Mechanisms of LPS-induced CD40 expression in human peripheral blood monocytic cells

Abstract

CD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified human peripheral blood monocytic cells (PBMCs) from healthy volunteers. Exposure to LPS induced increases in CD40 mRNA and protein expression on PBMCs. LPS stimulation caused IkappaBalpha degradation. Inhibition of NFkappaB activation abrogated LPS-induced CD40 expression. LPS stimulation also resulted in phosphorylation of mitogen-activated protein kinases, however, only Jun N-terminal kinase (JNK) was partially involved in LPS-induced CD40 expression. In addition, LPS exposure resulted in elevated interferon gamma (IFNgamma) levels in the medium of PBMCs. Neutralization of IFNgamma and IFNgamma receptor using specific antibodies blocked LPS-induced CD40 expression by 44% and 37%, respectively. In summary, LPS-induced CD40 expression on human PBMCs through activation of NFkappaB and JNK, and partially through the induction of IFNgamma production.

Fig 1. LPS exposure results in increase in CD40 expression on human monocytes

A, Human PBMCs were treated with 1 ng/ml LPS for 4 and 8 h. Cells were then lysed with TRIZOL reagent. RNA was extracted and reverse transcribed before further analysis of CD40 mRNA expression using quantitative PCR. CD40 mRNA levels were normalized using the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. B, Human PBMCs were treated with 0.001–1000 ng/ml LPS for 24 h. C, PBMCs were incubated with 1 ng/ml LPS for 6, 12, and 24 h. CD40 protein expression was measured with flow cytometry using isotype and anti-CD40 antibodies, respectively, as described in Materials and Methods. *P<0.05, compared to control (Ct).

Fig 2. NFκB activation is required for LPS-induced CD40 expression

A, PBMCs were treated with 1 ng/ml LPS for 0, 15, 30, 60, and 120 min. IκBα protein levels were measured with Western blotting using anti-IκBα antibody. B, PBMCs were pretreated with DMSO or 2 μM Bay11-7082 for 30 min prior to 1 ng/ml LPS stimulation for 24 h, respectively. CD40 expression (MFI) was measured with flow cytometry. * P<0.05, compared to vehicle (DMSO) LPS.

Fig 3. JNK is partially involved in LPS-induced CD40 expression

PBMCs were stimulated with 1 ng/ml LPS for 15–120 min. Cells were lysed with RIPA buffer. Supernatants of cell lysates were subjected to SDS-PAGE and immunoblotting. Phosphorylated JNK (A), p38 (B), and ERK (C) kinases were detected using phospho-specific antibodies. D, PBMCs were pretreated with DMSO (vehicle), 20 μM SB203580, 20 μM SP600125, or 20 μM PD98059 for 30 min, respectively, prior to 1 ng/ml LPS treatment for 24 h. CD40 expression was determined using flow cytometry. * P<0.05, compared to vehicle (DMSO) LPS. Data shown are representative of three separate experiments.

Fig 4. LPS-induced IFNγ production is involved in CD40 expression

A, PBMCs were treated with 0.01, 1, and 100 ng/ml LPS for 24 h, respectively. Supernatants of cell media were collected for measurement of IFNγ protein with ELISA. *P<0.05, compared to control (Ct). B, PBMCs were treated with 1 ng/ml LPS for 8 and 24 h, respectively. Supernatants of cell media were collected for measurement of IFNγ protein with ELISA. Increase in IFNγ protein release was expressed as fold over control. *P<0.05, compared to control (Ct) at the same time point. C, PBMCs were treated with 1 ng/ml LPS for 8 h prior to lysis with TRIZOL reagent. RNA was extracted and reverse transcribed before further analysis of IFNγ mRNA expression using quantitative PCR. D, PBMCs were pretreated with 20 μg/ml IgG (vehicle), anti-IFNγ, or anti-IFNγR for 2 h before treatment with 1 ng/ml LPS for 24 h, respectively. CD40 expression was determined and expressed as fold over control. *P<0.05, compared to vehicle LPS.