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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.

Agrawal S, Gupta S - J. Clin. Immunol. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic and Clinical Immunology, Medical Sciences I, C-240, University of California, Irvine, CA 92697, USA. sagrawal@uci.edu

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.

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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
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Fig1: 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

Mentions: Purified CD19+ B cells were stimulated with two different concentrations (determined by concentration kinetic studies) of Pam, ImQ, and CpG for 24 h. At the end of culture, cells were washed and stained with FITC-anti-CD80, PE-anti-CD86, PerCP-HLA-DR, and APC-anti-CD25 or isotype controls for 30 min on ice. Cells were then washed and examined for the expression of these activation and co-stimulatory antigens with multicolor flow cytometry using FACSCalibur. Ten thousand cells were acquired and analyzed by FlowJo software. Figure 1a displays a representative cytograph, and Fig. 1b shows cumulative data for fluorescence intensity (MFC#, mean±sd) from three separate experiments using three separate normal young subjects. All three TLR ligands significantly (P < 0.05-P < 0.001) upregulated expression of CD80, CD86, and CD25; ImQ did not upregulate HLA-DR expression (P > 0.05). It is also apparent that different TLR ligands upregulated activation antigens to different extent. CD80, HLA-DR, and CD25 were activated to a greater extent (P < 0.05) by Pam and CpG as compared to ImQ, whereas CD86 was upregulated to a greater extent (P < 0.05) by Pam and ImQ than by CpG.Fig. 1


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.

Agrawal S, Gupta S - J. Clin. Immunol. (2010)

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
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3064903&req=5

Fig1: 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
Mentions: Purified CD19+ B cells were stimulated with two different concentrations (determined by concentration kinetic studies) of Pam, ImQ, and CpG for 24 h. At the end of culture, cells were washed and stained with FITC-anti-CD80, PE-anti-CD86, PerCP-HLA-DR, and APC-anti-CD25 or isotype controls for 30 min on ice. Cells were then washed and examined for the expression of these activation and co-stimulatory antigens with multicolor flow cytometry using FACSCalibur. Ten thousand cells were acquired and analyzed by FlowJo software. Figure 1a displays a representative cytograph, and Fig. 1b shows cumulative data for fluorescence intensity (MFC#, mean±sd) from three separate experiments using three separate normal young subjects. All three TLR ligands significantly (P < 0.05-P < 0.001) upregulated expression of CD80, CD86, and CD25; ImQ did not upregulate HLA-DR expression (P > 0.05). It is also apparent that different TLR ligands upregulated activation antigens to different extent. CD80, HLA-DR, and CD25 were activated to a greater extent (P < 0.05) by Pam and CpG as compared to ImQ, whereas CD86 was upregulated to a greater extent (P < 0.05) by Pam and ImQ than by CpG.Fig. 1

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic and Clinical Immunology, Medical Sciences I, C-240, University of California, Irvine, CA 92697, USA. sagrawal@uci.edu

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.

Show MeSH
Related in: MedlinePlus