Mechanisms of serum potentiation of GM-CSF production by human airway smooth muscle cells

D. J. Lalor ,1,* B. Truong,1,* S. Henness,1 A. E. Blake,1 Q. Ge,2 A. J. Ammit,1 C. L. Armour,1 and J. M. Hughes1

1Respiratory Research Group, Faculty of Pharmacy and 2Department of Pharmacology, University of Sydney, New South Wales 2006, Australia

Submitted 5 April 2004 ; accepted in final form 25 June 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Inflammation and vascular leakage are prevalent in asthma. This study aimed to elucidate the mechanisms involved in serum potentiation of cytokine-induced granulocyte macrophage colony stimulating factor (GM-CSF) production by human airway smooth muscle cells and to identify possible factors responsible. Serum-deprived cells at low density were stimulated with TNF-{alpha} and IL-1{beta} for 24 h. Human AB serum (10%), inhibitors of RNA and protein synthesis or specific signaling molecules, or known smooth muscle mitogens were then added for 24 h. Culture supernatants were analyzed for GM-CSF levels, and cells were harvested to assess viability, cell cycle progression, GM-CSF-specific mRNA content, and p38 phosphorylation. Serum potentiated GM-CSF release when added before, together with (maximal), or after the cytokines. The potentiation involved both new GM-CSF-specific mRNA production and protein synthesis. The mitogens IGF, PDGF, and thrombin all potentiated GM-CSF release, and neutralizing antibodies for EGF, IGF, and PDGF reduced the serum potentiation. Inhibitor studies ruled as unlikely the involvement of p70S6kinase and the MAPK p42/p44, two signaling pathways implicated in proliferation, and the involvement of the MAPK JNK, while establishing roles for p38 MAPK and NF-{kappa}B in the potentiation of GM-CSF release. Detection of significant p38 phosphorylation in response to serum stimulation, through Western blotting, further demonstrated the involvement of p38. These studies have provided evidence to support p38 being targeted to interrupt the cycle of inflammation, vascular leakage and cytokine production in asthma.

granulocyte macrophage colony stimulating factor; serum; p38 mitogen-activated protein kinase; NF-{kappa}B


ASTHMA IS AN INFLAMMATORY disease of the airways. The chronic inflammation in asthmatic airways has been long known to cause changes in the airways. These changes have been shown to be both physical changes (11) and changes in the types of inflammatory cells and agents present in the airways (7).

Airway smooth muscle plays an important role in these changes. Increased airway smooth muscle bulk, due to both increased size and proliferation of the smooth muscle cells, is a major component of the documented airway remodeling (11). Moreover, airway smooth muscle can influence the local inflammatory environment by synthesis of chemokines and cytokines that modulate subsequent inflammatory cell recruitment and activation.

Vascular leakage also occurs in the airways of asthmatics (19). Agents found within human serum/plasma such as insulin-like growth factor (IGF) (14), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and thrombin have been shown to cause the proliferation of airway smooth muscle cells (ASMC) (17). Their effects on other airway smooth muscle functions, such as chemokine/cytokine production, are largely undetermined.

We have previously reported that exposure of human ASMC to human serum potentiates the amount of granulocyte macrophage colony stimulating factor (GM-CSF) released by these cells in response to stimulation with tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} in vitro (20). This potentiation of GM-CSF may be important in acute episodes of asthma as GM-CSF enhances the survival and activation of a number of key inflammatory cells, including eosinophils (21), stimulates the release of other cytokines, and changes the contraction profiles of airway smooth muscle (8). Recently, a potential role for GM-CSF in airway wall remodeling has also emerged. In airway smooth muscle it has been shown to induce synthesis of collagen and fibronectin and, while not affecting the amount of transforming growth factor (TGF)-{beta} secreted by the smooth muscle, to elevate the expression of TGF-{beta} receptors, thereby making the muscle more sensitive to the fibrogenic effects of TGF-{beta} (5). Elucidation of the mechanisms by which serum potentiates GM-CSF release may lead to the development of a novel treatment for acute asthmatic episodes and identification of the agent(s) within serum exerting this effect may also provide a new target for asthma treatment.

TNF-{alpha}- and IL-1{beta}-stimulated GM-CSF release comes as a result of activation of the transcription factor nuclear factor (NF)-{kappa}B (21). NF-{kappa}B can be activated by the mitogen-activated protein kinases (MAPKs) p38 and p42/p44 (2), which also play a role in the release of cytokines (13). These MAPKs, particularly p42/p44, are also involved in the transduction of signals from mitogens found in serum/plasma resulting in cell proliferation (17). Recently, it has been demonstrated that the cytokine-induced release of GM-CSF by human ASMC can be reduced by inhibition of the MAPK JNK, clearly indicating a role for JNK in the regulation of cytokine-induced GM-CSF release (16). To date, p70S6kinase in airway smooth muscle has been found to play a role in proliferation (reviewed in Ref. 1) but not synthetic activity of the cells.

