Christopher V. Carman

Essential role for the peroxiredoxin Prdx1 in erythrocyte antioxidant defence and tumour suppression.

Reactive oxygen species are involved in many cellular metabolic and signalling processes and are thought to have a role in disease, particularly in carcinogenesis and ageing. We have generated mice with targeted inactivation of Prdx1, a member of the peroxiredoxin family of antioxidant enzymes. Here we show that mice lacking Prdx1 are viable and fertile but have a shortened lifespan owing to the development beginning at about 9 months of severe haemolytic anaemia and several malignant cancers, both of which are also observed at increased frequency in heterozygotes. The haemolytic anaemia is characterized by an increase in erythrocyte reactive oxygen species, leading to protein oxidation, haemoglobin instability, Heinz body formation and decreased erythrocyte lifespan. The malignancies include lymphomas, sarcomas and carcinomas, and are frequently associated with loss of Prdx1 expression in heterozygotes, which suggests that this protein functions as a tumour suppressor. Prdx1-deficient fibroblasts show decreased proliferation and increased sensitivity to oxidative DNA damage, whereas Prdx1-null mice have abnormalities in numbers, phenotype and function of natural killer cells. Our results implicate Prdx1 as an important defence against oxidants in ageing mice.

A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them

The basic route and mechanisms for leukocyte migration across the endothelium remain poorly defined. We provide definitive evidence for transcellular (i.e., through individual endothelial cells) diapedesis in vitro and demonstrate that virtually all, both para- and transcellular, diapedesis occurs in the context of a novel “cuplike” transmigratory structure. This endothelial structure was comprised of highly intercellular adhesion molecule-1– and vascular cell adhesion molecule-1–enriched vertical microvilli-like projections that surrounded transmigrating leukocytes and drove redistribution of their integrins into linear tracks oriented parallel to the direction of diapedesis. Disruption of projections was highly correlated with inhibition of transmigration. These findings suggest a novel mechanism, the “transmigratory cup”, by which the endothelium provides directional guidance to leukocytes for extravasation.

Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target.

Cyclin D1 (CyD1) is a pivotal cell cycle–regulatory molecule and a well-studied therapeutic target for cancer. Although CyD1 is also strongly up-regulated at sites of inflammation, its exact roles in this context remain uncharacterized. To address this question, we developed a strategy for selectively silencing CyD1 in leukocytes in vivo. Targeted stabilized nanoparticles (tsNPs) were loaded with CyD1–small interfering RNA (siRNA). Antibodies to β7 integrin (β7 I) were then used to target specific leukocyte subsets involved in gut inflammation. Systemic application of β7 I-tsNPs silenced CyD1 in leukocytes and reversed experimentally induced colitis in mice by suppressing leukocyte proliferation and T helper cell 1 cytokine expression. This study reveals CyD1 to be a potential anti-inflammatory target, and suggests that the application of similar modes of targeting by siRNA may be feasible in other therapeutic settings.

SELECTIVE REGULATION OF GALPHA Q/11 BY AN RGS DOMAIN IN THE G PROTEIN-COUPLED RECEPTOR KINASE, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein α subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211–212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF4 −-dependent binding to G protein α subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF4 −. This revealed the specific ability of bovine brain Gαq/11 to bind to both GRK2 and GRK3 in an AlF4 −-dependent manner. In contrast, Gαs, Gαi, and Gα12/13 did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Gαq/11 could also bind to an N-terminal construct of GRK2, while no binding of Gαq/11, Gαs, Gαi, or Gα12/13 to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Gαq revealed significant binding of both GαqGDP/AlF4 − and Gαq(GTPγS), but not Gαq(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Gαq(R183C) but not wild type Gαq. In vitro analysis revealed that GRK2 possesses weak GAP activity toward Gαq that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Gαq-mediated activation of phospholipase C-β both in vitro and in cells, possibly through sequestration of activated Gαq. These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.

Regulation of T Cell Receptor Activation by Dynamic Membrane Binding of the CD3ɛ Cytoplasmic Tyrosine-Based Motif

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

G-protein-coupled receptors: turn-ons and turn-offs

Advances in the study of G-protein-coupled receptor regulation have provided novel insights into the role of G-protein-coupled receptor kinases and arrestins in this process. Of particular interest are recent studies that have dramatically expanded the known cellular functions of these molecules to include roles in receptor endocytosis and activation of MAP kinase signalling pathways.

Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1.

Silencing gene expression by RNAi is a powerful method for exploring gene function and validating drug targets and potentially for therapy. Lymphocytes and other primary blood cells are resistant to lipid-based transfection in vitro and are difficult to target in vivo. We show here that antibody-protamine fusion proteins targeting the human integrin lymphocyte function-associated antigen-1 (LFA-1) efficiently deliver siRNAs and specifically induce silencing in primary lymphocytes, monocytes, and dendritic cells. Moreover, a fusion protein constructed from an antibody that preferentially recognizes activation-dependent conformational changes in LFA-1 selectively targets activated leukocytes and can be used to suppress gene expression and cell proliferation only in activated lymphocytes. The siRNA-fusion protein complexes do not cause lymphocyte activation or induce IFN responses. K562 cells expressing latent WT or constitutively activated LFA-1 engrafted in the lungs of SCID mice are selectively targeted by intravenously injected fusion protein–siRNA complexes, demonstrating the potential in vivo applicability of LFA-1-directed siRNA delivery.

The receptor PD-1 controls follicular regulatory T cells in the lymph nodes and blood

CD4+CXCR5+Foxp3+ follicular regulatory T cells (TFR cells) inhibit humoral immunity mediated by CD4+CXCR5+Foxp3− follicular helper T cells (TFH cells). Although the inhibitory receptor PD-1 is expressed by both cell types, its role in the differentiation of TFR cells is unknown. Here we found that mice deficient in PD-1 and its ligand PD-L1 had a greater abundance of TFR cells in the lymph nodes and that those TFR cells had enhanced suppressive ability. We also found substantial populations of TFR cells in mouse blood and demonstrated that TFR cells in the blood homed to lymph nodes and potently inhibited TFH cells in vivo. TFR cells in the blood required signaling via the costimulatory receptors CD28 and ICOS but were inhibited by PD-1 and PD-L1. Our findings demonstrate mechanisms by which the PD-1 pathway regulates antibody production and help reconcile inconsistencies surrounding the role of this pathway in humoral immunity.

The primacy of affinity over clustering in regulation of adhesiveness of the integrin αLβ2

Dynamic regulation of integrin adhesiveness is required for immune cell–cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function–associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.

Endothelial Cells Proactively Form Microvilli-Like Membrane Projections upon Intercellular Adhesion Molecule 1 Engagement of Leukocyte LFA-1

Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants. ICAM-1 was rapidly relocalized to newly formed microvilli-like membrane projections in response to binding LFA-1 on leukocytes. These ICAM-1-enriched projections encircled the leukocytes extending up their sides and clustered LFA-1 underneath into linear tracks. Projections formed independently of VCAM-1/very late Ag 4 interactions, shear, and proactive contributions from the LFA-1-bearing cells. In the ICAM-1-bearing endothelial cells, projections were enriched in actin but not microtubules, required intracellular calcium, and intact microfilament and microtubule cytoskeletons and were independent of Rho/Rho kinase signaling. Disruption of these projections with cytochalasin D, colchicine, or BAPTA-AM had no affect on firm adhesion. These data show that in response to LFA-1 engagement the endothelium proactively forms an ICAM-1-enriched cup-like structure that surrounds adherent leukocytes but is not important for firm adhesion. This finding leaves open a possible role in leukocyte transendothelial migration, which would be consistent with the geometry and kinetics of formation of the cup-like structure.