CD40 signaling in macrophages induces activity against an intracellular pathogen independently of gamma interferon and reactive nitrogen intermediates

Abstract

Gamma interferon (IFN-gamma) is the major inducer of classical activation of macrophages. Classically activated mouse macrophages acquire antimicrobial activity that is largely dependent on the production of reactive nitrogen intermediates. However, protection against important intracellular pathogens can take place in the absence of IFN-gamma and nitric oxide synthase 2 (NOS2). Using Toxoplasma gondii as a model, we investigated if CD40 signaling generates mouse macrophages with effector function against an intracellular pathogen despite the absence of priming with IFN-gamma and lack of production of reactive nitrogen intermediates. CD40-stimulated macrophages acquired anti-T. gondii activity that was not inhibited by a neutralizing anti-IFN-gamma monoclonal antibody but was ablated by the neutralization of tumor necrosis factor alpha (TNF-alpha). Moreover, while the induction of anti-T. gondii activity in response to CD40 stimulation was unimpaired in macrophages from IFN-gamma(-/-) mice, macrophages from TNF receptor 1/2(-/-) mice failed to respond to CD40 engagement. In contrast to IFN-gamma-lipopolysaccharide, CD40 stimulation did not induce NOS2 expression and did not trigger production of reactive nitrogen intermediates. Neither N(G)-monomethyl-l-arginine nor diphenyleneiodonium chloride affected the induction of anti-T. gondii activity in response to CD40. Finally, macrophages from NOS2(-/-) mice acquired anti-T. gondii activity in response to CD40 stimulation that was similar to that of macrophages from wild-type mice. These results demonstrate that CD40 induces the antimicrobial activity of macrophages against an intracellular pathogen despite the lack of two central features of classically activated macrophages: priming with IFN-gamma and production of reactive nitrogen intermediates.

FIG. 1.

CD40 enhances the anti-T. gondii activity of mouse macrophages. Resident peritoneal macrophages from BALB/c mice were incubated with either isotype control or stimulatory anti-CD40 MAb as described in Materials and Methods. Macrophages were washed and challenged with T. gondii for 1 h. Monolayers were examined by light microscopy 1, 6, 12, and 18 h after addition of T. gondii. Results shown are representative of one out of four independent experiments.

FIG. 2.

Activated T cells induce macrophage anti-T. gondii activity through CD40-CD154 interaction. Peritoneal macrophages were infected with T. gondii (Tg) for 1 h, followed by removal of extracellular parasites and addition of either resting (Tr) or activated (Ta) T cells. Macrophages and T cells were cultured in the presence of anti-IFN-γ (A), anti-CD154 (B), or control MAbs. Results of one representative experiment out of four are shown.

FIG. 3.

CD40 induces anti-T. gondii activity in mouse macrophages independently of IFN-γ but requires TNF-α signaling. (A) Peritoneal macrophages from BALB/c mice were cultured with either isotype control or stimulatory anti-CD40 MAb in the presence of a neutralizing anti-IFN-γ or control MAbs. Monolayers were examined by light microscopy 18 h after addition of T. gondii. (B) Peritoneal macrophages from BALB/c and IFN-γ^−/− mice were cultured with either isotype control or stimulatory anti-CD40 MAbs. (C) Peritoneal macrophages from BALB/c mice were cultured with either isotype control or stimulatory anti-CD40 MAb in the presence of a neutralizing anti-TNF-α or control MAbs. (D) Peritoneal macrophages from B6/129 and TNFR1/2^−/− mice were cultured with either isotype control or stimulatory anti-CD40 MAb. Results of one representative experiment out of three are shown.

FIG. 4.

Only the CD40⁺ subpopulation of macrophages acquires anti-T. gondii activity in response to anti-CD40 MAb. Peritoneal macrophages cultured with anti-CD40 or control MAbs were infected with T. gondii expressing YFP. At 1 and 18 h postinfection, expression of membrane CD40 was analyzed using a biotinylated anti-CD40 MAb and Alexa 568-conjugated streptavidin. Cells were examined by confocal immunofluorescence microscopy. (A) Percentages of infected cells in CD40⁺ and CD40⁻ macrophages at 1 and 18 h postinfection are shown. (B) Representative fluorescence and differential interference contrast microphotograph taken 18 h postinfection reveals that CD40 stimulation causes a decrease in the percentage of infected cells among CD40⁺ macrophages. Bar, 5 μm. Results from one representative experiment out of three are shown.

FIG. 5.

Only purified CD40⁺ macrophages acquire anti-T. gondii activity in response to anti-CD40 MAb. Unsorted and sorted CD40⁺ and CD40⁻ bone marrow-derived macrophages were cultured with anti-CD40 or control MAbs. Monolayers were examined by light microscopy 18 h after T. gondii challenge. Results of one representative experiment out of three are shown.

FIG. 6.

CD40 induces macrophage anti-T. gondii activity independently of RNI. Panels A and B show that sorted CD40⁺ bone marrow-derived macrophages from BALB/c mice were incubated with either isotype control or stimulatory anti-CD40 MAb or with IFN-γ plus LPS with or without NMA as described in Materials sand Methods. (A) Cell-free supernatants were collected 24 h poststimulation with MAbs or IFN-γ plus LPS and were used to measure nitrate concentrations. (B) Monolayers were examined microscopically at 18 h postchallenge. (C) Peritoneal macrophages from B6/129 or NOS^−/− mice were incubated with either control or stimulatory anti-CD40 MAb. Monolayers were examined microscopically at 18 h postchallenge with T. gondii. Results of one representative experiment out of three are shown.

FIG. 7.

CD40 does not induce NOS2 expression in mouse macrophages. (A) Bone marrow-derived macrophages were cultured in CM alone or in the presence of isotype control or stimulatory anti-CD40 MAb or IFN-γ plus LPS. Total cell lysates were obtained after 24 h and were used to determine levels of NOS2 and actin by immunoblot staining. (B) RNA was obtained from bone marrow-derived macrophages after 6 h and used to generate cDNA. Levels of NOS2 and HPRT mRNA were determined by reverse transcription-PCR. Results of one representative experiment out of three are shown.