Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Serum cytokine/chemokine array in patients with AP. Serum levels of six chemokines, among 40 cytokines/chemokines investigated, were significantly altered in MAP and SAP patients. Serum levels of CCL21, CCL13, and CCL15 in MAP were significantly lower in patients than in controls. Serum levels of MIF were significantly lower in SAP patients than in MAP patients. Serum levels of CCL27 were significantly lower in SAP patients than in control patients. Serum levels of CXCL16 were significantly higher in SAP patients than in control patients. When Bonferroni method was adopted to correct multiple testing problem, only CXCL16 level was revealed to have a significant difference. MAP, mild acute pancreatitis; SAP, severe acute pancreatitis. Results were shown as mean ± SD.

Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R&D Systems).

Techniques:

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Cxcl16 expression in acute necrotizing pancreatitis. ( A ) Protocols of acute necrotizing pancreatitis by repeated injection of cerulein. Cerulein (100 ug/kg) was injected every 1 h 8 times on 2 consecutive days into C57BL/6 mice. Four to five mice were sacrificed at the indicated time points. ( B ) Serum amylase level and ( C ) pathology score of the respective time-points. ( D ) H&E sections in the pancreas of mice treated with acute necrotizing pancreatitis regimen at 0, 9, 24, and 33 h, respectively. The section at 33 h shows disrupted acinar architecture, vacuolization and necrosis of acinar cells, and inflammatory cell infiltration. ( E ) Pancreatic mRNA expressions of Cxcl16 , Tnfα , Il6 , and Cxcl2 were determined by quantitative RT-PCR analysis. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R&D Systems).

Techniques: Expressing, Injection, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Cxcl16 −/− mice were resistant to the development of necrotizing pancreatitis. Cerulein (100 ug/kg) was injected into Cxcl16 -intact ( WT ) and Cxcl16 -deficient ( Cxcl16 −/− ) mice (n = 5 in each group) every 1 h 8 times on 2 consecutive days as decribed in Fig. ( A ) Serum amylase levels and ( B ) pathology scores of WT and Cxcl16 −/− mice. ( C ) H&E sections from WT and Cxcl16 −/− mice at 24 h and 33 h. ( D ) Immunostained (Gr1, F4/80, and CD3) sections from WT and Cxcl16 −/− mice at 33 h. ( E ) Semiquantitative assessment of infiltartion of neutrophils, macrophages, and T cells. The number of neutrophils, macrophages, and T cells were counted in 5 high powered fields in each pancreas sections of WT and Cxcl16 −/− mice at 33 h. ( F ) MPO levels determined by ELISA in the pancreatic lysates of WT and Cxcl16 −/− mice at 33 h. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R&D Systems).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Acinar cell expression of Cxcl16. Cerulein (100 µg/kg) was injected into WT and Cxcl16 −/− mice every 1 h 8 times on 2 consecutive days as described in Fig. . ( A ) Cxcl16 immunostaining of pancreatic frozen sections from WT mice (0 and 33 h) and Cxcl16 −/− mouse (33 h). ( B ) Dual immunofluorescence of Cxcl16 (Alexa Fluor 488) and amylase (Alexa Fluor 594), or Cxcl16 (Alexa Fluor 594) and F4/80 (Alexa Fluor 488). Most of Cxcl16-expressing on the cell surface was positive for cytoplasmic amylase expression. ( C ) Macrophage-depleted AP model (Left, protocol). Clodronate liposomes (100 mg/kg) were injected at 24 h before the first cerulein injection. F4/80 and Cxcl16 mRNA expression in the pancreas at 33 h, relative to those of control mice at 0 h, was evaluated at 33 h. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R&D Systems).

Techniques: Expressing, Injection, Immunostaining, Immunofluorescence

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Induction of Ccl9 by Cxcl16 in necrotizing acute pancreatitis. ( A ) Cytokine/chemokine array using pancreatic lysates from C57BL/6 mice and Cxcl16 −/− mice treated with necrotizing pancreatitis regimen as described in Fig. . The relative expression of cytokines and chemokines is shown together with representative images. The data presented were obtained from WT and Cxcl16 −/− mice at 33 h, and WT on 0 h. ( B ) Ccl9 , Vcam-1 , and Ccl2 mRNA expression, relative to those of WT mice at 0 h, was assessed by qPCR in the pancreas of WT and Cxcl16 −/− mice at 33 h. ( C ) Ccl9 immunostaining of pancreatic frozen sections from WT mice at 0 h and 33 h, and Cxcl16 −/− mice at 33 h. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R&D Systems).

Techniques: Expressing, Immunostaining

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Expression of Ccl9 by pancreatic acinar cells. ( A ) Amylase secretion by the rat acinar cell line AR42J upon stimulation with various concentrations of cerulein. ( B ) Cxcl16 and Ccl9 mRNA expression by AR42J upon stimulation with cerulein. ( C ) Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −10 M) in combination with various concentrations of recombinant Cxcl16 protein (left). Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −7 M) in the presence of neutralizing anti-Cxcl16 Ab or control Ab (right). ( D ) Cxcl16 and Ccl9 mRNA expression by pancreatic acinar cells isolated from WT and Cxcl16 −/− mice upon stimulation with cerulein (10 −7 M). *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R&D Systems).

