Diferentovanje timocita u organ kulturi timusa odraslih pacova

To investigate the differences between thymocytes development in vivo and in vitro, thymus lobe fragments from 12-weeks old male Albino Oxford rats were cultivated over a 7-days period. In the controls and cultivated thymic lobes fragments were evaluated and the viability, apoptosis and cell cycle of thymocytes, as well as the histological characteristics of thymic tissue. Additionally, we analyzed the expression of CD4, CD8 and TCRαβ on thymocytes by flow cytometry. The obtained results showed that thymus cellularity decreased during cultivated time due to expanded apoptosis, decreased proliferation and the absence of progenitors reseeding thymus. The relative proportion of thymocyte subsets in the first 24 hours of culture remained similar as in the control. However, cultivation for 3 and 7 days modulated the relative proportions between thymoctye subsets. The percentage of DP TCRαβlow increased, DP TCRαβhi subset remained unchanged, both SP TCRαβhi subsets decreased while the same mature SP phenotype dominated in culture media. These results demonstrate that cultivated thymic fragments retain the capacity for T cell development, although cultivation modulates this process.Sa namerom da ispitamo razlike između in vivo i in vitro sazrevanja timocita gajili smo fragmente lobusa timusa poreklom od mužjaka Albino Oksford pacova, starih dvanaest nedelja, u vremenskom periodu od sedam dana. Nakon kultivacije određivani su vijabilnost, apoptoza i ćelijski ciklus timocita, kao i histološke osobine timusnog tkiva. Takođe je analizirano ispoljavanje markera diferentovanja CD4, CD8 and TCRαβ na površini timocita metodom tečne citofluorometrije. Dobijeni rezultati su ukazali da sedmodnevna kultivacija dovodi do smanjenja broja ćelija u timusu usled povećane apoptoze, smanjene proliferacije i odsustva ulaska progenitora timocita. Tokom prvih 24 sata kultivacije ne dolazi do promena u odnosima timocitnih populacija. Međutim, duže vreme kultivacije - 3 i 7 dana moduliše relativne odnose između timocitnih subpopulacija - povećava se procenat DP TCRαβlow, procenat DP TCRαβhi timocita ostaje nepromenjen, dok su procenti ćelija oba subseta SP TCRαβhi smanjeni, mada je prisustvo pomenutih SP subsetova dominantno u medijumu za kultivaciju. Navedeni rezultati pokazuju da kultivisani fragmenti timusnog tkiva zadržavaju sposobnost da podrže sazrevanje timocita u jednostruko pozitivne T ćelije, mada je diferentovanje timocita donekle modulisano kultivacijom

Fenotipske promene izazvane imunizacijom encefalitogenom menjaju funkcije peritonealnih makrofaga u dva soja pacova različite osetljivosti prema indukciji eksperimentalnog autoimunskog encefalomijelitisa (EAE).

We have investigated the phenotype of peritoneal cells and the functions of peritoneal macrophages obtained from experimental autoimmune encephalomyelitis (EAE)-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rat strains on days 1, 3 and 7 post immunization with encephalitogen. Resident peritoneal cells from immunized and non-immunized rats of both strains were subjected to flow cytometric analyzes and after adherence were tested for zymosan phagocytosis, hydrogen peroxide (H2O2) and nitric oxide (NO) production. In non-immunized rats, macrophages from the DA rat strain phagocytosed more zymosan but produced less H2O2 than cells from the AO strain, while both strains produced comparable amounts of NO. Immunization increased phagocytosis in DA rats' cells, but decreased both phagocytosis and H2O2 production in cells from AO rats. Overall higher phagocyte ability in DA rats was associated with a significantly larger population of ED1+ cells (macrophages and dendritic cells), in contrast to a more pronounced expression of ED2 antigen (resident macrophages) on cells from AO rats. Immunization also increased the expression of CD11b molecule on non-resident ED2-macrophages of DA, but not of AO rats. The early and subtle phenotype changes in peritoneal cells of both rat strains might mirror the mechanism contributing to their different sensitivity to the induction of autoimmunity.Ispitivan je fenotip peritonealnih ćelija, kao i funkcije peritonealnih makrofaga, izolovanih od pacova Dark Agouti (DA) soja osetljivog na indukciju eksperimentalnog autoimunskog encefalomijelitisa (EAE) i pacova Albino Oxford (AO) soja koji je rezistentan prema EAE-u, 1, 3. i 7. dana nakon imunizacije encefalitogenom. Rezidentne peritonealne ćelije su ispitivane metodom protočne citofluorometrije, a zatim je nakon adherence testirana njihova sposobnost fagocitoze čestica zimozana i kapacitet produkcije vodonik peroksida (H2O2) i azot monoksida (NO). U neimunizovanih pacova makrofage DA soja su intenzivnije fagocitovale čestice zimozana i imale nižu sposobnost produkcije H2O2 nego ćelije pacova AO soja, ali nije bilo sojnih razlika u sposobnosti produkcije NO. Imunizacija je dovela do povećanja fagocitne sposobnosti makrofaga DA pacova, ali i do smanjenja fagocitoze i produkcije H2O2 makrofaga pacova AO soja. Generalno veću sposobnost fagocitoze u DA pacova prati i značajno veća zastupljenost ED1+ ćelija (koje čine uglavnom makrofage i dendritične ćelije) nasuprot većoj zastupljenosti ED2 antigena (marker rezidentnih makrofaga) na ćelijama pacova AO soja. Imunizacija encefalitogenom je takođe dovela do povećanja ekspresije CD11b molekula na nerezidentnim ED2- ćelijama pacova DA, ali ne i AO soja. Rane i diskretne fenotipske promene na peritonealnim ćelijama pacova oba soja verovatno odslikavaju mehanizme koji doprinose njhovoj različitoj osetljivosti prema indukciji autoimunskih oboljenja

