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The Journal of Immunology, 2004, 173: 1417-1425.
Copyright © 2004 by The American Association of Immunologists

Production of Profibrotic Cytokines by Invariant NKT Cells Characterizes Cirrhosis Progression in Chronic Viral Hepatitis1

Claudia de Lalla*, Grazia Galli{ddagger}, Luca Aldrighetti{dagger}, Raffaella Romeo§, Margherita Mariani, Antonella Monno*, Sandra Nuti{ddagger}, Massimo Colombo§, Francesco Callea||, Steven A. Porcelli#, Paola Panina-Bordignon, Sergio Abrignani2,{ddagger}, Giulia Casorati2,* and Paolo Dellabona2,*

* Experimental Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, DIBIT, and {dagger} First Division of General Surgery, H. San Raffaele Scientific Institute, Milano, Italy; {ddagger} IRIS Research Center, Chiron Vaccines, Siena, Italy; § Department of Internal Medicine, University of Milan, Istituto di Ricovero e Cura a Carattere Scientifico Maggiore Hospital, Milano, Italy; Bioxell Spa, Milano, Italy; || Department of Pathology, Bambin Gesù Pediatric Hospital, Roma, Italy; and # Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Invariant (inv)NKT cells are a subset of autoreactive lymphocytes that recognize endogenous lipid ligands presented by CD1d, and are suspected to regulate the host response to cell stress and tissue damage via the prompt production of cytokines. We investigated invNKT cell response during the progression of chronic viral hepatitis caused by hepatitis B or C virus infection, a major human disease characterized by a diffused hepatic necroinflammation with scarring fibrotic reaction, which can progress toward cirrhosis and cancer. Ex vivo frequency and cytokine production were determined in circulating and intrahepatic invNKT cells from controls (healthy subjects or patients with nonviral benign or malignant focal liver damage and minimal inflammatory response) or chronic viral hepatitis patients without cirrhosis, with cirrhosis, or with cirrhosis and hepatocellular carcinoma. invNKT cells increase in chronically infected livers and undergo a substantial modification in their effector functions, consisting in the production of the type 2 profibrotic IL-4 and IL-13 cytokines, which characterizes the progression of hepatic fibrosis to cirrhosis. CD1d, nearly undetectable in noncirrhotic and control livers, is strongly expressed by APCs in cirrhotic ones. Furthermore, in vitro CD1d-dependent activation of invNKT cells from healthy donors elicits IL-4 and IL-13. Together, these findings show that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection, and suggest that these cells, possibly triggered by the recognition of CD1d associated with viral- or stress-induced lipid ligands, contribute to the pathogenesis of cirrhosis by expressing a set of cytokines involved in the progression of fibrosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Approximately 6% of the world population is chronically infected by hepatitis B (HBV)3 or C (HCV) viruses, the most common causes of chronic liver diseases, thus posing a serious public health problem (1, 2). A major difference between the two viruses is that a relevant fraction (60–80%) of immunocompetent individuals infected by HCV (2) and only 1–2% of those infected by HBV (1) progress into chronic infection, characterized by the persistent necroinflammation of the liver. The morphologic modifications of the liver induced by the chronic infection with either HCV or HBV are difficult to distinguish: in both cases, the chronic necroinflammatory damage evolves into a fibrotic reaction, as the result of the host attempt to repair the damaged parenchyma with a scar (3). In ~10–20% and in 25–40% of patients with chronic HCV and HBV infection, respectively (1, 4), liver fibrosis progresses into cirrhosis, characterized by the disruption of liver architecture with fibrous septa and parenchymal nodules (5). Cirrhosis may evolve into severe complications such as liver failure or primary hepatocellular carcinoma (HCC) in a significant fraction of patients (6). Stellate cells, a heterogeneous group of cells of neural-crest origin resident in the subendothelial space of Disse, have been identified as the principal fibrogenic cells of the liver (3). Following liver injury of any etiology, hepatic stellate cells undergo activation and differentiation into proliferative, fibrogenic, and contractile myofibroblasts (3). Stimuli inducing stellate cell activation, such as reactive oxygen intermediates or cytokines, can derive from injured hepatocytes, endothelial cells, Kupffer cells, and infiltrating leukocytes (3). Among the cytokines implicated in the fibrotic response of the liver, TGF-{beta}1, IL-4, and IL-13 appear to be the most potent ones (3, 7, 8). Factors concurring to (dis)regulate the fibrogenic response are still poorly understood, and cells responsible for the production of fibrogenic cytokines have not been univocally identified.

In mice, a peculiar T cell subset, defined as invariant (inv)NKT lymphocytes, is selectively enriched in liver, where it accounts for up to 25–30% of total infiltrating lymphocytes (9). invNKT cells are remarkably conserved, and express a semi-invariant TCR, constituted by the homologous invariant V{alpha}24-J{alpha}Q and V{alpha}14-J{alpha}18 rearrangements in humans and mice, respectively, paired with junctionally diverse V{beta}11 and V{beta}8.2, V{beta}7, and V{beta}2 TCR chain in humans and mice, respectively (10, 11). invNKT cells are restricted for the non-MHC-encoded class I-like CD1d Ag-presenting molecule (12), and can be activated selectively by the xenogeneic glycosphingolipid {alpha}-galactosylceramide ({alpha}-GalCer), which binds CD1d (13). Nevertheless, invNKT display a remarkable autoreactivity for some endogenous ligands, possibly polar lipids, presented by the CD1d expressed on APCs, and can be activated to produce cytokines even in the absence of {alpha}-GalCer (14). Owing to a number of phenotypic and functional features, comprising their homogenous clonal distribution, innate memory phenotype, and immediately triggered effector functions, invNKT cells are considered to be at the interface of innate and adaptive immunity (9). A variety of animal and human studies has implicated invNKT cells in the regulation of infectious (15), tumor (16), and autoimmune conditions (17) associated with inflammation, tissue damage, and repair. Furthermore, activation and production of IL-4 and IFN-{gamma} by intrahepatic invNKT cells have been correlated to the hepatic damage caused either by Con A administration (18) or by the genetic ablation of the suppressor of cytokine signaling-1 gene, also known as STAT-induced STAT inhibitor-1, a negative feedback molecule for cytokine signaling (19).

In light of these observations, we sought to characterize both circulating and intrahepatic human invNKT cells in chronic viral hepatitis, to understand whether they play a role at different stages of liver tissue disruption. Numbers and effector functions of invNKT cells from chronically HCV- or HBV-infected patients were directly compared with those of invNKT cells obtained from patients with a pauci-inflammatory, localized tissue damage represented by either benign or malignant intrahepatic lesions without chronic viral infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

Forty-two patients with serologically documented chronic HBV (n = 15) or HCV (n = 27) infection were divided in three groups according to disease severity, namely: 1) without cirrhosis (n = 10); 2) with cirrhosis (n = 10); and 3) with cirrhosis and primary HCC (n = 22) (Table I). All of the patients had elevated serum levels (>40 U/L) of alanine aminotransferase, and histological diagnosis of chronic hepatitis from at least 5 years. Staging of the disease progression was performed by hepatic biopsies, and the severity of tissue damage was evaluated on the basis of infiltrates activity and fibrosis based according to the Ishak-modified histology activity index (20) (Table I). Cirrhosis was assessed by the observation of micro- and/or macroregenerative nodules in biopsy specimens. All of the cirrhotic patients analyzed in this study were in a compensated phase of the disease (child A). The presence of HCC was documented by ultrasound and spiral triphasic computerized tomography scans and, in surgical patients, by the histological analysis of the removed mass.


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  		Table I. Quantitative characterization of T and invNKT cells in healthy donors and in patientsa

 
As controls, we enrolled 16 patients with hepatic pathologies not related with the hepatitis virus infection, known to associate with focal liver damage and minimal intrahepatic inflammatory response, namely: 1) benign lesions (n = 3: 1 acute intrahepatic lithiasis, 1 angioma, and 1 focal nodular hyperplasia); and 2) malignant lesions (n = 13: 1 cholangiocarcinoma, 2 primary HCC, 1 metastasis from gastric carcinoma, 1 metastasis from gastrointestinal stromal tumor, 1 metastasis from pancreatic cancer, and 7 metastasis from colorectal adenocarcinoma, all confirmed by histological analysis).

In both groups of patients, invNKT cells were obtained from peripheral blood and, in case of patients undergoing hepatic surgery, from liver. Surgical specimens were required because liver biopsies did not provide enough cells for reliable intracellular flow cytometry analysis of primary invNKT cells. invNKT cells from the peripheral blood of healthy donors were used as reference control (Table I).

For reasons not related to this study, all patients with chronic viral hepatitis had never received antiviral therapy or were off therapy from at least 6 mo before being analyzed, while patients with any form of neoplastic lesion were off therapy (regional or systemic) from at least 6 mo. The study was approved by the local ethical committee, and a written informed consent was obtained from all the patients studied.

Preparation of PBL and intrahepatic lymphocytes (IHL)

To obtain PBL, heparinized venous blood was separated by Ficoll-Hypaque (Pharmacia, Peapack, NJ) density gradient centrifugation. To purify IHL, surgical liver specimens were washed four times with PBS to get rid of contaminating blood, and then mechanically dissociated by cutting them into small fragments with sterile scalpels, followed by manual homogenization with a syringe plunger on a cell strainer with 70-µm-pore diameter (Falcon). The filtered cell suspension was collected in RPMI 1640 (Invitrogen Life Technologies) containing 5% of normal human serum (NHS) (Euroclone, Milan, Italy) and centrifuged for 20 min at 1500 rpm. Infiltrating mononuclear cells were separated from hepatocytes by Percoll (Invitrogen Life Technologies, Carlsbad, CA) density gradient centrifugation: the cell pellet was resuspended in 40% (v/v) Percoll in RPMI 1640 and layered onto 80% (v/v) Percoll in RPMI 1640. For ~10 g of tissue, 15 ml of 40 and 80% Percoll solutions was used. The gradient was centrifuged for 10 min at 2000 rpm, and IHL were collected at the interface between 40 and 80%, washed once in RPMI 1640 with 10% NHS, and counted.

Determination of invNKT frequency

invNKT frequency in PBL and IHL was determined by flow cytometry by staining cells with biotin-conjugated anti-V{alpha}24, FITC-conjugated anti-V{beta}11 (both produced in house) (10), and allophycocyanin-conjugated anti-CD3 (BD Biosciences, San Jose, CA) mAbs, followed by CyChrome-conjugated streptavidin (BD Biosciences). Cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences), and results were analyzed by using the CellQuest software (BD Biosciences). At least 2 x 106 gated lymphocytes were acquired for each sample.

