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Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34*

Open AccessPublished:November 19, 2010DOI:https://doi.org/10.1074/jbc.M110.196659
      The X-chromosomal GPR34 gene encodes an orphan Gi protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.

      Introduction

      G protein-coupled receptors (GPCR)
      The abbreviations used are: GPCR
      G protein-coupled receptor
      DTH
      delayed type hypersensitivity
      hiCap
      heat-inactivated acapsular C. neoformans serotype D strain CAP67
      lyso-PS
      lyso-phosphatidylserine
      P-lyso-PS
      1-palmitoyl-lyso-phosphatidylserine
      qPCR
      quantitative real time PCR analysis
      S-lyso-PS
      1-stearyl-lyso-phosphatidylserine
      TM
      transmembrane region
      mBSA
      methylated BSA.
      form the largest gene family among transmembrane receptors, including more than 900 genes in humans and other mammals (
      • Fredriksson R.
      • Schiöth H.B.
      ). A great number of stimuli, such as light, hormones, neurotransmitters, peptides, and nucleotides, activate the distinct receptors. Nonodorant receptors form about one-third of the GPCR repertoire. Although more than 200 non-odorant GPCR have been assigned to specific agonists and functions, about 155 so-called “orphan” GPCR (
      • Wigglesworth M.J.
      • Wolfe L.A.
      • Wise A.
      ) await identification of their physiological relevance. The importance of GPCR in controlling almost every physiological function makes this receptor family the most frequently used target for therapeutic drugs. Therefore, unveiling the function of orphan GPCR is a central issue in academic and industrial research.
      Among the five structurally different GPCR families (
      • Fredriksson R.
      • Schiöth H.B.
      ,
      • Fredriksson R.
      • Lagerström M.C.
      • Lundin L.G.
      • Schiöth H.B.
      ), the rhodopsin-like receptors form the largest in humans and other vertebrates. The rhodopsin-like family is divided further into subfamilies and groups. The P2Y12-like receptor group includes the ADP receptors P2Y12 and P2Y13, the UDP-glucose receptor P2Y14, and the orphan receptors GPR87, GPR82, and GPR34 (
      • Schöneberg T.
      • Hermsdorf T.
      • Engemaier E.
      • Engel K.
      • Liebscher I.
      • Thor D.
      • Zierau K.
      • Römpler H.
      • Schulz A.
      ). Apart from the ADP receptor P2Y12, which has a central role in platelet aggregation and is the therapeutic target of clopidogrel (
      • Foster C.J.
      • Prosser D.M.
      • Agans J.M.
      • Zhai Y.
      • Smith M.D.
      • Lachowicz J.E.
      • Zhang F.L.
      • Gustafson E.
      • Monsma Jr., F.J.
      • Wiekowski M.T.
      • Abbondanzo S.J.
      • Cook D.N.
      • Bayne M.L.
      • Lira S.A.
      • Chintala M.S.
      ,
      • Hollopeter G.
      • Jantzen H.M.
      • Vincent D.
      • Li G.
      • England L.
      • Ramakrishnan V.
      • Yang R.B.
      • Nurden P.
      • Nurden A.
      • Julius D.
      • Conley P.B.
      ), very little is known about the function of the other members of this group.
      GPR34, an orphan receptor of the P2Y12-like receptor group, was first discovered by mining GenBankTM for novel GPCR sequences and homology cloning and has been assigned to the human X chromosome (
      • Marchese A.
      • Sawzdargo M.
      • Nguyen T.
      • Cheng R.
      • Heng H.H.
      • Nowak T.
      • Im D.S.
      • Lynch K.R.
      • George S.R.
      • O'dowd B.F.
      ,
      • Schöneberg T.
      • Schulz A.
      • Grosse R.
      • Schade R.
      • Henklein P.
      • Schultz G.
      • Gudermann T.
      ). Phylogenetic studies revealed that GPR34 has been highly conserved over the past 450 million years of vertebrate evolution, and no GPR34-deficient vertebrate has been identified yet (
      • Schulz A.
      • Schöneberg T.
      ). To date, there is no report of GPR34 deficiency in humans, and sequencing of more than 100 worldwide samples of human genomic DNA revealed no functionally relevant alleles indicating the physiological importance of the gene (
      • Engemaier E.
      • Römpler H.
      • Schöneberg T.
      • Schulz A.
      ). GPR34 was, however, included in a microdeletion and breakpoints at the Xp11.4 locus in a Turner syndrome patient (
      • Boucher C.A.
      • Sargent C.A.
      • Ogata T.
      • Affara N.A.
      ) and mucosa-associated lymphoid tissue lymphoma (

      Novak, A., Akasaka, T., Manske, M., Gupta, M., Witzig, T., Dyer, M. J. S., Dogan, A., Remstein, E., Ansell, S., (2008) 50th Annual Meeting and Exposition, December 6–8, 2008, American Society of Hematology, Poster 2251, San Francisco.

