Identification of complement 5a-like receptor (C5L2) from astrocytes: characterization of anti-inflammatory properties

Journal of Neurochemistry, 2005, 92, 1140–1149

doi:10.1111/j.1471-4159.2004.02942.x

Identification of complement 5a-like receptor (C5L2) from astrocytes: characterization of anti-inflammatory properties Vitaliy Gavrilyuk,* Sergey Kalinin,* Brian S. Hilbush,  Andrew Middlecamp,  Susan McGuire,à Dale Pelligrino,* Guy Weinberg*,§ and Douglas L. Feinstein*,§ *Department of Anesthesiology, University of Illinois, Chicago, Illinois, USA  Digital Gene Technologies, Inc., La Jolla, California, USA àDepartment of Pathology, Loyola University Chicago, Maywood, Illinois, USA §VA Chicago Health Care System, Research & Development, Chicago, Illinois, USA

Abstract Brain inflammation is regulated by endogenous substances, including neurotransmitters such as noradrenaline (NA), which can increase anti-inflammatory genes. To identify NA-regulated, anti-inflammatory genes, we used TOGA (total gene expression analysis) to screen rat astrocyte-derived RNA. NA-inducible cDNA clone DST11 encodes an isoform of the complement C5a receptor (C5aR), with 39% identity at the amino acid level to the rat C5aR, and 56% identity to a recently described human C5aR variant termed C5L2 (complement 5a-like receptor). Quantitative PCR confirmed that in astrocytes, DST11 mRNA expression is increased by NA, whereas in vivo depletion of cortical NA reduced DST11 levels. Western blot analysis demonstrated basal and NA-induced expression of DST11 as a 45 kDa protein in primary

astrocytes cultures. Immunocytochemical staining of adult rat brain revealed DST11-immunoreactivity throughout brain, colocalized to neurons and astrocytes. In astrocytes, induction of nitric oxide synthase type 2 was increased by treatment with antisense oligonucleotides to DST11. Reducing DST11 expression also increased nuclear factor jB reporter gene, and decreased cAMP response element reporter gene activation. These results demonstrate that DST11 is a C5aR isoform expressed by glia and neurons, which is regulated by NA, and exerts anti-inflammatory functions. Changes in DST11 levels in diseased brain could therefore contribute to the progression of inflammatory damage. Keywords: Alzheimer’s disease, gene expression, Locus ceruleus, mRNA, nitric oxide, noradrenaline. J. Neurochem. (2005) 92, 1140–1149.

Accumulating evidence indicates that inflammatory mediators contribute to cell damage and death in neurological diseases including multiple sclerosis (MS), Alzheimer’s disease (AD), and Parkinson’s disease. Previous studies (Feinstein et al. 1993; Galea et al. 2003) have shown that the endogenous neurotransmitter noradrenaline (NA) reduces glial activation in vitro (Frohman et al. 1988; Feinstein 1998), suggesting that NA might serve as an endogenous modulator of brain inflammatory responses (Galea and Feinstein 1999). If so, then disturbances in NA concentration or signaling, as occurs in AD due to loss of noradrenergic locus ceruleus (LC) neurons (Mann 1983) or in MS (due to reduced levels of astrocytic b-adrenergic receptors) (De Keyser et al. 1999; Zeinstra et al. 2000) might contribute to increased, or sustained inflammation and damage. We confirmed the former possibility by showing that chemical lesion of the LC in rats increased the cortical inflammatory responses to aggregated amyloid beta (Heneka et al. 2002).

The molecular basis for the anti-inflammatory effects of NA is not well known. Although an increase in intracellular cAMP levels can activate inhibitory actions of transcription factors such as CCAAT-enhancer binding protein b or cAMP response

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Received September 16, 2004; revised manuscript received October 20, 2004; accepted October 22, 2004. Address correspondence and reprint requests to Vitaliy Gavrilyuk, Department of Anesthesiology, University of Illinois, 1819 West Polk Street, MC519, Chicago, IL, 60612, USA. E-mail: [email protected] Abbreviations used: AD, Alzheimer’s disease; C5aR, complement C5a receptor; C5L2, complement 5a-like receptor; CRE, cAMP response element; EAE, experimental autoimmune encephalomyelitis; EST, expressed sequence tag; GDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; IFN-c, interferon-c; IjB, inhibitor of NFjB; LC, locus ceruleus; MS, multiple sclerosis; NA, noradrenaline; NeuN, neuron-specific nuclear protein; NFjB, nuclear factor jB; NOS2, nitric oxide synthase type 2; QPCR, quantitative PCR; TOGA, total gene expression analysis; UTR, untranslated region.


