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The Journal of Immunology, 2006, 176: 5033-5040.
Copyright © 2006 by The American Association of Immunologists

IFN-{gamma} Primes RAW264 Macrophages and Human Monocytes for Enhanced Oxidant Production in Response to CpG DNA via Metabolic Signaling: Roles of TLR9 and Myeloperoxidase Trafficking

Yoshiyuki Adachi*, Andrei L. Kindzelskii{dagger}, Aaron R. Petty{dagger}, Ji-Biao Huang{dagger}, Nobuyo Maeda§, Satoshi Yotsumoto, Yasuaki Aratani||, Naohito Ohno* and Howard R. Petty1,{dagger},{ddagger}

* Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan; {dagger} Department of Ophthalmology and Visual Sciences and {ddagger} Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48105; § Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599; Department of Drug Delivery and Molecular Biopharmaceutics, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan; and || Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages and monocytes are activated by CpG DNA motifs to produce NO, which is enhanced dramatically by IFN-{gamma}. We hypothesize that synergistic cellular responses to IFN-{gamma} and CpG DNA are due to cross-talk between metabolic signaling pathways of leukocytes. Adherent RAW264.7 macrophages and human monocytes exhibited NAD(P)H autofluorescence oscillation periods of ~20 s. IFN-{gamma} increased the oscillatory amplitude, which was required for CpG DNA-mediated metabolic changes. These alterations in metabolic dynamics required the appropriate combinations of murine/human TLR9 and murine/human-specific CpG DNA. Other factors that also promoted an increase in metabolic oscillatory amplitude could substitute for IFN-{gamma}. Because recent studies have shown that the metabolic frequency is coupled to the hexose monophosphate shunt, and the amplitude is coupled to the peroxidase cycle, we tested the hypothesis that myeloperoxidase (MPO) participates in IFN-{gamma} priming for oxidant production. MPO inhibitors blocked cell responses to IFN-{gamma} and CpG DNA. In the absence of IFN-{gamma} exposure, the effects of CpG DNA could be duplicated by MPO addition to cell samples. Moreover, monocytes from MPO knockout mice were metabolically unresponsive to IFN-{gamma} and CpG DNA. NAD(P)H frequency doubling responses due to CpG DNA were blocked by an inhibitor of the hexose monophosphate shunt. Because NAD(P)H participates in electron trafficking to NO and superoxide anions, we tested oxidant production. Although CpG DNA alone had no effect, IFN-{gamma} plus CpG enhanced NO and reactive oxygen metabolite release compared with IFN-{gamma} treatment alone. We suggest that amplitude and frequency modulation of cellular metabolic oscillations contribute to intracellular signaling synergy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Toll-like receptors play key roles in adaptive and innate immune responses (1, 2, 3, 4). TLRs recognize conserved pathogen-associated molecular patterns, such as those associated with LPS, lipoteichoic acid, peptidoglycan, flagellin, and bacterial DNA (1, 2, 3, 4). TLRs are type I transmembrane proteins that bind their target pathogen-associated molecular patterns at the plasma membrane or, in the case of TLR7 and TLR9, at the luminal side of endosome membranes (1). These binding events lead to a cascade of poorly understood signals that stimulate innate immune responses, leading to pathogen elimination and the development of adaptive immunity.

In contrast to vertebrate DNA, bacterial and viral DNA contains unmethylated CpG motifs (5, 6, 7). CpG DNA is recognized by TLR9, which activates B cells, macrophages, NK cells, and dendritic cells, leading to the production of cytokines, chemokines, and Ig. In addition, macrophages produce NO in response to stimulation with CpG DNA, but only after priming with IFN-{gamma} (8). Gramzinski et al. (9) illustrated the synergistic interactions of CpG DNA and IFN-{gamma} by demonstrating that previous administration of CpG DNA conferred protection against malaria in an IFN-{gamma}- and IL-12-dependent fashion. However, the mechanisms of macrophage activation by CpG DNA are not fully understood.

The production of NO and reactive oxygen metabolite (ROM)2 is a key factor contributing to host defense, as both oxidative molecules and paracrine and autocrine messengers (10). ROM production begins with superoxide synthesis by the NADPH oxidase: 1/2 NADPH + O2 -> 1/2 NADP+ + 1/2 H+ + O2– (Equation 1). NO is produced by the reaction: L-arginine + NADPH + H+ + O2->NG-hydroxy-L-arginine + NADP+ + H2O and 2 NG-hydroxy-L-arginine + NADPH + H+ + O2->2 L-citruline + NADP+ + 2 H2O + 2 NO (Equation 2).

One factor likely to regulate oxidant production is the availability of the substrate NADPH, which donates electrons during NO and superoxide synthesis. We have recently shown that the amplitude and frequency of NAD(P)H oscillations are demodulated into the periodic production of superoxide and NO by leukocytes (11, 12, 13, 14, 15). Thus, more superoxide is produced when NAD(P)H concentration oscillations increase in amplitude, frequency, or both; in other words, the substrate concentration is an important determinant of product output. Recent experimental and computational studies have indicated that the frequency is controlled by the hexose monophosphate shunt (HMS), whereas the amplitude is independently controlled by the peroxidase cycle (15, 16). Hence, NADPH production may be a point where multiple cell regulatory circuits intersect. Recent studies from our laboratory have suggested that certain cytokine pairs may interact in such a fashion (17, 18). We now examine the nature of IFN-{gamma}/CpG DNA synergy in macrophages. Our results suggest that IFN-{gamma} promotes CpG DNA-mediated oxidant production by promoting the transfer of myeloperoxidase (MPO) to the cell surface, thereby activating the peroxidase cycle and permitting HMS activation by CpG DNA.


