3rd Jack Pepys Workshop on Asthma in the Workplace (Montreal, May 17-19 2007)

1. 3rd Jack Pepys Workshop on Asthma in the Workplace (Montreal, May 17-19 2007) An ATS/ERS report: 100 key questions and needs in occupational asthma (ERJ 2006; 27:607-14) PHYSIOPATHOLOGY AND MECHANISMS Questions 47-60 Cristina E. Mapp University of Ferrara, Italy

2. Question 47: is asthma heterogeneous? • YES, both between and within individuals • Phenotypic classification of Adult-onset asthma: • etiologic factors • clinical characteristics of the disease • Each method is largely descriptive and is a gross • oversemplification

3. More recently, classification of asthma according to the nature of the underlying airway inflammation • has been suggested • Four distinct phenotypes: • eosinophilic • neutrophilic • mixed inflammatory • paucigranulocytic asthma

4. Non-eosinophilic asthma • by using IS, BP, BW, and BAL • absence of airway eosinophilia • normal subepithelial layer thickness • a poor short-term response to treatment with • inhaled corticosteroids • Berry MA et al. Thorax 2007; Mar 13

5. Neutrophilic asthma Increased expression of the innate immune receptors toll-like receptor (TLR)2,TLR4 and CD14, and inflammatory cytokines IL-8, IL1β in induced sputum Innate immune activation Simpson JL et al. Thorax 2007; 62:211-8

6. Contiguos innate and adaptive immunity (temporally and physically) in asthma Purple arrows = unidirectional connections Red arrows= continual dialog Dashed arrow= inflammatory stimuli Sabroe I et al. AJRCCM 2007; 175:306-11

7. Question 48: timing of A, AHR, and inflammation baseline ventilation heterogeneity is a strong predictor of AHR, independent of airway inflammation in asthmatics, its relationship persists following anti-inflammatory therapy a= baseline b= following treatment closed circles =ICS treatment; open circles = no ICS treatment; triangles= CFC-BDP treated; squares= HFA-BDP treated subjects Downie SRT et al. Thorax published online 20 Feb 2007

8. Dual asthmatic responders: the increase in AHR persists 7 d after allergen challenge Changes from baseline in FEV1 (A) and methacholine PC20 24 h and 7 d after allergen challenge

9. The 24 h increase in airway wall inflammation resolves by 7 d, whereas the increase in markers of remodeling persist A: tenascin, B: procollagen III, procollagen I cells, HSP-47+ fibroblasts, E: eosinophils, F: macrophages, G: CD3+ T cells, H: neutrophils, at baseline, 24 h and 7 d after allergen challenge Kariyawasam HH et al. AJRCCM 2007; 175:896-904

10. AHR can be dissociated from cellular • inflammation • whilst remaining associated with sustained • airway remodeling • Measuring the degree of inflammation does • not allow insight into disease severity in terms of AHR

11.   Time course of exhaled NO after specific inhalation test (SIT) 180 ** SIT + * — SIT - Exposure * 120 * NO ppb * 60 0 0 1h 2h 3h 4h 5h 6h 7h 24h 48h 30 m' 7day 15m' Time * p<0.05 ** p<0.01 • Ferrazzoni S. et al. ERS 2005 • Barbinova L. Int Arch Environ Health 2006; • 79:387 : eNO increase 22 h after isocyanate challenge

12. After specific bronchial challenge with TDI Eosinophils in IS: ↑ 8-24 h ↓ 48 h in BP ↑ 48 h CD8+ in blood: ↑ 8 h ↑ 48 h (not all subjects) AHR: ↑ 8-24 h ↓ 1-4 wk

13. 1in 4 asthmatics have PAO and uncontrolled symptoms at 4 to 6 wk post- viral exacerbation; the phenomenon does not involve the influx of inflam- matory cells into the lower airways during exacerbation Induced-sputum samples during an acute exacerbation Wood et al. Chest 2007; 131:415-23

14. Question 49: what is the additional value of other • procedures (to determine the influence of • confounders in the interpretation of cytology) on • top of clinical history taking in subjects who • perform induced-sputum? • clinical history • statistical analysis • Particular attention in the presence of • neutrophilic airway inflammation

15. Questions 50 and 57: role of neutrophils • sputum neutrophils are usually elevated • during asthma exacerbation • in severe asthma • in older asthmatics • in smoking asthma patients • In OA • at diagnosis • after cessation of exposure in subjects with no • improvement

