Identification of naturally secreted soluble form of TL1A, a TNF-like cytokine.
TL1A, a TNF-like ligand, mediates signaling via its cognate receptor DR3, a death receptor whose activation was known to induce both death and survival factors. TL1A, like TNF, is also presumed to circulate as a homotrimeric soluble form. To identify soluble TL1A in the immune system, we have developed a quantitative ELISA by pairing a monoclonal antibody (MAb) with a biotinylated anti-TL1A polyclonal antibody (PAb) as capture and detection antibodies, respectively. The assay had a detection limit of 32 pg/ml. Overnight culture of human umbilical vein endothelial cells (HUVEC) expressed up to 160 pg/ml of TL1A, and that proinflammatory cytokines, IL-1 and TNF, significantly increased TL1A mRNA and soluble protein up to several folds in contrast to IL-6 and IL-11, which induced neither protein nor mRNA in the cells. IL-1 appeared to be a better stimulator of TL1A than TNF-alpha, and the induction of soluble TL1A in HUVEC in response to IL-1 was dose and time-dependent. We have also purified soluble TL1A from a large volume of IL-1 stimulated HUVEC conditioned medium using an anti-TL1A PAb-coupled affinity column. The protein eluted from the column was further reacted with anti-TL1A MAbs in Western blot: 30-kDa and 32-kDa under non-reducing and reducing conditions, respectively. Taken together, our results indicated that ELISA might be useful in studying soluble TL1A regulation in certain inflammatory conditions.
Kim
Sunghee
S
Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487, USA. skim@bsc.as.ua.edu
Zhang
Lurong
L
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007375:Interleukin-1 / Q000276:immunology / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D013997:Time Factors; D053380:Tumor Necrosis Factor Ligand Superfamily Member 15; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis* / Q000187:drug effects
D007375:Interleukin-1; D012333:RNA, Messenger; C117026:TNFSF15 protein, human; D053380:Tumor Necrosis Factor Ligand Superfamily Member 15; D014409:Tumor Necrosis Factor-alpha