| Abstract | Artritis potaknut antigenom je eksperimentalni mišji model reumatoidnog artritisa induciran metiliranim goveđim serumskim albuminom. Jedna od glavnih karakteristika upalnog zglobnog zbivanja je sinovijalna hiperplazija, pri čemu hiperplastična sinovija sadrži mezenhimne prethodnike čija je diferencijacija u osteoblaste, hondroblaste ili adipocite poremećena. Kako se zna da Fas može inhibirati diferencijaciju osteoblasta podrijetlom iz koštane srži, cilj je ove studije bio pokazati izažavaju li osteoblasti sinovijalnih mezenhimnih stanica uzgojenih in vitro receptor Fas, opisati ulogu receptora Fas u njihovoj apoptozi i diferencijaciji, te razjasniti in vivo ulogu nedostatnosti receptora Fas u razvoju artritisa potaknutog antigenom.
Osteoblasti sinovijalnog podrijetla slabo izražavaju receptor Fas, čija je aktivacija in vitro povećala broj apoptotičnih nezrelih osteoblasta, no taj je broj bio manji od udjela osteoblasta koji su izražavali Fas. Dodavanje protutijela anti-Fas nije potaklo apoptozu u zrelih osteoblasta. Usprkos ograničenom apoptotičnom djelovanju, aktivacija receptora Fas in vitro je značajno smanjila diferencijaciju osteoblasta sinovijalnoga podrijetla.
Za procjenu in vivo učinka nedostatnosti receptora Fas koristila sam mišji model artritisa potaknutog antigenom, koji sam izazvala u miševa divljega tipa i miševa s izbačenim genom za receptor Fas (Fas –/–). Zglobnu upalu izazvala sam injekcijom metiliranog goveđeg serumskog albumina u životinja prethodno hiperimuniziranih istim antigenom, a potom zglobne promjene uspoređivala s kontrolnim miševima injiciranim fiziološkom otopinom puferiranom fosfatima, i to četrnaest dana nakon intraartikularne injekcije. Artritis je u miševa divljega tipa izazvao značajno povećanje promjera koljenih zglobova u usporedbi s kontrolnim miševima, a to je povećanje izostalo u miševa Fas –/–. Histološka procjena pokazala je znatno upalno oštećenje u miševa divljeg tipa kojima je potaknut artritis, koje je također bilo puno slabijeg intenziteta u miševa Fas –/–.
Histomorfometrijska analiza jukstaartikularnih metafiza pokazala je smanjenje volumena trabekularne kosti, broja i širine koštanih gredica te povećanje odvojenosti koštanih gredica u miševa divljega tipa s artritisom u odnosu na kontrolnu skupinu miševa. Sličan je bio i nalaz mikro-kompjutorizirane tomografije koji je potvrdio smanjenje volumena trabekularne kosti i broja koštanih gredica te povećanje razdvojenosti koštanih gredica u odnosu na kontrole. Navedene promjene nisu opažene u Fas –/– miševa s artritisom, čiji su koštani parametri u skupini miševa s artritisom bili slični kontrolnoj skupini miševa. Artritis potaknut antigenom pojačao je osteoblastnu diferencijaciju sinovijalnih mezenhimnih prethodnika, te mezenhimnih prethodnika koštane srži u miševa divljega tipa, a u miševa Fas –/– je taj učinak opažen samo u mezenhimnih prethodnika koštane srži.
Temeljni zaključak ovoga istraživanja je da sustav Fas/ligand Fas ima ulogu u nadzoru nad brojem i diferencijacijacijom osteoblasta sinovijalnog podrijetla, pri čemu u manjoj mjeri time što potiče njihovu apoptozu, a značajnije inhibicijom osteoblastogeneze. Nedostatnost receptora Fas ima zaštitni učinak na lokalni i jukstaartikularni gubitak kosti u artritisu potaknutom antigenom. |
| Abstract (english) | Antigen induced arthritis is a murine experimental model of rheumatoid arthritis induced by methylated bovine serum albumin. One of the main characteristics of joint inflammation is synovial hyperplasia, whereas hyperplastic synovium in arthritis contains mesenchymal progenitors with reduced ability to differentiate into osteoblasts, chondroblasts or adipocytes. Since Fas has been shown to inhibit osteoblast differentiation, the purpose of this study was to determine whether synovia-derived osteoblasts express Fas in vitro; describe the role of Fas in apoptosis and differentiation of synovia-derived osteoblasts in vitro; as well as elucidate the in vivo role of Fas deficiency in antigen induced arthritis.
Fas receptor was weakly expressed on synovia-derived osteoblasts. Fas activation increased the number of apoptotic immature osteoblasts in vitro but the number was smaller than the proportion of Fas expressing osteoblasts. Addition of activating anti-Fas antibody did not induce apoptosis of mature osteoblasts. Despite the limited induction of apoptosis, Fas activation significantly inhibited differention od synovia-derived osteoblasts in vitro.
In order to assess the in vivo effect of Fas deficiency, I used the experimental model of antigen induced arthritis in wild-type mice and mice knocked-out for Fas gene (Fas –/–). Arthritis was induced by intraarticular injection of methylated bovine albumine into the knees of animals pre-immunized by the same antigen. The changes of knee joints were compared with control mice injected by phosphate buffered saline, 14 days after intraarticular injection. Knee diameters were significantly increased in wild-type mice with induced arthritis compared to their controls, while this increase was not significant in Fas –/– mice. Histology revealed pronounced inflammatory parameters in joints of wild-type mice with arthritis, while the inflammation in arthritis-induced Fas –/– mice was significantly less developed.
Histomorphometric analysis showed a decreased trabecular bone volume, trabecular number and thickness, as well as increased trabecular separation in wild-type mice with arthritis compared to controls. Additionally, micro-computerized tomography analysis showed that wild-type mice with arthritis had a decreased trabecular bone volume and trabecular number, as well as increased trabecular separation compared to controls. Described changes in trabecular bone were not found in Fas –/– mice with arthritis, whose bone parameters were similar to those of controls. In vitro osteoblast differentiation from both synovial mesenchymal cells and bone marrow cells was increased in wild-type mice with induced arthritis, whereas only osteoblast differentiation from bone marrow mesenchymal stromal cells was increased in arthritic Fas –/– mice.
The main conclusion of this study is that the Fas/FasL system regulates the number and differentiation od synovia-derived osteoblasts, which is only partially related to the induction of apoptotic cell death but dominantly to the inhibition of osteoblastogenesis. Fas deficiency has a protective role in local and juxtaarticular bone loss in antigen induced arthritis. |
