Background Growing evidence shows that interleukin 13 (IL-13) may play an essential role in the development of airway inflammation and bronchial hyper-responsiveness (BHR), two defining features of asthma. Although the underlying mechanisms remain unknown, a number of reports have shown that IL-13 may exert its deleterious effects in asthma by directly acting on airway resident cells, including epithelial cells and airway smooth muscle cells. In this report, we hypothesize that IL-13 may participate in the pathogenesis of asthma by activating a set of "pro-asthmatic" genes in airway smooth muscle (ASM) cells.
Background
Growing evidence shows that interleukin 13 (IL-13) may play an essential role in the development of airway inflammation and bronchial hyper-responsiveness (BHR), two defining features of asthma. Although the underlying mechanisms remain unknown, a number of reports have shown that IL-13 may exert its deleterious effects in asthma by directly acting on airway resident cells, including epithelial cells and airway smooth muscle cells. In this report, we hypothesize that IL-13 may participate in the pathogenesis of asthma by activating a set of "pro-asthmatic" genes in airway smooth muscle (ASM) cells.Methods Microarray technology was used to study the modulation of gene expression of airway smooth muscle by IL-13 and IL-13R130Q. TaqMan™ Real Time PCR and flow cytometry was used to validate the gene array data.
Methods
Microarray technology was used to study the modulation of gene expression of airway smooth muscle by IL-13 and IL-13R130Q. TaqMan™ Real Time PCR and flow cytometry was used to validate the gene array data.Results IL-13 and the IL-13 polymorphism IL-13R130Q (Arg130Gln), recently associated with allergic asthma, seem to modulate the same set of genes, which encode many potentially interesting proteins including vascular cellular adhesion molecule (VCAM)-1, IL-13Rα2, Tenascin C and Histamine Receptor H1, that may be relevant for the pathogenesis of asthma.
Results
IL-13 and the IL-13 polymorphism IL-13R130Q (Arg130Gln), recently associated with allergic asthma, seem to modulate the same set of genes, which encode many potentially interesting proteins including vascular cellular adhesion molecule (VCAM)-1, IL-13Rα2, Tenascin C and Histamine Receptor H1, that may be relevant for the pathogenesis of asthma.Conclusions The data supports the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma.
Conclusions
The data supports the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma.

Background Recent reports using murine models of allergic asthma have shown that the Th2 type cytokine IL-13 may play a critical role in the pathogenesis of asthma, either by regulating airway inflammation, mucus hyper-secretion or airway hyper-responsiveness [ 1 - 5 ]. Evidence suggests that the potential role of IL-13 in asthma may come from its aptitude to directly interact with airway resident cells, such as epithelial cells or airway smooth muscle (ASM) cells, as shown by the ability of IL-13 to stimulate a set of different pro-asthmatic genes including inflammatory cytokines such as thymus and activation-regulated chemokine (TARC), eotaxin, monocyte chemotactic protein-1 (MCP-1) as well as growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) [ 6 - 10 ]. The ability of IL-13 to modulate ASM responsiveness to G-protein coupled receptor (GPCR) agonists, either by increasing contractile agonist-evoked calcium responses [ 11 ], and/or by impairing ASM responsiveness to β2-adrenoceptor stimulation [ 6 ], may also explain, at least in part, the putative role of IL-13 in allergen-associated BHR reported in animal models [ 1 - 4 ]. Previous reports have shown that other cytokines such as tumor necrosis factor alpha (TNFα) or interleukin (IL)-1β, may also participate in airway hyper-responsiveness by modulating ASM responsiveness to contractile GPCR agonists [ 12 - 14 ]. These data strongly support the current concept that cytokine modulation of ASM, an effector cell thought to solely regulate bronchomotor tone [ 12 ], may play an important role in the development of airway inflammation and bronchial hyper-responsiveness, the two main features of asthma. The molecular mechanisms by which IL-13 induces "pro-asthmatic responses" in ASM have not been clearly established. Identifying the expression profile of "pro-asthmatic" genes activated by IL-13 in ASM cells may therefore provide new insight into the design of novel therapeutic approaches for asthma. Using complementary molecular approaches, we investigated the effect of IL-13 on the transcription of "pro-asthmatic" genes in human airway smooth muscle cells (HASMC). The effect of IL-13 was compared to that of IL-13R130Q, a naturally occurring isoform resulting in a change from glutamine to arginine residues in the coding region that is associated with high serum IgE levels [ 15 ]. Interestingly, no report has yet investigated whether both IL-13 and IL-13R130Q share the same or have different biological activities. We found that both IL-13 and IL-13R130Q stimulate the same set of important genes that encode for proteins which may be clinically relevant for regulating airway hyper-responsiveness, airway inflammation and airway remodeling, key characteristics of asthma.
