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.     

Expression of beta-chemokines and chemokine receptors in human fetal astrocyte and microglial co-cultures: potential role of chemokines in the developing CNS.

Chemokines play specific roles in directing the recruitment of leukocyte subsets into inflammatory foci within the central nervous system (CNS). The involvement of these cytokines as mediators of inflammation is widely accepted. Recently, it has become evident that cells of the CNS (astrocytes, microglia, and neurons) not only synthesize, but also respond functionally or chemotactically to chemokines. We previously reported developmental events associated with colonization of the human fetal CNS by mononuclear phagocytes (microglial precursors), which essentially takes place within the first two trimesters of life. As part of the array of signals driving colonization, we noted specific anatomical distribution of chemokines and chemokine receptors expressed during this period. In order to further characterize expression of these molecules, we have isolated and cultured material from human fetal CNS. We demonstrate that unstimulated subconfluent human fetal glial cultures express high levels of CCR2 and CXCR4 receptors in cytoplasmic vesicles. Type I astrocytes, and associated ameboid microglia in particular, express high levels of surface and cytoplasmic CXCR4. Of the chemokines tested (MIP-1alpha, MIP-1beta, MCP-1, MCP-3, RANTES, SDF-1, IL-8, IP-10), only MIP-1alpha, detected specifically on microglia, was expressed both constitutively and consistently. Low variable levels of MCP-1, MIP-1alpha, and RANTES were also noted in unstimulated glial cultures. Recombinant human chemokines rhMCP-1 and rhMIP-1alpha also displayed proliferative effects on glial cultures at [10 ng/ml], but displayed variable effects on CCR2 levels on these cells. rhMCP-1 specifically upregulated CCR2 expression on cultured glia at [50 ng/ml]. It is gradually becoming evident that chemokines are important in embryonic development. The observation that human fetal glial cells and their progenitors express specific receptors for chemokines and can be stimulated to produce MCP-1, as well as proliferate in response to chemokines, supports a role for these cytokines as regulatory factors during development.     

Upregulated expression of interleukin-8, RANTES and chemokine receptors in human astrocytic cells infected with HIV-1

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) affects primarily microglial cells and astrocytes. Infection of these latter cells occurs independently of CD4 and is characterised by preferential accumulation of 2 Kb mRNA, encoding mostly Nef, and by low levels of 4.5 and 9 Kb RNAs. We have investigated the potential role of chronic HIV infection of human astrocytic cells on the expression of pro-inflammatory cytokines, chemokines and their receptors by comparing the infected TH4-7-5 with its parental uninfected 85HG66 cell lines. Upregulated levels of tumour necrosis factor-alpha (TNF-alpha) and of certain chemokines, namely interleukin-8 (IL-8) and regulated upon activation normal T cell expressed and secreted (RANTES), were observed in the infected versus uninfected cells, whereas monocyte chemotactic protein-1 (MCP-1) was comparably expressed in both cell lines. This pattern of expression was confirmed in primary foetal astrocytes transiently transfected with HIV. In addition, CXCR1, CXCR2 and CCR2b, receptors for IL-8 and MCP-1, respectively, were also found to be upregulated in TH4-7-5 versus 85HG66. CXCR4, the receptor of stromal cell derived factor-1 (SDF-1) and co-receptor for syncytium inducing HIVs, was comparably expressed in infected and uninfected astrocytic cells, whereas CCR5 was not detected in either cell line. Furthermore, treatment of TH4-7-5 cells with TNF-alpha or IL-1beta stimulated RNA and protein secretion of IL-8, MCP-1, and RANTES as well as HIV expression. Thus, our findings suggest that HIV infection of astrocytic cells can contribute to the establishment of a chronic inflammatory state in the CNS, eventually resulting in HIV encephalitis, by increasing the secretion of pro-inflammatory cytokines, such as TNF-alpha and several chemokines. Overexpression of chemokine receptors including CCR2b, CXCR1 and CXCR2 in infected astrocytic cells may contribute to HIV-induced damage of the CNS via autocrine/paracrine activation of astrocytes.     