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Identification of C5L2 from astrocytes 1141

element binding protein (CREB), it is likely that modification of expression patterns plays a major role. For example, the suppression of nitric oxide synthase type 2 (NOS2) expression by NA in vitro involves reduction in nuclear factor jB (NFjB)-dependent NOS2-promoter transcription (Galea and Feinstein 1999), most likely due to an NA-dependent increase in inhibitory IjB (inhibitor of NF-jB) expression (Gavrilyuk et al. 2002). In vivo, NA depletion reduces heat shock protein 70 levels (Heneka et al. 2003), a protein that can reduce inflammatory induced damage. NA also increases peroxisome proliferator activated receptor gamma (PPARc) expression (Klotz et al. 2003), which could increase anti-inflammatory effects of any endogenous PPARc ligands (Feinstein 2003). It is therefore likely that NA increases expression of other as of yet unidentified anti-inflammatory proteins or receptors. To help identify genes that could contribute to the antiinflammatory effects of NA in brain, we used an automated cDNA display technology (TOtal Gene expression Analysis, TOGA) (Sutcliffe et al. 2000). Of the several candidate genes identified, clone DST11 was found to encode an isoform of the complement 5a receptor (C5aR), with strongest similarity to a recently described human variant termed C5L2 (Ohno et al. 2000). Complement anaphylatoxin receptors in brain have been the subject of recent interest (Osaka et al. 1999; Mukherjee and Pasinetti 2000; Van Beek et al. 2000; O’Barr et al. 2001; Barnum et al. 2002; Jauneau et al. 2003), in view of the fact that complement activation may play a role in the progression of neurodegenerative diseases, including AD (O’Barr and Cooper 2000; Mukherjee and Pasinetti 2000) and MS (Muller-Ladner et al. 1996; Nataf et al. 1998; Reiman et al. 2002). Our data demonstrate that DST11 is regulated by NA in vitro and in vivo, and that DST11 normally restricts inflammation, consistent with a role as an anti-inflammatory effector. Down regulation of DST11 expression could contribute to increased inflammatory damage in neurological conditions.

Materials and methods Reagents Cell culture reagents [Dulbecco’s modified Eagle’s medium, antibiotics and lipopolysaccharide (LPS, Salmonella typhimurium) were from Sigma (St. Louis, MO, USA). Fetal calf serum was from Atlanta Biological (Norcross, GA, USA). Human recombinant interleukin-1b (4 · 106 U/mg) was obtained from the NIH AIDs reagents program. Recombinant rat interferon-c (IFN-c: 4 · 106 U/ mg) and synthetic oligonucleotides were from Gibco (Gaithersburg, MD, USA). Taq polymerase and cDNA reagents were from Promega (Madison, WI, USA) and Gibco. RNA analysis Primary cultures of astrocytes were prepared from neonatal Sprague Dawley rats. These cultures are greater than 98% astrocytes as

assessed by staining for the astrocyte marker glial fibrillary acidic protein (GFAP); and less than 2% microglial determined by staining with the microglial marker ED1. When confluent, the media was replaced with Dulbecco’s modified Eagle’s medium containing 1% fetal calf serum (control) and either bacterial endotoxin LPS (1 lg/mL) and IFN-c (20 U/mL recombinant rat, ‘LI’), LI plus NA (100 lM), or NA alone. After 4 h incubation, total cytosolic RNA was prepared from the cells by repeated phenol : chloroform extractions and isopropanol precipitation. The four RNA groups were analyzed by TOGA as described (Sutcliffe et al. 2000). The DNA sequence of rat DST11 has GenBank Accession no. AY741660. Real-time polymerase chain reaction Total cytoplasmic RNA from cell or tissues was prepared from cells using TRIZOL reagent, according to manufacturer’s procedures (Invitrogen/Gibco); aliquots were converted to cDNA using random hexamer primers, and mRNA levels were estimated by quantitative touchdown PCR (QPCR). The primers used for QPCR were: 5¢-TTCACACTCCCCGCTCCTTG-3¢ and 5¢-CGGCACTTTCCTCTTCATCCTGTA-3¢ (DST11); 5¢-CTGCATGGAACAGTATAAGGCAAAC-3¢ and 5¢-GAGACAGTTTCTGGTCGATGTCATGA-3¢ (NOS2); and 5¢-GCCAGGTATGATGACATCAAGAAG-3¢ and 5¢-TCCAGGGGTTTCTTACTCCTTGGA-3¢ (glyceraldehyde-3phospate dehydrogenase). PCR conditions were denaturation at 94C for 10 s; annealing at 58–63C for 15 s; and extension at 72C for 20 s; followed by 2 min at 72C in a Corbett Rotorgene RealTime PCR unit (Corbett, Australia). The PCR mastermix contained SYBR Green (1 lL diluted 1 : 10 000 SybrGreen1 10 000 · concentrate, Molecular Probes, Eugene, OR, USA). Relative mRNA concentrations were calculated from the takeoff point (Ct) of reactions using manufacturer’s software. Melting curve analysis and agarose gel electrophoresis were performed to ensure production of single and correct sized products. Transfections Cells (astrocytes, C6 cells) were transfected with antisense or control oligonucleotides, or full length DST11 expression plasmids using lipofectamine (Gibco/Invitrogen) as described (Gavrilyuk et al. 2001) and according to manufacturer’s recommendations. For stable transfections, cells were co-transfected with pSV2Neo vector (Clontech, Palo Alto, CA, USA) and stable transfectants selected with 0.5 mg/mL of antibiotic G418. C6 cells stably transfected with the luciferase reporter gene in plasmid pGL2-basic (Promega) under control of four tandem copies of the NFjB or the cAMP response element (CRE) transcription elements were prepared as previously described (Gavrilyuk et al. 2002). Phosphorothiolated oligonucleotides were obtained from Invitrogen, and were: 5¢-TTCAGCATCCTGGCCCC-3¢ [antisense DST11 (aS-DST11)]; and 5¢-TGCCAACCCTATTCTCCTTCTT-3¢ or 5¢-TCCACAACCTTCGCTTCAAA-3¢ (controls). Luciferase measurements C6 reporter cell lines (C6-NFjB and C6-CRE) were lysed by addition of CHAPS buffer, broken open by freeze–thawing, and then shaken on a rotary shaker for 15 min at room temperature (at 22C). Aliquots (10–20 lL) containing equal amounts of protein (5–20 lg) were transferred to a white 96-well microplate, an equal


