Chemokine synthesis by airway smooth muscle cells (ASMC) may be an important process underlying inflammatory cell recruitment in airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Fractalkine (FKN) is a recently described CX3C chemokine that has dual functions, serving as both a cell adhesion molecule and a chemoattractant for monocytes and T cells, expressing its unique receptor, CX3CR1. We investigated FKN expression by human ASMC in response to the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ, the T helper 2-type cytokines IL-4, IL-10, and IL-13, and the fibrogenic cytokine transforming growth factor (TGF)-β. Neither of these cytokines alone had any significant effect on ASMC FKN production. Combined stimulation with IFN-γ and TNF-α induced FKN mRNA and protein expression in a time- and concentration-dependent manner. TGF-β had a significant inhibitory effect on cytokine-induced FKN mRNA and protein expression. Dexamethasone (10−8–10−6 M) significantly upregulated cytokine-induced FKN mRNA and protein expression. Finally, we used selective inhibitors of the mitogen-activated protein kinases c-Jun NH2-terminal kinase (JNK) (SP-610025), p38 (SB-203580), and extracellular signal-regulated kinase (PD-98095) to investigate their role in FKN production. SP-610025 (25 μM) and SB-203580 (20 μM), but not PD-98095, significantly attenuated cytokine-induced FKN protein synthesis. IFN-γ- and TNF-α-induced JNK phosphorylation remained unaltered in the presence of TGF-β but was inhibited by dexamethasone, indicating that JNK is not involved in TGF-β- or dexamethasone-mediated regulation of FKN production. In summary, FKN production by human ASMC in vitro is regulated by inflammatory and anti-inflammatory factors.
- airway inflammation
- chronic obstructive pulmonary disease
there is now substantial evidence suggesting that airway smooth muscle cells (ASMC), by virtue of their immunomodulatory functions, may be involved in regulating airway inflammatory responses in diseases such as asthma and chronic obstructive pulmonary disease (COPD). ASMC express adhesion molecules and synthesize many cytokines and chemokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, eotaxin, and RANTES. These mediators are critically implicated in the recruitment, survival, and activation of key effector cells, such as eosinophils, neutrophils, and T lymphocytes, in asthma and COPD (10).
Fractalkine (FKN), also known as CX3C ligand 1 (CX3CL1), is a novel chemokine belonging to the CX3C chemokine family. FKN is a multidomain molecule expressed on the cell surface and consists of a transmembrane region and a heavily glycosylated mucin-like stalk that extends from the cell surface, holding the chemokine domain at its tip (5). FKN also exists as a soluble glycoprotein that is generated by proteolytic cleavage of the full-length molecule at a membrane-proximal site (27, 32, 55). The physiological relevance of membrane and soluble FKN is not known; however, it is currently thought that membrane-associated FKN (m-FKN) predominantly mediates cell adhesion, whereas the soluble protein FKN (s-FKN) acts as a chemoattractant (22, 30). The unique receptor for FKN, CX3CR1, is expressed on monocytes (3), subsets of T lymphocytes (24, 45), mast cells (48), natural killer cells (33), dendritic cells (16), platelets (52), neurons, astrocytes, and microglial cells (31, 40, 42) and mediates both the adhesive and chemotactic functions of FKN.
FKN expression, in both the membrane and soluble forms, has been demonstrated in various nonhemopoietic cells following activation with proinflammatory cytokines such as IFN-γ, TNF-α, and IL-1β. These include vascular endothelial cells (34), bronchial, intestinal, and renal tubular epithelial cells (7, 25, 43), vascular smooth muscle cells (8, 9, 39, 46), dermal fibroblasts (21), astrocytes (57), and keratinocytes (54). A role for FKN-CX3CR1-mediated inflammation has been demonstrated in several inflammatory disorders such as rheumatoid arthritis (44, 56), atherosclerosis (13, 28), psoriasis (49), and various inflammatory conditions of the kidney, brain, and lung (12, 25). This chemokine-receptor pair has also been implicated in allergic inflammatory airway disease. Patients with allergic rhinitis or allergic asthma have increased levels of s-FKN in plasma and bronchoalveolar lavage fluid (BALF) and increased CX3CR1 function of peripheral CD4+ T lymphocytes compared with nonatopic healthy control patients. Furthermore, soluble FKN levels in BALF are significantly elevated following segmental allergen challenge in allergic asthmatic patients (50).
