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1 : T oluen e diisocy an ate (T DI) tr an sformin g gr ow th factor 1 (T GF 1) T GF r ecept or II (R II) -, *,, * =A b s t r a c t = E x p re s s io n o f tr an s f o rm in g g ro w th f ac t o r 1 (T G F 1) an d T G F re c e p t or II (R II) in airw ay m u c o s a of t olu e n e dii s o c y an a te (T D I) - in du c e d a s t h m a : re l ati on s h ip s w ith p e r s i s t e n t a s th m atic s y m pt o m s Soo- Keol Lee, M.D., Sun - Min Lee, M.D. *, Ki- B aik Hahm, M.D., Hyun - Ee Yim, M.D., Sun - Sin Kim, M.D. Dong - Ho Nahm, M.D. and Hae- Sim P ark, M.D. Department of A llergy and Clinical Immunology, P ulmonary Critical Care M edicine *, Gastroenterology and A natomical Pathology, Aj ou University School of M edicine, Suwon, K orea B ackg round : The underlying mechanism to explain the poor prognosis of T DI-induced asthma is unknown. We performed this study to evaluate the role of T GF 1 and its receptor, T FG receptor II (RII) in T DI- induced asthma. Meth ods : We applied immunohistochemistry with monoclonal antibodies to T GF 1 and RII in bronchial mucosa from 22 subjects with TDI- induced asthma (group I: 10 newly diagnosed, group II: 12 subjects with persistent asthma symptoms for more than 4 years after diagnosis) and 8 non-asthmatics undergoing pneumonectomy from lung tumor. T he expression was analyzed in 4 areas of bronchial tissue-epithelium (EP), vascular endothelium (VE), smooth muscle (SM), mucous gland (MG). The grade of intensity was presented from 0 to 3. Subepithelial basement memberane (SBM) and submucosal extracellular matrix (SECM) thickness were measured using an image analyzer. Serum specific IgE and IgG antibody levels to T DI- human serum albumin (HSA) conjugate were detected by ELISA. Re s ult s : Grade of T GF 1 expression was significantly higher in EP, VE and SM in group II : : :, 5, ( ) E - m ail : h adang.ajou.ac.kr (Internation al Congress of Allergology and Clinical Immunology )

2 Korean Journal of Medicine : Vol. 61, No. 6, 2001 than those of group I of TDI-induced asthma (p <0.05 respectively), with significant difference in SM only in RII expression. SBM and SECM thickness of TDI-induced asthma were significantly higher than those of non- asthmatics (p <0.05, respectively), while there was no significant difference between group I and II (p >0.05). Significant correlations were noted between asthma duration after diagonsis and intensity of T GF 1 expression in EP, VE and SM (p <0.05, respectively), with no significant correlations with RII expression (p >0.05). T GF 1 expression was significantly higher in EP, VE and SM in subjects with specific IgG antibody to TDI-HSA than those without it (p <0.05). Conclus ion : These findings suggest that T GF 1 may contribute to develop persistent asthma symptoms in T DI- induced asthma.(korean J Med 61: , 2001) Key W ords : T DI induced asthma; T GF- beta- 1; T GF receptor II; Asthma symptom % 1, 2 )., isocyanate toluene diisocyanate (T DI), 13% 1)., T DI 50%, 3 ). T DI (airway remodeling),, T DI,. T GF (profibrotic growth factor) 4-7 ), 7-9). T DI-, T GF 1 2 T GF,. 1. T DI - T DI- 10 ( I ), 4 5, 12 ( II ). 4. T DI 10 ),, T DI, T DI - PC20,, (PEFR monitoring). 2. 1) TDI T DI- 11) 24. T DI- De Vilbiss 646 nebulizer (Devilbiss, Somerset, PA) 10 FEV1. TDI (80:20= 2,4-form : 2,6-form, Aldrich, Milwaukee, Wis) 5 15,

