Toshio Oda

Pulmonary toxicity induced by intratracheal instillation of Asian yellow dust (Kosa) in mice

Asian yellow dust (Kosa) causes adverse respiratory health effects in humans. The objective of this study was to clarify the lung toxicity of Kosa. ICR mice (5 weeks of age) were administered intratracheally with Kosa samples-two samples from Maowusu desert and Shapotou desert, one sample consisted of Shapotou Kosa plus sulfate, and natural Asian dust (NAD) from the atmosphere of Beijing-at doses of 0.05, 0.10 or 0.20mg/mouse at four weekly intervals. The four Kosa samples tested had similar compositions of minerals and concentrations of elements. Instillation of dust particles caused bronchitis and alveolitis in treated mice. The magnitude of inflammation was much greater in NAD-treated mice than in the other particles tested. Increased neutrophils, lymphocytes or eosinophils in bronchoalveolar lavage fluids (BALF) of treated mice were dose dependent. The number of neutrophils in BALF at the 0.2mg level was parallel to the content of β-glucan in each particle. The numbers of lymphocytes and eosinophils in BALF at the 0.2mg level were parallel to the concentration of SO(4)(2-) in each particle. Pro-inflammatory mediators-such as interleukin (IL)-12, tumor necrosis factor-(TNF)-α, keratinocyte chemoattractant (KC), monocyte chemotactic protein (MCP)-l and macrophage inflammatory protein-(MIP)-lα in BALF-were greater in the treated mice. Specifically, NAD considerably increased pro-inflammatory mediators at a 0.2mg dose. The increased amounts of MlP-lα and TNF-α at 0.2mg dose corresponded to the amount of β-glucan in each particle. The amounts of MCP-l or IL-12 corresponded to the concentration of sulfate (SO(4)(2-)) at a 0.2mg dose. These results suggest that inflammatory lung injury was mediated by β-glucan or SO(4)(2-), which was adsorbed into the particles, via the expression of these pro-inflammatory mediators. The results also suggest that the variations in the magnitude of inflammation of the tested Kosa samples depend on the amounts of these toxic materials.

Purification and characterization of a (1 → 3)-β-d-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes

A novel (1-->3)-beta-D-glucan-binding protein (T-GBP) has been purified from the amoebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. It is a basic protein (pI 9.2) which appears to be a homotetramer composed of subunits with an apparent mol wt of 168000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL. T-GBP exerted an inhibitory effect on the (1-->3)-beta-D-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate. The binding of (1-->3)-beta-D-glucans to T-GBP was evaluated by measuring the residual amidolytic activity of the clotting enzyme, the product of the coagulation cascade, using Boc-Leu-Gly-Arg-4-nitroanilide as the chromogenic substrate. The binding specificity of a wide range of (1-->3)-beta-D-glucans and other polysaccharides towards T-GBP was expressed by the relative inhibition (%) of the activation of factor G, the first protease zymogen in the pathway, which is activated by binding to (1-->3)-beta-D-glucans. T-GBP was found to have a high affinity for linear (1-->3)-beta-D-glucans, e.g. pachyman, curdlan, and paramylon. It was able to bind to (1-->3)-beta-D-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast beta-D-glucan, and (1-->3),(1-->4)-beta-D-glucans such as lichenin and barley beta-D-glucan. Binding of pachyman to T-GBP was demonstrated by an enzyme-linked immunosorbent assay using a specific antibody (rabbit IgG) raised against T-GBP.

Candida soluble cell wall β-glucan facilitates ovalbumin-induced allergic airway inflammation in mice: Possible role of antigen-presenting cells

BackgroundAlthough fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble β-glucan from Candida albicans (CSBG) (Ohno et al., 2007). In the present study, the effects of CSBG exposure on airway immunopathology in the presence or absence of other immunogenic allergen was investigated in vivo, and their cellular mechanisms were analyzed both in vivo and in vitro.MethodsIn vivo, ICR mice were divided into 4 experimental groups: vehicle, CSBG (25 μg/animal), ovalbumin (OVA: 2 μg/animal), and CSBG + OVA were repeatedly administered intratracheally. The bronchoalveolar lavage cellular profile, lung histology, levels of cytokines and chemokines in the lung homogenates, the expression pattern of antigen-presenting cell (APC)-related molecules in the lung digests, and serum immunoglobulin values were studied. In vitro, the impacts of CSBG (0–12.5 μg/ml) on the phenotype and function of immune cells such as splenocytes and bone marrow-derived dendritic cells (BMDCs) were evaluated in terms of cell proliferation, the surface expression of APC-related molecules, and OVA-mediated T-cell proliferating activity.ResultsIn vivo, repeated pulmonary exposure to CSBG induced neutrophilic airway inflammation in the absence of OVA, and markedly exacerbated OVA-related eosinophilic airway inflammation with mucus metaplasia in mice, which was concomitant with the amplified lung expression of Th2 cytokines and IL-17A and chemokines related to allergic response. Exposure to CSBG plus OVA increased the number of cells bearing MHC class II with or without CD80 in the lung compared to that of others. In vitro, CSBG significantly augmented splenocyte proliferation in the presence or absence of OVA. Further, CSBG increased the expression of APC-related molecules such as CD80, CD86, and DEC205 on BMDCs and amplified OVA-mediated T-cell proliferation through BMDCs.ConclusionCSBG potentiates allergic airway inflammation with maladaptive Th immunity, and this potentiation was associated with the enhanced activation of APCs including DC.

