Allogeneic mast cell degranulation induced by histocompatibility antibodies: An in vitro model of transplantation anaphylaxis

l’I’.I.I.l~I.AK IAl 1tIJNOLOC.Y 20, 133-155 (1975)

Allogeneic Antibodies:

Mast

Cell

Degranulation

An in Vitro

Model

Induced

by Histocompatibility

of Transplantation

Anaphylaxis

hIouse mast cells could be specifically and immediately tlegranulatetl by incubation with an alloantiserum directed against histocompatibility antigens horne by the ma\t cells. The behaviour of the mast cells \vas similar to that during classical anal)hylaxis with respect to microscopic and ultramicroscopic morphology and kinetics. This hehaviour was different from that observed during mast cell lysis induced by complementfixing antibody. The phenomenon was immunologically specific and the responsible antigens were shown to be of the classical so-called “SI)” type and not to be I., antigens or linked to specific receptors ; neither were they allotypic in~rnulloglol)ulit~ determinants. The phenomenon was cotiil)lement-iudepentlent. It needed the integrit? of the Fc portion of antibodies. Not all classes of antibodies could elicit the tlegraw ulation process. Neither 19s nor IgG, antibodies were efficient, although able to inhillit the reaction through Fah competition for the antigen. IgG, could he implicated as tht responsible antibodies. Heat-labile anaphylactic antibodies bvith characteristics of Ig f<: ihut not identified as such) had some activity in early antisera. Although different it1 some way from the classical anaphylaxis to heterologous soluble proteins, this reaction phenomenon involving living could thus be considered as a new in 7vitro anaphylactic mast cells both as effector cells and as the source of antigens. It \vas therefore callctl direct allogeneic anaphylactic degranulation (DAAI) ). Together with the description of the PC.4 directed against transplantation antigens and that of the immulloallogetl~i~~ shock, DAAI) helps to outline the concept of transplantation anaphylaxis. It alw provides both a technique for the detection of anaphylactic alloantihodie> in trankplantation reactions alld a possible iu 7fitl.o model for the stutly of their I~iologic;tl activitirs.

I’rex+ms works have shown that passive ctttaneotts atiaphylaxis ( I’(‘:4 ) of a classical type could be induced in mice with a trnnsl)latttatioti atitigeti-;ttitil)c,tl\ system ( 1) and that allogeneic transplantativn sera injected ititravetiotislv to corresponding recipients induced an “immutioallogeneic shock” anaph\~lnctic itt nature (2), In view of the important role of mouse IgGt antilmdies in the intmunr~logical facilitation reaction (enhancement) (3-h) in atitilwtly dependent imtiittti0regulation (F-9) and possibly, in transplantation tcjlerance ( 10 ) it ux3 tltottght 1 The authors are members of the Associated Research ( CSRS. ERA 149).

Copyright 6 1975 by Academic Press, Inc. All rights of rqxoduction in any form reserved.

Team

to the Sational

C’cllter

of .i;ri~,~~titil

134

DAiiRON

ET AL.

interesting to study further the mechanism of transplantation anaphylaxis and to devise an in vitro tool allowing more quantitative studies. Mouse mast cells are known to bear at their surface both transplantation antigens (11) and Fc receptors for anaphylactic antibodies ( 1.2)) the activation of which leads to active degranulation ( 13-16). It was therefore conceivable that anaphylactic transplantation alloantibodies could bind themselves to mast cell transplantation antigens through their Fab specific binding sites and to the Fc receptors of the very same mast cells through their Fc reactive portion, thus triggering the degranulation process. This was indeed found to happen, and the phenomenon hereafter described has been called direct allogeneic anaphylactic degranulation (DAAD) . MATERIALS

AND METHODS

Animals Except for the C57BL/6 strain mice purchased from the CNRS breeding Center, Villejuif (France), all animals used in these experiments were bred in our own colonies. Inbred strains used were A/J, CBA, BALB/C, C57BL/Ks, and IC. Experimental mice were taken at the age of 2-3 months. Preparation

of Immune Sera. Immunization

Procedures

Anti-H-Z. Female mice of the CBA, C57BL/K s, and IC strains were immunized with the following A/J immunizing materials : Living spleen cells consisting of five intraperitoneal injections administered at days 0, 15, 30, 60, and 85. Five or eight recipients were given the equivalent in cells of one A/Jax spleen each time. Mice were exsanguinated by cardiac puncture 2 weeks after the last booster. Lyophilized spleen cells : Homogenized lyophilized whole spleens were injected intraperitoneally at a dose of one dry weight spleen equivalent for five recipients on days 0, 15, 30, 56, and exsanguination was performed on day 70. Tumor cells: Sa I or YAC cells were injected first subcutaneously at a dose of 3 X lo5 cells per mouse and the dose was subsequently increased to 10s, 7 x 106, and 3 x lo7 at days 20, 40, and 60, respectively. Exsanguination was performed on day 95. EL 4 lymphoma, maintained in the C57 B1/6 strain, was injected ip into BALB/C females at a dosage of 5 x 106, 107, and 2 x lo7 cells on days 0, 14, and 28, respectively, and mice were bled on day 35. All injections were made in physiological saline at volumes of 0.2-0.5 ml. Anti-ovalbumin. Ovalbumin was injected in physiological saline at a dose of 0.5 mg for the first ip injection and two boosters of 1 mg each at days 0, 14, and 28, respectively. In some cases, the first injection was supplemented with 0.15 ml of a suspension containing 3.5-5 X lo9 formolated Bordetella pertussis per milliliter (Institut Pasteur). Blood was collected on day 35. Monospecific anti-mouse Ig sera. Antisera against mouse IgG1, IgGz, IgGna, IgA, and IgM were raised in rabbits immunized with Ig precipitates (in complete adjuvants) obtained by precipitation with 33% ammonium sulphate. The starting material consisted of sera derived from BALB/C mice bearing the corresponding myeloma. The rabbit sera were then absorbed with other myeloma proteins until they became monospecific.

