Nitric oxide production by alveolar macrophages in response to house dust mite fecal pellets and the mite allergens,Der p 1 and Der p 2

BACKGROUND: Allergens are frequently found within or attached to particulate material. For example, house dust mite fecal pellets (HDMFP) contain the major mite allergens, exposure to which have been implicated in the development of asthma. Although several studies have examined the ability of purified allergens to generate inflammatory responses, few studies have investigated whether HDMFP per se, are biologically active. OBJECTIVE: Our objective was to examine the ability of whole HDMFP to stimulate nitric oxide (NO) release from alveolar macrophages. METHODS: The rat alveolar macrophage cell line, NR8383, was exposed to HDMFP, Der p 1, or Der p 2, and nitrite levels in the culture supernatants were measured with the Griess reagent. NO synthase mRNA expression was determined by RT-PCR. RESULTS: HDMFP stimulated the production of NO in a dose-dependent and time-dependent manner, with maximum NO levels measured after 48 hours of exposure. Inclusion of polymyxin B did not influence NO production, suggesting that LPS was not responsible for NO production. HDMFP-mediated NO release was down-modulated after treatment with N(G)-nitro-l-arginine methyl ester (L-NAME), dexamethasone, EDTA, and cysteine, but not heat treatment. Inducible nitric oxide synthase mRNA was observed 3 hours after HDMFP exposure, with maximum levels after 48 hours. Both purified Der p 1 and Der p 2 induced NO production, and inhibition of the cysteine protease activity of Der p 1 had little effect on NO production. CONCLUSIONS: HDMFP, Der p 1 and Der p 2 are potent inducers of NO. Neither LPS nor the enzymatic activity of Der p 1 was responsible for NO production observed.     

p-Cresol inhibits IL-12 production by murine macrophages stimulated with bacterial immunostimulant

p-Cresol, an end product of aromatic amino acids, is produced from food proteins by intestinal bacteria, and is detectable in blood and feces. Especially, blood and fecal levels of p-cresol are high in chronic renal failure (CRF) patients. Although it has been suggested that p-cresol is toxic in the body, the effect of p-cresol on immune responses has not yet been clarified. In this study, we investigated the effect of p-cresol on IL-12 production of macrophages stimulated with Lactobacillus casei strain Shirota (LcS) in vitro. Pre-incubation with p-cresol inhibited IL-12 p40 production of LcS-stimulated J774.1 cells, a murine macrophage-like cell line, in a dose-dependent manner. IL-12 p40 and p70 production of LcS-stimulated murine peritoneal macrophages was also inhibited by p-cresol. The inhibitory effect was not dependent on the cytotoxicity of p-cresol. These results indicate that blood and fecal p-cresol may have adverse effects on the host defense system in CRF patients.     

Cytokine responses to Der p 1 and Der p 7: house dust mite allergens with different IgE-binding activities

BACKGROUND: There is very limited information comparing T-cell responses to different house dust mite (HDM) allergens even though T cells are essential in the initiation and regulation of immunoglobulin (Ig) E synthesis and eosinophilia. OBJECTIVE: To compare the level of T-cell proliferation and cytokine production to the group 1 and group 7 HDM allergens which are known to have different IgE-binding capabilities. METHODS: Freshly isolated peripheral blood mononuclear cells (PBMC) from HDM-allergic and HDM-nonallergic donors were stimulated with the group 1 and group 7 allergens of Dermatophagoides pteronyssinus and the level of proliferation as well as IL-5 and IFNgamma production were measured. RESULTS: The proliferative and IL-5 production to the group 1 and group 7 allergens were equivalent despite the group 7 allergen's lower frequency of IgE-binding. However more IFNgamma was produced to Der p 7 than to Der p 1, particularly for the nonallergic donors. As expected IL-5 production was much higher for PBMC from the allergic donors than for cells from nonallergics; however, there was no difference in the level of T-cell proliferation between the donor groups. CONCLUSION: The relative importance of the individual HDM allergens is normally determined by measuring the frequency of IgE-binding to the allergen in sera from an allergic population. The equivalent increased IL-5 response of PBMC from allergic people to the group 1 and group 7 allergens despite the different IgE-inducing activity indicates that these allergens may be equally capable of contributing to an asthmatic response by inducing eosinophilia.     

