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Published ahead of print on June 26, 2003, doi:10.1164/rccm.200301-005OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 685-691, (2003)
© 2003 American Thoracic Society

Original Article

Bronchoalveolar Lavage Fluid Surfactant Protein-A and Surfactant Protein-D Are Inversely Related to Inflammation in Early Cystic Fibrosis

Terry L. Noah, Paula C. Murphy, Jorien J. Alink, Margaret W. Leigh, William M. Hull, Mildred T. Stahlman and Jeffrey A. Whitsett

Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina; University of Groningen, Groningen, The Netherlands; Children's Hospital Medical Center, Cincinnati, Ohio; and Departments of Pediatrics and Pathology, Vanderbilt University, Nashville, Tennessee

Correspondence and requests for reprints should be addressed to Terry L. Noah, M.D., CB#7220, Chapel Hill, NC 27599–7220. E-mail: terry_noah{at}med.unc.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pulmonary collectins surfactant protein (SP)-A and SP-D play important roles in innate lung defense, enhancing opsonization of microbes and limiting lung inflammatory responses. To quantify relationships among collectins, bacteria, and inflammation in early cystic fibrosis (CF) airway secretions, bronchoalveolar lavage fluids (BALFs) were collected from children undergoing clinically indicated bronchoscopy. Quantitative bacteriology, differential cell counts, and ELISA for SP-A and SP-D were assessed. Significantly increased numbers of neutrophils relative to bacteria were noted in BALF from CF compared with non-CF subjects. Although SP-A levels tended to be lower in CF compared with non-CF, this was only significant in the presence of bacterial infection. Among CF patients, SP-A concentrations in BALF were inversely related to inflammation, bacterial colony-forming units per milliliter, and age. SP-D levels were significantly decreased in CF patients, and SP-D was rarely detectable in the presence of infection. Among CF patients, SP-D correlated inversely with inflammation and bacterial colony-forming units per milliliter, and there was decreased immunostaining of BALF cells for SP-D in CF. Immunohistochemistry of CF autopsy lung sections for SP-A and SP-D confirmed their paucity at sites of infection and inflammation. We conclude that relative collectin deficiency occurs early in CF airways and is inversely related to inflammation in CF airways.

Key Words: surfactant protein A • surfactant protein D • inflammation • cystic fibrosis • neutrophil

Collectins are oligomeric, multivalent proteins sharing distinct collagen-like and calcium-dependent carbohydrate recognition domains. The collectins bind endotoxins and glycoconjugates on the surface of various viral, fungal, and bacterial pathogens (1). Recent studies suggest an important role for these factors in innate lung defense. The collectins surfactant protein-A (SP-A) and surfactant protein-D (SP-D) enhance opsonization of pathogens and modulate inflammatory responses by alveolar macrophages (1–4). Binding of collectins to microbial pathogens and to human phagocytes influences innate and acquired immune responses.

SP-A and SP-D are expressed by epithelial cells lining the respiratory tract. In humans, SP-A and SP-D are expressed by nonciliated respiratory epithelial cells, type II alveolar cells, and subsets of cells in submucosal glands (5). Genetically engineered mice with null mutations for SP-A and SP-D demonstrate poor clearance of bacterial, viral, and fungal pathogens. SP-A (-/-) and SP-D (-/-) mice have shown decreased macrophage uptake and clearance of bacteria, including group B streptococcus, Hemophilus influenzae, Pseudomonas aeruginosa, and Klebsiella pneumoniae (2, 6, 7), as well as several common respiratory viruses. Increased and prolonged lung inflammation was observed after exposure of SP-A and SP-D–null mice to various respiratory pathogens. Deficiency of either collectin leads to increased inflammation and decreased macrophage phagocytosis, and SP-D deficiency enhances oxidant production, whereas SP-A deficiency inhibits it (7). Both collectins modulate LPS-induced cellular responses through interactions with CD14, mediated by interactions at distinct binding sites (8). Thus, SP-A and SP-D play complementary but distinct roles in enhancing clearance of respiratory pathogens, and both limit infection-induced inflammation in the lung.