We investigated the production of FKN by human ASMC exposed to a range of cytokines in culture, and we also determined whether cytokine-induced FKN production is regulated by corticosteroids and by activation of the mitogen-activated protein kinases.
MATERIALS AND METHODS
All recombinant human cytokines, goat IgG anti-human FKN (AF-365), and biotinylated anti-human FKN (BAF365) polyclonal antibodies used for ELISA assays, mouse IgG1 phycoerythrin-conjugated anti-human FKN (IC365P, clone 51637), and isotype control (IC002P, clone 11711.11) monoclonal antibodies used for flow-cytometric assays, and IL-8 ELISA DuoSet were purchased from R&D Systems (Abingdon, UK). Fast Activated Cell Based ELISA (FACE p38 Chemi and FACE JNK Chemi) kits for measurement of p38 and JNK phosphorylation were purchased from Active Motif (Brussels, Belgium). GM-6001, GM-6001 negative control, SB-203580, and PD-98059 were purchased from Calbiochem (Nottingham, UK). SP-610025 was a kind gift from Celgene (San Diego, CA). Insulin-selenium-transferrin-X supplement was purchased from GIBCO, Invitrogen (Paisley, UK). Dexamethasone, actinomycin d-mannitol, and all other tissue culture reagents were purchased from Sigma (Paisley, UK). Primers for FKN and GAPDH were purchased from Sigma Genosys (Pampisford, Cambridgeshire, UK).
ASMC preparation and culture.
Human bronchial tissue was obtained from the lungs of patients undergoing lung transplantation or surgical resection for carcinoma. Airway smooth muscle was dissected out, and ASMC were established in culture as described previously (36). Briefly, bronchi were dissected free from the surrounding tissue, the epithelium was removed, and the underlying bands of smooth muscle were gently cut out from the surrounding connective tissue. The smooth muscle bundles were washed with Hanks’ balanced salt solution, placed in 1 ml of Dulbecco’s modified Eagle’s medium (DMEM) (supplemented with 1 mM sodium pyruvate, 2 mM l-glutamine, 1:100 nonessential amino acids, 20 U/l of penicillin, 20 μg/ml streptomycin, and 2.5 μg/ml amphotericin B) containing 1 mg/ml collagenase and maintained in a humidified atmosphere at 37°C in 5% CO2/95% air (vol/vol) for enzymatic dispersion for 1–2 h. The resulting cell suspension was centrifuged (200 g for 5 min), and the pellet was resuspended in supplemented DMEM containing 10% fetal bovine serum (FBS) and incubated at 37°C in 5% CO2/95% air (vol/vol). The medium was replaced every 5 days, and once the ASMC had grown to confluence, they were passaged and transferred into 175-mm2 tissue culture flasks. When examined by light microscopy, cultured ASMC displayed typical “hill and valley” growth patterns. Immunofluorescent staining for smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain was positive for >95% of cells in culture.
ASMC at passage 3–8 were grown to confluence in six-well tissue culture plates and then serum deprived for 24 h in DMEM containing 1 mM sodium pyruvate, 2 mM l-glutamine, 1:100 nonessential amino acids, antibiotics as described above, 1% insulin-transferrin-selenium-X supplement, and 0.1% bovine serum albumin (BSA). After serum deprivation, cells were incubated in fresh serum-free supplemented DMEM containing cytokines, drugs, and chemical inhibitors as indicated in individual experiments. All drugs and chemical inhibitors were added 2 h before the addition of cytokines. In experiments examining the effects of TGF-β on cytokine-induced FKN production, TGF-β was added 2 h before the addition of other cytokines.
Cytokine ELISA assays.
ELISA assays for measurement of soluble FKN and IL-8 in culture supernatants were performed according to the manufacturer’s instructions (R&D Systems, Abingdon, UK).
Flow cytometric analysis.
