Leukocyte Extravasation: An Immunoregulatory Role for α-l-Fucosidase? Simi Ali, Yvonne Jenkins, Maureen Kirkley, Athanasios Dagkalis, Ayyakkannu Manivannan, Isabel Joan Crane and This information is current as John A. Kirby of September 28, 2021. J Immunol 2008; 181:2407-2413; ; doi: 10.4049/jimmunol.181.4.2407 http://www.jimmunol.org/content/181/4/2407 Downloaded from

References This article cites 32 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/181/4/2407.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 28, 2021 *average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Leukocyte Extravasation: An Immunoregulatory Role for 1 ␣-L-Fucosidase?

Simi Ali,2* Yvonne Jenkins,* Maureen Kirkley,* Athanasios Dagkalis,† Ayyakkannu Manivannan,† Isabel Joan Crane,† and John A. Kirby*

Fucosylated oligosaccharides and glycoconjugates have been implicated in several biological events, including the cell- processes that mediate inflammation. ␣-L-Fucosidase (ALF) is an exoglycosidase that is involved in the hydrolytic degradation of ␣-L-fucose from glycoconjugates. In this study, we investigated the potential role of ALF in regulation of leukocyte migration. Measurement of transendothelial migration in response to CCL5 demonstrated that pretreatment of monocytic cells with ALF Treatment with ALF .(0.0374 ؍ to a greater extent than treatment of the endothelial monolayer (p (0.0004 ؍ reduced migration (p significantly reduced the adhesion of monocytic cells to immobilized P-.Fc. A murine model of experimental autoimmune uveitis was then used to show that treatment of splenic cells with ALF produced an 8.6-fold decrease in rolling and a 3.2-fold Downloaded from decrease in cell migration across the retinal vasculature. Further in vitro studies demonstrated that treatment of with the CCL3 or CCL5 increased the level of mRNA encoding ALF; this was accompanied by the detection of significant increases in both the 51- and 56-kDa components of ALF by Western blotting. Treatment of monocytic cells with ALF for 2 h .(0.002 ؍ significantly reduced the cell surface expression of CD31, with a further decrease in expression observed after5h(p Thus, CD31 and fucosylated ligands of P-selectin seem to be the candidates through which ALF mediates its effect in vitro. These data identify a previously unrecognized immunoregulatory role for ALF in late stages of inflammation. The Journal of Immu- http://www.jimmunol.org/ nology, 2008, 181: 2407–2413.

ucose can be linked through ␣ (1, 3), ␣ (1, 4), and ␣ (1, 6) There is a considerable degree of structural heterogeneity, both bonds to N-acetylglucosamine and through ␣ (1, 2) to ga- tissue specific and within tissues. This has been attributed to F lactose in a range of and glycolipids. Fuco- variations in the sialic acid content as well as the two different sylated oligosaccharides and glycoconjugates play roles in several alleles and polymorphisms within the FUC1 gene. The serum biological events, including the cell-to-cell adhesion processes that ALF activity in normal individuals is reported to be 381 U/ml Ϯ mediate inflammation, embryonic development, tumorigenesis/ 10.86 (4). by guest on September 28, 2021 metastasis, and Ag recognition. The important role played by The ALF enzyme is associated with a variety of biological func- fucose residues has led to an increasing interest in ␣-L-fucosi- tions. The significance of this enzyme in human catabolism is im- 3 dase (ALF) (␣-L-fucoside fucohydrolase; EC3.2.1.51), an exo- plied by the genetic neurovisceral storage disease, fucosidosis, glycosidase involved in the hydrolytic degradation of fucose- which results from an absence or gross deficiency of ALF; this containing components of glycoproteins (1), glycolipids, and allows accumulation of fucoglycoconjugates, which result in men- oligosaccharides (2). tal and motor retardation (5, 6). A syndrome previously known as Approximately 10–20% of total ALF activity occurs on the leukocyte adhesion deficiency II is now recognized as a general- surface of a range of human cell types, including hemopoietic, ized metabolic disease caused by severe hypofucosylation of gly- epithelial, and mesenchymal cells (1). Mammalian ALF are coconjugates, including selectin ligands (7). Further to this, alter- ϳ multimeric, containing subunits of 53 kDa; they ations in levels of ALF have been reported in the plasma and/or have a maximal activity between pH 4 and 6.5. At least five serum of patients with endometrial, hepatocellular, and colorectal major isoforms of the ALF enzyme have been identified (3). cancer (4, 8, 9). Several molecules involved in leukocyte transmigration, in- *Applied Immunobiology and Transplantation Group, Institute of Cellular Medicine, cluding selectin ligands, proteoglycans, , and CD31, Medical School, University of Newcastle Upon Tyne, Newcastle Upon Tyne, United are glycosylated and contain fucosylated moieties. L-selectin- Kingdom; and †Department of Ophthalmology, University of Aberdeen, Institute of Medical Sciences, Forresterhill, Aberdeen, United Kingdom dependent interactions mediate tethering and roll- Received for publication October 18, 2007. Accepted for publication June 13, 2008. ing before -dependent cell activation and transmigra- The costs of publication of this article were defrayed in part by the payment of page tion across high endothelial venules. On , L-selectin charges. This article must therefore be hereby marked advertisement in accordance is a carbohydrate-binding protein that binds to ligands on high with 18 U.S.C. Section 1734 solely to indicate this fact. endothelial venules; the function of these ligands is dependent on 1 This work was supported by grants from Northern Counties Kidney Research Fund the expression of carbohydrate 6-sulfo sialyl Lewis ϫ (sialyl-(2– and the Wellcome Trust-078892. 3)-galactopyranosyl-(1–4)-Nacetyl glucosamine-((1–3)-fucopyr- 2 Address correspondence and reprint requests to Dr. Simi Ali, University of New- X castle upon Tyne, The Medical School, Newcastle upon Tyne, NE2 4HH United anosyl) groups termed sLe or CD15s. The pivotal function of Kingdom. E-mail address: [email protected] fucosylation of L-selectin ligands has been established by genetic 3 Abbreviations used in this paper: ALF, ␣-L-fucosidase; EAU, experimental auto- studies in mice. Mice lacking fucosyltransferase VII or both fuco- immune uveoretinitis; PSGL-1, P-selectin glycoprotein ligand-1; SLO, scanning laser syltransferase VII and fucosyltransferase IV show a reduction of ophthalmoscopy. 80% or more than 95%, respectively, in lymphocyte homing to Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 lymph nodes (10). www.jimmunol.org 2408 ROLE OF ALF IN LEUKOCYTE EXTRAVASATION

