AJP - Lung Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 291: L244-L251, 2006. First published February 17, 2006; doi:10.1152/ajplung.00474.2005
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/L244    most recent
00474.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fayon, M.
Right arrow Articles by Marthan, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fayon, M.
Right arrow Articles by Marthan, R.

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Michael Fayon,1,2 Muriel Rebola,2 Patrick Berger,1 Sophie Daburon,3 Olga Ousova,1 Frédéric Lavrand,4 Boutchi Moukaïla,1 Wilfried Pujol,1 Jean Luc Taupin,3 André Labbé,5 Mathieu Molimard,1 and Roger Marthan1

1Laboratoire de Physiologie Cellulaire Respiratoire, Université Victor Segalen Bordeaux 2, and Institut National de la Santé et de la Recherche Médicale, E-356, Bordeaux; 2Centre Hospitalier Universitaire de Bordeaux, Hôpital Pédiatrique, Unité de Pneumologie Pédiatrique et Centre de Recherche, Bordeaux; 3Service d'Immunologie and 4Service de Chirurgie Pédiatrique Bordeaux; and 5Centre Hospitalier Universitaire de Clermont Ferrand, Service de Pneumologie et Reanimation Pédiatrique F. Clermond, Ferrand, France

Submitted 9 November 2005 ; accepted in final form 10 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca2+]i) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-3H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-{alpha}) for [Ca2+]i signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-{alpha}-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca2+]i response was enhanced twofold after incubation with LIF, whereas TNF-{alpha} increased the [Ca2+]i to U-46619 threefold. In TNF-{alpha}-exposed adult ASMC, [Ca2+]i responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca2+]i signaling.

calcium signaling; bronchial hyperreactivity; asthma

ASTHMA VERY OFTEN BEGINS IN childhood and encompasses airway inflammation, obstruction, and hyperreactivity. It is now widely accepted that airway smooth muscle (ASM) cells (ASMC) possess new functions, in addition to their classical role as end-effector cells mediating bronchomotor tone. These functions include the secretion of cytokines and matrix proteins, cytokine-induced expression of cell adhesion molecule migration, proliferation, and cell-to-cell interactions by direct interaction with immune cells (8, 17, 25). An integral part of this interaction is the release of a variety of cytokines that regulate cellular and molecular responses. Indeed, several cytokines at the mRNA or protein level have been detected within the airways of asthmatic subjects (6). These proinflammatory mediators may act directly or indirectly, in an autocrine manner, on ASMC. All of the above greatly contribute to the perpetuation of the chronic local inflammation and the structural remodeling process of asthmatic airways. This complex inflammatory response to injury may also involve the coordinated interaction between the nervous and immune systems (27). Leukemia inhibitory factor (LIF), a member of the IL-6 family of cytokines, has also been shown to be an integral component of the interface between nerves and the immune system (19). However, little is known about this cytokine in the context of normal lung function or indeed, inflammation, especially in immature humans and animals.

The asthmatic inflammatory process may influence calcium signaling. The study of Ca2+ signaling provides information regarding ASMC contractile activity (22). Stimulation of an ASMC by a bronchoconstrictor agent induces a rapid rise in intracellular Ca2+ concentration ([Ca2+]i), mostly related to the release of intracellular Ca2+ stores, to a peak level roughly 10 times higher than the resting level (2). The binding of ACh to a muscarinic M3 cholinoceptor activates a Gq/11 protein that, in turn, activates phopholipase C, inducing hydrolysis of phosphatidylinositol 4,5-bisphophate to produce inositol 1,4,5-triphosphate and 1,2-diacylglycerol. Inositol 1,4,5-triphosphate binds to its receptor to release Ca2+ from the sarcoplasmic reticulum (34). The increased [Ca2+]i results in the activation of the Ca2+/calmodulin-sensitive myosin light-chain kinase, and the subsequent phosphorylation of the regulatory myosin light chain, which results in the cross bridging between myosin and actin (2). The ability of TNF-{alpha} to alter the [Ca2+]i signals induced by a variety of agonists suggests that this cytokine may prime ASMC, with subsequent hyperresponsiveness to contractile agents (2).

Despite the extensive literature, very little is known of the intrinsic properties of immature ASM. It is thus important to specifically characterize the in vitro properties of the immature ASM in humans. Since epidemiological studies in asthma suggest that outcome in adults may be determined primarily in early childhood (23, 24, 36), we hypothesized that immature ASMC may contribute more toward the inflammatory process and airway remodeling than adult ASMC. The aim of the present study was thus to evaluate the secretory properties of cultured human neonatal ASMC (secretion of the neurotrophic cytokine LIF and production of its mRNA). Moreover, we studied the effect of LIF on DNA synthesis in ASMC, migration of human mast cells (HMCs), intracellular ASMC [Ca2+]i signaling, and contraction of isolated rat pup tracheae. Moreover, the effect of other inflammatory mediators (histamine, U-46619, bradykinin) and contractile agonists (ACh) on [Ca2+]i was also evaluated. The findings were compared with adult cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human ASMC

Isolation and culture of human neonatal and adult ASMCs. Human lung was obtained by postmortem forceps biopsy from premature (≥26 wk) and term neonates and infants who had died from extrapulmonary causes, and at thoracotomy in adults undergoing resection for pulmonary carcinoma, as previously described (4, 12). The time between the death of the neonates and infants and harvesting of the tissue was <2 h in all cases. Adult patients were nonatopic and did not report any clinical history of asthma. The tissue obtained (tracheae in neonates, and 3rd to 5th generation airways in adults) was used for cell cultures. Collection and use of lung specimens were in accordance with the ethics recommendations of Bordeaux University Hospital and French law, and our laboratory, which is authorized to conduct human and animal studies, obtained informed consent from the parents.

