Anuska V. Andjelkovic

IL-12– and IL-23–modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition

The interleukin (IL)-12p40 family of cytokines plays a critical role in the development of experimental autoimmune encephalomyelitis (EAE). However, the relative contributions of IL-12 and IL-23 to the pathogenic process remain to be elucidated. Here, we show that activation of uncommitted myelin-reactive T cells in the presence of either IL-12p70 or IL-23 confers encephalogenicity. Adoptive transfer of either IL-12p70– or IL-23–polarized T cells into naive syngeneic hosts resulted in an ascending paralysis that was clinically indistinguishable between the two groups. However, histological and reverse transcription–polymerase chain reaction analysis of central nervous system (CNS) tissues revealed distinct histopathological features and immune profiles. IL-12p70–driven disease was characterized by macrophage-rich infiltrates and prominent NOS2 up-regulation, whereas neutrophils and granulocyte–colony-stimulating factor (CSF) were prominent in IL-23–driven lesions. The monocyte-attracting chemokines CXCL9, 10, and 11 were preferentially expressed in the CNS of mice injected with IL-12p70–modulated T cells, whereas the neutrophil-attracting chemokines CXCL1 and CXCL2 were up-regulated in the CNS of mice given IL-23–modulated T cells. Treatment with anti–IL-17 or anti–granulocyte/macrophage-CSF inhibited EAE induced by transfer of IL-23–polarized, but not IL-12p70–polarized, cells. These findings indicate that autoimmunity can be mediated by distinct effector populations that use disparate immunological pathways to achieve a similar clinical outcome.

Monocyte chemoattractant protein-1 regulation of blood-brain barrier permeability

The present study was designed to elucidate the effects of the chemokine monocyte chemoattractant protein (MCP-1) on blood–brain barrier (BBB) permeability. Experiments were conducted under in vitro conditions (coculture of brain endothelial cells and astrocytes) to study the cellular effects of MCP-1 and under in vivo conditions (intracerebral and intracerebroventricular administration of MCP-1) to study the potential contribution of MCP-1 to BBB disruption in vivo. Our results showed that MCP-1 induces a significant increase in the BBB permeability surface area product for fluorescein isothiocyanate (FITC)-albumin under in vivo conditions, particularly during prolonged (3 or 7 days) exposure (0.096±0.008 versus 0.031±0.005 μL/g min in controls at 3 days, P<0.001). Monocyte chemoattractant protein-1 also enhanced (17-fold compared with control) the permeability of the in vitro BBB (coculture) model. At the cellular level, MCP-1 causes alteration of tight junction (TJ) proteins in endothelial cells (redistribution of TJ proteins determined by Western blotting and loss of immunostaining for occludin, claudin-5, ZO-1, ZO-2). Monocyte chemoattractant protein-1-induced alterations in BBB permeability are mostly realized through the CCR2 receptor. Absence of CCR2 diminishes any effect of MCP-1 on BBB permeability in vitro and in vivo. The permeability surface area product for FITC-albumin after 3 days exposure to MCP-1 was 0.096±0.006 and 0.032±0.007 μL/g min, in CCR2+/+ and CCR2−/− mice, respectively (P<0.001). Monocytes/macrophages also participate in MCP-1-induced alterations in BBB permeability in vivo. Monocytes/macrophages depletion (by clodronate liposomes) reduced the effect of MCP-1 on BBB permeability in vivo ∼2 fold. Our results suggest that, besides its main function of recruiting leukocytes at sites of inflammation, MCP-1 also plays a role in ‘opening’ the BBB.

Potential role of MCP-1 in endothelial cell tight junction `opening': signaling via Rho and Rho kinase

The expression of the monocyte chemoattractant protein-1 (MCP-1) receptor CCR2 by brain endothelial cells suggests that MCP-1 may have other functions than purely driving leukocyte migration into brain parenchyma during inflammation. This study examines one of these potential novel roles of MCP-1 regulation of endothelial permeability using primary cultures of mouse brain endothelial cells. MCP-1 induces reorganization of actin cytoskeleton (stress fiber formation) and redistribution of tight junction proteins, ZO-1, ZO-2 occludin and claudin-5, from the Triton X-100-soluble to the Triton X-100-insoluble fractions. These morphological changes are associated with a decrease in transendothelial electrical membrane resistance and an increase in [14C]inulin permeability. MCP-1 did not induce these events in brain endothelial cells prepared from mice genotype CCR2–/–. The Rho kinase inhibitor Y27632 and inhibition of Rho (C3 exoenzyme, and dominant negative mutant of Rho, RhoT19N) prevented MCP-1-induced stress fiber assembly, reorganization of tight junction proteins and alterations in endothelial permeability. In all, this suggests that a small GTPase Rho and Rho kinase have a pivotal role in MCP-1-induced junction disarrangement. These data are the first to strongly suggest that MCP-1, via CCR2 present on brain endothelial cells, contributes to increased brain endothelial permeability.

