Direct Intratracheal Instillation Research Articles

Sublethal pulmonary toxicity screening of silica nanoparticles in rats after direct intratracheal instillation.

The present aimed to characterize the toxicity of silica nanoparticles in Sprague Dawley rats and determine the dose levels for a repeated-dose toxicity study. Silica nanoparticles (SiO2, 20nm and 50nm) were administered as a single intratracheal instillation of standardized SiO2 20nm (low dose, 200µg/mL; high dose, 400µg/mL) and 50nm (low dose, 200µg/mL; high dose, 400µg/mL). Each group consisted of five male rats. We documented the mortality rate, clinical signs, body weight, bronchoalveolar lavage fluid analysis, hematological values, serum chemistry values, organ weight, gross findings at necropsy, and histopathological assessments. Rats treated with 200µg/mL and 400µg/mL SiO2 50nm exhibited a decreased mean corpuscular volume, while those treated with 400µg/mL of SiO2 50nm showed increases in absolute monocyte and absolute lymphocyte count as well as prothrombin time. In addition, rats treated with 400µg/mL SiO2 20nm and 50nm presented reduced creatinine, alanine aminotransferase, and sodium levels. Therefore, a single intratracheal instillation of SiO2 20nm and 50nm elicited no toxicity up to a dose of 400µg/mL, and the approximate lethal dose of this test substance exceeded 400µg/mL in male Sprague Dawley rats under the present experimental conditions.

Endoscopic atomization of mesenchymal stromal cells: in vitro study for local cell therapy of the lungs

Background aimsCell-based therapies of pulmonary diseases with mesenchymal stromal cells (MSCs) are increasingly under experimental investigation. In most of these, MSCs are administered intravenously or by direct intratracheal instillation. A parallel approach is to administer the cells into the lung by endoscopic atomization (spraying). In a previous study, the authors developed a flexible endoscopic atomization device that allows administration of respiratory epithelial cells in the lungs with high survival. MethodsIn this study, the authors evaluated the feasibility of spraying MSCs with two different endoscopic atomization devices (air and pressure atomization). Following atomization, cell viability was evaluated with live/dead staining. Subsequent effects on cytotoxicity, trilineage differentiation and expression of MSC-specific markers as well as on MSC metabolic activity and morphology were analyzed for up to 7 days. ResultsMSC viability immediately after spraying and subsequent metabolic activity for 7 days was not influenced by either of the devices. Slightly higher cytotoxicity rates could be observed for pressure-atomized compared with control and air-atomized MSCs over 7 days. Flow cytometry revealed no changes in characteristic MSC cell surface marker expression, and morphology remained unchanged. Standard differentiation into osteocytes, chondrocytes and adipocytes was inducible after atomization. ConclusionsIn the literature, a minimal survival of 50% was previously defined as the cutoff value for successful cell atomization. This is easily met with both of the authors’ devices, with more than 90% survival. Thus, there is a potential role for atomization in intrapulmonary MSC-based cell therapies, as it is a feasible and easily utilizable approach based on clinically available equipment.

Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.

During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent.

Changes in cytokine and nitric oxide secretion by rat alveolar macrophages after oral administration of bacterial extracts.

Oral administration of the bacterial immunomodulator Broncho-Vaxom (OM-85), a lysate of eight bacteria strains commonly causing respiratory disease, has been shown to enhance the host defence of the respiratory tract. In this study we examined the effect of orally administered (in vivo) OM-85 on stimulus-induced cytokine and nitric oxide secretion by rat alveolar macrophages in vitro. The results show that alveolar macrophages isolated from OM-85-treated rats secreted significantly more nitric oxide, tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta upon in vitro stimulation with lipopolysaccharide (LPS), whereas, in contrast, LPS-induced IL-6 secretion was significantly lower. The observed effects of in vivo OM-85 treatment on stimulus-induced cytokine secretion in vitro are not due to a direct effect of OM-85 on the cells, because in vitro incubation of alveolar macrophages with OM-85 did not result in altered activity, nor did direct intratracheal instillation of OM-85 in the lungs of rats result in altered alveolar macrophage activity in vitro. It is hypothesized that oral administration of OM-85 leads to priming of alveolar macrophages in such a way that immune responses are non-specifically enhanced upon stimulation. The therapeutic action of OM-85 may therefore result from an enhanced clearance of infectious bacteria from the respiratory tract due to increased alveolar macrophage activity.