Characterization of intracellular cytokines expressed by invNKT cells

Intracellular cytokine production was detected by four-color flow cytometry, as described (21), with some modifications. Briefly, IHL or PBL (1 x 107) were activated in RPMI 1640 with 10% NHS with 25 ng/ml PMA (Sigma-Aldrich, St. Louis, MO) and 1 µg/ml ionomycin (Sigma-Aldrich) for 1 h at 37°C in the presence of 5 U/ml human rIL-2 (Roche, Basel, Switzerland). Brefeldin was added at 10 µg/ml (Sigma-Aldrich), and incubation continued for an additional 45 min in case of IHL, and for an additional 90 min for PBL. Cells were then stained with biotin-conjugated anti-V{alpha}24, FITC-conjugated anti-V{beta}11, and allophycocyanin-conjugated anti-CD3 mAbs for 10 min on ice, followed by CyChrome-conjugated streptavidin for 5 min on ice. Cells were then fixed in 1% paraformaldehyde (Sigma-Aldrich) for 5 min on ice, and permeabilized for 15 min at 4°C with 0.2% saponin (S-7900; Sigma-Aldrich). To overcome possible low surface signals due to activation-dependent TCR down-regulation, mAbs specific for V{alpha}24, V{beta}11, and CD3 were also included during the intracellular staining step together with PE-conjugated mouse anti-IFN-{gamma}, or rat anti-IL-4 or anti-IL-13 (BD Biosciences). Cells were washed with 0.2% saponin and incubated with CyChrome-conjugated streptavidin in 0.2% saponin for 5 min on ice. Cells were analyzed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences), and results were analyzed by using the CellQuest software (BD Biosciences). At least 106 T cells were acquired for each sample.

Immunohistochemistry

Frozen liver serial sections (5 µm) were fixed in 4% paraformaldehyde in PBS for 10 min, washed twice in PBS with BSA (Sigma-Aldrich) for 5 min at room temperature (RT), and incubated in 0.03% H2O2 in PBS for 10 min at RT. After washing in PBS, sections were incubated in avidin/biotin-blocking reagents (Vector Laboratories, Burlingame, CA) for 15 min at RT to block nonspecific staining. Normal goat serum (Sigma-Aldrich) was then added at 0.05% in PBS for 1 h at RT. Each of the following purified anti-CD1d-51, CD1d-55, CD1d-42 (22), and anti-CD3 (BD Biosciences), mAb was added separately to each serial section at a final concentration of 10 µg/ml for 16 h at 4°C, followed by biotin-conjugated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) and streptavidin HRP. As the three anti-CD1d mAbs gave a comparable pattern of reactivity on frozen liver sections, we continued the study by using only the CD1d-42 (BD Biosciences). Sections were washed twice in PBS, incubated with biotin-conjugated goat anti-mouse IgG1 (5 µg/ml with 0.5% normal goat serum and 2% NHS in PBS) for 2 h at 4°C, washed twice, incubated with HRP-conjugated streptavidin (NovaRED substrate kit for peroxidase) (Vector Laboratories), following the manufacturer instructions, then rinsed in distilled water for 5 min, and counterstained with hematoxylin. Sections were prepared from independent liver specimens of 10 cancer patients with chronic viral hepatitis and 10 cancer patients without chronic viral hepatitis, and 3–4 sections for each of the 20 patients were analyzed.

Confocal laser microscopy

Frozen sections of liver specimens (5 µm) were fixed in 2% paraformaldehyde for 1 h at 4°C, washed twice in wash buffer (0.1 M MOPS, pH 7.4) for 5 min at RT, and then incubated with 50 mM NH4CL for 10 min at 4°C. After washing, the sections were incubated in 5% NHS for 20 min at RT, washed, and then with avidin/biotin-blocking reagents for 15 min at RT. After washing, sections were incubated with anti-CD1d-42 (IgG1) mAb at 10 µg/ml in dilution buffer (3% BSA and 5% human serum in 0.1 M MOPS, pH 7.4) for 2 h at RT, followed by a washing step. Biotin-conjugated goat anti-mouse IgG1 at 5 µg/ml was added in dilution buffer for 1 h at RT, followed by a washing step and then by PE-conjugated streptavidin (BD Biosciences) in dilution buffer for 30 min at RT. After washing, each of the following purified mAb was added to the sections for 2 h at RT: anti-CD20 (IgG2a; DakoCytomation, Glostrup, Denmark), CD68 (IgG2b; BD Biosciences) mAb at 10 µg/ml in dilution buffer, or anti-{alpha}-smooth muscle actin ({alpha}-SMA) mAb (IgG2a; Sigma-Aldrich) at the dilution suggested by the manufacturer. After washing, FITC-conjugated goat anti-mouse IgG2a or IgG2b antiserum (Southern Biotechnology Associates) was added at 5 µg/ml in dilution buffer for 1 h at RT. In the case of costaining for T cells or dendritic cells (DCs), anti-CD1d-42 was added first, followed by biotin-conjugated goat anti-mouse IgG1 and PE-conjugated streptavidin. The secondary Ab was blocked with purified nonimmune mouse IgG1 (BD Biosciences) before adding FITC-conjugated anti-CD3 (IgG1; BD Biosciences) at a final concentration of 10 µg/ml. In the case of costaining for DCs, anti-CD1d-42 was added first, followed by biotin-conjugated goat anti-mouse IgG1 and FITC-conjugated streptavidin. After blocking the secondary Ab with purified nonimmune mouse IgG1, PE-conjugated anti-DC-LAMP (IgG1; Beckman Coulter) was added at 10 µg/ml. Stained sections (from 5 to 10 per staining) were analyzed on a confocal laser microscope (MRC 1024 Bio-Rad or TCS SP2 Leica), and the images were acquired by Bio-Rad (Hercules, CA) Software Laser Sharp 2000 or Leica Confocal Software (Leica Microsystems, Deerfield, IL). To facilitate the readout of confocal laser imaging, red was assigned to CD1d and green to CD3, CD20, CD68, DC-lysosome-associated protein (DC-LAMP), and {alpha}-SMA; the resulting merge was yellow for molecular colocalization.

