Jonathan E. Phillips

CXCR2 antagonists for the treatment of pulmonary disease.

Chemokines have long been implicated in the initiation and amplification of inflammatory responses by virtue of their role in leukocyte chemotaxis. The expression of one of the receptors for these chemokines, CXCR2, on a variety of cell types and tissues suggests that these receptors may have a broad functional role under both constitutive conditions and in the pathophysiology of a number of acute and chronic diseases. With the development of several pharmacological, immunological and genetic tools to study CXCR2 function, an important role for this CXC chemokine receptor subtype has been identified in chronic obstructive pulmonary disease (COPD), asthma and fibrotic pulmonary disorders. Interference with CXCR2 receptor function has demonstrated different effects in the lungs including inhibition of pulmonary damage induced by neutrophils (PMNs), antigen or irritant-induced goblet cell hyperplasia and angiogenesis/collagen deposition caused by lung injury. Many of these features are common to inflammatory and fibrotic disorders of the lung. Clinical trials evaluating small molecule CXCR2 antagonists in COPD, asthma and cystic fibrosis are currently underway. These studies hold considerable promise for identifying novel and efficacious treatments of pulmonary disorders.

Tachykinin NK3 and NK1 receptor activation elicits secretion from porcine airway submucosal glands

We presently characterized the tachykinin receptor subtypes, using tachykinin receptor agonists and selective antagonists, that induce submucosal gland fluid flux (JG) from porcine tracheal explants with the hillocks technique. We also investigated the effects of the tachykinin receptor agonists on the electrophysiologic parameters of the tracheal epithelium in Ussing chambers. The NK1 tachykinin receptor agonist substance P (SP, 1 μM) and the NK3 tachykinin receptor agonist [MePhe7]neurokinin B ([MePhe7]NKB, 1 μM) induced gland fluid fluxes of 0.29±0.03 μl min−1 cm−2 (n=26) and 0.36±0.05 μl min−1 cm−2 (n=24), respectively; while the NK2 tachykinin receptor agonist [βAla8]neurokinin A (4‐10) ([βAla8]NKA (4‐10), 1 μM) had no effect on JG (n=10). The NK1 receptor antagonist CP99994 (1 μM, n=9) blocked 93% of the SP‐induced JG, whereas the NK3 receptor antagonist SB223412 (1 μM, n=12) had no effect on the SP‐induced JG. However, SB223412 (1 μM, n=9) blocked 89% of the [MePhe7]NKB‐induced JG while CP99994 (1 μM, n=10) did not affect the [MePhe7]NKB‐induced JG. The NK2 receptor antagonist SR48968 (1 μM) did not block the JG induced by either the NK1 (n=4) or NK3 (n=13) receptor agonists. The nicotinic ganglionic acetylcholine receptor antagonist hexamethonium (1 μM) and the muscarinic acetylcholine receptor antagonist atropine (1 μM) also decreased the NK3 receptor agonist‐induced JG by 67% (n=10) and 71% (n=12), respectively. The potential difference (PD), short‐circuit current (ISC), and membrane resistance (RM) of the porcine tracheal epithelial membranes were not significantly affected by any of the neurokinin agonists or antagonists (1 μM, basolateral) used in this study, although SP and [βAla8]NKA (4‐10) induced a slight transient epithelial hyperpolarization. These data suggest that NK1 and NK3 receptors induce porcine airway gland secretion by different mechanisms and that the NK3 receptor agonists induced secretion is likely due to activation of prejunctional NK3 receptors on parasympathetic nerves, resulting in acetylcholine‐release. We conclude that tachykinin receptor antagonists may have therapeutic potential in diseases with pathophysiological mucus hypersecretion such as asthma and chronic bronchitis.

Computer assisted measurement of airway gland secretions by the hillocks technique

We describe a computer-based image acquisition and analysis system designed for the quantitation of airway submucosal gland fluid flux by the hillocks technique. This technique is based on the detection of the increase in surface area of hillocks formed above gland duct openings by secretions captured under a thin layer of tantalum powder. The advantages of the system are: (a) ability to detect and measure individual submucosal gland secretions, (b) computer assisted data acquisition and analysis, and (c) decreased propagated error in hillock volume measurements. Test results of a practical implementation of the system demonstrate a swine tracheal submucosal gland flux induced by the muscarinic agonist acetylcholine (50 microM) of 0.65 microl/min/cm(2).

