Biomedical subjects

Alberto Visintin

Publications and source records attributed to Alberto Visintin.

At least 19 recordsLinked to original sources

PubMed2006

MD-2 expression is not required for cell surface targeting of Toll-like receptor 4 (TLR4).

The cell surface receptor complex formed by TLR4 and myeloid differentiation 2 (MD-2) is engaged when cells are exposed to LPS. Recent studies suggested that surface localization of functional mouse TLR4 (mTLR4) depends on the simultaneous expression of MD-2. As we did not observe a similar requirement, we conducted a comparative study of human TLR4 and mTLR4 surface expression in immune cells derived from the MD-2 knockout mouse and LPS-responsive cell lines and in cells that ectopically express TLR4. Our results indicate that in the human and mouse models, neither TLR4 function nor TLR4 surface targeting requires MD-2 coexpression. Accordingly, we report on one human cell line, which constitutively expresses functional TLR4 on the cell surface in the absence of MD-2 expression.

Animals↗

PubMed2006

MD-2.

Toll-like receptors (TLRs) are a small family of type-I glycoproteins that bind to and are activated by conserved non-self molecular signatures carried by microorganisms. Toll-like receptor 4 is triggered by most lipopolysaccharides (LPS). LPS is a complex amphipathic saccharolipidic glycan derived from Gram-negative bacteria. Unique among TLRs, TLR4 activity and interaction with its natural ligand(s) strictly depends on the presence of the extracellular adaptor MD-2. MD-2 is a small secreted glycoprotein that binds with cytokine-like affinities to both the hydrophobic portion of LPS and to the extracellular domain of TLR4. The interaction between MD-2 and LPS induces a triggering event on TLR4, which involves the molecular rearrangement of the receptor complex and its homotypic aggregation. In silico analysis suggests that MD-2 and MD-1 are paralogs derived from a common predecessor at the level of early vertebrates. In this review, we summarize the current state of knowledge concerning MD-2.

Amino Acid Sequence↗

PubMed2006

Trypsin-sensitive modulation of intestinal epithelial MD-2 as mechanism of lipopolysaccharide tolerance.

Intestinal epithelial cells (IEC) are constantly exposed to both high concentrations of the bacterial ligand LPS and the serine protease trypsin. MD-2, which contains multiple trypsin cleavage sites, is an essential accessory glycoprotein required for LPS recognition and signaling through TLR4. The aim of this study was to characterize the expression and subcellular distribution of intestinal epithelial MD-2 and to delineate potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD). Although MD-2 protein expression was minimal in primary IEC of normal colonic or ileal mucosa, expression was significantly increased in IEC from patients with active IBD colitis, but not in ileal areas from patients with severe Crohn's disease. Endogenous MD-2 was predominantly retained in the calnexin-calreticulin cycle of the endoplasmic reticulum; only a small fraction was exported to the Golgi. MD-2 expression correlated inversely with trypsin activity. Biochemical evidence and in vitro experiments demonstrated that trypsin exposure resulted in extensive proteolysis of endogenous and soluble MD-2 protein, but not of TLR4 in IEC, and was associated with desensitization of IEC to LPS. In conclusion, the present study suggests that endoplasmic reticulum-associated MD-2 expression in IBD may be altered by ileal protease in inflammation, leading to impaired LPS recognition and hyporesponsiveness through MD-2 proteolysis in IEC, thus implying a physiologic mechanism that helps maintain LPS tolerance in the intestine.

Cell Line↗

PubMed2006

Meningococcal porin PorB binds to TLR2 and requires TLR1 for signaling.