In vitro activation of invNKT cells

PBLs from healthy donors were cultured with 50 ng/ml {alpha}-GalCer (supplied to P.D. by Kirin Brewery, Gumna, Japan) in 40 U/ml human rIL-2 (Roche). At determined time points, cells were collected and restimulated with PMA + ionomycin and brefeldin A, as described above, followed by staining with anti-CD3, anti-V{alpha}24, and anti-V{beta}11 mAbs or with anti-CD3, anti-V{alpha}24, anti-IFN-{gamma}, and IL-4 or IL-13 mAbs to determine the frequency and intracellular cytokine expression of invNKT cells, respectively.

Statistical analysis

invNKT cell frequencies were evaluated with two-tailed Mann-Whitney U test; p values ≤0.01 were considered to be significant. invNKT frequencies producing intracellular cytokines were analyzed using two-tailed Student’s t test; p values ≤0.05 were considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Intrahepatic enrichment of invNKT cells in chronic viral hepatitis

We first determined the frequencies of invNKT cells among total T lymphocytes contained in PBL and, when available, IHL freshly isolated from: 1) patients with chronic viral hepatitis; 2) patients with benign or malignant hepatic lesions without chronic viral hepatitis; and 3) healthy donors. invNKT cells were identified as V{alpha}24+V{beta}11+ double-positive cells in the CD3+ gated region. In humans, this triple staining identifies the same CD1d-restricted invNKT cells that show positive staining with the CD1d-{alpha}-GalCer tetramers (23). As shown in Table I and Fig. 1a, the percentage of invNKT cells found in PBL of all the patients examined was largely comparable with those of healthy donors. Furthermore, unlike the situation in the mouse, human liver intrahepatic invNKT cells were also low in frequency (Table I and Fig. 1a), irrespective of the pathology affecting the target organ. However, whereas in patients with hepatic metastasis the frequency of intrahepatic invNKT was at most 2.30 times greater than that in PBL, a significantly higher value for this ratio (p = 0.00185) was found in chronic hepatitis patients with cirrhosis and HCC, with an invNKT frequency in the liver 10.43 times greater than that of their circulating counterparts (Fig. 1b).



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  		FIGURE 1. Increased numbers of intrahepatic invNKT cell in cirrhotic patients. a, Representative analysis of invNKT frequency in PBL and IHL from patients affected by hepatic metastasis from colon adenocarcinoma, or by chronic HCV infection with HCC, respectively. invNKT cells were identified as V{alpha}24+V{beta}11+ double-positive cells in the CD3+ gated region. b, Fold increase of intrahepatic over peripheral blood-derived invNKT cell frequencies, obtained by dividing the percentages of intrahepatic by that of peripheral blood-derived invNKT measured in each patient. The increase of intrahepatic over peripheral blood-derived invNKT cells in chronically inflamed was significantly different (p = 0.00185) from that found in noninflamed livers. For this study, we analyzed 12 patients with hepatic lesions without chronic viral hepatitis and 14 patients affected by chronic viral hepatitis with cirrhosis and HCC (n = 8 HCV; n = 6 HBV).

 
Taken together, these results indicated that, despite being in low percentage among IHL, invNKT are significantly enriched in chronically inflamed livers as compared with noninflamed ones.

invNKT cell switch to profibrotic cytokine production characterizes progression to cirrhosis

invNKT cells are implicated in the regulation of the response to cell stress and tissue damage through the production of diverse cytokines. The above data thus raised the question as to whether invNKT cells could be involved in some way in the progressive damage of liver tissue in the course of the chronic viral hepatitis. To characterize peripheral and intrahepatic invNKT cells, lymphocytes were isolated from peripheral blood of all categories of patients and from healthy donors (Table I). Total lymphocytes were immediately stimulated with PMA/ionomycin, and analyzed by flow cytometry for the intracellular expression of either IFN-{gamma}, or IL-4 or IL-13 within the invNKT or T cell subsets. The major fraction of peripheral blood-derived invNKT cells produced IFN-{gamma}, irrespective of whether they were obtained from healthy donors or patients, suggesting that this is the dominant cytokine made by this subset of NKT cells (Fig. 2). Nevertheless, there was some variation from one category of patients to the other, with patients with chronic viral hepatitis and cirrhosis notably exhibiting the highest frequencies of IFN-{gamma}-producing invNKT cells, and those with hepatic metastasis the lowest. Unlike IFN-{gamma}, however, IL-4 and IL-13 displayed a markedly different distribution among circulating invNKT cells in different patient groups. Both cytokines were in fact nearly undetectable in invNKT cells from healthy donors, from patients with hepatic lesions without chronic viral hepatitis, confirming recent data (24), and from patients with chronic viral hepatitis without cirrhosis. IL-4 and IL-13 were instead produced by significantly higher frequency of circulating invNKT cells from cirrhotic patients, independently of the concomitance with HCCs (Fig. 2). This finding suggested a substantial modification in the effector functions of circulating invNKT cells during the progression of chronic viral hepatitis to cirrhosis.



