Clinical and Experimental Allergy
Volume 38, September 2008
Editor’s Choice The Editor takes a closer look at some of this month’s articles Safety of inhaled steroids: watch the dose The inhaled route for lipophilic topically active steroids has transformed the management of airway disease providing a safe and effective alternative to systemic treatment with glucocorticoids. However their excellent safety record has led to a tendency to prescribe in doses that are higher than the risk-benefit ratio would warrant. The paper by Weatherall and colleagues (pp. 1451–1458) pointing out a dose response relationship between the risk of fractures and inhaled steroid dose is a timely reminder that these drugs are not without their dangers and the minimum dose to control airway inflammation should be used.
Ion channels: a new target for asthma The complexity of ion channel biology is bewildering to non-experts yet at the core of all cellular functions is a flow of ions across the cell membrane. It is axiomatic therefore that both the abnormalities in immune regulation and the disordered function in structural cells that underlies the asthma process are controlled by ion channels. In a novel and exciting observation Sel et al (pp. 1548–1558) have found that the channel TRPC6 which is highly expressed in the lung is involved in both airway hyperresponsiveness and ovalbumin induced inflammation, offering a new target for therapeutic intervention.
Histamine3 receptor: a new tune for an old mediator
Immunohistochemistry for histamine H3 receptor in human nasal mucosa with allergic rhinitis (see fig 2 in Suzuki et al, pp. 1476–1482).
Histamine is the grandfather of allergy-associated mediators but the discovery that there are at least four histamine receptors has opened up new lines of investigation and treatment. However our understanding of the role of the H3 and H4 receptors in allergic disease remains limited. This interesting study by Suzuki et al (pp. 1476–1482) makes the case for histamine receptors being present on mucus glands and being involved in mucus hypersecretion, an aspect of rhinitis not well controlled with H1 blockers. We await a new class of antihistamines for the allergy clinic.
Gene expression in allergy: CISH goes the Th2 cell With hens egg allergy as a model using gene chip technology, Nakajima and colleagues (pp. 1499–1506) have investigated the genes differentially expressed in Th2 cells. They found that the gene most closely associated with allergy to hens egg was CIS(H) a member of the suppressors of cytokine signalling (SOCS) family of signal transduction molecules. This is one of the first times that this molecule has been implicated in Th2 function and opens up a new avenue to regulate the activation of these key allergy related cells.
Cows milk allergy: soybean may not be the answer Cows milk allergy is a major cause of morbidity in infants and parents often turn to soybean as an alternative. In this study Curciarello et al (pp. 1559–1565) have investigated the cross-reactive components of conventional soybean and a cultivar (Raiden) which may be less allergenic. They found that while Raiden contains less crossreactive components than conventional soybean both strains contain a subunit-b-conglycinin which crossreacted with cows milk specific IgE antibodies. Back to the drawing board.
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This logo highlights the Editor’s Choice articles on the cover and the first page of each of the articles.
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Clinical and Experimental Allergy, 38, 1548–1558
doi: 10.1111/j.1365-2222.2008.03043.x
O R I G I N A L PA P E R
Experimental Models of Allergic Disease
c 2008 The Authors c 2008 Blackwell Publishing Ltd Journal compilation
Loss of classical transient receptor potential 6 channel reduces allergic airway response S. Sel1, B. R. Rostw1, A. O¨. Yildirimz, B. Sel, H. Kalwaw, H. Fehrenbachz, H. Renz, T. Gudermannw and A. Dietrichw Department of Clinical Chemistry and Molecular Diagnostics, Philipps—University Marburg, Marburg, Germany, wInstitute for Pharmacology and Toxicology,
Philipps—University Marburg, Marburg, Germany and zClinical Research Group, Chronic Airway Diseases, Medical Faculty, Philipps-University Marburg, Marburg, Germany
Clinical and Experimental Allergy
Correspondence: Alexander Dietrich, Institute for Pharmacology and Toxicology, Philipps—University Marburg, Karl-vonFrisch-Str.1, 35033 Marburg, Germany. E-mail:
[email protected] Cite this as: S. Sel, B. R. Rost, A. O¨. Yildirim. B. Sel, H. Kalwa, H. Fehrenbach, H. Renz, T. Gudermann and A. Dietrich, Clinical and Experimental Allergy, 2008 (38) 1548–1558.
Summary Background Non-selective cation influx through canonical transient receptor potential channels (TRPCs) is thought to be an important event leading to airway inflammation. TRPC6 is highly expressed in the lung, but its role in allergic processes is still poorly understood. Objective The purpose of this study was to evaluate the role of TRPC6 in airway hyperresponsiveness (AHR) and allergic inflammation of the lung. Methods Methacholine-induced AHR was assessed by head-out body plethysmography of wild type (WT) and TRPC6 / mice. Experimental airway inflammation was induced by intraperitoneal ovalbumin (OVA) sensitization, followed by OVA aerosol challenges. Allergic inflammation and mucus production were analysed 24 h after the last allergen challenge. Results Methacholine-induced AHR and agonist-induced contractility of tracheal rings were increased in TRPC6 / mice compared with WT mice, most probably due to compensatory up-regulation of TRPC3 in airway smooth muscle cells. Most interestingly, when compared with WT mice, TRPC6 / mice exhibited reduced allergic responses after allergen challenge as evidenced by a decrease in airway eosinophilia and blood IgE levels, as well as decreased levels of T-helper type 2 (Th2) cytokines (IL-5, IL-13) in the bronchoalveolar lavage. However, lung mucus production after allergen challenge was not altered by TRPC6 deficiency. Conclusions TRPC6 deficiency inhibits specific allergic immune responses, pointing to an important immunological function of this cation channel in Th2 cells, eosinophils, mast cells and B cells. Keywords airway hyperresponsiveness, B cells, eosinophils, IgE, IL-5, IL-13, ovalbumininduced experimental airway inflammation, Th2 cells, TRPC6 Submitted 6 September 2007; revised 9 April 2008; accepted 10 April 2008
Introduction The transient receptor potential (TRP) family of ion channels is a growing group of structurally and evolutionarily related cation channels comprised of several subfamilies including the TRPC, TRPM and TRPV classes of channels [1, 2]. TRP-type ion channels are assembled as homo- or heterotetramers of subunits, each spanning the plasma membrane six times. The founding members of this channel family are the insect TRP and TRPL channels, which are responsible for photoreceptor depolarization in 1
These authors contributed equally to the work.
response to light. Mammalian TRPCs (C stands for canonical or classical) are the closest mammalian structural relatives of insect TRPs [2]. Among the TRPC channels, TRPC3, 6 and 7 are 75% identical and gated by signal transduction pathways that activate C-type phospholipases (PLCs) [3, 4], as well as by direct exposure to diacylglycerols (DAG) [5]. TRPC3, 6 and 7 interact physically and, upon co-expression, co-assemble to form functional channels [6]. In contrast to members of other TRP families, the functional importance of most members of the TRPC subfamily is still poorly understood. A TRPC channel for which considerable evidence has accumulated for a
TRPC6 / mice show reduced allergic airway responses
specific role is TRPC6, which has been proposed to regulate smooth muscle function [7]. The TRPC6 cDNA was originally isolated from mouse brain, but TRPC6 mRNA is also highly expressed in the lung [8]. Although TRPC6 expression has been demonstrated in human airway smooth muscle cells and differentiated bronchial smooth muscle cells [9], as well as in pulmonary arterial smooth muscle cells [10], data for a physiological role of TRPC6 in lung are rare. Recently, we were able to demonstrate a unique physiological role of TRPC6 in the regulation of the acute hypoxic vasoconstriction by analysing a TRPC6-deficient mouse model [11]. These data were in clear contrast to the systemic vasculature, where up-regulation of TRPC3 was partially able to compensate for the TRPC6 deficiency resulting in higher contractility of aortic smooth muscle cells, as well as smooth muscle cells from cerebral arteries [12]. TRPC6 is also expressed in neutrophils [13], eosinophils [14] and T lymphocytes [15], which are involved in airway inflammation leading to pathophysiological processes like asthma and chronic obstructive pulmonary disease (COPD) in the lung. However, experimental data supporting a unique functional role of TRPC6 expressed in leucocytes [13–15] are lacking. For this reason, we analysed agonist-induced airway hyperresponsiveness (AHR) and allergic airway inflammation [16] in a TRPC6-deficient mouse model. While airway contractility was enhanced in TRPC6 / mice compared with wild-type (WT) mice, most probably because of up-regulation of TRPC3 in the lung, antigenspecific IgE blood levels, as well as Th2 cytokine (IL-5 and IL-13) levels in the bronchoalveolar lavage (BAL) were decreased in ovalbumin (OVA)-sensitized and -challenged TRPC6-deficient mice. Moreover, airway eosinophilia was decreased in TRPC6-deficient mice, while lung mucus production after allergic inflammation was not altered. In conclusion, we provide first experimental evidence that TRPC6 has a unique non-redundant role in allergic airway inflammation and may represent a new drug target for airway inflammatory diseases like asthma and COPD.
Material and methods
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Head-out body plethysmography Bronchial responsiveness (BR) to b-methacholine was essentially tested using head-out body plethysmography as described before [17]. Methacholine was aerosolized with a nebulizer in increasing concentrations from 0 to 200 mg/mL in phosphate-buffered saline (PBS) and the methacholine concentration that caused a 50% reduction in baseline midexpiratory airflow (MCh50 mg/mL) is reported. Assessment of airway smooth muscle responsiveness by analysis of the contractility of tracheal rings Airway smooth muscle responsiveness was assessed by stimulation with increasing concentrations of methacholine as described [18]. Isolation of smooth muscle cells and epithelial cells from murine tracheae Airway smooth muscle cells from murine tracheae were isolated as described for tracheae from guinea-pigs [19] and identified by their immunoreactivity with antibodies against a-smooth muscle actin. Epithelial cells were isolated and cultured on collagen-coated transwells as described [20] in MTEC medium [DMEM-Ham’s F12 (1 : 1 v/v) medium with penicillin/streptomycin] with a supplemental mix for airway epithelial cell growth medium (C-21060; Promocell, Heidelberg, Germany). Cultivation of cell lines and mast cells Cell lines EL4 (murine T lymphocytes; ATCC no. TIB-39), RAW 264.7 (murine macrophages; ECACC no. 85011428) and WEHI-231 (lymphoblasts from a mouse B cell lymphoma, ECACC no. 85022107) were cultured according to the instructions of the suppliers [American Tissue Culture Collection (ATCC), Manassas, VA, USA, and European Collection of Cell Cultures (ECAC), provided by SigmaAldrich, Taufkirchen, Germany]. First-strand cDNA of primary mast cells, which were cultured as described [21], was a kind gift from Dr R. Mailhammer (GSF-National Research Center for Environmet and Health, Munich, Germany).
Animals
Quantitative real-time polymerase chain reaction
TRPC6 / mice and their WT litter mates were bred and genotyped as described previously [12]. For allergic sensitization and airway allergen challenges, the targeted mutation was crossed 10 times into the BALB/c background to establish an inbred colony carrying the TRPC6 / allele. Age- and sex-matched mice were used in all experiments. All animal experiments were approved by the local animal care committee.
