Suppression of LPS-induced matrix-metalloproteinase responses in macrophages exposed to phenytoin and its metabolite,5-(p-hydroxyphenyl-), 5-phenylhydantoin

Peripheral blood mononuclear cells were obtained from commercially-available buffy coats (Oklahoma Blood Institute, Oklahoma City, OK, USA) derived from healthy donors by density gradient centrifugation using Ficoll-paque (Amersham, Uppsala, Sweden). Six independent cultures were obtained from 6 independent donors. Monocytes were isolated using CD14 MicroBeads (Miltenyi Biotec, Auburn, CA, USA) according to manufacturer's instructions and cultured as previously described [12, 20, 21]. Briefly, isolated monocytes were plated onto duplicate 12-well tissue culture-treated plates (BD Biosciences, San Jose, CA, USA) at a density of 5 × 10⁵ cells/cm² in serum-free DMEM with L-glutamine (Cellgro, Manassas, VA, USA) containing 50 μg/mL gentamicin (Sigma, St. Louis, MO, USA) at 37 C, 5% CO₂ to promote monocyte attachment. After 2 hours, heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) was added to a final concentration of 10%. Cells were >95% CD14+ as determined by FACS analysis (data not shown) prior to culture.

After 5 days, the media and non-adhered cells were removed and replaced with complete media (DMEM, 10% FBS, gentamicin) and incubated at 37 C, 5% CO₂. Media was replaced every 2 days. Experiments were initiated upon confirmation of macrophage differentiation after 7 days in culture [12, 20, 21]. Macrophages were used between day 7 and 10 and pretreated with either: 1) 15 μg/mL of PHT-Na+ (Sigma), (serum levels, [22‐24]), 2) 50 μg/mL PHT-Na+ (high dose), 3) 15 μg/mL PHT metabolite (Sigma), (5-(4'-hydroxyphenyl),5-phenylhydantoin, HPPH), or 4) 50 μg/mL HPPH for 1 hour. Untreated cells served as control cultures. Stock solutions of PHT-Na+ (150 mg/mL) were made in sterile deionized water while HPPH (150 mg/mL) solutions were made in DMSO. Each stock solution was further diluted prior to use. The total concentration of DMSO in cultures was always less than 0.05%. DMSO concentrations less than 0.1% have been reported not to affect cellular viability and function [25, 26]. Nevertheless, we confirmed these findings in preliminary studies exposing macrophage cultures to 0.05% DMSO (data not shown). To induce production of MMPs and proinflammatory cytokines, macrophages were challenged with 100 ng/mL purified LPS from the Gram-negative, periodontal pathogen, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans (Aa), serotype b, strain Y4, a kind gift from K. L. Kirkwood, University of South Carolina, USA) for 24 hours. Isolation and purification of Aa LPS has been previously described [27]. Previous studies have demonstrated that LPS from this organism is capable of inducing MMP and TIMP production [28‐30] and our preliminary studies determined that this concentration of LPS was capable of significantly inducing TNF-α levels in human primary macrophages and THP-1 cells induced for macrophage differentiation (data not shown).

After 24 hours, the media was collected, spun at 12,000 × g, transferred to fresh tubes and stored at -80 C until further use. Quantification of supernatant MMP and TIMP levels were determined using the Luminex 100 System (Luminex Co., Austin, TX, USA) and the Fluorokine MAP Multiplex Human MMP Panel and the Fluorokine MAP Human TIMP Multiplex Kit, respectively according to the manufacturer's instructions (both from R & D, Minneapolis, MN, USA). These kits measure levels of pro-, mature, and TIMP complexed MMPs. Six independent experiments were performed from cells derived from 6 different donors. The assays were performed in 96-well plates, as previously described [20]. For MMP determination, microsphere beads coated with monoclonal antibodies against MMP-1, MMP-2, MMP-3, MMP-9, MMP-12 were added to the wells. For TIMP determination, microsphere beads coated with monoclonal antibodies against TIMP-1, TIMP-2, TIMP-3, and TIMP-4 were added to the wells of a separate plate. To remain below the upper level of quantitation, samples containing LPS were diluted 10-fold prior to analysis. This dilution factor was based on our preliminary studies. Samples and standards were pipetted into wells, incubated for 2 hours with the beads then washed using a vacuum manifold (Millipore Corporation, Billerica, MA USA). Biotinylated secondary antibodies were added and incubation for 1 h. The beads were then washed and incubated for an additional 30 minutes with streptavidin conjugated to the fluorescent protein, R-phycoerythrin (streptavidin/R-phycoerythrin). The beads were washed and analyzed (a minimum of 50 per analyte) using the Luminex 100 system. The Luminex 100 measures the amount of fluorescence associated with R-phycoerythrin, reported as median fluorescence intensity of each spectral-specific bead allowing it to distinguish the different analytes in each well. The concentrations of the unknown samples (antigens in macrophage supernatants) were estimated from the standard curve using a third-order polynomial equation and expressed as pg/mL after adjusting for the dilution factor. Samples below the detection limit of the assay were recorded as zero. The minimum detectable concentrations for the assays were as follows: MMP-1: 4.4 pg/mL, MMP-2: 25.4 pg/mL, MMP-3: 1.3 pg/mL, MMP-9: 7.4 pg/mL, TIMP-1: 1.54 pg/mL, TIMP-2: 14.7 pg/mL, TIMP-3: 86 pg/mL and TIMP-4: 1.29 pg/mL. All values were standardized for total protein using the Bradford assay (Pierce, Thermo Scientific, Rockford, IL, USA) according to manufacturer's instructions. Briefly, culture supernatants were mixed with assay reagent and incubated for 10 minutes at room temperature in 96 well plates. Bovine serum albumin (BSA, Invitrogen) was used as a standard. The absorbance at 595 nm was read using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Values obtained from untreated control cultures were arbitrarily used as a baseline measure. The ratio, (control)/(supernatant protein value) was used to normalize each sample based on total protein.

Viability of macrophages was evaluated using the CellTiter 96 AQueous One Solution Cell Proliferation Assay [3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, MTS] assay according to the manufacturer's protocol (Promega, Madison, WI, USA). This colorimetric method can be used to determine the number of viable cells in proliferation or to evaluate cytotoxicity. Briefly, macrophages were cultured in triplicate in 96-well plates and treated with PHT, HPPH and LPS as described above. Unstimulated cells served as control cultures. After 24 h, the cells were incubated with MTS for 2 h at 37 C, 5% CO₂. The absorbance was read at 490 nm using a microplate reader.

Data were analyzed using a hierarchical multiple regression approach relative to LPS, drug and dose. The first tier sought to establish the validity of the positive control, LPS vs the negative control group. The second tier of this analysis was aimed at determining whether PHT or HPPH have an effect on MMP, TIMP, TNF-α and IL-6 levels. Finally, the third tier sought to contrast dose and compare one drug with another. Data were expressed as mean ± SEM and compared using a two-tailed Student's t test for correlated samples (GraphPad Prism, GraphPad Software, La Jolla, CA, USA). Results were considered statistically significant at p < 0.05.

To evaluate the effects of PHT and its metabolite, HPPH on macrophage MMP and TIMP levels, human monocyte-derived macrophages were pretreated for 1 hour with either 15 μg/mL or 50 μg/mL of these agents prior to challenge with LPS. Previous studies have determined that PHT plasma levels of 10-20 μg/mL are necessary to effectively maintain effective seizure control [22‐24]. Thus, the concentrations used in our study represent therapeutic as well as elevated levels of PHT permitting the evaluation of dose on MMP and TIMP production. To rule out the possibility that differences in supernatant levels of these readouts were due to decreased cell viability, we performed a viability assay on cells cultured in each condition. No significant differences were noted in the viability of cells exposed to LPS and either dose of PHT, HPPH, PHT/LPS or HPPH/LPS as determined by MTS assay. Further, we standardized the results of each analyte to total protein concentration for each condition using a Bradford assay. No differences were noted for any analyte examined in conditioned media from macrophage cultures treated with PHT or HPPH alone compared to control cultures (p > 0.05). As expected, LPS markedly induced supernatant MMP-1, MMP-3, MMP-9, TIMP-1 but not MMP-2 levels in our 6 independent cultures after a 24 hour exposure (Fig. 1A-D). Compared to untreated control cultures, LPS significantly increased secretion of MMP-1 despite the presence of either PHT or HPPH at any dose. This was similarly observed for MMP-3 levels with the exception of cultures pretreated with 15 μg/mL HPPH which despite elevated levels, did not reach statistical significance (p > 0.05). In contrast, exposure of macrophages to 50 μg/mL of either PHT or HPPH prior to LPS stimulation prevented a significant increase in MMP-9 and TIMP-1 (Fig. 1D and Fig. 2). Levels of MMP-9 and TIMP-1 remained near control levels despite the potent proinflammatory challenge thus demonstrating the ability of these agents to alter macrophage function. Compared to LPS alone, pretreatment with 50 μg/mL PHT significantly blunted LPS-induced levels of MMP-1 (p < 0.05). In cultures pretreated with 50 μg/mL HPPH, MMP-3 levels were not significantly different compared to LPS-only treated cultures (p > 0.05) although the trend for reduced supernatant levels of MMP3 was evident. However, exposure of macrophages to either 15 μg/mL or 50 μg/mL PHT prior to LPS stimulation significantly blunted supernatant MMP-3 levels (p < 0.01 and p < 0.001, respectively, Fig. 1C) compared to LPS-only treated cultures. Interestingly, a trend for higher levels of MMP-1 were noted in cultures treated with HPPH while MMP-3 levels were slightly elevated in cultures treated with either PHT and HPPH although neither reached statistical significance (p > 0.05) (Fig. 1, A, C).

Elevated levels (50 μg/mL) of PHT or HPPH significantly reduced MMP-9 and TIMP-1 levels compared to LPS-only treated cells (Fig. 1D and Fig. 2). The levels of these analytes remained near control values despite LPS challenge. Interestingly, HPPH but not PHT was associated with reduced levels of MMP-2 compared to LPS only, but this relationship was not statistically significant. MMP-12 and TIMPs-2-4 remained below levels of detection in all groups and cultures.

At 24 hours, supernatant levels of TNF-α and IL-6 were significantly increased by LPS compared to untreated controls (p < 0.001). Similar to MMP and TIMP levels, no significant differences in TNF-α and IL-6 levels were observed in supernatants exposed to either 15 or 50 μg/mL PHT and HPPH alone compared to untreated cultures although a trend for decreased levels of TNF-α was evident (Fig 3A). However, macrophage cultures pretreated with 50 μg/mL PHT prior to challenge with LPS showed a significant (p < 0.05) decrease in TNF-α levels compared to LPS only treated cultures. No difference was noted for 15 μg/mL of PHT or HPPH at either concentration (Fig. 3A). Regardless of dosage, pretreatment with PHT or HPPH prior to LPS challenge had no significant effect (p > 0.05) on IL-6 secretion when compared to LPS only treated cultures.