TLR2 plays a key role in the initiation of the cellular innate immune responses by a wide range of bacterial products. TLRs signaling, including TLR2 and its coreceptors TLR1 and TLR6, is mediated by a number of specific ligands. Although many of the TLR-mediated cell signaling pathways have been elucidated in the past few years, the molecular mechanisms that lead to cell activation are still poorly understood. In this study, we investigate the interaction of PorB from Neisseria meningitidis with TLR2 and describe the direct binding of a bacterial protein to TLR2 for the first time. Using labeled PorB, we demonstrate its binding to TLR2 both in its soluble form in vitro, and when it is over-expressed on the surface of human embryonic kidney 293 cells. We also show that TLR2-mediated binding of PorB is directly related to cellular activation. In addition, using 293 cells expressing the chimeric TLR2/TLR1 and TLR2/TLR6 complexes, we report the selectivity of PorB binding to the TLR2/TLR1 heterodimer, which is required for initiating signaling in transfected 293 cells and in murine B cells. Together, these data provide new evidence that TLR2 recognizes PorB through direct binding, and that PorB-induced cell activation is mediated by a TLR2/TLR1 complex.

Cell Line↗

PubMed2005

Localization of TLR2 and MyD88 to Chlamydia trachomatis inclusions. Evidence for signaling by intracellular TLR2 during infection with an obligate intracellular pathogen.

Chlamydia trachomatis is an obligate intracellular gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis. We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2.

The detection of Gram-negative LPS depends upon the proper function of the TLR4-MD-2 receptor complex in immune cells. TLR4 is the signal transduction component of the LPS receptor, whereas MD-2 is the endotoxin-binding unit. MD-2 appears to activate TLR4 when bound to TLR4 and ligated by LPS. Only the monomeric form of MD-2 was found to bind LPS and only monomeric MD-2 interacts with TLR4. Monomeric MD-2 binds TLR4 with an apparent Kd of 12 nM; this binding avidity was unaltered in the presence of endotoxin. E5564, an LPS antagonist, appears to inhibit cellular activation by competitively preventing the binding of LPS to MD-2. Depletion of endogenous soluble MD-2 from human serum, with an immobilized TLR4 fusion protein, abrogated TLR4-mediated LPS responses. By determining the concentration of added-back MD-2 that restored normal LPS responsiveness, the concentration of MD-2 was estimated to be approximately 50 nM. Similarly, purified TLR4-Fc fusion protein, when added to the supernatants of TLR4-expressing cells in culture, inhibited the interaction of MD-2 with TLR4, thus preventing LPS stimulation. The ability to inhibit the effects of LPS as a result of the binding of TLR4-Fc or E5564 to MD-2 highlights MD-2 as the logical target for drug therapies designed to pharmacologically intervene against endotoxin-induced disease.

Cell Line↗

PubMed2005

Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor homolog RP105.

Activation of Toll-like receptor (TLR) signaling by microbial signatures is critical to the induction of immune responses. Such responses demand tight regulation. RP105 is a TLR homolog thought to be mostly B cell specific, lacking a signaling domain. We report here that RP105 expression was wide, directly mirroring that of TLR4 on antigen-presenting cells. Moreover, RP105 was a specific inhibitor of TLR4 signaling in HEK 293 cells, a function conferred by its extracellular domain. Notably, RP105 and its helper molecule, MD-1, interacted directly with the TLR4 signaling complex, inhibiting its ability to bind microbial ligand. Finally, RP105 regulated TLR4 signaling in dendritic cells as well as endotoxin responses in vivo. Thus, our results identify RP105 as a physiological negative regulator of TLR4 responses.

Animals↗

PubMed2005

The apoptotic response to pneumolysin is Toll-like receptor 4 dependent and protects against pneumococcal disease.

Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Inhibition of TLR-4/MD-2 signaling by RP105/MD-1.

