title: Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dundee, DDI 5EH, UK. c.watts@dundee.ac.uk
authors.forename: M A
authors.initials: MA
authors.lastname: West
authors.forename: A R
authors.initials: AR
authors.lastname: Prescott
authors.forename: E L
authors.initials: EL
authors.lastname: Eskelinen
authors.forename: A J
authors.initials: AJ
authors.lastname: Ridley
authors.forename: C
authors.initials: C
authors.lastname: Watts
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D010873:Pinocytosis / Q000187:drug effects / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051567:Proto-Oncogene Proteins c-vav; D020764:cdc42 GTP-Binding Protein / Q000502:physiology; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018797:Cell Cycle Proteins; D008070:Lipopolysaccharides; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; C496619:Vav1 protein, mouse; C031815:toxB protein, Clostridium difficile; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins