Characterization of DNA-protein complexes by nanoparticle tracking analysis and their association with systemic lupus erythematosus.

Nanotechnology enables investigations of single biomacromolecules, but technical challenges have limited the application in liquid biopsies, for example, blood plasma. Nonetheless, tools to characterize single molecular species in such samples represent a significant unmet need with the increasing appreciation of the physiological importance of protein structural changes at nanometer scale. Mannose-binding lectin (MBL) is an oligomeric plasma protein and part of the innate immune system through its ability to activate complement. MBL also serves a role as a scavenger for cellular debris, especially DNA. This may link functions of MBL with several inflammatory diseases in which cell-free DNA now appears to play a role, but mechanistic insight has been lacking. By making nanoparticle tracking analysis possible in human plasma, we now show that superoligomeric structures of MBL form nanoparticles with DNA. These oligomers correlate with disease activity in systemic lupus erythematosus patients. With the direct quantification of the hydrodynamic radius, calculations following the principles of Taylor dispersion in the blood stream connect the size of these complexes to endothelial inflammation, which is among the most important morbidities in lupus. Mechanistic insight from an animal model of lupus supported that DNA-stabilized superoligomers stimulate the formation of germinal center B cells and drive loss of immunological tolerance. The formation involves an inverse relationship between the concentration of MBL superoligomers and antibodies to double-stranded DNA. Our approach implicates the structure of DNA-protein nanoparticulates in the pathobiology of autoimmune diseases.

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMß2.

The integrin receptor αMß2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of αMß2 signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human αMß2 headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H αM variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside-inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the αM subunit in an Mg2+-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the αM subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound αMß2 with iC3b, suggesting that it may represent a novel high-affinity proteinaceous αMß2-specific agonist. Taken together, our data suggest that the iC3b-αMß2 complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to αMß2.

Potentiating Tweezer Affinity to a Protein Interface with Sequence-Defined Macromolecules on Nanoparticles.

Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.

Complement Receptor 3 Forms a Compact High-Affinity Complex with iC3b.

Complement receptor 3 (CR3, also known as Mac-1, integrin αMß2, or CD11b/CD18) is expressed on a subset of myeloid and certain activated lymphoid cells. CR3 is essential for the phagocytosis of complement-opsonized particles such as pathogens and apoptotic or necrotic cells opsonized with the complement fragment iC3b and, to a lesser extent, C3dg. Although the interaction between the iC3b thioester domain and the ligand binding CR3 αM I-domain is structurally and functionally well characterized, the nature of additional CR3-iC3b interactions required for phagocytosis of complement-opsonized objects remains obscure. In this study, we analyzed the interaction between iC3b and the 150-kDa headpiece fragment of the CR3 ectodomain. Surface plasmon resonance experiments demonstrated a 30 nM affinity of the CR3 headpiece for iC3b compared with 515 nM for the iC3b thioester domain, whereas experiments monitoring binding of iC3b to CR3-expressing cells suggested an affinity of 50 nM for the CR3-iC3b interaction. Small angle x-ray scattering analysis revealed that iC3b adopts an extended but preferred conformation in solution. Upon interaction with CR3, iC3b rearranges to form a compact receptor-ligand complex. Overall, the data suggest that the iC3b-CR3 interaction is of high affinity and relies on minor contacts formed between CR3 and regions outside the iC3b thioester domain. Our results rationalize the more efficient phagocytosis elicited by iC3b than by C3dg and pave the way for the development of specific therapeutics for the treatment of inflammatory and neurodegenerative diseases that do not interfere with the recognition of noncomplement CR3 ligands.

Size-Selective Phagocytic Clearance of Fibrillar α-Synuclein through Conformational Activation of Complement Receptor 4.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Efficient data acquisition with three-channel centerpieces in sedimentation velocity.

