Alpha-L beta-2 integrin activation on MonoMac6 cells
Lab/Group: Weber Lab (RWTH Aachen University), Bernhagen Lab (RWTH Aachen University)
Related Journal & Article Information
Journal: Nature Medicine
Article Title: MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment
Introduction
In our study, we have discovered chemokine-like functions for the cytokine MIF such as induction of leukocyte arrest on endothelial cells. Cell adhesion requires the activation, i.e. by arrest chemokines, of integrins on the cell surface, which subsequently interact with adhesion molecules, such as VCAM-1 and ICAM-1. The latter interacts with the alpha-L beta-2 integrin. As MIF, like CXCL8, induced monocyte arrest on ICAM-1-expressing CHO cells1, we investigated the direct effect of MIF on integrin activation.
Materials
Reagents
MonoMac6 cells
327C: a monoclonal antibody, which binds to the activated extended conformation of the αLβ2 integrin
Secondary antibody, e.g. FITC-conjugated
Mg2+ and EGTA (ethylene glycol bis(2-aminoethyl-ether)-N,N,N'N'-tetraacetic acid) as positive control
4% paraformaldehyde in phosphate-buffered saline (PFA)
Assay buffer: 1× HBSS, 10 mmol/L HEPES, 0.5% bovine serum albumin/BSA
Equipment
For example using FACSCalibur (BD Biosciences, San Jose, CA)
Procedure
1. Keep MonoMac6 cells (5×105 cells/mL) on ice in assay buffer until use.
2. Add one volume of assay buffer containing 100 ng/mL MIF (final concentration: 50 ng/mL) or canonical chemokine plus 2 mM Ca2+/Mg2+ (final concentration: 1 mM Ca2+/Mg2+) and incubate at 37 ˚C up to 30 min.
3. Negative control: incubate one sample (5×105 cells/mL) without stimulus for 30 min at 37 ˚C.
4. Positive control: incubate one sample (5×105 cells/mL) with 3 mM Mg2+ and 1 mM EGTA
5. Withdraw samples at different times and add to one volume 4% PFA (final concentration: 2%)
6. Wash cells with PBS and incubate with the monoclonal antibody 327C (10 µg/mL) for 30 min.
7. Wash cells with PBS and incubate with anti-mouse-IgG FITC conjugate for another 30 min.
8. Measure the mean fluorescent intensity (MFI) of each sample via FACS as described by Shamri et al.2. The MFI corresponds with the integrin activation.
Troubleshooting
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Critical Steps
Work fast, because integrin activation already occurs within a minute after stimulus addition.
Anticipated Results
See for example Bernhagen et al. (Nat. Med. 2007).
References
1. Bernhagen, J. et al. MIF is a non-cognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat. Med. Epub ahead of print(2007).
2. Shamri, R. et al. Lymphocyte arrest requires instantaneous induction of an extended LFA-1 conformation mediated by endothelium-bound chemokines. Nat. Immunol. 6, 497-506 (2005).
Acknowledgements
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Keywords
integrins, chemokines, adhesion molecules, beta2-integrin