Decreased Expression of Intelectin 1 in the Human Airway Epithelium of Smokers Compared to Nonsmokers¹

Cigarette smoking is a major risk factor for respiratory tract infections, with both active and passive smoke exposure increasing the risk of infection (1, 2, 3, 4). The mechanism of this enhanced susceptibility is multifactorial and includes alteration in structural and immune defenses (2). Although most attention has been placed on the alteration of cellular and humoral immune responses in the respiratory tract to cigarette smoking, respiratory tract secretions also contain a large number of antimicrobial molecules participating in the innate immune response (5). Important components of these antimicrobial molecules are the lectins, proteins on cell surfaces that act as phagocytic receptors and play a role in the recognition of specific bacterial cell wall components (6, 7, 8, 9).

With this background, we used microarray analysis to screen the expression of 72 known lectins in the large and small airway epithelium of healthy nonsmokers, healthy smokers, smokers with lone emphysema with normal spirometry, and smokers with chronic obstructive pulmonary disease (COPD).³ The microarray screen identified a unique smoking-associated down-regulation of intelectin 1, a recently described 34-kDa lectin thought to play a protective role in the innate immune response and in mucosal defense (10, 11, 12).

Miroarray assessment of relative mRNA levels of large and small airway epithelium demonstrated a marked down-regulation of expression of intelectin 1 associated with smoking, and this observation was confirmed by TaqMan RT-PCR. Similar to the intestine, the airway epithelial expression of intelectin 1 was observed in secretory cells with qualitatively decreased expression in smokers, as confirmed by Western analysis that demonstrated reduced levels of intelectin 1 in airway epithelium of healthy smokers compared with nonsmokers. Decreased expression of intelectin 1 was also observed in the small airway epithelium of smokers with lone emphysema and normal spirometry and smokers with established COPD. In the context that there is a heightened susceptibility to infections associated with cigarette smoking, the finding of decreased expression of this defense molecule in the airway epithelium of smokers may suggest a role for this lectin contributing to defenses against respiratory tract infections.

Healthy nonsmokers, healthy chronic smokers, smokers with lone emphysema with normal spirometry, and smokers with established COPD were recruited as study volunteers using advertisements in local print media and assistance from the Division of Pulmonary and Critical Care Medicine outpatient clinic (Weill Cornell Medical College, New York, NY). The study population was evaluated under the auspices of the Weill Cornell National Institutes of Health General Clinical Research Center and approved by the Weill Cornell Medical College Institutional Review Board. Written informed consent was obtained from each volunteer before enrollment in the study. Individuals were determined to be phenotypically normal on the basis of clinical history and physical examination, routine blood screening tests, urinalysis, chest x-ray, electrocardiogram, and pulmonary function testing. Current smoking status was confirmed by history, venous carboxyhemoglobin levels, and urinalysis for levels of nicotine and its derivative cotinine. Smokers with established COPD were defined according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (13). Smokers with “lone emphysema with normal spirometry” were defined as those not fulfilling the GOLD criteria for COPD, with normal forced expiratory volume in 1 s (FEV₁), forced expiratory volume (FVC), FEV₁/FVC and total lung capacity, but with an abnormally low diffusion capacity and evidence of emphysema on chest computed tomography scans. All individuals were asked not to smoke for at least 12 h before bronchoscopy to exclude the acute effects of smoking on airway epithelial gene expression.

Epithelial cells from the large and small airways were collected using flexible bronchoscopy. Smokers were asked not to smoke the evening before the procedure. After achieving mild sedation and anesthesia of the vocal cords, a flexible bronchoscope (EB-1530T3; Pentax) was advanced to the desired bronchus. Large airway epithelial samples were collected by gentle brushing of 3rd to 4th order bronchi and small airway samples were collected from 10th to 12th order bronchi using methods previously described (14). Briefly, a 2-mm-diameter brush was advanced 7–10 cm distally from the 3rd order bronchus under fluoroscopic guidance. The distal end was wedged in a bronchus of similar size in the right lower lobe, and cells were collected by gently brushing this area. The large and small airway epithelial cells were subsequently collected separately in 5 ml of LHC8 medium (Invitrogen). An aliquot of this was used for cytology and the differential cell count and the remainder was processed immediately for RNA extraction. Total cell counts were obtained using a hemocytometer while differential cell counts were determined on sedimented cells prepared by centrifugation (Cytospin 11; Shandon Instruments) and stained with Diff-Quik (Baxter Healthcare).

Analyses were performed using Affymetrix microarray HG-U133 Plus 2.0 (54,675 probe sets) and associated protocols. Total RNA was extracted from epithelial cells using TRIzol (Invitrogen) followed by RNeasy (Qiagen) to remove residual DNA. This process yielded 2–4 μg of RNA per 10⁶ cells. An aliquot of each RNA sample was run on an Agilent Bioanalyzer (Agilent Technologies) to visualize and quantify the degree of RNA integrity. The concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). Three quality control criteria were used for an RNA sample to be accepted for further processing: 1) A₂₆₀/A₂₈₀ ratio between 1.7 and 2.3; 2) concentration within the range of 0.2 to 6 μg/ml; and 3) Agilent electropherogram displaying two distinct peaks corresponding to the 28S and 18S ribosomal RNA bands at a ratio of 28S/18S of >0.5 with minimal or no degradation. Double-stranded cDNA was synthesized from 3 μg of total RNA using the GeneChip one-cycle cDNA synthesis kit followed by cleanup with GeneChip sample cleanup module, in vitro transcription (IVT) reaction using the GeneChip IVT labeling kit, and cleanup and quantification of the biotin-labeled cRNA yield by spectrophotometric analysis. All kits were from Affymetrix. Hybridizations to test chips and to microarrays were performed according to Affymetrix protocols and microarrays were processed by the Affymetrix fluidics station and scanned with the Affymetrix GeneChip Scanner 3000 7G. To maintain quality, only samples hybridized to test chips with a 3′ to 5′ ratio of <3 were deemed satisfactory.

Captured images were analyzed using Microarray Suite version 5.0 (MAS 5.0) algorithm (Affymetrix) as previously described (15, 16, 17). The data were normalized using GeneSpring version 6.2 software (Agilent Technologies) as follows: 1) per array, by dividing raw data by the 50th percentile of all measurements; and 2) per gene, by dividing the raw data by the median expression level for all the genes across all arrays in a data set.

TaqMan real-time RT-PCR was performed on RNA samples from the large airways of 12 healthy nonsmokers and 17 healthy smokers. TaqMan real-time RT-PCR was also performed on small airway samples of 12 healthy nonsmokers and 11 healthy smokers, six smokers with lone emphysema with normal spirometry, and 10 smokers with established COPD that had been used for HG-U133 Plus 2.0 microanalyses. First, cDNA was synthesized from 2 μg of RNA in a 100-μl reaction volume using the TaqMan reverse transcriptase reaction kit (Applied Biosystems) with random hexamers as primers. Dilutions of 1:10 and 1:100 were made from each sample, and triplicate wells were run for each dilution. TaqMan PCRs were conducted using premade kits from Applied Biosystems and 2 μl of cDNA was used in each 25-μl reaction volume. The endogenous control was 18S ribosomal RNA and relative expression levels were determined using the ΔΔCt cycle threshold method (Applied Biosystems). The average relative expression level for nonsmokers (i.e., average ΔCt) was used as the calibrator. The rRNA probe was labeled with VIC and the probe for intelectin 1 was labeled with 6-carboxyfluorescein (FAM). The PCRs were run in an Applied Biosystems 7500 Sequence Detection System.

To determine the airway epithelial localization of intelectin 1 expression, biopsies were obtained by flexible bronchoscopy from the large airway epithelium of 10 healthy nonsmokers and 10 healthy smokers (18). For technical reasons it was not possible to obtain biopsies of small airway epithelium. Immunohistochemistry was conducted on paraffin-embedded endobronchial biopsies from large airways of nonsmokers and healthy smokers obtained by flexible bronchoscopy. Sections were deparaffinized and dehydrated through a series of xylenes and alcohol. To enhance staining, an Ag retrieval step was conducted by boiling the sections at 100°C for 20 min in citrate buffer solution (LabVision) followed by cooling at 23°C, for 20 min. Endogenous peroxidase activity was quenched using 0.3% H₂O₂ and blocking was performed with normal goat serum to reduce background staining. Samples were incubated with the primary rabbit polyclonal anti-intelectin 1 Ab 94-145 (1 μg/μl at 1/1000 dilution; Phoenix Pharmaceuticals) for 12 h at 4°C. Blocking with intelectin peptide 94-145 (Phoenix Pharmaceuticals) was used as a control. Vectastain Elite ABC kit (Vector Laboratories) and 3-amino-9-ethyl carbazole substrate kit (Vector Laboratories) were used to detect Ab binding, and the sections were counterstained with hematoxylin (Sigma-Aldrich) and mounted using GVA mounting medium (Zymed Laboratories). Bright field microscopy was performed using a Nikon Microphot microscope, and images were captured with an Olympus DP70 charge-coupled device camera.

Western analysis was used to quantitatively assess intelectin 1 protein expression in large airway epithelium from healthy nonsmokers and healthy smokers. Brushed large airway epithelial cells were obtained as described. Initially, the cells were centrifuged at 600 × g for 5 min at 4°C. The whole cells were lysed with red cell lysis buffer (CellLytic mammalian tissue lysis/extraction reagent; Sigma-Aldrich) followed by whole cell lysis buffer (ammonium chloride potassium (ACK) lysing buffer; Invitrogen), and protease inhibitor (Sigma-Aldrich) was added to the sample. The sample was centrifuged at 10,000 × g and the protein-containing supernatant was collected. The protein concentrations were assessed using a bicinchoninic acid (BCA) protein concentration kit (Pierce). Equal concentrations of protein (20 μg) mixed with SDS sample loading buffer (Bio-Rad) and reducing agent were loaded on Tris-glycine gels (Bio-Rad). Protein electrophoresis was conducted at 100 volts for 2 h at 23°C. Sample proteins were transferred (25 volts for 1 h at 4°C) to a 0.45-μm-thick polyvinylidene difluoride membrane (Invitrogen) using a Powerpack 300 power source (Bio-Rad) and Tris-glycine transfer buffer (Bio-Rad). After transfer, the membranes were blocked with 5% milk in PBS for 1 h at 23°C. The membranes were incubated with primary rabbit polyclonal anti-intelectin 1 Ab 94-145 and 163-199 (Phoenix Pharmaceuticals), as this was shown to provide greater signal intensity than using 1 Ab alone, at 1/1000 dilution for 2 h at 4°C. Recombinant intelectin protein (Phoenix Pharmaceuticals) was used as a positive control. Detection was performed using HRP-conjugated anti-rabbit Ab (1/2000 dilution; Santa Cruz Biotechnology) and the ECL reagent system using Hyperfilm ECL (GE Healthcare). To assess the Western analyses quantitatively the film was digitally imaged, maintaining exposure within the linear range of detection. The contrast was inverted, the pixel intensity of each band was determined, and the background pixel intensity for a negative area of the film of identical size was subtracted using MetaMorph image analysis software (Universal Imaging). The membrane was subsequently stripped and reincubated with HRP-conjugated anti-β-actin Ab (Santa Cruz Biotechnology) as a control for equal protein concentration.

HG-U133 Plus 2.0 microarrays were analyzed using GeneSpring software. Average expression values for intelectin 1 in large and small airway samples (HG-U133 Plus 2.0) were calculated from normalized expression levels for nonsmokers, healthy smokers, and smokers with lone emphysema with normal spirometry and those with established COPD. Statistical comparisons for microarray data were calculated using GeneSpring software and an associated two-tailed Student’s t test. Statistical comparisons for categorical data were achieved using a χ² test. All other statistical comparisons were calculated using a two-tailed (Welsh) t test.

All data has been deposited in the Gene Expression Omnibus (GEO) site (www.ncbi.nlm.nih.gov/geo) curated by the National Center for Bioinformatics (NCBI). Accession number for the data is GSE10006.

Large airway samples from nine healthy nonsmokers and 20 healthy smokers and small airway samples from a total of 60 individuals, including 13 healthy nonsmokers, 20 healthy smokers, 13 smokers with lone emphysema with normal spirometry, and 14 smokers with established COPD were analyzed with Affymetrix HG-U133 Plus 2.0 microarray (Table I). All healthy individuals had no significant prior medical history, including no history suggestive of asthma and a normal general physical examination. There were no differences between groups with regard to ancestral background (p > 0.05). For the large airways, there was a gender difference (p < 0.03) but no age difference (p > 0.4) between the nonsmoker and smoker groups. For the small airways, there were no differences with respect to gender (p > 0.5, among any of the groups), however there were age differences between the nonsmoker and the lone emphysema with normal spirometry and established COPD groups (p < 0.01) and between the smokers with lone emphysema with normal spirometry and the established COPD groups (p < 0.03). All individuals were HIV negative, with blood and urine parameters within normal ranges (p > 0.05 for all comparisons). Smokers had an average smoking history of 37 ± 3 packs per year, and urine nicotine and cotinine and venous blood carboxyhemoglobin levels confirmed the current smoking status of these individuals. Pulmonary function testing revealed normal lung function in healthy nonsmokers and healthy smokers (Table II). Smokers with lone emphysema with normal spirometry had no evidence of airflow obstruction but had an isolated decrease in diffusing capacity for carbon monoxide on pulmonary function tests together with evidence of emphysema on computed tomography scans of the chest, while those smokers with established COPD (GOLD guidelines I–III, mild to severe) had airflow obstruction values that met the criteria for COPD (13). Data from some of these subjects has already been reported in previous studies (14).

Airway epithelial cells were obtained by fiberoptic bronchoscopy and brushing of large (3rd to 4th order) and small (10th to 12th order) airways. The number of cells recovered ranged from 4.9 to 7.3 × 10⁶ (Table I). In all cases, ≥96% of cells recovered were epithelial cells. The various categories of airway epithelial cells were as expected from the large and small airways (14, 16, 19).

Of the 72 lectin family genes surveyed using the Affymetrix HG-U133 Plus 2.0 array and the criteria for the Affymetrix detection call of “present” in ≥50% of either healthy nonsmokers or healthy smokers, there were 22 lectin genes expressed in large airway epithelium of healthy nonsmokers and 24 lectin genes expressed in the large airway epithelium of healthy smokers (Table III; see supplemental data⁴ table I for all of the expression data for the large airway epithelium of healthy nonsmokers and healthy smokers for the 72 lectin family genes for which there are probe sets). In the small airway epithelium there were a total of 24 lectin family genes expressed in healthy nonsmokers and in healthy smokers. (Table IV; see supplemental data table II for all of the expression data for the small airway epithelium of healthy nonsmokers and healthy smokers for the 72 lectin family genes for which there are probe sets). Of the 22 lectin family genes expressed in the large airway epithelium of healthy nonsmokers, 21 of these were also expressed (using the same Affymetrix “present” call of ≥50%) in the small airway epithelium of healthy nonsmokers (along with three other lectin genes not expressed in the large airway epithelium), and 23 of the lectin genes expressed in the large airway epithelium of healthy smokers were also expressed in the small airway epithelium of healthy smokers along with one other lectin gene. There were four lectin genes expressed in the large airway epithelium of healthy smokers that were not expressed in the large airway epithelium of healthy nonsmokers, namely C-type lectin 10A, C-type lectin 4D, lectin galactoside-binding soluble 4, and selectin ligand interactor cytoplasmic 1. In the small airway epithelium there were two other lectin genes (C-type lectin 12A and killer cell lectin-like receptor subfamily K, member 1) expressed in the small airway epithelium of healthy smokers that were not expressed in the small airway epithelium of healthy nonsmokers.

Of all the lectin family genes expressed in the large and small airways, only intelectin 1 was significantly changed >2-fold in healthy smokers compared with healthy nonsmokers (Tables III and IV). As assessed using the microarrays, intelectin 1 was significantly down-regulated in healthy smokers compared with healthy nonsmokers in large airway epithelium (3.8-fold decrease; p < 0.01) and healthy smokers compared with healthy nonsmokers in the small airway epithelium (5.6-fold decrease; p < 0.01; Fig. 1).

