Upregulation of erbB receptors in rat brain after middle cerebral arterial occlusion

We have previously demonstrated that neuregulin-1 (NRG-1) is upregulated and is neuroprotective in ischemic brain injury, however the expression and localization of its receptors during ischemia has not been investigated. Therefore, we used a rat middle cerebral artery occlusion (MCAO) model to examine the distribution of erbB receptors following ischemic stroke. Like neuregulin-1, we observed a dramatic induction of erbB4 in the peri-infarct regions of the ipsilateral cortex 24 h following MCAO. Using Fluoro-Jade B (FJB) staining as a marker of neurodegeneration, erbB4 was upregulated in FJB-positive cells, suggesting that erbB receptors are induced in injured neurons. The increase in erbB receptors was seen in neurons and a subpopulation of macrophages/microglia. There was no erbB co-localization with GFAP-positive astrocytes. These results demonstrate that erbB receptors are upregulated in neurons and macrophages/microglia following ischemic stroke and may be involved in neuroprotection and repair.     

Cannabinoid receptor CB2 is expressed on vascular cells,but not astroglial cells in the post-mortem human Huntington's disease brain

Huntington's disease (HD) is an inherited neurological disease with motor, cognitive and psychiatric symptoms. Characterised by neuronal degeneration, HD pathology is initially apparent in the striatum and cortex. Considerable research has recently suggested that the neurological immune response apparent in brain injury and disease may provide a valuable therapeutic target. Cannabinoid CB2 receptors are localised and up-regulated on a number of peripheral immune cell types following inflammation and injury. However, their cellular location within the human brain during inflammation has not been well characterised.The present study shows CB2 is expressed in human post-mortem striatum in HD. Quantification revealed a trend towards an increase in CB2 staining with disease, but no significant difference was measured compared to neurologically normal controls. In HD striatal tissue, there is an up-regulation of the brains' resident immune cells, with a significant increase in GFAP-positive astrocyte staining at both grade 1 (685 ± 118%) and grade 3 (1145 ± 163%) and Iba1-positive microglia at grade 1 (299 ± 27%) but not grade 3 (119 ± 48%), compared to neurologically normal controls. Both cell types exhibit irregular cell morphology, particularly at higher grades.Using double-labelled immunohistochemistry CB2 receptors are demonstrated not to be expressed on microglia or astrocytes and instead appear to be localised on CD31-positive blood vessel endothelium and vascular smooth muscle. Co-expression analysis suggests that CB2 may be more highly expressed on CD31 positive cells in HD brains than in control brains. Contrasting with evidence from rodent studies suggesting CB2 glial cell localisation, our observation that CB2 is present on blood vessel cells, with increased CD31 co-localisation in HD may represent a new context for CB2 therapeutic approaches to neurodegenerative diseases.     

Cannabinoid receptors in microglia of the central nervous system: immune functional relevance

Microglia, resident macrophages of the brain, function as immune effector and accessory cells. Paradoxically, they not only play a role in host defense and tissue repair but also have been implicated in a variety of neuropathological processes. Microglia, in addition to exhibiting phenotypic markers for macrophages, express CB1 and CB2 cannabinoid receptors. Recent studies suggest the existence of a third, yet-to-be cloned, non-CB1, non-CB2 cannabinoid receptor. These receptors appear to be functionally relevant within defined windows of microglial activation state and have been implicated as linked to cannabinoid modulation of chemokine and cytokine expression. The recognition that microglia express cannabinoid receptors and that their activation results in modulation of select cellular activities suggests that they may be amenable to therapeutic manipulation for ablating untoward inflammatory responses in the central nervous system.     