      ,
      • Wlodarska I.
      • Tousseyn T.
      • De Leval L.
      • Ferreiro J.
      • Urbankova H.
      • Michaux L.
      • Dierickx D.
      • Wolter P.
      • Vandenberghe P.
      • Marynen P.
      • De Wolf-Peeters C.
      • Baens M.
      ).
      GPR34 shows a ubiquitous expression pattern in murine and human tissues (
      • Schöneberg T.
      • Schulz A.
      • Grosse R.
      • Schade R.
      • Henklein P.
      • Schultz G.
      • Gudermann T.
      ). More detailed analyses showed GPR34 expression in the myeloid progenitor cell line HL-60 in K562 cells, and WEHI-3B cells, the macrophage cell line RAW 264.1 (
      • Engemaier E.
      • Römpler H.
      • Schöneberg T.
      • Schulz A.
      ), and in the murine mast cell line P815. These findings suggest a granulocytic/monocytic expression pattern that is consistent with the ubiquitous expression pattern seen in tissues.
      Recently, several members of the P2Y12-like receptor group have been assigned to agonists, including nucleotide derivates and lipids (
      • Tabata K.
      • Baba K.
      • Shiraishi A.
      • Ito M.
      • Fujita N.
      ,
      • Nonaka Y.
      • Hiramoto T.
      • Fujita N.
      ,
      • Sugo T.
      • Tachimoto H.
      • Chikatsu T.
      • Murakami Y.
      • Kikukawa Y.
      • Sato S.
      • Kikuchi K.
      • Nagi T.
      • Harada M.
      • Ogi K.
      • Ebisawa M.
      • Mori M.
      ). Specifically, GPR34 was shown to be activated by lyso-phosphatidylserine (lyso-PS) in vitro. lyso-PS is generated by hydrolysis of membrane lipids through phospholipases A1 and A2 when apoptotic cells expose phosphatidylserine on their surface to these phospholipases (
      • Aoki J.
      • Nagai Y.
      • Hosono H.
      • Inoue K.
      • Arai H.
      ,
      • Park K.S.
      • Lee H.Y.
      • Kim M.K.
      • Shin E.H.
      • Jo S.H.
      • Kim S.D.
      • Im D.S.
      • Bae Y.S.
      ). lyso-PS is a potent activator of histamine release from mast cells (
      • Bellini F.
      • Viola G.
      • Menegus A.M.
      • Toffano G.
      • Bruni A.
      ). Furthermore, lyso-PS has been described as a growth inhibitor of T cells and as a chemotactic substance for fibroblasts and tumor cells (
      • Park K.S.
      • Lee H.Y.
      • Kim M.K.
      • Shin E.H.
      • Jo S.H.
      • Kim S.D.
      • Im D.S.
      • Bae Y.S.
      ,
      • Bellini F.
      • Viola G.
      • Menegus A.M.
      • Toffano G.
      • Bruni A.
      ,
      • Park K.S.
      • Lee H.Y.
      • Kim M.K.
      • Shin E.H.
      • Bae Y.S.
      ,
      • Lee S.Y.
      • Lee H.Y.
      • Kim S.D.
      • Jo S.H.
      • Shim J.W.
      • Lee H.J.
      • Yun J.
      • Bae Y.S.
      ). These findings suggest an involvement of GPR34 in cellular chemotaxis and immune response, but proof of this concept has yet to be obtained.
      We generated and characterized a GPR34-deficient (KO) mouse model with specific focus on immunological functions. We found no evidence that lyso-PS is a natural agonist of the murine and human GPR34. KO mice showed no major alterations in a wide range of tests. However, GPR34 deficiency leads to improper immune response upon antigen and pathogen challenge.