Absence of the Chemokine Receptor CCR2 Protects Against Cerebral Ischemia/Reperfusion Injury in Mice

Background and Purpose— The chemokine, monocyte chemoattractant protein-1 (CCL2), is a major factor driving leukocyte infiltration into the brain parenchyma in a variety of neuropathologic conditions associated with inflammation, including stroke. In addition, recent studies indicate that CCL2 and its receptor (CCR2) could have an important role in regulating blood-brain barrier (BBB) permeability. This study evaluated the role of the CCL2/CCR2 axis in regulating postischemic inflammation, BBB breakdown, and vasogenic edema formation. Methods— CCR2−/− and CCR2+/+ mice were subjected to focal transient cerebral ischemia. BBB permeability and brain edema formation were observed at days 1 and 5 of reperfusion by evaluating the product surface area for fluorescein isothiocyanate–albumin and measuring water and electrolyte contents. Immunohistochemistry was used to assess leukocyte infiltration. cDNA gene and protein arrays for inflammatory cytokines were used to assess inflammatory profiles in CCR2+/+ and CCR2−/− mice. Results— CCR2−/− mice had reduced infarct sizes and significantly reduced BBB permeability and brain edema formation in the affected ischemic hemisphere compared with CCR2+/+ mice. This reduction in injury was closely associated with reduced infiltration of not only monocytes but also neutrophils (7- and 4-fold decreases, respectively). In addition, CCR2−/− mice had reduced expression/production of inflammatory cytokines during reperfusion. Conclusions— These data suggest that inhibiting the CCL2/CCR2 axis affects brain reperfusion outcome by reducing brain edema, leukocyte infiltration, and inflammatory mediator expression.

Brain endothelial cell-cell junctions: how to "open" the blood brain barrier.

The blood-brain barrier (BBB) is a highly specialized structural and biochemical barrier that regulates the entry of blood-borne molecules into brain, and preserves ionic homeostasis within the brain microenvironment. BBB properties are primarily determined by junctional complexes between the cerebral endothelial cells. These complexes are comprised of tight and adherens junctions. Such restrictive angioarchitecture at the BBB reduces paracellular diffusion, while minimal vesicle transport activity in brain endothelial cells limits transcellular transport. Under normal conditions, this largely prevents the extravasation of large and small solutes (unless specific transporters are present) and prevents migration of any type of blood-borne cell. However, this is changed in many pathological conditions. There, BBB disruption (“opening”) can lead to increased paracellular permeability, allowing entry of leukocytes into brain tissue, but also contributing to edema formation. In parallel, there are changes in the endothelial pinocytotic vesicular system resulting in the uptake and transfer of fluid and macromolecules into brain parenchyma. This review highlights the route and possible factors involved in BBB disruption in a variety of neuropathological disorders (e.g. CNS inflammation, Alzheimer’s disease, Parkinson’s disease, epilepsy). It also summarizes proposed signal transduction pathways that may be involved in BBB “opening”.

Protein Kinase Cα-RhoA Cross-talk in CCL2-induced Alterations in Brain Endothelial Permeability

Monocyte chemoattractant protein-1 (MCP-1 or CCL2) regulates blood-brain barrier permeability by inducing morphological and biochemical alterations in the tight junction (TJ) complex between brain endothelial cells. The present study used cultured brain endothelial cells to examine the signaling networks involved in the redistribution of TJ proteins (occludin, ZO-1, ZO-2, claudin-5) by CCL2. The CCL2-induced alterations in the brain endothelial barrier were associated with de novo Ser/Thr phosphorylation of occludin, ZO-1, ZO-2, and claudin-5. The phosphorylated TJ proteins were redistributed/localized in Triton X-100-soluble as well as Triton X-100-insoluble cell fractions. Two protein kinase C (PKC) isoforms, PKCα and PKCζ, had a significant impact on this event. Inhibition of their activity using dominant negative mutants PKCα-DN and PKCζ-DN diminished CCL2 effects on brain endothelial permeability. Previous data indicate that Rho/Rho kinase signaling is involved in CCL2 regulation of brain endothelial permeability. The interactions between the PKC and Rho/Rho kinase pathways were therefore examined. Rho, PKCα, and PKCζ activities were knocked down using dominant negative mutants (T17Rho, PKCα-DN, and PKCζ-DN, respectively). PKCα and Rho, but not PKCζ and Rho, interacted at the level of Rho, with PKCα being a downstream target for Rho. Double transfection experiments using dominant negative mutants confirmed that this interaction is critical for CCL2-induced redistribution of TJ proteins. Collectively these data suggest for the first time that CCL2 induces brain endothelial hyperpermeability via Rho/PKCα signal pathway interactions.

