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Prolonged Expression of Interferon‐Inducible Protein‐10 in Ischemic Cortex After Permanent Occlusion of the Middle Cerebral Artery in Rat
Journal of Neurochemistry
Abstract
Focal cerebral ischemia elicits local inflammatory reaction as demonstrated by the accumulation of inflammatory cells and mediators in the ischemic brain. Interferon-inducible protein-10 (IP-10) is a member of the C-X-C chemokine family that possesses potent chemoattractant actions for monocytes, T cells, and smooth muscle cells. To investigate a potential role of IP-10 in focal stroke, we studied the temporal expression of IP-10 mRNA after occlusion of the middle cerebral artery in rat by means of northern analysis. IP-10 mRNA expression after focal stroke demonstrated a unique biphasic profile, with a marked increase early at 3 h (4.9-fold over control; p < 0.01), a peak level at 6 h (14.5-fold; p < 0.001) after occlusion of the middle cerebral artery, and a second wave induction 10–15 days after ischemic injury (7.2- and 9.3-fold increase for 10 and 15 days, respectively; p < 0.001). In situ hybridization confirmed the induced expression of IP-10 mRNA and revealed its spatial distribution after focal stroke. Immunohistochemical studies demonstrated the expression of IP-10 peptide in neurons (3–12 h) and astroglial cells (6 h to 15 days) of the ischemic zone. To explore further the potential role of IP-10 in focal stroke, we demonstrated a dose-dependent chemotactic action of IP-10 on C6 glial cells and enhanced attachment of rat cerebellar granule neurons. Taken together, the data suggest that ischemia induces IP-10, which may play a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neuron attachment/sprouting after focal stroke.
... It binds the CXCR3 receptor, which is shared with CXCL9, and is expressed during focal stroke in animal models [13]. CXCL10 plays a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neural attachment/sprouting following focal stroke [14,15]. In recent studies, CCL11, CCL5, and CXCL10 are associated with ischemic stroke independent of traditional cardiovascular risk factors [16]. ...
... CXCL10 and its specific receptor CXCR3 are expressed in focal stroke in animal models [13]. As previously mentioned, CXCL10 plays a pleiotropic role in prolonged leukocyte recruitment, astrocyte migration/activation, and neuron attachment/sprouting after focal stroke [14,15]. A previous study demonstrated the focal enhanced serum levels of CXCL10 in rat brains peaking at 1 h, a followed by a second peak at 3-6 h, and a third peak at 15 days after stroke [16]. ...
... There are not enough studies considering levels of CXCL9 in stroke; however, it is known to be involved in inflammatory disorders such as atherosclerosis and rheumatoid arthritis, and participation with other inflammatory factors is well studied [3,12,14,19,20]. Its beneficial role in muscle reconstruction along with other chemokines is also well known [20,21]. ...
Objective:
Inflammation plays a significant role in the development of ischemic stroke. CXC chemokines play pleiotropic roles in prolonged leukocyte locomotion, astrocyte migration/activation, and neural attachment/sprouting in response to focal stroke. In this study, we aimed to explore the changes in serum levels of three chemokines, C-X-C motif chemokine ligand 1 (CXCL1), C-X-C motif chemokine ligand 9 (CXCL9), and C-X-C motif chemokine ligand 10 (CXCL10), in ischemic stroke patients at the time of admission and before discharge from the hospital ward.
Materials and methods:
In this study, we recruited 43 unrelated ischemic stroke patients using an easy convenience method or accidental sampling which is a type of non-probability sampling that involves the sample being drawn from that part of the population that is close to hand. We also enrolled 50 genetically unrelated healthy controls showing no history of neurologic, cardiovascular, or inflammatory diseases. Serum levels of the considered chemokines were measured by enzyme-linked immunosorbent assay (ELISA) in patients and healthy controls.
Results:
No significant difference was observed in ischemic stroke patients following hospitalization and prior discharging from the hospital; however, there was a significant difference in serum levels of CXCL9 and CXCL10 between patients and healthy controls. We also found that the level of the chemokine was not related to gender or medical therapy. It appears that CXCL9 and CXCL10 are more predisposing factors and play a direct role in stroke considering that they were higher in patients than in healthy controls.
Conclusion:
We believe that this study might be used as a basis for further studies on more effective medication regimens to prevent the onset and subsequent complications of stroke. However, these mediators are useful diagnostic and prognostic tools rather than therapeutic tools.
... CXCL10 is a wellknown chemo-attractant for peripheral immune cells including specific subsets of NK cells and macrophages, dendritic cells and activated CD4 1 T-cells and is therefore implicated in the modulation of both innate-and adaptive immune responses under pathological conditions ( Neville et al., 1997; Liu et al., 2005). Interestingly, increased expression of CXCL10 in the CNS has been demonstrated in ischemia ( Wang et al., 1998), Alzheimer's disease (Xia et al., 2000; Galimberti et al., 2006), Multiple sclerosis (MS) ( Balashov et al., 1999;Sorensen et al., 1999;Simpson et al., 2000), and human immunodeficiency virus (HIV)-encephalitis ( Sanders et al., 1998;Cinque et al., 2005). Depending on the insult, CXCL10 expression was demonstrated in neurons ( Wang et al., 1998;Rappert et al., 2004) and in astrocytes surrounding primary lesions ( Wang et al., 1998;Balashov et al., 1999;Simpson et al., 2000; Xia et al., 2000;Omari et al., 2005;Tanuma et al., 2006), while CXCR3 expression was found in microglia ( Biber et al., 2001Biber et al., , 2002Dijkstra et al., 2004;Rappert et al., 2004;Li et al., 2006;Tanuma et al., 2006 De Haas et al., 2008) and in reactive astrocytes ( Simpson et al., 2000;Goldberg et al., 2001;Omari et al., 2005;Tanuma et al., 2006). ...
... Interestingly, increased expression of CXCL10 in the CNS has been demonstrated in ischemia ( Wang et al., 1998), Alzheimer's disease (Xia et al., 2000; Galimberti et al., 2006), Multiple sclerosis (MS) ( Balashov et al., 1999;Sorensen et al., 1999;Simpson et al., 2000), and human immunodeficiency virus (HIV)-encephalitis ( Sanders et al., 1998;Cinque et al., 2005). Depending on the insult, CXCL10 expression was demonstrated in neurons ( Wang et al., 1998;Rappert et al., 2004) and in astrocytes surrounding primary lesions ( Wang et al., 1998;Balashov et al., 1999;Simpson et al., 2000; Xia et al., 2000;Omari et al., 2005;Tanuma et al., 2006), while CXCR3 expression was found in microglia ( Biber et al., 2001Biber et al., , 2002Dijkstra et al., 2004;Rappert et al., 2004;Li et al., 2006;Tanuma et al., 2006 De Haas et al., 2008) and in reactive astrocytes ( Simpson et al., 2000;Goldberg et al., 2001;Omari et al., 2005;Tanuma et al., 2006). In vitro, CXCL10 has been shown to induce directed migration of microglia ( Biber et al., 2002;Rappert et al., 2002) and proliferation in astrocytes ( Flynn et al., 2003), suggesting that CXCL10 might be involved in the activation and/or recruitment of local glia cells under pathological conditions. ...
... Interestingly, increased expression of CXCL10 in the CNS has been demonstrated in ischemia ( Wang et al., 1998), Alzheimer's disease (Xia et al., 2000; Galimberti et al., 2006), Multiple sclerosis (MS) ( Balashov et al., 1999;Sorensen et al., 1999;Simpson et al., 2000), and human immunodeficiency virus (HIV)-encephalitis ( Sanders et al., 1998;Cinque et al., 2005). Depending on the insult, CXCL10 expression was demonstrated in neurons ( Wang et al., 1998;Rappert et al., 2004) and in astrocytes surrounding primary lesions ( Wang et al., 1998;Balashov et al., 1999;Simpson et al., 2000; Xia et al., 2000;Omari et al., 2005;Tanuma et al., 2006), while CXCR3 expression was found in microglia ( Biber et al., 2001Biber et al., , 2002Dijkstra et al., 2004;Rappert et al., 2004;Li et al., 2006;Tanuma et al., 2006 De Haas et al., 2008) and in reactive astrocytes ( Simpson et al., 2000;Goldberg et al., 2001;Omari et al., 2005;Tanuma et al., 2006). In vitro, CXCL10 has been shown to induce directed migration of microglia ( Biber et al., 2002;Rappert et al., 2002) and proliferation in astrocytes ( Flynn et al., 2003), suggesting that CXCL10 might be involved in the activation and/or recruitment of local glia cells under pathological conditions. ...
The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10-deficient (CXCL10(-/-) ) and CXCR3-deficient (CXCR3(-/-) ) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA-treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10(-/-) and CXCR3(-/-) OHSCs in response to a high (50 μM) NMDA-concentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3(-/-) and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region-specific role for CXCL10/CXCR3 signaling in neuron-glia and glia-glia interactions under pathological conditions.
... Aif-1 is a marker of microglial activation, which is upregulated in response to CNS injury (Schwab et al., 2001). Osteopontin (Spp-1) is secreted by a subpopulation of activated microglia in response to a variety of insults, including stroke and systemic KA administration (Ellison et al., 1998;Kim et al., 2002). In the P15 animals, Spp-1 expression is only significantly upregulated at the 24 h time point, while Aif-1 is not significantly upregulated at any time point. ...
... In addition to its functions in the chemotaxis of T-lymphocytes, natural killer cells, and monocytes, Cxcl10 causes astrocyte Frontiers in Cell and Developmental Biology frontiersin.org chemotaxis when upregulated in response to cortical ischemia (Wang et al., 1998). The other two significantly upregulated alpha chemokines in human tissue, IL-8 and Cxcl2, function primarily to stimulate neutrophil chemotaxis and extravasation (Zhang et al., 2001). ...
Acute brain insults trigger diverse cellular and signaling responses and often precipitate epilepsy. The cellular, molecular and signaling events relevant to the emergence of the epileptic brain, however, remain poorly understood. These multiplex structural and functional alterations tend also to be opposing - some homeostatic and reparative while others disruptive; some associated with growth and proliferation while others, with cell death. To differentiate pathological from protective consequences, we compared seizure-induced changes in gene expression hours and days following kainic acid (KA)-induced status epilepticus (SE) in postnatal day (P) 30 and P15 rats by capitalizing on age-dependent differential physiologic responses to KA-SE; only mature rats, not immature rats, have been shown to develop spontaneous recurrent seizures after KA-SE. To correlate gene expression profiles in epileptic rats with epilepsy patients and demonstrate the clinical relevance of our findings, we performed gene analysis on four patient samples obtained from temporal lobectomy and compared to four control brains from NICHD Brain Bank. Pro-inflammatory gene expressions were at higher magnitudes and more sustained in P30. The inflammatory response was driven by the cytokines IL-1β, IL-6, and IL-18 in the acute period up to 72 h and by IL-18 in the subacute period through the 10-day time point. In addition, a panoply of other immune system genes was upregulated, including chemokines, glia markers and adhesion molecules. Genes associated with the mitogen activated protein kinase (MAPK) pathways comprised the largest functional group identified. Through the integration of multiple ontological databases, we analyzed genes belonging to 13 separate pathways linked to Classical MAPK ERK, as well as stress activated protein kinases (SAPKs) p38 and JNK. Interestingly, genes belonging to the Classical MAPK pathways were mostly transiently activated within the first 24 h, while genes in the SAPK pathways had divergent time courses of expression, showing sustained activation only in P30. Genes in P30 also had different regulatory functions than in P15: P30 animals showed marked increases in positive regulators of transcription, of signaling pathways as well as of MAPKKK cascades. Many of the same inflammation-related genes as in epileptic rats were significantly upregulated in human hippocampus, higher than in lateral temporal neocortex. They included glia-associated genes, cytokines, chemokines and adhesion molecules and MAPK pathway genes. Uniquely expressed in human hippocampus were adaptive immune system genes including immune receptors CDs and MHC II HLAs. In the brain, many immune molecules have additional roles in synaptic plasticity and the promotion of neurite outgrowth. We propose that persistent changes in inflammatory gene expression after SE leads not only to structural damage but also to aberrant synaptogenesis that may lead to epileptogenesis. Furthermore, the sustained pattern of inflammatory genes upregulated in the epileptic mature brain was distinct from that of the immature brain that show transient changes and are resistant to cell death and neuropathologic changes. Our data suggest that the epileptogenic process may be a result of failed cellular signaling mechanisms, where insults overwhelm the system beyond a homeostatic threshold.
... Ischaemic stroke models and peripheral IL-1 administration have been shown to increase levels of CXCL10/interferon-inducible protein-10 in unstimulated splenocytes [26], activating its receptor CXCR3. CXCL10 has been shown to be important in central leukocyte recruitment in stroke models [34], particularly in ischaemic cortex [35], with elevated levels also seen in a variety of other diseases [35]. Alongside its role as a chemoattractant for monocytes and activated T lymphocytes, CXCL10 has been implicated in glial cell migration [36] -making its splenic upregulation in stroke models a point of particular interest [26] and raising the possibility of a role for CXCL10 in the coordination of leukocyte migration in response to stroke. ...
... The temporal expression profile of the chemoattractant CXCL1 following MCAO generation demonstrates a rapid transient increase, with a peak at 4 h, followed by a decline to baseline [33]. Mechanistically, this rapid rise may result in neutrophil mobilisation from the bone marrow, whilst the decline allows for generation of a central-peripheral chemokine gradient resulting in central neutrophil migration, which is supported by multiple studies demonstrating a delay in peak brain neutrophil levels relative to plasma expression of CXCL1 [34]. ...
Fenofibrate, a PPAR-α activator, has shown promising results as a neuroprotective therapy, with proposed anti-inflammatory and anti-oxidant effects. However, it displays poor blood-brain barrier permeability leading to some ambiguity over its mechanism of action. Experimentally induced brain injury has been shown to elicit a hepatic acute phase response that modulates leukocyte recruitment to the injured brain. Here, we sought to discover whether one effect of fenofibrate might include the suppression of the acute phase response (APR) following brain injury.
A 1-h intraluminal thread middle cerebral artery occlusion (MCAO) model followed by a 6-h reperfusion was performed in C57/BL6 mice. Quantitative reverse transcriptase-polymerase chain reaction was then used to measure hepatic expression of chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine ligand 10 (CXCL10) and serum amyloid A-1 (SAA-1), and immunohistochemical analysis was used to quantify brain and hepatic neutrophil infiltration following stroke.
The MCAO and sham surgery induced the expression of all three acute phase reactants. A 14-day fenofibrate pre-treatment decreased reactant production, infarct volume, and neutrophil recruitment to the brain and liver, which is a hallmark of the APR.
The data highlight a novel mechanism of action for fenofibrate and lend further evidence towards the promotion of its use as a prophylactic therapy in patients at risk of cerebral ischaemia. Further research is required to elucidate the mechanistic explanation underlying its actions.
... This result is in agreement with previous reports on CXCL10 expression in the rodent CNS. Significant elevation of this chemokine has been observed in diverse neuropathologies, including inflammatory diseases such as EAE, contusion injury, cerebral ischemia, and neurotoxicant-induced neurodegeneration [35][36][37][38][39]. ...
For a long time, astrocytes were considered a passive brain cell population. However, recently, many studies have shown that their role in the central nervous system (CNS) is more active. Previously, it was stated that there are two main functional phenotypes of astrocytes. However, nowadays, it is clear that there is rather a broad spectrum of these phenotypes. The major goal of this study was to evaluate the production of some inflammatory chemokines and neurotrophic factors by primary human astrocytes after pro- or anti-inflammatory stimulation. We observed that only astrocytes induced by inflammatory mediators TNFα/IL-1a/C1q produced CXCL10, CCL1, and CXCL13 chemokines. Unstimulated astrocytes and those cultured with anti-inflammatory cytokines (IL-4, IL-10, or TGF-β1) did not produce these chemokines. Interestingly, astrocytes cultured in proinflammatory conditions significantly decreased the release of neurotrophic factor PDGF-A, as compared to unstimulated astrocytes. However, in response to anti-inflammatory cytokine TGF-β1, astrocytes significantly increased PDGF-A production compared to the medium alone. The production of another studied neurotrophic factor BDNF was not influenced by pro- or anti-inflammatory stimulation. The secretory response was accompanied by changes in HLA-DR, CD83, and GFAP expression. Our study confirms that astrocytes differentially respond to pro- and anti-inflammatory stimuli, especially to inflammatory cytokines TNF-α, IL-1a, and C1q, suggesting their role in leukocyte recruitment.
