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. Author manuscript; available in PMC: 2022 Jul 1.
Published in final edited form as: Am J Transplant. 2021 Jan 4;21(7):2360–2371. doi: 10.1111/ajt.16417

Figure 4. Coculture of peripheral blood MNCs and MCs activates both cell types.

Figure 4.

Peripheral blood MNCs (top chamber) were grown in the presence or absence of MCs (bottom chamber) in a transwell containing serum free media for 24 hours. (A) MNCs cocultured with MC produced significantly higher levels of sIL-6R (86.20±30.82, n=3) compared to PBMCs grown alone (4.88±2.20, n=4, **p<0.01). MNCs treated with conditioned media also had an increase in sIL-6R generation that was lower in the coculture model (37.96±9.30, n=3, *p<0.05). (B) Both MNC cocultured with MCs and treated with MC-conditioned media had significant upregulation of ADAM17 mRNA (0.95±0.19 vs 1.875±0.77 vs 2.183±0.9, n=4, **p<0.001). (C) The addition of the ADAM17 inhibitor GW280264X reduced the amount of sIL-6R stimulated by MC-conditioned media by approximately 51% suggesting that membrane shedding was a significant source of media sIL-6R (13.32+0.25 vs 39.85+3.19 vs 26.22+1.47, n=4, ***p<0.001 ****p<0.0001). (D) The presence of MCs also induced a 50-fold increase in MNC IL-6 mRNA expression (1.158±0.14 versus 56.50±12.92, n=3–4, p=0.0008). (E) The lysates of MCs grown in the presence or absence of MNCs, 10 nM ruxolitinib and/or 0.1 μg/mL IL-6 neutralizing antibody were probed for phospho-STAT3 Tyr705. Coculture stimulated robust STAT3 phosphorylation consistent with IL-6 trans-signaling (*** p<0.001, n=4). This phosphorylation was blocked by inhibition of JAK and neutralization of IL-6.