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. 2011 Jan 21;286(3):2101-10.
doi: 10.1074/jbc.M110.196659. Epub 2010 Nov 19.

Altered immune response in mice deficient for the G protein-coupled receptor GPR34

Ines Liebscher  1 , Uwe Müller, Daniel Teupser, Eva Engemaier, Kathrin M Y Engel, Lars Ritscher, Doreen Thor, Katrin Sangkuhl, Albert Ricken, Antje Wurm, Daniel Piehler, Sandra Schmutzler, Herbert Fuhrmann, Frank W Albert, Andreas Reichenbach, Joachim Thiery, Torsten Schöneberg, Angela Schulz
Affiliations

Altered immune response in mice deficient for the G protein-coupled receptor GPR34

Ines Liebscher et al. J Biol Chem. .

Abstract

The X-chromosomal GPR34 gene encodes an orphan G(i) 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.

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Figures

FIGURE 1.
FIGURE 1.
Effect of P- and S-lyso-PS on different GPR34 orthologs in COS-7 cells. COS-7 cells were transiently co-transfected with GPR34 orthologs and human P2Y12 (control) with the chimeric G protein Gαqi4. Listed substances were applied in equal concentrations (10 μm). GFP-transfected cells were used as negative control, whereas the ADP-sensitive receptor P2Y12, stimulated with methyl-S-adenosine diphosphate (MeS-ADP), served as positive control. Results are means ± S.E. (n = 3), with each experiment performed in triplicate. IP, inositol phosphate.
FIGURE 2.
FIGURE 2.
Functional expression of GPR34 orthologs and mutants in yeast. A, yeast cells expressing the human GPR34 and carp GPR34 subtypes were incubated with different concentrations of P-lyso-PS and S-lyso-PS. Receptor activation-dependent growth was measured as A600 nm after 24 h. B, yeast cells expressing the human (h) GPR34 (hGPR34) and a constitutively active mutant (hGPR34-T264A) were cultured in histidine- and uracil-deficient medium. Receptor activation-dependent growth was measured as A600 nm over time. C, yeast cells expressing the human and carp GPR34 subtype 2a and two chimeric constructs were incubated with increasing concentrations of S-lyso-PS. Receptor activation-dependent growth was measured as A600 nm after 24 h. Details of the constructs and cell surface and total cellular expression levels are given in supplemental Table S15. Mean ± S.E. of one representative assay performed in triplicate is shown.
FIGURE 3.
FIGURE 3.
Histamine release from peritoneal mast cells. Peritoneal mast cells from WT and KO mice were obtained by peritoneal lavage and enriched by short term cultivation. Cells were stimulated with P-lyso-PS (10 μm), and histamine contents were measured in the pellet and supernatant. Results are shown for P-lyso-PS-stimulated cells in comparison with untreated mast cells. Histamine release in the supernatant is given in % of total histamine release in both pellet and supernatant. Results are means ± S.E. of a representative assay (total number of experiments = 3 and number of animals per genotype in each assay = 6), with each experiment performed in duplicate.
FIGURE 4.
FIGURE 4.
Chemotaxis of peritoneal monocytic cells. Cells were incubated with different concentrations of S-lyso-PS, and cell migration was determined as described under “Experimental Procedures.” The percentage of cells migrated into the lower chamber in relation to WT basal migration (set as 100% = 5.6 × 105 cells) is given. Results are means ± S.E. (number of animals per group = 5) with each experiment performed in duplicate. Differences to non-stimulated migration of WT cells were tested for significance with Student's t test: *, p < 0.05; **, p < 0.01.
FIGURE 5.
FIGURE 5.
Cell swelling is altered in retinal glial cells from GPR34-deficient mice. A, perfusion of retinal slices from WT and KO mice with a hypoosmolar solution (containing 60% of control osmolarity) in the absence (control) and presence of barium chloride (1 mm) resulted in a swelling of glial somata. Mean ± S.E. soma areas of retinal glial cells were measured after perfusion with a hypoosmolar solution for 4 min. Data are expressed in percent of the soma size recorded before hypoosmotic challenge (100%). Note that perfusion of retinal slices from WT animals with hypoosmolar solution for 4 min did not significantly increase the size of glial cell somata. However, hypoosmotic swelling of glial somata was evoked in the presence of barium chloride (1 mm). The bar diagrams display data obtained in 13–21 cells from retinae of two different animals each. B, same investigations were performed with isolated Müller cells (5–7 cells/bar). Significant differences to WT control: **, p < 0.01; ***, p < 0.001. Significant differences between KO and WT in the presence of barium chloride: ●●, p < 0.01; ●●●, p < 0.001.
FIGURE 6.
FIGURE 6.
Increased footpad swelling (DTH test) in GPR34-deficient mice. Mice were immunized with mBSA. Eight days post-infection, one footpad was injected with mBSA, and the other was injected with 0.9% NaCl solution (control). Footpad thickness was measured using a millimeter screw. The graph shows swelling of mBSA-injected footpad in relation to reference footpad over a period of 48 h. Results are means ± S.D. from two independent experiments with 10 animals per genotype per experiment. Swelling differences between KO and WT were tested for significance using Student's t test: *, p < 0.05; **, p < 0.01.
FIGURE 7.
FIGURE 7.
GPR34-deficient mice show an increased pathogen burden after infection with C. neoformans. WT and KO mice were intranasally infected with C. neoformans. Animals were sacrificed after 60 days, and lung, brain, and spleen were removed. Fungal burden was determined by counting of growing colonies on agar plates plated with organ suspensions (n = 4 animals per genotype). Means, 95% percentiles and ranges are given. *, p < 0.05; **, p < 0.01.
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