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Published ahead of print on August 1, 2008, doi:10.1165/rcmb.2007-0348OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 40, pp. 231-238, 2009
© 2009 American Thoracic Society
DOI: 10.1165/rcmb.2007-0348OC

Thy1 Up-Regulates FasL Expression in Lung Myofibroblasts via Src Family Kinases

Pazit Y. Cohen1, Raphael Breuer1,2 and Shulamit B. Wallach-Dayan1

1 Lung Cellular and Molecular Biology Laboratory, Institute of Pulmonary Medicine, Hadassah–Hebrew University Medical Center, Jerusalem, Israel; and 2 Department of Pathology, Boston University School of Medicine, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Raphael Breuer, M.D., Head, Institute for Pulmonary Medicine, Hadassah–Hebrew University Medical Center, POB 12000, Jerusalem, Israel 91120. E-mail: raffi{at}hadassah.org.il


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We have previously demonstrated that myofibroblasts from lungs with bleomycin-induced fibrosis overexpress FasL molecules. Two subpopulations of fibroblasts, distinguished by their expression of Thy1 molecules, have been shown in the lungs of both mice and humans. Thy1-mediated FasL induction has been reported in T cells through the use of anti-Thy1 (G7). We therefore sought to determine whether FasL expression in lung myofibroblasts is associated with and/or dependent on Thy1 expression, and to examine the underlying mechanism of regulation. We show that myofibroblast Thy1 expression is associated with increased FasL expression. Moreover, we directly show that Thy1 activation induces FasL up-regulation. Initially, Thy1 activation causes translocation of FasL to the membrane surface, and later induces de novo synthesis of FasL at the mRNA and protein levels. In contrast to Thy1 activation, TNF-{alpha} and IFN-{gamma} do not induce FasL myofibroblast up-regulation. Using Src family kinase (SFKs) inhibitor (PP2), we show the general involvement of SFKs in Thy1 signal transduction leading to FasL up-regulation; and, using specific siRNA, we show the particular involvement of Fyn, one protein in the SFK family. These results demonstrate that Thy1 in myofibroblasts is not just a marker, but is a functional protein that transmits signals into the cell, up-regulating its FasL expression.

Key Words: FasL • myofibroblast • Src • Thy-1



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We show that Thy1 in lung myofibroblasts is not only a cell marker, but also a functional protein that transmits signals into the cell, up-regulating the cell's FasL expression—a critical step in the development and persistence of lung fibrosis.

 
FasL is a cell surface molecule belonging to the TNF family (1), whose main function is to induce apoptosis through binding to the Fas receptor (2). The Fas/FasL interaction plays a role in the pathophysiology of several diseases, including idiopathic pulmonary fibrosis (IPF) (3). FasL is predominantly induced in immune cells during activation of the immune system (4, 5), but was also shown to be constitutively expressed on nonimmune cells, including immune-privileged tissues (6), and tumors (7). In fibroblasts, FasL up-regulation has been demonstrated in experimental murine bleomycin-induced scleroderma in the skin (8), and in skin allografts (9).

Because FasL is such a potent apoptosis mediator, surface expression of FasL must be tightly regulated. Studies show that FasL expression is regulated at several levels: transcriptional (6, 10) and post-translational (e.g., cleavage of surface FasL by metalloprotease) (11, 12). Several stimuli, such as retinoic acid (9), irradiation (13), and cytokines, induce FasL expression (14, 15). In T cells, FasL is also known to be up-regulated by cross-linking of TCR or Thy1 surface protein (16).

Thy1 is a 25- to 37-kD glycosylphosphatidylinositol (GPI)-anchored cell surface protein that belongs to the immunoglobulin-like gene super family (17). Thy1 is involved in T cell activation of both immunologic (18), and nonimmunologic functions (19). To mediate these effects, Thy1 signals through multiple pathways, including Src family kinases (SFKs) (18, 20). It has been shown that Thy1 associates with SFK members fyn, lyn, and lck (21, 22), which were shown to mediate FasL up-regulation by various stimuli, such as TCR (23) or irradiation (13).

Thy1 is expressed on various cell types, including fibroblasts (18). In the lungs of mice and humans, there are two subpopulations of fibroblasts, which are distinguished by the expression of Thy1 (24, 25). Thy1+ and Thy1– fibroblasts differ with respect to size and shape (25), expression of and response to cytokine and growth factors (19, 26, 27), resistance to apoptosis in response to collagen contraction (27), and expression of {alpha}–smooth muscle actin ({alpha}-SMA) (19, 26, 27), as well as cellular migration (28).

