<title>Abstract</title>Gland macrophages are primed for gland development and functions through interactions within their niche. However, the phenotype, ontogeny, and function of steady-state salivary gland (SG) macrophages remain unclear. We herein identified CD11c⁺ and CD11c⁻ subsets among CD64⁺ macrophages in steady-state murine SGs. CD11c⁻ macrophages were predominant in the SGs of embryonic and newborn mice and decreased with advancing age. CD11c⁺ macrophages were rarely detected in the embryonic period, but rapidly expanded after birth. CD11c⁺, but not CD11c⁻, macrophage numbers decreased in mice treated with a CCR2 antagonist, suggesting that CD11c⁺ macrophages accumulate from bone marrow-derived progenitors in a CCR2-dependent manner, whereas CD11c⁻ macrophages were derived from embryonic progenitors in SGs. CD11c⁺ and CD11c⁻ macrophages strongly expressed colony-stimulating factor (CSF)-1 receptor, the injection of an anti-CSF-1 receptor blocking antibody markedly reduced both subsets, and SGs strongly expressed CSF-1, indicating the dependency of SG resident macrophage development on CSF-1. The phagocytic activity of SG macrophages was extremely weak; however, the gene expression profile of SG macrophages indicated that SG macrophages regulate gland development and functions in SGs. These results suggest that SG CD11c⁺ and CD11c⁻ macrophages are developed and instructed to perform SG-specific functions in steady-state SGs.

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is one of the most common tumors of the head and neck region. The tumor suppressor gene p53 (TP53) is the most frequently mutated gene in OSCC and TP53 mutations are associated with decreased survival and resistance to chemotherapy in patients with OSCC. Therefore, therapeutic strategies targeting TP53 reactivation are required to effectively treat OSCC. In this study, we investigated the effect of various p53-reactivating small molecules (RITA, PRIMA-1, and CP-31398) on the proliferation of human OSCC cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) derived from human oral tissues bearing a mutant TP53 gene. MATERIALS AND METHODS: Apoptosis induction by RITA was assessed by measuring Annexin V and propidium iodide (PI)-positive cells using flow cytometry. p53 and murine double minute 2 (MDM2) phosphorylation and Bax expression were detected in the lysates of RITA-treated Ca9-22 cells using western blotting. RESULTS: RITA markedly inhibited the growth of Ca9-22, HSC-2, HSC-3, and HSC-4 cells. In Ca9-22 cells, RITA induced apoptosis and inhibited cell proliferation while increasing p53 phosphorylation and Bax expression; however, RITA did not induce MDM2 phosphorylation. CONCLUSION: The inhibitory effect of RITA on human OSCC cell proliferation is mediated by apoptosis induction through p53 and Bax.

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.

Among the bisphosphonates (BPs), nitrogen-containing BPs (N-BPs) have much stronger anti-bone-resorptive actions than non-N-BPs. However, N-BPs have various side-effects such as acute influenza-like reactions after their initial administration and osteonecrosis of the jawbones after repeated administration. The mechanisms underlying such effects remain unclear. To overcome these problems, it is important to profile the inflammatory nature of N-BPs. Here, we analyzed the inflammatory reactions induced in mouse ear-pinnas by the N-BPs alendronate (Ale) and zoledronate (Zol). We found the following. (i) Ale and Zol each induced two phases of inflammation (early weak and late strong ear-swelling). (ii) Both phases were augmented by lipopolysaccharides [LPS; cell-surface constituent of gram-negative bacteria (including oral bacteria)], but prevented by inhibitors of the phosphate transporters SLC20/34. (iii) Macrophages and neutrophils were involved in both phases of Ale+LPS-induced ear-swelling. (iv) Ale increased or tended to increase various cytokines, and LPS augmented these effects, especially that on IL-1β. (v) ATP was involved in both phases, and Ale alone or Ale+LPS increased ATP in ear-pinnas. (vi) The augmented late-phase swelling induced by Ale+LPS depended on both IL-1 and neutrophil extracellular traps (NETs, neutrophil-derived net-like complexes). (vii) Neutrophils, together with macrophages and dendritic cells, also functioned as IL-1β-producing cells, and upon stimulation with IL-1β, neutrophils produced NETs. (viii) Stimulation of P2X7 receptors by ATP induced IL-1β in ear-pinnas. (ix) NET formation by Ale+LPS was confirmed in gingiva, too. These results suggest that (1) N-BPs induce both early- and late-phase inflammation via ATP-production and P2X7-receptor stimulation, (2) N-BPs and LPS induce mutually augmenting responses both early and late phases via ATP-mediated IL-1β production by neutrophils, macrophages, and/or dendritic cells, and (3) NET production by IL-1β-stimulated neutrophils may mediate the late phase, leading to prolonged inflammation. These results are discussed in relation to the side effects seen in patients treated with N-BPs. This article is protected by copyright. All rights reserved.

OBJECTIVES: The aim of this study was to investigate the inflammatory roles of P2 purinergic receptor (P2R) signaling in oral squamous cell carcinoma (OSCC). METHODS: Human OSCC cell lines HSC-2, Ca9-22, and HO-1-u-1 were stimulated with P2R agonists. The concentration of interleukin (IL)-6 in culture supernatants was measured using an enzyme-linked immune sorbent assay. Expression levels of messenger RNAs (mRNAs) were analyzed using reverse transcription polymerase chain reaction. Phosphorylation of intracellular signaling molecules was analyzed using western blotting. RESULTS: HSC-2 cells expressed the mRNAs for P2X4-6 and all P2YRs. ATP or ADP induced significantly greater production of IL-6 by HSC-2 cells. Ca9-22 cells expressed mRNAs for P2X4-6 and all P2YRs except P2Y4. ATP or ADP induced the production of IL-6 by Ca9-22 cells, but the IL-6 concentration was much lower than that in HSC-2 cells. Although HO-1-u-1 cells expressed the mRNAs for P2X4-6 and all P2YRs, ATP or ADP did not induce IL-6 production. The production of IL-6 by HSC-2 cells stimulated with adenine nucleotides was significantly inhibited by P2R antagonists and a p38 mitogen-activated protein kinase inhibitor, but not by extracellular signal-related kinase or c-Jun N-terminal kinase inhibitors. The proinflammatory cytokine IL-1 significantly augmented P2R-induced IL-6 production by HSC-2 cells via the nuclear factor-κB signaling pathway. CONCLUSIONS: The present study suggests that P2Rs signaling and IL-1 synergistically induce chronic inflammation in OSCC. Because chronic inflammation is a well-known driving force of tumor progression, these results support therapeutic strategies that target P2Rs signaling in OSCC.

Bisphosphonates (BPs) are major anti-bone-resorptive drugs. Among them, the nitrogen-containing BPs (NBPs) exhibit much stronger anti-bone-resorptive activities than non-nitrogen-containing BPs (non-NBPs). However, BP-related osteonecrosis of the jaw (BRONJ) has been increasing without effective strategies for its prevention or treatment. The release of NBPs (but not non-NBPs) from NBP-accumulated jawbones has been supposed to cause BRONJ, even though non-NBPs (such as etidronate (Eti) and clodronate (Clo)) are given at very high doses because of their low anti-bone-resorptive activities. Our murine experiments have demonstrated that NBPs cause inflammation/necrosis at the injection site, and that Eti and Clo can reduce or prevent the inflammatory/necrotic effects of NBPs by inhibiting their entry into soft-tissue cells. In addition, our preliminary clinical studies suggest that Eti may be useful for treating BRONJ. Notably, Eti, when administered together with an NBP, reduces the latter's anti-bone-resorptive effect. Here, on the basis of the above background, we examined and compared in vitro interactions of NBPs, non-NBPs, and related substances with hydroxyapatite (HA), and obtained the following results. (i) NBPs bind rapidly to HA under pH-neutral conditions. (ii) At high concentrations, Eti and Clo inhibit NBP-binding to HA and rapidly expel HA-bound NBPs (potency Eti>>Clo). (iii) Pyrophosphate also inhibits NBP-binding to HA and expels HA-bound NBPs. Based on these results and those reported previously, we discuss (i) possible anti-BRONJ strategies involving the use of Eti and/or Clo to reduce jawbone-accumulated NBPs, and (ii) a possible involvement of pyrophosphate-mediated release of NBPs as a cause of BRONJ.

Nickel (Ni) is the most frequent metal allergen and induces Th1-dependent type-IV allergies. In local skin, epidermal Langerhans cells (LCs) and/or dermal dendritic cells (DCs) uptake antigens and migrate to draining lymph nodes (LNs). However, the subsets of antigen-presenting cells that contribute to Ni presentation have not yet been identified. In this study, we analyzed the Ni-binding capabilities of murine DCs using fluorescent metal indicator Newport Green. Elicitation of Ni allergy was assessed after intradermal (i.d.) injection of Ni-treated DCs into ear pinnae of Ni-sensitized mice. The Ni-binding capabilities of MHC class IIhi CD11cint migratory DCs were significantly stronger than those of MHC class IIint CD11chi resident DCs and CD11cint PDCA1+ MHC class IIint B220+ plasmacytoid DCs. Migratory DCs in skin-draining and mandibular LNs showed significantly stronger Ni-binding capabilities than those in mesenteric and medial iliac LNs. An i.d. injection of IL-1β induced the activation of LCs and dermal DCs with strong Ni-binding capabilities. Ni-binding LCs were detected in draining LNs after i.d. challenge with IL-1β and Ni. Moreover, an i.d. injection of Ni-treated DCs purified from skin-draining LNs elicited Ni-allergic inflammation. These results demonstrated that migratory DCs in skin-draining LNs have strong Ni-binding capabilities and elicit Ni allergy.

© 2020, Center for Academic Publications Japan. All rights reserved. Summary Biotin is a water-soluble B-complex vitamin that functions as a cofactor of five carboxylases. Because biotin-dependent carboxylases catalyze indispensable cellular metabolic functions, biotin deficiency is considered to be involved in various pathological conditions. Moreover, biotin supplementation shows pharmacological effects in vivo. However, the precise mechanisms by which biotin deficiency induces pathological conditions remain unclear. Although abnormal metabolites are used as indicators for biotin deficiency, few comprehensive analyses of total metabolites have been reported. In this study, we analyzed the metabolomic profiles of liver extracts prepared from biotin-sufficient (BS) and-defi-cient (BD) mice. Thirteen of 126 metabolites showed significantly different concentrations between liver extracts from BD and BS mice. The concentrations of 5 essential amino acids, Met, Val, Thr, Ile, and Leu, and 2 conditionally essential amino acids, Cys and Tyr were significantly lower in BD mice than in BS mice. Among these, the concentrations of sulfur-containing amino acids, Cys and Met, were more than 1.5-fold lower in BD mice. The concentrations of Met metabolites, such as S-adenosylmethionine and S-adenosylhomocysteine were not significantly different between the two groups. The concentrations of glutathione and its reaction intermediates γ-Glu-Cys tendency to be lower in BD mice. The present study revealed that biotin deficiency induces an abnormal amino acids composition, especially among sulfur-containing amino acids and provide important information on the effect of biotin as a pharmacological agent.

BACKGROUND: Sublingual immunotherapy (SLIT) is used for the treatment of type 1 allergies, such as allergic rhinitis. SLIT leads to tolerance against allergens possibly via the redirection of allergen-specific T helper 2 cells to T helper 1 cells and the generation of peripheral regulatory T (Treg) cells. However, the detailed mechanisms remain unclear. Systemic tolerance to orally administered antigens (oral tolerance) has been extensively investigated. Recent studies have recognized the central role of Treg cells and classical dendritic cells (cDCs) in oral tolerance development. HIGHLIGHT: This review focuses on recent advances in the understanding of the underlying mechanisms of SLIT compared with those of oral tolerance. The sublingual administration of soluble protein antigens has been reported to induce antigen-specific Treg cells in oral mucosa-draining submandibular lymph nodes in mice. The generation of Treg cells is critical for SLIT efficacy because the transfer of SLIT-induced Treg cells confers tolerance against the antigens. A large number of oral cDCs with the CD103-CD11b+ phenotype exert retinoic acid-producing activity and convert naïve CD4+ T cells into Foxp3+ Treg cells in vitro in a transforming growth factor-β-dependent and retinoic acid-dependent manner. Oral CD103-CD11b+ cDCs transport sublingual antigens to submandibular lymph nodes and induce antigen-specific Treg cells. Sublingual antigens enter the mucosa most likely by crossing the sublingual ductal epithelium and are captured by oral antigen-presenting cells, especially macrophages. CONCLUSION: Oral CD103-CD11b+ cDCs are specialized for the induction of Treg cells in mice; thus, targeting their human counterpart may enhance the therapeutic effects of SLIT.

