Genetic Susceptibility Chronic Beryllium Disease and Sarcoidosis Lee S. Newman, MD, MA, FCCP, FACOEM Prof essor of Prev entiv e Medicine and Biometrics and Prof essor of Medicine Univ ersity of Colorado Denv er/health Sciences Center and National Jewish Medical and Research Center 1 Outline 1) Overview Genetics and Exposure Granulomatous disorders, immunopathogenesis 2) Chronic Beryllium Disease (Berylliosis, CBD) Genetic epidemiology Functional implications Genes and bery llium exposure 3) Sarcoidosis Genetic epidemiology Functional implications Implications f or f inding the cause 2 Genetics and Exposure Environment and genes interact Genetic Background -- Environmental Cause Going beyond the lip-service Exposure circumstances poorly understood Workplace environments Opportunity to characterize exposures 3 The screen versions of these slides have full details of copyright and acknowledgements 1
Workplace Immunologic Disorders of Known Environmental Cause Asthma: Platinum salt sensitization and asthma HLA-DR3 x low level platinum exposure Hypersensitivity Pneumonitis: TNF-α promoter polymorphism x bird protein Chronic Beryllium Disease: HLA-DP x beryllium 4 Granulomatous Disorders Immune- mediated hypersensitivity disorders Pathologic response to w ide range of pathogens and antigens Causes Inf ectious agents and their antigens Organic antigens Autoantigens Metal antigens Idiopathic 5 Non-caseating Granulomas 6 The screen versions of these slides have full details of copyright and acknowledgements 2
Non-caseating Granuloma 7 Immunopathogenesis Immunologic over-reaction to unknown triggering antigen or antigens Adaptive immune response Innate immune response Pivotal roles for CD4+ T cells Cytokine cascade 8 Immunopathogenesis Naïve T cell Clonal Expansion Ag-specific CD4+ Differentiation APC Effector Central IFN-γ, IL-2, TNF-α IL-2 9 The screen versions of these slides have full details of copyright and acknowledgements 3
The Trimolecular Complex Antigen TCAR T cell APC MHC 10 Genetic Implications Likely suspects based on biology Antigen processing and presentation genes Antigen recognition genes Cytokine expression genes 11 Genetic Epidemiology of Granulomatous Disorders: Ch 6 12 The screen versions of these slides have full details of copyright and acknowledgements 4
Genetic Epidemiology of Granulomatous Disorders The HLA Complex DP β α DM β α LMP/TAP DQ β α DR β β α C4b Bf HSP 70 1 and 2 TNFα B C A C4a C2 TNFβ Class II Class III Class I 13 A Tale of Two Disorders Two kindred granulomatous disorders Chronic Beryllium Disease Known etiology Sarcoidosis Unknown etiology 14 Chronic Beryllium Disease (CBD) 15 The screen versions of these slides have full details of copyright and acknowledgements 5
CBD 16 Immunopathogenesis of CBD Alveolus Beryllium Antigen Lymphocyte Macrophage 17 HLA DPB1 Glu69 (vs K or R) 97% Cases vs. 30% Controls Higher frequency of 0201 alleles (Glu69) Richeldi (Science 1993) Expression of HLA-DPB1 polymorphisms coding for amino acid substitutions at variable regions C, D (CBD black; Be-exp white) 18 The screen versions of these slides have full details of copyright and acknowledgements 6
