Regulation of Leukocyte Recruitment to the Spleen and Peritoneal Cavity during Pristane-Induced Inflammation

Abstract

Chronic inflammation is associated with an increased number of leukocytes in the spleen, which are then redirected to the site of inflammation. However, it remains unknown how leukocyte recruitment is regulated. Herein, chronic inflammation was induced by intraperitoneal injection of pristane into mice. Leukocytes in the spleen or in the peritoneal cavity were quantified by flow cytometry. We found that the loss of IL-6 decreased macrophage recruitment to the spleen and the peritoneal cavity during pristane-induced inflammation. The loss of TNFα delayed the recruitment of neutrophils and macrophages to the spleen and inhibited the recruitment of neutrophils, macrophages, B cells, and T cells. The recruitment of neutrophils and macrophages into the spleen or peritoneal cavity was largely inhibited in the absence of LTα. The loss of TNFα receptor 1/2 resulted in reduced recruitment of neutrophils, macrophages, and dendritic cells into the spleen, but only neutrophil recruitment was inhibited in the peritoneal cavity. Similarly, a lack of B cells significantly impeded the recruitment of neutrophils, macrophages, and dendritic cells to the spleen. However, only macrophage recruitment was inhibited in the absence of T cells in the spleen. These data provide insight into the development of chronic inflammation induced by noninfectious substances.

Figure 1

Leukocyte responses to pristane treatment were dose dependent. Whole splenic cells were analyzed using flow cytometry after staining with antibodies specific for B220, TCRβ, CD11c, CD11b, and Gr-1 (a). The number of B cells (B220⁺), T cells (TCRβ⁺), dendritic cells (CD11c⁺), macrophages (CD11b⁺Gr1^low), or neutrophils (CD11b⁺/Gr1^hi) in the spleen was quantified (b). The difference between 100 μL of pristane treatment and 300 μL of pristane treatment in C57BL/6J mice is shown. (c) Peritoneal mesentery was collected from C57BL/6 mice treated with 300 μL of pristane and analyzed for the expression of cytokines. The difference between 300 μL pristane-treated and nontreated C57BL/6J mice is shown. Data represent two independent experiments. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 2

IL-6 deficiency decreased the recruitment of splenic macrophages in response to pristane treatment. (a, b) IL-6^−/− mice and WT mice were injected with 300 μL (8.3 × 10⁻⁴ mol) of pristane and sacrificed at three weeks thereafter. Single cell suspensions of the spleen (SP) and peritoneal cavity (PC) from pristane-injected WT and IL-6^−/− mice were prepared for cellularity analysis by flow cytometry. The total cell number and the number of each cell type were calculated, respectively. (c, d) The total number of cells or the number of each cell type in the PC was analyzed in the same way. The difference between WT and IL-6^−/− mice is shown. Data represent two independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 3

LTα loss inhibited the recruitment of both splenic neutrophils and macrophages in response to pristane treatment. (a, b) LTα^−/− mice and WT mice were injected with 300 μL (8.3 × 10⁻⁴ mol) of pristane and were then sacrificed at the indicated time points. The total number of cells and the number of each cell type were calculated. (c, d) The total number of cells or the number of each cell type in the PC was also analyzed. The difference between WT and LTα^−/− mice is shown. Data represent two independent experiments. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 4

Lack of TNFα delayed the recruitment of splenic neutrophils and macrophages in response to pristane treatment. (a, b) TNFα^−/− mice and WT mice were injected with 300 μL (8.3 × 10⁻⁴ mol) of pristane and were then sacrificed at the indicated time points. The total number of cells and the number of each cell type were calculated. (c, d) The total number of cells or the number of each cell type in the PC was also analyzed. The difference between WT and TNFα^−/− mice is shown. Data represent two independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 5

The absence of TNFR1/2 inhibited the recruitment of splenic neutrophils, macrophages, and dendritic cells in response to pristane treatment. (a, b) TNFR1/2^−/− mice and WT mice were injected with 300 μL (8.3 × 10⁻⁴ mol) of pristane and sacrificed at three weeks thereafter. Total number of cells and the number of each cell type were calculated. (c, d) Total number of cells and the number of each cell type in the PC were also analyzed. The difference between WT and TNFR1/2^−/− mice is shown. Data represent two independent experiments. ^∗p < 0.05.

Figure 6

B cell loss reduced the cellularity of both leukocytes and lymphocytes during pristane-induced splenic inflammation. (a, b) μMT mice and WT mice were injected with 300 μL (8.3 × 10⁻⁴ mol) of pristane and were then sacrificed at the indicated time points. The total number of cells and the number of each cell type were calculated. The difference between WT and μMT mice is shown. Data represent two independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.

Figure 7

T cell loss inhibits splenic macrophage recruitment during pristane-induced splenic inflammation. (a, b) LAT^−/− mice and WT mice were injected with 300 μL (8.3 × 10⁻⁴ mol) of pristane and sacrificed at three weeks thereafter. Total number of cells and the number of each cell type were calculated. The difference between WT and LAT^−/− mice is shown. Data represent two independent experiments. ^∗p < 0.05 and ^∗∗p < 0.01.