Summary
Lectins are innate immune defense proteins that recognize specific bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with increased risk of bacterial infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey expression of lectin genes in large airway epithelium from 9 nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change, p<0.05) in lectin gene expression among healthy smokers compared to nonsmokers except for a striking down regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in the cell walls of bacteria (large airway epithelium, p<0.01; small airway epithelium, p<0.01). This was confirmed by TaqMan RT-PCR in both large (p<0.05) and small airway epithelium (p<0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared to healthy nonsmokers (p<0.02). Finally, compared to healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema with normal spirometry (n= 13, p<0.01) and smokers with established COPD (n= 14, p<0.01). In the context that intelectin 1 is an epithelial molecule that likely plays a role in defense against bacteria, its down regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers, including those with COPD.
Introduction
Cigarette smoking is a major risk factor for respiratory tract infections, with both active and passive smoke exposure increasing the risk of infection (1-4). 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 by cigarette smoking, respiratory tract secretions contain a large number of antimicrobial molecules participating in the innate immune response (5). An important component of these antimicrobial molecules is the lectins, proteins on cell surfaces that act as phagocytic receptors, playing a role in the recognition of specific bacterial cell wall components (6-9).
With this background, we used microarray analysis to screen the expression of 72 known lectins in large and small airway epithelium of healthy nonsmokers, healthy smokers, smokers with lone emphysema with normal spirometry and smokers with chronic obstructive lung disease (COPD). 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 mucosal defense (10-12).
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, confirmed by Western analysis that demonstrated reduced levels of intelectin 1 in airway epithelium of healthy smokers compared to nonsmokers. Decreased expression of intelectin 1 was also observed in the small airway epithelium of smokers with lone emphysema with 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 the defenses against respiratory tract infections.
Methods
Study Population
Healthy nonsmokers, healthy chronic smokers and smokers with lone emphysema with normal spirometry and established COPD were recruited using local print media and from the Division of Pulmonary and Critical Care Medicine outpatient clinic as study volunteers. The study population was evaluated under the auspices of the Weill Cornell NIH 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, ECG and pulmonary function testing. Current smoking status was confirmed on history, venous carboxyhemoglobin levels and urinalysis for nicotine levels 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 sec (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 hr prior to bronchoscopy to exclude the acute effects of smoking on airway epithelial gene expression.
Collection of Airway Epithelial Cells
Epithelial cells from the large and small airways were collected using flexible bronchoscopy. Smokers were asked not to smoke the evening prior to the procedure. After achieving mild sedation and anesthesia of the vocal cords, a flexible bronchoscope (Pentax, EB-1530T3) was advanced to the desired bronchus. Large airway epithelial samples were collected by gentle brushing of the 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 to 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 collected by gently brushing this area. The large and small airway epithelial cells were subsequently collected separately in 5 ml of LHC8 medium (GIBO, Grand Island, NY). An aliquot of this was used for cytology and 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, Pittsburg, PA) and stained with DiffQuik (Baxter Healthcare, Miami, FL).
RNA Extraction and Microarray Processing
Analyses were performed using Affymetrix (Santa Clara, CA) microarray HG-U133 Plus 2.0 (54,675 probe sets) and associated protocols. Total RNA was extracted from epithelial cells using TRIzol (Invitrogen, Carlsbad, CA) followed by Rneasy (Qiagen, Valencia, CA) to remove residual DNA. This process yielded 2 to 4 μg RNA per 106 cells. An aliquot of each RNA sample was run on an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) to visualize and quantify the degree of RNA integrity. The concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). 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 2 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 clean-up and quantification of the biotin-labeled cRNA yield by spectrophotometric analysis. All kits were from Affymetrix (Santa Clara, CA). 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. In order to maintain quality, only samples hybridized to test chips with a 3′ to 5′ ratio of <3 were deemed satisfactory.
Microarray Data Analysis
Captured images were analyzed using Microarray Suite version 5.0 (MAS 5.0) algorithm (Affymetrix) as previously described (15-17). The data were normalized using GeneSpring version 6.2 software (Agilent Technologies, Palo Alto, CA) 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 RT-PCR Confirmation of Microarray Expression Levels
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, 6 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 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 PCR reactions were carried out using pre-made 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 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 labeled with FAM. The PCR reactions were run in an Applied Biosystems Sequence Detection System 7500.
