Center for Vaccinology, Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium1
Author for correspondence: Peter Vanlandschoot. Fax +32 9 240 36 54. e-mail Peter.Vanlandschoot{at}rug.ac.be
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Abstract |
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Introduction |
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Embedded in the viral membrane are three related viral membrane proteins, L, M and S, which share 226 carboxy-terminal amino acids. During HBV infection, non-infectious subviral lipoprotein HBV surface antigen (HBsAg) particles are produced in large quantities by the infected hepatocytes and are secreted into circulation, where concentrations of 50300 µg/ml are attained (Ganem, 1996 ). Like virions, these lipoprotein particles contain predominantly the S protein, smaller amounts of M protein and hardly any L protein. Recombinant expression of only the S protein in yeast, mammalian and insect cells demonstrated that this protein has the unique property to form these HBsAg particles, which contain up to 30% of cell-derived lipids. The reason for the existence of, or the possible advantage of, the production of HBsAg remains elusive, until today. It is, however, remarkable that, in both acutely and chronically infected persons, a cellular and humoral anti-HBsAg response is lacking, despite the presence of HBsAg (Milich, 1997
). Because anti-envelope antibodies are clearly detectable only in patients who recover from acute hepatitis and not in chronically infected subjects, these are thought to play a critical role in virus clearance.
The mechanism by which HBV establishes a persistent infection is at present still unclear. Studies with HBV transgenic mice led to the generally accepted idea that tolerance at the T cell level is an important underlying mechanism for the establishment of the persistent state, especially in neonates (Milich, 1997 ; Chisari & Ferrari, 1995
; Chisari, 1995
). However, defects in the antigen-presenting activity of dendritic cells, rather than functional defects in T or B cells are claimed to be responsible for the induction of HBV persistence (Akbar et al., 1993
; Kurose et al., 1997
). In vitro studies demonstrate a reduced capacity of PBMCs from chronically infected persons to produce cytokines upon stimulation with lipopolysaccharide (LPS) (Muller & Zielinski, 1990
, 1992
), while HBsAg inhibits the release of LPS-induced cytokines by human macrophages (Jochum et al., 1990
). Taken together, these results suggest that HBV infection or virus products may interfere with the normal function of antigen-presenting cells, like monocytes, macrophages and dendritic cells, which may add to the development of HBV persistence. To examine if and how HBsAg can influence the activity of monocytes, the physical interaction of HBsAg with PBMCs was studied by FACS, using biotinylated yeast-derived S particles. It is reported here that such particles bind almost solely to monocytes and not to T cells, while some interaction with B cells is observed. It is further shown that recombinant HBsAg (rHBsAg) particles not only inhibit LPS-induced secretion of IL-1
and TNF
, but also inhibit IL-2-induced secretion of IL-8.
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Methods |
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Biotinylation of rHBsAg.
rHBsAg was biotinylated using an enhanced chemiluminescent protein biotinylation module (RPN 2202, Amersham Pharmacia). 300 µl rHBsAg was mixed with 270 µl H2O, 30 µl 0·8 M bicarbonate buffer, pH 8·6, and 15 µl biotinylation reagent. The mixture was incubated at room temperature for 1 h, after which 24 µl 1 M Tris was added. Biotinylated rHBsAg (b-rHBsAg) was purified by gel filtration on a Sephadex G25 column using PBS. Fractions of 1 ml were collected and the two b-rHBsAg peak fractions, as determined by ELISA, were pooled.
ELISA.
Maxisorb 96-well plates (Nunc) were coated with rHBsAg or b-rHBsAg in PBS. The wells were blocked with 0·1% BSA in PBS, followed by washing three times (0·1% Triton X-100). HBsAg-specific monoclonal antibodies (MAbs) (1 µg/ml) or streptavidinhorseradish peroxidase were added and plates were incubated for 1 h at room temperature. MAbs were detected with goat anti-mouse or goat anti-human antibodies labelled with peroxidase. After three washes, 3,3',5,5'-tetramethylbenzidine (Sigma) was added and, 30 min later, the reaction was stopped with 1 N H2SO4.
Antibodies.