The aims of the current study were to elucidate some of the mechanisms by which serum potentiates human ASMC release of GM-CSF in response to stimulation with TNF-{alpha} and IL-1{beta} and to identify possible factors responsible. To do this, we examined the effect of serum/plasma-derived airway smooth muscle mitogens on cytokine-primed airway smooth muscle GM-CSF release and cell cycle progression. We also treated serum with neutralizing antibodies for EGF, IGF, and PDGF and explored their effects on GM-CSF release. Furthermore, we investigated whether new gene transcription as well as new protein synthesis are involved in the serum potentiation. Finally, we determined whether the new protein synthesis involved comes as a result of stimulation of synthetic or proliferative pathways by exploring the role of NF-{kappa}B, p38, p42/p44, JNK, and p70S6kinase in the cell signaling leading to the observed serum potentiation of GM-CSF release.


    MATERIALS
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 ABSTRACT
 MATERIALS
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Recombinant human TNF-{alpha}, IL-1{beta}, neutralizing antibodies to EGF (AF236), IGF-1 (AF-291-NA), and PDGF (AB-23-NA), and an isotype control (AB-108-C) were all obtained from R&D Systems (Minneapolis, MN). Cycloheximide, rapamycin, and PDGF were purchased from Sapphire Bioscience (Sydney, Australia). Actinomycin D and dimethyl fumarate (DMF) were purchased from Sigma Australia. MAPK inhibitors SB-203580 and PD-98059 and the negative congener SB-202474 were provided by Calbiochem (San Diego, CA), SP-600125 was purchased from A. G. Scientific (San Diego, CA). Monoclonal antibodies used for the Western blotting of p38 and phospho-p38 (Thr180/Tyr182) were supplied by Cell Signaling Technology (Beverly, MA). EGF and IGF were obtained from Invitrogen Life Technologies. Thrombin was obtained from Pfizer (Sydney, Australia). All reagents were reconstituted and stored according to the suppliers' instructions.

The Access RT-PCR kit, Random-Primed DNA labeling kit, and RNA Gel Extraction kit were obtained from Promega, Roche Diagnostics, and QIAGEN Australia, respectively. TRIzol was purchased from Invitrogen, Zetaprobe GT membrane for Northern blotting was obtained from Bio-Rad, and [32P]dCTP was purchased from Perkin-Elmer Australia. Capture (clone: BVD2-23B6) and detection (clone: BVD2-21C11) antibodies for GM-CSF were purchased from PharMingen. Streptavidin-horseradish peroxidase was purchased from Amersham Life Science (Buckingham, UK), and the TMB Microwell peroxidase substrate system from KPL (Gaithersburg, MD). DMEM, fetal bovine serum (FBS), L-glutamine, and trypsin-EDTA were all supplied by Thermo Trace (Melbourne, Australia). Penicillin, streptomycin, and amphotericin B were obtained from GIBCO. Human AB serum, insulin-transferrin-sodium selenite media supplement (ITS), nonessential amino acids (NEAA), and all other materials were purchased from Sigma Australia.


    METHODS
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 ABSTRACT
 MATERIALS
 METHODS
 RESULTS
 DISCUSSION
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Human ASMC

Human lung was obtained from patients undergoing either lung transplant or resection. Ethics approval for the use of human lung tissue was supplied by the Central Area Health Service and for this study by the Human Ethics Committee of the University of Sydney. Airway smooth muscle bundles were dissected free from surrounding tissues, and the cells were grown in culture at 37°C in a humidified 5% CO2 in air atmosphere, as previously described (10). Cells established in culture from each lung donor were checked for the presence of the airway smooth muscle contractile proteins {alpha}-smooth muscle actin and h-calponin by immunohistochemistry (12). At the time of plating, the average viability of each cell line was 96.2 ± 1.1% (average ± SE, n = 11) as tested by trypan blue dye exclusion testing.

Potentiation of Airway Smooth Muscle GM-CSF Release by Human Serum

The experimental protocol closely followed that used by Sukkar et al. (20). Briefly, ASMC from three to seven lung donors were plated down at a density of 5 x 104 cells/well in six-well plates (5.2 x 103 cells/cm2) in the presence of 2 ml of DMEM supplemented with 10% vol/vol heat-inactivated FBS, 2 mM glutamine, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B. To study the effect of serum later in the protocol, after 24 h the medium was removed and replaced with serum-free medium (phenol red-free DMEM supplemented with 2 mM glutamine, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, 0.25 µg/ml amphotericin B, 1% vol/vol NEAA, and ITS solution with a final concentration of 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium) for a further 24 h. At this point, the cells were stimulated with TNF-{alpha} and IL-1{beta} (both at 10 ng/ml) in fresh serum-free medium. After a further 24 h, the cytokine-containing culture medium was removed, and the cells were stimulated with 10% vol/vol human AB serum (in serum-free medium). After a further 24 h, culture supernatants were harvested and stored at –20°C for GM-CSF quantification, and cells were harvested with trypsin-EDTA for estimation of total cell number using Kimura Light stain and for cell viability using trypan blue dye exclusion.

In an additional series of experiments the above protocol was extended to include extra treatments where human serum (10% vol/vol in serum-free medium) was added first for 24 h, followed by the cytokines in serum-free medium for the final 24 h, or together with the cytokines for 24 h followed by serum-free medium for the final 24 h. In these experiments, supernatants were collected and stored after each 24-h treatment period for GM-CSF quantification.