In vivo responses of macrophages and myofibroblasts in the healing following isoproterenol-induced myocardial injury in rats

To clarify the relation between macrophage and myofibroblast involvement in various myocardial diseases, the authors investigated the kinetics of these cells in the healing (scar tissue formation) following isoproterenol-induced myocardial injury in rats. Alphasmooth muscle actin (-SMA) expressing myofibroblasts were seen at the border of the affected area and appeared in the greatest numbers on days 3–7 post-injection, followed by a gradual decrease by day 35. The peak on day 3 was consistent with the timing of the highest proliferative activity of myofibroblasts. The number of ED1-positive macrophages began to increase as early as day 1, reaching a peak on day 3 within the injured myocardium. The expansion of EDI-positive macrophages preceded an increased number of -SMA-positive myofibroblasts suggesting that myofibroblast proliferation and activation may be mediated by factors released by ED1-positive mcrophages in response to myocardial injury. The number of ED2-positive tissue-fixed, resident macrophages gradually increased from day 3 post-injection, and peaked on day 14, but the number of ED2-positive macrophages was consistently fewer than that of ED1-positive macrophages during the 35 day-observation period after the injection. The labelling index of the ED2-positive cells was maximal on day 14, indicative of local proliferation of resident macrophages. In the healing process after myocardial injury, EDI-positive macrophages increase markedly in the early stages; ED2-positive macrophages appear later.     

Expression of the cannabinoid CB2 receptor in the rat cerebellum: an immunohistochemical study

Reports of cannabinoid CB2 receptor protein in the brain have been ambiguous. We therefore tested for CB2 immunoreactivity in the rat brain using immunofluorescence. We detected CB2 labeling in fine fibers in the granule layer. This CB2 labeling did not co-localise with the astrocyte marker glial fibrillary acidic protein (GFAP) and, therefore, the CB2-positive fibers were not astrocytes and were possibly microglial or neuronal. Additionally, strong CB2 labeling was detected in capillary endothelia in the granule, Purkinje cell, and molecular layers. Our results suggest that the role of CB2 receptors in the brain may have been previously underestimated.     

Cannabinoid receptor and N-acyl phosphatidylethanolamine phospholipase D--evidence for altered expression in multiple sclerosis

Cannabinoids have been shown to have a beneficial effect in both animal models of multiple sclerosis (MS) and human disease, although the mechanisms of action are unclear. We examined expression of the major cannabinoid receptors [(CBRs) cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2)] and a key enzyme involved in synthesis of the endocannabinoid anandamide [N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] in autopsy brain samples from patients with MS. CB1 was expressed in neurons, injured axons, oligodendrocytes, macrophages/microglia, some astrocytes, endothelial cells, smooth muscle cells and pericytes. CB2 and NAPE-PLD were localized to cerebral endothelial cells, pericytes, smooth muscle cells, astrocytes and macrophages/microglia. NAPE-PLD immunoreactivity was also seen in neurons. Endothelial CB2 expression was greatest in chronic inactive plaques, and in areas was seen in segments of endothelium where the endothelial expression of adhesion molecules (VCAM-1 and ICAM-1) was focally undetectable, and was often expressed in areas of blood-brain barrier damage. Vascular density was increased in chronic active plaques and normal-appearing white matter compared with controls. These data support findings from animal models which suggest a role for the endocannabinoid system in the MS, particularly in the regulation of endothelial leukocyte adhesion and the cellular response to injury.     

Clinical implications of the involvement of tPA in neuronal cell death

 Tissue plasminogen activator (tPA), the serine protease that converts inactive plasminogen to the protease plasmin, was recently shown to mediate neurodegeneration in the mouse hippocampus. Mice deficient in tissue plasminogen activator (tPA) display a dramatic resistance to a paradigm of excitotoxic neuronal death that involves intrahippocampal injection of the excitotoxin. This model is thought to reproduce the mechanism of neuronal death observed during acute (such as ischemic stroke) and degenerative (such as amyotrophic lateral sclerosis) diseases of the nervous system. The requirement for the proteolytic activity of tPA to mediate neuronal death is acute in the adult mouse. Serine protease inhibitors, specific for tPA or the tPA/plasmin proteolytic cascade, are effective in conferring extensive neuroprotection following the excitotoxic injection. These findings suggest possible new ways for interfering with the neuronal death observed in the hippocampus as a result of excitotoxicity. In addition, tPA is produced in the hippocampus primarily by microglial cells, which become activated in response to the neuronal injury. Blocking microglial activation has been shown in other injury paradigms to protect against neuronal death, therefore suggesting another way to retard neurodegeneration in the CNS. Furthermore, after the insult has been inflicted and in the presence of a compromised blood-brain barrier macrophages (cells deriving from the same lineage as microglia) migrate into the brain, where they are thought to contribute to the neuronal cell loss by secreting neurotoxic molecules. If these macrophages/microglia expressed, however, a tPA inhibitor, rather than the possibly neurotoxic tPA, they might be able to protect the neurons from dying. Received: 1 October 1996 / Accepted: 27 January 1997     

