Heat Shock Proteins, Tolllike receptors and Innate Immunity

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Slide 1 : Heat Shock Proteins, Toll-Like Receptors and Innate Immunity Min-Fu Tsan, M.D., Ph.D. VAMC and Georgetown University Washington, DC June 19, 2007, Stanford University
Slide 2 : Heat Shock Proteins (HSPs) Phytogenetically conserved proteins present in all cells, both constitutively (cognate proteins) and under stressful conditions (Inducible forms) Classified based on related functions and molecular masses, i.e., small HSPs, HSP40, 60, 70, 90 and 110 Primarily function as molecular chaperones, but also function in antigen presentation and cross-presentation
Slide 3 : Cytokine functions of HSPs Since 1993, HSPs have been shown to be potent activators of the innate immune system, capable of inducing the production of cytokines by monocytes and macrophages, and the activation and maturation of dendritic cells. HSP cytokine functions are shown to be mediated by Toll-like receptors (TLRs) 2 & 4. HSPs: Chaperokines; Danger signals; and Endogenous ligands of TLRs
Slide 4 : HSPs: A Link between Innate and Adaptive Immunity HSPs (e.g., Hsp60, 70, 90 and gp96) activate macrophages to release pro-inflammatory cytokines, and induce maturation of dendritic cells via TLRs 2&4. HSPs present chaperoned peptides to T cells directly or after intracellular procession (cross-presentation) via MHC I & II molecules leading to activation of CD4+ and CD8+ T cells. [Srivastava P, Nat Rev Immunol 2: 185-95, 2002]
Slide 5 : “Induction of TNF? and MnSOD by Endotoxin: Role of Membrane CD14 and Toll-like Receptor-4” [Tsan et al., Am J Physiol 280: C1422-30, 2001] Induction of TNFa and Mn-superoxide dismutase (MnSOD) by lipopolysaccharide (LPS) in murine macrophages is mediated by CD14 and TLR4. “Can HSPs induce MnSOD?”
Slide 6 : Recombinant Human Hsp70(rhHsp70) StressGen Biotech. Corp., Victoria, BC, Canada: rhHsp70-1: #ESP-555, low endotoxin preparation (< 50 EU/mg). rhHsp70-2: #NSP-555, not tested for endotoxin, recommended for use in protein-binding assay Endotoxin contents (using chromogenic LAL Assay): rhHsp70-1: 4.1 + 0.2 EU/mg (1.4 pg/µg) rhHsp70-2: 577.0 + 74.2 EU/mg (0.2 ng/ µg) “rhHsp70-1 failed to induced not only MnSOD but also TNFa in murine macrophages!”
Slide 7 : Induction of TNFa Release from Murine Macrophages by rhHSP70 and LPS ? p < 0.05 vs. Control.. A C B ? ?
Slide 8 : LPS contamination is responsible for the induction of TNFa by rhHsp70-2[Gao B, Tsan MF. J Biol Chem 278: 174-9, 2003] rhHsp70-1 & 2 contained similar amounts of Hsp70 as determined using SDS gels and immunoblots. rhHsp70-1 & 2 had similar enzymatic activities as determined by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS by polymyxin B (PMB)-agarose column or direct addition of PMB to the incubation medium eliminated TNFa-inducing activity of rhHsp70-2. Addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in similar TNFa-inducing activity as that in rhHsp70-2.
Slide 9 : Recombinant Human Hsp60(rhHsp60) StressGen Biotech. Corp., Victoria, BC, Canada: rhHsp60-1: #ESP-540, low endotoxin preparation (< 50 EU/mg). rhHsp60-2: #NSP-540, not tested for endotoxin, recommended for use in protein-binding assay Endotoxin contents (using chromogenic LAL Assay): rhHsp60-1: 8.6 + 3.9 EU/mg (3.2 pg/µg) rhHsp60-2: 85.6 + 20.6 EU/mg (32 pg/ µg)
Slide 10 : Effect of rhHSP60 on TNF? Release by Murine Macrophages A B ? p < 0.05 vs. Control.. ? ?
Slide 11 : A B Effect of Polymyxin B on the Endotoxin Activity and TNFa-Inducing Activity of rhHsp60-2 and LPS
Slide 12 : * * * * * * rhHSP60 1 2 3 4 B C A Effect of Polymyxin B Agarose Column on Endotoxin Activity and TNFa-Inducing Activity of rhHSP60-2 rhHSP60-2 and PB fractions were tested at 5µg/ml. * p < 0.01 vs. rhHSP60-2. Lane 1: rhHSP60 Lane 3: PB-2 Lane 2: PB-1 Lane 4: PB-3
Slide 13 : rhHsp60 does not Induce TNFa release from Murine Macrophages[Gao B & Tsan MF. J Biol Chem 278: 22523-9, 2003] rhHsp60-1 & 2 contained similar amounts of Hsp60 as determined using SDS gels and immunoblots. rhHsp60-1 & 2 had similar ATPase activities. Approximately 50% of the rhHsp60-2 TNFa-inducing activity was due to LPS contamination and the rest was due to the contamination of LPS-associated molecule(s).
Slide 14 : Hsp60 & 70 do not Induce Cytokines in Murine Macrophages and Lymphocytes Using a gene expression array, rhHsp60-1 and rhHsp70-1 had no effect on any of the 96 common cytokine genes in murine macrophages. (Gao B & Tsan MF. BBRC: 317: 1149-1154, 2004) Similarly, rhHsp60-1 and rmHsp60 had no effect on any of the 113 common cytokine genes in murine splenocytes. (Wang Y, Gao, B & Tsan MF. Cytokine 32: 149-154, 2005)