3 Soo- Keol Lee, et al : Expression of transforming growth factor 1 (T GF 1) and T GF receptor II (R II) in airway mucosa of toluene diisocyanate (TDI)- induced asthma: relationships with persistent asthmatic symptoms, T LD- 1 (MDA Scientific, Jupiter, FL) T DI 20 ppb , 30 1, 7 FEV1 20%. 2) (right intermediate bronchus),,, paraffin (formalin- fixed and paraffin- embeded) 4 m probe (immunohistochemical method). 3) (Immunohistochemistry) 30 Histostain- plus kit (Zymed Lab. Inc., San Francisco, CA). 4 m (rehydration) 0.1N sodium citrate dihydrate 0.1 N sodium citrate 10. (100%) (70%) (ethanol) (rehydration) (methanol) 30% (H2O2) 1 endogenous peroxidase. (non- immune serum, reagent A) 1. 1:200 v/ v T GF 1 (Santa Cruz Bio., Santa Cruz, CA) T GF RII (Santa Cruz Bio., Santa Cruz, CA) (reagent B) 1, streptavidine- peroxidase (reagent C) 30. Substrate chromogen solution (reagent D), (cunter- staining) hematoxyline. 1. (epithelial layer), (smooth muscle), (vascular endothelium), (mucous glands), ) (subepithelial basement membrane) (submucosal extracellular matrix) hematoxyline- eosin (subepthelial basement membrane, SBE ), (submucosal extracellular matrix, SECM ) - Image pro plus (Media cybernetics, Silver spring, MD, USA). SBM SECM SBM (smooth muscle), ) TDI- (TDI-HSA) 12 )., 2.4 g T DI 90 ml 1% 5, 10, 20, 30, 40. Ammonium carbonate (2M) 4 3,000 g 20 cellulose membrane (molecular weight cut off 12 KD, Sigma Chemical Co., St. Louis, MO) 3 4L 0.1 M ammonium carbonate buffer. 20% trichloroacetic acid 1 M sodium carbonate 1. T DI Gutmann assay 13 ), Lowry 14 ). 6) TDI- IgG IgE IgG. 1 mg/ ml T DI- carbonate buffer (ph

4 : ) 96- well microtiter plate (Costar, Cambridge, MA) 1 well 1 g % T ween 20 (PBS) 3 3% bovine serum albumin PBS-T (3% BSA-PBST ) well % BSA- PBST 1: 500 well peroxidase IgG (Sigma, St. Louis, MO) 3% BSA- PBST 1: o- phenylenediamine dihydrochloride (Sigma, St. Louis, MO) well 100, H2SO4 ELISA reader 490 nm. IgE IgG, 5% BSA- PBST 1: 10, - IgE (Vector Lab., Burlingam, CA) 1: 500. Peroxidase streptavidine (Sigma, St. Louis, MO) 1: 2000 T MB (Sigma, St. Louis, MO). 20, 2 cut- off,. 7) mean SEM, Mann- Whitney U- test Spearman 's correlation test. 1) T DI- 22 T DI- 10 I, II,,, T DI,, IgE ( 1). ex- smoker pack- year. 2) TDI- I II TGF 1 TGF 2 ( 1, 2, 3, 4) 1 2 T GF 1 T GF 2 A T DI-, B 4 5. I, II T GF 1 ( 3) (epithelium, EP) vs , (vascular endothelium, VE) vs , (smooth muscle, SM) vs II I ( p <0.05), (mucous gland, MG) vs (p >0.05). T GF 2 ( 3) vs II T able 1. Comparis on of dermog raphic data betw een g roup I and II subjects Sex (m/ f) Age (yrs ) Atopy * Exposure duration to T DI (Mo) FEV1 (% predicted) Methacholine PC20 (mg/ ml) T otal IgE (IU/ ml) Group I (n=10) Group II (n=12) p value 4/ / / / Group I, 10 subjects biopsied at initial diagnosis; Group II, 12 subjects with persistent asthmatic symptoms (biopsy collected 65.8 months following the diagnosis); Atopy *, (presence/ absence); Mo, months; All values are presented as means SEM;, Not significant

5 6 : T oluene diisocyanate (T DI) transforming growth factor 1 (T GF 1) T GF receptor II (R II) Fig ure 1A Figure 1B F ig ure 1. Immunohistochemical staining of T GF 1 in airway mocosa of TDI- induced asthmatic patients. A. initial biopsy at diagnosis, B. follow biopsy at 4 or 5 years after the diagnosis Figure 2A Figure 2A Fig ure 2. Immunohistochemical staining of T GF receptor II in airway mocosa of TDI induced asthmatic patients. A. initial biopsy at diagnosis, B. follow biopsy at 4 or 5 years after the diagnosis Fig ure 3. Comparison of T GF 1 and receptor II expression in airway mucosa between group I (closed circle) and II (open circle) TDI induced asthmatic patients. Horizontal bars indicate mean values. A. epithelium, B. smooth muscle, C. vascular endothelium, D. mucous glands

6 Korean Journal of Medicine : Vol. 61, No. 6, 2001 I (p <0.05), vs , vs , vs ( p >0.05). I ( 4),, T GF 1 ( p <0.05). T GF 2 (p >0.05). 3) TDI- TGF 1 TGF 2 T GF vs , vs , vs , vs ( p >0.05). T GF vs , vs , vs , vs ( p >0.05). F ig ure 4. Changes of T GF 1 and receptor II expression at initial diagnosis (closed circle) and 4 5 years later (open circle) in 5 patients with persistent asthmatic symptoms in T DI- induced asthmatics. A. epithelium, B. smooth muscle, C. vascular endothelium, D. mucous glands Fig ure 5. Comparison of T GF 1 and receptor II expression in airway mucosa of TDI- induced asthma patients according to presence of serum specific IgG antibody to TDI- HSA conjugate. Closed circle- positive responders, open circle- negative responders. Horizontal bars indicate mean values. A. epithelium, B. smooth muscle, C. vascular endothelium, D. mucous glands

7 Soo- Keol Lee, et al : Expression of transforming growth factor 1 (T GF 1) and T GF receptor II (R II) in airway mucosa of toluene diisocyanate (TDI)- induced asthma: relationships with persistent asthmatic symptoms 4) S BM S ECM I, II SBM SECM, SBM vs m, SECM vs m (, p >0.05). SBM SECM, SBM vs (p <0.0001), SECM vs (p <0.05). 5) TDI- TGF 1 ( 5) T DI- IgG IgE (I 7, II 7 ) cut off IgE I 1, II 1, 12. IgG, I 1 (14.3%), II 5 (71.4%), 8. IgG,, T GF 1 ( p <0.05), (p >0.05). T GF 2,,, ( p >0.05). (subepithelial fibrosis), (hyperplasia and hypertrophy of submucosal glands and bronchial smooth muscle), (neovascularization) 15 )., 16 ), 17-19). T GF (profibrotic cytokine) (fibroblast) (extracellular matrix protein) 20, 2 1), 22 ), (chemoattractant) 23-25). T GF 1, T GF 1, 26, 27 ). T DI-, T GF 1 T GF 2, SBM SECM, 4 T GF 1, SBM SECM ),. II I., 32 ), T DI T DI T DI,.,. Saetta 33 ) T DI

8 : ,.,,. SBM. Chu 34 ) SBM,, 34, 35 ). SBM SECM 35). SBM, (smooth muscle hypertrophy/ hyperplasia), (neovascularization), (glandular hyperplasia),. T GF 1,, 17, 22 ),,. T GF 2, T GF,. T GF, 36 )., T DI- T GF 2,,. II I in situ hybridization. T GF 1. T GF 1 mrna 37, 38 ) T GF 1 mrna 39 )., T GF 1,, T DI-. 11) T DI- HSA IgG T DI-, T DI., IgG 40 ). IgG T GF 1. T DI- HSA IgG T GF 1 IgG. T DI T GF 1,. : TDI- 50%. TDI

