April 2011
Volume 52, Issue 5
Free
Cornea  |   April 2011
Lipopolysaccharide Regulation of Toll-Like Receptor-4 and Matrix Metalloprotease-9 in Human Primary Corneal Fibroblasts
Author Affiliations & Notes
  • Yuk Wong
    From the Division of Infection, Inflammation and Immunity, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; and
  • Claire Sethu
    From the Division of Infection, Inflammation and Immunity, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; and
  • Fethi Louafi
    From the Division of Infection, Inflammation and Immunity, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; and
  • Parwez Hossain
    From the Division of Infection, Inflammation and Immunity, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; and
    the Eye Unit, Southampton General Hospital, Southampton, United Kingdom.
  • Corresponding author: Parwez Hossain, Eye Unit, Southampton General Hospital, Tremona Road, Southampton, S016 6YD, UK; p.n.hossain@soton.ac.uk
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2796-2803. doi:10.1167/iovs.10-5459
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Yuk Wong, Claire Sethu, Fethi Louafi, Parwez Hossain; Lipopolysaccharide Regulation of Toll-Like Receptor-4 and Matrix Metalloprotease-9 in Human Primary Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2011;52(5):2796-2803. doi: 10.1167/iovs.10-5459.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Toll-like receptor 4 (TLR4) is a key component of the innate immune response related to microbial keratitis (MK). Pathways and downstream effectors relating to TLR signaling remain unknown in human bacterial MK. To this effect, by activating the TLR4 signaling cascade with lipopolysaccharide (LPS), the authors investigated whether TLR4, matrix metalloproteases (MMP)-2, MMP-9, and cytokine expression in diseased human primary corneal fibroblasts (CFs) were altered.

Methods.: Human primary CFs from patients with severe corneal ulceration were cultured in conjunction with healthy control CFs and treated with LPS derived from Pseudomonas aeruginosa.

Results.: TLR4, MMP-2, and MMP-9 were constitutively expressed in both ulcerated and control CFs. Diseased CFs showed greater responsiveness to LPS stimulation. TLR4 and MMP-9 expression was dose-dependently increased by LPS. MMP-2 expression was not affected by LPS. Analysis on cytokine expression revealed that IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFNγ, and TNFα expression increased after LPS treatment but only in diseased cells.

Conclusions.: TLR4 activation with LPS increases TLR4, MMP-9, and cytokine expression in CFs cultured from patients with microbial keratitis. Overexpression of these products may provide a local mechanism to eradicate bacterial infection but may also aid corneal ulceration and perforation.

Bacterial keratitis is a common cause of blindness. Studies have shown that the bacterium Pseudomonas aeruginosa is strongly associated with corneal ulceration 1,2 and is most prevalent among contact lens wearers and immunocompromised patients. 3 5 In severe cases, perforation of the cornea can occur, leading to permanent visual impairment or blindness. Bacterial toxins, increased infiltration of polymorphonuclear (PMN) leukocytes, and the release of matrix metalloproteases (MMPs, which digest extracellular matrices) all contribute to cell necrosis and ulcer formation. 
Toll-like receptors (TLRs) are a family of proteins that act as sensors for detecting microbial products and are involved with the early phase of host defense against pathogens. These receptors have been classically ascribed to immune cells such as macrophages and mast cells. However, TLR4 is also expressed on human corneal epithelial and fibroblast cells. TLR4, with the aid of accessory proteins, has the ability to recognize the bacterial cell wall product lipopolysaccharide (LPS). Activated TLRs can stimulate endothelial cells to recruit leukocytes and leak plasma proteins and complement into the infected regions. In addition, TLR4 can initiate the induction of gene transcription using a variety of intracellular signaling cascades that lead to the expression of immune and inflammatory genes. 6  
During inflammation or injury, resident stromal keratocytes transform into corneal fibroblasts (CFs), which play a key role in corneal remodeling and healing. Cytokine released from resident cells contributes to the progression of the inflammatory response and aids in the eradication of the infective agent. The activation of TLR4 in mice causes inflammation and increased MMP release in the eye. 7,8 In CFs, TLR4 signaling leads to the production of a number of products such as IL-8, MCP-1, 9 and ICAM-1 and to the phosphorylation of NF-κB. 10  
However, the relationship between TLR4 activation and the production of proinflammatory cytokines and MMPs in diseased human CFs has not been fully characterized. In this study, using diseased MK CFs (i.e., ulcerated CFs) isolated from human patients, we examined the connection between TLR4 signaling and the expression patterns of TLR4, MMP-2, MMP-9 and proinflammatory cytokines and chemokines (IL-1β, IL-2, IL-6, IL-8, IL-12p70, granulocyte colony–stimulating factor (GM-CSF), interferon-gamma (IFNγ), and tumor necrosis factor-alpha (TNFα). 
Materials and Methods
Patient Samples and Primary Keratocyte Cultures
Perforated ulcerated human corneal samples were collected from consenting patients (ethics number 06/Q1602/56) undergoing corneal grafts for microbial keratitis at Southampton General Hospital (Southampton, UK). Five patients with microbial keratitis with perforation (age range, 62–83 years) underwent corneal scraping as part of their clinical treatment. Cultures tested positive for Pseudomonas aeruginosa in three patients, and Gram-negative bacteria were identified on microscopy in the other two patients. Areas of corneal ulceration were marked during surgery, and cells within this region were isolated. Separate control samples were obtained from unused residual corneal material (donor age range, 66–80 years; n = 4; Bristol Eye Bank, Bristol, UK) and an additional control from a single patient undergoing routine keratoplasty for noninflammatory corneal disease (Fuch's dystrophy). Donors of the control corneas had no history of ocular inflammation and were similar in age and sex to the patients with the ulcerated corneas. Human material in this study was used in strict accordance with the principles of the Declaration of Helsinki. 
Corneal tissue samples were collected during surgery in sterile tubes containing corneal culture medium (CCM; DMEM/F12; Invitrogen-Gibco, Paisley, UK) supplemented with 5% fetal bovine serum, penicillin, streptomycin, gentamicin, and amphotericin B (Lonza Biologics, Tewkesbury, UK). Human primary stromal CFs were immediately isolated and cultured under sterile conditions. To extract the CFs, the epithelial layer of the corneal tissue was removed. The stromal layer was separated from the basement membrane using collagenase digestion (1 mg/mL, 4°C overnight). CFs from different persons were not pooled and remained as independent samples in all experiments. They were left to reach confluence (in CCM). Cells were then passaged into six-well plates (Greiner Bio-One, Stonehouse, UK) (with some containing sterilized coverslips for immunocytochemistry). Cells were left to reach 70% confluence before incubation in DMEM/F12 serum-free media (SFM) containing 0, 50, or 100 ng/mL commercially available LPS (L8643; Sigma-Aldrich, Poole, UK) for 24 hours. Given that P. aeruginosa was a common pathogen in our study, we opted for this LPS preparation rather than purer forms produced by other bacteria. Passage numbers in this study were kept below 5 to reduce any effect of cell differentiation, in accordance with previous studies. In addition, flow cytometry, morphology, and vimentin expression were performed on cells after passages 1 to 4 to test the purity of the cell cultures used. 
Flow Cytometry
CFs were eluted from confluent cultures using trypsin/EDTA (Invitrogen-Gibco) and centrifuged. The resultant cell pellet was resuspended in FACS buffer (1% BSA and 0.1% sodium azide) with 10% FCS and incubated (4°C, 30 minutes). At this stage, a number of isolated CFs were also spiked with monocyte-derived dendritic cells (DCs; produced from human blood mononuclear cells), which acted as our positive control for immune cells. Monocytes were cultured for 5 to 7 days in RPMI 1640 (with 10% FCS, 1000 U/mL, GM-CSF, and 1000 /mL IL-4 [Immunotools, Friesoythe, Germany]) to obtain immature DCs. All cells were then centrifuged, and the pellet was resuspended in 100 μL FACS buffer (with 10% FCS) and anti–human HLA-DR (15–9956 1:20, 4°C, 1 hour; eBiosciences, San Diego, CA). Cells were then washed with FACS buffer before cell morphology, and expression characteristics were analyzed using flow cytometry (FACSCalibur; Becton Dickinson) and acquisition software (CellQuest Pro; Becton Dickinson). 
qRT-PCR
After LPS stimulation (24 hours) in SFM, total RNA was extracted with a kit (RNeasy Minikit; Qiagen, Valencia, CA) in accordance with the manufacturer's instructions. RNA abundance was quantified using a spectrophotometer (NanoDrop ND-1000; Thermo Scientific, Wilmington, DE), and the samples were then normalized. Using random nanomers and oligo-dT primers, reverse transcription was performed to obtain complementary DNA according to the manufacturer's protocols (PrimerDesign, Southampton, UK). qRT-PCR was performed in conjunction with GAPDH using a PCR detection system (iCycler; Bio-Rad) in accordance with the manufacturer's recommendations (PrimerDesign). The TLR4 primer sequence was 5-CCTTCACTACAGAACTTTATTCC-3, and the antisense primer was 5-ACACCACAACAATCACCTTTCG-3. Results were analyzed using ratios of target gene mRNA to GAPDH mRNA (the comparative method). 
Western Blot Analysis
CFs were lysed in 100 μL RIPA buffer (Millipore, Hertfordshire, UK) with 10 μL protease inhibitor (Sigma-Aldrich) and lysates loaded at 15 μg/lane. Proteins were separated using 10% Bis-Tris gels (Invitrogen) under reduced conditions and were transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% nonfat milk in PBS with 0.1% Tween 20 (PBS-T) and incubated with rabbit anti-TLR4 (TP376, 1:1000, 4°C, 24 hours; AMS Biotechnology Ltd., Abingdon, UK), rabbit anti-MMP-2 (ab52756, 1:4000, 4°C, 24 hours; Abcam, Cambridge, UK), rabbit anti-MMP-9 (ab76003, 1:4000, 4°C, 24 hours; Abcam), or rabbit anti-β-actin (ab8227, 1:5000, 4°C, 24 hours; Abcam) and were washed five times in PBS-T before they were incubated with goat anti-rabbit-HRP (1:4000, room temperature [RT], 2 hours; Dako, Glostrup, Denmark). Blots were visualized using enhanced chemiluminescence (ECL Advance; GE Healthcare, Piscataway, NJ) and a molecular imager (Versadoc MP-4000; Bio-Rad). Protein expression was analyzed using image acquisition and analysis software (Quantity One; Bio-Rad). 
Gelatin Zymography
Gelatin zymography was used to detect the presence of active MMP-2 and MMP-9. Supernatants were collected from CFs treated with LPS (SFM, 24 hours). Samples were loaded at 15 μg/lane and were separated by a 10% SDS-PAGE gel containing 1 mg/mL gelatin. Sample proteins were renatured in 2.5% Triton X-100 (30 minutes, RT) and placed into substrate buffer (50 mM Tris, pH 7.5, 0.2 M NaCl, 5 mM CaCl2, 0.02% NaN3, and 0.02% Brij-35) for 15 minutes before they were replaced with fresh substrate buffer and incubated (20 hours, 37°C). Enzymatic activity was visualized (Coomassie Brilliant Blue R-250) and quantified using densitometry (Quantity One; Bio-Rad). Negative control zymograms were incubated in substrate buffer containing 20 mM EDTA, which is known to be a metalloprotease inhibitor. 
Immunocytochemistry
Fluorescence microscopy was used to assess the distribution of TLR and vimentin expression in cultured fibroblasts. Cells were washed with 0.1 M PBS and fixed in 4% paraformaldehyde (PFA; RT, 10 minutes). PFA was removed, and the fixed cells were washed twice with PBS to remove residual PFA. Cells were then blocked with distilled water containing 0.2% Triton X-100, 0.05 M Tris saline, and 5% normal goat serum before they were incubated with either mouse anti-vimentin (ab8069; 1:100, RT, 2 hours; Abcam) or rabbit anti-TLR4 (TP376; 1:100, 4°C, 24 hour; AMS Biotechnology Ltd.). Cells were washed in 0.1 M PBS and placed in either anti-mouse-FITC (1:500, RT, 2 hours; Vector Laboratories, Burlingame, CA) or anti-rabbit-FITC (1:500, RT, 2 hours; Vector Laboratories) before being washed and coverslipped (Fluoromount; Sigma-Aldrich). 
Analysis of Cytokine Expression
Cultured CFs (control and ulcerated) were treated with LPS (0, 50, and 100 ng/mL) in SFM for 24 hours when confluent. Cells were then lysed with RIPA buffer, normalized in BCA (0.5 mg/mL total protein), and plated in duplicate onto a proinflammatory cytokine multiplex plate (K11007B-1; Meso Scale Discovery, Gaithersburg, MD) according to manufacturer's instructions, and cytokine expression was analyzed (Sector Imager 6000; Meso Scale Discovery). 
Statistical Analysis
Each sample was derived from a different patient. The mean score for each LPS concentration and sample type was calculated. All samples were assessed at least in duplicate and were not pooled. The SEM was calculated by using the SD for each LPS concentration divided by the square root of n, where n represented the number of patient samples. Data were analyzed using the Mann-Whitney U test, with P < 0.05 considered statistically significant. 
Results
Purity of Human Primary Corneal Fibroblast Cultures
To exclude macrophage and other immune cell contamination from our cell lines, we used antibodies that detected markers expressed by major histocompatibility complex (MHC) class II cells. Flow cytometry analysis (Fig. 1) found cultured CFs to be negative for HLA-DR (MHC class II cells). 
Figure 1.
 
