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Published ahead of print on October 17, 2003, doi:10.1165/rcmb.2003-0282OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 585-593, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0282OC

Human Lung Fibroblasts Express Interleukin-6 in Response to Signaling after Mast Cell Contact

S. Matthew Fitzgerald, Steven A. Lee, H. Kenton Hall, David S. Chi and Guha Krishnaswamy

Division of Allergy and Clinical Immunology, Department of Internal Medicine, East Tennessee State University, Johnson City, Tennessee

Address correspondence to: Steven Matthew Fitzgerald, East Tennessee State University, Internal Medicine, P.O. Box 70622, Johnson City, TN 37614. E-mail: mattfitzgerald1{at}aol.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Asthma is a chronic inflammatory disease of the airways. Mast cell–derived cytokines may mediate both airway inflammation and remodeling. It has also been shown that fibroblasts can be the source of proinflammatory cytokines. In the human airways, mast cell–fibroblast interactions may have pivotal effects on modulating inflammation. To study this further, we cocultured normal human lung fibroblasts (NHLF) with a human mast cell line (HMC-1) and assayed for production of interleukin (IL)-6, an important proinflammatory cytokine. When cultured together, NHLF/HMC-1 contact induced IL-6 secretion. Separation of HMC-1 and NHLF cells by a porous membrane inhibited this induction. HMC-1–derived cellular membranes caused an increase in IL-6 production in NHLF. Activation of p38 MAPK was also seen in cocultures by Western blot, whereas IL-6 production in cocultures was significantly inhibited by the p38 inhibitor SB203580. IL-6 production in cocultures was minimally inhibited by a chemical inhibitor of nuclear factor-{kappa}B (Bay11), indicating that nuclear factor-{kappa}B may have a minimal role in signaling IL-6 production in mast cell/fibroblasts cocultures. Blockade of inter-cellular adhesion molecule-1, tumor necrosis factor-RI, and surface IL-1ß with neutralizing antibodies failed to significantly decrease IL-6 production in our coculture, indicating that other receptor–ligand associations may be responsible for this activation. These novel studies reveal the importance of cell–cell interactions in the complex milieu of airway inflammation.

Abbreviations: enzyme-linked immunosorbent assay, ELISA • electrophoretic mobility shift assay, EMSA • human mast cells-1, HMC-1 • intercellular adhesion molecule, ICAM • interleukin, IL • mitogen-activated protein kinase, MAPK • normal human lung fibroblasts, NHLF • nuclear factor-{kappa}B, NF-{kappa}B • phosphate-buffered saline, PBS • phenylmethylsulfonyl fluoride, PMSF • reverse transcriptase–polymerase chain reaction, RT-PCR • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the asthmatic airway, tissues are highly sensitive to antigenic challenge and respond by mediator production and inflammatory cell infiltration. Mast cells are a major contributor of this response. After cross-linking of high-affinity IgE receptors, mast cells can release a host of potent proinflammatory cytokines (1, 2). Histologically, mast cells are in close proximity to fibroblasts, and c-kit/stem cell factor interactions can prolong mast cell proliferation and survival. Fibroblasts themselves are capable of releasing cytokines after activation. Therefore, it is likely that mast cells and fibroblasts can mutually activate each other.

Mast cell–fibroblast interactions have been shown to be important in many cellular processes. Cairns and coworkers have demonstrated that tryptase isolated from human tissue caused increases in collagen type I synthesis from the human lung fibroblast cell line MRC-5 (3). Abe and colleagues showed similar results with human dermal fibroblasts (4). They also found that tryptase increases fibroblast proliferation in a concentration-dependent manner. Tryptase-induced proliferation of fibroblasts has been associated with protease-activated receptor-2 signaling. Protease-activated receptor-2 is localized more to lung fibroblasts than to dermal fibroblasts, and this may potentiate the cascade of fibroblasts hyperplasia in the asthmatic lung (5). Other studies demonstrate the importance of mast cell–fibroblast interactions in collagen gel contraction (6) and growth factor production (7). The reverse has also been demonstrated. Hogaboam and associates have shown that stem cell factor from fibroblasts induced eotaxin production in mast cells (8). Eotaxin being a potent chemoattractant for eosinophils, a major inflammatory cell associated with tissue damage and epithelial denudation. Mutual mast cell–fibroblast interactions leading to enhanced cytokine secretion can lead to increased inflammatory responses and could contribute to late phase airway inflammation. This could partly explain the persistence of asthma even in the absence of antigenic challenge, this process being mediated by novel cell–cell interactions regulated by cell surface molecules.

Several investigators have reported on the importance of mitogen-activated protein kinases (MAPKs) in fibroblast signaling as well. There are three major MAPKs: extracellular signal–regulated kinase, C-Jun-N-terminal kinase, and p38, all of which have been shown to be expressed in human fibroblasts. Suzuki and coworkers have shown that in fresh rheumatoid synovial fibroblasts, interleukin (IL)-6 and IL-8 production are dependent on p38 MAPK activation (9). It has also been shown that tissue samples taken from patients with idiopathic pulmonary fibrosis demonstrate p38 MAPK activation in epithelial, endothelial, smooth muscle, and fibroblast cells (10). Another important signaling molecule in fibroblasts is nuclear factor (NF)-{kappa}B. NF-{kappa}B is a transcription factor specific for proinflammatory gene activation. Located in the cytoplasm in an inactive dimer, NF-{kappa}B can be activated by many different stimuli and freed from its dormant state. Once free, NF-{kappa}B can translocate to the nucleus, where it binds {kappa}B domains of proinflammatory genes to initiate transcription. We have previously shown the activation of NF-{kappa}B in normal human lung fibroblasts by IL-1ß, tumor necrosis factor (TNF)-{alpha}, and macrophage contact (11).

