Alexander S. Easton

Modulation of blood-brain barrier permeability by neutrophils: in vitro and in vivo studies

The blood-brain barrier (BBB) restricts solute permeability across healthy cerebral endothelial cells. However, during inflammation, permeability is increased and can lead to deleterious cerebral edema. Neutrophils are early cellular participants in acute inflammation, but their effect on BBB permeability is unclear. To study this, neutrophils were applied in a resting and activated state to in vitro and in vivo models of the BBB. In vitro, human neutrophils (5 x 10(6)/ml) were activated with tumor necrosis factor (100 U/ml) and leukotriene B(4) (10(-7) mol/l). Untreated neutrophils reduced permeability across the human brain endothelial cell line hCMEC/D3. Activated neutrophils returned permeability to baseline, an effect blocked by the reactive oxygen scavengers superoxide dismutase (10 U/ml) and catalase (1000 U/ml). In vivo, human neutrophils (2.5 x1 0(5) in 4 microl) were injected into the striatum of anesthetized juvenile Wistar rats, and BBB permeability measured 30 min later. This was compared to control injections (4 microl) of vehicle (0.9% saline) and arachidonic acid (10(-3) mol/l). The injection generated a small hematoma around the injection tract (<3 microl). Untreated neutrophils induced significantly lower permeability in their vicinity than activated neutrophils, with a trend to lowered permeability compared to the vehicle control. Neither untreated nor activated neutrophils induced permeability increases, while arachidonic acid increased permeability as a positive control. This study further delineates the effect of neutrophils on the BBB, and demonstrates that resting neutrophils induce acute reductions in permeability while activated neutrophils have a neutral effect. The in vivo model reiterates some aspects of acute intracerebral hemorrhage.

Neutrophils and stroke - can neutrophils mitigate disease in the central nervous system?

Neutrophils are first responders to injury in inflammatory diseases of the central nervous system (CNS) such as ischemic stroke, trauma and intracerebral hemorrhage. Studies carried out in the last three decades showed that neutrophils have mixed effects in animal models of stroke. Some studies correlated the presence of neutrophils to injury. When neutrophil infiltration was reduced by targeting CD18 or intercellular adhesion molecule-1 (ICAM-1) this generated improved outcomes. However other studies showed that when neutrophil infiltration was stimulated prior to stroke, this reduced the burden of disease. Clinical trials did not show a benefit in stroke patients from neutrophil blockade. Neutrophils may be subject to a threshold effect. When they reach a critical ratio relative to the volume of injury in the CNS, they adopt an anti-inflammatory phenotype that is able to reduce disease. When neutrophil infiltration was stimulated by injecting a stroke site in the rat with the chemokine CXCL1, this resulted in reductions in vascular permeability. Similar reductions in permeability were modeled in tissue culture models, in which neutrophils were applied to monolayers of brain endothelial cells. Neutrophils blocked the permeability increases associated with oxygen-glucose deprivation in human brain endothelial monolayers. The evidence suggests that neutrophils might adopt a pro-inflammatory N1 phenotype or an anti-inflammatory N2 phenotype in the CNS depending on environmental cues. The N2 phenotype may be adopted when neutrophil numbers exceed a critical threshold. This suggests that strategies that promote neutrophil infiltration into stroke, and other CNS inflammatory diseases, could result in improved outcomes.

Evidence that tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits angiogenesis by inducing vascular endothelial cell apoptosis

Tumor necrosis factor (TNF) and its related ligands TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL) play roles in the regulation of vascular responses, but their effect on the formation of new blood vessels (angiogenesis) is unclear. Therefore, we have examined the effects of these ligands on angiogenesis modeled with primary cultures of human umbilical vein endothelial cells (HUVEC). To examine angiogenesis in the context of the central nervous system, we have also modeled cerebral angiogenesis with the human brain endothelial cell line hCMEC/D3. Parameters studied were bromodeoxyuridine (BrdU) incorporation and cell number (MTT) assay (to assess endothelial proliferation), scratch assay (migration) and networks on Matrigel (tube formation). In our hands, neither TRAIL nor FasL (1, 10, and 100 ng/ml) had an effect on parameters of angiogenesis in the HUVEC model. In hCMEC/D3 cells by contrast, TRAIL inhibited all parameters (10-100 ng/ml, 24h). This was due to apoptosis, since its action was blocked by the pan-caspase inhibitor zVADfmk (5 x 10(-5) mol/l) and TRAIL increased caspase-3 activity 1h after application. However FasL (100 ng/ml) increased BrdU uptake without other effects. We conclude that TRAIL has different effects on in vitro angiogenesis depending on which model is used, but that FasL is generally ineffective when applied in vitro. The data suggest that TRAIL primarily influences angiogenesis by the induction of vascular endothelial apoptosis, leading to vessel regression.

