ED1-positive Macrophages Research Articles

EFFECT OF POLYPHENOL‐CONTAINING AZUKI BEAN (VIGNA ANGULARIS) EXTRACT ON BLOOD PRESSURE ELEVATION AND MACROPHAGE INFILTRATION IN THE HEART AND KIDNEY OF SPONTANEOUSLY HYPERTENSIVE RATS

1. Hypertension is a major risk factor for myocardial infarction and renal damage, and it has also been shown to have pro-inflammatory actions that increase the formation of reactive oxygen species. Macrophage infiltration has been suggested to play a role in the pathogenesis of hypertension. Azuki beans are known to contain pro-anthocyanidins, a group of polyphenolic bioflavonoids with remarkable radical-scavenging activities in vitro. Therefore, the aim of the present study was to investigate the effect of polyphenol-containing azuki bean extract (ABE) on systolic blood pressure (SBP) and macrophage infiltration in the heart and kidney of spontaneously hypertensive rats (SHR). 2. Spontaneously hypertensive rats and control normotensive Wistar-Kyoto (WKY) rats were divided into two groups fed either 0 or 0.8% ABE in their diets. Tail SBP and macrophage kinetics in the heart and kidney were examined. 3. The SBP of the SHR group was higher than that of age-matched WKY rats throughout the treatment period. After 8 weeks of treatment, the increased SBP in ABE-treated SHR was significantly less than that in untreated SHR. 4. Nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH)-stimulated superoxide (O2-) production was enhanced in the kidney and heart in SHR and WKY rats compared with levels in the absence of NADH or NADPH. The NADPH-stimulated superoxide (O2-) levels in the kidney in untreated SHR was significantly higher than that in untreated WKY rats. The (O2-) levels in ABE-treated SHR were significantly decreased compared with the untreated SHR group. 5. In immunohistochemical analyses, the number of macrophages in the heart and in the glomeruli and tubulointerstitium of the kidney was significantly higher in ABE-untreated SHR than in ABE-untreated WKY rats. Conversely, there was a significant decrease in the number of macrophages in ABE-treated SHR compared with the untreated SHR. There were significant positive correlations between SBP and the number of ED1-positive macrophages in the heart and tubulointerstitial and glomerular areas of the kidney in WKY rats and SHR. 6. In conclusion, the results of the present study suggest that ABE attenuates the elevation of SBP and macrophage infiltration in the heart, as well as in the glomeruli and tubulointerstitium of the kidney, in our SHR model.

Macrophages, myofibroblasts and mast cells in a rat liver infected withCapillaria hepatica

We trapped a rat (Rattus norvegicus) infected with Capillaria hepatica. At necropsy, grossly yellowish-white nodules (2-3 mm in diameter) were noted to be scattered on the liver's surface. Microscopically, granulomatous and fibrotic nodules that contained the eggs and/or adult worms of Capillaria hepatica were detected in the liver. Septal fibrosis was diffusely formed throughout the liver. There were a number of ED1-positive macrophages located in the sinusoids of the pseudolobules. On the double staining, myofibroblasts and mast cells were generally observed within the fibrous septa with the mast cells in close proximity to the myofibroblasts. We suggest that the interactions between macrophages, myofibroblasts and mast cells play a role in the septal fibrosis observed in rats infected by Capillaria hepatica.

Angiotensin AT1 receptor antagonists exert anti‐inflammatory effects in spontaneously hypertensive rats

