| Abstract | Cilj istraživanja: Cilj je istraživanjabio utvrditi moguće promjene u raspodjeli i izražaju granulizina (GNLY) u leukocitnim subpopulacijama te u GNLY-posredovanoj citotoksičnosti stanica NK uz analizu popratnog unutarstaničnog izražavanja interferon gama (IFN-γ), interleukina (IL)-4, IL-15 i IL-17 u limfocitnim subpopulacijama periferne krvi bolesnika s aktivnim stadijem i remisijom psorijatičnog artritisa (PsA) u odnosu na skupinu zdravih ispitanika i bolesnika s osteoartritisom koljena (OA). Također je analizirana korelacija između vrijednosti srednjeg intenziteta fluorescencije (MFI) za GNLY s biokemijskim i kliničkim mjernim pokazateljima aktivnosti PsA – hsCRP, SE, HAQ (eng., Health Assessment Questionaire), PASI (eng., Psoriasis Area and Severity Index ili PASI score) te DAPSA (eng. Disease Activity index for PSoriatic Arthritis).Ispitanici i metode:u istraživanje je bilo uključeno 50 bolesnika s aktivnim PsA i 52 bolesnika s PsA u stadiju remisije, koji su zadovoljili uključne kriterije. Kontrolne skupine su sačinjavali bolesnici s osteoartritisom koljena (OA) - (24 bolesnika) i zdravi ispitanici - 20 osoba, koji su po spolu i životnoj dobi odgovarali bolesnicima s PsA. Uzorci periferne krvi (10 ml) sakupljani su nakon predhodno potpisane Informirane suglasnosti odobrene od strane Etičkog povjerenstva Medicinskog Fakulteta Sveučilišta u Rijeci i specijalne bolnice “Thalassotherapia-Opatija” iz Opatije. Suspenziju mononuklearnih stanica periferne krvi dobili smo postupkom naslojavanja krvnih uzoraka i centrifugiranjem na gradijentu gustoće. Zastupljenost GNLY-pozitivnih (+) stanica među mononuklearnim stanicama periferne krvi prikazali smo metodom imunocitokemije na uzorcima dobivenim centrifugiranjem na Cytospin centrifugi. Raspodjelu i udjele GNLY i citokina u mononuklearnim stanicama periferne krvi utvrđivali smo metodom istovremenog obilježavanja unutrastaničnih (GNLY ili pojedinog cikokina) i površinskih (CD14, CD1a, CD19, CD3 i/ili CD56) biljega metodom indirektne i direktne imunofluorescencije Stanicama NK-posredovanu citotoksičnost (sveukupnu smrtnost i apoptozu)prema K-562 liniji stanica humane eritrolukemije određivali smo u 18-satnom testu citotoksičnosti, nakon predhodnog postupka negativnog magnetskog izdvajanja stanica NK Uzorke smo očitavali na FACSCalibur protočnom citometru koristeći se CellQuestPro programom. Rezultate smo analizirali korištenjem računalnog programa WinMDI version 2.9. Statističke analize suučinjene uz pomoć programa Statistika 8.0. Razlike među grupama su ustanovljavane Kruskal-Wallis neparametrijskim testom, a zatim je statistička značajnost računata Mann-Whitney U testom s VIIrazinom statističke značajnosti podešene prema broju međusobnih usporedbi.Povezanost brojčanih varijabli, odnosno korelaciju između vrijednosti srednjeg intenziteta fluorescencije za GNLY (MFI) s biokemijskim i kliničkim mjerilima aktivnosti PsA, određivali smo metodom jednostruke linearne regresijske analize. Rezultati: Učestalost GNLY+ stanica se bitno ne razlikuju unutar ograde mononuklearnih stanica i unutar ograde limfocita periferne krvi (LPK) u svim ispitivanim skupinama. Postotak GNLY+ LPK iznosi do 25% u bolesnika s PsA, i nije se razlikovao obzirom na stadij bolesti, ali je statistički značajno bio niži u odnosu na OA.S druge strane, on je u bolesnika s aktivnim PsA bio uvijek viši nego učestalost GNLY+ stanica u zdravih ispitanika, zahvaljujući višim udjelima GNLY+ stanica unutar skupine limfocita T (p<0,001), stanica NKT (p<0,001) te obiju podskupina stanica NK - CD56+ (p<0,0001) i CD56+bright (p<0,0001). MFI za GNLY je statistički značajno viši u LPK bolesnika s aktivnim stadijem PsA u usporedbi s remisijom bolesti (p<0,01) i zdravim ispitanicima (p<0,001).Vrijednost MFI za granulizin ima vrlo dobru (r=0,41) i statistički značajnu povezanost (p<0,012) s mjerilom PASI za određivanje zahvaćenosti i jačine kožnih promjena kod psorijatične bolesti.Sveukupnacitotoksičnost i apoptoza stanica NK bolesnika s aktivnim stadijem PsA prema K-562 staničnoj liniji je statistički značajno veća, nego kod bolesnika s PsA u remisiji (p<0,01) i zdravih ispitanika (p<0,001) u omjerima od 6:1 do 50:1. Apoptoza K-562 stanica posredovanastanicama NK bolesnika s aktivnim PsA statistički je snažno potisnuta (p<0,01) primjenom protu-GNLY RC8 mAb ili protu-GNLY RF10 mAb, oboje usmjerenih na aktivi oblik GNLY molekule od 9kDa, što nije bio slučaj u testovima citotoksičnosti stanica NK bolesnika s PsA u remisji i zdravih ispitanika, gdje se središnji učinak blokade apoptoze ciljnih stanica postiže primjenom protu-perforinskog mAb. Protu-perforinsko mAb samostalno uklanja apoptozu uzrokovanu stanicama NK u bolesnika s aktivnom fazom bolesti do iste razine do koje i protu-GNLY RC8 mAb i RF10 mAb, ali ne postoji zbrajanje učinaka protu-perforinskog mAb i protu-GNLY mAb.Analizom uzoraka bolesnika s OA koljena uočili smo sličan obrazac raspodjele i udjela GNLY, uključivši i vrijednosti MFI za GNLY kao i kod bolesnika s PsA u aktivnom stadiju. U 18-satnom testu citotoksičnosti, NK-stanicama posredovana apoptoza K-562 stanica ne razlikuje se obimom značajno od vrijednosti u testovima sa zdravim ispitanicima, ali protu-GNLY mAb gotovo u cijelosti može potisnuti apoptozu bolesnika s OA za razliku od zdravih ispitanika. Isti učinak postiže se u prisutnosti protu-perforinskog mAb, a dodatak protu-granulizinskog mAb i protu- VIIIperforinskog mAbne utiče na daljnje smanjivanje apoptoze. Značajno niži udjeli IL-4+ limfocita T (p<0,01) i IL-4+ limfocita NKT(p<0,01) u bolesnika s PsA u aktivnom stadiju i bolesnika s OA u odnosu na zdrave ispitanike ukazuju na nedostatni TH2 odgovor tijekom aktiviranja upalnog procesa. Citokinsku prevlast TH1 odgovora dodatno upotpunjuje značajno viši udio IFN-γ+ stanica NK (p<0,01) i njezinih CD56+dim (p<0,01) i CD56+bright (p<0,01) povdvrsta – u bolesnika s aktivnim stadijem PsA u odnosu na bolesnike u remisiji PsA, na bolesnike s OA i zdrave ispitanike. U bolesnika s OA, udioIL-15+CD56+bright stanica NK te IL-17+CD56+dimstanica NK i IL-17+ limfocita T je statistički značajno veći (p<0,01 za sve skupine) u odnosu na zdrave ispitanike. Zaključak: Ovim smo istraživanjem ukazali na bogatu zastupljenost molekule apoptotičkog posrednika GNLY u citotoksičnim limfocitima periferne krvi bolesnika s PsA, osobito tijekom aktivnog stadija bolesti, kao i bolesnika s OA koljena u odnosu na zdrave ispitanike. Povišene vrijednosti MFI za GNLY u citotoksičnim limfocitima periferne krvi ove skupine bolesnika, osobito u stanicama NK, odražavaju prosječan broj molekula GNLY po pojedinoj stanic, a time iaktivacijski status tih stanica teobjašnjavaju njihovu snažno izraženu citotoksičnost, uključenu u patogenezu bolesti. GNLY je izravno uključen u tijek apoptoze ciljnih stanica i dio svog apoptotičkog učinka posreduje neovisno o perforinu u aktivnoj fazi bolesti PsA i u OA. U stadiju remisije PsA,GNLY ima neznatnu ulogu u apoptozi posredovanoj stanicama NK, jednaku kao u zdravim ispitanicima.Istaknuli smo prevlast TH1 imunosnog odgovora tijekom pogoršanja tijeka PsA i OA koljena, te otvorili mogućnost primjene mjerila MFI za GNLY u procjeni aktivnosti PsA. |
