The CD14 receptor does not mediate entry of Mycobacterium tuberculosis into human mononuclear phagocytes

Abstract

Prior reports have suggested that CD14 mediates uptake of Mycobacterium tuberculosis into porcine alveolar macrophages and human fetal microglia, but the contribution of CD14 to cell entry in human macrophages has not been studied. To address this question, we used flow cytometry to quantify uptake by human monocytes and alveolar macrophages of M. tuberculosis expressing green fluorescent protein. Neutralizing anti-CD14 antibodies did not affect bacillary uptake and the efficiency of bacillary entry was similar in THP-1 cells expressing low and high levels of CD14. However, most internalized bacteria were found in CD14+ but not in CD14− monocytes because M. tuberculosis infection upregulated CD14 expression. We conclude that: (1) CD14 does not mediate cellular entry by M. tuberculosis; (2) M. tuberculosis infection upregulates CD14 expression on mononuclear phagocytes, and this may facilitate the pathogen's capacity to modulate the immune response.

1 Introduction

Mycobacterium tuberculosis is a highly successful human pathogen, infecting one-third of the world's population and causing 1.9 million deaths annually [1]. A distinctive characteristic of M. tuberculosis is its capability to enter and replicate in macrophages. Elucidation of the mechanism by which M. tuberculosis enters macrophages will yield insight into a central feature of disease pathogenesis that may facilitate development of therapeutic interventions. Studies to date have revealed that M. tuberculosis can enter human macrophages through complement receptors, the mannose receptor, scavenger receptors and the surfactant protein A receptor [2,3].

CD14 is a membrane-bound glycoprotein of mononuclear phagocytes that is the receptor for lipopolysaccharide of Gram-negative bacteria and for the mycobacterial homolog lipoarabinomannan [4–6]. Limited evidence suggests that CD14 mediates uptake of M. tuberculosis into macrophages. Anti-CD14 antibodies inhibited phagocytosis of M. tuberculosis by fetal microglia [7] and uptake of Mycobacterium bovis by porcine alveolar macrophages [8]. To determine if CD14 is a receptor that facilitates entry of M. tuberculosis into human mononuclear phagocytes, we studied entry of M. tuberculosis expressing green fluorescent protein (GFP) into cells with variable degrees of CD14 expression.

2 Materials and methods

2.1 Isolation of mononuclear phagocytes

Blood was obtained from 10 healthy tuberculin-negative donors, peripheral blood mononuclear cells (PBMC) were centrifuged on Ficoll-Paque (Amersham Biosciences AB, Uppsala, Sweden) and CD14+ cells were isolated from the monocyte fraction with immunomagnetic beads conjugated to anti-CD14 (Miltenyi Biotech, Auburn, CA, USA).

Bronchoalveolar lavage was performed in three patients who had bronchoscopy for possible cancer. No patient had tuberculosis. Lavage was performed in the lung, where there was no bronchoscopic or radiographic evidence of cancer. Cell preparations that were >98% alveolar macrophages were obtained from bronchoalveolar lavage fluid, as described in [9].

2.2 Preparation of M. tuberculosis expressing GFP

M. tuberculosis H37Ra was transfected with a plasmid encoding the gene for GFP [10], with kanamycin as the selection marker. Transformants were selected on Middlebrook 7H10 medium with 0.5% glycerol and 50 µg ml⁻¹ of kanamycin. Single colonies of brightly fluorescent bacilli were picked and cultured in Middlebrook 7H9 broth with 0.2% glycerol and kanamycin for 5–10 days. Single-cell suspensions of M. tuberculosis were prepared as previously described [11] and were used to infect monocytes. The number of bacteria was estimated by counting in a Petroff–Hauser chamber. The inoculum was also plated on Middlebrook 7H10 medium, and mycobacterial viability was 65–70%.