Background
Recent reports using murine models of allergic asthma have shown that the Th2 type cytokine IL-13 may play a critical role in the pathogenesis of asthma, either by regulating airway inflammation, mucus hyper-secretion or airway hyper-responsiveness [ 1 - 5 ]. Evidence suggests that the potential role of IL-13 in asthma may come from its aptitude to directly interact with airway resident cells, such as epithelial cells or airway smooth muscle (ASM) cells, as shown by the ability of IL-13 to stimulate a set of different pro-asthmatic genes including inflammatory cytokines such as thymus and activation-regulated chemokine (TARC), eotaxin, monocyte chemotactic protein-1 (MCP-1) as well as growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) [ 6 - 10 ]. The ability of IL-13 to modulate ASM responsiveness to G-protein coupled receptor (GPCR) agonists, either by increasing contractile agonist-evoked calcium responses [ 11 ], and/or by impairing ASM responsiveness to β2-adrenoceptor stimulation [ 6 ], may also explain, at least in part, the putative role of IL-13 in allergen-associated BHR reported in animal models [ 1 - 4 ]. Previous reports have shown that other cytokines such as tumor necrosis factor alpha (TNFα) or interleukin (IL)-1β, may also participate in airway hyper-responsiveness by modulating ASM responsiveness to contractile GPCR agonists [ 12 - 14 ]. These data strongly support the current concept that cytokine modulation of ASM, an effector cell thought to solely regulate bronchomotor tone [ 12 ], may play an important role in the development of airway inflammation and bronchial hyper-responsiveness, the two main features of asthma. The molecular mechanisms by which IL-13 induces "pro-asthmatic responses" in ASM have not been clearly established. Identifying the expression profile of "pro-asthmatic" genes activated by IL-13 in ASM cells may therefore provide new insight into the design of novel therapeutic approaches for asthma.Using complementary molecular approaches, we investigated the effect of IL-13 on the transcription of "pro-asthmatic" genes in human airway smooth muscle cells (HASMC). The effect of IL-13 was compared to that of IL-13R130Q, a naturally occurring isoform resulting in a change from glutamine to arginine residues in the coding region that is associated with high serum IgE levels [ 15 ]. Interestingly, no report has yet investigated whether both IL-13 and IL-13R130Q share the same or have different biological activities. We found that both IL-13 and IL-13R130Q stimulate the same set of important genes that encode for proteins which may be clinically relevant for regulating airway hyper-responsiveness, airway inflammation and airway remodeling, key characteristics of asthma.Methods Cell Culture Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions and the tracheal muscle was isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCl 2 , 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105 μm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F12 medium (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT) 100 U/ml penicillin (Gibco), 0.1 mg/ml streptomycin (Gibco), and 2.5 μg/ml fungizone (Gibco). Aliquots of the cell suspension were plated at a density of 1.0 × 10 4  cells/cm 2 . The cells were cultured in Ham's F12 media supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and this was replaced every 72 h. Human ASM cells in subculture during the second through to fifth cell passages were used because, during these cell passages, the cells retain native contractile protein expression, as demonstrated by immunocytochemical staining for smooth muscle actin and myosin [ 16 ]. Unless otherwise specified, all chemicals used in this study were purchased from Sigma/Aldrich (St. Louis, MO). RNA isolation Total cellular RNA was isolated from IL-13 (50 ng/ml), IL-13R130Q (50 ng/ml) or control treated HASMC using the RNeasy mini kit (Qiagen, Inc. Valencia, CA) as per manufacturer's instructions. The IL-13 was purchased from R&D Systems (Minneapolis, MN) and the IL-13R130Q was generated in house at Centocor Inc. The quality and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer (South Plainfield, New Jersey). Samples that demonstrated high quality (ratio of 28S rRNA and 18S rRNA is greater than 1.7) were submitted for microarray analysis. Microarray Processing A complimentary DNA (cDNA) microarray, or DNA chip (Target B), containing a total of 8159 human gene cDNA clones from Research Genetics (IMAGE consortium, Huntsville, AL), Incyte Genomics (Santa Clara, CA) and internal sources was used in this study. All clones have been verified by DNA sequencing and are printed as 2 independent spots on a given chip. Duplicate chips were used for each RNA sample. Non-linear normalization between duplicate chips allowed each clone to be averaged to a single intensity value for each RNA sample. RNA amplification, probe synthesis and labeling, cDNA chip hybridization and washing were performed as described previously [ 17 ]. Agilent Image Scanner was used to scan the cDNA chips (Agilent Technologies, Palo Alto, CA). Fluorescence intensity for each feature of the array was obtained by using ImaGene software (BioDiscovery, Los Angeles, CA). Microarray data analysis In this study, fifty one-color cDNA microarrays were used to profile gene expression in human airway smooth muscle cells from 3 donors stimulated with IL-13, or its variant IL-13R130Q at 2 time points (6 hr and 18 hr). Untreated samples from the same group of donors were used as control. The samples being analyzed are listed in Table  1 . Table 1 Summary of number of samples from each donor and treatments Time Donor Untreated IL-13 IL-13R130Q 6 hr Donor 1* - - - Donor 2 1 2 2 Donor 3 1 2 2 18 hr Donor 1 1 2 2 Donor 2 1 2 2 Donor 3 1 2 2 *The samples from Donor 1 at the 6 hr time point were not included due to poor quality of RNA. Purified cDNA probes were hybridized to two microarrays, each containing two spots for each cDNA. Raw intensity data from the cDNA arrays were first normalized within each sample. Linear normalization and then nonlinear normalization was performed within each sample. Outlier intensity data points (greater than 1.4 fold away from the median of replicate measurements) were identified and removed from the data sets. The average intensity was generated by calculating the arithmetic mean of nonoutlier intensity values. The averaged intensity for each clone was further normalized across all samples. Chip-to-chip normalization was performed by dividing the averaged intensity of each clone by the 50.0 th  percentile of all measurements in that sample. The intensity of each clone was then normalized to the median intensity of that clone in the untreated control group. The normalized intensity was then log transformed. Using Partek Pro™ 5.1, sources of variance, such as batch effects, were identified by Principle Component Analysis (PCA) and appropriate factors were taken into account in the Analysis of Variance (ANOVA). ANOVA was performed to identify the genes that were differentially expressed by cytokine stimulation. Treatment (IL-13 and IL-13R130Q), time (6 hour and 18 hour), and donor (1, 2, and 3) were the three main effects considered in ANOVA. P-value cutoff was 0.05. Benjamini and Hochberg false discovery rate (FDR) was performed for multiple testing correction. After comparing the gene lists from IL-13 and IL-13R130Q treatments, it was clear that these two treatments resulted in the regulation of the same set of genes. Subsequently, samples from these two treatments were combined and regarded as replicates in ANOVA. Furthermore, outliner samples in the data set were detected by PCA and removed to improve the detection power. As an alternative approach, fold change comparisons (cutoff = 1.5 fold) between a treated condition and the control were carried out within each donor by using GeneSpring™ 6.2 [ 18 ]. A gene was considered as reliably detected in a given condition if more than half of the replicates representing the same condition had a raw expression intensity of more than 50, CV smaller than 25%, and raw intensity being generated from 2 or more of the duplicate spots representing the clone. A pair-wise comparison between a treatment and its untreated control was performed only on the genes that were reliably detected in at least one condition of the pair. The genes that showed at least 1.5 fold differential expression in two or more donors were identified for each cytokine treatment at a time. Reverse Transcription and Real Time PCR 1 μg of total RNA from each of the IL-13 (50 ng/ml) or IL-13R130Q (50 ng/ml) or control treated HASMC were used for the reverse transcription (RT) reaction. The RT reaction was performed as per protocol using TaqMan ®  RT reagents (Applied Biosystems) at 37°C for 120 min followed by 25°C for 10 min. Forty ng of cDNA per reaction were used in the Real Time PCR using the ABI Prism ®  7900 sequence detection system (Foster City, California). In the presence of AmpliTaq Gold DNA plolymerase (ABI biosystem, Foster City, California), the reaction was incubated for 2 min at 50°C followed by 10 min at 95°C. Then the reaction was run for 40 cycles at 15 sec, 95°C and 1 min, 60°C per cycle. Assays-on-Demand™ primers and probes (Applied Biosystems) were used in the PCR. The Real Time PCR data was analyzed using the standard curve method. Flow Cytometry Flow cytometry was performed as described previously with slight modifications [ 19 ]. Briefly, adherent cells treated with IL-13 for 24 hr were washed with PBS, detached by trypsinization (2 min, 37°C) and then washed with Ham's-F12 (10% FCS) media, centrifuged, and transferred to microfuge tubes (1.5 ml). Cells were incubated with anti-IL-13Rα2 (5 μg/ml, Santa Cruz Biotech) antibody followed by 1 hr incubation with a fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories; West Grove, PA). In parallel experiements, cells were incubated with the FITC-conjugated mouse anti-VCAM-1 antibody (2 μg/ml, Santa Cruz Biotech) for 1 h at 4°C. The cells were then centrifuged and resuspended in cold PBS in microfuge tubes. Samples were then analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). VCAM-1 and IL-13Rα2 levels were expressed as the increase in mean fluorescence intensity over un-stimulated cells.
