Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

1. Plating and Maintenance of Microvascular Endothelial Cells

1. Thaw and grow your microvascular endothelial cells according to the manufacturers supplied instructions. In this protocol, cells were grown in EGM-2 MV media (Lonza), and it is recommended that all experiments are performed within two weeks of thawing to minimize any variation due to passage number. Our experiments with HMVEC-Lung are performed between passages 4 to 9.
2. Day 1: When cells reach 80-90% confluency, trypsinize and resuspend at 120,000 cells per ml. Plate 36,000 cells (0.3 ml) per well into 48-well, polystyrene tissue culture plate(s). Include wells of HMVEC that will not be incubated with neutrophils in order to obtain a background fluorescence reading. Unless using 48-well plates specifically designed for fluorescence assays, alternating rows or columns will minimize any light contamination from neighboring wells. NOTE: Wells can be pre-coated with poly-lysine, collagen or fibronectin.
3. Day 2: Add fresh media.
4. Day 3: By phase contrast microscopy, confirm the cells are healthy (i.e. "cobblestone" appearance and little cell debris), and they are 100% confluent (Figure 1). If the cells are not 100% confluent, change the media every day until they are ready.
5. Once the HMVEC monolayers are ready for treatment, wash the cells once with Hank's balanced salt solution (HBSS) and add 0.3 ml of fresh EGM-2 MV media or media containing inflammatory agonist to the appropriate wells. In this protocol the HMVEC-Lung were treated with TNFα (100 ng/ml) (PeproTech, New Jersey) for 3 hr since this is a well-described activator of ECs ²¹. NOTE: It is recommended that you time your experiment so that your activated HMVEC cells (Step 4.1) and calcein AM labeled neutrophils (Step 3.5) are ready to use at the same time. It takes us approximately 2.5 hr to isolate and label neutrophils.

2. Neutrophil Isolation using Polymorphprep

1. Isolate neutrophils as described previously ²², or with a ready-made density gradient solution to isolate polymorphonuclear granulocytes from whole blood. Employing Polymorphprep following the protocol by Nuzzi, et al. 2007 results in yields of approximately 2-3 x 10⁶ neutrophils per ml of whole blood ²³. NOTE: To avoid excessive erythrocyte lysis, a dextran sedimentation to remove red blood cells may be performed prior to density gradient centrifugation ²⁴.
2. Bring the Polymorphprep density gradient solution to RT.
3. Collect 30 ml of whole blood from a healthy human volunteer in the presence of an anti-coagulant such as heparin, citrate or EDTA. The blood should be used within 2 hr, and kept between 18-22 °C.
4. Layer 5 ml of whole blood over 5 ml of the density gradient solution in a 15 ml conical tube (Figure 2A). Avoid altering volumes of blood and the density gradient solution as this may change the subsequent centrifugation conditions.
5. Centrifuge at 450 x g for 30 min at 18-22 °C. Make sure to slowly speed up and down the centrifuge (i.e. turn off the centrifuge brake), and balance the tubes well to prevent vibrations to help reduce activation of the neutrophils and prevent the mixing of layers. Longer centrifugation times or higher centrifugal force will result in the lower leukocyte layer, which contains neutrophils, to migrate further down towards to the pelleted red blood cells.
6. Aspirate the plasma and upper leukocyte band containing PBMCs to prevent the contamination of the layer containing neutrophils (Figure 2B).
7. Use a 1-2 ml serological pipet to remove the lower leukocyte layer containing neutrophils. Combine the neutrophil layers into 30 ml of PBS (without Ca²⁺ and Mg²⁺) in a 50 ml conical tube.
8. Bring the volume up to 50 ml with PBS (without Ca²⁺ and Mg²⁺) and centrifuge at 450 x g for 10 min at 18-22 °C.

Note: Steps 2.9 and 2.10 are optional but highly recommended.