IL-8 production and AP-1 transactivation induced by UVA in human keratinocytes: roles of D-alpha-tocopherol

Identification of individual response-signal pathway induced by UVA-irradiation is necessary for understanding photo-biological and -pathological mechanisms with respect to the prevention of UV-irradiated skin damage and aging. Here, we investigated the role of D-alpha-tocopherol in the regulation of IL-8 production and AP-1 binding activity in UVA-irradiated human keratinocytes. UVA dramatically upregulated IL-8 mRNA expression and protein secretion and enhanced the AP-1-DNA binding activity. These effects of UVA irradiation were effectively reduced by D-alpha-tocopherol in a dose-dependent manner. The human keratinocytes expressed various NAD(P)H oxidase components, gp91phox homologues Nox1, and p22phox, p47phox, p67phox, as well as NOXO1, suggesting that cellular stress induced by UVA included the activation of non-phagocytic NADPH oxidase system, leading to AP-1 transactivation and IL-8 expression. D-alpha-tocopherol significantly inhibited the NADPH oxidase activity and the formation of malondialdehyde-thiobarbituric acid under UVA exposure. These results demonstrated that D-alpha-tocopherol may be able to prevent the IL-8 upregulation and the increase in AP-1 activation induced by UVA irradiation through down-modulating cellular oxidative stress.     

Glycosylation of recombinant proforms of major house dust mite allergens Der p 1 and Der f 1 decelerates the speed of maturation

BACKGROUND: The efficient manufacture of recombinant Der p 1 and Der f 1 has been an important bottleneck in the study of house dust mite allergies and the development of applications for allergen engineering. While Der f 1 has only one N-glycosylation motif in the mature sequence, Der p 1 has two motifs, one in the prosequence and the other in the mature sequence. To test whether inefficient maturation of a recombinant Pro-Der p 1 versus Pro-Der f 1 is due to N-glycosylation, the maturation speed of N-glycosylation motif mutants was compared. METHODS: Expression vectors for the mutants, in which the motif in the Der p 1 prodomain was disrupted or a motif was created within the Der f 1 prodomain, were constructed by site-directed mutagenesis of preproforms with or without the motif within the mature portion. Culture supernatants of yeast Pichia pastoris transfectant cells containing proforms were buffer exchanged by gel filtration and incubated for maturation. Samples from the reactions were collected every 20 min and subjected to electrophoresis. The maturation speed was compared based on the band densities of the pro- and mature forms. RESULTS: Disruption of the motif in the mature portion decreased the productivity and accelerated the maturation. Maturation was also accelerated by disruption of the other motif in the Der p 1 prodomain and slowed down by introduction of the motif into the Der f 1 prodomain. CONCLUSIONS: Maturation systems using Pro-Der p 1 without the prodomain glycosylation are useful for the efficient preparation of a recombinant mature allergen. In addition, these results demonstrated that the maturation of cysteine protease could be controlled through glycosylation of the prodomain.     

A mite subversive: cleavage of CD23 and CD25 by Der p 1 enhances allergenicity

The dust mite Dermatophagoides pteronyssinus is a major cause of allergic disease in the Western world. Der p 1 is considered to be the most immunodominant allergen involved in the expression of IgE-mediated dust-mite hypersensitivity. Here, Farouk Shakib and colleagues suggest that it is the proteolytic effect of Der p 1 on CD23 and CD25 which makes it such a potent inducer of IgE synthesis.     

Association between mite allergen (Der p 1, Der f 1, Blo t 5) levels and microscopic identification of mites or skin prick test results in asthmatic subjects

BACKGROUND: Mite allergens have been involved in airway sensitization and allergic diseases. Immunoassays for the identification and quantifiction of house dust mite (HDM) allergens are useful to improve the knowledge of regional mite fauna and the remediation of mite allergens in allergic diseases. The present study analyzed the association between levels of HDM allergen and results of mite identification or skin prick test (SPT) in two different areas of Bahia, Brazil. METHODS: Forty-two asthmatic subjects from a rural area (group I; n = 21) and a slum (group II; n = 21) were evaluated through SPT with HDM allergens and had dust samples collected at their homes for mite identification and allergen measurements. RESULTS: Positive SPT to Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis allergens were observed in 42.9, 38.0 and 42.9% subjects from group I and in 47.6, 19.0 and 33.3% subjects from group II, respectively. D. pteronyssinus and B. tropicalis were identified in approximately 76 and 50% of samples from both groups, respectively. D. farinae was identified in 38.0 and 9.5% of samples from groups I and II, respectively (p < 0.005). Der p 1, Der f 1 and Blo t 5 detection were associated with mite identification (p < 0.05). Association between HDM allergen levels over 2 microg/g of dust and positive SPT occurred only with D. pteronyssinus (p < 0.0001). CONCLUSIONS: D. pteronyssinus was the most prevalent mite species in this study followed by B. tropicalis and D. farinae. Immunoassays done to measure mite allergens were associated with mite-species identification. We conclude that these three mite species must be included on panels for the diagnosis of allergic airway diseases in subjects living in such regions.     

