Studio dei meccanismi trasduzione del segnale che controllano l'attivazione integrinica nei leucociti

Il reclutamento leucocitario \ue8 un processo finemente modulato e svolge un ruolo fondamentale nel controllo della risposta immune. Il processo avviene attraverso una sequenza di pi\uf9 tappe controllate sia da molecole d\u2019adesione sia da fattori attivanti. Il riconoscimento dell\u2019endotelio vascolare da parte dei leucociti \ue8 tradizionalmente descritto come una sucessione di almeno tre eventi mediati da distinte famiglie di proteine (Fig1){1}. Le Selectine controllano il contatto iniziale (tethering) e il rotolamento (rolling) dei globuli bianchi circolanti sui carboidrati presentati dall\u2019endotelio{2}. Il thetering consiste in una iniziale e transiente adesione dei leucociti sull\u2019endotelio vasale, su leucociti gi\ue0 adesi o su piastrine; successivamente, l\u2019adesione si fa pi\uf9 stabile ed i leucociti cominciamo a rotolare. Il lento movimento della fase di rolling permette ai leucociti di interagire con i chemoattrattanti esposti sulla membrana delle cellule endoteliale. Successivamente, i chemoattrattanti (come, ad esempio, le chemochine) inducono un segnale intracellulare, attraverso recettori a sette domini transmembrana accoppiati a proteina trimeriche G (GPCRs), che causa l\u2019aumento dell\u2019avidit\ue0 integrinica (e quindi dell\u2019adesivit\ue0 dei leucociti) per il controligando endoteliale appartenente alla famiglia delle proteine con domini immunoglobulinici. Quest\u2019ultimo evento, che promuove l\u2019adesione stabile (firm adhesion) e la migrazione transendoteliale dei leucociti, \ue8 ora conosciuto come segnale \u201cinside-out\u201d{3}. Partendo da questo semplice schema, recentemente sono stati fatti numerosi progressi nella comprensione e nella revisione del fenomeno. Inanzitutto, sono state meglio caratterizzate le fasi di rolling e di tethering durante le quali si sono viste partecipare, oltre alle Selectine, anche le integrine. Cos\uec, il dogma secondo cui il rolling doveva essere un processo indipendente dell\u2019attivazione (integrinica) \ue8 stato inficiato dalla scoperta che le integrine \u3b14 (e in certe condizioni anche le \u3b22) potevano supportare il rolling prima dell\u2019attivazione indotta da chemochine {4,5}.Il rolling quindi \ue8 stato nuovamente distinto in lento e veloce includento la possibilit\ue0 che segnali intracellulari attivati da Selectine, e non solo da chemochine, possano giocare un ruolo nell\u2019attivazione integrinica sotto flusso{6}. Queste osservazioni portarono a pensare che, durante il rolling, i segnali attivati da Selectine, 6 sebbene non in grado di attivare completamente le integrine, partecipino alla loro \u201cpreattivazione\u201d (priming) per la sucessiva totale attivazione indotta da chemochine. Inoltre, negli ultimi anni \ue8 stata definitivamente identificata la modalit\ue0 di attivazione integrinica indotta da chemochine e responsabile dell\u2019arresto sotto flusso dei leucociti e sono stati ottenuti significativi avanzamenti nella comprensione degli eventi intracellulari che controllano l\u2019intero processo. Dal momento in cui Selectine, Integrine, chemoattrattanti e i loro recettori sono stati identificati e si sono trovati possedere profili di espressione leucocita-specifici, si \ue8 sviluppato il concetto di \u201carea-code\u201d (letteralmente \u201ccodice postale\u201d) tessuto-specifico {7-9}. In tale modello, le Selectine, le chemochine e le integrine generano una grande diversit\ue0 combinatoriale in base alle diverse coppie di selettina-carboidrato, chemochinarecettore e integrina-ligando imunoglobulinico presentate rispettivamente sul leucocita o sulla cellula endoteliale. Il leucocita per migrare in un certo tessuto o organo deve riconoscere il \u201ccodice\u201d espresso dal distretto vascolare del tessuto stesso. In assenza di \u201ccodice\u201d il leucocita rimane nel circolo sanguigno. Un leucocita che \ue8 in grado di rotolare e di aderire, ma non di migrare, non si accumula nel tessuto e torna nel circolo sanguigno senza svolgere la propria funzione. La superficie del vaso sanguigno \ue8 campionata in cerca dei corretti elementi che compongano un giusto codice. Se il codice \ue8 giusto il leucocita completer\ue0 la sequenza e migrer\ue0 nel tessuto, altrimenti ritorner\ue0 nel circolo. Queste scoperte hanno fornito di recente spunto per importanti applicazioni in campo biomedico. Negli organi linfoidi, le molecole d\u2019adesione PNAd e MAdCAM-1, insieme con i loro ligandi linfocitari e la coppia chemochina-recettore, creano un \u201ccodice\u201d specifico per la migrazione dei linfociti nativi (na\uefve) {2}. In ogni caso, ad oggi, nessun \u201ccodice assoluto\u201d di molecole specifiche per la migrazione in un esclusivo sito di migrazione \ue8 mai stato caratterizzato. Ad esempio, le mucine, Selectine, ed integrine come VLA-4 e LFA-1, sono state viste essere implicate nella migrazione di leucociti in diversi organi infiammati. Lo steso dicasi per le chemochine. Di fatto, esiste un considerevole livello di ridondanza e sovrapposizione, il che suggerisce che altri meccanimsi rendano il processo coerente e non abiguo. In realta\u2019 dati recenti suggeriscono che paramteri quantitativi come le caratteristiche emodinamiche del vaso sanguigno (cio\ue8 flusso lento contro flusso veloce), il livello di densit\ue0 (alto o basso) di molecole d\u2019adesione, l\u2019espressione di recettori per chemochine e, in fine, il momento di inizio del processo infiammatorio (presto Vs ritardato) possono specificatamente selezionare una sottopopolazione linfocitaria rispetto ad un\u2019altra durante l\u2019intero processo infiammatorio.Non disponibil