Statins Reduce Ambient Particulate Matter-Induced Lung Inflammation by Promoting the Clearance of Particulate Matter < 10 μm From Lung Tissues

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) suppress ambient particulate matter, 10 μm (PM(10) )-induced inflammatory response in vitro. The aim of this study was to determine the effect of statins on PM(10) -induced lung inflammation in vivo. New Zealand white rabbits were exposed to either PM(10) (1.0 mg/kg) or saline by direct intratracheal instillation three times a week for 4 weeks lovastatin 5.0 mg/kg/d. BAL fluid was assessed for cell counts and proinflammatory cytokine levels. Lung inflammation was quantified using immunohistochemical techniques and morphometric methods. Ex vivo phagocytosis assay of alveolar macrophages using PM 10 particles was performed. Distribution of PM(10) particles in lung tissues and draining lymph nodes was quantified morphometrically to evaluate the clearance of PM(10) particles. PM(10) exposure increased the production of IL-6 and IL-8, promoted the recruitment of macrophages and polymorphonuclear leukocytes into the lung, and activated these recruited leukocytes. Lovastatin significantly suppressed all these effects. Lovastatin increased the phagocytic activity of macrophages and promoted the migration of PM 10 -laden macrophages to the regional lymph nodes. Lovastatin attenuates the PM(10) -induced recruitment and activation of alveolar macrophages and polymorphonuclear leukocytes, reduces local proinflammatory cytokine production, and promotes the clearance of PM(10) particles from lung tissues to regional lymph nodes. These novel pleiotropic properties of statins are most likely to contribute to the downregulation of PM(10) -induced lung inflammation.

Pulmonary Endotoxin Tolerance Protects against Cockroach Allergen-Induced Asthma-Like Inflammation in a Mouse Model

Background: Compounds which activate the innate immune system, such as lipopolysaccharide, are significant components of ambient air, and extremely difficult to remove from the environment. It is currently unclear how prior inhalation of endotoxin affects allergen sensitization. We examined whether lung-specific endotoxin tolerance induction prior to sensitization can modulate the response to allergen. Methods: Endotoxin tolerance was induced by repeated intratracheal exposure to endotoxin. All mice were then sensitized and challenged by direct intratracheal instillation of cockroach allergen. Results: After allergen sensitization and challenge, endotoxin tolerant mice had significantly decreased airways hyperresponsiveness to methacholine challenge, which was confirmed by invasive lung function tests. Decreased goblet cell hyperplasia and mucus production were also found by histological assessment. Tolerant mice were protected from airway eosinophilia through the mechanism of reduced CCL11 and CCL24. Interestingly, endotoxin tolerant mice had only a modest reduction in cockroach-specific IgE; however, total IgE was significantly reduced. Conclusions: These data show that induction of endotoxin tolerance prior to sensitization protects against the hallmark features of asthma-like inflammation, and that transient modulation of innate immunity can have long-lasting effects on adaptive responses.

TLR4 is necessary for hyaluronan-mediated airway hyperresponsiveness after ozone inhalation.

