Inka Heiner

Jörg Eisfeld

Maike Warnstedt

Natalia Radukina

Eberhard Jüngling

Andreas Lückhoff
email: luckhoff@physiology.rwth-aachen.de

Endogenous ADP-ribose enables calcium-regulated cation currents through TRPM2 channels in neutrophil granulocytes

TRPM2 is a Ca 2+ -permeable cation channel gated by ADP-ribose (ADPR) from the cytosolic side. To test whether endogenous concentrations of intracellular ADPR are sufficient for TRPM2 gating in neutrophil granulocytes, we devised an HPLC protocol to determine ADPR contents in perchloric acid cell extracts. The reversed phase ion-pair HPLC protocol with a Mg 2+ containing isocratic eluent allows baseline resolution of one ADPR peak. Intracellular ADPR concentrations were about 5 µM in granulocytes and not significantly altered by stimulation with the chemoattractant peptide fMLP. We furthermore analyzed intracellular concentrations of cyclic ADPR (cADPR) with a cyclase assay involving enzymatic conversion of cADPR to NAD and fluorometric determination of NAD.

Intracellular cADPR concentrations were about 0.2 µM and not altered by fMLP. In patch-clamp experiments, ADPR (0.1 to 100 µM) was dialyzed into granulocytes to analyze its effects on wholecell currents characteristic for TRPM2, in the presence of a low (< 10 nM) or a high (1 µM) intracellular Ca 2+ concentration. TRPM2 currents were significantly larger in high than in low Ca 2+ (e.g. -225 ± 27.1 vs. -7 ± 2.0 pA/pF at 5 µM ADPR) but no currents at all were observed in the absence of ADPR (ADPR concentration ≤ 0.3 µM). cADPR (0.1, 0.3 and 10 µM) was without effect even in the presence of subthreshold ADPR (0.1 µM). We conclude that ADPR enables an effective regulation of TRPM2 by cytosolic Ca 2+ . Thus, ADPR and Ca 2+ in concert behave as a messenger system for agonist-induced influx of Ca 2+ through TRPM2 in granulocytes.

INTRODUCTION

TRPM2 is a non-selective cation channel expressed in a wide range of human tissues, including the brain, bone marrow, spleen and pancreas [START_REF] Nagamine | Molecular cloning of a novel putative Ca2+ channel protein (TRPC7) highly expressed in brain[END_REF][START_REF] Inamura | Response to ADP-ribose by activation of TRPM2 in the CRI-G1 insulinoma cell line[END_REF][START_REF] Uemura | Characterization of human and mouse TRPM2 genes: identification of a novel N-terminal truncated protein specifically expressed in human striatum[END_REF]. We have recently shown its expression in neutrophil granulocytes with RT-PCR analysis [START_REF] Heiner | Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD[END_REF]. Moreover, we have provided functional evidence for its expression by demonstrating channels in neutrophils [START_REF] Heiner | Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD[END_REF] that exhibit the same characteristic biophysical properties of channels found when TRPM2 is heterologously expressed in HEK 293 cells [START_REF] Perraud | ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology[END_REF]. Thus, TRPM2 is so far the only Ca 2+ -permeable channel in neutrophils identified by its functional properties that can be attributed to a particular channel protein.

Gating of TRPM2 channels can be induced by ADP-ribose (ADPR) [START_REF] Perraud | ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology[END_REF]. Further activators of TRPM2 that either alone, or in cooperation with ADPR, or through the formation of ADPR may lead to the opening of the channel include cyclic ADPR (cADPR) [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF], NAD [START_REF] Sano | Immunocyte Ca2+ influx system mediated by LTRPC2[END_REF][START_REF] Hara | LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death[END_REF], H 2 O 2 [START_REF] Hara | LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death[END_REF][START_REF] Wehage | Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose[END_REF], and Ca 2+ ions [START_REF] Mchugh | Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation[END_REF]. For gating by ADPR, the NUDT9 homology region of TRPM2 is decisive. Most likely, binding of ADPR to a defined part within the homology region is involved [START_REF] Kühn | Sites of the NUDT9-H domain critical for ADP-ribose activation of the cation channel TRPM2[END_REF]. The NUDT9 homology region is localized within the cytosolic C-terminal tail of TRPM2. Thus, TRPM2 may be activated by an endogenous intracellular ligand, raising the question whether ADPR has a role as a second messenger that regulates cation influx through TRPM2.

However, actual intracellular concentrations of ADPR are not known in granulocytes. When we started our study, no analytical method for the determination of ADPR levels had been reported. Furthermore, metabolic pathways through which the formation of ADPR may be induced are poorly understood, particularly with respect to their regulation. One possible source of ADPR in many cells are ADPR polymers, formed in the nucleus by Poly(ADPR) polymerases (PARPs). The polymers may be degraded to ADPR by glycohydrolases. PARP activation can be elicited by many factors, such as radiation, oxidative stress or some toxic substances. There have been many enzymes described with a PARP domain, numbered from PARP-1 to PARP-18 [START_REF] Ame | The PARP superfamily[END_REF]. The majority of PARP activity is attributed to PARP-1. Strikingly, neutrophil granulocytes are within the few cells that do not express PARP-1 [START_REF] Sanghavi | Caspase-mediated proteolysis during apoptosis: insights from apoptotic neutrophils[END_REF]. Therefore, activation of TRPM2 in neutrophils by ADPR derived from ADPR polymers seems unlikely. As alternative pathway for ADPR production, the membrane-associated protein CD38 may be considered in neutrophil granulocytes. This is an enzyme with glycohydrolase activity that degrades NAD and related compounds [START_REF] Schuber | Structure and enzymology of ADP-ribosyl cyclases: conserved enzymes that produce multiple calcium mobilizing metabolites[END_REF]. In the past, major relevance of CD38 has been attributed to its generation of cADPR [START_REF] Partida-Sanchez | Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo[END_REF]. cADPR is an established second messenger that mediates Ca 2+ release from intracellular stores by action on the ryanodine receptor, a Ca 2+ release channel in the membrane of calcium stores [START_REF] Guse | Novel hydrolysis-resistant analogues of cyclic ADP-ribose: modification of the "northern" ribose and calcium release activity[END_REF]. Moreover, cADPR in concentrations of 10 µM has been reported to potentiate the effects of ADPR [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF]. However, cADPR contributes only marginally to the CD38 products. Within the products of CD38-catalyzed metabolism of NAD, the overwhelming majority (96 %) is comprised by ADPR [START_REF] Howard | Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38[END_REF]. Thus, CD38 may be viewed as an ADPR synthase. Yet it has to be taken into account that CD38 is an ectoenzyme [START_REF] De Flora | Topology of CD38[END_REF] that catalyzes the conversion of extracellular Copyright 2006 Biochemical Society NAD to extracellular ADPR. At present, it has not been clarified whether neutrophil CD38 may provide intracellular ADPR formation as well or whether effective transport systems exist that shuffle the substrate and the product of CD38 through the plasma membrane.

The aim of the present study was a determination of the concentrations of ADPR and of cADPR in neutrophil granulocytes, to answer the question whether these concentrations are sufficient for TRPM2 gating. Both nucleotides, in the determined physiological concentrations, were then dialyzed into neutrophils and the currents through TRPM2 were monitored. Furthermore, we tested whether stimulation with the chemoattractant peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), known to induce Ca 2+ influx [START_REF] Krause | The calcium signal and neutrophil activation[END_REF], led to increases in the concentrations of the nucleotides.

We report the modification of an HPLC-based method, originally developed for the analysis of intracellular nucleotides [START_REF] Jüngling | Rapid assay of adenine nucleotides or creatine compounds in extracts of cardiac tissue by paired-ion reverse-phase high-performance liquid chromatography[END_REF], for ADPR determination, while cADPR was analyzed with the cyclase assay developed by Graeff and Lee [START_REF] Graeff | A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity[END_REF]. The levels of ADPR but not of cADPR were in the range that allow intracellular Ca 2+ an effective regulation of TRPM2 channels in cooperation with ADPR.

Since intracellular Ca 2+ concentrations are raised by fMLP-induced release from stores, ADPR and Ca 2+ in concert behave as messenger system for the induction of cation influx through TRPM2.

EXPERIMENTAL

Materials

Nucleotide standards, nucleotide pyrophosphatase from Crotalus adamentus venom, alkaline phosphatase, NADase from Neurospora crassa, ADP-ribosyl-cyclase, nicotinamide, alcohol dehydrogenase, resazurin, diaphorase and N-formyl-methionyl-leucyl-phenylalanine (fMLP) were from Sigma (Sigma, Taufkirchen, Germany), riboflavin-5'-monophosphate (FMN) and tri-noctylamine were from Fluka (Fluka, Sigma-Aldrich, Steinheim, Germany). 1,1,2trichlorotrifluoroethane was from Riedel-de Haën (Seelze, Germany). Perchloric acid (PCA), methanol (LiChrosolv) and tetrabutylammonium dihydrogensulfate (TBAHS) were from Merck (Darmstadt, Germany).

