OX6-positive Cells Research Articles

Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP(+)-induced neuronal toxicity.

Nitrative stress is a key component of the pathogenic process in Parkinson's disease (PD), but the relative roles of constitutive neuronal nitric oxide synthase (n-NOS) and inducible nitric oxide synthase (i-NOS) in glial cells remain unresolved. We have investigated the effects of a range of concentrations of the selective n-NOS inhibitor ARR17477, and the selective i-NOS inhibitor 1400W, on MPP(+)-induced cell death in foetal ventral mesencephalic (VM) dopaminergic cultures. MPP(+) induced a loss of TH-positive neurones accompanied by an increase in immunoreactivity for GFAP and OX-6 as markers of astrocytes and activated microglia, respectively, and induced i-NOS immunoreactivity. Unexpectedly, MPP(+) treatment did not induce 3-NT immunoreactivity in the cultures. ARR17477 and 1400W alone had no effect on the number of TH-positive cells or on the number of GFAP or OX-6 positive cells. ARR17477 did not prevent the MPP(+)-induced decrease in TH-positive neurones and had no effect on the increased number of GFAP- and OX-6-positive cells. By contrast, 1400W caused a concentration-dependent preservation of TH-positive neurones in the presence of MPP(+). It also significantly reduced the number of OX-6-immunoreactive cells and there was a small reduction in GFAP immunoreactivity. The results suggest a major role for i-NOS-mediated nitrative stress in microglia in MPP(+)-induced dopaminergic cell death and this may have important implications for developing neuroprotective strategies for PD.

Abstract 171: Effect of Long-Term Treatment With Fimasartan on Transient Focal Ischemia in Rat Brain

Background and purpose: Fimasartan is a novel angiotensin receptor blocker (ninth), and its safety and efficacy have been established as an antihypertensive drug. Anti-apoptotic or anti-inflammatory effects of angiotensin receptor blockers were reported. However, few studies have elaborated on the effects of long-term treatment with low-dose ARB on cerebral ischemia independent of blood pressure lowering. Methods: Noninvasive blood pressure monitoring was performed in Sprague-Dawley rats which 0.5 mg/kg of fimasartan or phosphate-buffered saline (PBS) were administered for 4 weeks. Fimasartan (0.5, 1 or 3 mg/kg) was administered intraorally for 4 weeks, and we induced ischemia-reperfusion injury in rats. Infarct volume and the number of TUNEL-positive apoptotic cells were measured at 7 days. Functional outcomes were evaluated by modified limb placing test. To identify the anti-inflammatory effect of fimasartan, the number of inflammatory cells (OX6-positive cells) was measured. To identify mediators, phospho-STAT3 and IkB proteins were measured via Western blot analyses at 48 hours. Results: Blood pressures were decreased after administration of 1 mg/kg or 3 mg/kg of fimasartan for 4 weeks, but were not decreased in 0.5 mg/kg group. All doses of fimasartan decrease infarct volumes, and low-dose fimasartan (0.5 mg/kg) maximally reduced infarct volume up to 70% of those of the control group ( p < 0.05). Quantitative analysis showed that the number of TUNEL-positive cells was remarkably decreased in the low-dose fimasartan group (622 ± 57 cell/mm 3 vs. 235 ± 48 cells/mm 3 ; p < 0.05). Low-dose fimasartan improved functional outcomes at 14 days (74% recovery in fimasartan group vs. 43% in control group; p < 0.05). The low-dose fimasartan group exhibited a lower number of OX6-positive cells (fimsartan group: 157 ± 17 cells/mm 3 ; control: 86 ± 11 cell/mm 3 ; p < 0.05). The IkB degradation as a marker of NF-kB activation, was delayed, and phospho-STAT3 was decreased in the low-dose fimasartan group ( p < 0.05). Conclusion: We demonstrate that long-term treatment with fimasartan improves neurological outcome in transient focal ischemia in rats. This protective effect of fimasartan is independent blood pressure lowering.