Innate immune responses to cytomegalovirus infection in the developing mouse brain and their evasion by virus-infected neurons

Cytomegalovirus (CMV) is the most frequent infectious cause of developmental brain disorders in humans. Here we show the role of innate immune responses caused by natural killer (NK) cells and nitric oxide (NO) derived from brain macrophages during murine CMV (MCMV) infection of the developing brain. Viral replication in the brain of newborn mice was significantly enhanced by administration of anti-asialo-GM1 antibody, specific for NK cells, or L-N6-(1-imminoethyl)-lysine, a specific inhibitor of NO synthase 2 (NOS2). These results suggest that NK cells and NO contribute to the viral clearance from the brain. At 3 days postinfection (dpi) MCMV early antigen (Ag)-positive cells were immunohistochemically detected in the periventricular area, where most of the positive cells were macrophages. At 7 dpi MCMV-Ag was found not only in cells of the periventricular area but also in neurons of the hippocampus and cortex. At 11 dpi MCMV-Ag disappeared from the periventricular area, but persisted in neurons. In the periventricular area, NK cells and NOS2-positive macrophages were associated with MCMV-Ag-positive cells. In contrast, there were very few NK cells and NOS2-positive macrophages around the MCMV-Ag-positive neurons. In situ hybridization for MCMV DNA demonstrated that positive signals were found mostly in the periventricular cells, and rarely in neurons. These results suggest that the innate immune responses are restricted to the virus-replicating cells, and do not affect MCMV-infected neurons. Therefore, evasion of the innate immune responses by MCMV-infected neurons may be an important factor in supporting the viral persistence in the developing brain.     

MHC-positive, ramified macrophages in the normal and injured rat peripheral nervous system

Summary Resident endoneurial macrophages form a prominent, but little recognized component of the PNS. We have studied immunocytochemically the distribution, morphology and immunophenotype of endoneurial macrophages in several normal peripheral nerves of the rat. In addition, we investigated the macrophage response following crush injury of the sciatic nerve.Resident endoneurial macrophages had a ramified morphology with processes oriented parallel to the long axis of nerve fibres. They were positive for several monocyte/macrophage markers such as ED1, ED2 and the recently-described MUC 101 and MUC 102 antibodies. They furthermore expressed the complement type three receptor, the CD4 antigen and MHC class I and II molecules. These results were consistent in all the peripheral nerves studied. In addition, 1000 rad of -irradiation led to a strong reduction in the number of MHC class II-positive ramified cells in the peripheral nerves similar to that observed in other peripheral organs such as the heart. A considerable percentage of resident macrophages in the PNS and/or their precursor cells are therefore radiosensitive and could be related to the lineage of dendritic cells.Following crush injury, ED1-3-, OX-42-, MUC 101- and MUC 102-positive round macrophages were observed from 24 h postlesion onward at the site of trauma. In the distal part, they were observed to form strings of round, foamy macrophages probably involved in myelin phagocytosis. In contrast, the number of MHC class II-positive resident macrophages was only slightly increased at the site of trauma and in the distal part. These cells transformed from a ramified to a round morphology, but did not appear as typical strings of foamy macrophages.These results demonstrate that the PNS is provided with a resident macrophage population analogous in many respects to microglial cells in the CNS. These constitutively MHC class II-positive PNS microglial-like cells could act as the major antigen-presenting cells in the peripheral nerve. They may thus constitute a local immune defense system of the PNS with a function similar to that of microglial cells in the CNS.On leave of absence from the Institute of Neurology, University of Verona, Italy.     

Nuclear retention of IL‐1α by necrotic cells: A mechanism to dampen sterile inflammation

Sterile inflammation is a host response to tissue injury that is mediated by damage‐associated molecular patterns released from dead cells. Sterile inflammation worsens damage in a number of injury paradigms. The pro‐inflammatory cytokine IL‐1α is reported to be a damage‐associated molecular pattern released from dead cells, and it is known to exacerbate brain injury caused by stroke. In the brain, IL‐1α is produced by microglia, the resident brain macrophages. We found that IL‐1α is actively trafficked to the nuclei of microglia, and hence tested the hypothesis that trafficking of IL‐1α to the nucleus would inhibit its release following necrotic cell death, limiting sterile inflammation. Microglia subjected to oxygen‐glucose deprivation died via necrosis. Under these conditions, microglia expressing nuclear IL‐1α released significantly less IL‐1α than microglia with predominantly cytosolic IL‐1α. The remaining IL‐1α was immobilized in the nuclei of the dead cells. Thus, nuclear retention of IL‐1α may serve to limit inflammation following cell death.     

A light and electron microscopic study of the CB1 cannabinoid receptor in monkey basal forebrain

The distribution of the CB1 cannabinoid receptor was studied in the monkey basal forebrain by immunocytochemistry and electron microscopy, using an antibody to the CB1 brain cannabinoid receptor. Large numbers of labelled neurons were observed in the medial septum, nucleus of the diagonal band, and the nucleus basalis of Meynert. The labelled neurons had dimensions similar to those of cholinergic neurons and were larger than those of GABAergic neurons. Double immunolabelling with an antibody to the synthetic enzyme for acetylcholine, choline acetyl transferase (ChAT) showed that CB1-positive neurons were also positive for ChAT, whilst electron microscopy confirmed that CB1-labelled neurons contained lipofuscin granules and dense clusters of rough endoplasmic reticulum, characteristic of cholinergic neurons. The dense labelling of cholinergic neurons for CB1 is interesting from the standpoint of neuroprotection. The CB1 receptor has been shown to couple in an inhibitory manner to voltage dependent calcium channels, and the dense labelling of CB1 in cholinergic neurons would therefore suggest that CB1 receptors could be important in limiting calcium influx through voltage dependent calcium channels in these neurons. This could serve to limit intracellular calcium concentrations, and consequent calcium mediated injury, in these neurons.     

Upregulation of phospholipase D1 in the spinal cords of rats with clip compression injury

This study examined phospholipase D 1 (PLD1) expression in the central nervous system following clip compression spinal cord injury (SCI) in Sprague-Dawley rats. After inducing SCI with a vascular clip, the expression of PLD1 in the affected spinal cord was analyzed by Western blot and immunohistochemistry. Western blot analysis showed that the expression of PLD1 gradually increased in the spinal cord on days 0.5, 1, 2, and 4 post injury. Immunohistochemistry showed that some cells, including neurons, astrocytes, and some inflammatory cells, were positive for PLD1 in the lesions at days 1 and 2 post injury. At day 4, the number of PLD1-positive cells in SCI lesions increased, largely matching the increases in ED1-positive macrophages and glial fibrillary acidic protein-positive astrocytes. At this time, macrophages expressed proliferating cell nuclear antigen in addition to PLD1. These results suggest that PLD1 expression is increased in injured spinal cords, and might be involved in the activation and proliferation of macrophages and astrocytes in SCI.     

Apoptotic neurodegeneration in the context of traumatic injury to the developing brain

Head trauma is the leading cause of death and disability in the pediatric population. Some recent studies on neuropathological and biochemical features of traumatic injury to the developing brain revealed interesting aspects and potential targets for future research. Trauma triggers both excitotoxic and apoptotic neurodegeneration in the developing rat brain. Apoptotic neurodegeneration occurs in a delayed fashion over several days and contributes in an age-dependent fashion to neuropathologic outcome following head trauma, with the immature brain being exceedingly sensitive. Biochemical studies indicate that both the extrinsic and the intrinsic apoptotic pathways are involved in pathogenesis of apoptotic cell death following trauma in the developing brain and that caspase inhibition ameliorates apoptotic neurodegeneration in an infant head trauma model. Given the major contribution of apoptotic neurodegeneration to neuropathologic outcome following trauma to the developing brain, interference with apoptotic pathways may comprise a potential therapeutic target in pediatric traumatic brain injury.     