Neutrophil isolation

Neutrophil granulocytes were prepared from peripheral blood of healthy volunteers as described [START_REF] Heiner | Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD[END_REF].

Cell extracts

Neutrophils (5 to 7 x 10 7 ) were stored in test tubes (15 ml, Greiner, Frickenhausen, Germany) at room temperature for up to 30 min and then kept at 37°C for 2 or 5 min. During this time, some aliquots of the cell suspensions were stimulated with fMLP (1 µM). Thereafter, the tubes were placed in an ice-salt bath for 3 min prior to centrifugation (1000 g, 4°C, 2 min). Pellets were resuspended in ice-cold 0.4 N perchloric acid, followed by three freeze-thaw-cycles (liquid nitrogen / 37°C) and short ultrasonic disruption. Cell debris was removed at 550 g for 5 min at 4°C. For ADPR determinations, supernatants were divided into two identical halves, to one of which an ADPR standard was added (spiking). Extracts were titrated to pH 5-6 with potassium carbonate. Precipitated potassium chlorate was removed by centrifugation (10,000 g, 4°C, 10 min). Finally, the supernatants were filtered through disposable 0.2 µm filters (Spin X, Corning Costar, Wiesbaden, Germany). For cADPR determinations, perchloric acid was removed by mixing 1 vol. of the supernatants with a solution containing 3 vol. of 1,1,2-trichlorotrifluoroethane and 1 vol. tri-n-octylamine and vortexing the mixture for 1 min. Following a centrifugation step for 10 min at 1500 g, the probes were maintained on ice until the aqueous phase easily separated from the organic phase. The aqueous layer containing the cADPR was removed and adjusted to a pH of 7-8 with identical volume of sodium phosphate (20 mM, pH 8).

Enzymatic degradation of ADPR

Neutralized extracts or standard ADPR were incubated with nucleotide pyrophosphatase (1.8 U/ml, 2 mM MgCl 2 , 37°C, 30 min). The enzyme was removed by centrifugation of the samples through Centrisart I filter devices (10 kDa MWCO, Sartorius, Göttingen, Germany).

HPLC Analysis

Neutralized neutrophil extracts were analyzed by reversed phase HPLC protocol modified from a procedure developed for the analysis of purine nucleotides in tissue extracts [START_REF] Jüngling | Rapid assay of adenine nucleotides or creatine compounds in extracts of cardiac tissue by paired-ion reverse-phase high-performance liquid chromatography[END_REF][START_REF] Folley | Separation of nucleotides by ion-pair, reversed-phase high-performance liquid chromatography: Use of Mg(II) and triethylamine as competing hetaerons in the separation of adenine and guanine nucleotides[END_REF]. A LaChrom Elite HPLC system (Merck, Hitachi) was used with a LiChrospher RP-8 column (250 x 4 mm, 5 µm particle size, Merck). A volume of 20-40 µl of the samples (corresponding to the extract of 2x10 6 to 4x10 6 cells) or standards were injected with an autosampler (LaChrom Elite L-2200, Merck, Hitachi) that kept the samples at 4°C. An isocratic eluent was used containing methanol (6 % w/v) and (in mM): KH 2 PO 4 130, TBAHS 3, MgSO 4 1; pH 5.9. The flow rate was 1.0 ml/min. Absorbance was measured at 270 nm with an UV-absorbance detector (LaChrom Elite L-2400, Merck, Hitachi). Data were processed by the EZChrom Elite data acquisition system. The recovery of the extraction was calculated by comparing an ADPR standard with spiked and non-spiked samples. cADPR assay cADPR concentrations were determined in standards and in cell extracts with the method described by Graeff and Lee ( [START_REF] Graeff | A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity[END_REF]), based on the conversion of cADPR to NAD with commercially available ADP-ribosyl-cyclase and the fluorometrical determination of the NAD concentration with the resazurin/resorufin indicator reaction. In short, 100 µl of the cell extracts were incubated overnight at room temperature in an enzyme mixture containing (final concentrations): nucleotide pyrophosphatase (0.44 U/ml), alkaline phosphatase (12.5 U/ml), NADase (0.0625 U/ml), MgCl 2 (2.5 mM), and sodium phosphate (20 mM, pH 8). Enzymes were removed by filtration with Vivaspin 500 filters (10 kDa MWCO, Vivascience, Hannover, Germany) and samples were recovered in the filtrate after centrifugation at 4°C and 3000 g for 30 min. Conversion of cADPR to NAD was initiated by incubating 100 µl of the probes with 50 µl of a reagent for 15 min at room temperature. The reagent contained ADP-ribosyl-cyclase (0.3 µg/ml), nicotinamide (30 mM), sodium phosphate (100 mM, pH 8). Afterwards, 100 µl of the cycling reagent were added, containing ethanol (2%), alcohol dehydrogenase (100 µg/ml), resazurin (20 µM), diaphorase (10 µg/ml), FMN (10 µM), nicotinamide (10 mM), BSA (100 µg/ml) and sodium phosphate (100 mM, pH 8). The samples were then put into glass tubes (0.3 ml, CS Chromatographie Service, Langerwehe, Germany) and the increase in the resorufin fluorescence (with excitation at 544 nm and emission at 590 nm) was monitored for 65 min in a fluorescence spectrophotometer (Cary Eclipse, Varian, Darmstadt, Germany). The cADPR concentration of the cell extracts was calculated from the fluorescence increase over time in comparison to that of cADPR standards. As negative controls, probes without cyclase addition were used.

cADPR purification

Since cADPR stock solutions contained about 20 % ADPR as contamination, ADPR was removed by incubation with nucleotide pyrophosphatase as described above for extracts.

Electrophysiology

Human granulocytes were studied with the patch-clamp technique in the whole-cell mode, using an EPC 9 amplifier equipped with a personal computer with Pulse 8.5 and X Chart software (HEKA, Lamprecht, Germany). Pipets were made of borosilicate glass; the pipet tips were coated with dental wax (Moyco, Philadelphia, USA) to reduce thermic noise for the resolution of single channel events in the whole-cell mode. The bath solution was as described [START_REF] Heiner | Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD[END_REF], with Na + as main cation sometimes replaced by 150 mM N-methyl-D-glucamine (NMDG). The pipet solution contained 145 mM Csglutamate, 8 mM NaCl, 2 mM MgCl 2 , 10 mM HEPES, pH 7.2 (CsOH) and the Ca 2+ concentration was adjusted to either <10 nM (10 mM Cs-EGTA) or 1 µM (0.886 mM CaCl 2 , 1 mM Cs-EGTA). Copyright 2006 Biochemical Society from voltage ramps (from -90 to +60 mV over 400 ms). These experiments were carried out at room temperature.

RESULTS

To verify whether the chosen HPLC system enabled quantitative determinations of ADPR concentrations, the column was loaded with standards containing either of the following nucleotides in various mixtures: ATP, ADP, AMP, adenosine, GTP, cADPR, NAD, nicotinamide, and ADPR.

These compounds were readily separated, resulting in defined peaks in the chromatograms that allowed the identification of each of them by their individual retention times (see supplementary Fig. 1). The area of the peak attributed to ADPR was linearly correlated with the ADPR content in the sample. The detection limit was in the range of 1 pmol ADPR.

Chromatograms of neutralized PCA extracts from neutrophil granulocytes displayed several more peaks than the standards, many of them not well separated (Fig. 1A). Importantly, however, there was one single baseline-resolved peak with the same retention time as that of ADPR standards.

To test for the specificity of this peak attributed to ADPR, we spiked the extracts with ADPR, i.e. we added ADPR to the extracts prior to neutralization with potassium carbonate, thereby increasing their ADPR content by a defined amount. Only 50 % of the volume of each extract was spiked, whereas the same volume of water was added to the other half. The two chromatograms of representative twin samples (spiked and not spiked) from neutrophils are compared in Fig. 1A. Spiking resulted in the specific enlargement of the ADPR peak. From a comparison of the peak area of the unspiked sample with that of an ADPR standard, the amount of ADPR in the unspiked sample after neutralization is calculated as 7.6 pmol. The comparison with the peak area of the spiked sample allows to calculate a recovery rate during the sample preparation of 98 %. The same recovery rate was found when five fold higher amounts of ADPR were used for spiking. As further test for the specificity of the ADPR peak, we incubated standards as well as twin samples of cell extracts with pyrophosphatase that converts ADPR to AMP and ribose-5-phosphate. Fig. 1B shows that pyrophosphatase incubation completely removed the ADPR peak, in standards and extracts alike. At the same time, the AMP peak was considerably enlarged, indicating that ADPR as well as other nucleotides were degraded to AMP.

