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CORRESPONDENCE Jesús Rivera-Nieves: jr3u{at}virginia.edu
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The inflammatory bowel diseases (ulcerative colitis [UC] and Crohn's disease [CD]) are chronic immune-mediated conditions that affect more than one million patients in North America and lead to more than $3.6 billion in productivity losses each year (1, 2). The inflammatory process is characterized by heavy leukocytic infiltration of the intestinal lamina propria (LP), leading to fibrosis and loss of function (3, 4). Although the etiology of the disease remains mostly unknown, modulation of the immune response by immunomodulators (e.g., azathioprine, 6-mercaptopurine), corticosteroids, and mAbs to TNF-
The recent characterization of mouse models that spontaneously develops chronic ileitis (i.e., the SAMP1/Yit and SAMP1/YitFc models) enabled us to study the trafficking pathways into the chronically inflamed small intestine (8–10). SAMP1/YitFc mice develop discontinuous, transmural chronic inflammation localized to the small intestine (8, 9) that closely recapitulates the human disease (10, 11). The LP is intensely infiltrated by effector T cells, which display an activated phenotype and adoptively transfer disease to SCID mice (9). However, unlike the CD45RBhigh transfer model (12), lymphocytes from SAMP1/YitFc mice predominantly induce ileitis and not colitis, suggesting an inherent capacity to recirculate preferentially to the small intestine (9). The resulting ileitis is also resistant to modulation by regulatory cells as cotransfer of CD45Rblow or CD25+ T cells, which abolishes colitis and has no effect on ileitis (13). In addition, although colitis responds to single adhesion molecule blockade (14–17), ileitis requires interference with two or more adhesion pathways (i.e., ICAM-1/VCAM-1, L-selectin/mucosal addressin cell adhesion molecule-1 [MAdCAM-1],
Gut-homing CD4+ T cells from SAMP1/YitFc mice coexpress not only
Specialized endothelial cells express glycoproteins that serve as L-selectin ligands in secondary lymphoid organs, including a heterogeneous group of glycans that share the peripheral node addressin (PNAd) epitope (25, 26). MAdCAM-1 (the endothelial ligand for
The potential role of L-selectin in trafficking to the inflamed small intestine prompted us to search for its associated intestinal endothelial ligands. A monoclonal antibody against PSGL-1, but not any other single antiadhesion molecule strategy, attenuated both the CD4+-induced and the spontaneous ileitis. Unexpectedly, not only the infiltrating leukocytes, but also endothelial cells from small intestine and mesenteric lymph node (MLN) expressed functional PSGL-1. Our results, therefore, suggest that PSGL-1 is an integral part of the address code for small intestinal homing and that it is critically involved in recruitment of leukocytes, including effector CD4+ T cells, into the LP of the chronically inflamed small intestine. 
improve disease symptoms and outcomes (3–6). Although UC affects strictly the large intestine, CD primarily involves the small intestine, predominantly the terminal ileum (chronic ileitis) (3, 4). Thus, the recirculating lymphocyte pool likely possesses a defined repertoire of adhesion molecules and chemokines ("address code") (7) that allows them to distinguish between the small and large intestine. However, the specific combination of molecules engaged by pathogenic cells to localize specifically to the small intestine has not been identified. 4 integrins) (18, 19). This is in keeping with clinical trials in which patients with CD receiving natalizumab (a mAb that targets the shared 4 moiety of both 4ß7 and 4ß1 integrins) achieved a clinical response, whereas specific blockade of the gut-homing integrin 4ß7 was not more efficacious than the placebo (20–22). 4ß7 and 4ß1 integrins, but also L-selectin (19). This L-selectinhi population produces copious amounts of TNF- and plays an important role in disease induction in SCID mice (19). L-selectin is involved in trafficking of naive and subpopulations of memory CD4+ T cells to intestinal sites (19, 23, 24), but the small intestinal endothelial L-selectin ligands are not known. Identification of endothelial ligands that may be preferentially used in inflammatory trafficking may prove valuable for the development of therapeutic agents, as L-selectin is ubiquitously expressed. 4ß7 integrin) is present in intestinal- and gastrointestinal-associated lymphoid tissues (27) and can also serve as an L-selectin ligand when appropriately glycosylated (28). P-selectin glycoprotein ligand 1 (PSGL-1) (29) can also bind L-selectin (expressed on most leukocytes), P-selectin (expressed on inflamed endothelial cells and activated platelets), and E-selectin (expressed on inflamed endothelial cells) (30, 31).