Although the main therapeutic effect of angiotensin AT1 receptor antagonists is to decrease blood pressure, they also exert anti-inflammatory effects in the cardiovascular system. However, the underlying mechanisms remain unclear. We investigated the inhibitory effect of AT1 antagonists on the chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor 2 (CCR2) in rat monocytes and aortas. Spontaneous hypertensive rats (SHRs) were treated with the AT1 antagonists losartan or telmisartan for 4 weeks, and Wistar-Kyoto rats (WKYs) were used as normotensive controls. Systolic arterial pressure was measured, and the number of macrophages in the aortic vessel wall was assessed by anti-ED-1 antibody immunolabelling. Compared with WKYs, SHRs showed significantly increased ED-1 positive macrophages in the aortic wall, which were decreased after high doses of losartan or telmisartan. Low doses of losartan did not improve blood pressure significantly as did the high doses, but markedly decreased macrophage infiltration in the vessel wall. AT1 antagonists, particularly at high doses, improved aortic remodeling in SHR. At the molecular level, AT1 antagonists attenuated the expression of MCP-1 and CCR2 in the aorta and peripheral blood monocytes and lowered the serum level of MCP-1. In addition, Western blotting showed that AT1 antagonists inhibited the phosphorylation of Akt in mouse monocytes. AT1 antagonism inhibited vessel wall inflammation and inhibition of PI3K/Akt may be involved in the modulation of the MCP-1/CCR2 system by AT1 antagonists in SHRs.

MCP-1 targeting inhibits muscularis macrophage recruitment and intestinal smooth muscle dysfunction in colonic inflammation

Upregulation of muscularis macrophage numbers and activities plays an important role in the intestinal dysmotility associated with intestinal inflammation. The present study aimed to clarify changes in population dynamics of intestinal muscularis macrophages during colonic inflammation and to test possible inhibitory actions of agents targeting monocyte chemoattractant protein-1 (MCP-1) on muscularis macrophage dynamics and motility disorder in the colonic inflammation elicited by 2,4,6-trinitrobenzene sulfonic acid. In the inflamed muscle layer, ED1 antibody-positive monocytes and monocyte-derived macrophages were increased, followed by increasing resident macrophages positively staining for ED2 antibody. Initiation of the ED1-positive macrophage dynamic is associated with MCP-1 mRNA expression. MCP-1 was expressed in both ED1- and ED2-positive macrophages after inflammation. Electromicroscopic analysis revealed that the cell-division phase of muscularis macrophages was seen only in the early stages of inflammation. In addition, ED1 and ED2 double-positive macrophages can be detected during inflammation. Treatment with dominant negative MCP-1 or neutralizing MCP-1 antibodies markedly inhibited numbers of both ED1- and ED2-positive macrophages. Inflammation-mediated dysmotility was partially recovered by treatment with neutralizing MCP-1 antibodies. These results suggest that the inflamed muscle layer is initially infiltrated by monocytes, which then differentiate and develop into muscularis-resident macrophages. These macrophages express MCP-1 for further recruitment of monocytes. MCP-1 may be one potential therapeutic target for inhibiting intestinal motility disorders in gut inflammation.

Increased invasion of ED-1 positive macrophages in both ipsi- and contralateral dorsal root ganglia following unilateral nerve injuries

There is an increasing evidence that unilateral nerve injury induces cellular and molecular changes in the associated DRG not only on the ipsilateral but also in the contralateral side. In this investigation, ED-1+ macrophages were quantified by image analysis in the naïve L5 DRG (nDRG) and compared with the ipsi- and contralateral ones 2 and 4 weeks after unilateral sciatic nerve ligature and ventral root transection (VRT). A few ED-1+ macrophages were found in nDRG but not closely associated with the neuronal bodies. In contrast, following nerve injuries ED-1+ macrophages and their processes were frequently located close neuronal bodies and became their satellite cells. Moreover, an increased number of ED-1+ cells was found in the ipsilateral DRG 2 weeks after unilateral sciatic nerve ligature or VRT, but no significant differences were measured between 2 and 4 weeks after both types of nerve lesion. Contralateral DRG displayed a significant enhanced number of ED-1+ cells no sooner than 4 weeks from sciatic nerve ligature. In contrast, VRT induced a significant increased invasion of the ED-1+ cells in the contralateral DRG as early as 2 weeks after operation. Our experiments indicate that a significantly higher number of ED-1+ macrophages remained in both ipsi- and contralateral DRG up to 4 weeks from nerve injury. Based on results from different models of nerve injury, we suggest that more than one mechanism operates to stimulate the invasion of ED-1+ macrophages into the DRG including retrograde transport of factors produced during Wallerian degeneration or their delivery by blood flow. Signaling for macrophage invasion into DRG contralateral to nerve injury may be mediated by lost motoneurons or by interneurones.

IL-12/IFN-γ/NO axis plays critical role in development of Th1-mediated experimental autoimmune encephalomyelitis

The importance of the IL-12/IFN-γ/nitric oxide (NO) axis in the pathogenesis of autoimmune diseases remains controversial. In parallel experiments, we explored the role of the IL-12/IFN-γ/NO axis in the development of MOG 35-55-induced experimental autoimmune encephalomyelitis (EAE) in mice lacking IL-12, IFN-γ receptor (IFN-γR) and inducible nitric oxide synthase (NOS2), respectively. In comparison with wide-type control mice, IL-12 −/−, IFN-γR −/− and NOS2 −/− mice displayed more severe clinical signs of EAE both in remission and at subsequent relapse. Given the relatively low IFN-γ production in IL-12 −/− mice and the lack of IFN-γ/IFN-γR signaling pathway in IFN-γR −/− mice, IL-12 −/−, IFN-γR −/− and NOS2 −/− mice with EAE exhibited low NO production. This correlated negatively with MOG 35-55-induced T cell proliferation. Both ED1-positive macrophages and CD4-positive T cells were increased in spinal cords from IL-12 −/−, IFN-γR −/− and NOS2 −/− compared to control mice. In vitro experiments demonstrate that spleen mononuclear cells from IL-12 −/−, IFN-γR −/− and NOS2 −/− mice with EAE present stronger migration capacity when compared to control mice. These results reveal that the IL-12/IFN-γ/NO axis plays a critical role in the development of MOG 35-55-induced EAE, possibly over failing NO production.

Increased phosphorylation of cyclic AMP response element-binding protein in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis

To investigate whether the phosphorylation of cyclic AMP response element-binding protein (CREB) is implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the change in the level of CREB phosphorylation was analyzed in the spinal cord of Lewis rats with EAE. Western blot analysis showed that the phosphorylation of CREB in the spinal cord of rats increased significantly at the peak stage of EAE compared with the controls ( p < 0.05) and declined significantly in the recovery stage ( p < 0.05). Immunohistochemistry showed that the phosphorylated form of CREB (p-CREB) was constitutively immunostained in few astrocytes and dorsal horn neurons in the spinal cord of normal rats. In the EAE-affected spinal cord, p-CREB was mainly found in ED1-positive macrophages at the peak stage of EAE, and the number of p-CREB-immunopositive astrocytes was markedly increased in the spinal cord with EAE compared with the controls. Moreover, p-CREB immunoreactivity of sensory neurons, which are closely associated with neuropathic pain, was significantly increased in the dorsal horns at the peak stage of EAE. Based on these results, we suggest that the increased phosphorylation of CREB in EAE lesions was mainly attributable to the infiltration of inflammatory cells and astrogliosis, possibly activating gene transcription, and that its increase in the sensory neurons in the dorsal horns is involved in the generation of neuropathic pain in the rat EAE model.

Osteopontin expression in activated glial cells following mechanical- or toxin-induced nigral dopaminergic cell loss

Osteopontin (OPN) is a glycosylated phosphoprotein that regulates both oxidative stress and inflammatory processes. OPN is present in the rat substantia nigra (SN) and both protein and mRNA levels are up-regulated following a pro-inflammatory insult produced by lipopolysaccharide. We now report on the effects of lesioning the SN using 6-hydroxydopamine (6-OHDA) and mechanical vehicle-induced lesioning on OPN expression. Intranigral administration of 6-OHDA induced a marked time-dependent loss of tyrosine hydroxylase (TH) positive nigral cells. Vehicle administration also produced a loss of TH positive cells. This was small compared to 6-OHDA and due to mechanical damage during surgery. 6-OHDA and mechanical-induced cell loss was accompanied by an increase in OPN protein and mRNA expression. Both 6-OHDA and mechanical lesions resulted in equivalent time-dependent increases in OX-42 positive microglial cells. However, the elevation was far less marked following mechanical damage compared to 6-OHDA-induced cell death. 6-OHDA lesioning induced a slow up-regulation of GFAP positive astroglial cells but this was not present following mechanical damage. Importantly, both 6-OHDA and mechanical lesions resulted in an up-regulation in ED1 positive macrophages of equivalent magnitude and time course. There was co-localisation of OPN with ED1 positive cells but not TH, OX-42 or GFAP cells following both toxin and mechanical lesions. Nigral TH positive cell death of toxin or mechanical origin increases OPN expression in parallel with the up-regulation of ED1 positive macrophages. The increase in OPN/ED1 expression is independent of the extent of cell death. OPN appears to be an important regulator of nigral cell survival through its association with inflammatory events and its manipulation may provide a means of achieving neuroprotection in Parkinson's disease.