After cell stimulation, the medium was removed, and adherent cells were washed once with ice-cold phosphate-buffered saline (PBS). Cells were removed from the plate by scraping and then fixed in 2% formaldehyde for 10 min on ice. After fixation, cells were washed twice with PBS, and ∼2 × 105–5 × 105 cells were incubated with phycoerythrin-conjugated anti-human FKN monoclonal antibody (2.5 μg/ml) or phycoerythrin-conjugated IgG1 isotype control antibody (2.5 μg/ml) in 100 μl of 0.1% BSA in PBS for 30 min on ice. At the end of the incubation period, 2 ml of PBS were added to the antibody solution, which was then underlaid with 300 μl of heat-inactivated FBS. Cells were then pelleted by centrifugation at 200 g for 10 min and resuspended in 0.1% BSA in PBS before flow cytometric analysis. A FACScan flow cytometer [Becton Dickinson Immunocytometry Systems (BIDS)] equipped with a 488-nm argon ion laser was used, and typically 10,000 events were acquired in the viable cell region of the forward light scatter/side light scatter plots. List-mode data files were analyzed using CELLQuest software (BIDS), and the fluorescence signal was calculated as the median fluorescence intensity of the gated ASMC population.
RNA extraction, RT-PCR.
Total RNA was extracted from ASMC using the RNeasy Mini Kit (Qiagen, West Sussex, UK) according to the manufacturer’s instructions. Single-stranded complementary DNA (cDNA) for a PCR template was synthesized from 0.5 μg of RNA using random hexamers (Promega) and AMV reverse transcriptase (Promega). Primers used were: FKN sense: 5′-AACTCGAAATGGCGGCACCTT-3′ and anti-sense: 5′-ATGAATTACTACCACAGCTCCG-3′; GAPDH sense: 5′-CCACCCATGGCAAATTCCATGGCA-3′ and anti-sense: 5′-TCTAGACGGCAG GTCAGGTCCACC-3′. The amplified cDNA products for FKN and GAPDH are 887 and 696 base pairs. Cycling conditions for PCR were as follows: 1× 94°C, 1 min; 26× (FKN) or 22× (GAPDH) 94°C, 0.5 min; 62°C, 0.5 min; 72°C, 0.5 min; and 1× 72°C, 1 min. The products were electrophoresed on a 1.5% agarose gel containing ethidium bromide. Relevant band intensities were quantified by scanning densitometric analysis using software from Ultra-Violet Products (UVP, Cambridge, UK).
Total RNA extraction and reverse transcription were performed as described above. Real-time PCR analysis was performed with Rotor Gene 3000 (Corbett Research) using SYBR Green PCR Master Mix Reagent Kit (Promega). Primers used were: FKN sense: 5′-CCTGTAGCTTTGCTCATCCACTATC-3′ and anti-sense: 5′-TCCAAGATGATTGCGCGTT-3′; GAPDH sense: 5′-GAAGATGGTGATGGGATTTC-3′ and anti-sense: 5′-GAAGGTGAAGGTCGGAGT-3′. Cycling conditions were as follows: step 1, 15 min at 95°C; step 2, 20 s at 94°C; step 3, 20 s at 60°C; and step 4, 20 s at 72°C, with repeat from step 2 to step 4 for 45 cycles. Data from the reaction were collected and analyzed by the complementary computer software (Corbett Research). Relative quantitations of gene expression were calculated with standard curves and normalized to GAPDH.
Measurement of p38 and JNK phosphorylation.
Measurement of phosphorylated p38 and JNK and total p38 and JNK protein was performed using FACE assays. ASMC were grown to confluence in 96-well plates and placed in fresh serum-free supplemented DMEM for 24 h before cytokine or drug treatment. FACE assays were performed according to the manufacturer’s instructions (Active Motif, Brussels, Belgium).
Data are presented as means ± SE. Comparison between groups was performed using one-way analysis of variance or paired t-tests. A P value of <0.05 was taken as significant.
Human ASMC synthesize FKN in response to combined stimulation with IFN-γ and TNF-α.
We examined FKN protein synthesis by human ASMC in response to several cytokines. Stimulation of ASMC with 10 ng/ml of IL-1β (n = 2), TNF-α (n = 6), or IFN-γ (n = 6) alone for 24 h did not induce detectable release of s-FKN into culture supernatants (data not shown). However, combined stimulation with IFN-γ and TNF-α for 24 h had a synergistic effect on ASMC s-FKN production (Fig. 1A). s-FKN release induced by IFN-γ and TNF-α was detectable as early as 4–8 h after stimulation and increased in a time-dependent manner for up to 60 h (Fig. 1B). Combined stimulation of ASMC with IL-1β and TNF-α (10 ng/ml each) or IL-1β and IFN-γ (10 ng/ml each) did not result in detectable s-FKN release (n = 2, data not shown).
