Localization of Burkholderia cepacia complex bacteria in cystic fibrosis lungs and interactions with Pseudomonas aeruginosa in hypoxic mucus.

The localization of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with Pseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc and P. aeruginosa bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined the growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as "biofilm-like structures." In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a P. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibit P. aeruginosa biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.

Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis.

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

Transcriptional responses of Mycobacterium tuberculosis to lung surfactant.

This study uses microarray analyses to examine gene expression profiles for Mycobacterium tuberculosis (Mtb) induced by exposure in vitro to bovine lung surfactant preparations that vary in apoprotein content: (i) whole lung surfactant (WLS) containing the complete mixture of endogenous lipids and surfactant proteins (SP)-A, -B, -C, and -D; (ii) extracted lung surfactant (CLSE) containing lipids plus SP-B and -C; (iii) column-purified surfactant lipids (PPL) containing no apoproteins, and (iv) purified human SP-A. Exposure to WLS evoked a multitude of transcriptional responses in Mtb, with 52 genes up-regulated and 23 genes down-regulated at 30min exposure, plus 146 genes up-regulated and 27 genes down-regulated at 2h. Notably, WLS rapidly induced several membrane-associated lipases that presumptively act on surfactant lipids as substrates, and a large number of genes involved in the synthesis of phthiocerol dimycocerosate (PDIM), a cell wall component known to be important in macrophage interactions and Mtb virulence. Exposure of Mtb to CLSE, PPL, or purified SP-A caused a substantially weaker transcriptional response (

Peripheral cell wall lipids of Mycobacterium tuberculosis are inhibitory to surfactant function.

The transmission of Mycobacterium tuberculosis (TB) requires extensive damage to the lungs to facilitate bacterial release into the airways, and it is therefore likely that the microorganism has evolved mechanisms to exacerbate its local pathology. This study examines the inhibitory effects of lipids extracted and purified chromatographically from TB on the surface-active function of lavaged bovine lung surfactant (LS) and a clinically relevant calf lung surfactant extract (CLSE). Total lipids from TB greatly inhibited the surface activity of LS and CLSE on the pulsating bubble surfactometer at physical conditions applicable for respiration in vivo (37 degrees C, 20 cycles/min, 50% area compression). Minimum surface tensions for LS (0.5 mg/ml) and CLSE (1 mg/ml) were raised from <1 mN/m to 15.7+/-1.2 and 18.7+/-1.3 mN/m after 5 min of bubble pulsation in the presence of total TB lipids (0.15 mg/ml). TB mixed waxes (0.15 mg/ml) and TB trehalose monomycolates (TMMs, 0.15 mg/ml) also significantly inhibited the surface activity of LS and CLSE (minimum surface tensions of 10-16 mN/m after 5 min of bubble pulsation), as did purified trehalose 6,6'-dimycolate (TDM, cord factor). Phosphatidylinositol mannosides (PIMs, 0.15 mg/ml) from TB had no inhibitory effect on the surface activity of LS or CLSE. Concentration dependence studies showed that LS was also inhibited significantly by total TB lipids at 0.075 mg/ml, with a smaller activity decrease apparent even at 0.00375 mg/ml. These findings document that TB contains multiple lipids that can directly impair the biophysical function of endogenous and exogenous lung surfactants. Direct inhibition by TB lipids could worsen surfactant dysfunction caused by plasma proteins or other endogenous substances induced by inflammatory injury in the infected lungs. TB lipids could also inhibit the effectiveness of exogenous surfactants used to treat severe acute respiratory failure in TB patients meeting criteria for clinical acute lung injury (ALI) or the acute respiratory distress syndrome (ARDS).

Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.