... The brain parenchyma concomitantly experiences its own inflammatory process with release of damage associated molecular patterns (DAMPs) from injured cells and activation of various immune cells such as astroglia and microglia that produce pro-inflammatory cytokines that have been associated with stroke severity, including IL-1b, IL-6 and TNFa (58,59). More so, this intra-parenchymal immune response has been shown to increase the expression of chemoattractants for monocytes and T cells (i.e., CXCL10, MCP-1) as well as Tregs (i.e., CCL20) among others (60)(61)(62)(63). Previous studies have found that the SCFAs Acetate, Propionate and Butyrate are able to decrease the secretion of TNFa and MCP-1 from cultured monocytes as well as TNFa-induced VCAM1 expression in endothelial cells (15,17). ...
Introduction
Short chain fatty acids (SCFA) are gut microbiota-derived metabolites that contribute to the gut-brain axis and may impact stroke outcomes following gut dysbiosis. We evaluated plasma SCFA concentrations against stroke severity parameters and identified SCFA-associated protein networks.
Methods
The Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC), a continuously enrolling tissue bank, was used to obtain stroke samples. Arterial blood distal and proximal to the thrombus was obtained from Acute Ischemic Stroke (AIS) Patients (n=53) during thrombectomy. Patient demographics, stroke presentation and outcome parameters were reported. The SCFAs were isolated from proximal plasma via chemical derivatization UHPLC coupled tandem mass spectrometry using electrospray ionization and multiple reaction monitoring. Proteomic levels for 184 cardioembolic and inflammatory proteins was quantified from systemic and intracranial plasma by Olink. Arterial blood from cerebrovascular patients undergoing elective neurointerventional procedures was used as controls.
Results
Acetate positively correlated with time from last known normal (LKN) and was significantly lower in stroke patients compared to control. Isobutyrate, Butyrate and 2-Methylbutyrate negatively correlated with %ΔNIHSS. Isobutyrate and 2-Methylbutyrate positively correlated with NIHSS discharge. SCFA concentrations were not associated with NIHSS admission, infarct volume, or edema volume. Multiple SCFAs positively associated with systemic and pro-inflammatory cytokines, most notably IL-6, TNF-α, VCAM1, IL-17, and MCP-1.
Conclusions
Plasma SCFA concentrations taken at time of stroke are not associated with stroke severity at presentation. However, higher levels of SCFAs at the time of stroke are associated with increased markers of inflammation, less recovery from admission to discharge, and worse symptom burden at discharge.
... It has been reported that IP-10/CXCR3 had an important part in the pathological process of stroke patients [25,26] and experimental cerebral ischemia [27][28][29][30][31]. The mRNA and protein expression of IP-10/CXCR3 was increased in a timedependent manner after permanent occlusion of the middle cerebral artery, suggesting IP-10/CXCR3 may be a potential novel therapeutic target in focal stroke [32,33]. That's exactly what have happened. ...
Background:
Infection is an important clinical complication facing stroke-patients and triples the risk of death within 30 days post-stroke via mechanisms which are poorly understood.
Aims:
We tried to explore the mechanisms that inflammation caused by infections aggravated the ischemic brain injury after middle cerebral artery occlusion (MCAO).
Methods:
We used lipopolysaccharide (LPS) as systemic inflammatory stimuli to explore the mechanisms of aggravated ischemic brain injury after Sprague-Dawley male rats subjected to MCAO. Brain damage was evaluated by cerebral blood perfusion, Longa-5 scores, infarct volume and edema degree. Systemic cytokine responses and inflammatory changes in the plasma and brain were analyzed by ELISA kit, RT2 Profiler™ PCR array, and quantitative real-time PCR. The differential genes were subjected to Gene Ontology enrichment analysis and protein-protein interaction (PPI) network construction.
Results:
Lipopolysaccharide profoundly aggravated the brain damage after 24 h post-MCAO. At the acute stage (ischemia/reperfusion 90 min/3 h), the brain homogenate gene expression of interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and Interferon gamma-induced protein 10 (IP-10) was significantly up-regulated and the contents in plasma and brain homogenate were significantly increased in MCAO and MCAO + LPS group. IP-10 was the only gene with significant difference between MCAO and MCAO + LPS group, which was also in an important position with degrees of ≥ 14 in PPI network.
Conclusions:
It was possible that trace LPS aggravated the ischemic brain injury by induction of excessive IP-10 secretion in the acute stage, leading to excessive inflammatory response, which consequently increased the infarct volume and edema degree 24 h post-MCAO.
... Accordingly, convincing evidence has shown the correlation of pro-and anti-inflammatory mediators, namely TNF-α, IL-1α, IL-1β, IL-6, and IL-10 plasma levels to clinical outcomes(Tuttolomondo et al. 2009;Basic Kes et al. 2008;Wytrykowska et al. 2016;Kwan et al. 2013;Ormstad et al. 2011). Besides, excretion of chemokines such as MCP-1, IP-10, and IL-8 plays a prominent role in inflammation progression due to leukocyte recruitment and modulation of blood-brain barrier permeability(Chen et al. 2003;Stamatovic et al. 2005;Seifert et al. 2014;Amin et al. 2017;Wang et al. 1998;Shaheen et al. 2018;Domac and Misirli 2008). ...
Ischaemic stroke represents one of the main causes of disability. According to the broad investigations, it is widely assumed that the contribution of inflammatory mediators is strongly involved in its pathogenesis. Hence, it seems that stroke treatment needs more efficient and inflammatory-targeted compounds to modulate inflammatory-related pathways. Such strategies paved the way to achieve better clinical outcomes along with conventional therapies. Boswellic acids (BAs), the main bioactive compounds of Boswellia sp. resin; are triterpenoids with well-documented anti-inflammatory properties. Compared with NSAIDs, BAs cross blood–brain barrier yet they do not cause serious gastrointestinal adverse effects. Considering BAs anti-inflammatory features, we conducted a randomized double-blind placebo-controlled pilot trial of these compounds as a supplementary therapy. This trial randomized 80 ischaemic stroke patients (40–80-years old) with a 4–20 score according to the National Institutes of Health Stroke Scale (NIHSS), within 72 h of neurological sign onset, in 1-month follow-up period. We assessed NIHSS as primary and plasma levels of TNF-α, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IFN-γ, IP-10, MCP-1, 8-isoprostane, and PGE2 as secondary outcomes. According to NIHSS evaluation, patients who were allocated to BA group had a significant recovery in neurological function during the 1-month follow-up, compared with the placebo. The levels of plasma inflammatory markers were significantly decreased in BA group after 7 days of intervention in TNF-α, IL-1β, IL-6, IL-8, and PGE2. As a preliminary controlled trial in ischaemic stroke, BAs could improve clinical outcome in the early phases of stroke along with promising changes in plasma inflammatory factors.
Clinical trial registrationhttps://www.irct.ir Unique identifier: IRCT20170315033086N5. IRCT is a primary registry in the WHO registry network (https://www.who.int/ictrp/network/primary/en/)
... CXCL10 is a chemokine highly inducible by IFN-g, and is a chemoattractant for NK cells in the brain [25]. Abundant evidence has indicated the increase of CXCL10 in rodent and human brains after ischemia [7,26,27]. Depletion of CXCL10 has been shown to abolish the brain infiltration of NK cells after ischemia, suggesting that CXCL10 attracts NK cells infiltrating into the ischemic brain [7]. In agreement with these findings, our current study showed that brain ischemia upregulated both the mRNA and protein levels of CXCL10 in rat brains, and these upregulations were robustly reduced by BHD. ...
Background/aims:
Natural killer (NK) cells are among the first immune cells that respond to an ischemic insult in human brains. The infiltrated NK cells damage blood-brain barrier (BBB) and exacerbate brain infarction. Buyang Huanwu Decoction (BHD), a classic Chinese traditional herbal prescription, has long been used for the treatment of ischemic stroke. The present study investigated whether BHD can prevent brain infiltration of NK cells, attenuate BBB disruption and improve ischemic outcomes.
Methods:
Transient focal cerebral ischemia was induced in rats by a 60-minute middle cerebral artery occlusion, and BHD was orally administrated at the onset of reperfusion, 12 hours later, then twice daily. Assessed parameters on Day 3 after ischemia were: neurological and motor functional deficits through neurological deficit score and rotarod test, respectively; brain infarction through TTC staining; BBB integrity through Evans blue extravasation; matrix metalloproteinase-2/9 activities through gelatin zymography; tight junction protein, nuclear factor-kB (NF-kB) p65 and phospho-p65 levels through Western blotting; NK cell brain infiltration and CXCR3 levels on NK cells through flow cytometry; interferon-γ production through ELISA; CXCL10 mRNA levels through real-time PCR; CXCL10 expression and p65 nuclear translocation through immunofluorescence staining.
Results:
BHD markedly reduced brain infarction, improved rotarod performance, and attenuated BBB breakdown. Concurrently, BHD attenuated the upregulation of matrix metalloproteinase-2/9 activities and the degradation of tight junction proteins in the ischemic brain. Infiltration of NK cells was observed in the ischemic hemisphere, and this infiltration was blunted by treatment with BHD. BHD suppressed brain ischemia-induced interferon-γ and chemokine CXCL10 production. Furthermore, BHD significantly reduced the expression of CXCR3 on brain-infiltrated NK cells. Strikingly, BHD did not affect NK cell levels or its CXCR3 expression in the spleen or peripheral blood after brain ischemia. The nuclear translocation of NF-kB p65 and phospho-p65 in the ischemic brain was inhibited by BHD.
Conclusion:
Our findings suggest that BHD prevents brain infiltration of NK cells, preserves BBB integrity and eventually improves ischemic outcomes. The inhibitory effects of BHD on NK cell brain invasion may involve its ability of suppressing NF-kB-associated CXCL10-CXCR3-mediated chemotaxis. Notably, BHD only suppresses NK cells and their CXCR3 expression in the ischemic brain, but not those in periphery.
... Furthermore, the inflammatory process is promoted by various pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β, produced by several subtypes of T cells (29). Chemokines, including CXCL-10, are secreted by the activated cells in ischemic regions to attract the inflammatory leukocytes into the region of infarction (30). Elevation of these inflammatory molecules clearly demonstrates detrimental effects on viable brain tissue (31). ...
The purpose of the present study was to explore the combination effect of FTY720 and vitamin E on cerebral ischemia. Astrocytes were isolated from newborn Sprague‑Dawley rats and were subjected to FTY720, vitamin E, or combination of the two. The astrocyte cultures were then exposed to oxygen‑glucose deprivation (OGD) to simulate an ischemic model in vitro. Cell viability, lactate dehydrogenase (LDH) leakage and cell apoptosis were detected following 12 h of exposure to OGD. In addition, the levels of tumor necrosis factor (TNF)‑α, interleukin (IL)‑6, IL‑1β, total antioxidant capacity, intercellular adhesion molecule (ICAM)‑1, vascular cell adhesion molecule (VCAM)‑1, chemokine (C‑X‑C motif) ligand (CXCL)‑10, heme oxygenase (HO)‑1 and superoxide dismutase (SOD)‑1 were measured. Pre‑treatment with FTY720 or vitamin E significantly elevated the cell viability and decreased LDH release and number of apoptotic cells. Combination treatment with FTY720 and vitamin E demonstrated a synergistic protective effect on OGD‑induced cell viability, toxicity and apoptosis. Pre‑treatment with FTY720 markedly reduced the release of IL‑1β, TNF‑α, IL‑6, ICAM‑1, VCAM‑1 and CXCL‑10, and pre‑treatment with vitamin E increased the levels of antioxidant, HO‑1 and SOD‑1. However, pre‑treatment with FTY720 combined with vitamin E revealed a synergistic effect. Pre‑treatment with FTY720 combined with vitamin E exerts synergistic neuroprotective effects in the simulated cerebral ischemia in vitro.
... Neuron-derived chemokines binds to chemokine receptor CXCR3 functionally expressed on microglia and astrocytes, and may therefore attract glial cells to the site of damage (Biber et al., 2002;Biber et al., 2001;Wang et al., 1998). ...
... [11][12][13] CXCR3 is critical to mediate the recruitment of activated T cells and microglia/macrophage. 10,14-17 CXCL10 expression is increased in the central nervous system in neurodegenerative diseases including Alzheimer's disease and multiple sclerosis. [18][19][20][21] The activation of CXCL10/CXCR3 axis has also been shown to promote microglia recruitment and induce neuronal cell death in several models of neurodegeneration. 22,23 The role of CXCL10/CXCR3 pathway in glaucomatous optic neuropathy is unknown. ...
Acute glaucoma is a leading cause of irreversible blindness in East Asia. The mechanisms underlying retinal neuronal injury induced by a sudden rise in intraocular pressure (IOP) remain obscure. Here we demonstrate that the activation of CXCL10/CXCR3 axis, which mediates the recruitment and activation of inflammatory cells, has a critical role in a mouse model of acute glaucoma. The mRNA and protein expression levels of CXCL10 and CXCR3 were significantly increased after IOP-induced retinal ischemia. Blockade of the CXCR3 pathway by deleting CXCR3 gene significantly attenuated ischemic injury-induced upregulation of inflammatory molecules (interleukin-1β and E-selectin), inhibited the recruitment of microglia/monocyte to the superficial retina, reduced peroxynitrite formation, and prevented the loss of neurons within the ganglion cell layer. In contrast, intravitreal delivery of CXCL10 increased leukocyte recruitment and retinal cell apoptosis. Inhibition of endoplasmic reticulum (ER) stress with chemical chaperones partially blocked ischemic injury-induced CXCL10 upregulation, whereas induction of ER stress with tunicamycin enhanced CXCL10 expression in retina and primary retinal ganglion cells. Interestingly, deleting CXCR3 attenuated ER stress-induced retinal cell death. In conclusion, these results indicate that ER stress-medicated activation of CXCL10/CXCR3 pathway has an important role in retinal inflammation and neuronal injury after high IOP-induced ischemia.
... In agreement with previous findings of increased cytokine and chemokine gene expression after stroke, 37,39,[90][91][92][93][94] we show significant upregulation of TNF, TNFR1, IL-1b, IL-6, CXCL1, CXCL10 and CCL2 mRNA levels in both mTNF Á/Á and mTNF wt/wt mice one day after pMCAO as compared to sham and naive mice. We did not find differences in gene expression between mTNF Á/Á and mTNF wt/wt mice in any of the genes investigated. ...
Microglia respond to focal cerebral ischemia by increasing their production of the neuromodulatory cytokine tumor necrosis factor (TNF), which exists both as membrane-anchored TNF (mTNF) and as cleaved soluble TNF (solTNF) forms. We previously demonstrated that TNF knockout mice display increased lesion volume after focal cerebral ischemia, suggesting that TNF is neuroprotective in experimental stroke. Here, we extend our studies to show that mice with intact mTNF, but no solTNF, display reduced infarct volumes at 1 and 5 days after stroke. This was associated with improved functional outcome after experimental stroke. No changes were found in the mRNA levels of TNF and TNF-related genes (TNFR1, TNFR2, TACE), pro-inflammatory cytokines (IL-1β, IL-6) or chemokines (CXCL1, CXCL10, CCL2), however, protein expression of TNF, IL-1β, IL-6 and CXCL1 was reduced in mTNF∆/∆ compared to mTNFwt/wt mice 1 day after experimental stroke. This was paralleled by reduced MHCII expression and a reduction in macrophage infiltration in the ipsilateral cortex of mTNF∆/∆ mice. Collectively, these findings indicate that mTNF mediates the protective effects of TNF signaling in experimental stroke, and therapeutic strategies specifically targeting solTNF could be beneficial in clinical stroke therapy.
... Interestingly, the herein observed increase in YKL-40 was not significantly correlated with levels of IL-8, IP-10 or MCP-1 suggesting that alternative mechanisms other than those orchestrating levels of MCP-1 and IL-8 may be controlling the levels of YKL-40 in the brain. Also, as IL-8, MCP-1 and IP-10 can be secreted by various cell types including endothelial cells [32,33], astrocytes [34], pericytes [35,36], microglia [37], oligodendrocytes [38,39] and to some extent by neurons [40,41] the overall quantified levels of those markers may as a pool from various sources not appropriately reflect the activation of specific cell populations. Of note, interleukin-1b (IL-1b) and tumor necrosis factor-α (TNF-α) were recently shown to be the main inducers of YKL-40 secretion in primary human astrocytes [23] and whilst many proinflammatory markers including MCP-1 [42] and IL-8 [43] are positively regulated by NF-κB signaling, IL-1b is decreased upon NF-κB activation [44]. ...
