Academic literature on the topic 'Chemokine-Triggered LFA-1'

Kindlin-3 is a key lymphocyte function–associated antigen-1 (LFA-1) coactivator deleted in leukocyte adhesion deficiency-III (LAD-III). In the present study, we investigated the involvement of this adaptor in lymphocyte motility and TCR-triggered arrest on ICAM-1 or on dendritic cells (DCs). Kindlin-3–null primary T cells from a LAD-III patient migrated normally on the major lymph node chemokine CCL21 and engaged in normal TCR signaling. However, TCR activation of Kindlin-3–null T lymphocytes failed to trigger the robust LFA-1–mediated T-cell spreading on ICAM-1 and ICAM-1–expressing DCs that is observed in normal lymphocytes. Kindlin-3 was also essential for cytoskeletal anchorage of the LFA-1 heterodimer and for microclustering of LFA-1 within ventral focal dots of TCR-stimulated lymphocytes spread on ICAM-1. Surprisingly, LFA-1 on Kindlin-3–null lymphocytes migrating over CCL21 acquired normal expression of an epitope associated with the conformational activation of the key headpiece domain, β I. This activated LFA-1 was highly responsive to TCR-triggered ICAM-1–driven stop signals in normal T cells locomoting on CCL21, but not in their Kindlin-3–null T-cell counterparts. We suggest that Kindlin-3 selectively contributes to a final TCR-triggered outside-in stabilization of bonds generated between chemokine-primed LFA-1 molecules and cell-surface ICAM-1.

Regulation of integrin activity enables leukocytes to circulate freely, avoiding inappropriate adhesion while maintaining the ability to adhere quickly at sites of infection or inflammation. This regulation involves at least two components: affinity for ligand and affinity-independent avidity effects such as lateral mobility. Using lymphocyte function associated antigen-1 (LFA-1) as a model, we investigated the role of integrin release from cytoskeletal motion constraints in response to the chemokine stromal cell-derived factor-1 (SDF-1α) in this process. All experiments were done in primary T cells to avoid nonphysiological activation processes often seen with the use of cell lines. We found that SDF-1α releases LFA-1 from cytoskeletal constraints as effectively as does cytochalasin D. The resultant increased diffusion is correlated with a robust increase in LFA-1-mediated adhesion under physiological shear stress. We further investigated the role of the highly conserved GFFKR sequence in the LFA-1 cytoplasmic domain. We report that the GFFKR sequence is both necessary and sufficient for regulation of the SDF-1α-triggered proadhesive release from cytoskeleton interactions. While this does not address the role of transient SDF-1α-induced conformational changes in the activation process, these results strongly suggest that any model of chemokine-induced LFA-1 activation must take into account chemokine-induced integrin lateral mobility. In addition, these results have ramifications for models of differential binding of LFA-1 to surface-bound vs. soluble intercellular adhesion molecule-1.

Abstract Rac GTPases are key regulators of leukocyte motility. In lymphocytes, chemokine-mediated Rac activation depends on the CDM adaptor DOCK2. The present studies addressed the role of DOCK2 in chemokine-triggered lymphocyte adhesion and motility. Rapid chemokine-triggered activation of both LFA-1 and VLA-4 integrins took place normally in DOCK2–/– T lymphocytes under various shear flow conditions. Consequently, DOCK2–/– T cells arrested normally on TNFα-activated endothelial cells in response to integrin stimulatory chemokine signals, and their resistance to detachment was similar to that of wild-type (wt) T lymphocytes. Nevertheless, DOCK2–/– T lymphocytes exhibited reduced microvillar collapse and lamellipodium extension in response to chemokine signals, ruling out a role for these events in integrin-mediated adhesion strengthening. Strikingly, arrested DOCK2–/– lymphocytes transmigrated through a CCL21-presenting endothelial barrier with similar efficiency and rate as wt lymphocytes but, unlike wt lymphocytes, could not locomote away from the transmigration site of the basal endothelial side. DOCK2–/– lymphocytes also failed to laterally migrate over multiple integrin ligands coimmobilized with chemokines. This is a first indication that T lymphocytes use 2 different chemokine-triggered actin remodeling programs: the first, DOCK2 dependent, to locomote laterally along apical and basal endothelial surfaces; the second, DOCK2 independent, to cross through a chemokine-bearing endothelial barrier.

Abstract Neutrophil adhesion and extravasation into tissue at sites of injury or infection depend on binding of the integrin lymphocyte function–associated antigen 1 (LFA-1) to ICAM-1 expressed on activated endothelial cells. The activation-dependent conformational change of LFA-1 to the high-affinity conformation (H+) requires kindlin-3 binding to the β2-integrin cytoplasmic domain. Here we show that genetic deletion of the known kindlin interactor integrin-linked kinase (ILK) impaired neutrophil adhesion and extravasation in the cremaster muscle and in a clinically relevant model of renal ischemia reperfusion injury. Using in vitro microfluidic adhesion chambers and conformation-specific antibodies, we show that knockdown of ILK in HL-60 cells reduced the conformational change of β2-integrins to the H+ conformation. Mechanistically, we found that ILK was required for protein kinase C (PKC) membrane targeting and chemokine-induced upregulation of its kinase activity. Moreover, PKC-α deficiency also resulted in impaired leukocyte adhesion in bone marrow chimeric mice. Mass spectrometric and western blot analyses revealed stimulation- and ILK-dependent phosphorylation of kindlin-3 upon activation. In summary, our data indicate an important role of ILK in kindlin-3–dependent conformational activation of LFA-1.

Abstract Continuous migration of B cells at the follicle contrasts with their stable arrest after encounter with antigen. Two main ligand/receptor pairs are involved in these cell behaviors: the chemokine CXCL13/chemokine receptor CXCR5 and antigen/BCR. Little is known regarding the interplay between CXCR5 and BCR signaling in the modulation of B-cell dynamics and its effect on B-cell activation. We used a 2-dimensional model to study B-cell migration and antigen recognition in real time, and found that BCR signaling strength alters CXCL13-mediated migration, leading to a heterogeneous B-cell behavior pattern. In addition, we demonstrate that CXCL13/CXCR5 signaling does not impair BCR-triggered immune synapse formation and that CXCR5 is excluded from the central antigen cluster. CXCL13/CXCR5 signaling enhances BCR-mediated B-cell activation in at least 2 ways: (1) it assists antigen gathering at the synapse by promoting membrane ruffling and lymphocyte function–associated antigen 1 (LFA-1)–supported adhesion, and (2) it allows BCR signaling integration in motile B cells through establishment of LFA-1–supported migratory junctions. Both processes require functional actin cytoskeleton and non-muscle myosin II motor protein. Therefore, the CXCL13/CXCR5 signaling effect on shaping B-cell dynamics is an effective mechanism that enhances antigen encounter and BCR-triggered B-cell activation.

We investigated the role of H-Ras in chemokine-induced integrin regulation in leukocytes. Stimulation of Jurkat T cells with the CXC chemokine stromal cell-derived factor-1α (SDF-1α) resulted in a rapid increase in the phosphorylation, i.e., activation of extracellular signal receptor-activated kinase (ERK) but not c-Jun NH2-terminal kinase or p38 kinase, and phosphorylation of Akt, reflecting phosphatidylinositol 3-kinase (PI3-K) activation. Phosphorylation of ERK in Jurkat cells was enhanced and attenuated by expression of dominant active (D12) or inactive (N17) forms of H-Ras, respectively, while N17 H-Ras abrogated SDF-1α-induced Akt phosphorylation. SDF-1α triggered a transient regulation of adhesion to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 mediated by lymphocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), respectively, and a rapid increase in LFA-1 binding to soluble ICAM-1.Ig, which was inhibited by D12 but not N17 H-Ras. Both D12 and N17 H-Ras abrogated the regulation of LFA-1 but not VLA-4 avidity, and impaired LFA-1–mediated transendothelial chemotaxis but not VLA-4–dependent transmigration induced by SDF-1α. Analysis of the mutant Jurkat J19 clone revealed LFA-1 with constitutively high affinity and reduced ERK phosphorylation, which were partially restored by expression of active H-Ras. Inhibition of PI3-K blocked the up-regulation of Jurkat cell adhesion to ICAM-1 by SDF-1α, whereas inhibition of mitogen-activated protein kinase kinase impaired the subsequent down-regulation and blocking both pathways abrogated LFA-1 regulation. Our data suggest that inhibition of initial PI3-K activation by inactive H-Ras or sustained activation of an inhibitory ERK pathway by active H-Ras prevail to abolish LFA-1 regulation and transendothelial migration induced by SDF-1α in leukocytes, establishing a complex and bimodal involvement of H-Ras.

