Modulation of the immune response to Mycobacterium tuberculosis during malaria/M. tuberculosis co-infection.

Tuberculosis (TB) causes significant morbidity and mortality on a global scale. The African region has 24% of the world's TB cases. TB overlaps with other infectious diseases such as malaria and HIV, which are also highly prevalent in the African region. TB is a leading cause of death among HIV-positive patients and co-infection with HIV and TB has been described as a syndemic. In view of the overlapping epidemiology of these diseases, it is important to understand the dynamics of the immune response to TB in the context of co-infection. We investigated the cytokine response to purified protein derivative (PPD) in peripheral blood mononuclear cells from TB patients co-infected with HIV or malaria and compared it to that of malaria- and HIV-free TB patients. A total of 231 subjects were recruited for this study and classified into six groups; untreated TB-positive, TB positive subjects on TB drugs, TB- and HIV-positive, TB- and malaria-positive, latent TB and apparently healthy control subjects. Our results demonstrate maintenance of interferon (IFN)-γ production in HIV and malaria co-infected TB patients in spite of lower CD4 counts in the HIV-infected cohort. Malaria co-infection caused an increase in the production of the T helper type 2 (Th2)-associated cytokine interleukin (IL)-4 and the anti-inflammatory cytokine IL-10 in PPD-stimulated cultures. These results suggest that malaria co-infection diverts immune response against M. tuberculosis towards a Th-2/anti-inflammatory response which might have important consequences for disease progression.

The homeostatic properties of the mannose receptor in health and disease

El estudio de la especificidad y distribución anatómica del Receptor de la Manosa (MR) y la caracterización fenotípica de ratones deficientes en el MR han permitido discernir la función de este receptor en condiciones fisiológicas normales y patológicas. En esta revisión se considera la biología del MR en tres contextos diferentes: A nivel molecular como miembro de la familia de receptores de manosa; como receptor de macrófagos y células dendríticas; y como un marcador de vasos linfáticos. Resultados recientes en estas tres áreas resaltan las propiedades únicas de este receptor y la necesidad de mantenerla mente abierta a la hora de establecer su función fisiológica (AU)

Research into the binding properties and anatomical distribution of the Mannose Receptor (MR), and the phenotypic characterization of MR deficient animals have provided highly valuable information regarding the role of the MR in health and disease. In this review, the biology of MR is considered in three different contexts: At the molecular level as a member of the mannose receptor family of proteins; as a macrophage and dendritic cell receptor; and as marker of lymphatic endothelia. Novel observations in these three areas highlight the unique properties of MR and the need to keep an open mind when assessing its physiological role (AU)

Macrophage receptors and immune recognition.

Macrophages express a broad range of plasma membrane receptors that mediate their interactions with natural and altered-self components of the host as well as a range of microorganisms. Recognition is followed by surface changes, uptake, signaling, and altered gene expression, contributing to homeostasis, host defense, innate effector mechanisms, and the induction of acquired immunity. This review covers recent studies of selected families of structurally defined molecules, studies that have improved understanding of ligand discrimination in the absence of opsonins and differential responses by macrophages and related myeloid cells.

Endogenous ligands of carbohydrate recognition domains of the mannose receptor in murine macrophages, endothelial cells and secretory cells; potential relevance to inflammation and immunity.

The macrophage mannose receptor (MR) has an established role in the phagocytosis of a wide range of microbes, and also functions in viral endocytosis, and clearance of a number of endogenous glycoproteins from the circulation. Its broad ligand specificity is mediated by tandemly linked carbohydrate recognition domains (CRDs). Recent studies suggest that binding or internalization of both natural and synthetic ligands of MR CRDs may modulate macrophage (MPhi ) function, for example to increase cidal capacity or cytokine synthesis. To identify endogenous ligands in the normal mouse we used an Fc-fusion protein (CRD4-7Fc) bearing four of the CRDs of MR. CRD4-7Fc recognized endocytic compartments of cultured MPhi, consistent with lysosomal enzymes being major ligands of MR. CRD4-7Fc also recognized MPhi and some endothelial cells in tissues, and intensely labeled secretory cells of the exocrine pancreas, salivary gland and thyroid. Strongly MR-positive interstitial cells were found in close proximity to the ligand-rich secretory cells, suggesting a role for MR in uptake of secretory glycoproteins, including thyroglobulin which was identified as a novel ligand in vitro. Endocytosis of these ligands by MR may have implications for tissue homeostasis and immunity, including antigen presentation, in secretory organs.

