1 Institute of Infectious Diseases, Warsaw Medical Academy, Poland
2 Municipal Hospital for Infectious Diseases, Warsaw, Poland
3 Department of Medicine, SC Johnson Bldg Sj3, Mayo Clinic Scottsdale, AZ 85259, USA
4 MaternalChild Virology Research Laboratory, University of Southern California, Los Angeles, CA 90033, USA
Correspondence
Tomasz Laskus
laskus.tomasz{at}mayo.edu
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ABSTRACT |
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INTRODUCTION |
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Within the population of PBMC, the cells harbouring replicating virus have been identified as belonging to monocytes/macrophages, as well as T-cell and B-cell lineages (Bain et al., 2001; Laskus et al., 2000a
; Sansonno et al., 1996
). Whether the infection of macrophages is consequential is currently unclear; it was reported that chronic infection by HCV is associated with an allostimulatory defect and impaired maturation of monocyte-derived dendritic cells (Auffermann-Gretzinger et al., 2001
; Bain et al., 2001
; Kanto et al., 1999
). However, it is impossible to differentiate between changes caused by virus replication itself and those due to factors released from other cells of the immune system and/or from infected liver. Furthermore, although the presence of replication of HCV in macrophages in patients with chronic infection is highly likely, it is unclear whether these cells are susceptible to infection in vitro. Moreover, it is unclear whether macrophage infection affects the infected cell. The current study suggests that primary human macrophages can be infected by HCV in vitro and that the ensuing infection leads to induction of tumour necrosis factor-
(TNF-
) and interleukin 8 (IL-8) in the infected cells.
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METHODS |
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Monocytes/macrophages were collected from 10 healthy donors. Specifically, they were all anti-HCV and anti-HIV negative in serum and HCV RNA, hepatitis B virus (HBV) DNA and HGV RNA negative both in serum and in unfractioned PBMC. To obtain monocytes/macrophages, PBMCs were isolated from 50 ml of blood by centrifugation over a density gradient (Ficoll-Hypaque, Pharmacia). Cells were removed from the upper band located at the plasma and resolving medium interface and washed three times with Mg2+- and Ca2+-free PBS (pH 7·4). The typical yield of PBMCs was 24x107; these cells were determined to be free of granulocytes and platelets. To separate monocytes/macrophages from other cells, PBMCs were resuspended in 10 ml RPMI 1640 medium containing 10 % foetal bovine serum (FBS) and incubated in plastic six-well cell culture dishes (Costar), 1·5 ml of suspension per well. After incubation at 37 °C for 1 h, non-adhering cells were removed by vigorous washing four times with Mg2+- and Ca2+-free PBS while adhering cells (approximately 2x105 cells per well) were maintained in RPMI 1640 (Gibco/BRL) with 10 % FBS. The typical purity of the cells was >95 % based on morphological features in light microscopy and flow cytometry staining analysis of detached cells using monoclonal anti-CD68 and anti-CD14 antibodies. After 24 h, the macrophages were incubated for 4 h with HCV-positive serum diluted 1 : 10 in RPMI 1640 with occasional agitation. Subsequently, the cells were washed 3x10 min with PBS and maintained in RPMI with 10 % FBS. The culture medium was changed every 34 days.
Each of the HCV-positive sera was incubated with three independent macrophage cultures from two different donors (six cultures total for each serum). RNA was extracted from macrophages after 1, 2 or 3 weeks of culture and stored at -80 °C until analysis. RNA was extracted from cells and 100 µl of serum by means of a modified guanidinium thiocyanate/phenol/chloroform technique using a commercially available kit (TRIZOL LS, Gibco/BRL) and dissolved in 20 µl of water. Four to five µl of this RNA solution was reverse transcribed as further described.
Strand-specific RT-PCR.