To confirm the results obtained from the microarray screen, TaqMan RT-PCR was conducted on RNA samples from large airways of 12 healthy nonsmokers and 17 healthy smokers and on RNA samples from small airways of 12 healthy nonsmokers and 11 healthy smokers. The TaqMan data confirmed that intelectin 1 was significantly down-regulated in large airways of healthy smokers (4.8-fold decrease; p < 0.05) compared with healthy nonsmokers and in the small airways of healthy smokers (14.7-fold decrease; p < 0.02) compared with healthy nonsmokers (Fig. 2).

The expression of intelectin 1 at the protein level was assessed with immunohistochemistry on endobronchial biopsy specimens from the large airways of healthy nonsmokers and healthy smokers. Positive staining for intelectin 1 was observed in secretory cells in nonsmokers only (Fig. 3). There was no positive staining for intelectin 1 observed in healthy smokers, consistent with the concept that the expression of intelectin 1 in airway epithelium is in secretory cells and that, qualitatively, there was less intelectin 1 protein expression in the large airways of healthy smokers compared with healthy nonsmokers. Because intelectin 1 was expressed in secretory cells, morphological analysis of cytopreparations revealed no difference in the number of secretory cells present in healthy nonsmokers and healthy smokers in large airway epithelium (p > 0.2) and small airway epithelium (p > 0.9) to account for the observed decrease in expression of intelectin 1 in healthy smokers. In addition, there was increased mRNA expression of MUC5AC in smokers compared with nonsmokers (p < 0.05; data not shown), indicating that while there may be increased secretory activity of existing goblet cells, there remains decreased expression of intelectin 1.

Western analysis conducted on large airway samples from healthy nonsmokers and healthy smokers was used to quantitatively assess intelectin 1 expression. Using multiple brushes from the airway, ∼20 μg total protein was obtained from each individual. Western analysis of this protein demonstrated decreased intelectin 1 protein expression in healthy smokers compared with healthy nonsmokers. Quantitative analysis revealed decreased intelectin 1 protein expression in healthy smokers compared with healthy nonsmokers (p < 0.02; Fig. 4).

Interestingly, in a manner similar to that of healthy smokers, intelectin 1 gene expression was down-regulated in the small airway epithelium of smokers with lone emphysema with normal spirometry (7.8-fold decrease; p < 0.01) and smokers with established COPD (8.4-fold decrease, p < 0.01) compared with healthy nonsmokers (Table V). There was no difference in mRNA expression levels of intelectin 1 among healthy smokers, smokers with lone emphysema with normal spirometry, and smokers with established COPD (p > 0.3 for all comparisons). This microarray observation was confirmed with TaqMan RT-PCR in six smokers with lone emphysema with normal spirometry and 10 smokers with established COPD. Intelectin 1 gene expression was down-regulated in smokers with lone emphysema with normal spirometry (7.9-fold decrease; p < 0.02) and smokers with established COPD (34.4-fold decrease; p < 0.02) compared with healthy nonsmokers. As was observed with the microarray data, there was no difference in intelectin 1 expression levels among the smoking groups (p > 0.2, all comparisons).

Cigarette smoking is associated with increased susceptibility to infections of the respiratory tract (4). An important part of defense against pathogens is the innate immune response including lectin family members, a group of diverse peptides that are involved in cell and pathogen interaction through specific carbohydrate recognition domains (6, 7, 8, 9). In the present study we asked the question: does smoking alter the gene expression pattern of lectins in the respiratory epithelium? The data demonstrate that, while many of the lectin family genes are expressed in the human large and small airway epithelium, the gene expression pattern of the vast majority is not altered by cigarette smoking. However, there is a marked decrease in gene expression levels of intelectin 1 in the large and small airway epithelium of healthy smokers compared with healthy nonsmokers. This was confirmed at the protein level by immunohistochemistry, demonstrating the expression of intelectin 1 in secretory cells with qualitatively reduced expression in healthy smokers compared with nonsmokers, and quantitatively reduced expression at the protein level by Western analysis. Interestingly, intelectin 1 gene expression was also down-regulated in the small airway epithelium of smokers with lone emphysema with normal spirometry and smokers with established COPD compared with healthy nonsmokers. This observation is a further example of the modulatory effects of smoking on defense mechanisms in the lung.