Total RNA and first-strand synthesis and real-time PCR was carried out as described [12]. The following primers pairs were used for the amplification of specific fragments from the first-strand synthesis: TRPC1: C1F (5 0 -TGG GCC CAC TGC AGA TTT CAA) and C1R (5 0 -AAG ATG GCC ACG TGC GCT AAG GAG); TRPC2: C2F (5 0 -TTG CCT CCC TCA TCT TCC TCA CCA) and C2R (5 0 -CCG CAA GCC CTC GAT CCA CAC CT), TRPC3: C3F (5 0 -AGC CGA GCC CCT GGA
c 2008
The Authors c 2008 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 38 : 1548–1558 Journal compilation
1550 S. Sel et al AAG ACA C) and C3R (5 0 -CCG ATG GCG AGG AAT GGA AGA C); TRPC4: C4F (5 0 -GGG CGG CGT GCT GCT GAT) and C4R (5 0 -CCG CGT TGG CTG ACT GTA TTG TAG); TRPC5: C5F (5 0 -AGT CGC TCT TCT GGT CTG TCT TT) and C5R (5 0 -TTT GGG GCT GGG AAT AAT G); TRPC6: C6F (5 0 GAC CGT TCA TGA AGT TTG TAG CAC) and C6R (5 0 -AGT ATT CTT TGG GGC CTT GAG TCC), TRPC7: C7F (5 0 -GTG GGC GTG CTG GAC CTG) and C7R (5 0 -AGA CTG TTG CCG TAA GCC TGA GAG) as well as glyceraldehydephosphate dehydrogenase (GAPDH): GAPDHF (5 0 -GTG AAG GTC GGT GTG AAC G) and GAPDHR (5 0 -GGT GAA GAC ACC AGT AGA CTC) and b-actin ACTF, 5 0 -CCA ACC GTG AAA AGA TGA CC, ACTR, 5 0 -GTG GTA CGA CCA GAG GCA TAC as reference genes. All primers were tested using diluted first-strand synthesis from brain RNA (10–1000fold) to confirm linearity of the reaction. Real-time PCR was performed using the 2 master mix from the Quantitect SYBR Green PCR kit (Qiagen, Hilden, Germany) containing a HotStar Taq polymerase, buffer, nucleotides, 5 mM MgCl2 (final 2.5 mM) and SYBR Green. Ten picomoles of each primer pair and 0.2 mL from the first-strand synthesis were added to the reaction mixture and PCR was carried out in a light-cycler apparatus (Roche, Mannheim, Germany) using the following conditions: 15 min of initial activation and 45 cycles of 12 s at 94 1C, 30 s at 50 1C, 30 s at 72 1C and 10 s at 80 1C each. Fluorescence intensities were recorded after the extension step at 80 1C after each cycle to exclude the fluorescence of primer dimers melting at temperatures lower than 80 1C. Samples containing primer dimers were excluded by melting curve analysis and identification of the products by agarose gel electrophoresis. Crossing points were determined by a software program. The relative gene expression was quantified using the formula: (2e(crossing point GAPDH crossing point X) ) 100 = % of reference gene expression. Ba21 influx experiments, design and electroporation of siRNA vectors expressing small interfering RNAs Cells were loaded with fura-2-acetoxymethylester and analysed as described previously [12, 22]. The design and sequences for the production of an siRNA homologous to TRPC3 are described elsewhere [12]. The unrelated siRNA (5 0 -ACC GAC GAG CAG CTA GAA C) was selected by its low homology to sequences of a genomewide screen. Plasmids containing the siRNA expression cassettes under the control of an H1 promoter were electroporated using a nucleofactor (Amaxa GmbH, Cologne, Germany) and the protocol and solutions for human aortic smooth muscle cells as described by the supplier. Allergic sensitization and airway allergen challenges TRPC6 / BALB/c mice and WT mice were sensitized to ovalbumin (OVA) by three intraperitoneal (i.p.) injections
of OVA adsorbed to aluminium hydroxide (Pierce Biotechnology, Rockford, IL, USA) and diluted in PBS (50 mg OVA grade VI per injection; Sigma, Tauf-Kirchen, Germany) on days 0, 14 and 21. Mice were challenged with 1% OVA aerosols in PBS on days 27, 28, and 29 before analysis on days 30 and 31. Sham sensitization was carried out with sterile aluminium hydroxide in PBS, and PBS aerosols were used as sham challenges. Measurements of ovalbumin-specific serum immunoglobulin levels Blood was drawn from the tail vein and OVA-specific Ig levels (IgE, IgG1 and IgG2a) were determined using ELISA techniques as described previously [23]. Isolation of bronchoalveolar lavage fluid and quantification of eosinophilia and cytokine levels Sensitized and airway allergen-challenged mice were killed 24–48 h after the last challenge, and the tracheas were cannulated. BAL was performed by flushing the lungs and airways three times with 800 mL of F12K Nutrient Mixture (Invitrogen, Carlsbad, CA, USA) supplemented with 15% foetal calf serum (FCS), 1% glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin (PAA ¨ Laboratories, Colbe, Germany). Cells were spun down onto glass slides with a cytospin (Thermo Electron Corp., Waltham, MA, USA), followed by staining with Diff-Quick (Dade, Marburg, Germany), to identify immune cells. The percentage of leucocytes was determined microscopically using standard cytological criteria. In cell-free lavage fluids, IL-5 (BD Biosciences, San Jose, CA, USA) and IL-13 (Invitrogen) were measured by ELISA according to the manufacturer’s instructions. In vitro ovalbumin restimulation of splenic mononuclear cells Splenic mononuclear cells (MNCs) were isolated and restimulated with OVA as described previously [23]. Total RNA and first-strand synthesis and real-time PCR was carried out as described [12]. Cytokine levels in cell culture supernatants were analysed as described above. Flow cytometric analysis of single-cell suspensions from the bone marrow, thymus and spleen The spleen and thymus from control and TRPC6 / mice were removed and placed in 60-mm culture dishes containing resuspension buffer (PBS supplemented with 1% FCS). Single-cell suspensions of the spleen and thymus were prepared by passing disrupted organs through a 70 mm mesh nylon filter. The bone marrow was flushed from the