We have recently demonstrated that myofibroblasts from bleomycin-treated mice overexpress FasL molecule, and act as effector cells that induce apoptosis in Fas+ epithelial cells (29) and lymphocytes (30) via Fas/FasL interaction (29). Moreover, we have demonstrated that FasL expression on lung myofibroblasts facilitates emergence of a phenotype of escape from in vivo immune surveillance as a mechanism for tissue fibrosis (30).

In this study we aimed to detect differences in FasL expression in the two myofibroblast subsets, as well as to study whether Thy1 has a role in the regulation of FasL expression in lung myofibroblasts, and by which mechanism.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animals
Eleven- to twelve-week-old, male, C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) and C57BL/6J Thy1-deficient mice were used (Provided by Dr. R.J. Morris, Nosten-Bertrand, M1996, Mayeux-Portas V 2000). All animal procedures were approved by the Hebrew University–Hadassah Medical School Animal Care Committee. Mice were housed in a specific pathogen–free environment.

Intratracheal Instillation
The intratracheal instillation (IT) techniques used in this analysis have been previously described by our lab (29, 30). Briefly, mice were anesthetized by injection of 0.05 to 0.07 ml of 40 mg/ml Ketalar intraperitoneally (Parke-Davis, Pontypool, Gwent, UK) and 0.5 mg/ml Droperidol (Janssen Pharmaceutica, Beerse, Belgium). The skin and subcutaneous tissues overlying the proximal portion of the trachea were exposed by a 5-mm transverse incision to allow for direct external visualization of the trachea. A metal cannula fitted to a tuberculin syringe was carefully passed into the trachea. A dose of 0.06 to 0.08 units of bleomycin (H. Lundbeck, Copenhagen, Denmark) dissolved in 0.1 ml of saline solution, or 0.1 ml of saline alone, was slowly injected. For confirmation of fibrosis, we performed assays of the extent of inflammation in bronchoalveolar fluid, and histopathology, as we have previously described in detail (30).

Lung Cell Isolation and Myofibroblast Culture
Techniques used in this research have been previously described by our lab (29, 30). Briefly, mice were killed with a lethal dose of pentobarbital. Lungs were removed, minced, and incubated (37°C, 5% CO2 air) for 45 minutes in PBS containing 1 mg/ml collagenase (C0130; Sigma-Aldrich, St. Louis, MO). After enzyme treatment, lung tissue was gently passed through a cell dissociation sieve (Sigma-Aldrich), then washed twice in PBS. For myofibroblast culture experiments, lung cells (LC) were resuspended in fibroblast culture medium (RPMI 1640; Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS), 100 µg/ml penicillin, 100 mg/ml streptomycin, 25 mM HEPES buffer, 2 mM L-glutamine, 50 µg/ml gentamicin sulfate, nonessential amino acids (Biological Industries, Beit Haemek, Israel), and 5 x l0–5 M 2-mercaptoethanol (Sigma-Aldrich). Cell cultures were incubated at 37°C in 5% humidified CO2. Typically, within 1 week of culture initiation, more than 95% of the cells are morphologically myofibroblasts.

Cells were passaged every 5 days by dissociating monolayers with a mild trypsin solution (Biological Industries). Fibroblasts obtained on passages 2 through 6, after initial cultures were established, were used.

Myofibroblast Thy1 and Cytokine Stimulation for Analysis by Flow Cytometry, Real-Time PCR, Western Blot, and Immunofluorescence
Subconfluent myofibroblasts were stimulated with anti-Thy1 G7, which have previously been shown to activate T cells (16), or with anti-Rat IgG2C{kappa} isotype control (PharMingen, San Diego, CA). Both stimulants were added to the myofibroblasts at differing concentrations ranging from 1 to 20 µg/ml, together with recombinant Protein G crosslinker (Sigma) at the same concentration. In other experiments, subconfluent myofibroblasts were stimulated with TNF-{alpha} (50–500 ng/ml) or IFN-{gamma} (50–200 µ/ml) (R&D Systems, Minneapolis MN) for various time periods (30 min, 1 h, 24 h, 48 h, 72 h).

RNA Isolation
Total cellular RNA was isolated from myofibroblasts in culture, using TriReagent (T9424; Sigma), according to the protocol supplied by the manufacturer. To assess RNA integrity and exclude DNA contamination, an aliquot of each sample was analyzed by 1% agarose gel electrophoresis. Purity and quantitation of RNA was assessed by spectrophotometer.

RT-PCR
RNA was reverse-transcribed to cDNA using an avian myeloblastosis virus-RT base protocol and random primers, as well as poly (dT) (Reverse Transcription System; Promega, Madison, WI). One microgram of each sample was uniformly used for reverse transcription. The cDNA was diluted in a final volume of 200 µl with nuclease-free water.