Richeldi, Science 1993 n= 33 CBD 44 Controls Richeldi, Am J Ind Med 1997 n= 6 CBD 121 Controls Wang, J Immunol 1999 n= 20 CBD 34 Controls Frequency of HLA DPB1 Glu 69 AUTHOR CBD BeS CONTROLS Maier, AJRCCM 2001 n= 103 CBD 52 BeS 122 Controls Wang, Tox 2001 n= 25 BeS 63 Controls Saltini, Eur Respir J 2001 n= 22 CBD 23 BeS 93 Controls Rossman, AJRCCM 2002 n= 25 CBD 23 BeS 82 Controls 97% NA 30% 83% NA 30% 95% NA 45% 85% 87% 38% NA 88% 38% 73% 39% 40% 84% 90% 47% 19 Genetic Epidemiology of CBD ILLUSTRATION: K. SUTLIFF 20 Newman, Science 1993 Other Genetic Risk Factors Less robust than Glu69 HLA-DR HLA-DR Arg74 (Saltini 2001) HLA-DRB1 (Rossman 2002) HLA-DR1: CBD 5% BeS 13% Controls 17% (Maier 2001) HLA-DQ HLA-DQB1 Gly 86: CBD 28% BeS 10% Controls 6% (Rossman 2002) -DQ6: CBD 33% BeS 20% Controls 23% (Maier 2001) TNF-alpha promoter polymorphism -308 A to T (Maier 2004) 21 The screen versions of these slides have full details of copyright and acknowledgements 7
Functional Significance of Genes in CBD CBD excellent opportunity to provide biological validity Mechanisms that can be investigated in vitro Antigen available Access to cells from affected organ, e.g. lung 22 Anti-DP mab Blocks BeSO 4 -Induced Proliferation of Be-Specific T Cell Line 100 % Inhibition 75 50 25 anti-dp (B7.21) anti-dr (L243) anti-dr LB3.1) anti-dq (SVPL3) 0 0.1 1.0 3.0 30 Antibody Concentration (µg/ml) 23 Fontenot et al. 2000 PNAS Inhibition of Be-Induced IFN-γ Expression with anti HLA-DP 100 100 75 75 % Inhibition 50 25 Anti-DP (B7.21) Anti-DR (LB3.1) Anti-DQ (SVLP3) % Inhibition 50 25 0 0.1 1 10 Concentration of Anti-class II mab (µg/ml) 0 30 3 0.3 30 30.3 30 30.3 Anti-DP Anti-DR Anti-DQ (µg/ml) 24 Fontenot et al. 2002 J Clin Invest The screen versions of these slides have full details of copyright and acknowledgements 8
Proliferation of CBD T Cell Line to BeSO 4 Presented by DP-Expressing Fibroblast 10 0 Thymidine Incorporation (CPM x 10-3 ) 7.5 5.0 2.5 anti-dr anti-dp 25 50 75 100 % Inhibition 0 No BeSO4 BeSO4 1 x 10-5 M 0 0.1 1.0 10 30 Antibody Concentration ( µg/ml) 25 Fontenot et al. 2000 PNAS Proliferation of CBD T Cell Line to BeSO 4 Presented by Different APCs 60 40 Control 1 Control 2 CPM x 10-3 Control 3 20 9.5 20 178 <2 Stimulator Cells DPB1 DRB1 Autologous LCL CBD Patient 4 Control 4 Control 5 *0201 *1101 *0201, *0301 *1302, *1502 *0201 *1302, *1502 *0401, *0402 *1101, 1103 *0401, *1701 *0701, 1101 *0301, *0401 *03, 1301 *0401 *1001 0 Unstimulated BeSO4 1 x 10-5 M 26 Fontenot et al. 2000 PNAS Polymorphic Amino Acid Residues of Selected HLA-DPB1 Alleles DPB1*0101 DPB1*0401 Ability to Present Be Amino Acid Position 7 8 9 11 17 33 35 36 55 56 57 65 67 68 69 76 84 85 86 87 - Y V Y G A E Y A A A E I E E K V D E A V - - L F - - - F - - - - - - - - M G G P M DPB1*0201 DPB1*0402 DPB1*0601 DPB1*1001 DPB1*1701 + - L F - - - F V D E - - - - E M G G P M + - L F - - - F V D E - - - - - M G G P M + - - - L - - F V D E D L - - E M - - - - + - - H L - - F V D E - - - - E - - - - - + - - H L - - F V D E D - - - E M - - - - 27 The screen versions of these slides have full details of copyright and acknowledgements 9
Modeling of HLA-DP based on HLA-DR structure 69 55 56 28 TNF-α levels are Associated with TNF Genotype 15000 12500 TNF-α pg/ml 10000 7500 5000 2500 0 TNF1 TNF2 Medians and interquartile ranges 25%, 75% and 10% and 90% limits, p=0.04 Maier, 2001 29 Adaptive Immunity Beryllium Exposure Innate Immunity Genetic Susceptibility HLA-DPB1 Glu69 Genetic Susceptibility TNF-α promoter Beryllium Antigen Presentation Beryllium-specific T Cell Recognition Oxidative Stress Beryllium-induced Cytokine Expression (TNF-α, IFN-γ) Beryllium Sensitization Unregulated Inflammatory Response Granuloma Formation and Fibrosis: Chronic Beryllium Disease 30 The screen versions of these slides have full details of copyright and acknowledgements 10