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femur and tibia with a 23-gauge needle. The remaining cell aggregates were removed by passing cell suspensions through a 70-mm nylon mesh filter. After washing, cells were resuspended in PBS at 1 107 cells/mL. One 106 cells were incubated in PBS containing 5% of rat serum for 5 min at room temperature and subsequently incubated with the appropriate antibody coupled to fluorescent dyes for 10 min at room temperature. After adding FACS-lysis buffer (BD Bioscience) and an additional incubation for 10 min at room temperature, cells were washed once and analysed using a FACSortTM flow cytometer and CELLQuestTM software (BD Bioscience). The following antibodies were used: fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD45, PE-conjugated rat anti-mouse CD45, PE-conjugated rat antimouse CD8, PerCP-conjugated rat anti-mouse CD45R, PerCP-conjugated rat anti-mouse CD4 and FITC-conjugated rat anti-mouse Ly-6G. All antibodies were purchased from BD Bioscience. Analysis of mucus production The lungs were formaldehyde fixed by instillation at a constant pressure of a 20-cm fluid column. Subsequently, the trachea was ligated, and the lungs were immersed in fixative. Organ slices, tissue block and fields of view were collected according to the principle of ‘systematic uniform random sampling’ in order to assure that the sample was representative of the whole organ. Paraffin-embedded lung sections were stained with Periodic acid–Schiff (PAS). The volume of PAS-stained epithelial mucus (Vmucus) per surface area of airway epithelial basal membrane (Sep) was determined by means of point and intersection counting using a computer-assisted stereology tool box [24] (CASTGrid 2.0, Olympus, Kobenhavn, Denmark) and subsequent calculation according to the formula: Vmucus/Sep = LpSPmucus/2SIep with SPmucus = sum of all points hitting mucus, SIep = sum of all intersections of test lines with epithelial basal membrane, and Lp = test-line length at final magnification. Statistics Results are presented as standard error of the mean (SEM). Student’s unpaired t-test was used to determine the significance of differences between groups. Results Increased airway responsiveness and contractility of tracheal rings in TRPC6 / mice We used head-out body plethysmography to monitor the respiratory pattern of mice exposed to increasing concentrations of methacholine, applied as aerosol, which acts on c 2008
Fig. 1. Analysis of pulmonary expiration rates and tracheal smooth muscle responsiveness of TRPC6 / and wild-type (WT) mice challenged with a cholinergic agonist. (a) Head-out body plethysmography of WT (black bars, n = 16) and TRPC6 / (grey bars, n = 17) mice. Mice were exposed to aerosols with increasing methacholine concentrations and expiration rates were analysed simultaneously. Methacholine concentrations that produced half-maximal expiration rates were calculated as described in ‘Material and methods’. (b) Force development of isolated tracheas from WT (black bars, n = 10) and TRPC6 / (grey bars, n = 9) mice were analysed under isometric conditions. Values were normalized to the contraction elicited by application of KCl. (c) One representative experiment from a WT (top) and a TRPC6 / tracheal ring (bottom) after application of the cholinergic agonist methacholine and the antagonist atropine is shown. All values are expressed as means SEM. Significant differences (Po0.05) between WT and TRPC6 / mice.
phospholipase C-coupled muscarinic acetylcholine receptors in airway smooth muscle cells. TRPC6-deficient mice displayed an increased responsiveness to methacholine and showed a 50% reduction in the midexpiratory airflow at a lower methacholine concentration than WT litter mates (Fig. 1a). In agreement with the in vivo findings, a significantly stronger contraction in response to 1 mM methacholine was detected in tracheal rings of TRPC6 / mice than in tracheal rings from control mice (Fig. 1b). The effect of methacholine was mediated by muscarinic receptors, because it was blocked by 1 mM atropine, an antagonist of muscarinic receptors (Fig. 1c). Similar results were obtained in the BALB/c background strain analysed below (data not shown).
The Authors c 2008 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 38 : 1548–1558 Journal compilation
1552 S. Sel et al Up-regulated TRPC3-levels in tracheal smooth muscle cells of transient receptor potential channel 6-deficient mice To gain an insight into possible molecular mechanisms for the increased AHR in TRPC6-deficient mice, we next quantified mRNA levels of all seven TRPCs in the lungs and brains of TRPC6 / and WT mice (Fig. 2). A realtime quantitative RT-PCR analysis of lung RNA showed the presence of PCR products (Fig. 2a, inset) for mRNAs of all TRPC family members, except TRPC5 and TRPC6. The latter was absent only in TRPC6 / mice. Most TRPCs were transcribed at similar levels in WT and TRPC6deficient mice. However, TRPC3 mRNA was elevated three- to fourfold in TRPC6 / mice (Fig. 2a). To identify the tissues and cells involved in the up-regulation of TRPC3 in the lung, we analysed tracheal smooth muscle
cells and epithelial cells from WT and TRPC6-deficient mice and found a two- to threefold higher expression of TRPC3 mRNA in TRPC6 / tracheal smooth muscle cells compared with WT cells (Fig. 2b). In clear contrast, all TRPC genes except for TRPC6 were transcribed at similar levels in RNA samples of epithelial cells (Fig. 2c) as well as pre-capillary pulmonary arterial smooth muscle cells [11] and endothelial cells (data not shown) from WT and TRPC6-deficient mice, excluding any compensatory up- and/or down-regulation of TRPC channels in these lung tissues. Expression of the CaV1.2a1 subunit gene of voltage-gated calcium channels was also not altered in tracheal smooth muscle cells (data not shown), excluding any compensatory up-regulation of these channels.
