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

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 1. The specific induction of TNF-a from THP-1 cells and their phenotypic changes by anti-moesin pAb treatment. (A) Left panel: dose- dependent stimulation of THP-1 cells by anti-moesin pAbs. THP-1 cells were cultured for 48 h in the presence of several concentrations of anti- moesin pAbs or human IgG from healthy individuals or anti-human CD43 mAb. TNF-a concentration in the culture supernatant was measured by ELISA. *P < 0.01, **P < 0.001. The figure shows the representative results of three independent experiments. Right panel: THP-1 cells were stained with FITC-labeled anti-moesin mAb (upper panel, filled histogram), anti-CD43 mAb (bottom panel, filled histogram) or isotype antibodies (open histogram). (B) The effect of anti-moesin pAb treatment on the surface marker expression on THP-1 cells. THP-1 cells were cultured for 48 h in the presence or absence of 5 lg ml1 of anti-moesin pAbs or isotype human IgG, and the expression levels of several cell surface proteins were determined by flow cytometry. Filled histogram, untreated cells; green, cells stained with isotype control antibodies; red, human IgG-stimulated cells; blue, anti-moesin pAb-stimulated cells. The figure shows the representative results of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Labeling, Marker, Expressing, Cytometry, Control

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 2. Correlation between TNF-a secretion levels induced by anti-moesin antibodies from THP-1 cells and the expression level of moesin protein on the cell surface. (A) Moesin expression by moesin shRNA-transfected and untransfected THP-1 cells: lysate of THP-1 cells were analyzed for moesin expression level using western blotting with a mouse anti-human moesin mAb (clone 38). The figure shows representative results of three independent experiments. (B) Moesin expression levels on the surface of shRNA moesin-transfected or untransfected THP-1 cells. THP-1 cells were stained with mouse anti-moesin FITC mAb (clone 38/87) and analyzed by flow cytometry. The figure shows representative data of three independent experiments. (C) THP-1 cells transfected with shRNA moesin (clones 3 and 7) or with shRNA NC as well as untransfected cells were cultured in the presence or absence of 5 lg ml1 of anti-moesin pAbs or isotype human IgG for 48 h and levels of TNF-a in the culture supernatants were determined by ELISA. (D) THP-1 cells transfected with shRNA moesin (clone 7) were stimulated with 100 ng ml1

