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P-177 Role of macrophages in regeneration of the liver
Posters/International Hepatology Communications 3 Suppl. (1995) $37-S169
P-177
RoLE
or
MACaOPHAGES
IN
R E G E N E R A T I O N O F THE LIVER
S.Hongo, K.Ohmura, T.Arima, T.Fukuchi, T.Nagura,H.Kiriyama,H.Takada,K.Matsumoto, Y.Shiratori Dept. of Gastro-Hepatology, Koshigaya Hospital, Dokkyo University, School of Medicine; Dept. of Int. Med (II), University of Tokyo, Japan To clarify the role of macrophages and their mediators during regeneration of the liver, the difference of liver regeneration in C3H/HeN (LPS-responsive) and C3H/HeJ (LPS-resistant) mice has been investigated. After 67% partial hepatectomy, an increase in the weight of regenerating liver, PCNA labelling index of hepatocytes, and TNF activity were reduced in C3H/HeJ mice, when compared with C3H/HeN mice. IL-6 activity in serum was enhanced in both strains of mice. Suppression of Kupffer cells by gadolinium chloride in C3H/HeN mice reduced the increase in both serum TNF and IL-6 levels, reduced PCNA labelling index, and disturbed liver regeneration. Previous administration of antibody against T N F reduced PCNA labelling index of hepatocytes in C3H/HeN mice. These results suggest that LPS-responsive Kupffer cells could play a role in the liver regeneration.
P-179
GROWTH-PROMOTING ACFIVITY OF TIMP-1 FOR HEPATOMA CELLS Y.Katano, Y.Fukuda, Y.Koyama, I.Nakano, F.Urano, M.Yamada, A.Marni, ICImada, O.Kato, M.Ebata, H.Toyoda, T.Hayakawa, *ICYamashita, **K.[wata, *T.Hayakawa 2nd Dept. of Internal Medicine, Nagoya Univ. School of Medicine, *Dept. of Biochemistry, School of Dentistry, Aichi-Gakuin Univ., Nagoya, and **Fuji Chemical [ndusldes,Ltd.,Takaoka,Japan Human tissue inhibitor of metalloproteinases-1 (TIMP-1) has a poteut growth-promoting activity for wide range of cells. In this study, we examined whether TIMP-1 promote the cell growth of hepatoma cell line HepG2. Methods HepG2 cells were incubated in serum-free RPMI1640. The concentration of TIMP-1 in the resultant nredia of HepG2 cells was determined by EIA. To examine the effect of TIMP-1 in serum, TIMP-1free fetal bovine serum (FBS) was prepared by the passage of FBS lhrnugh anti-TIMP-1 monoclonal antibody-Sepharose 4B affinity columns. The cell proliferation was assessed by counting the cell number or measuring the radioactivity of incorporated [ZH]thymidine. The effect of exogeneous purified recombinant TIMP-1 was examined in the 5-1000ng/ml range. Results HepG2 cells were found In secrete TIMP-1 in the serum-free media without stimulation. As compared with untreated FBS, removal of TIMP-1 from FBS showed to inhibit significantly DNA synthesis at 24 hours after incubation. However, at 36 hours after incubation, there was no difference between cell proliferation in the TIMP-l-free FBS nredia and that in the untreated FBS media. It is seemed that TIMP-1 produced by HepG2 cells effected cell proliferation, rTIMP-1 demonstrated a dose-dependent stimulalory effect. The maximal DNA synthesis was observed at the concentration of 500ng/ml and was 1.8-2.0 times the DNA synthesis in control. Conclusion These results suggest that TIMP-1 is one of the autocrine cell-growth factors for HepG2 cells.