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

Murine and human cytokines were obtained from R&D Systems. Salicylhydroxamic acid (SHA), 6-aminonicotinamide, p-hydroxybenzoic acid hydrazide, cyanide, hydroxyquinone, and native MPO were obtained from Sigma-Aldrich. Wortmannin was obtained from Calbiochem. IFN-{gamma} was obtained from E. Adolf (BD Biosciences). Dihydrotetramethylrosamine (H2TMRos; Molecular Probes), and diaminofluorescein-2 diacetate (Daiichi Kagaku Yakuhin) were obtained as indicated.

CpG DNA

Phosphorothioate-modified ODNs were custom synthesized by Sigma-Aldrich The following sequences were used: 1668 TCC ATG ACG TTC CTG ATG CT (murine CpG); TCC ATG AGC TTC CTG ATG CT (murine GpC); and 2006 TCG TCG TTT TGT CGT TTT GTC GTT (human CpG). In some cases, fluorescein or TAMRA was attached to these molecules. ODNs were negative for LPS, as measured by the Limulus assay (Sigma-Aldrich). Escherichia coli DNA and calf thymus DNA were purchased from Sigma-Aldrich.

Antibodies

Polyclonal rabbit Abs reactive with murine MPO was purchased from Chemicon International. An FITC-conjugated anti-human MPO Ab was purchased from Accurate Chemical. Abs were conjugated with tetramethylrhodamine isothiocyanate (TRITC) or FITC as described previously (13). For MPO staining, viable cells were labeled with 1 µg/ml FITC-conjugated anti-MPO at room temperature for 30 min, followed by washing and fluorescence microscopy.

Leukocyte isolation

Peripheral blood monocytes were obtained using two Ficoll-Hypaque solutions of different buoyant densities (Histopaque 1077 and 1119; Sigma-Aldrich) or Fico/Lite (Atlanta Biologicals) and centrifugation. Cells were washed twice by centrifugation, then resuspended in HBSS (Invitrogen Life Technologies). Cell adherence was used to further enrich monocytes. Trypan blue staining indicated that 95–99% of the cells were viable.

Cell culture

RAW264.7 macrophages were grown in RPMI 1640 containing 10% FCS and 1% penicillin G-streptomycin-amphotericin B (Invitrogen Life Technologies). For spectrophotometric assays, cells were grown in 24-well plates. For microscopy experiments, cells were grown for 24 h attached to glass coverslips.

Preparation of RAW264.7 transfectant

TLR9-expressing RAW264.7 transfectants were prepared using human TLR9 cDNA. It was amplified by KOD plus DNA polymerase (Toyobo) from a cDNA mixture of human monocytes. It was cloned into p3X FLAG CMV-14 (Sigma-Aldrich). The cloned plasmid was subjected to DNA sequencing (ABI PRISM 310; Applied Biosystems) to confirm sequence and identity. Stably transfected cells expressing TLR9 were established using an electroporator (gene pulser; Bio-Rad). Cells were screened for expression of the recombinant molecule by flow cytometric analysis. Levels of expression were confirmed by staining with biotin-conjugated Ab to FLAG-tag and Alexa488-conjugated streptavidin by flow cytometry.

MPO knockout mice

Monocytes from MPO knockout mice (19) and parental C57BL/6 mice were used.

Fluorescence microscopy

Cells were observed using an Axiovert fluorescence microscope (Zeiss) with mercury illumination interfaced to a computer using Scion image processing software (13). A narrow bandpass discriminating filter set (Omega Optical) was used with excitation at 485/22 nm and emission at 530/30 nm for FITC and excitation of 540/20 nm and emission at 590/30 nm for TRITC and TAMRA. Long-pass dichroic mirrors of 510 and 560 nm were used for FITC and TRITC (TAMRA), respectively. The fluorescence images were collected with an intensified charge-coupled device camera (Princeton Instruments).

Electrode configuration

Electric fields were applied as described previously (20, 21). Pulse application was performed manually to coincide with the trough in the NAD(P)H autofluorescence intensity for each cell under study.

NAD(P)H oscillations

NAD(P)H autofluorescence oscillations were detected as previously described (15, 16, 17). Briefly, a 365WB50 excitation filter, a 400-nm long-pass dichroic mirror, and a 450AF58 emission filter were used. A cooled high sensitivity photomultiplier tube in a D104 detection system (Photon Technology International) attached to a Zeiss microscope was used. Data were analyzed using Felix software (Photon Technologies).

