Proinflammatory and Th2 Cytokines Regulate the High Affinity IgE Receptor (FcεRI) and IgE-Dependant Activation of Human Airway Smooth Muscle Cells

Background The high affinity IgE receptor (FcεRI) is a crucial structure for IgE-mediated allergic reactions. We have previously demonstrated that human airway smooth muscle (ASM) cells express the tetrameric (αβγ2) FcεRI, and its activation leads to marked transient increases in intracellular Ca2+ concentration, release of Th-2 cytokines and eotaxin-1/CCL11. Therefore, it was of utmost importance to delineate the factors regulating the expression of FcεRI in human (ASM) cells. Methodology/Principal Findings Incubation of human bronchial and tracheal smooth muscle (B/TSM) cells with TNF-α, IL-1β or IL-4 resulted in a significant increase in FcεRI-α chain mRNA expression (p<0.05); and TNF-α, IL-4 enhanced the FcεRI-α protein expression compared to the unstimulated control at 24, 72 hrs after stimulation. Interestingly, among all other cytokines, only TNF-α upregulated the FcεRI-γ mRNA expression. FcεRI-γ protein expression remained unchanged despite the nature of stimulation. Of note, as a functional consequence of FcεRI upregulation, TNF-α pre-sensitization of B/TSM potentially augmented the CC (eotaxin-1/CCL11 and RANTES/CCL5, but not TARC/CCL17) and CXC (IL-8/CXCL8, IP-10/CXCL10) chemokines release following IgE stimulation (p<0.05, n = 3). Furthermore, IgE sensitization of B/TSM cells significantly enhanced the transcription of selective CC and CXC chemokines at promoter level compared to control, which was abolished by Lentivirus-mediated silencing of Syk expression. Conclusions/Significance Our data depict a critical role of B/TSM in allergic airway inflammation via potentially novel mechanisms involving proinflammatory, Th2 cytokines and IgE/FcεRI complex.


Introduction
Airway inflammation has been considered as a critical factor in the pathogenesis of allergic asthma, often associated with bronchial hyperresponsiveness and is correlated with disease severity [1]. The inflammation is mainly due to an increased number of activated T lymphocytes, mast cells, eosinophils, and neutrophils within the airway lumen and bronchial submucosa [1,2]. The CD4 + T cells have been demonstrated as the predominant cell type involved in the regulation of airway inflammation through the expression of T helper 2 (Th2) cytokines [2].
Besides these prototype inflammatory cells, however, airway smooth muscle (ASM) cells have been described recently as a rich source of proinflammatory cytokines, chemokines, and growth factors; and have been considered as key inflammatory determi-nants of asthma pertaining to their ability to contract in response to these mediators [3]. ASM cells can contribute directly to the pathogenesis of asthma by expressing cell adhesion and costimulatory molecules and by secreting multiple proinflammatory cytokines and chemokines that may perpetuate airway inflammation and the development of airway remodeling in vivo [4]. Therefore, it is now widely accepted that ASM actively participates in the pathogenesis of allergic asthma, by virtue of its role in airway inflammation, airway hyperresponsiveness and airway remodeling.
Several studies have led to novel insights into immune processes and the role of IgE in atopic diseases such as asthma [5]. Bronchial hyperresponsiveness has been shown to be associated with increased serum IgE levels and could be transferred from asthmatic to non-asthmatic subjects by IgE-rich serum administration [5]. The biological activities of IgE are mediated through the high affinity (FceRI) and the low affinity (FceRII or CD23) IgE receptors [6,7]. Initially discovered on mast cells and basophils which are involved in cellular degranulation, FceRI has been shown to be expressed by many inflammatory cells including Langerhans cells, dendritic cells, monocytes, eosinophils, platelets, neutrophils from allergic asthmatics, and bronchial epithelial cells [7,8]. Very recently, FceRI expression was detected in pinealocytes, the melatonin-secreting cells of the pineal gland, where it is proposed to provide a missing link between the neuroendocrine and the immune system in context of the neural control of allergic disorders [9].
Classically, TNF-a and IL-1b are prototype pro-inflammatory cytokines, which have been shown to play a central role in lung inflammation both in animal models and allergic patients [10,11,12,13]. Th-2 cytokines are also known to play a critical role in pathogenesis of allergic disorders, and have been investigated extensively in strategies targeting respiratory diseases [14]. In particular, IL-4, in combination with IL-13 is known to induce Th-2 humoral immune responses leading to IgE production by B-cells [6].
