Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways

Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways
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  Glycobiology  vol. 15 no. 5 pp. 519–527, 2005 doi:10.1093/glycob/cwi026 Advance Access publication on December 15, 2004 Glycobiologyvol. 15no. 5 © Oxford University Press 2004; all rights reserved. 519 Galectin-3 and soluble fibrinogen act in concert to modulate neutrophil activation and survival: involvement of alternative MAPK pathways Gabriela C. Fernández 1,3 , Juan M. Ilarregui 1,4 , Carolina J. Rubel 1,3 , Marta A. Toscano 4 , Sonia A. Gómez 3 , Macarena Beigier Bompadre 3 , Martín A. Isturiz 3 , Gabriel A. Rabinovich 4 , and Marina S. Palermo 1,2,3 3 División Inmunología, Instituto de Investigaciones Hematológicas, Aca-demia Nacional de Medicina, Buenos Aires, Argentina; 4 División Inmu-nogenética, Hospital de Clínicas “José de San Martín,” Universidad de Buenos Aires, Buenos Aires, Argentina Received on October 15, 2004; revised on December 7, 2004; accepted December 9, 2004 Galectin-3 (Gal-3), a member of a family of highly conservedcarbohydrate-binding proteins, has recently emerged as a novelcellular modulator at inflammatory foci. Here we investigatedthe effects of Gal-3 on central effector functions of humanneutrophils, including phagocytosis, exocytosis of secretorygranules, and survival. We examined the effects of Gal-3alone or in combination with soluble fibrinogen (sFbg), anextracellular mediator that plays a key role during the earlyphase of the inflammatory response through binding to inte-grin receptors. In addition we evaluated the intracellularsignals triggered by these mediators in human neutrophils.Human neutrophils incubated with recombinant Gal-3 aloneincreased their phagocytic activity and CD66 surface expres-sion. In contrast to the known antiapoptotic effect of Gal-3on many cellular types, Gal-3 enhanced PMN apoptotic rate.Preincubation with Gal-3 primed neutrophils to the effects of sFbg, resulting in a synergistic action on degranulation. Onthe other hand, Gal-3 and sFbg had opposite effects on PMNsurvival, and the simultaneous action of both agonists partiallycounteracted the proapoptotic effects of Gal-3. In addition,although sFbg induced its effects through the activation of theERKs, Gal-3 led to p38 phosphorylation. Disruption of this signaling pathway abrogated Gal-3-mediated modulationof neutrophil degranulation, phagocytosis, and apoptosis.Together, our results support the notion that Gal-3 and sFbgare two physiological mediators present at inflammatory sitesthat activate different components of the MAPK pathwayand could be acting in concert to modulate the functionalityand life span of neutrophils. Key words: apoptosis/galectins/inflammation/MAPK pathway/neutrophil activation Introduction Polymorphonuclear leukocytes (PMNs) circulate within thevasculature in a quiescent state, but during the early phaseof an inflammatory response they receive different signalsthat are able to induce their recruitment to the inflamma-tory foci, as well as regulate several effector functions. Theintegrin family plays an early role in this process throughbinding to specific ligands (Hynes, 1992). In this regard,CD11b/CD18 is a β 2  integrin that serves as a receptor forfibrinogen, complement factor C3bi, fibrin, and collagens(Altieri et al  ., 1990; Wright et al  ., 1983) and modulatesPMN locomotion, degranulation, phagocytosis, and apop-tosis (Rubel et al  ., 2001). Galectins, a growing family of mammalian lectins highlyconserved throughout animal evolution (Hirabayashi andKasai, 1993), have recently attracted the attention of immu-nologists as novel regulators of inflammation (Almkvist andKarlsson, 2004; Rabinovich et al  ., 2002, 2004). According totheir structure, these β -galactoside-binding proteins have beenclassified into proto type (galectins-1, -2, -5, -7, -10, -11, -13,-14, and -15), chimera type (galectin-3), and tandem repeattype (galectins-4, -6, -8, -9, and -12) (Hirabayashi and Kasai,1993; Rabinovich et al  ., 2004). They share remarkablesequence similarities in the carbohydrate recognition domain,and many family members preferentially recognize galactose-containing