Gene x Environment in CBD Many Glu69+ beryllium workers do not get Beryllium sensitization or CBD Some Glu69- beryllium workers get CBD Exposure related risk for BeS and CBD Model condition for understanding gene x environment interaction 31 Beryllium Machinist Risk for CBD: Relatively Higher Beryllium Exposures 32 Kreiss, 1996 Beryllium: Exposure x Glu69 (Richeldi Am J Ind Med 1997) Machinists Non-Machinists Total (0.9 µg/m3) (0.3 µg/m3) Glu69 Pos. 4/16 1/25 5/41 (25%) (4%) (12.2%) Glu69 Neg. 1/31 0/55 1/86 (3.2%) (0%) (1.2%) Total 5/47 (10.6%) 1/80 (1.3%) 6/127 (4.7%) 33 The screen versions of these slides have full details of copyright and acknowledgements 11
Non-caseating granuloma 34 Differential Diagnosis Environmental exposures Organic and inorganic antigens Hypersensitivity pneumonitis Metals Chronic beryllium disease Silicates 35 Differential Diagnosis Infectious agents Mycobacteria Fungi Bacteria Spirochetes Parasites Neoplasms Vasculitis Wegener s granulomatosis Churg-Strauss, Lymphomatoid granulomatosis Autoimmune disorders Lupus Primary biliary cirrhosis 36 The screen versions of these slides have full details of copyright and acknowledgements 12
Sarcoidosis Like CBD, except cause is not known Multisystem, inflammatory disorder Non-caseating granulomas are hallmark Many and varied clinical manifestations clinical presentation course prognosis 37 Worldwide Epidemiology Prevalence 1-40/100,000 Age-adjusted incidence rates 11/100,000 U.S. whites 36/100,000 U.S. African Americans All genders, races, ethnicities, ages Most often: Adults <40 Female Severity and clinical patterns vary by race 38 Diagnostic Criteria 1. Presence of consistent clinical and radiographic picture 2. Demonstration of non-caseating granulomas on biopsy 3. Exclusion of other conditions that produce similar pathology Statement on sarcoidosis. Am J Respir Crit Care Med (1999) Newman et al. N Engl J Med (1997) 39 The screen versions of these slides have full details of copyright and acknowledgements 13
Organ Involvement Sarcoidosis can affect most organs, predominantly Lungs Lymphatics Eyes Skin Liver Bone Heart 40 Clinical Presentations Incidental chest x-ray, asymptomatic Chronic Usually more gradual onset Acute Löfgren s syndrome Erythema nodosum Bilateral hilar lymphadenopathy Fever polyarthritis 41 The Challenge of Sarcoidosis Complex clinical phenotype Complex genetics Complex data on possible etiologies Hypothesis: HLA x Antigen = Clinical Subtype 42 The screen versions of these slides have full details of copyright and acknowledgements 14
Approach: Divide and Conquer Identify and isolate clinical phenotypes Find gene associations (case control; familial linkage) Seek antigens in subsets by phenotype/genotype 43 Genetic Suspects in Sarcoidosis Based on Mechanism HLA Chemokines Cytokines Other Appreciating the role of haplotypes 44 Clinical Subsets of Sarcoidosis Clarify Genetic Susceptibility Sarcoidosis in general Increased Risk: HLA-DR14, -DR12 associations Protection: HLA-DR1 and -DR4 (on HLA-DQB1*0301 haplotype) Uveitis Increased Risk: HLA-DR4, DR15 association HLA-DQB1*0601 + DRB1*0803 (Japanese) HLA-DRB1*15 + DQB1*0602 (Caucasian) HLA-DRB1*04 + DQB1*0301 (Both) Protection: Löfgren's HLA-DR3 HLA-DR3 (DRB1*0301-DQB1*0201) CCR2 45 The screen versions of these slides have full details of copyright and acknowledgements 15