2.3 Infection of mononuclear phagocytes with GFP-expressing M. tuberculosis

CD14+ monocytes were cultured in Teflon-coated PetriPERM plates (Heraeus Instruments, Hapkinton, MA, USA) at 10⁶ cells ml⁻¹ in RPMI-1640 (Gibco, Fredrick, MD, USA) with 10% heat-inactivated human serum. Under these conditions, cells remained non-adherent for 72–96 h, at which time CD14 was expressed on approximately 50% of the cells.

In some experiments, CD14+ monocytes were adhered to tissue culture plates and incubated for 7 days to obtain monocyte-derived macrophages prior to infection. Alveolar macrophages were cultured in the plates for 72–96 h prior to infection.

In some aliquots of THP-1 cells (ATCC TIB-202), <10% of cells was CD14+, and in other aliquots, 30% was CD14+. The latter was further enriched for CD14 by positive selection with anti-CD14-conjugated magnetic beads. We previously obtained stable transfectants of THP-1 cells containing either empty vector (pRc/RSV) or RSV containing wild-type human full-length CD14, which were cultured using previously published methods [12].

Monocytes, monocyte-derived macrophages, alveolar macrophages, THP-1 cells and THP-1 transfectants were washed three times with serum-free medium, and then infected with GFP-expressing M. tuberculosis at a multiplicity of infection of 20:1. Infected and uninfected control cells were incubated in RPMI-1640 without serum for 4–24 h.

2.4 Effect of anti-CD14 antibodies

Three anti-CD14 antibodies were used: MY4 (Coulter, Hialeah, FL, USA), 63D3 and 60bca [13,14]. A polyclonal anti-CD14 neutralizing antibody (R&D Systems, Minneapolis, MN, USA) produced in sheep was also used. Previous studies have shown that 5–25 µg ml⁻¹ of anti-CD14 monoclonal antibodies effectively blocks CD14 [7,8]. Anti-CD14 antibodies were added in concentrations of 5–35 µg ml⁻¹ to monocytes 60 min prior to infection, and the percentages of infected monocytes were determined after 4 and 24 h, as outlined below.

2.5 Cytofluorometric and fluorescent microscopy analysis

Aliquots of mononuclear phagocytes were washed and stained with phycoerythrin (PE)-labeled anti-CD14 antibodies (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). For each sample, 5×10⁵ cells were incubated with PE-labeled anti-CD14 antibodies at a final concentration of 20 µg ml⁻¹ for 20 min on ice. FACS buffer (phosphate-buffered saline with 10 g l⁻¹ bovine serum albumin and 0.1 g l⁻¹ sodium azide) was used to wash the cells and to dilute the reagents. After incubation with PE-labeled anti-CD14 antibodies, cells were washed three times, and fixed in 1% paraformaldehyde. Samples were analyzed in an EPICS C flow cytometer (Coulter). The isotype control was PE-labeled rat IgG (BD Pharmingen, San Diego, CA, USA). To determine the percentage of infected cells, we gated on live cells and measured the percentage of GFP-expressing cells.

For fluorescent microscopy, infected and uninfected control cells were washed with FACS buffer and fixed in 1% paraformaldehyde. A fluorescent microscope (Olympus BX40, Olympus America, Lake Success, NY, USA) was used to visualize the internalized bacilli, which were counted in a minimum of 300 cells.

2.6 Statistical analysis

Results are shown as the mean±standard error, and values were compared by the Student's t-test or the Wilcoxon rank-sum test, as appropriate.

3 Results

3.1 Effect of neutralizing antibodies on cellular entry by M. tuberculosis

Monocytes are often isolated from PBMC by adherence. However, adherent cell preparations may be contaminated with other cells. To avoid this problem, highly purified monocytes were isolated from PBMC by positive selection with anti-CD14-conjugated magnetic beads. These cells were 96–100% CD14+, as assessed by flow cytometry. After infection with GFP-expressing M. tuberculosis, fluorescent microscopy showed that >95% of the GFP-expressing M. tuberculosis was intracellular.