Activation of Toll-like receptor (TLR) signaling by microbial and host molecular signatures is critical to the induction of immune responses. Such signaling is, perforce, kept under tight control. We recently discovered a novel endogenous inhibitor of TLR-4 - RP105. Initially identified as a B-cell-specific molecule with a role in B-cell proliferation in response to RP105 mAb and LPS, RP105 is a TLR-4 homologue. Further, like TLR-4 whose surface expression and signaling depends upon co-expression of the secreted protein MD-2, surface expression of RP105 is dependent upon co-expression of the MD2 homologue, MD-1. Unlike the TLRs, however, RP105 lacks a signaling domain, having the apparent structure of a TLR inhibitor. Further, RP105 is not B-cell-specific; its expression directly mirrors that of TLR-4 on dendritic cells and macrophages. These considerations suggested a role for RP105 as a physiological inhibitor of TLR-4 signaling. Indeed, we have recently found that: (i) RP105 is a specific inhibitor of TLR-4 signaling in HEK293 cells; (ii) RP105/MD-1 interacts directly with TLR-4/MD-2, inhibiting the ability of this signaling complex to bind LPS; (iii) RP105 regulates TLR-4 signaling in dendritic cells and macrophages; and (iv) RP105 regulates in vivo responses to LPS.

Animals↗

PubMed2004

TLR9 signals after translocating from the ER to CpG DNA in the lysosome.

Microbial DNA sequences containing unmethylated CpG dinucleotides activate Toll-like receptor 9 (TLR9). We have found that TLR9 is localized to the endoplasmic reticulum (ER) of dendritic cells (DCs) and macrophages. Because there is no precedent for immune receptor signaling in the ER, we investigated how TLR9 is activated. We show that CpG DNA binds directly to TLR9 in ligand-binding studies. CpG DNA moves into early endosomes and is subsequently transported to a tubular lysosomal compartment. Concurrent with the movement of CpG DNA in cells, TLR9 redistributes from the ER to CpG DNA-containing structures, which also accumulate MyD88. Our data indicate a previously unknown mechanism of cellular activation involving the recruitment of TLR9 from the ER to sites of CpG DNA uptake, where signal transduction is initiated.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Targeting of HLA-DR molecules transduces agonistic functional signals in cutaneous melanoma.

The role of human leukocyte antigens (HLA) class II molecules in transducing intracellular signals in immune cells is well established. Solid tumors of different histotype can also express HLA class II antigens; however, their intracellular signaling ability is essentially unknown. Due to the frequent expression of HLA class II molecules in primary and metastatic lesions, cutaneous melanoma was utilized to investigate whether the engagement of HLA-DR molecules transduces functional intracellular signal(s). Triggering of HLA-DR molecules by the anti-HLA-DR monoclonal antibody (mAb) L243 induced a significant (P < 0.05) and dose-dependent growth-inhibition of metastatic melanoma cells Mel 120, as well as their homotypic aggregation. Furthermore, an increase in tyrosine phosphorylation of multiple cellular proteins with a molecular weight ranging from 66 to 130 kD, including p125 focal adhesion kinase, was observed. Lastly, the engagement of HLA-DR molecules by mAb L243 inhibited activator protein-1-DNA binding. Thus, HLA-DR molecules expressed on melanoma cells can transduce functional intracellular signals. This finding is consistent with evidences obtained in hematological malignancies, and suggests the potential usefulness of HLA-DR molecules to set-up new approaches of targeted therapy in metastatic melanoma.

Antibodies, Monoclonal↗

PubMed2004

Mechanisms of TLR9 activation.

Non-methylated CpG-motifs in bacterial or viral DNA are recognized by TLR9 as foreign. The activation of TLR9 by microbial DNA or synthetic oligonucleotides based on these motifs leads to the induction of innate immune responses. We have compared the subcellular localization of fluorescent versions of TLR9 and TLR4 and found that TLR9 is expressed in the endoplasmic reticulum while TLR4 is expressed on the plasma membrane. Fluorescently tagged bacterial DNA or CpG-DNA was observed to traffic to a tubular lysosomal compartment in human pDCs. In stimulated cells, TLR9 translocated to CpG-DNA or microbial DNA containing structures in the endosome, where TLR9 binds to DNA and initiates signaling.

Cell Membrane↗

PubMed2003

Targeted therapy of solid malignancies via HLA class II antigens: a new biotherapeutic approach?