Lack of immune cell contamination in cultured fibroblast cells. Flow cytometry analysis was performed on ulcerated (A) and control (B) fibroblast cells stained with HLA-DR. Analysis did not reveal any difference between unstained fibroblasts (C, E) and HLA-DR–treated fibroblasts (D), indicating the lack of MHC class II cells in fibroblast cultures. HLA-DR–positive cells were detected only when fibroblasts were spiked with human monocytes (acting as positive controls) before staining (F).
Figure 1.
 
Lack of immune cell contamination in cultured fibroblast cells. Flow cytometry analysis was performed on ulcerated (A) and control (B) fibroblast cells stained with HLA-DR. Analysis did not reveal any difference between unstained fibroblasts (C, E) and HLA-DR–treated fibroblasts (D), indicating the lack of MHC class II cells in fibroblast cultures. HLA-DR–positive cells were detected only when fibroblasts were spiked with human monocytes (acting as positive controls) before staining (F).
TLR4 Transcript Expression (qRT-PCR)
Data from our qRT-PCR (Fig. 2) revealed that TLR4 expression in control CFs increased significantly after LPS stimulation. Treatment at 100 ng/mL LPS did not significantly increase TLR4 expression in control CFs compared with the same group of cells not treated with LPS. In contrast, the same dose on ulcerated CFs significantly increased TLR4 expression (33 ± 21-fold) compared with their non-LPS counterparts. 
Figure 2.
 
Increase of TLR4 transcript in ulcerated fibroblast cells after LPS stimulation. Human primary CFs from five ulcer patients and five controls were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with 100 ng/mL LPS (24 hours, SFM). RNA was extracted, and qRT-PCR was performed. Fold change from baseline was significantly larger in ulcerated cells when treated with LPS (U0 vs. U100). In comparison, control cells were relatively less responsive to LPS than at baseline (C0 vs. C100; Mann-Whitney U test; n = 5; P = 0.008). Error bars indicate SEM.
Figure 2.
 
Increase of TLR4 transcript in ulcerated fibroblast cells after LPS stimulation. Human primary CFs from five ulcer patients and five controls were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with 100 ng/mL LPS (24 hours, SFM). RNA was extracted, and qRT-PCR was performed. Fold change from baseline was significantly larger in ulcerated cells when treated with LPS (U0 vs. U100). In comparison, control cells were relatively less responsive to LPS than at baseline (C0 vs. C100; Mann-Whitney U test; n = 5; P = 0.008). Error bars indicate SEM.
TLR4 Protein Expression
Western blot analysis (Fig. 3) revealed a dose-dependent response to rising LPS concentration in the ulcerated CF group. Analysis revealed that TLR4 expression increased by ∼4-fold when ulcerated CFs were stimulated with 100 ng/mL LPS. Changes to TLR4 expression after 50 ng/mL fell intermediately between vehicle control (0 ng/mL) and 100 ng/mL; however, at this concentration, changes in TLR4 expression were not significantly different from those of vehicle or 100 ng/mL. TLR4 expression appeared not to have changed at any LPS concentration in control CFs. Values were not significant in relation to one another, and results did not suggest a dose-dependent response. 
Figure 3.
 
TLR4 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and were run under reduced conditions. Blots were separately probed with rabbit anti-TLR4 (1:1000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and TLR4, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 3.
 
TLR4 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and were run under reduced conditions. Blots were separately probed with rabbit anti-TLR4 (1:1000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and TLR4, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Distribution of TLR4 Expression after LPS Stimulation
After we observed an increase in TLR4 expression in our ulcerated CFs, we next examined the cellular distribution of TLR4 using immunocytochemistry (Fig. 4). Fluorescence microscopy revealed that TLR4 staining was localized to the membrane and cytoplasm in both healthy and ulcerated corneal fibroblasts. When stimulated with LPS, both TLR4 cell types colocalized to the nuclear area, with ulcerated cells showing greater cell fragmentation and condensed nuclei. It was unclear whether TLR4 expression was localized specifically to endosomes. 
Figure 4.
 
TLR4 expression after LPS stimulation. Representative images of human primary CFs from five ulcer patients and five control patients cultured onto glass coverslips. Cells were incubated in SFM (24 hours, 37°C) and treated either before or after LPS (24 hours). Cells were fixed with 4% PFA (10 minutes, RT) and incubated with rabbit-anti-vimentin (1:100, RT, 2 hours) or rabbit anti-TLR4 (1:100, 4°C, 24 hours). All cells expressed vimentin (A). tj;2TLR4 expression was constitutively expressed on control human fibroblast cells (B) and with LPS stimulation (C). Ulcerated fibroblast cells also expressed LPS before (D) and after LPS stimulation (E). In both cell types, nuclear localization of TLR4 staining was observed with ulcer cells showing greater nuclear condensation and cell fragmentation. No signal was observed from fibroblast cells that received no primary antibodies (F).
Figure 4.
 
TLR4 expression after LPS stimulation. Representative images of human primary CFs from five ulcer patients and five control patients cultured onto glass coverslips. Cells were incubated in SFM (24 hours, 37°C) and treated either before or after LPS (24 hours). Cells were fixed with 4% PFA (10 minutes, RT) and incubated with rabbit-anti-vimentin (1:100, RT, 2 hours) or rabbit anti-TLR4 (1:100, 4°C, 24 hours). All cells expressed vimentin (A). tj;2TLR4 expression was constitutively expressed on control human fibroblast cells (B) and with LPS stimulation (C). Ulcerated fibroblast cells also expressed LPS before (D) and after LPS stimulation (E). In both cell types, nuclear localization of TLR4 staining was observed with ulcer cells showing greater nuclear condensation and cell fragmentation. No signal was observed from fibroblast cells that received no primary antibodies (F).
Effect of TLR4 Activation on MMP Expression and Activity
Western blot analysis on cell lysates confirmed the expression of proform MMP-2. LPS stimulation or the disease state did not play a role in regulating MMP-2 expression in either control or ulcerated CFs. Expression of MMP-2 in all cell groups was similar to each other (Fig. 5). In contrast, by using an antibody that detected both the proform and the active form of MMP-9, Western blot analysis revealed a dose-dependent increase to rising concentrations of LPS, with an increase in the concentration of active MMP-9 but not of the proform (Fig. 6). 
Figure 5.
 
MMP-2 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-2 (1:4000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-2, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 5.
 
MMP-2 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-2 (1:4000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-2, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 6.
 
MMP-9 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-9 (1:4000, 4°C, 24 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-9, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 6.
 
MMP-9 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-9 (1:4000, 4°C, 24 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-9, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Using gelatin zymography (Fig. 7), we investigated whether LPS had the potential to regulate the expression/excretion of active MMP-9 and MMP-2. Human primary CFs were stimulated for 24 hours with LPS (0–100 ng/mL) in SFM. Enzymatic activity of gelatinolytic MMPs were also measured in the presence or absence of 20 mM EDTA (a known inhibitor of MMPs). 
Figure 7.
 
Active MMP-9 expression was altered after LPS treatment. Human primary CFs were grown until confluent. Cells were incubated in SFM (24 hours, 37°C) and treated with LPS at different concentrations (SFM, 24 hours). Supernatant proteins were then normalized and loaded onto gelatin zymograms and were separated using electrophoresis. Metalloprotease activity was successfully blocked with 20 mM EDTA (Mann-Whitney U test). Error bars indicate SEM.
Figure 7.
 
Active MMP-9 expression was altered after LPS treatment. Human primary CFs were grown until confluent. Cells were incubated in SFM (24 hours, 37°C) and treated with LPS at different concentrations (SFM, 24 hours). Supernatant proteins were then normalized and loaded onto gelatin zymograms and were separated using electrophoresis. Metalloprotease activity was successfully blocked with 20 mM EDTA (Mann-Whitney U test). Error bars indicate SEM.
Gelatin zymograms from both ulcerated and control supernatants displayed bands at ∼92 kDa and ∼85 kDa. Control CFs excreted active MMP-9 at very low levels and were independent of LPS concentration. Basal expression/excretion of active MMP-9 was significantly higher in ulcerated CFs in comparison to controls (U0 vs. C0). In addition, LPS increased the excretion of active MMP-9 in a dose-dependent manner in ulcerated CFs. Increased excretion of MMP-9 of ∼20% and ∼70% from baseline was observed when LPS concentrations was increased to 50 ng/mL and 100 ng/mL (P < 0.75 and P < 0.05), respectively; this was not observed in the control group. 
To confirm that the observed bands were MMP-9, supernatant samples were run alongside human recombinant MMP-2 and MMP-9 in the presence or absence of 20 mM EDTA. Bands at the molecular weight that would be indicative of active MMP-2 were not observed using any of our supernatant samples, even though clear bands were observed with human recombinant MMP-2 (67 and 72 kDa). No degradation of the gels was observed when zymograms were incubated in EDTA. 
LPS Activation on Cytokine Expression
Cytokine expression in LPS-stimulated CFs were simultaneously analyzed using a proinflammatory cytokine plate (Meso Scale Discovery) (Fig. 8). Analysis of the plate revealed significant upregulation in a number of key cytokines. When compared with baseline (0 ng/mL LPS), stimulated CFs (100 ng/mL LPS) significantly increased IL-2 (by 4.4-fold), IL-8 (3.6-fold), IL-12p70 (4.6-fold), GM-CSF (5.5-fold), IFNγ (4.8-fold), and TNFα (5.1-fold). Expression of IL-1β and IL-6 was not altered by LPS. In contrast to ulcerated CFs, cytokine expression in control cells was, in general, not significantly altered from baseline. Only IL-1β and IL-10 were significantly altered after LPS stimulation. 
Figure 8.
 