Morphologic observation and pathogenesis investigation of regenerated sinusoidal endothelial cells in remodelling rat hepatic necrotic tissue

To investigate the morphological changes and regeneration mechanism of sinusoidal endothelial cell. Sixty male Wistar rats (bought from SLC company limited of Japan) were divided into three groups. Fifty of them belonged to experiment group, five rats belonged to untreated group, and the rest five ones belonged to normal saline treated group. The experiment group was then divided into ten subgroups. All the rats of the experiment group were killed under anaesthesia using aether at 12, 24, 36 hrs, and 2, 3, 5, 7, 8, 10 and 14 days subsequently after an one-off injection of dimethylnitrosamine (DMN) (50 mg/kg). The liver tissues, bone marrows and peripheral blood of the rats were taken out rapidly. All the tissues received with HE staining, immunohistochemistry staining and double immunofluorescence labelings, and they were observed under a light microscope and electron microscope. The livers, bone marrows and peripheral blood from the rats at 24 hrs to 14 days after an injection of DMN were examined by light microscopic, immunohistochemical, and ultrastructural methods. Small focal necrosis of the liver tissues was found at 12 hrs after the DMN injection, and gradually becomes more obvious from the 24 hrs. The most obvious necrosis, with lots of ED-1 (monocyte/phagocyte marker of rats) positive cells infiltration, was observed at 36 hrs. On the 2nd day and 3rd day after injection, the necrotic fragments and red cells were phagocyted by ED-1 positive macrophages. On the 5th day, some of the ED-1-positive cells were transformed from round to spindle in shape. On the 7th day, these cells contacted with residual reticulin fibers and became positive for SE-1, a marker of hepatic sinusoidal endothelial cells and Tie-1, an endothelial cell-specific surface receptor, associated with frequent occurrence of ED-1/SE-1 and ED-1/Tie-1 double positive spindle cells. On the 8th day, the histomorphology of liver tissue was similar with that on day 7, except that the range of the lesions had become smaller. On the 10th day, the regeneration of liver tissue increased, filling in the necrosis. On the 14th day, the necrotic tissues were almost replaced by regenerated liver tissues and thin bundles of central-to-central bridging fibrosis. 12 hrs after the DMN injection, bone marrow studies showed an increase in the number of ED-1 positive mononuclear cells, some of which were both BrdU/ED-1 positive. The number of ED-1 positive mononuclear cells reach their highest level at 36 hrs. These cells are morphologically similar to round mononuclear cells in bone marrows and could be found in the peripheral blood from 24 hrs to the 10 days. They reached their highest level in peripheral blood at the same time as in the bone marrow. These cells morphologically resembled ED-1 positive cells in necrotic tissues of the liver. These findings suggest that round mononuclear ED-1-positive cells proliferate first in the bone marrow after DMN treatment, reach necrotic areas of livers through circulation, and differentiate to sinusoidal endothelial cells. Namely, hepatic sinusoids in DMN-induced necrotic areas may partly be reorganized possibly by vasculogenesis.