Real-Time PCR
For TaqMan real-time PCR, FasL, 18S primers, and a TaqMan probe were purchased from Applied BioSystems (Foster City, CA). The probe sequences of the genes analyzed were: FasL, 5'-TGGCCCATTTAACAGGGAACCCCCA-3'; 18 s, 5'-ATTGGAGGGCAAGTCTGGTGCCAGC-3'.

The real time PCR reaction mixture contained 9 µl of the sample and 10 µl of 2x Taqman universal PCR master mix (Applied BioSystems), 1 µl of 20x mix of unlabeled PCR primers, and a fluogenic probe (5'FAM-dye labeled). An Applied Biosystems Prism 7000 Sequence Detection System was used with the default thermal cycling program (95°C for 10 min followed by 40 cycles of 95°C, 20 s, 60°C, 1 min). Reactions were performed in triplicate. The relative quantification method was used with {Delta}Ct calculated as Ct (target gene) – Ct (18 s gene). The relative quantity of the product was expressed according to the formula 2–{Delta}Ct.

Immunoblot Analysis
Cell samples were homogenized in RIPA buffer (1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS in Tris-buffered saline), including a protease inhibitor cocktail (Sigma-Aldrich) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Alternatively, for detection of phosphorylation, we used NP-40 lysis buffer (1% NP-40, 20 mM Tris pH 7.5, 150 mM Nacl, 0.1% NaN3), including a protease inhibitor cocktail, as well as Na vanadate (Sigma-Aldrich) and PMSF (Sigma-Aldrich).

Lysates were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. After electrophoresis, samples were transferred to a nitrocellulose membrane (Bio-Rad, Hercules CA). The membranes were blocked with 5% dry milk in Tris-buffered saline/Tween-20 (0.1%) (TBS-T) for 1 hour at room temperature, and washed with TBS-T. They were then incubated with polyclonal rabbit anti-mouse FasL antibody Q20 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA) in 5% dry milk TBS-T buffer for 1 hour, or with polyclonal rabbit anti-Phospho-Src family (Tyr416) antibody (1:1,000) (Cell Signaling Technology, Danvers, MA) in 5% BSA TBS-T buffer overnight at 4°C. The bolts were washed with TBS-T and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:1,000) (Jackson ImmunoResearch Laboratories, West Grove, PA) in 5% dry milk and TBS-T for 1 hour. Thereafter, blots were developed with the EZ-ECL chemiluminescence detection kit (Biological Industries) according to the manufacturer's instructions. β-actin (1:500) (Santa Cruz Biotechnology) or anti–Src-CT clone NL19 (1:1,000) (Upstate Biotechnology, Lake Placid, NY) was used for loading control.

SFK phosphorylation was determined by immunoprecipitation with polyclonal rabbit antibodies against Fyn, Yes, Lyn, Src, or Lck (Santa Cruz). Primary myofibroblast cultures were stimulated with 10 µg/ml G7 anti Thy1 mAb or control IgG isotype match for 10 minutes, harvested, and lysed in NP-40 lysis buffer, as described above. Supernatants containing equal amounts of protein (100 µg), were incubated for 18 hours at 4°C with 2 µg of the appropriate antibodies, followed by addition 100 µl of immobilized protein A gel slurry (Pierce, Rockford, IL) and further incubation at room temperature for 2 hours to perform immunoprecipitation (IP). Immunoprecipitates were then washed three times with IP buffer (25 mM Tris, 15 0 mM NaCl) pH 7.2, resuspended in 20 µl of sample buffer, heated to 95°C for 5 minutes, and subjected to SDS-PAGE and immunoblot using HRP-conjugated anti-p–Tyr Ab clone 4G10 (2 µg/ml) (Upstate Cell Signaling Solution, Lake Placid, NY), followed by ECL detection, as described above.

The FasL expression and phosphorylation level of SFKs was measured by scanning and quantitated by densitometry using TINA software (Raytest, Staubenhardt, Germany) after background subtraction.

Antibody Array
An antibody array (Hypomatrix, Worcester, MA) containing specific antibodies for detection of a range of signal transduction molecules and kinases was used to study the SFK-specific protein profile of phosphorylation in lung myofibroblasts after activation with anti-Thy1 mAb. Briefly, the antibody array was blocked with 5% dry milk in Tris-buffered saline/Tween-20 (0.1%) (TBS-T) for 1 hour at room temperature. The antibody array was then incubated for 2 hours at room temperature with lysates obtained, as described above, from primary myofibroblast cultures stimulated with 10 µg/ml G7 anti-Thy1 mAb or control IgG isotype match for 10 minutes. The membrane was washed with PBS and blotted with HRP-conjugated anti-Phosphotyrosine RC20 (2 µg/ml) (BD Transduction Laboratories, Franklin Lakes, NJ) for 2 hours at room temperature followed by ECL detection, and the phosphorylation level of SFKs proteins was quantitated. These steps are described above.

