Chronic alcohol consumption decreases the percentage and number of NK cells in the peripheral lymph nodes and exacerbates B16BL6 melanoma metastasis into the draining lymph nodes

Abstract

NK cells in the lymph nodes play important roles in inhibiting tumor metastasis into draining lymph nodes. Previously, we reported that chronic alcohol consumption interferes with NK cell trafficking from the bone marrow to the spleen. Herein, we found that alcohol consumption decreases the numbers of NK cells in lymph nodes. Adoptive transfer experiments indicated that continued exposure of donor splenocytes to alcohol inhibits NK but not T cell trafficking to lymph nodes. Alcohol did not negatively affect CCR7(+) and CXCR3(+) NK cells, but decreased the percentage and number of CD62L(+) NK cells in the spleen, which are an important source of NK cell trafficking into the lymph nodes. These data suggest that modulation of the microenvironment associated with alcohol consumption impairs the trafficking of NK cells to lymph nodes. The decreased number of NK cells in the lymph nodes was associated with increased melanoma metastasis into the draining lymph nodes.

Fig. 1

Effects of chronic alcohol consumption on NK cells in the LN. (A) and (B) Representative dot plots of gated NK cells in the LN cell population of water drinking mice (A) and alcohol consuming mice (B), respectfully. NK cells were identified by gating on the NK1.1⁺ cells that are CD3⁻ in this and subsequent figures. (C) Dot plot indicating annexin V stained cells in the LN from alcohol consuming mice. (D) Dot plot indicating BrdU⁺ LN cells from water drinking mice. (E) Total number of cells in LN. (F) Number of NK cells in LN. (G) Percentage of NK cells in the LN. (H) Percentage of annexin V⁺ NK cells. (I) Percentage of BrdU⁺ NK cells. Values are mean ± S.D. for groups containing 7–10 mice. The data are representative of three different experiments with similar findings. ETOH group different from Water group, **p<0.001. Water = Water drinking group. ETOH = Alcohol consuming group.

Fig. 2

Effects of chronic alcohol consumption on the expression of CCR7 in splenic NK cells and NK1.1⁻CD3⁺ cells. (A) Dot plot indicating gated NK cells (upper left region) and CD3⁺ T cells (lower right region) in the spleen of alcohol consuming mice. (B) Dot plot indicating CCR7⁺ NK cells in gated splenic NK cells of alcohol consuming mice. (C) Histogram of CCR7⁺ gated CD3⁺ T cells in the spleen of alcohol consuming mice. M1: CCR7^hi. M2: CCR7^int. (D) Percentage of CCR7⁺ NK cells in the spleen. (E) Percentage of CCR7^hi and CCR7^int CD3⁺ T cells in the spleen. Values are mean ± S.D. for groups containing 7–10 mice. The data are representative of two different experiments with similar findings. ETOH group different from Water group, *p<0.05. Water = Water drinking group. ETOH = Alcohol consuming group.

Fig. 3

Effects of chronic alcohol consumption on CXCR3⁺ and CD62L⁺ NK cells. (A) Percentage of CXCR3⁺ NK cells in the spleen, LN, and BM NK cells. (B) Percentage of CD62L⁺ NK cells in the spleen, LN, and BM NK cells. (C) Number of CD62L⁺ NK cells in the spleen, LN, and BM. (D) Percentage of CXCR3⁺CD3⁺ T cells in spleen, LN, and BM CD3⁺ T cells. (E) Percentage of CD62L⁺ CD3⁺ T cells in the spleen, LN, and BM CD3⁺ T cells. Values are mean ± S.D. for groups containing 7–10 mice. The data are representative of two different experiments with similar findings. Open bars, Water drinking group. Cross-hatched bars, Alcohol consuming group. Alcohol consuming group different from Water drinking group, *p<0.05. **p<0.001.

Fig. 4

Effects of chronic alcohol consumption on the trafficking and distribution of transferred NK and NK1.1⁻CD3⁺ T cells into the spleen, LN, and BM. Splenocytes from one water-drinking mouse were isolated, labeled with CFSE, These cells divided into two parts, and 2×10⁷ cells were injected intravenously into one alcohol consuming mice and one water drinking control mice via the lateral tail vein. In total 5 individual donor mice were utilized. Mice were euthanized at the indicated time points. Splenocytes, LN cells, and BM cells were isolated and stained with anti-mouse CD3-PE and anti-mouse NK1.1-PerCP and specific cell populations were analyzed by flow cytometry. (A) Number of CFSE⁺ NK cells in the LN. (B) Number of CFSE⁺NK cells in the BM. (C) Number of CFSE⁺NK cells in the spleen. (D) Number of CFSE⁺CD3⁺ T cells in the LN. (E) Number of CFSE⁺CD3⁺ T cells in the BM. (F) Number of CFSE⁺CD3⁺ T cells in the spleen. ETOH group different from Water group, *p<0.05. Water = Water consuming mice; ETOH = Alcohol consuming mice.

Fig. 5

Effect of chronic alcohol consumption on the percentage of splenic CD62L⁺ NK cells before and after adoptive splenocyte transfer. Splenocytes from one donor mouse were isolated, labeled with CFSE, divided into two parts, and 2×10⁷ cells in 200 µl of PBS were injected intravenously into one alcohol consuming and one water drinking mice via the lateral tail vein. In total 5 donor mice were utilized. Splenocytes were isolated 7 days after cell transfer and analyzed by flow cytometry. CFSE⁻, Percentage of CD62L⁺ NK cells that comprise the host NK cell population; CFSE⁺, Percentage of CD62L⁺ NK cells that comprise the adoptively transfer NK cell population. Values are mean ± S.D. ETOH group different from Water group, *p<0.05, **p<0.001. Water = Water consuming mice; ETOH = Alcohol consuming mice.

Fig. 6

Effects of chronic alcohol consumption on B16BL6 melanoma metastasis into the draining LN. Mice consumed alcohol for five months before tumor inoculation. B16BL6 melanoma cells then were inoculated subcutaneously into water drinking and alcohol consuming mice into the right hip area at a concentration of 10⁵ cells in 200 µl of CMF. Mice were euthanized three weeks after the tumor cell inoculation. The right inguinal LN were removed and evaluated for the presence of metastasis. The relative tumor burden was expressed as the percentage of tumor areas divided by the greatest cross-sectional area of the LN. The areas were measured using NIH ImageJ software. Each group contained 8 mice. ETOH group different from Water group, *p<0.03. Water = Water drinking group; ETOH = Alcohol consuming group.