Effects of the Chemokine CCL2 on Blood–Brain Barrier Permeability during Ischemia–Reperfusion Injury

The chemokine CCL2 is considered as one of the main effectors driving postischemic infiltration of monocytes into the brain parenchyma. New experimental data, however, suggest that CCL2 could also participate in blood–brain barrier (BBB) ‘opening’ during the transmigration of monocytes. The current study examines the role of CCL2 in regulating BBB permeability after ischemia in vitro. To address this issue, an in vitro BBB model (coculture of astrocytes and brain endothelial cells) was subjected to 5 h of oxygen glucose deprivation, followed by reoxgenation (in vitro ischemia/reperfusion (I/R)) for 0 to 48 h. During reperfusion, there was a biphasic enhancement of barrier permeability, with a 200-fold increase in barrier permeability to FITC-albumin at 6 h and a further period of disruption around 24 h. The latter coincided with increased secretion of CCL2 by both astrocytes and brain endothelial cells and increased levels of the CCL2 receptor, CCR2. Applying antisense oligonucleotide or neutralizing antibody to block CCL2 significantly decreased I/R-induced enhancement of BBB permeability (approximately twofold) and redistribution of tight-junction (TJ) proteins (occludin, zonula occluden-1, 2, claudin-5). Similarly, absence of CCR2 from endothelial cells caused stabilization of TJ complexes and decreased the permeability of brain endothelial barrier during in vitro I/R. These data suggest CCL2/CCR2 has an important role in regulating brain endothelial permeability and might be a potential novel therapeutic target for stroke.

Caveolae-mediated internalization of occludin and claudin-5 during CCL2-induced tight junction remodeling in brain endothelial cells.

Disturbance of the tight junction (TJ) complexes between brain endothelial cells leads to increased paracellular permeability, allowing leukocyte entry into inflamed brain tissue and also contributing to edema formation. The current study dissects the mechanisms by which a chemokine, CCL2, induces TJ disassembly. It investigates the potential role of selective internalization of TJ transmembrane proteins (occludin and claudin-5) in increased permeability of the brain endothelial barrier in vitro. To map the internalization and intracellular fate of occludin and claudin-5, green fluorescent protein fusion proteins of these TJ proteins were generated and imaged by fluorescent microscopy with simultaneous measurement of transendothelial electrical resistance. During CCL2-induced reductions in transendothelial electrical resistance, claudin-5 and occludin became internalized via caveolae and further processed to early (EEA1+) and recycling (Rab4+) endosomes but not to late endosomes. Western blot analysis of fractions collected from a sucrose gradient showed the presence of claudin-5 and occludin in the same fractions that contained caveolin-1. For the first time, these results suggest an underlying molecular mechanism by which the pro-inflammatory chemokine CCL2 mediates brain endothelial barrier disruption during CNS inflammation.

CCL2 regulates angiogenesis via activation of Ets-1 transcription factor

Although recent studies have suggested that CC chemokine CCL2 may directly affect the angiogenesis, the signaling events involved in such regulation remain to be determined. This study investigated a potential signal mechanism involved in CCL2-induced angiogenesis. Our in vitro and in vivo (hemangioma model of angiogenesis) experiments confirmed earlier findings that CCL2 can induce angiogenesis directly. Using a gene array analysis, CCL2 was found to induce expression of several angiogenic factors in brain endothelial cells. Among the most prominent was an up-regulation in Ets-1 transcription factor. CCL2 induced a significant increase in Ets-1 mRNA and protein expression as well as Ets-1 DNA-binding activity. Importantly, Ets-1 antisense oligonucleotide markedly abrogated in vitro CCL2-induced angiogenesis, suggesting that Ets-1 is critically involved in this process. Activation of Ets-1 by CCL2 further regulated some of Ets-1 target molecules including β3 integrins. CCL2 induced significant up-regulation of β3 mRNA and protein expression, and this effect of CCL2 was prevented by the Ets-1 antisense oligonucleotide. The functional regulation of Ets-1 activity by CCL2 was dependent on ERK-1/2 cascade. Inhibition of ERK1/2 activity by PD98509 prevented CCL2-induced increases in Ets-1 DNA-binding activity and Ets-1 mRNA expression. Based on these findings, we suggest that Ets-1 transcription factor plays a critical role in CCL2 actions on brain endothelial cells and CCL2-induced angiogenesis.

Inflammation and brain edema: new insights into the role of chemokines and their receptors.

Brain edema is associated with a variety of neuropathological conditions such as brain trauma, ischemic and hypoxic brain injury, central nervous system infection, acute attacks of multiple sclerosis, and brain tumors. A common finding is an inflammatory response, which may have a significant impact on brain edema formation. One critical event in the development of brain edema is blood-brain barrier (BBB) breakdown, which may be initiated and regulated by several proinflammatory mediators (oxidative mediators, adhesion molecules, cytokines, chemokines). These mediators not only regulate the magnitude of leukocyte extravasation into brain parenchyma, but also act directly on brain endothelial cells causing the loosening of junction complexes between endothelial cells, increasing brain endothelial barrier permeability, and causing vasogenic edema. Here we review junction structure at the BBB, the effects of pro-inflammatory mediators on that structure, and focus on the effects of chemokines at the BBB. New evidence indicates that chemokines (chemoattractant cytokines) do not merely direct leukocytes to areas of injury. They also have direct and indirect effects on the BBB leading to BBB disruption, facilitating entry of leukocytes into brain, and inducing vasogenic brain edema formation. Chemokine inhibition may be a new therapeutic target to reduce vasogenic brain edema.