Metabolic immunomodulation involving IL-1 has been investigated for unfavorable metabolic effects, including obesity, but a potentially favorable role for IL-1 remains unclear. Here, we find mechanistic interactions between working skeletal muscles and locally recruited neutrophils expressing IL-1β, which supports muscle performance through priming exercise-dependent GLUT4 translocation. Thus, during exercise, both IL-1α/β-deficient and neutrophil-depleted mice similarly exhibit increased fatigability associated with impaired muscle glucose homeostasis due to GLUT4 dysregulation. Deficiency of IL-1-producing neutrophils results in intrinsic abnormalities represented by aberrant Rac1 signaling and irregular GLUT4-storage vesicles, suggesting that these properties are maintained by local IL-1 produced by recruited neutrophils upon exercise, possibly on a daily basis. We propose that neutrophils are highly engaged in skeletal muscle performance via IL-1 regulation, which coordinates favorable inflammatory microenvironments supporting muscle glucose metabolism.

Background: Nickel (Ni) is the most frequent metal allergen and induces a TH1-dependent type-IV allergy. Although Ni2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. Objective: The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. Methods: BALB/cA mice were sensitized with an i.p. injection of NiCl2 and LPS. Ten days after sensitization, mice were challenged with NiCl2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Results: Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni2+. Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. Conclusions: These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo.

Bisphosphonates (BPs), with a non-hydrolysable P-C-P structure, are cytotoxic analogues of pyrophosphate, bind strongly to bone, are taken into osteoclasts during bone-resorption and exhibit long-acting anti-bone-resorptive effects. Among the BPs, nitrogen-containing BPs (N-BPs) have far stronger anti-bone-resorptive effects than non-N-BPs. In addition to their pyrogenic and digestive-organ-injuring side effects, BP-related osteonecrosis of jaws (BRONJ), mostly caused by N-BPs, has been a serious concern since 2003. The mechanism underlying BRONJ has proved difficult to unravel, and there are no solid strategies for treating and/or preventing BRONJ. Our mouse experiments have yielded the following results. (a) N-BPs, but not non-N-BPs, exhibit direct inflammatory and/or necrotic effects on soft tissues. (b) These effects are augmented by lipopolysaccharide, a bacterial-cell-wall component. (c) N-BPs are transported into cells via phosphate transporters. (d) The non-N-BPs etidronate (Eti) and clodronate (Clo) competitively inhibit this transportation (potencies, Clo&gt;Eti) and reduce and/or prevent the N-BP-induced inflammation and/or necrosis. (e) Eti, but not Clo, can expel N-BPs that have accumulated within bones. (f) Eti and Clo each have an analgesic effect (potencies, Clo&gt;Eti) via inhibition of phosphate transporters involved in pain transmission. From these findings, we propose that phosphate-transporter-mediated and inflammation/infection-promoted mechanisms underlie BRONJ. To treat and/or prevent BRONJ, we propose (i) Eti as a substitution drug for N-BPs and (ii) Clo as a combination drug with N-BPs while retaining their anti-bone-resorptive effects. Our clinical trials support this role for Eti (we cannot perform such trials using Clo because Clo is not clinically approved in Japan).

Dendritic cells (DCs) in lymphoid and non-lymphoid tissues are professional antigenpresenting cells that are essential for effective immunity and tolerance. However, the presence and characteristics of DCs in steady-state salivary glands (SGs) currently remain unknown. We herein identified CD64(-)CD11c(+) classical DCs (cDCs) as well as CD64(+) macrophages among CD45(+) MHC class II+ antigen-presenting cells in steady-state murine SGs. SG cDCs were divided into CD103(+)CD11b(-) and CD103(-)CD11b(+) cDCs. CD103(+)CD11b(-) cDCs expressed XCR1, CLEC9A, and interferon regulatory factor 8, whereas CD103(-)CD11b(+) cDCs strongly expressed CD172a. Both cDC subsets in SGs markedly expanded in response to the Flt3 ligand (Flt3L), were replenished by bone marrow-derived precursors, and differentiated from common DC precursors, but not monocytes. Furthermore, ovalbumin-pulsed SG CD103(+)CD11b(-) cDCs induced the proliferation of naive ovalbumin-specific CD8(+) T cells and production of interferon-gamma from proliferating T cells. SG CD103(+)CD11b(-) cDCs expanded by Flt3L in vivo exhibited the same properties. These results indicate that bona fide cDCs reside in steady-state murine SGs and cDCs with the CD103(+)CD11b(-) phenotype possess antigen cross-presenting capacity. Moreover, Flt3L enhances protective immunity by expanding cDCs. Taken together, SG cDCs might play an important role in maintaining immune homeostasis in the tissues.

© 2016 Intravenously injected lipopolysaccharides (LPS) rapidly induce pulmonary platelet accumulation (PPA) and anaphylaxis-like shock (ALS) in mice. Macrophages reportedly release catecholamines rapidly upon stimulation with LPS. Here, we examined the involvement of macrophage-derived catecholamines in LPS-induced PPA and ALS. A catecholamine or Klebsiella O3 (KO3) LPS was intravenously injected into mice, with 5-hydroxytryptamine in the lung being measured as a platelet marker. The tested catecholamines induced PPA, leading to shock. Their minimum shock-inducing doses were at the nmol/kg level. The effects of epinephrine and norepinephrine were inhibited by prazosin (α1 antagonist) and by yohimbine (α2 antagonist), while dopamine's were inhibited only by prazosin. Use of synthetic adrenergic α1- and/or α2-agonists, platelet- or macrophage-depleted mice, a complement C5 inhibitor and C5-deficient mice revealed that (a) α2-receptor-mediated PPA and shock depend on both macrophages and complements, while α1-receptor-mediated PPA and shock depend on neither macrophages nor complements, (b) the PPA and ALS induced by KO3-LPS depend on α1- and α2-receptors, macrophages, and complements, and (c) KO3-LPS-induced PPA is preceded by catecholamines decreasing in serum. Together, these results suggest the following. (i) Catecholamines may stimulate macrophages and release complement C5 via α2-receptors. (ii) Macrophage-derived catecholamines may mediate LPS-induced PPA and ALS. (iii) Moderate PPA may serve as a defense mechanism to remove excess catecholamines from the circulation by promoting their rapid uptake, thus preventing excessive systemic effects. (iv) The present findings might provide an insight into possible future pharmacological strategies against such diseases as shock and acute respiratory distress syndrome.

© 2017 Society for Mucosal Immunology. Sublingual immunotherapy (SLIT) is a safe and efficient treatment for type 1 allergies; however, the underlying immunological mechanisms, particularly the phenotype of oral antigen-presenting cells (APCs) responsible for the induction of regulatory T (Treg) cells, remain unclear. We show here that the sublingual application of ovalbumin (OVA) induced antigen-specific Foxp3 + Treg cells in draining submandibular lymph nodes (ManLNs). Oral APCs were classified into macrophages, classical dendritic cells (cDCs), and Langerhans cells by flow cytometry. A major subset of oral cDCs with the CD103 - CD11b + phenotype showed retinoic acid (RA)-producing activity and converted naive CD4 + T cells to Foxp3 + Treg cells in a transforming growth factor-β- and RA-dependent manner in vitro. In the ManLNs, migratory CD103 - CD11b + cDCs also showed RA-producing activity. After the sublingual application of fluorescent OVA, fluorescence was detected in oral macrophages in tissues, followed by migratory CD103 - CD11b + cDCs in ManLNs and migratory CD103 - CD11b + cDCs were the main APCs responsible for the induction of sublingual antigen-specific Treg cells. The transfer of OVA-SLIT-induced Treg cells suppressed the OVA-induced hypersensitivity response. These results suggest that oral CD103 - CD11b + cDCs transport sublingual antigens to draining ManLNs and induce antigen-specific Foxp3 + Treg cells, and, thus, provide a rationale for developing cDC-based therapeutic approaches in SLIT.

Bisphosphonates (BPs) are used against diseases with enhanced bone resorption. Those classed as nitrogen -containing BPs (N-BPs) exhibit much stronger anti-bone-resorptive effects than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs carry the risk of inflammatory/necrotic side effects. Depending on their side-chains, BPs are divided structurally into cyclic and non-cyclic types. We previously found in mice that etidronate and clodronate (both non-cyclic non-N-BPs) could reduce the inflammatory effects of all three N-BPs tested (cyclic and non-cyclic types), possibly by inhibiting their entry into soft-tissue cells via SLC20 and/or SLC34 phosphate transporters. Tiludronate is the only available cyclic non-N-BP, but its effects on N-BPs' side effects have not been examined. Here, we compared the effects of etidronate, clodronate, and tiludronate on the inflammatory effects of six N-BPs used in Japan [three cyclic (risedronate, zoledronate, minodronate) and three non-cyclic (pamidronate, alendronate, ibandronate)]. Inflammatory effects were evaluated in mice by measuring the hind-paw-pad swelling induced by subcutaneous injection of an N-BP (either alone or mixed with a non-N-BP) into the hind-paw-pad. All of six N-BPs tested induced inflammation. Etidronate, clodronate, and the SLC20/34 inhibitor phosphonoformate inhibited this inflammation. Tiludronate inhibited the inflammatory effects of all N-BPs except ibandronate and minodronate, which have higher molecular weights than the other N-BPs. The mRNAs of SLC20a1, SLC20a2, and SLC34a2 (but not of SLC34a1 and SLC34a3) were detected in the soft-tissues of hind-paw-pads. These results suggest that etidronate, clodronate, and phosphonoformate may act non-selectively on phosphate transporter members, while tiludronate may not act on those transporting N-BPs of higher molecular weights.

We previously reported that allergic responses to nickel (Ni) were minimal in mice deficient in the histamine-forming enzyme histidine decarboxylase (HDC-KO), suggesting an involvement of histamine in allergic responses to Ni. However, it remains unclear how histamine is involved in the process of Ni allergy. Here, we examined the role of histamine in Ni allergy using a murine model previously established by us. Mice were sensitized to Ni by intraperitoneal injection of a NiCl2-lipopolysaccharide (LPS) mixture. Ten days later, allergic inflammation was elicited by challenging ear-pinnas intradermally with NiCl2. Then, ear-swelling was measured. Pyrilamine (histamine H1-receptor antagonist) or cromoglicate (mast cell stabilizer) was intravenously injected 1 h before the sensitization or the challenge. In cell-transfer experiments, spleen cells from Ni-sensitized donor mice were intravenously transferred into non-sensitized recipient mice. In both sensitized and non-sensitized mice, 1 mM or more NiCl2 (injected into ear-pinnas) induced transient non-allergic inflammation (Ni-TI) with accompanying mast cell degranulation. LPS did not affect the magnitude of this Ni-TI. Pyrilamine and cromoglicate reduced either the Ni-TI or the ensuing allergic inflammation when administered before Ni-TI (at either the sensitization or elicitation step), but not if administered when the Ni-TI had subsided. Experiments on HDC-KO and H1-receptor-KO mice, and also cell-transfer experiments using these mice, demonstrated histamine's involvement in both the sensitization and elicitation steps. These results suggest that mast cell histamine-mediated Ni-TI promotes subsequent allergic inflammatory responses to Ni, raising the possibility that control of Ni-TI by drugs may be effective at preventing or reducing Ni allergy.