Cytokine expression in human cultured fibroblast cells after LPS stimulation. Human primary CFs from four ulcer patients and four control patients were cultured until confluent and treated with LPS in SFM (24 hours, 37°C). Cells were lysed in RIPA buffer and plated in duplicate at 0.5 mg/mL (total protein) onto a proinflammatory plate. Compared with baseline (0 ng/mL LPS) expression of IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFNγ, and TNFα in ulcerated fibroblast cells (dark gray bars) were significantly increased after 100 ng/mL LPS. In contrast, only IL-1b and IL-10 were significantly increased in control cells (light gray bars) after LPS stimulation. No change was seen with IL-6 in either cell group (Mann-Whitney U test; n = 4). *P < 0.05. Error bars indicate SEM.
Figure 8.
 
Cytokine expression in human cultured fibroblast cells after LPS stimulation. Human primary CFs from four ulcer patients and four control patients were cultured until confluent and treated with LPS in SFM (24 hours, 37°C). Cells were lysed in RIPA buffer and plated in duplicate at 0.5 mg/mL (total protein) onto a proinflammatory plate. Compared with baseline (0 ng/mL LPS) expression of IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFNγ, and TNFα in ulcerated fibroblast cells (dark gray bars) were significantly increased after 100 ng/mL LPS. In contrast, only IL-1b and IL-10 were significantly increased in control cells (light gray bars) after LPS stimulation. No change was seen with IL-6 in either cell group (Mann-Whitney U test; n = 4). *P < 0.05. Error bars indicate SEM.
Discussion
This study analyzed the downstream effects of TLR4 activation in ulcerated human CFs. TLR4 stimulation with LPS dose-dependently enhanced the expression of TLR4, MMP-9, IL-2, IL-8 IL-12p70, GM-CSF, IFNγ, and TNFα, but MMP-2 expression remained unchanged. 
Increased TLR4 expression in disease states such as corneal fungal keratitis has been observed. 11 As it does in fungal disease, we found that TLR4 expression increased after LPS stimulation and more strongly in diseased cells. The reasons for this differential change in expression of TLR4 compared with control CFs is unknown, but it may be that these cells require more concentrated doses of LPS or that they lack MD-2 9,12 to activate transcriptional changes. Thus, there is the possibility that some patients are more genetically susceptible to Gram-negative infection. 
Our immunohistochemistry observations showed distribution of TLR expression in both the cell membrane and the cytoplasm. After LPS stimulation, there was greater nuclear localization for TLR4 in both control and ulcer fibroblasts, with ulcer fibroblasts showing greater nuclear condensation and cell fragmentation. Similar observations have been made in other non–macrophage cells in which LPS activation of cytoplasmic TLR4 receptors results in complexing and activation of TLR-associated adaptor proteins (such as MyD88) with these TLR complexes, showing nuclear colocalization. Ulcerated fibroblasts showed a qualitatively greater level of cytoplasmic damage and nuclear condensation that likely represented the effect from pyroptosis that occurs from excessive TLR activation resulting in increased caspase-1 activity. 13,14 The observation that ulcerated fibroblasts showed lower fluorescent staining despite increased quantitative protein expression in Western blot analysis can be explained by the effects from the fixative process during immunohistochemistry (affecting already disrupted cells in the ulcer group), a process that is not required for Western blot analysis. 
Enhanced TLR4 expression in CFs would allow for a more effective, localized response during severe infections. In the mouse, LPS-induced activation of TLR4 increases neutrophil infiltration 15 and MMP-9 production 16 18 in a variety of cells, including CFs. In our study, LPS stimulation exaggerated MMP-9 production; such a response would exacerbate the preexisting infective condition and promote an environment for membrane dissolution and the generation of an ulcerative state. 
Unlike MMP-9, we could not detect any change in MMP-2 expression. MMP-2 expression occurs in nonpathologic corneal tissue 19 and is responsible for corneal tissue remodeling and healing. Increased MMP-2 expression has been described in other diseased states, such as burn injuries, 20,21 herpetic infections, and fungal keratitis. 22 MMP-2 expression in these disease states occurs after 3 days postinfection 23 and could explain the unaltered MMP-2 expression patterns observed in our study. 
In contrast, Yamamoto et al. 24 demonstrated that MMP-2, but not MMP-9, was endogenously expressed in cultured CFs. This discrepancy may be a consequence of experimental variations and patient selection. In our patient cohort, we are confident that our CFs express MMP-9 because we identified both the proform and the active form in our cells. 
Although the signaling observed might have occurred through a possible contaminant in the LPS, this seems unlikely for several reasons: the LPS we used was highly purified through gel filtration; any contaminants would have to have been highly specific to only the diseased cell type because control cells were not affected; other studies 25 27 that used the same LPS also suggest minimal input from contaminants; and TLR4 expression in our study was upregulated in CFs, making direct LPS stimulation seem the most likely explanation for the observed signaling outcome. 
Cytokine interactions are vital for the protection or the progression of diseased states. With the exception of IL-6 and IL-10, the cytokines expressed in this study can be regarded as destructive. Surprisingly, ulcer CFs increased expression of both IL-6 and IL-10; however, this may reflect a later protective function of CFs from further cellular damage during strong LPS stimulation (i.e., infections). 28 30  
A positive correlation between MIP-2 (functional mouse homologue to human IL-8) and IL-1β expression with PMN recruitment in mice has been found. 31,32 The neutralization of IL-1β expression promotes corneal healing and reduces PMN recruitment and has the ability to regulate IL-6 and IL-8 expression. 33 Similarly, IL-1β and IL-6 in this study share similar LPS response profiles. Here the expression peaked at 50 ng and declined at 100 ng, suggesting that the expression of both cytokines may be linked. 
Studies using mice strains that differed in their susceptibility to corneal ulceration found that IL-12 expression occurred in the mouse in susceptible mice (B6) but not in resistant mice (BALB/c) after P. aeruginosa infection. 34,35 However, the application of recombinant IL-12 in resistant mice led to a more susceptible phenotype, with concomitant corneal ulceration within 3 to 5 days. This is interesting because IL-12p70 was upregulated only in cells treated with LPS administration on previously ulcerated fibroblasts but not on LPS-treated control cells, suggesting that infected corneas and individual patients may have increased susceptibility to ulcer formation as a result of an increase in IL-12p70 expression. 
As a major regulator of macrophage and neutrophil function, GM-CSF promotes the release of proteolytic enzymes and oxygen-free radicals from neutrophils and has been implicated in a number of inflammatory disease states. Indeed, studies have shown that stimulation with IL-1α, IL-1β, and TNF-α can induce GM-CSF expression in human CFs. 36,37 A similar increase in these cytokines was present in ulcerated CFs and thus reflect a similar role here. 