In this study, we demonstrate a novel cell–cell contact mechanism of fibroblast activation by mast cells mediated by p38 MAPK. In our model, mast cell–fibroblast cocultures induce IL-6 production in a concentration- and time-dependent manner. IL-6 is a multifunctional cytokine produced by many cell types of the body. It has differentiation and antibody secreting effects on B cells as well as colony-stimulating effects on hematopoietic cells. IL-6 can also induce acute phase protein production from hepatocytes (12, 13). Serum levels of IL-6 have been shown to be elevated in patients with diseases such as arthritis, inflammatory bowel disease, and some cervical cancers (14–16). In the lung, IL-6 can stimulate the differentiation of B cells into IgE-secreting plasma cells. For its clinical significance as an immunosuppressant and its use as an anti-asthma drug, dexamethasone was used to test inhibition of IL-6 in mast cell–fibroblast cocultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue Culture
Human mast cell line (HMC-1) cells were grown in RPMI 1640 (Gibco BRL, Frederick, MD) supplemented with 11.1% FBS and 1% 1 M Hepes buffer solution (Rockville, MD), 50 mg/ml gentamycin, 0.05 M ß-mercaptoethanol, 1% L-glutamine, 1% sodium bicarbonate as described earlier (17). Normal human lung fibroblasts (NHLF; Clonetics-BioWhittaker, Walkersville, MD) were grown in fibroblast basal medium (Clonetics-BioWhittaker) at 5% CO2 at 37°C. Media was supplemented with 2% fetal bovine serum, human fibroblast growth factor-B (1.0 µg/ml), insulin (5 mg/ml), gentamicin, and amphotericin B. NHLF were cultured in 24-well culture plates at cell concentrations of 5 x 104 cells/well and incubated overnight. Supernatants were collected after 24 h and centrifuged to remove cellular debris. Coculturing of NHLF with HMC-1 cells was done in 24-well plates with 5 x 104 NHLF/well and 1 x 106 cells/ml of HMC-1 in 1-ml cultures of NHLF media. Dose-dependent coculturing of HMC-1 with NHLF were done at 0.1, 0.25, 0.50, 1, 2, and 3 x 106 HMC-1 per milliliter of media. Time-dependent experiments were done with NHLF seeded at 5 x 104 cells/well in a 24-well plate with HMC-1 added at 1 x 106 cells/ml in 1 ml of NHLF media. Supernatants were collected at 12, 24, 48, and 72 h, centrifuged, and assayed by enzyme-linked immunosorbent assay (ELISA). Cell separation experiments were done in six-well tissue culture plates with NHLF at 1 x 105 cells/well and HMC-1 at 1 x 106/ml with 0.4 µm track-etched membrane inserts to separate HMC-1 from NHLF (Falcon, Becton Dickinson, Franklin Lakes, NJ). Cellular derived membranes from NHLF and HMC-1 were cultured with HMC-1 and NHLF, respectively. NHLF were plated in 24-well plates at 5 x 104 NHLF/well and incubated with 75 and 100 µg/ml HMC-1–derived membranes. Whole cell HMC-1 cocultures were done for comparison. Likewise, HMC-1 were grown in 24-well plates in 1-ml cultures at 1 x 106 HMC-1/ml and then incubated with 75 and 100 µg/ml NHLF-derived membranes. Here, whole NHLF cocultures were used for comparison with membrane treatments. For HMC-1 and coculture conditioned media experiments, HMC-1 were grown in NHLF media and cocultures were incubated in NHLF media for 24 h before being harvested, centrifuged, and then added to pure NHLF cultures for 24 h. p38 MAPK inhibition studies were done with SB203580 (Calbiochem, San Diego, CA). NHLF were pretreated with SB203580 (10 µM) for 2 h before addition of HMC-1. For NF-{kappa}B inhibition studies, Bay 11 (1 µM, BAY-11–7082; Biomol Research Laboratories, Plymouth Meeting, PA) was added to NHLF cultures 1 h before addition of HMC-1. Bay 11 inhibits NF-{kappa}B by specifically blocking phosphorylation of I{kappa}B{alpha}, a regulator protein of NF-{kappa}B. Blocking experiments were done with anti-intercellular adhesion molecule (ICAM)-1 (BD Bioscience PharMingen, San Diego, CA), anti–TNF-RI (R&D Systems, Minneapolis, MN), and anti–IL-1ß (NCI Biological Resources Branch, Bethesda, MD). NHLF were pretreated with anti–ICAM-1 (10 µg/ml) and anti–TNF-RI (10 µg/ml) for 1 h, and HMC-1 were pretreated for 1 h with anti–IL-1ß (10 µg/ml). Dexamethasone (Sigma-Aldrich, St. Louis, MO) was added to NHLF cultures at 1 µM 48 h before addition of HMC-1. For all of these experiments, n = 3.

ELISA
IL-6 levels in cell-free supernatants were assayed by ELISA as previously described (11, 18–21) using commercially available kits (R&D Systems). Values were extrapolated or interpolated from a standard curve. Results were analyzed on an ELISA plate reader (Dynatech MR 5000 with supporting software).