Phenotypic characterization of mononuclear inflammatory cells following equine hydroxyapatite/collagen block grafting in rats

To measure the inflammatory changes associated with the implantation of an equine hydroxyapatite and collagen-containing block graft (eHAC block) in a rodent model system, an eHAC block graft was implanted subcutaneously in rats. Control groups included saline, turpentine oil, and human mineralized particulate allograft (hMPA). Animals were sacrificed and tissue samples obtained after three days, as well as after 1, 2, 4 and 8 weeks. A panel of immunologic probes was used to identify circulatory monocytic cells (ED1), resident mononuclear phagocytes (ED2), mononuclear phagocytes of lymphoid origin (ED3), expression of Ia antigen (OX6), T-cells (OX19), and B-cells (OX33). Immunocytochemical localization was performed and mononuclear cells localized with each immunologic probe counted. Rat sera obtained after eight weeks were used for nitrocellulose dot-blotting to assess circulating anti-equine immunoglobulins. Statistical analysis was performed using two-way analysis of variance, in conjunction with the Bonferroni correction to account for multiple comparisons. A transient increase in monocytes at 3 days and 1 week was observed in all groups, but was significantly higher in the turpentine control (P < 0.0001). A significant increase in the numbers of mononuclear cells detected with clones ED2 and ED3 was observed in specimens from the turpentine group, in contrast to the other groups in the 3 day to 4 week interval (P < 0.0001), as well as within all time periods (P < 0.0001). A statistically significant difference in numbers of ED3-positive cells was observed in the hMPA group compared to the saline and the eHAC block groups after one week (P < 0.0001). Significantly more OX6-positive cells were observed in the turpentine group, compared to other groups (3 days to 1 week; P < 0.0001). T-lymphocytes were essentially absent except for rats given turpentine (after 1 week). No B-lymphocyte response was found and none of the rats developed systemic anti-equine antibodies. These data indicate that a cellular immune response is not elicited following implantation with the eHAC block graft, which might serve as an alternative material for regenerative therapy.

Abstract 28: Detrimental Effect of Leptin on Intracerebral Hemorrhage via STAT3 signal pathway

Background: Leptin, one of the most alleged adipokines, is a key regulator of energy balance and body weight. Leptin exerts its tissue protective effect in ischemic injuries via anti-apoptotic mechanisms. However, few studies have elaborated on the association between leptin and tissue damages in intracerebral hemorrhage (ICH). Methods: We induced ICH in ICR mice via the infusion of collagenase, and recombinant leptin or PBS was administered intraperitoneally. To determine the effects of leptin deficiency, ICH-induction was done in wild-type (WT) or leptin-deficient Lep ob /Lep ob ( ob/ob ) mice. Brain water content, reflecting peri-hematomal edema, and hemorrhage volume were measured at 72 hours. The number of inflammatory cells (OX6-positive cells) was measured at 48 hours, and western blotting was performed at 24 hours. To identify the role of STAT3, a phosphorylated-STAT3 (pSTAT3) inhibitor, NCS74859 , was administered after inducing ICH in hyperleptinemic mice. To localize the working site of pSTAT3, we performed double-staining with a pSTAT3 antibody and a marker of neuron, astrocyte, or macrophage. Results: We compared two doses of leptin and the brain water contents were increased dose-dependently (ρ=0.53, p&lt;0.01). The water contents were 81.5±0.2% in leptin-injected group (8 mg/kg) and 80.1±0.3% in control group (p&lt;0.01). The water content of ob/ob mice was decreased, compared to WT mice in hemorrhagic hemispheres (WT mice: 79.7±0.6%, ob/ob mice: 78.8±0.6%; p&lt;0.05). Mean hemorrhage volumes were not different respectively between ob/ob and WT mice, and between leptin-injected and control groups. The leptin-injected group exhibited a higher number of OX6-positive cells (leptin-injected group: 129±13 cells/mm 3 ; control group: 88±10 cell/mm 3 ; p&lt;0.05). Western blotting revealed that the exogenous-leptin had affected an increase in the levels of pSTAT3, and the relative optical-density was higher, by 1.8-fold, in leptin-injected group than in control group (p&lt;0.05). After inhibiting pSTAT3 using NSC74859 , the brain water content of pSTAT3-inhibiting group was decreased, compared to non-inhibiting group (pSTAT3-inhibiting group: 79.8±0.8%, non-inhibiting group: 81.5±0.2%; p&lt;0.05). Ox6-positive cells were co-localized by pSTAT3, but in neurons and astrocytes, pSTAT3 was not detected. Conclusion: Our study documented that peri-hematomal edema and inflammation were increased by leptin via STAT3 signal pathway. These results may indicate the potentially detrimental role of leptin on ICH.

