Inhibition of dendritic cell-mediated antigen-specific Th1 cell responses by lidocaine <i>in vivo</i>.

<p>Bone marrow-derived dendritic cells were pulsed with OVA_323-339 in the presence of lidocaine or vehicle before being transferred into OT-II TcR transgenic mice (n = 3~4). (A and B) The frequencies of IFN- γ producers among Vα2⁺ cells. (C) The amounts of the indicated cytokines in the supernatant of splenocyte stimulated with OVA_323-339 were measured by ELISA. Data represent two independent experiments. Data shown are mean ± SEM. *<i>p</i><0.05.</p

Regulation of cytokines expression in dendritic cells in response to various TLR ligands by lidocaine.

<p>(A & B) Bone marrow-derived dendritic cells were stimulated with LPS (100 ng/ml), poly(I:C) (1 μg/ml) or R837 (1 μg/ml) in the presence of lidocaine (0.4 mg/ml) or vehicle. The amounts of IL–6 and TNF-α in the supernatant were measured by ELISA. (C) The mRNA levels of the indicated genes were analyzed by quantitative RT-PCR. Data represent at least two independent experiments. Data shown are mean ± SEM. *<i>p</i><0.05; **<i>p</i><0.01; ND, not detected.</p

Lidocaine inhibits dendritic cell-mediated Th1 cell differentiation while having little effects on dendritic cell-mediated Th2, Th17, regulatory T cell differentiation <i>in vitro</i>.

<p>Naïve CD4⁺ T cells were co-cultured with bone marrow-derived dendritic cells with Th1, Th17, Th2 or regulatory T cell differentiation conditioned-media or cultured with plate-coated anti-CD3 and anti-CD28 with supernatant of dendritic cells stimulated with LPS in the presence of lidocaine (0.2 mg/ml or indicated dose) or vehicle. (A-D) The frequencies of IFN-γ, IL–17, IL–4/5 or Foxp3 positive cells among CD4⁺ population were measured by flow cytometer. (E) The level of IFN-γ was measured using co-cultured supernatants from Th1 differentiation condition. Data represent at least two independent experiments. Data shown are mean ± SEM. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001; NS, not significant.</p

Lidocaine inhibits dendritic cell-mediated Th1 cell differentiation <i>in vitro</i>.

<p>Naïve CD4⁺ T cells were either co-cultured with bone marrow-derived dendritic cells in the presence of soluble anti-CD3 and LPS, or in anti-CD3, CD28 pre-coated plates in the presence of IL–2 and IL–12 for Th1 cell differentiation. Lidocaine was added at a concentration of 0.2 mg/ml. (A & B) The frequencies of IFNγ or IL–17 producing cells among CD4⁺ T cells. (C) The mRNA levels of the indicated genes. (D) The levels of IFN-γ in the cultured supernatants of naïve CD4⁺ T cells cultured with vehicle- or lidocaine-conditioned media. Data represent at least three independent experiments. Data shown are mean ± SEM. **<i>p</i><0.01; ***<i>p</i><0.001; NS, not significant.</p

Effects of lidocaine on the expression of various cytokines upon LPS stimulation.

<p>Bone marrow-derived dendritic cells were stimulated with 100 ng/ml of LPS in the presence of vehicle (EtOH) or 0.2 mg/ml lidocaine for 4 h and 24 h to examine mRNA expression and cytokine production, respectively. (A) The mRNA levels of the indicated genes were analyzed by quantitative RT-PCR. (B) The amounts of each cytokine produced were measured by ELISA. All experiments were performed at least three times. Data shown are mean ± SEM. *<i>p</i><0.05; ***<i>p</i><0.001; ND, not detected.</p

Lidocaine regulates the expression of cytokines and NF-κB signaling pathway in a dose-dependent manner.

<p>(A) Bone marrow-derived dendritic cells were stimulated with 100 ng/ml of LPS together with increasing concentrations of lidocaine for 4 h. The mRNA levels of the indicated genes were analyzed by quantitative RT-PCR. (B) Raw 264.7 cells were treated with increasing doses of lidocaine for 2 h and stimulated with LPS for 20 min. The expression of IκB-α was examined by western blot. All experiments were performed at least three times. Data shown are mean ± SEM. *<i>p</i><0.05; **<i>p</i><0.01.</p

Spontaneous germinal center responses in BXD2 mice.