We have previously demonstrated that human ASM (HASM) cells express the tetrameric (abc2) FceRI and its activation leads to transient increases in intracellular Ca2 + concentration, and release of IL-4, IL-5, IL-13 and the CC chemokine eotaxin-1/CCL11 [15]. Taking into account the proinflammatory and Th-2 cytokine milieu in allergic inflammation and expression of FceRI on ASM, it was imperative to examine the factors regulating the expression of FceRI in human ASM cells. FceRI-a-chain, a member of the immunoglobulin superfamily, contains the binding site for its ligand (IgE) while both b and c chains are responsible for the downstream signal propagation through the phosphorylation of their immunoreceptor tyrosine-based activation motif (ITAM) [7].
Here we demonstrate that TNF-a, IL-1b, and IL-4 induce the FceRI-a mRNA and protein expression in human bronchial/ tracheal smooth muscle (B/TSM) cells. The functional studies demonstrated that the IgE stimulation of cytokine pre-sensitized B/TSM cells significantly augmented the selective CC and CXC chemokine expression. Interestingly, Lentivirus mediated spleen tyrosine kinase (Syk) silencing abrogated the IgE sensitizationinduced transcription of selective CC and CXC chemokines at promoter level. Cumulatively, these results suggest the potentially novel mechanisms of FceRI regulation, portraying the critical role of B/TSM associated IgE/FceRI complex in allergic airway inflammation.

Preparation of Human Bronchial/Tracheal Smooth Muscle (B/TSM) Cells
Three sources of human airway smooth muscle cells were used. Both hTERT immortalized and primary human bronchial smooth muscle (HBSM) cells were prepared as described by us previously [18,19]. Primary human tracheal smooth muscle (HTSM) cells were obtained from macroscopically healthy segments of the trachea during post-mortem in Respiratory Hospital at Health Sciences Centre, Winnipeg, MB, Canada, and were isolated, cultured as we used for primary HBSM cells [15,18,19]. At confluence, primary B/TSM cells exhibited spindle morphology and a hill-and-valley pattern that is characteristic of smooth muscle in culture. Moreover, B/TSM cells at confluence retain smooth muscle-specific actin, SM22, and calponin protein expression and mobilize intracellular Ca2+ in response to acetylcholine, a physiologically relevant contractile agonist [19]. In all the experiments, primary B/TSM cells were used at passages 2-5, and hTERT cells at passages 10-17.

Cell stimulation
Sub-confluent B/TSM cells were growth arrested and synchronized by serum deprivation for 48 h in Ham's F-12 medium containing 5 mg/ml human recombinant insulin, 5 mg/ml human transferrin, 5 ng/ml selenium, and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). Cells were then stimulated in fresh FBS-free medium containing human recombinant IL-1b (10 ng/ml), TNF-a (10 ng/ml), IL-4 (10 ng/ml), IgE (1 or 10 mg/ ml) or vehicle (medium alone) for time periods specific to experiments, as mentioned below.

RNA isolation and RT-PCR
Serum-deprived confluent B/TSM cultures were harvested, and total cellular RNA was extracted using TRIzolH method (Invitrogen Canada Inc., Burlington, ON). Reverse transcription was performed by using 2 mg of total RNA in a first-strand cDNA synthesis reaction with High Capacity cDNA Reverse transcriptase kit as recommended by the supplier (Applied Biosystems, Foster City, CA, USA). Oligonucleotide primers were synthesized on the basis of the entire coding region of human FceRI-a (GenBank accession no. NM 002001.2) as follows: Forward primer 59-CTCCATTACAAATGCCACAGTTG-39 and Reverse primer 59-CACGCGGAGCTTTTATTACAGTA-39; and for human FceRI-c (GenBank accession no. NM004106) were: Forward primer, 59-CCA GCA GTG GTC TTG CTC TTA C-39 and reverse primer, 59-GCA TGC AGG CAT ATG TGA TGC C-39. Primers for human housekeeping gene, glyceraldhyde-3phosphate dehydrogenase (GAPDH) are forward primer 59-AGCAATGCCTCCTGCACCACCAAC-39 and reverse primer 59-CCGGAGGGGCCATCCACAGTCT-39. The PCR (FceRIa, 35 cycles; FceRI-c, 30 cycles; GAPDH, 25 cycles) was conducted in a thermal cycler 'Mastercycler' (Eppendorf Canada, Mississauga, ON). Each cycle included denaturation (94uC, 1 min), annealing (FceRI-a, 59uC, 1 min; FceRI-c, 64uC, 1 min and; GAPDH, 55uC, 1 min), and extension (72uC, 1 min 30 s). The initial denaturation period was 5 min, and the final extension was 10 min. The size of the amplified FceRI-a, -c and GAPDH fragment was 116 bp, 338 bp and 137 bp, respectively. GAPDH was amplified as internal control. Amplified products were analyzed by DNA gel electrophoresis in 1.5% agarose and visualized by ethidium bromide staining under ultraviolet illumination. The specificity of the amplified bands was confirmed by nucleic acid sequencing (data not shown).

