Dataset Information

Short cytoplasmic isoform of IL1R1/CD121a mediates IL1β induced proliferation of synovium-derived mesenchymal stem/stromal cells through ERK1/2 pathway.

ABSTRACT:

Objectives

IL1β enhances proliferation of synovial mesenchymal stem/stromal cells (synMSCs) although they don't express its receptor, IL1R1/CD121a, on the cell surface. This study was aimed to elucidate the underlying mechanisms of IL1β-mediated growth promotion.

Methods

Human synMSCs were isolated from the suprapatellar synovial membrane. Cell proliferation was measured by MTT. Flowcytometric analyses were performed for surface antigen expression. Intracellular signaling pathway was analyzed by western blotting, immunocytochemistry and Q-PCR.

Results

IL1β enhanced proliferation through IL1R1/CD121a because IL1 receptor antagonist (IL1Ra) completely inhibited it. Expression analyses indicated that a short isoform of IL1R1/CD121a is expressed in synMSCs. Immunocytochemistry indicated that IL1R1/CD121a was majorly localized to the cytoplasm. Western blotting indicated that IL1β induced delayed timing of the ERK1/2 phosphorylation and IκBα degradation in synMSCs. Q-PCR analyses for IL1β-target genes indicated that cyclin D was specifically downregulated by a MAPK/ERK inhibitor, U0126, but not by a NFκB inhibitor, TPCA-1. In contrast, the expression of inflammatory cytokines such as IL1α and IL6 are significantly decreased by TPCA-1 but less effectively decreased by U0126.

Conclusion

Our data indicated that the cytoplasmic IL1R1/CD121a transduced IL1β signal in synMSCs. And the growth-promoting effect of IL1β can be separated from its inflammatory cytokine-inducing function in synMSCs.

SUBMITTER: Tang G

PROVIDER: S-EPMC9133583 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Publications

Short cytoplasmic isoform of IL1R1/CD121a mediates IL1β induced proliferation of synovium-derived mesenchymal stem/stromal cells through ERK1/2 pathway.

Tang Guo G Asou Yoshinori Y Matsumura Etsuko E Nakagawa Yusuke Y Miyatake Kazumasa K Katagiri Hiroki H Nakamura Tomomasa T Koga Hideyuki H Komori Keiichiro K Sekiya Ichiro I Ezura Yoich Y Tsuji Kunikazu K

Heliyon 20220518 5

<h4>Objectives</h4>IL1β enhances proliferation of synovial mesenchymal stem/stromal cells (synMSCs) although they don't express its receptor, IL1R1/CD121a, on the cell surface. This study was aimed to elucidate the underlying mechanisms of IL1β-mediated growth promotion.<h4>Methods</h4>Human synMSCs were isolated from the suprapatellar synovial membrane. Cell proliferation was measured by MTT. Flowcytometric analyses were performed for surface antigen expression. Intracellular signaling pathway ...[more]

PMID: 35647352

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Project description:IntroductionTrauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3-7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less.MethodsThree potential assays were examined-assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2-72 h.ResultsSuppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h.DiscussionTakeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2-6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h.

Dataset Information

Short cytoplasmic isoform of IL1R1/CD121a mediates IL1β induced proliferation of synovium-derived mesenchymal stem/stromal cells through ERK1/2 pathway.

Objectives

Methods

Results

Conclusion

Publications

Short cytoplasmic isoform of IL1R1/CD121a mediates IL1β induced proliferation of synovium-derived mesenchymal stem/stromal cells through ERK1/2 pathway.

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