Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Summary

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Abstract

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro “vascular” models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.

Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNFα) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.

In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNFα-stimulated endothelium.

Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

Introduction

Inflammation is a protective response to microbial infection or tissue injury that requires tight regulation of leukocyte entry into and exit from the inflamed tissue to allow resolution^1,2. Cross-talk between endothelial cells (EC) that line blood vessels, circulating leukocytes and tissue-resident stromal cells is essential for coordinating this process³. However, uncontrolled recruitment of leukocytes and their ineffective clearance underpin the development of chronic inflammatory diseases⁴. Our current understanding of leukocyte recruitment in health and disease is incomplete and more robust models are needed to analyze this proces....

Protocol

1. Isolation and Culture of Primary Human Endothelial Cells and Mesenchymal Stem Cells

1. Isolation and culture of human umbilical vein endothelial cells (HUVEC):
   1. Put umbilical cord on a tray with paper towels and spray with 70% ethanol. Place in a tissue culture hood. Identify the vein and cannulate at both ends. Place a cable tie around the cannulated end to secure it.
   2. Wash out venous blood with PBS using a syringe. Fill syringe with air and pass through the vein to remove and discard the residual PBS.
   3. Thaw 10 mg/ml collagenase type Ia and dilute 1:10 in PBS (with calcium and magnesium chloride) to a final concentration of ....

Results

Initially, we analyzed the effect of stimulating EC with TNFα on the recruitment of neutrophils from flow using the Ibidi microslide model (Section 7 - 9). In the absence of TNFα, little if any neutrophils adhered to the endothelial monolayer (Figure 2A). This was expected, as untreated/resting EC do not express the necessary adhesion molecules (selectins) or chemokines to support binding^25,26. In contrast, cytokine-stimulation significantly increased neutrophil adhesion to th.......