Current theories of CF pathogenesis predict different predisposing "local environmental" conditions and sites of bacterial infection within CF airways. Here we show that, in CF patients with established lung disease, Pseudomonas aeruginosa was located within hypoxic mucopurulent masses in airway lumens. In vitro studies revealed that CF-specific increases in epithelial O(2) consumption, linked to increased airway surface liquid (ASL) volume absorption and mucus stasis, generated steep hypoxic gradients within thickened mucus on CF epithelial surfaces prior to infection. Motile P. aeruginosa deposited on CF airway surfaces penetrated into hypoxic mucus zones and responded to this environment with increased alginate production. With P. aeruginosa growth in oxygen restricted environments, local hypoxia was exacerbated and frank anaerobiosis, as detected in vivo, resulted. These studies indicate that novel therapies for CF include removal of hypoxic mucus plaques and antibiotics effective against P. aeruginosa adapted to anaerobic environments.

Phenotypic and transcriptomic analyses demonstrate interactions between the transcriptional regulators CtsR and Sigma B in Listeria monocytogenes.

Listeria monocytogenes sigma(B) positively regulates the transcription of class II stress response genes; CtsR negatively regulates class III stress response genes. To identify interactions between these two stress response systems, we constructed L. monocytogenes DeltactsR and DeltactsR DeltasigB strains, as well as a DeltactsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-beta-d-thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a DeltasigB strain, were assayed for motility, heat resistance, and invasion of human intestinal epithelial cells, as well as by whole-genome transcriptomic and quantitative real-time PCR analyses. Both DeltactsR and DeltactsR DeltasigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the DeltactsR strain when ctsR was expressed in trans. Although log-phase DeltactsR was not reduced in its ability to infect human intestinal cells, the DeltactsR DeltasigB strain showed significantly lower invasion efficiency than either the parent strain or the DeltasigB strain, indicating that interactions between CtsR and sigma(B) contribute to invasiveness. Statistical analyses also confirmed interactions between the ctsR and the sigB null mutations in both heat resistance and invasion phenotypes. Microarray transcriptomic analyses and promoter searches identified (i) 42 CtsR-repressed genes, (ii) 22 genes with lower transcript levels in the DeltactsR strain, and (iii) at least 40 genes coregulated by both CtsR and sigma(B), including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and sigma(B) play an important role in L. monocytogenes stress resistance and virulence.

Effects of autologous stromal cells and cytokines on differentiation of equine bone marrow-derived progenitor cells.

OBJECTIVE To develop an in vitro system for differentiation of equine B cells from bone marrow hematopoietic progenitor cells on the basis of protocols for other species. SAMPLE Bone marrow aspirates aseptically obtained from 12 research horses. PROCEDURES Equine bone marrow CD34+ cells were sorted by use of magnetic beads and cultured in medium supplemented with cytokines (recombinant human interleukin-7, equine interleukin-7, stem cell factor, and Fms-like tyrosine kinase-3), murine OP9 stromal cell preconditioned medium, and equine fetal bone marrow mesenchymal stromal cell preconditioned medium. Cells in culture were characterized by use of flow cytometry, immunocytofluorescence microscopy, and quantitative reverse-transcriptase PCR assay. RESULTS For these culture conditions, bone marrow-derived equine CD34+ cells differentiated into CD19+IgM+ B cells that expressed the signature transcription factors early B-cell factor and transcription factor 3. These conditions also supported the concomitant development of autologous stromal cells, and their presence was supportive of B-cell development. CONCLUSIONS AND CLINICAL RELEVANCE Equine B cells were generated from bone marrow aspirates by use of supportive culture conditions. In vitro generation of equine autologous B cells should be of use in studies on regulation of cell differentiation and therapeutic transplantation.

Identification of N-acylhomoserine lactones in mucopurulent respiratory secretions from cystic fibrosis patients.

Pseudomonas aeruginosa and species of the Burkholderia cepacia complex are the primary bacterial pathogens contributing to lung disease in patients with cystic fibrosis. Quorum sensing systems using N-acyl homoserine lactone (AHL) signal molecules are involved in the regulation of a number of virulence factors in these species. Extracts of mucopurulent respiratory secretions from 13 cystic fibrosis patients infected with P. aeruginosa and/or strains of the B. cepacia complex were fractionated using reverse-phase fast pressure liquid chromatography and analyzed for the presence of AHLs using a traI-luxCDABE-based reporter that responds to AHLs with acyl chains ranging between 4 and 12 carbons. Using this assay system, a broad range of AHLs were detected and identified despite being present at low concentrations in limited sample volumes. N-(3-oxo-dodecanoyl)-l-homoserine lactone, N-(3-oxo-decanoyl)-l-homoserine lactone and N-octanoyl-l-homoserine lactone (OHL) were the AHLs most frequently identified. OHL and N-decanoyl-l-homoserine lactone were detected in nanomolar concentrations compared to picomolar amounts of the 3-oxo-derivatives of the AHLs identified.