Three-channel 3D printed centerpieces with two sample sectors next to a joint solvent reference sector were recently described as a strategy to double the throughput of sedimentation velocity analytical ultracentrifugation experiments [Anal. Chem. 91 (2019) 5866-5873]. They are compatible with Rayleigh interference optical detection in commercial analytical ultracentrifuges, but require the rotor angles of data acquisition to be repeatedly adjusted during the experiment to record data from the two sample sectors. Here we present an approach to automate this data acquisition mode through the use of a secondary, general-purpose automation software, and an accompanying data pre-processing software for scan sorting.

Impact of Microbiota on Resistance to Ocular Pseudomonas aeruginosa-Induced Keratitis.

The existence of the ocular microbiota has been reported but functional analyses to evaluate its significance in regulating ocular immunity are currently lacking. We compared the relative contribution of eye and gut commensals in regulating the ocular susceptibility to Pseudomonas aeruginosa-induced keratitis. We find that in health, the presence of microbiota strengthened the ocular innate immune barrier by significantly increasing the concentrations of immune effectors in the tear film, including secretory IgA and complement proteins. Consistent with this view, Swiss Webster (SW) mice that are typically resistant to P. aeruginosa-induced keratitis become susceptible due to the lack of microbiota. This was exemplified by increased corneal bacterial burden and elevated pathology of the germ free (GF) mice when compared to the conventionally maintained SW mice. The protective immunity was found to be dependent on both eye and gut microbiota with the eye microbiota having a moderate, but significant impact on the resistance to infection. These events were IL-1ß-dependent as corneal IL-1ß levels were decreased in the infected GF and antibiotic-treated mice when compared to the SPF controls, and neutralization of IL-1ß increased the ocular bacterial burden in the SPF mice. Monocolonizing GF mice with Coagulase Negative Staphylococcus sp. isolated from the conjunctival swabs was sufficient to restore resistance to infection. Cumulatively, these data underline a previously unappreciated role for microbiota in regulating susceptibility to ocular keratitis. We predict that these results will have significant implications for contact lens wearers, where alterations in the ocular commensal communities may render the ocular surface vulnerable to infections.

Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3.

The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg(2+) ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15-25 µm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50-100 nm concentration of the drug reduced the adhesion by 20-40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists.

The cationic peptide LL-37 binds Mac-1 (CD11b/CD18) with a low dissociation rate and promotes phagocytosis.

As a broad-spectrum anti-microbial peptide, LL-37 plays an important role in the innate immune system. A series of previous reports implicates LL-37 as an activator of various cell surface receptor-mediated functions, including chemotaxis in integrin CD11b/CD18 (Mac-1)-expressing cells. However, evidence is scarce concerning the direct binding of LL-37 to these receptors and investigations on the associated binding kinetics is lacking. Mac-1, a member of the ß2 integrin family, is mainly expressed in myeloid leukocytes. Its critical functions include phagocytosis of complement-opsonized pathogens. Here, we report on interactions of LL-37 and its fragment FK-13 with the ligand-binding domain of Mac-1, the α-chain I domain. LL-37 bound the I-domain with an affinity comparable to the complement fragment C3d, one of the strongest known ligands for Mac-1. In cell adhesion assays both LL-37 and FK-13 supported binding by Mac-1 expressing cells, however, with LL-37-coupled surfaces supporting stronger cell adhesion than FK-13. Likewise, in phagocytosis assays with primary human monocytes both LL-37 and FK-13 enhanced uptake of particles coupled with these ligands but with a tendency towards a stronger uptake by LL-37.

The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage.

The formulation glatiramer acetate (GA) is widely used in therapy of multiple sclerosis. GA consists of random copolymers of four amino acids, in ratios that produce a predominantly positive charge and an amphipathic character. With the extraordinary complexity of the drug, several pharmacological modes-of-action were suggested, but so far none, which rationalizes the cationicity and amphipathicity as part of the mode-of-action. Here, we report that GA rapidly kills primary human T lymphocytes and, less actively, monocytes. LL-37 is a cleavage product of human cathelicidin with important roles in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45+ debris, derived from cell membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic contacts, which is critical for the lytic properties. In our study, SAXS showed that GA also forms this type of contacts. Taken together, our study offers new insight on the immunomodulatory mode-of-action of positively charged co-polymers. The comparison of LL-37 and GA highlights a consistent requirement of certain oligomeric and chemical properties to support cytotoxic effects of cationic polymers targeting human leukocytes.

