Saotomo Itoh

Staphylococcal Superantigen-Like Protein 3 Binds to the Toll-Like Receptor 2 Extracellular Domain and Inhibits Cytokine Production Induced by Staphylococcus aureus, Cell Wall Component, or Lipopeptides in Murine Macrophages

ABSTRACT Staphylococcal superantigen-like proteins (SSLs) are a family of exoproteins sharing structural similarity with superantigens, but no superantigenic activity. Corresponding host target proteins or receptors against a portion of SSLs in the family have been identified. In this study, we show that SSL3 specifically binds to Toll-like receptor 2 (TLR2) and inhibits the stimulation of macrophages by TLR2 ligands. An approximately 100-kDa protein was recovered by using recombinant His-tagged SSL3-conjugated Sepharose from the lysate of porcine spleen, and the protein was identified as porcine TLR2 by peptide mass fingerprinting analysis. The SSL3-conjugated Sepharose recovered human and mouse TLR2 but not TLR4 from human neutrophils and mouse macrophage RAW 264.7 cells, as well as a recombinant TLR2 extracellular domain chimera protein. The production levels of interleukin 12 (IL-12) from mouse macrophages treated with heat-killed Staphylococcus aureus and of tumor necrosis factor alpha (TNF-α) from RAW 264.7 cells induced by peptidoglycan or lipopeptide TLR2 ligands were strongly suppressed in the presence of SSL3. The mutation of consensus sialic acid-containing glycan-binding residues in SSL3 did not abrogate the binding ability to TLR2 or inhibitory activity on TLR2, indicating that the interaction of SSL3 with TLR2 was independent of the sialic acid-containing glycan-binding residues. These findings demonstrate that SSL3 is able to bind the extracellular domain of TLR2 and interfere with TLR2 function. The present study provides a novel mechanism of SSL3 in immune evasion of S. aureus via interfering with its recognition by innate immune cells.

Staphylococcal superantigen-like protein 10 (SSL10) binds to human immunoglobulin G (IgG) and inhibits complement activation via the classical pathway

Staphylococcal superantigen-like (SSL) proteins are a family of exoproteins that share structural similarity with staphylococcal superantigens but exhibit no superantigenic activity. It was previously reported that two members (SSL5 and SSL7) bound to serum components and cell adhesion molecules involved in host immune response; however, the other family members have not been functionally characterized. In this study, we attempted to isolate SSL10-binding proteins from human serum and found that recombinant His-tagged SSL10 bound two major polypeptides of approximately 50 and approximately 25 kDa after affinity purification and SDS-polyacrylamide gel electrophoresis. These polypeptides were identified as heavy and light chains of human IgG by peptide mass fingerprinting analysis. The specific interaction between recombinant SSL10 and human IgG was confirmed by far Western blot analysis using immobilized SSL10 and pull-down analysis using SSL10-conjugated Sepharose. Surface plasmon resonance analysis revealed that the dissociation equilibrium constant for the interaction between human IgG and recombinant SSL10 was estimated to be 220 nM. We also found that recombinant SSL10 inhibited the binding of complement component C1q to IgG-Sepharose and hemolysis of IgG-sensitized sheep erythrocytes via the classical complement activation pathway. These results suggest that SSL10 may play a role in the evasion of Staphylococcus aureus from the host immune system via interfering complement activation.

Staphylococcal Superantigen-Like Protein 5 Inhibits Matrix Metalloproteinase 9 from Human Neutrophils

ABSTRACT Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [KD] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (Ki = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.

Homotypic dimerization of the actin-binding protein p57/coronin-1 mediated by a leucine zipper motif in the C-terminal region

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/coronin-1. Recombinant p57/coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.

Transforming growth factor-β1 upregulates transcription of α3 integrin gene in hepatocellular carcinoma cells via ets-transcription factor-binding motif in the promoter region

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of α3β1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-β1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of α3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of α3β1 integrin by TGF-β1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-β1 induced the expression of α3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5′-flanking region of the mouse α3 integrin gene, and the results showed that the −260/−119 region (relative to the major transcription start site) contained elements responsive to TGF-β1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at −133 greatly decreased the promoter activity responding to TGF-β1 stimulation. The nuclear proteins extracted from TGF-β1-stimulated HepG2 cells yielded a larger amount of DNA–nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-β1 stimulates HepG2 cells to express a higher level of α3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.

Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: a role of lipid microdomains

P‐selectin glycoprotein ligand‐1 (PSGL‐1) is a mucin‐like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet‐leukocyte and endothelium‐leukocyte interactions. The treatment of neutrophils with a low concentration of IL‐8 induced the redistribution of PSGL‐1 to one end of the cell to form a cap‐like structure. We investigated the role of lipid microdomains in the redistribution of PSGL‐1 and its effect on the adhesive characteristics of IL‐8‐treated neutrophils. The redistribution of PSGL‐1 induced by IL‐8 was inhibited by cholesterol‐perturbing agents such as methyl‐β‐cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL‐1 was enriched in a low‐density fraction together with the GM1 ganglioside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X‐100 was used for the solubilization, PSGL‐1 was no longer recovered in the low‐density fraction, although GM1 ganglioside remained in the low‐density fraction. Furthermore, immunofluorescence microscopic observation demonstrated that the localization of PSGL‐1 differed from that of GM1 ganglioside, suggesting that PSGL‐1 is associated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neutrophils with IL‐8 increased the formation of microaggregates composed of neutrophils and activated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross‐linking of PSGL‐1 with antibodies. These results suggest that the association of PSGL‐1 with lipid microdomains is essential for its redistribution induced by IL‐8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P‐selectin.

Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

Background: Staphylococcal superantigen-like proteins (SSLs) share structural similarity with superantigens but no superantigenic activity. Their functions remained unclear. Results: SSL10 binds to prothrombin and factor Xa via the γ-carboxyglutamic acid domain. SSL10 inhibits the penultimate step of plasma clotting. SSL10 slightly inhibits clotting by coagulase. Conclusion: SSL10 inhibits blood coagulation. Significance: This work presents a novel function of SSLs, disturbing blood coagulation. The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10−7 m in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca2+ and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

Cytokine secretion from human monocytes potentiated by P-selectin-mediated cell adhesion.

Background/Aim: P-selectin is a carbohydrate-recognizing cell adhesion molecule expressed on activated platelets and endothelial cells. It plays a crucial role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. Cell adhesion mediated by P-selectin induces leukocyte activation, such as the generation of reactive oxygen species and the expression of blood coagulation factors. We assessed how P-selectin-mediated cell adhesion affects cytokine secretion from monocytes. Methods: Human peripheral blood monocytes were cultured in a plate that had been coated with P-selectin purified from human platelets, and cytokines released in the culture supernatant from monocytes were determined by ELISA. Results: The secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-12 and macrophage inflammatory protein-1β increased 3- to 10-fold in response to P-selectin compared with unstimulated monocytes. We next examined the effects of cytokine treatment of monocytes on their susceptibility to P-selectin. The secretion of TNF-α from monocytes in response to P-selectin was increased when monocytes were preincubated with granulocyte/macrophage colony-stimulating factor, monocyte chemotactic protein-1 or interferon-γ (IFN-γ); IFN-γ was the most effective in potentiating TNF-α secretion from monocytes. Conclusion: These results suggest that the interaction of monocytes with P-selectin plays an important role not only in their trafficking but also in the regulation of cytokine production by these cells.

Etiological role of cigarette smoking in rheumatoid arthritis: Nasal exposure to cigarette smoke condensate extracts augments the development of collagen-induced arthritis in mice

Cigarette smoking is a major environmental risk factor for rheumatoid arthritis (RA). However, the experimental bases supporting the etiological role of cigarette smoking in RA have not been fully provided. We have reported that cigarette smoke condensate (CSC), by means of subcutaneous injection into DBA/1J mice with collagen and complete Freunds adjuvant or intraperitoneal injection one day before immunization, augmented the development of arthritis in the mouse model of collagen type II-induced arthritis (CIA). However, these experimental procedures may not be appropriate for cigarette smoking. In this study, we nasally exposed mice to mainstream CSC and found that CSC augmented the induction and development of arthritis and antibody level against collagen. Histological examination confirmed the augmenting effect of CSC. These findings provide experimental bases supporting the etiological role of cigarette smoking in RA.

Monocyte differentiation induced by co-culture with tumor cells involves RGD-dependent cell adhesion to extracellular matrix

Macrophages that infiltrate tumor tissues, or tumor-associated macrophages (TAMs), affect the malignant behaviors of tumor cells. In this study, we attempted to induce monocytes to differentiate into TAM-like cells producing matrix metalloproteinases (MMPs) by co-culture with tumor cells. When human monocytes were co-cultured for 3-7 days with tumor cell lines, monocytes differentiated to produce MMP-9, accompanied by morphological changes. The in vitro cell invasion of MKN1 human gastric carcinoma cells into Matrigel membranes was promoted in the presence of differentiated monocytes, and the enhancement of cell invasion by differentiated monocytes was correlated with their MMP-9 productivity. The addition of an RGD (Arg-Gly-Asp) peptide to the culture significantly inhibited monocyte differentiation. The MMP-9 production from monocytes was diminished by the depletion of fibronectin from the conditioned media with gelatin-Sepharose, and potentiated by culturing them in fibronectin-coated plates. These results suggest that cell adhesion to the extracellular matrix plays a crucial role in monocyte differentiation into TAM-like cells.