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 (WHIM WT ) 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 WHIM WT cells. These findings identified GRK3 as a negative regulator of CXCL12-induced chemotaxis and as a candidate responsible for CXCR4 dysfunction in WHIM WT leukocytes. Consistent with this, we showed that GRK3 overexpression in both leukocytes and skin fibroblasts from 2 unrelated WHIM WT 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 WHIM WT disorder.

Correlation of CXCL12 Expression and FoxP3+ Cell Infiltration with Human Papillomavirus Infection and Clinicopathological Progression of Cervical Cancer

“…30 The WHIM syndrome (wart, hypogammaglobulinemia, infection, and myelokathexis syndrome), which is a syndrome characterized by disseminated HPVinduced warts, leukopenia due to bone marrow cell retention and other immune dysfunctions, has been correlated to a defect in CXCR4 receptor and CXCL12 signal transmission, thus confirming a major role of the cytokine in contributing to immune cell dysregulation and T cell-impaired trafficking in the context of HPVderived lesions. 31 Our study confirmed a significant relationship between HPV typing and CXCL12% area in both squamous and glandular neoplastic lesions, showing an increase of CXCL12% area in cases that were positive for both HPV6,11 and HPV16ϩ probes compared with cases that were negative for both probes. Interestingly, cases that were positive for only HPV6,11 demonstrated a higher CXCL12% area than those that were only HPV16ϩ positive.…”

Neutrophil Extracellular Traps Downregulate Lipopolysaccharide-Induced Activation of Monocyte-Derived Dendritic Cells

“…Phenotypic results were expressed as the relative mean fluorescence intensity (MFI), corresponding to the MFI of the specific label/MFI of the isotype control. CCR5, CCR7, and CXCR4 expression was analyzed with a LSR-Fortessa cytometer, and the results were displayed in MFI, as previously described (48). Cell viability was determined by labeling with annexin V and 7-aminoactinomycin D, following the manufacturer's instructions, followed by analysis on a FACSCalibur cytometer.…”

Imaging CXCR4 Signaling with Firefly Luciferase Complementation

“…Binding of CXCL12 to CXCR4 causes ligand-dependent conformational changes in the receptor, resulting in phosphorylation of the intracellular C-terminus of CXCR4 by protein kinase C and G protein-coupled receptor kinases (GRK) such as GRK3. 17 The phosphorylated receptor then recruits a member of the β-arrestin family of cytosolic scaffolding proteins, either β-arrestin-1 or β-arrestin-2. The interaction between activated chemokine receptor and β-arrestin causes internalization of the bound chemokine-receptor pair and regulates signaling through downstream effector molecules such as ERK1/2.…”