the airways of cf patients are initially colonized by viruses , fungi , or bacteria , including staphylococcus aureus , haemophilus influenzae , and klebsiella pneumonia . most patients later become infected with mucoid strains of pseudomonas aeruginosa and some with burkholderia cepacia . in cf patients , the number of neutrophils and the levels of cytokines such as tumor necrosis factor- ( tnf- ) , interleukin- ( il- ) 6 , and il-8 in the airways are increased compared to non - cf individuals [ 3 , 4 ] . cultured cf lung epithelial cells ( ib3 - 1 ) show downregulation of the anti - inflammatory cytokine il-10 and an exaggerated upregulation of il-8 in response to a variety of external stimuli , such as tnf- and bacterial products [ 5 , 6 ] . overproduction of il-8 is likely a major cause of excessive neutrophil infiltration , since il-8 is a potent chemoattractant for neutrophils . neutrophil migration in response to inflammatory stimuli requires cell adhesion molecules , such as intercellular adhesion molecule-1 ( icam-1 , also known as cd54 ) [ 8 , 9 ] . migration of neutrophils out of the vascular system occurs in distinct phases : rolling , firm adhesion , and transmigration . four types of cell adhesion molecules are involved in this process , namely , e - selectin , icam-1 , vascular cell adhesion molecule-1 ( vcam-1 ) , and platelet - endothelial cell adhesion molecule-1 ( pecam-1 ) . neutrophil rolling is the first step of the migration process and e - selectin is the key molecule involved in slowing down the circulating neutrophils . this step is critical to ensure firm adhesion of neutrophils to the endothelial cell layer . firm adhesion is mediated through icam-1 expressed on endothelial cells , which interacts with cd11a / cd18 ( lfa1 ) or cd11b / cd18 ( mac-1 ) as counter - receptors on neutrophils . the final phase of transmigration of neutrophils through the endothelium is triggered by pecam-1 and vcam-1 . currently , the mechanism by which neutrophils migrate to the airway lumen is unclear , but they are thought to travel through the intercellular space [ 12 , 13 ] . other cell adhesion molecules such as icam-2 and icam-3 are also involved in the migration of monocytes   or dendritic cells . icam-1 is a 114  kd inducible surface glycoprotein that belongs to the immunoglobulin superfamily   and it plays an important role in innate and adaptive immune responses . although the role of icam-1 in endothelial cells as well as in adaptive immunity [ 1720 ] is well established , the function of epithelial icam-1 during inflammation is not fully understood . since epithelial icam-1 is expressed on the airway lumen [ 2124 ] , a role for leukocyte transmigration is not expected . on the other hand , cell adhesion studies [ 25 , 26 ] indicate that epithelial icam-1 is important for leukocyte homing . because neutrophils and macrophages are enriched at the sites of injury or inflammation , it is possible that homing of these cells is part of the resolution of inflammation . among the adhesion molecules , icam-1 may play a more important role in the infiltration of leukocytes during airway inflammation . for example , hubeau et al . performed quantitative analysis of inflammatory cells infiltrating the cf airway mucosa in lung tissues collected at the time of transplantation and found that icam-1 , but not vcam-1 or e - selectin , was overexpressed on the epithelium surface . in addition , a recent in vitro study also showed that icam-1 is expressed in a higher percentage of cultured airway epithelial cell lines ( ib3 - 1 , c38 and beas-2b ) than other cell adhesion molecules , such as vcam-1 or e - selectin . interestingly , cf - deficient airway epithelial cells have a slightly higher basal level of icam-1 expression . upon stimulation with proinflammatory cytokine ( e.g. , tnf- or il-1 ) [ 29 , 30 ] or other stimulatory substances ( e.g. , lps ) , icam-1 expression is significantly induced in both human primary bronchial epithelial cultures and epithelial cell lines . this induction is mediated by activation of nuclear factor - kappa b ( nf-b ) signaling transduction pathway . in addition , icam-1 induction can also be mediated through the stat signaling pathway since ifn - gamma can significantly elevate its expression in epithelial cells . since epithelial icam-1 may be critical for neutrophil homing and epithelial killing , it is important to understand its regulation and function in airway epithelial cells in order to identify potential drug targets for the cf lung disease . the e26 transformation - specific ( ets ) family of transcription factors is characterized by a highly conserved 85 amino acid dna binding domain , which is known as the ets domain . the ets domain is usually located in the carboxyl - terminal region