Chemokines/chemokine receptors in the central nervous system and Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, and the fourth leading cause of death in the United States. Its pathological changes include amyloid beta deposits, neurofibrillary tangles and a variety of 'inflammatory' phenomenon such as activation of microglia and astrocytes. The pathological significance of inflammatory responses elicited by resident central nervous system (CNS) cells has drawn considerable attention in recent years. Chemokines belongs to a rapidly expanding family of cytokines, the primary function of which is control of the correct positioning of cells in tissues and recruitment of leukocytes to the site of inflammation. Study of this very important class of inflammatory cytokines may greatly help our understanding of inflammation in the progress of AD, as well as other neurodegenerative diseases. So far, immunoreactivity for a number of chemokines (including IL-8, IP-10, MIP-1beta, MIPalpha and MCP-1) and chemokine receptors (including CXCR2, CXCR3, CXCR4, CCR3, CCR5 and Duffy antigen) have been demonstrated in resident cells of the CNS, and upregulation of some of the chemokines and receptors are found associated with AD pathological changes. In this review, we summarize findings regarding the expression of chemokines and their receptors by CNS cells under physiological and pathological conditions. Although little is known about the potential pathophysiological roles of chemokines in CNS, we have put forward hypotheses on how chemokines may be involved in AD.     

Effects of pro-inflammatory cytokines in experimental spinal cord injury

Following injury to the spinal cord, secondary tissue damage leading to massive additional tissue loss and inflammatory reactions as well as scar formation takes place. The precise functions and effects of the inflammatory cells and their secreted factors are largely unclear. The present study investigates whether the exogenous local administration of pro-inflammatory cytokines to mice after spinal cord injury can influence these intrinsic processes. A mixture of murine recombinant interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor α (TNFα) was administered to the lesioned spinal cord of adult mice. These cytokines provoked an increased recruitment and activation of macrophages and microglial cells in the lesion area when administered 1 day post lesion. In contrast, when administered 4 days after the lesion, recruitment of macrophages was slightly increased while activation of microglia was decreased as compared to controls. The amount of tissue loss 7 days after trauma was smaller in the animals receiving the cytokine mixture than in the mice receiving Ringer control solution on day 4 after lesion. Thus the role of the inflammatory response in spinal cord injury seems to be complex and well regulated. Anti-inflammatory cytokines and factors probably also contribute to the outcome of the damage following injury to the spinal cord.     

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

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

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit chemokine production in activated microglia

Microglia react to even minor disturbances in CNS homeostasis and function as critical regulators of CNS inflammation. Activated microglia secrete inflammatory mediators such as cytokines and chemokines, which contribute to the pathophysiological changes associated with several neuroimmunologic disorders. Microglia-derived inflammatory chemokines recruit various populations of immune cells, which initiate and maintain the inflammatory response against foreign antigens. Entry and retention of activated immune cells in the CNS is a common denominator in a variety of traumatic, ischemic, and degenerative diseases. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two structurally related neuropeptides that function as potent anti-inflammatory factors in the periphery. Here we investigated the effects of VIP and PACAP on chemokine production by activated microglia. VIP and PACAP inhibit the expression of the microglia-derived CXC chemokines MIP-2 and KC, and of the CC chemokines MIP-1alpha, -1beta, MCP-1, and RANTES. The inhibition of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NFkB binding. The VIP/PACAP inhibition of both chemokine production and of NFkB binding is mediated through the specific receptor VPAC1 and involves a cAMP-dependent intracellular pathway. Of biological significance is the fact that the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the chemotactic activity generated by activated microglia for peripheral leukocytes, i.e., neutrophils, macrophages, and lymphocytes. Because reduction in the number and activation of infiltrating leukocytes represents an important factor in the control of inflammation in the CNS, VIP and/or PACAP released by neurons during an inflammatory response could serve as neuronal survival factors by limiting the inflammatory process.     