      EXPERIMENTAL PROCEDURES

      Materials

      If not stated otherwise, all standard substances were purchased from Sigma, Merck, and C. Roth GmbH (Karlsruhe, Germany). Cell culture material was obtained from Sarstedt (Nürnbrecht, Germany), and primers were purchased from Invitrogen. Primer sequences are provided in supplemental Table S1. For expression of GPR34 orthologs in yeast, the p416GPD vector (
      • Mumberg D.
      • Müller R.
      • Funk M.
      ) (kindly provided by Dr. Mark Pausch, Wyeth Research, Princeton, NJ) was used, and mammalian expression was performed using the pcDps vector (
      • Okayama H.
      • Berg P.
      ). Restriction enzymes were purchased from New England Biolabs (Frankfurt/Main, Germany). “Brain”-lyso-PS whose main component is stearyl-lyso-PS (S-lyso-PS) was obtained from Avanti Polar Lipids (Alabaster, AL).

      Methods

      Preparation and Purification of P-lyso-PS

      Because the P-lyso-PS used by Sugo et al. (
      • Sugo T.
      • Tachimoto H.
      • Chikatsu T.
      • Murakami Y.
      • Kikukawa Y.
      • Sato S.
      • Kikuchi K.
      • Nagi T.
      • Harada M.
      • Ogi K.
      • Ebisawa M.
      • Mori M.
      ) was no longer commercially available at Sigma or any other company, P-lyso-PS was synthesized by hydrolysis of 2-dipalmitoyl-sn-glycero-3-phospho-l-serine with phospholipase A2. P-lyso-PS was purified from the reaction mixture by extraction and thin layer chromatography (for further details see supplemental material).

      Cell Culture, Transfection, and Functional Assays

      The haploid Saccharomyces cerevisiae yeast strain MPY578t5 (provided by Dr. Mark Pausch) was used for the expression of GPR34 orthologs. Cells were transformed with plasmid DNA using electroporation as described previously (
      • Thor D.
      • Schulz A.
      • Hermsdorf T.
      • Schöneberg T.
      ). For expression in mammalian cells, COS-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM supplemented with 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin) at 37 °C in a humidified 5% CO2 incubator. Cyclic AMP measurements were performed as described previously (
      • Römpler H.
      • Stäubert C.
      • Thor D.
      • Schulz A.
      • Hofreiter M.
      • Schöneberg T.
      ) using the AlphaScreen® technology. Transient co-transfection experiments of COS-7 cells with the GPR34 constructs and the chimeric G protein Gαqi4 and inositol phosphate accumulation assays (
      • Berridge M.J.
      ) were essentially performed as described previously (
      • Schulz A.
      • Schöneberg T.
      ). All GPR34 constructs were introduced into the mammalian expression vector pcDps and double-tagged with an N-terminal HA tag and a C-terminal FLAG tag to monitor and quantify total cellular and plasma membrane expression using ELISA (
      • Böselt I.
      • Römpler H.
      • Hermsdorf T.
      • Thor D.
      • Busch W.
      • Schulz A.
      • Schöneberg T.
      ). Correctness of all PCR-derived constructs was verified by sequencing.

      Generation of a GPR34-deficient Mouse Strain

      The construction of the GPR34 conditional knock-out allele is shown in supplemental Fig. S1. The neomycin cassette was flanked by two loxP sites (
      • Sunahara R.K.
      • Dessauer C.W.
      • Whisnant R.E.
      • Kleuss C.
      • Gilman A.G.
      ), and a third loxP site was inserted into the 5′ region of Gpr34. Neomycin-resistant ES cells were screened for homologous recombination. Positive ES cell clones were injected into blastocysts. Chimeric offsprings were fertile and crossed into a C57BL/6 background. Correct homologous recombination was verified by sequencing of PCR products containing sequences of the 5′ and 3′ arms of the targeting constructs and the respective genomic flanking regions, which were not included in the targeting vector.
      The initial study described here was performed on complete GPR34-deficient mice. To obtain these animals, female heterozygous mice carrying the mutant loxP Gpr34-Neo cassette locus were bred with homozygous male EIIa-Cre mice. Correct deletion of the Gpr34 coding sequence and neomycin cassette was verified by PCR and locus sequencing. Resulting GPR34-deficient mice (referred to as KO mice) were backcrossed for 12 generations onto the C57BL/6 background. Animals were maintained in a controlled animal facility with 21 °C room temperature, 55% humidity, and a 12-h light/12-h dark cycle. All animal experiments were conducted in accord with accepted standards of humane animal care and approved by the respective regional government agency of the State of Saxony, Germany (TVV 43/07).
      Routine genotyping of KO and WT mice was performed by PCR using the primers loxP-GP34-2 sense, loxP-GP34-1-2 antisense, and loxP-GP34-3 antisense (supplemental Table S1). The genomic sequence was amplified from mouse tail DNA using a PCR protocol with the following conditions: 95 °C for 45 s, 60 °C for 45 s, and 72 °C for 45 s for 35 cycles followed by a 10-min extension at 72 °C. Amplification of the KO allele and the WT allele resulted in 270- and 385-bp fragments, respectively (supplemental Fig. S2).