Comparative analysis of normal versus CLL B-lymphocytes reveals patient-specific variability in signaling mechanisms controlling LFA-1 activation by chemokines

Activation of lymphocyte function-associated antigen-1 (LFA-1) by chemokines is fine-tuned by inside-out signaling mechanisms responsible for integrin-mediated adhesion modulation. In the present study, we investigated the possibility of qualitative variability of signaling mechanisms controlling LFA-1 activation in chronic lymphocytic leukemia (CLL) cells. We pursued a multiplexed comparative analysis of the role of the recently described chemokine-triggered rho-signaling module in human normal versus CLL B-lymphocytes. We found that the rho-module of LFA-1 affinity triggering is functionally conserved in normal B-lymphocytes. In contrast, in malignant B-lymphocytes isolated from patients with B-CLL, the role of the rho-module was not maintained, showing remarkable differences and variability. Specifically, RhoA and phospholipase D1 were crucially involved in LFA-1 affinity triggering by CXCL12 in all analyzed patients. In contrast, Rac1 and CDC42 involvement displayed a consistent patient-by-patient variability, with a group of patients showing LFA-1 affinity modulation totally independent of Rac1 and CDC42 signaling activity. Finally, phosphatidylinositol-4-phosphate 5-kinase isoform 1gamma (PIP5KC) was found without any regulatory role in all patients. The data imply that the neoplastic progression may completely bypass the regulatory role of Rac1, CDC42, and PIP5KC, and show a profound divergence in the signaling mechanisms controlling integrin activation in normal versus neoplastic lymphocytes, suggesting that patients with CLL can be more accurately evaluated on the basis of the analysis of signaling mechanisms controlling integrin activation. Our findings could potentially affect the diagnosis, prognosis, and therapy of CLL disorder

Rac1 selective activation promotes axonal regeneration after optic nerve crush in Brainbow mice.

<p>In Brainbow mice the injection of the AAV-Cre-GFP around the day of crush triggers a genetic recombination that leads to YFP expression (white false color) only in surviving neurons. We injected either the Tat-Rac1 mutants, WT or vehicle on the day of crush (day 0) and on day 2 and studied regeneration 15 days post lesion. A scheme of the treatment is in A. Nerves were acquired and studied by confocal microscopy. Single examples of whole mounted nerves after mosaic merge reconstruction are given in B to E, and are relative to vehicle (B), Rac1WT (C), L61F37A mutant (D) and L61Y40C mutant (E) double injections. The crush sites of B, C, D and E are enlarged in F, G, H and I respectively. By comparison of the panels it is clear that after treatment with L61F37A a higher number of axons is able to cross the crush site and run distally (D and H). Scale bars 100 µm.</p

Rac1 selective activation prevents the dendrite atrophy occurring after crush.

<p>Representative confocal maximum projections of YFPH mouse RGCs 15 days after crush and double injection of either vehicle or CA or DN mutants. A normal RGC is shown for comparison (A). After Rac1L61F37A and Y40C treatment (D and E respectively) the dendritic atrophy is prevented, whereas the same extent of degeneration was found in control (B) and after DN treatment (C). The plots relative to the Sholl analysis for the different treatments are shown in F–G: (F) the maximum number of intersections, the ramification index and the critical value were used for statistical evaluation (10 to 20 neurons per treatment), whereas (G) the N of intersections against the distance from the soma shows the morphological changes along the whole dendritic tree. Scale bar 20 µm. # p<0,01 versus the control, the L61F37A and the L61Y40C; *p<0,05 versus the indicated group (one way ANOVA followed by LSD post hoc test).</p

Quantification of axonal regeneration after optic nerve crush and Rac1 selective activation in Brainbow mice.