Astrocytes initiate inflammation in the injured mouse spinal cord by promoting the entry of neutrophils and inflammatory monocytes in an IL-1 receptor/MyD88-dependent fashion

CNS injury stimulates the expression of several proinflammatory cytokines and chemokines, some of which including MCP-1 (also known as CCL2), KC (CXCL1), and MIP-2 (CXCL2) act to recruit Gr-1⁺ leukocytes at lesion sites. While earlier studies have reported that neutrophils and monocytes/macrophages contribute to secondary tissue loss after spinal cord injury (SCI), recent work has shown that depletion of Gr-1⁺ leukocytes compromised tissue healing and worsened functional recovery. Here, we demonstrate that astrocytes distributed throughout the spinal cord initially contribute to early neuroinflammation by rapidly synthesizing MCP-1, KC, and MIP-2, from 3 up to 12 h post-SCI. Chemokine expression by astrocytes was followed by the infiltration of blood-derived immune cells, such as type I “inflammatory” monocytes and neutrophils, into the lesion site and nearby damaged areas. Interestingly, astrocytes from mice deficient in MyD88 signaling produced significantly less MCP-1 and MIP-2 and were unable to synthesize KC. Analysis of the contribution of MyD88-dependent receptors revealed that the astrocytic expression of MCP-1, KC, and MIP-2 was mediated by the IL-1 receptor (IL-1R1), and not by TLR2 or TLR4. Flow cytometry analysis of cells recovered from the spinal cord of MyD88- and IL-1R1-knockout mice confirmed the presence of significantly fewer type I “inflammatory” monocytes and the almost complete absence of neutrophils at 12 h and 4 days post-SCI. Together, these results indicate that MyD88/IL-1R1 signals regulate the entry of neutrophils and, to a lesser extent, type I “inflammatory” monocytes at sites of SCI.     

Effects of cytokine deficiency on chemokine expression in CNS of mice with EAE

Although both cytokines and chemokines have been implicated in the pathogenesis of clinical and histological EAE, their interactions in vivo have not yet been clearly established. To address this issue, we evaluated expression of chemokines and receptors in the CNS of wild-type control and cytokine deficient mice at the peak of EAE induced with MOG-35-55 peptide in CFA. Our results demonstrate that: 1) expression of most chemokines/receptors was drastically inhibited in TNF-alpha deficient mice, and was reflective of delayed onset and reduced severity of EAE; 2) distinct patterns of chemokine expression occurred in various other cytokine knockout mice that did not significantly affect expression of clinical EAE; 3) there was a strong association between expression of MIP-1alpha, MIP-2 and MCP-1 in CNS and overall severity of EAE in wild-type and cytokine knockout mice; and 4) among CNS infiltrating cells at the peak of EAE, macrophages and CD8+ T cells were the primary cellular source of most of the chemokines. Of note, we present evidence that TNF-alpha may be involved in regulating RANTES and MIP-1alpha, and that IL-4 may be involved in regulating MCP-1. Our results not only identify the cellular source of chemokines in CNS, but also implicate MIP-1alpha, MIP-2, and MCP-1 in controlling CNS inflammation and severity of EAE.     

Chemokine antagonist infusion attenuates cellular infiltration following spinal cord contusion injury in rat

Spinal cord injury is accompanied by an initial inflammatory reaction followed by secondary injury that is caused, in part, by apoptosis. Recruitment of leukocytes from the blood compartment to the site of inflammation in the injured spinal cord has been attributed to locally generated chemotactic agents (cytokines and chemokines). In addition to upregulation in the message levels of a number of chemokines, we have found up-regulation in the message levels of several chemokine receptors following spinal cord contusion injury. To reduce the inflammatory response after spinal cord injury, we have blocked the interaction of chemokine receptors with their ligands using the vMIPII chemokine antagonist. Using a rat model of spinal cord contusion injury, we show that continuous infusion of the antagonist for up to 7 days results in a decrease in infiltrating hematogenous cells at the site of injury. Histological evaluation ofthe tissue showed fewer activated macrophages at the site of injury. Concomitantly, reduced neuronal loss and gliosis were observed in the antagonist infused spinal cord. In addition, increased expression of Bcl-2 gene, an endogenous inhibitor of apoptosis, was seen in the antagonist infused spinal cord at 7 days post injury. Morphologically, staining with the bisbenzamide dye Hoechst 33342 showed significantly more apoptotic bodies in the vehicle compared to antagonist infused spinal cord. Our data suggest that chemokine antagonist infusion post-injury results in limiting the inflammatory response following spinal cord contusion injury, thereby attenuating neuronal loss, possibly due to decreased apoptosis. These findings support the contention that disrupting chemokine interactions with their receptors may be an effective approach in reducing the secondary damage after spinal cord injury.     

