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Chemokine receptors and adhesion molecules are used selectively for the transmigration of leukocytes across the blood-brain barrier (BBB) during neuroinflammation. We established an activated in vitro BBB (aIVBBB) using physiological concentrations of cytokines. We studied CCL5-driven migration as a model to determine how chemokine receptors and adhesion molecules regulate T-cell and monocyte migration across the aIVBBB. Increased expression of CCL5 and its receptors, CCR1 and CCR5 have been described in the perivascular space of multiple sclerosis (MS) lesions. Elucidating the determinants of CCL5-mediated mononuclear cell migration may clarify appropriate targets for therapeutic modulation in neuroinflammatory conditions. In response to CCL5, there was a significant increase in total mononuclear cell migration across the aIVBBB. Neutralizing monoclonal antibodies to CCR1 and CCR5 abrogated CCL5-driven transmigration, suggestive of non-redundant receptor usage in mononuclear cell migration to this chemokine in vitro. CCL5-driven transmigration was also dependent on alpha(4)beta(1) integrin/fibronectin connecting segment-1 (FN CS-1) and alpha(L)beta(2) integrin/intercellular adhesion molecule (ICAM-1) interactions. Monocyte migration to CCL5 was solely dependent on alpha(4)beta(1) integrin/FN CS-1 while T-cell migration required both alpha(L)beta(2) integrin/ICAM-1 and alpha(4)beta(1) integrin/FN CS-1 interactions. These findings provide plausible molecular targets for the selective inhibition of mononuclear cell trafficking during the acute immune effector phases of MS and other neuroinflammatory diseases.

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.

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.

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.

Cells generally maintain an asymmetric distribution of phospholipids across the plasma membrane bilayer, restricting the phospholipid, phosphatidylserine (PS), to the inner leaflet of the plasma membrane. When cells undergo apoptosis, this asymmetric transbilayer distribution is lost, bringing PS to the surface where it acts as a signal for engulfment by phagocytes. The fluorescent dye merocyanine 540 specifically stains the plasma membrane of apoptotic cells which have lost their asymmetric distribution of phospholipids. However, it also stains non-apoptotic macrophages, suggesting that phospholipid asymmetry may not be maintained in these cells, and thus that they may express PS on their surface. Here, the PS-binding protein, annexin V, was used to show that in fact normal macrophages do express PS on their surface. Furthermore, pre-treating macrophages with annexin V was found to inhibit phagocytosis of apoptotic thymocytes and thymocytes on which PS expression was artificially induced, but did not inhibit phagocytosis of latex beads or Fc receptor-mediated phagocytosis of opsonized erythrocytes. These results indicate that PS is constitutively expressed on the surface of macrophages and is functionally significant for the phagocytosis of PS-expressing target cells.

Expression of the aminophospholipid phosphatidylserine (PS) on the surface of apoptotic lymphocytes and lipid-symmetric erythrocytes triggers their phagocytosis by macrophages. Phagocytosis by both activated and unactivated macrophages, which utilize different recognition systems, can be blocked by certain monoclonal antibodies directed against the LPS receptor, CD14. Here we investigate the requirement for CD14 in the phagocytosis of both apoptotic thymocytes and lipid-symmetric erythrocytes by both activated and unactivated macrophages. We show that phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages is completely abolished when CD14 is removed from macrophages by cleaving its glycosylphosphatidylinositol tether with phospholipase C. This treatment also substantially reduces phagocytosis of apoptotic lymphocytes by both types of macrophages. Unactivated LR-9 mouse macrophages which are deficient in CD14 expression are completely unable to phagocytose either apoptotic thymocytes or lipid-symmetric erythrocytes. These results argue that CD14 is an absolute requirement for the phagocytosis of lipid-symmetric erythrocytes by both activated and unactivated macrophages, despite their different recognition systems, that CD14 contributes at least substantially to the phagocytosis of apoptotic lymphocytes by both activated and unactivated macrophages, and that activated macrophages may also possess an alternate, CD14-independent mechanism for phagocytosis of apoptotic lymphocytes.

Although different macrophages exploit different cell surface receptors to recognize apoptotic lymphocytes, indirect evidence suggested that the phosphatidylserine (PS) that appears on the surface of lymphocytes undergoing apoptosis participates in specific recognition by all types of macrophages. To test this possibility directly, annexin V, a protein that specifically binds to PS, was used to mask this phospholipid on the apoptotic cell surface. Preincubation of apoptotic lymphocytes with annexin V blocked phagocytosis by elicited mouse peritoneal macrophages, macrophages of the mouse J774 cell line and mouse bone marrow macrophages. Similarly, annexin V was able to inhibit phagocytosis of lipid-symmetric erythrocytes, another target cell upon which PS is exposed. Together these results demonstrate directly that macrophages of all types depend on the PS exposed on the surface of apoptotic lymphocytes for recognition and phagocytosis.

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.

Observational studies in multiple sclerosis (MS) demonstrated altered expression of chemokine receptors (CkRs) on comparable populations of mononuclear cells (e.g. CD 4 + / CD45RO + T-cells) in brain sections compared with blood. These findings raised questions about the regulation of CkRs on trafficking cells. Regulatory processes for CkRs are complex: examples include down-regulation following ligand engagement during migration and either up- or down-regulation following activation. Additionally, CkRs that mediate transmigration without being down-regulated will be selectively enriched on migrating cells in the inflammatory site. Finally, CkRs may act as functionally neutral markers of activated cells capable of undergoing transmigration. Clarifying CkR regulation may aid in the selection and application of antagonists for treating neuroinflammation. Mechanisms of receptor regulation during transmigration cannot be studied by descriptive methods. We evaluated CCR5 expression on CD14+ monocytes and CD3+ T-cells following CCL5-driven transmigration through an in vitro blood–brain barrier (IVBBB), as both T-cells and monocytes in MS lesions express CCR5. CCR5 expression was augmented on non-migrating CD14+ but not CD3+ cells, suggesting selective activation of monocytes by incubation in contact with endothelial cells. As proposed from observational studies, CCR5 was enriched on monocytes that migrated spontaneously in the absence of exogenous chemokine. Addition of the CCR5 ligand CCL5 to the lower chamber led to enhanced CD3+ T-cell migration. Interestingly, CCR5 was down-regulated on both CD14+ monocytes and CD3+ T cells during CCL5-driven migration. These results are distinct from those obtained in comparable studies of CCR2 and CXCR3, suggesting that the specifics for CkR expression should be studied for individual receptors on each leukocyte subpopulation during the design of strategies for pharmacological blockade in neuroinflammation.

Chemokines and chemokine receptors play a key role in the transmigration of leucocytes across the blood-brain barrier (BBB). CCR2 is the major receptor for CCL2, a potent monocyte and T cell chemoattractant. CCR2 and CCL2 have been consistently associated with a pathogenic role in experimental autoimmune encephalomyelitis, using knockout and transgenic mice, neutralizing antibodies, peptide antagonists and DNA vaccination. However, the significance of CCL2 and CCR2 in multiple sclerosis is enigmatic, because CCL2 levels are consistently decreased in the CSF of patients with this disease and other chronic neuroinflammatory conditions, despite abundant expression within lesional multiple sclerosis tissues. This study used an in vitro BBB model to test the hypothesis that CCL2 is removed from the extracellular fluid by CCR2-positive migrating cells as they cross the BBB, resulting in decreased CSF CCL2 levels. We showed that CCR2-positive T cells and monocytes migrated selectively across the in vitro BBB, and that CCL2 on the abluminal (tissue) side was consumed by migrating T cells and monocytes. Next, we used a new anti-CCR2 antibody to show that CCR2-positive mononuclear inflammatory cells could be readily detected in appropriate positive control tissues, but that CCR2+ cells were very infrequently found in multiple sclerosis lesions. We then showed that CCR2 receptor density on T cells and monocytes was specifically downregulated upon in vitro BBB transmigration in response to CCL2, but not irrelevant chemokines. These findings document a novel strategy for analysing chemokine receptor function in inflammatory CNS disease, and support the hypothesis that CCL2 is consumed by migrating inflammatory cells, which downregulate CCR2, as they cross the BBB.

CXCR3, the receptor for CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC, is preferentially expressed on activated Th1 T cells and has been predicted to play an important role in their trafficking. However, this simplistic view of the function of CXCR3 and its ligands has not been borne out by studies of disease models, including experimental autoimmune encephalomyelitis (EAE), using varied methods of receptor blockade, as well as knockout or transgenic mice. This review focuses on the current understanding of the enigmatic role of CXCR3 and its ligands in CNS inflammatory/autoimmune disorders. The conflicting results among varied models of CNS inflammation suggest complex and multiple roles for CXCR3 and its ligands in the pathogenesis of CNS inflammatory/autoimmune diseases. Thus, further study is needed to determine how CXCL10 neutralizing agents or CXCR3 receptor antagonists might be applied to treating human disease.

