Diane L. Rosin

IL-17 produced by neutrophils regulates IFN-γ–mediated neutrophil migration in mouse kidney ischemia-reperfusion injury

The IL-23/IL-17 and IL-12/IFN-gamma cytokine pathways have a role in chronic autoimmunity, which is considered mainly a dysfunction of adaptive immunity. The extent to which they contribute to innate immunity is, however, unknown. We used a mouse model of acute kidney ischemia-reperfusion injury (IRI) to test the hypothesis that early production of IL-23 and IL-12 following IRI activates downstream IL-17 and IFN-gamma signaling pathways and promotes kidney inflammation. Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and mAb neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in acute kidney IRI in mice. We further demonstrate that IL-17A produced by GR-1+ neutrophils was critical for kidney IRI in mice. Activation of the IL-12/IFN-gamma pathway and NKT cells by administering alpha-galactosylceramide-primed bone marrow-derived DCs increased IFN-gamma production following moderate IRI in WT mice but did not exacerbate injury or enhance IFN-gamma production in either Il17a-/- or Il17r-/- mice, which suggested that IL-17 signaling was proximal to IFN-gamma signaling. This was confirmed by the finding that IFN-gamma administration reversed the protection seen in Il17a-/- mice subjected to IRI, whereas IL-17A failed to reverse protection in Ifng-/- mice. These results demonstrate that the innate immune component of kidney IRI requires dual activation of the IL-12/IFN-gamma and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-gamma. These mechanisms might contribute to reperfusion injury in other organs.

Distribution of α2C‐adrenergic receptor‐like immunoreactivity in the rat central nervous system

The distribution of α2C‐adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70‐amino‐acid polypeptide portion of the third intracellular loop of the α2C‐AR fused to glutathione‐S‐transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]‐photoaffinity‐labeled α2‐AR and by immunohistochemical labeling of COS cells expressing the individual rat α2‐AR subtypes. In both cases the antibody recognized only the α2C‐AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, α2C‐AR‐like immunoreactivity (α2C‐AR‐LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of α2C‐LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

The chemokine receptors CCR2 and CX3CR1 mediate monocyte/macrophage trafficking in kidney ischemia–reperfusion injury

Chemokines and their receptors such as CCR2 and CX3CR1 mediate leukocyte adhesion and migration into injured tissue. To further define mechanisms of monocyte trafficking during kidney injury we identified two groups of F4/80-positive cells (F4/80(low) and F4/80(high)) in the normal mouse kidney that phenotypically correspond to macrophages and dendritic cells, respectively. Following ischemia and 3 h of reperfusion, there was a large influx of F4/80(low) inflamed monocytes, but not dendritic cells, into the kidney. These monocytes produced TNF-alpha, IL-6, IL-1alpha and IL-12. Ischemic injury induced in CCR2(-/-) mice or in CCR2(+/+) mice, made chimeric with CCR2(-/-) bone marrow, resulted in lower plasma creatinine levels and their kidneys had fewer infiltrated F4/80(low) macrophages compared to control mice. CX3CR1 expression contributed to monocyte recruitment into inflamed kidneys, as ischemic injury in CX3CR1(-/-) mice was reduced, with fewer F4/80(low) macrophages than controls. Monocytes transferred from CCR2(+/+) or CX3CR1(+/-) mice migrated into reperfused kidneys better than monocytes from either CCR2(-/-) or CX3CR1(-/-) mice. Adoptive transfer of monocytes from CCR2(+/+) mice, but not CCR2(-/-) mice, reversed the protective effect in CCR2(-/-) mice following ischemia-reperfusion. Egress of CD11b(+)Ly6C(high) monocytes from blood into inflamed kidneys was CCR2- and CX3CR1-dependent. Our study shows that inflamed monocyte migration, through CCR2- and CX3CR1-dependent mechanisms, plays a critical role in kidney injury following ischemia reperfusion.

Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow–derived cells

Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow-derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP-->WT, A2A-KO-->WT, or WT-->WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO-->WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT-->WT chimera. ATL146e reduced the induction of IL-6, IL-1beta, IL-1ra, and TGF-alpha mRNA in WT-->WT mice but not in A2A-KO-->WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells.

Ultrastructural localization of adenosine A2A receptors suggests multiple cellular sites for modulation of GABAergic neurons in rat striatum

Activation of adenosine A2A receptors (A2AR) has been shown to antagonize the function of D2 dopaminergic regulation of striatal γ‐aminobutyric acid (GABA)‐ergic output and, thus, locomotor activity. Adenosine A2A receptor immunoreactivity (A2A‐LI) has been localized to rat striatum by light microscopy by using a previously characterized human A2AR monoclonal antibody. In this study, we evaluated the localization of A2A‐LI and its colocalization with GABA immunoreactivity (GABA‐LI) in dorsolateral rat striatum by immunoelectron microscopy to further characterize the potential mechanism of purinergic control of striatal output. Ultrastructural analysis demonstrated A2A‐LI associated with the plasma membrane and cytoplasmic membranous structures of striatal neurons. A2A‐LI was prevalent in dendrites and dendritic spines (∼70% of total A2A‐profiles counted) and less prevalent in axons and axon terminals (23%), soma (3%), and glia (3%). Cellular elements exhibiting both A2A‐LI and GABA‐LI comprised 23% of the total profiles counted; colabeling was most common in dendrites. A2A‐LI was observed primarily at asymmetric synapses (n = 70) (both pre‐ and postsynaptically but predominantly in the postsynaptic element) and less frequently at symmetric synapses (n = 17). Of the 714 A2A‐immunoreactive profiles examined, 37% were apposed to GABA‐labeled profiles. The most common appositions were A2A‐labeled dendrites apposed to GABA‐immunoreactive dendrites (n = 132), axon terminals (n = 28), and somata (n = 22) and A2A‐labeled axons apposed to GABA‐labeled dendrites (n = 58), axon terminals (n = 14), and somata (n = 9). Our findings suggest that adenosine may play an important role in modulating excitatory input to striatal neurons and that A2AR may modulate GABAergic signaling at several cellular sites within the rat striatum. J. Comp. Neurol. 431:331–346, 2001.

Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2

Initially recognized for their importance in control of appetite, orexins (also called hypocretins) are neuropeptides that are also involved in regulating sleep, arousal, and cardiovascular function. Loss of orexin appears to be the primary cause of narcolepsy. Cells expressing the orexins are restricted to a discrete region of the hypothalamus, but their terminal projections are widely distributed throughout the brain. With the diversity of function and broad distribution of orexin terminals, it is not known whether the orexin cells constitute a homogeneous population. Because orexins produce neuroexcitatory effects, we hypothesized that orexin‐containing neurons are glutamatergic. In the present study we used digoxigenin‐labeled cRNA probes for the vesicular glutamate transporters, VGLUT1 and VGLUT2, for in situ hybridization studies in combination with immunohistochemical detection of orexin cell bodies in the hypothalamus. In general, cells in the hypothalamus expressed low levels of the vesicular glutamate transporters relative to other areas of the forebrain, such as the cortex and thalamus. Light labeling for VGLUT2 mRNA was detected in about 50% of the orexin‐immunoreactive neurons, and a much smaller percentage (≈13%) of orexin‐immunoreactive cells was found to express VGLUT1. Despite the fact that intense labeling for GAD67 mRNA was found in a large number of cells throughout the hypothalamus, none of the orexin‐immunoreactive cells was found to be GABAergic. These findings, showing that many of the orexin neurons are glutamatergic, are consistent with the neuroexcitatory effects of orexin but suggest that another neurochemical phenotype may define the remaining subset of orexin neurons. J. Comp. Neurol. 465:593–603, 2003.

A group of glutamatergic interneurons expressing high levels of both neurokinin-1 receptors and somatostatin identifies the region of the pre-Bötzinger complex.

