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The Journal of Immunology, 2007, 179, 6215 -6227
Copyright © 2007 by The American Association of Immunologists, Inc.
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Lipopolysaccharide, IFN-{gamma}, and IFN-beta Induce Expression of the Thiol-Sensitive ART2.1 Ecto-ADP-Ribosyltransferase in Murine Macrophages1

Shiyuan Hong*, Anette Brass{ddagger}, Michel Seman{dagger}, Friedrich Haag{ddagger}, Friedrich Koch-Nolte{ddagger} and George R. Dubyak2,*

* Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44120; {dagger} Institut National de la Santé et de la Recherche Médicale Unité 519, University of Rouen, Rouen, France; and {ddagger} Institute of Immunology, University Hospital, Hamburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nicotinamide adenosine dinucleotide (NAD) can act as a modulator of multiple immune and inflammatory responses when released into extracellular compartments. These actions of extracellular NAD are largely mediated by a family of mammalian ecto-ADP-ribosyltransferases (ARTs) that covalently modify target extracellular or cell surface proteins by transferring ADP-ribose to arginine or cysteine residues. In this study, we report that bone marrow-derived macrophages (BMDM) from BALB/c mice lack constitutive expression of any of the six murine ecto-ART subtypes, but selectively up-regulate ART2.1 in response to multiple proinflammatory mediators including agonists for TLR and type I and type II IFN. Stimulation of BMDM with LPS, IFN-beta, or IFN-{gamma} induced high expression of ART2.1, but not ART2.2, as a GPI-anchored cell surface ectoenzyme. ART2.1 expression in response to LPS was potentiated by inhibition of ERK1/2 signaling, but inhibited by blockade of the NF-{kappa}B, PI3K, and JAK-STAT pathways or the presence of neutralizing anti-IFN-beta. The catalytic function of the induced cell surface ART2.1 was strictly dependent on the presence of extracellular thiol-reducing cofactors, suggesting that in vivo activity of ART2.1-expressing macrophages may be potentiated in hypoxic or ischemic compartments. Consistent with the mutated art2a gene in C57BL/6 mice, LPS- or IFN-stimulated BMDM from this strain lacked expression of cell surface ART2 activity in the presence or absence of extracellular thiol reductants. Collectively, these studies identify ART2.1 as a new candidate for linking autocrine/paracrine activation of inflammatory macrophages to the release of NAD, a critical intracellular metabolite.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nicotinamide adenosine dinucleotide (NAD),3 an intracellular metabolite with critical roles in bioenergetics, can also act as a mediator or modulator of multiple immune and inflammatory responses (reviewed in Refs. 1 , 2) when released into extracellular compartments via cytolysis or nonlytic-regulated secretion (3, 4). Many extracellular NAD-induced changes in immune cell function are mediated by a family of mammalian ecto-ADP-ribosyltransferases (ARTs) that covalently modify target proteins by transferring the ADP-ribose moiety from NAD to arginine or cysteine residues (1, 2, 5, 6). The mammalian ecto-ART types include four human subtypes (ART1, 3, 4, 5) and six murine subtypes (ART1, 2.1, 2.2, 3, 4, 5) that are encoded by separate genes differentially expressed in particular tissues (6). Except for ART5, which is a secreted protein (7), all other ecto-ARTs are linked to the cell surface via a GPI-anchor (2).

The ART2 proteins expressed in murine T lymphocytes are among the best-characterized members of this ectoenzyme family at the genetic, biochemical, and cell physiological levels (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). Murine ART2 includes two homologous (80% sequence identity) isoforms, ART2.1 and ART2.2, encoded by separate but tandem genes, art2a and art2b, on chromosome 7; this general chromosome 7 locus also includes the genes encoding murine ART1 and ART5 (6). Both ART2.1 and ART2.2 can act as either ART or NAD-glycohydrolase. However, the activity of ART2.1 requires the presence of extracellular thiol-reducing agents, such as DTT or cysteine, to reversibly control oxidation of unique residues (Cys80 and Cys201) present in ART2.1 but not ART2.2 (19, 20). In the presence of micromolar extracellular NAD, ART2 enzymes modulate a variety of murine T lymphocyte functions by ADP-ribosylating multiple cell surface proteins including CD8, CD44, CD45, and the CD11a and CD18 chains of LFA-1 (12, 13). The ART2.2-mediated ADP-ribosylation of these various proteins inhibits T cell homotypic adhesion and modulates TCR signaling. The P2X7 purinergic receptor P2X7R of mouse T cells can be activated by ART2.2-mediated ADP-ribosylation, which induces phosphatidylserine exposure on T cell surfaces, increased shedding of CD62L, and acceleration of naive T cell death (21). This NAD-induced transactivation of P2X7R has been correlated with a decreased number of circulating CD4+CD25+ regulatory T cells in mice (22). ART2.2 is rapidly released from activated T cells by the action of endogenous metalloproteases or exogenously added phosphatidylinositol phospholipase C (PI-PLC) (11, 23). CD38, an ecto-NAD-glycohydrolase on B cells, controls the level of ADP-ribosylation of adjacent T cell surface proteins by limiting the availability of NAD as a substrate for ART2 (24). Circulating T cells from these CD38-deficient mice exhibit markedly reduced surface levels of CD62L, which is consistent with an enhanced in vivo activation of the P2X7R by ART2-mediated ADP-ribosylation.

The generation of double ART2.1/ART2.2 knockout mice has provided further insights regarding the biological significance of this enzyme (25). T cells from ART2 knockout mice are resistant to NAD-induced apoptosis, whereas mice lacking ART2 exhibit increased survival in a model of NK T cell-mediated autoimmune hepatitis (25, 26). Significantly, genetic background also affects expression of ART2.1 vs ART2.2 in different inbred mice strains (27, 28, 29). In BALB/c mice, both ART2.1 and ART2.2 are constitutively expressed in all subsets of naive T lymphocytes (30, 31). C57BL/6 mice, another widely used strain, express a mutated art2a gene that results in a premature stop codon in the transcribed ART2.1 mRNA and a consequent absence of functional ART2.1 protein (30). Thus, C57BL/6 T lymphocytes are phenotypically ART2.1-null but still express ART2.2 at high levels. Conversely, the art2b gene is inactive in New Zealand White (NZW) mice, and T cells from this strain lack functional ART2.2 and express only the extracellular thiol-dependent ART2.1 enzyme (27, 28, 32).

Only a few studies have investigated the possible expression or function of ecto-ART in the myeloid leukocytes (macrophages, monocytes, neutrophils) that comprise the other major group of immune/inflammatory effector cells. ART1 is present in an intracellular compartment in quiescent human neutrophils but its expression as a cell surface protein is rapidly up-regulated during neutrophil activation (33). The constitutive expression of ART3 mRNA and an LPS-induced expression of ART4 mRNA, but only minor ecto-ART enzyme activity, has been described in human monocytes (34, 35). In an early comparative analysis of murine immune effector cells, Okamoto et al. (12) reported that neither spleen macrophages nor peritoneal macrophages expressed functional cell surface ecto-ART activity.