      Morphological and Laboratory Chemical Characterization of GPR34-deficient Mice

      Litters of newborn KO and WT mice were followed in respect to genotype, gender, and vitality. The daily observation after birth further included measurement of weight and body length. At 3 months of age, mice were sacrificed. Organs, urine, and blood samples were taken for further examination. Histological slices (5 μm) were prepared from organs being fixed in 4% paraformaldehyde solution and embedded in paraffin wax. Slices were stained with hematoxylin and eosin. Blood cell counting from EDTA blood samples was performed automatically (Scil Vet abc; Scil Corp., Viernheim, Germany) and manually under a light microscope after May-Grünwald-Giemsa staining. Electrolytes, metabolites, enzymes, and hormones were analyzed in serum or, where appropriate, in whole blood, according to the guidelines of the German Society of Clinical Chemistry and Laboratory Medicine, using a Hitachi PPE-Modular analyzer (Roche Diagnostics). Acylcarnitine profiles were determined by electrospray ionization-MS/MS (API 2000, Applied Biosystems, Darmstadt, Germany) (
      • Müller P.
      • Schulze A.
      • Schindler I.
      • Ethofer T.
      • Buehrdel P.
      • Ceglarek U.
      ,
      • Ceglarek U.
      • Müller P.
      • Stach B.
      • Bührdel P.
      • Thiery J.
      • Kiess W.
      ). Blood glucose was measured using the Accu Check® device (Roche Diagnostics). Urine samples were obtained by direct urinary bladder puncture and tested for osmolality differences between the genotypes using a vapor pressure osmometer (Wescor®, Logan, UT).

      Behavioral Tests: Modified SHIRPA, Open Field Test, Light-Dark Test, and Hot Plate Test

      A modified SHIRPA protocol (
      • Masuya H.
      • Inoue M.
      • Wada Y.
      • Shimizu A.
      • Nagano J.
      • Kawai A.
      • Inoue A.
      • Kagami T.
      • Hirayama T.
      • Yamaga A.
      • Kaneda H.
      • Kobayashi K.
      • Minowa O.
      • Miura I.
      • Gondo Y.
      • Noda T.
      • Wakana S.
      • Shiroishi T.
      ,
      • Rogers D.C.
      • Fisher E.M.
      • Brown S.D.
      • Peters J.
      • Hunter A.J.
      • Martin J.E.
      ) was used to assess a number of motoric, sensoric, and autonomic functions of 8-week-old mice. The open field test and the light-dark test were performed as reported previously (
      • Plyusnina I.
      • Oskina I.
      ,
      • Crawley J.
      • Goodwin F.K.
      ,
      • Albert F.W.
      • Shchepina O.
      • Winter C.
      • Römpler H.
      • Teupser D.
      • Palme R.
      • Ceglarek U.
      • Kratzsch J.
      • Sohr R.
      • Trut L.N.
      • Thiery J.
      • Morgenstern R.
      • Plyusnina I.Z.
      • Schöneberg T.
      • Pääbo S.
      ) using automated measuring technology (TSE Systems, Bad Homburg, Germany). Activity of mice was recorded for a period of 5 min.
      In a hot plate test (Hot Plate 602001, TSE) the elapsed time until the first reaction of the mice to the heat stimulus (52 °C) was recorded. As end points, shaking or licking of one of the hind paws or jumping off the analgesia meter were used.

