Depressed CCL5 Expression in Human Pulmonary Tuberculosis

Ji-Sook Lee; Ki Hye Kim; Da-Youn Lee; Hong-Hee Choi; Hye-Mi Lee; Ji Woong Son; Tae-Hyun Paik; Eun-Kyeong Jo

doi:10.4167/jbv.2008.38.3.97

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Lee, Kim, Lee, Choi, Lee, Son, Paik, and Jo: Depressed CCL5 Expression in Human Pulmonary Tuberculosis

Original Article

J Bacteriol Virol 2008;38(3):97-107.

Published online: 18 January 2008

DOI: https://doi.org/10.4167/jbv.2008.38.3.97

Depressed CCL5 Expression in Human Pulmonary Tuberculosis

Ji-Sook Lee^1,², Ki Hye Kim^1,³, Da-Youn Lee³, Hong-Hee Choi^1,³, Hye-Mi Lee^1,³, Ji Woong Son⁴, Tae-Hyun Paik², Eun-Kyeong Jo^1,^3,^∗

¹Infection Signaling Network Research Center, Daejeon 301-747, Republic of Korea

³Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea

²Department of Microbiology, Daejeon 302-718, Republic of Korea

⁴Internal Medicine, College of Medicine, Konyang University, Daejeon 302-718, Republic of Korea

^∗Corresponding author Dr. Eun-Kyeong Jo, Department of Microbiology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-ku, Daejeon 301-747, Republic of Korea. Phone: +82-42-580-8243, Fax: +82-42-585-3686 e-mail: hayoungj@cnu.ac.kr

^∗∗ This work was supported by a grant of the Korea Science & Engineering Foundation through the Infection Signaling Network Research Center (R13-2007-020-01000-0) at Chungnam National University. The authors declare that they have no competing financial interests.

Revised 24 June 20088 July 2008 Accepted 16 July 2008

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

CCL5/regulated on activation, normal T expressed and secreted production (RANTES) is a principal CC chemokine, and can activate macrophages and Th1 lymphocytes, however, little is known about the CCL5 profiles associated with active tuberculosis (TB). In this study, we investigated the production of CCL5 by the peripheral blood mononuclear cells (PBMCs) of patients with active pulmonary TB after stimulation with Triton X-100 soluble proteins (TSP) or the 30-kDa antigen. The profiles of cytokines/chemokines [CXCL8/interleukin (IL)-8, IL-12 p40, and interferon (IFN)-γ] were also examined by PBMCs from TB patients, and compared with those obtained from healthy tuberculin reactors (HTR). Concordant with earlier studies, IFN-γ production was significantly depressed in the PBMCs from TB patients compared with those from HTR. In addition, the CCL5, but not CXCL8, levels in the PBMCs from TB patients were significantly depressed after stimulation for 18 hr compared to those in the PBMCs from HTRs. The CCL5 release was not significantly correlated with the release of IFN-γ in the cells from TB patients and HTRs. Further, inhibitor studies show that the 30-kDa- or TSP-induced CCL5 mRNA expression is sensitive to inhibitors of mitogen-activated protein kinase kinase (MEK) 1/2 and Janus kinase (JAK) 2, but not p38, pathway activation, suggesting a MEK1/2- or JAK2-based mechanism is responsible for modulating of the CCL5 expression in human PBMCs. Collectively, these data suggest that TB patients show depressed production of CCL5 secretion, which can be modulated by MEK- and JAK2-based transcriptional regulatory mechanisms, in response to the mycobacterial antigens.

Keywords: Tuberculosis, CCL5, Interferon-γ, 30-kDa antigen, TSP antigen

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Figure 1.

SDS-PAGE analysis of 30-kDa and TSP Ags of Mtb H37Rv. The 30-kDa Ag was purified from unheated concentrated culture filtrate of Mtb H37Rv by 0% to 40% ammonium sulfate precipitation, anion-exchange chromatography, and hydrophobic interaction chromatography. The TSP Ag was isolated from Mtb H37Rv by overnight solubilization with Triton X-100, 20% to 80% ammonium sulfate precipitation, three rounds of phase partitioning with Triton X-114, and three rounds of 0% to 80% ammonium sulfate precipitation. A, 30-kDa; B, TSP Ag.

Figure 2.

IFN-γ (A) and IL-12p40 (B) production in PBMC from TB patients and HTRs in response to the 30-kDa and TSP Ags. The production of IFN-γ and IL-12p40 by PBMC from TB patients and HTRs was determined after in vitro 96 hr (IFN-γ) or 18 hr (IL-12p40) stimulation with the 30-kDa and TSP Ags (1 μg/ml). Data are plotted as individual points on the graph (TB patients, n=20; HTRs, n=20). The protein levels of IFN-γ and IL-12p40 in culture supernatants were measured by specific ELISA. ∗, p<0.05; ∗∗, p<0.01.; ∗∗∗, p<0.001. Un, unstimulated.

Figure 3.

CCL5 (A) and CXCL8 (B) production in PBMC from TB patients and HTRs in response to the 30-kDa and TSP Ags. The production of CCL5 and CXCL8 by PBMC from TB patients and HTRs was determined after in vitro 18 hr stimulation with the 30-kDa and TSP Ags (1 μg/ml). Data are plotted as individual points on the graph (TB patient, n=20; HTR, n=20). The protein levels of CCL5 and CXCL8 in culture supernatants were measured by specific ELISA. ∗, p<0.05; ∗∗, p<0.01. Un, unstimulated.

Figure 4.

Correlation between IFN-γ and CCL5 or CXCL8 production in response to 30-kDa and TSP Ags in PBMC from TB patients and HTRs. The levels of each cytokine release after stimulation with the 30-kDa and TSP Ags were assayed by ELISA. Linear regression analyses were carried out to determine a possible correlation between CCL5 and IFN-γ production (for Panel A) and CXCL8 and IFN-γ production (for Panel B) in TB patients (n=12) and HTRs (n=18).

Figure 5.

Effect of specific inhibitors of MEK1/2, p38 MAPK, or JAK2 on the 30-kDa-or TSP-induced CCL5 mRNA expression in PBMCs. PBMCs from HTRs were preincubated in the presence or absence of AG490 (1, 5, 10 μM), U0126 (5, 10, 20 μM), or SB203580 (1, 5, 10 μM) for 1 hr, and then stimulated with the 30-kDa or TSP Ag (5 μg/ml for each Ag) for 6 hr. CCL5 mRNA expression was detected by semiquantitative RT-PCR analysis of cDNA by using the specific primers. The data shown are representative of four experiments. Un, unstimulated; D, DMSO control

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