P- and E- are expressed on activated endothelial cells with DPX mounting medium. Migrant cells were counted using high power and cooperatively mediate leukocyte rolling. They are membrane- microscopy (ϫ400) in four random fields on each filter. bound C-type lectins that bind to cell surface glycoconjugate li- Cell surface staining with fucose-specific lectin gands. P-selectin glycoprotein ligand-1 (PSGL-1), a mucin ex- pressed on leukocytes, is the best-characterized selectin ligand. THP-1 cells (200,000/ml) were washed twice with PBS containing 5% The receptor function of PSGL-1 depends on two posttranslational FCS. Cells were incubated with biotin-conjugated fucose-specific lectin from Tetragonolobus purpureas (50 ␮g/ml; Sigma-Aldrich) in HBSS events. One consists of the generation of O-linked glycan and in- buffer containing 1 mM CaCl2, 1 mM MgCl2,and1%FBSfor1hat37°C. cludes ␣ (1, 3) fucosylation and ␣ (2, 3) sialylation (such as sLeX). Cells were subsequently washed twice with HBSS buffer and resuspended The other involves sulfation of tyrosine residues. In vivo, PSGL-1 in 50 ␮l of HBSS buffer containing streptavidin-FITC-conjugated Ab (BD mediates leukocytes tethering to and rolling on P-selectin and sup- Pharmingen) for 30 min at 4°C. Following washing, the stained cells were analyzed by flow cytometry (FACSort; BD Biosciences). ports tethering to E-selectin (11, 12). Fucosylated proteoglycans Cells (100,000/ml) resuspended in RPMI 1640 containing 0.1% BSA also play a key role in CD11d/CD18-mediated adhesive interac- and 2% HEPES were treated with ALF (0.5-0.04 U/ml) for different time tions and the migration of polymorphonuclear leukocytes across periods (1–24 h). These cells were then stained with the fucose-binding the intestinal epithelium (13). Additionally, systemic treatment lectin, as described above. Trypan blue exclusion was assessed to ensure cell viability. Controls included cells stained with secondary Ab only. The with fucoidin, a sulfated polysaccharide containing L-fucose and stained cells were analyzed by flow cytometry (FACSort; BD Biosciences); L-fucose-4-sulfate, interferes with selectin receptor function and data analysis was performed using Lysis II software (BD Biosciences). inhibits leukocyte rolling (14–16). It is well established that the responsiveness of migratory in- Solid-phase adhesion assays

flammatory cells to chemokines is governed by the expression of Flat-profile 96-well ELISA plates (Linbro; MP Biomedicals) were coated Downloaded from chemokine receptors, but the processes involved in the resolution with goat anti-human IgG Fc Ab (Sigma-Aldrich) at 1 ␮g/well in PBS at of the inflammatory response are poorly defined. It is believed that 4°C for 18 h. Excess Ab was removed by washing twice with PBS, and ␮ chemokines are normally presented to intravascular leukocytes in human P-selectin.Fc fusion protein (1 g/well; R&D Systems) was added in HBSS buffer (Sigma-Aldrich) for an additional 18 h at 4°C. Excess the form of complexes bound to endothelial cell surface glycos- protein was washed twice with HBSS buffer, and nonspecific binding was aminoglycans. Significantly, it has been shown that the concentra- blocked for2hat37°C with HBSS containing 2% FBS.