Inhibition of New RNA and Protein Synthesis

To determine whether new RNA synthesis and new protein synthesis were involved in serum potentiation of GM-CSF release, specific inhibitors were added concurrently with human serum to ASMC from three to seven lung donors in the protocol described above. The inhibitor treatments included actinomycin D (0.01, 0.1, 0.5, 1.0, and 5.0 µg/ml), the highest concentration of its vehicle (DMSO 0.05% vol/vol), or cycloheximide (0.1, 0.5, and 1.0 µg/ml). The inhibitors were added to duplicate wells, and 24 h later, the culture medium was harvested and stored at –20°C for quantification of GM-CSF. Cells were again counted for total number and viability.

GM-CSF Gene Transcription

The role of new GM-CSF gene transcription in serum potentiation of GM-CSF release was examined in ASMC from three lung donors. The cells were seeded into 75-cm2 flasks at the same density of 5.2 x 103 cells/cm2 as used above. The protocol used to examine serum potentiation of ASMC GM-CSF release was followed. However, in this case, the cells were harvested at 4 h postexposure to serum using TRIzol at 0.1 ml/cm2, and total RNA was extracted according to the manufacturer's instructions. The RNA was then run on a 1% agarose formaldehyde gel, and the GM-CSF mRNA was quantified by Northern blot using GAPDH as a housekeeping gene (as described in Ref. 6). Northern blotting was performed using 5 µg of RNA. A probe for GM-CSF was prepared from human ASMC maximally stimulated with cytokines with the Access RT-PCR kit. The probe used was a region of the human GM-CSF gene (sequence accession number EO2287) spanning 195 base pairs from 348 to 542. It was constructed using the forward primer 5'-CTTCCTGTGCAACCCAGATT-3' and the reverse primer 5'-CTTGGTCCCTCCAAGATGAC-3'. The probe was then used in conjunction with a Random Primed DNA labeling kit and [32P]dCTP to quantify the amount of GM-CSF-specific mRNA present. All kits were used according to manufacturers' instructions.

Inhibition of Signaling Pathways

Experiments to explore the signaling pathways involved were conducted in ASMC from three to five lung donors. ASMC were plated into 24-well plates but at the same cell density (1 x 104 cells in 400 µl per well) as previously. Inhibitors were used on triplicate wells at: 30 nM rapamycin (an inhibitor of p70S6kinase) (18), 10 and 100 µM DMF (an inhibitor of NF-{kappa}B translocation) (23), 30 µM PD-98059 (an inhibitor of p42/p44 MAPK) (2), 10 µM SB-203580 (an inhibitor of p38 MAPK) (2), and 10 µM SP-600125 (a JNK inhibitor) (16) as previously reported. The negative control for MAPK inhibition studies, SB-202474, was also used at 10 µM (2). The inhibitors or their vehicle (0.1% DMSO) was added to the cells 30 or 60 min (DMF only) before the serum. For the studies using DMF, cell counts by trypan blue dye exclusion were conducted to assess cell viability.

Phosphorylation of p38 MAPK

Experiments were conducted as for the inhibitor studies with the following exceptions: cells were grown in 10-cm petri dishes. The studies with SB-203580 were conducted in duplicate and following 30-min incubation with SB-203580 or serum-free medium, cells were stimulated with 10% human serum for 15 min. Cells were lysed and analyzed by Western blotting using specific monoclonal antibodies against p38 and phospho-p38 (Thr180/Tyr182) performed as previously described (17).

Potentiation of GM-CSF Release by ASMC Mitogens

To establish the effect of ASMC mitogens on GM-CSF production a protocol mirroring that used to potentiate GM-CSF release by human serum was used, except that in place of adding serum, we added the mitogens PDGF, EGF, IGF, and thrombin (or their vehicles where appropriate) to ASMC from five to nine lung donors. The mitogen stocks were freshly diluted in serum free medium and added to wells of untreated or cytokine-primed ASMC at concentrations that had previously been shown to induce human ASMC proliferation: 40 ng/ml PDGF (10), 100 ng/ml EGF (17), 100 ng/ml IGF (14), and 0.1, 1.0, and 10 U/ml thrombin (22). A vehicle control of 1.0 µM acetic acid in serum-free medium was included for PDGF-AB and EGF. Culture supernatants were collected 24 h later for GM-CSF quantification, and the cells were harvested with trypsin-EDTA for cell cycle analysis.

Cell Cycle Analysis

To examine the cell cycle progression of mitogen-treated ASMC, the protocol previously published by Johnson et al. (12) was followed. Briefly, harvested ASMC were permeabilized and stained using a solution of 0.5% wt/vol saponin and 0.1% wt/vol bovine serum albumin in PBS containing 50 µg/ml propidium iodide and 50 µg/ml ribonuclease A. A FACSCalibur Sort (Becton Dickinson, Sydney, Australia) in conjunction with Cell Quest software (Becton Dickinson) was used to acquire the data from the stained cells. The resulting DNA profiles were analyzed using FL2 peak area, FL2 peak width, and Modfit software (Verity Software House, Topsham, ME) to determine the percentage of cells in each phase of the cell cycle.