A major difference in the revised diagnostic criteria for Alzheimer's disease (AD) is the incorporation of biomarkers to support a clinical diagnosis and allow the identification of preclinical AD due to AD neuropathological processes. However, AD-specific fluid biomarkers which specifically distinguish clinical AD dementia from other dementia disorders are still missing. Here we aimed to evaluate the disease-specificity of increased YKL-40 levels in cerebrospinal fluid (CSF) from AD patients with mild to moderate dementia (n = 49) versus Parkinson's disease (PD) (n = 61) and dementia with Lewy bodies (DLB) patients (n = 36), and non-demented controls (n = 44). Second we aimed to investigate whether altered YKL-40 levels are associated with CSF levels of other inflammation-associated molecules. When correcting for age, AD patients exhibited 21.3%, 27.7% and 38.8% higher YKL-40 levels compared to non-demented controls (p = 0.0283), DLB (p = 0.0027) and PD patients (p<0.0001). The AD-associated increase in YKL-40 was not associated with CSF P-tau, T-tau or Aβ42. No relationship between increased YKL-40 and levels of the astrocytic marker glial-fibrillary acidic protein (GFAP), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and interferon gamma-induced protein 10 (IP-10) could be identified. Our results confirm previous reports of an age-associated increased in CSF YKL-40 levels and further demonstrate increased CSF YKL-40 in AD patients versus non-demented controls and patients with DLB or PD. The increase in YKL-40 levels in the AD patients was unrelated to the established CSF AD biomarkers and the inflammatory markers GFAP, MCP-1, IP-10 and IL-8, proposing YKL-40 as a marker of yet to be identified AD-related pathological processes.
... Gleichzeitig werden Liganden des CXCR3 im Gehirnparenchym freigesetzt (Wang et al., 2000). Astrozyten und Neurone können den Liganden IP-10 nach Ischämie sekretieren (Wang et al., 1998). In der Peripherie wird CXCR3 auf Lymphozyten exprimiert und ist wichtig für das Homing dieser Zellen aus den LK (Cervantes- Barragán et al., 2012). ...
Nach einer Läsion im Gehirn kommt es trotz der Bildung autoreaktiver T-Zellen zu keiner autoimmunen Neuropathologie. Foxp3+ regulatorische T-Zellen (Tregs) vermitteln möglicherweise Immuntoleranz nach zerebraler Läsion. Deswegen wurde in dieser Studie die Rolle der Tregs 7, 14 und 30 Tage nach einem transienten Verschluss der mittleren Hirnarterie (MCAO), einem Modell für ischämischen Schlaganfall, und nach entorhinaler Kortexläsion (ECL) in der Maus untersucht. Durchflusszytometrisch wurde in beiden Modellen 14 und 30 Tage nach Läsion eine Akkumulation der Tregs in der ipsilateralen Hemisphäre beobachtet. Mikroskopisch wurden an der Läsion Zellkontakte der Tregs mit antigenpräsentierenden Zellen beobachtet. Weitere Experimente wurden ausschließlich nach MCAO durchgeführt. Am Tag 14 und 30 war in der ipsilateralen Hemisphäre eine Akkumulation der Mikroglia zu beobachten. Makrophagen und dendritische Zellen wurden an den Tagen 7, 14 und 30 detektiert. Am Tag 7 und 14 waren ipsilateral im Gehirn ca. 60 % der Tregs positiv für den Proliferationsmarker Ki-67. In zwei Versuchsansätzen wurden naive CD45RBhigh/CD4+ Zellen aus lymphatischen Organen von Foxp3EGFP Mäusen, mit Wildtyp T-Zellrezeptor (TCR), oder 2D2.Foxp3EGFP Mäusen, mit TCR spezifisch gegen Myelin-Oligodendrozyten-Glykoprotein, isoliert. Die Zellen wurden einen Tag vor MCAO in RAG1-/- Mäuse, welche keine adulten T- und B-Zellen besitzen, transferiert. Am Tag 14 nach MCAO war in den RAG1-/- Mäusen keine de novo Induktion Foxp3EGFP+ Tregs zu beobachten. CD25+ Tregs wurden durch die Injektion eines Antikörpers gegen CD25 depletiert, um deren Wirkung nach MCAO zu untersuchen. Nach Depletion konnte bis zu 27 Tage nach MCAO keine Veränderung des Läsionsvolumen und des Gangverhaltens beobachtet werden. In dieser Studie wurde im Gehirn eine späte Präsenz und Proliferation Foxp3+ Tregs nach Läsion nachgewiesen. Mikroglia und periphere Immunzellen sind langfristig an Immunvorgängen im lädierten Gehirn beteiligt.
... This included molecules involved in antigen presentation such as proteasomal components low molecular weight protein (LMP) 2 and LMP7, and classical and non-classical MHC class I molecules H2D b and Qa1 b . Also iNOS and IFN-γ inducible protein (IP-10), that are usually produced by macrophages/microglia in response to IFN-γ, but under some circumstances have been reported in neurons (Wang et al., 1998;Heneka & Feinstein, 2001), were elevated in Socs1 -/cultures. The enhanced responses to IFN-γ indicates that SOCS1 limits cytokine signaling in neurons in a manner similar to other cell types (Wormald et al., 2006). ...
... In experimental models of cerebral ischemia, like middle cerebral artery and common carotid artery occlusion in rats (MCAO), increased CNS expression of numerous chemokines has been reported, some of which are likely to be of glial origin. CCL2/MCP-1 and CXCL10/IP10 have been detected mainly in astrocytes (116,123,225), whereas CCL3/MIP-1␣ has been associated with reactive microglia (143,226). Increased expression of CCR1 in astrocytes and neurons and of CX3CR1 in microglia in ischemic tissue was also reported (143,161). ...
Chemokines are small secreted proteins that are essential for the recruitment and activation of specific leukocyte subsets at sites of inflammation and for the development and homeostasis of lymphoid and nonlymphoid tissues. During the past decade, chemokines and their receptors have also emerged as key signaling molecules in neuroinflammatory processes and in the development and functioning of the central nervous system. Neurons and glial cells, including astrocytes, oligodendrocytes, and microglia, have been identified as cellular sources and/or targets of chemokines produced in the central nervous system in physiological and pathological conditions. In this article, we provide an update of chemokines and chemokine receptors expressed by glial cells focusing on their biological functions and implications in neurological diseases.
... Similarly, IP-10 was detected in ischemic cerebral tissue of the mouse model examined ( Figure 3D,G), with the highest levels detected 12 hours after ischemia using RT-PCR. These results are consistent with the rat model of ischemia characterized by Wang et al. [31] (Figure 3H), and the decrease of NK cells infiltration in ischemic brain after IP-10 depletion in vivo prompt us to compare relationship between IP-10 and NK cells when stroke occurs, suggesting that IP-10 expression induces NK cell infiltration following cerebral ischemia ( Figure 3I,J). ...
Stroke is accompanied by a distinguished inflammatory reaction that is initiated by the infiltration of immunocytes, expression of cytokines, and other inflammatory mediators. As natural killer cells (NK cells) are a type of cytotoxic lymphocyte critical to the innate immune system, we investigated the mechanism of NK cells-induced brain injuries after cerebral ischemia and the chemotactic effect of IP-10 simultaneously.
NK cells infiltration, interferon-gamma (IFN-gamma) and IP-10 expression were detected by immunohistochemistry, immunofluorescence, PCR and flow cytometry in human and C57/BL6 wild type mouse ischemic brain tissues. The ischemia area was detected via 2,3,5-triphenyltetrazolium chloride staining. CXCR3 mean fluorescence intensity of isolated NK cells was measured by flow cytometry. The neuronal injury made by NK cells was examined via apoptosis experiment. The chemotactic of IP-10 was detected by migration and permeability assays.
In human ischemic brain tissue, infiltrations of NK cells were observed and reached a peak at 2 to 5 days. In a permanent middle cerebral artery occlusion(pMCAO) model, infiltration of NK cells into the ischemic infarct region reached their highest levels 12 hours after ischemia. IFN-gamma-positive NK cells and levels of the chemokine IP-10 were also detected within the ischemic region, from 6 hours up to 4 days after pMCAO was performed, and IFN-gamma levels decreased after NK cells depletion in vivo. Co-culture experiments of neural cells with NK cells also showed that neural necrosis was induced via IFN-gamma. In parallel experiments with IP-10, the presence of CXCR3 indicates that NK cells were affected by IP-10 via CXCR3, and the effect was dose-dependent. After IP-10 depletion in vivo, NK cells decreased. In migration assays and permeability experiments, disintegration of the blood-brain barrier (BBB) was observed following the addition of NK cells. Moreover, in the presence of IP-10 this injury was aggravated.
All findings support the hypothesis that NK cells participate in cerebral ischemia and promote neural cells necrosis via IFN-gamma. Moreover, IP-10 intensifies injury to the BBB by NK cells via CXCR3.
... Increased expression in infarcted brain [ 49 , 67 ], which is maintained along time [ 68 ]. ...
Chemokines act mainly in guiding leukocyte migration along the endothelium. Apart from angiogenesis or neuronal survival, chemokines are involved in damage and repair of brain tissue after ischemic stroke. We studied the presence of chemokines directly in neurons and brain blood vessels that were obtained by means of laser microdissection from human ischemic brains. Using multiple ELISA Searchlight® array we evaluated nine chemokines (CCL1−5, CCL11, CCL17, CCL22, and CXCL8) in microdissected samples. We found higher levels of CCL1 and CCL2 in neurons than in vessels; CCL5 and CCL22 were decreased in the infarcted areas.
The same ELISA array was performed in plasma samples from stroke patients. We explored the temporal profile of circulating chemokines from admission to 90 days after the cerebrovascular event, and found that only CCL22 showed significant changes along time and that these changes negatively correlated with neurological severity. When neurological outcome was assessed in the hyperacute phase of stroke no associations were found.
From our study, we can conclude that these chemokines do not perform a clear role of outcome biomarkers. Further studies are necessary to assess which mechanisms underlie the association of chemokines with the neurological state at distinct time points since the differences found here could be reflecting the dual role of chemokines in neuroinflammation.
... IP-10 and MCP-1 can also be released by human fetal and simian adult astrocytes in response to IFNy [74]. Astrocytes and microglia have increased expression of IP-10 in several infectious and neurotoxic contexts including AD, ischemia and LPSchallenge [44,[75][76][77]. There is also evidence to suggest that not only glial cells but neuronal cells too can release chemokines to attract T cells into the brain parenchyma [78]. ...
The chemokine Interferon gamma-induced protein 10 (IP-10) and human leukocyte antigen (HLA) are widely used indicators of glial activation and neuroinflammation and are up-regulated in many brain disorders. These inflammatory mediators have been widely studied in rodent models of brain disorders, but less work has been undertaken using human brain cells. In this study we investigate the regulation of HLA and IP-10, as well as other cytokines and chemokines, in microglia, astrocytes, pericytes, and meningeal fibroblasts derived from biopsy and autopsy adult human brain, using immunocytochemistry and a Cytometric Bead Array. Interferonγ (IFNγ) increased microglial HLA expression, but contrary to data in rodents, the anti-inflammatory cytokine transforming growth factor β1 (TGFβ1) did not inhibit this increase in HLA, nor did TGFβ1 affect basal microglial HLA expression or IFNγ-induced astrocytic HLA expression. In contrast, IFNγ-induced and basal microglial HLA expression, but not IFNγ-induced astrocytic HLA expression, were strongly inhibited by macrophage colony stimulating factor (M-CSF). IFNγ also strongly induced HLA expression in pericytes and meningeal fibroblasts, which do not basally express HLA, and this induction was completely blocked by TGFβ1, but not affected by M-CSF. In contrast, TGFβ1 did not block the IFNγ-induced increase in IP-10 in pericytes and meningeal fibroblasts. These results show that IFNγ, TGFβ1 and M-CSF have species- and cell type-specific effects on human brain cells that may have implications for their roles in adult human brain inflammation.
... 2011, 55, 1-12 dysregulated gut homeostasis. Experimental evidence has demonstrated up-regulation of the chemokine IP-10 in ileitis [14], brain ischemia [48] and adipocyte maturation [49]. In the present study, we have shown that CAPE abolishes expression of both TNF-and LPS-induced IP-10 expression in embryonic fibroblasts from TNF DARE/1 mice. ...
Caffeic acid phenethyl ester (CAPE) is an active constituent of honeybee propolis inhibiting nuclear factor (NF)-κB. The aims of our study were to provide new data on the functional relevance and mechanisms underlying the role of CAPE in regulating inflammatory processes at the epithelial interface in the gut and to determine the structure/activity relationship of CAPE.
CAPE significantly inhibited TNF-induced IP-10 expression in intestinal epithelial cells. Using various analogues, we demonstrated that substitution of catechol hydroxyl groups and addition of one extra hydroxyl group on ring B reversed the functional activity of CAPE to inhibit IP-10 production. The anti-inflammatory potential of CAPE was confirmed in ileal tissue explants and embryonic fibroblasts derived from TNF(ΔARE/+) mice. Interestingly, CAPE inhibited both TNF- and LPS-induced IP-10 production in a dose-dependent manner, independently of p38 MAPK, HO-1 and Nrf2 signaling pathways. We found that CAPE did not inhibit TNF-induced IκB phosphorylation/degradation or nuclear translocation of RelA/p65, but targeted downstream signaling events at the level of transcription factor recruitment to the gene promoter.
This study reveals the structure-activity effects and anti-inflammatory potential of CAPE in the intestinal epithelium.
... Chemokines and their G protein-coupled receptors are key regulators of leukocyte trafficking to sites of injury. Ischemia activates chemokine signals in brain tissue, including CCL2 (Kim et al. 1995), CCL5 (Offner et al. 2006a), CXCL2 (Yamasaki et al. 1995), CXCL8 (Montecucco et al. 2010) and CXCL10 (Wang et al. 1998) that promote expression of adhesion molecules by vascular endothelial cells. MOG-reactive splenocytes expressed striking differences in chemokine receptor levels that are essential for the infiltration, recruitment and localization of migrating leukocytes to the perivascular compartment (Horuk 2001; Prinz and Priller 2010). ...
Although inflammatory responses increase stroke severity, the role of immune cells specific for central nervous system (CNS) antigens remains controversial. Disruption of the blood-brain barrier (BBB) during stroke allows CNS antigens to leak into the peripheral circulation and enhances access of circulating leukocytes to the brain, including those specific for CNS antigens such as myelin oligodendrocyte glycoprotein (MOG) that can induce experimental autoimmune encephalomyelitis (EAE). We here demonstrate for the first time that myelin reactive splenocytes specific for MOG transferred into severe combined immunodeficient (SCID) mice can migrate into the infarct hemisphere of recipients subjected to 60 min middle cerebral artery occlusion (MCAO) and 96 h reperfusion; moreover these cells exacerbate infarct volume and worsen neurological deficits compared to animals transferred with naïve splenocytes. These findings indicate that autoimmunity in the CNS can exert detrimental injury on brain cells and worsen the damage from ischemic stroke.
This chapter discusses an important aspect of neuroinflammatory process, namely the infiltration of both resident and various types of hematogenous immune cells that invade the ischemic area at different stages of the pathological process. Many of the pathological processes involved in ischemia have been investigated. The necrotic process is a major starting point for neuroinflammatory processes. Cytokines are involved in the pathophysiology of focal cerebral ischemia. Within hours after induction of middle cerebral artery occlusion (MCAO) prominent expression of the proinflammatory cytokine interleukin-1β (IL-1β) has been observed. A distinct group of small inflammatory cytokines, called chemokines, plays an important role in the infiltration of hematogenous immune cells and the attraction of local microglia in the ischemic area. Both local and hematogenous immune cells seem involved in the pathology of ischemia. The time-related expression of chemokines in the ischemic area and their specific chemoattractant activity are the subject of this chapter.
Traumatic optic neuropathy (TON) is an acute injury of the optic nerve secondary to trauma. Loss of retinal ganglion cells (RGCs) is a key pathological process in TON, yet mechanisms responsible for RGC death remain unclear. In a mouse model of TON, real-time noninvasive imaging revealed a dramatic increase in leukocyte rolling and adhesion in veins near the optic nerve (ON) head at 9 hours after ON injury. Although RGC dysfunction and loss were not detected at 24 hours after injury, massive leukocyte infiltration was observed in the superficial retina. These cells were identified as T cells, microglia/monocytes, and neutrophils but not B cells. CXCL10 is a chemokine that recruits leukocytes after binding to its receptor C-X-C chemokine receptor (CXCR) 3. The levels of CXCL10 and CXCR3 were markedly elevated in TON, and up-regulation of CXCL10 was mediated by STAT1/3. Deleting CXCR3 in leukocytes significantly reduced leukocyte recruitment, and prevented RGC death at 7 days after ON injury. Treatment with CXCR3 antagonist attenuated TON-induced RGC dysfunction and cell loss. In vitro co-culture of primary RGCs with leukocytes resulted in increased RGC apoptosis, which was exaggerated in the presence of CXCL10. These results indicate that leukocyte recruitment in retinal vessels near the ON head is an early event in TON and the CXCL10/CXCR3 axis has a critical role in recruiting leukocytes and inducing RGC death.
