Genes and Immunity (2010) 11, 384–396 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 www.nature.com/gene ORIGINAL ARTICLE Variations in Gnai2 and Rgs1 expression affect chemokine receptor signaling and the organization of secondary lymphoid organs IY Hwang, C Park, KA Harrision, NN Huang and JH Kehrl B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Ligand bound chemoattractant receptors activate the heterotrimeric G-protein Gi to stimulate downstream signaling pathways to properly position lymphocytes in lymphoid organs. Here, we show how variations in the expression of a chemokine receptor and in two components in the signaling pathway, Gai2 and RGS1, affect the output fidelity of the signaling pathway. Examination of B cells from mice with varying numbers of intact alleles of Ccr7, Rgs1, Gnai2, and Gnai3 provided the basis for these results. Loss of a single allele of either Gnai2 or Rgs1 affected CCL19 triggered chemotaxis, whereas the loss of a single allele of Ccr7, which encodes the cognate CCL19 receptor, had little effect. Emphasizing the importance of Gnai2, B cells lacking Gnai3 expression responded to chemokines better than did wild-type B cells. At an organismal level, variations in Rgs1 and Gnai2 expression affected marginal zone B-cell development, splenic architecture, lymphoid follicle size, and germinal center morphology. Gnai2 expression was also needed for the proper alignment of MOMA-1 þ macrophages and MAdCAM-1 þ endothelial cells along marginal zone sinuses in the spleen. These data indicate that chemoattractant receptors, heterotrimeric G-proteins, and RGS protein expression levels have a complex interrelationship that affects the responses to chemoattractant exposure. Genes and Immunity (2010) 11, 384–396; doi:10.1038/gene.2010.27; published online 27 May 2010 Keywords: heterotrimeric G-protein; RGS protein; chemotaxis; calcium flux; spleen; marginal zone Introduction ing as evidenced by depressed chemotaxis, defective homing to lymph nodes, poor adherence to lymph node Chemoattractants help to recruit and position lympho- high endothelial venules, and decreased motility within cytes and dendritic cells in lymphoid organs and lymph node follicles.8,9 inflammatory sites.1–3 Most lymphocyte chemoattrac- GPCRs such as chemokine receptors exist in multiple tants and chemokines signal through G-protein-coupled dynamic states including ligand bound, inactive, and receptors (GPCRs) that use the heterotrimeric G-protein G-protein coupled, which influence G-protein activation 3 Gi to activate downstream effectors. The binding of and subsequent downstream signaling. A recent study ligand activates receptors triggering Gai subunits to used parameter variation and sensitivity analysis exchange GTP for GDP, resulting in the dissociation of to examine the ligand- and cell-specific parameters, the Ga subunit from its associated Gbg heterodimer. The which determine cellular responses in a dynamic model 12 release of Gi-associated Gbg subunits is necessary for of GPCR signaling. Not surprisingly, the most impor- 4,5 triggering directional migration. As Ga subunits tant factor was the ability of the ligand to trigger an possess an intrinsic GTPase activity, GTP hydrolysis active receptor conformation, but in addition, several leads to the re-assembly of heterotrimeric G-protein cell-specific parameters strongly correlated with G- causing signaling to cease.6,7 Lymphocytes express two protein activation. The three most important in rank 8,9 À/À members of the Gai subfamily, Gai2 and Gai3. Gnai3 order were G-protein concentration, Ga GTPase activity, mice were reportedly without a phenotype10; however, and receptor expression.12 Notably, these results indicate more recently, a defect in the early seeding of the thymus that the expression of a GPCR or G-protein several-fold by progenitors has been observed.11 Gnai2À/À mice above or below endogenous levels could result in exhibit defective lymphocyte chemokine receptor signal- responses inconsistent with those measured in endogen- ous systems, thereby providing a caveat for the inter- pretation of results from transfection studies. Also, Correspondence: Dr JH Kehrl, B-Cell Molecular Immunology small variations in cell-specific parameters may actually Section, Laboratory of Immunoregulation, National Institute of change a ligand-induced positive response to a negative Allergy and Infectious Diseases, National Institutes of Health, 9000 one.12 Rockville Pike, Building 10, Rm. 11B08, Bethesda, MD 20892, USA. E-mail: [email protected] Because of the ease of assessing chemokine receptor Received 14 December 2009; revised and accepted 16 March 2010; expression by flow cytometry, immunologists have published online 27 May 2010 focused on those levels as a measure of chemokine Chemoattractant signaling and secondary lymphoid organs IY Hwang et al 385 responsiveness largely ignoring two other potentially have used have been backcrossed 3–6 generations