In this study, we report that bone-marrow derived murine macrophages (BMDM) lack constitutive expression of any of the six murine ecto-ART subtypes, but robustly and selectively up-regulate the thiol-sensitive ART2.1 enzyme in response to activation by multiple proinflammatory mediators, including TLR agonists and type I and II IFN. Collectively, these studies identify ART2.1 as a new candidate for linking autocrine/paracrine activation of inflammatory macrophages to the release of NAD, a critical intracellular metabolite.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

NAD, etheno-NAD ({epsilon}-NAD), and ADP-ribose were from Sigma-Aldrich. LPS (Escherichia coli serotype O1101:B4) was from List Biological Laboratories. Other TLR ligands included PAM3C4SK (P3CSK; InvivoGen) and imiquimod (R837; InvivoGen). Recombinant murine IFN-{gamma} was from Boehringer Mannheim Biochemica; recombinant murine IFN-beta and neutralizing Ab against IFN-beta were from U.S. Biologicals. Pharmacological inhibitors of various kinases included: U0126 (Calbiochem), SB203580 (Biomol), SP600125 (Calbiochem), wortmannin (Sigma-Aldrich), BAY 11-7085 (Biomol), and AG490 (Calbiochem). Bacterial PI-PLC was from Sigma-Aldrich. Hybridoma cells expressing the 1G4 mouse mAb specific for {epsilon}-adenosine were a gift from Dr. R. Santella (Columbia University, New York, NY). These hybridoma cells were cultured in RPMI 1640 for 7 days and the spent medium containing 1G4 mAb was fractionated on mAb TRAPII-protein G columns (Pharmacia); the affinity purified mAb was concentrated to 3 mg/ml. Mouse mAb KL295 against MHC class II was a gift from Dr. C. Harding (Case Western Reserve University, Cleveland, OH). Monoclonal Abs directed against mouse ART2.1 or ART2.2 were generated and used as described recently (36). Goat polyclonal anti-actin (sc1615) Ab and all HRP-conjugated secondary Abs were from Santa Cruz Biotechnology. BALB/c, C57BL/6, or NZW mice were purchased from Taconic Farms and The Jackson Laboratory. All experiments and procedures involving mice were approved by the Institutional Animal Use and Care Committees of Case Western Reserve University or Hamburg University Hospital.

Cells

These studies used BMDM and splenocytes isolated from BALB/c, C57BL/6, or NZW mice. Mice were euthanized by CO2 inhalation. BMDM were isolated based on a modification of a previously described protocol (37). Femurs and tibia were removed from the euthanized mouse and briefly sterilized in 70% ethanol, and PBS was used to wash out the marrow cavity plugs. The bone marrow cells were resuspended in DMEM (Sigma-Aldrich) supplemented with 25% L cell-conditioned medium, 15% calf serum (HyClone Laboratories), 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen Life Technologies). Cells were plated onto 150-mm dishes and cultured in the presence of 10% CO2. After 5–9 days, the resulting BMDM were detached with PBS containing 5 mM EDTA and 4 mg/ml lidocaine, replated into 6- or 12-well plates, and used within 2 wk. Spleens from euthanized mice were cut into small pieces and pressed through nylon mesh to generate single cell suspensions; the mononuclear leukocyte fraction was depleted of erythrocytes, granulocytes, and debris by gradient centrifugation on Histopaque-1077 (Sigma-Aldrich). In some cases, spleen CD4+ T cells were separated from the spleen B cells by fractionation using Dynal Biotech magnetic beads coated with goat anti-mouse Ig.

RT-PCR analyses

Total RNA was extracted using TRIzol (Sigma-Aldrich) and 3 µg of RNA were primed with oligo(dT) primers (Promega) at 65°C for 10 min and incubated with AMV reverse transcriptase (Roche) at 42°C for 60 min. Primer pairs selective for the six subtypes of murine ecto-ARTs were designed as following: ART1 (forward) 5'-CAGCTTTGCCGCCATGGAGAAGGC-3', (reverse) 5'-GCCTGGTACTACCACTCATACC-3'; ART2 (forward) 5'-GAGGACAGAGACCCAGCTGCC-3', (reverse) 5'-GACCGAGGAGAACCACAAGGAACAG-3'; ART2.1 (forward) 5'-ATCCACAGAAGCCTTAATGAG-3', (reverse) 5'-CTACGGCTCAGCAAGAGTAA-3'; ART2.2 (forward) 5'-CCTCGCTATAGATTTTAACAG-3', (reverse) 5'-CTACGGCTCAGCAAGAGTAA-3'; ART3 (forward) 5'-ATGAAGATGGGACATTTTGAAATGGTCAC-3', (reverse) 5'-TCTGGACTTCCTGTGGGATCCC-3'; ART4 (forward) 5'-GATGGCGCTGTGGCTTCCAGGAGG-3', (reverse) 5'-AGCAGCTCCTTTAAAAAGGAGCCAG-3'; and ART5 (forward) 5'-AGGATGATTCTGGAGGATCTGCTGATG-3', (reverse) 5'-CTGCTTCCTGCAGCCGTTCAAAGCCC-3'.

Induction of other inflammatory response genes were assayed with the following primers: murine IL-1beta (forward) 5'-CCAGGATGAGGACATGAGCACC-3', (reverse) 5'-TTCTCTGCAGACTCAAACTCCAC-3'; murine inducible NO synthase (iNOS) (forward) 5'-ACGGAGAAGCTTAGATCTGGAGCAGAAGTG-3', (reverse) 5'-CTGCAGGTTGGACCACTGGATCCTGCCGAT-3'; murine IFN receptor factor-1 (IRF-1) (forward) 5'-TTAGCCCGGACACTTTCTCTGATGG-3', (reverse) 5'-GTCCCCTCGAGGGCTGTCAATCTCT-3'; and murine IFN-beta (forward) 5'-CTTCTCCACCACAGCCCTCTC-3', (reverse) 5'-CCCACGTCAATCTTTCCTCTT-3'. GAPDH was assayed as a housekeeping gene product and normalization signal for cDNA amplification using the primers (forward) 5'-GGGTGGAGCCAAACGGGTC-3' and (reverse) 5'-GGAGTTGCTGTTGAAGTCGCA-3'. PCR was routinely performed using 1/100 (for ARTs, iNOS, IFN-beta, and IRF-1), 1/1000 (for IL-1beta), or 1/10,000 (for GAPDH) dilutions of the reverse transcriptase reactions in 20-µl reaction volumes and cycling conditions optimized for selective amplifications. PCR conditions were as follows: ART2.1 and ART2.2 at 92°C, 1 min; 60°C, 1 min; 72°C, 2 min for 35 cycles; ART1, ART3, ART5, GAPDH, and IL-1beta at 94°C, 30 s; 60°C, 30 s; 72°C, 2 min for 35 cycles; ART4 at 94°C, 30 s; 64°C, 30 s; 72°C, 3 min for 35 cycles; ART2 at 94°C, 20 s; 70°C, 30 s; 72°C, 3 min for 3 cycles; ART2 at 94°C, 20 s; 65°C, 30 s; 72°C, 3 min for 3 cycles; ART2 at 94°C, 20 s; 60°C, 30 s; 72°C, 3 min for 30 cycles; IFN-beta at 94°C, 30 s; 58°C, 30 s; 72°C, 1 min for 35 cycles; IRF-1 at 94°C, 45 s; 61°C, 45 s; 72°C, 1.5 min for 25 cycles; and iNOS at 95°C, 60 s; 55°C, 90 s; 72°C, 3 min for 40 cycles. The sizes of target amplicons were as follows: ART1 at 1375 bp, ART2 888 bp, ART2.1 560 bp, ART2.2 567 bp, ART3 1178 bp, ART4 914 bp, ART5 1004 bp, IL-1beta 450 bp, IFN-beta 346 bp, IRF-1 434 bp, iNOS 654 bp, and GAPDH 532 bp. The PCR amplicons were separated by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining; the resulting fluorescence images were recorded with a Bio-Rad Gel Doc 1000 system.