The pre‐Bötzinger complex (pre‐BötC) is a physiologically defined group of ventrolateral medullary neurons that plays a central role in respiratory rhythm generation. These cells are located in a portion of the rostral ventrolateral medulla (RVLM) that is difficult to identify precisely for lack of a specific marker. We sought to determine whether somatostatin (SST) might be a marker for this region. The rat pre‐BötC area was defined as a 500‐μm‐long segment of ventrolateral medulla coextensive with the ventral respiratory group. This region was identified by juxtacellular labeling of neurons with respiratory‐related activity and by its location rostral to the phrenic premotor neurons. It contained most of the SST‐ir neuronal somata of the RVLM. These cells were small (107 μm2) and expressed high levels of preprosomatostatin mRNA. They were strongly neurokinin 1 receptor (NK1R)‐ir and were selectively destroyed by saporin conjugated with an NK1R agonist (SSP‐SAP). Most SST‐ir neurons (>90%) contained vesicular glutamate transporter 2 (VGLUT2) mRNA, and terminals immunoreactive for SST and VGLUT2 protein were found in their midst. Few SST‐ir neurons contained GAD‐67 mRNA (<1%) or preproenkephalin mRNA (6%). Retrograde labeling experiments demonstrated that over 75% of the SST‐ir neurons project to the contralateral pre‐BötC area, but none projects to the spinal cord. In conclusion, the RVLM contains many neurons that express preprosomatostatin mRNA. A subgroup of these cells contains high levels of SST and NK1R immunoreactivity in their somata. These glutamatergic interneurons identify a narrow region of the RVLM that appears to be coextensive with the pre‐BötC of adult rats. J. Comp. Neurol. 455:499–512, 2003.

Vesicular glutamate transporter DNPI/VGLUT2 is expressed by both C1 adrenergic and nonaminergic presympathetic vasomotor neurons of the rat medulla

The main source of excitatory drive to the sympathetic preganglionic neurons that control blood pressure is from neurons located in the rostral ventrolateral medulla (RVLM). This monosynaptic input includes adrenergic (C1), peptidergic, and noncatecholaminergic neurons. Some of the cells in this pathway are suspected to be glutamatergic, but conclusive evidence is lacking. In the present study we sought to determine whether these presympathetic neurons express the vesicular glutamate transporter BNPI/VGLUT1 or the closely related gene DNPI, the rat homolog of the mouse vesicular glutamate transporter VGLUT2. Both BNPI/VGLUT1 and DNPI/VGLUT2 mRNAs were detected in the medulla oblongata by in situ hybridization, but only DNPI/VGLUT2 mRNA was present in the RVLM. Moreover, BNPI immunoreactivity was absent from the thoracic spinal cord lateral horn. DNPI/VGLUT2 mRNA was present in many medullary cells retrogradely labeled with Fluoro‐Gold from the spinal cord (T2; four rats). Within the RVLM, 79% of the bulbospinal C1 cells contained DNPI/VGLUT2 mRNA. Bulbospinal noradrenergic A5 neurons did not contain DNPI/VGLUT2 mRNA. The RVLM of six unanesthetized rats subjected to 2 hours of hydralazine‐induced hypotension contained tenfold more c‐Fos‐ir DNPI/VGLUT2 neurons than that of six saline‐treated controls. c‐Fos‐ir DNPI/VGLUT2 neurons included C1 and non‐C1 neurons (3:2 ratio). In seven barbiturate‐anesthetized rats, 16 vasomotor presympathetic neurons were filled with biotinamide and analyzed for the presence of tyrosine hydroxylase immunoreactivity and/or DNPI/VGLUT2 mRNA. Biotinamide‐labeled neurons included C1 and non‐C1 cells. Most non‐C1 (9/10) and C1 presympathetic cells (5/6) contained DNPI/VGLUT2 mRNA. In conclusion, DNPI/VGLUT2 is expressed by most blood pressure‐regulating presympathetic cells of the RVLM. The data suggest that these neurons may be glutamatergic and that the C1 adrenergic phenotype is one of several secondary phenotypes that are differentially expressed by subgroups of these cells. J. Comp. Neurol. 444:207–220, 2002.