ALLOGENEIC

Goat anti-mouse for control tests. Fractionation

MAST CELLDEGRANI-LATIOK

Ig sera were pm-chased from the Lleipar

135

Company and nsetl

of Sera

On S+lzadr.v G-200. Mouse sera were fractionated on Pharmacia cohm~ns (Pharmacia, Fine Chemicals), 1.5 x 100 cm, at a flow rate of 24 mi/hr. The flow was controlled by a peristaltic Autoanaiyser pump. Individual fractions containing 6 ml each were examined using a spectrophotometer at 280 nm and the ascendent and descendent parts of the optical density curve, corresponding to the 19. 7, and 5s peaks, were collected and concentrated over a Diaflo Amicon 14 1 membrane. On 1)&1E-cfllulosr. The ion exchange fractionation on DEAE-cellulose was clone with a linear continuous moiarity gradient. The starting buffer was 0.02 J/ (XaCl-Tris, pH 8.2), the final one, 1.0 ~$1. Fractions corresponding to ionic strengths ranging between 0.02 and 0.03 ~51( ex_ 1jresseti in XaCi equivalence ) , 0.03 and 0.04 YU. 0.05 and 0.06 M, etc., were pooled and concentrated on a Diaflo Xmicon 11 1 filter. In most experiments only the fractions corresponding to a molarity up to 0.1 iz;r were taken for experimental purposes. I’rr,twvtion

of F(ab’)2

Fractions

(17)

Iniiiiunogiobuii~is were first precipitated with 339, ammonium suipiiate. The pepsin digestion was carried out, using a method described by Nisonoff (18) slightly modified by us, in an acetate buffer 0.1 :V at pH 4.5. The enzyme/protein ratio was l/30. The F(ab’)2 fragments were separated from undigested Ig moiecuies by filtration through a Sephadex G 75 column. The presence of F( al)‘) 2 and of undigested Ig was estimated using antisera specific for either Fab or i-c (IgG1 or IgGZ j portions of the Ig molecule. IIz~aiuntion

of tlzr Ig Contents

in Sun

and St~t~nl Frm-fions

The method consisted in testing by double diffusion in agarose the Ig-containing material against the corresponding monospecific anti-mouse Ig antiserum placed in the central well. A standard sample was used as reference. In Vitro Annplzylaxis Prcpavation of peritoneal cells. Inbred 2-3-month-old mice were killed and exsanguinated by decapitation. They were then injected with 2 ml of HEPESbuffered RPM1 medium (Gibco, Grand Island Biological Company ) intra1)eritoneaiiy. After gentle massage, the peritoneal cavity was opened and the cell suspension collected in S-ml plastic tubes. Peritoneal ceils were washed twice 1)) 2OOg centrifugation for 10 min and resuspended in fresh RPM1 medium at an average concentration of 5-10 X loo peritoneal cells per milliliter; 0.25-1111 samples of ceil suspension were warmed at 37°C for 20 min without shaking. Inczrbation. Anti-ozuhntin anaphylaris (AOfl ). Anti-ovaibumin serum was warmed at 37°C for 5 min and 20 ~1 were then added to the ceil suspension. Sentitization was performed by a lo-min incubation at 37°C (except for the kinetic experiments done without any sensitization period) and 20 ~1 of an ovaibumin solution, previously warmed at 37°C for 5 min, was introduced for challenge. The mixturr was further incubated at 37°C for 5 min.

136

DAERON

ET AL.

Degranulation by comfiound 48180. Fifty microliters of a previously warmed 48/80 solution (Burroughs Wellcome and Co., Tuckahoe, N.Y.) were introduced into the cell suspensions. Mast cells from different strains exhibited different sensitivities to 48/80 degranulation. BALB/C mast cells were among the more resistant ones. In this strain, 5 pg of 48/80 in 50 ~1 were added to 0.25 ml of the cell suspension. Incubation lasted for 5 min. DAAD. The outline of the tests is illustrated in Fig. 1. Alloantiserum was warmed at 37°C for 5 min and 50 ~1 were added to the peritoneal cells. The reaction was allowed to take place during a 5-min incubation at 37°C. The reaction was stopped by placing the tubes in an ice bath. After a lo-min rest, a drop (0.03 ml) of 0.1% toluidine blue (bleu toluidine, R. A. L. Kiihlmann, Paris) was introduced into each sample. The staining of mast cells only began when the suspensions were removed from ice. The tubes were then randomized after their numbers had been hidden. Cells were observed under a Zeiss light microscope with a 250X magnification. The degranulation percentages were established according to the morphological criteria described in Results by blind readings in casual order. Such a procedure minimized the subjectiveness of the method. Results are presented with the 95% confidence limits for each point. Antibody

Activity

Assays

Specific anti-H-2 antibodies were titrated using the following methods : Hewzagglzrtination tests were performed essentially using Gorer and Mikulska’s human serum-dextran technique as described in an earlier paper ( 19). Hemolysis was evaluated in the presence of absorbed rabbit normal serum as a source of complement. The technique described by Hildemann (20) was quantified using a Beckman spectrocolorimeter for measuring of hemoglobin contents in supernatants of RBC. A RBC sample lysed with bicarbonate distilled water was taken as a 100% lysis reference. The Yr cytotoxicity method was performed essentially according to Wigzell (21) using YAC tumor cells at A/JAX targets and EL 4 cells in the BALB/C anti-C57 BL 6 combination. The anti-H-2 passive cutaneous anaphylaxis (PCA) test consisted briefly, in sensitizing the skin of mice, isogenic to the antibody producing strain, by id injections of 0.05 ml of immune sera or fractions; 2.5 hr later an iv injection of 0.5% Evans blue (0.2 ml) was followed by the supernatant of 2-5 mg of H-2 target antigens solubilized as described by Kinsky ct al. (1). Electron Microscopy Itnmediately after the reaction, suspensions of peritoneal cells (20 X 10” cells/ ml) were fixed in 4% glutaraldehyde with 0.2 M cacodylate buffer (pH 7.2) for 60 min at 4°C. After rinsing in the same buffer with 8% sucrose, the pellets were postfixed in 2% osmium tetroxide with 0.1 M cacodylate buffer and 10% sucrose for 60 min at 4°C; they were then washed in 0.2 M cacodylate buffer with 8% sucrose, dehydrated in ethanol, and embedded in Epon (22). Thin sections were cut on a Porter-Blum MT2 ultramicrotome of LKB Ultrotome III and stained with 5% uranyl acetate and lead citrate. Observations were carried out on an RCA EMU 3 H electron microscope at 50 kV.

ALLOGENEIC

31.4%

CELL

FIG. 1. Sequence of operations

1.37

DEGRANUL.2TIOS

in the 1).4;\D

te>t

RESULTS The results bear on three aspects : tlescription, tra~~splantatioll antigen specificity. ant1 anaph~lactic mechanism of the phenomenon. I. ljP.siriptio~~ of the Plzrnonrenon Peritoneal cells collected from normal A/JAX mice were illcuhatett with immune ’ I’ivmg alloantiserum raised against A/JAX or tyophilized cells. After staining of the mast cells with totuidine blue, the percentage of tlegranulated cells uxs coaluated by btintl readings under a tight microscope. N’hereas only a small percentage of degranutation was seen in control su~t~ensioiis incubated \vithout any serum. consistent mast cell ttegranutation couttt he ol)servett with either anti-A/JAX spleen cells (CR.4 anti-A/JAS. C57 13I
4 : 2 j I 2 P

90 80 70 60 50 40 30 20

*

10 oi

FIG. 2. Dependenceof DA.4D of serum added under a volume suspension.

on serum dilution. Dilutions

indicate the initial concentration of 0.05 ml to a tube containing 0.25 ml of a peritoneal cell

138

DABRON

ET AL.