Lectins are present on cell surfaces as phagocytic receptors or in the plasma as opsonins or agglutinins (6, 7, 8, 9). In the innate immune response, bacterial carbohydrate chains are recognized by lectins, with each lectin being specific to the type of carbohydrate residue it recognizes (6, 20). The mannose receptors bind to materials containing terminal mannosyl residues such as zymosan, which enhances their clearance by phagocytes, while the collectins and the ficolins are soluble lectins that function as opsonins or agglutinins for bacteria (21). Many of these biological defense lectins have an affinity for mannose or N-acetyl carbohydrates present on the cell surfaces of pathogens (21). Animal lectins also include a group that have an affinity for galactose (galectins) that generally participate in cell differentiation, apoptosis, recognition of tumor Ags, and the uptake of glycosylated proteins such as aged proteins (8).

Intelectin 1 is a recently described 313-aa galactose-binding lectin that recognizes specific bacterial cell wall components (20) not recognized by other lectins. It is known to be constitutively expressed in the small intestine, where cellular expression has been localized to Paneth cells and secretory or goblet-type cells (10, 22). Consistent with this, we observed that the expression of intelectin 1 in the airway epithelium is in secretory cells. Intelectin 1 has a Ca²⁺-dependent affinity for d-pentose and d-galactofuranosyl residues present in bacterial and fungal cell walls and protozoal parasites, but not in mammalian cells, suggestive of a role in pathogen recognition (10, 20). Intelectin 1 is up-regulated in the small intestines of mice exposed to the intestinal nematode Trichinella spiralis, where its isoform, intelectin 2, is also induced in small intestine epithelium (12, 23). Studies in mice have also demonstrated up-regulation of intelectin 1 in the small intestine in response to Trichuris muris infection (24), in the liver of rainbow trout after infection with Listonella (25), and grass carp Ctenopharyngodon idella in response to LPS injection (26). Intelectin 1 is a Th2-driven antimicrobial protein. Mice that can effect appropriate worm expulsion from the intestine when infected with T. muris exhibit immune polarization toward a Th2 response and demonstrate up-regulation of intelectin 1 in their small intestines. Susceptible (AKR) mice exhibit a Th1 response, no increase in expression of intelectin in small intestine, and delayed worm expulsion (24).

Intelectin 1 is also expressed in omental adipose tissue and there is decreased expression of intelectin 1 in the omental fat of individuals with Crohn’s disease (11, 27, 28). Given that this disease is characterized by transmural intestinal inflammation, the absence of intelectin 1 suggests further derangement of the immune system and perhaps altered immune responses to infection, facilitating the process of transmural inflammation (11).

In the present study, intelectin 1 was observed to be markedly down-regulated in the bronchial epithelium of healthy smoker and smokers with lone emphysema with normal spirometry and smokers with established COPD. This is in contrast to the observation that intelectin 1 is up-regulated at the gene expression level in the bronchial epithelium of individuals with asthma, in association with the up-regulation of IL-13 (29). Interestingly, overexpression of intelectin 1 in the lung has been demonstrated at the mRNA level in mice exposed to Nippostrongylus brasiliensis infection (30) and at the protein level in pleural mesothelioma and ovarian and colon carcinoma (31). Relevant to lung infections, intelectin 1 binds to galactofuranosyl residues that are present in the cell walls of bacteria such as Nocardia, Mycobacterium, and Streptococcus, pathogens that, while not observed in our study population, are observed more commonly in individuals who smoke than nonsmokers (2).

In the context that cigarette smoking is associated with an increased susceptibility to many infections of the lower respiratory tract, the finding of decreased expression of intelectin 1 in the large and small airways of smokers is further evidence of the modulatory effects of smoking on innate defense, which may contribute to the multifactorial increase in susceptibility to infections that has been observed in cigarette smokers; however, further work is needed to advance this observation and determine whether there will be therapeutic advantages to correcting the levels of intelectin 1 in the lung.

We thank T. Raman, R. H. Hubner, and Barbara Ferris for technical assistance, Philip Leopold and Rui Wang for helpful discussion, and J. Xiang from the Weill Cornell Microarray Core Facility and T. Virgin-Bryan and N. Mohamed for help in preparing this manuscript.

The authors have no financial conflict of interest.