Sonia L. Carlson

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.

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.

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.

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.

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.

Platelet-activating factor induces cell death in cultured astrocytes and oligodendrocytes: Involvement of caspase-3

The biologically active lipid metabolite, platelet‐activating factor (PAF), is thought to contribute to inflammatory processes and tissue damage in a variety of central nervous system (CNS) injuries. In previous studies, we found that after contusion spinal cord injury, treatment with a PAF antagonist led to significantly increased white matter tissue sparing as well as decreased mRNA levels for pro‐inflammatory cytokines. Some studies suggest that PAF can also have toxic effects on neurons in vitro. Few studies, however, have examined the effects of PAF on glial cells of the CNS. In the present study, the potential for PAF to act as a toxin to cultured astrocytes was examined. Also investigated were the effects of PAF on oligodendrocytes at two different stages of development. Treatment with 0.02–2 μM PAF for 72 h resulted in significant levels of cell death in both cell types (P < 0.05), an effect that was blocked by the PAF receptor antagonists, WEB 2170 and BN 52021. To investigate PAF‐induced glial cell death further, we looked for activation of the enzyme, caspase‐3, which can be indicative of apoptosis. Immunocytochemistry demonstrated that PAF at all concentrations caused activation of caspase‐3 at 24, 48, and 72 h after treatment in both cell types. Caspase‐3‐dependent cell death was further confirmed using knockout mice (−/−) deficient in the caspase‐3 gene. Toxicity was lost when astrocytes (−/−) were exposed to 0.02–2 μM PAF (P < 0.01). Oligodendrocytes (−/−) were not susceptible to toxicity at 2 μM PAF (P < 0.001). The results demonstrate that the pro‐inflammatory molecule, PAF, induces cell death in cultured CNS glial cells and that this effect is, in part, dependent on caspase‐3 activation. GLIA 38:228–239, 2002.

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.

PAF antagonist treatment reduces pro-inflammatory cytokine mRNA after spinal cord injury.

Platelet-activating factor (PAF) is a pro-inflammatory molecule which contributes to secondary damage after spinal cord injury (SCI). To test if PAF contributes to cytokine induction following SCI, female Long-Evans rats were pretreated with the PAF antagonist WEB 2170 prior to receiving a contusion injury at spinal cord level T10 using the NYU impactor. RNase protection assay (RPA) analysis revealed that IL-1α mRNA peaked at 1 h post-injury while IL-1β and IL-6 mRNA levels were higher and peaked at 6 h. TNF-α mRNA was almost undetectable. All mRNA levels approached baseline by 24 h. Treatment with WEB 2170 (1 mg/kg, i.p.) 15 min prior to injury significantly decreased mRNA levels for all three cytokines at 6 h post-injury, but not at 1 h post-injury. These results demonstrate a role for PAF in proinflammatory cytokine induction after SCI.

[8] Preparation, characterization, and use of human and rodent lymphocytes, monocytes, and neutrophils

Publisher Summary This chapter describes some simple, concise protocols for the preparation, characterization, and use of leukocytes. The model systems used to examine the role of the nervous and endocrine systems in degenerative and inflammatory disease processes have ranged from simple in vitro proliferative assays to complex in vivo systems. Much of the progress that has been made in understanding the mechanisms of neuroimmunomodulation has come from in vitro studies using primary cultures of leukocytes. The chapter also describes methods that can be used to prepare leukocytes for in vitro studies, as well as methods for lymphocyte activation and analysis of second-messenger production. Several factors should be considered when designing a set of experiments. One of the most important is context.

[23] Modulation of leukocyte adhesion, migration, and homing by neurotransmitters and neuropeptides

Publisher Summary The effectiveness of the immune system is largely dependent on the mobile nature of leukocytes and the chance interaction of lymphocytes and antigen. Thus, lymphocytes are constantly recirculating between the blood and tissues as part of immune surveillance. For leukocytes (lymphocytes, monocytes, neutrophils) in the blood to enter a tissue or inflammatory site, the leukocytes must adhere to specialized endothelial cells found in postcapillary venules called high endothelial cells (HEC). These specialized endothelial cells are more cuboidal in shape than other endothelial cells and express specific adhesion molecules that can bind to adhesion molecules expressed on leukocytes. Among the techniques that are helpful in studying the effect of the nervous system on lymphocyte homing are methods to examine lymphocyte migration and homing in vivo and methods to examine lymphocyte binding to endothelial cells in vitro .