Detection of oxidant pericellular release

Pericellular release of ROMs from single cells was monitored as previously described (12). Briefly, adherent neutrophils were surrounded in 2% gelatin containing 100 ng/ml H2TMRos, hydroethidine (HE), or decay-accelerating factor. Oxidants released by the cells entered the gelatin matrix, where they reacted with these probes, which were then detected by fluorescence microscopy.

Spectrophotometric assay for NO

Macrophages (106/ml) were placed on culture plates and treated with recombinant murine IFN-{gamma} (10 U/ml) and with human CpG DNA for 24 h. Cell-free culture supernatants were collected for NO measurement. NO release was determined by assaying supernatants for nitrite content. Briefly, 40 µl of cell-free supernatant was reacted for 10 min at room temperature with an equal volume of Greiss reagent (1% sulfanilamide, 0.1% naphthylethylene diaminedihydrochloride, and 2.5% phosphoric acid) as previously described (22). ODs were measured at 540 nm. Nitrite content was quantitated by comparison with a standard curve generated using sodium nitrite (0–100 µM).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CpG DNA staining of cells

This study tests the hypothesis that IFN-{gamma} and CpG DNA synergize via metabolic pathways to enhance oxidant production. We examined the association of murine and human CpG DNA with RAW264.7 cells, RAW264.7/H8 (hTLR9-positive) cells, and human monocytes. TAMRA-modified murine and human CpG DNA were used. RAW264.7 and RAW264.7/H8 cells were incubated with 1 µg/ml TAMRA-conjugated CpG DNA at room temperature for 30 min. Both RAW264.7 vector control cells (Fig. 1, a and b) and RAW264.7/H8 (Fig. 1, c and d) were stained with murine and human CpG DNA. Purified human peripheral blood monocytes were also analyzed. These cells could be stained with both murine CpG DNA and human CpG DNA. CpG DNA may bind to TLR9 as well as other binding proteins, thereby staining intracellular and plasma membrane compartments (23, 24, 25). Hence, these immunostimulatory DNA molecules stain appropriate cells, suggesting that differential responses are not due to cell association.


Figure 1
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  		FIGURE 1. CpG DNA binding to RAW264.7 cells and human monocytes. Cells were stained using TAMRA-conjugated mouse CpG DNA 1668 (a, c, and e) or TAMRA-conjugated human CpG DNA 2006 (b, d, and f). Representative fluorescence micrographs are shown. RAW264.7 cells (a and b), H8/RAW264.7 cells (c and d), and human monocytes (e and f) were stained with both murine and human CpG DNA (c and d). Magnification, x820. n = 3.

 
IFN-{gamma} primes macrophages for CpG DNA-mediated metabolic frequency doubling

Our previous studies (11, 12, 13, 14, 15, 16, 17) suggest that metabolic frequency doubling is a hallmark of leukocyte activation that is closely tied to HMS activation. For example, FMLP, LPS, and immune complexes increase metabolic oscillation frequency, whereas the HMS inhibitor, 6-AN, and certain anti-inflammatory agents prevent these changes (16). Although frequency changes were not observed when RAW264.7 cells were incubated with murine or human CpG DNA alone, based upon previous physiological studies (8, 9) we hypothesized that signaling events associated with IFN-{gamma} exposure synergize with CpG DNA to yield outputs that neither molecule alone possessed. Because previous studies established that IFN-{gamma} primes leukocytes (26), we first exposed cells to IFN-{gamma} (4 h at 25 U/ml). We have previously shown that exposure to IFN-{gamma} leads to high amplitude metabolic oscillations (17, 21), as illustrated in Fig. 2d vs control (row 1). Addition of murine CpG DNA to IFN-{gamma}-treated RAW264.7 macrophages led to frequency doubling within 3 min (Fig. 2e), although no effect was observed for human CpG DNA, which served as a negative control (Fig. 2f). We next examined the effects of CpG DNA on RAW264.7 transfectants expressing hTLR9. No effects were observed during incubation with either murine or human CpG DNA alone (Fig. 2, m–o). However, IFN-{gamma} pretreatment led to metabolic changes in response to murine and human CpG DNA (Fig. 2, p–r). Because these cells express both forms of TLR9, this result is expected. This suggests that the transfected human TLR9 is competent to active metabolic changes in RAW264.7 cells.


Figure 2
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  		FIGURE 2. Representative kinetic traces illustrating the effects of IFN-{gamma} exposure on metabolic oscillations of mock transfectant A6/RAW264.7 and hTLR9 transfectant H8/RAW264.7 macrophages. These traces show NAD(P)H autofluorescence intensity (ordinate) vs time (abscissa); to conserve space, only a few oscillations are shown. Polarized cells were studied on glass slides at 37°C. Untreated cells demonstrated NAD(P)H oscillations with a period of ~20 s (trace a). NAD(P)H oscillations were not affected by treatment with human or murine CpG DNA (traces b, c, n, and o). However, the addition of IFN-{gamma} (25 U/ml, 4 h) increased the NAD(P)H amplitude (traces d–f and p–r). Addition of mouse CpG DNA to IFN-{gamma}-primed A6/RAW264.7 macrophages increased the metabolic oscillation frequency (trace e), whereas IFN-{gamma} and human CpG DNA did not (trace f). However, in the IFN-{gamma}-primed H8/RAW264.7 hTLR9 transfectant macrophage, the addition of either mouse CpG DNA or human CpG DNA resulted in increased frequency (traces q and r). Similar priming effects for CpG DNA-mediated metabolic changes were noted for PMA (g–i and s–v) and melatonin (j–l and v–x). a–u, n = 9; j–x, n = 3.

