In a study published in Nature Communications, the research team led by Prof. CHEN Jianfeng from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences collaborated with Prof. LIN Changdong from Shanghai YangZhi Rehabilitation Hospital, Tongji University demonstrated that second timescale activation of integrins can be achieved via chemokine-induced T cell membrane-proximal external Ca2+ concentration drop, which bridges the gap between initial chemokine stimulation and slow integrin inside-out activation, ensuring immediate lymphocyte arrest and subsequent diapedesis on the right spot.
The homing of lymphocytes from the bloodstream to lymphoid organs and inflamed tissues is essential to immune surveillance and host defense. The correct location of lymphocyte extravasation at a specific target site is determined by the transition from rolling to firm arrest, which is triggered by the activation of integrins, such as αLβ2, upon chemokine stimulation. Chemokines are expressed at high levels at lymphocyte homing target sites, inducing integrin activation that facilitates lymphocyte arrest on and trans-migration across the endothelium under both physiological and pathological conditions.
The binding of chemokines to their receptors on lymphocytes induces the activation of intracellular signalling and then promotes talin binding to the integrin β subunit cytoplasmic domain, which triggers integrin activation via inside-out signalling.
Notably, the classical inside-out activation of integrin by chemokines needs several minutes. Considering lymphocytes roll in postcapillary venules at a relatively high speed ranging from 30-100 μm/s and the slow integrin activation by chemokine-triggered inside-out signalling, a quicker integrin activation is required to mediate the rapid arrest of rolling lymphocytes.
Integrins are metalloproteins and their functions are strictly dependent on and regulated by free Ca2+ and Mg2+ that physiologically exist in serum at millimolar-level. Previous in vitro studies have revealed that Ca2+ keeps integrins in an inactive state via binding to a metal ion binding site named ADMIDAS in integrin β subunit I (βI) domain, and removal of extracellular Ca2+ induces integrin quick activation in seconds. Although the quick activation of integrin by removal of extracellular Ca2+ has been reported for decades, no evidence shows that this mechanism can work in vivo because blood Ca2+ concentration is relatively stable.
Notably, chemokines can induce rapid and robust Ca2+ flux, which makes us speculate that Ca2+ flux may result in a transient Ca2+ drop in the membrane-proximal external region of lymphocytes, and subsequently induces quick activation of integrins.
In this work, researchers generated a membrane-anchored external Ca2+ biosensor named calcium-measuring organelle-entrapped protein indicator (CEPIAexternal) to monitor T cell membrane-proximal external Ca2+ concentration ([Ca2+]ex) and used quantitative ratiometric fluorescence resonance energy transfer (FRET) imaging to assess the distance between the cytoplasmic domains of αL and β2 subunits, which can indicate integrin αLβ2 activation in real-time.
A mouse model (R26-LSL-CEPIAexternal;Itgal-LSL-Clover;Itgb2-LSL-mRuby2;CD4-Cre) was established to express these two biosensors in T cellssimultaneously. Using both the isolated T cells and intravital imaging, researchers demonstrated that chemokine CCL25-triggered Ca2+ influx induces [Ca2+]ex drop in 6 seconds from physiological concentration 1.2 mM to 0.3 mM, a critical extracellular Ca2+ threshold for inducing αLβ2 activation, and further to the lowest level 0.09 mM within 28 seconds, triggering rapid αLβ2 activation and T cell arrest.
The conventional chemokine-induced inside-out activation of integrins occurs after the rapid integrin activation by [Ca2+]ex drop. Talin knockdown inhibited the slow inside-out activation of αLβ2 but not [Ca2+]ex drop-triggered αLβ2 quick activation. Blocking Ca2+ influx significantly suppressed T cell rolling-to-arrest transition and homing to skin lesions in mouse psoriasis model, thus alleviating skin inflammation.
In summary, this study reveals that second timescale activation of integrins can be achieved via chemokine-induced Ca2+ transients on T cell surface, which update the current understanding of lymphocyte homing cascade by filling the gap between initial chemokine stimulation and slow inside-out activation of integrins. This mechanism enables lymphocytes promptly stick to the spot where they encounter chemokines, ensuring the precise homing of lymphocytes to the target sites.
Reference: https://www.nature.com/articles/s41467-024-50464-0
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