Cystic fibrosis (CF) is characterized by recurrent or chronic airway infections with bacteria, including H. influenzae, Staphylococcus aureus, and P. aeruginosa. We and others have recently shown that bronchoalveolar lavage fluids (BALFs) from young CF patients contain a high number of neutrophils relative to bacterial quantity when compared with non-CF disease control subjects (9, 10). Some studies have suggested that CF airway epithelial cells may overproduce cytokines, including the neutrophil chemoattractant interluekin-8, as a result of cystic fibrosis transmembrane regulator dysfunction (11); however, this appears to depend on the model system studied (12), raising the possibility that other pathways could contribute to a relative neutrophil excess in CF.

Several recent investigations have quantified SPs in CF airway secretions. Hull and colleagues (13) found that in infants with CF, BALF SP-A levels were normal and increased somewhat with infection. In contrast, subsequent reports in older children and adults with CF consistently found decreased SP-A or SP-D levels in respiratory secretions (14, 15). The presence of proteolytic degradation products of SP-A supports the concept that inflammation may play a role in decreasing collectin function in the CF airway (16). Potential relationships between collectins and inflammation have not been quantitatively explored.

In this study, relationships among collectins, bacteria, and inflammation in early CF airway secretions were assessed. Our results support the concept that relative deficiencies of SP-A and SP-D may develop early in life in these patients. Some of the results of these studies have been reported previously in the form of an abstract (17).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Subjects
BALFs were collected from children with CF and other conditions who were undergoing clinically indicated bronchoscopy. Choice of area to lavage and volume of lavage fluid instilled was at the discretion of the clinician performing the procedure and followed our clinical routine practice. Thus, location of bronchoalveolar lavage typically targeted the most affected area, either on chest radiograph or visually at bronchoscopy. The volume instilled was 1 ml/kg per aliquot x 2–3 (minimum aliquot of 10 ml). Children were excluded if they were using systemic or inhaled antiinflammatory agents within 2 weeks before bronchoscopy. Children with known immunodeficiency were excluded. All children with CF had elevated sweat chloride and/or diagnostic CF genotype. Recent antibiotic use is common before clinically indicated bronchoscopy in CF, and thus, this was not considered an exclusion factor; however, all patients were off antibiotics for at least 48 hours before procedures, as per our clinical routine. Preliminary results suggested that CF BALFs, which were positive for acid-fast bacilli, had a tendency for unusually high SP-A levels, and a recent study reported that mycobacterial infection induces expression of SP-D in A549 cells (18); thus, subjects with acid-fast bacilli–positive BALF were excluded.

Children with tracheostomy are often colonized with bacteria and are susceptible to recurrent bacterial tracheobronchitis (19). The subgroup of patients with tracheostomy was thus analyzed separately from the other non-CF control subjects, as a control population with known susceptibility to bacterial chronic airways infection (i.e., similar in that respect to CF). These children were undergoing clinically indicated bronchoscopy for tracheostomy evaluations. BALF cultures were done in these children at the discretion of the clinician, as surveillance for bacterial pathogens, to guide treatment of recurrent infections. This study was approved by the University of North Carolina School of Medicine's Committee on the Protection of the Rights of Human Subjects.

Initial Processing of BALF
An aliquot of BALF was immediately processed for total cell count excluding red blood cells using a hemocytometer. Cytocentrifuged BALF cells were fixed and stained with modified Wright stain (Hema-3; Fisher Scientific, Pittsburgh, PA) for differential cell counts. An aliquot of unprocessed BALF was frozen at -80°C, and the remainder of BALF was centrifuged at 500 g x 10 minutes to pellet cells. Pelleted cells were fixed for later use in immunostaining assays as described later in this article. The supernatant was frozen at -80°C for later use in ELISA assays as described later here.

BALF Assays
Quantitative analysis of SP-A and SP-D was performed by double-antibody capture ELISA as described previously (20–22). For SP-A, goat polyclonal anti-human SP-A and rabbit anti-goat antibody were used as first and second antibodies, respectively. For SP-D, first and second antibodies were generated in guinea pig and rabbit to mouse SP-D purified from a GM-CSF -/-, SP-A -/- null mutant mouse. These antibodies detect SP-D as a single band of 43 kD by Western blot of BALF from patients with alveolar proteinosis. Microtiter wells were conditioned overnight at 40°C with 0.1 M of NaHCO3 or coated with the first antibody. The wells were then washed twice with wash buffer consisting of 0.01 M of Trs-HCl (pH7.4) and 0.05% Tween and then blocked with assay buffer containing 5mg/ml of bovine serum albumin. The BALF samples and standards were diluted in phosphate-buffered saline (PBS) containing 0.5% Nonidet P-40, added to the wells, and incubated for 1–2 hours at 37°C. Wells were washed three times and then incubated with the appropriate second antibody for 1–2 hours. The plates were then washed again and incubated with peroxidase-conjugated goat anti-rabbit IgG (Calbiochem, La Jolla, CA) for 1 hour at 37°C. Color reaction was developed at room temperature using the orthophenylene diamine substrate system. Each assay plate included a standard curve, generated with the appropriate SP-A or SP-D purified from patients with pulmonary alveolar proteinosis and four serial dilutions of each clinical sample. All samples were measured in quadruplicate using a microtiter plate spectrophotometer at an outside diameter of 490 nm.