Binding properties of the mannose receptor.

A comprehensive approach to the study of mannose receptor (MR) biology has unveiled an unexpected level of complexity and stresses the importance of post-translational modifications and gene regulation in the analysis of protein function. The existence of endogenous tissue ligands for the MR highlights the need to reduce MR expression in antigen presenting cells and/or to regulate T cell stimulation after presentation of MR ligands, in order to avoid autoimmunity. This regulation might be achieved by down modulation of the antigen presenting cell stimulatory capacity upon MR ligation. In macrophages there are conflicting evidence regarding the outcome of MR recognition. These results are not unexpected if endogenous mannosylated and sulphated self-antigens, that need to be shielded from the immune system, are being eliminated through this receptor. The presence of counter receptors for the cysteine rich (CR) domain of the MR in specialized myeloid cells in lymphoid organs adds a new dimension to this system. It opens the possibility for a delivery pathway for MR carbohydrate recognition domains (CRDs) ligands that needs to be investigated further.

Mannose receptor and scavenger receptor: two macrophage pattern recognition receptors with diverse functions in tissue homeostasis and host defense.

In this report we have reviewed our recent data which suggest a new function for MR in antigen delivery in lymphoid organs, together with highlighting three recent discoveries from our laboratory concerning the role of SR-A in adhesion, phagocytosis of apoptotic cells and protection from endotoxic shock in mice. The diversity of functions mediated by each receptor demonstrates there is much yet to be discovered about how macrophages use their cell surface receptors to 'see' the external environment, and yet perform a wide range of strictly regulated functions.

Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway.

Antigens such as MUC1 coupled to oxidized mannan lead to rapid and efficient MHC class I presentation to CD8+ cells and a preferential T1 response; after reduction there is class II presentation and a T2 immune response. We now show that the selective advantage of the oxidized mannan-MUC1 is due to the presence of aldehydes and not Schiff bases, and that oxidized mannan-MUC1 binds to the mannose and not scavenger receptors and is internalized and presented by MHC class I molecules 1,000 times more efficiently than when reduced. After internalization there is rapid access to the class I pathway via endosomes but not lysosomes, proteasomal processing and transport to the endoplasmic reticulum, Golgi apparatus and cell surface. Aldehydes cause rapid entry into the class I pathway, and can therefore direct the subsequent immune response.

Macrophage lectins in host defence.

Macrophage lectins contribute to host defence by a variety of mechanisms. The best characterised, mannose receptor (MR) and complement receptor three (CR3), are both able to mediate phagocytosis of pathogenic microbes and induce intracellular killing mechanisms. The regulation of the effector functions induced via MR is complex, and may involve both host and microbial factors. Therefore, MR is likely to play a dynamic role in the response to infection; it may act as a classical pattern recognition receptor in phagocytosis, whereas other poorly characterised factors may make a more decisive contribution to its function in physiologic settings. In contrast, the lectin site of CR3 appears to lack host-derived ligands and may be a true pattern recognition receptor. Further studies are required to evaluate the roles of other macrophage lectins in recognition of and responses to microbes.

Cell-specific glycoforms of sialoadhesin and CD45 are counter-receptors for the cysteine-rich domain of the mannose receptor.