When neutrophil granulocytes were stimulated with fMLP (1 µM, 2 min) prior to acidic precipitation, the ADPR concentrations in the extracts were not significantly (paired t-test) changed (Fig. 2; n = 6 experiments with extracts from stimulated vs. control cells). There was a tendency for a small reduction of ADPR contents by fMLP (Fig. 2B), in the range of the resolution of the method.

Similar results were obtained after a stimulation of 5 min (data not shown). For comparison, we performed experiments on HEK 293 cells stimulated with hydrogen peroxide (10 mM, 10 min, n = 3). The HPLC data so far define the amounts of ADPR present in a given number of neutrophil granulocytes. When these figures are to be used for a calculation of intracellular ADPR concentrations, some uncertainties exist, most notably the cell size and the ADPR distribution space within the cells. The cell volume of neutrophil granulocytes has been estimated as 350 fl [START_REF] Jankowski | A noninvasive fluorimetric procedure for measurement of membrane potential. Quantification of the NADPH oxidase-induced depolarization in activated neutrophils[END_REF]. We derived similar values from the determination of the cytocrit of granulocyte suspensions. When this volume is assumed along with a homogenous intracellular ADPR distribution, the concentration of ADPR is 5.0 ± 1.8 µM (mean ± SEM) in unstimulated neutrophil granulocytes and 3.9 ± 1.8 µM after fMLP stimulation. We estimate that these values have an uncertainty factor of 0.5 to 6, i.e. the actual concentrations may be in the range from 2 to 30 µM.

To test for the functional significance of these ADPR concentrations for TRPM2 gating in neutrophil granulocytes, we performed patch-clamp experiments in which ADPR was infused into granulocytes through the patch pipet. We considered that intracellular Ca 2+ is a cofactor of ADPRdependent TRPM2 gating [START_REF] Mchugh | Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation[END_REF]. However, this finding has only been obtained with heterologously expressed TRPM2 channels which does not necessarily indicate that the same holds true in granulocytes. Therefore, the experiments were performed with two different Ca 2+ concentrations in the pipet solution, either a very low one achieved by addition of EGTA (10 mM) to a nominally Ca 2+free solution (Ca 2+ concentration < 10 nM), or a concentration corresponding to that in stimulated cells, i.e. about 1 µM accomplished by a mixture of EGTA and CaCl 2 (see Experimental).

When ADPR (5 µM) was infused into granulocytes in the presence of low Ca 2+ , the opening of a few channels was induced that could be resolved within the total whole-cell currents (Fig. 3A).

The extremely long (several seconds) open times and the single channel current amplitudes (consistent with the reported conductance of 50-70 pS) are characteristic for TRPM2 channels [START_REF] Perraud | ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology[END_REF] and provide strong evidence that ADPR gates TRPM2 channels in neutrophil granulocytes. Sizeable whole-cell currents, rather than only some single channel openings, were induced in granulocytes when ADPR (even at 1 µM) was infused along with Ca 2+ (Fig. 3B). No single channel events can be resolved within large whole-cell currents. However, since the exchange of the cytosolic solutes with that of the pipet fluid is a time-dependent process, the currents typically developed gradually before a plateau was reached. During the initial phase of the current development, the consecutive opening of single channels was evident, again with the characteristic properties of TRPM2 (Fig. 3B). Whole-cell currents were blocked by the large cation NMDG (Fig. 3) reported to be mostly impermeant to TRPM2 [START_REF] Wehage | Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose[END_REF].

Experiments with higher ADPR concentrations (30 or 100 µM) demonstrated again a critical role of Ca 2+ . Although ADPR evoked whole-cell currents in the absence of Ca 2+ , the currents were strikingly larger in amplitude in the presence of Ca 2+ (1 µM) in the pipet solution (Fig. 4). The I-V Copyright 2006 Biochemical Society relation of the currents, with either Na + or NMDG + as main extracellular cation, was characteristic for TRPM2 currents [START_REF] Heiner | Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD[END_REF][START_REF] Perraud | ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology[END_REF][START_REF] Wehage | Activation of the cation channel long transient receptor potential channel 2 (LTRPC2) by hydrogen peroxide. A splice variant reveals a mode of activation independent of ADP-ribose[END_REF].

Importantly, gating of TRPM2 channels was not evoked by Ca 2+ alone. When a pipet solution was used that contained the high Ca 2+ concentration (ADPR) but no ADPR, no single channel events and no increases in the whole-cell currents were observed (Fig. 3C). Thus, the presence of ADPR is essential for the effects of Ca 2+ on TRPM2. The dependence of currents in granulocytes on the concentration of ADPR, in the presence of either a high or a low intracellular Ca 2+ concentration, is summarized in Fig. 5. A minimal ADPR concentration of 1 µM was required to induce currents in the presence of high Ca 2+ . At higher ADPR concentrations, a saturation of the effects was observed. Over the whole ADPR concentration range studied, considerably and significantly lower currents were found for low than for high Ca 2+ .

A modulator of ADPR-induced TRPM2 activation may be cADPR, implicated to be a second messenger regulated by receptor-dependent stimulation in other cells [START_REF] Guse | Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose[END_REF]. The cADPR concentration reported to enhance TRPM2 currents was 10 µM. In our HPLC conditions, cADPR produces a peak at about 2.8 min retention time. For the range of this retention time, Fig. 6 shows an expanded view of two chromatograms of cell extracts, one from fMLP-stimulated cells and one from resting cells, along with a chromatogram of a cADPR standard. The two extracts produced almost identical chromatograms (in the shown retention time range) but no peak at the expected retention time of cADPR. We cannot exclude that one of the neighbouring peaks may represent cADPR; in this case, an fMLP-induced concentration increase could be ruled out. More likely, cADPR is not present intracellularly in sufficient concentrations to be resolved with our HPLC conditions. Therefore, we resorted to the enzymatic cADPR assay based on the conversion of cADPR to NAD and the fluorometrical determination of NAD [START_REF] Graeff | A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity[END_REF] to assess intracellular cADPR concentrations. With standards nominally containing 0-10 nM cADPR, a calibration curve was obtained in which the cADPR concentration was linearly related to the increase in resorfin fluorescence (correlation coefficient r = 0.92, see supplementary Fig. 2). The cADPR concentrations found in extracts were between 1 and 9 nM (n = 6). From the number of cells in each extract and the estimated cytosolic volume, the intracellular concentration was calculated to be 221 ± 84 nM. Aliquots of the same granulocyte preparations were stimulated with fMLP (1 µM for 3 min), resulting in an intracellular concentration of 152 ± 93 nM (n = 6). When cADPR was added to cell extracts in amounts corresponding to a raise in the intracellular concentration by 50 nM, a concentration increase in the expected range was found in extracts from unstimulated as well as from fMLP-stimulated cells. Since we discovered later (see below) that the standards may have contained, to a sizeable part, ADPR rather than cADPR, the actual intracellular cADPR concentrations are likely to be about 25 % lower.

Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183

Copyright 2006 Biochemical Society

As in the case of ADPR, we wished to examine whether cADPR in physiological concentrations had effects on whole-cell currents. First, we repeated the experiments performed by Kolisek et al. [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF] in TRPM2-transfected HEK 293 cells in granulocytes, i.e. we used a weakly Ca 2+buffered pipet solution with 10 µM cADPR. In contrast to Kolisek et al., we observed large currents (-200.8 ± 10.6 pA/pF, n = 3) as if ADPR rather than cADPR were infused (Fig. 7A). Similar results

were obtained with cADPR (10 µM) in our standard high Ca 2+ pipet solution. This prompted us to test for the purity of our cADPR solutions with an HPLC analysis (inset Fig. 7A) that demonstrated considerable contamination with ADPR that may be conservatively estimated to be at least 25 %, even when cADPR solutions are freshly prepared from cADPR salts. These results were confirmed in all of several cADPR batches tested. Therefore, the currents induced in our experiments with nominally 10 µM cADPR are explained by contaminating ADPR. To analyze possible effects of cADPR, cADPR stock solutions were incubated with a nucleotide pyrophosphatase, resulting in the complete conversion of ADPR to AMP (inset Fig. 7B). It was confirmed that pyrophosphatase-treated cADPR stock remained free of ADPR for several days after freezing. With pipet solutions prepared from ADPR-free cADPR, no currents were induced in granulocytes (n = 4, data not shown), not even when ADPR was added in a subthreshold concentration (0.1 µM; n = 4; Fig. 7B). Since AMP has been reported as an inhibitor of TRPM2 currents [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF], although only at high concentrations (0.5 mM), we furthermore tested AMP (5 µM which slightly exceeds the concentrations used in the experiments with ADPR-free cADPR) in the presence of ADPR (5 µM) which did not noticeably alter ADPRinduced TRPM2 currents in granulocytes (n = 4; Fig. 7C). Finally, we tested cADPR in physiological concentrations. When cADPR was infused in concentrations of 0.1 µM (n = 7) or 0.3 µM (n = 6), along with ADPR in a subthreshold concentration (i.e. 0.1 µM), no current was induced in any experiment.