Localization of Intelectin 1 Expression in Human Airway Epithelium
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 carried out on paraffin embedded endobronchial biopsies from large airways of nonsmokers and healthy smokers obtained at flexible bronchoscopy. Sections were deparaffinized and dehydrated through a series of xylenes and alcohol. To enhance staining an antigen retrieval step was carried out by boiling the sections at 100°C, 20 min in citrate buffer solution (Labvision, Fremont, CA) followed by cooling at 23°C, 20 min. Endogenous peroxidase activity was quenched using 0.3 % H202 and blocking was performed with normal goat serum to reduce background staining. Samples were incubated with the primary rabbit polyclonal anti-intelectin 1 antibody 94-145 (1 μg/μl at 1/1000 dilution, Phoenix Pharmaceuticals, Phoenix, AZ), 12 hr, 4°C. Blocking with intelectin peptide 94-145 (Phoenix Pharmaceuticals) was used as a control. Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) and 3-amino-9-ethyl carbazole substrate kit (Vector Laboratories) were used to detect antibody binding and the sections were counter stained with hematoxylin (Sigma-Aldrich, St. Louis, MO) and mounted using GVA mounting medium (Zymed, San Francisco, CA). Brightfield microscopy was performed using a Nikon Microphot microscope and images were captured with an Olympus DP70 CCD camera.
Western Analysis
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, 5 min, 4°C. The whole cells were lysed with red cell lysis buffer (Cell Lytic Mammalian Tissue Lysis/Extraction reagent, Sigma-Aldrich) followed by whole cell lysis buffer (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 collected. The protein concentrations were assessed using a bicinchoninic acid (BCA) protein concentration kit (Pierce, Rockford, IL). Equal concentration of protein (20 μg) mixed with SDS Sample Loading Buffer (Bio-Rad, Hercules, CA) and reducing agent was loaded on Tris-glycine gels (Bio-Rad). Protein electrophoresis was carried out at 100V, 2 hr, 23°C. Sample proteins were transferred (25V, 1hr, 4°C) to a 0.45 μm thick PVDF 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 1hr, 23°C. The membranes were incubated with primary rabbit polyclonal anti-intelectin 1 antibodies 94-145 and 163-199 (Phoenix Pharmaceuticals), as this was shown to provide greater signal intensity than using 1 antibody alone, at 1:1000 dilution, 2hr, 4°C. Recombinant intelectin protein (Phoenix Pharmaceuticals) was used as a positive control. Detection was performed using horseradish peroxidase-conjugated anti-rabbit antibody (1:2,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) and the Enhanced Chemiluminescent reagent (ECL) system (GE, Healthcare, Pittsburgh, PA) 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 determined, and the background pixel intensity for a negative area of the film of identical size subtracted using MetaMorph image analysis software (Universal Imaging, Downingtown, PA). The membrane was subsequently stripped and reincubated with horseradish peroxidase-conjugated anti-β actin antibody (Santa Cruz Biotechnology) as a control for equal protein concentration.
Statistical Analysis
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 established COPD. Statistical comparisons for microarray data were calculated using GeneSpring software and associated two-tailed Student's t test. Statistical comparisons for categorical data were achieved using Chi-squared test. All other statistical comparisons were calculated using a two-tailed (Welsh) t-test.
Web Deposition of Data
All data has been deposited in the Gene Expression Omnibus (GEO) site (http://www.ncbi.nlm.nih.gov/geo), curated by the National Center for Bioinformatics (NCBI). Accession number for the data is GSE 10006.
Results
Study Population
Large airway samples from 9 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 IA). 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 nonsmoker and lone emphysema with normal spirometry and established COPD groups (p<0.01) and between smokers with lone emphysema with normal spirometry and 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 pack-yr, and urine nicotine and cotinine, and venous blood carboxyhemoglobin levels confirmed current smoking status of these individuals. Pulmonary function testing revealed normal lung function in healthy nonsmokers and healthy smokers (Table 1B). 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 I-III) had airflow obstruction values that met criteria for COPD (13). Data from some of these subjects has already been reported in previous studies (14).
Table IA.
