Cross-talk between neural and immune receptors provides a potential mechanism of homeostatic regulation in the gut mucosa.

The relationship between elements of the immune system and the nervous system in the presence of bacteria has been addressed recently. In particular, the sensory vanilloid receptor 1 (transient receptor potential cation channel subfamily V member 1 (TRPV1)) and the neuropeptide calcitonin gene-related peptide (CGRP) have been found to modulate cytokine response to lipopolysaccharide (LPS) independently of adaptive immunity. In this review we discuss mucosal homeostasis in the gastrointestinal tract where bacterial concentration is high. We propose that the Gram-negative bacterial receptor Toll-like receptor 4 (TLR4) can activate TRPV1 via intracellular signaling, and thereby induce the subsequent release of anti-inflammatory CGRP to maintain mucosal homeostasis.

Study of the motor corticospinal system in the developing rat fetus: comparison of Wistar and normal and hydrocephalic HTx rats.

The motor corticospinal system can be identified from day E14 in Wistar and HTx fetuses. There are no significant anatomical differences between the two species of rats. In addition, in day E17 Wistar and HTx fetuses cell counts in the cortical mantle (cortical plate, intermediate zone and germinal matrix) are similar. However, in day E20 fetuses there are significant differences in the number of cells in the cortical mantle of the hydrocephalic HTx fetuses compared to that in the Wistar and normal HTx fetuses, their total number of cells being reduced compared to that of the normal HTx and Wistars. Breakdown of the numbers of cells in the different layers shows that in the hydrocephalics there is a significant reduction in the number of cells in the germinal matrix and intermediate zone but, although the number of cells is also reduced in the cortical plate, the reduction is not significant. Measurements of the anterior/posterior width of the pyramid show that its growth is almost complete by day E17 and that on day E20 the measurements are similar in Wistar and normal and hydrocephalic HTx fetuses. These findings suggest that it is only cells generated after day E17 that are missing from the cortex of day E20 hydrocephalic rats. It is known that the motor corticospinal tract axons arise from pyramidal cells in layers 6, 5 and 4 of the cortical plate. These layers are generated earlier than layers 3 and 2 and are almost certainly in place by day E17 and account for why motor corticospinal tract function is spared in younger animals with established hydrocephalus.

The effect of Bcr-Abl protein tyrosine kinase on maturation and proliferation of primitive haematopoietic cells.

BACKGROUND: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate. MATERIALS AND METHODS: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry. RESULTS: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells. CONCLUSIONS: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation.

Neural immunoregulation: emerging roles for nerves in immune homeostasis and disease.

In this review, James Downing and Jaleel Miyan outline emerging evidence for neural mechanisms that contribute to specific categories of host defence. Involvement of direct innervation in the adaptive control of immunological responses complements an established view of neuroendocrine-immune modulation. The challenge remains to understand the integrative and homeostatic functions of 'hardwiring' of peripheral immune effector sites, its bearing on disorder and potential for therapeutic modification.

Neuropeptide control of bone marrow neutrophil production is mediated by both direct and indirect effects on CFU-GM.

Noradrenaline- and peptide-containing nerve fibres project into the bone marrow and terminate in association with stromal cells and within the parenchyma. Peptidergic nerve terminals are also associated with antigen-processing and -presenting cells throughout the body and have been shown to be important in leucocyte trafficking and wound healing, as well as haemopoiesis. Here, we tested the in vivo effects of deleting the peripheral neuropeptide network on haemopoiesis and also investigated whether the target cell population for these substances was myeloid progenitor cells (colony-forming unit-granulocyte/macrophage, CFU-GM). Deletion of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) by capsaicin abrogates normal blood cell production. These neuropeptides produced significant stimulation of colony formation from unfractionated bone marrow and elicited production of soluble factors capable of stimulating highly enriched CFU-GM. CGRP also had a direct stimulatory effect on highly enriched CFU-GM. Noradrenaline elicited factors that inhibited colony formation and had no direct effect on CFU-GM. We conclude that the neuropeptides form the positive arm of a neural control system and that noradrenaline acts as a negative regulator.

Neuropeptide control of bone marrow neutrophil production. A key axis for neuroimmunomodulation.

