A new β2 integrin activation reporter mouse reveals localized intra- and extra-vascular neutrophil integrin activation in vivo

Abstract β2 integrins (LFA-1, Mac-1, CD11c-CD18, and CD11d-CD18) are leukocyte-specific adhesion receptors that play critical roles in leukocyte recruitment, as well as other immunological processes such as phagocytosis and immunological synapse formation. Adhesion of leukocytes to other cells such as endothelial cells are regulated by integrin affinity changes for their ligands (“activation”). Human β2 integrin activation can be detected by reporter antibodies including mAb24 and KIM127. No such activation epitopes are known in mouse β2 integrins. Because of the lack of mouse β2 integrin activation reporter antibodies, nothing is known about β2 integrin activation in vivo. Here, we generated a humanized β2 integrin knockin mouse by targeting the human β2 integrin coding sequence into the mouse Itgb2 locus. We show that this enables imaging of β2 integrin activation using the KIM127 (extension conformation) and mAb24 (high affinity) reporter antibodies. Human β2 pairs with the mouse integrin α chains, yielding normal expression of the β2 integrins LFA-1, Mac-1 and CD11c-CD18 in all major leukocyte populations. Using a CXCL1-induced acute inflammation model, we uncovered the dynamics and subcellular localization of β2 integrin activation in arresting neutrophils in vivo in venules of the mouse cremaster muscle. Activated integrins in arresting neutrophils in vivo are concentrated at the interface of neutrophils and the endothelium at the rear side of neutrophils facing against the blood flow. In a high-dose lipopolysaccharide (LPS) model, we found that β2 integrins are activated in association with elevated neutrophil adhesion in lung and liver. Thus, these mice, for the first time, enable studies into β2 integrin activation in vivo. This work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L.) and a Postdoctoral Fellowship (19POST34450228 to L.W.) from the American Heart Association, USA.