Immunology 1976 31 689
Competitive inhibition of passive sensitization of mouse mast cells by IgE A BIOASSAY FOR MOUSE AND RAT IGE*
ANNIE PROUVOST-DANON & ANNIE ABADIE Laboratoire de Midecine Expe'rimentale, College de France, Paris, France
Received 3 November 1975; accepted for publication 6 May 1976
Summary. Possibility of inhibition of an efficient in vitro IgE-sensitization system was studied. The sensitization of mouse peritoneal mast cells with an anti-ovalbumin IgE-rich fraction of serum, as tested by ovalbumin-induced degranulation, was inhibited by previous incubation with antisera of another or of no specificity. Fractionation and other experiments showed that the inhibiting activity correlated with IgE content. IgG1 did not seem to have an effect. Sensitization was also inhibited by rat myeloma IgE, 50 ng giving a 50 per cent inhibition. Plots of the logarithms of rat and mouse IgE concentration vs their inhibitory effect on sensitization gave two parallel linear curves, indicating that mouse and rat IgE compete for the same receptor sites. It was thus possible to use this system as a sensitive bioassay for both mouse and rat IgE levels and, by comparing inhibition by mouse IgE to that by a known rat IgE standard, to obtain not only relative data but absolute mouse IgE levels. This, and also a better discrimination of IgE doses, was the major advantage of this bioassay in relation to the equally sensitive anti-IgE degranulation tests. *
INTRODUCTION Previous results had shown that in vitro sensitization of mouse peritoneal mast cells could be obtained only with purified reaginic antibodies, suggesting that non-specific y-globulin of the reaginic class interferes by competition for a receptor site on the cells with the reaginic antibodies (Prouvost-Danon & Binaghi, 1970a). Mouse reaginic antibodies were further identified as pertaining to the IgE class (Prouvost-Danon, Binaghi, Rochas & Boussac-Aron, 1972). It was also possible to obtain in vitro passive sensitization of mouse mast cells with non-purified reaginic serum, when this contained a high concentration of reaginic (IgE) antibodies (ProuvostDanon, 1973). Cross-sensitization between rat and mouse IgE and mast cells has been demonstrated (Prouvost-Danon, Wyczolkowska, Binaghi & Abadie, 1975): sensitization of mouse mast cells being readily obtained by rat total IgE for anti-IgE challenge, but only irregularly by rat reaginic antibodies for specific antigen challenge; furthermore, the sensitization of the mast cells, which usually takes about 2 h (Prouvost-Danon et al., 1975; Bach, Brashler & Strechschulte, 1973) was much faster when either a purified antibody (Prouvost-Danon 1971) or the total IgE vs anti-IgE system (Prouvost-Danon et al., 1975) was used. All these data, as well as those of other authors (Ishizaka, Ishizaka & Terry, 1967; Stanworth,
Previous account of these data was given at the Workshop
on 'Immune Mechanisms involved in Triggering Mast Cells' at the 2nd European Immunological Congress, Amsterdam,
16-19 September 1975. Correspondence: Dr Annie Prouvost-Danon, Laboratoire de M6decine Experimentale, College de France, 11 place Marcelin Berthelot, 75231 Paris, Cedex 05, France.
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Annie Prouvost-Danon & Annie Abadie
Humphrey, Bennich & Johansson, 1967; Stanworth, 1973; Jarrett, Orr & Riley, 1971) indicate a competition for mast cells receptors between different IgE molecules. The present study was undertaken in order to study the conditions of this competition and its efficacy in preventing sensitization. The results show that the high degree and fast rate of in vitro sensitization of mouse peritoneal mast cells by mouse reaginic IgE antibodies for specific antigen-induced degranulation, can be readily inhibited by very low concentrations of either mouse or rat IgE molecules of other or no known specificity. The system may constitute a sensitive bioassay for measuring mouse and rat IgE.
MATERIALS AND METHODS Animals Swiss mice from Centre d'Elevage et de Selection du Centre National de la Recherche Scientifique, Orleans-la-Source, or Copacabana strain (ProuvostDanon & Binaghi, 1970b) were used. SpragueDawley rats were employed. Immunizations. Antisera
Copacabana mice were immunized by two or more i.p. injections with 1 pg ovalbumin (Serva 5 x crist.) or 1 pg haemocyanin, mixed with 1 mg aluminium hydroxide gel, given 2-4 weeks apart, after the method of Levine & Vaz (1970). This system produced sera with high titres of IgE antibodies. Other mice were immunized by several i.p. injections of 1 mg haemocyanin mixed with Freund's complete adjuvant, in order to obtain high concentration of IgG1 antibodies. The antisera obtained were fractionated by DEAE-Cellulose chromatography as previously reported (Prouvost-Danon et al. 1972) the IgE fraction being obtained by elution at 0 05 M phosphate buffer. Rat myeloma protein IgE (IR162) and goat anti-rat IgE were kindly furnished by Dr Bazin (University of Leuven, Brussels) (Bazin, Querinjean, Beckers, Heremans & Dessy, 1974). The IR162 protein was titrated by Dr Bazin as containing 5.7 mg IgE/ml (personal communication). For simplicity's sake, our tables and figure are presented and data calculated assuming the IR162 protein solution to contain 5 mg IgE/ml.
