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545 Surface characterization of ige binding cells in peripheral blood assessed by flow cytometry and sorting
543
544
CELLULAR CONTROL OF PREFERENTIAL IgE INDUCTION BY TOBACCO ANTIGEN. Carol Baum MD; Paul Szabo PhD, Gregory Siskind MD, Carl Becker MD, Carolyn Weinberg BA, Tova Francus PhD, New York, New York. Cigarette smoking has been associated with respiratory and cardiovascular disease. Tobacco glycoprotein (TGP), a Rutin(rich compound extractable from tobacco leaves and cigarette smoke, preferentially induces a long-lasting IgE response in mice injected intradermally. Also, mice injected with R-bovine serum albumin(BSA), preferentially synthesize IgE antibodies to BSA. Cell transfer studies were done to determine whether this is due to the induction of suppressor T Lethally cells (Ts) for other isotypes. irradiated LAFl mice were reconstituted with spleen cells from (a) naive mice, (b) BSA immunized mice, (c) R-BSA immunized mice; or a mixture of cells from naive and (d) BSA or (e) R-BSA immunized mice. After intraperitoneal immunization with BSA, the recipients were assayed for splenic anti-BSA plaque forming cells and anti-BSA hemagglutinating Ab. No difference was seen between recipients of spleens from mice immunized with BSA or R-BSA. IgE responses in mice have been shown to be associated with THZ-helper cells that produce interleukin 4 (IL4) but not IL2. Within 24 hrs of culture with TGP we detected IL4 mRNA in murine spleen cells while no IL2 was detected in the supernatants of such cultures. Overall our data support the hypothesis that the preferential IgE response to TGP results from the activation of TH2 rather than TS activity.
545
INDUCTION OF PceR2/CD23 ON NORMAL HUMANMOMOCYTHS (!4$2) BY HUMANRECOMBINANT INTRRLRURIN-4 (IL-4). D. Vercelli, H.H. Jabara. B.W. Lee, R.S. Geha, and D.Y.M. Leung. Department of Pediatrics, Harvard Medical School, Boston, MA. An increase in the frequency of peripheral blood MB bearing PC receptors for IgE (FccR2/ CD23) has been observed in atopic conditions. In order to define signals able to induce the expression of FceR2/CD23 we have tested a variety of recombinant lymphokines. FcoR2 induction was detected by flow cytometry using anti-CD23 monoclonal antibodies (mAbs) as well as soluble IgE. In 11 experiments, the mean baseline FcER~ expression on purified normal MQ was 522. Incubation with rIL-4 (50 U/ml), induced a significant increase in the expression of FceR2 (mean X FcER~ cells = 3023; p < 0.001).
546
TION OF I-
SORTING.Jann. Ahlstedt.
. .
CEt t S IN
Wfan
Pharmacra -. Diagnostics AB, 751 82
Uppsala, Sweden. Pheripheral blood cells were obtained from allergic and non-allergic blood donors. A significant (pcO.01 n=lZ) correlation was obtained between average cell surface bound IgE and total IgE in serum. Double staining, using fluorescein conjugated a-IgE and a phycoerythrine conjugated secondary antibody, was performed on DeriDheral blood cells after incubating the cells with IQE and’ monoclonal antibodies against different cell surface markers (OKT 9. a-CD 38. a-HLA-DR. a-CD 23, a-CD 21, a-CD 15, a:CD 5, a-CD 8, a-CD 4, a-CD 3, a-CD 19, a-Leu 19). The IgE binding cell population was split into two fractions and sorted onto glass slides for Wright’s staining and morphological evaluation. In normal healthy individuals double staining of all IgE positive cells was obtained only with a-CD 38. Partial binding of the IgE positive ceils was obtained with OKT 9, a-CD 15, a-CD 21 and a-CD 23. For one patient, selected for high IgE values, we found a large fraction of IgE bearing cells which also bound a-CD 21. Subsequent sorting of the IgE positive cells showed that a-CD 21 did not label the basophilic cell population. In conclusion, a-Cd 38 bound to all IgE positive cells whereas, OKT 9, a-CD 15, a-CD 21 and a-CD 23 only bound partially.