We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domain of the mannose receptor, binds to marginal zone metallophilic macrophages (Mo) and B cell areas in the spleen and to subcapsular sinus Mo in lymph nodes of naive mice (CR-Fc(+) cells). Several CR-Fc ligands were found in spleen and lymph node tissue lysates using ligand blots. In this paper we report the identification of two of these ligands as sialoadhesin (Sn), an Mo-specific membrane molecule, and the leukocyte common antigen, CD45. CR-Fc bound selectively to Sn purified from spleen and lymph nodes and to two low molecular weight isoforms of CD45 in a sugar-dependent manner. CR-Fc binding and non-binding forms of Sn, probably derived from CR-Fc(+) and CR-Fc(-) cells respectively, were selected from spleen lysates. Analysis of the glycan pool associated with the CR-Fc-binding form revealed the presence of charged structures resistant to sialidase, absent in the non-binding form, that could correspond to sulfated structures. These results confirm the identification of the CR region of the mannose receptor as a lectin. We also demonstrate that the same glycoprotein expressed in different cells of the same organ can display distinct sugar epitopes that determine its binding properties.

A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein.

Dendritic cells (DCs) are known to activate naive T cells to become effective helper cells. In addition, recent evidence suggests that DCs may influence naive B cells during the initial priming of antibody responses. In this study, using three-color confocal microscopy and three-dimensional immunohistograms, we have observed that in the first few days after a primary immunization, cells with dendritic morphology progressively localize within primary B cell follicles. These cells were identified by their ability to bind a fusion protein consisting of the terminal cysteine-rich portion of the mouse mannose receptor and the Fc portion of human immunoglobulin (Ig)G1 (CR-Fc). In situ, these CR-Fc binding cells express major histocompatibility complex class II, sialoadhesin, and CD11c and are negative for other markers identifying the myeloid DC lineage, such as (CD11b), macrophages (F4/80), follicular DCs (FDC-M2), B cells (B220), and T cells (CD4). Using CR-Fc binding capacity and flow cytometry, the cells were purified from the draining lymph nodes of mice 24 h after immunization. When injected into naive mice, these cells were able to prime T cells as well as induce production of antigen-specific IgM and IgG1. Furthermore, they produced significantly more of the lymphocyte chemoattractant, macrophage inflammatory protein (MIP)-1alpha, than isolated interdigitating cells. Taken together, these results provide evidence that a subset of DCs enters primary follicles, armed with the capacity to attract and provide antigenic stimulation for T and B lymphocytes.

Mannose receptor and its putative ligands in normal murine lymphoid and nonlymphoid organs: In situ expression of mannose receptor by selected macrophages, endothelial cells, perivascular microglia, and mesangial cells, but not dendritic cells.

The mannose receptor (MR) has established roles in macrophage (Mphi) phagocytosis of microorganisms and endocytic clearance of host-derived glycoproteins, and has recently been implicated in antigen capture by dendritic cells (DCs) in vitro. MR is the founder member of a family of homologous proteins, and its recognition properties differ according to its tissue of origin. Given this heterogeneity and our recent discovery of a soluble form of MR in mouse serum, we studied the sites of synthesis of MR mRNA and expression of MR protein in normal mouse tissues. We demonstrate that synthesis and expression occur at identical sites, and that mature Mphi and endothelium are heterogeneous with respect to MR expression, additionally describing MR on perivascular microglia and glomerular mesangial cells. However, MR was not detected on DCs in situ, or on marginal zone or subcapsular sinus Mphi, both of which have MR-like binding activities. We also compared expression of MR to the binding of a recombinant probe containing the cysteine-rich domain of MR. We show that MR and its putative ligand(s) are expressed at nonoverlapping sites within lymphoid organs, consistent with a transfer function for soluble MR. Therefore, in addition to endocytic and phagocytic roles, MR may play an important role in antigen recognition and transport within lymphoid organs.

Potential role of the mannose receptor in antigen transport.

Potential endogenous ligands for the cysteine rich domain of the murine mannose receptor (MR) have been detected in marginal zone metallophilic macrophages in spleen and subcapsular sinus macrophages in lymph nodes of naive mice by immunohistochemistry using a Fc chimeric protein. Additional labelling was observed in follicular dendritic cells and migratory dendritic cells in immunised animals. Based on this labelling pattern and the identification of a soluble form of the MR in macrophage-conditioned media and mouse serum, we propose a novel role for this receptor in antigen transport.

A functional soluble form of the murine mannose receptor is produced by macrophages in vitro and is present in mouse serum.

A soluble form of the mannose receptor (sMR) has been found in conditioned medium of primary macrophages in vitro and in mouse serum. sMR was released as a single species, had a smaller size than the cell-associated form, and accumulated in macrophage-conditioned medium, in a cytokine-regulated manner, to levels comparable with those found for cell-associated mannose receptor. Pulse-chase experiments showed that sMR production in culture occurred by constitutive cleavage of pre-existing full-length protein. A binding assay was developed to determine the sugar specificity of sMR and its ability to interact with pathogens and particulate antigens (i.e. Candida albicans and zymosan). Protease inhibitor studies suggested that sMR was produced by cleavage of an intact mannose receptor by a matrix metalloprotease or ADAM metalloprotease. A role for sMR in the immune response is proposed based on its binding properties, regulation by cytokines, and the previous discovery of putative ligands for the cysteine-rich domain of the mannose receptor in lymph nodes and spleen.

Characterization of the African swine fever virus structural protein p14.5: a DNA binding protein.

The gene encoding the structural protein p14.5 of African swine fever virus (ASFV) has been mapped and sequenced. This gene, designated E120R, is located in the Sa/l H/EcoRl E restriction fragment of the ASFV genome and is predicted to encode a protein of 120 amino acids with a molecular weight of 13.4 kDa. Northern-blot analysis showed that E120R is transcribed at late times during the viral replication cycle. The E120R gene product has been expressed in Escherichia coli, purified, and used as an antigen for antibody production. The antiserum anti-pE120R recognized a protein in infected cell extracts with an apparent molecular mass of 14.5 kDa, named p14.5. This antiserum also detected protein p14.5 in purified virus particles. Protein p14.5 is synthesized late in infection and is located in viral factories. Immunoprecipitation analysis and binding-assay experiments have shown that protein p14.5 interacts with a protein that could correspond to the major structural protein p72. Purified protein p14.5 interacts with DNA in a sequence-independent manner. It binds to both single-stranded and double-stranded DNA. A possible role of protein p14.5 in the encapsidation of ASFV DNA is suggested.

Fc chimeric protein containing the cysteine-rich domain of the murine mannose receptor binds to macrophages from splenic marginal zone and lymph node subcapsular sinus and to germinal centers.

Ligands for the cysteine-rich (CR) domain of the mannose receptor (MR) were detected by incubating murine tissues with a chimeric protein containing CR fused to the Fc region of human IgG1 (CR-Fc). In naive mice, CR-Fc bound to sialoadhesin+, F4/80low/-, macrosialin+ macrophages (M phi) in spleen marginal zone (metallophilic M phi) and lymph node subcapsular sinus. Labeling was also observed in B cell areas of splenic white pulp. Western blotting analysis of spleen and lymph nodes lysates revealed a restricted number of molecules that interacted specifically with CR-Fc. In immunized mice, labeling was upregulated on germinal centers in splenic white pulp and follicular areas of lymph nodes. Kinetic analysis of the pattern of CR-Fc labeling in lymph nodes during a secondary immune response to ovalbumin showed that CR ligand expression migrated towards B cell areas, associated with cells displaying distinctive dendritic morphology, and accumulated in developing germinal centers. These studies suggest that MR+ cells or MR-carbohydrate-containing antigen complexes could be directed towards areas where humoral immune responses take place, through the interaction of the MR CR domain with molecules expressed in specialized macrophage populations and antigen transporting cells.

Macrophage membrane molecules: markers of tissue differentiation and heterogeneity.

