Barbara Tucky

Human cerebrospinal fluid central memory CD4+ T cells: Evidence for trafficking through choroid plexus and meninges via P-selectin

Cerebrospinal fluid (CSF) from healthy individuals contains between 1,000 and 3,000 leukocytes per ml. Little is known about trafficking patterns of leukocytes between the systemic circulation and the noninflamed CNS. In the current study, we characterized the surface phenotype of CSF cells and defined the expression of selected adhesion molecules on vasculature in the choroid plexus, the subarachnoid space surrounding the cerebral cortex, and the cerebral parenchyma. Using multicolor flow cytometry, we found that CSF cells predominantly consisted of CD4+/CD45RA-/CD27+/CD69+-activated central memory T cells expressing high levels of CCR7 and L-selectin. CD3+ T cells were present in the choroid plexus stroma in autopsy CNS tissue sections from individuals who died without known neurological disorders. P- and E-selectin immunoreactivity was detected in large venules in the choroid plexus and subarachnoid space, but not in parenchymal microvessels. CD4+ T cells in the CSF expressed high levels of P-selectin glycoprotein ligand 1, and a subpopulation of circulating CD4+ T cells displayed P-selectin binding activity. Intercellular adhesion molecule 1, but not vascular cell adhesion molecule 1 or mucosal addressin cell adhesion molecule 1, was expressed in choroid plexus and subarachnoid space vessels. Based on these findings, we propose that T cells are recruited to the CSF through interactions between P-selectin/P-selectin ligands and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 in choroid plexus and subarachnoid space venules. These results support the overall hypothesis that activated memory T cells enter CSF directly from the systemic circulation and monitor the subarachnoid space, retaining the capacity to either initiate local immune reactions or return to secondary lymphoid organs.

Expression of CCR7 in multiple sclerosis: Implications for CNS immunity

It is unclear how immune cells traffic between the lymphoid compartment and the central nervous system (CNS), which lacks lymphatic vessels and is shielded by the blood–brain barrier. We studied the expression of CCR7, a chemokine receptor required for migration of T cells and dendritic cells (DCs) to lymphoid organs, in the CNS of patients with multiple sclerosis (MS) to gain insight into pathways for CNS immune cell trafficking. Inflamed MS lesions contained numerous CCR7+ myeloid cells expressing major histocompatibility complex class II, CD68 and CD86, consistent with maturing DCs. CCR7+ DCs also were identified in cerebrospinal fluid (CSF). These observations suggested that the afferent limb of CNS immunity is comprised, in part, of DCs, which are generated within the CNS and migrate to deep cervical lymph nodes through the CSF after antigen capture. Ninety percent of CSF T cells expressed CCR7 and CSF from patients with MS was relatively depleted of CCR7‐negative effector‐memory T cells. In contrast, all T cells in parenchymal MS lesions lacked CCR7, indicating local retention and differentiation of central‐memory T cells upon restimulation by antigen within the CNS. These data suggested that the efferent limb of CNS immunity is executed by central‐memory T cells, which enter CSF directly from the circulation. Ann Neurol 2004

T-cells in the cerebrospinal fluid express a similar repertoire of inflammatory chemokine receptors in the absence or presence of CNS inflammation: Implications for CNS trafficking

It is believed that chemokines and their receptors are involved in trafficking of T‐cells to the central nervous system (CNS). The aim of the current study was to define the expression on cerebrospinal fluid (CSF) T‐cells of six chemokine receptors associated with trafficking to sites of inflammation. Flow cytometry was used to detect chemokine receptor expression. We observed that CD3+T‐cells in the CSF express a restricted array of inflammatory chemokine receptors, specifically CXCR3, CCR5 and CCR6, but little CCR1‐3. This repertoire was independent of the presence of CNS inflammation, since comparable findings were obtained in patients with multiple sclerosis (MS) and individuals with non‐inflammatory neurological diseases. The enrichment of CCR5+T‐cells in the CSF could largely be explained by higher frequency of CD4+/CD45RO+T‐cells in this compartment. In contrast, CD4+/CD45RO+T‐cells expressing CXCR3 were significantly enriched in CSF as compared with blood. Similar levels of CCR6+/CD3+T‐cells were observed in blood and CSF, while levels of CCR2+/CD3+T‐cells were lower in CSF than in blood. The CSF was virtually devoid of CCR5+/CXCR3‐ T‐cells, suggesting that the expression of CCR5 alone is not sufficient for the trafficking of CD3+T‐cells to the CSF. We hypothesize that CXCR3 is the principal inflammatory chemokine receptor involved in intrathecal accumulation of T‐cells in MS. Through interactions with its ligands, CXCR3 is proposed to mediate retention of T‐cells in the inflamed CNS.

Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

Endothelial cells that functionally express blood brain barrier (BBB) properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs) and human umbilical vein endothelial cells (HUVECs). With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER) and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

Imaging Correlates of Leukocyte Accumulation and CXCR4/CXCL12 in Multiple Sclerosis

OBJECTIVE To compare leukocyte accumulation and expression of the chemokine receptor/ligand pair CXCR4/CXCL12 in magnetic resonance imaging-defined regions of interest (ROIs) in brains from patients with chronic multiple sclerosis. We studied the following ROIs: normal-appearing white matter (NAWM); regions abnormal only on T2-weighted images (T2 only); and regions abnormal on T2- and T1-weighted images with an abnormal magnetization transfer ratio (T2/T1/MTR). DESIGN Case-control study. SETTING Cleveland Clinic. PATIENTS Brain tissue was acquired from 5 patients with secondary progressive multiple sclerosis (MS) and 5 nonneurological controls. INTERVENTION Magnetic resonance imaging pathological correlations were performed on the 5 cases. Based on imaging characteristics, 30 ROIs were excised. MAIN OUTCOME MEASURE Using immunohistochemical analysis, we evaluated myelin status, leukocyte accumulation, and CXCR4/CXCL12 expression in the MS ROIs and white matter regions from the 5 nonneurological controls. RESULTS Eight of 10 T2/T1/MTR regions were chronic active or chronic inactive demyelinated lesions, whereas only 2 of 10 T2-only regions were demyelinated and characterized as active or chronic active lesions. Equivalent numbers of CD68+ leukocytes (the predominant cell type) were present in myelinated T2-only regions as compared with NAWM. Parenchymal T cells were significantly increased in T2/T1/MTR ROIs as compared with T2-only regions and NAWM. Expression of CXCR4 and phospho-CXCR4 were found on reactive microglia and macrophages in T2-only and T2/T1/MTR lesions. CXCL12 immunoreactivity was detected in astrocytes, astrocytic processes, and vascular elements in inflamed MS lesions. CONCLUSIONS Inflammatory leukocyte accumulation was not increased in myelinated MS ROIs with abnormal T2 signal as compared with NAWM. Robust expression of CXCR4/CXCL12 on inflammatory elements in MS lesions highlights a role of this chemokine/receptor pair in central nervous system inflammation.

Chemokine receptors on infiltrating leucocytes in inflammatory pathologies of the central nervous system (CNS)

Haematogenous leucocytes enter the central nervous system (CNS) during diverse disorders of varied aetiologies. Understanding the trafficking cues that mediate CNS leucocyte infiltration might promote the development of flexible and selective means to modulate inflammation to achieve clinical benefit. The trafficking machinery of leucocytes has been elucidated during the past decade and consists of cell‐surface adhesion molecules, chemoattractant cytokines (chemokines) and their receptors. Recent work in our laboratory characterized chemokine receptors found on T lymphocytes and monocytes in brain sections from subjects with one pathological subtype of multiple sclerosis (MS), an immune‐mediated inflammatory demyelinating disease. In these tissues, the types 1 and 5 CC chemokine receptors (CCR1 and CCR5) were detected on perivascular monocytic cells whereas only CCR5 was present on parenchymal macrophages. The type 3 CXC chemokine receptor (CXCR3) was present on virtually all CD3‐positive T cells. In the current study, we evaluated the expression of these receptors on the infiltrating cells present in cases of other inflammatory CNS disorders including those of dysimmune, infectious, neoplastic, and vascular aetiology. Perivascular and parenchymal monocytic cells expressed CCR1 in all cases and CXCR3 was consistently present on a substantial proportion of CD3+ T cells. The occurrence of CCR5 on parenchymal macrophages was much less uniform across the varied disorders. These data implicate CCR1 in monocyte infiltration of the CNS and are consistent with reports of studies in CCR1‐deficient mice. CXCR3 is also likely to play a role in accumulation  of  T  cells  in  the inflamed  CNS.  By  contrast, our findings suggest that regulation of CCR5 on phagocytic macrophages may be contingent on the lesion environment.