Transfection
Small interference RNA (siRNA) against Fyn, or nontargeting siRNA, were purchased from Qiagen (Cat #SI01007398 and 1027281, respectively; Valencia CA). For electroporation, the cells were harvested and washed twice with ice-cold phosphate-buffered saline (PBS) (Mg2+,Ca2+ free) and resuspended in solution R (transfection kit; Digital Bio Technology, Seoul, Korea, cat #MPK-1096). Ten picomoles of siRNA was added to 12 µl of cell suspension containing 0.5 x 106 lung cells. The mixture was then subjected to a single pulse from a micoporator apparatus (Digital Bio Technology). After shocking, the samples were added to growth medium with 10% FCS. FasL protein expression analysis after Thy1 activation was performed 24 hours after siRNA transfection.

Immunofluorescence Staining
Myofibroblasts were cultured on 0.2-mm-thick coverslips and grown to 80% confluence. The cells were stimulated with {alpha}Thy1 (G7) mAb or IgG isotype control for 1 hour (for FasL) and then fixed with cold methanol for 10 minutes at –20°C. Nonspecific binding was blocked by immersing the cells in 10% FBS/PBS at room temperature for 30 minutes. After washing with PBS, the cells reacted with polyclonal rabbit anti-mouse FasL antibody Q20 (1:100; Santa Cruz Biotechnology) for 30 minutes at 37°C, followed by FITC-conjugated anti-Rabbit (Jackson ImmunoResearch Laboratories) for 45 minutes at 4°C. The nuclei were stained with 1 µg/ml propidium iodide (Sigma-Aldrich) for 5 minutes at room temperature. After washing, cells were mounted onto glass slides with 3% DABCO glycerol/PBS solution, and observed through a 410 laser scanning confocal microscope (Carl Zeiss AG, Oberkochen, Germany) attached to a Zeiss Axiovert 135 M inverted microscope.

Flow Cytometry
After stimulation, cells were harvested by rubber policeman. Myofibroblasts (0.5 x 106) were incubated in fluorescence-activated cell sorter (FACS) buffer (PBS, 3% FCS) with 1 µg/ml anti-FasL PE-conjugated antibody MLF3 (PharMingen), or with phycoerythrin (PE)-conjugated 53–1.2 anti Thy1 mAb, for 30 minutes at room temperature. The cells were washed with FACS buffer and analyzed or sorted by flow cytometry (FACStar; Becton Dickinson, Mountain View, CA). For intracellular staining, cells were suspended in SAPONIN buffer (PBS, 1% BSA, 1% Saponin, 1 mM Hepes) for permeability.

Measurement of Nitric Oxide
After cytokine stimulation, nitric oxide (NO) production was monitored by measuring NO2– levels in culture media using the Griess reagent system (Promega, Madison, WI), according to the manufacturer's protocol. Absorbance was measured at 540 nm after incubating the culture media with Griess reagent.

Statistical Analysis
Comparison of FasL mRNA levels between IgG and anti-Thy1 treatment groups was assessed using the nonparametric Wilcoxon rank-sum test.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
The Association between Thy1 and FasL Up-Regulation in Bleomycin-Treated Lung Myofibroblasts
We determined whether FasL up-regulation in lung myofibroblasts is associated with Thy1 expression. FasL expression was analyzed in Thy1+ and Thy1– subpopulations, previously sorted from a total population of myofibroblasts, isolated from the lungs of wild-type (WT) C57BL/6 mice 14 days after intratracheal bleomycin (bleo)- or saline (control)-treatment. Flow cytometry analysis, using PE-conjugated anti-FasL mAb, demonstrated that FasL expression is significantly higher in the Thy1+ compared with the Thy1– myofibroblast subpopulation, in bleomycin-treated compared with control mice (Figure 1).


Figure 1
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  		Figure 1. Association between Thy1 and FasL up-regulation. Lung cells were isolated from bleomycin-treated or control saline-treated mice 14 days after intratracheal instillation (IT). Primary lung myofibroblasts were obtained, and fluorescence-activated cell sorting (FACS) was performed using phycoerythrin (PE)-conjugated anti-Thy1 (CD90) mAb to obtain Thy1– and Thy1+ subsets (see inserts). Flow cytometry analysis using PE-conjugated anti-FasL mAb shows varying distribution of FasL between Thy1 subsets. Results are representative of two separate experiments with similar results.

 
FasL Up-Regulation on Myofibroblasts Is Thy1-Dependent
To test the critical role Thy1 plays on myofibroblast FasL up-regulation, we assessed changes in FasL expression in myofibroblasts isolated from the lungs of bleomycin-treated WT and Thy1-deficient mice. Flow cytometry analysis showed increased FasL expression in myofibroblasts from WT mice compared with those from Thy1-deficient mice (Figure 2).


