1 Department of Internal Medicine I, Johannes Gutenberg University, Langenbeckstrasse 1, 55131 Mainz, Germany
2 Department of Internal Medicine II, University of Freiburg, Germany
3 Department of Virology, Humboldt University, Berlin, Germany
4 Department of Internal Medicine II, Klinikum Grosshadern, University of Munich, Germany
Correspondence
Wulf O. Böcher
boecher{at}mail.uni-mainz.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of our study was to assess the functional consequences of this MDDC phenotype in patients with various courses of chronic hepatitis B. Because isolation of DC from peripheral blood is hampered by the very low numbers and immature differentiation stage of circulating DC precursors (Banchereau & Steinman, 1998), a well established protocol to generate DC in vitro from monocytes by culture with IL4 and GM-CSF (Palucka et al., 1998
; Romani et al., 1994
) has been employed to study phenotype and function of MDDC from patients with chronic hepatitis B, healthy controls and donors with spontaneously resolved acute HBV infection (RHB).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
MDDC culture.
Donor PBMC were incubated at 12x106 cells per well in 6-well plates for 60 min in PBS. After removal of non-adherent cells, the remaining cells were incubated in serum-free MDDC medium [X-Vivo 15 (Biowhittaker) supplemented with 800 U ml1 GM-CSF (Leukomax) and 1000 U ml1 IL4 (Strathmann Biotech)] at 37 °C. Cells were fed every 2 days with fresh medium. After 7 days, non-adherent immature MDDC were harvested. In maturation experiments, MDDC were cultured for an additional 2 days in X-Vivo 15 medium supplemented with IL1, TNF-
(both: 10 ng ml1, R&D Systems; each: 1000 U ml1), IL6 (Strathmann Biotech; 1000 U ml1) and Prostaglandin (Pg) E2 (Sigma; 1 mg ml1).
Flow cytometry of surface markers.
Expression of surface markers was analysed by flow cytometry using conjugated monoclonal mouse-anti-human antibodies: FITC-anti-CD1a, FITC-anti-CD3, FITC-anti-CD40, FITC-anti-CD83, PE-anti-CD14, PE-anti-CD86, PE-anti-HLA DR and CY-Chrome-anti-CD19 (Pharmingen). Analysis was performed on a FACScan (Becton Dickinson) utilizing the CellQuest software. Quadrants were set according to staining with the respective isotype controls.
T cell stimulation (ELISpot, proliferation assay).
MDDC from patients and controls were harvested at day 7, before assessment by enzyme-linked immunospot assays (ELISpot) as described (Böcher et al., 1999). PVDF-membrane-bottomed 96-well plates (Millipore) were coated with 10 µg per well of mAb against IFN-
(MabTech) in carbonate coating buffer. Patient and control MDDC were seeded in triplicates (5x104 per well) together with 1x105 per well CD4+ Th cells. Phytohaemagglutinine-stimulated MDDC/T cell co-cultures served as positive controls. MDDC were irradiated (3000 rad) and washed twice before use. After incubation for 40 h, cells were discarded and the plates were washed in PBS-0·05 % Tween and incubated with biotinylated anti-IFN-
mAb (1 : 1000; MabTech). After washing, plates were incubated with HRP-Extravidin, washed again and incubated with AEC solution (both Sigma). The staining was stopped by rinsing under water and red spots were counted as single spot forming cells (SFC). Frequencies of alloreactive T cells were calculated from the SFC in immature MDDC/T cell co-cultures corrected for the background SFC of MDDC monocultures.
Mixed lymphocyte reactions (MLR) were performed by 4 days of co-incubation of immature patient and control MDDC in the indicated numbers with 105 per well allogeneic PBMC, before plates were pulsed with 0·25 µCi (9·25 kBq) per well [3H]thymidine and read by liquid scintillation technique.
For stimulation of the HBc-specific Th cell clone, PBMC from 4 HLA DR7-positive chronic HBV patients and healthy controls each was stored frozen and thawed simultaneously, cultured for 7 days and pulsed with medium or HBc antigen as described above. A monoclonal anti-DR antibody (10 µg ml1; Pharmingen) was added to analyse HLA restriction. MDDC (5x104 per well) and clonal T cells (1x105 per well) were co-incubated in triplicates in 96-well plates for 4 days and pulsed with 0·25 µCi per well [3H]thymidine for a further 18 h before the incorporated radioactivity was measured by liquid scintillation technique. The results were expressed as antigen specific stimulation index (SI) calculated as the ratio of stimulated and unstimulated cultures. Baseline counts were similar for patient and control cultures (range: 215700 c.p.m. and 180560 c.p.m., respectively).