Increased basal cation entry in TRPC6-deficient tracheal smooth muscle cells
Fig. 2. Quantitative analysis of mRNA levels by real-time PCR of total RNA from mouse lungs (a), tracheal smooth muscle cells (b) and epithelial cells (c). Total RNA was prepared from wild-type (WT; lung n = 7, brain n = 4, black bars) and TRPC6 / mice (TRPC6 / ; lung n = 6, brain n = 5, grey bars), reverse-transcribed and products of the first-strand synthesis were analysed for the presence of amplification products obtained with the primer pairs described in ‘Material and methods’. Reaction products were analysed by agarose gel electrophoresis (a, inset). mRNAs coding for TRPCs (main panels) were quantified with the aid of a light cycler. Values are presented as percentage of reference mRNA expression. Significant differences (P o 0.05) between WT and TRPC6 / mice.
To prove, whether up-regulation of TRPC3 mRNAs in tracheal smooth muscle cells of TRPC6-deficient mice is correlated with an increased contractility of airway smooth muscle, smooth muscle cells from the tracheae were isolated and Ba21 influx was monitored in the presence of 100 mM CdCl2 in fura-2-loaded single cells. Basal influx of Ba21 ions was profoundly increased in smooth muscle cells isolated from TRPC6-deficient mice compared with WT cells (Fig. 3a). Cultured smooth muscle cells of TRPC6 / mice electroporated with plasmids carrying an established siRNA expression cassette under the control of an H1 promoter directed against the TRPC3 mRNA plus GFP [12] or plasmids carrying an expression cassette for an unrelated siRNA plus GFP were further analysed. Expression of TRPC3-siRNA in these cells resulted in decreased basal activity (Fig. 3b) compared with TRPC6 / cells expressing an unrelated siRNA, as already described for smooth muscle cells of the aorta and cerebral arteries [12]. These data favour a model of TRPC3/6 heteromeric channels in these tissues where TRPC6 inhibits a constitutively active TRPC3 channel activity [22]. In clear contrast to these results, we could not detect any changes in the expression of TRPC3 in splenic MNCs from TRPC6 / mice compared with WT cells by quantitative PCR (Fig. 3c). Expression of TRPC3- and TRPC6-mRNAs was also compared in freshly isolated mast cells as well as in cell lines derived from B cells, T cells and macrophages. In these cell types, TRPC3 mRNA levels were already significantly higher than TRPC6 mRNA levels (Fig. 3d), making it improbable that TRPC6 suppresses the basal channel activity of TRPC3 [22] in these cells. Therefore, it is rather unlikely that similar effects as outlined in Fig. 3b result from a possible up-regulation of TRPC3- in TRPC6deficient cells.
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tages of leucocyte subpopulations extracted from these tissues were not significantly different as shown in Fig 4. There was no difference in the total cell counts from the spleen and thymus between control and TRPC6 / mice (data not shown).
Decreased ovalbumin-specific serum immunoglobulin E levels in TRPC6-deficient mice compared with wild-type mice To analyse a possible role of TRPC6 in experimental airway inflammation, the TRPC6 mutation was bred into a BALB/c background strain, because BALB/c mice develop the highest type I hypersensitivity responses to OVA and represent an established mouse model of allergic airway inflammation (reviewed in [16]). WT and TRPC6 / BALB/c mice were sensitized and challenged with OVA. Sham-treated animals were exposed to PBS only. As expected, OVA-sensitized and -challenged mice produced significantly higher levels of OVA-specific IgE and IgG1. However, IgE levels were reduced in TRPC6 / mice (Fig. 5a), while IgG1 and IgG2a levels were not significantly different in TRPC6 / mice compared with WT mice (Figs 5b and c). Total IgE levels were not significantly different in OVA-sensitized and -challenged TRPC6 / mice compared with WT mice (data not shown). Fig. 3. Ba21 influx in isolated smooth muscle cells from wild-type (WT) and TRPC6 / (TRPC6 / ) mice (a, b) and quantitative analysis of TRPC3- and TRPC6-mRNA levels in splenic mononuclear cells (c) mast cells and cell lines derived from B-, T cells and macrophages (d). (a) Ba21 influx in isolated smooth muscle cells from tracheae of WT and TRPC6 / mice. Isolated cells were loaded with fura-2 and basal Ba21 influx was assessed by the increase in fluorescence ratios. After application of EGTA (2 mM) and CdCl2 (100 mM) in a nominally Ca21-free solution, Ba21 (2 mM) was added to the bath solution. The black (WT, n = 59 cells from five mice) and grey (TRPC6 / , n = 14 cells from five mice) lines represent the calculated means, whereas the light grey areas indicate SEM. (b) Ba21 influx in cultured smooth muscle cells from tracheae of TRPC6 / mice electroporated with a pSUPER-based vector expressing the green fluorescent protein (GFP) and a TRPC3-specific small interfering (si) RNA (siRNA TRPC3/GFP; n = 14 cells from five mice) or an unrelated siRNA (siRNA unrelated; n = 14 cells from five mice). (c, d) Quantitative analysis of TRPC3 and TRPC6 mRNA levels by real-time PCR of total RNA from splenic mononuclear cells (c) and isolated murine mast cells, as well as cell lines derived from T cells, B cells and macrophages (d). Data are expressed as means SEM.
