Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions
Summary
Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host’s inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.
Abstract
The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.
Introduction
Since the vascular endothelium is in direct contact with the circulating blood, it is uniquely situated to initiate a rapid inflammatory response during infection or injury. Endothelial cells (ECs) express pattern recognition receptors that recognize a variety of conserved bacterial components and danger molecules, and receptors for host inflammatory mediators such as TNFα. Activation of these receptors induces ECs to secrete cytokines (e.g. IL-6, IL-8, CXCL1 and CCL2), and to upregulate adhesion molecules (e.g. E/P-selectin, VCAM-1 and ICAM-1) at their cell surface 1,2. These molecules all facilitate the localization of leukocytes to sites of infection and injury in order to clear the host of the infectious agents and initiate tissue repair 3,4.The neutrophil response to infection involves a well-coordinated interplay between the vascular endothelium and the responding neutrophils. Upon EC activation, IL-8 is secreted and forms an intravascular gradient on the endothelium that allows neutrophils to home in to the site of infection or injury 5,6. E/P-selectins mediate neutrophil capture and rolling through relatively weak associations with glycomolecules on the neutrophil cell surface. These interactions, along with IL-8 binding to its cognate receptors, facilitate the robust, integrin-mediated attachment and eventual arrest of neutrophils on the endothelial cell surface 7-10. After arrest, neutrophils can migrate out of the vasculature to the specific sites of infection to directly eliminate pathogens, generate neutrophil extracellular traps to prevent the spread of pathogens, promote wound healing and release additional factors that recruit other leukocytes such as monocytes, macrophages and dendritic cells 11-17.
Described in this report is an in vitro method to quantitate neutrophil adherence to microvascular ECs after activation by the host inflammatory mediator TNFα. This assay is designed to assess the activation of ECs, and not neutrophils. Primary human neutrophils are first isolated using density gradient separation, and are then labeled with calcein acetoxymethyl (AM). Esterases within the live cells hydrolyze calcein AM to the highly fluorescent calcein molecule with an excitation of 492-495 nm and emission of 513-516 nm 18. The fluorescently-labeled neutrophils are then incubated with EC monolayers, and non-adherent neutrophils are subsequently removed. The fluorescence of the remaining, bound neutrophils is then measured using a fluorescence spectrophotometer, and calculated as a percent of total neutrophil fluorescence input per well. This method has the additional advantage that the bound calcein-labeled neutrophils used in spectrophotometry can be directly visualized using fluorescence microscopy to give a more qualitative read out of EC activation. Since this assay is performed under static conditions, only the very initial events that occur in the neutrophil adhesion cascade will be assessed. This is confirmed in this report using E-selectin blocking antibodies to show that neutrophil adherence to TNFα-treated human lung microvascular EC (HMVEC-Lung) monolayers is drastically reduced when the interaction with E-selectin is disrupted.
In addition to TNFα, we have successfully used this assay to determine the extent of human umbilical vein EC (HUVEC) activation by the Toll-like receptor 1/2 agonists peptidoglycan-associated lipoprotein (PAL), murein lipoprotein (MLP) and Pam3Cys and HMVEC-Lung activation by Pam3Cys 19,20. In addition, we successfully used this assay with kinase inhibitors and after RNAi-mediated knockdown of surface and cytoplasmic proteins in HMVEC-Lung, suggesting that this methodology is compatible with a variety of biochemical and screening assays 20. In summary, this assay provides an easy to use, reproducible, more functional way to access the extent of EC activation by inflammatory mediators in vitro.
Protocol
Representative Results
Discussion
The most critical steps for a successful neutrophil / microvascular endothelial cell adhesion assay are: 1) Use of low passage number (<9), healthy endothelial cells; 2) Maintaining the isolated neutrophils at a low density (i.e. <5 x 106 cells / ml) and using them within two hours of isolation; and 3) Fastidious washing of the calcein AM-labeled neutrophils and use of the calcein AM-labeled neutrophils in a timely fashion to minimize EC contamination and loss of calcein from the neutroph…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the UCSF Department of Anesthesia and Perioperative Care.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| HMVEC-Lung | Lonza | CC-2527 | |
| EGM-2 MV | Lonza | CC-3202 | |
| HBSS | Life Technologies | 14175-095 | Can be substituted with any vendor |
| 48-well Tissue Culture Plates | BD Falcon | 353078 | |
| PBS (without phenol red) | UCSF Cell Culture Facility | CCFAL001 | Can be substituted with any vendor |
| D-PBS (without Ca2+ and Mg2+ and phenol red) | UCSF Cell Culture Facility | CCFAL003 | Can be substituted with any vendor |
| RPMI-1640 (without phenol red) | Life Technologies | 11835-030 | |
| Polymorphprep | Axis-Shield | 1114683 | |
| calcein AM | Life Technologies | C3099 | (0.995 mM) stock soln |
| Trypan Blue Solution | Sigma-Aldrich | T8154 | |
| 40 μM filters | VWR | 21008-949 | Can be substituted with any vendor |
| FLUOstar OPTIMA Spectrophotometer | BMG LABTECH | – |
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