Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats.

BACKGROUND: In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour's response to IR has not been addressed. METHODS: We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro. RESULTS: CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs. CONCLUSIONS: These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

Expression of CXCR3 and its ligands CXCL9, -10 and -11 in paediatric opsoclonus-myoclonus syndrome.

Opsoclonus-myoclonus syndrome (OMS) is a neuroinflammatory disorder associated with remote cancer. To understand more clearly the role of inflammatory mediators, the concentration of CXCR3 ligands CXCL10, CXCL9 and CXCL11 was measured in 245 children with OMS and 81 paediatric controls using enzyme-linked immunosorbent assay (ELISA), and CXCR3 expression on CD4(+) T cells was measured by flow cytometry. Mean cerebrospinal fluid (CSF) CXCL10 was 2·7-fold higher in untreated OMS than controls. Intrathecal production was demonstrated by significantly different CXCL10 CSF : serum ratios. The dichotomized 'high' CSF CXCL10 group had higher CSF leucocyte count (P = 0·0007) and B cell activating factor (BAFF) and CXCL13 concentrations (P < 0·0001). CSF CXCL10 did not correlate with clinical severity or relapse using grouped data, although it did in some patients. Among seven types of immunotherapy, including rituximab or chemotherapy, only adrenocorticotrophic hormone (ACTH) monotherapy showed reduced CSF CXCL10, but prospective longitudinal studies of ACTH combination therapies indicated no reduction in CXCL10 despite clinical improvement (P < 0·0001). CXCL10 concentrations were 11-fold higher in CSF and twofold higher in serum by multiplexed fluorescent bead-based immunoassay than enzyme-linked immunosorbent assay, but the two correlated (r = 0·7 and 0·83). In serum, no group differences for CXCL9 or CXCL11 were found. CXCR3 expression on CD4(+) T cells was fivefold higher in those from CSF than blood, but was not increased in OMS or altered by conventional immunotherapy. These data suggest alternative roles for CXCL10 in OMS. Over-expression of CXCL10 was not reduced by clinical immunotherapies as a whole, indicating the need for better therapeutic approaches.

Altered hippocampal synaptic transmission in transgenic mice with astrocyte-targeted enhanced CCL2 expression.

Elevated expression of neuroinflammatory factors in the central nervous system (CNS) contributes to the cognitive impairment in CNS disorders such as injury, disease and neurodegenerative disorders. However, information on the role of specific neuroimmune factors in normal and abnormal CNS function is limited. In this study, we investigated the effects of chronic exposure to the chemokine CCL2 on hippocampal synaptic function at the Schaffer collateral-CA1 synapse, a synapse that is known to play an important role in cognitive functions such as memory and learning. Synaptic function was measured in vitro using hippocampal slices obtained from transgenic mice that express elevated levels of CCL2 in the CNS through astrocyte expression and their non-transgenic littermate controls. Extracellular field potential electrophysiological recordings showed a significant reduction in the magnitude of synaptic responses in hippocampal slices from the CCL2 transgenic mice compared with slices from non-transgenic littermate controls. Two forms of short-term synaptic plasticity (post-tetanic potentiation and short-term potentiation) thought to be important cellular mechanisms of short-term memory were enhanced in hippocampal slices from CCL2 transgenic mice compared to non-transgenic hippocampal slices, whereas long-term synaptic plasticity (LTP), which is critical to long-term memory formation, was not altered. Western blot analysis of hippocampus from the CCL2 transgenic mice and non-transgenic mice showed no change in level of neuronal specific enolase, a neuronal specific protein, GFAP, an astrocyte specific protein, and several synaptic proteins compared with non-transgenic littermate controls. These results show that CCL2, which is known to be chronically produced at elevated levels within the CNS in a number of CNS disorders, can significantly alter hippocampal function and implicate a role for CCL2 in the cognitive dysfunction associated with these CNS disorders.

Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis.