In summary, we have shown that TLR4, MMP-9 (but not MMP-2), and proinflammatory cytokine expression in human CFs can be modulated with LPS. These changes occurred predominantly in ulcerated CFs. Therefore, taken together, TLR4 in CFs may play an important role in host defense in the early stages of infection, providing a platform for pathogen eradication through MMP-9 production and TLR4 activation. During severe infection, bacterial LPS binding onto aberrant overexpression of TLR4 could swing the production of MMP-9 and cytokines from a protective to a destructive nature, leading to corneal ulceration and necrosis. In addition to changes to TLR4 and MMP production, we found concurrent changes to the balance of protective or destructive cytokines, and our results show similarities to animal models of bacterial keratitis. Thus, by dampening TLR4 signaling, it may be possible to reduce corneal tissue damage in human bacterial keratitis and to provide a more adequate clinical resolution to the diseased state. 
Footnotes
 Supported by the Gift of Sight, the Royal College of Surgeons of Edinburgh, the TFC Frost Foundation, and the British Council for the Prevention of Blindness.
Footnotes
 Disclosure: Y. Wong, None; C. Sethu, None; F. Louafi, None; P. Hossain, None
The authors thank Linda Hazlett for her suggestions in preparing the manuscript. 
References
Kernacki KA Hobden JA Hazlett LD Fridman R Berk RS . In vivo bacterial protease production during Pseudomonas aeruginosa corneal infection. Invest Ophthalmol Vis Sci. 1995;36:1371–1378. [PubMed]
Kernacki KA Goebel DJ Poosch MS Hazlett LD . Early TIMP gene expression after corneal infection with Pseudomonas aeruginosa . Invest Ophthalmol Vis Sci. 1998;39:331–335. [PubMed]
Fleiszig SM Efron N . Conjunctival flora in extended wear of rigid gas permeable contact lenses. Optom Vis Sci. 1992;69:354–357. [CrossRef] [PubMed]
Fleiszig SM Efron N Pier GB . Extended contact lens wear enhances Pseudomonas aeruginosa adherence to human corneal epithelium. Invest Ophthalmol Vis Sci. 1992;33:2908–2916. [PubMed]
Willcox MD . Pseudomonas aeruginosa infection and inflammation during contact lens wear: a review. Optom Vis Sci. 2007;84:273–278. [CrossRef] [PubMed]
Takeda K Akira S . Toll receptors and pathogen resistance. Cell Microbiol. 2003;5:143–153. [CrossRef] [PubMed]
Miyajima S Akaike T Matsumoto K . Matrix metalloproteinases induction by pseudomonal virulence factors and inflammatory cytokines in vitro. Microb Pathog. 2001;31:271–281. [CrossRef] [PubMed]
Ikema K Matsumoto K Inomata Y . Induction of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs correlates with outcome of acute experimental pseudomonal keratitis. Exp Eye Res. 2006;83:1396–1404. [CrossRef] [PubMed]
Kumagai N Fukuda K Fujitsu Y Lu Y Chikamoto N Nishida T . Lipopolysaccharide-induced expression of intercellular adhesion molecule-1 and chemokines in cultured human corneal fibroblasts. Invest Ophthalmol Vis Sci. 2005;46:114–120. [CrossRef] [PubMed]
Fukuda K Kumagai N Yamamoto K Fujitsu Y Chikamoto N Nishida T . Potentiation of lipopolysaccharide-induced chemokine and adhesion molecule expression in corneal fibroblasts by soluble CD14 or LPS-binding protein. Invest Ophthalmol Vis Sci. 2005;46:3095–3101. [CrossRef] [PubMed]
Jin X Qin Q Lin Z Chen W Qu J . Expression of toll-like receptors in the Fusarium solani infected cornea. Curr Eye Res. 2008;33:319–324. [CrossRef] [PubMed]
Zhang J Kumar A Wheater M Yu FS . Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness. Immunol Cell Biol. 2009;87:141–148. [CrossRef] [PubMed]
Hornef MW Normark BH Vandewalle A Normark S . Intracellular recognition of lipopolysaccharide by toll-like receptor 4 in intestinal epithelial cells. J Exp Med. 2003;198:1225–1235. [CrossRef] [PubMed]
Brough D Rothwell NJ . Caspase-1-dependent processing of pro-interleukin-1beta is cytosolic and precedes cell death. J Cell Sci. 2007;120:772–781. [CrossRef] [PubMed]
Sun Y Pearlman E . Inhibition of corneal inflammation by the TLR4 antagonist eritoran tetrasodium (E5564). Invest Ophthalmol Vis Sci. 2009;50:1247–1254. [CrossRef] [PubMed]
Opdenakker G Masure S Grillet B Van DJ . Cytokine-mediated regulation of human leukocyte gelatinases and role in arthritis. Lymphokine Cytokine Res. 1991;10:317–324. [PubMed]
Opdenakker G Masure S Proost P Billiau A Van DJ . Natural human monocyte gelatinase and its inhibitor. FEBS Lett. 1991;284:73–78. [CrossRef] [PubMed]
Van RM Norga K Masure S . The cytokine-protease connection: identification of a 96-kD THP-1 gelatinase and regulation by interleukin-1 and cytokine inducers. Cytokine. 1991;3:231–239. [CrossRef] [PubMed]
Fini ME Cui TY Mouldovan A Grobelny D Galardy RE Fisher SJ . An inhibitor of the matrix metalloproteinase synthesized by rabbit corneal epithelium. Invest Ophthalmol Vis Sci. 1991;32:2997–3001. [PubMed]
Matsubara M Zieske JD Fini ME . Mechanism of basement membrane dissolution preceding corneal ulceration. Invest Ophthalmol Vis Sci. 1991;32:3221–3237. [PubMed]
Matsubara M Girard MT Kublin CL Cintron C Fini ME . Differential roles for two gelatinolytic enzymes of the matrix metalloproteinase family in the remodelling cornea. Dev Biol. 1991;147:425–439. [CrossRef] [PubMed]
Dong X Shi W Zeng Q Xie L . Roles of adherence and matrix metalloproteinases in growth patterns of fungal pathogens in cornea. Curr Eye Res. 2005;30:613–620. [CrossRef] [PubMed]
Yang YN Bauer D Wasmuth S Steuhl KP Heiligenhaus A . Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis. Exp Eye Res. 2003;77:227–237. [CrossRef] [PubMed]
Yamamoto K Kumagai N Fukuda K Fujitsu Y Nishida T . Activation of corneal fibroblast-derived matrix metalloproteinase-2 by tryptase. Curr Eye Res. 2006;31:313–317. [CrossRef] [PubMed]
Raoust E Balloy V Garcia-Verdugo I Touqui L Ramphal R Chignard M . Pseudomonas aeruginosa LPS or flagellin are sufficient to activate TLR-dependent signaling in murine alveolar macrophages and airway epithelial cells. PLoS One. 2009;4:e7259. [CrossRef] [PubMed]
Berclaz PY Carey B Fillipi MD . GM-CSF regulates a PU.1-dependent transcriptional program determining the pulmonary response to LPS. Am J Respir Cell Mol Biol. 2007;36:114–121. [CrossRef] [PubMed]
Zhang Z Louboutin JP Weiner DJ Goldberg JB Wilson JM . Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by Toll-like receptor 5. Infect Immun. 2005;73:7151–7160. [CrossRef] [PubMed]
Durum SK Oppenheim JJ . Proinflammatory cytokines and immunity. In: Paul WE ed. Fundamental Immunology. 3rd ed. New York: Raven Press; 1993:801–835.
Xue ML Willcox MD Lloyd A Wakefield D Thakur A . Regulatory role of IL-1beta in the expression of IL-6 and IL-8 in human corneal epithelial cells during Pseudomonas aeruginosa colonization. Clin Exp Ophthalmol. 2001;29:171–174. [CrossRef]
Cole N Krockenberger M Bao S Beagley KW Husband AJ Willcox M . Effects of exogenous interleukin-6 during Pseudomonas aeruginosa corneal infection. Infect Immun. 2001;69:4116–4119. [CrossRef] [PubMed]
Kernacki KA Barrett RP Hobden JA Hazlett LD . Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection. J Immunol. 2000;164:1037–1045. [CrossRef] [PubMed]
Yan XT Tumpey TM Kunkel SL Oakes JE Lausch RN . Role of MIP-2 in neutrophil migration and tissue injury in the herpes simplex virus-1-infected cornea. Invest Ophthalmol Vis Sci. 1998;39:1854–1862. [PubMed]
Thakur A Barrett RP McClellan S Hazlett LD . Regulation of Pseudomonas aeruginosa corneal infection in IL-1 beta converting enzyme (ICE, caspase-1) deficient mice. Curr Eye Res. 2004;29:225–233. [CrossRef] [PubMed]
Huang X McClellan SA Barrett RP Hazlett LD . IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production. J Immunol. 2002;168:5756–5763. [CrossRef] [PubMed]
Hazlett LD Rudner XL McClellan SA Barrett RP Lighvani S . Role of IL-12 and IFN-gamma in Pseudomonas aeruginosa corneal infection. Invest Ophthalmol Vis Sci. 2002;43:419–424. [PubMed]
Cubitt CL Lausch RN Oakes JE . Differential regulation of granulocyte-macrophage colony-stimulating factor gene expression in human corneal cells by proinflammatory cytokines. J Immunol. 1994;153:232–240. [PubMed]
Duan R Remeijer L van Dun JM Osterhaus AD Verjans GM . Granulocyte macrophage colony-stimulating factor expression in human herpetic stromal keratitis: implications for the role of neutrophils in HSK. Invest Ophthalmol Vis Sci. 2007;48:277–284. [CrossRef] [PubMed]
Figure 1.
 
