The roles of galectin-3 in monocyte and lymphocite lineages

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.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