Identification with MRI of the pleura as a major site of the acute inflammatory effects induced by ovalbumin and endotoxin challenge in the airways of the rat.

Inflammatory effects in the rat lung have been investigated, non-invasively by MRI, at early time points (3 and 6 h) after ovalbumin (OA) or endotoxin (LPS) challenges. Six hours after challenge with OA, a strong, even inflammatory signal was present around the periphery of the lung in a region corresponding to the pleura. Histological analysis confirmed the presence of marked edema associated with the pleural cavity of OA-treated animals. Lower levels of pleural edema were observed in MRI and histological evaluation of LPS-treated animals and no abnormality was observed in actively sensitized and naïve, saline-treated groups. Diffuse edematous signals were detected in the lung 3 and 6 h after challenge with OA or LPS; the signal volumes were larger at both time points following OA instillation. Bronchoalveolar lavage (BAL) fluid analysis performed 6 h after challenge revealed increased levels of protein and greater cellular activation in OA- than in LPS-treated animals. Furthermore, increased levels of peribronchial edema were found by histology 6 h after OA. BAL fluid and histological assessments demonstrated that the inflammatory signals were due to edema and not mucus as no significant changes in BAL mucin concentrations or differences in goblet cells were identified between OA or LPS challenge and their respective vehicle groups. Our data show that MRI is able to detect, non-invasively, inflammatory signals in both the lung and the pleura in spontaneously breathing animals, highlighting its potential to study the consequences of pulmonary insults on both sites.

Characterization of cigarette smoke-induced inflammatory and mucus hypersecretory changes in rat lung and the role of CXCR2 ligands in mediating this effect.

Repetitive, acute inflammatory insults elicited by cigarette smoke (CS) contribute to the development of chronic obstructive pulmonary disease (COPD), a disorder associated with lung inflammation and mucus hypersecretion. Presently, there is a poor understanding of the acute inflammatory mechanisms involved in this process. The aims of this study were to develop an acute model to investigate temporal inflammatory changes occurring after CS exposure. Rats were exposed to whole body CS (once daily) generated from filtered research cigarettes. Initial studies indicated the generation of a neutrophilic/mucus hypersecreting lung phenotype in <4 days. Subsequent studies demonstrated that just two exposures to CS (15 h apart) elicited a robust inflammatory/mucus hypersecretory phenotype that was used to investigate mechanisms driving this response. Cytokine-induced neutrophil chemoattractants (CINCs) 1-3, the rat growth-related oncogene-alpha family homologs, and IL-1beta demonstrated time-dependent increases in lung tissue or lavage fluid over the 24-h period following CS exposure. The temporal changes in the neutrophil chemokines, CINCs 1-3, mirrored increases in neutrophil infiltration, indicative of a role in neutrophil migration. In addition, a specific CXCR2 antagonist, SB-332235, effectively inhibited CS-induced neutrophilia in a dose-dependent manner, supporting this conclusion. This modeling of the response of the rat airways to acute CS exposure indicates 1) as few as two exposures to CS will induce a phenotype with similarities to COPD and 2) a novel role for CINCs in the generation of this response. These observations represent a paradigm for the study of acute, repetitive lung insults that contribute to the development of chronic disease.

Nucleotide-mediated mucin secretion from differentiated human bronchial epithelial cells.

Most current cell-based models for examining the regulation of mucin secretion demonstrate low signal-to-noise ratios, making experimental manipulation and data interpretation difficult. Using adenosine triphosphate (ATP) as a mucin secretagogue, we have developed a model of agonist-induced mucin secretion in differentiated human bronchial epithelial cells. Mucin secretory signals were estimated using enzyme-linked lectin assay, and typical signals of 300-400% of baseline were observed in response to a 30-min exposure to ATP (100 microM). ATP and uridine triphosphate equipotently stimulated mucin secretion consistent with mediation via P2Y2 receptor activation. Suramin and AR-C118925XX, a competitive P2Y2 receptor antagonist, inhibited adenosine 5'-o-(3-thiotriphosphate) (ATP-gammaS)-induced mucin secretion. A selective Gq G-protein antagonist (GP-ANT)-2A completely abrogated ATP-gammaS-induced mucin secretion. Pertussis toxin and the G(i/o)-specific, GP-ANT-2, had no effect. The phospholipase C inhibitor, D609, and the protein kinase C inhibitor, calphostin C, substantially inhibited ATP-gammaS-induced mucin secretion. Phorbol myristate acetate also stimulated mucin secretion in a calphostin C-sensitive manner. ATP-gammaS-induced mucin secretion was inhibited by the Ca2+ chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid tetra (acetoxymethyl) ester. Ionomycin and thapsigargin both stimulated mucin secretion. Our data are broadly consistent with known G-protein-coupling and downstream signaling events associated with the P2Y2 receptor. The exceptional signal-to-noise ratios obtained using this model have permitted clear evaluation of the involvement of these mechanisms in agonist-induced mucin secretion from differentiated human bronchial epithelial cells.

