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In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Published on: January 6, 2014
In vitro neutrophil transepithelial migration
Winston Y Lee1, Alex C Chin, Susan Voss
1Division of Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.
Abstract:
Polymorphonuclear leukocyte (PMN) transmigration into tissues is a highly regulated process and plays a central role in host defense. In inflammatory human diseases such as ulcerative colitis and Crohn's disease, the infiltration of intestinal mucosa by large numbers of PMNs contributes to epithelial pathophysiology. The sequence of events that fine-tune PMN migration across epithelial cells is not well-understood. In this chapter, we describe a method to study PMN transmigration across intestinal epithelial T84 monolayers using a modified Boyden chamber system. This in vitro model system consists of three main components: the epithelium, purified PMN, and a chemoattractant gradient. Intestinal epithelial cells are cultured as inverted monolayers on permeable filter supports to facilitate the study of PMN transmigration in the physiologically relevant basolateral-to-apical direction. PMNs are isolated from human blood using dextran sedimentation followed by Ficoll density gradient centrifugation. PMN transmigration is elicited using N-formyl-methionyl-leucyl-phenylalanine gradients and is quantified by assaying for myeloperoxidase activity. The advantages of this model are its reductionist approach and the fact that the system can be easily manipulated. Studies using this model system will shed more light on the mechanisms regulating PMN responses in acute inflammatory diseases.
Insights
This study presents a new in vitro model to investigate polymorphonuclear leukocyte (PMN) migration across intestinal epithelial cells. This method aids in understanding inflammatory diseases like ulcerative colitis and Crohn's disease.
Area of Science:
- Immunology
- Gastroenterology
- Cell Biology
Background:
- Polymorphonuclear leukocyte (PMN) transmigration is crucial for host defense but contributes to epithelial pathophysiology in inflammatory diseases.
- The precise mechanisms regulating PMN migration across intestinal epithelium are not fully understood.
Purpose of the Study:
- To describe a novel in vitro method for studying PMN transmigration across intestinal epithelial T84 monolayers.
- To provide a reductionist and manipulable model system for investigating PMN responses in inflammatory conditions.
Main Methods:
- Utilized a modified Boyden chamber system with inverted T84 intestinal epithelial monolayers on permeable supports.
- Isolated human PMNs from blood via dextran sedimentation and Ficoll density gradient centrifugation.
- Elicited PMN transmigration using N-formyl-methionyl-leucyl-phenylalanine gradients and quantified via myeloperoxidase activity.
Main Results:
- Successfully established an in vitro model for studying PMN transmigration in a physiologically relevant basolateral-to-apical direction.
- The model allows for controlled investigation of PMN migration mechanisms.
- Quantification of transmigration was achieved through myeloperoxidase activity assays.
Conclusions:
- The developed in vitro model offers a valuable tool for dissecting the complex mechanisms of PMN transmigration.
- This research will advance our understanding of PMN roles in acute inflammatory diseases affecting the intestine.