tion of ALF and soluble glycosaminoglycans increases in Grave’s THP-1 cells (100,000/ml) resuspended in RPMI 1640 containing 0.1% http://www.jimmunol.org/ disease (17), suggesting the potential for cleavage of proinflam- BSA and 2% HEPES were treated with ALF (0.08 U/ml; 8 h), and un- treated cells were incubated in the same medium for 8 h. Viability of treated matory chemokine complexes and selectin ligands from the endo- cells was 95%; equal number of viable cells from control and treated group thelial cell surface. was used in subsequent experiment. THP-1 cells (2 ϫ 106/ml) were labeled Based on these observations, we hypothesized that ALF plays an with 2Ј,7Ј-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein tetrakis (ace- important regulatory role in the later stages of inflammation. A toxymethyl) ester (Sigma-Aldrich) at 20 ␮g/ml for 15 min at 37°C. After ϫ 5 series of experiments was designed to examine the role of ALF in washing by centrifugation, the cells were resuspended in HBSS, 5 10 cells were added to each assay well, and the plates were centrifuged at leukocyte migration both in vitro and in vivo. Transendothelial 60 ϫ g for 2 min at 37°C. assays were used to determine the functional signifi- Following incubation of the assay plates at 37°C for 1 h, the nonadher- cance of ALF in regulating leukocyte trafficking in vitro. A murine ent cells were removed by mechanical oscillation and gentle washing with by guest on September 28, 2021 experimental autoimmune uveoretinitis (EAU) model was then warm HBSS, and the remaining, adherent T cells were lysed by the addi- tion of 2% Triton X-100 (Sigma-Aldrich). The quantity of released fluo- used to assess the role of ALF in leukocyte rolling and tissue rochrome was measured using a plate-format fluorometer (Fluostar Op- infiltration in vivo. tima; BMG Labtech). In vivo assay of leukocyte extravasation Materials and Methods Induction of EAU. Female B10R.III mice, 12–20 wk old (Medical Re- Cell culture search Facility, University of Aberdeen), were treated humanely according THP-1 monocytic cells were supplied by European Cell Culture Collection to the Animals (Scientific Procedures) Act (United Kingdom). EAU was induced with a s.c. 50-␮l injection into each thigh of 25 ␮g of peptide and expanded in RPMI 1640 medium with 10% FCS, 100 U/ml penicillin, Ͼ 100 ␮g/ml streptomycin, and 1 ␮g/ml folic acid (all from Sigma-Aldrich). 161–180 (SGIPYIISYLHPGNTILHVD; purity 85%; Sigma Genosys) of EAhy926, a human aortic endothelial cell hybridoma created by fusion of the human interphotoreceptor retinoid-binding protein emulsified 1:1 HUVECs with human lung carcinoma-derived cell line A549 (18), was with CFA (H37Ra; Difco Laboratories) (19, 20). Animals were ob- grown in DMEM medium (Sigma-Aldrich) supplemented with FCS (10%), served using a slit-lamp for clinical evaluation (grading) of the eye at 2 mM glutamine, penicillin, and streptomycin, as above. day 11 postimmunization. Leukocyte preparation. A single-cell suspension of was pre- pared 11 days after sensitization for EAU. Erythrocytes were lysed with Transendothelial chemotaxis assay 0.75% (w/v) NH4Cl in 17 mM Tris buffer (pH 7.2), and the remaining EAhy926 cells (5 ϫ 104) were seeded onto 3-␮m-pore Falcon Transwell leukocytes were washed twice in RPMI 1640 medium. The cells were then filters and allowed to grow to confluency; the endothelial monolayers were resuspended in 10 ml of medium supplemented with 0.1% (w/v) BSA at 6 then treated for1hat37°C with either no ALF, an optimal 0.08 U/ml ALF 1 ϫ 10 cells/ml, and some samples were incubated at 37°C with 0.08 U/ml 5 (Sigma-Aldrich), or 0.08 U/ml ALF, which had been denatured by heating ALF per 5 ϫ 10 cells for optimal time. The cells were then pelleted, to 100°C for 5 min. resuspended in 5 ml of RPMI 1640 plus 0.1% BSA, and incubated with 40 THP-1 cells were assayed for their ability to migrate through a 3-␮m- ␮g/ml calcein-AM (Molecular Probes) at 37°C for 30 min with gentle pore-diameter filter toward the basal compartment containing either no mixing. Cells were spun down and resuspended in 150 ␮l of medium. CCL5 or 100 ng/ml CCL5 (PeproTech). THP-1 cells were stimulated for Tracking cells with scanning laser ophthalmoscopy (SLO). Eleven days 16 h using 300 U/ml IFN-␥, and then 5 ϫ 105 cells were added to each well after sensitization, mice of equivalent EAU severity were anesthetized and ␮ and incubated for2hat37°C with 5% CO2. For some experiments, mono- fitted with contact lenses, as described (21). A total of 100 l of 0.05% cytic THP-1 cells and EAhy926 cells were also treated with 0.08 U/ml ALF (v/v) sodium fluorescein (Sigma-Aldrich) was injected via the tail vein, for 1 h. Following the removal of excess cells from both the upper and followed by 1 ϫ 107 calcein-AM-labeled cells in 150 ␮l of medium. For lower chambers, the upper surface of each filter was carefully wiped with each eye, three regions of interest containing one to three veins/venules a clean swab. The filters were then fixed in 100% cold methanol for 1 h. were observed by SLO, and images were recorded for at least 15 min after After fixation, the filters were stained with Gill’s hematoxylin (Sigma- cell injection (21). Video analysis was conducted off-line, as described Aldrich) for 30 min, followed by a 5-min wash in Scott’s tap water (166 elsewhere (21). Rolling leukocytes and those not interacting with the en-

mM MgSO4, 24 mM NaHCO3). Sequential washing and dehydration using dothelium were counted in each venule (22–24). The rolling efficiency was increasing concentrations of ethanol were then performed before mounting calculated as the percentage of labeled rolling cells among the total number The Journal of Immunology 2409 of labeled cells that entered a venule. The sticking efficiency was deter- sion cell line EAhy926 was grown on the upper surface of the mined as the percentage of labeled cells becoming firmly adherent for at membrane, which contained 3-␮m pores. Fig. 1 shows a visual least 20 s compared with the total number of labeled cells that rolled within representation of cell morphology following treatment with ALF. the venule during the same time interval. Fig. 1a shows the normal chemotactic response toward 100 ng/ml Whole retinal mounts for confocal microscopy. Fifty minutes after the injection of labeled resting cells and SLO, the anesthetized mice were CCL5 in the lower chamber; treatment of the monocytic cells with injected via the tail vein with 100 ␮l of Evans blue solution (2% (w/v) in 0.08 U/ml ALF markedly decreased migration (Fig. 1b), but no PBS; Sigma-Aldrich). After 10 min, animals were killed by terminal an- decrease in cell viability was observed. These findings suggest that esthesia, and the eyes were immersed in 2% (w/v) paraformaldehyde for the cell morphology following treatment with ALF remains gen- 1 h. The retinas were removed, washed twice in PBS for 15 min, spread on clean glass slides, and mounted with the vitreous side upward (25). The erally unchanged, whereas migration is reduced to basal levels. mounts were examined using a confocal laser scanning microscope, LSM Dose-response assays using 0–0.1 U/ml ALF revealed that the 510 (Carl Zeiss). prior incubation of THPs with 0.08 U/ml produced a maximal inhibition of the transmonolayer migration of monocytic THP-1 Northern hybridization analysis cells (Fig. 1c). The addition of 100 ng/ml CCL5 and 0.08 U/ml Total RNA from control and stimulated cells was extracted using RNAzol ALF decreased migration to background levels. ϩ B (Ambion), according to the manufacturer’s protocol. Poly(A) RNA was To examine whether both the THP-1 migratory cells and the en- purified using Oligotex mRNA kits (Qiagen), as recommended by the man- ufacturer. RNA samples were electrophoresed in formaldehyde-containing, dothelial monolayers were sensitive to treatment with the acid hydro- denaturing agarose gels and blotted onto Hybond XL nitrocellulose (Am- lase, a parallel experiment was conducted in which either the model ersham Biosciences). A quantity amounting to 25 ␮g of total RNA was or the migratory monocytic cells were treated individu- loaded onto the gel. Probes were prepared using 25 ng of PCR product as ally or together with 0.08 U/ml ALF. As with previous experiments, Downloaded from ␣32 template and [ P]dCTP (Amersham Biosciences) as label. Excess nu- an optimal 100 ng/ml CCL5 was added to the lower chamber during cleotides were removed from the probes by purification using Sephadex G-50 nick columns (Amersham Biosciences). Blots were prehybridized for the chemotaxis assay. The results demonstrated that compared with 4 h at 50°C in high SDS Church buffer (7% SDS, 50% formamide, 25% the controls, migration was markedly inhibited in all cases in which 20ϫ SSC, 5% 1 M sodium phosphate (pH 7), 10% 50ϫ Denhardt’s solu- ALF was added (Fig. 1d). Furthermore, compared with the basal mi- ϫ tion, and 1% 10 N-lauroyl sarcosine (Sigma-Aldrich)) containing 100 gration levels, pretreatment of the monocytic cells with ALF produced ␮g/ml salmon sperm DNA for blocking. Hybridization was performed for