Clues from Familial Study Schürmann et al. (AJRCCM 2001) Sarcoidosis familial linkage study 3p21: chemokine receptors CCR2, CCR5 Subsequent studies of polymorphisms in sarcoidosis clinical subtypes 46 Combined Effect of HLA-DR3,CCR2 Sum of -DR3 CCR2 hap 2 Controls n=171 Non Löfgren n=92 Löfgren n=43 Total 0 77 (61%) 45 (36%) 4 (3%) 126 1 70 (61%) 35 (31%) 9 (8%) 114 2 22 (37%) 12 (20%) 26 (43%) 60 3/4 2 (33%) 0 4 (67%) 6 47 Spagnolo et al, 2003 TNF-α -307A promoter polymorphism Carrier Frequencies in Löfgren's Controls n = 576 38% Non- Löfgren s Sarcoidosis Löfgren s Syndrome n = 180 38% n = 41 78% Sato & Grutters, AJRCCM 2003 48 The screen versions of these slides have full details of copyright and acknowledgements 16
Löfgren's syndrome: Population Attributable Risk Genetic basis > fully explained by three strong genetic associations HLA DR*0301-DQB1*0201 TNF 307A haplotype CCR2 haplotype 2 Complex disease in w hich genetics resolved Goal: the rest of sarcoidosis CCR5, BTNL2, TGF-Beta, others 49 Implications of Genetics for Discovering Cause of Sarcoidosis Can understanding genetic basis of a condition like Löfgren's lead to causative agent? Back to immunology and biochemisty Characterizing the T cell antigen receptor Isolating the HLA-antigen complex 50 T Cell Antigen Receptor Usage in Löfgren s Syndrome Suggests Specific Antigen Oligoclonal AV2S3+ T cells in bronchoalveolar lavage - only in sarcoidosis patients, not controls Only in DR17 + sarcoidosis patients, - often w ith Löfgren s syndrome Express high levels of activation markers Associate with disease activity and good prognosis 51 The screen versions of these slides have full details of copyright and acknowledgements 17
Strategy to find Cause of Löfgren s Grunewald and colleagues: Collect BAL cells of HLA-DR17+ patients Separate HLA-DR molecules Elute peptides from HLA-DR molecules 52 Strategy to Isolate and Test Antigen: Immunologic Proof Separate these peptides (HPLC) Determine peptide sequences (mass spectrometry) Identify the protein(s) Synthesize peptides and test for T cell stimulatory capacity Selective stimulation of AV2S3+ T cells 53 Conclusions Importance of well-designed, population-based studies of genotype Care and attention to clinical phenotype Take clues from mechanism Confirm gene function in disease state Use exposure to detect genetic susceptibility Use genetic susceptibility to pursue exposure Expect complex diseases to have complex genetic answers 54 The screen versions of these slides have full details of copyright and acknowledgements 18
References: Beryllium Saltini, C. et al. (1989) Maintenance of alveolitis in patients with chronic beryllium disease by beryllium-specific helper Tcells. N. Engl. J. Med. 320, 1103 1109. Richeldi, L. et al. (1993) HLA-DPB1 glutamate 69: a genetic marker of beryllium disease. Science 262, 242 244 Newman LS. (1993) To Be2+ or not to Be2+: immunogenetics and occupational exposure. Science. 262:197-8 Kreiss, K. et al. (1996) Machining risk of beryllium disease and sensitization with median exposures below 2 mg/m 3. Am. J. Ind. Med, 30, 16-25. Richeldi, L. et al. (1997) Interaction of genetic and exposure factors in the prevalence of berylliosis. Am. J. Ind. Med. 32, 337 340 Tinkle, S.S. et al. (1997) Beryllium induces IL-2 and IFN-g in berylliosis. J. Immunol. 