DISCUSSION

In the present study, we provide measurements of intracellular ADPR concentrations, an activator of the Ca 2+ -permeable cation channel TRPM2, in neutrophil granulocytes. As main finding, we report that the ADPR concentrations are in the range that allow intracellular Ca 2+ an effective regulation of TRPM2 gating. Thus, even though ADPR concentrations were not increased by stimulation of the cells and Ca 2+ by itself is inactive on TRPM2, ADPR and Ca 2+ in combination can act as a messenger system. This messenger system is capable to induce Ca 2+ influx through TRPM2 channels in response to stimuli like fMLP that lead to release of Ca 2+ from intracellular stores. In contrast, no evidence was obtained for a regulation of TRPM2 by cADPR.

Measurements of ADPR became possible after we modified a reversed phase ion-pair HPLC protocol devised for the measurements of intracellular purine nucleotides [START_REF] Jüngling | Rapid assay of adenine nucleotides or creatine compounds in extracts of cardiac tissue by paired-ion reverse-phase high-performance liquid chromatography[END_REF]. An isocratic eluent was used that offers the advantage that small concentration changes can be more precisely assessed Copyright 2006 Biochemical Society than with a gradient elution. The separation of ADPR from ATP and ADP became possible due to the addition of Mg 2+ as additional counterion in the eluent [START_REF] Folley | Separation of nucleotides by ion-pair, reversed-phase high-performance liquid chromatography: Use of Mg(II) and triethylamine as competing hetaerons in the separation of adenine and guanine nucleotides[END_REF]. The protocol provides the baseline resolution of an ADPR peak, not only in standards but in extracts from granulocytes as well. A similar HPLC method, not known to us at the start of our study, was recently reported [START_REF] Gasser | Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC[END_REF] and used to analyze ADPR concentrations in Jurkat cells [START_REF] Gasser | Activation of T cell calcium influx by the second messenger ADP-ribose[END_REF]. In our protocol, the preparation of the samples from PCA extracts involves fewer and less time-consuming steps; the almost complete recovery rate indicates that no ADPR is lost during the preparation. The experiments with nucleotide pyrophosphatase provide further proof for the specificity of the ADPR peak, even though other nucleotides are certainly substrates for this enzyme.

From the ADPR contents measured in the cell extracts, a range of likely ADPR concentrations may be deduced under consideration of several uncertainties. A relatively safe assumption is the cell size of 350 fl, meaning that the average intracellular concentration is about 5 µM. However, the distribution space of ADPR within the cell is largely unknown. Thus, the cytosolic concentration facing TRPM2 could be several times larger. Fortunately, our data indicate that no matter whether the actual ADPR concentration may be 2 or 30 µM, the relevance of ADPR remains the same. This is because over this concentration range (and beyond), the concentration-effect curve of ADPR on TRPM2 currents reveals a uniform requirement for Ca 2+ of ADPR-dependent TRPM2 gating (see Fig. 6). In the absence of Ca 2+ , ADPR induced a few channel openings but no sizeable whole-cell currents. However, almost maximal TRPM2 currents were evoked in the presence of 1 µM Ca 2+ when ADPR was infused at concentrations as low as 1 µM. Further experiments demonstrate the essential role of ADPR for TRPM2 currents. Infusion of an ADPR-free solution prohibited any currents. This is explained by a rapid dilution of endogenous ADPR when the cell is dialyzed with the pipet fluid in the whole-cell configuration [START_REF] Pusch | Rates of diffusional exchange between small cells and a measuring patch pipette[END_REF]. In this context, it is noteworthy that a pipet solution with 0.1 µM ADPR was without effect on TRPM2 even in the presence of 1 µM Ca 2+ , demonstrating that dilution down to and below this level occurs in patch-clamp experiments without ADPR.

We did not obtain a full concentration-effect curve for Ca 2+ but confined the analysis to two concentrations, one extremely small and the other one in the range of peak intracellular concentrations after stimulation with fMLP. In between, accurate adjustment of Ca 2+ concentrations with EGTA as chelator is problematic due to the equilibrium constant of EGTA/Ca 2+ . Attempts to buffer Ca 2+ in the concentration range of resting or mildly stimulated cells may result in severe errors deriving from small inaccuracies of the calculated EGTA and CaCl 2 amounts, aggravated by small calcium contaminations of other salts and not completely quantifiable effects of other ions. Moreover, the buffering is weak in this Ca 2+ concentration range. It is expected that other Ca 2+ concentrations may exert intermediate effects in comparison to those observed with the two extreme concentrations but an exact quantification cannot be provided at present.

On the basis of our measurements of physiological ADPR levels, the cooperativeness of Copyright 2006 Biochemical Society cytosolic Ca 2+ concentration due to release of Ca 2+ from intracellular stores [START_REF] Krause | The calcium signal and neutrophil activation[END_REF], we propose that ADPR in neutrophil granulocytes renders TRPM2 a Ca 2+ dependent Ca 2+ entry pathway that is activated in consequence of a PI response. In this model, there is no need for a stimulated ADPR formation because Ca 2+ acts at basal ADPR concentrations. These basal concentrations, however, are essential because Ca 2+ has no effect on TRPM2 by its own. Ca 2+ influx through TRPM2 is a selfenhancing process, in line with the fact that fMLP leads to an irreversible activation of neutrophil granulocytes. Taken together, ADPR and Ca 2+ in concert mediate the effects of extracellular ligands like fMLP. Thus, ADPR and Ca 2+ as pair behave like a second messenger system, exhibiting relevant increases of the one player (Ca 2+ ) that becomes effective by the other one (ADPR).

These considerations on the ADPR / Ca 2+ messenger system do not include the possibility of ADPR concentration increases confined to small areas around the cell membrane and, especially, TRPM2 channels. One prominent enzyme that could be responsible for the formation of ADPR in neutrophil granulocytes is CD38 that exhibits the paradoxical property of being an ectoenzyme [START_REF] De Flora | Topology of CD38[END_REF].

At present, one can only hypothesize whether CD38 in the plasma membrane has a role as source of intracellular ADPR, which would necessitate the presence of a transport of NAD (the substrate) to the outside and of ADPR (the product) to the inside [START_REF] Schuber | Structure and enzymology of ADP-ribosyl cyclases: conserved enzymes that produce multiple calcium mobilizing metabolites[END_REF]. However, if such transport systems exist, spatially increased ADPR concentrations would appear likely. The experimental demonstration of localized ADPR peaks would enlarge our view on the second messenger role of the ADPR / Ca 2+ system but would probably not dramatically diminish the importance of Ca 2+ because ADPR even at concentrations considerably higher than the average physiological ones is far more effective in the presence than in the absence of Ca 2+ .

Other intracellular compounds may contribute to the Ca 2+ -modulated activation of TRPM2 by ADPR. Most notably, cADPR and NAD may act as enhancers and AMP as inhibitor [START_REF] Kolisek | Cyclic ADP-ribose and hydrogen peroxide synergize with ADP-ribose in the activation of TRPM2 channels[END_REF]. However, the required concentrations of cADPR were quite high. Kolisek et al used up to 1 mM cADPR, when the compound stimulated TRPM2 by its own, whereas a potentiation of ADPR-induced currents was reported by 10 µM cADPR. Already our HPLC analysis of cell extracts allows to exclude intracellular concentrations of 10 µM. This was confirmed with the cycling assay that revealed a concentration range of about 0.2 µM. Our patch-clamp experiments in granulocytes with 10 µM cADPR were complicated by ADPR contamination of the commercially available cADPR. After enzymatic removal of ADPR, no effect of cADPR could be confirmed, not even in the presence of sub-threshold ADPR concentrations.

Stimulation with fMLP did not lead to an increase in intracellular cADPR concentrations;

there was rather a tendency towards a decrease. However, since cADPR levels were at the detection limit of the method, small and possibly spatially limited concentration increases cannot be excluded. Copyright 2006 Biochemical Society Ca 2+ influx in response to fMLP [START_REF] Partida-Sanchez | Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose[END_REF]. These findings would be in line with Ca 2+ release and subsequent Ca 2+ -induced Ca 2+ influx as well as with direct activation of Ca 2+ entry pathways. On the basis of our experiments, a direct effect of cADPR on TRPM2 channels in neutrophil granulocytes appears unlikely.