<p>Brainbow mouse optic nerves were crushed at day 0 and, after one or more injections of either Rac1 mutants, WT or vehicle, were dissected at 15 or 30 days post crush in order to investigate regeneration. A scheme of the different treatments is given in A. Nerves were studied by confocal microscopy and the results of the regeneration study are plotted in B, C and D. Only the double injection of L61F37A was able to increase the average number of axons crossing the crush site per 100 µm of nerve z-section (B) at 15 days post lesion. The number of regenerating axons is higher than control also after 2 and 5 injections of L61F37A at 30 dpl. The data at 15 days are confirmed also by the analysis of length distribution in the entire distal stump (C). The same analysis at 30 days (D) revealed that, despite after 2 and 5 L61F37A injections the total number of regenerating neurons are similar, the repetitive treatment resulted in longer axons. Since we found no differences between the various vehicle injection protocols of treatment at 15 and 30 days, we put together the data of the controls on the same column/curve (n = 6 and 8). Data are mean ±SEM. N is in brackets. ^#p<0.01, *p<0.05 by ANOVA (LSD post hoc test).</p

Phosphorylation of Pak1 and upregulation of ERK1/2 after injections of Rac1 mutants.

<p>Retina sections were immunostained by antibodies against the pan-specific and the phosphorylated form of Pak1 (T212), ERK1/2 and JNK. Retinas were dissected and immunostained 3 days after optic nerve crush and ivit treatment with either vehicle, or Rac1WT, or L61F37A or L61Y40C. Some representative relevant images are shown in A to J, N and O. (A–B) Colocalization of phospho-Pak1 (Pak1-p, red) and βIII tubulin (green) indicates that the L61F37A resulted in Pak1-p increase in RGCs, confirmed also by the lack of colocalization with GFAP (C–D). (E–F) Colocalization of phospho-ERK (ERK1/2-p, red) and GFAP (green) indicates that the L61Y40C mutant activated ERK1/2 in retinal glial cells. (G–H) The lack of colocalization between MAP2 (blue) and ERK1/2-p after Y40C treatment indicates that this protein is not activated in neurons. A control is also shown (I–J) where ERK1/2-p positivity is very low. (N–O) Histag staining after L61Y40C treatment showed a clear positivity in RGCs, meanwhile the ERK1/2-p positivity pattern was still glial-like. (K) Semi-quantitative expression of total and phosphorylated level of Pak1, ERK1/2, and JNK measured in correspondence of the ganglion cell layer and normalized on the signal of the normal eye (n = 9 to 15 confocal stacks from 3 to 6 animals). (L) Ratio between the normalized intensity of phosphorylated and total forms of Pak1, ERK1/2 and JNK gives an indication about the degree of kinase activity. Scale bar 30 µm. GCL: ganglion cell layer. (M) We hypothesize that L61F37A effect on survival might be related to the increased expression of ERK1/2 and increased Pak1 phosphorylation in neurons. Meanwhile, L61Y40C is most likely boosting survival through the activation of ERK1/2 in astrocytes.</p

JAK tyrosine kinases promote hierarchical activation of Rho and Rap modules of integrin activation

Item does not contain fulltextLymphocyte recruitment is regulated by signaling modules based on the activity of Rho and Rap small guanosine triphosphatases that control integrin activation by chemokines. We show that Janus kinase (JAK) protein tyrosine kinases control chemokine-induced LFA-1- and VLA-4-mediated adhesion as well as human T lymphocyte homing to secondary lymphoid organs. JAK2 and JAK3 isoforms, but not JAK1, mediate CXCL12-induced LFA-1 triggering to a high affinity state. Signal transduction analysis showed that chemokine-induced activation of the Rho module of LFA-1 affinity triggering is dependent on JAK activity, with VAV1 mediating Rho activation by JAKs in a Galphai-independent manner. Furthermore, activation of Rap1A by chemokines is also dependent on JAK2 and JAK3 activity. Importantly, activation of Rap1A by JAKs is mediated by RhoA and PLD1, thus establishing Rap1A as a downstream effector of the Rho module. Thus, JAK tyrosine kinases control integrin activation and dependent lymphocyte trafficking by bridging chemokine receptors to the concurrent and hierarchical activation of the Rho and Rap modules of integrin activation

Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency

Rationale: Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. Objectives: We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses. Methods: We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study. Measurements and Main Results: We found that chemoattractant-induced activation of β1 and β2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of β1 and β2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient. Conclusions: Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type–selective leukocyte adhesion deficiency disease, providing new insights into CF pathogenesis