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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 2. IL-6 trans-signaling stimulates prolonged STAT3 phosphorylation via a JAK2-dependent mechanism in human BAL-derived MCs.

Figure 2.

(A) Confluent cultures of pulmonary tissue-derived fibroblasts and BAL-derived MC were grown in serum free media for 24 hours. The media was removed and IL-6 and sIL-6R concentrations were measured by ELISA. Tissue-derived fibroblasts secreted approximately one-tenth of the IL-6 (44.75±20.49pg/mL, n=3) produced by BAL-derived MCs cells (474.5±74.11pg/mL, n=6, p<0.0001). (B) Neither tissue-derived fibroblasts or BAL-derived MCs secrete significant amounts of sIL-6R (0.2749±0.24 pg/mL, n=4 and 0.4600±0.25 pg/mL, n=8 respectively). (C) MCs were treated with vehicle, 50 ng/mL IL-6, 200 ng/mL sIL-6R or the combination of IL-6 and sIL-6R for 30 minutes. Representative western blot and quantification of STAT3 Tyr705 phosphorylation. sIL-6R alone stimulates a 7.5-fold increase in STAT3 Tyr705 phosphorylation while the combination of IL-6 and sIL-6R stimulates approximately 15-fold increase in STAT3 Tyr705 phosphorylation compared to vehicle control (n=6, ***p=0.001). (D) Reduction in endogenously produced IL-6 by addition of an IL-6 neutralizing antibody completely blocks sIL-6R-stimulated STAT3 phosphorylation (0.824±0.54 vs 11.707±7.53 vs 0.400±0.93, ***p < 0.001, ****p<0.0001, n=7). (E) IL-6/sIL-6R treatment induces significant prolonged STAT3 Tyr705 phosphorylation (n=3, *p<0.05 **p<0.01 compared to time 0). (F) MCs grown on coverslips were treated with either vehicle or the combination of IL-6 and sIL-6R for one hour and then labeled by immunofluorescence phospho-STAT3 Tyr705 and DAPI (n=3, representative images shown). IL-6/sIL-6R stimulated phospho-STAT3 was predominantly observed within the nucleus of the cell. MCs were co-treated with IL-6/sIL-6R and various concentrations of the JAK inhibitors (G) ruxolitinib, (H) NVP-BSK805 and (I) WHI-P154. Ruxolitinib (JAK 1/2 pan inhibitor; JAK1 IC50 3.3 nM, JAK2 IC50 2.8 nM) and NVP-BSK805 (JAK 2 specific inhibitor; JAK1 IC50 31.6nM, JAK2 IC50 0.5nM) were able to block IL-6/sIL-6R-mediated STAT3 phosphorylation in a dose dependent manner. WHI-P154 (JAK 3 inhibitor; JAK3 IC50 1.8μM) had no effect on IL-6/sIL-6R stimulated STAT3 phosphorylation (n=3, representative images shown).