16. In vitro IgE can delay programmed cell death of neutrophils from allergic asthmatics and this may possibly contribute to neutrophilic inflammation in atopic asthma IgE could influence biological function of neutrophils Neutrophils from AAs express FcRI and carry surface-bound IgE. Freshly isolated neutrophils from two (nos. 1 and 2) were analyzed for expression of FcRI (A), surface-bound IgE (B), and CD16 (C) by FACS. Figure is representative of eight separate experiments. Saffar AS et. J Immunol 2007; 178:2535-41

17. Neutrophils fit both in allergic and nonallergic asthma

18. Question 51: can skin absorption lead to • respiratory sensitization? • Vanoirbeek JA et al. Toxicol Sci 2004; 80:310-21(TDI) • Plitnick LM et al. Toxicology 2005; 207:487-99 (isocyanates) • Selgrade M et al.Toxicol Sci 2006; 94:108-17 (DNCB, isoc.) • Vanoirbreek JA et al. J Allergy Clin Immunol 2006; 117: 1090-7 (TMA, DNCB) • Tarkowski M et al. Am J Physiol Lung Cell Mol Physiol 2007; 292:L207-14 (TDI) • Sun LZ ert al. Scand J Immunol 2007; 65:118-25 (TDI) • dermal exposure causes respiratory • hypersensitivity

19. isocyanate skin exposure can occur at work • it is likely that such exposure can contribute to the • development of isocyanate asthma, presumably by • inducing systemic sensitization • Bello D et al. Environ Health Perspect 2007; 115:328-35 • a reasonable advice: avoid skin contact with chemical sensitizers in the workplace to prevent chemical-induced asthma • Vanoirbreek JA et al. J Allergy Clin Immunol 2006; 117:1090-7

20. ….. a provocative hypothesis dermal contact with polyurethane-containing medical materials in early life may be involved in dysregulation of the neonatal immune system and could predispose infants to the development of childhood asthma Krone CA et al. Med Sci Monit 2003; 9:HY39-43

21. Question 52: are animal models useful in understanding the pathogenesis of OA? animal models are useful, but at the moment, they do not provide enough information to predict the ability of a chemical to act as a sensitizer and to fully understand the mechanims involved in OA

22. Animal models of chemical-induced asthma • Th2 or mixed Th1 and Th2 responses • stimulatory effects on bronchial epithelial • cells • production of proinflammatory cytokines • and chemokines • a protective role of PPARgamma • JACI 2006; 117;1090-7 Exp Lung Res 2006; 32:245-62 • J Immunol 2006; 177:5248-57

23. The PPARgamma is a ligand-activated • transcription factor belonging to the • nuclear hormone receptor superfamily • Is expressed on dendritic cells, • eosinophils, macrophages, and T cells • PPAR ligands are thought to exert anti- • inflammatory effects by negatively • regulating the expression of pro- • inflammatory genes • PPAR agonists to prevent and treat asthma

24. Question 53: many questions (outcome, pH, tolerance…..) IS is a feasible, repeatable, non-invasive method to measure airways pH (with a cut-off value of 7.3: sensitivity 72.1% and specificity 100% to distinguish asthmatics from healthy subjects) Correlation between the levels of control of asthma (ACQ score) and pH and percentage of eosinophils Kodric M et al. Am J Respir Crit Care Med 2007; 175:905-10

25. Questions 53-54: LMWA and the immune system One example: diisocyanates although many authors have focused their research on the mechanisms involved in LMWA-induced OA many questions such as: the outcome when inhaled, how long do stay in the airways, how does the immune system recognizes a LMWA and generates a specific response, can tolerance occur, are still incompletely understood

26. Diisocyanate-induced asthma is a non IgE-mediated disease at least in subjects in whom specific IgE antibodies to diisocyanates are undectable Jones MG et al. JACI 2006; 117:663-9

27. Question 55: is appropriate the concept of “united airways”? • YES • upper and lower airways may be considered a • unique entity • allergic rhinitis is a risk factor for the occurrence • and severity of asthma • rhinoconjunctivitis is a risk factor for HMWA- • induced OA

28. Rhinitis - Sinusitis Eosinophils IL-5 Basophils Mast cells degranulation Nasal allergen challenge Loss of filtration efficiency Allergen Postnasal drip Reflex Nose-bronchi Precursors Cytokines Bone marrow Viral infection Eosinophils ICAM-1 VCAM-1 Bronchial allergen challenge Asthma

