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S L Carlson

Publications and source records attributed to S L Carlson.

At least 19 recordsLinked to original sources
PubMed1998

Development of immune hyperinnervation in NGF-transgenic mice.

Sympathetic innervation of lymphoid tissues is localized to specific tissue compartments, but little is known of the "factors" that are important in establishing this pattern during development. Numerous studies have shown interactions of nerve growth factor (NGF) with the immune system, which may include modulation of immune innervation. We previously have shown that NGF transgenic mice, which overexpress NGF in skin and not immune tissues, have a dramatic hyperinnervation of splenic marginal zone and peripheral lymph node medulla and capsule. The purpose of the current studies was to determine if the presence of elevated NGF would alter immune system development and the process of sympathetic ingrowth. The results show that the splenic innervation in NGF transgenics gradually diverged from controls during the first two postnatal weeks, with the greatest change occurring between postnatal days 13 and 16 when the splenic organization was reaching the adult pattern. In contrast, the peripheral lymph nodes were hyperinnervated at an earlier age. mesenteric lymph nodes never diverged from the normal pattern. NGF levels in transgenic spleen were much higher than controls at postnatal days 1 and 2, when little innervation was present, and declined as the tissue matured, possibly because of NGF uptake by the ingrowing sympathetic fibers. This suggests that immune tissues are capable of concentrating NGF, which in turn may modulate the level of innervation by the sympathetic nervous system.

Animals↗
PubMed1998

Acute inflammatory response in spinal cord following impact injury.

Numerous factors are involved in the spread of secondary damage in spinal cord after traumatic injury, including ischemia, edema, increased excitatory amino acids, and oxidative damage to the tissue from reactive oxygen species. Neutrophils and macrophages can produce reactive oxygen species when activated and thus may contribute to the lipid peroxidation that is known to occur after spinal cord injury. This study examined the rostral-caudal distribution of neutrophils and macrophages/microglia at 4, 6, 24, and 48 h after contusion injury to the T10 spinal cord of rat (10 g weight, 50 mm drop). Neutrophils were located predominantly in necrotic regions, with a time course that peaked at 24 h as measured with assays of myeloperoxidase activity (MPO). The sharpest peak of MPO activity was localized between 4 mm rostral and caudal to the injury. Macrophages/microglia were visualized with antibodies against ED1 and OX-42. Numerous cells with a phagocytic morphology were present by 24 h, with a higher number by 48 h. These cells were predominantly located within the gray matter and dorsal funiculus white matter. The number of cells gradually declined through 6 mm rostral and caudal to the lesion. OX-42 staining also revealed reactive microglia with blunt processes, particularly at levels distant to the lesion. The number of macrophages/microglia was significantly correlated with the amount of tissue damage at each level. Treatments to decrease the inflammatory response are likely to be beneficial to recovery of function after traumatic spinal cord injury.

Animals↗
PubMed1998

A rapid and sensitive assay for measuring mitochondrial metabolic activity in isolated neural tissue.

In the present study, we used the oxidation-reduction sensitive dye Alamar Blue, a fluorometric/colorimetric indicator of metabolic activity, as a tool for examining mitochondrial function in rat spinal cord synaptosomes. At 15 min following incubation, Alamar Blue fluorescence levels were found to increase by 3-fold, and could be detected in samples containing as little as 25 microg of protein. Alamar Blue is non-toxic, making it possible to obtain measures of the metabolic rate and the maximal functional capacity of mitochondria in a single sample. The findings of this study demonstrate that Alamar Blue fluorescence levels increased in a near linear fashion when samples were measured every 15 min for a period of 1 h. To document that the changes in Alamar Blue fluorescence are directly related to mitochondrial function, synaptosomes were pre-incubated with antimycin A (10 microM) or malonate (50 mM), both of which are potent inhibitors of mitochondrial function. Pretreatment with either compound significantly reduced the Alamar Blue fluorometric signal at all time points examined. These results provide evidence that Alamar Blue is a valuable analytical tool for examining mitochondrial function in synaptosomal preparations from neural tissue. Moreover, the properties of Alamar Blue are such that it provides a more sensitive and simpler indicator compared to indicators used in existing assays.

Animals↗
PubMed1998

Variability of peripheral blood lymphocyte beta-2-adrenergic receptor density in humans.

