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Cellular and Molecular Bioengineering

, Volume 11, Issue 2, pp 91–98 | Cite as

An Experimentally Determined State Diagram for Human CD4+ T Lymphocyte CXCR4-Stimulated Adhesion Under Shear Flow

  • Nicholas R. Anderson
  • Dooyoung Lee
  • Daniel A. Hammer
Article

Abstract

Introduction

The leukocyte adhesion cascade is important for the maintenance of homeostasis and the ability of immune cells to access sites of infection and inflammation. Despite much work identifying the molecular components of the cascade, and numerous simulations to predict the relationship between molecule density, identity, and adhesion, these relationships have not been measured experimentally.

Methods

Using surfaces functionalized with recombinant ICAM-1 and/or E-selectin along with immobilized SDF-1α, we used a flow chamber to measure rates of tethering, rolling and arrest of primary naïve human CD4+ T lymphocytes on different surface densities of ligand.

Results

Cells required a minimum level of ligand density to progress beyond tethering. E-selectin and ICAM-1 were found to have a synergistic relationship in promoting cell arrest. Surfaces with both ligands had the highest levels of arrest, while surfaces containing only E-selectin hindered the cell’s ability to progress beyond rolling. In contrast, surfaces of ICAM-1 allowed only tethering or arrest. Cells maintained constant rolling velocity and time to stop over large variations in surface density and composition. In addition, surface densities of only O(101) sites/µm2 allowed for rolling while surface densities of O(102) sites/µm2 promoted arrest, approximately equal to previously determined simulated values.

Conclusions

We have systematically and experimentally mapped out the state diagram of T cell adhesion under flow, directly demonstrating the quantitative requirements for each dynamic state of adhesion, and showing how multiple adhesion molecules can act in synergy to secure arrest.

Keywords

E-selectin ICAM-1 SDF-1α CXCL12α Flow chamber Site density 

Notes

Acknowledgments

This work was supported by National Institutes of Health Grants to DAH AI082292 and GM123019.

Conflicts of interest

Nicholas R. Anderson, Dooyoung Lee, and Daniel A. Hammer declare that they have no conflicts of interest.

Ethical Standards

No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

Supplementary material

12195_2018_519_MOESM1_ESM.eps (446 kb)
Supplementary material 1 (EPS 446 kb). Supplementary Fig. 1: Determination of site densities using an IgG1 probe. Protein A/G and SDF-1α were co-immobilized in polystyrene microwells and incubated with IgG1. Site density was determined by comparing to fresh dilutions of an AlexaFluor 488-tagged anti-human IgG1 hinge antibody. Results shown are mean ± SE of three independent experiments
12195_2018_519_MOESM2_ESM.eps (4.4 mb)
Supplementary material 2 (EPS 4524 kb). Supplementary Fig. 2: Extended state diagrams of the fraction of cells undergoing (A) tethering, (B) rolling, or (C) firm arrest, including surfaces with high site densities. Red dots indicate experimentally tested points. All experiments were performed at a calculated wall shear rate of 100 s−1
12195_2018_519_MOESM3_ESM.eps (2.5 mb)
Supplementary material 3 (EPS 2541 kb). Supplementary Fig. 3: Comparison between cell spreading after arrest on (A) E-selectin only surfaces and (B) surfaces with E-selectin and ICAM-1. Both images are the end of 10 min flow chamber experiments and are representative of repeated experiments. Orange arrowheads point to arrested cells that are not spread and green arrowheads identify arrested cells that have spread. Surfaces containing only E-selectin show fewer arrested cells than surfaces containing both E-selectin and ICAM-1. Cells arrested on E-selectin surfaces also did not spread efficiently, shown by the fewer cells marked with green. Cells not marked by arrowheads are rolling or tethering during the moment this frame was taken
12195_2018_519_MOESM4_ESM.eps (1.8 mb)
Supplementary material 4 (EPS 1792 kb). Supplementary Fig. 4: Calculated surface showing the effect of ICAM-1 and E-selectin densities on the distance to stop. Red dots indicate experimentally tested points. All experiments were performed at a calculated wall shear rate of 100 s−1
12195_2018_519_MOESM5_ESM.eps (999 kb)
Supplementary material 5 (EPS 999 kb). Supplementary Fig. 5: Comparison of adhesion on surfaces containing and lacking SDF-1α. Surfaces were functionalized with a 1:1 molar mixture of E-selectin/Fc:ICAM-1/Fc chimeras at an overall site density of 1250 sites/µm2 with and without SDF-1α. Surfaces were compared for their ability to support tethering, rolling, and arrest. Surfaces with SDF-1α showed a rate of arrest approximately 2.5 times higher than surfaces lacking SDF-1α, highlighting the importance of chemokine to cause arrest. Results are the average from three different experiments consisting of duplicate surfaces.

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Copyright information

© Biomedical Engineering Society 2018

Authors and Affiliations

  1. 1.Department of Chemical and Biomolecular EngineeringUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Department of BioengineeringUniversity of PennsylvaniaPhiladelphiaUSA
  3. 3.Applied BioMath, LLCLincolnUSA

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