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. Author manuscript; available in PMC: 2013 Apr 1.
Published in final edited form as: J Immunol. 2012 Feb 24;188(7):3447–3457. doi: 10.4049/jimmunol.1102211

Figure 1. Ligation of ITAM-associated β2 integrins and FcγRs inhibits proximal IFNAR signaling.

Figure 1

Primary human monocyte-derived macrophages were plated onto control FBS-coated wells, fibrinogen (Fb) coated wells, or IgG coated wells for the indicated times, and then IFN-α (1000 U/mL) was added for an additional 2 h to assess induction of IFN-α response genes (A,B,G) or 15 min to assess phosphorylation of Jak1 and STAT1 (C,D,F).

A, Macrophages were plated for 2 and 4 h prior to addition of IFN-α. mRNA expression was measured by qPCR, and results are presented as mean ± SD of triplicate wells normalized relative to GAPDH mRNA. Data are representative of at least 3 independent experiments.

B, Cumulative data from macrophages plated for 2 h prior to addition of IFN-α. mRNA expression was measured by qPCR and normalized relative to GAPDH mRNA. Results are presented as mean ± SEM of 11 independent donors. Statistical analysis was performed using the 2-tailed, paired Student t test, p = 0.0203.

C and D, Macrophages were plated for 6 h prior to addition of IFN-α. Whole cell lysates were immunoblotted with Abs against phospho-STAT1 (Y701) and STAT1 (C) and Abs against phospho-JAK1 (Y1022/1023) and JAK1 (D). Data are representative of at least 3 independent experiments.

E, Cell surface expression of both subunits of the type I IFN receptor was measured by flow cytometry. Macrophages were plated for 3 or 4 h. Flow cytometry data from an experiment showing strong downregulation of IFNAR1 are shown (top panels) and cumulative data from 6 independent experiments are shown in the table (bottom). Percent inhibition was calculated by subtracting isotype control staining and comparing IFNAR1 MFI of control and Fb-stimulated cells.

F, Macrophages were plated for 1.5 h on control or IgG coated wells prior to addition of IFN-α. Whole cell lysates were immunoblotted with Abs against phospho-STAT1 (Y701) and STAT1. Data are representative of at least 3 independent experiments.

G, Macrophages were plated for 1.5 h on control or IgG coated wells prior to addition of IFN-α. mRNA expression was measured by qPCR, and results are presented as mean ± SD of triplicate wells normalized relative to GAPDH mRNA. Data are representative of at least 3 independent experiments.

H, Macrophages were stimulated for 4 h with zymosan (10 ug/mL) prior to addition of IFN-α. Whole cell lysates were immunoblotted with Abs against phospho-STAT1 (Y701) and STAT1. Data are representative of 3 independent experiments.