A nerve-goblet cell association promotes allergic conjunctivitis through rapid antigen passage.

The penetration of allergens through the epithelial layer is the initial step in the development of allergic conjunctivitis. Although pollinosis patients manifest symptoms within minutes after pollen exposure, the mechanisms of the rapid transport of the allergens remain unclear. In the present study, we found that the instillation of pollen shells rapidly induces a large number of goblet cell-associated antigen passages (GAPs) in the conjunctiva. Antigen acquisition by stromal cells, including macrophages and CD11b+ dendritic cells, correlated with surface GAP formation. Furthermore, a substantial amount of antigen was transported to the stroma during the first 10 minutes of pollen exposure, which was sufficient for the full induction of an allergic conjunctivitis mouse model. This inducible, rapid GAP formation and antigen acquisition were suppressed by topical lidocaine or trigeminal nerve ablation, indicating that the sensory nervous system plays an essential role. Interestingly, pollen shell-stimulated GAP formation was not suppressed by topical atropine, suggesting that the conjunctival GAPs and intestinal GAPs are differentially regulated. These results identify pollen shell-induced GAP as a therapeutic target for allergic conjunctivitis.

Development of mouse model for oral allergy syndrome to identify IgE cross-reactive pollen and food allergens: ragweed pollen cross-reacts with fennel and black pepper.

Oral allergy syndrome (OAS) is an IgE-mediated immediate food allergy that is localized to the oral mucosa. Pollen food allergy syndrome (PFAS), a pollinosis-associated OAS, is caused by cross-reactivity between food and pollen allergens. However, we need to more precisely understand the underlying pathogenesis of OAS/PFAS. In the present study, we developed a method to comprehensively identify cross-reactive allergens by using murine model of OAS and protein microarray technology. We focused on lip angioedema, which is one of the most common symptoms of OAS, and confirmed that mast cells reside in the tissues inside the lower lip of the mice. Interestingly, when the food allergen ovalbumin (OVA) was injected inside the lower lip of mice with high levels of OVA-specific IgE followed by an intravenous injection of the Evans blue dye, we found immediate dye extravasation in the skin of the neck in a mast cell-dependent manner. In addition, the degree of mast cell degranulation in the oral cavity, reflecting the severity of oral allergic responses, can be estimated by measuring the amount of extravasated dye in the skin. Therefore, we used this model of OAS to examine IgE cross-reactive allergens in vivo. Protein microarray analysis showed that serum IgE from mice intraperitoneally sensitized with ragweed pollen, one of the major pollens causing pollinosis, bound highly to protein extracts from several edible plants including black peppercorn and fennel. We confirmed that the levels of black pepper-specific IgE and fennel-specific IgE were significantly higher in the serum from ragweed pollen-sensitized mice than in the serum from non-sensitized control mice. Importantly, analysis of murine model of OAS showed that the injection of black pepper or fennel extract induced apparent oral allergic responses in ragweed pollen-sensitized mice. These results indicate IgE cross-reactivity of ragweed pollen with black pepper and fennel. In conclusion, we developed mouse model of OAS to identify IgE cross-reactive pollen and food allergens, which will help understand the pathogenesis of OAS/PFAS.

Pollen shells and soluble factors play non-redundant roles in the development of allergic conjunctivitis in mice.

PURPOSE: We aimed to clarify the role of particulate allergen exposure to the conjunctiva in the development of allergic conjunctivitis. METHODS: We administered ragweed pollen suspension, pollen extract, pollen shell, particulate air pollutants, and their combinations to the mouse conjunctiva five days a week without prior sensitization. Clinical signs were scored. Histological changes, cellular infiltrations, mRNA expressions, lymph node cell recall responses, and serum immunoglobulin levels were assessed. Immune cell-depleting antibodies and ST2 knockout mice were used to investigate the cellular and molecular requirements. RESULTS: Pollen suspension, but not the extract or shell alone, induced robust eosinophilic conjunctivitis, accompanied by a proliferative response of epithelial cells. A combination of pollen extract and shell completely restored eosinophil accumulation. In addition, eosinophilic conjunctivitis was induced by a mixture of particulate air pollutants and pollen extract. Mechanistically, eosinophil accumulation was ameliorated by deficiency of the IL-33 receptor ST2 and abolished by depleting CD4+ T cells. Pollen shells, but not the extract, induced IL-33 release from conjunctival epithelial cells in vivo. CONCLUSIONS: Our results indicate the non-redundant roles for the allergens' particulate properties and soluble factors in the development of allergic conjunctivitis.

