FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2100431176> ?p ?o ?g. }

• W2100431176 endingPage "4680" @default.
• W2100431176 startingPage "4669" @default.
• W2100431176 abstract "Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) γ release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNα production in professional APC. Neutralization of IFNα as well as the absence of functional IFNαβ receptor on APC and T cells interfered with Hsp60-mediated IFNγ secretion in antigen-dependent T cell activation, strongly suggesting that IFNα represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNγ release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling. Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) γ release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNα production in professional APC. Neutralization of IFNα as well as the absence of functional IFNαβ receptor on APC and T cells interfered with Hsp60-mediated IFNγ secretion in antigen-dependent T cell activation, strongly suggesting that IFNα represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNγ release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling. Activation of antigen-presenting cells (APC) 2The abbreviations used are: APC, antigen-presenting cells; HSP, heat shock protein; LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; DC, dendritic cells; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; FCS, fetal calf serum; PEC, peritoneal exudate cells; bmDC, bone marrow-derived dendritic cells; FACS, fluorescence-activated cell sorter; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; PE, phycoerythrin; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. such as dendritic cells (DC) and macrophages is a critical step in the initiation of innate as well as adaptive immune responses and is known to be induced by pathogen-associated molecular pattern (PAMP) molecules such as bacterial lipopolysaccharide (LPS) and other endotoxins. These molecules are recognized by pattern recognition receptors (PRR) like members of the conserved Toll-like receptor (TLR) family (1.Janeway Jr., C.A. Medzhitov R. Annu. Rev. Immunol. 2002; 20: 197-216Crossref PubMed Scopus (6246) Google Scholar, 2.Iwasaki A. Medzhitov R. Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3368) Google Scholar). In the last years, several members of the heat shock protein (HSP) family including Hsp60 have been described to modulate APC functions and to stimulate immune responses in vitro and in vivo (3.Breloer M. Fleischer B. von Bonin A. J. Immunol. 1999; 162: 3141-3147PubMed Google Scholar, 4.Breloer M. Dorner B. More S.H. Roderian T. Fleischer B. von Bonin A. Eur. J. Immunol. 2001; 31: 2051-2059Crossref PubMed Scopus (88) Google Scholar, 5.Moseley P. Immunopharmacology. 2000; 48: 299-302Crossref PubMed Scopus (185) Google Scholar, 6.Osterloh A. Meier-Stiegen F. Veit A. Fleischer B. Von Bonin A. Breloer M. J. Biol. Chem. 2004; 279: 47906-47911Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Therefore, HSP have been suspected to function as endogenous danger signals to the immune system (4.Breloer M. Dorner B. More S.H. Roderian T. Fleischer B. von Bonin A. Eur. J. Immunol. 2001; 31: 2051-2059Crossref PubMed Scopus (88) Google Scholar, 7.Chen W. Syldath U. Bellmann K. Burkart V. Kolb H. J. Immunol. 1999; 162: 3212-3219PubMed Google Scholar, 8.Wallin 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 (498) Google Scholar, 9.Gallucci S. Matzinger P. Curr. Opin. Immunol. 2001; 13: 114-119Crossref PubMed Scopus (1011) Google Scholar). HSP are highly conserved and ubiquitously expressed proteins that are normally hidden within the cell and function as molecular chaperons of nascent or aberrantly folded proteins in different cellular compartments (10.Bukau B. Horwich A.L. Cell. 1998; 92: 351-366Abstract Full Text Full Text PDF PubMed Scopus (2435) Google Scholar, 11.Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3130) Google Scholar). HSP are up-regulated and released from cells upon various cellular stresses and necrotic cell death (12.Barreto A. Gonzalez J.M. Kabingu E. Asea A. Fiorentino S. Cell. Immunol. 2003; 222: 97-104Crossref PubMed Scopus (108) Google Scholar, 13.Lang A. Benke D. Eitner F. Engel D. Ehrlich S. Breloer M. Hamilton-Williams E. Specht S. Hoerauf A. Floege J. von Bonin A. Kurts C. J. Am. Soc. Nephrol. 2005; 16: 383-391Crossref PubMed Scopus (47) Google Scholar). Furthermore, stress-induced cell surface expression of HSP like Hsp60, which is normally localized within the mitochondria playing an essential role in the folding of imported mitochondrial proteins has been observed (14.Belles C. Kuhl A. Nosheny R. Carding S.R. Infect. Immun. 1999; 67: 4191-4200Crossref PubMed Google Scholar, 15.Vendetti S. Cicconi R. Piselli P. Vismara D. Cassol M. Delpino A. J. Exp. Clin. Cancer Res. 2000; 19: 329-334PubMed Google Scholar, 16.Pfister G. Stroh C.M. Perschinka H. Kind M. Knoflach M. Hinterdorfer P. Wick G. J. Cell Sci. 2005; 118: 1587-1594Crossref PubMed Scopus (156) Google Scholar, 17.Sapozhnikov A.M. Ponomarev E.D. Tarasenko T.N. Telford W.G. Cell Prolifer. 1999; 32: 363-378Crossref PubMed Scopus (62) Google Scholar). Extracellular Hsp60 has been shown to induce the maturation of human and murine DC and macrophages indicated by an up-regulation of co-stimulatory cell surface molecules and the production of the proinflammatory cytokines IL-1, IL-6, IL-12, and TNFα (7.Chen W. Syldath U. Bellmann K. Burkart V. Kolb H. J. Immunol. 1999; 162: 3212-3219PubMed Google Scholar, 18.Kol A. Lichtman A.H. Finberg R.W. Libby P. Kurt-Jones E.A. J. Immunol. 2000; 164: 13-17Crossref PubMed Scopus (473) Google Scholar, 19.Flohe S.B. Bruggemann J. Lendemans S. Nikulina M. Meierhoff G. Flohe S. Kolb H. J. Immunol. 2003; 170: 2340-2348Crossref PubMed Scopus (199) Google Scholar). Moreover, Hsp60 has been shown to enhance IFNγ production in antigen-dependent T cell activation (4.Breloer M. Dorner B. More S.H. Roderian T. Fleischer B. von Bonin A. Eur. J. Immunol. 2001; 31: 2051-2059Crossref PubMed Scopus (88) Google Scholar, 6.Osterloh A. Meier-Stiegen F. Veit A. Fleischer B. Von Bonin A. Breloer M. J. Biol. Chem. 2004; 279: 47906-47911Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), an effect that was mainly ascribed to the release of IL-12 by APC (20.More S.H. Breloer M. von Bonin A. Int. Immunol. 2001; 13: 1121-1127Crossref PubMed Scopus (57) Google Scholar, 21.Breloer M. More S.H. Osterloh A. Stelter F. Jack R.S. Bonin Av A. Int. Immunol. 2002; 14: 1247-1253Crossref PubMed Google Scholar). The receptors that have been proposed to be responsible for Hsp60-mediated immune effects are CD14 (18.Kol A. Lichtman A.H. Finberg R.W. Libby P. Kurt-Jones E.A. J. Immunol. 2000; 164: 13-17Crossref PubMed Scopus (473) Google Scholar) and members of the TLR family, namely TLR4 (22.Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1368) Google Scholar, 23.Vabulas R.M. Ahmad-Nejad P. da Costa C. Miethke T. Kirschning C.J. Hacker H. Wagner H. J. Biol. Chem. 2001; 276: 31332-31339Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar) and TLR2 (23.Vabulas R.M. Ahmad-Nejad P. da Costa C. Miethke T. Kirschning C.J. Hacker H. Wagner H. J. Biol. Chem. 2001; 276: 31332-31339Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar, 24.Zanin-Zhorov A. Nussbaum G. Franitza S. Cohen I.R. Lider O. FASEB J. 2003; 17: 1567-1569Crossref PubMed Scopus (163) Google Scholar). The receptor complex consisting of the glycosylphosphatidylinositol-anchored CD14 co-receptor and the TLR4 signaling receptor is known to mediate LPS signaling (25.Triantafilou M. Triantafilou K. Trends Immunol. 2002; 23: 301-304Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar), whereas TLR2 is a receptor for bacterial lipoproteins and lipoteichoic acid (26.Janeway Jr., C.A. Medzhitov R. Curr. Biol. 1999; 9: R879-R882Abstract Full Text Full Text PDF PubMed Google Scholar, 27.Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2800) Google Scholar, 28.Kirschning C.J. Schumann R.R. Curr. Top. Microbiol. Immunol. 2002; 270: 121-144Crossref PubMed Scopus (175) Google Scholar). The Hsp60 preparations, however, that have been used in earlier studies were expressed in Escherichia coli and, therefore, were likely to be contaminated with bacterial endotoxins. For this reason, it could not be excluded that the observed effects were due to contaminating bacterial structures, especially LPS, rather than the Hsp60 protein itself, although controls like heat sensitivity and polymyxine B insensitivity of Hsp60 versus LPS were included (8.Wallin 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 (498) Google Scholar). Employing eukaryotic cell lines expressing the murine Hsp60 as a membrane-bound cell surface protein we have shown that Hsp60 enhances IFNγ production in antigen-dependent T cell activation in an endotoxin-free environment, clearly demonstrating that Hsp60 possesses an intrinsic immunostimulatory potential (6.Osterloh A. Meier-Stiegen F. Veit A. Fleischer B. Von Bonin A. Breloer M. J. Biol. Chem. 2004; 279: 47906-47911Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). On the other hand, this endotoxin-free Hsp60 did not induce TNFα production in APC, an effect that was described to be mediated by contaminating LPS in the recombinant E. coli-expressed Hsp60 preparations used in earlier studies (29.Gao B. Tsan M.F. J. Biol. Chem. 2003; 278: 22523-22529Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 30.Gao B. Tsan M.F. Biochem. Biophys. Res. Commun. 2004; 317: 1149-1154Crossref PubMed Scopus (64) Google Scholar). In addition, also Hsp70-mediated cytokine secretion in APC has been ascribed to contaminating bacterial endotoxins (31.Bausinger 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, 32.Gao B. Tsan M.F. J. Biol. Chem. 2003; 278: 174-179Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar) and it was suggested that HSP such as Hsp70 and Hsp90 bind bacterial LPS and modulate LPS signaling (25.Triantafilou M. Triantafilou K. Trends Immunol. 2002; 23: 301-304Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 33.Byrd C.A. Bornmann W. Erdjument-Bromage H. Tempst P. Pavletich N. Rosen N. Nathan C.F. Ding A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5645-5650Crossref PubMed Scopus (176) Google Scholar, 34.Triantafilou K. Triantafilou M. Dedrick R.L. Nat. Immunol. 2001; 2: 338-345Crossref PubMed Scopus (357) Google Scholar). Recently, the stress protein gp96 was shown to bind different TLR agonists including LPS, thereby enhancing the biological effect of the associated PAMP (35.Warger T. Hilf N. Rechtsteiner G. Haselmayer P. Carrick D.M. Jonuleit H. van Landenberg P. Rammensee H.-G. Nicchitta C.V. Radsak M.P. Schild H. J. Biol. Chem. 2006; 281: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Interestingly, also Hsp60 has been shown to bind LPS and to enhance LPS-induced TNFα production in a macrophage cell line (36.Habich C. Kempe K. van der Zee R. Rumenapf R. Akiyama H. Kolb H. Burkart V. J. Immunol. 2005; 174: 1298-1305Crossref PubMed Scopus (80) Google Scholar) indicating that Hsp60 may influence LPS signaling. Therefore, the present study was performed to dissect the immunological functions of Hsp60, LPS, and Hsp60/LPS complexes. We show that Hsp60 exclusively binds to professional APC but not to T- or B-lymphocytes. Thereby, Hsp60 colocalizes with the CD14 receptor as well as LPS binding sites. Furthermore, we confirm that Hsp60 specifically binds bacterial LPS and show that both molecules synergistically stimulate innate and adaptive immune responses indicated by enhanced IL-12p40 production in APC and IFNγ release in antigen-dependent T cell