Changes of cell immunity in severe brain injured patients

Cilj istraživanja: Infekcije su vodeći uzrok pobola i smrtnosti bolesnika s teškom ozljedom mozga. Do danas još uvijek nije u potpunosti razjašnjen mehanizam odgovoran za povećanu sklonost infekcijama u ovih bolesnika. Cilj ovog istraživanja je bio utvrditi najčešće infekcije i njihove uzročnike u bolesnika s teškom ozljedom mozga, ispitati promjene stanične imunosti, poglavito udjela leukocitnih subpopulacija, izražavanje citotoksičnog posrednika perforina, aktivnost stanica NK i izražaj pojedinih citokina u različitim imunokompetentnim stanicama periferne krvi bolesnika s teškom ozljedom mozga u ranom razdoblju nakon ozljede. Ispitanici, materijal i metode: U istraživanje je bilo uključeno 40 bolesnika s teškom ozljedom mozga u dobi od 18. do 75. godine života. Zdravi i dobrovoljni davaoci krvi su bili kontrolna skupina. Demografski, klinički i laboratorijski podatci sakupljani su i analizirani su za svakog bolesnika. Svakom bolesniku s teškom ozljedom mozga 1., 4., 7. i 10. dana nakon ozljede uzimali smo uzorak trahealnog aspirata, krvi i mokraće te slali na mikrobiološku analizu. Prvog, 4. i 7. dana nakon ozljede svakom bolesniku uzimali smo 20 ml heparinizirane venske krvi za imunološka istraživanja. Mononuklearne stanice periferne krvi izdvojili smo centrifugiranjem na gradijentu gustoće, te višestrukim obilježavanjem površinskih biljega metodom izravne imunofluorescencije analizirali njihov fenotip i stupanj aktivacije protočnom citometrijom. Izdvojene mononuklearne stanice obilježavali smo istovremeno protutijelima na površinske biljege kako bi utvrdili njihovu pripadnost pojedinim leukocitnim populacijama, permeabilizirali njihovu membranu te metodom izravne i neizravne imunofluorescencije unutarstanično obilježavali perforin, interleukin (IL)-4, IL-15, IFNγ (interferon gama), TNFα (engl. tumor necrosis factor alpha, čimbenik tumorske nekroze alfa) korištenjem odgovarajućih monoklonskih protutijela, te smo stanice analizirali na protočnom citometru. Uzorci koji su bili predviđeni za unutarstanično obilježavanje citokina prethodno smo stimulirali s PMA (engl. phorbol myristate acetat), ionomicinom i monenzinom radi prikazivanja citokina u stanici. Imunocitokemiju smo koristili za prikazivanje molekule perforina i citokina IL-15 u preparatima svježe izdvojenih mononuklearnih stanica periferne krvi. Metodom pozitivne i negativne magnetske separacije iz mononuklearnih stanica periferne krvi izdvojili smo stanice NK i određivali njihovu citotoksičnu aktivnost prema K-562 NK osjetljivoj staničnoj liniji korištenjem PKH-26 (red) dvosatnog testa citotoksičnosti uz očitavanje protočnim citometrom. Rezultati: U 55% bolesnika s teškom ozljedom mozga došlo je do razvoja infekcije (pneumonije, sepse i infekcije mokraćnih putova) s najvećom učestalosti pojave 4. dana nakon ozljede. Najčešći uzročnici pneumonije bili su Staphylococcus aureus, Pseudomonas aeruginosa i Escherichia colli. Sepsa je bila najčešće uzrokovana bakterijom Straphylococcus aureus, dok su najčešći uzročnici infekcije mokraćnih putova bili bakterije Enterococcus faecalis i Escherichia colli, te gljivca Candida albicans. Četvtog dana nakon ozljede u perifernoj krvi bolesnika s teškom ozljedom mozga zabilježili smo statistički značajano smanjenje postotka limfocita T, osobito CD3+CD8+CD56- subpopulacije, smanjenje udjela stanica NK-T, stanica NK i njihovih CD3-CD56+dim i CD3-CD56+bright subpopulacija. Postotak limfocita B nije se značajno mijenjao u perifernoj krvi bolesnika s teškom ozljedom mozga, dok je udio monocita značajno porastao 1. dana nakon ozljede u odnosu na kontrolnu skupinu i smanjio se 7. dana. Inficirani bolesnici s teškom ozljedom mozga imali su statistički značajno smanjenje udjela limfocita T i stanica NK 4. dana nakon ozljede u odnosu na 1. dan i kontrolnu skupinu. U bolesnika s teškom ozljedom mozga zabilježili smo vrlo niski udio proupalnih citokina (IFNγ, TNFα, IL-15) i prevlast IL-4 nad IFNγ u svim leukocitnim subpopulacijama. Stimulacija monocita periferne krvi bolesnika s teškom ozljedom mozga s različitim koncentracijama lipopolisaharida (LPS) potiče stvaranje IFNγ 1. i 7. dana nakon ozljede kao i u kontrolnoj skupini, ali nema učinka 4. dana nakon ozljede. Statistički značajno smanjenje postotka perforin pozitivnih limfocita, uključujući limfocite T, stanice NK i stanice NK-T, zabilježeno je u perifernoj krvi bolesnika s teškom ozljedom mozga 4. dana nakon ozljede. Sedmog dana nakon ozljede povećao se udio perforin pozitivnih limfocita s citotoksičnim fenotipom (CD3+CD8+CD56- limfociti T, stanice NK i stanice NK-T) u odnosu na 1. dan. Izdvojene stanice NK iz periferne krvi bolesnika s teškom ozljedom mozga imaju statistički značajno manju citotoksičnost prema NK osjetljivoj staničnoj liniji K-562 4. i 7. dana nakon ozljede u odnosu na 1. dan i kontrolnu skupinu. Četvrtog dana nakon ozljede u bolesnika s teškom ozljedom mozga nađena je pozitivna korelacija između GCS, kao pokazatelja težine ozljede mozga, te postotka limfocita T i stanica NK, kao i između GCS i postotka perforin pozitivnih stanica, uključujući CD3+ perforin+ i CD56+ perforin+ stanice. Zaključak: Visoka učestalost infekcija u bolesnika s teškom ozljedom mozga u ranom razdoblju nakon ozljede