IL-6 Gene Expression by Reverse Transcriptase–Polymerase Chain Reaction
Gene expression for IL-6 was assessed using reverse transcriptase–polymerase chain reaction (RT-PCR) as previously described (11, 22, 23). RNA was extracted by an RNAzol technique from cultured cells. Briefly, total cellular RNA was extracted from cultured cells (NHLF at 1 x 106 cells/plate and HMC-1 at 1 x 106/ml) by the addition of 1.0 ml of RNAzol B (Tel-Test, Inc., Friendswood, TX). The suspension was shaken for 1 min and centrifuged at 12,000 x g for 15 min at 4°C. The aqueous phase was washed twice with 0.8 ml phenol:chloroform (1:1, vol/vol), and once with 0.8 ml of chloroform. Each time, the suspension was centrifuged at 12,000 x g for 15 min at 4°C. An equal volume of isopropanol was added to the aqueous phase, and the preparation refrigerated at -20°C overnight. The samples were then centrifuged at 12,000 x g for 30 min at 4°C and the RNA pellet washed with 1.0 ml 75% ethanol. The RNA pellet was air-dried and suspended in 20 µl of DEPC-treated water. RNA was quantitated by optical density readings at 260 nm, equalized to 1,000 ng/µl, and electrophoresed in ethidium bromide–stained agarose gels at 1,000 ng/well to determine the integrity of the 28S and 18S RNA bands. First-strand cDNA was synthesized in the presence of murine leukemia virus reverse transcriptase (2.5 U/µl); 1 mM each of the nucleotides dATP, dCTP, dGTP, and dTTP; RNase inhibitor (1 U/µl); 10x PCR buffer (500 mM KCl, 100 mm Tris-HCl, pH 8.3); and MgCl2 (5 mM), using oligo(dT)16 (2.5 µM) as a primer. The preparation was incubated at 42°C for 20 min in a DNA thermocycler (Perkin-Elmer Corp., Norwalk, CT) for reverse transcription. PCR amplification was done on aliquots of the cDNA in the presence of MgCl2 (1.8 mM), each of dNTPs (0.2 mM), and AmpliTaq polymerase (1 U/50 µl), and paired cytokine-specific primers (0.2 nM of each primer) to a total volume of 50 µl. PCR consisted of 1 cycle of 95°C for 2 min, 45 cycles of 95°C for 45 s, 60°C for 45 s, and 72°C for 1 min 30 s, and lastly, 1 cycle of 72°C for 10 min. Fourteen microliters of the amplified products were subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide. IL-6 bands were compared with predicted base pair migration distances from a PhiX 174 Hae III DNA marker (Promega, Madison, WI).

Cellular Membrane Extraction
Cellular membranes were extracted by a previously described method (11, 24) . Resting NHLF and HMC-1 cells were collected separately and suspended in 1 ml of TKM hypotonic buffer (Tris-HCL 50 mM [pH 7.5], KCl 25 mM, MgCl2 5 mM), incubated in a TKM-phenylmethylsulfonyl fluoride (PMSF) solution for 20 min on ice, and dounce homogenized. In addition to dounce homogenization, cell membranes were broken with ultrasound on a micro-ultrasonic cell disrupter (Kontes, Vineland, NJ). Cell membranes were separated by high-speed centrifugation (120,000 x g at 10°C) in a 73% and 35% STKM sucrose solution. After 1 h, the 35/73% interface was removed, placed in a new tube, and suspended in 1x TKM-PMSF. Finally, membranes were centrifuged at 10°C at 45,000 x g for 30 min, resuspended in 100 µl phosphate-buffered saline (PBS), rinsed again in 50 µl PBS, protein content measured with bicinchonic acid assay by Pierce (Pierce Chemical, Rockford, IL), and stored at -80°C. NHLF and HMC-1 membranes were added to HMC-1 and NHLF cultures, respectively, at 75 and 100 µg/ml. Because resting cell membranes produced significant increases in IL-6 production, activated cellular membranes were not used. One million HMC-1 yields ~ 100 µg of cellular derived membranes.

Extraction of Nuclear Proteins
Nuclear proteins were extracted from NHLF by a method previously described, with modifications (11, 25). NHLF were trypsinized from 100 x 20 mm plates at 2.0 x 106 cells/plate, washed three times in PBS, and collected in a 1.5-ml microcentrifuge tube. Added to this was 100 µl ice-cold hypotonic buffer: 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM PMSF, 1 µM aprotinin, 1 µM pepstatin, 14 µM leupeptin, 50 mM NaF, 30 mM ß-glycerophosphate, 1 mM Na3VO4, and 20 mM p-nitrophenyl phosphate. Cells were incubated on ice for 30 min and vortexed after addition of 6.25 µl of 10% Nonidet P-40. After 2 min of centrifugation at 30,000 x g, supernatants were kept at -80°C while the pellets were collected and vortexed every 20 min for 3 h in 60 µl of a hypertonic salt solution: 20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 12 mM DTT, 1 mM PMSF, 1 µM aprotinin, 1 µM pepstatin, 14 µM leupeptin, 50 mM NaF, 30 mM ß-glycerophosphate, 1 mM Na3VO4, and 20 mM p-nitrophenyl phosphate. This solution, containing nuclear proteins, was assayed for total protein concentration by the bicinchonic acid protein assay reagent.