Horseshoe Crab Factor G: A New Heterodimeric Serine Protease Zymogen Sensitive to (1→3)-β-D-Glucan

Horseshoe crabs (or limulus), as well as other invertebrate animals, do not have an ordinary immune system like mammals. Although they prefer to live in clean oceans, there are still a number of bacteria in their environment. In order to defend themselves from such microorganisms, they have developed a unique and sophisticated defense system in their hemolymph.

The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen

Background: Factor C is autocatalytically activated on lipopolysaccharides. Results: The N-terminal Arg of factor C is essential for the autocatalytic activation. Conclusion: The N-terminal Arg of factor C is required to be in sufficiently close vicinity for the autocatalytic activation. Significance: The recombinant factor C with restricted and homogeneous N-glycans may be useful for further structure-function studies. Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.

Limulus clotting factor B

Publisher Summary This chapter describes the limulus clotting factor B. Horseshoe crab hemocytes are highly sensitive to gram-negative bacterial endotoxins, which are lipopolysaccharides (LPS); exposure to LPS results in activation of intracellular coagulation systems. In the Japanese horseshoe crab, Tachypleus tridentatus , the coagulation system consists of three intracellular serine protease zymogens—namely, proclotting enzyme, factor B, and factor C, in addition to the fibrinogen-like substance, coagulogen. In the presence of LPS, a LPS-sensitive serine protease zymogen, factor C, is autocatalytically activated. The resulting clotting enzyme converts soluble coagulogen to an insoluble coagulin gel. Like the mammalian coagulation and complement systems, this cascade reaction is propagated by limited proteolysis. These zymogens and coagulogen constitute the cascade pathway responsible for the LPS-mediated activation.

Soluble cell wall β-glucan of candida induces/enhances apoptosis and oxidative stress in murine lung

The bioactivity of cell wall component(s) of fungi has not been fully investigated, especially in vivo. We have successfully extracted a soluble cell wall β-glucan from C. albicans (CSBG), and shown that pulmonary exposure to CSBG induces lung inflammation in mice. CSBG-induced lung inflammation was concomitant with the nuclear translocation of signal transducer and activator of transcription (STAT)6 and enhanced lung expression of various cytokines and chemokines. However, the effects of CSBG on the murine respiratory system and their mechanisms have not been fully investigated. In this study, we further explored the effects of pulmonary exposure to CSBG on lung pathophysiology in terms of the induction of apoptosis and enhancement of oxidative stress. ICR mice were intratracheally instilled with vehicle, CSBG, or structurally degraded products of CSBG by formic acid (DEG-CSBG), and 24 h later, the lungs were isolated and evaluated for apoptosis by the TUNEL assay and oxidative stress by immunohistochemistry of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a proper marker of the oxidative DNA damage. In another experiment, the mice were sacrificed and lungs were isolated 4 h after the instillation with vehicle or CSBG for evaluation of transcriptional level of heme oxygenase (HO)-1, a stress responsive protein. Pulmonary exposure to CSBG significantly induced apoptosis and enhanced the formation of 8-OHdG in the lung parenchyma as compared to vehicle or DEG-CSBG exposure. CSBG significantly induced HO-1 in the lung. Taken together, CSBG induces/enhances apoptosis and oxidative stress in the lung in association with lung inflammation/injury.

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade

Background: It is not known whether LPS is required for the activation of coagulation factor B. Results: Factor B is an LPS-binding zymogen activated by α-factor C in an LPS-dependent manner. Conclusion: The clip domain of factor B has an important role in localizing factor B to LPS. Significance: Horseshoe crab coagulation is an ideal model for understanding proteolytic cascades. Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by β-factor C, in an LPS-dependent manner and that β-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C.

Genetic engineering approach to develop next-generation reagents for endotoxin quantification

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.