| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Internal Medicine,*
University of
Bonn, Bonn, and the Department of Hepatology and
Gastroenterology,
Medizinische Hochschule
Hannover, Hannover, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recently, it has become evident that a sufficient immune response depends on an efficient T cell activation via co-stimulatory molecules.1-4 The interaction of CD80 (B7-1) and CD86 (B7-2) with their counter-receptors on T lymphocytes (CD28 and CD152) provides a particularly potent co-stimulatory signal, which amplifies the response of T cells. Although antigen presentation followed by co-stimulation induces full T-cell activation, such antigen presentation that lacks co-stimulatory signals results in tolerance or anergy for the specific antigen.5-7 CD80 is a 44- to 60-kd member of the immunoglobulin superfamily with a limited expression on professional APCs such as macrophages, dendritic cells, and activated B cells. CD86 is a 75- to 115-kd cell surface glycoprotein with 25% amino acid homology to CD80. It also has a restricted expression on APCs.
Regulation of co-stimulation may also involve the CD40 molecule. CD40 is a 45- to 48-kd glycoprotein, which interacts with its natural ligand CD154 (gp39) on lymphocytes and which can be expressed on a great variety of different cells.8-10 A favored hypothesis suggests that recognition of the MHC-antigen complex by T cells leads to the induction of CD154 in these T cells. Subsequently, the simultaneous engagement of the MHC-antigen complex by the T-cell receptor and CD40 by CD154 up-regulates the expression of the co-stimulatory molecules CD80 and CD86 in the APCs.11-14
Following the hypothesis that the delicate balance between hepatic immune tolerance and immune stimulation could be deranged in FHF, we studied the expression of the co-stimulatory and immunoactivating molecules CD80, CD86, and CD40 as well as their ligands in patients with FHF. Furthermore, the time cascade of CD80/CD86 and CD40 expression was examined after inducing of experimental FHF in BALB/c mice by exposure to either galactosamine and lipopolysaccharide (GalN/LPS) or galactosamine and tumor necrosis factor (GalN/TNF).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Explant tissue from 18 patients with FHF was included in the study. All patients fulfilled the King's College criteria15 for urgent transplantation. The etiology of hepatic failure was hepatitis B (n = 10), cryptogenic (n = 5), 5-methoxy-3,4-methylene-dioxymethamphetamine (MDMA) toxicity (n = 1), and autoimmune hepatitis (n = 2). The liver tissue from 30 patients with chronic liver disease (CLD); —nine cases of chronic persistent hepatitis B, five of chronic active hepatitis B, six of chronic hepatitis C, five of primary biliary cirrhosis, three of primary sclerosing cholangitis, and two of autoimmune chronic active hepatitis) as well as from two patients with fulminant Wilson's disease and from three patients with acute hepatitis (HAV, n = 2; HCV, n = 1)—were included as disease controls. Non-tumor-bearing liver tissue obtained during the resection of hepatic malignancies in four patients and wedge biopsies from five donor livers at the time of hepatectomy served as normal controls (NCs).
Immunostaining Procedures
Liver tissue was immediately embedded in Tissue Tek OCT compound (Miles Laboratories, Naperwille, IL) and snap-frozen in liquid nitrogen. Frozen tissue was kept at -80°C until examined. Sections of 5 to 7 µm were stained by an indirect immunoperoxidase technique. In brief, endogenous peroxidase activity was blocked by 0.03% H2O2/NaNO3 (peroxidase blocking reagent, Dako, Carpinteria, CA). The sections were incubated with mouse monoclonal primary antibodies in phosphate-buffered saline (PBS)/1% fetal calf serum (FCS) in a moist chamber at room temperature for 90 minutes. After washing in PBS, peroxidase-coupled goat anti-mouse antibody (Dianova, Hamburg, Germany) was applied for 30 minutes. Any bound antibody was detected with 3-amino-9-ethylcarbazole (Sigma Chemicals, Munich, Germany). All sections were then counterstained with Meyer's hematoxylin.
