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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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Measurement of changes in [Ca+2]i stimulated by chemokine exposure. A. Comparison of B cells from different mice. B cells purified from the spleens of wild type and mice with varying alleles of Rgs1 and Gnai2 were prepared and then incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12 or CXCL13 (100 or 1000 ng/ml, respectively). Changes in [Ca+2]i were monitored over 3 min. The data was analyzed with SOFT max Pro 5.2 and is shown as fluorescent counts and the y-axis labeled as Lm1. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. B. Comparison of [Ca+2]i stimulated by different chemokine concentrations with wild type or Rgs1−/− B cells. Wild type or Rgs1−/− B cells were stimulated with increasing concentrations of CXCL12 or CXCL13 as indicated. The data was analyzed with SOFT max Pro 5.2 and transformed with Graph Pad Prism and a linear regression analysis performed to fit the curves. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. Statistical significance was calculated using Mann-Whitney t-test compared with wild type (Rgs1+/+Gnai2+/+), (*, p<0.0001).
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Figure 3: Measurement of changes in [Ca+2]i stimulated by chemokine exposure. A. Comparison of B cells from different mice. B cells purified from the spleens of wild type and mice with varying alleles of Rgs1 and Gnai2 were prepared and then incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12 or CXCL13 (100 or 1000 ng/ml, respectively). Changes in [Ca+2]i were monitored over 3 min. The data was analyzed with SOFT max Pro 5.2 and is shown as fluorescent counts and the y-axis labeled as Lm1. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. B. Comparison of [Ca+2]i stimulated by different chemokine concentrations with wild type or Rgs1−/− B cells. Wild type or Rgs1−/− B cells were stimulated with increasing concentrations of CXCL12 or CXCL13 as indicated. The data was analyzed with SOFT max Pro 5.2 and transformed with Graph Pad Prism and a linear regression analysis performed to fit the curves. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. Statistical significance was calculated using Mann-Whitney t-test compared with wild type (Rgs1+/+Gnai2+/+), (*, p<0.0001).

Mentions: Exposure of B lymphocytes elicits a rapid increase in intracellular calcium [Ca2+]i which is mediated by Gβγ stimulation of phospholipase β and blocked by pre-treatment with pertussis toxin. Although the increase in [Ca2+]i apparently contributes little to lymphocyte chemotaxis, it provides an easy and rapidly accessible measure of heterotrimeric G-protein activation. Therefore, we examined chemokine induced changes in [Ca2+]i using B cells from the various mouse strains. We found that the loss of one allele of Rgs1 enhanced the [Ca2+]i response to CXCL12 and CXCL13, both the peak level and duration, while the loss of both alleles resulted in a further increase (Figure 3). In contrast to previous experiments with T cells where the loss of one allele of Gnai2 substantially decreased CXCL12 induced increase in [Ca2+]I,9 the loss of one allele in B cells had little effect on the CXCL12 induced increase in [Ca2+]i although the CXCL13 induced response was impaired. Similar to T cells the loss of both alleles of Gnai2 in B cells severely compromised the CXCL12 induced increases in [Ca2+]i and as well the CXCL13 triggered response. The double heterozygotic B cells had a phenotype most similar to the mice that lacked an Rgs1 allele and the double knock-out mice had a surprisingly good [Ca2+]i response, a result consistent with a partial re-coupling to Gq. Together these results indicate that in B lymphocytes chemokine receptor signaling induced changes in [Ca2+]i and chemotaxis are sensitive to changes in Gαi2 and RGS1 expression.


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)

Measurement of changes in [Ca+2]i stimulated by chemokine exposure. A. Comparison of B cells from different mice. B cells purified from the spleens of wild type and mice with varying alleles of Rgs1 and Gnai2 were prepared and then incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12 or CXCL13 (100 or 1000 ng/ml, respectively). Changes in [Ca+2]i were monitored over 3 min. The data was analyzed with SOFT max Pro 5.2 and is shown as fluorescent counts and the y-axis labeled as Lm1. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. B. Comparison of [Ca+2]i stimulated by different chemokine concentrations with wild type or Rgs1−/− B cells. Wild type or Rgs1−/− B cells were stimulated with increasing concentrations of CXCL12 or CXCL13 as indicated. The data was analyzed with SOFT max Pro 5.2 and transformed with Graph Pad Prism and a linear regression analysis performed to fit the curves. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. Statistical significance was calculated using Mann-Whitney t-test compared with wild type (Rgs1+/+Gnai2+/+), (*, p<0.0001).
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Figure 3: Measurement of changes in [Ca+2]i stimulated by chemokine exposure. A. Comparison of B cells from different mice. B cells purified from the spleens of wild type and mice with varying alleles of Rgs1 and Gnai2 were prepared and then incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12 or CXCL13 (100 or 1000 ng/ml, respectively). Changes in [Ca+2]i were monitored over 3 min. The data was analyzed with SOFT max Pro 5.2 and is shown as fluorescent counts and the y-axis labeled as Lm1. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. B. Comparison of [Ca+2]i stimulated by different chemokine concentrations with wild type or Rgs1−/− B cells. Wild type or Rgs1−/− B cells were stimulated with increasing concentrations of CXCL12 or CXCL13 as indicated. The data was analyzed with SOFT max Pro 5.2 and transformed with Graph Pad Prism and a linear regression analysis performed to fit the curves. Each experimental value is the mean of three determinations. The experiment was performed three times with similar results. Statistical significance was calculated using Mann-Whitney t-test compared with wild type (Rgs1+/+Gnai2+/+), (*, p<0.0001).
Mentions: Exposure of B lymphocytes elicits a rapid increase in intracellular calcium [Ca2+]i which is mediated by Gβγ stimulation of phospholipase β and blocked by pre-treatment with pertussis toxin. Although the increase in [Ca2+]i apparently contributes little to lymphocyte chemotaxis, it provides an easy and rapidly accessible measure of heterotrimeric G-protein activation. Therefore, we examined chemokine induced changes in [Ca2+]i using B cells from the various mouse strains. We found that the loss of one allele of Rgs1 enhanced the [Ca2+]i response to CXCL12 and CXCL13, both the peak level and duration, while the loss of both alleles resulted in a further increase (Figure 3). In contrast to previous experiments with T cells where the loss of one allele of Gnai2 substantially decreased CXCL12 induced increase in [Ca2+]I,9 the loss of one allele in B cells had little effect on the CXCL12 induced increase in [Ca2+]i although the CXCL13 induced response was impaired. Similar to T cells the loss of both alleles of Gnai2 in B cells severely compromised the CXCL12 induced increases in [Ca2+]i and as well the CXCL13 triggered response. The double heterozygotic B cells had a phenotype most similar to the mice that lacked an Rgs1 allele and the double knock-out mice had a surprisingly good [Ca2+]i response, a result consistent with a partial re-coupling to Gq. Together these results indicate that in B lymphocytes chemokine receptor signaling induced changes in [Ca2+]i and chemotaxis are sensitive to changes in Gαi2 and RGS1 expression.

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