Plasma cells and interleukin-4 in chronic bronchitis and COPD. Jie Zhu, Yu Sheng Qiu, Monica Valobra, Shengyang Qiu, Swati Majumdar, Dean

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1 Plasma cells and interleukin-4 in chronic bronchitis and COPD Jie Zhu, Yu Sheng Qiu, Monica Valobra, Shengyang Qiu, Swati Majumdar, Dean Matin, Virginia De Rose and Peter K. Jeffery Online Data Supplement 43

2 On Line Repository for Methods Subjects: Each patient had <10% reversibility or < 200 ml increase of FEV1 in response to inhaled β 2 -agonist. Atopic patients or patients with a history of wheezing, asthma, allergic rhinitis or any allergic disease were excluded from the study. None had received antibiotics or inhaled or oral corticosteroids within the preceding month. Tissue processing The tissues were obtained in collaboration with the departments of Respiratory Diseases at University of Turin (Italy) and Ghent University Hospital (Belgium). Only grossly normal lobar and segmental bronchi (i.e. airways with mucus-secreting glands and supportive cartilage plates). The airways selected were well away from circumscribed tumor and not blocked by tumor. The tissues were freshly fixed in 10% buffered formaldehyde, processed to paraffin wax, sectioned 5µm thick and stained with haematoxylin-eosin (H.E) in order to visualize morphology first before conducting counts of the inflammatory cells. Histochemistry staining for plasma cells Modified Unna-Pappenheim methyl green-pyronin stain was used. The procedures described in Carleton s Histological Technique were closely followed (E1). Briefly, the sections were deparaffinized, rehydrated to distilled water and were stained in methyl green pyronin solution. The slides were rinsed in differentiating solution and then quick-rinse in CNP30. The sections were mounted with DPX. Using the technique, the cytoplasm of plasma cells was stained pinkish red. Immunohistochemistry 44

3 The alkaline phosphatase anti-alkaline phosphatase (APAAP) was used to label CD45+ inflammatory cells, mast cells and IL-4 protein using anti-human CD45, leucocyte commom antigen mab (DAKO M0701), anti-human tryptase mab (DAKO M7052) and anti-human IL-4 monoclonal antibody (Chemicon International, Inc, Temecula, CA MAB1029), respectively, as previously described (E2). The sections were counterstained with Harris s haematoxylin mounted in Aqua per mounting medium. The avidin-biotinylated peroxidase complex (ABC) technique was used to identify plasma cells, CD4+, and CD8+ lymphocytes as previously described but with minor modification (E3). Briefly, following protein K (for plasma cells) and microwave (for CD8 cells) antigen retrieval, the sections were incubated with 0.3% hydrogen peroxide (H 2 O 2 ) in distilled water and then immunostained for 60 minutes with appropriately diluted (1 in 50 in PBS) mouse anti-human monoclonal antibodies (mab) to CD4 antigen (Novocastra NCL-CD4-1F6), CD8 antigen (DAKO Cambridge, UK M7103), CD20 antigen (Novocastra NCL-CD20-L26) and plasma cell specific antigens (Novocastra Newcastle, UK NCL-PC). Sections were washed and incubated for a further 30 minutes with biotinylated rabbit anti-mouse immunoglobulins (DAKO Cambridge, E0464) diluted 1 in 300. Sections were then washed and incubated for 50 minutes with avidin complexed with biotinylated peroxidase (DAKO K0355). After a further wash, bound peroxidase was detected as a dark brown product following 10 minutes incubation with 0.03% H 2 O 2 and 0.5 mg/ml diaminobenzidine (DAB). The sections were counterstained with Harris s haematoxylin to provide morphological detail, then mounted in DPX. 45

