Complex association patterns for inflammatory mediators in induced sputum from subjects with asthma.

BACKGROUND: The release of various inflammatory mediators into the bronchial lumen is thought to reflect both the type and degree of airway inflammation, eosinophilic Th2, and Th9, or neutrophilic Th1, and Th17, in patients with asthma. AIMS: We investigated whether cytokines and chemokines differed in sputum from subjects with more severe compared with milder asthma and whether unbiased factor analysis of cytokine and chemokine groupings indicates specific inflammatory pathways. METHODS: Cell-free supernatants from induced sputum were obtained from subjects with a broad range of asthma severity (n = 158) and assessed using Milliplex® Cytokines/Chemokine kits I, II and III, measuring 75 individual proteins. Each cytokine, chemokine or growth factor concentration was examined for differences between asthma severity groups, for association with leucocyte counts, and by factor analysis. RESULTS: Severe asthma subjects had 9 increased and 4 decreased proteins compared to mild asthma subjects and fewer differences compared to moderate asthma. Twenty-six mediators were significantly associated with an increasing single leucocyte type: 16 with neutrophils (3 interleukins [IL], 3 CC chemokines, 4 CXC chemokines, 4 growth factors, TNF-α and CX3CL1/Fractalkine); 5 with lymphocytes (IL-7, IL-16, IL-23, IFN-α2 and CCL4/MIP1ß); IL-15 and CCL15/MIP1δ with macrophages; IL-5 with eosinophils; and IL-4 and TNFSF10/TRAIL with airway epithelial cells. Factor analysis grouped 43 cytokines, chemokines and growth factors which had no missing data onto the first 10 factors, containing mixes of Th1, Th2, Th9 and Th17 inflammatory and anti-inflammatory proteins. CONCLUSIONS: Sputum cytokines, chemokines and growth factors were increased in severe asthma, primarily with increased neutrophils. Factor analysis identified complex inflammatory protein interactions, suggesting airway inflammation in asthma is characterized by overlapping immune pathways. Thus, focus on a single specific inflammatory mediator or pathway may limit understanding the complexity of inflammation underlying airway changes in asthma and selection of appropriate therapy.

eQTL of bronchial epithelial cells and bronchial alveolar lavage deciphers GWAS-identified asthma genes.

BACKGROUND: Genome-wide association studies (GWASs) have identified various genes associated with asthma, yet, causal genes or single nucleotide polymorphisms (SNPs) remain elusive. We sought to dissect functional genes/SNPs for asthma by combining expression quantitative trait loci (eQTLs) and GWASs. METHODS: Cis-eQTL analyses of 34 asthma genes were performed in cells from human bronchial epithelial biopsy (BEC, n = 107) and from bronchial alveolar lavage (BAL, n = 94). RESULTS: For TSLP-WDR36 region, rs3806932 (G allele protective against eosinophilic esophagitis) and rs2416257 (A allele associated with lower eosinophil counts and protective against asthma) were correlated with decreased expression of TSLP in BAL (P = 7.9 × 10(-11) and 5.4 × 10(-4) , respectively) and BEC, but not WDR36. Surprisingly, rs1837253 (consistently associated with asthma) showed no correlation with TSLP expression levels. For ORMDL3-GSDMB region, rs8067378 (G allele protective against asthma) was correlated with decreased expression of GSDMB in BEC and BAL (P = 1.3 × 10(-4) and 0.04) but not ORMDL3. rs992969 in the promoter region of IL33 (A allele associated with higher eosinophil counts and risk for asthma) was correlated with increased expression of IL33 in BEC (P = 1.3 × 10(-6) ) but not in BAL. CONCLUSIONS: Our study illustrates cell-type-specific regulation of the expression of asthma-related genes documenting SNPs in TSLP, GSDMB, IL33, HLA-DQB1, C11orf30, DEXI, CDHR3, and ZBTB10 affect asthma risk through cis-regulation of its gene expression. Whenever possible, disease-relevant tissues should be used for transcription analysis. SNPs in TSLP may affect asthma risk through up-regulating TSLP mRNA expression or protein secretion. Further functional studies are warranted.

Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-beta1, TGF-beta2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on alpha-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts.