Cytokine secretion by MDDC.
Supernatants of MDDC were stimulated or not for 48 h with varying doses of HBcAg (4 and 20 µg ml1; Diasorin) or LPS (0·1, 1, 10 µg ml1; Sigma) used to assess the secretion of TNF-, IL10, and IL12p70 by ELISA according to the manufacturer's instructions (OptEIA; Pharmingen). Background values of unstimulated MDDC cultures were between 10 and 60 pg ml1 and 0 and 50 pg ml1 for IL12 and IL10, respectively, and did not differ between donor groups.
HBV-DNA and antigen expression.
CD14+ monocytes, CD19+ B cells and CD4+ or CD8+ T cells were separated from PBMC using immunomagnetic beads for the respective surface marker (Miltenyi Biotech). In some experiments, MDDC were enriched further by immunomagnetic depletion of CD14+, CD19+ and CD8+ cells. Cellular DNA was isolated from 1·0 to 2·5x106 cells by the Qiagen method (Qiagen), resuspended in a total volume of 100 µl distilled water and the DNA content was quantified photometrically. Cellular and serum HBV-DNA was detected quantitatively using the HBV-Amplicor assay according to manufacturer's instructions (Roche Diagnostics).
For expression studies of HBs antigen, the pellet of at least 2x106 MDDC was lysed in lysis buffer of 1 % Triton X, 1 mM PMSF (Roche Diagnostics; #1697498), 50 mM Tris in 150 mM NaCl (pH 8·0) and stored at 20 °C until analysis in a commercial HBsAg ELISA according to manufacturer's instructions (ETI-MAK-3; Diasorin Biomedica).
Statistics.
The Mann-Whitney test for non-parametric data were calculated utilizing the StatView software (SAS Institute). P-values<0·05 were regarded as significant. The MLR proliferation data were analysed as summary measures between patients and controls using the student t-test for impaired data.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
The transcription of HBV genes in MDDC from a total of nine CAH and ISC patients was analysed by ELISA and uncovered the expression of HBsAg in MDDC from two CAH patients, both with high serum DNA levels (2x107 and 1·2x1010 VGE ml1), while MDDC from a further five CAH and one ISC patients were HBsAg-negative, probably due to low-level viral transcription or the low amount of protein available from the MDDC lysate.
Alloreactive Th cell stimulation
In previous studies, MLR have been used to study the T cell stimulatory capacity of MDDC from HBV patients. Because allostimulation depends on the degree of HLA mismatch between stimulator and responder cells, immature MDDC and responder T cells from a rather large number of donors were assessed in our study: MDDC derived from six healthy individuals or six HBV patients were co-incubated with a panel of separated CD4+ Th cells or PBMC derived from five allogeneic healthy donors and the frequencies of alloreactive T cells were analysed by IFN--ELISpot or proliferation assay. In contrast to the previous studies, both sets of experiments did not reveal significant differences between patient and control MDDC in stimulating alloreactive IFN-
secretion (Fig. 3
a) or proliferation (P>0·05; Fig. 3b
), arguing strongly against a reduced T cell stimulation by patient DC.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The central mechanisms of DC-mediated T cell stimulation after antigen uptake and processing by the APC comprise the engagement of the antigen-specific T cell receptor by peptide loaded MHC molecules, interaction of costimulatory molecules with their receptors on the T cell and the secretion of cytokines. Our FACS analyses revealed a significantly reduced expression of CD40 and HLA DR and a slight reduction of CD86 on MDDC from ISC and CAH patients. This rather immature phenotype is in concordance with Wang et al. (2001), whereas Beckebaum et al. (2002)
using a different protocol to generate MDDC found only the expression of CD80 and CD86 reduced. In our study, a reduced expression of CD86 and HLA DR was detected even years after resolution from self-limited HBV infection, while CD40 was strongly induced on RHB-MDDC indicating a permanent influence of the virus on MDDC phenotype. However, the reduced expression of costimulatory molecules of immature patient MDDC was reversed after cytokine induced maturation and was not associated with reduced allostimulation of healthy Th cells in IFN-
secretion and proliferation assays, indicating the lack of functional relevance of this finding. Although this is in contrast to previous reports of reduced allostimulation by MDDC from HBV patients (Beckebaum et al., 2002
; Wang et al., 2001
), different protocols to generate MDDC, heterogeneous patient populations and the examination of only one or two healthy responder cell donors are important limitations of these reports. Moreover, a reduced allostimulation by DC would indicate a general T cell stimulation failure of chronic HBV patients that would contrast to the clinical immune competence of chronic HBV patients.