4 We used an irrelevant antibody, mouse IgG1, Kappa (MOPC21 Sigma, Dorset, UK M7894), as a replacement for the primary mab as the negative control procedure for both APAAP and ABC methods. Preparation of crna probes Digoxigenin (Dig)-labelled antisense and sense complementary ribonucleic acid (crna) probes were generated from complementary deoxyribonucleic acid (cdna) according to a published method, which has been described previously (E2, E3). The sizes (base pairs), vectors and sources of the cdna probes were as follows: IL-4 (318bp; PGEM-1), IL-5 (412bp; PGEM-IV; both from Glaxo Wellcome Biomedical Research, Geneva, Switzerland) and TNF-α (500bp; PGEM-3Z; R & D Systems Europe Ltd, Oxon UK). Immunohistochemistry/non-isotopic in situ hybridization double-staining: The EnVision TM +horseradish peroxidase (HRP) kit (Dako K4006) was first applied for the labelling of plasma cells, mast cells and CD4+ cells following the kits suppler's instruction. HRP was detected using diaminobenzidine (DAB). Positive was shown a golden brown pigment. Non-isotopic in situ hybridization was then carried out as the protocol described previously (E3) to detect cytoplasmic mrna for IL-4, IL-5 and TNF-α. Detection was carried out by addition of 5-bromo-4- chloro-3-indolyl phosphate/nitroblue tetrazolium (NBT/BCIP) substrate (Roche Diagnostics Limited, Sussex, UK). The positive signals were showed as dark blue. The slides were counterstained with nuclear fast red, then mounted in Aqua perm mounting medium. 46

5 Immunofluorescence double staining The sections were deparaffinized, rehydrated. Following protein K (for plasma cells) and microwave (for CD20 and CD68 cells) antigen retrieval, and washed in PBS then incubated with 10% normal rabbit serum for plasma cells in PBS for 10 min. In the first sequence the sections incubated with first primary mab to plasma cell specific antigens, CD20 (Novocastra) and CD68 (Dako M0876) antigens for 1 hour and then incubated tetramethylrhodamine isothiocyanate isomer R (TRITC) conjugated rabbit anti-mouse IgG (Dako R0270) for plasma cells mab and fluorescein isothiocyanate isomer (FITC) conjugated rabbit anti-mouse F(ab ) 2 (Dako F0313) for anti-cd20 or -CD68 mabs for 1 hour, respectively. After the first indirect labelling, free binding sites of the first primary mab were saturated by incubation for 30 min with unconjugated rabbit anti-mouse immunoglobulin (Dako Z0259) to block false binding of the secondary antibody to the first primary mab in the next sequence. The sections were then incubated with 15% normal mouse serum for 20 min to saturate any open antigen-binding site on the conjugated secondary antibody, so that binding of the second primary mab in the next sequence were prevented. The secondary indirect staining were performed using anti-il-4 mab (Genzyme Cambridge UK ) and anti-plasma cell specific antigen mab for 1.5 hour and then FITC or TRITC conjugated rabbit anti-mouse F(ab ) 2 or IgG for 1 hour. Single staining was carried out as a control procedure, using an irrelevant antibody, mouse IgG1, Kappa (MOPC21 Sigma, Dorset, UK M7894) instead of first or second primary layer followed by both secondary antibodies. Replacing both first and secondary primary antibodies with the irrelevant antibody and continuation with both secondary antibodies was 47

6 performed as a negative control. Immune-complexed sections were counterstained with DAPI and mounted with aqueous mounting media. Images were acquired using a Leica TCS-SP confocal microscope with lasers giving excitation wavelengths of nm, 488 nm, 568 nm (Leica Microsystems UK Ltd, Milton Keynes, UK). Quantification, variability, data presentation and statistics: Areas of epithelium, interstitial areas in the subepithelium (a zone between the external edge of the reticular basement membrane and inner aspect of bronchial muscle), areas of submucosal mucus-secreting gland (a line was drawn around areas of gland including inter-acinar interstitium, secretory acini and ducts), and remainder of the airway wall (excluding cartilage) of each entire section were assessed using Apple Macintosh computer Image 1.5 software (Apple Mac, Cupertino,CA, USA). Plasma cells, tryptase, CD4 and CD45 immuno-positive cells and double IHC/ISH stained cells were counted using a Leitz Dialux 20 light microscope (Leitz Wetzlar, West Germany) at 200 magnification using an eyepiece graticule divided into 100 squares in order to ensure no overlap of fields of view. A similar eyepiece graticule was used at 400 magnification to point count and assess the percentage of epithelium containing AB-PAS positive mucosubstances (E4). The points overlying Alcian blue-pas positive cytoplasm areas of surface, submucosal gland acinar and glandular duct epithelial cells were counted as positive, points overlying the unstained areas counted as negative. Points overlying nuclei of the epithelial cells were excluded. The entire cross section of lobar or segmental bronchial ring was assessed for each subject. There were no significant differences in the bronchial diameter (calculated from measures of luminal 48

