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Published ahead of print on October 9, 2003, doi:10.1164/rccm.200304-558OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 174-179, (2004)
© 2004 American Thoracic Society

Airway Inflammation and Infection in Congenital Bilateral Absence of the Vas Deferens

Marita Gilljam, Yuri Moltyaner, Gregory P. Downey, Roslyn Devlin, Peter Durie, André M. Cantin, Julian Zielenski and D. Elizabeth Tullis

Department of Respiratory Medicine and Allergology, Sahlgrenska University Hospital, Göteborg University, Göteborg, Sweden; Division of Respirology, Department of Medicine, University of Sherbrooke, Sherbrooke, Quebec; Adult Cystic Fibrosis Centre, and Diagnostic Laboratories and Pathobiology, St Michael's Hospital; Toronto General Hospital Research Institute; Research Institute, Hospital for Sick Children; and Division of Respirology, Department of Medicine, Department of Pediatrics, and Department of Medical and Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

Correspondence and requests for reprints should be addressed to Elizabeth Tullis, M.D., St. Michael's Hospital, 30 Bond Street, Toronto, Ontario, Canada M5B 1W8. E-mail: tullise{at}smh.toronto.on.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In cystic fibrosis (CF), airway disease begins early in life. Bacteria and elevated levels of neutrophils and inflammatory mediators have been detected in bronchoalveolar lavage (BAL) fluid from infants with CF. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) are common in men with congenital bilateral absence of the vas deferens (CBAVD) and it has been suggested that this syndrome represents a mild form of CF. We hypothesized that men with CBAVD also have subclinical pulmonary disease. Bronchoscopy with BAL, viral and quantitative bacterial cultures, and analyses of total and differential cell count, cytokines, and free neutrophil elastase was performed in eight men with CBAVD, who had mutations in the CFTR and intermediate or elevated sweat chloride levels, and in four healthy control subjects. There was light growth of Staphylococcus aureus in one of eight men with CBAVD, and small numbers of opportunistic gram-negative bacteria in six of eight men with CBAVD and in one control subject. BAL cell counts and neutrophil elastase were within the normal range. Interleukin-8 and tumor necrosis factor-{alpha} levels were higher for men with CBAVD than for control subjects. These data suggest that mutations in the CFTR in men with CBAVD, in addition to causing infertility, lead to subclinical bacterial pulmonary infection and inflammation consistent with mild CF.

Key Words: bronchoalveolar lavage • cystic fibrosis • cystic fibrosis transmembrane conductance regulator • infection • inflammation

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). CF has a complex phenotype with variable disease severity and with manifestations in many organs (1). Involvement of the respiratory tract, however, is the dominant clinical feature and progressive pulmonary disease accounts for most of the morbidity and virtually all the mortality in patients with CF. The CFTR gene product is a transmembrane glycoprotein known to function as a chloride channel in epithelial cells. Absence or dysfunction of CFTR leads to alterations in the microenvironment of the lung that are manifest early in life as airway inflammation. Reported abnormalities in the CF lung include altered fluid and ionic fluxes across the respiratory epithelium, excessive lumenal mucus and dehydration of the mucus, diminished mucociliary clearance, altered patterns of epithelial surface glycosylation, and diminished activity of bactericidal factors (2, 3).

Bronchoscopy studies in infants have shown elevated levels of inflammatory markers in bronchoalveolar lavage (BAL) fluid, with or without the presence of bacteria, as early as 4 weeks of age (4, 5). Inflammation of the respiratory tract is extensive in CF but it remains unclear whether it is a consequence or cause of infection (1, 6). Mutations in CFTR facilitate infection by a restricted group of microorganisms, particularly Staphylococcus aureus and Haemophilus influenzae, but eventually most patients with CF become chronically infected with Pseudomonas aeruginosa (7, 8). Some variation of CF disease may be related to the different functional consequences of diverse mutations in CFTR, of which more than 1,000 have been described to date (9), or to modifier gene effects or environmental influence (10).