1G4 mAb-based ART activity assay and Western blot protocols

ART activity in intact BMDM or splenocytes was assayed using a Western blot protocol based on the 1G4 mAb specific for etheno-adenosine, which permits detection of proteins containing covalently associated etheno-ADP-ribose moieties (38). Intact cells in 6-well plates were transferred to fresh DMEM containing 15% calf serum, and then treated (37°C) for 0–60 min with 1–100 µM {epsilon}-NAD (as an NAD surrogate) in the presence of 1 mM ADP-ribose to prevent reversal of ADP-ribosylation reactions by cell surface ectonucleotidases. Where indicated, the test medium was additionally supplemented with 0.1–10 mM DTT or 0.5–4 mM L-cysteine. The ADP-ribosylation reactions were terminated by aspiration of the test medium, rapid washing of cells with ice-cold PBS, and immediate lysis in 200 µl of PBS containing 1% Triton X-100 (Sigma-Aldrich), 2 mM DTT, 2 µg/ml leupeptin, 100 µg/ml PMSF, and 2.5 µg/ml aprotinin. After 20 min on ice, lysates were centrifuged at 15,000 x g for 10 min and the supernatants were transferred to new tubes. Aliquots of lysate were mixed with 4x SDS gel loading buffer and boiled for 5 min. A 60-µl processed samples was separated by 12% SDS-PAGE gel. Proteins were electrophoretically blotted (24 V for 54 min) onto polyvinylidene difluoride membranes (Millipore) in Tris-glycine buffer (9.09 g of Tris, 43.2 g of glycine, 600 ml of methanol, 2.4 L of H2O). The membrane was blocked by 4% nonfat milk in immunoblot buffer (10 mM Tris (pH 7.4), 0.9% NaCl, 0.05% Tween 20, and 1 mM EDTA) for 1 h and then incubated with primary Abs (1G4 at 75 µg/ml; anti-actin at 0.4 µg/ml) at 4°C for 12–18 h. The membrane was washed five times with immunoblot buffer, incubated for 60 min with appropriate HRP-conjugated secondary Ab, and again washed five times with immunoblot buffer before development using chemiluminescent reagents (SuperSignal; Pierce). Chemiluminescence signals were recorded using Eastman Kodak x-ray film. After the 1G4-based Western blot analysis, membranes were stripped of bound Ab and reprobed with anti-actin Ab (sc1615 at 1/1000) as a protein-loading control. Where indicated, stripped membranes were additionally probed with anti-MHC class II KL295 mAb at 1/2500.

FACS analyses

BMDM and splenocytes were stained with fluorochrome-conjugated Abs against CD40, CD80, and MHC class II (BD Biosciences). Staining for ART2.1 was performed with mAbs Gugu2-53, Gugu2-32, and Gugu2-44 (rat IgG2a, 1 µg/ml) for 30 min at 4°C followed by PE-conjugated anti-mouse IgG (1/100; The Jackson Laboratory) for 30 min at 4°C (36). Monoclonal Ab NONI-63 (rat IgG2a, anti-human ART4) was used as an isotype control. Stained cells were washed and analyzed on a FACSCalibur using the CellQuest software (BD Biosciences). Gating was performed on living cells on the basis of propidium iodide exclusion. Etheno-ADP-ribosylation of cell surface proteins was monitored as previously described (38) following incubation of cells for 10 min with 50 µM {epsilon}-NAD using Alexa Fluor 488-conjugated, etheno-adenosine-specific mAb 1G4.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Murine macrophages lack constitutive expression of ART1–ART5, but up-regulate ART2 in response to proinflammatory activation

RT-PCR analysis revealed that none of the five major murine ART subtypes (ART1–ART5) were constitutively expressed in BMDM (Fig. 1A) from BALB/c mice. Hearts from the same mice expressed ART1, 3, 4, and 5, whereas their spleens contained high levels of ART2 and lower amounts of ART4 mRNA (Fig. 1A). In contrast to their lack of constitutively expressed ART subtypes, BMDM stimulated with 100 ng/ml LPS for 24 h selectively accumulated ART2 mRNA but not ART1, 3, 4, or 5 (Fig. 1A). The up-regulation of ART2 was correlated with LPS-mediated induction of the IL-1beta gene that is otherwise transcriptionally silent in nonactivated macrophages (39).


Figure 1
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  		FIGURE 1. Murine macrophages lack constitutive expression of ART1–ART5 but up-regulate ART2 in response to proinflammatory activation by LPS. A, Expression of ART1, 2, 3, 4, or 5 mRNA was assayed by RT-PCR in naive or LPS-primed (100 ng/ml LPS, 24 h) BALB/c BMDM, as well as in freshly isolated heart or spleen from a BALB/c mouse. B, BALB/c BMDM were stimulated with 100 ng/ml LPS for the indicated times before extraction and RT-PCR analysis of ART2, IL-1beta, and GAPDH mRNA content. C, BALB/c BMDM were stimulated with the indicated concentrations of LPS for 24 h before extraction and RT-PCR analysis of ART2, IL-1beta, and GAPDH mRNA content. All results are representative of observations from two or more independent experiments.

 
Fig. 1, B and C, respectively, illustrates the induction times and LPS concentration range required for optimal ART2 expression in BMDM. Short-term (4 h) priming with LPS (100 ng/ml) resulted in only minor ART2 mRNA accumulation that contrasted with the maximal induction of IL-1beta expression. ART2 transcripts were detectable within 8 h, but continued to progressively accumulate over the next 16 h. The art2 gene expression in BMDM was characterized by a bell-shaped response to LPS concentration. Measurable accumulation of ART2 mRNA was induced by 0.1 ng/ml LPS and progressively increased as the LPS concentration was elevated to 100 ng/ml. However, a further increase in LPS concentration to 1000 ng/ml resulted in reduced induction of ART2 but not IL-1beta.

LPS induction of ART2 expression involves the NF-{kappa}B, ERK, and PI3K signaling pathways

Signals triggered by LPS-occupied TLR4 receptors include the ERK, JNK, and p38 MAPK kinases, the PI3K/Akt pathway, and transcriptional networks based on I{kappa}K/NF-{kappa}B and JAK/STAT cascades (40, 41). BALB/c BMDM were incubated with 100 ng/ml LPS in the presence of various inhibitors of these signaling pathways for 24 h before RT-PCR analysis. Blockade of the NF-{kappa}B pathway by BAY-11-7085, an I{kappa}K inhibitor, markedly decreased ART2 induction by LPS, as well as the stimulated IL-1beta expression (Fig. 2A). LPS-induced art2 gene expression was potentiated by pharmacological inhibition of the ERK1/2 pathway using the MEK1 inhibitor U0126, but was inhibited by the wortmannin-mediated suppression of PI3K/Akt signaling (Fig. 2B). Significantly, U0126 and wortmannin had opposite effects on the LPS-induced regulation of IL-1beta vs ART2 expression, indicating that the observed effects on ART2 were unlikely to reflect nonspecific actions on the general transcriptional machinery or mRNA stability. Neither the ART2 nor the IL-1beta expression stimulated by LPS was significantly affected by SP600125, a widely used JNK inhibitor, or SP203580, which suppresses p38 MAPK activity.


