Leptin activates human B cells to secrete TNF-α, IL-6, and IL-10 via JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathway

Abstract

Leptin, one of the adipokines, functions as a hormone and a cytokine. In this investigation, we show for the first time that leptin, in a concentration-dependent manner, activates human peripheral blood B cells to induce secretion of IL-6, IL-10, and TNF-α. Leptin increased B cells expressing CD25 and HLA-DR. Leptin induces phosphorylation of Janus activation kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), p38 mitogen-activated protein kinase (p38MAPK), and extracellular signal-regulated kinase (ERK1/2). Furthermore, leptin-induced cytokine secretion by B cells was blocked by inhibitors of JAK2, STAT3, p38MAPK, and ERK1/2. These data demonstrate that leptin activates human B cells to secrete cytokines via activation of JAK2/STAT3 and p38MAPK/ERK1/2 signaling pathways, which may contribute to its inflammatory and immunoregulatory properties.

Fig. 1

Leptin stimulates human B cells to secrete IL-6, TNF-α, and IL-10. B cells were purified from MNCs from healthy young donors by CD19 beads (>97% CD19+) and incubated with various concentrations of human recombinant leptin for 24 h, and supernatants were collected and analyzed by ELISA assay. Figure 2 shows data from three subjects (mean ± SD). Leptin, in a concentration-dependent manner, induced significant secretion of TNF-α (a), IL-6 (b), and IL-10 (c). Leptin did not induce secretion of IL-1β (d)

Fig. 2

Leptin activates B cells. Purified B cells were stimulated in the presence or absence of 50 ng/ml of human recombinant leptin for 24 h, and then stained with CD25, and CD69, CD86, and HLA-DR antibodies and isotype controls, and analyzed by multicolor flow cytometry. Data are from three different subjects. Leptin increased significantly (*P < 0.05) proportions of B cells with expression of CD25 and HLA-DR (a). However, no significant difference was observed on the density of any of the activation molecules as determined by mean flourescence intensity (MFI) on B cells (b)

Fig. 3

Leptin activates B cells via JAK2, STAT3, ERK1/2, and p38MAPK. Purified B cells from young healthy subjects were cultures in the presence or absence of leptin (50 ng/ml) for 10 min. Cell lysates were prepared, and western blotting was performed using native and phospho anti-JAK2, anti-STAT3, anti-ERK1/2, and anti-p38 MAPK antibodies. Actin was used as a loading control. Bands were scanned, and volumes were calculated by densitometry. Quantification was performed by a ratio between signaling molecule and actin. Leptin induced phosphorylation of JAK2 and STAT3 (a), and of p38 MAPK and ERK1/2 (b)

Fig. 4

Inhibitors of JAK2/STAT3, p38MAPK, and ERK1/2 inhibit leptin-induced secretion of cytokines. Purified B cells were incubated with 50 ng/ml of leptin in the presence or absence of inhibitors of ERK1/2 (1 μM SB203580), JAK2 (25 μM Tyrphostin AG490), STAT3 (WP1066), or p38 MAPK inhibitor (12.5 μM U0126) for 24 h, and cytokines were measured in the supernatants by ELISA assay. Each inhibitor significantly blocked leptin-induced secretion of IL-6, TNF-α, and IL-10 (* = P < 0.05, **P < 0.001)