Introduction

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The ability of lymphocytes to arrest under conditions of vascular flow enables their movement into both normal lymphoid tissues and sites of inflammation. Lymphocyte recruitment in the vasculature is regulated by the differential expression and activation of homing receptors (selectins and integrins) on lymphocytes that interact with counter-receptors of the Ig superfamily on high endothelial venules (HEVs) (Bargatze and Butcher, 1993; Bargatze et al., 1995). This interaction mediates a multistep process, involving rolling and tethering of leukocytes to endothelial ligands, rapid activation of integrins by locally released chemokines, stable adhesion of activated integrins to endothelial ligands, and transendothelial migration through the vessel wall (Bargatze et al., 1995; Warnock et al., 2000). Although all integrins expressed on leukocytes can mediate firm adhesion, ₄₇ and ₄₁ are members of a small subset of integrins that can also mediate rolling (Berlin et al., 1995).

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1), expressed on HEVs of Peyer's patch and other gut-associated lymphoid tissues (GALTs), is the principal ligand for ₄₇, an integrin highly expressed on gut-homing memory lymphocytes (Berlin et al., 1993; Shyjan et al., 1996; Briskin et al., 1997). ₄₇ binds to MAdCAM-1 with higher affinity than to VCAM-1 or the CS-1 subdomain of human fibronectin. Although both ₄₇ and ₄₁ can bind VCAM-1 and CS-1, ₄₁ does not bind MAdCAM-1 (Berlin et al., 1993). Both L-selectin and ₄₇ mediate the initial attachment and rolling of lymphocytes by interacting with MAdCAM-1, whereas ₄₇ also mediates the firm adhesion of lymphocytes via this ligand (Rott et al., 1996).

Lymphocyte trafficking in the GALT not only enables normal immune responses (Butcher and Picker, 1996) but also contributes to unwanted inflammation (Podolsky and Fiocchi, 2000). Gut inflammation can induce dramatic changes in the extent and selectivity of lymphocyte recruitment to the gut wall (Briskin et al., 1997; Picarella et al., 1997). For example, the expression of MAdCAM-1 can be up-regulated by as much as 5-fold on blood vessels at sites of intestinal inflammation (Briskin et al., 1997), and proinflammatory cytokines facilitate the recruitment of lymphocytes and other leukocytes to sites of active inflammation (Podolsky and Fiocchi, 2000). The tissue-specific distribution of MAdCAM-1 and selective interaction with gut-homing memory lymphocytes expressing ₄₇ suggest a contributing role of this ligand-receptor pair to inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

Blockade of ₄₇ and MAdCAM-1 with antibodies defines their role in models of inflammatory bowel disease. Monoclonal antibodies (mAbs) directed against ₄ or ₄₇ block lymphocyte homing to intestinal sites in naïve mice (Hamann et al., 1994), and mAbs against ₇ or MAdCAM-1 reduce inflammation in mouse models of colitis (Picarella et al., 1997; Kato et al., 2000). In the cotton-top tamarin, which spontaneously develops colitis, an anti-₄₇ mAb effectively resolved the established colitis (Hesterberg et al., 1996). Perhaps the strongest argument for the importance of ₄ integrins in mediating inflammation of the gut is derived from recent clinical trials with Antegren (anti-₄; Elan/Biogen, Cambridge, MA). In a blinded placebo-controlled phase II trial of 248 patients with moderate-to-severe Crohn's disease, Antegren administered at a single dose of 3 mpk i.v. resulted in a 46% remission rate after 6 weeks, versus 27% remission with placebo (Ghosh et al., 2001).

We have identified a dual ₄₇/₄₁ antagonist by evaluating the ability of the compound to block the binding of human or murine ₄₇-expressing cells to soluble human or murine MAdCAM-Ig. Small molecule antagonists of ₄₇ that block the static adhesion of human ₄₇-expressing cells to the CS-1 subdomain of human fibronectin, human VCAM-Ig, human MAdCAM-Ig, or murine MAdCAM-Ig have been described previously (Shroff et al., 1996, 1998; Carson et al., 1997; Harriman et al., 1999; Martin et al., 1999). The ability of ₄₇ antagonists to block the binding of murine ₄₇-expressing cells to soluble murine MAdCAM-Ig under static conditions has also been reported (Martin et al., 1999), but the ability of compounds to block ligand binding under in vitro or in vivo shear flow conditions has not been examined. We used in vitro shear flow assays to quantitate the adhesion of both human and murine ₄₇-expressing cells to human and murine MAdCAM-Ig, and we characterized the ability of compounds to inhibit adhesion to HEVs in an in vivo model.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Antibodies and Cell Lines

