Cross-Presentation of Soluble and Cell-Associated Antigen by Murine Hepatocytes Is Enhanced by Collectrin Expression.

Cross-presentation is a modular series of intracellular events dictating the internalization and subsequent MHC class I (MHC I) display of extracellular Ags. This process has been defined in dendritic cells and plays a fundamental role in the induction of CD8+ T cell immunity during viral, intracellular bacterial, and antitumor responses. Herein, acute viral infection of murine liver with adenovirus, a model for intrahepatic cross-presentation, confirms hepatocytes directly contribute to cross-presentation of Ags and priming the pool of naive CD8+ T cells within the liver microenvironment. Processing of soluble and cell-associated Ags into peptide displayed by MHC I is however defective in hepatocytes lacking collectrin, an intracellular chaperone protein that localizes within the endoplasmic reticulum-Golgi intermediate compartment. Loss of hepatic collectrin expression leads to the diminished cross-priming and expansion of cytolytic antiviral CD8+ T cells. This study demonstrates that collectrin positively regulates processing of engulfed Ags into MHC I:peptide complexes within hepatocytes. Collectrin-mediated cross-presentation supports intrahepatic adaptive antiviral immune responses and may lead to insights into the nature of how the liver acts as a primary site of CD8+ T cell activation.

Loss of collectrin, an angiotensin-converting enzyme 2 homolog, uncouples endothelial nitric oxide synthase and causes hypertension and vascular dysfunction.

BACKGROUND: Collectrin is an orphan member of the renin-angiotensin system and is a homolog of angiotensin-converting enzyme 2, sharing ≈50% sequence identity. Unlike angiotensin-converting enzyme 2, collectrin lacks any catalytic domain. Collectrin has been shown to function as a chaperone of amino acid transporters. In rodents, the renal expression of collectrin is increased after subtotal nephrectomy and during high-salt feeding, raising the question of whether collectrin has any direct role in blood pressure regulation. METHODS AND RESULTS: Using a susceptible genetic background, we demonstrate that deletion of collectrin results in hypertension, exaggerated salt sensitivity, and impaired pressure natriuresis. Collectrin knockout mice display impaired endothelium-dependent vasorelaxation that is associated with vascular remodeling, endothelial nitric oxide synthase uncoupling, decreased nitric oxide production, and increased superoxide generation. Treatment with Tempol, a superoxide scavenger, attenuates the augmented sodium sensitivity in collectrin knockout mice. We report for the first time that collectrin is expressed in endothelial cells. Furthermore, collectrin directly regulates l-arginine uptake and plasma membrane levels of CAT1 and y(+)LAT1 amino acid transporters in endothelial cells. Treatment with l-arginine modestly lowers blood pressure of collectrin knockout mice. CONCLUSIONS: Collectrin is a consequential link between the transport of l-arginine and endothelial nitric oxide synthase uncoupling in hypertension.

Imaging flow cytometry elucidates limitations of microparticle analysis by conventional flow cytometry.

Microparticles (MPs) are submicron vesicles released from cell membranes in response to activation, cell injury, or apoptosis. The clinical importance of MPs has become increasingly recognized, although no standardized method exists for their measurement. Flow cytometry (FCM) is the most commonly used technique, however, because of the small size of MPs, and the limitations of current FCM instrumentation, accurate identification is compromised by this methodology. We decided to investigate whether the use of FCM combined with imaging, such as is possible with the ImagestreamX imaging FC (ISX), would be a more sensitive approach to characterizing MPs. Combining FCM with imaging eliminates some of the limitations demonstrated by conventional FCM, whereas also providing morphological confirmation and the ability to distinguish true single events from aggregates and cell debris. The detection limit of standard nonspecialized FCM is suboptimal when compared to ISX. Evaluating MPs below 0.200 µm and sizing remain a challenge as some MPs remain below the detection limit of ISX. Standardized calibrators, that more closely reflect the physical characteristics of MPs, need further development.

Preexposure of murine macrophages to CpG-containing oligonucleotides results in nuclear factor kappaB p50 homodimer-associated hyporesponsiveness.

BACKGROUND: DNA containing the CpG motif is associated with immunomodulation of the innate immune response. Preexposure of macrophages to CpG DNA elicits a hyporesponsiveness to subsequent lipopolysaccharide (LPS) stimulation. We tested the hypothesis that this effect is due to decreased nuclear translocation of nuclear factor kappaB (NF-kappaB). METHODS: Murine macrophage-like RAW 264.7 cells were incubated with 1.5 microg/mL CpG-containing oligonucleotides (CpG ODN) for 0.5 to 9 hours followed by restimulation with 1 microg/mL LPS for 20 minutes. Some cells were cotransfected with an NF-kappaB sensitive luciferase reporter construct and a control beta-gal plasmid. Cytoplasmic and nuclear extracts were assayed for NF-kappaB by electrophoretic mobility shift assay and supershift assays, for NF-kappaB, IkappaB and phospho-IkappaB by Western blot, for luciferase activity, and for p38, c-Jun NH(2)-terminal kinase, and extracellular signal-related kinase activity assay. RESULTS: NF-kappaB functional activity was decreased as demonstrated by luciferase activity assay in the prolonged CpG ODN pretreatment groups. Unlike endotoxin tolerance, CpG ODN preexposure increased cytoplasmic phospho-IkappaB-alpha and did not abrogate mitogen-activated protein kinase activity. CONCLUSIONS: In macrophages, exposure to CpG DNA increases expression of the inhibitory p50 NF-kappaB homodimer and decreases NF-kappaB activity without inhibition of IkappaB kinases. Mitogen-activated protein kinase activity remains intact. Understanding these interactions between different toll receptor ligands may provide insight into novel therapeutic modalities.

