Patrick Kitabgi

Chemokines: a new class of neuromodulator?

Chemokines are not only found in the immune system or expressed in inflammatory conditions: they are constitutively present in the brain in both glial cells and neurons. Recently, the possibility has been raised that they might act as neurotransmitters or neuromodulators. Although the evidence is incomplete, emerging data show that chemokines have several of the characteristics that define neurotransmitters. Moreover, their physiological actions resemble those of neuromodulators in the sense that chemokines usually have few effects by themselves in basal conditions, but modify the induced release of neurotransmitters or neuropeptides. These findings, together with the pharmacological development of agonists and antagonists that are selective for chemokine receptors and can cross the blood–brain barrier, open a new era of research in neuroscience.

Neuroanatomical distribution of CXCR4 in adult rat brain and its localization in cholinergic and dopaminergic neurons

Accumulating evidence supports a role of chemokines and their receptors in brain function. Up to now scarce evidence has been given of the neuroanatomical distribution of chemokine receptors. Although it is widely accepted that chemokine receptors are present on glial cells, especially in pathological conditions, it remains unclear whether they are constitutively present in normal rat brain and whether neurons have the potential to express such chemokine receptors. CXCR4, a G protein‐coupled receptor for the chemokine stromal cell‐derived factor‐1 (SDF‐1/CXCL12) was reported to have possible implications in brain development and AIDS‐related dementia. By dual immunohistochemistry on brain sections, we clearly demonstrate that CXCR4 is constitutively expressed in adult rat brain, in glial cells (astrocytes, microglia but not oligodendrocytes) as well as in neurons. Neuronal expression of CXCR4 is mainly found in cerebral cortex, caudate putamen, globus pallidus, substantia innominata, supraoptic and paraventricular hypothalamic nuclei, ventromedial thalamic nucleus and substantia nigra. Using confocal microscopy, a differential distribution of CXCR4 in neuronal perikarya and dendrites can be observed according to the brain structure. Furthermore, this work demonstrates for the first time the coexistence of a chemokine receptor with classical neurotransmitters. A localization of CXCR4 is thus observed in neuronal cell bodies expressing choline acetyltransferase‐immunoreactivity in the caudate putamen and substantia innominata, as well as in tyrosine hydroxylase‐positive neurons in the substantia nigra pars compacta. In conclusion, the constitutive neuronal CXCR4 expression suggests that SDF‐1/CXCL12 could be involved in neuronal communication and possibly linked up with cholinergic and dopaminergic neurotransmission and related disorders.

Highly regionalized distribution of stromal cell‐derived factor‐1/CXCL12 in adult rat brain: constitutive expression in cholinergic, dopaminergic and vasopressinergic neurons

The stromal cell‐derived factor‐1 (SDF‐1)/CXCL12 and its receptor CXCR4 are key modulators of immune functions. In the nervous system, SDF‐1/CXCL12 is crucial for neuronal guidance in developing brain, intercellular communication and the neuropathogenesis of acquired immunodeficiency syndrome. However, cerebral functions of SDF‐1/CXCL12 in adult brain are poorly understood. The understanding of its role in the adult brain needs a detailed neuroanatomical mapping of SDF‐1/CXCL12. By dual immunohistochemistry we demonstrate that this chemokine is constitutively expressed not only in astrocytes and microglia but also in neurons, in discrete neuroanatomical regions. Indeed, neuronal expression of SDF‐1/CXCL12 is mainly found in cerebral cortex, substantia innominata, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra and oculomotor nuclei. Moreover, we provide the first evidence that SDF‐1/CXCL12 is constitutively expressed in cholinergic neurons in the medial septum and substantia innominata and in dopaminergic neurons in substantia nigra pars compacta and the ventral tegmental area. Interestingly we also show, for the first time, a selective co‐localization of SDF‐1/CXCL12 with vasopressin‐expressing neurons in the supraoptic and paraventricular hypothalamic nuclei. In addition, in the lateral hypothalamic area, SDF‐1/CXCL12 was found to be located on melanin concentrating hormone‐expressing neurons. Altogether, these original data suggest that SDF‐1/CXCL12 could be a modulatory neuropeptide regulating both central cholinergic and dopaminergic systems. In addition, a key role for SDF‐1/CXCL12 in neuroendocrine regulation of vasopressin‐expressing neurons represents an exciting new field of research.

Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats

A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP‐1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP‐1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP‐1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co‐localized with pain‐related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium‐dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2‐induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.

Constitutive expression of CCR2 chemokine receptor and inhibition by MCP-1/CCL2 of GABA-induced currents in spinal cord neurones

In the CNS, immune‐like competent cells (microglia and astrocytes) were first described as potential sites of chemokine synthesis, but more recent evidence has indicated that neurones might also express chemokines and their receptors. The aim of the present work was to investigate further, both in vivo and in vitro, CC Chemokine Family Receptor 2 (CCR2) expression and functionality in rat spinal cord neurones. First, we demonstrated by RT–PCR and western blot analysis that CCR2 mRNA and protein were present in spinal extracts. Furthermore, we showed by immunolabelling that CCR2 was exclusively expressed by neurones in spinal sections of healthy rat. Finally, to test the functionality of CCR2, we used primary cultures of rat spinal neurones. In this model, similar to what was observed in vivo, CCR2 mRNA and protein were expressed by neurones. Cultured neurones stimulated with Monocyte Chemoattractant Protein‐1 (MCP‐1)/CCL2, the best characterized CCR2 agonist, showed activation of the Akt pathway. Finally, patch‐clamp recording of cultured spinal neurones was used to investigate whether MCP‐1/CCL2 could modulate their electrophysiological properties. MCP‐1 alone did not affect the electrical properties of spinal neurones, but potently and efficiently inhibited GABAA‐mediated GABAergic responses in these neurones. These data constitute the first demonstration of a modulatory role of MCP‐1 on GABAergic neurotransmission and contribute to our understanding of the roles of CCR2 and MCP‐1/CCL2 in spinal cord physiology, in particular with respect to nociceptive transmission, as well as the implication of this chemokine in neuronal adaptation or dysfunction during neuropathy.

Highly regionalized neuronal expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) in rat brain: Evidence for its colocalization with neurotransmitters and neuropeptides

The monocyte chemoattractant protein‐1 (MCP‐1/CCL2) and its receptor CCR2 are key modulators of immune functions. In the nervous system, MCP‐1/CCL2 is implicated in neuroinflammatory pathologies. However, cerebral functions of MCP‐1/CCL2 under normal conditions are still unclear. In this study, using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and specific rat MCP‐1 enzyme‐linked immunosorbent assay (ELISA) approaches, we observed that MCP‐1/CCL2 mRNA and protein were expressed in different punched regions of the normal rat central nervous system. Immunohistochemical studies further revealed that this chemokine is constitutively expressed not only in astrocytes but also in neurons, in discrete neuroanatomical regions. Neuronal expression of MCP‐1/CCL2 is mainly found in the cerebral cortex, globus pallidus, hippocampus, paraventricular and supraoptic hypothalamic nuclei, lateral hypothalamus, substantia nigra, facial nuclei, motor and spinal trigeminal nuclei, and gigantocellular reticular nucleus and in Purkinje cells in the cerebellum. Moreover, we obtained the first evidence that MCP‐1/CCL2 is constitutively expressed in cholinergic neurons, notably in the magnocellular preoptic and oculomotor nuclei, and in dopaminergic neurons of the substantia nigra pars compacta. In addition, in the lateral hypothalamic area, MCP‐1/CCL2 co‐localized with melanin‐concentrating hormone‐expressing neurons. Interestingly, we demonstrate a co‐localization of MCP‐1/CCL2 with vasopressin in magnocellular neuronal cell bodies and processes in the supraoptic and paraventricular hypothalamic nuclei, as well as in processes in the internal layer of the median eminence and in the posterior pituitary. Taken together, our data suggest that MCP‐1/CCL2 could act as a modulator of neuronal activity and neuroendocrine functions. J. Comp. Neurol. 489:275–292, 2005.