Methods
Cell Culture Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions and the tracheal muscle was isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCl 2 , 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105 μm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F12 medium (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT) 100 U/ml penicillin (Gibco), 0.1 mg/ml streptomycin (Gibco), and 2.5 μg/ml fungizone (Gibco). Aliquots of the cell suspension were plated at a density of 1.0 × 10 4  cells/cm 2 . The cells were cultured in Ham's F12 media supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and this was replaced every 72 h. Human ASM cells in subculture during the second through to fifth cell passages were used because, during these cell passages, the cells retain native contractile protein expression, as demonstrated by immunocytochemical staining for smooth muscle actin and myosin [ 16 ]. Unless otherwise specified, all chemicals used in this study were purchased from Sigma/Aldrich (St. Louis, MO).
Cell Culture
Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions and the tracheal muscle was isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCl 2 , 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105 μm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham's F12 medium (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT) 100 U/ml penicillin (Gibco), 0.1 mg/ml streptomycin (Gibco), and 2.5 μg/ml fungizone (Gibco). Aliquots of the cell suspension were plated at a density of 1.0 × 10 4  cells/cm 2 . The cells were cultured in Ham's F12 media supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and this was replaced every 72 h. Human ASM cells in subculture during the second through to fifth cell passages were used because, during these cell passages, the cells retain native contractile protein expression, as demonstrated by immunocytochemical staining for smooth muscle actin and myosin [ 16 ]. Unless otherwise specified, all chemicals used in this study were purchased from Sigma/Aldrich (St. Louis, MO).RNA isolation Total cellular RNA was isolated from IL-13 (50 ng/ml), IL-13R130Q (50 ng/ml) or control treated HASMC using the RNeasy mini kit (Qiagen, Inc. Valencia, CA) as per manufacturer's instructions. The IL-13 was purchased from R&D Systems (Minneapolis, MN) and the IL-13R130Q was generated in house at Centocor Inc. The quality and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer (South Plainfield, New Jersey). Samples that demonstrated high quality (ratio of 28S rRNA and 18S rRNA is greater than 1.7) were submitted for microarray analysis.
RNA isolation
Total cellular RNA was isolated from IL-13 (50 ng/ml), IL-13R130Q (50 ng/ml) or control treated HASMC using the RNeasy mini kit (Qiagen, Inc. Valencia, CA) as per manufacturer's instructions. The IL-13 was purchased from R&D Systems (Minneapolis, MN) and the IL-13R130Q was generated in house at Centocor Inc. The quality and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer (South Plainfield, New Jersey). Samples that demonstrated high quality (ratio of 28S rRNA and 18S rRNA is greater than 1.7) were submitted for microarray analysis.Microarray Processing A complimentary DNA (cDNA) microarray, or DNA chip (Target B), containing a total of 8159 human gene cDNA clones from Research Genetics (IMAGE consortium, Huntsville, AL), Incyte Genomics (Santa Clara, CA) and internal sources was used in this study. All clones have been verified by DNA sequencing and are printed as 2 independent spots on a given chip. Duplicate chips were used for each RNA sample. Non-linear normalization between duplicate chips allowed each clone to be averaged to a single intensity value for each RNA sample. RNA amplification, probe synthesis and labeling, cDNA chip hybridization and washing were performed as described previously [ 17 ]. Agilent Image Scanner was used to scan the cDNA chips (Agilent Technologies, Palo Alto, CA). Fluorescence intensity for each feature of the array was obtained by using ImaGene software (BioDiscovery, Los Angeles, CA).
Microarray Processing
A complimentary DNA (cDNA) microarray, or DNA chip (Target B), containing a total of 8159 human gene cDNA clones from Research Genetics (IMAGE consortium, Huntsville, AL), Incyte Genomics (Santa Clara, CA) and internal sources was used in this study. All clones have been verified by DNA sequencing and are printed as 2 independent spots on a given chip. Duplicate chips were used for each RNA sample. Non-linear normalization between duplicate chips allowed each clone to be averaged to a single intensity value for each RNA sample.RNA amplification, probe synthesis and labeling, cDNA chip hybridization and washing were performed as described previously [ 17 ]. Agilent Image Scanner was used to scan the cDNA chips (Agilent Technologies, Palo Alto, CA). Fluorescence intensity for each feature of the array was obtained by using ImaGene software (BioDiscovery, Los Angeles, CA).Microarray data analysis In this study, fifty one-color cDNA microarrays were used to profile gene expression in human airway smooth muscle cells from 3 donors stimulated with IL-13, or its variant IL-13R130Q at 2 time points (6 hr and 18 hr). Untreated samples from the same group of donors were used as control. The samples being analyzed are listed in Table  1 . Table 1 Summary of number of samples from each donor and treatments Time Donor Untreated IL-13 IL-13R130Q 6 hr Donor 1* - - - Donor 2 1 2 2 Donor 3 1 2 2 18 hr Donor 1 1 2 2 Donor 2 1 2 2 Donor 3 1 2 2 *The samples from Donor 1 at the 6 hr time point were not included due to poor quality of RNA. Purified cDNA probes were hybridized to two microarrays, each containing two spots for each cDNA. Raw intensity data from the cDNA arrays were first normalized within each sample. Linear normalization and then nonlinear normalization was performed within each sample. Outlier intensity data points (greater than 1.4 fold away from the median of replicate measurements) were identified and removed from the data sets. The average intensity was generated by calculating the arithmetic mean of nonoutlier intensity values. The averaged intensity for each clone was further normalized across all samples. Chip-to-chip normalization was performed by dividing the averaged intensity of each clone by the 50.0 th  percentile of all measurements in that sample. The intensity of each clone was then normalized to the median intensity of that clone in the untreated control group. The normalized intensity was then log transformed. Using Partek Pro™ 5.1, sources of variance, such as batch effects, were identified by Principle Component Analysis (PCA) and appropriate factors were taken into account in the Analysis of Variance (ANOVA). ANOVA was performed to identify the genes that were differentially expressed by cytokine stimulation. Treatment (IL-13 and IL-13R130Q), time (6 hour and 18 hour), and donor (1, 2, and 3) were the three main effects considered in ANOVA. P-value cutoff was 0.05. Benjamini and Hochberg false discovery rate (FDR) was performed for multiple testing correction. After comparing the gene lists from IL-13 and IL-13R130Q treatments, it was clear that these two treatments resulted in the regulation of the same set of genes. Subsequently, samples from these two treatments were combined and regarded as replicates in ANOVA. Furthermore, outliner samples in the data set were detected by PCA and removed to improve the detection power. As an alternative approach, fold change comparisons (cutoff = 1.5 fold) between a treated condition and the control were carried out within each donor by using GeneSpring™ 6.2 [ 18 ]. A gene was considered as reliably detected in a given condition if more than half of the replicates representing the same condition had a raw expression intensity of more than 50, CV smaller than 25%, and raw intensity being generated from 2 or more of the duplicate spots representing the clone. A pair-wise comparison between a treatment and its untreated control was performed only on the genes that were reliably detected in at least one condition of the pair. The genes that showed at least 1.5 fold differential expression in two or more donors were identified for each cytokine treatment at a time.
