Effects of endotoxin on neonatal pig Kupffer cells

Abstract

Kupffer cells (KC) were isolated by sequential perfusion of neonatal pig livers with buffers and collagenase and purified by density gradient separation using arabinogalactan. Pulmonary Alveolar Macrophages (PAM) were recovered by broncho-alveolar lavage from the same pigs for comparison and to serve as control macrophages for the assays performed. Activity of the two cell types was determined on an equal number of cells using in-vitro methods to evaluate phagocytosis, bactericidal activity, production of superoxide anion (O2), tumor necrosis factor alpha (TNF-a) and nitric oxide (NO). Both KC and PAM demonstrated similar phagocytic activity of 125-iodoxyuridine-labeled S. aureus. PAM produced more O2* as measxired by their ability to reduce cytochrome-C and killed more S. aureus as measured by reduction of MTT. Both PAM and KC produced TNF-a when incubated for a period of four hours with lipopolysaccharide (LPS) or opsonized zymosan. Neither KC nor PAM produced nitric oxide when incubated in vitro with LPS or zymosan. These results suggest that KC from neonatal pigs are immunologically active, and that there are inherent differences in the two macrophage populations, probably enhancing their roles in the different organs. In an additional study, the effects of endotoxin prefreatment on the activity of KC and PAM, and hepatic microsomal enzyme activity was investigated. Neonatal pigs were injected with LPS intraperitoneally at a dose of 50 ng/kg body weight. Rectal temperature taken 5 hours post-administration of LPS was significantly higher (p<0.05) than that of the control animals. KC from LPS treated pigs had higher bactericidal and phagocytic activity (p<0.05, p< 0.01 respectively) and a higher production of O2* (p< 0.05) than KC from control animals. PAM from both groups had similar bactericidal and phagocytic activity, and O2' production. Hepatic microsomal enzyme activity of aniline hydroxylase was not affected by pretreatment of neonatal pigs with LPS. Tetracyclines (TC) have been shown to have anti-inflammatory effects in addition to the antimicrobial action. The effects of in-vivo administration of chlortetracycline (CTC) on ex-vivo perfused pig livers was investigated. The retention and clearance of Salmonella choleraesuis (SCS), and production of C-reactive protein (CRP), and haptoglobin (HPG) by whole livers were studied. In addition, CTC modulation of production of TNF-a by pig Kupffer cells was studied. Pigs were dosed orally with CTC for three days, and given LPS 24 hours before removal of the liver. Salmonella retention and clearance by the livers of pigs fed CTC was lower (p< 0.01 and p<0.05 respectively) than the control livers. An increase in CRP and HPG by the liver after a three-hour perfusion was observed. Further, CTC decreased the production TNF-a by cultured Kupffer cells incubated in-vitro with LPS. CTC may thus assxmie more subtle roles than just killing bacteria. It's modulation of production of TNF-a suggests a potential for attenuating the acute phase response.