Figure 1.
Total serum IgE in donor mice along with surface markers and cytokine production by B-lymphocytes of the auricular lymph nodes.
Serum and lymphocytes from the auricular lymph nodes were obtained of mice dermally treated with vehicle (DVeh) or TDI (DTDI). The lymphocytes were stained with anti-CD19 to identify B lineage cells and for different surface markers and co-stimulatory molecules. CD19+-lymphocytes were co-stained with anti-CD23 and anti-IgD to distinguish between follicular and marginal zone B-lymphocytes (A); co-stained with CD5 to distinguish between B1- (B1a) and B2-lymphocytes (B) and co-stained with MHCII, CD86, CD80 and CD40 to characterize the antigen presentation capacity of the B-lymphocytes (C). Lymphocytes of auricular lymph nodes were cultured in vitro for 5 hours with PMA, Ca2+ ionophore and monensin. Anti-CD19 was used to identify B lineage cells and the percentage B-lymphocytes staining intracellularly for the cytokines IL-4, IFN-γ and IL-10 was assessed. Figure 1 D shows representative dot plots of intracellular cytokine expression in B-lymphocytes from TDI-sensitized mice. The percentage of the total B-lymphocytes expressing cytokines is quantified in graph E. In the serum of Dveh or DTDI mice, total IgE levels were measured (F). Data are presented as means ± SEM, n = 4-5, * p < 0.05 and *** p < 0.001.
Figure 2.
Production of an asthma-like response in naïve wild type BALB/c mice after having received B-lymphocytes.
Experimental groups are DTDIRVeh, DTDIRTDI, DTMARVeh, DTMARTMA and DTDIRTMA. D represents donor (D) animals that received dermal applications of TDI (DTDI) or TMA (DTMA) on days 1 and 8. Their B-lymphocytes were transferred into naïve recipient (R) mice which received a challenge with vehicle (RVeh), TDI (RTDI) or TMA (RTMA) three days after the transfer. Airway resistance (R), after increasing concentrations of methacholine (0-10 mg/ml), was measured using a forced oscillation technique, 22 hours after the challenge. Figure 2 A reflects the airway hyperreactivity (AHR) to increasing concentrations of methacholine after transferring B-lymphocytes of auricular lymph nodes into wild type BALB/c mice. Macrophages and neutrophils were identified in the BAL fluid (B) and in lung tissue (D) 24 hours after the challenge. Total serum IgE was measured (C). Figure 2 E, F, G and H represent AHR of the experiment assessing the transfer of the B-lymphocytes of TMA sensitized mice, the specificity of the B-lymphocytes to TDI, the transfer of serum and the transfer of B-lymphocytes of the spleen, respectively. Data are presented mean ± SEM, n = 4-10 per group, * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the DTDIRVeh group (A, B, C, G and H) and with DTMARTMA (E). Symbols: (↑) inflammation and (▲) epithelial damage.
Figure 3.
Transferring B-lymphocytes leads to an asthma-like response after TDI challenge in B-KO and SCID BALB/c mice.
Airway methacholine reactivity was measured after transferring B-lymphocytes in B-KO (A) or SCID (F) mice. Macrophages and neutrophils were identified in the BAL fluid (B and G) and in lung tissue (D, E and H, I) 24 hours after the challenge. Total IgE was assessed in serum of B KO mice. Experimental groups for the adoptive transfer setup are identical to those of Figure 2 (DTDIRVeh and DTDIRTDI). Data are presented as means ± SEM, n = 5-8 per group, * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the DTDIRVeh group. Symbols: (↑) inflammation and (▲) epithelial damage.
Figure 4.
Transferred B-lymphocytes are present in the lungs of TDI challenged wild type BALB/c mice.
Freshly isolated B-lymphocytes of the auricular lymph nodes of TDI-sensitized mice were labeled with DAPI and SNARF-1 carboxylic acid acetate and transferred into naïve wild type BALB/c mice. 5x106 labeled B-lymphocytes were transferred. Three days after the transfer mice were challenged with TDI and cryostat sections were made. Experimental groups for the adoptive transfer setup are identical to those of Figure 2 (DTDIRVeh and DTDIRTDI). Figure C shows the merged picture of the DAPI (A) and SNARF-1 (B) staining.