Microarray data analysis
In this study, fifty one-color cDNA microarrays were used to profile gene expression in human airway smooth muscle cells from 3 donors stimulated with IL-13, or its variant IL-13R130Q at 2 time points (6 hr and 18 hr). Untreated samples from the same group of donors were used as control. The samples being analyzed are listed in Table  1 .Summary of number of samples from each donor and treatments*The samples from Donor 1 at the 6 hr time point were not included due to poor quality of RNA.Purified cDNA probes were hybridized to two microarrays, each containing two spots for each cDNA. Raw intensity data from the cDNA arrays were first normalized within each sample. Linear normalization and then nonlinear normalization was performed within each sample. Outlier intensity data points (greater than 1.4 fold away from the median of replicate measurements) were identified and removed from the data sets. The average intensity was generated by calculating the arithmetic mean of nonoutlier intensity values. The averaged intensity for each clone was further normalized across all samples. Chip-to-chip normalization was performed by dividing the averaged intensity of each clone by the 50.0 th  percentile of all measurements in that sample. The intensity of each clone was then normalized to the median intensity of that clone in the untreated control group. The normalized intensity was then log transformed.Using Partek Pro™ 5.1, sources of variance, such as batch effects, were identified by Principle Component Analysis (PCA) and appropriate factors were taken into account in the Analysis of Variance (ANOVA). ANOVA was performed to identify the genes that were differentially expressed by cytokine stimulation. Treatment (IL-13 and IL-13R130Q), time (6 hour and 18 hour), and donor (1, 2, and 3) were the three main effects considered in ANOVA. P-value cutoff was 0.05.Benjamini and Hochberg false discovery rate (FDR) was performed for multiple testing correction. After comparing the gene lists from IL-13 and IL-13R130Q treatments, it was clear that these two treatments resulted in the regulation of the same set of genes. Subsequently, samples from these two treatments were combined and regarded as replicates in ANOVA. Furthermore, outliner samples in the data set were detected by PCA and removed to improve the detection power.As an alternative approach, fold change comparisons (cutoff = 1.5 fold) between a treated condition and the control were carried out within each donor by using GeneSpring™ 6.2 [ 18 ]. A gene was considered as reliably detected in a given condition if more than half of the replicates representing the same condition had a raw expression intensity of more than 50, CV smaller than 25%, and raw intensity being generated from 2 or more of the duplicate spots representing the clone. A pair-wise comparison between a treatment and its untreated control was performed only on the genes that were reliably detected in at least one condition of the pair. The genes that showed at least 1.5 fold differential expression in two or more donors were identified for each cytokine treatment at a time.Reverse Transcription and Real Time PCR 1 μg of total RNA from each of the IL-13 (50 ng/ml) or IL-13R130Q (50 ng/ml) or control treated HASMC were used for the reverse transcription (RT) reaction. The RT reaction was performed as per protocol using TaqMan ®  RT reagents (Applied Biosystems) at 37°C for 120 min followed by 25°C for 10 min. Forty ng of cDNA per reaction were used in the Real Time PCR using the ABI Prism ®  7900 sequence detection system (Foster City, California). In the presence of AmpliTaq Gold DNA plolymerase (ABI biosystem, Foster City, California), the reaction was incubated for 2 min at 50°C followed by 10 min at 95°C. Then the reaction was run for 40 cycles at 15 sec, 95°C and 1 min, 60°C per cycle. Assays-on-Demand™ primers and probes (Applied Biosystems) were used in the PCR. The Real Time PCR data was analyzed using the standard curve method.
Reverse Transcription and Real Time PCR
1 μg of total RNA from each of the IL-13 (50 ng/ml) or IL-13R130Q (50 ng/ml) or control treated HASMC were used for the reverse transcription (RT) reaction. The RT reaction was performed as per protocol using TaqMan ®  RT reagents (Applied Biosystems) at 37°C for 120 min followed by 25°C for 10 min. Forty ng of cDNA per reaction were used in the Real Time PCR using the ABI Prism ®  7900 sequence detection system (Foster City, California). In the presence of AmpliTaq Gold DNA plolymerase (ABI biosystem, Foster City, California), the reaction was incubated for 2 min at 50°C followed by 10 min at 95°C. Then the reaction was run for 40 cycles at 15 sec, 95°C and 1 min, 60°C per cycle. Assays-on-Demand™ primers and probes (Applied Biosystems) were used in the PCR. The Real Time PCR data was analyzed using the standard curve method.Flow Cytometry Flow cytometry was performed as described previously with slight modifications [ 19 ]. Briefly, adherent cells treated with IL-13 for 24 hr were washed with PBS, detached by trypsinization (2 min, 37°C) and then washed with Ham's-F12 (10% FCS) media, centrifuged, and transferred to microfuge tubes (1.5 ml). Cells were incubated with anti-IL-13Rα2 (5 μg/ml, Santa Cruz Biotech) antibody followed by 1 hr incubation with a fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories; West Grove, PA). In parallel experiements, cells were incubated with the FITC-conjugated mouse anti-VCAM-1 antibody (2 μg/ml, Santa Cruz Biotech) for 1 h at 4°C. The cells were then centrifuged and resuspended in cold PBS in microfuge tubes. Samples were then analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). VCAM-1 and IL-13Rα2 levels were expressed as the increase in mean fluorescence intensity over un-stimulated cells.