FIG. 3. Optical Microscope Morphology and Ultrastructural Aspects of Mast Cells in DAAD and Other Conditions. l-4: Light microscopy (toluidine blue staining). 1: Control A/JAX normal mast cell: The plasma membrane is smooth and refringent (X2%0). 2-4: A/JAX mast cells after incubation with CBA anti A/JAX serum (DAAD) = progressive stages of degranulation. 2 : The plasma membrane is less refringent and has a rough aspect (X2500). 3 : Appearance of extracellular granules (X2500). 4: Ultimate stage of degranulation = plasma membrane can be no longer seen (X2500). 5-13 : Electron microscopy. 5 : Control BALB/C unaltered

ALLOGENEIC

MAST

CELL

I~EGKAN~7LA’l’10N

130

Toluidine blue stained the cell content uniformly, except the nuclear region which looked slightly less dense than the peripheral cytoplasm. Granules were distinguishable neither inside nor outside the cell. More than 9075 (usually 93-98% 1 of such normal mast cells were found in control preparations. M’hen incubated with alloantibodies directed against antigens of the mast cells strain, numerous mast cells appeared markedly altered ; the number altered u:as asually correlated with the intensity of the alterations in individual cells. This heterogeneity in the morphological changes of cells led LIS to define five progressive stages of alteration from normal (stage 0) to entirely degranulated cells ( stage 1) A small number of cells had lost their round shape, which 1)ecame more polygonal (stage 1 ). Cells of this type being as numerous in controls as in exl)erimental preparations. they were operationally considered as nondegra~l~~latetl cells. The first detectable sign of degranulation affected the plasma membrane which. on a few spots, was less refringent and exhibited a rough aspect. In these spots, granules could be distinguished as small bulgings of the membrane (stage 2-Fig. .1-2 ) X11 obvious degranulation was characterized by the appearance of clearly extracellular granules, still in contact with the plasma membrane. The whole plasma membranr had a rough surface, giving the cell a morula aspect (stage 3-Fig. 3-3 1. In tht ultimate stage of degranulation almost the whole content of granules seemed to ha\e been expelled from the mast cell whose plasma membrane could no longer lje sew. The released granules remained gathered near the cell. so that even entirely tlcgranulated cells could be recognized ( stage l--Fig. 5-C ) The number of mast cells in each of the five niorl~hological stages was tletermine~l by blind counts of 300-500 mast cells in each sample. A line. passing thro~~gh the third class (stage 2) was arbitrarily chosen, beycmtl which the cells were considered as tlegranulated and the degranulation percentage was thus established 1)~ adding the numbers of cells in 3 and 4, plus half the numbrr of cells in 2. According to this procedure. the spontaneous degranulation in control preparations constantly reprcsented less than lo%, usually between 2 and 7/j: This spontaneous degranulatioil percentage was substracted from the degranulation percentage in experimental preparations. so that the results could be considered to represent experimentall\; induced tlegranulation. No morpliological differences could be detected between degranulation produced by DAA, anti-ovalbumin anaphylaxis, and compound 48/80. _____._ ___~~~-~~--_.____ --- ~--ma.st cell. Cytoplasmic granules show an homogeneous dense dark appearance ( x7000 1. O-10 : RALB/C mast cells after incubation (5 min) with CBA anti-A/JAX serum ( 11.4.4 I) ). 0 : Stan! intracellular granules exhibit a decrease in density, a floculent appearance. and a large perigranular space (X10,500). 7 : Cytoplasniic vacuolation alItI appearance of altered extracellular granules (X5650). 8 and 9: Numerous hroken projections and large empty vacuolez. Note presence of unaffected intracellular granules ( X6000 ). 10 : .4 strongly degranulated maat cell, main untrastructural changes of DAAD are seen = intracellular granule alteration, cytoplasmic vacuolation and numerous extracellular floculent granules ( X4500). 11 : l{AI.U/(I mast cell after 10 min sensitization with CBA anti-ovalbumin serum and 5 min contact nit11 ovalhumin. Note essentially the same features as in DA.4D (~6000). 12: BAT,B/C mast cell after treatment with compound 48/80. A strongly degranulated mast cell showing the same mast cell after a cytotoxic reaction (incubation bvitb granular chang-es (X6000). 13 : BALB/C a CB.4 anti-A/JAX serum plus complement). Main alterations involved essentially the I-rll ilztcylritj = plasma membrane is disrupted, cytoplasmic and nuclear matrices show a spongy appearance. Altered and unaltered granules are dispersed throughout remnants of cytqllasnr (~7000). However granules are not expelled out of the ~~11.

140

DAERON

ET AL.

Electron microscope study. Three different in vitro systems were compared. Peritoneal cells (PC) were collected from normal BALB/C mice and, within the same experiment, incubated with either CBA anti-A/JAX antibodies, or CBA antiovalbumin serum plus ovalbumin, or 48/80 compound. The degranulation percentages, estimated in samples of the different preparations by counts under a light microscope were the following : PC + CBA anti-A/JAX serum 61% PC + anti-ovalbumin serum + ovalbumin 84 PC + 48/80 59 PC + anti-ovalbumin serum 10 PC + ovalbumin 3 PC alone 3. Identical morphological characteristics were found in any of the three control preparations (Fig. 3-5). Mast cells could be easily recognized as large round or oval cells containing numerous well-outlined dense granules. Cytoplasm could be seen between the granules and contained cytoplasmic organelles. The nucleus, when apparent, was of irregular shape, working in between the granules which embossed it. Dense chromatin extended into the periphery of the nucleus. The mast cells were surrounded by a continuous plasma membrane, fitting closely round the granules, so that it exhibited dented outlines. All granules were homogeneous in shape, size, and density. A very small number of them were surrounded by a clear perigranular space. After incubation with appropriate histocompatibility antibodies, mast cells exhibited various degrees of alteration. No change could be observed affecting either the macrophages or the lymphocytes of the peritoneal suspension. In mast cells, only the granules and the plasma membrane were altered. No alteration of the cytoplasm or of the nucleus was seen. In a number of mast cells, a few granules (generally in the periphery of the cell) looked swollen and exhibited a loss of density. The granular matrix had a heterogeneous flocculent appearance. The perigranular space was often enlarged. Sometimes, intracellular granules seemed to be open to the outside of the cell. Very few extracellular granules could be observed (Fig. 3-6). Other mast cells were more obviously altered. They contained a greater number of clear swollen granules, some of them being fused together inside vacuoles. More granules were seen outside the cell. These extracellular granules all had a light flocculent appearance and were clearly devoid of membrane, resembling the granules found in the vacuoles (Fig. 3-7). In strongly degranulated mast cells, a great number of extracellular granules were seen. However, the plasma membrane did not appear disrupted and the process of exocytosis could still be seen working (Figs. 3-7 through 3-10) a number of intracellular granules remaining to be expelled. Numerous fragments of ridges surrounded the cells but they seemed still to contain cytoplasm and they could not be clearly seen as free particles. In anti-ovalbumin anaphylaxis (Fig. 3-11) and in 48/80 degranulation (Fig. 3-12) the mast cells exhibited the same morphological changes as in DAAD (Fig. 3-10). This type of degranulation alteration of mast cells was firmly compared with complement-dependent cytotoxicity toward mast cells as targets. The cells were incubated, in the presence of complement, with a CBA antiA/JAX DEAE fraction known to contain IgG:! immunoglobulins and to elicit serocytoxicity and hemolysis, but almost no DAAD (‘fraction no. 1, Fig. 12).