A preliminary comparison of results from sonicated BALF versus BALF supernatants showed no significant difference for SP-A or SP-D. Incubation of BALF with EDTA did not increase detectable SP-A or SP-D. Thus, the majority of detectable SP-A and SP-D in BALF using these assays appeared to be free in supernatants. All ELISA data reported in this article are for cell-free BALF supernatants.

Immunohistochemical Staining of BALF Cells and Lung Tissue
Pelleted BALF cells were embedded in paraffin, and 5-µ sections were placed on slides. Sections were deparaffinized in a series of xylene and graded ethanols, rehydrated with Tris-buffered saline (TBS), and then blocked with 20% normal goat serum for 2 hours. Tissue sections were incubated overnight at 4°C with the primary antibody (polyclonal rabbit anti-human SP-A; Chemicon, Temecula, CA) diluted 1/2000 in TBS-triton. The next day sections were washed with TBS. The secondary antibody, Biotin-SP–conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson Immunoresearch Laboratories, West Grove, PA), was applied for 30 minutes at room temperature and diluted 1:2,500 in TBS-triton. Again, sections were washed with TBS. Then avidin:biotinylated enzyme complex alkaline phosphatase (Vector Laboratories, Burlingame, CA) was used at a 1:100 dilution in TBS-triton for 30 minutes at room temperature. After washing with TBS, sections were pretreated with levamisol and diluted 1:100 in 100 mM of Tris-HCl, pH 8.2–8.5. The substrate Vector Red was used to detect the avidin:biotinylated enzyme complex alkaline phosphatase according to the manufacturer's instructions. The sections were counterstained with Mayer's hematoxylin and saturated aqueous Li2CO3. For negative control slides, the primary antibody was replaced by normal rabbit serum, used at the same concentration as the primary antibody. The same protocol was followed when staining for SP-D, using polyclonal rabbit anti-mouse SP-D (Chemicon) as the primary antibody.

The sections were covered with PBS/glycerine 1:1, a nonpermanent aqueous mounting media, and then covered with a glass cover slip. The slides were viewed under oil at x100 on a phase-contrast microscope. Positive staining was observed in macrophages and to a lesser degree in neutrophils. A semiquantitative scoring index, adapted from the method described by Colombo and Hallberg for lipids (23), was used to measure the positivity of staining of cells. Two hundred consecutive nonepithelial cells were assessed in blinded fashion under a grid, along a standardized pattern. Cells without any red staining of the cytoplasm were scored 0. Cells with 0–25% stained were scored 1 (A); 25–50%, 2 (B); 50–75%, 3 (C); and 75–100%, 4 (D) (Figure 1) . The final score was determined by this formula: S = (1 x A) + (2 x B) + (3 x C) + (4 x D).



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  		Figure 1. Bronchoalveolar lavage fluid (BALF) cells immunostained for surfactant protein (SP)-D (A) and assay control (B), illustrating semiquantitative scoring index (see the text for details of the scoring system).

 
Lung tissue was also obtained at autopsy from four patients with CF, age 7 months to 24 years. Sections were stained with hematoxylin and eosin, and immunohistochemistry was performed for SP-A and SP-D using rabbit polyclonal antibodies as described previously here.