Flow Cytometry
Flow cytometry was performed as described previously with slight modifications [ 19 ]. Briefly, adherent cells treated with IL-13 for 24 hr were washed with PBS, detached by trypsinization (2 min, 37°C) and then washed with Ham's-F12 (10% FCS) media, centrifuged, and transferred to microfuge tubes (1.5 ml). Cells were incubated with anti-IL-13Rα2 (5 μg/ml, Santa Cruz Biotech) antibody followed by 1 hr incubation with a fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories; West Grove, PA). In parallel experiements, cells were incubated with the FITC-conjugated mouse anti-VCAM-1 antibody (2 μg/ml, Santa Cruz Biotech) for 1 h at 4°C. The cells were then centrifuged and resuspended in cold PBS in microfuge tubes. Samples were then analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). VCAM-1 and IL-13Rα2 levels were expressed as the increase in mean fluorescence intensity over un-stimulated cells.Results IL-13 regulates gene expression of HASMCs IL-13 may exert its deleterious effects in asthma by directly altering gene expression in airway resident cells such as epithelial cells or ASM cells [ 5 - 7 , 20 ]. In order to determine which genes are regulated by IL-13 in airway smooth muscle cells, we employed the cDNA microarray technology. We also wanted to ascertain if the effect of IL-13R130Q, a naturally occurring isoform of IL-13 and associated with high serum IgE levels [ 15 ], was any different than IL-13 in terms of modulating gene expression. The concentrations of IL-13 (10–100 ng/ml) used in our study were shown previously to stimulate gene expression in human ASM cells [ 7 , 8 , 10 ], although the  in vivo  relevance of these particular concentrations remains unknown. Three donors were used and two types of analyses were carried out (Fold change analysis; Statistical Analysis). Both IL-13 and IL-13R130Q generated a similar expression profile i.e., genes regulated by IL-13 were the same as those regulated by IL-13R130Q at the 1.5 fold cutoff. Table  2  lists genes of interest that were identified from analyzing the data and divides them into one of three categories. Genes involved in all three characteristics of asthma (airway inflammation, remodeling and bronchial hyper-responsiveness) were identified. Of particular interest are vascular cellular adhesion molecule (VCAM)-1, Tenascin C, IL-13Rα2 and Histamine Receptor H1. Table 2 Summary of genes up regulated by IL-13 and IL-13R130Q. Category Gene(s) Fold change Airway Inflammation Adhesion Molecules VCAM-1 ↑  2 fold ALCAM Selectin P ligand Laminin B1 Chemokines Chemokine Ligand 2 Chemokine Ligand 11 Chemokine Ligand 26 Chemokine Ligand 27 Cytokine receptors IL-13 Rα2 ↑  1.6 fold Interleukin 1 receptor Airway Remodeling Extracellular matrix Tenascin C ↑  2 fold Tenascin R Collagen Type I Collagen Type VI Collagen Type III Fibulin 1 CD44 Cell proliferation Pim-1 eEF1A Cytokines PDGFC Retinoic acid Receptor Interferon beta 1 Bronchial Hyper-responsiveness Cytoskeletal constituants Vimentin Tropomyosin 1 Tropomyosin 2 Actin Calcium regulators Phospholipase D Calreticulin hGIRK1 TRPC4 TRPC6 Sphingosine kinase 1 Rho GDP dissociation inhibitor FKBP1A Receptor Histamine H1 receptor ↑  1.3 fold The fold changes correspond to the genes in bold. Real Time PCR validation TaqMan™ Real Time PCR was used to validate VCAM1, IL-13Rα2, Tenascin C and Histamine Receptor H1. As shown in Figure  1A , VCAM1 was upregulated between 2 and 2.5 fold upon IL-13 or IL-13R130Q treatment at the 6 and 18 hour time points in both donors. This is comparable to the microarray data (Table  2 ). In Figure  1B , IL-13Rα2 mRNA is upregulated with IL-13 or IL-13R130Q. However, the upregulation is more pronounced at the 18 hour time point compared to 6 hour. In Figure  2A  Tenascin C is upregulated with IL-13 and IL-13R130Q and in Figure  2B , Histamine Receptor H1 shows an upregulation of about 1.5 fold in both donors at both time points and with both treatments. Again, this is comparable to the microarray data (Table  2 ). Figure 1 Real Time PCR (Taqman ® ) analysis showing the level of A) VCAM1 B) IL-13Rα2 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6. Figure 2 Real Time PCR (Taqman ® ) analysis showing the level of A) Tenascin C and B) Histamine receptor H1 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6. Validation of VCAM-1 and IL-13Rα2 at the protein level In order to validate the modulatory effect of IL-13 on VCAM-1 and IL-13Rα2 genes at their protein level, flow cytometry was performed to confirm the up regulation of VCAM-1 and IL-13Rα2 in HASMC by IL-13. As shown in Figure  3  and  4 , IL-13 (10–100 ng/ml, 24 hr) differentially stimulates the expression of VCAM-1, with levels increasing in a dose-dependent manner, while IL-13Rα2 levels were identical at 10, 30 and 50 ng/ml. At 100 ng/ml IL-13, VCAM-1 and IL-13Rα2 levels were significantly increased by 20% and 35% over basal, respectively (n = 3, p < 0.05). Increases in VCAM-1 and IL-13Rα2 proteins by IL-13 nicely correlate with changes in mRNA levels (Figure  1A  and Figure  1B ), suggesting that the IL-13 regulates the expression of inflammatory proteins via transcriptional mechanisms. Figure 3 Effect of IL-13 on VCAM-1 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. VCAM-1 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. *P < 0.05 significant from untreated cell. Figure 4 Effect of IL-13 on IL-13Rα2 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. IL-13Rα2 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. *P < 0.05 significant from untreated cell.
Results
IL-13 regulates gene expression of HASMCs IL-13 may exert its deleterious effects in asthma by directly altering gene expression in airway resident cells such as epithelial cells or ASM cells [ 5 - 7 , 20 ]. In order to determine which genes are regulated by IL-13 in airway smooth muscle cells, we employed the cDNA microarray technology. We also wanted to ascertain if the effect of IL-13R130Q, a naturally occurring isoform of IL-13 and associated with high serum IgE levels [ 15 ], was any different than IL-13 in terms of modulating gene expression. The concentrations of IL-13 (10–100 ng/ml) used in our study were shown previously to stimulate gene expression in human ASM cells [ 7 , 8 , 10 ], although the  in vivo  relevance of these particular concentrations remains unknown. Three donors were used and two types of analyses were carried out (Fold change analysis; Statistical Analysis). Both IL-13 and IL-13R130Q generated a similar expression profile i.e., genes regulated by IL-13 were the same as those regulated by IL-13R130Q at the 1.5 fold cutoff. Table  2  lists genes of interest that were identified from analyzing the data and divides them into one of three categories. Genes involved in all three characteristics of asthma (airway inflammation, remodeling and bronchial hyper-responsiveness) were identified. Of particular interest are vascular cellular adhesion molecule (VCAM)-1, Tenascin C, IL-13Rα2 and Histamine Receptor H1. Table 2 Summary of genes up regulated by IL-13 and IL-13R130Q. Category Gene(s) Fold change Airway Inflammation Adhesion Molecules VCAM-1 ↑  2 fold ALCAM Selectin P ligand Laminin B1 Chemokines Chemokine Ligand 2 Chemokine Ligand 11 Chemokine Ligand 26 Chemokine Ligand 27 Cytokine receptors IL-13 Rα2 ↑  1.6 fold Interleukin 1 receptor Airway Remodeling Extracellular matrix Tenascin C ↑  2 fold Tenascin R Collagen Type I Collagen Type VI Collagen Type III Fibulin 1 CD44 Cell proliferation Pim-1 eEF1A Cytokines PDGFC Retinoic acid Receptor Interferon beta 1 Bronchial Hyper-responsiveness Cytoskeletal constituants Vimentin Tropomyosin 1 Tropomyosin 2 Actin Calcium regulators Phospholipase D Calreticulin hGIRK1 TRPC4 TRPC6 Sphingosine kinase 1 Rho GDP dissociation inhibitor FKBP1A Receptor Histamine H1 receptor ↑  1.3 fold The fold changes correspond to the genes in bold.