Techniques: Expressing, Recombinant, Isolation

Journal: Scientific Reports

Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

doi: 10.1038/s41598-018-27200-y

Figure Lengend Snippet: Therapeutic effects of neutralizing Cxcl16 Ab in the necrotizing pancreatitis model. ( A ) Injection protocol of anti-Cxcl16 Ab in necrotizing pancreatitis model. ( B ) Serum amylase level, ( C ) representative H&E sections, and pathology score of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( D ) Gr1 immunostained sections and neutrophil counts of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( E ) Pancreatic Ccl9 mRNA, pancreatic Ccl9 protein, and serum Ccl9 protein levels in control Ab or anti-Cxcl16 Ab-treated mice at 39 h. n = 5 in each group. *p < 0.05 Results were shown as mean ± SD.

Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R&D Systems).

Techniques: Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CXCR4 inhibition in human pancreatic and colorectal cancers induces an integrated immune response

doi: 10.1073/pnas.2013644117

Figure Lengend Snippet: The effect of CXCL12-stimulated CXCR4 on chemokine receptor-mediated migration of human immune cells. ( Left ) The coexpression of CXCR4 with ( A ) CXCR1, ( B ) CXCR3, ( C ) CXCR5, ( D ) CXCR6, and ( E ) CCR2 on human immune cell lines was evaluated by flow cytometry after staining with antibodies specific for the relevant chemokine receptors. Gray peaks indicate isotype controls. ( Center ) The effect of CXCL12-stimulation of CXCR4 on the chemotactic responses of A CXCR1-coexpressing Jurkat T lymphoblastoid cells to CXCL8, ( B ) CXCR3-coexpressing HSB2DP T lymphoblastoid cells to CXCL10, ( C ) CXCR5-coexpressing Raji B lymphoblastoid cells to CXCL13, ( D ) CXCR6-coexpressing Jurkat T lymphoblastoid cells to CXCL16, and ( E ) CCR2-coexpressing Molm13 monocytoid cells to CCL2 was assessed by including CXCL12 in the upper chamber (blue) and the other chemokines in the lower chamber (red) in the Boyden two-chamber assay. ( Right ) The chemotaxis assays were performed with the five cell lines when the placement of the chemokines in the Boyden chambers was reversed. Bar diagrams display mean and SEM (n = 3-4). Statistical analysis by Student’s t test: *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: The concentration of each chemokine used is as following: CXCL8 (R&D Systems, 208-IL), 20 ng/mL; CXCL10 (R&D Systems, 266-IP), 1,000 ng/mL; CXCL13 (R&D Systems, 801-CX), 1,000 ng/mL; CXCL16 (R&D Systems, 976-CX), 50 ng/mL; CCL2 (R&D Systems, 279-MC), 200 ng/mL

Techniques: Migration, Flow Cytometry, Staining, Boyden Chamber Assay, Chemotaxis Assay

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 1 | TWEAK (tumor necrosis factor–like weak inducer of apoptosis) induces renal CXCL16 expression in vivo. (a) Quantification of CXCL16 mRNA by real-time quantitative reverse transcription-PCR in kidneys from mice 4 h after the administration of TWEAK and/or parthenolide. *Po0.003 vs. control. #Po0.05 vs. TWEAK. Data were normalized with murine glyceraldehyde-3-phosphate dehydrogenase mRNA. (b) Quantification of CD3-positive cells 4 h after TWEAK or/and parthenolide injection. *Po0.009 vs. control. #Po0.01 vs. TWEAK. Mean±s.e.m. of six animals per group. h.p.f, high power field.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, In Vivo, Reverse Transcription, Control, Injection

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 2 | Localization of TWEAK (tumor necrosis factor–like weak inducer of apoptosis)-induced renal CXCL16 expression in vivo. In control kidneys CXCL16 located mainly to tubules. In TWEAK-injected animals, an increased number of CXCL16- expressing tubules is noted. CXCL16 immunohistochemistry. Original magnification 400. Pictures are representative of six animals per group.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, In Vivo, Control, Injection, Immunohistochemistry

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 3 | TWEAK (tumor necrosis factor–like weak inducer of apoptosis) neutralization decreases CXCL16 and CXCR6 mRNA expression in experimental tubulointerstitial inflammation (TII). (a) During experimental TII, CXCL16 mRNA is increased. TWEAK neutralization decreased kidney CXCL16 mRNA (real-time quantitative reverse transcription-PCR (qRT-PCR)). *Po0.001 vs. control, **Po0.01 vs. TII, #Po0.05 vs. TII þ IgG. Data were normalized with 18s eukaryotic ribosomal RNA. (b) During experimental TII, CXCR6 mRNA is increased. TWEAK neutralization decreased kidney CXCR6 mRNA (real-time qRT-PCR). *Po0.01 vs. control, **Po0.03 vs. TII. Data were normalized with 18s eukaryotic ribosomal RNA. (c) Histological assessment confirmed that cellular tubulointerstitial injury was decreased by anti-TWEAK antibodies. *Po 0.001 vs. control, **Po0.003 vs. TII, #Po0.003 vs. TII þ IgG. Mean±s.e.m. of eight animals per group.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Neutralization, Expressing, Reverse Transcription, Quantitative RT-PCR, Control

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 4 | Localization of CXCL16 expression in experimental tubulointerstitial inflammation (TII). Note tubular cell localization of increased CXCL16 expression in mice with TII, when compared with healthy control or mice with TII treated with anti- TWEAK (tumor necrosis factor–like weak inducer of apoptosis). Immunohistochemistry. Original magnification 400. Pictures representative of eight animals per group.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Control, Immunohistochemistry