Impact of immunomodulatory oligodeoxynucleotides on cytokine production in the lipopolysaccharide-stimulated human whole blood model.

BACKGROUND: In studying potential immunotherapeutics for sepsis, we used a lipopolysaccharide (LPS)-stimulated whole blood model to test the immunomodulating capacity of cytosine-phospho-guanine oligodeoxynucleotides (CpG ODNs). We hypothesized that CpG ODNs would have considerable counterinflammatory effects on LPS-induced cytokine production. METHODS: We administered 4 micromol/L of CpG ODNs (2216, D19, 2006, K3, or 1668) or guanine-phospho-cytosine (GpC) ODNs (control D or control K) immediately after LPS (10 ng/mL) stimulation of heparinized human whole blood. Samples were incubated for 1, 3, 6, 12, and 24 hours. Media and LPS were used as negative and positive controls. Cell-free supernatants were obtained and evaluated for interferon gamma (IFN-gamma), interleukin (IL)-12(p40), tumor necrosis factor alpha, IL-6, IL-10, IFN-alpha, IL-8, and IL-1beta by ELISA. RESULTS: Compared to LPS alone, significantly reduced levels of IFN-gamma, IL-12(p40), tumor necrosis factor alpha, and IL-6 were associated with both CpG and GpC ODNs administration to LPS-stimulated whole blood. IL-10 levels were concomitantly increased. However, IFN-alpha generation was CpG specific as was increased IL-8 levels. Lastly, only 2216 was associated with decreased IL-1beta levels. CONCLUSIONS: CpG ODNs and GpC ODNs in the LPS-stimulated whole blood model demonstrate differential counterinflammatory effects, but only CpG ODNs were associated with proinflammatory cytokine production. With further examination, we may find that these observed immunomodulatory differences could potentially be exploited for therapeutic benefit.

Primary human hepatocytes in spheroid formation to study hepatitis C infection.

BACKGROUND: Hepatitis C (HCV) is a worldwide health problem, affecting nearly 170 million people. Current models for studying Hepatitis C have focused primarily on the use of poorly permissive cell lines and viral constructs, because of the lack of a suitable animal model or an in vitro system for studying functional infection. As hepatocytes are the primary reservoir for the virus in vivo, we report on a model using primary human hepatocytes cultured in spheroid formation. MATERIALS AND METHODS: The hepatocytes were harvested from uninfected liver resections and cultured as spheroids (that promotes a differentiated phenotype) or monolayers. Spheroids expressed the putative receptors CD81 and human scavenger receptor B1 in a variable pattern throughout the culture period. Samples were inoculated with infectious HCV serum, and HCV RNA was detected using RT-PCR. RNA was detected in the cells and culture medium by 3 days and 5 days after inoculation, respectively. Selection of HVR1 variants occurred in a differential pattern based on culture technique, suggesting that viral selection was dependent on host phenotype. Detection of NS5A by Western blot analysis of infected samples and immunofluorescence for HCV core protein was seen only in infected spheroids. CONCLUSION: The use of spheroid formation to study Hepatitis C is associated with the establishment of HVR1 selection and functional infection. This represents a promising alternative model to study Hepatitis C.

HCV infection of the transplanted liver: changing CD81 and HVR1 variants immediately after liver transplantation.

The second envelope protein at hypervariable region 1 (HVR1) has been implicated in contributing to hepatitis C virus (HCV)-host cell interactions and CD81 (a multifunctional protein) has been demonstrated to act as a cell surface receptor for HCV and may interact directly with HVR1. The purpose of the current study was to determine if certain HVR1 quasispecies variants more effectively associate with and infect allografts after liver transplantation than other HVR1 variants and whether CD81 receptor expression changes after transplantation. Blood and allograft samples were obtained from the peritransplant period in seven patients. Clones of RT-PCR product were directly sequenced to identify HVR1 quasispecies variants. Explanted liver and serial allograft biopsies in recipients with HCV were examined by immunohistochemistry (IHC) for CD81 expression. Examination of HVR1 sequences demonstrated that only a fraction of the quasispecies variants recovered from each patient's blood sampled immediately prior to transplantation associated with and infected the allografts. Genetic diversity at HVR1 decreased with reperfusion but did not significantly decrease with infection. Expression of CD81 varied during the immediate post-transplant period. In conclusion, HVR1 quasispecies variants differentially associate with, and infect allografts, after liver transplantation. Additionally, allografts express variable amounts of CD81 after transplantation.

E2 quasispecies specificity of hepatitis C virus association with allografts immediately after liver transplantation.

It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus-liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end-stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase-polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants.