CD14 Mediates Cross Talk between Mononuclear Cells and Fibroblasts for Upregulation of Matrix Metalloproteinase 9 by Borrelia burgdorferi

Patients.Serum was obtained from patients who met the Centers for Disease Control and Prevention clinical criteria for the diagnosis of acute Lyme disease. Serum was also obtained from healthy donors. Written informed consent was obtained from each study subject. Human experimentation guidelines of the U.S. Department of Health and Human Services and of the Boston University and Boston Medical Center institutional review boards were followed in the conduct of this research.

Antibodies and recombinant proteins.Monoclonal anti-human MMP-9, monoclonal anti-human MMP-2, monoclonal anti-human CD14 antibodies, recombinant MMP-9, recombinant MMP-2, and recombinant CD14 (rCD14) (endotoxin level, <0.1 ng per 1 μg of the cytokines, determined by the Limulus amebocyte lysate method) were purchased from R&D Systems (Minneapolis, MN). Polyclonal antibody against Toll-like receptor 2 (TLR2), polyclonal antibody against TLR4, polyclonal antibody against CD14, normal human immunoglobulin G1 (IgG1), and mouse IgG1 were purchased from Santa Cruz Biotechnology (Santa Cruz).

Culture of spirochetes.Low passages (passages 2 to 5) of B. burgdorferi strain N40 or B31 were cultured in Barbour-Stoenner-Kelly medium (Sigma). All spirochetes were grown to logarithmic phase, harvested by centrifugation, washed three times, and resuspended in cell culture medium.

Cell culture.U937 cells (ATCC CRL-1593.2; ATCC, Manassas, VA) were cultured in RPMI 1640 medium supplemented with 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/liter glucose, 1.5 g/liter sodium bicarbonate, and 10% fetal bovine serum (FBS). The human fibroblasts (ATCC SCRC-1042.2) were cultured in Dulbecco's modified Eagle medium with 4.5 g/liter glucose, 1.5 g/liter sodium bicarbonate, and 10% FBS. The cells of three to four passages at 80% confluence were changed to fresh medium containing 10% FBS or 1% FBS and subjected to various treatments.

Zymography.Gelatin zymography was performed using 10% Tris-glycine gels with 0.1% gelatin incorporated as a substrate according to the manufacturer's instructions (Invitrogen). The supernatants from the cultured cells were diluted with deionized water and sodium dodecyl sulfate sample buffer (2×) without heating prior to loading into gels. For inhibition studies 1,10-phenanthroline (10 mM) was included in the developing buffer prior to overnight incubation at 37°C. Gels were stained with Coomassie blue (0.5% [wt/vol]) and destained using an acetic acid destaining solution. Gelatinase activities were visualized as clear bands indicating proteolysis of the substrate. Zymographic results were confirmed by three or more replicate experiments. The Kodak Scientific Image System (Eastman Kodak) was used to quantify the data.

Isolation of RNA.Total RNA from the cultured cells was isolated using TRIZOL (Life Technologies) according to the manufacturer's instructions. Briefly, the cultured cells were lysed with TRIZOL reagent and were then mixed with chloroform, incubated on ice for 5 min, and centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase containing the RNA was transferred to a fresh tube and the RNA precipitated with isopropyl alcohol and centrifuged. The pellet containing the RNA was washed with ethanol and dissolved in RNase-free water. The amount and purity of the RNA were determined by measurement of optical density 260/280 and RNA electrophoresis on denaturing agarose gels.

MMP mRNA determination.Human MMP Nonrad-GEArrays (SuperArray Inc.) was used to analyze the mRNA expression of MMPs. Total RNA from the cultured cells was converted to biotinylated cDNA probes by reverse transcription with a deoxyribonucleoside triphosphate mix containing biotin-16-2′-deoxy-uridine-5′-triphosphate (Biotin-dUTP). Biotinylated cDNA probes were hybridized to gene-specific cDNA fragments spotted on the membranes. The GEArray membrane was then blocked with GEAblocking solution and incubated with alkaline phosphatase-conjugated streptavidin. The relative expression levels of multiple MMP genes were detected by chemiluminescence using the alkaline phosphatase substrate, CDP-Star. The membrane was exposed to X-ray film for various lengths of time to determine the abundances of different transcripts. Semiquantitative expressions of genes were done by using Quantity One Software (Bio-Rad) and normalized with the internal controls, glyceraldehyde-3-phosphate dehydrogenase or β-actin.

