Stable Expression and Characterization of an Optimized Mannose Receptor.

The mannose receptor (MR) is a macrophage surface receptor that recognizes pathogen associated molecular patterns (PAMPs) from a diverse array of bacterial, fungal and viral pathogens. Functional studies of the MR are hampered by the scarcity of human cell lines that express the receptor. Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties. Although several laboratories have reported transient and stable expression of MR from plasmids, preliminary data from our laboratory suggests that these plasmids produce a protein that lacks critical domains and is often not stable over time. In this current report we describe the generation and characterization of a novel human codon-optimized system for transient and stable MR expression. Rare codons and sequences that contribute to mRNA instability were modified to produce mRNA that is qualitatively and quantitatively improved. Confocal imaging of the transient and stably expressed optimized receptor demonstrates a distribution consistent with previous reports. To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells. We show that three molecules participate in the engagement and internalization of S. aureus. MR was found to colocalize with Toll-like receptor 2 (TLR2) and Rab5 following exposure to pHrodo-stained S. aureus, suggesting cooperation among the three molecules to engage and internalize the bacterial particle. This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.

The School for Science and Math at Vanderbilt: An Innovative Research-Based Program for High School Students.

The School for Science and Math at Vanderbilt (SSMV) is an innovative partnership program between a Research I private university and a large urban public school system. The SSMV was started in 2007 and currently has 101 students enrolled in the program, with a total of 60 students who have completed the 4-yr sequential program. Students attend the SSMV for one full day per week during the school year and 3-6 wk in the summers following their ninth- to 11th-grade years, with each grade of 26 students coming to the Vanderbilt campus on a separate day. The research-based curriculum focuses on guiding students through the process of learning to develop questions and hypotheses, designing projects and performing analyses, and communicating results of these projects. The SSMV program has elevated the learning outcomes of students as evidenced by increased achievement scores relative to a comparison group of students; has provided a rigorous research-based science, technology, engineering, and mathematics elective curriculum that culminates in a Summer research internship; has produced 27 Intel and Siemens semifinalists and regional finalists over the past 4 yr; and has supported the development of writing and communication skills resulting in regional and national oral presentations and publications in scientific journals.

Interaction of members of the heat shock protein-70 family with the macrophage mannose receptor.

The macrophage MR has been the subject of investigation for over 20 years, and several important physiological functions have been described. However, the molecular mechanisms that regulate MR signaling and trafficking during these processes still remain elusive. The focus of the current paper was to identify potential cellular MR-interacting proteins. An initial screen of binding proteins in MR-expressing cells was performed using coimmunoprecipitation, followed by identification of matching peptide sequences using proteomics and MS. The major class of binding proteins identified belonged to the heat shock family of proteins. The specific interaction of the MR with HSP70 family members was validated by Western blot analysis, ligand binding assays, and intracellular colocalization using confocal microscopy. Additional studies indicated that inhibition of the HSP BiP by treatment of cells with EGCG reduced BiP interaction with and surface expression of the MR. Studies of possible motifs within the cytoplasmic tail of the receptor suggested that a juxtamembrane dibasic sequence may contribute to the interaction with BiP. These findings suggest that the molecular association of the MR with HSP70 family members via the receptor cytoplasmic tail may contribute to MR trafficking in macrophages.

Characterization of functional mannose receptor in a continuous hybridoma cell line.

BACKGROUND: The mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor. RESULTS: In the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin. CONCLUSIONS: The 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions.

Pulmonary surfactant: an immunological perspective.

Pulmonary surfactant has two crucial roles in respiratory function; first, as a biophysical entity it reduces surface tension at the air water interface, facilitating gas exchange and alveolar stability during breathing, and, second, as an innate component of the lung's immune system it helps maintain sterility and balance immune reactions in the distal airways. Pulmonary surfactant consists of 90% lipids and 10% protein. There are four surfactant proteins named SP-A, SP-B, SP-C, and SP-D; their distinct interactions with surfactant phospholipids are necessary for the ultra-structural organization, stability, metabolism, and lowering of surface tension. In addition, SP-A and SP-D bind pathogens, inflict damage to microbial membranes, and regulate microbial phagocytosis and activation or deactivation of inflammatory responses by alveolar macrophages. SP-A and SP-D, also known as pulmonary collectins, mediate microbial phagocytosis via SP-A and SP-D receptors and the coordinated induction of other innate receptors. Several receptors (SP-R210, CD91/calreticulin, SIRPalpha, and toll-like receptors) mediate the immunological functions of SP-A and SP-D. However, accumulating evidence indicate that SP-B and SP-C and one or more lipid constituents of surfactant share similar immuno-regulatory properties as SP-A and SP-D. The present review discusses current knowledge on the interaction of surfactant with lung innate host defense.