activation. On the other hand, we observe that Hsp60 stimulates IFNα production in APC, an effect that is not induced by LPS and not further enhanced by Hsp60-LPS complexes. Furthermore, we show that IFNα release as well as expression of functional IFNαβ receptor on APC and T cells is important in Hsp60- but not LPS-mediated stimulation of T cell activation. Thus, Hsp60 and LPS differentially stimulate leukocyte functions. Taken together, our results reveal different mechanisms by which Hsp60 can modulate immune responses in the absence or presence of LPS: (i) Hsp60 enhances antigen-dependent T cell activation in an endotoxin-free environment (6.Osterloh A. Meier-Stiegen F. Veit A. Fleischer B. Von Bonin A. Breloer M. J. Biol. Chem. 2004; 279: 47906-47911Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) whereby IFNα, which is released by APC upon Hsp60 stimulation, is one mediator. (ii) Hsp60 functions as a LPS carrier protein that enhances LPS-induced TLR4 signaling in APC and as a consequence augments LPS-mediated T cell activation. Cell Culture− 8–10-Week-old female DO11.10 TCR transgenic mice expressing a TCR specific for OVA323–339/H2-Ad (37.Hosken N.A. Shibuya K. Heath A.W. Murphy K.M. O'Garra A. J. Exp. Med. 1995; 1821579Crossref PubMed Scopus (677) Google Scholar), C57BL/6, BALB/c mice, and BALB/c-IL-12p40-/- mice (38.Magram J. Connaughton S.E. Warrier R.R. Carvajal D.M. Wu C.Y. Ferrante J. Stewart C. Sarmiento U. Faherty D.A. Gately M.K. Immunity. 1996; 4: 471Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar) were bread in the animal facilities of the Bernhard-Nocht-Institute for Tropical Medicine and the Universitaets-Klinikum Eppendorf in Hamburg, Germany. IFNαβR-/- (39.Muller U. Steinhoff U. Reis L.F. Hemmi S. Pavlovic J. Zinkernagel R.M. Aguet M. Science. 1994; 264: 1918-1921Crossref PubMed Scopus (2021) Google Scholar) and IFNβ-/- (40.Erlandsson L. Blumenthal R. Eloranta M.L. Engel H. Alm G. Weiss S. Leanderson T. Curr. Biol. 1998; 8: 223-226Abstract Full Text Full Text PDF PubMed Google Scholar) mice were generated on Sv129 and backcrossed to C57BL/6 (41.Gerlach N. Schimmer S. Weiss S. Kalinke U. Dittmer U. J. Virol. 2006; 80: 3438-3444Crossref PubMed Scopus (51) Google Scholar). IFNαβR-/- and IFNβ-/- mice were bred at the Paul Ehrlich Institute, Langen, Germany, and the Helmholtz Center for Infection Research, Braunschweig, Germany. T cells from DO11.10 mice (DO11.10 T cells termed hereafter), C57BL/6 and IFNαβR-/-, MHC II+ cells, and B cells from BALB/c mice were purified from spleens by magnetic cell sorting using the Pan T cell isolation kit, the MHC II depletion kit, and the Pan B cell isolation kit (Miltenyi Biotec, Germany) according to the manufacturer’s protocol. Cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum (FCS), HEPES, and 10 mm l-glutamine. Peritoneal exudate cells (PEC) were induced by intraperitoneal injection of 500 μl of thioglycolate into BALB/c mice and isolated by peritoneal lavage after 5 days. Bone marrow-derived dendritic cells (bmDC) were obtained from purified BALB/c bone marrow cells that were cultured in RPMI1640/10% FCS supplemented with 20 ng/ml granulocyte monocyte colony-stimulating factor and harvested after 9 days of culture. Transfection of COS1−Eukaryotic COS1 cells were transiently transfected using the FuGENE 6 transfection reagent (Roche Applied Science, Germany) according to the manufacturer’s protocol. In brief, COS1 cells were plated into 6-well culture plates. 