PERSPECTIVE
Chinks in the Armor of the Airway
Pseudomonas Infection in the Cystic Fibrosis Lung

Thomas W. Ferkol and Dwight C. Look

Departments of Pediatrics and Medicine, Washington University School of Medicine, St. Louis, Missouri

Defective expression or function of the cystic fibrosis transmembrane conductance regulator (CFTR) in airway lumen and submucosal gland epithelial cells leads to persistent and damaging lung infection that begins early in life and causes the majority of the morbidity and mortality in patients with cystic fibrosis (CF) (1). Respiratory infections do not appear to be a consequence of altered pulmonary development because lungs of neonates with CF are structurally normal (with the exception of acinar distension of submucosal glands) as well as sterile (2). However, at some point after birth, the respiratory tract becomes colonized with characteristic bacterial species. Initially, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli are frequently isolated from young patients with CF, but these organisms eventually surrender the lung to Pseudomonas aeruginosa (3). The isolation of mucoid strains of P. aeruginosa from the respiratory tract is virtually pathognomonic for this disease, and its early acquisition is associated with a poorer prognosis (4). Once acquired, P. aeruginosa is virtually impossible to eradicate from the CF lung, even with aggressive use of inhaled or systemic antibiotics. Unfortunately, the precise mechanisms by which abnormal CFTR leads to persistent pulmonary infection and the almost singular vulnerability to P. aeruginosa in patients with CF remain unclear.

The vulnerability of CF patients to chronic pulmonary infection with P. aeruginosa has focused research on identification of Pseudomonas-specific interactions with the CF airway that explain this predilection. One possibility for the predisposition to infection with P. aeruginosa could be increased adherence of this organism to airway epithelial cells of CF patients (5). P. aeruginosa bacilli adhere in greater numbers to epithelial cells of patients homozygous for the F508 CFTR mutation (6), and this appears to be secondary to increased asialylated glycoconjugate receptors at the epithelial surface (7). Bacterial adhesins that are components of P. aeruginosa pili attach specifically to asialylated glycosphingolipid receptors containing an N-acetylgalactosamine-galactose binding moiety, such as gangliotetraosylceramide (asialo-GM₁), which are increased on respiratory epithelial cells from CF patients (8). Increased attachment of the Pseudomonas bacilli to the cystic fibrosis airway leads to impaired clearance, and binding via pili and other bacterial structures promotes inflammation by provoking airway epithelial cells to release mediators such as interleukin-8 (9). However, the clinical significance of this mechanism for Pseudomonas-epithelial cell interaction is uncertain because increased binding to asialylated receptors is relatively modest and not bacteria-specific (10).

Another proposal to explain the predilection of P. aeruginosa for the CF lung focuses on the capacity of respiratory epithelial cells to participate in clearance of the bacterium. In this model, normal airway epithelial cells internalize P. aeruginosa and then desquamate to eliminate the organism from the airway (11). Moreover, CFTR may actually serve as the receptor for binding and internalization of P. aeruginosa in this system, and airway clearance through this process may be decreased by CFTR mutation (12). Accordingly, decreased ingestion of P. aeruginosa at the epithelial surface may permit establishment of Pseudomonas infection in the CF lung, but the actual role of epithelial cells as "phagocytes" and its relevance to innate defense of the airway in vivo is unclear (13).

Since the neutrophil is the predominant inflammatory cell in the CF airway and central to bacterial killing (14, 15), others have concentrated on Pseudomonas interactions with these professional phagocytes. P. aeruginosa is a potent stimulus for neutrophil influx into the lungs, yet the bacteria has virulence factors, such as a quorum sensing system for biofilm generation, that allow it to evade neutrophil killing (16). Epithelial cells containing a mutant CFTR may amplify neutrophil recruitment through constitutive and bacteria-induced release of inflammatory mediators that is disproportionate to the infectious stimulus (17). Neutrophil proteases that normally degrade outer membrane proteins of bacteria are released into the CF airway (14, 20, 21). High extracellular concentrations of proteases such as neutrophil elastase damage the lung and can interfere with antibacterial defenses through digestion of proteins that promote opsonic phagocytosis such as immunoglobulins, complement components, and receptors (22, 23). Taken together, it is possible that P. aeruginosa is best adapted for the CF airway because it not only survives, but also can persist and thrive in the face of established pulmonary inflammation. Still, these virulence factors do not clearly explain why Pseudomonas colonization occurs in the first place.

