Anti-inflammatory and immunomodulatory effects of Biodentine on human periapical lesion cells in culture.

AIM: To evaluate the effect of Biodentine eluate on cytotoxicity and production of pro- and anti-inflammatory cytokines and osteodestructive/osteoprotective cytokines in cultures of human periapical lesion cells. METHODOLOGY: Conditioned Biodentine Medium (CBM) was prepared according to ISO 10993-12, by incubating Biodentine in RPMI medium (0.2 g mL-1 ) for 3 days at 37 °C. CBM contained both released microparticles and leachable soluble components. Inflammatory cells were isolated from 22 human periapical lesions after apicectomy or tooth extraction, by collagenase/DNase digestion, and cultured in several dilutions of CBM. The composition of periapical lesion cells was determined by morphological criteria, cytotoxicity was quantified by MTT and flow cytometric apoptosis/necrosis assays, whereas the levels of produced cytokines in cell culture supernatants were measured by flow cytometry and ELISA. Student t-test and Friedman test with Dunn's post-test were used for comparison of parametric and nonparametric variables, respectively. RESULTS: Undiluted (100%), 75% and 50% CBM were cytotoxic for periapical lesion cells due to induction of both necrosis (100% CBM) and apoptosis (75% and 50% CBM). Noncytotoxic concentrations of CBM (25%) inhibited the production of pro-inflammatory cytokines: TNF-α, (P < 0.005); IL-1ß (P < 0.01); IL-6 (P < 0.005) and chemokines IL-8: (P < 0.005); MCP-1 (P < 0.005), stimulated the production of anti-inflammatory cytokine (IL-10; P < 0.005), Th2 cytokines: IL-4, IL-5 and IL-33 (all P < 0.01), and IL-17A (P < 0.01). The concentration of CBM (12.5%) inhibited the production of IL-6 (P < 0.05), IL-8 (P < 0.01) and MCP-1 (P < 0.005) and augmented the production of IL-10 (P < 0.05). No significant effects on Th1-related cytokines (IFN-γ and IL-12) and IL-23 were detected with 25% and 12.5% CBM concentrations. Both CBM concentrations inhibited the production of osteolytic receptor activator of nuclear factor kappa-Β ligand (RANKL), dose dependently (P < 0.005 and P < 0.01, respectively). Higher CBM concentrations decreased RANKL/osteoprotegerin (OPG) ratio (P < 0.05), without significant influence on the levels of osteoprotective OPG. CONCLUSION: Biodentine possesses immunomodulatory properties by suppressing pro-inflammatory and augmenting anti-inflammatory cytokines. Together with the reduction of osteodestructive mediators, this novel root-end filling cement could be beneficial for healing and bone reparation after the surgical treatment of periapical lesions.

The correlation between lipid peroxidation in different brain regions and the severity of lindane-induced seizures in rats.

The aim of this study was to investigate the dynamics of lipid peroxidation and the possible correlation between lipid peroxidation in different brain regions and behavioral manifestations in lindane-induced seizures in rats. Male Wistar rats were divided into the following groups: 1. control, saline-treated group; 2. dimethylsulfoxide (DMSO)-treated group; 3. lindane-treated group (8 mg/kg), intraperitoneally. Animals were sacrificed 0.5 or 4 h after treatment and the malondialdehyde level and superoxide dismutase (SOD) activity were determined in various brain regions spectrophotometrically. Behavioral changes were classified according to the descriptive scale (0--no response, 1--head nodding, lower jaw twitching; 2--myoclonic body jerks, bilateral forelimb clonus with full rearing; 3--progression to generalized clonic convulsions followed by tonic extension of fore- and hind limbs and tail; 4--status epilepticus). A significant rise in the malondialdehyde level was detected in the cerebral cortex, hippocampus, and thalamus of lindane-treated animals 0.5 and 4 h after administration (P < 0.05). SOD activity (total and mitochondrial) was significantly decreased in the hippocampus and the cortex of lindane-treated animals at both time points (P < 0.05). An initial fall in SOD activity was detected in the thalamus 4 h after lindane administration (P < 0.05). A positive correlation between seizure severity and the malondialdehyde level was found in the hippocampus at both time points (P < 0.01). These results suggest that lipid peroxidation may contribute to the neurotoxic effects of lindane in early acute lindane intoxication and that behavioral manifestations correlate with lipid peroxidation in the hippocampus of lindane-treated rats.