ALLOCl<~EIC

MAST

CELL

141

DEGRANL-LhTIOS

20 10 0

FIG.

3. Comparative

xyd..-.

015

curves of DAAI>

15

30

60

and anti-ovalhumin

allaphylaxis

killetics.

Mast cells were all markedly altered (Fig. 3-13 ). The plasma membranes were completely destroyed. The nuclei were heavily damaged : Numerous “holes” were seen in the chromatin, the outlines of which were irregular. Only fragments of cytoplasm remained in the cells. The granules were also altered : Some of them had the flocculent appearance seen in degranulated cells, others seemed to have lost their matrix but to have kept their membrane, others still had the dense homogeneous aspect of normal intracellular granules, However. and most importantly, they had not been expelled from the cells : They remained gathered together in spite of the absence of any plasma membrane. These aspects of cell lysis were thus clearly tlif’ferent from those ol~scr~~ed ill tlegranulated mast cells. Kinetics of f)AA/). When identical samples of peritoneal cells were incubatt.tl with the same dose of CBA anti-A/JAX serum for \:arious periods of time. a great \.ariation in the percentage of degranulatetl mast cells could be observed. Almost immediately the reaction reached its utmost intensity which began to decrease after the fifth minute (Fig. 4). An anti-ovalbumin anaphylactic reaction was achieved within the same experiment with samples of the same cell suspension. In order to enable us to compare it with DAAD, the anti-ovalbumin reaction was performed by using a CBA antiovalbumin serum, which was added to the cells simultaneously with ovaIbumin dilution. The kinetic curves of both phenomena presented a striking similarity ( Fig. -I ) The same relationship with time was observed in DAAD performed \vitll Cl:;‘i anti-A/JAX serum either on A/JAX or BALB/C mast cells. Thus, morphological changes induced by a transplantation immune >crimi appeared to affect mast cells within the very first seconds of incubation, similarly to the conventional anaphylactic degranulation ( 13 ) The decrease in the number oi tlegranulated cells after the fifth minute of incubation L\:as unexpected and bvill l)e discussed later. II.

Transplantation

Antigen

Specificity

of the Plzenonzenopz

The specificity of DAAD was investigated by studying the cross reactivity of given alloantisera on mast cells from different strains of inbred mice. Absorption experiments were also performed.

142

DAkRON

Linkage

of

the

phenomenon

to H-2

ET AL.

antigenic

specificities.

Anti-A/JAX

Sera

were able to degranulate not only A/JAX mast cells but also mast cells from other allogeneic strains, provided that they possessed the H-2 determinants against which the serum was directed. As a consequence, CBA anti-A/JAX serum could degranulate A/JAX, BALB/C, and C57 BL/Ks mast cells which share H-2 specificities recognized by CBA anti-A/JAX serum. Similarly, C57 BL/Ks anti-A/JAX serum could degranulate A/JAX and CBA mast cells which both bore the same H-2 specificities, with respect to C57 BL/Ks anti-A/JAX serum. Conversely, no degranulation was observed when CBA anti-A/JAX serum was incubated with CBA mast cells. Moreover, C57 BL/Ks anti-A/JAX serum did not degranulate either C57 BL/Ks or BALB/C mast cells where none of the H-2 specificities recognized by the serum was present. Anti-A/JAX tumor cells sera had the same specificities as anti-A/JAX spleen cells sera. Absorption on specific spleen cells inhibited the degranulation activity. Absence of dependence on Ia antigens. In order to test the role of putative antibodies that would be directed against surface antigens coded by the Ir region, anti-A/JAX CBA serum was absorbed on A/JAX erythrocytes (known to bear H-2, but not Ia antigens), A/JAX erythrocytes were washed and separated from leukocytes by centrifugation, the last one on Ficoll/Triosil, then washed again three times. One milliliter of anti-A/JAX CBA immune serum, diluted one-tenth, was absorbed on 0.3 ml of packed purified A/JAX erythrocytes. This was repeated three times. After each absorption the hemagglutination and mast cell degranulating power of the immune serum was observed. Its complement-dependent cytotoxic power was also studied before and after absorption. It was found that erythrocytes bearing the relevant H-2 specificities were able to remove all degranulating activity after the first absorption. “Pseudo-absorptions” on CBA erythrocytes had no effect (Table 1). Neither Ia antigens nor antigens linked to lymphocyte receptors appear therefore to be involved in the DAAD phenomenon. Absence of dependence on immunoglobulin allotypic antigens. Since cells (spleen cells, sarcoma I, EL 4 leukemia) injected to induce allogeneic antisera are known to have immunoglobulins at their surface and peritoneal cells, especially mast cells, might also have the same type of immunoglobulins at their surface, the TABLE

1

DEGRANULATING ACTIVITY OF CBA ANTI-A/JAX IMMUNE SERUM. ABSORPTION BY ERYTHROCYTES BEARING THE RELEVANT H-Z DETERMINANTS Treatment

of CBA Anti-A/JAX immune serum

Properties Hemagglutinationa

of serum preparations

Direct allogeneic degranulation* on A/JAX mast cells

Untreated Absorbed 1 time on A/JAX erythrocytes 2 times erythrocytes 3 times erythrocytes “Absorbed” 1 time on CBA erythrocytes 2 times erythrocytes 3 times erythrocytes

on BALB /C mast cells 72

10,000 160 x 10.000 10,000 10.000

0 Reverse of dilution. ) Percentage of specifically degranulated mast cells. Under our experimental reactive than A/JAX for anaphylactic degranulation. c Percentage of specifically hlue cells (trypan blue).

