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

Question 47: is asthma heterogeneous?

YES, both between and within individuals Phenotypic classification of Adult-onset asthma:

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etiologic factors

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clinical characteristics of the disease Each method is largely descriptive and is a gross oversemplification

More recently, classification of asthma according to the nature of the underlying airway inflammation has been suggested Four distinct phenotypes:

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eosinophilic

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neutrophilic

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mixed inflammatory

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paucigranulocytic asthma

Non-eosinophilic asthma by using IS, BP, BW, and BAL

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absence of airway eosinophilia

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normal subepithelial layer thickness

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a poor short-term response to treatment with inhaled corticosteroids Berry MA et al. Thorax 2007; Mar 13

Neutrophilic asthma Increased expression of the innate immune receptors toll-like receptor (TLR)2,TLR4 and CD14, and inflammatory cytokines IL-8, IL1 β in induced sputum Innate immune activation Simpson JL et al. Thorax 2007; 62:211-8

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

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

Dual asthmatic responders: the increase in AHR persists 7 d after allergen challenge Changes from baseline in FEV 1 (A) and methacholine PC 20 challenge 24 h and 7 d after allergen

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

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AHR can be dissociated from cellular inflammation

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whilst remaining associated with sustained airway remodeling Measuring the degree of inflammation does not allow insight into disease severity in terms of AHR

Time course of exhaled NO after specific inhalation test (SIT) 180 SIT + SIT -

** *

120 Exposure

* * *

60 0 Time

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Ferrazzoni S. et al. ERS 2005 Barbinova L. Int Arch Environ Health 2006; * p<0.05 79:387 : eNO increase 22 h after isocyanate challenge ** p<0.01

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

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

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?

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clinical history

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statistical analysis Particular attention in the presence of neutrophilic airway inflammation

Questions 50 and 57: role of neutrophils sputum neutrophils are usually elevated

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during asthma exacerbation in severe asthma in older asthmatics in smoking asthma patients In OA

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at diagnosis after cessation of exposure in subjects with no improvement

IgE could influence biological function of neutrophils In vitro IgE can delay programmed cell death of neutrophils from allergic asthmatics and this may possibly contribute to neutrophilic inflammation in atopic asthma 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

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Saffar AS et. J Immunol 2007; 178:2535-41

Neutrophils fit both in allergic and nonallergic asthma

Question 51: can skin absorption lead to respiratory sensitization?

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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

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isocyanate skin exposure can occur at work

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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

….. 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

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

Animal models of chemical-induced asthma

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Th2 or mixed Th1 and Th2 responses

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stimulatory effects on bronchial epithelial cells

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production of proinflammatory cytokines and chemokines

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a protective role of PPARgamma

JACI 2006; 117;1090-7 Exp Lung Res 2006; 32:245-62 J Immunol 2006; 177:5248-57

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The PPARgamma is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily

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Is expressed on dendritic cells, eosinophils, macrophages, and T cells

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PPAR ligands are thought to exert anti inflammatory effects by negatively regulating the expression of pro inflammatory genes

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PPAR agonists to prevent and treat asthma

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

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

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

Question 55: is appropriate the concept of “united airways”?

YES

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upper and lower airways may be considered a unique entity

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allergic rhinitis is a risk factor for the occurrence and severity of asthma

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rhinoconjunctivitis is a risk factor for HMWA induced OA

Nasal allergen challenge Rhinitis - Sinusitis

Viral infection Loss of filtration efficiency Cytokines Postnasal drip Reflex Nose-bronchi Allergen Precursors Bone marrow

Eosinophils IL-5 Basophils Mast cells degranulation Eosinophils ICAM-1 VCAM-1 Bronchial allergen challenge Asthma

There is a gradient of T-cell activation (blood and induced sputum)

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ENA: a decrease of Th1, an increase of Th2 and Treg after SIT ( tolerating profile )

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OA: an increase of both Th1 and Th2, a decrease of Treg cells after SIT ( inflammatory profile )

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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

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

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

Several studies indicate that CD8 T cells

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do not exhibit a fixed phenotype

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can differentiate into functionally distinct subsets

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uptake by DCs is sufficient for their initial proliferative response to protein allergens

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sustained expansion and functional maturation of the CD8 response most likely requires signals from CD4 T cells or Toll-like receptor ligation

CD8 and occupational asthma

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Animal and human data show a mixed Th1/Th2 activation with implication of the CD8+ T cells in OA

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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

Questions 59-60: specific to compounds (diisocyanates)

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Physiologic levels of GSH protect against HDI exposure

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Type I hypersensitivity mechanism in 44% of TDI asthma by using vapor TDI-albumin conjugates

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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

Isocyanate asthma and oxidative stress

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glutathione S-transferase polymorphisms

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protective role of airway GSH

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protective role of PPARgamma agonists

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PPARgamma modulates reactive oxygen species (ROSs) generation and activates the redox-sensitive transcription factor nuclear-kappaB and hypoxia-inducible factor 1alpha

Question 60: asthma and hairdressers

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sensitization is more related to individual hypersusceptibility

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an immunologic mechanism is strongly suggested but not definitely demonstrated

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diagnosis is based on standardized SIC Moscato G and Galdi E. Curr Opion Allergy Clin Immunol 2006; 6:91-5

Certain, or a large body of Uncertain or limited evidence evidence

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asthma is heterogeneous skin and respiratory sensitization

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timing of OA, AHR and AI usefulness of animal models is different

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neutrophils fit both in allergic mechanisms of recognition of LMWA and nonallergic asthma by the immune system

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upper and lower airways as a role of GSH and a low pH in asthma unique entity

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role of DCs in asthma

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CD8+ T cells exhibit different phenotypes

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CD8+ T cells play a role in OA