158, 518 526 55 References: Beryllium Fontenot, A.P. et al. (1998) Expansi ons of T-cell subsets expressing particular T cell receptor variable regions in chronic beryllium disease. Am. J. Respir. Cell Mol. Biol. 18, 581 589 Fontenot, A.P. et al. (1999) Identificati on of pathogenic T cells in patients with beryllium-induced lung disease. J. Immunol. 163, 1019 1026 Fontenot, A.P. et al. (2000) Beryllium presentati on to CD4+ T cells underlies disease suscepti bility HLA-DP alleles in chronic beryllium disease. Proc. Natl. Acad. Sci. U. S. A. 97, 12717 12722 Fontenot, A.P. et al. (2001) Chronic beryllium disease: T cell recognition of a metal presented by HLA-DP. Clin. Immunol. 100, 4 14. 56 References: Beryllium Wang, Z. et al. (2001) Beryllium sensitivity is linked to HLA-DP genotype. Toxicology 165, 27 38 Amicosante, M. et al. (2001) Beryllium binding to HLA-DP molecule carrying the marker of susceptibility to berylliosis glutamate Β69. Hum. Immunol. 62, 686 693 Maier, L.A. et al. (2001) High beryllium-stimulated TNF-a is associated with the -308 TNF-α promoter polymorphism and with clinical severity in chronic beryllium disease. Am. J. Respir. Crit. Care Med. 164, 1192 1199 Fontenot, A.P. et al. (2002) Target organ localization of memory CD4+ T cells in patients with chronic beryllium disease. J. Clin. Invest. 110, 1473 1482. Rossman, M.D. et al. (2002) Human leukocyte antigen class II amino acid epitopes: susceptibility and progression markers for beryllium hypersensitivity. Am. J. Respir. Crit. Care Med. 165, 788 794 57 The screen versions of these slides have full details of copyright and acknowledgements 19
References: Beryllium Maier, L.A. et al. (2003) Influence of MHC class II in susceptibility to beryllium sensitization and chronic beryllium disease. J. Immunol. 171, 6910 6918 Newman, L.S. and Maier, L.A. (2003) Beryllium disease. In Interstitial Lung Disease (Schwarz, M.I. and King, T.E., Jr., eds), pp. 435 451, B.C. Decker Inc. Scott, B.L. et al. (2003) Potenti al binding models of beryllium with class II major histocompati bility complex HLA-DP: a combined theoretical and structural database study. J. Inorg. Biochem. 94, 5 13. McCanlies, E.C. et al. (2004) The association between HLA- DPB1Glu 69 and chronic beryllium disease and beryllium sensitization. Am. J. Ind Med. 46, 95 103 58 References: Beryllium Sawyer RT, et al. (2004) Beryllium-induced tumor necrosis factor-alpha production by CD4+ T cells is mediated by HLA-DP. Am J Respir Cell Mol Biol. 31:122-30. Chou, Y.K. et al. (2005) Activation pathways implicate anti-hla-dp and anti-lfa-1 antibodies as lead candidates for intervention in chronic berylliosis. J. Immunol. 174, 4316 4324 Fontenot AP, Maier LA (2005) Genetic susceptibility and immunemediated destruction in beryllium-induced disease. Trends in Immunology 26:543-49. Pott, G.B. et al. (2005) Frequency of beryllium-specific, TH1-type cytokine-expressing CD4+ T cells in patients with beryllium-induced disease. J. Allergy Clin. Immunol. 115, 1036 1042 Fontenot, A.P. et al (2005). Frequency of beryllium-specific, central memory CD4+ T cells in blood determines proliferative response. J. Clin. Invest. 115:2886-93. 