Regulation of Permeability Across the Blood-Brain Barrier

The blood-brain barrier refers to the very low permeability across microvessels in the Central Nervous System (CNS), created by the interaction between vascular endothelial cells and surrounding cells of the neurovascular unit. Permeability can be modulated (increased and decreased) by a variety of factors including inflammatory mediators, inflammatory cells such as neutrophils and through alterations in the phenotype of blood vessels during angiogenesis and apoptosis. In this chapter, some of these factors are discussed as well as the challenge of treating harmful increases in permeability that result in brain swelling (vasogenic cerebral edema).

Bevacizumab diminishes experimental autoimmune encephalomyelitis by inhibiting spinal cord angiogenesis and reducing peripheral T-cell responses.

Abstract Angiogenesis in the animal model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE) is regulated by vascular endothelial growth factor (VEGF) and angiopoietin-2. We determined whether VEGF blockade with the anti-VEGF monoclonal antibody bevacizumab could inhibit angiogenesis and affect peripheral pathogenic immune responses in EAE. Mice treated with bevacizumab from the time of onset of clinical signs showed reduced clinical and pathologic scores. Bevacizumab suppressed angiogenesis and reduced angiopoietin-2 expression at Day 21 but had no effect on VEGF upregulation at Day 14. Messenger RNA levels for the angiogenesis-related protein CD105 were increased at Day 14. Bevacizumab reduced vascular permeability in the spinal cord at Day 14 and Day 21. In peripheral lymph nodes, it induced retention of CD4-positive T cells and inhibited T-cell proliferation. It also reduced mononuclear cell infiltration into spinal cord and the relative proportion of T cells. Isolated lymphoid cells showed reduced secretion of the T-helper 17 (Th-17) cell cytokine interleukin 17 and the Th-1 cytokine interferon-&ggr;. When bevacizumab was added to naive T cells or to antigen-stimulated T cells from mice with untreated EAE in vitro, it had no effect on proliferation or the secretion of interleukin 17 or interferon-&ggr;. These data indicate that bevacizumab ameliorates vascular and T-cell responses during EAE, but its effects on T cells may be indirect, possibly by suppressing angiogenesis.

Amyloid β-related Angiitis of the Central Nervous System: Report of 3 Cases

OBJECTIVE Amyloid-β (Aβ) related angiitis (ABRA) is a recently described clinicopathological entity characterized by cerebrovascular Aβ deposition and arteritis. Cerebral Aβ deposition is commonly present in cerebal amyloid angiopathy (CAA) and Alzheimers disease (AD) but is rarely associated with inflammatory infiltration of vessel walls. Our objective is to help clarify the clinical spectrum, radiographic findings, response to treatment, and outcomes of ABRA. The neuropathological relationship between ABRA, cerebral amyloid angiopathy, and Alzheimers disease is discussed. METHODS We present three cases of ABRA managed at a tertiary care centre. RESULTS All three patients presented with seizures and cognitive dysfunction; one had multifocal neurologic findings. Brain biopsies revealed inflammatory arteritis with Aβ deposits in the vessel walls. All were treated with steroids and cyclophosphamide. Two had favorable outcomes and one stabilized but with severe residual neurologic disability. CONCLUSIONS ABRA is an unusual but likely under-recognized and potentially treatable disorder. As in other reported cases, our findings suggest that many patients respond favorably to immunosuppressive therapy. We believe that all biopsy specimens consistent with primary angiitis of the central nervous system (CNS) should be further examined for vascular Aβ deposition.

Neutrophils block permeability increases induced by oxygen glucose deprivation in a culture model of the human blood-brain barrier.

Experimentally, oxygen glucose deprivation (OGD) has been widely used to mimic the environmental conditions present during cerebral ischemia-reperfusion (IR) injury. OGD is known to increase permeability across cultured cerebral endothelial cells, which models the effect of IR on permeability across the blood-brain barrier (BBB); however, studies have yet to be performed in a human model. The effect of neutrophils on the increase in BBB permeability associated with IR injury has yet to be modeled in vitro. To address these questions, the human brain endothelial cell line hCMEC/D3 was exposed to OGD with reoxygenation, and permeability was measured for a range of OGD exposure times (1-24h). One hour of exposure to OGD induced a reversible increase in permeability, unassociated with cytotoxicity (assessed from lactate dehydrogenase release). However, 12-24h OGD exposures induced sustained increases in permeability associated with cytotoxicity. The 1h permeability increase was inhibited with the nitric oxide synthase inhibitors l-NAME (10(-)(7)mol/l) and 1400W (10(-)(7)mol/l). Neutrophils (5x10(6)/ml) blocked the permeability increase associated with 1h OGD, whether applied during or after OGD exposure. Permeability remained low if neutrophils were activated with leukotriene (Lt)B(4) (10(-)(7)mol/l) or exposed to a transendothelial gradient of LtB(4), while neutrophil activation with phorbyl myristate acetate (4x10(-)(8)mol/l) induced a small increase. Neutrophils had no effect on the permeability increase induced by 12h OGD exposure. This study finds that OGD induces reversible increases in permeability linked to nitric oxide synthesis in a human culture model of the BBB and shows that neutrophils mitigate permeability increases in this context.