Statistical Analysis
Intergroup comparisons for more than two groups were made using one-way analysis of variance after normalization of data by log transformation, with Dunnett post-testing for subgroup comparisons. For two-group comparisons, Student's t test was used. All tests were run on the software program Prism (GraphPad, San Diego, CA). A p < 0.05 was considered significant throughout.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of Study Population
Clinical characteristics of the subjects studied are shown in Table 1 . BALF from 46 CF patients and 31 non-CF patients were obtained from subjects meeting criteria outlined previously here, from January 2001 to February 2002. There was no significant difference in ages between these groups. For CF patients, the indication for bronchoscopy was persistent exacerbation of cough in children unable to expectorate sputum for culture. For non-CF control subjects, there were a variety of indications, with symptoms of upper airway obstruction being the most common. BALF were obtained from 17 patients with chronic tracheostomy, a group with increased susceptibility for recurrent bacterial airway infections. This group was slightly older than the non-CF control subjects (p = 0.057), but ages were not significantly different from the CF group.


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  		TABLE 1. Clinical characteristics of study subjects

 
The volume of lavage fluid instilled was greater in CF subjects (27 ± 2 ml) than in either non-CF control subjects (22 ± 1 ml) or tracheostomy subjects (22 ± 2 ml), likely because of the (nonsignificant) tendency toward older ages in the CF group (Table 1). The proportion of instilled fluid returned as BALF was the same in CF (46 ± 2%) and non-CF control subjects (48 ± 2%) and was slightly lower in the tracheostomy group (41 ± 3%).

Relationship of Bacteria to Neutrophils in BALF
Among children with CF, 25 were infected (more than 5 x 104 bacterial cfu/ml) with the following pathogens: S. aureus (n = 13), Stenotrophomonas maltophilia (n = 8), P. aeruginosa (n = 6), H. influenzae (n = 6), Moraxella catarrhalis (n = 3), and Burkholderia cepacia (n = 2). Among non-CF control subjects, 12 were infected with the following pathogens: M. catarrhalis (n = 9), H. influenzae (n = 7), S. pneumoniae (n = 3), and P. aeruginosa (n = 1). Multiple pathogens were isolated from several subjects in each group. Total bacterial pathogens in eight of the tracheotomized patients were more than 5 x 104 cfu/ml total bacterial pathogens isolated from BALF. The pathogens isolated were H. influenzae (n = 5), P. aeruginosa (n = 5), M. catarrhalis (n = 2), and S. aureus (n = 1); again, several BALF contained multiple pathogens.

There was a significantly positive relationship between neutrophils per milliliter BALF and bacterial cfu/ml BALF for both CF and non-CF samples. Although the slopes of linear regression lines for neutrophils versus bacteria did not differ significantly between these groups, the line for CF was significantly elevated compared with non-CF (p < 0.001), as shown in Figure 2 . Thus, for a given quantity of bacteria, there were significantly greater numbers of neutrophils for CF patients. For the tracheostomy group, there were fewer data and no statistically significant trend relative to either the CF or non-CF control groups (data not shown).



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  		Figure 2. Neutrophils as a function of bacterial cfu/ml BALF, for patients with cystic fibrosis (CF) (closed circles; n = 44) and those without CF (open circles; n = 29). The solid line indicates linear regression for CF and dashed line for non-CF. The y-intercept for CF is significantly elevated compared with that of non-CF (p = 0.001).

 
SP-A and SP-D in BALF
For uninfected subjects, SP-A did not differ among CF, non-CF control subjects, and tracheostomy patients (Figure 3) . For infected subjects, there was a significant decrease for CF compared with non-CF control subjects but no significant difference for children with tracheostomy (Figure 3). Among infected CF subjects, BALF SP-A did not differ between P. aeruginosa–negative and P. aeruginosa–positive subjects (p = 0.69). There was a tendency for decreased SP-D among uninfected CF children (Figure 4) , but this was not statistically significant. However, SP-D was usually undetectable in CF BALF in the presence of infection (Figure 4). Among infected CF subjects, BALF SP-D did not differ between P. aeruginosa–negative and P. aeruginosa–positive subjects. Children with tracheostomy also had significantly decreased SP-D compared with non-CF control subjects in the presence of infection (Figure 4), although levels were not as consistently undetectable as in the infected CF group.



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  		Figure 3. Concentration of SP-A in BALF from patients without CF (N; open bars), those with CF (CF; solid bars), and children with tracheostomy (T; hatched bars). Bars represent mean ± SEM. Data are also broken down into those from infected (+) (5 x 104 or more bacterial cfu/ml BALF) and uninfected (-) (less than 5 x 104 bacterial cfu/ml BALF) patients. *p < 0.05 versus non-CF (N).

