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. 2012 Apr;5(4):1005-10.
doi: 10.3892/mmr.2012.751. Epub 2012 Jan 12.

Lipopolysaccharide and dose of nicotine determine the effects of nicotine on murine bone marrow-derived dendritic cells

Su Xian Hu  1 , Hua Xiu Sui, Hao Jie Jin, Xiao Yan Ni, Xiao Xing Liu, Mao Qiang Xue, Ye Zhang, Feng Guang Gao
Affiliations

Lipopolysaccharide and dose of nicotine determine the effects of nicotine on murine bone marrow-derived dendritic cells

Su Xian Hu et al. Mol Med Rep. 2012 Apr.

Abstract

The reported effects of nicotine on dendritic cells (DCs) are controversial. To investigate the factors which determine the effects of nicotine on DCs, immature dendritic cells (imDCs) induced from murine bone marrow were treated with different doses of nicotine with or without lipopolysaccharides (LPS). The morphology and expression of the co-stimulatory molecules CD80, CD86, CD40 and CD54 were observed and determined by microscopy and flow cytometry, respectively. The results showed that, firstly, nicotine treatment promoted the development of DC precursors into imDCs with a semi-mature phenotype revealed by a higher expression of CD11c and more branched projections. Secondly, lower doses of nicotine (16.5 ng/ml), but not higher (200 µg/ml), up-regulated the expression of the co-stimulatory molecules CD80, CD40 and CD54 on imDCs. Co-administration of LPS and nicotine revealed differential effects on co-stimulatory molecule expression on imDCs. Thirdly and importantly, treatment with lower doses of nicotine (16.5 ng/ml) did not augment expression of the CD80, CD86, CD40 and CD54 molecules in mature DCs. Fourthly and interestingly, high doses of nicotine (more than 165 µg/ml) revealed pro-apoptotic activity but lower doses of nicotine (16.5-0.165 ng/ml) achieved an anti-apoptotic effect on imDCs. All data presented here indicate that the controversial effects of nicotine on DCs may be due to the LPS of the nicotinic environment and the dose of nicotine used.