RESULTS
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Abstract
RESULTS
DISCUSSION
MATERIALS AND METHODS
References
PSGL-1 plays a critical role in chronic murine ileitis
We have recently shown that L-selectin plays an important role in adoptively transferred chronic ileitis (19), a disease induced by pathogenic CD4+ T cells (9). To identify relevant L-selectin ligands within the microcirculation of the terminal ileum, we designed a series of therapeutic studies. SCID mice adoptively transferred with CD4+ T cells isolated from donor SAMP1/YitFc mice develop severe ileitis within 6 wk (9, 18). The PNAd epitope (shared by several L-selectin ligands) was expressed within the MLN of inflamed SCID mice, but not in those from their noninflamed counterparts (Fig. 1 A). Therefore, 6 wk after adoptive transfer, we targeted PNAd using the mAb MECA-79 alone (not depicted) or in combination with anti–MAdCAM-1 (MECA-367) for 3 d (Fig. 1 B) to determine whether PNAd was the critical small intestinal endothelial ligand. This treatment provided no therapeutic benefit (active index = 4.5 ± 0.7, chronic index = 4.4 ± 0.9) compared with mice treated with isotype antibodies (active index = 4.1 ± 0.6, chronic index = 4.9 ± 0.6).
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4ß7 integrin and for L-selectin when appropriately glycosylated (27, 28). The mAb MECA-367 interferes with 4ß7 binding, whereas MECA-89 blocks interactions with L-selectin (32). However, MAdCAM-1 does not appear to be the critical small intestinal endothelial ligand, as there was no therapeutic benefit after MECA-89 antibody treatment alone or when combined with MECA-367 (active index = 4.8 ± 0.7, chronic index = 5.7 ± 0.7) compared with isotype-treated controls (active index = 5.3 ± 0.8, chronic index = 5.2 ± 0.9) (unpublished data). We tested whether PSGL-1, a known pan-selectin ligand, could be the endothelial counterpart responsible for the therapeutic effect of L-selectin blockade. Using the function-blocking mAb 4RA10 (33), we targeted PSGL-1 alone or combined with anti–MAdCAM-1 mAb MECA-367 in inflamed SCID mice. Animals were administered three doses of mAb/s every other day, 6 wk after adoptive transfer, as the ileitis reached maximum severity. 4RA10 reduced both active and chronic inflammatory indices (active index = 3.4 ± 0.4, chronic index = 3.8 ± 0.5) compared with isotype-treated controls (active index = 5.6 ± 0.3, chronic index = 5.5 ± 0.4, P < 0.01) (Fig. 1 C). Additional MAdCAM-1 blockade did not significantly improve the effect (4RA10/MECA 367; active index = 2.8 ± 0.4, chronic index = 2.7 ± 0.6; the p-value vs. 4RA10 was not significant).
PSGL-1 blockade attenuated spontaneous chronic ileitis
To investigate whether a similar therapeutic effect could be achieved on the spontaneous ileitis of SAMP1/YitFc mice, we administered three doses of the 4RA10 mAb (200 µg) every other day to 10-wk-old mice, which at that age exhibit robust ileitis. Prior studies had shown that, at this time point, the disease is refractory to all antiadhesion strategies evaluated (18). However, 4RA10 reduced the villous distortion index by 30% (P < 0.005) (Fig. 2 A), the active inflammatory index (granulocytic infiltrates) by 70% (P < 0.0001) (Fig. 2 B), and the chronic inflammatory index (lymphocytic/monocytic infiltrates) by 50% (P < 0.0005) (Fig. 2 C) compared with isotype-treated controls (Fig. 2, black bars). The total inflammatory index was reduced by 50% (P < 0.0001) (Fig. 2 D) and restoration of the villous and crypt architectures was also observed in the 4RA10-treated mice (Fig. 2, E and F). No other single or combined antiadhesion molecule strategy has shown comparable therapeutic benefit (18). The therapeutic effect of anti–PSGL-1 mAb in SAMP1/Yit mice has been independently verified by Inoue et al. (34).