Beta2-adrenergic receptor (betaAR) density on peripheral blood lymphocytes has been used as an index to reflect the betaAR state of the body. Lymphocytes betaARs are unequally distributed among lymphocyte subpopulations, with the highest density on CD8+ cells and the lowest on CD4+ cells. Thus, the measurement of peripheral blood lymphocyte betaAR density could vary with changes in CD4+ and CD8+ cell concentrations. We examined the individual and intersubject variance of betaAR density and lymphocyte subpopulations over time in 10 normal subjects, studied on 3 to 5 different d always at approximately 9:00 A.M. over a 4- to 12-wk period. Peripheral blood lymphocytes were isolated and beta2-adrenergic receptor density was determined by specific binding of [125I]-(-)iodopindolol, and lymphocyte subpopulations were measured by flow cytometry. Average receptors per lymphocyte were 776 +/- 183. Whereas the absolute values of CD4+% and CD8+% cell concentrations varied little in individual subjects (coefficient of variation 9.5% and 11.1%, respectively), the individual betaAR variance was greater (coefficient of variation 22.4%). However there was a significant correlation between betaAR and CD4+% and CD8+% cell concentration (correlation coefficients: -0.58, p < 0.001; +0.51, p < 0.001, respectively). This information is relevant to interpretations of changes in peripheral betaAR in humans.

Adrenergic beta-Agonists↗
PubMed1997

T cell receptor-mediated signaling is defective in T cells obtained from patients with primary intracranial tumors.

It has been well established that patients with malignant glioblastomas exhibit T cell anergy. In this report, we further investigate the nature of this T cell anergy. The results demonstrate that tumor size but not location correlates with decreased mitogen or anti-CD3 mAb responsiveness of T cells obtained from patients. Stimulation of the TCR/CD3 complex on these patients' T cells revealed defects in early transmembrane signaling. Both PHA and anti-CD3 mAb activated PBL and T cells obtained from patients exhibited a marked decrease in the tyrosine phosphorylation of a number of proteins. In particular, decreased phosphorylation of pp100 and phospholipase Cgamma1 (PLCgamma1) was observed. In addition, PLCgamma1 and p56(lck) protein levels were dramatically reduced in T cells obtained from patients harboring a glioma. In contrast, the protein levels of p59(fyn) were normal or only slightly reduced in T cells obtained from patients with gliomas. Quantitation of free intracellular calcium concentrations ([Ca2+]i) after mitogen (PHA) stimulation or ionomycin treatment of T cells obtained from patients revealed that they mobilize less calcium than do T cells obtained from normal subjects. Stimulation of T cells obtained from patients with PMA and ionomycin, which should bypass the requirement for PLCgamma1 activation as well as directly activate the p21(ras) signaling pathway, did not restore the proliferative capacity of these T cells to normal levels. These results indicate that the anergy observed in T cells obtained from these patients is a consequence of one or more defects in the early transmembrane signaling events associated with TCR/CD3 stimulation.

Brain Neoplasms↗
PubMed1997

Surface expression, polarization, and functional significance of CD73 in human intestinal epithelia.

During active intestinal inflammation polymorphonuclear leukocytes (PMN) transmigrate into the lumen and release 5'-AMP (J. Clin. Invest. 1993. 91:2320-2325). 5'-AMP is converted to adenosine by the apical epithelial surface with subsequent activation of electrogenic Cl- secretion (the basis of secretory diarrhea) via apical A2b adenosine receptors (J. Biol. Chem. 1995. 270:2387-2394). Using a polarized human intestinal epithelial monolayer (T84), we now characterize the basis of the observed conversion of 5'-AMP to adenosine required for this paracrine signaling pathway. An inhibitor of the ecto-5'-nucleotidase CD73, alpha, beta-methylene ADP (AOPCP), inhibited epithelial Cl- secretory responses to 5'-AMP, but not to authentic adenosine. Confocal immunofluorescent microscopy revealed CD73 to be surface expressed on both model and natural human intestinal epithelia. Expression was about sixfold greater on the apical cell surface as assessed biochemically by selective cell surface biotinylation, and morphologically by immunofluorescence. Treatment with phosphotidylinositol specific-phospholipase C (PI-PLC) released 95% of apical CD73, indicating that the intestinal CD73 possesses a glycosylphosphatidylinositol (GPI) anchor. Neither adenosine nor 5'-AMP stimulation induced intact T84 cells to shed surface CD73. The bulk of apical CD73 ( approximately 60%) was released from the cell surface by treatment with 1% Triton X-100 (TX-100) at 4 degrees C, but such release was not affected by pretreatment with ligand or by prior, antibody-mediated cross-linking of CD73. Subsequent analyses showed that the subpool of CD73 released by TX-100 at 4 degrees C was not truly solubilized, but rather represented TX-100-induced release of CD73-containing membrane fragments. These membrane fragments displayed light density on sucrose gradients characteristic of detergent insoluble glycosphingolipid-rich membrane domains (DIGs)/ caveolae, were solubilized by n-octyl glucoside (NOG, 1%) at 4 degrees C, and contained caveolin. These data indicate that human intestinal epithelia express CD73, which is apically polarized and targeted to microdomains with DIGs/caveolae characteristics. CD73 likely participates in translating paracrine, PMN-derived 5'-AMP signals to the authentic effector adenosine. These studies define CD73 as central to PMN-mediated intestinal Cl- secretion, the major directacting mechanism by which PMN induce intestinal epithelial Cl- secretion.

