anti β1 integrin function blocking antibody  (Developmental Studies Hybridoma Bank)

Journal: Gut

Article Title: Neutrophils interact with cholangiocytes to cause cholestatic changes in alcoholic hepatitis

doi: 10.1136/gutjnl-2020-322540

Figure Lengend Snippet: (A) Analysis of RNA-seq transcriptome data with ingenuity pathways analysis (IPA) software identifies the six signalling pathways that are significantly altered in the NHC cell line when co-cultured with neutrophils. Bars represent −log(p value) of significance level for each pathway; orange indicates upregulated and blue indicates downregulated pathways. (B) Volcano plot of RNA-seq analysis of NHC proteins that are affected by exposure of the cells to neutrophils. RNA was extracted from the NHC cell line alone (n=3) and co-cultured with neutrophils (n=4), and both groups were analysed by Affymetrix High-Throughput Transcriptomics Array. A total of 5818 mRNA genes were differentially expressed (grey circles), including plasma membrane proteins (orange circles) and integrin β1 (ITGB1) (blue circle). (C) RT-PCR demonstrates that ITGB1 is more heavily expressed than ITGB3 or ITGA5 in the NHC cell line (n=4). mRNA expression levels were normalised to ACTB. (D) Representative immunofluorescence images of NHCs stained for ITGB1 (green). Hoechst 33342 is used to stain the nuclei (blue). Scale bar: 10 μm.

Article Snippet: Functional blocking interventions To inhibit the function of ITGB1 in cholangiocytes, an anti-β1-integrin function blocking antibody (AIIB1, Developmental Studies Hybridoma Bank) was used.

Techniques: RNA Sequencing, Software, Cell Culture, High Throughput Screening Assay, Clinical Proteomics, Membrane, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence, Staining

Journal: Gut

Article Title: Neutrophils interact with cholangiocytes to cause cholestatic changes in alcoholic hepatitis

doi: 10.1136/gutjnl-2020-322540

Figure Lengend Snippet: Human neutrophils increase the expression of (A) the pro-inflammatory cytokine interleukin-6 (IL-6), (B) the pro-inflammatory chemokines CXCL1 and (C) the pro-inflammatory chemokine CXCL8 in the normal human cholangiocyte (NHC) cell line. IL-6, CXCL1 and CXCL8 expression each is significantly increased in the NHC cell line co-cultured with neutrophils from healthy control subjects, and the increase is even greater when co-cultured with neutrophils from patients with AH. Data represent mean ± SEM (n=4), *p<0.05. Lipopolysaccharides (LPS) increases the expression of (D) IL-6, (E) CXCL1 and (F) CXCL8 in the NHC cell line in a dose-dependent manner. Data represent mean ± SEM (n=4), (**p<0.01; ***p<0.0001). Note that the increase in CXCL8 expression is much more than that of IL-6 or CXCL1 in response to either neutrophils or LPS. (G) CXCL8 is responsible for recruiting neutrophils to NHCs. Left: representative differential interference contrast (DIC) and CMTMR fluorescence images of neutrophil migration assay (left). Neutrophils (polymorphonuclear neutrophil (PMN)) were stained with CMTMR CellTracker (red) and co-cultured with NHCs in the presence of either LPS or CXCL8 antagonist. Recombinant CXCL8 was used as a positive control for neutrophil migration. LPS and recombinant CXCL8 each stimulate migration of neutrophils through Transwell membranes, and the CXCL8 antagonist inhibits LPS-induced neutrophil migration. Original magnification ×20. Right: quantitative assessment of neutrophil migration across a permeable Transwell chamber. Data represent mean ± SEM (n=5), ***p<0.0001. (H) Proposed mechanism by which neutrophils interact with cholangiocytes to cause cholestasis in alcoholic hepatitis. Endotoxin (LPS) stimulates cholangiocytes to produce IL-6, CXCL1 and CXCL8, thus recruiting neutrophils to bile ducts. Vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on the neutrophils interact with integrin β1 (ITGB1) on cholangiocytes, which triggers RAC1 signalling to phosphorylate JNK. Phospho-JNK (pJNK) then enters the nucleus to phosphorylate the transcription factor c-Jun/AP-1, which in turn binds to AP1 sites on the promoter of the ITPR3 gene to suppress expression of ITPR3. Loss of ITPR3 expression results in loss of Ca2+-mediated biliary bicarbonate secretion, which contributes to cholestasis.

Article Snippet: Functional blocking interventions To inhibit the function of ITGB1 in cholangiocytes, an anti-β1-integrin function blocking antibody (AIIB1, Developmental Studies Hybridoma Bank) was used.

Techniques: Expressing, Cell Culture, Control, Fluorescence, Migration, Staining, Recombinant, Positive Control