Strand specificity of our RT-PCR for the detection of the 5' untranslated region (5' UTR) HCV RNA negative strands was ascertained by conducting cDNA synthesis at high temperature using the thermostable enzyme Tth. The sensitivity and strand specificity of this reaction was established using synthetic RNA as templates. A detailed description of our strand-specific assay and sequence of employed primers was published previously (Laskus et al., 1997, 1998
). In brief, the cDNA was generated in 20 µl of reaction mixture containing 50 pM of sense primer, 1x RT buffer (Perkin Elmer), 1 mM MnCl2, 200 µM (each) dNTP, and 5 U Tth (Perkin Elmer). After 20 min at 65 °C, Mn2+ was chelated with 8 µl of 10x EGTA chelating buffer (Perkin Elmer), 50 pM of antisense primer were added and the volume was adjusted to 100 µl, and the MgCl2 concentration was adjusted to 2·2 mM. The amplification was performed in a Perkin Elmer GenAmp PCR System 9600 thermocycler as follows: initial denaturing for 1 min at 94 °C, 50 cycles of 94 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s followed by a final extension at 72 °C for 7 min. Twenty µl of the final product was analysed by agarose gel electrophoresis and Southern hybridization with a 32P-labelled internal oligoprobe. For the detection of positive strand, the primers were added in reverse order.
The strand-specific assay was capable of detecting approximately 100 genomic equivalent (equiv.) molecules of the correct strand while unspecifically detecting about 108 genomic equiv. of the incorrect strand. The addition of 1 µg of total cellular RNA extracted from human tissues would lower the sensitivity of the reaction by no more than one log, while the specificity of the assay was not affected. Thus, the assay was capable of detecting between 102 and 103 virus genomic equiv. in 1 µg of RNA. The sensitivity and specificity of our assay for the detection of the positive strand was identical to that for the detection of the negative strand. Titres were determined by analysing 10-fold serial dilutions of the RNA template. The titres were calculated by assuming that the end-point dilution contains 102 genomic equiv. when tested with the Tth-based assay. Specificity controls, which consisted of serial dilutions of synthetic template, were included in each series of experiments.
Standard RT-PCR.
MMLV RT-based amplification of the 5' UTR has been described in detail previously (Laskus et al., 1998). This assay was capable of detecting approximately 10 genomic equiv. of the correct synthetic template but was not strand specific. Similarly to Tth-based assay, the addition of cellular RNA would slightly lower the sensitivity by up to one log. The established detection limit was approximately 10100 genomic equiv. per 1 µg of total RNA. In serum the approximate detection limit was 100 genomic equiv. per 1 ml. The non-structural (NS) 5B region was amplified by RT-PCR as described previously (Laskus et al., 2001
).
Appropriate measures, described elsewhere (Laskus et al., 1997, 1998
), were employed to prevent and detect contamination. Nested protocols, which are prone to carry over contamination, were not used for detection purposes. All RT-PCR runs included positive controls, consisting of end-point dilutions of respective RNA strands, and negative controls, which included macrophages from uninfected subjects and normal sera.
Analysis of HCV quasispecies.
The analysis was conducted on the stable 5' UTR and relatively stable NS5B region because a small number of expected virus variants within quasispecies allows for reliable comparison, and we have previously found that variations in the 5' UTR may correlate with extrahepatic replication (Laskus et al., 1998, 2000a
). In addition, comparison of the highly variable E2 region may be unreliable due to selective adsorption by human cells of virus quasispecies variants differing in the E2 region (Laskus et al., 2000b
). For the purpose of sequence comparison, nested protocols were used to maximize the yield of PCR product. Amplification of the 5' UTR and NS5B regions was conducted with RT-PCR assay as previously described (Laskus et al., 1998
, 2001
).
HCV quasispecies were compared by the single-strand conformation polymorphism (SSCP) assay as described previously (Laskus et al., 1998), with minor modifications. In brief, PCR products were purified with a DNA-binding resin system (Wizard PCR, Promega) and resuspended in 50 µl of water. Next, 24 µl of the purified product was diluted in 15 µl of low ionic strength solution (10 % saccharose, 0·5 % bromophenol blue, 0·5 % xylene cyanol), denatured by heating at 97 °C for 3 min, immediately cooled on ice and subjected to non-denaturing 8 % PAGE in 1x Tris/borate/EDTA buffer with 400 V applied for 56 h at a constant temperature of 25 °C. The bands were visualized with silver staining (Silver Stain, Promega). This assay enables detection of minor variants representing 3 % of the whole population (Laskus et al., 1998
).
