Ibuprofen inhibits cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion.

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.     

Parallel improvement of sodium and chloride transport defects by miglustat (n-butyldeoxynojyrimicin) in cystic fibrosis epithelial cells

Cystic fibrosis, an autosomal recessive disease frequently diagnosed in the Caucasian population, is characterized by deficient Cl- transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A second major hall-mark of the disease is Na+ hyperabsorption by the airways, mediated by the epithelial Na+ channel (ENaC). In this study, we report that in human airway epithelial CF15 cells treated with the CFTR corrector miglustat (n-butyldeoxynojyrimicin), whole-cell patch-clamp experiments showed reduced amiloride-sensitive ENaC current in parallel with a rescue of defective CFTR Cl- channel activity activated by forskolin and genistein. Similar results were obtained with cells maintained in culture at 27 degrees C for 24 h before electrophysiology experiments. With monolayers of polarized CF15 cells, short-circuit current (Isc) measurements also show normalization of Na+ and Cl- currents. In excised nasal epithelium of cftr(F508del/F508del) mice, like with CF15 cells, we found normalization of amiloride-sensitive Isc. Moreover, oral administration of miglustat (6 days) decreased the amiloride-sensitive Isc in cftr(F508del/F508del) mice but had no effect on cftr-/- mice. Our results thus show that rescuing the trafficking-deficient F508del-CFTR by miglustat down-regulates Na+ absorption. A miglustat-based treatment of CF patients may thus have a beneficial effect both on Cl- and Na+ transports.     

Expression of CFTR from a ciliated cell-specific promoter is ineffective at correcting nasal potential difference in CF mice

Successful gene therapy will require that the therapeutic gene be expressed at a sufficient level in the correct cell type(s). To improve the specificity of gene transfer for cystic fibrosis (CF) and other airway diseases, we have begun to develop cell-type specific promoters to target the expression of transgenes to specific airway cell types. Using a FOXJ1 promoter construct previously shown to direct transgene expression specifically to ciliated cells, we have generated transgenic mice expressing human cystic fibrosis transmembrane conductance regulator (CFTR) in the murine tracheal and nasal epithelia. RNA analysis demonstrated levels of CFTR expression is greater than or equal to the level of endogenous mouse CFTR. Immunoprecipitation and western blotting demonstrated the production of human CFTR protein, and immunochemistry confirmed that CFTR was expressed in the apical region of ciliated cells. The transgenic animals were bred to CFTR null mice (Cftr(tm1Unc)) to determine if expression of CFTR from the FOXJ1 promoter is capable of correcting the airway defects in Cl(-) secretion and Na(+) absorption that accompany CF. Isolated trachea from neonatal CF mice expressing the FOXJ1/CFTR transgene demonstrated a correction of forskolin-stimulated Cl(-) secretion. However, expression of human CFTR in ciliated cells of the nasal epithelia failed to significantly change the nasal bioelectrics of the CF mice.     

Respiratory damage caused by long-term inhalation of high concentration of sulphur-dioxide in dogs

It has been tried in this study to produce chronic obstructive bronchitis in dogs by exposing them to high concentrations of SO2. The study was extended to 175 days and during that period the animals were exposed to 300-500 ppm SO2 for a total of 435 h. None of the three dogs developed serious respiratory damage. Most of the symptoms were transitory. Histological examinations showed increased number of goblet cells in trachea, main- and lobar bronchi and a slight alteration of the bronchial glands.     

Dynamic activation of cystic fibrosis transmembrane conductance regulator by type 3 and type 4D phosphodiesterase inhibitors