conditions

Serocytotoxirityc on A/JAX spleen cells 94.5

N”D”

E

NOD ND 89

N”D ND 88

BALBIC

mast cells are more

ALLOGENEIC

MAST

CELL

143

IIEGKANVLATJON

DAAD might conceivably have been the consequence of an allotype-anti-;tllot~pc reaction at the surface of mast cells. Two types of experiments were performed to test that possibility : the first compared the effects of diluting the anti-A Cl::1 immune serum in normal A and in normal CBA sera, both giving the same results on mast cell degranulation as dilutions in PBS. The second experiment consisted in performing ininiunoabsorptions of the antiLL CBA immune serum on polymerized A/JAX itnInunoglobttlilIs or, as controls. ott polymerized CBA imn~unoglobulins. Ko activity Mhxtsoever ws absorbed 1)~ this procedure (Table 2). The antigen-antibody system responsible for the degranulating activity of trattsplantation immune sera appears therefore to be independent of the Ix system and of Tg aliotypes and to be linked to H-2 antigens in the classical sense. For practical reasons, cross reactions were occasionally used : ,4 certain number of experiments \\:ere performed within triangular strain combinations. III.

.-lna~hylactir

Nature

of the Phenomenon

In addition to the parallelism of DAAD with anti-ovalbumin anaphylaxis for their morphology and their kinetics, the anaphylactic nature of DAAD was ascertained by the following five sets of experiments. C‘owplrwent independence of DAAD. Although DAAD could be achieved b! DEAE or Sephadex G-200 fractions in the absence of any source of fresh serum. the possible participation of complement in the degranulation phenomenon whets induced by nonfractionated immune sera was investigated further. DAAD was performed with A/JAX mast cells and CBA anti-A/JAX serum. Il”ithin the same experiment, the activity of nonheated serum and serum heated at 56°C for 1 hr in the presence or absence of fresh normal CBA serum as a sottrcc of complement were compared. As can be seen in Fig. 5. heated immune serum exhibited a limited (97%) c1ecrease in its activity. This decrease could not be rest(Jred by the addition of fresh normal CBA serttm. DAAD. thus, did not involve the activation of the complement system.

DEX;HANLILATIN(~ ACTIVITY OF CBA ANTI-A/JAX OF IMMUNOABSORPTION ON POLYMERIZED

Treatment

of CBA Anti-A/JA.X immune serum

Untreated Absorbed on polymerized A/JAX immunoglobulins “Absorbed” on polymerized CBA immunoglobulins Eluate from A/JAX immunoabsorbant Eluate from CBA immunoabsorbant 0 Not determined.

IMMUNE

A/JAX

SWUM. I,ACK OF ISFLCENCI IMMI’NO(.I.~BI:I.INS

Degranulating activity on A/JAX at the following dilutions

mast cells

--

l/20

l/5

1 /lO

18 19

16 19

19

19.5

0

N DfL

sn

0.5

ND

KD

9 11 7

144

DABRON

ET AL.

CBA m-A/J SERUM: Non heated tkatedW,T.~~~ SO. 7c-

$60. 150~ i 40

$30 Yj20 10

01 I o in

+ RPM1 + CBA rormol +CBA nwmd

~cr..mheated swum

FIG. 5.Complement independence of DAAD. Heating resulted in a slight decrease of activity. The addition did not modify the reaction.

(1 hr at 56°C) the immune serum only of normal serum either fresh or heated

Independence front chemical mediators of other peritoneal exudate cel!s. Since lymphocytes and macrophages in the peritoneal suspensions bore the same antigens as the mast cells, it had to be investigated whether these cells could participate in the degranulation process. The extremely short period of time required for the reaction to be achieved (almost maximum after a few seconds) made such an intervention of other peritoneal cells in DAAD unlikely. Neither antibody-dependent cell-mediated cytotoxicity (23) nor the antibody-induced release of soluble mediators from lymphoid cells (24) are known to be efficient within incubation periods of a few minutes. Moreover, changes were not detected in the ultrastructure of either the macrophages or the lymphocytes of the peritoneal cell suspension submitted to the serum effect, arguing against the possible release of materials from injured cells. However, to exclude such a possibility, we looked for a hypothetical release of nonspecific soluble substances which could cause mast cells to degranulate. If, when incubated with specific alloantibodies, lymphoid cells release degranulating substances which mediate DAAD, supernatants from such a mixture should degranulate both allogeneic and syngeneic mast cells, the intensity of the reaction being a function of the number of lymphoid cells. Increasing numbers of C57 BL/6 spleen cells were incubated for 5 min with a BALB/C anti-EL 4 DEAE fraction known to contain IgGr and IgGs antibodies. The supernatants were then tested for their ability to degranulate C57 BL/6 and BALB/C mast cells during a 5-min incubation. 70 % z : 60 1% - 40 I g 30 i 20 d-10 O-

FIG, 6. Effect of supernatant from BALB/C anti-EL 4 DEAE fraction incubated C57 BL/6 spleen cells, on allogeneic and syngeneic mast cells. The only effect of incubation the absorption of specific antibodies. No effect was observed on syngeneic mast cells.

with was

1 45

0

10

FIG. 7. Positive correlation between the DAz4D and anti-ovalbumin anaphylactic reactions of mast cells from different mouse colonies and strains. A/Jax SA : A/Jax colony from the HApita Saint-Antoine, Paris. A/Jax VJ: A/J ax colony from the CNRS Breeding Center, Yillejuif. A/Jax OR: A/Jax colony from the CNRS Breeding Center Orlkans. Each point is plotted a$ a correlation between DAAD and anti-ovalbumin anaphylaxis, both being tested ou the samr peritoneal cell suspension. Horizontal and vertical bars represent the confidence limits of both tests. Each line joins the point established Lvithin one experiment.

