C57Bl/6 J mice (WT), and congenic ASIC3-/- and AISC1-/- mice on a C57B1/6 J background were bred at the University of Iowa or the University of California San Diego Animal Care Facility. The ASIC3-/- and ASIC1-/- mice strains have been previously described and characterized in multiple studies
[7, 8, 28, 29]. Male mice, 9 to 10 weeks of age, were used in these studies. All experiments using animals were approved by Animal Care and Use Committee at the University of Iowa and the University of California San Diego, and conducted in accordance with National Institutes of Health (NIH) guidelines. Use of synoviocytes from human subjects was approved by the Institutional Review Board in the Human Subjects Office at the University of Iowa and in the Human Subjects Protection Program at the University of California San Diego.
Fibroblast-like synoviocyte (FLS) isolation and culture
Preparation of FLS from mice
FLS were isolated from WT, ASIC3-/- and ASIC1-/- mice according to previously published methods
[14, 19]. Mice were euthanized with sodium pentobarbital (100 mg/kg, ip) and the limbs excised above the knee and elbow joints. After rinsing the excised limbs in ethanol, the knee, ankle and elbow joints were stripped of muscle and tendon. The remaining bone and tissue were finely minced and incubated in 0.5 mg/ml Type VIII collagenase (Sigma, St. Louis, MO, USA) in RPMI (Invitrogen, Carlsbad, CA, USA) at 37°C for 2 h. The tissue was pelleted at 1,200 rpm and the supernatant discarded. The pellet was rinsed twice in high-glucose DMEM (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with penicillin, streptomycin, 10% FBS, and 0.5% gentamycin (Cellgro, Manassas, VA, USA), resuspended, plated onto T75 culture flasks and incubated at 37°C with 5% CO2. FLS were grown until >80 to 90% confluent before passaging. FLS were at passage 3 to 4 for use in western blot, (Ca2+)i, mRNA expression studies, and Live/Dead assays.
Preparation of human synovial tissue and FLS
This study was approved by the Institutional Review Board of University of California, San Diego School of Medicine and informed consent was obtained from all participants. Synovial tissue was obtained from patients with RA at the time of total joint replacement or synovectomy as previously described. The diagnosis of RA conformed to American College of Rheumatology 1987 revised criteria. The samples were processed for cell culture. The synovium was minced and incubated with 1 mg/ml collagenase type VIII (Sigma) in serum-free RPMI 1640 (Gibco BRL, Grand Island, NY, USA) for 1 h at 37°C, filtered, extensively washed, and cultured in DMEM (Gibco BRL) supplemented with 10% FBS (Gemini Bio Products, Calabasas, CA, USA), penicillin, streptomycin, gentamicin, and glutamine, in a humidified 5% CO2 atmosphere. Cells were allowed to adhere overnight, non-adherent cells were removed, and adherent FLS were split at 1:3 when 70 to 80% confluent. FLS were used from passage 3 through 9 during which time they are a homogeneous population of cells (<1% CD11b-positive, <1% phagocytic, and <1% FcγRII- and FcγRIII-receptor-positive). FLS were cultured and used at 80% confluence. Cells were synchronized in 0.1% FBS for 24 h before the addition of the appropriate stimulus.
External physiological pH solutions used for Ca2+ imaging contained 120 mM NaCl, 5 mM KCl,1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM MES; the pH of each solution was adjusted with tetramethylammonium hydroxide, and osmolarity was adjusted with tetramethylammonium chloride
. Cells were plated at 30,000 cells/dish in poly-L-lysine (Sigma) coated 35-mm glass-bottom petri dishes (MatTek Corp., Ashland, MA, USA), and grown for 24 h in 10% FBS, antibiotic-supplemented DMEM and subsequently serum-starved (0.1% FBS, antibiotic-supplemented DMEM) 24 h prior to testing.
