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Involvement of High Mobility Group Box 1 (HMGB1) In Dendritic Cell Differentiation and Activation of Eosinophils
S252 Abstracts
975
An Endogenous Danger Signal, Uric Acid, Attracts and Activates Human Eosinophils T. Kobayashi, H. Kita; Mayo Clinic Rochester, Rochester, MN. RATIONALE: Uric acid (UA) is an important endogenous danger signal released from injured cells during inflammation and infection. Generation of UA is also implicated in the effects of an authentic Th2 adjuvant, aluminum hydroxide. Because eosinophils are often localized at the sites of Th2type chronic inflammation, we hypothesized that eosinophils may respond to UA. METHODS: We studied the effects of UA on eosinophils isolated from normal individuals. Eosinophils were incubated with monosodium urate (MSU) or UA at 0.001-10 mg/ml. Eosinophil migration was examined by a TranswellÒ system. Cytokines and eosinophil-derived neurotoxin (EDN) released into the supernatants were measured by Bio-Plex and ELISA. RESULTS: Both MSU and UA induced eosinophil migration with maximal effects at 0.1 mg/ml. MSU also induced IL-8 production; the effect was observed as low as 0.003 mg MSU/ml, reached a peak at 0.1 mg MSU/ml, and decreased with higher concentrations. Other cytokines and chemokines, including IL-1b, IL-6, IL-17, IFN-g, MCP-1, MIP-1a, MIP-1b, TNF-a, G-CSF, and GM-CSF, were produced by eosinophils incubated with MSU; the IL-6 and MIP-1b responses were particularly robust. In contrast, UA did not induce any of these cytokines. MSU and UA at higher concentrations (e.g. 1 mg/ml) induced EDN release from eosinophils in a calcium-dependent manner. CONCLUSIONS: UA stimulates eosinophils to migrate, to produce various cytokines, and to release granule protein(s). Such multiple responses of eosinophils to a danger signal, UA, may explain the self-perpetuating nature of chronic inflammation in certain human diseases, such as asthma.
976
TUESDAY
Eosinophil Regulatory Activity in Models of Virus-Induced Inflammation J. T. Kelly, K. E. Fox, E. H. Lucey, S. K. Mathur; University of Wisconsin, Madison, WI. RATIONALE: Airway eosinophilia is a predictor of asthma exacerbations and a marker of asthma severity. Viral infections, including rhinovirus, account for the majority of asthma exacerbations. The role of eosinophils (EOS) in virus-induced inflammation remains to be defined. We chose to study human EOS regulatory activity in altering host responses to virus stimulation through interactions with peripheral blood mononuclear cells (PBMCs), monocytes, and epithelial cells (ECs). We have previously shown significant increases in IL-13 and IFN-gamma in co-culture experiments of EOS with CD4 1 T-cells. The synthetic double-stranded RNA polyinosinic: polycytidylic acid (poly I: C) was used as a virus analogue for the co-culture of eosinophils with PBMCs and monocytes. METHODS: Using an in vitro co-culture system, purified human peripheral blood EOS were incubated with primary human PBMCs or monocytes and stimulated by poly I: C at 25 mcg/ml for 48 hours. EOS were also incubated with primary ECs and BEAS-2B cells for 24 hours. Supernatants from the co-cultures were collected and analyses of cytokine production were performed by ELISA. RESULTS: Co-culture of EOS with PBMCs stimulated with poly I: C in vitro significantly increased IL-13 expression (p 5 0.010.) Co-culture of EOS with monocytes stimulated by poly I: C significantly increased IL1beta expression (p 5 0.008). Preliminary experiments suggest that incubation of EOS with primary ECs decreases IL-8 expression. CONCLUSIONS: The presence of EOS alters in vitro cytokine expression of PBMCs, monocytes and ECs. Thus, EOS may affect host response to viral infection in asthma by altering cytokine production of inflammatory cells as well as epithelial cells.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
977