Immunoprecipitation of 1251-labelled MQ and the MQ cell line U937 with CD23-specific mAbs or IgE showed a 45 kD band identical to the 45 kD band precipitated by the same reagents from the FccR2(+) RPM1 8866 B cell line. Fc~R2/cD23 was fully expressed after a 24 hr incubation with rIL-4. The effect of IL-4 was specific: none of, the other ILs tested (IL-l, IL-2, IL-3, BSFZ, GMCSF and IFN-x could induce FcsR2/CD23, either alone or in various combinations. rINF-# did not inhibit the IL-4-induced expression of FcoR/CD23. Our results show that rIL-4 specifically induces FceR2iCD23 on normal human Mp. An increased secretion of IL-4 may thus be responsible for the high percentage of circulating M$3 bearing FccR2 observed in allergy.
304
ALLERGENINDUCED Fc-RECEPTORS FOR IgE (FcaRk ON HUMAN T LYMPHOCYTES. J.C. Prinz. X. Baur, J. Ring. N. Endres. E.P. Rieber, ~Munich, FRG. FcaR+ T lymphocytes are supposed to be involved in the regulation of the IgE response. In order to determine the conditions for FcsR expression on human T lymphocytes we have stimulated PBMC of patients allergic 1) to phospholipase A2 (PLAa) from bee venom or 2) to hemoglobin of chironomide masks (Chb) from fishfodder with the specific allergens, with control antigens and different lectins. The expression of FceR on the activated T cells was monitored during 6 days in a triple marker analysis, using monoclonal antibodies (MAB) raised against the human low affinity FcaR (CD23) in our laboratory, and fluorochrome labeled MABs against T and B cell antigens . No FcsR+ T cells were detected among resting PBMC. After stimulation with PHA 7,6+6% (day 3), with Con A 0,6+0,7% (day 2), with PWM 0,8+0,8% (day 3) of the total T cells expressed FcaR. After stimulation of PBMC from allergic donors with the respective allergens PLAz 2,54 2,5% and Chb 7,8+1,2% (day 6) FceR+ T cells were observed, after stimulation with the control antigens tetanus toxoid 0,6+0,8% (day 4) and tuberculin 0,2+0,6% (day 3). All allergen induced FceR+ T cells were CD4+, whereas lectin induced F&R+ T cells were either CD4+ or CD8+. Thus, FcsR are transiently expressed as activation antigens on T cells. They are preferentially induced by stimulation of PBMC from allergic patients with the respective allergens. The role of these cells in the IgE response can now be studied.
544
CELLULAR CONTROL OF PREFERENTIAL IgE INDUCTION BY TOBACCO ANTIGEN. Carol Baum MD; Paul Szabo PhD, Gregory Siskind MD, Carl Becker MD, Carolyn Weinberg BA, Tova Francus PhD, New York, New York. Cigarette smoking has been associated with respiratory and cardiovascular disease. Tobacco glycoprotein (TGP), a Rutin(rich compound extractable from tobacco leaves and cigarette smoke, preferentially induces a long-lasting IgE response in mice injected intradermally. Also, mice injected with R-bovine serum albumin(BSA), preferentially synthesize IgE antibodies to BSA. Cell transfer studies were done to determine whether this is due to the induction of suppressor T Lethally cells (Ts) for other isotypes. irradiated LAFl mice were reconstituted with spleen cells from (a) naive mice, (b) BSA immunized mice, (c) R-BSA immunized mice; or a mixture of cells from naive and (d) BSA or (e) R-BSA immunized mice. After intraperitoneal immunization with BSA, the recipients were assayed for splenic anti-BSA plaque forming cells and anti-BSA hemagglutinating Ab. No difference was seen between recipients of spleens from mice immunized with BSA or R-BSA. IgE responses in mice have been shown to be associated with THZ-helper cells that produce interleukin 4 (IL4) but not IL2. Within 24 hrs of culture with TGP we detected IL4 mRNA in murine spleen cells while no IL2 was detected in the supernatants of such cultures. Overall our data support the hypothesis that the preferential IgE response to TGP results from the activation of TH2 rather than TS activity.
545
INDUCTION OF PceR2/CD23 ON NORMAL HUMANMOMOCYTHS (!4$2) BY HUMANRECOMBINANT INTRRLRURIN-4 (IL-4). D. Vercelli, H.H. Jabara. B.W. Lee, R.S. Geha, and D.Y.M. Leung. Department of Pediatrics, Harvard Medical School, Boston, MA. An increase in the frequency of peripheral blood MB bearing PC receptors for IgE (FccR2/ CD23) has been observed in atopic conditions. In order to define signals able to induce the expression of FceR2/CD23 we have tested a variety of recombinant lymphokines. FcoR2 induction was detected by flow cytometry using anti-CD23 monoclonal antibodies (mAbs) as well as soluble IgE. In 11 experiments, the mean baseline FcER~ expression on purified normal MQ was 522. Incubation with rIL-4 (50 U/ml), induced a significant increase in the expression of FceR2 (mean X FcER~ cells = 3023; p < 0.001).
