Yan-Yan Yu, Chong-Wen Si, Xiu-Lan Tian, Qun He, Hai-Peng Xue, ¹Department of Infectious Diseases, ²Department of Pathology, The First Teaching Hospital, Beijing Medical University,
Beijing 100034, China
Dr. Yan-Yan Yu, female, born on 1962-12-14 in Lanzhou City, Gansu Province, China, graduated from Beijing Medical University as a Ph.D. in 1994, now associate professor of infectious diseases majoring infectious diseases, having two papers published.
Supported by National Natural Science Foundation of China, No.39170704.
Correspondence to: Dr. Yan-Yan Yu, Department of Infectious Diseases, The First Teaching Hospital, Beijing Medical University, Beijing 100034, China
Telephone: +86-10-66171122 ext 2362,  Fax. +86-10-66176450
Received: 1998-05-18

Subject headings: hepatitis; liver necrosis; tumor necrosis factor; interleukins; interferon

Yu YY, Si CW, Tian XL, He Q, Xue HP.Effect of cytokines on liver necrosis.World J Gastroenterol, 1998;4(4):311-313

Abstract
AIM: To investigate the effect of cytokines on the liver necrosis.

METHODS: rIL (interleukin)-1, rIL-6, rIFN (interferon), rTNF (tumor necrosis factor) α with or without D-galactosamine (D-GAL) were injected into the abdominal cavity of mice separately. ALT, TBIL (total bilirubin) and histological changes were observed.

RESULTS: There was no effect on hepatocyte of normal mice after injection of rIL-1, rIL-6, rIFN alone or together. The serum total bilirubin (TBIL) and liver necrosis of mice increased after rTNFα, rIL-6 or rIFN were used separately with D-GAL. The TBIL level (μmol/L)was 46.19±10.62, 44.55±12.9 and 41.94±14.9, higher than that caused by D-GAL alone (TBIL, 26.67μmol/L±11.14μmol/L). The serum TBIL of mice and the degree of liver necrosis increased after injection of IL-1, IL-6 with D-GAL and rTNFα.

CONCLUSION: Cytokines, like IL-1, IL-6, IFNγ and TNFαjoined in the process of hepatocyte necrosis.They can enhance the degree of liver necrosis induced by D-GAL.

INTRODUCTION
Immune system in human body is a net system and so are the effects of cytokines, which can be affected by other cytokines. They promote or inhibit each other, TNFα can aggravate hepatic necrosis^［1］, and its effect is also influenced by other cytokines. The levels of IL-1, IL-6 and IFN in plasma of hepatitis patients were increased after infection^［2-6］. We investigated the effect of multicytokines to understand the synergistic action of multicytokines.

MATERIALS AND METHODS
Material and animal
Animal Ninety-six Balb/c male mice weighing 20g each were used. They were provided by the Center of Experiment Animal of Chinese Academy of Medical Sciences.

Reagent rIL-1 (10⁵U/ml), rIL-6 (10⁵u/ml),rIFN (5×10⁵U/ml) andrTNFα (5×10⁵U/ml) were provided by the Chinese Academy of Military Medical Sciences. D-GAL were derived from Department of Chemistry, Chongqing Medical University.

Method
The effect of rIL-1, rIL-6, rTNFα and rIFN on normal mice Thirty mice were selected and divided into 5 groups. rIL-1 (10 ⁶U/kg), rIL-6 (10⁶U/kg), rIFN (5×10⁶U/kg) andrTNFα (5×10⁵U/kg) were injected into the abdominal cavity of mice separately and normal saline was used for the control. TBIL and ALT were detected 48 hours after the injection. The pathologic changes of the liver were observed.
The combined effect of TNFα with IL-1 or IL-6 or IFN on liver Eighteen mice were selected and divided into 3 groups. TBIL and ALT were detected 48 hours after injection ofrTNFα with rIL-1, or rIL-6, orrIFN-separately into the abdominal cavity. Liver histological changes were observed at the same time.
The aggravating effect of rIL-1, rIL-6, rIFN and rTNFα separately in liver necrosis Thirty mice were divided into 5 groups. D-GAL (1.5g/kg), or D-GAL with rIL-1 (5×10⁴U/kg) or rIL-6 (5×10⁴U/kg) or rIFN (2.5×10⁵U/kg) or rTNFα (1×10⁴U/kg) were injected separately into abdominal cavity of mice. Serum TBIL and pathologic changes in liver were compared with the control after 48 hours.The rest 18 mice were divided into 3 groups. D-GAL and rTNFα with rIL-1 or rIL-6 or rIFN (the same dose as above) were injected into the abdominal cavity of mice separately.
ALT and TBIL detection Beckman Biochemistry Machine was used to detect the serum ALT and TBIL. Methods of liver biopsy^［7］ Hepatic tissues were taken and fixed in Bovin′s fluid. After dehydration they were embedded in paraffin wax, and sections were made, and stained by HE. We marked mild degeneration as - or ±, spot necrosis as +, small piece necrosis as ++ , sublarge necrosis as +++ , large necrosis as ++++.
Statistical analysis χ² test was used to compare the difference among groups.