Mouse anti-human CD3FITC (clone SK7), CD14FITC (clone MP9), CD19FITC (clone 4G7), IgG1FITC isotype control and streptavidinphycoerythrin (StrepPE) antibodies were purchased from Becton Dickinson. Mouse anti-human CD14FITC (clone My4) and IgG2bFITC isotype control antibodies were purchased from Immunotech. Human anti-HBsAg clones F47B and F9H9 were a kind gift from Lia Sillekens (Centraal Laboratorium van de Bloedbank, Amsterdam). Human MAb anti-a was developed in the laboratory. Mouse anti-d and anti-y were a kind gift from DiaSorin.
Cells.
Human PBMCs were isolated from buffy coats using FicollHypaque (density=1·077 g/ml, Nycomed Pharma) centrifugation. Cells were stored in liquid nitrogen. Phorbol 12-myristate 13-acetate treatment (25 ng/ml) (PMA, Sigma) was performed for 4 h in cRPMI (RPMI 1640 containing 10% foetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin and 20 µM -mercaptoethanol). THP-1 cells were grown in cRPMI. To induce differentiation, 100 nM 1,25-dihydroxyvitamin D3 (1,25-VitD3, Calbiochem) was added for 24 or 48 h. Cultured cells were detached mechanically or by using non-enzymatic cell dissociation buffer (Sigma), washed twice with 2% non-heat-inactivated human AB serum (HS, Bio-Whitaker) in Hank's Balanced Salt Solution (Gibco BRL) (2% HSHBSS) and stained as described below.
Staining of cells.
PBMCs were thawed and washed twice with 2% HSHBSS. Approximately 106 cells were incubated with b-rHBsAg in 200 µl 2% HSHBSS for 1 h on ice. After two washes with the same solution, cells were incubated with StrepPE and/or FITC-labelled antibodies in 2% HSHBSS for 1 h on ice. After two washes, cells were resuspended in 1 ml 2% HSHBSS or PBS containing propidium iodide (PI) and analysed on a FACScan flow cytometer (Becton Dickinson). Dead cells, which incorporated PI, were gated out of analysis. At least 5000 cells were counted per analysis. Fluorescence (530 nm for FITC and 580 nm for PE) was measured. Median fluorescence was determined in each case. Signals were acquired in a logarithmic mode for Fl1 (FITC) and Fl2 (PE). Threshold levels were set according to negative (StrepPE only) and isotypic controls.
LPS treatment of THP-1 cells and PBMCs.
THP-1 cells (5x105) were treated for 24 h with 100 nM 1,25-VitD3. After washing, the cells were incubated in cRPMI either with or without 10 or 50 ng/ml LPS (Escherichia coli 0111:B4, Sigma), to which 0, 0·1, 1, 10 or 50 µg/ml rHBsAg was added. In separate experiments, 106 PBMCs were incubated in cRPMI either with or without 10 or 50 ng/ml LPS to which 0, 0·1, 1, 10, 25 or 50 µg/ml rHBsAg was added. Cell supernatants were collected after 24 h and tested for the presence of IL-1 and TNF
.
IL-2 treatment of PBMCs.
Approximately 106 PBMCs were incubated in cRPMI either with or without 1000 U/ml IL-2 (Eurocetus), after which, 0, 1, 10, 25 or 50 µg/ml rHBsAg was added. Cell supernatants were collected after 24 h and tested for the presence of IL-8.
Cytokine determination.
The concentrations of IL-1, TNF
and IL-8 in the cell supernatants were determined using commercially available kits (Bioscource) according to the manufacturer's instructions.
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Results |
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Divalent cations reduce binding of b-rHBsAg to monocytes
Divalent cations that are present at millimolar concentrations in serum can often modulate the interaction between ligands. Therefore, the effect of Ca2+ and Mg2+ on the binding of b-rHBsAg to PBMCs was investigated. The addition of increasing amounts of Ca2+ and Mg2+ caused reduced binding of b-rHBsAg (Fig. 5a); the addition of 5 mM EDTA to the mixture restored attachment. The addition of Ca2+ and Mg2+ after binding had no effect (Fig. 5a
). Reduced binding was also observed when only Ca2+ or only Mg2+ was added (data not shown). These experiments were all performed in 2% HS in 50 mM TrisHCl and 150 mM NaCl instead of HBSS to prevent acidification when adding 5 mM EDTA.
