Interaction of TLR2 and TLR4 Ligands with the N-terminal Domain of Gp96 Amplifies Innate and Adaptive Immune Responses

Abstract

Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (≥50 μg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses. Activation of dendritic cells by ligands for Toll-like receptors (TLR) is a crucial event in the initiation of innate and adaptive immune responses. Several classes of TLR ligands have been identified that interact with distinct members of the TLR-family. TLR4 ligands include lipopolysaccharide derived from different Gram-negative bacteria and viral proteins. Recent reports have demonstrated the TLR-mediated activation of dendritic cells by heat shock proteins (HSPs). However, doubts were raised as to what extent this effect was due to lipopolysaccharide contaminations of the HSP preparations. We re-examined this phenomenon using Gp96 or its N-terminal domain, nominally endotoxin-free (<0.5 enzyme units/mg). As described previously, innate immune cells are activated by Gp96 at high concentrations (≥50 μg/ml) but not at lower concentrations. However, preincubation of low amounts of Gp96 with TLR2 and TLR4 ligands at concentrations unable to activate dendritic cells by themselves results in the production of high levels of proinflammatory cytokines, up-regulation of activation markers, and amplification of T cell activation. Our results provide significant new insights into the mechanism of HSP-mediated dendritic cell activation and present a new function of HSPs in the amplification of dendritic cell activation by bacterial products and induction of adaptive immune responses. HSPs 3The abbreviations used are: HSP, heat shock protein; DC, dendritic cells; BMDC, bone marrow-derived dendritic cells; TLR, Toll-like receptor; CFSE, carboxyfluorescein diacetate succimide ester; EU, enzyme units; LPS, lipopolysaccharide; APC, antigen-presenting cell; LAL, limulus amoebocyte lysate; FITC, fluorescein isothiocyanate; NTD, N-terminal domain; PMN, polymorphonuclear neutrophilic granulocytes; GM-CSF, granulocyte-macrophage colony-stimulating factor; FACS, fluorescence-activated cell sorter; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL, interleukin; CTL, cytotoxic T cells; RANTES, regulated on activation, normal T cell expressed and secreted; KC, keratinocyte derived-chemokine; MCP-1, monocyte chemoattractant protein-1. are a family of highly conserved molecules that have the potential to execute several functions. First of all, they act as molecular chaperones involved in the folding of newly synthesized proteins as well as in degradation of misfolded proteins (1Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1919) Google Scholar, 2Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3776) Google Scholar). In addition, HSPs have been reported to fulfill immunological functions. These include the ability to associate with peptides derived from intracellular protein degradation and to induce adaptive immune responses against these peptides (3Schild H. Arnold-Schild D. Lammert E. Rammensee H.G. Curr. Opin. Immunol. 1999; 11: 109-113Crossref PubMed Scopus (86) Google Scholar, 4Srivastava P. Nat. Rev. Immunol. 2002; 2: 185-194Crossref PubMed Scopus (887) Google Scholar). Based on these features, HSPs have been proposed to be involved in a process called cross-priming, which describes the uptake of antigens by professional APCs, the processing and presentation of these antigens by major histocompatibility complex molecules, and the subsequent activation of naive T cells (5Bevan M.J. J. Exp. Med. 1976; 143: 1283-1288Crossref PubMed Scopus (770) Google Scholar, 6Arnold D. Faath S. Rammensee H. Schild H. J. Exp. Med. 1995; 182: 885-889Crossref PubMed Scopus (295) Google Scholar). A crucial event for this process to take place efficiently is the receptor-mediated interaction of HSPs with professional APCs, first reported in 1999 (7Arnold-Schild D. Hanau D. Spehner D. Schmid C. Rammensee H.G. de la Salle H. Schild H. J. Immunol. 1999; 162: 3757-3760Crossref PubMed Google Scholar). Only receptor-mediated uptake of HSP-peptide complexes allows peptide representation by major histocompatibility complex molecules (8Singh-Jasuja H. Toes R.E. Spee P. Munz C. 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Da Costa C. Rammensee H.G. Wagner H. Schild H. J. Biol. Chem. 2002; 277: 20847-20853Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 22Vabulas 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 (808) Google Scholar, 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: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar). This finding immediately raised concerns as to whether or not HSP molecules themselves or HSP-contaminating endotoxins are responsible for the effects observed (24Wallin 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 (505) Google Scholar). This concern was legitimate because all HSP preparations contained low, but detectable, amounts of endotoxin. Nevertheless, several control experiments, including pretreatment of HSP molecules with polymyxin B, which neutralizes LPS, suggested that HSP molecules and not endotoxins were responsible for the biological activity observed (24Wallin 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 (505) Google