Discussion

Here we describe two in vitro “vascular” models for studying the recruitment of circulating neutrophils by inflamed endothelium. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We have previously observed a dose-dependent increase in neutrophil adhesion to and transmigration through TNFα-stimulated EC^9,29. We also describe how both models can be adapted to study the effects of stromal ce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase Type Ia	Sigma	C2674	Dilute in 10 ml PBS to get a final concentration of 10 mg/ml. Store at -20 °C in 1 ml aliquots.
Dulbecco's PBS	Sigma	D8662	With calcium and magnesium chloride. Keep sterile and store at RT.
1X Medium M199	Gibco	31150-022	Warm in 37 °C water bath before use.
Gentamicin sulphate	Sigma	G1397	Store at 4 °C. Add to M199 500 ml bottle.
Human epidermal growth factor	Sigma	E9644	Store at -20 °C in 10 µl aliquots.
Fetal calf serum (FCS)	Sigma	F9665	FCS must be batch tested to ensure the growth and viability of isolated EC. Heat inactivate at 56 °C. Store in 10 ml aliquots at -20 °C.
Amphotericin B	Gibco	15290-026	Potent and becomes toxic within a week so fresh complete HUVEC medium must be made up every week. Store at -20 °C in 1 ml aliquots.
Hydrocortisone	Sigma	H0135	Stock is in ethanol. Store at -20 °C in 10 µl aliquots.
Collagenase Type II	Sigma	C6885	Dilute stock in PBS to a final concentration of 100 mg/ml. Store at -20 °C in 100 µl aliquots.
Hyaluronidase	Sigma	H3631	Dilute stock in PBS to a final concentration of 20,000 U/ml. Store at -20 °C in 100 µl aliquots.
100 µm cell strainer for 50 ml centifuge tube	Scientific Lab Supplies (SLS)	352360	Other commercially available cell strainers (e.g. Greiner bio-one) can also be used.
DMEM low glucose	Biosera	LM-D1102/500	Warm in 37 °C water bath before use.
Penicillin/Streptomycin mix	Sigma	P4333	Store at -20 °C in 1 ml aliquots.
25 cc tissue culture flask	SLS	353109
75 cc tissue culture flask	SLS	353136
Bone marrow mesenchymal stem cells vial	Lonza	PT-2501	Store in liquid nitrogen upon arrival. Cells are at passage 2 upon arrival but are designated passage 0. Exapand to passage 3 and store in liquid nitrogen for later use.
Mesenchymal stem cell growth medium (MSCGM)	Lonza	PT-3001	Warm in 37 °C water bath before use. For Cell Tracker Green staining use medium without FCS.
EDTA (0.02%) solution	Sigma	E8008	Store at 4 °C. Warm in 37 °C water bath before use.
Trypsin solution	Sigma	T4424	Store at -20 °C in 2 ml aliquots. Thaw at RT and use immediately.
Cryovials	Greiner bio-one	2019-02	Keep on ice before adding before adding cell suspension.
Mr. Frosty Freezing Container	Nalgene	5100-0001	Store at RT. When adding cryovials with cells store at -80 °C for 24 hr before transfering cells to liquid nitrogen.
Ibidi u-Slide VI (0.4), T/C treated, sterile	Ibidi	IB-80606	Alternative models include glass capillaries, Cellix Biochips (www.cellixltd.com), BioFlux Plates (www.fluxionbio.com/bioflux/) and GlycoTech parallel plate flow chambers (http://www.glycotech.com/apparatus/parallel.html).
Cell tracker green dye	Life technologies	C2925	Store in 5 µl aliquots at -20 °C. Dilute in 5 ml prewarmed (at 37 °C) MSCGM.
Cell counting chambers	Nexcelom	SD-100	Alternatively a haemocytometer can be used.
Cellometer auto T4 cell counter	Nexcelom	Auto T4-203-0238
Tumor necrosis factor α (TNFα)	R&D Systems	210-TA-100	Dilute stock in PBS to a final concentration of 100,000 U/ml. Store at -80 °C in 10 µl aliquots.
6-well, 0.4 µm PET Transwell filters	SLS	353090
K2-EDTA in 10ml tubes	Sarstedt		Store at RT.
Histopaque 1119	Sigma	11191	Store at 4 °C. Warm to RT before use.
Histopaque 1077	Sigma	10771	Store at 4 °C. Warm to RT before use.
10 ml round bottomed tube	Appleton Woods	SC211 142 AS
7.5% BSA Fraction V solution	Life technologies	15260-037	Store at 4 °C.
20 ml Plastipak syringes	BD falcon	300613
5 ml Plastipak syringes	BD falcon	302187
2 ml Plastipak syringes	BD falcon	300185
3M hypo-allergenic surgical tape 9 m x 2.5 cm	Micropore	1530-1	Use to secure the syringe tap onto the wall of the perspex chamber.
Silicon rubber tubing, internal diameter/external diameter (ID/OD) of 1/3 mm (thin tubing)	Fisher Scientific	FB68854	Cut silicon tubing to the appropriate size. All tubing leading directly to the electronic microvalve must be thin.
Silicon rubber tubing ID/OD of 2/4 mm (thick tubing)	Fisher Scientific	FB68855
Portex Blue Line Manometer tubing	Smiths	200/495/200	Tubing leading to the syringe pump.
3-way stopcock	BOC Ohmeda AB
Glass 50 ml syringe for pump	Popper Micromate	550962	Must be primed prior to use by removing any air bubbles.
Glass coverslip	Raymond A Lamb		26 x 76 mm coverslips made to order. Lot number 2440980.
Parafilm gasket	American National Can Company		Cut a 26 x 76 mm piece of parafilm using an aluminium template and cut a 20 x 4 mm slot into it using a scalpel 10a. Gasket thickness is approximately 133 µm.
Two perspex parallel plates	Wolfson Applied Technology Laboratory		Specially designed chamber consisting of parallel plates held together by 8 screws. The lower plate has a viewing slot cut out in the middle and a shallow recess milled to allow space for the coverslip, filter and gasket. The upper perspex plate has an inlet and outlet hole positioned over the flow channel.
Electronic 3-way microvalve with min. dead space	Lee Products Ltd.	LFYA1226032H	Electronically connected to a 12 volt DC power supply.
Syringe pump for infusion/withdrawal (PHD2000)	Harvard Apparatus	70-2001	Set the diameter to 29 mm and refill (flow) rate.
L-shaped connector	Labhut	LE876	To attach to the inlet and outlet ports onto the Ibidi microslide channel.
Video camera	Qimaging	01-QIC-F-M-12-C	Connected to a computer which enables digitall videos to be recorded.
Image-Pro Plus 7.0	Media Cybernetics	41N70000-61592	For data analysis. Manually tag cells displaying the different behaviors. Track cells for analysis of rolling and migration velocities.
Refer to product datasheets for details on hazards of using the reagents described here.