Immunohistochemical Characterization of Macrophages and Myofibroblasts in α-Naphthylisothiocyanate (ANIT)–Induced Bile Duct Injury and Subsequent Fibrogenesis in Rats

To investigate pathogenesis of post-bile duct (BD) injury fibrosis, interlobular BD epithelial injury was induced in male F344 rats by a single IP injection of α-naphthylisothiocyanate (75 mg/kg body weight) and rats were observed for 12 days. On days 1 to 2, cholangiocytes were injured and desquamated. On days 3 to 5, the affected BD began to regenerate, showing positive staining for CK19 and vimentin. On days 5 to 9, fibrotic areas gradually developed around regenerating BD in Glisson's sheath. These consisted of cells positive for vimentin, desmin, and α-SMA; vimentin- and desmin-positive cells were increased in early stage (days 1-3), whereas α-SMA-positive cells appeared in mid (days 4-7) and late stages (days 8-12), although there were cells coexpressing these cytoskeletons. On day 12, BD regeneration almost completed, with reduced fibrosis. Macrophages positive for ED2 (CD163) increased transiently in early stage, whereas those reacting to ED1 (CD68), OX6 (MHC II), and SRA-E5 (CD204) showed a consistent increase throughout the experiment. Interestingly, OX6-positive cells were limited to Glisson's sheath, whereas SRA-E5-positive cells were seen exclusively along sinusoids of hepatic lobules. MCP-1 mRNA increased significantly in early stage. This study shows that macrophages exhibiting different immunophenotypes and distributions participate in post-BD injury fibrosis associated with myofibroblasts expressing various mesenchymal cytoskeletons.

Immunohistochemical analyses of the kinetics and distribution of macrophages, hepatic stellate cells and bile duct epithelia in the developing rat liver

Non-parenchymal cells in the liver consist mainly of Kupffer cells, hepatic stellate (HS) cells and cholangiocytes. To establish base-line data and clarify the nature, this study investigated immunohistochemically the kinetics of these cell populations in developing liver of F344 rats. Samples were collected from fetuses on days 18 and 20, neonates on days 1, 4, 8, 15, and 21, and adults at weeks 5–35. ED1 (CD68)-positive macrophages showed highest number as early as fetal day 18, and then decreased gradually until adulthood. The numbers of macrophages reacting to ED2 (CD163), OX6 (MHC II), and SRA-E5 (scavenger receptor A, CD204) increased after birth (early neonates), and ED2- and SRA-E5-positive cell numbers were maintained until adulthood, but OX6-positive cell number decreased at late stages of neonates and adulthood. ED2- and SRA-E5-positive cells appeared along sinusoids, indicating Kupffer cells, whereas OX6-positive cells were limited in Glisson's sheath. Vimentin-positive HS cells were seen consistently from fetuses to adulthood. Desmin- and glial fibrillary acidic protein (GFAP)-positive HS cells tended to be seen in fetuses and early stages of neonates. HS cells reacting to α-smooth muscle actin (α-SMA) were not detectable. Cholangiocytes, reacting to cytokeratin 19 and AE1/AE3, began to be seen on fetus day 18 with faint reaction, and interlobular bile ducts were completed in Glisson's sheath by neonatal day 8. This study shows that there are heterogeneous macrophage populations and that HS cells can show various cytoskeletal proteins in rat hepatogenesis.

Epithelial Cell Rests of Malassez and OX6‐Immunopositive Cells in the Periodontal Ligament of Rat Molars: A Light and Transmission Electron Microscope Study

It has been shown that human and cat epithelial cell rests of Malassez (ERM) consist of heterogeneous cell populations. Immunohistochemical and immunoelectron microscopic analyses have verified the presence of neuroendocrine and Merkel-like cells in both of these epithelia. During experimental orthodontic tooth movement, immunocompetent cells have also been found in the vicinity of ERM in rat periodontal ligament (PDL), but have not been characterized in normal rat PDL. The aim of this study was to investigate the presence and distribution of MHC class II antigen presenting cells by using OX6 antibody in ERM of rat molars by light and transmission electron microscopy. Immunohistochemical and immunoelectron microscopic observations of rat maxillary molars confirmed the presence of OX6-positive cells in contact with ERM. Some immunopositive cytoplasmic processes containing vesicles interdigitated with cells of the Malassez epithelial clusters. Based on these findings it can be concluded that immunocompetent cells are localized close to Malassez epithelial clusters in normal rat PDL. Furthermore, the ultrastructural evidences indicate a possible interaction between the epithelial and immunocompent cells and suggest morphological and functional properties for ERM.