saccharide ligands. Galectin-3 (Gal-3) has beenreported to regulate different inflammatory cell types, and tar-geted mutation of  gal-3  gene results in an attenuated inflam-matory response following immunological challenge (Colnot et al  ., 1998; Hsu et al  ., 2000). Gal-3 activates mast cells andbasophils (Zuberi et al  ., 1994), potentiates lipopolysaccharide-induced IL-1 production from monocytes (Jeng et al  ., 1994),and induces monocyte-macrophage chemotaxis (Sano et al  .,2000) and phagocytosis (Sano et al  ., 2003). Regarding theinfluence of Gal-3 on neutrophil physiology, this proteinbinds to the surface of PMN through CD66b (Almkvist et al  .,2001; Feuk-Lagerstedt et al  ., 1999; Yamaoka et al  ., 1995),induces cell aggregation (Timoshenko et al  ., 2003), promotesPMN adhesion to laminin (Kuwabara and Liu, 1996), andpromotes extravasation in response to infection (Sato et al  .,2002). Although it has been reported that Gal-3 activates theNADPH-oxidase in primed PMNs (Karlsson et al  ., 1998;Yamaoka et al  ., 1995), the effects of Gal-3 on central effectormechanisms during an inflammatory process, includingphagocytosis, exocytosis of granule content (degranulation),and life span, have not yet been explored.An inflammatory situation in vivo exposes the cell to awide variety of mediators, either in a simultaneous orsequential manner, which could reciprocally potentiate orcounteract their effects. Moreover, accumulating evidence has 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed; e-mail:   b  y g u e  s  t   onM a r  c h 1 2  ,2  0 1 4 h  t   t   p :  /   /   gl   y c  o b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  G.C. Fernández et al. 520raised the concept that the pattern of activation in severalcells, including neutrophils, is dependent on the activation of alternative intracellular pathways. Thus the final outcome inthe modulation of neutrophil activation and survival willdepend on the balance of the intracellular signals triggered bythose agents. Following this assumption, we investigated theintracellular signaling pathway elicited by Gal-3 in PMNs andanalyzed the physiological effects derived from the interplaybetween Gal-3 and soluble fibrinogen (sFbg) as an integrin-dependent stimulus. SFbg activates human PMNs through aCD11b-dependent mechanism and was selected for our studyon the basis of recent observations reporting the effect of thisearly inflammatory mediator in the up-regulation of CD66bsurface expression, which acts as a major receptor for Gal-3 inPMNs (Almkvist et al  ., 2001). In this context, we undertookthis work to study the interplay between Gal-3 and sFbg inthe modulation of neutrophil physiology and the intracellularsignals triggered by these mediators. Results Effect of Gal-3 on neutrophil phagocytosis Because PMNs are the main phagocytic cells and the effectof rhGal-3 on this central capacity of PMNs has not beenpreviously studied, erythrophagocytosis was assayed usingPMNs against optimally sensitized target cells. As shown inFigure 1, phagocytosis was significantly increased by 4 µ g/mlof recombinant human Gal-3 (rhGal-3), and lower concen-trations were not effective at modulating this function.Furthermore, when PMNs were previously incubated for 15min with lactose (30 mM), rhGal-3 (4 µ g/ml) was not able toaffect basal values of phagocytosis, suggesting that the modu-lation of phagocytosis by Gal-3 depends on the carbohydrate-binding properties of this protein. Because sFbg also increasesFc-dependent phagocytosis (Rubel et al  ., 2001), we tested theeffect of adding both stimuli together. Preincubation witheither sFbg (6 µ M) or rhGal-3 (4 µ g/ml) did not induce ahigher increase compared with each stimulus added separ-ately (Figure 1), indicating no additive or synergistic action of rhGal-3 and sFbg on PMN-mediated phagocytosis. Influence of Gal-3 on neutrophil degranulation Neutrophil degranulation is an important inflammatoryevent secondary to PMN activation that can be studiedphenotypically by measuring the up-regulation of the mem-brane marker CD66b. This molecule resides in the specificgranules of resting PMNs and appears on the cell surface uponstimulation (Stocks et al  ., 1995). As depicted in