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

https://doi.org/10.1016/j.vetimm.2007.10.009Get rights and content

Abstract

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.

Introduction

In chickens, as in mammals, absolute numbers and relative proportions of circulating leukocytes are important indicators of the state of the immune system, and are also representative of the pattern of leukocyte distribution in the body (Latimer and Bienzle, 2000). Additionally, identification of the morphological profiles of leukocytes is an important tool in understanding their response to various challenges, including stress (Maxwell and Robertson, 1998, Latimer and Bienzle, 2000). Similar to mammalian immune cells, lymphoid (B and T lymphocytes) and non-lymphoid (mononuclear phagocytes and granulocytes) cells, provide to the bird specific and non-specific immunological defences respectively. The avian heterophil is the predominant polymorphonuclear leukocyte and the main granulocyte leukocyte that mediates acute inflammation (Latimer et al., 1988, Harmon, 1998) and functions as a phagocyte against bacterial infection (Powell, 1987, Kogut et al., 1998). Lymphocytes respond to a variety of stimuli and move to secondary lymphoid organs where antigen presentation may take place (Powell, 1987).

There is evidence in chickens that bacterial stressors cause an elevation in plasma corticosterone concentrations (Trout et al., 1996, Nakamura et al., 1998) and increased H/L ratio, due to leukopenia (lymphopenia) and heterophilia (Harmon, 1998). Interestingly, an increase in H/L ratio characterises stress in birds (Davison and Flack, 1981, Gross and Siegel, 1983) caused by a variety of environmental factors, such as feed or water deprivation, malnutrition, injury, extreme temperatures, poor housing, overcrowding, and exposure to stress hormones introduced parenterally or in the diet (Thaxton and Siegel, 1970, McFarlane and Curtis, 1989, Maxwell et al., 1992, Thaxton and Puvadolpirod, 2000, Post et al., 2003, Mumma et al., 2006). The available evidence suggests that chickens respond to environmental stressors with a large number of behavioural, hormonal and physiological changes. However, comparative ultrastructural alterations of chicken leukocytes induced by corticosterone and LPS have not been documented before. Information concerning the morphological alterations in chicken heterophils and lymphocytes that accompany an increase in the H/L ratio is lacking.

Our recent studies attempted to explain and differentiate the effects of corticosterone and LPS on chicken leukocytes (Shini, 2006, Shini et al., 2007). We conducted a series of experiments and observed that, although both corticosterone and bacterial endotoxin increased the proportion of heterophils to lymphocytes, the mechanism that operates behind these changes remains to be explained. In particular, examination of the ultrastructural morphology of these cells could facilitate studies on the effects of corticosterone on the host at the molecular level (Shini et al., 2007). In the present paper, we report the effects of dietary corticosterone and LPS on the proportions and ultrastructural characteristics of chicken heterophils and lymphocytes with the aim of elucidating and differentiating morphological changes that are linked to an increase in H/L ratio in corticosterone- and LPS-stressed birds.

Section snippets

Birds and housing

The experiments were conducted with female chicks, selected at random from a single flock of commercial Hy-Line brown laying hens. Three weeks before the experiments began, birds were tagged and placed in an environmentally controlled house, in stainless steel batteries, each holding six birds. The light regimen was 17 h of light (05.00–22.00 h), and temperature was kept between 22 ± 2 °C during the experimental period. A standard starter diet, and water was provided ad libitum. To facilitate

Results

The effects of corticosterone and LPS on plasma corticosterone concentrations and H/L ratios (mean ± SEM) are presented in Fig. 1, Fig. 2, respectively. Baseline corticosterone concentrations did not differ among treatment groups. In corticosterone-treated birds, there was a significant increase in plasma corticosterone concentrations 1 h after the treatment began, and remained elevated throughout the study when compared to control and LPS-treated chickens (Fig. 1). Corticosterone increased (p < 

Discussion

It has been known for many years that immune cells are influenced by glucocorticoids induced in response to pathogens and environmental stressors. In chickens, the major stress hormone is corticosterone (Harvey et al., 1984, Siegel, 1995). Corticosterone is essential for an adequate physiological and behavioural response to acute stressful events. If the stress response is inadequate or excessive and/or prolonged, birds may survive but suffer adverse consequences in growth, metabolism,

Conclusion

This study demonstrated that both corticosterone and LPS caused an elevation in plasma corticosterone concentrations and activated immune cell response systems. When chickens were exposed to corticosterone, plasma corticosterone levels increased which in turn upregulated immune cell proportions in peripheral blood and bone marrow, and altered their morphology. Exposure to LPS mediated a wide variety of physiological, immunological, and behavioural changes, probably through the stimulation of

Acknowledgments

We thank Rick Webb, Robin Webb and Rob Gould from the University of Queensland Centre for Microscopy and Microanalysis for their technical assistance. SS is the recipient of a postdoctoral fellowship from the University of Queensland. PK is supported by the Biotechnology and Biological Sciences Research Council, UK.

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