ϭ http://www.jimmunol.org/ 16 h at 50°C, and detection was performed by x-ray film exposure. a greater decrease in cell migration ( p 0.0004) than pretreatment of the endothelial monolayer ( p ϭ 0.0374). Furthermore, combined pre- Preparation of cell lysates treatment of monocytes and endothelial cells seems to have an addi- THP-1 cells were washed in sterile 0.01% PBS, and then serum starved for tive effect on transmigration. The decrease in migration compared 24 h. The cells were washed and supplemented with 2 mg/ml BSA or 2 with individual treatment of monocytes and endothelial cells was sig- mg/ml BSA and 100 ng/ml CCL5 or CCL3 (PeproTech). Cells were in- nificant ( p ϭ 0.0255 and p ϭ 0.0195, respectively). Controls for basal cubated at 37°C for 24 h or in subsequent experiments for 0, 2, 4, 16, or migration in ALF-pretreated monocytes and endothelial cells in ab- 24 h, respectively. Following incubation, cells were resuspended in lysis sence of CCL5 were not significantly different from untreated cells buffer (dH2O, 150 mM NaCl, 1% Nonidet P-40, 50 mM Tris, 0.2 mM ␮ (data not shown). NaVO4, 1 mM DTT, and 25 g/ml each of aprotinin, leupeptin, and pep- statin). Lysates were incubated at 4°C with agitation for 30 min, followed To control for the possibility that the migratory process was by guest on September 28, 2021 by centrifugation at 12,000 ϫ g for 2 min. An equal volume of glycerol affected by agents present in the ALF suspension buffer, further (Sigma-Aldrich) was added to each sample. Protein estimation was per- chemotaxis experiments were performed. Fig. 1e demonstrates that formed on samples using the bicinchoninic acid protein assay kit (Pierce). compared with samples containing CCL5 alone, the additional SDS-PAGE and Western blot analysis of ALF using THP-1 cells presence of denatured ALF did not significantly decrease cell mi- Ͼ An optimum concentration of protein (20 ␮g) was loaded onto 10% SDS- gration ( p 0.4). PAGE and transferred to a Hybond P nitrocellulose membrane (Amersham Biosciences). Western blot analysis was performed using 5% nonfat milk Cell surface staining with fucose-specific lectin following ALF in TBS with 0.5% Tween 20 for blocking. The membrane was incubated treatment of THP-1 cells with primary Ab Mab72 specific for ALF (University of Iowa) at 4°C overnight at a concentration of 1:50. This was followed by incubation with Labeling experiments were performed to examine the effect of goat anti-rat HRP-conjugated Ab (Santa Cruz Biotechnology) for 1 h at fucosidase treatment on binding of fucose-specific lectin (T. pur- room temperature with shaking. Reactions were developed using the Pierce ECL chemiluminescence substrate kit. pureas) to THP-1 cells. Suspension of THP-1 cells was treated with fucosidase (0.08 U/ml) for 1, 2, 8, and 16 h, respectively, Flow cytometric detection of cell surface ALF and CD31 followed by binding with lectin. Untreated cells strongly labeled To quantify the membrane-bound fraction of ALF, flow cytometry was per- with the lectin (Fig. 2). In contrast, ALF treatment for 8 and 16 h formed using polyclonal rabbit anti-ALF, provided by M. Pa´ez de la Cadena demonstrated a significant reduction in labeling of THP-1. How- (Universidad de Vigo, Vigo, Spain) (1). THP-1 cells were labeled with an ever, incubation for 1 and 2 h with 0.08 U/ml ALF did not show optimal concentration of primary Ab at 4°C for 30 min, washed, and coun- significant reduction in binding of lectin (data not shown). The terstained with a FITC-conjugated goat anti-rabbit Ig (Sigma-Aldrich) for an additional 20 min. CD31 was labeled using a murine mAb (clone JC/70a; enzyme-treated cells were 95 and 85% viable following 8- and Abcam) for 30 min before washing and counterstaining with an appropriate 16-h incubation based on trypan blue exclusion assay. fluorochrome-conjugated goat anti-mouse IgG reagent (Sigma-Aldrich). Ab specificity was controlled with preimmune rabbit serum or an isotype- Effect of ALF treatment on adhesion of THP-1 cells to matched, irrelevant mAb (DakoCytomation). In both cases, the stained cells were analyzed by flow cytometry (FACSort; BD Biosciences); data analysis P-selectin was performed using Lysis II software (BD Biosciences). To determine whether the decrease in transmigration following ALF treatment is due to the effect of enzyme on selectin ligands, Results we conducted adhesion experiments for ALF-treated THP-1 cells The role of ALF in cellular migration to P-selectin.Fc. For these experiments, THP-1 cells were treated To determine whether ALF has the potential to modify the extrav- with 0.08 U/ml ALF, followed by labeling with 2Ј,7Ј-bis(2-car- asation process, a series of chemotaxis experiments was per- boxyethyl)-5-(6)-carboxyfluorescein tetrakis. As a control, a par- formed. In these experiments, a monolayer of the endothelial fu- allel experiment was performed in which cells were untreated 2410 ROLE OF ALF IN LEUKOCYTE EXTRAVASATION