5'-Nucleotidase↗
PubMed1997

Catecholamine modulation of lymphocyte homing to lymphoid tissues.

Lymphocyte migration is an essential process for immune surveillance and for promoting cell-cell interactions necessary to generate an immune response. This report examined whether catecholamine prestimulation would alter the pattern of lymphocyte homing to spleen and lymph nodes in mice as determined by tracking fluorescently labeled cells. The results of cell sorter analysis showed that catecholamine-pretreated cells had increased accumulation in spleen and lymph nodes 1 and 2 h after i.v. injection. In addition, microscopic analysis showed that labeled cells migrated from the splenic red pulp to T-cell regions of the white pulp over a 2-h time course. Within the lymph nodes, labeled cells localized predominantly to the pericortex. Additional studies examined the migration of lymphocytes to lymphoid tissues of NGF-transgenic mice that have sympathetic hyperinnervation of spleen and peripheral lymph nodes. In contrast to the studies above, migration of T-cells from control mice to lymphoid tissues of the hyperinnervated mice was not different than that in control mice in most tissues. The accumulation of lymphocytes in lymphoid tissues is a balance between the influx of newly migrated cells and efflux back into the circulation. The studies in this report lend support to other studies showing catecholamine modulation of lymphocyte migration and homing, but it is a complex process about which much has yet to be understood.

Adrenergic beta-Agonists↗
PubMed1997

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation.

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 +/- 5% fall in transepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 +/- 2% (P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 microM ML-7 or 40 microM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 (P < 0.01), which was inhibited by ML-9 (P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa (P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

Animals↗
PubMed1996

Effects of NGF overexpression on anatomical and physiological properties of sympathetic postganglionic neurons.

To examine the effects of increased target derived nerve growth factor (NGF) on the sympathetic nervous system, the superior cervical ganglion was characterized in transgenic mice overexpressing NGF in keratinized epithelium (e.g. skin, tongue and oral cavity). In these mice NGF overexpression was achieved via expression of an NGF transgene driven by the K14 keratin promoter. This promoter is expressed at approximately embryonic day 11 and thereafter expressed constitutively in the adult. This expression results in supranormal levels of NGF in targets of sympathetic postganglionic neurons prior to the period of programmed cell death. Examination of postnatal day 6 (PN6) and adult transgenic mice shows ca. 2.5-fold increase in neuron number in the superior cervical ganglion (SCG). Analysis of SCG neuronal size revealed a dramatic hypertrophy in the transgenic mice that is present by PN6 and is maintained in the adult. Intracellular physiological measurements of control superior cervical ganglia identified two distinct types of neurons identified on the basis of their response to depolarizing current; 'phasic' neurons fire a single action potential while 'tonic' neurons fire continuously. In adult transgenic mice the phasic neurons were 102% larger than control phasic neurons while the tonic neurons only increased 44% relative to controls. The hypertrophy of sympathetic ganglia in the transgenic mice was correlated with an increased innervation of skin and dorsal root ganglia, structures that either express the transgene or concentrate NGF produced by the skin.

Animals↗
PubMed1996

Catecholamines decrease lymphocyte adhesion to cytokine-activated endothelial cells.

Numerous studies have shown that catecholamines can modulate lymphocyte migration. This effect may be mediated in part by modulation of lymphocyte-endothelial cell interactions, which is dependent on adhesion molecules expressed on both of these cells. Our results show that catecholamines decreased T-cell binding to IL-1 activated endothelial cells in vitro. The decrease in adhesion was not mediated by a change in adhesion molecule expression as LFA-1 and VLA-4 expression on T-cells and ICAM-1 and VCAM-1 expression on endothelial cells were not changed by catecholamine stimulation. T-cells flatten and enlarge the area of surface contact as they adhere to endothelial cells. Image analysis of the number of T-cells bound and the amount of cell spreading over several time points suggests that catecholamines alter the kinetics of T-cell-endothelial cell adhesion. These results support the hypothesis that catecholamines can alter lymphocyte-endothelial interactions in vivo, which in turn would affect lymphocyte migration.

