Sandra Keir

Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)1 and sensory neuropeptides in the guinea-pig respiratory system

Electrophysiological studies within the lung have documented the presence of heterogenous groups of afferent fibers composed of Adelta and C-fibers and studies of somatosensory nerves within the skin reveal a complex pattern of distribution of sensory neuropeptides and transient receptor potential vanilloid (TRPV)1 positive nerves. However, the anatomical location of these different subpopulations of nerves within the lung has not been extensively studied. In the present study we have demonstrated that TRPV1 axons represented only a small proportion of the total number of PGP9.5 staining nerves within guinea-pig tracheal epithelium and only half the number of TRPV1 axons was immunopositive for substance P. In contrast, most TRPV1 positive neurones found within guinea-pig intrapulmonary airways were found to co-localize with sensory neuropeptides substance P and calcitonin gene-related peptide within and beneath the epithelium, around blood vessels, within airway smooth muscle and alveoli, indicative of heterogeneity of TRPV1 positive axons throughout the airways. However, in the smooth muscle layer of the trachea there was evidence of substance P and calcitonin gene-related peptide containing nerves that did not stain for TRPV1. We also demonstrated a complete loss of TRVP1 positive axons in the trachea and intrapulmonary airways and associated loss of bronchoconstriction induced by capsaicin, in animals chronically treated with capsaicin. However, some neuropeptide immunoreactive axons remained in the smooth muscle layer of capsaicin-treated animals which could represent the small subset of neuropeptide containing fibers which do not co-localize with TRPV1. We have provided evidence of heterogeneity of TRPV1 positive nerve fibers, including fibers characterized by lack of co-localization with neuropeptides in various regions of the airways and the existence of neuropeptide containing fibers that were not TRPV1 positive in guinea-pigs.

Protective effect of a PAR2-activating peptide on histamine-induced bronchoconstriction in guinea-pig

Protease activated receptor‐2 (PAR2) is a seven transmembrane domain G protein coupled receptor proteolytically activated. PAR2, together with other PARs, can be also activated by peptides mimicking the sequence of the receptor tethered ligand. We have evaluated the effect of systemic administration of a peptide activating PAR2 (PAR2‐AP, SLIGRL) on histamine‐induced increase in lung resistances in the guinea‐pig. Intravenous administration of PAR2‐AP (1 mg kg−1) significantly inhibited histamine‐induced increase in lung resistance in a time‐dependent fashion that was not abolished by indomethacin or vagotomy. Bronchoprotective effect of PAR2‐AP was not reversed by the cyclo‐oxygenase inhibitor, indomethacin, the nitric oxide synthetase inhibitor, L‐NAME, nor by the non‐selective beta‐antagonist, propranolol. Indomethacin augmented the bronchoconstriction to histamine which was inhibited by PAR2‐AP. Furthermore, in vagotomized animals, the bronchial hyper‐responsiveness to histamine was significantly reduced, and in these circumstances, PAR2‐AP still retained the capacity to provide bronchoprotection against histamine. PAR2‐AP also produced a modest reduction in histamine‐induced protein leakage in trachea and upper bronchi. Our results indicated that PAR2 might have a bronchoprotective role in the guinea‐pig in vivo independent of prostaglandin or nitric oxide release.

RhoA signaling through platelet P2Y1 receptor controls leukocyte recruitment in allergic mice

BACKGROUND Clinical studies reveal platelet activation in patients with asthma, allergic rhinitis, and eczema. This is distinct from platelet aggregation, which is critical for the maintenance of hemostasis and in which a role for platelet purinergic receptors is well documented. However, purines are also essential for inflammatory cell trafficking in animal models of allergic lung inflammation, which are known to be platelet dependent, yet the role of purines in the platelet activation accompanying inflammation is unknown. OBJECTIVES We investigated whether the involvement of purine activation of platelets during allergic inflammation is distinct from purine involvement in platelet aggregation. METHODS BALB/c mice were sensitized to ovalbumin and subsequent airway ovalbumin challenge. Bronchoalveolar lavage fluid was analyzed for inflammatory cells, and blood samples were assessed for platelet activation. The role of platelet purinergic receptors and associated signaling mechanisms (RhoA) were assessed. RESULTS P2Y₁, but not P2Y₁₂ or P2X₁, antagonism inhibited pulmonary leukocyte recruitment. The formation of platelet-leukocyte complexes in vivo and platelet/P-selectin-dependent polymorphonuclear cell migration in vitro were exclusively platelet P2Y₁ receptor dependent. Furthermore, platelet P2Y₁ activation resulted in RhoA activity in vivo after allergen challenge, and RhoA signaling in platelets through P2Y₁ stimulation was required for platelet-dependent leukocyte chemotaxis in vitro. Leukocyte recruitment in thrombocytopenic mice remained suppressed after reinfusion of platelets pretreated with a P2Y₁ antagonist or a Rho-associated kinase 1 inhibitor, confirming the crucial role of platelet P2Y₁ receptor and subsequent activation of RhoA. CONCLUSION RhoA signaling downstream of platelet P2Y₁, but not P2Y₁₂, represents a clear dichotomy in platelet activation during allergic inflammation versus hemostasis.