Because the CFTR is a chloride channel, several investigators have worked to more directly link abnormal airway salt and water transport with respiratory infection. Early theories proposed that failure of chloride secretion through CFTR and/or associated sodium hyperabsorption through the epithelial sodium channel results in desiccation of endobronchial secretions (24). The thickened, dehydrated secretions obstruct and prevent elimination of bacteria from the airway. This failure of mucociliary clearance then permits establishment of bacterial colonization. However, direct measurements of mucociliary function in the airways of patients with CF have not always shown impaired clearance (27), and defective mucociliary clearance alone does not explain the propensity for P. aeruginosa infection in the CF lung.

More recently, it has been proposed that acquisition of airway infection is due to a breech in the innate defenses of the CF airway that is directly linked to abnormal CFTR function. Investigators have shown that low concentrations of P. aeruginosa are eradicated when applied to the apical surface of normal human airway epithelial cells grown in differentiated cultures or when mixed with airway surface fluid from normal human bronchial xenografts, whereas bacteria continue to grow when epithelial cells from CF patients are used in these models (28, 29). Also, airway surface fluid collected from CF epithelium has antimicrobial activity when collected in water, while high salt concentrations added to normal airway surface fluid block this effect. This antibacterial activity appears to be mediated by human beta-defensins (29, 30), but the function of other components of airway defense, like antimicrobial enzymes secreted from submucosal glands, are also hindered by the abnormally high ionic composition of epithelial lining fluid that might be present in the CF lung (31). Consequently, Pseudomonas bacilli deposited in the CF airway are not effectively cleared, permitting the organism to secure a foothold in the lung and provoke an inflammatory response. Not all investigators have found CF airway surface fluid to have elevated ionic concentrations relative to normal fluid, which is central to this hypothesis (25, 26, 32). In addition, altered antimicrobial activity of airway surface fluid with bacterial specificity that would explain vulnerability to P. aeruginosa has not been demonstrated.

In this issue, an interesting twist is added to the list of potential defects in innate defense in the CF airway (Figure 1). Using a bronchial xenograft system for culture of differentiated human airway epithelial cells from patients without or with CF, Bals and colleagues demonstrate that antimicrobial activity in airway surface liquid is decreased in CF samples (33). Although abnormal airway surface liquid activity for microbial killing has been demonstrated previously in CF epithelia (28, 29), the factor (or factors) detected by Bals and coworkers that participates in killing of P. aeruginosa appears different from known endogenous antibacterial agents because full activity in CF samples is not restored by lowering the salt concentration. The reason why other studies have not identified reduced antimicrobial activity in CF airway secretions when studied at low salt concentrations is unclear, but could be due to differences in the assay systems or source of airway liquid (31). The identity of this antimicrobial activity is currently unknown, as is the mechanism by which a defective CFTR gene affects its function. It is also unclear if this less salt-sensitive antimicrobial activity kills other respiratory pathogens. However, it is reassuring that this activity can be restored in CF cells by expression of wildtype CFTR using an adenoviral vector. Accordingly, identification and characterization of this activity may provide new leads toward understanding altered innate immunity and/or patterns of bacterial infection in patients with CF.

View larger version (37K): [in this window] [in a new window]   Figure 1.   Secreted innate airway defenses are critical in preventing infection in the normal lung. The epithelium generates an array of antimicrobial agents including polymeric immunoglobulins, collectins, antiproteases, antibacterial enzymes, and -defensins to protect the host from pathogens. Airway defenses are breached early in the life of patients with CF, which may be related to impaired function of these antimicrobial agents through their degradation by proteases or inactivation by the high salt content of airway surface fluid.

Although it has been more than a decade since the gene responsible for CF was identified and cloned (34), the specific link between mutant CFTR and persistent airway infection has been difficult to identify. This difficulty likely reflects the complex, multitiered, and redundant nature of innate airway defense. Nevertheless, the novel concepts that have been proposed to explain the pathogenesis of airway infection in the CF lung have led directly to greater insight into innate and adaptive immunity in the normal airway. Understanding how the airway combats infection and why this response is altered in disease is crucial for the development of therapeutic strategies to restore airway defense to a fully functional status in CF and other pulmonary diseases.