Catecholamines↗
PubMed1995

cAMP accumulation in T-cells inhibits anti-CD3 monoclonal antibody-induced actin polymerization.

The results presented in this report offer a novel explanation for how stimulation of the beta-adrenergic receptor (beta AR) inhibits the ability of T cells to proliferate after interaction with immobilized anti-CD3 monoclonal antibody (mAb). Accordingly, T cells binding to immobilized anti-CD3 mAb but not anti-CD4 mAb undergo time-dependent F-actin assembly with concomitant formation of pseudopodia. This process is completely inhibited in the presence of isoproterenol (ISO) indicating that stimulation of the beta AR on T cells interferes with the biochemical processes responsible for the assembly of actin. To confirm these observations, we quantitated the formation of F-actin in T cells stimulated with immobilized anti-CD3 mAb in the presence of cAMP elevating agents. The results show that stimulation of the beta AR on T-cells, as well as the addition of forskolin or dibutyryl cAMP, abrogates the formation of F-actin.

Actins↗
PubMed1995

Signal transduction by cholera toxin: processing in vesicular compartments does not require acidification.

In the polarized human intestinal epithelial cell line T84, signal transduction by cholera toxin (CT) follows a complex series of events in which CT enters the apical endosome and moves through multiple vesicular compartments before it activates adenylate cyclase. As with processing of many other surface ligands, it has been suggested that CT must enter acidic vesicles to exert its downstream effects. To determine if intravesicular pH may regulate signal transduction by CT, we examined the cAMP-dependent Cl- secretory response [short-circuit current (Isc)] to CT in T84 cell monolayers treated with chloroquine (500 microM), methylamine (50 mM), NH4Cl (10 mM), nigericin (4 microM), or bafilomycin A1 (1 microM). Each of these reagents collapsed intravesicular pH gradients as confirmed by accumulation of acridine orange within subcellular compartments of living T84 cells imaged by confocal epifluorescence microscopy. Both acidotropic amines and nigericin inhibited the cAMP-dependent Cl secretory response in T84 cells. However, none of these reagents specifically affected adenylate cyclase itself or coupling of adenylate cyclase with the heterotrimeric guanosinetriphosphatase Gs as judged by the secretory response to the adenosine 3',5'-cyclic monophosphate (cAMP) agonists vasoactive intestinal peptide (VIP), forskolin, or 8-bromo-cAMP. In vitro enzyme-linked immunosorbent assay showed that CT binding to ganglioside GM1 was not dependent on pH between 5.0 and 10. Maximal Isc elicited by apical CT relative to maximal Isc elicited by VIP was not affected by pretreatment with chloroquine, methylamine, NH4Cl, or bafilomycin AI. Nigericin was the only reagent to inhibit CT-induced Isc (5 +/- 2% maximal response to VIP). The data indicate that low intravesicular pH will have little or no effect on CT association with its membrane receptor GM1, or on subsequent processing/signal transduction events.

Acids↗
PubMed1995

NGF modulates sympathetic innervation of lymphoid tissues.

Immune tissues are known to be innervated by the sympathetic nervous system, but little is known of what directs the innervation to specific tissue compartments. This report examines the sympathetic innervation of immune tissues in transgenic mice that overexpress nerve growth factor (NGF) in skin and other epithelial structures. NGF transgenic mice exhibited dramatic hyperinnervation in the splenic marginal zone, and the medulla and capsule of peripheral lymph nodes. In contrast, the transgenic mesenteric lymph nodes showed no hyperinnervation. This difference correlated with the location of these nodes; peripheral lymph nodes drain skin where the transgene was expressed while mesenteric lymph nodes drain non-transgene-expressing structures. In addition, the level of innervation correlated with the level of NGF peptide content as assayed by ELISA (3- and 13-fold increase in transgenic spleen and axillary lymph nodes, respectively; no increase in mesenteric nodes) and immunocytochemistry. RT-PCR showed that the NGF transgene was not being expressed in the immune tissues, suggesting that immune tissues can concentrate transgene-produced NGF. It was also demonstrated that the change in innervation had functional consequences. The mitogen response to concanavalin A (ConA) by spleen cells was decreased in the transgenics suggesting that elevated catecholamines or NGF can modulate the proliferative response of these cells. These mice demonstrate that NGF can modulate the sympathetic innervation and function of the immune system.

Animals↗
PubMed1994

Enhancement of beta-adrenergic-induced cAMP accumulation in activated T-cells.