Abstract Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, are essential for normal stem/progenitor cell movement, adherence, and retention within the bone marrow environment. Two mechanisms through which BCR-ABL1 are thought to disrupt CXCR4-mediated chemotactic responses have been described in leukemia: the inhibition of CXCR4 expression (Geay et al. 2005, Cancer Res.) and intra-cellular signaling defects without modification of CXCR4 expression (Salgia et al. 1999, Blood; Ptasznik et al. 2002, J. Exp. Med.). These opposing mechanisms suggest that the actual situation is more complex and that new signaling paradigms are needed. To address this, we studied the effects of BCR-ABL1 on SDF-1-dependent, integrin-mediated, migration and adhesion of hematopoietic precursors. Stimulation of BCR-ABL1(−) hematopoietic cells with SDF-1 showed reduced cell adherence to surfaces coated with ICAM-1 (a ligand for the LFA-1 integrin), which was associated with down-regulated expression of activation-dependent epitopes of the β2 integrin, LFA-1, on hematopoietic cells. Inhibition of Lyn expression with siRNA prevented the SDF-1-triggered down-regulation of LFA-1 and cell adherence, indicating that CXCR4 inhibited the function of LFA-1 through Lyn. Expression of BCR-ABL1 in these cells resulted in increased expression of activation-dependent epitopes of LFA-1 and prevented SDF-1-dependent regulatory effects on both LFA-1 affinity and ICAM-1 adherence. Also, expression of BCR-ABL1 prevented Lyn-mediated regulation of cell adhesion to ICAM-1 as well as Lyn-mediated regulation of LFA-1 affinity. These results indicate that BCR-ABL1 constitutively increases the affinity of the LFA-1 integrin to its ligand ICAM-1, locking the integrin into an “active” conformation. The net result is the loss of responsiveness of LFA-1 to SDF-1-induced ‘inside-out’ signaling involving CXCR4 and Lyn kinase. Because in our experiments BCR-ABL1 had no significant effect on the expression of CXCR4 in Mo7e cells, transfected with low and high amounts of p210-BCR-ABL, or in primary BCR-ABL(+) cells from CML blast crisis patients (n=3), we conclude that BCR-ABL1 inhibits CXCR4-triggered ‘inside-out’ integrin signaling rather than CXCR4 expression. Taken together, we propose that BCR-ABL1 disrupts the signaling link between the chemokine receptor, CXCR4 and the β2 integrin LFA-1 so as to inhibit normal SDF-1-mediated chemotaxis and adhesion in hematopoietic cells.

Leukocyte and platelet integrins rapidly alter their affinity and adhesiveness in response to various activation (inside-out) signals. A rare leukocyte adhesion deficiency (LAD), LAD-III, is associated with severe defects in leukocyte and platelet integrin activation. We report two new LAD cases in which lymphocytes, neutrophils, and platelets share severe defects in β1, β2, and β3 integrin activation. Patients were both homozygous for a splice junction mutation in their CalDAG-GEFI gene, which is a key Rap-1/2 guanine exchange factor (GEF). Both mRNA and protein levels of the GEF were diminished in LAD lymphocytes, neutrophils, and platelets. Consequently, LAD-III platelets failed to aggregate because of an impaired αIIbβ3 activation by key agonists. β2 integrins on LAD-III neutrophils were unable to mediate leukocyte arrest on TNFα-stimulated endothelium, despite normal selectin-mediated rolling. In situ subsecond activation of neutrophil β2 integrin adhesiveness by surface-bound chemoattractants and of primary T lymphocyte LFA-1 by the CXCL12 chemokine was abolished. Chemokine inside-out signals also failed to stimulate lymphocyte LFA-1 extension and high affinity epitopes. Chemokine-triggered VLA-4 adhesiveness in T lymphocytes was partially defective as well. These studies identify CalDAG-GEFI as a critical regulator of inside-out integrin activation in human T lymphocytes, neutrophils, and platelets.

Abstract Our studies focus on the pathways that restrict homing of specific subsets of immune cells, and thereby fine-tune the immune response at specific lymphoid and peripheral tissues. Here, we report that CCL2 (at picomolar [pM] levels) renders both murine and human T cells defective in their ability to develop CCR7-triggered activation of LFA-1– and LFA-1–mediated adhesion strengthening to endothelial ICAM-1 both in vitro and in vivo. CCL2 also attenuated lymphocyte chemotaxis toward lymph node chemokines. Consequently, low-dose CCL2 inhibited lymphocyte homing to peripheral lymph nodes but did not affect lymphocyte trafficking through the spleen. Impaired homing of lymphocytes to peripheral lymph nodes resulted in attenuated progression of both asthma and adjuvant arthritis. Thus, pM levels of circulating CCL2 can exert global suppressive effects on T-cell trafficking and differentiation within peripheral lymph nodes, and may be clinically beneficial as an anti-inflammatory agent.

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (αMβ2) but not lymphocyte function–associated antigen-1 (LFA-1; αLβ2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1α in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1α were confirmed by expression of αM or αL in αL-deficient Jurkat cells. Moreover, expression of chimeras containing αL and αM cytoplasmic domain exchanges indicated that α cytoplasmic tails conferred the specific mode of regulation. Coexpressing αM or chimeras in mutant Jurkat cells with a “gain of function” phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the αL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of β2 integrins. Our data suggest that a specific regulation of β2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the α subunit cytoplasmic domains.