All analysed products were sequenced directly using Perkin Elmer ABI 377 automatic sequencer. HCV genotypes were determined by direct sequencing of the NS5B region (Simmonds et al., 1993). HCV RNA was quantified using branched DNA assay (Quantiplex HCV-RNA 2.0, Roche).
Cytokine assays.
Concentrations of cytokines TNF-, IL-1
, IL-6, IL-8, IL-10 and IL-12p70 were measured in cell culture supernatants using a commercially available Cytometric Bead Array (BD). In this assay soluble analytes are captured on particles and then measured using a fluorescence-based detection system and flow cytometry analysis (Cook et al., 2001
; Oliver et al., 1998
).
The mRNAs of the above cytokines were quantified using the multiplex PCR (MPCR) kit marketed by Maxim Biotech, Inc. When the initial analysis suggested the presence of quantitative mRNA differences, analysis was repeated using Dual Quantitative PCR (Maxim Biotech, Inc.), which coamplifies only the housekeeping gene and the gene of interest. Amplified products were fractioned on an agarose gel, stained with ethidium bromide and quantified using the Bio-Rad UV camera and analysis software (Quantity One version 4.1.1, Bio-Rad). For comparison, gene expressions were normalized against the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative percentage of target specific gene expression was calculated as follows: X/Yx100 %=normalized target specific gene expression, where X=target specific gene expression level and Y=housekeeping gene expression level.
For confirmation of the above assay, which is largely semi-quantitative, transcripts of TNF- and IL-8 were quantified by real-time RT-PCR with SYBR Green I detection. The assays were performed with a LightCycler (Roche) with single-round amplification. Reaction mixtures included 2 µl of Fast Start DNA Master SYBR Green I (Roche), 4 mM MgCl2, 0·5 µM concentration of each primer and 2 µl of the template to a total volume of 20 µl. After initial incubation at 95 °C for 10 min, 40 cycles of amplification were performed each cycle consisting of 30 s at 95 °C, 5 s at 55 °C, and 30 s at 72 °C. Specificity of the signal was checked by melting curve analysis and target specific gene expression was normalized against histone mRNA (H3F3A). The following primers were used: H3F3A (sense 5'-CCACTGAACTTCTGATTCGC-3'; antisense 5'-GCGTGCTAGCTGGATGTCTT-3'), IL-8 (sense 5'-GCCAAGGAGTGCTAAAGA-3'; antisense 5'-CTTCTCCACAACCCTCTG-3'), TNF-
(sense 5'-GGCTCCAGGCGGTGCTTGTTC-3'; antisense 5'-AGACGGCGATGCGGCTGATG-3'). NCBI accession numbers for H3F33A, IL-8 and TNF-
genes, which were the basis for designing of the primers, were NM_002107, BC013615, and AY214167, respectively.
Statistical analysis was performed using the SPSS version 9.0 package. Means were compared by the non-parametric MannWhitney U test and paired data were compared by Wilcoxon signed rank sum test. Proportions were analysed by Fisher's exact test.
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RESULTS |
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To determine whether infection is reproducible, sera #14 and #15 were used to infect macrophages from two additional donors (Table 1). Virus positive strand was detectable in all cell cultures and virus negative strand was detectable in the majority of the samples suggesting that the implicated sera were infectious for macrophages from different donors. Furthermore, HCV RNA was detected in supernatant from every cell culture infected with sera #14 and #15 although the titre was low (5x1025x103 genomic equiv. ml-1). To determine the ratio of positive to negative strands, 10-fold serial dilutions of RNA extracted from macrophages incubated with serum #14 were tested for the presence of positive and negative HCV RNA by the Tth-based RT-PCR. The titre of virus negative strand was found to be one log lower than the titre of the positive strand (data not shown).