Scholar). However, modifications of the protocols used for HSP purification were able to dramatically reduce the levels of contaminating and these HSP preparations were not able to activate APCs but as for biological activity B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). were with a in which endotoxin-free HSPs not activate professional APCs, but at the concentrations of to in HSP preparations were also not to induce activation Hilf N. Singh-Jasuja H. Braedel S. P. Rammensee H.G. Schild H. 2003; PubMed Scopus Google Scholar). by a during Gp96 in which levels as with that Gp96 in the this to the that the interaction of Gp96 with the biological activity of that Gp96 molecules in a and B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). with a low of in the presence or of Gp96, observed a of the biological activity of by Gp96 with to the production of proinflammatory cytokines, and the up-regulation of to the of TLR ligands to observed that in to LPS, Gp96 also dramatically the biological activity of the TLR2 the Gp96 activation of in significant activation of These provide for the observed in the biological of More a new function of Gp96 molecules in the amplification of by TLR2 and ligands that be for of innate immune responses during bacterial purification was to the described D. Faath S. Rammensee H. Schild H. J. Exp. Med. 1995; 182: 885-889Crossref PubMed Scopus (295) Google with modifications B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In a of the cell cell in was in and cell the was for at was and with a and and was by on A were with and was on a and by using protein were a of in Gp96 in a at were the of by on described by B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google was for the purification of Gp96 used in were to by in or by for at were in of the as described the was with of and of and and of of Gp96 was as by of was by limulus amoebocyte limulus amoebocyte was as in the which is in with the by the S. and the activation of a in the Gp96 was with the the and at for was with the and was at the and the is in the As used which is the used for of the dendritic of the Gp96 preparations were in a from to Gp96 and for Gp96 using the procedure by B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the of Gp96 was to the described by A. Nicchitta C.V. T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In a was expressed in was by on contaminating was in and at was by the of and was by against endotoxin-free was by limulus amoebocyte of was at the molecular is TLR4 was from TLR2 was from was with with from and with the from protein and the of were from peptide was synthesized by and by were used for by and were from was from and were from the of the of by were by Wagner which the from the T. A. R. C. M.A. Eur. J. Immunol. PubMed Scopus Google Scholar), were by for are for a T cell receptor the peptide from the protein on They were with to were in with the of the of of polymorphonuclear neutrophilic were from from as described using Hilf N. Singh-Jasuja H. Braedel S. P. Rammensee H.G. Schild H. 2003; PubMed Scopus Google Scholar). cells were by a from in was with of cells was by of were from bone to protocols (21Vabulas 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 (427) Google Scholar, K. M. N. H. M. S. S. R.M. J. Exp. Med. 1992; PubMed Scopus Google Scholar). of on of on of was was with were by cells were in with and the was and cells were for at in of of PMN, or the cells were in for activation. were with Gp96, N-terminal Gp96 in the of TLR ligands to Gp96, a preincubation was for at to the of activation, were for were and used for of were in the cell by protocols or by the were from and by They were used to the production by was by of by ELISA, all and were from was the at of was using enzyme that with the of the with the was at of using the from of T in were activated with Gp96, LPS, in for of was as described the was to the peptide was the for at but T cell receptor T cells from the of a were with carboxyfluorescein diacetate succimide for at in the cell was in at a of and the cells were by of surface activation was on and In a from the intracellular production was In the T cells were to cell the the cells were with peptide A to for of the the cells were and in for and intracellular were in all with a and Gp96 been demonstrated to activate BMDC cell function S. Binder R.J. Ramalingam T. Srivastava P.K. Immunity. 2001; 14: 303-313Abstract Full Text Full Text PDF PubMed Scopus (949) Google Scholar, 18Binder R.J. Anderson K.M. Basu S. Srivastava P.K. J. Immunol. 2000; 165: 6029-6035Crossref PubMed Scopus (188) Google Scholar). In it was reported that this activity was of contaminating levels B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). concentrations to in HSP preparations were also not for BMDC activation Hilf N. Singh-Jasuja H. Braedel S. P. Rammensee H.G. Schild H. 2003; PubMed Scopus Google Scholar). for these is that and Gp96 associate with and that this interaction to the BMDC to As Gp96 been demonstrated to function as protein B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), whether such the to were to the of Gp96 and concentrations to production of the proinflammatory and to the of a As in amounts of Gp96 activation of in a at high concentrations by the production of and the and as well as by the production of low amounts of This was also for the production of and by the cell and for the of by not lower concentrations the effects of Gp96 on cell were We for LPS, on the effects on this cell is involved in innate and adaptive immune responses. activation was observed at concentrations of the of in levels of and at production was was observed for the production of using and Gp96 at concentrations unable to induce activation for and for Gp96, in and production was were However, preincubation of with Gp96 for at a significant of the or production Gp96 This was by the up-regulation of molecules and CD40 and also by the of and KC, the production of was not this in amounts of with amounts of Gp96 and As in observed a production that was at of in the presence of Gp96 was for the production of and a was at of the effect of Gp96 was also observed for the and but not for the production of observed the amplification of activation, whether Gp96 the to TLR with different TLR ligands at concentrations that induce a activation of as by the production of low amounts of proinflammatory and of was in the presence or of amounts of As in the different TLR ligands the TLR2 a significant of the production of and in the presence of Gp96 by was for not These results were by the in the of and CD40 not As observed for the of the preincubation of with Gp96 was for the induction of not activation of by depends on the of TLR2 S. B. P. A. 1999; PubMed Scopus Google Scholar). Gp96 was to its effects mainly TLR4 (21Vabulas 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 (427) Google Scholar), to whether or not TLR4 to the of the BMDC activation in the presence of this from and with or as a control in the presence or of in from with production to or in the presence of Gp96 but were able to to was from were with from the presence of Gp96 not from these to to LPS, the was results were for the production of not These results that low Gp96 not BMDC activation on its and not with the of the whether the presence of with the of the We concentrations of to complexes for at and the activation of derived from which are unable to to As Gp96 to the purification to reduce levels or low concentrations of were not able to induce BMDC activation preincubation of Gp96 and a BMDC activation, which with of concentrations of of at BMDC activation but with complexes for the of was BMDC be observed whether the Gp96 or the N-terminal to Immunol. 1992; Full Text PDF PubMed Scopus Google was responsible for the observed low amounts of with different concentrations of Gp96, or as a control protein and As in Gp96 and but not the control protein were able to the by from in a As observed of the was not to whether or not the amplification of the TLR2 and TLR4 into adaptive immune responses. These were by the significant of production observed in the in and naive T cells from a T cell receptor for were with and with activated with or at low concentrations in the presence or of As in or BMDC activation in the presence of Gp96 in a significant of as by the of the and in to activation as by the production of As to be and production were by the up-regulation of and molecules and the of with activated by or in the presence of Gp96 the in surface activation by was to or and not to with a control peptide not HSP-mediated activation of cells of the innate immune been described by and it was that this family of molecules to that and cell (3Schild H. Arnold-Schild D. Lammert E. Rammensee H.G. Curr. Opin. Immunol. 1999; 11: 109-113Crossref PubMed Scopus (86) Google Scholar, 4Srivastava P. Nat. Rev. Immunol. 2002; 2: 185-194Crossref PubMed Scopus (887) Google Scholar, H. Rammensee H.G. Nat. Immunol. 2000; 1: PubMed Scopus Google Scholar, P. Nat. Rev. Immunol. PubMed Scopus Google Scholar). However, this effect been by the finding that HSP preparations used were with low amounts of endotoxins H. D. S. J.P. J.P. S. Haeuw C. de la S.H. Hanau D. Eur. J. Immunol. 2002; PubMed Scopus Google Scholar, B. J. PubMed Scopus Google Scholar). be able to this on the function of HSPs in the activation of innate immune responses As demonstrated previously, that high amounts of Gp96 are able to induce activation of innate immune such as or B. J.P. Nicchitta C.V. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Hilf N. Singh-Jasuja H. Braedel S. P. Rammensee H.G. Schild H. 2003; PubMed Scopus Google Scholar). However, preincubation of Gp96 at concentrations with the TLR or at concentrations also unable to induce BMDC activation in activation as by the of production and of a and TLR is for the induction of BMDC activation levels and In to the production of proinflammatory and also a significant in the production of the and Gp96 was with or but of Gp96 on its the production of RANTES, KC, and low amounts of at concentrations A and the and to be with Gp96 and and not In addition, the interaction of endotoxins with HSPs not biological activity but also with by polymyxin as suggested H. D. S. J.P. J.P. S. Haeuw C. de la S.H. Hanau D. Eur. J. Immunol. 2002; PubMed Scopus Google and the of polymyxin to the effects of different HSP preparations (21Vabulas 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. 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