Most men with CF are infertile because of obstructive azoospermia due to bilateral absence of the vas deferens (11–13). Mutations in CFTR are also common in otherwise healthy men with congenital bilateral absence of the vas deferens (CBAVD) presenting with infertility (14–16). In addition, the 5-thymidine (5T) variant of the CFTR intron 8 polypyrimidine T-tract length is frequently present in men with CBAVD (17–19). The 5T variant causes less efficient splicing of CFTR exon 9 compared with the more common variants 7T and 9T, resulting in reduced CFTR expression. Thus, it has been suggested that CBAVD may represent a mild form of CF (20).

We have previously reported a high frequency of CFTR mutations in healthy men with CBAVD, diagnosed during investigation for infertility (21). Most of these men had intermediate or elevated sweat chloride levels but normal chest radiographs and spirometry (P Durie, DE Tullis, unpublished data). We hypothesized that these men may have early, subclinical pulmonary disease with inflammation and/or infection, not evident by routine pulmonary function testing or radiologic investigations. Some of the results of these studies have been previously reported in abstracts (22, 23).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Healthy men with CBAVD, previously enrolled in a genotype/phenotype study (21), with intermediate or elevated sweat chloride levels and one or two CFTR gene mutations, were recruited. Healthy men were recruited as control subjects. Smokers and those with current airway infection, allergy to lidocaine, or treatment with antiinflammatory agents were excluded. The study was approved by the St. Michael's Hospital (Toronto, ON, Canada) research ethics board and all subjects gave written informed consent.

Procedures
All subjects had spirometry, a chest radiograph, white blood cell count with differential, and serum alpha-1 antitrypsin.

Before bronchoscopy, midazolam and atropine sulfate were given intravenously for sedation and to minimize secretions. The pharynx was anesthetized with nebulized 2% lidocaine. A flexible bronchoscope was inserted through a mouthpiece and the larynx and tracheobronchial tree were anesthetized with directly instilled 1% lidocaine. The bronchoscope was wedged into the right middle lobe and BAL was performed by instillation of three 50-ml aliquots of prewarmed (37°C) sterile, isotonic saline. The fluid was removed by gentle manual suction, using sterile polypropylene syringes. The procedure was repeated in the left upper lobe.

Aliquots from each lobe were used for quantitative bacterial and viral cultures. After Gram staining, the remainder of the aliquot was homogenized with an equal volume of BAL fluid and Sputagest (Mast Diagnostics, Merseyside, UK). Three 1:10 dilutions were made from the original homogenate and each was plated, using a 0.01-ml loop, on four agar plates (Burkholderia cepacia select agar [Oxoid, Basingstoke, UK], MacConkey agar without crystal violet, Fildes medium [24], and blood agar). All pathogens were identified and enumerated quantitatively, and susceptibility testing was done. Unusual glucose nonfermenting gram-negative bacteria were identified at the Toronto Public Health Laboratory (Toronto, ON, Canada). Burkholderia cepacia strains were typed at the Hospital for Sick Children and at the B. cepacia reference laboratory (25). BAL fluid was inoculated onto monolayers of MK and MRC-5 cells in shell vials to screen for influenza A and B, parainfluenza, respiratory syncytial virus, herpes simplex virus, cytomegalovirus, and rhinovirus.

The remaining BAL fluid was filtered into polypropylene tubes. One milliliter of fluid was removed for cell and differential count, and viability testing. After centrifugation at 250 x g, the supernatant was centrifuged at 12,000 x g for 10 minutes at 4°C and pipetted into microcentrifuge tubes containing phenylmethylsulfonyl fluoride. EDTA was added to a final concentration of 1 mM. The samples were frozen at –70°C until analysis was done in batches. Tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-6, IL-8, and IL-10 levels in BAL fluid were quantified using commercially available ELISA kits (Cytoscreen; Biosource International, Camarillo, CA). Neutrophil elastase (NE) was quantitated with an EnzChek elastase assay kit (Molecular Probes, Eugene, OR).