RATIONALE: Asthmatic airway remodelling is characterized by myofibroblast hyperplasia and subbasement membrane collagen deposition. We hypothesized that cytokines and growth factors implicated in asthmatic airway remodelling are increased in bronchoalveolar lavage (BAL) fluid of asthmatics after segmental allergen challenge (SAC), and that these growth factors and cytokines increase alpha-smooth muscle actin (alpha-SMA) and collagen III synthesis by human lung fibroblasts (HLFs). METHODS: Transforming growth factor (TGF)-beta1, TGF-beta2, IL-4 and IL-13 levels were measured in BAL fluid from 10 asthmatics and 9 non-asthmatic controls at baseline and then 1 day, 1 week and 2 weeks after SAC. Confluent cultures of HLFs were stimulated by exogenous addition of TGF-beta1, TGF-beta2, IL-4 or IL-13 (concentration range 0.01-10 ng/mL) over 48 h. Collagen III was measured in culture supernates and alpha-SMA in cell lysates by Western blot. RESULTS: At baseline, there was no difference in BAL fluid concentrations of TGF-beta1, IL-4 and IL-13 between asthmatics and controls; however, non-asthmatics had higher concentrations of total TGF-beta2. In asthmatics, BAL fluid concentrations of all four factors increased significantly 1 day after SAC. TGF-beta1, TGF-beta2 and IL-13 concentrations returned to baseline by 1 week after SAC, but BAL fluid IL-4 concentration remained elevated for at least 2 weeks. TGF-beta1, TGF-beta2 and IL-4 significantly increased alpha-SMA in fibroblasts, but only IL-4 caused corresponding increases in collagen III synthesis. IL-13 had no direct effects on collagen III synthesis and alpha-SMA expression. CONCLUSIONS: Because IL-4 caused a dose-dependent increase in alpha-SMA and collagen III synthesis, it may be an important cytokine mediating asthmatic airway remodelling. TGF-beta1 and TGF-beta2 may also play a role in airway remodelling by stimulating phenotypic change of fibroblasts to myofibroblasts. Additionally, collagen III synthesis appears to be independent of myofibroblast phenotype and is apparently regulated by different growth factors and cytokines.

Interactions of allergens and irritants in susceptible populations in producing lung dysfunction: implications for future research.

Environmental agents, when applied in combination or sequentially, can induce a wide variety of adverse health effects in humans. To determine the effects of sequential allergen challenge and acid exposure on human bronchial epithelial cell function, we subjected normal, nonallergic control and ragweed-allergic individuals to bronchoscopic segmental ragweed challenge in vivo. We harvested bronchial epithelial cells by brush biopsy both before challenge and 24 hr after challenge and exposed cells to an acid stress in vitro (pH 5 for 3 hr), followed by a 1-hr recovery period at normal pH. In normal, nonallergic subjects, segmental allergen challenge produced no effects on ciliary activity; pH 5 exposure produced reduced ciliary activity (a decrease in the percent of the initially active area), with significant recovery after cells were returned to a normal pH. Ciliary activity from allergic subjects was also inhibited by pH 5 exposure; however, activity was not recovered when cells were placed in medium of normal pH. Ciliary activity in allergics who developed a stress response postantigen challenge, as determined by an induction of the 27 kDa stress (heat shock) protein, displayed no ciliary dysfunction when exposed to a pH 5 stress. In this case, a stress sufficient to provoke a heat shock (stress) protein (HSP) response (but not one that produced more severe lung injury and did not provoke an HSP response) protected cells from a subsequent acid stress. Because of our observations and recent findings reported in the literature, we suggest that in order to define the wide variety of health effects of environmental agents, control as well as at-risk populations should be studied and the ability to define potentially beneficial as well as detrimental effects should be built into the experimental design. Inclusion of different and novel end points also should be considered.

Kinetics of IL-10 production after segmental antigen challenge of atopic asthmatic subjects.

