Journal: Mediators of Inflammation

Article Title: CD4 + T Cells Promote IgG Production in MHC-Independent and ICAM-1-Dependent Manners in Pristane-Induced Lupus Mice

doi: 10.1155/2022/9968847

Figure Lengend Snippet: Serum immunoglobulin levels in pristane-induced lupus-like mice. Sera were collected before the injection and at 6 months post-PBS or pristane treatment. The levels of total IgG (a), total IgM (b), IgG2a (c), and anti-P0 (d), anti-SnRNP (e), and anti-dsDNA (f) autoantibodies in the serum were determined by an ELISA assay. Furthermore, the kidneys were collected when the mice were sacrificed at 6 months after PBS/pristane injection. The deposition of immune complexes (g) in the kidney was detected by the immunofluorescence assay. (h) Statistic analysis of immune complexes in the kidneys. Data were pooled from three independent experiments with 5 mice per group in each experiment. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: 100 μ L diluted samples were added to the wells, and the plates were incubated at RT for 2 h. After washing 3 times with PBST, the wells were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (H+L) (for total IgG, anti-dsDNA, and anti-Sn-RNP), HRP-conjugated goat anti-mouse IgM (SouthernBiotech), HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech), or alkaline phosphatase- (AP-) conjugated goat anti-mouse IgG2a Abs (Rockland, Gilbertsville, PA, USA), respectively, at RT for 2 h. Tetramethylbenzidine (TMB) (BD Biosciences, San Diego, CA, USA) solution (for HRP) or VISIGLO AP CHEMILUM SUBSTRATE (Sigma) solution (for AP) (100 μ L/well) was added.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Mediators of Inflammation

Article Title: CD4 + T Cells Promote IgG Production in MHC-Independent and ICAM-1-Dependent Manners in Pristane-Induced Lupus Mice

doi: 10.1155/2022/9968847

Figure Lengend Snippet: CD4 + T cells are more inclined to promote IgG production in pristane-induced lupus-like mice. Serum from PBS or pristane-treated C57BL/6 and TCR α −/− mice (on a C57BL/6 background) were subjected to the determination of total IgM/IgG and IgG subtype IgG1/IgG2a by the ELISA assay. OD 450 value was detected to represent IgM (a), IgG (b), IgG1 (c), and IgG2a (d) levels in the serum of the mice. CD4 + T cells sorted from PBS and pristane-treated C57BL/6 mice were i.v. injected into TCR α −/− mice (1 × 10 5 cells/body), respectively, and the serum of TCR α −/− mice were collected 14 days after CD4 + T cells were transferred. The levels of total IgM/IgG (e) and IgG subtype IgG1/IgG2a (f) in the serum were determined by ELISA. Each group had at least six mice for the analysis. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: 100 μ L diluted samples were added to the wells, and the plates were incubated at RT for 2 h. After washing 3 times with PBST, the wells were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (H+L) (for total IgG, anti-dsDNA, and anti-Sn-RNP), HRP-conjugated goat anti-mouse IgM (SouthernBiotech), HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech), or alkaline phosphatase- (AP-) conjugated goat anti-mouse IgG2a Abs (Rockland, Gilbertsville, PA, USA), respectively, at RT for 2 h. Tetramethylbenzidine (TMB) (BD Biosciences, San Diego, CA, USA) solution (for HRP) or VISIGLO AP CHEMILUM SUBSTRATE (Sigma) solution (for AP) (100 μ L/well) was added.

Techniques: Enzyme-linked Immunosorbent Assay, Injection

Journal: Mediators of Inflammation

Article Title: CD4 + T Cells Promote IgG Production in MHC-Independent and ICAM-1-Dependent Manners in Pristane-Induced Lupus Mice

doi: 10.1155/2022/9968847

Figure Lengend Snippet: Activated CD4 + T cells can promote IgG production in an MHC-independent way. CD4 + T cells were sorted from PBS and pristane-treated BALB/c mouse (6 months later) by MACS. 3 × 10 4 CD4 + T cells were cultured with 9 × 10 4 CD4 − splenocytes derived from wild-type BALB/c or TCR α −/− mice (on a C57BL/6 background). Total IgM (a, b) and IgG (c, d) levels in the supernatants at different time points (days 2, 4, 6, 8, and 12) were quantified by ELISA. Each group had at least six mice for the analysis. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: 100 μ L diluted samples were added to the wells, and the plates were incubated at RT for 2 h. After washing 3 times with PBST, the wells were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (H+L) (for total IgG, anti-dsDNA, and anti-Sn-RNP), HRP-conjugated goat anti-mouse IgM (SouthernBiotech), HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech), or alkaline phosphatase- (AP-) conjugated goat anti-mouse IgG2a Abs (Rockland, Gilbertsville, PA, USA), respectively, at RT for 2 h. Tetramethylbenzidine (TMB) (BD Biosciences, San Diego, CA, USA) solution (for HRP) or VISIGLO AP CHEMILUM SUBSTRATE (Sigma) solution (for AP) (100 μ L/well) was added.