Because s-FKN may be generated by proteolytic cleavage of the membrane-associated molecule (m-FKN), we examined whether stimulation with IFN-γ and TNF-α led to accumulation of m-FKN. Flow cytometric analysis of ASMC stimulated with IFN-γ and TNF-α (20 ng/ml each) demonstrated m-FKN expression that reached maximal levels 16 h after stimulation. Although m-FKN expression began to decrease after 16 h, expression levels at 24 and 48 h were still above baseline (Fig. 2).
Finally, we examined the effects of IFN-γ and TNF-α on FKN mRNA expression. Stimulation of ASMC with IFN-γ (10 ng/ml) or TNF-α (10 ng/ml) alone for 4–60 h did not induce significant FKN mRNA expression (n = 3, data not shown). However, FKN mRNA expression was observed following combined stimulation with IFN-γ and TNF-α (10 ng/ml each). FKN mRNA expression was detectable 4 h after stimulation, reached maximal levels by 16 h, and was sustained for up to 60 h (Fig. 3).
Effect of matrix metalloproteinase inhibition on ASMC s-FKN release.
To determine whether s-FKN is the cleavage product of m-FKN, we examined IFN-γ- and TNF-α-stimulated s-FKN release in the presence or absence of the broad-spectrum hydroxamic acid inhibitor of matrix metalloproteinases, GM-6001, or a structurally similar compound that serves as a negative control (GM-6001 negative control, Calbiochem). Neither GM-6001 (0.1–10 μM, n = 4) nor the negative control compound (0.1–10 μM, n = 2) inhibited IFN-γ- and TNF-α-stimulated s-FKN release (data not shown). Furthermore, GM-6001 did not increase IFN-γ- and TNF-α-stimulated m-FKN expression as determined by flow cytometry (Fig. 2).
Modulation of cytokine-induced ASMC s-FKN release by the Th2-type cytokines IL-4, IL-10, and IL-13.
We determined whether ASMC synthesize FKN protein in response to the Th2-type cytokines IL-4, IL-10, and IL-13 and also determined whether these cytokines influence FKN production induced by IFN-γ and TNF-α. Stimulation of ASMC with 10 ng/ml of IL-4 (n = 1), IL-10 (n = 1), or IL-13 (n = 5) for 24 h did not induce s-FKN release into culture supernatants (data not shown). In addition, stimulation of ASMC with 10 ng/ml of IL-4, IL-10, or IL-13 combined with 10 ng/ml of either TNF-α or IFN-γ did not result in significant s-FKN production (n = 2–5 cell donors, data not shown). Finally, we examined the effects of the Th2-type cytokines on s-FKN release induced in response to combined IFN-γ and TNF-α stimulation. We found that neither one of these cytokines had any modulatory effects on IFN-γ and TNF-α stimulated s-FKN release (n = 3 to 5, data not shown).
Effects of TGF-β on ASMC FKN production.
We determined whether TGF-β induces ASMC to produce FKN and, in addition, whether this cytokine modulates IFN-γ- and TNF-α-induced FKN production. Stimulation of ASMC with TGF-β (10 ng/ml) alone for 24 h did not induce detectable s-FKN protein release into culture supernatants (n = 8, data not shown). Combined stimulation with TGF-β and IFN-γ (10 ng/ml each) or TGF-β and TNF-α (10 ng/ml each) for 24 h also did not result in detectable s-FKN release (n = 3, data not shown). However, TGF-β had a significant inhibitory effect on IFN-γ- and TNF-α-stimulated s-FKN release, causing approximately a 50% reduction in s-FKN production. The inhibitory effects of TGF-β were evident 16 h after stimulation and were sustained for up to 60 h (Fig. 4A). Also, flow cytometric studies showed that TGF-β inhibits IFN-γ- and TNF-α-stimulated m-FKN expression. The mean fluorescence intensity (MFI) of cells stimulated with IFN-γ and TNF-α in the presence of TGF-β was reduced by ∼50% compared with cells treated with IFN-γ and TNF-α only (MFI values 10.22 vs. 5.20) (Fig. 2).