BACKGROUND: IL-10 is an anti-inflammatory cytokine made by lymphocytes, monocytes-macrophages, and eosinophils, and it may have an important role in regulating the asthmatic inflammatory response. IL-10 levels have been reported to be reduced in asthmatic airways, potentially contributing to more intense inflammation. OBJECTIVE: We sought to determine whether IL-10 levels were deficient in patients with mild asthma compared with controls and to determine whether IL-10 levels were associated with the resolution of eosinophilic inflammation. METHODS: We quantified IL-10 levels in the bronchoalveolar lavage (BAL) fluid (ELISA), BAL cells (quantitative immunocytochemistry), purified alveolar macrophages-monocytes studied ex vivo (ELISA), before (day 1) and after (24 hours [day 2], 1 week [day 9], and 2 weeks [day 16]) segmental antigen challenge (SAC), and investigated the effect of glucocorticoid treatment on ex vivo macrophage-monocyte IL-10 production. RESULTS: IL-10 levels were significantly higher in the BAL fluid of mild asthmatic subjects who demonstrated a dual reaction (both early and late) after whole lung ragweed inhalation challenge compared with nonallergic, nonasthmatic control subjects before and 24 hours and 1 week after SAC. Macro-phages-monocytes obtained before and after SAC from asthmatic patients also secreted increased amounts of IL-10 ex vivo than those from controls. Dexamethasone did not significantly change spontaneous IL-10 secretion from macrophages-monocytes in vitro. Quantitative immunocytochemical analysis of BAL cells demonstrated increased IL-10 in macrophages 24 hours after SAC and a similar trend in eosinophils. CONCLUSION: IL-10 is not deficient in mild asthma. Furthermore, BAL IL-10 levels are significantly higher in asthmatic subjects with a dual response than in control subjects before and after SAC. The increase in IL-10 was coincident with the initial increase in BAL eosinophils, although BAL eosinophilia persisted after IL-10 levels had returned to baseline, suggesting that the increased IL-10 levels could not promptly terminate this localized eosinophilic response.

HSP27 elevated in mild allergic inflammation protects airway epithelium from H2SO4 effects.

Inflammation in allergic individuals is hypothesized to elevate stress proteins [heat shock proteins (HSP)] in airway epithelium, which may protect cells from further adverse conditions. Allergic, either asthmatic or not, and normal volunteers participated in a 2-day segmental allergen challenge bronchoscopic procedure. Bronchial epithelium was obtained before and after challenge. Epithelium was exposed to medium with H2SO4 (pH5), returned to medium at pH 7.4, and finally harvested for Western blotting with anti-27-kDa HSP (HSP27) antibody. Prechallenge epithelium of all subjects had significantly inhibited ciliary function by H2SO4 (pH 5) conditions (P < 0.001); only epithelium of normals recovered (P = 0.02). Allergic subjects with mild inflammation (< 50 micrograms/ml increase in albumin in bronchoalveolar lavage) had significantly increased HSP27 postchallenge (P = 0.01) and little ciliary dysfunction at pH 5, whereas subjects with severe inflammation (> 50 micrograms/ml increase in albumin) had little change in HSP27 and significant ciliary inhibition (P = 0.02). Normal epithelium had similar trends in HSP27 and equivalent inhibition of ciliary activity at pH 5 before and after allergen challenge. These data indicate that mild inflammation to allergen elevates HSP27 stress protein levels, thereby potentially protecting epithelial function from additional adverse conditions.

Kinetics of the development and recovery of the lung from IgE-mediated inflammation: dissociation of pulmonary eosinophilia, lung injury, and eosinophil-active cytokines.

Events occurring up to 16 d after antigen challenge were characterized using a novel protocol employing four bronchoscopies, two segmental antigen challenge (SAC) procedures (on Days 1 and 2), and six bronchoalveolar lavages (BALs) (on Days 1, 2, 9, and 16) in three groups: ragweed allergic asthmatics with dual phase airway reactions (AA-D), allergic asthmatics with a single early airway reaction (AA-S), and nonallergic nonasthmatic control subjects. In AA-D subjects, SAC produced a marked eosinophilic inflammatory response at 24 h associated with eosinophil degranulation (eosinophil cationic protein [ECP] in BAL fluid) and lung injury, which largely resolved by Day 16. When the second antigen-challenged segment (SAC performed on Day 2) was lavaged 7 d after challenge (Day 9), a persistent pulmonary eosinophilia was noted accompanied by minimal elevations in ECP and albumin. Eosinophil-active cytokines showed unique patterns: interleukin-5 (IL-5) increased in the antigen segment on Day 2 then returned to baseline after 7 d; granulocyte-macrophage colony-stimulating factor (GM-CSF) peaked at Day 2 but was persistently elevated throughout Day 16 in antigen segments, and increased in control segments at late time points; IL-3 levels were constant and similar in antigen and control segments. Changes were specific to AA-D subjects in comparison with control subjects. Elements of the IgE-mediated pulmonary inflammatory response differ markedly in their development and resolution.

Beta 2-agonist-elevated stress response in human bronchial epithelial cells in vivo and in vitro.