Effects of BAL Fluid on Neutrophil Elastase Release
Peripheral blood neutrophils, isolated by plasma–Percoll gradients from healthy donors, were incubated with BAL fluid from normal control subjects and from men with CBAVD and release of elastase was quantified (26).

Statistical Analysis
The Mann–Whitney rank-sum test was used for comparison between CBAVD and control subjects and the Wilcoxon signed-rank test was used for comparison between the right middle lobe and left upper lobe. A p value < 0.05 was considered significant (27). The computer software StatView (SAS Institute, Cary, NC) was used for all analyses.

See the online data supplement for more detailed description of the methods.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Demographic and Clinical Data
We performed bronchoscopy on eight men with CBAVD and on four control subjects. Demographic information is summarized in Table 1 . On the basis of elevated sweat chloride values or abnormal nasal chloride conductance, five of eight met the current diagnostic criteria for CF (28) although none had two CF-causing mutations. Parameters used to determine abnormal chloride conductance on nasal potential difference (PD) were the 99% confidence limits for 18 normal control subjects at our center (7.65–22.6 mV). The group with CBAVD (39 ± 5 years; range, 31–45 years) was older than the control group (29 ± 2 years; range, 26–30 years). All patients, with the exception of Patient 8, were healthy with no reported pulmonary symptoms except for a history of mild seasonal allergic rhinitis in one patient. Seven patients had a normal chest radiograph and high-resolution chest computed tomography scan, and no excessive airway secretions or obvious signs of inflammation were noted at the time of bronchoscopy. Patient 8, despite a normal chest radiograph, had evidence of mild bronchiectasis by high-resolution chest computed tomography scan. His FEV1 was at the lower limit of normal and the airways were inflamed, with mucosal edema and purulent secretions noted at bronchoscopy.


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  		TABLE 1. Demographic data for men with congenital bilateral absence of the vas deferens

 
White blood cell count with differential, alpha-1 antitrypsin, and erythrocyte sedimentation rate were normal for all patients with CBAVD and control subjects.

Analysis of Bronchoalveolar Lavage Fluid
The return of the BAL was slightly higher for control subjects (56 ± 10%) as compared with patients with CBAVD (45 ± 9%). The viability of cells recovered by BAL was 98 ± 1% with fewer than 10 epithelial cells per low-power field for all subjects. No significant differences were noted between samples taken from the right middle lobe and left upper lobe for all subjects. With the exception of Patient 8, the BAL total and differential cell counts were within the normal range (Table 2) . Patient 8 had increased levels of total cells, a higher percentage and absolute number of neutrophils, and elevated levels of IL-8, TNF-{alpha}, IL-6, and NE and lower levels of IL-10 in BAL fluid in comparison with other subjects.


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  		TABLE 2. Bronchoalveolar lavage cell count

 
Microbiological Analysis
Potential pathogens were isolated from BAL fluid from six of eight men with CBAVD (Table 3) , but bacterial counts were all less than 105 cfu/ml. Organisms identified included Staphylococcus aureus (n = 1), Burkholderia cepacia (n = 2), Burkholderia cepacia fungorum (n = 1), Burkholderia gladioli (n = 2), Brevundimonas diminuta (n = 1), and Ralstonia pickettii (n = 1). One of the B. cepacia strains was typed as genomovar IV (B. stabilis). BAL fluid from one control subject yielded a light growth of R. pickettii. No viruses were isolated.


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  		TABLE 3. Bronchoalveolar lavage cultures, right middle lobe

 
Cytokine and Chemokine Levels Elevated in Patients with CBAVD
IL-8 and TNF-{alpha} levels were higher for patients with CBAVD than for control subjects, even when Patient 8 was excluded (Mann–Whitney, p < 0.02). However, except for Patient 8, all were within the normal range (Table 4 and Figure 1) . NE levels in BAL fluid were at the lower limits of detection for CBAVD and control subjects: 136 ± 55 and 124 ± 43 ng of protein per milliliter of BAL fluid. Incubation of neutrophils from normal donors with BAL fluid from men with CBAVD and control subjects induced release of 236 ± 117 and 150 ± 66 ng of NE per milliliter, respectively (p = 0.08), with the highest value, 436 ng/ml, for Patient 8.


