Mycobacterial Lipomannan Induces Matrix Metalloproteinase-9 Expression in Human Macrophagic Cells through a Toll-Like Receptor 1 (TLR1)/TLR2- and CD14-Dependent Mechanism

Induction of MMP-9 from human macrophages in response to mycobacterial LM.

Mycobacterium kansasii and Mycobacterium chelonae are two opportunistic pathogens that are particularly virulent in immunocompromised patients and are frequently associated with skin lesions and serious pulmonary diseases (14, 15, 18, 26). The capacities of various LM isolated from M. kansasii (LM_Mk), M. chelonae (LM_Mc), and Mycobacterium bovis BCG (LM_BCG; sharing the same structure of LM from M. tuberculosis [24]) for triggering the MMP-9 gene expression was first investigated by reverse transcription (RT)-PCR analysis on differentiated THP-1 cells in serum-free medium. LM from M. chelonae (ATCC 19538), M. kansasii (PHR1 901, a clinical isolate), and M. bovis BCG (Pasteur strain 1173P2) were purified as described previously (12, 13). The endotoxin content of each preparation was <20 pg lipopolysaccharide (LPS)/10 μg as determined by the chromogenic Limulus lysate assay (QCL1000; BioWhittaker, Walkersville, MD). Human promonocytic leukemia THP-1 cells (European Collection of Cell Cultures no. 88081201) were differentiated with 50 nM 1,25-dihydroxy-vitamin D3 (Calbiochem, Darmstadt, Germany) for 72 h (34). To determine the effects of LM on the expression levels of the MMP-9 and TIMP-1 genes, 5 × 10⁶ differentiated THP-1 cells were incubated in 12-well plates in RPMI-glutamine serum-free medium with either increasing LM concentrations or 100 ng/ml of LPS from Escherichia coli 055B5 (Sigma) used as a positive control of MMP-9 induction. Following 24-h incubation at 37°C, total RNA was purified from activated cells by using the Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany), according to the manufacturer's instructions. Reverse transcription was performed with 5 μg of total RNA, in presence of M₁MLVRT reverse transcriptase (Promega, Madison, WI), oligo(dT) primers, deoxynucleotide triphosphate, and RNasin. PCR amplification was performed by using primer pairs (Eurogentec) designed for the specific detection of human MMP-9, TIMP-1, and GAPDH genes, according to the method of Devy et al. (6). The first-strand sequence amplification was performed with GoTaq polymerase (Promega). Each amplification reaction consisted of an initial denaturation at 94°C and multiple cycles (23, 23, and 17 cycles at 68°C, 65°C, and 69°C for MMP-9, TIMP-1, and GAPDH genes, respectively) and a primer extension step at 72°C. PCR products (208, 627, and 256 bp for MMP-9, TIMP-1, and GAPDH genes, respectively) were separated by electrophoresis on a 2% agarose gel, visualized by ethidium bromide staining, and then analyzed with the Bio-Rad GelDoc analysis system (Bio-Rad, Italy).

As illustrated in Fig. 1A, each LM type was found to up-regulate MMP-9 mRNA synthesis in a dose-dependent manner, whereas in unstimulated cells, no specific product was detected. The induction of MMP-9 mRNA was maximal with 10 μg/ml of LM. LM_Mk, LM_Mc, and LM_BCG displayed similar MMP-9-inducing activities. Expression levels of GAPDH mRNA, a housekeeping gene product included as an internal control, remained unchanged, regardless of the concentration of LM used.

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FIG. 1.

(A) MMP-9 gene (MMP-9) expression in differentiated THP-1 cells in response to LM. Differentiated human promonocytic THP-1 cells were incubated for 24 h with 0.1 to 20 μg/ml of LM purified from M. kansasii (KanLM), M. chelonae (CheLM), or M. bovis BCG (BCGLM) in RPMI-glutamine serum-free medium. LPS from E. coli 055B5 (100 ng/ml) was included as a positive control. Levels of expression of MMP-9 and GAPDH mRNA were determined by RT-PCR as described in the text. GAPDH was used as a constitutively expressed marker. The ratio of fluorescence intensities of MMP-9 and GAPDH (GAPDH) gene PCR products are plotted in the bar graphs. Results are representative of at least three independent experiments. Bar charts are expressed as mean values ± standard errors of the means (SEM). The levels of statistical significance of differences in comparison with unstimulated cells were calculated by using the Student t test (one asterisk indicates a P value of <0.01). (B) Effect of anti-TNF-α neutralizing antibodies on MMP-9 mRNA expression in differentiated THP-1 cells stimulated with LM. Differentiated THP-1 cells were stimulated for 24 h with LM_Mk and LM_Mc in the absence or presence of 5 μg/ml anti-TNF-α neutralizing antibodies (Abs). Levels of expression of MMP-9 and GAPDH mRNA were determined by RT-PCR and expressed as described above. Results are representative of three independent experiments. The statistical significance of differences in comparison with untreated cells by neutralizing Abs was calculated by using the Student t test (two asterisks, P < 0.05; ns, nonsignificant value). (C) MMP-9 gene expression in human primary macrophagic cells in response to LM. Human macrophages derived from blood monocytic cells in the presence of macrophagic colony stimulating factor were incubated for 24 h in RPMI-glutamine serum-free medium and stimulated with either 10 μg/ml of each LM subtype or 100 ng/ml of E. coli 055B5 lipopolysaccharide (LPS). Levels of expression of MMP-9 and GAPDH mRNA were determined by RT-PCR and expressed as described above. Data are representative of one experiment. Similar results were obtained from at least three independent experiments performed with different blood donors. The statistical significance of differences in comparison with unstimulated cells was calculated by using the Student t test (two asterisks, P < 0.05).