 


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  		Figure 4. Concentration of SP-D in BALF from patients without CF (N; open bars), those with CF (CF; solid bars), and children with tracheostomy (T; hatched bars). Bars represent mean ± SEM. Data are also broken into those from infected (+) (5 x 104 or more bacterial cfu/ml BALF) and uninfected (-) (less than 5 x 104 bacterial cfu/ml BALF) patients. *p < 0.05 versus non-CF (N).

 
SP-A and SP-D in Immunostained BALF Cells
Variably positive immunostaining for SP-A was found in BALF cells (macrophages and neutrophils) from both CF and control subjects. In general, staining of neutrophils did not appear as intense as that in macrophages. Using the semiquantitative method described previously here, there were no significant differences in intensity of staining between CF and non-CF samples (data not shown). As for SP-A, more intense staining for SP-D was generally observed in macrophages than in neutrophils. However, there was less staining of BALF cells for SP-D from CF versus non-CF samples, both in the presence and absence of infection (Figure 5) .



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  		Figure 5. Immunostaining index for SP-D in BALF cells from patients without CF (N; open bars), those with CF (CF; solid bars). Bars represent mean ± SEM. Data are also broken down into those from infected (+) (5 x 104 or more bacterial cfu/ml BALF) and uninfected (-) (less than 5 x 104 bacterial cfu/ml BALF) patients. *p < 0.05 versus non-CF (N).

 
Effects of Inflammation, Bacterial Quantity, and Age on SP-A and SP-D
An increased proportion or number of neutrophils appeared to be associated with a decreased quantity of collectins in BALF of children with CF. BALF with greater than 50% neutrophils on differential cell counts or more than 5 x 105 neutrophils/ml had significantly lower SP-A (Figures 6A and 6C) , an effect not seen for non-CF control subjects. A high percentage of neutrophils was associated with lower SP-D levels in BALF for both CF and non-CF subjects (Figure 6B), whereas high neutrophil counts did not associate with further decrease in low SP-D among children with CF (Figure 6D).



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  		Figure 6. Association of neutrophilic inflammation with SP levels in BALF from patients without CF (N; open bars), those with CF (CF; solid bars). (A) SP-A levels in BALF with low (0–50%) or high (51–100%) fraction of neutrophils; (B) SP-D levels in BALF with low (0–50%) or high (51–100%) fraction of neutrophils; (C) SP-A levels in BALF with low (0–500 x 103) or high (more than 500 x 103) neutrophils/ml BALF; (D) SP-D levels in BALF with low (0–500 x 103) or high (more than 500 x 103) neutrophils/ml BALF. Bars represent mean ± SEM. *p < 0.05 versus "low."

 
For CF patients, both high quantity of bacteria (more than 5 x 105 cfu/ml BALF) and older age (more than 48 months) were associated with significantly decreased SP-A levels (p = 0.046 and 0.01, respectively). SP-D was very significantly decreased (p < 0.01) in the presence of higher bacterial quantity, but age was not a significant correlate. In tracheostomy patients, for whom fewer data were available, there was no statistically significant impact of percentage of neutrophils, neutrophils/ml BALF, bacterial quantity, or age on BALF levels of either of the SPs (data not shown).

When the SP-A and SP-D immunostaining indices in BALF cells were analyzed in relationship to the same factors, a similar pattern was found. A high percentage of neutrophils was the only factor associated with decreased SP-A staining of BALF cells and only in the CF group (p = 0.047). In children with CF, a high percentage of neutrophils (p = 0.005), high neutrophils/ml BALF (p = 0.002), high bacterial cfu/ml BALF (p = 0.004), and older age (p = 0.009) were all strongly associated with decreased SP-D staining of BALF cells. In non-CF control subjects, only high neutrophils/ml BALF was significantly associated with decreased SP-D (p = 0.048).

Immunostaining for SP-A and SP-D in End-stage CF
Lung tissue from a CF patient with end-stage lung disease was immunohistochemically stained for SP-A and SP-D (Figure 7) . In areas of the lung with severe inflammation involving macrophages and neutrophils, there was little staining for either SP-A or SP-D (Figures 7A and 7B). However, areas with lesser inflammation showed positive cytoplasmic staining of alveolar epithelium for SP-A (Figures 7C and 7E) and positive staining for SP-D along apical aspects of alveolar epithelium (Figures 7D and 7F). Findings were similar in lung tissue from four patients, ages 7 months to 24 years, who were available for study.



