Abstract
Interleukin 34 (IL-34) constitutes a cytokine that shares a common receptor, colony-stimulating factor-1 receptor (CSF-1R), with CSF-1. We recently identified a novel type of monocytic cell termed follicular dendritic cell-induced monocytic cells (FDMCs), whose differentiation depended on CSF-1R signaling through the IL-34 produced from a follicular dendritic cell line, FL-Y. Here, we report the functional mechanisms of the IL-34-mediated CSF-1R signaling underlying FDMC differentiation. CRIPSR/Cas9-mediated knockout of the Il34 gene confirmed that the ability of FL-Y cells to induce FDMCs completely depends on the IL-34 expressed by FL-Y cells. Transwell culture experiments revealed that FDMC differentiation requires a signal from a membrane-anchored form of IL-34 on the FL-Y cell surface, but not from a secreted form, in a direct interaction between FDMC precursor cells and FL-Y cells. Furthermore, flow cytometric analysis using an anti-IL-34 antibody indicated that IL-34 was also expressed on the FL-Y cell surface. Thus, we explored proteins interacting with IL-34 in FL-Y cells. Mass spectrometry analysis and pulldown assay identified that IL-34 was associated with the molecular chaperone 78-kDa glucose-regulated protein (GRP78) in the plasma membrane fraction of FL-Y cells. Consistent with this finding, GRP78-heterozygous FL-Y cells expressed a lower level of IL-34 protein on their cell surface and exhibited a reduced competency to induce FDMC differentiation compared with the original FL-Y cells. These results indicated a novel GRP78-dependent localization and specific function of IL-34 in FL-Y cells related to monocytic cell differentiation.
Highlights
Interleukin 34 (IL-34) constitutes a cytokine that shares a common receptor, colony-stimulating factor-1 receptor (CSF1R), with CSF-1
We recently identified a novel type of monocytic cell termed follicular dendritic cell-induced monocytic cells (FDMCs), whose differentiation depended on Colony-stimulating factor-1 receptor (CSF-1R) signaling through the IL-34 produced from a follicular dendritic cell line, FL-Y
We found that a membrane-anchored form, but not a secreted from, of IL-34 produced from FL-Y cells was involved in monocytic cell differentiation, the secreted form of IL-34 detected in culture medium could exhibit a stimulating activity through CSF-1R
Summary
To further confirm the IL-34 – dependent differentiation of FDMCs, we first established IL-34 KO FL-Y cells by using the CRISPER/Cas system to eliminate IL-34 expression. Flow cytometric analysis showed that the IL-34 –Nst protein was detectable on the cell surface of FL-Y–IL-34 –Nst cells using either the anti–IL-34 Ab or anti–Strep-tag mAb and that the expression level of IL-34 on FL-Y–IL-34 –Nst cells was significantly higher than that on the original FL-Y cells (Fig. 3B). Consistent with the data from flow cytometric analysis, Western blotting analysis showed that IL-34 –Nst protein was detectable in the plasma membrane fraction of FL-Y–IL-34 –Nst cells but not in that of IL-34 KO FL-Y cells (Fig. 4A). To examine whether the reduced expression of GRP78 abrogates FL-Y–induced FDMC differentiation, we cultured T cell– depleted splenocytes on PFA-treated FL-Y cells for 12 days to induce FDMCs. The number of CD11bϩ cells generated on GRP78-heterozygous FL-Y cells was reduced to approximately half of that on WT FL-Y cells (Fig. 5F). These results clearly indicated that GRP78 promotes IL-34 expression
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