N-acetyl cysteine inhibits IL-1α release from murine keratinocytes induced by 2-hydroxyethyl methacrylate.

The hydrophilic compound 2-hydroxyethyl methacrylate (HEMA) is a major component of dental bonding materials, and it enhances the binding of resin-composites to biomolecules. However, HEMA is a well-known contact sensitizer. We reported previously that intradermal injection of HEMA induces the production of IL-1 locally in the skin. Keratinocytes are the first barrier against chemical insults and constitutively express IL-1α. In this study, we analyzed whether HEMA induces the production of inflammatory cytokines from murine keratinocyte cell line Pam212 cells. We demonstrated that HEMA induced the release of 17-kDa mature IL-1α and caused cytotoxicity. The activity of calpain, an IL-1α processing enzyme, was significantly higher in HEMA-treated cells. The thiol-containing antioxidant N-acetyl cysteine (NAC) inhibited HEMA-induced IL-1α release but not cytotoxicity. NAC inhibited intracellular calpain activity and reactive oxygen species (ROS) production induced by HEMA. NAC post-treatment also inhibited IL-1α release and intracellular ROS production induced by HEMA. Furthermore, HEMA-induced in vivo inflammation also inhibited by NAC. NAC inhibited polymerization of HEMA through adduct formation via sulfide bonds between the thiol group of NAC and the reactive double bond of HEMA. HEMA-induced IL-1α release and cytotoxicity were also inhibited if HEMA and NAC were pre-incubated before adding to the cells. These results suggested that NAC inhibited IL-1α release through decreases in intracellular ROS and the adduct formation with HEMA. We concluded that HEMA induces IL-1α release from skin keratinocytes, and NAC may be a promising candidate as a therapeutic agent against inflammation induced by HEMA.

Lipopolysaccharide Priming Exacerbates Anaphylatoxin C5a-Induced Anaphylaxis in Mice.

Anaphylaxis is a serious allergic or hypersensitivity reaction with a sudden onset that can be life-threatening or fatal. Previous studies have highlighted two pathways of anaphylaxis in mice. One is the classical immunoglobulin E (IgE)-mediated pathway that involves mast cells and histamine. The other is an alternative IgG-mediated pathway that involves basophils, monocytes/macrophages, neutrophils, and the platelet-activating factor (PAF). However, little is known about the mechanism by which complement anaphylatoxins contribute to the induction of anaphylaxis. Infection is a cofactor that potentially amplifies the risk of anaphylaxis. Here, we showed that priming with a lipopolysaccharide (LPS), which mimics bacterial infection, exacerbates anaphylatoxin C5a-induced anaphylaxis in mice. LPS plus C5a-induced anaphylaxis was mediated by histamine and lipid mediators, especially PAF. Cell depletion experiments demonstrated that LPS plus C5a-induced anaphylaxis depended on monocytes/macrophages, basophils, and neutrophils. These results suggest that C5a is a potent inducer of anaphylaxis in bacterial infections. Remarkably, the molecular and cellular mediators of LPS plus C5a-induced anaphylaxis are mostly shared with IgE- and IgG-mediated anaphylaxis. Therefore, combined inhibition of histamine and PAF may be beneficial as a second-line treatment for severe anaphylaxis.

IL-33 induces histidine decarboxylase, especially in c-kit+ cells and mast cells, and roles of histamine include negative regulation of IL-33-induced eosinophilia.

OBJECTIVE AND METHODS: IL-33 is present in endothelial, epithelial, and fibroblast-like cells and released upon cell injury. IL-33 reportedly induces mast-cell degranulation and is involved in various diseases, including allergic diseases. So, IL-33-related diseases seem to overlap with histamine-related diseases. In addition to the release from mast cells, histamine is newly formed by the induction of histidine decarboxylase (HDC). Some inflammatory and/or hematopoietic cytokines (IL-1, IL-3, etc.) are known to induce HDC, and the histamine produced by HDC induction is released without storage. We examined the involvement of HDC and histamine in the effects of IL-33. RESULTS: A single intraperitoneal injection of IL-33 into mice induced HDC directly and/or via other cytokines (including IL-5) within a few hours in various tissues, particularly strongly in hematopoietic organs. The major cells exhibiting HDC-induction were mast cells and c-kit+ cells in the bone marrow. HDC was also induced in non-mast cells in non-hematopoietic organs. HDC, histamine, and histamine H4 receptors (H4Rs) contributed to the suppression of IL-33-induced eosinophilia. CONCLUSION: IL-33 directly and indirectly (via IL-5) induces HDC in various cells, particularly potently in c-kit+ cells and mature mast cells, and the newly formed histamine contributes to the negative regulation of IL-33-induced eosinophilia via H4Rs.