Angiogenesis is regulated by angiopoietins during experimental autoimmune encephalomyelitis and is indirectly related to vascular permeability.

Abstract The regulation of angiogenesis was studied over the course of the animal model of multiple sclerosis, acute experimental autoimmune encephalomyelitis (EAE) in mice using immunohistochemistry. During EAE, angiogenesis peaked 21 days after disease induction, with significant increases in gray matter and adjacent to the leptomeninges. Angiogenesis correlated with clinical and pathologic scores. Spinal cord expression of angiopoietin 1 (Ang-1) by neurons and glia was reduced at Day 14, but expression by inflammatory cells restored earlier levels at Day 21. Angiopoietin 2 expression increased markedly at Day 21 and was mostly associated with inflammatory cells. Levels of the angiopoietin receptor Tie-2 were reduced at Day14, but recovered by day D21. Double labeling demonstrated Ang-1 expression on infiltrating CD3-positive T cells; Ang-2 was expressed by monocytes/macrophages. During EAE, the expression of vascular endothelial growth factor peaked at Day 14 and began to decrease by Day 21. Double labeling showed expression of Tie-2 andvascular endothelial growth factor receptor 2 but not Ang-2 in blood vessels at Day 21. Vascular permeability increased early in EAE, butwas reduced by Day21. Although individual values did notcorrelate with angiogenesis, the volume of permeable tissue showed a weak positive correlation with angiogenesis. These temporal changes in angiogenic factors suggest an integral role during EAE-related angiogenesis.

Human Brain Chemokine and Cytokine Expression in Sepsis: A Report of Three Cases.

BACKGROUND Sepsis is a systemic response to infection that can affect brain function by inducing resident cells (including astrocytes and microglia) to generate brain chemokines and cytokines. However, there are few studies on the human brain. Since this information may shed further light on pathogenesis, our study objective was to measure the expression of 36 chemokines and cytokines in autopsied brain from 3 cases of sepsis and 10 controls, and to relate this to astrocyte and microglial activation. METHODS The right frontal pole was removed at autopsy and chemokine and cytokine expression measured by multiplexed enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction (qPCR). Immunohistochemistry and image analysis were carried out to determine the expression of glial fibrillary acidic protein (GFAP), a marker of activated astrocytes, and CD68 and CD45, markers of activated microglial cells. RESULTS Concentrations of the chemokines CXCL8, CXCL10, CXCL12, CCL13 and CCL22 were increased in pooled data from the three cases of sepsis (p<0.05); however, their messenger RNA (mRNA) expression was unaltered. CXCL13, CXCL1, CXCL2, CCL1, CCL2, CCL8, CCL20, (interleukin) IL-16, IL-1β and (tumour necrosis factor) TNF concentrations showed increases in two of three sepsis cases. Additionally, individual sepsis cases showed increases in mRNA expression for HDAC (histone deacetylase) 6 and EIF (eukaryotic translation initiation factor) 4A2. Brain GFAP expression was significantly increased (p<0.05) in pooled data from the three sepsis cases. Individual sepsis cases showed increases in CD68 or CD45 expression. CONCLUSIONS These expression patterns add to our understanding of the pathogenesis of sepsis and its effects on the brain.

Murine T cell activation is regulated by surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide)

Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) binds to glycosaminoglycans (GAGs) and has been shown to influence their function, and the function of proteoglycans (complexes of GAGs linked to a core protein). T cells synthesize, secrete and express GAGs and proteoglycans which are involved in several aspects of T cell function. However, there are as yet no studies on the effect of GAG-binding agents such as surfen on T cell function. In this study, surfen was found to influence murine T cell activation. Doses between 2.5 and 20 μM produced a graduated reduction in the proliferation of T cells activated with anti-CD3/CD28 antibody-coated T cell expander beads. Surfen (20 mg/kg) was also administered to mice treated with anti-CD3 antibody to activate T cells in vivo. Lymphocytes from surfen-treated mice also showed reduced proliferation and lymph node cell counts were reduced. Surfen reduced labeling with a cell viability marker (7-ADD) but to a much lower extent than its effect on proliferation. Surfen also reduced CD25 (the α-subunit of the interleukin (IL)-2 receptor) expression with no effect on CD69 expression in T cells treated in vivo but not in vitro. When receptor activation was bypassed by treating T cells in vitro with phorbyl myristate acetate (10 ng/ml) and ionomycin (100 ng/ml), surfen treatment either increased proliferation (10 μM) or had no effect (2.5, 5 and 20 μM). In vitro treatment of T cells with surfen had no effect on IL-2 or interferon-γ synthesis and did not alter proliferation of the IL-2 dependent cell line CTLL-2. The effect of surfen was antagonized dose-dependently by co-treatment with heparin sulfate. We conclude that surfen inhibits T cell proliferation in vitro and in vivo. When T cell receptor-driven activation is bypassed surfen had a neutral or stimulatory effect on T cell proliferation. The results imply that endogenous GAGs and proteoglycans play a complex role in promoting or inhibiting different aspects of T cell activation.