Monocyte chemoattractant protein (MCP)-1 plays a critical role in innate immunity by directing the migration of monocytes into inflammatory sites. Recent data indicated a function for this chemokine in adaptive immunity as a regulator of T cell commitment to T helper cell type 2 (Th2) effector function. Studies in a Th1-dependent animal model, experimental autoimmune encephalomyelitis (EAE), showed that MCP-1 was highly expressed in the central nervous system (CNS) of affected rodents, and MCP-1 antibodies could block relapses of the disease. Mice deficient for the major MCP-1 receptor, CC chemokine receptor (CCR)2, did not develop EAE after active immunization but generated effector cells that could transfer the disease to naive wild-type recipients. We analyzed EAE in mice deficient for MCP-1 to define the relevant ligand for CCR2, which responds to murine MCP-1, MCP-2, MCP-3, and MCP-5. We found that C57BL/6 MCP-1-null mice were markedly resistant to EAE after active immunization, with drastically impaired recruitment of macrophages to the CNS, yet able to generate effector T cells that transferred severe disease to naive wild-type recipients. By contrast, adoptive transfer of primed T cells from wild-type mice into naive MCP-1-null recipients did not mediate clinical EAE. On the SJL background, disruption of the MCP-1 gene produced a milder EAE phenotype with diminished relapses that mimicked previous findings using anti-MCP-1 antibodies. There was no compensatory upregulation of MCP-2, MCP-3, or MCP-5 in MCP-1-null mice with EAE. These results indicated that MCP-1 is the major CCR2 ligand in mice with EAE, and provided an opportunity to define the role of MCP-1 in EAE. Compared with wild-type littermates, MCP-1-/- mice exhibited reduced expression of interferon gamma in draining lymph node and CNS and increased antigen-specific immunoglobulin G1 antibody production. Taken together, these data demonstrate that MCP-1 is crucial for Th1 immune responses in EAE induction and that macrophage recruitment to the inflamed CNS target organ is required for primed T cells to execute a Th1 effector program in EAE.

Interferon (IFN) beta acts downstream of IFN-gamma-induced class II transactivator messenger RNA accumulation to block major histocompatibility complex class II gene expression and requires the 48-kD DNA-binding protein, ISGF3-gamma.

Interferon (IFN) gamma, a cardinal proinflammatory cytokine, induces expression of the gene products of the class II locus of the major histocompatibility complex (MHC), whereas IFN-alpha or -beta suppresses MHC class II expression. The mechanism of IFN-beta-mediated MHC class II inhibition has been unclear. Recently, a novel factor termed class II transactivator (CIITA) has been identified as essential for IFN-gamma-induced MHC class II transcription. We studied the status of IFN-gamma-induced CIITA messenger RNA (mRNA) accumulation and CIITA-driven transactivation in IFN-beta-treated cells and used cell lines that had defined defects in the type I IFN response pathway to address the roles of IFN signaling components in the inhibition of MHC class II induction. IFN-beta treatment did not suppress IFN-gamma-induced accumulation of CIITA mRNA. After cells were stably transfected with CIITA, endogenous MHC class II genes were constitutively expressed, and MHC class II promoters, delivered by transfection, were actively transcribed in CIITA-expressing cells. Expression of these promoters was significantly impaired by pretreatment with IFN-beta. These results suggest that IFN-beta acts downstream of CIITA mRNA accumulation, and acts in part by reducing the functional competence of CIITA for transactivating MHC class II promoters. IFN stimulated gene factor 3 (ISGF3) gamma was essential for IFN-beta to mediate inhibition of MHC class II induction, regardless of whether MHC class II transcription was stimulated by IFN-gamma or directly by CIITA expression. Results of these experiments suggest that inhibition of MHC class II in IFN-beta-treated cells requires expression of gene(s) directed by the ISGF3-IFN-stimulated response element pathway, and that these gene product(s) may act by blocking CIITA-driven transcription of MHC class II promoters.

CSF neutrophils are implicated in the development of vasospasm in subarachnoid hemorrhage.