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  		FIGURE 2. Increased production of profibrotic cytokines by circulating invNKT from cirrhotic patients. Intracellular IFN-{gamma}, IL-4, and IL-13 accumulation in peripheral invNKT cells obtained from: healthy donors (n = 15), patients with hepatic metastasis from colorectal cancer without chronic viral infection (n = 10), or patients with chronic viral hepatitis at progressive disease stages (no cirrhosis, n = 10; cirrhosis, n = 10; cirrhosis and HCC, n = 10). Total PBL were stimulated with PMA, ionomycin, and brefeldin A, and quadruple stained with anti-V{alpha}24, anti-V{beta}11, anti-CD3, and either anti-IFN-{gamma} or IL-4- or IL-13-specific mAbs. Intracellular cytokine expression by invNKT was determined after gating on CD3+V{alpha}24+V{beta}11+ cells. a, Representative dot plots from healthy donors and individual patients are shown. Numbers indicate the percentage of invNKT cells expressing the given cytokine. b, Overall percentages of circulating invNKT cells from healthy donors and patients expressing the given intracellular cytokine. Statistically significant difference between the percentage of invNKT cells expressing a given cytokine in chronic viral hepatitis vs nonchronic viral hepatitis patients is indicated by asterisks, with the following values: *, p = 0.001; **, p = 0.0001; and ***, p = 0.00001.

 
Because the effector functions of T lymphocytes are displayed in the target organs, we also compared the pattern of cytokines produced by primary intrahepatic invNKT cells obtained from patients with hepatic malignant lesions without chronic viral hepatitis, or patients with chronic viral hepatitis, cirrhosis, and HCC. As shown in Fig. 3, the majority of intrahepatic invNKT cells from both groups of patients produced IFN-{gamma}, but only those from cirrhotic patients with HCC exhibited significant expression of IL-4 and IL-13.



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  		FIGURE 3. Increased production of profibrotic cytokines by intrahepatic invNKT from cirrhotic patients. Intracellular IFN-{gamma}, IL-4, and IL-13 accumulation in intrahepatic invNKT cells obtained from patients with hepatic metastasis from colorectal cancer without chronic viral infection (n = 5), or patients affected by chronic viral infection with cirrhosis and HCC (n = 5). Total IHL were stimulated and stained, as described in Fig. 2. a, Representative dot plots from invNKT cells from individual patients are shown. Numbers indicate the percentage of cells expressing the given cytokine. b, Overall percentage of intrahepatic invNKT cells from the two groups of patients expressing the given intracellular cytokine. Statistically significant difference between the percentage of invNKT cells expressing a given cytokine in chronic viral hepatitis vs nonchronic viral hepatitis patients is indicated by asterisks, with the following values: *, p = 0.01; **, p = 0.001; and ***, p = 0.0001.

 
Interestingly, circulating and intrahepatic conventional {alpha}{beta} T cells also showed a significantly increased frequency of IL-4-producing cells in cirrhotic patients (data not shown), confirming that progression toward cirrhosis is accompanied by the up-regulation of type 2 cytokines in conventional T cells (25, 26).

These data therefore show that invNKT cells modify substantially their effector potential with the progression to cirrhosis of chronic viral hepatitis, although independently of the presence of an intrahepatic neoplasm.

CD1d is strongly expressed in cirrhotic liver

To assess whether invNKT could undergo a TCR-specific activation in the liver, we determined the level of expression and cellular distribution of CD1d in liver sections, obtained from patients with chronic viral hepatitis at different stages of activity and fibrosis (27), as well as from patients with hepatic metastasis from gastrointestinal neoplasia, displaying little or no liver inflammation and fibrosis. Serial sections from liver tissues were analyzed for the presence of infiltrating T cells, identified with anti-CD3 mAb, as a direct measure of the degree of chronic inflammation, and for CD1d expression (Fig. 4). In noncirrhotic livers with benign lesions or metastasis from colorectal cancer, there was nearly any inflammatory T cell infiltrate within the parenchyma. CD1d expression was extremely rare too, and apparently confined within the infrequent lymphoid aggregates found in the intact parenchyma (Fig. 4, a and b). In patient with hepatic metastasis, however, some CD1d expression was present in the proximity of the fibrotic tissue surrounding the neoplastic lesion, in which some infiltrating T lymphocytes were also present, suggesting some degree of reactivity around the lesion (data not shown). By contrast, in chronically inflamed livers, the expression of CD1d was strong, and it increased in parallel with the progression of the inflammatory infiltrate and fibrosis, as shown by the representative immunohistochemistry analysis of hepatic tissue obtained from patients with chronic viral hepatitis and associated fibrosis with or without cirrhosis (Fig. 4, c–h). CD1d expression was localized in portal inflammatory infiltrates (c–f), as depicted by the presence of infiltrating T lymphocytes (d and f), as well as within the fibrous septa surrounding the cirrhotic lobules (g). The pattern of CD1d expression was quite comparable in chronic HBV- or HCV-infected livers (data not shown).