Unaltered percentage of leucocytes, T-helper cells, cytotoxic T cells, CD4/CD81/ T cells, granulocytes and B cells in TRPC6 / mice To identify any differences in leucocyte subpopulations, we performed flow-cytometric analysis of immune cells from the bone marrow, thymus and spleen from control (TRPC61/ and WT) and TRPC6 / mice. The percen c 2008
T-helper type 2 cytokines are reduced in ovalbuminsensitized and -challenged TRPC6 / mice Th2 cytokines were analysed in the BAL from OVAsensitized and -challenged TRPC6 / mice and WT mice, as well as from control mice from both genotypes treated with PBS. As expected, OVA-sensitized and -challenged mice showed significantly higher levels of Th2 cytokines than sham-treated animals. However, in TRPC6-deficient mice, cytokines like IL-5 and IL-13 were decreased compared with WT mice (Figs 6a and b) and in clear contrast to IL-10 levels, which were not significantly different (data not shown). IL-5 and IL-13 levels of OVA re-stimulated MNCs from spleens of TRPC6 / and WT mice were also significantly reduced compared with WT mice, while IFNg levels as well as IL-12 levels from all cells were below the detection limit (data not shown). Decreased eosinophilia in TRPC6-deficient mice The percentages of lymphocytes, eosinophils, neutrophils and macrophages were determined in BAL samples. After OVA sensitization and challenge, the percentage of all immune cells analysed (Figs 7a–d), with the exception of macrophages (Fig. 7c), increased significantly compared with PBS-treated control animals. While the fractions of
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Fig. 4. Flow cytometric analysis of single-cell suspensions from the bone marrow, thymus and spleen of TRPC6 / (n = 4) and control mice (n = 4). Data are expressed as means SEM.
lymphocytes and neutrophils were not significantly different in WT and TRPC6 / mice (Figs 7b and d), the fraction of eosinophils was significantly lower in TRPC6 / mice compared with WT mice (Fig. 7a). This decreased eosinophilia is not due to a general reduction in the number of immune cells in WT and TRPC6 / mice, because flow-cytometric immunophenotyping of the spleen, thymus and bone marrow cells showed no differences regarding the relative numbers of different immune cells of the granulocyte lineages as well as B and T lymphocytes (Fig. 4). The reason why the fraction of macrophages in BAL samples decreased after OVA sensitization and challenge, as well as the reason for the increased macrophage fraction in TRPC6 / mice compared with WT mice are unknown.
Fig. 5. Immunoglobulin levels in ovalbulin (OVA)-sensitized and challenged TRPC6 / (grey bars, n = 7) and wild type (WT) (black bars, n = 7) mice (a–c). OVA-specific IgE (a), IgG1 (b) and IgG2a (c) serum levels were determined by ELISA on day 31. Data are expressed as means SEM. Po0.005 levels in TRPC6 / mice vs. WT mice.
Mucus production is not altered by transient receptor potential channel 6 deficiency Because IL-13 stimulates lung epithelial cells to produce mucus and TRPC6 / mice produced low levels of IL-13, we set out to analyse mucus production in OVA-sensitized and -challenged TRPC6-deficient and WT mice. Although mucus production was significantly increased in OVAsensitized and -challenged mice (Figs 8a and b), no difference in mucus production was detected between OVA-sensitized and -challenged TRPC6 / and WT mice (Fig. 8b). Discussion
Fig. 6. Levels of T-helper type 2 (Th2) cytokines in the bronchoalveolar lavage (BAL) from ovalbulin (OVA)-sensitized and -challenged TRPC6 / (grey bars, n = 7) and wild type (WT) (black bars, n = 7) mice and after OVA restimulation of splenic mononuclear cells. IL-5 (a) and IL-13 levels (b) were determined in cell-free BAL fluids. Data are expressed as means SEM. Significant difference (Po0.05) between WT and TRPC6 / mice.
The current study explores the potential roles of TRPC6 in allergic airway inflammation. Despite some reports demonstrating TRPC6 expression in immune cells (reviewed in [25]) and some evidence regarding a contribution of TRPC6 to COPD [13], a role of TRPC6 in allergic airway inflammation has not been addressed experimentally previously. We used a TRPC6-deficient mouse model and a wellestablished OVA sensitization/challenge protocol [18] to
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Fig. 7. Percentages of eosinophils (a), neutrophils (b), macrophages (c) and lymphocytes (d) in the bronchoalveolar lavage (BAL) from ovalbulin (OVA)sensitized and -challenged TRPC6 / (grey bars, n = 7) and wild type (WT) (black bars, n = 7) mice. Samples were taken 24 h after the last challenge. The percentage of stained cells was determined microscopically using standard cytological criteria. The values are expressed as means SEM. Significant difference (Po0.05) between WT and TRPC6 / mice.
analyse the different effects of allergic airway inflammation. Interestingly, methacholine-induced smooth muscle contractility as well as methacholine-induced airway responsiveness was increased in TRPC6 / mice compared with WT mice. This phenotype may be explained by upregulation of the closely related TRPC3 in lung tissues including smooth muscle cells, which overcompensates the TRPC6 deficiency, a phenomenon already described in other tissues containing smooth muscle cells [12]. These data raised the intriguing question as to whether TRPC6deficient immune cells show similar processes. To analyse immunological responses in a well-established mouse model of type-I allergy [16], the TRPC6 mutation was bred into a BALB/c background. Most interestingly, immunological responses like production of Th2 cytokines and IgE as well as eosinophilia were reduced in OVA-sensitized and -challenged TRPC6-deficient mice compared with control mice. While eosinophils express TRPC6 and TRPV1 [14], the expression of TRPC3 [27] and/ or TRPC6 [15] was reported in T-lymphocytes (reviewed in [25]). Obviously, our results support an important role of TRPC6 in both cell types. After OVA sensitization and challenge, antigen-presenting cells (APCs) induce T cell differentiation to Th cells (Th1 and Th2), triggering an immune response c 2008
against OVA antigens very similar to allergic airway inflammation. A Th1/Th2 disequilibrium in favour of Th2 cells then leads to increased production of IL-4, IL-5 and IL-13, which contribute to the allergic processes [28]. TRPC6 expression was demonstrated in human peripheral T lymphocytes by RT-PCR and Western blotting with a TRPC6-specific antibody [15], while in the Jurkat T cell line, data are contradictory [15, 27]. However, both reports agree on the expression of TRPC3 in peripheral T lymphocytes and Jurkat T cells [15, 27]. We were able to detect TRPC6 and TRPC3 in a cell line derived from T cells (Fig. 3d) as well as in splenic MNCs from WT mice, but could not detect any changes in the expression of TRPC3 in splenic MNCs from TRPC6 / mice by quantitative PCR (Fig. 3c). For this reason, a compensatory up-regulation in splenic MNCs appears to be unlikely. Because Th2 cytokines arise not only from Th2 T cells but also from mast cells and eosinophils, our functional data support the concept of a unique role of TRPC6 in these cells, because Th2 cytokines were reduced in OVA-sensitized and -challenged TRPC6-deficient mice compared with WTmice. TRPC6 deficiency may result in reduced numbers of Th2 cells, mast cells and/or eosinophils, as well as reduced production of cytokines from these cells. Future experiments in our laboratories will determine the
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Fig. 8. Mucus production in the lungs of ovalbulin (OVA)-sensitized and -challenged TRPC6 / (grey bars, n = 7) and wild type (WT) (black bars, n = 7) mice. (a) Representative photomicrographs of periodic acid-Schiff (PAS)-stained airway cross-sections are shown. (b) Volumes of PAS-stained epithelial mucus (Vmucus) per surface area of airway epithelial basal membrane (Sep) were determined by means of point and intersection counting as described in ‘Material and methods’. Data are expressed as means SEM.