Figure 2
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  		FIGURE 2. LPS induction of ART2 expression involves the NF-{kappa}B, ERK, and PI3K pathways. A, BALB/c BMDM were stimulated with or without LPS (100 ng/ml) in the additional presence or absence of 20 µM BAY-11-7085 for 24 h before extraction and RT-PCR analysis of ART2, IL-1beta, and GAPDH mRNA content. B, BALB/c BMDM were stimulated with or without LPS (100 ng/ml) in the additional presence or absence of 2 µM wortmannin, 1 µM SB203580, 10 µM SB600125, or 10 µM U0126 for 24 h before extraction and RT-PCR analysis of ART2, IL-1beta, and GAPDH mRNA content. C, Parallel samples of BALB/c BMDM were removed (–M-CSF) from the standard M-CSF-supplemented macrophage growth medium or were transferred to fresh M-CSF-supplemented medium (+M-CSF). After 24 h, each set of cells was stimulated with or without LPS (100 ng/ml) in the additional presence or absence of 10 µM U0126 for another 24 h before extraction and RT-PCR analysis of ART2 and GAPDH mRNA content. D, BALB/c BMDM were transferred to M-CSF-free medium (24 h) and then stimulated with or without 0.2 µg/ml PAM3C4SK (P3CSK) or 5 µg/ml R837 (ligands for TLR2 or TLR7/8, respectively) in the absence or presence of 10 µM U0126 for 24 h before extraction and RT-PCR analysis of ART2 and GAPDH mRNA content. All results are representative of observations from two or more independent experiments.

 
The experiments in Figs. 1 and 2, A and B, involved direct addition of LPS to the M-CSF-supplemented growth medium used to generate these BMDM. However, LPS regulation of various genes in macrophages can be positively or negatively modulated by the presence of M-CSF (42). In the experiment illustrated in Fig. 2C, we tested whether the presence of M-CSF might attenuate basal expression of ART2 and/or modulate the ability of LPS to induce this gene product. This particular set of ART2 RT-PCR analyses was performed using a reduced amount of cell-derived cDNA for visualization of positive or negative effects of M-CSF on accumulation of ART2 mRNA; this underlies the weak ART2 signal in the LPS-stimulated BMDM extracts in Fig. 2C relative to those illustrated in Fig. 2, A and B. Although removal of M-CSF per se did not elicit accumulation of ART2 mRNA, it did enhance the induction of ART2 elicited by LPS (Fig. 2C). Moreover, the combined removal of M-CSF plus inclusion of U0126 further increased the LPS-dependent accumulation of ART2 mRNA. Thus, all subsequent experiments described in this study were routinely performed using BMDM transferred to M-CSF-free medium during stimulation with LPS or other proinflammatory mediators.

Fig. 2D shows that the effects of LPS on ART2 expression could be mimicked by the lipopeptide PAM3C4SK, a synthetic pathogen-associated molecular pattern (PAMP) ligand for TLR2, or the antiviral imiquimod R837, a synthetic agonist for TLR7/8, which are physiologically targeted by bacterial RNAs. As with LPS, inclusion of U0126 further potentiated the ART2 mRNA accumulation elicited by these pathogen-associated molecular patterns. These findings indicate that ART2 induction in macrophages can be initiated via activation of multiple TLR signaling pathways targeted by a variety of proinflammatory pathogen-associated molecular pattern ligands.

LPS selectively induces expression of the thiol-sensitive ART2.1 isoform in murine macrophages, with analysis of mRNA, protein, and cell surface enzyme activity

Murine ART2 activity is represented by two homologous isoforms, ART2.1 and ART2.2, encoded by the tandem art2a and art2b genes on chromosome 7. Given that T lymphocytes from BALB/c mice constitutively express both isoforms (Fig. 3A), we tested whether both were also LPS-inducible gene products in BMDM using previously described primer sets that permit selective amplification of ART2.1 vs ART2.2 cDNA. Although LPS induced both ART2.1 and ART2.2, the relative accumulation of ART2.1 transcripts was much greater in either the absence or presence of U0126 (Fig. 3A). Note that BMDM yielded much stronger bands for ART2.1 than for ART2.2, whereas the inverse holds for BALB/c splenocytes that are known to express higher levels of ART2.2 than ART2.1 (28, 29). We verified that the ART2.2 primers did not weakly cross-amplify ART2.1 cDNA by performing identical RT-PCR analyses using RNA from splenic lymphocytes of NZW mice that are natural art2b knockouts (32).


Figure 3
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  		FIGURE 3. LPS selectively up-regulates the thiol-sensitive ecto-ART2.1 isoform in macrophages. Analysis of mRNA and cell surface enzyme activity is shown. A, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 ng/ml LPS in the additional presence or absence of 10 µM U0126 for 24 h before extraction and RT-PCR analysis of total ART2 (using primers that amplify both ART2.1 and ART2.2 cDNA), ART2.1, ART2.2, IL-1beta, and GAPDH mRNA content. RNA from freshly isolated BALB/c splenocytes (which constitutively express both ART2.1 and ART2.2) or freshly isolated NZW splenocytes (which constitutively express only ART2.1) were analyzed in parallel by the same RT-PCR protocols. B, Schematic illustration of the 1G4 mAb-based ecto-ART activity assay. C, Freshly isolated intact BALB/c splenocytes were stimulated with or without 50 µM {epsilon}-NAD plus 1 mM ADP-ribose for 15 min at 37°C before extraction for SDS-PAGE and Western blot analysis of 1G4 reactive etheno-ADP-ribosylated proteins. D, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 ng/ml LPS in the additional presence or absence of 10 µM U0126 for 24 h (priming incubation). The primed BMDM were then stimulated with or without 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the additional presence or absence of 2 mM DTT for 15 min at 37°C (test incubation) before extraction for SDS-PAGE and Western blot analysis of 1G4 reactive etheno-ADP-ribosylated proteins. The blot was then stripped and reprobed with anti-actin as a protein loading control. E, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with 100 ng/ml LPS in the additional presence of 10 µM U0126 for 24 h (priming incubation). The LPS-primed cells were treated with or without 1 U of PI-PLC at 37°C for an additional 1 h (PI-PLC incubation), followed by removal of the extracellular medium supernatant (SN) and replacement with fresh medium lacking PI-PLC. Both the cells and extracellular supernatant were separately incubated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the additional presence of 2 mM DTT for 15 min at 37°C (test incubation) before Western blot analysis as described in D. All results are representative of observations from two or more independent experiments.

 
To investigate whether the LPS-initiated ART2.1 or ART2.2 mRNAs were translated into functional cell surface ectoenzymes in intact BMDM, we used a Western blot assay based on the 1G4 mAb specific for etheno-adenosine, which permits detection of proteins containing covalently associated etheno-ADP-ribose moieties (38). This experiment involves brief incubation of intact cells with {epsilon}-NAD (as an NAD surrogate) in the presence of exogenous-free ADP-ribose to minimize catabolism of NAD/{epsilon}-NAD substrates and reversal of ADP-ribosylation reactions by other cell surface ectoenzymes. (This is analogous to including ATPase and protein phosphatase inhibitors when assaying protein phosphorylation). Fig. 3B shows the schematic of this assay and Fig. 3C illustrates the robust etheno-ADP-ribosylation of multiple cell surface proteins observed in BALB/c splenic lymphocytes that are known to constitutively express ART2.2 (and to a lesser extent, also ART2.1) as a cell surface ectoenzyme (28, 29).

