Natural Killer Cell Transcript 4 promotes the development of SjÓ§gren's syndrome via activation of Rap1 on B cells.

Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal Natural Killer Cell Transcript 4 (NK4) as a novel regulator that promotes the development of autoimmune disorders. NK4 was significantly elevated in samples from patients with SjÓ§gren's Syndrome (SS). SS patients show elevated NK4 levels. There is a strong and positive correlation between the increased levels of NK4 and the duration of SS. Interestingly, transgenic expression of NK4 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the NK4 transgenic mice. The autoimmune phenotypes became more severe in older mice. Moreover, after NK4 transfection, human naïve B cells were activated and memory B cells differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies.NK4 regulated the differentiation and activation of B cells through activating Rap1 activity. NK4 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells. Collectively, these findings identify NK4 as a promoter of the development of autoimmune disorders through its roles on B cells. Therefore, NK4 may be a novel therapeutic target for the treatment of autoimmune diseases.

miR-590-3p Alleviates diabetic peripheral neuropathic pain by targeting RAP1A and suppressing infiltration by the T cells.

BACKGROUND: MicroRNAs play a crucial role in diabetic peripheral neuropathic pain (DPNP). miR-590-3p is a novel miRNA and involved in multiple diseases. However, the pathological mechanism of miR-590-3p in DPNP needs to be elucidated. MATERIALS AND METHODS: The db/db mice and db/m mice were selected to mimic diabetes and control, respectively, for in vivo studies. The miR-590-3p agomir was injected into db/db mice and pain-related behavioral tests were performed. The interaction of miR-590-3p with target gene was confirmed by dual-luciferase reporter assay. The expression of target gene was determined by qRT-PCR and western blot assay. The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: miR-590-3p was down-regulated in diabetic peripheral neuropathy mice. More importantly, miR-590-3p agomir alleviated pain-related behavior, reduced TNF-α, IL-1ß and IL-6 concentrations, and inhibited neural infiltration by immune cells in db/db mice. Interestingly, RAP1A was predicted to be the target of miR-590-3p by Targetscan, and was actually regulated by miR-590-3p. Finally, the rescue experiments proved that overexpression of RAP1A partially abrogated the suppressive impact of miR-590-3p on T cells proliferation and migration. CONCLUSION: miR-590-3p ameliorates DPNP via targeting RAP1A and inhibiting T cells infiltration, indicating that exogenous miR-590-3p may be a potential candidate for clinical treatment of DPNP.

HS1 deficiency impairs neutrophil recruitment in vivo and activation of the small GTPases Rac1 and Rap1.

Neutrophil extravasation is a critical step of the innate immune system's response to inflammation. This multistep process is tightly regulated by adhesion and signaling molecules in the endothelium and neutrophils. Activation of the ß2 integrin LFA-1 is critical for adhesion of leukocytes to postcapillary venules. This step requires coordinated activation of signaling pathways in chemokine-stimulated neutrophils, including GTPase activation and cytoskeletal remodeling, leading to conformational changes in LFA-1. Hematopoietic cell-specific lyn substrate 1 (HS1) is a cortactin-related and leukocyte-specific actin-binding protein (ABP) that regulates several processes in various immune cells. It has been shown in vitro that HS1 is important for neutrophil chemotaxis and transendothelial migration of NK cells, but its role in neutrophil extravasation in vivo has not been investigated yet. Intravital microscopy of CXCL1-stimulated cremaster venules revealed an increased rolling velocity and reduced neutrophil adhesion and transmigration in HS1 knockout (KO) mice. CXCL1-induced rapid neutrophil arrest in vivo and adhesion under flow conditions in vitro were also reduced significantly. Whereas random motility of neutrophils was unaffected, chemotaxis toward a CXCL1 gradient was reduced in the absence of HS1. Further analysis of the underlying mechanisms demonstrated that HS1 controls CXCL1-induced activation of the small GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras-related protein 1 (Rap1), thus supporting LFA-1-mediated neutrophil adhesion. Importantly, with the use of Rac1 KO neutrophils, we could show that Rac1 acts upstream of Rap1. Our results establish HS1 as an important regulator of proper Rac1 and Rap1 activation and neutrophil extravasation.

Dual functions of Rap1 are crucial for T-cell homeostasis and prevention of spontaneous colitis.