Effect of acute lindane and alcohol intoxication on serum concentration of enzymes and fatty acids in rats.

This study examines possible synergistic effects of lindane and ethanol on inducing liver injury and serum fatty acid derangement in adult male Wistar rats. When administered together, ethanol and lindane-induced even more pronounced increase of alanine aminotransferase (165 +/- 10 U/L) and gamma-glutamyltranspeptidase activity (10.3 +/- 0.6 U/L) than after isolated administration of either substance. In addition, separate administration of lindane and ethanol was followed by a significant decrease of linoleic acid level in the serum (301 +/- 38 mg/L, 276 +/- 35 mg/L vs. 416 +/- 48 mg/L). However, when ethanol administration was followed by lindane injection, serum linoleic acid was at the similar level found in the control group (516 +/- 62 mg/L). Ethanol-treated rats that received lindane 30 min after ethanol administration have shown a marked increase of palmitic (421 +/- 27 mg/L) and linolic acid level (43 +/- 5 mg/L) in comparison with rats that have been treated only with ethanol (316+/-26 mg/L for palmitic and 32 +/- 2 mg/L for linolic acid) or lindane (295 +/- 26 mg/L for palmitic and 301 +/- 38 mg/L for linolic acid). Linolic acid level was significantly greater in comparison with control group (29 +/- 1 mg/L). In conclusion, this study found enough evidence to support the hypothesis that acute ethanol intoxication potentiates lindane-induced liver injury and enhances lipid derangement.

Immunomodulatory effect of xylazine, an alpha(2) adrenergic agonist, on rat spleen cells in culture.

Xylazine is an adrenergic alpha(2) agonist, which is used in veterinary medicine as a sedative and anesthetic agent. In this work we found that xylazine administered in vivo at a dose of 2.5 mg/kg enhanced spleen cell proliferation and interleukin 2 (IL-2) production in cultures stimulated with concanavalin A (Con A), whereas doses of 10 and 25 mg/kg were inhibitory. A similar stimulatory (10 microM) and inhibitory (50-500 microM) effect on splenocyte proliferation and IL-2 production was observed in vitro. Clonidine, another alpha(2) adrenergic agonist, only had a stimulatory proliferative effect on splenocytes. Yohimbine, an alpha(2) adrenergic antagonist, abrogated the stimulatory action of both clonidine and xylazine, but not the suppressive proliferative activity of xylazine in vitro. The inhibited proliferation of splenocytes to Con A correlated with increased apoptosis of T cells. The apoptosis was not blocked by yohimbine or antibodies to Fas and Fas-L. N-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, enhanced proliferation of splenocytes to Con A, partly abrogated the inhibitory effect of xylazine in the proliferation assay, and, only at high concentration (1000 microM), partly suppressed apoptosis of lymphocytes. The enhancing effect of L-NAME on the Con A-induced proliferation of splenocytes correlated with decreased NO production. However, decreased NO production observed in cultures with xylazine was followed by both decreased lymphocyte proliferation and apoptosis. Cumulatively, these results suggest that the immunosuppressive properties of xylazine on splenocytes in vitro are due to increased apoptosis of lymphocytes, predominantly involve NO-independent pathways, and are probably independent of its action through alpha(2) adrenoreceptors.

Comparison of signaling pathways involved in apoptosis of a thymocyte hybridoma triggered by a rat thymic medullary epithelial cell line, dexamethasone or T-cell receptor cross-linking.

