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Variations in Gnai2 and Rgs1 expression affect chemokine receptor signaling and the organization of secondary lymphoid organs.

Hwang IY, Park C, Harrision KA, Huang NN, Kehrl JH - Genes Immun. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Ligand bound chemoattractant receptors activate the heterotrimeric G-protein G(i) 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, Galpha(i2) 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(+) macrophages and MAdCAM-1(+) 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.

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Comparison of Ccl19 triggered chemotaxis of B cells from wild type, Ccr7+/−, and Ccr7−/− mice. (a) CCR7 expression on wild type and mutant mice. Flow cytometry with an isotype control and CCR7 antibody. Spleens B cells were purified from wild type, Ccr7+/−, and Ccr7−/− mice. Results are representative of 4 experiments done. (b) Mean Fluorescent intensity (MFI) of CCR7 expression on B cells from wild type and mutant mice. Data representative of one of four experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with MFI of CCR7 of CCR7+/+. (***, p<0.0001) (c) CXCL12 mediated Chemotaxis. A standard chemotaxis assay was performed with splenic B cells. The results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. A standard chemotaxis assay was performed with splenic B cells. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (**, p<0.01 and ***, p<0.001) (d) CXCL19 mediated chemotaxis. The Results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (***, p<0.001)
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Figure 5: Comparison of Ccl19 triggered chemotaxis of B cells from wild type, Ccr7+/−, and Ccr7−/− mice. (a) CCR7 expression on wild type and mutant mice. Flow cytometry with an isotype control and CCR7 antibody. Spleens B cells were purified from wild type, Ccr7+/−, and Ccr7−/− mice. Results are representative of 4 experiments done. (b) Mean Fluorescent intensity (MFI) of CCR7 expression on B cells from wild type and mutant mice. Data representative of one of four experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with MFI of CCR7 of CCR7+/+. (***, p<0.0001) (c) CXCL12 mediated Chemotaxis. A standard chemotaxis assay was performed with splenic B cells. The results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. A standard chemotaxis assay was performed with splenic B cells. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (**, p<0.01 and ***, p<0.001) (d) CXCL19 mediated chemotaxis. The Results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (***, p<0.001)

Mentions: Since we had observed changes in chemokine responsiveness in B cells lacking one allele of Rgs1 or one allele of Gnai2 we examined the consequences of the loss of one allele of Ccr7 on B cell responsiveness to the chemoattractant Ccl19. Varying Ccr7 expression, hence Ccr7 mediated signaling, is known to help B cells position themselves properly in the B cell follicle.23 We isolated B cells from wild type, Ccr7+/−, and Ccr7−/− littermates derived from a heterozygote cross and tested for their ability to respond in a standard chemotaxis assay. Surprisingly we found that the lack of one allele of Ccr7, which caused a greater than 50% reduction in CCR7 expression as assessed by flow cytometry, had only a modest impact of B cell chemotaxis to the Ccl19. B cells from Ccr7+/− mice responded to low concentrations of CCL19 as well as did the wild type control mice although they showed a modest decrease (20%) at higher concentrations of CCL19 (Figure 5). As expected B cells from Ccr7−/− mice failed to respond to CCL19, but they responded well to CXCL12 even surpassing the wild type B cell responses particularly with lower concentrations of chemokine. In addition, the Ccr7−/− B cells responded better to CXCL13 than did the wild type B cells (data not shown). Thus, the lack of one allele of Rgs1 or Gnai2 affected Ccl19 triggered chemotaxis more than did the loss of a single allele of Ccr7. This argues that small changes in chemokine receptor expression may not be translated into significant changes in chemokine responsiveness while similar magnitude changes in Gnai2 and Rgs1 expression can impact chemokine responsiveness.


Variations in Gnai2 and Rgs1 expression affect chemokine receptor signaling and the organization of secondary lymphoid organs.

Hwang IY, Park C, Harrision KA, Huang NN, Kehrl JH - Genes Immun. (2010)

Comparison of Ccl19 triggered chemotaxis of B cells from wild type, Ccr7+/−, and Ccr7−/− mice. (a) CCR7 expression on wild type and mutant mice. Flow cytometry with an isotype control and CCR7 antibody. Spleens B cells were purified from wild type, Ccr7+/−, and Ccr7−/− mice. Results are representative of 4 experiments done. (b) Mean Fluorescent intensity (MFI) of CCR7 expression on B cells from wild type and mutant mice. Data representative of one of four experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with MFI of CCR7 of CCR7+/+. (***, p<0.0001) (c) CXCL12 mediated Chemotaxis. A standard chemotaxis assay was performed with splenic B cells. The results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. A standard chemotaxis assay was performed with splenic B cells. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (**, p<0.01 and ***, p<0.001) (d) CXCL19 mediated chemotaxis. The Results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (***, p<0.001)
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Figure 5: Comparison of Ccl19 triggered chemotaxis of B cells from wild type, Ccr7+/−, and Ccr7−/− mice. (a) CCR7 expression on wild type and mutant mice. Flow cytometry with an isotype control and CCR7 antibody. Spleens B cells were purified from wild type, Ccr7+/−, and Ccr7−/− mice. Results are representative of 4 experiments done. (b) Mean Fluorescent intensity (MFI) of CCR7 expression on B cells from wild type and mutant mice. Data representative of one of four experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with MFI of CCR7 of CCR7+/+. (***, p<0.0001) (c) CXCL12 mediated Chemotaxis. A standard chemotaxis assay was performed with splenic B cells. The results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. A standard chemotaxis assay was performed with splenic B cells. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (**, p<0.01 and ***, p<0.001) (d) CXCL19 mediated chemotaxis. The Results are mean and standard error of sextuplet samples from one experiment and are shown as % specific migration. Results are representative of one of 3 experiments performed. Statistical significance was calculated using Mann-Whitney t-test compared with CCR7+/+. (***, p<0.001)
Mentions: Since we had observed changes in chemokine responsiveness in B cells lacking one allele of Rgs1 or one allele of Gnai2 we examined the consequences of the loss of one allele of Ccr7 on B cell responsiveness to the chemoattractant Ccl19. Varying Ccr7 expression, hence Ccr7 mediated signaling, is known to help B cells position themselves properly in the B cell follicle.23 We isolated B cells from wild type, Ccr7+/−, and Ccr7−/− littermates derived from a heterozygote cross and tested for their ability to respond in a standard chemotaxis assay. Surprisingly we found that the lack of one allele of Ccr7, which caused a greater than 50% reduction in CCR7 expression as assessed by flow cytometry, had only a modest impact of B cell chemotaxis to the Ccl19. B cells from Ccr7+/− mice responded to low concentrations of CCL19 as well as did the wild type control mice although they showed a modest decrease (20%) at higher concentrations of CCL19 (Figure 5). As expected B cells from Ccr7−/− mice failed to respond to CCL19, but they responded well to CXCL12 even surpassing the wild type B cell responses particularly with lower concentrations of chemokine. In addition, the Ccr7−/− B cells responded better to CXCL13 than did the wild type B cells (data not shown). Thus, the lack of one allele of Rgs1 or Gnai2 affected Ccl19 triggered chemotaxis more than did the loss of a single allele of Ccr7. This argues that small changes in chemokine receptor expression may not be translated into significant changes in chemokine responsiveness while similar magnitude changes in Gnai2 and Rgs1 expression can impact chemokine responsiveness.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: B-Cell Molecular Immunology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Ligand bound chemoattractant receptors activate the heterotrimeric G-protein G(i) 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, Galpha(i2) 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(+) macrophages and MAdCAM-1(+) 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.

Show MeSH
Related in: MedlinePlus