Study Population of Airway Epithelial Samples1
| Large airways2 |
Small airways2 |
||||||
|---|---|---|---|---|---|---|---|
| Parameter | Healthy non- smokers |
Healthy smokers |
Healthy non- smokers |
Healthy smokers |
Lone emphysema with normal spirometry smokers |
COPD smokers |
|
| n | 9 | 20 | 13 | 20 | 13 | 14 | |
| Sex (male/female) | 9/0 | 12/8 | 11/2 | 15/5 | 10/3 | 13/1 | |
| Age (yr) | 44 ± 3 | 47 ± 1 | 42 ± 2 | 45 ± 1 | 51 ± 2 | 58 ± 2 | |
| Race (B/W/H/A)3 | 3/6/0/0 | 13/7/0/0 | 6/5/2/0 | 14/6/0/0 | 8/4/1/0 | 4/7/2/1 | |
| Smoking history (pack-yr) |
0 | 35 ± 4 | 0 | 34 ± 4 | 35 ± 6 | 44 ± 7 | |
| Urine nicotine (ng/ml) |
Negative | 849 ± 222 | Negative | 425 ± 117 | 833 ± 271 | 734 ± 255 | |
| Urine cotinine (ng/ml) |
Negative | 934 ± 190 | Negative | 1093 ± 195 | 1410 ± 408 | 1185 ± 312 | |
| Venous CO-Hb4 | 0.3 ± 0.9 | 2.8 ± 0.5 | 0.9 ± 0.3 | 2.9 ± 0.5 | 1.8 ± 0.5 | 3.1 ± 0.5 | |
| Epithelial cells | |||||||
| Total number recovered ×106 |
6.5 ± 0.5 | 4.9 ± 1.0 | 5.5 ± 0.7 | 6.4 ± 1.3 | 7.3 ± 1.6 | 5.4 ± 1.0 | |
| % epithelial | 99 ± 1 | 99 ± 1 | 99 ± 1 | 99 ± 1 | 99 ± 1 | 96 ± 1 | |
| % inflammatory | 1 ± 1 | 1 ± 1 | 1 ± 1 | 1 ± 1 | 1 ± 1 | 4 ± 1 | |
| Differential cell count | |||||||
| Ciliated | 51 ± 4 | 42 ± 3 | 78 ± 7 | 75 ± 10 | 81 ± 3 | 71 ± 4 | |
| Secretory | 8 ± 1 | 10 ± 2 | 7 ± 3 | 7 ± 3 | 4 ± 1 | 9 ± 4 | |
| Basal | 23 ± 1 | 27 ± 2 | 7 ± 2 | 9 ± 4 | 7 ± 3 | 2 ± 1 | |
| Undifferentiated | 17 ± 3 | 20 ± 3 | 8 ± 4 | 9 ± 4 | 7 ± 1 | 2 ± 1 | |
Data are presented as mean ± standard error.
Five of the 9 healthy nonsmokers, from which large airway epithelium were obtained, were also the source of the healthy nonsmoker small airway epithelium; 14 of the 20 healthy smokers, from which large airway epithelium was obtained, were also the source of the healthy smoker small airway epithelium.
B=Black, W=White, H=Hispanics of Latin American origin, A=Asian.
Venous carboxyhemoglobin, a secondary marker of current smoking; nonsmokers, normal value <1.5%.
Table IB.
Pulmonary Function Parameters in the Study Population1
| Large airways |
Small airways |
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Healthy nonsmokers |
Healthy smokers |
Healthy nonsmokers |
Healthy smokers | Smokers with lone emphysema with normal spirometry |
Smokers with COPD |
|||||||
| Parameter | Pre | Post | Pre | Post | Pre | Post | Pre | Post | Pre | Post | Pre | Post |
| FVC | 101 ± 3 | ND | 108 ± 3 | 110 ± 3 | 105 ± 3 | ND | 107 ± 3 | 108 ± 3 | 100 ± 3 | 99 ± 4 | 94 ±7 | 100 ± 7 |
| FEV1 | 100 ± 5 | ND | 108 ± 3 | 112 ± 3 | 106 ± 2 | ND | 108 ± 3 | 111 ± 4 | 95 ± 4 | 96 ± 3 | 73 ± 7 | 76 ± 6 |
| FEV1/FVC % observed |
78 ± 1 | ND | 83 ± 1 | 83 ± 1 | 82 ± 2 | ND | 82 ± 1 | 83 ± 1 | 78 ± 1 | 79 ± 1 | 60± 3 | 61 ± 3 |
| TLC | 98 ± 3 | ND | 101 ± 3 | ND | 97 ± 2 | ND | ND | 100 ± 3 | ND | 94 ± 4 | ND | 111 ± 5 |
| DLCO | 97 ± 3 | ND | 93 ± 2 | ND | 96 ± 2 | ND | ND | 94 ± 2 | ND | 64 ± 2 | ND | 72 ± 5 |
FVC = forced vital capacity; FEV1 = forced expiratory volume in first second; TLC = total lung capacity; DLCO = diffusing capacity for carbon monoxide; all data are presented as % predicted except for FEV1/FVC presented as % observed. Pre = pre-bronchodilator parameters, Post = post-bronchodilator parameters, ND = not done.
Sampling of Airway Epithelium
Airway epithelial cells were obtained by fiberoptic bronchoscopy and brushing of the 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 IA). 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).
Expression of Lectin Family Members in the Airway Epithelium
Of the 72 lectin family genes surveyed using the Affymetrix HG-U133 Plus 2.0 array and the criteria of Affymetrix Detection Call of Present (P call) 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 II; see Supplemental Data 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 III; 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 P call of ≥50%) in the small airway epithelium of healthy nonsmokers (along with 3 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 4 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 2 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.
Table II.