Bisphosphonates (BPs) are used against diseases involving increased bone-resorption. Among BPs, nitrogen containing BPs (N-BPs) have much stronger anti-bone-resorptive effects than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs carry the risk of inflammatory/necrotic effects, including osteonecrosis of jawbones. When injected into mouse ear-pinnas, N-BPs induce inflammatory/necrotic effects within the ear-pinna. We previously found that (a) the non-N-BPs clodronate and etidronate can reduce such side effects of N-BPs, and (b) phosphonoformate (an inhibitor of the phosphate transporters SLC20 and SLC34) can reduce the inflammatory/necrotic effects of zoledronate (the N-BP with the highest reported risk of side effects). However, it is not clear (i) whether phosphonoformate can reduce the side effects of other N-BPs, too, and (ii) whether other phosphonocarboxylates have such inhibitory effects. Here, using the mouse ear-pinna model, we compared the effects of etidronate, clodronate, and four phosphonocarboxylates on the inflammatory/necrotic effects of N-BPs of the alkyl type (alendronate) or cyclic type (zoledronate and minodronate). Like phosphonoformate, the other three phosphonocarboxylates protected against the inflammatory/necrotic effects of all the N-BPs. The protective potencies were clodronate&gt;etidronate&gt;phosphonoacetate&gt;phosphonoformate&gt; phosphonopropionate&gt;phosphonobutyrate. With a similar order of potencies, these agents reduced the amount of H-3-alendronate retained within the ear-pinna after its injection therein. The mRNAs of SLC20 and SLC34 were detected in untreated ear-pinnas. These findings suggest that the inhibition of phosphate transporters by phosphonocarboxylates, as well as by etidronate and clodronate, might be a useful preventive strategy against the side effects of both alkyl- and cyclic-type N-BPs.

Bisphosphonates (BPs) are typical anti-bone-resorptive drugs, with nitrogen-containing BPs (N-BPs) being stronger than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs have inflammatory/necrotic effects, while the non-N-BPs clodronate and etidronate lack such side effects. Pharmacological studies have suggested that clodronate and etidronate can (i) prevent the side effects of N-BPs in mice via inhibition of the phosphate transporter families SLC20 and/or SLC34, through which N-BPs enter soft-tissue cells, and (ii) also inhibit the phosphate transporter family SLC17. Vesicular transporters for the pain transmitters glutamate and ATP belong to the SLC17 family. Here, we examined the hypothesis that clodronate and etidronate may enter neurons through SLC20/34, then inhibit SLC17-mediated transport of glutamate and/or ATP, resulting in their decrease, and thereby produce analgesic effects. We analyzed in mice the effects of various agents [namely, intrathecally injected clodronate, etidronate, phosphonoformic acid (PFA; an inhibitor of SLC20/34), and agonists of glutamate and ATP receptors] on the nociceptive responses to intraplantar injection of capsaicin. Clodronate and etidronate produced analgesic effects, and these effects were abolished by PFA. The analgesic effects were reduced by N-methyl-D-aspartate (agonist of the NMDA receptor, a glutamate receptor) and α,β-methylene ATP (agonist of the P2X-receptor, an ATP receptor). SLC20A1, SLC20A2, and SLC34A1 were detected within the mouse lumbar spinal cord. Although we need direct evidence, these results support the above hypothesis. Clodronate and etidronate may be representatives of a new type of analgesic drug. Such drugs, with both anti-bone-resorptive and unique analgesic effects without the adverse effects associated with N-BPs, might be useful for osteoporosis.

© 2016 The Pharmaceutical Society of Japan. Bisphosphonate (BP)-related osteonecrosis of the jaw (BRONJ) can occur when enhanced bone-resorptive diseases are treated with nitrogen-containing BPs (N-BPs). Having previously found, in mice, that the non-N-BP etidronate can (i) reduce the inflammatory/necrotic effects of N-BPs by inhibiting their intracellular entry and (ii) antagonize the binding of N-BPs to bone hydroxyapatite, we hypothesized that etidronate-replacement therapy (Eti-RT) might be useful for patients with, or at risk of, BRONJ. In the present study we examined this hypothesis. In each of 25 patients receiving N-BP treatment, the N-BP was discontinued when BRONJ was suspected and/or diagnosed. After consultation with the physician-in-charge and with the patient's informed consent, Eti-RT was instituted in one group according to its standard oral prescription. We retrospectively compared this Eti-RT group (11 patients) with a non-Eti-RT group (14 patients). The Eti- RT group (6 oral N-BP patients and 5 intravenous N-BP patients) and the non-Eti-RT group (5 oral N-BP patients and 9 intravenous N-BP patients) were all stage 2-3 BRONJ. Both in oral and intravenous N-BP patients (particularly in the former patients), Eti-RT promoted or tended to promote the separation and removal of sequestra and thereby promoted the recovery of soft-tissues, allowing them to cover the exposed jawbone. These results suggest that Eti-RT may be an effective choice for BRONJ caused by either oral or intravenous N-BPs and for BRONJ prevention, while retaining a level of anti-bone-resorption. Eti-RT may also be effective at preventing BRONJ in N-BP-treated patients at risk of BRONJ. However, prospective trials are still required.

4. Nagai, Yasuhiro; Shiraishi, Daisuke; Tanaka, Yukinori; Nagasawa, Yuya; Ohwada, Shyuichi; Shimauchi, Hidetoshi; Aso, Hisashi; Endo, Yasuo; Sugawara, Shunji: Transportation of sublingual antigens across sublingual ductal epithelial cells to the ductal antigen-presenting cells in mice. Clinical &amp; Experimental Allergy, 45(3) : 677-86, 2014. (DOI: 10.1111/cea.12329) (IF = 4.789)

Resin monomers (RMs) are inflammatory agents and are thought to cause allergic contact dermatitis (ACD). However, mouse models are lacking, possibly because of the weak antigenicities of RMs. We previously reported that inflammatory substances can promote the allergic dermatitis (AD) induced by intradermally injected nickel (Ni-AD) in mice. Here, we examined the effects of RMs on Ni-AD. To sensitize mice to Ni, a mixture containing non-toxic concentrations of NiCl2 and an RM [either methyl methacrylate (MMA) or 2-hydroxyethyl methacrylate (HEMA)] was injected intraperitoneally or into ear-pinnae intradermally. Ten days later, a mixture containing various concentrations of NiCl2 and/or an RM was intradermally injected into ear-pinnae, and ear-swelling was measured. In adoptive transfer experiments, spleen cells from sensitized mice were transferred intravenously into non-sensitized recipients, and 24 h later NiCl2 was challenged to ear-pinnae. Whether injected intraperitoneally or intradermally, RM plus NiCl2 mixtures were effective in sensitizing mice to Ni. AD-inducing Ni concentrations were greatly reduced in the presence of MMA or HEMA (at the sensitization step from 10 mM to 5 or 50 mu M, respectively, and at the elicitation step from 10 mu M to 10 or 100 nM, respectively). These effects of RMs were weaker in IL-1-knockout mice and in macrophage-depleted mice. Cell-transfer experiments in IL-1-knockout mice indicated that both the sensitization and elicitation steps depended on IL-1. Challenge with an RM alone did not induce allergic ear-swelling in mice given the same RM + NiCl2 10 days before the challenge. These results suggest that RMs act as adjuvants, not as antigens, to promote Ni-AD by reducing the AD-inducing concentration of Ni, and that IL-1 and macrophages are critically important for the adjuvant effects. We speculate that what were previously thought of as RM-ACD might include ACD caused by antigens other than RMs that have undergone promotion by the adjuvant effects of RMs.

Periodontal disease is a chronic inflammatory disease that results in the breakdown of the tooth-supporting tissues, and can ultimately lead to resorption of the alveolar bone. Recently, several studies have shown a close relationship between increased interleukin-18 (IL-18) levels and the pathogenesis of chronic periodontitis, a major cause of tooth loss. However, it has yet to be shown whether chronic periodontitis results from or causes an increase in IL-18 after bacterial infection. In the present study, we investigated how IL-18 overexpression relates to periodontal disease using IL-18 transgenic (Tg) mice. IL-18Tg and wild-type mice were inoculated intraorally with Porphyromonas (P.) gingivalis, which has been implicated in the etiology of chronic periodontitis. Seventy days after P. gingivalis infection, alveolar bone loss and gingival cytokine levels were assessed using histo-morphological analysis and enzyme-linked immuno-absorbent assay, respectively. Periodontal bone loss was evoked in IL-18Tg mice, but not in wild-type mice. Interestingly, levels of bone-resorptive cytokines, including IL-1 alpha, IL-1 beta, tumor necrosis factor-alpha, and IL-6, were unchanged in the gingival tissues of IL-18Tg mice infected with P. gingivalis, although levels of interferon gamma (a proinflammatory T-helper 1 cytokine) decreased. RT-PCR analysis showed elevated expression of mRNAs for receptor activator of nuclear factor kappa-B ligand (a key stimulator of osteoclast development and activation) and CD40 ligand (a marker of T cell activation) in the gingiva of IL-18Tg mice infected with P. gingivalis. We conclude that increased IL-18 in the gingival tissues evokes chronic periodontitis after bacterial infection, presumably via a T cell-mediated pathway.

従来、全身性アナフィラキシーはIgE、肥満細胞、ヒスタミンに依存すると考えられてきた(古典経路)。しかし、マウスモデルを用いた最近の研究から、IgGを介した代替経路の存在が明らかとなった。代替経路は血小板活性化因子(PAF)に依存し、単球/マクロファージや好塩基球、好中球が関与し得る。意外なことに、能動的全身性アナフィラキシー(ASA)は主に代替経路により引き起こされることが分かってきた。これらの知見がヒトの場合にも当てはまるかどうか、今後の研究が期待される。(著者抄録)

Diseases involving enhanced bone-resorption (e.g., osteoporosis) are widely treated with bisphosphonates (BPs). BPs are of two types: the nitrogen-containing BPs (N-BPs) and the non-nitrogen-containing BPs (non-N-BPs). N-BPs have much stronger anti-bone-resorptive effects than non-N-BPs, and N-BPs can exert inflammatory and necrotic effects, including osteonecrosis of jawbones. Minodronate, an N-BP, was approved in 2009 in Japan for osteoporosis. Its anti-bone-resorptive effect is comparable to that of zoledronate, the N-BP with the strongest anti- bone-resorptive effect and the highest risk of side effects yet reported. Unlike other N-BPs, minodronate has an analgesic effect, and no serious side effects have been documented. Here, to examine whether minodronate lacks inflammatory and/or necrotic effects, we used mice (since the N-BPs tested so far induce such effects in mice with potencies that parallel those reported in humans). To facilitate comparison with previous studies, we gave a single systemic (intraperitoneal) or local (ear pinna) injection of minodronate (or another N-BP). We measured the systemic responses (weight of thoracic exudate, number of inflammatory cells in the peritoneal cavity, and spleen weight) or local responses (area of inflamed skin and incidence of necrosis). Anti-bone-resorptive effects were evaluated by X-ray analysis of tibias following intraperitoneal injection. Minodronate's anti-bone-resorptive effect and its inflammatory and necrotic effects were as great as, or greater than those of zoledronate. Moreover, in cultured human periodontal ligament cells, the cytotoxicity of minodronate was significantly greater than that of zoledronate. These results suggest that caution may be needed with minodronate in clinical use, as with other N-BPs.

The anti-bone-resorptive effects (ABREs) of nitrogen-containing BPs (NBPs) are much more powerful than those of non-nitrogen-containing BPs (nonNBPs). However, NBPs have inflammatory and necrotic side effects (INSEs) including osteonecrosis of jaw bones. Here, we examined antagonism between BPs and structurally related substances in terms of ABREs and INSEs. Our findings suggest that a certain type(s) of transporters or related mechanisms may be involved in the intracellular uptake of NBPs, leading to INSEs.

Few adequate animal models exist for metal-allergies. However, we previously found that lipopolysaccharide (LPS) promote metal-allergies in mice, and we thereby established a reproducible murine metal-allergy model. Information concerning cross-reactivity among metals is still insufficient, especially lacking the studies in vivo. Here, we used this murine model to examine allergic cross-reactions among some metals. In the experiments using ultra-pure metals (Ni, Pd, Co, Cr, and Cu), Ni and Pd cross-reacted, and their minimum allergy-inducing concentrations were similar. However, there were no cross-reactions among other tested metals. Surprisingly, in the experiments using low-purity metals, all the tested metals (Ni, Pd, Co, Cr, and Cu) cross-reacted. Our findings suggest that there is cross-reactivity between Ni and Pd in the tested metal (Ni, Pd, Co, Cr, and Cu) and high-purity metal salts should be considered for use in clinical patch-testing.