5 μg of pFM92 or pFM92-mHsp60 (6.Osterloh A. Meier-Stiegen F. Veit A. Fleischer B. Von Bonin A. Breloer M. J. Biol. Chem. 2004; 279: 47906-47911Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 42.Gunning P. Leavitt J. Muscat G. Ng S.Y. Kedes L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4831-4835Crossref PubMed Scopus (667) Google Scholar) vector DNA and 6 μl of FuGENE 6 reagent were added to 100 μl of RPMI1640 without FCS and incubated for 30 min. Cell culture medium was replaced by 2 ml of RPMI1640, 10% FCS and the transfection mixture was added for 24 h. Expression of cell surface Hsp60 was monitored by FACS staining as described earlier (6.Osterloh A. Meier-Stiegen F. Veit A. Fleischer B. Von Bonin A. Breloer M. J. Biol. Chem. 2004; 279: 47906-47911Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Reagents−Low-endotoxin recombinant human Hsp60 (named hHsp60 hereafter) was obtained from Loke Diagnostics APs (Denmark; batch number B02-141205) and contained <2 endotoxic units of LPS/mg of protein as determined by limulus amoebocyte lysate assay (BioWhittaker). For binding studies hHsp60 and BSA control protein (Sigma) were labeled with fluorescein isothiocyanate (FITC) or Alexa 647 using the protein labeling kits from Molecular Probes. Proteins were labeled according to the manufacturer’s protocol. Unlabeled E. coli lipopolysaccharide (LPS) (strain 055:B5) and Alexa 488-labeled LPS of the same strain were purchased from Sigma (Germany). 3H-LPS (E. coli strain K12 LCD25) was obtained from List Biological Laboratories (Canada). OVA323–339 peptide was synthesized by JPT (Germany). Antibodies−Hsp60-specific antibody clones LK-1 and 4B9 were obtained from Stressgen (number SPA806) and Dianova (number MA3-012) as was mouse IgG2a isotype antibody. Rat anti-mouse CD14 was purchased from BD Pharmingen and TRITC- or PE-labeled goat anti-mouse as well as FITC-labeled goat anti-rat secondary antibodies and PE- or FITC-labeled antibodies against CD11c, CD11b, B220, CD4, and CD8 were purchased from Dianova. Alexa 488-conjugated rabbit anti-FITC antibody was obtained from Molecular Probes and DAPI was purchased from Sigma. Neutralizing polyclonal rabbit anti-IFNα (number 32100-1) and anti-IFNβ serum (number 32400-1) as well as rabbit IgG control serum were obtained from R&D Systems (Germany). Binding Studies−To analyze whether LPS binds to Hsp60, 3 × 104 pFM92-mHsp60-transfected COS1 cells expressing the murine Hsp60 protein as a cell surface molecule or mock transfected COS1 cells that obtained the pFM92 control vector were incubated on ice with 500 ng/ml 3H-LPS in 200 μl of culture medium in a 96-well plate for 45 min. To test for specificity the binding of 3H-LPS was blocked by addition of either 15 μg/ml anti-Hsp60 antibody (clone 4B9) or 5 μg/ml unlabeled LPS for 45 min on ice before addition of 3H-LPS. Afterward cells were harvested and cell-bound radioactivity was detected. For binding of Hsp60 to PEC and bmDC, 1 × 106 BALB/c-derived PEC or bmDC were incubated on ice with either 30 μg/ml FITC-labeled or unlabeled hHsp60. Afterward, cells that obtained unlabeled hHsp60 were treated with 30 μl of Cohn II fraction (Sigma) and stained with Hsp60-specific antibody (clone LK-1, 1:100 in PBS), TRITC-labeled goat anti-mouse secondary antibody (1:400 in PBS) and DAPI (1:1000 in PBS). After staining cells were fixed in PBS/1% paraformaldehyde (PFA), centrifuged onto glass slides, and covered with anti-FADE solution (BiomedDia, Germany). In addition, BALB/c PEC that had been incubated with 15 μg/ml of hHsp60 were centrifuged onto glass slides before staining. After overnight drying cells were fixed with acetone for 5 min and dried again for 1 h. After Fc block with 50 μl of Cohn II fraction cells were stained with mouse anti-Hsp60 antibody (LK-1, 1:100), rat anti-CD14 antibody (1:100), TRITC-labeled goat anti-mouse (1:400), and FITC-labeled goat anti-rat (1:200) secondary antibodies. FITC staining of CD14 was further enhanced by addition of Alexa 488-labeled anti-FITC (1:200). In addition, cells were stained with DAPI (1:1000) and finally covered with anti-FADE solution. Furthermore, BALB/c-derived PEC were incubated in chamber slides (Nunc, Wiesbaden, Germany). After adherence overnight at 37 °C dead cells were washed out and cells were incubated alone or with 15 μg/ml hHsp60 for 45 min at 37 °C. Afterward cells were washed and fixed in ice-cold acetone/methanol (1:1) at -20 °C for 10 min. After drying, cells were blocked by addition of 200 μl of Cohn II fraction with PBS, 1% BSA (1:1) for 20 min. Alexa 488-conjugated LPS (1:100) was added for 30 min and cells were stained with DAPI, anti-Hsp60 (clone LK-1) and TRITC-labeled goat anti-mouse antibody as described before. Dose-dependent binding of Hsp60 to BALB/c spleen cells was analyzed by incubating 2 × 106 cells with 0.4, 2, 10, 40, or 200 μg/ml hHsp60-Alexa 647 for 30 min on ice in 50 μl of culture medium. To identify cell populations in spleen that bind Hsp60, 2 × 106 BALB/c spleen cells were incubated 30 min on ice either alone, with 10 μg/ml BSA-Alexa 647 or 10 μg/ml hHsp60/Alexa 647 in 100 μl of culture medium. Binding of Hsp60 was competed by incubating cells with 20, 200, or 400 μg/ml unlabeled hHsp60 30 min prior to addition of hHsp60-Alexa 647. For further stainings, Fc receptors were blocked by addition of 30 μl of Cohn II fraction for 20 min and PE- or FITC-labeled CD11c-, CD11b-, B220-, CD4-, or CD8-specific antibodies (1:200) were added for 30 min. Cells were analyzed by FACS whereby two different gates were used: gate R1 that mainly contains CD4+, CD8+, and B220+ lymphocytes, and gate R2 that contains the majority of CD11c+ and CD11b+ cells (data not shown). 6 × 105 cells were detected. Cellular Assays−All assays were performed in RPMI1640 supplemented with 10% FCS, HEPES, and l-glutamine. For stimulation of APC, 1 × 105 BALB/c-derived PEC or bmDC were incubated in 96-well round-bottom plates either alone or with the indicated amounts of hHsp60 or LPS. In addition, cells were treated with a combination of LPS, hHsp60, or BSA or a combination of the same amounts of these proteins that had been preincubated with LPS for 2 h at 37 °Cto allow complexation. For T cell stimulation, 1 × 105 purified T cells from DO11.10 mice were co-cultured with 5 × 104 BALB/c-derived PEC or purified BALB/c B cells and activated with 0.5–1 μg/ml OVA323–339 peptide. In parallel experiments complexation of hHsp60 and LPS was inhibited by addition of 2 μg/ml anti-Hsp60 antibody (clone 4B9) during the preincubation period. As a control 2 μg/ml of isotype antibody (mouse IgG2a) was used. IFNα and IFNβ were neutralized by the addition of 2.3 kilounits of polyclonal rabbit anti-IFNα or anti-IFNβ serum. Control cultures received 2 μg/ml isotype antibodies or serum. Alternatively, instead of soluble recombinant hHsp60, 2 × 103 or 1 × 104 of transfected COS1 cells expressing cell surface Hsp60 were used. Thereby, the indicated amounts of COS1 cells were added alone or preincubated with LPS for 2 h at 37 °C before addition of 1 × 105 of DO11.10 T cells, 5 × 104 of PEC, and OVA peptide. Cytokine Quantification−Cytokines were detected after 24 or 48 h of culture. IL-12p40 was quantified by standard sandwich ELISA. 96-Well Maxisorb plates (Nunc, Roskilde, Denmark) were coated with 2 μg/ml anti-IL-12p40/70 (clone C15.6) at 4 °C for 24 h. Plates were blocked with PBS containing 1% BSA for 2 h at 37 °C and washed three times with PBS containing 0.05% Tween 20. Culture supernatants and a standard of recombinant IL-12 were added to the coated plates and incubated at 4 °C for 24 h. After 6 washes, 1 μg/ml biotinylated anti-IL-12p40/70 (clone C17.8) was added as detection antibody and incubated at room temperature for 1 h. Following 6 washes, a 1:10000 dilution of peroxidase-conjugated streptavidine (Amersham Biosciences) in PBS/0.1% BSA was added for 30 min at room temperature. Plates were washed 6 times and developed with 300 μg/ml tetramethylbenzidine, diluted in 0.1 m NaH2PO4, pH 5.5, containing 0.003% H2O2. The reaction was stopped by addition of 25 μl of 2 m H2SO4, and OD at 450 nm was measured immediately. All antibodies and recombinant cytokine standards were obtained from BD Pharmingen. IFNγ and IFNα content in the supernatants were determined employing the IFNγ DuoSet ELISA development system and the mouse IFNα ELISA kit from R&D Systems according to the manufacturer’s protocols. Hsp60 Binds to Macrophages and DC but Not to B and T Lymphocytes−It has been shown that Hsp60 modulates APC as well as T and B cell activation (4.Breloer M. Dorner B. More S.H. Roderian T. Fleischer B. von Bonin A. Eur. J. Immunol. 2001; 31: 2051-2059Crossref PubMed Scopus (88) Google Scholar, 6.Osterloh A. Meier-Stiegen F. Veit A. Fleischer B. Von Bonin A. Breloer M. J. Biol. Chem. 2004; 279: 47906-47911Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 7.Chen W. Syldath U. Bellmann K. Burkart V. Kolb H. J. Immunol. 1999; 162: 3212-3219PubMed Google Scholar, 22.Ohashi K. Burkart V. Flohe S. Kolb H. J. Immunol. 2000; 164: 558-561Crossref PubMed Scopus (1368) Google Scholar). To mediate a stimulatory effect it has to be assumed that Hsp60 binds to the cells via specific receptors. Therefore, we identified cell populations that bind Hsp60 to elucidate which cells might be able to respond to Hsp60 in a direct way. For this purpose BALB/c spleen cells were incubated with Alexa 647-labeled human Hsp60 (hHsp60-Alexa 647) or BSA (BSA-Alexa 647) as a control. Cells were subsequently stained against different cellular marker molecules and analyzed by flow cytometry (FACS). Fig. 1A shows that Hsp60 binds to subpopulations of CD11b+ and CD11c+ cells. About 35% of the CD11c+ cells and 21% of the CD11b+ cells were positive for hHsp60-Alexa 647, whereas the control protein BSA-Alexa 647 did not bind to these cells. Furthermore, Fig. 1 shows that hHsp60 does not bind to CD4+ or CD8+ T and B220+ B lymphocytes. Similar binding studies were also performed using unlabeled hHsp60 whereby bound hHsp60 was detected with Hsp60-specific antibody leading to the same results (data not shown). Binding of hHsp60-Alexa 647 could be inhibited by preincubation of spleen cells with unlabeled hHsp60. The addition of 20 μg/ml hHsp60, a 2-fold excess, already reduced the amount of hHsp60-Alexa 647 binding spleen cells from 18 to 7% and binding could be further inhibited using higher concentrations (20- and 40-fold excess) of unlabeled hHsp60 (Fig. 1B, left and middle). As shown before, the subpopulation of 18% of Hsp60-binding spleen cells can already be detected with 10 μg/ml Hsp60-Alexa 647 (Fig. 1B, left) and does not increase using higher concentrations of labeled Hsp60 (data not shown). However, the mean fluorescence intensity of this Hsp60-binding spleen cell fraction reaches a plateau when using more than 40 μg/ml hHsp60-Alexa 647 (Fig. 1B, right). These results show that binding of Hsp60 to spleen cells is saturable and specific. In addition, we analyzed binding of Hsp60 to PEC and bmDC by FACS (data not shown) and fluorescent microscopic analysis of cytospins. Fig. 2 shows that hHsp60 binds to the cell surface of bmDC as well as PEC (Fig. 2, A and B). Interestingly, Hsp60 appears to concentrate in distinct membrane regions that cannot be ascribed to antibody-in" @default.
• W2100431176 created "2016-06-24" @default.
• W2100431176 creator A5000856059 @default.
• W2100431176 creator A5008486712 @default.
• W2100431176 creator A5011269591 @default.
• W2100431176 creator A5032178490 @default.
• W2100431176 creator A5038700330 @default.
• W2100431176 date "2007-02-01" @default.
• W2100431176 modified "2023-09-30" @default.
• W2100431176 title "Synergistic and Differential Modulation of Immune Responses by Hsp60 and Lipopolysaccharide" @default.
• W2100431176 cites W1504160997 @default.
• W2100431176 cites W1553853809 @default.
• W2100431176 cites W1585651039 @default.
• W2100431176 cites W1591714343 @default.
• W2100431176 cites W1602279356 @default.
• W2100431176 cites W1899375067 @default.
• W2100431176 cites W1950448919 @default.