7 perimeter) between the three smoker groups (mean±sem: AS = 4.24±0.58, CB = 3.98±0.48, CB+AO = 4.25±0.49). Slides were coded to avoid observer bias. To test the consistency of quantification and the inherent variation of repeated counts, one section was selected and counted five times over the period of the entire study. The sections, which had been hybridized with the sense (negative) probe, were examined first in order to detect and be aware of only background signal. The coefficient of variation (CV = SD/mean 100) was used to express the error of repeat counts in a single section of a patient with CB. The errors for repeat counts for the numbers of plasma cells in the subepithelium and submucosal glands by one observer (JZ) were 4.6% and 5.3% respectively. The errors for repeat point counts for the percentage of positive surface and glandular epithelium for Alcian blue-pas staining were 6.2% and 6.9%, respectively. The data for single staining of plasma cells, mast cells, CD4+ and CD45+ cells, are expressed as number of positive cells per mm 2 of: (i) epithelium (ii) subepithelial zone, (iii) gland or (iv) bronchial smooth muscle and (v) remainder of airway wall. The counts of IL-4 and IL-5 mrna+ cells are expressed as number of positive cells per mm 2 tissue for three zones of the airway wall: (i) the subepithelium (ii) that area of the section containing mucus-secreting gland acini or ducts and (iii) the remainder of airway wall. Data for double staining for plasma or mast or CD4+ cells /IL-4 mrna, plasma or mast or CD4+ cells /IL-5 mrna and mast cells/tnf-α mrna are expressed as percentage of double stained cells of the total number of positive cells stained for plasma, mast and CD4+ cells, respectively, or each of cytokines in the 49

8 subepithelial zone and interstitium between mucus-secreting gland acini or surrounding gland ducts. 50

9 Reference List E1. Drury RAB and Wallington EA. Chapter 7, General staining procedures, p Carleton's Histological Techniques. Oxford New York Toronto: Oxford University Press; 1980, p E2. Zhu J, Kilty I, Granger H, Gamble E, Qiu YS, Hattotuwa K, Elston W, Liu WL, Oliva A, Pauwels RA, Kips JC, De Rose V, Barnes N, Yeadon M, Jenkinson S, Jeffery PK. Gene expression and immuno-localization of 15-lipooxygenase isozymes in the airway mucosa of smokers with chronic bronchitis. Am J Resp Cell Mol Biol. 2002; 27: E3. Zhu J, Majumdar S, Qiu YS, Ansari T, Oliva A, Kips JC, Pauwels RA, De Rose V, and Jeffery PK: IL-4 and IL-5 gene expression and inflammation in the mucus-secreting glands and subepithelial tissue of smokers with chronic bronchitis: lack of relationship with CD8+ cells. Am J Resp Crit Care Med. 2001; 164: E4. Aherne WA, Dunnill MS. Point counting and morphometry, In: Aherne WA, Dunnill MS, editors. Morphometery. London: Arnold E;1982. p