Real-time RT-PCR.Real-time reverse transcription-PCR (RT-PCR) was performed with a TaqMan ABI 7000 sequence detection system (Applied Biosystems) using the TaqMan One-Step RT-PCR Master Mix reagents kit according to the manufacturer's instructions. 18S rRNA proprietary fluorescent dye-minor groove binder (VIC-MGB; Applied Biosystems) served as an internal control to assess the overall cDNA content. The oligonucleotide primers (300 nM) and probes (250 nM) were used for this study. PCR primers and TaqMan probes specific for the target molecules were designed using Primer Express software (Applied Biosystems). MMP-9 primers were as follows: 391F, 5′-GATCCAAAACTACTCGGAAGACTTG-3′; 454R, 5′-GAAGGCGCGGGCAAA-3′; MMP-9 probe, 417T, 6FAM-CGCGGGCGGTGATTGACGAC-TAMRA. CD14 primers were as follows: 879F, 5′-CGCTCCGAGATGCATGTG-3′; 938R, 5′-AGCCCAGCGAACGACAGAT-3′; CD14 probe, 6FAM-CCAGCGCCCTGAACTCCCTCT-TAMRA. TLR2 primers were as follows: 2085F, 5′-CTACTGGGTGGAGAACCTTATGGT-3′; 2160R, 5′-CCGCTTATGAAGACACAACTTGA-3′; TLR2 probe, 6FAM-CAGGAGCTGGAGAACTTCAATCCCCC-TAMRA.

Thermal cycling parameters for use with the TaqMan One-Step RT-PCR Master Mix reagents kit were as follows: reverse transcription, 48°C for 30 min; AmpII Taq Gold activation, 95°C for 10 min; there were 40 PCR cycles. A PCR cycle consisted of heating to 95°C for 15 s to denature and 60°C for 1 min for annealing/extension. Relative gene expression was calculated manually from the exported results, following the instructions of the ABI PRISM 7000 sequence detection system's manufacturer.

Western blot analysis.The cell lysates or supernatants from the cultured cells were electrophoresed on a 4 to 20% sodium dodecyl sulfate-polyacrylamide gel and electrophoretically transferred to polyvinylidene fluoride membranes. Membranes were blocked overnight at 4°C with 5% nonfat milk in TBST (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1.5 mM MgCl₂, 0.05% Tween 20) and incubated with the primary antibodies for 1 h. After three washes in TBST (10 min for each wash), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies in TBST. Immunoreactivity was detected using an enhanced chemiluminescence system (Amersham).

Enzyme-linked immunosorbent assay (ELISA).Human MMP-9 (total) and sCD14 (R&D Systems, Minneapolis, MN) were measured by immunoassay according to the manufacturer's instructions. Briefly, the serum was diluted 200-fold for sCD14 and 100-fold for MMP-9 using a calibrator diluent. The optical density of each well was determined within 30 min, using a microplate reader set to 450 nm and 540 nm.

Statistics.The results were presented as means ± standard deviations and were analyzed using the Student t test. Statistical significance was determined to be a P value of <0.05.

Concentrations of MMP-9 and sCD14 in serum from the patients with acute Lyme disease.The concentration of MMP-9 and/or sCD14 in serum from patients with acute Lyme disease was determined by ELISA. As shown in Fig. 1, the concentrations of both of MMP-9 (Fig. 1A) and sCD14 (Fig. 1B) were significantly elevated for patients with acute Lyme disease compared to levels for healthy controls (P < 0.05).

• Open in new tab
• Download powerpoint

FIG. 1.

Concentrations of MMP-9 and sCD14 in serum from patients with acute Lyme disease. (A) Concentration of MMP-9 in serum from patients with acute Lyme disease (LD) (n = 43) compared to that for healthy controls (n = 29), determined by using ELISA. *, P < 0.05. (B) Concentration of sCD14 from the patients with acute Lyme disease (n = 8) compared to that for healthy controls (n = 8), determined by using ELISA. *, P < 0.05. ELISAs were repeated twice in duplicate.

Upregulation of MMP-9 by stimulation of B. burgdorferi in U937 cells and fibroblasts.As assessed by gelatin zymography, the upregulation of the 92-kDa protein by B. burgdorferi was time and concentration dependent in U937 cells. The activity of the 72-kDa protein was not significantly different in the presence or absence of B. burgdorferi (Fig. 2A and B). Western blot analysis demonstrated that not only was the expression of MMP-9 (92 kDa) in the supernatants collected from U937 cells elevated (Fig. 2C), the expression of MMP-9 (92 kDa) was also increased in U937 cell lysates, as determined using monoclonal antibody against MMP-9 (Fig. 2D) and monoclonal antibody against MMP-2 (for MMP-2, 72 kDa, data not shown). β-Actin was used as a control (Fig. 2E). All gelatinolytic activity was inhibited when zymography was performed in the presence of 10 mM 1,10-phenanthroline, a specific inhibitor of MMPs. Stimulation of MMP-9 production by B. burgdorferi in U937 cells was not affected by 50 μg/ml polymyxin B, an inhibitor of LPS activation (data not shown).

• Open in new tab
• Download powerpoint

FIG. 2.

