Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors - PubMed (original) (raw)

Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors

S Tsuji et al. Infect Immun. 2000 Dec.

Free PMC article

Abstract

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Cell surface phenotypes of DC exposed to BCG-CWS. iDC were prepared by culture for either 3, 6, or 9 days and were also incubated for an additional 2 days in medium with GM-CSF (500 IU/ml) and BCG-CWS (15 μg/ml). Broken lines in the histograms show nonspecific fluorescence by subclass control MAb. Fluorescence for the indicated antigens on DC before and after BCG-CWS treatment is shown by bold lines and shaded areas, respectively. This experiment was repeated three times with similar results, and representative results are shown.

FIG. 2

FIG. 2

Levels of surface markers on iDC with and without treatment with reagents. iDC were incubated for 2 days in medium containing GM-CSF (500 IU/ml) and the following materials: GM-CSF alone (none) (▨), emulsion buffer (15 μl/ml) (□), TNF-α (100 IU/ml) (▩), IL-1β (100 ng/ml) (formula image), BCG-CWS (15 μg/ml) (■), heat-killed BCG (iDC/bacterium ratio = 1:1) (formula image), or viable BCG (iDC/bacterium ratio = 1:1) (▩). Values are expressed as the mean fluorescence intensities measured by a flow cytometer, and the mean values of subclass control MAbs were negligible (∼3 to 4). This experiment was repeated three times with similar results, and representative results are shown.

FIG. 3

FIG. 3

Cytokine production by iDC and DC treated with BCG-CWS. Monocytes were cultured in medium containing GM-CSF and IL-4, and the medium was collected every 3 days (0-3, 3-6, 6-9). iDC cultured for 3, 6, or 9 days were incubated in medium with GM-CSF (500 IU/ml) and BCG-CWS (15 μg/ml), and each medium was collected after 2 days (3-5, 6-8, 9-11). The concentrations of each cytokine in the medium (IL-1β [□], TNF-α [■], IL-6 [▨], and IL-12 p40 [▩]) were determined by ELISA. Values are the means ± standard deviations (SD) of triplicate determinations. The concentrations of IL-12 p40 are on the right vertical axis.

FIG. 4

FIG. 4

The levels of CD83 in the lower-well DC in the transwell system. iDC were incubated for 2 days in a transwell apparatus with GM-CSF (500 IU/ml) and the reagents indicated in the figure. The cells in the lower well were harvested, and levels of CD83 were measured by a flow cytometer. Values are expressed as the mean fluorescence intensity (open bars, levels of CD83; closed bars, background levels by subclass control MAb). Emulsion buffer is abbreviated EB. These experiments were repeated three times with similar results, and representative results are shown.

FIG. 5

FIG. 5

Allogeneic MLR using iDC and DC treated with BCG-CWS. iDC were incubated for 2 days in medium with GM-CSF plus IL-4 (○) or GM-CSF plus BCG-CWS (●). These DC were irradiated and cultured for 4 days with allogeneic lymphocytes, and [3H]thymidine incorporation was measured. Values are expressed as the means ± SD of triplicate determinations. Similar experiments were repeated twice, and representative results are shown.

FIG. 6

FIG. 6

Flow-cytometric analysis of phagocytosis of FITC–BCG-CWS by iDC. iDC were incubated for 0.5 or 7 h in medium with GM-CSF (500 IU/ml) and FITC–BCG-CWS (15 μg/ml). Thin lines show self-fluorescence of cells, and bold lines reflect fluorescence of FITC-BCG-CWS particles which are bound and/or phagocytosed. Total fluorescence intensities (panels a and c) and those quenched with trypan blue (panels b and d) are shown. This experiment was repeated three times with similar results, and representative results are shown.

FIG. 7

FIG. 7

Signaling of BCG-CWS via TLR. Thioglycolate-elicited peritoneal macrophages from wild-type (1; ■), TLR2-deficient (2; □), TLR4-deficient (3; ▩), or TLR2- and TLR4-deficient (4) mice were cultured with IFN-γ (30 U/ml) in the presence or absence of BCG-CWS (10 μg/ml) for 24 h. Concentrations of TNF-α in the culture supernatants were measured by ELISA. ND, not detected. This experiment was repeated twice with similar results, and representative results are shown.

Similar articles

Cited by

References

    1. Alexandroff A B, Jackson A M, O'Donnell M A, James K. BCG immunotherapy of bladder cancer: 20 years on. Lancet. 1999;353:1689–1694. - PubMed
    1. Aliprantis A O, Yang R, Mark M R, Suggett S, Devaux B, Radolf J D, Klimpel G R, Godowski P, Zychlinsky A. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science. 1999;285:736–739. - PubMed
    1. Azuma I, Thomas D W, Adam A, Ghuysen J M, Bonaly R, Petit J F, Lederer G. Occurrence of N-glycolylmuramic acid in bacterial cell walls. Biochim Biophys Acta. 1970;208:444–451. - PubMed
    1. Azuma I, Ribi E E, Meyer T J, Zbar B. Biologically active components from mycobacterial cell walls. I. Isolation and composition of cell wall skeleton and component P3. J Natl Cancer Inst. 1974;52:95–101. - PubMed
    1. Azuma I, Okumura H, Saiki I, Tanio Y, Kiso M, Hasegawa A, Yamamura Y. Adjuvant activity of 6-amino-6-deoxy-muramyldipeptides and their acylamino derivatives on the induction of delayed hypersensitivity to azobenzenearsonate-N-acetyl-l-tyrosine in guinea pigs. Infect Immun. 1981;32:1305–1308. - PMC - PubMed

MeSH terms

Substances

LinkOut - more resources