The tracheae and bronchi were washed three times with sterile DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (antimycotic-antibiotic solution; GIBCO). Segments of airways were dissected free from the surrounding parenchyma in the same medium at room temperature. The epithelium was removed to expose the underlying bands of smooth muscle, which were then gently separated from the underlying connective tissue in small bundles. Smooth muscle bands were cut into squares measuring 1–2 mm2 and transferred into six-well culture plates (~1–2 explants/well) and covered with a minimal amount of medium and 10% (vol/vol) FCS (GIBCO), supplemented with 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (Sigma Chemical), 1% (vol/vol) nonessential amino acid mixture (Sigma Chemical), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (antimycotic-antibiotic solution; GIBCO). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2-95% air. The medium was replenished every 3 days. Cells were passaged once they reached confluence (after 4–8 wk), and passages 2–5 were used for experimentation. Cells were rinsed twice with HBSS (GIBCO) and then passaged with trypsin-EDTA (GIBCO).

Cell characterization by immunocytochemistry. To assess the purity of the cells, an immunocytochemical method was employed using an indirect immunofluorescence technique (4). Briefly, after fixation of ASMC in methanol, anti-{alpha} actine (clone 1A4, Sigma), anti-myosin (clone hSM-V, Sigma), anti-epithelial cell cytokeratin (Sigma), anti-endothelial cell factor VIII (Dako), and anti-fibroblast cellular surface protein (clone 1B10 [PDB] Sigma) primary antibodies were used to confirm that the cells were ASMC. All human ASMCs stained positively for smooth muscle actin and myosin. An irrelevant antibody served as a control.

Analysis of LIF levels in ASMC culture supernatant. To assess whether the ASMC secreted LIF, TNF-{alpha} (100 ng/ml) was added to the medium after the cells had been starved in insulin, transferrin, and selenium (ITS) medium for 24 h. The supernatant was then harvested every day thereafter (days 1–5), stocked on ice, and immediately frozen (–80°C). The LIF assay was performed as previously described (29) using a colorimetric ELISA. Briefly, specific high-titer polyclonal Ig were obtained by intra-lymph node immunization of rabbits with recombinant human (rh) LIF. The same batch of purified anti-LIF Ig was used for the coating (capture antibody) and for the quantitative detection of the bound cytokine (soluble biotinylated antibodies). The sensitivity of this ELISA is 12 pg/ml of LIF (28).

RNA extraction and real-time PCR. We extracted total RNA from cultured ASMC, 600 µl, of TRIzol, according to the manufacturer's instructions (GIBCO, Invitrogen, Cergy Pontoise, France). RNA integrity (the integrity of the 28S and 18S fractions of rRNA) was verified by gel electrophoresis. The concentration of total RNA was measured by GeneQuant RNA/DNA calculator (Amersham Pharmacia). The reverse transcription reaction was carried out as mentioned previously (4). Real-time PCR was performed with a Rotor-Gene 2000 (Corbett Research; Morlake, Sidney, Australia) as described earlier (4). All specific primers were designed by using oligo primer analysis software (Oligo 6.6, Molecular Biology Insights, Cascade, CO) and ordered from Sigma-Genosys (Sigma-Genosys, Cambridge, UK). The primer pair sequences are indicated in Table 1. The efficiency of all of the PCR reactions was >90%. The specificity of the amplified products was examined in 2% agarose gel containing ethidium bromide. The accuracy of the normalization of real-time PCR data was verified by geometric averaging of four internal control genes (see above), according to a previously described method (38).


View this table:
[in this window]
[in a new window]
  		Table 1. Specific primer-pairs used for gene-expression analysis by real-time reverse transcription PCR

 
DNA synthesis assay. Once harvested by trypsinization, cells were grown in 96-well plates (2,000 cells/well), starved for 24 h (serum-free DMEM supplemented with 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, 0.5 µg/ml BSA, 4.7 µg/ml linoleic, and oleic acid) (ITS solution, Sigma Chemical), 2 mM L-glutamine (GIBCO), 1 mM sodium pyruvate (Sigma Chemical), 1% (vol/vol) nonessential amino acid mixture (Sigma Chemical), 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B (antimycotic-antibiotic solution, GIBCO), and incubated for 1–5 days with medium, 10% FCS, ITS, or PDGF-AA (15 ng/ml diluted in ITS medium). After the incubation period, 0.5 mCi [methyl-3H]thymidine (Amersham) was added to each well for an additional 8 h. Cell proliferation was then arrested by placing the culture plates in a 20°C freezer. Cells were harvested on a 0.7-mm-pore glass fiber filter and counted in scintillant on a 2000CA TriCarb liquid scintillation analyzer (4).