In conclusion, our study provides evidence for a physiological role of TRPM2 gating by ADPR in neutrophil granulocytes. The role in these cells is different from that proposed for Jurkat cells [START_REF] Gasser | Activation of T cell calcium influx by the second messenger ADP-ribose[END_REF] that display slight increases in their ADPR content in response to concavalin A, interpreted as a second messenger role of ADPR. In granulocytes, the regulatory function of ADPR depends on its cooperation with Ca 2+ . A second messenger role can be attributed only to ADPR and Ca 2+ in concert. In effect, basal levels of ADPR enable activation by Ca 2+ of TRPM2 which will induce further increases in the cytosolic Ca 2+ concentration due to Ca 2+ influx. This mechanism may be associated with the irreversible phase of neutrophil activation. Future studies in other cells may be on the question whether a decrease in ADPR concentrations is used to terminate Ca 2+ influx through TRPM2. (B) In contrast to (A), the pipet solution was prepared from a cADPR stock solution incubated with nucleotide pyrophosphatase (2.7 U/ml, 20 min, 37 °C), resulting in the complete conversion of ADPR to AMP (inset). ADPR was then added to the pipet solution at a sub-threshold concentration (0.1 µM).

The absence of currents under these conditions was confirmed in 3 further experiments.

  http://www.stanford.edu/~cpatton/maxc.html). The weakly Ca 2+ -buffered pipet solution contained 50 µM Cs-EGTA. ADPR was added from a stock solution to final concentrations of 0.1 to 100 µM. cADPR was added from a freshly prepared stock solution to final nominal concentrations of either 10, 0.3 or 0.1 µM. Neutrophils were held at -60 mV and current-voltage (I-V) relations were obtained Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183

  ADPR and Ca 2+ , and the previous well-established findings that fMLP induced increases in the Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183

Figure 1

 1 Figure 1 Chromatograms of samples prepared by PCA extraction from neutrophil granulocytes (A) One extract (black) was spiked with ADPR (13 pmol). ADPR in extracts displayed the same retention time as ADPR in standards (shown in the supplementary Fig. 1 displaying an HPLC analysis of a mixture of several standard nucleotides). The ADPR content in the non-spiked extract (grey chromatogram) was calculated as 7.6 pmol. The inset shows an enlarged section of the chromatogram that contained the ADPR peak. (B) Effects of pyrophosphatase incubation on the ADPR peak in extracts (and in standards, see inset). Note that the AMP peak is considerably enlarged by pyrophosphatase treatment, indicating that ADPR is not an exclusive substrate of the enzyme. The minor differences in the retention time in both panels are due to different columns (although of the same type) and different equilibration times with elution buffer (1 vs. 2 days) in the chromatographies.

Figure 2 ADPR

 2 Figure 2 ADPR content in neutrophil granulocytes at basal conditions or after stimulation with fMLP (A) Chromatograms from two extracts of granulocytes from one donor. Cells were stimulated with fMLP (1 µM for 2 min; black chromatogram) or were not stimulated (control, gray chromatogram).(B) Summary of the fMLP effects on the ADPR content. Data (mean ± SEM) are from six granulocyte preparations from different donors in which 50 % of the cells were stimulated with fMLP and 50 % were used as unstimulated controls.

Figure 3

 3 Figure 3 Whole-cell currents in neutrophil granulocytes induced by low ADPR concentrations and the effects of intracellular Ca 2+ The pipet solution contained ADPR (5 or 1 µM) in the experiments shown in A and B and was free of ADPR in C. The intracellular (pipet) Ca 2+ concentration was low (< 10 nM) in (A) and high (1 µM) in B and C. The holding potential was -60 mV. The Na + in the bath was replaced by the impermeable cation NMDG during time periods indicated by horizontal bars. The enlarged current tracings in the insets of A and B (scale bars 5 pA and 10 s, respectively) show single-channel openings resolved at low whole-cell current amplitudes.

Figure 4

 4 Figure 4 Whole-cell currents in neutrophil granulocytes induced by high ADPR concentrations and the effects of intracellular Ca 2+ The pipet solution contained 30 µM ADPR (A-C) or 100 µM ADPR (D) in the presence of either a low (<10 nM) or high (1 µM) Ca 2+ concentration. The Na + in the bath was replaced by the impermeable cation NMDG during time periods indicated by horizontal bars in A and D. Currentvoltage relations in low Ca 2+ (B) and in high Ca 2+ (C) were recorded with voltage ramps at time points indicated with numbers in A. Note the different scaling of the ordinate in B and C.

Figure 5

 5 Figure 5 Current densities in dependence of the ADPR concentration, in either low or high Ca 2+ Current densities (mean ± SEM) at low Ca 2+ (open circles) were significantly (Mann-Whitney U-test) lower than at high Ca 2+ (closed circles) for each ADPR concentration. The number of experiments is indicated for each experimental condition.

Figure 6

 6 Figure 6 Expanded view of HPLC chromatograms of extracts and a standard around the cADPR peakThe standard (dotted line) contained nominally 16 µM cADPR. The two almost identical chromatograms were from two extracts from cells from one donor. One extract was from cells stimulated with fMLP (1 µM for 2 min; black), the other one from unstimulated cells (grey).

Figure 7

 7 Figure 7 Effect of cADPR on whole-cell currents in neutrophil granulocytes in the presence of 1 µM intracellular Ca 2+ (A) The pipet solution that contained nominally 10 µM cADPR was prepared from a cADPR stock solution considerably contaminated with ADPR, as shown with HPLC (inset). Inward currents developing during infusion of contaminated cADPR were blocked by NMDG present in the bath during time periods indicated by horizontal bars.

Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183

Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183 Copyright 2006 Biochemical Society

This treatment diminished the ADPR contents by about 60 % and strikingly altered several other Biochemical peaks, noticeably those attributable to ATP, ADP, and to further unidentified compounds (data not shown).

Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183

Biochemical Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183

Journal Immediate Publication. Published on 9 May 2006 as manuscript BJ20060183

Such increases could indirectly be relevant for TRPM2 if cADPR induced Ca 2+ release from stores in granulocytes. Interestingly, 8-bromo-cADPR, a membrane permeant antagonist of cADPR, inhibited Biochemical

ACKNOWLEDGEMENTS

We thank Ilinca Ionescu for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Project B5 of SFB542)


Abas H Laftah

Naveen Sharma

Matthew J Brookes

Andrew T Mckie

Tariq H Iqbal Robert J Simpson

Dr Chris Tselepis
email: c.tselepis@bham.ac.uk

Tumour necrosis factor alpha causes hypoferraemia and reduced intestinal iron absorption in mice

Keywords: Iron, Tumour necrosis factor alpha, anaemia, murine, Ireg1, ferritin Abbreviations-ACD-Anaemia of chronic disease, DMT1-divalent metal transporter

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The anaemia of chronic disease (ACD) or anaemia of chronic inflammation, as it is also commonly referred to, has long perplexed clinicians. [START_REF] Locke | The copper and nonhemoglobinous iron contents of the blood serum in disease[END_REF] It is usually characterised by a normochromic normocytic anaemia with hypoferraemia and hyperferritinaemia in the face of adequate body iron stores. [START_REF] Weiss | Pathogenesis and treatment of anaemia of chronic disease[END_REF] The pathogenesis of ACD is linked to increased circulating levels of pro-inflammatory cytokines found in chronic inflammatory disorders. [START_REF] Means | Pathogenesis of the anemia of chronic disease: a cytokinemediated anemia[END_REF] Cytokines such as tumour necrosis factor alpha (TNF-α), interleukin 1 (IL1), interleukin 6 (IL6) and interferon gamma (INF γ) have a number of diverse actions on the erythropoietic and reticuloendothelial system and have all been implicated in the pathogenesis of ACD. [START_REF] Weiss | Anaemia of chronic disease[END_REF] The role of IL6 in ACD is related to its ability to stimulate hepcidin expression causing hypoferraemia by iron sequestration within the reticuloendothelial system and reducing intestinal iron absorption. [START_REF] Andrews | Anemia of inflammation: the cytokine-hecidin link[END_REF] The hepatic antimicrobial peptide hepcidin has previously been measured in increased concentrations in the urine of patients with ACD secondary to sepsis. [START_REF] Nemeth | Hepcidin, a putative mediator of anemia of inflammation, is a type II acute phase protein[END_REF][START_REF] Kemna | Novel urine hepcidin assay by mass spectrometry[END_REF] Nemeth et al demonstrated that media from endotoxin (LPS) stimulated macrophages induced hepcidin production from cultured human hepatocytes, an effect which was reversed by the addition of IL6 neutralising antibodies. [START_REF] Nemeth | IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin[END_REF] Both in this study and a further study by Kemna et al [START_REF] Kemna | Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS[END_REF] infusion of IL6 or its induction by LPS infusion led to increased hepcidin levels and resultant hypoferraemia in healthy human subjects.

Interestingly, neither study demonstrated an induction in hepatic hepcidin expression by TNF-α.

There is considerable evidence in the literature supporting a role for TNF-α in the anaemia of both chronic inflammation and malignancy. [START_REF] Spivak | The anaemia of cancer: death by a thousand cuts[END_REF][START_REF] Bertero | Anemia of chronic disorders in systemic autoimmune diseases[END_REF] In the context of rheumatoid arthritis anti-TNF-α therapy has been shown to improve haemoglobin concentrations in affected patients compared to controls. [START_REF] Davis | Anaemia of chronic disease in rheumatoid arthritis: in vivo effects of tumour necrosis factor α blockade[END_REF] In another study, bone marrow aspirates from rheumatoid arthritis patients had low levels of haematopoietic progenitor CD34 cells and increased rates of erythroid cell apoptosis. [START_REF] Papadaki | Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumor necrosis factor-α antibody treatment[END_REF] These effects were reversed by in-vitro treatment with anti-TNF-α antibodies. In two murine studies TNF-α administration caused hypoferraemia. [START_REF] Tanaka | Recombinant human tumor necrosis factor depresses serum iron in mice[END_REF][START_REF] Alvarez-Hernandez | Induction of hypoferremia and modulation of macrophage iron metabolism by tumor necrosis factor[END_REF] In the latter work this was attributed to increased iron sequestration within macrophages. A potential mechanism for macrophage iron sequestration was proposed by Ludewicz et al [START_REF] Ludwiczek | Cytokine-mediated regulation of iron transport in human monocytic cells[END_REF] who demonstrated that TNF-α stimulation upregulated the cellular iron import protein divalent metal transporter 1 (DMT1) and reduced the iron exporter iron regulated protein 1 (Ireg1) in a human monocyte cell line.

The majority of work studying the effects of pro-inflammatory cytokines in ACD has focused on the erythropoietic and reticuloendothelial systems. The direct effects of cytokines on small bowel iron absorption have been less well studied. Over the past decade the key proteins involved in inorganic iron trafficking across the small bowel epithelium have been identified [START_REF] Sharma | The emerging role of the liver in iron metabolism[END_REF]. Briefly ferric iron is thought to be reduced to the ferrous form by duodenal cytochrome b (dcytb) at the brush border and is imported into the enterocyte by DMT1. Once in the cell iron may be stored as ferritin or exported across the basolateral membrane by Ireg1 which works in conjunction with the copperlinked oxidase hephaestin generating ferric iron which is transported in the plasma largely bound to transferrin.

In a previous study we demonstrated TNF-α produced rapid changes in the expression and localisation of DMT1, Ireg1 and ferritin in both in-vitro and ex-vivo enterocyte model systems. [START_REF] Sharma | A role for tumour necrosis factor alpha in human small bowel iron transport[END_REF] This ultimately led to iron sequestration within enterocytes and a reduction of iron export across a Caco-2 cell monolayer. In a similar study, Johnson et al [START_REF] Johnson | Tumour necrosis factor alpha regulates iron transport and transporter expression in human intestinal epithelial cells[END_REF] demonstrated reduced DMT1 expression in Caco-2 cells following 72 hours of TNF-α exposure. Interestingly, a previous murine study had shown that small intestinal intra-epithelial lymphocytes produced TNF-α in response to dietary iron, an effect which appeared important in preventing hepatic iron overload. [START_REF] Ten Elshof | Gamma delta intraepithelial lymphocytes drive tumor necrosis factor-alpha responsiveness to intestinal iron challenge: relevance to hemochromatosis[END_REF] This led us to propose that local intestinal responsiveness to TNF-α could provide enterocyte targets allowing reduced iron absorption in the face of systemic TNF-α excess in chronic inflammation. The in-vitro effects observed were hepcidin independent. We therefore wished to extend this study to see if the same effects could be observed in a whole organism.

In this current study, utilising wild type mice, we investigated the effects of TNF-α on small intestinal iron transporter expression and localisation as well as iron absorption. In addition we have studied the effects of TNF-α on iron levels in serum, spleen and liver with concomitant hepcidin expression.

Materials and Methods

6 week old CD1 male mice (weights 28-32g) (Charles Rivers, Margate, Kent), were subject to intra peritoneal injection with either 0.15M NaCl (control) or TNF-α (10ng/mouse) in 0.15M NaCl. At 3 or 24 hrs post injection mice were anaesthetised and sacrificed by cervical dislocation. Duodenum, liver, spleen and serum were then collected. Serum iron analysis was performed at Birmingham City Hospital, Dept of Biochemistry, whilst small bowel, spleen and liver were divided equally into three portions for: i) RNA extraction, ii) protein extraction iii) immunohistochemistry. All animal experiments were performed under the authority of a UK Home Office licence.

Mice were fed CRM diet (Scientific Diet Supplies [SDS], Witham, Essex, United Kingdom).

Real Time PCR

RNA was extracted from small bowel, liver and spleen specimens using Trizol reagent with 1µg of RNA subject to reverse transcription utilising a reverse transcription kit (Promega). cDNA was then subject to Real Time PCR as described previously [START_REF] Tselepis | Upregulation of the oncogene cmyc in Barrett's adenocarcinoma: induction of c-myc by acidified bile acid in vitro[END_REF].

Briefly, all reactions were performed using 18S ribosomal RNA as an internal standard (PE Biosystems, Roche, USA). Each reaction was performed in triplicate and contained one of the following sets of probes and primers; i) DMT1 (Probe 5' FAM CTG CAT TCT GCC TTA GTC AAG TCT AGA CAG CTA AAC TAMRA 3'), Forward Primer

Western Blotting

Whole Duodenum was cut longitudinally and the epithelium removed using a glass slide. The extract was then processed for Western blotting as previously described [START_REF] Tselepis | Upregulation of the oncogene cmyc in Barrett's adenocarcinoma: induction of c-myc by acidified bile acid in vitro[END_REF] with monoclonal antibodies against either DMT1 (5µg/ml, NRAMP24-A ADI, USA), Ireg1 (5µg/ml MTP11-A ADI, USA), ferritin (1:500 ab16875, Abcam, UK), TFR1

(1:500 A11130, Zymed, USA) or Cytokeratin 19 (1:2,000 IF-15, Oncogene Research Products USA), the last for normalisation of epithelial protein loading. Immunoreactive bands were then subject to densitometry using software NIH Image 1.62. Prior to reprobing with the different antibodies the membranes were incubated in stripping buffer containing 2% SDS, 100 mM mercaptoethanol in 62.5 mM Tris-HCL pH 6.8 for 30 min at 70 °C.

Immunocytochemistry

7µm sections of small bowel were processed for immunohistochemistry as previously described. Briefly, sections were dewaxed and then incubated in 0•1% Tween 20 containing 1mM EDTA pH 8•0 for 16 hrs at 65 0 C. Sections were then incubated for 1 hr with either DMT1 (10µg/ml, NRAMP24-A ADI, USA), Ireg1 (10 µg/ml MTP11-A ADI, USA) or ferritin (1:500 ab16875, Abcam, UK). Immunoreactivity was detected utilising the avidin-biotinylated secondary antibody method (DAKO ABC kit, DAKO, USA) and visualised with DAB, (diaminobenzidine) reagent. Sections were counterstained with haemotoxylin. Omission of primary antibody was employed as a negative control. Stained sections were scored independently by three observers (SH, CT and TI) with regards cellular localisation. Images were visualised from paraffin sections using a Nikon Eclipse E600 microscope and digital image taken using a Nikon DXM1200F camera (Surrey, UK). Nikon ACT-1 version 2.62 software was used for image acquisition (Surrey, UK).

Perl's staining

Paraffin sections were dewaxed and incubated in 1% HCl containing 1% ferrous cyanate for 20 minutes. Sections were washed and counterstained with neutral red prior to visualisation. Images were visualised from paraffin sections using a Nikon Eclipse E600 microscope and digital image taken using a Nikon DXM1200F camera (Surrey, UK). Nikon ACT-1 version 2.62 software was used for image acquisition (Surrey, UK).

Radiolabeled iron uptake assay

The length of the duodenum was tied off at both ends and the duodenal segment prewashed with 0.15M NaCl at 37 o C. The segment was then injected with 250µM FeCl 3 (Fe:nitrilotriacetate, 1:2) containing 5µCi of radiolabelled iron ([ 59 Fe]Cl 3, specific activity 185 GBq/g, PerkinElmer, UK) in physiological media (125mM NaCl, 3.5mM KCl, 1mM CaCl 2 , 10mM MgSO 4 , 10mM D-glucose in 16mM HEPES-NaOH buffer, pH 7.4). The chelator nitrilotriacetate was chosen as it has previously been used in iron absorption studies and has been shown to form relatively stable complexes with iron. [START_REF] Laftah | Effect of hepcidin on intestinal iron absorption in mice[END_REF][START_REF] Bates | The kinetics and mechanism of iron (3) exchange between chelates and transferrin. I. The complexes of citrate and nitrilotriacetic acid[END_REF] Following a 10 minute incubation, the duodenal segment was removed and the remaining carcass was subject to γ ray counting using a high-resolution bulk sample counter. The count measured was termed mucosal transfer.

Statistics

All experimental errors are shown as 2 standard errors of the mean. Statistical significance was calculated by use of the unpaired Student's t test using SPSS version 10.0 (SPSS Inc, USA). Significance was accepted at P<.05.

Results

Determination of serum iron levels in control and TNFα treated mice

Analysis of serum iron levels in both 3 hr and 24 hr TNF-α treated mice demonstrated a significant decrease in serum iron compared to control mice P<0.05 (Figure 1).

TNF-α mediated repression of iron transfer across small bowel mucosa

No significant change in mucosal iron transfer was observed after 3hr stimulation with TNF-α. (Figure 2). However, a longer exposure to TNF-α (24hrs) did significantly reduce mucosal transfer of iron compared to control mice (p<0.05).

Iron transporter mRNA expression following TNF-α exposure

Consistent with our previous studies Real Time PCR analysis revealed a significant increase in DMT1, Ireg1 and ferritin mRNA in 3 hr TNF-α treated mice, which, with the exception of ferritin, had returned to baseline level by 24 hrs (Figure 3A-C).

As hepcidin release has been shown to be a key mediator of the systemic response of iron metabolism to inflammation we examined whether hepcidin was also modulated by TNF-α. Livers and spleens from these mice were removed, RNA extracted and Real Time PCR performed (Figure 4). In both 3 hr and 24 hr TNF-α treated mice there was no significant modulation in hepatic hepcidin mRNA levels (Figure 4A). In contrast splenic hepcidin levels appeared to be significantly repressed following 24 hour TNF-α exposure (p<0.05) (Figure 4B).

Iron transporter protein expression following TNF-α exposure

To determine whether these transcriptional changes were mirrored at the protein level western blotting was performed. In keeping with the mRNA data, at 3 hours there was a significant increase in DMT1 and Ireg1 protein expression although these had returned to baseline by 24 hours (Figure 5). There was a comparative delay in ferritin expression with protein levels only becoming significant at 24 hours. There was no difference in the protein levels of TFR1 in mouse enterocytes at either time point eliminating this as a potential route of iron ingress into these cells.

Immunolocalisation of iron transporter proteins

Immunohistochemical studies furthermore showed alterations in the localisation of these proteins (Figure 6). Ferritin in control mice was localised predominantly in the apical portion of the villous enterocytes whilst in the treated animals immunoreactivity throughout the enterocyte was apparent. No obvious difference was observed in the crypts where ferritin immunoreactivity was weak and diffuse. The transporter DMT1 was localised as anticipated to the apical border of the villi in both control and TNF-α treated mice and in all mice only very weak patchy cytoplasmic staining in the crypts was observed.

In control mice there was evidence of both apical and basal cytoplasmic staining of Ireg1 in the villous enterocytes with the latter lost following TNF-α treatment. In the crypts of control mice Ireg1 was predominantly preserved on the basolateral borders.

This was almost completely lost with only diffuse cytoplasmic immunoreactivity evident 24 hours after TNF-α treatment.

Perl's staining of small bowel, spleen and liver

To examine whether these changes in expression and localisation of ferritin, DMT1 and Ireg1 lead to an increase in enterocyte iron loading Perl's staining was performed on duodenum from both control and TNF-α treated mice (Figure 7). In the villi of control and 3hr TNF-α treated mice there was no discernable staining, however in the 24 hr TNF-α treated mice Prussian blue staining was clearly observed in the apical poles of the villous enterocytes. In none of the mice was there any evidence of crypt Prussian blue staining.

To determine whether iron loading was confined to the small bowel we chose to examine the two major tissue stores of iron, the spleen and liver (Figure 7). In the spleen there was evidence of iron loading in both the 3hr and 24 hr TNF-α treated mice whilst there was no evidence of iron loading in the liver at either time point.

Discussion

In a previous investigation using a Caco-2 cell model we demonstrated TNF-α modulates iron transport ultimately leading to inhibition of iron export from enterocytes. [START_REF] Sharma | A role for tumour necrosis factor alpha in human small bowel iron transport[END_REF] These effects were local and hepcidin-independent. Our findings added to the evidence for a physiological role for TNF-α in small bowel iron absorption and also suggested a potential mechanism for the inhibition of iron absorption in the context of systemic inflammation. [START_REF] Ten Elshof | Gamma delta intraepithelial lymphocytes drive tumor necrosis factor-alpha responsiveness to intestinal iron challenge: relevance to hemochromatosis[END_REF] To further clarify the effects of TNF-α on iron regulation a murine model was utilised in the current investigation. In addition to assessing the effect of TNF-α on iron transporter expression and localisation in enterocytes we examined the simultaneous effects on liver and spleen.

In the present study we have been able to demonstrate a rapid fall in serum iron 3 hours after TNF-α injection which was co-incident with an increase in splenic iron loading. It is likely this effect was caused by increased retention of iron by monocytes in response to TNF-α, as previously alluded to by others. [START_REF] Alvarez-Hernandez | Induction of hypoferremia and modulation of macrophage iron metabolism by tumor necrosis factor[END_REF][START_REF] Ludwiczek | Cytokine-mediated regulation of iron transport in human monocytic cells[END_REF] As expected there was little or no parenchymal hepatic iron deposition in response to TNF-α.

As in our earlier in-vitro study [START_REF] Sharma | A role for tumour necrosis factor alpha in human small bowel iron transport[END_REF] an early induction in both duodenal DMT1 and Ireg1 was followed by later fall to baseline levels along with a late up-regulation of ferritin. The induction of ferritin by TNF-α is a recognised phenomenon and is likely to be an adaptive immune response to bacteraemia. [START_REF] Torti | Regulation of ferritin genes and protein[END_REF] However, the relevance of the early induction of DMT1 and Ireg1 is less obvious. Others have shown a similar increase in DMT1 expression in bronchial epithelial cells in response to TNF-α, IFN-γ and LPS. [START_REF] Wang | TNF, IFN-gamma, and endotoxin increase expression of DMT1 in bronchial epithelial cells[END_REF] This increase in DMT1 expression was also paralleled by increased ferritin expression. The authors of that study speculated that the increase in DMT1 expression may be a local effect to "de-toxify" bronchial mucosa by reducing the access of potential micro-organisms to iron in the respiratory tract. Whereas the early induction of iron importing proteins at the intestinal brush border seen in our study may seem counter intuitive, it remains possible that this early response is a similar attempt at local detoxification of small bowel luminal contents. Of note, despite the early up-regulation Copyright 2006 Biochemical Society of both DMT1 and Ireg1 message and protein no significant change in mucosal transfer of iron was seen at 3 hours in TNF-α treated compared to control mice. This implies that in enterocytes at least, the early induction in DMT1 and Ireg1 protein is non-functional and is supported by the observation that there was no discernible enterocyte iron staining at 3 hours.

The mechanisms underlying the observed effects of TNF-α on iron transporter expression are unclear. Interestingly, the 5' untranslated region of both ferritin and Ireg1 genes reveal putative binding sites for the transcription factors NF-κB and Sp1.

Previous work has already implicated Sp1 in the activation of ferritin expression. [START_REF] Tsuji | Activation of the ferritin H enhancer, FER-1, by the cooperative action of members of the AP1 and Sp1 transcription factor families[END_REF] Recently two studies in fibroblasts and monocytic cell lines respectively, have demonstrated a co-operative effect of Sp1 and NF-κB in the TNF-α dependent transcription of monocyte chemo-attractant protein 1. [START_REF] Boekhoudt | Communication between NF-kappa B and Sp1 controls histone acetylation within the proximal promoter of the monocyte chemoattractant protein 1 gene[END_REF]28] It is interesting to speculate whether a similar mechanism could be at play in our study. In contrast, the DMT1 promoter region lacks a NF-κB binding domain but does contain a putative IFNγ response element. TNF-α has previously been shown to stimulate IFN-γ production in a number of cell types [29] and it is possible that the effects observed on DMT1 expression may be mediated by secondary IFN-γ release. It is also possible that the increase in DMT1 and Ireg1 mRNA may be secondary to IRP mediated post transcriptional stabilisation. However, IRP mediated stabilisation would normally lead to reciprocal changes in DMT1 and Ireg1 expression.