Upregulation of MMP-9 by stimulation of B. burgdorferi in U937 cells. U937 cells were incubated in the presence or absence of B. burgdorferi (10⁶ organisms/ml) for 0 to 72 h or of B. burgdorferi (0 to 10⁶ organisms/ml) for 48 h. The activities of 92-kDa and 72-kDa proteins with stimulation by B. burgdorferi were analyzed by zymography in a time (A) or concentration (B) pattern (St, standard). Western blotting was performed with monoclonal antibody against human MMP-9 (92 kDa) or with monoclonal antibody against human MMP-2 (72 kDa; data not shown) in the supernatants (equal volume, 10 μl/well) (C) or in the cell lysates (D). β-Actin was used as a control (E). mRNA expression of MMP-9 in U937 cells with B. burgdorferi was assayed using real-time RT-PCR (F) or a targeted gene array of MMPs (G). *, P < 0.01; n = 3. Zymography and Western blot experiments were repeated three times, and representative experiments are shown. Real-time RT-PCR and targeted gene array experiments were repeated three times, and data presented are mean values for three separate experiments performed in duplicate.

The mRNA expression of MMP-9 was also significantly increased in U937 cells in the presence of B. burgdorferi, as determined using real-time RT-PCR (Fig. 2F) and human MMP Nonrad-GEArrays (Fig. 2G).

Functional activity of MMP-9 was not detected in fibroblasts in the presence or absence of B. burgdorferi by using gelatin zymography and a targeted gene array (data not shown). However, MMP-9 mRNA expression was detected when the more-sensitive technique, real-time RT-PCR, was used. The expression of MMP-9 was not changed in fibroblasts with B. burgdorferi, using real-time RT-PCR (Fig. 3). Nonetheless, when fibroblasts were incubated with the conditional medium (CM) (0.5 ml/ml) from the supernatants of U937 cells with B. burgdorferi, the expression of MMP-9 was significantly increased compared to results for fibroblasts with normal cultured medium (1 ml) (Fig. 3). Furthermore, MMP-9 expression in fibroblasts with conditional medium (0.5 ml/ml) was much higher when B. burgdorferi was added than for fibroblasts with normal cultured medium (1 ml) in the presence of B. burgdorferi (Fig. 3). However, there was no effect in the activity of MMP-9 in fibroblasts when the supernatants from U937 cells without B. burgdorferi were used.

• Open in new tab
• Download powerpoint

FIG. 3.

mRNA expression of MMP-9 in fibroblasts. Fibroblasts were incubated in the presence or absence of supernatants of cultured U937 cells with B. burgdorferi (CM, 0.5 ml/ml) in the presence or absence of B. burgdorferi. mRNA expression of MMP-9 was analyzed by real-time RT-PCR. HF, human fibroblasts; Bb, Borrelia burgdorferi. For HF plus CM versus HF only, *, P < 0.05; for HF plus CM plus Bb versus HF plus CM or HF plus Bb, **, P < 0.01; n = 5. The real-time RT-PCR experiments were repeated five times, and data presented are mean values for five separate experiments performed in duplicate.

Regulation of CD14 expression by stimulation of B. burgdorferi in U937 cells and fibroblasts.We assessed whether the expression of MMP-9 in U937 cells and fibroblasts with B. burgdorferi involved a CD14 signaling pathway. The U937 cells and fibroblasts were incubated in the presence or absence of B. burgdorferi. In Western blot analysis, not only the expression of mCD14 (55 kDa) (Fig. 4A) but also the expression of sCD14 (48 kDa) (Fig. 4C and D, equal volume of 10 μl/well of supernatants) in U937 cells was elevated in the presence of B. burgdorferi in a time- and dose-dependent manner. β-Actin was used as a control (Fig. 4B). The expression pattern of CD14 showed the same time-dependent trend as that of MMP-9 by stimulation of B. burgdorferi.

• Open in new tab
• Download powerpoint

FIG. 4.

Upregulation of CD14 by stimulation of B. burgdorferi in U937 cells. Western blotting was performed with cell lysates of U937 with or without B. burgdorferi (10⁶ organisms/ml) using monoclonal antibody against human CD14 (55 kDa; membrane CD14) (A) or β-actin (B). Western blotting was also performed with supernatants of U937 cells with or without B. burgdorferi with a time-dependent (C) or concentration-dependent (D) pattern (48 kDa; sCD14) (equal volume of 10 μl/well of supernatants). RNA from treated U937 cells was isolated. mRNA expression of CD14 (E) or TLR2 (F) was determined in U937 cells with or without B. burgdorferi, using real-time RT-PCR. *, P < 0.01; n = 3. Western blot experiments were repeated three times, and representative experiments are shown. Real-time RT-PCR experiments were repeated three times, and data presented are mean values of three separate experiments performed in duplicate.