Figure 2, Fig. 1. Effect of rhGal-3 on erythrophagocytosis. PMNs (2.5 ×  10 6 /ml) were incubated for 60 min at 37°C in medium (control), sFbg (6 µ M), or 15 min with rhGal-3 (0.4 and 4 µ g/ml), in the absence or presence of lactose (30 mM). After washing, phagocytosis assay was performed employing target cells opti-mally sensitized (IgG anti-sheep erythrocytes). Phagocytosis was evaluated after 30 min as described in Materials and methods . Data are expressed as the arithmetic mean ±  SEM of five independent experiments using cells from dif-ferent donors in each experiment. *  p < 0.05 compared with control or Gal-3 in the presence of 30 mM lactose. Fig. 2. Effect of rhGal-3 on CD66b expression as an indicator of PMN degranulation. PMNs (2.5 ×  10 6 /ml) were incubated for 60 min at 37°C in medium (control), sFbg (6 µ M), or 15 min with rhGal-3 (0.4 and 4 µ g/ml). Then cells were centrifuged, washed, and stained with a specific anti-CD66b mAb as described in Materials and methods . ( A  and B ) Representative histograms of CD66b expression after different treatments are shown. The shaded histograms represent staining with isotype controls. The thin lines represent a control indicating basal levels of CD66b expression in PMN. The  y  and x  axes represent cell number and fluorescence intensity (FL-1) respectively. ( A ) PMNs were incubated with different concentrations of rhGal-3 and rhGal-3 (4 µ g/ml) plus lactose (30 mM). ( B ) Hatched histograms represent PMNs incubated with sFbg (6 µ M) alone, or in combination with rhGal-3 (4 µ g/ml). ( C ) Each bar represents the mean ±  SEM of eight independent experiments using cells from different donors in each experiment. PMNs incubated with rhGal-3 (4 µ g/ml) in the presence of lactose (30 mM) is also shown. *  p < 0.01 com-pared with control; #  p < 0.05 compared to sFbg- or rhGal-3-treated cells.   b  y g u e  s  t   onM a r  c h 1 2  ,2  0 1 4 h  t   t   p :  /   /   gl   y c  o b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  Modulation of neutrophil physiology by galectin-3 521incubation for 15 min with rhGal-3 (0.4 and 4 µ g/ml) wassufficient to induce a dose-dependent up-regulation of CD66b expression. Prolonged incubation up to 90 min didnot show differences compared with 15-min exposure (datanot shown). As a control, preincubation with 30 mM lac-tose prevented Gal-3-induced degranulation. In addition,pretreatment with sFbg (6 µ M) for 60 min, which signifi-cantly increases CD66 membrane expression, did not mod-ify the effects of rhGal-3. Conversely, 15 min preincubationwith rhGal-3 synergistically enhanced the expression of CD66b induced by sFbg. Modulation of neutrophil apoptosis by Gal-3 and sFbg  The role of Gal-3 on cell survival has been controversial.Intracellularly this protein has shown antiapoptotic activityin several cell types, including T cells and tumors (Moon etal  ., 2001; Yang et al  ., 1996), whereas exogenously addedGal-3 promoted apoptosis of T cell lines and primary T lym-phocytes (Fukumori et al  ., 2003). However, the effects of thisprotein on PMN survival have not yet been examined. Onthis versatile population, different activating stimuli, includ-ing sFbg, are able to inhibit the apoptotic program (Colotta et al  ., 1992; Rubel et al  ., 2001). On the other hand, othermediators such us TNF- α  or IL-6 accelerate the PMN apop-totic rate (Afford et al  ., 1992; Takeda et al  ., 1993). Therefore,we investigated whether rhGal-3 could modulate neutrophillifespan in addition to triggering PMN activation. Apoptosiswas assessed by flow cytometry after 24 h of cell cultureaccording to the frequency of subdiploid nuclei followingpermeabilization and propidium iodide (PI) staining. Asshown in Figure 3, preincubation with rhGal-3 (0.4 µ g/ml) for15 min enhanced the neutrophil apoptotic population from~30% (control) to 50%. On the other hand, sFbg reducedPMN apoptosis to ~15%, as has been previously reported(Rubel et al  ., 2001), and the combined treatment of rhGal-3and sFbg, independent of the order of incubation, resulted ina partial blockade of the proapoptotic effect induced byexogenous Gal-3. This effect was dependent on the carbohy-drate-binding properties of this protein because it