Because the LPS-primed BMDM strongly expressed ART2.1, which has been characterized as a thiol-dependent enzyme (19), we assayed these cells (following a 24-h LPS priming step) for etheno-ADP-ribosylation of cell surface proteins in either the absence or presence of 2 mM extracellular DTT (Fig. 3D). In the absence of LPS priming, naive BMDM lacked measurable ecto-ART activity with or without DTT in the test medium. Likewise, no accumulation of etheno-ADP-ribosylated proteins was observed when LPS-primed BMDM were acutely incubated with {epsilon}-NAD in the absence of DTT. In contrast, costimulation of these cells with both {epsilon}-NAD and DTT produced a robust etheno-ADP-ribosylation of multiple cell surface proteins. Consistent with the measurements of ART2 mRNA, inclusion of U0126 during the LPS priming step further increased the expression of the thiol-dependent ART activity on the BMDM cell surface. These data also indicate that surface activity of the thiol-independent ART2.2 isoform was not detectable despite the LPS-induced accumulation (albeit modest) of ART2.2 transcripts. It should be stressed that the assay of cell surface etheno-ADP-ribosylation in splenic T cells illustrated in Fig. 3C was performed in the absence of DTT as a cofactor and the expression was not significantly changed by the presence of the thiol reductant, consistent with higher expression of ART2.2 vs ART2.1 in splenocytes (28, 29).

Acute treatment of LPS-primed (plus U0126) BMDM with bacterial PI-PLC, followed by removal of the extracellular supernatant, before assaying ecto-ART activity greatly diminished the thiol-dependent etheno-ADP-ribosylation of surface proteins (Fig. 3E). Conversely, significant ART activity was present in the extracellular supernatant from the PI-PLC-treated, but not untreated, BMDM. These observations are consistent with 1) the induced expression of ART2.1 as a GPI-anchored ectoenzyme, and 2) the ability of the PI-PLC-cleaved ART2.1 to retain enzymatic activity following release as a soluble protein into the extracellular medium.

Use of the 1G4 mAb in FACS-based analyses (Fig. 4, left columns) indicated that LPS treatment (100 ng/ml; 20 h) induced significant thiol-dependent, cell surface ART activity in ~40% of the BMDM population. Despite this robust ART2.1 activity, these LPS-primed BMDM were only weakly stained with a rat mAb (Gugu2-53), which recognizes a common epitope on ART2.1 and ART2.2 (36). Inclusion of U0126 during the LPS treatment markedly increased the percentage of ART-expressing cells, the 1G4-dependent mean fluorescence per cell, and the number of Gugu2-53-positive cells. For comparison, FACS analyses of other macrophage cell surface proteins revealed constitutive expression of CD80 in the absence of LPS and a modest elevation in the LPS-primed cells. Consistent with previous studies, expression of the CD40 member of the TNFR family was very low in control BMDM but strongly up-regulated upon stimulation with LPS. In contrast to its effects on ART2 surface expression, the additional presence of U0126 did not potentiate LPS-dependent up-regulation of CD40.


Figure 4
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  		FIGURE 4. FACS analysis of ART2.1 enzyme activity and protein expression in control and LPS-primed macrophages. BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 ng/ml LPS in the absence or presence of 10 µM U0126 for 20 h. Aliquots of the control and primed cells were incubated for 10 min with 50 µM {epsilon}-NAD in the absence or presence of 2 mM DTT and then stained with Alexa Fluor 488-conjugated, etheno-adenosine-specific mAb 1G4. Parallel aliquots of control and primed cells were stained with Gugu2-53 anti-ART2.1/ART2.2 mAb, anti-CD40 mAb, or anti-CD80 mAb as described in Materials and Methods. The number in each panel represents the calculated mean fluorescent intensity of the two cell populations shown in the histograms.

 
Functional characterization of the cell surface ART2.1 activity in LPS-primed BMDM

The experiments in Fig. 3 suggested that the thiol-independent ART2.2 was not significantly expressed as a cell surface enzyme in LPS-primed BMDM despite the modest accumulation of ART2.2 mRNA. This suggested that ART2.2 protein might be inefficiently translated, poorly targeted to the cell surface, or preferentially retained in an intracellular membrane compartment similar to the trafficking of ART1 in quiescent human neutrophils (33). However, analysis of ART activity in whole cell lysates prepared from LPS-primed BMDM indicated a strict dependence on the presence of DTT similar to that observed in the intact cell assays (Fig. 5A). This indicated that little, if any, ART2.2 protein accumulates in any cellular compartment of these BMDM. The relative accumulation of ADP-ribosylated surface proteins observed in intact cell experiments progressively increased in a concentration-dependent manner as extracellular DTT concentration was increased from 0 to 2 mM (Fig. 5B). Time course experiments revealed that the thiol-dependent ART2.1 acted very rapidly to catalyze massive ADP-ribosylation of multiple cell surface proteins within minutes after the pulsed addition of {epsilon}-NAD (Fig. 5C). Finally, given that DTT is a nonphysiologic sulfhydryl reagent, we verified that its ability to support the ecto-ART2.1 activity could be mimicked by extracellular cysteine, a physiologically relevant thiol-reducing agent (Fig. 5D).


Figure 5
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  		FIGURE 5. Functional characterization of the cell surface ART2.1 activity in LPS-primed BMDM. A–D, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 ng/ml LPS in the additional presence or absence of 10 µM U0126 for 24 h (priming incubation). A, Primed BMDM were used directly as intact cells (Cell Surface) or were first lysed in 1% Triton X-100 buffer (Cell Lysates) and then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the presence or absence of DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. B, Primed BMDM were stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the additional presence of the indicated concentrations of DTT at 37°C for 15 min before Western blot analysis as described in Fig. 3. C, Primed BMDM were stimulated with a substrate mix of 50 µM {epsilon}-NAD, 1 mM ADP-ribose, and 2 mM DTT for the indicated times at 37°C before Western blot analysis as described in Fig. 3. D, Primed BMDM were stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the additional presence of the indicated concentrations of cysteine or 2 mM DTT at 37°C for 15 min before Western blot analysis as described in Fig. 3. All results are representative of observations from two or more independent experiments.

 
IFN-{gamma} induces expression of ART2.1 in murine BMDM

We observed that the AG490 JAK inhibitor markedly suppressed LPS-dependent ART2 mRNA accumulation, whereas IFN-{gamma} mimicked the stimulatory effect of LPS (Fig. 6A); these data indicated that IFN/JAK/STAT signaling cascades likely regulate ART2 expression in murine BMDM. As with LPS treatment, IFN-{gamma} elicited accumulation of both ART2.1 and ART2.2 transcripts, but with the former as the quantitatively predominant product (Fig. 6B). In contrast to the slower induction of ART2 expression observed in LPS-treated cells (Fig. 1), increased levels of ART2.1 and ART2.2 mRNA were detectable within 2 h; ART2.1 but not ART2.2 transcripts continued to increase over the next 12 h. Similar to LPS-primed cells, no accumulation of ART1, 3, 4, or 5 mRNA was observed in BMDM stimulated with IFN-{gamma} (Fig. 6C). The effects of IFN-{gamma} on induction of ART2.1 mRNA showed a bell-shaped dose response with a maximal effect at 10 U/ml. Induction of ART2.1 mRNA by IFN-{gamma} was accompanied by induction of iNOS and IRF-1, two other IFN-{gamma}-inducible gene products (Fig. 6D), albeit with different dose responses.


