Leukocyte analysis from WHIM syndrome patients reveals a pivotal role for GRK3 in CXCR4 signaling
Karl Balabanian, … , Fernando Arenzana-Seisdedos, Françoise Bachelerie
Karl Balabanian, … , Fernando Arenzana-Seisdedos, Françoise Bachelerie
Published February 14, 2008
Citation Information: J Clin Invest. 2008;118(3):1074-1084. https://doi.org/10.1172/JCI33187.
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Research Article Immunology

Leukocyte analysis from WHIM syndrome patients reveals a pivotal role for GRK3 in CXCR4 signaling

Abstract

Leukocytes from individuals with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome, a rare immunodeficiency, and bearing a wild-type CXCR4 ORF (WHIMWT) display impaired CXCR4 internalization and desensitization upon exposure to CXCL12. The resulting enhanced CXCR4-dependent responses, including chemotaxis, probably impair leukocyte trafficking and account for the immunohematologic clinical manifestations of WHIM syndrome. We provided here evidence that GPCR kinase-3 (GRK3) specifically regulates CXCL12-promoted internalization and desensitization of CXCR4. GRK3-silenced control cells displayed altered CXCR4 attenuation and enhanced chemotaxis, as did WHIMWT cells. These findings identified GRK3 as a negative regulator of CXCL12-induced chemotaxis and as a candidate responsible for CXCR4 dysfunction in WHIMWT leukocytes. Consistent with this, we showed that GRK3 overexpression in both leukocytes and skin fibroblasts from 2 unrelated WHIMWT patients restored CXCL12-induced internalization and desensitization of CXCR4 and normalized chemotaxis. Moreover, we found in cells derived from one patient a profound and selective decrease in GRK3 products that probably resulted from defective mRNA synthesis. Taken together, these results have revealed a pivotal role for GRK3 in regulating CXCR4 attenuation and have provided a mechanistic link between the GRK3 pathway and the CXCR4-related WHIMWT disorder.

Authors

Karl Balabanian, Angélique Levoye, Lysiane Klemm, Bernard Lagane, Olivier Hermine, Julie Harriague, Françoise Baleux, Fernando Arenzana-Seisdedos, Françoise Bachelerie

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Figure 8

Consequences of GRK3 expression or knock-down on CXCL12-promoted chemotaxis.

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Consequences of GRK3 expression or knock-down on CXCL12-promoted chemota...
(A) Leukocytes from healthy (CTRL#1) and P3 subjects were nucleoporated with 5 μg of either pcDNA1 (vector) or plasmid encoding GRK3 cDNA and assayed 15 h after transfection for chemotaxis in response to CXCL12. (B) IL-2–expanded leukocytes that contained >95% CD25+ blasted T cells, as evaluated by flow cytometry, from independent healthy individuals were nucleoporated with 5 μg SCR or GRK3 siRNAs. Two days after transfection, leukocytes were tested for their ability to migrate in response to CXCL12 (right panel). Transmigrated cells recovered in the lower chamber were stained with mAbs specific for CD3 and CD4 antigens and counted by flow cytometry. Results, expressed as a percentage of input CD4+-gated T cells that migrated to the lower chamber, are representative of those obtained in CD8+-gated T cells. GRK3 transcript levels were evaluated by quantitative PCR and normalized to those of IDUA (B, left panel). Inhibition or increase of GRK3 expression had no effect on cell surface expression of CXCR4. Results are from 1 representative determination of 2 performed in triplicate (A) or are means ± SD of 3 independent determinations performed in duplicate (B, right panel) or in triplicate (B, left panel). *P < 0.05; **P < 0.005 compared with leukocytes transfected with vector or SCR siRNAs.