Topical Gynura procumbens as a Novel Therapeutic Improves Wound Healing in Diabetic Mice.

Nonhealing wounds are major socioeconomic challenges to healthcare systems worldwide. Therefore, there is a substantially unmet need to develop new drugs for wound healing. Gynura procumbens, a herb found in Southeast Asia, may be an effective therapeutic for nonhealing diabetic wounds. The aim of this study was to evaluate the efficacy of G. procumbens on wound healing in the diabetic milieu. G. procumbens extract was obtained using 95% ethanol and its components were determined by thin layer chromatography. Diabetes was induced in mice using streptozotocin. We found that G. procumbens extract contained stigmasterol, kaempferol and quercetin compounds. Topical application of G. procumbens on the wounded skin of diabetic mice accelerated wound healing and induced the expression of angiogenin, epidermal growth factor, fibroblast growth factor, transforming growth factor and vascular endothelial growth factor. Furthermore, G. procumbens promoted in vitro wound healing and enhanced the migration and/or proliferation of human endothelial cells, fibroblasts, keratinocytes and mast cells cultured in diabetic conditions. Finally, G. procumbens promoted vascular formation in the diabetic mice. To the best of our knowledge, this is the first study that evaluates in vivo wound healing activities of G. procumbens and activation of cells involved in wound healing process in diabetic conditions. The findings that G. procumbens accelerates wound healing and activates cells involved in the wound healing process suggest that G. procumbens might be an effective alternative therapeutic option for nonhealing diabetic wounds.

The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide-dependent neutrophil recruitment.

Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b-/- mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b+ inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.

A water-soluble derivative of propolis augments the cytotoxic activity of natural killer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis, a resinous material collected from numerous plants by honeybees, has historically been used as a health-promoting food. Recently, due to its potential anti-tumor effects, use of propolis has been proposed as an adjuvant therapy to chemotherapy; however, the effects of propolis on immune responses remain unclear. AIM OF THE STUDY: In this study, we examined the effects of the oral ingestion of propolis on natural killer (NK) cell activity, which is important in immune surveillance against cancer and viral infections. In addition, we assessed the effects of the major components of the water-soluble powder derivative of propolis (WPP). MATERIALS AND METHODS: C57BL/6 (B6) wild-type (WT) and RAG 2-deficient (RAG-/-) mice and BALB/c WT, interferon (IFN)-γ-deficient (IFN-γ-/-), IFN-γ receptor-deficient (IFN-γR-/-) and RAG-/- mice were orally administered WPP or its major components. NK cell populations and cytotoxic activity were then examined by flow cytometry and 51Cr release assay, respectively. RESULTS: While the cytotoxic activity of NK cells was increased following administration of 100 mg/kg/day of WPP for 7 days or 200 or 500 mg/kg/day of WPP for 4 days in WT mice, the proportions of NK cell populations were unaltered. Similar activation of NK cell cytotoxicity was observed when RAG-/-, but not IFN-γ-/- or IFN-γR-/-, mice were orally administered 200 mg/kg/day of WPP for 4 days. Oral ingestion of artepillin C or p-coumaric acid, but not drupanin, augmented NK cell cytotoxicity in a manner similar to WPP and to the mixture of these three components. CONCLUSION: These results suggest that oral ingestion of WPP enhances NK cell cytotoxic activity, but not proliferation, in a manner dependent on IFN-γ and without the contribution of acquired immune responses. Further, artepillin C or p-coumaric acid, but not drupanin, may be the components responsible for this augmentation of NK cell cytotoxicity. These findings suggest the possible utility of WPP as a therapeutic for prevention of cancer development and against viral infection through NK cell activation.

Interferon-γ-Mediated Natural Killer Cell Activation by an Aqueous Panax ginseng Extract.

Panax ginseng extracts are used in traditional herbal medicines, particularly in eastern Asia, but their effect on natural killer (NK) cell activity is not completely understood. This study aimed to examine the effects of P. ginseng extracts on the cytotoxic activity of NK cells. We orally administered P. ginseng extracts or ginsenosides to wild-type (WT) C57BL/6 (B6) and BALB/c mice and to B6 mice deficient in either recombination activating gene 2 (RAG-2) or interferon-γ (IFN-γ). We then tested the cytotoxic activity of NK cells (of spleen and liver mononuclear cells) against NK-sensitive YAC-1 cells. Oral administration of P. ginseng aqueous extract augmented the cytotoxicity of NK cells in WT B6 and BALB/c mice and in RAG-2-deficient B6 mice, but not in IFN-γ-deficient B6 mice. This effect was only observed with the aqueous extract of P. ginseng. Interestingly, the ginsenosides Rb1 and Rg1 did not augment NK cell cytotoxicity. These results demonstrated that the aqueous P. ginseng extract augmented NK cell activation in vivo via an IFN-γ-dependent pathway.

Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion.

Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils.

Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice.

Plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of a major fibrinolytic factor, tissue-type plasminogen activator, can both promote and inhibit angiogenesis. However, the physiologic role and the precise mechanisms underlying the angiogenic effects of PAI-1 remain unclear. In the present study, we report that pharmacologic inhibition of PAI-1 promoted angiogenesis and prevented tissue necrosis in a mouse model of hind-limb ischemia. Improved tissue regeneration was due to an expansion of circulating and tissue-resident granulocyte-1 marker (Gr-1(+)) neutrophils and to increased release of the angiogenic factor VEGF-A, the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated increased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization and genetic knockout studies indicated that both the improved tissue regeneration and the increase in circulating and ischemic tissue-resident Gr-1(+) neutrophils depended on the activation of tissue-type plasminogen activator and matrix metalloproteinase-9 and on VEGF-A and FGF-2. These results suggest that pharmacologic PAI-1 inhibition activates the proangiogenic FGF-2 and VEGF-A pathways, which orchestrates neutrophil-driven angiogenesis and induces cell-driven revascularization and is therefore a potential therapy for ischemic diseases.

Cupressaceae pollen grains modulate dendritic cell response and exhibit IgE-inducing adjuvant activity in vivo.

Pollen is considered a source of not only allergens but also immunomodulatory substances, which could play crucial roles in sensitization and/or the exacerbation of allergies. We investigated how allergenic pollens from different plant species (Japanese cedar and Japanese cypress, which belong to the Cupressaceae family, and birch, ragweed, and grass) modulate murine bone marrow-derived dendritic cell (DC) responses and examined the effect of Cupressaceae pollen in vivo using mice. DCs were stimulated with pollen extracts or grains in the presence or absence of LPS. Cell maturation and cytokine production in DCs were analyzed by flow cytometry, ELISA, and/or quantitative PCR. Pollen extracts suppressed LPS-induced IL-12 production and the effect was greatest for birch and grass. Without LPS, pollen grains induced DC maturation and cytokine production without IL-12 secretion and the response, for which TLR 4 was dispensable, was greatest for the Cupressaceae family. Intranasal administration of Cupressaceae pollen in mice induced an elevation of serum IgE levels and airway eosinophil infiltration. Coadministration of ovalbumin with Cupressaceae pollen grains induced ovalbumin-specific IgE responses associated with eosinophil infiltration. The results suggest that modulation of DC responses by pollen differs among the plant families via (1) the promotion of DC maturation and cytokine production by direct contact and/or (2) the inhibition of IL-12 production by soluble factors. The strong DC stimulatory activity in vitro and IgE-inducing activity in mice support the clinical relevance of Cupressaceae pollen to allergies in humans.

FcepsilonRIalpha gene -18483A>C polymorphism affects transcriptional activity through YY1 binding.

Three frequent genetic polymorphisms in the human high-affinity IgE receptor alpha-subunit (FcepsilonRIalpha) were shown to be associated with allergic disorders and/or total serum IgE levels in allergic patients. Two of these were previously demonstrated to affect FcepsilonRIalpha expression while the third -18483A>C (rs2494262) has not yet been subjected to functional studies. We hypothesized that the -18483A>C variant affects transcriptional activity of the FcepsilonRIalpha distal promoter in monocytes in which FcepsilonRIalpha transcription is driven through that regulatory region. Indeed, we confirmed preferential binding of the YY1 transcription factor to the -18483C allele, resulting in lower transcriptional activity when compared with the -18483A allele.

NADPH oxidase activity in allergenic pollen grains of different plant species.

Pollen is an important trigger of allergic diseases. Recent studies have shown that ragweed pollen NAD(P)H oxidase generates reactive oxygen species (ROS) and plays a prominent role in the pathogenesis of allergies in mouse models. Here, we demonstrated that allergenic pollen grains showed NAD(P)H oxidase activity that differed in intensity and localization according to the plant families. The activity occurred at the surface or in the cytoplasm in pollen of grasses, birch, and ragweed; in subpollen particles released from ragweed pollen; and at the inner surface or in the cytoplasm but not on the outer wall, which was sloughed off after the rupture, of pollen of Japanese cedar and Japanese cypress. The activity was mostly concentrated within insoluble fractions, suggesting that it facilitates the exposure of tissues to ROS generated by this enzyme. The extent of exposure to pollen-generated ROS could differ among the plant families.