Razlike u edemu šape pacova indukovanom konkanavalinom a u zavisnosti od soja - uticaj histaminskih H1 i H2 receptora

The present study tests the hypothesis that the difference in the intensity of paw edema found between the Dark Agouti (DA) and Albino Oxford (AO) rat strains originates from the distinct participation of histamine, serotonin and their corresponding receptors in Concanavalin A (Con A)-induced inflammation. DA and AO male rats were intraplantarly injected with specific receptor antagonists prior to Con A, and the intensity of inflammation was determined by measuring the paw diameter. Our results have showed that histamine H1 and H2 receptor antagonists reduced the Con A-induced paw edema in DA rats, while serotonin 5HT3 receptor antagonist diminished the inflammation in both DA and AO rat strains. The calcium channel blocker did not change Con A-induced inflammation. Strain differences in the intensity and kinetics of inflammation observed between the DA and AO rats are most likely defined by the diversity of mediators released and their receptors activated upon Con A injection.Testirana je hipoteza da razlike u intenzitetu inflamatornog edema šape indukovanog konkanavalinom A u pacova Dark Agouti (DA) i Albino Oxford (AO) soja potiču od različitog doprinosa histamina i serotonina i njihovih odgovarajućih receptora. Mužjaci pacova DA i AO soja su intraplantarno tretirani antagonistima specifičnih receptora pre izazivanja inflamacije konkanavalinom A i intenzitet inflamacije je praćen merenjem dijametra šape. Naši rezultati su ukazali da antagonisti histaminskih H1 i H2 receptora smanjuju edem šape indukovan konkanavalinom A u DA pacova, dok antagonist serotoninskih 5HT3 receptora smanjuje edem šape u oba soja pacova. Blokator kalcijumskih kanala ne utiče na inflamaciju izazvanu konkanavalinom A. Razlike u intenzitetu i kinetici inflamatornog odgovora indukovanog konkanavalinom A između DA i AO sojeva su najverovatnije posledica razlika u oslobođ enim medijatorima i aktivaciji odgovarajućih receptora nakon injekcije konkanavalina A

Strain differences in peritoneal macrophage activity and susceptibility to experimental allergic encephalomyelitis induction in rats

The objective of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (EAE). Rats of EAE-susceptible Dark Agouti and EAEresistant Albino Oxford strain were immunized with guinea pig spinal cord homogenate (DA(GPSC) and AO(GPSC)), while non-immunized rats served as controls (DA(NIM), AO(NIM))- On day 15 after immunization rats were sacrificed and their peritoneal macrophages tested for adherence capacity, zymosan phagocytosis and respiratory burst. Macrophages from AO(NIM) rats exhibited lower adherence capacity and higher phagocytosis and H2O2 production when compared to macrophages from DA(NIM) rats. Immunization with GPSC decreased adherence and phagocytosis and increased H2O2 production in macrophages from AO rats, but did not influence these activities in macrophages from DA rats. The results from the present study suggest that inflammatory activities of macrophages from AO rats could be considered as regulatory mechanisms connected with the resistance to EAE induction

NPY suppressed development of experimental autoimmune encephalomyelitis in Dark Agouti rats by disrupting costimulatory molecule interactions

Neuropeptide Y (NPY) suppressed clinical experimental autoimmune encephalomyelitis (EAE) and reduced numbers of CD28+, CD11b+ and CD80+ cells among spinal cord infiltrating cells at the peak of disease in Dark Agouti rat strain. Suppression of EAE was accompanied by the reduced expression of costimulatory CD80 and CD86 molecules on ED1+ macrophages and OX62+ dendritic cells in draining lymph nodes during the inductive phase of EAE. An inhibitor of dipeptidyl peptidase 4, an enzyme which terminates the action of NPY on 11 receptor subtype, did not sustain the suppressive effect of NPY on the EAE development, suggesting involvement of Y2 and Y5 receptors. (C) 2012 Elsevier B.V. All rights reserved

Neuropeptide Y modulates functions of inflammatory cells in the rat: Distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocito-chemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay. (C) 2011 Elsevier Inc. All rights reserved