Studies with Neutralizing Antibodies to Growth Factors

To determine the contribution of EGF, IGF, and PDGF to the serum potentiation of GM-CSF release from cytokine-primed cells, we used neutralizing antibodies to the growth factors. The protocol was as for the initial experiments examining the potentiating effects of serum except that the medium containing 10% human antibody serum was preincubated with a neutralizing antibody to EGF (2.5, 5, and 10 µg/ml), IGF (1.25, 2.5, 5, and 10 µg/ml), or PDGF (at 5, 10, and 20 µg/ml) for 15–30 min at 37°C and then added to triplicate well cultures of ASMC from three to five lung donors. AB serum treated with an irrelevant antibody of the same isotype was used as a control. The cells were left to incubate for 24 h, and then the supernatants were collected for GM-CSF quantification.

GM-CSF Quantification

The GM-CSF concentration of the culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Capture and detection antibodies for GM-CSF were used at a concentration of 1 and 0.25 µg/ml, respectively. ELISA was conducted according to the protocol supplied with these antibodies. The limit of detection for the ELISA studies was 15.6 pg/ml.

Data Analysis

Data were analyzed using the StatView statistical package. Analysis of variance was used together with Fisher's paired least significant difference to compare each treatment on the outcome measures (GM-CSF release, cell number, cell viability, mRNA/GAPDH, p38 phosphorylation, and %S/G2+M). A value of P < 0.05 was considered significant. Potentiation of GM-CSF secretion by serum or the growth factors was defined as a significant increase in GM-CSF secretion by cytokine-primed ASMC over their baseline secretion of GM-CSF in the same period.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Potentiation of GM-CSF Release by Human Serum

In this study we have extended our previously reported findings that human serum potentiated GM-CSF release by cytokine-primed human ASMC when added after the cytokine treatment period (20). Here we demonstrate that a 24-h treatment with serum potentiated total GM-CSF release over a 48-h period, irrespective of whether the serum was added the day before, together with, or the day after the ASMC were primed with the cytokines TNF-{alpha} and IL-1{beta} for 24 h. Changes in GM-CSF release in response to the different serum treatments are summarized in Fig. 1. The GM-CSF release induced by the cytokines alone was 750.4 ± 231.2 pg/ml (mean ± SE) over the 48-h period. Serum caused the greatest potentiation of this release if it was added together with the cytokines (ninefold) and increases of 3- and 5.4-fold when added before and after the cytokine treatment period, respectively (Fig. 1).



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Fig. 1. Treatment with serum caused a significant increase in the amount of granulocyte macrophage colony stimulating factor (GM-CSF) released over a 48-h period by cytokine-treated human airway smooth muscle cells (ASMC). After a 24-h plating down period, human ASMC were serum-starved for 24 h before being treated for 24 h with the cytokines TNF-{alpha} (10 ng/ml) and IL-1{beta} (10 ng/ml) (Cyt) and/or 10% vol/vol human AB serum (Ser) or serum-free medium (–) on treatment days 1 and 2 as shown. Values are expressed as means ± SE, n = 3–7; Cyt+Ser, added at the same time; *significant difference from cytokine-treated cells, P < 0.02.

 
Inhibition of RNA and Protein Synthesis

Inhibition of new RNA synthesis. Actinomycin D added over a range of concentrations (0.5, 1.0, and 5.0 µg/ml) to cytokine-primed cells to inhibit RNA synthesis significantly reduced, in a concentration-related manner, the amount of GM-CSF produced over the following 24-h serum treatment period compared with the positive control. These actinomycin D treatments inhibited the amount of GM-CSF produced by 60.00 ± 11.44, 78.00 ± 5.57, and 81.93 ± 4.17%, respectively (n = 4, P < 0.001; Fig. 2A). At two lower concentrations, 0.01 and 0.1 µg/ml, actinomycin D reduced the expression of GM-CSF by 20.09 ± 6.03 and 35.38 ± 12.41%, neither of which was significant compared with the positive control (n = 3, P = 0.316 and P = 0.090). The vehicle for actinomycin D, DMSO, at a concentration of 0.05% (the highest concentration at which it was present), reduced the amount of GM-CSF produced by the ASMC by 30.27 ± 7.65% (n = 4, P = 0.001). The effect of the actinomycin D, at the two highest concentrations, was significantly greater (>50%) than that of the vehicle (P < 0.001). Addition of actinomycin D did not have a significant effect on total cell number or cell viability (data not shown).



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Fig. 2. Effect of inhibition of RNA synthesis by actinomycin D (A) and protein synthesis by cycloheximide (B) on the serum potentiation of GM-CSF release by cytokine-pretreated human ASMC. Values are means ± SE, n = 4. *P < 0.0031 vs. positive control (10% human AB serum), {dagger}P < 0.0001 vs. vehicle control (0.05% vol/vol DMSO).