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 6 | Increased tubular fibroblast growth factor– inducible 14 (Fn14) and CXCL16 expression is associated with interstitial inflammatory infiltrates in human tubulointerstitial inflammation (TII). (a) CXCL16 immunohistochemistry. CXCL16 is observed in renal tubules (arrows) and in surrounding inflammatory infiltrates (arrowheads) in a case of TII secondary to glomerular injury (rapidly progressive glomerulonephritis), but not in minimal change disease (MCD). (b) Fn14 immunohistochemistry. Fn14 is observed in the same renal tubules (arrows) that were stained for CXCL16 in serial sections of the same case of TII, but not in MCD. Original magnifications 200 and 400. Control for the technique is shown in Supplementary Figure S3 online. Representative images of two patients with MCD, and two with rapidly progressive glomerulonephritis.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Immunohistochemistry, Staining, Control

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 7 | Increased tubular fibroblast growth factor–inducible 14 (Fn14) and CXCL16 expression is associated with interstitial inflammatory infiltrates in human acute tubulointerstitial nephritis. (a) CXCL16 immunohistochemistry. CXCL16 is observed in renal tubules (arrows) and in surrounding inflammatory infiltrates (arrowheads). (b) Fn14 immunohistochemistry. Fn14 is observed in renal tubules (arrows) of the same case. Original magnification 400.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Immunohistochemistry

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 8 | TWEAK (tumor necrosis factor–like weak inducer of apoptosis) modulates CXCL16 expression in cultured renal tubular cells. (a) MCT cells were stimulated with 100 ng/ml TWEAK. Quantification of CXCL16 mRNA expression (real-time quantitative reverse transcription-PCR). Expression level at 6 h control was considered to be 100%. Mean±s.e.m. of three independent experiments. *Po0.04 vs. control 6 h; #Po0.05 vs. control 24 h. (b) MCT cells were pretreated for 1 h with 10 mmol/l parthenolide and stimulated with TWEAK for 3 h. The nuclear factor-kB inhibitor parthenolide prevented TWEAK-induced CXCL16 upregulation. Mean±s.e.m. of three independent experiments. *Po0.05 vs. vehicle; #Po0.05 vs. vehicle þ TWEAK. Data were normalized with murine glyceraldehyde-3-phosphate dehydrogenase mRNA. (c) MCT cells stimulated with TWEAK for 6 h or 24 h were analyzed for CXCL16 surface expression by flow cytometry. Mean±s.e.m. of three independent experiments. *Po0.03 vs. control. (d) NP-1 cells were treated with 100 ng/ml TWEAK for 24 h. Soluble and cellular CXCL16 were quantified by ELISA. Mean±s.e.m. of three independent experiments. *Po0.05 vs. control.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Reverse Transcription, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 9 | Confocal microscopy detection of CXCL16 and CXCR6 expression in cultured tubular cells. Cells were stimulated with 100 ng/ml TWEAK (tumor necrosis factor–like weak inducer of apoptosis) or vehicle (control) for 24 h. (a) Constitutive CXCR6 (green) and inducible CXCL16 (red) expression in proximal tubular MCT cells. Original magnification 40. (b) Constitutive CXCR6 and inducible CXCL16 expression in proximal tubular MCT cells. Note the increased peripheral location of CXCL16 following TWEAK with areas of overlap (yellow). Original magnification 40, zoom 7. (c) Constitutive CXCR6 and inducible CXCL16 expression in distal tubular NP1 cells. Original magnification 40, zoom 7. Indirect immunofluorescence using anti-CXCR6 with secondary Alexa Fluor 488–conjugated antibody (green) and anti-CXCL16 antibodies with secondary Alexa Fluor 633–conjugated antibody (red). Nuclei were stained with 40-6-diamidino-2-phenylindole (blue). Images are representative of three independent experiments.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Confocal Microscopy, Expressing, Cell Culture, Control, Immunofluorescence, Staining

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 10 | CXCL16 does not modulate tubular cell survival or proliferation. (a) Tubular MCT cell death and (b) proliferation were assessed by flow cytometry of DNA content after culture for 24 h in the presence of 100 ng/ml TWEAK and different concentrations of CXCL16. Hypodiploid cells were considered apoptotic and cells in S/G2/M phase were considered proliferating. Mean±s.e.m. of four independent experiments. *Po0.02 vs. control.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Flow Cytometry, Control

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 11 | CXCL16 modulates TWEAK (tumor necrosis factor–like weak inducer of apoptosis)-induced tubular inflammation. MCT cells were pretreated with 50 ng/ml CXCL16 for 1 h, and then stimulated with TWEAK for 6 h or 24 h. (a) ICAM-1 mRNA, *Po0.02 vs. control 6 h, **Po0.03 vs. control 24 h, #Po0.05 vs. TWEAK 6 h, ##Po0.05 vs. TWEAK 24 h. (b) MCP-1 mRNA, *Po0.01 vs. control 6 h, **Po0.03 vs. control 24 h, #Po0.05 vs. TWEAK 6 h. (c) RANTES mRNA, *Po0.01 vs. control 6 h, **Po0.03 vs. control 24 h, ##Po0.05 vs. TWEAK 24 h. Real-time quantitative reverse transcription-PCR. Values for mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Expression level at 6 h control was considered to be 100%. Mean±s.e.m. of four independent experiments.