Chemotaxis assay. The migration of HMC-1 cells against recombinant LIF was assayed by using a 48-well micro-chemotaxis (Boyden) chamber (Neuroprobe, Cabin John, MD), as described previously (4). Nitrocellulose filters (150-µm thick, pore size of 8 µm) (Millipore, St. Quentin en Yvelines, France) were coated overnight at room temperature with 100 µg/ml fibronectin (Sigma) and then air-dried for 30 min. Migration of HMC-1 cells (3 x 105 cells/ml) was assessed in response to 0.2, 2, 20, and 200 ng/ml rhLIF (Chemicon International). Each experimental condition was processed in triplicate. After 60 min at 37°C and 5% CO2, nonmigrant cells were removed from the upper surface, and the filter was then fixed in ethanol, stained with Harris hematoxylin, and mounted in Permount (Fisher, Elancourt, France). The number of migrant cells present in the whole surface of each well (i.e., 8 mm2) was counted by using a quantitative method of color recognition described previously (5). The spontaneous migration of mast cells with either supernatant from unstimulated human ASMCs or DMEM served as controls for human ASMC supernatant or cytokine chemotaxis, respectively, and was referred to as 100% migration. Control experiments showed that mast cells that had migrated through remained attached to the filter, as no cell was detected in the fluid phase of the lower chamber. In addition, chemokinesis was differentiated from chemotaxis by placing attractants at the same concentrations in the upper wells.

Intracellular Ca2+ signaling. Once harvested by trypsinization, cells were placed on 14-mm circular glass plates and starved for 3 days in ITS medium. This medium was supplemented during the last 48 h (stimulated cells) or not (control cells) with the proinflammatory cytokine TNF-{alpha} (100 ng/ml) or LIF (5 ng/ml). Cells were then rinsed and incubated for 30 min at room temperature in a physiological saline solution with 2 mM Ca2+ (composition in mM: 130 NaCl; 5.6 KCl; 2 CaCl2; 10 HEPES; 11.1 glucose; 1 MgCl2) containing 1 µM of esterified indo-1 (indo-1 AM). Four different contractile agonists (histamine, bradykinin, ACh, and U-46619, a thromboxane A2-agonist), all at an identical concentration of 10–5 or 10–3 M, were microejected in the immediate vicinity of the cells. The agonist-induced variations in [Ca2+]i in each cell was recorded by microspectrofluorimetry using an epifluorescent microscope (Diaphot 300, Nikon), according to a previously described technique (33).

Rat Pups

Isometric contraction. Our laboratory has the approval of the Animal Ethics Committee of our Institution to house rats and conduct these experiments. Pathogen-free Wistar rat pups obtained from Janvier, France, were housed with their nursing mother during the entire experimental time. They were killed at the age of 12 days. The tracheae were dissected and cut into 5-mm-long segments (1 trachea studied per pup). Tracheal rings were incubated with LIF (5 ng/ml) or DMEM media alone for 24 h. Isometric contraction measurements were then performed, according to a previously described method (9). After rings were set at optimal resting tension, maximal contraction was elicited by the administration of ACh (10–3 M). All solutions were freshly prepared on the day of the experiment.

Statistical analysis. All values quoted are the means ± SE of separate experiments, performed on different days with different cell cultures. Regarding the secretion of LIF, each value corresponded to a different experiment. The [Ca2+]i relative peak amplitude corresponds to the difference between the maximal absolute peak and the resting [Ca2+]i. Statistical analysis was performed using the software package, NCSS 6.0.21 (Kaysville, UT). Data were analyzed for statistical significance using, as appropriate, the unpaired Student t-test, the Mann and Whitney U-test, or a one-way or multiple ANOVA. A P value <0.05 was considered significant.

Reagents. ACh, bradykinin, histamine, U-46619, and carbachol were purchased from Sigma-Aldrich Chimie (Lyon, France). Human TNF-{alpha}, IL-6, and IL-1beta were purchased from R&D Systems Europe (Lille, France), and rhLIF from Chemicon International.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Demographic Data

The demographic data of included infants and adults are shown Table 2.


View this table:
[in this window]
[in a new window]
  		Table 2. Patient characteristics

 
LIF Release

Detectable amounts of LIF were found in the supernatant of cultured immature and adult ASMC. Baseline levels after 24-h incubation in ITS medium was 112.6 ± 53.3 and 411.8 ± 170.3 pg/ml, in adult (n = 9) and immature (n = 6) ASMC, respectively (P = 0.037). At all study times, LIF secretion was enhanced after the addition of TNF-{alpha} (100 ng/ml) to the medium (Fig. 1). There was a 14-fold increase by adult ASMC and 24-fold increase by immature ASMC after 5 days of stimulation by the proinflammatory cytokine. In one case in immature ASMC, LIF secretion was increased by more than a power of 102.