 
Inhibition of new protein synthesis. After treatment of the cytokine-primed cells with cycloheximide at each of three concentrations (0.1, 0.5, and 1.0 µg/ml) to inhibit protein synthesis, the amount of GM-CSF released by the ASMC in response to serum treatment for 24 h was significantly decreased (P < 0.0001, n = 4). As shown in Fig. 2B, the decrease in GM-CSF was concentration related, with the three concentrations of cycloheximide reducing GM-CSF release by 51.08 ± 9.52, 82.05 ± 2.26, and 88.34 ± 1.75%, respectively. Addition of cycloheximide did not have a significant effect on total cell number or cell viability (data not shown).

GM-CSF Gene Transcription

Studies conducted into the amount of GM-CSF-specific mRNA transcribed by the ASMC (Fig. 3, A and B) showed that, at 4 h, the cytokine-primed, serum-stimulated cells contained 2.10 times the quantity of mRNA specific for GM-CSF compared with the cytokine-stimulated cells (n = 3, P < 0.0001). This was 18.37 times the amount in unstimulated cells (n = 3, P < 0.0001).



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Fig. 3. Comparison of the amount of GM-CSF-specific mRNA and protein produced after a 4-h serum treatment of cytokine pretreated ASMC from 3 lung donors. A: representative Northern blot of GM-CSF-specific mRNA and mRNA for the housekeeping gene GAPDH to control for initial gel loading. B: quantification of GM-CSF-specific mRNA synthesis in response to the treatments. C: GM-CSF protein release in response to the treatments. Unstim, untreated ASMC; *P < 0.0005 (n = 3) compared with cells treated with cytokine only.

 
From the same experiments, supernatants were harvested from the cells and analyzed for GM-CSF content. These studies showed that at 4 h poststimulation with serum there was 2.07 times more GM-CSF present in the supernatant of cells that had been secondarily stimulated with serum when compared with cytokine-primed cells (n = 3, P = 0.0004) and 8.54 times more than the unstimulated cells (n = 3, P < 0.0001; Fig. 3C).

Inhibition of Signaling Pathways

Addition of 30 nM rapamycin, an inhibitor of p70S6kinase, had no significant effect on the amount of GM-CSF produced by cytokine-primed cells treated with serum for 24 h when compared with the positive control (P = 0.258, n = 3; see Fig. 4A).



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Fig. 4. Effect of inhibition of the p70S6kinase by rapamycin (A), p42/p44 MAPK by PD-98059 and JNK MAPK by SP-600125 (B), the p38 MAPK by SB-203580 (C), and the transcription factor NF-{kappa}B by dimethyl fumarate (DMF, D) on the serum potentiation of GM-CSF release over 24 h by cytokine-pretreated ASMC. Values are means ± SE, n = 4. *P < 0.01 vs. positive control (10% human AB serum), {dagger}P < 0.05 vs. vehicle control (0.05% vol/vol DMSO), {ddagger}P < 0.01 vs. negative control (SB-202474).

 
Similarly, 10 µM of SP-600125, an inhibitor of the MAPK JNK caused no significant decrease in GM-CSF produced by cytokine-primed cells treated with serum for 24 h when compared with the positive control (P = 0.887, n = 3; see Fig. 4B).

Inhibition of the p42/p44 MAPK by PD-98059 at 30 µM resulted in a significant decrease of 32.36 ± 11.99% in the amount of GM-CSF produced by cytokine-primed cells treated with serum for 24 h when compared with the positive control (P = 0.0092, n = 3; Fig. 4B). However, a similar decrease (36.22 ± 4.50%; P = 0.0042, n = 3) in GM-CSF was achieved by treating the cells with 0.1% vol/vol DMSO, the vehicle in which PD-98059 was dissolved (Fig. 4B). This was not different from the reduction caused by the PD-98059 (P = 0.737, n = 3).

In contrast to these observations, addition of the p38 MAPK inhibitor SB-203580 to the cell cultures caused a 62.08 ± 4.42% decrease in the amount of GM-CSF released in response to stimulation with cytokines followed by 10% human serum (P < 0.0001, n = 3). In addition, SB-203580 inhibition of GM-CSF production was greater than that of its vehicle, 0.1% vol/vol DMSO, (P = 0.033, n = 3) and its negative congener, SB-202474 (P = 0.0098, n = 3). These results are summarized in Fig. 4C.

The inhibitor of NF-{kappa}B translocation, DMF, at a concentration of 10 µM with a preincubation of 60 min, did not induce a significant reduction in the amount of GM-CSF released by cytokine-primed cells treated with serum for 24 h when compared with 0.05% vol/vol DMSO, the vehicle control. However, at a concentration of 100 µM, DMF reduced the amount of GM-CSF released by 94.53 ± 2.42% (Fig. 4D). This result was significant (P < 0.0001, n = 4) compared with both the positive control and its vehicle. In fact, 100 µM DMF reduced the level of GM-CSF released from serum-stimulated cytokine-primed cells to that released by cytokine-primed cells in the absence of serum (5.47 ± 2.42 vs. 10.22 ± 2.52% positive control; P = 0.6736, n = 4). DMF treatment at this concentration did not have a significant effect on the total number or viability of the ASMC (data not shown).