The following purified monoclonal antibodies were obtained from BD PharMingen (San Diego, CA): 4B4 (mouse anti-human ₁), FIB27 (rat anti-mouse ₇ that cross-reacts with human ₇), DATK32 (rat anti-mouse ₄₇), Ha2/5 (hamster anti-rat ₁ that cross-reacts with murine ₁; Mendrick and Kelly, 1993), MEL-14 (rat anti-mouse L-selectin), and isotype controls (hamster IgM, rat IgG2b, and rat IgG2a). HP2/1 (mouse anti-human ₄) was obtained from Beckman Coulter, Inc. (Fullerton, CA). PS/2 (rat anti-mouse ₄) (Miyake et al., 1991) and a matched isotype control (rat anti-human Ras Ab) were supplied by LigoCyte (Bozeman, MT). The following cell lines were used: RPMI-8866 cells (human B cell line) obtained from J. Wilkins (University of Manitoba, Winnipeg, MB, Canada), TK-1 cells (murine T cell line) obtained from I. Weissman (Stanford University, Stanford, CA) (Holzmann and Weissman, 1989), and Jurkat (human T cell line) and RBL-2H3 cells (rat mucosal-type mast cell line) from American Type Culture Collection (Manassas, VA).

Expression and Purification of Cellular Adhesion Molecule-Immunoglobulin Fusion Proteins

Human MAdCAM-Ig. Domains 1 and 2 of human MAdCAM-1 (GenBank no. U43628) were amplified by PCR using human small intestinal cDNA (Invitrogen, Carlsbad, CA) as a template and the following primer sequences: 5'-PCR primer, 5'-ATTAGGAATTCGCCACCATGGATTTCGGACTGGCCCTCCTGCTGG-3'; and 3'-PCR primer, 5'-AATTGGGATCCACTTACCTGTGGAGGTCGGGCTGTGCAGGACG- GGGATG-3'. PCR was performed in the presence of 10% DMSO with KlenTaq (CLONTECH, Palo Alto, CA) in a thermocycler (MJ Research, Waltham, MA) by using 40 cycles with the following parameters: 45 s at 94°C, 45 s at 60°C, and 90 s at 72°C. The resulting PCR product of 660 bp was digested with EcoRI and BamHI and ligated into a pIg (R & D Systems, Minneapolis, MN) expression vector. The pIg vector contains the genomic fragment that encodes the hinge region, CH2 and CH3 of human IgG1 (GenBank no. Z17370), and the fragment encoding human MAdCAM-1 (hMAdCAM-1) was ligated proximal to the IgG1 region. The sequence of the resulting hMAdCAM-1 fragment fused to human IgG1 was verified using Sequenase (U.S. Biochemical Corp., Cleveland, OH). The fragment encoding the entire MAdCAM-Ig fusion was subsequently excised from the pIg vector with EcoRI and NotI and ligated to pcDNA3.1/neo (Invitrogen). The resulting vector, pcDNA3.1/neo-MAdCAM-Ig, was transfected into CHOKI cells (CCL61; American Type Culture Collection) by electroporation and grown under selection with 0.7 mg/ml G418 (Invitrogen). Culture supernatants from single cell clones were assayed by Ig enzyme-linked immunosorbent assay, and a high expressing clone (1 µg/ml) was adapted to CHO-SFM II serum-free media (Invitrogen) for large-scale expression. hMAdCAM-Ig was purified from crude culture supernatants by affinity chromatography on protein A/G Sepharose and dialyzed into 50 mM sodium phosphate buffer, pH 7.6.

Murine MAdCAM-Ig. The entire extracellular domain of murine MAdCAM-1 (domain 1, domain 2, mucin, and domain 3) (GenBank no. L21203) was amplified by PCR using murine small intestinal cDNA (Invitrogen) as a template and the following primer sequences: 5'-PCR primer, 5'-CCGAGATATCGCCACCATGGAATCCATCCTGGCCCTCCTG-3'; and 3'-PCR primer, 5'-CCTTGGATCCACTTACCTGTGGTGGAGGAGGAATTCGGGGTCA-3'. PCR was performed in the presence of 10% DMSO with KlenTaq (CLONTECH) in a thermocycler (MJ Research) by using 30 cycles with the following parameters: 1 min at 94°C, 1 min at 60°C, and 2 min at 72°C. The resulting PCR product of 1095 bp was digested with EcoRV and BamHI and ligated into a pIg (R & D Systems) expression vector. After verification of the sequence, the fragment encoding the entire MAdCAM-Ig fusion was excised from the pIg vector with EcoRI and NotI and ligated into pCMV-I/IRES/GFP puro vector (Cell & Molecular Technologies, Inc., Phillipsburg, NJ). The resulting vector carrying the gene for GFP was used to transfect CHOKI cells that were grown in the presence of 4 µg/ml puromycin. Transfected cells were sorted by flow cytometry on the basis of their high GFP expression, and the GFP expression was followed during subsequent culture, before the isolation of single cell clones. Culture supernatants from single cell clones were assayed by Ig enzyme-linked immunosorbent assay and immunoblot using anti-murine MAdCAM-1 mAb (MECA-367; BD PharMingen). A high expressing clone (1 µg/ml) was adapted to CHO-SFM II serum-free media (Invitrogen) for large-scale expression, and mMAdCAM-Ig was purified as described for hMAdCAM-Ig.