Using an in vitro co-culture assay we found that a rat medullary thymic epithelial cell (TEC) line (TE-R2.5) induces apoptosis of the BWRT8 thymocyte hybridoma (TH) (CD4(hi)CD8(low) alphabetaTCR(hi)). TH apoptosis induced by this TEC line was predominantly mediated by direct cell-cell contacts and was potentiated by cross-linking of the T cell receptor (TCR) by R73 monoclonal antibody (mAb). Dexamethasone (Dx) also triggered TH apoptosis but inhibited death of these cells induced by TE-R2.5 cells or immobilized R73 mAb. The TEC-induced apoptosis was independent of the LFA-1/ICAM-1 interaction but partly depended on a novel 29 kDa molecule expressed on TE-R2.5 cells. All three types of TH apoptosis were followed by the cleavage of poly-(ADP-ribose)-polymerase and were blocked by a caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F.PKC stimulation by phorbol myristate acetate interfered with the TH apoptosis induced by TE-R2.5 and Dx, but did not modulate the effect of R73 mAb. On the contrary, inhibition of calcineurin with cyclosporine A did not influence the apoptosis induced by TE-R2.5 and Dx, but completely prevented the R73-triggered TH cell death. The TE-R2.5-mediated BWRT8 apoptosis was suppressed by Na-orthovanadate, an inhibitor of protein tyrosine phosphatases (PTP) as well as by genistein, a protein tyrosine kinase (PTK) inhibitor, while both compounds potentiated the effect of Dx. Blocking PTP, but not PTK decreased the proapoptotic effect of R73 mAb. These results, including those using a BWRT8 subclone (BWRT8-MDP.2) which is resistant to TCR-triggered apoptosis, but sensitive to apoptosis stimulated by TE-R2.5 and Dx, indicate that TE-R2.5-induced TH apoptosis in our model is different from apoptosis in other TEC co-culture models, published so far.

Immunosine (7-thia-8-oxoguanosine) acts as a cofactor for proliferation of T cells.

Immunosine (7-thia-8-oxoguanosine) is a novel guanosine analogue showing immunostimulatory activity both in vivo and in vitro. This compound acts on different components of the immune system including B cells, natural killer (NK) cells and antigen-presenting cells (APC). However, its influence on functions of T cells is poorly understood. In this work we studied the effect of immunosine on proliferation of total rat splenocytes and purified T cells triggered by different mitogens and the mechanisms involved. The results demonstrate that immunosine significantly stimulates proliferation of T cells. The effect was dose-dependent and also depended on concentrations of specific stimulators. Maximal stimulation was seen using 250 microM immunosine. The stimulatory effect of immunosine on lymphocyte proliferation triggered by Concanavalin A (Con A) correlated with increased interleukin 2 (IL-2) production and upregulation of the IL-2 receptor alpha (IL-2Ralpha) expression. The dependency of T-cell proliferation on IL-2/IL-2R was confirmed using neutralizing anti-IL-2Ralpha monoclonal antibodies (mAbs). Higher concentrations of immunosine in the presence of optimal concentrations of Con A (5 microg/mL) inhibited proliferation of T cells. A similar stimulatory effect of immunosine on proliferation of purified T cells and IL-2 production was observed using an anti-T-cell receptor (TCR) mAb and a combination of anti-TCR mAb and IL-2. However, the guanosine analogue did not significantly modulate proliferation of T cells triggered by IL-2 alone. When the combination of phorbol myristate acetate (PMA) and ionomycin was used for T-cell stimulation different results were obtained. Under lower cell stimulation immunosine significantly potentiated T-cell proliferation, expression of IL-2Ralpha and IL-2 production. In the presence of suboptimal stimulation the compound stimulated T-cell proliferation and IL-2Ralpha expression, whereas under maximal stimulation an enhancing effect on IL-2 production was seen. Since direct stimulatory effect of immunosine on T-cell growth in culture was rather weak it can be postulated that the compound acts as a cofactor for T-lymphocyte proliferation.