was pre-vented by 30 mM lactose (Figure 3). Intracellular signaling pathways triggered by Gal-3 in PMNs In neutrophils, several inflammatory stimuli trigger mitogen-actived protein kinase (MAPK) phosphorylation (McLeish et al  ., 1998; Nick et al  ., 1997; Rane et al  ., 1997). The obser-vation that activation of these kinases play a pivotal role inregulating the lifespan of these inflammatory cells,together with our recent findings demonstrating that sFbginhibits PMN apoptosis through an extracellular signal-regulated kinase (ERK)-dependent pathway (Rubel et al  .,2002), prompted us to investigate the activation of theMAPK pathway on incubation with rhGal-3. Phosphoryla-tion of p38 and ERK following exposure of PMNs to rhGal-3was investigated by flow cytometry and western blot analy-sis, respectively. As shown in Figure 4A, p38 phosphoryla-tion was clearly induced 10 min after addition of rhGal-3,returning to basal levels after 15 min. On the other hand,western blot analysis using a specific anti-phosphoERKmonoclonal antibody (mAb), revealed that rhGal-3 didnot induce a significant phosphorylation of ERK1/2 in Fig. 3. Modulation of neutrophil apoptosis by rhGal-3. PMNs (2.5 ×  10 6 /ml) were incubated for 60 min at 37°C in medium (control), sFbg (6 µ M), or 15 min with rhGal-3 (0.4 µ g/ml), in the absence or presence of lactose (30 mM). All samples were washed and cultured for 24 h at 37°C. Then, the percentage of apoptotic cells was determined by PI staining. ( A ) Representative histograms showing the percentage (M1) nuclei with subdiploid DNA content after 24 h of incubation. ( B ) Results are expressed as the mean ±  SEM of seven independent experiments using cells from different donors in each experiment. *  p < 0.01 compared with control; #  p < 0.01 compared with rhGal-3 treatment.   b  y g u e  s  t   onM a r  c h 1 2  ,2  0 1 4 h  t   t   p :  /   /   gl   y c  o b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  G.C. Fernández et al. 522periods ranging from 5 to 30 min (Figure 4B, lanes 2 to 5).As a positive control of ERK activation, PMNs from thesame donor were incubated 2 min with N-formyl-methionyl-leveyl-phenylelanine (FMLP) (10  –7  M) (Figure 4B, lane 6).Furthermore, preincubation with PD98059, an inhibitor of ERK1/2 phosphorylation, abolished phosphorylation of ERK, confirming the specificity of the antibody and effec-tiveness of the inhibitor (Figure 4, lane 7). In contrast tothese observations, we previously demonstrated the abilityof sFbg to activate ERK1/2 and not p38 in a time-dependentmanner (Rubel et al  ., 2002), suggesting that Gal-3 andsFbg activate different intracellular pathways to modu-late neutrophil activation and survival. Modulation of Gal-3 effects by MAPK inhibitors To determine the involvement of the MAPK cascade inGal-3-induced modulation of PMN functionality, we invest-igated the effects of pharmacological inhibitors of ERKand p38 MAPK pathways in Gal-3-induced degranulation,enhancement of erythrophagocytosis, and apoptosis rate of PMN. For this purpose, we used PD98059, an inhibitor of MEK1 and MEK2 kinases, which are responsible for MAPKERK1/2 phosphorylation (Alessi et al  ., 1995), and SB203580,a phamacological inhibitor of p38 MAPK (Cuenda et al  .,1995). The effects of these inhibitors on sFbg-inducedincreased degranulation and phagocytosis and decreasedapoptosis were previously studied (Rubel et al  ., 2002).Briefly, the effects of sFbg on those functions were shownto be abolished by PD98059 and not modified by SB203580(MFI CD66 expression: sFbg = 185 ±  25, sFbg + PD98059= 78 ±  9 (  p  < 0.005); % phagocytosis: sFbg = 62 ±  4, sFbg +PD98059 = 40 ±  2 (  p  < 0.05); % apoptosis: sFbg = 18 ± 2,sFbg + PD98059 = 40 ±  4 (  p  < 0.005). As shown in Figure 5(A, B, and C), 30 µ M SB203580 significantly decreased theeffects of rhGal-3 on phagocytosis, apoptosis, and CD66bmembrane expression. On the other hand, 50 µ M PD98059was not able to modify rhGal-3 effects, except CD66bexocytosis, which was partially blocked by this pharmaco-logical inhibitor. The ability of PD98059 to inhibit ERKactivation was confirmed by western blot analysis onFMLP-activated PMN (Figure 4B, lane 7).Taken together, these data indicate that