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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 chemokine responses of B cells prepared from wild type and Gnai3−/− mice. A. Chemotaxis assays. B cells purified from wild-type (littermate controls) and Gnai3−/− mice were subjected to a 2 h chemotaxis in response to different concentrations of CXCL12, CCL19, or CXCL13 as indicated. Results are mean and standard error of sextuplet samples from three experiments and are shown as % specific migration. Statistical significance was calculated using Mann-Whitney t-test compared with Rgs1+/+Gnai2+/+. (***, p<0.001) B. Measurement of changes in [Ca+2]i. B cells purified from the spleens of wild type (littermate controls) and Gnai3−/− mice were prepared and incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12, CCL19, or CXCL13 (100, 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.
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Figure 4: Comparison of chemokine responses of B cells prepared from wild type and Gnai3−/− mice. A. Chemotaxis assays. B cells purified from wild-type (littermate controls) and Gnai3−/− mice were subjected to a 2 h chemotaxis in response to different concentrations of CXCL12, CCL19, or CXCL13 as indicated. Results are mean and standard error of sextuplet samples from three experiments and are shown as % specific migration. Statistical significance was calculated using Mann-Whitney t-test compared with Rgs1+/+Gnai2+/+. (***, p<0.001) B. Measurement of changes in [Ca+2]i. B cells purified from the spleens of wild type (littermate controls) and Gnai3−/− mice were prepared and incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12, CCL19, or CXCL13 (100, 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.

Mentions: B and T lymphocyte chemotaxis, lymph nodes homing, lymph node egress, thymus egress, and positioning within lymph node organs are all sensitive to pertussis toxin treatment, which ADP ribosylates the Gαi subunits Gαi1, Gαi2, Gαi3, and Goα but not Gzα. Mature B and T lymphocytes predominantly express Gαi2 and Gαi3, but not Gαi1. Lymphocytes lacking Gαi2 exhibit defects in all of the above with the exception of lymph node and thymus egress although sphingosine 1-phosphate (S1P) mediated lymphocyte chemotaxis is markedly reduced in the absence of Gαi2.8,9,22. Some residual chemotaxis has been noted with the Gnai2−/− lymphocytes, however, it is insensitive to pertussis toxin treatment.8,9 Together these data argue that Gαi3 cannot mediate lymphocyte CXCR4, CXCR5, or CCR7 triggered chemotaxis. The inability of Gαi3 to substitute for Gαi2 in lymphocyte chemotaxis despite the elevated Gαi3 expression is somewhat surprising. To directly examine the role of Gαi3 in B lymphocytes we performed chemotaxis assays and examined chemokine induced changes in [Ca2+]i using B cells prepared from Gnai3−/− mice. The percentage of chemokine-responsive cells from the Gnai3−/− mice equaled or exceeded the number from controls (Figure 4). In addition the increase in [Ca2+]ielicited by the CXCL12, CXCL13, and CCL19 exceeded the levels achieved with wild type control B cells (Figure 4). These results indicate that in contrast to Gαi2, Gαi3 is not needed for B lymphocytes to respond to chemokines, and that in its absence, Gαi2 may more efficiently couple to chemoattractant receptors.


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 chemokine responses of B cells prepared from wild type and Gnai3−/− mice. A. Chemotaxis assays. B cells purified from wild-type (littermate controls) and Gnai3−/− mice were subjected to a 2 h chemotaxis in response to different concentrations of CXCL12, CCL19, or CXCL13 as indicated. Results are mean and standard error of sextuplet samples from three experiments and are shown as % specific migration. Statistical significance was calculated using Mann-Whitney t-test compared with Rgs1+/+Gnai2+/+. (***, p<0.001) B. Measurement of changes in [Ca+2]i. B cells purified from the spleens of wild type (littermate controls) and Gnai3−/− mice were prepared and incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12, CCL19, or CXCL13 (100, 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.
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Related In: Results  -  Collection

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Figure 4: Comparison of chemokine responses of B cells prepared from wild type and Gnai3−/− mice. A. Chemotaxis assays. B cells purified from wild-type (littermate controls) and Gnai3−/− mice were subjected to a 2 h chemotaxis in response to different concentrations of CXCL12, CCL19, or CXCL13 as indicated. Results are mean and standard error of sextuplet samples from three experiments and are shown as % specific migration. Statistical significance was calculated using Mann-Whitney t-test compared with Rgs1+/+Gnai2+/+. (***, p<0.001) B. Measurement of changes in [Ca+2]i. B cells purified from the spleens of wild type (littermate controls) and Gnai3−/− mice were prepared and incubated for 1 h at 37°C in the calcium assay loading buffer before adding CXCL12, CCL19, or CXCL13 (100, 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.
Mentions: B and T lymphocyte chemotaxis, lymph nodes homing, lymph node egress, thymus egress, and positioning within lymph node organs are all sensitive to pertussis toxin treatment, which ADP ribosylates the Gαi subunits Gαi1, Gαi2, Gαi3, and Goα but not Gzα. Mature B and T lymphocytes predominantly express Gαi2 and Gαi3, but not Gαi1. Lymphocytes lacking Gαi2 exhibit defects in all of the above with the exception of lymph node and thymus egress although sphingosine 1-phosphate (S1P) mediated lymphocyte chemotaxis is markedly reduced in the absence of Gαi2.8,9,22. Some residual chemotaxis has been noted with the Gnai2−/− lymphocytes, however, it is insensitive to pertussis toxin treatment.8,9 Together these data argue that Gαi3 cannot mediate lymphocyte CXCR4, CXCR5, or CCR7 triggered chemotaxis. The inability of Gαi3 to substitute for Gαi2 in lymphocyte chemotaxis despite the elevated Gαi3 expression is somewhat surprising. To directly examine the role of Gαi3 in B lymphocytes we performed chemotaxis assays and examined chemokine induced changes in [Ca2+]i using B cells prepared from Gnai3−/− mice. The percentage of chemokine-responsive cells from the Gnai3−/− mice equaled or exceeded the number from controls (Figure 4). In addition the increase in [Ca2+]ielicited by the CXCL12, CXCL13, and CCL19 exceeded the levels achieved with wild type control B cells (Figure 4). These results indicate that in contrast to Gαi2, Gαi3 is not needed for B lymphocytes to respond to chemokines, and that in its absence, Gαi2 may more efficiently couple to chemoattractant receptors.

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