Regulation of chemokine receptor expression in human microglia and astrocytes

It has been proposed that the positioning of mobile cells within a tissue is determined by their overall profile of chemokine receptors. This study examines the profiles of chemokine receptors expressed on resting and activated adult human microglial cells, astrocytes and a microglial cell line, CHME3. Microglia express highest levels of CXCR1, CXCR3 and CCR3. Astrocytes also have moderate levels of CXCR1 and CXCR3, and some CCR3, while both cell types also expressed CCR4, CCR5, CCR6, CXCR2, CXCR4 and CXCR5 at lower levels. Activation of the cells with the inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) increased the expression of some but not all receptors over a period of 24 h. Microglia showed moderate enhancement of receptor expression, while astrocytes responded particularly strongly to TNFalpha with enhanced CXCR3, CCR3 and CXCR1. However, the migratory and proliferative responses of the microglia and astrocytes to the same chemokine were different, with microglia migrating and astrocytes proliferating in response to CXCL10. The data indicates a mechanism by which activated microglia and astrocytes become selectively more sensitive to inflammatory chemokines during CNS disease, and the paper discusses which of the many chemokines present in CNS would have priority of action on microglia and astrocytes.     

Expression of chemokines and their receptors in human and simian astrocytes: evidence for a central role of TNF alpha and IFN gamma in CXCR4 and CCR5 modulation

Chemokines are key mediators of the selective migration of leukocytes that occurs in neurodegenerative diseases and related inflammatory processes. Astrocytes, the most abundant cell type in the CNS, have an active role in brain inflammation. To ascertain the role of astrocytes during neuropathological processes, we have investigated in two models of primary cells (human fetal and simian adult astrocytes) the repertoire of chemokines and their receptors expressed in response to inflammatory stimuli. We demonstrated that, in the absence of any stimulation, human fetal and simian adult astrocytes express mRNA for receptors APJ, BOB/GPR15, Bonzo/CXCR6, CCR2, CCR3, CCR5, CCR8, ChemR23, CXCR3/GPR9, CXCR4, GPR1, and V28/CX3CR1. Moreover, TNFalpha and IL-1beta significantly increase BOB/GPR15, CCR2, and V28/CX3CR1 mRNA levels in both models. Furthermore, TNFalpha and IFNgamma act synergistically to induce expression of the major coreceptors for HIV infection, CXCR4 and CCR5, at both the mRNA and protein levels in human and simian astrocytes, whereas CCR3 expression was not affected by cytokine treatment. Finally, TNFalpha/IFNgamma was the most significant cytokine combination in leading to a pronounced upregulation in a comparable, time-dependent manner of the production of chemokines IP-10/CXCL10, RANTES/CCL5, MIG/CXCL9, MCP-1/CCL2, and IL-8/CXCL8. In summary, these data suggest that astrocytes serve as an important source of chemokines under the dependence of a complex cytokine regulation, and TNFalpha and IFNgamma are important modulators of chemokines and chemokine receptor expression in human as well as simian astrocytes. Finally, with the conditions we used, there was no difference between species or age of tissue.     

Expression of binding sites for beta chemokines on human astrocytes

Astrocytes are major sources of chemokines and are thus critical effectors of central nervous system (CNS) inflammation. However, it is as yet unclear whether these cells, like leukocytes, also possess receptors for chemokines (CCRs). To address this issue, we utilized a novel fluorescence approach to detect qualitatively and quantitatively binding sites for biotinylated derivatives of the beta chemokines monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) on cultured human fetal astrocytes. Both chemokines were found to bind to the surface of astrocytes in a specific and saturable manner and with the high-affinity typical of these chemokines' binding to leukocyte CCRs. Binding of labeled MCP-1 and of labeled MIP-1alpha was antagonized by the respective unlabeled homologue but not by the unlabeled heterologous chemokine. Binding of labeled MCP-1 was also inhibited by unlabeled MCP-3, both of which are ligands for CCR2. In a parallel manner, binding of labeled MIP-1alpha was first shown to be attenuated by unlabeled RANTES, both of which recognize CCR1 and CCR5, and then separately antagonized by MCP-3 and MIP-1beta, which bind to CCR1 and CCR5, respectively. Finally, binding of both labeled chemokines was observed to be modulated in response to astrocyte stimulation by proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), further indicating that these binding sites are subject to regulation and, thus, likely to be physiologically responsive. Collectively, these results indicate that binding sites exhibiting characteristics of chemokine receptors exist on human astrocytes. Such sites might function in the recruitment of both astrocytes and leukocytes to specified brain regions during physiological and pathophysiological processes.     

Experimental stroke induces massive, rapid activation of the peripheral immune system.