The key cytokines provided by APC determining the induced Th cell response towards a Th1 or Th2 cell cytokine pattern are TNF-, IL10 and IL12 (Trinchieri & Scott, 1994
) that are secreted by DC in response to various viral, protozoal or bacterial products (Ahuja et al., 1998
; Hilkens et al., 1997
; Macatonia et al., 1995
; von Stebut et al., 1998
). In HBc-antigen stimulated MDDC cultures, MDDC derived from CAH or ISC patients indeed showed a reduced secretion of IL12 and IL10 compared with RHB and HBV-naive controls, corresponding to previous findings under different stimulation conditions (Beckebaum et al., 2002
; Wang et al., 2001
). In contrast, after LPS stimulation the secretion of both cytokines was intact, precluding a general cytokine secretion defect.
In order to assess, whether this HBc-specific cytokine secretion failure might have functional implications on HBc-specific T cell stimulation, the response of an HBc-specific Th cell clone to stimulation by HLA DR matched MDDC from HBV patients and controls was studied. The finding of identical proliferation and IFN- secretion by the Th1 cell clone in co-cultures with MDDC from chronic HBV patients or controls ruled out a significant stimulation failure of patient MDDC and rather argues for a virus-specific responder Th cell defect.
HBV might cause these phenotypic and functional alterations by direct infection of DC precursors in the blood or bone marrow, as suggested by our PCR and ELISA studies and the report of Beckebaum et al. (2002), demonstrating the expression of HBV-DNA, particles and antigens in MDDC from HBV patients. However, whether DC are a site of HBV replication or gene expression is not clear yet, but from our studies the proof of HBV in MDDC seems to be irrelevant for the function of DC, particularly in the light of an otherwise fully competent immune system of chronic HBV patients.
Finally, our data, just as those of previous studies, have to be interpreted in consideration of using an in vitro model to generate DC from monocytic precursors. Although this differentiation pathway has been reported in vitro and in vivo (Randolph et al., 1998, 1999
), artefacts cannot be excluded because the resulting cell population might contain different DC and non-DC populations. Thus, ex vivo studies on purified DC are under way to assess whether these data are applicable to DC subpopulations in vivo.
In conclusion, our data suggest the infection of myeloid DC by the HBV leading to minor phenotypic and functional alterations with reduced expression of costimulatory molecules and IL10/IL12 secretion under certain experimental conditions. However, the Th cell stimulatory capacity of MDDC from chronically-infected HBV patients was unaffected by these changes, indicating that mechanisms other than a DC defect led to the well described antiviral T cell failure in chronic hepatitis B.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Auffermann-Gretzinger, S., Keeffe, E. B. & Levy, S. (2001). Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection. Blood 97, 31713176.
Bain, C., Fatmi, A., Zoulim, F., Zarski, J. P., Trepo, C. & Inchauspe, G. (2001). Impaired allostimulatory function of dendritic cells in chronic hepatitis C infection. Gastroenterology 120, 512524.[Medline]
Banchereau, J. & Steinman, R. M. (1998). Dendritic cells and the control of immunity. Nature 392, 245252.[CrossRef][Medline]
Beckebaum, S., Cicinnati, V. R., Dworacki, G. & 8 other authors (2002). Reduction in the circulating pDC1/pDC2 ratio and impaired function of ex vivo-generated DC1 in chronic hepatitis B infection. Clin Immunol 104, 138150.[CrossRef][Medline]
Bertoletti, A. & Ferrari, C. (2003). Kinetics of the immune response during HBV and HCV infection. Hepatology 38, 413.[CrossRef][Medline]
Böcher, W. O., Herzog-Hauff, S., Schlaak, J., Meyer zum Büschenfeld, K. H. & Löhr, H. F. (1999). Kinetics of hepatitis B surface antigen-specific immune responses in acute and chronic hepatitis B or after HBs vaccination: stimulation of the in vitro antibody response by interferon-. Hepatology 29, 238244.[CrossRef][Medline]
Chisari, F. V. & Ferrari, C. (1995). Hepatitis B virus immunopathogenesis. Annu Rev Immunol 13, 2960.[CrossRef][Medline]
Engelmayer, J., Larsson, M., Subklewe, M., Chahroudi, A., Cox, W. I., Steinman, R. M. & Bhardwaj, N. (1999). Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion. J Immunol 163, 67626768.