relevance of each of the above-mentioned cells to the described phenotype in TRPC6-deficient mice. Along these lines, decreased IgE levels in OVA-sensitized and -challenged TRPC6-deficient mice compared with WT mice might be explained by reduced Th2 cell activation of B lymphocytes, because TRPC6 expression is not detectable in the latter cells (Fig. 3c) and was not reported so far (reviewed in [25]). Mediators such as IL-5 control the infiltration process by regulating the maturation of eosinophils in the bone marrow, priming them for recruitment, and by regulating the attraction of eosinophils into tissues. Eosinophils
contribute to the initiation and maintenance of allergic airway diseases [26]. They are recruited to allergic inflammatory sites, and eosinophil accumulation into the inflamed lung is associated with both acute and chronic phases of allergic airway inflammation [29]. The number of eosinophils within the allergic tissue depends primarily on the extent of cell infiltration during the acute inflammatory response. Cytokines such as IL-5 are known to enhance eosinophil survival and thus contribute to the persistence of eosinophils at sites of allergic inflammation [30]. For this reason, the reduced eosinophilic infiltration into the BAL can be explained by two different
c 2008 The Authors c 2008 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 38 : 1548–1558 Journal compilation
TRPC6 / mice show reduced allergic airway responses
mechanisms that are not mutually exclusive. First, lower IL-5 levels from Th2 cells may lead to decreased eosinophil maturation in the bone marrow and consequently in their release into the blood (reviewed in [31, 32]) and, second, TRPC6 deficiency may decrease eosinophil adhesion and subsequent infiltration into the lung tissue. Very recently, evidence was provided that lysophosphatidylcholine (LPC) regulates b2-integrin-mediated adhesion of eosinophils by a non-store-operated Ca21 channel [14]. Moreover, only TRPC6 and TRPV1 of the TRPC, M and V families were found to be expressed in eosinophils. Because capsaicin, a TRPV1 agonist, was not able to increase the intracellular calcium concentration ([Ca21]i) in eosinophils, the authors concluded that LPC causes a non-store-operated Ca21 influx via TRPC6, which subsequently activates CD11b/CD18 of the b2-integrin subfamily to cause eosinophil adhesion [14]. Only specific down-regulation of TRPC6 in eosinophils may answer the question, as to which of the mechanism(s) is/are required for the TRPC6-dependent eosinophilia in inflammatory diseases like allergic airway inflammation. The pathogenesis of allergic airway inflammation is composed of two phases: (i) AHR and (ii) subsequent airway remodelling. To investigate the latter, we also analysed goblet hyperplasia and resulting mucus hypersecretion in airway epithelial cells. As IL-13 levels are responsible for mucus production [33] and TRPC6 is expressed in airway epithelial cells [9], we expected a decreased mucus production in TRPC6 / mice. Much to our surprise, we were not able to detect altered mucus production and goblet cell hyperplasia in OVA-sensitized and -challenged TRPC6-deficient mice as compared with WT mice. The TRPC6-dependent IL-13 level may not play a role in mucus production in airway epithelial cells during acute allergic airway inflammation, because temporary dissociation of IL-13-mediated mucus hypersecretion from AHR was described recently [34]. However, as mucus production and goblet hyperplasia are more pronounced in chronic airway inflammation, further studies need to be performed to investigate whether there is TRPC6-dependent mucus production in a mouse model of chronic airway inflammation [35]. In conclusion, our data point to a unique and nonredundant role of TRPC6 in Th2 cells, mast cells and/or eosinophils that results in reduced Th2 cytokine production and decreased airway eosinophilia in OVA-sensitized and -challenged TRPC6-deficient mice. Acknowledgements We wish to thank Winfried Lorenz, Tanja Rausch, Anja Spieß-Naumann and Susanne Ziegler for excellent technical assistance. The expertise of Drs Udo Herz and Khai Vi in head-out body-plethysmography and the analysis of the contractility of tracheal rings is greatly acknowledged. c 2008
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We would like to thank Dr Reinhard Mailhammer (GSF National Research Center for Environment and Health, Munich, Germany) for his gift of mast cell cDNA.