Electrophoretic Mobility Shift Assay
Nuclear translocation of NF-{kappa}B was analyzed by the electrophoretic mobility shift assay (EMSA). Briefly, 7 µg of nuclear protein were added to 2 µl of 10x binding buffer (50 µg/ml double-stranded poly dI-dC, 10 mM Tris HCl pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 1 mM MgCl2, and 10% glycerol), and 35 fmol of double-stranded NF-{kappa}B consensus oligonucleotides end labeled with {gamma}-P32 ATP. The reaction mixture was incubated at room temperature for 20 min and analyzed by eletrophoresis on a 5% nondenaturing polyacrylamide gel. The gel was then dried on a Gel-Drier (Bio-Rad Laboratories, Hercules, CA) and exposed to Kodak X-ray film at -80°C. Cold competition studies were done with 7 µg of nuclear protein from NHLF/HMC-1 cocultures. Seven micrograms of the nuclear protein were aliquoted into three microfuge tubes, and hot NF-{kappa}B, cold NF-{kappa}B, and cold AP-2 consensus oligonucleotides were added. After 10 min of incubation at room temperature, hot NF-{kappa}B was added to the tubes with cold NF-{kappa}B and AP-2. The samples were incubated at room temperature for another 10 min before being run on a 5% nondenaturing polyacrylamide gel.

Western Blot Analysis
Cytoplasmic extracts in hypotonic buffer extracted from cocultures at 90 min were used to analyze phosphorylated p38 MAPK expression by Western blot. Briefly, 10 µg of sample was diluted 1:2 with Laemmli buffer (Bio-Rad) and boiled for 5 min in a sand bath. The resultant sample was then run in a Bio-Rad Modular Mini Electrophoresis System on a 10% polyacrylamide gel for 1 h and then transferred to a 0.2 µm nitrocellulose membrane (Bio-Rad) for 1 h. The blot was then removed and incubated in blocking buffer (1% bovine serum albumin, 10 mM Tris pH 7.4, 100 mM NaCl, and 0.1% Tween) for 1 h at 25°C with gentle agitation. Phospho-p38 MAPK (Thr180/Tyr182) rabbit anti-human polyclonal antibody (Calbiochem) was diluted 1:1,000 in blocking buffer and incubated on the blot overnight at 4°C with gentle agitation. The next day, the primary antibody was removed and the blot was washed every 10 min for 30 min with agitation in wash buffer (10 mM Tris pH 7.4, 100 mM NaCl, and 0.1% Tween). After this, the blot was incubated in horseradish peroxidase conjugate antibody (mouse/human adsorbed anti-rabbit; Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:5,000 in blocking buffer. The blot remained in the secondary antibody for 1 h at 25°C. The blot was then washed with wash buffer for 30 min and covered with Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) for 5 min. Fisher brand polyvinyl chloride wrap (Fisher Scientific, Atlanta, GA) was used to cover the blot before exposing it to acetate transparency film (Kodak, Rochester, NY). The blot was exposed for 60 s before being developed. Nonphosphorylated p38 MAPK (Abcam, Cambridge, UK) was used as a loading control.

Statistical Analysis
All experiments were done in triplicate. All values are given as the mean ± SD. Statistical analysis was done using the Student's t test and Statistica version 5 computer software (StatSoft, Inc Tulsa, OK). A P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NHLF/HMC-1 Cocultures Produce Significant Amounts of IL-6
Coculturing of NHLF with HMC-1 led to a significant increase in IL-6 production. As detected by ELISA in 24 h cell-free supernatants, IL-6 levels in NHLF and HMC-1 controls was 5.05 ± 0.410 pg/ml and 0.402 ± 0.16 pg/ml, respectively. IL-6 levels in NHLF plus HMC-1 (1 x 106 HMC-1/ml) cocultures was 309.5 ± 23.2 pg/ml (P < 0.00003 compared with both controls) (Figure 1A). NHLF/HMC-1 cocultures also demonstrated a dose-dependent response dependent on the number of HMC-1 cultured with a constant number of NHLF (Figure 1B). NHLF control produced 33.37 ± 4.61 pg/ml of IL-6, and HMC-1 control produced 0.640 ± 0.271 pg/ml. IL-6 levels of NHLF/HMC-1 cocultures were 44.77 ± 6.27 pg/ml for 0.1 x 106 HMC-1 cells/ml (A), 67.90 ± 2.78 pg/ml for 0.25 x 106 HMC-1 cells/ml (B) (P < 0.0004 compared with NHLF and HMC-1 control), 102.92 ± 28.0 pg/ml for 0.5 x 106 HMC-1 cells/ml (C) (P < 0.02 compared with NHLF and HMC-1 control), 299.25 ± 16.40 pg/ml for 1 x 106 HMC-1 cells/ml (D) (P < 0.0005 compared with NHLF and HMC-1 control), 326.21 ± 12.97 pg/ml for 2 x 106 HMC-1 cells/ml (E) (P < 0.0005 compared with NHLF and HMC-1 control), and 349.20 ± 47.0 pg/ml IL-6 for 3 x 106 HMC-1 cells/ml (F) (P < 0.0005 compared with NHLF and HMC-1 control). NHLF/HMC-1 cocultures release IL-6 protein in a time-dependent manner over 72 h (Figure 1C). NHLF were cocultured with HMC-1 at 1 x 106 HMC-1/ml and harvested at 12, 24, 48, and 72 h for assay by ELISA. NHLF alone produced small amounts of IL-6 over the time course: 30.26 ± 0.21 pg/ml at 12 h, 50.28 ± 2.30 pg/ml at 24 h, 44.89 ± 0.24 pg/ml at 48 h, and 98.75 ± 2.82 pg/ml at 72 h. HMC-1 produced very small amounts of IL-6: 0.355 ± 0.50 pg/ml at 12 h, 0.695 ± 0.86 pg/ml at 24 h, 1.56 ± 0.56 pg/ml at 48 h, and 0.981 ± 1.39 pg/ml at 72 h. When HMC-1 were cocultured with NHLF, however, IL-6 production significantly rose in as little as 12 h and began to plateau at 48–72 h (205.6 ± 28.6 pg/ml at 12 h [P < 0.001 compared with both controls at 12 h], 234.42 ± 23.8 pg/ml at 24 h [P < 0.001 compared with both controls at 24 h], 309.2 ± 3.37 pg/ml at 48 h [P < 0.0001 compared with both controls at 48 h], and 310.3 ± 25.4 pg/ml at 72 h[P < 0.0001 compared with both controls at 72 h]). We chose 24 h for convenience and based on the findings of significant IL-6 induction at this time point.





