| Abstract | Uvod i cilj istraživanja: Metalotioneini (MT) su ubikvitarna obitelj malih unutarstaničnih proteina, molekularne mase 6–10 kDa, koja je raspostranjena u mnogim organizmima, ako i u stanicama sisavaca, a razlikuju se tri izoforme molekule MT-a (MT-I, II i III). Imaju sposobnost vezivanja različitih metala, te sudjeluju u brojnim fiziološkim i patofiziološkim procesima primajući i otpuštajući metale, a ovisno o tkivnim potrebama mogu vršiti preraspodjelu tkivnih koncentracija metala koje služe kao kofaktori za mnoge stanične enzime regulirajući time, za život organizma, važne i neophodne biološke procese. Proteini toplinskog šoka su ubikvitarni, evolucijski konzervirani proteini, esencijalni za život stanice, te ih s punim pravom tako nazivamo jer njihova razina izražaja znatno raste u stanici tijekom povišenja temperature. Glavni predstavnik proteina toplinskog šoka unutar grupe hsp90 je glukoza–related protein 94 (grp94) ili glikoprotein 96 (gp96). U promijenjenim uvjetima u stanici nastaju denaturirani proteini koji zbog svojih hidrofobnih domena teže stvaranju proteinskih agregata, stanicu štite upravo proteini toplinskog šoka zahvaljujući njihovom svojstvu da djeluju kao „chaperoni“. Ovi proteini imaju sposobnost imunomodulacije, tj. poticanja specifičnog imunološkog odgovora pri čemu se različiti peptidi i antigeni mogu predočiti kao kompleks gp96-peptid na površini antigen predočnih stanica, nakon čega dolazi do aktivacije pomagalačkih i citotoksičnih limfocita T čega dolazi do poticanja imunološkog odgovora protiv peptida iz navedenog komplek protiv peptida iz navedenog kompleksa.
S obzirom da su MT i gp96 stresni proteini, cilj istraživanja je bio utvrditi obrazac izražaja MT-I i MT-II te gp96 na proteinskoj i genskoj razini u različitim organima eksperimentalnih životinja (jetra, slezena, timus, bubreg, pluća, mozak), kao i u modelima tkivnih oštećenja (mi smo istražili u: stresu, intoksikaciji benzinskim parama i u primjeru upalnog modela autoimunog poremećaja, tj. u eksperimentalnom alergijskom encefalomijelitisu EAE-u). Budući da MT proteini vežu odnosno otpuštaju metale, usporedit ćemo njihov tkivni izražaj sa tkivnom koncentracijom metala. Budući da gp96 protein ima imunomodulacijska svojstva, usporedit ćemo razinu njegovog izražaja u modelu stresa i intoksikacije sa promjenama imunofenotipa i citotoksičnosti mononuklearnog limfatičnog sustava jetre i slezene.
Materijal i metode: Istraživanja su provedena na C57/BL6 soju miševa i DA soju ženki štakora starosti 2-3 mjeseca (za model EAE-a). Promjene proteinskog izražaja utvrđene su imunohistokemijski na parafinskim tkivnim rezovima, upotrebom monoklonskih protutijela I DAKO EnVision + System kita. Promjene izražaja gena za MT-I i gp96 utvrdili smo klasičnom lančanom reakcijom polimeraze (PCR). Koncentracije tkivnih metala određene su spektrometrijski. Imunofenotipizacija limfatičkih stanica izvedena je metodom izravne imunoflorescencije i analizom protočnom citometrijom, kojom je detektiran i test citotoksičnosti limfocita.
Rezultati: U modelu stresa i intoksikacije utvrđen je porast razine izražaja MT-I i MT-II te gp96 u jetri, slezeni, timusu, bubregu, plućima i mozgu na proteinskoj razini uz pojačanu transkripcijsku aktivnost gena za MT-I i gp96. Istovremeno je zabilježen pad koncentracija Zn i Cu iona u navedenim organima. U modelu EAE-a utvrđen je porast izražaja MT-I i II i gp96 na proteinskoj razini u organima središnjeg živčanog sustava (mozak, vratna i lumbalna leđna moždina) kao i u jetri. Porast razine izražaja MT-I i II u EAE-u je bio u korelaciji s porastom razine tkivne koncentracije Zn i Cu u jetri i organima središnjeg živčanog sustava tijekom različitih faza kroničnog EAE-a. Porast izražaja gp96 je praćen porastom udjela NK i NKT stanica u jetri i slezeni pokusnih životinja izloženih stresu i intoksikaciji. Istodobno, mononuklearne limfatičke stanice jetre i slezene stekle su veći stupanj citotoksične aktivnosti protiv singeničnih antigena.