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  		FIGURE 4. CD1d expression in livers with or without chronic viral hepatitis. Frozen liver serial sections were stained with anti-CD1d (a, c, e, and g)- or anti-CD3 (b, d, f, and h)-specific mAbs. A representative immunohistochemistry analysis of hepatic tissues from patients with: a and b, hepatic metastasis from colon adenocarcinoma without chronic viral hepatitis. Only rare CD1d-expressing cells are observed in the liver parenchyma (a, arrow and inset), localized in a modest lymphoid aggregate related to intrahepatic blood flow stasis (b); c and d, chronic HBV hepatitis with fibrosis and HCC, without cirrhosis (27). Shown is a portal inflammatory infiltrate (PI). Both CD1d- and CD3-expressing cells are in the portal infiltrate, but not in the liver parenchyma; e–h, chronic HBV hepatitis with active cirrhosis and HCC (27). Shown are a portal inflammatory infiltrate (PI) with surrounding fibrosis (e and f), or a portal fibrosis (PF) with fibrous septa (FS) and cirrhotic nodules (CN) (g and h). CD1d is expressed by cells within the portal infiltrate, as well as by cells within the fibrous areas. At variance with T cells, no CD1d-expressing cells are present in the liver parenchyma. (Original magnification x10.)

 
Collectively, these data show that CD1d is highly expressed in the liver of chronic viral hepatitis patients with progressing fibrosis. CD1d is not only distributed within the portal inflammatory infiltrates, but also in fibrotic tissue, suggesting that it might be a molecular signature of the tissue damage progression.

CD1d is expressed by APCs infiltrating liver parenchyma and on cells juxtaposed to hepatic stellate cells

To obtain a more precise definition of CD1d-expressing cells in the cirrhotic liver, confocal laser microscopy was performed on sections by costaining with CD1d and one of the following markers: CD3 for T cells, CD20 for B cells, CD68 for macrophages, DC-LAMP for DC, and {alpha}-SMA for stellate cells (3). The costaining experiments showed that CD1d and CD3 were expressed by different cells within the portal inflammatory infiltrate (Fig. 5, a–c). By contrast, a conspicuous fraction of B cells, either in the periportal lymphoid aggregates (Fig. 5, d–f) or scattered in the fibrotic areas (data not shown), coexpressed CD1d. In the portal infiltrate, the majority of CD68+ macrophages coexpressed CD1d (Fig. 5, g–i), as well as a sizeable fraction of CD1d molecules colocalized with DC-LAMP on DCs, mainly in portal lymphoid aggregates (Fig. 5, j–l). However, most of CD68+ macrophages contained within either the fibrous septa or the hepatic parenchyma did not express CD1d, while DC-LAMP+ DCs were substantially absent from these two locations (data not shown). We also determined whether stellate cells, the principal fibrogenic cell of the liver, expressed CD1d. Costaining of cirrhotic liver sections with CD1d and {alpha}-SMA did not reveal coexpression of the two molecules within the same cells (Fig. 5, m–o). Interestingly, though, cells expressing either molecule were found in close proximity and specifically situated within the fibrous septa juxtaposed to the regenerating lobule, supporting a possible indirect effect of CD1d recognition by invNKT cells in the activation of bystander stellate cells.



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  		FIGURE 5. In the cirrhotic liver, CD1d is expressed by DC, B cells, macrophages, and unidentified cells juxtaposed to stellate cells. Shown are representative cases of chronic HBV infection with active cirrhosis and HCC. Sections were stained with anti-CD1d mAb in red (a, d, g, j, and m) and one of the following markers in green: CD3 for T cells (b), CD20 for B cells (e), CD68 for macrophages (h), DC-LAMP for DC cells (k), and {alpha}-SMA for stellate cells (n). Colocalization is shown in yellow (c, f, i, l, and o): CD1d is almost completely excluded from CD3+ T cells in portal infiltrates (a–c), and very infrequent colocalization is observed (arrowhead). Note that CD1d is expressed on cells often surrounding biliary ductules (c, inset). CD1d is heavily coexpressed with CD20 by B cells in lymphoid aggregates flanking the fibrous septa (d–f). At the interface between fibrous septa and lymphoid aggregate CD1d is expressed by CD68+ macrophages (g–i). A small fraction of CD1d molecules colocalizes with DC-LAMP (j–l) on DC cells in lymphoid aggregates. CD1d+ cells are also closely juxtaposed to stellate cells within fibrous septa (m–o). (Original magnification x40.)

 
In any of the examined tissue sections, however, we were unable to find CD1d expression on hepatocytes, at a variance with what has been reported in the mouse (28, 29).

Together, these findings document that APCs express high levels of CD1d in parallel with the progression of inflammation and tissue damage.

In vitro activated invNKT cells acquire the effector functions found in cirrhosis

The increasing expression of CD1d in the liver during chronic viral hepatitis could be responsible for the sustained Ag-dependent stimulation of invNKT cells, accounting for the cytokine switch documented in cirrhotic patients. We therefore tested whether invNKT cells could be induced to switch toward the production of fibrogenic cytokines, upon activation in vitro with the specific CD1d ligand {alpha}-GalCer and low doses of IL-2. As shown in Fig. 6a, primary invNKT cells from the peripheral blood of healthy donors modified progressively their cytokine pattern, going from the expected high IFN-{gamma}, low IL-4, and no IL-13 production found immediately after their purification, to the high IFN-{gamma}, high IL-4, and high IL-13 production found as early as 1 wk of culture, and reminiscent of the one exhibited by primary invNKT cells from cirrhotic patients. Interestingly, in vitro activated invNKT cells expressed either IFN-{gamma} alone, or both IFN-{gamma} and IL-4 or IL-13, indicating that these cells assume either a type 1 or a type 0 cytokine profile, but only rarely a pure type 2 one (Fig. 6b). The cytokine pattern found in primary invNKT cells from cirrhotic patients could thus reflect a previous Ag-driven activation event in vivo.