Effects of lipopolysaccharide on the appearance of macrophage populations and fibrogenesis in cisplatin-induced rat renal injury

Macrophages play an important role in renal interstitial fibrosis via production of transforming growth factor- β1 (TGF- β1) and tumor necrosis factor- α (TNF- α); these fibrogenic factors mediate induction of myofibroblastic cells capable of producing extracellular matrices. We investigated the effects of lipopolysaccharide (LPS), a macrophage activator, on the appearance of macrophage populations and subsequent fibrogenesis in cisplatin (CDDP)-induced rat renal lesions. In keeping with the progression of interstitial fibrosis, α-smooth muscle actin ( α-SMA)-immunopositive myofibroblastic cell number began to increase on day 4 and continued gradually until day 16 after CDDP injection. Cells immunoreactive for ED1 (for exudate macrophages), ED2 (for resident macrophages) and ED3 (for activated resident macrophages) showed the highest number on day 4 or day 7, and thereafter, the numbers were gradually decreased up to day 16. On the other hand, the number of cells immunoreactive for OX6 (rat MHC class II-recognizing antibody) was increased on day 7 and remained elevated up to day 16. LPS was injected on day 7 after CDDP injection when the greatest number of ED1-positive macrophages were present. In CDDP/LPS-injected rats, the numbers of macrophages reacting to ED1, ED2, ED3, and OX6 were higher than those in CDDP-injected rats during the observation period between days 7 and 16; ED3- and OX6-positive cells were more prominently increased than ED1- and ED2-postive cells. By RT-PCR analysis, the expression of TGF- β1 and TNF- α mRNAs in CDDP/LPS-injected rats on day 7 was markedly increased in contrast to those in CDDP-injected rats. These findings indicate that LPS treatment enhanced the macrophage expression of fibrogenic factors. However, there was no marked difference in the fibrogenesis between CDDP/LPS- and CDDP-injected rats. These findings suggest that the macrophage populations appearing in CDDP-induced rat renal lesions should be investigated further, to address the complicated pathogenesis of renal interstitial fibrosis.

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) (300mg/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.

Regional Differences in the Distribution and Type of Immunocompetent Cells in the Rat Oral Mucosae

The distribution and type of immunocompetent cells were investigated in rat oral mucosae using immunocytochemistry and enzyme histochemistry, focusing on histological structures. We used two antibodies, OX6 and ED1, whichrecognize the rat Ia-antigen and macrophage/monocyte lineage, respectively. Enzymatic histochemistry for acid phosphatase (ACPase) activity and adenosine triphosphatase (ATPase) activity was also employed to identify macrophages and Langerhans cells, respectively. Many OX6-immunopositive cells, dendritic or irregular in shape, were recognizable in the lamina propria of oral mucosae: some cells extended their dendritic processes into the epithelial layer of the buccal and sublingual mucosae. Dendritic cells within the epithelium showed intense ATPase reaction, indicating they could be categorized as Langerhans cells. A small number of ED1-positive cells existed in the lamina propria, but none were present in the epithelial cell layer. Double staining either with OX6 and ED1 or OX6 and ACPase made it possible to divide the immunocompetent cells in the lamina propria into three types: the OX6-positive cells without ED1 or ACPase-reaction, the OX6-negative cells with ED1 and ACPase-reactions, and the OX6-ED1/ACPase-co-expressing cells, each of which possessed characteristic ultrastructural features demonstrated by immunoelectron microscopy. Taking these findings together with previous reports, these three types of cells were regarded as a dendritic-like cell, a macrophage without antigen-presentation ability, and a macrophage with antigen-presentation ability, respectively. There were regional differences in the distribution and density of these immunocompetent cells; they were densely distributed in order of the buccal and sublingual mucosae, the palatal mucosa, and the dorsal surface of tongue. The region-specific distribution and density of the immunocompetent cells might be due to the histological structure of each oral mucosa, suggesting the presence of different immune-defense systems among each portion of the oral mucosae.