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  		TABLE 4. Cytokine levels in bronchoalveolar lavage from patients with congenital bilateral absence of the vas deferens and from control subjects

 


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  		Figure 1. IL-8 and TNF-{alpha} in bronchoalveolar lavage fluid supernatant from the right middle lobe (RML) of eight men with congenital bilateral absence of the vas deferens and four healthy control subjects. Data are presented in log scale.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We observed subtle evidence of pulmonary dysfunction in men with CBAVD presenting for evaluation of infertility but without pulmonary symptoms. Staphylococcus aureus or gram-negative bacteria were cultured in BAL fluid from the majority of patients with CBAVD despite no or very mild symptoms suggestive of airway disease. In contrast to patients with classic CF, the BAL fluid from these men with CBAVD revealed evidence of a much more muted inflammatory response. We speculate that the mutations in CFTR present in these men cause more subtle defects in CFTR but still predispose to colonization of the airways with pathogenic bacteria. In at least one of these patients, this was associated with evidence of asymptomatic bronchiectasis as determined by a high-resolution chest computed tomography scan. Whether the remainder of patients, in the face of airway colonization/infection by pathogenic bacteria, will eventually develop morphologic evidence of airways damage, is unknown and will require prolonged follow-up.

Previous studies in infants with classic CF, diagnosed through screening programs, have provided evidence of neutrophilic airway inflammation, potentially in the absence of bacteria, even in infants as young as 6 weeks of age (4). Mutations in CFTR have been hypothesized to result in primary dysfunction of the inflammatory response. In contrast to an earlier study, Armstrong's group concluded that inflammation is a response to current or previous infection with S. aureus and respiratory viruses (5). There was a relationship between infection and IL-8 and percent neutrophils, but only in subjects with bacterial counts exceeding 105 cfu/ml. Both studies included non-CF infant control subjects. More recent studies of slightly older infants with CF have demonstrated the similar finding that there is a close relationship between infection and inflammation (29–31). Dakin and coworkers found pathogens exceeding 105 cfu/ml in the BAL fluid of 8 of 22 infants with CF (32). This was associated with a highly significant increase in neutrophils and IL-8 compared with the 6 of 22 subjects with a lower degree of infection (less than 105 cfu/ml) and the 8 of 22 subjects with no bacteria (less than 10 cfu/ml). However, even patients with lower levels of infection or negative cultures had more neutrophils and IL-8 than did non-CF control infants from other studies. It seems that even minor degrees of infection can stimulate an excessive inflammatory response in people with CF.

Contamination, Colonization, Infection, or Normal Flora?
We chose to use the first 50-ml portion of the BAL fluid for bacterial cultures because our primary purpose was to identify even light growth of possible pathogens. Contamination of the bronchoscope as it traverses the oropharynx can produce misleading microbiological culture results, as shown in one study in which only 1 of 10 volunteers had sterile BAL cultures (33). The likelihood of a false-positive result, that is, bacterial growth originating from upper airway flora, is higher if more than one organism belonging to the normal nasopharyngeal flora is found in low numbers (less than 104 cfu/ml), and if the BAL sample contains more than 1% squamous epithelial cells (4, 34). However, there may be a relationship between pathogenic bacteria present in the oropharynx and subsequent development of infection of the lower airways and lung parenchyma as shown in critically ill patients (35). With specific reference to CF, some studies have shown a high correlation of bacteria species between oropharyngeal and BAL cultures (36, 37). In addition, it has been argued that more than 3% squamous cells present in BAL fluid may be an indicator of bronchial inflammation (38) and squamous metaplasia may represent airway remodeling. Nevertheless, B. diminuta, R. pickettii, B. cepacia, and B. gladioli are not part of the normal oropharyngeal flora (39) and indeed are potentially pathogenic inasmuch as they may cause severe infection in immunocompromised patients and in patients with CF (40). Human pathogenic strains of B. cepacia are not necessarily distinct from environmental strains and their acquisition from an environmental source may be one explanation for their presence in BAL cultures despite no known contact with patients with CF (41).

