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Chemokines modulate their own expression and matrix metalloproteinase secretion in human hepatic stellate cells
Category 3: Molecular
I
745
and cell biology (gene expression,
SERUM LAMININ LEVELS IN NON ALCOHOLIC STEATOHEPATITIS
I
778
signalling, fibrosis)
85
CHEMOKINES MODULATE THEIR OWN EXPRESSlON AND MATRIX METALLOPROTEINASE SECRETlON IN HUMAN HEPATIC STELLATE CELLS
P. Di Rocco, A. Grieco, A. Bianco, A. Caprodossi, L. Miele, G.L. Rapaccini, M. Pompili, A.V. Greco, G.B. Gasbarrini. Internal
E. Efsen I, F. Marra ‘, CM. Lloyd. ’ Dip. Medicina Interna, Univ. of
Medicine, Catholic University of the Sacred Heart, Rome, Italy
Florence, Italy; Leukocyte Biology, BMS, Imperial College, Lmdon,
Non alcoholic steatohepatitis (NASH) is characterized by macrovescicular steatosis, inllammation and variable presence of fibrosis. NASH can evolve into progressive liver disease and cirrhosis. The importance of fibrosis in the natural history of virus-related chronic active hepatitis is well known. Our aim was to evaluate the presence of fibrosis in patients with NASH, through the measurement of serum laminin levels, widely accepted as fibrosis marker. We studied 16 patients with histological diagnosis of NASH (age: 37 f 9 yrs, M/F: 12/4, AST: 41 f 15 III/l, ALT 74 f 34 Xl/I), 16 patients with HCV related chronic active hepatitis (CAH) (age: 50 f 9 yrs, M/F: 1016, AST: 93 f 22 IU/l, ALT: 154 f 35 II-l/l) and 16 patients with cirrhosis (age 55 f 8 yrs, M/F: 8/4, Child B: 10/16, Child C: 606, AST: 50 f 43 III/l, ALT: 60 f 51 IU/l). Eight volounteers (age: 3 1 f 8 yrs, M/F: 5/3) served as healthy controls. The fibrosis (Knodell score) was minimal in NASH patients (range O-l) and mild-severe in CAH (range l-3). Serum laminin was measured by Quantimatrix Human Laminin ELISA Kit (Chemicon International Inc.). In the NASH group mean levels of serum laminin were 141.61 f 27.64 @ml, in the CAH group 124.35 f 26.80 ng/ml, in the cirrhotic group 150.11 f 20.91 &ml. In the healthy controls serum laminin was not detectable. The evidence of similar serum laminin levels in NASH, in CAH, and in cirrhosis suggests that a clear fibrotic activity can be observed even in the early stage of NASH.
Hepatic stellate cells (HSC) play a crucial role during hepatic fibrogenesis. Upon liver injury, HSC acquire a myofibroblast-like phenotype which secretes extracellular matrix (ECM) components and cytokines, including chemokines. The CC-chemokine monocyte chemotactic protein-l (MCP-1) is a chemoattractant for monocytes and T-lymphocytes and contributes to the formation and maintenance of the hepatic inflammatory response. Degradation of ECM is controlled by matrix metalloproteinases (MMPs). Aim: To investigate the autoregulation of the chemokine system and the possible modulation of MMPs by MCP-1 in human HSC. Results: HSC were stimulated with MCP-1, the RNA harvested and chemokine gene expression assessed using ribonuclease protection assay. Exposure of HSC to MCP-1 increased gene expression of interleukin-8 and MCP-1 itself with a peak at 12 h, this rise was sustained at 24 h but decreased after 48 h. To test whether the rise in RNA levels was associated with increased protein secretion, HSC were exposed to MCP-1 for 24 h, washed, and incubated 48 h. A two-fold increase in MCP-1 secretion was observed in comparison to basal levels. Unstimulated HSC expressed MMP-2, and incubation with MCP-1 increased this expression as evaluated by zymography. Conclusion: In human HSC, MCP-1 stimulates its own expression and that of other chemokines, and induces MMP-2 activity. These data indicate that chemokine networks involving HSC may be relevant for the pathophysiology of liver fibrogenesis.