Nerve fibers project into the bone marrow and terminate in association with stromal cells. Nerve terminals are also associated with antigen-processing and -presenting cells throughout the body and have been shown to be important in leukocyte trafficking and wound healing as well as hemopoiesis. Here we show that neuropeptide input to the bone marrow is vital to normal granulopoiesis and that deletion of the neuropeptides, substance P, and calcitonin gene-related peptide (CGRP), with the neurotoxin, capsaicin, abrogates normal blood cell production. Norepinephrine, neurokinins a and 2, and vasoactive intestinal peptide all have inhibitory effects on in vitro CFU-GM colony formation. Substance P, neurokinin 1, nerve growth factor, and CGRP have stimulatory effects on CFU-GM. Furthermore, in vitro experiments show that, apart from CGRP, all the neuroactive substances we tested operate through effects on accessory cells, stimulating the release of regulatory molecules that have a direct effect on purified CFU-GM.

Flt3 ligand can promote survival and macrophage development without proliferation in myeloid progenitor cells.

Flt3 ligand elicits a variety of effects on early hemopoietic progenitors by occupying its cognate receptor, Flt3, a member of the type III tyrosine kinase receptor family. The cytokines macrophage colony-stimulating factor (M-CSF) and stem cell factor (SCF) bind to related members of this tyrosine kinase receptors family, c-fms and c-kit, respectively. The relative effects of the cytokines M-CSF, SCF, and Flt3L on the proliferation and development of the late myeloid progenitors granulocyte-macrophage colony-forming cells (GM-CFC) were investigated. Distinct biologic responses were stimulated by ligand binding to these different tyrosine kinase receptors in enriched GM-CFC. M-CSF stimulated GM-CFC to proliferate and develop into macrophages. SCF, on the other hand, stimulated GM-CFC to develop into neutrophils. Flt3 ligand had a relatively small proliferative effect on enriched GM-CFC compared to SCF and M-CSF and had no ability to either stimulate colony formation or synergize with these two cytokines in promoting DNA synthesis, colony formation, or expansion in liquid culture. Flt3 ligand, however, was capable of maintaining the clonogenic potential of GM-CFC and acted as an anti-apoptotic agent as assessed using the Annexin-V apoptosis assay. GM-CFC cultured in Flt3 ligand eventually formed macrophages and neutrophils in liquid culture. Labeling with the membrane-associated cell tracker dye PKH26 indicated that the majority of the enriched GM-CFC responded to Flt3 ligand by undergoing limited proliferation and macrophage development, whereas other cells survived but did not proliferate and differentiate into macrophages. Thus, Flt3 ligand promoted survival and stimulated development without proliferation in primary-enriched myeloid progenitor cells.

Coordinated host defense through an integration of the neural, immune and haemopoietic systems.

Interactions between the neural and immune systems exist through humoral factors operating via the hypothalamic-pituitary-adrenal axis and cytokines acting over a relatively long distance. Anatomical evidence also suggests direct, hard-wired pathways of interaction and control through innervation of lymphoid organs and peripheral sites involved in host defense, including the thymus, spleen, lymph nodes, and skin. Recent evidence has demonstrated: 1) neural control of the bone marrow haemopoietic system, 2) interactions between peripheral nerve endings in the skin and epidermal Langerhans cells, and 3) peripheralization of leukocytes in the initial stages of stress. This leads us to propose that the nervous system is involved in host monitoring and coordination of host defense systems. If the brain is to have appropriate control of host defense mechanisms it must have: (a) afferent inputs monitoring host defense status, (b) efferent control pathways that modulate primary reactions to infection and damage, (c) efferent activation pathways to the myeloid defense system while the specific, lymphoid immune system is activated, and (d) inhibition of the proliferative lymphocytic response if the infection has been dealt with. We are investigating whether such a model, which allows for control and coordination of both the initial myeloid defense system and of the acquired immune response, is observed in mammals.

Cell death in the brain of the HTx rat.

The structural changes in the hydrocephalic brain suggest that cell death must be a significant factor in the loss of cortical mantle thickness and cell numbers. In the developing hydrocephalic brain a similar conclusion might also be reached. We present data suggesting that in the developing hydrocephalic HTx rat brain, there are at least two processes that underlie the structural deficit in the cortex. The first is abnormal development of the cortex probably resulting from a combination of the genetic defect and blockage of CSF circulation in the fetus. The second is a direct consequence of raised intracranial pressure and is manifested in a lack of development of glial cells in the neonatal brain and in the loss of myelination and neurones as pressure rises after birth, particularly after skull plate fusion. We find little evidence of increased cell death by apoptosis, recording instead a decrease in apoptotic index. There is evidence that necrosis must have occurred.