Xylazine, an alpha 2-adrenergic agonist, modulates proliferation of rat thymocytes in vivo and in vitro.

The effect of xylazine, an alpha 2-adrenergic agonist, on proliferation of rat thymocytes in vivo and in vitro was examined. It was found that the agonist administered to rats in vivo at doses of 2.5 mg/kg and 5 mg/kg stimulated thymocyte proliferation to suboptimal (0.625 microgram/ml) concentrations of concanavalin A (Con A). A similar effect was confirmed in vitro when lower concentrations of xylazine (5 microM) were added to cultures of thymic cells from intact animals in the presence of both suboptimal and optimal (2.5 micrograms/ml) Con A concentrations. Higher doses in vivo (25 mg/kg) and in vitro (50 microM, 100 microM and 250 microM) significantly inhibited proliferation of thymocytes to Con A. The phenomenon was followed by a decrease in interleukin-2 (IL-2) production (in vivo and in vitro) and down-regulation of IL-2 receptor alpha (IL-2R alpha) expression (in vitro). The exogenous IL-2 completely restored the inhibitory effect of xylazine in vivo on thymocyte proliferation. However, a minimal influence of the cytokine on the xylazine-inhibited thymocyte proliferation in vitro was observed. Stimulatory effect of xylazine on proliferation of thymocytes was probably mediated through alpha 2-adrenoreceptors since it was blocked by yohimbine, an alpha 2-adrenoreceptor antagonist. It seems that the pathways involved in inhibition of thymocyte proliferation by xylazine are more complex because the xylazine-suppressed thymocyte proliferation was potentiated by yohimbine.

Proliferation of spleen cells in culture stimulated by 7-thia-8-oxoguanosine: evidence that both B- and T-cells are the targets of its action.

7-thia-8-oxoguanosine (immunosine) is a nucleoside analog showing efficient antiviral activity in rodent models as a consequence of enhancement of the immune response. However, little is known about the mechanisms of its action. In this work the effect of immunosine on proliferation of mouse and rat splenocytes in culture was studied. It was found that the compound stimulated proliferation of lymphocytes in a dose-dependent manner without any additional stimuli. The effect is predominantly mediated by interleukin-2 (IL-2) as judged by increased IL-2 production, upregulation of IL-2 receptor alpha (IL-2R alpha) expression and by significant inhibition (60-75%) of cell proliferation by anti-IL-2R alpha monoclonal antibodies (mAbs). Immunosine also stimulated proliferation both of T- and B-cells purified by immunomagnetic sorting. The response of B-cells was much higher than that of T-cells. The stimulatory effect of immunosine on both lymphocyte subpopulations was further increased by the addition of enriched splenic antigen-presenting cells or purified dendritic cells. Proliferation of purified T-cells to immunosine was also significantly potentiated by an anti-alpha beta T-cell receptor mAb (R 73). All these data suggest that T-, B- and accessory cells in splenic cultures are the targets of immunosine action.

Dynamics of oxidative/nitrosative stress in mice with methionine-choline-deficient diet-induced nonalcoholic fatty liver disease.

Insulin resistance, oxidative stress, and proinflammatory cytokines play a key role in pathogenesis of nonalcoholic fatty liver disease (NAFLD). The aim of our study was to investigate the dynamics of oxidative/nitrosative stress in methionine-choline-deficient (MCD) diet -induced NAFLD in mice. Male C57BL/6 mice were divided into following groups: group 1: control group on standard diet; group 2: MCD diet for 2, 4, and 6 weeks (MCD2, MCD4, and MCD6, respectively). After treatment, liver and blood samples were taken for histopathology, alanine- and aspartate aminotransferase, acute phase reactants, and oxidative/nitrosative stress parameters. Liver malondialdehyde level was higher in all MCD-fed groups versus control group (p < 0.01), while nitrites + nitrates level showed a progressive increase. The activity of total superoxide dismutase and its isoenzymes was significantly lower in all MCD-fed groups (p < 0.01). Although catalase activity was significantly lower in MCD-fed animals at all intervals (p < 0.01), the lowest activity of this enzyme was evident in MCD4 group. Liver content of glutathione was lower in MCD4 (p < 0.05) and MCD6 group (p < 0.01) versus control. : Ferritin and C-reactive protein serum concentration were significantly higher only in MCD6 group. Our study suggests that MCD diet induces a progressive rise in nitrosative stress in the liver. Additionally, the most prominent decrease in liver antioxidative capacity is in the fourth week, which implies that application of antioxidants would be most suitable in this period, in order to prevent nonalcoholic steatohepatitis but not the initial NAFLD phase.

Influence of aging on ethanol-induced oxidative stress in digestive tract of rats.

Aging and ethanol induce oxidative stress due to increased prooxidant production and decreased antioxidative capacity. The aim was to investigate the influence of aging on oxidative stress in liver, stomach and pancreas in acute ethanol intoxication. Adult (3 months) and old (18 months) male Wistar rats were divided into the following groups: control (control group rats aged 3 months (C3) and control group rats aged 18 months (C18)) and ethanol-treated groups (ethanol-treated 3-month-old rats (E3) and ethanol-treated 18-month-old rats (E18)). Ethanol was administered in five doses of 2 g/kg at 12-h intervals by orogastric tube. Tissue samples were collected for the determination of oxidative stress parameters. Malondialdehyde (MDA) concentration was increased in all the experimental groups and investigated organs versus C3 group ( p < 0.01). The highest MDA level was observed in the stomach in E18 group when compared with C18 and E3 groups ( p < 0.01). Activity of total superoxide dismutase (SOD) and its isoenzymes (copper-/zinc-SOD and manganese-SOD) in E18 group was significantly decreased when compared with E3 and C18 groups ( p < 0.01). Nitrates and nitrites (NO x ) concentration was increased in stomach and pancreas for all the groups when compared with C3 group ( p < 0.01). Hepatic, gastric and pancreatic NO x level was significantly increased in E18 group when compared with E3 group ( p < 0.01). Moreover, level of NO x in liver and pancreas in E18 group was significantly increased when compared with C18 group ( p < 0.01). Aging potentiates ethanol-induced oxidative stress in liver, stomach and pancreas due to increased lipid peroxidation and nitrosative stress and decreased antioxidative tissue capacity.

Regulatory T-cells in periapical lesions.

CD4(+)CD25(hi)Foxp3(+) regulatory T-cells (Tregs) are of crucial importance in regulating the immune response, including the control of any defense against infection. Their presence in periapical lesions has not been demonstrated, as yet. We hypothesized that Tregs infiltrate periapical lesions, where they inhibit T-cell proliferation. The aim of this study was to characterize Tregs in periapical lesions by confocal microscopy, flow cytometry, and functional assays. We showed that CD4(+)CD25(hi)Foxp3(+) cells in periapical lesions expressed IL-10 and TGF-beta. Their frequency was significantly higher than in peripheral blood and correlated with the levels of TGF-beta and IL-10 in culture supernatants of periapical lesion mononuclear cells. Tregs inhibited the proliferation of responder T-cells in vitro, at least in part, by stimulating the production of IL-10. These findings suggest that CD4(+)CD25(hi)Foxp3(+) cells in periapical lesions may play regulatory roles in controlling local immune/inflammatory processes.

Immunomodulatory activity of IL-27 in human periapical lesions.