the p38 MAPKpathway is critically involved in Gal-3-induced modulationof PMN functionality and survival. Discussion In this study we observed novel effects of exogenouslyadded Gal-3 on PMN that contributes to the activationpattern of circulating neutrophils. Gal-3 induces anincrease in antibody-dependent erythrophagocytosis, mod-ulates PMN degranulation as shown by up-regulated sur-face expression of CD66, and influences PMN survival.Gal-3-mediated modulation of PMN functions was depen-dent on the carbohydrate-binding activity of this lectin,because preincubation with lactose was able to preventthese effects.The observation that Gal-3 induces PMN activation isconsistent with the ability of this protein to stimulate super-oxide production (Karlsson et al  ., 1998; Yamaoka et al  .,1995), to enhance adhesiveness to extracellular matrix pro-teins (Kuwabara and Liu, 1996) and to promote extravasa-tion in response to infectious agents (Sato et al  ., 2002).Several studies have reported the requirement of previouspriming with bacterial derivates to observe Gal-3-mediatedeffects on PMN (Faldt et al  ., 2001; Karlsson et al  ., 1998).Although we have demonstrated that Gal-3 has directeffects on purified PMNs, it is possible that the purificationprocedure and fetal bovine serum (FBS)-containing mediacould prime PMNs, as has been previously demonstrated(Haslett et al  ., 1985; Rubel et al  ., 2001). On the other hand,pretreatment with Gal-3 markedly enhanced the effects of sFbg on degranulation, whereas preincubation with sFbgdid not modify the effects of Gal-3 on PMN response.Although the molecular mechanisms responsible for theinduction of this sensitized state are still unclear, a review of the current literature makes it evident that several signalswork in concert to modulate PMN physiology (Hallett and Fig. 4. rhGal-3 induces MAPK activation in PMNs. ( A ) PMNs (2.5 ×  10 6 /ml) were incubated at 37°C with medium or rhGal-3 (0.4 µ g/ml) for the time periods indicated. Then cells were centrifuged, washed, and stained with a specific anti-phospho-p38 mAb as described in Materials and methods . Data are the mean ±  SEM of five independent experiments using cells from different donors in each experiments. *  p < 0.05 compared with controls. ( B ) Western blot analysis of ERK1/2 phosphorylation. PMNs (2.5 ×  10 6 /ml) were incubated at 37°C with medium alone (lane 1) or rhGal-3 (0.4 µ g/ml) for the indicated time periods (lanes 2–5). As controls, PMNs were incubated with FMLP (10  –7  M) (lane 6) or FMLP plus the PD98059 inhibitor (lane 7). Cells were then centrifuged, washed, and lysed in the presence of a protease inhibitor cocktail and analyzed by western blot analysis as described in Materials and methods . Densitometric analysis of the intensity of each band (expressed as arbitrary units) is also shown (lower panel).   b  y g u e  s  t   onM a r  c h 1 2  ,2  0 1 4 h  t   t   p :  /   /   gl   y c  o b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  Modulation of neutrophil physiology by galectin-3 523Lloyds, 1995). Suggested mechanisms include signalingevents, such as tyrosine phosphorylation, increase in intrac-ellular free calcium, and exposure of new receptors. In thisregard, it has been previously documented that Gal-3increases CD11b-dependent adhesion to extracellular matrixproteins (Matarrese et al  ., 2000; Ochieng et al  ., 1998).Moreover, cross-linking of CD66, which has been postulatedto be a major Gal-3 receptor in PMNs (Feuk-Lagerstedt etal  ., 1999; Kuwabara and Liu, 1996), has been shown toaugment adhesion to fibrinogen (Stocks et al  ., 1996). Thusan alternative explanation for the synergistic effects of Gal-3and sFbg may be that Gal-3 increases avidity of CD11b,which has been postulated to act as a receptor for sFbg onthe cell surface of PMNs (Altieri et al  ., 1990; Wright et al  .,1988) . However, we cannot rule out other potential mech-anisms, which might be also operating to increase the res-ponsiveness of PMN to fibrinogen.Although Gal-3 sensitized PMNs to sFbg effects, pre-treatment with sFbg did not affect PMN response to Gal-3effects. Almkvist and colleagues (2001) demonstrated thatlipopolysaccharide primes PMNs to Gal-3 effects throughCD66b