Peritoneal mast cell degranulation differently affected thioglycollate-induced macrophage phenotype and activity in Dark Agouti and Albino Oxford rats

Aims: Macrophages are heterogeneous population of inflammatory cells and, in response to the microenvironment, become differentially activated. The objective of the study was to explore macrophage effector functions during different inflammatory conditions in two rat strains. Main methods: We have investigated the effects of in vivo treatment with mast cell-degranulating compound 48/80 and/or thioglycollate on peritoneal macrophage phagocytosis and capacity to secrete hydrogen peroxide (H2O2), tumor necrosis factor-alpha (INF-alpha) and nitric oxide (NO) in Dark Agouti (DA) and Albino Oxford (AO) rat strains. Besides, fresh peritoneal cells were examined for the expression of ED1, ED2 and CD86 molecules. Key findings: In thioglycollate-elicited macrophages, increased proportion of ED1 + cells was accompanied with elevated phagocytosis of zymosan (DA strain), whereas increased expression level of CD86 molecule on ED2 + macrophages matched elevated secretory capacity for H2O2, TNF-alpha and NO (AO rats). Although mast cell degranulation induced by compound 48/80 increased the percentages of ED2 + macrophages in both rat strains, the proportion of ED2 + cells expressing CD86 molecule was decreased and increased in DA and AO rats, respectively. Furthermore, in DA strain compound 48/80 diminished macrophage secretion of NO, but stimulated all macrophage functions tested in AO strain. If applied concomitantly, the compound 48/80 additively increased macrophage activity induced by thioglycollate in AO rats. Significance: Macrophages from DA and AO rat strains show different susceptibility to mediators released from mast cells, suggesting that strain-dependant predisposition(s) toward particular activation pattern is decisive for the macrophage efficacy in response to inflammatory agents. (c) 2013 Elsevier Inc. All rights reserved

Methionine-enkephalin modulation of hydrogen peroxide (H2O2) release by rat peritoneal macrophages involves different types of opioid receptors

We investigated the involvement of specific types of opioid receptors in methionine-enkephalin (MET)-induced modulation of hydrogen peroxide (H2O2) release by rat macrophages primed with sub-optimal concentrations of phorbol myristate acetate (PMA). Peritoneal macrophages in vitro treated with different concentrations of MET were tested for H2O2 release in phenol red assay. In the antagonistic study macrophages were treated with MET and one opioid receptor antagonist, or combination of MET and two or three opioid receptor antagonists. MET decreased H2O2 release in eight individual macrophage samples, and increased it in 10 samples. The increase of H2O2 release induced by MET in macrophages was blocked with combination of opioid receptor antagonists specific delta(1,2) and mu receptors, as well as with combination of antagonists specific for delta(1,2) and kappa opioid receptors. MET-induced decrease of the H2O2 release in macrophages was prevented by opioid receptor antagonists specific for delta(1,2) or mu receptors, and also with combination of two or three opioid receptor antagonists. MET-induced enhancement of H2O2 release was mediated via delta(1) or delta(2) opioid receptor subtypes, or by mu-kappa opioid receptor functional interactions, while MET-induced suppression involved functional interactions between delta(1) and mu, delta(2) and mu, or delta(1) and kappa opioid receptors. It is possible that individual differences in basal or induced macrophage capacity to produce H2O2 might shape the repertoire of opioid receptors expression and in that way pre-determine the direction of MET-induced changes after the in vitro treatment. (c) 2007 Elsevier Ltd. All rights reserved

The effects of corticosterone and beta-endorphin on adherence, phagocytosis and hydrogen peroxide production of macrophages isolated from Dark Agouti rats exposed to acute stress

Background: Given that stressful experiences can change the reaction to a subsequent exposure to stress, we tested the in vitro effects of the stress mediator corticosterone and the opioid peptide beta-endorphin on the function of macrophages isolated from control rats and from rats exposed to electric tail shock stress (ES) or a stress-witnessing procedure (SW) 24 h earlier. Methods: Peritoneal macrophages isolated from control and stressed rats of the Dark Agouti (DA) strain were treated in vitro with corticosterone or beta-endorphin and tested for adherence, phagocytosis and hydrogen peroxide release. Results: ES diminished adherence and SW decreased phagocytosis. The suppressive effect of corticosterone on phagocytosis was absent in rats exposed to ES and SW, while the suppressive effect of beta-endorphin on adherence was not observed in rats exposed to SW. ES and SW did not affect H2O2 release, neither directly nor indirectly by changing macrophage response to corticosterone and beta-endorphin in this test. Conclusions: In DA rats early macrophage activation steps, i.e. adherence and phagocytosis, were more sensitive to stress than their effector function, corresponding to H2O2 production. We suggest that neuroendocrine mediators of stress that converge on macrophages might have changed specific macrophage receptors or postreceptor events and alter their response to artificial stressors, represented by corticosterone and beta-endorphin in vitro. Copyright (C) 2008 S. Karger AG, Basel