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Levels of soluble P- and E-selectins increased in inflamed SAMP1/YitFc mice, but pan-selectin antagonism did not attenuate chronic ileitis
Pan-selectin antagonism through targeted blockade of PSGL-1 (a pan-selectin ligand) is an obvious potential mechanism for the therapeutic effect of 4RA10. Therefore, we first examined whether P- and E-selectins played a role in the development of ileitis by measuring the levels of soluble selectins in serum as the disease progressed from 4 to 28 wk. Both soluble P- and E-selectin levels increased from 4 to 8 wk during the early stages of the disease and reached a plateau thereafter (Fig. 4 A). This correlation between soluble serum selectin levels and severity of inflammation supported a role for both P- and E-selectins in the development of the disease and justified a therapeutic study in which all selectins were simultaneously targeted. However, targeting all three selectins in 10-wk-old SAMP1/YitFc mice offered no therapeutic benefit (Fig. 4 B), suggesting that pan-selectin antagonism was not the sole mechanism of the disease-attenuating effect of mAb 4RA10.
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WT bone marrow chimeric miceWT chimeric mice lacked leukocyte-derived PSGL-1(Fig. 7 A), but continued to express PSGL-1 within small intestinal endothelium (Fig. 7 B). Real-time RT-PCR of PSGL-1–/–WT chimeric mice tissues localized PSGL-1 mRNA within the duodenum, jejunum, ileum, and MLN but not within the kidney, spleen, liver, bone marrow, or heart. Peripheral blood leukocytes from control mice reconstituted with wild-type bone marrow (WTWT) expressed PSGL-1 on leukocytes (Fig. 7 A), as well as PSGL-1 mRNA in all organs surveyed, commensurate with their leukocyte content (Fig. 7 C, black bars). PSGL-1–/– tissues, used as negative control, lacked PSGL-1 mRNA (representative PSGL-1–/– MLN shown, Fig. 7 C). Collectively, these data demonstrate that venular endothelial cells in the small intestine and MLN express functional PSGL-1.
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ARE mice that, like SAMP1/YitFc mice, develop chronic ileitis (39). Decreased incorporation of radioactivity was observed throughout the small intestine and colon of PSGL-1–deficient TNFARE mice; however, these differences reached statistical significance only in the MLN and ileum (P < 0.05). No differences in recruitment were seen in the liver, kidney, or colon (Fig. 8).
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50–80 µm in diameter, where engaged cells (Fig. 9 B, red arrows) could be identified as they advanced much slower than their free-flowing counterparts (Fig. 9 B, blue arrowheads). A significant reduction in the number of rolling cells (70%) was observed after anti–PSGL-1 antibody injection (Fig. 9 C). As the rolling cells lacked PSGL-1, the effect of the antibody must be attributed to interference with endothelial PSGL-1 interactions.
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The mAb MECA-79 recognizes several sialomucins (PNAd) that, when properly glycosylated, serve as L-selectin ligands. Functionally, MECA-79 blocks lymphocyte interactions with HEVs in vitro and short-term lymphocyte homing to lymph nodes in vivo (25). Its role in inflammatory trafficking has been shown in the inflamed pancreas (40, 41) and lacrimal glands (42). MECA-79 binds to HEV-like vessels in the organized lymphoid aggregates present in chronically inflamed tissues (26). Although we demonstrate PNAd expression in SCID mice with ileitis, no beneficial therapeutic effect was seen by MECA-79 administration.
MAdCAM-1, the 4ß7 integrin endothelial ligand expressed in intestine and gastrointestinal-associated lymphoid tissues (27), serves as an L-selectin ligand when appropriately glycosylated (7, 27, 28). MAdCAM-1 is constitutively expressed in postcapillary venules of intestinal LP, MLN, and Peyer's patches HEVs (27, 43), and is aberrantly expressed in the inflamed pancreas (39). Furthermore, MAdCAM-1 is up-regulated in the chronically inflamed small and large intestine of patients with UC and CD, where it is likely induced by TNF- (43, 44). The mAb MECA-367 binds to the first Ig domain of MAdCAM-1 and interferes with 4ß7 binding, whereas MECA-89 binds to the second Ig domain, which interferes with L-selectin interactions as a result of its proximity to the mucinous portion of MAdCAM-1 (32). If MAdCAM-1 mediated critical L-selectin interactions in the gut, we would expect that combined MECA-367/MECA-89 treatment would provide the same benefit as combined MECA-367/MEL-14 (19). However, administering MECA-89 together with MECA-367 provided no added therapeutic benefit.
PSGL-1 binds to L-selectin (29), E-selectin, and P-selectin (30, 31). The most comprehensive survey of PSGL-1 expression to date identified bone marrow–derived cells as the primary site of expression, whereas expression by endothelial cells was limited to small venules and capillaries of pathological tissues (i.e., benign prostatic hypertrophy and fibrocystic disease of the breast) (45). PSGL-1 binding to selectins is regulated by fucosyltransferases, core 2-glucosaminyltransferases, sialyltransferases, and sulfotransferases, which are constitutively expressed in myeloid cells but regulated in lymphocytes. Thus, effector memory cells of the Th1 type bind selectins better than Th2 polarized CD4+ T cells (30, 46–48). Endothelial cells also express most, if not all, of these glycosyltransferases (49), suggesting that all of the machinery necessary to express functional selectin binding activity is present.