	Footnotes

Address correspondence to: Thomas W. Ferkol, Washington University School of Medicine, Department of Pediatrics, Campus Box 8208, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: ferkol_t{at}kids.wustl.edu

(Received in original form May 31, 2001).

Acknowledgments: The authors' work is supported by grants from the National Institutes of Health, Cystic Fibrosis Foundation, and American Lung Association.

	References

1. FitzSimmons, S. C.. 1993. The changing epidemiology of cystic fibrosis. J. Pediatr. 122: 1-9 [Medline].

2. Sturgess, J., and J. Imrie. 1982. Quantitative evaluation of the development of tracheal submucosal glands in infants with cystic fibrosis and control infants. Am. J. Pathol. 106: 303-311 [Abstract].

3. Cystic Fibrosis Foundation. 1995 patient registry annual data report. Bethesda, MD.

4. Henry, R. L., C. M. Mellis, and L. Petrovic. 1992. Mucoid Pseudomonas aeruginosa is a marker of poor survival in cystic fibrosis. Pediatr. Pulmonol. 12: 158-161 [Medline].

5. Saiman, L., G. Cacalano, D. Gruenert, and A. Prince. 1992. Comparison of adherence of Pseudomonas aeruginosa to respiratory epithelial cells from cystic fibrosis patients and healthy subjects. Infect. Immun. 60: 2808-2814 [Abstract/Free Full Text].

6. Zar, H., L. Saiman, L. Quittell, and A. Prince. 1995. Binding of Pseudomonas aeruginosa to respiratory epithelial cells from patients with various mutations in the cystic fibrosis transmembrane regulator. J. Pediatr. 126: 230-233 [Medline].

7. Dosanjh, A., W. Lencer, D. Brown, D. A. Ausiello, and J. L. Stow. 1994. Heterologous expression of F508 CFTR results in decreased sialylation of membrane glycoconjugates. Am. J. Physiol. (Cell Physiol.) 266: C360-C366 [Abstract/Free Full Text].

8. Saiman, L., and A. Prince. 1993. Pseudomonas aeruginosa pili bind to asialoGM1 which is increased on the surface of cystic fibrosis epithelial cells. J. Clin. Invest. 92: 1875-1880 .

9. DiMango, E., H. J. Zar, R. Bryan, and A. Prince. 1995. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin. Invest. 96: 2204-2210 .

10. Krivan, H. C., D. D. Roberts, and V. Ginsburg. 1988. Many pulmonary pathogenic bacteria bind specifically to the carbohydrate sequence GalNAc1-4Gal found in some glycolipids. Proc. Natl. Acad. Sci. USA 85: 6157-6161 [Abstract/Free Full Text].

11. Pier, G. B., M. Grout, T. S. Zaidi, J. C. Olsen, L. G. Johnson, J. R. Yankaskas, and J. B. Goldberg. 1996. Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 271: 64-67 [Abstract].

12. Pier, G. B., M. Grout, and T. S. Zaidi. 1997. Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc. Natl. Acad. Sci. USA 94: 12088-12093 [Abstract/Free Full Text].

13. Chroneos, Z. C., S. E. Wert, J. L. Livingston, D. J. Hassett, and J. A. Whitsett. 2000. Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo. J. Immunol. 165: 3941-3950 [Abstract/Free Full Text].

14. Konstan, M. W., K. A. Hilliard, T. M. Norvell, and M. Berger. 1994. Bronchoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation. Am. J. Respir. Crit. Care Med. 150: 448-454 [Abstract].

15. Danel, C., S. C. Erzurem, N. G. McElvaney, and R. G. Crystal. 1996. Quantitative assessment of the epithelial and inflammatory cell populations in large airways of normals and individuals with cystic fibrosis. Am. J. Respir. Crit. Care Med. 153: 362-368 [Abstract].

16. Singh, P. K., A. L. Schaefer, M. R. Parsek, T. O. Moninger, M. J. Welsh, and E. P. Greenberg. 2000. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407: 762-764 [Medline].

17. Van Heeckeren, A., R. Walenga, M. W. Konstan, T. Bonfield, P. B. Davis, and T. Ferkol. 1997. Excessive inflammatory response of cystic fibrosis mice to bronchopulmonary infection with Pseudomonas aeruginosa. J. Clin. Invest. 100: 2810-2815 [Medline].