CXCR3, the receptor for CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC, is preferentially expressed on activated Th1 T cells and has been predicted to play an important role in their trafficking. However, this simplistic view of the function of CXCR3 and its ligands has not been borne out by studies of disease models, including experimental autoimmune encephalomyelitis (EAE), using varied methods of receptor blockade, as well as knockout or transgenic mice. This review focuses on the current understanding of the enigmatic role of CXCR3 and its ligands in CNS inflammatory/autoimmune disorders. The conflicting results among varied models of CNS inflammation suggest complex and multiple roles for CXCR3 and its ligands in the pathogenesis of CNS inflammatory/autoimmune diseases. Thus, further study is needed to determine how CXCL10 neutralizing agents or CXCR3 receptor antagonists might be applied to treating human disease.

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.

Chemokines and their receptors may be implicated in leukocyte ingress into brain during inflammation observed during the course of multiple sclerosis (MS). To address receptor modulation on CD4+ memory T lymphocytes during diapedesis, we used an in vitro model of the blood-brain barrier (BBB). We found that only memory (CD45RO+) cells transmigrated and type 3 CXC chemokine receptor (CXCR3) was enriched on transmigrated cells. CXCR3 depletion of the input population did not affect transmigration capability. CXCR3 reemerged on CXCR3 depleted cells independently of endothelial cell exposure, but was susceptible to incubation at 4 degrees C, indicating receptor recycling. We propose that CXCR3 serves as a surface marker for cells that have the capacity to cross the BBB, but does not play an essential role in extravasation.

The central nervous system (CNS) has long been described as an immunologically privileged organ. However, evidence is accumulating that the CNS is more adequately described as immune competent, albeit atypical. Leukocyte trafficking into the CNS in response to inflammation, as in peripheral organs, occurs in several distinct steps. As with all leukocyte trafficking, the overall process is governed in part by chemokines. The CNS has unique anatomic and physiologic attributes, including the blood-brain barrier. These characteristic features of the CNS interact with a distinct array of chemokines, expressed both constitutively and in response to inflammation. In this review, we consider the potential sites of chemokine action in guiding leukocyte migration. The process of extravasation can usefully be regarded as comprising individual steps. This review is focused on the need to consider these stages separately, and the elucidation of mechanisms at each stage is outlined.

Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient phagocytosis. Monocytes, the precursors of macrophages, do not express PS on their surface and do not efficiently phagocytose apoptotic cells. We report here that PS appears on the surface of both human monocytic U937 cells and primary human monocytes as they differentiate in culture and acquire the ability to phagocytose apoptotic thymocytes. Phagocytosis was blocked by pretreating either the apoptotic target or the phagocyte with annexin V to mask PS and was CD14-dependent. Expression of PS, like other events characteristic of differentiating monocytes such as Mac-1 expression, was independent of the agent used to induce differentiation and was insensitive to the addition of caspase inhibitors. These results demonstrate that PS is expressed on monocytes as part of their differentiation program and is independent of apoptosis.

Erythrocytes have an asymmetric distribution of phospholipids across the bilayer of their plasma membranes, maintained by an ATP-dependent aminophospholipid translocase, and dissipated by activation of a non-specific lipid flipsite. Loss of asymmetry provokes recognition by the reticuloendothelial system. In vitro, enhanced phagocytosis of erythrocytes with a symmetric bilayer can be inhibited by artificial lipid vesicles made of phosphatidylserine (PS), indicating that macrophages recognize the PS that appears on the erythrocyte surface upon loss of asymmetry. It is becoming increasingly clear that these same fundamental membrane structure/function relationships established in the erythrocyte paradigm also apply to lymphocytes. All evidence suggests that lymphocytes maintain an asymmetric transbilayer distribution of phospholipids in their plasma membranes, maintained by an aminophospholipid translocase. Asymmetry is lost as part of the program of cell death, by down-regulation of the translocase and activation of the non-specific lipid flipsite, exposing PS on the cell surface. That PS exposure has functional consequences is demonstrated by the ability of artificial lipid vesicles containing PS to inhibit enhanced phagocytosis of apoptotic lymphocytes by macrophages. However, other signals besides PS are also involved in recognition of apoptotic lymphocytes. Studies with other inhibitors indicate that macrophages also utilize integrin-mediated and lectin-like recognition systems, although each is restricted to either unactivated or activated macrophages. These results indicate that although many fundamental features of recognition by the reticuloendothelial system may be analogous in erythrocytes and lymphocytes, the signals for recognition of apoptotic lymphocytes are more complex and involve multiple recognition systems.