9 6 : T oluene diisocyanate (T DI) transforming growth factor 1 (T GF 1) T GF receptor II (R II) T GF 1 T GF 2 TDI-. : 22 T DI- ( I - 10, II : 4 12 ) 8, T GF 1 T GF 2. T DI- IgG IgE (ELISA). T GF 1 T GF 2, (epthelium, EP), (vascular endothelium, VE), (smooth muscel, SM) (mucous glands, MG), 0 3. Subepithelial basement membrane (SBM) submucosal extracellular matrix (SECM) image analyzer. : T GF 1, II I ( p <0.05), T GF 2 II I (p <0.05). SBM SECM TDI- ( p <0.05), ( p >0.05)., T GF 1 T DI- ( p <0.05), T GF 2 (p >0.05). T DI- IgG T GF 1, ( p <0.05). : T DI- T GF 1. R E F E R E N C E S 1) Park HS, Park JN, Kim JW, Kim SK. Clinical and immunological evaluation of isocyanate- exp osed workers. J K or Sci 7: , ) Kim YY, Cho SH, Yoon HJ, Min KU, Back DM, Jung KC. Isocyanate- induced occupational asthma in K orea. K orean J Intern M ed 47: , ) Park HS, Nahm DH. P rognostic factors f or toluene diisocyanate- induced occupational asthma af ter removal from exp osure. Clin Exp A llergy 27: ,1997 4) Kovacs EJ, Dipietro LA. F igbrogenic cytokines and connective tissue production. FA SEB J 8: , ) Khalil N, O'Connor RN, Unruh HW, Warren PW, Flanders KC, Kemp A, Bereznay OH, Greenberg AH. Increased p roduction and immunohistochemical location of transf orming growth factor- beta in idiopathic p ulmonary fibrosis. A m J R esp ir Cell M ol B iol 5: , ) Corrin B, Butcher D, McAnulty BJ, Dubois RM, Black CM, Laurent GJ, Harrison NK. Immunohistochemical localization of transforming growth factor- beta 1 in the lungs of patients with systemic sclerosis, cryp togenic fibrosing alveolitis and other lung disorders. H istopathology 24: , ) Border WA, Noble NA. T ransf orming growth factor beta in tissue f ibrosis. N Engl J M ed 331: , ) Elovic A, Wong DT, Weller PF, Matossian K, Galli SJ. Exp ression of transf orming growth factors-alpha and beta 1 m essenger RN A and p roduct by eosinophils in nasal p olyps. J A llergy Clin Immunol 93: , ) Ohno I, Lea RG, Flanders KC, Clark D, Banwatt D, Dolovich J, Denburg J, Harley CB, Gauldie J, Jordana M. E osinophils in chronically inf lamed human upp er airway tissues exp ress transform ing growth factor beta 1 gene (T GF beta 1). J Clin Invest 89: , ),,,,,. T oluene diisocyanate (T DI) TDI- IgG IgE. 19: , ) Park HS, Kim HY, Nahm DH, Son JW, Kim YY. Sp ecif ic Ig G, but not sp ecific IgE, antibodies to toluene diisocyanate-human serum albumin conj ugate are associated with TDI bronchop rovocation test results. J A llergy Clin Immunol 104: , ) Son MS, Lee M, Kim YT, Yoon JK, Park HS. H eterogeneity of IgE resonse to toluene diisocyanate(tdi)- HSA conj ugate by ELISA in TDI - induced occupational asthma patients. J K orean M ed Sci 13: , ) Modesto PR, Pesce AJ. Use of tolylene diisocyanate