BACKGROUND: Cerebral vasospasm is a significant cause of morbidity in patients after aneurysmal subarachnoid hemorrhage (aSAH). There are few effective treatments. The search for new treatments has focused predominantly on dilating cerebral blood vessels. Growing evidence supports a role for inflammation in its pathogenesis but no potential target for intervention has emerged. METHODS: CSF and clinical information from patients with aSAH were collected. Additionally, tyrosine modifications by stable isotope dilution HPLC with online tandem mass spectrometry were quantified in CSF samples. RESULTS: We report an association between neutrophil accumulation in the cerebrospinal fluid of patients with aSAH and the development of vasospasm. In particular, CSF neutrophil content of >62% on the third day after aSAH is an independent predictor of the later development of vasospasm (OR 6.8, 95% CI 2.0-23.3, P = 0.002). Further, activity of myeloperoxidase and NADPH oxidase is elevated in aSAH suggesting a role for modification of CSF proteins by reactive oxidant species. CONCLUSIONS: Neutrophil percentage is an independent predictor of vasospasm in aSAH patients, days prior to its onset suggesting a role of neutrophils in vasospasm. The activity of neutrophil enzymes is also increased suggesting a mechanism for blood vessel damage. Inflammation mediated by neutrophils is a potential target for therapies in vasospasm. More study is necessary to determine the mechanism by which neutrophils damage cerebral blood vessels.

TNF-alpha mediates SDF-1 alpha-induced NF-kappa B activation and cytotoxic effects in primary astrocytes.

Stromal-derived cell factor-1 alpha (SDF-1 alpha; CXCL12) and its receptor, CXCR4, are constitutively expressed on neuroepithelial cells and are believed to be involved in both development and pathological processes, such as AIDS-associated neurologic disorders. Here, we demonstrate that SDF-1 alpha activates NF-kappa B, stimulates production of chemokines and cytokines, and induces cell death in primary astrocytes, effects that depend on ongoing secretion of TNF-alpha. SDF-1 alpha upregulated TNF-alpha mRNA and protein secretion, as well as TNF receptor 2 expression. TNF-alpha treatment mimicked SDF-1 alpha induction of NF-kappa B, IL-1 alpha/beta, and RANTES, as well as cell death; neutralizing antibodies against TNF-alpha opposed these responses. We also found that SDF-1 alpha activated Erk1 and Erk2 (Erk1/2) MAPK in a biphasic fashion. Early Erk1/2 activation was stimulated directly by SDF-1 alpha and late activation was mediated by TNF-alpha. PD98059 suppression of early Erk1/2 activation correlated with reduction of SDF-1 alpha-induced TNF-alpha expression. Late Erk1/2 activation was involved in TNF-alpha-stimulated NF-kappa B activation and cytokine induction. SDF-1 alpha was induced in reactive CXCR4-positive astrocytes near axotomized spinal cord motor neurons, consistent with autocrine SDF-1/CXCR4 signaling in these cells. We propose that these novel effects of SDF-1 alpha are relevant to the pathogenic and developmental roles of SDF-1 alpha in the CNS.

Neutrophil infiltration, glial reaction, and neurological disease in transgenic mice expressing the chemokine N51/KC in oligodendrocytes.

Chemokines (pro-inflammatory chemoattractant cytokines) are expressed in pathological conditions of the central nervous system (CNS). Previous studies suggested that the CNS is relatively resistant to leukocyte diapedesis after chemokine injection, leaving their functional role unresolved. The CNS function of N51/KC, a neutrophil-selective chemokine, was addressed by expressing N51/KC under control of the myelin basic protein (MBP) promoter in transgenic (tg) mice (MBP-N51/KC mice). CNS-specific N51/KC expression produced remarkable neutrophil infiltration into perivascular, meningeal, and parenchymal sites, demonstrating that this chemokine exerts the multiple functions in vivo required to recruit leukocytes into the CNS. MBP-N5 1/KC mice represent an incisive model for the molecular dissection of neutrophil entry into the CNS. Unexpectedly, MBP-N51/KC mice developed a neurological syndrome of pronounced postural instability and rigidity at high frequency beginning at 40 days of age, well after peak chemokine expression. 68/182 mice in one tg fine were found dead before one year of age, with prominent neurological symptoms premortem in 26 (38%). Florid microglial activation and blood-brain barrier disruption without dysmyelination were the major neuropathological alterations. Late-onset neurological symptoms in MBP-N51/KC mice may indicate unanticipated consequences of CNS chemokine expression.

Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients.

Chemokines direct tissue invasion by specific leukocyte populations. Thus, chemokines may play a role in multiple sclerosis (MS), an idiopathic disorder in which the central nervous system (CNS) inflammatory reaction is largely restricted to mononuclear phagocytes and T cells. We asked whether specific chemokines were expressed in the CNS during acute demyelinating events by analyzing cerebrospinal fluid (CSF), whose composition reflects the CNS extracellular space. During MS attacks, we found elevated CSF levels of three chemokines that act toward T cells and mononuclear phagocytes: interferon-gamma-inducible protein of 10 kDa (IP-10); monokine induced by interferon-gamma (Mig); and regulated on activation, normal T-cell expressed and secreted (RANTES). We then investigated whether specific chemokine receptors were expressed by infiltrating cells in demyelinating MS brain lesions and in CSF. CXCR3, an IP-10/Mig receptor, was expressed on lymphocytic cells in virtually every perivascular inflammatory infiltrate in active MS lesions. CCR5, a RANTES receptor, was detected on lymphocytic cells, macrophages, and microglia in actively demyelinating MS brain lesions. Compared with circulating T cells, CSF T cells were significantly enriched for cells expressing CXCR3 or CCR5. Our results imply pathogenic roles for specific chemokine-chemokine receptor interactions in MS and suggest new molecular targets for therapeutic intervention.

Mice overexpressing chemokine ligand 2 (CCL2) in astrocytes display enhanced nociceptive responses.

Recent findings demonstrate that chemokines, and more specifically CC chemokine ligand 2 (CCL2 or monocyte chemoattractant protein-1), play a major role in pain processing. In the present study, we assess nociceptive responses of mice that overexpressed CCL2 under control of glial fibrillary acidic protein promoter (CCL2 tg). In models of acute nociception CCL2 tg mice demonstrated significantly enhanced nociceptive behavior relative to wild-type controls in responses to both thermal (hot plate) and chemical (formalin test) stimulus modalities. There were no differences in mechanical allodynia in the partial sciatic nerve ligation model, in terms of either magnitude or duration of the allodynic response; however, both groups responded to the maximal extent measurable. In a model of inflammatory pain, elicited by intraplantar administration of complete Freund's adjuvant (CFA), CCL2 tg mice displayed both greater edema and thermal hyperalgesia compared with control mice. In control mice, edema and hyperalgesia returned to baseline values 5-7 days post CFA. However, in CCL2 tg mice, thermal hyperalgesia was significantly different from baseline up to 3 weeks post CFA. Parallel to these enhanced behavioral responses CCL2 serum levels were significantly greater in CCL2 overexpressing mice and remained elevated 7 days post CFA. Consequently, proinflammatory cytokine mRNA expression (IL-1beta, IL-6, and TNFalpha) levels were greater in skin, dorsal root ganglia (DRG), and spinal cord, whereas the anti-inflammatory cytokine (IL-10) level was lower in skin and DRG in CCL2 overexpressing mice than in control mice. Taken together with data from CCR2-deficient mice, these present data confirm a key role of CCL2/CCR2 axis in pain pathways and suggest that inhibiting this axis may result in novel pain therapies.

Chemokines in immune-mediated inflammation of the central nervous system.

Inflammatory cell recruitment to the central nervous system (CNS) is a cardinal feature of physiological and pathological processes, including multiple sclerosis (MS). Despite recent progress, the soluble signals that attract inflammatory cells from the vascular compartment into the CNS parenchyma remain obscure. We favor the hypothesis that chemoattractant cytokines termed 'chemokines' are uniquely important for mediating leukocyte entry into CNS tissues during immune-mediated inflammation. Three lines of evidence supporting this hypothesis will be reviewed. The first regards expression of chemokines in animal models of immune-mediated CNS inflammation and in the human disease, multiple sclerosis. The second line of evidence involves interventional studies of chemokine blockade in such model disorders. The third line of evidence comprises function of chemokines in the CNS, as analysed in transgenic mice. Investigation of CNS chemokine function will enhance our understanding of leukocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.