$81
P-178 DIFFERENT SUGAR CHAINS OF AFP PRODUCED IN ACUTE HEPATITIS AND CHRONIC LIVER DISEASES K. T a k e t a , M. Liu, H. Y a m a r n o t o , M. O g a t a , S. I k e d a l ; A. O h m o r i 2 ; K. A k a m a t s u 3 ; C. Sekiya4; H. T a g a 5 1Dept. P u b l i c H e a l t h , O k a y a m a Univ. Med. School; O k a y a m a ; 2Shigei M e d . Res. H o s p . , O k a y a m a ; 3 3 r d D e p t . M e d . , E h i m e Univ. S c h o o l Med.; 4 3 r d D e p t . M e d . , A s a h i k a w a M e d . C o l l e g e , A s a h i k a w a ; 5 T u m o r Lab., T o k y o , Japan Lentil l e c t i n ( L C A ) - r e a c t i v e a - f e t o p r o t e i n (AFP)-L3 a n d erythroagglu-tinating phytohemagglutinin(E-PHA)reactive AFP-P4/AEP-P5 increase in fulminant hepatitis as in h e p a t o c e l l u l a r c a r c i n o m a . In t h i s s t u d y , AFP-L3 a n d AFP-P4/AFP-P5 w e r e a n a l y z e d b y l e c t i n a f f i n i t y e l e c t r o p h o r e s i s o n s e r a f r o m p a t i e n t s w i t h a c u t e h e p a t i t i s A, B, C and E without encephalopathy and compared with those in patients with chronic hepatitis and cirrhosis having r e l a t i v e l y h i g h levels o f s e r u m AFP. P r o p o r t i o n s o f AFP-L3 a n d AFP-P4 w e r e i n c r e a s e d in a c u t e h e p a t i t i s as i n fulminant hepatitis, while those in patients with chronic hepatitis and cirrhosis were low despite their higher s e r u m levels o f AFP. AFP-L3 a n d A F P - P 4 / A F P - P 5 in a c u t e h e p a t i t i s w e r e h i g h f r o m t h e b e g i n n i n g o f AFP rise. T h e results indicate that undifferentiated hepatocytes resulting from liver injury in acute hepatitis are r e s p o n s i b l e f o r t h e i n c r e a s e s in AFP-L3 a n d A F P - P 4 / A F P P5, a n d t h u s t h e m e c h a n i s m o f AFP p r o d u c t i o n i n a c u t e h e p a t i t i s is d i f f e r e n t f r o m t h a t o f c h r o n i c h e p a t i t i s a n d cirrhosis.
P-180
EFFECT OF CENTRAL TYHROTROPINRELEASING HORMONE (TRH) ON HEPATIC PROLIFERATION M. Yoneda, S. Yokohama, K. Tamori, Y. Sato, K. Nakamura, A. Kimuru, M. Fujita, K. Baba, H. Saitoh, K. Akiyama and 1. Makino Second Dept of Medicine, Asahikawa Medical College, Japan TRH is widely distributed in the central nervous system, especially in the dorsal vagal complex which is important site for autonomic nervous system, and plays a role in central regulation of gastric functions. Liver is richly innervated and vagus is known to be involved in hepatic regeneration. However, nothing is known about a role of central neuropeptides on hepatic proliferation. Purpose: To investigate the effect of central TRH on hepatic proliferation. Methods: Hepatic proliferation was assessed by thymidine incorporation into hepatic DNA. Male Wistar rats (200-220 g) were injected with either TRH analog, RX 77368 (1, 5. 10 or 100 ng), or saline intracisternally or intravenously. ~H-thymidine (20 ,uCi/100 g) was injected intraperitoneally 6, 12, 24, 48 or 72 h after the peptide injection. Rats were decapitated and liver was removed 2 h after ~H-thymidine injection. Liver sample was homegenized with phosphate buffer and thymidine incorporation into hepatic DNA was assesed by Schneider's method. Results: Hepatic proliferation was stimulated by intracisternat TRH analog (10 rig) with peak response at 24 h after the peptide reJection and returned to basal at 72 h (Mean_+ SEM; cpm/mg DNA: 0 h 92+_ 6; 6 h 131 _+ l h 12h 195 _+ 42; 24 h 693 _+ 78; 48 h 378 + 81; 72 h 120 _+ 27; n=5-8). This stimulatory effect by central TRH on hepatic proliferation was dose-related ranging from 5 ng to 100 rig. Stimulauon of hepatic proliferation by central TRH was abolished by hepatic branch vagotomy, atropine and indomethacin but not by L-NAME (ntric oxide inhibitor). Intravenous TRH (100 ng) did not influence hepatic proliferation. Conclusion: TRH acts in the brain to stimulate hepatic proliferation through vagal, muscafinic and prostaglandin pathways but not through nitric oxide. These results suggests the central regulation of hepatic proliferation by neuropeptides.