Ozone is a common environmental air pollutant that contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced airway hyperresponsiveness, remain poorly understood. We have previously reported that toll-like receptor 4 (TLR4)-deficient animals are protected against ozone-induced airway hyperresponsiveness (AHR) and that hyaluronan (HA) mediates ozone-induced AHR. However, the relation between TLR4 and hyaluronan in the airway response to ozone remains unexplored. We hypothesized that HA acts as an endogenous TLR4 ligand for the development of AHR after ozone-induced environmental airway injury. TLR4-deficient and wild-type C57BL/6 mice were exposed to either inhaled ozone or intratracheal HA and the inflammatory and AHR response was measured. TLR4-deficient mice have similar levels of cellular inflammation, lung injury, and soluble HA levels as those of C57BL/6 mice after inhaled ozone exposure. However, TLR4-deficient mice are partially protected from AHR after ozone exposure as well as after direct intratracheal instillation of endotoxin-free low molecular weight HA. Similar patterns of TLR4-dependent cytokines were observed in the bronchial alveolar lavage fluid after exposure to either ozone or HA. Exposure to ozone increased immunohistological staining of TLR4 on lung macrophages. Furthermore, in vitro HA exposure of bone marrow-derived macrophages induced NF-kappaB and production of a similar pattern of proinflammatory cytokines in a manner dependent on TLR4. Our observations support the observation that extracellular matrix HA contributes to ozone-induced airways disease. Furthermore, our results support that TLR4 contributes to the biological response to HA by mediating both the production of proinflammatory cytokines and the development of ozone-induced AHR.

Cyclooxygenase-2/Prostaglandin D2/CRTH2 Pathway Mediates Double-Stranded RNA-Induced Enhancement of Allergic Airway Inflammation

Respiratory RNA viruses responsible for the common cold often worsen airway inflammation and bronchial responsiveness, two characteristic features of human asthma. We studied the effects of dsRNA, a nucleotide synthesized during viral replication, on airway inflammation and bronchial hyperresponsiveness in murine models of asthma. Intratracheal instillation of poly I:C, a synthetic dsRNA, increased the airway eosinophilia and enhanced bronchial hyperresponsiveness to methacholine in OVA-sensitized, exposed rats. These changes were associated with induction of cyclooxygenase-2 (COX-2) expression and COX-2-dependent PGD2 synthesis in the lungs, particularly in alveolar macrophages. The direct intratracheal instillation of PGD2 enhanced the eosinophilic inflammation in OVA-exposed animals, whereas pretreatment with a dual antagonist against the PGD2 receptor-(CRTH2) and the thromboxane A2 receptor, but not with a thromboxane A2 receptor-specific antagonist, nearly completely eliminated the dsRNA-induced worsening of airway inflammation and bronchial hyperresponsiveness. CRTH2-deficient mice had the same degree of allergen-induced airway eosinophilia as wild-type mice, but they did not exhibit a dsRNA-induced increase in eosinophil accumulation. Our data demonstrate that COX-2-dependent production of PGD2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenetic factors responsible for the dsRNA-induced enhancement of airway inflammation and responsiveness.

Mapping alveolar binding sites in vivo using phage peptide libraries.

Targeting lung tissue is nonselective due in part to the lack of specific cell-surface receptors identified on target lung cells. We used in vivo phage display to identify a panel of peptides that can bind selectively to lung epithelial cells with less binding to nonepithelial cells. By direct intratracheal instillation of phage libraries into the lung, we isolated and identified 143 individual phage clones. Three phage clones revealed enhanced binding to the lung in vitro and in vivo. These three identified peptides were synthesized and demonstrated selective binding to epithelial cells in lung tissue versus the control peptide. Further, the peptides specifically bound to freshly isolated type II alveolar epithelial cells compared with Hep2 cells. The results suggest that the airway phage display approach could be exploited for analyzing the molecular diversity in the lower respiratory tract.