The results of the indirect immunoperoxidase technique were confirmed by a double-step immunoenzymatic labeling using immune complexes of alkaline phosphatase and monoclonal anti-phosphatase (APAAP). After incubating sections with the primary antibody, rabbit anti-mouse antibodies were applied to the slides for 25 minutes (Dako). Thereafter, APAAP complexes were detected with naphthol-AS biphosphate/neufuchsin (Sigma Chemicals).16
Monoclonal Antibodies
To ensure the specificity of monoclonal antibodies (MAbs) we stained two different antibodies for the detection of CD40, CD86, and CD28 expression. Both 5C3 (Dianova) and anti-CD40 (Genzyme, Cambridge, MA) react with the 45- to 48-kd type 1 integral membrane glycoprotein CD40 found on peripheral blood and tonsillar B cells. Both Fun-1 (Dianova) and IT2.2 (Dianova) recognize different epitopes on the 75-kd cell surface protein CD86 (B7-2) expressed primarily on monocytes and activated B cells. Both Leu 28 (Becton Dickinson, Heidelberg, Germany) and anti-CD28 (Pharmingen, San Diego, CA) detect the disulfide-linked 44-kd CD28 antigen. CD8+ lymphocytes were stained with Leu 2a (Becton Dickinson). MAb BB1 (CD80/B7-1) (Becton Dickinson) reacts with a 44- to 60-kd protein on B7-transfected mouse cells identical to CD80. MAb TRAP1 (Pharmingen) reacts with CD154 (CD40 ligand, gp39), a 39-kd type II membrane glycoprotein expressed on activated T cells.
We performed immunohistochemistry on mice with rat anti-mouse CD80 (1G10, Pharmingen, San Diego, CA), rat anti-mouse CD86 (GL1, Pharmingen), rat anti-mouse CD40 (3/23, Pharmingen), and rat anti-mouse CD8 (53-6.7, Pharmingen).
As isotype controls, irrelevant mouse monoclonal antibodies (IgG1 and IgG2; Becton Dickinson) were used in human and rat MAbs (Pharmingen) were used in the mouse models.
Serial Sections and Double Stainings
To identify different parenchymal and nonparenchymal cell types, we carried out serial liver slides and double stainings. We used anti-cytokeratin (KL-1; Immunotech, Hamburg, Germany; positive reaction to hepatocytes and bile ducts), anti-CD31 (JC/70A; Dako; positive reaction to endothelial cells) and anti-liver sinusoidal endothelium (HM15/3; Monosan, Uden, The Netherlands; positive reaction to SECs), CD68 (PGM1; Dako; positive reaction to macrophages), and anti-smooth muscle actin (1A4; Dako; positive reaction to Ito cells). We performed double stainings with FITC-conjugated anti-CD31 and anti-CD68 antibodies. After performing the immunoperoxidase reaction, we incubated the sections for 20 minutes with irrelevant IgG1 and IgG2 antibodies to block any possible free binding site of the secondary antibody before the FITC-conjugated antibody was applied for 30 minutes. Analysis of double-labeling experiments was done by bright-field and fluorescent photomicrographs on a Leica DMLB fluorescence microscope with a MPS60 photo camera.
D-Galactosamine-LPS and D-Galactosamine-TNF Mouse Model
Six- to eight-week-old pathogen-free male BALB/c mice were
obtained from the Animal Research Institute of the Medizinische
Hochschule Hannover. All of the experiments were started between 8 and
10 a.m. and were performed in agreement with the German Legal
requirements. Either 10 µg/kg LPS or 10 µg/kg TNF-
were injected
intravenously with 700 mg/kg D-galactosamine. The livers of
the animals were removed 4, 8, and 12 hours after treatment and frozen
in liquid nitrogen for further analysis. To confirm lethality of the
chosen GalN/LPS and GalN/TNF doses, one mouse each was treated without
further intervention and died between 12 and 24 hours.
