Figure 1.
High MCP-2/CCL8 abundance in TB-PEs.
(A) Diagram showed that PEs from both TB and non-TB patients were applied to protein chip assays to screen differentially expressed cytokines. Data were subsequently confirmed by ELISA. (B) Differentially expressed proteins in PEs from TB patients and non-TB patients. (C–D) ELISA of IFN-γ (C) or MCP-2/CCL8(D) in the PEs from TB or non-TB patients. (E) ELISA of MCP-2/CCL8 protein in the peripheral blood from TB patients with or without PEs and healthy donors. (F) Receiver operating characteristic curve showing the area under curve (AUC) of MCP-2/CCL8 and IFN-γ. **, p<0.001, *, p<0.05.
Table 1.
Protein array analysis of cytokine abundance in PEs from TB patients and non-TB patients (Values stand for relative intensity of fluorescence).
Figure 2.
Higher percentage of CCR5-expressing CD4+ T cells in TB-PEs.
(A) Representative FACS detection of CD3+, CD4+ and CD8+ cells in PEs from TB patients. (B–D) Representative FACS detection of the expression of CCR5(B), CCR1 (C) or CCR2 (D) on CD3+, CD4+ and CD8+ cells from TB-PEs. (E–G) Graph shows the comparison of the percentage of CCR5 (E), CCR1 (F) or CCR2 (G) expressing CD3+,CD4+ and CD8+ cells between TB-PEs and malignant PEs (n = 6). All the fluorescent antibodies are purchased from ebioscience (San Diego, CA). The fluorescent antibodies are used according to the manual.*, p<0.05
Figure 3.
Mycobacteria-induced MCP-2/CCL8 expression in macrophages.
(A–B) Real-time PCR detection of the production of mcp-2/ccl8 mRNA in murine macrophage cell line Raw264.7 infected with either M. bovis BCG (A) or M. tuberculosis H37Rv (B) at MOI 5 for indicated times; (C–D) Real-time PCR detection of the production of mcp-2/ccl8 mRNA in primary peritoneal macrophages treated with either M. bovis BCG (C) or M. tuberculosis H37Rv (D) at MOI 5 for indicated times; (E–F) ELISA of MCP-2/CCL8 protein in THP-1, a human acute monocytic leukemia cell line, infected with either M. bovis BCG (E) or M. tuberculosis H37Rv (F) at MOI 5 for indicated times. (G–H) ELISA of MCP-2/CCL8 protein in human PBMC monocyte-derived macrophages (MDM) infected with either M. bovis BCG (G) or M. tuberculosis H37Rv (H) at MOI 5 for indicated times. Data shown are representative of at least three independent experiments. **, p<0.001, *, p<0.05.
Figure 4.
Mycobacteria-induced expression of MCP-2/CCL8 is regulated by p38 and PI3K.
(A-B) Western blot of the signaling pathways including p38, JNK, ERK, Akt, NF-κB in mouse primary peritoneal macrophages infected with either M. bovis BCG (A) or M. tuberculosis H37Rv (B) at MOI 5 for indicated time. Data are representative of three independent experiments. (C) ELISA detection of MCP-2/CCL8 protein in THP-1 cells infected with M. bovis BCG at MOI 5 for 24 h in the presence of different kinase inhibitors, SB203580 (10 µM), SP600125 (10 µM), PD98059 (10 µM), LY294002 (10 µM), BAY11-7082 (10 µM). (D) ELISA detection of MCP-2/CCL8 protein in human PBMC monocyte-derived macrophages (MDM) infected with M. tuberculosis H37Rv at MOI 5 for 24 h in the presence of different kinase inhibitors as indicated in (C). **, p<0.001, *, p<0.05.
Figure 5.
Mycobacteria-induced expression of MCP-2/CCL8 through TLR2.
(A-B) Real-time PCR of mcp-2/ccl8 mRNA in primary peritoneal macrophages from wildtype, TLR2−/−, TLR3−/−, or TLR4−/− mice infected with either M. bovis BCG (A) or M. tuberculosis H37Rv (B) at MOI 5 for 24 h. (B) Western blot of phosphorylation of Akt and p38 in primary peritoneal macrophages from wildtype or TLR2 KO mice infected with M. bovis BCG at MOI 5 for indicated times. Data shown are representative of three independent experiments. (D) Densitometric analysis of three independent experiments as in (C). Data shown are the mean±sem of three independent experiments. **, p<0.001, *, p<0.05.
Figure 6.
Diagram depicting recruitment of T lymphocytes expressing CCR5 in TB-PEs by mycobactera-induced expression of MCP-2/CCL8 in macrophages.
Induction of MCP-2/CCL8 in macrophages by mycobacteria involves activation of TLR2/PI3K/Akt and p38 signaling. Highly expressed MCP-2/CCL8 in the TB-PEs may be responsible for the homing of CD4+ T lymphocytes into the infection site by activating the highly expressed CCR5, the primary receptor used by MCP-2/CCL8.
Table 2.
Primers used for quantitative RT-PCR analysis.