Blackwell Science, LtdOxford, UKCEIClinical and Experimental Immunology1365-2249Blackwell Publishing Ltd, 2003 1341 7077 Original Article B. Singh et al. MTSA-10 down-regulates B7·1 and NO
Clin Exp Immunol 2003; 134:70–77
doi:10.1046/j.1365-2249.2003.02258.x
Intracellular expression of Mycobacterium tuberculosis-specific 10-kDa antigen down-regulates macrophage B7·1 expression and nitric oxide release B. SINGH*‡, G. SINGH*‡, V. TRAJKOVIC*†‡ & P. SHARMA* *Immunology Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India, and †Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Belgrade, Serbia
(Accepted for publication 22 July 2003)
SUMMARY To explore the role of the 10-kDa Mycobacterium tuberculosis-specific secreted antigen (MTSA-10 or CFP-10) in modulation of macrophage function, J774 macrophages were transfected stably with DNA encoding MTSA-10. Compared to normal or mock-transfected controls, MTSA-10-expressing macrophages had markedly lower levels of co-stimulatory molecule B7·1 on their surface, while the expression of B7·2 and ICAM-1 was not affected. MTSA-transfected cells also produced significantly less microbicidal free radical nitric oxide (NO) upon stimulation with interferon (IFN)- g, lipopolysaccharide or M. tuberculosis cell lysate. Western blot analysis revealed the absence of tyrosine-phosphorylated protein slightly larger than 112 kDa in MTSA-transfected macrophages. Moreover, the treatment of control J774 cells with protein tyrosine kinase inhibitor genistein completely mimicked the effects of transfection with MTSA-10, selectively down-regulating NO and B7·1, but not B7·2 or ICAM-1 expression. The observed MTSA-10-mediated block of B7·1 expression and NO release might contribute to the suppression of antimycobacterial response in tuberculosis. Keywords
B7·1
CFP-10
IFN-g
M. tuberculosis
INTRODUCTION Mycobacterium tuberculosis is a highly successful intracellular pathogen well known for its ability to persist inside macrophages and cause chronic disease in susceptible individuals. The prevention of phagosome maturation into an acidic, hydrolytic compartment with microbicidal activity seems to present a major survival strategy of M. tuberculosis [1,2]. However, additional mechanisms for evading the immune response are also employed by mycobacteria, including down-regulation of the protective T cell response through interference with antigen processing and presentation, and the expression of co-stimulatory molecule B7 in infected macrophages [3-5]. An important feature of mycobacterial subversion of the host immune functions is also the induction of macrophage unresponsiveness to interferon (IFN)- g [6,7], a T cell cytokine crucial for optimal macrophage activation and subsequent synthesis of bactericidal molecules such as oxygen and nitrogen radicals [8,9]. Correspondence: Dr P. Sharma, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India. E-mail:
[email protected] ‡These authors contributed equally to this paper.
70
MTSA-10
macrophages
nitric oxide
The proteins secreted actively by M. tuberculosis into the culture medium (culture filtrate proteins: CFP) represent possible candidates for mycobacterial down-regulation of macrophage function. Such a notion is based on the findings that only live, but not dead M. tuberculosis, can prevent phagosomal-lysosomal fusion and macrophage B7 expression [5,10,11]. Among CFP constituents is the recently discovered 10-kDa protein M. tuberculosis secreted antigen (MTSA-10), designated originally CFP-10 [12]. MTSA-10 is one of the major antigens recognized by M. tuberculosis-specific human T and B cells [13–17], and it induces strong delayed type hypersensitivity response in M. tuberculosis-infected guinea pigs [13,18,19]. MTSA-10 is missing in M. bovis BCG, and therefore represents an ideal candidate for diagnostic test that will discriminate between infected and BCG-vaccinated people [19,20]. However, as it is specific for M. tuberculosis, MTSA-10 might also contribute to its unique ability for down-regulation of macrophage activity and subsequent impairment of the protective T cell response. We have reported recently that MTSA-10 can bind to the surface of macrophages and profoundly modulate their function, causing partial unresponsiveness to induction of nitric oxide (NO) release [21]. As a continuation of this line of research, the present study shows that MTSA-10-transfected J774 macrophages are less © 2003 Blackwell Publishing Ltd
MTSA-10 down-regulates B7·1 and NO capable of NO production, as well as B7·1 expression, indicating that intracellular presence of MTSA-10 might also contribute to the loss of macrophage antimycobacterial function in tuberculosis.
MATERIALS AND METHODS Reagents Dulbeco’s modified Eagle medium (DMEM), fetal calf serum (FCS) and lipofectin reagent were all from Invitrogen Life Technologies (Carlsbad, CA, USA). Retroviral pLNCX2 vector and PT67 cell line were from Clontech/BD Biosciences (Palo Alto, CA, USA). Griess reagent, murine recombinant IFN-g, genistein, SB203580, G418, HRPO-conjugated antimouse or antirabbit IgG, as well as biotinylated antiphosphotyrosine (PT-66), antiphosphoserine (PSR-45) and antiphosphothreonine (PTR-8) antibodies were all purchased from Sigma (St Louis, MO, USA). FITClabelled antimouse B7·1 and B7·2, antimouse CD32/CD16 (Fc block), biotinylated antimouse ICAM-1 and streptavidin-FITC were from BD Pharmingen (San Diego, CA, USA), while U0126 was obtained from Promega (Madison, WI, USA). Recombinant MTSA-10 was expressed in Escherichia coli [lipopolysaccharide (LPS) concentration
