Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
Assessment of Chemokine (Interleukin -8) and
Interferon–γ (IFN- γ) among Autoimmune
Hepatitis patients in association with certain
viral infections.
Raheem Tuama O. AL–Mammori
Dr . Azhar Emran AL-Thahab
Clinical immunology. Babylon Science College
Immunology. Babylon Science College
Dr . Alaa Sadeq Al-Awad
Consultant physician. Babylon Medicine college
Email :- raheemtoama@yahoo.com
Abstract :Thirty five (35) patients clinically diagnosed as autoimmune hepatitis (AIH) attending
Gastrointestinal (GIT) center in Merjan medical city and (30 matched age person ) as a control group
enrolled in this study in the period between January and August 2012 . Specific anutoantibodies done
to confirm diagnosis and classify of AIH types such as (ANA, ASMA, LKM, LSP) IL-8, IFN- γ, and
hepatitis screen for HCV infection also done . The result show that female is more susceptible to
infected with AIH rather than male, and young age group is more susceptible for infection than other
age group. Highly significant increased ( P<0.01 ) of cytokines (IL-8) in compareson with control as
well as highly significant of IFN- γ. The major group of patients at type 1 AIH and other types are less.
Patients may present without circulating autoantibodies, referred to as (autoantibody-negative
autoimmune hepatitis) or type 4 AIH gave significant group of AIH. Cytokines level important to
monitored the severity of damage and whether the patients with good or bad prognosis, so that
recommended to study other different types of cytokines for Th1, Th2, and NK cell activators to predict
of ability to producing pro-inflammatory and anti-inflammatory mediators as well as ability to
treatment of such diseases.
Introduction :Autoimmune hepatitis is a chronic hepatitis characterized by immunologic and
auto immunologic features, generally including the presence of circulating autoantibodies and a high total serum globulin, usually confined to the IgG
fraction (Krawitt EL, 2006). The main circulating autoantibodies, are antinuclear
antibodies (ANA), anti-smooth muscle antibodies (ASMA), anti-liver kidney
microsome antibodies (ALKM) and anti liver cytosol antibody (ALC).( Manna, et al
2010). Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the
liver characterized by auto antibodies, association with HLA DR3 or DR4 and a
favorable response to immunosuppressive treatment. The detection of non organ and
liver related auto antibodies remains the hallmark for the diagnosis of the disease in
the absence of viral, metabolic, genetic, and toxic etiology of chronic hepatitis or
hepatic injury. (Macfarline et al, 2002). AIH is characterized by the presence of
specific autoantibodies against liver and kidney microsomal antigens (LKM) and/or
auto antibodies against liver cytosol antigen (LC). Anti-LKM-1 and anti-LKM-3
autoantibodies are also detected in some patients with chronic hepatitis C (HCV) and
chronic hepatitis D (HDV). (Manns, MP, and Strasburg, 2001). Some viral enzymes
can inhibit the replication of human peripheral blood mononuclear cells in vitro and
up regulate the production of TNF- alpha, IL-1 B, IL-6, IFN- γ , IL-8 and IL-10. It is
also enhanced the ability of natural killer cells to lyses target cells. HCV and EBV
patients trigger such a wide range of autoimmune diseases.( Lerner et al . 2004) .
IFN- inhibits the activity of Th2 cells, there by inhibiting antibody synthesis. It is
also the most potent activator of macrophages. It stimulates MHC class II expression
on macrophage lineage cells and endothelial cells. IFN- γ used as antiviral,
antiprolefreative and immunomodulary effects. (Frances F., Marchall B.D., 2009)
303
ANA characterize classical , type 1 Autoimmune hepatitis ( AIH) , ASMA are also
frequently detected in type 1 as well as p-ANCA in some cases , Although AMAs are
specific and sensitive diagnostic markers for primary Biliary cirrhoses (PBC) also can
be detected in about 15% of AIH patients in association with type 1, and type 3 with
specific liver proteins . Liver Kidney Microsome ( LKM ) is more specific , can be
subdivided into two subtypes (2a,and 2b( if the patients have HCV antibody). Type 4
is association with different cytokines causing the immune damage rather than
antibodies or negative auto antibody type, it appear as lymphocytes infiltration and
ductal obstruction in liver cell . (Manns, MP 1992 ).
Classification of different types of AIH according to Autoantibodies associated with
the disease present below in this table:AIH type ANA LKM SLP ASMA AMA HCV p-ANCA Therapy
+
±
±
±
Immunosuppression
Type 1
+
Immunosuppression
Type2a
Type2b
Type 3
Type 4
-
+
-
+
-
-
+
-
+
-
-
Immunosuppression
Immunosuppression
Immunosuppression
AIH= Autoimmune Hepatitis, ANA = Antinuclear antibody, LKM- Liver Kidney Microsome , SLP= Specific
liver Protein , ASMA = Anti Smooth Muscle Antibody , AMA = AntiMitoconderail Antibody , HCV = Hepatitis
C virus, p-ANCA= pre cytoplasmic Anti neutrophil Cytoplasmic antibody . ( John BH, 2001,Czaic
2012) ) .
Autoimmune hepatitis affect young to middle aged women, many of whom
(60%) have associated autoimmune disorders such as diabetes mellitus, thyroditis and
glomerulonephritis. Smooth muscle antibodies (ASMA) are not specific for
autoimmune hepatitis and are found as temporary in up to 80% of patients with viral
infections. However high titer IgG antibodies to smooth muscle are classically found
in autoimmune hepatitis. The target antigen is actin, a cytoskeleton protein.
Antibodies to liver and kidney microsomes (LKM)occur in proportion of patients.
(Johnson PJ , 1997)
Patients and methods :Thirty five (35) patients clinically diagnosed as autoimmune hepatitis (AIH)
attending Gastrointestinal (GIT) center in Merjan medical city and (30 matched age
person ) as a control group enrolled in this study in the period between Junuary and
Augest 2012 . Specific anutoantibodies done to confirm diagnosis and classify of AIH
types such as (ANA, ASMA, LKM, LSP) done by using Indirect immunoflourescent
technique, Depending on Principle and manual procedure of Euroimmune
Company(Enger ,W. 2000). IL-8 , IFN- γ, and hepatitis screen for (Hepatitis viruses
C) also done by ELISA technique .Depending on Principle and manual procedure of
BioSource Company (Moore ,et al 1993).
Result and Discussion :The result shows that age groups (20 -29 y, 40 -49 y) have higher percent (11: 35
=31.5%, 9 : 35 = 25.7% ) , while other age groups have lower percent as shown in
(Figure 1 ), The ratio of male to female ( M:F=1: 2.4) it mean the female more
affected with AIH rather than male . From this result can conclude that the young age
group is more susceptible for infected by Autoimmune hepatitis than other age group,
that might caused by individual , hormonal and environmental activity for patients as
well as active immune response .
304
Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
Figure ( 1 ) Age range Distribution of AIH patients .
The (Figure 2) shows that s a highly significant increased (P<0.01) of cytokine
(IL-8) in compareson with control. The mean result of patients and control
(486.8±(SE. 65.4 )for test samples and 298.9± 47.6 for control ). This result might
show active liver cell damage and bad prognosis of disease. Increased IL-8 level
constituent with it functions because it (IL-8) small ,inducible ,secreted cytokines or
Chemoattractant or super family of low molecular weight proteins. Play an important
role as pathogenic mediators in several pathogenesis, inflammatory and autoimmune
diseases. (Frances F. and Marchall B.D. 2009). IL-8 serum levels were significantly
increased in CLD patients, especially in end stage cirrhosis. Interestingly, patients
with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated
with liver function, inflammatory cytokines and non invasive fibrosis markers.(Tritto
,et al 2011). Intrahepatic IL-8 could only be associated to neutrophil infiltration in
patients with primary biliary cirrhosis (PBC). In non cholestatic cirrhosis, increased
IL-8 levels were associated with hepatic macrophage accumulation. Moreover,
monocyte derived macrophages from CLD patients, especially the non classical
CD16+ subtype, displayed enhanced IL-8 secretion in vitro .( Clement ,et al.2010).
Interleukin -8 in AIH
Concentration pg/ml
14
12.49
12
10
6.2
8
6
4
2
0
1
2
patients
group
Figure ( 2) The Concentration of IL-8 ( pg/ml ) in AIH patients and control.
AIHsignificant
patients increased
2= Control
The (Figure 3) Shows that1=
highly
(P<0.01) in IFN-γ of AIH
patients in comparison with control. The mean result of patients ± SE ( 930 pg/ml ±
305
54.1 ) and ( 415 pg/ml ± 20.1 of Control ). This result might show that the interferon
activity is associated with viral activity that might be act as underline cause for the
liver cell damage and causing the Autoimmune hepatitis. There is some patients
having chronic infection with hepatitis C virus, as well as specific auto antibodies for
liver tissues. IFN- inhibits the activity of Th2 cells, thereby inhibiting antibody
synthesis. It is also the most potent activator of macrophages. It stimulates MHC class
II expression on macrophage lineage cells and endothelial cells. IFN- γ used as
antiviral, antiprolefreative and immunomodulary effects. (Frances F., Marchall B.D,
2009).
The pathology of autoimmune hepatitis, the immunity mechanism of T-helper 1
(Th1) and Th2 cells was recently evaluated. Serum cytokines obtained from children
with autoimmune hepatitis for a better understanding of the mechanism. Serum
interferon (IFN- γ) and interleukin-4 (IL-4) were rarely detected. In contrast the IFNγ /B-actins mRNA levels were high. Autoimmune hepatitis is a Th1-predominant
state, therefore immune modulation therapies that target the control of Th1 cytokines
should be used. ( Kawashima , et al 2008 ).
Gamma Interferun in AIH
Concentration pg/ml
930
1000
800
415
600
400
200
0
1
2
Paitient group
1= AIH patients 2= Control
Figure (3) Concentration of IFN- γ ( pg/ml) in AIH patients and Control .
This result might shows a highly association between increased of cytokines level
and progressive damage of liver tissues as well as auto antibody progressive. Presence
of Hepatitis C is highly associated with AIH rather than other types of hepatitis virus.
Presence of certain Auto Antibodies is the hallmark that might give pin point
diagnosis for AIH as well as make an important role in classification to specific
subtypes. By using IFAT procedure, and different tissue substrate (Hep -20 for
ANA,AMA ) Mouse stomach Kidney(MSK) for (ASMA ,LKM and SLP). There are
many auto antibodies diagnosed at different concentration. The main concentration for
each antibody is equal to moderate level at titer (320) .Table ( 1) Show the
distribution of these antibodies with certain types. The result shows that type 1 (13 :35
= 37.2%) is the major type of AIH in the present study, type 2a ((9:35 = 25.7%) LKM
antibody without hepatitis C infection) in the second stage and in the third stage is
type 4 ((7: 35 = 20%) gave negative auto antibodies , and the proper diagnosis is done
306
Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
depending on clinical criteria and histological section). Presence of Hepatitis C with
Anti LKM antibody is belonging type 2b( 4:35 = 11.4%), LKM antibody give pin
point diagnosis for AIH , liver cell damage either directly by this antibody or
association with viral infection (Hepatitis C virus). Only 2 patients have SLP ( 2:35 =
5.7%) at type 3 AIH. (Figure 3) show the profile pictures of different types of
autoantibodies .
Table ( 1) Distribution of Auto antibodies among Different types of AIH patients.
Auto
Statistic
AIH type.
Total
Antibodies
type 1 type 2 A type 2 B type 3 Type 4
6
2
2
7
17
Negative
ANA
AMA
ASMA
LKM
SLP
Positive
Negative
Positive
Negative
Positive
Negative
Positive
Negative
Positive
13
11
2
3
10
13
13
-
3
8
1
9
9
9
-
2
4
4
4
4
-
2
2
2
2
7
7
7
7
-
The auto antibodies are not believed to be involved in the pathogenesis of
autoimmune hepatitis and are not predictive of histologic severity or treatmentresponse (Mehendiratta et al (2009). Their main role is to identify patients with
autoimmune hepatitis and thereby point the way toward appropriate therapy and form
the basis for a classification . Patients may present with autoimmune hepatitis without
circulating auto antibodies, referred to as (autoantibody negative autoimmune
hepatitis). Although infrequent, their course and response to treatment appear to be
identical to autoantibody positive patients (Czaic 2012).
307
18
32
3
23
12
22
13
33
2
Anti Mitochondrial Ab.(AMA,
Hep-20 substrate )
Specific Liver Protein Antibody
( MSK Substrate )
Antinuclear Ab.(ANA = Hep -20 )
Anti Smooth Muscle Ab.
(ASMA = MSK substrate )
Anti Liver Kidney Microsome (LKM = MSK substrate).
Figure ( 3) The profile pictures of Auto Immune Antibodies of AIH .