 
We also tested the effects of PMA (10–8 M) and melatonin (50 µg/ml), two reagents known to prime leukocytes (27, 28), on RAW264.7 responses to CpG DNA. These reagents increase the amplitude of metabolic oscillations (Fig. 2, g, j, s, and v). When combined with PMA, human CpG DNA promoted high amplitude, high frequency metabolic responses of RAW264.7 cells in a species-specific fashion (Fig. 2, h, i, k, l, t, u, w, and x). Because melatonin requires HMS activity to activate high frequency oscillations (16), coincubation of melatonin and CpG DNA is required to activate cells (Fig. 2, j–l and v–x).

IFN-{gamma} primes monocytes for CpG DNA-mediated metabolic changes

The studies described above show that IFN-{gamma} increases the amplitude of NAD(P)H oscillations in RAW264.7 macrophages and that IFN-{gamma} can synergize with CpG DNA to double metabolic oscillation frequency. Although alterations in metabolic amplitude and frequency have been noted in human cells (11, 12, 13, 14, 15, 16, 17), it remains possible that these synergistic effects on metabolic signaling are limited to this transformed cell line. To test this possibility, we exposed human monocytes to human IFN-{gamma} or other priming stimuli as well as to murine or human CpG DNA (Fig. 3). As described above for RAW264.7 cells, murine and human CpG DNA had no effect on human monocytes (Fig. 3, a–c). However, after preincubation with human IFN-{gamma} for 4 h at 25 U/ml, the NAD(P)H oscillations increased in amplitude (Fig. 3d) (17, 21). Incubation of IFN-{gamma}-primed human monocytes with human CpG increased the frequency of metabolic oscillations (Fig. 3f), but murine CpG DNA had no effect (Fig. 3e). Previous studies (29) suggested that IL-12 treatment synergizes with CpG DNA to promote cellular responses. To test for IL-12 synergy, monocytes were incubated for 60 min with 50 ng/ml IL-12, followed by addition of murine or human CpG DNA. In this case, high frequency metabolic responses were noted with human, but not murine, CpG DNA (Fig. 3i). The effects of PMA (10–8 M) and melatonin (50 µg/ml) on CpG DNA responses were also examined. When combined with human CpG DNA, these priming molecules triggered high amplitude, high frequency metabolic responses of human monocytes (Fig. 3, l and o). Human CpG DNA can cooperate with several priming factors to alter the metabolic phenotype of human monocytes.


Figure 3
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  		FIGURE 3. Representative kinetic traces of NAD(P)H autofluorescence oscillations of human monocytes in the presence of buffer (Nil; left), mouse CpG DNA (center), and human CpG DNA (right) are shown. Both IFN-{gamma} and IL-12 increase the amplitude of metabolic oscillations 3-fold (traces d and g). Although mouse CpG alone or in combination with priming reagents had no effect (traces b, e, h, k, and n), human CpG added to IFN-{gamma}-, IL-12-, PMA-, or melatonin-primed cells led to an increase in metabolic frequency (traces f, i, l, and o). a–l, n = 9; m–o, n = 3.

 
Artificial metabolic amplitude modulation reconstitutes CpG DNA-mediated frequency doubling

Although the above studies correlate IFN-{gamma}, IL-12, PMA, and melatonin-mediated priming with CpG DNA-mediated frequency doubling, it is unclear whether this change requires amplitude modulation. To explore this issue, we heightened NAD(P)H amplitudes by a physical, instead of a chemical, means. Cells were exposed to a frequency- and phase-matched electric field that heightens NAD(P)H oscillatory amplitudes in the absence of receptor ligation (20, 21). Pulsed direct current electric fields (2 x 10–3 V/m with 20-ms duration) were applied to cells at troughs of NAD(P)H autofluorescence intensity. The NAD(P)H oscillatory amplitude markedly increased in the presence of an appropriate phase-matched electric field. Approximately 2 or 3 min after murine CpG DNA addition, the metabolic frequency doubled ({tau} = ~10 s; Fig. 4e); this was not observed in the absence of high amplitude oscillations (Fig. 4b) or when CpG DNA was replaced with GpC DNA (Fig. 4f). When the electric field was terminated, the high amplitude, high frequency oscillations rapidly reverted to the low amplitude, low frequency state (data not shown). However, human CpG DNA had no effect on murine cells (data not shown). We suggest that metabolic amplitude modulation in the absence of priming factors or altered gene expression is sufficient to synergize with CpG DNA to elicit metabolic changes.


Figure 4
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  		FIGURE 4. Synergy of CpG DNA and electric fields in metabolic oscillation frequency doubling. In the absence of applied electric fields, buffer, CpG, and GpC DNA had no effect on metabolic oscillations of RAW264.7 cells (a–c). A phase-matched electric field was applied at NAD(P)H troughs (arrows, d–f), leading to enhanced NAD(P)H amplitudes. When mouse CpG DNA was added, the NAD(P)H oscillation frequency was increased 2-fold, with a corresponding 2-fold increase in pulsed electric field application. GpC DNA had no effect (f). n = 4.

 
CpG DNA metabolic effects are sensitive to inhibitors

Cellular pathways promoting CpG DNA-mediated metabolic responses in the presence of IFN-{gamma} were examined. Pharmacologic agents affecting cell activation were tested. Wortmannin, a PI3K inhibitor, inhibits CpG DNA-mediated activation of TLR9 (30). When wortmannin was added at 1 µM, the frequency-doubling effects of murine CpG DNA on RAW264.7 macrophages and H8/RAW264.7 transfectants were inhibited (Fig. 5, g and i). It also blocked the ability of human TLR9-positive H8/RAW264.7 cells to respond to human CpG DNA (Fig. 5j). Wortmannin had a parallel effect on the ability of human monocytes to respond to human CpG DNA (Fig. 5l) and a similar effect on IL-12-mediated priming of CpG responses (data not shown). Hence, signal-active reagents interfering with this pathway also block the metabolic activation of cells. Wortmannin is not acting directly on the metabolic amplitude pathways, but is presumably influencing activation of the frequency changes by blocking the delivery of CpG DNA to a stimulatory cell compartment.


Figure 5
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  		FIGURE 5. Effects of wortmannin and 6-AN on metabolic oscillations of cells. RAW264.7 and H8/RAW264.7 cells were treated with IFN-{gamma}, IFN-{gamma} plus 1 µM wortmannin, or IFN-{gamma} plus 5 µg/ml 6-AN. Both RAW264.7 and H8/RAW264.7 cells primed with IFN-{gamma} experienced doubling of metabolic oscillation frequency in the presence of mouse CpG (traces a and c). However, IFN-{gamma}-primed mock RAW264.7 cells did not experience an increase in frequency in the presence of hCpG DNA (trace b), whereas IFN-{gamma}-primed H8/RAW264.7 cells in the presence of hCpG DNA undergo frequency doubling (trace d). Mock transfectant RAW264.7 cells primed with IFN-{gamma} and treated with wortmannin (traces g and h) or with 6-AN (traces m and n) do not undergo metabolic frequency oscillation changes in the presence of mouse CpG or human CpG. H8/RAW264.7 cells primed with IFN-{gamma} and treated with wortmannin (traces i and j) or 6-AN (traces o and p) also do not undergo metabolic frequency oscillation change in response to mouse CpG or human CpG. Human monocytes were also exposed to mouse CpG DNA (traces e, k, and q) or human CpG DNA (f, l, and r). mouse CpG DNA. Addition of hCpG (trace f) to IFN-{gamma}-primed monocytes resulted in doubling of the frequency of the NAD(P)H autofluorescence oscillations compared with monocytes in the presence of IFN-{gamma} alone (trace e). Adding 1 µM signaling inhibitor wortmannin (trace l) or 5 µg/ml 6-AN (trace r) inhibited this increase. a–d, n = 12; e–r, n = 4.

 
Experimental and computational studies indicate that the high frequency metabolic oscillations are associated with the HMS and oxidant production (11, 12, 13, 14, 15, 16, 17, 31). To examine the HMS’s role in CpG DNA-mediated activation of high frequency metabolic oscillations, cells were treated with 6-AN (32). RAW264.7 cells were first incubated with IFN-{gamma}, then with 5 µg/ml 6-AN. Subsequent stimulation with murine CpG DNA did not elicit metabolic frequency doubling (Fig. 5n). Similarly, treatment of H8/RAW264.7 cells with murine or human CpG DNA did not effect metabolic oscillations (Fig. 5, q and r). Moreover, human monocytes did not respond to human CpG DNA (Fig. 5, l and x). 6-AN exhibited parallel effects on IL-12/human CPG DNA synergy in monocytes (data not shown). The HMS plays a key role in the induction of high frequency metabolic oscillations.

Role of MPO activity in priming cell responses to CpG DNA

We have shown that the high amplitude metabolic oscillations of monocytes and neutrophils can be traced to metabolic feedback cycles, which are triggered by deposition of MPO and NADPH oxidase into the same cell compartment (16). Because heightened metabolic amplitudes synergize with CpG DNA, we hypothesized that MPO trafficking may participate in priming cells to respond to CpG DNA. To test this idea, we first examined the effect of IFN-{gamma} on cell surface expression of MPO using direct immunofluorescence microscopy. To avoid confounding the total expression with the cell surface expression, live cells were examined. RAW264.7 macrophages incubated with buffer alone for 4 h. were not stained with anti-MPO Ab (Fig. 6a) or with a negative control rabbit IgG (data not shown). Incubation of RAW264.7 cells with murine CpG DNA and monocytes with human CpG DNA did not result in MPO surface expression (Fig. 6, b, f, and j). When incubated with murine IFN-{gamma} for 4 h at 37°C, RAW264.7 cells were stained about their periphery with an anti-murine MPO Ab (Fig. 7c), but not a negative control reagent (data not shown). Similarly, incubation of H8/RAW264.7 cells with murine IFN-{gamma} and incubation of monocytes with human IFN-{gamma} led to the expression of surface-accessible MPO (Fig. 6, g and k). Furthermore, CpG DNA had no effect on the expression of MPO by IFN-{gamma}-primed cells (Fig. 6, d, h, and l). Hence, cell surface MPO expression accompanies metabolic sensitivity to CpG DNA.