For a drug to be effectivein vivo
in-vitroprotection must be translated to studies of both global (transient forebrain ischemia) to discern “cellular” neuroprotection (cytoprotection) and focal ischemia to evaluate “parenchymal” neuroprotection (infarct reduction). Drug concentrations that suggest that the mechanism is activein vitromust translate to drug levels that achieve cytoprotection and infarct volume reductions which are not only significant but sustained (indefatigable). Initiation of experimental therapy should be delayed in order to have clinical relevance. Most importantly, the “effective dose” plasma level reached in rodents to achieve a neuroprotective effect must be matched in humans, in the absence of overt toxicity.
To date there are a number of shortcomings with “cytoprotective drugs” - in rats, in particular, their inability to protect white matter and striatum. The drugs have to be given early and the cytoprotective dose in rats has yet to be achieved in humans.
This review covers mechanisms of cell death following both focal and global ischemia such as excitotoxicity, acid and Ca’ overload, free radical-mediated injury, inflammation and apoptosis.
Drugs which block NMDA and AMPA receptors, as well as downstream events, have now made it to the clinic and they will be critically reviewed. Hypothermia following both global and focal ischemia is protective and at least for global ischemia has now been translated two successful clinical trials in patients resuscitated from cardiac arrest. For the first time neuroprotection has been demonstrated in patients who achieved better neurological outcomes. The review will finish with some rules for the translation of experimental work to clinical trials.
The superfamily of chemokines (chemoattractant cytokines) consists of a broad array of polypeptides of diverse biological actions and structures (Fig. l).The current number of chemokines exceeds 50 related proteins. These proteins range in size from 68–120 amino acids (in the mature form) and can be segregated into at least four structural branches: C, C-C, C-X-C, and CXXXC according to variations in a shared cysteine motif. The largest branch, i.e., the C-C or β-chemokines, has nearly twenty members in humans while the smallest branch, the C class, has only one (Oppenheim et al. 1991; Rollins 1997; Mantovani 1999; Kelner et al. 1994; Pan et al. 1997). The C-X-C, or the β-chemokine branch can be further subdivided by structure and function into proteins containing the amino acid motif E-L-R-C-X-C (the majority) and those few that do not have the E-L-R motif adjunct to C-X-C. Structural distinctions of the different branches of the superfamily of chemokines have been shown to parallel general (though not absolute) distinctions in their biological activities (Fig. 2). For example, most C-X-C chemokines (those with E-L-R) are chemoattractants for neutrophils but not for monocytes, whereas C-C chemokines generally attract monocytes and lymphocytes, but not neutrophils. Basophils and eosinophils are also affected by C-C chemokines. The C chemokine appears thus far to be lymphocyte-specific.
Stroke is a highly complex process, involving cerebrovascular and parenchymal tissues through the interaction of multiple mechanisms. This chapter outlines a complex cascade of biochemical and associated electrophysiological mechanisms involved in ischaemic brain injury. Several experimental approaches to the study of neuronal ischaemic injury have produced evidence suggesting that neuronal death may be mediated by the effects of excitatory neurotransmitters. In addition to these processes, there are a number of microcirculatory processes that precede or occur during these processes, which may exacerbate injury. Also fundamental to the microvascular response is the reorganization of the neurovascular unit following tissue hypoxia and inflammatory activation. Adaptive inflammatory response in the context of ischaemia reperfusion can be either interrupted or pathologically perpetuated, resulting in attenuation or an amplification of the initial pathological stimulus. In contrast to experimental studies, neuroprotective therapies assessed in clinical trials have been unequivocally negative, despite evidence in experimental studies of positive results related to the effects of therapies targeting specific mechanisms on traditional outcomes such as histological injury. The timing of specific interventions may be critical to the development of significant neuroprotective, anti-inflammatory therapies. Establishing the importance of the early steps in the inflammatory process involved within the cerebrovasculature will be critical to understanding secondary tissue injury following stroke. This edition first published 2014
This chapter discusses the role of microglial chemokines and chemokine receptors in central nervous system (CNS) inflammation. Microglia are invariably activated during CNS pathology and expresses macrophage-specific surface markers. Microglia contribution to the synthesis of chemokines in the CNS during pathological conditions has also been presented in this chapter. Microglia, therefore, are a substantial part of the chemokinergic network within the CNS. Since most of the endogenous cells of the CNS express receptors for chemokines, a broad spectrum of chemokines in CNS physiology and pathophysiology has been proposed. Chemokines are most likely involved in development and patterning of the brain. Furthermore, chemokines may contribute to CNS synaptic transmission and plasticity. Chemokine signaling in the CNS is involved in physiological as well as pathological processes. Since the scope of functions of chemokines and their receptors has broadened considerably to extend beyond leukocyte-chemoattraction, microglial chemokines, and chemokine receptors might be important factors in controlling the activity of microglia. The identification of the precise relationship of chemokines and microglial activity and the determination, whether microglial activation induced by chemokines will have protective or pathogenic consequences, will be the future challenge.
Chemokines (chemoattractant cytokines) play a vital role in the development and function of the immune system. In addition to this well characterized role, recent evidence has suggested chemokines may be produced by both neurons and glial cells in response to various stimuli, thus implicating chemokines in a number of neurological disorders, including ischemic stroke, trauma, Alzheimer’s disease, multiple sclerosis and brain tumors.
The first studies on expression of chemokines and their receptors in the central nervous system (CNS) appeared several years ago and since that time many papers were published increasing our knowledge in that field. Recent studies are concentrated mostly on involvement of chemokines and chemokine receptors in neurodegeneration and neuroprotection. There are evidences that chemokines may directly initiate neurodegeneration through activation of their receptors on the surface of neurons or indirectly through activation of microglia which in turn may secrete neurotoxic mediators damaging neuronal cells. There are also evidences suggesting that chemokines and chemokine receptors are also involved in neuroproiection. So far only two chemokines, CX3CL1 (fractalkine) and CXCL12 (SDF-1 - stromal cell-derived factor-1) have been shown to be expressed constitutively in the CNS. However, expression of many chemokine receptors including, CXCR2, CXCR4, CCR1, CCR3, CCR4, CCR5, CCR9/10, CX3CR1 i DARC has been detected on the surface of neuronal cell. Based on presented in this review studies it may be concluded that direct interaction between some chemokine receptors and chemokines or other chemokine receptor ligands may be important for development of neurodegeneration and/or neuroprotection. The detailed mechanisms of those processes are still not well known. This is confirmed by the high number of inconsistent results in current scientific literature so the further studies are necessary in that field.
Study of the molecular mechanisms involved in the cerebral infiltration of lymphocytes T in the Parkinson's disease
Support is provided for the hypothesis that activated leukocytes, especially monocytes/macrophages, contribute to cerebral white matter damage in extremely low gestational age newborns. Much of the evidence is indirect and comes from analogies to brain diseases in adults, and from models of brain damage in adult and newborn animals. If the recruitment of circulating cells to the brain contributes to white matter damage in extremely low gestational age newborns, then minimizing the transendothelial migration of circulating cells by pharmacological manipulation might prevent or reduce the occurrence of neonatal white matter damage and the disabilities that follow.
Astroglia are the most prevalent cell type in the human central nervous system (CNS) and perform important roles in normal tissue homeostasis, during pathological events and following trauma. Astroglial-derived chemokines have important neurotrophic effects and are important to CNS immunocompetence and response to injury, in part, due to their direct role in leukocyte and microglial cell recruitment. However, while ethanol is known to induce CNS pathologies and to be peripherally immunosuppressive, ethanol effects on chemokine expression in human astroglia are essentially unknown. We have demonstrated that chemotaxis of human U937 leukocytic cells, across a 0.5 μm pore polycarbonate transmembrane insert, is induced in response to culture media collected from 10 μg/ml lipopolysaccharide (LPS)+10 ng/ml interleukin (IL)-1β-stimulated A172 human astroglia cells. The involvement of the chemokine CXCL10 (also known as interferon-γ inducible protein or IP-10) in astroglial-induced chemotaxis of U937 cells has been indicated, as chemotaxis can be reduced by an anti-CXCL10 neutralizing antibody. Interestingly, chemotaxis of U937 cells, in response to astroglial-exposed media, is reduced when astroglia are chronically (9 days) exposed to 50 mM ethanol before stimulation with LPS+IL-1β. Furthermore, we observed that LPS+IL-1β-stimulated CXCL10 production is inhibited in human A172 astroglia exposed to chronic 50 mM ethanol. Thus, alterations in astroglial CXCL10 expression may disrupt CNS immunocompetence and play an important role in ethanol-induced CNS pathologies.
We investigated the neuroprotective properties of single doses of minocycline and ozagrel when administered prior to stroke. Male Sprague-Dawley rats were assigned randomly to one of the following groups: (1) control (Con) group (n=10), (2) minocycline (Mino) group (n=10), (3) sodium ozagrel (SO) group (n=10). Rats were treated with a single dose of minocycline or ozagrel at 30min before stroke. A middle cerebral artery occlusion (MCAO) was made at 30min after drug administration and reperfusion was done. The rats were subjected to a neurobehavioral test at days 1, 3 and 7 after MCAO. The cerebral ischemic volume was quantified by MetaMorph imaging software after TTC staining. The neuronal cell survival and astrocytes expansion were assessed by the NeuN and GFAP immunohistofluorescence staining. Apoptosis was detected by the TUNEL assay. We statistically analyzed and compared the results with each other. Mino and SO groups had neuroprotective effect and showed a better behavioral performance of adhesive removal and treadmill test at 7 days after stroke. Mino and SO groups also showed a smaller infarct volume than control group at 7 days after stroke. Immunohistofluorescence staining showed a higher number of NeuN positive cells, lower activated astrocytes in GFAP and a lower apoptosis in TUNEL staining. This study showed that single doses of minocycline and ozagrel prior to stroke had neuroprotective effects. These agents will be useful not only in post-stroke therapy but also in stroke prevention in several cerebrovascular procedures like carotid endarterectomy, bypass procedure, endovascular angioplasty, thromboembolectomy or thrombolysis.
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The peripheral inflammatory response, as a proxy for the acute-phase response (a known mechanism for ischemic preconditioning), and non– damage-producing transient ischemia must exist together in humans if this candidate mechanism confers ischemic tolerance. The present study was aimed at determining whether the peripheral inflammatory response (ie, elevated white blood cell, neutrophil, and monocyte counts) exists in transient ischemic attack (TIA) and stroke patients at the time of emergency room admission. The null hypothesis was tested for the variables of the peripheral inflammatory response between the mean of the laboratory normal population versus stroke and TIA patients. A retrospective review of 1041 medical records yielded 12 first-time TIA patients and 34 first-time stroke patients with no confounding evidence of other inflammatory processes. In both groups, neutrophil and monocyte percentages were significantly higher than the laboratory means (in TIA cases: neutrophils, 67.9% [12.67%], P .001; monocytes, 8.2% [2.7%], P .020; in stroke cases: neutrophils, 64.9% [9.1%], monocytes, 7.7% [1.6%]; both P .001). Absolute neutrophil count was significantly higher than the laboratory mean for the stroke cases (5.13 [1.88] K/UL; P .022). Lymphocyte percentages and absolute lymphocyte count in both groups were significantly and abnormally lower than the laboratory mean (in TIA cases, 21.7% [10.5%] and 1.4 [0.6] K/UL, respectively; in stroke cases, 24.7% [8.4%] and 1.9 [0.7] K/UL, respectively ; all P .001). No other absolute counts were significant. These findings suggest that the peripheral inflammatory response exists in transient ischemia, which hypothetically does not damage brain tissue, as well as in stroke (or permanent ischemia), which is known to produce brain tissue damage. Key Words: Inflammatory response—transient ischemic attacks—acute phase response.
Activation of the zinc-finger transcription factor early growth response (Egr)-1, initially linked to developmental processes, is shown here to function as a master switch activated by ischemia to trigger expression of pivotal regulators of inflammation, coagulation and vascular hyperpermeability. Chemokine, adhesion receptor, procoagulant and permeability-related genes are coordinately upregulated by rapid ischemia-mediated activation of Egr-1. Deletion of the gene encoding Egr-1 strikingly diminished expression of these mediators of vascular injury in a murine model of lung ischemia/reperfusion, and enhanced animal survival and organ function. Rapid activation of Egr-1 in response to oxygen deprivation primes the vasculature for dysfunction manifest during reperfusion. These studies define a central and unifying role for Egr-1 activation in the pathogenesis of ischemic tissue damage.
Background:
2-chloro-N(6) -cyclopentyladenosine (CCPA) was proven to be a protective factor in ischemic reperfusion injury. The purpose of this study was to determine how CCPA would affect the single tissue layers of the adipocutaneous flap.
Methods:
Seventy male Wistar rats were divided into 5 experimental groups. Samples were taken of the area of flap necrosis and the wound margin after classical or pharmacological preconditioning on the fifth postoperative day. All samples were fixed in formaldehyde, embedded in paraplast, and analyzed in 3- to 4-μm sections (hemalaun-eosin stain and light microscopy).
Results:
In general, wound healing was alike and remained unaffected by the experimental design. The most sensitive part of the flap during preconditioning is the subcutis. The number of neutrophils and of plasma cells is reduced significantly (p < .05).
Conclusion:
CCPA has an effect on each tissue layer of the flap. Subcutis became apparent as the most sensitive layer. CCPA influences complement pathway and neutrophils directly and indirectly.
A short duration of ischemia (i.e., ischemic preconditioning) was shown to result in significant tolerance to subsequent ischemic injury. Since previous reports suggest that interleukin-1β (IL-1β) may be involved in both ischemic damage and neuroprotection, the present work examined the expression of IL-1β mRNA in cortical brain tissue after an established preconditioning (PC) stimulus known to produce significant brain tolerance to focal stroke after 1–7 days. Significant induction of IL-1β mRNA was observed in the ipsilateral cortex at 6 hr (87 ± 9 copies of the mRNA per microgram of brain tissue compared to 16 ± 5 copies in sham-operated samples, P < 0.001, n = 4) and 8 hr (46 ± 4 copies, P < 0.01, n = 4) after PC by means of real-time Taqman polymerase chain reaction (PCR). The peak expression of IL-1β mRNA after PC was significantly (P < 0.01) lower than that after permanent occlusion of the middle cerebral artery (MCAO), i.e., 87 ± 9 and 546 ± 92 copies of RNA per microgram tissue at peak levels for PC and focal stroke, respectively. Immunohistochemistry studies revealed a parallel induction of IL-1β in the ipsilateral cortex after PC. The maximal expression of IL-1β was observed during the first week post-PC, showing marked parallelism with the duration of ischemic tolerance. These data suggest that the significant but low levels of IL-1β induction after PC may contribute to ischemic brain tolerance. J. Neurosci. Res. 59:238–246, 2000 © 2000 Wiley-Liss, Inc.
Chemokines are a family of proteins associated with the trafficking of leukocytes in physiological immune surveillance and inflammatory cell recruitment in host defence. They are classified into four classes based on the positions of key cystiene residues: C, CC, CXC, and CX3C. Chemokines act through both specific and shared receptors that all belong to the superfamily of G-protein-coupled receptors. Besides their well-established role in the immune system, several recent reports have demonstrated that these proteins also play a role in the central nervous system (CNS). In the CNS, chemokines are constitutively expressed by microglial cells, astrocytes, and neurons, and their expression can be increased after induction with inflammatory mediators. Constitutive expression of chemokines and chemokine receptors has been observed in both developing and adult brains, and the role played by these proteins in the normal brain is the object of intense study by many research groups. Chemokines are involved in brain development and in the maintenance of normal brain homeostasis; these proteins play a role in the migration, differentiation, and proliferation of glial and neuronal cells. The chemokine stromal cell-derived factor 1 and its receptor, CXCR4, are essential for life during development, and this ligand–receptor pair has been shown to have a fundamental role in neuron migration during cerebellar formation. Chemokine and chemokine receptor expression can be increased by inflammatory mediators, and this has in turn been associated with several acute and chronic inflammatory conditions. In the CNS, chemokines play an essential role in neuroinflammation as mediators of leukocyte infiltration. Their overexpression has been implicated in different neurological disorders, such as multiple sclerosis, trauma, stroke, Alzheimer's disease, tumor progression, and acquired immunodeficiency syndrome-associated dementia. An emerging area of interest for chemokine action is represented by the communication between the neuroendocrine and the immune system. Chemokines have hormone-like actions, specifically regulating the key host physiopathological responses of fever and appetite. It is now evident that chemokines and their receptors represent a plurifunctional family of proteins whose actions on the CNS are not restricted to neuroinflammation. These molecules constitute crucial regulators of cellular communication in physiological and developmental processes.