Statistical Analysis
CD80-, CD86-, CD28-, CD40-, and CD8-positive cells were counted in at least six randomly selected fields at 400-fold magnification. CD154-positive cells were counted in 100 visual fields at 400-fold magnification. The results for each patient are given as the mean of all evaluated visual fields. The results of each patient group are given as mean ± SD. Using the SPSS PC+ software package, we analyzed the differences between the groups for statistical significance by the nonparametric Mann-Whitney U test. Pearson's correlation coefficients were also calculated.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In patients with FHF, several patterns of hepatocyte damage could
be discerned. Six patients (1, 2, 3, 7, 9, and 18) had a complete loss
of hepatocytes in their specimens, whereas CD31-positive, CD68-negative
SECs were preserved. In 11 patients with FHF, 75% to 95% of
hepatocytes were destroyed. Single surviving hepatocytes were found in
5 of these 11 patients (4, 8, 12, 14, and 16). In the six other
patients (5, 6, 10, 11, 13, and 15), surviving hepatocytes formed
islets of intact hepatic structure, which were sharply limited by dense
infiltrates of macrophages (Figure 1C)
.
No inflammatory cells were seen within these islets. One patient (17)
had an intact hepatic structure with dense mononuclear infiltrates. Two
patients with fulminant hepatitis from Wilson's disease showed
complete cirrhosis with minimal necrosis and only very few inflammatory
infiltrates. These two patients were studied as a separate group.
|
In all examined patients with FHF, a markedly increased number of
both CD80- and CD86-positive cells was noted (data are given as number
of cells per visual field (VF) at 400-fold magnification: CD80,
108 ± 37 cells/VF; CD86, 112 ± 36 cells/VF) as compared
with patients with CLD (CD80, 23 ± 9 cells/VF, P
< 0.0001; CD86, 22 ± 9 cells/VF, P < 0.0001)
and NCs (CD80, 16 ± 7 cells/VF; CD86, 14 ± 6 cells/VF,
P < 0.0001) (Figures 1, A–C and 3, A and B
; Table 1
). CD80/CD86 expression of patients with
fulminant Wilson's disease, acute hepatitis, or chronic active
hepatitis was not different from CLD. In FHF, most of these cells were
CD68-positive macrophages, which constituted the predominant cell type
in the liver infiltrates of FHF. In contrast, infiltrates in chronic
liver disease were dominated by lymphocytes with only few interposed
CD80- and CD86-positive macrophages. FHF was unique in that there was
strong immunoreactivity of CD80/CD86 that was also found on a part of
the SECs, as identified by morphological characteristics and positive
reaction to the monoclonal antibody CD31 in serial sections (Figure 2, A–C)
and double-staining experiments
(Figure 2, D and E)
. No CD80 or CD86 immunoreactivity could be observed
on hepatocytes, bile ducts, or Ito cells (data now shown). In FHF with
islets of surviving hepatocytes, SECs within the islets were
consistently negative for CD80 and CD86, as were all the SECs in
the specimens from CLD and NCs (Figures 1C and 3, A and B
).
|
|
|
). Interestingly, in the islets
of surviving hepatocytes, no infiltration of CD28-positive lymphocytes
was noted.
|
Significantly higher numbers of CD40-positive cells were found in
FHF than in control groups (FHF, 156 ± 68 cells/VF; CLD, 50
± 5 cells/VF, P < 0.001; NC, 28 ± 17 cells/VF,
P < 0.001), comprising macrophages, lymphocytes, SECs,
Kupffer cells, hepatocytes, and bile duct epithelia. CD40
immunoreactivity was not found on hepatocytes in NCs, whereas 32
± 38% of hepatocytes were positive in CLD, and 63 ± 25% of
hepatocytes were positive in FHF. Immunoreactivity for CD40 was
detected on bile ducts and bile duct proliferates in FHF (100 ±
0%), in CLD (89 ± 23%), and in NCs (48 ± 18%).
