The effects of methylprednisolone and mitoxantrone on CCL5-induced migration of lymphocytes in multiple sclerosis.

OBJECTIVES: Chemokines are involved in migration of inflammatory cells to the central nervous system (CNS) in multiple sclerosis (MS). The aim of this study was the analysis of the impact of MS treatment on CCL5-induced migration of leukocyte subpopulations. MATERIAL AND METHODS: Migration of lymphocytes and monocytes from blood of MS patients treated with methylprednisolone (MP) or mitoxantrone (MTX) was analysed in a chemotaxis chamber. RESULTS: CCL5-induced migration of lymphocytes from untreated MS patients was significantly increased over controls. The treatment of MS with MP and MTX reduced this chemotaxis. The plasma level of CCL5 was increased in MS patients before treatment and was also significantly decreased in the treatment of MS with MP and MTX. CONCLUSIONS: This observation supports the hypothesis that in MS, chemokine CCL5 may induce migration of leukocytes to the CNS and suggests that treatment of the disease with MP and MTX may reduce this migration.

Expression of chemokine receptors CCR7 and CCR8 in the CNS during ChREAE.

Chemokines and their receptors are important players in organism homeostasis, development and immune response to inflammatory stimuli. It has been recently confirmed that they are also involved in the development of several autoimmune diseases. In this study, we analysed the expression of two recently identified CC chemokine receptors, CCR7 and CCR8, in the central nervous system (CNS) and in peripheral tissues during chronic relapsing experimental autoimmune encephalomyelitis (ChREAE) -- an animal model of the human demyelinating disease multiple sclerosis (MS). We observed upregulation of both chemokine receptors in the CNS during the first and second attacks of ChREAE, whereas disease remission was characterized by a lower expression of those receptors. An analysis of the kinetics of CCR7 and CCR8 expression in the CNS during the first attack of the disease showed a constant increase in the first few days after the onset of clinical signs. This expression correlated with the clinical severity of ChREAE. CCR7-positive mononuclear cells were detected mostly in perivascular inflammatory cuffs in the CNS. In peripheral tissues (the spleen and kidneys) expression of both receptors was not upregulated during active ChREAE. These findings suggest that CCR7 and CCR8 may play a significant role in the pathogenesis of EAE and probably MS.

Chemokine upregulation follows cytokine expression in chronic relapsing experimental autoimmune encephalomyelitis.

Chronic relapsing experimental autoimmune encephalomyelitis (ChREAE) is an autoimmune disease of the central nervous system (CNS) induced by CNS myelin components. In the early active stage, both ChREAE and multiple sclerosis (MS) are characterized by the presence of perivascular inflammatory cuffs disseminated in the CNS. There is growing evidence that chemoattractant cytokines (chemokines) play an important role in this process. The main goal of the present study was to analyse the hypothesis that chemokine expression in the CNS during autoimmune inflammation is regulated by proinflammatory cytokines. To address this concept, we analysed temporal relations between chemokine and cytokine expression during ChREAE. Phasic upregulation of gene expression for chemokines T-cell activation gene 3 (TCA-3)/CCL1, monocyte chemoattractant protein-1 (MCP-1)/CCL2, macrophage inflammatory protein-1 alpha (MIP-1alpha)/CCL3, MIP-1beta/CCL4, regulated on activation normal T cell expressed and secreted (RANTES)/CCL5 and MIP-2/CXCL2-3 as well as cytokines tumour necrosis factor-alpha (TNF-alpha), -beta, LT-beta, interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1) in the CNS was observed during attacks of ChREAE. Expression of cytokines TNF-beta and LT-beta preceded, and the expression of TGF-beta1 followed chemokine upregulation. Our results suggest that chemokine expression during CNS autoimmune inflammation may be regulated by some proinflammatory cytokines.

[Gene therapy in neurological diseases]. / Terapia genowa w chorobach neurologicznych.

Gene therapy is a new therapeutic method which uses molecular biology techniques to modulate expression of genes involved in disease pathogenesis. Gene therapy has evolved rapidly in the last ten years. In that time several experimental models of metabolic and degenerative neurological disorders, brain tumours, stroke, epilepsy, motor neuron disease, multiple sclerosis and muscle dystrophy were treated with some positive results by gene therapy techniques. The first clinical trials are currently ongoing in certain metabolic diseases, brain tumours and limb girdle dystrophy. DNA vaccination is also used in clinical trials. It seems that gene therapy will become soon the alternative method of treatment of those neurological diseases where current methods of treatment are not effective.

Chemokines and chemokine receptors in inflammation of the nervous system: manifold roles and exquisite regulation.

This article focuses on the production of chemokines by resident glial cells of the nervous system. We describe studies in two distinct categories of inflammation within the nervous system: immune-mediated inflammation as seen in experimental autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS) and post-traumatic inflammation. We provide evidence that chemokines play a role in amplifying the inflammatory reaction in EAE (and, probably, MS). In the context of neural trauma, chemokines appear to be primary stimuli for leukocyte recruitment. Strikingly, expression of monocyte chemoattractant protein (MCP)-1 and interferon-gamma-inducible protein-10 (IP-10) are largely restricted to astrocytes or other glial cells in these diverse pathological states. The remainder of the review focuses on studies that address the molecular mechanisms which underlie transcriptional regulation of three astrocyte-derived chemokines: MCP-1, IP-10 and beta-R1/interferon-gamma-inducible T-cell chemoattractant (I-TAC). Based on these studies, we propose that the complex promoters of these genes are marvelously organized for flexible and efficient response to challenge. In the case of MCP-1, several different stimuli can elicit gene transcription, acting through a conserved mechanism that includes binding of inducible transcription factors and recruitment of the constitutive factor Sp1. For IP-10 and beta-R1/I-TAC, it appears that efficient gene transcription occurs only in highly inflammatory circumstances that produce aggregates of simultaneous stimuli. These characteristics, in turn, mirror the expression patterns of the endogenous genes: MCP-1 is expressed under a variety of circumstances, while IP-10 appears primarily during immune-mediated processes that feature exposure of resident neuroglia to high levels of inflammatory cytokines.

CXC chemokine receptors expression during chronic relapsing experimental autoimmune encephalomyelitis.

Chemokines are small proinflammatory cytokines that possess the ability to stimulate migration of inflammatory cells towards the tissue site of inflammation. Previous reports showed that several chemokines may be involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of autoimmune central nervous system (CNS) inflammation. Inflammatory cells respond to chemotactic chemokine gradient through the chemokine receptors (ChRs). The goal of this study was to analyze expression of ChRs belonging to CXC subfamily during different stages of chronic relapsing EAE. We found significantly increased expression of CXCR2 and CXCR4 in the spinal cord during the first and second disease attacks. The kinetics of this expression in CNS and blood suggests that CXCR2 is expressed by leukocytes migrating from the blood, but CXCR4 is expressed mainly by CNS parenchymal cells. Those results support the interpretation that chemokine-chemokine receptor interactions may play an important role in the development of CNS autoimmune inflammation.

[Chemokines in central nervous system pathology]. / Udzial chemokin w patologii osrodkowego ukladu nerwowego.

Chemokines are a family of proinflammatory cytokines which are able to stimulate directional migration of leukocytes in vitro and in vivo. Because of this feature chemokines may be potent mediators of inflammatory processes. Numerous observations indicate that chemokines may be involved in the pathogenesis of autoimmune and infectious inflammation within the central nervous system (CNS). Moreover, there are many reports showing increased expression of some chemokines in experimental models of brain mechanical injury and ischaemia. Several lines of cultured CNS cells are able to produce chemokines in vitro. All those data suggest that chemokines are important mediators of CNS pathology and that they can be a promising target for future therapy of neurological diseases.

Treatment with BBB022A or rolipram stabilizes the blood-brain barrier in experimental autoimmune encephalomyelitis: an additional mechanism for the therapeutic effect of type IV phosphodiesterase inhibitors.

We examined the treatment effects of two structurally distinct phosphodiesterase type IV (PDE IV) inhibitors, BBB022 and rolipram, in murine and rat models of experimental autoimmune encephalomyelitis (EAE). Based on our data, we propose a mechanism of action which may supplement immunomodulatory effects of PDE IV inhibitors. In particular, PDE inhibitors promote elevation of intracellular cAMP levels, increasing the electrical resistance of endothelial monolayers by stabilizing intercellular junctional complexes. Such an effect on central nervous system (CNS) vascular endothelium has the potential to reduce disease severity in EAE, because both inflammatory cells and humoral factors readily cross a disrupted blood-brain barrier (BBB). In this report, we demonstrate the capacity of BBB022 and rolipram to decrease clinical severity of EAE. further, PDE IV inhibitors significantly reduced BBB permeability in the spinal cords of mice with EAE. These results provide evidence that PDE IV-inhibitors may exert therapeutic effects in EAE by modifying cerebrovascular endothelial permeability, reducing tissue edema as well as entry of inflammatory cells and factors.

