-755). None of the two rhHsp70 preparations contained the constitutively expressed Hsp70 (Hsc70). Both rhHsp70 preparations were assayed for their abilities to bind and hydrolyze ATP. The ESP-555 preparation was the low endotoxin preparation containing <50 EU (endotoxin units)/mg of rhHsp70 as determined using the Limulus Amebocyte lysate (LAL) assay, which was recommended for use in assays requiring low endotoxin. The NSP-555 preparation was not tested for endotoxin levels and was used in protein-binding assays. For the purpose of this report, the low endotoxin preparation, ESP-555, was designated rhHsp70–1, whereas the NSP-555 preparation was designated rhHsp70–2. Protein-free LPS (from JM83 E. coli K-12, rough strain) was kindly provided by Dr. John E. Somerville of Bristol-Myers Squibb Co. Before use, LPS was dissolved in sterile, for with a and with B No. cell and ATP were purchased from B-agarose catalog No. was from Western were purchased from murine macrophages (from were in medium with of macrophages were by into macrophages were in at the the with the the were with or rhHsp70 and/or LPS in of medium containing for at the of the were and by at for 5 in a TNFα content of the was determined by a using the TNFα No. to the were in In rhHsp70 and LPS were for in a to the in macrophages were with or polymyxin B for at the addition of rhHsp70 or LPS to LPS. The endotoxin activities of rhHsp70 and LPS preparations were determined using the to the The rhHsp70 preparations and from polymyxin B-agarose of from Using were by using gels by with Coomassie Blue or by Western using an to recombinant human Hsp70 No. by system as B. T. J. Cell 2002; PubMed Scopus Google Scholar). of rhHsp70 of the Coomassie SDS gels or were using the system was as B. Biosca J. Craig E.A. Greene L.A. Eisenberg E. J. Biol. Chem. 1991; 266: 19565-19571Abstract Full Text PDF PubMed Google Scholar). the uncoating contained μg/ml clathrin-coated vesicles containing clathrin provided by Greene and Eisenberg of the of 1 and an system of and The were at for and vesicles in the were by at 1 for in a from the vesicles in the was by Coomassie Blue and The activities of were expressed as of in rhHsp70–2 was using polymyxin B-agarose to of of polymyxin B-agarose were into and in were with 5 of by of of rhHsp70–2 at μg/ml was column and at for The column was with in The column were by SDS stained with Coomassie and as were expressed as the of were determined using a F. for and and and Scholar), and a of was used to The reported of cytokine release by rhHsp70 was similar to the of LPS, through the CD14 and TLR-4 receptor signal transduction pathway (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar, 15Asea 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, 16Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 17Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar). Because of by LPS is also CD14 and TLR-4 J. 2001; PubMed Google Scholar), we to rhHsp70 could also induce the of LPS, we the highly low endotoxin preparation of observed to that rhHsp70–1 not not induce also to induce TNFα production by RAW264.7 murine macrophages not Failure to induce TNFα release from macrophages by rhHsp70–1 was in to the reported of For this we to the of both rhHsp70–1 and the less purified rhHsp70–2 preparation to rhHsp70 could in induce TNFα production by shown in 1 rhHsp70–1 at 1 as with not an in TNFα release by murine In contrast, rhHsp70–2 at the same concentration induced a in TNFα release to a similar extent as induced by ng/ml LPS. response studies that rhHsp70–1 at concentrations up to 5 μg/ml to induce TNFα rhHsp70–2 at a concentration as low as μg/ml induced a release of TNFα 1 LPS at a concentration as low as ng/ml induced a release of TNFα from murine macrophages 1 were two for the rhHsp70 had the TNFα release by the observed of was due to present in the rhHsp70–2 rhHsp70 have TNFα-inducing of rhHsp70–1 to induce TNFα release by macrophages was due to the of rhHsp70 in the rhHsp70–1 two we determined the as as endotoxin of the rhHsp70–1 and rhHsp70–2 shown in A and of rhHsp70–1 and rhHsp70–2 that both preparations were identical in rhHsp70 protein molecular and ability to with an anti-rhHsp70 antibody. In both preparations had similar molecular chaperone activities as determined by the uncoating of clathrin-coated vesicles In contrast, rhHsp70–2 contained a content of endotoxin rhHsp70–1 as determined using the shown in the endotoxin activity of rhHsp70–1 was 0.2 that of rhHsp70–2 was and that of E. was Thus, the endotoxin content in the rhHsp70–2 preparation was that in the rhHsp70–1 The LPS concentration in the rhHsp70–1 was 1.4 whereas it was 0.2 ng/μg in the rhHsp70–2. shown in 1 the concentration of LPS present in 1 μg/ml 0.2 was to the observed TNFα release from macrophages by rhHsp70–2. the contaminating LPS in the rhHsp70–2 preparation was responsible for its TNFα-inducing we used polymyxin B-agarose to remove LPS from rhHsp70–2. The concentration of rhHsp70 in the from polymyxin B-agarose was determined using and The same of rhHsp70 present in the polymyxin B column as in the rhHsp70–2 preparation through the column were used to the endotoxin activity as as the TNFα-inducing shown in the polymyxin B column of the endotoxin activity from rhHsp70–2. The endotoxin activity of rhHsp70–2 the polymyxin B column was whereas it was and in the two of the polymyxin B polymyxin B column of the TNFα-inducing activity of rhHsp70–2 Thus, polymyxin B column LPS from the rhHsp70–2 preparation with its TNFα-inducing In similar studies (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar, 15Asea 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, 17Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar), polymyxin B has been into the