10 E Figure legends Figures E1A-F Double immunofluorescence staining to identify co-localization of CD20+ B-lymphocytes or CD68+ monocytes/macrophages and IL-4 protein in the submucosal compartment of bronchi from a patient with CB: immunopositive A) CD 20+ and D) CD68+ cells are illustrated with green FITC fluorescence alone. B,E) IL-4 protein positivity stained by anti-human IL-4 antibody is shown by Texas red fluorescence alone. Cells showing co-expression of either C) CD20+ cells or F) CD68+ cells with IL-4 protein are fluorescing yellow (I,e, a mixture of the two colors). Nuclei are counter stained blue with DAPI (internal scale bars = 20 µm). Figure E2. Double immunofluorescence stain confirming that mouse anti-human monoclonal antibodies to plasma cell specific antigens do not cross react with CD20+ B-lymphocytes in the submucosal compartment of bronchi from a patient with CB: plasma cells are illustrated with Texas red (arrows) and CD20+ B-lymphocytes shown by green FITC fluorescence (arrow heads). No yellow fluorescent double-labelled cells can be seen. Nuclei are counter stained blue with DAPI (internal scale bar = 10 µm). Figure E3A & B. Alcian blue-periodic Acid Schiff staining with diastase digestion to indentify acidic and neutral mucopolysaccharides stained blue or purple red, respectively in A) surface and B) glandular epithelium of bronchi from a patient with CB (internal scale bar = 20 µm). 52

11 Table E1 Characteristics of subjects with chronic bronchitis and chronic obstructive pulmonary disease Subjects No. Sex (M/F) Age (Yr) Smoking (Pack/yr) FVC ( %) FEV1 (%) FEV1/FVC (%) AS 1 M M M M M M M M M M M Mean SEM CB 12 M M M M M M M M M M M 80 Ex-smoker Mean SEM CB+COPD 23 M M M M M M M M M M Mean SEM * 3.5 Definition of abbreviations: AS = asymptomatic smokers; CB = chronic bronchitis; COPD = chronic obstructive pulmonary disease; SEM = standard error of mean. *P < compared to AS and CB groups, respectively, (unpaired Student s t test). 60.5*

12 Table E2 Median (range) of inflammatory cells in epithelium, subepithelium, glands and muscles: a comparison within or between tissue compartments Cell AS CB CB + AO type Ep S-Ep Gland Muscle Ep S-Ep Gland Muscle Ep S-Ep Gland Muscle Plasma cells 0 (0-0) 38 (4-107) 41 (10-136) 0 (0-0) 0 (0-0) 110 (44-235) 213 (76-313) 0 (0-0) 0 (0-0) 55 (20-177) 118 (18-224) 0 (0-0) Mast cells CD4+ cells 21 (0-39) 9 (0-47) 65 (39-247) 77* (12-118) 35 (15-97) 38* (23-91) 47 װ (29-86) 6 (0-51) 19 (0-124) 12 (0-58) 204 (61-348) 63* (29-137) 34 (21-64) 46* (17-95) 50 װ (30-140) 8 (0-24) 26 (0-95) 9 (0-76) 115 (43-259) 87* (35-191) 34 (19-78) 62* (20-88) 33 װ (0-57) 10 (2-21) CD8+ cells 123 (30-312) 75 (16-187) 41 (5-113) 2 (0-15) 130 (49-391) 99 (33-253) 36 (12-70) 3 (0-19) 109 (8-352) 109 (10-280) 31 (4-99) Abbreviations: Ep = epithelium; S-Ep = sub-epithelium. *P < 0.01 vs epithelial and muscle areas of AS, CB and CB+AO, respectively; P < vs mast cells, CD4+ cells and plasma cells in epithelial area of AS, CB and CB+AO, respectively; P< 0.01 vs plasma cells, CD4+ and CD8+ cells in subepithelial area of CB; P < 0.05 or 0.01 vs mast cells, CD4+ and CD8+ cells in gland area of CB and CB+AO, respectively. װ P< 0.01 vs plasma cells, CD4+ and CD8+ cells in muscle area in each of three groups. 5(0-27) 54

13 Table E3 Fold increases in the numbers of inflammatory cells in different compartments of the large airways of CB: a comparison with AS subjects and those with CB+AO Cell type Comparison with AS Comparison with CB+COPD Ep Sub- Ep Gland Ep Sub-Ep Gland CD45+ cells Plasma cells Mast cells CD4+ cells CD8+ cells Abbreviations: Ep = Epithelium, AS = Asymptomatic smoker, CB = Chronic bronchitis, AO= airflow obstruction 55

14 Figures E1A-F 56

15 Figure E2 57

16 Figure E3A&B 58