      RNA Isolation, Microarray Expression Analysis, and Real Time PCR

      Hearts were removed from five WT and KO mice at an age of 3 months. Total RNA was extracted from the tissues using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA was further purified with RNeasy kits (Qiagen, Hilden, Germany) according to the RNA clean-up protocol. For microarray analysis, RNA integrity and concentration were quantified on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) using the RNA 6.000 LabChip kit (Agilent Technologies) according to the manufacturer's instructions. Microarray expression analysis using GeneChip® Mouse Genome Arrays 430A 2.0 and data analysis were performed as described previously (
      • Schliebe N.
      • Strotmann R.
      • Busse K.
      • Mitschke D.
      • Biebermann H.
      • Schomburg L.
      • Köhrle J.
      • Bär J.
      • Römpler H.
      • Wess J.
      • Schöneberg T.
      • Sangkuhl K.
      ).
      For quantitative real time PCR analysis (qPCR), total RNA was reversely transcribed (Superscript II, Invitrogen) with oligo(dT) primer. cDNA from 500 ng of total RNA was subjected to qPCR using Platinum-SYBR Green® qPCR Supermix (Invitrogen), 0.6 μm forward and reverse primers, and 100 nm ROXTM (5-carboxy-X-rhodamine, passive reference dye). Oligonucleotide primers (supplemental Table S2) were designed with the Primer3 software (
      • Rozen S.
      • Skaletsky H.J.
      ) to flank intron sequences. qPCR was performed by an MX 3000P instrument (Stratagene, La Jolla, CA) using the following protocol: 2 min at 50 °C, 2 min at 95 °C and 50 cycles of 15 s at 95 °C and 30 s at 60 °C. A product melting curve was recorded to confirm the presence of a single amplicon. The correct amplicon size and identity were additionally confirmed by agarose gel electrophoresis and restriction enzyme cleavage or sequencing. Standard curves with serial dilutions of cDNA were generated for each primer pair to assert linear amplification. Threshold cycle (Ct) values were set within the exponential phase of the PCR. After normalization to β2-microglobulin, ΔCt values were used to calculate the relative expression levels (
      • Livak K.J.
      • Schmittgen T.D.
      ). Gene regulation was statistically evaluated by subjecting the ΔΔCt values derived from matched littermate samples to a two-sided Student's t test assuming equal variances. Gene regulation ratios are given as 2ΔΔCt values.

      Mast Cell Degranulation Assay and Histamine Concentration Measurements

      Peritoneal mast cells were obtained by peritoneal lavage from 3-month-old mice and cultured in RPMI 1640 medium (Sigma). A degranulation assay was performed as described previously (
      • Sugo T.
      • Tachimoto H.
      • Chikatsu T.
      • Murakami Y.
      • Kikukawa Y.
      • Sato S.
      • Kikuchi K.
      • Nagi T.
      • Harada M.
      • Ogi K.
      • Ebisawa M.
      • Mori M.
      ). Briefly, peritoneal mast cells were adjusted to equal numbers per reaction assembly and stimulated with P-lyso-PS (10 μm). After incubation for 20 min, suspensions were centrifuged, and supernatant and pellet were resuspended separately in 1 ml of Ca2+/Mg2+-free Tyrode's buffer. Histamine was extracted using bis-(2-ethylhexyl)-phosphoric acid in heptane. For quantification through a fluorescence detector, derivatization of histamine was performed with o-phthaldialdehyde. After filtration and degassing of the individual extracts, histamine concentration was obtained by high performance liquid chromatography (HPLC) (KNAUER, Berlin, Germany) with a stationary phase consisting of phenyl on silica gel (Nucleosil®, MACHEREY-NAGEL GmbH & Co. KG, Düren, Germany) and a mobile phase with a sodium dihydrogen phosphate buffer/acetonitrile gradient of 5–50%. Chromatograms were recorded and analyzed by Chromgate® version 3.1 software (KNAUER, Berlin, Germany- based on EZChrom Elite®).