Figure 6
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  		FIGURE 6. IFN-{gamma} induces expression of ART2.1 mRNA in murine BMDM. A, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with or without IFN-{gamma} (100 U/ml) or LPS (100 ng/ml) in the additional presence or absence of 10 µM AG490 for 24 h before extraction and RT-PCR analysis of ART2 and GAPDH mRNA content. B, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with or without IFN-{gamma} (100 U/ml) for the indicated times before extraction and RT-PCR analysis of total ART2, ART2.1, ART2.2, and GAPDH mRNA content. C, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with or without IFN-{gamma} (100 U/ml) for 24 h before extraction and RT-PCR analysis of ART1, 2 (total), 3, 4, 5, iNOS, and GAPDH mRNA content. RNA from freshly isolated BALB/c heart and spleen tissue was analyzed in parallel by the same RT-PCR protocols. D, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with or without the indicated concentrations IFN-{gamma} for 24 before extraction and RT-PCR analysis of total ART2, ART2.1, ART2.2, iNOS, IRF-1, and GAPDH mRNA content. All results are representative of observations from two or more independent experiments.

 
As with LPS-primed cells, cell surface ART activity in IFN-{gamma}-primed BMDM required the presence of DTT as a necessary extracellular cofactor (Fig. 7A). Comparison of BMDM treated for 12 h with either IFN-{gamma} or LPS in the absence of U0126 revealed that IFN-{gamma} elicited a more robust induction than LPS of the thiol-dependent cell surface ART activity (Fig. 7A). The intensity and number of etheno-ADP-ribosylated protein bands in the IFN-{gamma}-induced cells increased as the {epsilon}-NAD substrates were elevated from the micromolar to submillimolar range (Fig. 7B). FACS analysis of 1G4 and anti-ART2.1 mAb staining in intact BMDM verified IFN-{gamma} as an inducer of both cell surface ART2.1 protein expression and functional cell surface ART activity (Fig. 7C). However, the 1G4 staining (mean fluorescence intensity per cell) of either the IFN-{gamma}-treated or LPS-treated BMDM was markedly lower than that observed in T cells. As noted for LPS-primed BMDM (Figs. 4 and 7C), IFN-{gamma}-treated macrophages were only weakly stained with anti-ART2.1 relative to the anti-ART2.1 reactivity of BALB/c T cells. Note that the strong 1G4 staining observed in T cells occurred also in the absence of DTT, consistent with the high level of ART2.2 protein expressed on the surface of these cells. No cell surface ART2.2 protein was apparent in BMDM primed with either IFN-{gamma} or LPS and this observation contrasted with the high ART2.2 staining of T cells. The IFN-{gamma}-induced activation of these BMDM was further confirmed by the increased expression of CD40 and MHC class II. Consistent with the Western blot data, these FACS-based measurements verified that etheno-ADP-ribosylation of surface proteins in BMDM induced with IFN-{gamma} or LPS required the presence of DTT as a cofactor.


Figure 7
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  		FIGURE 7. Expression and function of ART2.1 is inducible by IFN-{gamma} in murine macrophages. A, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without IFN-{gamma} (100 U/ml) or LPS (100 ng/ml) for 12 h (priming incubation). The primed BMDM were then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the presence or absence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. B, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without IFN-{gamma} (100 U/ml) for 12 h (priming incubation). The primed BMDM were then stimulated with 1 mM ADP-ribose and the indicated concentrations of {epsilon}-NAD in the presence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. C, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 ng/ml LPS or 100 U/ml IFN-{gamma} for 24 h. Aliquots of the control and primed cells were incubated for 10 min with 50 µM {epsilon}-NAD with or without 2 mM DTT and then stained with Alexa Fluor 488-conjugated, etheno-adenosine-specific mAb 1G4. Parallel aliquots of control and primed cells with stained with a mix of with a mix of ART2.1-specific rat mAbs Gugu2-32 and Gugu2-44, ART2.2-specific rat mAb Nika-102, or with mAbs against CD40, or anti-MHC class II as described in Materials and Methods. The number shown in each panel represents the calculated mean fluorescent intensity of the two cell population shown in the histograms. For comparison, CD4+ splenic/lymph node T cells isolated from BALB/c mice were similarly assayed for constitutive expression of ART2 activity (by 1G4 mAb following a 10 min incubation with 50 µM {epsilon}-NAD) and ART2.1 or ART2.2 protein. All results are representative of observations from two or more independent experiments.

 
IFN-beta induces expression of ART2.1 in murine BMDM

The slow time course, bell-shaped concentration-response, and sensitivity to the AG490 JAK inhibitor, which characterized LPS-activated ART2 expression (Figs. 1 and 6A), suggested that induction of this gene may additionally reflect indirect regulation via the actions of diverse proinflammatory and anti-inflammatory gene products produced in response to LPS. Multiple studies have established that LPS induces the production and release of IFN-beta, which then acts as an autocrine mediator of JAK/STAT-based transcriptional cascades (43, 44, 45). Thus, we tested whether IFN-beta also regulates expression of ART2.1 or other ART subtypes. As with LPS or IFN-{gamma}, IFN-beta treatment of BALB/c BMDM induced strong expression of ART2.1 but no expression of the other five murine ART genes including ART2.2 (Fig. 8A). ART2.1 mRNA was apparent within 4 h after IFN-beta exposure and there was progressive induction of these transcripts over the next 20 h (Fig. 8B). This time course of ART2.1 mRNA accumulation was distinct from that describing iNOS with the latter gene product peaking at 4 h and then declining to prestimulus levels by 24 h. The IFN-beta-stimulated accumulation of both ART2.1 and iNOS transcripts was preceded by induction of the IRF-1 transcription factor, which was maximal within 2 h. In contrast to LPS or IFN-{gamma}, IFN-beta did not induce accumulation of ART2.2 transcripts. Consistent with this predominant expression of ART2.1, ecto-ART activity in the IFN-beta-treated BMDM was strictly dependent on the presence of DTT as an extracellular cofactor (Fig. 8C). This IFN-beta-induced activity was detectable within 2 h but was greatly increased at 8 h and thereafter (Fig. 8D). These effects were dose-dependent with threshold activation by ~2 U/ml IFN-beta (Fig. 8E).


Figure 8
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  		FIGURE 8. IFN-beta induces expression and function of ART2.1 in murine BMDM. A, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with or without IFN-beta (100 U/ml) for 24 h before extraction and RT-PCR analysis of ART1, 2 (total), ART2.1, ART2.2, 3, 4, 5, IRF-1, and GAPDH mRNA content. RNA from freshly isolated BALB/c heart and spleen tissue was analyzed in parallel by the same RT-PCR protocols. B, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with or without IFN-beta (100 U/ml) for the indicated times before extraction and RT-PCR analysis of ART2 (total), ART2.1, ART2.2, iNOS, IRF-1, and GAPDH mRNA content. C, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without IFN-beta (100 U/ml) for 24 h (priming incubation). The primed BMDM were then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the absence or presence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. D, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without IFN-beta (100 U/ml) for the indicated times (priming incubation). The primed BMDM were then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the presence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. E, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without the indicated concentrations of IFN-beta for 24 h (priming incubation). The primed BMDM were then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the presence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. All results are representative of observations from two or more independent experiments.