Microbial components stimulate innate immunity via Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and/or IL-1. We recently reported that in mice, Escherichia coli lipopolysaccharide (LPS, TLR4-ligand) promotes allergic responses to nickel (Ni) at both the sensitization and elicitation steps. Here, we examined in mice the effects of administering other microbial or inflammatory materials at the Ni-sensitization step. A mixture of 1 mM NiCl2 and a test solution was injected into BALB/c mice intraperitoneally (0.1 ml/10 g body weight), and 10 days later 5 mM NiCl2 was challenged intradermally into the ear pinnas of the mice (20 mu l/ear). The following preparations or substances exhibited adjuvant activities: Prevotella intermedia LPS. Saccharomyces cerevisiae mannan, a synthetic muramyl dipeptide (NOD2-stimulating cell-wall component of bacteria), Pam(3)Cys-SKKKK (TLR2-stimulating synthetic peptide), poly I:C (TLR3-stimulating double-stranded RNA), concanavalin A (a typical T-cell mitogen and T-cell-mediated hepatitis-inducer), heat-killed Propionibacterium acnes (Gram-positive bacterium that causes pimples and induces macrophage-mediated experimental hepatitis), and nitrogen-containing bisphosphonates (chemicals stimulating IL-1 production). Unexpectedly, P. intermedia LPS, which displayed the most potent adjuvant activity among the tested preparations, was effective in TLR4-dysfunctional mutant mice, but not in TLR2-deficient mice, whereas the reverse was true for S. cerevisiae mannan. These results suggest that (i) for the establishment of Ni-allergy in mice, stimulation of innate immunity (including TLRs, NLRs, IL-1 production, and/or other factors) may be important at the sensitization step, and (ii) P. intermedia may produce a substance(s) that potently promotes Ni-allergy via stimulation of TLR2. (C) 2011 Elsevier B.V. All rights reserved.

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment + ConA induced hepatitis. In mice deficient in IL-1 alpha and IL-1 beta, (but not in TNF alpha), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types. (C) 2011 Elsevier B.V. All rights reserved.

During the course of experiments on neutrophil depletion, we happened to find that intravenous injection of antineutrophil monoclonal antibodies (RB6-8C5 and 1A8) induces anaphylaxis-like shock in mice pretreated with lipopolysaccharide. The shock reaction depends largely on neutrophils and Toll-like receptor 4, and significantly on macrophages. Complement C5 may be involved in the shock reaction. We expect that elucidation of behavior of neutrophils may contribute to understand the pathology of septic shock.

For analysis of allergen incorporation mechanism, OVA solution was applied under tongue of the mice, and immunostaining was performed on the sublingual tissues using anti-OVA antibody as primary antibody in immunohistology. We found that the OVA antigen localized around the sublingual lamina propria. To establish of sublingual immunotherapy for anaphylactic shock, sensitized mice that onset anaphylactic shock were vaccinated by sublingual route. Anaphylactic symptom tended to improve by sublingual vaccination.

Mesenchymal stem cells show tolerogenic property. It is also known that periodontal tissues contain many kinds of mesenchymal stem cells, such as the dental pulp stem cells, dental follicle cells, and periodontal ligament stem cells. In this study, we attempted to establish mesenchymal stem cells from the human periodontal tissues and investigated the immunosuppressive function of the cells. A fibroblast-like cell line named H1C1 was established by limiting dilution method and the cell line had abilities to differentiate into adipocytes, osteocytes, and chondrocytes; therefore, we termed H1C1 as the human mesenchymal stem cells. When human PBMC were cocultured with Hid, the population of regulatory T cells (Tregs) was increased compared with monoculture of PBMC, and neutralization of B7-H3 suppressed this induction. These results suggest that the human oral mesenchymal stem cells such as H1C1 have the abilities to suppress immunoreactions by costimulatory molecules and increasing regulatory T cells.

Muramyldipeptide (MDP) is the minimum essential structure responsible for the immunoadjuvant activity of peptidoglycan, and it is known to be a ligand of nuclear-binding domain 2 (NOD2). MDP augments the activity of lipopolysaccharide (LPS), but the mechanism underlying this effect is unclear. Here, we used mice to examine the effects of MDP on endotoxin-shock and the LPS-induced productions of cytolcines. Intravenously injected MDP augmented LPS-induced hypothermia, although mice deficient in IL-1 alpha beta and/or tumor necrosis factor (TNF)-alpha did not exhibit this response. MDP also increased pro-IL-1 beta in tissues, downregulated the expression of suppressor of cytokine signaling (SOCS) I, and augmented the LPS-induced productions of TNF-alpha, IL-12 p40, and IFN-gamma. Moreover, by performing in vivo and in vitro experiments, we obtained evidence that macrophages were involved in these effects of MOP. These results suggest that two different mechanisms may underlie the above augmenting effect of MDP namely stimulation of pro-IL-1 beta production and downregulation of SOCS1 in tissues, which are involved in macrophages.

Nitrogen-containing bisphosphonates (NBPs) have powerful antibone-resorptive effects (ABREs), but they induce unexpected side effect, osteonecrosis of jaw-bones (ONJ). The mechanism underlying NBP-associated ONJ is unclear. Zoledronate, the strongest NBP, exhibits the highest incidence of ONJ. We previously found that clodronate and etidronate (non-NBPs) inhibit NBP-induced inflammation. Here, we found the following. (a) Subcutaneous injection of zoledronate into ear-pinnas induced inflammation and then necrosis at these sites. These effects of zoledronate were strongest among NBPs tested, while non-NBPs lacked these effects. (b) Coinjection of clodronate or etidronate reduced the amount of zoledronate retained within the ear tissue and reduced the inflammatory and necrotic effects of zoledronate. (c) When zoledronate and clodronate were intraperitoneally injected, clodronate little affected the ABRE of zoledronate, as well as those of other NBPs. In contrast, etidronate markedly reduced the ABRE of zoledronate. Notably, etidronate reduced the ABRE of zoledronate even when it was injected 16 h after the injection of zoledronate. These results suggest that (a) clodronate and etidronate may inhibit the entry of NBPs into cells related to inflammation and/or necrosis and prevent NBPs' side effects, (b) clodronate could be useful as a combination drug with NBPs for preventing their side effects while retaining their ABREs, (c) etidronate (but not clodronate) may competitively inhibit the binding of NBPs to bone hydroxyapatite (BHA), and this reagent may reduce NBPs that have already accumulated within bones, and (d) etidronate, if used as a substitution drug for NBPs, may be effective at treating or preventing NBP-associated ONJ.

Few adequate animal models for metal allergies have existed so far. However, we found that lipopolysaccharide (LPS) promotes and augments metal allergies in mice and established a murine model close to the actual situations. Here, we investigated the effects of LPS on metal concentrations and on cross-reactions in this murine model. LPS markedly reduced the threshold concentration at both sensitization and elicitation steps in Ni allergy and at its cross-reactions. Our findings suggest that the bacterial milieu is a critically important factor leading to metal allergies.

Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-alpha production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-alpha levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-alpha were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-alpha expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-alpha production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-kappa B family or AP-1. The TNF-alpha induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-alpha production or that biotin excess down-regulates TNF-alpha production, suggesting that biotin status may influence inflammatory diseases.

Histamine is an important mediator in immune responses, but it is unclear whether periodontal tissues express histamine receptors and are able to respond to histamine. We hypothesized that histamine, inflammatory cytokines, and bacterial components released in inflamed periodontal tissues may be synergistically involved in periodontitis. The present study showed that human gingival fibroblasts mainly express histamine receptor H1R, and responded to histamine to produce interleukin (IL)-8. Stimulation of gingival fibroblasts with tumor necrosis factor-alpha, IL-1 alpha, and lipopolysaccharide markedly induced IL-8 production, and the IL-8 production was synergistically augmented in the presence of or pre- treatment with histamine. Selective inhibitors of mitogen- activated protein kinases (MAPKs), nuclear factor (NF)-kappa B, and phospholipase C (PLC) significantly inhibited the synergistic effect. These results indicate that histamine induces IL-8 production from gingival fibroblasts through H1R, and synergistically augments the inflammatory stimuli by amplification of the MAPK and NF-kappa B through H1R-linked PLC. Abbreviations used: HDC, histidine decarboxylase; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; HR, histamine receptor; PLC, phospholipase C; MAPK, mitogen- activated protein kinase; NF, nuclear factor; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; R, receptor; TLR, Toll-like receptor; alpha-MEM, alpha-minimum essential medium; FCS, fetal calf serum; RT-PCR, reverse-transcriptase polymerase chain-reaction; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; LDH, lactate dehydrogenase.

Microbial infection is thought to modulate allergic disorders, and we previously demonstrated that not only mast cells (which release histamine), but also platelets are involved in the anaphylaxis induced in mice sensitised to ovalbumin (OVA). Here, we examined the effects of a lipopolysaccharide (LPS) from the oral bacterium Preuotellu intermedia (Pi) on OVA-induced anaphylaxis. Upon intraperitoneal co-injection of Pi-LPS plus OVA into BALB/c mice, the Pi-LPS displayed a potent adjuvant effect comparable to that of alum (a standard adjuvant) in terms of its abilities to induce both anaphylactic shock and histamine -release following an antigen (OVA) -challenge. Moreover, an injection of Pi-LPS given to OVA+alumsensitised mice shortly before an OVA-challenge augmented the shock-response. This LPS-pretreatment did not affect histamine -release, but did augment pulmonary platelet accumulation. Histamine was not by itself causal for shock-induction in sensitised mice. These results suggest that oral bacteria and/or their constituents (such as LPS) may help to sensitise the host to an antigen or exacerbate the host's allergic reactions ("aggravation effect"), probably by enhancing the platelet response to the antigen OVA. (C) 2007 Elsevier Ltd. All rights reserved.

IL-18, an important regulator of immune responses, is expressed in activated macrophages and also in nonimmune cells, such as keratinocytes and epithelial cells. Increased levels of serum IL-18 are reported in patients with a wide variety of diseases, but it is unclear which type of cell is the major source of serum IL-18. Here, we showed that the administration of liposomes encapsulating clodronate (Clo-lip) in mice selectively depleted F4/80(+) phagocytic macrophages in the liver and spleen. Serum levels of mature IL-18 with 18 kDa were increased markedly in mice treated with Propionibacterium acnes and LPS, whereas administration of Clo-lip and gadolinium chloride, another widely used macrophage inactivator, showed no obvious effect on serum IL-18 levels, which were marginal in the liver, lung, and spleen and more pronounced in the intestines, especially in the duodenum. Treatment with P. acnes alone induced IL-18 more than twofold in each organ, and P. acnes and LPS induced a marked increase in IL-18 levels in the liver and spleen but decreased in the intestines. The administration of Clo-lip showed only a marginal effect on the IL-18 levels in these organs. Furthermore, serum levels of liver enzymes and TNF-alpha and liver injury (necrotic change and granuloma formation) induced by P. acnes and LPS were reduced moderately by Clo-lip. These results suggest that phagocytic macrophages do not actively contribute to the induction of serum IL-18 and liver injury in mice treated with P. acnes and LPS.

Background Few adequate murine models exist for metal allergies, it being especially difficult to induce Ni allergy in mice. Objective We examined the effect of lipopolysaccharide (LPS) on allergies to Ni and other metals in mice. Methods Ten days after sensitization with a metal salt and LPS, the ears were challenged with the same metal salt. Results LPS+NiCl2 (1 mM) was effective at sensitizing mice to Ni, LPS being effective at very low concentrations whether injected intradermally or intraperitoneally. The ear-swelling response to Ni was more severe and more rapid in C57BL/6 mice than in BALB/c mice. In mast-cell-deficient mice, TNF-alpha-deficient mice, and interestingly even in nude (T cell deficient) mice, NiCl2+LPS induced a Ni allergy similar in degree to that in the respective control mice, but it induced Ni allergy only weakly in TLR4-mutant mice, macrophage-depleted mice, and IL-1-deficient mice. The activity of the histamine-forming enzyme histidine decarboxylase (HDC) in the ears increased in parallel with ear swelling, and HDC-deficient mice were resistant to ear swelling. Challenge with NiCl2+LPS augmented ear swelling (vs. NiCl2 alone). LPS induced effective sensitization to other metals (Cr, Co, Pd, or Ag). Conclusions These results indicate that in mice, LPS is a very important inducer of metal allergies, and potently promotes them (dependent on both innate immunity and HDC induction in cells other than mast cells). We discussed the idea that the bacterial environment is important for the establishment of metal allergies and for their provocation, and that the current thinking (including the contribution of T cells) should be reappraised in future studies.

Lactoferrin (Lf) is a member of the transferrin family of iron-binding anti-bacterial proteins, present in most exocrine secretions, such as saliva, and plays an important role in mucosal defense. In this study, we identified small Lf peptides with Con A low-affinity in the parotid saliva of chronic periodontitis patients by Con A two-dimensional immunoelectrophoresis, Con A affinity chromatography and Western blotting using anti-human Lf polyclonal Ab. N-terminal amino acid sequencing of the four Con A low-affinity Lf peptides confirmed them to be fragments of intact U. The detection ratio of the proteinase 3 (PR3)-like activity was elevated in the parotid saliva of periodontitis patients and was associated with the severity of clinical symptoms. PR3 protein was also detected in the parotid saliva of periodontitis patients, and PR3, but not human leukocyte elastase and cathepsin G, degraded intact Lf. Con A low-affinity saliva Lf peptides showed no anti-bacterial activity against Escherichia coli, and had a reduced iron-chelating capacity. Con A low-affinity saliva Lf peptides, PR3-treated Lf preparation and two of four synthetic polypeptides induced the production of interleukin IL-6, monocyte chemoattractant protein-1 and IL-8, and the activation of NF-kappa B in human oral epithelial HSC-2 cells. Furthermore, concentrations of the Lf peptides in the parotid saliva of periodontitis patients were increased with a correlation to the severity of clinical symptoms. These results suggest that Lf in the parotid saliva of periodontitis patients was degraded into small peptides by the PR3-like activity with the capability to induce inflammatory mediators. (c) 2006 Elsevier Ltd. All rights reserved.

Toll-like receptor (TLR) family members are pattern-recognition receptors and very important molecules in innate immunity. Although TLRs are originally type I transmembrane receptors, soluble forms of TLRs are detected in human plasma and milk. This study showed that soluble TLR2 (sTLR2) is detected in human parotid saliva. Western blotting with anti-TLR2 antibodies (Abs) showed that three polypeptides are detected as sTLR2 with molecular weights of 55, 40 and 27 kDa, respectively. Parotid saliva neutralized the binding of anti-TLR2 polyclonal Ab to cell-surface TLR2 on THP-1, a human monocytic cell line. Inummohistochemical analysis revealed that TLR2 is expressed in serous and interlobular ductal cells of human salivary gland. Human salivary gland cell lines, AZA3 and HSY, constitutively expressed TLR2. Parotid saliva augmented IL-8 production of THP-1 cells stimulated with a synthetic TLR2 ligand, Pam(3)Cys-Ser-(LyS)(4) (PaM3CSK4). Depletion of sCD14 from parotid saliva by immunoprecipitation eliminated the augmentation of IL-8 production, indicating that the augmentable effects depended on sCD14 in parotid saliva. On the other hand, preincubation of Pam(3)CSK(4) with parotid saliva abrogated the augmentation of IL-8 production, indicating that sTLR2 in saliva bound to PaM3CSK4 and neutralized its function. These results suggest that parotid saliva modulates the TLR2-mediated immune responses with binary mechanisms via sTLR2 and sCD14 in the oral cavity. (c) 2006 Elsevier Ltd. All rights reserved.

Nitrogen-containing bisphosphonates (NBPs) are powerful anti-bone-resorptive drugs, but they frequently induce various inflammatory side effects. Recent clinical applications have disclosed an unexpected new side effect, jaw-bone necrosis and exposure. In vitro studies suggest that the inflammatory effects of NBPs are due to V gamma 2V delta 2 T-cells, stimulated directly and/or indirectly [the latter via isopentenylpyrophosphate (IPP) in the mevalonate pathway]. Rats and mice, however, lack V gamma 2V delta 2 T-cells, yet NBPs still induce necrotic and inflammatory reactions. In mice, NBPs induce IL-1-dependent inflammatory reactions, such as inductions of histidine decarboxylase (HDC, the histamine-forming enzyme) in the liver, lung, spleen, and bone marrow, an increase in granulocytic cells in the peritoneal cavity, pleural exudation, and splenomegaly. Here, we examined the involvement of IPP, TNF, macrophages, and T-cells in the inflammatory actions of alendronate (a typical NBP) in mice. Various statins (mevalonate-synthesis inhibitors) suppressed the alendronate-induced HDC inductions, while mevalonate itself augmented such inductions. IPP injection also induced HDC. Like IL-1-deficient mice, TNF-deficient mice were resistant to alendronate-stimulated HDC induction. Alendronate-stimulated HDC inductions were significantly weaker in macrophage-depleted mice and in nude mice than in control mice. Similar, though generally less clear-cut, results were obtained when other alendronate-induced inflammatory reactions were examined. These results suggest that (i) inhibition of the mevalonate pathway causes and/or modifies at least some inflammatory actions of alendronate in mice, (ii) in addition to IL-1, TNF is also involved in the inflammatory actions of alendronate, and (iii) alendronate may act on a variety of cells, including macrophages and T-cells. (c) 2006 Elsevier B.V. All rights reserved.

Background: Previous findings suggest that antigen challenge (AC) may induce histidine decarboxylase (HDC) in cells other than mast cells (MCs) via MC-derived IL-1. We examined this hypothesis. Methods: Mice were sensitized to ovalbumin. After the sensitization, an AC was delivered intravenously. Results: In control mice, AC markedly induced HDC at a postanaphylactic time in the liver, lung, spleen, and ears. In MC-deficient W/W-v mice, AC also induced HDC, although the effect was weaker than in control mice. AC increased IL-1 in the tissues, the pattern being similar in W/W-v and control mice. AC induced HDC similarly in IL-1-deficient and control mice. In control mice, AC decreased histamine in the tissues ( except the liver) for several hours. Conclusion: ( 1) AC induces HDC in both MC-dependent and MC-independent ways. ( 2) AC induces IL-1 mostly in non-MCs, but this IL-1 is not a prerequisite for the induction of HDC by AC. ( 3) HDC induction may contribute to the replenishment of the reduced pool of MC histamine in the anaphylactic period. ( 4) In the case of MC-dependent HDC induction, AC may stimulate MCs in such a way as to induce HDC within the MCs themselves, and/or AC-stimulated MCs may stimulate HDC induction in other cells, which will need to be directly identified in future studies. Copyright (c) 2007 S. Karger AG, Basel.

Elastase activity and concanavalin A (Con A) low affinity bovine lactoferrin (bLf) molecule were detected in mammary gland secretions (MGSs) from mammary glands (MGs) with clinical staphylococcal mastitis. Changes in clinical symptoms correlated with increases in both elastase activity and the concentration of Con A low-affinity Lf in MGSs from mastitic MGs. Bovine Lf treated with elastase (elastase-Lf) showed various small bLf molecules and the same image on Con A two-dimensional immunoelectrophoresis as low Con A affinity bLf in MGSs. We confirmed the presence of four common bLf peptides for the elastase-bLf and low Con A affinity bLf molecules in mastitic MGSs, and synthesized four peptides. Strong mRNA expression of interleukin-6 (IL-6), tumor necrosis factor a (TNF alpha), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) was induced in bovine mammary epithelial cells on stimulation with low Con A affinity bLf, elastase-bLf, and GQRDLLFKDSAL, a synthesis bLf peptide based on nuclear factor kappa B (NF kappa B) activation. These results suggest that bLf was cleaved by elastase, and that this cleavage changed the physical function of Lf. Our results indicate that elastase induced production of low Con A affinity bLf, including the bLf peptide GQRDLLFKDSAL, and had an inflammatory effect on staphylococcal mastitis.

It has been suggested that macrophages and multinucleated giant cells are responsible for phagocytosis of resorbable calcium phosphate (CaP) compounds implanted in bone defects. However, function of macrophages around the CaP, if continuously exposed to various concentration of extracellular calcium ions ([Ca2+](0)), is still unknown. The present study showed that when resorbable octacalcium phosphate was implanted in mouse calvaria, macrophage-like cells were observed around the implant during bone formation. Then, experiments were designed to investigate whether secretion of bone morphogenetic protein 2 (BMP-2) is enhanced by extracellular calcium ions in a macrophage cell line (J774A.1) in vitro. The mRNA expression and the secretion of BMP-2 in J774A.1 cells were significantly increased when incubated in the medium with [Ca2+](0) up to 14 mM. The promotion of mRNA expression was maintained even when incubated with a small amount of minute CaP crystals. The present results suggest that [Ca2+](0), above physiological concentration may stimulate macrophages to induce osteogenic cytokine, such as BMP-2, for bone formation by osteoblasts. (c) 2006 Elsevier Inc. All rights reserved.

Oral epithelium might be the first barrier against oral bacteria in periodontal tissue. We hypothesized that oral epithelium is endowed with innate immune receptors for bacterial components, which play roles in host defense against bacterial infection without being accompanied by excessive inflammatory responses. We found clear expression of Toll-like receptor (TLR) 4 as well as TLR2, and strong expression of NOD1 and NOD2 in normal oral epithelial tissues by immunohistochemical analysis. We also showed that primary oral epithelial cells in culture expressed these molecules using PCR, flow cytometry, and immunostaining. In inflamed oral epithelium, cell-surface localizations of TLR2 and TLR4 were more clearly observed than in healthy tissue. Upon stimulation with synthetic ligands for these receptors, the expression of beta-defensin 2 was markedly up-regulated. These findings indicate that these molecules in oral epithelial cells are functional receptors that induce antibacterial responses.

Nitrogen-containing bisphosphonates (N-BPs), powerful anti-bone-resorptive drugs, have inflammatory side effects, while histamine is not only an inflammatory mediator, but also an immuno-modifier. In murine models, a single intraperitoneal injection of an N-BP induces various inflammatory reactions, including the induction of the histamine-forming enzyme histidine decarboxylase (HDC) in tissues important in immune responses (such as liver, lungs, spleen, and bone marrow). Lipopolysaccharide (LPS) and the proinflammatory cytokines IL-1 and TNF are also capable of inducing HDC. We reported previously that in mice, (i) the inflammatory actions of N-BPs depend on IL-1, (ii) N-BP pretreatment augments both LPS-stimulated IL-1 production and HDC induction, and (iii) the co-administration of clodronate (a non-N-BP) with an N-BP inhibits the latter's inflammatory actions (including HDC induction). Here, we add the new findings that (a) pretreatment with alendronate (a typical N-BP) augments both IL-1- and TNF-induced HDC elevations, (b) LPS pretreatment augments the alendronate-induced HDC elevation, (c) co-administration of clodronate with alendronate abolishes these augmentations, (d) alendronate does not induce HDC in IL-1-deficient mice even if they are pretreated with LPS, and (e) alendronate increases IL-1 p in all tissues tested, but not in the serum. These results suggest that (1) there are mutual augmentations between alendronate and immuno-stimulants (IL-1, TNF, and LPS) in HDC induction, (2) tissue IL-10 is important in alendronate-stimulated HDC induction, and (3) combination use of clodronate may have the potential to reduce the inflammatory effects of alendronate (we previously found that clodronate, conveniently, does not inhibit the anti-bone-resorptive activity of alendronate). (c) 2005 Elsevier Inc. All rights reserved.