Clinical experimental stroke induces injurious local brain inflammation. However, effects on the peripheral immune system have not been well characterized. We quantified mRNA and protein levels for cytokines, chemokines, and chemokine receptors (CCR) in brain, spinal cord, peripheral lymphoid organs (spleen, lymph node, blood, and cultured mononuclear cells from these sources), and blood plasma after reversible middle cerebral artery occlusion (MCAO) or sham treatment in male C57BL/6 mice. Middle cerebral artery occlusion induced a complex, but organ specific, pattern of inflammatory factors in the periphery. At both 6 and 22 h after MCAO, activated spleen cells from stroke-injured mice secreted significantly enhanced levels of TNF-alpha, IFN-gamma, IL-6, MCP-1, and IL-2. Unstimulated splenocytes expressed increased chemokines and CCR, including MIP-2 and CCR2, CCR7 and CCR8 at 6 h; and MIP-2, IP-10, and CCR1 and CCR2 at 22 h. Also at 22 h, T cells from blood and lymph nodes secreted increased levels of inflammatory cytokines after activation. As expected, there were striking proinflammatory changes in postischemic brain. In contrast, spinal cord displayed suppression of all mediators, suggesting a compensatory response to intracranial events. These data show for the first time that focal cerebral ischemia results in dynamic and widespread activation of inflammatory cytokines, chemokines, and CCR in the peripheral immune system.     

Peripheral chemokine receptors, their ligands, cytokines and Alzheimer's disease

Cerebral inflammation as well as systemic immunological alterations has been reported in Alzheimer's disease (AD). We aimed to determine whether spontaneous and mitogen stimulated production of peripheral blood mononuclear cell (PBMC) cytokines, chemokines and chemokine receptors in clinically diagnosed patients with AD were unregulated. PBMC were purified from AD patients and from healthy controls. Supernatants were analyzed for cytokine levels by ELISA methods. mRNA expression was determined by RT-PCR. Expression of chemokine receptors CCR2 and CCR5 was determined by cytofluorimetric analysis. Both CCR5 and CCR2 expression were increased in AD patients respect to control subjects and the expression of CCR2 and CCR5 was more frequent on CD4+ and less frequent on CD8+ cells. Levels of Th1-type cytokine IFNgamma and chemokine RANTES were increased and levels of Th2-type cytokine IL-4 and chemokine MCP-1 were reduced in AD patients compared with those of control subjects. Acetylcholinesterase inhibitor pyridostigmine bromide (AChEI)-therapy reduced CCR2, CCR5, RANTES and IFNgamma expression and production in AD patients. CCR5, CCL5/RANTES, CCL2/MCP-1 and IFNgamma expression and production were increased in PBMC treated with amyloid-beta1-42. Addition of AChEI to PBMC suppresses CCL5/RANTES and IFNgamma. The observed patterns of cyto-chemokine involvement strengthen the questions regarding the inflammatory theory in AD, and raise a pathophysiologic role for selective alteration of cyto-chemokine network.     

Chemokine

Accumulation and activation of mononuclear cells (lymphocytes and monocytes) in the CNS is one of the crucial steps in the pathogenesis of multiple sclerosis (MS). Chemokines and their receptors govern physiological and pathological leukocyte trafficking and may also be pertinent in hematogenous leukocyte infiltration of the CNS. Due to broad pharmacological interest in the chemokine system, peptide antagonists and small molecular antagonists are now available for clinical therapeutic trials. For the treatment of MS in particular, the chemokine receptors CCR1, CCR2, CCR5, and CXCR3 are possible targets in a chemokine-based therapeutic approach. In this review, we summarize current knowledge of the roles of chemokines and chemokine receptors in the pathogenesis of MS. Furthermore, options for possible therapeutic intervention through the chemokine system are outlined. Clinical studies in MS patients applying this knowledge are expected soon.     

Developmental expression patterns of CCR5 and CXCR4 in the rhesus macaque brain.

Emerging data indicate that chemokine receptors on neurons and glia in the central nervous system (CNS) play a role in normal CNS development, intercellular communication, and the neuropathogenesis of AIDS. To further understand chemokine receptors in the brain and explore their potential role in HIV neuropathogenesis, particularly in pediatrics, we examined the regional and cellular distribution of CCR5 and CXCR4 in normal fetal, neonatal, and adult rhesus macaques. CCR5 and CXCR4 were detected by immunohistochemistry and immunofluorescence within the cytoplasm of subpopulations of neurons in the neocortex, hippocampus, basal nuclei, thalamus, brain stem, and cerebellum and by flow cytometry on the surface of neurons and glia. Interestingly, expression of CCR5 and CXCR4 increased significantly (p<0.05) from birth to 9 months of age. We further characterize this dynamic developmental pattern of CCR5 and CXCR4 expression in resident cells of the CNS.     