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volume of luciferase substrate (Steady Glo reagent, Promega) was added, and the luminescence measured in a microplate luminometer (Rosys-Anthos, Hombrechtikon, Switzerland). Nitric oxide synthase type 2 induction and nitrite measurements Astrocyte NOS2 expression was induced by incubating cells in Dulbecco’s modified Eagle’s medium containing 1% fetal calf serum and 1 lg/mL LPS, except for the initial studies done to isolate RNA for TOGA in which recombinant rat IFN-c (20 U/mL) was also added to provide a robust inflammatory stimulus. NOS2 expression in C6 cells, which do not respond to LPS alone (Feinstein et al. 1994), was induced by incubating cells with LPS (1 lg/mL) together with 20 U/mL of recombinant rat IFN-c. For both cell types, nitrite accumulation in the cell culture media (an index of NO production from NOS2) was determined by mixing 80 lL of culture medium with ½ volume of Griess reagent as described (Gavrilyuk et al. 2001), and measuring absorbance at 540 nm. Antibody production An anti-DST11 antibody (D11-1) was prepared by Biosource Inc. (Hopkinton, MA, USA). Briefly, a 15-residue peptide LRDLQDEEESAVTKV, derived from near the C-terminus of the DST11 coding region, was synthesized, initiated with an N-terminal acetlyated-cysteine, HPLC purified, conjugated with key hole limpet carrier, and used to immunize rabbits. Titers of anti-DST11 antibodies were measured in blood samples from immunized rabbits after each booster immunization. After 3 months, animals were killed, serum collected, and antibody affinity purified. The selectivity of the antibody DST11-1 was confirmed by enzyme-linked inmmunosorbent assay and dot blotting using the immunogen peptide (data not shown). Western blotting Cells or tissue samples were lysed in 8 M urea or CHAPS buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5% CHAPS) with protease inhibitors (Sigma) and sonicated. Aliquots were mixed with sample buffer, boiled, and an aliquot used for measurement of protein concentration using the Bradford reagent and bovine serum albumin as standard. Equal amounts of protein (between 10 and 20 lg of each sample) were loaded into wells and separated through 10% polyacrylamide sodium dodecyl sulfate gels, and transferred to polyvinylidene difluoride membranes by semi-dry electrophoresis. Coomassie blue staining of gels after transfer and Colloidal Gold Total Protein Stain (Bio-Rad, Hercules, CA, USA) staining of membranes after transfer confirmed equal transfer of samples. Membranes were blocked with Tris-buffered saline containing 5% dry milk, rinsed with Tris-buffered saline, then incubated with primary antibodies in Tris-buffered saline plus Tween (0.05%) and bovine serum albumin (0.2%) overnight at 4C. The primary antibody was removed, membranes washed, and peroxidase-labeled goat secondary antibodies added for 2 h. Following further washes with Tris-buffered saline plus Tween, bands were visualized with ECL Western Blotting Analysis System (Amersham, Pierce, Rockford, IL, USA). Gels or X-ray films were scanned with Alpha Imager system (Alpha Innotech, Inc., San Leandro, CA, USA) and optical density of corresponding bands was determined with ImageJ software from NIH (http://rsb.info.nih.gov/ij/).

Immunocytochemistry Brains were fast frozen in isopentane at )20C, then 10-lm sections prepared, mounted to slides (Colorfrost/Plus Fisherbrand), and fixed in 80% ethanol at )20C for 10 min. Double-labeling was done to co-localize DST11 with GFAP and NeuN (neuron-specific nuclear protein). Tissues were washed with phosphate-buffered saline, blocked with bovine serum albumin and a mixture of two antibodies were applied overnight at 4C (rabbit polyclonal DST111, 1 : 200 dilution; together with mouse monoclonal anti-GFAP (Chemicon, diluted 1 : 500) or mouse monoclonal anti-NeuN (Chemicon, diluted 1 : 500). After washing, sections were incubated for 2 h with a mixture of two fluorescent conjugated secondary antibodies [fluorescein anti-rabbit (Vector, Burlingame, CA, USA) together with Alexa594 conjugated anti-rat or anti-mouse] at 1 : 200 dilutions. Slides were washed with phosphate-buffered saline and covered using Vectashield mounting medium (Vector). Stained tissues were examined with a Zeiss Axiophot 2 microscope, equipped with a Hamamatsu C5810 camera. Co-localization was confirmed by confocal microscopy using a Carl Zeiss LSM510 equipped with a 63 · water immersion objective. The 488 nm and 568 nm beams from an argon-krypton laser were used for excitation and the emission from FITC, Alexa594 was detected through a LP505 and LP585 filter, respectively. The two different fluorochromes were scanned sequentially by using multitracking function to avoid bleedthrough among different fluorescent dyes.