Phosphorylation of p38

Cytokine-primed cells stimulated with serum showed a distinct phosphorylation of the p38 MAPK (Fig. 5A). Compared with this effect, unstimulated cells (36.63 ± 6.40% of serum-induced phosphorylation), cytokine-primed cells (49.91 ± 4.81% serum-induced phosphorylation), or cells stimulated with serum in the presence of SB-203580 (70.47 ± 5.26% serum-stimulated phosphorylation) showed a significantly lower level of p38 phosphorylation (P < 0.0001, P < 0.0001, and P = 0.0003, n = 3 respectively; Fig. 5B).



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Fig. 5. Effect of serum stimulation on the phosphorylation of p38 MAPK in cytokine-primed cells from 3 lung donors. A: representative Western blot of phospho-p38 and p38. B: comparison of the degree of phosphorylation of p38 in response to the treatments. Values are means ± SE, n = 3. *P < 0.0003 vs. serum-stimulated cells.

 
Potentiation of GM-CSF Release by ASMC Mitogens

Levels of GM-CSF released from ASMC treated with growth factors alone for 24 h under the experimental conditions described above were <4% of the positive control (cytokine-primed cells treated with 10% human serum) and not significantly different from those detected in the unstimulated cells (2.6 ± 1.0% positive control). All growth factors (EGF, IGF, PDGF, and thrombin) caused increases in GM-CSF release by ASMC that had been pretreated with cytokines (Fig. 6, A and B). GM-CSF release from cytokine-primed cells in the presence of these growth factors was 60–80% of that with 10% human serum (positive control). IGF (P = 0.0133, n = 5) significantly increased GM-CSF release by 2.8-fold compared with the cytokine control (Fig. 6A). Although EGF caused a significant increase in release compared with the cytokine control (P = 0.0043, n = 5), it was not significantly different (P = 0.079, n = 5) from release in the presence of its vehicle (1.0 µM acetic acid). However, PDGF did significantly increase (P = 0.0133, n = 5) GM-CSF release twofold over the vehicle control (1.0 µM acetic acid, Fig. 6A). Thrombin at 0.1, 1.0, and 10 units/ml also caused significant (P < 0.05, n = 5–9) increases in GM-CSF release from cytokine-treated cells. The levels released in the presence of thrombin at 10 units/ml were 80% of those in the presence of human serum (Fig. 6B).



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Fig. 6. Effects of a 24-h treatment with the mitogens insulin-like growth factor (IGF), epidermal growth factor (EGF), platelet-derived growth factor-AB (PDGF) (A), or thrombin (B) on GM-CSF release from ASMC pretreated with the cytokines TNF-{alpha} (10 ng/ml) and IL-1{beta} (10 ng/ml). Values are means ± SE, n = 5. serum, 10% human AB serum; *P < 0.02 vs. 0 (cytokine pretreatment only); {dagger}P < 0.02 vs. the vehicle control for EGF and PDGF (1.0 µM acetic acid).

 
Effects of Serum and Growth Factors on Cell Cycle Progression

Treatment of cytokine-primed ASMC with 10% human serum for 24 h induced a twofold increase in the number of ASMC in the S/G2+M phases of the cell cycle (Table 1). The growth factors had no significant effect on unstimulated or cytokine-stimulated ASM cell cycle progression over the 24-h period. However, although the growth factors did not significantly alter the DNA profiles of the ASMC, a trend was evident in cytokine-pretreated cells that received PDGF or thrombin, with increases of 6.4 and 6.3%, respectively, in cells progressing into S/G2+M, compared with cytokine-primed cells receiving no growth factors (Table 1).


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Table 1. Effect of a 24-h exposure to mitogens or human serum on cell cycle progression of unstimulated and cytokine-primed ASMC

 
Effect of Neutralizing Antibodies for Growth Factors on Serum Potentiation

To assess the role of the individual growth factors in the human serum potentiation of ASMC GM-CSF release, medium containing 10% human serum was treated with a range of concentrations of neutralizing antibodies for EGF, IGF, or PDGF before its addition to the ASMC. Interestingly, all three neutralizing antibodies significantly reduced human serum potentiation of GM-CSF release (Fig. 7), whereas an irrelevant antibody of the same isotype had no significant effect over the same concentration ranges (Fig. 7A). The EGF neutralizing antibody caused significant concentration-related reductions in the potentiation, with GM-CSF release being only 36.6 ± 2.5% of the serum control (P < 0.0001, n = 4) in the presence of the highest concentration (10 µg/ml) of antibody used (Fig. 7B). The IGF neutralizing antibody caused a significant but similar reduction to ~43% of control at concentrations of 5 µg/ml or higher (P < 0.001, n = 4; Fig. 7C). The PDGF neutralizing antibody also caused significant concentration-related reductions in the serum potentiation, reducing GM-CSF release to 46.8 ± 5.63% of control at 20 µg/ml (P < 0.0001, n = 5; Fig. 7D).



