| Abstract | Galektini-1 i -3 moduliraju mnoge stanične procese, a regulacijom migracije, fagocitoze i degranulacije
upalnih stanica, te sinteze citokina i medijatora upale, ovi su lektini uključeni u sve faze upalnih reakcija
uroĎenog i stečenog imunosnog odgovora. Djelovanje mu izrazito ovisi o lokalizaciji i tipu stanica, no galektin-1
općenito se smatra jakim protu-upalnim signalom, dok galektin-3 uglavnom potiče upalu. Ovim radom ispitana
je uloga galektina-3 u fiziologiji limfocita, te uloga i ekspresija galektina-1 i -3 u monocita, te klasično i
alternativno diferenciranih i aktiviranih makrofaga.
Humani limfociti i monociti izolirani su iz krvi zdravih dobrovoljnih davatelja. Uzgojem u hranidbenom
mediju uz dodatak granulocitno-makrofagnog, odnosno makrofagnog faktora stimulacije rasta kolonija, monociti
su diferencirani u makrofage tipa M1 i M2. Diferencirani makrofagi M1 aktivirani su klasično, dodatkom
lipopolisaharida i interferona-γ, ili alternativno u fenotip M2a/M2c, dodatkom interleukina (IL)-4/IL-10.
UtvrĎena je ekspresija galektina-1 i -3 na genskoj i proteinskoj razini, te njihova prisutnost na membrani stanica.
U hranidbenom mediju spomenutih stanica odreĎena je koncentracija galektina-3. UtvrĎen je učinak egzogeno
dodanog galektina-3 na fiziologiju limfocita, monocita i aktiviranih makrofaga.
Dobiveni rezultati upućuju na to da diferencijaciju i polarizaciju humanih monocita u klasično (M1) i
alternativno (M2a/M2c) aktivirane makrofage prate izrazite promjene razine ekspresije i proteolitičkog kidanja
galektina-3. Ekspresija i sekrecija galektina-3 usko su regulirane i znatno se razlikuju u klasično, u odnosu na
alternativno aktivirane makrofage, dok u galektina-1 razlike u razini ekspresije nisu toliko naglašene.
Zamijećene su izrazite razlike ekspresijskih profila i kidanja galektina-3 u istih podtipova makrofaga podrijetlom
od različitih donora krvi. U klasično aktiviranih makrofaga, temeljem intenziteta ekspresije membranskog
galektina-3, zamijećene su dvije odvojene populacije stanica. Humani monociti pokazuju izrazito velik kapacitet
vezanja egzogeno dodanog galektina-3, dok su u aktiviranih makrofaga receptori tog proteina u potpunosti
zasićeni. Egzogeno dodani galektin-3 ne utječe na izlučivanje citokina limfocita i aktiviranih makrofaga.
Razlike u razini i obrascu ekspresije galektina-3 u različito diferenciranih/aktiviranih makrofaga u odnosu na
galektin-1 su značajne. Stoga, specifičan uzorak ekspresije i sekrecije ovog proteina u diferenciranih i aktiviranih
makrofaga pridonosi boljem razumijevanju uloge i regulacije galektina-3 u tim stanicama. Novi uvid u biološke
karakteristike različito diferenciranih i aktiviranih makrofaga, te limfocita baca svjetlo na galektin-3 kao
modulator ovih stanica. |
| Abstract (english) | Galectins-1 and -3 modulate many cellular processes, and through regulation of cell migration, phagocytosis,
immune cell degranulation and modulation of cytokine and inflammatory mediator production, these lectins are
intimately involved in all phases of inflammatory reactions of innate and acquired immunity. Galectin-1 is
generally considered a strong anti-inflammatory and galectin-3 a pro-inflammatory signal, but their effects
tremendously vary with respect to their localization and the cell type. In this work we address the role of
galectin-3 in the physiology of lymphocytes and the role and expression of galectins-1 and -3 in human
monocytes and classically or alternatively differentiated and activated macrophages.
Human lymphocytes and monocytes were isolated from the blood of healthy donors and differentiated into
M1 and M2 subtype of macrophages by cultivation in culture media supplemented with granulocyte-macrophage
or macrophage colony-stimulating factor, respectively. M1 macrophages were activated classically by
lipopolysaccharide and interpheron-γ or alternatively into M2a/M2c phenotypes by interleukin (IL)-4/IL-10,
respectively. The expression of galectins-1 and -3 was determined on gene and protein levels, and the amount of
galectins bound to the cell membranes was estimated. Culture media were probed for secreted galectin-3. The
effect of exogenously added galectin-3 on the physiology of lymphocytes, monocytes and activated macrophages
was investigated.
Obtained results imply that differentiation and activation of monocytes into classically (M1) and alternatively
(M2a/M2c) activated macrophages is followed by marked changes of expression and proteolitic cleavage of
galectin-3. Expression and secretion of galectin-3 was tightly regulated and significantly differed among
classically and alternatively activated macrophages, while the differences of galectin-1 expression profiles were
not pronounced. Significant differences in galectin-3 expression profiles were observed between the same
macrophage subtypes obtained from different blood donors. Moreover, classically activated macrophages
polarize into two distinct populations with respect to the expression of membrane galectin-3. Human monocytes
exhibited high amount of free galectin-3 receptors, while on both types of activated macrophages the receptors
were fully saturated. Exogenously added galectin-3 does not affect cytokine secretion of lymphocytes or
activated macrophages.
Galectin-3 is more distinctive descriptor of macrophage differentiation/activation than galectin-1. Its specific
expression and secretion pattern in M1 vs. M2a/M2c macrophages contributes to better understanding of its role
and regulation in these cells and provides a new insight on biological characteristics of these cells. |