Bone marrow innervation regulates cellular retention in the murine haemopoietic system.

An anatomical analysis of the innervation of murine femora revealed intimate association of haemopoietic and stromal cells with nerve fibres. The mechanical denervation of these femora resulted in significant mobilization of cells into the peripheral blood within 24h. There was a decrease in femoral cellularity and analysis of the type of cells mobilized also revealed that there was an increase in progenitor cells in the peripheral blood. In non-splenectomized mice these progenitor cells were quickly cleared from the circulation. Chemical sympathectomy of mice with 6-hydroxydopamine resulted in decreased bone marrow cellularity without a change in bone marrow or peripheral blood progenitor cell numbers, nor the sustained rise in peripheral leucocytes observed with whole nerve denervation. These observations argue for selective control of mobilization by the nervous system and also indicate possible control of proliferation within the bone marrow. We conclude that the innervation has an important role in the maintenance of the blood-marrow interface, control of peripheral blood cell numbers, and mobilization of colony forming cells into the periphery.

Effects of hydrocephalus on nitric oxide neurones in the brain of the HTx rat.

The effect of hydrocephalus on the nitric oxide system in the brain of the HTx rat brain was investigated. We used NADPH-diaphorase histochemistry to stain neurones containing the enzyme, nitric oxide synthase. Comparison of normal and hydrocephalic HTx rat brains showed a reduction in stained neurones and a greatly depressed staining intensity in those that were labelled in the hydrocephalic brain. Also, there was an apparent reduction in number and length of radiating fibres from stained neurones. In addition to these changes we also observed a clear association between stained neurones and the microvasculature of the brain. This suggests that changes in the activity of these neurones may have a significant impact on the normal perfusion of the brain.

The functional outcome of shunting H-Tx rat pups at different ages.

"Normal", untreated hydrocephalic and CSF shunted hydrocephalic Texas (H-Tx) rat pups at different ages and Sprague-Dawley controls were tested for spatial learning and memory acquisition in an 8-arm radial maze. Shunting 5 day old hydrocephalic pups improved their performance to the level of that of "normal" litter-mates, whereas shunting at 10 days produced no improvement, and the performance of these animals was comparable to that of the untreated hydrocephalics. None of the H-Tx rats, whether treated, untreated or "normal", performed as well as the Sprague-Dawley controls. These findings suggest that shunting prior to skull plate fusion, which occurs at approximately the tenth post-natal day, is capable of protecting brain function involved in memory and learning. The poor functional scores achieved by the "normal" as well as the treated and untreated H-Tx animals compared to the control Sprague-Dawleys, raises the possibility that the H-Tx rat has brain deficits in addition to those caused by hydrocephalus.

Induction of ACTH- and TNF-alpha-like molecules in the hemocytes of Calliphora vomitoria (Insecta, Diptera).

Three basic cell types are described in the hemolymph of newly enclosed adult Calliphora vomitoria: prohemocytes, plasmatocytes, and granular cells. In addition, anucleate cellular fragments with some inclusions are observed. Cell division is found only in the prohemocytes. Plasmatocytes and granular cells are capable of in vitro bacterial phagocytosis and take part in capsule formation. Adrenocorticotropin hormone (ACTH)-like molecules are found in both cell types. The staining of the plasmatocytes is related to the functional activation of the cells. Indeed, positive staining is only observed during capsule formation, suggesting an induction of ACTH expression. The behaviour of tumor necrosis factor (TNF)-alpha-like molecules is similar. In conclusion, these data provide further support for the close, evolutionary relationship between the immune and the neuroendocrine systems.

Innervation of dipteran eclosion muscles: ultrastructure, immunohistochemistry, physiology and death.

The thoracic eclosion muscles of flies die by cytotoxic attack under neural control. We have investigated the innervation, ultrastructure and immunohistochemistry of the ventral eclosion muscle of Glossina. Two neurons located in the thoracic ganglion innervate this muscle. One of these is immunoreactive for serotonin and does not provide motor innervation. It appears to terminate near the attachment of an immunocyte involved in the dismantling of the muscle. The neuromuscular junction has features that distinguish it from any other chemical junction. A narrow, 3 nm gap separates pre- and post-synaptic membranes and this apparently acts to limit diffusion into and out of the junction. The immunocyte may use neuromuscular innervation as a path-finder to all muscle fibres and may even receive direct input from this source. Neuromuscular transmission is probably chemical as decreasing temperature results in decreasing amplitude of the (graded) muscle potential.