Biologically active recombinant forms of a major house dust mite group 1 allergen Der f 1 with full activities of both cysteine protease and IgE binding

BACKGROUND: Group 1 allergens from mite faeces, Der f 1 and Der p 1, are the most significant in-door allergens. Therefore, they are the most important component in the standardization of house dust mite extract for diagnosis and allergen-specific immunotherapy (AIT). Although their cDNAs have been cloned, efforts to prepare biologically active recombinant forms in expression systems using bacteria or yeast have failed. OBJECTIVE: Our purpose is to establish an efficient system to prepare recombinant Der f 1(rDer f 1), identical in quality to native Der f 1. METHODS: The preproforms of Der f 1 and a mutant N53Q, whose consensus motif for N-glycosylation was disrupted, were expressed in yeast Pichia pastoris. Cysteine protease activity and IgE reactivity were analysed using synthetic substrates and by RAST-EIA, respectively. RESULTS: The proforms of the two rDer f 1 molecules were efficiently secreted into culture medium. Their prosequences were removed autocatalytically by dialysis against acidic buffer. Although the wild-type rDer f 1 was more highly glycosylated than native Der f 1, N53Q had almost the same apparent molecular weight as native Der f 1 on SDS-PAGE. Both the protease and IgE binding activities of the mature rDer f 1 molecules were the same as those of native Der f 1, whereas the proforms had no or markedly reduced activities. CONCLUSION: The efficient system to prepare active rDer f 1s established in this study is useful for diagnosis and standardized AIT for house dust mite allergy. Furthermore, the system would be a tool for analysis of IgE epitopes, determination of tertiary structure, allergen engineering for safer and more effective AIT, resolving the relation between the enzymatic activity and pathogenesis, and the development of therapeutic inhibitors.     

Molecular basis of the synergistic production of IL-1 receptor antagonist by human neutrophils stimulated with IL-4 and IL-10

In this study, we report that the release of IL-1 receptor antagonist (IL-1ra) from IL-4-stimulated neutrophils is markedly enhanced in the presence of IL-10. We also show that up-regulation of IL-1ra release by IL-10 in IL-4-stimulated neutrophils takes place through IL-1ra mRNA stabilization and enhancement of IL-1ra de novo synthesis. Furthermore, we report that the ability of IL-10 to up-regulate IL-1ra mRNA expression in IL-4-treated neutrophils requires 5-6 h and it is preceded by the acquisition of the capacity to activate Stat3 tyrosine phosphorylation. This latter response to IL-10 was strictly dependent on the levels of expression of IL-10R1, which were in fact significantly increased by IL-4 in cultured neutrophils via a signaling pathway sensitive to the serine/threonine kinase inhibitor H-7. Collectively, our data emphasize the central role of IL-10R1 expression in regulating cell responsiveness to IL-10. In addition, the fact that IL-10 strongly up-regulates IL-1ra production in IL-4-activated neutrophils uncovers a novel mechanism whereby IL-10 and IL-4 cooperate to negatively modulate the inflammatory responses.     

Syk-dependent ERK activation regulates IL-2 and IL-10 production by DC stimulated with zymosan

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL-2 and IL-10 response in DC and emphasises the importance of Syk-coupled pattern recognition receptors such as dectin-1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors.     

A sensitive fluorescent assay for measuring the cysteine protease activity of Der p 1, a major allergen from the dust mite Dermatophagoides pteronyssinus.

The potent allergenicity of Der p 1, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is thought to be related to its cysteine protease activity. Therefore, there is considerable interest in developing a sensitive assay for measuring Der p 1 activity to screen for specific inhibitors. This study demonstrates for the first time that the activity of Der p 1 can be measured conveniently in a continuous rate assay with the fluorogenic substrate Boc-Gln-Ala-Arg-AMC (K(m) = 280 microM and kcat/K(m) = 4.6 x 10(3)/M/s).     