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  		Figure 1. (A) NHLF/HMC-1 cocultures induced a significant rise in IL-6 levels. Cell coculture supernatants were harvested after 24 h and assayed for IL-6 by ELISA. *P < 0.00003 compared with both controls. (B) Increasing numbers of HMC-1 in NHLF/HMC-1 cocultures exhibited a dose-dependent response in the amount of IL-6 produced. HMC-1 at A = 0.1 x 106 HMC-1/ml, B = 0.25 x 106 HMC-1/ml, C = 0.5 x 106 HMC-1/ml, D = 1 x 106 HMC-1/ml, E = 2 x 106 HMC-1/ml, F = 3 x 106 HMC-1/ml were cultured with NHLF for 24 h and harvested for IL-6 by ELISA. *P < 0.0004, **P < 0.02, and ***P < 0.0005 compared with NHLF and HMC-1 controls. (C) Kinetics of IL-6 production in NHLF/HMC-1 cocultures. NHLF were cocultured with HMC-1 (1 x 106 cells/ml) for 12, 24, 48, and 72 h and assayed for IL-6 production by ELISA. *P < 0.001 compared with both controls at their respective time point; **P < 0.0001 compared with both controls at their respective time point. Values are means ± SD for triplicate samples, n = 3.

 
Cell–Cell Contact Is Essential for IL-6 Production
Coculture of NHLF with HMC-1 produced increased amounts of IL-6. To investigate whether direct cell-to-cell contact is needed for this activation, NHLF and HMC-1 were incubated in the same well but separated from contact by a porous membrane. When the NHLF and HMC-1 were separated by a 0.4-µm porous membrane, IL-6 production was decreased to 64.2 ± 10.9 pg/ml as compared with that of the coculture (293.9 ± 9.3 pg/ml, P < 0.00001) (Figure 2A).





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  		Figure 2. (A) Separation of NHLF and HMC-1 by a porous membrane decreased IL-6 production. NHLF were cultured with HMC-1 in direct contact and separated by a 0.4 µm porous membrane for 24 h and then harvested for IL-6 by ELISA. *P < 0.00001 compared with both controls and **P < 0.00001 compared with coculture. (B) HMC-1–derived cellular membranes induced IL-6 production in NHLF in a dose-dependent manner. HMC-1 membranes at 75 and 100 µg/ml induced a dose-dependent rise in IL-6 production as detected by ELISA in 24 h supernatants. *P < 0.000003 compared with NHLF and HMC-1 controls, **P < 0.00004 compared with NHLF control. (C) Coculture-conditioned and HMC-1–conditioned media do not further activate NHLF to produce IL-6. Cocultures and HMC-1 were incubated for 24 h in NHLF media and then centrifuged and added to pure NHLF cultures. HMC-1–conditioned media was added at 1:10 and 1:100 dilutions. *P < 0.001 compared with NHLF and HMC-1 control, **P < 0.0005 compared with NHLF + HMC-1. Values are means ± SD for triplicate samples, n = 3.

 
To test which cell is the main producer of this IL-6, cellular derived membranes from resting NHLF and HMC-1 were prepared and incubated in HMC-1 and NHLF cultures, respectively. NHLF cultured alone produced 25.7 ± 4.6 pg.ml of IL-6, whereas HMC-1 alone produced no IL-6 (0.00 ± 0.00 pg/ml). When HMC-1 membranes were added to NHLF cultures at 75 and 100 µg/ml, IL-6 production was increased to 58.6 ± 21.3 pg/ml and 151.7 ± 9.8 pg/ml (P < 0.00004), respectively (Figure 2B). However, when NHLF-derived membranes were added to HMC-1 cultures, IL-6 production did not increase over control levels (data not shown).