Figure 2
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  		Figure 2. Dependence of FasL on Thy1 expression. Primary myofibroblasts from bleomycin-treated wild-type and Thy1-deficient mice (Day 14) were cultured and stained using PE-conjugated anti-FasL, and analyzed by FACS. Results are representative of two separate experiments.

 
Thy1 Activation Induces FasL Up-Regulation in Lung Myofibroblasts
To test whether myofibroblast FasL expression is a result of a direct Thy1 activation, primary lung myofibroblasts isolated from WT and Thy1-deficient C57BL/6 mice were stimulated with G7 anti-Thy1 mAb (1–20 µg/ml), which has been previously shown to induce FasL expression in T cells (16). After Thy1 activation, cells were stained with PE-conjugated anti-FasL mAb. Flow cytometry analysis showed that FasL expression was enhanced by Thy1 stimulation in a dose-dependent manner in WT (Figure 3A), but not in Thy1-deficient myofibroblasts (Figure 3B).


Figure 3
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  		Figure 3. Thy1 activation induces FasL overexpression. (A) Primary myofibroblasts from untreated C57BL/6 mice were stimulated with different concentrations of G7 anti-Thy mAb (1–20 µg/ml) with the help of protein G-crosslinker (1–20 µg/ml) for 1 hour. Expression of FasL, as assessed by flow cytometry using PE-conjugated anti-FasL. The percentage of FasL expression exposed to each G7 anti-Thy1 concentration is indicated. (B) Primary myofibroblasts from Thy1-deficient mice were stimulated with G7 anti-Thy mAb (5 µg/ml) with the help of protein G-crosslinker (5 µg/ml) for 1 hour. Expression of FasL, as assessed by flow cytometry using PE-conjugated anti-FasL

 
IFN-{gamma} and TNF-{alpha} Cytokines Do Not Induce FasL Up-Regulation in Lung Myofibroblasts
IFN-{gamma} and TNF-{alpha} have been shown to be the main cytokines inducing FasL expression (14, 15). To assess whether these cytokines play a role in lung myofibroblast FasL expression, primary myofibroblasts isolated from WT and Thy1-deficient naïve mice were stimulated with IFN-{gamma}, TNF-{alpha}, or a combination of IFN-{gamma} and TNF-{alpha}, for 24 hours. Despite nitrite (NO2–) production following myofibroblast activation by these cytokines (Figure 4, insert), no significant increase in FasL expression was detected in WT (Thy1+) (Figure 4) or Thy1-deficient (Thy1–) myofibroblasts (data not shown). Activation for various periods of time—1 hour to 24 hours, or even a 72-hour period—using different cytokine concentrations (IFN-{gamma} 50–200 µ/ml, or TNF-{alpha} 50–500 ng/ml) did not induce FasL up-regulation (data not shown). The RAW macrophage cell line served as a positive control for FasL induction by these cytokines (Figure 4B).


Figure 4
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  		Figure 4. IFN-{gamma} and TNF-{alpha} cytokines do not induce FasL up-regulation in myofibroblasts. (A) Primary myofibroblasts from C57BL/6 mice were stimulated with IFN-{gamma} (100 U/ml), TNF-{alpha} (50 ng/ml), or IFN-{gamma} (100 U/ml) plus TNF-{alpha} (50 ng/ml) for 24 hours. Expression of FasL, as assessed by flow cytometry using PE-conjugated anti-FasL. Insets: nitrite production after cytokine stimulation. (B) Expression of FasL in positive-control macrophage RAW cell line assessed under the same conditions. In each graph, the solid line represents untreated (UNTR) control cells and the dotted line represents cells stimulated with the different cytokines. Results are representative of four separate experiments with similar results.

 
Lung Myofibroblast Thy1 Activation Mediates FasL Translocation to the Cell Surface
Thy1 antibody (5 µg/ml) mediated activation of myofibroblast showed overexpression of cell surface FasL at 10, 20, 30, and 60 minutes. Dose–response data at 60 minutes is presented (Figure 3). To assess whether this rapid Thy1-induced FasL overexpression on the cell membrane results from a mechanism of translocation of cytosolic FasL from the cytosol to the surface cell membrane, rather than from the up-regulation of gene transcription or protein synthesis (see Figure 6), cellular FasL distribution was studied 1 hour after anti-Thy1 stimulation. More intense cell membrane staining was detected in Thy1-activated myofibroblasts compared with IgG controls (Figure 5A). Similarly, FACS analysis of Thy1-activated myofibroblasts showed increased staining of membranal FasL, with no difference in total (membranous and cytoplasmatic) FasL staining (Figure 5B).