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Fig. 7. Serum potentiation of GM-CSF production by cytokine pretreated ASMC is not affected by an irrelevant antibody with the same isotype (n = 3–8, A) but is reduced by neutralizing antibodies for EGF (*P < 0.02 vs. the positive control of 10% human AB serum, n = 3–4; B), IGF (*P < 0.001 vs. positive control, n = 3–5; C), and PDGF (*P < 0.001 vs. positive control, n = 5; D). Values are means ± SE.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study we have extended our previous findings (20) and demonstrated for the first time some of the mechanisms by which serum potentiates cytokine-induced GM-CSF release from human ASMC. We have shown that both new protein production and new gene transcription are involved. Further findings specifically demonstrate that the increase in GM-CSF secretion due to serum stimulation is regulated at the transcriptional level. Interestingly, we have provided evidence that signaling via the MAPK p42/p44 or p70S6kinase molecules, important in ASMC proliferation (reviewed in Ref. 1), or via the MAPK JNK, which has a role in cytokine induced GM-CSF release (16), is not implicated, but that the MAPK p38 and the transcription factor NF-{kappa}B are involved. Furthermore, we have also demonstrated, for the first time, that the mitogens IGF, PDGF, and thrombin have a significant potentiating effect on IL-1{beta}- and TNF-{alpha}-induced GM-CSF release from human ASMC before having a significant effect on cell cycle progression. Finally, we have shown that the growth factors EGF, IGF, and PDGF were significant contributors to the potentiating effect of serum.

Inflammation and vascular leakage are both important processes in acute asthmatic attacks (19). Previous research conducted by our group showed that serum potentiates the release of the cytokine GM-CSF by human ASMC in response to TNF-{alpha} and IL-1{beta} (20). This was a finding confirmed and extended by the present study. It may be hypothesized that this serum potentiation is responsible for some of the inflammation seen in vivo during an acute attack of asthma. The determination of the mechanism through which serum is acting and the particular components of serum exerting this effect may provide a potential treatment target in an acute exacerbation of asthma.

Under our experimental conditions, a 24-h serum treatment caused both increased synthesis of GM-CSF and the initiation of proliferation by the ASMC. The latter was demonstrated by an increase in the number of cells moving out of the resting or G0/G1 phase of the cell cycle into the S/G2+M phases, although a significant increase in cell number was not yet observable. Exploring the effects of known proliferative agents contained within serum on GM-CSF production was therefore interesting. This was especially so in light of a recent report showing that, with a much longer exposure time, some mitogens have the ability to both induce GM-CSF secretion by and cause cell cycle progression of ASMC (3).

The effects of a number of mitogens, EGF, IGF, PDGF, and thrombin, on ASMC function were investigated in this study. IGF, PDGF, and thrombin were shown to increase the amount of GM-CSF produced by cytokine-primed ASMC, whereas EGF, PDGF, and IGF were shown to contribute to the potentiating action of serum. The mitogens alone induced little release of GM-CSF over a 24-h period. Additionally, the mitogens failed to cause any significant progression through the cell cycle in that period under the same conditions. This demonstrates a separation of the proliferative and synthetic functions of ASMC and suggests that the activity of an ASMC may depend on the environmental conditions that the cell is exposed to. That is, the same stimulus, for example the mitogens used in these experiments, may have different effects on ASMC depending on the preexisting condition of the cells as well as the period of time for which these conditions prevail.

Rapamycin has previously been shown to inhibit growth factor-induced p70S6kinase activity and proliferation in ASMC at the concentration used in this study (18). Our results showing that rapamycin had no effect on serum potentiation of GM-CSF production are consistent with serum also exerting its action through synthetic rather than proliferative pathways. Although the p70S6kinase pathway is not the only proliferative pathway, this finding lends more weight to the above suggestion that ASMC have a synthetic and a proliferative nature and that the two need not coincide.

The potentiating effects of the ASMC mitogens may be important physiologically. All the mitogens are present in the airways (4, 15, 24). However, their levels in the airways of asthmatics are likely to vary depending on the degree of inflammatory cell activation and vascular leakage occurring locally. The growth factors studied and thrombin are of particular interest as, under inflammatory conditions, a wide range of concentrations of thrombin potentiated ASMC GM-CSF production and all three growth factors contributed significantly to serum potentiation of its production. Thus the findings reported here are consistent with these ASMC mitogens playing an important role in amplifying the locally sustained inflammation occurring in the airways of asthmatics, particularly during periods of vascular leakage.

Examination of the effects of the ASMC mitogens on the release of GM-CSF by cytokine-primed cells provided evidence that the stimulatory effect of serum comes as a result of a concerted effect of these mitogens. Each of the mitogens was shown to potentiate GM-CSF release from cytokine-stimulated cells. In the case of EGF, however, this effect was not deemed significant compared with its vehicle. It is possible that at higher concentrations the effects of EGF may become significant. In addition, antibodies to PDGF, IGF, and EGF all showed an ability to inhibit the potentiating effects of serum. It is of note that the inhibitory effect of no one antibody alone was sufficient to completely ablate the potentiation caused by serum. These data suggest a concerted, and perhaps synergistic, effect of the mitogens. It is not obvious from the data whether this concerted action comes as a result of stimulating one common pathway or whether an array of mitogen-activated pathways may be involved in the mitogen potentiation of GM-CSF. What is clear from the data is that inhibition of NF-{kappa}B translocation is sufficient to negate the effects of serum. It is apparent then, that if these agents are causing the effects of serum by stimulating different pathways, these messages must combine to cause an increase in NF-{kappa}B activation. Additionally, a large proportion of the potentiating effect of serum is inhibited by blocking p38 signaling. This indicates that even if individual mitogens are signaling via different pathways, the effects of the physiologically important stimulus, serum, can be reduced by p38 inhibition.