59 References: Sarcoidosis Grunewal d, J. et al. (1992) Restricted Va2.3 gene usage by CD4+ T lymphocytes in bronchoal veol ar lavage fluid from sarcoidosi s patients correlates with HLA-DR3. Eur. J. Immunol. 22, 129 135 Grunewal d, J. et al. (1994) T-cell receptor variable region gene usage by CD4 and CD8 T cells in bronchoal veolar lavage fluid and peripher al blood of sarcoidosis patients. Proc. Natl. Acad. Sci. U. S. A. 91, 4965 4969 Grunewald, J. et al. (1995) Restricted usage of T cell receptor Vα/Jα gene segments with different nucleoti de but identical amino acid sequences in HLA-DR3+ sarcoidosi s patients. Mol. Med. 1, 287 296 Newman, L.S. et al. (1997) Sarcoidosis. N. Engl. J. Med. 336, 1224 1234. 60 The screen versions of these slides have full details of copyright and acknowledgements 20
References: Sarcoidosis Schurmann M, et al. (2001) Results from a genome- wide search for predisposing genes in sarcoidosis.am J Respir Crit Care Med. 164:840-6. Katchar K et al. (2001) Highly activated T-cell receptor AV2S3(+) CD4(+) lung T-cell expansi ons in pulmonar y sarcoidosis. Am J Respir Crit Care Med. 163:1540-5 Sato H et al. (2002) HLA-DQB1*0201: a marker for good prognosis in British and Dutch patients with sarcoidosis. Am J Respir Cell Mol Biol. 27:406-12. Grutters JC et al. (2002) Increased frequency of the uncommon tumor necrosis factor -857T allele in British and Dutch patients with sarcoidosis. Am J Respir Crit Care Med. 165:1119-24 61 References: Sarcoidosis Planck A, et al. (2003) T-lymphocyte activity in HLA-DR17 positive patients with active and clinically recovered sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis. 20:110-7 Spagnolo P et al. (2003) C-C chemoki ne receptor 2 and sarcoidosis: association with Löfgren's syndrome. Am J Respir Crit Care Med. 168:1162-6 Rossman MD et al. (2003) HLA-DRB1*1101: a significant risk factor for sarcoidosis in blacks and whites. Am J Hum Genet. 73:720-35. Newman LS et al. (2004) A case control etiologic study of sarcoidosis: environmental and occupati onal risk factors. Am J Respir Crit Care Med. 170:1324-30 62 References: Sarcoidosis Spagnolo P et al. (2005) C-C chemoki ne receptor 5 gene variants in relation to lung disease in sarcoidosis. Am J Respir Crit Care Med. 172:721-8 Valentonyte R, et al. (2005) Study of C-C chemoki ne receptor 2 alleles in sarcoidosis, with emphasis on family-based analysis. Am J Respir Crit Care Med. 171:1136-41. Iannuzzi MC et al. (2005) Genome- wide search for sarcoidosis susceptibility genes in African Americans. Genes Immun. 6:509-18 Valentonyte R et al. (2005) Sarcoidosis is associated with a truncating splice site mutation in BTNL2. Nat Genet. 37:357-64. 63 The screen versions of these slides have full details of copyright and acknowledgements 21
References: Other Newman Taylor AJ, et al (1999) Interacti on of HLA phenotype and exposure intensity in sensitization to complex platinum salts. Am J Respir Crit Care Med. 160:435-8 Camarena A et al. (2001) Major histocompati bility complex and tumor necrosis factor-alpha polymor phisms in pigeon breeder's disease. Am J Respir Crit Care Med. 163:1528-33. Selman M et al. (2005) Gene Expressi on Profiles Distinguish Idiopathic Pulmonar y Fibrosis from Hypersensiti vity Pneumonitis. Am J Respir Crit Care Med. (in press) 64 65 The screen versions of these slides have full details of copyright and acknowledgements 22