Immunology 1977 33 51
Concanavalin A induced histamine release from human basophils in vitro
A. M. MAGRO & H. BENNICH New York State Kidney Disease Institute, and Department ofMicrobiology aid Immunology, Albany Medical College Albany, New York and Department of Molecular Biology, University of Aarhus, Aarhus, Denmark
Received 11 October 1976; acceptedfor publication 9 December 1976
Querinjean, Beckers, Heremans & Dessy, 1974) reagin has been characterized as an antibody of the IgE class. Concanavalin A a globulin isolated from the jack bean (Canavalia ensiformis) (Sumner, 1919) is a mitogenic lectin which has the capacity to induce histamine release from mast cells (Keller, 1973) and basophils (Hook, Dougherty & Oppenheim, 1974). The induction of histamine release by Con-A raised the interesting possibility that Con-A was activating mast cells and basophils by interacting at sites on the membrane independent of the membrane bound IgE. The fact that Con-A is mitogenic to thymus derived (T) lymphocytes (Janossy & Greaves, 1971; Andersson, Sjoberg & Moller, 1972) and the speculation that mast cells and basophils are derivatives of a T-cell precursor (Burnet, 1975; Ishizaka, Okudaira, Mauser & Ishizaka, 1976) could be considered supportive of the hypothesis that Con-A induced histamine release is not IgE mediated. However, Keller (1973) concluded that in rat mast cells Con-A induced histamine release is IgE mediated. His data show that mast cells from rats immunized to produce high titres of reaginic antibody released to Con-A whereas mast cells from non-immunized rats did not. The more recent report (Sullivan, Greene & Parker, 1975) showed that in the presence of the potentiator of histamine release phosphatidyl serine (Goth, Adams & Knoohuizen, 1971) Con-A does
Summary. The site of interaction for concanavalin A (Con-A)-induced histamine release from human basophils was studied in vitro. Blocking the epsilonone determinant (D-. 1) of IgE with high concentrations of monomer (Fab) anti-Dj1 does not significantly inhibit the quantity of histamine released by suboptimum concentrations of Fc specific anti-IgE. This indicates that the monomer anti-Dj1 does not have the capacity to sterically hinder the bridging of all of the determinants in the C.3 and CA domains (Fc'-e'region) of IgE. The monomer anti-Dj1 does effectively inhibit release induced by suboptimum concentrations of Con-A. The data indicate that for suboptimum concentrations, Con-A activation is IgE mediated and takes place in the proximity of Dj1 and not at the membrane receptor for IgE.
INTRODUCTION
Antigen-induced histamine release from basophils and mast cells is mediated by reaginic antibody. In humans (Ishizaka, Ishizaka & Hornbrook, 1967; Ishizaka & Ishizaka, 1967) and rats (Bazin, Correspondence: Dr A.M. Magro, New York State Kidney Disease Institute, 120 New Scotland Avenue, Albany, N.Y. 12208, U.S.A.
51
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A. M. Magro & H. Bennich
have the capacity to induce histamine release from mast cells of non-immunized rats. This report has promoted speculation that Con-A activation in rat mast cells is not necessarily IgE mediated. In human basophils, the evidence that Con-A induced histamine release is IgE mediated is also equivocal. The reports (Magro, 1974a; 1974b), which show that the blocking of the Fc portion of IgE with monomer anti-FcIgE has the ability to inhibit Con-A-induced histamine release from human basophils, indicate that Con-A is activating in the proximity of the Fc portion of IgE. However, it is currently believed that IgE is bound by its Fc portion to a receptor in the membrane of the histamine releasing cell. The monomer anti-FcIgE blocking data cannot resolve the question of whether Con-A is interacting directly with the Fc portion of IgE or whether it is interacting with the membrane receptor for IgE. The possibility of Con-A interacting at occupied membrane receptors for IgE cannot be excluded for inhibition of Con-Ainduced release by the monomer anti-FcIgE could be due to a steric hindrance phenomenon, where the monomer is preventing the Con-A from interacting with the IgE receptor. The fact that human IgE is well characterized (Bennich & Johansson, 1971; Bennich & Von Bahr-Lindstrom, 1974) can be put to advantage in order to resolve whether Con-A is interacting with the receptor for IgE, or the IgE directly when it induces histamine release from human basophils. The epsilon chains which form the Fc portion of IgE consist of three domains: C.2, C.3 and CA4. The C,2 domain is closest to the Fab portion of the IgE molecule and the CA4 domain incorporates the carboxyl terminal region of the molecule. It is currently believed that the membrane receptor for IgE does not bind in the vicinity of the C.2 domain. It appears that the cytotropic activity of the Fc portion of IgE is a property of the CA4 domain or the C,3 and CA4 domains in cooperation (Dorrington & Bennich, 1973). As antigen the Fc portion of IgE has a major antigenic determinant, historically defined as DJ1 (Bennich & Johansson, 1971) within the C,2 domain of the molecule. This study was initiated to determine whether monomer antibodies specific for DJ1 can inhibit Con-A induced histamine release. Inhibition by monomer anti-Del would indicate that con A activation takes place in the proximity of DJ1 which is the Fc determinant farthest removed from the IgE receptor.
MATERIALS AND METHODS
Buffers and cell preparations Human leucocytes were obtained by venipuncture from blood donors considered atopic in that they have a history of allergies and show an in vitro response to several allergens. The leucocytes were aspirated from the leucocyte-rich supernatant obtained from a sedimentation of the whole blood in the presence of dextran and EDTA (Lichtenstein & Osler, 1964). The supernatant was centrifuged to remove platelets and plasma and the isolated leucocytes were washed twice and then resuspended in a tris-buffered (pH 74) solution, which was 120 mm Na+, 2-2 mm K+, 1 mM2+, 0 7 mm Ca2+ and 0 03 Y. human serum albumin. Reagents
F(ab')2-e fragments were produced by peptic digestion of the IgE (ND) (Johansson and Bennich, 1967) for 8 h in 0-1 m sodium acetate buffer pH 4 5 at 37°. The F(ab')2-e was purified by application to a Sephadex G-150 column followed by chromatography on DEAE-Sephadex A-50 (Bennich & Johansson, 1967). Anti-De1 was prepared from specific rabbit anti-IgE by immunosorption to F(ab')2-e. The eluted anti-Del showed no detectable activity against Fc'. When the anti-D81 was tested against F(ab')2-e and Fc", a reaction of identity was obtained indicating that anti-Del obtained in this manner only reacts with determinants in the C22 region. Monomer anti-Del and monomer anti-FcIgE were obtained by papain digestion (Porter, 1959). The digests were applied to a Sephadex G-100 column in order to separate the monomers from intact antibodies. Crystalized Con-A was provided by Pharmacia (Piscataway, New Jersey). Estimates of molar concentration were based on a mol. wt of 150,000 for intact anti-Dj1 and intact anti-FcIgE, 71,000 for Con-A (Olson & Liener, 1971), and 50,000 daltons for monomer anti-Del and monomer anti-FcIgE. Reaction mixtures and histamine release Reagents and inducing agents were added followed by the cells all at 40. The additions were made into 12 x 75 mm Falcon plastic tubes (2052) to a volume of 0 3 ml. The reaction mixtures were then placed in a 37° bath and incubated for 60 min. Following the incubation the volume was adjusted to 0 5 ml by the addition of cold tris EDTA. Subsequently,
Con-A bridging of sugar moieties at D.5 the tubes were centrifuged and the supernatants decanted for assay. Total available histamine was determined by analysis of supernatants from tubes in which the cells were lysed by incubating in 0 3 N perchloric acid. Typically, there were 1-2 x 106 leucocytes per tube yielding a total quantity of histamine equivalent to 50-100 ng histamine base per ml. Blank tubes containing buffer or monomers in the absence of inducing agent were less than 5 % of the total histamine content. Histamine assay The histamine was assayed by an automated spectrofluorometric technique (Siraganian, 1974). The extraction and fluorometric procedures are a modified version of the techniques as reported by Shore, Burkhalter & Cohn, (1959). The percent histamine released was calculated in excess of the blank as follows: per
cent release
=
100 x
sample -blank
complete - blank
In experiments in which duplicates were done they were averaged. For histamine release in excess of 10%/ the mean deviation divided by the mean was less than 0 08, which implies the data points have a precision within ± 8% about the mean. Completes were usually performed in quintuplet and they showed a precision within ± 5% about the mean.