FIGURE 1. Transendothelial chemotaxis assay. A monolayer of the endothelial fusion cell line EAhy926 was grown on the upper surface of the membrane, which contained 3-␮m pores. THP-1 cells were assayed for their ability to migrate. Fol- lowing migration for 2 h, the filters were fixed in methanol and stained with Gill’s hematoxylin. Mi- grant cells were counted using high power micros- copy (ϫ400) in four random fields on each filter. a, THP-1 cells migrating (as small densely stained cells) in response to CCL5 (100 ng/ml). b, THP-1 cells treated with 0.08 U/ml ALF and allowed to migrate in response to 100 ng/ml CCL5. Results are representative of three similar experiments. c, Dose response to determine the optimal concentration of ALF. THP-1 cells were treated for 1 h with 0.02–0.1 U/ml ALF, and then added to the upper compart- ment of the chemotaxis chamber to assess migration through a monolayer of EAhy926 endothelial cells Downloaded from in presence or absence of 100 ng/ml CCL5. d, THP-1 cells or EAhy926 were treated for 1 h with an optimal 0.08 U/ml human ALF. Enzyme-treated cells were then rinsed, and THP-1 cells were added to the upper compartment of the chemotaxis chamber system. e, The endothelial monolayers were treated http://www.jimmunol.org/ with 0.08 U/ml ALF, no ALF, or 0.08 U/ml ALF, which had been denatured by heating to 100°C for 5 min. THP-1 cells were assayed for their ability to mi- grate through a 3-␮m pore diameter filter toward the basal compartment containing either no CCL5 or an optimal 100 ng/ml CCL5. EC ϭ Endothelial cell monolayer; mono ϭ migratory monocytic cell line. ;p Ͻ 0.001 ,ءءء .Bars show mean values Ϯ SEM .p Ͻ 0.05 ,ء ;p Ͻ 0.01 ,ءء by guest on September 28, 2021

(Fig. 3). ALF treatment significantly reduced THP-1 cell adhesion to P-selectin.Fc ( p ϭ 0.0004).

Effect of ALF treatment on the trafficking of splenocytes in vivo in the retinal vasculature To examine the functional consequences of ALF treatment under shear flow, an in vivo study was conducted using a single-cell

FIGURE 2. Cell surface staining with fucose-specific lectin following ALF treatment of THP-1. Representative flow cytometric analysis showing binding of T. purpureas lectin to ␣-L-fucose structures. THP-1 cells were FIGURE 3. Adhesion of THP-1 cells to P-selectin.Fc following treatment treated with fucosidase (0.08 U/ml) for 8 and 16 h, respectively. Following with ALF. Analysis of the adhesion of untreated THP-1 cells or following this, cells were incubated with lectin (50 ␮g/ml) for1hat37°C, followed treatment with ALF (0.08 U/ml) to P-selectin.Fc. Control consists of wells by staining with FITC-conjugated Ab. Black histogram indicates untreated with goat anti-human IgG-Fc Ab with labeled cells only. A total of 500,000 cells, whereas dark gray and pale gray are cells treated with ALF for 8 and cells was added per well, and the experiment shows mean data from three- 16 h, respectively. Representative of three independent experiments. assay wells. Results are representative of two independent experiments. The Journal of Immunology 2411

FIGURE 5. Northern blot analysis using the human monocytic cell line THP-1 to examine regulation of ALF following stimulation with 100 ng/ml CCL5 or CCL3 for 24 h. RNA samples were electrophoresed in formal- dehyde-denaturing agarose gels and blotted onto nitrocellulose; 25 ␮gof total RNA was loaded onto each gel. Probes were prepared using DNA amplified from differentially expressed bands using 25 ng of the PCR prod- ␣32

uct as template with [ P]dCTP. The results are representative of three Downloaded from separate experiments.

thermore, the ability of the splenocytes to migrate through retinal vasculature to penetrate the retinal tissue was also inhibited fol- lowing treatment of migratory cells with the fucosidase, resulting ϭ

in a 3.5-fold decrease in infiltration (Fig. 4c; p 0.0119). http://www.jimmunol.org/

Examining the regulation of the ALF following stimulation with chemokine To determine whether ALF plays a natural role in regulation of inflammation, the human monocytic cell line THP-1 was stimu- lated for various time periods with a prototypical inflammatory chemokine CCL5 at 100 ng/ml. Northern blot analysis was performed to examine regulation of the gene encoding ALF. Fig. 5 shows the densitometric analysis of the by guest on September 28, 2021 Northern blots and demonstrates that there was a 2.2-fold increase in FIGURE 4. Effect of ALF on rolling of cells on retinal vasculature. a, Representative single frame from video analysis of labeled splenocytes passing the expression of ALF following 24-h stimulation with CCL5 and a through retinal venules. b, Analysis of the proportion of rolling cells that were 1.8-fold increase in expression following 24-h stimulation with defined by visible interaction with the vessel wall and reduced velocity com- CCL3. The Northern membranes were also subsequently probed for pared with nonrolling cells. Rolling and noninteracting splenocytes were GAPDH, to ensure equal loading (data not shown). counted in each venule. The percentage of rolling cells among the total number To further examine the regulation of ALF at the protein level, of labeled splenocytes that entered a venule was calculated for untreated and Western blot analysis was performed; this revealed two bands at 56 ALF-treated splenocytes. Three eyes were used for each test, with rolling and and 51 kDa, which showed increased expression after 4 h (data not adherence being measured in at least three venules; the results are presented as shown) and 24 h (Fig. 6). Densitometric analysis (normalized to an Ϯ mean values SEM. c, Analysis of retinal infiltration by labeled splenocytes. ␣-tubulin loading control) was performed, which yielded values for Following cell infusion (50 min), 100 ␮l of 2% (w/v) Evans blue (Sigma- OD; this showed that after 24-h stimulation with CCL5, the 51- and Aldrich) was injected via the tail vein and allowed to bind for 10 min. The eyes were harvested and fixed in 2% (w/v) paraformaldehyde (Agar Scientific) for 56-kDa bands were up-regulated 2.4- and 3.2-fold, respectively. 1 h. Untreated and ALF-treated samples were observed using a confocal scan- ALF is usually found as a soluble component of the lysosomes ning laser imaging system fitted with krypton/argon lasers (Zeiss LSM510; and antiserum. However, it has also been reported that it can exist Carl Zeiss). Three eyes were used for each test, and results presented as mean associated with plasma membrane (10–20% of the total cellular values Ϯ SEM. fucosidase activity) in a range of cell types, including monocytes (1). Therefore, to determine whether stimulation with chemokines suspension from mice immunized (11 days postimmu- altered the distribution of membrane-bound and cytoplasmic ALF, nization) with interphotoreceptor retinoid-binding protein peptide a flow cytometric analysis was performed. Although ALF was de- to induce EAU. Cells were divided into two groups, and one group tected on the cell surface, no significant change in cell surface was treated with ALF. To ensure the cleavage of fucose residues expression was observed following stimulation with CCL5 for 30 present on the surface of splenocytes, the cells were stained with min, 4 h, or 24 h (data not shown). a biotinylated fucose-specific lectin (T. purpureas agglutinin) fol- lowing treatment with ALF to validate removal from the cell sur- Modification of cell surface CD31 by treatment with ALF face (data not shown). The process of transendothelial migration involves a series of ad- Results from SLO (Fig. 4a) revealed that the treatment of hesion molecules together with their respective receptors; ALF is splenocytes with ALF had a highly significant effect on both the known to degrade fucosylated moieties. Because the fucosylated rolling and retinal infiltration of cells. Following treatment with molecule CD31 (PECAM-1) is central to the process of leukocyte ALF (0.08 U/5 ϫ 105 cells), an 8.6-fold decrease ( p Ͻ 0.0001) in migration, immunofluorescence flow cytometry was used to mea- rolling compared with untreated cells was observed (Fig. 4b). Fur- sure the surface expression of CD31 on monocytic THP-1 and 2412 ROLE OF ALF IN LEUKOCYTE EXTRAVASATION

Discussion ALF is a ubiquitous acid hydrolase that is normally found as a soluble component of lysosomes and plasma, but up to 20% of ALF activity is associated with the cell surface (1). This enzyme can process fucosylated residues that normally play a role in sta- bilizing intercellular adhesion, potentially regulating cell traffick- ing in health and disease. Furthermore, ALF from some sources has also been shown to have unique property to perform the syn- thesis of complex oligosaccharides by transglycosylation (26). This might provide a valuable approach for synthesis of fucose containing oligosaccharides because ␣-glycosynthases are difficult to obtain. The current study was designed to assess the possibility that ALF can modulate inflammation by reducing the interaction between fucoslyated adhesion molecules that normally support leukocyte extravasation. The ability of the monocytic cell line, THP-1, to migrate through an endothelial cell monolayer in response to the chemo- kine CCL5 was tested in a model system. Dose-response studies Downloaded from showed that the presence of 0.08 U/ml ALF inhibited leukocyte migration to a background level. A comparison of treatment of either the endothelial cells or the monocytes showed that both cell FIGURE 6. Western blot analysis of ALF using THP-1. A, An optimum types express migration-critical residues that were inhibited by concentration of 20 ␮g of protein was loaded onto 10% SDS-PAGE and ALF. However, the slightly greater inhibition produced by treat- transferred to a nitrocellulose membrane. Western blot analysis was per- ment of the monocytes suggests that fucosylated ligands may play formed using 5% nonfat milk in TBS with 0.5% Tween 20 for blocking. http://www.jimmunol.org/ a dominant role on these cells. Furthermore, combined pretreat- The membrane was incubated with primary Ab Mab72 ALF at a concen- tration of 1:50. This was followed by incubation with goat anti-rat HRP- ment of endothelial and THP-1 cells had an additive effect on conjugated Ab. Lane 1, Cells were treated with 2 mg/ml BSA for 24 h; lane inhibition of transmigration. 2, cells were treated with 2 mg/ml BSA and 100 ng/ml CCL5 for 24 h. The Using flow cytometry, we identified a significant decrease in the results are representative of three separate experiments. binding of fucose-specific lectin following ALF treatment. Inter- estingly, treatment of both the monocytic and endothelial cells for 1 h with 0.8 U/ml ALF was sufficient to reduce cell migration to a basal level. However, a significant reduction in the binding of the EaHY.926 cells up to 24 h after the addition of 0.08 U/ml ALF. CD31 expression was examined at each time point using flow cy- lectin was only evident after treatment for 8 and 16 h. It is difficult by guest on September 28, 2021 tometric analysis. to compare the results with previous observations due to variability EAhy926 cells demonstrated no significant difference in CD31 in the source of enzyme and lectins/Abs used (27). expression following ALF treatment for 2 h; however, at 5 h, there PSGL-1, originally identified as a ligand for P-selectin, can bind was a 7% increase in cell surface expression ( p ϭ 0.0001). The all three selectins and has a proadhesive function in many inflam- expression of CD31 declined dramatically between 5 and 16 h, and matory settings (28). L-selectin is constitutively expressed by most gradually increased by 24 h such that there was no significant leukocytes, whereas the other members of selectin family, P- and difference between 0 and 24 h. Following treatment with ALF, E-selectin, are expressed by activated endothelium. Acting in co- THP-1 cells demonstrated decreased expression of CD31 between operation with L-selectin, P-selectin mediates the initial interaction 0and5h(p ϭ 0.002), but Ab binding then gradually increased between circulating leukocytes and endothelial cells that produces (Fig. 7). The level of CD31 expression was significantly lower at characteristic rolling of leukocytes on endothelium. To assess the 24 h compared with 0 h with p value of 0.006. contribution of fucose residues on selectin binding, solid-phase adhesion experiments were conducted. The current study was con- sistent with previous observations that fucosylation of appropriate carbohydrate determinants is critical for selectin ligand generation (13). Furthermore, treatment of THP-1 cells with ALF resulted in 53% decrease in the adhesion of THP-1 cells to P-selectin.Fc. To further examine the role of ALF in vivo, we used an EAU model, induced by immunization of mice with retinal Ags (20). The blood-retina barrier is breached in EAU, allowing both T lymphocytes and monocytes to move into the retina (29). The pretreatment of splenocytes from sensitized animals with ALF resulted in a profound decrease in the ability of these cells to roll along the surface of the retinal endothelium, and also reduced the number of cells that undergo diapedesis and penetrate the retinal tissues. Homophilic interactions between CD31 (PECAM-1) on the sur- FIGURE 7. Examination of CD31 Ab binding site following ALF treat- face of inflammatory leukocytes and CD31 within the junctions ment of THP-1 and EAhy926 cells. THP-1 and EAhy926 cells were incubated with 0.08 U/ml ALF (0–24 h). Using THP-1 and EAhy926 cells labeled with between endothelial cells allow the passage of leukocytes across an appropriate isotype control, a positivity marker was set at 3%. Cell popu- confluent endothelium (30), with Ab blockade of CD31 reducing lations that were gated beyond this threshold were defined as positive for the transendothelial migration by up to 90% (31). To identify a further expression of CD31. Eahy926 is denoted by Œ, and THP-1 by ●. potential mechanism by which ALF can mediate its effect, a series The Journal of Immunology 2413 of studies was performed to measure the potential of this enzyme 8. Ayude, D., J. Fernandez-Rodriguez, F. J. Rodriguez-Berrocal, V. S. Martinez- to degrade CD31. Investigation of the nature of the N-linked gly- Zorzano, A. de Carlos, E. Gil, and M. Paez de La Cadena. 