Different from other models of colitis that may be successfully treated by blockade of single adhesion molecules (14–17), the spontaneous chronic ileitis of SAMP/Yit mice is refractory to single and to many combined antiadhesion molecule strategies (i.e., MAdCAM-1, 4ß7 integrin, ICAM-1, VCAM-1, 4 integrin, combined ICAM-1/VCAM-1, and ICAM-1/4) (18). In contrast, the current data shows that targeting PSGL-1 alone ameliorates ileitis. This effect cannot be solely explained by the expected pan-selectin antagonism as combined blockade of all three selectins failed to attenuate ileitis. Expression of PSGL-1 on ileal endothelium provides a plausible explanation of why blockade of a single adhesion molecule is effective. First, PSGL-1 blockade prevents entry of functional PSGL-1–expressing (Th1-polarized) (30, 46–48) and L-selectin–expressing (naive and subpopulations of memory CD4+ T cells) into MLN and ileal LP (19, 23), where endothelial PSGL-1 acts as an "addressin" for small intestinal homing of L-selectin–expressing cells. Second, anti–PSGL-1 interferes with recruitment of PSGL-1+ granulocytes and monocytes, as well as those expressing L-selectin, which might use endothelial PSGL-1 as a ligand. Third, blocking PSGL-1 prevents secondary leukocyte tethering to P-selectin on adherent platelets, as well as PSGL-1/P-selectin- and/or PSGL-1/L-selectin–mediated leukocyte–leukocyte and leukocyte–platelet interactions. P-selectin–PSGL-1 interactions are known to exacerbate atherosclerosis (50) and dextran sulfate sodium–induced colitis (51). Ligation of PSGL-1 on activated Th1 cells may also induce rapid apoptosis of these cells, which are known to be pivotal for induction and maintenance of ileitis (52).
Collectively, our data demonstrate that PSGL-1 is constitutively expressed on endothelial cells of the MLN and small intestine. In this location, endothelial PSGL-1 may act as an addressin to support homing of L-selectin–expressing leukocytes and memory T cells into the small intestinal LP and MLN. Because the SAMP1/YitFc model shares many features of the human disease, it would be reasonable to expect that PSGL-1 may represent a viable therapeutic target for the treatment of patients with CD.
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MATERIALS AND METHODS |
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TopAbstractRESULTSDISCUSSIONMATERIALS AND METHODSReferences |
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C3SnSmn.CB17-Prkdcscid/J SCID mice (6–8 wk old) (The Jackson Laboratory) were purchased and housed at the University of Virginia vivarium under specific pathogen-free conditions for 1 wk before SAMP1/YitFc CD4+ T cell adoptive transfer and thereafter. PSGL-1–/– and E-, P-, L-selectin–deficient mice, both on C57BL/6 background were generated as described previously (31, 37).
TNFARE mice were generated as described previously (39), backcrossed to C57BL/6J mice (18 generations), and kept under specific pathogen-free conditions. The C57BL/6J genetic background did not alter the localization, time course, or severity the intestinal inflammation (unpublished data). TNFARE+/–/PSGL-1–/– were generated by crossing TNFARE+/– mice to PSGL-1–/– mice. The double mutant mice were obtained after two backcrosses. PSGL-1 deficiency was confirmed by PCR and flow cytometry of peripheral leukocytes.
SAMP.B6-MHC congenic mice for alleles from the C57BL/6J (B6) chromosome 17 were generated by introgression of a B6 interval defined by the microsatellite loci D17Mit16 (31.3 Mb) and D17Mit221 (87.9 Mb) onto the SAMP genetic background using a marker-assisted selection ("speed congenic") protocol. This interval included the entire major histocompatibility complex, converting the new congenic strain to the H-2b haplotype from H-2k. This change renders SAMP.B6-MHC congenic mice as suitable recipients for bone marrow from C57BL/6J (B6) mice and PSGL-1–/– mice on this background, without altering the intestinal phenotype (35). All animal handling procedures were approved by the University of Virginia Health Sciences Center institutional committee for animal use.
Tissue collection and histological analyses.