Human VCAM-Ig. Domains 1 and 2 of human VCAM-1 (GenBank no. M30257) were amplified by PCR using the human VCAM-1 cDNA (R & D Systems) as a template and the following primer sequences: 3'-PCR primer, 5'-AATTATAATTTGATCAACTTACCTGTCAATTCTTTTACAGCCTGCC-3'; and 5'-PCR primer, 5'-ATAGGAATTCCAGCTGCCACCATGCCTGGGAAGATGGTCG-3'. PCR was performed for 30 cycles using the following parameters: 1 min at 94°C, 2 min at 55°C, and 2 min at 72°C using a DNA thermal cycler (model 480; PerkinElmer Instruments, Norwalk, CT). The resulting PCR product of 650 bp was digested with EcoRI and BclI and ligated into a pIg (R & D Systems) expression vector. After verification of the sequence, the fragment encoding the entire VCAM-Ig fusion was excised from the pIg vector with EcoRI and NotI and ligated to pCI-neo (Promega, Madison, WI). The resulting vector, pCI-neo/VCAM-Ig, was transfected into CHOKI cells by calcium phosphate DNA precipitation and grown under selection with 0.4 mg/ml G418. Culture supernatants from single cell clones were assayed for the ability to support Jurkat cell adhesion, and a high expressing clone (1 µg/ml) was adapted to CHO-SFM II serum-free media. VCAM-Ig was purified as described for hMAdCAM-Ig.

Ligand Binding Assays for ₄₇ and ₄₁

A ligand binding assay for ₄₇ was performed by incubating RPMI-8866 cells (7.5 × 10⁵ cells/well) or TK-1 cells (1 × 10⁶ cells/well) with <200 pM iodinated human or murine MAdCAM-Ig in a 96-well filter binding format. A ligand binding assay for ₄₁ has been described previously (Hagmann et al., 2001) and was performed by incubating Jurkat cells (5 × 10⁵ cells/well) or RBL-2H3 cells (4 × 10⁴ cells/well) with <100 pM iodinated VCAM-Ig in a 96-well filter binding format. Purified VCAM-Ig and MAdCAM-Ig were labeled with ¹²⁵I using Bolton Hunter reagent and purified using high-performance liquid chromatography gel filtration chromatography. Specific radioactivities were in excess of 1100 Ci/mmol. Compounds were evaluated by incubating radioligand compound (prepared in DMSO; <1% DMSO final concentration), cells, and binding buffer (25 mM HEPES, 150 mM NaCl, 3 mM KCl, 2 mM glucose, and 0.1% bovine serum albumin, with 1 mM MnCl₂, pH 7.4) at 25°C for 30 min (₄₁ assays) or 45 min (₄₇ assays) in a 96-well multiscreen MHVBN filtration plate (Millipore, Bedford, MA). After filtration and a single wash with binding buffer, the filtration plates were dried and transferred to adaptor plates (Packard BioScience, Meriden, CT). After adding 100 µl of Microscint-20 (Packard Bioscience) to each well, the plates were sealed, placed on a shaker for 1 min, and counted on a Packard BioScience TopCount. Wells containing cells + radioligand + 1 µM compound or DMSO alone served as controls to calculate 100 and 0% inhibition, respectively.

Quantitative FACS Analysis

A total of 10⁶ RPMI-8866 or Jurkat cells were incubated for 30 min on ice in FACS buffer (phosphate-buffered saline with Ca²⁺/Mg²⁺, 5% fetal bovine serum, 100 µg/ml goat IgG, and 0.05% sodium azide) containing saturating levels of the following polyethylene-conjugated antibodies: FIB504 rat anti-mouse ₇ (2.4 µg/ml; cross-reacts with human ₇), MAR4 mouse anti-human ₁ (80 µg/ml), 9F10 mouse anti-human ₄ (10 µg/ml), and mIgG1 isotype and rIgG2a isotype controls. Similarly, a total of 10⁶ TK-1 or murine mesenteric lymph node (MLN) lymphocytes were incubated for 30 min on ice in FACS buffer with Fc block (10 µg/ml; BD PharMingen) containing DATK32-PE rat anti-mouse ₄₇ (10 µg/ml) or rIgG2a-PE isotype control antibodies. All polyethylene-conjugated antibodies were obtained from BD PharMingen. Cells were washed in FACS buffer and resuspended in FACS buffer containing 1 µg/ml propidium iodide. Cells were analyzed by FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ). Standardized quantum R-PE microbeads (Flow Cytometry Standards Corp., Fishers, IN) were analyzed by flow cytometry and used to create a calibration curve that relates mean fluorescence intensities to molecules of equivalent soluble fluorescence for use in calculating receptor density values.