RESULTS
The effect of multicytokines on normal liver of mice
rTNFα, rIL-1, rIL-6 and rIFN were injected into the abdominal cavity of normal mice separately. The changes of their hepatic function are shown in Table 1 . And the results of their hepatic histologic examination were - or ±. There was no difference from normal control. There was no difference in TBIL and ALT either between each group, indicating that the injection of rTNFα, rIL-1, rIL-6 and rIFN have no effects on liver function and liver histology of normal mice.

Table 1 The changes of liver function after cytokines injection in mice (mean±SD )

F=08 between groups, P＞0.05

The effect of rTNFα with rIL-1 or rIL-6 or rIFN on normal mice rTNFα with rIL-1, or rIL-6, or rIFNγ were injected into the abdominal cavity of normal mice. The changes of their liver function are shown in Table 2 . There was no difference in TBIL and ALT among the groups, and no difference in liver histology compared with normal control. So, it is obvious that rTNFα with rIL-1 or rIL-6 or rIFNγ had no effect on liver function and liver histology of normal mice.

Table 2 The combined effect of rTNFα with rIL-1 or rIL-6 or rIFNγ on liver function of normal mice (mean±SD)

F=0.8, between groups, P＞0.05.

The effect of cytokines on mice liver
DGal combined with cytokines were injected separately into the abdominal cavity of mice. The results of liver function and hepatic histology are shown in Table 3. The level of TBIL in different groups of cytokine was higher than that of controls. The TBIL level in groups of D+rIL-6,D+rIFN and D+rTNFα rose significantly compared with group D, and hepatic necrosis worsened. There was no difference in TBIL level between group D+rIL-1 and group D.

Table 3 The effect of D-GAL with cytokines on TBIL and histological changes of liver

^aP＜0.05, vs group D GAL.

Table 4 The effects of DGal and rTNFα with rIL-1, rIL-6, rIFN- separately on TBIL and hepatic cell of mice

^a P＜0.05, vs group of D+rTNFα.DGal and rTNFα with rIL-1, or rIL-6 or rIFNγ were injected into the abdominal cavity of mice. The results of liver function and hepatic histology are shown in Table 4 . The degree of hepatic necrosis was more severe and the level of TBIL was higher in group D+T+1 and group D+T+6 than in group D+T. There was no significant difference between group D+T+IFNand group D+T. It is suggested that D+T can raise the level of TBIL and the degree of hepatic necrosis. And adding, rIL-1 and rIL-6 can increase the level of TBIL and ALT and enhance the degree of liver necrosis, but no difference with rIFNγ.

DISCUSSION
Our experimental results showed that various cytokines, such as TNFα, IL-1, IL-6 and rIFN alone or together have no effect on liver. But TNFα, rIL-6 and rIFNγ can enhance the degree of liver lesions caused by D-Gal and rIL-1 and rIL-6 can aggravate liver necrosis caused by TNFα.
      Lipopolysaccharide (LPS) is a very strong inducer of many cytokines. It can induce IL-1, IL-6,TNFα and IFN and so on, ^［8,9］ e.g., hepatitis B can induce TNFα in hepatocytes^［10］. Besides increased TNFα in patients with viral hepatitis, the level of many other cytokines was elevated, for example IL-1, IL-6 and IFN. The effect of any cytokine could be affected by other cytokines. IFN can induce the production of TNFα^［11］, which produced cytokines including IL-1 and IL-6. So, not only TNFα, but many other cytokines affect the liver function when infections appear. Our results demonstrated the role of multiple cytokines in hepatic necrosis. There might be many routes for multiple cytokines to worsen the hepatic damage in hepatitis. One of the routes is local Shwartzman reaction. TNFα and LPS can induce Shwartzman as either sensitizing agent or stimulating agent^［12］. TNFα together with IL-1, IL-6 can aggravate hepatic necrosis and bleeding. They can spoil the cell membrane by inducing indirectly serine protease and by activating phosphatase A₂^［13］. Cytokines could not induce hepatic lesions in mice with normal liver function. The susceptibility to cytokines was increased after injection of D-GAL. D-GAL, actimycin D and mitomycin C are hepatocyte-specific inhibitors of RNA synthesis. The toxic effect of cytokine can increase dramatically when cytokines injected with DGAL^［14］. The catabolism and anabolism of protein in hepatocytes are affected in hepatopathy and the toxic effect of cytokine can reveal in hepatitis.
      It can be supposed that viral infection and bacteriotoxemia in patients with hepatitis can induce a large amount of cytokines and aggravate liver necrosis through their interaction, resulting in severe hepatitis. To study the effect of multicytokines in patients with hepatitis is of great importance to understand the mechanism of hepatic necrosis and to search for effective means for prevention and treatment of liver necrosis.

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