Rapid changes in shape and number of MHC class II expressing cells in rat airways after Mycoplasma pulmonis infection

Mycoplasma pulmonis infection in rodents causes a chronic inflammatory airway disease with a strong immunological component, leading to mucosal remodeling and angiogenesis. We sought to determine the effect of this infection on the shape and number of dendritic cells and other major histocompatibility complex (MHC) class II expressing cells in the airway mucosa of Wistar rats. Changes in the shape of subepithelial OX6 (anti-MHC class II)-immunoreactive cells were evident in the tracheal mucosa 2 days after intranasal inoculation with M. pulmonis. By 1 week, the shape of the cells had changed from stellate to rounded (mean shape index increased from 0.42 to 0.77). The number of OX6-positive cells was increased 6-fold at 1 week and 16-fold at 4 weeks. Coincident with these changes, many columnar epithelial cells developed OX6 immunoreactivity, which was still present at 4 weeks. We conclude that M. pulmonis infection creates a potent immunologic stimulus that augments and transforms the OX6-immunoreactive cell population in the airways by changing the functional state of airway dendritic cells, initiating an influx of MHC class II expressing cells, and activating expression of MHC class II molecules by airway epithelial cells.

Immunohistochemical study on pulpal response in rat molars after cavity preparation by Er:YAG laser

While Er:YAG laser systems are in extensive use for caries removal and cavity preparation, the effects of such treatment on pulp tissue remain unclear. This study evaluates these systems using immunohistochemical methods and compares the results with information gained from treatment using conventional burs. Cervical cavities were prepared in the upper first molars of rats, using either an Er:YAG laser or a conventional tungsten-carbide bur. At intervals of 5 min, 6 h, 12 h, 1 d, 3 d and 7 d after cavity preparation, the teeth were processed for immunohistochemical analyses of tissue non-specific alkaline phosphatase, OX6-positive major histocompatibility complex class II antigen-expressing cells and PGP 9.5-immunoreactive nerve fibers. DNA fragmentation was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Tissue non-specific alkaline phosphatase was observed mainly in the subodontoblastic layer under the cavity lesion, from 5 min, in both groups. The immunoreactivity was more pronounced in the laser group, but by 7 d no significant differences were recognizable. At 12 h, TUNEL-positive cells were detected around the odontoblastic layer in both groups. From 3 d to 7 d, a limited number of positive cells were still visible in the group that underwent standard treatment. Clear similarities in the distribution patterns of OX6-immunopositive cells and PGP 9.5-immunoreactive nerve fibers were also noted. From 12 h to 1 d, OX6-positive cells accumulated along the pulp-dentin border, extending their processes into the dentinal tubules. Numerous bead-like PGP 9.5-immunoreactive nerve fibers were observed under the odontoblastic layer at 7 d. These results demonstrated that there was no appreciable difference in the manner in which pulp tissue responded to treatment with either Er:YAG laser or a conventional drill. This would seem to indicate the usefulness of the Er:YAG laser system in the removal of caries and cavity preparation.

Participation of different macrophage populations and myofibroblastic cells in chronically developed renal interstitial fibrosis after cisplatin-induced renal injury in rats.