Zaključak: Porast izražaja MT-I i MT-II, te gp96 proteina u limfatičkim organima, kao i organima koji sudjeluju u detoksikaciji i u organima središnjeg živčanog sustava u modelu stresa, intoksikacije i EAE-a ukazuje na njihovo citoprotektivno i imunomodulacijsko djelovanje, koje je potvrđeno preraspodjelom tkivne koncentracije metala i imunomodulacijskim promjenama u kojima se ističe aktivacija autoreaktivnih klonova (porast udjela NKT stanica i njihove citotoksične aktivnosti protiv ciljnih stanica). |
| Abstract (english) | Introduction and objectives: Metallothioneins (MTs) are ubiquitous family of small intracellular proteins, the molecular mass of 60-10 kDa, widespread in many organisms, as well as in mammalian cells, and we can differ three isoforms of MT (MT-I, II and III). These proteins have an affinity for binding different metals, and participate in various physiological and pathophysiological processes in a way that the receiving and releasing a variety of metals, depending on the needs of the tissues can redistribute tissue concentration of metals that serve as cofactors for many enzymes regulating cell time for the life of the organism important and essential biological processes. Heat shock proteins are ubiquitous, evolutionarily conserved proteins, essential for living cells, because their expression levels significantly augmented in the cell during the temperature increase. The main representative in a group of the heat shock protein Hsp90 is glucose-related protein 94 (grp94) or glycoprotein 96 (gp96). In conditions when the cell resulting denatured proteins due to their hydrophobic domains have a tendency to create protein aggregates, cells protect precisely heat shock protein due to their capacity to act as a “chaperone”. These proteins are capable of immunomodulation and promotion of a specific immune response due to the fact that different peptides and antigens can be presented as a complex of gp96-peptide on the surface of antigen presenting cells followed by activation of helper T lymphocytes and cytotoxic T lymphocytes against peptides from this complex.
The aim of this study was to determine the form expressed MT-I and MT-II and gp96 on protein and gene level in various organs of experimental animals (liver, spleen, thymus, kidney, lung, brain), and in models of tissue damage from which we chose the stress, intoxication with gasoline vapors and model of inflammatory autoimmune disorders, and this is an experimental allergic encephalomyelitis (EAE). Since the MT proteins have a capacity to bind/release metals, we compare their tissue expression with the tissue concentration of metals. As the gp96 protein has immunomodulatory properties, we compare the level of its expression in a model of stress and intoxication with changes in immunophenotype and with cytotoxicity mononuclear lymphatic system of the liver and spleen.
Materials and methods: Studies were conducted on C57/BL6 strain of mice and female DA strain rats aged 2-3 months (for a model of EAE). Changes in protein expression were determined immunohistochemically on paraffin tissue sections, using monoclonal antibodies and DAKO Envision + System kit. Changes in gene expression for MT-I and gp96 were determined by the classical polymerase chain reaction (PCR). Tissue metal concentrations IX
were determined by spectrophotometer. Immunophenotypic analyses of mononuclear lymphatic cells were performed by direct immunofluorescent staining and flow cytometry, which was also used for functional cytotoxicity assay.
Results: In the model of stress and intoxication we found rising levels of expression MT-I and MT-II and gp96 in the liver, spleen, thymus, kidney, lung and brain of the protein level and increased transcriptional activity of the gene for MT-I and gp96. At the same time a decline recorded and the concentrations of Zn and Cu ions in these organs. In the EAE model we have also found increased expression MT-I and II, as well as, the gp96 protein level in the organs of the central nervous system (brain, cervical and lumbar spinal cord) and in the liver. The increase in expression levels of MT-I, MT-II in EAE was correlated with increased levels of tissue concentration of Zn and Cu in the liver and organs of the central nervous system during various stages of chronic EAE-a. The increased expression of gp96 was accompanied by the increase of NK and NKT cells in the liver and spleen of experimental animals exposed to stress and intoxication. Simultaneously, mononuclear lymphatic cells of the liver and spleen have gained a greater degree of cytotoxic activity against syngeneic antigens.
Conclusion: The increased expression of MT-I and MT-II and gp96 protein in the lymphatic organs, the organs involved in detoxification or in the organs of the central nervous system in a model of stress, intoxication, and EAE indicate their cytoprotective and immunomodulatory activities, as confirmed by redistribution tissue concentrations of metals and immunomodulatory changes of the dominating activation of autoreactive clones (increased concentration of NKT cells and their cytotoxic activity against target cells). |
