Alpha 1-acid glycoprotein-induced tumor necrosis factor-α secretion of human monocytes is enhanced by serum binding proteins and depends on protein tyrosine kinase activation

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Abstract

The acute phase protein, α1 acid glycoprotein (AGP), stimulated human mononuclear cells as well as monocytes to secrete tumor necrosis factor-α (TNFα) which was demonstrated by ELISA, RT-PCR and functional assays. AGP-induced TNFα secretion of monocytes was enhanced in the presence of human plasma and inhibited by protein kinase inhibitors, indicating it is serum and tyrosine kinase dependent. The activation of tyrosine kinase in AGP-stimulated monocytes was further confirmed by immunoblotting of tyrosine phosphorylated proteins of monocytes at different time after AGP stimulation. Furthermore, several serum proteins such as C3, sCD14 and IgG were able to bind to AGP and enhanced TNFα secretion of human monocytes induced by AGP. Taken together, these results suggest serum proteins binding to AGP enhance its ability to stimulate human monocytes to secrete pro-inflammatory cytokines through a tyrosine kinase dependent pathway.

Introduction

Human alpha 1-acid glycoprotein (AGP) is an acute phase protein produced by hepatocytes in response to inflammatory cytokines such as TNFα, IL-1 and IL-6 (Baumann et al., 1993). AGP is a highly glycosylated, polyanionic protein with molecular weight about 40 KD. In normal human blood, the level of AGP is about 0.2–1 mg/ml whereas during the acute phase of inflammation, it rises three to four times normal range (Eap and Baumann, 1993) and undergoes extensive glycosylation change (De Graaf et al., 1993; Brinkman-van der Linden et al., 1996). Different glycosylation forms of AGP have been associated with different immunomodulatory activities of AGP (Eap and Baumann, 1993; Shiyan and Bovin, 1997). On the one hand, AGP can activate monocytes, induce T cell proliferation (Singh and Fudenberg, 1986) and enhance TNFα, IL-1, and IL-6 secretion (Boutten et al., 1992; Drenth et al., 1996; Su and Yeh, 1996). On the other hand, it can also induce the production of IL-1 receptor antagonist (Tilg et al., 1993) and cause immunosuppression (Bennett and Schmid, 1980). Therefore, AGP has both pro-inflammatory and anti-inflammatory effects that other acute phase proteins and IL-6 do (Tilg et al., 1993, Tilg et al., 1997). The dual immunomodulatory effects of AGP in vitro may indicate it plays an important role in the regulation of immune response. However, transgenic mice that over-produce AGP have a normal immune response (Dewey et al., 1990). The precise biological functions of AGP are, therefore, unclear.

AGP can interact with different drugs (Kremer et al., 1988; Turk et al., 1996) and circulating immunocomplex (Croce and Segal-Eiras, 1996). In addition, the interaction of AGP with bacterial LPS has been suggested to explain its protection from sepsis (Moore et al., 1997). However, it is unclear why pretreatment of mice with AGP is able to protect them from TNFα-induced lethality (Libert et al., 1994). AGP specifically inhibits TNFα-induced apoptosis of hepatocytes in vivo (Van Molle et al., 1997) but it is unable to protect hepatoma cells from TNF-induced apoptosis in vitro (Van Molle et al., 1997). Since a low dose of TNFα can protect mice against subsequent lethal challenge of TNFα and TNFα is increased in AGP-stimulated human monocytes (Su and Yeh, 1996), it is possible that the protective effect of AGP in vivo is mediated through TNFα secretion or some other cytokines induced by AGP stimulated monocytes. Therefore, it is necessary to understand more about the effect of AGP on monocytes. In this study, we sought to determined the mechanisms by which AGP stimulate human monocytes to secrete TNFα and focused on its serum dependency and signal transduction.

Section snippets

Isolation of mononuclear cells and monocytes

The peripheral blood mononuclear cells (PBMC) were isolated from normal blood donors by Ficoll–Hypaque gradient sedimentation. In some experiment as indicated, the monocyte-enriched preparation was developed by plating PBMC (4×106 cells/ml) onto plastic tissue culture wells with 10% heat-inactivated fetal calf serum (FCS) in RPMI 1640. After 2 h of incubation, nonadherent cells were removed by washing three times with Hanks' balance salt medium. The adherent cells, which were mostly monocytes

TNFα and IL-1β secretion of AGP-stimulated PBMC or monocytes is enhanced in the presence of sera

The amount of TNFα and IL-1β in the supernatants of PBMC cultures after 24 h of incubation with different doses of AGP in vitro is shown in Fig. 1. In the absence of AGP, the resting PBMC produced few TNFα in serum-free conditions while only low levels of TNFα (50–60 pg/ml) was produced in the presence of 10% human plasma which contained about 70 μg/ml AGP. The amount of TNFα was increased in the presence of AGP in a concentration-dependent manner. AGP at 1000 μg/ml (serum-free) induced over

Discussion

TNFα is an important proinflammatory cytokine produced by activated monocytes and has been shown to have multiple biological effects, such as causing tumor necrosis, septic shock and inflammation (Beutler and Cerami, 1988; Fiers, 1991). The expression of TNFα is under both transcriptional and translational control (Schindler et al., 1990). Previously, we demonstrated that TNFα secretion of human monocytes induced by AGP is dependent on de novo synthesis of TNFα mRNA (Su and Yeh, 1996). In this

Acknowledgements

This work was supported by grants NSC 87-2314-B-006-094 from the National Science Council, Taiwan, R.O.C. We thank Tainan blood center for providing human blood and plasma.

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