Intravenous injection of Klebsiella O3 lipopolysaccharide (LPS) into BALB/c mice induces an anaphylaxis-like shock within minutes. Using 5-hydroxytryptamine as a marker for platelets, we previously suggested that a rapid platelet accumulation in the liver and lung precedes the shock, and that a complement-dependent platelet-degradation is involved in the shock. Here, we examined (i) the effect of platelet-depletion (using an anti-platelet monoclonal antibody) on the shock and (ii) the contribution of macrophages to the platelet-accumulation in those organs. LPS-induced platelet-accumulations in the liver and lung were confirmed by immunostaining. In platelet-depleted mice, the shock was largely prevented. The number of F4/80-positive macrophages was much greater in liver than in lung, and the hepatic macrophages were largely lost in mice given clodronate-encapsulated liposomes. In mice treated with such liposomes, both the LPS-induced accumulation of platelets in the liver (but not in the lung) and the shock were largely prevented, and repopulation of hepatic macrophages restored these LPS-induced responses. These results suggest that (i) platelets are indeed involved in the shock, (ii) Kupffer cells mediate the hepatic platelet accumulation, and (iii) preventing this hepatic accumulation can largely prevent rapid shock being induced by LPS (at the dose used here).

Intravenous injection of Klebsiella O3 lipopolysaccharide (LPS) into BALB/c mice induces an anaphylaxis-like shock within minutes. Using 5-hydroxytryptamine as a marker for platelets, we previously suggested that a rapid platelet accumulation in the liver and lung precedes the shock, and that a complement-dependent platelet-degradation is involved in the shock. Here, we examined (i) the effect of platelet-depletion (using an anti-platelet monoclonal antibody) on the shock and (ii) the contribution of macrophages to the platelet-accumulation in those organs. LPS-induced platelet-accumulations in the liver and lung were confirmed by immunostaining. In platelet-depleted mice, the shock was largely prevented. The number of F4/80-positive macrophages was much greater in liver than in lung, and the hepatic macrophages were largely lost in mice given clodronate-encapsulated liposomes. In mice treated with such liposomes, both the LPS-induced accumulation of platelets in the liver (but not in the lung) and the shock were largely prevented, and repopulation of hepatic macrophages restored these LPS-induced responses. These results suggest that (i) platelets are indeed involved in the shock, (ii) Kupffer cells mediate the hepatic platelet accumulation, and (iii) preventing this hepatic accumulation can largely prevent rapid shock being induced by LPS (at the dose used here). © 2005 Elsevier B.V. All rights reserved.

Activated neutrophils produce serine proteases, which activate cells through proteaseactivated receptor 2 (PAR2). As proteinase 3 (PR3) induces the secretion of interleukin (IL)-18 from epithelial cells in combination with lipopolysaccharide (LPS) in vitro, we examined whether nentrophils, serine proteases, and PAR2 are involved in the induction of serum IL-18 and IL-18-dependent liver injury in mice treated with heat-killed Propionibacterium acnes and LPS. LPS-induced serum IL-18 levels in P. acnes- mice were reduced significantly by anti-Gr-1 injection (depletion of neutrophils and macrophages) but not by a macrophage "suicide" technique, using liposomes encapsulating clodronate. The IL-18 induction was decreased significantly by coadministration of a serine protease inhibitor [Nafamostat mesilate (FUT-175)] with LPS. Serum levels of tumor necrosis factor alpha and liver enzymes induced by P. acnes and LPS were abolished by anti-Gr-1 treatment, and concomitantly, liver injury (necrotic change and granuloma formation) and Gr-1(+) cell infiltration into the liver were prevented by the treatment. A deficiency of PAR2 in mice significantly impaired IL-18 induction by treatment with P. acnes and LPS, and only slight pathological changes in hepatic tissues occurred in the PAR2-deficient mice treated with P. acnes and LPS. Furthermore, coadministration of exogenous murine PR3 or a synthetic PAR2 agonist (ASKH95) with LPS in the anti-Gr-1-treated mice restored the serum IL-18 levels to those in control mice treated with P. acnes and LPS. These results indicate that nentrophil recruitment and PAR2 activation by neutrophil serine proteases are critically involved in the induction of IL-18 and IL-18-dependent liver injury in vivo.

Among the bisphosphonates, the nitrogen-containing bisphosphonates have much stronger anti-bone-resorptive activities than bisphosphonates containing no nitrogen, but nitrogen-containing bisphosphonates mostly have inflammatory side effects. Our previous murine-model experiments with a single intraperitoneal bisphosphonate injection demonstrated that (i) nitrogen-containing bisphosphonates induce various inflammatory reactions via an IL-1-dependent mechanism, (ii) alendronate (an nitrogen-containing bisphosphonate) produces a clear sclerotic line in the tibia that is easily detectable by radiography a few weeks later (tentatively called the bisphosphonate line, a useful marker for the anti-bone-resorptive activities of bisphosphonates), and (iii) clodronate (a non-nitrogen-containg bisphosphonate) reduces the inflammatory reactions induced by alendronate but does not reduce the bisphosphonate line formation induced by alendronate. We compared the effects of clodronate, aspirin and dexamethasone on the inflammatory reactions induced by alendronate (40 mu mol/kg) (induction of the histamine-forming enzyme, accumulation of pleural exudate and splenomegaly) and on the bisphosphonate line formation induced by alendronate (0.1 mu mol/kg). The effects of aspirin (833 mu mol/kg) were weak. However, like clodronate, dexamethasone (10 mu mol/kg, injected 5 min. after alendronate), strongly inhibited the alendronate-induced inflammatory reactions but did not reduce the alendronate-induced bisphosphonate line formation. Alendronate produced normal bisphosphonate lines in IL-1-deficient mice, too. These results suggest that clodronate and/or dexamethasone may be suitable for preventing or reducing the inflammatory side effects of nitrogen-containing bisphosphonates while preserving their powerful anti-bone-resorptive activities (although in practice the known side effects of dexamethasone may limit its use), and that the anti-bone resorptive activities of nitrogen-containing bisphosphonates are not influenced by IL-1.

To investigate the role of human gingival fibroblasts (HGF) in periodontitis, we examined the expression of interleukin-2 receptor (IL-2R) and IL-15R subunits in HGF from normal and inflamed regions and the role of endogenous IL-15 in IL-2-mediated signaling. Normal HGF expressed IL-2Rβ and common γ-chain (γc) but not IL-2Rα or IL-15Rα, whereas inflamed HGF expressed IL-2Rα, IL-15Rα, IL-2Rβ and γc. Exogenous IL-2 and IL-15 induced production of monocyte chemoattractant protein-1 (MCP-1) but not IL-8 in normal HGF, and induced the production of both chemokines in inflamed HGF. Both HGF constitutively transcribed 48aa-IL-15 isoform, and the isoform existed as a membrane-bound form. Pretreatment with anti-IL-15 neutralizing mAb completely inhibited the production of MCP-1 induced by IL-2 and IL-15 and IL-2-induced phosphorylation of Janus tyrosine kinase 1 and 3 in HGF. The pretreatment and RNA interference targeted to IL-15 mRNA resulted in total inhibition of the IL-2Rβ and γc expression at mRNA and protein levels. Furthermore, inhibition of nuclear factor (NF)-κB abrogated the expression of IL-2Rβ and γc, and IL-2-induced-nuclear translocation of NF-κB was completely inhibited by the RNA interference in HGF. These results suggest that endogenous membrane-bound IL-15 sustains recruitment of IL-2Rβ and γc through activation of NF-κB in HGF. © 2005.

Among the bisphosphonates (BPs), the aminobisphosphonates (aminoBPs) have much stronger bone-resorption-inhibitory activities (BRIAs) than nonaminobisphosphonates (nonaminoBPs). However, aminoBPs have inflammatory effects. We previously reported that in mice: (i) all aminoBPs tested (10-40 mu mol/kg) induced various inflammatory reactions (including induction of histidine decarboxylase), whereas clodronate (a non-aminoBP) (10-160 mu mol/kg) inhibited these reactions; and (ii) a clear sclerotic line (tentatively called the BP line) was detectable in the tibia by radiography a few weeks after a single injection of either alendronate (a typical aminoBP) (1.6 mu mol/kg) or clodronate (160 mu mol/kg), and this BP-line formation (a marker for the BRIAs of BPs) was not reduced in mice given both alendronate and clodronate. In this study, using this murine model, we compared clodronate, etidronate (another typical non-aminoBP), alendronate, etidronate + alendronate, and clodronate + alendronate in terms of their inflammatory effects and/or BP-line formation. For BP-line formation, 480 mu mol/kg etidronate was needed (single injection). At 160 mu mol/kg, etidronate inhibited the histidine decarboxylase induction, but not the other inflammatory reactions induced by alendronate. However, etidronate (unlike clodronate) also inhibited alendronate-induced BP-line formation (even at 40 mu mol/kg). Etidronate (160 mu mol/kg) also inhibited the physicochemical changes in the tibia induced by six, weekly injections of alendronate. Therefore, depending on the dose, etidronate can inhibit alendronate's inflammatory actions and its BRIA. These results, together with those reported previously, suggest that a strategy utilizing clodronate (but not etidronate) plus an aminoBP might prevent or reduce the inflammatory side effects induced by aminoBPs while preserving their powerful BRIAs. We discuss the mechanisms underlying the antagonism between aminoBPs and non-aminoBPs.

Within a few minutes of an intravenous injection of a lipopolysaccharide (LPS) into mice, platelets accumulate, largely in the lung. At higher doses, LPS induces rapid shock (within 10 min), leading to death within I h. This type of shock differs from so-called endotoxin shock, in which shock signs and death occur several hours or more later. Here, we found that platelet depletion (by a monoclonal anti-platelet antibody) prevented LPS-induced rapid shock, but increased delayed lethality. In Japan, glycyrrhizin (GL), a compound isolated from licorice, is daily and slowly infused intravenously into chronic hepatitis C patients. A single bolus intravenous injection into mice of GL (200 mg/kg or less) shortly before (or simultaneously with) LPS injection reduced the pulmonary platelet accumulation and the severity of the rapid shock, and prevented death in both the early and later periods. GL itself, at 400 mg/kg, produced no detectable abnormalities in the appearance or activity of mice. Intraperitoneal injection of aspirin or dexamethasone had only marginal effects on LPS-induced platelet responses and lethality. These results suggest that platelets play important roles in the development of both the rapid and delayed types of shock induced by LPS. Although the mechanism by which GL suppresses platelet responses and delayed lethality remains to be clarified, GL might provide a strategy for alleviating the acute respiratory distress syndrome seen in sepsis. Our results may also support the proposal by Cinatl et al. [Cinatl J, Morgenstern B, Bauer G, Chandra P, Ravenau H, Doerr HW. Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 2003; 361: 2045-6.] that GL may be an effective drug against severe acute respiratory syndrome. (C) 2004 Elsevier B.V. All rights reserved.

It has recently been shown that human salivary glands constitutively express CD14, an important molecule in innate immunity, and that a soluble form of CD14 is secreted in saliva. The concentration of CD14 in parotid (a serous gland) saliva was comparable to that in normal serum and 10-fold the amount in whole saliva, although the physiological function of saliva CD14 remained unclear. Actinobacillus actinomycetemcomitans is a periodontopathic bacterium and is able to invade oral epithelial cells. The present study showed that upon exposure to live A. actinomycetemcomitans Y4 for 2 h, human oral epithelial HSC-2 cells produced interleukin-8 (IL-8) for a further 24 h and whole saliva augmented the production induced by A. actinomycetemcomitans Y4. Parotid saliva showed a more pronounced effect on the production of IL-8 than whole saliva. Neither saliva preparation itself had IL-8-inducing activity. Parotid saliva exhibited antibacterial activity against a low concentration of A. actinomycetemcomitans Y4, but recombinant CD14 did not show the activity. The internalization of A. actinomycetemcomitans Y4 into HSC-2 cells was inhibited by cytochalasin B, indicating that the process was actin dependent, and depletion of CD14 from parotid saliva inhibited the invasion and, as a consequence, inhibited production of IL-8. Furthermore, human recombinant CD14 augmented invasion and IL-8 production. These results suggest that saliva CD14 promoted the invasion of oral epithelial cells by A. actinomycetemcomitans and consequently augmented the production of IL-8, playing an important role in innate immunity in the oral cavity.