Besides their well-established role of inducing cell migration in the immune system, it is now proven that chemokines and
their receptors also play a role in the central nervous system (CNS). The best known central action is the ability of chemokines
to act as immunoinflammatory mediators. Indeed, they regulate leukocyte infiltration in the brain during inflammatory and
infectious diseases. Recent studies, however clearly demonstrate that chemokines and their receptors are expressed by glial
and neuronal cells in the CNS, where they are involved in neurotransmission. The purpose of this chapter is to summarize the
latest information concerning the role of chemokines in various central functions. The first part will focus on the expression
of chemokines and their receptors in the CNS with the emphasis on neuronal expression. In the second part, we will discuss
the involvement of chemokines and their receptors in different spinal and supraspinal mechanisms. In conclusion, a better
understanding of the implication of chemokines in cell communication will lead to: (1) the identification of new pathways
for neuron-neuron and/or glia-glia and/or neuron-glia cross-talk relevant to both normal brain function and neuroinflammatory
and neurodegenerative pathologies; (2) the development of new therapeutic approaches for yet untreatable nervous diseases.
Key wordsChemokine-Chemokine receptor-Central nervous system-Neuromodulation-Neuronal activity-CXCL12/CXCR4-CCL2/CCR2-CCL4/CCR5
Recruitment of inflammatory cells is known to drive the secondary damage cascades that are common to injuries of the central nervous system (CNS). Cell activation and infiltration to the injury site is orchestrated by changes in the expression of chemokines, the chemoattractive cytokines. Reducing the numbers of recruited inflammatory cells by the blocking of the action of chemokines has turned out be a promising approach to diminish neuroinflammation and to improve tissue preservation and neovascularization. In addition, several chemokines have been shown to be essential for stem/progenitor cell attraction, their survival, differentiation and cytokine production. Thus, chemokines might indirectly participate in remyelination, neovascularization and neuroprotection, which are important prerequisites for CNS repair after trauma. Moreover, CXCL12 promotes neurite outgrowth in the presence of growth inhibitory CNS myelin and enhances axonal sprouting after spinal cord injury (SCI). Here, we review current knowledge about the exciting functions of chemokines in CNS trauma, including SCI, traumatic brain injury and stroke. We identify common principles of chemokine action and discuss the potentials and challenges of therapeutic interventions with chemokines.
After central nervous system (CNS) trauma, axons have a low capacity for regeneration. Regeneration failure is associated with a muted regenerative response of the neuron itself, combined with a growth-inhibitory and cytotoxic post-injury environment. After spinal cord injury (SCI), resident and infiltrating immune cells (especially microglia/macrophages) contribute significantly to the growth-refractory milieu near the lesion. By targeting both the regenerative potential of the axon and the cytotoxic phenotype of microglia/macrophages, we may be able to improve CNS repair after SCI. In this review, we discuss molecules shown to impact CNS repair by affecting both immune cells and neurons. Specifically, we provide examples of pattern recognition receptors, integrins, cytokines/chemokines, nuclear receptors and galectins that could improve CNS repair. In many cases, signaling by these molecules is complex and may have contradictory effects on recovery depending on the cell types involved or the model studied. Despite this caveat, deciphering convergent signaling pathways on immune cells (which affect axon growth indirectly) and neurons (direct effects on axon growth) could improve repair and recovery after SCI. Future studies must continue to consider how regenerative therapies targeting neurons impact other cells in the pathological CNS. By identifying molecules that simultaneously improve axon regenerative capacity and drive the protective, growth-promoting phenotype of immune cells, we may discover SCI therapies that act synergistically to improve CNS repair and functional recovery.
Using the suppression subtractive hybridization (SSH) strategy for differential gene cloning, we identified the induced expression of a rat homologue to murine and human monocyte chemotactic protein-3 (MCP-3) in ischemic brain. The 2.4-kilobase rat MCP-3 gene features high homology in gene structure and sequence to murine MCP-3. The temporal expression of MCP-3 mRNA was examined in brain tissue rendered ischemia by permanent or temporary occlusion of the middle cerebral artery (MCAO). A marked increase in MCP-3 mRNA was observed 12 h post-ischemia, with 49-fold and 17-fold increase (n=4, p<0.01) over control in the permanent or temporary MCAO, respectively. Significant induction of MCP-3 in the ischemic cortex was sustained up to 5 days after ischemic injury. The profile of MCP-3 mRNA induction paralleled leukocyte infiltration and accumulation that occur after focal stroke, suggesting a role for MCP-3 in recruiting these inflammatory cells into the ischemic tissue. Molecular cloning of rat MCP-3 should provide a valuable tool, as demonstrated in the present work, for the investigation of MCP-3 expression and function in rat disease models.
Chemo-attractant cytokines, now known as chemokines, comprise the largest and most diverse subset of cytokines identified to date. Chemokines are characterized by their capacity to bring about the directional migration and activation of leucocytes and other somatic cell types during inflammation; cell-mediated immune reactions; to regulate cell adhesion, angiogenesis, leukocyte trafficking and homing, as well as lymphopoiesis and hematopoiesis. Chemokines are produced by a wide variety of leukocytes and other cell types in response to inflammatory agents, antigens, and endogenous cytokines. Studies of the in vivo effects of neutralizing antibodies and homologous deletions of chemokine genes reveal that chemokines play a central role in host defense against infectious organisms. Chemochines are relevant in normal central nervous system (CNS) physiology and development, as well as in the pathogenesis of diverse conditions such as tumor metastasis, riperfusion injuries and stroke. Chemokine structure, expression in CNS, and systematic classification schemes are presented according to (1) their constitutive expression and (2) their inducibility in response to inflammatory stimuli. The classification of chemokine receptor expression in specific CNS cells and their principal intracellular signal transduction pathways are also included, in addition to their various physiological roles. Consideration is given to the fascinating hypothesis that chemokines may influence neural and glial cell migration and proliferation during CNS development. The immuno-competence of the CNS, including inflammatory cell recruitment into the CNS as a function of chemokines in the evolution of pathological and host defense processes, is explored for head trauma, brain ischemia and trauma, AIDS dementia, Alzheimer’s disease, and brain tumor progression.
The importance of cytokines and the complement system in the propagation and maintenance of the brain inflammatory response to injury are emphasised. Much data supports the case that ischemia and trauma elicit an inflammatory response in the injured brain. This inflammatory response consists of mediators (cytokines, complement activation, chemokines and adhesion molecules) followed by cells (neutrophils early after the onset of brain injury and then a later monocyte infiltration). De novo up-regulation of pro-inflammatory cytokines, chemokines and endothelial-leukocyte adhesion molecules occurs soon following focal ischemia and trauma and at a time when the tissue injury is evolving. The significance of this brain inflammatory response and its contribution to brain injury is now better understood. In this review, we discuss the role of TNFα and IL-1β in traumatic and brain injury and associated inflammation, and the co-operative actions of the complement system, chemokines and adhesion molecules in this process. Celluar stress and cellular stress signalling is key to the neurodegenerative process in brain injury. Therefore, we also address novel approaches to target cytokines and reduce the brain inflammatory response, and thus brain injury, in stroke and neurotrauma. The mitogen-activated protein kinase (MAPK), p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Stroke-induced p38 enzyme activation in the brain has been demonstrated, and treatment with p38 MAPK inhibitors can provide a significant reduction in infarct size, neurological deficits and increased inflammatory cytokines/proteins expression produced by focal stroke. p38 MAPK inhibition can also provide direct protection of cultured brain tissue to in vitro ischemia. This robust neuroprotection that can be produced by inhibition of p38 MAPK signalling emphasizes a significant opportunity for targeting MAPK pathways CNS injury/disease. Many examples of the roles of inflammation, cellular oxidative stress and MAPK signalling in Psychiatric and Neurodegenerative Diseases are also provided. As a whole, the available data suggests that inflammation, cellular stress and p38 MAPK signalling are important in nervous disease pathologies and that inhibition of cellular stress signalling should be considered for improving outcome in many CNS diseases.
After brain ischemia, significant amounts of adenosine 5'-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y(1) receptor (P2Y(1)R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y(1)R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y(1)R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y(1)R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y(1)R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y(1)R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.
Myelin-laden macrophages reside within the CNS, the CSF and in the CNS-draining lymph nodes during MS and EAE, suggesting migration of these macrophages between these compartments and interaction with other cells. Since chemokines and their receptors are pivotal for leukocyte trafficking, we addressed whether myelin ingestion affects chemotaxis of mouse macrophages in vitro. Myelin ingestion enhanced expression of CCR7 and CXCR3 on macrophages and migration towards CCL21 and CXCL10. Furthermore, myelin-laden macrophages released chemoattractants resulting in enhanced migration of myeloid cells in vitro. Our data demonstrate that myelin-laden macrophages have increased motility and suggest trafficking between anatomical compartments in vivo.
Reduced risk and severity of stroke in adult females is thought to depend on normal endogenous levels of estrogen, a well-known neuroprotectant and immunomodulator. In male mice, experimental stroke induces immunosuppression of the peripheral immune system, characterized by a reduction in spleen size and cell numbers and decreased cytokine and chemokine expression. However, stroke-induced immunosuppression has not been evaluated in female mice. To test the hypothesis that estradiol (E2) deficiency exacerbates immunosuppression after focal stroke in females, we evaluated the effect of middle cerebral artery occlusion on infarct size and peripheral and CNS immune responses in ovariectomized mice with or without sustained, controlled levels of 17-beta-E2 administered by s.c. implant or the putative membrane estrogen receptor agonist, G1. Both E2- and G1-replacement decreased infarct volume and partially restored splenocyte numbers. Moreover, E2-replacement increased splenocyte proliferation in response to stimulation with anti-CD3/CD28 Abs and normalized aberrant mRNA expression for cytokines, chemokines, and chemokine receptors and percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells observed in E2-deficient animals. These beneficial changes in peripheral immunity after E2 replacement were accompanied by a profound reduction in expression of the chemokine, MIP-2, and a 40-fold increased expression of CCR7 in the lesioned brain hemisphere. These results demonstrate for the first time that E2 replacement in ovariectomized female mice improves stroke-induced peripheral immunosuppression.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
In previous studies, we have used histological methods to characterize cellular changes, and validated the use of the myeloperoxidase
(MPO) activity assay to quantitate increased neutrophil infiltration in ischemic stroke. We also identified increased leukotriene
B4 (LTB4) binding sites as a potential marker for neutrophil infiltration into, focal ischemic tissue. However, these studies were
conducted at only one time-point, 24 h after ischemia. In the present study, we examined the full time-course of MPO activity
and LTB4 receptor binding following middle cerebral artery occlusion (MCAO) made permanently (PMCAO) or transiently (160 min followed
by reperfusion; TMCAO) in spontaneously hypertensive rats, and compared the results to previously characterized histologic
changes in these models. Ischemic and contralateral (control) cortical tissue samples were assayed for MPO (U/g wet wt) and
[3H]LTB4 receptor binding (fmol/mg protein). Following PMCAO, MPO activity significantly increased as early as 12 h and continued
to increase over the next 5 d (p<0.05). Following TMCAO, MPO activity was significantly elevated already after only 6 h of reperfusion and also continued
to increase over the next 5 d of reperfusion (p<0.05). LTB4 receptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5–5dp<0.01). However, by 15 d post-MCAO, LTB4 receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB4 binding was associated with the significant gliosis that was characterized previously to persist in these models. The time-course
of increased MPO activity and initially increased LTB4 binding post-MCAO correspond very well to our previous histological data that characterized the time-course for leukocyte
infiltration under these conditions. Therefore, the increased MPO activity over time was associated with initial neutrophil
and later mononuclear cell infiltration into ischemic tissue in these models. In addition, the present studies utilized histochemical
analysis to demonstrate peroxidase activity in macrophages within the cerebral infarct following MCAO, thus validating that
MPO activity originates from the later infiltrating mononuclear cells in addition to the early infiltrating neutrophils that
had been previously characterized in the same manner. TMCAO produces a significantly larger and earlier increase in ischemic
cortex MPO activity and a similar later increase in MPO activity compared to PMCAO treatment. Clearly, reperfusion of cerebral
tissue following ischemia greatly exacerbates the degree of cerebral tissue inflammation. These biochemical assays, especially
the MPO activity assay, have now been validated for quantitating the early and late phases of the cerebral inflammatory reaction
to tissue injury.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
An IFN-gamma-inducible protein, IP-10, has previously been described to belong to a gene family of chemotactic and mitogenic proteins, associated with inflammation and proliferation. Biochemical characterization of this predicted protein has been pursued through the development of polyclonal monospecific antisera to recombinant protein and synthetic peptides. These reagents establish that the IP-10 protein is secreted from a variety of cells (endothelial, monocyte, fibroblast, and keratinocyte) in response to IFN-gamma. Posttranslational processing occurs in the biosynthesis of this protein, resulting in a 6-7-kD species, which may reflect COOH-terminal cleavage. Pulse-chase studies indicate that this processing is a rapid event in the primary cell lines studied, completed in the 30-min labeling period. A model is presented for the processing and secondary structure of this protein. In an accompanying study, Kaplan, et al. using these antisera, demonstrate that the IP-10 protein is associated, in vivo, with a delayed-type hypersensitivity response.
Marked increases in the brain expression of interleukin (IL)-1 have been reported in rats after permanent occlusion of a large cerebral artery. Interactions between endothelial cells and leukocytes have been implicated in the pathogenesis of several types of ischemic injury to the myocardium and other organs. In this study we asked whether inhibiting the effects of IL-1 would affect the outcome of an experimental brain infarct. Adult male Wistar rats (n = 13) with permanent occlusion of the middle cerebral artery were given IL-1 receptor antagonist. A second group (n = 13) with the same type of brain injury was given a placebo. A third group, subjected to a sham operation, was given either IL-1 receptor antagonist (n = 2) or a placebo (n = 2). Experiments were terminated after either 24 hours or 7 days. Compared with the control group, animals treated with IL-1 receptor antagonist improved their neurological score (P < 0.05), experienced less pronounced changes in body weight (P < 0.05), and had fewer necrotic neurons (P < 0.001) and fewer leukocytes in the ischemic hemisphere (P < 0.001) as well as a smaller area of pallor (P < 0.05) in the ischemis hemisphere. The results suggest that inhibiting the proinflammatory effects of IL-1 with a receptor antagonist is an effective way of influencing the leukocyte responses elicited by an arterial occlusion. Such leukocyte inhibition seemingly attenuates the number of necrotic neurons resulting from the occlusion of a large brain artery.
In this study, we demonstrate that the CXC family of chemokines displays disparate angiogenic activity depending upon the
presence or absence of the ELR motif. CXC chemokines containing the ELR motif (ELR-CXC chemokines) were found to be potent
angiogenic factors, inducing both in vitro endothelial chemotaxis and in vivo corneal neovascularization. In contrast, the CXC chemokines lacking the ELR motif, platelet factor 4, interferon -inducible protein 10, and monokine induced by -interferon, not only failed to induce significant in vitro endothelial cell chemotaxis or in vivo corneal neovacularization but were found to be potent angiostatic factors in the presence of either ELR-CXC chemokines or
the unrelated angiogenic factor, basic fibroblast growth factor. Additionally, mutant interleukin-8 proteins lacking the ELR
motif demonstrated potent angiostatic effects in the presence of either ELR-CXC chemokines or basic fibroblast growth factor.
In contrast, a mutant of monokine induced by -interferon containing the ELR motif was found to induce in vivo angiogenic activity. These findings suggest a functional role of the ELR motif in determining the angiogenic or angiostatic
potential of CXC chemokines, supporting the hypothesis that the net biological balance between angiogenic and angiostatic
CXC chemokines may play an important role in regulating overall angiogenesis.
IP-10 is a member of the -C-X-C-chemokine superfamily of proinflammatory cytokines whose secretion is induced by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). To date no function has been described for IP-10. We have genetically engineered tumor cells to secrete high levels of murine IP-10 and demonstrate that while IP-10 has no effect on the growth of these tumor cells in culture, it elicits a powerful host-mediated antitumor effect in vivo. The IP-10 antitumor response is T lymphocyte dependent, non-cell autonomous, and appears to be mediated by the recruitment of an inflammatory infiltrate composed of lymphocytes, neutrophils, and monocytes. These results document an important biologic property of IP-10 and raise the possibility that some of the T cell-directed effects of IFN-gamma and LPS may be mediated by this chemokine.