Involvement of High Mobility Group Box 1 (HMGB1) In Dendritic Cell Differentiation and Activation of Eosinophils C. Straub, K. Pazdrak, S. J. Stafford, A. Kurosky; The University of Texas Medical Brnach, Galveston, TX. RATIONALE: Emerging evidence suggests an immunoregulatory role of eosinophils in allergic inflammation and certain parasitic and viral infections. Our proteomic studies of non-activated and activated eosinophils identified the expression and release of HMGB1. HMGB1 plays a prominent role in immunoregulatory cell activation including granulocyte activation and T-cell and dendritic cell activation. We sought to determine whether eosinophil-derived HMGB1 contributes to the activation of eosinophils and the differentiation of monocytes toward dendritic cells. METHODS: Cell survival rates and expression of CD69 were determined after stimulation of eosinophils with GM-CSF, rHMGB1, and various HMGB1 inhibitors. Dendritic cell differentiation was determined after co-culture of monocytes with media from eosinophil cultures followed by assessment of CD80 and CD86 upregulation. RESULTS: Eosinophils stimulated with either GM-CSF or rHMGB1 showed significantly higher viability and expression of CD69. Pretreatment of eosinophils with glycyrrhizin, a specific inhibitor of HMGB1 activity, partially inhibited GM-CSF-induced prolongation of survival as well as upregulation of CD69. Incubation of monocytes with media from either eosinophil cultures or recombinant HMGB1 resulted in a significant upregulation of CD80 and CD86, comparable to that observed with GM-CSF stimulation. This upregulation was significantly inhibited by treatment with glycyrrhizin or anti-RAGE2 antibody. CONCLUSIONS: Our studies demonstrate for the first time the contribution of HMGB1 to eosinophil activation and establish the involvement of eosinophil-derived HMGB1 in dendritic cell function. These findings indicate a significant immunoregulatory role for eosinophils and provide a novel mechanism for the characterization of eosinophil-associated pathologies.
978
Evaluation Of Cytokine Profiles In The Secretion From Eosinophilic Otitis Media H. Uchimizu1, Y. Matsuwaki1, M. Kato2, H. Moriyama1; 1The Jikei University School of Medicine, Tokyo, Japan, 2Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Japan. RATIONALE: Eosinophilic otitis media (EOM) is an intractable disease having a remarkably viscous secretion and an accumulation of numerous eosinophils in both the middle ear secretion and the mucosa. Recent studies suggest that association with both adult onset bronchial asthma and eosinophilic rhinosinusitis. EOM causes the sensorineural hearing loss and occasionally causes the deafness. In this study, we examined the most important factors of eosinophils inflammation the middle ear of EOM. METHODS: The middle ear secretion samples were collected and stored at -20 degree from twelve patients of EOM and nine patients of secretary otitis media (SOM), as controls. Multiple cytokines in the secretion were measured by the Bio-Plexä Human Cytokine 27-Plex panel (Bio-Rad Laboratories, Hercules, CA). Eosinophil derived neurotoxin (EDN) and neutrophil elastase were measured by EDN ELISA kit (MBL, Nagoya, Japan) and human PMN elastase ELISA (Bender MedSystems, Vienna, Austria), respectively. RESULTS: The concentrations of EDN, IL-5, IL-1b, MIP-1a, G-CSF, IL1ra, IL-4, IFN-g and MIP-1b (lining up in order of smaller p-value) in the secretion from EOM were significantly higher than those from SOM (p < 0.01). On the other hand, the concentration of IP-10 in the secretion from SOM was significantly higher than that from EOM. In EOM, a strong correlation with EOM was found in order of RANTES, IL-13, eotaxin and IL-2. CONCLUSIONS: EDN, IL-5, IL-1b, MIP-1a, G-CSF, IL-1ra, IL-4, IFNg and MIP-1b are likely involved in the pathogenesis of EOM, compared with SOM. RANTES, IL-13, eotaxin and IL-2 are most important factors of eosinophilic inflammation in the middle ear of EOM.
975
An Endogenous Danger Signal, Uric Acid, Attracts and Activates Human Eosinophils T. Kobayashi, H. Kita; Mayo Clinic Rochester, Rochester, MN. RATIONALE: Uric acid (UA) is an important endogenous danger signal released from injured cells during inflammation and infection. Generation of UA is also implicated in the effects of an authentic Th2 adjuvant, aluminum hydroxide. Because eosinophils are often localized at the sites of Th2type chronic inflammation, we hypothesized that eosinophils may respond to UA. METHODS: We studied the effects of UA on eosinophils isolated from normal individuals. Eosinophils were incubated with monosodium urate (MSU) or UA at 0.001-10 mg/ml. Eosinophil migration was examined by a TranswellÒ system. Cytokines and eosinophil-derived neurotoxin (EDN) released into the supernatants were measured by Bio-Plex and ELISA. RESULTS: Both MSU and UA induced eosinophil migration with maximal effects at 0.1 mg/ml. MSU also induced IL-8 production; the effect was observed as low as 0.003 mg MSU/ml, reached a peak at 0.1 mg MSU/ml, and decreased with higher concentrations. Other cytokines and chemokines, including IL-1b, IL-6, IL-17, IFN-g, MCP-1, MIP-1a, MIP-1b, TNF-a, G-CSF, and GM-CSF, were produced by eosinophils incubated with MSU; the IL-6 and MIP-1b responses were particularly robust. In contrast, UA did not induce any of these cytokines. MSU and UA at higher concentrations (e.g. 1 mg/ml) induced EDN release from eosinophils in a calcium-dependent manner. CONCLUSIONS: UA stimulates eosinophils to migrate, to produce various cytokines, and to release granule protein(s). Such multiple responses of eosinophils to a danger signal, UA, may explain the self-perpetuating nature of chronic inflammation in certain human diseases, such as asthma.
976
TUESDAY
Eosinophil Regulatory Activity in Models of Virus-Induced Inflammation J. T. Kelly, K. E. Fox, E. H. Lucey, S. K. Mathur; University of Wisconsin, Madison, WI. RATIONALE: Airway eosinophilia is a predictor of asthma exacerbations and a marker of asthma severity. Viral infections, including rhinovirus, account for the majority of asthma exacerbations. The role of eosinophils (EOS) in virus-induced inflammation remains to be defined. We chose to study human EOS regulatory activity in altering host responses to virus stimulation through interactions with peripheral blood mononuclear cells (PBMCs), monocytes, and epithelial cells (ECs). We have previously shown significant increases in IL-13 and IFN-gamma in co-culture experiments of EOS with CD4 1 T-cells. The synthetic double-stranded RNA polyinosinic: polycytidylic acid (poly I: C) was used as a virus analogue for the co-culture of eosinophils with PBMCs and monocytes. METHODS: Using an in vitro co-culture system, purified human peripheral blood EOS were incubated with primary human PBMCs or monocytes and stimulated by poly I: C at 25 mcg/ml for 48 hours. EOS were also incubated with primary ECs and BEAS-2B cells for 24 hours. Supernatants from the co-cultures were collected and analyses of cytokine production were performed by ELISA. RESULTS: Co-culture of EOS with PBMCs stimulated with poly I: C in vitro significantly increased IL-13 expression (p 5 0.010.) Co-culture of EOS with monocytes stimulated by poly I: C significantly increased IL1beta expression (p 5 0.008). Preliminary experiments suggest that incubation of EOS with primary ECs decreases IL-8 expression. CONCLUSIONS: The presence of EOS alters in vitro cytokine expression of PBMCs, monocytes and ECs. Thus, EOS may affect host response to viral infection in asthma by altering cytokine production of inflammatory cells as well as epithelial cells.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
977
Involvement of High Mobility Group Box 1 (HMGB1) In Dendritic Cell Differentiation and Activation of Eosinophils C. Straub, K. Pazdrak, S. J. Stafford, A. Kurosky; The University of Texas Medical Brnach, Galveston, TX. RATIONALE: Emerging evidence suggests an immunoregulatory role of eosinophils in allergic inflammation and certain parasitic and viral infections. Our proteomic studies of non-activated and activated eosinophils identified the expression and release of HMGB1. HMGB1 plays a prominent role in immunoregulatory cell activation including granulocyte activation and T-cell and dendritic cell activation. We sought to determine whether eosinophil-derived HMGB1 contributes to the activation of eosinophils and the differentiation of monocytes toward dendritic cells. METHODS: Cell survival rates and expression of CD69 were determined after stimulation of eosinophils with GM-CSF, rHMGB1, and various HMGB1 inhibitors. Dendritic cell differentiation was determined after co-culture of monocytes with media from eosinophil cultures followed by assessment of CD80 and CD86 upregulation. RESULTS: Eosinophils stimulated with either GM-CSF or rHMGB1 showed significantly higher viability and expression of CD69. Pretreatment of eosinophils with glycyrrhizin, a specific inhibitor of HMGB1 activity, partially inhibited GM-CSF-induced prolongation of survival as well as upregulation of CD69. Incubation of monocytes with media from either eosinophil cultures or recombinant HMGB1 resulted in a significant upregulation of CD80 and CD86, comparable to that observed with GM-CSF stimulation. This upregulation was significantly inhibited by treatment with glycyrrhizin or anti-RAGE2 antibody. CONCLUSIONS: Our studies demonstrate for the first time the contribution of HMGB1 to eosinophil activation and establish the involvement of eosinophil-derived HMGB1 in dendritic cell function. These findings indicate a significant immunoregulatory role for eosinophils and provide a novel mechanism for the characterization of eosinophil-associated pathologies.
978
Evaluation Of Cytokine Profiles In The Secretion From Eosinophilic Otitis Media H. Uchimizu1, Y. Matsuwaki1, M. Kato2, H. Moriyama1; 1The Jikei University School of Medicine, Tokyo, Japan, 2Gunma Prefectural Institute of Public Health and Environmental Sciences, Maebashi, Japan. RATIONALE: Eosinophilic otitis media (EOM) is an intractable disease having a remarkably viscous secretion and an accumulation of numerous eosinophils in both the middle ear secretion and the mucosa. Recent studies suggest that association with both adult onset bronchial asthma and eosinophilic rhinosinusitis. EOM causes the sensorineural hearing loss and occasionally causes the deafness. In this study, we examined the most important factors of eosinophils inflammation the middle ear of EOM. METHODS: The middle ear secretion samples were collected and stored at -20 degree from twelve patients of EOM and nine patients of secretary otitis media (SOM), as controls. Multiple cytokines in the secretion were measured by the Bio-Plexä Human Cytokine 27-Plex panel (Bio-Rad Laboratories, Hercules, CA). Eosinophil derived neurotoxin (EDN) and neutrophil elastase were measured by EDN ELISA kit (MBL, Nagoya, Japan) and human PMN elastase ELISA (Bender MedSystems, Vienna, Austria), respectively. RESULTS: The concentrations of EDN, IL-5, IL-1b, MIP-1a, G-CSF, IL1ra, IL-4, IFN-g and MIP-1b (lining up in order of smaller p-value) in the secretion from EOM were significantly higher than those from SOM (p < 0.01). On the other hand, the concentration of IP-10 in the secretion from SOM was significantly higher than that from EOM. In EOM, a strong correlation with EOM was found in order of RANTES, IL-13, eotaxin and IL-2. CONCLUSIONS: EDN, IL-5, IL-1b, MIP-1a, G-CSF, IL-1ra, IL-4, IFNg and MIP-1b are likely involved in the pathogenesis of EOM, compared with SOM. RANTES, IL-13, eotaxin and IL-2 are most important factors of eosinophilic inflammation in the middle ear of EOM.