Real-time RT-PCR analysis
The FceRI-a and GAPDH standards were prepared using PCR-amplified cDNA from a human basophilic cell line (KU812, ATCCH # CRL-2099 TM ). PCR products were isolated from 2% w/v agarose gel using QIAEX II Agarose Gel Extraction kit (Qiagen Inc., Mississauga, ON, Canada). The primers used were same as we used for RT-PCR, as mentioned above. The amount of extracted cDNA was quantified by spectrophotometry and expressed as copy number. A serial dilution was used to generate each standard curve. Real-time quantitative PCR was carried out by ABI 7500 Real-Time PCR System and analyzed by 7500 System SDS software version 1.3.1 (Applied Biosystems, Foster City, CA, USA), following manufacturer's instructions. Product specificity was determined by melting curve analysis and by visualization of PCR products on agarose gels. Calculation of the relative amount of each cDNA species was performed according to standard protocols. Briefly, the amplification of FceRI-a gene in stimulated cells was calculated first as the copy number ratio of FceRI-a to GAPDH, and then expressed as normalized values of fold increase over the value obtained with unstimulated (control) cells.

Immunoprecipitation and Western blot
B/TSM FceRIa-chain protein expression was analyzed by immunoprecipitation and Western blotting with minor modifications from the protocol described earlier [8]. Briefly, B/TSM cells were lysed for 30 min 4uC in NP-40 lysis buffer supplemented with a cocktail of protease inhibitors (2 mM sodium orthovanadate, 1 mM phenyl-methylsulfonylfluoride, 10 mg/ml leupeptin, 0.15 units/ml approtinin, 1 mg/ml pepstatin A) (Sigma-Aldrich) and centrifuged for 20 min to remove nuclei. Cell lysates from B/TSM or basophilic cell line KU812 (positive control) were pre-cleared with protein G sepharose-coated beads (Amersham-Pharmacia) for 2 h at 4uC in a rotating mixer, followed by incubation with protein G sepharose-coated beads conjugated with 2 mg/ml of murine anti-human FceRIa mAb 15/1 or isotype mouse IgG1 monoclonal antibody (MOPC 21) for 16 h at 4uC. Immuno-complexes were then pelleted by centrifugation and washed six times with the wash buffer (PBS/1% NP40). For immunoblotting, samples were separated on SDS polyacrylamide gel (11-13%) and electrotransferred onto PVDF membrane (Millipore, Mississauga, ON). The membrane was blocked at RT for 2 hrs with 5% Blotto, (Santa Cruz Biotechnology, CA, USA), incubated with rabbit antihuman FceRIa-chain polyclonal Ab (Upstate Biotechnology, Inc., Lake Placid, NY) (1 mg/ml) at room temperature for 2 h, followed by secondary antibody HRP-goat anti-rabbit IgG (H+L) prepared in TBST (1:5000). The blots were developed by enhanced chemiluminescence as recommended by the supplier (Amersham Pharmacia, ON). A band non-specific to FceRI in the same gel was used as loading control. On the other hand, 15 mg of B/TSM and KU812 cell lysates were directly loaded onto 14% SDS polyacrylamide gel (11-13%) and electro-transferred onto PVDF membrane. The membrane was blocked at RT for 2 hrs with 5% Blotto, incubated with goat anti-human FceRI-c polyclonal Ab (K-16 clone) (Santa Cruz, CA) prepared in TBST (1:500) at RT for 2 h, followed by secondary antibody HRP-rabbit anti-goat IgG whole molecule (Sigma-Aldrich) prepared in TBST (1:5000), and the blots were developed as described above. After stripping, the blots were probed for GAPDH and used as a loading control. The corresponding values of pre-stained protein molecular weight marker were scaled to the FceRI-a and -c protein bands. The intensity of FceRI-c bands was determined by using AlphaEase FC software version 3.1.2 relative to control loading levels.