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  		Figure 7. Immunohistochemical analysis of SP-A and SP-D expression in surgically resected, end-stage CF lung. Paraffin sections were immunostained for SP-A (A, C, and E) or SP-D (B, D, and F) using an immunoperoxidase detection system and rabbit polyclonal antibodies generated to isolated, deglycosylated human SP-A or mouse SP-D. Little to no staining for either protein was detected in inflamed alveolar regions (A and B; macrophages at arrows). Detectable levels of both SP-A (C and E) and SP-D (D and F) were present in alveolar epithelium in noninflamed regions. SP-A staining in alveolar epithelium was cytoplasmic, whereas SP-D staining was strongest along the apical aspect of these cells. Findings are representative of four autopsy specimens from CF patients who were 7 months to 24 years old.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have found decreased concentrations of collectins in BALF from young CF patients, particularly in relationship to bacterial infection and inflammation. BALF SP-A was decreased only in the presence of bacterial counts greater than 5 x 104 cfu/ml BALF and decreased with age in CF patients. In contrast, SP-D was decreased in all CF patients as a group and was virtually undetectable in the presence of bacterial infection, regardless of age. Our findings are thus consistent with previously published data from patients with CF demonstrating decreased SP-A and SP-D in airway secretions (14–16), but perhaps normal SP-A levels at the earliest stages of disease (13).

Mechanisms that may account for these data include (1) increased degradation of collectins related to persistent inflammation, (2) increased consumption of SP-A and SP-D related to phagocytosis in the presence of chronic infection, (3) decreased collectin production by respiratory epithelial cells, or (4) decreased contribution to SP-A and SP-D pools related to obstruction of bronchioles or submucosal gland ducts. The relationship of our observations to each of these potential mechanisms is discussed in the next paragraphs.

Our CF study population manifested "excessive" neutrophil response to bacteria (Figure 2), as reported in previous studies (9, 10). A significant association between low SP-A and SP-D concentrations and high proportion or numbers of neutrophils in BALF was observed, suggesting that neutrophilic inflammation may be either a cause or effect of collectin deficiency. A similar pattern was found when staining indices for SP-A and SP-D were analyzed in relation to inflammation. In a murine model of asthma, allergen-induced bronchial inflammation induced transient deficiencies of both SP-A and SP-D (24). BALF SP-A was decreased in patients with lung injury and inflammation, such as ARDS (25) and gastroesophageal reflux and aspiration (26). von Bredow and colleagues (16) reported an increase in proteolysis of SP-A in CF BALF, suggesting that proteases released by macrophages or activated neutrophils may contribute to SP-A deficiency. These authors suggested that it is possible that measurement of immunoreactive SP-A may thus overestimate functional SP-A in CF airways.

Taken together, these studies suggest that inflammation may contribute to collectin deficiency in the CF lung. Our observation of low or absent immunostaining for SP-A and SP-D in markedly inflamed areas of end-stage CF lung (Figure 7) is consistent with this hypothesis. A higher proportion of neutrophils was associated with low SP-D in both CF patients and non-CF control subjects (Figure 6). However, there appeared to be less correlation between SP-A and inflammation in the non-CF control subjects, suggesting that the shorter term presence of bacteria and inflammation are not sufficient to result in decreased SP-A in the airways. We speculate that the persistence of bacteria over long periods, with time-dependent exhaustion of factors such as protease inhibitors in the airway environment, may be necessary to produce the inverse relationships between collectins and neutrophil-dominated inflammation we measured in CF patients.

SP-A and SP-D promote phagocytosis and clearance of bacteria by macrophages and neutrophils by enhancing opsonization, resulting in uptake of the bacterial/collectin complex (1–4). Herbein and Wright (27) reported a marked increase in association of SP-D with neutrophils 1 hour after treatment of rats with LPS. Thus, rapid uptake by phagocytes in the presence of large numbers of bacteria represents a potential mechanism for decreased collectins in airway secretions. The inverse relationship between SP-D and bacterial cfu/ml BALF that we observed is consistent with this concept (Figure 3D). Although staining for immunoreactive proteins is likely to reflect a complex equilibrium between rates of uptake and intracellular degradation, it might be predicted that immunostaining of BALF phagocytes for SP-A and SP-D would increase under these circumstances. However, we observed no overall change in CF BALF cell immunostaining for SP-A compared with control subjects and a significant decrease in staining of BALF cells for SP-D among children with CF (Figure 7). These results paralleled the trends that we observed for collectin levels in BALF supernatants and suggest diminished production or increased degradation of collectins rather than increased uptake by phagocytes.

