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CD30 expression and apoptosis in human blood eosinophils
Abstracts
J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 1
Gene Expression Analysis in Human Peripheral Eosinophils by Various Cytokine Stimuli Shinji Kagaya*, Ryoichi Hashida*, Kaoru Ogawa*, Masami Miyagawa*, Yuji Sugita*, Kenji Matsumoto& T Katsunumag Akira Akasawag, Hirohisa SaitoY “Genox Research, Incorporated, Kawasaki-shi, Kanagawa, Japan ONational Children’s Medical Research Center, Setagaya-ku Tokyo, Japan YNational Children’s Hospital, Setagaya-ku Tokyo, Japan Eosinophils play important roles in allergic diseases. However, careful transcriptional analysis of these cells has not been accomplished. We reported that by the real-time quantitative PCR technique (ABI7700), the expression of some genes, such as CD44, granulocyte-macrophage colonystimulating factor (GM-CSF) receptor, and IL-3 receptor, but not IL-5 receptor, was shown to be remarkably enhanced in peripheral blood eosinophils of patients with atopic dermatitis compared with those of healthy volunteers. To clarify these mechanisms, peripheral blood eosinophils of healthy volunteers were isolated, adhered to BSA coated dishes, and stimulated by various cytokines. The expression of some genes was determined by the AB17700. Expression of GM-CSF receptor alpha was increased by stimulation with IL-5, IL-4, or IFN gamma dose-dependently. Expression of GM-CSF receptor beta was stimulated by IL-5, IFN gamma, or GM-CSF. IL-5 receptor expression was not increased by any cytokine stimuli, indicating that in vitro eosinophil gene expression by such cytokine stimuli were well-correlated with results of analyses of peripheral blood eosinophils in pathological conditions of atopic dermatitis patients.
690
691
CD30 Expression
and Apoptosis
in Human
Blood Eosinophils
Abdo Ibrahim Berro, Devendra KAgrawal Creighton University, Omaha, NE Eosinophils play a critical role in the pathogenesis and severity of allergic diseases including asthma. Several reports have established that Type 2 cytokines, such as IL-5, increase the survival of eosinophils in the lungs of allergic asthmatic subjects. Therefore, identifying the surface molecules and molecular mechanisms underlying the regulation of eosinophils apoptosis could facilitate the development of therapies aiming at limiting the role of eosinophils in allergy and asthma. CD30, also known as IQ-1 antigen, is a member of the tumor necrosis factor receptor (TNF-R) superfamily that also includes Fas/APO-1, TNF-RI and 2, and TRAIL receptors. Exact role of CD30 is still unclear, but CD30 has been linked to thymic negative selection of thymocytes. It has been suggested that CD30 is a negative regulator for CD8+ T cells cytotoxicity. In this study we examined the expression of CD30 and its role in the apoptosis of blood eosinophils. Human eosinophils were purified from allergic and atopic asthmatic subjects (purity >98%, viability >99%) and CD30 expression was examined by both flow cytometry using FITC-conjugated anti-CD30 antibodies and Western Blotting before and after the induction of apoptosis. Eosinophils expressed CD30 molecule, and there was significantly increased CD30 expression in serum-deprived (for 24 hours) eosinophils, which were undergoing apoptosis, as measured by Annexin V-labeling. We are currently examining the role of CD30 molecule in eosinophils and its relationship with programmed cell death in eosinophils. However, to our knowledge this is the first report on the presence of CD30 molecule in eosinophils.
S229
Serum Tryptase levels as a Possible Marker of Myeloproliferative Disease in a Subset of Patients With tlypereosinophilic Syndrome Amy D Klion, Melissa Law, Pierre Noel, Thomas B Nutman National Institute of Health, Bethesda, MD Hypereosinophilic syndrome (HES), as defined by the presence of eosinophilia of unknown etiology (>1500/mm3 for >6 months) and evidence of end organ damage, is a heterogeneous group of disorders of unknown etiology. Despite comparable levels of eosinophilia at presentation. some patients remain stable over many years time, whereas others experience rapid deterioration with progression to lymphoma or leukemia. Recent studies have demonstrated that some HES patients have clonal populations of lymphocytes or eosinophils detectable in the peripheral blood; however, the etiology of HES remains unclear in a majority of patients. Since serum tryptase levels, a specific marker of mast cell-mediated disease, are elevated in some patients with myeloproliferative disorders, we examined the utility of serum tryptase in identifying a population of HES patients with an underlying myeloproliferative disorder. Serum tryptase levels were elevated (geometric mean 34, range 22.3 to 47.5, normal range: 0 to 11.5) in 5/l 0 patients with HES, but in none of 9 patients with comparable eosinophil levels secondary to parasitic infection (n=4), lymphoma (n=l) or other eosinophilic disorders (n=4). Four of the five patients with elevated tryptase (and none of the patients with normal tryptase levels) have had progression of end organ involvement despite therapy, and all 5 have dysplastic eosinophils and eosinophilic precursors on peripheral smear. Bone marrow biopsies in these 5 patients were notable only for a marked increase in eosinophils and eosinophil progenitors, and cytogenetic and immunophenotyping studies were normal. Serial tryptase levels continued to rise (from 27.1 to 35.6 to 47.5) in one patient who experienced progression of disease despite a decrease in eosinophilia from 7,500/mm3 to 15OO/mm3 over a 7 month period. These findings suggest that a subset of patients with HES have a proliferative disorder involving cells of the early myeloid lineage. Serum tryptase may be useful in identifying these patients and in monitoring their response to therapy.