Given that we observed an inhibitory effect of TGF-β on IFN-γ- and TNF-α-induced FKN protein synthesis, we determined whether these effects occurred at the level of FKN gene expression. As expected, stimulation of ASMC with TGF-β (10 ng/ml) alone for 4–60 h did not induce FKN mRNA expression (n = 5, data not shown). TGF-β, however, did reduce IFN-γ- and TNF-α-stimulated FKN mRNA expression. The inhibitory effects of TGF-β on FKN gene expression were delayed, becoming evident 16 h after stimulation (Fig. 3).
Comparison of the effects of TGF-β on IFN-γ- and TNF-α-induced IL-8 release by ASMC.
We examined whether the regulatory effects of TGF-β extend to other ASMC-derived chemokines induced by IFN-γ and TNF-α. IL-8 was measured in the same supernatants that were used for assay of s-FKN production. The effects of TGF-β on IFN-γ and TNF-α induced IL-8 release were in direct contrast to its effects on s-FKN release. We found that TGF-β significantly potentiates IFN-γ- and TNF-α-stimulated IL-8 release (Fig. 4B).
Modulation of cytokine-induced ASMC FKN production by the corticosteroid dexamethasone.
We examined whether cytokine-induced FKN production by human ASMC is susceptible to regulation by corticosteroids. Contrary to its usual inhibitory effects on inflammatory mediator expression, we found that dexamethasone upregulates ASMC FKN production. Stimulation of ASMC with IFN-γ and TNF-α (10 ng/ml each) in the presence of dexamethasone increased FKN mRNA expression, cell-associated m-FKN, as well as s-FKN release into culture supernatants. Dexamethasone-induced increases in FKN mRNA were evident as early as 4 h after stimulation and were sustained for up to 60 h after stimulation (Fig. 3). Dexamethasone-mediated increases in s-FKN release were evident 16 h after stimulation and remained at an elevated level for up to 60 h after stimulation. The effects of dexamethasone were concentration dependent, and significant potentiation of s-FKN release was observed at concentrations between 0.01 and 1 μM (Fig. 5). Flow cytometric studies show that cytokine-stimulated m-FKN expression was significantly increased in the presence of dexamethasone (Fig. 2). The MFI of cells stimulated with IFN-γ and TNF-α (20 ng/ml each) in the presence and absence of dexamethasone (1 μM) was 14.7 ± 1.8 (SE) and 8.7 ± 0.5 (SE) (n = 5, P < 0.05), respectively. Treatment of ASMC with dexamethasone (1 μM) alone had no effect on FKN gene or protein expression.
Effect of actinomycin D on IFN-γ- and TNF-α-stimulated FKN mRNA expression in the presence and absence of dexamethasone.
Given that increased FKN protein synthesis in the presence of dexamethasone was associated with increased FKN mRNA expression, we determined whether potentiation of FKN production in the presence of dexamethasone was due to increased mRNA stability. To do this, we examined the rate of FKN mRNA degradation following transcriptional arrest with the transcriptional blocker actinomycin D. In these studies, cells were stimulated with IFN-γ and TNF-α (10 ng/ml each) in the presence or absence of dexamethasone (10−6 M) for 16 h, as this was the time point of maximal FKN mRNA expression (Fig. 3). The rate of FKN mRNA degradation in the presence of actinomycin D (10 μg/ml) was then monitored for up to 8 h by quantitative real-time PCR. At 16 h after stimulation, IFN-γ- and TNF-α-induced FKN mRNA expression was significantly elevated in the presence of dexamethasone [256.5 ± 61.26 (SE) % increase compared with cells stimulated with IFN-γ and TNF-α only; n = 6, P ≤ 0.05]. The rate of FKN mRNA degradation in the presence of actinomycin D, however, was not affected by dexamethasone, suggesting that increased FKN mRNA expression in the presence of dexamethasone is not due to an effect on mRNA stability (Fig. 6).
Role of ERK, p38, and JNK in cytokine-induced ASMC FKN production and regulation by TGF-β and dexamethasone.
We determined whether IFN-γ- and TNF-α-stimulated FKN production is mediated by p38, ERK, and JNK protein kinases. Treatment of ASMC with the ERK inhibitor PD-98059 had no effect on IFN-γ- and TNF-α-stimulated s-FKN release. SP-610025, an inhibitor of JNK, and SB-203580, an inhibitor of p38, caused significant inhibition of IFN-γ- and TNF-α-stimulated s-FKN release at concentrations of 25 and 20 μM, respectively (Fig. 7). We also investigated the effect of SP-610025 (25 μM) on m-FKN expression by flow cytometry. The MFI of cells stimulated with IFN-γ and TNF-α in the presence of SP-610025 was reduced by ∼35% compared with cells treated with IFN-γ and TNF-α only (MFI values 10.22 vs. 6.65) (Fig. 2).