Anti-mouse IgE antiserum was prepared as previously reported (Prouvost-Danon et al., 1972) by immunization of rats with rat peritoneal mast cells previously sensitized with mouse reaginic preparations and thoroughly washed. Anti-IgE serum was made specific by absorption with a DEAE-cellulose 0-1 M elution fraction. Control antiserum was prepared by parallel immunization of rats with rat mast cells treated in the same way but without addition of mouse reagins. Passive cutaneous anaphylaxis Mouse and rat IgE antibodies were titrated by passive cutaneous anaphylaxis (PCA) in rats (Mota, 1964; Mota & Wong, 1969) using a 24-h latent period for skin sensitization. Mouse IgGI antibodies were titrated by means of a short-latence (2 h) PCA test in mouse (Ovary, 1964; Prouvost-Danon & Binaghi 1970b). Verifications of the titres were obtained by repeat testing after heating at 560 to inactivate IgE antibodies. In vitro sensitization and degranulation of peritoneal mast cells Peritoneal mast cells were collected from normal animals, about 2 months old, washed twice by centrifugation and resuspended in a final volume of 0-2 ml in RPMI 1640 medium (Grand Island Biological Company) buffered at pH 6 9 with 0 005 M Hepes. In vitro passive sensitization of peritoneal mast cells was performed by incubation at 37° for various periods of time with dilutions of a reagin-rich (IgE) DEAE 0 05 M fraction of serum obtained from mice immunized with a repeated dose (1 pg) of ovalbumin and referred to as 'sensitizing fraction' (SF). The cell suspension was then thoroughy washed by at least three centrifugations and finally resuspended in 0-2 ml of the same medium. The specific antigen (1 ug ovalbumin in a volume of 10 p1) was added after temperature re-equilibration for 10 min at 370 and incubation was continued at 370 for a further 6 min. For the experiments concerned with inhibition of sensitization, the peritoneal mast cells were first incubated with a test preparation having no anti-
ovalbumin antibody specificity, then sensitization proceeded as previously described by the addition of the SF. In some experiments there was, besides the washing at the end of the sensitization step, also a washing (at least three centrifugations) between the
Inhibition ofpassive sensitization by IgE addition of the inhibiting preparation and that of the SF. Experiments were also performed for testing sensitization by total IgE; rat mast cells were incubated with dilutions of mouse IgE, then washed and degranulation induced by various dilutions of anti-mouse IgE and conversely mouse mast cells were incubated with dilutions of rat IgE, washed, and degranulation was induced by anti-rat IgE. At the end of the reaction the mast cells were stained with toluidine blue as described (ProuvostDanon, Silva Lima & Queiroz Javierre, 1966) for observation and counting of the degranulated and intact cells. It has been shown in previous papers (Prouvost-Danon, Peixoto & Queiroz Javierre, 1966; Prouvost-Danon & Binaghi, 1970a) that degranulation provides a parallel measure to that of histamine release of the degree of the anaphylactic sensitization of the mouse mast cells. Results were expressed as percent degranulated in relation to total mast cells. About 100 (from 50 to 450) mast cells were counted for each measurement. In various experiments, we have used duplicate-tube tests and performed several countings for each test. Statistical significance of the results was calculated by x2 test (Lison, 1958). The variation between duplicate testings and/or countings was small and never significant. Some representative results are given in the tables. Controls were performed throughout all the experiments: cells with no antigen, or no antibody, but otherwise treated in the same manner, gave minimal degranulation (0-2 per cent); this has been considered as negligible and has not been reported in the tables. RESULTS Sensitization of mouse peritoneal mast cells Assays were performed to study the sensitization of mouse peritoneal mast cells with the anti-ovalbumin sensitizing fraction (SF) as described in the Materials and Methods section. SF contained a high concentration of IgE antibodies (PCA titre = 5000) and a low concentration of IgGl antibodies (PCA = 500). Table 1 shows that SF produces not only a high, but also a fast sensitization of the mouse mast cells. The SF was thus considered satisfactory for use in assays to study the inhibition processes of IgE sensitization.
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Table 1. Kinetics of sensitization of mouse peritoneal mast cells by anti-ovalbumin IgE sensitizing fraction (SF) Time of sensitization (min) SF dilution
1/10 1/100
5
10
15
30
54 36
52 49
54 54
63 49
Total mast cells counted for each test from 54-101. No significant differences between different times of sensitization (P> 0-05). The results are expressed as percentage degranulation, induced by 1 pg ovalbumin.
Inhibition of sensitization A mouse anti-haemocyanin anti-serum obtained by hyperimmunization as described in the Materials and Methods section and refered to as anti-haemocyanin serum (AHS) was used for the inhibition experiments. In contrast to SF it contained a high concentration of IgG1 antibodies (2 h PCA titre = 5000) and a low IgE PCA titre (250). In the following experiment (Table 2) two dilutions of the antihaemocyanin serum were added to the mast cells suspension at time zero. The sensitizing fraction (SF diluted to 1/100) was added either simultaneously, or 30 min later, total time of incubation of the cells before washing being 60 min. The data show that the anti-haemocyanin serum at a 1/20 dilution produces a statistically significant (P < 0 01) inhibition. The inhibition is stronger when the anti-haemocyanin serum is added 30 min before the sensitizing fraction. Table 2. Effect of a mouse anti-haemocyanin serum on the mouse mast cells sensitization induced by the anti-ovalbumin IgE sensitizing fraction (SF)
Time of addition of SF in relation to AHS AHS Control
1/200 1/20
Simultaneous
30 min after
60 67
49 40
29*
6*
AHS = Mouse anti-haemocyanin serum. Sensitization by SF diluted to 1/100. The results are expressed as percentage degranulation, induced by 1 pg ovalbumin. Total mast cells counted for each test from 93-143. Duplicate counting gave no significant differences (P- 0 7). * Significant differences in relation to controls (P< 0-01)
Annie Prouvost-Danon & Annie Abadie
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Nature of the inhibiting agent Further experiments were performed to verify the nature of the inhibiting agent contained in the anti-haemocyanin serum (Table 3). The mast cells were first incubated with the inhibiting agent for 30 min, then SF was added for a further 30 min. The results indicated a strict correlation between the presence of the inhibiting activity and that of IgE activity after DEAE fractionation of the antihaemocyanin serum, or after heating at 560. Furthermore, washing the mast cells by three successive centrifugations, between addition of the inhibiting agent and that of the sensitizing fraction did not affect the ability to inhibit; showing that the inhibiting factor firmly binds to the mouse mast cells. Furthermore, the fractionation and heating experiments show that in the absence of IgE, there was no inhibiting activity, even in the presence of high concentrations of IgG1.
Table 3. Nature of the inhibiting agent
Inhibiting agent Experimental preparation
IgE PCA Dilution titre
Control AHS* Idem heated 560 Control DEAE fractions 0-02 M 0 05 M 0-1
M
Control AHS followed by cell washing
Percentage degranulation 49 4t 50 65
250 0
1/10 1/10
0 250 0
1/10 1/10
64t 0 63 26
250
1/10
0t
1/5
Inhibiting agent incubated for 30 min; then addition of 1/100 SF for 30 min. Control without inhibiting agent. Degranulation induced by 1 jug ovalbumin. Total mast cells counted for each test from 50 to 113. * AHS = anti-haemocyanin serum.
t Significant differences in relation
to controls
Table 4. Action of anti-mouse IgE on rat mast cells sensitized by mouse IgE
Sensitizing agent Percentage Expt no.