RESULTS The effect of increasing concentrations of monomer upon the histamine releasing capacity of intact anti-Del The induction of histamine release which is IgE mediated requires the crosslinking or bridging of neighbouring IgE molecules by the inducing agent. Thus intact antibodies directed against IgE evoke a histamine releasing response (Ishizaka, Ishizaka, Johansson & Bennich, 1969) whereas the monomers of these antibodies fail to elicit a response and inhibit histamine release by the intact antibodies (Ishizaka & Ishizaka, 1969). The in vitro dose response follows a profile where the quantity of histamine released ascends through a maximum and then descends with increasing concentrations of inducing agent. From the profile the concentrations
anti-D.1
53
of inducing agent can be divided into three regions: suboptimum, optimum and supraoptimum. The quantity of bridging of the membrane-bound IgE increases with increasing concentrations of inducing agent until along the descending portion of the curve the cells are rendered unresponsive by excess bridging (Magro & Alexander, 1974). Monomer antibodies, produced by papain digestion, have the capacity to bind determinants but not bridge. Through a process of site competition they will decrease the quantity of bridging of the intact antibodies. To ascertain if the monomer anti-Del has the capacity to bind at the epsilon-one determinant of IgE the effect of increasing concentrations of monomer anti-Dj1 upon the histamine releasing capacity of intact anti-D,1 was determined. The inset ofFig. 1 illustrates thehistaminerelease dose-response for intact anti-Dj1 and the points A and B indicate the suboptimum and supraoptimum concentrations used to obtain the data of curve A and curve B respectively. It can be seen from curve A that increasing concentrations of monomer anti-Dj1 inhibit the quantity of histamine released by the suboptimum concentration of intact anti-D.I. As the monomer successfully competes for the D.1 sites the quantity of bridging is lessened until the probability of a bridge is reduced below the activating level. However, for the supraoptimum concentration of anti-Del (curve B), the cells are excessively bridged, and the increasing concentrations of the monomer, although decreasing the quantity of the bridging, brings the cells toward a state of optimal bridging and enhances the histamine release. Fig. 1 demonstrates that the monomer antiD81 has a high enough affinity for the epsilon-one determinant to successfully compete with both suboptimum and supraoptimum concentrations of the intact anti-D81. A comparison of the effect of increasing concentrations of monomer anti-D81 upon the histamine releasing capacity of intact anti-FcIgE and Con-A Fc-specific anti-IgE activates basophils to release histamine by bridging determinants within the C,2, C,3 and CA domains of the IgE molecule. D81 is within the C,2 domain. The membrane receptor for IgE does not bind at C.2, but in the proximity of the CA4 domain or the C83 and CA domains in cooperation. Anti-FcIgE, by having the capacity to bridge determinants within the C,3 and CA4 domains
54
A. M. Magro & H. Bennich A
50p -
-B 0
30p
r I)
10
oo'' 9
8
7
6
-Log1o[monomer (Fab) anti-Dc 1] (mol/)
Figure 1. The effect of increasing concentrations (abscissa) of monomer anti-D81 upon histamine release induced by: curve A: a fixed suboptimum concentration (10-10 M) of intact anti-Del; curve B: a fixed supraoptimum concentration (10-8 M) of intact anti-Dj1. Inset: dose-response for intact anti-D.1 induced release.
of IgE interacts within the vicinity of the membrane receptor for IgE. If monomer anti-D81 has the ability to inhibit the histamine-releasing capacity of
anti-FcIgE, it would indicate that the blocking radius of the monomer is such that it inhibits interactions within the C.3 and CA domains and that there is a high plausibility of the monomer sterically hindering interactions in the proximity of the membrane receptor for IgE. However, curves A and C of Fig. 2 show that increasing concentrations of monomer anti-De1 do not significantly affect the histamine releasing capacity of a near optimum (curve A) or a suboptimum (curve C) concentration of Fc specific anti-IgE. It can be seen that the blocking of the epsilon-one determinant within the C.2 domain does not sufficiently reduce the quantity of bridging within the C.3 and/or CA4 domains to significantly reduce the quantity of histamine released. Since the monomer anti-Dj1 does not inhibit anti-FcIgE induced release, blocking of interactions at the membrane receptors for IgE by the monomer is not likely. The monomer antiDC1 should decrease the quantity of anti-FcIgE bridging within the C.2 domain. The slight decrease in the quantity of histamine released in curves A and C of Fig. 2 could be indicative of this. However, if a small number of bridges suffice to activate a releasing unit of histamine (Magro, 1975; Lawson,
70-
2
501
vr
E IL
301
10 o
8
7
-Loglo[monomer
(Fob)
6
anti-D.1] (maul1)
Figure 2. The effect of increasing concentrations (abscissa) of monomer anti-DI upon histamine release induced by: M) curve A: a fixed near optimum concentration (5 6 x 10oof intact anti-FcIgE; curve B: a fixed near optimum concentration (2-5 x 10-8 M) of Con-A; curve C: a fixed suboptimum concentration (3.1 xlO-10 M) of intact antiFcIgE; curve D: a fixed suboptimum concentration (1-4 x 10`8 M) of Con-A.
Fewtrell, Gomperts & Raff, 1975), it is possible that when bridging within the C.2 domain is totally prevented, the quantity of bridging within parts of the CQ3 and CA4 domains of IgE, by the anti-FcIgE,
even
Con-A bridging of sugar moieties at D1l is of a degree that the probability of activating the histamine releasing units is not significantly reduced. Con A has the capacity to bind polysaccharides and glycoproteins which contain sugar moieties having the C-3, C-4 and C-6 hydroxyl groups of the D-arabino hexopyranosyl ring system (Goldstein, Hollerman & Smith, 1965). At physiological pH Con-A is an equilibrium mixture of identical subunits consisting primarily of tetramers having the capacity to bridge two or more sugar moieties (Mcubbin & Kay, 1971; Becker, Reeke & Edelman, 1971). This ability to bind and bridge specific pyranosyl type sugar moieties is the primary physicochemical property of con-A which renders it active as a histamine releasing agent. In order to resolve whether con-A is bridging sugar moieties at the membrane receptor for IgE or whether it is bridging sugar moieties directly attached to IgE, the effect of blocking D81 upon the histamine releasing capacity of con-A is determined. Curves B and D of Fig. 2 show that increasing concentrations of monomer anti-D81 effectively inhibit histamine release induced by a near optimum (curve B), and a suboptimum (curve D) concentration of Con-A. The data imply that the histamine releasing Con-A interactions are in the proximity of Del. In Fig. 2, the data of curves B and D in conjunction with curves A and C suggest that when Con-A induces histamine release there is a low probability of Con-A bridging sugar moieties at the membrane receptor for IgE.
