cytokines are mediators of information transfer between cells , and in this way , they regulate physiological and pathological mechanisms in the body . they are potentially of use in diagnosis . as a result of its function during inflammatory processes and regeneration , interleukin-6 ( il-6 ) it is produced by lymphoid cells and by a variety of non - lymphoid cells , including endothelial cells , epithelial cells and fibroblasts ( kishimoto et al . for example , tumour necrosis factor- , interleukin-1 and catecholamines stimulate the synthesis of il-6 , whereas glucocorticoids suppress it ( papanicolaou et al . the target cells of il-6 are lymphoid cells and other cells such as hepatocytes and bone marrow cells ( barton 1997 ; papanicolaou et al . 1998 ) . il-6 acts on its target cells via different transduction pathways , including those that involve protein kinase c , camp / protein kinase a and the calcium release pathway ( kamimura et al . release of il-6 occurs in cases of infection , trauma and neoplasia , and its functions range from induction of acute - phase proteins to cell growth and differentiation ( jakab and kalabay 1998 ; barrientos et al . a role is described for il-6 , for example , in different autoimmune and inflammatory diseases . in addition , il-6 appears to have a function in different diseases of bones and liver ( linker - israeli et al . in hepatic diseases of humans , increased serum concentrations of il-6 have been found in cases of hepatitis , hepatic tumours and regeneration of the liver after different insults ( lacour et al . given its function in different disease mechanisms , il-6 could be an important parameter in clinical pathology to describe the aetiology , severity and course of diseases , and to determine the prognosis . in this study , the serum concentration of il-6 was measured in dogs with different diseases of the liver . the aim was to investigate the association of different liver diseases and the serum concentration of il-6 . furthermore , the presence of a correlation between the degree of liver disease and the serum concentration of il-6 was investigated . finally , the serum concentration of il-6 associated with hepatic disease was compared with that in cases of extra - hepatic inflammatory disease , tumours and metabolic diseases . the diagnosis of liver disease was based on the anamnesis , the clinical investigation , clinical pathological examination [ alanine transferase ( alt ) ; aspartate transferase ( ast ) ; glutamate dehydrogenase ( gldh ) ; alkaline phosphatase ( alp ) ; fasting bile acids ( ba ) ; bilirubin ( bili ) and albumin ( alb ) ] , ultrasound investigation and histopathological diagnosis from a biopsy . the definitive diagnosis was based on the histological investigation , and the disease was classified according to the recommendations of the world small animal veterinary association ( wsava liver standardization group 2006 ) . the il-6 values of these dogs were compared with those of 18 clinically healthy dogs . these dogs were presented for vaccination and other routine examinations and had no abnormalities in anamnesis , clinical investigation , clinical biochemistry ( alt , ast , gldh , alp , ba , alb , bili , creatinine , urea , phosphorus , calcium and glucose ) and haematology ( e.g. white blood cells , packed cell volume ) . furthermore , no abnormalities were found in the liver during ultrasound investigation of these dogs . both groups were compared with a group of 30 dogs with extra - hepatic diseases . ten dogs of this group had diabetes mellitus , ten dogs had extra - hepatic inflammation and ten dogs had an extra - hepatic tumour . blood samples were collected from the cephalic vein with a 22-g needle and were harvested in a plastic serum tube . the samples were kept at room temperature for 15  min to allow them to clot and were then centrifuged ( 10  min , 3,000g ) . analysis of alt , ast , gldh , alp , fasting ba , bili and alb was performed according to standardised procedures using an analyser ( konelab , thermo fisher scientific ) and commercial kits . the serum samples were stored for no longer than 3  months before measurements were made . the concentration of il-6 was measured using the canine il-6 duoset elisa ( catalogue number dy1609 ; r&d systems , inc . ) . the analysis included plate preparation and assay procedure and was performed according to the manufacturer s elisa protocol . finally , the microtitre plate was