As shown by Fig. 6, the only effect of previous incubation (Jf antibodies bvitll lymphoid cells was actually a decrease in the degranulation of allogeneic mast cells, corresponding to the absorption of specific antibodies on incubated cells. S(I dcgrandation of syngeneic mast cells Was iiidnced KhateWr the Iiml~Jer (Jf slJleei1 cells used for previous incubation was. ~1s was explained earlier. a giveit Ccl1 receptor dependence of the plzeno~rwnon. serum was able to degranulate mast cells from different strains, provided that the!~ bore the antigenic specificities against which the serum \vas directed, In order to clarify the other determining cellular factors in\olved in the reaction, the abilit\ of CBA anti-A/JAX serum to degranulate mast cells from different strains (all of them bearing every H-2 specificity recognized by the serum) was studied ant1 compared, When incubated with the same serum, A/JAX mast cells from tllrrc different breedings, BALB/C and CS7 BI,/Ks mast cells, exhibitetl tlifferent responses. A possible explanation of such discrepancies could reside in differences affecting the mimbers of accessible receptor sites for the Fc end of the anaphylactic atitibodies. Evidence of the validity of this hypothesis was obtained by observing the same discrepancies when the same cells were challenged \z.ith CR.4 ;Inti-ovall~~~n~iii serum plus ovalbumin. .4s a rule. a linear relationship between the ability of mast cells t(J achie\,e i j4.41) and o\albulnin/anti-ovalbumin anaphylaxis could be observed. \\‘hereas 15..\1,1:/C mast cells constantly strongly reacted in both s!,stems. A/JAS and C.5i I’,I./Ks mast cells gave weak reactions in either system ( I;ig. 7 1, These results led us to conclude that these two phenomena were passing througll a common pathway-the binding of antibody molecules on tile receptor sites for I;c on the mast cells-and that these receptors could be more or less mumerous 011mast cells from different strains. Moreover. differences could be found between mast cells \vithin the same original strain from different breedings. One possible interpretation would be that naturalI\, occurring antibodies (IgE ?) could saturate these receptors. as suggestetl 1)). t IW fnllowing experiment.

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antibodies. F(ab’)Z fragments FIG. 8. Biological properties of pepsin-treated IC anti-A/Jax cause neither detectable DAAD nor hemolytic effects although noticeable hemagglutination was preserved. The peak of undigested antibodies was active in all three tests.

When mast cells were collected from CBA mice immunized against Sa I tumor cells, they lost an equal proportion of their ability to achieve both DAAD (induced by C57 BL/Ks anti-A/JAX serum) and anti-ovalbumin anaphylaxis when compared with nonimmunized mice. Taken together, these data were thus consistent with the conclusion that DAAD was dependent on the same receptor sites on mast cells as classical anaphylaxis. Necessity of the antibody Fc portion. The results reported above strongly suggested that DAAD involved the receptor sites for the Fc end of cytophilic antibodies on the mast cell membrane. To ensure the validity of this statement, induction of degranulation was tried by incubating A/JAX mast cells with F(ab’)2 fragments prepared from pepsin-digested IC anti-A/JAX antibodies. The F(ab’)2 fragments were isolated from nondigested immunoglobulins, by gel filtration through a Sephadex G-75 column. The fraction obtained no longer reacted with anti-Fc serum but still gave a precipitation line with anti-Fab serum in a double diffusion test. When tested for its anti-A/JAX biological activities, this fraction was shown to be active for hemagglutination but to have lost its hemolytic properties. Moreover, it had also lost its DAAD activity (Fig. 8). Therefore, degranulation was not the consequence merely of combination between the antigens of the cell surface and the Fab ends of antibodies. DAAD also needed a more complex phenomenon, involving the interactions of the Fc ends of antibodies with specific receptor sites on the mast cell membrane. As a consequence it could be predicted that restricted classes on antibodies would achieve DAAD. Responsible antibody classes. Contrary to the classical anaphylaxis where only restricted classes of antibodies can interact with mast cells, in DAAD, antibodies of every class are able to bind on the cell surface, provided the latter possess the corresponding transplantation antigens. An important question was thus to know whether DAAD was mediated only by anaphylactic antibodies or if other antibody classes could also induce the phenomenon. (a) 19s vwsus 7s antibodies. Various sera were fractionated by gel filtration through a Sephadex G-200 column, as described in material and methods. The 19, 7, and 5S fractions were examined for their biological properties. In one of these experiments, 1.5 ml of CBA anti-A/JAX serum was mixed with 0.5 ml of CBA anti-ovalbumin serum. The mixture was then submitted to gel filtration and the concentrated fractions were tested for their anti-ovalbumin anaphylactic properties by means of the 2 hr 30 min PCA test in CBA mice, and

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ANAPHYLACTIC PRQPERTIES f,F SEPHADEX C200 FRACTION5 Mtxture of CBA anti-A/J +CBA ant,.Ovoltumm 5er-o

0.800160

t\ f, 1 i \

FIG. 9. Coincidence of anti-ovalbumin PCA and DAAD obtained from a mixture of anti-ovalbumin and anti-A/Jax with the 7S peak.

activities in Sephadex G 200 fractions CBA sera. Both activities are linked

for their DAAD activity of BALB/C mast cells. As shown by Fig. 9, DAAD was clearly associated with the 7 S peak. No significant effect was exhibited by the 19 S fractions. PCA activity, evaluated by the measure of extravasated dye areas, was exactly superimposed with the DAAD curve. In other experiments, DAAD was compared with j’Cr serocytotoxicity in the 19 and 7S fractions of three sera collected after one, two, or three injections of allogeneic cells. Figure 10 illustrates one of these experiments, carried out in the BALB/C anti-EL 4 system. Serocytotoxicity was evaluated with EL 4 leukemia cells as targets, DAAD was performed on C57 BL/6 mast cells. No significant DAAD was found with either the 19 or 7S fractions from normal serum or from the sera collected after one or two injections of EL 4 cells. After the third immunization, whereas the 7S fractions exhibited obvious activity, the 19s fraction was devoid of any effect. Neither normal serum nor the serum of the first immunization had any cytotoxic activity in any of the fractions tested. After the second injection, only the 7S fraction exerted significant cytotoxicity, and after the third injection, all the fractions were consistently efficient. When DAAD was compared with cytotoxicity, clear differences could be observed in the fractions of the hyperimmune serum. Whereas DAAD was only associated with the 7S peak, cytotoxicity could be induced by any of the 19s or the 7S fractions.

FIG. 10. Comparative study of DAAD and Tr cytotoxicity of 19 and 7S fractions obtained from BALB/C sera either normal or after one, two, and three injections of EL 4 cells. While cytotoxicity was found in both 19 and 7s fractions, DAAD was restricted to the 7s peak.

148

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50-

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FK. 11. Inhibitory effect of mast cells preincubation with BALB/C anti EL 4 19s and IgGs specific antibodies (Ab) on DAAD elicited with a fraction giving a standard (75%) degranulation. Normal BALB/C immunoglobulins (Ig) obtained in identical conditions did not inhibit significantly DAAD.