Multiple low-affinity interactions support binding of human osteopontin to integrin αXß2.

Integrin α(X)ß(2) (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. α(X)ß(2) has been reported to bind a large number of structurally unrelated ligands, often with a shared molecular character in the presence of polyanionic stretches in poorly folded proteins or glucosaminoglycans. Nevertheless, it is unclear what chemical sources of polyanionicity enable the binding by α(X)ß(2). Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin α(X)ß(2). Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by α(X)ß(2) remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of α(X)ß(2) and OPN, its fragments, or caseins are weak, with dissociation constants higher than 10(-5)M but with high apparent stoichiometries. From comparison with cell adhesion studies, the discrimination between α(X)ß(2) ligands and non-ligands appears to rely on these apparent stoichiometries in a way, which involves glutamate rather than aspartate side chains. Surprisingly, the extensive, negatively charged phosphorylation of OPN is not contributing to α(X)ß(2) binding. Furthermore, synchrotron radiation circular spectroscopy excludes that the phosphorylation affects the general folding of OPN. Taken together, our quantitative analyses reveal a mode of ligand recognition by integrin α(X)ß(2), which seem to differ in principles considerably from other OPN receptors.

Structural insight on the recognition of surface-bound opsonins by the integrin I domain of complement receptor 3.

Complement receptors (CRs), expressed notably on myeloid and lymphoid cells, play an essential function in the elimination of complement-opsonized pathogens and apoptotic/necrotic cells. In addition, these receptors are crucial for the cross-talk between the innate and adaptive branches of the immune system. CR3 (also known as Mac-1, integrin αMß2, or CD11b/CD18) is expressed on all macrophages and recognizes iC3b on complement-opsonized objects, enabling their phagocytosis. We demonstrate that the C3d moiety of iC3b harbors the binding site for the CR3 αI domain, and our structure of the C3d:αI domain complex rationalizes the CR3 selectivity for iC3b. Based on extensive structural analysis, we suggest that the choice between a ligand glutamate or aspartate for coordination of a receptor metal ion-dependent adhesion site-bound metal ion is governed by the secondary structure of the ligand. Comparison of our structure to the CR2:C3d complex and the in vitro formation of a stable CR3:C3d:CR2 complex suggests a molecular mechanism for the hand-over of CR3-bound immune complexes from macrophages to CR2-presenting cells in lymph nodes.

Effect of Polarization on Airway Epithelial Conditioning of Monocyte-Derived Dendritic Cells.

Airway epithelial cells (AECs) form polarized barriers that interact with inhaled allergens and are involved in immune homeostasis. We examined how monocyte-derived dendritic cells (MDDCs) are affected by contact with the airway epithelium. In traditional setups, bronchial epithelial cell lines were allowed to polarize on filter inserts, and MDDCs were allowed to adhere to the epithelial basal side. In an optimized setup, the cell application was reversed, and the culture conditions were modified to preserve cellular polarization and integrity. These two parameters were crucial for the MDDCs' immunoregulatory properties; thus, previous observations obtained using traditional setups should be considered with caution. Using the optimized setup, AEC conditioning of MDDCs led to increased expression of programmed death 1 ligand 1, immunoglobulin-like transcript 3, CD40, CD80, and CD23. This increased expression was accompanied by decreased secretion of monocyte chemotactic protein 1 and eotaxin and donor-variable effects on IL-12 and IL-10 secretion. Conditioning varied between maturation states and depended partly on direct contact between AECs and MDDCs. The setup allowed MDDCs on the basal side of the epithelium to sample allergens administered to the apical side. Allergen uptake depended on polarization and the nature of the allergen. AEC conditioning led to decreased birch allergen-specific proliferation of autologous T cells and a trend toward decreased secretion of the Th2-specific cytokines IL-5 and IL-13. In conclusion, we determined that AEC conditioning favoring cellular integrity leads to a tolerogenic MDDC phenotype, which is likely to be important in regulating immune responses against commonly inhaled allergens.