Renal protective effects of pitavastatin on spontaneously hypercholesterolaemic Imai Rats

Independent of their lipid-lowering effects, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have renal protective effects on various models of progressive renal diseases, therefore, additional therapeutic advantages have been considered. In the present study, using spontaneously hypercholesterolaemic Imai rats, we examined the protective effects of pitavastatin on renal injuries and the oxidative modification of the low-density lipoprotein (LDL) and high-density lipoprotein (HDL), since oxidized lipoproteins are speculated to be involved in the mechanism of this rat strain's renal injuries. Male Imai rats were treated with pitavastatin (n = 11) at a dose of 100 mg/kg diet or received no specific therapy as controls (n = 11) from 10 to 22 weeks of age. Body weight, urinary protein excretion and serum constituents were evaluated every 4 weeks. At the end of the study, the effects of pitavastatin on the susceptibility of serum LDL and HDL to oxidation, and renal histology were examined. Pitavastatin treatment did not affect hyperlipidaemia, but significantly reduced proteinuria and preserved creatinine clearance deterioration. At the end of the study, lag times for LDL and HDL oxidation were prolonged by the treatment of pitavastatin to 126 and 153%, respectively, compared with the controlled group. The glomerulosclerosis index (SI) for untreated controlled rats was significantly higher than that for the pitavastatin-treated group. An immunohistochemistry study showed significantly lower numbers of ED-1 positive macrophages in the glomeruli and interstitium in pitavastatin-treated rats compared with those controlled. Pitavastatin treatment prevented renal injuries in Imai rats independent of lipid-lowering effects. Prevention of oxidative modification of LDL and HDL may play an important role on the beneficial effects of pitavastatin treatment.

Phagocytotic Activation of Muscularis Resident Macrophages Inhibits Smooth Muscle Contraction in Rat Ileum

Intestinal muscularis resident macrophages distributed in myenteric region may play an important role in the immunological host defense against infection. In this study, we investigated the phagocytic stimulation of resident macrophages on cyclooxygenase-2 (COX-2) expression and smooth muscle contraction in the small intestine of rat. After the injection of FITC-dextran to rat, phagocytosed macrophages could be detected in the myenteric plexus. FITC-positive macrophages were also immunostained with COX-2 antibody. The number of COX-2 immunopositive cells increased in a time-dependent manner reaching its maximum at 4 hr after the injection, which then decreased gradually but considerable number of cells were still remained on 7 days. The injection of FITC-dextran, however, did not change the population of ED2-positive resident macrophages even on 7 days. Production of PGE2 was significantly higher in the dextran treated tissue as compared to control tissue. In the smooth muscle tissue phagocytosed dextran, carbachol-induced contraction was significantly decreased. The suppression of the carbachol-induced contraction was completely restored by COX inhibitor, indomethacin. Finally we demonstrated that, in freshly isolated macrophage cells, addition of dextran induced a slow and sustained increase in intracellular Ca2+ concentration. These results indicate that phagocytotic activation of muscularis resident macrophages induces COX-2 gene expression and then results in production of PGE2 to suppress the smooth muscle contractile activity.

Orazipone, a locally acting immunomodulator, ameliorates intestinal radiation injury: A preclinical study in a novel rat model

Intestinal radiation injury (radiation enteropathy) is relevant to cancer treatment, as well as to radiation accidents and radiation terrorism scenarios. This study assessed the protective efficacy of orazipone, a locally-acting small molecule immunomodulator. Male rats were orchiectomized, a 4-cm segment of small bowel was sutured to the inside of the scrotum, a proximal anteperistaltic ileostomy was created for intraluminal drug administration, and intestinal continuity was re-established by end-to-side anastomosis. After three weeks postoperative recovery, the intestine in the "scrotal hernia" was exposed locally to single-dose or fractionated X-radiation. Orazipone (30 mg/kg/day) or vehicle was administered daily through the ileostomy, either during and after irradiation, or only after irradiation. Structural, cellular, and molecular aspects of intestinal radiation toxicity were assessed two weeks after irradiation. Orazipone significantly ameliorated histologic injury and transforming growth factor-beta immunoreactivity levels, both after single-dose and fractionated irradiation. Intestinal wall thickness was significantly reduced after single-dose and nonsignificantly after fractionated irradiation. Mucosal surface area and numbers of mast cells were partially restored by orazipone after single-dose irradiation. This work (1) demonstrates the utility of the ileostomy rat model for intraluminal administration of response modifiers in single-dose and fractionated radiation studies; (2) shows that mucosal immunomodulation during and/or after irradiation ameliorates intestinal toxicity; and (3) highlights important differences between single-dose and fractionated radiation regimens.