Figure 1
View larger version (29K):
[in this window]
[in a new window]
  		Fig. 1. Absolute values of leukemia inhibitory factor (LIF) in adult and immature human airway smooth muscle cell (ASMC) culture supernatant. Results are means ± SE. LIF after incubation in insulin, transferrin, and selenium (ITS) medium (open bars) or with TNF-{alpha} (100 ng/ml) (shaded bars) was determined in the culture medium by ELISA [immature (no hatching), n = 4; adult (hatched bars) n = 9]. *P < 0.05 (ANOVA).

 
LIF mRNA Expression

In immature ASMC stimulated by TNF-{alpha} (100 ng/ml), LIF mRNA was upregulated in a sustained manner, i.e., 3–24 h after the addition of TNF-{alpha} (n = 3). In contrast, adult ASMC mRNA levels decreased from hour 3 to 24 (n = 5) (Fig. 2).


Figure 2
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. LIF mRNA after incubation of ASMC in TNF-{alpha}. Results are means ± SE. LIF mRNA after incubation in ITS medium (open bars) or with TNF-{alpha} (100 ng/ml) (shaded bars) was determined in the culture medium by ELISA [immature (no hatching), n = 3; adult (hatched bars) n = 5]. *P < 0.05 (Mann-Whitney U-test).

 
DNA Synthesis

Cultured cells assayed 1 and 5 days in ITS medium showed similar levels of incorporation of [methyl-3H]thymidine: day 1: 440.9 ± 147.4 vs. 454.1 ± 49.9; day 5: 449.1 ± 89.4 vs. 387.6 ± 81.0 in immature (n = 5) and adult (n = 5) ASMC, respectively (not significant).

Incubation with LIF (20 ng/ml) showed a modest but significant 21% increase in adult ASMC (n = 5) DNA synthesis on day 5 only, while only a nonsignificant 12% increase could be observed in immature ASMC (n = 5).

Migration of HMC-1 Cells

The effect of exogeneous rhLIF alone on migration of HMC-1 cells was negligible (% values in ITS medium): 109.2 ± 1.0, 103.0 ± 11.2, 104.5 ± 14.1, and 105.9 ± 6.2% at a LIF concentration of 0.2, 2, 20, and 200 ng/ml, respectively (n = 4 per concentration).

[Ca2+]i

In LIF. Incubation of immature ASMC in 5 ng/ml LIF greatly enhanced [Ca2+]i response to ACh, compared with control conditions (ITS medium) (Fig. 3). Baseline [Ca2+]i increased 59% [317.8 ± 26.5 nM (n = 18) vs. 505.8 ± 33.9 nM (n = 24) in ITS medium and LIF, respectively (P < 0.001)]. Relative [Ca2+]i peak increased more than twofold (248%) from 210.4 ± 22.9 to 522.0 ± 43.6 nM (P < 0.001), and time to maximal peak decreased twofold from 5.19 ± 0.51 to 2.47 ± 0.18 s (P < 0.001). There was a significant difference in baseline [Ca2+]i and time to maximal [Ca2+]i peak after ACh stimulation in immature and adult ASMC (100 ± 13 nM and 14.8 ± 0.8 s, respectively) (Fig. 3).


Figure 3
View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Intracellular calcium concentration ([Ca2+]i) after 2 days of incubation with or without LIF. Results are means ± SE. ASMC were incubated for 48 h in ITS medium alone (immature, n = 18; mature, n = 53) or with LIF (5 ng/ml; immature, n = 24). They were then stimulated by 10–5 M (adult) or 10–3 M (immature) ACh. A: representative [Ca2+]i response curve in immature ASMC in ITS medium (dotted line) or LIF (solid line). B: baseline (left), relative peak (middle), and time to peak (right) [Ca2+]i responses. *P < 0.001 (ANOVA).

 
After incubation with TNF-{alpha}. After 3 days of incubation with the proinflammatory cytokine TNF-{alpha}, immature cells showed a trend toward greater relative [Ca2+]i peak after stimulation by the four contractile agonists studied compared with control cells (Fig. 4). However, the enhanced response reached statistical significance after stimulation by U-46619 only (184.8 ± 66.6 vs. 66.2 ± 15.4 nM, n = 15 per agonist, P < 0.01, in stimulated and control cells, respectively). Mature cells responded significantly to ACh only (400.8 ± 122.1 vs. 64.5 ± 26.3 nM, n = 15 per agonist, P < 0.01). This response in adults was much more marked compared with controls (sixfold increase) than in immature cells (twofold increase).


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4. Relative [Ca2+]i peak after 3 days of incubation with or without TNF-{alpha}. Results are means ± SE. ASMC were incubated for 72 h in ITS medium alone (n = 15 per experimental condition) or with TNF-{alpha} (100 ng/ml). They were then stimulated by 10–5 M histamine, bradykinin, ACh, or U-46619. **P < 0.01 (Student t-test).

 
Contractility of Rat Pup Isolated Tracheae

As shown in Fig. 5, there was a 48% increase in maximal isometric force generated by 10–3 M ACh after stimulation by LIF [1,391 ± 159 mg (LIF, n = 11) vs. 941 ± 177 mg (control group, n = 10); P < 0.05].