9. Aspirate the supernatant. Resuspend the neutrophils in 8 ml of sterile water for 30 sec to lyse red blood cells, immediately add the cell suspension to 2 ml of 5x PBS (without Ca²⁺ and Mg²⁺) and gently mix by hand. DO NOT incubate cells in water for longer than 30 sec since significant neutrophil death can occur.
10. Centrifuge at 250 x g for 5 min at 18-22 °C.
11. Wash the cells once with 10 ml of PBS (without Ca²⁺ and Mg²⁺) and centrifuge again at 250 x g for 5 min at 18-22 °C.
12. Resuspend the neutrophils in 10 ml of RPMI-1640 (without phenol red) and count the live cells using the trypan blue dye exclusion method. Avoid prolonged periods of cell densities greater than 5 x 10⁶ per ml since this may result in activation of the neutrophils.

3. Neutrophil Labeling with Calcein AM

1. 6 x 10⁵ neutrophils are used per well of a 48-well plate.
2. After counting, transfer the resuspended cells to a 50 ml conical tube and dilute the neutrophils to 2-4 x 10⁶ cells per ml with phenol red-free RPMI-1640.
3. Add calcein AM stock solution to 3 μM (i.e. 2.98 μl of calcein AM stock (1 mg/ml) per ml of media) and mix well by gently flicking the tube. Greater than 5 μM calcein AM will result in leakage from the neutrophils. Note: Non-fluorescent calcein AM is cleaved within the cells by endogenous esterases to produce the highly fluorescent molecule calcein (i.e. dead cells will not fluoresce).
4. Cover the tube with aluminum foil and incubate for 30 min at 37 °C in the dark. Note: Longer incubation times may result in more calcein being liberated from the neutrophils over the course of the adhesion assay.
5. Centrifuge at 450 x g for 5 min at 18-22 °C, and wash the neutrophils 2x with 10 ml of RPMI-1640 (without phenol red; pre-warmed to 37 °C). After resuspending the cells for the second wash, first pass the neutrophils though a 40 μM sterile filter to remove clumps and then centrifuge once more at 450 x g for 5 min at 18-22 °C.
6. Resuspend the neutrophils in RPMI 1640 (without phenol red; pre-warmed to 37 °C) at 2 x 10⁶ neutrophils per ml.

4. Neutrophil Binding Assay

1. Wash the HMVEC monolayers 3x with 0.5 ml of filter-sterilized (0.22 μm) RPMI 1640 (without phenol red) containing 3% BSA per well.
2. Aspirate the media and add 6 x 10⁵ calcein-labeled neutrophils (0.3 ml of cell suspension), or 0.3 ml of RPMI 1640 (without phenol red) without neutrophils, to the appropriate wells of the 48-well plate. Note: This is the equivalent of 8 x 10⁵ neutrophils per cm².
3. Incubate for 20 min in a 37 °C, 5% CO₂ incubator.
4. Total Neutrophil Fluorescence (PRE wash): Measure the fluorescence intensity of each well with an excitation wavelength of 485 nm and an emission wavelength of 520 nm (fluorescein filter set) in a FLUOstar, or equivalent, spectrophotometer. Perform this step just prior to the incubation end point. Note: In this protocol, calcein excitation and detection of the emitted light were performed from the top of the uncovered wells; however, both can also be performed from the bottom of the wells.
5. Wash the wells containing HMVEC monolayers and neutrophils 5x with 0.5 ml per well of PBS (w/ Ca²⁺ and Mg²⁺). Remove the media and washes by inverting the plate, and patting gently onto paper towels to remove the remaining liquid.
6. Add back 0.3 ml of RPMI 1640 (without phenol red) per well.
7. Adherent Neutrophil Fluorescence (POST wash): Measure the fluorescence intensity of each well as in Step 4.4.
8. Determine the percent adherence and relative adherence per well:

Note: At this stage, images of the calcein-labeled neutrophils can be obtained by using a fluorescence capable microscope equipped with standard fluorescein filters. The images in Figure 4 were obtained on an Olympus IX51 inverted microscope equipped with a Retiga 2000R camera (Q Imaging) using Q-Capture Pro7 software (Q Imaging).

Wyniki

In order to obtain reliable, reproducible results using a neutrophil binding assay, it is essential that the health and the confluency of the microvascular endothelial cells are optimal on the day of the assay, as illustrated with HMVEC-Lung in Figure 1. In addition, it is imperative that low passage number microvascular ECs are used (i.e. less than 9 passages), and accordingly, we recommend performing all experiments within two weeks of thawing. The health of the neutrophils is also of utmost i...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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	-