546
TION OF I-
SORTING.Jann. Ahlstedt.
. .
CEt t S IN
Wfan
Pharmacra -. Diagnostics AB, 751 82
Uppsala, Sweden. Pheripheral blood cells were obtained from allergic and non-allergic blood donors. A significant (pcO.01 n=lZ) correlation was obtained between average cell surface bound IgE and total IgE in serum. Double staining, using fluorescein conjugated a-IgE and a phycoerythrine conjugated secondary antibody, was performed on DeriDheral blood cells after incubating the cells with IQE and’ monoclonal antibodies against different cell surface markers (OKT 9. a-CD 38. a-HLA-DR. a-CD 23, a-CD 21, a-CD 15, a:CD 5, a-CD 8, a-CD 4, a-CD 3, a-CD 19, a-Leu 19). The IgE binding cell population was split into two fractions and sorted onto glass slides for Wright’s staining and morphological evaluation. In normal healthy individuals double staining of all IgE positive cells was obtained only with a-CD 38. Partial binding of the IgE positive ceils was obtained with OKT 9, a-CD 15, a-CD 21 and a-CD 23. For one patient, selected for high IgE values, we found a large fraction of IgE bearing cells which also bound a-CD 21. Subsequent sorting of the IgE positive cells showed that a-CD 21 did not label the basophilic cell population. In conclusion, a-Cd 38 bound to all IgE positive cells whereas, OKT 9, a-CD 15, a-CD 21 and a-CD 23 only bound partially.
Immunoprecipitation of 1251-labelled MQ and the MQ cell line U937 with CD23-specific mAbs or IgE showed a 45 kD band identical to the 45 kD band precipitated by the same reagents from the FccR2(+) RPM1 8866 B cell line. Fc~R2/cD23 was fully expressed after a 24 hr incubation with rIL-4. The effect of IL-4 was specific: none of, the other ILs tested (IL-l, IL-2, IL-3, BSFZ, GMCSF and IFN-x could induce FcsR2/CD23, either alone or in various combinations. rINF-# did not inhibit the IL-4-induced expression of FcoR/CD23. Our results show that rIL-4 specifically induces FceR2iCD23 on normal human Mp. An increased secretion of IL-4 may thus be responsible for the high percentage of circulating M$3 bearing FccR2 observed in allergy.
304
ALLERGENINDUCED Fc-RECEPTORS FOR IgE (FcaRk ON HUMAN T LYMPHOCYTES. J.C. Prinz. X. Baur, J. Ring. N. Endres. E.P. Rieber, ~Munich, FRG. FcaR+ T lymphocytes are supposed to be involved in the regulation of the IgE response. In order to determine the conditions for FcsR expression on human T lymphocytes we have stimulated PBMC of patients allergic 1) to phospholipase A2 (PLAa) from bee venom or 2) to hemoglobin of chironomide masks (Chb) from fishfodder with the specific allergens, with control antigens and different lectins. The expression of FceR on the activated T cells was monitored during 6 days in a triple marker analysis, using monoclonal antibodies (MAB) raised against the human low affinity FcaR (CD23) in our laboratory, and fluorochrome labeled MABs against T and B cell antigens . No FcsR+ T cells were detected among resting PBMC. After stimulation with PHA 7,6+6% (day 3), with Con A 0,6+0,7% (day 2), with PWM 0,8+0,8% (day 3) of the total T cells expressed FcaR. After stimulation of PBMC from allergic donors with the respective allergens PLAz 2,54 2,5% and Chb 7,8+1,2% (day 6) FceR+ T cells were observed, after stimulation with the control antigens tetanus toxoid 0,6+0,8% (day 4) and tuberculin 0,2+0,6% (day 3). All allergen induced FceR+ T cells were CD4+, whereas lectin induced F&R+ T cells were either CD4+ or CD8+. Thus, FcsR are transiently expressed as activation antigens on T cells. They are preferentially induced by stimulation of PBMC from allergic patients with the respective allergens. The role of these cells in the IgE response can now be studied.