Intestinal epithelia (T84) possess basolateral ligands for CD11b/CD18-mediated neutrophil adherence.
The process of active intestinal inflammation can be modeled by the migration of neutrophils [polymorphonuclear leukocytes (PMN)] across monolayers of the human intestinal cell line T84. We now evaluate the polarity of early adhesive events occurring between human PMN and ligands on intact T84 monolayers. Transient Ca2+ depletion (tr-Ca-depl) of confluent monolayers grown on permeable supports (2 mM EDTA, 12 min, 37 degrees C) resulted in disruption of tight junctions separating apical and basolateral domains (resistance fell from 973 +/- 35 to 57.4 +/- 3.8 omega.cm2 after 12 min). The columnar T84 phenotype was not grossly altered by tr-Ca-depl. Access to basolateral membrane proteins from the apical surface after tr-Ca-depl was confirmed by enzyme-linked immunosorbant assay and immunofluorescence, using antibodies to the basolateral proteins E-Cadherin and beta 1-integrin. The effects of tr-Ca-depl on early events in PMN adhesion were next assessed. Neutrophils (2 x 10(6)) were applied to the apical surface of monolayers followed by stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (100 nM, 5 min, 37 degrees C) and quantitation. In comparison with control monolayers, tr-Ca-depl increased subsequent PMN adhesion (1.7 +/- 0.24 vs. 3.1 +/- 0.28 x 10(4) adherent PMN/monolayer; P < 0.005), which was inhibited 80% by anti-CD11b/CD18 antibodies. These experiments suggest that PMN adhere to subjunctional ligands on T84 cells in a CD11b/CD18-dependent fashion.
Parkos
C A
CA
Department of Pathology, Harvard Medical School, Boston, Massachusetts.
Colgan
S P
SP
Bacarra
A E
AE
Nusrat
A
A
Delp-Archer
C
C
Carlson
S
S
Su
D H
DH
Madara
J L
JL
D018821:CD18 Antigens / Q000502:physiology*; D002118:Calcium / Q000172:deficiency; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D016764:Cell Polarity; D004553:Electric Conductivity; D000939:Epitopes; D006801:Humans; D016023:Integrins / Q000502:physiology; D007365:Intercellular Junctions / Q000502:physiology; D007413:Intestinal Mucosa / Q000378:metabolism*; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000502:physiology*; D009504:Neutrophils / Q000502:physiology*; D013997:Time Factors
D018821:CD18 Antigens; D000939:Epitopes; D016023:Integrins; D008024:Ligands; D016177:Macrophage-1 Antigen; D002118:Calcium