Lipid-soluble components of honeybee-collected pollen exert antiallergic effect by inhibiting IgE-mediated mast cell activation in vivo.

It was shown previously that bee-collected pollen (bee pollen, BP), inhibited in vitro murine mast cell activation. This study further analysed the antiallergic effect of BP in vivo by measuring cutaneous mast cell activation using a passive cutaneous anaphylaxis reaction. Daily oral administration of BP to mice, dose-dependently reduced the cutaneous mast cell activation elicited by IgE and specific antigens. Administration of BP also reduced the plasma concentration of malondialdehyde (MDA), an indicator of lipid peroxidation. The inhibitory effect of BP was mostly in a lipid- but not in water-soluble fraction. The HPLC analysis of isoflavones in BP revealed that genistein was a major isoflavone. However, administration of genistein alone at the concentration found in BP, did not show an inhibitory effect as observed in whole BP, suggesting that component(s) other than genistein would be responsible for the inhibitory effect of BP. These results first reveal that lipid-soluble components of BP exert an antiallergic action by inhibiting the FcåRI-mediated cutaneous mast cell activation.

Fusion of OTT to BSAC results in aberrant up-regulation of transcriptional activity.

OTT/RBM15-BSAC/MAL/MKL1/MRTF-A was identified as a fusion transcript generated by t(1;22)(p13;q13) in acute megakaryoblastic leukemia. Previous studies have shown that BSAC (basic, SAP, and coiled-coil domain) activates the promoters containing CArG boxes via interaction with serum response factor, and OTT (one twenty-two) negatively regulates the development of megakaryocytes and myeloid cells. However, the mechanism by which OTT-BSAC promotes leukemia is largely unknown. Here we show that OTT-BSAC, but not BSAC or OTT strongly activates several promoters containing a transcription factor Yin Yang 1-binding sequence. In addition, although BSAC predominantly localizes in the cytoplasm and its nuclear translocation is considered to be regulated by the Rho-actin signaling pathway, OTT-BSAC exclusively localizes in the nucleus. Moreover, OTT interacts with histone deacetylase 3, but this interaction is abolished in OTT-BSAC. Collectively, these functional and spatial changes of OTT and BSAC caused by the fusion might perturb their functions, culminating in the development of acute megakaryoblastic leukemia.

Procyanidin C1 from apple extracts inhibits Fc epsilon RI-mediated mast cell activation.

BACKGROUND: Polyphenol-enriched fractions, which are extracted from unripe apples (Rosaceae, Malus spp.), consisting of procyanidins (polymers of catechins) are known to have an anti-allergenic effect on patients with various allergic diseases. Although it has been reported that apple extracts inhibit histamine release from mast cells, the molecular mechanisms for this anti-allergenic effect are not well understood. To elucidate the molecular mechanisms by which apple extracts induce their anti-allergenic effects, the effects of purified apple extract components on high-affinity receptors for IgE (Fc epsilon RI)-mediated mast cell activation were investigated. METHODS: The anti-allergic effect of oral administration of apple procyanidin extracts on passive cutaneous anaphylactic responses of BALB/c mice was assessed. We evaluated the effects of procyanidin C1 (PC1) [epicatechin-(4beta-->8)-epicatechin-(4beta-->8)-epicatechin], a component of the procyanidin fraction, on mouse bone-marrow-derived mast cell degranulation, cytokine production, protein tyrosine phosphorylation and on the generation of intracellular reactive oxygen species (ROS) of cells stimulated by Fc epsilon RI cross-linking in vitro. RESULTS: In an in vivo study, oral administration of the procyanidin fraction suppressed the mast-cell-dependent allergic reaction. In in vitro studies, PC1 dose-dependently decreased Fc epsilon RI-mediated degranulation and cytokine production of mast cells. Furthermore, PC1 inhibited tyrosine phosphorylation of Syk and linker for activation of T cells, and the ROS generation in stimulated mast cells. CONCLUSIONS: PC1 suppresses Fc epsilon RI-mediated mast cell activation by inhibiting intracellular signaling pathways. These observations provide evidence for the anti-allergenic effects of the procyanidin-enriched apple extract.

Immunomodulating Activity of Agaricus brasiliensis KA21 in Mice and in Human Volunteers.

We performed studies on murine models and human volunteers to examine the immunoenhancing effects of the naturally outdoor-cultivated fruit body of Agaricus brasiliensis KA21 (i.e. Agaricus blazei). Antitumor, leukocyte-enhancing, hepatopathy-alleviating and endotoxin shock-alleviating effects were found in mice. In the human study, percentage body fat, percentage visceral fat, blood cholesterol level and blood glucose level were decreased, and natural killer cell activity was increased. Taken together, the results strongly suggest that the A. brasiliensis fruit body is useful as a health-promoting food.

