Related Experiment Videos
Adhesive interactions between fibroblasts and polymorphonuclear neutrophils in vitro
1Department of Thoracic Medicine, National Heart and Lung Institute, University of London, United Kingdom.
Abstract:
Although the in vivo interaction between polymorphonuclear neutrophils (PMN) and fibroblasts may be important, these pathways have not been well studied. We have investigated the adherence of PMN to monolayers of human fetal lung fibroblasts, using a microtiter plate assay based upon the uptake by cells of the vital stain Rose Bengal. Stimulation with phorbol myristate acetate (PMA) caused a significant increase of adherence over basal levels which was rapid in onset and plateaued at 5 min. Adhesion was dependent on the leucocyte integrin family of glycoproteins, notably on Mac-1, since monoclonal antibodies toward the beta chain (CD18) and alpha chain (CD11b) of Mac-1 almost completely suppressed PMA-induced PMN adhesion (88% and 77% inhibition, respectively). Adhesion was also inhibited by the peptides RGDS and GRGDS (24.2% and 26.6%, respectively using 1 mM peptide). Prestimulation of fibroblasts for longer time periods (5 and 24 h) with interleukin 1 alpha and tumor necrosis factor alpha, but not transforming growth factor beta, also resulted in a significant increase in adhesion of unstimulated PMN (after 24 h preincubation, 10 U/ml IL1 alpha stimulated adhesion by 179% of control, 500 U/ml TNF alpha by 157%). This indicated that there are both PMN- and fibroblast-dependent pathways for PMN adhesion. Components of the extracellular matrix of fibroblasts do not appear to play important roles in the adhesion process since addition of fibronectin and type IV collagen, or of purified antibodies to fibronectin and types I and IV collagen, did not affect PMA-induced PMN adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Polymorphonuclear neutrophils (PMN) adhesion to fibroblasts is mediated by Mac-1 integrins and cytokine-induced pathways. Fibroblast extracellular matrix components do not significantly influence this interaction, highlighting distinct adhesion mechanisms.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- The interaction between polymorphonuclear neutrophils (PMN) and fibroblasts is crucial in inflammatory responses but remains poorly understood.
- Investigating PMN-fibroblast interactions can elucidate mechanisms of tissue repair and inflammation.
Purpose of the Study:
- To investigate the adherence mechanisms of PMN to human fetal lung fibroblasts.
- To identify the molecular players and pathways involved in PMN-fibroblast adhesion.
Main Methods:
- Utilized a microtiter plate assay measuring Rose Bengal dye uptake to quantify PMN adherence to fibroblast monolayers.
- Employed phorbol myristate acetate (PMA) for PMN stimulation and assessed the role of Mac-1 integrins using specific monoclonal antibodies (anti-CD18, anti-CD11b).
- Investigated the effect of fibroblast prestimulation with cytokines (IL-1α, TNF-α, TGF-β) and extracellular matrix components (fibronectin, collagen) on PMN adhesion.
Main Results:
- PMA stimulation significantly increased PMN adherence within 5 minutes, indicating a rapid response.
- PMN adherence was highly dependent on the Mac-1 integrin (CD11b/CD18), with antibodies inhibiting adhesion by 88% and 77%.
- Fibroblast prestimulation with IL-1α and TNF-α (but not TGF-β) enhanced unstimulated PMN adhesion, demonstrating fibroblast-dependent pathways. Extracellular matrix components did not affect adhesion.
Conclusions:
- PMN adherence to fibroblasts involves both PMN-dependent (Mac-1) and fibroblast-dependent (cytokine-mediated) pathways.
- The findings highlight the complex interplay between immune cells and structural cells in inflammatory processes.
- Extracellular matrix proteins of fibroblasts are not primary mediators of PMN adhesion in this model.