Coating of titanium implants with collagen, RGD peptide and chondroitin sulfate

Coating of orthopaedic implants with extracellular bone matrix components was performed to enhance bone healing. Titanium pins of 0.8mm diameter were coated with type I collagen (Ti/Coll), RGD peptide (Ti/RGD) or type I collagen and chondroitin sulfate (Ti/Coll/CS). Uncoated pins (Ti) served as control. The pins were inserted as intramedullary nails into the tibia of male adult Wistar rats. Six specimens of each group were retrieved at 4, 7, 14 and 28 days. All implants healed uneventfully without adverse reactions. ED 1-positive macrophages appeared in higher numbers around Ti/RGD at day 4 and around Ti at day 14 after implantation (p < 0.05). TRAP-positive osteoclasts and precursors were abundant around Ti/Coll/CS at day 7 (p < 0.05). A significant increase in osteopontin-positive osteoblasts was seen around Ti/Coll/CS implants at days 7 and 14, and around Ti/RGD at day 14 (p < 0.05). At day 28, 62% of Ti, 76% of Ti/Coll, 85%* of Ti/RGD and 89%* of Ti/CoIl/CS (*p < 0.05) implants were covered with newly formed lamellar bone. The addition of extracellular matrix components significantly enhances bone remodelling in the early stages of bone healing around Ti implants, eventually leading to increased new bone formation at the implant surface after 4 weeks.

Molecular insights of the injured lesions of rat spinal cords: Inflammation, apoptosis, and cell survival

Spinal cord injury (SCI) is a devastating neurologic injury with functional deficits. In the acute phase, which starts at the moment of the injury and extends over the first few days, numerous pathological processes begin. In this study, we made several additional advances to broaden our understanding of SCI-induced gene expression changes. We examined changes at multiple time points: 0, 6, 24, 48, and 72 h after injury, with the latter time period being added. Also, we utilized multiple analysis methods such as real-time RT-PCR, Western blot, and immunohistochemistry to increase confidence in our candidate gene and molecular processes. From the pool of information, we generated profiles of expression changes and molecular mechanisms of several injury processing. Early stages after the injury are characterized by the strong upregulation of genes involved in transcription, inflammation, and signaling proteins, and a general downregulation of neural function-related genes. In addition, edema of the spinal cord develops, and metabolic disturbances involving intra-neuronal Ca 2+ accumulation occur. This translates into a general failure of normal neural functions and a stage of signal shock that lasts for a few days in experimental rat models. Traumatic injury to the spinal cord also leads to a strong inflammatory response with the recruitment of peripherally derived immature cells, such as ED1-positive macrophages. After the trauma, apoptotic cell death continues, and scarring and demyelination accompany Wallerian degeneration. Strong expression of transcription factors of the Janus-activated kinase (JAK) and signal transducer and activator of transcription (STAT) family represents an early attempt of spinal cord repair and regeneration. Our study allowed us to conclude that combined therapeutic strategies for enhanced recovery should be performed until the chronic phase of the injury in areas distal to the lesion epicenter of spinal cords.

Immunohistochemical study of caveolin-1 in the sciatic nerves of Lewis rats with experimental autoimmune neuritis

The expression of caveolin-1 and the related molecule endothelial nitric oxide synthase (eNOS) was analyzed in the sciatic nerves of Lewis rats with experimental autoimmune neuritis (EAN). Western blot analysis showed that caveolin-1 significantly increased in the sciatic nerves with EAN upon initiation of cell infiltration during the early and peak stages (days 10 and 14 post-immunization, p.i.) and declined thereafter. The pattern of eNOS expression over the course of EAN largely matched that of caveolin-1. Immunohistochemistry showed that in EAN lesions, intense caveolin-1 immunostaining occurred in ED1-positive macrophages as well as in vessels, while the caveolin-1 immunoreaction was reduced in Schwann cells in the inflammatory lesions. Consequently, we postulated that caveolin-1 expression increased in the sciatic nerves with EAN; this possibly mediated either molecular trafficking or nitric oxide generation partly through the activation of eNOS in vascular endothelial cells, as well as in inflammatory macrophages in EAN and/or cellular apoptosis of inflammatory cells.