Mechanism of adenosine-induced airways obstruction in allergic guinea pigs.

Inhaled adenosine induces airway obstruction in asthmatic but not healthy subjects, a phenomenon that is also observed in various animal species when they are immunised to a relevant antigen, but which does not occur in naïve animals. The purpose of this study was to investigate the mechanisms of airway responsiveness to adenosine receptor agonists in anaesthetised allergic guinea pigs. Inhaled adenosine 5′‐monophosphate (AMP), the A1‐selective adenosine receptor agonist N6‐cyclopentyladenosine (CPA) and ovalbumin all caused airway obstruction in allergic guinea pigs, but not naïve animals, as assessed by changes in total lung resistance. In contrast, the A2a‐selective (CGS 21680; 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxoamido adenosine) and A3‐selective (IB‐MECA; 1‐deoxy‐1‐[6‐[[3‐iodophenyl)‐methyl]amino]‐9H‐purin‐9‐yl]‐N‐methyl‐β‐D‐ribofuranuronamide) adenosine receptor agonists failed to elicit airway obstruction in passively sensitised guinea pigs. Airway obstruction induced by AMP or CPA was not inhibited by the H1 receptor antagonist, mepyramine (1 mg kg−1) in passively sensitised guinea‐pigs. In contrast, airway obstruction to ovalbumin was significantly inhibited by this antagonist. Airway obstruction induced by AMP and CPA was significantly inhibited in sensitised animals chronically treated with capsaicin. In contrast, airway obstruction to ovalbumin was not inhibited by this treatment. Airway obstruction induced by AMP, CPA and ovalbumin was significantly inhibited following bilateral vagotomy or pharmacological treatment with atropine (2 mg kg−1). Airway obstruction to CPA was inhibited by the adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX: 0.1–1 mg kg−1). In contrast, airway obstruction to ovalbumin was not inhibited by this treatment. These observations provide evidence indicating that AMP and CPA may induce airway obstruction in sensitised guinea pigs by a mechanism unrelated to histamine release from mast cells, but is mediated via an adenosine A1‐receptor‐dependent mechanism. The inhibition of AMP‐ and CPA‐induced airway obstruction by atropine, capsaicin and bilateral vagotomy suggests a neuronal‐dependent mechanism with the particular involvement of capsaicin‐sensitive nerves.

Airway responsiveness in an allergic rabbit model

BACKGROUND Animal models of allergy and bronchial hyperresponsiveness (BHR) are useful in researching pulmonary diseases and evaluating drug effects on the airways. Neonatally immunised rabbits exhibit several features of asthma as adults, including early and late airway responses following antigen challenge, oedema and inflammatory cell infiltration into the lung, BHR to inhaled histamine and methacholine (compared with naïve rabbits) and exacerbations of BHR following antigen challenge. Therefore this model can be used to investigate the underlying mechanisms of BHR and for the preclinical evaluation of new drugs for the treatment of asthma. AIM To describe the characteristics of airway responses in a rabbit model of allergic inflammation and to evaluate the relationship between skin test reactivity to antigen, airway inflammation and BHR. METHODS New Zealand White rabbits were neonatally immunised against Alternaria tenius. At 3 months of age, airway responsiveness was measured to aerosolised histamine, methacholine or allergen. Bronchoalveolar lavage (BAL) was performed and cell counts recorded. Direct skin tests were performed to assess skin reactivity to allergen and passive cutaneous anaphylaxis (PCA) tests were performed to determine titres of circulating IgE. RESULTS In a population of allergic rabbits, allergen challenge induced a significant bronchoconstriction, airway inflammation and BHR. Skin test responsiveness to allergen did not correlate with various indices of pulmonary mechanics e.g. baseline sensitivity to methacholine and histamine, or allergen-induced BHR. In contrast, skin test responsiveness did predict airway inflammation as assessed by measurements of eosinophil recruitment to the lung. CONCLUSION The allergic rabbit is a useful model to further our understanding of allergic diseases. Recording lung function using a minimally invasive procedure allows repeated measurements to be made in rabbits longitudinally, and each animal may thus be used as its own control. Our observations do not support the use of skin responsiveness to allergen as a predictor of airway sensitivity as we observed no correlation between skin sensitivity and airway responsiveness to inhaled histamine, methacholine or allergen. However, skin reactivity did predict airway inflammation as assessed by measurements of eosinophil recruitment to the lung. Our results also further highlight the likely dissociation between airway inflammation and BHR.