mobilization. In our system, pretreatment withsFbg was not effective at modulating responsiveness of PMN to Gal-3, although sFbg successfully increased CD66bexpression.A remarkable set of data from the present work is theobservation that Gal-3, broadly described as a moleculewith antiapoptotic and proliferative effects on cells of dif-ferent srcin (Inohara et al  ., 1998; Moon et al  ., 2001; Yang et al  ., 1996), has proapoptotic activity when added toPMNs. In this sense, Gal-3 exogenously added to humanlung fibroblastic cells stimulated DNA synthesis as well ascell proliferation (Inohara et al  ., 1998); Gal-3 expressionhas been associated with neoplastic progression and meta-static potential on several tumor cells (Takenaka et al  .,2004). Furthermore, in synovial mononuclear leukocytesfrom arthritic patients, the up-regulation of Gal-3 expressionhas been associated with the inhibition of apoptosis (Harjacek et al  ., 2001). Moreover, Gal-3 has been demonstrated to bealso involved in B cell differentiation and survival (Acosta-Rodriguez et al  ., 2004; Hoyer et al  ., 2004). In contrast, wehave demonstrated that Gal-3, exogenously added toPMNs at low doses of 0.4 µ g/ml, induces a strong proapop-totic signal. In this sense, a recent report suggests thatextracellular Gal-3 can act as a proapototic signal in humanT leukemia cell lines, human peripheral blood mononuclearcells, and activated mouse T cells (Fukumori et al  ., 2003).Thus the assumption that Gal-3 can act either as an anti- orproapoptotic molecule is worthwhile to be discussed interms of different variables, such as the target cell type, sub-cellular localization of this protein, activation state of thetarget cells, and the balance of intracellular signals that canpotentiate or counteract its effects.The MAPK cascade is known to participate in multiplecellular functions, such as degranulation, locomotion, pro-liferation, differentiation, and survival (Lewis et al  ., 1998).The MAPKs are phosphorylated on threonine-tyrosine res-idues by distinct MAPK kinases. ERK1/2 are activated bya variety of growth factors and play a critical role inmitogenesis (Robinson and Cobb, 1997). JNK and p38 aretypically activated by cellular stress or proinflammatorycytokines that are known to induce cell death (Raingeaud et al  ., 1995), although recent studies have also demon-strated their activation by hematopoietic growth factors(Foltz et al  ., 1997). For a better understanding of the mechanisms involvedin neutrophil activation, it is of central interest to investigatethe functional relevance of signaling molecules. In thepresent work, we showed for the first time that Gal-3 stimu-lates p38 MAPK activity in a time-dependent manner with amaximum activity at 10 min, whereas there is no significant Fig. 5. Gal-3 mediated modulation of PMN physiology by MAPK inhibitors. PMNs (2.5 ×  10 6 /ml) were preincubated 30 min with medium (control), PD98059 (50 µ M), or SB203580 (30 µ M), followed by stimula-tion for 15 min with the indicated concentration of rhGal-3 at 37°C. ( A ) Erythrophagocytosis. After washing, phagocytosis assay was performed and evaluated as described in Materials and methods . Data are expressed as the arithmetic mean ±  SEM of five independent experiments using cells of different donors in each experiment. *  p < 0.01 compared with control; #  p < 0.01 compared with rhGal-3. ( B ) Apoptosis. All samples were washed and cultured for 24 h at 37°C. Then the percentage of apoptotic cells was determined by PI staining. Results are expressed as the mean ±  SEM of five independent experiments using cells from different donors in each experiment. *  p < 0.01 compared with control; #  p < 0.01 compared with rhGal-3 treatment. ( C ) CD66 membrane expression. Cells were washed and stained with a specific anti-CD66b mAb as described in Materials and methods . Data represent the mean ±  SEM of MFI from four different donors. *  p < 0.001 compared with control; #  p < 0.001 and **p < 0.05 compared with rhGal-3 treatment.   b  y g u e  s  t   onM a r  c h 1 2  ,2  0 1 4 h  t   t   p :  /   /   gl   y c  o b  . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om
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