Document a4xew22KqmbbYjOnQbe9KzD9y

& > (-/$ / Workshop on measurement of in vitro IgE synthesis and regulation of IgE synthesis Judith G. Massicot, Ph.D., and Kimishige Ishizaka, M.D., Ph.D. Bethesda and Baliimore, Md. Twenty years have passed since the isolation of IgE, the antibody responsible for immediate-type hyper sensitivity. This discovery led investigators to search for an understanding of the mechanisms regulating the synthesis of IgE with the ultimate goal being the treat ment, and perhaps even prevention, of allergic dis eases via the regulation of IgE synthesis. Many distinguished and diligent scientists have been and currently are engaged in studies in this field. Significant strides have been made, but the research is hampered by some technical problems, one of the most significant being the accurate measurement of low levels of IgE synthesis in vitro. On May 17, 1985, the Workshop on Measurement of in vitro IgE Syn thesis and Regulation of IgE Synthesis was held at the National Institutes of Health in Bethesda, Md. The goal of this workshop was to provide a forum where those pursuing work in the field of IgE regu lation could (I) present their work, (2) discuss com mon problems, (3) talk about possible solutions to these technical difficulties, and (4) come to an agree ment on future goals for these studies. Dr. Ishizaka gave an overview of the field, and 11 other investi gators gave short presentations. A condensed version of each presentation is included in this article. Ap proximately 20 other interested persons, from all over the world, attended and joined in the discussions. T CELL FACTORS INVOLVED IN THE ISOTYPE-SPECIFIC REGULATION OF THE IgE RESPONSE AND APPROACHES TO CONTROL IgE FORMATION Kimishige Ishizaka, M.D., Ph.D., The Johns Hopkins School of Medicine Baltim ore, Md. IgE synthesis is regulated, like other antibody classes, via antigen-specific T cells, both helper and tfic National IriMiiulr of Allergy an j Inlcilim i'. Di'-tNi'S'.. Asthma and Allergy Hiaocii. Ik 'tliCMla, Md . and The Julius Hi'fikins t hiisrrsiiy Selioot o! M iM ii'im-. I Vpatimc! id M '`i!u<irr and M ii'lutnoli'gv. Ualiimnu- . Md KtvI'Otfd for [miH k m Uhji I Kv !('. IUX5 AsWj'icd for [niMn/aiain Jan X. JVSfi. Kcpiint requests' Judith Massiewl, t'h I) .. Asthma ami Allergy flraneh, National Institute o f Allergy and lnleeiious Diseases, Wcuv.envd lildg . Room 752, lledtestla, M D 2nx*i2 544 A b b revia tio n s u sed PF: Potentiating factor SF: S uppressive factor BF: B inding factor G EF: G lycosylation-enhancing factor G IF; G ylcosylation-inhibiting factor PB M N C : Peripheral blood m o n o n u clear cell PW M : Pokcw ecd m itogen id d d io ly p e R IA : R adioim m unoassay T C S : T cell sup ern atan ts A ThCC: A llorcaclivc helper T cell clone E B V : E pstcin-B arr virus C on A; C oncanavalin A R: R eceptor ( suppressor. However, there also functions a unique mechanism that is selective for the IgE isotype. Dr. Ishizaka and his coworkers during the last 6 years have examined this phenomenon at the cellular`and molecular levels in the rodent systems and most re cently have evidence that a similar mechanism is at work in man. His keynote lecture summarized what is known regarding T cell factors involved in isotypespccific regulation of IgE synthesis and possible clin ical applications. Table 1 lists selected influences on IgE produc tion. These indicated the possibility of an isotypespcciiic mechanism. Using Nippostrongyhts-\nfeeted rats, they found soluble factors with affinity for IgE that were derived from W 3/25* (Lyt 1+) T'cells. Depending on when the cells were taken, the factors either enhanced or suppressed IgE synthesis. Both IgE-PF and IgE-SF have similar molecular weights (13,000 to 15,000 dallons). Both are glycoproteins hut contain different oligosaccharides. These IgE-BFs could also be obtained after injec tions of complete Freund's adjuvant or Bordctella per- vaccine Genetic factors as well as low-dose X l a y s pia\ .i mlo in determining the nature of the ;.:i MF ie. *. ,\: !e!: l1!