Figure 5
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  		Figure 5. Thy1 initially induces FasL translocation to the cell membrane surface. (A) Primary myofibroblasts were stimulated with G7 anti-Thy1 mAb (5 µg/ml) or control IgG isotype match for 1 hour. Myofibroblasts were fixed and stained with anti-FasL mAb, and subjected to confocal microscopy analysis. (B) Flow cytometry analysis of FasL staining on cell membranes (upper panels), and for both cell membrane and cytoplasm (lower panels), using permeabilization buffer. Results are representative of three separate experiments.

 

Figure 6
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  		Figure 6. Kinetics of FasL expression after Thy1 activation. (A) Relative expression of FasL mRNA normalized to 18 s in primary myofibroblasts stimulated with G7 anti-Thy1 mAb (5 µg/ml), or control IgG isotype match, for the time indicated: 30 minutes, 1 hour, or 6 hours. FasL was detected by real-time PCR. Results are expressed as fold change related to IgG treatment. *P < 0.001. (Paired values of Thy1 and IgG treatment were compared by the Wilcoxon rank-sum test.) (B) Lysates obtained from primary myofibroblasts were stimulated with G7 anti-Thy1 mAb (5 µg/ml) or control IgG isotype match at 1 hour, 24 hours, or 72 hours, and subjected to SDS PAGE and immunoblotted using anti-FasL antibody (upper row). The immunoblot was stripped and reblotted with β-actin antibody (lower row). The numbers under the blot represent the ratio of FasL/β-actin. Results are representative of three separate experiments with similar results.

 
Thy1-Mediated FasL Transcriptional and Translational Regulation
To assess whether Thy1 is also involved in the regulation of FasL at the transcriptional and/or translational level, we periodically measured both FasL mRNA and protein levels from 30 minutes to 72 hours after anti-Thy1 stimulation. FasL mRNA levels were up-regulated only after 1 hour of stimulation (Figure 6A), and protein levels increased at 24 hours (Figure 6B).

These results demonstrate that Thy1 activation leads to rapid membranal FasL overexpression by FasL translocation from the cytosol, whereas at a later stage Thy1 activation induces de novo synthesis of FasL mRNA and protein.

Thy1 Mediates FasL Up-Regulation via SFKs
SFKs are nonreceptor tyrosine kinases that are known to mediate Thy1 signaling in T cells (20). To study SFK involvement in Thy1 signal transduction, leading to FasL up-regulation in lung myofibroblasts, we assessed Src phosphorylation (normalized to total Src) after Thy1 stimulation. Thy1 stimulation induced Src phosphorylation (Figure 7A). Moreover, pretreatment of myofibroblasts with Src kinase inhibitor (PP2) inhibited Thy1-induced FasL up-regulation, as assessed by flow cytometry (Figure 7B).


Figure 7
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  		Figure 7. Thy1 signaling via Src kinase. (A) Lysates obtained from myofibroblasts previously stimulated for 10 minutes with G7 anti-Thy1 mAb (5 µg/ml) or control IgG isotype match, were subjected to SDS PAGE and immunoblotting using anti-p-Src (phosphorylated-Src) antibody. The blot was stripped and reblotted with total Src antibody. The numbers under the blot represent the ratio of p-Src/total Src. Results are representative of three separate experiments. (B) Myofibroblasts were pretreated with 10 µM PP2 (selective inhibitor of the Src family of protein tyrosine kinases) for 16 to 24 hours and then stimulated with G7 anti-Thy1 mAb for 1 hour. Flow cytometry analysis of FasL expression was performed in myofibroblasts after IgG (control, dark gray line), G7 anti-Thy1 (light gray line), or G7 anti-Thy1, pretreated with PP2 (black line). Results are representative of three separate experiments.

 
Thy1-Induced Up-Regulation of FasL Is Mediated by Fyn
We then determined which of the Src family kinases is involved in Thy1-induced up-regulation of FasL in lung myofibroblasts. For this purpose we initially assessed the extent of phosphorylation on different SFKs after Thy1 activation. Lung myofibroblasts were stimulated with 10 µg/ml G7 anti-Thy1 mAb or IgG control. Whole cell lysates were subjected to an array of antibodies against SFK proteins and further immunoblotted with antityrosine residue phosphorylation (p-Tyr)-specific antibody to detect the state of phosphorylation. Among SFKs, only phosphorylation of Fyn significantly increased after Thy1 activation (Figure 8A). This result was confirmed by immunoprecipitation using anti-Fyn mAb, followed by immunoblotting with anti-phosphotyrosine antibody (Figure 8B).