Immunogenicity of twenty peptides representing epitopes of the hepatitis B core and surface antigens by IFN-γ response in chronic and resolved HBV.

BACKGROUND: Patients with chronic hepatitis B virus infection (CHB) usually mount a modest T cell response against HBV epitopes. In order to determine immunogenic epitopes of HBV recognized by HBV-specific T cells, previous studies focused on previously confirmed HBV epitopes and assessed the T cell response by the number of HBV-specific T cells by IFN-γ ELISPOT. METHODS: We studied T cell functionality by combined in silico methods predicting HBV-specific epitopes and experimental investigations on the recognition of these epitopes. 30 chronic CHB patients and 10 patients with resolved HBV (RHB) were included in the study. We identified epitopes from the literature and by in silico analysis. These were evaluated for immunogenicity by use of synthetic peptides representing the epitopes through exposure to PBMCs from patients with CHB or RHB by IFN-γ ELISPOT. The number of IFN-γ producing cells (SFC), mean spot size (MSS) and stimulation index (SI) were recorded. RESULTS: The frequency of HBV-specific T cells producing IFN-γ after stimulation with HBV epitopes was similar in CHB and RHB patients. CHB patients had a higher MSS SI than RHB patients. Patients not carrying the HLA-A2 genotype had higher SFC SI and MSS SI. Patients with HLA-A11 had higher MSS SI compared to non- HLA-A11 allele patients. HBeAg-positive patients had a lower MSS SI, and none of the HBeAg positive patients had the HLA-A11 genotype. We found 3 immunogenic epitopes not described previously. CONCLUSION: IFN-γ ELISPOT-determined MSS is an efficient marker for T cell recognition of epitopes. This experimental measure showed the in silico analysis for epitope prediction to be a valuable tool in future studies on HLA genotypes and HBV epitopes. This way our study now points to previously unappreciated consequences of carrying the HLA-A11 allele in terms of stronger immunity to HBV.

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: interactions with human ß2 integrins and erythrocytes.

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)ß(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express ß(2) integrin. Cells expressing α(M)ß(2) (CD11b/CD18) or α(X)ß(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)ß(2). Erythrocytes, which do not express ß(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the ß(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)ß(2) and α(X)ß(2) as novel receptors for LtxA.

Sialic acid residues are essential for cell lysis mediated by leukotoxin from Aggregatibacter actinomycetemcomitans.

Leukotoxin (LtxA) from Aggregatibacter actinomycetemcomitans is known to target and lyse ß2-integrin-expressing cells such as polymorphonuclear leukocytes and macrophages. LtxA is an important virulence factor that facilitates chronic inflammation and is strongly associated with a fast-progressing form of periodontitis caused by the JP2 clone of the bacterium. Here, we show that sialic acid residues are important for LtxA-induced cell lysis, regardless of whether the cell express ß2-integrin or not. Clearly, removal of sialic acid groups significantly reduces a ß2-integrin-specific LtxA-induced lysis. Moreover, sialic acid presented on alternative proteins, such as, for instance, on erythrocytes that do not express ß2-integrin, also makes the cells more sensitive to LtxA. The data also illustrate the importance of the negative charge in order for the sialic acid to associate LtxA with the membrane. Removal of sialic acid is in itself sufficient to significantly reduce the negative charge on the erythrocytes. Moreover, we found that on human erythrocytes there is a positive association between the sensitivity to LtxA and the amount of negative charge caused by sialic acid. Interestingly, these features are not shared by all RTX toxins, since α-hemolysin from Escherichia coli induced cell lysis of both ß2-integrin-expressing and nonexpressing cells and this lysis is independent of the presence of sialic acid residues. In conclusion, LtxA not only is cytotoxic to ß2-integrin-expressing cells but can potentially initiate cell lysis in all cells that present a sufficient density of sialic acid groups on their plasma membrane.

