Hydrogen peroxide inhibits IL-12 p40 induction in macrophages by inhibiting c-rel translocation to the nucleus through activation of calmodulin protein

Abstract

Although the antimicrobial activity of reactive oxygen species (ROSs) is well defined, the role of ROSs in regulating the immune response of the body is not well understood. We now provide evidence that hydrogen peroxide (H 2 O IntroductionAccumulated damage due to reactive oxygen species (ROSs) has been suggested to occur during aging 1,2 and more clearly demonstrated during the respiratory burst of phagocytes. 3,4 Hydrogen peroxide (H 2 O 2 ), is a primary component of ROSs produced in large amounts by macrophages and granulocytes and is a mediator of innate immunity against the invading pathogens. 5,6 H 2 O 2 is known to function as a second messenger 7,8 and regulates activation of many important transcription factors, such as nuclear factor-B (NF-B) and activator protein-1 (AP-1), 9,10 that control the inducible expression of genes regulating macrophage-effector functions and cytokine signaling. [11][12][13] The macrophage-induced cytokines determine the fate of the subsequent T-cell responses as type 1 14,15 or type 2. 16,17 Thus, the possibility exists that H 2 O 2 may alter the T-cell immune responses by affecting the macrophageeffector responses. The function of ROSs may not be restricted to its antimicrobial activity alone, 18 but ROSs may also play an important role in regulating the immune environment in the body. Such situations may arise during certain pathophysiologic conditions such as tuberculosis. 19,20 We earlier reported that activated macrophages from the Bruton tyrosine kinase (btk)-deficient mice (CBA/N) produced lower levels of ROSs as compared to the wild-type mice (CBA/J). 21 However, in contrast to CBA/J macrophages, the IL-12 induction was significantly higher in macrophages from CBA/N mice with T-cell responses biased toward type 1. 21,22 Although the contribution of btk enzyme in controlling the signal transduction cascades responsible for ROS production independent of IL-12 signal may not be ruled out, it is possible that btk targets the ROS pathway to influence IL-12 production. However, the exact mechanism by which ROSs down-regulate IL-12 production is not well understood. We now provide evidence that H 2 O 2 /ROSs affect the signaling events important for IL-12 production, leading to downregulation of the same in activated macrophages. Materials and methods MiceBALB/c mice were bred and maintained in the animal facility of Indian Immunologicals (IIL; Hyderabad, India). All mice were 6 to 12 weeks old and experimental protocol was approved by the Institutional Review Committee for care and usage of animals of Indian Immunologicals. Macrophage stimulation assayThe peritoneal exudate cells (PECs) were harvested by injecting 4% thioglycolate broth as described elsewhere. 23 The RAW 264.7 macrophages were obtained from NCCS (National Centre for Cell Science, Pune, India) and maintained in Dulbecco minimal essential medium (DMEM; Invitrogen, Grand Island, NY) containing 10% fetal calf serum and antibiotics (DMEM-10). The macrophages were plated at a density of 3 ϫ 10 6 cells/mL and ...

Glutathione-Redox Balance Regulates c-rel–Driven IL-12 Production in Macrophages: Possible Implications in Antituberculosis Immunotherapy

“…Membranes were washed and incubated with either antimouse or anti-rabbit Ig coupled to HRP (Sigma-Aldrich). Bound enzyme was detected by ECL following the manufacturer's protocol (GE Healthcare, Little Chalfont, U.K.) as described earlier (21). Equal loading of protein was confirmed by measuring the b-actin level in the same extracts by IB.…”

“…An EIA method involving competitive binding of anti-CaM mAb was used to measure cytoplasmic CaM levels as described earlier (21,22). Briefly, RAW 264.7 macrophages were either left untreated or treated with various concentrations of NAC for 1 h. In some experiments, RAW 264.7 macrophages were pretreated with 10 mM SB203580 for 30 min followed by treatment with 20 mM NAC.…”

“…The reaction was terminated using 1 N H 2 SO 4 , and the absorbance values were measured at 490 nm. CaM levels in the test samples were expressed as the fold change over the untreated control (21).…”

“…Culture supernatants were harvested after 48 h to estimate levels of IL-12 p40/p70, IL-10, and TNF-a cytokines by two-site sandwich enzyme immunoassays (EIAs) (BD Biosciences, San Jose, CA) as described earlier (20,21). The c-rel or phospho-IkBa or CaM or phospho-p38 (pp38) MAPK levels were measured after 45-60 min of NAC treatment or at indicated time points by immunoblotting (IB), EIA, or flow cytometry.…”

“…Nuclear extracts were prepared from nonidet P-40-lysed cells as described earlier (20,21) to detect the levels of p50, p65 and c-rel. Equal amounts of the extracts were separated by 10% SDS-PAGE.…”