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  		FIGURE 6. invNKT cells from healthy donors produce IL-4 and IL-13 upon CD1d-dependent activation in vitro. invNKT cells from the peripheral blood of healthy donors were activated in vitro with the CD1d-restricted ligand {alpha}-GalCer in the presence of 40 U/ml, and their pattern of intracellular cytokine expression was determined by four-color flow cytometry at the indicated time points, as described in Materials and Methods. One representative experiment of three is reported. a, Both frequency of invNKT cells expressing either IFN-{gamma}, or IL-4 or IL-13, and of invNKT cells among total T cells are indicated. Day 0 of culture indicates primary invNKT cells, activated ex vivo with PMA + ionomycin and brefeldin A. b, Intracellular cytokines produced by invNKT cells at day 21 of activation. Total lymphocytes were activated with PMA + ionomycin and brefeldin A, stained with anti-CD3 and anti-V{alpha}24 mAbs, permeabilized, and stained with anti-IFN-{gamma} and anti-IL-4 or anti-IFN-{gamma} and anti-IL-13. The percentage of cells coexpressing each pair of cytokines is shown in the quadrants. All of the cells stained by anti-CD3 and anti-V{alpha}24 mAbs also expressed V{beta}11 (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have found that invNKT cells respond to the progressive liver damage caused by chronic hepatitis virus infection. invNKT cells are in fact significantly enriched, relative to peripheral blood, in the chronically inflamed organs as compared with the noninflamed ones. Moreover, invNKT cells undergo a substantial modification in their effector potential, which characterizes the cirrhotic state, consisting in the capacity to produce IL-4 and IL-13 in addition to IFN-{gamma}. These functional changes in invNKT cells are present in both chronic HBV and HCV infections, suggesting that these cells respond to factors released as a consequence of tissue damage rather than to viral Ags.

Our findings confirm that the human liver harbors significantly less invNKT cells than the mouse one (23, 24, 30, 31, 32), suggesting the possibility that the two species differ in some major molecular pathways involved in the migration and/or the accumulation of invNKT cells in this organ. Furthermore, the data presented in this study underscore the migratory capacity of invNKT cells toward inflammatory stimuli, a property described for these cells in animal models of bacterial infection as well as in primary biliary cirrhosis, a human autoimmune disease characterized by chronic inflammation state of the liver (32, 33). In line with this property, invNKT cells constitutively express functional receptors for inflammatory chemokines (34), and the expression of the receptors for the inflammatory chemokines IFN-{gamma}-inducible protein-10 and liver and activation-regulated chemokine is maintained in the cells isolated from the cirrhotic livers (C.d.L., P.D., and G.C., unpublished data).

Migration and accumulation of invNKT cells into inflammatory sites do not require the presence of CD1d molecules (33); however, CD1d expression at the inflammation site may be crucial for the display of effector functions of these cells locally. invNKT cells have been defined "autoreactive by design" (35), as they recognize endogenous ligands, presumably polar lipids, bound to CD1d. These endogenous ligands could be unleashed in target organs by signals triggered by the inflammatory stimuli and/or cell death, two conditions fulfilled in chronic viral hepatitis. Accordingly, we have found a strong expression of CD1d in chronically inflamed livers, and documented its increase with the progression of both fibrosis and parenchyma damage. The colocalization studies show that CD1d is indeed found mainly on B cells and macrophages infiltrating the portal space or along the fibrous septa, or on DC in the portal space. Because several studies show that invNKT cells recognize in vitro DC and B cells in a self-reactive, CD1d-specific manner (21, 36, 37, 38), it is conceivable that this interaction may very well occur in vivo. In light of these considerations, we propose that the cytokine switch in invNKT cells observed in cirrhosis would reflect the sustained activation of these cells consequent to the described high CD1d expression, possibly in association with inflammation-induced CD1d ligands. Moreover, a reciprocal influence between invNKT cells and APCs has been described and may also take place in chronically inflamed liver; for instance, invNKT cell-dependent activation of B cells might take part in the pathogenesis of both cryoglobulinemia and B cell lymphoma, which occurs in some chronic HCV infections. The observed switch of invNKT cells infiltrating the cirrhotic liver to produce IL-4 and IL-13, two important growth factors implicated in the invNKT-dependent help of B cells (21), would further support a possible pathogenetic role of invNKT cells in these HCV-related immunopathologies.

We cannot discount also the possibility that environmental factors present in the chronically inflamed liver may facilitate the cytokine change occurring in these cells. An interesting candidate could be IL-18, which can induce type 2 cytokines in murine invNKT cells, concomitant to the maintenance of sustained IFN-{gamma} production, in the presence of invTCR engagement and of limiting amount of IL-12 (39). IL-18 is indeed found abundantly expressed during chronic viral hepatitis and cirrhosis (40), supporting its possible role as type 2 cytokine inducers in human invNKT cells.