 
LPS regulation of ART2.1 involves both potentiation via autocrine IFN-beta signaling and suppressive effects on IFN induction of ART2.1

The ability of LPS to trigger accumulation of IFN-beta mRNA in BALB/c BMDM suggested a likely role for IFN-beta as an autocrine mediator of LPS-induced ART2.1 expression (Fig. 9A). Although IFN-beta mRNA levels were elevated within 2 h after LPS, longer incubation is likely required for extracellular IFN-beta protein to reach threshold concentrations for autocrine stimulation of the type I IFN receptor. This was supported by the ability of a neutralizing anti-IFN-beta Ab to dose-dependently attenuate the up-regulation of cell surface ART2.1 activity elicited during a 24-h LPS exposure (Fig. 9B). Control experiments (data not shown) indicated that a nonimmune isotype control Ig did not suppress induction of ART2.1 activity in response to LPS. Consistent with the more rapid accumulation of ART2 mRNA and protein in response to IFNs vs LPS, cells treated for 12 h with IFN-beta or IFN-{gamma} expressed higher ART2.1 activity than cells incubated with LPS for 12 h (Fig. 9C). However, costimulation of BMDM for 12 h with IFN-beta plus LPS or IFN-{gamma} plus LPS resulted in markedly lower levels of ART activity that approximated levels observed with LPS priming alone (Fig. 9C). This result suggests a possible role for LPS-induced expression of regulatory factors, such as the SOCS proteins (46, 47), which act as negative modulators of JAK/STAT signaling.


Figure 9
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  		FIGURE 9. LPS regulation of ART2.1 involves both potentiation via autocrine IFN-beta signaling and suppressive effects on IFN-induction of ART2.1. A, BALB/c BMDM were transferred to M-CSF-free medium and stimulated with or without IFN-beta (100 U/ml) for the indicated times before extraction and RT-PCR analysis of IFN-beta and GAPDH mRNA content. B, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with 100 ng/ml IFN-beta in the additional presence of the indicated concentrations of neutralizing anti-IFN-beta Ab for 24 h (priming incubation). The primed BMDM were then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the presence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. C, BALB/c BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 U/ml IFN-beta alone, 100 U/ml IFN-{gamma} alone, 100 ng/ml LPS alone, or costimulatory mixtures of the IFN plus LPS for 12 h (priming incubation). The primed or coprimed BMDM were then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the presence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. All results are representative of observations from two or more independent experiments.

 
Inducible expression of ART2.2 does not compensate for the lack of functional ART2.1 in C57BL/6 macrophages

C57BL/6 mice are null for functional ART2.1 protein due an art2a mutation that results in a premature stop codon in ART2.1 transcripts (30). Given our observations that only ART2.1 was expressed as an inducible ectoenzyme in inflammatory BALB/c BMDM, we tested whether increased ART2.2 expression might compensate for the lack of functional ART2.1 in C57BL/6 BMDM. LPS induced accumulation of total ART2 mRNA in these BMDM, and this accumulation was increased in the presence of U0126 (Fig. 10A). In contrast to the findings with BALB/c BMDM (Fig. 3A), ART2.2 mRNA was more abundant than ART2.1 mRNA in the C57BL/6 macrophages. As expected, splenic lymphocytes from the same C57BL/6 mice constitutively expressed high levels of ART2.2, but only low levels of ART2.1 mRNA (28, 29, 30). Significantly, neither thiol-independent nor thiol-dependent ecto-ART activity could be measured in intact C57BL/6 BMDM primed with LPS, IFN-beta, or IFN-{gamma} (Fig. 10, C and D). In contrast, robust, thiol-independent etheno-ADP-ribosylation of multiple surface proteins was readily observed when splenic lymphocytes from these mice were assayed under identical conditions (Fig. 10B). Thus, expression of ART2.2 as an LPS- or IFN-inducible cell surface ectoenzyme in BMDM does not compensate for the loss of functional ART2.1 in the C57BL/6 genetic background.


Figure 10
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  		FIGURE 10. Inducible expression of ART2.2 does not compensate for the lack of functional ART2.1 in C57BL/6 macrophages primed with LPS, IFN-{gamma}, or IFN-beta. A, C57BL/6 BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 ng/ml LPS in the additional presence or absence of 10 µM U0126 for 24 h before extraction and RT-PCR analysis of total ART2 (using primers that amplify both ART2.1 and ART2.2 cDNA), ART2.1, ART2.2, IL-1beta, and GAPDH mRNA content. RNA from freshly isolated C57BL/6 splenocytes (which constitutively express both ART2.1 and ART2.2) was analyzed in parallel by the same RT-PCR protocols. B, Freshly isolated intact C57BL/6 splenocytes were stimulated with 50 µM {epsilon}-NAD plus 1 mM ADP-ribose for 15 min in additional presence or absence of 2 mM DTT at 37°C before Western blot analysis as described in Fig. 3. C, C57BL/6 BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 ng/ml LPS in the additional presence or absence of 10 µM U0126 for 24 h (priming incubation). The primed BMDM were then stimulated with or without 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the additional presence or absence of 2 mM DTT for 15 min at 37°C (test incubation) before Western blot analysis as described in Fig. 3. D, C57BL/6 BMDM were transferred to M-CSF-free medium and then stimulated with or without 100 U/ml IFN-beta or 100 U/ml IFN-{gamma} for 24 h (priming incubation). The primed BMDM were then stimulated with 50 µM {epsilon}-NAD and 1 mM ADP-ribose in the additional presence or absence of 2 mM DTT at 37°C for 15 min (test incubation) before Western blot analysis as described in Fig. 3. All results are representative of observations from two or more independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have identified the thiol-dependent, GPI-anchored ART2.1 ectoenzyme as an LPS- and IFN-inducible gene product in murine macrophages. Our data also indicate that LPS induction of ecto-ART2.1 in macrophages is mediated, in large part, via an autocrine pathway involving IFN-beta (Figs. 8 and 9). IFN-{gamma}, a type II T cell-derived IFN, independently induced ART2.1 expression and function in macrophages (Figs. 6 and 7). The requirement for a highly regulated and selective expression of ART2.1 by inflammatory mediators in BMDM stands in sharp contrast to the constitutive expression of both ART2.1 and ART2.2 in freshly isolated spleen T lymphocytes (Figs. 3, 4, 6, and 7). Whether ART2.1 is constitutively expressed by resident naive macrophages within in vivo tissues is a significant question for future experiments.

The negative or positive effects of various ERK, PI3K, NF-{kappa}B, and JAK inhibitors on LPS induction of ART2.1 may reflect 1) direct roles for these signaling pathways in transcriptional regulation of ART2.1, 2) indirect roles in the regulation of IFN-beta expression by LPS, or 3) possible nonselective actions of these pharmacological reagents. However, the ability of U0126, an inhibitor of the MEK/ERK pathway to increase rather than suppress LPS induction of ART2.1 at the mRNA (Fig. 2), protein (Fig. 4), and activity (Fig. 3) levels was particularly noteworthy. Although we did not explore the mechanism underlying this apparent suppressive effect of active ERK on ART2.1 expression, Saito et al. (48) recently described a similar ability of U0126 to increase LPS-induced expression of IL-12p40 in RAW264.7 macrophages. That effect was linked to suppressed binding of the ERK-dependent transcription factor GAP-12 to the GA-12 repressor element in the promoter of the IL-12p40 gene.

