Dendritic cells: New roles for Cdc42 and Rac in antigen uptake?

Journal of immunology (Baltimore, Md. : 1950), 2005

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 signif...

The Journal of Immunology, 2001

To explore the function of Rho in DC, exoenzyme C3 from Clostridium botulinum was used as a specific inhibitor of Rho. Treatment of DC with C3 (DC/C3) resulted in profound morphological changes by losing dendrites and emerging of shrunk membrane processes that were in parallel with marked reduction of polymerized actin in the marginal area. Inactivation of Rho-associated coiled coil-containing kinase (p160ROCK) by a specific ROCK inhibitor Y-27632 also led to disappearance of dendrites of DC with retaining large membrane expansions. In scanning electron microscopy, untreated DCs interacted with CD4 ؉ T cells more efficiently than DC/C3. Conjugate formation assay showed that the number of DCs associated with CD4 ؉ T cells was 2-fold higher in untreated DCs than that of DC/C3. Alloantigen-presenting capacity of DC/C3 was significantly suppressed in a dose-dependent manner. Because C3 treatment did not affect the surface expression of HLA, costimulatory, and adhesion molecules of DC, we examined cytokine production of DC and naive CD4 ؉ T cells to further elucidate the inhibitory mechanism of MLR. Unexpectedly, DC/C3 increased IL-12 production after LPS stimulation. Naive CD4 ؉ T cells cocultured with DC/C3 produced the increased percentage of IFN-␥producing cells, whereas the percentage of IL-2-producing T cells was decreased. These results demonstrate that Rho GTPase in DC controls both characteristic shape and immunogenic capacity.

Current Biology, 2000

Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells. We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis. Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.

Immunity, 2009

A unique subpopulation of spleen dendritic cells (DCs) that express the CD8 surface marker efficiently present phagocytosed antigens to CD8+ T lymphocytes in a process called “crosspresentation,” which initiates cytotoxic immune responses. We now show that the small GTPase Rac2 plays a critical role in antigen crosspresentation selectively in this DC subpopulation. In CD8+ DCs, Rac2 determines the subcellular assembly of the NADPH oxidase complex (NOX2) to phagosomes, whereas in CD8− DCs, Rac1 mediates the assembly of NOX2 at the plasma membrane. In the absence of Rac2, the production of reactive oxygen species (ROS) in DC-phagosomes was abolished, the phagosomal pH dropped, and the efficiency of antigen crosspresentation was reduced. We conclude that the activity of Rac1 and 2 control crosspresentation in DC subpopulations through the regulation of phagosomal oxidation and pH.

Blood, 2005

To better understand the influence of cytoskeletal regulation on dendritic cell (DC) function in vivo, the Rho guanosine triphosphatase (GTPase) Rac1 was selectively inhibited in DCs in transgenic (Tg) mice. Although transgene expression did not interfere with the migratory capacities of DC in vivo, a decreased uptake of fluorescent probes was observed. Interestingly, the absence of full Rac1 function most strongly affected the development and function of CD8+ DCs. Apoptotic cell uptake was severely reduced in Tg mice, impairing subsequent DC-mediated cross-presentation and priming of bacteria-specific T-cell responses. These findings highlight a special role for Rac1 in the capacity of CD8+ DCs to endocytose apoptotic cells and prime T cells via cross-presentation.

Frontiers in immunology, 2017

Antigen processing for presentation by major histocompatibility complex class II (MHCII) molecules requires the latter to travel through the endocytic pathway together with its chaperons: the invariant chain (Ii) and DM. Nevertheless, the nature of the compartments where MHCII molecules travel to acquire peptides lacks definition regarding molecules involved in intracellular vesicular trafficking, such as Rab small GTPases. We aimed to define which Rab proteins are present during the intracellular transport of MHCII, DM, and Ii through the endocytic pathway on their route to the cell surface during dendritic cell (DC) maturation. We examined, by means of three-color confocal microscopy, the association of MHCII, DM, and Ii with Rab5, Rab7, Rab9, and Rab11 during the maturation of bone marrow-derived or spleen DC in response to LPS as an inflammatory stimulus. Prior to the stage of immature DC, MHCII migrated from diffuse small cytoplasmic vesicles, predominantly Rab5+Rab7- and Rab5+...

Haematologica, 2010

We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration.

Febs Letters, 2006

Critical changes occurring in Rac-1 molecule, a cytoskeleton organizing small GTPase associated with cell ruffling, have been analyzed in dendritic cells (DCs) derived from monocytes cultured with granulocyte-macrophage colony-stimulating factor and IFN-α or IL-4. Although with different kinetics, both agents induced activation of Rac-1 molecule and, more importantly, an upregulation of both protein expression and mRNA transcription. These findings strengthen

2004

To better understand the influence of cytoskeletal regulation on dendritic cell (DC) function in vivo, the Rho guanosine triphosphatase (GTPase) Rac1 was selectively inhibited in DCs in transgenic (Tg) mice. Although transgene expression did not interfere with the migratory capacities of DC in vivo, a decreased uptake of fluorescent probes was observed. Interestingly, the absence of full Rac1 function most strongly affected the development and function of CD8 ؉ DCs. Apoptotic cell uptake was severely reduced in Tg mice, impairing subsequent DC-mediated crosspresentation and priming of bacteriaspecific T-cell responses. These findings highlight a special role for Rac1 in the capacity of CD8 ؉ DCs to endocytose apoptotic cells and prime T cells via crosspresentation. (Blood. 2005

American Journal of Immunology, 2010

Problem statement: Dendritic Cells (DCs) integrate responses of innate and adaptive immune system via transmitting pro-inflammatory and regulatory signals to naïve cells within draining lymph nodes. DCs have high capacity for endocytosis, both Clathrin-Dependent (CDE) and Caveolin-Dependent (CvDE). Approach: We have recently shown that ATP-hydrolysis by the multifunctional transporter protein RLIP76 is the primary determinant of the rate of CDE. Activation of CDE is necessary for DCs to internalize and process exogenous antigens. Thus, in present studies we investigated the role of RLIP76 in antigen uptake, maturation and T cell priming capacity of monocyte-derived DCs. Results: Using flow cytometry, we demonstrate variable surface expression of RLIP76 on immature DCs generated from peripheral blood mononuclear cells from healthy individuals. Studies on the effect of anti-RLIP76 antibodies on endocytosis of DC-specific C-type Lectin Receptors (CLRs) (DC-SIGN and DEC-205) and on co-localization of these receptors with MHC-class II were conducted using confocal microscopy. RLIP76-specific antibodies suppressed the maturation of DCs and the expression of typical activation markers and co-stimulatory and adhesion molecules including CD83, HLA-DR, HLA-ABC, CD40, CD80 and CD38. They also inhibited the stimulating potency of DCs in allogeneic Mixed Leukocyte Reaction (allo-MLR). Conclusions: We conclude that RLIP76 is necessary for activation, membrane trafficking and functional maturation of DCs. Further exploration of the role of RLIP76 in DC biology is warranted and may provide promising means in DC-based immunotherapies.