Tumor necrosis factor alpha (TNF-α) was previously shown to stimulate secretion of MMP-9 in monocytes/macrophages (20). Although LM is a poor TNF-α-inducing factor in the absence of serum (35), we examined the contribution of low levels of TNF-α released in culture supernatants to induced MMP-9 induction. Hence, differentiated THP-1 cells were stimulated for 24 h at 37°C with 1 or 10 μg/ml of LM_Mc and LM_Mk, in fetal calf serum-free RPMI medium, and in the absence or presence of 5 μg/ml of TNF-α-neutralizing goat polyclonal antibodies (R&D Systems, Minneapolis, MN). Neutralization of TNF-α reduced only weakly the secretion of MMP-9 with at most 22% inhibition (Fig. 1B). These results suggest that the vast majority of MMP-9 induced by LM in absence of serum is secreted through a TNF-α-independent mechanism.

The MMP-9-inducing capacity of LM was next examined on human blood monocyte-derived macrophages. Human peripheral blood mononuclear cells were isolated by density gradient centrifugation on Lymphoprep separation medium (Nycomed, Oslo, Norway), and adherent monocytes were collected after lymphocyte depletion. Purity of the population was evaluated by flow cytometry analysis (FACScalibur cytometer; Becton Dickinson). Macrophages were derived from monocytes in the presence of 100 ng/ml macrophagic colony-stimulating factor for 48 h in RPMI-glutamine supplemented with 10% heat-inactivated fetal calf serum. Macrophages were then stimulated with 10 μg/ml of LM for 24 h and processed for RT-PCR analysis, as described above. Figure 1C shows that all LM subtypes were potent MMP-9-inducing factors in primary human macrophages. Although LM_BCG, LM_Mc, and LM_Mk display subtle structural differences pertaining mainly to the nature of fatty acids bound to anchor and the linkage and the degree of substitution in the mannopyranosyl side chains of the mannan core (2, 24), they behave similarly in up-regulating MMP-9 synthesis, suggesting that the MMP-9-inducing activity is a common feature to all mycobacterial LMs.

The amount of MMP-9 protein released in culture supernatants of differentiated THP-1 cells (2 × 10⁵ cells/well) was next quantified by enzyme-linked immunosorbent assay, after 48 h of cell incubation with either LM_Mk or LM_Mc in serum-free RPMI medium. Microtiter plates were coated overnight at 4°C with aliquots of cell supernatants, blocked with phosphate-buffered saline (PBS)-0.05% Tween 20-1% bovine serum albumin, and then incubated with biotin-conjugated anti-MMP-9 antibodies (1/500 dilution; Santa Cruz Biotechnology, CA), prior to the addition of extravidin-conjugated peroxidase (1/1,000 dilution) for 30 min. Detection was performed with o-phenylenediamine-dihydrochloride and by reading the absorbance at 490 nm. MMP-9 concentrations in culture supernatants were quantified according to a standard curve generated with human recombinant pro-MMP-9 (R&D Systems, Minneapolis, MN) at concentrations ranging from 2 to 30 ng/ml. LM induced the secretion of MMP-9 in a dose-dependent manner, and the levels of MMP-9 secreted from cells stimulated with LM_Mk and LM_Mc were approximately six and five times higher, respectively, than that from unstimulated cells (Fig. 2A).

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FIG. 2.

Secretion of MMP-9 in the culture supernatant of differentiated THP-1 cells stimulated with LM. LM_Mk, KanLM; LM_Mc, CheLM. (A) Differentiated THP-1 cells were stimulated with increasing concentrations of LM_Mk or LM_Mc in RPMI-glutamine medium. Culture supernatants were collected after 48 h and assayed by enzyme-linked immunosorbent assay for MMP-9 secretion. Data presented are from one representative experiment out of three independent experiments with similar results. Results are expressed as mean values ± standard deviations of triplicate wells. The P value for the comparison between untreated and stimulated cells was <0.01. (B) Release of the MMP-9 protein form was investigated by detecting the gelatinase activity by zymography analysis. Differentiated THP-1 cells were incubated with 10 μg/ml of LM_Mk and LM_Mc, for 48 h. Control cells (−) were left unstimulated. (C) Western blot analysis using specific anti-human MMP-9 antibodies. Data are representative of four independent experiments for zymography and two experiments for Western blot analysis.

MMP-9 is secreted from macrophages as a latent precursor form of 92-kDa pro-MMP-9, in which the propeptide can be cleaved by proteases, generating the mature 84-kDa MMP-9 form (11, 36). Both the pro-MMP-9 form and the mature form can be detected by gelatin zymography analysis. This technique was applied to determine the identity of the MMP-9 form released in response to LM stimulation. Therefore, the supernatants of differentiated THP-1 cells activated for 48 h with 10 μg/ml of either LM_Mk or LM_Mc were loaded on a 7.5% sodium dodecyl sulfate gel impregnated with 2 mg/ml gelatin and processed according the procedure described by Leber and Balkwill (17). Densitometric analysis of proteolytic bands was performed by using the Image1.61 analysis program. Figure 2B shows the gelatinolytic activity found in the supernatant of cells treated with LM_Mc or LM_Mk, characterized by intense bands comigrating with the recombinant 92-kDa pro-MMP-9, while the mature 84-kDa form remained undetectable. The presence of pro-MMP-9 was also confirmed by Western blot analysis (Fig. 2C). For this aim, proteins present in the conditioned medium obtained after 48 h of culture with LM were precipitated with cold ethanol, lyophilized, and resuspended in sodium dodecyl sulfate-Laemmli loading buffer, as reported by Zhang et al. (40). Samples were then electrophoresed on a 10% polyacrylamide gel and transferred onto nitrocellulose membrane, which was subsequently blocked with PBS-0.1% Tween 20 buffer containing 5% nonfat dry milk, probed with primary mouse immunoglobulin G (IgG) antibodies of anti-MMP-9 recognizing both the pro-MMP-9 and the cleaved forms (1/250; Santa Cruz Biotechnology) in PBS-0.05% Tween 20-0.1% bovine serum albumin for 2 h, and incubated with horseradish peroxidase-conjugated anti-mouse IgG antibodies (1/4,000; Santa Cruz). Detection of MMP-9 was done using the enhanced chemiluminescence detection system (Amersham). Altogether, zymography and Western blot analysis indicate that LM stimulation induces only the release of the latent pro-MMP-9 form. Whether LM also regulates the activity of extracellular serine proteases secreted from immune cells at the inflammatory sites which convert the latent pro-MMP-9 to the mature MMP-9 remains an attractive hypothesis that remains to be elucidated.

Induction of MMP-9 gene by LM is mediated through a TLR1/TLR2- and CD14-dependent pathway.

In an attempt to identify receptors present on the surface of differentiated THP-1 cells that recognize LM and transmit a positive signal for MMP-9 expression, we evaluated the effects of various neutralizing antireceptor antibodies, known to inhibit biological activities of microbial products (21, 29, 31, 35), on MMP-9 synthesis. Differentiated cells were pretreated for 30 min at 37°C with 5 μg/ml of neutralizing antibodies (purchased from BD Biosciences, BD Pharmingen, San Diego, CA, or HBT, Uden, The Netherlands), and then 1 μg/ml of LM_Mk was added to the cells for 24 h to allow MMP-9 gene expression. The corresponding isotype controls were used as negative controls. As illustrated in Fig. 3, pretreatment with anti-TLR2 (mouse IgG2a clone TL2-1), anti-TLR1 (mouse IgG1κ clone GD2.F4), and anti-CD14 (mouse IgG1 clone MEM-18) antibodies inhibited MMP-9 gene expression by 48%, 37%, and 66%, respectively. Surprisingly, the anti-mannose receptor antibody (mouse IgG1κ clone 19), previously shown to interfere with MMP-9 gene expression elicited by mannose lipoarabinomannan of M. tuberculosis (31), displayed only a residual inhibitory effect (8%). Antibodies raised against TLR4 (mouse IgG2aκ clone HTA 125), a potent LPS-responsive receptor, failed to inhibit MMP-9 gene expression. Incubation with the corresponding control isotype antibodies did not alter MMP-9 mRNA synthesis (data not shown). Altogether, these results suggest the participation of several pattern recognition receptors, in particular, TLR1/TLR2 and CD14, in the signaling pathway leading to MMP-9 induction. These results are consistent with previous studies demonstrating that macrophage activation by LM_Mk or LM_Mc is mediated by TLR2 and CD14 but is independent of TLR6 and TLR4 (2, 29, 35). Interestingly, the TLR1/TLR2 heterodimer triggers, through the adaptor protein MyD88, the activation of several signal transduction molecules, leading to the activation of NF-κB and mitogen-activated protein kinases in macrophages (33). Rivera-Marrero et al. (31) demonstrated that induction of MMP-9 by mannose lipoarabinomannan from M. tuberculosis required the transcriptional activation of AP-1 through mitogen-activated protein kinases. According to these authors, the addition of PD-98059, a MEK1 inhibitor, resulted in a total inhibition of MMP-9 induction (31). In preliminary experiments, we have tested the effect of PD-98059 and observed that, at 50 μM, this molecule strongly inhibited LM-induced MMP-9 expression in differentiated THP-1 cells (not shown), suggesting that MEK1 may participate in this transduction signal.