P-177
RoLE
or
MACaOPHAGES
IN
R E G E N E R A T I O N O F THE LIVER
S.Hongo, K.Ohmura, T.Arima, T.Fukuchi, T.Nagura,H.Kiriyama,H.Takada,K.Matsumoto, Y.Shiratori Dept. of Gastro-Hepatology, Koshigaya Hospital, Dokkyo University, School of Medicine; Dept. of Int. Med (II), University of Tokyo, Japan To clarify the role of macrophages and their mediators during regeneration of the liver, the difference of liver regeneration in C3H/HeN (LPS-responsive) and C3H/HeJ (LPS-resistant) mice has been investigated. After 67% partial hepatectomy, an increase in the weight of regenerating liver, PCNA labelling index of hepatocytes, and TNF activity were reduced in C3H/HeJ mice, when compared with C3H/HeN mice. IL-6 activity in serum was enhanced in both strains of mice. Suppression of Kupffer cells by gadolinium chloride in C3H/HeN mice reduced the increase in both serum TNF and IL-6 levels, reduced PCNA labelling index, and disturbed liver regeneration. Previous administration of antibody against T N F reduced PCNA labelling index of hepatocytes in C3H/HeN mice. These results suggest that LPS-responsive Kupffer cells could play a role in the liver regeneration.
P-179
GROWTH-PROMOTING ACFIVITY OF TIMP-1 FOR HEPATOMA CELLS Y.Katano, Y.Fukuda, Y.Koyama, I.Nakano, F.Urano, M.Yamada, A.Marni, ICImada, O.Kato, M.Ebata, H.Toyoda, T.Hayakawa, *ICYamashita, **K.[wata, *T.Hayakawa 2nd Dept. of Internal Medicine, Nagoya Univ. School of Medicine, *Dept. of Biochemistry, School of Dentistry, Aichi-Gakuin Univ., Nagoya, and **Fuji Chemical [ndusldes,Ltd.,Takaoka,Japan Human tissue inhibitor of metalloproteinases-1 (TIMP-1) has a poteut growth-promoting activity for wide range of cells. In this study, we examined whether TIMP-1 promote the cell growth of hepatoma cell line HepG2. Methods HepG2 cells were incubated in serum-free RPMI1640. The concentration of TIMP-1 in the resultant nredia of HepG2 cells was determined by EIA. To examine the effect of TIMP-1 in serum, TIMP-1free fetal bovine serum (FBS) was prepared by the passage of FBS lhrnugh anti-TIMP-1 monoclonal antibody-Sepharose 4B affinity columns. The cell proliferation was assessed by counting the cell number or measuring the radioactivity of incorporated [ZH]thymidine. The effect of exogeneous purified recombinant TIMP-1 was examined in the 5-1000ng/ml range. Results HepG2 cells were found In secrete TIMP-1 in the serum-free media without stimulation. As compared with untreated FBS, removal of TIMP-1 from FBS showed to inhibit significantly DNA synthesis at 24 hours after incubation. However, at 36 hours after incubation, there was no difference between cell proliferation in the TIMP-l-free FBS nredia and that in the untreated FBS media. It is seemed that TIMP-1 produced by HepG2 cells effected cell proliferation, rTIMP-1 demonstrated a dose-dependent stimulalory effect. The maximal DNA synthesis was observed at the concentration of 500ng/ml and was 1.8-2.0 times the DNA synthesis in control. Conclusion These results suggest that TIMP-1 is one of the autocrine cell-growth factors for HepG2 cells.