Because IFN-γ and TNF-α have synergistic effects on ASMC FKN production and because we could demonstrate a role for JNK and p38 in IFN-γ- and TNF-α-induced FKN production, we determined whether synergistic activation of these MAP kinases (MAPK) by IFN-γ and TNF-α may be a possible mechanism underlying their synergistic effects on FKN production. We measured phosphorylation of JNK and p38 by FACE and chemiluminescent detection. Cells were treated with IFN-γ and TNF-α (10 ng/ml each), either alone or in combination, for various time periods up to 16 h. As expected, TNF-α induced phosphorylation of JNK and p38, and this was maximal 15–30 min after stimulation. IFN-γ did not induce phosphorylation of JNK or p38 and had no effect on TNF-α-induced phosphorylation of these MAPK (Fig. 8). The total protein content of both JNK and p38 remained unchanged under all treatment conditions (data not shown).
We also determined whether regulation of cytokine-induced FKN production by TGF-β and dexamethasone is associated with inhibition or potentiation, respectively, of cytokine-induced JNK phosphorylation. Cells were pretreated with TGF-β (10 ng/ml) or dexamethasone (10−6 M) for 2 h and then stimulated with IFN-γ and TNF-α (10 ng/ml each) for 30 min. TGF-β had no effect on IFN-γ- and TNF-α-induced JNK phosphorylation [58,208.8 ± 8,017.432 relative light units (RLU) vs. 57,256.8 ± 7,367.463 RLU in the absence and presence of TGF-β, n = 4, P ≥ 0.05]. Dexamethasone, however, had a small but significant inhibitory effect on IFN-γ- and TNF-α-induced JNK phosphorylation (58,208.750 ± 8,017.432 RLU vs. 46,811 ± 6,183.3 RLU in the absence and presence of dexamethasone, n = 4, P ≤ 0.05). TGF-β and dexamethasone had no direct effect on JNK phosphorylation, and the total protein content of JNK remained unchanged under all treatment conditions (data not shown).
We describe FKN mRNA and protein expression following combined IFN-γ and TNF-α stimulation of human ASMC in culture and demonstrate regulation of FKN mRNA and protein expression by TGF-β and corticosteroids. TGF-β had a marked inhibitory effect on cytokine-induced FKN mRNA and protein expression, whereas dexamethasone markedly potentiated cytokine-induced FKN mRNA and protein expression. The Th2 cytokines IL-4, IL-10, and IL-13 did not induce ASMC FKN production and had no modulatory effects on cytokine-stimulated FKN production. In addition, FKN production was dependent on the phosphorylation activity of JNK and p38, but not of ERK MAPK induced by combined IFN-γ and TNF-α stimulation. Our study suggests that FKN is another ASMC-derived chemokine that may contribute to the inflammatory response in airway diseases such as asthma and COPD.
FKN production required synergistic interactions between the IFN-γ and TNF-α signaling pathways as neither of these cytokines alone had any significant effect. Synergistic effects of IFN-γ and TNF-α on FKN production have previously been described in human vascular smooth muscle cells and astrocytes (39, 46, 57). FKN production in other cell types, such as epithelial cells, endothelial cells, fibroblasts, and keratinocytes, can be induced by IFN-γ, TNF-α, or IL-1β stimulation alone (7, 9, 21, 25, 26, 34, 41, 43, 54). We could not demonstrate any effect of IL-1β on ASMC FKN production, either alone or in combination with TNF-α or IFN-γ. Thus FKN production in human ASMC is more strictly regulated than that in other cell types. Although IFN-γ and TNF-α interact synergistically to induce FKN production, this cytokine combination has variable effects on other ASMC-derived chemokines. IL-8, RANTES, and eotaxin production by human ASMC is induced by TNF-α but not IFN-γ. Furthermore, although IFN-γ potentiates TNF-α-induced RANTES release, it has no synergistic effects on TNF-α-induced IL-8 or eotaxin release (11, 35, 36, 51). Thus the relative presence or absence of IFN-γ and TNF-α in the inflammatory milieu may direct the pattern of ASMC chemokine synthesis and hence the type of inflammatory cells recruited.