Figure 6
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  		FIGURE 6. MPO expression on RAW cells and human monocytes after stimulation with CpG DNA, IFN-{gamma}, or both. A6/RAW264.7 (a–d), hTLR9 transfectant H8/RAW264.7 macrophages (e–h), and human monocytes (i–l) were stimulated with mouse CpG DNA 1668 (a, e, and i), human CpG DNA 2006 (b, f, and j), IFN-{gamma} (c, g, and k), or the combination of IFN-{gamma} with mouse CpG DNA (d and h) or human CpG DNA (l), respectively. To avoid labeling intracellular MPO, viable cells were stained with anti-MPO Abs. Although minimal staining was found in the presence of CpG DNAs alone, substantial labeling was found after incubation with IFN-{gamma}. Magnification, x820. n = 4.

 

Figure 7
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  		FIGURE 7. Effect of MPO inhibition on the synergistic effects of IFN-{gamma} and CpG DNA. A representative kinetic trace of a single cell treated with reagents at different times is shown. Cells were initially treated with IFN-{gamma} for 4 h at 37°C. High amplitude oscillations were observed (left side). Mouse CpG DNA was added at the time indicated, which resulted in an increase in the metabolic frequency. The MPO inhibitor, SHA, was then added at the indicated time point. Both amplitude and frequency rapidly decayed to unstimulated levels. Thus, MPO appears to play a key role in mediating IFN-{gamma}/CpG DNA synergy. n = 3.

 
We next examined MPO’s functional role in metabolic activation of leukocytes. RAW264.7 cells were treated with 50 mM SHA, an MPO inhibitor, for 20 min at 37°C. Although SHA had no effect on untreated control cells, it blocked the high amplitude metabolic oscillations of cells. Fig. 7 shows a representative kinetic trace illustrating the effects of CpG DNA and SHA addition. Cells were first incubated with IFN-{gamma}. CpG DNA addition leads quickly to metabolic frequency doubling (Fig. 7). SHA was then added to the sample at a final concentration of 50 mM. As this figure illustrates, the high amplitude/high frequency metabolic oscillations quickly reverted to low amplitude/low frequency oscillations. A panel of MPO inhibitors, including p-hydroxybenzoic acid hydrazide, hydroxyquinone, and cyanide, had a similar effect (data not shown). Similar effects were noted for human monocytes (data not shown). This suggests that MPO participates in these high amplitude metabolic oscillations.

To confirm MPO’s role in metabolic amplitudes, additional strategies were used. If MPO surface expression were sufficient to initiate metabolic synergy, then it should be possible to reconstitute the metabolic effect by adding exogenous MPO to cells. To do this, we added purified MPO (10 µg/ml) to untreated cells. As expected, the metabolic oscillatory amplitude increased dramatically (Fig. 8, d, h, and i). When MPO-treated RAW264.7 cells or monocytes were exposed to murine or human CpG DNA, respectively, the metabolic changes (Fig. 8, c, d, g, h, k, and l) paralleled those found after IFN-{gamma} stimulation (Fig. 3f). Moreover, if MPO, a highly cationic protein, were removed from the cell surface using a brief rinse with a hypertonic salt solution (400 mM NaCl), NAD(P)H amplitudes returned to normal (data not shown), indicating that its continued presence is required. Several lines of evidence support the idea that MPO trafficking contributes to the high amplitude metabolic oscillations required for CpG DNA synergy.


Figure 8
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  		FIGURE 8. Reconstitution of metabolic effects of CpG DNA using exogenous MPO. Mock transfectant A6/RAW264.7 (a–d), hTLR9 transfectant H8/RAW264.7 macrophages (e–h), and human monocytes (i–l) were studied. Cells stimulated with mouse CpG DNA 1668 (a and e) or human CpG (hCpG) DNA 2006 (i) alone had no effect. However, IFN-{gamma}-primed cells (b, f, and j) expressed high amplitude and high frequency metabolic oscillations in the presence of the appropriate CpG DNA. Addition of exogenous MPO caused high amplitude oscillations (d, h, and l), and MPO plus CpG DNA promoted high amplitude and frequency metabolic oscillations in cells (c, g, and k). n = 5.