Do chemokines mediate leukocyte recruitment in post-traumatic CNS inflammation?

Hematogenous leukocytes infiltrate the CNS after inflammatory stimuli, including infection, mechanical trauma and excitotoxic neuronal necrosis. However,the role of leukocytic inflammation in promoting or hindering tissue repair is poorly understood. Identification of signals that lead to leukocyte recruitment and activation is essential for the designing of interventions that modulate inflammation, thus improving neurological outcome. Chemokines are small pleiotropic chemoattractant cytokines whose target specificity suggests an important role in determining the cellular composition of inflammatory infiltrates. Chemokine expression profiles in the CNS during autoimmune and post-traumatic inflammation correlate well with the composition of leukocyte infiltrates, and expression studies in systems such as transgenic mice, suggest that chemokines have potent functional attributes in CNS physiology. We propose that selective chemokine expression by CNS cells is crucial for post-traumatic leukocyte accumulation.

Elevated levels of the chemokine GRO-1 correlate with elevated oligodendrocyte progenitor proliferation in the jimpy mutant.

The dysmyelinating mutant jimpy (jp) arises from a point mutation in the mouse gene encoding proteolipid protein and is characterized by severe dysmyelination attributable to oligodendrocyte death. This mutant was used to investigate the regulation of oligodendrocyte progenitor proliferation in the postnatal spinal cord. At postnatal day 18, jp spinal cord contained a three- to eightfold greater number of proliferating oligodendrocyte progenitor cells than did wild-type (wt) spinal cord. Increased proliferation in jp spinal cord was accompanied by a twofold increase in the number of progenitor cells. Semiquantitative reverse transcriptase-PCR revealed no change in the level of mRNA encoding the platelet-derived growth factor A, transforming growth factor-beta, or insulin-like growth factor-I, all of which have been implicated as regulators of proliferation and differentiation of oligodendrocyte progenitor cells. There was, however, a 17-fold increase in the level of mRNA encoding the chemokine GRO-1 and a 5- to 6-fold increase in GRO-1 protein in the jp spinal cord. Double immunofluorescence labeling revealed elevated levels of GRO-1 in reactive astrocytes in jp spinal cord white matter. In vitro studies indicated that extracts from jp spinal cord stimulated oligodendrocyte progenitor proliferation. Furthermore, removal of GRO-1 from jp extracts by immunoprecipitation reduced the proliferation of progenitor cells to a level similar to that achieved by wt extracts. These findings suggest a novel mechanism by which proliferation of oligodendrocyte progenitor cells is regulated in the postnatal spinal cord in response to insult.

P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase.

Leukocyte infiltration in the CNS after trauma or inflammation is triggered in part by upregulation of the chemokine, monocyte chemoattractant protein-1 (MCP-1), in astrocytes. However the signals that induce the upregulation of MCP-1 in astrocytes are unknown. We have investigated the roles for ATP P2X7 receptor activation because ATP is an intercellular signaling transmitter that is released in both trauma and inflammation and P2X7 receptors are involved in immune system signaling. Astrocytes in primary cell culture and acutely isolated from the hippocampus were immunopositive for P2X7 receptors. In astrocyte cultures, application of the selective P2X7 agonist, benzoyl-benzoyl ATP (Bz-ATP), activated MAP kinases extracellular signal receptor-activated kinase 1 (ERK1), ERK2, and p38. Purinergic antagonists depressed this activation with a profile suggesting P2X7 receptors. Bz-ATP also increased MCP-1 expression in cultured astrocytes, and again P2X7 antagonists prevented this increase. Blocking either the ERK1/ERK2 or the p38 pathway (with PD98059 or SB203580, respectively) significantly inhibited Bz-ATP-induced MCP-1 expression. Coapplication of both antagonists caused a greater depression. We also tested the roles for ATP receptor activation in inducing MCP-1 upregulation in corticectomy, an in vivo model of trauma. This model of cortical trauma was previously shown to increase MCP-1 expression in vivo principally in astrocytes. Suramin, a wide-spectrum purinergic receptor antagonist, significantly depressed the rapid (3 hr) trauma-induced increase in MCP-1 mRNA. These data indicate that purinergic transmitter receptors in astrocytes are important in regulating chemokine synthesis. The regulation of MCP-1 in astrocytes by ATP may be important in mediating communication with hematopoietic inflammatory cells.