The results obtained from these two experiments suggested that the 19s antibodies were unable to mediate DAAD. However, these results only offered negative data to support this conclusion. A more direct positive proof was obtained by the following experiment. If 19s antibodies are not involved in the DAAD phenomenon, they are probably able to bind to the antigens of the cell surface. One way to prove their lack of effectiveness clearly was to demonstrate that, when bound to the mast cell membrane, they could inhibit the reaction induced by active antibodies, by masking part of the accessible antigens. C57 BL/6 peritoneal cells were therefore incubated for 20 min at 37°C with either 19s antibodies isolated from BALB/C anti-EL 4 serum or 19s immunoglobulins from a normal BALB/C serum or with RPM1 medium. They were then submitted to a second incubation for 5 min with a BALB/C anti-EL 4-DEAE fraction, known to contain IgGr and IgGz immunoglobulins. No degranulation was obtained by the preincubation of the cells with either 19s antibodies or 19s immunoglobulins followed by a 5-min incubation with RPM1 medium. Figure 11 shows that the preincubation of the cells with the 19s immunoglobulins from normal serum gave no significant inhibition of the reaction whereas the preincubation with the 19 S specific antibodies inhibited 36% of the DAAD induced by the second incubation with active antibodies. It can be concluded from these experiments, that although the 19 S antibodies were able to bind to the antigens of the cell surface by their Fab end, their Fc end could not be recognized by the receptor sites specific to the Fc end of anaphylactic antibodies and were thus unable to trigger the DAAD reaction. (b) IgGr ~e~‘sus lgGn antibodies. Numerous sera were fractionated by DEAE cellulose ion exchange. Sera included anti-A/JAX sera (CBA anti-living spleen cells serum, CBA anti-lyophilized spleen cells serum, CBA anti-YAC 222 lymphoma cells serum, C57 BL/Ks anti-living spleen cells serum, C57 BL/Ks anti-YAC 222 serum) and BALB/C anti-EL 4 serum. Fractions were pooled and concentrated as described in Material and Methods and were then tested for their immunoglobulin contents and for their biological activities. When a CBA or a BALB/C serum was fractionated, the first one or the first two fractions contained only IgG2 immunoglobulins, as detected by double diffusion in agarose. With the third frac-

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1‘&(I

FIG. 12. DEAE-fractionated CB,4 anti-A/Jax serum. Notice fraction 1 containing detectable and containing detectIgG, and giving cytotoxicity and fractions Z-6 giving in addition DAAD able IgG,. The same serum fractionated after having been heated at 56°C for 2.5 hr shows that heat labile degranulating activities were present in fractions 2-4.

tion. appeared the IgGl immunoglobulins which thus could not be isolated free of IgGa immunoglobulins. When a C57 BL/Ks serum was fractionated, IgGl appeared as early as the first fraction. In this strain, no pure IgG, could be obtained. With fractions from CBA or BALB/C sera, DAA4D acti\:ity was found in those fractions containing IgGl immunoglobulins while the fractions containing IgG, free of IgGl were unable to induce DAAD. This was found to be true in the several combinations explored from this point of view : CBA serum anti-A/JAX spleen cells tested on A/JAX mast cells (Fig. 12)) BALB/C serum anti-EL 3 cells tested on C57 BL/G mast cells, and C.57 BL/Ks serum anti-YXC 222 tested on A/JAX mast cells. So link was ever found between DAAD and either IgLI- or IgA-containing fractions. Fractionated sera were also examined for their other biological activities, including hemagglutination (achieved by the noncomplement-fixing and the coiiiplement-fixing (achieved by the complement-fixing antibodies j, hemolysis and “‘Cr cytotoxicity antibodies), and in one instance anti-H-2 PCA and immunoallogeneic shock (2) (achieved by the noncomplement-fixing antibodies). The DAAD curves were compared with the curves of these activities in the different fractions. Figure 12 shows the example of a fractionated C57 BL/Ks anti-YAC 222 serum. DAAD was performed on A/JAX mast cells. It can be seen that \vhereas the hemagglutillation activity was widely spread over all the fractions, the DAAD peak was restricted to the first fractions, clearly distinct from the hemolysis peak. 1f:ith the fractionated CBA anti-A/JAX serum the DAAD curve raised from OF in the first fraction up to its maximum intensity in the following fractions (Fig. 12 ) . On the contrar!,. the cytotoxicitv curve, immediately reached its maximum in the first fraction. Comparable risults were obtained in other similar experiments. Although the PCA and the immunoallogeneic shock could not be easily quantified, a good correlation was observed between these activities and D.\AD. From these data, it thus appeared that I>XAI) leas associated with Ig(;l immunoglobulins and complement independent biological activities. Moreover. no correlation was found with the presence of IgG, immunoglobulins and DAAI) was clearly dissociated from the complement-dependent biological activities. In order to obtain direct evidence of the inability of IgG, antibodies to mediate DAAD, we looked for inhibition of the degranulation by the previous incubation of peritoneal cells with IgG2, using the same reasoning as thp one usetl to eliminate

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any participation of 19 S antibodies. C57 BL/6 peritoneal cells were incubated for 20 min at 37°C with either IgGz antibodies isolated from BALB/C anti-EL 4 serum or IgGz immunoglobulins from normal BALB/C serum or RPM1 medium. Cells were washed once and incubated for 5 min at 37°C with a BALB/C antiEL 4 fraction known to contain IgGl and IgG2 antibodies. No degranulation was obtained by either IgGz antibodies or IgGz immunoglobulins, whether it was by preincubation alone or by incubation alone. Preincubation of the cells with IgG, immunoglobulins from normal serum gave no inhibition of the reaction whereas preincubation with anti-EL 4 IgGz antibodies inhibited 46% of the DAAD induced by active antibodies (Fig. 11) . Taken together, these data constituted good evidence of the inability of IgG2 antibodies to elicit DAAD. (c) Possible role of IgE antibodies. Since DAAD could not be induced by complement-fixing IgM and IgGz antibodies, even by a complement-independent mechanism, the best candidates for the active antibodies appeared as the noncomplement-fixing IgGl antibodies. However, it is now well established that two different classes of antibodies are able to mediate anaphylactic phenomena in mice (2.5, 28-30). IgGl antibodies are known to be heat stable and to immediately sensitize mouse mast cells in vitro by means of transient and weak fixation (28). On the contrary, IgE antibodies are destroyed by heating at 56°C and can slowly sensitize mast cells during incubation periods in the range of hours by firm fixation on the receptor sites (28-30). In order to look for a possible role of IgE antibodies in DAAD we compared the DAAD percentages induced by a given serum, before and after it was heated at 56°C for 1 hr at least. When hyperimmune sera were so treated, only a few percent of activity was lost by heating (Figs. 5 and 12). However, when sera were collected 10 days after one injection of allogeneic cells, a great part of their DAAD activity could be lost by heating. CBA mice were immunized by one subcutaneous injection of 5 X lo6 living Sa I cells. Samples of blood were collected on days 3, 5, 10, 14, 18, 26, and 33 by cutting the tail artery, and the serum of each sample was examined for DAAD (performed on A/TAX mast cells) before and after heating at 56°C for 1 hr.