Results

Total cytosolic RNA (Fig. 1a) was prepared from primary rat astrocytes that had been treated for 4 h with a robust inflammatory stimulus (LPS plus IFN-c, ‘LI’), NA (100 lM) alone, LI plus NA, or untreated (none). RT-PCR (Fig. 1b) confirmed that LI increased NOS2 mRNA levels, which were decreased by co-treatment with NA. Incubation with NA alone caused a small increase in NOS2 mRNA expression. In the same samples, levels of GDH mRNA (Fig. 1c) were not significantly modified by LI and/or NA. QPCR showed LI increased NOS2 mRNA 10-fold, which was reduced approximately 50% by the presence of NA (Fig. 1d). These RNAs were processed in parallel for TOGA analysis, and TOGA Portal software was used to identify mRNAs whose expression was increased at least twofold by treatment with NA. Of the 16 700 mRNAs identified, 230 (1.4%) of the genes were detected to be induced at least twofold by NA. From that group we removed any genes that were also induced by LI, or by LI and NA together. This left nine genes that were specifically increased by NA. Of those, sequence analysis of clone DST11 revealed similarity to members of the G-protein coupled receptor family, and we therefore selected this clone for further analysis. BLAST analysis identified several rat expressed sequence tags (ESTs) identical to DST11. The complete insert of the longest EST (Accession no. BI293718) together with the data of the original DST11 insert provided 1321 bp, consisting of 134 bp of- 5¢-UTR, 1034 bp of coding region and 150 bp of


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RNA NOS2 GDH NOS2 MRNA level

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10 5 0

none

LI

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NA

Stimulus Fig. 1 Anti-inflammatory effect of noradrenaline (NA) in astrocytes. Analysis of nitric oxide synthase type 2 (NOS2) and glutamate dehydrogenase (GDH) mRNA levels in astrocytes treated 4 h with lipopolysaccharide (1 lg/mL) plus interferon-c (20 U/mL) (LI); noradrenaline 100 lM, NA; LI plus NA; or media only (none). (a) Nondenaturing gel electrophoresis of 1.0 lg of total cytosolic RNA used for total gene expression analysis. (b) RT-PCR for NOS2; (c) RT-PCR for GDH. The gels shown are representative of results from more than 10 similar studies. (d) Quantitative PCR analysis shows a 10-fold increase in relative NOS2 mRNA levels following 4 h incubation with LI, which was reduced approximately 40% (to sixfold increase) by the presence of NA. The data is mean ± SEM of results from three different RNA samples in each group, and is the relative NOS2 mRNA level normalized to values obtained for GDH mRNA levels.

3¢-UTR. Database searching revealed that the DST11 protein (344 residues) is a member of the C5aR family, having 34% identity and 51% similarity to rat C5aR (Accession no. P97520) (Fig. 2). The DST11 protein has high identity (86%) with a mouse protein (NM_176912), and both the mouse and rat proteins are similar to the recently identified human C5L2 cDNA (56% identity, and 69% similarity), a paralog of C5aR

(Ohno et al. 2000). The proteins encoded by DST11 and by mouse NM_176912 are also paralogs of C5aR. Despite only moderate similarity to human C5L2, DST11 (as well as mouse NM_176912) are likely the rodent homologs, as they share several features with other members of the C5L2 family that distinguish this family from other complement receptors, including: (i) lack of a DRY motif at residues 137– 139; (ii) lack of NPXXY sequence at residues 293–297; (ii) a short third intracellular loop lacking key polar residues; and (iv) preservation of residues key for C5a binding (Cain and Monk 2002). The regulation of DST11 expression in primary astrocytes was confirmed by real time QPCR (Fig. 3a). DST11 mRNA was detected at low levels in unstimulated primary rat astrocytes, was not significantly altered by incubation with LPS, but was increased between two and threefold upon incubation with NA (100 lM for 4 h). Incubation of astrocytes with LPS plus IFN-c did not lead to any further induction compared to LPS alone (data not shown). Interestingly, co-incubation with LPS together with NA did not lead to an increase in DST11 mRNA. DST11 mRNA was expressed at basal levels in most tissues examined (Fig. 3b), with highest levels observed in heart and spleen, and lowest levels in intestine. This distribution is similar to that described for mouse C5L2 (Okinaga et al. 2003), although it differs slightly from that of human C5L2, which showed relatively higher expression in kidney and liver, and lower expression in lung (Okinaga et al. 2003). DST11 protein expression was examined using a polyclonal antibody (D11-1) raised against a C-terminal peptide of the predicted protein (see Fig. 2 for location). D11-1 sera, but not pre-immune sera, strongly stained primary astrocytes (Fig. 4a). D-11 recognized a protein band of approximately 45 kDa in whole cell lysates from primary astrocytes (Fig. 4b), which was increased about twofold after 4 h treatment with NA (Fig. 4c). As the case for the mRNA,