Figure 5
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 5. Contractility of isolated rat pup tracheae to ACh. Results are means ± SE. Tracheal rings were incubated with LIF (5 ng/ml) (n = 11) or DMEM media alone (n = 10) for 24 h. Maximal relaxation was elicited by the administration of ACh (10–3 M). *P < 0.05 (Mann-Whitney U-test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have shown that TNF-{alpha}-stimulated immature ASMC massively produce LIF mRNA and protein, although this proinflammatory neurotrophic cytokine moderately increases DNA synthesis in adult ASMC only and has no relevant chemotactic effect on HMCs. The main demonstrable effect of LIF involved Ca2+ signaling and isometric airway contractility to ACh in immature, isolated rat trachea. The proinflammatory cytokine TNF-{alpha} did not significantly enhance Ca2+ signaling to ACh in immature ASMC, as opposed to mature cells. However, [Ca2+]i was greatly enhanced in immature cells after stimulation by the thromboxane analog U-466619. This work, together with our own previous work, thus suggests that immature ASMC are already in a primed state and that increased LIF secretion may be one of the triggers of this chain reaction.

The ASMC is at the center of a dynamic asthmatic response and plays an important role in the hyperresponsiveness and remodeling that occur in the asthmatic airway, by subserving inflammation-driven fibro-proliferative remodeling of the airway wall, including increased muscle mass, matrix protein deposition in the muscle layer, submucosa, and adventia (16). This results from its contractile and migratory properties, as well as its active participation in the inflammatory response via the autocrine production of cytokines and chemokines (37). The IL-6 family of cytokines, which includes IL-6 itself, LIF, IL-11, oncostatin M, cardiotropin 1, and ciliary neutrophic factor, as well as other cytokines, plays an important role in airway inflammation (11). As an example, oncostatin M causes eotaxin-1 release from ASM, in synergy with IL-4 and IL-13, by a mechanism involving STAT-3 (11). To the best of our knowledge, the secretion of LIF by human neonatal ASMCs has not been studied. In the present study, we have demonstrated that cytokine is massively released and may enhance the inflammatory response. Our investigations into the deleterious effect of exogenous LIF showed that it greatly increases airway signaling and contractility to the cholinergic agonist ACh.

Ca2+ plays a central role in the activation of many cellular processes, including the most relevant end point in ASM physiology: contraction. For this reason, the cytosolic concentration of Ca2+ is tightly controlled. Ca2+ mobilization and signaling is a postreceptor event that is closely related to the muscarinic neural system and phosphoinositide hydrolysis. It may be induced by contractile agonists, whose effect is also modulated by the ASMC inflammatory microenvironment. Indeed, when stimulated by Th2-type cytokines, airways adopt a proasthmatic phenotype of altered hyperresponsiveness (15). The autocrine-induced changes in ASM responsiveness are attributed to altered receptor-coupled transmembrane signaling in the sensitized ASM, resulting in perturbed expression and release of second-messenger molecules that regulate ASM contraction and relaxation (15). Cyclic ADP-ribose (cADPr), an endogenous activator of the ryanodine receptor channel in mammalian cells, has been shown to modulate agonist-induced Ca2+ responses in ASM cells (10, 39). In addition, cADPr-mediated Ca2+ release appears to play an important role in the "nonspecific" increased ASM responsiveness to contractile agonists in cytokine-treated cells, a characteristic finding of asthma (10, 39). Furthermore, other signaling molecules, such as Rho/Rho kinase and phosphodiesterase, also contribute to bronchial hyperresponsiveness (3). In the present study, we have demonstrated that LIF enhances baseline and relative peak [Ca2+]i response to ACh in moderately serum-deprived immature ASMC (cells that are reacquiring their contractile protein content). This response is significantly greater than what is observed in adult ASMC. The mechanisms involved in this increased [Ca2+]i response to LIF require further studies.

However, a number of studies have investigated the role of the proinflammatory cytokine TNF-{alpha} on ASMC contraction. TNF-{alpha} may act directly by altering the G-protein-mediated signal transduction (activation of pathways dependent on protein synthesis) (2, 3, 18), or the intracellular Ca2+ pumps (decreased sequestering of Ca2+ into intracellular stores by interfering with the sarco-endoplasmic reticulum Ca2+-ATPases), rather than by modifying the expression of contractile agonist receptors (2). The inflammatory process may render ASMC hyperresponsive to contractile agents, which might be, at least in part, mediated through activation of the Ras/Raf/MEK/MAPK pathway (40). In another study, it has been shown that the augmentation of ACh-induced contractile response is mediated, at least in part, by synthesis of protein, such as RhoA, through activation of p42/44, but not p38 MAPK (35). Additionally, TNF-{alpha} caused significant augmentation of CD38 expression (a cell surface protein that catalyzes the synthesis and degradation of cADPr), ADP-ribosyl cyclase activity, and Ca2+ responses to ACh, bradykinin, and thrombin (10). In the present study, TNF-{alpha} significantly increased the [Ca2+]i response to U-46619 in immature ASMC and showed a trend toward a greater response when stimulated by the other inflammatory mediators such as bradykinin. Adult ASMC also showed a marked response to ACh after being primed by TNF-{alpha}. TNF-{alpha} is an interesting candidate mediator in airway inflammatory diseases, considering that high levels of this substance are found in asthmatic airways (1). It results from infiltration of the airways by mast cells, monocytes, and epithelial cells (7). Importantly, the number of mast cells within the ASM bundles themselves in an asthmatic airway is increased (4). This would potentially result in a greater concentration of TNF-{alpha} within the vicinity of the ASM bundle. TNF-{alpha} has thus the potential to modulate the interactions between cells present in the inflamed airways of a patient with asthma and, therefore, to contribute to the regulation of airway inflammation and remodeling (7).

