: 61 6 2001 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 : 2001 1 29 : 2001 8 3 :, 5, (442-749) E - m ail : h spark @m adang.ajou.ac.kr 2000 4. 52. 17 (Internation al Congress of Allergology and Clinical Immunology ). - 623 -
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 61:623-633, 2001) Key W ords : T DI induced asthma; T GF- beta- 1; T GF receptor II; Asthma symptom 2.9 13% 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, - 624 -
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. 1 10 3, 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) 4 12. (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), 0 3. 4) (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 400 10. SECM SBM (smooth muscle), 400 10. 5) 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 - 625 -
: 61 6 496 2001 9.6) 96- well microtiter plate (Costar, Cambridge, MA) 1 well 1 g 4 18. 0.05% T ween 20 (PBS) 3 3% bovine serum albumin PBS-T (3% BSA-PBST ) well 350 3. 3% BSA- PBST 1: 500 well 50 37 2. peroxidase IgG (Sigma, St. Louis, MO) 3% BSA- PBST 1:5000 37 1 30. o- phenylenediamine dihydrochloride (Sigma, St. Louis, MO) well 100, 10 100 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, 4 5 12 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)- 0.55 0.19 vs 1.83 0.17, (vascular endothelium, VE)- 0.35 0.15 vs 1.21 0.14, (smooth muscle, SM)- 0.39 0.20 vs 1.91 0.25 II I ( p <0.05), (mucous gland, MG) 0.83 0.31 vs 0.89 0.18 (p >0.05). T GF 2 ( 3) 0.40 0.16 vs 1.32 0.27 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/ 6 41.9 1.3 2/ 8 90.5 14.2 76.8 0.8 2.69 1.70 335.8 143.0 7/ 5 44.6 2.2 4/ 8 84.0 0.20 91.9 6.5 1.78 1.55 245.7 55.1 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 - 626 -
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 - 627 -
Korean Journal of Medicine : Vol. 61, No. 6, 2001 I (p <0.05), - 1.35 0.21 vs 1.58 0.28, - 1.00 0.15 vs 1.25 0.25, - 1.25 0.21 vs 0.75 0.23 ( p >0.05). I 4 5 5 ( 4),, T GF 1 ( p <0.05). T GF 2 (p >0.05). 3) TDI- TGF 1 TGF 2 T GF 1-1.25 0.19 vs 1.31 0.24, - 0.82 0.14 vs 1.19 0.21, - 1.23 0.24 vs 1.23 0.23, - 0.87 0.16 vs 1.13 0.23 ( p >0.05). T GF 2-1.48 0.18 vs 1.31 0.27, - 1.14 0.15 vs 1.44 0.26, -0.88 0.19 vs 0.88 0.23, -0.97 0.16 vs 1.38 0.35 ( 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 - 628 -
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 11.28 0.96 vs 11.73 1.24 m, SECM 285.33 23.76 vs 272.14 33.65 m (, p >0.05). SBM SECM, SBM 11.540.80 vs 5.350.54 (p <0.0001), SECM 279.6 19.2 vs 183.4 32.3 (p <0.05). 5) TDI- TGF 1 ( 5) T DI- IgG IgE 22 14 (I 7, II 7 ). 20 2 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. 28-31),. II I., 32 ), T DI T DI T DI,.,. Saetta 33 ) T DI-. - 629 -
: 61 6 496 2001 10 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-. - 630 -
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