Sinusoidal CD40 immunoreactivity was observed in eight of nine NCs as
well as in all specimens from patients with FHF and CLD. Infiltrating
macrophages and Kupffer cells were consistently CD40 positive in all
groups (Figure 4)
. In contrast to
abundant hepatic CD40 immunoreactivity, numbers of CD154-positive
lymphocytes were comparatively low (Table 1)
. However, still
significantly more CD154-positive lymphocytes were detected in FHF
(0.53 ± 0.32 cells/VF) than in CLD (0.05 ± 0.05 cells/VF,
P < 0.005) and NCs (0.003 ± 0.004 cells/VF,
P < 0.005).
|
). Animal Models
In the animal models, mice were killed 4, 8, and 12 hours after injection of GalN/LPS or GalN/TNF, respectively. Microscopic examination showed preserved liver architecture throughout the whole study period. However, 12 hours after treatment, focal edema and hepatocyte swelling was observed. The numbers of infiltrating macrophages and lymphocytes did not reach the extent seen in human fulminant hepatic failure. With respect to CD80, CD86, and CD40 expression, untreated mice matched the physiological findings in healthy man; a faint CD80, CD86, and CD40 expression was exclusively restricted to some Kupffer cells. SECs, bile ducts, and hepatocytes were negative for CD80, CD86, and CD40. Only a low number of CD8-positive lymphocytes was observed in the control livers (0.4 per visual field).
In both murine models, a time-dependent up-regulation of CD80 and CD86
expression was noted (Figure 5, A–D)
.
The numbers of CD80- and CD86-positive Kupffer cells increased 4 hours
after induction of liver failure in each model. At 8 hours, additional
expression of CD80 and CD86 was noted on SECs in focal areas. At 12
hours, the numbers of CD80- and CD86-positive cells had further
increased, and focal edema and swelling of hepatocytes became apparent
particularly in areas with CD80/86-positive SECs. However, CD86
staining proved stronger than CD80 staining in each cell type
throughout the study. In parallel to CD80/CD86 expression, the CD40
immunoreactivity increased on Kupffer cells, and an increase in
immunoreactivity also became detectable 8 hours after induction of
liver failure (Figure 5, E–H)
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In FHF, this situation is changed dramatically. Our results demonstrate a marked increase in the numbers of CD80- and CD86-positive cells in FHF not only including macrophages but also SECs. Thus, antigens reaching the liver in FHF are presented not only by a higher number of macrophages but also by SECs. An expression of abundant co-stimulatory molecules may indicate that now both cell types have become competent in efficient co-stimulation. Thus, portal antigens may no longer be tolerated but instead trigger potent immunoactivation that contributes to further hepatocyte damage in FHF. In line with these observations, we found a significant increase in lymphocytes that express the CD80/CD86 counter-receptor CD28. Furthermore, there was a close correlation between the total number of CD80/CD86-positive cells and CD8-positive lymphocytes. Interestingly, within islets of surviving hepatocytes, a normal pattern of CD80 and CD86 expression exclusively on Kupffer cells was linked with a complete absence of CD28-positive and CD8-positive lymphocytes. It is tempting to speculate that these hepatocytes survived because control of immune activation had been maintained locally within the islets.
A collapse of liver tissue in FHF is unlikely to account for the higher numbers of CD80/CD86-positive cells, because their numbers were still increased when corrected for the number of portal tracts per visual field (data not shown).
Nevertheless, our data in human livers cannot exclude the possibility that expression of co-stimulatory molecules may follow extensive hepatocellular necrosis, as serial specimens are not available in FHF in man. Thus, we examined the temporal relationship of CD80 and CD86 expression in the GalN/LPS and in the GalN/TNF mouse models of FHF. In both of these well characterized mouse models we could demonstrate up-regulation of CD80 and CD86 expression on Kupffer cells and SECs. This up-regulation occurred before the onset of massive hepatocyte necrosis and hepatic infiltration by inflammatory cells. The results in the animal models therefore indicate that CD80 and CD86 up-regulation is involved as an early step in the pathogenesis of FHF.
The interaction of CD40 with its ligand CD154 is believed to be a key step in an up-regulation of CD80 and CD86 expression in APCs and has a strong impact on B-cell activation, the initiation of antigen-specific T-cell responses, and macrophage development.8,21,22 In liver tissue from patients with FHF we found markedly enhanced expression of CD40 on macrophages, Kupffer cells, SECs, hepatocytes, and bile ducts as well as an increased frequency of CD154-positive lymphocytes. This observation of enhanced CD40 expression in humans is confirmed in the murine models, where we could demonstrate an early and strong expression of CD40 on macrophages and SECs that accompanies the induction of CD80 and CD86. Up-regulation of CD40 occurred before tissue damage but apparently did not precede CD80/CD86 induction. However, hepatocytes and bile ducts were CD40 negative in mice in contrast to humans where these also stained positive in cases of chronic active liver disease or FHF.