      Chemotaxis Assay

      A chemotaxis assay was performed using transwell plates (Greiner Bio-One, Solingen, Germany) as described previously (
      • Park K.S.
      • Lee H.Y.
      • Kim M.K.
      • Shin E.H.
      • Jo S.H.
      • Kim S.D.
      • Im D.S.
      • Bae Y.S.
      ,
      • Bae Y.S.
      • Yi H.J.
      • Lee H.Y.
      • Jo E.J.
      • Kim J.I.
      • Lee T.G.
      • Ye R.D.
      • Kwak J.Y.
      • Ryu S.H.
      ). Cells were harvested by peritoneal lavage with PBS, centrifuged, and resuspended in DMEM (plus 0.5% FBS, penicillin/streptomycin) for each mouse separately. A dry non-coated polycarbonate filter (8-μm pore size) was placed in a cavity of a 24-well plate, and the upper chamber was filled with 150 μl of cell suspension (3.3 × 106 cells/ml). Cells were incubated overnight at 37 °C to allow adhesion onto the membrane. The next day, different concentrations of S-lyso-PS in a volume of 800 μl were applied to the lower chamber. After incubation for 4 h at 37 °C, non-migratory cells on the upper surface of the filter were removed by scraping, and migrated adherent cells on the lower surface of the filter were fixed and stained with the hemacolor set (Merck). The stained cells were counted in four randomly chosen high power fields (×40) for each well. The number of migrated nonadherent cells in the medium of the lower chamber was determined using a Neubauer counting cell chamber.

      Glial Swelling Test

      GPR34 is highly expressed in glial cells (
      • Bédard A.
      • Tremblay P.
      • Chernomoretz A.
      • Vallières L.
      ) such as astrocytes and microglia (detailed expression data from own studies, see supplemental Table S3). One function of glial cells in brain and retina is the compensation of osmotic imbalances. To assess whether GPR34 is involved in this specific glial function. a retinal glia swelling test was performed. To monitor volume changes of retinal glial cells in response to hypotonic challenge, the somata of glial cells in the inner nuclear layer of retinal slices or the somata of isolated single cells were focused. Filter stripes with the retinal slices (about 1 mm thick) were transferred to recording chambers and kept submerged in extracellular solution. The chambers were mounted on the stage of an upright confocal laser scanning microscope (LSM 510 Meta; Zeiss, Oberkochen, Germany). All experiments were performed at room temperature (20–23 °C). Retinal slices or isolated cells were loaded with the vital dye Mitotracker Orange (10 μm), which has been shown to stain somata of Müller glial cells selectively in the inner nuclear layer of retinal tissues (
      • Uckermann O.
      • Iandiev I.
      • Francke M.
      • Franze K.
      • Grosche J.
      • Wolf S.
      • Kohen L.
      • Wiedemann P.
      • Reichenbach A.
      • Bringmann A.
      ). After an incubation time of 3 min, slices or cells were continuously perfused with extracellular solution at a flow rate of 2 ml/min, and recordings were made with an Achroplan 63×/0.9 water immersion objective. The pinhole was set at 172 μm; the thickness of the optical section was adjusted to 1 μm. Mitotracker Orange was excited at 543 nm with an HeNe laser, and emission was recorded with a 560-nm long pass filter. Images were obtained with an x-y frame size of 256 × 256 pixels (73.1 × 73.1 μm). In the course of the experiments, the Mitotracker Orange-stained somata of Müller glial cells were recorded at the plane of their maximal extension. To ensure that the maximum soma area was precisely measured, the focal plane was continuously adjusted in the course of the experiments.

      Delayed Type Hypersensitivity (DTH) Response

      The DTH response test to methylated BSA (mBSA; Sigma) was performed according to Nambu et al. (
      • Nambu A.
      • Nakae S.
      • Iwakura Y.
      ). At day 1, mice were immunized intradermally with 200 μl of 2 mg/ml mBSA emulsified with complete Freund's adjuvant. After 8 days, mice were challenged intradermally in one footpad with 20 μl of 5 mg/ml mBSA in 0.9% NaCl solution and an equal volume of solely 0.9% NaCl solution in the other footpad. Footpad swelling was measured every 12 h over a total time period of 48 h using a millimeter screw (Kroeplin GmbH, Schlüchtern, Germany). DTH reaction was determined as difference between thickness of mBSA- and NaCl solution-injected footpad.
      At the end of the experiment, mice were sacrificed, and primary spleen cell cultures were established. Thus, spleen tissue of the individual mice was disintegrated through a 100-μm cell strainer to obtain a single cell suspension in PBS. After erythrocyte lysis with Gey's solution, cells were resuspended (5 × 106 cells/ml in Iscove's medium with glutamine, 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin), seeded into 24-well plates, and stimulated with different concentrations of mBSA for 72 h. Cytokine concentrations in supernatants of untreated and mBSA-treated cells were measured in a multiplex assay (Th1/Th2-Kit, Bio-Rad) according to the manufacturer's instructions. For IL-1β and IL-6 measurements, an ELISA kit (BioLegend, Fell, Germany) was used according to the manufacturer's protocol.