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Effect of monocyte differentiation state on the attachment of b-rHBsAg
THP-1 cells, a pre-monocytic cell line, differentiate towards a monocytic cell type by treatment with 1,25-VitD3. This differentiation is easily detected by the expression of CD14. Binding of b-rHBsAg to this pre-monocytic cell line and 1,25-VitD3-differentiated THP-1 cells was investigated. CD14 expression was detected using two different specific MAbs, clones P9 and My4. Both antibodies were used because they recognize different forms of CD14, which may differ in expression levels on monocytes and monocytic cell lines (Pedron et al., 1995 ). Undifferentiated THP-1 cells showed no detectable expression of CD14 and did not bind b-rHBsAg (Fig. 6a
). 1,25-VitD3-differentiated THP-1 cells expressed CD14 molecules that were recognized by both antibodies. These differentiated cells did bind b-rHBsAg (Fig. 6a
), which demonstrates that only monocytes in a certain maturation state bind rHBsAg.
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Effect of non-biotinylated rHBsAg on the function of monocytes
Because rHBsAg interacts specifically with monocytes, the effect of rHBsAg on the function of these cells was investigated (Fig. 7). Monocytes were activated with LPS or IL-2. First, THP-1 cells were incubated for 24 h with 100 nM 1,25-VitD3, washed and incubated either with or without LPS in the presence of different concentrations of rHBsAg. Culture supernatant was collected after 24 h of incubation and tested for the presence of IL-1
and TNF
. rHBsAg alone did not induce any detectable cytokine production, while LPS induced the secretion of both cytokines (Fig. 7
). High levels of TNF
were produced, while lower concentrations of IL-1
were detected. The highest cytokine levels were obtained with 50 ng/ml LPS. In the presence of rHBsAg, LPS-induced cytokine production decreased in a dose-dependent manner.
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The effect of rHBsAg on IL-2-induced cytokine production was studied with PBMCs from four donors. As shown in Table 1, non-stimulated PBMCs already produced IL-8, the levels of which increased by adding IL-2. In the presence of rHBsAg, IL-8 production of both stimulated and non-stimulated PBMCs decreased in a dose-dependent manner.
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Discussion |
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To find a possible biological function for this rHBsAgreceptor interaction, the effect of rHBsAg on LPS- and IL-2-induced activation of monocytes was investigated. One of the most potent activators of monocytes is LPS, which induces the secretion of several cytokines, such as IL-1, IL-6, IL-12 and TNF
. rHBsAg particles themselves did not induce any of these cytokines, while LPS-induced secretion of IL-1
and TNF
was reduced in the presence of rHBsAg. Using macrophages and plasma-purified HBsAg, identical results for TNF
have been reported previously. However, in contrast to our results, human macrophages produced IL-1
in response to HBsAg (Jochum et al., 1990
). A second potent activator of monocytes is IL-2, which, among several other activities, increases the secretion of cytokines like IL-8, IL-6 and TNF
. As shown previously (Bosco et al., 1997
), blood monocytes already secreted IL-8 when cultured without IL-2. This production was downregulated by rHBsAg. More importantly, IL-2-induced IL-8 secretion was reduced in the presence of rHBsAg.
Viruses have long been viewed as simple genetic parasites that use the host cellular machinery to propagate themselves. However, it has become clear that the co-existence of these pathogens and their hosts have shaped the immune system and resulted in a surprising diversity of virus strategies to manipulate different cellular and immune regulatory systems. Viruses have targeted cellular cytokine production and cytokine receptor-signalling pathways, apoptotic pathways, cell growth and activation pathways, MHC-restricted antigen presentation pathways and humoral immune responses (Alcami et al., 2000 ; Tortorella et al., 2000
). Our results suggest strongly that monocytes express a receptor that is recognized by HBsAg. Engagement of this receptor, through interaction with a serum protein, suppresses the activity of monocytes. These observations suggest that HBV produces HBsAg in excess amounts to interfere with the normal function of antigen-presenting cells.
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Acknowledgments |
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References |
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Received 18 January 2002;
accepted 8 February 2002.