Longitudinal study of chemokine receptor expression on peripheral lymphocytes in multiple sclerosis: CXCR3 upregulation is associated with relapse

The interaction between chemokines and their receptors leads to selective recruitment of cells to foci of inflammation. Cross-sectional studies have reported significantly different expression of chemokine receptors CXCR3, CCR5 and CCR2 on peripheral blood lymphocytes in multiple sclerosis (MS) compared with controls. Cells expressing these receptors are likely to play a pathogenic role as suggested by studies of experimental autoimmune encephalomyelitis. Also, immunogenetic studies of nonfunctional CCR5 receptors in MS patients, due to 32delta deletion, demonstrated a delay in time to next relapse. The aims of this study were to detect any changes in the serial expression of chemokine receptors CCR2, CCR3, CCR5 and CXCR3 on peripheral blood CD4+ lymphocytes from patients with MS and to correlate the changes with relapses. Upregulation of CXCR3 expression on peripheral blood CD4+ lymphocytes was associated with all relapses and CCR5 expression was significantly affected with all relapses. Clinical recovery, with or without intravenous methylprednisolone treatment, coincided with the return of CXCR3 towards baseline in all but one case. Fluctuation in the expression of CXCR3 and CCR5 was also significantly greater in clinically stable patients with MS compared with controls, which may be due to subclinical disease activity. These findings provide further support for the view that CXCR3 and CCR5 antagonists could have a therapeutic value in MS.     

Expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) characterised by perivascular inflammatory cell infiltrates and plaques of demyelination. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The action of chemokines requires the expression of their complementary chemokine receptors by their target cells. We have examined the expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue using single- and double-labelling immunocytochemistry techniques. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In chronic active MS lesions CCR2, CCR3 and CCR5 were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present, but we also noted CCR3 and CCR5 on astrocytes in five of the 14 cases of MS investigated, particularly associated with processes around vessels and at the glia limitans. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localised around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS.     

Expression pattern and cellular sources of chemokines in primary central nervous system lymphoma

The expression pattern of a subset of chemokines and their corresponding receptors was investigated in primary central nervous system lymphomas (PCNSL). The tumor cells consistently expressed CXCR4, CXCL12, CXCR5, and CXCL13, both at mRNA and protein levels. Cerebral endothelial cells were positive for CXCL12 and CXCL13, while reactive astrocytes and microglial cells expressed CXCL12, CCR5, and CCR6. Inflammatory T cells in PCNSL were characterized by CCR5 and CCR6 positivity. Taken together, our data indicate a cell type-specific repertoire of chemokine and chemokine receptor expression in PCNSL suggesting that chemokine-mediated interactions facilitate crossing of the blood-brain barrier as well as intracerebral dissemination of PCNSL cells. In addition, chemokines expressed by tumor cells may contribute to induction of reactive glial changes and influence the composition of inflammatory infiltrates in PCNSL. Therefore, cell type specific expression of distinct chemokine profiles likely plays a role in the pathogenesis of PCNSL and may contribute to their characteristic histological appearance.     

17 beta-estradiol inhibits cytokine, chemokine, and chemokine receptor mRNA expression in the central nervous system of female mice with experimental autoimmune encephalomyelitis

Cytokines and chemokines govern leukocyte trafficking, thus regulating inflammatory responses. In this study, the anti-inflammatory effects of low dose 17 beta-estradiol were evaluated on chemokine, chemokine receptor, and cytokine expression in the spinal cords (SC) of BV8S2 transgenic female mice during acute and recovery phases of experimental autoimmune encephalomyelitis (EAE). In EAE protected mice, 17 beta-estradiol strongly inhibited mRNA expression of the chemokines RANTES, MIP-1 alpha, MIP-2, IP-10, and MCP-1, and of the chemokine receptors CCR1, CCR2 and CCR5 at both time points. Conversely, ovariectomy, which abrogated basal 17 beta-estradiol levels and increased the severity of EAE, enhanced the expression of MIP-1 alpha and MIP-2 that were over-expressed by inflammatory mononuclear cells in SC. 17 beta-estradiol inhibited expression of LT-beta, TNF-alpha, and IFN-gamma in SC, but had no effect on IL-4 or IL-10, indicating reduced inflammation but no deviation toward a Th2 response. Interestingly, elevated expression of CCR1 and CCR5 by lymph node cells was also inhibited in 17 beta-estradiol treated mice with EAE. Low doses of 17 beta-estradiol added in vitro to lymphocyte cultures had no direct effect on the activation of MBP-Ac1-11 specific T cells, and only at high doses diminished production of IFN-gamma, but not IL-12 or IL-10. These results suggest that the beneficial effects of 17 beta-estradiol are mediated in part by strong inhibition of recruited inflammatory cells, resulting in reduced production of inflammatory chemokines and cytokines in CNS, with modest effects on encephalitogenic T cells that seem to be relatively 17 beta-estradiol insensitive.     

