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 2

Selective alteration of CXCR4 functioning in WHIMWT cells.

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Analysis of CXCR4 expression in WHIMWT-derived cells. (A...
(A) EBV-B cells from CTRL and P4 individuals were nucleoporated with 5 μg of plasmids encoding CCR5, CCR7, CXCR2, CXCR4, or CXCR5 cDNA and assayed 15 h after transfection for chemotaxis using a Transwell system in response to the cognate chemokine (left section). Concentration-dependent migration of B cells expressing CXCR4 is shown in response to CXCL12 (right section). Results are expressed as a percentage of input B cells that migrated to the lower chamber. (B) Fibroblasts expressing CXCR4 (left panel) and B cells expressing either CXCR4 (middle panel) or CXCR5 (right panel) were tested for CXCL12- or CXCL13-triggered actin polymerization using FITC-phalloidin as a probe for intracellular F-actin. Arrows indicate chemokine stimulation. The baseline level of unstimulated cells was set as 100% (dotted line). (C) Cell surface expression of distinct chemokine receptors in control (CTRL#1) and WHIMWT fibroblasts treated with 200 nM of the cognate agonist for 45 min at 37°C. Results indicate the amount of receptors that remains at the cell surface after incubation with agonist. Receptor expression at the surface of cells incubated in medium alone was set as 100%. Following transfection, expression levels of the different chemokine receptors were comparable at the surface of all unstimulated cell types as determined by flow cytometry (data not shown). Results represent the means ± SD of 3–5 independent experiments (A and C) or are representative of 3 independent determinations (B). *P < 0.05; **P < 0.005 compared with healthy subjects.