TLR1/2, TLR7, and TLR9 signals directly activate human peripheral blood naive and memory B cell subsets to produce cytokines, chemokines, and hematopoietic growth factors

Abstract

Recently, it has been reported that using multiple signals, murine and human B cells secrete several cytokines with pro-inflammatory and immunoregulatory properties. We present the first comprehensive analysis of 24 cytokines, chemokines, and hematopoietic growth factors production by purified human peripheral blood B cells (CD19+), and naive (CD19+CD27-) and memory (CD19+CD27+) B cells in response to direct and exclusive signaling provided by toll-like receptor (TLR) ligands Pam3CSK (TLR1/TLR2), Imiquimod (TLR7), and GpG-ODN2006 (TLR9). All three TLR ligands stimulated B cells (CD19+) to produce cytokines IL-1α, IL-1β, IL-6, TNF-α, IL-13, and IL-10, and chemokines MIP-1α, MIP-1β, MCP-1, IP-10, and IL-8. However, GM-CSF and G-CSF production was predominantly induced by TLR2 agonist. Most cytokines/chemokines/hematopoietic growth factors were predominantly or exclusively produced by memory B cells, and in general, TLR2 signal was more powerful than signal provided viaTLR7 and TLR9. No significant secretion of eotaxin, IFN-α, IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-7, IL-15, IL-17, IL-12p40, IL-12p70, and TNF-β (lymphotoxin) was observed. These data demonstrate that human B cells can be directly activated viaTLR1/TLR2, TLR7, and TLR9 to induce secretion of cytokines, chemokines, and hematopoietic growth factors and suggest a role of B cells in immune response against microbial pathogenesis and immune homeostasis.

Fig. 1

TLR-induced activation of B cells. Purified B cells were stimulated by 5 μg/ml and 10 μg/ml of TLR1/TLR2 (Pam), TLR7 (ImQ), and TLR9 (CpG) ligands for 24 h and co-stimulatory and activation molecules were analyzed by multicolor flow cytometry. a. Representative cytoflourograph. b. Cumulative data from three experiments from three healthy individuals

Fig. 2

TLR-induced cytokines (interleukins) production by B cells. Purified B cells were stimulated as in Fig. 1. Supernatants were collected and examined for cytokine production by multiplex biomarker immunoassay. Data are expressed as mean±sd from three separate experiments. All TLRs, although to different levels, induced secretion of proinflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α (a). Immunoregulatory IL-10 (b) IL-13 were also secreted by B cells; however, IL-13 secretion was secreted in low amounts and almost exclusively by TLR1/TLR2 stimulation (c)

Fig. 3

TLR-induced production of chemokines. Purified B cells were stimulated as in Fig. 1. Supernatants were collected and examined for chemokines production. All three TLRs induced secretion of MIP-1α and IP-10 (CXCL3); however, secretion of MIP-1β, MCP-1, and IL-8 by various TLR ligands was variable

Fig. 4

TLR-induced production of hematopoietic growth factors by B cells. Purified B cells were stimulated as in Fig. 1. Supernatants were collected and examined for the production of G-CSF and GM-CSF by multiplex biomarker immunoassay. Both G-CSF and GM-CSF were produced predominantly in response to TLR1/TLR2 agonist

Fig. 5

TLR-induced cytokines (interleukins) produced by naive and memory B cells. Purified B cells were further separated into naive (CD27-) and memory (CD27+) B cells and stimulated with TLR ligands as described in Fig. 1. Data are expressed as mean±sd from three separate experiments. All four pro-inflammatory cytokines (IL-1α, IL-1β, TNF-α, and IL-6) were produced predominantly by memory B cells, in response to all three TLRs ligands (a). Immunoregulatory IL-10 was produced by both naive and memory B cells by all three TLRs; however, significantly (P < 0.01) more by memory B cells in response to TLR1/TLR2 stimulation (b). In contrast, similar levels of immunoregulatory IL-13 were produced by naive and memory B cells and almost exclusively by TLR1/TLR2 stimulation (c)

Fig. 6

TLR-induced production of chemokines by naive and memory B cells. Purified B cells were further separated into naive (CD27-) and memory (CD27+) B cells and stimulated with TLR ligands as described in Fig. 1. All TLRs induced chemokines production by both naive and memory B cells

Fig. 7

TLR-induced production of hematopoietic growth factors by naive and memory B cells. Purified B cells were further separated into naive (CD27-) and memory (CD27+) B cells and stimulated with TLR ligands as described in Fig. 1. TLR1/TLR2 and TLR7 induced G-CSF and GM-CSF secretion almost exclusively by memory B cells. TLR9 stimulations did not induce secretion of either growth factor