mogla bi se objasniti smanjenjem postotka stanica s citotoksičnim fenotipom, smanjenim udjelom perforina u limfocitnih subpopulacijama i njihove citotoksične aktivnosti, te stvaranja proupalnih citokina.Objectives: Infections are leading causes of increased morbidity and mortality of severe brain injured patients. The mechanism underlying the susceptibility to the infections is still unexplained. The purpose of the study was to identify the most common infection and their infection agents in severe brain injured patients, and to analyze the changes in cell-mediated immunity, especially the frequency of leukocytes subpopulations, expression of cytotoxic mediator perforin, activity of NK cells and expression of cytokines in different immune cells in peripheral blood of severe brain injured patients. Patients, material and methods: Forty severe brain injured patients between age of 18 and 75 years we included in the study. Healthy volunteers served as controls. Demographic, clinical and laboratory data were collected and analyzed for each patient. The sample of endothacheal aspirate, blood and urine were taken for each severe brain injured patient and sent for further microbiologic analysis on day 1, 4, 7 and 10 after the injury. The sample of 20 ml of heparinised peripheral blood was taken form each severe brain injured patient on day 1, 4 and 7 after the injury and used for further immunological investigations. Peripheral blood mononuclear cells were obtained after gradient centrifugation and their phenotype and activation levels were analyzed by simultaneous multiple staining of surface antigens using direct immunoflourescency and flow cytometry analyzes. Isolated mononuclear cells were stained simultaneously by monoclonal antibody against surface antigens, specific for different leukocyte populations, and were further permeabilised for intracellular staining of perforin, interleukin (IL)-4, IL-15, IFNγ (interferon gamma), TNFα (tumor necrosis factor alpha). The labelled cells were analysed by flow cytometry. Cell samples that were scheduled for intracellular cytokine staining, were previously stimulated with PMA, ionomycine and monensim which are necessary for cytokine visualisation. Immunocytochemitstry was used for visualisation of perforin molecule and IL-15 cytokine in the samples of freshly isolated peripheral blood mononuclear cells. CD56+ NK cells were isolated from peripheral blood mononuclear cells by positive and negative magnetic cell separation method. The cytotoxic activity of separated CD56+ NK cells against NK sensitive cell K-562 cell line was analysed by PKH-26 (red) cell linker kit and flow cytometry. Results: Infection (pneumonia, sepsis, uroinfection) occurred in 55% of severe brain injured patients with the highest incidence on day 4 after the injury. The most common infection agents of pneumonia were Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia colli. Sepsis was caused by bacteria Staphylococcus aureus, while uroinfection was caused by bacteria Enterococcus faecalis and Escherichia colli, and fungus Candida albicans. On day 4 after the injury statistically significant decrease of peripheral blood lymphocyte T, their CD3+CD8+CD56- subpopulation, as well as percentage of NK-T cell, NK cells including their CD3-CD56+dim and CD3-CD56+bright subpopulation in peripheral blood of severe brain injured patients was observed. The frequency of B lymphocytes in peripheral blood of severe brain injured patients did not change basically during the investigated period, while the frequency of monocytes increased on day 1 in comparison to healthy controls and decreased on day 7. Infected patients with severe brain injury had statistically significant decrease of T lymphocytes and NK cell on day 4 after the injury. Low frequency of proinflammatry cytokines (IFNγ, TNFα, IL-15), as well as domination of IL-4 over IFNγ were observed in all leukocytes populations in severe brain injured patients. LPS stimulate IFNγ production by monocytes in peripheral blood of healthy controls and severe bran injured patients on day 1 and 7, but not on day 4 after the injury. Statistically significant decrease of perforin-positive lymphocytes including T, natural killer (NK) and NKT cells was observed in peripheral blood of severe brain injured patients on day 4 after the injury. On day 7, perforin expression was restored in lymphocyte of cytotoxic phenotype (CD8+ T lymphocytes, NK cells, and NKT cells) compared with day 1. Isolated CD56+ NK cells for peripheral blood of severe brain injured patients showed statistically significant lower cytotoxic activity against NK sensitive cells line K-562 on day 4 and 7 when compared to day 1 and healthy controls. On day 4 after the injury positive correlation was found between GCS, as a measure of severity of brain injury, and percentage of T lymphocytes, and NK cells, as well as between GCS and all perforin positive cells, including CD3+ perforin+ and CD56+ perforin+ cells. Conclusion: High incidence of infection in severe brain injured patients in early posttraumatic period could be explained by diminished percentage of cells with cytotoxic phenotype, decrease in frequency in their perforin expression and cytotoxic activity as well as proinflammatory cytokine production