The diseases of cystic fibrosis, chronic obstructive pulmonary disease (COPD), and chronic bronchitis are characterized by mucus-congested and inflamed airways. Anti-inflammatory agents that can simultaneously restore or enhance mucociliary clearance through cystic fibrosis transmembrane conductance regulator (CFTR) activation may represent new therapeutics in their treatment. Herein, we report the activation of CFTR-mediated chloride secretion by phosphodiesterase (PDE) 4 inhibitors in T84 monolayer using (125)I anion as tracer. In the absence of forskolin, the iodide secretion was insensitive to PDE4 inhibitor L-826,141 [4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-ethyl]-3-methylpyridine-1-oxide], roflumilast, or to PDE3 inhibitor trequinsin. However, these inhibitors potently augmented iodide secretion after forskolin stimulation, with efficacy coupled to the activation states of adenylyl cyclase. The iodide secretion from PDE3 or PDE4 inhibition was characterized at first by a prolonged efflux duration, followed by progressively elevated peak efflux rates at higher inhibitor concentrations. Paralleled with an increased phosphor-cAMP response element-binding protein formation, the CFTR activation dissociated from a global cAMP elevation and was blocked by H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide]. 2-(4-Fluorophenoxy)-N-[(1S)-1-(4-methoxyphenyl)ethyl]nicotinamide, a stereoselective PDE4D inhibitor, augmented iodide efflux more efficiently than its less potent (R)-isomer. The peak efflux from maximal PDE4 and PDE3 inhibition matched that from full adenylyl cyclase activation. These data suggest that PDE3 and PDE4 (mainly PDE4D) form the major cAMP diffusion barrier in T84 cells to ensure a compartmentalized CFTR signaling. Together with their potent anti-inflammatory properties, the potentially enhanced airway mucociliary clearance from CFTR activation may have contributed to the efficacy of PDE4 inhibitors in COPD and asthmatic patients. PDE4 inhibitors may represent new opportunities to combat cystic fibrosis and other respiratory diseases in future.     

The amiloride-inhibitable Na+ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways.

Cystic fibrosis (CF) airway cells, besides their well-known defect in cAMP-dependent Cl- conductance, are characterized by an enhanced Na+ conductance. In this study we have examined the Na+ conductance in human respiratory tract by measuring transepithelial voltage and resistance (Vte, Rte) and by assessing membrane voltages (Vm) of freshly isolated airway epithelial cells from CF and non-CF patients. Basal amiloride inhibitable (10 micromol/liter) equivalent short circuit current (Isc = Vte/Rte) was significantly increased in CF compared with non-CF tissues. After stimulation by forskolin (10 micromol/liter) a significant depolarization of Vm corresponding to the cAMP-dependent activation of a Cl- conductance was observed in non-CF but not in CF airway cells. In non-CF tissue but not in CF tissue the effects of amiloride and N-methyl-D-glucamine on Vm were attenuated in the presence of forskolin. Also the amiloride-inhibitable Isc was significantly reduced by forskolin (1 micromol/liter) and isobutylmethylxanthine (IBMX; 100 micromol/liter) only in non-CF tissue. We conclude that cystic fibrosis transmembrane conductance regulator acts as a downregulator of epithelial Na+ channels in human airways. This downregulation of epithelial Na+ channels is absent in CF airways, leading to hyperabsorption and to the characteristic increase in mucus viscosity.     

Reversible acquired tracheobronchomalacia of a combined crescent type and saber-sheath type

A case of tracheobronchomalacia (TBM) in a 71-year-old woman, who had suffered a cough syncope, is reported. It was a combination of both the crescent type (the posterior membranous portion of trachea or bronchus protrudes into the lumen) and the saber-sheath type (the lateral cartilaginous wall of trachea or bronchus protrudes into the lumen). In this patient, acute bronchitis had developed superimposed upon a chronic bronchitis in addition to age-related regressive changes of the trachea and bronchus. A TBM due to acute inflammation can be reversible, but aggressive airway management as well as medical treatment of the underlying inflammation are critical to a successful outcome.     

Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa.

In cystic fibrosis (CF), defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells and submucosal glands results in chronic pulmonary infection with Pseudomonas aeruginosa. The pulmonary infection incites an intense host inflammatory response, causing progressive suppurative pulmonary disease. Mouse models of CF, however, fail to develop pulmonary disease spontaneously. We examined the effects of bronchopulmonary infection on mice homozygous for the S489X mutation of the CFTR gene using an animal model of chronic Pseudomonas endobronchial infection. Slurries of sterile agarose beads or beads containing a clinical isolate of mucoid P. aeruginosa were instilled in the right lung of normal or CF mice. The mortality of CF mice inoculated with Pseudomonas-laden beads was significantly higher than that of normal animals: 82% of infected CF mice, but only 23% of normal mice, died within 10 d of infection (P = 0.023). The concentration of inflammatory mediators, including TNF-alpha, murine macrophage inflammatory protein-2, and KC/N51, in bronchoalveolar lavage fluid in CF mice 3 d after infection and before any mortality, was markedly elevated compared with normal mice. This inflammatory response also correlated with weight loss observed in both CF and normal littermates after inoculation. Thus, this model may permit examination of the relationship of bacterial infections, inflammation, and the cellular and genetic defects in CF.     