Induction of Gas6 protein in CCl4-induced rat liver injury and anti-apoptotic effect on hepatic stellate cells

The protein product of the growth arrest-specific gene 6 (Gas6) is a secreted ligand for tyrosine kinase receptors, among which Axl is the most widely distributed and displays the highest affinity for Gas6. The Gas6/Axl signaling pathway has been increasingly implicated in growth and survival processes occurring during development and tissue repair. In liver, after an acute or chronic injury, repair involves macrophages and hepatic stellate cells (HSC) activated into myofibroblastic cells (HSC/MFB), which produce cytokines and matrix proteins. We investigated the expression and the role of Gas6 and its receptor Axl in liver repair. Three days after CCl4-induced liver injury in the rat, we detected the expression of Gas6 in ED1-positive macrophages as well as in desmin-positive HSC, which accumulated in injured areas. Axl, the high-affinity receptor for Gas6, was detected in macrophages, HSC, and HSC/MFB. In vitro, expression of gamma-carboxylated Gas6 was strongly induced in HSC along with their transformation into myofibroblasts, and it exerted an anti-apoptotic effect on both HSC and HSC/MFB mediated by the Axl/PI3-kinase/Akt pathway. In conclusion, Gas6 is a survival factor for these cells and we suggest that Gas6, secreted by macrophages and HSC/MFB in vivo after liver injury, promotes HSC and HSC/MFB survival and might support transient HSC/MFB accumulation during liver healing.

Intra- and extraneuronal changes of immunofluorescence staining for TNF-alpha and TNFR1 in the dorsal root ganglia of rat peripheral neuropathic pain models.

1. Several lines of evidence suggest that cytokines and their receptors are initiators of changes in the activity of dorsal root ganglia (DRG) neurons, but their cellular distribution is still very limited or controversial. Therefore, the goal of present study was to investigate immunohistochemical distribution of TNF-alpha and TNF receptor-1 (TNFR1) proteins in the rat DRG following three types of nerve injury. 2. The unilateral sciatic and spinal nerve ligation as well as the sciatic nerve transection were used to induce changes in the distribution of TNF-alpha and TNFR1 proteins. The TNF-alpha and TNFR1 immunofluorescence was assessed in the L4-L5 DRG affected by nerve injury for 1 and 2 weeks, and compared with the contralateral ones and those removed from naive or sham-operated rats. A part of the sections was incubated for simultaneous immunostaining for TNF-alpha and ED-1. The immunofluorescence brightness was measured by image analysis system (LUCIA-G v4.21) to quantify immunostaining for TNF-alpha and TNFR1 in the naive, ipsi- and contralateral DRG following nerve injury. 3. The ipsilateral L4-L5 DRG and their contralateral counterparts of the rats operated for nerve injury displayed an increased immunofluorescence (IF) for TNF-alpha and TNFR1 when compared with DRG harvested from naive or sham-operated rats. The TNFalpha IF was increased bilaterally in the satellite glial cells (SGC) and contralaterally in the neuronal nuclei following sciatic and spinal nerve ligature. The neuronal bodies and their SGC exhibited bilaterally enhanced IF for TNF-alpha after sciatic nerve transection for 1 and 2 weeks. In addition, the affected DRG were invaded by ED-1 positive macrophages which displayed simultaneously TNFalpha IF. The ED-1 positive macrophages were frequently located near the neuronal bodies to occupy a position of the satellites. 4. The sciatic and spinal nerve ligature resulted in an increased TNFR1 IF in the neuronal bodies of both ipsi- and contralateral DRG. The sciatic nerve ligature for 1 week induced a rise in TNFR1 IF in the contralateral DRG neurons and their SGC to a higher level than in the ipsilateral ones. In contrast, the sciatic nerve ligature for 2 weeks caused a similar increase of TNFR1 IF in the neurons and their SGC of both ipsi- and contralateral DRG. The spinal nerve ligature or sciatic nerve transection resulted in an increased TNFR1 IF located at the surface of the ipsilateral DRG neurons, but dispersed IF in the contralateral ones. In addition, the SGC of the contralateral in contrast to ipsilateral DRG displayed a higher TNFR1 IF. 5. Our results suggest more sources of TNF-alpha protein in the ipsilateral and contralateral DRG following unilateral nerve injury including macrophages, SGC and primary sensory neurons. In addition, the SGC and macrophages, which became to be satellites, are well positioned to regulate activity of the DRG neurons by production of TNF-alpha molecules. Moreover, the different cellular distribution of TNFR1 in the ipsi- and contralateral DRG may reflect different pathways by which TNF-alpha effect on the primary sensory neurons can be mediated following nerve injury.