55
C3, and C4 domains it does not allow the monomer anti-D81 to interact at D.I. The descending portion of the dose-response of the inset of Fig. 1 reveals that when most of the D81 sites are cross-linked by the intact anti-D81 the cells are rendered unresponsive by excess bridging. Anti-FcIgE has specificity to bridge determinants at D.1. At a near optimum concentration, the anti-FcIgE is not excessively bridging at D81 for as the inset of Fig. 1 shows the cells would be rendered unresponsive. The determination of whether the addition of intact anti-D81 in excess concentration renders the cells unresponsive to a near optimum concentration of intact antiFcIgE would ascertain if the anti-Dj1 can successfully compete for sites with the anti-FcIgE. The data in Fig. 3 display the effect of increasing concentrations of intact anti-Dj1 upon the histamine releasing capacity of a near optimum concentration of intact anti-FcIgE. Curve A of Fig. 3 is the dose '70, cx
- 50 F c
0
8 0
30
C w
0 _G
.-IF The effect of increasing concentrations of intact
anti-D.1 upon the histamine releasing capacity of a near optimum concentration of intact anti-FcIgE The ability of monomer anti-D81 to inhibit release induced by the intact anti-D81 (Fig. 1) indicates that the monomer can successfully compete with the intact antibodies for the epsilon-one sites. Curves A and C of Fig. 2 established that high concentrations of the monomer anti-D81 have little effect upon the releasing capacity of intact anti-FcIgE. If it is assumed that the monomer anti-DJ1 blocks most of the sites at DJ1 in the presence of antiFcIgE then the data of Fig. 2 indicate that the monomer does not sterically hinder all interactions within the C.3 and CA domains. However, an alternate interpretation of the data of curves A and C of Fig. 2 is that the affinity of the anti-FcIgE is such that when it bridges determinants within C,2,
co '12
11
10
9
8
7
-Logqopintoct anti-D,l ] (mol/l) Figure 3. Curve A: normal dose response for intact anti-D81 induced release; curve B: the effect of increasing concentrations of intact anti-D8l (abscissa) upon release induced by a fixed near optimum concentration (10-10 M) of intact
anti-FcIgE.
response for the increasing concentrations of antiDC1 alone. Curve B of Fig. 3 shows the quantity of histamine released when the concentrations of anti-Dj1, indicated by the abscissa, were added to the fixed near optimum concentration of antiFcIgE. It is evident from the graph that the cells in the reaction mixtures containing the near optimum concentration of anti-FcIgE are rendered unresponsive when the concentrations of intact antiDj1 are increased past the optimum into the in-
56
A. M. Magro & H. Bennich
hibitory region. The data indicate that a near optimum concentration of anti-FcIgE does not prevent the higher concentrations of anti-D.1 from excessively bridging at DJ1. Since it was demonstrated by the data of Fig. 1 that high concentrations of monomer anti-De1 can successfully compete with intact anti-D.1, suboptimum concentrations of intact anti-FcIgE are not apt to prevent high concentrations of monomer anti-D,1 from binding at the epsilon-one sites. A comparison of the effect of increasing concentrations of monomer anti-FcIgE upon the histamine releasing capacity of Con-A and intact anti-FcIgE The ability of monomer anti-FcIgE to inhibit histamine release induced by con A and intact anti-FcIgE occurs via two different mechanisms. The monomer anti-FcIgE being derived from a papain digestion of the intact anti-FcIgE retains the ability to bind the same determinants as the intact antibody (Porter, 1959). Therefore, the monomer anti-FcIgE inhibits the intact anti-FcIgE by a process of site competition. In contrast, Con-A binding is very specific for certain pyranosyl type sugar moieties. Consequently, the monomer antiFcIgE inhibits the binding and the histamine releasing capacity of Con-A not by site competition, but by steric hindrance. The proximity of the two different binding sites is an important parameter to the efficiency of a steric hindrance phenomenon. A disparity in the ability of monomer anti-FcIgE to inhibit Con-A induced histamine release when compared with its ability to inhibit intact antiFcIgE induced release would indicate that the sugar moieties to which Con-A binds are removed from the antigenic determinants of the Fc portion of IgE. However, the data of Fig. 4 display that the ability of monomer anti-FcIgE to inhibit Con-A induced release is not significantly different from its ability to inhibit release induced by intact antiFcIgE. Although the data of Fig. 4 has related concentrations of Con-A and anti-FcIgE of equal histamine releasing capacity, anti-FcIgE induces histamine release at much lower concentrations than Con-A. This implies that anti-FcIgE binds either with a higher affinity than Con-A and/or it binds at a greater number of sites on the IgE molecule. Therefore, it is not possible to equate absolutely the efficiency of the monomer anti-FcIgE in inhibiting
0
30
\
E
i'\% 0
"8
7
6
-Loglo[monomer (Fab) anti- FcIgE] (mol/ I) Figure 4. The effect of increasing concentrations (abscissa) of monomer anti-FcIgE upon histamine release induced by: curve A: a fixed near optimum concentration (10-1o M) of intact anti-FcIgE; curve B: a fixed near optimum concentration (7-8 x 1o-8 M) of Con-A; curve C: a fixed suboptimum concentration (4-4 x 10-8 M) of Con-A; curve D: a fixed suboptimum concentration (56 x 1O-11 M) of intact antiFcIgE.
Con-A and anti-FcIgE induced histamine release. These considerations aside, the data of Fig. 4 are not contradictory to or incompatible with the hypothesis that Con-A is bridging sugar moieties directly attached to a localized region of the IgE molecule when it induces histamine release. DISCUSSION The use of mitogens as probes has been a productive area of investigation leading to workable models whereby some basic mechanisms of cellular activation have been formulated. The finding that the T-cell mitogen Con-A induces histamine release, coupled with recent speculations that mast cells and basophils are derived from T-cell precursors, raised expectations that Con-A could be used to elucidate some basic parameters of non-IgE-mediated stimulatory signals in these cells. The usefulness of Con-A as a probe to investigate the existence of non-IgE activating receptors, is dependent upon whether Con-A as an inducing agent is bridging sugar moieties directly attached to IgE. If Con-A activates histamine releasing cells by crosslinking membrane bound IgE, then Con-A would have no particular advantage over specific allergen or anti-IgE in revealing information about the existence or nature
Con-A bridging of sugar moieties at Del of activating receptors other than IgE. For a given concentration of Con-A, the probability that Con-A will interact with a glycoprotein is dependent upon the type and density of available sugar moieties within the glycoprotein molecule. Con A binds sugar moieties with varying ability showing the highest affinity for the D-mannosides (Goldstein et al., 1965). A high content of the proper pyranosyl-type sugar moieties within IgE, particularly mannose, would be supportive of the direct IgE bridging hypothesis. Baenziger, Kornfeld & Kochwa (1974) were able to isolate a high mannose oligosaccharide unit from a pronase digestion of IgE (PS) (Ogawa, Kochwa, Smith, Ishizaka & McIntyre, 1969). Although the pronase digestion did not permit a localization of the glycopeptide fragments in terms of the Porter-Edelman model of immunoglobulins, the report demonstrates that there are regions of the epsilon chain which are high in mannose. The antigenic determinants of IgE (ND) being well defined and localized, were put to advantage in this present report to investigate on a molecular level the region of interaction when Con-A induces histamine release from human basophils. This report has illustrated that monomer anti-Dj1, which binds determinants confined to the C.2 domain of IgE, effectively inhibits Con-A but not intact antiFcIgE induced histamine release. The inability of monomer anti-D,1 to inhibit anti-FcIgE induced release indicates that the monomer's blocking radius is of a dimension less than the tertiary structure of the Fc portion of IgE. A blocking radius of submolecular dimensions, centered at C,2, having the capacity to inhibit Con-A induced release, indicates there is a low probability that Con-A is activating basophils at sites far removed from C82. The fact that monomer anti-D81 can inhibit Con-A induced release does not exclude the possibility that Con-A has the ability to interact in domains neighbouring C.2. Chemical analyses demonstrate that the carbohydrate side chains of IgE are localized within the Cj1, C,2 and C,3 domains of the epsilon chain (Bennich & von Bahr Lindstrom, 1974). All three of these domains contain mannose, and unless the tertiary structure of membrane bound IgE is such that it renders the sugar moieties within the C,1 and C.3 domains unavailable to suboptimum concentrations of Con-A, the data imply the sugar moities in these domains are sufficiently near Dj1 so that the presence of monomer anti-Dj1 will inhibit Con-A binding to them.
57
The epsilon one determinant is not more than one domain removed from any of the carbohydrate content of the IgE molecule. Although the domains of the Fc portion are not arranged in a linear configuration, it would be interesting to determine if monomer antibodies directed against determinants two domains removed from C82 could inhibit Con-A induced release. This question could be answered with antibodies directed against determinants confined to the CA domain. Unfortunately, the cyanogen bromide fragment of IgE, compromising the CA4 region, is unstable and specific antibodies directed against this region are difficult to isolate. It should be mentioned that during the course of these experiments (data not shown) it was determined that monomer anti-D82, which is directed against determinants of the combined domains C83 and CA4, was an effective inhibitor of both Con-A and intact anti-FcIgE induced histamine release. This data indicated that monomer antibodies directed against a combination of the C,3 and CA4 domains, pile up enough monomer to have a blocking radius in excess of the Fc portion of IgE and offered no particular advantage over monomer anti-FcIgE in localizing the site of interaction of Con-A. The data in this report are in consistent with existing evidence concerning the structure and composition of IgE, the properties of Con-A, and the mechanisms of IgE mediated release and inhibition. The report substantiates the premise that Con-A induced histamine release from human basophils is IgE mediated and further shows that the primary site of activation is in the proximity of the epsilon-one determinant of IgE.
ACKNOWLEDGMENTS This work was supported in part by National Institute of Health Grant no. l-MO-1-RR-00749-04, and Svenska Livforsakringsbolagens nimnd f6r medicinsk forskning. REFEREN CES ANDERSSON J., SJOBERG 0. & MOLLER G. (1972) Mitogens as probes for immunocyte activation and cellular cooperation. Transplant. Rev. 11, 131. BAENZIGER J., KORNFELD S. & KOCHWA S. (1974) Structure
of the carbohydrate units of IgE immunoglobulin 1.
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Over-all composition, glycopeptide isolation, and structure of the high mannose oligosaccharide unit. J. biol. Chem. 249, 1889. BAZIN H., QUERINJEAN P., BECKERs A., HEREMANS J.I. & DEssy F. (1974) Transplantable immunoglobulinsecreting tumors in rats IV. Sixty-three IgE-secreting immunocytoma tumors. Immunology, 26, 713. BECKER J.W., REEKE G.N., JR & EDELMAN G.M. (1971) Location of the saccharide binding site of concanavalin A. J. biol. Chem. 246, 6123. BENNICH H. & JOHANSSON S.G.O. (1967) Gamma globulins structure and control of biosynthesis. Nobel Symposium 3 (ed. by J. Killander) p. 199. Almqvist & Wiksell, Stockholm. BENNICH H. & JOHANSSON S.G.O. (1971) Structure and function of human immunoglobulin E. Advance. Immunol. 13, 1. BENNICH H. & VON BAHR-LINDSTROM (1974) Structure of immunoglobulin E (IgE). Progress in Immunology II I (ed. by L. Brent & J. Holborow) p. 49 North-Holland Publishing Company, Amsterdam. BURNET F.M. (1975) Possible identification of mast cells as special post-mitotic cells. Med. Hypoth. 1, 3. DORRINGTON K.J. & BENNICH H. (1973) Thermally induced structural changes in immunoglobulin E. J. biol. Chem. 248, 8378. GOLDSTEIN I.J., HOLLERMAN G.E. & SMITH E.E. (1965) Protein-carbohydrate interaction. II. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry, 4, 876. GOTH A., ADAMS H.R. & KNOOHUIZEN M. (1971) Phosphatidyl serine: selective enhancer of histamine release. Science, 173, 1034. HOOK W.A., DOUGHERTY S.F. & OPPENHEIM J.J. (1974) Histamine release from hamster mast cells and human basophils by non-specific mitogens. Fed. Proc. 33, 1000 (abstract). ISHIZAKA K. & ISHIZAKA T. (1967) Identification of yE antibodies as a carrier of reaginic activity. J. Immunol. 99, 1187. ISHIZAKA K. & ISHIZAKA T. (1969) Immune mechanisms of reversed type reaginic hypersensitivity. J. Immunol. 103, 588. ISHIZAKA K., ISHIZAKA T. & HORNBROOK A.A. (1967) Allergen-binding activity of yE, yG and yA antibodies in sera from atopic patients. J. Immunol. 98, 490. ISHIZAKA T., ISHIZAKA K., JOHANSSON S.G.O. & BENNICH H. (1969) Histamine release from human leucocytes by anti-yE antibodies. J. Immunol. 102, 884. ISHIZAKA T., OKUDAIRA H., MAUSER E. & ISHIZAKA K. (1976) Development of rat mast cells in vitro I. Differentiation of mast cells from thymus cell. J. Immunol. 116, 747.
JANOSSY G. & GREAVES M.L. (1971) Lymphocyte activation. I. Response of T and B Lymphocytes to phytomitogens. Clin. exp. Immunol. 9, 483. JOHANSSON S.G.O. & BENNICH H. (1967) Immunological studies of an atypical (myeloma) immunoglobulin. Immunology, 13, 381. KELLER R. (1973) Concanavalin A, as model 'antigen' for the in vitro detection of cell-bound reaginic antibody in the rat. Clin. exp. Immunol. 13, 139. LICHTENSTEIN L.M. & OSLER A.G. (1964) Studies on the mechanisms of hypersensitivity phenomena IX. Histamine release from human leucocytes by ragweed pollen antigen. J. exp. Med. 120, 507. LAWSON P., FEWTRELL C., GOMPERTS B. & RAFF M.C. (1975) Anti-immunoglobulin-induced histamine secretion by rat peritoneal mast cells studied by immunoferritin electron microscopy. J. exp. Med. 142, 391. MAGRO A.M. (1974a) Evidence for IgE involvement in Con A induced histamine release from human basophils in vitro. Nature (Lond.), 249, 512. MAGRO A.M. (1974b) In vitro studies of concanavalin-Ainduced histamine release from human basophils: Excess bridging in the inhibitory region of the doseresponse curve. Int. Arch. Allergy, 47, 433. MAGRO A.M. (1975) Evidence for one hit activation for the in vitro release of histamine from human basophils. Immunochemistry, 12, 389. MAGRO A.M. & ALEXANDER A. (1974) Histamine release: In vitro studies of the inhibitory region of the doseresponse curve. J. Immunol. 112, 1762. MCUBBIN W.D. & KAY C.M. (1971) Molecular weight studies on concanavalin A. Biochem. Biophys. Res. Commun. 44, 101. OGAWA M., KOCHWA S., SMITH C., ISHIZAKA K. & MCINTYRE O.R. (1969) Clinical aspects of IgE myeloma. N. Engl. J. Med. 281, 1217. OLSON M.O.J. & LIENER I.E. (1971) Some physical and chemical properties of concanavalin A, the phytohemagglutinin of the jack bean. Biochemistry, 6, 105. PORTER R.R. (1959) The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem. J. 73, 118. SHORE P.A., BURKHALTER A. & CHON V.H. (1959) A method for the fluorometric assay of histamine in tissue. J. Pharmacol. Exp. Ther. 127, 182. SIRAGANIAN R.P. (1974) An automated continuous-flow system for the extraction and fluorometric analysis of histamine. Analyt. Biochem. 57, 383. SULLIVAN T.J., GREENE W.C. & PARKER C.W. (1975) Concanavalin A-induced histamine release from normal rat mast cells. J. Immunol. 115, 278. SUMNER J.B. (1919) The globulins of the jack bean, canavalia ensiformis. J. biol. Chem. 37, 137.