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

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

Similar pattern of MCP-1 expression in spinal cords and eyes of Lewis rats with experimental autoimmune encephalomyelitis associated anterior uveitis

Monocyte chemoattractant protein-1 (MCP-1) is a member of the CC chemokine family responsible for the recruitment of T cells that have been found during inflammation of the spinal cord in experimental autoimmune encephalomyelitis (EAE) in Lewis rats immunized with myelin basic protein (MBP). Lewis rats injected with MBP also developed anterior uveitis (AU), which coincided with the onset of EAE. In the present studies, we examined the expression and distribution of MCP-1 in the eye and spinal cord during disease and compared it to the expression of Th1 cell type cytokines. Initially, MCP-1 expression was detected at the preclinical phase in the iris/ciliary body and lumbar spinal cord and increased during the course of EAE/AU. Mononuclear infiltrating cells and endothelial cells and astrocytes of the CNS could be identified as a source of MCP-1 by in situ hybridization. Kinetics of expression of Th1 characteristic cytokines such as IL-2 and IFNγ was in agreement with the expression of MCP-1 chemokine. Moreover, induction of the gene expression of MCP-1 seemed to occur earlier than that of MIP-2, and it correlated with increasing disease severity. MCP-1 seems to contribute to the initial recruitment of inflammatory cells into both the tissues of the eye and CNS over the course of disease. J. Neurosci. Res. 50:531–538, 1997. © 1997 Wiley-Liss, Inc.     

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

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

Microglial voltage-gated proton channel Hv1 in spinal cord injury

After spinal cord injury,microglia as the first responders to the lesion display both beneficial and detrimental characteristics.Activated microglia phagocyte and eliminate cell debris,release cytokines to recruit peripheral immune cells to the injury site.Excessively activated microglia can aggravate the secondary damage by producing extravagant reactive oxygen species and pro-inflammatory cytokines.Recent studies demonstrated that the voltage-gated proton channel Hv1 is selectively expressed in microglia and regulates microglial activation upon injury.In mouse models of spinal cord injury,Hv1 deficiency ameliorates microglia activation,resulting in alleviated production of reactive oxygen species and pro-inflammatory cytokines.The reduced secondary damage subsequently decreases neuronal loss and correlates with improved locomotor recovery.This review provides a brief historical perspective of advances in investigating voltage-gated proton channel Hv1 and home in on microglial Hv1.We discuss recent studies on the roles of Hv1 activation in pathophysiological activities of microglia,such as production of NOX-dependent reactive oxygen species,microglia polarization,and tissue acidosis,particularly in the context of spinal cord injury.Further,we highlight the rationale for targeting Hv1 for the treatment of spinal cord injury and related disorders.     

Tumor necrosis factor α and transforming growth factor β upregulate astrocyte expression of monocyte chemoattractant protein-1

Astrocytes participate in the pathophysiology of central nervous system (CNS) inflammatory disease. Astrocyte expression of adhesion molecules, cytokines, and major histocompatibility complex antigens may contribute to these inflammatory processes. In addition, recent data suggested that astrocytes may be a source of monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a member of the chemokine family of small cytokines and functions both as a chemoattractant as well as a stimulator of monocytes. To further characterize the role of astrocytes in CNS inflammation, we examined the effect of inflammatory cytokines on MCP-1 expression by astrocytes. Results of these studies demonstrate that the pro-inflammatory cytokine tumor necrosis factor alpha (TNFa) upregulates MCP-1 message and protein expression. The pleiotropic cytokine transforming growth factor beta (TGFβ) also stimulated MCP-1 expression. When astrocytes were exposed to both cytokines simultaneously, an additive effect on MCP-1 message, but not MCP-1 protein expression, was observed. These data suggest that TNFa and TGFβ, each present during CNS inflammatory disease, may upregulate the expression of MCP-1 which, in turn, may function to both recruit monocytes to the site of inflammation as well as to activate those monocytes already present in an inflammatory lesion.     