I 753
SERUM LEVELS OF INTERLEUKIN 6 AND OF INTERLEUKIN 6 RECEPTOR IN PATIENTS WITH HEPATOCELLULAR CARCINOMA
L. Giannitrapani, G. Montalto, M. Notarbartolo, L. Virruso, M. Soresi, A. Carroccio, N. D’Alessandro, M. Cervello. Internal Medicine, University of Palermo, Italy
Interleukine 6 (IL-6) is a mutlifunctional cytokine that plays a central role in the host immune defense, hemopoiesis and acute phase reaction. In this study we have evaluated the serum levels of IL-6 and of sIL-6R in a group of patients with liver cirrhosis (LC) and in a group of patients with HCC-LC associated, and we compared them with a control group. The study was undertaken on 167 subjects divided into three groups. Group I included 83 patients with HCC; they were divided into 3 stages according to Okuda’s staging. Group II included 42 patients with LC, divided into 3 classes according to Child-Pugh’s classification. HCC and LC patients were all anti-HCV-associated. Control group included 42 healthy subjects recruited from blood donors. Serum IL-6 levels in the HCC group were significantly higher than those in the LC and control groups (p < 0.0001). Patients with LC had higher, but not significantly, serum values of IL-6 than controls. No significant difference was found for sIL-6R levels among the 3 groups. When HCC patients were divided into the 3 stages according to Okuda classification, there was a significant increase of both IL-6 and sIL-6R values towards stage II and III, p < 0.02 and p < 0.0005 respectively, as evaluated by the Spearman’s correlation coefficient. Moreover, stage III HCC patients had IL-6 values significantly higher than stage II (p < 0.02) and stage I (p < 0.003) patients. The comparison of HCC and LC patients, divided into 3 classes according to Child-Pugh, showed significantly higher values in the HCC group than in LC group of the same class (p < 0.01). In conclusion IL-6 serum levels in patients with HCC were higher than in patients with LC and controls, suggesting an increased production of this cytokine by the neoplastic cells.
I
791
UK
COMPARATIVE STUDY OF C-MYC mRNA IN CHRONIC ACTIVE HEPATlTlS, CIRRHOSIS, AND HCC
M. Bortolami, C. Venturi, R. Scalerta, S. Bacchetti, C. Carlotto, F. Farinati. Dept. of Surgical and Gastroenterological Sciences, University of Padua, Italy
The biological significance of c-myc in HCC is not well known. HCV infection is considered a risk factor for HCC development. Aims: to evaluate the expression of c-myc in different stages of liver disease and to determine whether HCV infection modulates the expression of c-myc. Methods: We examined 50 liver samples of patients with HCV-related disease: 33 chronic active hepatitis (CAH), 3 cirrhosis, and 7 cirrhosis with HCC and the corresponding HCC tissue. Twenty-one specimens from HCV-ve patients: 5 CAH, 8 cirrhosis with HCC and the corresponding HCC tissue, were considered as a control group. The c-myc mRNAs were detected by semiquantitative comparative RT-PCR. Results: CAH expressed the lowest level of c-myc mRNAs. HCV+ve livers showed lower levels of transcripts than HCV-ve (p = 0.015). Statistical significant differences were observed between CAH and cirrhosis whether HCV+ve (p = 0.003) or HCV-ve (p = O.OCOO8). Cirrhotic livers presented the highest levels either in the presence and in the absence of HCC (p = 0.016). Pericancerous cirrhotic livers expressed higher c-myc mRNAs than HCC itself (p = 0.02). In HCC no difference according to etiology was found. Conclusions: c-myc levels in liver tissues are related to the’progression of liver damage from CAH to cirrhosis and to HCC. In HCV+ve patients c-myc expression is delayed, possibly in correlation with the very long time lag from infection to cancer development. (Supported by MURST funds 980533231ooO5/98).