*' m d e l e t e d whenever IgH svnihoM.s is vun.uived. am! Igli-Nr is lownd when the lei! icspuii'.e is selectively stippiessed. I'llis suggested that the IgF-files were invoked in the regulation of the IgJi antibody response in vivo.1 v o lu m e 7? NUMBER 4 Workshop on measurement of in vitro IgE synthesis 545 The same T ceJJs have the capacities to produce both IgE-PF and JgE-SF. The environment of the cells determines the nature of the IgE-BF. The major dif ference between the factors is in their carbohydrate moieties. IgE-PFhasbolh N-linked, mannose-richoligosaccharide and O-Jinked oligosaccharide with both oligosaccharides having sialic acid as the terminal sugar residues. IgE-SF contains O-linkcd oligosac charide with galactose --* jV-acctyl galactosamine as the terminal sugar. T cells also produced 60K and 30K IgE-BF, some of which either suppressed or enhanced the IgE re sponse. Recently, Drs. Kevin Moore and Chris M ar tens (in DNAX Institute of Molecular Biology) in collaboration with Dr. Ishizaka's group, cloned the gene for rat IgE-BFs, and four cDNA clones were obtained.1Transfection of COS7 monkey kidney cells by each cDNA clone resulted in formation of IgEBFs. None of them suppressed IgE synthesis; how ever, IgE-BF derived from two clones selectively po tentiated it. The IgE-BFs derived from clone 8.3 con sisted of two species, one 60,000 and another 11,000 to 12,000 daltons. Both potentiated the IgE response. Since the cDNA were constructed from messenger RNA of cells that form IgE-SF, these results suggested that IgE-PF and IgE-SF share a common structural gene and that posttranslalion glycosylation events de cide the difference. Nucleotide sequencing of cDNA clone 8.3 revealed a coding region for 556 amino acids, corresponding in size to the 60K IgE-BF. Thus, the 1IK factor must be a cleavage product. It is hoped that gene cloning will elucidate the structural basis of the function of IgE-BFs. The effective substance to induce the factor for mation is an inlerfcron-like substance. However, this inducer substance does not determine the nature of the IgE-BFs formed. The activities of the factors are controlled by two T cell factors that either enhance or inhibit the W-glycosylation of IgE-BFs. GEF is derived from a subset of Lyt 1* T cells. Thus, when Fc R+ T cells are stimulated in the presence of GEF, these cells selectively form glycosylated IgE-BF that potentiates the IgE response. The same Fc R + T cells selectively form IgE-SF when the cells were stimu lated by imluccrs in the presence of GIF. In the mouse the major source of GIF is Lyi 2*. I-J * antigen-spe cific suppressor T eel Is. GIF. with :t molecular eight ol about 15 K. is a fragment ol phosphor. I.tied lipom vtabu. It inhibits /V-glycosylation ol" IgE-BI;s via inactivniion ol phos pholipase. Meanwhile, GEF' has serine piotense ac tivity and is a kallikrein-like enzyme. Under physi ologic conditions, the balance between GEF and GIF appears to determine the nature of IgE-BFs formed. Hie production of IgE-SF is always accompanied by TABLE 1. Correlation between the IgE response end selective formation of IgE- . potentiating factor and IgE-suppressive factor Influence on IgE production IgE response IgE-BF Infection with nematodes 14 days 8 days Adjuvant treatment Bordetella pertussis vaccine Complete Freund's adjuvant Antigen priming Aluminum hydroxide Complete Freund's adjuvant Genetic factor BDF, mice SJL mice Irradiation with x-ray or cyclohexamide treatment t <-) T i (+) (-) (+) (-) r PF SF PF SF PF SF PF SF PF f Indicates enhancement of IgE response; i indicates suppression of IgE response; ( + indicates IgE antibody formation; and ( --Vindicates no IgE response. the formation of GIF, whereas GEF is detected along with IgE-PF. This same principle may even explain genetic differences. Those mice that are poor IgE producers have cells that constituiively form GIF, whereas those strains that are high IgE producers have cells constituiively forming GEF.1 IgE-BFs are produced by peripheral blood lympho cytes of ragweed-sensitive patients when the cells are incubated with specific antigen and homologous IgE. With the use of peripheral blood T cells of patients with hyper-IgE syndrome or with atopic dermatitis, Saryan et al.j were able to demonstrate the release of an lgE-spccific potentiating factor. Alternatively, they found, in the serum of normal individuals with very low IgE levels, an IgE-SF. Thus, human T cells can form IgE-PF and IgE-SF essentially the same as those found in rodent systems. ^ Dr. Ishizaka has constructed human T cell hybri- domas that produce IgE-BFs on incubation with hu man IgE.4Since human IgE-BFs have affinity not only for human IgE but also for rat IgE. their effect on IgE synthesis can be determined by use of rat MLN cells. With T cells from a subject with no known allergies and a very low serum IgH level, the IgE-BFs produced by the liyhndumas suppiessed Igi e. mheris. With ihc use of another human I' cell iiybiidoma. I66A2, the nature of the IcH-BFn formed could be switched from PF to SF. depending on whether in cubated with bradykinin (which enhances glyco- sylation) or with (ilF. Thus, a common median- Iy*' i i. ir: i1 I1* 546 M assicot and Ishizaka J. ALLERGY CUN. IM M UN O L APRIL 1986 ism appears to regulate the IgE-BFs in man and rodents. The genes encoding human IgE-BFs are now being cloned by Drs. Moore