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha

In this study, we investigated the roles of CD4 T cell cytokines IL-17 and IL-17F in GM-CSF production from lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not induce GM-CSF release from LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1beta- and TNF-alpha-induced GM-CSF mRNA expression and production, whereas IL-17F had an enhancing effect on IL-1beta-induced GM-CSF production, but a marked inhibitory effect on TNF-alpha-induced secretion. GM-CSF production was further enhanced with the combination of three cytokines IL-1beta, TNF-alpha and IL-17 or IL-17F. Additionally, when Th1 or Th2 cytokine was combined with IL-1beta or TNF-alpha, both Th1 and Th2 cytokines had a modest stimulatory effect on TNF-alpha-induced GM-CSF production, whereas IL-4 and IFN-gamma profoundly attenuated IL-1beta-induced secretion. Moreover, the regulation by IL-17 plus Th1 or Th2 cytokine of GM-CSF production from LMVECs treated with IL-1beta or TNF-alpha was dependent on the concentration of IL-17. Our findings indicate that IL-17 and IL-17F play a differential regulatory role in GM-CSF production by LMVECs stimulated with IL-1beta and/or TNF-alpha, which is sensitive to Th1 and Th2 cytokine modulation.     

Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1

BACKGROUND: House dust mite allergen Der p 1 is a cysteine peptidase. Previously, we have suggested that the proteolytic activity of this allergen may contribute to asthma by damaging the barrier formed by the airways epithelium. OBJECTIVE: The present study applied novel techniques to compare changes in permeability with quantitative events in tight junctions (TJs) and desmosomes (DMs) of epithelial cells exposed to Der p 1. METHODS: Confluent monolayers of Madin-Darby canine kidney (MDCK) and 16HBE14o-human bronchial epithelial cells were used as experimental models. Permeability was estimated from mannitol clearance. Digital imaging with quantification of TJs and DMs was achieved by fluorescent antibody staining and 2-photon molecular excitation microscopy (2PMEM). Biochemical changes in TJs were studied by immunoblotting, radiolabelling and immunoprecipitation. RESULTS: Der p 1 caused a time-dependent breakage of TJs and reduction in their content of the protein ZO-1. Reduction in ZO-1 immunofluorescence at TJs occurred with a small increase in the amount of diffuse, cytoplasmic immunoreactive ZO-1 staining. Morpho-logical changes in TJs occurred in synchrony with increases in epithelial permeability. DM puncta increased both in size and intensity of staining. Immunoblotting demonstrated that the disruption of TJ morphology was associated with cleavage of ZO-1 and occludin. Cells recovered from allergen exposure by de novo synthesis of occludin. CONCLUSION: Der p 1 could contribute to sensitization and allergic responses by degrading the function of the airway epithelial barrier.     

GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha.

The role of mucosal fibroblasts in intestinal inflammatory reactions is not known. In this study, we demonstrate that fibroblasts grown from histologically normal human duodenal biopsy tissues expressed mRNA genes for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) when stimulated with lipopolysaccharide (LPS) or IL-1 alpha. The increased mRNA expression of GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and IL-8 in response to IL-1 alpha and LPS stimulation was time- and dose-dependent. In contrast, IL-10 was weakly expressed when fibroblasts were stimulated with LPS, IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha), but the expression was enhanced in the presence of cycloheximide combined with optimal concentrations of LPS, IL-1 alpha or TNF-alpha, IL-1 alpha was a more potent stimulator than LPS for GM-CSF, IL-6, IL-8 and IL-10 expression, but not for IL-1 alpha and IL-1 beta. Increased GM-CSF, IL-6 and IL-8 gene expression was associated with the production of cytokine proteins in culture supernatant, but IL-1 alpha and IL-1 beta remained undetectable. Dexamethasone suppressed both gene expression and protein production of GM-CSF, IL-6 and IL-8 when fibroblasts were exposed to IL-1 alpha. TNF-alpha stimulated the release of GM-CSF, IL-6 and IL-8 and, combined with IL-1 alpha, cytokine production was enhanced synergistically. Finally, both LPS and IL-1 alpha up-regulated ICAM-1 and VCAM-1 gene expression. These findings implicate duodenal fibroblasts in the initiation and/or regulation of intestinal inflammation.     