Nonviral gene delivery to the lung with copolymer-protected and transferrin-modified polyethylenimine

Polyethylenimine (PEI) has been shown to efficiently mediate topical gene transfer to the lungs after either direct intratracheal instillation or nebulisation. Recently, the protection of polyplexes with novel copolymers of poly(ethylene glycol) (PEG) via electrostatic interaction has been reported. In this study, such coated PEI polyplexes were investigated for their stability and interaction with human plasma and bronchoalveolar lavage fluid (BALF). Further, their potential for gene delivery to the mouse lungs in vivo was examined. Plasma protein and mucin adsorption was effectively inhibited when polyplexes were coated with the novel copolymers. Gene transfer efficiency of the coated PEI polyplexes decreased as compared with uncoated PEI polyplexes when administered intratracheally to the lung. The higher the molecular weight of the copolymerized PEG was, the stronger the observed gene transfer reduction. Gene transfer decreased presumably due to reduced interaction of the coated gene vectors with the cell surface. To circumvent this problem, transferrin was combined with PEI/DNA polyplexes for specific binding to the cell surface. In this case, gene transfer efficiency decreased. Gene transfer of the copolymer-protected and transferrin-modified gene vectors increased as compared with the copolymer-protected gene vectors alone but did not reach the level of uncoated gene vectors. These data show that copolymers could be used to effectively shield polyplexes from interaction with components of the airway surface liquid (ASL). Increased gene delivery was found upon transferrin modification of the coated PEI polyplexes suggesting a targeting effect.

Use of perfluorocarbon (fluorinert) to enhance reporter gene expression following intratracheal instillation into the lungs of Balb/c mice: Implications for nebulized delivery of plasmids

Perfluorocarbons combine high respiratory gas dissolving capabilities with extreme chemical and biological inertness and therefore offer an attractive option as an excipient in the area of pulmonary therapeutics. Perfluorocarbons have also been shown to ‘float’ mucus, because of their high densities (1.9–2.5 g/mL), which may hold potential in gene delivery for cystic fibrosis patients, in terms of enhancing penetration through highly viscous mucus and thereby providing access to target epithelial cells to correct the gene defect. Additionally, their low surface tension allows for better dispersion. A commonly available perflurocarbon, heptacosafluorotributylamine (Fluorinert), was used to deliver either plasmid DNA (pDNA) alone or cationic‐lipid‐complexed plasmid DNA to the lungs of Balb/c mice by direct intratracheal instillation. The complexes consisted of supercoiled (SC) plasmid DNA (4.7 Kb, 0.625mg/mL) and lipid (ethyldimyristoyl phosphatidylcholine [EDMPC]/cholesterol [1:1 mole ratio], with pDNA (3:1mg pDNA/mM EDMPC in 20mM Tris‐HCl pH 8.0) expressing chloramphenicol acetyl transferase (CAT) or β‐galactosidase (β‐Gal). pDNA alone was supplemented with 14% w/v Fluorinert. Cationic lipid/pDNA complexes were supplemented with 3, 8, and 14% w/v Fluorinert. Results showed that the CAT expression from pDNA alone was enhanced 24× using 14% w/v Fluorinert, whereas that from the cationic‐lipid‐formulated pDNA was enhanced 7× using 14% w/v Fluorinert. Immunohistochemistry showed that β‐Gal expression was primarily from epithelial cells and not from F4/80 or MAC3 antigen‐stained cells (predominantly macrophages), indicating efficient delivery. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1336–1344, 2001

Enhanced mortality from perfluorocarbon administration in a rat model of kerosene aspiration.

Aspiration of low-viscosity hydrocarbons may lead to fulminant pneumonitis and acute respiratory distress syndrome. Animal and human studies suggest that partial liquid ventilation with perfluorocarbon improves gas exchange and pulmonary function in acute respiratory failure. The objective of this investigation was to determine the effect of intratracheal perfluorocarbon administration and a brief period of partial liquid ventilation on survival in a rat model of severe hydrocarbon aspiration. Two randomized, non-blinded, controlled experiments were performed. First, male Wistar rats (n = 12) were given 0.3 mL/kg kerosene via direct intratracheal instillation and after 5 minutes were randomized to partial liquid ventilation or standard gas ventilation (control) groups. Partial liquid ventilation rats (n = 6) received 20 mL/kg of pre-oxygenated FC-77 intratracheally and positive-pressure gas ventilation (FiO2 = 1.0), and control rats (n = 6) received positive-pressure gas ventilation alone. Animals were observed for survival and 7-day mortality was compared using the Fisher Exact test. The study was then repeated in 12 additional animals using a 15-minute interval between kerosene instillation and treatment (PLV vs control). Mortality was significantly greater in the partial liquid ventilation group (5 of 6; 83%) as compared to the control group (1 of 6; 17% [p = 0.039]). Results were identical in the repeat study. All animals that died succumbed from acute respiratory failure within 24 hours of kerosene instillation. In this rat model of severe kerosene aspiration, intratracheal perfluorocarbon administration and a brief period of partial liquid ventilation proved detrimental and significantly increased mortality.