$81
P-178 DIFFERENT SUGAR CHAINS OF AFP PRODUCED IN ACUTE HEPATITIS AND CHRONIC LIVER DISEASES K. T a k e t a , M. Liu, H. Y a m a r n o t o , M. O g a t a , S. I k e d a l ; A. O h m o r i 2 ; K. A k a m a t s u 3 ; C. Sekiya4; H. T a g a 5 1Dept. P u b l i c H e a l t h , O k a y a m a Univ. Med. School; O k a y a m a ; 2Shigei M e d . Res. H o s p . , O k a y a m a ; 3 3 r d D e p t . M e d . , E h i m e Univ. S c h o o l Med.; 4 3 r d D e p t . M e d . , A s a h i k a w a M e d . C o l l e g e , A s a h i k a w a ; 5 T u m o r Lab., T o k y o , Japan Lentil l e c t i n ( L C A ) - r e a c t i v e a - f e t o p r o t e i n (AFP)-L3 a n d erythroagglu-tinating phytohemagglutinin(E-PHA)reactive AFP-P4/AEP-P5 increase in fulminant hepatitis as in h e p a t o c e l l u l a r c a r c i n o m a . In t h i s s t u d y , AFP-L3 a n d AFP-P4/AFP-P5 w e r e a n a l y z e d b y l e c t i n a f f i n i t y e l e c t r o p h o r e s i s o n s e r a f r o m p a t i e n t s w i t h a c u t e h e p a t i t i s A, B, C and E without encephalopathy and compared with those in patients with chronic hepatitis and cirrhosis having r e l a t i v e l y h i g h levels o f s e r u m AFP. P r o p o r t i o n s o f AFP-L3 a n d AFP-P4 w e r e i n c r e a s e d in a c u t e h e p a t i t i s as i n fulminant hepatitis, while those in patients with chronic hepatitis and cirrhosis were low despite their higher s e r u m levels o f AFP. AFP-L3 a n d A F P - P 4 / A F P - P 5 in a c u t e h e p a t i t i s w e r e h i g h f r o m t h e b e g i n n i n g o f AFP rise. T h e results indicate that undifferentiated hepatocytes resulting from liver injury in acute hepatitis are r e s p o n s i b l e f o r t h e i n c r e a s e s in AFP-L3 a n d A F P - P 4 / A F P P5, a n d t h u s t h e m e c h a n i s m o f AFP p r o d u c t i o n i n a c u t e h e p a t i t i s is d i f f e r e n t f r o m t h a t o f c h r o n i c h e p a t i t i s a n d cirrhosis.
P-180
EFFECT OF CENTRAL TYHROTROPINRELEASING HORMONE (TRH) ON HEPATIC PROLIFERATION M. Yoneda, S. Yokohama, K. Tamori, Y. Sato, K. Nakamura, A. Kimuru, M. Fujita, K. Baba, H. Saitoh, K. Akiyama and 1. Makino Second Dept of Medicine, Asahikawa Medical College, Japan TRH is widely distributed in the central nervous system, especially in the dorsal vagal complex which is important site for autonomic nervous system, and plays a role in central regulation of gastric functions. Liver is richly innervated and vagus is known to be involved in hepatic regeneration. However, nothing is known about a role of central neuropeptides on hepatic proliferation. Purpose: To investigate the effect of central TRH on hepatic proliferation. Methods: Hepatic proliferation was assessed by thymidine incorporation into hepatic DNA. Male Wistar rats (200-220 g) were injected with either TRH analog, RX 77368 (1, 5. 10 or 100 ng), or saline intracisternally or intravenously. ~H-thymidine (20 ,uCi/100 g) was injected intraperitoneally 6, 12, 24, 48 or 72 h after the peptide injection. Rats were decapitated and liver was removed 2 h after ~H-thymidine injection. Liver sample was homegenized with phosphate buffer and thymidine incorporation into hepatic DNA was assesed by Schneider's method. Results: Hepatic proliferation was stimulated by intracisternat TRH analog (10 rig) with peak response at 24 h after the peptide reJection and returned to basal at 72 h (Mean_+ SEM; cpm/mg DNA: 0 h 92+_ 6; 6 h 131 _+ l h 12h 195 _+ 42; 24 h 693 _+ 78; 48 h 378 + 81; 72 h 120 _+ 27; n=5-8). This stimulatory effect by central TRH on hepatic proliferation was dose-related ranging from 5 ng to 100 rig. Stimulauon of hepatic proliferation by central TRH was abolished by hepatic branch vagotomy, atropine and indomethacin but not by L-NAME (ntric oxide inhibitor). Intravenous TRH (100 ng) did not influence hepatic proliferation. Conclusion: TRH acts in the brain to stimulate hepatic proliferation through vagal, muscafinic and prostaglandin pathways but not through nitric oxide. These results suggests the central regulation of hepatic proliferation by neuropeptides.