La rapida induzione dell’affinità integrinica è un processo dinamico cruciale nel reclutamento leucocitario, che è controllato da complessi meccanismi molecolari di segnale intracellulare indotti da chemochine. Le small GTPasi della famiglia di rho e rap sono certamente le molecole di segnale più studiate in questo contesto; dati recenti da noi ottenuti hanno evidenziato inoltre un ruolo importante delle proteine tirosin-chinasi della famiglia delle Jak (Jak PTKs) che agiscono da regolatori a monte delle small GTPasi. Gli scambiatori di nucleotidi guanosinici (GEFs-Guanosine Exchange Factors) sono i principali attivatori diretti delle small GTPasi e quindi rappresentano le molecole canditate più probabili per chiarire il legame funzionale tra Jak PTKs e il modulo della rho. In questo studio abbiamo dimostrato il ruolo regolatorio concorrente di quattro differenti rho-GEFs Vav1, Sos1, Arhgef1 e Dock2 nella modulazione dell’affinità dell’integrina LFA-1 e nella conseguente adesione cellulare di linfociti T umani primari stimolati con la chemochina CXCL12. La ridotta espressione di queste quattro molecole porta ad una minore induzione dell’affinità dell’integrina LFA-1 e ad una ridotta adesione all’ICAM-1 in condizioni statiche e sotto flusso. Da notare, l’attivazione di queste quattro proteine, indotta da CXCL12, è mediata dalle Jak PTKs e avviene in un intervallo di tempo coerente con la rapida induzione dell’affinità integrinica da chemochine. Inoltre l’attivazione di RhoA e Rac1 è strettamente dipendente dall’attività di Vav1, Sos1, Arhgef1 e Dock2. Complessivamente in questo studio abbiamo identificato e caratterizzato dettagliatamente il ruolo regolatorio di quattro rho-GEFs nell’adesione mediata da LFA-1 indotta da CXCL12, fornendo una descrizione completa dei meccanismi molecolari di segnale esistenti tra Jaks e modulo della rho. Analizzando i nostri dati da un punto di vista quantitativo, abbiamo riscontrato alcune differenze tra queste proteine osservando un ruolo più marcato per Vav1 e Sos1 in confronto a quello di Arhgef1 e Dock2. Questo diverso coinvolgimento di molteplici rho-GEFs con apparentemente la stessa funzione può avvalorare la nuova interpretazione quantitativa dei meccanismi di trasduzione del segnale, dove la complessità a livello molecolare è essenziale per generare un sistema flessibile in grado di rispondere efficientemente a differenti condizioni ambientali.
The rapid integrin affinity up-regulation is a crucial dynamic process in leukocyte recruitment that is controlled by a complex inside-out signalling pathway induced by chemokines. Small GTP binding proteins of rap and rho family are certainly the most studied signaling molecules involve in this pathway; in addition our recent data identified Jak PTKs as new upstream regulator of these small GTPases. Considering that Guanosine Exchange Factors (GEFs) are the main direct activators of small GTPases, they represent obvious molecule candidates to fill out the functional gap between Jak PTKs and rho-module. In this study we show the concurrent regulatory role of four rho specific GEFs Vav1, Sos1, Arhgef1 and Dock2 in CXCL12-induced LFA-1 affinity triggering and mediated-adhesion in human T lymphocytes. A reduced expression of these four molecules resulted in an impaired chemokine-induced LFA-1 affinity up-regulation and in a reduced cell adhesion to ICAM-1 in static and under-flow conditions. Importantly, CXCL12-activation of these four proteins is mediated by Jak PTKs and occurs in a time frame coherent with LFA-1 affinity triggering by chemokine. Moreover the activation of RhoA and Rac1 is strictly dependent on Vav1, Sos1, Arhgef1 and Dock2 activity. Collectively in this study we identified and fully characterized the role of four rho-GEFs in CXCL12-induced LFA-1 mediated adhesion providing a comprehensive signalling link between Jak PTKs and rho-module. Considering our results from a quantitative point of view, we observed some variability in the relative regulatory role of these proteins, with a major role for Vav1 and Sos1 with respect to Arhgef1 and Dock2 activity. This variable involvement of multiple rho-GEFs with apparently the same function may support the new emergent quantitative-concurrency view of signal transduction in which this complexity in mechanisms controlling integrin activation is essential to generate a very flexible signalling system able to efficiently respond to a variety of environmental conditions.