= 4). Introduction Macrophages play an important part in innate and adaptive immunity as professional phagocytes by internalizing and degrading pathogens (1, 2). They recognize pathogens that are opsonized by specific Abs, through specific receptors, such as Fc receptors (FcRs) and match receptor-3 (CR3; also known MS417 as Mac pc-1 or CD11b/18 integrin). Ligation of these receptors is definitely accompanied by activation of specific signaling pathways and cytoskeletal changes, such as filamentous actin (F-actin) redistribution, as well as production of superoxide anion, proinflammatory cytokines, and chemokines such as TNF-, IL-1, and IL-8 (2). Macrophages also play an important part in a variety of physiological and pathological processes by phagocytosing apoptotic cells. They recognize these cells by using additional receptors, including class A and B scavenger receptors, CD36, and CD14 (2). This process is definitely accompanied by production of anti-inflammatory cytokines and mediators such as TGF-, IL-10, and PGE2 (2). Galectin-3 is definitely a member of large family of -galactosideCbinding animal lectins (3). It is secreted by numerous cell types including monocytes, macrophages, and epithelial cells (4, 5). The released protein can function as an extracellular molecule to activate cells (6C11), mediate cell-cell and cell-ECM relationships (12C14), and induce migration of monocytes, macrophages (15), and endothelial cells (16), probably by binding to relevant receptors through lectin-carbohydrate relationships. More recently, cell surface galectin-3 was implicated in restricting T cell receptor clustering and thus negatively regulating T Rabbit Polyclonal to Tau (phospho-Thr534/217) cell activation (17). This protein is also abundantly present inside the cells and offers been shown to play important roles in some biological reactions through its intracellular actions (18). For example, it has been identified as a factor in pre-mRNA splicing MS417 (19) and as a regulator of the MS417 cell cycle (20) and apoptosis (21, 22), although the precise mechanisms of action are still undetermined. Galectin-3 expression is definitely highly upregulated when monocytes differentiate into macrophages (6) and are downregulated when these cells differentiate into dendritic cells (23), suggesting that this protein might have functions specifically associated with particular differentiation phases in the monocyte cell lineage. The recent findings that galectin-3 is definitely a major component of phagosomes inside a mouse macrophage cell collection (24) and of exosomes derived from a dendritic cell collection (25) further suggest that this protein may be involved in endocytosis and antigen demonstration. Here, we have studied the part of endogenous galectin-3 in the phagocytic function of macrophages by using galectin-3Cdeficient (mice were generated as explained previously (26) and backcrossed to C57BL/6 mice for nine decades. Recombinant mouse GM-CSF was provided by Kirin Brewery Co. Ltd. (Maebashi, Japan). Sheep reddish blood cell (srbc) suspension was from ICN Biomedicals Inc. (Aurora, Ohio, USA). Goat antiCgalectin-3 Ab (6) and mouse antiCmouse rbc mAb 34-3C (IgG2a) (27) have been explained previously. Rabbit polyclonal anti-srbc Ab was from Sigma-Aldrich (St. Louis, Missouri, USA). Rat antiCmouse FcR (CD16/CD32) mAb 2.4G2 and FITC-conjugated annexin V were from BD Pharmingen (San Diego, California, USA). Alexa 488C and FITC-conjugated mouse anti-goat IgG, TOTO-3, rhodamine-phalloidin, NBD-phallacidin, and DiD oil (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate) were purchased from Molecular Probes Inc. (Eugene, Oregon, USA). Rhodamine-conjugated donkey anti-goat IgG and F(ab)2 fragment of mouse anti-rat IgG were from Jackson ImmunoResearch Laboratories Inc. (Western Grove, Pennsylvania, USA). RPMI 1640 was purchased from Invitrogen (Grand Island, New York, USA). Preparation of monolayer ethnicities of macrophages. Mouse bone marrowCderived macrophages (BMM) were prepared as adherent ethnicities using previously explained methods (28), except that recombinant mouse GM-CSF (10 ng/ml) was used instead of L-cellCconditioned medium. More than 95% of the cells from both wild-type and mice showed the characteristic appearance of macrophages when examined by light microscopy following Wright staining. Preparation of IgG-opsonized srbcs and apoptotic thymocytes. Opsonization of srbcs was performed by treating the cells having a subagglutinating concentration of anti-srbc Ab for 30 minutes at 37C with mild agitation. Thymocytes were harvested from your thymus of 3- to 6-week aged mice. Apoptosis was induced by treating these cells with 1.