read using a microtitre reader ( slt spectra , tecan ) at 450  nm ( correction wavelength 540  nm ) . the livers were examined for size , shape and structure . using a biopsy needle ( true - cut , surgivet ) and guided by ultrasound , the dogs were investigated under general anaesthesia ( levomethadone , l - polamivet , intervet ; xylazine , rompun , bayer ; isoflurane , isoflurane , abbott ) . the histopathological diagnosis corresponded to the recommendations of the wsava liver standardization group ( 2006 ) . group 1 comprised clinically healthy dogs ; group 2 , dogs with acute hepatitis ; group 3 , chronic hepatitis of mild degree ; group 4 , chronic hepatitis of moderate degree ; group 5 , liver tumours ; group 5a , primary liver tumours ; group 5b , secondary liver tumours ; group 6 , extra - hepatic inflammation ; group 7 , extra - hepatic tumours and group 8 , diabetes mellitus . the statistical analysis was performed with the programme sas system , univariate procedure , version 8.1 ( sas institute inc . ) . the level of significance was p  <  0.05 . the precision of the il-6 elisa was determined using three serum samples on which ten consecutive runs were performed during a single day . the day - to - day precision was determined using three serum samples that were evaluated once daily for 5  days . the diagnosis of liver disease was based on the anamnesis , the clinical investigation , clinical pathological examination [ alanine transferase ( alt ) ; aspartate transferase ( ast ) ; glutamate dehydrogenase ( gldh ) ; alkaline phosphatase ( alp ) ; fasting bile acids ( ba ) ; bilirubin ( bili ) and albumin ( alb ) ] , ultrasound investigation and histopathological diagnosis from a biopsy . the definitive diagnosis was based on the histological investigation , and the disease was classified according to the recommendations of the world small animal veterinary association ( wsava liver standardization group 2006 ) . the il-6 values of these dogs were compared with those of 18 clinically healthy dogs . these dogs were presented for vaccination and other routine examinations and had no abnormalities in anamnesis , clinical investigation , clinical biochemistry ( alt , ast , gldh , alp , ba , alb , bili , creatinine , urea , phosphorus , calcium and glucose ) and haematology ( e.g. white blood cells , packed cell volume ) . furthermore , no abnormalities were found in the liver during ultrasound investigation of these dogs . both groups were compared with a group of 30 dogs with extra - hepatic diseases . ten dogs of this group had diabetes mellitus , ten dogs had extra - hepatic inflammation and ten dogs had an extra - hepatic tumour . blood samples were collected from the cephalic vein with a 22-g needle and were harvested in a plastic serum tube . the samples were kept at room temperature for 15  min to allow them to clot and were then centrifuged ( 10  min , 3,000g ) . analysis of alt , ast , gldh , alp , fasting ba , bili and alb was performed according to standardised procedures using an analyser ( konelab , thermo fisher scientific ) and commercial kits . the serum samples were stored for no longer than 3  months before measurements were made . the concentration of il-6 was measured using the canine il-6 duoset elisa ( catalogue number dy1609 ; r&d systems , inc . ) . the analysis included plate preparation and assay procedure and was performed according to the manufacturer s elisa protocol . finally , the microtitre plate was read using a microtitre reader ( slt spectra , tecan ) at 450  nm ( correction wavelength 540  nm ) . the livers were examined for size , shape and structure . using a biopsy needle ( true - cut , surgivet ) and guided by ultrasound , three to four tissue samples were taken from each structural lesion for further examination . the tissue core specimens were 0.51.0  cm long and 2  mm thick . in some cases the dogs were investigated under general anaesthesia ( levomethadone , l - polamivet , intervet ; xylazine , rompun , bayer ; isoflurane , isoflurane , abbott ) . the histopathological diagnosis corresponded to the recommendations of the wsava liver standardization group ( 2006 ) . group 1 comprised clinically healthy dogs ; group 2 , dogs with acute hepatitis ; group 3 , chronic hepatitis of mild degree ; group 4 , chronic hepatitis of moderate degree ; group 5 , liver tumours ; group 5a , primary liver tumours ; group 5b , secondary liver tumours ; group 6 , extra - hepatic