Characterization of proteases, proteins, and eicosanoid-like substances in soluble extracts from allergenic pollen grains.

BACKGROUND: Pollen is an important trigger of seasonal rhinitis, conjunctivitis, and/or allergic asthma, and an exacerbating factor in atopic dermatitis. Pollen grains contain allergen proteins, enzymes, and bioactive lipid mediators, the latter two possibly involved in the pathogenesis of allergic diseases through IgE-independent mechanisms. METHODS: We analyzed the patterns of release of endopeptidases from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and birch, ragweed, and two grasses, Kentucky blue and cultivated rye, using synthetic substrates, class-specific inhibitors, and zymography. The proteins released were analyzed by gel electrophoresis. Eicosanoid-like substances were measured by enzyme-linked immunosorbent assays for prostaglandin E(2) and leukotriene B(4). RESULTS: Major fractions of proteins, eicosanoid-like substances, and at least one molecular species of serine endopeptidase were released into phosphate-buffered saline from the pollen grains at 37 degrees C within 25 min or 60 min without sonication. In the Cupressaceae/Taxodiaceae family, sonication was necessary for the release of other proteins and another serine endopeptidase. In birch, ragweed, and the grasses, most of the serine and cysteine endopeptidases were released without sonication. Proteases released within 25 min digested gelatin and/or casein differently among plant species. CONCLUSIONS: Grains of allergenic pollen release proteases, which can digest not only short synthetic substrates but also protein substrates, along with eicosanoid-like substances and proteins. The release of these components could contribute to the formation of a microenvironment optimum for initiation of the sensitization or the exacerbation of pollen allergy in tissues exposed to pollen grains.

Inhibitory effect of honeybee-collected pollen on mast cell degranulation in vivo and in vitro.

Bee-collected pollen (bee pollen [BP]) has been used as a folk medicine for centuries against various diseases, including allergy. There is no study elucidating how BP exerts such an anti-allergic effect. Since mast cells play a central role in the pathogenesis of various allergic diseases, we investigated the effect of BP on mast cell activation elicited by the Fc immunoglobulin E (IgE) receptor (Fc epsilon RI)-mediated pathways. The in vivo effect of orally administered BP on cutaneous mast cell activation was examined by passive cutaneous anaphylaxis reaction. In vitro mast cell degranulation and IgE binding to mast cells and the status of protein tyrosine phosphorylation were examined using bone marrow-derived mast cells. Daily oral administration of BP to mice significantly reduced the cutaneous mast cell activation elicited by IgE and specific antigens. BP also reduced in vitro mast cell degranulation and tumor necrosis factor-alpha production by inhibiting IgE binding to Fc epsilon RI on mast cells. The inhibitory effect of BP on mast cell degranulation by preventing IgE binding was confirmed by the reduced levels of protein tyrosine phosphorylation, which occurred as downstream events in activated mast cells via Fc epsilon RI. These results first revealed that the anti-allergic action of BP was exerted by inhibiting the Fc epsilon RI-mediated activation of mast cells, which plays important roles, not only in the early phase, but also in the late phase of allergic reactions.

Protease activity of allergenic pollen of cedar, cypress, juniper, birch and ragweed.

BACKGROUND: Pollen is an important trigger of allergic rhinitis, conjunctivitis, and/or asthma, and an exacerbating factor in atopic dermatitis. Although it is proposed that protease activity from allergen sources, such as mites, enhances allergenicity, little information is available on that from relevant allergenic pollens such as Japanese cedar and Japanese cypress pollens, which are the major cause of pollinosis in Japan. METHODS: We analyzed the protease activities derived from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and white birch and short ragweed, using synthetic substrates and class-specific inhibitors. RESULTS: We found that the pollen of the three members of the Cupressaceae/Taxodiaceae family contained serine protease activity, that the pollen of white birch and short ragweed contained not only serine protease activity but also cysteine protease activity, that all five types of pollen tested contained at least one other type of serine protease, whose sensitivity to a serine protease-specific inhibitor was relatively low, and that the content and releasability of the pollen-derived proteases differed according to the plant families. CONCLUSIONS: Clinically relevant allergenic pollens tested in the present study can release serine and/or cysteine endopeptidases. Information on the spectrum of the endopeptidase activities from these allergenic pollen grains will be useful for investigating their contribution to the pathogenesis of allergies.