Hilkens, C. M., Kalinski, P., de Boer, M. & Kapsenberg, M. L. (1997). Human dendritic cells require exogenous interleukin-12-inducing factors to direct the development of naive T-helper cells toward the Th1 phenotype. Blood 90, 19201926.
Jonuleit, H., Kuhn, U., Muller, G., Steinbrink, K., Paragnik, L., Schmitt, E., Knop, J. & Enk, A. H. (1997). Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur J Immunol 27, 31353142.[Medline]
Jung, M. C., Diepolder, H. M., Spengler, U. & 7 other authors (1995). Activation of a heterogeneous hepatitis B (HB) core and e antigen-specific CD4+ T-cell population during seroconversion to anti-HBe and anti-HBs in hepatitis B virus infection. J Virol 69, 33583368.[Abstract]
Kanto, T., Hayashi, N., Takehara, T., Tatsumi, T., Kuzushita, N., Ito, A., Sasaki, Y., Kasahara, A. & Hori, M. (1999). Impaired allostimulatory capacity of peripheral blood dendritic cells recovered from hepatitis C virus-infected individuals. J Immunol 162, 55845591.
Kunitani, H., Shimizu, Y., Murata, H., Higuchi, K. & Watanabe, A. (2002). Phenotypic analysis of circulating and intrahepatic dendritic cell subsets in patients with chronic liver diseases. J Hepatol 36, 734741.[CrossRef][Medline]
Lew, Y. Y. & Michalak, T. I. (2001). In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus. J Virol 75, 17701782.
Löhr, H. F., Pingel, S., Böcher, W. O., Bernhard, H., Herzog-Hauff, S., Rose-John, S. & Galle, P. R. (2002). Reduced virus specific T helper cell induction by autologous dendritic cells in patients with chronic hepatitis B - restoration by exogenous interleukin-12. Clin Exp Immunol 130, 107114.[CrossRef][Medline]
Macatonia, S. E., Hosken, N. A., Litton, M. & 7 other authors (1995). Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 154, 50715079.
Palucka, K. A., Taquet, N., Sanchez-Chapuis, F. & Gluckman, J. C. (1998). Dendritic cells as the terminal stage of monocyte differentiation. J Immunol 160, 45874595.
Randolph, G. J., Beaulieu, S., Lebecque, S., Steinman, R. M. & Muller, W. A. (1998). Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 282, 480483.
Randolph, G. J., Inaba, K., Robbiani, D. F., Steinman, R. M. & Muller, W. A. (1999). Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity 11, 753761.[Medline]
Romani, N., Gruner, S., Brang, D. & 7 other authors (1994). Proliferating dendritic cell progenitors in human blood. J Exp Med 180, 8393.[Abstract]
Servet-Delprat, C., Vidalain, P. O., Bausinger, H., Manie, S., Le Deist, F., Azocar, O., Hanau, D., Fischer, A. & Rabourdin-Combe, C. (2000). Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells. J Immunol 164, 17531760.
Stoll-Becker, S., Repp, R., Glebe, D., Schaefer, S., Kreuder, J., Kann, M., Lampert, F. & Gerlich, W. H. (1997). Transcription of hepatitis B virus in peripheral blood mononuclear cells from persistently infected patients. J Virol 71, 53995407.[Abstract]
Trinchieri, G. & Scott, P. (1994). The role of interleukin 12 in the immune response, disease and therapy. Immunol Today 15, 460463.[CrossRef][Medline]
von Stebut, E., Belkaid, Y., Jakob, T., Sacks, D. L. & Udey, M. C. (1998). Uptake of Leishmania major amastigotes results in activation and interleukin 12 release from murine skin-derived dendritic cells: implications for the initiation of anti-Leishmania immunity. J Exp Med 188, 15471552.
Wang, F. S., Xing, L. H., Liu, M. X., Zhu, C. L., Liu, H. G., Wang, H. F. & Lei, Z. Y. (2001). Dysfunction of peripheral blood dendritic cells from patients with chronic hepatitis B virus infection. World J Gastroenterol 7, 537541.[Medline]
Received 29 March 2004;
accepted 18 June 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |