Regulation of haptoglobin secretion by recombinant bovine cytokines in primary cultured bovine hepatocytes

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Abstract

We examined the regulation of haptoglobin (Hp) secretion in primary cultured bovine hepatocytes using recombinant bovine (rb) proinflammatory cytokines. The concentrations of Hp in the supernatant of cultured hepatocytes after incubation with rb interleukin (IL)-6, rb tumor necrosis factor (TNF)-α, rbIL-1β or rbIFN-γ alone or with combinations of two of these cytokines were measured by ELISA. The rbIL-6, rbTNF-α and rbIL-1β increased Hp synthesis, but rbIFN-γ did not, and rbIL-6 was the most effective Hp inducer among these cytokines. The Hp secretion was accelerated synergistically by combined treatment with rbIL-6 and rbTNF-α, whereas it remained unchanged with a combination of rbIL-6 and rbIL-1β. In contrast, the combination of rbIL-6 and rbIFN-γ downregulated Hp secretion. In conclusion, IL-6 is the principal cytokine in Hp secretion in bovine hepatocytes in vitro, and its activity may be regulated by other cytokines.

Introduction

Acute phase proteins (APPs) play important roles in the innate immunity against infection, inflammation, trauma and tissue damage [1]. The APPs involve production of the acute phase reactants and simultaneous suppression of normal export proteins through reprioritization of hepatic protein synthesis. This phenomenon is principally under the control of the cytokines and glucocorticoids acting on the hepatocytes [2], [3], [4]. Analysis of the regulation of APP synthesis is important for understanding local and systemic host defense mechanisms.

Hepatic APPs can be classified into two major types by their regulators. Type 1 APP is regulated by interleukin (IL)-1 and tumor necrosis factor (TNF), whereas type 2 APP is regulated by IL-6 [1], [2]. Species differences in the qualitative and quantitative patterns of APPs mean that haptoglobin (Hp) is classified as a type 1 APP in the rat, but as a type 2 APP in humans [1], [4], [5]. In rats, IL-1- and TNF-mediated stimulation of type 1 APP genes is synergistically enhanced by IL-6 [4]. IL-1 does not stimulate any of the type 2 APP genes, nor does it enhance the effects of IL-6; rather, if there is any influence, it is inhibitory [6]. In cattle, Hp is not detected during normal conditions, but it increases more than 100-fold during the acute phase of inflammation [7], [8], although the serum level of Hp increases only a few fold in rats and humans after an inflammatory stimulus [3]. Thus, Hp synthesis in bovine hepatocytes may be regulated by different mechanisms from those in the other species such as rodents and humans [9].

In cattle, Hp could be the most useful marker of mastitis [10] or respiratory diseases [11]. We reported that bovine Kupffer cells produce the proinflammatory cytokines TNF-α, IL-1 and IL-6 following stimulation with lipopolysaccharide (LPS) [12]. After activation of hepatocyte receptors by proinflammatory cytokines, the secretion of APPs is upregulated in rodents and humans [1]. However, the regulation of Hp synthesis in bovine hepatocytes in the cytokine network is still unclear. The aim of the present study was to examine the effects of single and combined administration of the recombinant bovine (rb) proinflammatory cytokines IL-1β, TNF-α, IFN-γ and IL-6 on the secretion of Hp as a positive APP, albumin as a negative APP. Further the potential use of Hp as a marker of hepatic function [13], and lactate dehydrogenase (LDH) release as a marker of hepatocyte injury in primary cultured bovine hepatocytes was investigated.

Section snippets

Recombinant bovine cytokines

Recombinant bovine IL-6 was expressed in a baculovirus vector–insect cell system, as reported previously [14], and purified to homogeneity from the culture supernatant with Superose 12HR 10/30 (Pharmacia, Uppsala, Sweden) HPLC (FPLC) and immunoaffinity column chromatography. The amount of IL-6 protein was determined by a protein assay reagent (Bio-Rad, Hercules, CA) with γ-globulin used as a standard. Recombinant bovine TNF-α and recombinant bovine IL-1β, produced in a Brevibacillus chosinensis

Effects of cytokines on Hp and albumin secretion in bovine hepatocytes

Fig. 1 shows the effects of each cytokine on Hp secretion in bovine hepatocytes. While cultured bovine hepatocytes secreted detectable amounts of Hp (60 ng/mL) into the culture medium in the absence of any stimulus. The concentration of Hp (520 ng/mL) in the medium increased about 9-fold (P<0.01) by the addition of 1 ng/mL IL-6. TNF-α and IL-1β at a dose of 1000 U/mL for both cytokines also increased approximately 2-fold the secretion (100 and 140 ng/mL, respectively) of Hp (P<0.01). In contrast,

Discussion

In cattle, Hp shows the most marked change among APPs during the inflammatory process [7], [8]. In the present experiment, albumin secretion in bovine hepatocytes was reduced by IL-6, IL-1β, TNF-α and IFN-γ in vitro. Secretion of Hp was increased by IL-6, IL-1β and TNF-α, but not by IFN-γ. In particular, IL-6 induced 5–10-fold increases in Hp, which was considerably more than observed for TNF-α, IL-1β and IFN-γ.

Type 1 APP is regulated by IL-1 and TNF, whereas type 2 APP is regulated by IL-6 in

Acknowledgements

This work was supported by project Grant no. RCP-4310-2002 from the Ministry of Agriculture, Forestry and Fisheries of Japan.

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