Increased Expression of Osteopontin in the Heart Tissue of Lewis Rats with Experimental Autoimmune Myocarditis

The expression of osteopontin (OPN) in the hearts of rats with experimental autoimmune myocarditis (EAM) was evaluated. Western blot analysis showed that OPN was significantly increased in the hearts with EAM compared with those of complete Freund's adjuvant (CFA) immunized control. Immunohistochemically, OPN was weakly expressed in the cardiomyocytes in the heart with normal and CFA immunized controls. In EAM lesions, OPN was intensely immunostained in some inflammatory cells, mainly ED1 positive macrophages. These findings suggest that OPN is significantly increased in EAM lesions and that OPN mediates the inflammatory process in the course of rat EAM model.

Expression of CD44 Adhesion Molecule in Rat Testis with Ischemia/Reperfusion Injury

The expression pattern of CD44 was studied in the rat testis following ischemia/reperfusion (I/R) injury to elucidate the possible role of the CD44 adhesion molecule in acute experimental testicular torsion. Western blot analysis showed that CD44 expression began to increase significantly 24 hr after reperfusion, compared with the normal control; the increased expression persisted until 96 hr after I/R. Immunohistochemistry showed that, in the normal testis, CD44 was constitutively expressed mainly in ED2-positive resident macrophages in the interstitial space. After I/R, the majority of inflammatory cells in the interstitial space surrounding the damaged tubules were ED1-positive macrophages that were CD44-positive. These findings suggest that the significant increase in CD44 expression that occurs during the delayed phase after reperfusion originates from infiltrating macrophages possibly in anticipation of the migration and adhesion of additional macrophages into the affected testis.

Increased expression of caveolin-1 and -2 in the hearts of Lewis rats with experimental autoimmune myocarditis

The expression of caveolin-1, -2 and -3 was studied in the hearts of rats with experimental autoimmune myocarditis (EAM), to elucidate the involvement of caveolins in the pathogenesis of EAM. Western blot analysis showed that levels of caveolin-1 and -2 were significantly increased in the hearts of rats with EAM on day 14 post-immunization (pi), as compared to the hearts of normal controls (p < 0.05, normal controls vs. EAM). Caveolin-3 is already at a high level in control animals, so it does not increase further.Immunohistochemistry showed that caveolin-1 was expressed mainly in ED1-positive macrophages and in some cardiomyocytes and vessels in the EAM lesions. Caveolin-2 was expressed constitutively in the vascular endothelial cells of normal hearts, and its expression was enhanced in EAM rats, as compared with the normal control group. Caveolin-3 was expressed constitutively in the plasma membranes of cardiomyocytes, but not in the vascular endothelial cells and inflammatory cells in the EAM lesions. Our results suggest that the expression of caveolin-1 and -2 is increased in EAM lesions and that the increased expression of caveolin-1 stimulates second signals in affected cells, such as macrophages and some cardiomyocytes, in EAM rats.