After rinsing with pH 7.4 external buffer, cells were loaded with the Ca2+-sensitive fluorescent indicator Oregon Green BAPTA-1 AM (OGB-1) (Invitrogen, 8 μg/ml (made from a 2.5 μg/μl DMSO stock), containing 0.013% pluronic F-127, (Invitrogen, diluted from a 20% DMSO stock) in pH 7.4, 1 h, room temperature) as we previously described
: 10 ml of pH and treatment solutions were introduced into the culture dish at a rate of 100 μl/s through a syringe pump (Harvard Apparatus PHD2000, Holliston, MA). Solution was simultaneously removed from the opposite side of the dish by vacuum aspiration.
Fluorescence was measured on a 150-s time course before, during, and after application of pH solutions. An Olympus IX81 motorized inverted microscope and integrated Intelligent Imaging Innovation’s Slidebook software, v.4.1 was used to capture images. Analysis was done using Image J (NIH) to measure the change of intensity in a constant, defined area of the cell during each pH application. Each session started with pH 7.4 infusion for 20 s, followed by 2 minutes of acidic pH. Between pH solutions, pH 7.4 was again infused. All pH solutions were applied in increasing acidic pH to each culture dish with pH 7.4 infused between each solution. Percent change in (Ca2+)i was quantified using the formula:
where F is the fluorescence intensity at any given time point and F0 is fluorescence intensity under control conditions (pH = 7.4, calculated by averaging the intensities recording during the first 20s of each time course). The area under the curve during the 2-minute time period was calculated using the percent-change scores; zero represented no change in area and a positive number represented an increase in calcium intensity.
For Ca2+-free pH solutions calcium chloride was eliminated from the external buffers and NaCl adjusted osmolarity to 123 mM. For (Ca2+)i blockade, 30 μM cyclopiazonic acid (CPA, Sigma-Aldrich) was added to the cultures after OGB-1 loading, 5 minutes prior to Ca2+ imaging (12 μM CPA was included in treatment during Ca2+imaging). In the IL-1β study, 1 ng/ml IL-1β (R and D Systems, Minneapolis, MN, USA) was included in 0.5% FBS serum-starved media 24 h prior to Ca2+ imaging.
Two cell culture lines were used for testing FLS from the WT, ASIC3-/- and ASIC1-/-, Ca2+source study, and the IL-1β study. Two cell cultures lines from control (for example, WT) and experimental groups (for example, ASIC1-/-) were always run on the same day to control for differences due to loading or imaging. The profiles of percent change (% Δ) as a function of time for all viable cells in a group were averaged and compared, taken as a percent change from the WT average at pH 5 or compared to maximum intensity for each cell.
Amiloride (100, 250, 500 μM; Sigma) was added to cultured human FLS, after OGB-1 loading, 10 minutes prior to and during calcium imaging. Percent change in (Ca2+)i was quantified using the formula:
where F is the fluorescence intensity at any given time point and F0 is fluorescence intensity under control conditions. Three cell culture lines were used for RA FLS study under each condition.
Quantitative real-time polymerase chain reaction (qPCR)
FLS were plated at 100,000 cells/well in a 6-well tissue culture plate and grown for 24 h in 10% FBS/DMEM at 37°C with 5% CO2 and subsequently incubated in 0.5% FBS/DMEM for 24 h with or without 2 ng/ml IL-1β (Calbiochem, San Diego, CA, USA). FLS were then incubated in 0.5% FBS/DMEM pH 7.4 or pH 6.0 for 24 h. Cells were then lysed in RNA STAT-60 (Tel-Test, Friendswood, TX, USA). FLS RNA isolation and qPCR was performed as previously described
. Using cDNA, mRNA expression of IL-6, metalloproteinases (MMPs) and ASICs was measured by TaqMan Gene Expression Assay (Applied Biosystems). The threshold cycle (Ct) values were used to calculate the number of cell equivalents using a standard complementary DNA curve as previously described
. The data were normalized to the expression of HPRT (hypoxanthine guanine phosphoribosyltransferase) and the results were expressed as relative expression units. We examined mRNA expression for IL-6, MMP-3 and MMP-13 from WT and ASIC3-/- FLS with and without treatment with IL-1β (n = 3 preparations/group), and for ASIC3 and ASIC1 in WT FLS with and without IL-1β at pH 7.4 (n = 4 preparations/group). Different cell culture lines were used for each group (for example, WT, ASIC3-/-).
