Serotonin 5-HT(3) receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT(3) receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT(3)A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT(3)A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT(3)A receptor subunits do not originate from the subtypes of primary afferent fibres that bind IB4 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres.
Chemokine-directed leukocyte migration is a critical component of all innate and adaptive immune responses. The atypical chemokine receptor ACKR2 is expressed by lymphatic endothelial cells and scavenges pro-inflammatory CC chemokines to indirectly subdue leukocyte migration. This contributes to the resolution of acute inflammatory responses in vivo. ACKR2 is also universally expressed by innate-like B cells, suppressing their responsiveness to the non-ACKR2 ligand CXCL13, and controlling their distribution in vivo. The role of ACKR2 in autoimmunity remains relatively unexplored, although Ackr2 deficiency reportedly lessens the clinical symptoms of experimental autoimmune encephalomyelitis induced by immunization with encephalogenic peptide (MOG35–55). This was attributed to poor T-cell priming stemming from the defective departure of dendritic cells from the site of immunization. However, we report here that Ackr2-deficient mice, on two separate genetic backgrounds, are not less susceptible to autoimmunity induced by immunization, and in some cases develop enhanced clinical symptoms. Moreover, ACKR2 deficiency does not suppress T-cell priming in response to encephalogenic peptide (MOG35–55), and responses to protein antigen (collagen or MOG1–125) are characterized by elevated interleukin-17 production. Interestingly, after immunization with protein, but not peptide, antigen, Ackr2 deficiency was also associated with an increase in lymph node B cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that enhances T helper type 17 (Th17) cell development and survival. Thus, Ackr2 deficiency does not suppress autoreactive T-cell priming and autoimmune pathology, but can enhance T-cell polarization toward Th17 cells and increase the abundance of GM-CSF+ B cells in lymph nodes draining the site of immunization.
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