In line with our results, primary human invNKT cells from healthy donors have been found biased as a whole toward Th1 cytokine profile; however, the CD4+ and CD4– invNKT subsets exhibit a Th0 and Th1 cytokine profile, respectively (41, 42). We could not find significant variation in the CD4+ vs CD4– subset distribution in cirrhotic patients compared with healthy donors (C. de Lalla, unpublished results). Thus, the increased frequency of Th0 invNKT cells found in cirrhotic patients cannot be simply related to an expansion of the CD4+ subset.

Reversible qualitative changes in invNKT cells have been described in patients with advanced prostate cancer (43), or in association with the clinical progression of multiple myeloma (44). In both tumors, however, the change consisted in the loss of IFN-{gamma}, with the acquisition of IL-4 in the prostate cancer only, indicating that the modification of the invNKT cell effector functions in these pathologies is substantially different from those found in chronically infected liver with cirrhosis. This would indicate certain plasticity in invNKT cell responses, which might not univocally differentiate toward an invariable effector phenotype.

What are the possible effects of type 2 cytokines produced by invNKT cells on the pathogenesis of cirrhosis? The pathologic hallmark of cirrhotic progression is the sustained inflammatory liver damage, with necrotic tissue replaced by the host wound healing response accompanied by the disordered attempt to regenerate the hepatic lobule. We propose that the production of type 2 cytokines by invNKT cells at a peculiar stage of the chronic viral disease plays an active role in promoting this scenario. One of the most significant functions of IL-4 and, especially, of IL-13 is in fact the promotion of wound healing and fibrosis, to counteract necroinflammatory responses (7). IL-4 and/or IL-13 lead to increased deposition of collagen fibers in the extracellular matrix by inducing the expression of the enzymes arginase I and II, responsible for the synthesis of ornithine-derived proline, an essential precursor for the production of collagen, with the concomitant suppression of NO generation (7). IL-13 is a potent direct inducer of collagen production in fibroblasts (7); moreover, it exerts an indirect profibrotic role by inducing TGF-{beta}, which is a major profibrotic cytokine (7). Blocking IL-13 action with a soluble chimeric IL-13R-Fc molecule indeed counteracts the fibrotic reaction occurring in the liver of mice infected by Schistosoma mansoni, a clear example of hepatic inflammation and fibrotic response evolving from an early type 1 into a sustained type 2 cytokine-type response (7). IL-4, in contrast, acts primarily as an indirect profibrotic signal, possibly by regulating the expression of TGF-{beta} (8), although it may stimulate synthesis of the extracellular matrix proteins by human dermal fibroblasts in vitro (45).

As already discussed, a peculiar aspect of the hepatic fibrosis is that it entails the activation of stellate cells, which are the main profibrotic cells in the liver (3). Our confocal analysis rules out CD1d expression on stellate cells, yet it reveals the expression of CD1d in some unidentified cells situated in close contact with stellate cells. The recognition of CD1d expressed on these yet unidentified cells could trigger invNKT cells to produce type 2 profibrotic cytokines, which in turn act on the adjacent stellate cells to activate, with other factors, their fibrogenic program.

The cytokine switch in circulating invNKT cells is already present in cirrhotic patients who have not yet developed primary HCC, and is maintained when HCCs develop, suggesting that the acquisition of IL-4 and IL-13 production by invNKT cells precedes the tumor onset. Recently, IL-13 produced by CD1d-dependent NKT cells was shown to induce a TGF-{beta}-dependent suppression of antisarcoma immune response in a mouse model (46). Thus, the invNKT cell-dependent cytokine network that we implicate in the cirrhosis progression of chronic viral hepatitis might also play an immunosuppressive role, favoring in some way the development of HCC.

In conclusion, our findings reveal a possible pathogenic role of CD1d-dependent invNKT in chronic viral hepatitis: consistent with their sentinel role in the immune system, invNKT cells may be activated by CD1-dependent stimulation to halt the tissue injury through the production of type 2 cytokines. The persistent production of type 2 cytokines, however, may trigger excessive healing and contribute to the progression of the fibrotic process toward cirrhosis and, possibly, also to HCC outgrowth. Targeting type 2 cytokines could thus be of clinical benefit in liver cirrhosis.


   Footnotes
 
1 This study was supported by grants from Human Frontier Science Program (to P.D.), Associazione Italiana Ricerca Cancro (to G.C. and P.D.), Italian Ministry of Health (Ricerca Finalizzata, to G.C.), and European Union (QLK2-CT-201-01205 to S.A., G.C., and P.D.). Back

2 Address correspondence and reprint requests to Dr. Sergio Abrignani, IRIS Research Center, Chiron Vaccines, Via Fiorentina 1, 53100 Siena, Italy (E-mail address: sergio_abrignani{at}chiron.com); Dr. Giulia Casorati, DIBIT, H. San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy (E-mail address: casorati.giulia{at}hsr.it); or Dr. Paolo Dellabona, DIBIT, H. San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy (E-mail address: dellabona.paolo{at}hsr.it) Back

3 Abbreviations used in this paper: HBV, hepatitis B virus; {alpha}-GalCer, {alpha}-galactosylceramide; {alpha}-SMA, {alpha}-smooth muscle actin; DC, dendritic cell; DC-LAMP, DC-lysosome-associated protein; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; IHL, intrahepatic lymphocyte; invNKT, invariant NKT; NHS, normal human serum; RT, room temperature. Back

Received for publication February 20, 2004. Accepted for publication April 28, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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