Expression of Lectin Family Genes in Large Airway Epithelium of Healthy Smokers Compared to Healthy Nonsmokers1
| Probe set ID | Gene symbol |
Gene title | Nonsmoker % present |
Smoker % present |
Smoker/ nonsmoker |
p value |
|---|---|---|---|---|---|---|
| 209732_at | CLEC2B | C-type lectin domain family 2, member B | 100 | 100 | −1.24 | 0.520 |
| 220132_s_at | CLEC2D | C-type lectin domain family 2, member D | 89 | 85 | 1.03 | 0.380 |
| 219947_at | CLEC4A | C-type lectin domain family 4, member A | 100 | 75 | −1.02 | 0.923 |
| 1552773_at | CLEC4D | C-type lectin domain family 4, member D | 22 | 55 | 1.22 | 0.532 |
| 221698_s_at | CLEC7A | C-type lectin domain family 7, member A | 100 | 95 | 1.45 | 0.059 |
| 207420_at | CLEC10A | C-type lectin domain family 10, member A | 0 | 75 | 1.44 | 0.052 |
| 221019_s_at | COLEC12 | collectin sub-family member 12 | 89 | 70 | 1.03 | 0.878 |
| 223597_at | ITLN1 | intelectin 1 | 56 | 20 | −3.77 | 0.002 |
| 206785_s_at | KLRC1 | killer cell lectin-like receptor subfamily C, member 1 |
100 | 95 | −1.56 | 0.156 |
| 220646_s_at | KLRF1 | killer cell lectin-like receptor subfamily F, member 1 |
56 | 65 | 1.01 | 0.965 |
| 205821_at | KLRK1 | killer cell lectin-like receptor subfamily K, member 1 |
89 | 40 | −1.18 | 0.374 |
| 201105_at | LGALS1 | lectin, galactoside-binding, soluble, 1 | 100 | 80 | −1.52 | 0.085 |
| 208949_s_at | LGALS3 | lectin, galactoside-binding, soluble, 3 | 100 | 100 | −1.23 | 0.005 |
| 200923_at | LGALS3BP | lectin, galactoside-binding, soluble, 3 | 100 | 100 | −1.40 | 0.054 |
| 204272_at | LGALS4 | lectin, galactoside-binding, soluble, 4 | 44 | 50 | −1.26 | 0.492 |
| 206400_at | LGALS7 | lectin, galactoside-binding, soluble, 7 | 100 | 95 | −1.17 | 0.552 |
| 208934_s_at | LGALS8 | lectin, galactoside-binding, soluble, 8 | 100 | 100 | −1.28 | 0.088 |
| 203236_s_at | LGALS9 | lectin, galactoside-binding, soluble, 9 | 100 | 100 | 1.00 | 0.996 |
| 224629_at | LMAN1 | lectin, mannose-binding, 1 | 100 | 85 | 1.13 | 0.173 |
| 200805_at | LMAN2 | lectin, mannose-binding 2 | 100 | 95 | −1.14 | 0.396 |
| 221274_s_at | LMAN2L | lectin, mannose-binding 2-like | 100 | 100 | −1.10 | 0.234 |
| 229111_at | MASP2 | mannan-binding lectin serine peptidase 2 | 100 | 100 | −1.02 | 0.876 |
| 201373_at | PLEC 1 | plectin 1 | 100 | 90 | 1.06 | 0.605 |
| 228869_at | SLIC1 | selectin ligand interactor cytoplasmic-1 | 33 | 55 | −1.04 | 0.857 |
| 204563_at | SELL | selectin L | 89 | 70 | −1.51 | 0.173 |
| 209879_at | SELPLG | selectin P ligand | 100 | 75 | −1.25 | 0.021 |
Listed are all lectin family genes considered “expressed” based on the criteria of present in ≥50% samples of either or both healthy nonsmokers or healthy smokers; among the 26 lectin family genes expressed, only intelectin 1 was significantly changed >2.0 fold, p<0.05 in smokers compared to nonsmokers. For the complete microarray data set for all lectin family genes for healthy nonsmokers and healthy smokers, see Supplementary Data Table I.
Table III.