In the current study at 24 hours expression levels of DMT1 and Ireg1 returned to baseline, whilst ferritin remained significantly induced. As in our previous in-vitro observations the ultimate effect of TNF-α was to cause enterocyte iron loading and inhibition of iron absorption, which was independent of hepatic hepcidin expression.

We propose these effects are secondary to the induction of intracellular ferritin and the post-translational re-localisation of Ireg1 away from its basolateral compartment. [30] has demonstrated that hepcidin also exerts its major effect by binding to Ireg1 and causing re-localisation and loss of function of this iron exporter. It is intriguing, therefore, that although TNF-α and hepcidin appear to act independently in our model their ultimate targets and effects are likely to be similar. Interestingly their study also revealed that the transcriptional control of Ireg1 in splenic macrophages was IL6 and hepcidin independent and that hepcidin was also synthesised in the spleen.

Recently work by Nemeth et al

The relevance of splenic hepcidin production is unknown. In our study we have demonstrated that TNF-α causes a significant repression in splenic hepcidin mRNA expression at 24 hours. It appears, therefore, that TNF-α is able to cause hypoferraemia independent of both hepatic and splenic hepcidin expression.

In summary we provide evidence that TNF-α can potentially cause ACD by the inhibition of small bowel iron absorption in addition to its documented effects on erythropoeisis and the reticuloendolethial system. This appears to be independent of hepcidin but involves effects on enterocyte ferritin expression and Ireg1 localisation.

The anaemia of chronic disease is an important clinical problem with significant prognostic implications. To date, there have been few direct clinical strategies to treat this problem. It has been proposed that hepcidin antagonists may be developed in the future therapy for this condition. [START_REF] Weiss | Anaemia of chronic disease[END_REF] In addition to this, TNF-α antagonists which are in widespread clinical use in conditions such as Crohn's disease and rheumatoid arthritis present a possible future therapy for the anaemia of chronic disease.

TNFα stimulation

At 3 hours there is significant increased expression of DMT1 and Ireg1 protein (P<0.05). This is not maintained for 24 hours. At 24 hours there is an increase in ferritin levels (p<0.05). There was no change seen in TFR1 expression. Cytokeratin 19 (CK19) was employed to ensure normalisation of epithelial loading. Ferritin in control mice was localised predominantly to the apical pole of the villous enterocytes whilst in the TNF-α treated mice immunoreactivity was observed throughout the cytoplasm of all villous enterocytes. Ferritin immunoreactivity was weak and diffuse in all crypts of both control and TNF-α treated mice. DMT1 was localised to the apical border of the villi in both control and TNF-α treated mice as anticipated. However, in all mice only very weak patchy cytoplasmic staining was observed in the crypts.

Ireg1 was observed in both apical and basal cytoplasmic compartments in control mice whilst in TNF-α treated mice there was mostly apical cytoplasmic staining with little evidence of basal immunoreactivity. In the crypts of control mice Ireg1 was preserved on the basolateral borders. This basolateral immunoreactivity was almost completely lost in the TNF-α treated mice with only diffuse cytoplasmic immunoreactivity evident.

(Original magnification x 60).

( 5 '

 5 -AGC TGT CAT CAT GCC ACA CAA C -3') and Reverse Primer (5'-GCT TCT CGA ACT TCC TGC TTA TTG 3'), ii) Ireg1 (Probe 5' FAM AGG ATT GAC CAG TTA ACC AAC ATC TTA GCC CC TAMRA 3'), Forward Primer (5'-AGC AAA TAT GAA TGC CAC AAT ACG-3') and Reverse Primer (5'-CAA ATG TCA TAA TCT GGC CAA CAG-3'), iii) ferritin (Probe 5'FAM CCA ACG AGG TGG CCG AAT CTT CCT T TAMRA 3'), Forward Primer (5'-GGA ACA TGC TGA GAA ACT GAT GAA-3') and Reverse Primer (5'-CAT CAC AGT CTG GTT TCT TGA TAT CC-3'), or iv) hepcidin (HEPC1) (Probe 5'-FAM CCT GAG CAG CAC CAC CTA TCT CCA TCA-3'), Forward Primer (5'-CTG TCT CCT GCT TCT CCT CCT T-3') and Reverse Primer (5'-CTG CAG CTC TGT AGT CTG TCT CAT C-3') in the presence of 1X Mastermix (PE Biosystems), 50 nM 18S 5' and 3' primers and 200 nM 18S probe (5' VIC, 3'-TAMRA labelled) and 0.25 µl of cDNA (equivalent to 12.5ng of reverse transcribed RNA) in a 25 µl reaction. Reactions without cDNA were included as negative controls. Gene expression was normalised to the 18S and represented as ∆Ct values. For each sample the mean of the three ∆Ct values was calculated. Comparison of gene expression between control and treated samples was derived from subtraction of control ∆Ct values from treatment ∆Ct values to give a ∆∆Ct value and relative gene expression was calculated as 2 -∆∆Ct . Relative gene expression is normalised to 1.0 (100%) of controls. Each experiment was performed in triplicate.

Biochemical

  Journal Immediate Publication. Published on 27 Mar 2006 as manuscript BJ20060215 Copyright 2006 Biochemical Society Liu et al [31] have shown that LPS causes a post-translational down-regulation of Ireg1 in murine splenic macrophages, which is IL6 and hepatic hepcidin dependent.

28

  Ping D, Boekhoudt, G. Zhang, F. Morris, A. Philipsen, S. Warren, S.T. and Boss, J.M. (2000) Sp1 binding is critical for promoter assembly and activation of the MCP-1 gene by tumor necrosis factor. J. Biol. Chem. 275, 1708-1714 29 Nagano, Y. (1977) Endotoxin induction of interferon. Tex. Rep. Biol. Med. 35, 105-110 30 Nemeth, E. Tuttle, M.S. Powelson, J. Vaughn, M.B. Donovan, A. Ward, D.M. Ganz, T. and Kaplan, J. (2004) Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 306, 2090-2093 31 Liu, X.B. Nguyen, N.B. Marquess, K.D. Yang, F. and Haile, D.J. (2005) Regulation of hepcidin and ferroportin expression by lipopolysaccharide in splenic macrophages. Blood Cells Mol. Dis. 35, 47-56

Figure 1 TNFFigure 2

 12 Figure 1 TNF-α causes a decrease in serum iron levels 6 week old male CD1 mice were subject to intra peritoneal injection with either 0.15M saline (control, n=6) or recombinant murine TNF-α (10 ng/mouse, n=6). At 3 or 24 hrs post intra peritoneal injection blood was collected and serum iron levels determined. * (p<0.05). Error bars= 2 standard errors of the mean

Figure 3

 3 Figure 3 Modulation of DMT1, Ireg1, ferritin and hepcidin mRNA levels by TNFα.6 week old CD1 male mice were injected either with 0.15M saline (Control) or 10ng/mouse of TNF-α. Mice were either sacrificed at 3 hrs or 24hrs post injection and

Figure 4

 4 Figure 4 Modulation of hepcidin mRNA levels by TNF-α. 6 week old CD1 male mice were injected either with 0.15M saline (Control) or 10ng/mouse of TNF-α. Mice were either sacrificed at 3 hrs or 24hrs post injection and hepatic (A) and splenic (B) mRNA levels of hepcidin were determined (n=6 for each time point). *(p<0.05). Error bars= 2 standard errors of the mean.

Figure 5

 5 Figure 5 Protein expression of DMT1, Ireg1, ferritin and TFR1 in response to

Figure 6

 6 Figure 6 Immunolocalisation of ferritin, DMT1 and Ireg1 in control and TNF-α challenged mice. Control (n=6) and 24hr TNF-α treated mice (n=6) were sacrificed and whole duodenum processed into paraffin blocks, sectioned and subject to immunohistochemistry with antibodies to DMT1 (NRAMP24A), Ireg1 (MTP11-A) and ferritin (ab16875).

Figure 7 Enterocyte

 7 Figure 7 Enterocyte Iron loading in TNF-α treated (24 hr) mice Control mice showed no evidence of Prussian blue staining in duodenum, spleen or liver. In both 3hr and 24hr TNF-α treated mice an abundance of Prussian staining was observed in both duodenal enterocytes and the spleen. However, no hepatic iron loading was observed in TNF-α treated mice. (Original Magnification x40).

Figure 1 Figure 2 Figure 3

 123 Figure 1

Figure 6 Figure 7 Biochemical

 67 Figure 6

Biochemical Journal Immediate Publication. Published on 27 Mar 2006 as manuscript BJ20060215

Acknowledgements

-This work was supported by grants from the BBSRC (BBS/B/10781) and Sandwell and West Birmingham NHS Trust R&D fund.