The human FKN gene has been mapped to a region of chromosome 16 (16q13) that also encodes two other chemokine genes, MDC (macrophage-derived chemokine; CCL22) and TARC (thymus- and activation-regulated chemokine; CCL17) (28). TARC is a selective chemoattractant for Th2 type CD4+ T lymphocytes expressing its receptor, CCR4, and there is strong evidence suggesting a role for TARC in allergic asthma (37, 38, 53). Like FKN, TARC is produced by human ASMC in culture; however, MDC is not. In contrast to FKN release, TARC release by ASMC only occurs after combined stimulation with TNF-α and IL-4 or TNF-α and IL-13 (20). Thus, although FKN, MDC, and TARC are linked chromosome 16q13 chemokines, their expression by human ASMC appears to be differentially regulated.
TGF-β is a potent fibrogenic cytokine, stimulating extracellular matrix and growth factor production, and has been implicated in structural remodeling of the airway wall in asthma. TGF-β, however, also modulates immune cell function and may act as either a pro- or anti-inflammatory mediator (18). The immunomodulatory effects of TGF-β also extend to airway structural cells, including ASMC. TGF-β stimulates ASMC to synthesize IL-6, IL-11, and IL-8 and induces ASMC prostanoid synthesis via induction of cyclooxygenase-2 expression (19, 23). Although we could not demonstrate any direct stimulatory effects of TGF-β on FKN production, we found that it attenuated IFN-γ- and TNF-α-stimulated FKN mRNA and protein expression. Interestingly, in the same culture supernatants, we showed that TGF-β significantly augments IFN-γ- and TNF-α-induced IL-8 production, demonstrating differential regulatory effects of TGF-β on ASMC chemokine synthesis. Recently, it was shown that TGF-β released from tryptase-activated ASMC is chemotactic for mast cells (6). This, together with our findings, suggests that TGF-β may be critically implicated in an inflammatory pathway regulating ASMC-mediated inflammatory cell recruitment in the airways.
Corticosteroids are the most effective anti-inflammatory drugs for the treatment of asthma, and previous studies have demonstrated inhibitory effects of corticosteroids on ASMC cytokine and chemokine synthesis (35, 36). Paradoxically, we observed marked upregulation of cytokine-stimulated FKN mRNA expression and protein synthesis in the presence of the corticosteroid dexamethasone. This was most likely caused by an increase in the rate of FKN mRNA transcription rather than an increase in mRNA stability, as dexamethasone had no effect on the rate of FKN mRNA degradation in the presence of the transcriptional blocker actinomycin D. Interestingly, it has also been demonstrated that corticosteroids potentiate IL-1β-induced stem cell factor (SCF) production in human lung fibroblasts. The mechanism of corticosteroid-mediated potentiation was due to increased SCF mRNA transcription as well as increased mRNA stability. In addition, corticosteroid-mediated potentiation of IL-1β-induced SCF promoter activity required activation of functional NF-κB and glucocorticoid receptor responsive element (GRE)-like sites in the SCF promoter, indicating that an interaction between these regulatory elements leads to increased SCF mRNA transcription (14, 15). It is possible that corticosteroid-mediated potentiation of cytokine-induced FKN production is mediated by a similar mechanism. Indeed, previous studies have reported a requirement for NF-κB in FKN production (1, 8, 17), and we have also found that NF-κB activation is required for IFN-γ and TNF-α induced FKN production in human ASMC (Sukkar, unpublished observation). Furthermore, using DNA homology analysis, we have discovered the presence of putative NF-κB and GRE DNA binding sites within 1 kb of the start site of transcription in the FKN gene (Bhavsar PK, unpublished observation). We are currently investigating the possibility that dexamethasone-mediated potentiation of FKN production involves functional activation and cooperation of these regulatory elements in the FKN gene.
Although ASMC-derived FKN, by virtue of its cell adhesive and chemotactic functions, is likely to be involved in the propagation of airway inflammatory responses, the upregulation of muscle-derived FKN by dexamethasone leads to the speculation that FKN may be implicated in steroid-mediated suppression of airway inflammation. There is evidence to suggest that FKN has anti-inflammatory activity. FKN suppresses LPS-induced TNF-α and IL-6 secretion in microglial cultures in vitro (42, 58). Furthermore, in rat models of neuroinflammation induced by cerebral injection of LPS, neutralization of endogenous brain FKN with specific antibodies was associated with potentiation of LPS-induced release of inflammatory mediators, including TNF-α and 8-isoprostane, a marker of oxidative stress (59).