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FIG. 3.

Inhibition of the MMP-9 expression by blocking pattern recognition receptors. Differentiated THP-1 cells were pretreated with 5 μg/ml of anti-CD14, anti-TLR1, anti-TLR2, anti-mannose receptor, or control isotype antibodies for 30 min at 37°C, prior to the addition of LM_Mk (1 μg/ml). After 24 h of incubation, expression levels of MMP-9 and GAPDH mRNA were determined by RT-PCR analysis as described above. Results are expressed as percentages of the MMP-9/GAPDH mRNA expression ratio obtained with LM_Mk. The ratio values obtained in the absence of antibodies were arbitrarily placed at 100%. Data presented represent the mean values ± SEM of three independent experiments. Statistical analyses were performed by using Student's t test to compare antireceptor Ab-treated cells with nontreated cells (two asterisks, P < 0.05; ns, nonsignificant).

Modulation of TIMP-1 transcription by LM.

Alteration in the relative expression of TIMPs has been ascribed in many lung disorders characterized by inflammation, tissue fibrosis, and destruction (1, 5). TIMP-1 is an endogenous key regulating molecule, produced by macrophages, which forms a bimolecular complex with MMP-9 to inactivate the enzyme (1). TIMP-1 mRNA expression levels were examined in differentiated THP-1 cells stimulated with LM, following a procedure similar to that described for MMP-9 mRNA expression. In contrast to MMP-9 mRNA, TIMP-1 mRNA was constitutively expressed in unstimulated THP-1 cells (Fig. 4). However, transcription of the TIMP-1 gene was significantly inhibited after 24 h of exposure to 10 μg/ml of LM. Stimulation with either LM_Mc or LM_Mk resulted in a reproducible 40% or 33% decrease in TIMP-1 gene expression compared to unstimulated cells. Altogether, disruption of the MMP-9/TIMP-1 gene expression balance by increasing of pro-MMP-9 and inhibiting TIMP-1 expression results in a net activation of MMP-9.

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FIG. 4.

TIMP-1 mRNA expression in differentiated THP-1 cells in response to LM. Differentiated THP-1 cells were incubated for 24 h with 10 μg/ml of either LM_Mk (KanLM) or LM_Mc (CheLM) in RPMI-glutamine serum-free medium. Expression levels of TIMP-1 and GAPDH mRNA were determined by RT-PCR analysis. The ratios of the fluorescence intensities of TIMP-1 and GAPDH PCR products are plotted in the bar graph. Results are representative of at least three independent experiments. Bar charts are expressed as mean values ± SEM (statistical analyses were performed by using Student's t test [two asterisks, P < 0.05]).

The present study demonstrates for the first time that LM, which represent major cell wall lipoglycans, are potent pro-MMP-9-inducing factors from human macrophages. Overall, these results suggest that LM may play a major functional role in the mycobacterial immunopathology through the induction of MMP-9. This may be a critical step towards controlling pathogen growth and influencing the outcome of the infection as well as host tissue injury. Recent work suggests that MMP-9 participates directly in tissue destruction in tuberculosis patients (28). However, whether LM contributes to tissue remodeling and damage processes occurring during the course of mycobacterial infection requires further work. In vivo administration of LM in mice followed by immunohistochemical detection of MMP-9 and TIMP-1 will help to define the eventual participation of LM in tissue damage.