Concanavalin A induced histamine release from human basophils in vitro
A. M. MAGRO & H. BENNICH New York State Kidney Disease Institute, and Department ofMicrobiology aid Immunology, Albany Medical College Albany, New York and Department of Molecular Biology, University of Aarhus, Aarhus, Denmark
Received 11 October 1976; acceptedfor publication 9 December 1976
Querinjean, Beckers, Heremans & Dessy, 1974) reagin has been characterized as an antibody of the IgE class. Concanavalin A a globulin isolated from the jack bean (Canavalia ensiformis) (Sumner, 1919) is a mitogenic lectin which has the capacity to induce histamine release from mast cells (Keller, 1973) and basophils (Hook, Dougherty & Oppenheim, 1974). The induction of histamine release by Con-A raised the interesting possibility that Con-A was activating mast cells and basophils by interacting at sites on the membrane independent of the membrane bound IgE. The fact that Con-A is mitogenic to thymus derived (T) lymphocytes (Janossy & Greaves, 1971; Andersson, Sjoberg & Moller, 1972) and the speculation that mast cells and basophils are derivatives of a T-cell precursor (Burnet, 1975; Ishizaka, Okudaira, Mauser & Ishizaka, 1976) could be considered supportive of the hypothesis that Con-A induced histamine release is not IgE mediated. However, Keller (1973) concluded that in rat mast cells Con-A induced histamine release is IgE mediated. His data show that mast cells from rats immunized to produce high titres of reaginic antibody released to Con-A whereas mast cells from non-immunized rats did not. The more recent report (Sullivan, Greene & Parker, 1975) showed that in the presence of the potentiator of histamine release phosphatidyl serine (Goth, Adams & Knoohuizen, 1971) Con-A does
Summary. The site of interaction for concanavalin A (Con-A)-induced histamine release from human basophils was studied in vitro. Blocking the epsilonone determinant (D-. 1) of IgE with high concentrations of monomer (Fab) anti-Dj1 does not significantly inhibit the quantity of histamine released by suboptimum concentrations of Fc specific anti-IgE. This indicates that the monomer anti-Dj1 does not have the capacity to sterically hinder the bridging of all of the determinants in the C.3 and CA domains (Fc'-e'region) of IgE. The monomer anti-Dj1 does effectively inhibit release induced by suboptimum concentrations of Con-A. The data indicate that for suboptimum concentrations, Con-A activation is IgE mediated and takes place in the proximity of Dj1 and not at the membrane receptor for IgE.
INTRODUCTION
Antigen-induced histamine release from basophils and mast cells is mediated by reaginic antibody. In humans (Ishizaka, Ishizaka & Hornbrook, 1967; Ishizaka & Ishizaka, 1967) and rats (Bazin, Correspondence: Dr A.M. Magro, New York State Kidney Disease Institute, 120 New Scotland Avenue, Albany, N.Y. 12208, U.S.A.
51
52
A. M. Magro & H. Bennich
have the capacity to induce histamine release from mast cells of non-immunized rats. This report has promoted speculation that Con-A activation in rat mast cells is not necessarily IgE mediated. In human basophils, the evidence that Con-A induced histamine release is IgE mediated is also equivocal. The reports (Magro, 1974a; 1974b), which show that the blocking of the Fc portion of IgE with monomer anti-FcIgE has the ability to inhibit Con-A-induced histamine release from human basophils, indicate that Con-A is activating in the proximity of the Fc portion of IgE. However, it is currently believed that IgE is bound by its Fc portion to a receptor in the membrane of the histamine releasing cell. The monomer anti-FcIgE blocking data cannot resolve the question of whether Con-A is interacting directly with the Fc portion of IgE or whether it is interacting with the membrane receptor for IgE. The possibility of Con-A interacting at occupied membrane receptors for IgE cannot be excluded for inhibition of Con-Ainduced release by the monomer anti-FcIgE could be due to a steric hindrance phenomenon, where the monomer is preventing the Con-A from interacting with the IgE receptor. The fact that human IgE is well characterized (Bennich & Johansson, 1971; Bennich & Von Bahr-Lindstrom, 1974) can be put to advantage in order to resolve whether Con-A is interacting with the receptor for IgE, or the IgE directly when it induces histamine release from human basophils. The epsilon chains which form the Fc portion of IgE consist of three domains: C.2, C.3 and CA4. The C,2 domain is closest to the Fab portion of the IgE molecule and the CA4 domain incorporates the carboxyl terminal region of the molecule. It is currently believed that the membrane receptor for IgE does not bind in the vicinity of the C.2 domain. It appears that the cytotropic activity of the Fc portion of IgE is a property of the CA4 domain or the C,3 and CA4 domains in cooperation (Dorrington & Bennich, 1973). As antigen the Fc portion of IgE has a major antigenic determinant, historically defined as DJ1 (Bennich & Johansson, 1971) within the C,2 domain of the molecule. This study was initiated to determine whether monomer antibodies specific for DJ1 can inhibit Con-A induced histamine release. Inhibition by monomer anti-Del would indicate that con A activation takes place in the proximity of DJ1 which is the Fc determinant farthest removed from the IgE receptor.
MATERIALS AND METHODS
Buffers and cell preparations Human leucocytes were obtained by venipuncture from blood donors considered atopic in that they have a history of allergies and show an in vitro response to several allergens. The leucocytes were aspirated from the leucocyte-rich supernatant obtained from a sedimentation of the whole blood in the presence of dextran and EDTA (Lichtenstein & Osler, 1964). The supernatant was centrifuged to remove platelets and plasma and the isolated leucocytes were washed twice and then resuspended in a tris-buffered (pH 74) solution, which was 120 mm Na+, 2-2 mm K+, 1 mM2+, 0 7 mm Ca2+ and 0 03 Y. human serum albumin. Reagents
F(ab')2-e fragments were produced by peptic digestion of the IgE (ND) (Johansson and Bennich, 1967) for 8 h in 0-1 m sodium acetate buffer pH 4 5 at 37°. The F(ab')2-e was purified by application to a Sephadex G-150 column followed by chromatography on DEAE-Sephadex A-50 (Bennich & Johansson, 1967). Anti-De1 was prepared from specific rabbit anti-IgE by immunosorption to F(ab')2-e. The eluted anti-Del showed no detectable activity against Fc'. When the anti-D81 was tested against F(ab')2-e and Fc", a reaction of identity was obtained indicating that anti-Del obtained in this manner only reacts with determinants in the C22 region. Monomer anti-Del and monomer anti-FcIgE were obtained by papain digestion (Porter, 1959). The digests were applied to a Sephadex G-100 column in order to separate the monomers from intact antibodies. Crystalized Con-A was provided by Pharmacia (Piscataway, New Jersey). Estimates of molar concentration were based on a mol. wt of 150,000 for intact anti-Dj1 and intact anti-FcIgE, 71,000 for Con-A (Olson & Liener, 1971), and 50,000 daltons for monomer anti-Del and monomer anti-FcIgE. Reaction mixtures and histamine release Reagents and inducing agents were added followed by the cells all at 40. The additions were made into 12 x 75 mm Falcon plastic tubes (2052) to a volume of 0 3 ml. The reaction mixtures were then placed in a 37° bath and incubated for 60 min. Following the incubation the volume was adjusted to 0 5 ml by the addition of cold tris EDTA. Subsequently,
Con-A bridging of sugar moieties at D.5 the tubes were centrifuged and the supernatants decanted for assay. Total available histamine was determined by analysis of supernatants from tubes in which the cells were lysed by incubating in 0 3 N perchloric acid. Typically, there were 1-2 x 106 leucocytes per tube yielding a total quantity of histamine equivalent to 50-100 ng histamine base per ml. Blank tubes containing buffer or monomers in the absence of inducing agent were less than 5 % of the total histamine content. Histamine assay The histamine was assayed by an automated spectrofluorometric technique (Siraganian, 1974). The extraction and fluorometric procedures are a modified version of the techniques as reported by Shore, Burkhalter & Cohn, (1959). The percent histamine released was calculated in excess of the blank as follows: per
cent release
=
100 x
sample -blank
complete - blank
In experiments in which duplicates were done they were averaged. For histamine release in excess of 10%/ the mean deviation divided by the mean was less than 0 08, which implies the data points have a precision within ± 8% about the mean. Completes were usually performed in quintuplet and they showed a precision within ± 5% about the mean.