The panel waived the requirement of 6 months of disease activity to establish
chronicity, expanded the histologic spectrum to include lobular hepatitis, and
reaffirmed the non viral nature of the disease. The panel also designated incompatible
histologic features, such as cholestatic histology, the presence of bile duct injury, and
ductopenia. The term autoimmune hepatitis was selected to replace terms such as
autoimmune liver disease and autoimmune chronic active hepatitis. (Johnson PJ,
McFarlane, 1993). Cytokines level important to monitored the severity of damage and
whether the patients with good or bad prognosis , so that recommended to study other
different types of cytokines for Th1 ,Th2 and NK cell activators to predict of ability to
producing proinflammatory and anti-inflammatory mediators as well as ability to
treatment of such diseases .
References :1. Clement S, Pascarella S, Conzelmann S, Gonelle-Gispert C, Guilloux K, (2010) The
hepatitis C virus core protein indirectly induces alpha-smooth muscle actin expression in
hepatic stellate cells via interleukin-8. J Hepatol 52: 635–643.
2. Czaja AJ. ( 2012 ) Autoantibody-negative autoimmune hepatitis. Dig Dis Sci; 57:610.
3. Enger ,W. (2000) : The use of laboratory tests in the diagnosis of SLE. J.Clin. Pathol. 53:
424-432.Cited in Euroimmune , Medizinische labrodiagnostika AG.(2012) .E-mail
euroimmune@euroimmune.de.internet.www.euroimmune.de.
4. Frances F., Marchall B.D., (2009) . Overview of Immunodiagnostic Studies : Amanual of
Laboratory and Diagnostic Tests . 8th edd. Lippincott Williams and Wilkins . pp( 650 –
663).
308
Journal of Babylon University/Pure and Applied Sciences/ No.(1)/ Vol.(22): 2012
College of Science/Babylon University Scientific Conference
5. John. B.H. ( 2001 ) , Clinical diagnosis and management by laboratory methods , 2 nd .
edd. W.B. Sanders.
6. Johnson PJ , (1997) Treatment of Autoimmune hepatitis .Gut 41, 3-4 .
7. Johnson PJ, McFarlane IG.( 1993 ): Meeting report: International Autoimmune Hepatitis
Group. Hepatology. Oct;18(4):998-1005.
8. Krawitt EL.( 2006) Autoimmune hepatitis. N Engl J Med; 354:54.
9. Kawashima H, Kato N, Ioi H, Nishimata S, Watanabe C, Kashiwagi Y, Takekuma
K, Hoshika A, Szenborn L, Bergman K. (2008) mRNA expression of T-helper 1, T-helper
2 cytokines in autoimmune hepatitis in childhood. Pediatr Int. 2008 Jun;50(3):284-6.
10. Lerner AM , Beqaj SH , Deeter RG, Fitzgorald JT (( 2004)) . IgM serum antibodies to
Epstein Barr virus are uniquely present in a subset of patients with chronic fatigue
syndrome . Journal :in vivo , 18 (2); 101 -6 .
11. Manns MP,( 1992) Autoimmune diseases of the liver ,Clin. Lab. Med. ; 12:25-40.
12. Manns MP, Czaja AJ, Gorham JD, (2010 ) Diagnosis and management of autoimmune
hepatitis. Hepatology; 51:2193.
13. Manns MP, Strassburg CP: (2001) Autoimmune hepatitis: clinical challenges.
Gastroenterology , 120:1502-1517.
14. McFarlane IG ( 2002) Definition and classification of autoimmune hepatitis. Semin Liver
Dis , 22:317-324.
15. Mehendiratta V, Mitroo P, Bombonati A, (2009)Serologic markers do not predict
histologic severity or response to treatment in patients with autoimmune hepatitis. Clin
Gastroenterol Hepatol; 7:98.
16. Moore K.W. Ogarra A. DE. Waal –Malefyt R. Vieira P. ( 1993) Interleukins Annu. Rev.
Immunol, 11 : 165 -190 .Cited in BioSource Europe S.A. (2011 ),Rue De I Indousterie 8B-1400 .Nivelles – Belguim .
17. Tritto G, Bechlis Z, Stadlbauer V, Davies N, Frances R, (2011) Evidence of Neutrophil
Functional Defect Despite Inflammation in Stable Cirrhosis. J Hepatol.
309