CC and CXC Chemokines ELISA from cytokine presensitized B/TSM cell supernatants
In order to study the functional significance of cytokine stimulated FceRIa-chain upregulation, primary human B/TSM cells were grown until 65% confluency in 12-well culture plates and 48 h serum-deprived cells were then stimulated for 48 h with recombinant human IL-1b, TNF-a, IL-4 (10 ng/ml each) or vehicle (medium alone). The supernatants were removed and cells were washed twice with serum-free Ham's F12 media. Thereafter, the cells in each treatment group were either left unstimulated (medium alone) or stimulated with mIgG1 (1 mg/ml) or recombinant human IgE (1 mg/ml) for 24 h under the similar culture conditions. The culture supernatants were then collected, centrifuged at 1200 rpm for 7 min at 4uC to remove cellular debris and stored at 280uC until future use. Immunoreactive eotaxin-1/CCL11, IL-8/CXCL8, IP-10/CXCL10 (10 kDa interferon-gamma-induced protein), RANTES/CCL5 (Regulated upon Activation, Normal T-cell Expressed, and Secreted), and TARC/CCL17 (Thymus and activation-regulated chemokine) released into B/TSM culture supernatants were quantified using ELISA with matched Abs according to basic laboratory protocol provided by the manufacturer (R&D Systems, Minneapolis, MN, USA). Chemokine proteins were quantified in reference to serial dilutions of recombinant standards falling within the linear part of the standard curve for each specific chemokine sample measured. The sensitivity limits of these chemokine assays are 5 pg/ml for eotaxin-1/CCL11, 3 pg/ml for IL-8/CXCL8, 7.8 pg/ml for IP-10/CXCL10, 7.8 pg/ml for RANTES/CCL5 and 7.8 pg/ml for TARC/CCL17. Each data point represents readings from three separate assays.

Lentiviral vector transduction in B/TSM cells
For short-hairpin RNA (shRNA)-induced gene silencing studies, pseudotyped lentiviral vector (clone Id: V2LHS_153702) expressing specific Syk shRNA was obtained from Open-Biosystems (Huntsville, AL). 293T cells used for virus production and titration, were cultured in Iscove's modified Dulbecco's medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin/glutamate (PSG) (Gibco, Grand Island, NY). Lentivirus were generated using 293T cell lines and viral titer was determined by counting the puromycin resistant colonies, as described elsewhere [21]. A control shRNA unrelated to Syk sequence (scramble shRNA) was used as a transduction control. For silencing Syk protein expression, B/TSM cells were transduced at a multiplicity of infection (MOI) of 10 in the presence of polybrene (8 mg/ml). In brief, cells were exposed to recombinant lentivirus for 2 hr at 37uC, medium replaced and cultured for additional 72 hrs. Transduced cells were selected with puromycin. The average transduction efficiency was more than 95% as determined by FACS using the turbo-green fluorescent protein (tGFP) as the marker for cell sorting. Viability of the transduced cells undergoing experiment was .98% as assessed by trypan blue dye after completion of the experiment.
Briefly, 20 ml of cell lysate was mixed with 100 ml of Luciferase Assay Reagent II and firefly luciferase activity was first recorded. Then, 100 ml of Stop-and-Glo Reagent was added, and Renilla luciferase activity was measured. All values were normalized to Renilla luciferase activity and expressed relative to the control transfected non-stimulated cells.