In Vitro Shear Flow Adhesion Assay

An in vitro shear flow system adapted from published methods (Berlin et al., 1993) was used to evaluate the ability of compounds to block the binding of human RPMI-8866 cells or murine MLN lymphocytes (2 × 10⁶ cells/ml) isolated from BALB/c mice to capillary tubes coated with murine or human MAdCAM-Ig, respectively. Cells at a density of 6 × 10⁶ cells/ml were preincubated with test compounds (0.06-6 µM; final DMSO <1%), neutralizing mAb, or isotype control mAb at 0.6 µM in buffer (HBSS without Ca²⁺/Mg²⁺, 20 mM HEPES, 2 mM Mn²⁺, and 2% human serum, pH 7.0) for 10 min at 37°C. A 500-µl cell suspension was then injected into a closed loop flow system that contained 2.5 ml of assay buffer (HBSS with Ca²⁺/Mg²⁺, 20 mM HEPES, and 2% human serum, pH 7.0). Murine assays included preincubation of cells with anti-L-selectin at 0.6 µM to block L-selectin-dependent rolling due to interactions with the mucin domain of mMAdCAM-Ig. A silicone tubing loop and roller pump were used to circulate cells through a MAdCAM-Ig-coated capillary tube mounted on an inverted microscope stage. MAdCAM-Ig was titrated (5, 10, and 50 µg/ml), and 50 µg/ml was selected for use because it supported 50 to 150 interacting cells/field after 15 min of continuous shear flow. The shear rate was 2 dynes/cm² for the first 5 min and 1.2 dynes/cm² for the last 10 min, simulating physiological shear flow, where noninteracting lymphocytes have a midline velocity of 4000 µm/s in murine HEVs (Bargatze et al., 1995). Cells were monitored for 15 min by videomicroscopy, and the number of adherent cells was determined at 1-min intervals by analysis of individual frames using either a customized Proteo-Flow computer analysis package (LigoCyte Pharmaceuticals) or manual counting directly from the monitor screen. Control adhesion for individual experiments was based on the isotype or 1% DMSO control treatment. Data are represented as area under the curve (AUC) from time 0 to 15 min. Data were normalized by calculating percentage of control adhesion within each experiment, with two to six tests evaluated per treatment.

Interaction of Lymphocytes with High Endothelial Venules in Murine Peyer's Patches

Compounds were evaluated for their ability to block the interactions of lymphocytes with high endothelial venules in murine Peyer's patches using an intravital microscopy system adapted from published methods (Bargatze et al., 1995). In brief, murine MLN lymphocytes were isolated from normal donor BALB/c mice and fluorescently labeled with rhodamine (MitoTracker Orange CM-H₂-TMRos) or fluorescein (carboxy-4',5'-dimethylfluorescein diacetate) purchased from Molecular Probes (Eugene, OR). After labeling, cells were incubated in EDTA buffer (HBSS without Ca²⁺/Mg²⁺, 10 mM HEPES, and 2 mM EDTA) for 10 min at 25°C, washed in buffer (HBSS without Ca²⁺/Mg²⁺ and 10 mM HEPES), resuspended in activating buffer (HBSS without Ca²⁺/Mg²⁺, 10 mM HEPES, and 2 mM Ca²⁺/Mn²⁺), and treated with anti-L-selectin Ab at 200 µg/ml for 5 min for rhodamine-labeled cells to maintain both a saturating in vitro and in vivo concentration of mAb and at 50 µg/ml for 5 min before i.v. injection for fluorescein-labeled cells to provide a saturating in vitro concentration. Syngeneic recipient BALB/c mice were anesthetized and prepared for intravital microscopy by abdominal incision to exteriorize the small intestines, and a selected Peyer's patch was gel mounted and positioned for epifluorescence videomicroscopy. The protocol used to evaluate the effect of mAb or compounds on the binding of murine lymphocytes to Peyer's patch HEVs by intravital microscopy is described in Scheme 1.

View larger version (11K): [in this window] [in a new window]   Scheme 1.   The protocol used to evaluate the effect of mAb or compounds on the binding of murine lymphocytes to Peyer's patch HEVs by intravital microscopy.

At time 10 min, rhodamine-labeled cells were injected by i.v. bolus into the tail vein and monitored for 10 min to document control adhesion events in each animal. The same high endothelial field was monitored during the entire time course. Compounds were prepared in HBSS containing 1% DMSO and 2% polyethylene glycol-400. Compounds (0.01-10 mpk) or mAbs (10 mpk) were then dosed by i.v. injection 5 min before the i.v. injection of 1.5 × 10⁷ fluorescein-labeled MLN lymphocytes. In some experiments, fluorescein-labeled cells were also incubated with compounds (100 µM) for 10 min before injection. After monitoring the adhesion of fluorescein-labeled cells for 10 min, anti-₄ (PS/2; 10 mpk) was injected i.v., and cell adhesion was monitored for another 10 min to quantitate ₄-independent baseline adhesion as a negative control. A single HEV field containing multiple venules (3-5) was evaluated for each animal. Interacting cells were determined at 1-min intervals by analysis of individual frames using either a customized computer analysis package (LigoCyte Pharmaceuticals) or manual counting directly from the monitor screen to obtain statistically significant values when evaluating four to six mice per treatment group.

The ability of mAbs or compounds to reverse established lymphocyte interactions with murine Peyer's patch HEVs was evaluated as described in Scheme 2. For experiments involving only fluorescein-labeled cells, these cells were preincubated with anti-L-selectin mAb at 200 µg/ml for 5 min before i.v. injection to maintain both a saturating in vitro and in vivo concentration of mAb. At time 14 to 4 min, 1.5 × 10⁷ fluorescein-labeled MLN lymphocytes were injected by i.v. bolus into the tail vein and monitored for 10 min to document control adhesion events in each animal. Compounds (10 mpk) or mAbs (10 mpk) were then dosed by i.v. injection, and adhesion events were monitored for another 10 min. For compound-treated animals, anti-₄ (PS/2; 10 mpk) was dosed by i.v. injection 10 min after compound treatment, and cell interactions were monitored for another 10 min. Interacting cells were quantitated as described above.

View larger version (9K): [in this window] [in a new window]   Scheme 2.   The protocol used to evaluate the ability of mAbs or compounds to reverse established lymphocyte interactions with murine Peyer's patch HEVs.

Synthesis of Compounds

Compound 1, N-{N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl}-(D)-phenylalanine, and compound 2, N-{N-[(3-chlorobenzene)sulfonyl] azetidine-2-(S)-carboxyl}-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine, were synthesized as described previously (Durette et al., 1998a,b; Hagmann et al., 2001; Kopka et al., 2002). TR14035, N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine, was synthesized as described in Sircar et al. (1999a). The structures of the compounds are shown in Fig. 1.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Structure of antagonists of 47/41 integrins. The structures of TR14035 (Sircar et al., 1999a), compound 1 (Durette et al., 1998a,b; Hagmann et al., 2001), and compound 2 (Kopka et al., 2002) are shown.

Pharmacokinetic Analysis of Compounds

Female BALB/c mice (20-23 g; n = 4-6/group) were dosed intravenously with compounds prepared in HBSS containing 1% DMSO and 2% polyethylene glycol-400. At the end of each experiment, terminal blood samples were taken for the determination of compound concentrations in plasma. Blood was collected into EDTA after euthanasia at either 15 or 20 min postinjection, and plasma was prepared. Plasma samples were immediately acidified by combining 200 µl of plasma with 50 µl of 0.5 M formate buffer (21.5 ml of 88% formic acid/liter of distilled H₂O; pH 3.0 adjusted with NaOH), snap frozen on dry ice, and stored at 20°C. Compounds were purified from plasma samples by solid phase extraction (OASIS cartridge; Waters, Milford, MA), and concentrations of compounds were determined by liquid chromatography-tandem mass spectrometry (API 3000; PerkinElmer Sciex, Foster City, CA). Plasma values for compounds are represented as mean ± S.E.M. in nanomolar concentration.

To determine the disappearance of compound in the blood of rodents, male Sprague-Dawley rats (150-200 g; n = 4-6/group) were dosed intravenously with compounds at 1 mpk as described above, and the plasma concentration of compound was calculated at various time points to determine the time when 50% of the compound had disappeared from the blood (t_1/2 in min).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Antagonists Block Both ₄₁ and ₄₇ Integrins on Human and Rodent Cells. To determine whether a potent antagonist of ₄₁ (Fig. 1; compound 1) (Hagmann et al., 2001) was also capable of blocking ₄₇, an assay was developed to measure the binding of MAdCAM-1 to ₄₇. RPMI-8866, a human B cell line, was demonstrated by flow cytometry to express high levels of ₄₇ (60,000 ₄₇ receptors/cell) but low levels of ₄₁ (4,000 ₄₁ receptors/cell), and was therefore chosen to evaluate the binding of ₄₇ to soluble ¹²⁵I-hMAdCAM-Ig in the presence of the activating divalent cation Mn²⁺. The concentration of ¹²⁵I-hMAdCAM-Ig used for the binding assay was maintained at <200 pM, based on an IC₅₀ of 1200 pM for competition by unlabeled MAdCAM-Ig. Anti-₄ and anti-₇ mAbs blocked ¹²⁵I-MAdCAM-Ig binding with IC₅₀ values of 110 and 40 ng/ml, respectively, whereas anti-₁ did not block at concentrations up to 3.3 µg/ml, demonstrating the specificity of the interaction for ₄₇ (data not shown).

View this table: [in this window] [in a new window]   TABLE 1 Activity of antagonists of 47/41 integrins in human and rodent in vitro ligand binding assays Compounds were tested using a 10-point titration in human and rodent 47 and 41 ligand binding assays to determine compound potency and specificity. Assays for 47 measured the binding of the human RPMI-8866 B cell line to 125I-human MAdCAM-Ig and the binding of the murine TK-1 T cell line to 125I-murine MAdCAM-Ig in suspension in the presence of Mn2+. Assays for 41 measured the binding of the human Jurkat T cell line and the rat RBL-2H3 cell line to 125I-human VCAM-Ig in suspension in the presence of Mn2+. IC50 (nM) values represent the average of two to four independent experiments, with the exception of the data generated using RBL-2H3 cells, which were directly compared to those for Jurkat cells in the same single experiment. The ranges of IC50 values are shown in parentheses, and all values were within 95% confidence limits.

TR14035 Is More Potent than compound 1 in Its Ability to Block ₄₇ Adhesion to MAdCAM-Ig under in Vitro Shear Flow Conditions. Because ₄ integrins contribute to lymphocyte attachment and rolling under physiological flow, compounds that blocked binding of soluble ligands were further tested for their ability to block the binding of human ₄₇-expressing cells to hMAdCAM-Ig-coated capillary tubes under in vitro shear flow (Fig. 2, A and B). The assay was modified from systems described previously for ₄₇-dependent adhesion under flow (Berlin et al., 1993) by using RPMI-8866 cells and the MAdCAM (domain 1 and domain 2)-Ig fusion protein. Cells were preincubated with neutralizing mAb or compounds in the presence of 2 mM Mn²⁺ for 10 min at 37°C before injection into the capillary tube adhesion chamber, and the final concentration of Mn²⁺ in the flow chamber was 0.33 mM. Anti-₇ mAb completely blocked adhesion at concentrations between 1 nM (data not shown) and 0.1 µM (20 µg/ml, 97% inhibition) (Fig. 2B). Anti-₄ mAb was similarly potent, inhibiting adhesion by 99% at 0.1 µM (Fig. 2, A and B). Anti-₁ mAb did not significantly block adhesion (33% inhibition) at concentrations up to 1 nM (200 ng/ml) (data not shown), but did inhibit by 86% at 0.1 µM (20 µg/ml) (Fig. 2B). Although there is no apparent explanation for the blockade of adhesion at the higher concentration of anti-₁, the much higher potency of anti-₇ indicates that human RPMI-8866 cells bind human MAdCAM-Ig-coated capillary tubes in an ₄₇-dominant manner under shear flow. TR14035 at 1 µM blocked adhesion of RPMI-8866 cells to MAdCAM-Ig by 100% (Fig. 2, A and B), with an approximate IC₅₀ of 0.01 µM (Fig. 2B). In contrast, compound 1 at 1 µM did not significantly block adhesion (Fig. 2, A and B), indicating that TR14035 was >100-fold more potent than compound 1 in conditions of shear flow.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Inhibition of human 47 adhesion to human MAdCAM-Ig under physiological shear flow. Compounds were evaluated for their ability to block the binding of human 47-expressing cells to hMAdCAM-Ig-coated capillary tubes under in vitro shear flow, as described under Materials and Methods. Compounds or mAbs (anti-4, HP2/1; anti-7, FIB27; anti-1, 4B4; or isotype antibody) were preincubated with RPMI-8866 cells for 10 min in the presence of Mn2+ before injection into the shear flow chamber. A, data from a representative experiment are plotted as time in minutes versus total number of adhesion events per field. B, dose response of an 47/41 antagonist. Data are represented as percentage of control adhesion area under the curve ± standard error of the mean (AUC ± S.E.M.) from time 0 to 15 min. Statistics were generated with PRISM, where P < 0.001 () and P < 0.01 (), compared with the vehicle control. Values represent the average of three to six independent experiments per treatment group.

View larger version (49K): [in this window] [in a new window]   Fig. 3.   Inhibition of murine 47 adhesion to murine MAdCAM-Ig under physiological shear flow. Compounds were evaluated for their ability to block the binding of murine 47-expressing cells to mMAdCAM-Ig-coated capillary tubes under in vitro shear flow, as described under Materials and Methods. Compounds or mAbs (anti-47, DATK32; anti-4, PS/2; anti-1, Ha2/5; or isotype Ab at 0.1 µM) were preincubated with murine MLN lymphocytes for 10 min in the presence of Mn2+ before injection into the shear flow chamber. A, data from a representative experiment are plotted as time in minutes versus total number of adhesion events per field. Two untreated controls (1% DMSO) are shown that represent the variation observed in this model. B, dose response of 47/41 antagonists. Data are represented as percentage of control adhesion area under the curve ± standard error of the mean (AUC ± S.E.M.) from time 0 to 15 min. Statistics were generated with PRISM, where P < 0.001 () and P < 0.05 (). Values represent the average of three to six independent experiments per treatment group.

