A key component of the inflammatory response is the recruitment and extravasation of leukocytes into perivascular tissue. This process requires leukocyte interaction with vascular endothelium and consists of a multistep process including the capture of circulating leukocytes, subsequent leukocyte rolling, arrest, firm adhesion, and transmigration (Figure 1).
This complex process initially involves the recognition and interaction of endothelial cell adhesion molecules with their specific ligands on leukocytes. The capture and the removal of the leukocytes from the flowing blood, as well as the subsequent rolling of the leukocyte along the vessel wall, are due to the reversible binding of selectins which are found on both the leukocytes and the endothelial cell surface. Arrest and firm adhesion of leukocytes to endothelium is dependent on the activation of b2 integrins including Mac-1 or LFA-1 on the leukocyte cell surface, followed by interaction with endothelial cell proteins belonging to the immuno-globulin superfamily such as ICAM-1.
We have used gene regulatory networks to identify novel regulatory hubs genes involved in the inflammatory response in human umbilical endothelial cells (HUVECs). To assess the potential role of these genes we have used a leukocyte adhesion assay to model this complex process and looked at the response of the endothelium following RNAi knockdown of the selected genes. We have used primary peripheral blood mononuclear cells (PBMCs) and a number of leukocyte cell lines (HL60, JY, and U937) which differ in their adhesion ligands.
All measurements were done using a multimode microplate reader from BMG LABTECH.
Materials & MethodsThe materials were purchased from the following manufacturers:
- BMG LABTECH microplate reader
- siRNA on-target reagents from Dharmacon, Perbio
- Cell tracker Green dye from Invitrogen Microplates, black 96-well from Greiner
- Tissue culture media from Lonza
- HL60.Ast and JY cell lines from ECACC
- TNFa recombinant protein from R&D systems
Whilst incubating the HUVECs with TNFa, the HL60 cells (or other leukocyte cells) were labelled with cell tracker green (2.5 μM in PBS) for 25 min at 37°C following the manufactures instructions. After labelling and washing the HL60 were resuspended in EGM2 and then added to all wells (1x105/well) except the negative control wells to which unlabelled cells were added.
The plates were incubated for 1 hr to allow leukocyte adhesion and then washed gently x3 with PBS at RT to remove any unbound cells. Fluorescence in each well was then quantified using the BMG LABTECH microplate reader (Excitation 485 nm and emission 520 nm) with replicates of 5 per sample. Knockdown of the TNFRSF1A was used as a positive control.
Results & Discussion
Initial results demonstrated that there was a linear relationship between cell number and fluorescence (Figure 2).
Preliminary adhesion assays were then set up using HUVECs that were transfected with siRNA for luciferase (siControl) and TNFRSF1A (positive control). 48 hrs after transfection the cells were treated with TNFa (0-10 ng/mL) for 4 hrs prior to the addition of cell tracker green labelled HL60 cells. Following 1 hr incubation and gently washing to remove unattached HL60, the fluorescence was quantified using the BMG LABTECH microplate reader in fluorescence mode with bottom optic measurement. Results shown in Figure 3 demonstrate that increasing the concentration of TNFa causes increased adhesion of HL60 to the HUVEC monolayer in both untreated and siControl KD conditions. However, following siRNA knockdown of the TNFRSF1A, there is no significant effect of TNFa on HL60 adhesion. This would be predicted as this receptor is required for TNFa signalling.
QPCR studies confirm that the knockdown efficiency was >90%. This is a very valuable positive control for this assay and was used for all subsequent studies. The well scanning mode of the BMG LABTECH microplate reader is helpful when scaling this assay up to use in 48 well plates as it ensures accurate quantification due to the possible uneven distribution of the HL60 cells. This function also provides a good visual output for the assay in a 96-well plate as shown in Figure 4 below.
The results were compared with fluorescent microscopy analysis of the wells (data not shown). This showed a very similar pattern but use of the microplate reader enables rapid analysis of multiple plates rather than the time-consuming image capture and quantification by microscopy.
The results show good reproducibility for a complex biological assay and provide a method for rapid screening of this aspect of the inflammatory process. We have utilized this method to screen a number of potential inflammatory markers and it provides a very easy to use technique which can also be adapted for primary neutrophils or PBMC if a more “in vivo-like” assay is required.
Cell Tracker Green is a registered trademark of Invitrogen