Chemokine expression in GKO mice (lacking interferon-gamma) with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) considered to be an animal model for multiple sclerosis (MS). The detailed mechanism that specifies accumulation of inflammatory cells within the CNS in these conditions remains a subject of active investigation. Chemokines including IP-10, GRO-alpha, MCP-1 are produced in EAE tissues selectively by parenchymal astrocytes, but the regulatory stimuli that govern this expression remain undetermined. The unexpected occurrence of increased EAE susceptibility in Balb/c GKO mice (lacking IFN-gamma) offered an opportunity to examine the spectrum of chemokine expression during immune-mediated inflammation in the absence of a single regulatory cytokine. We found that chemokines MCP-1 and GRO-alpha were upregulated in the CNS of mice with EAE despite the GKO genotype. IP-10, which is highly expressed in the CNS of mice with an intact IFN-gamma gene and EAE, was strikingly absent. In vitro experiments confirmed that IFNgamma selectively stimulates astrocytes for IP-10 expression. These results indicate that IP-10 is dependent upon IFN-gamma for its upregulation during this model disease, and document directly that astrocyte expression of chemokines during EAE is governed by pro-inflammatory cytokines.

Expression of chemokines RANTES, MIP-1alpha and GRO-alpha correlates with inflammation in acute experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an investigator-initiated disorder that serves as an animal model for the common human demyelinating disease multiple sclerosis. Both diseases are typified by disseminated perivascular and submeningeal cuffs in the central nervous system (CNS). It was shown recently that chemokines are integral to the pathogenesis of EAE. In the present study we analyzed the gene expression of three chemokines, RANTES, MIP-1alpha and GRO-alpha, at the onset of acute EAE, and correlated that expression with the intensity of inflammatory changes in the CNS. We showed that all three chemokines are upregulated simultaneously with symptom onset of acute EAE, and that chemokine expression correlates with the intensity of inflammation in the CNS. This consistent relationship supports the hypothesis that chemokines are relevant to leukocyte accumulation in CNS parenchyma.

Tumor necrosis factor-alpha induced pathology in the rat brain: characterization of stereotaxic injection model.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine of cytotoxic and proinflammatory properties. It is believed to play an important role in inflammatory and demyelinization processes in the central nervous system (CNS). The aim of this study was to investigate what pathological changes could be produced by a stereotaxic administration of TNF-alpha in vivo into rat brain. Specimens were evaluated after staining with hematoxylin and eosin (H & E), Kluver-Barrera staining and using immunocytochemical methods. Disturbances of the blood-brain barrier (BBB) were analyzed as well as inflammatory infiltrates, changes in neurons, astrocytes and myelin. TNF-alpha injected in vivo into a rat brain caused a prominent inflammatory reaction in the cerebral meninges and a local cytotoxic brain edema when compared with the control group. Moreover, disturbances of the BBB premeability, infiltration of blood-borne macrophages in the area of the cytokine injection and early arising astrogliosis were observed. These results suggest that TNF-alpha can be an important mediator of inflammatory processes in the CNS.

Expression of monocyte chemoattractant protein (MCP-1) and nitric oxide synthase-2 following cerebral trauma.

Traumatic injury to the brain initiates multiple interrelated processes that involve parenchymal, vascular, and infiltrating inflammatory cells. Nitric oxide (NO) and chemokines have been implicated as regulators of the central nervous system injury response. Following a cryogenic lesion of the cerebral cortex in mice, mRNA for NO synthase (NOS)-2 was detected by reverse transcriptase polymerase chain reaction ipsilaterally 12 h after injury and persisted for 2 weeks. While mRNA was also detected contralaterally, the time course of expression was shorter (1 week). By immunohistochemistry, NOS-2 protein was initially detected ipsilaterally 12 h after injury in infiltrating inflammatory cells. Astroglial cells expressed NOS-2 from 24 to 72 h after injury. The expression of monocyte chemoattractant protein (MCP-1) mRNA peaked at 6 h on the lesion side, remained for 24 h and then declined by 48 h. On the unlesioned side, MCP-1 mRNA was expressed to a much lesser extent and had declined by 24 h. The up-regulation of MCP-1 was relatively specific as a closely related mRNA encoding IP-10 was not significantly increased. These findings implicate a role for NOS-2 and MCP-1 as potential regulators of cellular events following cryogenic cerebral trauma.

Synchronous synthesis of alpha- and beta-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis.

Chemokines are secreted peptides that exhibit selective chemoattractant properties for target leukocytes. Two subfamilies, alpha- and beta-chemokines, have been described, based on structural, genetic, and functional considerations. In acute experimental autoimmune encephalomyelitis (EAE), chemokines are up-regulated systemically and in central nervous system (CNS) tissues at disease onset. Functional significance of this expression was supported by other studies; intervention with an antichemokine antibody abrogated passive transfer of EAE, and chemokines expressed in brains of transgenic mice recruited appropriate leukocyte populations into the CNS compartment. Chemokine expression in the more relevant circumstance of chronic EAE has not been addressed. We monitored the time course and cellular sources of chemokines (monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha, interferon-gamma-inducible protein of 10 kd, KC, and regulated on activation, normal T-cell expressed and secreted cytokine) in CNS and peripheral tissues during spontaneous relapses of chronic EAE. We found coordinate chemokine up-regulation in brain and spinal cord during clinical relapse, with expression confined to CNS tissues. Monocyte chemoattractant protein-1, interferon-gamma-inducible protein of 10 kd, and KC were synthesized by astrocytic cells, whereas macrophage inflammatory protein-1 alpha and regulated on activation, normal T-cell expressed and secreted cytokine were elaborated by infiltrating leukocytes. The results demonstrate stringent regulation of multiple chemokines in vivo during a complex organ-specific autoimmune disease. We propose that chemokine expression links T-cell antigen recognition and activation to subsequent CNS inflammatory pathology in chronic relapsing EAE.

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.

Chemokine monocyte chemoattractant protein-1 is expressed by astrocytes after mechanical injury to the brain.

By 24 h after mechanical trauma to the cerebral cortex, astroglial reaction begins and injury sites are infiltrated by activated mononuclear phagocytes derived from blood-borne monocytes and endogenous microglia. There is little information about cellular interactions between astrocytes and leukocytes during this process. We previously showed that murine astrocytes produce chemokines including monocyte chemoattractant protein-1 (MCP-1) during experimental autoimmune encephalomyelitis. In this study, we asked whether astrocytes produce MCP-1 in the absence of immune mediated inflammation. To address this question, we analyzed the time course and cellular source of MCP-1 in mouse brain after penetrating mechanical injury, with particular focus on early time points before histologic detection of infiltrating mononuclear phagocytes. We observed sharply increased steady state levels of MCP-1 mRNA within 3 h after nitrocellulose membrane stab or implant injury to the adult mouse brain, and MCP-1 protein elevations were documented at 12 h postinjury. In situ hybridization combined with immunohistochemistry for the glial fibrillary acidic protein astrocyte marker showed that astrocytes were the cellular source of MCP-1 mRNA at these early time points after mechanical brain injury. Stab injury to the neonatal brain evoked neither MCP-1 expression nor astrogliosis. These results demonstrate that chemokine gene expression comprises one component of the astrocyte activation program. The data are consistent with a role for MCP-1 in the central nervous system inflammatory response to trauma.

In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis.

Reactive astrogliosis is a prominent pathological feature of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. It is characterized by hypertrophy of astrocytes with increased content of glial fibrillary acidic protein (GFAP.) Studies of reactive astrocytes in acute experimental autoimmune encephalomyelitis have been complicated by the observation that the diffuse increase in GFAP immunohistochemical staining at the onset of central nervous system inflammation does not parallel the gradual increase in GFAP content probably because tissue edema enhances GFAP immunostaining. To characterize changes in GFAP expression, we performed in situ hybridization at 3- to 7-day intervals during the course of acute murine experimental autoimmune encephalomyelitis. We found a biphasic course of GFAP expression: an early phase of astrocyte reaction surrounding perivascular inflammatory cuffs and submeningeal infiltrates at the onset of central nervous system inflammation and clinical disease and a later phase of increased GFAP mRNA expression in regions of demyelination during resolution of inflammation and clinical improvement. IP-10, a member of a family of proinflammatory chemoattractant cytokines called chemokines, was expressed by astrocytes in a similar distribution as those expressing increased GFAP mRNA in the early phase of inflammation but was no detected in astrocytes in the later phase of activation. These results indicate that location and function of reactive astrocytes may vary during the course of immune-mediated demyelination.