The human cytokine interferon-inducible protein 10 (IP-10) is a small glycoprotein secreted by activated T cells, monocytes, endothelial cells, and keratinocytes, and is structurally related to a family of chemotactic cytokines called chemokines. Although this protein is present in sites of delayed-type hypersensitivity reactions and lepromatous leprosy lesions, the biological activity of IP-10 remains unknown. We report here that recombinant human IP-10 stimulated significant in vitro chemotaxis of human peripheral blood monocytes but not neutrophils. Recombinant human IP-10 also stimulated chemotaxis of stimulated, but not unstimulated, human peripheral blood T lymphocytes. Phenotypic analysis of the stimulated T cell population responsive to IP-10 demonstrated that stimulated CD4+ and CD29+ T cells migrated in response to IP-10. This resembles the biological activity of the previously described T cell chemoattractant RANTES. Using an endothelial cell adhesion assay, we demonstrated that stimulated T cells pretreated with optimal doses of IP-10 exhibited a greatly enhanced ability to bind to an interleukin 1-treated endothelial cell monolayer. These results demonstrate that the IP-10 gene encodes for an inflammatory mediator that specifically stimulates the directional migration of T cells and monocytes as well as potentiates T cell adhesion to endothelium.
Interferon-inducible protein (IP)-10 is a small glycoprotein member of a family of chemotactic cytokines structurally related to interleukin-8. We have recently described the induction of IP-10 mRNA in mouse mesangial cells stimulated with lipopolysacharide, interferon-gamma, and tumor necrosis factor-alpha. To further evaluate a possible role for this chemokine in renal injury, we have studied IP-10 in an experimental model of nephrosis induced in rats by adriamycin. High levels of glomerular IP-10 mRNA expression and glomerular and tubulointerstitial IP-10 protein were seen on day 21, coinciding with maximal proteinuria, glomerular tumor necrosis factor mRNA expression, and interstitial cellular infiltrates. Maintenance on a low protein diet not only delayed the appearance of proteinuria and interstitial cellular infiltrate but also decreased glomerular IP-10 mRNA expression. Isolated normal glomeruli and cultured glomerular epithelial and mesangial cells from normal rats expressed IP-10 mRNA upon stimulation with 100 U/ml interferon or 1 microgram/ml lipopolysaccharide for 3 hours. IP-10 mRNA expression was also inducible by lipopolysaccharide and cytokines in NRK 49F renal interstitial fibroblasts and, to a lesser extent, in NRK 52E tubular epithelial cells. Furthermore, IP-10 protein was inducible in murine mesangial cells. We conclude that IP-10 is highly inducible in vitro and in vivo in resident glomerular and tubulointerstitial cells. IP-10 may participate in the modulation of renal damage in experimental nephrosis.
We have recently established an animal model of adult respiratory distress syndrome (ARDS)-like microvascular lung injury elicited by infusion of human interleukin-2 (IL-2). Based on the pronounced, transcriptional upregulation of multiple pro-inflammatory mediators in IL-2-induced ARDS, differential display was applied to search for potentially novel genes in this paradigm of lung injury. Differential display on total lung RNA derived from IL-2-challenged rats presented a highly reproducible 3'-UTR fragment profile in which a band (approximately 250 bp), termed B1, was strongly induced. B1 cDNA sequence exhibited 99.14% homology to the 3'-UTR of mob-1, a recently cloned gene belonging to the C-X-C chemokine superfamily. Furthermore, Northern blot analysis showed that IL-2-induced pulmonary mob-1 mRNA was expressed at time points before the onset of lung injury and suppressed after TNF-alpha inhibition. These data imply that lung mob-1 is a novel, highly inducible gene in a clinically relevant model of ARDS and, based on its identification as a chemokine, could participate in the development of lung injury.
The endothelin family of 21-amino acid peptides represents the most potent series of known vasoconstrictors and contributes to the local regulation of vascular homeostasis. Four distinct isoforms—ET-1, ET-2, ET-3, and vasoactive intestinal contractor or ET-β)—which differ in amino acid substitutions, have been identified to date and show widespread distribution in mammalian tissue. Vascular endothelial cells produce only ET-1, but ET-1, ET-2, and ET-3 are present and/or active in brain. Neuropeptides function as neuromodulators or local hormones, which oversee the central nervous system regulation of cardiovascular function. In addition, endothelin is found both in posterior and anterior pituitary, regulating anterior pituitary function by eliciting inositol trisphosphate and diacylglycerol production, calcium mobilization and influx, and gonadotropin secretion. Endothelin distributes in a nonvascular pattern in brain, and high densities of endothelin-binding sites are found in the granule cell layer of the cerebellum. This chapter discusses some systems of both primary cultured cells and established neural cell lines for endothelin receptors and their biochemical significance.
Publisher Summary This chapter focuses on interleukin-8 (IL-8) and related chemotactic cytokines—namely, CXC and CC chemokines. IL-8 is the best known member of a new class of cytokines that are widely studied because of their ability to attract and activate leukocytes, and their potential role as mediators of inflammation. IL-8 was originally isolated from the culture supernatants of stimulated human blood monocytes and was identified as a protein of 72 amino acids with a molecular weight of 8383. The three-dimensional structure of IL-8 has been studied by nuclear magnetic resonance spectroscopy and X-ray crystallography. In concentrated solution, and on crystallization, IL-8 is present as a dimer. The first CC chemokine was identified after cloning by differential hybridization from human tonsillar lymphocytes and was termed LD78. The CC and CXC chemokines are similar in size and have an overall structure that is characterized by the two intrachain disulfide bonds, short N-terminal and long C-terminal sequences. It discusses the role of chemokines in pathology with skin inflammation because psoriasis was the first disease to be linked to overproduction of IL-8. Several independent studies document the occurrence of high levels of IL-8 in the synovial fluid of inflamed joints of patients with different forms of rheumatic diseases, osteoarthritis, and gout.
: The expression of interleukin-1β (IL-1β) mRNA in the cerebral cortex, hippocampus, striatum, and thalamus of rats was studied after transient forebrain ischemia. IL-1β mRNA was not detected in all these regions of sham-operated control rats. IL-1β mRNA was induced after transient forebrain ischemia and reached a detectable level in all regions examined 15 min after the start of recirculation. The induction of IL-1β mRNA had a few peaks, that is, peaks were observed at 30 and 240 min in the four regions examined, and another peak was observed at 90 min in the striatum. One day after the start of recirculation, IL-1β mRNA levels were markedly decreased, but even 7 days after that, IL-1β mRNA was found at very low levels in all regions examined. The amounts of c-fos and β-actin mRNAs on the same blots were also examined. The induction of c-fos mRNA was transient and had only one peak in all regions examined, whereas the levels of β-actin mRNA in these regions were fairly constant throughout the recirculation period. Thus, we provide the first evidence for a characteristic expression of IL-1β mRNA in several brain regions after transient forebrain ischemia.
Interferons are a family of proteins first identified by their ability to induce cellular resistance to infection by many viruses. In addition to the antiviral properties it shares with the alpha- and beta-interferons, gamma-interferon (IFN-gamma), a lymphokine secreted by activated T cells, activates macrophages, stimulates B cells, increases fibroblast and endothelial cell resistance to many non-viral intracellular parasites and modulates cell-surface proteins central to immune cell regulation1-13. To identify molecules involved in the IFN-gamma response and characterize their modulation, we have isolated genes that are induced following recombinant IFN-gamma treatment of U937 cells, a histiocytic lymphoma cell line with monocytic characteristics14,15. We report here the molecular cloning and characterization of a gene regulated by rIFN-gamma in U937 cells as well as in human mononuclear cells, fibroblasts and endothelial cells. Messenger RNA from this gene is induced within 30 min of rIFN-gamma treatment and demonstrates maximal (>30-fold) accumulation within 5 h. Increased transcription is partly responsible for this accumulation. This gene encodes a protein of relative molecular mass (Mr) 12,378 which has significant amino-acid homology to platelet factor-4 and beta-thromboglobulin, two chemo-tatic proteins released by platelets on degranulation. This IFN-gamma-inducible protein may be a member of a family of proteins involved in the inflammatory process.
The expression of intercellular adhesion molecule 1 (ICAM-1) was studied in rat focal ischemic cortex. A significant increase in ICAM-1 mRNA expression in the ischemic cortex over levels in contralateral (nonischemic) site was observed by means of Northern blot analysis following either permanent or temporary occlusion with reperfusion of the middle cerebral artery (PMCAO or MCAO with reperfusion) in spontaneously hypertensive rats. In the ischemic cortex, levels of ICAM-1 mRNA increased significantly at 3 h (2.6-fold, n = 3, P < 0.05), peaked at 6 to 12 h (6.0-fold, P < 0.01) and remained elevated up to 5 days (2.5-fold, P < 0.05) after PMCAO. The profile of ICAM-1 mRNA expression in the ischemic cortex following MCAO with reperfusion was similar to that following PMCAO, except that ICAM-1 mRNA was significantly increased as early as 1 h (6.3-fold, n = 3, P < 0.05) and then gradually reached a peak at 12 h (12-fold, P < 0.01) after reperfusion. ICAM-1 mRNA expression in ischemic cortex following PMCAO was significantly greater in hypertensive rats than in two normotensive rat strains. Immunostaining using anti-ICAM-1 antibodies indicated that upregulated ICAM-1 expression was localized to endothelial cells of intraparenchymal blood vessels in the ischemic but not contralateral cortex. The data suggest that an upregulation of ICAM-1 mRNA and protein on brain capillary endothelium may play an important role in leukocyte migration into ischemic brain tissue.
Proinflammatory cytokines mediate brain injury in experimental studies. This study was undertaken to analyze the production of proinflammatory cytokines in experimental contusion. A brain contusion causing delayed edema was mimicked experimentally in rats using a weight-drop model. Intracerebral expression of the cytokines interleukin (IL)—1β, tumor necrosis factor—α (TNFα), IL-6, and interferon-γ (IFNγ) was studied by in situ hybridization and immunohistochemistry. The animals were killed at 6 hours or 1, 2, 4, 6, 8, or 16 days postinjury. In the injured area, no messenger (m)RNA expression was seen during the first 2 days after the trauma. On Days 4 to 6 posttrauma, however, strong IL-1β, TNFα, and IL-6 mRNA expression was detected in mononuclear cells surrounding the contusion. Expression of IFNγ was not detected. Immunohistochemical double labeling confirmed the in situ hybridization results and demonstrated that mononuclear phagocytes and astrocytes produced IL-1β and that mainly astrocytes produced TNFα.
The findings showed, somewhat unexpectedly, a late peak of intracerebral cytokine production in the injured area and in the contralateral corpus callosum, allowing for both local and global effects on the brain. An unexpected difference in the cellular sources of TNFα and IL-1β was detected. The cytokine pattern differs from that seen in other central nervous system inflammatory diseases and trauma models, suggesting that the intracerebral immune response is not a uniform event. The dominance of late cytokine production indicates that many cytokine effects are late events in an experimental contusion. Different pathogenic mechanisms may thus be operative at different times after brain injury.
Chemokines direct the trafficking of white blood cells in immune surveillance, playing a key role in inflammatory and infectious diseases such as AIDS. All chemokines studied so far are secreted proteins of relative molecular mass approximately 7K-15K and fall into three families that are defined by a cysteine signature motif: CXC, CC and C (refs 3, 6, 7), where C is a cysteine and X any amino-acid residue. We report here the identification and characterization of a fourth human chemokine type, derived from non-haemopoietic cells and bearing a new CX3C fingerprint. Unlike other chemokine types, the polypeptide chain of the human CX3C chemokine is predicted to be part of a 373-amino-acid protein that carries the chemokine domain on top of an extended mucin-like stalk. This molecule can exist in two forms: either membrane-anchored or as a shed 95K glycoprotein. The soluble CX3C chemokine has potent chemoattractant activity for T cells and monocytes, and the cell-surface-bound protein, which is induced on activated primary endothelial cells, promotes strong adhesion of those leukocytes. The structure, biochemical features, tissue distribution and chromosomal localization of CX3C chemokine all indicate that it represents a unique class of chemokine that may constitute part of the molecular control of leukocyte traffic at the endothelium.
Interleukin-1 (IL-1) synthesis in the brain is stimulated by mechanical injury and IL-1 mimics some effects of injury, such as gliosis and neovascularization. We report that neuronal death resulting from focal cerebral ischaemia (middle cerebral artery occlusion, 24 h) is significantly inhibited (by 50%) in rats injected with a recombinant IL-1 receptor antagonist (IL-1ra, 10 micrograms, icv 30 min before and 10 min after ischaemia). Excitotoxic damage due to striatal infusion of an NMDA-receptor agonist (cis-2,4-methanoglutamate) was also markedly inhibited (71%) by injection of the IL-1ra. These data indicate that endogenous IL-1 is a mediator of ischaemic and excitotoxic brain damage, and that inhibitors of IL-1 action may be of therapeutic value in the treatment of acute or chronic neuronal death.
Studies conducted in many laboratories over the past several years have resulted in the identification and initial characterization of a large superfamily of structurally and functionally related inflammatory cytokines. This superfamily currently includes 14 distinct members: platelet factor 4, beta-thromboglobulin, neutrophil activating peptide-1/interleukin-8, gro, IP-10, mig, ENA-78, macrophage inflammatory proteins-1 alpha and -1 beta, monocyte chemoattractant protein-1/JE, RANTES, HC-14, C-10, and I-309. Although numerous biological activities have been assigned to these molecules, a common theme is their ability to stimulate the chemotactic migration of distinct sets of cells, including neutrophils, monocytes, lymphocytes, and fibroblasts. Accumulating evidence indicates that these molecules play important roles in mediating cell recruitment and activation necessary for inflammation and the repair of tissue damage.
The extent to which polymorphonuclear leukocytes and monocytes/macrophages contribute to the pathobiology of cerebral ischemia and stroke is an issue of long-standing contradiction and controversy. Recent developments in the ability to selectively modify leukocyte adhesion with antiadhesion antibodies and the potential clinical application of this therapeutic approach have spurred a resurgence of experimental studies examining the role of leukocytes in cerebral ischemia and stroke.
We review studies examining leukocyte accumulation, initiation of thrombosis, and exacerbation of ischemic brain injury in stroke, and we examine other proposed contributions of leukocytes to cerebrovascular pathophysiology.
The importance of specific characteristics of a given ischemia model and of underlying stroke risk factors in determining the degree of leukocyte involvement and effectiveness of therapies directed against these cells is discussed.
A review of the sensitivity of genetically hypertensive rats to cerebral ischemia was presented together with original data describing the systematic comparison of the effects of focal ischemia (permanent and temporary with reperfusion) performed in hypertensive and normotensive rats (i.e., blood pressures verified in conscious instrumented rats). Microsurgical techniques were used to isolate and occlude the middle cerebral artery (MCAO) of spontaneously hypertensive (SHR), Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats at the level of the inferior cerebral vein. Following permanent (24 h) MCAO, persistent and similar decreases in local microvascular perfusion (i.e., to 15.6 +/- 1.7% of pre-MCAO levels) were verified in the primary ischemic zone of the cortex for all strains using Laser-Doppler flowmetry. A contralateral hemiplegia that occurred following MCAO, evidenced by forelimb flexion and muscle weakness, was greater in SHR (neurological grade = 2.0 +/- 0.1) than SD (1.0 +/- 0.4) or WKY (0.7 +/- 0.4) rats (N = 7-9, p less than 0.05). SHR also exhibited sensory motor deficits following MCAO compared to sham-operation, with decreased normal placement response of the hindlimb (% normal = 20 vs. 83, N = 23-30, p decreased rota-rod (41 +/- 7 vs. 126 +/- 19 on rod, N = 10-15, p less than 0.05) and balance beam (25 +/- 5 vs. 116 +/- 29 s on beam, N = 5-7, p less than 0.05) performance. However, an index of general motor activity was not affected by permanent MCAO. Triphenyltetrazolium-stained forebrain tissue analyzed by planimetry revealed a significantly larger and more consistent cortical infarction in SHR (hemispheric infarction = 27.9 +/- 1.5%) compared to SD (15.4 +/- 4.1%) and WKY (4.0 +/- 2.4%) rats (N = 7-9, p less than 0.05), occupying predominantly the frontal and parietal areas. Also, a significant degree of ipsilateral hemispheric swelling (4.6 +/- 0.9%, N = 7-9, p less than 0.05) and increased brain water content (78.4 +/- 0.3% to 80.4 +/- 0.2%, N = 8-9, p less than 0.05) was identified in SHR that was not observed in SD or WKY rats. A novel model of temporary MCAO also was evaluated in the hypertensive and normotensive rat strains. Initially, the effect of increasing MCAO-time followed by 24 h reperfusion in SHR was studied. During temporary MCAO (20 to 300 min), persistent and stable decreases in local microvascular perfusion (i.e., to 15-20% of pre-MCAO levels) were verified in the primary ischemic zones of the cortex.(ABSTRACT TRUNCATED AT 400 WORDS)
The effect of simvastatin (MK-733), a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on the migration of cultured porcine smooth muscle cells (SMCs) was investigated in modified Boyden chambers. Platelet-derived growth factor (PDGF) stimulated the SMC migration dose dependently. MK-733 inhibited the migration response induced by PDGF with an IC50 value of 2 microM. Supplementation with mevalonate restored the migration response inhibited by MK-733 but the addition of low-density-lipoprotein (LDL) did not change the response. Another HMG-CoA reductase inhibitor, pravastatin (CS-514), also reduced the migration response. However its potency was far less than that of MK-733. MK-733 also inhibited the SMC migration stimulated by fibrinogen. These results suggest that non-sterol metabolite(s) of mevalonate, possibly prenylated proteins, are involved in a migration signaling pathway and that HMG-CoA reductase inhibitors are effective in the prevention of the formation of intimal hyperplasia in atherosclerosis.
Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non-washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli.
Recently, we have isolated and characterized a set of cDNA clones which encode lipopolysaccharide-inducible proteins in murine peritoneal macrophages. Here, we report the sequence and identification of one of these cDNAs previously termed C7. Nucleotide sequence analysis revealed an open reading frame encoding a predicted polypeptide composed of 98 amino acids, which contained a 21 amino acid residue signal peptide, indicating approximately 9 kDa of mature protein. The deduced protein sequence showed homology (67% identity, 77% considering conservative amino acid changes) with the human INF gamma-inducible gene IP-10, a member of the recently described superfamily of chemotactic and mitogenic proteins which includes platelet factor 4, monocyte-derived neutrophil chemotactic factor (NAF, NAP-1, IL-8), and MGSA/gro/KC. Thus C7 would appear to represent the murine homologue of the human IP-10 gene or a very closely related gene.
The use of physiologically regulated, reproducible animal models is crucial to the study of ischemic brain injury--both the mechanisms governing its occurrence and potential therapeutic strategies. Several laboratory rodent species (notably rats and gerbils), which are readily available at relatively low cost, are highly suitable for the investigation of cerebral ischemia and have been widely employed for this purpose. We critically examine and summarize several rodent models of transient global ischemia, resulting in selective neuronal injury within vulnerable brain regions, and focal ischemia, typically giving rise to localized brain infarction. We explore the utility of individual models and emphasize the necessity for meticulous experimental control of those variables that modulate the severity of ischemic brain injury.
In an anesthetized canine model in which ischemia was induced by incremental air embolism, 16 animals were exposed to 1 hr of ischemia and monitored for 10 min (n = 4), 60 min (n = 6), or 240 min (n = 6). Fourteen animals were observed for corresponding periods without being subjected to ischemia 70 min (n = 4), 120 min (n = 4), or 300 min (n = 6). Autologous granulocytes were labeled with 111In and reinfused just before ischemia. At the conclusion of each experiment, a 14C-iodoantipyrine autoradiographic blood flow study was performed. Granulocyte accumulation measured by gamma scintigraphy (cpm/gm) occurred in the injured hemisphere of ischemic animals at 60 min in anterior brain segments and at 240 min in anterior, middle, and posterior segments. By means of a double-label autoradiography technique, clustering of punctate granulocyte images was detected in regions of low flow or heterogeneous flow in half of the animals at both 60 min and 240 min postischemia. Granulocyte clustering did not occur in the autoradiograms of nonischemic animals. The results implicate granulocyte participation in the acute phase of ischemic brain injury and signal a convergence of hemostatic and inflammatory processes during the immediate postischemic period.
Polymorphonuclear leukocytes have been implicated in the development of the "no-reflow" phenomenon after focal cerebral ischemia and reperfusion. To further understand the role of granulocytes in microvascular occlusions, the responses of the granulocyte-endothelial cell adhesion molecules P-selectin and intercellular adhesion molecule-1 during middle cerebral artery ischemia and reperfusion were examined in a primate model.
Twelve adolescent male baboons were used for 2-hour middle cerebral artery occlusion (n = 3) or for 3-hour occlusion with 1-hour (n = 3), 4-hour (n = 3), and 24-hour (n = 3) reperfusion, and three separate unoperated primates served as controls. A quantitative immunohistochemical study of the microvascular distribution of P-selectin and intercellular adhesion molecule-1 was performed using 10-microns frozen sections from basal ganglia analyzed with computerized light microscopy video imaging.
Significant (P < .05) persistent upregulation of P-selectin (beginning during ischemia) and transient upregulation of intercellular adhesion molecule-1 (at 1 and 4 hours of reperfusion) were observed on endothelium of selected post-capillary microvessels of the ischemic lenticulostriate artery territory. Platelet accumulation also occurred in this territory and was responsible for a significant proportion of the nonendothelial P-selectin signal at 24 hours after reperfusion.
Focal cerebral ischemia/reperfusion stimulates endothelial P-selectin and intercellular adhesion molecule-1 expression in brain microvessels in the ischemic zone, which may contribute to enhanced leukocyte adherence and persistent activation.
Leukocyte infiltration from circulating blood into ischemic brain tissue contributes significantly to ischemic injury. The role of adhesion molecules in leukocyte attachment and infiltration in ischemic tissue has been emphasized. The aim of the present study was to evaluate whether endothelial-leukocyte adhesion molecule-1 (ELAM-1 or E-selectin) mRNA expression is upregulated in focal brain ischemia.
Northern blot analysis with the use of poly(A) RNA isolated from the ischemic and nonischemic rat cortex at 2 and 12 hours after permanent occlusion of the middle cerebral artery (PMCAO) was used to examine ELAM-1 mRNA expression. The temporal expression profile of ELAM-1 mRNA in the ischemic cortex was further evaluated with the use of a quantitative reverse transcription and polymerase chain reaction technique.
A very low level of ELAM-1 mRNA was detected in the sham-operated cortex or in the nonischemic cortex. The expression of ELAM-1 mRNA in the focal ischemic cortex was significantly induced by PMCAO, reaching a peak level at 12 hours (6.9-fold increase compared with sham surgery cortical samples, P < .01) and remained elevated for up to 2 days (3.3-fold increase, P < .01) after PMCAO.
The demonstration of upregulated ELAM-1 mRNA expression after focal stroke suggests that ELAM-1 may play an important role in leukocyte infiltration into the ischemic brain and that ELAM-1 may provide a potential therapeutic target in ischemic stroke. However, the demonstration of translated ELAM-1 and its cellular localization in the ischemic tissue is required when specific antibodies become available.
The expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) mRNAs was significantly increased in the rat ischemic cortex following temporary occlusion of the middle cerebral artery (TMCAO) with reperfusion. Northern blot analysis demonstrated that the induction of TNF-alpha and IL-1 beta mRNAs occurred as early as 1 h after reperfusion, exhibiting a 4.6-fold increase (p < 0.05, n = 4) and 6.8-fold increase (p < 0.05, n = 4) in the ischemic cortex over control, respectively. TNF-alpha mRNA reached its peak at 3 h (8.0-fold, p < 0.05), whereas IL-1 beta mRNA reached its peak at 6 h (29.5-fold, p < 0.05). Both cytokine mRNA levels remained elevated for up to 2 d after reperfusion. In contrast to the time course of these cytokine mRNAs, c-fos and zif268 mRNAs, two early response genes, displayed a greater and earlier time-response profile. The early induction of c-fos and zif268 mRNAs in temporary brain ischemia with reperfusion suggests their roles in transcriptional regulation. The later concomitant expression of TNF-alpha and IL-1 beta suggests that these cytokines play an important role in the inflammatory response associated with focal ischemia.
Previously we demonstrated that focal cerebral ischemia results in an increased expression of several cytokines/chemokines that precede the infiltration of leukocytes into the ischemic cortex after focal stroke induced by occlusion of the middle cerebral artery (MCAO). Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant specific for monocytes. The aim of the present study was to examine whether MCP-1 messenger RNA (mRNA) is expressed in ischemic brain tissue after MCAO.
The expression of MCP-1 mRNA in the ischemic cortex was first identified by means of a sensitive reverse transcription and polymerase chain reaction technique. The time course of expression of MCP-1 mRNA in the ischemic and nonischemic cerebral cortex after both permanent MCAO and temporary MCAO (160 minutes) with reperfusion was then examined by means of Northern blot analysis.
Almost no expression of MCP-1 mRNA was found in the sham-operated or nonischemic (contralateral) cortex. A significant increase in MCP-1 mRNA expression in the ischemic cortex was observed after either permanent or temporary MCAO. MCP-1 mRNA was elevated at 6 hours (4.4-fold increase over sham; n = 4), reached its highest expression from 12 hours to 2 days (22.7-fold at the peak level; P < .01), and remained elevated up to 5 days (5.6-fold; P < .01) after permanent MCAO. The profile of MCP-1 mRNA expression in the ischemic cortex after MCAO with reperfusion was similar to that of permanent MCAO except that MCP-1 mRNA was increased earlier (ie, 12.5-fold increase at 3 hours; n = 4; P < .01). Also, MCP-1 mRNA expression in the ischemic cortex after permanent MCAO was significantly greater in hypertensive rats than in two normotensive rats (n = 4; P < .05).
The demonstration of induced MCP-1 mRNA expression early after focal ischemia suggests that MCP-1 may represent a locally expressed monocyte chemoattractant that plays an important role in monocyte infiltration into ischemic tissue and therefore may contribute to the tissue injury in ischemic stroke. Further studies must concentrate on identifying the induced expression of MCP-1 and its cellular localization in the ischemic brain when the appropriate antibodies become available.
Transforming growth factor-beta 1 (TGF-beta 1) is a pleiotropic peptide growth factor. The expression of TGF-beta 1 mRNA in the focal ischemic cortex of rats was studied by means of Northern hybridization. A moderately low level of constitutively expressed TGF-beta 1 mRNA was detected following sham-surgery or in the contralateral (nonischemic) cortex. A significant increase of TGF-beta 1 mRNA level in the ischemic cortex was observed at 2 days (3.2-fold increase compared to sham-operated animals, p < 0.01, n = 4) following permanent occlusion of the middle cerebral artery (PMCAO). The elevated TGF-beta 1 mRNA expression was plateaued for up to 15 days (3.6-fold increase, p < 0.01) following PMCAO. This temporal profile for TGF-beta 1 mRNA expression in focal stroke was significantly delayed compared to that of TNF-alpha, IL-1 beta and IL-6 mRNA expressions as demonstrated previously which peaked at 12 h and decreased to almost basal levels by 5 days following PMCAO. Interestingly, the TGF-beta 1 mRNA expression profile was remarkably parallel with that of monocyte/macrophage accumulation in the ischemic cortex, as well as with the increased formation of extracellular matrix in the focal ischemic brain. These data suggest that TGF-beta 1 may play a role in anti-inflammatory process and in tissue remodeling following ischemic brain injury.
From human PBMC stimulated with PHA, we have isolated cDNA clones encoding a novel cytokine named SCM-1, which is significantly related to the CC and the CXC chemokines but has only the 2nd and the 4th of the four cysteines conserved in these proteins. Its gene is also distinctly mapped to human chromosome 1. SCM-1 is strongly induced in human PBMC and Jurkat T cells by PHA stimulation. Among various human tissues, SCM-1 is expressed most strongly in spleen. SCM-1 is found to be 60.5% identical to lymphotactin, a recently described murine lymphocyte-specific chemokine, which also retains only two cysteines. SCM-1 and lymphotactin may thus represent the human and murine prototypes of a novel C or gamma type chemokine family.
Intercellular adhesion molecule-1 (ICAM-1) is a glycoprotein expressed on endothelial cells that facilitates leukocyte adhesion. To test the hypothesis that reduction of leukocytes in an ischemic lesion reduces ischemic brain damage, we measured the effect of administration of an anti-ICAM-1 monoclonal antibody on ischemic brain damage after transient middle cerebral artery occlusion in the rat. ICAM-1 expression increased in the ischemic lesion, and the lesion volume was significantly reduced by 41% in the anti-ICAM-1 antibody group compared with the control group (p < 0.05). Numbers of polymorphonuclear leukocytes (PMNs) were significantly reduced in the cortices of the anti-ICAM-1 antibody group compared with the control animals (p < 0.05). Our data indicate that administration of anti-ICAM-1 antibody results in a significant reduction of ischemic brain damage concomitant with a reduction of PMNs in the lesion after transient focal cerebral ischemia in the rat.
In this study, the cytokine-producing profile of progenitor T cells (pro-T cells) was determined. During screening of a complementary
DNA library generated from activated mouse pro-T cells, a cytokine designated lymphotactin was discovered. Lymphotactin is
similar to members of both the Cys-Cys and Cys-X-Cys chemokine families but lacks two of the four cysteine residues that are
characteristic of the chemokines. Lymphotactin is also expressed in activated CD8+ T cells and CD4-CD8- T cell receptor alpha
beta + thymocytes. It has chemotactic activity for lymphocytes but not for monocytes or neutrophils. The gene encoding lymphotactin
maps to chromosome one. Taken together, these observations suggest that lymphotactin represents a novel addition to the chemokine
superfamily.
Interleukin-8 (IL-8) is a chemokine produced by a variety of cell types involved in atherogenesis and is chemotactic for neutrophils and lymphocytes. A recent study has shown that IL-8 is angiogenic and induces proliferation and chemotaxis of endothelial cells. The present study was undertaken to find out whether IL-8 is also mitogenic and chemotactic for vascular smooth muscle cells. IL-8 induced a concentration-dependent (0.1 to 10 nmol/L) stimulation of DNA synthesis and cell proliferation in both human and rat aortic smooth muscle cells. In addition, IL-8 stimulated smooth muscle cells to produce prostaglandin E2, which can inhibit IL-8-induced smooth muscle cell proliferation. In the presence of indomethacin (5 mumol/L), IL-8 (1 nmol/L) stimulated an increase in human and rat aortic smooth muscle cell number during a 3-day period of incubation by 61 +/- 16% and 59 +/- 7% (n = 4), respectively. IL-8 also increased DNA synthesis in human and rat aortic smooth muscle cells by 98 +/- 10% and 151 +/- 27% (n = 5), respectively. Moreover, IL-8 stimulated rat aortic smooth muscle cell migration by 20-fold over the control value, with an EC50 value of 0.83 nmol/L; this chemotactic activity of IL-8 was also potentiated by indomethacin. Exposure of smooth muscle cells to IL-8 caused rapid and transient expression of the immediate-early genes c-fos and zif268 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine with diverse proinflammatory actions, including endothelial leukocyte adhesion molecule expression. Since leukocytes infiltrate into ischemic brain lesions, the present study was conducted to examine whether TNF-alpha messenger RNA (mRNA) and peptide are expressed in the brain after experimental focal stroke and before leukocyte accumulation.
TNF-alpha mRNA and protein expression were monitored in the ischemic and nonischemic cerebral cortex of rats after focal ischemia produced by permanent middle cerebral artery occlusion. The effect of TNF-alpha administered by microinjection into the brain cortex on leukocyte adherence to brain capillaries was also studied.
Induction of TNF-alpha mRNA, normalized to a standard reference rat macrophage TNF-alpha mRNA, was detected as early as 1 hour after middle cerebral artery occlusion. TNF-alpha mRNA was elevated by 3 hours (29 +/- 6% versus 2 +/- 1% in sham-operated rats) only in the ischemic cortex, with peak expression at 12 hours (104 +/- 8%; P < .01). Five days after middle cerebral artery occlusion, TNF-alpha mRNA levels in ischemic cortex were still significantly elevated (38 +/- 5%; P < .05). Also, TNF-alpha mRNA expression was greater in the ischemic cortex of spontaneously hypertensive rats than in normotensive rats (P < .05). Double-labeling, immunohistochemical studies revealed the presence of TNF-alpha protein localized within nerve fibers in the evolving infarct at 6 and 12 hours after ischemia and further expression in the tissues immediately adjacent to the infarct 24 hours after ischemia. After 5 days, the neuronally localized peptide had diminished greatly, but macrophages located within the infarcted tissues were immunoreactive. Cortical microinjections of TNF-alpha (10 ng in 1 microL) produced a significant neutrophil adherence/accumulation in capillaries and small blood vessels 24 hours later.
