Effects of 6-hydroxydopamine on the development of the immune system in chickens.

It has been suggested that the sympathetic nervous system communicates with lymphocytes expressing cell surface receptors for neurotransmitters such as norepinephrine (NE), on the basis of the finding that neurotransmitters modify immune responses in mammalian species. We confirmed that chicken lymphocytes in the brusa of Fabricius, thymus and spleen expressed beta-adrenergic receptor (beta-AR) mRNA from embryonic day (E) 10 and that intracellular cAMP level was elevated by NE, suggesting that lymphocytes express functional beta-AR on their surface at an early embryonal stage. To clarify whether the nervous system is involved in the development of the immune system, the effects of 6-hydroxydopamine (6-OHDA), one of sympathectomizing agents, on chicken lymphocytes was investigated. A single injection of 6-OHDA at a dose of 400 microg into a chicken embryo was carried out at E7 or 14 (as referred to E7 group and E14 group, respectively). NE level and the relative proportion of Bu-1a(+), CD4(+) and CD8(+) cells in the spleen of 3-week-old chickens were not altered by 6-OHDA treatment. However, the proliferative responses and expression of IL-2 mRNA in spleen cells cultured with pokeweed mitogen were reduced in E7 group compared with those of control. Furthermore, in CD8(+) spleen cells of E14 group of 3-week-old chickens, the expression of beta-AR mRNA and the relative increase of intracellular cAMP stimulated with NE were significantly decreased. These results suggest that the sympathetic nervous system affects the development of the immune system.

Histochemical and immunohistochemical localisation of nitrergic neuronal and non-neuronal cells in the bursa of Fabricius of the chicken.

The bursa of Fabricius of 24 chickens, 2-4 weeks old, was investigated to determine the distribution of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) staining and nitric oxide synthase (NOS) antigen, by use of NADPH-d histochemistry and NOS immunohistochemistry, respectively. NADPH-d reaction product was localised in neuronal cell bodies and nerve fibres. The stained cell bodies were predominantly in the different bursal compartments; some occurred in the wall and between the lymphoid follicles, and some in the immediate vicinity of blood vessels. Stained nerve fibres travelled mostly with blood vessels, but many were also observed in the connective tissue of the bursa, independent of blood vessels, and some in contact with the lymphoid follicle and interfollicular epithelium. In addition to neuronal profiles, the interfollicular epithelium of the plicae, and the vascular endothelium were densely stained, and lymphocytes were moderately labelled. NOS immunoreactivity was found in neuron-like cells and fibres which were confirmed to be neuronal in adjacent sections stained with antibodies raised against neuron-specific enolase. It was also detected in interfollicular epithelium and lymphocytes but not in vascular endothelium. The distribution patterns of NADPH-d and NOS suggest that nitric oxide (NO) may play a role in the regulation of the secretory activity of interfollicular epithelium and the blood flow through the bursa.

Age-related changes of the noradrenergic and acetylcholinesterase reactive nerve fibres innervating the pigeon bursa of Fabricius.

Age-dependent changes in the innervation of the pigeon (Columba livia, L.) bursa of Fabricius, from hatching to 120 days of age, were studied by fluorescence-histochemical and neurochemical methods for demonstrating noradrenergic and acetylcholinesterase (AChE)-reactive nerve fibres respectively. The distribution of both nerve fibre types was largely perivascular. Furthermore, a few isolated nerve fiber profiles were observed beneath the bursal epithelium, in the interfollicular septa and in the follicular cortex. No nerve fibre profiles reaching the medulla of the lymphoid follicles were observed. In addition to nerve fibres, AChE reactive neuron-like cells were encountered within the capsule and interfollicular septa. AChE reactivity was also found in dendritic-like cells localized in the cortical and cortico-medullary border. No changes in the density of perivascular noradrenergic innervation were noticeable during the ages studied, whereas the density of AChE-reactive fibres supplying vessels reached the adult pattern at 30 days, and then remained unvaried. The density of non-perivascular nerve fiber profiles, specially the AChE reactive type, increased until 30 days, remained unchanged until 75 days and then increased with aging (90-120 days). The interrelationship between the autonomic nervous system and the immune system is discussed.

The neuro-B cell link of peptidergic innervation in the Bursa Fabricii.

The Bursa Fabricii, restricted to birds, specifically provides the microenvironment for B-cell maturation. The presence of nerve fibers containing immunopotent neuropeptides in immune organs opens interesting perspectives on the understanding of neuroimmune communication. As an organ for the development of only B-lymphocytes is not known in mammals, the contribution of a peptidergic innervation to the microenvironment of B-cells is not known. Therefore, we studied the peptidergic innervation of the Bursa Fabricii as an organ of B-cell maturation. Four different neuropeptides were found in nerve fibers of the Bursa Fabricii: tachykinins (TK), vasoactive intestinal peptide (VIP), galanin (GAL), and calcitonin gene-related peptide (CGRP). All these neuropeptides were distributed throughout the different bursal compartments, with the exception of the medulla of the bursal follicles, where the early stages of lymphocyte maturation are found. Double immunofluorescent studies revealed three different fiber populations: One containing coexisting VIP and GAL, the second TK and CGRP, and the last TK and no CGRP. Some of the TK-ir fibers may contain coexisting VIP. As demonstrated by double labeling for neuropeptides and lymphocytes, these heterologous fiber populations were found adjacent to B-cells in the follicle cortex. In addition, VIP-ir fibers were seen in association with macrophages. The origins of the fiber populations and the possible functional implications of our findings are discussed.

A contribution to the study of lymphopoiesis in the bursa of Fabricius in Gallus domesticus.

It has been proposed that the anatomical connection between the bursa of Fabricius and the cloaca is the pathway for unknown intestinal factors which are necessary for the induction of normal bursal lymphopoiesis. It has also been suggested that normal lymphopoiesis occurs only if nerve and vascular connections are intact. Experiments were performed to test these hypotheses. To test the influence of the intestinal contents, the bursal stalk was cut on the 16th day of incubation or at hatching. In this way, contact between the bursa and the intestinal flow in embryos was avoided and bacterial contamination of the bursa at hatching was also avoided. No change in the bursal follicles was observed. To study the influence of the nervous system on bursal lymphopoiesis, the bursa was isolated from the cloaca at hatching, and by maintaining vascularization the bursa was sutured to the peritoneum of the abdominal wall after scratching the contact surfaces. Once a new vascular network was established, one fragment of bursa was completely isolated from its normal anatomical site, causing interruption of the blood vessels and nerves of the bursa. The histological appearance of the bursa was not changed. It would appear that the integrity of the anatomical relation between bursa and cloaca and an intact nerve supply is not necessary for normal lymphopoiesis to occur in the bursa of Fabricius. In contrast, sufficient vascularization appears to be essential.