To shed some light on the mechanisms behind renal fibrogenesis, the present study immunohistochemically investigated the participation of different macrophage populations and myofibroblastic cells in rat renal interstitial fibrosis developed chronically after repeated injection of cisplatin (2 mg/kg body weight, once weekly for 7 weeks). During the 19-week recovery period after the final injection, fibrotic lesions progressively developed in the corticomedullary junction, with the greatest level at post-final injection (FPI) week 5, and then the lesions were gradually repaired by PFI week 19, indicative of a healing process. In conformity with the development of fibrotic lesions, the number of myofibroblastic cells reacting with an anti-alpha-smooth muscle actin antibody was increased, with a peak at PFI week 3, and collagens (types I, III, and IV), fibronection, and laminin were excessively accumulated in these areas. Interstitial cells forming the fibrotic lesions showed mitotic activity at the early stages, whereas they disappeared by apoptosis in the healing process. A large number of cells reacting with an antibody of ED1 (for exudate macrophages), ED2 (for resident macrophages), or OX6 (for major histocompatibility complex class II-presenting macrophages and interstitial dendritic cells) had already appeared at PF1 week 1, and then their numbers increased, with a peak at PFI weeks 7, 3, and 9 in ED1-, ED2-, and OX6-positive cells, respectively. Thereafter, the number of ED1- and ED2-positive cells decreased, whereas the number of OX6-positive cells persisted at a high level until PFI week 19. In the healing process, clusters of lymphocytes were present, the development of which might have been related to OX6-positive cells. The present study demonstrated that chronically developing rat renal interstitial fibrosis might be produced by the complicated mechanisms evoked by interactions between different macrophage populations and myofibroblastic cells, because macrophages show heterogeneous functions depending on microenvironmental factors.

Different distribution patterns of the two mannose 6-phosphate receptors in rat liver.

Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glisson's capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glisson's capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue.

Temporal changes in the distribution and number of macrophage-lineage cells in the periodontal membrane of the rat molar in response to experimental tooth movement

In order to evaluate the possible role of macrophages in the remodelling of periodontal tissue in response to tooth movement, temporal changes in the number and distribution of macrophage-lineage cells in the periodontal membrane of the rat molar tooth after experimental tooth movement were examined immunohistochemically using four anti-rat monoclonal antibodies: ED1 (anti-monocyte/macrophage-lineage cells and dendritic cells), ED2 (anti-resident macrophages), KI-M2R (anti-tissue macrophages), and OX6 (anti-classII molecules). The right maxillary first molar tooth of Wistar rats was moved mesially by a closed-coil spring for 1, 3, 5, or 7 days. Sham-treated rats wearing an inactivated appliance for each experimental period and entirely untreated rats were used as controls. Alternate horizontal serial cryostat sections were cut and incubated with antibodies to ED1, ED2, KI-M2R, and OX6. In addition, cells immunopositive for each monoclonal antibody in the periodontal membrane during tooth movement were analysed on the tension and pressure sides. In the control rats, large numbers of cells positively stained with each monoclonal antibody were distributed throughout the periodontal membrane surrounding the distobuccal root. At 1 day after experimental tooth movement, the number of immunopositive cells obtained with all four monoclonal antibodies decreased as compared with those of the control on the mesial/pressure side. During the later experimental time periods, ED1- and OX6-positive cells in the periodontal membrane of this side were significantly increased in number compared with controls, whereas the density and distribution pattern of cells positive with ED2 or KI-M2R remained unchanged. On the mesial/pressure side, which underwent hyalinization, a marked accumulation of OX6- and ED1-reactive cells, but not of ED2- or KI-M2R-reactive cells, was frequently observed in the area of the hyalinized tissue at 5–7 days after the start of tooth movement. On the distal/tension side, no particular change in the distribution of immunopositive cells obtained with any antibody was detected throughout the experimental periods, with the exception that there was a significant increase in the number of ED1-positive cells and in of OX6-positive cells at 1 and 7 days, respectively, after the start of tooth movement. These results suggest that after the start of tooth movement OX6- and ED1-positive cells, which are mostly exudative macrophages, but not ED2- and KI-M2R-positive cells, i.e., resident macrophages, may be actively engaged in bone resorption and the remodelling of tissues on the pressure side of the periodontal membrane.

Localization and density of myeloid leucocytes in the periodontal ligament of normal rat molars