Local expression of cytokines in idiopathic inflammatory myopathies

H. Lepidi, V. Frances, D. Figarella-Branger, C. Bartoli, A. Machado-Baeta & J-F. Pellissier (1998) Neuropathology and Applied Biology , 24, 73–79
Local expression of cytokines in idiopathic inflammatory myopathies
The idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM), are regarded as autoimmune diseases. They are characterized by chronic lymphocytic and macrophagic infiltration in muscle tissue. Of particular importance in understanding the immune response to IIM is the specific pattern of locally produced cytokines. Frozen muscle tissues from IIM (5 DM, 3 PM, and 1 IBM) were used to investigate the cytokine responses. The RT-PCR technique was instrumental to determine the pattern of expression of pro-inflammatory (IL-1β, IL-6, TNF-α), Th1 (IFN-γ IL-2), and Th2 (IL-4) cytokines. Immunohistochemistry was also used to localize morphologically IFN-γ and IL-4. Our results show that pro-inflammatory cytokines and Th1 cytokines are mainly expressed in IIM. The accumulation of mononuclear inflammatory cells and the inflammatory syndrome in IIM are probably related in part to the production of pro-inflammatory cytokines. Moreover, the pattern of local cytokine expression is consistent with a Th1 immune response related to autoimmune diseases.     

Induction of human immunodeficiency virus type 1 replication in human glial cells after proinflammatory cytokines stimulation: Effect of IFNγ, IL1β, and TNFα on differentiation and chemokine production in glial cells

Although evidence for human immunodeficiency virus 1 (HIV-1) presence in the central nervous system (CNS) of infected patients is well established, the intensity of viral replication within the brain is not usually known. In vitro, human embryonic microglial cells internalized HIV-1 through a CD4-dependent pathway but were not permissive to viral replication. We observed that HIV replication was induced when CNS cell cultures were stimulated for 14 days by a combination of proinflammatory cytokines including IFNγ, IL1β, and TNFα. After long-term cytokine stimulation, morphologically differentiated glial cells appeared, in which HIV-1 tat antigen was detected after infection. Thus, variations in the stage of maturation/activation of CNS cells under inflammatory conditions probably play a major role in facilitating massive production of HIV-1. We then studied the effect of prolonged cytokine stimulation on the secretion of inflammatory mediators by glial cells. An early increased secretion of prostaglandin F2α and chemokines (RANTES>>MIP-1α>>MIP-1β) was observed, due to both microglia and astrocytes. In contrast to persistent PGF2α production, an extinction of RANTES and MIP-1β but not of MIP-1α secretion occurred during the 14 days of stimulation and was inversely correlated with the ability of glial cells to replicate HIV-1. The study of the secretory factors produced in response to a persistent inflammation could provide a better understanding of the modulation of HIV replication in glial cells. GLIA 23:304–315, 1998. © 1998 Wiley-Liss, Inc.     

