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380 Increases in circulating CD34+ hemopoietic progenitors in allergen-induced late asthmatic responses
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 1, PART 3 377
Analysis of Recombinant Human Tumor Necrosis F a c t o r - o r - I n d u c e d C D 4 E x p r e s s i o n on H u m a n Eosinophils. M H o s s a i n M S L Y O k u b o M D , S Horic
Abstracts
379
M D , M Sekilzuchi M D , M a t s u m o t o , J a p a n It is reported that various cytokines are involved in allergic diseases. Several newly expressed surface antigens on bnman eosinophils are reported in sputum and hronchoalveolar lavage fluid. We examined CIM expression on human eosinophils by tumor necrosis factor-alpha(TNFct) stimulation, We further examined regulating mechanisms of CIM expression and effector function of CD4. Purified eosinophils were obtained by magnetic cell separation system using anti-CDl6 monoclonal antibody. Eosinophils were cultured with various concentrations of rhTNF-ct with or without drugs for 24 hr. After culture, CD4 expression on eosinophils were analysed using flow cytometry, rhTNF-ct at a concentration of 0.1 ng/mi and at 12 hr culture significantly induced CIM expression on eosinophils, rhTNF-ctinduced CD4 expression was shown in a dose-and time-dependent fashion, rhTNF-ct-induced CD4 expression was significantly inhibited by 10-6 M cycloheximide, 10 -6 M dexamethasone or 10.6 M herbimycin A. Recombinant human interferon-gamma(rhlFN-y) inlfibited rhTNF- ct-induced CD4 expression in a dose-dependent fashion above 10U/ml. Cross-linking of CD4 on eoalnophils did not evoke eosinophil protein X(EPX) release as eosinophil degranulation. It is suggested that CD4 synthesis on cosinophils is dependent on tyrosine kinase activity and de novo protein synthesis. Furthermore, it is suggested that crossJinking of newly expressed CD4 molecule does not mediate eosinophil degranulation.
378
EosinophiI-T Lymphocyte Interaction: Induction o f T C e l l P r o l i f e r a t i o n via C D 4 0 . K Lim MD. R Geha MD. PF Weller. MI), Boston, MA Eosinophils, important effector granulocytes involved in allergic inflammatory diseases, also are capable of influencing lymphocyte activities. Eosinophils were isolated using the MACS system, and T lymphocytes were isolated by monocyte adherence to plastic and passage through a T cell enrichment column. Freshly isolated eosinophils from nonallergic donors were negative by flow cytometry for CD40 expression, whereas freshly isolated eosinophils from patients with idiopathic hypereosinophilic syndrome (HES) and those with histories of allergy were positive for CD40 (n=10). The following cytokine combinations failed to induce CD40 on eosinophils from normal nonatopic donors: GM-CSF, GM-CSF+IFN-3,, GM-CSF +TNF-ct, GM-CSF+IL-4, GM-CSF+IL7, GM-CSF+IL10, GM-CSF+ IL-2, IL-3+IL-4, IL3+IL10, IL3+IL-7, and IL-4+IL-5. To evaluate whether ceil surface activation might induce eosinophils to express CD40, mAbs against HLA-DR, CD54, CD58, and CD69 were added and crosslinked. These, as well as crosslinked sIgA and PMA+ionomycin, failed to induce CD40 expression on eosinophils. Using soluble CD3 stimulation and autologous eosinophils as accessory ceils, highly purified T lymphocytes from allergic donors proliferated in the presence of eosinophils. This eosinophil-dcpendent T cell proliferation was inhibited by mAbs against CD40, CD54, CD58 and not by rsCTLA-4lg. Addition of rsCD40 was inhibitory confirming that CD40 on eosinophils is responsible for the T lymphocyte stimulation. T cell proliferation was not accompanied by IL-2 production detectable by ELISA. In summary, CD40 is present on eosinot~hils from donors with HES or allergy and may function to induce T lymphocyte proliferation, a previously unrecognized effect of CD40 ligand engagement.
380
277
Effects of dexamethasone, cyclosporine A and rapamyein on eosinophil degranulation and survival. O. Men~. S. YinE. B. Assoufi. R. Moabel. and A.B. Kay. London UK We examined the effects of rapamycin (Rapa), eyclosporine A (CsA) and dexamethasone (Dex) on survival and degranulation of eosinophils (Eos). These drugs are known to inhibit eytokine transcription, cytokine-dependent biological activities and degranulation events. Eos wore isolated from atopio subjects using MACS method, and cultured with various conditions. Viability of Eos was assessed by flow eytometry after staining6with pr~pidium iodide. All three compounds (I0" to i0" M) inhibited rhlL-5induced survival of Eos in a time and dosedependent fashion. However, the effects of Rapa and CsA, although significant, were relatively weak. Addition of Rapa and CsA 24h later the commencement of the culture gave similar results to incubation from time 0. IL-5 significantly enhanced ECP release by serum coated beads. In the absence of IL-5, no significant effects of the drugs on degranul~tion of Eos were observed. Rape and CsA (i0 T M and I0 "7 M) gave weak, but significant inhibition of IL-5enhanced ECP release. Those data suggest that the drugs which influence cytokine gene expression in T-cells also effect eytokineenhanced degranulation and survival of Eos.
INCREASES
IN
CIRCULATING
CD34 +
HEMOPOIETIC
PROGENITORS IN ALLERGEN-INDUCED LATE ASTHMATIC RESPONSES R SclmfiPhD. L Wood. R Watson. MD Inman MD PhD, PM O'Bvrne MB. JA Denbur~ MD. McMaster University, Hamilton, Ontalio, Canada. The role of circulating hemopoietio progenitors in the development of airways inflammation in asthma was investigated. A group of I l stable asthmatiosubjects (1~ agonist only as needed) had inhalation provocation tesL,~ with allergen (Ag). Blood (PB) samples were taken before and at 611 and 24h post-Ag; low density non-adherent mononuolear cells (NAMNC) were collected by centrifugation of PB on 65% Percoll density gradients. Progenitors were enumerated by a newly developed method for FACS analysis ofCD34, a "pan-progenitor"cell marker, as well as by 14 day methylcellulose colony forming (CFU) assays. In subjects who developed late asthmatic responses following Ag inhalation (n=6), a significant increase in PB CD34+ cells was detected at 24h post-Ag compared with pre-Ag (10 482 4. 4524 (M ± SEM) vs. 4332 4- 1639 CD34+cells/l 06NAMNC; p
Analysis of Recombinant Human Tumor Necrosis F a c t o r - o r - I n d u c e d C D 4 E x p r e s s i o n on H u m a n Eosinophils. M H o s s a i n M S L Y O k u b o M D , S Horic
Abstracts
379
M D , M Sekilzuchi M D , M a t s u m o t o , J a p a n It is reported that various cytokines are involved in allergic diseases. Several newly expressed surface antigens on bnman eosinophils are reported in sputum and hronchoalveolar lavage fluid. We examined CIM expression on human eosinophils by tumor necrosis factor-alpha(TNFct) stimulation, We further examined regulating mechanisms of CIM expression and effector function of CD4. Purified eosinophils were obtained by magnetic cell separation system using anti-CDl6 monoclonal antibody. Eosinophils were cultured with various concentrations of rhTNF-ct with or without drugs for 24 hr. After culture, CD4 expression on eosinophils were analysed using flow cytometry, rhTNF-ct at a concentration of 0.1 ng/mi and at 12 hr culture significantly induced CIM expression on eosinophils, rhTNF-ctinduced CD4 expression was shown in a dose-and time-dependent fashion, rhTNF-ct-induced CD4 expression was significantly inhibited by 10-6 M cycloheximide, 10 -6 M dexamethasone or 10.6 M herbimycin A. Recombinant human interferon-gamma(rhlFN-y) inlfibited rhTNF- ct-induced CD4 expression in a dose-dependent fashion above 10U/ml. Cross-linking of CD4 on eoalnophils did not evoke eosinophil protein X(EPX) release as eosinophil degranulation. It is suggested that CD4 synthesis on cosinophils is dependent on tyrosine kinase activity and de novo protein synthesis. Furthermore, it is suggested that crossJinking of newly expressed CD4 molecule does not mediate eosinophil degranulation.
378
EosinophiI-T Lymphocyte Interaction: Induction o f T C e l l P r o l i f e r a t i o n via C D 4 0 . K Lim MD. R Geha MD. PF Weller. MI), Boston, MA Eosinophils, important effector granulocytes involved in allergic inflammatory diseases, also are capable of influencing lymphocyte activities. Eosinophils were isolated using the MACS system, and T lymphocytes were isolated by monocyte adherence to plastic and passage through a T cell enrichment column. Freshly isolated eosinophils from nonallergic donors were negative by flow cytometry for CD40 expression, whereas freshly isolated eosinophils from patients with idiopathic hypereosinophilic syndrome (HES) and those with histories of allergy were positive for CD40 (n=10). The following cytokine combinations failed to induce CD40 on eosinophils from normal nonatopic donors: GM-CSF, GM-CSF+IFN-3,, GM-CSF +TNF-ct, GM-CSF+IL-4, GM-CSF+IL7, GM-CSF+IL10, GM-CSF+ IL-2, IL-3+IL-4, IL3+IL10, IL3+IL-7, and IL-4+IL-5. To evaluate whether ceil surface activation might induce eosinophils to express CD40, mAbs against HLA-DR, CD54, CD58, and CD69 were added and crosslinked. These, as well as crosslinked sIgA and PMA+ionomycin, failed to induce CD40 expression on eosinophils. Using soluble CD3 stimulation and autologous eosinophils as accessory ceils, highly purified T lymphocytes from allergic donors proliferated in the presence of eosinophils. This eosinophil-dcpendent T cell proliferation was inhibited by mAbs against CD40, CD54, CD58 and not by rsCTLA-4lg. Addition of rsCD40 was inhibitory confirming that CD40 on eosinophils is responsible for the T lymphocyte stimulation. T cell proliferation was not accompanied by IL-2 production detectable by ELISA. In summary, CD40 is present on eosinot~hils from donors with HES or allergy and may function to induce T lymphocyte proliferation, a previously unrecognized effect of CD40 ligand engagement.
380
277
Effects of dexamethasone, cyclosporine A and rapamyein on eosinophil degranulation and survival. O. Men~. S. YinE. B. Assoufi. R. Moabel. and A.B. Kay. London UK We examined the effects of rapamycin (Rapa), eyclosporine A (CsA) and dexamethasone (Dex) on survival and degranulation of eosinophils (Eos). These drugs are known to inhibit eytokine transcription, cytokine-dependent biological activities and degranulation events. Eos wore isolated from atopio subjects using MACS method, and cultured with various conditions. Viability of Eos was assessed by flow eytometry after staining6with pr~pidium iodide. All three compounds (I0" to i0" M) inhibited rhlL-5induced survival of Eos in a time and dosedependent fashion. However, the effects of Rapa and CsA, although significant, were relatively weak. Addition of Rapa and CsA 24h later the commencement of the culture gave similar results to incubation from time 0. IL-5 significantly enhanced ECP release by serum coated beads. In the absence of IL-5, no significant effects of the drugs on degranul~tion of Eos were observed. Rape and CsA (i0 T M and I0 "7 M) gave weak, but significant inhibition of IL-5enhanced ECP release. Those data suggest that the drugs which influence cytokine gene expression in T-cells also effect eytokineenhanced degranulation and survival of Eos.
INCREASES
IN
CIRCULATING
CD34 +
HEMOPOIETIC
PROGENITORS IN ALLERGEN-INDUCED LATE ASTHMATIC RESPONSES R SclmfiPhD. L Wood. R Watson. MD Inman MD PhD, PM O'Bvrne MB. JA Denbur~ MD. McMaster University, Hamilton, Ontalio, Canada. The role of circulating hemopoietio progenitors in the development of airways inflammation in asthma was investigated. A group of I l stable asthmatiosubjects (1~ agonist only as needed) had inhalation provocation tesL,~ with allergen (Ag). Blood (PB) samples were taken before and at 611 and 24h post-Ag; low density non-adherent mononuolear cells (NAMNC) were collected by centrifugation of PB on 65% Percoll density gradients. Progenitors were enumerated by a newly developed method for FACS analysis ofCD34, a "pan-progenitor"cell marker, as well as by 14 day methylcellulose colony forming (CFU) assays. In subjects who developed late asthmatic responses following Ag inhalation (n=6), a significant increase in PB CD34+ cells was detected at 24h post-Ag compared with pre-Ag (10 482 4. 4524 (M ± SEM) vs. 4332 4- 1639 CD34+cells/l 06NAMNC; p