Accumulation of nano-sized particles in a murine model of angiogenesis.

PURPOSE: To evaluate the ability of nm-scaled iron oxide particles conjugated with Azure A, a classic histological dye, to accumulate in areas of angiogenesis in a recently developed murine angiogenesis model. MATERIALS AND METHODS: We characterised the Azure A particles with regard to their hydrodynamic size, zeta potential, and blood circulation half-life. The particles were then investigated by Magnetic Resonance Imaging (MRI) in a recently developed murine angiogenesis model along with reference particles (Ferumoxtran-10) and saline injections. RESULTS: The Azure A particles had a mean hydrodynamic diameter of 51.8 ± 43.2 nm, a zeta potential of -17.2 ± 2.8 mV, and a blood circulation half-life of 127.8 ± 74.7 min. Comparison of MR images taken pre- and 24-h post-injection revealed a significant increase in R2(*) relaxation rates for both Azure A and Ferumoxtran-10 particles. No significant difference was found for the saline injections. The relative increase was calculated for the three groups, and showed a significant difference between the saline group and the Azure A group, and between the saline group and the Ferumoxtran-10 group. However, no significant difference was found between the two particle groups. CONCLUSION: Ultrahigh-field MRI revealed localisation of both types of iron oxide particles to areas of neovasculature. However, the Azure A particles did not show any enhanced accumulation relative to Ferumoxtran-10, suggesting the accumulation in both cases to be passive.

Interleukin 20 protein locates to distinct mononuclear cells in psoriatic skin.

We previously demonstrated that mRNA for the pro-inflammatory cytokine interleukin 20 (IL-20) is expressed in suprapapillary keratinocytes of lesional psoriatic skin (LS). Here, we describe the distribution of IL-20 protein and the identity of the IL-20-positive cells in LS. We found that the main part of IL-20 immunoreactivity is present in mononuclear cells of the dermal papillae, and that the IL-20-positive cells located in the papillae were langerin+, CD1a+, CD4+ and CD303+. These cells might be immature dendritic cell. In situ hybridization for IL-20 mRNA on non-LS, ex vivo stimulated with IL-1ß revealed a colocalization between IL-20 mRNA and the keratinocyte marker CK14. No IL-20 mRNA was detected in the dermal mononuclear cells. Our results suggest that IL-20 is produced by keratinocytes, released into the epidermis and then possibly taken up by papillary mononuclear cells. Our study supports that IL-20 is involved in the pathogenesis of psoriasis.

The role of nanometer-scaled ligand patterns in polyvalent binding by large mannan-binding lectin oligomers.

Mannan-binding lectin (MBL) is an important protein of the innate immune system and protects the body against infection through opsonization and activation of the complement system on surfaces with an appropriate presentation of carbohydrate ligands. The quaternary structure of human MBL is built from oligomerization of structural units into polydisperse complexes typically with three to eight structural units, each containing three lectin domains. Insight into the connection between the structure and ligand-binding properties of these oligomers has been lacking. In this article, we present an analysis of the binding to neoglycoprotein-coated surfaces by size-fractionated human MBL oligomers studied with small-angle x-ray scattering and surface plasmon resonance spectroscopy. The MBL oligomers bound to these surfaces mainly in two modes, with dissociation constants in the micro to nanomolar order. The binding kinetics were markedly influenced by both the density of ligands and the number of ligand-binding domains in the oligomers. These findings demonstrated that the MBL-binding kinetics are critically dependent on structural characteristics on the nanometer scale, both with regard to the dimensions of the oligomer, as well as the ligand presentation on surfaces. Therefore, our work suggested that the surface binding of MBL involves recognition of patterns with dimensions on the order of 10-20 nm. The recent understanding that the surfaces of many microbes are organized with structural features on the nanometer scale suggests that these properties of MBL ligand recognition potentially constitute an important part of the pattern-recognition ability of these polyvalent oligomers.