Lipopolysaccharide-induced Synthesis of IL-1beta, IL-6, TNF-alpha and TGF-beta by Peripheral Blood Mononuclear Cells

Jung, Sung Hwan; Park, Choon Sik; Kim, Mi Ho; Kim, Eun Young; Chang, Hun Soo; Ki, Shin Young; Uh, Soo Taek; Moon, Seung Hyuk; Kim, Yong Hoon; Lee, Hi Bal

Abstract

BACKGROUND: Endotoxin (LPS lipopolysaccharide), a potent activator of immune system, can induce acute and chronic inflammation through the production of cytokincs by a variety of cells, such as monocytes, endothelial cells, lymphocytes, eosinophils, neutrophils and fibroblasts. LPS stimulate the mononucelar cells by two different pathway, the CD14 dependent and independent way, of which the former has been well documented, but not the latter. LPS binds to the LPS-binding protein (LBP), in serum, to make the LPS-LBP complex which interacts with 0D14 molecules on the mononuclear cell surface in peripheral blood or is transported to the Ussues. In case of high concentration of LPS, LPS can stimulate directly the macrophages without LBP. We investigated to detect, the generation of proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF-alpha, and fibrogenic cytokine, TGF-beta, by peripheral blood mononuclear cells (PBMC) after LB'S stimulation under serum-free conditions, which lacks LBPs.
METHODS
PBMC were obtained by centrifugation on Ficoll Hypaque solution of peripheral venous bloods from healthy normal subjects, then stimulated in the presence of LPS (0.1 microgram/mL to 100 microgram/mL). The activities of IL-1, IL-6, TNF, and TGF-betawere measured by bioassaies using cytokines - dependent proliferating or inhibiting cell lines. The cellular sources producing the cytokines was investigated by immunohistochemical stains and in situ hybridization.
RESULTS
PBMC started to produce IL-6, TNF-alpha and TGF-beta in 1 hr, 4 hrs and 8hrs, respectively, after LPS stimulation The production of IL-6, TNF-alpha and TGF-beta continuously increased 96 His after stimulation of LPS. The amount of production was 19.8 ng/ml of IL-6 by 105 PEMC, 4.1 ng/mL of TNF by 106 PBMC and 344 pg/mL of TGF-betaby 2 x 106 PBMC. The immunoreactivity to IL-6, TNF-alpha and TGF-betawere detected on monocytes in LPS-stimulated PBMC. Some of lymphocytes showed positive immunoreactivity to TGF-beta. Double immunohistochemical stain showed that IL-1beta, IL-6, TNF-alpha expression was not associated with CD14 postivity on monocytes. IL-1beta, IL-6, TNF-alpha and TGF-/betamRNA expression worn same as observed in immunoreactivity for each cytokines. CONCLUISON: When monocytes are stimulted with LPS under serum-free conditions, IL-6 and TNF-alphaare secreted in early stage of inflammation. In contrast, the secretion of TGF-beta arise in the late stages and that N maintained after 96 his. The main cells releasing lL-1beta, IL-6, INF-alpha and TGF-beta are monocytes, but also lymphocytes can secret TGF-beta.