I
745
and cell biology (gene expression,
SERUM LAMININ LEVELS IN NON ALCOHOLIC STEATOHEPATITIS
I
778
signalling, fibrosis)
85
CHEMOKINES MODULATE THEIR OWN EXPRESSlON AND MATRIX METALLOPROTEINASE SECRETlON IN HUMAN HEPATIC STELLATE CELLS
P. Di Rocco, A. Grieco, A. Bianco, A. Caprodossi, L. Miele, G.L. Rapaccini, M. Pompili, A.V. Greco, G.B. Gasbarrini. Internal
E. Efsen I, F. Marra ‘, CM. Lloyd. ’ Dip. Medicina Interna, Univ. of
Medicine, Catholic University of the Sacred Heart, Rome, Italy
Florence, Italy; Leukocyte Biology, BMS, Imperial College, Lmdon,
Non alcoholic steatohepatitis (NASH) is characterized by macrovescicular steatosis, inllammation and variable presence of fibrosis. NASH can evolve into progressive liver disease and cirrhosis. The importance of fibrosis in the natural history of virus-related chronic active hepatitis is well known. Our aim was to evaluate the presence of fibrosis in patients with NASH, through the measurement of serum laminin levels, widely accepted as fibrosis marker. We studied 16 patients with histological diagnosis of NASH (age: 37 f 9 yrs, M/F: 12/4, AST: 41 f 15 III/l, ALT 74 f 34 Xl/I), 16 patients with HCV related chronic active hepatitis (CAH) (age: 50 f 9 yrs, M/F: 1016, AST: 93 f 22 IU/l, ALT: 154 f 35 II-l/l) and 16 patients with cirrhosis (age 55 f 8 yrs, M/F: 8/4, Child B: 10/16, Child C: 606, AST: 50 f 43 III/l, ALT: 60 f 51 IU/l). Eight volounteers (age: 3 1 f 8 yrs, M/F: 5/3) served as healthy controls. The fibrosis (Knodell score) was minimal in NASH patients (range O-l) and mild-severe in CAH (range l-3). Serum laminin was measured by Quantimatrix Human Laminin ELISA Kit (Chemicon International Inc.). In the NASH group mean levels of serum laminin were 141.61 f 27.64 @ml, in the CAH group 124.35 f 26.80 ng/ml, in the cirrhotic group 150.11 f 20.91 &ml. In the healthy controls serum laminin was not detectable. The evidence of similar serum laminin levels in NASH, in CAH, and in cirrhosis suggests that a clear fibrotic activity can be observed even in the early stage of NASH.
Hepatic stellate cells (HSC) play a crucial role during hepatic fibrogenesis. Upon liver injury, HSC acquire a myofibroblast-like phenotype which secretes extracellular matrix (ECM) components and cytokines, including chemokines. The CC-chemokine monocyte chemotactic protein-l (MCP-1) is a chemoattractant for monocytes and T-lymphocytes and contributes to the formation and maintenance of the hepatic inflammatory response. Degradation of ECM is controlled by matrix metalloproteinases (MMPs). Aim: To investigate the autoregulation of the chemokine system and the possible modulation of MMPs by MCP-1 in human HSC. Results: HSC were stimulated with MCP-1, the RNA harvested and chemokine gene expression assessed using ribonuclease protection assay. Exposure of HSC to MCP-1 increased gene expression of interleukin-8 and MCP-1 itself with a peak at 12 h, this rise was sustained at 24 h but decreased after 48 h. To test whether the rise in RNA levels was associated with increased protein secretion, HSC were exposed to MCP-1 for 24 h, washed, and incubated 48 h. A two-fold increase in MCP-1 secretion was observed in comparison to basal levels. Unstimulated HSC expressed MMP-2, and incubation with MCP-1 increased this expression as evaluated by zymography. Conclusion: In human HSC, MCP-1 stimulates its own expression and that of other chemokines, and induces MMP-2 activity. These data indicate that chemokine networks involving HSC may be relevant for the pathophysiology of liver fibrogenesis.
