Abstract
The α4 integrin, α4β7, plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of α4β1 and α4β7,N-(2,6-dichlorobenzoyl)-(l)-4-(2′,6′-bis-methoxyphenyl)phenylalanine (TR14035) andN-{N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl}-(d)-phenylalanine (compound 1), were tested for their ability to block the binding of α4β7-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human α4β7to an 125I-MAdCAM-Ig fusion protein with IC50values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to α4β1 or α4β7 on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human α4β7-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC50 values of 0.1 and 1 μM, respectively. Intravital microscopy was used to quantitate α4-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn2+ to activate α4β7 binding to ligand, anti-α4 monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-β1 mAb did not block adhesion, demonstrating that this interaction was dependent on α4β7. TR14035 blocked adhesion to HEVs [ED50 of 0.01–0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, α4β7/α4β1antagonists blocked α4β7-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.
Footnotes
- Abbreviations:
- HEV
- high endothelial venule
- MAdCAM-1
- mucosal addressin cell adhesion molecule-1
- GALT
- gut-associated lymphoid tissue
- mAb
- monoclonal antibody
- mpk
- milligrams per kilogram
- VCAM-1
- vascular cell adhesion molecule-1
- PCR
- polymerase chain reaction
- DMSO
- dimethyl sulfoxide
- bp
- base pair(s)
- GFP
- green fluorescent protein
- FACS
- fluorescence-activated cell sorting
- MLN
- murine mesenteric lymph node
- HBSS
- Hanks' balanced salt solution
- Ab
- antibody
- AUC
- area under the curve
- LFA-1
- lymphocyte function antigen-1
- ICAM
- intercellular adhesion molecule
- compound 1
- N-{N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl}-(d)-phenylalanine
- compound 2
- N-{N-[(3-chlorobenzene)sulfonyl] azetidine-2-(S)-carboxyl}-(l)-4-(2′,6′-bis-methoxyphenyl)phenylalanine
- TR14035
- N-(2,6-dichlorobenzoyl)-(l)-4-(2′,6′-bis-methoxyphenyl)phenylalanine
- Received November 28, 2001.
- Accepted March 11, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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