Slide 15 : Microbial Cell-Wall Contaminants in Peptides: A potential Source of Physiological Artifacts [Majde JA. Peptides 14: 629-32, 1992] “Microbial cell wall products (MCWP) are ubiquitous contaminants of materials that are not processed using aseptic good manufacturing practice conditions employed by the pharmaceutical industry.” “A simple MCWP control procedure can be used to test heat-susceptible peptides from any source: simple heat inactivation. Boiling peptide X for at least 30 min will denature most larger peptides but will leave the MCWP contaminants unaffected.”
Slide 16 : Ruling out LPS Contamination Two most common methods: Simple heat inactivation with the assumption that LPS is heat resistant Use polymyxin B (PMB) to inhibit the biological activities of LPS
Slide 17 : Endotoxin is Heat Resistant Richard Pfeifer’s original observation: heat-inactivated Vibrio cholearae were still capable of inducing irreversible shock in experimental animals. Pyrogenic effect of LPS is resistant even to autoclaving. Dry heat at 250oC for 1-2 h or at 180oC for 4 h is required to render a substance pyrogen-free.
Slide 18 : Cytokine-Inducing Effect of HSPs is Heat-Sensitive
Slide 19 : A B LPS is Heat Sensitive! * * * * * * * p < 0.05 vs. non-heated.
Slide 20 : Effect of Duration of Boiling on the TNFa-Inducing Activity of LPS[LPS, 200 ng/ml was heated for 15, 30 or 60 min, 0.1 to 10 ng/ml of control or boiled LPS were then tested.]
Slide 21 : The Heat Sensitivity of Cytokine-Inducing Effect of Lipopolysaccharide [Gao B, Wang Y & Tsan MF. J Leukoc Biol 80: 359-66, 2006] Boiling LPS for 15 min inactivated ~90% of TNFa-inducing activity. Heat-induced inactivation of LPS activities were not due to adherence of boiled LPS to container wall or aggregation of boiled LPS. Boiled LPS retained its ability to bind PMB. Boiling LPS reduced LPS aggregate sizes as determined by native PAGE. Boiling also inactivated the TNFa-inducing activity of diphosphoryl lipid A (DPLA). Heat-induced inactivation of DPLA was not due to its conversion to monophosphoryl lipid A (MPLA).
Slide 22 : Toll-Like Recptors (TLRs) Mammalian homologues of Drosophila Toll protein Members of IL-1R superfamily: cytoplasmic Toll/IL-1R (TIR) domain and extracellular leucine-rich repeat domain (LRR) Important role in innate host defense against invading microbes by recognizing conserved motifs of microbial origins, i.e., pathogen-associated molecular patterns (PAMPs)
Slide 23 : TLR Ligands Based on PAMPs, TLRs can be grouped into at least three families: TLR2 (TLR1/2 & TLR2/6) & TLR4: Lipid-based ligands, e.g., bacterial lipoproteins, lipoteichoic acid, (TLR2), LPS (TLR4) TLR3, 7, 8 & 9: Bacterial and viral nucleic acids, e.g., dsRNA (TLR3), ssRNA (TLR7 & 8), non-methylated CpG DNA (TLR9) TLR5 &11: Microbial proteins, e.g., flagellin (TLR5), profilin (murine TLR11) [No ligand has been identified for TLR10]
Slide 24 : Reported Endogenous Ligands of TLRs [Tsan MF & Gao B. J Endotoxin Res, 13: 6-14, 2007] __________________________________________________________ TLRs Endogenous Molecules __________________________________________________________ TLRs2&4 Hsp60, Hsp70,Gp96; HMGB1; Urate crystal TLR4 Fibrinogen; Surfactant protein-A; Fibronectin extra domain A; Heparan sulfate; Soluble hyaluronan; ß-Defensin 2-fusion protein (rmDF-2/rfv); Minimally modified low density lipoprotein (mmLDL); Pancreatic elastase; Hsp22; aA crystallin TLR7 RNA immune complex TLR9 Chromatin immune complex; DNA Immune complex ____________________________________________________________
Slide 25 : Hypotheses for Endgoenous Ligands of Immune Receptors Danger Model (Matzinger, 1994 vs. Self-Non-Self Model, Janeway, 1989) Surveillance Model (Johnson et al., 2003) Hydrophobicity Model (Seong & Matzinger, 2004)
Slide 26 : PAMP Contamination as Putative Endogenous Ligands of TLRs [Tsan MF & Gao B. J Endotxin Res, 13: 6-14, 2007] HSPs: Hsp60, Hsp70, Hsp90 & gp96 Pancreatic elastase (thought to be responsible for systemic inflammatory response syndrome associated with severe acute pancreatitis) C-reactive protein (thought to a mediator of atherosclerosis and atherothrombotic events) Surfactant protein A High mobility group box 1 (HMGB1) protein (thought to be a late mediator of endotoxic shock)
Slide 27 : Reasons for failure to recognize PAMP contamination Failure to use highly purified preparations free of PAMP contamination Failure to recognize the heat sensitivity of LPS Failure to consider contamination by PAMPs other than LPS
Slide 28 : Avoiding Designation of PAMP Contamination as Putative Endogenous Ligands of TLRs Use only highly purified preparations that are essentially free of LPS contamination Measure endotoxin activity using LAL assay Do not use heat sensitivity as a criterion to rule out LPS contamination Use Polymyxin B (PMB) to inhibit LPS activity Use endotoxin removal system (e.g., PMB column) to re-purify the preparations No readily available methods to rule out contamination of PAMPs other than LPS

 


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