Fig. 2 Amino acid lineup of DST11 with mouse and human counterparts. Rat DST11 and mouse C5L2 (Accession no. NM_176912) are 56% identical to human C5L2 (Accession no. AB038237). Mouse C5L2 is 86% identical to rat DST11. Asterisks indicate the location of the peptide sequence used for antibody D11-1 generation. Lineups were made using ClustalW 1.8 multiple sequence alignment software available at http://www.searchlauncher. bcm.tmc.edu


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Fig. 3 DST11 mRNA expression in astrocytes and tissues (a) Steady state DST11 mRNA levels were determined by quantitative PCR (QPCR) in primary astrocytes treated 4 h with lipopolysaccharide (1 lg/mL) (L); noradrenaline (NA, 100 lM); L plus NA; or media only (none). Data are the relative expression levels compared to control, and are mean ± SD values from three experiments. (b) DST11 mRNA levels were determined by QPCR in indicated tissues from adult rat. CTX, frontal cortex; CB, cerebellum; Hrt, heart; Spl, spleen; Liv, liver; Mus, smooth muscle; Int, small intestine. Data are mean ± SD of three experiments.

stimulation with LPS did not increase the DST11 protein; however, the DST11 protein was increased by the combination of LPS plus NA. The basis for the discrepancy between the effects of LPS and LPS/NA on DST11 mRNA vs. protein levels is not yet understood, but suggests that these inflammatory modulators can influence DST11 protein synthesis and/or stability as well as mRNA levels. In normal adult rat the D11-1 antibody revealed immunoreactive cells in most brain regions examined (Fig. 5a), including frontal cortex, hippocampus, and cerebellum. In the cortex, D11-1 staining co-localized with NeuN and some GFAP stained cells. D11-1 also co-localized with NeuN staining in the hippocampus and cerebellum, and in these regions co-localization with GFAP positive staining cell body and radial glial fibers was also observed. Colocalization to white matter astrocytes, to cerebellar radial glial fibers, and to cortical neurons was confirmed by confocal microscopy (Fig. 5b). To determine if NA regulated DST11 expression in vivo, we treated adult rats with the neurotoxin DSP4, which

Fig. 4 DST11 protein expression in primary astrocyte cultures (a) Primary astrocytes were stained with antibody D-11 or with preimmune sera. (b) Whole cell lysates were prepared from primary astrocytes treated for 4 h with lipopolysaccharide (1 lg/mL) (L), lipopolysaccharide plus 25 lM noradrenaline (L/NA); NA alone, or control (C), and analyzed by western blot analysis using rabbit polyclonal antiDST11 antibody D11-1, immunostaining for two samples for each treatment group. (c) Densitometric analysis (mean ± SD of three experiments) of band intensities assessed by ImageJ software.

selectively destroys LC noradrenergic neurons and depletes cortical NA levels (Heneka et al. 2002). Following DSP4 treatment, a neural inflammatory response was initiated by peripheral injection of LPS. DST11 mRNA was detected in frontal cortex of control animals (non-DSP4-treated) and in contrast to in vitro studies in astrocytes, its expression was increased (65 ± 8% vs. control) following peripheral injection of LPS (Fig. 6). This may be due to up-regulation in other cell types (microglia or neurons). DSP4-treatment reduced basal DST11 mRNA expression (to 33 ± 1% of control values). In DSP4-treated animals, LPS injection still increased DST11 mRNA approximately 40% vs. DSP4-treatment alone. The reduction of DST11 mRNA levels by DSP4 treatment (reduced NA) is consistent with the converse in vitro observations that NA increased DST11 mRNA expression. A functional role for DST11 was tested by examining astrocytic responses to treatment with LPS after DST11


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(a)

DST11 / NeuN

DST11 / GFAP

FC

HP

CB

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DST11 / NeuN

DST11 / GFAP

Fig. 5 DST11 protein is expressed in neurons and radial glia in vivo. (a) Adult rat brain sagittal sections were double-labeled with antibody D11-1 together with anti-NeuN (left panels) or anti-GFAP (right panels). FC, frontal cortex; HP, hippocampus; CB, cerebellum. No significant staining was detected when primary antibodies were omitted, or if antibody D-11 was pre-incubated with DST11 peptide (data not shown). (b) Colocalization was confirmed by confocal microscopy using a Zeiss laser-scanning confocal system (Axiover 100M) in multitracking mode to prevent interference of the dyes. Left panel costaining for DST11 (green) and NeuN (red); right panel costaining for DST11 (green) and GFAP (red).

levels were reduced by antisense treatment (Fig. 7a). In nonstimulated astrocytes, low levels of the NOS2 mRNA were detected, and this level was slightly increased (up to twofold) by antisense treatment to DST11. Incubation with LPS strongly increased NOS2 mRNA levels (20–50-fold compared to control cells), and this increase was further potentiated (2.5 ± 0.6-fold) by prior antisense treatment (Fig. 7b). Antisense treatment also led to an increase in LPS-dependent nitrite production from astrocytes (Fig. 7c)

Fig. 6 DST11 expression is decreased following depletion of cortical noradrenaline (NA). Adult rats were treated with the selective neurotoxin DSP4 to lesion the locus ceruleus and deplete cortical NA levels. After 5 weeks, they were given an injection of lipopolysaccharide (LPS, 1 mg/kg; i.p.) or saline to induce an inflammatory response in brain and frontal cortex RNA prepared 1 day later. Quantitative PCR revealed that DSP4 treatment reduced DST11 levels to roughly 33% of control values (**p < 0.0001), whereas LPS increased DST11 mRNA levels in both control (***p < 0.0005) and DSP4-treated animals (*p < 0.005). Data is the fold-increase vs. control samples and is the mean ± SD from n ¼ 3 animals per group.

measured after 24 and 48 h; however, after 72 h this effect was lost. Similar findings were observed in C6 cells, in which antisense treatment to DST11 significantly reduced DST11 protein levels (Fig. 7d) and increased LPS/IFN-c dependent nitrite production (Fig. 7e). Measurements of GDH mRNA levels in the same samples revealed no significant changes due to antisense treatment or incubation with LPS/IFN-c or NA (data not shown). To begin to examine the molecular basis for regulation of NOS2 expression by DST11, we examined its modulation of the activation of an NFjB reporter gene (Fig. 8). In C6-pNFjB cells transfected with a control oligonucleotide (directed against a distinct TOGA cDNA clone), LPS/IFN-c induced activation of the NFjB luciferase reporter gene, whereas prior antisense treatment to DST11 increased the magnitude (about twofold) of that activation. Conversely, overexpression of full length DST11 in C6-pNFjB cells (Fig. 8b) significantly reduced NFjB reporter gene activation. Antisense treatment to DST11 also reduced activation of a CRE-driven reporter gene (Fig. 8c), suggesting that DST11 can modulate intracellular cAMP signaling, which is known to regulate NFjB activation (Galea and Feinstein 1999). Discussion

In this study, TOGA identified clone DST11 as an mRNA whose expression is increased in primary rat astrocytes by NA, and this induction was confirmed by QPCR analysis.


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Fig. 7 DST11 knockdown increases nitric oxide synthase type 2 (NOS2) expression. Primary astrocytes (a–c) or C6 cells (d, e) were transfected with an antisense oligonucleotide to DST11 (‘aS-DST11’) or with a control oligonucleotide (‘Luc’, directed against bacterial luciferase). After 1 day, cells were incubated to induce NOS2 expression [none or 1 lg/mL lipopolysaccharide (LPS) for astrocytes; none or 1 lg/mL LPS plus 20 U/mL interferon-c (IFN-c) for C6 cells]. (a) RT–PCR analysis for DST11 mRNA in primary astrocytes 24 h after activation. The gel shows results for two samples in each group, and similar findings were found in two other experiments. (b) Quantitative PCR analysis of NOS2 mRNA levels in primary astrocytes 24 h after activation. Data is fold-increase vs. control (no LPS; control transfected) values (set to 1). *p < 0.05 (as-DST11 vs. Luc-transfected, unpaired t-test, n ¼ 3 experiments. (c) Control (filled bars) and asDST11-transfected (empty bars) astrocytes were treated with LPS and nitrite levels in the media measured at the indicated time points. Data is mean ± SEM nmoles nitrite produced per well. *p < 0.05; **p < 0.005 control vs. as-DST11 transfected cells, one way ANOVA, Bonferroni post hoc, n ¼ 3. (d) Western blot analysis for DST11 (top gel) and b-tubulin (bottom gel) levels in C6 cell whole cell lysates measured 1 day after transfection with control (Luc) or as-DST11 oligonucleotides. The gels show two lanes for each treatment group. (e) Control (filled bars) and as-DST11-transfected (empty bars) C6 cells were stimulated with LPS and IFN-c, and nitrite accumulation in the media measured after 24 h. Data is mean ± SEM nmoles nitrite produced per well. **p < 0.005 control vs. as-DST11 transfected cells, unpaired t-test, n ¼ 3 experiments.