Increase of NG2-positive cells associated with radial glia following traumatic spinal cord injury in adult rats

In the CSN including the spinal cord, NG2 proteoglycan is a marker of oligodendrocyte progenitors. To elucidate the dynamics of the endogenous neural stem (progenitor) cells in adult rats with spinal cord injury (SCI), we examined an immunohistochemical analysis of NG2, GFAP, and 3CB2, a specific marker of radial glia (RG). SD rats were divided into a SCI group (n = 25) and a sham-operated group (n = 5). In the injury group, laminectomy was performed at Th11–12 and contusive compression injury was created by applying a weight of 30 g for 10 min. Rats were sacrificed at 24 h, and 1, 4, 8 and 12 weeks post-injury. Frozen 20-μ m sections of tissue 5 and 10 mm rostral and caudal to the epicenter of injury were prepared. Immunohistochemistry was performed using antibodies against NG2, GFAP and 3CB2. At 4 weeks after injury, NG2-positive glial cells arose from below the pial surface as bipolar cells with processes extending throughout the entire white matter. NG2 expression peaked at 4 weeks after injury, showing a 7-fold increase compared to the 24 h after injury. The NG2-positive cells with processes which increased in the white matter of the spinal cord were GFAP-positive and also co-localized with 3CB2 antigen. The pattern of NG2 expression of these cells was temporally and spatially different from the pattern of NG2 expression that accumulated around the hemorrhagic and necrotic epicenter. These results suggest that NG2 positive cells which derived from subpial layer, may have some lineage to RG after SCI in adult rodents. The first and second authors contributed equally to this work.     

Involvement of Disabled-2 protein in the central nervous system inflammation following experimental cryoinjury of rat brains

Disabled-2 (Dab-2) functions in the mitogenic signal transduction pathway, and is expressed in a variety of tissues. We investigated the roles of Dab-2 expression in the rat brain following experimental cryoinjury in relation to central nervous system (CNS) inflammation. Western blot analysis showed that Dab-2 expression increased significantly (p < 0.001) in the frontal cortex 4-14 days after cryoinjury, and declined slightly thereafter. Immunohistochemistry showed that Dab-2 immunostaining occurred in most of the vessels in the control cerebral cortex. After cryoinjury, Dab-2 was localized in the majority of inflammatory cells (especially in ED1-positive macrophages) in the core and periphery, as well as in vessels. These findings suggest that Dab-2 is involved in the inflammation that follows CNS injury through the migration of activated inflammatory cells in the rat brain.     

2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL-1 human eosinophilic leukemia cells and human peripheral blood eosinophils

2-arachidonoylglycerol (2-AG) is an endogenous cannabinoid receptor ligand. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, abundantly expressed in the brain, and the CB2 receptor, expressed in various lymphoid tissues such as the spleen. The CB1 receptor has been assumed to play an important role in the regulation of synaptic transmission, whereas the physiological roles of the CB2 receptor remain obscure. In this study, we examined whether the CB2 receptor is present in human eosinophils and found that the CB2 receptor is expressed in human peripheral blood eosinophils. In contrast, human neutrophils do not contain a significant amount of the CB2 receptor. We then examined the effect of 2-AG on the motility of eosinophils. We found that 2-AG induces the migration of human eosinophilic leukemia EoL-1 cells. The migration evoked by 2-AG was abolished in the presence of SR144528, a CB2 receptor antagonist, or by pretreatment of the cells with pertussis toxin, suggesting that the CB2 receptor and Gi/o are involved in the 2-AG-induced migration. The migration of EoL-1 cells induced by 2-AG was suggested to be a result of chemotaxis. In contrast to 2-AG, neither anandamide nor free arachidonic acid elicited the migration. Finally, we examined the effect of 2-AG on human peripheral blood eosinophils and neutrophils and found that 2-AG induces migration of eosinophils but not neutrophils. These results suggest that the CB2 receptor and its endogenous ligand 2-AG may be closely involved in allergic inflammation accompanied by the infiltration of eosinophils.     