The two groups did not differ with respect to the distribution of HCV genotypes: type 1b was dominant as it was present in 12 out of 15 (80 %) and in 8 out of 11 (73 %) infective and non-infective sera, respectively. The second prevalent genotype was 3a and it was present in two sera from each group (Table 1). However, the infective sera had a higher mean virus load than the non-infective sera (7·3x106 versus 1·9x106 copies ml-1) and this difference was statistically significant (P<0·02). An important role of virus titre is further suggested by sera dilution experiments. Both sera #14 and #15, while infectious at dilution 1 : 10, did not infect macrophages when diluted 1 : 1000. Presence of HIV coinfection could have facilitated HCV infection as the mean HCV RNA load was lower in HIV positive sera transmitting infection when compared to HIV-negative sera transmitting infection (2·2x106 versus 9·8x106 copies ml-1). However, as the HIV-coinfected group was small, this difference did not reach statistical significance. HIV itself did not infect macrophages as all culture supernatants tested negative for the p24 antigen.
Analysis of HCV RNA in macrophage cultures by SSCP and sequencing
In the next step virus sequences amplified from cultured macrophages were compared by SSCP with sequences amplified from respective serum samples. In the vast majority of cases, SSCP band patterns between serum and cultured macrophages were indistinguishable, suggesting the presence of identical virus variant. However in four cases, virus sequences present in the macrophage cultures were different than those amplified from the infective serum either in the 5' UTR or NS5B region. These differences were not present in 1-week culture and were found only in macrophages grown for the duration of 23 weeks. In cases where virus negative strand could be amplified, its SSCP band pattern was congruent with the positive strand, suggesting that it indeed represented a virus replicative intermediary. Fig. 1 shows SSCP analysis in the example case of serum #14 where the band patterns for serum- and macrophage-derived virus sequences for both 5' UTR and NS5B regions were identical, and all four cases with dissimilar band patterns in either of these regions.
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Secretion and expression of cytokines by infected macrophages
To determine whether infection of macrophages in vitro entails any biological effect, we measured the concentrations of TNF-, IL-1
, IL-6, IL-8, IL-10, and IL-12p70 in cell culture supernatants at 2 weeks. The timing was not chosen arbitrarily: in 1-week cultures we could not determine whether the infection was successful or not, as evidenced by the common presence of HCV RNA in cases where no later cultures tested positive (Table 1
), while in the 3-week cultures the cells were already undergoing normal death. Altogether, 63 2-week culture supernatants were available for analysis. The concentrations of IL-1
, IL-6, IL-12p70 were below the cut-off value of the assay in 9095 % of the analysed samples and these cytokines did not undergo any further analysis. However, as shown in Fig. 4
(A), the concentrations of TNF-
and IL-8 were significantly higher in cases with putative HCV infection compared to uninfected cases. Similarly, IL-10 concentration was also higher in supernatants from infected macrophages, but this difference did not reach statistical significance (P=0·07).
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To determine whether there are differences in cytokine transcription levels, mRNAs for TNF-, IL-1
, IL-6, IL-8, IL-10 and IL-12 were quantified using the multiplex PCR (MPCR). Except for TNF-
and IL-8, mRNAs for other cytokines were amplified only occasionally and were therefore not analysed further. For quantification purposes, TNF-
and IL-8 mRNA was RT-PCR amplified using Dual Quantitative PCR, which coamplifies only the housekeeping gene and the gene of interest. The mRNA for the cellular enzyme GAPDH was monitored as an internal control to allow comparison of transcript levels between macrophages incubated with infective and heat-inactivated sera. Under the conditions used, both TNF-
and IL-8 gene expression was in a linear range as determined on serial dilutions of the template. Sufficient quality amplification allowing for analysis was available in all 16 HCV-positive cases and in 9 out of 12 HCV-negative controls. As seen in Fig. 5
, the quantity of amplified mRNA was lower in heat-inactivated serum cultures than in paired non-inactivated serum macrophage cultures. These differences were even more pronounced when transcripts were analysed by real-time RT-PCR. Again, parallel analysis using HCV-negative sera did not reveal significant differences between activated and inactivated samples, suggesting that the observed differences were not related to the process of heating per se (Fig. 5
).
