Chemokines and the Signaling Modules Regulating Integrin Affinity

Integrin-mediated adhesion is a general concept referring to a series of adhesive phenomena including tethering–rolling, affinity, valency, and binding stabilization altogether controlling cell avidity (adhesiveness) for the substrate. Arrest chemokines modulate each aspect of integrin activation, although integrin affinity regulation has been recognized as the prominent event in rapid leukocyte arrest induced by chemokines. A variety of inside-out and outside-in signaling mechanisms have been related to the process of integrin-mediated adhesion in different cellular models, but only few of them have been clearly contextualized to rapid integrin affinity modulation by arrest chemokines in primary leukocytes. Complex signaling processes triggered by arrest chemokines and controlling leukocyte integrin activation have been described for ras-related rap and for rho-related small GTPases. We summarize the role of rap and rho small GTPases in the regulation of rapid integrin affinity in primary leukocytes and provide a modular view of these pro-adhesive signaling events. A potential, albeit still speculative, mechanism of rho-mediated regulation of cytoskeletal proteins controlling the last step of integrin activation is also discussed. We also discuss data suggesting a functional integration between the rho- and rap-modules of integrin activation. Finally we examine the universality of signaling mechanisms regulating integrin triggering by arrest chemokines

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking

Identification of Jak PTK-regulated rho-specific GEFs involved in activation of lymphocyte adhesion

La rapida induzione dell\u2019affinit\ue0 integrinica \ue8 un processo dinamico cruciale nel reclutamento leucocitario, che \ue8 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 pi\uf9 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 pi\uf9 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\u2019affinit\ue0 dell\u2019integrina 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\u2019affinit\ue0 dell\u2019integrina LFA-1 e ad una ridotta adesione all\u2019ICAM-1 in condizioni statiche e sotto flusso. Da notare, l\u2019attivazione di queste quattro proteine, indotta da CXCL12, \ue8 mediata dalle Jak PTKs e avviene in un intervallo di tempo coerente con la rapida induzione dell\u2019affinit\ue0 integrinica da chemochine. Inoltre l\u2019attivazione di RhoA e Rac1 \ue8 strettamente dipendente dall\u2019attivit\ue0 di Vav1, Sos1, Arhgef1 e Dock2. Complessivamente in questo studio abbiamo identificato e caratterizzato dettagliatamente il ruolo regolatorio di quattro rho-GEFs nell\u2019adesione 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 pi\uf9 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 pu\uf2 avvalorare la nuova interpretazione quantitativa dei meccanismi di trasduzione del segnale, dove la complessit\ue0 a livello molecolare \ue8 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

ESAT-6 and HspX improve the effectiveness of Bacille Calmette-Guerin to induce human dendritic cells-dependent T cells and natural killer cells activation.

The limited efficacy of Bacillus Calmette-Guerin (BCG) vaccination against some forms of tuberculosis is partly due to a missing expression of critical immunogenic proteins. We hypothesized that addition of ESAT-6 and HspX Mycobacterium Tuberculosis (Mtb) antigens could ameliorate the BCG ability to activate human dendritic cells (DC), that play an essential role in immune response. Here we report that BCG showed a weak ability to induce DC maturation, cytokine release and the subsequent CD4+ lymphocytes and NK cells activation. Addition of single ESAT-6 or HspX to BCG-stimulated DC did not significantly improve these processes. However, simultaneous addition of ESAT-6 and HspX enhanced BCG-dependent DC maturation and IL-12, IL-1β, IL-23, IL-6 and TNFα release. Moreover, DC incubated with BCG in presence of both ESAT-6 and HspX elicited IFN-γ release by CD4+ lymphocytes, and increased IFN-γ secretion and CD69 cytolysis marker expression in NK cells. These effects were inhibited by IL-12-blocking antibodies. A specific TLR2-blocking antibody decreased IL-12 release by BCG-stimulated DC incubated with ESAT-6 and HspX, as well as IFN-γ secretion by CD4+ lymphocytes co-cultured with these cells. Moreover, HspX and ESAT-6 improved the capacity of BCG-treated DC to induce the expression of the memory phenotype marker CD45RO in naïve CD4+ T cells. Our results indicate that ESAT-6 and HspX cooperation enhances the ability of BCG to stimulate human DC, that become able to induce T lymphocytes and NK cells-mediated immune responses through TLR2-dependent IL-12 secretion. Therefore ESAT-6 and HspX represent good candidates for improving the effectiveness of vaccination with BCG

JAK2 tyrosine kinase mediates integrin activation induced by CXCL12 in B-cell chronic lymphocytic leukemia

Chemokines participate to B-cell chronic lymphocytic leukemia (B-CLL) pathogenesis by promoting cell adhesion and survival in bone marrow stromal niches and mediating cell dissemination to secondary lymphoid organs. In this study we investigated the role of JAK protein tyrosine kinases (PTK) in adhesion triggering by the CXC chemokine CXCL12 in normal versus CLL B-lymphocytes. We demonstrate that CXCL12 activates JAK2 in normal as well as CLL B-lymphocytes, with kinetics consistent with rapid adhesion triggering. By using complementary methodologies of signal transduction interference, we found that JAK2 mediates CXCL12-triggered activation of lymphocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins. We also show that JAK2 mediates the activation of the small GTP-binding protein RhoA, in turn controlling LFA-1 affinity triggering by CXCL12. Importantly, comparative analysis of 41 B-CLL patients did not evidence JAK2 functional variability between subjects, thus suggesting that JAK2, differently from other signaling events involved in adhesion regulation in B-CLL, is a signaling molecule downstream to CXCR4 characterized by a conserved regulatory role. Our results reveal JAK2 as critical component of chemokine signaling in CLL B-lymphocytes and indicate JAK inhibition as a potentially useful new pharmacological approach to B-CLL treatment

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells

The regulatory role of protein tyrosine kinases in β1- and β2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment

Effect of HspX and ESAT-6 on BCG-elicited DC maturation.

<p>DCs were treated with Mtb as a positive control and with BCG alone or combined with HspX and ESAT6. Cells were collected after 24(A) Histograms illustrate CD83, CD86 and HLA-DR surface expression in CD1a⁺ cells and the MFI. Filled histograms represent the control, open histograms indicate treated cells. One of four different experiments is presented. (B) Bar graphs show the CD83, CD86 and HLA-DR MFI value of the four experiments expressed as fold change (MFI FC) over control (CTRL). Statistical analysis: CTRL vs BCG alone; BCG alone vs BCG plus HspX and ESAT6; ns P>0.05, *P<0.05.</p

TLR2 is involved in IL-12-dependent IFN-γ secretion by CD4⁺ cells co-cultured with ESAT-6, HspX and BCG-treated DCs.

<p>DCs cultured in the absence (filled bars) or presence (open bars) of 5 µg/ml TLR2-blocking antibody were treated for 24 hrs with Mtb, BCG alone or combined with HspX and ESAT6, 10 µg/ml Pam3CSK4 (Pam3) or 100 ng/ml LPS. (A) Supernatants were collected and IL-12 release was analyzed by ELISA. Results are the mean value+SD of four experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P>0.05, *P<0.05, ***P<0.001. (B) DCs were co-cultured with autologous CD4⁺ T lymphocytes. After 7 days, culture supernatants were collected and analyzed by ELISA for IFN-γ release. Results are the mean+SD of three experiments. Statistical analysis: antibody-treated vs antibody-untreated cells, ns P>0.05, *P<0.05.</p