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BIO 5051 Foundations in Immunology

This document discusses the fundamental aspects of inflammation as a response to microbial infections, allergens, and tissue injury. It covers local and systemic features of inflammation, including upregulation of adhesion molecules, increased blood flow, and phagocytosis, as well as fever and the acute phase response. Key transcription factors in the immune system such as GR, NF-kB, NFAT, and others are highlighted, emphasizing their roles in mediating both pro-inflammatory and anti-inflammatory gene expression.

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BIO 5051 Foundations in Immunology

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  1. BIO 5051Foundations in Immunology Signaling in lymphocytes (transcription factors) November 04, 2005 Robert H. Arch arch@wustl.edu phone 747-4681

  2. Inflammation • response to?microbial infections and tissue injury • local features include? upregulation of adhesion molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation • systemic features include? fever and acute phase response • mediated and resolved by? a large array ofsoluble factors, cell surface molecules, and enzymes 2003 0390

  3. Inflammation • response to infections, allergens and tissue injury • local features include? upregulation of adhesion molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation • systemic features include? acute phase response and fever • mediated and resolved by? a large array ofsoluble factors, cell surface molecules, and enzymes 2003 0390

  4. Inflammation • response to infections, allergens and tissue injury • local features include upregulation of adhesion molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation • systemic features include? acute phase response and fever • mediated and resolved by? a large array ofsoluble factors, cell surface molecules, and enzymes 2003 0390

  5. Inflammation • response to infections, allergens and tissue injury • local features include upregulation of adhesion molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation • systemic features include acute phase response and fever • mediated and resolved by? a large array ofsoluble factors, cell surface molecules, and enzymes 2003 0390

  6. Inflammation • response to infections, allergens and tissue injury • local features include upregulation of adhesion molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation • systemic features include acute phase response and fever • mediated and resolved by a large array of soluble factors, cell surface molecules and enzymes 2003 0390

  7. dexamethasone cortisol, hydrocortisone Glucocorticoids activate transcriptionof anti-inflammatory genes HSP-90 HSP-90 Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54 2005 0536

  8. Acetylation of core histones regulates gene repression and transcription Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54 2005 0533

  9. Glucocorticoids inhibit transcriptionof pro-inflammatory genes Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54 2005 0535

  10. Transcription factorsof the immune system • GR glucocorticoid receptor • NF-kB nuclear factor kappa B • NFAT nuclear factor of activated T cells • AP-1 activating protein-1 • STAT signal transducer and activator of transcription • GATA-3 (A/T)GATA(A/G) consensus binding motif • T-bet T box expressed in T cells • p53 tumor suppressor p53 • Smad Sma/Mad (C. elegans/Drosophila) 2003 0349

  11. Transcription factorsof the immune system • GR glucocorticoid receptor • NF-kB nuclear factor kappa B • NFAT nuclear factor of activated T cells • AP-1 activating protein-1 • STAT signal transducer and activator of transcription • GATA-3 (A/T)GATA(A/G) consensus binding motif • T-bet T box expressed in T cells • p53 tumor suppressor p53 • Smad Sma/Mad (C. elegans/Drosophila) 2003 0349

  12. Nuclear factor kappa B (NF-kB) • first described as a nuclear factor in B cells that binds to a 10 bp region of the k intronic enhancer and is pivotal for Ig k light chain transcription • can be found in the cytoplasm of most cell types • family of dimeric transcription factors • monomers have 300 aa Rel homology region required for dimerization, DNA binding, and interaction with inhibitor proteins (IkB) • release from IkB results in nuclear translocation 2003 0357

  13. Li and Verma (2002), Nature Rev. Immunol. 2:725-34 • RHD: Rel-homology domainTD: transactivation domainN: nuclear localization signalLZ: leucine zipperGRR: glycine-rich regionANK: ankyrin repeats • transcriptionally active:p65/p50, p65/p65, p50/c-Rel • transcriptionally inactive:p50/p50, p52/p52 • p100/p52 and l05/p50 are precursors • processing (signal-dependent and -independent pathways?) is ATP-dependent, requires poly-ubiquitination of IkB, and can be blocked by proteasome inhibitors 2003 0379

