Small rho GTPases regulate antigen presentation in dendritic cells

Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity. However, the molecular mechanisms maintaining DC function remain to be elucidated. In this study, we report on the role of small Rho GTPases: Cdc42, Rac1, and RhoA in the regulation of DC adherence, Ag presentation, migration, chemotaxis, and endocytosis. Murine DC were transfected with vaccinia virus-based constructs, encoding dominant-negative or constitutively active (ca) mutant forms of Rho GTPases. We demonstrate that Cdc42 plays a major role in the regulation of DC adhesion, because caCdc42-transfected DC had significant up-regulation of adhesion to extracellular matrix, which was blocked by the Rho GTPase inhibitor toxin B (ToxB). In contrast, caRho-transfected DC only modestly elevated DC adhesion, and caRac had no effect. Additionally, caCdc42 and caRho increased the ability of DC to present OVA peptide to specific T cells. This effect was abrogated by ToxB. Activation of Cdc42 in DC significantly inhibited spontaneous and chemokine-induced DC migration. Furthermore, uptake of dextran 40 by DC was significantly enhanced by Rho GTPase activators cytotoxic necrotizing factor 1 and PMA, and reduced by ToxB. caCdc42 also increased endocytotic activity of DC, whereas dominant-negative Cdc42 blocked it. Thus, Rho GTPases Cdc42, RhoA, and Rac1 regulate DC functions that are critical for DC-mediated immune responses in vivo.     

Regulation of endocytic traffic by rho family GTPases

Endocytosis is a complicated yet highly efficient process that involves the uptake and processing of cargoes, ranging from small molecules, to activated signalling receptors, to whole microorganisms. Regulation of endocytic pathways is poorly understood. Recent evidence suggests that the Rho GTPase family of signalling proteins is intimately involved in endocytic traffic, providing novel insights into the control mechanisms that govern this process.     

Interferon regulatory factor 8 mediates tumor-induced inhibition of antigen processing and presentation by dendritic cells

Introduction  Suppression of dendritic cells (DCs) is a crucial mechanism by which tumor cells escape immune recognition and elimination. We have recently reported that MHC class I antigen processing machinery (APM) component expression in human DCs is down-regulated by tumor-derived gangliosides. However, the molecular mechanisms underlying this abnormality were not identified. Thus, the aim of this work was to analyze the role of interferon regulatory factor 8 (IRF-8) in APM protein expression and the antigen presenting capacity of DCs developed in the tumor microenvironment. Results  We demonstrate that the expression of several MHC class I APM components, including delta, MB-1, LMP-10, ERp57, and tapasin, is significantly decreased in murine DCs generated in the presence of prostate cancer cells. APM component down-regulation was associated with decreased ability of DCs to present model antigen to antigen-specific T cells. Notable, impaired antigen-presenting activity of DCs co-cultured with tumor cells was accompanied by decreased levels of IRF-8. Transduction of DCs with the silencing RNA for the IRF-8 gene also led to reduced expression of APM components in DCs and decreased antigen presenting function. Conclusion  Together, our data suggest that tumor-induced inhibition of antigen processing and presenting function of DCs is mediated by IRF-8, a member of the interferon regulatory factor family. These results provide a new molecular target for optimizing the generation of efficient DC vaccines for cancer therapy.     

Regulation of rho GTPases by crosstalk and neuronal activity in vivo

Proper development of neurons depends on synaptic activity, but the mechanisms of activity-dependent neuronal growth are not well understood. The small GTPases, RhoA, Rac, and Cdc42, regulate neuronal morphogenesis by controlling the assembly and stability of the actin cytoskeleton. We report an in situ method to determine endogenous Rho GTPase activity in intact Xenopus brain. We use this method to provide evidence for crosstalk between Rho GTPases in optic tectal cells. Moreover, crosstalk between the Rho GTPases appears to affect dendritic arbor development in vivo. Finally, we demonstrate that optic nerve stimulation regulates Rho GTPase activity in a glutamate receptor-dependent manner. These data suggest a link between glutamate receptor function, Rho GTPase activity, and dendritic arbor growth in the intact animal.     

Small rho GTPases and cholesterol biosynthetic pathway intermediates in African swine fever virus infection

The integrity of the cholesterol biosynthesis pathway is required for efficient African swine fever virus (ASFV) infection. Incorporation of prenyl groups into Rho GTPases plays a key role in several stages of ASFV infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. We found that Rho GTPase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions. Furthermore, abundant defective virions reached the plasma membrane, and filopodia formation in exocytosis was abrogated. Rac1 was activated at early ASFV infection stages, coincident with microtubule acetylation, a process that stabilizes microtubules for virus transport. Rac1 inhibition did not affect the viral entry step itself but impaired subsequent virus production. We found that specific Rac1 inhibition impaired viral induced microtubule acetylation and viral intracellular transport. These findings highlight that viral infection is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell; this modulation results critical for virus morphogenesis and in turn, triggers cytoskeleton remodeling, such as microtubule stabilization for viral transport during early infection.     

Opposite effects of rho family GTPases on engulfment of apoptotic cells by macrophages

The efficient engulfment of apoptotic cells by professional or nonprofessional phagocytes is critical to maintain mammalian homeostasis. To identify molecules involved in the engulfment of apoptotic cells, we established a retrovirus-based expression cloning system coupled with the engulfment assay. By screening a cDNA library of a mouse macrophage cell line, we identified two small GTPase family members (RhoG and Rab5) that enhanced the engulfment of apoptotic cells. By examining other small GTPase family members, we found that Rac1 enhanced the engulfment of apoptotic cells, whereas RhoA inhibited the process. Accordingly, the expression of a dominant-negative form of RhoG or Rac1 in primary macrophage cultures severely reduced the ability of the macrophages to engulf apoptotic cells, and a dominant-negative form of RhoA enhanced the process. These results indicated that the efficient engulfment of apoptotic cells requires the concerted action of small GTPase family members. We demonstrated previously that NIH3T3 cells expressing the alphav beta3 integrin efficiently engulf apoptotic cells in the presence of milk fat globule epidermal growth factor 8 via a phosphatidylserine-dependent mechanism. The dominant-negative form of RhoG or Rac1 inhibited this process, which suggested RhoG and Rac1 are also involved in the integrin-mediated engulfment.     

Prostanoids play a major role in the primary tumor-induced inhibition of dendritic cell differentiation

Production of immunosuppressive factors is one of the mechanisms by which tumors evade immunosurveillance. Soluble factors hampering dendritic cell (DC) development have recently been identified in culture supernatants derived from tumor cell lines. In this study, we investigated the presence of such factors in 24-h culture supernatants from freshly excised solid human tumors (colon, breast, renal cell carcinoma, and melanoma). While primary tumor-derived supernatant (TDSN) profoundly hampered the in vitro DC differentiation from CD14(+) plastic-adherent monocytes or CD34(+) precursors (based on morphology and CD1a/CD14 phenotype), the effects of tested tumor cell line-derived supernatants were minor. Cyclooxygenase (COX)-1- and COX-2-regulated prostanoids present in the primary TDSN were found to be solely responsible for the observed hampered differentiation of monocyte-derived DC (MoDC). In contrast, both prostanoids and IL-6 were found to contribute to the TDSN-induced inhibition of DC differentiation from CD34(+) precursor cells. While the addition of TDSN during differentiation interfered with the ability of CD34-derived DC to stimulate a primary allogeneic T cell response, it actually increased this ability of MoDC. These opposite effects were correlated to different effects of the TDSN on the expression levels of CD86 and HLA-DR on the DC from the different precursor origins. Although TDSN increased the T cell-stimulatory capacity of MoDC, TDSN inhibited the IL-12 production and increased the IL-10 production of MoDC, thus skewing them to a type-2 T cell-inducing phenotype. In conclusion, this study demonstrates that primary tumors negatively impact DC development and function through COX-1 and -2 regulated factors, whereas tumor-derived cell lines may lose this ability upon in vitro propagation.     

Microbial toxins and the glycosylation of rho family GTPases

Large clostridial cytotoxins act on cells by glycosylating low molecular mass GTPases using nucleotide-sugars as the sugar donor. These toxins are important virulence factors in human and animal diseases, but are also valuable cell biology tools. Recent findings shed some light on their mode of action and provide new insights into the structure/activity relationship of these bacterial toxins.     

Regulation of actin ring formation by rho GTPases in osteoclasts

Actin ring formation is a prerequisite for osteoclast bone resorption. Although gelsolin null osteoclasts failed to exhibit podosomes, actin ring was observed in these osteoclasts. Wiscott-Aldrich syndrome protein (WASP) was observed in the actin ring of gelsolin null osteoclast. Osteoclasts stimulated with osteopontin simulated the effects of Rho and Cdc42 in phosphatidylinositol 4,5-bisphosphate (PIP2) association with WASP as well as formation of podosomes, peripheral microfilopodia-like structures, and actin ring. To explore the potential functions of Rho and Cdc42, TAT-mediated delivery of Rho proteins into osteoclasts was performed. Although Rho and Cdc42 are required for actin ring formation, transduction of either one of the proteins alone is insufficient for this process. Addition of osteopontin to osteoclasts transduced with Cdc42Val12 or transduction of osteoclasts with both RhoVal14 and Cdc42Val12 augments the formation of WASP-Arp2/3 complex and actin ring. Neomycin, an antibiotic, blocked the effects of osteopontin or TAT-RhoVal14 on PIP2 interaction with WASP. WASP distribution was found to be cytosolic in these osteoclasts. Depletion of WASP by short interfering RNA-mediated gene silencing blocked actin polymerization as well as actin ring formation in osteoclasts. These results suggest that Rho-mediated PIP2 interaction with WASP may contribute to the activation and membrane targeting of WASP. Subsequent interaction of Cdc42 and Arp2/3 with WASP may enhance cortical actin polymerization in the process of actin ring formation in osteoclasts.     

Comparative analysis of dendritic cells transduced with different anti-apoptotic molecules: sensitivity to tumor-induced apoptosis

BACKGROUND: Tumors develop mechanisms to escape recognition by the immune system. It has recently been demonstrated that tumors cause apoptotic death of key immune cells, including the major antigen-presenting cells, dendritic cells (DC). Elimination of DC from the tumor environment significantly diminishes development of specific immunologic responses. We have recently demonstrated that tumor-induced DC apoptosis could be prevented by overexpression of the anti-apoptotic molecule Bcl-x(L). The aim of this study was to identify extrinsic and intrinsic tumor-induced apoptotic pathways in DC by targeting different anti-apoptotic molecules, including FLIP, XIAP/hILP, dominant-negative procaspase-9 and HSP70. METHODS: Murine bone marrow derived DC were transduced with adenoviral vectors carrying different anti-apoptotic molecules and co-incubated with tumor cells in a Transwell system. Apoptosis of DC was assessed by Annexin V and PI staining. RESULTS: We have demonstrated that adenoviral infection of DC with genes encoding different anti-apoptotic molecules exhibits different degrees of resistance to melanoma-induced apoptosis. Furthermore, we have shown that anti-apoptotic molecules other than the Bcl-2 family of proteins are able to protect DC and prevent tumor-induced apoptosis in DC. CONCLUSIONS: The results show that tumor-induced apoptosis of DC is not limited to the mitochondrial pathway of cell death and open additional possibilities for targeted molecular protection of DC longevity in cancer. Therefore, effective protection of DC from tumor-induced apoptosis may significantly improve the efficacy of DC-based therapies for cancer.     

Skin-derived dendritic cells can mediate deletional tolerance of class I-restricted self-reactive T cells

Skin-draining lymph nodes contain a number of dendritic cell (DC) subsets of different origins. Some of these are migratory, such as the skin-derived epidermal Langerhans cells and a separate dermal DC subset, whereas others are lymphoid resident in nature, such as the CD8+ DCs found throughout the lymphoid tissues. In this study, we examine the DC subset presentation of skin-derived self-Ag by migratory and lymphoid-resident DCs, both in the steady state and under conditions of local skin infection. We show that presentation of self-Ag is confined to skin-derived migrating DCs in both settings. Steady state presentation resulted in deletional T cell tolerance despite these DCs expressing a relatively mature phenotype as measured by traditional markers such as the level of MHC class II and CD86 expression. Thus, self-Ag can be carried to the draining lymph nodes by skin-derived DCs and there presented by these same cells for tolerization of the circulating T cell pool.     

Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments

Incubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes sensitive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: 1) in vitro endosomes were osmotically insensitive; and 2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1-10 min at 37 degrees C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate-conjugated dextran (FITC-dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC-dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10-min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose ("very early" endosomes) and the other not ("early" endosomes). After a brief uptake (1-3 min), postincubation of CHO cells in 0.25 M sucrose-containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.     

Presynaptic actions of endocannabinoids mediate alpha-MSH-induced inhibition of oxytocin cells

We recently showed that central injections of alpha-melanocyte-stimulating hormone (alpha-MSH) inhibits oxytocin cells and reduces peripheral release of oxytocin, but induces oxytocin release from dendrites. Dendritic oxytocin release can be triggered by agents that mobilize intracellular calcium. Oxytocin, like alpha-MSH, mobilizes intracellular calcium stores in oxytocin cells and triggers presynaptic inhibition of afferent inputs that is mediated by cannabinoids. We hypothesized that this mechanism might underlie the inhibitory effects of alpha-MSH. To test this, we recorded extracellularly from identified oxytocin and vasopressin cells in the anesthetized rat supraoptic nucleus (SON). Retrodialysis of a CB1 cannabinoid receptor antagonist to the SON blocked the inhibitory effects of intracerebroventricular injections of alpha-MSH on the spontaneous activity of oxytocin cells. We then monitored synaptically mediated responses of SON cells to stimulation of the organum vasculosum of the lamina terminalis (OVLT); this evoked a mixed response comprising an inhibitory component mediated by GABA and an excitatory component mediated by glutamate, as identified by the effects of bicuculline and 6-cyano-7-nitroquinoxaline-2,3-dione applied to the SON by retrodialysis. Application of CB1 receptor agonists to the SON attenuated the excitatory effects of OVLT stimulation in both oxytocin and vasopressin cells, whereas alpha-MSH attenuated the responses of oxytocin cells only. Thus alpha-MSH can act as a "switch"; it triggers oxytocin release centrally, but at the same time through initiating endocannabinoid production in oxytocin cells inhibits their electrical activity and hence, peripheral secretion.     

A novel type of myosin implicated in signalling by rho family GTPases.

A novel widely expressed type of myosin (fifth unconventional myosin from rat: myr 5) from rat tissues, defining a ninth class of myosins, was identified. The predicted amino acid sequence of myr 5 exhibits several features not found previously in myosins. The myosin head domain contains a unique N-terminal extension and an insertion of 120 amino acids at a postulated myosin-actin contact site. Nevertheless, myr 5 is able to bind actin filaments in an ATP-regulated manner. The head domain is followed by four putative light chain binding sites. The tail domain of myr 5 contains a region which coordinates two atoms of zinc followed by a region that stimulates GTP hydrolysis of members of the ras-related rho subfamily of small G-proteins. Myr 5 therefore provides the first direct link between rho GTPases which have been implicated in the regulation of actin organization and the actin cytoskeleton. It is also the first unconventional myosin for which a tail binding partner(s), namely members of the rho family, has been identified.     

Effects of d-allose on the endocytic activity of dendritic cells and the subsequent stimulation of T cells

We examined the effects of a rare sugar, d-allose, which is 6-carbon monosaccharide, on endocytosis and T cell stimulation by dendritic cells (DCs). The endocytosis of BCG-anti-BCG immune complexes by DCs markedly decreased in d-allose-containing medium. Co-culture with T cells (mixed leukocyte reaction, MLR) of DCs, which had been exposed to BCG in d-allose-supplemented medium, induced apoptosis of CD4⁺ T cells in a manner dependent on d-allose concentration. After the MLR, DCs cultured in the medium with d-allose expressed less CD40 and more Fas ligands than those cultured without d-allose. It was suggested that the functions of DCs, internalization, processing and the subsequent antigen presentation to T cells, are down-regulated via the action of d-allose.     

Tumor-infiltrating dendritic cells are potent antigen-presenting cells able to activate T cells and mediate tumor rejection

Dendritic cells (DC) are potent inducers of immune responses. DC have been shown to infiltrate tumors, but very little is known about the functional status of these naturally occurring tumor-infiltrating DC (TIDC). In this study, the status and function of TIDC from several types of mouse melanoma were investigated in detail. CD11c+/MHC II+ cells, consistent with a DC phenotype, were found in all of transplantable or spontaneous melanomas studied. These TIDC were predominantly myeloid (CD11c+/CD8alpha-/B220-) in nature with small numbers of plasmacytoid (CD11c+/B220+). TIDC had an intermediate maturation phenotype with some expression of costimulatory molecules and the capacity to take up particles. Upon culture overnight ex vivo, the TIDC markedly up-regulated the expression of costimulatory molecules and also increased IL-12 production. Importantly, such ex vivo-matured TIDC pulsed with OVA were able to migrate to lymph nodes, to activate naive OVA-specific CD4+ and CD8+ T cells, and to confer protection against a challenge with OVA-expressing tumor cells. In conclusion, melanomas are infiltrated by functional DC that can act as fully competent APC. These APC have the potential to be manipulated and may therefore represent a promising target for cancer immunotherapy.     

Preformed Ω-profile closure and kiss-and-run mediate endocytosis and diverse endocytic modes in neuroendocrine chromaffin cells

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