<p>(<b>A</b>) Auto-reactive autoantibody levels against double-strand DNA and histone in the sera of WT and BXD2 mice at the age of 6 months or older were measured by ELISA. (<b>B</b>) Immunofluorescence imaging of PNA⁺ (red) germinal center area of the spleen from WT control or BXD2 mice (×4 magnification). (<b>C</b>) The number of GC per spleen section in the WT and BXD2 mice was enumerated by fluorescence microscopy. (<b>D</b>) Flow cytometry analysis of the percentage and number of GL7⁺Fas⁺ germinal center B cells in the WT and BXD2 mice at the age of 6 months or older. Cells were gated on B220⁺ B cells. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.</p

Positive correlation of Tfh cells, not Th17 cells with outputs of GC responses in BXD2 mice.

<p>Linear regression analysis of the frequency of Th17 cells (<b>A</b>) or Tfh cells (<b>D</b>) with that of germinal center B cells, and Th17 cells (<b>B</b>) or Tfh cells (<b>E</b>) with dsDNA specific autoantibody levels. Linear regression analysis of the number of Th17 cells (C) or Tfh cells (F) with that of germinal center B cells. Pearson correlation coefficients (r²) between the percent of T helper cell subset and of germinal center B cells or those of T helper cell subset and dsDNA specific autoantibodies levels are indicated in each graph.</p

Regulation of Autoimmune Germinal Center Reactions in Lupus-Prone BXD2 Mice by Follicular Helper T Cells

<div><p>BXD2 mice spontaneously develop autoantibodies and subsequent glomerulonephritis, offering a useful animal model to study autoimmune lupus. Although initial studies showed a critical contribution of IL-17 and Th17 cells in mediating autoimmune B cell responses in BXD2 mice, the role of follicular helper T (Tfh) cells remains incompletely understood. We found that both the frequency of Th17 cells and the levels of IL-17 in circulation in BXD2 mice were comparable to those of wild-type. By contrast, the frequency of PD-1⁺CXCR5⁺ Tfh cells was significantly increased in BXD2 mice compared with wild-type mice, while the frequency of PD-1⁺CXCR5⁺Foxp3⁺ follicular regulatory T (Tfr) cells was reduced in the former group. The frequency of Tfh cells rather than that of Th17 cells was positively correlated with the frequency of germinal center B cells as well as the levels of autoantibodies to dsDNA. More importantly, CXCR5⁺ CD4⁺ T cells isolated from BXD2 mice induced the production of IgG from naïve B cells in an IL-21-dependent manner, while CCR6⁺ CD4⁺ T cells failed to do so. These results together demonstrate that Tfh cells rather than Th17 cells contribute to the autoimmune germinal center reactions in BXD2 mice.</p></div

IL-21-producing CXCR5⁺CD4⁺ T cells of BXD2 mice, not IL-17-producing CCR6⁺CD4⁺ T cells, provide B cell help for IgG production.

<p>(<b>A</b>) CXCR5⁺ CD4⁺ T cells and CCR6⁺ CD4⁺ T cells were sorted and subjected to intracellular cytokine staining. (<b>B</b>) Quantitative RT-PCR analysis of Tfh or Th17 cell related gene expression in the CXCR5⁺ and CCR6⁺ CD4⁺ T cells from BXD2 mice (<b>C</b>) Naïve B cells (B220⁺IgD⁺GL7^-) from BXD2 mice were co-cultured with CXCR5⁺ or CCR6⁺ CD4⁺ T cells from BXD2 mice for 7days. The total IgG levels were measured by ELISA (<b>D</b>) Different cytokine blocking reagents, isotype control antibody (Iso Hu-Fc), Rat anti mouse IL-17A antibody (α-IL-17A), or recombinant mouse IL-21 receptor Fc chimera (IL-21R-Fc) were added (10 μg/ml, every other day) into the cell culture described in (<b>C</b>) and the levels of total IgG were measured by ELISA. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ^###p < 0.001 comparing 10:10–10:5 ratio of B:T co-culture condition.</p