The phenotypic distribution and density of macrophage-associated antigen-expressing cells in the periodontal ligament (PDL) of normal rat mandibular first molars was evaluated by immunohistochemistry, and an attempt made to identify dendritic cells (DCs) by immunoelectron microscopy. Cells immunopositive to ED1 (a general macrophage marker) were widely distributed throughout the PDL and were most common around blood vessels. A small number of T lymphocytes and OX62 (anti-veiled cells and γδ T cells)-positive DC-like cells were also found. The relative density of cells immunopositive to ED9 (CD14), OX42 (CD11b), OX6 (anti-class II MHC molecules), ED2 (anti-tissue-resident macrophages), 8A2 (CD11c) and WT.1 (CD11a) varied in the mesial, distal and periapical regions of the distal root and the furcal region. This finding suggests that there are several subpopulations of ED1-positive cells which express various combinations of these markers. Immunoelectron microscopy revealed that a small, but distinct, subpopulation of ED1- and OX6-positive cells did have a DC-like ultrastructure, although the majority of these cells were identified as macrophages. The DC-like cells were characterized by poorly developed lysosomal structures and an absence of phagocytic vesicles. It was concluded that the normal rat PDL is equipped with heterogeneous populations of macrophages with regional variations in density. The DC-like cells may function as antigen-presenting cells.

Dendritic cells in the rat pituitary gland evaluated by the use of monoclonal antibodies and electron microscopy.

A detailed analysis of the difference in the localization and the immunoreactivity for various surface markers among folliculo-stellate cells, macrophages, and dendritic cells was performed using immunohistochemistry and electron microscopy of the rat pituitary gland. The folliculo-stellate cells were selectively labeled by an antiserum against S100 protein. The majority of dendritic cells were immunoreactive for the MHC class II (Ia) antigen (OX6) and/or the dendritic cell antibodies (OX62). The main population of macrophages was positive for the macrophage antibodies (ED1, ED2, and/or OX42). The cellular density of adenohypophyseal macrophages was significantly lower than that of folliculo-stellate cells and of dendritic cells. All the neurohypophyseal microglial cells were labeled with OX42, while the mAb OX6 labeled a small population of cells different from the cells identified by OX42 in the neurohypophysis. Double-immunoperoxidase staining for ED1 and OX6 revealed that positively stained cells could be classified into ED1+OX6-, ED1+OX6+, and ED1-OX6+ cells. Double staining with OX62 and OX6 mAbs showed that about 60% of the OX6+ cells were also immunolabeled with OX62 in the anterior lobe: OX62 detects a subpopulation of dendritic cells but does not recognize macrophage populations. Furthermore, double staining for S100 and OX6 resulted in no S100+ OX6+ cells. At the electron-microscopic level, reaction products for OX6 were confirmed in the cell membrane and labeled cells were distinguished from macrophages and folliculo-stellate cells by distinctive short, broad cytoplasmic processes and the rare presence of cytoplasmic organelles. Such cytological characteristics of the OX6-positive cells in the pituitary gland are similar to dendritic cells. Our results suggest that resident dendritic cells and folliculo-stellate cells are two different main components of interstitial cells in the pituitary gland.

Microglial reactions to retrograde degeneration of tracer-identified thalamic neurons after frontal sensorimotor cortex lesions in adult rats.

Thalamic neuronal degeneration after neocortical lesions involve both anterograde and retrograde components. This study deals with the thalamic microglial response after neocortical aspiration lesions, using fluorogold fluorescent prelabeling, to identify retrogradely degenerating thalamocortical neurons, combined with histochemical or immunohistochemical staining of microglial cells. Adult male Wistar rats were injected with the retrograde fluorescent tracer fluorogold, in the right sensorimotor cortex (forepaw area) in order to retrogradely label thalamic neurons projecting to this area. After 1 week, the fluorogold injection site was removed by aspiration, axotomizing at the same time the thalamic projection neurons now retrogradely labeled with fluorogold. After 3, 7, 14, and 28 days the animals were killed and processed for nucleoside diphosphatase histochemistry or complement type 3 receptor immuno-histochemistry and class I and II major histocompatibility complex immunohistochemistry using OX42, OX18, and OX6 antibodies. The histological analysis showed a prominent and progressive nucleoside diphosphatase-, OX42-, and OX6-positive microglial cell response in the ventrolateral, posterior, and ventrobasal thalamic nuclei with ongoing retrograde and anterograde neuronal degeneration. Initially the reactive microglia had a bushy morphology and were succeeded by ameboid microglia and microglial cluster cells as the reaction progressed. However, in the reticular thalamic nucleus, which suffered exclusively anterograde neuronal degeneration, a different picture was seen with only bushy microglia. The neurons undergoing retrograde degeneration in the ventrolateral, posterior, and ventrobasal thalamic nuclei were retrogradely labeled by the fluorogold tracer. Individual nucleoside diphosphatase-, OX42-, or OX6-positive microglial cells extended long cytoplasmic processes surrounding fluorogold-labeled neurons and had in some cases apparently phagocytized these. Several microglial cells were thus double-labeled with nucleoside diphosphatase or OX42 and fluorogold. In addition, small nucleoside diphosphatase-positive, fluorogold-labeled perivascular cells were observed in the neocortex near the fluorogold-injected and ablated neocortical areas and in the ipsilateral thalamus. This study demonstrates: (1) that the microglial response to thalamic degeneration after neocortical lesion is graded with a limited reaction to the well-known massive anterograde axonal degeneration and a more extended reaction to the axotomy-induced retrograde cell death; and (2) that also perivascular cells and possibly macrophages may contribute to this reaction, as seen by uptake of fluorogold from axotomized neurons in the degenerating thalamic nuclei.