Cysteine proteinases (gingipains) from Porphyromonas gingivalis are considered key virulence factors of severe periodontitis and host immune evasion. Since expression of intercellular adhesion molecule-1 (ICAM-1) on gingival epithelium is indispensable in polymorphonuclear leukocyte (PMN) migration at the site of periodontitis, we examined the effects of gingipains on the expression of ICAM-1 on human oral epithelial cell lines (KB and HSC-2) by flow cytometry and Western blotting. We found that three purified forms of gingipains efficiently reduced ICAM-1 expression on the cells in a time- and dose-dependent manner. Gingipains reduced the expression on fixed cells and degraded the ICAM-1 in the cell membranes, indicating that the reduction resulted from direct proteolysis. They then disturbed the ICAM-1-dependent adhesion of PMNs to the cells. These results indicate that gingipains cleave ICAM-1 on oral epithelial cells, consequently disrupting PMN-oral epithelial cell interaction, and are involved in immune evasion by the bacterium in periodontal tissues.

To investigate the role of human gingival fibroblasts (HGF), the major constituents of gingival tissue in periodontal inflammatory disease, the expression of interleukin-2 receptor (IL-2R) et, beta and gamma chains was examined. Immunohistochemistry showed a pronounced accumulation of CD8(+) T cells in the inflamed lamina propria of gingival tissue from patients with adult periodontitis. HGF express IL-2Rbeta and IL-2Rgamma at mRNA and protein levels, but the expression of IL-2Ralpha could not be detected, as assessed by reverse transcriptase-polymerase chain reaction and flow cytometry. IL-2Rbeta, and -gamma expressed on HGF were functionally active, as addition of neutralizing anti-IL-2Rbeta and -gamma antibodies caused inhibition of the IL-2-induced production of monocyte chemoattractant protein-1 (MCP-1), and addition of IL-2 induced phosphorylation of Janus tyrosine kinase 3, which is critical in signaling through IL-2Rgamma in HGF. The IL-2-induced MCP-1 production was significantly inhibited by pretreatment with neutralizing antibody to IL-15. Addition of IL-2 also induced a marked up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of HGF, which in turn, significantly augmented the adhesion of human neutrophils, which were inhibited by an anti-ICAM-1 antibody. These results suggest that HGF express functional IL-2Rbetagamma, respond to IL-2 from infiltrated T cells, and actively participate in the inflammatory process in the periodontal region and that IL-15 produced by HGF sustains IL-2-mediated signaling in HGF.

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin-8 (IL-8) responses of the cells to Salmonella lipopolysaccharide, a water-soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll-like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR-related molecules, i.e. TLR2, TLR4, MD-2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL-8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL-8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2- and TLR4-independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti-CD14 MAb.

Lipopolysaccharide (LPS) and interferon (IFN)-gamma synergistically induced interleukin-8 (IL-8) production in human monocytic THP-1 cells. IFN-gamma-primed THP-1 cells produced higher levels of IL-8 on stimulation with LPS than non-primed cells and the level correlated with duration of priming up to 24 h, although the level of IL-8 induced was most comparable to that induced by costimulation with LPS and IFN-gamma. Unstimulated THP-1 cells were shown by flow cytometry to be practically devoid of membrane CD14 (mCD14). LPS and IFN-gamma enhanced mCD14 and Toll-like receptor (TLR) 4 expression in THP-1 cells, respectively, and co-stimulation with LPS and IFN-gamma induced higher levels of mCD14 and TLR4 expression than stimulation with either agent alone. LPS and IFN-gamma alone each augmented MD-2 and MyD88 mRNA expression in THP-1 cells, and costimulation with LPS and IFN-gamma markedly enhanced MD-2 and MyD88 mRNA expression in the cells compared to those with either LPS or IFN-gamma alone. Anti-CD14 and anti-TLR4 monoclonal antibodies almost completely inhibited IL-8 production induced by LPS plus IFN-gamma in THP-1 cells. These findings suggest that combined stimulation of THP-1 cells with LPS and IFN-gamma up-regulate mCD14, TLR4, MD-2 and MyD88 expression by these cells, which might be involved in synergistic IL-8 production by the cells.

The strong association of acute guttate psoriasis and streptococcal throat infection has suggested a role for streptococcal antigens in the pathogenesis of psoriasis. We have reported that psoriatic peripheral blood mononuclear cells (PBMCs) showed significantly lower responses to cytoplasmic membrane-associated protein (CAP) isolated from group A P-hemolytic streptococci, a kind of streptococcal superantigen. The objectives were to evaluate the abnormal cytokine production by psoriatic PBMCs to streptococcal superantigen, CAP. We compared the production of four different cytokines, i.e. IL-4, IL-5, IL-10, and IFN-gamma, by PBMCs between psoriatic patients and healthy controls after stimulation with CAP or two different staphylococcal superantigens, staphylococcal enterotoxin A (SEA) or E (SEE). When PBMCs were stimulated with CAP, the production of IL-10 was significantly lower by psoriatic PBMCs than by those from healthy controls, whereas those of IL-4, IL-5, or IFN-gamma were not different between the two groups. Such a significant decrease in IL-10 production by psoriatic PBMCs was not observed when they were stimulated with staphylococcal superantigens. Flow cytometric analysis of intracytoplasmic IL-10 demonstrated defective IL-10 production by psoriatic PBMCs in both CD3+ T cells and CD14+ monocytes. There was a significant positive correlation between IFN-gamma production by PBMCs and the proliferation of Vbeta8+ T cells preferentially stimulated by CAP. These data demonstrating the defective IL-10 production by psoriatic PBMCs stimulated with streptococcal superantigen seem to explain why only psoriatic patients evolve sustained and Th-1 deviated skin lesions after streptococcal upper respiratory infection.

Arginine-specific cysteine proteinases (gingipains-R) from periodontopathic Porphyromonas gingivalis cleaved CD14, a bacterial pattern recognition receptor, on human gingival fibroblasts (HGF). Consequently, gingipains-R reduced lipopolysaccharide-induced interleukin-8 production by HGF, indicating that gingipains-R inhibited CD14-dependent HGF activation and are involved in immune evasion by the bacterium in periodontal tissues.

Gamma interferon (IFN-gamma)-primed human gingival fibroblasts (HGF) have been shown to produce higher levels of interleukin-8 (IL-8) upon stimulation with bacterial products and inflammatory cytokines than nonprimed controls. In this study, we examined whether priming of HGF with IFN-gamma up-regulates IL-8 production by the cells in response to purified lipopolysaccharide (LPS). The priming effect of IFN-gamma was clearly observed in the high-CD14-expressing (CD14(high)) HGF but not in the low-CD14-expressing (CD14(low)) HGF. The CD14(high) HGF were most effectively primed with IFN-gamma (1,000 IU/ml) for 72 h. To elucidate the mechanism of the priming effects of IFN-gamma for the LPS response by HGF, we examined whether IFN-gamma regulated expression of CD14, Toll-like receptor 2 (TLR2), TLR4, MD-2, and MyD88, all of which are molecules suggested to be associated with LPS signaling. In CD14(high) HGF, IFN-gamma markedly up-regulated CD14 and MyD88 but not TLR4 protein and MD-2 mRNA expression, while in CD14(low) HGF, IFN-gamma slightly increased MyD88 and scarcely affected CD14, TLR4 protein, and MD-2 mRNA levels. LPS-induced IL-8 production by IFN-gamma-primed CD14(high) HGF was significantly inhibited by monoclonal antibodies (MAbs) against CD14 and TLR4, but not by an anti-TLR2 MAb. These findings suggested that IFN-gamma primed CD14(high) HGF to enhance production of IL-8 in response to LPS through augmentation of the CD14-TLR system, where the presence of membrane CD14 was indispensable for the response of HGF to LPS.

Black-pigmented anaerobic bacteria, such as Porphyromonas gingivalis and Prevotella intermedia, are amongst the predominant bacteria in periodontal pockets and have been implicated in periodontal diseases. To elucidate the roles of gingival keratinocytes, which are the first cells encountered by oral bacteria in periodontal diseases, human gingival keratinocytes in primary culture were stimulated with cell-surface components of P. gingivalis and Pr. intermedia. A glycoprotein fraction from Pr. intermedia (PGP) clearly augmented the release of interleukin-8, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, as determined by enzyme-linked immunosorbent assay. This PGP also induced expression of intercellular adhesion molecule-1 (ICAM-1), as determined by flow cytometry. The augmentation of mRNA expression for these molecules was also confirmed by reverse transcription PCR. In contrast, lipopolysaccharide (LPS) from Pr. intermedia and Escherichia coli was completely inactive in these assays. LPS fraction and purified fimbriae from P gingivalis exhibited weak activities. Cytokine production and ICAM-1 expression by gingival keratinocytes might cause accumulation and activation of neutrophils in the epithelium and, therefore, may be involved in the initiation and development of inflammation in periodontal tissues.

The cytokine-inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll-like receptors (TLRs). Tumor necrosis factor alpha (TNF-alpha) production by monocytes was markedly induced in a dose-dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 mug/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF-alpha production than respective preparations from the hyphal form. Only slight TNF-alpha production was induced by the S. cerevisiae glucan. The TNF-alpha production triggered by reference LPS and purified fungal mannans required the presence of LPS-binding protein (LBP), and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan-LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

IL-18, a potent IFN-gamma -inducing cytokine, is expressed by various nonimmune cells as well as macrophages, suggesting that it has important physiological and immunological roles. The present study focused on the mechanism of active IL-18 induction from human oral epithelial cells. The epithelial cells and the cell lines constitutively express IL-18 mRNA and the 24-kDa precursor form of IL-18. Bioactive IL-18 exhibiting IFN-gamma -inducing activity was detected in the supernatant of the cells on costimulation with neutrophil proteinase 3 (PR3) and LPS for 24 h after IFN-gamma -priming for 3 days. An active 18-kDa form of IL-18 was detected in lysate and supernatant of the cells only after the above treatment and the induction was inhibited by cycloheximide and by serine proteinase inhibitors. After the treatment, lactate dehydrogenase activity was not detected in the cell culture supernatant, and PR3 was detected only in the membrane and not in cytoplasm fractions of the cells. Caspase-1 was not detected in the cells even after the treatment and the IL-18 induction was not inhibited by a caspase-1 inhibitor. These results suggest that the PR3-mediated induction of bioactive IL-18 secretion from oral epithelial cells in combination with LPS after IFN-gamma -priming occurred via a caspase-1-independent pathway, and provide new insight into the possible involvement of a neutrophil proteinase in the induction of bioactive IL-18 in oral inflammation such as periodontitis.

Lipopolysaccharide (LPS) preparations from gram-negative black-pigmented bacteria such as Porphyromonas gingivalis and Prevotella intermedia activate cells from non-LPS-responsive C3H/HeJ mice, but it is still unclear whether this activity is due to the unique structure of LPS or to a minor component(s) responsible for the activity in the preparation. A nonendotoxic glycoprotein with bioactivity against cells from C3H/HeJ mice was purified from a hot phenol-water extract of P. intermedia ATCC 25611 and designated Prevotella glycoprotein (PGP). Treatment of human monocytic THP-1 cells with 22-oxyacalcitriol (OCT) induced maturation and marked expression of CD14 on the cells, but the cells constitutively expressed Toll-like receptor 2 (TLR2) and TLR4 on the cells irrespective of the treatment. PGP induced a high level of interleukin-8 production at doses of 100 ng/ml and higher in OCT-treated THP-I cells compared with Salmonella LPS, and the production was significantly inhibited by anti-CD14 and anti-TLR2 but not anti-TLR4 antibodies. Consistent with this, TLR2-deficient murine macrophages did not respond to PGP. It was also shown that PGP activity on the THP-1 cells was LPS-binding protein dependent and was inhibited by a synthetic lipid A precursor IV,. These results indicate that PGP activates monocytic cells in a CD14- and TLR2-dependent manner.

Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4-12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-alpha -inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin 8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like tells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IVA, an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-I cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4 dependent manner, similar to LPS.