Intracellular signals, delivered in professional antigen-presenting cells following the engagement of major histocompatibility complex (MHC) class II molecules, activate a variety of cellular functions that also contribute to efficient antigen presentation. As far as human malignancies, the signaling ability of human leukocyte antigens (HLA) class II molecules is a rather well-characterized event in hematologic tumors; in contrast, very limited evidences are available in solid neoplasias of different histotypes that may constitutively express HLA class II antigens. Among solid malignancies, a significant proportion of human cutaneous melanomas have been shown to express HLA class II molecules, and cutaneous melanoma undoubtedly represents a 'model disease' to investigate tumor immunobiology, to unveil the molecular basis underlying the interactions between neoplastic cells and host's immune system, and ultimately to set up new bio-immunotherapeutic approaches. Upcoming preclinical evidences unveil a signaling potential of HLA-DR antigens expressed on melanoma cells, and suggest for the clinical implication of HLA class II molecules as novel therapeutic targets. Therefore, in this review, we will focus on the emerging role of HLA class II antigens as intracellular signal transducing elements in neoplastic cells of the melanocytic lineage, emphasizing their foreseeable role in targeted therapy of human melanoma and potentially of HLA class II antigens-positive tumors of different histology.

Antigen-Presenting Cells↗

PubMed2003

LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF.

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction.

Three cell-surface proteins have been recognized as components of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS): CD14, Toll-like receptor-4 (TLR4), and MD-2. Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation. These studies clarify how MD-2 functions: we found that MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. MD-2 must be bound to TLR4 on the cell surface before binding can occur. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction. TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and identify MD-2 as an attractive target for pharmacological intervention in endotoxin-mediated diseases.

Amino Acid Sequence↗

PubMed2003

The role of disulfide bonds in the assembly and function of MD-2.

MD-2 is a secreted glycoprotein that binds to the extracellular domain of Toll-like receptor 4 (TLR4) and is required for the activation of TLR4 by lipopolysaccharide (LPS). The protein contains seven Cys residues and consists of a heterogeneous collection of disulfide-linked oligomers. To investigate the role of sulfhydryls in MD-2 structure and function, we created 17 single and multiple Cys substitution mutants. All of the MD-2 mutant proteins, including one totally lacking Cys residues, were secreted and stable. SDSPAGE analyses indicated that most Cys residues could participate in oligomer formation and that no single Cys residue was required for oligomerization. Of the single Cys substitutions, only C95S and C105S failed to confer LPS responsiveness on TLR4 when mutant and TLR4 were cotransfected into cells expressing an NF-kappaB reporter plasmid. Surprisingly, substitution of both C95 and C105 partially restored activity. Structural analyses revealed that C95 and C105 formed an intrachain disulfide bond, whereas C95 by itself produced an inactive dimer. In contrast to the cotransfection experiments, only WT MD-2 conferred responsiveness to LPS when secreted proteins were added directly to TLR4 reporter cells. Our data are consistent with a model in which most, possibly all sulfhydryls lie on the surface of a stable MD-2 core structure where they form both intra- and interchain disulfide bridges. These disulfide bonds produce a heterogeneous array of oligomers, including some species that can form an active complex with TLR4.

Amino Acid Substitution↗

PubMed2003

The LPS receptor generates inflammatory signals from the cell surface.

Bacterial lipopolysaccharides (LPSs) are recognized in mammals by a receptor complex composed of CD14, Toll-like receptor 4 (TLR4), and MD-2. The mechanism of TLR4 function remains to be elucidated. We constructed chimeric TLR molecules C-terminally fused to fluorescent proteins and stably expressed these chimeric constructs in cells. Confocal microscopy revealed TLR4 to be expressed on the plasma membrane and the Golgi apparatus. Time-lapse confocal imaging showed rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane. Membrane TLR4 engagement by antibody was sufficient to induce signaling and pharmacological disruption of the Golgi did not affect cellular responses to LPS. Thus, LPS signaling commences after LPS recognition by surface-expressed TLR4 independent of LPS trafficking to the Golgi.

Cell Line↗

PubMed2002

Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction.

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.

Adaptor Proteins, Signal Transducing↗