Expression of Lectin Family Genes in Small Airway Epithelium of Healthy Smokers Compared to Healthy Nonsmokers1
| Probe set ID | Gene symbol |
Gene title | Nonsmoker % present |
Smoker % present |
Smoker/ nonsmoker |
p value |
|---|---|---|---|---|---|---|
| 209732_at | CLEC2B | C-type lectin domain family 2, member B | 92 | 100 | −1.21 | 0.383 |
| 1560156_at | CLEC2D | C-type lectin domain family 2, member D | 77 | 60 | −1.13 | 0.545 |
| 219947_at | CLEC4A | C-type lectin domain family 4, member A | 92 | 95 | −1.07 | 0.714 |
| 221698_s_at | CLEC7A | C-type lectin domain family 7, member A | 92 | 85 | 1.11 | 0.649 |
| 206682_at | CLEC10A | C-type lectin domain family 10, member A | 54 | 75 | 1.25 | 0.361 |
| 1552398_a_at | CLEC12A | C-type lectin domain family 12, member A | 31 | 60 | 1.70 | 0.125 |
| 221019_s_at | COLEC12 | collectin sub-family member 12 | 69 | 75 | 1.13 | 0.324 |
| 223597_at | ITLN1 | intelectin 1 | 69 | 40 | −5.60 | 0.001 |
| 206785_s_at | KLRC1 | killer cell lectin-like receptor subfamily C | 69 | 85 | 1.56 | 0.131 |
| 220646_s_at | KLRF1 | killer cell lectin-like receptor subfamily F | 62 | 45 | −1.20 | 0.147 |
| 205821_at | KLRK1 | killer cell lectin-like receptor subfamily K | 46 | 50 | 1.25 | 0.279 |
| 201105_at | LGALS1 | lectin, galactoside-binding, soluble, 1 | 100 | 100 | −1.53 | 0.174 |
| 208949_s_at | LGALS3 | lectin, galactoside-binding, soluble, 3 | 100 | 100 | −1.14 | 0.048 |
| 200923_at | LGALS3BP | lectin, galactoside-binding protein 3 | 100 | 100 | −1.20 | 0.088 |
| 204272_at | LGALS4 | lectin, galactoside-binding, soluble, 4 | 69 | 50 | −1.20 | 0.480 |
| 206400_at | LGALS7 | lectin, galactoside-binding, soluble, 7 | 85 | 100 | 1.16 | 0.619 |
| 208934_s_at | LGALS8 | lectin, galactoside-binding, soluble, 8 | 100 | 100 | −1.16 | 0.114 |
| 203236_s_at | LGALS9 | lectin, galactoside-binding, soluble, 9 | 100 | 100 | −1.11 | 0.392 |
| 224629_at | LMAN1 | Lectin, mannose-binding, 1 | 100 | 100 | −1.15 | 0.224 |
| 200805_at | LMAN2 | lectin, mannose-binding 2 | 100 | 100 | 1.04 | 0.612 |
| 221274_s_at | LMAN2L | lectin, mannose-binding 2-like | 100 | 100 | −1.22 | 0.054 |
| 229111_at | MASP2 | Mannan-binding lectin serine peptidase 2 | 100 | 100 | −1.06 | 0.047 |
| 216971_s_at | PLEC1 | plectin 1 | 100 | 90 | −1.44 | 0.147 |
| 204563_at | SELL | selectin L | 85 | 75 | −1.15 | 0.100 |
| 209879_at | SELPLG | selectin P ligand | 100 | 85 | −1.48 | 0.005 |
| 228869_at | SLIC1 | selectin ligand interactor cytoplasmic-1 | 69 | 65 | −1.09 | 0.697 |
Listed are all lectin family genes considered “expressed” based on the criteria of present in ≥50% samples of either or both healthy nonsmokers or healthy smokers; among the 26 lectin family genes expressed, only intelectin 1 was significantly changed >2.0 fold, p<0.05 in smokers compared to nonsmokers. For the complete microarray data set for all lectin family genes for healthy nonsmokers, healthy smokers, smokers with lone emphysema with normal spirometry and smokers with established COPD (GOLD I-III) see Supplementary Data Table II.
Down-regulation of Intelectin 1 in Large and Small Airway Epithelium of Healthy smokers
Of all of the lectin family genes expressed in the large and small airways, only intelectin 1 was significantly changed >2-fold in healthy smokers compared to healthy nonsmokers (Table II and Table III). As assessed using the microarrays, intelectin 1 was significantly down-regulated in healthy smokers compared to healthy nonsmokers in large airway epithelium (3.8-fold decrease, p<0.01) and healthy smokers compared to healthy nonsmokers in the small airway epithelium (5.6-fold decrease, p<0.01; Figure 1).
Figure 1.
Intelectin 1 gene expression levels in large and small airway epithelium. A. Average normalized gene expression levels of intelectin 1, as assessed using microarray HG-U133 Plus 2.0, in large airway epithelium of 9 healthy nonsmokers and 20 healthy smokers. The ordinate shows the average normalized gene expression levels for intelectin 1. B. Average normalized gene expression levels of intelectin 1, as assessed using microarray HG-U133 Plus 2.0, in small airway epithelium of 13 healthy nonsmokers and 20 healthy smokers. The ordinate shows the average normalized gene expression levels for intelectin 1. Error bars represent standard error.
To confirm the results obtained from the microarray screen, TaqMan RT-PCR was carried out 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 to healthy nonsmokers and in the small airways of healthy smokers (14.7 fold decrease, p<0.02) compared to healthy nonsmokers (Figure 2).
Figure 2.
TaqMan RT-PCR confirmation of intelectin 1 gene expression changes in large and small airway epithelium. A. TaqMan confirmation of changes in intelectin 1 gene expression levels in large airways of 12 healthy nonsmokers and 17 healthy smokers. B. TaqMan confirmation of changes in intelectin 1 gene expression levels in small airways of 12 healthy nonsmokers and 11 healthy smokers. The ordinate shows average gene expression levels and error bars represent standard error.