of the protein as a winged helix - turn - helix structural motif and binds to purine - rich dna that has a core consensus sequence of ggaa / t- within the promoter and enhancer regions of target genes . ets transcriptional factors act as both positive and negative regulators of gene expression in various biological processes , such as cellular proliferation , differentiation , apoptosis , metastasis , hematopoiesis , and angiogenesis . although many of ets family members are expressed in nonepithelial cells , such as hematopoietic and endothelial cells , ese-1 is mainly expressed in epithelial - rich tissues , such as lung , kidney , stomach , small intestine , colon , pancreas , trachea , salivary gland , prostate gland , mammary gland , uterus , and skin , but it can be upregulated in nonepithelial cells by proinflammatory cytokines such as tnf- and il-1. our previous work has shown that ese-1 can be highly induced in epithelial cells by inflammatory cytokines . in this study , we investigated the regulation of icam-1 expression by ese-1 . here we demonstrate that the expression of both icam-1 and ese-1 is upregulated in human bronchial epithelial cells ( beas2b ) , cf cells ( ib3 - 1 ) , and lung cancer cells ( a549 ) by inflammatory cytokines . we also show that icam-1 expression is downregulated upon ese-1 knockdown in a549 cells and that ese-1 regulates the icam-1 expression at the transcriptional level . finally , we demonstrate that the downregulation of icam-1 by knocking down ese-1 in a549 cells results in a reduced capacity of a549 cells to interact with hl-60 cells . human bronchial epithelial cells ( beas-2b ) , human lung carcinoma cells ( a549 ) , and cf bronchi epithelial cells ( ib3 - 1 ) were cultured in dulbecco 's modified eagle 's medium ( dmem ) medium ( invitrogen ) supplemented with 10% fetal bovine serum ( fbs ) . recombinant human tumor necrosis factor- ( tnf- ) and interleukin-1 ( il-1 ) were obtained from r&d systems ( minneapolis , mn ) and reconstituted in phosphate buffered saline ( pbs ) containing 0.1% bovine serum albumin ( bsa ) . for cytokines stimulation , ese-1 gene was knocked down in a549 cells using a shrna helper - dependent adenoviral vector expressing two shrnas from murine u6 gene promoter as described previously . a549 cells were seeded at 2  10 cells per well in 6-well plates overnight , and then cells were transduced at 4060% confluency with viral vector at 2500  particles / cell ( or 50  moi ) under serum - free conditions for two hours , followed by addition of media to a final concentration of 10% fbs . cells were then collected for protein extraction or rna isolation at desired time points . for cell transfection and cotransfection experiments , beas-2b cells were seeded in 6-well plates and transfected at 5060% confluency by using polyfect transfection reagent ( qiagen ) according to the manufacturer 's protocol . after 24  h , cells were harvested and luciferase activity was measured using a dual lucifcerase kit ( promega ) and a luminometer ( eg&g berthold , badwildbad , germany ) as described previously . cells were lysated and rnas were extracted by using rna spin mini kit ( ge healthcare ) according to manufacturer 's instructions . total rna ( 1  g ) was reverse transcribed using random hexamers and superscript ii reverse transcriptase ( invitrogen ) , and then the resulting template ( 20  ng cdna ) was used for each real - time pcr reaction ( abi prism 7700 , applied biosystems ) . primers for human ese-1 and icam-1 were purchased from acgt , toronto . for relative quantification , pcr signals were compared between groups after normalization with gapdh as an internal reference . primers used were hicam1-f : ggaaggtgtatgaactgagcaa , hicam1-r : ggagtccagtacacggtga , hese1-f : ggcgtcttcaagttcctgcg , hese1-r : ctcccgtttgtagtagtacct , hgapdh - f : gaaggtgaaggtcggagtc , hgapdh - r : gaagatggtgatgggatttc . cell lysates were run on an 8% sds - polyacrylamide gel at 10  g each lane and transferred to nitrocellulose membrane ( bio - rad ) following electrophoresis . the membrane was then blocked with tris - buffered saline with tween-20 ( tbst ) containing 5% milk and probed with goat anti - human icam-1 antibody ( r&d system , minneapolis , usa ) at 1  :  1000 dilution and rabbit anti - human ese-1 antibody ( r&d system , minneapolis , usa ) at 1  :  5000 dilution . rabbit anti - human gapdh antibodies ( trevigen , gaithersburg , usa ) were used at 1  :  3000 as a protein loading control . the horseradish peroxidase - conjugated secondary antibodies for rabbit anti - goat igg were from kirkegaard & perry laboratories and for goat anti - rabbit igg were from bio - rad laboratories . detection of proteins was performed with enhanced chemiluminescence reagents ( amersham pharmacia biotech , baie - d'urfe , ca ) . at day 0 , 1  10 a549 cells were seeded on a 10  cm dish and virus vector particles were added at 100  moi ( 5000 particles per cell ) for 1 hour in serum - free media . fetal bovine serum was then added to final concentration of 10% . at day 5 , the treated cells were harvested and seeded at 250,000 to each well of a-24-well plate . the reason for waiting for 5 days was to minimize the influence of inflammatory cytokines induced by the viral transduction . for visualization of cell - cell interactions , hl-60 cells were labeled with carboxyfluorescein succinimide ester ( cfse ) at 5  m in the cell culture for 5  min at 37c and then washed 3 times with rpmi 1640 . the labeled hl-60 cells were put back in the cell culture condition for 24 hours . the a549 cells were then stimulated with il-1 and tnf- each at 10  ng / ml for 6 hours and labeled hl-60 cells were activated with 12-o - tetradecanoylphorbol-13-acetate ( tpa ) at 5  ng / ml for 4 hours . after washing both cells , 1 million hl-60 cells in 0.5  ml of rpmi 1640 were added to each well of the treated a549 cells under cell culture conditions . following adhesion for 1 hour , nonattached hl-60 cells were washed away with 1x pbs for 3 times at room temperature . cells were fixed with 1% paraformaldehyde in 1x pbs for 10  min and washed 3 times with 1x pbs . data were analyzed by using mann - whitney u test ( kruskal - wallis test for more than two groups ) . to examine whether both ese-1 and icam-1 are upregulated under proinflammatory conditions , beas-2b , ib3 - 1 , and a549 cells were stimulated with a combination of proinflammatory cytokines tnf- and il-1 ( 10  ng / ml each ) for 2 hours , and their mrna levels were assessed by qrt - pcr . as shown in figures 1(a ) and 1(b ) , levels of mrna for both ese-1 and icam-1 were increased significantly ( except for ese-1 expression in a549 cells ) . we also observed a higher level of ese-1 mrna expression in a549 cells than beas-2b and ib3 - 1 cells . to further investigate at which time point ese-1 and icam-1 start responding to cytokine stimulation , we completed a time course study . as shown in figures 1(c ) and 1(d ) , the ese-1 mrna expression peaked at 2 hours after stimulation in a549 cells while icam-1 mrna expression reached a plateau at 4 hours after stimulation . since both ese-1 and icam-1 mrna expression were upregulated when stimulated by proinflammatory cytokines and given that ese-1 is a transcription factor , we questioned whether icam-1 expression is transcriptionally regulated by ese-1 . we used a helper - dependent adenovirus ( hd - ad ) vector to knockdown ese-1 gene expression in a549 cells and examined the changes in icam-1 expression . we selected a549 cells for the knockdown experiment , as these cells constitutively express relatively high levels of ese-1 , at both mrna and protein levels . as expected , we found a significant reduction of ese-1 mrna following hd - ad vector transduction ( figure 2(b ) ) . interestingly , icam-1 expression was also downregulated compared to the empty vector ( c4hsu ) control group ( figure 2(c ) ) . we examined ese-1 and icam-1 expression at days 4 and 5 after transduction because viral vectors alone can induce the production of proinflammatory cytokines that can last for more than two days after viral transduction . we also used longer time points for cytokine stimulation in protein analysis ( 16  h ) than in mrna analysis ( 2  h ) , as changes in protein expression lag that of the mrna . to further investigate the regulatory mechanism , we developed an icam-1 promoter reporter assay to examine the role of ese-1 in the regulation of icam-1 expression . as shown in figure 3(a ) , we made the reporter construct by inserting a 1.4  kb human icam-1 promoter in front of the luciferase reporter gene in pgl-3-basic vector ( promega ) . beas-2b cells were cotransfected with the icam-1 promoter luciferase reporter plasmid and pcdna3-ese-1 or pcdna3-empty as an empty vector control . beas-2b cells are noncancerous human bronchial epithelial cells which express minimal basal levels of ese-1 , allowing us to specifically overexpress the protein by transfection . twenty - four hours after cotransfection , the luciferase activity was measured and as shown in figure 3(b ) , the level of luciferase activity was increased in the ese-1 overexpression group compared to empty vector control group , suggesting that ese-1 regulates icam-1 expression at the transcriptional level . since icam-1 is involved in cell - cell interactions , we performed a cell adhesion assay to investigate whether knockdown of ese-1 gene could affect cellular adhesion . we found that , following transduction with ese-1-rnai viral vector , a549 cells had less binding of hl-60 cells compared