These various protein kinase cascades could also modulate the STAT and/or IRF transcription factors that are likely involved in the induction of ART2.1 by IFNs. We also observed (Fig. 9C) that cotreatment of macrophages with LPS plus IFN-{gamma} or LPS plus IFN-beta induced lower ART2.1 expression than with either IFN alone. This suggests that LPS induction of negative modulators of JAK/STAT signaling, such as SOCS proteins or protein inhibitors of activated STAT, might attenuate the extent of IFN-induced ART2.1 expression (46). Previous studies have indicated that prolonged (>4 h) stimulation of murine macrophages with LPS leads to an accumulation of SOCS3 that markedly reduces the activation of STAT1 by IFN-{gamma} (47). Complex positive and negative input into from multiple inflammatory mediators may underlie our FACS data (Figs. 4 and 7D) showing significant expression of cell surface ART2.1 protein and activity on only a subfraction (~50%) of the induced BMDM population.

Catalytic activity of the inducible ART in intact macrophages was absolutely dependent on the presence of extracellular thiol-reducing agents as cofactors. The nonphysiological DTT was used as a thiol cofactor in most experiments, but we verified that extracellular cysteine also supported the induced ART2.1 activity (Fig. 5D). Although both ART2.1 and ART2.2 act as ecto-ARTs, only ART2.1 requires the presence of extracellular thiol cofactors to modulate a reactive cysteine uniquely present at position 201 in ART2.1 (19, 20). Interestingly, in the three-dimensional structure of rat ART2.2, the side chains of Ser60 and Phe181, i.e., the residues corresponding to the extra cysteine residues in ART2.1, are in close proximity, suggesting that the two cysteines may form a labile disulfide bridge in ART2.1 (Fig. 11) (49, 50). There is growing appreciation that reversible oxidation/reduction of allosteric disulfide bonds and/or reactive cysteine residues in the extracellular domains of various cell surface proteins, including channels, receptors, and ectoenzymes, can be functionally significant (51, 52).


Figure 11
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  		FIGURE 11. An allosteric disulfide bond between Cys80 and Cys201 in mouse ART2.1. Illustrative (A) and surface (B) models of rat ART2.2 with NAD bound in the active site crevice (PDB database, entry code 1OG3, (49 50 )). The side chains of residue Ser60 and Phe181 are shown as stick models. At the corresponding positions, mouse ART2.1 carries cysteine residues unique in the ART family. The activity of ART2.1 is strictly dependent on the presence of thiol-reducing agents, suggesting that an allosteric disulfide bond might be formed in ART2.1 at this position. Note that differences in the residue numbering of mouse ART2.1 and rat ART2.2 result from inclusion (mouse) or exclusion (rat) of the 20 N-terminal residues of the signal peptide.

 
Changes in the redox state of extracellular protein-associated cysteine residues can be directly regulated by the local concentration of free cysteine or other small molecule sulfhydryl (thiol) donors. Extracellular thiols in circulating plasma include 8–10 µM cysteine, ~40 µM cystine, and 2–4 µM glutathione (51). Significantly, the concentration of extracellular cysteine within inflammatory loci can rise to submillimolar levels due to increased release and degradation of glutathione by activated macrophages (20, 53). Depending on local oxygenation, the accumulated cysteine will be variably oxidized to cystine to set the local redox ratio of cysteine to disulfide cystine, and this ratio will be increased in interstitial compartments with low oxygen tension as occurs in ischemic or hypoxic tissue. In addition to the effects of free cysteine on the local thiol redox ratio, certain proteins secreted from inflammatory leukocytes or other cells can act as extracellular sulfhydryl oxidoreductases. These proteiin include macrophage inhibitory factor (54), the IFN-{gamma}-inducible lysosomal thiol reductase GILT (55), and protein disulfide isomerase (56, 57). The critical dependence of ART2.1 activity on local thiol cofactors, and the likely presence of these proteins within the vicinity of ART2.1 expressing cells, raises intriguing possibilities regarding the in vivo context for the ART2.1-dependent functions of immune effector cells. Such scenarios may involve highly synergistic regulation of ART2.1 activity by both regional oxygen tension and local accumulation of extracellular sulfhydryl oxidoreductases within inflammatory loci.

Our experiments leave open the formal possibility that expression of ART2.2 in macrophages may also be induced by inflammatory stimuli albeit involving additional layers of regulation. LPS and IFN-{gamma}, but not IFN-beta, induced a modest accumulation of ART2.2 mRNA (Figs. 3, 6, and 8). However, none of these inflammatory mediators elicited the production of measurable ART2.2-type enzyme activity on the cell surface or within intracellular compartments of BALB/c BMDM (Figs. 3D and 7A) or C57BL/6 BMDM (Fig. 10). BALB/c and C57BL/6 mice are known to show markedly different responses to infection by Listeria, Yersinia, Leishmania, trypanosomes and other pathogens (58, 59, 60, 61). It is conceivable that some of these differences are a consequence of the differential expression of the thiol-sensitive ART2.1 by BALB/c and C57BL/6 macrophages.

Our data indicate that a wide range of cell surface proteins are ADP-ribosylated when LPS- or IFN-induced macrophages are stimulated with extracellular NAD in the presence of thiol cofactors. LFA-1, the CD11a/CD18 integrin (62, 63), is a well-characterized substrate for the constitutively expressed ART2.2 in murine T cells with consequent effects on homotypic adhesion and trafficking (9, 12). Likewise, the {alpha}7 integrin subunit of laminin-binding integrins is a preferred substrate for ART1 in skeletal muscle cells (64, 65, 66). It is noteworthy that the protein bands most intensely labeled (Figs. 3, 5, and 7–9) by {epsilon}-NAD in the ART2.1-expressing macrophages are in the 100–150 kDa range of integrins. Other possible ART target proteins (12, 21, 67) known or potentially expressed by macrophages include CD27, a member of the TNFR family (68), CD43, a sialoglycoprotein implicated in the growth of mycobacteria (69), CD44, a hyaluronan receptor and negative regulator of TLR signaling (70), CD45, a transmembrane tyrosine phosphatase (71), the P2X7 purinoceptor, a key mediator of ATP-induced release of IL-1 (72), and the secreted antimicrobial peptide defensin-1 (73). Identifying the major protein targets of the inducible ART2.1 in macrophages are obvious next steps. Likewise, determining the consequences of these posttranslational modifications on key macrophage functions, such as phagocytosis, Ag presentation, production of reactive oxygen/nitrogen species, and secretion of additional cytokines, is an important goal for future experiments.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
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 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported Grant GM36387 (to G.R.D.) from the National Institutes of Health and Grant No. 310/6 (to F.H. and F.K.-N.) from The Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. George R. Dubyak, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44120. E-mail address: george.dubyak{at}case.edu Back

3 Abbreviations used in this paper: NAD, nicotinamide adenosine dinucleotide; ART, ADP-ribosyltransferase; BMDM, bone marrow-derived macrophage; NZW, New Zealand White; PI-PLC, phosphatidylinositol phospholipase C; IRF, IFN response factor; iNOS, inducible NO synthase. Back

Received for publication April 11, 2007. Accepted for publication August 20, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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S. Hong, N. Schwarz, A. Brass, M. Seman, F. Haag, F. Koch-Nolte, W. P. Schilling, and G. R. Dubyak
Differential Regulation of P2X7 Receptor Activation by Extracellular Nicotinamide Adenine Dinucleotide and Ecto-ADP-Ribosyltransferases in Murine Macrophages and T Cells
J. Immunol., July 1, 2009; 183(1): 578 - 592.
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