 
To provide additional compelling evidence to support MPO’s role in CpG DNA-mediated metabolic signaling, we performed experiments using MPO knockout mice (19). Monocytes were obtained from the peripheral blood of control and MPO knockout mice. After incubation with buffer or IFN-{gamma} for 4 h at 37°C, cells were examined using quantitative microfluorometry. Although adherent control monocytes incubated with buffer exhibited low amplitude metabolic oscillations (Fig. 9a), cells incubated with IFN-{gamma} demonstrated high amplitude oscillations (Fig. 9c), as illustrated above for RAW264.7 cells. IFN-{gamma}-primed monocytes responded to murine CpG DNA with metabolic frequency doubling (Fig. 9e). Adherent MPO knockout monocytes did not demonstrate high amplitude metabolic oscillations after incubation with IFN-{gamma} (Fig. 9d) and were unresponsive to murine CpG DNA (Fig. 9f). Collectively, the translocation, inhibitor, reconstitution with purified MPO, and MPO knockout data indicate that MPO plays a key role in CpG-mediated leukocyte activation.


Figure 9
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  		FIGURE 9. Metabolic amplitude changes and IFN-{gamma}/CpG DNA synergy are not observed in leukocytes of MPO knockout (k-o) mice. Peripheral blood monocytes were isolated from normal (a, c, and e) and MPO knockout (b, d, and f) C57BL/6 mice. After incubation with IFN-{gamma} for 4 h at 37°C, cells from control mice exhibited high amplitude oscillations and responded to treatment with mouse CpG DNA with an increase in metabolic frequency. Cells from MPO knockout mice, however, were unresponsive to IFN-{gamma} and IFN-{gamma} plus mouse CpG DNA (d and f, respectively). n = 3.

 
IFN-{gamma} and CpG DNA synergize to increase oxidant production

To test the physiological role of metabolic amplitude and frequency changes, we measured oxidant production in response to IFN-{gamma} and/or CpG DNA. Single-cell assays were performed (11, 12, 13, 14, 15, 16, 17) to allow direct comparisons of amplitude and frequency changes in substrate (NADPH) and products (ROMs and NO). The initial rate of ROM production was determined by the generation of fluorescence by the oxidation of H2-TMRos or HE. When adherent RAW264.7 or H8/RAW264.7 cells were treated with buffer or appropriate CpG DNA, no significant effects on ROM production were noted (Fig. 10, A and C). IFN-{gamma} increased the ROM production rate. Moreover, when IFN-{gamma} was combined with specific CpG DNA, the ROM release rate was increased dramatically (Fig. 10, A and C). Similar effects were noted for adherent peripheral blood monocytes from C57BL/6 mice in the presence of buffer, IFN-{gamma}, and/or murine CpG DNA (Fig. 10E). However, when monocytes from MPO knockout mice were used, no effect of IFN-{gamma} and/or murine CpG DNA was noted (Fig. 10G). This confirms that MPO is important in both the metabolic and functional changes in the cells. These ROM responses were evident in murine and human cells incubated with appropriate CpG DNA and IFN-{gamma} (Fig. 10, E and I). Similar results were obtained using HE, which is specific for superoxide (data not shown), and the NO-sensitive indicator, decay-accelerating factor (Fig. 10, B, D, F, H, and J). Therefore, a dramatic difference in leukocyte activation exists between CpG DNA-treated cells in the presence and the absence of IFN-{gamma}.


Figure 10
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  		FIGURE 10. Representative kinetic traces showing the release of ROMs and NO from individual cells. Cells from RAW264.7 cells (A), H8/RAW264.7 macrophages (B), monocytes from control mice (C), monocytes from MPO knockout mice (D), and human monocytes (E) were evaluated. In these experiments, cells were treated with buffer alone (a), CpG DNA alone (b), IFN-{gamma} alone (c), or IFN-{gamma} and species-matched CpG DNA (d). This single-cell assay demonstrates the marked synergy of IFN-{gamma} and CpG DNA in ROM and NO release and the essential role of MPO. A–D, n = 9; E and F, n = 4; G and H, n = 3; I and J, n = 12.

 
Finally, we performed a series of conventional NO production assays to confirm the validity of our observations. Experiments were performed on RAW264.7 cells after incubation for 4 h at 37°C with buffer or IFN-{gamma}. Primed or control cell samples were incubated with CpG DNA (Fig. 11a) or, as a control, GpG DNA (Fig. 11b). As this illustrates, NO production, as judged by nitrite formation, was dependent upon the dose of CpG DNA and was greatly augmented by IFN-{gamma}. However, negative control GpC DNA had no effect on NO production. Thus, NO production is enhanced by exposure to IFN-{gamma} and CpG DNA.


Figure 11
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  		FIGURE 11. NO production by RAW264.7 macrophages. After IFN-{gamma} priming, cells were treated with CpG DNA (a) or GpC DNA (b). In both experiments cells were examined after IFN-{gamma} treatment with ({diamond}) and without ({blacksquare}) addition of CpG DNA or GpC DNA. CpG DNA dramatically enhanced NO production in the presence of IFN-{gamma}. n = 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The pioneering experiments of Tokunaga et al. (33) and Krieg et al. (34) demonstrated that bacterial DNA was immunostimulatory and that specific nucleotide sequences containing unmethylated CpG dinucleotides were responsible for their functional activity. TLR9 was subsequently recognized as a receptor for CpG DNA (35). Internalization of CpG DNA and intracellular trafficking play key roles in signaling (24, 29). Much research has focused upon the roles of adaptor proteins and downstream kinases that lead to activation of AP-1 and NF-{kappa}B transcription factors. Although superoxide and NO are intercellular signaling molecules and directly participate in host defense, little effort has been focused on understanding the mechanism of CpG DNA-mediated oxidant release (8, 36). We propose a mechanistic model of CpG DNA-mediated TLR9-dependent ROM and NO production based upon the cooperation of two metabolic pathways, the HMS and peroxidase cycle.