18. Tabary, O., S. Escotte, J. P. Couetil, D. Hubert, D. Dusser, E. Puchelle, and J. Jacquot. 2000. High susceptibility for cystic fibrosis human airway gland cells to produce IL-8 through the IB kinase  pathway in response to extracellular NaCl content. J. Immunol. 164: 3377-3384 [Abstract/Free Full Text].

19. Kube, D., U. Sontich, D. Fletcher, and P. B. Davis. 2001. Proinflammatory cytokine responses to P. aeruginosa infection in human airway epithelial cell lines. Am. J. Physiol. (Lung Cell. Mol. Physiol.) 280: L493-L502 [Abstract/Free Full Text].

20. Meyer, K. C., and J. Zimmerman. 1993. Neutrophil mediators, Pseudomonas, and pulmonary dysfunction in cystic fibrosis. J. Lab. Clin. Med. 121: 654-661 [Medline].

21. Belaaouaj, A., K. S. Kim, and S. D. Shapiro. 2000. Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. Science 289: 1185-1188 [Abstract/Free Full Text].

22. Fick, R. B., G. P. Naegel, S. U. Squier, R. E. Wood, J. B. L. Gee, and H. Y. Reynolds. 1984. Proteins of the cystic fibrosis respiratory tractfragmented immunoglobulin G opsonic antibody causing defective opsonophagocytosis. J. Clin. Invest. 74: 236-248 .

23. Tosi, M. F., H. Zakem, and M. Berger. 1990. Neutrophil elastase cleaves C3bi on opsonized Pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch. J. Clin. Invest. 86: 300-308 .

24. Tomkiewicz, R. P., E. M. App, J. G. Zayas, O. Ramirez, N. Church, R. C. Boucher, M. R. Knowles, and M. King. 1993. Amiloride inhalation therapy in cystic fibrosisinfluence on ion content, hydration, and rheology of sputum. Am. Rev. Respir. Dis. 148: 1002-1007 [Medline].

25. Knowles, M. R., J. M. Robinson, R. E. Wood, C. A. Pue, W. M. Mentz, G. C. Wager, J. T. Gatzy, and R. C. Boucher. 1997. Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J. Clin. Invest. 100: 2588-2595 [Medline].

26. Matsui, H., B. R. Grubb, R. Tarran, S. H. Randell, J. T. Gatzy, C. W. Davis, and R. C. Boucher. 1998. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95: 1005-1015 [Medline].

27. Yeates, D. B., J. M. Sturgess, S. R. Kahn, H. Levison, and N. Aspin. 1976. Mucociliary transport in trachea of patients with cystic fibrosis. Arch. Dis. Child. 51: 28-33 [Abstract].

28. Smith, J. J., S. M. Travis, E. P. Greenberg, and M. J. Welsh. 1996. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid [published erratum appears in Cell 1996;87::355]. Cell 85: 229-236 [Medline].

29. Goldman, M. J., G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff, and J. M. Wilson. 1997. Human -defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88: 553-560 [Medline].

30. Bals, R., X. Wang, Z. Wu, T. Freeman, V. Bafna, M. Zasloff, and J. M. Wilson. 1998. Human -defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. J. Clin. Invest. 201: 874-880 .

31. Travis, S. M., B. D. Conway, J. Zabner, J. J. Smith, N. N. Anderson, P. K. Singh, E. P. Greenberg, and M. J. Welsh. 1999. Activity of abundant antimicrobials of the human airway. Am. J. Respir. Cell Mol. Biol. 20: 872-879 [Abstract/Free Full Text].

32. Matsui, H., C. W. Davis, R. Tarran, and R. C. Boucher. 2000. Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia. J. Clin. Invest. 105: 1419-1427 [Medline].

33. Bals, R., D. J. Weiner, R. L. Meegalla, F. Accurso, and J. M. Wilson. 2001. Salt-independent abnormality of antimicrobial activity in cystic fibrosis airway surface fluid. Am. J. Respir. Cell Mol. Biol. 25: 21-25 [Abstract/Free Full Text].

34. Riordan, J. R., J. M. Rommens, B. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J. Chou, M. L. Drumm, M. C. Iannuzzi, F. S. Collins, and L. Tsui. 1989. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073 [Abstract/Free Full Text].