Mitogen-activated protein kinases and NFkappaB are involved in SP-A-enhanced responses of macrophages to mycobacteria.

BACKGROUND: Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFkappaB in mediating the enhanced signaling in response to BCG in the presence of SP-A. METHODS: Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 microg of SP-A to 5 x 105 BCG for 30 min at 37 degrees C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined. RESULTS: Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFkappaB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IkappaB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively. CONCLUSION: These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFkappaB.

Virus glycosylation: role in virulence and immune interactions.

The study of N-linked glycosylation as it relates to virus biology has become an area of intense interest in recent years due to its ability to impart various advantages to virus survival and virulence. HIV and influenza, two clear threats to human health, have been shown to rely on expression of specific oligosaccharides to evade detection by the host immune system. Additionally, other viruses such as Hendra, SARS-CoV, influenza, hepatitis and West Nile rely on N-linked glycosylation for crucial functions such as entry into host cells, proteolytic processing and protein trafficking. This review focuses on recent findings on the importance of glycosylation to viral virulence and immune evasion for several prominent human pathogens.

The Virtual Scientist: connecting university scientists to the K-12 classroom through videoconferencing.

The Vanderbilt University Center for Science Outreach (CSO) connects university scientists to the K-12 community to enhance and improve science education. The Virtual Scientist program utilizes interactive videoconference (IVC) to facilitate this connection, providing 40-50 sessions per academic year to a national audience. Scientists, defined as research faculty members, clinicians, postdoctoral fellows, graduate and medical students, and professional staff, participate through conventional volunteer recruitment and program announcements as well as outreach partnership efforts with other Vanderbilt centers. These experts present 30- to 45-min long, grade-appropriate content sessions from the CSO IVC studio or their own laboratory. Teachers register for sessions via an on-line application process. After the session, teachers, students, and experts are requested to complete an anonymous on-line evaluation that addresses both technical- and content-associated issues. Results from 2003 to the present indicated a favorable assessment for a promising program. Results showed that 69% of students (n = 335) and 88% of teachers (n = 111) felt that IVC improved access to scientists, whereas 97% of students (n = 382) and teachers (n = 126) and 100% of scientists (n = 23) indicated that they would participate in future videoconferences. Students and teachers considered that the Virtual Scientist program was effective [76% (n = 381) and 89% (n = 127), respectively]. In addition, experts supported IVC as effective in teaching [87% (n = 23)]. Because of the favorable responses from experts, teachers, and students, the CSO will continue to implement IVC as a tool to foster interactions of scientists with K-12 classrooms.

HIV-1 Tat interaction with cyclin T1 represses mannose receptor and the bone morphogenetic protein receptor-2 transcription.

Macrophage transcription is significantly altered by HIV-1 infection. HIV Tat, an immediate-early product of the viral lifecycle, interacts with host transcription factors to alter host gene expression. We have previously shown that Tat represses transcription from the mannose receptor (MR) and the bone morphogenetic protein receptor-2 (BMPR2) promoters. The current study shows that transcriptional repression of these receptors involves Tat interaction with cyclin T1. Assays using U937 human monocytic cells transiently expressing MR or BMPR2 promoter-luciferase constructs demonstrated equal repression by one- and two-exon Tat gene products. A mutant Tat expression vector encoding Tat protein lacking the cyclin T1 binding domain failed to inhibit MR and BMPR2 promoter activities. Over-expression of cyclin T1 in the presence of wild-type Tat resulted in recovered activity from both promoters. Finally, two inhibitors of cyclin-dependent kinase 9 (a dominant negative CDK9 and flavopiridol) repressed activity from the MR and BMPR2 promoters.

Novel collectin/C1q receptor mediates mast cell activation and innate immunity.

Mast cells play a critical role in innate immunity, allergy, and autoimmune diseases. The receptor/ligand interactions that mediate mast cell activation are poorly defined. The alpha2beta1 integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1 (MMP-1), endorepellin, and several viruses, has been implicated in normal developmental, inflammatory, and oncogenic processes. We recently reported that alpha2 integrin subunit-deficient mice exhibited markedly diminished neutrophil and IL-6 responses during Listeria monocytogenes- and zymosan-induced peritonitis. Peritoneal mast cells require alpha2beta1 integrin expression for activation in response to pathogens, yet the ligand and molecular mechanisms by which the alpha2beta1 integrin induces activation and cytokine secretion remain unknown. We now report that the alpha2beta1 integrin is a novel receptor for multiple collectins and the C1q complement protein. We demonstrate that the alpha2beta1 integrin provides a costimulatory function required for mast cell activation and cytokine secretion. This finding suggests that the alpha2beta1 integrin is not only important for innate immunity but may serve as a critical target for the regulation of autoimmune/allergic disorders.

HIV-1 TAT represses transcription of the bone morphogenic protein receptor-2 in U937 monocytic cells.

The bone morphogenetic protein receptor-2 (BMPR2) is a member of the transforming growth factor-beta receptor family and is expressed on the surface of several cell types including endothelial cells and macrophages. Recently, a cause for familial primary pulmonary hypertension (FPPH) has been identified as mutations in the gene encoding BMPR2. Three forms of pulmonary hypertension (PH) exist, including PPH, FPPH, and PH secondary to other etiologies (sporadic PH) such as drug abuse and human immunodeficiency virus (HIV) infection. It is interesting that these subtypes are histologically indistinguishable. The macrophage is a key target cell for HIV-1, significantly altering macrophage cell function upon infection. HIV-1 trans-activator of transcription (Tat), an immediate-early product of the HIV-1 lifecycle, plays an important role in mediating HIV-induced modulation of host cell function. Our laboratory has previously shown that Tat represses mannose receptor transcription in macrophages. In the current study, we examined activity from the BMPR2 promoter in the macrophage cell line U937 and potential regulation by Tat. Transfection of U937 cells with BMPR2 promoter-reporter constructs revealed dose-dependent repression of BMPR2 promoter activity in the presence of Tat. Experiments using truncations of the BMPR2 promoter localized Tat-mediated repression to the first 208 bases of the promoter. Decreased BMPR2 transcription resulted in altered downstream signaling. Similar to mothers against decapentaplegics (SMAD) phosphorylation and SMAD6 expression, in response to BMP2 treatment, were down-regulated after Tat treatment. Finally, HIV-1 infection and treatment with Tat protein of the U937 human monocytic cell line resulted in a decreased, endogenous BMPR2 transcript copy number.

HIV-1 Nef mediates post-translational down-regulation and redistribution of the mannose receptor.

Human immunodeficiency virus (HIV) has derived a variety of means to evade the host immune response. HIV-derived proteins, including Tat, Nef, and Env, have all been reported to decrease expression of host molecules such as CD4 and major histocompatibility complex I, which would assist in limiting viral replication. The mannose receptor (MR) on the surface of macrophages and dendritic cells (DC) has been proposed to function as an effective antigen-capture molecule, as well as a receptor for entering pathogens such as Mycobacterium tuberculosis and Pneumocystis carinii. Regulation of this receptor would therefore benefit HIV in removing an additional arm of the innate immune system. Previous work has shown that MR function is reduced in alveolar macrophages from HIV-infected patients and that surface MR levels are decreased by the HIV-derived protein Nef in DC. In addition, several laboratories have shown that CD4 is removed from the surface of T cells in a manner that might be applicable to decreased MR surface expression in macrophages. In the current study, we have investigated the role of Nef in removing MR from the cell surface. We have used a human macrophage cell line stably expressing the MR as well as human epithelial cells transiently expressing CD4 and a unique CD4/MR chimeric molecule constructed from the extracellular and transmembrane domains of CD4 and the cytoplasmic tail portion of the MR. We show that the MR is reduced on the cell surface by approximately 50% in the presence of Nef and that the MR cytoplasmic tail can confer susceptibility to Nef in the CD4/MR chimera. These data suggest that the MR is a potential intracellular target of Nef and that this regulation may represent a mechanism to further cripple the host innate immune system.

