Unravelling fatty liver haemorrhagic syndrome: 2. Inflammation and pathophysiology.

To study the role of inflammation in the pathophysiology of the fatty liver haemorrhagic syndrome (FLHS), mature laying hens were treated with oestrogen (ß-oestradiol-17-dipropionate or E2) and challenged with lipopolysaccharide (LPS). Oestrogen injections induced FLHS, but the incidence and severity of the condition was increased with a combination of E2 & LPS. Hepatic mRNA levels of the genes encoding key regulators of inflammation, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interleukin-18 (IL-18), were evaluated. The expression of IL-6 mRNA in hepatocytes of all treated groups (E2, LPS and E2 & LPS hens) was elevated from 6-fold to 56-fold (P < 0.01), when compared to baseline and control values, with the highest fold change at 3 h post-treatment. The mRNA levels for IL-1ß were better expressed at 24 h post-treatments with E2, LPS and E2 & LPS. The expression of IL-18 mRNA in the liver tissue was lower than IL-1ß and IL-6 mRNA in all treated birds. At 24 h post-treatment, total white blood cell (WBC) counts and fibrinogen levels were elevated (P < 0.05) in E2-, LPS- and E2- & LPS-treated hens. Histologically, livers of hens from E2- and E2- & LPS-treated groups revealed inflammatory alterations with areas showing mononuclear aggregations, vacuolar fatty degeneration of hepatocytes, and increased sinusoidal congestion and haemorrhages. It was concluded that liver lipid accumulation and injury were associated with incidences of local (hepatic) and systemic inflammation, which could have initiated liver blood vessel and capsule rupture and, subsequently, the onset of FLHS.

Unravelling fatty liver haemorrhagic syndrome: 1. Oestrogen and inflammation.

Previous studies have implicated oestrogen as a factor in the induction of fatty liver haemorrhagic syndrome (FLHS). In this study, a refined laying hen model was employed to permit further investigations. Intramuscular (i.m.) injections of exogenous oestrogen as ß-estradiol-17-dipropionate (E2) (5 mg/kg BW) were given every 4 days for 20 days to 30-week-old hens fed either ad libitum or with restricted feed intake. Elevated (P < 0.01) plasma oestrogen concentrations produced significant macroscopic and microscopic hepatic alterations. Hens in the E2-treated ad libitum fed (EAL) group experienced a higher incidence of FLHS than hens in the E2-treated restricted feed intake group, showing that birds with a higher feed intake are more at risk of developing FLHS. Histological examination of livers revealed that hens in the E2-treated ad libitum fed group had consistent and severe fat infiltration in the liver, and fat vacuolization within hepatocytes. Fat accumulation and fat droplets were found not only in the cytoplasm of hepatocytes but also in liver sinusoids. White blood cell counts and fibrinogen concentrations were altered (P < 0.01) in hens treated with E2 when compared with controls. Plasma fibrinogen concentrations were altered over time, and correlated with white blood cell counts (Pearson's correlation r = 0.96; P = 0.001). Hens treated with E2 had increased (P < 0.01) levels of cholesterol and triglycerides, confirming that E2 induced hypercholesterolaemia and hypertriglyceridaemia. It was concluded that E2 successfully induced FLHS in hens, with typical systemic and hepatic events resulting from a disturbance in lipid metabolism and chronic low-grade inflammation.

Cytokine and chemokine gene expression profiles in heterophils from chickens treated with corticosterone.

In chickens, corticosterone is the end-product of stress. However, the nature of the immune response to elevated plasma corticosterone concentrations at the molecular level has not yet been characterised. We recently demonstrated that exposure to corticosterone in drinking water for 1 week significantly upregulates mRNA expression levels for the pro-inflammatory interleukins (IL)-1beta, IL-6, IL-18 and the pro-inflammatory chemokine CCLi2 in chicken lymphocytes, particularly 3 h after the treatment started. In the present study, we investigated cytokine and chemokine mRNA expression levels in circulating heterophils of chickens, and show that at 3 h post initial treatment with corticosterone in drinking water (20 mg/1L) the mRNA expression levels for IL-1beta, IL-6, IL-10, IL-12alpha and IL-18 are upregulated. The mRNA expression levels for IL-6, IL-10 and IL-18 correlate with plasma corticosterone concentration and total heterophil counts. Corticosterone downregulated the expression levels of all pro-inflammatory cytokines at 24 h and 1 week post-treatments. Repeated treatment with corticosterone upregulated mRNA expression levels of transforming growth factor-beta4 and the chemokine CCL16. These data indicate that cytokine and chemokine gene expression signatures in chicken heterophils can be altered during stress and therefore could be used as an indicator of stress.