Histamine acts via H4-receptor stimulation to cause augmented inflammation when lipopolysaccharide is co-administered with a nitrogen-containing bisphosphonate.

OBJECTIVE AND METHODS: Nitrogen-containing bisphosphonates (NBPs, anti-bone-resorptive agents) have inflammatory side-effects. Alendronate (Ale, an NBP) intradermally injected into mouse ear-pinnae together with LPS (bacterial cell-wall component) induces augmented ear-swelling that depends on IL-1 and neutrophils. Using this model, we examined histamine's involvement in Ale + LPS-induced inflammation. RESULTS: Ale increased histamine in ear-pinnae by inducing histidine decarboxylase (HDC). This induction was augmented by LPS. In HDC-deficient mice, such augmented ear-swelling was not induced. At peak-swelling, 74.5% of HDC-expressing cells were neutrophils and only 0.2% were mast cells (MCs). The augmented swelling was markedly reduced by a histamine H4-receptor (H4R) antagonist, but not by an H1R antagonist. In MC-deficient mice, unexpectedly, Ale + LPS induced prolonged ear-swelling that was augmented and more persistent than in normal mice. MCs highly expressed H4Rs and produced MCP-1(inflammatory cytokine that recruits macrophages) and IL-10 (anti-inflammatory cytokine) in response to an H4R agonist. CONCLUSION: Histamine produced by HDC-induction mainly in infiltrated neutrophils stimulates H4Rs, leading to augmented Ale + LPS-induced ear-swelling via MCP-1 production by MCs. Since MCP-1 is produced by other cells, too, the contribution of MCs and their H4Rs to augmented ear-swelling is partial. In the later phase of the swelling, MCs may be anti-inflammatory via IL-10 production.

Release of Nitrogen-Containing Bisphosphonates (NBPs) from Hydroxyapatite by Non-NBPs and by Pyrophosphate.

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.

Nitrogen-containing bisphosphonates and lipopolysaccharide mutually augment inflammation via adenosine triphosphate (ATP)-mediated and interleukin 1ß (IL-1ß)-mediated production of neutrophil extracellular traps (NETs).

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 pinnae 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 (LPSs; cell-surface constituent of gram-negative bacteria, including oral bacteria), but prevented by inhibitors of the phosphate transporters of solute carrier 20/34 (SLC20/SLC34); (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 interleukin 1ß (IL-1ß); (v) adenosine triphosphate (ATP) was involved in both phases, and Ale alone or Ale+LPS increased ATP in ear pinnae; (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-1ß-producing cells, and upon stimulation with IL-1ß, neutrophils produced NETs; (viii) stimulation of the purinergic 2X7 (P2X7) receptors by ATP induced IL-1ß in ear pinnae; (ix) NET formation by Ale+LPS was confirmed in gingiva, too. These results suggest that (i) N-BPs induce both early-phase and late-phase inflammation via ATP-production and P2X7 receptor stimulation; (ii) N-BPs and LPS induce mutually augmenting responses both early and late phases via ATP-mediated IL-1ß production by neutrophils, macrophages, and/or dendritic cells; and (iii) NET production by IL-1ß-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. © 2021 American Society for Bone and Mineral Research (ASBMR).

Migratory dendritic cells in skin-draining lymph nodes have nickel-binding capabilities.

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-1ß 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-1ß 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.

[Basic Studies on the Mechanism, Prevention, and Treatment of Osteonecrosis of the Jaw Induced by Bisphosphonates].

Since the first report in 2003, bisphosphonate-related osteonecrosis of the jaw (BRONJ) has been increasing, without effective clinical strategies. Osteoporosis is common in elderly women, and bisphosphonates (BPs) are typical and widely used anti-osteoporotic or anti-bone-resorptive drugs. BRONJ is now a serious concern in dentistry. As BPs are pyrophosphate analogues and bind strongly to bone hydroxyapatite, and the P-C-P structure of BPs is non-hydrolysable, they accumulate in bones upon repeated administration. During bone-resorption, BPs are taken into osteoclasts and exhibit cytotoxicity, producing a long-lasting anti-bone-resorptive effect. BPs are divided into nitrogen-containing BPs (N-BPs) and non-nitrogen-containing BPs (non-N-BPs). N-BPs have far stronger anti-bone-resorptive effects than non-N-BPs, and BRONJ is caused by N-BPs. Our murine experiments have revealed the following. N-BPs, but not non-N-BPs, exhibit direct and potent inflammatory/necrotic effects on soft-tissues. These effects are augmented by lipopolysaccharide (the inflammatory component of bacterial cell-walls) and the accumulation of N-BPs in jawbones is augmented by inflammation. N-BPs are taken into soft-tissue cells via phosphate-transporters, while the non-N-BPs etidronate and clodronate inhibit this transportation. Etidronate, but not clodronate, has the effect of expelling N-BPs that have accumulated in bones. Moreover, etidronate and clodronate each have an analgesic effect, while clodronate has an anti-inflammatory effect via inhibition of phosphate-transporters. These findings suggest that BRONJ may be induced by phosphate-transporter-mediated and infection-promoted mechanisms, and that etidronate and clodronate may be useful for preventing and treating BRONJ. Our clinical trials support etidronate being useful for treating BRONJ, although additional clinical trials of etidronate and clodronate are needed.