Statistical analysis
All the data were obtained from experiments performed three or more times. Statistical analysis was performed by using GraphPad Prism Software Version 3.02 for Windows (GraphPad Software Software, San Diego, CA, USA). Association between chemokine expression levels in the subgroups and cytokine stimulation effect on FceRI expression were studied using Mann-Whitney U test. P values ,0.05 were considered statistically significant.   [15,26]. In the present study, human B/TSM cells stimulated with IL-1b, TNF-a, or IL-4 showed significantly enhanced FceRI-a mRNA expression ( Figure 1A). In contrast to cytokine stimulation, the basal FceRI-a mRNA expression was uniform and unaffected by time of culture. FceRI-a mRNA increased expression was then confirmed by quantitative real-time RT-PCR analysis. As shown in Figure 1B, B/TSMCs stimulated with TNF-a, and IL-1b upregulated the FceRI-a transcript expression by 45.064.5-, and 28.263.8-fold, respectively, compared to unstimulated cells at 2 h. Interestingly, the mRNA expression was downregulated at 6 h but again gained peak at 20 h; whereas TNF-a inducing the maximum expression (32. 462.1-fold). Notably, IL-4 stimulation steadily upregulated the FceRI-a mRNA expression (2.4460.28fold, 2 h; 2.4360.29-fold, 6 h; and 2.7960.7-fold, 20 h) compared to the control in B/TSM cells ( Figure 1B). On the other hand, the mRNA expression for FceRI-c chain was upregulated by TNFa only at 2 h ( Figure 1A). Collectively, this data suggest that proinflammatory and Th-2 cytokines can potentially regulate the transcription of FceRI in B/TSM cells.

TNF-a, IL-4 upregulate the FceRI-a protein expression in human B/TSMCs
To investigate the effect of IL-1b, TNF-a, and IL-4 stimulation on FceRI-a and -c protein expression, serum-deprived B/TSM cells were stimulated with TNF-a, IL-1b, or IL-4 and subjected to immunoprecipitation and Western blot. As demonstrated in Figure 2A, TNF-a and IL-4 stimulation enhanced the ,45 KDa (referred to be the intracellular) [7,8] FceRI-a protein expression at 24 h compared to unstimulated or IL-1b-stimulated B/TSMCs. Immunoprecipitation from basophilic cell line KU812 also revealed a positive band at ,45 KDa (Figure 2A).
We then explored the effect of chronic (72 h) cytokine stimulation on FceRI-a protein expression. As shown in Figure 2B, TNF-a, and IL-4 stimulation for 72 h led to the upregulation of FceRI-a protein in B/TSM cells. TNF-a stimulation remarkably augmented the ,45 KDa intracellular and 60-65 KDa (referred to be as surface chain) [27] FceRI-a protein compared to unstimulated B/TSM cells ( Figure 2B). Chronic IL-4 stimulation also augmented the FceRI-a protein expression, both for ,45 KDa and ,60-65 KDa bands (Figure 2A and B). In contrast to FceRI-a and TNF-mediated FceRI-c mRNA upregulation, no significant upregulation in protein was observed ( Figure 2C and D). As expected, KU812 cell lysate revealed both FceRIa intracellular and surface, and FceRI-c protein bands and served as a positive control. Importantly, immunoprecipitation with isotype control antibody mIgG1-MOPC21 did not show any conspicuous bands of FceRI-a either of ,45 KDa or 60-65 KDa at 24 or 72 h stimulation. Collectively, our data demonstrate that TNF-a and IL-4 upregulate the FceRI-a chain protein expression in B/TSM cells.
TNF-a pre-sensitization augments the eotaxin-1/CCL11, IL-8/CXCL8, IP-10/CXCL10, RANTES/CCL5 but not TARC/ CCL17 release in B/TSM following IgE stimulation We then investigated the functional consequences of FceRI-a protein upregulation by TNF-a, or IL-4 in B/TSM cells. Since we have previously demonstrated that IgE-dependent activation of B/ TSM cells induces the CCL11 release [15], it was enticing to assess whether TNF-a or IL-4-mediated FceRI-a protein upregulation followed by IgE stimulation can augment the chemokines release by B/TSM cells. Therefore, B/TSM cells were first sensitized with TNF-a, IL-1b, or IL-4 for 48 h, washed and then stimulated with IgE, mIgG1 (MOPC21) or left unstimulated for 24 h in fresh medium. CC (CCL11, CCL5, and