with c4hsu empty vector transduced group ( figure 4 ) . in response to environmental perturbations , airway epithelia produce and release a variety of inflammatory cytokines , including tnf- , il-1 , il-6 , and il-8 . a number of studies have shown that the epithelium of patients who have airway inflammatory diseases is structurally and functionally altered , and that bronchial epithelial cells that are isolated from patients with asthma or cystic fibrosis ( cf ) express increased levels of cytokines ( il-8 , il-25 ) [ 40 , 41 ] . it is thus important to fully understand the gene regulation in airway epithelium in order to alleviate airway inflammatory diseases . in this study , we show that both ese-1 and icam-1 mrnas are upregulated in lung epithelial cell lines after treatment with tnf- and il-1. however , while both genes can be activated by the nf-b , we questioned whether there was any regulatory relationship between the two . the regulation of icam-1 gene expression is primarily at the transcriptional level by several signaling pathways including protein kinase c ( pkc ) , mitogen activated protein ( map ) kinase ( jnk , erk , and p38 ) , and nf-b . the human icam-1 gene promoter contains binding sites for many transcription factors , including ap-1 , c / ebp , ets , nf-b , stat , and sp1 . since ese-1 is a member of ets family transcription factors which shares dna binding sites , we decided to examine whether ese-1 regulated icam-1 expression . to investigate the relationship between these two genes , we used helper - dependent adenovirus vector containing shrna - ese-1 to knockdown ese-1 . we observed that icam-1 expression in a549 cells was indeed decreased after knocking down ese-1 . to further investigate on the mechanism of this relationship between ese-1 and icam-1 , we used icam-1 promoter luciferase reporter assays to determine whether this upregulation was at the transcription level . upon cotransfecting the luciferase reporter plasmid with an ese-1 gene expressing plasmid into beas-2b cells , we observed an increase in luciferase activity when ese-1 gene was overexpressed compared to the cells cotransfected with an empty vector plasmid . however , we noted that the level of upregulation was not dramatic , but under chronic inflammatory conditions , a small change in icam-1 levels may have a significant impact on disease progression . we also performed a cell adhesion assay to investigate whether knocking down ese-1 could affect cell adhesion of epithelial cells . we show that , following transduction with ese-1-rnai vector , a549 cells exhibited less binding of hl-60 cells compared to groups of transduced with c4hsu empty vector or untransduced . this suggests that knocking down ese-1 in a549 cells causes downregulation of icam-1 gene expression which in turn results in a reduced binding interaction between hl-60 and a549 cells . although our finding of ese-1 regulating icam-1 expression in this work is interesting , there are limitations to its clinical applications . first , ese-1 knocking down was carried out only in a549 cells , not in primary cf lung epithelial cells . future studies on this regulation in other model systems will be important for the development of effective anti - inflammatory strategies suitable for therapeutic intervention of inflammatory conditions such as cf . since ese-1 is an transcription factor , it could be a potential drug target for screening small molecules that inhibit its expression .
cystic fibrosis ( cf ) patients suffer from chronic airway inflammation with excessive neutrophil infiltration . 
 migration of neutrophils to the lung requires chemokine and cytokine signaling as well as cell adhesion molecules , such as intercellular adhesion molecule-1 ( icam-1 ) , which plays an important role in mediating adhesive interactions between effector and target cells in the immune system . in this study 
 , we investigated the relationship between icam-1 and epithelium - specific ets - like transcription factor 1 ( ese-1 ) and found that icam-1 expression is upregulated in cell lines of cf ( ib3 - 1 ) as well as non - cf ( beas-2b and a549 ) epithelial origin in response to inflammatory cytokine stimulation . 
 since ese-1 is highly expressed in a549 cells without stimulation , we examined the effect of ese-1 knockdown on icam-1 expression in these cells . 
 we found that icam-1 expression was downregulated when ese-1 was knocked down in a549 cells . 
 we also tested the effect of ese-1 knockdown on cell - cell interactions and demonstrate that the knocking down ese-1 in a549 cells reduce their interactions with hl-60 cells ( human promyelocytic leukemia cell line ) . 
 these results suggest that ese-1 may play a role in regulating airway inflammation by regulating icam-1 expression .