Similarly, hyperresponsiveness to ACh after incubation with LIF has been demonstrated in the present study in an isolated airway rat model. This is compatible with the findings regarding human ASMC Ca2+ homeostasis, although the experimental conditions are not entirely comparable (Ca2+ measurements were carried out in cells following incubation with LIF for 48 h, while contractility of tracheal rings was measured following 24-h incubation). The interaction between LIF and the cholinergic system suggests a role for LIF in the regulation of neurotransmitter and neurotransmitter receptor phenotype, particularly muscarinic receptors. LIF is an integral component of the interface between nerves and the immune system (19). Evidence is emerging that this cytokine may also play an important role in regulating the neural-immune system interaction during acute inflammatory insult and the subsequent healing and restitution process. In guinea pig airways, it amplifies the contractile response to tachykinins (20). LIF may thus act as a mediator of bidirectional cross talk between neural tissue and the immune system (21). This is of importance, since convincing experimental work in a rat model has highlighted the importance of neurogenic inflammation in acute viral bronchiolitis and its role in subsequent inflammation and reactive airways disease, which may lead to asthma (26).

There is also increasing evidence to support the hypothesis that LIF and related cytokines play both direct and indirect roles in the proliferative response of epithelial and mesenchymal cells (19). In our study, however, the effect of LIF on ASMC DNA synthesis and mast cell migration was very modest. This may be due to the fact that LIF does not induce STAT-3-dependent gene transcription (11). An indirect action via the potentiation of muscarinic M3-receptor stimulation is also a possibility. Notewothy, muscarinic stimulation enhances ASM proliferation to peptide growth factors in vitro (13). It is also likely that the deleterious effect of LIF on ASMC function may be potentiated by the combined presence of other mediators, growth factors, or cytokines (11). For example, in eosinophils, LIF does not appear to influence chemotaxis per se, although it markedly upregulates eosinophil migration in response to classical agonists such as platelet-activating factor, as well as substance P (19). Similarly, the PDGF-dependent proliferation of nontransformed human fibroblasts, vascular smooth muscle cells, and mesengial cells involves the action of IL-6 (32). Once released, LIF may exert a number of autocrine and paracrine effects.

The fact that immature ASMC exhibits enhanced responses to inflammatory mediators and cytokines, albeit in a different manner compared with adults, may have implications for the early life environment. Numerous studies have shown the potential influence of in utero cytokine levels on the risk of atopy (30, 31). It is also known that the cytokine-driven autocrine signaling mechanism in ASM may be triggered by either Fc-receptor activation in the atopic (IgE-mediated) sensitized state or by ASM exposure to specific viral respiratory pathogens, most notably including rhinovirus (14). Rhinovirus 16 elicits proasthmatic changes in ASM responsiveness that are attributed solely to binding of the virus to its host receptor (ICAM-1) on the ASM cell surface (14). The present study adds further evidence to the risk of early increased airway hyperresponsiveness and remodeling in the presence of a proinflammatory ambiance.

The main limitation of the study lies in the fact that this is not a true ontogenetic study, since only two age groups were studied: neonates and adults. This precludes the extrapolation of our results to infants and preschool children for whom we have the greatest gap in our knowledge. Moreover, the airway generations from which the smooth muscle was taken were different in the two age groups (cultured cells from tracheae in neonates vs. intrapulmonary airways in adults). This is due to the major difficulties in obtaining human lung tissue. The conclusions have thus to be interpreted with caution. Nonetheless, from the results, one can gain an insight into the properties of immature human ASMC.

In conclusion, we supply evidence suggesting that human ASMC significantly secrete LIF (immature >> adults) upon stimulation by the proinflammatory cytokine TNF-{alpha}. Immature ASMC exhibit increased [Ca2+]i responses to LIF and the thromboxane analog U-46619, as well as increased airway contractility to LIF, while Ca2+ signaling in mature ASMC was more marked in the presence of ACh. Taken together, the data from our study indicate that immature ASMC are primed to amplify inflammation in the airways.