IL-13 regulates gene expression of HASMCs
IL-13 may exert its deleterious effects in asthma by directly altering gene expression in airway resident cells such as epithelial cells or ASM cells [ 5 - 7 , 20 ]. In order to determine which genes are regulated by IL-13 in airway smooth muscle cells, we employed the cDNA microarray technology. We also wanted to ascertain if the effect of IL-13R130Q, a naturally occurring isoform of IL-13 and associated with high serum IgE levels [ 15 ], was any different than IL-13 in terms of modulating gene expression. The concentrations of IL-13 (10–100 ng/ml) used in our study were shown previously to stimulate gene expression in human ASM cells [ 7 , 8 , 10 ], although the  in vivo  relevance of these particular concentrations remains unknown.in vivoThree donors were used and two types of analyses were carried out (Fold change analysis; Statistical Analysis). Both IL-13 and IL-13R130Q generated a similar expression profile i.e., genes regulated by IL-13 were the same as those regulated by IL-13R130Q at the 1.5 fold cutoff. Table  2  lists genes of interest that were identified from analyzing the data and divides them into one of three categories. Genes involved in all three characteristics of asthma (airway inflammation, remodeling and bronchial hyper-responsiveness) were identified. Of particular interest are vascular cellular adhesion molecule (VCAM)-1, Tenascin C, IL-13Rα2 and Histamine Receptor H1.Summary of genes up regulated by IL-13 and IL-13R130Q.Airway InflammationVCAM-12 foldIL-13 Rα21.6 foldAirway RemodelingTenascin C2 foldBronchial Hyper-responsivenessHistamine H1 receptor1.3 foldThe fold changes correspond to the genes in bold.Real Time PCR validation TaqMan™ Real Time PCR was used to validate VCAM1, IL-13Rα2, Tenascin C and Histamine Receptor H1. As shown in Figure  1A , VCAM1 was upregulated between 2 and 2.5 fold upon IL-13 or IL-13R130Q treatment at the 6 and 18 hour time points in both donors. This is comparable to the microarray data (Table  2 ). In Figure  1B , IL-13Rα2 mRNA is upregulated with IL-13 or IL-13R130Q. However, the upregulation is more pronounced at the 18 hour time point compared to 6 hour. In Figure  2A  Tenascin C is upregulated with IL-13 and IL-13R130Q and in Figure  2B , Histamine Receptor H1 shows an upregulation of about 1.5 fold in both donors at both time points and with both treatments. Again, this is comparable to the microarray data (Table  2 ). Figure 1 Real Time PCR (Taqman ® ) analysis showing the level of A) VCAM1 B) IL-13Rα2 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6. Figure 2 Real Time PCR (Taqman ® ) analysis showing the level of A) Tenascin C and B) Histamine receptor H1 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6.
Real Time PCR validation
TaqMan™ Real Time PCR was used to validate VCAM1, IL-13Rα2, Tenascin C and Histamine Receptor H1. As shown in Figure  1A , VCAM1 was upregulated between 2 and 2.5 fold upon IL-13 or IL-13R130Q treatment at the 6 and 18 hour time points in both donors. This is comparable to the microarray data (Table  2 ). In Figure  1B , IL-13Rα2 mRNA is upregulated with IL-13 or IL-13R130Q. However, the upregulation is more pronounced at the 18 hour time point compared to 6 hour. In Figure  2A  Tenascin C is upregulated with IL-13 and IL-13R130Q and in Figure  2B , Histamine Receptor H1 shows an upregulation of about 1.5 fold in both donors at both time points and with both treatments. Again, this is comparable to the microarray data (Table  2 ).Validation of VCAM-1 and IL-13Rα2 at the protein level In order to validate the modulatory effect of IL-13 on VCAM-1 and IL-13Rα2 genes at their protein level, flow cytometry was performed to confirm the up regulation of VCAM-1 and IL-13Rα2 in HASMC by IL-13. As shown in Figure  3  and  4 , IL-13 (10–100 ng/ml, 24 hr) differentially stimulates the expression of VCAM-1, with levels increasing in a dose-dependent manner, while IL-13Rα2 levels were identical at 10, 30 and 50 ng/ml. At 100 ng/ml IL-13, VCAM-1 and IL-13Rα2 levels were significantly increased by 20% and 35% over basal, respectively (n = 3, p < 0.05). Increases in VCAM-1 and IL-13Rα2 proteins by IL-13 nicely correlate with changes in mRNA levels (Figure  1A  and Figure  1B ), suggesting that the IL-13 regulates the expression of inflammatory proteins via transcriptional mechanisms. Figure 3 Effect of IL-13 on VCAM-1 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. VCAM-1 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. *P < 0.05 significant from untreated cell. Figure 4 Effect of IL-13 on IL-13Rα2 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. IL-13Rα2 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. *P < 0.05 significant from untreated cell.