Anti-IgE
Nature
IgE PCA Titre
Dilution
degranulation
1
1/100 1/100 1/1000 1/1000
SF SF
5000
1/100
77t
5000
1/100
54t
SF
5000
1/100
5000
1/100
5000
1/50 1/200 1/800
7
4
control*
2
1/100 1/100 1/1000 1/1000 1/1000
SF SF
1/3200 AHS fractions 0 05 M 0-02 M
250 0
1/50 1/200 1/800 1/10
11 11 4 6 5
41t t 32 t § 28t t 10§
21t** 10 14 8
One hour of incubation with sensitizing agent, then washing, followed by addition of antimouse IgE. Total mast cells counted for each test from 61-105 in expt 1, and from 127-213 in expt 2. * Control: rat anti-rat mast cells control serum. t P< 0-01 in relation to controls without sensitizing agent. t § P< 0-01 between themselves. ** Difference not significant in relation to SF 1/800 (P 0 25) but significant in relation to respectively SF 1/50, 1/200 and 1/3200 (P< 0-01).
(P 0-2). * Significant difference (P< 0-01) in relation to controls without IR162. t Significant differences (P< 0 01) between successive dilutions. B
Control IR162 Heated IR162 IR162 followed
2
6 1000 400 200 100
Experimental IgE dose Percentage Percentage (ng) degranulationt inhibition preparation
by cell washing Control IR162
Heated 1R162 3
Control IR162 AHS diluted
-
1000 100 1000 1000 -
200 100 66 50 1000 200 -
100 50 25
1/40 1/80 1/160 1/320
28
0t 61
26
4t
100 79 7
86
44
-
2t§** 13t§ 21t
95 70 52 43 18 0
25t**
36 46 31-3
7-3tt§
7-6 50-5
22t 6-7t§**
30 78 5 47-3 31 0
15-5t §
16 5t §
216$** 31-4
Total mast cells counted from 66-133 in expts 1 and 2; from 321-454 in expt 3. In expt 3, results from duplicate tests with duplicate countings did no differ more than 1 or 2 points from means. These differences were not significant (P- 0 8). * Inhibiting agent (dose in ng added to 0-2 ml) incubated with mast cells for 30 min then addition of 1/100 SF for 30 min. Control without inhibiting agent. t Degranulation induced by 1 pg ovalbumin. P< 0 01 in relation to controls. § ** P< 0 01 between themselves.
Annie Prouvost-Danon & Annie Abadie
694 1/320
equivalent by use of a standard curve with rat myeloma IgE IR162.
Dilutions of mouse AHS 1/80 1/40 1/160 /
c
0
ti :3 c
X,"
2 751-
C" (L) _0
0 c
0
50k
-e _r_
.s
cu Ot
.2c
25k
R
/
Q)
v CL
a
12-5
,
50 100 25 Rat IgE dose (ng)
200
Figure 1. A bioassay for rat and mouse IgE; the experimental conditions and the data are those presented in Table 6 (expts 2 and 3). Rat IR162 myeloma IgE in nanograms per X) from expt. 0-2 ml: (A A) from expt 2; (X 3; Mouse anti-haemocyanin serum (AHS) in dilutions: (0 0) from expt 3. ---
readily inhibits SF-induced mouse mast cells sensitization. The inhibiting capacity is destroyed by previous heating of rat IR162 for 5 h at 560. The inhibition is observed even when washing the mast cells between the inhibition and sensitization steps. The dilutions of rat myeloma IgE IR162 able to inhibit sensitization of mouse peritoneal mast cells seem of the same order as those able to sensitize
them to the action of anti-rat IgE, as shown in previous paper (Prouvost-Danon et al., 1975) and the present one (Tables 5 and 6). A bioassay for measuring mouse and rat IgE In order to assess this system as a possible one for the evaluation of concentrations of rat or mouse total IgE, further experiments were performed to verify the effect of various dilutions of either rat myeloma IgE or mouse anti-haemocyanin serum on the mouse mast-cell sensitization by SF. Table 6 (expts 2 and 3) and Fig. 1 show the results of two representative experiments. A plot of the logarithms of the IR162 protein doses versus their effect on degranulation, gave a linear transformation of the data. Furthermore, the slopes of inhibition by rat myeloma IgE or by mouse IgE containing serum are not significantly different. Fifty per cent inhibition was achieved when a total of 50 ng of rat IgE was added to the reaction mixture (0-2 ml total volume). The amount of total IgE in a test serum might thus be calculated directly in rat IgE
IgE levels in some mouse and rat sera As an example, some sera were tested in this system. Pooled-normal serum from 2-month-old Swiss mice were found to contain 0-6 jug IgE/ml. The anti-haemocyanin hyperimmune serum was calculated as containing 20 pg IgE/ml (Table 6 and Fig. 1). Reaginic serum (PCA :1000) from mice immunized by two low doses (1 pg) of haemocyanin showed an IgE level of 50 ,g/ml. A pooled-normal serum from adult rats was found to contain 0 8 pg/ml and rat anti-haemocyanin serum (PCA:81) 1V5 pg IgE/ml. Sera from mice infected by Trichinella gave increased values which were in good agreement with those obtained by Perrudet-Badoux et al. (1976) by the use of an immuno-enzymatic method adapted from Guesdon & Avrameas (1974). DISCUSSION Our results show that in vitro mouse mast-cell sensitization by a reagnic serum is readily inhibited by a mouse antiserum with a different antibody specificity (Table 2). Experiments performed to identify the inhibiting factor (Table 3) (fractionation, or heating of inhibiting serum to destroy its binding capacity to mast cells) all indicated that the inhibiting capacity corresponded to the IgE content of the preparation. Rat myeloma IgE was also able to inhibit the mouse mast-cells sensitization (Table 6) suggesting that rat and mouse IgE compete for the same receptor site on the mouse mast cells. Some authors, (Stanworth et al., 1967; Stanworth, 1973) had previously demonstrated that in vivo (PK or PCA) human- or monkey-passive sensitization by reagins could be inhibited by competition with myeloma IgE, high doses of competing IgE being necessary in these conditions. Jarrett et al. (1971) similarly showed, in rats, that passive sensitization processes by a reaginic serum could be inhibited by a second reagin directed against another antigen. This paper provides the results of a study of IgE competition for mouse peritoneal mast cells in an in vitro passive sensitization, which permitted more quantitative and sensitive data to be obtained. By plotting the inhibition observed against the logarithm of the dose of rat myeloma IgE a linear-