692
693
Withdrawn
J ALLERGY CLIN IMMUNOL VOLUME 109, NUMBER 1
Gene Expression Analysis in Human Peripheral Eosinophils by Various Cytokine Stimuli Shinji Kagaya*, Ryoichi Hashida*, Kaoru Ogawa*, Masami Miyagawa*, Yuji Sugita*, Kenji Matsumoto& T Katsunumag Akira Akasawag, Hirohisa SaitoY “Genox Research, Incorporated, Kawasaki-shi, Kanagawa, Japan ONational Children’s Medical Research Center, Setagaya-ku Tokyo, Japan YNational Children’s Hospital, Setagaya-ku Tokyo, Japan Eosinophils play important roles in allergic diseases. However, careful transcriptional analysis of these cells has not been accomplished. We reported that by the real-time quantitative PCR technique (ABI7700), the expression of some genes, such as CD44, granulocyte-macrophage colonystimulating factor (GM-CSF) receptor, and IL-3 receptor, but not IL-5 receptor, was shown to be remarkably enhanced in peripheral blood eosinophils of patients with atopic dermatitis compared with those of healthy volunteers. To clarify these mechanisms, peripheral blood eosinophils of healthy volunteers were isolated, adhered to BSA coated dishes, and stimulated by various cytokines. The expression of some genes was determined by the AB17700. Expression of GM-CSF receptor alpha was increased by stimulation with IL-5, IL-4, or IFN gamma dose-dependently. Expression of GM-CSF receptor beta was stimulated by IL-5, IFN gamma, or GM-CSF. IL-5 receptor expression was not increased by any cytokine stimuli, indicating that in vitro eosinophil gene expression by such cytokine stimuli were well-correlated with results of analyses of peripheral blood eosinophils in pathological conditions of atopic dermatitis patients.
690
691
CD30 Expression
and Apoptosis
in Human
Blood Eosinophils
Abdo Ibrahim Berro, Devendra KAgrawal Creighton University, Omaha, NE Eosinophils play a critical role in the pathogenesis and severity of allergic diseases including asthma. Several reports have established that Type 2 cytokines, such as IL-5, increase the survival of eosinophils in the lungs of allergic asthmatic subjects. Therefore, identifying the surface molecules and molecular mechanisms underlying the regulation of eosinophils apoptosis could facilitate the development of therapies aiming at limiting the role of eosinophils in allergy and asthma. CD30, also known as IQ-1 antigen, is a member of the tumor necrosis factor receptor (TNF-R) superfamily that also includes Fas/APO-1, TNF-RI and 2, and TRAIL receptors. Exact role of CD30 is still unclear, but CD30 has been linked to thymic negative selection of thymocytes. It has been suggested that CD30 is a negative regulator for CD8+ T cells cytotoxicity. In this study we examined the expression of CD30 and its role in the apoptosis of blood eosinophils. Human eosinophils were purified from allergic and atopic asthmatic subjects (purity >98%, viability >99%) and CD30 expression was examined by both flow cytometry using FITC-conjugated anti-CD30 antibodies and Western Blotting before and after the induction of apoptosis. Eosinophils expressed CD30 molecule, and there was significantly increased CD30 expression in serum-deprived (for 24 hours) eosinophils, which were undergoing apoptosis, as measured by Annexin V-labeling. We are currently examining the role of CD30 molecule in eosinophils and its relationship with programmed cell death in eosinophils. However, to our knowledge this is the first report on the presence of CD30 molecule in eosinophils.
S229
Serum Tryptase levels as a Possible Marker of Myeloproliferative Disease in a Subset of Patients With tlypereosinophilic Syndrome Amy D Klion, Melissa Law, Pierre Noel, Thomas B Nutman National Institute of Health, Bethesda, MD Hypereosinophilic syndrome (HES), as defined by the presence of eosinophilia of unknown etiology (>1500/mm3 for >6 months) and evidence of end organ damage, is a heterogeneous group of disorders of unknown etiology. Despite comparable levels of eosinophilia at presentation. some patients remain stable over many years time, whereas others experience rapid deterioration with progression to lymphoma or leukemia. Recent studies have demonstrated that some HES patients have clonal populations of lymphocytes or eosinophils detectable in the peripheral blood; however, the etiology of HES remains unclear in a majority of patients. Since serum tryptase levels, a specific marker of mast cell-mediated disease, are elevated in some patients with myeloproliferative disorders, we examined the utility of serum tryptase in identifying a population of HES patients with an underlying myeloproliferative disorder. Serum tryptase levels were elevated (geometric mean 34, range 22.3 to 47.5, normal range: 0 to 11.5) in 5/l 0 patients with HES, but in none of 9 patients with comparable eosinophil levels secondary to parasitic infection (n=4), lymphoma (n=l) or other eosinophilic disorders (n=4). Four of the five patients with elevated tryptase (and none of the patients with normal tryptase levels) have had progression of end organ involvement despite therapy, and all 5 have dysplastic eosinophils and eosinophilic precursors on peripheral smear. Bone marrow biopsies in these 5 patients were notable only for a marked increase in eosinophils and eosinophil progenitors, and cytogenetic and immunophenotyping studies were normal. Serial tryptase levels continued to rise (from 27.1 to 35.6 to 47.5) in one patient who experienced progression of disease despite a decrease in eosinophilia from 7,500/mm3 to 15OO/mm3 over a 7 month period. These findings suggest that a subset of patients with HES have a proliferative disorder involving cells of the early myeloid lineage. Serum tryptase may be useful in identifying these patients and in monitoring their response to therapy.
692
693
Withdrawn