incubation medium to the observed is due to LPS. we also tested the of polymyxin B in the incubation In macrophages were with polymyxin B at μg/ml rhHsp70–2 or LPS was to the incubation the we used an of LPS with an endotoxin activity to that found in 1 μg/ml rhHsp70–2. shown in polymyxin B the TNFα-inducing activities of both LPS and rhHsp70–2. The suggest that the observed TNFα-inducing of rhHsp70–2 was due to the contaminating LPS. this were the that the addition of LPS at a concentration found in rhHsp70–2 to the rhHsp70–1 in the same TNFα-inducing activity as the rhHsp70–2. shown in rhHsp70–1 at 1 μg/ml had TNFα-inducing the addition of LPS to the rhHsp70–1 at a final concentration of 0.2 ng/ml resulted in a similar TNFα-inducing activity as that of 0.2 ng/ml LPS or 1 μg/ml rhHsp70–2. of the of LPS is its heat studies show that the TNFα-inducing activity of rhHsp70 was whereas that of LPS was (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar, 15Asea 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, 16Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 17Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar). was that the TNFα-inducing activity of rhHsp70 could not have been due to the of contaminating LPS. shown in we also demonstrated that the TNFα-inducing activity of rhHsp70–2 at 1 not that of LPS at was sensitive to in a for The demonstrated heat of rhHsp70–2 in TNFα release in to that the TNFα-inducing activity of rhHsp70–2 was due to the contaminating LPS. the that LPS at low such as is and that heat by is not to an observed is due to LPS. shown in the addition of 0.2 ng/ml LPS to rhHsp70–1 resulted in a TNFα-inducing similar to that of 0.2 ng/ml LPS or 1 μg/ml rhHsp70–2 demonstrated in the TNFα-inducing of the of LPS and rhHsp70–1 was as as that of the rhHsp70–2. of rhHsp70 using that heat of Hsp70 from the rhHsp70–1 and rhHsp70–2 Thus, it was that heat the rhHsp70 with LPS from the in the observed of TNFα-inducing the of in the heat of LPS, we LPS at two ng/ml and and determined the endotoxin activity and TNFα-inducing activity of 0.2 ng/ml LPS and shown in ng/ml LPS in a for its endotoxin activity as as its TNFα-inducing LPS at μg/ml also its endotoxin activity and TNFα-inducing to a Thus, LPS at low concentrations is highly in the current demonstrated that rhHsp-70 not induce TNFα release from murine macrophages and that the TNFα-inducing activity of the rhHsp70–2 preparation was entirely due to the contaminating LPS. was from the the highly purified rhHsp70 preparation with an LPS content of 1.4 pg/μg was unable to induce TNFα release from macrophages at concentrations up to 5 whereas the less purified rhHsp70 preparation at 1 with a LPS content of 0.2 ng/μg was able to induce TNFα release to the same extent as that induced by 0.2 ng/ml LPS of rhHsp70–1 to induce TNFα release was not due to defective physical properties since rhHsp70–1 and rhHsp70–2 contained identical Hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 A and Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles of LPS from rhHsp70–2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFα-inducing activity of rhHsp70–2 and the addition of LPS at the concentration found in rhHsp70–2 to rhHsp70–1 resulted in the same TNFα-inducing activity as observed with rhHsp70–2 Using rhHsp70 from the same as the we used in the current Biotechnologies a number of reported that rhHsp70 induced TNFα production by monocytes and macrophages (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar, 15Asea 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, 16Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 17Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar). with the of by (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar), of the which rhHsp70 preparation from StressGen was used in the Using the less purified preparation of rhHsp70 or (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google that their rhHsp70 preparation had an endotoxin activity of demonstrated that the rhHsp70 preparation could induce TNFα and interleukin-6 production by murine macrophages and human monocytes at rhHsp70 concentrations of μg/ml. The endotoxin activities present in μg/ml e.g. to that of ng/ml were to induce the cytokine production as demonstrated in the current studies 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, 16Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. 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Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 17Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1254) Google Scholar). the activity of has also been shown to be Immunol. 2002; PubMed Scopus Google Scholar). In the current study, we show that the results of heat may the concentrations of LPS used to shown in ng/ml LPS was used to TNFα release from murine heat of LPS to have TNFα in 1 LPS at ng/ml was to induce TNFα release from murine Thus, in for of LPS at be LPS to induce TNFα the that TNFα-inducing activity of LPS was in that LPS at the same concentration found in μg/ml rhHsp70–2 it was resulted in an in TNFα-inducing the observed TNFα-inducing activity of rhHsp70–2 was In the current study, we demonstrated that of the contaminating LPS from rhHsp70–2 by polymyxin B-agarose column or by of macrophages with polymyxin B the TNFα-inducing activity of In contrast, observed of or with polymyxin B activity of the rhHsp70 (12Dybdahl B. Wahba A. Lien E. Flo T.H. Waage A. Qureshi N. Sellevold O.F.M. Espevik T. Sundan A. Circulation. 2002; 105: 685-690Crossref PubMed Scopus (325) Google Scholar, 15Asea A. Kraeft S-K. Kurt-Jones E.A. Stevenson M.A. Chen L.B. Finberg R.W. Koo G.C. Calderwood S.K. 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Gao et al. (Sat,) studied this question.