The mRNA expression of CD14 was also significantly increased from U937 cells in the presence of B. burgdorferi, using real-time RT-PCR (Fig. 4E). Moreover, the expression of TLR2 was not significantly changed from U937 cells in the presence of B. burgdorferi (Fig. 4F). However, the expression of CD14 (mCD14 or sCD14) in fibroblasts was not found in the presence or absence of B. burgdorferi (data not shown).

Effect of anti-CD14 antibody on upregulation of MMP-9 by B. burgdorferi in U937 cells and fibroblasts.To determine whether the upregulation of MMP-9 by B. burgdorferi was associated with the CD14 signaling pathway specifically, the U937 cells and/or fibroblasts were cultured with or without anti-CD14 antibody (monoclonal or polyclonal antibodies). When anti-CD14 antibody was added to U937 cells, the upregulation of MMP-9 by B. burgdorferi was partially blocked in a concentration-dependent manner (Fig. 5A and B).

• Open in new tab
• Download powerpoint

FIG. 5.

Effect of anti-CD14 antibody on production of MMP-9. (A) Gelatinolytic activity of MMP-9 in U937 cells. Lane 1, medium control; lanes 2 to 7, Borrelia burgdorferi (Bb), 10⁶ organisms/ml (lane 2, Bb only; lane 3, Bb plus mouse IgG [3 μg/ml]; lane 4, Bb plus monoclonal anti-CD14 antibody [50 ng/ml]; lane 5, Bb plus monoclonal anti-CD14 antibody [100 ng/ml]; lane 6, Bb plus monoclonal anti-CD14 antibody [1 μg/ml]; lane 7, Bb plus monoclonal anti-CD14 antibody [3 μg/ml]). Zymography experiments were repeated three times, and representative experiments are shown. (B) Quantitative analysis of activity of MMP-9 from values in all three separate experiments, using Kodak digital image analysis software. For 6 and 7 versus 2, *, P < 0.01; n = 3.

As mentioned above, not only the expression of mCD14 (Fig. 4A) but also the expression of sCD14 (Fig. 4C and D) in U937 cells was elevated in the presence of B. burgdorferi in a time- and dose-dependent manner as determined by Western blotting. When fibroblasts were incubated with CM (containing CD14), the expression of MMP-9 was significantly increased. Furthermore, the expression of MMP-9 was much higher when B. burgdorferi was added than for fibroblasts with normal cultured medium. This effect was completely blocked by anti-CD14 antibody (Fig. 3).

Recombinant CD14 directly activates MMP-9 enzymatic activity in fibroblasts.To determine whether the upregulation of MMP-9 in fibroblasts was associated with the expression of CD14, we incubated the fibroblasts with or without rCD14 (endotoxin level, <0.1 ng per 1 μg of the cytokines, determined by the LAL method). Indeed, the activity of MMP-9 was significantly elevated when fibroblasts were preincubated with rCD14 itself, and the activity of MMP-9 was much increased, especially in the presence of rCD14 and with B. burgdorferi, as determined by zymography (Fig. 6A and B). Expression of MMP-9 was also significantly upregulated by rCD14 as determined using real-time RT-PCR (Fig. 6C). The effect was completely abolished by anti-CD14 antibody (Fig. 6A, B, and C). MMP-9 production by rCD14 with or without B. burgdorferi in fibroblasts was not affected by 50-μg/ml polymyxin B, an inhibitor of LPS activation.

• Open in new tab
• Download powerpoint

FIG. 6.

Recombinant sCD14 directly activated MMP-9 enzymatic activity in human fibroblasts (HF). (A) Gelatinolytic activity of MMP-9 in HF in the presence or absence of recombinant CD14 (rCD14) and/or anti-CD14 antibody. Lane 1, HF medium control; lane 2, HF plus Borrelia burgdorferi (Bb); lane 3, HF plus Bb plus rsCD14 (1 μg/ml) plus anti-CD14 (3 μg/ml); lane 4, HF plus rCD14 (1 μg/ml); lane 5, HF plus rCD14 (1 μg/ml) plus Bb; lane 6, HF plus rCD14 (1 μg/ml) plus Bb plus mouse IgG (3 μg/ml). Zymography experiments were repeated four times, and representative experiments are shown. (B) Quantitative analysis of activity of MMP-9 from values in all four separate experiments, using Kodak digital image analysis software. 5 versus 4 or 1, **, P < 0.01; 4 versus 1, *, P < 0.05; n = 4. (C) mRNA expression of MMP-9 in fibroblasts with or without recombinant CD14 and/or anti-CD14 antibody. HF plus rsCD14 plus Bb versus HF plus rsCD14 or HF only, **, P < 0.01; HF plus rCD14 versus HF only, *, P < 0.05; n = 5. Real-time RT-PCR experiments were repeated five times, and data presented are mean values for five separate experiments performed in duplicate.