  14. Chen et al. (1998), Nature 391:410-3 • RHD: Rel-homology domainTD: transactivation domainN: nuclear localization signalLZ: leucine zipperGRR: glycine-rich regionANK: ankyrin repeats • transcriptionally active:p65/p50, p65/p65, p50/c-Rel • transcriptionally inactive:p50/p50, p52/p52 • p100/p52 and l05/p50 are precursors • processing (signal-dependent and -independent pathways?) is ATP-dependent, requires poly-ubiquitination of IkB, and can be blocked by proteasome inhibitors 2003 0379

  15. Li and Verma (2002), Nature Rev. Immunol. 2:725-34 • RHD: Rel-homology domainTD: transactivation domainN: nuclear localization signalLZ: leucine zipperGRR: glycine-rich regionANK: ankyrin repeats • transcriptionally active:p65/p50, p65/p65, p50/c-Rel • transcriptionally inactive:p50/p50, p52/p52 • p100/p52 and l05/p50 are precursors • processing (signal-dependent and -independent pathways?) is ATP-dependent, requires poly-ubiquitination of IkB, and can be blocked by proteasome inhibitors 2003 0379

  16. NF-kB inhibitors (IkB) • IkBa, IkBb, IkBg, IkBd, IkBe, Bcl-3 • central ankyrin repeat mediate interaction with rel-homology domains of NF-kB proteins • N-terminal domain is phosphorylated in response to NF-kB activating signals • phosphorylation of two conserved Ser residues is required for ubiquitylation and degradation • C-terminal PEST domain involved in basal turnover 2003 0384

  17. NF-kB activation by LTbR vs. TNFR-I Yilmaz et al., (2003) EMBO J. Vol 22:121-30 2003 267

  18. adhesion molecules intercellular adhesion molecule-1 (ICAM-1) vascular cell adhesion molecule-1 (VCAM-1) E-selectin cytokines tumor necrosis factor a (TNF-a) interleukin-1b interleukin-6 interleukin-11 granulocyte-macrophage colony stimulating factor (GMCSF) chemokines interleukin-8 CCL3 (macrophage inflammatory protein (MIP)-1a ) CCL7 (monocyte chemotactic protein (MCP)-3) CCL5 (RANTES) CCL11 (eotaxin) enzymes inducible nitric oxide synthase (iNOS) cyclooxygenase-2 (COX-2) cytosolic phospholipase A2 5-lipidoxygenase (5-LOX) anti-apoptotic proteins TNFR-associated factors (TRAF) 1 and TRAF2 cellular inhibitor of apoptosis (c-IAP) 1 and c-IAP2 bcl-2 homologues AI/Bfl-1 and bcl-xL NF-kB and IkB family members IkBa NF-kB1 (p105/p50) NF-kB2 (p100/p52) RelB NF-kB-regulated genes 2003 0358

  19. A20 CYLD A central role for ubiquitinin multiple signalling pathways Chen (2005). Nature Cell Biol. 7:758-65 2005 0532

  20. The NF-kB signalling pathways IL-1R, TLR BAFF-R, LTbR, CD40 TNFRs canonical non-canonical modified from Chen (2005). Nature Cell Biol. 7:758-65 2005 0531

  21. ikka-/- and ikkb-/- • ikka-/-mice die post-natally due to multiple morphological defects; shiny taut skin prevents emergence of fore- and hind-limbs, absence of ears, truncation of head, skeletal abnormalities • ikkb-/-mice die between E12.5 and E14.5 as a result of fetal hepatocyte apoptosis; embryonic lethality is rescued by crossing with TNFR-I-/-and TNF-a-/- animals

  22. IKKa-/- Hu et al. (1999), Science 284:316 2003 0350

  23. IKKa-/- Hu et al. (1999), Science 284:316 2003 0350

  24. IKKb-/- Li et al. (1999), J. Exp. Med. 189:1839 2003 0351

  25. Li and Verma (2002), Nature Reviews 2:725 2002 006

  26. ikba-/- • normal embryonic development, but mice die7-10 days post-natally due to severe widespread dermatitis and granulocytosis (delayed in DKO) • increased expression of distinct pro-inflammatory cytokines and factors associated with granulocyte recruitment, such as TNF-a, G-CSF and VCAM • not all genes induced by NF-kB are upregulated

  27. Doi et al. (1997), J. Exp. Med. 185:953 rela-/- • embryonic lethality between E15 and E16 due to fetal hepatocyte apoptosis induced by TNF-a • embryonic lethality can be rescued by crossing with TNFR-I-/- and TNF-a-/- animals • reconstitution of SCID mice with fetal hepatocytes revealed defects in mitogen-induced proliferation and isotype switching but normal lymphopoiesis

  28. rela-/- • embryonic lethality between E15 and E16 due to fetal hepatocyte apoptosis induced by TNF-a • embryonic lethality can be rescued by crossing with TNFR-I-/- and TNF-a-/- animals • reconstitution of SCID mice with fetal hepatocytes revealed defects in mitogen-induced proliferation and isotype switching but normal lymphopoiesis

  29. p50-/- (NFKB1-/-) • despite nearly ubiquitous expression and its role as major partner of p65 (Rel A), which is essential for embryogenesis, surprisingly normal development • although not essential for hematopoiesis, multiple defects in functions of immune system

  30. Histone acetylation regulatesNF-kB-induced transcription Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54 2005 0534

  31. Activating protein 1 (AP-1) • family of dimeric transcription factors • expressed at low levels • usually constitutively bound to their DNA sites • rapid changes of complex composition upon stimulation of cells due to de novo synthesis • phosphorylation by MAPK, e.g., c-Jun N-terminal kinase (JNK), strongly enhances transactivating capacity • play crucial roles in cell proliferation, apoptosis and oncogenesis 2003 0355

  32. Signals leading toIL-2 expression in CD4+ cells Foletta et al (1998) J. Leukoc. Biol. 63:139. 2004 0474

  33. Interactions between AP-1 proteins and other transcription factors Foletta et al (1998) J. Leukoc. Biol. 63:139. 2004 0475

  34. Co-operative DNA bindingof NFAT and AP-1 proteins Monomeric NFAT and heterotrimeric AP-1 transcription factors havelow affinity for their respectivebinding sites. Interactions betweenNFAT and AP1 stabilize theNFAT-AP1-DNA complex. Fig 11.24 Lodish et al. Molecular Cell Biology 2004 0471

  35. Nuclear factor of activated T cells (NFAT) • first identified in T cells as rapidly inducible nuclear factor binding to the IL-2 promoter • family of transcription factors related to NF-kB • expressed in most cells of the immune system, including lymphocytes, mast cells, basophils,NK cells and endothelial cells • target genes include cytokines, cell surface receptors, signaling proteins and transcription factors 2003 0381

  36. The NFAT family renal atrophy and lack of tonicity-responsive gene expression modified from Macián et al. (2001) Oncogene 20:2476. 2004 0472

  37. Signal transduction byCa2+, calcineurin and NF-AT Crabtree (1999) Cell 96:611. 2004 0473

  38. Signal transduction byCa2+, calcineurin and NF-AT Macian (2005) Nature Reviews Immunology 5, 472-84. 2004 0473

  39. Macián et al. (2001) Oncogene 20:2476.

  40. Analysis of NFAT1 Phosphorylation.Okamura et al. (2000) Mol. Cell 6:539. ST1 ST4 ST5 ST2 ST8

  41. The SRR-1 Region Regulates the Active Conformation of NFAT1.Okamura et al. (2000) Mol. Cell 6:539.

  42. Macián et al. (2001) Oncogene 20:2476.

  43. O’Shea et al. (2004), Nature Rev. Drug Disc. 3:555-64 O’Shea et al. (2004), Nature Rev. Drug Disc. 3:555-64 JAK/STAT signal transduction Janus kinases • Jak1 • Jak2 • Jak3 • Tyk2 Signal transducer andactivator of transcription • Stat1 • Stat2 • Stat3 • Stat4 • Stat5a • Stat5b • Stat6 Benekli et al. (2003), Blood 101:2940-54 2003 0366

  44. STAT1 is activated by IFNg McBride et al. (2000), EMBO J. 19:6196-206 2003 0376

  45. STAT domain structureand protein binding sites Levy and Darnell. (2003),Nature Rev. Mol. Cell Biol. 3:651-62 2003 0368

  46. Leptomycin B inhibits nuclear exportof STAT1 McBride et al. (2000), EMBO J. 19:6196-206 2003 0372

  47. Intracellular localization ofSTAT1 DNA binding mutant McBride et al. (2000), EMBO J. 19:6196-206 2003 0373

  48. Identification of STAT1 nuclear export signal McBride et al. (2000), EMBO J. 19:6196-206 2003 0373

  49. Effect of NES placement outside ofthe STAT1 DNA biding domain McBride et al. (2000), EMBO J. 19:6196-206 2003 0374

  50. Grogan and Locksley (2002), Curr. Opin. Immunol. 14:366

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