RESULTS The effect of increasing concentrations of monomer upon the histamine releasing capacity of intact anti-Del The induction of histamine release which is IgE mediated requires the crosslinking or bridging of neighbouring IgE molecules by the inducing agent. Thus intact antibodies directed against IgE evoke a histamine releasing response (Ishizaka, Ishizaka, Johansson & Bennich, 1969) whereas the monomers of these antibodies fail to elicit a response and inhibit histamine release by the intact antibodies (Ishizaka & Ishizaka, 1969). The in vitro dose response follows a profile where the quantity of histamine released ascends through a maximum and then descends with increasing concentrations of inducing agent. From the profile the concentrations
anti-D.1
53
of inducing agent can be divided into three regions: suboptimum, optimum and supraoptimum. The quantity of bridging of the membrane-bound IgE increases with increasing concentrations of inducing agent until along the descending portion of the curve the cells are rendered unresponsive by excess bridging (Magro & Alexander, 1974). Monomer antibodies, produced by papain digestion, have the capacity to bind determinants but not bridge. Through a process of site competition they will decrease the quantity of bridging of the intact antibodies. To ascertain if the monomer anti-Del has the capacity to bind at the epsilon-one determinant of IgE the effect of increasing concentrations of monomer anti-Dj1 upon the histamine releasing capacity of intact anti-D,1 was determined. The inset ofFig. 1 illustrates thehistaminerelease dose-response for intact anti-Dj1 and the points A and B indicate the suboptimum and supraoptimum concentrations used to obtain the data of curve A and curve B respectively. It can be seen from curve A that increasing concentrations of monomer anti-Dj1 inhibit the quantity of histamine released by the suboptimum concentration of intact anti-D.I. As the monomer successfully competes for the D.1 sites the quantity of bridging is lessened until the probability of a bridge is reduced below the activating level. However, for the supraoptimum concentration of anti-Del (curve B), the cells are excessively bridged, and the increasing concentrations of the monomer, although decreasing the quantity of the bridging, brings the cells toward a state of optimal bridging and enhances the histamine release. Fig. 1 demonstrates that the monomer antiD81 has a high enough affinity for the epsilon-one determinant to successfully compete with both suboptimum and supraoptimum concentrations of the intact anti-D81. A comparison of the effect of increasing concentrations of monomer anti-D81 upon the histamine releasing capacity of intact anti-FcIgE and Con-A Fc-specific anti-IgE activates basophils to release histamine by bridging determinants within the C,2, C,3 and CA domains of the IgE molecule. D81 is within the C,2 domain. The membrane receptor for IgE does not bind at C.2, but in the proximity of the CA4 domain or the C83 and CA domains in cooperation. Anti-FcIgE, by having the capacity to bridge determinants within the C,3 and CA4 domains
54
A. M. Magro & H. Bennich A
50p -
-B 0
30p
r I)
10
oo'' 9
8
7
6
-Log1o[monomer (Fab) anti-Dc 1] (mol/)
Figure 1. The effect of increasing concentrations (abscissa) of monomer anti-D81 upon histamine release induced by: curve A: a fixed suboptimum concentration (10-10 M) of intact anti-Del; curve B: a fixed supraoptimum concentration (10-8 M) of intact anti-Dj1. Inset: dose-response for intact anti-D.1 induced release.
of IgE interacts within the vicinity of the membrane receptor for IgE. If monomer anti-D81 has the ability to inhibit the histamine-releasing capacity of
anti-FcIgE, it would indicate that the blocking radius of the monomer is such that it inhibits interactions within the C.3 and CA domains and that there is a high plausibility of the monomer sterically hindering interactions in the proximity of the membrane receptor for IgE. However, curves A and C of Fig. 2 show that increasing concentrations of monomer anti-De1 do not significantly affect the histamine releasing capacity of a near optimum (curve A) or a suboptimum (curve C) concentration of Fc specific anti-IgE. It can be seen that the blocking of the epsilon-one determinant within the C.2 domain does not sufficiently reduce the quantity of bridging within the C.3 and/or CA4 domains to significantly reduce the quantity of histamine released. Since the monomer anti-Dj1 does not inhibit anti-FcIgE induced release, blocking of interactions at the membrane receptors for IgE by the monomer is not likely. The monomer antiDC1 should decrease the quantity of anti-FcIgE bridging within the C.2 domain. The slight decrease in the quantity of histamine released in curves A and C of Fig. 2 could be indicative of this. However, if a small number of bridges suffice to activate a releasing unit of histamine (Magro, 1975; Lawson,
70-
2
501
vr
E IL
301
10 o
8
7
-Loglo[monomer
(Fob)
6
anti-D.1] (maul1)
Figure 2. The effect of increasing concentrations (abscissa) of monomer anti-DI upon histamine release induced by: M) curve A: a fixed near optimum concentration (5 6 x 10oof intact anti-FcIgE; curve B: a fixed near optimum concentration (2-5 x 10-8 M) of Con-A; curve C: a fixed suboptimum concentration (3.1 xlO-10 M) of intact antiFcIgE; curve D: a fixed suboptimum concentration (1-4 x 10`8 M) of Con-A.
Fewtrell, Gomperts & Raff, 1975), it is possible that when bridging within the C.2 domain is totally prevented, the quantity of bridging within parts of the CQ3 and CA4 domains of IgE, by the anti-FcIgE,
even
Con-A bridging of sugar moieties at D1l is of a degree that the probability of activating the histamine releasing units is not significantly reduced. Con A has the capacity to bind polysaccharides and glycoproteins which contain sugar moieties having the C-3, C-4 and C-6 hydroxyl groups of the D-arabino hexopyranosyl ring system (Goldstein, Hollerman & Smith, 1965). At physiological pH Con-A is an equilibrium mixture of identical subunits consisting primarily of tetramers having the capacity to bridge two or more sugar moieties (Mcubbin & Kay, 1971; Becker, Reeke & Edelman, 1971). This ability to bind and bridge specific pyranosyl type sugar moieties is the primary physicochemical property of con-A which renders it active as a histamine releasing agent. In order to resolve whether con-A is bridging sugar moieties at the membrane receptor for IgE or whether it is bridging sugar moieties directly attached to IgE, the effect of blocking D81 upon the histamine releasing capacity of con-A is determined. Curves B and D of Fig. 2 show that increasing concentrations of monomer anti-D81 effectively inhibit histamine release induced by a near optimum (curve B), and a suboptimum (curve D) concentration of Con-A. The data imply that the histamine releasing Con-A interactions are in the proximity of Del. In Fig. 2, the data of curves B and D in conjunction with curves A and C suggest that when Con-A induces histamine release there is a low probability of Con-A bridging sugar moieties at the membrane receptor for IgE.