The taxonomy of these pathogenic gram-negative bacteria is complex and has evolved as the methods for identification have improved. Ralstonia pickettii previously was considered a member of the B. cepacia complex and indeed is still frequently misidentified as B. cepacia (42). It is difficult to differentiate between the B. cepacia genomovars as well as to distinguish the B. cepacia complex from related species (25, 41). The Toronto Adult Cystic Fibrosis Centre has a high prevalence of infection with B. cepacia, but almost exclusively with the highly transmissible, genomovar IIIa, cable pili-positive strain. This is clearly distinct from the isolates recovered from the men with CBAVD and from the control patient.

The presence of potentially pathogenic bacteria from normal volunteers is of concern. In the present study, R. pickettii was recovered from one normal volunteer. It is of interest that this individual had contact with many patients with CF from Toronto and the surrounding area. To our knowledge, there are no studies that have investigated the existence of bacteria in the lower airways of relatives of patients with CF and CF healthcare workers. However, transmission of B. cepacia to non-CF, critically ill patients has been described (43).

In our series, four patients demonstrated bacterial growth of 104 cfu/ml, and more than one organism was identified in two patients. Three of these patients (Patients 3, 5, and 8) met the current criteria for a diagnosis of CF (28) and we conclude that the findings represent early CF airway infection.

Inflammatory Response
BAL cell counts and differential, and IL-8 and TNF-{alpha} levels, were within the normal range of values previously reported in normal nonsmoking subjects (44). However, statistical analysis revealed significant differences between patients with CBAVD and control subjects for IL-8 and TNF-{alpha}, suggesting mild inflammation. In contrast to studies of infants with CF, we did not find the same excessive inflammatory response. Even the highest levels of IL-8 seen were markedly lower than in these other studies. Our small study population precluded analysis of relationship between pathogen colony-forming units per milliliter and level of inflammation. The only patient with obvious bronchitis also had neutrophilic airway inflammation with elevated levels of inflammatory cytokines, and a lower level of IL-10 in concert with growth of B. cepacia and S. aureus.

It is likely that NE and IL-8 play a key role in the self-perpetuating inflammatory process on the CF lung epithelial surface (4, 45–53). Correlation between age and IL-8 levels has been observed, although there were overlapping values between patients with CF and control subjects (46). When NE and anti-NE capacity in the epithelial lining fluid were measured, children and adults with CF had higher levels of NE and lower anti-NE capacity compared with healthy adults, but there were several children as old as 7 years with undetectable NE levels (48). We observed higher NE release after incubation of neutrophils from normal donors with BAL from men with CBAVD than from control subjects. We postulate that this is due to the presence of yet unidentified substances that activate neutrophils to release elastase from primary granules. These substances may be lipid mediators, peptides, or chemokines or cytokines that can induce neutrophil activation and are stable in aqueous solution under the storage conditions (–70°C) used in these experiments.

IL-10 is a regulatory cytokine that decreases inflammatory responses and T cell stimulation and is produced by bronchial epithelial cells. Levels of IL-10 are known to be diminished in CF (54) and low levels were present in Patient 8 in our current study.