Cationic lipid-mediated gene delivery to murine lung: correlation of lipid hydration with in vivo transfection activity.

A panel of lipidic tetraalkylammonium chlorides has been prepared and screened in studies of both lipid hydration and in vivo mouse transfection. The effect of cationic lipid structure on liposome surface hydration was determined using differential scanning calorimetry. Increases in headgroup steric bulk and the inclusion of cis-unsaturation in the hydrophobic domain led to greater lipid hydration, indicative of a decrease in lipid polar domain associations. Cationic lipids containing hydrogen-bonding functionality in the polar domain exhibited a corresponding decrease in observed lipid hydration, indicative of an increase in lipid polar domain associations. To explore a potential correlation of the hydration data with transfection activity, we examined the in vivo transfection activity of the lipid panel by direct intratracheal instillation of cationic liposome-DNA complexes into BALB/c mice. The more active transfection agents were the lipids that featured headgroup structures promoting close polar domain association in combination with fatty acyl cis-unsaturation. The hydration data suggest that the more effective transfection lipids for mouse lung delivery are those possessing the greatest imbalance between the cross-sectional areas occupied by the polar and hydrophobic domains.

Exogenous surfactant enhances the delivery of recombinant adenoviral vectors to the lung.

Somatic gene therapy for pulmonary diseases must be accomplished in vivo, requiring the spread of a gene transfer vector across a vast expanse of respiratory epithelium. Surfactant, a naturally occurring protein and lipid mixture used to treat the respiratory distress syndrome of prematurity, disperses rapidly and evenly throughout the lung. We employed exogenous bovine surfactant (Survanta beractant) as a carrier vehicle for pulmonary delivery of a recombinant adenovirus expressing beta-galactosidase (beta-Gal). Rats treated with an adenovirus-beractant mixture demonstrated more uniform lobar distribution of transgene expression than rats treated with the same amount of virus in saline. Tissue homogenates were examined for quantitative beta-Gal expression by reaction with o-nitrophenol beta-n-galactopyranoside (ONPG). The degree of beta-Gal activity was affected by both the volume and type of carrier used to deliver the virus. At low volumes (0.5 ml, 1.3 ml/kg), beractant-treated animals demonstrated significantly greater pulmonary beta-Gal activity than saline-treated animals (p < 0.002) and untreated controls. At high volume (1.2 ml, 4 ml/kg), average beta-Gal activity was similar between groups treated with beractant or saline, but was more variable within the saline treated group. Higher volumes of delivery medium were associated with increased levels of beta-Gal expression regardless of the carrier used. Survanta was well tolerated by the animals and did not affect the duration of transgene expression. Exogenous beractant provides a useful medium for delivering recombinant adenoviruses to the lung when diffuse distribution of transgene expression is desired.

Activation of a distinct subpopulation of pulmonary macrophages following exposure to biological response modifiers.