IL-27, a cytokine with pro-inflammatory and anti-inflammatory properties, is a new member of the IL-6/IL-12 family, whose function in periapical lesions is unknown. We hypothesized that the production of IL-27 and its effect depend upon the type of immune/inflammatory response and clinical presentation of periapical lesions. We tested this hypothesis by studying the expression and function of IL-27 in human periapical lesions, both in situ and in culture. Immunohistochemistry demonstrated the strongest expression of IL-27 by endothelial cells and mononuclear phagocytes. Its production by periapical lesion mononuclear cells (PL-MNC), especially in symptomatic lesions, was significantly higher compared with that in peripheral blood MNC and correlated with the frequency of CD14(+) and CD3(+) cells. Exogenous IL-27 stimulated Th1 and down-regulated Th17 cytokine production by PL-MNC from symptomatic lesions, but down-regulated Th1 and Th2 responses in asymptomatic lesions. These findings suggest that IL-27 is an immunomodulatory cytokine in periapical lesions, with complex biological effects.

Liver antioxidant capacity in the early phase of acute paracetamol-induced liver injury in mice.

The aim of our study was to investigate the relationship between liver antioxidant capacity and hepatic injury in the early phase of acute paracetamol intoxication in mice. Male Swiss mice were divided into groups: (1) control, that received saline, (2) paracetamol-treated group (300 mg/kg intraperitoneally). Animals were sacrificed 6, 24 and 48 h after treatment. Oxidative stress parameters were determined in blood and liver samples spectrophotometrically. Liver malondialdehyde and nitrite + nitrate level were significantly increased 6 h after paracetamol administration in comparison with control group (p < 0.05). Paracetamol induced a significant reduction in total liver superoxide dismutase (SOD) and copper/zinc SOD activity at all time intervals (p < 0.01). However, manganese SOD activity was significantly increased within 6 h (p < 0.01), while its activity progressively declined 24 and 48 h after paracetamol administration in comparison with control group (p < 0.01). Content of sulfhydryl groups in the liver was increased 24 h after paracetamol administration (p < 0.05), while its level was decreased within next 24 h when compared to control animals (p < 0.01). Our data showed that liver antioxidant capacity increases in first 24 h of paracetamol-induced liver injury were in correlation with manganese SOD activity and increase in level of sulfhydryl groups.

Ethanol suppressed seizures in lindane-treated rats. Electroencephalographic and behavioral studies.

This study examines the effects of ethanol on lindane-induced seizures in rats. The animals were divided into following groups: 1. saline, 2. DMSO (dimethylsulfoxide), 3. lindane dissolved in DMSO in the dose of 4, 6 or 8 mg/kg (L(4), L(6) and L(8) groups, respectively), 4. ethanol 2 g/kg administered 30 min prior to lindane (protected groups AL(4), AL(6) and AL(8)) and 5. ethanol alone (2 g/kg). In order to determine ethanol concentration in plasma, blood samples were collected by cardiac puncture 30 and 60 min after ethanol injection. For EEG and power spectra recordings, electrodes were implanted into the skull. The lindane treatment resulted in a dose-dependent increase of seizure incidence and severity. The rats displayed severe seizure patterns characterized by high voltage spike-wave complexes, poly-spikes and sleep-like patterns in EEG, while the power spectra were intensively elevated in comparison to the corresponding controls. Ethanol alone led to increased EEG power spectra, which became dominant in the range of 0-4 Hz. For evaluation of anticonvulsant ethanol action we compared latency to seizure, incidence and seizure severity (scale from 0 to 4) in the examined groups. Ethanol diminished seizure incidence in AL(4) and AL(6) groups, decreased intensity of convulsions, and prolonged duration of latency period in AL(8) group. We observed suppression of the EEG signs of lindane-provoked epileptiform activity in AL(4) and AL(6), but not in AL(8) group. These results suggest that ethanol acted protectively on lindane-induced seizures and suppressed behavioral and epileptic EEG spiking activity.

Characterization of antigen-presenting cells in human apical periodontitis lesions by flow cytometry and immunocytochemistry.