The accumulation of s-FKN in culture supernatants of cytokine-stimulated vascular smooth muscle cells is mediated by proteolytic cleavage of the membrane-associated molecule (25). Thus stimulation with IFN-γ and TNF-α in the presence of batimastat, a broad-spectrum hydroxamic acid inhibitor of zinc-dependent proteinases, leads to increased expression of m-FKN and a concomitant decrease in the level of s-FKN, suggesting metalloproteinase-induced cleavage of m-FKN (39). The compound GM-6001 is another broad-spectrum hydroxamic acid inhibitor of metalloproteinases and inhibits FKN cleavage induced by TNF-α in vascular smooth muscle cells (9). We found that stimulation of ASMC with IFN-γ and TNF-α in the presence of GM-6001 had no effect on membrane expression of FKN or the level of soluble FKN generated in culture supernatants. Therefore, GM-6001-sensitive metalloproteinases are not involved in FKN cleavage in human ASMC.
ASMC secretory responses of certain cytokines and chemokines are, in part, mediated by activation of the MAPK pathway (2, 4, 29, 47). MAPK are a family of serine/threonine kinases, and at least three major groups that differ in their substrate specificity have been characterized: ERK, JNK, and p38 MAPK. In this study, we show that IFN-γ- and TNF-α-stimulated s-FKN release is suppressed by SP-600125, an inhibitor of JNK, and SB-203580, an inhibitor of p38 MAPK, but not by PD-98059, which is an inhibitor of ERK1/2 activation. In our previous characterization of the effects of SP-600125 on IL-1β- and TNF-α-stimulated RANTES, IL-8, and GM-CSF production in human ASMC (47), we demonstrated that SP-610025 attenuates IL-1β- or TNF-α- induced RANTES, IL-8, and GM-CSF release by selective inhibition of JNK activity. Similarly, the effect of SB-203580 on cytokine-induced mediator release has also been evaluated in human cultured ASMC. SB-203580 specifically inhibited cytokine-induced p38 kinase activity at concentrations up to 10 μM and only partially inhibited JNK and ERK activity at 100 μM (29). It is unlikely that inhibition of JNK activity contributed to the inhibitory action of SB-203580 on s-FKN release observed at the 20 μM concentration used in this study.
Because we could demonstrate a role for p38 and JNK in cytokine-induced FKN production, we determined whether regulatory effects at the level of these MAPK contribute to the synergistic effects of IFN-γ and TNF-α and the inhibitory and potentiating effects of TGF-β and dexamethasone, respectively, on FKN production. IFN-γ did not induce phosphorylation of p38 or JNK, nor did it modulate TNF-α-induced phosphorylation of these MAPK, indicating that synergistic interactions between IFN-γ and TNF-α are not due to increased p38 or JNK phosphorylation. We also demonstrated that inhibition of FKN production by TGF-β is not due to an inhibitory effect on cytokine-induced JNK-phosphorylation. Interestingly, whereas dexamethasone acts to potentiate FKN production, we observed a small, but significant inhibitory effect of dexamethasone on IFN-γ- and TNF-α-induced JNK phosphorylation. Thus, although dexamethasone modulates JNK phosphorylation levels, this does not provide a mechanism for the observed increase in FKN production by dexamethasone. Indeed, further studies are required to elucidate the mechanisms regulating FKN production in ASMC.
In conclusion, IFN-γ and TNF-α induce ASMC FKN expression, and this represents a potential new mechanism by which ASMC may regulate airway inflammatory responses. Furthermore, the inhibition of FKN release by TGF-β and its enhancement by corticosteroids indicate that FKN can be divergently modulated by various inflammatory and anti-inflammatory factors.
This work was funded by a Programme Grant from the Wellcome Trust.
The authors thank Dr. Andreas Ludwig for helpful advice on flow cytometric analysis of membrane FKN. The authors also thank Dr. Andrew Nicholson at the Royal Brompton Hospital, London, for the supply of human lung tissue.
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- Copyright © 2004 the American Physiological Society