To completely remove HMC-1 cellular influence on NHLF cultures, HMC-1–conditioned media at two dilutions was added to NHLF cultures. Coculture-conditioned media was also added to NHLF cultures to test for additional effects on IL-6 production (Figure 2C). HMC-1 conditioned media at 1:10 and 1:100 dilution added to NHLF cultures failed to significantly increase IL-6 production over NHLF control levels (33.3 ± 2.7 and 24.0 ± 0.3 pg/ml for 1:10 and 1:100 dilutions, respectively, compared with 27.1 ± 3.6 for NHLF control). IL-6 production in NHLF treated with HMC-1–conditioned media was still significantly less than whole cell NHLF/HMC-1 cocultures, however (P < 0.0005). Coculture-conditioned media had no additional effects on IL-6 production in NHLF (224.2 ± 6.3 pg/ml for NHLF + coculture conditioned media and 222.9 ± 2.0 pg/ml for NHLF + HMC-1).

IL-6 Gene Expression and NF-{kappa}B Are Increased in Cocultures
NHLF and HMC-1 were incubated separately and together for 6 h and then harvested for RNA. Analysis of IL-6 gene transcripts by RT-PCR was then performed. Hypoxanthine phophoribosyltransferase (HPRT), an enzyme important for purine synthesis and nervous system function, was used as a housekeeping gene. After 6 h, IL-6 gene transcription was increased markedly in NHLF/HMC-1 cocultures as compared with NHLF or HMC-1 alone (Figure 3). Studies also showed NF-{kappa}B activation after 2 h of coculture (Figure 4A). Cold competition studies were done to determine the specificity of the NF-{kappa}B binding. Here, the NF-{kappa}B band from the NHLF/HMC-1 cocultures disappears when preincubated with cold NF-{kappa}B before hot NF-{kappa}B is added. Samples were also preincubated with cold AP-2, a nonspecific nuclear binding protein, before hot NF-{kappa}B was added. This band did not disappear, indicating that nuclear binding is specific for our NF-{kappa}B oligonucleotide (Figure 4B).



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  		Figure 3. NHLF/HMC-1 cocultures increased IL-6 gene expression. NHLF and HMC-1 were cultured separate and together for 6 h and harvested for analysis by RT-PCR. HPRT was used as a housekeeping gene to ensure equal loading.

 


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  		Figure 4. (A) NHLF/HMC-1 cocultures increase activation of NF-{kappa}B. NHLF and HMC-1 were incubated separately and together for 2 h and assayed for NF-{kappa}B translocation by EMSA. (B) Cold competition for NF-{kappa}B oligonucleotide. Nuclear extracts from NHLF/HMC-1 cocultures were labeled with hot NF-{kappa}B oligo, cold NF-{kappa}B oligo followed by hot NF-{kappa}B oligo, and cold AP-2 oligo followed by hot NF-{kappa}B oligo. Preincubation with cold NF-{kappa}B oligo before hot oligo was added eliminated the NF-{kappa}B band. Preincubation with cold AP-2 oligo before hot NF-{kappa}B oligo was added did not eliminate the NF-{kappa}B band. These results show specificity of the NF-{kappa}B oligonucleotide for {kappa}B binding domains in our nuclear extracts.

 
Role of p38 MAPK in Signaling IL-6 Production in Cocultures
Because of its importance in cytokine signaling, phosphorylated p38 MAPK was assayed by Western blot. Neither cell type alone, NHLF nor HMC-1, exhibited p38 MAPK activation. When the two cell types were incubated together, however, the presence of phosphorylated p38 MAPK was detectable (Figure 5A). Unphosphorylated (nonactive) p38 MAPK was used as a loading control. To test the functionality of this p38, a specific inhibitor of p38 MAPK activation, SB203580, was used to test the effects on IL-6 protein production in cocultures. After pretreatment for 2 h with SB203580, IL-6 production in NHLF/HMC-1 coculture was decreased from 299.3 ± 16.4 pg/ml to 33.0 ± 9.1 pg/ml (P < 0.00002). SB203580 also had an inhibitory effect on IL-6 production in NHLF control (33.4 ± 4.6 pg/ml without the inhibitor versus 7.6 ± 0.9 pg/ml with the inhibitor (Figure 5B).




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  		Figure 5. (A) Western blot was used to detect the presence of phosphorylated p38 MAPK. Coculturing of NHLF and HMC-1 induced phosphorylation of p38 in NHLF/HMC-1 cocultures. Nonactive, unphosphorylated p38 was used to ensure equal loading. (B) SB203580 decreased IL-6 production from NHLF–HMC-1 cocultures. NHLF were pretreated for 2 h with SB203580 before being incubated with HMC-1. Cell supernatants were harvested after 24 h and assayed for IL-6 levels by ELISA. *P < 0.00002 compared with NHLF and HMC-1 controls, **P < 0.0007 compared with NHLF control, ***P < 0.00002 compared with NHLF/HMC-1 coculture. Values are means ± SD for triplicate samples, n = 3.

 
Role of NF-{kappa}B Activation in IL-6 Production from Cocultures
To examine the role NF-{kappa}B plays in inducing IL-6 protein production from NHLF/HMC-1 cocultures, we inhibited NF-{kappa}B with a chemical inhibitor, Bay 11. Preincubation of NHLF with Bay 11 (1 µM) for 1 h inhibited IL-6 production in NHLF/HMC-1 cocultures by 17%. Higher concentrations of Bay 11 had toxic effects on NHLF. NHLF and HMC-1 alone produced little IL-6 (31.5 ± 4.6 pg/ml and 0.604 ± 0.37 pg/ml, respectively), whereas NHLF + HMC-1 produced significantly higher levels of IL-6 (290.0 ± 9.7 pg/ml, P < 0.00002 compared with both controls). Bay 11 inhibited IL-6 production in NHLF control and NHLF/HMC-1 cocultures. NHLF control levels of IL-6 went from 31.5 ± 4.6 pg/ml to 11.7 ± 1.3 pg/ml with Bay 11 (P < 0.002), whereas that of NHLF/HMC-1 cocultures went from 290.0 ± 9.7 pg/ml to 241.3 ± 1.4 pg/ml with Bay 11 (P < 0.02) (Figure 6). These data indicate that IL-6 production in NHLF/HMC-1 cocultures may be only partially mediated by the NF-{kappa}B pathway.