AIM: To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. METHODOLOGY: Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. RESULTS: Antigen-presenting cells (HLA-DR(+) cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR(+) CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR(+) CD14(+)), dendritic cells (DC) (HLA-DR(+) CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR(+) CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR(+) cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P < 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P < 0.05). CONCLUSIONS: Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells.

Two novel monoclonal antibodies reactive with different components of the rat thymic epithelium.

Two novel monoclonal antibodies (mAbs) (PT10B7 and PT13D11) have been raised against molecules of rat thymic epithelial cells (TEC). Streptavidin-biotin immunoperoxidase staining and double immunofluorescence using these mAbs and anti-cytokeratin (CK) antibodies showed that PT10B7 and PT13D11 mAbs bound to different components of rat TEC. PT10B7 mAb reacted with cortical and a subset of medullary TEC, whereas PT13D11 mAb labeled subcapsular/perivascular and most medullary TEC, including TE-R 2.5 TEC line of medullary origin. Their staining patterns were different from those seen using mAbs to CK10, CK18 and CK19 polypeptides and other anti-rat TEC mAbs produced so far. The differences in immunoreactivity of these two mAbs on rat thymus during ontogeny and on other epithelial cells of adult rats were also seen. Namely, PT13D11 stained ectoderm-derived epithelia, whereas PT10B7 stained some cells of simple epithelia. Cumulatively, these results reveal a fine phenotypic heterogeneity within rat thymic epithelium.

Bidirectional interactions between thymocytes and thymic epithelial cell lines in vitro.

In vitro interactions of thymocytes and thymocyte hybridomas with cortical (R-TNC.1) and medullary (TE-R 2.5) rat thymic epithelial-cell (TEC) lines were studied. It was found that the cortical line had better adhesion capability. It bound exclusively immature CD4+ CD8+ alpha beta TCRlo thymocytes, induced apoptosis of a subset of these cells, and stimulated proliferation of the BWRT 1 (CD4- CD8- alpha beta TCR-) hybridoma. The medullary line bound both immature and mature thymocytes, decreased their apoptosis, and induced apoptosis of the BWRT 8 (CD4+ CD8lo alpha beta TCRhi) hybridoma. Thymocyte differently modulated cytokine production by TEC lines, upregulating the secretion of IL-1 by R-TNC.1 and IL-6 by TE-R 2.5 cells. Finally, coculture of thymocytes with TEC lines resulted in different patterns of protein-tyrosine phosphorylation in thymocytes. These results show the existence of mutual bidirectional interactions between thymocytes and TEC lines in vitro, but these processes differed depending on phenotypic characteristics and origin of TEC lines used.

Adhesion molecules involved in the binding and subsequent engulfment of thymocytes by a rat thymic epithelial cell line.

A rat thymic epithelial cell (TEC) line (R-TNC.1) was established from a long-term TEC culture. Based on its ultrastructure, phenotype and cytokeratin profile, this line was characterized as a type of cortical TEC. R-TNC.1 cells had nursing activity which was manifested by the binding and subsequent engulfment of thymocytes. The role of adhesion molecules involved in these processes was studied extensively using a coculture of resting thymocytes and unstimulated or interferon-gamma (IFN-gamma)-stimulated R-TNC.1 cells. It was found that a number of adhesion molecules, such as CD2, CD4, CD8, LFA-1, CD18, ICAM-1 and Thy-1, was partly involved in the nursing activity. The effect of monoclonal antibodies (mAb) to these molecules depended on the incubation time and stimulation of R-TNC.1 cells. The inhibitory effect of mAb to CD2, LFA-1, CD18 and ICAM-1 on thymocyte engulfment was higher than their effect on thymocyte binding to the R-TNC.1 line. In addition, a LFA-1/CD18-dependent/ICAM-1-independent adhesion pathway was identified when unstimulated R-TNC.1 cells with minimal expression of ICAM-1 were used. The combination of inhibitory mAb did not completely abrogate the nursing activity of the R-TNC.1 line, suggesting the possible involvement of some other adhesion molecules.