CCL17) and CXC (CXCL8 and CXCL10) chemokines released in supernatants were then measured by ELISA. Since IL-1b did not induce a marked FceRIa protein expression (Figure 2A and B), it is plausible that it would not affect the subsequent chemokine release following IgE stimulation. Interestingly, TNF-a pre-sensitized B/TSM cells released significantly elevated (p,0.05, n = 3) levels of eotaxin-1/CCL11, IL-8/CXCL8, IP-10/CXCL10, and RANTES/CCL5 following IgE stimulation compared to IgE-unstimulated cells ( Figure 3A, B, C and D). Moreover, IL-4 pre-sensitization followed by IgE stimulation also enhanced the eotaxin-1/CCL11 release significantly (p,0.01, n = 3, Figure 3A). We, however, did not see any significant change in TARC/CCL17 release in TNF-a, IL-1b, or IL-4 pre-sensitized and IgE stimulated B/TSM cells (data not shown), suggesting the selective nature of FceRI activationinduced chemokine expression. These results, therefore, suggest that TNF-a, in particular, upregulates the FceRI-a expression as considerably that subsequent stimulation of B/TSM with IgE engages and activates the FceRI strongly, leading to enhanced selective chemokine release. Lentivirus mediated Syk-silencing abrogates the IgEinduced transcriptional activation of human eotaxin-1/ CCL11, IL-8/CXCL8, IP-10/CXCL10, and RANTES/CCL5 promoters in B/TSM cells In the light of the above findings, we further investigated whether IgE-induced chemokine expression involved the FceRI activation. In inflammatory cells such as mast cells, FceRI activation triggers many signaling pathways including the phosphorylation of FceRI-b and -c by Lyn kinase, followed by the activation of Syk through its recruitment to FceRI [7]. Activation of Syk is crucial for FceRI downstream signals propagation including phosphorylation of phospholipase Cc, calcium mobilization, degranulation, and proinflammatory cytokine/chemokine release [7,26]. These observations suggest that blocking/silencing the Syk expression might be a useful strategy to investigate the FceRI activation in B/TSM cells. To inhibit the Syk expression, B/TSM cells were transduced with a pseudotyped lentiviral vector expressing specific Syk-shRNA. Mock and scramble sequence were used as negative controls. As shown in Figure 5A, more than 95% of the lentivirus-transduced cells were tGFP positive by FACS analysis. Transduction of cells with Syk-shRNA clone resulted in a highly significant and reproducible decrease in Syk expression, as shown by Western blotting ( Figure 5B). However, transduction with the control scramble shRNA failed to reduce Syk expression in B/TSM cells ( Figure 5B). To determine if the transcriptional activation of chemokine expression by IgE is affected in the absence of Syk, stably Syksilenced B/TSM cells were transiently transfected with wild-type promoters for earlier studied CC and CXC chemokines and stimulated with IgE, IL-1b, mIgG1 or left unstimulated. As shown in Figure 5C, D, E and F, IgE-induced chemokines promoter activity over unstimulated control was completely abrogated (eotaxin-1/CCL11, 1.3060.01 to 0.9460.14-fold; IL-8/CXCL8, 1.2160.01 to 1.0860.07-fold; IP-10/CXCL10, 1.2260.01 to 1.0160.07-fold, and RANTES/CCL5, 1.3160.02 to 0.9460.09fold) in Syk-shRNA-transduced compared to the scramble-shRNAtransduced B/TSM cells. However, in contrast, lentivirus-mediated Syk-silencing in B/TSM cells did not affect the IL-1b-induced promoter activity for IL-8/CXCL8 and IP-10/CXCL10 ( Figure 5D and E). Moreover, although eotaxin-1/CCL11 and RANTES/ CCL5 promoter activity was slightly decreased, it was still significant compared to the unstimulated control in IL-1b-stimulated Syksilenced B/TSM cells ( Figure 5C and F). The isotype mIgG1 (MOPC21) stimulation did not affect the promoter activity for any of the chemokines in either Syk-shRNA or scramble-shRNA-transduced B/TSM cells ( Figure 5). Taken together, these results suggest that the IgE-sensitization of B/TSM cells induce the chemokine expression at least via involving Syk activity and the respective chemokine promoter activation.