Immunohistochemical Assessment of Intelectin 1 Expression
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 (Figure 3). There was no positive staining for intelectin 1 observed in healthy smokers, consistent with the concept that 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 to healthy nonsmokers. Since intelectin 1 is expressed in secretory cells, morphological analysis of cytopreparations reveals 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 is increased mRNA expression of MUC5AC in smokers compared to 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.
Figure 3.
Immunohistochemistry assessment of large airway epithelium for expression of intelectin 1 in healthy nonsmokers and healthy smokers. A. Healthy nonsmokers. Shown are 2 examples with rabbit anti-human intelectin 1 antibody and blocking peptide as a control. B. Healthy smokers. Shown are 2 examples with rabbit anti-human intelectin 1 antibody and blocking peptide as a control. Bar= 20 μm
Western Analysis of Intelectin 1 Protein Expression
Western analysis carried out on large airway samples from healthy nonsmokers and healthy smokers was employed to quantitatively assess intelectin 1 expression. Using multiple brushes from the airway, approximately 20 μg total protein was obtained from each individual. Western analysis of this protein demonstrated decreased intelectin 1 protein expression in healthy smokers compared to healthy nonsmokers. Quantitative analysis revealed decreased intelectin 1 protein expression in healthy smokers compared to healthy nonsmokers (p<0.02, Figure 4).
Figure 4.
Western analysis of intelectin 1 protein expression in large airway epithelium of nonsmokers and healthy smokers. A. Upper panel - intelectin 1 protein expression in nonsmokers (lanes 1-3) and smokers (lanes 4-6). Lower panel - same gel probed with anti β-actin antibody; 20 μg protein loaded per well. B. Ratio of intelectin 1 to β-actin. The ratio for intelectin 1 to β actin is represented on the ordinate for nonsmoker and smoker bands. Error bars represent the standard error.
Intelectin 1 Gene Expression in Smokers with Lone Emphysema with Normal Spirometry and Established COPD
Interestingly, in a manner similar to 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 to healthy nonsmokers (Table IV). 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 6 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 to 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).
Table IV.
Down-regulation of Expression of Intelectin 1 in the Small Airway Epithelium of Healthy Smokers, Smokers with Lone Emphysema with Normal Spirometry and Smokers with Established COPD Compared to Healthy Nonsmokers.
| Microarray HG U133 Plus 2.0 |
TaqMan RT-PCR |
|||||
|---|---|---|---|---|---|---|
| Phenotype1 | Average normalized gene expression level |
Fold-change compared to healthy nonsmokers |
p value |
Average expression level |
Fold- change |
p value |
| Healthy nonsmokers |
8.80 | _ | _ | 2.50 | _ | _ |
| Healthy smokers |
1.57 | −5.6 | <0.01 | 0.17 | −14.7 | <0.01 |
| Smokers with lone emphysema with normal spirometry |
1.14 | −7.8 | <0.01 | 0.32 | −7.9 | <0.02 |
| Smokers with established COPD |
1.05 | −8.4 | <0.01 | 0.07 | −34.4 | <0.02 |
See Methods for criteria.
Discussion
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 (6-9). In the present study we asked the question: does smoking alter the gene expression pattern of lectins in the respiratory epithelium? The data demonstrates 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 to 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 to 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 to healthy nonsmokers. This observation is a further example of the modulatory effects of smoking on defense mechanisms in the lung.
Lectins, Intelectin and Host Defense
Lectins are present on cell surfaces as phagocytic receptors or in the plasma as opsonins or agglutinins (6-9). In the innate immune response, bacterial carbohydrate chains are recognized by lectins, with each lectin being specific in 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 to galactose (galectins) that generally participate in cell differentiation, apoptosis, recognition of tumor antigens and the uptake of glycosylated proteins such as aged proteins (8).
Intelectin 1 is a recently described 313 amino acid 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 the 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 intestine 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 lipopolysaccharide injection (26). Intelectin 1 is a Th2 driven antimicrobial protein. Mice that can effect appropriate worm expulsion from the intestines, when infected with Trichuris muris, exhibit immune polarization towards a Th2 response and demonstrate upregulation of intelectin 1 in their small intestines. Susceptible (AKR) mice exhibit a Th1 response, no increase in expression of intelectin in small intestine and have 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). 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).
Smoking-induced Down-regulation of Intelectin 1 in the Airway Epithelium
In the present study, intelectin 1 was observed to be markedly down regulated in the bronchial epithelium of healthy smokers and smokers with lone emphysema with normal spirometry and 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, over expression of intelectin 1 in the lung has been demonstrated at the mRNA level in mice exposed to Nippostronggylus brasiliensis infection (30) and at the protein level in pleural mesothelioma, 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, Mycobacteria 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 if there will be therapeutic advantages to correcting the levels of intelectin 1 in the lung.
Supplementary Material
Acknowledgments
We thank T. Raman, R.H. Hubner and Barbara Ferris for technical assistance, Philip Leopold and Rui Wang for helpful discussion, J. Xiang from the Weill Cornell Microarray Core Facility and T. Virgin-Bryan and N. Mohamed for help in preparing this manuscript.