Prospects for understanding immune-endocrine interactions in the chicken.

Despite occupying the same habitats as mammals, having similar ranges of body mass and longevity, and facing similar pathogen challenges, birds have a different repertoire of organs, cells, molecules and genes of the immune system when compared to mammals. In other words, birds are not "mice with feathers", at least not in terms of their immune systems. Here we discuss differences between immune gene repertoires of birds and mammals, particularly those known to play a role in immune-endocrine interactions in mammals. If we are to begin to understand immune-endocrine interactions in the chicken, we need to understand these repertoires and also the biological function of the proteins encoded by these genes. We also discuss developments in our ability to understand the function of dendritic cells in the chicken; the function of these professional antigen-presenting cells is affected by stress in mammals. With regard to the endocrine system, we describe relevant chicken pituitary-adrenal hormones, and review recent findings on the expression of their receptors, as these receptors play a crucial role in modulating immune-endocrine interactions. Finally, we review the (albeit limited) work that has been carried out to understand immune-endocrine interactions in the chicken in the post-genome era.

Differential alterations in ultrastructural morphology of chicken heterophils and lymphocytes induced by corticosterone and lipopolysaccharide.

Birds are continuously confronted by a large number of stressors, including pathogens. Despite their variety, all stressors induce an elevation in plasma corticosterone concentration, and consequently increase heterophil to leukocyte (H/L) ratio. In order to evaluate and differentiate effects of endocrine (non-bacterial) and bacterial stress on the proportions and ultrastructural characteristics of chicken leukocytes, a series of experiments were conducted with seven-week old chickens exposed either to dietary corticosterone or to intravenous (i.v.)-injected lipopolysaccharide (LPS). Samples were taken for haematological, endocrine, and electron microscopy examination. Administration of corticosterone and LPS significantly elevated plasma corticosterone concentrations and increased H/L ratios. Electron microscopy observations indicated changes in heterophil size, shape, and granulation, and lymphocyte cytoplasmic characteristics. Immature heterophils were observed in the peripheral blood, suggesting that corticosterone and LPS both stimulate an earlier release of heterophils from bone marrow and enhance their influx into blood circulation. The LPS induced a degenerative morphology and the destruction of lymphocytes, whereas corticosterone appeared to stimulate their redistribution rather than destruction. The results indicate that exposure to corticosterone or LPS similarly increase H/L ratios, but differentially alter the ultrastructure of heterophils and lymphocytes. Elucidation of the mechanisms that cause such changes may play an important role in distinguishing between a nonimmune and immune stress challenge at the molecular level.

Biological response of chickens (Gallus gallus domesticus) induced by corticosterone and a bacterial endotoxin.

Experiments were conducted with chickens exposed to corticosterone and lipopolysaccharide (LPS) from Escherichia coli, with the aim of evaluating and differentiating their effects on endocrine, metabolic and immune response. Both, corticosterone and LPS significantly elevated plasma corticosterone concentrations and increased heterophil to lymphocyte (H/L) ratios 1 h, 3 h and 24 h post-treatments. Repeated exposure to corticosterone caused a prolonged elevation of plasma corticosterone concentration and H/L ratio. Data on blood metabolites demonstrated that corticosterone stimulated hyperglycaemia, hypercholesterolemia and hypertriglyceridemia. In contrast, LPS induced hypocholesterolemia and hypotriglyceridemia at 24 h post-injection. Weight gain and relative weight of the spleen and bursa were reduced in chickens treated with corticosterone. The LPS did not show any significant effect on weekly weight gain, but stimulated an increase in the relative weight of the spleen. Corticosterone initially stimulated antibody responsiveness to infectious bronchitis virus (IBV) vaccination, but thereafter the titres decreased. This was in contrast to LPS which depressed the antibody titre to IBV vaccination. It was concluded that the biological response of chickens induced by corticosterone could be differed from the response to LPS. The major difference occurred in metabolic, growth and immune activities. It appears that, both corticosterone and LPS differently alter physiological, metabolic and immunological responses of chickens through an activation of different molecular components (cytokines and chemokines) and neuroendocrine-immune network systems.