Mucociliary clearance is enhanced in rat models of cigarette smoke and lipopolysaccharide-induced lung disease.

In this study, the authors describe a new technique enabling the rapid assessment of mucociliary clearance (MCC) in rats and characterize this aspect of innate host defense in 2 animal models of bronchitis. Following instillation into the airways, fluorescent microspheres were rapidly cleared over 24 hours, with 60% to 80% of clearance occurring within 4 hours. On a background of airway neutrophilia and mucus hypersecretion, induced by either lipopolysaccharide or cigarette smoke, MCC was significantly enhanced. This reserve capacity in the MCC system will need to become overwhelmed in order to model the clinically observed impairment of lung mucus clearance in an animal system.

Noninvasive detection of endotoxin-induced mucus hypersecretion in rat lung by MRI.

Using magnetic resonance imaging (MRI), we detected a signal in the lungs of Brown Norway rats after intratracheal administration of endotoxin [lipopolysaccharide (LPS)]. The signal had two components: one, of diffuse appearance and higher intensity, was particularly prominent up to 48 h after LPS; the second, showing an irregular appearance and weaker intensity, was predominant later. Bronchoalveolar lavage fluid analysis indicated that generalized granulocytic (especially neutrophilic) inflammation was a major contributor to the signal at the early time points, with mucus being a major factor contributing at the later time points. The facts that animals can breathe freely during data acquisition and that neither respiration nor cardiac triggering is applied render this MRI approach attractive for the routine testing of anti-inflammatory drugs. In particular, the prospect of noninvasively detecting a sustained mucus hypersecretory phenotype in the lung brings an important new perspective to models of chronic obstructive pulmonary diseases in animals.

Pulmonary inflammation monitored noninvasively by MRI in freely breathing rats.

A detailed analysis has been carried out of the correlation between the signals detected by MRI in the rat lung after allergen or endotoxin challenge and parameters of inflammation determined in the broncho-alveolar lavage (BAL) fluid. MRI signals after allergen correlated highly significantly with the BAL fluid eosinophil number, eosinophil peroxidase activity and protein concentration. Similar highly significant correlations were seen when the anti-inflammatory glucocorticosteroid, budesonide, manifested against allergen. In contrast, following endotoxin challenge, mucus was the sole BAL fluid parameter that correlated significantly with the long lasting signal detected by MRI. Since edema is an integral component of pulmonary inflammation, MRI provides a noninvasive means of monitoring the course of the inflammatory response and should prove invaluable in profiling anti-inflammatory drugs in vivo. Further, the prospect of noninvasively detecting a sustained mucus hypersecretory phenotype in the lung brings an important new perspective to models of chronic obstructive pulmonary diseases.

Development and validation of a lectin-based assay for the quantitation of rat respiratory mucin.

The significance of a mucus hypersecretory phenotype to the pathogenesis of airways disease is controversial, but increasing evidence suggests that it can negatively impact upon patient health status. A critical aspect of developing our understanding of the role of mucus hypersecretion in disease is the development and appropriate use of methods permitting detection and quantitation of mucins in biological samples. However, the unique biophysical and biochemical properties of this class of glycoproteins do not lend themselves to routine quantitation. Individual pure mucins are not commercially available, the reactivity profile of commonly used reagents is generally not well characterized and assay development and validation is rarely covered adequately in the literature. Therefore quantitation of mucin in biological samples relies upon careful histochemical and biochemical characterization and partially purified mucin preparations. The absence of tools considered essential for assay development in other areas means that this characterization process does not generally lead to proof of selective detection of mucin, but rather to a level of confidence that mucin is detected and defined contaminants are not. This chapter provides an example of the process of development and validation of a lectin-based assay for quantitation of mucin in untreated complex biological samples.