Depressed CCL5 Expression in Human Pulmonary Tuberculosis

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)-gamma] were also examined by PBMCs from TB patients, and compared with those obtained from healthy tuberculin reactors (HTR). Concordant with earlier studies, IFN-gamma 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-gamma 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.

Elevated Levels of Interferon-inducible Protein-10 (IP)-10/CXCL10, but not of Interferon-gamma, in Patients with Pulmonary Tuberculosis

Mycobacterial strains are potent inducers of cytokines/chemokines by mononuclear phagocytes, which constitute an important cellular component of the first line of defense in the innate immune system. Interferon (IFN)-gamma-inducible protein (IP-10 or CXCL10) is a potent chemoattractant; however, little is known about the IP-10 profiles attributable to the Th1 regulation associated with active tuberculosis (TB). In this study, we investigated the production of IP-10, interleukin (IL)-12 p40, and IFN-gamma by the peripheral blood mononuclear cells (PBMCs) of patients with active pulmonary TB in response to in vitro stimulation with Triton X-100 soluble proteins (TSPs) or the 30-kDa antigen. The TSP antigens used in the present study were isolated and purified from Mycobacterium tuberculosis H37Rv (virulent strain), M. tuberculosis H37Ra (avirulent strain), and Mycobacterium bovis BCG. The results were compared with those obtained for healthy tuberculin reactors (HTRs). Concordant with earlier studies, IFN-gamma production was significantly depressed in the PBMCs from TB patients compared with those in the HTR group. However, the IP-10 levels in the PBMCs from TB patients were significantly elevated 18 h after stimulation compared to those in the PBMCs from HTRs. IP-10 release was correlated in a significant manner with the release of IFN-gamma in the HTRs, but this was not the case for the TB patients. Collectively, these data suggest that TB patients show altered regulation of Th1-driving cytokine and chemokine production in response to a variety of mycobacterial antigens.

Identification and Functional analysis of Gene Expression in Mycobacterium tuberculosis-infected Human Monocytic Cells Under Hypoxic Conditions

Mycobacterium tuberculosis-induced granulomatous lesions, particularly those undergoing central caseation, are known as hypoxic. To analyze the host genes associated with hypoxic conditions from cells infected with M. tuberculosis, we performed GeneChip analyses on mRNA from M. tuberculosis H37Rv-treated human monocytic THP-1 cells cultured in oxygen-depleted status for 18 h. The expression of 99 genes was altered, including those involved in intracellular signaling, energy production, and protein metabolism, as revealed by stringent microarray data analysis. Most notably, mRNA expression of chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) was significantly up-regulated in M. tuberculosis-infected cells under hypoxic conditions. We further analyzed the CCL20 expression in peripheral blood mononuclear cells (PBMCs) and monocyte derived macrophages (MDMs) from healthy controls and TB patients. A comparative analysis has revealed that the mRNA and protein expression of CCL20 were prominently up-regulated in PBMCs, and MDMs from TB patients, compared with healthy controls. Collectively, these data show that the gene expression of CCL20 was up-regulated in M. tuberculosis H37Rv-infected human monocytic THP-1 cells cultured in hypoxic conditions. In addition, the production of CCL20 is substantially increased in cells from TB patients than in healthy controls, suggesting an important role of CCL20 in the immunopathogenesis during TB infection.

CD40-CD40 Ligand Interactions in the Production of IL-12 and IFN-gamma by Tuberculous Pleural Mononuclear Cells

BACKGROUND: Our previous study showed that purified protein derivative (PPD)- stimulated pleural mononuclear cells (PMC) from tuberculous pleurisy (Tbp) produced significantly more IFN-gamma (10- to 70-fold) after in vitro PPD stimulation than freshly isolated pleural cells from malignant pleurisy. The present study was designed to determine whether blocking the CD40-CD40 ligand (CD40L) interaction decreases IFN-gamma production by altering IL-12 levels. METHODS: IL-12 and IFN-gamma production after neutralizing anti-CD40L antibody treatment was compared to the efficacy of anti-CD80, anti-CD86, and a combination of anti-CD80 and CD86 (CD80+86) monoclonal antibodies (mAb). These activities were measured by enzyme-linked immunosorbent assays (ELISAs) and reverse transcription-polymerase chain reaction (RT-PCR), after in vitro stimulation with PPD antigen (Ag). RESULTS: Neutralization of CD80, CD86 and CD80+86 did not decrease IFN-gamma and IL-12 production in Tbp-PMC, whereas neutralization of CD40L significantly depressed IL-12 p40 and IFN-gamma. In addition, neutralization of CD40L completely inhibited IL-12 p40 and IFN-gamma mRNA expression. CONCLUSION: The CD40-CD40L interaction might play a major role in IL-12 and IFN-gamma production in Tbp-PMC, thus contributing to protective immunity in human tuberculosis.

CD40-CD40 Ligand Interactions in the Production of IL-12 and IFN-gamma by Tuberculous Pleural Mononuclear Cells

BACKGROUND: Our previous study showed that purified protein derivative (PPD)- stimulated pleural mononuclear cells (PMC) from tuberculous pleurisy (Tbp) produced significantly more IFN-gamma (10- to 70-fold) after in vitro PPD stimulation than freshly isolated pleural cells from malignant pleurisy. The present study was designed to determine whether blocking the CD40-CD40 ligand (CD40L) interaction decreases IFN-gamma production by altering IL-12 levels. METHODS: IL-12 and IFN-gamma production after neutralizing anti-CD40L antibody treatment was compared to the efficacy of anti-CD80, anti-CD86, and a combination of anti-CD80 and CD86 (CD80+86) monoclonal antibodies (mAb). These activities were measured by enzyme-linked immunosorbent assays (ELISAs) and reverse transcription-polymerase chain reaction (RT-PCR), after in vitro stimulation with PPD antigen (Ag). RESULTS: Neutralization of CD80, CD86 and CD80+86 did not decrease IFN-gamma and IL-12 production in Tbp-PMC, whereas neutralization of CD40L significantly depressed IL-12 p40 and IFN-gamma. In addition, neutralization of CD40L completely inhibited IL-12 p40 and IFN-gamma mRNA expression. CONCLUSION: The CD40-CD40L interaction might play a major role in IL-12 and IFN-gamma production in Tbp-PMC, thus contributing to protective immunity in human tuberculosis.

Increased IL-12 , but Depressed IL-18 Production after In Vitro Stimulation with a 30-kDa Mycobacterial Antigen in Tuberculous Pleural Mononuclear Cells

In this study, we investigated interleukin (IL)-18 and IL-12 following in vitro stimulation with either the 30-kDa or purified protein derivative (PPD) antigens (Ag) of pleural mononuclear cells from 12 cases of tubercular pleurisy (TB-PMC) and 8 cases of malignant pleurisy (MG-PMC). Ag-stimulated TB-PMC produced significantly more IL-12 than did MG-PMC and the levels correlated with those of IFN - gamma. Although elevated IL-18 levels were found in freshly isolated pleural fluids, in vitro IL-18 production in response to either Ag was dramatically decreased in TB-PMC. Pro-IL-18 mRNA was detected before and after Ag stimulation in TB patients. Supernatants from the Ag-stimulated TB-PMC significantly suppressed IL-18 production in normal peripheral blood mononuclear cells (PBMC) and primary malignant cells over an 18 h incubation period. In addition, this suppressive activity was not inactivated by either heat or trypsin. Our findings imply that modulation of IL-12 and IL-18 levels may contribute to the Th1 elevation induced in human TB-P VIC by the 30-kDa and PPD antigens.