Monoclonal antibodies directed against murine macrophage differentiation antigens provide tools to characterize novel glycoproteins, their expression by different macrophage subpopulations in situ and regulation by cytokines and other agents in vitro. Studies are in progress to determine ligands for these molecules, and to establish possible functions. We summarize current knowledge of the following molecules: F4/80, a glycoprotein with homology to the G-protein linked transmembrane 7 hormone receptor family; macrosialin, a member of the lysosomal-associated membrane protein [lamp] family with a macrophage-specific mucin-like extracellular domain also present in its human homologue, CD68; sialoadhesin, a sialic acid binding lectin with multiple Ig superfamily domains; mannosyl receptor, a lectin-like molecule with multiple C-type lectin domains; type 3 complement receptor, a beta 2 integrin involved in cell migration and adhesion; scavenger receptor, a transmembrane homotrimer with a collagenous domain.

Mapping and investigation of the role in pathogenesis of the major unique secreted 35-kDa protein of rabbitpox virus.

Following infection, many secreted poxvirus proteins are able to modulate the host immune response through interactions with cytokines or components of the complement pathway. A comparison of the secreted protein profiles from cells infected with vaccinia Western Reserve (VV-WR), cowpox virus Brighton strain, or rabbitpox virus (RPV) showed an abundant 35-kDa protein present only in the supernatants from RPV-infected cells. The gene encoding this protein was identified and mapped by N-terminal sequencing of the protein. Examination of the predicted amino acid sequence showed it to be identical to the 35-kDa secreted protein of the Lister strain of vaccinia virus described by Patel et al. (1990, J. Gen. Virol. 71, 2013-2021). The counterpart of this gene in the commonly studied VV-WR strain is truncated and encodes a 7.5-kDa protein under control of the well-characterized p7.5 promoter. While nonessential for replication in cell culture, conservation of this gene in at least two orthopoxvirus strains suggested that this protein might play an important role in vivo. Following intranasal inoculation of Balb/c mice at several doses (10(3), 10(4), or 10(5) PFU), a mutant of RPV lacking a functional 35-kDa gene (RPV delta 35) appeared to induce an earlier onset and more severe illness at low, sublethal doses (10(3) PFU) than was observed with wild-type (wt) RPV. At higher doses (10(4) or 10(5) PFU), the behavior of wt RPV and RPV delta 35 became indistinguishable and the overall LD50 values were similar. Intradermal infection of rabbits simultaneously, at separate sites, with RPV and RPV delta 35 showed no gross or microscopic differences between either primary skin lesions or viremic extension of each virus into the lungs. Therefore, this abundant secreted protein does not appear to play a major role in the virulence of the virus.

The ps/hr gene (B5R open reading frame homolog) of rabbitpox virus controls pock color, is a component of extracellular enveloped virus, and is secreted into the medium.

Wild-type rabbitpox virus (RPV) produces red hemorrhagic pocks on the chorioallantoic membranes (CAMs) of embryonated chicken eggs. Like the crmA (SPI-2) gene of cowpox virus, disruption of the RPV ps/hr gene results in a mutant which produces white pocks on the CAMs. An examination of the properties of the RPV(ps/hr) mutant in cell culture also reveals a significantly reduced host range, defined as the inability to form plaques, compared with wild-type virus. One of several cell types on which RPV(ps/hr) mutants fail to produce plaques is chicken embryo fibroblasts, cells which have been traditionally used to propagate spontaneously arising white pock mutants isolated from CAMs. The inability of the RPV(ps/hr) mutant to form plaques in chicken embryo fibroblasts correlates with a failure of a low multiplicity of infection to spread to neighboring cells and to form extracellular enveloped virus (EEV), although the formation and yields of infectious intracellular naked virus appear relatively normal. The gene product of the ps/hr gene, initially synthesized as a 45-kDa glycoprotein, is found as a component of EEV, but not intracellular naked virus, and as a smaller, secreted soluble protein of 35 kDa. Production of the secreted 35-kDa protein was found to be independent of any viral morphogenesis, suggesting two distinct pathways for release of the ps/hr gene product from the cell, i.e., as a component of the EEV particle and as a separately secreted glycoprotein.