Cytokine profiles for peripheral blood lymphocytes from patients with active pulmonary tuberculosis and healthy household contacts in response to the 30-kilodalton antigen of Mycobacterium tuberculosis

Abstract

Patients with active tuberculosis (TB) have a stronger humoral but a poorer cellular immune response to the secreted 30-kDa antigen (Ag) of Mycobacterium tuberculosis than do healthy household contacts (HHC), who presumably are more protected against disease. The basis for this observation was studied by examining the Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-gamma])- and Th2 (IL-10 and IL-4)-type cytokines produced in response to the 30-kDa Ag by peripheral blood mononuclear cells (PBMC) from patients with active pulmonary TB (n = 7) and from HHC who were tuberculin (purified protein derivative) skin test positive (n = 12). Thirty-kilodalton-Ag-stimulated PBMC from TB patients produced significantly lower levels of IFN-gamma (none detectable) than did those from HHC (212 +/- 73 pg/ml, mean +/- standard error) (P < 0.001). Likewise, 30-kDa-Ag-stimulated PBMC from TB patients failed to express IFN-gamma mRNA by reverse transcription-PCR, whereas cells from HHC expressed the IFN-gamma gene. In contrast, 30-kDa-Ag-stimulated PBMC from TB patients produced significantly higher levels of IL-10 (403 +/- 80 pg/ml) than did those from HHC (187 +/- 66 pg/ml) (P < 0.013), although cells from both groups expressed the IL-10 gene. IL-2 and IL-4 were not consistently produced, and their genes were not expressed by 30-kDa-Ag-stimulated cells from either TB patients or HHC. After treatment with antituberculous drugs, lymphocytes from four of the seven TB patients proliferated and three of them expressed IFN-gamma mRNA in response to the 30-kDa Ag and produced decreased levels of IL-10.

Figures

FIG. 1

Blastogenic (A) and serologic (B) responses to the 30-kDa Ag in TB patients and HHC. (A) PBMC from patients with active TB and HHC that were PPD skin test positive were stimulated with the 30-kDa Ag (2 μg/ml) for 6 days, and incorporation of [3H]thymidine was measured. Results are expressed as SIs. An SI of >3 was considered a positive response. The baseline counts without Ag were 660 ± 398 cpm for TB patients and 678 ± 271 cpm for HHC. (B) ELISA was performed on serum samples from TB patients and PPD-positive HHC for reactivity to the 30-kDa Ag. Results are expressed in OD units. An OD of >0.2 was considered a positive result.

FIG. 2

Detection of IgG subclasses directed against the 30-kDa Ag. Sera from patients with active TB were analyzed by ELISA for specific IgG subclass reactivity to 30-kDa Ag. Results are presented as mean OD units ± SE.

FIG. 3

IFN-γ and IL-10 production in response to the 30-kDa Ag. PBMC from TB patients and PPD-positive HHC were stimulated with the 30-kDa Ag for 48 h. Supernatants of cells were assayed for IFN-γ and IL-10 by ELISA. Results are expressed as mean ± SE in picograms per milliliter.

FIG. 4

Expression of IFN-γ mRNA by 30-kDa-Ag-stimulated PBMC. PBMC from HHC and patients with TB were stimulated with the 30-kDa Ag, and RNA was extracted after 48 h. RT-PCR was performed as described in Materials and Methods. RT-PCR products were visualized by staining with ethidium bromide. Lanes: 1, negative control for the extraction (all reagents without DNA from cells); 2, lysates of PBMC from HHC amplified for β actin; 3, lysates of PBMC from HHC stimulated with the 30-kDa Ag and amplified for IFN-γ; 4, lysates of PBMC from TB patients amplified for β actin; 5, lysates of PBMC from a TB patient stimulated with the 30-kDa Ag and amplified with IFN-γ; 6, molecular weight markers.