Functional effects of the inhibition of the cysteine protease activity of the major house dust mite allergen Der p 1 by a novel peptide-based inhibitor.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is an important source of allergens, which can cause allergic conditions. The cysteine protease activity of Der p 1 may enhance the potency of this major mite allergen through cleavage of CD23 and CD25 from the surface of immune cells, IgE independent mast cell activation, increases in epithelial cell permeability and inactivation of an endogenous serine protease inhibitor. Inhibition of the enzymatic activity of Der p 1 may therefore be of therapeutic benefit. OBJECTIVE: To examine the activity of PTL11028, a newly developed Der p 1 inhibitor, in a range of assays that directly or indirectly measure Der p 1 protease activity and to compare its activity to endogenous cysteine protease inhibitors. METHODS: The proteolytic activities of purified Der p 1 or HDM extract and inhibitory properties of PTL11028 were examined through cleavage of an artificial peptidyl substrate, cleavage of CD23 from human B cells and permeability studies on primary human bronchial epithelial cells. RESULTS: PTL11028 is a highly potent and specific Der p 1 inhibitor, being effective against both purified protease and Der p 1 within HDM extract. PTL11028 can completely inhibit Der p 1-mediated CD23 cleavage from human B cells and also reduces HDM-induced human bronchial epithelial cell permeability by 50%. Der p 1 is potently inhibited by cystatin A and to a lesser extent by cystatins C and E/M. CONCLUSION: PTL11028 is a highly potent and selective irreversible inhibitor of the cysteine protease activity of Der p 1, an activity that may be modulated in vivo by some human cystatins. PTL11028 prevents the Der p 1-mediated cleavage of CD23 from human B cells and significantly reduces HDM-induced permeabilization of the epithelial barrier. PTL11028 is an important tool to examine the biological effects of Der p 1 in a range of in vitro and in vivo model systems.     

Acute ethanol exposure inhibits macrophage IL-6 production: role of p38 and ERK1/2 MAPK

Acute ethanol consumption has been linked to an increase in infectious complications in trauma and burn patients. Ethanol modifies production of a variety of macrophage-derived immunoregulatory mediators. Lipopolysaccharide (LPS), a potent stimulator of inflammatory responses in macrophages, activates several intracellular signaling pathways, including mitogen-activated protein kinases (MAPK). In the current study, we investigated the effect of acute ethanol exposure on in vivo activation of p38 and extracellularly regulated kinases 1 and 2 (ERK1/2) MAPK in murine macrophages and the corresponding, LPS-stimulated interleukin (IL)-6 production. We demonstrated that a single dose of ethanol transiently down-regulated p38 and ERK1/2 activation levels (3-24 h after treatment) and impaired IL-6 synthesis. Ethanol-related reduction in IL-6 production was not further affected by the presence of inhibitors of p38 and ERK1/2 (SB 202190 and PD 98059, respectively). These results demonstrate that acute ethanol exposure can impair macrophage IL-6 production and indicate that this effect may result from ethanol-induced alterations in intracellular signaling through p38 and ERK1/2.     

Analysis of the structure and allergenicity of recombinant pro- and mature Der p 1 and Der f 1: major conformational IgE epitopes blocked by prodomains

BACKGROUND: The major house dust mite group 1 allergens Der p 1 and Der f 1, which belong to the papain-like cysteine protease family, are the most potent of indoor allergens. However, little information is available on the location of IgE epitopes. OBJECTIVE: We investigated the allergenicities of recombinant proforms and mature forms of Der p 1 and Der f 1 to compare them with natural Der p 1 and Der f 1 and to obtain information on the conformational IgE-binding epitopes. METHODS: Secreted pro-Der p 1 and pro-Der f 1 and their mutants without hyperglycosylation expressed in yeast were converted to mature forms. We purified the proforms and mature forms and analyzed their apparent molecular sizes and secondary structures by means of gel-filtration and circular dichroism analysis and their allergenicities by means of assays for IgE binding, IgE-binding inhibition, and basophil histamine release. The tertiary structure of pro-Der f 1 was predicted by molecular modeling. RESULTS: The recombinant mature forms exhibited similar molecular sizes, secondary structures, and allergenicities as their natural types. On the other hand, their proforms exhibited different secondary structures and less allergenicities than the mature forms in all sera and volunteers tested. Molecular modeling revealed that the prosegment is anchored at the prosegment-binding loop and the substrate-binding cleft on the surface of the mature portion. CONCLUSIONS: Our studies indicate that the prodomains of Der p 1 and Der f 1 reduce allergenicity and that the major conformational IgE epitopes commonly found in a broad population of patients exist within the 2 regions blocked by the prosegments. Recombinant Der p 1 and Der f 1 and the findings in the present study will be the basis for allergen standardization and the design of safer and more effective allergen vaccines.