Inhibition ofpassive sensitization by IgE regression curve was obtained (Fig. 1) which was parallel to that obtained by plotting the logarithm of the mouse IgE dose, thus permitting a comparison of the dose of IgE of one species with that of another. The system therefore provided a sensitive bioassay for estimating total IgE levels in sera either from rat or mouse, by comparing inhibition data with the rat myeloma IgE IR162 standard curve. The addition of 50 ng rat IgE in this system provided a 50 per cent inhibition. The system was therefore sensitive enough to permit the measurement of IgE levels comparable to those contained in normal sera from adult animals. Normal sera from 2-month-old animals from different pools were thus shown to contain from 0 5 to 2 ,g IgE/ml. There was a good agreement with the values reported by other authors (Carson, Metzger & Bazin, 1975; Perrudet-Badoux et al., 1976). It must be kept in mind, however, that the bioassay of IgE, either by a competitive test or by anti-IgE test, is based on the binding capacity of IgE for mast cells. After preservation of the sera at -20°, there is sometimes a loss of the binding capacity of the 'Fc fragment'. The present bioassay only provides an estimation of the IgE which is functionally intact. The anti-IgE system (Tables 4 and 5) afforded an alternative bioassay model for estimating IgE levels. It was as sensitive (Tables 4 and 5) (Prouvost-Danon et al., 1975) as the inhibition test. Furthermore, it permitted the comparison of IgE antibodies of any specificity. However, it was somewhat more difficult to obtain in this system a good discrimination between the effect of successive dilutions of IgE (Tables 4 and 5). Moreover, the inhibition test had the major advantage of permitting the comparison of mouse IgE with a known rat IgE standard, and thus of converting relative values of mouse IgE into absolute values, into equivalents of rat IgE in micrograms. Although this is not necessary for the present purposes, the sensitivity of the inhibition bioassay could be increased by decreasing the content of non-specific IgE in the sensitizing preparation by specific purification of the antibody. For the measurement of anti-ovalbumin IgE antibodies, it would be necessary to use a sensitizing system of another specificity. In contrast, no significant inhibition was obtained by incubation with preparations containing very high concentrations of mouse IgG1 (Table 3). This confirms the previous suggestion (ProuvostDanon & Binaghi, 1970a) that IgE and IgG1
695
molecules would react with different receptor sites on the mouse mast cells. This seems to differ from what happens in other animal species, as reported by various authors (Bach, Bloch & Austen, 1973; Revoltella & Ovary, 1973; Malley, Baecher, Porter & Gerding, 1974; Stanworth & Smith, 1973). Further experiments are in progress with regard to this question. It has been shown by several authors (Binaghi, Liacopoulos, Halpern & Liacopoulos-Briot, 1962; Halpern, Liacopoulos, Liacopoulos-Briot, Binaghi & van Neer, 1959; Mongar & Schild, 1960) that the velocity of sensitization of guinea-pig tissues is directly proportional to the concentration of sensitizing antibodies and inversely proportional to that of the non specific immunoglobulin pertaining to the same class. Similar data were observed in the case of reaginic antibodies: thus PCA latence in rats could be shortened from 24 h to 30 min by using a high concentration (Mota, 1964) or specifically purified (Binaghi, 1968) antibodies. In vitro passive sensitization, or fixation of reaginic IgE antibodies on rat or mouse peritoneal mast cells, was shown optimally to require about 1-2 h (Prouvost-Danon et al., 1975; Bach et al., 1973). However, previous experiments (Prouvost-Danon 1971; Bach & Brashler, 1973; Prouvost-Danon et al., 1975) as well as the present ones (Table 1) show that, when using a reagin-rich preparation, the in vitro passive sensitization of normal mast cells from young adult mice (2 months old) can be obtained in a very short time. We are now looking into the kinetics of sensitization in various experimental conditions, as when the receptor sites on the mast cells have been previously covered with IgE molecules and there is a need to dislodge previously bound IgE by IgE present in suspension medium.
ACKNOWLEDGMENT This work was supported in part by the Institut National de la Sante et de la Recherche Medicale, France (contract no. ATP 1.73.16). REFER ENCES BACH M.K., BLOCH K.J. & AUSTEN K.F. (1971) IgE and IgGa antibody-mediated release of histamine from rat
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peritoneal cells. II. Interaction of IgE and IgGa at the target cell. J. exp. Med. 133, 772. BACH M.K. & BRASHLER J.R. (1973) On the nature of the presumed receptor for IgE on mast cells. II. Demonstration of the specific binding of IgE to cell-free particulate preparations from rat peritoneal mast cells. J. Immunol. 111, 324. BACH M.K., BRASHLER J.R. & STRECHSCHULTE D.J. (1973) Isolation of hapten-specific antibodies from reagin rat sera and their use for the preparation of rat peritoneal cells for antigen-mediated histamine release. Immunochemistry, 10, 305. BAZIN H., QUERINJEAN P., BECKERS A., HEREMANS J.F. & DESSY F. (1974) Transplantable immunoglobulinsecreting tumours in rats. IV. Sixty three IgE secreting immunocytoma tumours. Immunology, 26, 713. BINAGHI R., LIACOPOULOS P., HALPERN B.N. & LIACOPOULOSBROIT M. (1962) Interference of non-specific gammaglobulins with passive in vitro anaphylactic sensitization of isolated guinea pig intestine. Immunology, 5, 204. BINAGHI R. (1968) Biochemistry ofthe Acute Allergic Reactions, (ed. by K.F. Austen and E.L. Becker), p. 295. Blackwell Scientific Publications, Oxford. CARSON D., METZGER H. & BAZIN H. (1975) A simple radio-immunoassay for the measurement of human and rat IgE levels by ammonium sulfate precipitation. J. Immunol. 115, 561. GUESDON J.L. & AVRAMEAS S. (1974) An immunoenzymatic method for measuring low concentrations of antigens by simple radial diffusion. Immunochemistry, 11, 595. HALPERN B., LIACOPOULOS P., LIAcoPouLos-BRIoT M., BINAGHI R. & VAN NEER F. (1959) Patterns of in vitro sensitization of isolated smooth muscle tissues with antibody. Immunology, 2, 351. ISHIZAKA K., ISHIZAKA T. & TERRY W.D. (1967) Antigenic structure of gamma E globulin and reaginic antibody. J. Immunol. 99, 849. JARRETT E.E.E., ORR T.S.C. & RILEY P. (1971) Inhibition of allergic reactions due to competition for mast cells sensitization sites by two reagins. Clin. exp. Immunol. 9, 585. LEVINE B.B. & VAZ N.M. (1970) Effects of combinations of inbred strain, antigen and antigen dose on immune responsiveness and reagin production in the mouse. Int. Arch. Allergy, 39, 156. LISON L. (1958) Statistique Applique ai la Biologie Exp~rimentale p. 291. Gauthiers-Villars, Paris. MALLEY A., BAECHER L., PORTER G. & GERDING R. (1974) Competitive inhibition of reagin-mediated histamine release by a human IgG2 myeloma protein. Int. Arch. Allergy, 47, 194. MONGAR J.S. & SCHILD H.O. (1960) A study of the mechanism of passive sensitization. J. Physiol. 150, 546.