The Listeria monocytogenes prfAP2 promoter is regulated by sigma B in a growth phase dependent manner.

Listeria monocytogenes prfA, encoding positive regulatory factor A, is transcribed from three promoters (prfAP1, prfAP2, and PplcA). The prfAP2 promoter was previously proposed to be sigma B (sigma(B))-dependent. This hypothesis was tested by creating prfA promoter-gus transcriptional fusions in both L. monocytogenes wild-type (wt) and DeltasigB backgrounds and then measuring (i) beta-glucuronidase (GUS) activities; (ii) gus mRNA transcript levels; and (iii) the presence or absence of GUS in cells by immunofluorescence staining. prfAP2-directed expression increased as the wt L. monocytogenes strain entered stationary phase, whereas prfAP2-directed expression was greatly reduced in the DeltasigB strain, confirming both growth phase- and sigma(B)-dependent transcription of prfAP2. We conclude that prfAP2 is directly regulated by sigma(B).

Alternative sigma factor sigmaB is not essential for listeria monocytogenes surface attachment.

Listeria monocytogenes is a foodborne pathogen frequently isolated from the food processing environment. Multiple lines of evidence suggested a possible role for the L. monocytogenes alternative transcription factor sigma B (sigmaB) in surface attachment and biofilm formation. Therefore, through plate count and microscopic techniques, the L. monocytogenes 10403S strain and an otherwise isogenic deltasigB strain were tested for attachment to stainless steel. Analysis of microscopic images revealed that after 72 h at 24 degrees C under static conditions the tested L. monocytogenes strains attached uniformly to surfaces as single cells. Both strains were capable of rapid attachment (i.e., numbers of attached cells were essentially the same after either 5 min or 24 h of incubation). Numbers of attached deltasigB cells were significantly lower than those of the wild-type strain after 48 and 72 h of incubation at 24 degrees C (P = 0.001). Similar numbers of the deltasigB strain attached to stainless steel regardless of temperature (24 or 37 degrees C); however, deltasigB cells attached at higher relative numbers in the presence of 6% NaCl after 48 and 72 h. Furthermore, in the presence of Pseudomonas fluorescens, similarly high numbers of wild-type and deltasigB cells attached to the surfaces, forming mixed biofilms. Our data suggest that sigmaB is not required for initial surface attachment of L. monocytogenes.

A bacterial cell to cell signal in the lungs of cystic fibrosis patients.

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.

Disposition and intracellular levels of moxifloxacin in human THP-1 monocytes in unstimulated and stimulated conditions.

Moxifloxacin uptake by human THP-1 monocytes was passive and initially linear and reached equilibrium after approximately 4 h. High intracellular concentrations were achieved and intracellular/extracellular [I/E] ratios were between 1925 and 4575 for the lowest concentration of 0.004 microg/ml at pH 7.4 and 6.9. The uptake of moxifloxacin was reduced by sodium fluoride, -azide, -cyanide, low temperature and low pH. However, the uptake was not affected by any of the ion channel blockers. Adenosine demonstrated marginal competition with moxifloxacin for uptake suggesting a nucleoside transporter may be involved. The sodium-ATPase pump when blocked, also retarded moxifloxacin uptake at 2 and 4 h. This I/E ratio was high compared with other macrolides and indicateed that the monocyte may contain sufficient moxifloxacin levels to conduct the antibiotic throughout systemic circulation to infection sites. Efflux from THP-monocytes was essentially complete after 2 h indicating no long term sequestering of the antibiotic occurred. Disposition of the antibiotic within the THP-1 monocytes showed large amounts present in the nucleus and cytoplasm in stimulated and unstimulated cells. Increased amounts of the drug were found in the THP-1 monocytes as well as the endoplasmic reticulum and the isolated phagosomes after stimulation by zymogen A, Staphylococcus aureus or Streptococcus pneumoniae.