2000. Value of the serum ␣-L-fucosidase activity in the diagnosis of colorectal cancer. Oncology 59: cans in Chinese hamster ovary cell-expressed CD31 has already 310–316. revealed the presence of sialylated bi-, tri-, and tetra-antennary 9. Giardina, M. G., M. Matarazzo, R. Morante, A. Lucariello, A. Varriale, V. Guardasole, and G. De Marco. 1998. Serum ␣-L-fucosidase activity and early compounds, of which 62% are fucosylated at the core GlcNAc detection of hepatocellular carcinoma: a prospective study of patients with cir- residue (32). The current study demonstrated CD31 expression by rhosis. Cancer 83: 2468–2474. both endothelial and monocytic cell lines. ALF does have an effect 10. Homeister, J. W., A. D. Thall, B. Petryniak, P. Maly, C. E. Rogers, P. L. Smith, R. J. Kelly, K. M. Gersten, S. W. Askari, G. Cheng, et al. 2001. The ␣(1,3) fuco- on CD31 expression on EAhy926 endothelial cells at several of syltransferases FucT-IV and FucT-VII exert collaborative control over selectin-de- the time points that were observed; however, after 24-h expo- pendent leukocyte recruitment and lymphocyte homing. Immunity 15: 115–126. sure, the CD31 levels return to the basal level. THP-1 cells 11. Xia, L., M. Sperandio, T. Yago, J. M. McDaniel, R. D. Cummings, S. Pearson-White, K. Ley, and R. P. McEver. 2002. P-selectin glycoprotein li- treated under the same conditions show a decline in CD31 expres- gand-1-deficient mice have impaired leukocyte tethering to E-selectin under flow. sion between 0 and 5 h. The difference in the level of CD31 ex- J. Clin. Invest. 109: 939–950. 12. Yang, J., T. Hirata, K. Croce, G. Merrill-Skoloff, B. Tchernychev, E. Williams, pression between the two cell types might be attributed to the R. Flaumenhaft, B. C. Furie, and B. Furie. 1999. Targeted gene disruption dem- location of CD31 molecule. It is possible that the cleavage of fu- onstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selec- cose residues, contained within the CD31 molecules, within the tin-mediated but not E-selectin-mediated rolling and migration. J. Exp. Med. 190: 1769–1782. junction of an endothelial monolayer, would be more difficult to 13. Zen, K., Y. Liu, D. Cairo, and C. A. Parkos. 2002. CD11b/CD18-dependent access than those that are expressed on cell surface of migratory interactions of with intestinal epithelium are mediated by fucosylated cells. Significantly, the potential of a CD31 mAb to label these proteoglycans. J. Immunol. 169: 5270–5278. 14. Granert, C., J. Raud, X. Xie, L. Lindquist, and L. Lindbom. 1994. Inhibition of cells was reduced by pretreatment of the cells with ALF, suggest- leukocyte rolling with polysaccharide fucoidin prevents pleocytosis in experi- Downloaded from ing degradation of a fucose-containing epitope that may be critical mental meningitis in the rabbit. J. Clin. Invest. 93: 929–936. for transendothelial migration. 15. Linnemann, G., K. Reinhart, U. Parade, A. Philipp, W. Pfister, E. Straube, and W. Karzai. 2000. The effects of inhibiting leukocyte migration with fucoidin in The possibility of regulation of ALF expression at both the a rat peritonitis model. Intens. Care Med. 26: 1540–1546. mRNA and protein levels was investigated. Interestingly, follow- 16. Giblin, P. A., S. T. Hwang, T. R. Katsumoto, and S. D. Rosen. 1997. Ligation of ␤ L-selectin on T lymphocytes activates 1 integrins and promotes adhesion to ing treatment with CCL5 (100 ng/ml), the gene product that coded fibronectin. J. Immunol. 159: 3498–3507. for ALF was up-regulated by more than 2-fold. A similar increase 17. Komosinska-Vassev, K., K. Olczyk, E. M. Kozma, K. Winsz-Szczotka, http://www.jimmunol.org/ in expression (1.8-fold) was produced by treatment with 100 ng/ml P. Olczyk, and G. Wisowski. 2003. Graves’ disease-associated changes in the serum lysosomal glycosidases activity and the glycosaminoglycan content. Clin. CCL3. To examine the protein expression of ALF, THP-1 cells Chim. Acta 331: 97–102. were stimulated with 100 ng/ml CCL5 for 4 and 24 h; ALF bands 18. Edgell, C. J., C. C. McDonald, and J. B. Graham. 1983. Permanent cell line were identified at 51 and 56 kDa by Western blotting, which is expressing human factor VIII-related established by hybridization. Proc. Natl. Acad. Sci. USA 80: 3734–3737. consistent with previous reports (1, 3, 5). Treatment with CCL5 19. Silver, P. B., C. C. Chan, B. Wiggert, and R. R. Caspi. 