MOTA I. (1964) The mechanisms of anaphylaxis. I. Production and biological properties of mast cell sensitizing antibody. Immunology, 7, 681. MOTA I. & WONG D. (1969) Homologous and heterologous passive cutaneous anaphylactic activity of mouse antisera during the course of immunization. Life Sci. 8, 813. OVARY Z. (1964) Passive cutaneous anaphylaxis. Ackroyd: CIOMS Symposium on Immunological Methods, pp. 259-283. Blackwell Scientific Publications, Oxford. PERRUDET-BADoux A., BINAGHI R. & BoussAc-ARON Y. (1976) Production of different classes of immunoglobulins in rats infested with Trichinella spinalis. Immunochemistry, 13, 443. PROUVOST-DANON A. (1971) Purification and biological properties of mouse reaginic antibody. Ciencia e Cultura, 23, 433. PRouvosT-DANoN A. (1973) Characterization of mouse IgE and mast cell reactions. Mechanisms in allergy: reaginmediated hypersentivity, (ed. by L. Goodfriend, A.L. Sehon and R.P. Orange), p. 271-285. Dekker, New York. PRouvosT-DANoN A. & BINAGHI R. (1970a) In vitro sensitization of mouse peritoneal mast cells with reaginic antibody. Nature (Lond.), 228, 66. PRouvosT-DANON A. & BINAGHI R. (1970b) Reaginic antibody in adult and young mice: production and biological properties. Int. Arch. Allergy, 38, 648. PRouvosT-DANON A., BINAGHI R., ROCHAS S. & BOUSSACARON Y. (1972) Immunochemical identification of mouse IgE. Immunology, 23, 481. PROUVOST-DANON A., PEIXOTO J.M. & QUEIROZ-JAVIERRE M. (1966) Release of 5-hydroxytryptamine from mouse peritoneal mast cells during active and passive anaphylactic reactions in vitro. Life Sci. 5, 1867. PRouvosT-DANON A., SILVA LIMA M. & QUEIROZ-JAvIERRE M. (1966) Active anaphylactic reaction in mouse peritoneal mast cells in vitro. Life Sci. 5, 289. PRouvosT-DANoN A., WYCZOLKOWSKA J., BINAGHI R. & ABADIE A. (1975) Mouse and rat IgE. Cross sensitization of mast cells and antigenic relationships. Immunology, 29, 151. REVOLTELLA R. & OVARY Z. (1973) Competitive effect of homologous IgG or Fc in sensitization of rabbits for PCA mediated by reaginic antibody. J. Immunol. 111, 698. STANWORTH D.R. (1973) Immediate hypersensitivity: the Molecular Basis of the Allergic Response. (Frontiers of Biology), volume 28, p. 334. STANWORTH D.R., HUMPHREY J.H., BENNICH H. & JOHANSSON S.G.O. (1967) Specific inhibition of the PrausnitzKustner reaction by an atypical human myeloma protein. Lancet, ii, 330. STANWORTH D.R. & SMITH A.K. (1973) Inhibition of reagin mediated PCA reactions in baboons by the human IgG4 subclass. Clin. Allergy, 3, 37.
Competitive inhibition of passive sensitization of mouse mast cells by IgE A BIOASSAY FOR MOUSE AND RAT IGE*
ANNIE PROUVOST-DANON & ANNIE ABADIE Laboratoire de Midecine Expe'rimentale, College de France, Paris, France
Received 3 November 1975; accepted for publication 6 May 1976
Summary. Possibility of inhibition of an efficient in vitro IgE-sensitization system was studied. The sensitization of mouse peritoneal mast cells with an anti-ovalbumin IgE-rich fraction of serum, as tested by ovalbumin-induced degranulation, was inhibited by previous incubation with antisera of another or of no specificity. Fractionation and other experiments showed that the inhibiting activity correlated with IgE content. IgG1 did not seem to have an effect. Sensitization was also inhibited by rat myeloma IgE, 50 ng giving a 50 per cent inhibition. Plots of the logarithms of rat and mouse IgE concentration vs their inhibitory effect on sensitization gave two parallel linear curves, indicating that mouse and rat IgE compete for the same receptor sites. It was thus possible to use this system as a sensitive bioassay for both mouse and rat IgE levels and, by comparing inhibition by mouse IgE to that by a known rat IgE standard, to obtain not only relative data but absolute mouse IgE levels. This, and also a better discrimination of IgE doses, was the major advantage of this bioassay in relation to the equally sensitive anti-IgE degranulation tests. *
INTRODUCTION Previous results had shown that in vitro sensitization of mouse peritoneal mast cells could be obtained only with purified reaginic antibodies, suggesting that non-specific y-globulin of the reaginic class interferes by competition for a receptor site on the cells with the reaginic antibodies (Prouvost-Danon & Binaghi, 1970a). Mouse reaginic antibodies were further identified as pertaining to the IgE class (Prouvost-Danon, Binaghi, Rochas & Boussac-Aron, 1972). It was also possible to obtain in vitro passive sensitization of mouse mast cells with non-purified reaginic serum, when this contained a high concentration of reaginic (IgE) antibodies (ProuvostDanon, 1973). Cross-sensitization between rat and mouse IgE and mast cells has been demonstrated (Prouvost-Danon, Wyczolkowska, Binaghi & Abadie, 1975): sensitization of mouse mast cells being readily obtained by rat total IgE for anti-IgE challenge, but only irregularly by rat reaginic antibodies for specific antigen challenge; furthermore, the sensitization of the mast cells, which usually takes about 2 h (Prouvost-Danon et al., 1975; Bach, Brashler & Strechschulte, 1973) was much faster when either a purified antibody (Prouvost-Danon 1971) or the total IgE vs anti-IgE system (Prouvost-Danon et al., 1975) was used. All these data, as well as those of other authors (Ishizaka, Ishizaka & Terry, 1967; Stanworth,
Previous account of these data was given at the Workshop
on 'Immune Mechanisms involved in Triggering Mast Cells' at the 2nd European Immunological Congress, Amsterdam,
16-19 September 1975. Correspondence: Dr Annie Prouvost-Danon, Laboratoire de M6decine Experimentale, College de France, 11 place Marcelin Berthelot, 75231 Paris, Cedex 05, France.
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Humphrey, Bennich & Johansson, 1967; Stanworth, 1973; Jarrett, Orr & Riley, 1971) indicate a competition for mast cells receptors between different IgE molecules. The present study was undertaken in order to study the conditions of this competition and its efficacy in preventing sensitization. The results show that the high degree and fast rate of in vitro sensitization of mouse peritoneal mast cells by mouse reaginic IgE antibodies for specific antigen-induced degranulation, can be readily inhibited by very low concentrations of either mouse or rat IgE molecules of other or no known specificity. The system may constitute a sensitive bioassay for measuring mouse and rat IgE.
MATERIALS AND METHODS Animals Swiss mice from Centre d'Elevage et de Selection du Centre National de la Recherche Scientifique, Orleans-la-Source, or Copacabana strain (ProuvostDanon & Binaghi, 1970b) were used. SpragueDawley rats were employed. Immunizations. Antisera
Copacabana mice were immunized by two or more i.p. injections with 1 pg ovalbumin (Serva 5 x crist.) or 1 pg haemocyanin, mixed with 1 mg aluminium hydroxide gel, given 2-4 weeks apart, after the method of Levine & Vaz (1970). This system produced sera with high titres of IgE antibodies. Other mice were immunized by several i.p. injections of 1 mg haemocyanin mixed with Freund's complete adjuvant, in order to obtain high concentration of IgG1 antibodies. The antisera obtained were fractionated by DEAE-Cellulose chromatography as previously reported (Prouvost-Danon et al. 1972) the IgE fraction being obtained by elution at 0 05 M phosphate buffer. Rat myeloma protein IgE (IR162) and goat anti-rat IgE were kindly furnished by Dr Bazin (University of Leuven, Brussels) (Bazin, Querinjean, Beckers, Heremans & Dessy, 1974). The IR162 protein was titrated by Dr Bazin as containing 5.7 mg IgE/ml (personal communication). For simplicity's sake, our tables and figure are presented and data calculated assuming the IR162 protein solution to contain 5 mg IgE/ml.
Anti-mouse IgE antiserum was prepared as previously reported (Prouvost-Danon et al., 1972) by immunization of rats with rat peritoneal mast cells previously sensitized with mouse reaginic preparations and thoroughly washed. Anti-IgE serum was made specific by absorption with a DEAE-cellulose 0-1 M elution fraction. Control antiserum was prepared by parallel immunization of rats with rat mast cells treated in the same way but without addition of mouse reagins. Passive cutaneous anaphylaxis Mouse and rat IgE antibodies were titrated by passive cutaneous anaphylaxis (PCA) in rats (Mota, 1964; Mota & Wong, 1969) using a 24-h latent period for skin sensitization. Mouse IgGI antibodies were titrated by means of a short-latence (2 h) PCA test in mouse (Ovary, 1964; Prouvost-Danon & Binaghi 1970b). Verifications of the titres were obtained by repeat testing after heating at 560 to inactivate IgE antibodies. In vitro sensitization and degranulation of peritoneal mast cells Peritoneal mast cells were collected from normal animals, about 2 months old, washed twice by centrifugation and resuspended in a final volume of 0-2 ml in RPMI 1640 medium (Grand Island Biological Company) buffered at pH 6 9 with 0 005 M Hepes. In vitro passive sensitization of peritoneal mast cells was performed by incubation at 37° for various periods of time with dilutions of a reagin-rich (IgE) DEAE 0 05 M fraction of serum obtained from mice immunized with a repeated dose (1 pg) of ovalbumin and referred to as 'sensitizing fraction' (SF). The cell suspension was then thoroughy washed by at least three centrifugations and finally resuspended in 0-2 ml of the same medium. The specific antigen (1 ug ovalbumin in a volume of 10 p1) was added after temperature re-equilibration for 10 min at 370 and incubation was continued at 370 for a further 6 min. For the experiments concerned with inhibition of sensitization, the peritoneal mast cells were first incubated with a test preparation having no anti-
ovalbumin antibody specificity, then sensitization proceeded as previously described by the addition of the SF. In some experiments there was, besides the washing at the end of the sensitization step, also a washing (at least three centrifugations) between the
Inhibition ofpassive sensitization by IgE addition of the inhibiting preparation and that of the SF. Experiments were also performed for testing sensitization by total IgE; rat mast cells were incubated with dilutions of mouse IgE, then washed and degranulation induced by various dilutions of anti-mouse IgE and conversely mouse mast cells were incubated with dilutions of rat IgE, washed, and degranulation was induced by anti-rat IgE. At the end of the reaction the mast cells were stained with toluidine blue as described (ProuvostDanon, Silva Lima & Queiroz Javierre, 1966) for observation and counting of the degranulated and intact cells. It has been shown in previous papers (Prouvost-Danon, Peixoto & Queiroz Javierre, 1966; Prouvost-Danon & Binaghi, 1970a) that degranulation provides a parallel measure to that of histamine release of the degree of the anaphylactic sensitization of the mouse mast cells. Results were expressed as percent degranulated in relation to total mast cells. About 100 (from 50 to 450) mast cells were counted for each measurement. In various experiments, we have used duplicate-tube tests and performed several countings for each test. Statistical significance of the results was calculated by x2 test (Lison, 1958). The variation between duplicate testings and/or countings was small and never significant. Some representative results are given in the tables. Controls were performed throughout all the experiments: cells with no antigen, or no antibody, but otherwise treated in the same manner, gave minimal degranulation (0-2 per cent); this has been considered as negligible and has not been reported in the tables. RESULTS Sensitization of mouse peritoneal mast cells Assays were performed to study the sensitization of mouse peritoneal mast cells with the anti-ovalbumin sensitizing fraction (SF) as described in the Materials and Methods section. SF contained a high concentration of IgE antibodies (PCA titre = 5000) and a low concentration of IgGl antibodies (PCA = 500). Table 1 shows that SF produces not only a high, but also a fast sensitization of the mouse mast cells. The SF was thus considered satisfactory for use in assays to study the inhibition processes of IgE sensitization.
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Table 1. Kinetics of sensitization of mouse peritoneal mast cells by anti-ovalbumin IgE sensitizing fraction (SF) Time of sensitization (min) SF dilution
1/10 1/100
5
10
15
30
54 36
52 49
54 54
63 49
Total mast cells counted for each test from 54-101. No significant differences between different times of sensitization (P> 0-05). The results are expressed as percentage degranulation, induced by 1 pg ovalbumin.
Inhibition of sensitization A mouse anti-haemocyanin anti-serum obtained by hyperimmunization as described in the Materials and Methods section and refered to as anti-haemocyanin serum (AHS) was used for the inhibition experiments. In contrast to SF it contained a high concentration of IgG1 antibodies (2 h PCA titre = 5000) and a low IgE PCA titre (250). In the following experiment (Table 2) two dilutions of the antihaemocyanin serum were added to the mast cells suspension at time zero. The sensitizing fraction (SF diluted to 1/100) was added either simultaneously, or 30 min later, total time of incubation of the cells before washing being 60 min. The data show that the anti-haemocyanin serum at a 1/20 dilution produces a statistically significant (P < 0 01) inhibition. The inhibition is stronger when the anti-haemocyanin serum is added 30 min before the sensitizing fraction. Table 2. Effect of a mouse anti-haemocyanin serum on the mouse mast cells sensitization induced by the anti-ovalbumin IgE sensitizing fraction (SF)
Time of addition of SF in relation to AHS AHS Control
1/200 1/20
Simultaneous
30 min after
60 67
49 40
29*
6*
AHS = Mouse anti-haemocyanin serum. Sensitization by SF diluted to 1/100. The results are expressed as percentage degranulation, induced by 1 pg ovalbumin. Total mast cells counted for each test from 93-143. Duplicate counting gave no significant differences (P- 0 7). * Significant differences in relation to controls (P< 0-01)
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Nature of the inhibiting agent Further experiments were performed to verify the nature of the inhibiting agent contained in the anti-haemocyanin serum (Table 3). The mast cells were first incubated with the inhibiting agent for 30 min, then SF was added for a further 30 min. The results indicated a strict correlation between the presence of the inhibiting activity and that of IgE activity after DEAE fractionation of the antihaemocyanin serum, or after heating at 560. Furthermore, washing the mast cells by three successive centrifugations, between addition of the inhibiting agent and that of the sensitizing fraction did not affect the ability to inhibit; showing that the inhibiting factor firmly binds to the mouse mast cells. Furthermore, the fractionation and heating experiments show that in the absence of IgE, there was no inhibiting activity, even in the presence of high concentrations of IgG1.
Table 3. Nature of the inhibiting agent
Inhibiting agent Experimental preparation
IgE PCA Dilution titre
Control AHS* Idem heated 560 Control DEAE fractions 0-02 M 0 05 M 0-1
M
Control AHS followed by cell washing
Percentage degranulation 49 4t 50 65
250 0
1/10 1/10
0 250 0
1/10 1/10
64t 0 63 26
250
1/10
0t
1/5
Inhibiting agent incubated for 30 min; then addition of 1/100 SF for 30 min. Control without inhibiting agent. Degranulation induced by 1 jug ovalbumin. Total mast cells counted for each test from 50 to 113. * AHS = anti-haemocyanin serum.
t Significant differences in relation
to controls
Table 4. Action of anti-mouse IgE on rat mast cells sensitized by mouse IgE
Sensitizing agent Percentage Expt no.
Anti-IgE
Nature
IgE PCA Titre
Dilution
degranulation
1
1/100 1/100 1/1000 1/1000
SF SF
5000
1/100
77t
5000
1/100
54t
SF
5000
1/100
5000
1/100
5000
1/50 1/200 1/800
7
4
control*
2
1/100 1/100 1/1000 1/1000 1/1000
SF SF
1/3200 AHS fractions 0 05 M 0-02 M
250 0
1/50 1/200 1/800 1/10
11 11 4 6 5
41t t 32 t § 28t t 10§
21t** 10 14 8
One hour of incubation with sensitizing agent, then washing, followed by addition of antimouse IgE. Total mast cells counted for each test from 61-105 in expt 1, and from 127-213 in expt 2. * Control: rat anti-rat mast cells control serum. t P< 0-01 in relation to controls without sensitizing agent. t § P< 0-01 between themselves. ** Difference not significant in relation to SF 1/800 (P 0 25) but significant in relation to respectively SF 1/50, 1/200 and 1/3200 (P< 0-01).
(P 0-2). * Significant difference (P< 0-01) in relation to controls without IR162. t Significant differences (P< 0 01) between successive dilutions. B
Control IR162 Heated IR162 IR162 followed
2
6 1000 400 200 100
Experimental IgE dose Percentage Percentage (ng) degranulationt inhibition preparation
by cell washing Control IR162
Heated 1R162 3
Control IR162 AHS diluted
-
1000 100 1000 1000 -
200 100 66 50 1000 200 -
100 50 25
1/40 1/80 1/160 1/320
28
0t 61
26
4t
100 79 7
86
44
-
2t§** 13t§ 21t
95 70 52 43 18 0
25t**
36 46 31-3
7-3tt§
7-6 50-5
22t 6-7t§**
30 78 5 47-3 31 0
15-5t §
16 5t §
216$** 31-4
Total mast cells counted from 66-133 in expts 1 and 2; from 321-454 in expt 3. In expt 3, results from duplicate tests with duplicate countings did no differ more than 1 or 2 points from means. These differences were not significant (P- 0 8). * Inhibiting agent (dose in ng added to 0-2 ml) incubated with mast cells for 30 min then addition of 1/100 SF for 30 min. Control without inhibiting agent. t Degranulation induced by 1 pg ovalbumin. P< 0 01 in relation to controls. § ** P< 0 01 between themselves.
Annie Prouvost-Danon & Annie Abadie
694 1/320
equivalent by use of a standard curve with rat myeloma IgE IR162.