onto important cell-specific parameters, G-protein levels and C57BL/6 background and have thrived normally. Double Ga GTPase activity, the later which largely depends on heterozygote crosses were bred to generate mice with the the presence of RGS proteins in the system.13 Increas- varying alleles of Rgs1 and Gnai2. The double knockout ingly, attention has been focused on RGS proteins as a mice exhibited the same smaller stature as did the new class of pharmaceutical targets.14 In this study, we Gnai2À/À mice (Figure 1). Analysis of the frequency of the examined the consequences of altering Gai2 and RGS1 different genotypes obtained from these crosses revealed levels on B-cell responses to three different chemokines an increased representation of the wild-type Gnai2 allele CXCL12, CXCL13, and CCL19 by using B cells from mice in the offspring. The most over represented genotype with one or two disrupted alleles of Rgs1 or Gnai2 and among the mice was Rgs1 þ /À/Gnai2 þ / þ , whereas the from mice that have various combinations of disrupted Rgs1 allele status had little effect on the survival of the and wild-type alleles of these two genes.8,15 In addition, mice lacking both Gnai2 alleles. There was also we compared the responsiveness of B cells from wild- the suggestion that the lack of one allele of Gnai2 type mice to those with only one intact allele of Ccr7. Our negatively impacted the survival of the mice. results indicate that the RGS1/Gai2 ratio is an important parameter to consider in assessing B-cell chemokine Varying levels of Gnai2 and Rgs1 expression affected B-cell responsiveness. They also provide some insights into the responses to chemokines role of G-protein signaling in the organization of the In lymphocytes, the loss of a single allele of Gnai2 B splenic lymphoid architecture. reduced Gai2 levels 50%, whereas Gai3 levels increased a similar amount. A loss of both alleles of Gnai2 resulted in a several-fold increase in Gai3 levels compared with controls.8 The loss of Gnai3 has been reported to Results 17 not significantly impact Gai2 expression levels and the À/À The generation of mice for analysis and the impact of genotype Gnai3 lymphocytes we used had similar Gai2 expres- on early mouse viability sion as did littermate controls (data not shown). The On a C57BL/6 background, the Rgs1À/À mice bred and disruption of a single allele of Rgs1 reduced Rgs1 mRNA thrived similar to wild-type mice, whereas the Gnai2À/À expression 50% (data not shown). To determine the mice bred poorly or not at all and were maintained as relative importance of Gnai2 and Rgs1 expression on heterozygotes. Litters from heterozygotic crosses gener- chemokine receptor signaling in B lymphocytes, we ated lower than expected numbers of Gnai2À/À mice. An prepared splenic B cells from mice with various earlier study had reported that Gnai2 þ /À intercrosses disrupted alleles of Gnai2 and Rgs1 and tested them produced 9.7% Gnai2À/À mice versus the expected 25%. using standard chemotaxis assays to CXCL12, CCL19, A significant loss of Gnai2À/À mice was reported to occur and CXCL13. The specific migration to three different perinatally.16 In our colony, Gnai2À/À mice are smaller concentrations of chemokine was measured. We found than their wild-type littermates and often die before 6 that loss of one allele of Rgs1 increased responses to all months of age of varying causes. The Gnai3À/À mice we three chemokines and the loss of a second allele further Figure 1 Intercross of Rgs1 þ /À and Gnai2 þ /À mice. Photographs of representative wild-type, Gnai2À/À, and Rgs1À/À/Gnai2À/À C57/BL6 mice. The result of genotyping mice from double heterozygote crosses is shown below the photographs. The percentage of each of the different genotypes is shown and the predicted frequency of the genotype is given in parentheses. The results are from genotyping 342 mice. The P-values are significantly different for each observed genotype number versus expected genotype number (Po0.001 by w2-tests). Genes and Immunity Chemoattractant signaling and secondary lymphoid organs IY Hwang et al 386 Figure 2 Comparison of B cells prepared from the different genotypes in standard chemotaxis assays. B cells purified from wild type and mice with varying intact alleles of Rgs1 and Gnai2 were subjected to a 2-h chemotaxis in response to different concentrations of CXCL12, CCL19, or CXCL13 as indicated. The percentages of cells responding to are shown. The results are mean and standard error of sextuplet samples from four experiments and are shown as percentage-specific migration. Statistical significance was calculated using Mann–Whitney t-test compared with Rgs1 þ / þ Gnai2 þ / þ (*Po0.05, **Po0.01, and ***Po0.001). Specific migration is the percentage cells responding to chemokine minus the percentage cells that spontaneously migrate in the absence of chemokine. increased the specific migration of B lymphocytes measure of heterotrimeric G-protein activation.