CC chemokine receptor 8 in the central nervous system is associated with phagocytic macrophages

CC chemokine receptor 8 (CCR8) has been detected in vitro on type 2 helper and regulatory lymphocytes, which might exert beneficial functions in multiple sclerosis (MS) and on macrophages and microglia, possibly promoting tissue injury in MS lesions. To discriminate the relevant expression pattern in vivo, we defined the cell types that expressed CCR8 in MS lesions and determined the relationship of CCR8 expression and demyelinating activity. CCR8 was not expressed on T cells but was associated with phagocytic macrophages and activated microglia in MS lesions and directly correlated with demyelinating activity. To identify factors associated with CCR8 expression, the study was extended to other central nervous system (CNS) pathologies. CCR8 was consistently expressed on phagocytic macrophages and activated microglia in stroke and progressive multifocal leukoencephalopathy, but not expressed on microglia in pathologies that lacked phagocytic macrophages such as senile change of the Alzheimers type. CCR8 was up-regulated by macrophage differentiation and activating stimuli in vitro. In summary CNS CCR8 expression was associated with phagocytic macrophages and activated microglial cells in human CNS diseases, suggesting that CCR8 may be a feasible target for therapeutic intervention in MS. CCR8 expression may also indicate a selective program of mononuclear phagocyte gene expression.

α4 Integrin/FN-CS1 mediated leukocyte adhesion to brain microvascular endothelial cells under flow conditions

Insights into sequential leukocyte-endothelial interactions during leukocyte trafficking have been obtained through experiments using human umbilical vein endothelial cells (HUVEC) under flow conditions. To investigate leukocyte-brain endothelial cell interactions, we developed a dynamic in vitro system, using Transfected Human Brain Microvascular Endothelial Cells (THBMEC) and a parallel plate flow chamber. Human peripheral blood mononuclear cells (PBMC) were perfused across confluent THBMEC cultures at a velocity that approximates the rate found in human brain capillaries. Leukocyte-THBMEC interactions were visualized by phase-contrast microscopy, and images were captured on a CCD camera. To simulate inflammatory conditions, we activated THBMEC with the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), which up-regulated chemokine and adhesion molecule expression in THBMEC without affecting the distribution of immunoreactivity for tight junction-associated proteins. PBMC adhesion was enhanced by cytokine-mediated activation of THBMEC. G protein-coupled receptor (GPCR) activation was essential for leukocyte-THBMEC interaction, as pertussis toxin (PTX) treatment of PBMC abrogated PBMC adhesion to activated THBMEC. The anti-alpha4 integrin antibody, natalizumab, infused into MS patients, significantly reduced the adhesion of their ex vivo PBMC to activated THBMEC under flow conditions. Further study showed that alternatively spliced fibronectin containing the CS1 region (FN-CS1), but not Vascular Cell Adhesion Molecule type 1 (VCAM-1), was the ligand of alpha4 integrin on activated THBMEC. Blocking FN-CS1 abrogated PBMC adhesion on activated THBMEC, while anti-VCAM-1 antibodies had no effect. These results established a novel in vitro dynamic BBB model. We also demonstrated the dependence of leukocyte-endothelial interactions in this model on alpha4 integrins and FN-CS1.

Comparison of ventricular and lumbar cerebrospinal fluid T cells in non-inflammatory neurological disorder (NIND) patients

The aim of the present study was to define the cellular composition of ventricular, as compared with lumbar, cerebrospinal fluid (CSF) in patients with non-inflammatory neurological disorders (NIND). We addressed this issue by determining the cellular composition of lumbar CSF from patients with normal pressure hydrocephalus (NPH) who were undergoing lumbar CSF drainage during evaluation for shunting procedures, and evaluating ventricular CSF from a subset of these who underwent subsequent placement of ventriculoperitoneal shunts. We determined the cellular composition of lumbar CSF from 18 patients with NPH, and found that the leukocyte differentials, and relative proportions of CD4+ and CD8+ central memory (TCM), effector memory (TEM) and naive cell (TNaive) populations, were equivalent to those found previously in studies of CSF from patients with NIND. We further evaluated cells in the ventricular CSF of five patients who had previously undergone lumbar drainage. Leukocyte differential counts, as well as CD4+ and CD8+ TCM, TEM, and TNaive proportions, were equivalent in matched ventricular and lumbar CSF samples. These observations support the hypothesis that leukocytes enter the CSF in a selective fashion, at its site of formation in the choroid plexus. The results implicate CSF T cells in the immune surveillance of the central nervous system.

CXCL12-Induced Monocyte-Endothelial Interactions Promote Lymphocyte Transmigration Across an in Vitro Blood-Brain Barrier

An in vitro model of the human blood-brain barrier provides insights into how chemokine receptors regulate the transmigration of leukocytes into brain tissue. Going with the Flow White blood cells traffic ceaselessly throughout the body, using blood vessels as their conduits. They also migrate into inflamed tissues to defend the host against microbes or to repair damaged tissue. However, in many human diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, or type 1 diabetes, white blood cells invade apparently healthy uninfected tissues and cause injury. Recent research has identified the molecular regulators (chemokine receptors and their chemokine ligands; adhesion molecules) of white blood cell transmigration out of the blood vessels and into tissues. Because there are 100 or so such molecules (used selectively when specific types of white blood cells transmigrate), it is important to identify those molecules that are most directly involved in harmful inflammation. Chemokine receptors are molecules on white blood cell surfaces that receive signals to guide cells into tissue, and they change as white blood cells transmigrate across different vessel walls. In a new study, Man and colleagues have devised an in vitro model of a specialized vessel wall of the human brain termed the blood-brain barrier (BBB). They use this elegant model to study how chemokine receptors influence and are influenced by the transmigration of white blood cells across a human BBB-like endothelial cell layer. First, the authors coaxed a special type of human endothelial cell to form a BBB-like layer in a dual perfusion chamber. Then, they allowed human white blood cells to flow across the layer at a flow rate approximating that found in brain capillaries. Some white blood cells flowed across the layer and out of the device, whereas others transmigrated across the BBB-like endothelial layer into the lower chamber of the device. The researchers wanted to establish how chemokine receptor expression by white blood cells would alter as the cells transmigrated across the BBB. They studied a chemokine receptor termed CXCR4, which is expressed on almost all white blood cells. When they added the triggering molecule for CXCR4 to their in vitro system, unexpectedly, they found that only one cell type, monocytes, showed altered CXCR4 expression. CXCR4 appeared to deliver signals to monocytes, which empowered these cells to assist other white blood cells such as T and B cells to cross the BBB. This surprising result opens up new vistas for understanding how white blood cells and vessel wall endothelial cells “talk” to each other in inflamed tissues and should spur progress for identifying the best targets for blocking harmful inflammation in the brain. The accumulation of inflammatory cells in the brain parenchyma is a critical step in the pathogenesis of neuroinflammatory diseases such as multiple sclerosis (MS). Chemokines and adhesion molecules orchestrate leukocyte transmigration across the blood-brain barrier (BBB), but the dynamics of chemokine receptor expression during leukocyte transmigration are unclear. We describe an in vitro BBB model system using human brain microvascular endothelial cells that incorporates shear forces mimicking blood flow to elucidate how chemokine receptor expression is modulated during leukocyte transmigration. In the presence of the chemokine CXCL12, we examined modulation of its receptor CXCR4 on human T cells, B cells, and monocytes transmigrating across the BBB under flow conditions. CXCL12 stimulated transmigration of CD4+ and CD8+ T cells, CD19+ B cells, and CD14+ monocytes. Transmigration was blocked by CXCR4-neutralizing antibodies. Unexpectedly, CXCL12 selectively down-regulated CXCR4 on transmigrating monocytes, but not T cells. Monocytes underwent preferential CXCL12-mediated adhesion to the BBB in vitro compared with lymphocytes. These findings provide new insights into leukocyte-endothelial interactions at the BBB under conditions mimicking blood flow and suggest that in vitro BBB models may be useful for identifying chemokine receptors that could be modulated therapeutically to reduce neuroinflammation in diseases such as MS.