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Figures

Figure 1
Figure 1
Nicotine promotes murine DC differentiation. DCs induced from murine bone marrow were treated with 16.5 ng/ml of nicotine for 12 h, then the morphology of DCs was observed by microscopy and CD11c expression was determined by flow cytometry. (A) DC morphology and CD11c expression on day 4, day 4 stimulated with nicotine and day 6. (B) Histographic presentation of percentages of the expression of CD11c (p=0.0002, imDC day 4 vs. imDC day 4 + Ni; p=0.0005, imDC day 4 vs. imDC day 6; p=0.0013, imDC day 4 + Ni vs. imDC day). (C) Histographic presentation of MFI on expression of CD11c (p=0.0009, imDC day 4 vs. imDC day 4 + Ni; p<0.0001, imDC day 4 vs. imDC day 6; p=0.0154, imDC day 4 + Ni vs. imDC day). A representative flow cytometric analysis out of 3 is shown; Student's t-test. Ni, nicotine.
Figure 2
Figure 2
Lower doses of nicotine up-regulate the expression of co-stimulatory molecules on imDCs. imDCs on day 4 induced from murine bone marrow were treated with 16.5 ng/ml, 25 and 200 μg/ml of nicotine for 12 h, and the expression of CD80, CD86, CD40 and CD54 on DCs was determined by flow cytometry. (A) Histographic presentation of CD80 expression on imDCs. (B) CD80 expression on imDCs (p=0.0256, imDC control vs. Ni 16.5 ng/ml; p=0.0004, Ni 16.5 ng/ml vs. Ni 200 μg/ml). (C) Histographic presentation of CD86 expression on imDCs. (D) CD86 expression on imDCs (p=0.0098, imDC control vs. Ni 16.5 ng/ml; p=0.0011, imDC control vs. Ni 25 μg/ml; p=0.0437, imDC control vs. Ni 200 μg/ml; p=0.0010, Ni 25 μg/ml vs. 200 μg/ml). (E) Histographic presentation of CD40 expression on imDCs. (F) CD40 expression on imDCs (p=0.0240, imDC control vs. Ni 16.5 ng/ml; p=0.0459, imDC control vs. Ni 25 μg/ml; p<0.0001, imDC control vs. Ni 200 μg/ml; p<0.0010, Ni 25 μg/ml vs. Ni 200 μg/ml). (G) Histographic presentation of CD54 expression on imDCs. (H) CD54 expression on imDCs (p=0.0013, imDC control vs. Ni 16.5 ng/ml; p<0.0001, Ni 16.5 ng/ml vs. Ni 25 μg/ml; p<0.0001, imDC control vs. Ni 200 μg/ml). A representative flow cytometry analysis out of 3 was shown; Student's t test. Ni, nicotine.
Figure 3
Figure 3
Lower doses of nicotine up-regulate the expression of surface molecules on imDCs in the presence of LPS. imDCs were treated with 16.5 ng/ml, 25 and 200 μg/ml of nicotine in the presence of 100 ng/ml LPS for 12 h on day 4, and then the expression levels of CD80, CD86, CD40 and CD54 on DCs were determined by flow cytometry. (A) CD80 expression on imDCs with different doses of nicotine and LPS stimulation (p<0.0001, imDC control vs. Ni 16.5 ng/ml; p<0.0001, imDC control vs. Ni 200 μg/ml; p<0.0001, Ni 16.5 ng/ml vs. Ni 25 μg/ml; p<0.0001, Ni 25 μg/ml vs. Ni 200 μg/ml). (B) CD86 expression on imDCs with different doses of nicotine and LPS stimulation (p<0.0001, imDC control vs. Ni 16.5 ng/ml; p<0.0001, Ni 16.5 ng/ml vs. Ni 25 μg/ml; p=0.0032 and Ni 25 μg/ml vs. Ni 200 μg/ml). (C) CD40 expression on imDCs with different doses of nicotine and LPS stimulation (p<0.0001, imDC control vs. Ni 16.5 ng/ml; p<0.0001, imDC control vs. Ni 25 μg/ml; p=0.0004, imDC control vs. Ni 200 μg/ml; p=0.0440, Ni 16.5 ng/ml vs. Ni 200 μg/ml; p=0.0252, Ni 25 μg/ml vs. Ni 200 μg/ml). (D) CD54 expression on imDCs with different doses of nicotine and LPS stimulation (P<0.0001, imDC control vs. Ni 16.5 ng/ml; p<0.0001, imDC control vs. Ni 25 μg/ml; p<0.0001, imDC control vs. Ni 200 μg/ml). A representative flow cytometric analysis out of 3 is shown; Student's t test. Ni, nicotine.
Figure 4
Figure 4
The doses of nicotine determine the expression of surface molecules on maDCs. At day 4, imDCs induced from murine bone marrow were treated with 10 ng/ml LPS for a further 4 days and were considered as mature DCs (maDCs). maDCs were stimulated with 16.5 ng/ml, 25 and 200 μg/ml of nicotine for 14 h, and the expression levels of CD80, CD86, CD40 and CD54 were determined by flow cytometry. (A) Histographic presentation of CD80 expression on maDCs. (B) CD80 expression on maDCs (p=0.0256, imDC control vs. Ni 16.5 ng/ml; p=0.0004, Ni 16.5 ng/ml vs. Ni 200 μg/ml). (C) Histographic presentation of CD86 expression on maDCs. (D) CD86 expression on maDCs (p=0.0098, imDC control vs. Ni 16.5 ng/ml; p=0.0011, imDC control vs. Ni25 μg/ml; p=0.0437, imDC control vs. Ni 200 μg/ml; p=0.0010, Ni 25 μg/ml vs. Ni 200 μg/ml). (E) Histographic presentation of CD40 expression on maDCs. (F) CD40 expression on maDCs (p=0.0240, imDC control vs. Ni 16.5 ng/ml; p=0.0459, imDC control vs. Ni 25 μg/ml; p<0.0001, imDC control vs. Ni 200 μg/ml; p<0.0010, Ni 25 μg/ml vs. Ni 200 μg/ml). (G) Histographic presentation of CD54 expression on maDCs. (H) CD54 expression on maDCs (p=0.0013, imDC control vs. Ni 16.5 ng/ml; p<0.0001, Ni 16.5 ng/ml vs. Ni 25 μg/ml; p<0.0001, imDC control vs. Ni 200 μg/ml). A representative flow cytometry analysis out of 3 is shown; Student's t test. Ni, nicotine.
Figure 5
Figure 5
The pro- or anti-apoptotic effects of nicotine on imDCs are dependent on the dose of nicotine. On day 4, imDCs were treated with different doses of nicotine for a further 12 h, and the viability of imDCs was determined by flow cytometry using FITC-labeled Annexin V and propidium iodide (PI). (A) Histographic presentation of flow cytometric data. (B) Histographic presentation of A. p<0.0001, imDC control vs. Ni 1.65 mg/mm, 16.5 ng/ml; p<0.0001, imDC control vs. 165 μg/ml; p=0.0046, imDC control vs. Ni 16.5 ng/ml; p=0.0405, imDC control vs. Ni 0.165 ng/ml. Data represent the mean ± SEM, n=4. A representative flow cytometric analysis out of 3 is shown; Student's t test. Ni, nicotine.
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