Figure 8
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  		Figure 8. Specific Fyn siRNA inhibits Thy1-induced FasL up-regulation. (A) After incubation with 10 µg/ml G7 anti-Thy1 mAb or control IgG, lung myofibroblast lysates were subjected to antibody array detecting SFK phosphorylation. Quantitative analysis of the phosphorylation was preformed by computerized optical densitometry of dots on the array after background subtraction. Optical densitometry was measured as arbitrary units. Results are expressed as fold change related to IgG treatment. (B) Immunoprecipitates of G7 anti-Thy1 mAb or control IgG-treated myofibroblast lysates were created using anti-Src family kinase mAbs (e.g., anti–c-Src, anti-Fyn, anti-Lyn, anti-Lck, anti–c-Yes). Immunoprecipitates were analyzed by immunoblotting with anti–p-Tyr residual Ab (upper row). Reblotting of immunoblots with antibodies against the same set of Src family kinases served as a control (lower row). (C) Flow cytometry analysis of FasL expression was compared in control/IgG versus G7 anti-Thy1 mAb-treated myofibroblasts transfected with Fyn or control-nontargeting siRNA. Control (dark gray line), G7 anti-Thy1 previously transfected with nontargeting siRNA (light gray line); G7 anti-Thy1 previously transfected with Fyn siRNA (black line). Results are representative of two separate experiments.

 
To further examine the critical role Fyn plays in FasL up-regulation by Thy1, we down-regulated Fyn expression using specific siRNA. Fyn or control (nontargeting) siRNA transfected myofibroblasts were activated by anti-Thy1 mAb or control IgG. After Thy1 activation, FasL expression was assessed by FACS analysis. The induction of FasL up-regulation by Thy1 activation was reduced in Fyn siRNA-transfected cells compared with control siRNA-transfected cells (Figure 8C).


   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We have recently demonstrated that myofibroblasts from lungs of patients with IPF, as well as those from bleomycin-treated mice, overexpress FasL molecules (29). Lung myofibroblasts act as effector cells that induce apoptosis of Fas+ epithelial cells (29) and lymphocytes (30) via Fas/FasL interaction. Moreover, we have demonstrated that FasL expression on lung myofibroblasts facilitates emergence of a phenotype of escape from in vivo immune surveillance as a mechanism for tissue fibrosis (30).

IFN-{gamma} and TNF-{alpha} have previously been shown to be the main cytokines inducing FasL expression (14, 15). In myofibroblasts, these cytokines cause increased nitrite production (NO2–), but not FasL expression (Figure 4). The absence of FasL up-regulation by IFN-{gamma} and TNF-{alpha} has also been reported by others in dermal fibroblasts (9) and human tracheal epithelial cell lines (31). This indicates that another factor is responsible for the up-regulation of FasL in fibroblasts.

In the lung, there are two subpopulations of fibroblasts, which are distinguished by expression of Thy1 (24, 25). We asked whether Thy1 expressed on lung fibroblasts has a role in FasL up-regulation. We show FasL expression on Thy1-negative myofibroblasts isolated from the total lung fibroblast population of untreated WT mice by FACS cell sorting, indicating that Thy1 is not necessary for a basal expression of FasL (Figure 1). However, after bleomycin treatment, FasL was up-regulated in Thy1-positive but not in Thy1-negative myofibroblasts isolated from the lungs of WT mice after sorting (Figure 1); further, FasL was up-regulated in myofibroblasts isolated from WT (Thy1+) mice compared with Thy1-deficient (Thy1–) mice (Figure 2). This indicates that Thy1 is essential for the up-regulation of FasL after stimulation through a bleomycin-induced milieu. Nevertheless, not all Thy1+ myofibroblasts from bleomycin-treated mice express FasL. On the basis of in vitro experiments showing that the proportion of cells expressing FasL increased in a dose-dependent manner after Thy1 activation (Figure 3), we assume that not all Thy1+ primary myofibroblasts from bleomycin-treated mice were activated in the in vivo milieu, and therefore some do not express FasL. This further strengthens the fact that the up-regulation of FasL on lung myofibroblasts correlates with Thy1 molecule expression only after activation, either directly by agonist G7 anti-Thy1 mAb, or as a result of the bleomycin-induced milieu.

The ligand of Thy1 has not yet been identified. However, it has been shown that human polymorphonuclear leukocytes (PMN) express putative Thy1 ligand, which is implicated in their binding to fibroblasts that express Thy1 (32). We have previously shown that lung injury 1 to 3 days after IT bleomycin is characterized by an influx of inflammatory cells, including PMN leukocytes (33). We therefore speculate that these cells may bind to myofibroblast Thy1 molecules, inducing lung myofibroblast activation. The increase of FasL expression on lung myofibroblasts isolated from bleomycin-treated mice at Days 3, 7, and 14 after intratracheal instillation, but not at Day 1 (29), strengthens this speculation.

Thy1 requires crosslinking to transmit a signal. No physiologic Thy1 crosslinking mechanism has been described. However, we hypothesize that Thy1 ligand assembles in homodimers or trimers, and can thus crosslink the Thy1 molecule, as has been shown in the TNF ligand family (34).