These results represent the first demonstration that focal cerebral ischemia in rats results in elevated TNF-alpha mRNA and protein in ischemic neurons. The neuronal expression of peptide appears to facilitate the infiltration of inflammatory cells that can further exacerbate tissue damage in cerebral ischemia and might contribute to increased sensitivity and risk in focal stroke.
Cytokine-induced neutrophil chemoattractant (CINC), originally identified as a chemoattractant in rat kidney epithelial cells, is related to human 'gro' and murine 'KC'. The proteins encoded by these genes belong to the chemokine alpha superfamily, most of which have neutrophil chemotactic activity. Since brain chemokines may play a significant role in neutrophil accumulation in cerebral ischemia which can contribute to the extent of tissue injury in stroke, we examined the expression of CINC mRNA in the cerebral cortex of rats subjected to focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). Significant CINC mRNA expression was observed in the ipsilateral (ischemic) cortex from 6 h (17.3 +/- 3.7%, n = 6, P < 0.05) to 24 h (32.1 +/- 3.7%, n = 5, P < 0.01) with a peak at 12 h (43.9 +/- 3.7%, n = 6, P < 0.01) after MCAO. Five days post-MCAO, CINC mRNA levels were no longer elevated. No significant CINC mRNA expression was observed in the contralateral (control) cortex. These studies suggest that message for the neutrophil chemoattractant CINC is induced early in brain tissue subjected to ischemia, and therefore supports the possibility that brain-derived chemokines support the infiltration of circulating inflammatory cells following focal stroke.
Postischemic cerebral inflammation may contribute to ischemic cell damage. The CD11b/18 (Mac-1) integrin mediates stimulated neutrophil binding to endothelia. We therefore investigated the effect of administration of an anti-Mac-1 monoclonal antibody on cerebral ischemic cell damage in the rat.
Rats (n = 10) were subjected to 2 hours of middle cerebral artery occlusion; the anti-Mac-1 antibody was administered at a dose of 2 mg/kg i.v. at 1 hour of reperfusion and 1 mg/kg i.v. at 22 hours of reperfusion or an isotype-matched control antibody (n = 10) was administered using the same experimental protocol. Rats were killed at 46 hours of reperfusion, and brain sections were stained with hematoxylin and eosin for histological evaluation. In a separate population of rats given either vehicle (n = 8) or anti-Mac-1 antibodies (n = 9), intraparenchymal neutrophils were measured by means of a myeloperoxidase assay.
The lesion volume was significantly smaller (28%) in the anti-Mac-1 antibody group compared with the vehicle control group (P < .01). Numbers of intraparenchymal polymorphonuclear cells were significantly reduced (P < .05) in the cortex of the anti-Mac-1 antibody group compared with the vehicle control group.
Our data demonstrate that administration of anti-Mac-1 antibody 1 hour after onset of reperfusion results in significant reductions of ischemic cell damage and intraparenchymal neutrophils after transient (2-hour) focal cerebral ischemia in the rat.
Interleukin-1 beta is a proinflammatory cytokine produced by blood-borne and resident brain inflammatory cells. The present study was conducted to determine if interleukin-1 beta mRNA was produced in the brain of rats subjected to permanent focal ischemia.
Rat interleukin-1 beta cDNA, synthesized from stimulated rat peritoneal macrophage RNA by reverse transcription and polymerase chain reaction and cloned in plasmid Bluescript KS+, was used to evaluate the expression of interleukin-1 beta mRNA in cerebral cortex from spontaneously hypertensive rats and normotensive rats subjected to permanent middle cerebral artery occlusion. Interleukin-1 beta mRNA was quantified by Northern blot analysis and compared with rat macrophage RNA standard. To correct for gel loading, blots were also analyzed with cyclophilin cDNA, which encodes an abundant, conserved protein that was unchanged by the experimental conditions.
Interleukin-1 beta mRNA produced in the ischemic zone was significantly increased from 6 hours to 120 hours, with a maximum of 211 +/- 24% of interleukin-1 beta reference standard, ie, 0.2 ng stimulated rat macrophage RNA, mRNA compared with the level in nonischemic cortices (4 +/- 2%) at 12 hours after ischemia (P < .01; n = 6). Interleukin-1 beta mRNA at 12 hours after ischemia was markedly elevated in hypertensive rats over levels found in two normotensive rat strains. Neurological deficits were also apparent only in the hypertensive rats.
Brain interleukin-1 beta mRNA is elevated acutely after permanent focal ischemia and especially in hypertensive rats. These data suggest that this potent proinflammatory and procoagulant cytokine might have a role in brain damage following ischemia.
The results of several experimental studies of focal ischemia and anecdotal observations suggest that leukocytes may contribute to the injury initiated by an arterial occlusion. The timing and the nature of leukocyte responses in evolving brain infarcts (either human or experimental) are incompletely characterized. This is a study of experimental brain lesions in 96 Wistar rats that underwent occlusion of a large intracranial artery for variable intervals ranging between 30 minutes and 7 days. The experimental model, based on the occlusion of a middle cerebral artery ostium via the insertion of a nylon monofilament through the external carotid artery, does not require opening the skull; therefore, the inflammatory response is not influenced by the effects of craniotomy and changes in intracranial pressure are only those induced by the ischemic lesion. All 96 animals having the same type of arterial occlusion developed an ischemic brain lesion (limited to the territory of the corresponding artery) that evolved into an area of extensive neuronal necrosis over a period of 6 to 12 hours followed by pan-necrosis (infarct) approximately 60 hours later. In this study, leukocytes (in particular polymorphonuclear cells) were detected in the microvessels (capillaries and venules) of the ischemic hemisphere as early as 30 minutes after the arterial occlusion. Numbers of intravascular neutrophils peaked at 12 hours, whereas intraparenchymal granulocytes were most numerous at 24 hours; a few granulocytes were visible in the brain infarct as late as day 7. Circulating monocytes were first detected within the capillaries/venules of the ischemic area after 4 to 6 hours. Platelet aggregates were more abundant in the arterial than the venous side of the circulation, and luminal obstruction of arteries by platelet aggregates became noticeable only 48 hours after the arterial occlusion. Fibrin thrombi were conspicuous for their absence. These observations provide the background for studies that will attempt to unravel the relationship between the biological responses of leukocytes and neuronal necrosis secondary to focal ischemia.
IFN-gamma is a potent macrophage activator and induces a number of early and delayed genes. crg-2, the presumed murine homologue of human IP-10, belongs to a family of proinflammatory chemokines and is induced as an immediate early gene in response to IFN-gamma in macrophages. In contrast, class II MHC or Ia genes which are essential for Ag presentation are induced as a delayed response to IFN-gamma. We studied the expression of crg-2 and compared it with Ia in astrocytes and microglia of the central nervous system since, like macrophages, these cells can also produce a number of cytokines, express Ia molecules, and present Ag. We showed that crg-2 mRNA was induced in astrocytes and microglia by IFN-gamma as well as a paramyxovirus, Newcastle disease virus (NDV). Crg-2 protein was detected in the cytoplasm and in the supernatants of IFN-gamma-treated astrocytes and microglia. IFN-gamma and NDV or UV irradiated-NDV (UV-NDV) also induced Ia mRNA in these cells. The kinetics of expression of crg-2 and Ia mRNA were compared in the same systems. While crg-2 mRNA appeared within 2 h and reached a maximum in 6 to 8 h, Ia mRNA was not detected before 8 h. Cycloheximide superinduced crg-2 mRNA induced by IFN-gamma or UV-NDV but it abolished Ia mRNA induction by the same stimuli. The induction of crg-2 in astrocytes and microglia likely contributes to the development of immune-mediated inflammation in response to viruses or in autoimmune diseases of the central nervous system.
The dynamics of leukocyte infiltration in human cerebral ischemia were studied using technetium-99m hexamethylpropyleneamine oxime (99mTc HMPAO)-labeled leukocyte brain single-photon emission computed tomography (SPECT).
Twenty-two patients diagnosed as having hemispheric ischemic stroke were examined with 99mTc HMPAO brain SPECT for cerebral blood flow study and 99mTc HMPAO-labeled leukocyte brain SPECT for the study of leukocyte infiltration. Three patients with chronic hemispheric ischemic stroke received one examination. Nineteen patients with acute hemispheric ischemic stroke received their initial examination within the first after onset. Follow-up examinations were performed at intervals of 1-3 weeks whenever possible.
In patients with chronic hemispheric ischemic stroke, leukocyte infiltration was not seen in areas of perfusion defect. In patients with acute hemispheric ischemic stroke, leukocyte infiltration was seen in areas of perfusion defect during the acute stage, which persisted for no less than 5 weeks after onset and then declined.
A new method to study and monitor the process of leukocyte infiltration in acute cerebral ischemia using 99mTc HMPAO-labeled leukocyte brain SPECT is described. This method shows that leukocyte infiltration in acute hemispheric ischemic stroke is a dynamic process that persists for no less than 5 weeks and then declines.
In response to interleukin 1 or tumor necrosis factor, human synovial cells and fibroblasts expressed several genes encoding known chemotactic factors or related proteins. Transcripts for interleukin 8 (IL-8), gro/MGSA, pAT 464, IP-10, pAT 744 and Monocyte Chemotactic and Activating Factor (MCAF) accumulated rapidly in IL-1 and TNF-treated cells. The inhibition of protein synthesis led to the superinduction of IL-8 and gro/MGSA mRNAs in IL-1, but not in TNF-treated cells. Thus, IL-1 and TNF are likely to regulate the expression of these mRNAs by different mechanisms. Important cell-specific differences in mRNA accumulation characterized the expression of chemotactic factor genes. Moreover, only a subset of the same genes was activated in quiescent cells stimulated by serum. Therefore, genes encoding closely related proteins each had a distinct pattern of expression. continuous stimulation of fibroblasts and synovial cells with IL-1 resulted in high and prolonged expression of IL-8 and gro/MGSA mRNAs. These results extend the list of chemotactic factor genes expressed by mesenchymal cells in vitro and suggest a pivotal role for these cells in processes such as chronic inflammation.
Development and resolution of the lesion produced by occlusion of the middle cerebral artery (MCAO) was studied through quantitative planimetry and histologic/immunohistochemical techniques. MCAO, performed in spontaneously hypertensive rats (SHR), initially (1-3 days) produced large, consistent cerebral cortical infarctions and an increase in ipsilateral hemispheric size (i.e., swelling) quantitated by planimetry on 2,3,5-triphenyltetrazolium chloride (TTC)-stained gross tissue sections. These initial changes correlated well with changes identified from 2 h to 3 days using hematoxylin and eosin stained histologic tissue sections and immunohistochemical techniques including: the progressive development of a cortical area of pan necrosis, infiltration of neutrophils into infarcted tissues, and activation of astroglia. During the initial 2 days following MCAO, glial fibrillary acidic protein immunoreactive cells increased in number and became larger and more intensely fluorescent medial to the cortical infarct. At 5 to 15 days, both the infarct and the ipsilateral hemisphere decreased in size. These changes correlated with the presence of abundant macrophages, and cavitation of the lesion along its medial border. Also during this period, a loose connective tissue matrix formed along the superficial aspect of the infarct. This connective tissue contained fibroblasts, extracellular matrix immunoreactive for laminin and collagen, capillary buds indicating neovascularization, and abundant macrophages. By the final timepoint (30 days), necrotic tissue could no longer be detected in either gross or histologic tissue sections, the inflammatory infiltrate had resolved, and the connective tissue was removed.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute neutrophil (PMN) recruitment to postischemic cardiac or pulmonary tissue has deleterious effects in the early reperfusion period, but the mechanisms and effects of neutrophil influx in the pathogenesis of evolving stroke remain controversial. To investigate whether PMNs contribute to adverse neurologic sequelae and mortality after stroke, and to study the potential role of the leukocyte adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of stroke, we used a murine model of transient focal cerebral ischemia consisting of intraluminal middle cerebral artery occlusion for 45 min followed by 22 h of reperfusion. PMN accumulation, monitored by deposition of 111In-labeled PMNs in postischemic cerebral tissue, was increased 2.5-fold in the ipsilateral (infarcted) hemisphere compared with the contralateral (noninfarcted) hemisphere (P < 0.01). Mice immunodepleted of neutrophils before surgery demonstrated a 3.0-fold reduction in infarct volumes (P < 0.001), based on triphenyltetrazolium chloride staining of serial cerebral sections, improved ipsilateral cortical cerebral blood flow (measured by laser Doppler), and reduced neurological deficit compared with controls. In wild-type mice subjected to 45 min of ischemia followed by 22 h of reperfusion, ICAM-1 mRNA was increased in the ipsilateral hemisphere, with immunohistochemistry localizing increased ICAM-1 expression on cerebral microvascular endothelium. The role of ICAM-1 expression in stroke was investigated in homozygous null ICAM-1 mice (ICAM-1 -/-) in comparison with wild-type controls (ICAM-1 +/+). ICAM-1 -/- mice demonstrated a 3.7-fold reduction in infarct volume (P < 0.005), a 35% increase in survival (P < 0.05), and reduced neurologic deficit compared with ICAM-1 +/+ controls. Cerebral blood flow to the infarcted hemisphere was 3.1-fold greater in ICAM-1 -/- mice compared with ICAM-1 +/+ controls (P < 0.01), suggesting an important role for ICAM-1 in the genesis of postischemic cerebral no-reflow. Because PMN-depleted and ICAM-1-deficient mice are relatively resistant to cerebral ischemia-reperfusion injury, these studies suggest an important role for ICAM-1-mediated PMN adhesion in the pathophysiology of evolving stroke.
Interferon-inducible protein-10 (IP-10) is a member of the C-X-C chemokine family. Using mRNA differential display, we isolated a rat homologue to murine and human IP-10 from lipopolysaccharide-stimulated carotid arteries. Our studies demonstrated that IP-10 is a potent mitogenic and chemotactic factor for vascular smooth muscle cells, the critical features of smooth muscle cells for their contribution to the pathogenesis of atherosclerosis and restenosis. IP-10 induced a concentration-dependent stimulation of DNA synthesis, cell proliferation, and cell migration of rat aortic smooth muscle cells. A concentration- and time-dependent IP-10 mRNA induction was observed in lipopolysaccharide- or interferon-gamma-stimulated, but not interleukin-1beta- or tumor necrosis factor-alpha-stimulated smooth muscle cells. A marked synergistic effect on IP-10 mRNA expression was observed when smooth muscle cells were challenged with interferon-gamma together with interleukin-1beta or tumor necrosis factor-alpha. Furthermore, IP-10 mRNA expression was induced in the rat carotid artery after balloon angioplasty. The mitogenic and chemotactic features of IP-10 for smooth muscle cells, along with its discrete induction in cultured vascular smooth muscle cells and in carotid arteries after balloon angioplasty (neointima formation) suggest that IP-10 may play an active and distinct role in vascular remodeling processes.
Oligodendrocyte responses in vitro to platelet-derived growth factor (PDGF) include proliferation, survival, migration, and changes in cell morphology and molecular expression. Studies of mixed glial cultures established that astrocytes secrete PDGF; thus astrocytes are considered to be key regulators of oligodendrocyte development in vitro. We previously demonstrated PDGF alpha receptor mRNA expression by oligodendrocyte progenitors and preoligodendrocytes during postnatal development of rat cerebral cortex. In the present study, we have mapped the spatial and temporal expression of PDGF A-chain ligand mRNA and alpha receptor mRNA to determine if the cell-cell interactions that form the basis for PDGF regulation of oligodendrocyte development in vitro are also present in vivo. By in situ hybridization (ISH) we demonstrate that at embryonic day 17 (E17) cells expressing receptor mRNA (PDGFR alpha +) are initially in the subventricular zone, at a distance from cells expressing ligand mRNA (PDGF+) in the cortical plate. By E20 PDGFR alpha + cells are found throughout the corpus callosum and cortical gray matter. PDGF+ cells are restricted to the cortical plate prenatally and only appeared in the corpus callosum postnatally. Combined immunocytochemistry and ISH demonstrated the PDGF+ cells colocalized with neurofilament, but not with GFAP. These data establish that PDGF is expressed by neurons during PDGFR alpha + oligodendrocyte progenitor migration from the subventricular zone to the corpus callosum and gray matter. Furthermore, neurons continue to express PDGF during the generation and differentiation of appropriate numbers of oligodendrocytes needed to myelinate axons as the nervous system matures.