55
C3, and C4 domains it does not allow the monomer anti-D81 to interact at D.I. The descending portion of the dose-response of the inset of Fig. 1 reveals that when most of the D81 sites are cross-linked by the intact anti-D81 the cells are rendered unresponsive by excess bridging. Anti-FcIgE has specificity to bridge determinants at D.1. At a near optimum concentration, the anti-FcIgE is not excessively bridging at D81 for as the inset of Fig. 1 shows the cells would be rendered unresponsive. The determination of whether the addition of intact anti-D81 in excess concentration renders the cells unresponsive to a near optimum concentration of intact antiFcIgE would ascertain if the anti-Dj1 can successfully compete for sites with the anti-FcIgE. The data in Fig. 3 display the effect of increasing concentrations of intact anti-Dj1 upon the histamine releasing capacity of a near optimum concentration of intact anti-FcIgE. Curve A of Fig. 3 is the dose '70, cx
- 50 F c
0
8 0
30
C w
0 _G
.-IF The effect of increasing concentrations of intact
anti-D.1 upon the histamine releasing capacity of a near optimum concentration of intact anti-FcIgE The ability of monomer anti-D81 to inhibit release induced by the intact anti-D81 (Fig. 1) indicates that the monomer can successfully compete with the intact antibodies for the epsilon-one sites. Curves A and C of Fig. 2 established that high concentrations of the monomer anti-D81 have little effect upon the releasing capacity of intact anti-FcIgE. If it is assumed that the monomer anti-DJ1 blocks most of the sites at DJ1 in the presence of antiFcIgE then the data of Fig. 2 indicate that the monomer does not sterically hinder all interactions within the C.3 and CA domains. However, an alternate interpretation of the data of curves A and C of Fig. 2 is that the affinity of the anti-FcIgE is such that when it bridges determinants within C,2,
co '12
11
10
9
8
7
-Logqopintoct anti-D,l ] (mol/l) Figure 3. Curve A: normal dose response for intact anti-D81 induced release; curve B: the effect of increasing concentrations of intact anti-D8l (abscissa) upon release induced by a fixed near optimum concentration (10-10 M) of intact
anti-FcIgE.
response for the increasing concentrations of antiDC1 alone. Curve B of Fig. 3 shows the quantity of histamine released when the concentrations of anti-Dj1, indicated by the abscissa, were added to the fixed near optimum concentration of antiFcIgE. It is evident from the graph that the cells in the reaction mixtures containing the near optimum concentration of anti-FcIgE are rendered unresponsive when the concentrations of intact antiDj1 are increased past the optimum into the in-
56
A. M. Magro & H. Bennich
hibitory region. The data indicate that a near optimum concentration of anti-FcIgE does not prevent the higher concentrations of anti-D.1 from excessively bridging at DJ1. Since it was demonstrated by the data of Fig. 1 that high concentrations of monomer anti-De1 can successfully compete with intact anti-D.1, suboptimum concentrations of intact anti-FcIgE are not apt to prevent high concentrations of monomer anti-D,1 from binding at the epsilon-one sites. A comparison of the effect of increasing concentrations of monomer anti-FcIgE upon the histamine releasing capacity of Con-A and intact anti-FcIgE The ability of monomer anti-FcIgE to inhibit histamine release induced by con A and intact anti-FcIgE occurs via two different mechanisms. The monomer anti-FcIgE being derived from a papain digestion of the intact anti-FcIgE retains the ability to bind the same determinants as the intact antibody (Porter, 1959). Therefore, the monomer anti-FcIgE inhibits the intact anti-FcIgE by a process of site competition. In contrast, Con-A binding is very specific for certain pyranosyl type sugar moieties. Consequently, the monomer antiFcIgE inhibits the binding and the histamine releasing capacity of Con-A not by site competition, but by steric hindrance. The proximity of the two different binding sites is an important parameter to the efficiency of a steric hindrance phenomenon. A disparity in the ability of monomer anti-FcIgE to inhibit Con-A induced histamine release when compared with its ability to inhibit intact antiFcIgE induced release would indicate that the sugar moieties to which Con-A binds are removed from the antigenic determinants of the Fc portion of IgE. However, the data of Fig. 4 display that the ability of monomer anti-FcIgE to inhibit Con-A induced release is not significantly different from its ability to inhibit release induced by intact antiFcIgE. Although the data of Fig. 4 has related concentrations of Con-A and anti-FcIgE of equal histamine releasing capacity, anti-FcIgE induces histamine release at much lower concentrations than Con-A. This implies that anti-FcIgE binds either with a higher affinity than Con-A and/or it binds at a greater number of sites on the IgE molecule. Therefore, it is not possible to equate absolutely the efficiency of the monomer anti-FcIgE in inhibiting
0
30
\
E
i'\% 0
"8
7
6
-Loglo[monomer (Fab) anti- FcIgE] (mol/ I) Figure 4. The effect of increasing concentrations (abscissa) of monomer anti-FcIgE upon histamine release induced by: curve A: a fixed near optimum concentration (10-1o M) of intact anti-FcIgE; curve B: a fixed near optimum concentration (7-8 x 1o-8 M) of Con-A; curve C: a fixed suboptimum concentration (4-4 x 10-8 M) of Con-A; curve D: a fixed suboptimum concentration (56 x 1O-11 M) of intact antiFcIgE.
Con-A and anti-FcIgE induced histamine release. These considerations aside, the data of Fig. 4 are not contradictory to or incompatible with the hypothesis that Con-A is bridging sugar moieties directly attached to a localized region of the IgE molecule when it induces histamine release. DISCUSSION The use of mitogens as probes has been a productive area of investigation leading to workable models whereby some basic mechanisms of cellular activation have been formulated. The finding that the T-cell mitogen Con-A induces histamine release, coupled with recent speculations that mast cells and basophils are derived from T-cell precursors, raised expectations that Con-A could be used to elucidate some basic parameters of non-IgE-mediated stimulatory signals in these cells. The usefulness of Con-A as a probe to investigate the existence of non-IgE activating receptors, is dependent upon whether Con-A as an inducing agent is bridging sugar moieties directly attached to IgE. If Con-A activates histamine releasing cells by crosslinking membrane bound IgE, then Con-A would have no particular advantage over specific allergen or anti-IgE in revealing information about the existence or nature
Con-A bridging of sugar moieties at Del of activating receptors other than IgE. For a given concentration of Con-A, the probability that Con-A will interact with a glycoprotein is dependent upon the type and density of available sugar moieties within the glycoprotein molecule. Con A binds sugar moieties with varying ability showing the highest affinity for the D-mannosides (Goldstein et al., 1965). A high content of the proper pyranosyl-type sugar moieties within IgE, particularly mannose, would be supportive of the direct IgE bridging hypothesis. Baenziger, Kornfeld & Kochwa (1974) were able to isolate a high mannose oligosaccharide unit from a pronase digestion of IgE (PS) (Ogawa, Kochwa, Smith, Ishizaka & McIntyre, 1969). Although the pronase digestion did not permit a localization of the glycopeptide fragments in terms of the Porter-Edelman model of immunoglobulins, the report demonstrates that there are regions of the epsilon chain which are high in mannose. The antigenic determinants of IgE (ND) being well defined and localized, were put to advantage in this present report to investigate on a molecular level the region of interaction when Con-A induces histamine release from human basophils. This report has illustrated that monomer anti-Dj1, which binds determinants confined to the C.2 domain of IgE, effectively inhibits Con-A but not intact antiFcIgE induced histamine release. The inability of monomer anti-D,1 to inhibit anti-FcIgE induced release indicates that the monomer's blocking radius is of a dimension less than the tertiary structure of the Fc portion of IgE. A blocking radius of submolecular dimensions, centered at C,2, having the capacity to inhibit Con-A induced release, indicates there is a low probability that Con-A is activating basophils at sites far removed from C82. The fact that monomer anti-D81 can inhibit Con-A induced release does not exclude the possibility that Con-A has the ability to interact in domains neighbouring C.2. Chemical analyses demonstrate that the carbohydrate side chains of IgE are localized within the Cj1, C,2 and C,3 domains of the epsilon chain (Bennich & von Bahr Lindstrom, 1974). All three of these domains contain mannose, and unless the tertiary structure of membrane bound IgE is such that it renders the sugar moieties within the C,1 and C.3 domains unavailable to suboptimum concentrations of Con-A, the data imply the sugar moities in these domains are sufficiently near Dj1 so that the presence of monomer anti-Dj1 will inhibit Con-A binding to them.