| Abstract (english) | Objectives:The objective of the present study was to investigate possible changes in distribution and expression of granulysin (GNLY) in peripheral blood mononuclear cells (PBMC) as well as in the GNLY-mediated cytotoxicity of NK cells along with the analyses of concomitant intracellular expression of interferon-gama (IFN-γ), interleukin (IL)-4, IL-15 and IL-17 in peripheral blood lymphocytes (PBL) of the patients with active psoriatic arthritis (PsA) and PsA in remission in relation to healthy controls and patients with knee osteoarthritis (OA). The correlation between the value of mean fluorescence intensity for GNLY (MFI) and biochemical and clinical parameters of PsA activity assessment – hsCRP, ESR, HAQ (Health Assessment Questionaire), PASI (Psoriasis Area and Severity Index or PASI score), and DAPSA (Disease Activity index for PSoriatic Arthritis) was analyzed. Patients and methods:A total of 50 patients who met the criteria for active PsA and 52 patients who fulfilled the criteria for the disease in remission were enrolled for the study. The control groups were consisted of patients with knee osteoarthritis (OA) - 24 patients and healthy volunteers - 20 examinees who were matched by age and sex to the PsA groups. Peripheral blood samples (10 ml) were collected from the examinees following signing of The Informed Consent approved by the Ethics Committee of Medical Faculty Rijeka and Special Hospital “Thalassotherapia-Opatija”. Suspension of PBMC was prepared through overlaying blood samples onto Lymphoprep and centrifuging them on density gradient. GNLA positive cells (GNLY+) cells among PBMC were demonstrated by means of immunocytochemistry following preparation of the cells on cytospin centrifuge. In attempt to determine distribution and proportions of GNLY+ cells among PBMC, simultaneous detection of intracellular antigens (GNLY or respective cytokine) and of surface antigens (CD14, CD1a, CD19, CD3 and/or CD56) was performed by means of direct and indirect immunofluorescence. The NK cells-mediated cytotoxicity against K-562 cells of human erithroleukaemia (overall death and apoptosis) was determined in 18-hours cytotoxicity assay, following prior negative magnetic selection procedure of NK cells. The samples were harvested and anaylzed by FACSCalibur using CellQuestPro software. The resusults were analyzed with WinMDI software, version 2.9. Statistical analysis were done with Statistica 8.0 software. The differences between the gropus were determined with Kruskal-Wallis non-parametric test and statistical significance was calculated with Mann-Whitney U test with the levels of significance adjusted according to the number of reciprocal Xcomparisons. The correlation of numeric variables, specifically between MFI level for GNLY with biochemical and clinical parameters of PsA activity assessment was determined by simple linear regression analyses. Results: The frequency of GNLY+ cells is not different within the gate of PBMC and within the gate of PBL in all study groups. The percentage GNLY+ PBL amounts up to 25% in the patients with PsA and was not different with the respect to the disease stage, but it was statistically significantlly lower compared to the OA group. Still, the percentage of GNLY+ PBL was allways higher in patients with active PsA compared to healthy examinees, due to higher proprotions of GNLY+ cells among T cells (p<0,001), NKT cells (p<0,001) and both NK cells subpopulations - CD56+dim NK cells (p<0,0001) and CD56+bright NK cells (p<0,0001). The MFI for GNLY was statistically higher