      Pulmonary Infection with Cryptococcus neoformans

      Local infection of mice with C. neoformans was carried out as described previously (
      • Kleinschek M.A.
      • Muller U.
      • Brodie S.J.
      • Stenzel W.
      • Kohler G.
      • Blumenschein W.M.
      • Straubinger R.K.
      • McClanahan T.
      • Kastelein R.A.
      • Alber G.
      ,
      • Müller U.
      • Stenzel W.
      • Köhler G.
      • Werner C.
      • Polte T.
      • Hansen G.
      • Schütze N.
      • Straubinger R.K.
      • Blessing M.
      • McKenzie A.N.
      • Brombacher F.
      • Alber G.
      ). Prior to infection, the encapsulated C. neoformans strain 1841, serotype D, was incubated overnight in Sabouraud dextrose medium (2% glucose, 1% peptone; Sigma) at 30 °C. Twenty μl of a 2.5 × 104 cells/ml Cryptococcus cell suspension (500 colony-forming units) were administered into one nostril. During this procedure, mice were intraperitoneally anesthetized with a 1:1 mixture of 10% ketamine (100 mg/ml; Ceva Animal Health) and 2% xylazine (20 mg/ml; Ceva Animal Health).
      Infected mice were observed daily for any signs of morbidity. After 60 days, animals were sacrificed, and lung, spleen, and brain were removed under sterile conditions. All organs were weighed, and equal organ parts were homogenized with an Ultra-Turrax® (T8; Ika-Werke, Staufen, Germany) in 1 ml of PBS. Serial dilutions of homogenates were administered on Sabouraud-dextrose agar plates, and grown colonies were counted after 72 h of incubation at 30 °C.
      For cytokine measurements, spleens of sacrificed animals were pooled in groups, and primary spleen cell culture was performed as described above. Cells were incubated with medium (control) or the heat-inactivated acapsular C. neoformans serotype D strain CAP67 (hiCap) (1 × 107 cells/ml) for 72 h at 30 °C. Subsequently, cytokine concentrations in supernatants were determined in a multiplex assay (Th1/Th2-Kit, Bio-Rad).

      FACS Analysis

      To monitor macrophages by fluorescence, KO mice were crossed into a mouse line expressing enhanced GFP under control of the CX3CR1 receptor promoter (
      • Jung S.
      • Aliberti J.
      • Graemmel P.
      • Sunshine M.J.
      • Kreutzberg G.W.
      • Sher A.
      • Littman D.R.
      ). The background of both mouse strains was C57BL/6. For experiments, WT and KO animals heterozygous for the CX3CR1 receptor were used. Spleens from naive and mBSA-immunized WT and KO animals were collected individually and disintegrated through a 100-μm nylon cell strainer (BD Biosciences). Erythrocytes were lysed with Gey's solution, and after centrifugation leukocytes were resuspended in FACS/wash buffer (PBS containing 1% FBS and 0.1% NaN3). After Fc-block (FcR blocking reagent, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany), cells were counted, and 105 cells per staining reaction were incubated with labeled antibodies to identify different spleen cell populations. The following antibodies were used: anti-CD3ϵ, -CD8, -Gr1, and -CD11c (BD Biosciences); anti-CD4, -B220, -CD86, and -F4/80 (eBiosciences, Frankfurt, Germany); anti-CD11b (CALTAG, Buckingham, UK), anti-Nkp46 (R&D Systems, Wiesbaden-Nordenstadt, Germany); and anti-CD117 (Miltenyi Biotech GmbH). Measurement of stained cells was performed in a 96-well format with a flow cytometer (FACSCalibur with HTS loader, BD Biosciences).

      Acknowledgments

      We are very grateful to C. Deng and J. Wess for their initial help in generating the transgenic mouse. We especially thank Susann Lautenschläger for excellent technical assistance and help in different methods. We thank Rainer Strotmann and Evi Kostenis for functional testing of GPR34 in Fura-2 calcium measurements and the Epic system (Corning Glass), respectively. We thank K. Krohn (core unit “DNA Technologies,” IZKF Leipzig) for performing the microarray measurements.

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