Techniques: Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: CD4 + T Cells Promote IgG Production in MHC-Independent and ICAM-1-Dependent Manners in Pristane-Induced Lupus Mice

doi: 10.1155/2022/9968847

Figure Lengend Snippet: ICAM-1 attenuates IgG production in the coculture of CD4 + T cell and B cell interaction from PBS or pristane-treated mice. CD4 + T cells and B cells were sorted from PBS and pristane-treated BALB/c mouse (6 months later) by MACS, respectively. 3 × 10 4 CD4 + T cells were cultured with 9 × 10 4 B cells derived from PBS or pristane mice (on a BALB/c background). (a) Flow cytometric analysis of ICAM-1 expression on CD4 + T cells. (b) Increased expression levels of ICAM-1 on CD4 + T cells from lupus-like mice. (c–f) Total IgG (c), IgM (d), IgG1 (e), and IgG2a (f) levels in the supernatants of different coculture groups (T+B, T+B+ISO, and T+B+anti-ICAM-1) were quantified by the ELISA assays. Each group had at least five mice for the analysis. Data are represented by mean of OD 450 values with bar of SEM from five mice. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: 100 μ L diluted samples were added to the wells, and the plates were incubated at RT for 2 h. After washing 3 times with PBST, the wells were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (H+L) (for total IgG, anti-dsDNA, and anti-Sn-RNP), HRP-conjugated goat anti-mouse IgM (SouthernBiotech), HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech), or alkaline phosphatase- (AP-) conjugated goat anti-mouse IgG2a Abs (Rockland, Gilbertsville, PA, USA), respectively, at RT for 2 h. Tetramethylbenzidine (TMB) (BD Biosciences, San Diego, CA, USA) solution (for HRP) or VISIGLO AP CHEMILUM SUBSTRATE (Sigma) solution (for AP) (100 μ L/well) was added.

Techniques: Cell Culture, Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: CD4 + T Cells Promote IgG Production in MHC-Independent and ICAM-1-Dependent Manners in Pristane-Induced Lupus Mice

doi: 10.1155/2022/9968847

Figure Lengend Snippet: ICAM-1 attenuates IgG production in the coculture of in vitro activated CD4 + T cell and B cells. Wildtype CD4 + T cells and B cells were sorted from 6- to 8-week-old BALB/c mice by MACS, respectively. CD4 + T cells were stimulated by anti-CD3 and anti-CD28 antibodies for 72 hours. Then, 3 × 10 4 activated CD4 + T cells were cultured with 9 × 10 4 B cells. (a) Flow cytometric analysis of ICAM-1 expression on CD4 + T cells. (b) Increased expression levels of ICAM-1 on in vitro activated CD4 + T cells. (c–f) Total IgG (c), IgM (d), IgG1 (e), and IgG2a (f) levels in the supernatant with or without anti-ICAM-1 blocking antibodies were determined by ELISA. (g, h) Determination of differentiated B cells after the coculture. Each group had at least five mice for the analysis. ∗∗ p < 0.01; ∗∗∗∗ p < 0.0001.

Article Snippet: 100 μ L diluted samples were added to the wells, and the plates were incubated at RT for 2 h. After washing 3 times with PBST, the wells were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-mouse IgG (H+L) (for total IgG, anti-dsDNA, and anti-Sn-RNP), HRP-conjugated goat anti-mouse IgM (SouthernBiotech), HRP-conjugated goat anti-mouse IgG1 (SouthernBiotech), or alkaline phosphatase- (AP-) conjugated goat anti-mouse IgG2a Abs (Rockland, Gilbertsville, PA, USA), respectively, at RT for 2 h. Tetramethylbenzidine (TMB) (BD Biosciences, San Diego, CA, USA) solution (for HRP) or VISIGLO AP CHEMILUM SUBSTRATE (Sigma) solution (for AP) (100 μ L/well) was added.

Techniques: In Vitro, Cell Culture, Expressing, Blocking Assay, Enzyme-linked Immunosorbent Assay