IL-10 receptor and SOCS3 expression levels increase with gestational age.

<p>Fetal type II cells were isolated at different gestational ages (E17–E19). Samples were processed and analyzed by real-time PCR for IL-10 receptor mRNA expression (<b>A</b>), by Western blot for IL-10 receptor protein abundance (<b>B</b>) and by real-time PCR for SOCS3 gene expression (<b>C</b>). <i>Upper panels</i> in <b>B</b> are representative blots. (*P<0.05 and **P<0.05 versus E17).</p

Mechanical stretch decreases IL-10 receptor and IL-10 gene expression and increases SOCS3.

<p>E18 type II epithelial cells were exposed to 20% cyclic stretch for 24 hours. (<b>A</b>) Samples were analyzed by real-time PCR for IL-10 receptor mRNA expression (n = 5, *P<0.0001, **P<0.0001 vs their respective controls). (<b>B</b>) By Western blot for IL-10 R1 protein abundance (n = 4, *P<0.05 vs control). (<b>C</b>) By real-time PCR for IL-10 gene expression (n = 4, *P<0.05). (<b>D</b>) By real-time PCR for SOCS3 gene expression (n = 3, *P<0.05).</p

SOCS3 decreases IL-10 secretion and increases IL-6 release in fetal type II cells exposed to mechanical stretch.

<p><b>A)</b> E18 type II epithelial cells were transfected with nothing (negative), no target or siRNA for SOCS3 [1 µM] and exposed to 20% mechanical stretch for 24 h. Supernatant was collected and IL10 concentration was determined by ELISA (n = 5, *P<0.05 vs their respective controls). The <i>upper panel</i> shows a representative Western blot demonstrating efficient inhibition of SOCS3 protein in samples treated with siRNA SOCS3 at 1 µM. <b>B)</b> E18 type II cells were processed as described in <b>A</b> and exposed to mechanical stretch for 24 h to measure release of IL-6 by ELISA (n = 4, *P<0.05 vs control; **P<0.05 vs stretch no target). <b>C)</b> E18 type II cells were infected with adenovirus expressing SOCS3 at multiplicity of infection (MOI) of 15 or negative control green fluorescent protein (GFP) at the same MOI. Two days later, monolayers were exposed to 20% stretch for 24 h. Supernatants were collected and IL-6 concentration was quantified by ELISA (n = 4, *P<0.05 vs control; **P<0.05 vs stretch GFP). The <i>upper panel</i> shows a representative Western blot showing increased expression of SOCS3 protein in samples infected with adenovirus encoding SOCS3.</p

Effect of mechanical stretch on IL-10 signaling proteins in wild-type cells.

<p>E18 type II epithelial cells were exposed to 20% cyclic stretch for 24 h. Samples were analyzed by Western blot using phospho-JAK1 (<b>A</b>), phospho-TYK2 (<b>B</b>), phospho-STAT3 (<b>C</b>) and total SOCS3 (<b>D</b>) antibodies. Blots were reprobed with total antibodies and GAPDH (for SOCS3) to control for protein loading. <i>Top panels</i> are representative Western blots. Results are from 4 independent experiments. (*P<0.05 vs controls).</p

Effect of mechanical stretch on IL-10 and IL-6 release into the supernatant of type II epithelial cells.

<p><b>A)</b> E18 type II cells were exposed to 20% cyclic stretch for 24 hours. Supernatant was collected and measured for concentrations of IL-10 by ELISA, as described in methods. Results were normalized to the cell lysate concentration in each sample (n = 5, *P<0.02 vs control). <b>B)</b> Samples were processed as above, except that IL-6 release was also measured in type II cells isolated from IL-10 knockout mice (n = 3, *P<0.05 vs control wild-type; n = 3, **P<0.05 vs control IL-10 KO).</p