The most well-known stimulus of FasL up-regulation in lymphocytes is T cell receptor (TCR), which is a more potent activator than Thy1 (16). However, in cells that lack TCR but express GPI surface protein (35), FasL up-regulation may be induced through activation of GPI proteins, including Thy1, in a manner resembling the process in lung myofibroblasts.

We further show that Thy1 activation in lung myofibroblasts initially causes translocation of FasL from cell cytoplasm to the cell membrane surface (Figure 5), and only later induces a de novo synthesis of FasL at the mRNA and protein levels (Figure 6). Our findings in lung myofibroblasts are consistent with previous studies in lymphocytes, showing that FasL is stored in specialized secretory lysosomes that deliver it to the surface of the cell membrane after activation (36). Activated myofibroblasts may use a mechanism for their recently demonstrated cytotoxic function (29, 30), and for escape from immune surveillance (30) that is similar to the mechanism employed by lymphocytes with cytotoxic functions that respond promptly to extracellular stimuli (36).

Cell surface FasL can be cleaved by metalloproteases (11, 12), thus synthesis of new protein is necessary to maintain high levels of FasL expression at the cell surface. Indeed, we demonstrate a later elevation of the FasL mRNA and protein levels (Figure 6), allowing myofibroblasts to maintain FasL expression at the cell surface.

In addition to its contribution to the cytotoxic function of lymphocytes (2) and myofibroblasts (29, 30), FasL can also regulate cell proliferation (37, 38) and autocrine apoptosis (39) of lymphocytes, and proliferation of neighboring Fas-expressing cells (40). It may play similar roles vis a vis myofibroblasts. These possible functions of FasL expressed on myofibroblasts require further study.

SFKs are nonreceptor tyrosine kinases, known to mediate Thy1 signaling in T cells (20). In fibroblasts, it has been shown that, at baseline, Thy1– cells have an increased number of activated SFKs compared with Thy1+ cells (28). However, only in Thy1+ fibroblasts was there increased phosphorylation of SFKs in response to TSP-1/hep I (41). Here, we show directly that Thy1 activation induces SFK phosphorylation (Figure 7A). Moreover, we show that SFKs are responsible for the up-regulation of FasL expression in myofibroblasts by Thy1 (Figure 7B).

Our findings of SFK phosphorylation after Thy1 activation are supported by similar findings after TSP-1/hep I stimulation (41). The involvement of Src-like protein tyrosine kinases (PTK) with FasL induction was also shown in T lymphocytes after TCR engagement (42).

As mentioned above, FasL stored in secretory lysosomes is released to the cell surface upon cell stimulation (36, 43). The targeting motif for secretory lysosome localization was identified as a proline-rich region (PRD) of FasL cytoplasmic tail (44). It has been shown that the PRD of FasL binds Fgr, Fyn, and Lyn, but not Lck tyrosine kinases, leading to phosphorylation of FasL. Loss of phosphorylation reduces internalization of FasL into secretory lysosome (45). Consistent with these findings, we show that Fyn was the only member of the SFK family with increased phosphorylation after myofibroblast Thy1 activation by G7 anti-Thy1 mAb (Figures 8A and 8B). Moreover, we show that Fyn down-regulation, using specific Fyn siRNA, reduced Thy1-induced FasL expression in lung myofibroblasts (Figure 8C).

The predominant histopathologic finding of IPF is myofibroblast accumulation and collagen deposition in the extracellular matrix (46, 47). Activated fibroblasts (myofibroblasts) are the key cellular source of the excessive extracellular matrix (ECM) deposition in lung fibrosis (48). Previous studies focused on the association between Thy1 expression and extracellular matrix production, or fibroblast proliferation and migration (19, 26, 28, 49). We show an additional role for Thy1, which is up-regulation of FasL. Through this up-regulation, Thy1 indirectly induces a cytotoxic cell phenotype in myofibroblasts (29, 30), and may also alter the rate of lung myofibroblast proliferation.

In summary, we show here that in lung myofibroblasts, Thy1 is a functional protein, which transmits into the cell signals that up-regulate the cell's FasL expression. This step may be critical in the development and persistence of lung fibrosis.


   Acknowledgments
 
The authors thank Anita Kol for her assistance in performing the experimental work described above, and Shifra Fraifeld for her editorial assistance in preparing this manuscript.


   Footnotes
 
This study was supported by the Israel Science Foundation, the David Shainberg Fund, and the Israel Lung Association (Tel Aviv, Israel).

Originally Published in Press as DOI: 10.1165/rcmb.2007-0348OC on August 1, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 23, 2007

Accepted in final form July 30, 2008


   References
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 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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