After initially demonstrating that the observed increase in GM-CSF release following serum exposure required production of both new protein and mRNA, we extended these findings to show that there was also a significant increase in production of GM-CSF-specific mRNA. These findings indicate that the observed serum potentiation of GM-CSF release comes as a result of an increase in the signal to transcribe the GM-CSF gene.

The MAPKs p38, p42/p44, and JNK have been shown to be involved in a number of processes in human airway smooth muscle, including proliferation and cytokine release. ASMC proliferation in response to known mitogens such as PDGF and EGF is dependent on activation of the p42/p44 MAPK (17). Results from other studies indicate that the regulation of cytokine-mediated cytokine release also involves the MAPKs. The release of several cytokines, GM-CSF, regulated on activation normal T cells expressed and secreted (RANTES), and eotaxin, is dependent on the activation of the MAPKs in a complex fashion (9). Hallsworth et al. (9) showed that GM-CSF release was dependent on the activation of p42/p44 but was suppressed by the activation of p38, and more recently Oltmanns et al. (16) demonstrated the involvement of JNK in the TNF-{alpha}- and IL-1{beta}-induced expression of GM-CSF, IL-8, and RANTES. These few examples demonstrate the complexity of cell responses to activation of the MAPKs.

In light of the various signals that are conducted through the MAPK families p38, p42/p44, and JNK, it was of interest to determine whether they were involved in the cell signaling leading to serum potentiation of GM-CSF release. We used pharmacological agents at concentrations previously demonstrated to specifically inhibit these MAPKs in human ASMC (2, 16) to do this. It is intriguing that we have found evidence for p38, but not p42/p44 or JNK, to be involved in the cell signaling leading to serum potentiation of GM-CSF release. The exact effect of this signaling through p38 remains unclear. The role of p38, therefore, will become an interesting area of study.

NF-{kappa}B activation has been shown to be of importance in the production of GM-CSF (21). Through the use of DMF, which has been used on different cell types such as normal dermal fibroblasts to inhibit the translocation of NF-{kappa}B (23), we have been able to significantly decrease the amount of GM-CSF released by cytokine-primed ASMC in response to human serum. This finding, combined with the observed increases in GM-CSF message and protein, lends weight to the suggestion that components found within human serum are causing the potentiation of ASMC GM-CSF through increasing the transcription of the GM-CSF gene.

Previously it has been suggested that p38 and p42/p44 can exert their actions either via NF-{kappa}B-dependent or -independent pathways (2). We have demonstrated that both p38 and NF-{kappa}B are involved in the cell signaling leading to the serum potentiation of GM-CSF release and that preventing NF-{kappa}B translocation markedly reduces the serum potentiation. Our findings are consistent with p38 exerting its action via an NF-{kappa}B-mediated mechanism. This observation also helps to explain the lack of involvement of JNK in these signaling processes, since JNK activation results in downstream phosphorylation of c-Jun and subsequent activation of the transcription factor activator protein-1 (16).

We have provided a picture of the mitogens involved in and the mechanisms leading to the serum potentiation of GM-CSF release by airway smooth muscle. Serum was shown to advance the cell cycle of the ASMC, whereas the known mitogens (EGF, IGF, PDGF, and thrombin) failed to do so under the above experimental conditions yet still caused a potentiation of GM-CSF release by cytokine-primed ASMC. EGF, IGF, and PDGF were shown to contribute significantly to the potentiating effect of human serum. An increase in both gene transcription and protein production was required for the serum potentiation of GM-CSF release. Findings from studies analyzing the mRNA produced by the cells showed that GM-CSF-specific mRNA levels were increased by stimulation with serum and that these same cells also showed increased GM-CSF protein secretion, suggesting that the effect of serum was at the transcriptional level. The involvement of the proliferative pathway p70S6kinase and the MAPKs p42/p44 and JNK were ruled unlikely, whereas the importance of p38 and NF-{kappa}B were highlighted. Given that the p38 pathway is involved in the serum potentiation of GM-CSF release, further studies should investigate ways in which it might be targeted to interrupt the cycle of inflammation, vascular leakage, and cytokine production in asthma.


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This work was funded by the National Health and Medical Research Council of Australia, the Ramaciotti Foundations of New South Wales, and the University of Sydney Sesqui R and D scheme.


    ACKNOWLEDGMENTS
 
We thank the theatre and pathology staff of the Sydney metropolitan teaching hospitals for the supply of human lung tissue and the collaborative effort of the cardiopulmonary transplant team at St Vincent's Hospital.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Hughes, Faculty of Pharmacy A15, Univ. of Sydney, NSW 2006 Australia (E-mail: margh{at}pharm.usyd.edu.au)

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.

* D. J. Lalor and B. Truong contributed equally to this work. Back


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