ABSTRACT
OBJECTIVES: B-cell depletion time after rituximab (RTX) treatment is prolonged in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) compared with other autoimmune diseases. We investigated central and peripheral B-cell development to identify the causes for the defect in B-cell reconstitution after RTX therapy. METHODS: We recruited 91 patients with AAV and performed deep phenotyping of the peripheral and bone marrow B-cell compartment by spectral flow and mass cytometry. B-cell development was studied by in vitro modelling and the role of BAFF receptor by quantitative PCR, western blot analysis and in vitro assays. RESULTS: Treatment-naïve patients with AAV showed low transitional B-cell numbers, suggesting impaired B-lymphopoiesis. We analysed bone marrow of treatment-naïve and RTX-treated patients with AAV and found reduced B-lymphoid precursors. In vitro modelling of B-lymphopoiesis from AAV haematopoietic stem cells showed intact, but slower and reduced immature B-cell development. In a subgroup of patients, after RTX treatment, the presence of transitional B cells did not translate in replenishment of naïve B cells, suggesting an impairment in peripheral B-cell maturation. We found low BAFF-receptor expression on B cells of RTX-treated patients with AAV, resulting in reduced survival in response to BAFF in vitro. CONCLUSIONS: Prolonged depletion of B cells in patients with AAV after RTX therapy indicates a B-cell defect that is unmasked by RTX treatment. Our data indicate that impaired bone marrow B-lymphopoiesis results in a delayed recovery of peripheral B cells that may be further aggravated by a survival defect of B cells. Our findings contribute to the understanding of AAV pathogenesis and may have clinical implications regarding RTX retreatment schedules and immunomonitoring after RTX therapy.
ABSTRACT
AIMS: Being central part of the DNA repair machinery, DNA-dependent protein kinase (DNA-PK) seems to be involved in other signalling processes, as well. NOR1 is a member of the NR4A subfamily of nuclear receptors, which plays a central role in vascular smooth muscle cell (SMC) proliferation and in vascular proliferative processes. We determined putative phosphorylation sites of NDA-PK in NOR1 and hypothesized that the enzyme is able to modulate NOR1 signalling and, this way, proliferation of SMC. METHODS AND RESULTS: Cultured human aortic SMC were treated with the specific DNA-PK inhibitor NU7026 (or siRNA), which resulted in a 70% inhibition of FCS-induced proliferation as measured by BrdU incorporation. Furthermore, FCS-stimulated up-regulation of NOR1 protein as well as the cell-cycle promoting proteins proliferating cell nuclear antigen (PCNA), cyclin D1, and hyperphosphorylation of the retinoblastoma protein were prevented by DNA-PK inhibition. Co-immunoprecipitation studies from VSM cell lysates demonstrated that DNA-PK forms a complex with NOR1. Mutational analysis and kinase assays demonstrated that NOR1 is a substrate of DNA-PK and is phosphorylated in the N-terminal domain. Phosphorylation resulted in post-transcriptional stabilization of the protein through prevention of its ubiquitination. Active DNA-PK and NOR1 were found predominantly expressed within the neointima of human atherosclerotic tissue specimens. In mice, inhibition of DNA-PK significantly attenuated neointimal lesion size 3 weeks after wire-injury. CONCLUSION: DNA-PK directly phosphorylates NOR-1 and, this way, modulates SMC proliferation. These data add to our understanding of vascular remodelling processes and opens new avenues for treatment of vascular proliferative diseases.