Validation of VCAM-1 and IL-13Rα2 at the protein level
In order to validate the modulatory effect of IL-13 on VCAM-1 and IL-13Rα2 genes at their protein level, flow cytometry was performed to confirm the up regulation of VCAM-1 and IL-13Rα2 in HASMC by IL-13. As shown in Figure  3  and  4 , IL-13 (10–100 ng/ml, 24 hr) differentially stimulates the expression of VCAM-1, with levels increasing in a dose-dependent manner, while IL-13Rα2 levels were identical at 10, 30 and 50 ng/ml. At 100 ng/ml IL-13, VCAM-1 and IL-13Rα2 levels were significantly increased by 20% and 35% over basal, respectively (n = 3, p < 0.05). Increases in VCAM-1 and IL-13Rα2 proteins by IL-13 nicely correlate with changes in mRNA levels (Figure  1A  and Figure  1B ), suggesting that the IL-13 regulates the expression of inflammatory proteins via transcriptional mechanisms.Discussion Recent evidence using various experimental approaches such as gene-deficient mice, soluble inhibitors or transgene overexpressing IL-13 in the airways have highlighted the critical role of IL-13 in the pathogenesis of allergic asthma, possibly due to its ability to regulate goblet cell metaplasia, mucus hypersecretion and airway hyper-responsiveness [ 1 , 3 , 21 ]. Few reports that used microarray analyses applied to cultured human ASM cells demonstrated that IL-13 differentially regulates a number of important genes that are relevant for the pathogenesis of asthma [ 7 , 10 ]. Although the functional relevance of such gene microarray analyses remains yet uncertain, these studies strongly suggest that IL-13 may be involved in the pathogenesis of asthma by directly modulating physiological responses of the ASM. Compared to the previous gene microarray reports [ 7 , 10 ], we did confirm the physiological relevance of the microarray data using two different experimental approaches. At least for four different genes, VCAM-1 (an adhesion protein), IL-13Rα2, Histamine H1 receptor (a G protein-coupled receptor) and Tenascin C (an extracellular matrix glycoprotein), there was a close correlation between the data obtained from gene microarray with those obtained by real time PCR analyses. In addition, we showed that IL-13 stimulates the expression of VCAM-1 and IL-13Rα2 at the protein level, showing the physiological relevance of the gene array data. It is interesting to note that no difference in gene expression profile were noticeable between cells exposed to IL-13 or IL-13R130Q, an IL-13 polymorphism recently found to be associated with elevated serum and allergen-specific IgE [ 15 , 22 ]. Our report is the first to suggest that this particular IL-13 polymorphism is equally effective as IL-13 in the transcriptional regulation of the genes examined in the present study. Our present study further supports the concept that IL-13 regulates the expression of different "pro-asthmatic" genes that are potentially important in the regulation of all three key features of asthma, i.e., airway inflammation, airway remodeling and bronchial hyper-responsiveness (for details see Table  2 ). Previous reports using cultured ASM cells also demonstrated that IL-13 can stimulate the expression of other pro-inflammatory proteins, such as eotaxin [ 8 , 23 , 24 ], TARC [ 9 ] or VEGF [ 25 ] and tenascin [present report and [ 10 ]]. Upregulation of tenascin C and R, glycoproteins that contribute to extracellular matrix structure [ 26 ], may play an important role in airway remodeling, a characteristic of chronic asthma. The stimulatory role of IL-13 on VCAM-1 may be important in asthma since VCAM-1 has been regarded as a key player in the development of airway inflammation [ 27 ]. The ability of IL-13 to increase the expression of different G-protein coupled receptors (GPCR), such as Histamine H1 [present report and [ 10 ]] or CysLT1 receptor [ 28 ] represents one potential mechanism by which IL-13 promotes airway hyper-responsiveness to GPCR agonists previously described both  in vivo  [ 1 , 3 , 21 ] or  in vitro  in isolated airways preparations [ 11 , 29 , 30 ]. Additional studies are needed to determine whether IL-13 also modulates ASM responsiveness to Histamine. The receptor complex by which IL-13 regulates cellular function comprises the IL-13Rα1, which binds IL-13 and forms a complex with the IL-4Rα to initiate signal transduction via the JAK/STAT6 pathway [ 31 ]. IL-13Rα2, the other cell surface protein binds IL-13 with high affinity but the complex is not functionally active. One previous report using transgenic mice showed that overexpressing IL-13 in the airways induced a marked increase in both IL-13Rα1 and IL-13Rα2 mRNA levels, mostly in epithelial cells and macrophages [ 32 ]. Our study is the first to show that the expression of IL-13Rα2 is also transcriptionally increased by IL-13 in ASM cells. Although the functional significance of such regulation remains unknown, it is possible that the newly induced IL-13Rα2 could function as a decoy receptor to limit IL-13 signaling in ASM cells. Additional studies are needed to further support this hypothesis.
Discussion
Recent evidence using various experimental approaches such as gene-deficient mice, soluble inhibitors or transgene overexpressing IL-13 in the airways have highlighted the critical role of IL-13 in the pathogenesis of allergic asthma, possibly due to its ability to regulate goblet cell metaplasia, mucus hypersecretion and airway hyper-responsiveness [ 1 , 3 , 21 ]. Few reports that used microarray analyses applied to cultured human ASM cells demonstrated that IL-13 differentially regulates a number of important genes that are relevant for the pathogenesis of asthma [ 7 , 10 ]. Although the functional relevance of such gene microarray analyses remains yet uncertain, these studies strongly suggest that IL-13 may be involved in the pathogenesis of asthma by directly modulating physiological responses of the ASM. Compared to the previous gene microarray reports [ 7 , 10 ], we did confirm the physiological relevance of the microarray data using two different experimental approaches. At least for four different genes, VCAM-1 (an adhesion protein), IL-13Rα2, Histamine H1 receptor (a G protein-coupled receptor) and Tenascin C (an extracellular matrix glycoprotein), there was a close correlation between the data obtained from gene microarray with those obtained by real time PCR analyses. In addition, we showed that IL-13 stimulates the expression of VCAM-1 and IL-13Rα2 at the protein level, showing the physiological relevance of the gene array data. It is interesting to note that no difference in gene expression profile were noticeable between cells exposed to IL-13 or IL-13R130Q, an IL-13 polymorphism recently found to be associated with elevated serum and allergen-specific IgE [ 15 , 22 ]. Our report is the first to suggest that this particular IL-13 polymorphism is equally effective as IL-13 in the transcriptional regulation of the genes examined in the present study.Our present study further supports the concept that IL-13 regulates the expression of different "pro-asthmatic" genes that are potentially important in the regulation of all three key features of asthma, i.e., airway inflammation, airway remodeling and bronchial hyper-responsiveness (for details see Table  2 ). Previous reports using cultured ASM cells also demonstrated that IL-13 can stimulate the expression of other pro-inflammatory proteins, such as eotaxin [ 8 , 23 , 24 ], TARC [ 9 ] or VEGF [ 25 ] and tenascin [present report and [ 10 ]]. Upregulation of tenascin C and R, glycoproteins that contribute to extracellular matrix structure [ 26 ], may play an important role in airway remodeling, a characteristic of chronic asthma. The stimulatory role of IL-13 on VCAM-1 may be important in asthma since VCAM-1 has been regarded as a key player in the development of airway inflammation [ 27 ]. The ability of IL-13 to increase the expression of different G-protein coupled receptors (GPCR), such as Histamine H1 [present report and [ 10 ]] or CysLT1 receptor [ 28 ] represents one potential mechanism by which IL-13 promotes airway hyper-responsiveness to GPCR agonists previously described both  in vivo  [ 1 , 3 , 21 ] or  in vitro  in isolated airways preparations [ 11 , 29 , 30 ]. Additional studies are needed to determine whether IL-13 also modulates ASM responsiveness to Histamine.in vivoin vitroThe receptor complex by which IL-13 regulates cellular function comprises the IL-13Rα1, which binds IL-13 and forms a complex with the IL-4Rα to initiate signal transduction via the JAK/STAT6 pathway [ 31 ]. IL-13Rα2, the other cell surface protein binds IL-13 with high affinity but the complex is not functionally active. One previous report using transgenic mice showed that overexpressing IL-13 in the airways induced a marked increase in both IL-13Rα1 and IL-13Rα2 mRNA levels, mostly in epithelial cells and macrophages [ 32 ]. Our study is the first to show that the expression of IL-13Rα2 is also transcriptionally increased by IL-13 in ASM cells. Although the functional significance of such regulation remains unknown, it is possible that the newly induced IL-13Rα2 could function as a decoy receptor to limit IL-13 signaling in ASM cells. Additional studies are needed to further support this hypothesis.Conclusions These data further support the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma. Additional studies are clearly needed to define the transcriptional regulation of the different "pro-asthmatic" genes by IL-13, which may lead to novel therapeutic approaches for the treatment of allergic asthma.
Conclusions
These data further support the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma. Additional studies are clearly needed to define the transcriptional regulation of the different "pro-asthmatic" genes by IL-13, which may lead to novel therapeutic approaches for the treatment of allergic asthma.Authors' contributions FS, LL, YA and RAP participated in the conception and design of the study. FS coordinated the study and along with YA drafted the manuscript. OT performed the RNA isolation and flow cytometry experiments. CH and KL performed the microarray data analysis. MB performed the Real Time PCR experiments. DG and BA proof read the manuscript. All authors read and approved the final manuscript.
Authors' contributions
FS, LL, YA and RAP participated in the conception and design of the study. FS coordinated the study and along with YA drafted the manuscript. OT performed the RNA isolation and flow cytometry experiments. CH and KL performed the microarray data analysis. MB performed the Real Time PCR experiments. DG and BA proof read the manuscript. All authors read and approved the final manuscript.


Clinical Research  faces perhaps a certain grain of confusion. Often, it is mistakenly reduced to case report. Caveats of even greater risks may be hidden in the secrets of patents and interests of manufacturers covertly promoting their products. Thus, a book clearly stating what a clinical trial is, well located within the scientific frame, appears as a significant contribution enhancing the importance and complexity of XXIst Century human health.Clinical ResearchThe book is preceded by two editions (1981 and 1985) and well backed by the experience and prestige of its authors.  Clinical Research , a branch of  Applied Research , finds in clinical trials the most definitive tool for evaluation of its actual applicability. The book is divided in 19 chapters, each setting up unequivocally the  Fundamental Point  to be treated, as for example the introductory Chapter 1:  A properly planned and executed clinical trial is a powerful experimental technique for assessing the effectiveness of an intervention . A  clinical trial  compares the effect and value of intervention(s) against a control in human beings.  Intervention  refers either to a drug, a procedure or a technological device, thus implying that biomedical engineering scientists and professionals might be involved in it.  Clinical Research  always presents early stages where the laboratory work is mandatory. This book emphasizes the concept all along, so placing the clinical trial in its proper hierarchical perspective. There is laboratory work during drug, device and/or procedure development, animal studies and early tests in small number of human beings. Case reports, typical of the hospital, find their place as a warning of adverse effects or as a flag of an unexpected beneficial effect.Clinical ResearchApplied ResearchFundamental PointA properly planned and executed clinical trial is a powerful experimental technique for assessing the effectiveness of an interventionclinical trialInterventionClinical ResearchIn Chapter 2 the authors emphasize that a clinical trial must have a primary question. It should be carefully selected, as any subsidiary question must be, too. Otherwise, the investigators may lose the track. Chapter 3 deals with the study population, underlying the importance of unambiguous eligibility criteria, the latter having a direct influence on the recruitment of participants and, thus, on the final number involved in the project (which may end up with not enough data). Chapter 4 outlines the basic study design; it stresses the demand of a control group and the need of randomization for assigning participants to control and intervention groups.An extremely important subject is the randomization process, essential to avoid any possible bias (Chapter 5). Several forms are presented: simple, blocked and stratified. More elaborate procedures make use of adaptive methods. There are examples and an appendix with an algorithm of general applicability. Bias (a systematic error) is one of the main concerns; thus, a double-blind design is essential (Chapter 6). Chapter 7,  Sample Size , addresses the question of how many subjects is enough. The trial must have sufficient statistical power to detect differences between groups. Therefore, calculation of sample size with provision for adequate levels of significance is essential. Several situations with their respective examples are presented making of this pages an invaluable tool. Unfortunately, several equations have been printed in such a way that there is confusion on whether some parameters are factors or exponents.Sample SizeBaseline refers to the status of participants before the start of the intervention (Chapter 8), while Chapter 9 deals with the recruitment of participants. The latter depends on developing a careful plan with multiple strategies. Chapter 10 enters into data collection and quality control, wisely underlining that no study can be better than the quality of its data. Monitoring and even auditing are thus necessary. The history of new medical interventions abounds in sad experiences. Hence, adverse effects, if any at all, must be searched and assessed. Chapter 11 clearly probes into this complex aspect. Sadly, perhaps due to sheer enthusiasm (and I do no want to hint possible commercial interests because the idea alone crosses a risky line), often it has played a secondary role in clinical trials, as stated by the authors.Chapter 12, by Michelle J. Naughton and Sally A. Shumaker, catches the assessment of health-related quality of life, that is, trying to elucidate if the intervention improved, say, physical, psychological and social functioning, perception of well-being and health status, personal productivity, sexual life, sleep disturbances and other parameters. Not an easy task, indeed, although in certain extreme situations (as when life is supported by mechanical means and conscience is no longer present) may lead into deep controversies.The remaining seven chapters deal, respectively, with participant adherence to the program, survival analysis, monitoring response variables (37 pages), issues in data analysis (38 pages full of statistical considerations), closeout (that is, how to properly terminate the study), reporting and interpreting of results (one of the most difficult steps because recommendations will be produced that may end up in massive use of the intervention), and finally a chapter devoted to multicenter trials.The book provides an exceedingly good collection of references so that the interested reader can deepen in a particular subject. All in all, I would highly recommend it, to the physician, basic investigator and biomedical engineer. For the beginner, instead, it may be a little hard for some parts require good knowledge of statistics. It is no doubt excellent material for a course.