Article Snippet: MCT murine proximal tubular epithelial cells were cultured in RPMI 1640, and NP-1 murine distal tubular epithelial cells were cultured in DMEM supplemented with decomplemented fetal bovine serum (10%), glutamine (2 mmol/l), and penicillin/streptomycin (100 U/ml; LONZA, Verviers, Belgium) in 5% CO2 at 37 1C.4,51 Both cell lines were originated from the kidneys of SJL mice in the University of Pennsylvania and obtained from Eric G Neilson and Frank Strutz, respectively.52 Recombinant human soluble TWEAK (Millipore, Massachusetts, MA) was used at 100 ng/ml, based on prior dose–response studies in these cells.3 Cell culture and in vivo experiments suggest that human and murine TWEAK have similar actions on murine renal cells.3,34,37,53 This is not unexpected as the TWEAK and Fn14 amino-acid sequences are remarkably conserved throughout evolution, and cross-reactivity between human and mice has been documented biochemically and in other cell systems.54 Recombinant mouse CXCL16 was obtained from R&D Systems (Minneapolis, MN).

Techniques: Control, Reverse Transcription, Expressing

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 1. Circulating CXCL16 concentrations in the patients with sepsis were increased. (A) Soluble levels of serum CXCL16 in 55 septic patients and 30 healthy controls. (B) The dynamics of soluble CXCL16 levels in the serum of patients with sepsis. (C) Soluble CXCL16 levels in the serum collected from patients with septic shock and patients without shock on day of ICU admission. (D) Soluble CXCL16 levels in the serum collected from septic survivors and nonsurvivors. Nonparametric ManneWhitney U test or KruskaleWallis test followed by Dunn's multiple comparisons post test was performed to analyze results between groups. Horizontal bars represent median values, and dots represent individual participants.

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 2. Soluble serum CXCL16 levels at admission correlated with disease severity in the patients with sepsis. (A) Correlation of soluble CXCL16 levels with Sequential Organ Failure Assessment (SOFA) scores in septic patients. (B) Correlation of soluble CXCL16 levels with white blood cells (WBC) in septic patients. (C) Correlation of soluble CXCL16 levels with C- reactive protein (CRP) levels in septic patients. Spearman's correlation coefficient was performed to analyze the correlation between the levels of CXCL16 and SOFA scores, WBC, or CRP levels. Horizontal bars represent median values, and dots represent individual participants.

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 3. Receiving operating characteristic (ROC) curve analysis of CXCL16, SOFA score, PCT, and CRP at admission for predicting 28-day mortality in septic patients. Area under the ROC curve: 0.717 ([95% CI] 0.497e0.939) for CXCL16; 0.849 ([95% CI] 0.715e0.982) for SOFA score; 0.617 ([95% CI] 0.429e0.805) for PCT; and 0.521 ([95% CI] 0.338e0.705) for CRP.

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques:

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 4. CXCL16 worsened cecal ligation and puncture (CLP)-induced nonsevere sepsis. (A) Survival of CLP mice (n ¼ 12 per group) after treatment with recombinant CXCL16 protein in the absence or presence of CLP-induced nonsevere sepsis. Murine recombinant CXCL16 protein was administered intraperitoneally at 0.5e1.0 mg/injection immediately after nonsevere CLP, and PBS was used as a control. Comparison between groups was performed using KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.001 when compared with septic mice treated with PBS control. (B) Representative examples of hematoxylin and eosinestained lung, liver, and kidney tissues from nonsevere CLP mice (n ¼ 6) treated with or without recombinant CXCL16 (0.5 mg/injection). (C) Histological scores for lung, liver, and kidney in nonsevere CLP mice (n ¼ 6) treated with or without recombinant CXCL16 (0.5 mg/injection). p < 0.05, p < 0.001when compared with septic mice treated with PBS control (ManneWhitney U test). (D) ALT, AST, LDH, and creatinine levels in nonsevere CLP mice (n ¼ 6), treated with or without recombinant CXCL16 (0.5 mg/injection). p < 0.001 when compared with septic mice treated with PBS control (ManneWhitney U test).

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques: Ligation, Recombinant, Injection, Control, Comparison

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 5. Treatment with recombinant murine CXCL16 enhanced inflammatory response in CLP-induced nonsevere sepsis. (A) Numbers of leukocytes including neutrophils, monocytes/macrophages and lymphocytes in peritoneal lavage fluid (PLF) of septic mice treated with or without recombinant murine CXCL16 (0.5 mg/injection). (B) Cytokine and chemokine levels in PLF and blood from septic mice (n ¼ 5) treated with or without recombinant murine CXCL16 (0.5 mg/injection) at 24 h after nonsevere CLP. p < 0.05, p < 0.01, p < 0.001 when compared with septic mice treated with PBS control (ManneWhitney U test).