and Martens. Mixtures of some cDNA clones have been used to transfect COS7 cells and produce human IgE-BFs. If the nature of IgE-BFs is determined by the bal ance between GEF and GIF, administration of GIF might be effective to suppress the IgE response. GIF has been purified from culture filtrates of a hybridoma cell and injected into mice. It completely suppressed both The IgE and IgG primary antibody response to DNP-OA. GIF is also effective in suppressing ongoing IgE antibody synthesis.3 Gene cloning of GIF is now in progress. Many hurdles remain to be cleared before clinical applica tion. However, it is hoped that basic mechanisms dis covered in animal systems will lead to new approaches to regulating the IgE antibody response in allergic patients in the not too distant future. MULTICENTER PILOT STUDY OF METHODS TO MEASURE IgE PROTEIN IN CELLCULTURE SUPERNATANTS*-" R. M. Helm, Ph.D., Mayo Clinic and Foundation Rochester, M inn. Numerous immunoassays have been used to mea sure low levels of IgE in supernatants from cultured PBMNCs. Published articles are at variance as to whether IgE is produced by such cells either spon taneously or following mitogen stimulation. To eval uate the sensitivity, specificity, and precision of IgE immunoassays being used by various investigators, a multicenter collaborative study was organized in which Iyophilized test samples were distributed to 22 laboratories. Sensitivity was tested by including samples con taining tissue-culture medium alone or medium spiked with 0.05 ng/ml, 0.25 ng/ml, or 0.50 ng/mi of polyclonal IgE (United States reference standard). Only 13 of 22 laboratories were able to quantitate IgE in the 0i5 ng/ml sample, and only five were able in the 0.25 ng/ml and 0.05 ng/ml samples. Specificity was assayed by including samples con taining 5 ng/ml of polyclonal IgE (unheated or heated at 56 C for 4 hours), IgE (YU), and IgE (ND). All but one or two laboratories were able to quantitate the IgE; however, the values for the polyclonal IgE were in the I to 3 ng/ml range. Half of (lie luboiatorics were unable to measuie the heated pol\clonal IgE sample, indicating their antisera were recognizing heat-labile determinants on the e chain. The ability to quantitate IgE in ccll-culturc super natants was examined by including a sample from a 12-day, pokeweed mitogen-stimulated PBMNC cul ture. Control samples included culture medium with pokeweed mitogen alone, culture medium from un stimulated cells, and culture medium from stimulated cells that had been freeze-thawed five times on day 0. To test the ability of the IgE assays to quantitate IgE in the presence of large quantities of IgG, a stim ulated cell-culture supernatant was spiked with 10 ng/ml of polyclonal IgE. Eighteen laboratories mea sured IgE in the stimulated cultures (values ranging from 0.25 ng/ml to 6.5 ng/ml) with >70% in the lower part of the range. Only those measuring, the higher values found a net increase in IgE over control samples. Most were able to quantitate the IgE in the spiked sample. To assess precision, coded duplicate test samples containing 0.5 ng/ml of polyclonal IgE were included. Only 50% of the participants could measure IgE in both samples, and frequently the values were neither precise nor accurate. In this study a number of different assays were used, including RIA, enzyme immunoassay, and particlecounting immunoassay. Each used their own anti-IgE preparations and their own IgE standards. The accu racy, precision, sensitivity, and specificity of the as says were not dependent on the type of assay. It was concluded that there was a wide variation in the sen sitivity and precision of the assays used to quantitate low levels of IgE. Only three laboratories obtained results consistent with de novo IgE production by the mitogen-stimulated PBMNCs that were used in this study. ANTIBODIES TO IDIOTYPIC DETERMINANTS OF HUMAN IgE MYELOMA PROTEINS REACT WITH POLYCLONAL IMMUNOGLOBULIN IN SUPERNATANTS OF POKEWEED MITOGEN-STIMULATED CELLS AND CAUSE AN OVERESTIMATION OF IgE SYNTHESIS IN VITRO7-9*10 Hans L. Spiegelberg, M.D., Scripps Clinic^ and Research Foundation La Jolla, Cafif. Several years ago Dr. Spiegelberg discovered a subpopulation of lymphocytes carrying Fc receptors for IgE. The number of such cells is increased in patients with allergic diseases. He speculated that these cells may be involved in the regulation of IgE synthesis and at tempted to study the effect of Fc, icccptor-positive B and T cells mi FWM-induced IgE synthesis in \itro. The ability of human PBMNC to synthesize IgE in vitro in response to FWM is controversial. To de termine whether the con Hiding results obtained by different laboratories could be, in pan, the result of