Chemokines in neurological disease models: correlation between chemokine expression patterns and inflammatory pathology.

We have recently determined chemokine expression profiles in a variety of models of neural trauma and immune-inflammation. The results indicate the following: (1) Chemokine expression in posttraumatic inflammation is generally restricted to the monocyte chemoattractant MCP-1, and occurs before hematogenous cell entry into neural tissues. Therefore MCP-1 is an excellent candidate for a mediator of leukocyte recruitment in these settings. (2) Chemokine expression in immune-inflammation is diverse and includes both alpha- and beta-chemokines. Chemokine production can be attributed to parenchymal and infiltrating cell populations. Early signs of inflammation precede chemokine expression, which is believed to exert the crucial function of amplifying the immune-mediated inflammatory reaction. These observations provide a basis for evaluating model neurological disorders in transgenic mice that express chemokines ectopically or in mice that are deficient in chemokine ligands or receptors as a consequence of gene targeting. Ultimately, a clear definition of roles of chemokines and their receptors in neurological diseases will suggest rational intervention.

Evidence for the contribution of adult neurogenesis and hippocampal cell death in experimental cerebral malaria cognitive outcome.

Cognitive dysfunction is a major sign of cerebral malaria (CM). However, the underlying mechanisms of CM cognitive outcome remain poorly understood. A body of evidence suggests that adult neurogenesis may play a role in learning and memory processes. It has also been reported that these phenomena can be regulated by the immune system. We hypothesized that memory dysfunction in CM results from hippocampal neurogenesis impairment mediated by the deregulated immune response during the acute phase of CM. C57Bl/6 mice were infected with Plasmodium berghei ANKA (PbA) strain, using a standardized inoculation of 10(6) parasitized erythrocytes. Long-term working memory was evaluated using the novel object recognition test. The mRNA expression of brain-derived neurotrophic factor (BDNF), tropomyosin-receptor-kinase (TRK-B) and nerve growth factor (NGF) in the frontal cortex and hippocampus was estimated by real-time polymerase chain reaction (PCR). The protein levels of cytokine interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and CCL11 and neurotrophins BDNF and NGF were determined using a cytometric bead array (CBA) kit or enzyme-linked immunosorbent assay. Cell viability in the hippocampus was analyzed by Confocal Microscopy. Neurogenesis in the dentate gyrus was determined through quantification of doublecortin (DCX) positive cells. PbA-infected mice presented working memory impairment on day 5 post-infection. At this same time point, CM mice exhibited a decrease in DCX-positive cells in the dentate gyrus in parallel with increased cell death and elevated inflammatory cytokines (IL-6, TNF-α, IFN-γ and CCL11) in the hippocampus and frontal cortex. A significant reduction of BDNF mRNA expression was also found. IL-6 and TNF-α correlated negatively with BDNF and NGF levels in the hippocampus of CM mice. In summary, we provide further evidence that neuroinflammation following PbA-infection influences neurotrophin expression, impairs adult hippocampal neurogenesis and increases hippocampal cell death in association with memory impairment following CM course. The current study identified potential mediators of memory impairment in CM.

Distribution of immunoglobulin superfamily members ICAM-1, -2, -3, and the beta 2 integrin LFA-1 in multiple sclerosis lesions.