Recent studies report high baseline levels of stress (heat shock) proteins in bronchial epithelial cells from asthmatic individuals. The promoter of the gene encoding the 72-kDa heat shock protein has an element responsive to cAMP, which may be affected by beta-agonists. This study examined stress protein levels in subjects enrolled in a segmental lung allergen challenge study to determine whether beta-agonist medication could contribute to a stress response. Subjects were divided on the basis of no premedication (n = 17), metered dose inhalations of albuterol (n = 24), or placebo inhalation (n = 3) prior to bronchoscopy. Levels of the inducible stress protein Hsp72 and constitutive Hsp73 were quantitated in bronchial epithelial cells from brush biopsy of allergic nonasthmatic, allergic asthmatic, and normal individuals. Mean levels were increased significantly (p < 0.003 and p < 0.004, respectively) in those subjects who received albuterol premedication. No significant differences were found between clinical groups of individuals or for placebo inhalation vs nonpremedication. Albuterol in vitro increased the levels of Hsp72 and Hsp73 in epithelial cells from either nonpremedicated or placebo-treated donors; the Hsp72 levels correlated linearly with increased albuterol concentration (r = 0.81, p < 0.01). Therefore, beta-agonists elevate or prolong an elevated stress response in epithelial cells, possibly through cAMP-mediated effects.

IL-1 beta release from cultured bronchial epithelial cells and bronchoalveolar lavage cells from allergic and normal humans following segmental challenge with ragweed.

This work investigated whether interleukin 1 beta (IL-1 beta) release from epithelial cells is modulated by antigen challenge in vivo, and inflammatory cells in vitro. Bronchial epithelial cells were obtained before and after segmental allergen challenge in allergic and normal individuals, and were cultured with and without autologous bronchoalveolar lavage (BAL) cells. IL-1 beta in culture medium was quantitated by enzyme-linked immunoabsorbent assay (ELISA). Pre-challenge IL-1 beta levels from epithelial cells were similar in allergic (4.4 +/- 0.8 pg/ml, n = 32) and normal (6.8 +/- 1.7 pg/ml, n = 17) subjects. IL-1 beta levels were significantly elevated from epithelium with BAL cell co-culture vs without co-culture in both subject groups (allergic, 13.2 +/- 2.3 pg/ml, P = 0.006; normal: 16.4 +/- 4.0 pg/ml, P = 0.007). Post-challenge IL-1 beta from epithelial cells without BAL cells was increased in both groups, but significant only for allergic subjects (P = 0.003). Post-challenge IL-1 beta from epithelial with BAL cells changed little for allergic subjects (13.8 +/- 2.4 pg/ml), and increased for normal subjects (20.0 +/- 4.8 pg/ml). Decreased production of tumour necrosis factor alpha (TNF-alpha) was observed in allergic subjects (day 1: 447 pg/ml vs day 2: 258 pg/ml). Moreover, pre-challenge release of TNF-alpha and IL-1 beta levels from epithelial + BAL cells correlated well for allergic (r = 0.84) and normal subjects (r = 0.6), but post-challenge release correlated only in normal subjects (r = 0.90). These results indicate that bronchial epithelial cells and BAL cells interact, modulating release of these inflammatory cytokines.

Two types of bacteria adherent to bovine respiratory tract ciliated epithelium.

Two hundred sixty tracheas were obtained from a Philadelphia abattoir under permit from the Department of Agriculture; the tracheas were excised from predominantly Holstein calves of both sexes that weighed approximately 250 kg. Tracheas were transported in normal saline to the laboratory at Thomas Jefferson University, Philadelphia, Pennsylvania. Evidence of bacteria adherent to the tracheal epithelium was found in specimens from 20/24 of these tracheas. The epithelium from each of five tracheas was placed in glutaraldehyde fixative for transmission electron microscopic examination. Epithelium from each of 12 other tracheas was placed in formaldehyde fixative for light microscopic examination. Microscopically, 13 of these 17 bovine tracheal epithelia were observed to contain bacteria located longitudinally parallel to and between cilia and microvilli of ciliated cells. Preparations of ciliary axonemes isolated from the epithelium of seven additional bovine tracheas also contained these bacteria in sections viewed by a transmission electron microscope. These bacteria had two different ultrastructural morphologies: filamentous with a trilaminar-structured cell wall and short with a thick, homogeneously stained cell wall beneath a regularly arrayed surface layer. The short bacillus had surface carbohydrates, including mannose, galactose, and N-acetylgalactosamine, identified by lectin binding. The filamentous bacillus was apparently externally deficient in these carbohydrates. Immunogold staining revealed that the filamentous bacillus was antigenically related to cilia-associated respiratory (CAR) bacillus, which has been identified in rabbit and rodent species. Significantly decreased numbers of cilia were obtained from tracheal epithelium heavily colonized by the filamentous bacilli, suggesting a pathologic change in ciliated cells.