Our work has identified key factors in CpG DNA-mediated oxidant production, including the HMS, CpG DNA trafficking (wortmannin sensitivity), correct species-specific pairing of CpG DNA and TLR9, and the peroxidase cycle. ROM and NO production require NADPH as an electron source. One key source of NADPH production is the HMS. The ability of CpG DNA, in the presence of IFN-{gamma}, to promote a doubling in metabolic frequency strongly indicates HMS activation. To directly demonstrate the role of HMS, we treated cells with 6-AN. The results showed that the HMS was required to yield a CpG DNA-specific cell response in cell metabolism and oxidant release. CpG DNA alone could enter cells (Fig. 1), but had no effect on metabolism or oxidant production. Thus, CpG DNA cannot directly stimulate the HMS, but requires complementary or synergistic interactions with other cellular systems.

Four lines of evidence support the concept that MPO participates in leukocyte CpG DNA responses. Because MPO translocation is required for activation of the peroxidase cycle (16), we tested the translocation of MPO to the cell surface after priming with IFN-{gamma}, which would permit chemical communication between the oxidase and peroxidase. Using a vital cell staining protocol to avoid intracellular MPO labeling, we detected MPO translocation to the cell surface. Because MPO is a highly cationic protein, its attachment to the cell surface is likely to be electrostatic in nature. MPO surface staining and high amplitude oscillations can be reversed by washing cells with a high ionic strength buffer, suggesting a link between these observations. Thus, IFN-{gamma} priming yields conditions that would support the peroxidase cycle. We next eliminated IFN-{gamma} priming by directly adding MPO to adherent cells. Exogenous MPO delivery to cells was sufficient to promote high amplitude NAD(P)H oscillations and reconstitute the stimulatory effects of CpG DNA. Not only does this provide another line of evidence to support the role of MPO translocation in CpG DNA/IFN-{gamma} synergy, but it suggests that such synergies may be induced in vivo in the neighboring primed cells of unprimed macrophages. Hence, MPO and its trafficking are important in CpG DNA-mediated activation of cell metabolism.

Two additional lines of evidence support the unconventional role of MPO in priming CpG DNA-mediated cell activation. MPO inhibitors block high amplitude metabolic oscillations and the effects of CpG DNA on metabolism. For example, the kinetic experiment in Fig. 7 shows that after IFN-{gamma} priming to induce high amplitude metabolic oscillations, CpG DNA addition causes frequency doubling and that both are inhibited by addition of an MPO inhibitor. This is consistent with a dynamic mechanism in which MPO is required for high amplitude NAD(P)H oscillations that are, in turn, required for the high frequency oscillations induced by CpG DNA. To provide additional definitive data regarding MPO’s role in cell stimulation, peripheral blood monocytes were isolated from control and MPO knockout mice. Although monocytes from control mice responded to the IFN-{gamma} priming and CpG DNA treatment, MPO knockout mice were unable to respond to either reagent. This striking difference provides the fourth line of evidence supporting the role of MPO in priming CpG DNA responses. It also suggests that MPO trafficking/activation of the peroxidase cycle may contribute to leukocyte priming for ROM and NO release.

Although we have identified intracellular pathways participating in CpG DNA/IFN-{gamma} synergy, several biochemical details remain to be clarified. We have not yet established the mechanism by which the HMS couples with the peroxidase cycle. It is known that LPS concentrations below the threshold necessary for HMS activation can synergize with CpG DNA to activate the HMS (37). In contrast to LPS, IFN-{gamma} synergy with CpG DNA is closely related to NADPH amplitude changes. How might these amplitude changes couple to the HMS? One possibility is that the trough of NADPH itself contributes to HMS stimulation. For example, the high NADP+ concentration that exists during the NADPH autofluorescence trough is the substrate for the HMS and therefore would be expected to drive the shunt toward the product. Because glucose-6-phosphate dehydrogenase is strongly inhibited by its product, NADPH (38), the reduction in inhibition during the autofluorescence trough will also accelerate the HMS. These events, when coupled to the poor activating ability of CpG DNA, may create conditions sufficient to activate the shunt. Emerging computational tools (15) will probably help in addressing mechanistic details.


   Acknowledgments
 
We thank Yoshihiko Ikeda and Takashi Ishii for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 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 Address correspondence and reprint requests to Dr. Howard R. Petty, Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, 1000 Wall Street, Ann Arbor, MI 48105. E-mail address: hpetty{at}umich.edu Back

2 Abbreviations used in this paper: ROM, reactive oxygen metabolite; HMS, hexose monophosphate shunt; H2TMRos, dihydrotetramethylrosamine; MPO, myeloperoxidase; SHA, salicylhydroxamic acid; TRITC, diaminofluorescein-2 diacetate; HE, hydroethidine. Back

Received for publication April 13, 2005. Accepted for publication January 19, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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