Dendritic cells phagocytose and are activated by Treponema pallidum

Abstract

Cell-mediated immune processes play a prominent role in the clinical manifestations of syphilis, a sexually transmitted disease of humans caused by spirochetal bacterium Treponema pallidum. The immune cell type that initiates the early immune response to T. pallidum thus far has not been identified. However, dendritic cells (DCs) are the first immune-competent cells to encounter antigens within skin or mucous membranes, the principal sites of early syphilitic infection. In the present study, immature DC line XS52, derived from murine skin, was utilized to examine T. pallidum-DC interactions and subsequent DC activation (maturation). Electron microscopy revealed that T. pallidum was engulfed by DCs via both coiling and conventional phagocytosis and was delivered to membrane-bound vacuoles. The XS52 DC line expressed surface CD14 and mRNA for Toll-like receptors 2 and 4, molecules comprising important signaling components for immune cell activation by bacterial modulins. Both T. pallidum and a synthetic lipopeptide (corresponding to the 47-kDa major membrane lipoprotein) activated the XS52 DC line, as indicated by the secretion of interleukin-12 (IL-12), IL-1beta, tumor necrosis factor alpha, and IL-6 and elevated surface expression of CD54. The combined data support the contention that DCs stimulated by T. pallidum and/or its proinflammatory membrane lipoproteins are involved in driving the cellular immune processes that typify syphilis.

FIG. 1

XS52 cells express mRNA for tlr2 and tlr4. (A) Total RNA was extracted from RAW cells and XS52 cells and analyzed by Northern blotting with radioactive probes specific for either tlr2 (TLR2) or tlr4 (TLR4). Membranes were rehybridized with a radioactive probe for GAPDH to ensure equal loading of RNA in each gel lane. X-ray film was exposed to membranes for various periods of time to detect radioactive signals (tlr2, 2 h; tlr4, 2 days; GAPDH, 1 h). (B) Detection of tlr2 and tlr4 mRNA in RAW cells and XS52 cells by RT-PCR. DNase-treated RNA (50 ng) was used as the template in each reaction. PCR products in lanes 1, 4, 7, and 10 were amplified with TLR2-specific primers. PCR products in lanes 2, 5, 8, and 11 were amplified using TLR4-specific primers. Products in lanes 3, 6, 9, and 12 were amplified with GAPDH-specific primers. + and −, reaction mixtures containing and lacking reverse transcriptase (RT), respectively. DNA fragment size markers are indicated on the left.

FIG. 2

Time-dependent association of T. pallidum with XS52 cells. T. pallidum cells were incubated with XS52 cells at a ratio of 100 spirochetes per DC for the indicated times. Ethanol-fixed samples were probed with a monoclonal antibody specific for the abundant 47-kDa lipoprotein of T. pallidum, followed by staining with a FITC-conjugated goat anti-mouse secondary antibody. Arrowheads, spirochetes or spirochetal antigens associated with XS52 DCs. Results are representative of three independent experiments. Magnification, ×400.

FIG. 3

Phagocytosis of T. pallidum by XS52 cells. T. pallidum cells were incubated with XS52 cells at a ratio of 1,000 spirochetes/cell for 24 h. Samples were prepared for thin-section (80 nm) TEM and negatively stained with lead citrate and uranyl acetate. (A and B) Two examples of conventional phagocytosis. (C) Overlapping coiling phagocytosis. (D) Overlapping coiling phagocytosis in the process of internalizing T. pallidum. (E) The initial pseudopod of rotating coiling phagocytosis surrounding a spirochete. (F) A pseudopod whorl of rotating coiling phagocytosis. (G) Three pseudopod whorls of rotating coiling phagocytosis. (H) Two organisms internalized in what appears to be a membrane-bound vacuole. Bars: 100 (A to F) 200 (G), and 50 nm (H).

FIG. 4

IL-12p40 production by immature DCs stimulated with T. pallidum or the synthetic lipopeptide. XS52 cells were incubated with increasing amounts of either virulent treponemes (A) or a synthetic lipopeptide modeled after the 47-kDa membrane lipoprotein of T. pallidum (47-L) (B). Times examined are denoted in panel A. Cell supernatants were then assayed for cytokine production by capture ELISA. Neg, negative control (either TEx [A] or the 47 hexapeptide [B]) equivalent to the highest dose of stimulus, assayed after 24 h of incubation. Shown are the means ± standard errors of duplicate determinations from two (A) and three (B) independent experiments.

FIG. 5

IL-1β production by immature DCs stimulated with T. pallidum or the synthetic lipopeptide. XS52 cells were incubated with increasing amounts of either T. pallidum (A) or synthetic lipopeptide (47-L) (B). Times examined are denoted in panel A. Cell supernatants were then assayed for cytokine production by capture ELISA. Neg, negative control (either TEx [A] or the 47 hexapeptide [B]) equivalent to the highest dose of stimulus, assayed after 24 h of incubation. Shown are the means ± standard errors of duplicate determinations for two (A) and three (B) independent experiments.

FIG. 6

TNF-α production by immature DCs stimulated with T. pallidum or the synthetic lipopeptide. XS52 cells were incubated with increasing amounts of either T. pallidum (A) or synthetic lipopeptide (47-L) (B). Times examined are denoted in panel A. Cell supernatants were then assayed for cytokine production by capture ELISA. Neg, negative control (either TEx [A] or the 47 hexapeptide [B]) equivalent to the highest dose of stimulus, assayed after 24 h of incubation. Shown are the means ± standard errors of duplicate determinations for two (A) and three (B) independent experiments.

FIG. 7

IL-6 production by immature DCs stimulated with T. pallidum or the synthetic lipopeptide. XS52 cells were incubated with increasing amounts of either T. pallidum (A) or synthetic lipopeptide (47-L) (B). Times examined are denoted in panel A. Cell supernatants were then assayed for cytokine production by capture ELISA. Neg, negative control (either TEx [A] or the 47 hexapeptide [B]) equivalent to the highest dose of stimulus, assayed after 24 h of incubation. Shown are the means ± standard errors of duplicate determinations for two (A) and three (B) independent experiments.

FIG. 8

Upregulation of CD54 on immature DCs stimulated with either T. pallidum or the synthetic lipopeptide. XS52 cells were stimulated for 24 h, stained with a FITC-conjugated anti-CD54 monoclonal antibody, and then analyzed by flow cytometry. (A) LPS (40 ng/ml) was included as a positive control. (B) T. pallidum (Tp) (1,000 spirochetes/cell), but not TEx, induced CD54. (C) The synthetic lipopeptide (47-L) (32 μg/ml), but not the 47 hexapeptide control (47-HX), induced CD54. NA, no application (unstimulated cells). Results are representative of two independent experiments.