Dilutions of mouse AHS 1/80 1/40 1/160 /
c
0
ti :3 c
X,"
2 751-
C" (L) _0
0 c
0
50k
-e _r_
.s
cu Ot
.2c
25k
R
/
Q)
v CL
a
12-5
,
50 100 25 Rat IgE dose (ng)
200
Figure 1. A bioassay for rat and mouse IgE; the experimental conditions and the data are those presented in Table 6 (expts 2 and 3). Rat IR162 myeloma IgE in nanograms per X) from expt. 0-2 ml: (A A) from expt 2; (X 3; Mouse anti-haemocyanin serum (AHS) in dilutions: (0 0) from expt 3. ---
readily inhibits SF-induced mouse mast cells sensitization. The inhibiting capacity is destroyed by previous heating of rat IR162 for 5 h at 560. The inhibition is observed even when washing the mast cells between the inhibition and sensitization steps. The dilutions of rat myeloma IgE IR162 able to inhibit sensitization of mouse peritoneal mast cells seem of the same order as those able to sensitize
them to the action of anti-rat IgE, as shown in previous paper (Prouvost-Danon et al., 1975) and the present one (Tables 5 and 6). A bioassay for measuring mouse and rat IgE In order to assess this system as a possible one for the evaluation of concentrations of rat or mouse total IgE, further experiments were performed to verify the effect of various dilutions of either rat myeloma IgE or mouse anti-haemocyanin serum on the mouse mast-cell sensitization by SF. Table 6 (expts 2 and 3) and Fig. 1 show the results of two representative experiments. A plot of the logarithms of the IR162 protein doses versus their effect on degranulation, gave a linear transformation of the data. Furthermore, the slopes of inhibition by rat myeloma IgE or by mouse IgE containing serum are not significantly different. Fifty per cent inhibition was achieved when a total of 50 ng of rat IgE was added to the reaction mixture (0-2 ml total volume). The amount of total IgE in a test serum might thus be calculated directly in rat IgE
IgE levels in some mouse and rat sera As an example, some sera were tested in this system. Pooled-normal serum from 2-month-old Swiss mice were found to contain 0-6 jug IgE/ml. The anti-haemocyanin hyperimmune serum was calculated as containing 20 pg IgE/ml (Table 6 and Fig. 1). Reaginic serum (PCA :1000) from mice immunized by two low doses (1 pg) of haemocyanin showed an IgE level of 50 ,g/ml. A pooled-normal serum from adult rats was found to contain 0 8 pg/ml and rat anti-haemocyanin serum (PCA:81) 1V5 pg IgE/ml. Sera from mice infected by Trichinella gave increased values which were in good agreement with those obtained by Perrudet-Badoux et al. (1976) by the use of an immuno-enzymatic method adapted from Guesdon & Avrameas (1974). DISCUSSION Our results show that in vitro mouse mast-cell sensitization by a reagnic serum is readily inhibited by a mouse antiserum with a different antibody specificity (Table 2). Experiments performed to identify the inhibiting factor (Table 3) (fractionation, or heating of inhibiting serum to destroy its binding capacity to mast cells) all indicated that the inhibiting capacity corresponded to the IgE content of the preparation. Rat myeloma IgE was also able to inhibit the mouse mast-cells sensitization (Table 6) suggesting that rat and mouse IgE compete for the same receptor site on the mouse mast cells. Some authors, (Stanworth et al., 1967; Stanworth, 1973) had previously demonstrated that in vivo (PK or PCA) human- or monkey-passive sensitization by reagins could be inhibited by competition with myeloma IgE, high doses of competing IgE being necessary in these conditions. Jarrett et al. (1971) similarly showed, in rats, that passive sensitization processes by a reaginic serum could be inhibited by a second reagin directed against another antigen. This paper provides the results of a study of IgE competition for mouse peritoneal mast cells in an in vitro passive sensitization, which permitted more quantitative and sensitive data to be obtained. By plotting the inhibition observed against the logarithm of the dose of rat myeloma IgE a linear-
Inhibition ofpassive sensitization by IgE regression curve was obtained (Fig. 1) which was parallel to that obtained by plotting the logarithm of the mouse IgE dose, thus permitting a comparison of the dose of IgE of one species with that of another. The system therefore provided a sensitive bioassay for estimating total IgE levels in sera either from rat or mouse, by comparing inhibition data with the rat myeloma IgE IR162 standard curve. The addition of 50 ng rat IgE in this system provided a 50 per cent inhibition. The system was therefore sensitive enough to permit the measurement of IgE levels comparable to those contained in normal sera from adult animals. Normal sera from 2-month-old animals from different pools were thus shown to contain from 0 5 to 2 ,g IgE/ml. There was a good agreement with the values reported by other authors (Carson, Metzger & Bazin, 1975; Perrudet-Badoux et al., 1976). It must be kept in mind, however, that the bioassay of IgE, either by a competitive test or by anti-IgE test, is based on the binding capacity of IgE for mast cells. After preservation of the sera at -20°, there is sometimes a loss of the binding capacity of the 'Fc fragment'. The present bioassay only provides an estimation of the IgE which is functionally intact. The anti-IgE system (Tables 4 and 5) afforded an alternative bioassay model for estimating IgE levels. It was as sensitive (Tables 4 and 5) (Prouvost-Danon et al., 1975) as the inhibition test. Furthermore, it permitted the comparison of IgE antibodies of any specificity. However, it was somewhat more difficult to obtain in this system a good discrimination between the effect of successive dilutions of IgE (Tables 4 and 5). Moreover, the inhibition test had the major advantage of permitting the comparison of mouse IgE with a known rat IgE standard, and thus of converting relative values of mouse IgE into absolute values, into equivalents of rat IgE in micrograms. Although this is not necessary for the present purposes, the sensitivity of the inhibition bioassay could be increased by decreasing the content of non-specific IgE in the sensitizing preparation by specific purification of the antibody. For the measurement of anti-ovalbumin IgE antibodies, it would be necessary to use a sensitizing system of another specificity. In contrast, no significant inhibition was obtained by incubation with preparations containing very high concentrations of mouse IgG1 (Table 3). This confirms the previous suggestion (ProuvostDanon & Binaghi, 1970a) that IgE and IgG1
695
molecules would react with different receptor sites on the mouse mast cells. This seems to differ from what happens in other animal species, as reported by various authors (Bach, Bloch & Austen, 1973; Revoltella & Ovary, 1973; Malley, Baecher, Porter & Gerding, 1974; Stanworth & Smith, 1973). Further experiments are in progress with regard to this question. It has been shown by several authors (Binaghi, Liacopoulos, Halpern & Liacopoulos-Briot, 1962; Halpern, Liacopoulos, Liacopoulos-Briot, Binaghi & van Neer, 1959; Mongar & Schild, 1960) that the velocity of sensitization of guinea-pig tissues is directly proportional to the concentration of sensitizing antibodies and inversely proportional to that of the non specific immunoglobulin pertaining to the same class. Similar data were observed in the case of reaginic antibodies: thus PCA latence in rats could be shortened from 24 h to 30 min by using a high concentration (Mota, 1964) or specifically purified (Binaghi, 1968) antibodies. In vitro passive sensitization, or fixation of reaginic IgE antibodies on rat or mouse peritoneal mast cells, was shown optimally to require about 1-2 h (Prouvost-Danon et al., 1975; Bach et al., 1973). However, previous experiments (Prouvost-Danon 1971; Bach & Brashler, 1973; Prouvost-Danon et al., 1975) as well as the present ones (Table 1) show that, when using a reagin-rich preparation, the in vitro passive sensitization of normal mast cells from young adult mice (2 months old) can be obtained in a very short time. We are now looking into the kinetics of sensitization in various experimental conditions, as when the receptor sites on the mast cells have been previously covered with IgE molecules and there is a need to dislodge previously bound IgE by IgE present in suspension medium.
ACKNOWLEDGMENT This work was supported in part by the Institut National de la Sante et de la Recherche Medicale, France (contract no. ATP 1.73.16). REFER ENCES BACH M.K., BLOCH K.J. & AUSTEN K.F. (1971) IgE and IgGa antibody-mediated release of histamine from rat
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