Role of chemokines in CNS health and pathology: a focus on the CCL2/CCR2 and CXCL8/CXCR2 networks

Chemokines and their receptors have crucial roles in the trafficking of leukocytes, and are of particular interest in the context of the unique immune responses elicited in the central nervous system (CNS). The chemokine system CC ligand 2 (CCL2) with its receptor CC receptor 2 (CCR2), as well as the receptor CXCR2 and its multiple ligands CXCL1, CXCL2 and CXCL8, have been implicated in a wide range of neuropathologies, including trauma, ischemic injury and multiple sclerosis. This review aims to overview the current understanding of chemokines as mediators of leukocyte migration into the CNS under neuroinflammatory conditions. We will specifically focus on the involvement of two chemokine networks, namely CCL2/CCR2 and CXCL8/CXCR2, in promoting macrophage and neutrophil infiltration, respectively, into the lesioned parenchyma after focal traumatic brain injury. The constitutive brain expression of these chemokines and their receptors, including their recently identified roles in the modulation of neuroprotection, neurogenesis, and neurotransmission, will be discussed. In conclusion, the value of evidence obtained from the use of Ccl2- and Cxcr2-deficient mice will be reported, in the context of potential therapeutics inhibiting chemokine activity which are currently in clinical trial for various inflammatory diseases.     

Interactions between HIV-1 gp120, chemokines, and cultured adult microglial cells

HIV dementia (HIVD), a disease that is apparently mediated by neurotoxins and viral proteins secreted by HIV infected microglia, is characterized neuropathologically by an increased number of activated microglia in the brains of affected individuals. Consequently, the rational design of potential therapeutic strategies should take into account the mechanisms that lead to microglial activation and to their increased prominence in the adult brain. In this regard, one leading hypothesis proposes that microglia are recruited to specific sites in the central nervous system (CNS) as a result of interactions between microglial chemokine receptors and chemokines, or even the viral glycoprotein gp120, which binds chemokine receptors in the process of cellular entry. Adult microglia express the functional chemokine receptors CCR5 and CXCR4 molecules that mediate chemotaxis in these and other cell types. We determined that purified adult microglial cultures contain a heterogeneous population with respect to their ability to respond to the alpha- and beta-chemokines, SDF1alpha, and MIP-1beta. A mean of 14.6% of the microglia assayed responded to both alpha- and beta-chemokines (CCR5(+)CXCR4(+) phenotype); 45.4% of microglia were phenotyped as CCR5(+)CXCR4(-); 12.9% of the microglia were CXCR4(+)CCR5(-); and 27.0% of microglia did not respond to either chemokine. No increase in intracellular calcium levels was seen in the vast majority of microglia exposed to the soluble HIV envelope protein, gp120, or to HIV envelope (gp120/gp41) expressed on MLV virus pseudotypes. However, exposure of microglia to soluble fractalkine or to other chemokines resulted in an intracellular calcium flux. Our results raise the possibility of microglial heterogeneity with respect to their response to chemokines, and indicate that any effects due to gp120 are likely to be considerably less robust than the response of microglia to the natural ligands of their chemokine receptors, for example SDF1alpha and MIP-1beta.     

Regulation and function of central nervous system chemokines

In this paper, we discuss the potential involvement of a new family of cytokines, termed chemokines, in CNS inflammatory pathology. Chemokines are a family of proinflammatory cytokines which are able to stimulate target-cell-specific directional migration of leukocytes. Because of this feature, chemokines may be potent mediators of inflammatory processes. We have previously reported observations indicating that chemokines may be involved in the process of lesion formation during autoimmune inflammation within CNS, and, in particular, are likely participants in the process of influx of inflammatory cells into the CNS parenchyma. We observed also that mechanical injury of brain and subsequent post-traumatic inflammation may in part be mediated by chemokines. Chemokines undoubtedly co-operate with cell-associated adhesion molecules during recruitment of leukocytes from blood to CNS. The sequential expression of soluble and membrane-bound signals for leukocyte migration is an intricate process that can be interrupted by a variety of strategies. Our data suggest that chemokines may represent a promising target for future therapy of inflammatory conditions, including CNS inflammation resulting from varied insults.