Essential roles of monocytes in stimulating human peripheral blood mononuclear cells with Lactobacillus casei to produce cytokines and augment natural killer cell activity

Abstract

We examined the effect of a probiotic strain, Lactobacillus casei strain Shirota, on cytokine production and natural killer (NK) cell activity in human peripheral blood mononuclear cells (PBMNC). The cellular mechanisms of immunoregulation by L. casei strain Shirota were also investigated. L. casei strain Shirota stimulated PBMNC to secrete interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10. However, depletion of monocytes from PBMNC eliminated the induction of these cytokines. L. casei strain Shirota was phagocytosed by monocytes and directly stimulated them to secrete IL-12, TNF-alpha, and IL-10. IFN-gamma production was diminished by the addition of anti-IL-12 antibody to the PBMNC cultures. Purified T cells, but not NK cells, produced IFN-gamma effectively when stimulated with L. casei strain Shirota in the presence of monocytes, indicating that monocytes triggered by L. casei strain Shirota help T cells to produce IFN-gamma through secreting IL-12. In addition, NK cell activity and CD69 expression on NK cells increased after cultivation of PBMNC with L. casei strain Shirota. When monocytes were depleted from PBMNC, L. casei strain Shirota did not enhance NK cell activity. These results demonstrate that monocytes play critical roles in the induction of cytokines and following the augmentation of NK cell activity during the stimulation of human PBMNC with L. casei strain Shirota.

FIG. 1.

Cytokine production by PBMNC stimulated with L. casei strain Shirota. (A) PBMNC (2 × 10⁵ cells/well) were cultured in medium alone or with L. casei strain Shirota (LcS) (0.1 to 100 μg/ml), and the levels of IL-12, TNF-α, and IL-10 on day 3 and IFN-γ on day 6 in culture supernatants were determined by ELISA. Data are expressed as means ± standard deviations (SD) of individual values from six subjects. (B) PBMNC were cultured with L. casei strain Shirota (10 μg/ml) for 1, 3, and 6 days, and the levels of cytokines in culture supernatants were determined. Data are values from six subjects. Each symbol represents values of individual subjects. *, P < 0.05; **, P < 0.01 (versus the group with L. casei strain Shirota at 0 μg/ml).

FIG. 2.

Monocytes phagocytose L. casei strain Shirota and change in morphology. Purified monocytes (2 × 10⁵ cells) were cultured on collagen type I-coated cover glasses with L. casei strain Shirota (10 μg/ml) for 24 h and stained with Giemsa solution (A). Monocytes were cultured in the presence (B) or absence (C) of L. casei strain Shirota for 72 h. Phagocytosis of bacteria by monocytes and morphology of monocytes were observed by light microscopy. Original magnifications, ×1,000 (A) and ×200 (B and C).

FIG. 3.

L. casei strain Shirota stimulates monocytes to secrete IL-12, TNF-α, and IL-10. PBMNC, monocytes (Mono), and monocyte-depleted cells (Mono-de) were cultured with L. casei strain Shirota (LcS) (0.1 to 100 μg/ml) at a density of 2 × 10⁵ cells/well, and the levels of IL-12, TNF-α, and IL-10 on day 3 and IFN-γ on day 6 in culture supernatants were determined by ELISA. Data are expressed as means ± SD of individual values for four subjects. Each symbol represents values for individual subjects. *, P < 0.05 versus the PBMNC group with L. casei strain Shirota at matched concentrations.

FIG. 4.

L. casei strain Shirota-induced IFN-γ production is mediated by IL-12. PBMNC (2 × 10⁵ cells/well) were cultured with L. casei strain Shirota (1 μg/ml) in the presence of anti-IL-12 antibody or isotype-matched control antibody (Cont Ab) for 3 and 6 days, and the levels of IFN-γ in culture supernatants were determined by ELISA. Data are expressed as means ± SD of individual values for six subjects. Each symbol represents values for individual subjects. *, P < 0.05.

FIG. 5.

IFN-γ is produced mainly by T cells. PBMNC, T-cell-depleted cells (T-de), and NK cell-depleted cells (NK-de) were cultured with L. casei strain Shirota (1 μg/ml) at a density of 2 × 10⁵ cells/well. Purified T cells and NK cells at a density of 1 × 10⁵ cells/well were cultured with L. casei strain Shirota in the absence (−) or presence of irradiated (3,000 rad) PBMNC (Irr-PBMNC) (2 × 10⁵ cells/well). The levels of IL-12 on day 3 and IFN-γ on day 6 in culture supernatants were determined by ELISA. Data are expressed as means ± SD of individual values for four subjects. Each symbol represents values of individual subjects. *, P < 0.05 versus the PBMNC group; #, P < 0.05 versus the irradiated-PBMNC group.

FIG. 6.

Monocytes are essential for IFN-γ production by T cells. PBMNC (2 × 10⁵ cells/well) were cultured with L. casei strain Shirota (1 μg/ml). Purified monocytes (Mono) (1 × 10⁵ cells/well) were cultured with L. casei strain Shirota in the absence (−) or presence (+) of purified T cells or NK cells (1 × 10⁵ cells/well). The levels of IL-12 on day 3 and IFN-γ on day 6 in culture supernatants were determined by ELISA. Data are expressed as means ± SD of individual values for four subjects. Each symbol represents values of individual subjects. *, P < 0.05 versus the PBMNC group; #, P < 0.05 versus the Mono group.

FIG. 7.

L. casei strain Shirota and other bacteria induce IL-12 secretion, augment NK cell activity, and enhance CD69 expression on NK cells. PBMNC were cultured in the absence (Med) or presence of L. casei strain Shirota (LcS), L. acidophilus (La), or B. breve (Bb) at a concentration of 1 μg/ml for 6 days, and viable cells were then collected. The cultured cells as well as freshly isolated cells (Fresh) were assayed for NK cell activity (n = 9) and CD69 expression on CD56⁺ NK cells (n = 5). LU were calculated as described in Materials and Methods. The levels of IL-12 on day 3 in culture supernatants (n = 9) were determined by ELISA. Data are expressed as means ± SD of individual values. Each symbol represents values of individual subjects. *, P < 0.05 versus the Fresh group; **, P < 0.01 versus the Fresh group; #, P < 0.05 versus the L. casei strain Shirota group; ##, P < 0.01 versus the L. casei strain Shirota group; ++, P < 0.01 versus the Med group.

FIG. 8.

Monocytes are important for augmentation of NK cell activity. PBMNC, monocyte-depleted cells (Mono-de), and T-cell-depleted cells (T-de) were cultured in the absence (Med) or presence of L. casei strain Shirota (LcS) (1 μg/ml) for 6 days, and viable cells were then collected. The cultured cells as well as freshly isolated cells (Fresh) were assayed for NK cell activity. Data at an effector/target ratio of 12.5 are expressed as means ± SD of individual values for six subjects. Each symbol represents values of individual subjects. *, P < 0.05; **, P < 0.01.