• W2100431176 cites W1963770237 @default.
• W2100431176 cites W1971521446 @default.
• W2100431176 cites W1986974750 @default.
• W2100431176 cites W1994043407 @default.
• W2100431176 cites W2000109838 @default.
• W2100431176 cites W2000175632 @default.
• W2100431176 cites W2004829503 @default.
• W2100431176 cites W2005570998 @default.
• W2100431176 cites W2011678998 @default.
• W2100431176 cites W2027855860 @default.
• W2100431176 cites W2032942842 @default.
• W2100431176 cites W2050812895 @default.
• W2100431176 cites W2051662558 @default.
• W2100431176 cites W2057121293 @default.
• W2100431176 cites W2057809738 @default.
• W2100431176 cites W2063053561 @default.
• W2100431176 cites W2063557216 @default.
• W2100431176 cites W2071923005 @default.
• W2100431176 cites W2072064957 @default.
• W2100431176 cites W2073204639 @default.
• W2100431176 cites W2083064282 @default.
• W2100431176 cites W2089559330 @default.
• W2100431176 cites W2090640877 @default.
• W2100431176 cites W2097556922 @default.
• W2100431176 cites W2098901296 @default.
• W2100431176 cites W2102403552 @default.
• W2100431176 cites W2104901317 @default.
• W2100431176 cites W2105077859 @default.
• W2100431176 cites W2108266076 @default.
• W2100431176 cites W2113553995 @default.
• W2100431176 cites W2118275423 @default.
• W2100431176 cites W2119999468 @default.
• W2100431176 cites W2122702136 @default.
• W2100431176 cites W2133449490 @default.
• W2100431176 cites W2141501589 @default.
• W2100431176 cites W2141895910 @default.
• W2100431176 cites W2145196632 @default.
• W2100431176 cites W2151777859 @default.
• W2100431176 cites W2152375607 @default.
• W2100431176 cites W2155900510 @default.
• W2100431176 cites W2160396729 @default.
• W2100431176 cites W2162440021 @default.
• W2100431176 cites W2168778458 @default.
• W2100431176 doi "https://doi.org/10.1074/jbc.m608666200" @default.
• W2100431176 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17164250" @default.
• W2100431176 hasPublicationYear "2007" @default.
• W2100431176 type Work @default.
• W2100431176 sameAs 2100431176 @default.
• W2100431176 citedByCount "90" @default.
• W2100431176 countsByYear W21004311762012 @default.
• W2100431176 countsByYear W21004311762013 @default.
• W2100431176 countsByYear W21004311762014 @default.
• W2100431176 countsByYear W21004311762015 @default.
• W2100431176 countsByYear W21004311762016 @default.
• W2100431176 countsByYear W21004311762017 @default.
• W2100431176 countsByYear W21004311762019 @default.
• W2100431176 countsByYear W21004311762020 @default.
• W2100431176 countsByYear W21004311762021 @default.
• W2100431176 countsByYear W21004311762022 @default.
• W2100431176 countsByYear W21004311762023 @default.
• W2100431176 crossrefType "journal-article" @default.
• W2100431176 hasAuthorship W2100431176A5000856059 @default.
• W2100431176 hasAuthorship W2100431176A5008486712 @default.
• W2100431176 hasAuthorship W2100431176A5011269591 @default.
• W2100431176 hasAuthorship W2100431176A5032178490 @default.
• W2100431176 hasAuthorship W2100431176A5038700330 @default.
• W2100431176 hasBestOaLocation W21004311761 @default.
• W2100431176 hasConcept C104317684 @default.
• W2100431176 hasConcept C121332964 @default.
• W2100431176 hasConcept C123079801 @default.
• W2100431176 hasConcept C146621620 @default.
• W2100431176 hasConcept C185592680 @default.
• W2100431176 hasConcept C203014093 @default.
• W2100431176 hasConcept C205260736 @default.
• W2100431176 hasConcept C24890656 @default.
• W2100431176 hasConcept C2778754761 @default.
• W2100431176 hasConcept C55493867 @default.
• W2100431176 hasConcept C68991219 @default.
• W2100431176 hasConcept C86803240 @default.
• W2100431176 hasConcept C8891405 @default.
• W2100431176 hasConcept C93226319 @default.

• next