Disposition and intracellular activity of azithromycin in human THP-1 acute monocytes.

Uptake of [14C]-azithromycin into THP-1 human monocytes was determined at pH 7.4, 6.8 or 5.5 over 4-log antibiotic concentrations for 24 h under a number of conditions. Stimulation of cells was with bacteria, latex beads, lipopolysaccharide (LPS), or zymogen A. Subcellular organelle disposition was determined after isolation by ultracentrifugation or sucrose gradients. Hydrolytic enzyme activities and mediators of intracellular inflammation (IL-1, IL-6, IL-8, and TNFalpha) were assessed. Azithromycin uptake into human THP-1 monocytes was initially linear achieving approximately 2% of the extracellular concentration. At pH 7.4, uptake was both passive- and carrier-mediated, but as the pH became more acidic, the uptake was exclusively passive. The intracellular concentration was not pH-dependent over 24 h. Uptake was dependent upon temperature but not the presence of foetal calf serum. Intracellular disposition in zymogen A-stimulated and unstimulated cells was throughout all compartments of the cell, but was higher in the nucleus and cell sap. Phagosomes of stimulated cells contained higher level of the antibiotic. Efflux from THP-1 monocytes was complete between 3 and 4 h. After 1 h treatment with zymogen A, THP-1 monocytes demonstrated an increase in intracellular acidity, protein kinase C, SOD and NAG activities, and NO, H(2)O(2), TNFalpha and IL-1 release over the 1st h. After 2-4 h the pH became alkaline, activities of NADPH reductase, NAG and cathepsin were reduced, and the release of NO, H(2)O(2), TNFalpha and IL-6 were suppressed. Protein synthesis and killing of the bacteria was evident in bacteria kept in monocyte-free medium and those phagocytized by the THP-1 monocytes moderately at 2 h, but more significantly at 24 h. The early killing of the bacteria appears to be a cidal mechanism whereas later, a standard bacteriostatic mechanism was evident. Nevertheless, suppression of these chemical mediators and hydrolytic enzyme activities would reduce the infection and the spread to adjacent areas.

Effects of moxifloxacin in zymogen A or S. aureus stimulated human THP-1 monocytes on the inflammatory process and the spread of infection.

Antimicrobial agents have been reported to exhibit immunomodulatory and anti-inflammatory activities, both in vivo and in vitro (e.g., in human lymphocytes, macrophages and monocytes). The effects of moxifloxacin on cytokine immunomodulatory mediators, free radical generation and hydrolytic enzyme activities in zymogen A-stimulated human THP-1 monocytes were evaluated. An increase in c-AMP levels, protein kinase C activity, and the release of nitric oxide and hydrogen peroxide with a decrease in pH occurred within the first hour. Further, the effects of moxifloxacin were reduced by agents which blocked the oxygen burst, lysosome-phagosome fusion, and the energy generation within the cell. After 4 h, there was a decrease in NAG and cathepsin D activities, lipid peroxidation and the release of pro-inflammatory cytokines. These data indicate that moxifloxacin may modify the acute-phase inflammatory responses through inhibition of cytokine release in monocytes. Moxifloxacin inhibited the release of TNFalpha, IL-1, IL-6, and IL-8 in a concentration-dependent manner across a range of 0.004 to 4 microg/mL. After 4 h, there was a decrease in the release of these cytokines, thus interfering with the inflammation process to reduce infection and its spread. The effects of moxifloxacin appear initially to activate monocytes to kill bacteria through the innate immune process by releasing ROS and lysosomal hydrolytic enzymes as well as phagocytosis of the organism. At a later time the bacteria are killed through a Bacterialstatic mechanism of protein synthesis inhibition and there is a reversal of the effects of moxifloxacin on cytokine release, free radical generation and hydrolytic enzymes so that lipid peroxidation and tissue destruction by the infection process is suppressed.

Effects of alatrofloxacin, the parental prodrug of trovafloxacin, on phagocytic, anti-inflammatory and immunomodulation events of human THP-1 monocytes.