Afferent and efferent connections of the rat retrotrapezoid nucleus.

The rat retrotrapezoid nucleus (RTN) contains candidate central chemoreceptors that have extensive dendrites within the marginal layer (ML). This study describes the axonal projections of RTN neurons and their probable synaptic inputs. The ML showed a dense plexus of nerve terminals immunoreactive (ir) for markers of glutamatergic (vesicular glutamate transporters VGLUT1–3), γ‐aminobutyric acid (GABA)‐ergic, adrenergic, serotonergic, cholinergic, and peptidergic transmission. The density of VGLUT3‐ir terminals tracked the location of RTN chemoreceptors. The efferent and afferent projections of RTN were studied by placing small iontophoretic injections of anterograde (biotinylated dextran amine; BDA) and retrograde (cholera toxin B) tracers where RTN chemoreceptors have been previously recorded. BDA did not label the nearby C1 cells. BDA‐ir varicosities were found in the solitary tract nucleus (NTS), all ventral respiratory column (VRC) subdivisions, A5 noradrenergic area, parabrachial complex, and spinal cord. In each target region, a large percentage of the BDA‐ir varicosities was VGLUT2‐ir (41–83%). Putative afferent input to RTN originated from spinal cord, caudal NTS, area postrema, VRC, dorsolateral pons, raphe nuclei, lateral hypothalamus, central amygdala, and insular cortex. The results suggest that 1) whether or not the ML is specialized for CO2 sensing, its complex neuropil likely regulates the activity of RTN chemosensitive neurons; 2) the catecholaminergic, cholinergic, and serotonergic innervation of RTN represents a possible substrate for the known state‐dependent control of RTN chemoreceptors; 3) VGLUT3‐ir terminals are a probable marker of RTN; and 4) the chemosensitive neurons of RTN may provide a chemical drive to multiple respiratory outflows, insofar as RTN innervates the entire VRC. J. Comp. Neurol. 499:64–89, 2006.

Immunohistochemical localization ofα2a-adrenergic receptors in catecholaminergic and other brainstem neurons in the rat

alpha 2-Adrenergic receptors mediate a large portion of the known inhibitory effects of catecholamines on central and peripheral neurons. Molecular cloning studies have established the identity of three alpha 2-adrenergic receptor genes from several species that encode the A, B and C subtypes of the receptor. The rat alpha 2A-adrenergic receptor, as defined by sequence similarity, is the orthologue of the human alpha 2A-adrenergic receptor. In this paper, we report the development of rabbit antisera directed against a portion of the third intracellular loop of the rat alpha 2A-adrenergic receptor and the histochemical localization of alpha 2A-adrenergic receptor-like immunoreactive material in the brainstem and spinal cord of the adult rat. Our antisera detected alpha 2A-adrenergic receptor-specific punctate staining associated with neuronal perikarya. alpha 2A-adrenergic receptor-like immunoreactivity was widely, but heterogeneously, distributed in the brainstem and spinal cord, predominantly in areas involved in the control of autonomic function. Double labelling with antisera to tyrosine hydroxylase or phenylethanolamine-N-methyl-transferase revealed that alpha 2A-adrenergic receptor-like immunoreactivity is present in most, perhaps all, noradrenergic and adrenergic cells of the brainstem. alpha 2A-Adrenergic receptor-like immunoreactivity was detected in a small percentage of the dopaminergic cells of the A9 and A10 groups. This study provides the first description of the specific immunohistochemical localization of alpha 2A-adrenergic receptors using a subtype-specific polyclonal antibody. The results support the view that alpha 2-adrenergic receptors are involved in central cardiovascular control and suggest that the catecholaminergic autoreceptors of central noradrenergic and adrenergic neurons are the A subtype of the alpha 2-adrenergic receptors.