Recipient iNOS but not eNOS deficiency reduces luminal narrowing in tracheal allografts

Chronic airway rejection is characterized by prolonged inflammation, epithelial damage, and eventual luminal obliterative bronchiolitis (OB). In cardiac allografts, the inducible nitric oxide synthase (iNOS) promotes acute rejection but paradoxically reduces neointimal formation, the hallmark of chronic rejection. The specific roles of NOS isoforms in modulating lymphocyte traffic and airway rejection are not known. Using a double lumen mouse tracheal transplant model, tracheal grafts from B10.A (allo) or C57BL/6J (iso) mice were transplanted into cyclosporine-treated wild-type (WT) iNOS(-/-) or endothelial NOS (eNOS)(-/-) recipients. OB was observed in WT tracheal allografts at 3 weeks (53 +/- 2% luminal occlusion vs. 17 +/- 1% for isografts, P < 0.05) with sites of obstructive lesion formation coinciding with areas of CD3(+) CD8(+) T cell-rich lymphocytic bronchitis. In contrast, allografts in iNOS(-/-) recipients exhibited reductions in local expression of proinflammatory chemokines and cytokines, graft T cell recruitment and apoptosis, and luminal obliteration (29 +/- 2%, P < 0.05 vs. WT allografts). Recipient eNOS deficiency, however, suppressed neither chemokine expression, lymphocyte infiltration, nor airway occlusion (54 +/- 2%). These data demonstrate that iNOS exacerbates luminal obliteration of airway allografts in contrast with the known suppression by iNOS of cardiac allograft vasculopathy. Because iNOS(-/-) airways transplanted into WT allograft hosts are not protected from rejection, these data suggest that iNOS expressed by graft-infiltrating leukocytes exerts the dominant influence on airway rejection.     

Correction of the apical membrane chloride permeability defect in polarized cystic fibrosis airway epithelia following retroviral-mediated gene transfer.

We are studying the introduction and expression of the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA into cultured human airway epithelial cells as a model for gene therapy of cystic fibrosis. In this paper, we show that the chloride transport defect at the apical membrane is corrected in vitro in differentiated ion-transporting CF airway epithelial cells that exhibit polarized properties similar to those found in vivo. Using a retroviral vector containing a copy of the normal CFTR cDNA, we infected cultures of proliferating, cystic fibrosis CFT1 cells and found that correction was maintained following differentiation into a polarized epithelial sheet. At least partial correction of the Cl- transport defect was preserved in CFT1 cells for periods of up to 6 months without selection for maintenance of the retroviral provirus. These results suggest that it may be feasible to target proliferating cells in the lung using retroviral vectors for treatment of CF lung disease.     

Regulation of sodium absorption by canine tracheal epithelium.

To regulate the quantity of respiratory tract fluid, the airway epithelium either secretes chloride, Cl-, or reabsorbs sodium, Na+. Many secretagogues inhibit Na+ absorption, but the decrease may result from a fall in the electrochemical gradient for Na+ absorption. We examined regulation of Na+ absorption independent of Cl- secretion, by bathing canine tracheal epithelium in Cl--free, gluconate Ringers solution. Prostaglandin E2, 2-chloroadenosine, and isoproterenol increased short-circuit current (Isc) and the rate of Na+ absorption. In contrast, indomethacin, which inhibits endogenous prostaglandin production, decreased Isc. These agents regulate cellular levels of cAMP; direct addition of 8-Br-cAMP also acutely increased Isc. We examined chronic regulation of Na+ absorption in cell monolayers grown on permeable supports in serum-free media. Exposure to aldosterone for two days increased baseline Isc by 50% and the amiloride-inhibitable current by 55%. These data indicate that Na+ absorption is both acutely and chronically regulated in the airway epithelium.     

Effect of D-alanine methionine enkephalin amide on ion transport in rabbit ileum.