   ACKNOWLEDGMENTS
 
The authors are grateful to Béatrice Martinez and Huguette Crevel for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Fayon, Service de Pneumologie Pédiatrique, Hôpital Pellegrin-Enfants, Place Amélie Raba Léon, 33076 Bordeaux Cedex, France (E-mail: michael.fayon{at}chu-bordeaux.fr; michael.fayon{at}wanadoo.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Amrani Y, Krymskaya V, Maki C, and Panettieri RA Jr. Mechanisms underlying TNF-{alpha} effects on agonist-mediated calcium homeostasis in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 273: L1020–L1028, 1997.[Abstract/Free Full Text]
  2. Amrani Y and Panettieri RA Jr. Cytokines induce airway smooth muscle cell hyperresponsiveness to contractile agonists. Thorax 53: 713–716, 1998.[Free Full Text]
  3. Amrani Y, Tliba O, Deshpande DA, Walseth TF, Kannan MS, and Panettieri RA Jr. Bronchial hyperresponsiveness: insights into new signaling molecules. Curr Opin Pharmacol 4: 230–234, 2004.[CrossRef][ISI][Medline]
  4. Berger P, Girodet PO, Begueret H, Ousova O, Perng DW, Marthan R, Walls AF, and Tunon de Lara JM. Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis. FASEB J 17: 2139–2141, 2003.[Abstract/Free Full Text]
  5. Berger P, Lavallee J, Rouiller R, Laurent F, Marthan R, and Tunon-de-Lara JM. Assessment of bronchial inflammation using an automated cell recognition system based on colour analysis. Eur Respir J 14: 1394–1402, 1999.[Abstract]
  6. Berkman N, Krishnan VL, Gilbey T, Newton R, O'Connor B, Barnes PJ, and Chung KF. Expression of RANTES mRNA and protein in airways of patients with mild asthma. Am J Respir Crit Care Med 154: 1804–1811, 1996.[Abstract]
  7. Burgess JK, Blake AE, Boustany S, Johnson PR, Armour CL, Black JL, Hunt NH, and Hughes JM. CD40 and OX40 ligand are increased on stimulated asthmatic airway smooth muscle. J Allergy Clin Immunol 115: 302–308, 2005.[CrossRef][ISI][Medline]
  8. Chung KF. Airway smooth muscle cells: contributing to and regulating airway mucosal inflammation? Eur Respir J 15: 961–968, 2000.[Abstract]
  9. Denis D, Fayon MJ, Berger P, Molimard M, De Lara MT, Roux E, and Marthan R. Prolonged moderate hyperoxia induces hyperresponsiveness and airway inflammation in newborn rats. Pediatr Res 50: 515–519, 2001.[ISI][Medline]
  10. Deshpande DA, Walseth TF, Panettieri RA, and Kannan MS. CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness. FASEB J 17: 452–454, 2003.[Abstract/Free Full Text]
  11. Faffe DS, Flynt L, Mellema M, Moore PE, Silverman ES, Subramaniam V, Jones MR, Mizgerd JP, Whitehead T, Imrich A, Panettieri RA Jr, and Shore SA. Oncostatin M causes eotaxin-1 release from airway smooth muscle: synergy with IL-4 and IL-13. J Allergy Clin Immunol 115: 514–520, 2005.[CrossRef][ISI][Medline]
  12. Fayon M, Ben-Jebria A, Elleau C, Carles D, Demarquez JL, Savineau JP, and Marthan R. Human airway smooth muscle responsiveness in neonatal lung specimens. Am J Physiol Lung Cell Mol Physiol 267: L180–L186, 1994.[Abstract/Free Full Text]
  13. Gosens R, Bos IS, Zaagsma J, and Meurs H. Protective effects of tiotropium bromide in the progression of airway smooth muscle remodeling. Am J Respir Crit Care Med 171: 1096–1102, 2005.[Abstract/Free Full Text]
  14. Grunstein MM, Hakonarson H, Whelan R, Yu Z, Grunstein JS, and Chuang S. Rhinovirus elicits proasthmatic changes in airway responsiveness independently of viral infection. J Allergy Clin Immunol 108: 997–1004, 2001.[Medline]
  15. Hakonarson H and Grunstein MM. Autocrine regulation of airway smooth muscle responsiveness. Respir Physiol Neurobiol 137: 263–276, 2003.[CrossRef][ISI][Medline]
  16. Halayko AJ and Amrani Y. Mechanisms of inflammation-mediated airway smooth muscle plasticity and airways remodeling in asthma. Respir Physiol Neurobiol 137: 209–222, 2003.[CrossRef][ISI][Medline]
  17. Howarth PH, Knox AJ, Amrani Y, Tliba O, Panettieri RA Jr, and Johnson M. Synthetic responses in airway smooth muscle. J Allergy Clin Immunol 114: S32–S50, 2004.[CrossRef][Medline]
  18. Huang CD, Ammit AJ, Tliba O, Kuo HP, Penn RB, Panettieri RA Jr, and Amrani Y. G-protein-coupled receptor agonists differentially regulate basal or tumor necrosis factor-alpha-stimulated activation of interleukin-6 and RANTES in human airway smooth muscle cells. J Biomed Sci 12: 763–776, 2005.[Medline]
  19. Knight D. Leukaemia inhibitory factor (LIF): a cytokine of emerging importance in chronic airway inflammation. Pulm Pharmacol Ther 14: 169–176, 2001.[CrossRef][Medline]
  20. Knight D, McKay K, Wiggs B, Schellenberg RR, and Bai T. Localization of leukaemia inhibitory factor to airway epithelium and its amplification of contractile responses to tachykinins. Br J Pharmacol 120: 883–891, 1997.[CrossRef][ISI][Medline]