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Expressing, shRNA, Transfection, Western Blot, Staining, Cytometry, Clone Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 3. The anti-moesin pAb-induced activation of the ERK1/2 pathway in THP-1 cells and monocytes. (A) pAb-induced ERK1/2 phosphorylation. THP-1 cells were stimulated with anti-moesin pAbs for the indicated time and their lysates were subjected to western blotting with antibodies specific to phosphorylated ERK1/2 (phospho ERK1/2). The figure shows representative results of three independent experiments. (B) ERK1/2 activation in monocytes. The monocytes were incubated in the presence or absence of anti-moesin pAbs or IgG of healthy donors for the indicated time and the activation of ERK1/2 in cell lysates was determined as described above. The figure shows the representative results of three independent experiments using monocytes from one of the three individuals tested. Effect of an ERK1/2 inhibitor on the TNF-a secretion induced by anti-moesin pAbs in monocytic cells. THP-1 cells (C) or monocytes (D) were pre-incubated for 2 h in the presence or absence of PD98059 and then stimulated with anti-moesin pAbs for 48 h and the TNF-a levels in the supernatants were determined by ELISA and expressed as the means 6 SDs of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 4. Effect of anti-moesin pAb Fab fragments on THP-1 cells. (A) Binding of anti-moesin Fab fragments to moesin on THP-1 cells. THP-1 cells were incubated with anti-moesin pAb Fab fragments or Fab fragments of isotype human IgG followed by staining with FITC- conjugated anti-Fab fragments of human IgG. Filled histogram, cells stained with anti-moesin pAb Fab fragments; open histogram, cells stained with Fab fragments of human IgG. One representative result of three experiments is shown. (B) Effect of Fab fragments on THP-1 cells. THP-1 cells were cultured in the presence of intact anti-moesin pAbs isolated from AA patients or Fab fragments of anti-moesin pAbs or Fab fragments of IgG derived from healthy individuals for 48 h and TNF-a levels in culture supernatant were determined by ELISA. (C) Effect of Fab fragments on anti-moesin pAb TNF-a secretion. THP-1 cells were cultured in the presence of increasing concentrations of Fab fragments of anti-moesin pAbs or Fab fragments of isotype human IgG for 2 h followed by stimulation with intact anti-moesin pAbs for 48 h. The amount of TNF-a induced by anti-moesin pAbs in the absence of Fab fragments was designed as 100%. Data presented in (B and C) are the means 6 SDs of three independent experiments. *P < 0.01.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Binding Assay, Incubation, Staining, Cell Culture, Isolation, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 5. Effect of moesin cross-linking on TNF-a secretion from THP-1 cells. (A) Binding of F(ab#)2 fragments of anti-moesin pAbs to moesin on the surface of THP-1 cells. THP-1 cells were incubated with anti-moesin pAb F(ab#)2 fragments or F(ab#)2 fragments of isotype human IgG followed by staining with FITC-conjugated anti-F(ab#)2 fragments of human IgG. Open histogram, F(ab#)2 fragment of IgG; filled histogram, F(ab#)2 fragments of anti-moesin pAbs. The figure shows a representative result of three independent experiments. (B) Effect of anti-human IgG F(ab#)2 fragments cross-linking antibodies. THP-1 cells were cultured in the presence of 5 lg ml1 of anti-moesin pAb F(ab#)2 fragments or F(ab#)2 of isotype human IgG for 30 min and thereafter 5 lg ml1 of goat anti-human IgG F(ab#)2 fragment-specific antibody was added. After 48 h of incubation, the levels of TNF-a secreted in culture supernatant were determined by ELISA. Data represent the means 6 SDs of three independent experiments. *P < 0.01, **P < 0.001. (C) ERK1/2 phosphorylation by anti-moesin pAb F(ab#)2 fragments. Lysates of THP-1 cells stimulated with anti-moesin pAb F(ab#)2 fragments or F(ab#)2 fragments of isotype human IgG in the presence of cross-linking anti-F(ab#)2 human IgG or with intact anti-moesin pAbs were analyzed by western blotting to detect phosphorylated ERK1/2. The figure shows the representative results of three independent experiments. (D) Effect of ERK1/2 inhibitor: THP-1 cells were treated for 2 h with increasing concentrations of PD98059 or with dimethyl sulfoxide followed by stimulation with cross-linked anti-moesin pAb F(ab#)2 fragments. The TNF-a level in the culture supernatant was determined by ELISA. Data represent the means 6 SDs of three independent experiments.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Binding Assay, Incubation, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot

Journal: International immunology

Article Title: Anti-moesin antibodies derived from patients with aplastic anemia stimulate monocytic cells to secrete TNF-alpha through an ERK1/2-dependent pathway.

doi: 10.1093/intimm/dxp058

Figure Lengend Snippet: Fig. 6. The effect of anti-moesin pAbs on the activation state of moesin proteins in monocytic cells. (A) The phosphorylation of moesin. THP-1 cells were stimulated with anti-moesin pAbs for the indicated time and their lysates were subjected to western blotting with antibodies specific to phosphorylated moesin proteins (phospho moesin). (B) Phosphorylation of moesin in monocytes. The monocytes were incubated in the presence or absence of anti-moesin pAbs or IgG of healthy donors for the indicated time and the phosphorylation of moesin in cell lysates was determined as described above. (A) shows the representative results of three independent experiments while (B) shows the representative results of three independent experiments using monocytes from one of the three individuals tested.

Article Snippet: The following antibodies and reagents were used in this study: anti-CD40 mAb (clone 82111), isotype mouse IgG1 and mouse IgG2b (R&D Systems, Minneapolis, MN, USA), FITC-labeled goat anti-mouse IgG (BD PharMingen), mouse anti-phospho ERK1/2 mAb (#9106) and rabbit anti-phosphoezrin/radixin/moesin polyclonal antibody (pAb) (#3142) were purchased from cell signaling technology; anti-total ERK1/2, anti-active p38 and JNK rabbit pAbs were purchased from Promega.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Incubation