Is CBAVD a Form of Mild CF?
According to a consensus conference (28), diagnosis of cystic fibrosis is based on clinical symptoms typical of CF in association with laboratory evidence of CFTR malfunction. The R117H mutation should not be considered a CF-causing mutation unless combined with the 5T variant or evidence of CFTR malfunction demonstrated by sweat test or nasal PD measurements (28). In our study population, three of the four men with infertility who carried the R117H mutation in association with the 7T mutation had independent clinical or laboratory evidence of CFTR malfunction with elevated sweat chlorides and/or abnormal nasal PD. In addition to the T-tract length, it is possible that environmental factors or modifier genes play a role in determining whether patients with R117H mutations presenting with CBAVD eventually will develop multiorgan disease (10). Nontypical CF disease sometimes develops late in life as described in an elderly woman with the {Delta}F508/R117H mutation (unknown T-tract) (55). Nasal PD is listed as one of the diagnostic tests for CF (28), but it has not been standardized as a diagnostic tool. Thus, its role in diagnosing patients with unusual mutations or borderline sweat chloride levels is uncertain (56). In our study, nasal PD measurements were abnormal for three of eight men with CBAVD, four of whom had normal or intermediate sweat chlorides. The combination of bilateral absence of the vas deferens, airway bacteriology, and evidence by nasal PD of malfunctioning CFTR is a strong indicator that some patients with CBAVD may have a mild form of CF. Patients with obvious CF disease have previously been detected in cohorts of infertile men (57, 58). We were able to detect evidence of early airway changes in men with CBAVD who had normal spirometry and chest radiography.

Limitations of the Study
This study involved evaluation of a small number of patients and therefore has several inherent limitations. To validate these results would require repetition of this type of study in a larger population sample. It may prove difficult to recruit men with CBAVD because of fear of being labeled as having CF and reluctance to have bronchoscopy performed. It would also be of interest to repeat the study in a larger group of control subjects including those with and without close contact with patients with CF.

Conclusions
The results of the current study suggest that selected ("mild") mutations in the CF gene carried by men with CBAVD predispose to bacterial airway infection but with a milder inflammatory response than usually seen in classic CF. It is possible that if pulmonary infection and inflammatory injury to the lung progress in these men, a clinical syndrome more compatible with usual CF lung disease will become evident. Only observation of these patients for a prolonged period of time, perhaps decades, will help determine their risk of developing symptomatic pulmonary disease over time. Future studies will also be required to determine the most appropriate tests that will allow the identification of bacterial infection and inflammation as a diagnostic aid in atypical CFTR-related disease. We suggest that these patients should be monitored on a regular basis in an experienced CF center, with attention to infection control measures to prevent person-to-person spread of bacteria.


   Acknowledgments
 
The authors acknowledge Sheelagh Martin (patient recruitment), Dr. Vera Cherepanov (cytokine analyses), Barbara Hooper (assistance at bronchoscopy), Dr. Uma Sajjan (typing of B. cepacia), and Dr. Francis Jamieson (identification of unusual glucose-nonfermenting gram-negative bacteria).


   FOOTNOTES
 
Supported by operating grants from the National Institutes of Health (P50 DK49096-06 SCOR) to P.D., G.P.D., D.E.T., and J.Z., from the Canadian Institutes of Health Research to G.P.D., and from the Canadian Cystic Fibrosis Foundation to P.D. G.P.D. holds the R. Fraser Elliott Chair in Transplantation Research from the Toronto General Hospital of the University Health Network, and a Canada Research Chair in Respiration from the Canadian Institutes of Health Research. M.G. was supported by the Canadian Cystic Fibrosis Foundation, the Swedish Medical Society, the Gothenburg Medical Society, the Swedish Cystic Fibrosis Association, and the Swedish Heart and Lung Foundation.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: M.G. has no declared conflict of interest; Y.M. has no declared conflict of interest; G.P.D. has no declared conflict of interest; R.D. has shares in Bristol Myers Squibb, her spouse has shares in Merck and Co. Inc. and Medco Health and the aggregate total is less than $10,000; P.D. has no declared conflict of interest; A.M.C. has no declared conflict of interest; J.Z. has no declared conflict of interest; D.E.T. has no declared conflict of interest.

Received in original form April 22, 2003; accepted in final form October 6, 2003


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 METHODS
 RESULTS
 DISCUSSION
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