There is circumstantial evidence that expression of CD80/CD86 or CD40 conveys potent immunoactivating properties to the cells that express these molecules (overview in Ref. 7 ). Blocking these pathways with a CTLA-4Ig fusion protein or CD154 antibodies can prevent autoimmune disease, autoantibody production, antigen presentation, T cell activation, T cell-B cell interaction and clonal expansion of effector T-cell populations19,23-29 as well as rejection in most transplant models.30-34 Therefore, it is quite likely that also in human FHF, an up-regulation of CD80/CD86 and CD40 will result in a potent activation of immunocompetent cytotoxic effector cells. This concept is further supported by recent experiments by Adachi and co-workers.35 Blocking the CD80/CD86-CD28 or the CD40-CD154 pathways with neutralizing CD80 and CD86 or CD154 antibodies prevented liver cell damage in their concanavalin A mouse model of fulminant hepatitis.
In summary, our observations provide evidence for abnormally up-regulated antigen presentation by nonparenchymal liver cells as an early step in the immune-mediated pathogenesis of FHF. In particular, expression of co-stimulatory molecules is markedly enhanced and may trigger and maintain the inflammatory processes leading to massive hepatocyte damage. Novel therapeutic strategies targeted at blocking these co-stimulatory molecules might therefore be a promising novel approach to the treatment and prevention of FHF in man.
|
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported in part by a grant from the BONFOR Forschungsförderung.
Accepted for publication March 13, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
leads to autoimmunity in transgenic mice. Proc Natl Acad Sci USA 1994, 91:5138–5142
This article has been cited by other articles:
 |
|
 |
|
  A. Nolan, M. Weiden, A. Kelly, Y. Hoshino, S. Hoshino, N. Mehta, and J. A. Gold CD40 and CD80/86 Act Synergistically to Regulate Inflammation and Mortality in Polymicrobial Sepsis Am. J. Respir. Crit. Care Med., February 1, 2008; 177(3): 301 - 308. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q Zhou, M Hennenberg, J Trebicka, K Jochem, L Leifeld, E Biecker, T Sauerbruch, and J Heller Intrahepatic upregulation of RhoA and Rho-kinase signalling contributes to increased hepatic vascular resistance in rats with secondary biliary cirrhosis Gut, September 1, 2006; 55(9): 1296 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Wuensch, R. H. Pierce, and I. N. Crispe Local Intrahepatic CD8+ T Cell Activation by a Non-Self- Antigen Results in Full Functional Differentiation J. Immunol., August 1, 2006; 177(3): 1689 - 1697. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Leifeld, K. Fink, G. Debska, M. Fielenbach, V. Schmitz, T. Sauerbruch, and U. Spengler Anti-Apoptotic Function of Gelsolin in Fas Antibody-Induced Liver Failure in Vivo Am. J. Pathol., March 1, 2006; 168(3): 778 - 785. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A H Lau, A de Creus, L Lu, and A W Thomson Liver tolerance mediated by antigen presenting cells: fact or fiction? Gut, August 1, 2003; 52(8): 1075 - 1078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Dory, H. Echchannaoui, M. Letiembre, F. Ferracin, J. Pieters, Y. Adachi, S. Akashi, W. Zimmerli, and R. Landmann Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb -tsA58 mice J. Leukoc. Biol., July 1, 2003; 74(1): 49 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Petroff, L. Chen, T. A. Phillips, D. Azzola, P. Sedlmayr, and J. S. Hunt B7 Family Molecules Are Favorably Positioned at the Human Maternal-Fetal Interface Biol Reprod, May 1, 2003; 68(5): 1496 - 1504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. F. Schwabe, B. Schnabl, Y. O. Kweon, and D. A. Brenner CD40 Activates NF-{{kappa}}B and c-Jun N-Terminal Kinase and Enhances Chemokine Secretion on Activated Human Hepatic Stellate Cells J. Immunol., June 1, 2001; 166(11): 6812 - 6819. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