10 Korean Journal of Medicine : Vol. 61, No. 6, 2001 for the preparation of a peroxidase- labelled antibody conj ugate: quantitation of the amount of diisocyanate bound. B iochim B iophys A cta 295: , ) Lowry OH, Rosenbrough N, Lewis FA, Randall RJ. P rotein measurement with the F olin phenol reagent. J B iol Chem 193: , ) Arm JP, Lee T H. The pathology of bronchial asthma. A dv Immunol 51: , ) Wiggs BR, Bosken C, Pare PD, James A, Hogg JC. A model of airway narrowing in asthma and in chronic obstructive p ulm onary disease. A m R ev R esp ir Dis 145: , ) Aubert JD, Dalal BI, Bai T R, Roberts CR, Hayashi S, Hogg JC. T ransf orming growth factor beta 1 gene exp ression in human airways. Thorax 49: , ) Aubert JD, Hayashi S, Hards J, Bai TR, Pare PD, Hogg JC. P latelet- derived growth factor and its recep tor in lungs f rom patients with asthma and chronic airflow obstructiion. A m J Physiol 266:L , ) Ohno I, Nitta Y, Yamauchi K, Hoshi H, Honma M, Woolley K, O'Byrne P, Dolovich J, Jordan M, T amura G. E osinophils as a p otential source of p lateletderived growth factor B - chain (PD GF -B ) in nasal p olyp osis and bronchial asthma. A m J R esp ir Cell M ol B iol 13: , ) Raghu C, Masta S, Meyers D, Narayanan AS. Collagen synthesis by normal and fibrotic lung fibroblasts and the effect of transforming growth factor- beta. A m R ev R esp ir Dis 140:95-100, ) Raghow R. R ole of transf orming growth factor- beta in repair and fibrosis. Chest 99:61S- 65S, ) Magnan A, Retornaz F, T sicopoulos A, Brisse J, van Pee D, Gosset P, Chamlian A, T onnel AB, Vervloet D. A ltered compartmentalization of transf orming growth factor- beta in asthmatic airways. Clin Exp A llergy 27: , ) Wahl SM, Hunt DA, Wakefield LM, McCarthy- Francis N, Wahl LM, Roberts AB, Sporn MB. T ransforming growth factor typ e beta induces monocytes chemotaxis and growth factor p roduction. P roc N atl A cad Sci U S A 84: , ) Postlewaite AE, Keski- Oja EJ, Moses HL, Kang AH. Stimulation of the chem otactic m igration of human fibroblasts by transf orming growth factor- beta. J Exp M ed 165: , ) Gruber BL, Marchese MJ, Kew RR. T ransf orming growth factor-beta mediates mast cell chemotaxis. J Immuol 152: , ) Ohno I, Nitta Y, Yamauchi K, Hoshi H, Honma M, Woolley K, O'Byrne P, T amura G, Jordan M, Shirato K. Transforming growth factor 1 (T GF 1) gene exp ression by eosinophils in asthmatic airway inf lammation. A m J R esp ir Cell M ol B iol 15: , ) Minshall EM, Leung DY, Martin RJ, Song YL, Cameron L, Ernst P, Harrid Q. E osinphil-associated T GF -1 mrna expression and airway fibrosis in bronchial asthma. A m J R esp ir Cell M ol B iol 17: , ) Vignola AM, Kips J, Bousquet J. T issue remodeling as a f eature of p ersistent asthma. J A llergy Clin Immunol 105: , ) Yates S, Carter P, Heinly T, Lieberman P. A ccelerated irreversal decline in lung f unction in asthmatics. J A llergy Clin Immunol 99:S147, ) van Essen-Zandvliet EE, Hughes MD, Waalkens HJ, Duiverman EJ, Pocock SJ, Kerrebijn KF. Eff ects of 22 months of treatment with inhaled corticosteroids and/ or beta-2-agonist on lung f unction, airway resp onsiveness, and symp toms in children with asthma: the Dutch Chronic N onsp ecif ic Lung Disease Study Group. A m R ev R esp ir Dis 146: , ) Hoshino M, Nakamura Y, Sim JJ, Yamashiro Y, Uchida K, Hosaka K, Isogai S. Inhaled corticosteroid reduced lamina reticularis of the basement membrane by modulation of insulin- like growth factor (IGF)-I expression in bronchial asthma. Clin Exp A llergy 28: , ) Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. B ronchial biopsies in asthma: an ultra- structural, quantitative study and correlation with hyp ereactivity. A m R ev R esp ir Dis 140: , ) Saetta M, Maestrelli P, T urato G, Mapp CE, Milani G, Pivirotto F, Fabbri LM, Di Stefano A. A irway wall remodeling after cessation of exp osure to isocyanate in sensitiz ed asthmatic subj ects. A m J R esp ir Crit Care M ed 151: , ) Chu HW, Halliday JL, Martin RJ, Leung DY, Szefler SJ, Wenzel SE. Collagen dep osition in large airway may not diff erentiate severe asthma from milder f orms of the disease. A m J R esp ir Crit Care M ed 158: , ) Vignola AM, Chanez P, Chiappara G, Merendino A, Pace E, Rizzo A, la Rocca AM, Bellia V, Bonsignore G, Bousquet J. T ransforming growth factor- beta expression in mucosal biopsies in asthma and chronic bronchitis. A m J R esp ir Crit Care M ed 156: , ) Munger JS, Huang X, Kawasatsu H, Griffiths MJD, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C,

11 Soo- Keol Lee, et al : Expression of transforming growth factor 1 (T GF 1) and T GF receptor II (R II) in airway mucosa of toluene diisocyanate (TDI)- induced asthma: relationships with persistent asthmatic symptoms Matthay MA, Rifkin DB, Sheppard D. The integrin alpha v beta 6 binds and activates latent T GF 1: a mechanism f or regulating p ulmonary inf lammation and f ibrosis. Cell 96: , ) Khalil N, Whitman C, Zuo L, Danielpour D, Greenberg A. R egualtion of alveolar macrophage transf orming growth factor- beta secretion by corticosteroids in bleomycin- induced pulmonary inf lammation in the rat. J Clin Invest 92: , ) AyanlarBatuman O, Ferrero AP, Diaz A, Jimenez SA. R egulation of transf orming growth factor- beta 1 gene expression by glucocorticoids in normal human T lymphocytes. J Cllin Invest 88: , ) Jeffery PK, Gldfrey RW, Adelroth E, Nelson F, Rogers A Johansson SA. Eff ects of treatm ent on airway inflammation and thickening of basem ent membrane reticular collagen in asthma. A m R ev R esp ir D is 145: , ) Park HS, Kim HY, Suh JH, Kim SS, Lee SK, Nahm DH. Sp ecific IgE and Ig G antibodies to toluene diisocyanate (TDI)- human serum albumin (HSA ) conj ugate : usef ul markers for p redicting long - term prognosis in TDI -induced asthma. Clin Exp A llergy 2001 (in p ress)

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