The effect of topical cyclosporin on conjunctiva-associated lymphoid tissue (CALT).

Topical cyclosporin A is increasingly being used in the treatment of ocular surface immune-mediated disorders. The availability of the drug in oil-based vehicles or collagen shields has restricted its use because of ocular irritation or blurring of vision. Although topical cyclosporin is being used more frequently, its effect on the immunocompetent cells of the conjunctiva is not known. Our aim was to study the effect of cyclosporin instillation on the immunocomponent cells of conjunctiva-associated lymphoid tissue (CALT) of Lewis rat, using a novel method of topical drug delivery. A suspension of collagen bits impregnated with cyclosporin A was instilled into eyes of Lewis rats for 4 days (group 1) or 8 days (group 2). Control rats (group 3) received the suspension without cyclosporin. Frozen sections of eyelids and conjunctiva were immunostained with the following monoclonal antibody markers: W3/13 (CD3), W3/25 (CD4, macrophages), OX-8 (CD8), MARD-3 (B cells), ED1, ED2 (macro/monocytes), OX-6 (class II MHC, Ia) and OX-39 (CD25, IL-2 receptor). Intraepithelial (IE) and substantia propria cells for each subset were counted and expressed as numbers per section. By day 8, intraepithelial and substantia propria cells for all the above markers, except B cells, showed a significant reduction in numbers. The p values were < 0.02 for W3/13 (CD3), W3/25 (CD4), OX-8 (CD8), OX-39 (CD25) (IE only), ED1, ED2 and OX-6 positive cells. Goblet cells of control animals showed strong positive reaction with OX-39 (CD25) antibody. This was completely abolished following 8 days of topical cyclosporin. This study demonstrated that topical cyclosporin A induces a marked reduction in numbers of all subtypes of immunocompetent cells in the conjunctival epithelium and substantia propria.

Expression of class II MHC molecules in the rat pineal gland during development and effects of treatment with carbon tetrachloride.

Cells expressing major histocompatibility complex (MHC) class II (Ia) antigen have been examined during the development of rat pineals and in the pineal gland of adult rats treated with carbon tetrachloride. Cells positive for MHC class II are first detected in the pineal gland of the 7-day-old rat. These positive cells increase in number gradually during development, MHC class II immunoreactivity reaching adult levels at 4 weeks after birth. The MHC class II antigen is intensely labeled on the cell surface, and labeled cells are distributed throughout the organ, several positive cells being gathered into groups. The positive cells are small (7-12 microm in diameter), irregular in shape, and frequently exhibit one or more processes. At the electron-microscopic level, the cytoplasm of positive cells contains few organelles, variously sized empty vacuoles, and a few electron-dense lysosome-like structures. Pinealocytes with synaptic ribbons have been found adjacent to immunoreactive cells. Double-immunoperoxidase staining for MRC OX6, MRC OX42, and ED1 results in OX6(-)/ED1(+)/OX42(+), OX6(-)/ED1(-)/OX42(+), and OX6(+)/ED1(-)/OX42(- )cells. These findings suggest that OX6-positive cells in the pineal can be considered as peripheral dendritic cells. The number of cells expressing MHC class II (Ia) antigen significantly increases in the pineal gland of rats after treatment with carbon tetrachloride (P<0.005). Our results indicate that at least some of the OX6-positive cells migrate into the gland from the circulation under these conditions.