Mice were anesthetized and killed at the times required by the experimental design. The MLN was identified at the confluence of the mesenteric vasculature and harvested. The distal ilea (10 cm) were resected, opened, rinsed of debris, oriented from distal to proximal, and pinned longitudinally in corkboard. Tissues were fixed in 10% buffered formalin or Bouin's, embedded in paraffin, and cut into 3–5 µm sections. Resulting sections were stained with hematoxylin and eosin. Histological assessment of ileal inflammation was performed by a single pathologist in a blinded fashion using a standardized semi-quantitative scoring system as described previously (18).
Immunohistochemistry.
Mice were injected with mAbs 4RA-10 against PSGL-1, MECA-79 against PNAd or isotype control mAb (IgG1, IgM, respectively) i.p. MLN and ilea were harvested 30 min after mAb injection and snap frozen, and sections (5 µm) were cut on a cryostat (Microm HM505N). Secondary staining was conducted with rabbit anti–rat antibody HRP using either VIP purple or DAB as substrates and methyl green or hematoxylin as counterstains, respectively (Vector Laboratories). Nonspecific binding was reduced using normal mouse serum (Sigma-Aldrich). Tissues from mice injected with isotype antibody served as controls.
Determination of serum soluble P- and E-selectin levels.
Serum levels of soluble P- and E-selectins were determined by ELISA per the manufacturer's instructions from mice of the indicated ages (R&D Systems).
Intestinal LP lymphocyte and endothelial cell isolation.
MLN were aseptically removed at the time of necropsy. Single cell suspensions were obtained by gently pressing the MLN against a 100-µm cell strainer. LP mononuclear cell isolation was performed as described previously (19).
Cell culture.
For enrichment of adherent endothelial cells, LP mononuclear cells were cultured at 37°C in six-well flat-bottom plates at 106 cells/ml in complete medium (RPMI 1640 with 10% FBS, 2 mM L-glutamine and 1% penicillin/streptomycin) for 2 h or leukocytes were depleted using CD45+ magnetic beads.
T cell and endothelial cell enrichment and separation.
Enriched CD4+ T cell fractions were obtained by incubation with anti-CD4–bound magnetic beads and sorted into discrete populations, using a magnetic cell–sorting system. Endothelial cell fractions were enriched by depleting the LP mononuclear cell fraction of CD45+ cells using anti-CD45 magnetic beads (Miltenyi Biotec) following manufacturer's instructions. Flow cytometry confirmed 95–97% purity of CD4+ T cell fractions and 98% purity of CD45 negative fraction, which was further purified by FACS sorting.
CD4+ T cell adoptive transfer.
SAMP1/YitFc (30–40 wk old) mice MLN were harvested and rendered into a single cell suspension, followed by positive selection for CD4 using magnetic beads. CD4+ lymphocytes (105/mouse) were injected i.p. or i.v. into 6–8-wk-old SCID mice (The Jackson Laboratory). Neither the selection protocols (positive or negative) nor the route of injection (i.p. or i.v.) altered the severity or time course of disease. Mice were housed in a barrier facility and fed irradiated, standard chow. After 5 wk, the adoptively transferred mice showed ileitis with moderate to severe LP leukocyte infiltration and architectural changes (villous and crypt distortion, goblet cell hyperplasia, and hypertrophy of the muscularis propia).
In vivo homing.
The in vivo migration of lymphocytes was analyzed as described previously (53). In brief, magnetically enriched CD4+/CD62L+ lymphocytes were labeled with 20 µCi chromium 51/ml. A total of 50 x 106 lymphocytes suspended in 0.5 ml were injected i.p. into 8–12-wk-old C57BL/6J (n = 3) or PSGL-1–/– mice (n = 3). Mice were killed after 72 h and the distribution of radioactivity in different organs was measured. Each sample was counted to 3% statistical error. The mean values and their standard error were determined from three animals per strain.
Flow cytometry and cell sorting.
Fluorescently tagged monoclonal antibodies reactive with PSGL-1 (PE-labeled 2PH1; BD Biosciences) or P-sel human Ig-G chimera (2 µg/106 cells; BD Biosciences) or control CD4-human IgG chimera (2 µg/106 cells, a gift from J.B. Lowe, University of Michigan, Ann Arbor, MI) preincubated with allophycocyanin-labeled anti–human IgG Fab fragments (Zenon Human IgG labeling kit; Invitrogen) were subsequently incubated with cells in suspension for 20 min, including FITC-labeled anti-CD4 (GK1.5) for gating of MLN cells or PERCP anti-CD45 (30-F11) and FITC or allophycocyanin–anti-CD31 (390) (BD Biosciences) for gating of intestinal endothelial cells. Cells were fixed with 1% paraformaldehyde and three-to-four color analyses were performed using the FACS Calibur system (Becton Dickinson Immunocytometry Systems). Further analyses were performed using FLOWJo software (Tree Star Inc.). Endothelial cells were sorted using a FACSVantage sorter.