Ciliated respiratory epithelial surface changes after formaldehyde exposure.

The investigation sought to identify alterations of specific ciliated epithelial surface components after exposure to formaldehyde (HCHO) levels that decrease respiratory ciliary function. Bovine tracheae were reacted with an analog of N-hydroxysuccinimidobiotin to label epithelial surface-accessible components before exposure to HCHO. The tracheae were then exposed to 0, 16, 33, and 66 micrograms HCHO/cm2 epithelial surface for 30 min. Cilia were isolated from the epithelium, separated into membrane and internal axonemal portions, analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and either stained to detect proteins or transblotted to detect biotin-labeled components. Densitometric analysis of axoneme proteins showed a decrease in the total amount extracted with increased HCHO concentration, including axoneme-specific proteins, dynein, and tubulin. However, biotinylated proteins in the axoneme fractions proportionately increased. Membrane fractions showed little change in protein with increasing HCHO concentration. The majority of these is not biotin-labeled and thus not surface-accessible components. Biotinylated material in the membrane fractions showed a significant decrease with increased HCHO concentration, particularly of bands at 92, 98, and 105 kD. These data suggest that increasing HCHO exposure reduces both extractable ciliary axonemes and detergent-soluble surface components, possibly by stabilizing respiratory epithelial membranes. This process apparently strengthens association of certain surface components with the internal axoneme, thereby reducing subsequent solubilization in detergent.

Inhibition and recovery of mammalian respiratory ciliary function after formaldehyde exposure.

Formaldehyde (HCHO) has been reported to impair mucociliary clearance. The present investigation using rabbit and porcine tracheal explants in vitro examined (1) the impairment of ciliary activity, an essential component of mucociliary clearance; (2) the reversibility of ciliary dysfunction after HCHO exposure; and (3) the mechanism by which ciliary activity is reduced by HCHO. HCHO treatment of rabbit tracheal rings significantly decreased zones of active ciliated epithelium in direct proportion to concentration and exposure duration. There was also a significant concentration-dependent reduction of ciliary beat frequency. Removal of HCHO permitted recovery of zones of ciliary activity to normal beat frequencies; greater inhibitory concentrations of HCHO required greater time for return of function. Treatment of porcine tracheae with increasing concentrations of HCHO for time periods inhibitory to rabbit ciliary activity correspondingly reduced the yield of cilia extractable from treated epithelium. Furthermore, the specific activity of ATPase of extracted ciliary axonemes was diminished with increasing HCHO concentration, indicating loss of function. A recovery period following identical exposures of the porcine tracheae to the lower HCHO concentrations resulted in normal yields of functionally intact ciliary axonemes. Similarly, a recovery period after the highest HCHO concentration produced more functional axonemes than obtained from exposed tracheae without a recovery period, although less than normal yields. Therefore, ciliary dysfunction elicited by a defined range of HCHO concentrations is reversible. The yield and functional integrity of ciliary axonemes from epithelium exposed to HCHO with a recovery period are significantly greater than those without such a recovery period, suggesting an alteration and subsequent repair of epithelial surface components following HCHO exposure.

Identification of surface components of mammalian respiratory tract cilia.

Cilia isolation methods were modified to retain respiratory tract ciliary membranes and to identify accessible surface components. Prior to isolation of cilia, halves of cow tracheae were treated with the extended spacer arm analog of N-hydroxysuccinimido-biotin (NHS-LC-biotin) to label accessible membrane constituents. Mechanical disruption of the epithelium and substitution of CHAPS for Triton X-100 provided a good yield of cilia with membranes and with minimal contamination. Subsequent extraction of these cilia with Triton X-100 solubilized the membranes and released soluble matrix proteins. Proteins of membrane + matrix and axoneme fractions were analyzed after electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The major biotin-labeled components in the membrane + matrix fraction were 105, 98, and 92 kd, were glycosylated, and remained with reconstituted, pelleted membrane vesicles along with the major non-biotinylated protein at 51 kd. Other membrane + matrix proteins at 126 and 76 kd bound streptavidin even from nonlabeled trachea, but remained soluble. Several biotin-labeled proteins distinct from those in the membrane fraction remained with Triton X-100-extracted axonemes. Streptavidin-colloidal-gold (SAG) particles appeared to bind randomly along the length of cilia. The peripheral join between A and B microtubules was a predominant nonspecific location of SAG on axonemes. Axonemes with biotin label also bound significant numbers of SAG to outer dynein arms, confirming the streptavidin reaction with separated proteins on transfers. These results suggest close association of the membrane with the axoneme in respiratory tract cilia and a membrane composition somewhat different from protozoan cilia.