In end-stage CF lung, we noted decreased immunostaining of alveolar epithelium and little staining in macrophages for both SP-A and SP-D in inflamed regions, whereas positive staining for both proteins was detected in noninflamed or regenerating alveolar epithelium (Figure 7). These findings support the association of inflammation with reduction in SP-A and SP-D. Likewise, decreased SP-A mRNA was observed in regions of severe infection and inflammation but was readily detectable by in situ hybridization in regions without inflammation (data not shown), suggesting focally decreased expression may contribute to the reduced SP levels.

If relative collectin deficiency is caused by enhanced proteolytic degradation or decreased production mediated by chronic infection and inflammation, similar changes in SP-A and SP-D might be expected in non-CF patients with chronic airways infection. Children with tracheostomy often harbor bacteria, including P. aeruginosa (19), which we found in significant quantities in BALF from five of eight infected tracheotomized children. In these non-CF children, we found that SP-D (but not SP-A) levels in BALF were decreased, but only in association with bacterial infection. The ratios of BALF neutrophils to bacteria were similar to those in CF for some but not all tracheotomized children (data not shown). We speculate that in this group of patients, infection/inflammation may only intermittently reach levels of chronicity or intensity required to cause collectin deficiency. Further investigation of other conditions involving bronchiectasis, such as primary ciliary dyskinesia, will be required to define relationships among collectin concentrations, inflammation, and infection in non-CF patients.

Our data suggest that SP-D may be more sensitive than SP-A to the effects of chronic inflammation or infection. For both CF patients and tracheotomized patients, SP-D was more significantly decreased in the face of infection than SP-A. The reason for this discrepancy cannot be determined from our data but could be related to relative susceptibility to the effects of chronic inflammation on synthesis, proteolytic degradation, or both. Because SP-A and SP-D do not have identical effects on inflammatory processes, a difference in their availability could have functional consequences in the airway. For example, LeVine and colleagues (7) found that bacterial killing by alveolar macrophages in SP-D (-/-) mice was associated with increased oxidant production, whereas in SP-A (-/-) mice, oxidant production was decreased. SP-D deficiency might therefore enhance the proinflammatory environment in the CF lung.

In summary, our data support the concept that chronic, infection-induced inflammation is linked to marked deficiency of SP-D and age-related decline of SP-A in relatively young children with CF. Based on these results and on a growing body of clinical research and work in experimental and animal models, it may be hypothesized that chronic airway infection in CF elicits persistent inflammation, which in turn increases degradation of collectins and perhaps inhibits production of collectins by respiratory epithelial cells (28). The resulting collectin deficiency may exacerbate neutrophil-dominated inflammation by inhibiting clearance of bacteria via macrophage and neutrophil phagocytosis while increasing production of oxidants (7). Thus, it is possible that the levels of collectins in the CF airway are insufficient for suppression of inflammation and bacterial endotoxin clearance, ultimately leading to bronchiectasis. Correction of the observed collectin deficiency in the airways may represent a reasonable therapeutic strategy for inhibiting pulmonary inflammation and infection in the CF lung.


   Acknowledgments
 
The authors thank Dr. Mary E. Gray and Dr. Susan Wert of Cincinnati Children's Medical Center for consultation regarding immunohistochemistry. They also thank Ms. Luisa Brighton, Ms. Jackie Kylander, and Ms. Kim Burns for technical assistance.


   FOOTNOTES
 
Supported by the Cystic Fibrosis Foundation (NOAH00Z0) and the University of North Carolina Center for Environmental Medicine, Asthma and Lung Biology (US EPA CR817643; Dr. Philip A. Bromberg, Director).

Conflict of Interest Statement: T.L.N. has no declared conflict of interest; P.C.M. has no declared conflict of interest; J.J.A. has no declared conflict of interest; M.W.L. has no declared conflict of interest; W.M.H. has no declared conflict of interest; M.T.S. has no declared conflict of interest; J.A.W. has no declared conflict of interest.

Received in original form January 2, 2003; accepted in final form June 22, 2003


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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