To determine if CD14 plays a role in mediating entry of M. tuberculosis into mononuclear phagocytes, we incubated monocytes with or without each of the monoclonal anti-CD14 antibodies, MY4, 63D3 or 60bca, a mixture of the three monoclonal antibodies, or with a neutralizing polyclonal anti-CD14 antibody for 60 min, prior to infection with GFP-expressing bacilli. Four and 24 h later, the percentage of infected (GFP+) monocytes was determined. The percentages of infected monocytes in the absence or in the presence of anti-CD14 antibodies were similar (Fig. 1). Isotype control antibodies did not affect the percentage of infected monocytes (data not shown). Serum-free medium was used to infect the cells, so that complement receptors should not play a significant role in mediating intracellular entry of M. tuberculosis under these conditions.

Because previous studies using microglia and porcine alveolar macrophages suggested that CD14 mediated intracellular entry of mycobacteria [7,8], we considered the possibility that CD14 played a role in differentiated macrophages but not in monocytes. Therefore, monocyte-derived macrophages from three individuals were infected with GFP-expressing M. tuberculosis, in the presence or absence of the anti-CD14 antibody MY4, and the percentage of infected cells was measured by flow cytometry. Anti-CD14 did not affect entry of M. tuberculosis into the monocyte-derived macrophages (Fig. 2A). Alveolar macrophages were obtained from three persons, and the percentages of infected cells were counted, using a fluorescence microscope. Anti-CD14 antibodies did not affect the percentages of infected cells (Fig. 2B). The MY4 antibody was functional, as it effectively blocked TNF-α production by monocytes in response to lipopolysaccharide, a process that is CD14-dependent (5828±64 pg ml⁻¹ versus 1865±73 pg ml⁻¹ of TNF-α in the absence and presence of anti-CD14 antibody, respectively). These results are consistent with previously published results on the effects of MY4 on TNF-α production [15].

3.2 Entry of M. tuberculosis into mononuclear phagocytes expressing variable degrees of CD14

As an alternative means to evaluate the role of CD14 in entry of M. tuberculosis into mononuclear phagocytes, we isolated THP-1 cells expressing low (7%) or high (55%) levels of CD14. Four and 24 h after infection with M. tuberculosis, the percentages of infected cells were similar in THP-1 cells expressing low or high levels of CD14, suggesting that CD14 is not required for cellular entry of M. tuberculosis (Fig. 3A).

To more definitively demonstrate that CD14 is not required for entry of M. tuberculosis into mononuclear phagocytes, we studied stable transfectants of THP-1 cells containing either full-length CD14 or an empty vector (RSV) which were 96% and 4% CD14+, respectively. After 4 and 24 h, the percentages of infected cells in CD14-THP-1 transfectants and RSV-THP-1 transfectants were comparable (Fig. 3B).

3.3 Effect of M. tuberculosis infection on CD14 expression by monocytes

The findings above indicate that CD14 does not enhance entry of M. tuberculosis into mononuclear phagocytes. Nevertheless, others have suggested that M. bovis-infected alveolar macrophages express higher levels of CD14 than uninfected macrophages [8]. In addition, 4 h after infection of monocytes from seven donors with GFP-expressing M. tuberculosis, 34±9% of the CD14+ cells was infected with M. tuberculosis, compared to 5.5±2.7% of CD14- cells (P=0.01, data not shown). To determine if M. tuberculosis infection enhances expression of CD14, we isolated CD14+ monocytes from PBMC of three donors. These cells were 99% CD14+, but after incubation for 3–4 days in Teflon-coated plates, CD14 expression was reduced to 50±7%. These monocytes were then exposed to GFP-expressing M. tuberculosis, which infected a subpopulation of the monocytes. After 4 h, cells were stained with PE-labeled anti-CD14, and the percentage of CD14+ infected and uninfected cells was determined by flow cytometry. Prior to infection, 50±7% of the cells was CD14+. After 4 h, this percentage rose to 89±3% in the infected cells, but fell to 22±6% in the uninfected cells (P=0.004, Fig. 4). These findings indicate that M. tuberculosis infection upregulates CD14 expression. We also measured CD14 expression in monocytes from three additional donors. Half of the monocytes were infected with GFP-labeled M. tuberculosis and half were uninfected. Twenty-four hours later, mean CD14 expression by infected and uninfected cells was 79% and 59%, respectively. In addition, fluorescence microscopy confirmed enhanced expression of CD14 after infection of monocytes with M. tuberculosis (data not shown).