Constitutive neuronal expression of CCR2 chemokine receptor and its colocalization with neurotransmitters in normal rat brain: Functional effect of MCP-1/CCL2 on calcium mobilization in primary cultured neurons

Chemokines and their receptors are well described in the immune system, where they promote cell migration and activation. In the central nervous system, chemokine has been implicated in neuroinflammatory processes. However, an increasing number of evidence suggests that they have regulatory functions in the normal nervous system, where they could participate in cell communication. In this work, using a semiquantitative immunohistochemistry approach, we provide the first neuroanatomical mapping of constitutive neuronal CCR2 localization. Neuronal expression of CCR2 was observed in the anterior olfactory nucleus, cerebral cortex, hippocampal formation, caudate putamen, globus pallidus, supraoptic and paraventricular hypothalamic nuclei, amygdala, substantia nigra, ventral tegmental area, and in the brainstem and cerebellum. These data are largely in accordance with results obtained using quantitative autoradiography with [125I]MCP‐1/CCL2 and RT‐PCR CCR2 mRNA analysis. Furthermore, using dual fluorescent immunohistochemistry we studied the chemical phenotype of labeled neurons and demonstrated the coexistence of CCR2 with classical neurotransmitters. Indeed, localization of CCR2 immunostaining is observed in dopaminergic neurons in the substantia nigra pars compacta and in the ventral tegmental area as well as in cholinergic neurons in the substantia innominata and caudate putamen. Finally, we show that the preferential CCR2 ligand, MCP‐1/CCL2, elicits Ca2+ transients in primary cultured neurons from various rat brain regions including the cortex, hippocampus, hypothalamus, and mesencephalon. In conclusion, the constitutive neuronal CCR2 expression in selective brain structures suggests that this receptor could be involved in neuronal communication and possibly associated with cholinergic and dopaminergic neurotransmission and related disorders. J. Comp. Neurol. 492:178–192, 2005.

Effects of neurotensin on isolated intestinal smooth muscles

Abstract The effects of neurotensin were investigated in intestinal smooth muscle preparations. Neurotensin relaxed the rat ileum, and contracted the guinea-pig ileum and taenia. Neurotensin induced a biphasic response (relaxation followed by contraction) in the contracted guinea-pig ileum. In all systems, half-maximal effects were obtained with 4–5 nM neurotensin and maximal responses with 30–60 nM; tachyphylaxis occurred with higher concentrations. Tetrodotoxin did not affect the responses to neurotensin in the rat ileum and the guinea-pig taenia. Tetrodotoxin abolished the contraction or the contraction phase (but not the relaxation phase) of the biphasic response induced by neurotensin in the guinea-pig ileum. Atropine partially inhibited the neurotensin-induced contraction in the guinea-pig ileum. These results suggest that neurotensin acts on intestinal smooth muscle both directly (relaxation of the rat and guinea-pig ileum, and contraction of the guinea-pig taenia) and through a nerve-mediated, partly cholinergic, process (contraction of the guinea-pig ileum).

Neurotensin contracts the guinea-pig longitudinal ileal smooth muscle by inducing acetylcholine release

Neurotensin contracted the isolated longitudinal smooth muscle strip of the guinea-pig ileum, but was only 22% as effective as histamine. Neostigmine (0.1 micrometer) increased the effectiveness of neurotensin 4-fold. Atropine (0.1 micrometer) completely inhibited the contracting effect of neurotensin measured in the presence of neostigmine, whereas hexamethonium (0.1 micrometer) was without effect. It is concluded that in the longitudinal smooth muscle of the guinea-pig ileum, the contracting effect of neurotensin is mediated by the release of acetylcholine from nerve endings in response to a postganglionic stimulation of cholinergic nerve fibers.

Long term exposure to the chemokine CCL2 activates the nigrostriatal dopamine system: a novel mechanism for the control of dopamine release

Accumulating evidence show that chemokines can modulate the activity of neurons through various mechanisms. Recently, we demonstrated that CCR2, the main receptor for the chemokine CCL2, is constitutively expressed in dopamine neurons in the rat substantia nigra. Here we show that unilateral intranigral injections of CCL2 (50 ng) in freely moving rats increase extracellular concentrations of dopamine and its metabolites and decrease dopamine content in the ipsilateral dorsal striatum. Furthermore, these CCL2 injections are responsible for an increase in locomotor activity resulting in contralateral circling behavior. Using patch-clamp recordings of dopaminergic neurons in slices of the rat substantia nigra, we observed that a prolonged exposure (>8 min) to 10 nM CCL2 significantly increases the membrane resistance of dopaminergic neurons by closure of background channels mainly selective to potassium ions. This leads to an enhancement of dopaminergic neuron discharge in pacemaker or burst mode necessary for dopamine release. We provide here the first evidence that application of CCL2 on dopaminergic neurons increases their excitability, dopamine release and related locomotor activity.