Lack of immune cell contamination in cultured fibroblast cells. Flow cytometry analysis was performed on ulcerated (A) and control (B) fibroblast cells stained with HLA-DR. Analysis did not reveal any difference between unstained fibroblasts (C, E) and HLA-DR–treated fibroblasts (D), indicating the lack of MHC class II cells in fibroblast cultures. HLA-DR–positive cells were detected only when fibroblasts were spiked with human monocytes (acting as positive controls) before staining (F).
Figure 1.
 
Lack of immune cell contamination in cultured fibroblast cells. Flow cytometry analysis was performed on ulcerated (A) and control (B) fibroblast cells stained with HLA-DR. Analysis did not reveal any difference between unstained fibroblasts (C, E) and HLA-DR–treated fibroblasts (D), indicating the lack of MHC class II cells in fibroblast cultures. HLA-DR–positive cells were detected only when fibroblasts were spiked with human monocytes (acting as positive controls) before staining (F).
Figure 2.
 
Increase of TLR4 transcript in ulcerated fibroblast cells after LPS stimulation. Human primary CFs from five ulcer patients and five controls were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with 100 ng/mL LPS (24 hours, SFM). RNA was extracted, and qRT-PCR was performed. Fold change from baseline was significantly larger in ulcerated cells when treated with LPS (U0 vs. U100). In comparison, control cells were relatively less responsive to LPS than at baseline (C0 vs. C100; Mann-Whitney U test; n = 5; P = 0.008). Error bars indicate SEM.
Figure 2.
 