Western blot analysis
FLS were plated in six-well plates at 200,000 cells/well, and grown 24 h and then incubated in 0.1% FBS/DMEM for 24 h before addition of stimulating factors. Western blot analysis was performed as described previously
. After treatments (see below), protein was extracted with lysis buffer (50 mM HEPES, 150 mM NaCl, 25 mM MgCl2, 1 mM EDTA, 10% Glycerol, 1% tritonX-100, 20mMβ-glycerophosphate, 10 mM NaF, 1 mM Na3VO4, supplemented with Complete Proteinase inhibitors (Roche Applied Science, Indianapolis, IN, USA)) and protein concentration determined using the Micro BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Samples containing 25 μg of protein were resolved on Invitrogen NuPAGE 4% to 12% Bis Tris gels and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% non-fat milk, incubated with primary Ab (all from Cell Signaling Technology, Boston, MA, USA) at 4°C overnight, followed by horseradish peroxidase-conjugated secondary Ab for 1 h. Membranes were developed and imaged using the UVP Bioimaging systems and density of the band was quantified using Image J software (NIH). β-actin was used as a loading control and used as an internal control, and all blots were normalized to β-actin. Data were normalized to the response at pH 7.4. All western blot data analyzed included controls and experimental data for each condition. Data are expressed as the ratio compared to the control. Each blot contained three replicates of control and three replicates of experimental conditions. Each control and experimental condition was run in three separate primary cell cultures for a total of nine preparations for each condition (n = 9/condition).
FLS were plated onto 12 mm poly-L-lysine-coated round coverslips in 24-well tissue culture plates at 15,000 cells/well and incubated for 24 h in DMEM complete medium at 37°C in an atmosphere of 5% CO2. FLS were then incubated for 24 h in 0.5% FBS/DMEM serum starved medium IL-1β, 1 ng/ml.
To determine Ca2+ dependency for cell death, FLS were treated 1 h at 37°C in 0.5% FBS/DMEM with or without BAPTA-AM (1 μM, pH 7.4), and then treated for 24 h in 0.5% FBS/DMEM of pH 7.4 or pH 6.0. To determine if p-ERK contributes to the cell death, FLS were treated 1 h with PD98059 (10 μM, pH 7.4) and then 24 h in 0.5% FBS/DMEM of pH 6.0 with PD98059 (10 μM).
Live/Dead assays (Invitrogen) were performed as described previously
 using a Live/Dead Viability/Cytotoxicity kit (L-3224; Invitrogen), 0.5 mM calcein AM, and 0.5 mM ethidium homodimer 1. Stained cells were then mounted on slides with aqueous CMF-1 mounting medium (Electron Microscopy Sciences, Hatfield, PA)) and imaged with an Olympus BX51 fluorescence microscope with a spot camera. Image J software (NIH) was used to merge and quantify live and dead cells. Data are expressed as the percentage of dead cells. Each experimental condition was analyzed in three different primary cell cultures. An average number of 713 ± 28 cells were counted for each primary cell culture in each condition.
Data are expressed as mean ± standard error of the mean (SEM). (Ca2+)i in response to different pH in FLS from WT, ASIC1-/- and ASIC3-/- FLS were analyzed by repeated-measures analysis of variance (ANOVA) followed by Tukey’s post hoc test for differences between groups. Differences in Ca2+imaging, western Blots, qPCR, and Live-Dead assays for individual experiments were analyzed with one-way ANOVA followed by Tukey’s post-hoc test for group differences. P-values less than 0.05 were considered significant.