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques: Recombinant, Injection, Control

Journal: Microbes and infection

Article Title: Prognostic and pathogenic role of CXC motif ligand 16 in sepsis.

doi: 10.1016/j.micinf.2021.104882

Figure Lengend Snippet: Fig. 6. Anti-CXCL16 antibody ameliorated cecal ligation and puncture (CLP)-induced severe sepsis. C57BL/6 mice were subjected to severe CLP, and then 10 mg of anti-mouse CXCL16 monoclonal antibody was injected intraperitoneally at 2 h or 6 h after severe CLP. IgG2A control antibody was delivered in a similar way. (A) Survival of septic mice (n ¼ 12 per group) following administration with antieCXCL16 antibody (2 h after CLP) or control antibody after severe CLP. Comparison between groups was performed by KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.05 when compared with septic mice treated with IgG control. (B) Sur- vival of septic mice (n ¼ 12 per group) following administration with antieCXCL16 antibody (6 h after CLP) or control antibody after severe CLP. Comparison between groups was performed by KaplaneMeier analysis followed by log-rank tests. Results are representative of three independent experiments. p < 0.05 when compared with septic mice treated with IgG control. (C) Representative examples of hematoxylin and eosinestained lung, liver, and kidney tissues from septic mice (n ¼ 6) treated with or without anti-CXCL16 neutralizing antibodies (2 h after CLP). (D) Histological scores for lung, liver, and kidney in septic mice (n ¼ 6) treated with or without anti-CXCL16 neutralizing antibodies (2 h after CLP). p < 0.05, p < 0.01 when compared with septic mice treated with isotypical IgG control (ManneWhitney U test). (E) ALT, AST, LDH, and creatinine levels in septic mice (n ¼ 6) treated with or without anti- CXCL16 neutralizing antibodies (2 h after CLP). p < 0.05, p < 0.01 when compared with septic mice treated with isotypical IgG control (ManneWhitney U test).

Article Snippet: The Neutralization Dose (ND50) is typically 0.15e0.6 mg/mL in the presence of 7.5 ng/mL recombinant mouse CXCL16 chemokine domain (R&D systems, Catalog # 503-CX-025)).

Techniques: Ligation, Injection, Control, Comparison

Journal: Structure (London, England : 1993)

Article Title: Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding

doi: 10.1016/j.str.2020.03.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For this report, human recombinant His-tagged FcγR receptors I, IIA/B and IIIA (R&D systems) were labeled with Alexa Fluor 647.

Techniques: Recombinant, Isolation, Expressing, Concentration Assay, Antibody Labeling, Plasmid Preparation, Software

Journal: PLoS Biology

Article Title: Intravascular Immune Surveillance by CXCR6 + NKT Cells Patrolling Liver Sinusoids

doi: 10.1371/journal.pbio.0030113

Figure Lengend Snippet: (A) Binding of CXCL16 by splenocytes and liver leukocytes from cxcr6 gfp/+ and cxcr6 gfp/gfp mice. Splenocytes and liver leukocytes from cxcr6 gfp/+ (+/−) and cxcr6 gfp/gfp (−/−) mice were stained with PE-conjugated CD1d tetramer loaded with α-GalCer (αGC), PerCp-conjugated anti-CD3 antibodies and CXCL16-Fc fusion protein, and Cy5-conjugated goat anti-human Fc. The two black curves represent duplicate samples from two different cxcr6 gfp/+ mice. (B) Thymocytes, splenocytes, and liver leukocytes from cxcr6 gfp/+ and cxcr6 gfp/gfp mice were stained with PE-conjugated CD1d tetramer loaded with α-GalCer and biotinylated anti-NK1.1 followed by PerCp-conjugated streptavidin and APC-conjugated anti-CD3 antibodies. Left panels are gated on CD3 + T cells and represent GFP fluorescence intensity on CD1d/α-GalCer-reactive T cells and on conventional (tetramer-negative) T cells. The right panels are gated on CD3 − cells and represent GFP fluorescence intensity on NK1.1 + CD3 − NK cells. Numbers are percent obtained from a representative experiment among six performed. (C) Liver sinusoidal endothelial cells express CXCL16. Sections (10 μm thick) of PFA-fixed liver from wild-type mice (top panel, left), cxcr6 gfp/+ mice (top panel, right), and Tie2-GFP mice (bottom panel) were stained with rat monoclonal antibody against mouse CXCL16 (Clone 10H7, IgG2a), or with an isotype control, followed by goat anti-rat antibody (F[ab′]2) conjugated to Cy3. (D) GFP hi CD1d-restricted T cells within liver sinusoids. Sections (7 μm thick) of PFA-fixed liver from cxcr6 gfp/+ mice were stained with rabbit polyclonal serum against caveolin-1 followed by goat anti-rabbit antibody conjugated to Cy3. (E) Flow cytometry analysis of cells harvested by perfusion of cxcr6 gfp/+ liver with cold PBS–ethylenediamine tetra-acetic acid.

Article Snippet: Recombinant murine CXCL16, SDF-1, and fractalkine were purchased from R & D Systems (Minneapolis, Minnesota, United States).

Techniques: Binding Assay, Staining, Fluorescence, Control, Flow Cytometry

Journal: PLoS Biology

Article Title: Intravascular Immune Surveillance by CXCR6 + NKT Cells Patrolling Liver Sinusoids

doi: 10.1371/journal.pbio.0030113

Figure Lengend Snippet: (A) Flow cytometry analysis of liver leukocytes from GFP knock-in mice after overnight culture. Dot plots represent GFP signal versus CD1d-α-GalCer-tetramer staining. Histogram plots represent Cell Tracker 633 labeling gated on tetramer-positive cells, indicating no cell division during the course of the culture. (B) Survival of liver NKT cells from cxcr6 gfp/+ and cxcr6 gfp/gfp mice. Duplicate samples of 5 × 10 4 CD1d-reactive T cells/well were incubated for each condition and time point. Histograms represent the percentage of viable, CD1d-α-GalCer-tetramer-positive GFP hi cells, as determined by flow cytometry at the indicated time points. The proportion of CXCL16-cultured cells from control cxcr6 gfp/+ mice was set at 100%. Values are mean ± standard deviation from three independent experiments. (C) Flow cytometry analysis of liver leukocyte apoptosis in culture. The percentage of cells binding Annexin V was determined by flow cytometry at the indicated time points. Results are representative of two independent experiments. A representative analysis at 10 h is shown on the right, with the percentage of CD1-αGC-tetramer-positive and Annexin V–positive cells indicated.