among PBL in patients with active PsA compared to the remission group (p<0,01) and healthy examenees (p<0,001). The MFI for GNLY demonstrated very good (r=0,41) and statistically significant correlation (p<0,012) with PASI score for assessment of skin psoriasis severity. Overall cytotoxicity and apoptosis of NK cells in patients with active PsA against K-562 cells is statisticaly significantly higher compared to the remission group (p<0,01) and healthy examinees (p<0,001) in ratios ranging from 6:1 to 50:1. The NK-cell mediated apoptosis in patients with active PsA against K-562 cells was statisticaly significantly diminished (p<0,01) by the anti-GNLY RC8 mAb or anti-GNLY RF10 mAb, both directed against the active form of GNLY (9 kDa) and that was not so in the cytotoxicity assays with patients in the remission group or healthy volunteers where the key decrease in apoptosis of the target cells was achieved by anti-perforin mAb. Anti-perforin mAb diminishes by itself NK cell-mediated apoptosis in patients with active PsA to the same level as anti-GNLY RC8 mAb or anti-GNLY RF10 mAb but there was no additive effect to the combination of both mAbs. In the knee OA patients we observed the very same pattern of GNLY distribution and GNLY proportions, including the MFI levels for GNLY as they were in patients with active PsA. Cytotoxicity assays revealed thatNK cell-mediated apoptosis in OA patients against K-562 cells is not different in extent compared to healthy examinees, but anti-GNLY mAb may almost entirely diminish apoptosis of the target cells compared to healthy examenees. Very same effect is achieved in the presence of anti-perforin mAb and addition of anti-GNLY mAb and anti-perforin mAb exerts no further effect on apoptosis. Significantly lower percentages of IL-4+ T cells (p<0,01) and IL-4+ NKT cells(p<0,01) cells in patients with active PsA and OA patients XIcompared to healthy examenees suggest insufficient TH2 responses during activation of inflammatory arthritis. The predominance of TH1 cytokines is further corroborated with higher proportion ofIFN-γ+NK cells (p<0,01) and its CD56+dim (p<0,01) and CD56+bright (p<0,01) subpopulations in patients with active PsA, compared to the remission group, the OA group and healthy volunterees. In patients with knee OA, the proportion of IL-15+CD56+bright NK cells and IL-17+CD56+dim NK cells as well as IL-17+ T cells is statisticaly significantly higher (p<0,01 in all groups) compared to healthy examenees. Conclusion:With this studywe emphasizedthe abundant expression of apoptotic mediator, granulysin, among the cytotoxic PBL in patients with active PsA, particularly in the course of active disease, as well as in patients with knee OA, compared to healthy volonterees. A higher MFI level for GNLY in cytotoxic PBL, particularly in NK cells of patients with active disease, represents the average number of GNLY molecules per cell and therefore, mirrors activation status of these cells and explains for their increased cytotoxicity which plays part in the disease progression. GNLY seems to be directly involved in the apoptotic death of target cells and exhibits its effects at least partly through perforin-independent manner in patients with active PsA and patient with knee OA. In the remission of PsA GNLY has a marginal role in the NK-cell mediated apoptosis, as well as among healthy volonterees. We stressed the predominance of TH1 immune responses in the course of exacerbation of PsA and OA, and proposed the new avenue in attempting to assess the activity of PsA by means of MFI for granulysin. |