inflammation ; group 7 , extra - hepatic tumours and group 8 , diabetes mellitus . the statistical analysis was performed with the programme sas system , univariate procedure , version 8.1 ( sas institute inc . ) . the level of significance was p  <  0.05 . the precision of the il-6 elisa was determined using three serum samples on which ten consecutive runs were performed during a single day . the day - to - day precision was determined using three serum samples that were evaluated once daily for 5  days . the dogs with liver disease were divided into five groups , depending on their histopathological diagnoses . six dogs had acute hepatitis , 13 dogs had chronic hepatitis and 16 dogs had a liver tumour . the six dogs with acute hepatitis could be divided into three cases of acute purulent hepatitis , one case of hepatitis associated with adenovirus infection , one case associated with parvovirus infection and one case associated with leptospirosis . the serum concentrations of il-6 in this group varied between 1 and 202  pg / ml , with an average of 102  pg / ml ( fig .  1 ) . median , maximum and first and third quartiles of il-6 serum concentrations in groups 1 to 8 ( group 1  =  healthy controls , group 2  =  acute hepatitis , group 3  =  mild chronic hepatitis , group 4  =  moderate chronic hepatitis , group 5  =  liver tumours [ group 5a  =  primary liver tumours , group 5b  =  secondary liver tumours ] , group 6  =  extra - hepatic inflammation , group 7  =  extra - hepatic tumours and group 8  =  diabetes mellitus ) minimum , median , maximum and first and third quartiles of il-6 serum concentrations in groups 1 to 8 ( group 1  =  healthy controls , group 2  =  acute hepatitis , group 3  =  mild chronic hepatitis , group 4  =  moderate chronic hepatitis , group 5  =  liver tumours [ group 5a  =  primary liver tumours , group 5b  =  secondary liver tumours ] , group 6  =  extra - hepatic inflammation , group 7  =  extra - hepatic tumours and group 8  =  diabetes mellitus ) the group with chronic hepatitis consisted of 13 dogs , of which eight had a mild degree of hepatitis and five had a moderate degree . reactive hepatitis , as a reaction of an extra - hepatic disease , was excluded by histopathology ; those dogs were not considered in this study . the average concentration in this group was 25  pg / ml ( fig .  1 ) . in addition , two cases of haemangiosarcoma , two cases of fibrosarcoma and three cases of lymphoma were also included in the study . the range of il-6 concentrations in this group was from 3 to180  pg / ml , with an average concentration of 58  pg / ml ( fig .  2 ) . the average concentration of il-6 in dogs with a primary liver tumour ( hepatocellular carcinoma , cholangiocellular carcinoma ) was 31  pg / ml and that of dogs with secondary liver tumours ( lymphoma , haemangiosarcoma , fibrosarcoma ) was 91  pg / ml ( fig .  3 ) . median , maximum and first and third quartiles of il-6 serum concentrations in groups 2 to 5 . median , maximum and first and third quartiles of il-6 serum concentrations in groups 5a and 5b . significant differences are indicated by an asterisk minimum , median , maximum and first and third quartiles of il-6 serum concentrations in groups 2 to 5 . significant differences are indicated by asterisks minimum , median , maximum and first and third quartiles of il-6 serum concentrations in groups 5a and 5b . significant differences are indicated by an asterisk we compared these data between the liver disease groups and also compared them with data from healthy controls . the serum concentrations of il-6 in dogs with different liver diseases were highly significantly ( p  <  0.001 ) increased compared with those of clinically healthy dogs . the difference between acute and chronic hepatitis was also significant ( p  <  0.05 ) . on the other hand , there was no significant difference between dogs with mild and moderate chronic hepatitis ( fig .  2 ) . the serum concentrations of il-6 in dogs with different liver tumours were highly significantly increased compared with those of healthy dogs ( p  <  0.001 ; fig .  1 ) . dogs with secondary liver tumours had significantly higher serum concentrations of il-6 than dogs with primary liver tumours ( p  <  0.01 ; fig .  3 ) . there was no significant difference between dogs with acute hepatitis and dogs with primary and secondary liver tumours ( p  >  0.05 ; fig . 2 ) ; however , the difference between dogs with chronic hepatitis and those with secondary liver tumours was highly significant ( p  <  0.001 ) . to detect differences in the serum concentration of il-6 between dogs with intra- and extra - hepatic inflammatory diseases or tumours , we compared the dogs with liver disease in this study with dogs that had extra - hepatic diseases . this group of dogs included ten dogs with an extra - hepatic inflammatory process ; two dogs in this group had prostatitis and eight had pyometra . a further ten dogs with an extra - hepatic tumour were included in this comparison , comprising five cases of mammary carcinoma , three cases of splenic haemangiosarcoma , one case of bronchial carcinoma and one case of carcinoma of the stomach . finally , all data were compared with those from a group of ten dogs with diabetes mellitus that had no inflammatory or neoplastic disease . no significant difference could be found between dogs with acute hepatitis and those with extra - hepatic inflammation ( p  >  0.05 ) ; in contrast , the serum concentrations of il-6 in dogs with chronic hepatitis were significantly lower than those in dogs with extra - hepatic inflammation ( p  <  0.01 ) . the serum concentration of il-6 in dogs with intra- or extra - hepatic tumours showed no significant difference ( p  >  0.05 ) . however , dogs with an extra - hepatic inflammation or tumour had highly significantly increased serum concentration of il-6 compared with dogs with diabetes mellitus ( p  <  0.001 ; fig .  1 ) . finally the intra- and inter - assay precision of the il-6 assay was 2.9% and 5.9% , respectively . the measurement of cytokines in diseased dogs is a new field in clinical pathology . given their function as mediators , cytokines can help to identify disease mechanisms , to describe the course of disease and to assess prognosis . this study focused on il-6 in dogs with liver disease because il-6 has been shown to be critical for the acute - phase response , for protection against hepatic injury and for liver regeneration ( taub 2005 ; berasain et al . no healthy dog was found with a serum concentration above 1  pg / ml . unfortunately , there is no study in which a reference interval for il-6 in dogs has been calculated . however , in human studies with healthy controls , comparable serum concentrations of about 1  pg / ml were reported ( benoy et al . 2008 ; salamon et al . 2008 ) . given this very low reference range , the elevations of il-6 associated with different diseases can be recognised easily . the results of this study showed that liver diseases of different aetiologies were associated with elevated serum concentrations of il-6 . this is in agreement with the results of investigations in humans , which have involved measurement of il-6 in the serum of patients with different neoplastic diseases , including metastases , and those with non - neoplastic liver diseases . in both types of diseases , the serum concentrations of il-6 are increased compared with those of controls ( matzaraki et al . 2007 ; the serum concentration of il-6 in cases of acute hepatitis is significantly higher than that in chronic hepatitis . the release of different cytokines , including il-6 , indicates the early beginning of an inflammatory process ( eklund 2009 ) . measurement of a significant difference in the level of il-6 between acute and chronic liver disease may help to differentiate between these processes . the results of this study show that il-6 could be helpful for identification of acute hepatitis in dogs ; however , the number of dogs with acute hepatitis in this study was low , so further studies are recommended with a larger number of patients . unfortunately , there was no significant difference in the level of il-6 between patients with mild and moderate chronic hepatitis . in human patients with chronic hepatitis , the serum concentration of il-6 correlates with the degree of fibrosis ( migita et al . given that all the dogs with chronic hepatitis in this study had a low degree of inflammation and no or only mild fibrosis , dogs with severe chronic hepatitis and fibrosis should be observed in further studies to investigate the correlation between the serum concentration of il-6 and the degree of hepatitis . all dogs with hepatitis in this study , both acute and chronic , had comparable serum concentrations of il-6 to dogs with inflammatory diseases outside the liver , such as prostatitis and pyometra . the concentration of il-6 in dogs with chronic hepatitis was lower than in the control group with extra - hepatic inflammatory processes . one reason for this could be a lower degree of inflammation in the group with chronic hepatitis . the dogs with liver tumours in this study also had elevated serum concentrations of il-6 . the levels were comparable to those in cases of acute hepatitis but higher than in cases of chronic hepatitis . an explanation for this could be the acute initiation of inflammation by mediators in hepatic tumours and the resulting destruction of the liver parenchyma . it could also be caused by attempts of the liver to regenerate its tissue during neoplastic alteration . elevation of il-6 has also been reported in humans with liver tumours of different aetiologies ( goydos et al . studies of liver tumours and serum concentrations of il-6 in dogs have not been published previously . it is interesting that the concentrations of il-6 in dogs with secondary liver tumours were significantly higher than those in dogs with primary liver tumours . in cases of lymphoma , the release of il-6 from lymphoid tumour cells could explain this observation . in humans , serum concentrations of il-6 are increased in patients with lymphoma , and the levels fall during chemotherapy ( serebriakov et al . 1998 ) . to confirm that non - inflammatory and non - neoplastic diseases have little influence on the serum concentration of il-6 , we measured il-6 in cases of diabetes mellitus and compared these results with those from all cases of inflammation or tumour . like the healthy controls , the dogs with diabetes mellitus have very low concentrations of il-6 in the serum . it can be concluded that the absence of inflammatory processes is associated with a lack of il-6 activation . on the other hand , in humans , secondary inflammation associated with diabetes mellitus can induce elevations of il-6 ( goldberg 2009 ) . this observation requires investigation in further studies . in conclusion , the results of our study show that il-6 is measurable in dogs and that diseases of the liver are associated with elevations of this cytokine . differentiation between acute and chronic hepatitis may be possible , but the assessment of the degree of hepatitis is not possible in milder cases . dogs with liver tumours of different aetiologies have elevated serum concentrations of il-6 , and those with secondary liver tumours showed significantly higher levels than those with primary tumours or inflammatory diseases . the reason for this is not clearly understood , but production of il-6 by the tumour cells could be one explanation . further studies of il-6 and its involvement in liver disease in dogs are necessary to investigate the prognostic use of il-6 in patients with liver diseases .
cytokines are part of pathogenesis in many diseases 
 . their measurement could be interesting for diagnostic purposes . 
 one cytokine which participates in different inflammatory and neoplastic diseases is interleukin-6 ( il-6 ) . 
 the aim of this study was to investigate the il-6 serum concentration in dogs with different liver diseases to show if there is any association between the cytokine serum level and the disease aetiology or the degree of the disease . 
 il-6 was measured in dogs with acute hepatitis , chronic hepatitis of different degrees and primary and secondary liver tumours . 
 the data were compared with clinically healthy dogs and dogs with extra - hepatic diseases . for measurement , a commercial elisa kit ( r&d systems ) was used . 
 compared with clinically healthy dogs and dogs with diabetes mellitus , all dogs with an intra- or extra - hepatic inflammatory or neoplastic disease have increased serum levels of il-6 . dogs with acute hepatitis have significantly increased il-6 serum concentrations compared with dogs with chronic hepatitis ( p  <  0.05 ) . 
 no significant difference between mild and moderate chronic hepatitis exists ( p  >  0.05 ) . 
 dogs with secondary liver tumours have significantly increased il-6 serum concentrations in comparison to dogs with primary liver tumours ( p  <  0.01 ) , but both groups have comparable il-6 serum concentration to dogs with extra - hepatic tumours . 
 measurement of il-6 serum concentration may help differentiate between acute and chronic hepatitis and between primary and secondary liver tumours . 
 further information about the aetiology of the liver disease can not be obtained by measuring il-6 in the serum .