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DST11 is constitutively and widely expressed in most tissues and brain regions. Immunocytochemical staining detected DST11 throughout the rodent brain in both glia and neurons. The effect of NA on DST11 in vitro was replicated in vivo,

where NA depletion caused a reduction in DST11 levels. Functionally, antisense oligonucleotide and over-expression studies show that DST11 normally plays an anti-inflammatory role, at least in astrocytes. Together, these data suggest an anti-inflammatory role for DST11 in normal brain, and hence that perturbations of brain NA levels could reduce DST11 expression and thus contribute to increased inflammatory responses. Our data show that DST11 codes for a 344 amino acid G-protein coupled receptor (GPCR) member of the complement C5aR family. C5a is a chemoattractant for most myeloid cell types and T and B cells (Barnum 2002). Upon binding to C5aR, C5a causes signal transduction activation and inflammatory responses (Osaka et al. 1999; Mukherjee and Pasinetti 2000; O’Barr and Cooper 2000; Monsinjon et al. 2003). C5aR has been identified on most cell types in brain, including oligodendrocytes, neurons and glia (Sayah et al. 1997; Nataf et al. 1998; Osaka et al. 1999; O’Barr et al. 2001). The role of C5a in brain is slowly being unraveled (see Barnum 2002 for review), and it has been implicated to play a role in AD (Mukherjee and Pasinetti 2000; O’Barr and Cooper 2000), stroke (Van Beek et al. 2000; Barnum et al. 2002), excitotoxicity (Osaka et al. 1999; Mukherjee and Pasinetti 2001), and MS (Nataf et al. 1998; Reiman et al. 2002). The existence of additional C5aR family members with distinct expression patterns and biological functions may help explain the role of C5a in these diseases.


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Fig. 8 DST11 modulates NFjB and CRE promoter activation. C6 glioma cells stably transfected with the nuclear factor jB (NFjB) promoter construct driving the luciferase reporter gene were transfected with (a) antisense oligonucleotide to DST11 (aS) or a control (Ctl) oligonucleotide; or (b) full length DST11 cDNA (DST) or vector alone (Ctl). After 1 day, the cells were stimulated with lipopolysaccharide (1 lg/mL) plus interferon-c (20 U/mL) and luciferase activity measured after 4 h. (c) C6 cells stably transfected with the cAMP response element (CRE) promoter driving the luciferase reporter gene were transfected with antisense to DST11 (aS) or control oligonucleotides (Ctl) and after 1 day stimulated with 25 lM NA to induce CRE activity. Data is luciferase activity relative to control transfected cells, and is the mean ± SD of 3–6 experiments. *p < 0.05 vs. control, unpaired t-test.

DST11 has high similarity to a previously identified mouse cDNA (NM_176912), which was concluded to be the mouse equivalent of the human C5L2 complement receptor (Okinaga et al. 2003). C5L2 was first cloned from human dendritic cells using degenerate PCR primers directed against conserved regions in the second and seventh transmembrane regions of chemoattractant receptors (Ohno et al. 2000). C5L2, called GPR77, was also identified by data base mining for novel G-protein coupled receptors (Lee et al. 2001). C5L2 is a member of the C5a receptor subfamily, with highest similarity to the receptors for C5a (58%) and C3a (55%). The exact function of C5L2 remains unclear; however, is has both similarities and differences to C5aR. Structurally, many GCPRs have a conserved arginine residue present in the DRY motif of the third transmembrane region that is important for G protein activation. In C5L2, this arginine is replaced with a leucine residue, suggesting that C5L2 may not couple strongly to G proteins (Okinaga et al. 2003). C5L2, like C5aR, binds the C5a peptide with high affinity, but unlike C5aR it has significantly higher affinity (10–50-fold) for the less potent ligand des-arginated C5a (C5adR) (Cain and Monk 2002; Kalant et al. 2003). In addition, C5aR, but not C5L2 activation increases intracellular Ca2+ and causes degranulation, and C5L2 is not internalized (Cain and Monk 2002). These properties, and the fact that cells from C5aR-deficient mice (which express C5L2) do not show inflammatory responses to C5a led to the suggestion that the main function of C5L2 is to modulate C5aR function by scavenging C5a (Okinaga et al. 2003). However, observations that C5L2 binds the desarginated forms of C3a and C4a with high affinity suggests other functions for C5L2 (Kalant et al. 2003). C3a des-Arg, also