  21. Knight DA, D'Aprile AC, Spalding LJ, Goldie RG, and Thompson PJ. Leukaemia inhibitory factor (LIF) upregulates excitatory non-adrenergic non-cholinergic and maintains cholinergic neural function in tracheal explants. Br J Pharmacol 130: 975–979, 2000.[Medline]
  22. Marthan R, Hyvelin JM, Roux E, and Savineau JP. [Electrophysiology and calcium signalling in human bronchial smooth muscle]. Therapie 54: 79–83, 1999.[ISI][Medline]
  23. Martinez FD, Wright AL, Taussig LM, Holberg CJ, Halonen M, and Morgan WJ. Asthma and wheezing in the first six years of life. The Group Health Medical Associates. N Engl J Med 332: 133–138, 1995.[Abstract/Free Full Text]
  24. Morgan WJ, Stern DA, Sherrill DL, Guerra S, Holberg CJ, Guilbert TW, Taussig LM, Wright AL, and Martinez FD. Outcome of asthma and wheezing in the first six years of life: follow-up through adolescence. Am J Respir Crit Care Med 172: 1253–1258, 2005.[Abstract/Free Full Text]
  25. Panettieri RA Jr. Cellular and molecular mechanisms regulating airway smooth muscle proliferation and cell adhesion molecule expression. Am J Respir Crit Care Med 158: S133–S140, 1998.[ISI][Medline]
  26. Piedimonte G. Contribution of neuroimmune mechanisms to airway inflammation and remodeling during and after respiratory syncytial virus infection. Pediatr Infect Dis J 22: S66–S74, 2003.[CrossRef][ISI][Medline]
  27. Piedimonte G, Hegele RG, and Auais A. Persistent airway inflammation after resolution of respiratory syncytial virus infection in rats. Pediatr Res 55: 657–665, 2004.[CrossRef][Medline]
  28. Pitard V, Lorgeot V, Taupin JL, Aubard Y, Praloran V, and Moreau JF. The presence in human serum of a circulating soluble leukemia inhibitory factor receptor (sgp190) and its evolution during pregnancy. Eur Cytokine Netw 9: 599–605, 1998.[Medline]
  29. Pitard V, Taupin JL, Miossec V, Blanchard F, Cransac M, Jollet I, Vernallis A, Hudson K, Godard A, Jacques Y, and Moreau JF. Production and characterization of monoclonal antibodies against the leukemia inhibitory factor low affinity receptor, gp190. J Immunol Methods 205: 177–190, 1997.[CrossRef][Medline]
  30. Prescott SL, King B, Strong TL, and Holt PG. The value of perinatal immune responses in predicting allergic disease at 6 years of age. Allergy 58: 1187–1194, 2003.[CrossRef][ISI][Medline]
  31. Prescott SL, Taylor A, King B, Dunstan J, Upham JW, Thornton CA, and Holt PG. Neonatal interleukin-12 capacity is associated with variations in allergen-specific immune responses in the neonatal and postnatal periods. Clin Exp Allergy 33: 566–572, 2003.[CrossRef][ISI][Medline]
  32. Roth M, Nauck M, Tamm M, Perruchoud AP, Ziesche R, and Block LH. Intracellular interleukin 6 mediates platelet-derived growth factor-induced proliferation of nontransformed cells. Proc Natl Acad Sci USA 92: 1312–1316, 1995.[Abstract/Free Full Text]
  33. Roux E, Guibert C, Savineau JP, and Marthan R. [Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity. Br J Pharmacol 120: 1294–1301, 1997.[CrossRef][ISI][Medline]
  34. Roux E, Molimard M, Savineau JP, and Marthan R. Muscarinic stimulation of airway smooth muscle cells. Gen Pharmacol 31: 349–356, 1998.[CrossRef][ISI][Medline]
  35. Sakai H, Otogoto S, Chiba Y, Abe K, and Misawa M. Involvement of p42/44 MAPK and RhoA protein in augmentation of ACh-induced bronchial smooth muscle contraction by TNF-{alpha} in rats. J Appl Physiol 97: 2154–2159, 2004.[Abstract/Free Full Text]
  36. Sears MR, Greene JM, Willan AR, Wiecek EM, Taylor DR, Flannery EM, Cowan JO, Herbison GP, Silva PA, and Poulton R. A longitudinal, population-based, cohort study of childhood asthma followed to adulthood. N Engl J Med 349: 1414–1422, 2003.[Abstract/Free Full Text]
  37. Shore SA. Direct effects of Th2 cytokines on airway smooth muscle. Curr Opin Pharmacol 4: 235–240, 2004.[CrossRef][Medline]
  38. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, and Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: 34.1–34.11, 2002.
  39. White TA, Kannan MS, and Walseth TF. Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle. FASEB J 17: 482–484, 2003.[Abstract/Free Full Text]
  40. Yang CM, Chien CS, Wang CC, Hsu YM, Chiu CT, Lin CC, Luo SF, and Hsiao LD. Interleukin-1beta enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth-muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway. Biochem J 354: 439–446, 2001.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/L244    most recent
00474.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fayon, M.
Right arrow Articles by Marthan, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fayon, M.
Right arrow Articles by Marthan, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.