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DISCUSSION |
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While infection of monocytes/macrophages in HCV-positive patients is likely, its consequences for cell function are unclear. It was reported that monocyte-derived dendritic cells from patients with chronic HCV infection demonstrate an allostimulatory defect (Bain et al., 2001; Kanto et al., 1999
). However, a direct relationship between virus replication and dendritic cell function could not be established in these studies. In the current study, in which primary human macrophages were infected in vitro and therefore the effects of factors unrelated to direct virusmacrophage interaction were likely to be eliminated, we found that exposure of primary macrophages to HCV-positive sera resulted in enhanced production of TNF-
and IL-8 and their respective mRNAs. Induction of these proinflammatory cytokines could be regarded as additional evidence for the susceptibility of human macrophages to HCV infection in vitro and suggests that such an infection is consequential to the cell.
TNF- is a major component of the immune system involved in the control of virus infection through direct antiviral activity, usually in association with interferon-
, and the induction of apoptosis (Herbein & O'Brien, 2000
). It is made mainly by monocytes and macrophages and its production was reported to increase when isolated human macrophages were infected with such RNA viruses as respiratory syncytial virus (Becker et al., 1991
), or influenza A virus (Lehmann et al., 1996
). Thus, production of TNF-
seems to be a natural response of macrophages to virus infection.
IL-8 was originally described as a neutrophil chemotactic factor, but subsequent studies demonstrated its various effects on T-cells and monocytes (Mukaida, 2000). There is growing evidence that infection with various viruses, or transfection with viral gene products, can induce IL-8 in different cells. For example, human cytomegalovirus (HCMV) infection can induce IL-8 production by concurrent activation of two distinct transcription factors, nuclear factor (NF)-
B and activator protein (AP)-1 (Murayama et al., 1997
); similar mechanisms of IL-8 gene transactivation were described for human T-cell leukaemia virus type 1 Tax protein and hepatitis B virus X protein (Mahe et al., 1991
; Mori et al., 1998
). Interestingly, increased serum levels of IL-8 were recently found in chronic hepatitis C patients (Polyak et al., 2001b
) and the same group of researchers demonstrated that expression of HCV non-structural 5A (NS5A) protein in human cells induced IL-8 mRNA and protein probably through a mechanism similar to that described previously for HCMV (Polyak et al., 2001a
). Because monocytes are one of the main producers of IL-8 in vivo, they could be an important source of increased IL-8 serum levels in chronic hepatitis C patients. Notably, IL-8 itself may directly promote virus replication. It was reported that IL-8 enhances HCMV and HIV-1 replication, probably by acting through its receptors CXCR1, and CXCR1 and CXCR2, respectively (Lane et al., 2001
; Murayama et al., 1994
). The effect on virus replication could also be indirect, as IL-8 was reported to reduce the antiviral activities of IFN-
. This inhibitory action on IFN-
antiviral activity was associated with reduced 2',5'-A oligoadenylate synthetase activity, a pathway well correlated with the antiviral action of IFN-
(Khabar et al., 1997
). The latter mechanism has been postulated to promote HCV persistence and resistance to IFN therapy (Polyak et al., 2001b
).
It has been long known that HCV RNA levels are higher in HIV-coinfected subjects and this phenomenon has been largely attributed to the HIV-induced immunosuppression with subsequent impairment of the containment effects of the immune system. However, several studies suggest a more direct interaction: HCV RNA levels were reported to be more closely associated with HIV RNA levels than with CD4+ T cell count (Thomas et al., 2001), and HIV seroconversion in HCV-infected patients is often associated with a burst of enhanced HCV replication (Beld et al., 1998
). Interestingly, it was also reported that HCV infection is associated with higher HIV RNA levels and more rapid HIV disease progression (Daar et al., 2001
; Greub et al., 2000
). As both HIV and HCV induce IL-8 production, which in turn could enhance their replication, it could be speculated that IL-8 could play some role in HCV/HIV interactions.
In summary, our data suggest that human macrophages may be susceptible to HCV infection in vitro, although the level of ensuing replication seems to be small. Macrophage infection was associated with induction of TNF- and IL-8 cytokines and their respective mRNAs.
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ACKNOWLEDGEMENTS |
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Received 7 July 2003;
accepted 15 September 2003.