known as acylation-stimulating protein (ASP) stimulates triglyceride synthesis in cells expressing C5L2, suggesting that C5L2 may mediate the effects of this peptide. In contrast, C5a and C5a des-Agr, which appear to bind at a site on C5L2 distinct from C3 des-Arg, do not stimulate triglyceride synthesis (Kalant et al. 2003). Our in vitro data show that antisense treatment of astrocytes to reduce DST11 levels increased NOS2 induction, whereas overexpression in C6 cells reduced NOS2 expression. These findings are consistent with results using an NFjB reporter gene, whose activation was increased by reducing DST11 expression, and reduced by increasing expression. Previous studies from our laboratory and others have shown that increasing intracellular cAMP reduces glial proinflammatory gene expression (Galea and Feinstein 1999). It has been long known that C5aR activation is coupled to inhibitory Gi protein activation (Vanek et al. 1994; Shum et al. 1995), and a recent study in primary astrocytes showed that binding of C5a to C5aR on astrocytes reduced cAMP levels (Sayah et al. 2003). This raises the possibility that the effects of modifying DST11 expression on cAMP signaling are due in part to scavenging C5a (or some other ligand) away from C5aR. Consistent with this, antisense treatment to reduce DST11 levels led to a reduction in CRE-dependent promoter activation, reflecting lower cAMP levels. The demonstration that DST11 can regulate NFjB as well as CRE-dependent gene expression, both key factors in modulating overall patterns of inflammatory gene expression, points to an important role for this receptor in inflammation. Interestingly, our results using antisense treatment were done in the absence of exogenously added ligands (such as C5a), and therefore suggest that astrocytes may normally produce an endogenous C5aR or DST11 ligand. While the identity of such a ligand needs to be determined, astrocytes have been shown to produce complement components including C5 (Gasque et al. 1993) and there is evidence that some cell types (including macrophages) can generate C5a from C5 using a mechanism distinct from the complement cascade (Huber-Lang et al. 2002). Finally, although published data suggests that DST11 (or C5L2) is not constitutively active, and does not couple functionally to G-proteins (Okinaga et al. 2003), we propose that DST11 acts primarily to scavenge C5a (or other endogenous ligand) away from existing C5aR (Fig. 9). Our data show that DST11 is increased by NA in vitro, and conversely is decreased in adult rat brain when cortical NA levels are reduced. In both cases, constitutive DST11 expression was observed, indicating further regulation by NA. Our in vivo data suggests that the normally present levels of cortical NA maintain DST11 expression, thus contributing to an ‘anti-inflammatory’ environment. Similarly, in vitro in the absence of exogenously added NA, DST11 levels are low, and the cells are receptive to inflammatory stimuli. NA


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1148 V. Gavrilyuk et al.

Fig. 9 Schematic to illustrate anti-inflammatory actions of DST11. Binding of LPS or of proinflammatory cytokines (such as IL-1b) to their cognate receptors (CD14/Tlr4; IL-1bR, respectively) initiates a phosphorylation cascade ultimately leading to degradation of inhibitory IjB proteins and activation of NFjB and NOS2 gene expression. This activation pathway has previously been demonstrated to be suppressed by elevation of intracellular cAMP (by binding of NA to b-AR, or of other ligands to G-protein coupled receptors activating Gs). Concurrently, we hypothesize that glial activation induces, or increases, endogenous release of a C5aR ligand (C5a), which upon binding to C5aR reduces intracellular cAMP levels and therefore potentiates NFjB activation and NOS2 expression. DST11, normally present in cells but at low levels, is increased by NA (or other cAMP elevating agents). Binding of C5a to DST11 reduces C5aR activation, increases cAMP levels, and thereby reduces NFjB activation and NOS2 induction. b-AR, b-adrenoceptor; C5aR, complement C5a receptor; GPCRs, G-protein coupled receptors; IjB, inhibitor of NFjB; IL-1b, interleukin-1b; IL-1bR, interleukin-1b receptor; LPS, lipopolysaccharide; NO, nitric oxide; NOS2, nitric oxide synthase type 2; Tlr4, toll-like receptor 4.

addition increases DST11 levels (and other anti-inflammatory genes) and thereby increases the overall ‘anti-inflammatory’, or ‘refractory’ state of the cells. However, further characterization of DST11 regulation by NA in neurons and microglia is required before any strong conclusions regarding in vivo patterns of expression can be made. Our results are consistent with previous reports showing that cAMP can increase C5aR family members (Burg et al. 1996; Rubin et al. 1996). We have previously shown that the inflammatory response in brain to LPS or to aggregated amyloid beta is increased following lesion of the LC and accompanying reduction in cortical NA levels (Heneka et al. 2002), and that this increase is due, in part, to LC-induced loss of inhibitory proteins including IjBa and IjBb (Heneka et al. 2003). The current findings suggest that changes in other potential ‘antiinflammatory’ proteins also occur following LC loss, which could exacerbate inflammatory responses. Loss of an antiinflammatory complement receptor such as DST11 may therefore further exacerbate the consequences of increased C3aR and C5aR levels reported to occur in AD (Strohmeyer et al. 2000). A role for complement, and in particular C3a and C5a, in demyelinating disease has also been proposed (MullerLadner et al. 1996; see Barnum 2002 for review). Nataf et al. (1998) first showed that C5aR is increased in brain

during the course of experimental autoimmune encephalomyelitis (EAE), the animal model for MS, suggesting a role for this receptor, and its ligand, in demyelinating diseases. Recent studies using C5aR deficient mice, however, showed no difference in disease onset or severity of actively induced EAE (Reiman et al. 2002), although deletion of C3aR reduced clinical symptoms (Boos, Wetsal, Barnum, unpublished observations). Although we have not yet examined DST11/C5L2 expression in EAE or MS tissues, evidence that NA signaling is disturbed in these diseases (Zeinstra et al. 2000; Galea et al. 2003), suggests that changes in DST11 may contribute to inflammatory damage in demyelinating disease. Acknowledgements We wish to thank Anthony Sharp, Rick Ripper, and Patricia Murphy for technical assistance, and Cinzia Dello Russo and Jose Munoz for help with cell culture studies. We thank Peter Monk for advise. This work was supported in part by grants from NINDS (NS44945-01), the VA Health Care Division, and the American Alzheimer’s Association.

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