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  		Figure 6. Bay 11, an inhibitor of NF-{kappa}B, decreased IL-6 production in NHLF/HMC-1 cocultures. Bay 11 with 1 µM added to NHLF 1 h before addition of HMC-1 was able to minimally but significantly decrease the amount of IL-6 detected in 24-h supernatants per ELISA. *P < 0.00002 compared with both controls, **P < 0.002 compared with NHLF control, ***P < 0.02 compared with NHLF/HMC-1 coculture. Values are means ± SD for triplicate samples, n = 3.

 
Dexamethasone Inhibits IL-6 Production
Dexamethasone, a drug widely used in the treatment of asthma, was also included to investigate the inhibition of IL-6 production. Synthetic glucocorticoids, like dexamethasone, work by ultimately stabilizing the NF-{kappa}B/I{kappa}B complex by inducing I{kappa}B gene transcription. They may also function through various metabolic pathways not yet understood. Preincubation of NHLF cultures with dexamethasone (1 µM) for 48 h before HMC-1 were added significantly reduced IL-6 production. NHLF alone produced 116.1 ± 11.8 pg/ml of IL-6 and HMC-1 alone produced 1.5 ± 0.2 pg/ml of IL-6. Dexamethasone decreased IL-6 production in NHLF/HMC-1 cocultures from 483.0 ± 8.1 pg/ml to 395.3 ± 22.3 pg/ml (P < 0.05 compared with NHLF + HMC-1) (Figure 7). Dexamethasone treatment of NHLF alone reduced IL-6 production, but this was not significant from NHLF control.



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  		Figure 7. Dexamethasone decreased IL-6 production in NHLF/HMC-1 cocultures. Dexamethasone, a synthetic glucocorticoid, added to NHLF cultures 48 h before addition of HMC-1 decreased IL-6 production. Supernatants were harvested after 24 h and assayed for IL-6 by ELISA. *P < 0.0008 compared with NHLF and HMC-1 controls **P < 0.05 compared with NHLF/HMC-1 coculture. Values are means ± SD for triplicate samples, n = 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inflammation in the airway is orchestrated by a number of different cytokines working together and separately. These cytokines are produced by resident cells of the lungs as well as infiltrating inflammatory cells. The exact mechanisms by which these cytokines work and how they are orchestrated by the host cells of the lung are not completely understood. We do know, however, that cell-to-cell contact between lung cells and the infiltrating cells of the immune system plays a major role in the pathogenesis of inflammation and airway remodeling, the irreversible structural damage caused by repeated inflammatory insult. Among these infiltrating cells are macrophages, eosinophils, and mast cells, all of which are seen in increased numbers in the asthmatic lung (26). As we have previously shown, macrophage–fibroblast interactions lead to increased granulocyte macrophage-colony stimulating factor (GM-CSF) gene expression and protein production (11). GM-CSF is a potent activator of eosinophils.

Here we have shown that mast cell–fibroblast interactions produce significantly higher amounts of IL-6 than control cells. IL-6 is a mediator of many inflammatory processes of the human body, including B-cell maturation and differentiation as well as acute phase protein production and hematopoietic effects on stem cells (12, 13). Of interest to the lung, IL-6 can cause class switching of B cells into IgE-producing plasma cells. By cross-linking high-affinity IgE receptors (Fc{epsilon}R1) on mast cells, IgE attached to antigen can cause a cascade of inflammatory events in the bronchi. Mast cells produce preformed and newly synthesized mediators that are ready to be released within minutes upon activation. These mediators include cytokines, chemokines, leukotrienes, and proteases.

We have found that the coculturing of normal human lung fibroblasts with a leukemic mast cell line, HMC-1, increased IL-6 secretion into the supernatant after 24 h (Figure 1A). The IL-6 production in cocultures was positively correlated to the number of HMC-1 in NHLF/HMC-1 cocultures and began to plateau at 1 x 106 HMC-1/ml (Figure 1B). A time-dependent release of IL-6 was also seen over 72 h in our study. Twelve-hour incubation supernatants from NHLF/HMC-1 cocultures had significant amounts of IL-6 compared with NHLF and HMC-1 alone. This increase of IL-6 production progressed and began to plateau at 48–72 h (Figure 1C).