A distinct subpopulation of tissue-associated pulmonary macrophages (TAPM) displayed tumoricidal activity towards syngeneic and xenogeneic targets following in vitro incubation with N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). This subpopulation, as well as, the predominant population of freely lavagable alveolar macrophages destroyed allogeneic targets following a similar incubation with either 6-0-stearoyl MDP (S-MDP) or recombinant interferon-gamma (IFN-gamma). IFN-gamma-induced in vivo tumoricidal activation of both populations of pulmonary macrophage was most effective when delivered either intravenously or via osmotic minipump infusion and least effective when administered by direct intratracheal instillation. The separate populations also displayed in vivo activation in response to liposome-encapsulated i.v. administered S-MDP. Under comparable conditions, IFN-alpha was not nearly as effective. Metabolic activation of TAPM, assessed by the release of increased levels of superoxide free radicals during phagocytosis, occurred following 24 hr exposure to S-MDP or lipopolysaccharide. Incorporation of these agents into multilamellar vesicle liposomes further augmented the release of superoxide observed at 24 hrs. Our results collectively demonstrated that a subpopulation of lung macrophage, a tissue-associated pulmonary macrophage, may be activated to a tumoricidal state and to release pronounced levels of oxygen free radicals following either in vitro or in situ treatment with several biological response modifiers.

Acute Lung Injury Induced by Phospholipase A2: Structural and Functional Changes

On the basis of the observation that serum levels of phospholipase A2 (PLA2) are elevated in pancreatitis and systemic sepsis, and the association of these conditions with the subsequent development of acute lung injury, the present investigation examined the structural and physiologic consequences of intratracheal administration of PLA2 to adult male rats. Rats received direct intratracheal instillation of either control vehicle or 40,000 units/kg of PLA2 repurified from Naja naja venom. Animals treated with PLA2 showed higher cumulative mortality (33% versus 0%, n = 79; p less than 0.01) than did their control littermates. The PLA2-treated animals showed histologic evidence of acute lung injury characterized by interstitial and alveolar edema, accumulation of inflammatory cells, and alveolar wall thickening, which reached maximal severity 48 h after enzyme instillation. Forty-eight hours after PLA2 administration experimental animals had lower arterial oxygen tensions (73.9 +/- 7.66 mm Hg versus 96.7 +/- 2.52 mm Hg, mean +/- SEM; p less than 0.01), higher alveolar-arterial oxygen gradients (35.3 +/- 6.3 mm Hg versus 18.8 +/- 1.42 mm Hg, p less than 0.01), and higher wet-dry lung weight ratios (5.08 +/- 0.26, mean +/- SEM, n = 7 versus 3.29 +/- 0.08, n = 3; p less than 0.002) than did control animals. Lung lavage from experimental animals 48 h after PLA2 instillation showed increased total cell counts [(26.6 +/- 5.04) x 10(6) cells versus (4.69 +/- 1.48) x 10(6) cells; p less than 0.01], an increased percentage of neutrophils (34.2 +/- 4.6% versus 1.25 +/- 0.25%, mean +/- SEM; p less than 0.01), and increased protein concentrations in lavage fluid (0.38 +/- 0.06 mg/ml, mean +/- SEM, n = 4 versus 0.27 +/- 0.02 mg/ml, n = 5; p less than 0.05). The histologic and physiologic abnormalities had largely resolved by 240 h. These results suggest that PLA2 may be a potent mediator of lung inflammation and that intratracheal administration of PLA2 to adult rats may provide a useful experimental model of acute lung injury.

Human eosinophil major basic protein induces airway constriction and airway hyperresponsiveness in primates.

We have examined the effects of direct intratracheal instillation of purified eosinophil granule proteins on pulmonary function and airway responsiveness in primates. The results of this study show for the first time that installation of major basic protein (MBP) directly into the trachea of primates results in a significant and dose-related increase in airway responsiveness to inhaled methacholine. Furthermore, MBP and eosinophil peroxidase (EPO) induce a transient bronchoconstriction immediately after instillation that resolves by 1 h postinstillation. In contrast, instillation of other eosinophil granule proteins had no effect on airway responsiveness or pulmonary function. These data indicate a direct role of the eosinophil in the pathogenesis of airway hyperresponsiveness. We suggest that the MBP of human eosinophils has an effector role in the pathogenesis of airway hyperresponsiveness which may involve active interaction with resident airway tissue cells. MBP may also mediate altered lung function in various inflammatory lung diseases associated with pulmonary eosinophilia.