Astrocyte-T cell crosstalk regulates region-specific neuroinflammation

During multiple sclerosis (MS), an inflammatory and neurodegenerative disease of the central nervous system (CNS), symptoms, and outcomes are determined by the location of inflammatory lesions. While we and others have shown that T cell cytokines differentially regulate leukocyte entry into perivascular spaces and regional parenchymal localization in murine models of MS, the molecular mechanisms of this latter process are poorly understood. Here, we demonstrate that astrocytes exhibit region-specific responses to T cell cytokines that promote hindbrain versus spinal cord neuroinflammation. Analysis of cytokine receptor expression in human astrocytes showed region-specific responsiveness to Th1 and Th17 inflammatory cytokines. Consistent with this, human and murine astrocytes treated with these cytokines exhibit differential expression of the T cell localizing molecules VCAM-1 and CXCR7 that is both cytokine and CNS region-specific. Using in vivo models of spinal cord versus brain stem trafficking of myelin-specific T cells and astrocyte-specific deletion strategies, we confirmed that Th1 and Th17 cytokines differentially regulate astrocyte expression of VCAM-1 and CXCR7 in these locations. Finally, stereotaxic injection of individual cytokines into the hindbrain or spinal cord revealed region- and cytokine-specific modulation of localizing cue expression by astrocytes. These findings identify a role for inflammatory cytokines in mediating local astrocyte-dependent mechanisms of immune cell trafficking within the CNS during neuroinflammation.     

Molecular mechanisms involved in T cell migration across the blood–brain barrier

Summary. In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood–brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood–spinal cord and blood–brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that α4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood–brain and blood–spinal cord barrier based on our in vitro and in vivo investigations.     

Prior exposure to repeated morphine potentiates mechanical allodynia induced by peripheral inflammation and neuropathy

Opioids, such as morphine, induce potent analgesia and are the gold standard for the treatment of acute pain. However, opioids also activate glia, inducing pro-inflammatory cytokine and chemokine production, which counter-regulates the analgesic properties of classical opioid receptor activation. It is not known how long these adverse pro-inflammatory effects last or whether prior morphine could sensitize the central nervous system (CNS) such that responses to a subsequent injury/inflammation would be exacerbated. Here, multiple models of inflammation or injury were induced two days after morphine (5 mg/kg b.i.d., five days , s.c.) to test the generality of morphine sensitization of later pain. Prior repeated morphine potentiated the duration of allodynia from peripheral inflammatory challenges (complete Freund’s adjuvant (CFA) into either hind paw skin or masseter muscle) and from peripheral neuropathy (mild chronic constriction injury (CCI) of the sciatic nerve). Spinal cord and trigeminal nucleus caudalis mRNAs were analyzed to identify whether repeated morphine was sufficient to alter CNS expression of pro-inflammatory response genes, measured two days after cessation of treatment. Prior morphine elevated IL-1β mRNA at both sites, MHC-II and TLR4 in the trigeminal nucleus caudalis but not spinal cord, but not glial activation markers at either site. Finally, in order to identify whether morphine sensitized pro-inflammatory cytokine release, spinal cord was isolated two days after morphine dosing for five days , and slices stimulated ex vivo with lipopolysaccharide. The morphine significantly induced TNFα protein release. Therefore, repeated morphine is able to sensitize subsequent CNS responses to immune challenges.     

MIP-1alpha and MCP-1 Induce Migration of Human Umbilical Cord Blood Cells in Models of Stroke

Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein (MIP-1alpha) are implicated in monocyte infiltration into the central nervous system (CNS) under pathological conditions. We previously showed that in vivo human umbilical cord blood cells (HUCB) migrate toward brain injury after middle cerebral artery occlusion (MCAO). We hypothesized that MCP-1 and MIP-1alpha may participate in the recruitment of HUCB towards the injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), and 24 hours later the production of MCP-1 and MIP-1alpha in the brain was examined with immunohistochemistry, ELISA, and western blotting. The chemotactic effect of MCP-1 and MIP-1alpha, and the expression of MCP-1 receptor CCR2 and MIP-1alpha receptor CCR1, CCR5 on the surface of HUCB were also examined. MCP-1 and MIP-1alpha expression were significantly increased in the ischemic hemisphere of brain, and significantly promoted HUCB cell migration compared to the contralateral side. This cell migration was neutralized with polyclonal antibodies against MCP-1 or MIP-1alpha. Also chemokine receptors were constitutively expressed on the surface of HUCB cells. The data suggested that the increased chemokines in the ischemic area can bind cell surface receptors on HUCB, and induce cell infiltration of systemically delivered HUCB cells into the CNS in vivo.