Figure 4

Immunoassay of cytokine release and cytokine effect on tryptase, chymase, and carboxypeptidase expression of 8 wk HuMC by FACS and QPCR. Effect of rhIL-1β and rhIL-6 blocking antibodies. A) Immunoassay for the presence of IL-1β, IL-6, IL-8, IL-12 and SCF in supernatants of 8 wk HuMC treated for 72 hr with rhSCF, rhSCF and LPS, and rhSCF and PGN; B) Left-sided graph – HuMC numbers expressing tryptase, chymase, and carboxypeptidase following 72 hr in culture with 10 ng/ml rhSCF alone, and with 150 pg/ml rhIL1-β, 25 pg/ml rhIL-6, 5000 pg/ml rhIL-8, and 25 pg/ml rhIL-12. For 10 ng/ml rhSCF alone, the mean ± SEM of percentages of protease positive HuMC are: tryptase 91.50 ± 3.13%, chymase 92.20 ± 3.11%, carboxypeptidase 95.73 ± 1.86%, tryptase/chymase 87.32 ± 3.76% and tryptase/carboxypeptidase 85.70 ± 4.77%. For 10 ng/ml rhSCF and rhIL1-β, rhIL-6, rhIL-8, and rhIL-12, the mean ± SEM of percentages of protease positive HuMC are: tryptase 94.99 ± 0.83%, chymase 97.23 ± 0.35%, carboxypeptidase 97.62 ± 0.50%, tryptase/chymase 89.93 ± 3.87% and tryptase/carboxypeptidase 89.80 ± 4.23%. Results are shown as the mean ± SEM of 3 separate experiments performed in duplicate. Right-sided graph – HuMC numbers expressing tryptase, chymase, and carboxypeptidase following 72 hr in culture with 10 ng/ml rhSCF and 100 ng/ml LPS, and with 0.5 μg/ml anti-rhIL-1β and 1 μg/ml anti-rhIL-6. Goat blocking antibodies are matched with goat isotype IgG control. For 10 ng/ml rhSCF and LPS, the mean ± SEM of percentages of protease positive HuMC are: tryptase 92.30 ± 1.30%, chymase 98.22 ± 0.35%, carboxypeptidase 95.53 ± 1.22%, tryptase/chymase 91.40 ± 2.77%, and tryptase/carboxypeptidase 91.43 ± 1.91%. For 10 ng/ml rhSCF, LPS, anti-rhIL-1β and anti-rhIL-6, the mean ± SEM of percentages of protease positive HuMC are: tryptase 90.60 ± 1.37%, chymase 94.71 ± 0.45%, carboxypeptidase 92.12 ± 1.22%, tryptase/chymase 88.30 ± 2.31%, and tryptase/carboxypeptidase 87.11 ± 3.05%. Results are shown as the mean ± SEM of 2 separate experiments performed in duplicate. C) Left-sided graph – QPCR for tryptase and chymase expression of HuMC cultured with rhSCF alone, and with IL-1β, IL-6, IL-8 and IL-12; Right-sided graph – QPCR for tryptase and chymase expression of HuMC cultured with rhSCF alone, and with LPS. Results are shown as the fold increase over rhSCF alone, and are the mean ± SEM of n = 3 separate experiments performed in triplicate.