IL-4 modulates transcriptional control of the mannose receptor in mouse FSDC dendritic cells.

The mannose receptor is a 175 kDa protein found on the surface of macrophages and dendritic cells whose functions include clearance of extracellular hydrolases, internalization of pathogens, and antigen capture. Receptor expression is closely linked to the functional state of these cells and is regulated by cytokines. Previous work has shown that treatment of macrophages and dendritic cells with interleukin-4 leads to increased mannose receptor expression. We have examined the mechanism of this IL-4-mediated up-regulation in the murine dendritic cell line FSDC. IL-4 increased mannose receptor activity, protein, and mRNA. The mannose receptor promoter was functional in FSDCs using transient transfection assays, and IL-4 treatment increased promoter activity 2.6-fold. The responsive region was localized to the proximal 228 bp. Electrophoretic mobility shift assays detected an IL-4-inducible protein that bound to the mannose receptor promoter at a site spanning the region between -147 and -108 bp. The sequence TTAC(N)4CACC (-135 and -124 bp) is similar to the IL-4 response region in the Fc receptor II. Mutation of the flanking TT and CC in this motif blocked IL-4 responsiveness and binding of the IL-4-induced mannose receptor binding protein. This protein does not appear to be STAT6 since neither an anti-STAT6 antibody nor a STAT6 consensus oligonucleotide altered factor binding.

The recycling of apolipoprotein E and its amino-terminal 22 kDa fragment: evidence for multiple redundant pathways.

A portion of apolipoprotein E (apoE) internalized by hepatocytes is spared degradation and is recycled. To investigate the intracellular routing of recycling apoE, primary hepatocyte cultures from LDL receptor-deficient mice and mice deficient in receptor-associated protein [a model of depressed expression of LDL receptor-related protein (LRP)] were incubated with human VLDL containing 125I-labeled human recombinant apoE3. Approximately 30% of the internalized intact apoE was recycled after 4 h. The N-terminal 22 kDa fragment of apoE was also resecreted, demonstrating that this apoE domain contains sufficient sequence to recycle. The 22 kDa fragment has reduced affinity for lipoproteins, suggesting that apoE recycling is linked to the ability of apoE to bind directly to a recycling receptor. Finally, apoE was found to recycle equally well in the presence of brefeldin A, a drug that blocks transport from the endoplasmic reticulum and leads to collapse of the Golgi stacks. Our studies demonstrate that apoE recycling occurs 1) in the absence of the LDL receptor or under conditions of markedly reduced LRP expression; 2) when apoE lacks the carboxyl-terminal domain, which allows binding to the lipoprotein; and 3) in the absence of an intact Golgi apparatus. We conclude that apoE recycling occurs through multiple redundant pathways.

Surfactant protein A enhances Mycobacterium avium ingestion but not killing by rat macrophages.

Mycobacterium avium complex (MAC) is a significant cause of opportunistic infection in patients with acquired immunodeficiency syndrome. Although the major route of entry of MAC is via the gastrointestinal tract, MAC can infect humans through the respiratory tract and eventually encounter alveolar macrophages within the lung. Once in the lung, MAC can potentially interact with surfactant protein A (SP-A), an important component of the pulmonary innate-immune response. Previous work on other pulmonary pathogens including Mycobacterium bovis Bacillus Calmette-Guerin (BCG) suggests that SP-A participates in promoting efficient clearance of these organisms by alveolar macrophages. In the present study, we investigated the role of SP-A in clearance of MAC by cultured rat macrophages. SP-A bound to MAC organisms and enhanced the ingestion of the mycobacteria by macrophages. Infection of macrophages with SP-A-MAC complexes induced the production of nitric oxide (NO) and tumor necrosis factor-alpha. However, intracellular survival of MAC was not altered by preopsonization with SP-A. In addition, inhibitors of inducible NO synthase did not alter MAC clearance. These results suggest that SP-A can bind to and enhance the uptake of MAC by alveolar macrophages, similar to previous findings with BCG and Mycobacterium tuberculosis.However, unlike BCG and other pulmonary pathogens that are cleared effectively in the presence of SP-A via a NO-dependent pathway, macrophage-mediated clearance of MAC is not enhanced by SP-A.