The presence of enkephalins in the intestine and the use of opiates to treat diarrheal diseases suggests that enkephalins may affect intestinal ion transport. Using isolated rabbit ileal mucosa, we found that leucine enkephalin, methionine enkephalin, and D Ala2-methionine enkephalin amide (D Ala2-Met E) decreased the short circuit current (Isc) and potential difference although the effect of D Ala2-Met E was more pronounced and prolonged. D Ala2-Met E increased net sodium (+1.27 +/- 0.5 mu eq/cm2h), and chloride absorption (+2.33 +/- 0.4), and increased tissue conductance by 37%. Although the effect of enkaphalin on ion transport is opposite that of cyclic AMP, D-Ala2-Met had no effect on basal or vasoactive intestinal polypeptide-stimulated cyclic AMP levels. The effect of D-Ala2-Met E on Isc was blocked by naloxone, suggesting the involvement of specific opiate receptors. Tetrodotoxin completely blocked the decrease in Isc induced by D-Ala2-Met E but not by epinephrine, inferring that enkephalins are preganglionic neurotransmitters. The effect of D-Ala2-Met E on Isc was not blocked by phentolamine, haloperidol, or pretreatment of animals with 6-hydroxydopamine, suggesting that enkephalin does not affect the Isc by stimulating the release of alpha-adrenergic or dopaminergic agonists. D-Ala2-Met E also decreased the Isc in the presence of carbachol and bethanechol, indicating that enkephalin does not inhibit the release of acetylcholine. Further, up to 10 mu M atropine had no effect on the Isc. These studies demonstrate that enkephalins stimulate intestinal ion transport and may do so by stimulating (or inhibiting) the release of a nonadrenergic, noncholinergic neurotransmitter.     

P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport

Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl(-) secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K(+) channels. We therefore speculated that a basolateral P2Y(6) receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y(6) receptor using crypt [Ca(2+)](i) measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y(6)elevates cAMP, we coexpressed the P2Y(1) or P2Y(6) receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl(-) currents. In oocytes expressing the P2Y(1) receptor, ATP transiently activated the endogenous Ca(2+)-activated Cl(-) current, but not CFTR. In contrast, in oocytes expressing the P2Y(6)receptor, UDP transiently activated the Ca(2+)-activated Cl(-) current and subsequently CFTR. CFTR Cl(-) currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y(6) receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca(2+)](i) and cAMP. In support of these data P2Y(6) receptor stimulation differentially activates CFTR in Xenopus oocytes.     

Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus

We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.     

支气管内光学相干断层成像技术评估兔创伤性气道狭窄类型的诊断价值

目的 使用支气管内光学相干断层成像技术（EB-OCT）获得兔创伤性气道狭窄模型的图像,评估EB-OCT在判断良性气道狭窄类型中的诊断价值。方法 建立黏膜破坏联合软骨破坏的兔创伤性气道狭窄模型,建模成功12只,获取12只气道狭窄兔术后2、4、6和8周支气管镜、EB-OCT图像特点和气管组织病理学图像,测量EB-OCT和病理图像匹配部分软骨层及非软骨层的厚度,并比较两者的相关性。结果 12只建模成功的气道狭窄兔的EB-OCT图像特点：低信号反射的软骨层结构,从断裂、不连续到软骨膜逐渐修复;EB-OCT诊断气管结构层次：黏膜破坏3例,黏膜破坏及软骨破坏9例,与组织病理学比较,差异无统计学意义（P > 0.05）。EB-OCT和病理图像匹配部分软骨层及非软骨层的厚度呈线性正相关,相关曲线分别是：软骨层Y = 3.7498+0.9717X,r = 0.9992（P < 0.01）;非软骨层Y = -9.4328 + 0.9935X,r = 0.9999（P < 0.01）。结论 EB-OCT可以很好地观察到良性气道狭窄中气管黏膜破坏和/或软骨破坏以及两者的破坏程度,具有病理组织学的诊断优势。     

Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated Cl(-) channel that regulates other epithelial transport proteins by uncharacterized mechanisms. We employed a yeast two-hybrid screen using the COOH-terminal 70 residues of CFTR to identify proteins that might be involved in such interactions. The alpha1 (catalytic) subunit of AMP-activated protein kinase (AMPK) was identified as a dominant and novel interacting protein. The interaction is mediated by residues 1420-1457 in CFTR and by the COOH-terminal regulatory domain of alpha1-AMPK. Mutations of two protein trafficking motifs within the 38-amino acid region in CFTR each disrupted the interaction. GST-fusion protein pull-down assays in vitro and in transfected cells confirmed the CFTR-alpha1-AMPK interaction and also identified alpha2-AMPK as an interactor with CFTR. AMPK is coexpressed in CFTR-expressing cell lines and shares an apical distribution with CFTR in rat nasal epithelium. AMPK phosphorylated full-length CFTR in vitro, and AMPK coexpression with CFTR in XENOPUS: oocytes inhibited cAMP-activated CFTR whole-cell Cl(-) conductance by approximately 35-50%. Because AMPK is a metabolic sensor in cells and responds to changes in cellular ATP, regulation of CFTR by AMPK may be important in inhibiting CFTR under conditions of metabolic stress, thereby linking transepithelial transport to cell metabolic state.     

Macromolecular complexes of cystic fibrosis transmembrane conductance regulator and its interacting partners

The cystic fibrosis transmembrane conductance regulator (CFTR) is the product of the gene mutated in patients with cystic fibrosis (CF). CFTR is a cAMP-regulated chloride channel localized primarily at the apical or luminal surfaces of epithelial cells lining the airway, gut, exocrine glands, etc., where it is responsible for transepithelial salt and water transport. CFTR chloride channel belongs to the superfamily of the ATP-binding cassette (ABC) transporters, which bind ATP and use the energy to drive the transport of a wide variety of substrates across extra- and intracellular membranes. A growing number of proteins have been reported to interact directly or indirectly with CFTR chloride channel, suggesting that CFTR might regulate the activities of other ion channels, receptors, or transporters, in addition to its role as a chloride conductor. The molecular assembly of CFTR with these interacting proteins is of great interest and importance because several human diseases are attributed to altered regulation of CFTR, among which cystic fibrosis is the most serious one. Most interactions primarily occur between the opposing terminal tails (N- or C-) of CFTR and its binding partners, either directly or mediated through various PDZ domain-containing proteins. These dynamic interactions impact the channel function as well as the localization and processing of CFTR protein within cells. This review focuses on the recent developments in defining the assembly of CFTR-containing complexes in the plasma membrane and its interacting proteins.     

Lower respiratory tract lactoferrin and lysozyme arise primarily in the airways and are elevated in association with chronic bronchitis

Lactoferrin and lysozyme are proteins found in high concentrations on mucosal surfaces, and they have activities potentially important for the modulation of inflammation. To investigate whether these proteins might contribute to the modulation of the intraluminal airway inflammation associated with chronic bronchitis, lactoferrin and lysozyme were measured in bronchoalveolar lavage (BAL) fluid from 22 subjects with chronic bronchitis and, for comparison, with 10 symptom-free smokers and 16 normal subjects. As a further control, transferrin, a protein structurally homologous to lactoferrin but not known to arise in airway epithelial cells, was also measured. BAL was performed by sequentially instilling and retrieving five 20 ml aliquots of normal saline solution into each of three sites. Analyzing the first aliquots separately from the later four provided fluid that was enriched for airway contents. The concentration of lactoferrin (11.83 +/- 2.86 micrograms/ml vs 0.68 +/- 0.18 micrograms/ml, p less than 0.00001), and lysozyme (6.75 +/- 1.51 micrograms/ml vs 0.52 +/- 0.09 microgram/ml, p less than 0.00001), but not transferrin (3.22 +/- 0.38 microgram/ml vs 2.68 +/- 0.24 micrograms/ml, p = 0.55) was higher in the bronchial sample lavage fluid, suggesting an airway origin for lactoferrin and lysozyme. In subjects with chronic bronchitis, bronchial sample lactoferrin (23.1 +/- 0.5 micrograms/ml) and lysozyme (12.6 +/- 3.5 micrograms/ml) were elevated compared with the normal subjects' lactoferrin (1.9 +/- 0.5 micrograms/ml, p less than 0.0001) and lysozyme (0.77 +/- 0.22 microgram/ml, p less than 0.0001) and the symptom-free smokers' lactoferrin (4.1 +/- 0.8 micrograms/ml, p = 0.005) and lysozyme (4.9 +/- 1.3 micrograms/ml, p = 0.02). Transferrin concentrations did not demonstrate the same relationships. Finally, when the content of bronchial sample lactoferrin and lysozyme were compared with the content of bronchial sample neutrophils, poor correlations were found, which may imply an airway epithelial origin for the two proteins. Thus lactoferrin and lysozyme appear to arise in the lower respiratory tract within the airways and their levels are elevated in association with chronic bronchitis. This suggests that lactoferrin and lysozyme may contribute to the modulation of airway inflammation in chronic bronchitis.