4 Discussion

The data in this report suggest that: (1) the CD14 receptor does not play an essential role for entry of M. tuberculosis to human mononuclear phagocytes; (2) M. tuberculosis infection upregulates CD14 expression on mononuclear phagocytes, and this may represent an important mechanism by which the pathogen modulates the immune response.

The question of whether mycobacteria utilize the CD14 receptor to enter mononuclear phagocytes is controversial. Anti-CD14 antibodies inhibit uptake of M. tuberculosis by human fetal microglia, which are derived from monocyte precursors and are phagocytic [7]. In addition, M. bovis preferentially infects porcine alveolar macrophages that express high levels of CD14, and anti-CD14 antibodies inhibit bacillary entry [8]. In contrast, Reiling and colleagues reported that macrophages from CD14+/+ and CD14−/− mice are equally susceptible to entry by Mycobacterium avium[16]. We found that the efficiency of bacillary entry was similar in THP-1 cells expressing low and high levels of CD14, and three different neutralizing monoclonal antibodies to CD14 and a neutralizing polyclonal anti-CD14 antibody did not reduce the capacity of M. tuberculosis to infect monocytes, monocyte-derived macrophages or alveolar macrophages. We believe that the most likely explanation for these results is that CD14 is not an essential receptor for entry of M. tuberculosis into human mononuclear phagocytes. However, we cannot formally exclude the possibility that all the mononuclear phagocytes that we studied expressed very low levels of CD14, and that these low levels were sufficient to mediate mycobacterial entry.

Several factors may explain why our results differ from those of other studies evaluating members of the M. tuberculosis complex. First, the rate of entry of M. tuberculosis into microglia is much slower than that into monocytes or alveolar macrophages [7], suggesting that the receptors used for bacillary entry differ in these cell populations. Second, Khanna and colleagues studied entry of M. bovis into porcine cells, whereas we evaluated entry of M. tuberculosis into human cells [8]. These species differences may explain differences in receptors used by mycobacteria to enter phagocytic cells. For example, closely related Chlamydia species show differential usage of mannose receptors to enter macrophages [17]. We demonstrated that the avirulent H37Ra strain of M. tuberculosis did not utilize CD14 to enter human mononuclear phagocytes. However, our findings may also apply to virulent strains, as both CD14-deficient and wild-type mice have comparable levels of infection in the lungs after aerosol infection with H37Rv [18].

Although our results did not suggest that CD14 was necessary for M. tuberculosis to enter human monocytes, 4 h after infection the majority of infected cells were CD14+. This may imply that M. tuberculosis infection upregulated surface CD14 expression in infected monocytes. CD14 binds to the mannose-rich polysaccharide lipoarabinomannan containing highly branched arabinofuranosyl side chains, which is a major component of the mycobacterial cell envelope of Mycobacterium smegmatis[6]. Lipoarabinomannan in M. tuberculosis strains is a homologous structure that is capped with terminal mannose residues [19]. Mannosylated lipoarabinomannan stimulates significant production of the immunosuppressive cytokine transforming growth factor (TGF)-β, but not of the pro-inflammatory cytokines such as TNF-α and IL-6 [20]. In patients with tuberculosis, enhanced TGF-β production markedly inhibits T-cell responses to M. tuberculosis[21], and TGF-β enhances mycobacterial growth in monocytes [22]. It is intriguing to speculate that upregulation of CD14 expression by M. tuberculosis may contribute to the immunopathogenesis of tuberculosis by facilitating interactions with mannosylated lipoarabinomannan, leading to increased TGF-β production and suppression of the immune response.

Acknowledgements

This study was supported by the Cain Foundation for Infectious Disease Research and the Center for Pulmonary and Infectious Disease Control (CPIDC). P.F.B. holds the Margaret E. Byers Cain Chair for Tuberculosis Research.

References