Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions

Abstract

Leukocyte adhesion deficiency (LAD)-III is associated with homozygous stop codon mutations in Kindlin-3, the hematopoietic member of the Kindlin family of integrin coactivators. In addition, a subgroup of LAD-III patients has a homozygous splice junction mutation in and reduced expression of the Rap-1 guanine nucleotide exchange factor, CalDAG-GEFI (CDGI). In this study, we compared the adhesive properties of the leukocyte function-associated antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4) integrins in both primary and activated leukocytes derived from these 2 LAD-III subgroups. Primary lymphocytes lacking both Kindlin-3 and CDGI lost all firm T-cell receptor-stimulated LFA-1 adhesiveness, in contrast to LAD-III lymphocytes deficient in Kindlin-3 alone. Effector T cells expanded from all tested LAD-III variants expressed normal CDGI, but lacked Kindlin-3. These Kindlin-3-null effector T cells exhibited total loss of inside-out IntroductionIntegrins constitute the major and largest family of cell adhesion receptors. 1 In hematopoietic cells, these heterodimers rapidly undergo dramatic allosteric conformational changes in response to various activation signals. 2 Talins are key integrin activators implicated in these processes in essentially all integrin-containing cell types. 3 Activated integrins are essential for platelet aggregation, firm leukocyte adhesiveness to vascular endothelium, and lymphocyte arrest on antigen-presenting cells. 4 Recent evidence suggests that Kindlins, a group of 3 structurally related adaptors, cooperate with talin in activating integrins in different cell types through binding to distinct motifs on the short tails of the integrin ␤ subunits. [5][6][7][8] In contrast to Kindlins-1 and -2, Kindlin-3 expression is restricted to the hematopoietic system. 7 Whereas loss of talin1 is embryonically lethal, deletion of Kindlin-3 is not; 7 yet, deletion of Kindlin-3 in mice results in severe defects in platelet and leukocyte integrin activation and in reduced lymphocyte counts, 7,9 osteoporosis, 7 and abnormal erythrocyte function, which is ultimately fatal. 10 Although a recent study has directly implicated murine Kindlin-3 in neutrophil and monocyte integrin adhesiveness to inflamed endothelium in vitro and in vivo, 9 the role of Kindlin-3 in inside-out (chemokine-mediated) and outside-in (ligand-induced) activation of lymphocyte integrins has remained unexplored.Leukocyte adhesion deficiency (LAD)-III is a rare autosomal recessive syndrome, which is manifested as a combined defect in ␤ 3 , ␤ 2 , and ␤ 1 integrin activation in platelets, neutrophils, and lymphocytes. 11 We previously reported 3 LAD-III cases from families of Turkish origin, associated with a homozygous splice junction mutation that results in defective expression of a key Rap-1-specific guanine nucleotide exchange factor (Rap-1/2 GEF), CalDAG-GEFI (CDGI), in platelets, neutrophils, and resting lymphocytes. 12 We recently showed that these patients suffer from an additional mutation, a homozygous nonsense stop ...

Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells

“…Despite this efficient arrest of Kindlin-3-deficient effector T cells at high VCAM-1 levels, firm adhesion on recombinant VCAM-1 and endothelial cells was reduced. These findings are in line with a previous report showing that chemokine-stimulated Kindlin-3-deficient LAD-III effector T cells show transient adhesion, but not stable adhesion, to VCAM-1 under flow conditions (28). Furthermore, these findings suggest that α4β1 on Kindlin-3-deficient effector T cells can adopt an affinity state sufficient for the production of weak α4β1-VCAM-1 interactions.…”

Kindlin-3 Is Essential for the Resting α4β1 Integrin-mediated Firm Cell Adhesion under Shear Flow Conditions

“…5), suggesting the important role of kindlin-3 in triggering extended (high affinity) conformation of ␣4 integrins. The results are consistent with previous reports that kindlin-3 is required for the induction of the high affinity conformation of ␣L␤2 (31,59). Interestingly, kindlin-3 has been shown to have little effect on the affinity of purified monomeric ␣IIb␤3 integrin in a cell-free system (60).…”

“…Previous study shows that ␣4␤1 in kindlin-3-null lymphocytes retains intrinsic rolling adhesions to VCAM-1 and exhibits partial defects in chemokine-stimulated adhesiveness to VCAM-1 (31). Moreover, it has been reported that kindlin-3-deficient lymphocytes, although deficient in optimal firm adhesions, still are able to use their residual integrin adhesiveness to enter tissues (50).…”

“…Furthermore, it has been reported that integrin ␤1 tails have higher binding affinity for kindlin-3 than ␤3 tails in a cell-free system (31,45). Thus, it is possible that kindlin-3 regulates the ligand binding of different integrins (␣4␤1 and ␣IIb␤3) via distinct mechanisms.…”

“…In addition, loss of kindlin-3 expression accounts for the rare autosomal human disease named leukocyte adhesion deficiency type III (28,29). Lymphocytes derived from leukocyte adhesion deficiency type III patients showed defective activation of ␣L␤2 and ␣4␤1 integrins upon phorbol 12-myristate 13-acetate stimulation and showed impaired cell spreading and migration (26,30,31). Besides the critical role of kindlin-3 in inducing integrin activa-tion through inside-out signaling, it is also reported that kindlin-3 binds the resting integrin ␣4␤7 at a high level, and the dissociation of kindlin-3 from ␤7 tail specifically increases the binding affinity of ␣4␤7 to mucosal vascular addressin cell adhesion molecule-1 but suppresses VCAM-1 binding (18).…”