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Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface
Published on: November 2, 2011
ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18)
M S Diamond1, D E Staunton, A R de Fougerolles
1Committee on Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02115.
Insights
Intercellular adhesion molecule (ICAM)-1 binds to Mac-1, a leukocyte integrin. This interaction is crucial for adhesion between stimulated neutrophils and endothelial cells, clarifying Mac-1
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- The interaction between leukocyte integrins and adhesion molecules is vital for immune cell trafficking.
- While lymphocyte function-associated antigen (LFA)-1 binding to intercellular adhesion molecule (ICAM)-1 is established, Mac-1's interaction with ICAM-1 remains controversial.
- Understanding these interactions is key to elucidating inflammatory and immune responses.
Purpose of the Study:
- To investigate and clarify the controversial interaction between the Mac-1 integrin and ICAM-1.
- To determine if ICAM-1 serves as a counter-receptor for Mac-1.
- To assess the role of the Mac-1/ICAM-1 interaction in neutrophil-endothelial cell adhesion.
Main Methods:
- Utilized multiple cell binding assays with purified Mac-1, ICAM-1, and transfected cell lines.
- Employed immunoaffinity purification of Mac-1 and ICAM-1.
- Conducted reciprocal binding assays and two-color fluorescence cell conjugate experiments.
Main Results:
- Stimulated endothelial cells expressing ICAM-1 bound to purified Mac-1, an interaction inhibited by antibodies to Mac-1 and ICAM-1.
- Transfected cells expressing ICAM-1 showed specific, dose-dependent binding to Mac-1, distinct from LFA-1 binding characteristics.
- Neutrophil binding to endothelial cells was dependent on ICAM-1, Mac-1, and LFA-1.
Conclusions:
- Intercellular adhesion molecule (ICAM)-1 is confirmed as a counter-receptor for the Mac-1 integrin.
- The Mac-1/ICAM-1 receptor pair contributes to the adhesion between stimulated neutrophils and endothelial cells.
- This finding resolves controversy and clarifies a significant mechanism in leukocyte adhesion.
Abstract:
While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.