Footnotes
These studies were supported, in part, by R01 HL074326; P50 HL084936; M01RR00047; and Will Rogers Memorial Fund, Los Angeles, CA.
Publisher's Disclaimer: “This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.”
References
- 1.Almirall J, Gonzalez CA, Balanzo X, Bolibar I. Proportion of community-acquired pneumonia cases attributable to tobacco smoking. Chest. 1999;116:375–379. doi: 10.1378/chest.116.2.375. [DOI] [PubMed] [Google Scholar]
- 2.Arcavi L, Benowitz NL. Cigarette smoking and infection. Arch. Intern. Med. 2004;164:2206–2216. doi: 10.1001/archinte.164.20.2206. [DOI] [PubMed] [Google Scholar]
- 3.Bensenor IM, Cook NR, Lee IM, Chown MJ, Hennekens CH, Buring JE, Manson JE. Active and passive smoking and risk of colds in women. Ann. Epidemiol. 2001;11:225–231. doi: 10.1016/s1047-2797(00)00214-3. [DOI] [PubMed] [Google Scholar]
- 4.Murin S, Bilello KS. Respiratory tract infections: another reason not to smoke. Cleve. Clin. J Med. 2005;72:916–920. doi: 10.3949/ccjm.72.10.916. [DOI] [PubMed] [Google Scholar]
- 5.Rogan MP, Geraghty P, Greene CM, O'Neill SJ, Taggart CC, McElvaney NG. Antimicrobial proteins and polypeptides in pulmonary innate defence. Respir. Res. 2006;7:29. doi: 10.1186/1465-9921-7-29. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kilpatrick DC. Animal lectins: a historical introduction and overview. Biochim. Biophys. Acta. 2002;1572:187–197. doi: 10.1016/s0304-4165(02)00308-2. [DOI] [PubMed] [Google Scholar]
- 7.Cambi A, Koopman M, Figdor CG. How C-type lectins detect pathogens. Cell Microbiol. 2005;7:481–488. doi: 10.1111/j.1462-5822.2005.00506.x. [DOI] [PubMed] [Google Scholar]
- 8.Liu FT. Regulatory roles of galectins in the immune response. Int. Arch. Allergy Immunol. 2005;136:385–400. doi: 10.1159/000084545. [DOI] [PubMed] [Google Scholar]
- 9.Tosi MF. Innate immune responses to infection. J Allergy Clin. Immunol. 2005;116:241–249. doi: 10.1016/j.jaci.2005.05.036. [DOI] [PubMed] [Google Scholar]
- 10.Komiya T, Tanigawa Y, Hirohashi S. Cloning of the novel gene intelectin, which is expressed in intestinal paneth cells in mice. Biochem. Biophys. Res. Commun. 1998;251:759–762. doi: 10.1006/bbrc.1998.9513. [DOI] [PubMed] [Google Scholar]
- 11.Schaffler A, Neumeier M, Herfarth H, Furst A, Scholmerich J, Buchler C. Genomic structure of human omentin, a new adipocytokine expressed in omental adipose tissue. Biochim. Biophys. Acta. 2005;1732:96–102. doi: 10.1016/j.bbaexp.2005.11.005. [DOI] [PubMed] [Google Scholar]
- 12.Pemberton AD, Knight PA, Gamble J, Colledge WH, Lee JK, Pierce M, Miller HR. Innate BALB/c enteric epithelial responses to Trichinella spiralis: inducible expression of a novel goblet cell lectin, intelectin-2, and its natural deletion in C57BL/10 mice. J Immunol. 2004;173:1894–1901. doi: 10.4049/jimmunol.173.3.1894. [DOI] [PubMed] [Google Scholar]
- 13.From the Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2007 http://www.goldcopd.org/Guidelineitem.asp?l1=2&l2=1&intId=989 (Last accessed 3/28/07)
- 14.Harvey BG, Heguy A, Leopold PL, Carolan BJ, Ferris B, Crystal RG. Modification of gene expression of the small airway epithelium in response to cigarette smoking. J Mol. Med. 2007;85:39–53. doi: 10.1007/s00109-006-0103-z. [DOI] [PubMed] [Google Scholar]
- 15.Hackett NR, Heguy A, Harvey BG, O'Connor TP, Luettich K, Flieder DB, Kaplan R, Crystal RG. Variability of antioxidant-related gene expression in the airway epithelium of cigarette smokers. Am. J Respir. Cell Mol. Biol. 2003;29:331–343. doi: 10.1165/rcmb.2002-0321OC. [DOI] [PubMed] [Google Scholar]
- 16.Heguy A, Harvey BG, O'Connor TP, Hackett NR, Crystal RG. Sampling-dependent up-regulation of gene expression in sequential samples of human airway epithelial cells. Mol. Med. 2003;9:200–208. doi: 10.2119/2003-00051.crystal. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Kaplan R, Luettich K, Heguy A, Hackett NR, Harvey BG, Crystal RG. Monoallelic up-regulation of the imprinted H19 gene in airway epithelium of phenotypically normal cigarette smokers. Cancer Res. 2003;63:1475–1482. [PubMed] [Google Scholar]
