Lipopolysaccharide, IFN-γ, and IFN-β Induce Expression of the Thiol-Sensitive ART2.1 Ecto-ADP-Ribosyltransferase in Murine Macrophages

Abstract

Extracellular NAD modulates multiple immune and inflammatory responses via the activity of a family of ecto‐ADP‐ribosyltransferases (ARTs) that covalently modify target proteins at arginine or cysteine residues. Functional ARTs have been identified thus far in neutrophils and T cells but not macrophages. This study investigates the expression and function of ARTs by RT‐PCR and western blot analysis in primary bone‐marrow‐derived macrophages (BMDM) isolated from Balb/c mice. Although naive BMDM did not express any of the 6 murine ART genes at the mRNA or protein level, stimulation with lipopolysaccharide (LPS) or ligands for other Toll‐like receptors induced robust expression of ART2.1 as a GPI‐anchored cell surface ecto‐enzyme. ART2.1 expression was potentiated by pharmacological inhibition of ERK1/2 pathways but inhibited by pharmacological blockade of NFkappaB‐ or PI3K‐ signaling pathways. Significantly, catalytic enzyme activity of the inducible ART2.1 was strictly dependent on the presence of extracellular thiol reducing molecules such as cysteine or dithiolthreitol. These data indicate that modulation of immune function by NAD‐dependent ART2.1 activity in macrophages may be synergistically regulated by extracellular redox environments that occur during hypoxia or ishchemia. Support: NIH‐GM‐36387

Basal and inducible expression of the thiol-sensitive ART2.1 ecto-ADP-ribosyltransferase in myeloid and lymphoid leukocytes

“…Taq DNA polymerase was from New England BioLabs. 1G4 mouse monoclonal antibody was prepared as previously described [16]. Goat polyclonal anti-actin (sc1615) antibody and all HRPconjugated secondary antibodies were from Santa Cruz Biotechnology.…”

“…For BMDM or BMDC, femurs and tibia were isolated from the euthanized mouse and the marrow cavity plugs were washed out by sterile PBS. BALB/c BMDM were expanded and grown in the presence of M-CSF as previously described [16]. For generation of BMDC, bone marrow cells were cultured in DMEM (Sigma-Aldrich) supplemented with 2 ng/ml GM-CSF (PeproTech), 10% calf serum (HyClone Laboratories), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen Life Technologies) in 150-mm dishes, in the presence of 10% CO 2 at 37°C.…”

“…The resulting PCR amplicons were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining. The primers and PCR conditions for the six murine ectoARTs, iNOS, IRF-1, IFN-β and GAPDH were identical to those previously described [16]. Additionally, CD38 was used as a marker for the B cell population with the primer set: forward: 5′-GCAACATCACAAGAGAAGACTACGC-3′; reverse: 5′-ACACACTGAAGAAACCTGGCAGGCC-3′.…”

“…1G4 mAb-based ART activity assay and Western blot protocols ART activity was assayed in BMDC, BMDM, freshly isolated tissue macrophages, splenic T lymphocytes, splenic B lymphocytes, or BW5147 thymoma cells using the 1G4 antibody [26] that detects etheno-ADP-ribosylated proteins as previously described [16]. Briefly, intact cells were transferred to basic salt solution (BSS) containing 130 mM NaCl, 5 mM KCl, 1.5 mM CaCl 2 , 1 mM MgCl 2 , 25 mM HEPES (pH 7.5), 5 mM glucose and 1% BSA, supplemented with the indicated concentrations of ε-NAD substrate and 1 mM ADP-ribose (ADP-R) plus or minus 2 mM dithiothreitol (DTT).…”

“…We have recently reported that bone-marrow-derived macrophages (BMDM) lack significant expression of any of the murine ecto-ART subtypes, but selectively upregulate ART2.1 (but not ART2.2 or other subtypes) in response to multiple inflammatory mediators [16]. In contrast, freshly isolated T cells from the same mice basally express both ART2.1 and ART2.2 [17][18][19][20][21][22][23].…”