Alatrofloxacin functions similar to other fluoroquinolone antibiotics in that it not only has antibiotic activity to kill invading organisms by interfering with DNA synthesis, it possesses immunosuppressive activity. In the first hour after bacteria have been phagocytosed by THP-1 monocytes, the drug activates a lytic mechanism involving the release of c-AMP, tumor necrosis factor (TNFalpha), interleukin-1 (IL-1), IL-6 and nitric oxide, with elevations in lysosomal hydrolytic enzyme activities. This effect reverses between 2 and 4 h. At this time, all of these inflammatory processes are returned to normal values or below suggesting that alatrofloxacin reduces the spread of infection and destruction of tissue related to inflammation.

A 3-D airway epithelial cell and macrophage co-culture system to study Rhodococcus equi infection.

We developed a 3-D equine bronchial epithelial cell (BEC) culture that fully differentiates into ciliary beating and mucus producing cells. Using this system, we evaluated how mucus affects the phagocytic activity of macrophages. Adult horse monocyte-derived macrophages were incubated with Rhodococcus equi for 4h either in the mucus layer of in vitro generated airway epithelium or on collagen coated membranes. Using light and electron microscopy, we noted that the number of macrophages with intracellular bacteria, and the number of intracellular bacteria per macrophage were lower in the presence of mucus. TNFα measurements revealed that the presence of BECs promoted TNFα production by R. equi-infected macrophages; a decrease in TLR-2 (involved in R. equi recognition) and an increase in EGF-R (involved in mucin production) mRNA expression were also noted. Interestingly, when foal macrophages were added to foal BECs, we made the opposite observation, i.e. many macrophages were loaded with R. equi. Our in vitro bronchial system shows great potential for the identification of mechanisms how BECs and mucus play a role in phagocyte activation and bacterial clearance. Further studies using this system will show whether the airway environment in the foal responds differently to R. equi infection.

Equine bronchial epithelial cells differentiate into ciliated and mucus producing cells in vitro.

We describe a method for creating differentiated equine bronchial epithelial cell cultures that can be used for in vitro studies including airway disease mechanisms and pathogen-host interactions. Our method is based on the culturing of human tracheobronchial epithelial cells at an air-liquid interface (ALI) in specific serum-free, hormone-supplemented medium. Bronchial epithelial cells are isolated and grown on T-Clear® insert membranes. Within 2 to 3 wk, cells differentiate into ciliated and mucus producing cells as demonstrated by confocal and electron microscopy. Furthermore, the demonstration of the two major gel-forming mucin species, Muc5ac and Muc5b, in our bronchial epithelial cell culture system validates this method for studies of respiratory tract disease of the horse.

Evaluation of the pathogenicity of Listeria spp. in Caenorhabditis elegans.

Caenorhabditis has proven to be a useful model for studying host-pathogen interactions as well as the ability of nematodes to serve as vectors for the dispersal of foodborne pathogens. In this study, we evaluated whether C. elegans can serve as a host for Listeria spp. While there was an effect of growth media on C. elegans killing, C. elegans exposed to L. monocytogenes and L. innocua pregrown in Luria-Bertani medium showed reduced survival when compared to nonpathogenic E. coli OP50, while L. seeligeri showed survival similar to E. coli OP50. In a preference assay, C. elegans preferred E. coli over L. monocytogenes and L. innocua, but showed no preference between L. monocytogenes and L. innocua. A gentamicin assay indicated that L. monocytogenes did not persist within the C. elegans intestinal tract. Our findings that L. monocytogenes and L. innocua strains tested have equally deleterious effects on C. elegans and that L. monocytogenes did not establish intestinal infection conflict with other recently published results, which found intestinal infection and killing of C. elegans by L. monocytogenes. Further studies are thus needed to clarify the interactions between L. monocytogenes and C. elegans, including effects of environmental conditions and strain differences on killing and intestinal infection.

A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms.

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an approximately 5 x 10(3) strain PAO1 P. aeruginosa. Although bacteria grew to 10(10) cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical-chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.

Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia.

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.