Agonist stimulation of the beta-adrenergic receptor on T-cells results in the production of cAMP, which has been correlated with modulation of T-cell function. In previous studies, we have demonstrated that the mitogen PHA can synergistically enhance the accumulation of cAMP in T-cells in response to the agonist isoproterenol. In this report we have investigated the mechanisms by which dual stimulation of T-cells acts to synergistically enhance cAMP accumulation. The results demonstrate that increasing the levels of intracellular calcium with ionomycin or thapsigargin enhanced isoproterenol-induced cAMP accumulation in T-cells. In contrast, PHA enhanced isoproterenol-induced cAMP by a calcium-independent mechanism as evidenced by stimulation with isoproterenol plus PHA in calcium-free medium. Further studies revealed that PHA prevented both sequestration of the beta-adrenergic receptor and its dissociation from Gs protein in response to isoproterenol stimulation. In contrast, PHA did not prevent the functional uncoupling of the beta-adrenergic receptor from adenylyl cyclase, suggesting that additional mechanisms are likely involved. In summary, these studies demonstrate that dual receptor signalling of T-cells increases cAMP accumulation and offers a potential mechanism for catecholamine modulation of T-cell function.

Calcium↗
PubMed1990

Fluorescence histochemical techniques for catecholamines as tools in neurobiology.

Formaldehyde-induced and glyoxylic-acid-induced fluorescence histochemistry permits the tissue localization of catecholamines in the central nervous system (CNS) and peripheral nervous system (PNS), and in culture. Counterstains such as ethidium bromide provide excellent background identification of specific innervated regions in both the CNS and the periphery. Use of fluorescence histochemistry with immunocytochemistry can elucidate catecholamine-peptide relationships. Gelatin-ink perfusion used with fluorescence histochemistry permits the investigation of neuro-vascular relationships and documentation of vascular and parenchymal compartmentation of innervation. Combined use of fluorescence histochemistry and retrograde tracing methods demonstrates the specific cellular sources of innervation of target regions. Micropunch neurochemical analysis provides quantitative data for correlation with fluorescence histochemistry within a target region of innervation, and microspectrofluorometric analysis provides a semi-quantitative evaluation of the amount of fluorophore within a target region or within specific subcellular compartments such as the cell body or terminals.

Animals↗
PubMed1989

Neurotransmitter-lymphocyte interactions: dual receptor modulation of lymphocyte proliferation and cAMP production.

Stimulation of the beta-adrenergic receptor on lymphocytes can decrease the proliferative response of these cells to mitogens. We have found that simultaneous stimulation of T cells with the beta-adrenergic agonist isoproterenol and mitogens (phytohemagglutinin (PHA) and OKT3 monoclonal antibody) results in a 2- to 4-fold increase in cAMP production compared to cells exposed to isoproterenol alone. Mitogens alone have little effect on cAMP synthesis, but do activate the phosphatidylinositol (PI) cycle, suggesting that interactions may be occurring between the second messenger systems resulting in a cAMP synergy. Further experiments suggest that calcium may be involved in inducing the cAMP synergy observed in T cells. It is proposed that the synergy between beta-adrenergic and mitogenic stimulation of T cells for cAMP may be involved in the mechanism of catecholamine modulation of lymphocyte function.

Cyclic AMP↗
PubMed1987

Noradrenergic sympathetic innervation of the spleen: V. Acute drug-induced depletion of lymphocytes in the target fields of innervation results in redistribution of noradrenergic fibers but maintenance of compartmentation.

Sympathetic noradrenergic fibers follow the vasculature into the white pulp of the spleen and branch from the periarteriolar plexuses into T lymphocyte zones. These lymphocytes, reported to express adrenergic receptors, are contacted directly by norepinephrine (NE) terminals and are putative targets of the locally released NE. Although this splenic innervation has been studied extensively, the functional interdependence of the lymphocytes and the sympathetic innervation is not well understood. To assess the effect of acute lymphocyte loss on the splenic innervation, T and B lymphocytes were depleted through treatment with cyclophosphamide (CY) or hydrocortisone acetate (HC). Despite reductions in spleen weight and cellularity, the total NE content (pmol) per spleen did not change. However, the NE concentration increased in the treated spleens. Although the general compartmental organization of the noradrenergic fibers in the treated spleens was similar to that of controls, the NE fibers redistributed and increased in density around the smaller central arteries in lymphocyte depleted spleens. The accommodation of these NE fibers to the changing environment of the white pulp suggests that the innervation to the spleen remains chemically stable despite a large disruption to the normal splenic milieu, but is capable of plasticity in the face of a shrinking white pulp.

Animals↗