Interleukin-1 and histamine are essential for inducing nickel allergy in mice.

BACKGROUND: We previously reported that (a) lipopolysaccharide (LPS) is a potent adjuvant for inducing Nickel (Ni) allergy in mice at both the sensitization and elicitation steps, (b) LPS induces Interleukin-1 (IL-1) and histidine decarboxylase (HDC, the histamine-forming enzyme), and IL-1 induces HDC, (c) Ni allergy is induced in mast cell-deficient, but not IL-1-deficient (IL-1-KO) or HDC-KO mice. OBJECTIVE: To examine the roles of IL-1 and HDC (or histamine) and their interrelationship during the establishment of Ni allergy. METHODS: Ni (NiCl2 ) 1 mmol/L containing IL-1ß and/or histamine was injected intraperitoneally (sensitization step). Ten days later, test substance(s) were intradermally injected into ear pinnas (elicitation step), and ear swelling was measured. RESULTS: In wild-type mice, Ni + LPS or Ni + IL-1ß injection at sensitization step followed by Ni alone at elicitation step induced Ni allergy. In IL-1-KO, injection of Ni + IL-1ß (but not Ni + histamine) was required at both sensitization and elicitation steps to induce Ni allergy. In HDC-KO, Ni + IL-1ß + histamine at sensitization step followed by Ni + histamine at elicitation step induced Ni allergy. In histamine H1 receptor-deficient mice, IL-1ß induced HDC, but was ineffective as an adjuvant for inducing Ni allergy. In wild-type mice, injection into ear pinnas of Ni 10 mmol/L alone or Ni 1 mmol/L + LPS induced IL-1ß, HDC and a prolonged swelling of ear pinnas. In non-sensitized mice, injection of IL-1ß by itself into ear pinnas in IL-1-KO mice induced prolonged ear swelling. Ni augmented IL-1 production (both IL-1α and IL-1ß) and HDC induction in wild-type mice sensitized to Ni. CONCLUSIONS: In mice: (a) for inducing Ni allergy, IL-1 is essential at both the sensitization and elicitation steps, and HDC induction is involved in the effect of IL-1, (b) stimulation of H1 receptor is also essential for inducing Ni allergy at both sensitization and elicitation steps, and (c) the 'sensitization to Ni' state may be a state where tissues are primed for augmented production of IL-1α and/or IL-1ß in response to Ni. (within 300 words, now 300).

Augmentation of Lipopolysaccharide-Induced Production of IL-1α and IL-1ß in Mice Given Intravenous Zoledronate (a Nitrogen-Containing Bisphosphonate) and Its Prevention by Clodronate (a Non-nitrogen-containing Bisphosphonate).

Bisphosphonates (BPs) bind strongly to bone and exhibit long-acting anti-bone-resorptive effects. Among BPs, nitrogen-containing BPs (N-BPs) have far stronger anti-bone-resorptive effects than non-N-BPs. However, N-BPs induce acute inflammatory reactions (fever, arthralgia and myalgia, etc.) after their first injection. The mechanisms underlying these side effects remain unclear. Zoledronate (one of the most potent N-BPs) is given intravenously to patients, and the side-effect incidence is reportedly the highest among N-BPs. Our murine experiments have clarified that (a) intraperitoneally injected N-BPs induce various inflammatory reactions, including a production of interleukin-1 (IL-1) (a typical inflammatory cytokine), and these inflammatory reactions are weak in IL-1-deficient mice, (b) subcutaneously injected N-BPs induce inflammation/necrosis at the injection site, (c) lipopolysaccharide (LPS; a cell-wall component of Gram-negative bacteria) and N-BPs mutually augment their inflammatory/necrotic effects, (d) the non-N-BP clodronate can reduce N-BPs' inflammatory/necrotic effects. However, there are few animal studies on the side effects of intravenously injected N-BPs. Here, we found in mice that (i) intravenous zoledronate exhibited weaker inflammatory effects than intraperitoneal zoledronate, (ii) in mice given intravenous zoledronate, LPS-induced production of IL-1α and IL-1ß was augmented in various tissues, including bone, resulting in them increasing in serum, and (iii) clodronate (given together with zoledronate) prevented such augmentation and enhanced, slightly but significantly, zoledronate's anti-bone-resorptive effect. These results suggest that infection may be a factor promoting the acute inflammatory side effects of N-BPs via augmented production of IL-1 in various tissues (including bone), and that clodronate may be useful to reduce or prevent such side effects.