Rap1-GTP activates leukocyte function-associated antigen-1 (LFA-1) to induce arrest on the high endothelial venule (HEV). Here we show that Rap1-GDP restrains rolling behaviours of T cells on the peripheral lymph node addressin (PNAd), P-selectin and mucosal addressin cell adhesion molecule-1 (MadCAM-1) by inhibiting tether formation. Consequently, Rap1 deficiency impairs homing of naive T cells to peripheral lymph nodes, but accelerates homing of TH17 and TH1 cells to the colon, resulting in spontaneous colitis with tumours. Rap1-GDP associates with and activates lymphocyte-oriented kinase, which phosphorylates ERM (ezrin, radixin and moesin) in resting T cells. Phosphomimetic ezrin reduces the rolling of Rap1-deficient cells, and thereby decreases their homing into the colon. On the other hand, chemokines activate Rap1 at the plasma membrane within seconds, and Rap1-GTP binds to filamins, which diminishes its association with the ß2 chain of LFA-1 and results in LFA-1 activation. This Rap1-dependent regulation of T-cell circulation prevents the onset of colitis.

Regulatory T cell-derived adenosine induces dendritic cell migration through the Epac-Rap1 pathway.

Dendritic cells (DC) are one target for immune suppression by regulatory T cells (Treg), because their interaction results in reduced T cell stimulatory capacity and secretion of inhibitory cytokines in DC. We show that DC in the presence of Treg are more mobile as compared with cocultures with conventional CD4(+) T cells and form DC-Treg aggregates within 2 h of culture. The migration of DC was specifically directed toward Treg, as Treg, but not CD4(+) T cells, attracted DC in Boyden chambers. Treg deficient for the ectonucleotidase CD39 were unable to attract DC. Likewise, addition of antagonists for A2A adenosine receptors abolished the formation of DC-Treg clusters, indicating a role for adenosine in guiding DC-Treg interactions. Analysis of the signal transduction events in DC after contact to Treg revealed increased levels of cAMP, followed by activation of Epac1 and the GTPase Rap1. Subsequently activated Rap1 localized to the subcortical actin cytoskeleton in DC, providing a means by which directed locomotion of DC toward Treg is facilitated. In aggregate, these data show that Treg degrade ATP to adenosine via CD39, attracting DC by activating Epac1-Rap1-dependent pathways. As a consequence, DC-Treg clusters are formed and DC are rendered less stimulatory. This adenosine-mediated attraction of DC may therefore act as one mechanism by which Treg regulate the induction of immune responses by DC.

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector.

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P(2)) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2).

Deficiency of Rap1-binding protein RAPL causes lymphoproliferative disorders through mislocalization of p27kip1.

RAPL (an alternative spliced form of Rassf5) is a critical Ras-related protein1 (Rap1) effector that regulates lymphocyte adhesion. Here, we have shown that in addition to this previously described function, RAPL also negatively controls lymphocyte proliferation and prevents autoimmunity and lymphoma. RAPL-deficient mice experienced age-related lupus-like glomerulonephritis and developed B cell lymphomas. RAPL-deficient lymphocytes showed hyperproliferation by enhanced S phase entry after antigen receptor ligation. Compared to wild-type cells, RAPL-deficient naive lymphocytes had a 2- to 3-fold increase in Cdk2 kinase activity with a cytoplasmic mislocalization of the cyclin-dependent kinase inhibitor p27(kip1). RAPL was found to suppress the phosphorylation of p27(kip1) on serine 10 (S10) and promoted p27(kip1) nuclear translocation. An S10A mutation in p27(kip1) corrected its cytoplasmic accumulation, reduced hyperproliferation in RAPL-deficient lymphocytes, and suppressed glomerulonephritis and development of B cell lymphoma. Thus, RAPL serves as a checkpoint for S phase entry to prevent lymphoproliferative disorders through the spatial regulation of p27(kip1).

HPK1 competes with ADAP for SLP-76 binding and via Rap1 negatively affects T-cell adhesion.

The hematopoietic progenitor kinase 1 (HPK1) signals into MAPK and NFκB pathways downstream of immunoreceptors, but enigmatically is a negative regulator of leukocytes. Here, we report a novel role for HPK1 in regulating the activation of the adhesion molecule leukocyte function-associated antigen-1 (LFA-1). Upon TCR stimulation, mediated by binding of adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76, a ternary complex composed of ADAP/55-kDa src kinase associated phosphoprotein (SKAP-55) and RIAM translocates to the membrane and causes membrane recruitment of the active small GTPase Ras-related protein 1 (Rap1). Active Rap1, via its binding to RapL (regulator for cell adhesion and polarization enriched in lymphoid tissues), mediates LFA-1 integrin activation. We show here that HPK1, which also binds SLP-76, compete with ADAP for SLP-76 binding. In addition, HPK1 dampens Rap1 activation, resulting in decreased LFA-1 activity. Analysis of HPK1-deficient T cells revealed increased ADAP recruitment to SLP-76 and elevated Rap1 activation in those cells, leading to increased adhesion to ICAM-1 and cell spreading. Altogether, these results describe a novel function for HPK1 in linking TCR signaling to cell adhesion regulation and provide a mechanistic explanation for the negative regulatory role of HPK1 in T-cell biology.

Sustained T cell Rap1 signaling is protective in the collagen-induced arthritis model of rheumatoid arthritis.

OBJECTIVE: Defective activation of T cell receptor-proximal signaling proteins, such as the small GTPase Rap1, is thought to contribute to the pathologic behavior of rheumatoid arthritis (RA) synovial T cells. This study was undertaken to determine whether maintaining Rap1 signaling in murine T cells modifies disease onset or severity in collagen-induced arthritis (CIA). METHODS: CIA experiments were conducted using wild-type and RapV12-transgenic mice, which express an active mutant of Rap1 in the T cell compartment. Mice were assessed using macroscopic, microscopic, and radiologic measures, and serum levels of anticollagen antibodies were measured by enzyme-linked immunosorbent assay. Phenotypic and functional characterization of wild-type and RapV12-transgenic T cells under homeostatic conditions and during disease onset was performed by flow cytometry. RESULTS: Disease incidence and severity, synovial infiltration, joint destruction, and anticollagen antibody production were significantly reduced in RapV12-transgenic mice. Although the numbers and percentages of CD3+, CD4+, and CD8+ (naive, effector, and memory) T cells, Treg cells, and Th17 cells were equivalent in wild-type and RapV12-transgenic mice, a significant decrease in the percentage of tumor necrosis factor α-secreting CD8+ T cells was observed in RapV12-transgenic mice during CIA. RapV12-transgenic T cells also inefficiently expressed inducible costimulator and CD40L costimulatory proteins involved in B cell immunoglobulin class switching. CONCLUSION: Our findings indicate that maintenance of T cell Rap1 signaling in murine T cells reduces disease incidence and severity in CIA, which are associated with specific defects in T cell effector function. Therefore, the restoration of Rap1 function in RA synovial T cells may have therapeutic benefit in RA.

Zoledronic acid, an aminobisphosphonate, modulates differentiation and maturation of human dendritic cells.

Zoledronic acid (ZOL), an effective nitrogen-containing bisphosphonate against excessive bone loss, has been shown affecting the function of cells of both innate and acquired immunity. In this study, we tested the effect of ZOL on differentiation and maturation of human myeloid dendritic cells (DC). When ZOL (1.1 to 10 microM) was added to the culture of starting monocytes, but not to immature DC, the recovery rate of DC was markedly reduced in a concentration-dependent manner. The mature DC differentiated in the presence of ZOL had fewer and shorter cell projections. ZOL treatment affected DC differentiation and maturation in terms of lower expression of CD1a, CD11c, CD83, CD86, DC-SIGN, HLA-DR, and, in contrast, higher expression of CD80. IL-10 production by DC was inhibited by ZOL treatment whereas IL-12p70 secretion remained unchanged. Interestingly, ZOL augmented the allostimulatory activity of DC on naive CD4(++)CD45(+)RA(++) T cells in terms of their proliferation and interferon-gamma production. Addition of geranylgeraniol abrogated the effect of ZOL on DC differentiation and prenylation of Rap1A. It suggests that ZOL redirects DC differentiation toward a state of atypical maturation with allostimulatory function and this effect may go through prevention of Rap1A prenylation.

Rap1 activation is required for Fc gamma receptor-dependent phagocytosis.

Phagocytosis of IgG-opsonized microbes via the Fc gamma receptor (Fc gammaR) requires the precise coordination of a number of signaling molecules, including the low-molecular mass GTPases. Little is known about the Ras-family GTPase Rap1 in this process. We therefore investigated its importance in mediating Fc gammaR-dependent phagocytosis in NR8383 rat alveolar macrophages. Pulldown of active Rap1 and fluorescence microscopic analysis of GFP-RalGDS (Ral guanine dissociation stimulator)-transfected macrophages revealed that Rap1 is indeed activated by Fc gammaR crosslinking. Inhibition of Rap1 activity, both by Rap1GAP (GTPase-activating protein) expression and liposome-delivered blocking Ab, severely impaired the ability of cells to ingest IgG-opsonized targets. Fc gammaR-induced Rap1 activation was found to be independent of both cAMP and Ca(2+), suggesting a role for the second messenger-independent guanosine exchange factor, C3G. This was supported by the facts that 1) liposome-delivered blocking Ab against C3G inhibited both Fc gammaR-dependent phagocytosis and Rap1 activation, and 2) both active Rap1GTP and C3G were found to translocate to the phagosome. Taken together, our data demonstrate a novel role for Rap1 and its exchange factor C3G in mediating Fc gammaR-dependent phagocytosis.

The hematopoietic isoform of Cas-Hef1-associated signal transducer regulates chemokine-induced inside-out signaling and T cell trafficking.

Leukocyte migration and trafficking is dynamically regulated by various chemokine and adhesion molecules and is vital to the proper function of the immune system. We describe a role for the Cas and Hef-1-associated signal transducer in hematopoietic cells (Chat-H) as a critical regulator of T lymphocyte migration, by using lentivirus-mediated RNA interference (RNAi). Impaired migration of Chat-H-depleted cells coincided with defective inside-out signaling shown by diminished chemokine-induced activation of the Rap-1 GTPase and integrin-mediated adhesion. Localization of Chat-H to the plasma membrane, association with its binding partner Crk-associated substrate in lymphocytes (CasL), and Chat-H-mediated CasL serine-threonine phosphorylation were required for T cell migration. These results identify Chat-H as a critical signaling intermediate acting upstream of Rap1 to regulate chemokine-induced adhesion and migration.

Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis.

Extravasation of leukocytes is a crucial process in the immunological defense. In response to a local concentration of chemokines, circulating leukocytes adhere to and migrate across the vascular endothelium toward the inflamed tissue. The small guanosinetriphosphatase Rap1 plays an important role in the regulation of leukocyte adhesion, polarization, and chemotaxis. We investigated the role of a guanine nucleotide exchange protein for Rap1 directly activated by cAMP (Epac1) in adhesion and chemotaxis in a promonocytic cell line and in primary monocytes. We found that Epac1 is expressed in primary leukocytes, platelets, CD34-positive hematopoietic cells, and the leukemic cell lines U937 and HL60. Epac activation with an Epac-specific cAMP analog induced Rap1 activation, beta1-integrin-dependent cell adhesion, and cell polarization. In addition, activated Epac1 enhanced chemotaxis of U937 cells and primary monocytes. Similar to activation of Epac1, stimulation of cells with serotonin to induce cAMP production resulted in Rap1 activation, increased cell adhesion and polarization, and enhanced chemotaxis. The effects of serotonin on U937 cell adhesion were dependent on cAMP production but could not be blocked by a protein kinase A inhibitor, implicating Epac in the regulation of serotonin-induced adhesion. In summary, our work reveals the existence of previously unrecognized cAMP-dependent signaling in leukocytes regulating cell adhesion and chemotaxis through the activation of Epac1.

Semaphorin-3A is expressed by tumor cells and alters T-cell signal transduction and function.

An important aspect of tumor progression is the ability of cancer cells to escape detection and clearance by the immune system. Recent studies suggest that several tumors express soluble factors interfering with the immune response. Here, we show that semaphorin-3A (Sema-3A), a secreted member of the semaphorin family involved in axonal guidance, organogenesis, and angiogenesis, is highly expressed in several tumor cells. Conditioned media of Sema-3A-transfected COS-7 cells or human recombinant Sema-3A inhibited primary human T-cell proliferation and cytokines production under anti-CD3 plus anti-CD28 stimulating conditions. Sema-3A also inhibited the activation of nonspecific cytotoxic activity in mixed lymphocyte culture (MLC), as measured against K-562 cells. In contrast, suppression of Sema-3A in tumor cells with a small interfering RNA (siRNA) augmented T-cell activation. The inhibitory effect of Sema-3A in T cells is mediated by blockade of Ras/mitogen-activated protein kinase (MAPK) signaling pathway. The presence of Sema-3A increased the activation of the Ras family small GTPase Rap1 and introduction of the dominant-negative mutant of Rap1 (Rap1N17) blunted the immunoinhibitory effects of Sema-3A. These results suggest that Sema-3A inhibits primary T-cell activation and imply that it can contribute to the T-cell dysfunction in the tumor microenvironment.

Protease inhibitors prevent the protein kinase A-dependent loss of Rap1 GTPase from the particulate fraction of COS1 cells.

Immunoblotting with a monoclonal Rap1 antibody, we found that elevation of cyclic AMP, with forskolin and IBMX or CPT-cAMP, led to a rapid reduction in the levels of Rap1 protein associated with particulate, nuclear/perinuclear fractions from PC12 and COS1 cells. In contrast, cytoplasmic levels of Rap1 remained constant following cyclic AMP stimulation. To gain independent confirmation that cyclic AMP promoted loss of Rap1 in nuclear/perinuclear fractions we used a polyclonal Rap1 antibody, which gave similar results to the monoclonal antibody. This demonstrated that the loss in Rap1 immunoreactivity was not due to phosphorylation-dependent changes that alter immunorecognition. The reduction in Rap1 levels was blocked by PKA inhibitors and by a Rap1 serine to alanine PKA-phosphorylation site mutant (S180A). Peptide inhibitors of the proteasome, cathespin, and calpain II also inhibited the decrease in Rap1 levels, indicating that proteolytic degradation may contribute to maintaining Rap1 levels in the nuclear/perinuclear fraction of cells.