I 753
SERUM LEVELS OF INTERLEUKIN 6 AND OF INTERLEUKIN 6 RECEPTOR IN PATIENTS WITH HEPATOCELLULAR CARCINOMA
L. Giannitrapani, G. Montalto, M. Notarbartolo, L. Virruso, M. Soresi, A. Carroccio, N. D’Alessandro, M. Cervello. Internal Medicine, University of Palermo, Italy
Interleukine 6 (IL-6) is a mutlifunctional cytokine that plays a central role in the host immune defense, hemopoiesis and acute phase reaction. In this study we have evaluated the serum levels of IL-6 and of sIL-6R in a group of patients with liver cirrhosis (LC) and in a group of patients with HCC-LC associated, and we compared them with a control group. The study was undertaken on 167 subjects divided into three groups. Group I included 83 patients with HCC; they were divided into 3 stages according to Okuda’s staging. Group II included 42 patients with LC, divided into 3 classes according to Child-Pugh’s classification. HCC and LC patients were all anti-HCV-associated. Control group included 42 healthy subjects recruited from blood donors. Serum IL-6 levels in the HCC group were significantly higher than those in the LC and control groups (p < 0.0001). Patients with LC had higher, but not significantly, serum values of IL-6 than controls. No significant difference was found for sIL-6R levels among the 3 groups. When HCC patients were divided into the 3 stages according to Okuda classification, there was a significant increase of both IL-6 and sIL-6R values towards stage II and III, p < 0.02 and p < 0.0005 respectively, as evaluated by the Spearman’s correlation coefficient. Moreover, stage III HCC patients had IL-6 values significantly higher than stage II (p < 0.02) and stage I (p < 0.003) patients. The comparison of HCC and LC patients, divided into 3 classes according to Child-Pugh, showed significantly higher values in the HCC group than in LC group of the same class (p < 0.01). In conclusion IL-6 serum levels in patients with HCC were higher than in patients with LC and controls, suggesting an increased production of this cytokine by the neoplastic cells.
I
791
UK
COMPARATIVE STUDY OF C-MYC mRNA IN CHRONIC ACTIVE HEPATlTlS, CIRRHOSIS, AND HCC
M. Bortolami, C. Venturi, R. Scalerta, S. Bacchetti, C. Carlotto, F. Farinati. Dept. of Surgical and Gastroenterological Sciences, University of Padua, Italy
The biological significance of c-myc in HCC is not well known. HCV infection is considered a risk factor for HCC development. Aims: to evaluate the expression of c-myc in different stages of liver disease and to determine whether HCV infection modulates the expression of c-myc. Methods: We examined 50 liver samples of patients with HCV-related disease: 33 chronic active hepatitis (CAH), 3 cirrhosis, and 7 cirrhosis with HCC and the corresponding HCC tissue. Twenty-one specimens from HCV-ve patients: 5 CAH, 8 cirrhosis with HCC and the corresponding HCC tissue, were considered as a control group. The c-myc mRNAs were detected by semiquantitative comparative RT-PCR. Results: CAH expressed the lowest level of c-myc mRNAs. HCV+ve livers showed lower levels of transcripts than HCV-ve (p = 0.015). Statistical significant differences were observed between CAH and cirrhosis whether HCV+ve (p = 0.003) or HCV-ve (p = O.OCOO8). Cirrhotic livers presented the highest levels either in the presence and in the absence of HCC (p = 0.016). Pericancerous cirrhotic livers expressed higher c-myc mRNAs than HCC itself (p = 0.02). In HCC no difference according to etiology was found. Conclusions: c-myc levels in liver tissues are related to the’progression of liver damage from CAH to cirrhosis and to HCC. In HCV+ve patients c-myc expression is delayed, possibly in correlation with the very long time lag from infection to cancer development. (Supported by MURST funds 980533231ooO5/98).