Article Snippet: Recombinant murine CXCL16, SDF-1, and fractalkine were purchased from R & D Systems (Minneapolis, Minnesota, United States).

Techniques: Flow Cytometry, Knock-In, Staining, Labeling, Incubation, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Inflammation

Article Title: KANK4 Regulates CXCL16 Glycosylation Through TMEM260 to Modulate Microglial Activation in Sepsis-associated Encephalopathy

doi: 10.1007/s10753-026-02481-y

Figure Lengend Snippet: KANK4 regulated CXCL16 glycosylation and microglial activation via TMEM260. Microglia were isolated from the hippocampus of mice in Sham, SAE, SAE + AAV-NC, and SAE + AAV-KANK4 groups, followed by the following analyses: (A) IHC was performed to detect CXCL16 protein expression. Microglia were isolated from the Sham and SAE groups for the following assays: (B) Western blot analysis was conducted to assess CXCL16 glycosylation levels ( n = 3). (C) Co-IP was carried out to examine the interaction between TMEM260 and CXCL16. BV2 microglial cells were treated with LPS to establish a cell model. Control and LPS groups were subjected to the following analyses: (D) Western blot analysis was applied to detect CXCL16 expression using both anti-CXCL16 and anti-His-CXCL16 antibodies ( n = 3). (E) Co-IP was conducted to validate the interaction between TMEM260 and CXCL16. (F) Western blot was used to assess O-mannosylation of CXCL16 protein in LPS-treated microglia ( n = 3). (G) Effect of CXCL16 glycosylation site mutations on its glycosylation level. Wild-type (WT) and site-specific mutant (S137A, S117A, S139A) CXCL16 plasmids were transfected into microglia. CXCL16 glycosylation was assessed by Western blot ( n = 3). (H) Impact of CXCL16 glycosylation site mutation (S117A) on the expression of inflammatory factors. In an LPS-induced microglial inflammation model, cells were transfected with either WT or glycosylation-site mutant (S117A) CXCL16 plasmid. The levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in the cells were measured by ELISA. BV2 cells were transfected for TMEM260 overexpression or knockdown. The groups included Control, si-NC, si-TMEM260, NC-OE, and TMEM260-OE, and the following analyses were performed: (I) Western blot analysis was used to detect the expression of TMEM260 and CXCL16 proteins ( n = 3). (J) CHX assay was performed to evaluate the stability of CXCL16 protein. LPS-treated BV2 cells were used to conduct KANK4 overexpression and TMEM260 knockdown rescue experiments, with six groups: Control, LPS, LPS + OE-NC, LPS+KANK4-OE, LPS+KANK4-OE + si-NC, and LPS+KANK4-OE + si-TMEM260. The following analysis was conducted: (K) Western blot analysis was performed to measure CXCL16 protein expression in BV2 cells ( n = 3). For overexpression of TMEM260 and CXCL16 in LPS-treated BV2 cells, the groups included: Control, LPS, LPS + OE-NC, LPS+TMEM260-OE, LPS+TMEM260-OE + OE-NC, and LPS+TMEM260-OE+CXCL16-OE. The following analyses were carried out: (L) Western blot analysis was conducted to detect TMEM260 and CXCL16 protein levels in each group ( n = 3). (M) CCK-8 assay was applied to evaluate microglial cell viability across groups. (N) IF staining was performed to assess the expression of Iba-1 (green) and CD11b (red), indicating microglial activation status (Scale bar = 50 μm). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. LPS/LPS + OE-NC/LPS+TMEM260-OE + OE-NC; ns, no significant difference vs. LPS/Control

Article Snippet: CXCL16 protein levels were measured by Western blot (antibody: Proteintech, 60123-1-Ig, 1:1000), and the band intensity was quantified using ImageJ.

Techniques: Glycoproteomics, Activation Assay, Isolation, Expressing, Western Blot, Co-Immunoprecipitation Assay, Control, Mutagenesis, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Over Expression, Knockdown, CCK-8 Assay, Staining

Journal: Arthritis and rheumatism

Article Title: Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints.

doi: 10.1002/art.21004

Figure Lengend Snippet: Figure 1. Expression of CXCR6 on T cells from patients with rheu- matoid arthritis (RA). A, Fluorescence-activated cell sorting results. While a small percentage of peripheral blood T cells from RA patients (RA blood) express CXCR6, this chemokine receptor is widely expressed by RA synovial fluid (SF) T cells. T cells were stained for CXCR6 and CD3. The quadrants indicate the expression above the background. Dead cells were excluded by gating on propidium iodide– negative cells. Representative examples are shown. B, Percentage of CXCR6 T cells in control blood (C blood) (n 10), RA blood (n 4), and RA SF (n 6). Values are the mean and SD. P 0.01. C, Expression of CXCL16 mRNA in control (C) synovial tissue and in synovia from RA patients as determined by reverse transcriptase– polymerase chain reaction (PCR) using specific primers. Tonsil mRNA (T) and water (W) were used as positive and negative controls, respectively. Results shown are representative of control synovia and RA synovia. D, Quantification of CXCL16 mRNA demonstrating significantly enhanced expression in RA synovia (n 6) compared with control synovia (n 4). Expression was determined by quantita- tive real-time PCR. CXCL16 levels were normalized to the levels of the housekeeping gene GAPDH. P 0.05 versus controls.