Discussion
Several studies have investigated the regulation of high affinity IgE receptor (FceRI) expression in inflammatory cells both in vivo and in vitro, and a correlation has been established between the serum IgE levels and the FceRI expression on cell surface [28,29]. However, IgE is not the only factor governing FceRI expression, since mast cells from IgE-deficient (IgE 2/2 ) mice express low levels of FceRI [30]. Therefore, it is indeed apparent that the basal FceRI expression is under the control of some other regulatory mechanisms. Moreover, although the ASM cells were recently shown to respond to IgE through the expression of a tetrameric FceRI complex (abc2) [15], factors that modulate the FceRI expression by ASM remain unknown. In the present study, we demonstrated that proinflammatory (TNF-a) and Th2 (IL-4) cytokines upregulate the FceRI-a expression in cultured human B/TSM cells both at transcript and protein level. This was strikingly a novel finding as despite being central proinflammatory mediators in allergic inflammation [10,11,12,13], TNF-a and IL-1b have not previously been investigated in the context of FceRI modulation in any cell type. Interestingly, IgE engagement of FceRI on TNF-a pre-sensitized B/TSM cells significantly augmented the eotaxin-1/CCL11, IL-8/CXCL8, IP-10/CXCL10, and RANTES/CCL5 but not TARC/CCL17 release. In addition, IgE sensitization of B/TSM cells induced the gene expression for eotaxin-1/CCL11, IL-8/ CXCL8, IP-10/CXCL10, and RANTES/CCL5 at promoter level, which was completely abrogated upon Lentivirus-mediated Syk silencing. Our data underline the fact that proinflammatory and Th2 cytokine-induced FceRI regulation in B/TSM cells could significantly contribute to the airway inflammation via potentially novel mechanisms involving IgE/FceRI complex.
TNF-a is a prototype proinflammatory cytokine which has been proposed to exert deleterious effects directly on airway smooth muscle, including its most recently explored roles in (i) activation of transient receptor potential channel 3 (TRPC3) leading to abnormal store-operated calcium influx, and (ii) upregulation of CD38 which regulates intracellular calcium and plays a role in airway hyperresponsiveness [31,32]. Both TNF-a and IL-1b, independently, are known to induce IL-8/CXCL8 and RANTES/CCL5 release in ASM [33]. IL-8/CXCL8 is a chemoattractant for neutrophils, eosinophils, and to a lesser extent, T-lymphocytes, while RANTES/CCL5 acts as a chemoattractant for T-cell, eosinophils and monocytes. TNF-a also induced the release of IP-10/CXCL-10, a potent chemokine for activated T cells, NK cells and mast cells; and its expression is differentially modulated by vitamin D in human ASM cells [34]. In the present study, TNF-a stimulation augmented both FceRI-a transcript and protein expression in B/TSM cells. Of note, we did not incubate the cultured B/TSM cells with cytokines and IgE simultaneously as it could mask the effect of IgE. Indeed, we first pre-sensitized the B/TSM cells with cytokines and then engaged the receptor with IgE stimulation. Interestingly, TNF-a presensitization (and thus FceRI upregulation) followed by IgE stimulation augmented the selective eotaxin-1/CCL11, IL-8/ CXCL8, IP-10/CXCL10, and RANTES/CCL5 chemokines release, compared to IgE non-stimulated control. This was quite a plausible observation at functional level since IgE could engage the TNF-upregulated FceRI by a higher magnitude than basal FceRI expressed on B/TSM cells. Surprisingly, IL-1b enhanced the FceRI-a mRNA expression and had slight but not significant effect on FceRI-a protein upregulation. In accordance with this, IgE stimulation following IL-1b pre-sensitization did not augment the chemokine release compared to IgE-unstimulated cells. This may be explained by the involvement of post-transcriptional regulatory mechanisms in FceRI protein expression in response to IL-1b stimulation, and thus low magnitude of IgE-mediated signaling. Taken together, our observations essentially provide another mechanism by which TNF-a contribute to airway inflammation, mainly amplifying the FceRI-mediated ASM activation, and ultimately recruiting inflammatory cells through multiple chemokine expression in airways.
FceRI-c chain was shown to be downregulated alongwith FceRI-a chain by TGF-b1 in bone marrow derived mast cells (BMMC) [35], and has been demonstrated to be the limiting factor governing FceRIa surface expression in dendritic cells (DCs) [36]. However, we found that only FceRI-c transcript but not protein was upregulated by TNF-a. Lack of FceRI-c protein upregulation by TNF, IL-1b or IL-4 may suggest some post-transcriptional regulatory mechanisms modulating the FceRI-c protein translation. Since B/TSM express all FceRI subunits (abc2) [15], possibility of different regulatory mechanisms in B/TSM FceRI  The Lentiviral transduction efficiency was more than 95% for both scramble-shRNA and Syk-shRNA as determined by FACS using the tGFP as the marker for cell sorting. (B) Lentiviral Syk-shRNA transduction in B/TSM cells silenced the Syk expression efficiently as observed by Western blotting. Syk-and scramble-transduced human B/TSM cells were then transiently transfected with luciferase reporter constructs driven by wild-type promoters for human (C) eotaxin-1/CCL11, (D) IL-8/CXCL8, (E) IP-10/CXCL10, or (F) RANTES/CCL5; stimulated with mIgG1-MOPC21, IgE, or IL-1b for 12 h; and respective chemokine promoter activity was measured as mentioned in Materials and Methods. Data were normalized according to the Renilla luciferase activity, and presented as fold-increase over unstimulated control. Data represents the mean6SD of three separate experiments. Mann-Whitney U test was performed to analyze the differences between the samples. *P,0.05, **P,0.01 compared to unstimulated control. doi:10.1371/journal.pone.0006153.g005 than trimeric DC (ac2) [36] cannot be denied. Moreover, since FceRI-c chain is also shared by Fc gamma receptor subtypes expressed by B/TSM [37], additional regulatory mechanisms may be in force in controlling FceRI-c expression. Detailed studies are therefore required to delineate the regulation and role of FcRc in amplification or stabilization, if any, of FceRI surface expression in B/TSM cells.