To investigate the role of direct cell-to-cell contact in our system, we separated the two cell types by seeding the fibroblasts in the bottom of a six-well plate and then added the HMC-1 separated by a 0.4-µm porous membrane. When the two cell types were separated from direct contact, the amount of IL-6 detected in the supernatant after 24 h was significantly less than for that of direct cell–cell contact (Figure 2A). The amount of IL-6 in the separated coculture was still significantly greater than NHLF or HMC-1 control levels. This does not completely rule out the role of soluble mediator production in the activation of IL-6 in our model, but it does show the importance of direct cell-to-cell contact for IL-6 production. To determine the cell type responsible for the bulk of the IL-6 production, membranes from resting NHLF and HMC-1 were extracted and incubated with whole HMC-1 and NHLF, respectively. Although NHLF-derived membranes did not activate HMC-1 to produce IL-6, HMC-1–derived membranes had a dose-dependent effect on NHLF IL-6 production (Figure 2B). To completely remove HMC-1 cellular influence from NHLF, HMC-1–conditioned media at two dilutions (1:10 and 1:100) was added to pure NHLF cultures. Neither dilution of HMC-1–conditioned media caused a significant rise in IL-6 production from NHLF (Figure 2C). NHLF/HMC-1 coculture-conditioned media was also added to pure NHLF cultures to test additional effects on IL-6 production. IL-6 production with coculture conditioned media was found not to be significantly different from actual NHLF/HMC-1 cocultures (Figure 2C).

We also looked at transcription of the IL-6 gene by RT-PCR. RNA was extracted from cocultures at 6 h and subjected to RT-PCR analysis. Coculturing of NHLF with HMC-1 induced increased expression of the IL-6 mRNA (Figure 3). Activation of NF-{kappa}B was also assayed in our coculture. Translocation of activated NF-{kappa}B to the nucleus of NHLF cocultured with HMC-1 was increased after 2 h of coculturing (Figure 4A). Cold competition studies with NF-{kappa}B were also done to show specificity of NF-{kappa}B binding (Figure 4B).

For its significance in cytokine signaling, p38 MAPK activity was assayed by Western blot. Phosphorylated p38, the activated form of p38 MAPK, was induced in our coculture and was inhibited by a specific inhibitor, SB203580 (Figure 5A). Sano and coworkers have previously shown that p38 and extracellular signal–regulated kinase are involved in angiotensin II–mediated IL-6 production in cardiac fibroblasts, and its induction was independent of NF-{kappa}B (27). Others have also shown the activation of p38 MAPK in the induction of IL-6 in fibroblasts or fibroblast-like synoviocytes (9, 28–30). In our model, pretreatment of NHLF with SB203580 for 2 h decreased IL-6 production to prestimulus levels, indicating that p38 is a major signaling pathway for coculture-induced IL-6 production (Figure 5B). It is uncertain whether several signals converge on the activation of p38 or if there is one receptor-mediated signal which activates p38 in our coculture. Further studies are needed to identify this pathway and are out of the scope of this paper.

An inhibitor of NF-{kappa}B, Bay 11, was used to block IL-6 production to see how much of the IL-6 signal may come through NF-{kappa}B. ELISA results showed that Bay 11 at 1 µM subtly but significantly reduced IL-6 levels in NHLF/HMC-1 cocultures, indicating that IL-6 transcription may be partially regulated by NF-{kappa}B (Figure 6). Bay 11 works by inhibiting inhibitory {kappa}B (I{kappa}B) phosphorylation. In the cytoplasm, NF-{kappa}B is bound to I{kappa}B, and upon phosphorylation of I{kappa}B by certain signaling pathways, NF-{kappa}B is freed from its complex and is able to translocate to the nucleus, where it initiates transcription of inflammatory genes. These data suggest that NF-{kappa}B may play a minimal role in IL-6 signaling in NHLF/HMC-1 cocultures.

Preliminary experiments looking at blocking IL-6 signaling by using antibodies to specific cell surface molecules like ICAM-1, TNF-RI, and IL-1ß were negative (data not shown). Other protein–protein or receptor–ligand interactions in combination or separately may mediate IL-6 activation. Further experiments are needed to delineate this activation cascade and are the focus of future research in our laboratory. For its clinical significance in the treatment of moderate to severe persistent asthma, dexamethasone was used to investigate inhibition of IL-6 protein production in NHLF/HMC-1 cocultures. Corticosteroids like dexamethasone work by inducing I{kappa}B gene transcription, which will later stabilize the NF-{kappa}B dimer and prevent activation. NHLF pretreated for 48 h with 1 µM dexamethasone was able to significantly inhibit IL-6 protein production as detected in 24-h supernatants (Figure 7). These data parallel the Bay 11 data and further suggest that IL-6 production in NHLF/HMC-1 cocultures is minimally dependent on NF-{kappa}B. Due to the extended preincubation time with dexamethasone, which is needed for its complete effect, baseline IL-6 levels in NHLF were higher than previously seen. Dexamethasone, at concentrations used in this study, had no morphologic or toxic effects on NHLF.

These data give insight into the pathogenesis behind cell–cell mediated inflammation. Contact of resident airway cells with infiltrating inflammatory cells of the immune system may be one way in which activation of the airway occurs. Fibroblasts are known to secrete proinflammatory cytokines including IL-6, IL-8, GM-CSF, and transforming growth factor-ß. Mast cells are known mediators of allergic disease and themselves also secrete a host of vaso- and bronchoactive cytokines. Mast cells have also been found in increased numbers in the asthmatic lung. Knowing that IL-6 induction in fibroblasts by mast cell contact is mediated through p38 MAPK may lead to further drug targets.


   Acknowledgments
 
This study was supported by NIH grants AI-43310 and HL-63070, the Rondal Cole Foundation, and the Chair of Excellence in Medicine (State of Tennessee Grant 20233), the Cardiovascular Research Institute, and the Research Development Committee, East Tennessee State University.

Received in original form July 28, 2003

Received in final form October 8, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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