1999. The requirement for increased protein expression after 4 h (data not shown), with stim- pertussis to induce EAU is strain-dependent: B10.RIII, but not B10.A mice, de- ulation for 24 h increasing expression of both products by between velop EAU and Th1 responses to IRBP without pertussis treatment. Invest. Oph- thalmol. Visual Sci. 40: 2898–2905. 2- and 3-fold. 20. Jiang, H. R., X. Wei, W. Niedbala, L. Lumsden, F. Y. Liew, and J. V. Forrester. In conclusion, this study shows that the acid hydrolase, ALF, is 2001. IL-18 not required for IRBP peptide-induced EAU: studies in gene-defi- by guest on September 28, 2021 cient mice. Invest. Ophthalmol. Visual Sci. 42: 177–182. potentially up-regulated by chemokines in the later stages of in- 21. Xu, H., A. Manivannan, K. A. Goatman, J. Liversidge, P. F. Sharp, flammation. The function of this enzyme includes cleavage of fu- J. V. Forrester, and I. J. Crane. 2002. Improved leukocyte tracking in mouse cosylated residues from adhesion molecules such as sLeX and retinal and choroidal circulation. Exp. Eye Res. 74: 403–410. ␣ 22. Vajkoczy, P., M. Laschinger, and B. Engelhardt. 2001. 4--VCAM-1 CD31, which play an important role in intravascular leukocyte binding mediates G protein-independent capture of encephalitogenic T cell blasts rolling and subsequent extravasation. The potential for inflamma- to CNS white matter microvessels. J. Clin. Invest. 108: 557–565. tory chemokines to up-regulate the expression of ALF is consistent 23. Ley, K., and P. Gaehtgens. 1991. Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules. Circ. Res. with activation of a natural regulatory loop, resulting in a gradual 69: 1034–1041. diminution of the potential of blood-borne leukocytes to penetrate 24. Xu, H., A. Manivannan, H. R. Jiang, J. Liversidge, P. F. Sharp, J. V. Forrester, ␥ the endothelium at sites of existing inflammation. and I. J. Crane. 2004. Recruitment of IFN- -producing (Th1-like) cells into the inflamed retina in vivo is preferentially regulated by P-selectin glycoprotein li- gand 1:P/E-selectin interactions. J. Immunol. 172: 3215–3224. Disclosures 25. Chan-Ling, T. 1997. Glial, vascular, and neuronal cytogenesis in whole-mounted The authors have no financial conflict of interest. cat retina. Microsc. Res. Tech. 36: 1–16. 26. Berteau, O., J. Bielicki, A. Kilonda, D. Machy, D. S. Anson, and L. Kenne. 2004. ␣-L-fucosidases: exoglycosidases with unusual transglycosylation properties. References Biochemistry 43: 7881–7891. 1. Cordero, O. J., A. Merino, M. Paez de la Cadena, B. Bugia, M. Nogueira, 27. Yuan, K., D. Kucik, R. K. Singh, C. M. Listinsky, J. J. Listinsky, and J. E. Vinuela, V. S. Martinez-Zorzano, A. de Carlos, and F. J. Rodriguez-Berrocal. G. P. Siegel. 2008. Alterations in human breast cancer adhesion-motility in re- 2001. Cell surface human ␣-L-fucosidase. Eur. J. Biochem. 268: 3321–3331. sponse to changes in cell surface glycoproteins displaying ␣-L-fucose moieties. 2. Fukushima, H., J. R. de Wet, and J. S. O’Brien. 1985. Molecular cloning of a Int. J. Oncol. 32: 797–807. cDNA for human ␣-L-fucosidase. Proc. Natl. Acad. Sci. USA 82: 1262–1265. 28. Veerman, K. M., M. J. Williams, K. Uchimura, M. S. Singer, J. S. Merzaban, 3. Johnson, S. W., S. Piesecki, R. F. Wang, I. Damjanov, and J. A. Alhadeff. 1992. S. Naus, D. A. Carlow, P. Owen, J. Rivera-Nieves, S. D. Rosen, and Analysis of purified human liver ␣-L-fucosidase by Western-blotting with lectins H. J. Ziltener. 2007. Interaction of the selectin ligand PSGL-1 with chemokines and polyclonal and monoclonal antibodies. Biochem. J. 282: 829–834. CCL21 and CCL19 facilitates efficient homing of T cells to secondary lymphoid 4. Abdel-Aleem, H., A. Ahmed, A. M. Sabra, M. Zakhari, M. Soliman, and organs. Nat. Immunol. 8: 532–539. H. Hamed. 1996. Serum ␣ L-fucosidase enzyme activity in ovarian and other 29. Crane, I. J., H. Xu, C. Wallace, A. Manivannan, M. Mack, J. Liversidge, female genital tract tumors. Int. J. Gynaecol. Obstet. 55: 273–279. G. Marquez, P. F. Sharp, and J. V. Forrester. 2006. Involvement of CCR5 in the 5. Aviles, M., I. Abascal, J. A. Martinez-Menarguez, M. T. Castells, S. R. Skalaban, passage of Th1-type cells across the blood-retina barrier in experimental auto- J. Ballesta, and J. A. Alhadeff. 1996. Immunocytochemical localization and bio- immune uveitis. J. Leukocyte Biol. 79: 435–443. chemical characterization of a novel plasma membrane-associated, neutral pH 30. Liao, F., H. K. Huynh, A. Eiroa, T. Greene, E. Polizzi, and W. A. Muller. 1995. optimum ␣-L-fucosidase from rat testis and epididymal spermatozoa. Biochem. J. Migration of monocytes across endothelium and passage through 318: 821–831. involve separate molecular domains of PECAM-1. J. Exp. Med. 182: 1337–1343. 6. Landing, B. H., G. N. Donnell, O. S. Alfi, H. G. Neustein, F. A. Lee, G. Ng, 31. Muller, W. A., S. A. Weigl, X. Deng, and D. M. Phillips. 1993. PECAM-1 is required W. R. Bergren, and P. Sturgeon. 1976. Fucosidosis: clinical, pathologic, and for transendothelial migration of leukocytes. J. Exp. Med. 178: 449–460. biochemical studies of five patients. Adv. Exp. Med. Biol. 68: 147–165. 32. Newton, J. P., A. P. Hunter, D. L. Simmons, C. D. Buckley, and D. J. Harvey. 7. Hirschberg, C. B. 2001. Golgi nucleotide sugar transport and leukocyte adhesion 1999. CD31 (PECAM-1) exists as a dimer and is heavily N-glycosylated. Bio- deficiency II. J. Clin. Invest. 108: 3–6. chem. Biophys. Res. Commun. 261: 283–291.