Article Snippet: Serial dilutions of recombinant human CXCL16 (R&D Systems) were used to obtain a standard curve.

Techniques: Expressing, Fluorescence, FACS, Staining, Control, Reverse Transcription, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Arthritis and rheumatism

Article Title: Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints.

doi: 10.1002/art.21004

Figure Lengend Snippet: Figure 2. Enhanced expression of CXCL16 protein within rheumatoid arthritis (RA) synovia. CXCL16 was expressed in the thin synovial lining of healthy synovia (A) (arrowhead) but was much more pronounced in the hypertrophic lining of RA synovia (B). Many cells present within the sublining of the synovia of patients with RA expressed high levels of CXCL16 (B and C). Note that (cleaved) CXCL16 was also associated with filaments of the extracellular matrix (B). Staining of serial sections indicated that expression of CXCL16 (E) correlated with the presence of CD68 synovial macrophages (F). We also detected CXCL16 within some vessels (G) (arrow). Staining of serial sections for CD31 confirmed that these cells were endothelial cells (H). Many lymphocyte aggregates contained CXCL16 cells (I) (arrows). Analysis of stained serial sections suggested that these CXCL16 cells were CD68 macrophages (J) (arrows) amidst CD45RO memory lymphocytes (K). Cryosections were stained for CXCL16 (A–C, E, G, and I), CD68 (F and J), CD31 (H), CD45RO (K), or matched control antibodies (D and I). All sections were counterstained with hematoxylin. Sections shown are representative of synovial tissue from 10 RA patients and 5 controls. (Original magnification 400 in A–F and I–L; 630 in G and H.)

Article Snippet: Serial dilutions of recombinant human CXCL16 (R&D Systems) were used to obtain a standard curve.

Techniques: Expressing, Staining, Control

Journal: Arthritis and rheumatism

Article Title: Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints.

doi: 10.1002/art.21004

Figure Lengend Snippet: Figure 3. Strongly increased expression of CXCL16 by macrophages (M ) after exposure to RA SF or tumor necrosis factor (TNF). A, Peripheral blood monocytes were differentiated into macrophages and analyzed by flow cytometry. CD14 monocyte/macrophages were gated and stained with anti-CXCL16 (bold lines) or isotype-matched control antibodies (thin lines). Dead cells were excluded by gating on propidium iodide–negative cells. B, Mean percentages of CXCL16 macrophages at the time points indicated. C, Increasing levels of transmembrane CXCL16 on macrophages, with addition of increasing concentrations of RA SF or TNF to freshly isolated monocytes. On day 2, the percentage of CXCL16,CD14 macrophages was determined by flow cytometry. The mean and SD percentages from 2 donors are shown. Dead cells were excluded by gating on propidium iodide–negative cells. D, Significant levels of cleaved CXCL16 released by cultured macrophages. By sandwich enzyme-linked immunosorbent assay (ELISA), the supernatants of macrophages (MAC) or peripheral blood lymphocytes (PBL) cultured for 1, 2, or 3 days were analyzed for the presence of cleaved CXCL16. Values are the mean. E, TNF-induced expression of cleaved CXCL16. Culture supernatants of day-2 macrophages were analyzed by sandwich ELISA. Representative results of 2–5 experiments are shown. P 0.05; P 0.01; P 0.001 versus unstimulated group. See Figure 1 for other definitions.

Article Snippet: Serial dilutions of recombinant human CXCL16 (R&D Systems) were used to obtain a standard curve.

Techniques: Expressing, Flow Cytometry, Staining, Control, Isolation, Cell Culture, Sandwich ELISA, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints.

doi: 10.1002/art.21004

Figure Lengend Snippet: Figure 4. Reduced CXCL16 expression upon successful anti–tumor necrosis factor (anti-TNF) treatment of rheumatoid arthritis (RA) patients. Before anti-TNF treatment, CXCL16 was strongly expressed within RA synovia (A and C). Expression of CXCL16 was severely reduced in clinically responding patients (B), but not in nonresponding patients (D). Synovia were isolated before (A and C) and after (B and D) anti-TNF treatment. All cryosections were stained for CXCL16 and counterstained with hematoxylin. Control stainings were negative. Results are representative of 3 responding and 3 nonresponding patients. (Original magnification 100.)

Article Snippet: Serial dilutions of recombinant human CXCL16 (R&D Systems) were used to obtain a standard curve.

Techniques: Expressing, Isolation, Staining, Control

Journal: Arthritis and rheumatism

Article Title: Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints.

doi: 10.1002/art.21004

Figure Lengend Snippet: Figure 5. Enhanced expression of the CXCL16 protease ADAM-10 by RA synovial macrophages (M ). ADAM-10–negative peripheral blood monocytes were differentiated into macrophages and analyzed by flow cytometry (A). CD14 monocyte/macrophages were gated and stained with anti–ADAM-10 (bold lines) or isotype-matched control antibodies (thin lines). Dead cells were excluded by gating on propidium iodide–negative cells. Addition of RA SF to macrophages (day 3) increased their expression of ADAM-10 (B). After 2 days the percentage of ADAM-10,CD14 macrophages was determined by flow cytometry. The mean and SD percentages from 2 donors are shown. Dead cells were excluded by gating on propidium iodide–negative cells. P 0.05 versus controls. Synovial tissue from controls showed expression of both CXCL16 (C) and its protease ADAM-10 (D) in the thin synovial lining. In contrast, expression of both CXCL16 (F) and ADAM-10 (G) was highly enhanced in the hypercellular synovial lining of RA synovial tissue. Frozen sections were stained for CXCL16 (C and F), ADAM-10 (D and G), or a matched control antibody (E), and counterstained with hematoxylin. Representative stainings are shown. See Figure 1 for other definitions. (Original magnification 400.)