Porphyromonas gingivalis induces the production of interleukin-31 by human mast cells, resulting in dysfunction of the gingival epithelial barrier.

Interleukin (IL)-31 is important for innate immunity in mucosal tissues and skin, and increased IL-31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL-31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL-31 expression in the gingival tissues of wild-type mice but not in those of mast cell-deficient mice. The P. gingivalis-induced IL-31 production by human mast cells occurred through the activation of the JNK and NF-κB signalling pathways and was dependent on the P. gingivalis lysine-specific protease gingipain-K. P. gingivalis infection induced IL-31 receptor α and oncostatin M receptor ß expression in human gingival epithelial cells. Notably, the P. gingivalis-induced IL-31 production by mast cells led to the downregulation of claudin-1, a tight junction molecule, in gingival epithelial cells, resulting in an IL-31-dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL-31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.

Mouse Model of Hydroquinone Hypersensitivity via Innate and Acquired Immunity and its Promotion by Combined Reagents.

We established a mouse model of contact hypersensitivity (CHS) to hydroquinone (HQ), a widespread chemical in our environment. HQ was painted onto flanks; then, HQ was challenged by painting onto ear pinnas on days 7 and 14. The CHS after the second challenge was markedly greater than that after the first challenge. Both challenges increased thymic stromal lymphopoietin and T helper type 2 cytokines in ear pinnas, whereas IFN-γ (typical T helper type 1 cytokine) was decreased, despite an increase in IL-18 (typical IFN-γ inducer). In nude mice (T cell-reduced), although a first challenge induced CHS, a second challenge did not augment it. In severe combined immunodeficient, severe combined immunodeficient-beige, and IL-1-deficient mice, CHS was not induced. However, CHS was inducible in severe combined immunodeficient-beige mice after transfer of natural killer cells from HQ-sensitized normal mice. Tretinoin (used for enhancing the skin-whitening effect of HQ) and resin monomers (used to prevent polymerization of HQ) lowered the HQ concentration needed to establish sensitization to HQ. The augmented CHS after a second challenge was reduced by JNJ7777120, dexamethasone, suplatast tosilate (T helper type 2-cytokine inhibitor), and anti-thymic stromal lymphopoietin antibody. These results suggest that (i) thymic stromal lymphopoietin, IL-1, and T and/or natural killer cells are important in establishing and augmenting CHS to HQ and (ii) inflammatory chemicals may promote CHS to HQ as adjuvants.

Mast cell histamine-mediated transient inflammation following exposure to nickel promotes nickel allergy in mice.

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.

Inflammatory and necrotic effects of minodronate, a nitrogen-containing bisphosphonate, in mice.

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.

Metal allergens induce nitric oxide production by mouse dermal fibroblasts via the hypoxia-inducible factor-2α-dependent pathway.

Nickel (Ni) has been shown to be one of the most frequent metal allergens. We have already reported a murine metal allergy model with pathogen-associated molecular patterns (PAMPs) as adjuvants. Interleukin (IL)-1ß plays a critical role in our mouse model. Because nonimmune cells, including fibroblasts, play important roles in local allergic inflammation, we investigated whether Ni induces inflammatory responses in mouse dermal fibroblasts (MDF). We also analyzed the synergistic effects between Ni, PAMPs, and IL-1ß. MDF stimulated with Ni produced a significantly higher amount of nitric oxide (NO) in a dose-dependent manner. NO production was augmented by costimulation with IL-1ß but not with PAMPs. On the other hand, IL-1ß or PAMPs induced a significantly higher amount of IL-6 production by MDF, but no augmentation was detected in the presence of Ni. A specific inhibitor for inducible nitric oxide synthase (iNOS) inhibited Ni-induced NO production. iNOS mRNA expression was significantly higher in MDF stimulated with Ni, IL-1ß, or both. A specific inhibitor for hypoxia-inducible factor (HIF)-2α, but not HIF-1α, inhibited NO production. Another frequent metal allergen, cobalt, also induced iNOS expression and NO production by MDF via the HIF-2α-dependent pathway. The inhibitor for iNOS augmented ear swelling in Ni allergy mouse model. On the other hand, HIF-2α inhibitor attenuates allergic inflammation. These results indicate that metal allergens induce NO production in MDF via the HIF-2α-dependent pathway and IL-1ß augments NO production, which suggests that the NO induced by metal allergens plays a pathological role in metal allergies.