Manual and automated leukocyte differentiation in bronchoalveolar lavage fluids from rodent models of pulmonary inflammation

Manual total and differential leukocyte counting in bronchoalveolar lavage fluids (BALF) by visual microscopy is a standard means of evaluating airway inflammation and the anti-inflammatory properties of therapeutics in various animal models of lung disease. The manual cell counting method derives total leukocyte counts from BALF with a hemocytometer, and cell differentials (mononuclear, neutrophil, eosinophil) are calculated from the percentage of each cell type taken from a count of at least 200 cells on a stained cytocentrifuge preparation of the BALF cells. These manual methods are time-consuming and have inherent error–variability. The ADVIA 120 Hematology System is an automated analyzer designed to perform total and differential leukocyte analysis of blood. With the light scattering, cell lysis resistance, and cytochemical staining data from a BALF sample processed by the ADIVA, a BALF total leukocyte count and differential analysis is provided in approximately 30xa0s. In order to correlate automated BALF leukocyte counting by the ADVIA 120 Hematology System with manual counting, we developed a manual red blood cell lysing and white blood cell staining technique for BALF cells similar to the process used by the ADVIA. Significant correlations for BALF white blood cells were obtained for the manual (microscopic analysis) and the automated (ADVIA) methods. Comparison of manual and automated cell counts also generates the same conclusions about anti-inflammatory drug efficacy. Both manual and automated cell counting methods agree that 3xa0mg/kg orally administered dexamethasone inhibited cigarette-smoke-induced total BALF cell counts by ∼65% in mice and 42xa0μg/kg fluticasone propionate delivered by nose-only inhalation inhibited allergen-induced total BALF cells by 77% in rats. The use of the ADVIA to perform total and differential leukocyte counts in BALF will save time spent manually counting cells and this instrument will standardize the analysis of white blood cells across the laboratories currently using various manual counting preparations and procedures.

Nmur1−/− mice are not protected from cutaneous inflammation

Neuromedin U (Nmu) is a neuropeptide expressed primarily in the gastrointestinal tract and central nervous system. Previous reports have identified two G protein-coupled receptors (designated Nmur1 and Nmur2) that bind Nmu. Recent reports suggest that Nmu mediates immune responses involving mast cells, and Nmur1 has been proposed to mediate these responses. In this study, we generated mice with an Nmur1 deletion and then profiled the responses of these mice in a cutaneous inflammation model utilizing complete Freunds adjuvant (CFA). We report here that mice lacking Nmur1 had normal inflammation responses with moderate changes in serum cytokines compared to Nmur1(+/+) littermates. Although differences in IL-6 were observed in mice lacking Nmu peptide, these mice exhibited a normal response to CFA. Our data argues against a major role for Nmur1 in mediating the reported inflammatory functions of NmU.

Introduction to the pulmonary research group's symposium upper airway disease: Clinical and research perspectives

The following seven articles in this issue of Pulmonary Pharmacology and Therapeutics represent the proceedings of the Spring 2006 symposium entitled Upper Airway Disease: Clinical and Research Perspectives. The symposium was organized by the Pulmonary Research Group (PRG) and took place at the Asthma and Allergy Center, Johns Hopkins University, Baltimore, Maryland on May 4, 2006. The symposium speakers reviewed our current knowledge of upper airway diseases and addressed key questions about definitions, basic biological mechanisms and novel therapies. The upper airways extend from the nares to the larynx and are continuous with the lower airways, which begin at the trachea and extend to the alveoli. Dr. Jacqueline Corey (1) presented and discussed clinical techniques for measurement of nasal airway obstruction, a common clinical problem. Although the upper and lower respiratory tract have many similarities, the congestion observed in the upper respiratory tract is due to the dilation of the erectile vascular tissue present around the septum and turbinates while erectile tissue is absent in the lower airway. Since the vasculature and nervous system are major components in the generation of nasal symptoms, Dr. James Baraniuk (2) discussed the crucial role the components of the nasal erectile apparatus, such as the arteriovenous anatomoses, venous sinusoids and veins, play in the regulation the efflux of blood out of nasal sinusoids, as well as the provocative role that nociceptive nerve receptors and ion channels, in particular the TRPV1, play in nasal mucosal inflammation. Dr. Michel Corboz (3) provided evidence in animal models that a selective a2-adrenoceptor agonist-induced preferential constriction of nasal venous capacitance vessels and also produced nasal decongestion without a significant effect on blood pressure, which is typically observed with non-selective a-adrenoceptor decongestants. The authors therefore proposed that a2-adrenoceptors provide an effective and safer target for decongestion in all types of rhinitis, allergic and non-allergic. Dr. Thomas TaylorClark (4) presented an overview of the contribution of histamine (H)1, H2, and H3 receptors on nasal blockage, defined as occlusion of the nasal cavities. When the basal sympathetic tone that maintains decongestion is reduced