IL-4 on the other hand, is a prototype Th2 cytokine which in combination with IL-13, induce the class switch-recombination from IgG to IgE by B-cells [6]. IL-4 plays a critical role in atopic diseases and is also known to cause a marked increase in eotaxin-1/CCL11 release in ASM cells [38,39]. In our present study, interestingly, we found that IL-4 significantly enhances the surface and intracellular FceRI-a protein expression in B/TSM cells. This was quite interesting as there is a steady-state FceRI-a mRNA expression under IL-4 stimulation, suggesting the coherent translation of FceRI-a transcript into the protein at 24 h, 72 h after stimulation ( Figure 1B, 2A and B). Our data, therefore, strongly supports the previously observed positive role of IL-4 in the transcription of FceRIa-chain in human mast cells, eosinophils from atopic dermatitis patients, human dendritic cells, and human neutrophils [8,40,41,42].
Classical paradigm entails the IgE binding to FceRI as a 'passive sensitization' step in the mast cell activation and requires the multivalent antigens for cross-linking of FceRI-bound IgE [7]. However, recent reports highlight the IgE-mediated spectrum of effects including the pro-survival effects on mast cells, monocytes, and asthmatic neutrophils through binding to FceRI [43,44,45,46]. Moreover, IgE alone (i.e. sensitization) induced the expression of multiple cytokines (e.g. IL-6, TNF-a, IL-4 and IL-13) and activated signaling pathways by phosphorylation of several kinases such as Erks, p38, JNK and PKB in normal murine BMMC [44]. In this line, after exploring the FceRI regulation by proinflammatory and Th-2 cytokines and subsequent IgEmediated B/TSM activation, we tested our hypothesis of effect of IgE sensitization alone on ASM synthetic function. Interestingly, we found that IgE sensitization induces multiple, selective CC (eotaxin-1/CCL11, RANTES/CCL5) and CXC (IL-8/CXCL8, IP-10/CXCL10) chemokines expression in B/TSM cells at least at promoter level.
In inflammatory cells, activation of Syk is crucial for IgE crosslinking-induced FceRI downstream signals propagation including phosphorylation of phospholipase Cc, calcium mobilization, and degranulation [7,26]. Although details still remain to be investigated, the initial signaling events in non cross-linking model (i.e. IgE sensitization alone) of FceRI activation include the activation of Lyn and Syk leading to the activation of ERK in mast cells [47,48]. Interestingly, in our study, lentivirus-mediated Syk shutdown in B/TSM cells completely abrogated the CC and CXC chemokine promoter activity, suggesting the involvement and requirement of Syk activity in IgE sensitization-induced chemokine expression. These results essentially provide a proof-ofprinciple and increase our understanding that at least initial signaling events of IgE sensitization-induced FceRI activation are similar, if not the same, in ASM and mast cells. Altogether, our data suggest that blocking or silencing Syk, among others, could present a useful therapeutic strategy for allergic inflammatory disorders.
Collectively, our data demonstrate that proinflammatory (TNFa) and Th2 (IL-4) cytokines modulate the FceRI expression in B/ TSM, which can further augment the selective CC and CXC chemokine release following IgE exposure. Moreover, IgE sensitization of B/TSM cells induces multiple chemokine gene expression, via involving at least Syk activity and the respective gene promoter activation. Our data, therefore, highlight the fact that proinflammatory and Th2 cytokines directly or indirectly manifest the allergic inflammation, also by regulating FceRI expression, hence amplifying the IgE/FceRI associated reactions.