29. There is a gradient of T-cell activation (blood and • induced sputum) • ENA: a decrease of Th1, an increase of Th2 and • Treg after SIT (tolerating profile) • OA: an increase of both Th1 and Th2, a decrease • of Treg cells after SIT (inflammatory profile) • OR: an increase of Th2 cells, absence of Th1 • activation and of Treg cell decrease after SIT • (intermediate profile) • Mamessier E et al. Allergy 2007; 62:162-9

30. Questions 56-58: role of cells….. Dendritic cells are crucial for sensitization to inhaled Ag and in established inflammation; plasmacytoid DCs: tolerance; myeloid DCs: immunity Immune regulation by DCs in the lung. Under steady-state conditions (A), in the absence of accompanying danger signals in the lung, inhaled antigens are picked up by mDCs and pDCs, which take the antigen to the mediastinal nodes. Here partially mature mDCs induce a short-lived boost of division in antigen-specific T cells, but these T cells fail to differentiate into effector cells and die. Some T cells might also differentiate into Tregs. Plasmacytoid DCs in the draining node influence the generation of T effector cells from dividing T cells, probably by giving negative signals (indoleamine 2,3-dioxygenase [IDO] and programmed death ligand [PDL] 1) to T cells and mDCs. At the same time, they also generate Tregs. Under inflammatory conditions (B), mDCs arrive in the draining node as fully mature cells. Antigen-specific T cells undergo proliferation, this time generating effector cells. On the other hand, pDCs also acquire a mature phenotype and prime antigen-specific T cells to become effector cells as well. ICOSL, Inducible costimulator ligand. Hammad H and Lambrecht BN. J Allergy Clin Immunol 2006;118:331-6

31. By using BAL in allergic asthmatics, both myeloid and plasmacytoid DCs accumulate in the airway lumen 24 hours after allergen challenge Bratke K, et al. Thorax 2007; 62: 168-75 By using IS it is possible to analyze human airway DCs (0.5% of viable sputum cells, HLA-DR+) McCarthy NE et al. Clin Exp Allergy 2007; 37:72-82

32. Several studies indicate that CD8 T cells • do not exhibit a fixed phenotype • can differentiate into functionally distinct • subsets • uptake by DCs is sufficient for their initial proliferative response to protein allergens • sustained expansion and functional maturation • of the CD8 response most likely requires signals from CD4 T cells or Toll-like receptor ligation

33. CD8 and occupational asthma • Animal and human data show a mixed • Th1/Th2 activation with implication of the • CD8+ T cells in OA • In OA subjects, specific inhalation challenge • induces a mixed Th2/Th1 response and CD8+ • cells are the main producers of IFN-gamma • Allergy 2007; 62:162-9

34. Questions 59-60: specific to compounds • (diisocyanates) • Physiologic levels of GSH protect against HDI exposure • Type I hypersensitivity mechanism in 44% of TDI asthma by using vapor TDI-albumin conjugates • Autoantibodies to CK18 and CK19 as serologic • markers in TDI asthma • Clin Exp Allergy 2005; 35:352-7 • J Allergy Clin Immunol 2006; 118:885-91 • Yonsei Med J 2006; 47:773-81

35. Isocyanate asthma and oxidative stress • glutathione S-transferase polymorphisms • protective role of airway GSH • protective role of PPARgamma agonists • PPARgamma modulates reactive oxygen • species (ROSs) generation and activates • the redox-sensitive transcription factor • nuclear-kappaB and hypoxia-inducible • factor 1alpha

36. Question 60: asthma and hairdressers • sensitization is more related to individual • hypersusceptibility • an immunologic mechanism is strongly • suggested but not definitely demonstrated • diagnosis is based on standardized SIC • Moscato G and Galdi E. Curr Opion Allergy Clin Immunol • 2006; 6:91-5

37. Certain, or a large body of Uncertain or limited evidence • evidence • asthma is heterogeneous skin and respiratory sensitization • timing of OA, AHR and AI usefulness of animal models • is different • neutrophils fit both in allergic mechanisms of recognition of LMWA • and nonallergic asthma by the immune system • upper and lower airways as a role of GSH and a low pH in asthma • unique entity • role of DCs in asthma • CD8+ T cells exhibit different • phenotypes • CD8+ T cells play a role in OA