An analog of 1 alpha ,25-dihydroxyvitamin D-3, 22-oxyacalcitriol (OCT), differentiated human monocytic THP-1 and U937 cells to express membrane CD14 and rendered the cells responsive to bacterial cell surface components, Both THP-1 and U937 cells expressed Toll-like receptor 4 (TLR4) on the cell surface and TLR4 mRNA in the cells, irrespective of OCT treatment. In contrast, OCT-treated U937 cells scarcely expressed TLR2 mRNA, while OCT-treated THP-1 cells expressed this transcript. Muramyldipeptide (MDP) by itself exhibited only a weak ability to induce secretion of inflammatory cytokines such as interleukin-8 (IL-8) in the OCT-differentiated THP-1 cells but showed marked synergistic effects with Salmonella lipopolysaccharide (LPS) or lipoteichoic acid (LTA) from Staphylococcus aureus, both of which exhibited strong activities. Combinatory stimulation with LPS plus LTA did not show a synergistic effect on OCT-differentiated THP-1 cells. Similar results were observed in OCT-differentiated U937 cells, although combination experiments were carried out only with MDP plus LPS. Anti CD14 monoclonal antibody (MAb) MY4, anti-TLR4 MAb HTA125, and the synthetic lipid A precursor LA-14-PP almost completely inhibited the IL-8-inducing activities of LTA as well as LPS on OCT-treated THP-1 cells, but these treatments increased MDP activity. OCT-treated THP-1 cells primed,vith MDP exhibited enhanced production of IL-8 upon stimulation with LPS, while the cells primed,vith LPS showed no change in production upon stimulation with MDP. MDP up-regulated mRNA expression of an adapter molecule to TLRs, MyD88, to an extent similar to that for LPS in OCT-treated THP-1 cells. These findings suggested that LTA as well as LPS activated human monocytic tells in a CD14- and TLR4-dependent manner, whereas MDP exhibited activity in a CD14-, TLR4-, and probably TLR2-independent manner and exhibited synergistic and priming effects on the cells for cytokine production in response to various bacterial components.

To investigate the mechanisms underlying T-cell responses during superantigen (SAg) stimulation, we analysed the effects of SAg on CD27 expression with or without lipopolysaccharide (LPS) as a novel regulator of T-cell function. CD27 is expressed on the majority of resting peripheral blood T cells (CD27(low)). Activation of T cells by SAg induces high levels of CD27 surface expression (CD27(high)) accompanied with simultaneous CD30 receptor expression. After prolonged activation in vitro, the level of CD27 expression became intermediate. The effects of LPS on down-regulation of CD27(high) expression on CD30(+) T cells were dose-dependent. Separating LPS-stimulated monocytes from T cells by mechanical dispersion abolished its inhibitory effect, indicating the requirement for direct interactions between monocytes and T cells. We also found that SAg up-regulated CD80 expression on CD14(+) monocytes and LPS inhibited SAg-induced CD80 expression after 24 hr of stimulation. Up-regulation of CD152 (CTLA-4) was selective, since it was found to be preferentially expressed on the CD30(+) population. Competitive experiments using soluble blocking peptides showed that addition of CD28 or CD80 peptide recovered LPS-induced down-regulation of CD27(high) expression on CD30(+) T cells. These observations suggested that the presence of low levels of CD80 on monocytes may partially inhibit CD27 expression due to inefficient delivery of positive signals via CD28/CD80 interaction, and that the increased levels of CD80 enhance the inhibition through interactions with CD152 which is expressed at the highest levels after 48 hr of activation.

A superantigen (Streptococcus pyogenes mitogen-2; SPM-2) that stimulates human helper T cells bearing unique types of variable domains of T-cell receptor beta-chain (TCR V beta) was isolated from the culture supernatant of S. pyogenes strain T12. The active molecule isolated by diethylaminoethyl (DEAE)-cellulose chromatography and isoelectric focusing was a protein with a molecular weight (MW) of 29 000 and isoelectric point (pI) of 6.0. This new superantigen was found to activate preferentially V beta 4(+), 7(+), and 8(+) T cells, whereas recombinant streptococcal pyrogenic exotoxin A and C activated V beta 12(+) and V beta 2(+) T cells, respectively, as determined by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. This proliferative response was significantly inhibited by anti-HLA-DR monoclonal antibody, and required paraformaldehyde-fixed antigen-presenting cells (APC), indicating that this action is dependent on major histocompatibility complex (MHC) class II molecules without processing. Analysis of the amino-terminal amino acid sequence of the molecule failed to find any identical or significantly homologous proteins. We have previously reported that cytoplasmic membrane-associated protein (CAP), a streptococcal superantigen isolated from the cell membranes of S. pyogenes T12 strain, stimulated mainly V beta 8(+) T cells. Both SPM-2 and CAP preferentially stimulated helper T cells, and rabbit antiserum against SPM-2 completely neutralized the T-cell-stimulating activities of CAP, suggesting that SPM-2 and CAP belong to a family of streptococcal mitogenic proteins. The SPM-2 activity with stimulation of V beta 8(+) T cells was detected extensively in the culture fluids of group A streptococci, but not in those of other streptococcal species, including groups B and D streptococci, and most of the activities detected were completely inhibited by anti-SPM-2 serum. These results indicate that SPM-2 may be a newly discovered superantigen molecule, which can be commonly synthesized by group A streptococci.

Tumor cells undergo self-destruction when incubated with cytotoxic T-cells (CTL) consistent with the observation that suppression of target protein synthesis causes resistance to apoptosis. Resistance to CTL is also induced by stress, suggesting that pathways exist suppressing apoptosis. Here we examine whether stress induced lysis resistance to CTL and tumor necrosis factor a involves stress proteins GRP78 and GRP94. We show that inhibition of GRP78 synthesis by transfection of cells with grp78 antisense vector pRSV-78WO leads to inability to induce resistance to CTL or tumor necrosis factor a. Resistance induced in untransfected cells is reversible upon stress removal and correlates with GRP78 rephosphorylation, consistent with the notion that phosphorylated GRP78 is nonfunctional. The possibility that GRP78 plays a role in defense against CTL mediated apoptosis is supported by the finding that CTL but not CD4+ cells express a high level of unphosphorylated GRP78. © 1993, American Association for Cancer Research. All rights reserved.

Binding of cytotoxic T lymphocytes (CTL) to specific targets induces cytoskeletal movements in the effector cell followed by delivery of the lethal hit which ultimately results in target cell lysis. The question whether movement of the cytoskeleton in CTL are obligatory for delivery of the lethal hit is not resolved. Here we report that the CTX-B subunit of cholera toxin which is devoid of the catalytic CTX-A subunit inhibits CTL function. Inhibition was found not to be due to interference with TCR expression, CTL-target conjugate formation, target induced transmembrane signalling or secretion of BLT-esterase. CTX-B however does interface with F-actin patch formation at the effector target binding site and inhibits reorientation of the microtubule organizing center and Golgi apparatus towards the target binding site. It is concluded that interference with cytoskeletal movements is responsible for inhibition of cytolysis pointing to an important role of the cytoskeleton in the lytic reaction. © 1993.

The two competitive inhibitors of ADP-ribosylation, nicotinamide and 3-aminobenzamide. have been reported to interfere with TNF-induced cell apoptosis, and there is evidence that they inhibit killer-induced target cell lysis as well. There are very few drugs known to specifically interfere with target apoptosis induced by killer cells. We therefore sought to explore the effects these inhibitors have on CTL-mediated cell lysis. Here we show that TcR-mediated transmembrane signaling in CTL, measured by Ca2+ mobilization and generation of inositol phosphates, is inhibited by nicotinamide. The possibility that all cell functions are suppressed by the drug is excluded by the finding that constitutive secretion of BLT serine esterase is not inhibited, whereas stimulated secretion of this enzyme is suppressed. We also show that nicotinamide does not interfere with CTL target cell binding or reorientation of the Golgi apparatus toward the target binding site. It is concluded that nicotinamide inhibits transmembrane signaling in CTL and thereby interferes with delivery of the lethal hit to targets. © 1991.

The reactions that lead to target cell lysis by cytotoxic T cells (CTL) are despite intensive investigations poorly understood. To examine the relative roles effectors and targets play in the lytic reaction, protein synthesis in either CTL or targets was inhibited before assay of lysis. We show, in agreement with previous results, that de novo protein synthesis is not necessary in either effectors or targets during the cytolytic reaction. However, activation of CTL requires protein synthesis. Activated CTL respond to protein synthesis inhibitors with a cycling of activity, a result that is interpreted to be consistent with a stimulus secretion mechanism. Treatment of targets with protein synthesis inhibitors prior to incubation with CTL leads to a very rapid and irreversible loss of lytic susceptibility. It is shown that the decrease in lysability is not due to lack of proper CTL target interaction: MHC class I antigens are expressed on drug-treated targets and these cells serve as cold targets in competitive inhibition experiments. Moreover, drug-treated targets trigger transient Ca2+ mobilization and generation of inositol phosphates in CTL. It is therefore concluded that drug-treated targets are able to trigger CTL function but lack a component that is required for their successful lysis. © 1990.

The ability of lethally irradiated C57BL/6 mice to acutely reject H-2d bone marrow is due to a lymphocyte population that is NK1+, ASGM1+, CD4-, CD8-, CD3+. Transfer of spleen cells from C57BL/6 mice expressing these antigens into nonresponder 129 mice adoptively transfers the ability to reject H-2d marrow grafts. The specificity of this rejection maps to the H-2D major histocompatibility complex (MHC) region. Transplantation of high doses of H-2d marrow into C57BL/6 overrides the acute rejection mechanism leading to graft survival. During growth of the graft, a cytolytic activity develops that is due to ASGM1+, CD8+ cytolytic T lymphocytes (CTLs) with H-2Ld specificity. The possibility that the ASGM1+, CD8+ CTLs are descendents of the CD3+, NK1+, ASGM1+, CD8- cells responsible for acute rejection is investigated by adoptive cell transfer experiments. We show that beige mice that lack NK1+ cells as well as the ability to acutely reject H-2d marrow fail to generate specific CTLs after transplantation with a high dose of H-2d marrow. Transfer of highly purified NK1+ cells from B6.PL-Ly-2a/Ly-3a (Lyt-2.1) into beige mice together with H-2d marrow leads to generation of Lyt-2.1 CTLs from donor NK1+ cells. These results show that specific CTLs are generated from NK1+ cells during acute marrow graft rejection. © 1990 Springer-Verlag.

The effects of stress on four tumor cell lines are analyzed in view of the possibility that stress protects tumor cells against immune attack. We show that stress causes resistance to CTL and TNF in two cell lines. Induction of resistance is time dependent and reversible and not due to failure of killer cells to interact with stressed targets. It is shown that stress induces stress proteins concomitant with induction of resistance to killer cells and TNF. Moreover experiments are presented suggesting that resistance to either immune effector is due to independent mechanisms. The conclusion that stress can induce mechanisms in targets that interfere with the action of TNF as well as with target lysis following a lethal hit by CTL is discussed.

SATOH, J., SHINTANI, S., NOBUNAGA, T., SUGAWARA, S., MAKINO, S., TOYOTA, T. and GOTO, Y. NOD <i>Mice with High Incidence of Type 1 Diabetes are not T</i> <i>Lymphocytopenic</i>. Tohoku J. exp. Med., 1988, <b>155</b> (2), 151-158 - An autoimmune pathogenesis has been indicated in insulin-dependent (type 1) diabetes mellitus (IDDM). Previously we reported that non-obese diabetic (NOD) mice as an animal model of spontaneously developing IDDM were immunologically characterized by T lymphocytopenia and impaired cellular immunities. The cumulative incidence of diabetes in the T lymphocytopenic female NOD mice was 10-20% by 24 weeks of age. On the other hand, the incidence of diabetes are 80-90% in the female NOD/Shi-Sendai (S), in whom proportion of lymophocyte subsets has not been known yet. Therefore, we examined the spleen cells of female NOD/ Shi-S and female NOD/Shi with high incidence of diabetes, and of female Jcl: ICR as a control. Cell numbers, populations of T cells (Thy 1.2⁺, Lyt-1⁺ and Lyt-2⁺), B cells (surface-Ig⁺), NK cells (acialo GM₁⁺) and responsiveness to Concanavalin A were analyzed as immunological parameters. In contrast to the T lymphocytopenic NOD, these immunological parameters were not impaired in the NOD/Shi-S and NOD/Shi in comparison to those of Jcl: ICR. The results indicate that there may be a positive association between the incidence of diabetes and T cell number and functions in female NOD mice.