Article Snippet: Serial dilutions of recombinant human CXCL16 (R&D Systems) were used to obtain a standard curve.

Techniques: Expressing, Flow Cytometry, Staining, Control

Journal: Arthritis and rheumatism

Article Title: Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints.

doi: 10.1002/art.21004

Figure Lengend Snippet: Figure 6. Cleaved CXCL16 overexpression in RA SF and attraction of SF T cells. A, Expression of cleaved CXCL16 in RA SF (n 17) was compared with that in SF from controls (n 2) or from patients with osteoarthritis (OA; n 5). CXCL16 concentrations were measured by sandwich enzyme-linked immunosorbent assay. Values are the mean and SD. P 0.02. B, Loss of cell surface–expressed CXCR6 on RA SF T cells induced by activation with cleaved CXCL16. The level of CXCR6 expressed by unstimulated T cells was set at 100%. C, SF T cells from RA patients are attracted by soluble CXCL16. Migration was concentration dependent and reached an optimum at 10 ng/ml. Migration of RA SF T cells was determined with a Transwell migration assay. Migrating cells were counted and stained for CD3 and CXCR6. Representative results from 3 experiments are shown. Values are the mean and SD. See Figure 1 for other definitions.

Article Snippet: Serial dilutions of recombinant human CXCL16 (R&D Systems) were used to obtain a standard curve.

Techniques: Over Expression, Expressing, Sandwich ELISA, Activation Assay, Migration, Concentration Assay, Transwell Migration Assay, Staining

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) CXCL16 mRNA expression in the kidney during MuPyV infection. Expression is shown as fold change relative to sham infected samples. Data are from three independent experiments (n = 10-11). (B) Expression of NKCC2 [marks the ascending loop of Henle ], CD8, and CXCL16 in epithelia in sham-infected and 8 dpi kidneys; bottom right photomicrographs are merged images. Representative of two independent experiments. (C) CXCL16 expression in sham-infected, 4 dpi, and 8 dpi kidney lysates (left). Western blot image is representative of two independent experiments with each lane indicating protein lysate from kidneys of individual mice. Protein band intensity quantification for sCXCL16 was normalized to β-actin and analyzed by ImageLab and normalized to the loading control (right). Data are combined from two independent experiments (n = 3-5). (D) Experimental design of in vivo CXCL16 mAb administration. Mice were administered 250 µg of CXCL16 mAb or control rat IgG every two days from days 4-14 post infection and euthanized at 15 dpi. Numbers of CD45 mAb i.v.-negative, CD8 + CD44 + D b -LT359 tetramer + T cells and CD4 + CD44 + T cells in kidneys and spleens of infected mice given anti-CXCL16 or control rat IgG. Data were analyzed by one-way ANOVA (A and C) and by multiple Mann-Whitney tests (D).

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Infection, Western Blot, Control, In Vivo, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: (A) Dense lymphoplasmacytic infiltrate surrounding and infiltrating cortical tubules (H&E, x400). (B) Immunohistochemical staining for Large T antigen in nuclei of tubular epithelium (x250). (C) Expression of CXCR6 on infiltrating CD8 + cells. Photomicrographs (right) are enlarged images in the white squares (left); bottom right are merged images. No 1 o , no primary antibody. (D) Log-fold change of CXCL16 and CXCR6 across four independent studies, each comparing KTx biopsies from patients with PVAN and stable graft function. The error bars indicate the 95% confidence interval of log-fold change, with horizontal dashed grey line indicating log-fold change = 0, or no difference. GSE120495 is an RNA-seq study, while remaining studies are microarray based. Given the heterogeneity of the data, a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. The combined p-values for CXCL16 = 0.000337 and CXCR6 = 2.72x10 -05 . ns, non-significant at adjusted p-value > 0.05; *, statistical significance at adjusted p-value ≤ 0.05; **, statistical significance at adjusted p-value ≤ 0.01; ***, statistical significance at adjusted p-value ≤ 0.001.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Microarray

Journal: PLOS Pathogens

Article Title: The CXCR6-CXCL16 axis mediates T cell control of polyomavirus infection in the kidney

doi: 10.1371/journal.ppat.1012969

Figure Lengend Snippet: Summary of differential gene expression of CXCL16 and CXCR6 in four studies of KTx biopsies with PVAN. Because of the heterogeneity of the data (i.e., one RNA-seq, three microarray), a Cauchy combination method with uniform weights was employed to integrate the p-values across these studies. P-values significant after false discover rate (FDR) correction are bolded and italicized in the Adj. P-value column. Cauchy combined p-values < 0.05 are bolded and italicized.

Article Snippet: For CXCL16 neutralization, mice were infected with MuPyV and received 250 μg of anti-mouse CXCL16 mAb (clone 12-81, BioXCell) i.p. on alternate days from days 4-14 post infection or control rat IgG (Jackson ImmunoResearch Laboratories).

Techniques: Gene Expression, Microarray