Increase of TLR4 transcript in ulcerated fibroblast cells after LPS stimulation. Human primary CFs from five ulcer patients and five controls were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with 100 ng/mL LPS (24 hours, SFM). RNA was extracted, and qRT-PCR was performed. Fold change from baseline was significantly larger in ulcerated cells when treated with LPS (U0 vs. U100). In comparison, control cells were relatively less responsive to LPS than at baseline (C0 vs. C100; Mann-Whitney U test; n = 5; P = 0.008). Error bars indicate SEM.
Figure 3.
 
TLR4 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and were run under reduced conditions. Blots were separately probed with rabbit anti-TLR4 (1:1000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and TLR4, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 3.
 
TLR4 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and were run under reduced conditions. Blots were separately probed with rabbit anti-TLR4 (1:1000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and TLR4, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 4.
 
TLR4 expression after LPS stimulation. Representative images of human primary CFs from five ulcer patients and five control patients cultured onto glass coverslips. Cells were incubated in SFM (24 hours, 37°C) and treated either before or after LPS (24 hours). Cells were fixed with 4% PFA (10 minutes, RT) and incubated with rabbit-anti-vimentin (1:100, RT, 2 hours) or rabbit anti-TLR4 (1:100, 4°C, 24 hours). All cells expressed vimentin (A). tj;2TLR4 expression was constitutively expressed on control human fibroblast cells (B) and with LPS stimulation (C). Ulcerated fibroblast cells also expressed LPS before (D) and after LPS stimulation (E). In both cell types, nuclear localization of TLR4 staining was observed with ulcer cells showing greater nuclear condensation and cell fragmentation. No signal was observed from fibroblast cells that received no primary antibodies (F).
Figure 4.
 
TLR4 expression after LPS stimulation. Representative images of human primary CFs from five ulcer patients and five control patients cultured onto glass coverslips. Cells were incubated in SFM (24 hours, 37°C) and treated either before or after LPS (24 hours). Cells were fixed with 4% PFA (10 minutes, RT) and incubated with rabbit-anti-vimentin (1:100, RT, 2 hours) or rabbit anti-TLR4 (1:100, 4°C, 24 hours). All cells expressed vimentin (A). tj;2TLR4 expression was constitutively expressed on control human fibroblast cells (B) and with LPS stimulation (C). Ulcerated fibroblast cells also expressed LPS before (D) and after LPS stimulation (E). In both cell types, nuclear localization of TLR4 staining was observed with ulcer cells showing greater nuclear condensation and cell fragmentation. No signal was observed from fibroblast cells that received no primary antibodies (F).
Figure 5.
 
MMP-2 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-2 (1:4000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-2, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 5.
 
MMP-2 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-2 (1:4000, 4°C, 48 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-2, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 6.
 
MMP-9 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-9 (1:4000, 4°C, 24 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-9, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 6.
 
MMP-9 expression after LPS stimulation. Human primary CFs were cultured until confluent. Cells were then incubated in SFM (24 hours, 37°C) and were treated with LPS. Cells were lysed in RIPA buffer and run under reduced conditions. Blots were separately probed with rabbit anti-MMP-9 (1:4000, 4°C, 24 hours) and rabbit β-actin (1:5000, 4°C, 24 hours). Results are shown as the ratio difference between β-actin and MMP-9, with the background subtracted (Mann-Whitney U test). Error bars indicate SEM.
Figure 7.
 
Active MMP-9 expression was altered after LPS treatment. Human primary CFs were grown until confluent. Cells were incubated in SFM (24 hours, 37°C) and treated with LPS at different concentrations (SFM, 24 hours). Supernatant proteins were then normalized and loaded onto gelatin zymograms and were separated using electrophoresis. Metalloprotease activity was successfully blocked with 20 mM EDTA (Mann-Whitney U test). Error bars indicate SEM.
Figure 7.
 
Active MMP-9 expression was altered after LPS treatment. Human primary CFs were grown until confluent. Cells were incubated in SFM (24 hours, 37°C) and treated with LPS at different concentrations (SFM, 24 hours). Supernatant proteins were then normalized and loaded onto gelatin zymograms and were separated using electrophoresis. Metalloprotease activity was successfully blocked with 20 mM EDTA (Mann-Whitney U test). Error bars indicate SEM.
Figure 8.
 
Cytokine expression in human cultured fibroblast cells after LPS stimulation. Human primary CFs from four ulcer patients and four control patients were cultured until confluent and treated with LPS in SFM (24 hours, 37°C). Cells were lysed in RIPA buffer and plated in duplicate at 0.5 mg/mL (total protein) onto a proinflammatory plate. Compared with baseline (0 ng/mL LPS) expression of IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFNγ, and TNFα in ulcerated fibroblast cells (dark gray bars) were significantly increased after 100 ng/mL LPS. In contrast, only IL-1b and IL-10 were significantly increased in control cells (light gray bars) after LPS stimulation. No change was seen with IL-6 in either cell group (Mann-Whitney U test; n = 4). *P < 0.05. Error bars indicate SEM.
Figure 8.
 
Cytokine expression in human cultured fibroblast cells after LPS stimulation. Human primary CFs from four ulcer patients and four control patients were cultured until confluent and treated with LPS in SFM (24 hours, 37°C). Cells were lysed in RIPA buffer and plated in duplicate at 0.5 mg/mL (total protein) onto a proinflammatory plate. Compared with baseline (0 ng/mL LPS) expression of IL-2, IL-8, IL-10, IL-12p70, GM-CSF, IFNγ, and TNFα in ulcerated fibroblast cells (dark gray bars) were significantly increased after 100 ng/mL LPS. In contrast, only IL-1b and IL-10 were significantly increased in control cells (light gray bars) after LPS stimulation. No change was seen with IL-6 in either cell group (Mann-Whitney U test; n = 4). *P < 0.05. Error bars indicate SEM.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×