Structure and mass of mammalian respiratory ciliary outer arm 19S dynein.

Mammalian respiratory ciliary outer arm dyneins isolated as the major ATPase peak migrating at 19S on sucrose density gradients were examined by transmission electron microscopy of negatively stained samples and scanning transmission electron microscopy of unstained samples. The predominant discrete particle structure observed was composed of two globular heads apparently connected by amorphous or indistinct material. The heads were either circular or slightly elliptical of mean 13 +/- 1 X 10 +/- 2 nm dimensions. The mass of this structure averaged 1.22 +/- 0.34 million daltons with the individual globular heads averaging 310 +/- 77 kilodaltons (kD). Negative staining revealed that one or both of the globular heads often contained a central accumulation of stain measuring 2.5 +/- 1 nm across. A second type of structure, appearing with lesser frequency in the 19S fraction than in the unfractionated dynein preparation loaded onto the sucrose gradient, was a single globular head of 13 +/- 1 X 10 +/- 2 nm often with 2 +/- 1 nm centrally accumulated stain and with or without an appendage. This one-headed particle thus resembled one-half of the two-headed particle. Mass measurements were lower, however, for isolated, single globular heads, averaging 220 +/- 111 kD. A third type of particle observed was a ring-like structure with 4 +/- 1 nm centrally accumulated stain and without appendages. The ring structure was slightly larger in diameter, 14 +/- 1 nm, and had a greater peripheral accumulation of negative stain than either of the one- or two-headed particles, suggesting that it was not derived therefrom.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous production of rhamnolipids, 2-alkyl-4-hydroxyquinolines, and phenazines by clinical isolates of Pseudomonas aeruginosa.

Of 72 clinical isolates of Pseudomonas aeruginosa examined for simultaneous production of secondary metabolites, 86% produced 2-alkyl-4-hydroxyquinolines, 75% produced rhamnolipids, and 58% produced phenazines, including pyocyanin. Whereas isolates producing two or one constituted smaller groups, 39% released all three metabolites. Metabolite production did not appear to influence site of infection.

The effect of purified human eosinophil major basic protein on mammalian ciliary activity.

Eosinophil granulocyte infiltration in a variety of lung disorders may directly damage local tissue by release of granule contents. A principal constituent of eosinophil granules is the major basic protein (MBP). Previous light and electron microscopic observations have indicated that guinea pig and human MBP produce detachment of tracheal epithelial cells and cessation of ciliary activity. To quantitate the damage, selected regions of the epithelial surface of rabbit tracheal explants were videotaped before and after treatment with human MBP. Tapes were analyzed for ciliary beat frequency and the extent of zones along the epithelial surface displaying ciliary activity. The MBP at 0.1 mg/ml reduced beat frequency and significantly reduced the measured zones of ciliary activity. Also, MBP at 0.7 mg/ml significantly reduced beat frequency and the measured zone of ciliary activity on the epithelial surface. Beat frequency was lowered by 27% within 10 min, with only 1% further decrease by 60 min. The zones of ciliary activity on the epithelium were continuously decreased throughout the 60 min to 29% of the initially active zone. To examine whether MBP was capable of direct inhibition of ciliary activity, isolated porcine tracheal ciliary axonemes, the structural organelles of individual cilia, were treated with human MBP. Concentrations above 67 micrograms/ml of MBP were completely inhibitory to reactivated isolated axonemes, 67 micrograms/ml stopped activity within 10 min, and 27 micrograms/ml stopped activity within 15 min. Pretreatment of isolated axonemes with increasing concentrations of MBP resulted in decreasing ATPase activity. These effects were not attributable to pH alteration.(ABSTRACT TRUNCATED AT 250 WORDS)