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. Author manuscript; available in PMC: 2013 Oct 15.
Published in final edited form as: J Immunol. 2012 Sep 12;189(8):4088–4103. doi: 10.4049/jimmunol.1200777

Figure 5. LPS induces mA3 in an IFNαR-independent but TLR4-dependent manner.

Figure 5

(A) PBMCs obtained from IFNαR−/− mice were stimulated with 100 units/ml of endotoxin-free recombinant IFNα for 4 hours, 1μg/ml LPS for 6 hours, or vehicle. After stimulation, RNA was extracted from cells followed by cDNA synthesis and examination of mA3 mRNA levels. (B) Age and weight-matched IFNαR−/− and IFNαR+/+ mice (n=5) on C57BL/6 background were bled to obtain PBMCs for pre-stimulation sample. Mice were then given 200μg of LPS via the IP route. Twenty four hours later, mice were bled to obtain PBMCs which was used to examine mRNA expression level by qPCR. (C and D) PBMCs obtained from age and weight-matched IFNαR−/− and IFNαR+/+ mice were stimulated with 1μg/ml of LPS or vehicle for 6 hours or pre-treated with polymixin B (50μg/ml) or vehicle for 10 minutes and stimulated with LPS for 6 hours. After stimulation, cells were used to evaluate mA3 transcript level by qPCR. (E) Immortalized BMDM from C57BL/6 mice were pre-treated with CLI-095 (1μg/ml) for 10 minutes followed by stimulation with 1000 units/ml of endotoxin-free recombinant IFNα, 1μg/ml LPS, or vehicle for 4 or 6 hours. Cells were used for RNA extraction, cDNA synthesis and qPCR analysis of mA3 mRNA expression level. (F) Immortalized BMDM from WT and TLR4−/− mice in C57BL/6 background were stimulated with 1000 units/ml of endotoxin-free recombinant IFNα, 1μg/ml LPS, or vehicle for 4 or 6 hours followed by RNA extraction. RNA was reverse transcribed and cDNA used for qPCR analysis of mA3 mRNA levels. (G) Primary BMDM was obtained from bone marrow stem cells of C3H/HeN and C3H/HeJ mice. Cells were treated with endotoxin-free recombinant IFNα (1000 units/ml), LPS (1μg/ml), or vehicle for 4 or 6 hours respectively followed by RNA extraction, cDNA synthesis and qPCR analysis of mA3 mRNA levels. (H to L) Immortalized BMDM obtained from WT, MyD88−/−, and TRIF−/− mice in C57BL/6 background were stimulated with 1000 units/ml of endotoxin-free recombinant IFNα, 1μg/ml LPS, or vehicle for 4 or 6 hours followed by RNA extraction. RNA was reverse transcribed and cDNA used for qPCR analysis of (H, I, J, and L) mA3 mRNA levels and (K) TNFα transcripts. Data are normalized to GAPDH and presented as fold change relative to vehicle, LPS, or WT cells. Error bars are standard error, * is significance with p value less than 0.05 and ** is significance with p value less than 0.01. Experiments were repeated at least three different times with similar results.