References 1 Nilius B, Owsianik G, Voets T, Peters JA. Transient receptor potential cation channels in disease. Physiol Rev 2007; 87:165–217. 2 Venkatachalam K, Montell C. TRP Channels. Annu Rev Biochem 2007; 76:387–417. 3 Dietrich A, Kalwa H, Rost BR, Gudermann T. The diacylgylcerolsensitive TRPC3/6/7 subfamily of cation channels: functional characterization and physiological relevance. Pflugers Arch 2005; 451:72–80. 4 Dietrich A, Mederos y Schnitzler M, Kalwa H, Storch U, Gudermann T. Functional characterization and physiological relevance of the TRPC3/6/7 subfamily of cation channels. Naunyn Schmiedebergs Arch Pharmacol 2005; 371:257–65. 5 Hofmann T, Obukhov AG, Schaefer M, Harteneck C, Gudermann T, Schultz G. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 1999; 397:259–63. 6 Hofmann T, Schaefer M, Schultz G, Gudermann T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc Natl Acad Sci USA 2002; 99:7461–6. 7 Dietrich A, Chubanov V, Kalwa H, Rost BR, Gudermann T. Cation channels of the transient receptor potential superfamily: their role in physiological and pathophysiological processes of smooth muscle cells. Pharmacol Ther 2006; 112:744–60. 8 Boulay G, Zhu X, Peyton M et al. Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J Biol Chem 1997; 272:29672–80. 9 Corteling RL, Li S, Giddings J, Westwick J, Poll C, Hall IP. Expression of transient receptor potential C6 and related transient receptor potential family members in human airway smooth muscle and lung tissue. Am J Respir Cell Mol Biol 2004; 30:145–54. 10 Wang J, Shimoda LA, Sylvester JT. Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle. Am J Physiol Lung Cell Mol Physiol 2004; 286:L848–58. 11 Weissmann N, Dietrich A, Fuchs B et al. Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange. Proc Natl Acad Sci USA 2006; 103:19093–8. 12 Dietrich A, Mederos YSM, Gollasch M et al. Increased vascular smooth muscle contractility in TRPC6 / mice. Mol Cell Biol 2005; 25:6980–9. 13 Li S, Gosling M, Poll C. Determining the functional role of TRPC channels in primary cells. Pflugers Arch 2005; 451:43–52. 14 Zhu X, Learoyd J, Butt S et al. Regulation of eosinophil adhesion by lysophosphatidylcholine via a non-store-operated Ca21 channel. Am J Respir Cell Mol Biol 2007; 36:585–93. 15 Gamberucci A, Giurisato E, Pizzo P et al. Diacylglycerol activates the influx of extracellular cations in T-lymphocytes independently of intracellular calcium-store depletion and possibly
The Authors c 2008 Blackwell Publishing Ltd, Clinical and Experimental Allergy, 38 : 1548–1558 Journal compilation
1558 S. Sel et al
16 17
18
19
20
21
22
23
involving endogenous TRP6 gene products. Biochem J 2002; 364:245–54. Herz U, Renz H, Wiedermann U. Animal models of type I allergy using recombinant allergens. Methods 2004; 32:271–80. Neuhaus-Steinmetz U, Glaab T, Daser A et al. Sequential development of airway hyperresponsiveness and acute airway obstruction in a mouse model of allergic inflammation. Int Arch Allergy Immunol 2000; 121:57–67. Herz U, Braun A, Ruckert R, Renz H. Various immunological phenotypes are associated with increased airway responsiveness. Clin Exp Allergy 1998; 28:625–34. Devore-Carter D, Morway PF, Weiss EB. Isolation and characterization of guinea-pig tracheal smooth muscle cells that retain differentiated function in long-term subculture. Cell Tissue Res 1988; 251:325–31. You Y, Richer EJ, Huang T, Brody SL. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am J Physiol Lung Cell Mol Physiol 2002; 283:L1315–21. Hultner L, Szots H, Van Snick J, Moeller J, Welle M, Dormer P. Interleukin 3 mediates interleukin 6 production in murine interleukin 3-dependent hemopoietic cells. Growth Factors 1989; 2:43–51. Dietrich A, Mederos y Schnitzler M, Emmel J, Kalwa H, Hofmann T, Gudermann T. N-linked protein glycosylation is a major determinant for basal TRPC3 and TRPC6 channel activity. J Biol Chem 2003; 278:47842–52. Blumer N, Sel S, Virna S et al. Perinatal maternal application of Lactobacillus rhamnosus GG suppresses allergic airway inflammation in mouse offspring. Clin Exp Allergy 2007; 37: 348–57.
24 Weibel ER, Hsia CC, Ochs M. How much is there really? Why stereology is essential in lung morphometry. J Appl Physiol 2007; 102:459–67. 25 Schwarz EC, Wolfs MJ, Tonner S et al. TRP channels in lymphocytes. Handb Exp Pharmacol 2007; 179:445–56. 26 Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol 2006; 24:147–74. 27 Philipp S, Strauss B, Hirnet D et al. TRPC3 mediates T-cell receptor-dependent calcium entry in human T-lymphocytes. J Biol Chem 2003; 278:26629–38. 28 Ngoc PL, Gold DR, Tzianabos AO, Weiss ST, Celedon JC. Cytokines, allergy, and asthma. Curr Opin Allergy Clin Immunol 2005; 5:161–6. 29 Kay AB. Asthma and inflammation. J Allergy Clin Immunol 1991; 87:893–910. 30 Yamaguchi Y, Suda T, Ohta S, Tominaga K, Miura Y, Kasahara T. Analysis of the survival of mature human eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils. Blood 1991; 78:2542–7. 31 Rosenberg HF, Phipps S, Foster PS. Eosinophil trafficking in allergy and asthma. J Allergy Clin Immunol 2007; 119:1303–10. 32 Trivedi SG, Lloyd CM. Eosinophils in the pathogenesis of allergic airways disease. Cell Mol Life Sci 2007; 64:1269–89. 33 Wills-Karp M. Interleukin-13 in asthma pathogenesis. Immunol Rev 2004; 202:175–90. 34 Yang M, Rangasamy D, Matthaei KI et al. Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness. J Immunol 2006; 177:5595–603. 35 Wegmann M, Fehrenbach H, Fehrenbach A et al. Involvement of distal airways in a chronic model of experimental asthma. Clin Exp Allergy 2005; 35:1263–71.
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