- 18.Willemse BW, ten Hacken NH, Rutgers B, Postma DS, Timens W. Association of current smoking with airway inflammation in chronic obstructive pulmonary disease and asymptomatic smokers. Respir. Res. 2005;6:38. doi: 10.1186/1465-9921-6-38. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Breeze RG, Wheeldon EB. The cells of the pulmonary airways. Am. Rev. Respir. Dis. 1977;116:705–777. doi: 10.1164/arrd.1977.116.4.705. [DOI] [PubMed] [Google Scholar]
- 20.Tsuji S, Uehori J, Matsumoto M, Suzuki Y, Matsuhisa A, Toyoshima K, Seya T. Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall. J Biol. Chem. 2001;276:23456–23463. doi: 10.1074/jbc.M103162200. [DOI] [PubMed] [Google Scholar]
- 21.Holmskov U, Thiel S, Jensenius JC. Collections and ficolins: humoral lectins of the innate immune defense. Annu. Rev. Immunol. 2003;21:547–578. doi: 10.1146/annurev.immunol.21.120601.140954. [DOI] [PubMed] [Google Scholar]
- 22.Wrackmeyer U, Hansen GH, Seya T, Danielsen EM. Intelectin: a novel lipid raft-associated protein in the enterocyte brush border. Biochemistry. 2006;45:9188–9197. doi: 10.1021/bi060570x. [DOI] [PubMed] [Google Scholar]
- 23.Pemberton AD, Knight PA, Wright SH, Miller HR. Proteomic analysis of mouse jejunal epithelium and its response to infection with the intestinal nematode, Trichinella spiralis. Proteomics. 2004;4:1101–1108. doi: 10.1002/pmic.200300658. [DOI] [PubMed] [Google Scholar]
- 24.Datta R, deSchoolmeester ML, Hedeler C, Paton NW, Brass AM, Else KJ. Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite. Infect. Immun. 2005;73:4025–4033. doi: 10.1128/IAI.73.7.4025-4033.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Gerwick L, Corley-Smith G, Bayne CJ. Gene transcript changes in individual rainbow trout livers following an inflammatory stimulus. Fish. Shellfish. Immunol. 2007;22:157–171. doi: 10.1016/j.fsi.2006.04.003. [DOI] [PubMed] [Google Scholar]
- 26.Chang MX, Nie P. Intelectin gene from the grass carp Ctenopharyngodon idella: cDNA cloning, tissue expression, and immunohistochemical localization. Fish. Shellfish. Immunol. 2006 doi: 10.1016/j.fsi.2006.09.012. [DOI] [PubMed] [Google Scholar]
- 27.Yang RZ, Lee MJ, Hu H, Pray J, Wu HB, Hansen BC, Shuldiner AR, Fried SK, McLenithan JC, Gong DW. Identification of omentin as a novel depot-specific adipokine in human adipose tissue: possible role in modulating insulin action. Am. J Physiol Endocrinol. Metab. 2006;290:E1253–E1261. doi: 10.1152/ajpendo.00572.2004. [DOI] [PubMed] [Google Scholar]
- 28.de Souza Batista CM, Yang RZ, Lee MJ, Glynn NM, Yu DZ, Pray J, Ndubuizu K, Patil S, Schwartz A, Kligman M, Fried SK, Gong DW, Shuldiner AR, Pollin TI, McLenithan JC. Omentin Plasma Levels and Gene Expression are Decreased in Obesity. Diabetes. 2007 doi: 10.2337/db06-1506. [DOI] [PubMed] [Google Scholar]
- 29.Kuperman DA, Lewis CC, Woodruff PG, Rodriguez MW, Yang YH, Dolganov GM, Fahy JV, Erle DJ. Dissecting asthma using focused transgenic modeling and functional genomics. J Allergy Clin. Immunol. 2005;116:305–311. doi: 10.1016/j.jaci.2005.03.024. [DOI] [PubMed] [Google Scholar]
- 30.Voehringer D, Stanley SA, Cox JS, Completo GC, Lowary TL, Locksley RM. Nippostrongylus brasiliensis: Identification of intelectin-1 and -2 as Stat6-dependent genes expressed in lung and intestine during infection. Exp. Parasitol. 2007 doi: 10.1016/j.exppara.2007.02.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Wali A, Morin PJ, Hough CD, Lonardo F, Seya T, Carbone M, Pass HI. Identification of intelectin overexpression in malignant pleural mesothelioma by serial analysis of gene expression (SAGE) Lung Cancer. 2005;48:19–29. doi: 10.1016/j.lungcan.2004.10.011. [DOI] [PubMed] [Google Scholar]
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