A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.     

Targeting CB(2) receptor as a neuroinflammatory modulator in experimental autoimmune encephalomyelitis

During immune mediated demyelinating lesions, the endocannabinoid system is involved in the pathogenesis of both neuroinflammation and neurodegeneration through different mechanisms. Here, we explored the cellular distribution of cannabinoid 2 receptor (CB(2)R) in the central nervous system (CNS) and detected the level of CB(2)R expression during experimental autoimmune encephalomyelitis (EAE) by RT-PCR, Western blot and immunostaining. Our results show that CB(2)R was expressed in neurons, microglia and astrocytes. During EAE, the expression of CB(2)R in spinal cord rose slowly at days 9 and 17 post immunization (p.i.), and elevated rapidly at day 28 p.i., while the expression of CB(2)R in spleen elevated rapidly and got a plateau at days 17 and 28 p.i. Only the increase of CB(2)R expression in spinal cord demonstrated a significant difference when compared to control mice immunized with complete Freund's adjuvant (CFA). The selective CB(2)R antagonist (SR144528) exacerbated EAE clinical severity accompanied by weight loss. SR144528 inhibited the expression of CB(2)R, but increased the expression of CB(1)R in brain, spinal cord and spleen. The administration of SR144528 declined interferon-γ, IL-17, IL-4, IL-10, IL-1β, IL-6 and tumor necrosis factor-α, but increased CX3CL1 in brain and/or spinal cord. In contrast, IL-17 and MCP-1 were increased, while CX3CL1 was decreased in splenic mononuclear cells as compared to vehicle controls. These results indicate that manipulation of CB(2)R may have therapeutic value in MS, but its complexity remains to be considered and studied for further clinical application.     

Emergence of different macrophage populations in hepatic fibrosis following thioacetamide-induced acute hepatocyte injury in rats

Macrophages may play a role in fibrogenesis. The kinetics and distribution of different macrophage populations were investigated immunohistochemically in hepatic lesions following acute hepatocyte injury induced in F344 rats by a single injection of thioacetamide (TAA) (300 mg/kg body weight, intraperitoneally). Hepatocyte degeneration or necrosis induced by TAA occurred mainly in the perivenular areas of hepatic lobules as early as post-injection (PI) days 1 and 3; fibrotic lesion development began in the damaged areas on day 1, and peaked on day 5; thereafter (PI days 7 and 10), the fibrotic areas decreased and were replaced by regenerated hepatocytes on PI days 15 and 20, indicating a remodelling process. In this rat model, the number of macrophages reacting with ED1 antibody (specific for exudate macrophages), ED2 (recognizing cell membrane antigens of resident macrophages, including Kupffer cells) and OX6 (recognizing MHC class II antigens expressed in antigen-presenting macrophages and dendritic cells) began to increase on PI day 1, peaking on PI day 3. The numbers gradually decreased on PI days 5 and 7; however, the statistically significant increase was maintained in respect of ED1-positive cells up to PI day 20, whereas no significant increase in ED2- and OX6-positive cells remained from PI day 10 onwards. Interestingly, of the ED1-, ED2- and OX6-positive cells, the OX6-positive cells were the least numerous. ED1- and OX6-positive cells appeared exclusively in the injured perivenular areas, whereas ED2-positive cells were present mainly in the mid-zonal areas and in smaller numbers in the perivenular areas. These findings indicated differences in kinetics and distribution between macrophage populations appearing in hepatic fibrosis. In addition, RT-PCR revealed that mRNA expression of osteopontin, a factor for induction and maintenance of macrophages in inflammation, was markedly increased on PI days 5, 7 and 10, suggesting a role in the pathogenesis of hepatic fibrosis.