Real-time RT PCR.
RT-PCR primers and Taqman probes were designed using Beacon Designer V3.0 software (Premier Biosoft). The primers were designed to amplify WT PSGL-1 mRNA but not mRNA from PSGL-1–/– mice (truncated protein). Total RNA was isolated using RNAeasy Mini-Kit (QIAGEN) with DNase treatment (Rnase-Free Dnase Kit; QIAGEN). Reverse transcription was performed using 500 ng of RNA with an Omniscript RT Kit (QIAGEN) and oligo-dT primers. The primers were tested for specificity by standard PCR using cDNA made from WT mice and PSGL-1–/– mice. Reaction conditions were optimized and 2 µL of sample cDNA was multiplexed with GAPDH and PSGL-1 primers on an iCycler iQ Real-Time Detection System (QIAGEN). Values were determined using the iCycler iQ Real-Time Detection System Software v3.0a (QIAGEN). The corresponding values were normalized to GAPDH. The PSGL-1 primers used were: forward 5'-CTTCCTTGTGCTGCTGACCAT-3', reverse 5'-TCAGGGTCCTCAAAATCGTCATC-3' and probe 5'-CCAACCACCTGCC TCCGTTCCCG-3'.
Therapeutic interventions.
mAbs (200 µg each) against the following: PNAd (MECA-79, IgM), PSGL-1 (4RA-10, IgG1), MAdCAM-1 (MECA-367 or MECA 89, rat IgG 2a), P-selectin (RB40-34, IgG1), E-selectin (10E9.6, IgG2A), L-selectin (MEL-14, IgG2a), or irrelevant corresponding isotype control mAb were injected i.p. every other day for 3 d, 5 wk after adoptive transfer of SCID mice or at 10 wk of age for SAMP1/YitFc mice. Mice were killed 16–18 h after the last dose. MECA-367, MECA 79, and MECA-89 were obtained from the American Type Culture Collection. 4RA10 was obtained from D. Vestweber (University of Munster, Munster, Germany). Antibodies were produced and purified by HPLC from hybridoma supernatants at the University of Virginia Biomolecular Core Facility.
Generation of bone marrow chimeric mice.
C57BL/6J bone marrow transplant recipients were irradiated twice with 600 rad before and 4–6 h after injection of donor cells. Bone marrow cells were harvested in RPMI 1640 containing 10% fetal calf serum. 7.5 x 106 cells/500 µl were injected into the tail veins of recipient mice. Mice were killed 6–10 wk after transplantation.
Intravital microscopy.
C57BL/6J mice were prepared for intravital microscopy as per previously described methods (54). A 1.5-cm incision was made along the linea alba and the cecum and terminal ileum were exposed. The intestinal serosa was covered with plastic wrap and superfused with bicarbonate-buffered saline containing 100 nM isoproterenol. CFSE-labeled cells (10–20 x 106/200 µl PBS) from PSGL-1–/– mice were injected through a jugular vein catheter and the serosal vessels in which rolling cells could be visualized by stroboscopic epifluorescence microscopy were selected for monitoring. All monitored vessels were 30–80 µm in diameter. Two to three vessels per mouse were selected and rolling cells were counted before and after antibody injection. The number of rolling cells before 4RA10 was normalized to 100% and the percent reduction after 4RA10 treatment was calculated.
Statistics.
Statistical analyses for flow cytometry and inflammatory indices were performed using the two-tailed unpaired Student's t test. Data were expressed as mean and standard error of the mean. Statistical significance was set at P < 0.05.
Online supplemental material.
Supplemental Videos 1 and 2 depict the interactions between CFSE-labeled PSGL-1–/– cells and intestinal endothelium before and after injection of mAb 4RA10; they are available at http://www.jem.org/cgi/content/full/jem.20052530/DC1.
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Acknowledgments |
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ARE breeding pairs. This work was supported by grants from the National Institutes of Health (nos. DK-57880 and DK-067254) and by a Harold Amos grant from the Robert Wood Johnson Foundation.
The authors have no conflicting financial interests.
Submitted: 21 December 2005
Accepted: 22 February 2006
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References |
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