57
The epsilon one determinant is not more than one domain removed from any of the carbohydrate content of the IgE molecule. Although the domains of the Fc portion are not arranged in a linear configuration, it would be interesting to determine if monomer antibodies directed against determinants two domains removed from C82 could inhibit Con-A induced release. This question could be answered with antibodies directed against determinants confined to the CA domain. Unfortunately, the cyanogen bromide fragment of IgE, compromising the CA4 region, is unstable and specific antibodies directed against this region are difficult to isolate. It should be mentioned that during the course of these experiments (data not shown) it was determined that monomer anti-D82, which is directed against determinants of the combined domains C83 and CA4, was an effective inhibitor of both Con-A and intact anti-FcIgE induced histamine release. This data indicated that monomer antibodies directed against a combination of the C,3 and CA4 domains, pile up enough monomer to have a blocking radius in excess of the Fc portion of IgE and offered no particular advantage over monomer anti-FcIgE in localizing the site of interaction of Con-A. The data in this report are in consistent with existing evidence concerning the structure and composition of IgE, the properties of Con-A, and the mechanisms of IgE mediated release and inhibition. The report substantiates the premise that Con-A induced histamine release from human basophils is IgE mediated and further shows that the primary site of activation is in the proximity of the epsilon-one determinant of IgE.
ACKNOWLEDGMENTS This work was supported in part by National Institute of Health Grant no. l-MO-1-RR-00749-04, and Svenska Livforsakringsbolagens nimnd f6r medicinsk forskning. REFEREN CES ANDERSSON J., SJOBERG 0. & MOLLER G. (1972) Mitogens as probes for immunocyte activation and cellular cooperation. Transplant. Rev. 11, 131. BAENZIGER J., KORNFELD S. & KOCHWA S. (1974) Structure
of the carbohydrate units of IgE immunoglobulin 1.
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A. M. Magro & H. Bennich
Over-all composition, glycopeptide isolation, and structure of the high mannose oligosaccharide unit. J. biol. Chem. 249, 1889. BAZIN H., QUERINJEAN P., BECKERs A., HEREMANS J.I. & DEssy F. (1974) Transplantable immunoglobulinsecreting tumors in rats IV. Sixty-three IgE-secreting immunocytoma tumors. Immunology, 26, 713. BECKER J.W., REEKE G.N., JR & EDELMAN G.M. (1971) Location of the saccharide binding site of concanavalin A. J. biol. Chem. 246, 6123. BENNICH H. & JOHANSSON S.G.O. (1967) Gamma globulins structure and control of biosynthesis. Nobel Symposium 3 (ed. by J. Killander) p. 199. Almqvist & Wiksell, Stockholm. BENNICH H. & JOHANSSON S.G.O. (1971) Structure and function of human immunoglobulin E. Advance. Immunol. 13, 1. BENNICH H. & VON BAHR-LINDSTROM (1974) Structure of immunoglobulin E (IgE). Progress in Immunology II I (ed. by L. Brent & J. Holborow) p. 49 North-Holland Publishing Company, Amsterdam. BURNET F.M. (1975) Possible identification of mast cells as special post-mitotic cells. Med. Hypoth. 1, 3. DORRINGTON K.J. & BENNICH H. (1973) Thermally induced structural changes in immunoglobulin E. J. biol. Chem. 248, 8378. GOLDSTEIN I.J., HOLLERMAN G.E. & SMITH E.E. (1965) Protein-carbohydrate interaction. II. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry, 4, 876. GOTH A., ADAMS H.R. & KNOOHUIZEN M. (1971) Phosphatidyl serine: selective enhancer of histamine release. Science, 173, 1034. HOOK W.A., DOUGHERTY S.F. & OPPENHEIM J.J. (1974) Histamine release from hamster mast cells and human basophils by non-specific mitogens. Fed. Proc. 33, 1000 (abstract). ISHIZAKA K. & ISHIZAKA T. (1967) Identification of yE antibodies as a carrier of reaginic activity. J. Immunol. 99, 1187. ISHIZAKA K. & ISHIZAKA T. (1969) Immune mechanisms of reversed type reaginic hypersensitivity. J. Immunol. 103, 588. ISHIZAKA K., ISHIZAKA T. & HORNBROOK A.A. (1967) Allergen-binding activity of yE, yG and yA antibodies in sera from atopic patients. J. Immunol. 98, 490. ISHIZAKA T., ISHIZAKA K., JOHANSSON S.G.O. & BENNICH H. (1969) Histamine release from human leucocytes by anti-yE antibodies. J. Immunol. 102, 884. ISHIZAKA T., OKUDAIRA H., MAUSER E. & ISHIZAKA K. (1976) Development of rat mast cells in vitro I. Differentiation of mast cells from thymus cell. J. Immunol. 116, 747.
JANOSSY G. & GREAVES M.L. (1971) Lymphocyte activation. I. Response of T and B Lymphocytes to phytomitogens. Clin. exp. Immunol. 9, 483. JOHANSSON S.G.O. & BENNICH H. (1967) Immunological studies of an atypical (myeloma) immunoglobulin. Immunology, 13, 381. KELLER R. (1973) Concanavalin A, as model 'antigen' for the in vitro detection of cell-bound reaginic antibody in the rat. Clin. exp. Immunol. 13, 139. LICHTENSTEIN L.M. & OSLER A.G. (1964) Studies on the mechanisms of hypersensitivity phenomena IX. Histamine release from human leucocytes by ragweed pollen antigen. J. exp. Med. 120, 507. LAWSON P., FEWTRELL C., GOMPERTS B. & RAFF M.C. (1975) Anti-immunoglobulin-induced histamine secretion by rat peritoneal mast cells studied by immunoferritin electron microscopy. J. exp. Med. 142, 391. MAGRO A.M. (1974a) Evidence for IgE involvement in Con A induced histamine release from human basophils in vitro. Nature (Lond.), 249, 512. MAGRO A.M. (1974b) In vitro studies of concanavalin-Ainduced histamine release from human basophils: Excess bridging in the inhibitory region of the doseresponse curve. Int. Arch. Allergy, 47, 433. MAGRO A.M. (1975) Evidence for one hit activation for the in vitro release of histamine from human basophils. Immunochemistry, 12, 389. MAGRO A.M. & ALEXANDER A. (1974) Histamine release: In vitro studies of the inhibitory region of the doseresponse curve. J. Immunol. 112, 1762. MCUBBIN W.D. & KAY C.M. (1971) Molecular weight studies on concanavalin A. Biochem. Biophys. Res. Commun. 44, 101. OGAWA M., KOCHWA S., SMITH C., ISHIZAKA K. & MCINTYRE O.R. (1969) Clinical aspects of IgE myeloma. N. Engl. J. Med. 281, 1217. OLSON M.O.J. & LIENER I.E. (1971) Some physical and chemical properties of concanavalin A, the phytohemagglutinin of the jack bean. Biochemistry, 6, 105. PORTER R.R. (1959) The hydrolysis of rabbit y-globulin and antibodies with crystalline papain. Biochem. J. 73, 118. SHORE P.A., BURKHALTER A. & CHON V.H. (1959) A method for the fluorometric assay of histamine in tissue. J. Pharmacol. Exp. Ther. 127, 182. SIRAGANIAN R.P. (1974) An automated continuous-flow system for the extraction and fluorometric analysis of histamine. Analyt. Biochem. 57, 383. SULLIVAN T.J., GREENE W.C. & PARKER C.W. (1975) Concanavalin A-induced histamine release from normal rat mast cells. J. Immunol. 115, 278. SUMNER J.B. (1919) The globulins of the jack bean, canavalia ensiformis. J. biol. Chem. 37, 137.