Antagonists of ₄₁/₄₇ Block ₄₇-Dependent Lymphocyte Interactions with Murine Peyer's Patch High Endothelial Venules. To quantitate ₄₇ adhesion under shear flow conditions in vivo, we used in situ videomicroscopic analysis to measure the adhesion of fluorescently labeled murine MLN lymphocytes to Peyer's patch HEVs expressing murine MAdCAM-1. It has been previously reported that anti-₄, anti-₇, and anti-MAdCAM-1 mAbs block adhesion in this model when administered up to 10 min after the injection of the labeled cells (Bargatze et al., 1995). Because other molecular mechanisms than the ₄₇-MAdCAM interaction may contribute to adhesion events at longer time intervals, the evaluation of adhesion was limited to a 10-min interval after injecting rhodamine- or fluorescein-labeled cells. As described under Materials and Methods, results for each mouse (Table 2) were based on a positive control segment (binding of rhodamine-labeled cells that were pretreated with Mn²⁺ and anti-L-selectin before i.v. injection), an experimental segment (binding of fluorescein-labeled cells that were pretreated with Mn²⁺ and anti-L-selectin before i.v. injection into mice that had been predosed with mAb or compound 5 min before cell injection), and a negative control segment (binding of fluorescein-labeled cells after the i.v. injection of anti-₄ mAb to quantitate ₄-independent baseline adhesion). We confirmed that the interaction between MLN lymphocytes and Peyer's patch HEVs was dependent on ₄₇, because anti-₄ mAb at 10 mpk blocked adhesion by 95%, and anti-₁ mAb at 10 mpk failed to block adhesion (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Antagonists of 47/41 block 47-dependent lymphocyte interactions with murine Peyer's patch high endothelial venules In dosing regimen A, mice received an i.v. injection of compound or mAb 5 min before the injection of fluorescein-labeled cells, which were preincubated with 100 µM compound or mAb for 10 min. In dosing regimen B, the in vitro preincubation step was eliminated, and mice received only an i.v. injection of compound or mAb. Percentage of control represents the experimental results (fluorescein lymphocytes) divided by the positive control for each mouse (rhodamine lymphocytes). Statistics were evaluated with PRISM. Values represent the average of four to six mice per treatment group.

Anti-₄ mAb and TR14035 Can Reverse Established ₄₇-Dependent Lymphocyte Interactions with Murine Peyer's Patch High Endothelial Venules. To determine whether established adhesion events can be reversed in vivo, we administered anti-₄ mAb or TR14035 between 10 and 14 min after the injection of fluorescein-labeled cells and quantitated adhesion for an additional 10 min (Fig. 4, A and B). By 5 to 10 min after the cells were injected, adhesion reached a plateau. New adhesive interactions presumably did not occur after this time, because Mg²⁺ in plasma gradually exchanged with Mn²⁺ on ₄₇. Cation exchange inactivates ₄₇ and leads to a mixed population of activated (high-affinity) and unactivated (low-affinity) forms of the receptor (Bargatze et al., 1995). Within 5 min after anti-₄ mAb was injected, the number of adhesion events declined to 46% of control values, and further declined to 19% of control values by 10 min (Fig. 4A; Table 3) Within 5 min after administration of TR14035 (10 mpk i.v.), the number of adhesion events declined to 48%, but did not decline further (Fig. 4B; Table 3). In addition, ₄-dependent rolling was completely blocked after administering anti-₄ mAb, but not after administering TR14035 (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Anti-4 mAb and TR14035 can reverse established 47-dependent lymphocyte interactions with murine Peyer's patch high endothelial venules. Fluorescein-labeled lymphocytes were preincubated with anti-L-selectin mAb at 200 µg/ml for 5 min, injected i.v. at t = 0, and allowed to adhere for 10 to 14 min (A and B) or 4 min (C and D). Anti-4 mAb (A and C; 10 mpk i.v.) or TR14035 (B and D; 10 mpk i.v.) was administered as indicated by the arrows. Adhesion of fluorescein-labeled cells (), adhesion after injection of anti-4 mAb (), and adhesion after injection of TR14035 (). Data in each panel show results from a single representative experiment of two to four independently repeated experiments. A full statistical analysis of all the data obtained in these studies is described in Table 3.

View this table: [in this window] [in a new window]   TABLE 3 Activity of anti-4 and TR14035 in reversing established 47-dependent lymphocyte interactions with murine Peyer's patch high endothelial venules In dosing regimen A, mice received an i.v. injection of anti-4 mAb or TR14035 between 10 and 14 min after the injection of fluorescein-labeled cells. In dosing regimen B, mice received an i.v. injection of anti-4 mAb or TR14035 4 min after the injection of fluorescein-labeled cells. In dosing regimen C, mice received an i.v. injection of TR14035 4 min after the injection of fluorescein-labeled cells, and then mice received an i.v. injection of anti-4 mAb 10 min after the injection of TR14035. Percentage of control at 5 or 10 min represents the experimental results (adhesion events after i.v. injection with mAb or compound) divided by the positive control for each mouse (adhesion events for fluorescein lymphocytes were quantitated 10 min after i.v. injection). Statistics were evaluated with PRISM for treatment groups that included three to four independent experiments, and a range of values is shown in parentheses for the treatment group that included less than three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our studies confirm previous reports that TR14035 is a potent antagonist of both ₄₁ and ₄₇ (IC₅₀ values ranging from 2 to 46 nM) (Martin et al., 1999; Sircar et al., 1999a,b), and further demonstrate that compound 1 is a potent antagonist of both integrins. A study comparing the ability of TR14035 to block the adhesion of ₄₇ or ₄₁ to CS-1 immobilized on a plate concluded that the compound was 9 times more potent against ₄₇ than ₄₁ (IC₅₀ of 5 versus 46 nM for ₄₁) (Martin et al., 1999). In contrast, we found that TR14035 was 7 times more potent in blocking ₄₁ binding to soluble VCAM-1 (IC₅₀ of 0.11 nM) than in blocking ₄₇ binding to soluble MAdCAM-1 (IC₅₀ of 0.75 nM). Differences in the ligands used to evaluate the potency of TR14035 against ₄₁ and ₄₇ may contribute to the discrepancies in the IC₅₀ values. To compare the activity of TR14035 against ₄₁ and ₄₇ binding to the same ligand, we tested the ability of the compound to block the adhesion of ₄₇- or ₄₁-expressing cells to CS-1 immobilized on a plate and found similar potencies against both ₄₇ (IC₅₀ of 16 nM) and ₄₁ (IC₅₀ of 12 nM) (L. A. Egger and U. Kidambi, unpublished observations). Taken together, the results clearly indicate that TR14035 is active against both ₄₁ and ₄₇, with potencies for each dependent on the ligand used and the format of the assay.

The ability of the compounds to inhibit binding of human and rodent ₄₇ and ₄₁ to soluble ligands in suspension was measured to test for possible species-dependent shifts in potency. Species differences in ligand specificity have been reported for other integrins. For example, murine lymphocyte function antigen-1 (LFA-1; _L2) binds human ICAM-2, but not ICAM-1, whereas human LFA-1 binds not only human ICAM-1 and ICAM-2 but also murine ICAM-1 (Johnston et al., 1990). Species specificity for the binding of LFA-1 to ICAM has been mapped to the _L subunit of LFA-1 (Johnston et al., 1990), suggesting that structural differences exist within the ligand binding sites of the same integrins from different species. Previous studies have shown that ₄₁ and ₄₇ can bind ligands across species (Hession et al., 1992; Shyjan et al., 1996), consistent with the primary structural homology for human and rodent ₄, ₁, and ₇ (76, 92, and 87%, respectively) (Takada et al., 1989; Jiang et al., 1992; Cervella et al., 1993). A high degree of structural homology also extends to the ligands for ₄ integrins: human and rodent VCAM-1 have 85% sequence identity, and the ₄ integrin binding motif IDS is completely conserved (Briskin et al., 1996). Although the overall homology between full-length human and murine MAdCAM-1 is only 39%, the first two N-terminal Ig-like domains are 57% identical, and the ₄ integrin binding motif LDT in domain 1 of MAdCAM-1 is completely conserved (Shyjan et al., 1996). In this report, compounds blocked the binding of human or rodent ₄₇ and ₄₁ to soluble ligands in suspension with similar potencies, suggesting that the affinity of the receptors for compounds was similar for human or rodent ₄.

Differences in the activities of TR14035 and compound 1 against ₄₇ on human and rodent cells emerged in in vitro shear flow assays. TR14035 was approximately 10-fold more potent in the assay with human cells than with mouse cells, whereas the reverse was true for compound 1. Variations in receptor density on human and mouse cells may contribute to the species differences in the potency of the compounds. As measured by quantitative FACS analysis, the receptor density of ₄₇ on murine lymphocytes was 1000 receptors/cell, 60-fold lower than the receptor density of ₄₇ on RPMI-8866 cells (60,000 receptors/cell). Despite the lower density of ₄₇ on murine lymphocytes, a robust interaction (90-200 inter