Fiona E. McCann

Identification of an Intestinal Heme Transporter

Dietary heme iron is an important nutritional source of iron in carnivores and omnivores that is more readily absorbed than non-heme iron derived from vegetables and grain. Most heme is absorbed in the proximal intestine, with absorptive capacity decreasing distally. We utilized a subtractive hybridization approach to isolate a heme transporter from duodenum by taking advantage of the intestinal gradient for heme absorption. Here we show a membrane protein named HCP 1 (heme carrier protein 1), with homology to bacterial metal-tetracycline transporters, mediates heme uptake by cells in a temperature-dependent and saturable manner. HCP 1 mRNA was highly expressed in duodenum and regulated by hypoxia. HCP 1 protein was iron regulated and localized to the brush-border membrane of duodenal enterocytes in iron deficiency. Our data indicate that HCP 1 is the long-sought intestinal heme transporter.

Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells.

IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55−/− but not p75−/− mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55−/− mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.

Protocol for the induction of arthritis in C57BL/6 mice

Collagen-induced arthritis is a well-validated, but strain-dependent mouse model of rheumatoid arthritis, with H-2q and H-2r strains showing the greatest degree of susceptibility. This protocol describes the induction of arthritis in the C57BL/6 strain (H-2b), which forms the genetic background of the majority of genetically modified strains. This protocol involves purification of type II collagen from chicken sternums, immunization of mice, clinical assessment of arthritis and analysis of T- and B-cell responses to type II collagen. Key aspects of the protocol are the need to use chicken collagen for immunization and the importance of avoiding aggressive behavior in males. The incidence of arthritis varies from 50 to 80% and is milder than the classical collagen-induced arthritis model. This procedure takes ∼3 months to complete.

Apremilast, a novel PDE4 inhibitor, inhibits spontaneous production of tumour necrosis factor-alpha from human rheumatoid synovial cells and ameliorates experimental arthritis.

IntroductionType 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis.MethodsCells liberated from tissue excised from arthritic joints of RA patients were cultured in the presence of increasing concentrations of apremilast for 48 hours and spontaneous tumour necrosis factor-alpha (TNFα) production was analysed in culture supernatants by ELISA. In addition, arthritis was induced in BALB/c and DBA/1 mice by passive transfer of anti-type II collagen mAb and immunisation with type II collagen, respectively. Mice with established arthritis received 5 or 25 mg/kg apremilast and disease severity was monitored relative to mice receiving vehicle alone. At the end of the study, paws were removed and processed for histopathological assessment. Behavioural effects of apremilast, relative to rolipram, were assessed in naïve DBA/1 mice using an automated activity monitor (LABORAS).ResultsApremilast dose dependently inhibited spontaneous release of TNFα from human rheumatoid synovial membrane cultures. Furthermore, apremilast significantly reduced clinical score in both murine models of arthritis over a ten day treatment period and maintained a healthy joint architecture in a dose-dependent manner. Importantly, unlike rolipram, apremilast demonstrated no adverse behavioural effects in naïve mice.ConclusionsApremilast is an orally available PDE4 inhibitor that reduces TNFα production from human synovial cells and significantly suppresses experimental arthritis. Apremilast appears to be a potential new agent for the treatment of rheumatoid arthritis.

Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D.

Cell surface proteins major histocompatibility complex (MHC) class I–related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein–tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.

The size of the synaptic cleft and distinct distributions of filamentous actin, ezrin, CD43, and CD45 at activating and inhibitory human NK cell immune synapses.

In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains.

ADAP–SLP-76 Binding Differentially Regulates Supramolecular Activation Cluster (SMAC) Formation Relative to T Cell–APC Conjugation

T cell–APC conjugation as mediated by leukocyte function-associated antigen-1 (LFA-1)–intercellular adhesion molecule (ICAM)-1 binding is followed by formation of the supramolecular activation cluster (SMAC) at the immunological synapse. The intracellular processes that regulate SMAC formation and its influence on T cell function are important questions to be addressed. Here, using a mutational approach, we demonstrate that binding of adaptor adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation relative to conjugation. Although mutation of the YDDV sites (termed M12) disrupted SLP-76 SH2 domain binding and prevented the ability of ADAP to increase conjugation and LFA-1 clustering, M12 acted selectively as a dominant negative (DN) inhibitor of pSMAC formation, an effect that was paralleled by a DN effect on interleukin-2 production. ADAP also colocalized with LFA-1 at the immunological synapse. Our findings identify ADAP–SLP-76 binding as a signaling event that differentially regulates SMAC formation, and support a role for SMAC formation in T cell cytokine production.

The Activating NKG2D Ligand MHC Class I-Related Chain A Transfers from Target Cells to NK Cells in a Manner That Allows Functional Consequences

Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.

Selective Tumor Necrosis Factor Receptor I Blockade Is Antiinflammatory and Reveals Immunoregulatory Role of Tumor Necrosis Factor Receptor II in Collagen‐Induced Arthritis

Tumor necrosis factor (TNF) signals via 2 receptors, TNFR type I (TNFRI) and TNFRII, with distinct cellular distribution and signaling functions. In rheumatoid arthritis (RA), the net effect of TNFR signaling favors inflammatory responses while inhibiting the activity of regulatory T cells. TNFRII signaling has been shown to promote Treg cell function. To assess the relative contributions of TNFRI and TNFRII signaling to inflammatory and regulatory responses in vivo, we compared the effect of TNF blockade, hence TNFRI/II, versus TNFRI alone in collagen‐induced arthritis (CIA) as a model of RA.

New developments in the management of psoriasis and psoriatic arthritis: a focus on apremilast

Psoriasis is a chronic inflammatory skin disease, most commonly resulting in the occurrence of red and silver scaly plaques. About 30% of psoriasis sufferers develop psoriatic arthritis (PsA), a disorder that presents with additional joint inflammation and other clinical features. At present, the most effective treatment for moderate and severe psoriasis and PsA are biologics such as antitumor necrosis factor alpha therapy. Biologics are costly and typically require repeated injections; hence, the development of novel, orally available, small molecular inhibitors that are less expensive to produce is highly desirable. The phosphodiesterase 4 inhibitor apremilast is a small molecular inhibitor that acts by increasing cyclic adenosine monophosphate levels, ultimately suppressing tumor necrosis alpha production. Apremilast has been tested in a number of psoriasis and PsA pilot and Phase II trials to evaluate its efficacy and safety. More recently, three larger double-blinded, and randomized multicenter studies demonstrate that apremilast is efficacious in the treatment of psoriasis and PsA, with significantly higher numbers of apremilast-treated patients achieving endpoints of a 75% reduction compared to baseline in Psoriasis Area and Severity Index (PASI-75) or American College of Rheumatology-20 scores, relative to placebo. This encouraging data, along with a tolerable incidence of mild to moderate adverse events, has led to the initiation of several large Phase III trials that aim to further validate apremilast as a treatment for psoriasis and PsA. Here, we provide an overview of the current treatments for psoriasis and PsA, and summarize the findings from multiple Phase II clinical trials where the effects of apremilast in the treatment of psoriasis and PsA patients have been investigated.