Chemoattractant receptors and lymphocyte egress from extralymphoid tissue: changing requirements during the course of inflammation.

Memory/effector T cells traffic efficiently through extralymphoid tissues, entering from the blood and leaving via the afferent lymph. During inflammation, T cell traffic into the affected tissue dramatically increases; however, the dynamics and mechanisms of T cell exit from inflamed tissues are poorly characterized. In this study, we show, using both a mouse and a sheep model, that large numbers of lymphocytes leave the chronically inflamed skin. Many T cells capable of producing IFN-γ and IL-17 also entered the draining afferent lymph, demonstrating that memory/effector T cells egress from sites of inflammation. Whereas efficient egress from acutely inflamed skin required lymphocyte-expressed CCR7, chronic inflammation promoted significant CCR7-independent exit as well. Lymphocyte exit at late time points of inflammation was sensitive to pertussis toxin but was only partially affected by the drug FTY720, implying the contribution of alternative chemoattractant receptors other than spingosine 1-phosphate receptor 1. Our data show that CCR7 is an important receptor for lymphocyte egress from both resting and inflamed extralymphoid tissues, but that alternative exit receptors come into play during chronic inflammation.

Gamma motor neurons express distinct genetic markers at birth and require muscle spindle-derived GDNF for postnatal survival.

BACKGROUND: Gamma motor neurons (gamma-MNs) selectively innervate muscle spindle intrafusal fibers and regulate their sensitivity to stretch. They constitute a distinct subpopulation that differs in morphology, physiology and connectivity from alpha-MNs, which innervate extrafusal muscle fibers and exert force. The mechanisms that control the differentiation of functionally distinct fusimotor neurons are unknown. Progress on this question has been limited by the absence of molecular markers to specifically distinguish and manipulate gamma-MNs. Recently, it was reported that early embryonic gamma-MN precursors are dependent on GDNF. Using this knowledge we characterized genetic strategies to label developing gamma-MNs based on GDNF receptor expression, showed their strict dependence for survival on muscle spindle-derived GDNF and generated an animal model in which gamma-MNs are selectively lost. RESULTS: In mice heterozygous for both the Hb9::GFP transgene and a tau-lacZ-labeled (TLZ) allele of the GDNF receptor Gfralpha1, we demonstrated that small motor neurons with high Gfralpha1-TLZ expression and lacking Hb9::GFP display structural and synaptic features of gamma-MNs and are selectively lost in mutants lacking target muscle spindles. Loss of muscle spindles also results in the downregulation of Gfralpha1 expression in some large diameter MNs, suggesting that spindle-derived factors may also influence populations of alpha-MNs with beta-skeletofusimotor collaterals. These molecular markers can be used to identify gamma-MNs from birth to the adult and to distinguish gamma- from beta-motor axons in the periphery. We also found that postnatal gamma-MNs are also distinguished by low expression of the neuronal nuclear protein (NeuN). With these markers of gamma-MN identity, we show after conditional elimination of GDNF from muscle spindles that the survival of gamma-MNs is selectively dependent on spindle-derived GDNF during the first 2 weeks of postnatal development. CONCLUSION: Neonatal gamma-MNs display a unique molecular profile characterized by the differential expression of a series of markers - Gfralpha1, Hb9::GFP and NeuN - and the selective dependence on muscle spindle-derived GDNF. Deletion of GDNF expression from muscle spindles results in the selective elimination of gamma-MNs with preservation of the spindle and its sensory innervation. This provides a mouse model with which to explore the specific role of gamma-fusimotor activity in motor behaviors.

Synaptic pathology, altered gene expression, and degeneration in photoreceptors impacted by drusen.

PURPOSE: Drusen are risk factors for age-related macular degeneration and have been shown to negatively impact cells of the RPE and retina. In this study, the effects of drusen on the synaptic machinery of retinal photoreceptors are investigated. METHODS: Human donor eye tissue containing retina, RPE, and choroid was processed for confocal immunofluorescence microscopy, laser capture microdissection, and light and electron microscopy. Tissue sections were immunostained with a panel of antibodies to synapse-associated proteins. Populations of photoreceptors over drusen and normal populations of photoreceptors were microdissected from fresh frozen tissue, RNA was purified, and quantitative PCR was performed to compare relative levels of gene expression. RESULTS: The number of photoreceptor synaptic terminals is reduced in regions of the outer plexiform layer over drusen, synaptic proteins are mislocalized in photoreceptor cells, and synaptic terminals are often observed within the outer nuclear layer. Photoreceptors over drusen also increase expression of the stress response proteins apolipoprotein E and alphaB-crystallin. Abnormal immunolabeling patterns are not restricted to photoreceptors directly over drusen but are also observed in cells flanking drusen. Gene expression analysis confirms reductions in the expression of genes coding for synapse-associated proteins and signal transduction proteins and increases in the expression of apolipoprotein E and alphaB-crystallingene transcripts. Ultrastructural analysis of photoreceptor synaptic terminals over drusen reveals significant abnormalities, and cell counts show a reduction in photoreceptor density directly over, and lateral to, drusen of all sizes. CONCLUSIONS: Photoreceptors overlying and flanking drusen exhibit morphologic and biochemical signs of degeneration. The expression of synapse-associated proteins decreases in photoreceptor synaptic terminals, whereas the expression of stress-response proteins increases. Reductions in photoreceptor cell densities over, and flanking, drusen suggest that these degenerative effects eventually result in the death of photoreceptors.

Drusen-associated degeneration in the retina.

PURPOSE: Drusen are variably sized extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. They are commonly found in aged eyes, however, numerous and/or confluent drusen are a significant risk factor for age-related macular degeneration. The purpose of this study was to investigate the impact of drusen on overlying cells of the retina. METHODS: Tissue containing retina and RPE/choroid was dissected from human donor eyes, embedded in agarose, and sectioned at 100 micro m using a vibratome. Sections were immunostained with a panel of antibodies that labeled glial cells, first-, second-, and third-order retinal neurons and processed for confocal microscopy. RESULTS: Retinal cells that overlie both soft and hard drusen exhibited numerous structural and molecular abnormalities. Normally detectable only in the outer segments of rod photoreceptors, rod opsin immunolabeling was also observed in the inner segment, cell body, axon, and axon terminal of photoreceptors that overlie drusen. Labeling with this antibody also revealed the deflection and shortening of rod inner and outer segments. Cone photoreceptors displayed similar structural abnormalities, as well as a decrease in cone opsin immunoreactivity. Drusen-associated abnormalities in the synaptic terminals of photoreceptor cells were also observed. In addition, an increase in intermediate filament protein immunoreactivity (vimentin and glial fibrillary acidic protein) was observed within Müller glial cells in areas of retina overlying drusen. Both soft and hard drusen were associated with a similar spectrum of effects in both macular and extramacular regions. Second- and third-order neurons, including bipolar, horizontal, amacrine, and ganglion cells all appeared unaffected. The structural and molecular abnormalities observed in photoreceptors and Müller glial cells were confined to retinal regions directly overlying and immediately adjacent to drusen; more distant retinal regions appeared unperturbed. Remarkably, significant abnormalities were observed over small subclinical drusen. CONCLUSIONS: Retinal cells overlying both soft and hard drusen exhibit structural and molecular abnormalities indicative of photoreceptor degeneration and Müller glial activation. These abnormalities resemble the degenerative effects common to many forms of retinal degeneration, but are confined to areas directly overlying drusen. This suggests that photoreceptor cell function is compromised as a consequence of drusen formation.