To identify potential molecular substrates for leukocyte trafficking and activation in multiple sclerosis (MS) brain, we determined the immunocytochemical distribution of the beta, integrin lymphocyte-function-associated antigen-1 (LFA-1) and its major ligands, intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 in MS tissue. Colocalization of these adhesion molecules with lineage-specific markers was analyzed by dual-labeling immunocytochemistry and confocal microscopy. ICAM-1 and ICAM-2 were detected on endothelial cells, and ICAM-3 immunoreactivity was restricted to infiltrating leukocytes. In control brain, 10% of glucose transporter-1 positive vessels contained ICAM-1 immunoreactivity on their luminal surface and 21% were ICAM-2-positive. A significant increase in ICAM-1-positive vessels was found in MS brains. This increase was greater in MS lesions (81% of vessels) than in nonlesion areas (37% of vessels). A significant increase in ICAM-1-positive vessels was found in encephalitis (55% of vessels) but not in Parkinson's (17% of vessels) brains. The percentage of vessels expressing ICAM-2 was not increased in MS, encephalitis, or Parkinson's brains. Both ICAM-3 and LFA-1 were detected on the vast majority of infiltrating lymphocytes and monocytes in and near MS lesions, and these cells were often closely apposed to each other. In addition, LFA-1 was detected on activated microglia located close to the edge of demyelinating lesions. ICAM-3-positive leukocytes were often closely apposed to LFA-1-positive microglia. These results suggest a role for ICAM-1, -2, and LFA-1 in the transendothelial migration of leukocytes into MS brain and a role for ICAM 3/LFA-1 interactions in the activation of lymphocytes, monocytes, and microglia in MS lesions.

Role of chemokines, neuronal projections, and the blood-brain barrier in the enhancement of cerebral EAE following focal brain damage.

The role of focal brain damage as a trigger for autoimmune inflammation in multiple sclerosis (MS) is unclear. In this study we examine mechanisms by which experimental autoimmune encephalomyelitis (EAE) is enhanced by focal brain damage. EAE was produced in Lewis rats by footpad inoculation; focal brain damage, in the form of a cortical cryolesion (cryolesion-EAE), was induced 8 days post-inoculation (d.p.i.). The distribution of inflammation and chemokine production in cryolesion-EAE and EAE-only were compared. Inflammation in the brain, measured by immunocytochemistry for T lymphocytes (W3/13) and microglial activation (MHC class II -OX6), was significantly enhanced in cryolesion-EAE 11-15 d.p.i. (p < 0.01-0.05) but by 20-40 d.p.i., equated with EAE-only. Inflammation in cryolesion-EAE related to breakdown of the blood-brain barrier (BBB) at the site of the cryolesion and also to the corticospinal tracts and thalamus, reflecting the afferent and efferent neuronal connections with the cryolesioned cortex. Semiquantitative RT/PCR dot-blot hybridization assay showed a 6-fold increase in mRNA for specific chemokines in the brain in cryolesion-EAE at 9 d.p.i. (MCP-1) and 11 d.p.i. (MCP-1 and MCP-5) with no significant increase in RANTES, GRO-alpha, or MIP-1alpha. By 14 d.p.i., the levels of MCP-1 and MCP-5 mRNA equated with EAE-only animals. These results suggest that enhancement and location of autoimmune inflammation in the brain following focal cortical injury initially involve chemokines such as the macrophage chemoattractants MCP-1 and MCP-5, and the activities of afferent and efferent neuronal connections with the site of damage. By analogy, similar factors may modulate or reactivate autoimmune inflammation in MS.

Do chemokines mediate inflammatory cell invasion of the central nervous system parenchyma?

Inflammatory cell recruitment into the central nervous system (CNS) is a critical step in the response to diverse insults, including infection, trauma and infarction, as well as immune-mediated disorders such as multiple sclerosis (MS). Despite considerable advances in understanding immune surveillance and antigen recognition in the CNS, the signals resulting in parenchymal inflammation are incompletely understood. Members of a novel family of chemo-attractant cytokines, the chemokines, are made in the CNS and are emerging as likely mediators of inflammatory cell migration into the CNS.