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FIG. 14. Evolution of heat-stable and heat-labile IIAAD immunized by a single injection of SaI i\ cells. The restricted to day 10 after immunization.

15 1

responses in the serum of CI3x mice appearance of heat labile adirity is

Heat labile activity was evaluated by substracting the degranulation percentage induced by heated serum (heatstable DAAD) from the degranulation percentage induced by the same nonheated sertm7. It appeared (Fig. 14) that the heat-labile response was weak and transient, restricted to the lo-day period. The heat-stable response could be clearly dissociated from the heat-labile one : it wts of higher magnitude, could be detected over a long period of time from the 14th day on, and reached its maximum intensity by day 26. Therefore, the heating of the serum did not nonselectively destroy part of the antibodies, but preferentially destroyed those antibodies which were synthesized during the early part of the response. Since the IgE antibody response is known to be transient and early (28) these data firmly suggest the role of TgE transplantation antibodies in early DAAD. Additional evidence that the heat labile antibodies represented a particular population of molecules was provided by the following experiment. A hyperimmune CL\ anti-A/JAX serum was divided into two parts, one of which being heated at 56°C for 2.5 hr. They were then both fractionated by DEAE cellulose ion exchange and similar fractions were identically pooled and concentrated. D4AD was performed with each of the fractions on A/JAX mast cells. and the fractions from the nonheated serum were compared with the corresponding ones from the heated serum. It could be seen (Fig. 12) that the heated serum had lost part of its DAAD activity in a restricted number of fractions which could be assumed to contain the heat-labile antibodies. DISCUSSIOK This work describes and studies a new phenomenon in the field of both transplantation and cellular immunology : mast cells may be triggered in vitro for their physiological function (i.e., the release of mediators through the degranulation process) by the direct action of anaphylactic antibodies directed against tranaplantation antigens borne by the mast cells themselves. This phenomenon was called therefore direct allogeneic anaphylactic degranulation (DAAD) . Iwmzmo!ogical

Specificity

of the DAAD

The necessary and sufficient condition by an immune serum is that this serum directed against transplantation antigens by utilizing different strain combinations

Plzenomwon

for mast cells to be directly degranulatetl should contain (anaphylactic) antibodies borne by the mast cells. This is shown and specific absorptions. The responsible

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allogeneic system appears to correspond to the classical H antigens, sometimes called SD (serologically determined). It does not appear to be due to antigens of the Ia system since the responsible antibodies are rapidly and completely absorbed by the corresponding erythrocytes that are known to bear H-2 (and some non-H-2)) but no Ia specificities. For the same reason, it is not due to anti-lymphocyte receptor antibodies. Neither is it due to immunoglobulin allotypes since neither dilution of immune sera in target strain normal serum nor immunodbsorption on the same polymerized normal sera decreases the degranulating capacity of the specific immune sera. It is not clearly established whether H-2 specificities alone are responsible for the phenomenon or if non-H-2 specificities actually play a role, since no coisogenic strain was utilized in the study. However, the very fact that the degranulation was seen if, and only if, corresponding H-2 targets were available on mast cells in rather strong evidence favoring H-2 as the responsible antigens. Anaphylactic

Nature of the Phenonzenon

This essential feature is established by six types of experimental evidence. 1. The morphological study of the phenomenon both at the optical and at the electron microscopic levels shows a strict identity between DAAD and classical in vitro anaphylactic mast cell degranulation obtained by others (31-34) and by us with, for instance, the ovalbumin-anti-ovalbumin system. 2. Kinetics of the phenomenon were identical for DAAD and ovalbumin anaphylactic degranulation. The significance of the decreased percentage of degranulated mast cells with increasing time of incubation is not clear. However, the following facts favor a “regranulation” process after a physiological (therefore nonlethal) degranulation : No decrease in the total proportion of mast cells to the total population was observed in relation to the incubation time; no sign of cell lysis or even injury was noted even with the electron microscope; mast cells after prolonged incubation with the corresponding serum offered a lighter and rather unhomogeneous staining suggestive of the presence of new granules. 3. The phenomenon is C-independent and it differs strikingly both morphologically and in its kinetics from (?-induced lysis of mast cells by means of C’-fixing antibodies. 4. It is not secondary to an action on peritoneal cells other than mast cells followed by the release of a mediator that would be the cause of the degranulation. 5. It depends on the presence at the surface of the mast cells of available Fc receptors for anaphylactic antibodies. Mast cells from different but H-2 compatible strains (or even from similar strain but bred in different laboratories), used for DAAD and ovalbumin anaphylactic degranulation, showed a marked correlation between the two systems in the percentage of degranulated cells. Since the common point between the two systems is the sensitization process (i.e., the interaction between anaphylactic Fc and Fc receptor) this correlation may be due to the number of available receptors differing in different inbred strains (35). Evidence for this possibility is provided by the fact that preimmunization of the mast cell donor by a foreign antigen resulted in a parallel decrease in the percentage of degranulation in the two systems, presumably due to a competition for Fc receptors (12) between actively induced and passively administered antibodies. 6. Similar again to classical anaphylactic degranulation, DAAD requires the inter-

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a

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b

DEGRAX

153

I’LA’I‘IOS

C

FIG. IS. Three possibilities for allogeneic mast-cell &granulation. ( a). Soluble histocompatibility antigens. (b), Membrane histocompatibility antigens of other cells. (c), Tdernhrane histocompatibility antigens of the mast-cell itself.

vention of intact anaphylactic antibodies (IgGI and possibly, but to a lesser extellt. IgE). IgiU, IgGz, and F(ab’)a are inactive; they even proved to be inhibitory, as expected from a competition for the antigenic sites. It would therefore seem unreasonable to avoid the conclusion that this direct allogenic degranulation phenomenon is an expression of in zlifro anaphylaxis. It is therefore justified to name it direct allogeneic anaphylactic degranulation (Dt\AD).

Classical anaphylactic mast cell degranulation invol\-es two steps (29‘). One involves the interaction of the antibody Fab parts with the (soluble) specific antigen, the other one the interaction of the antibody Fc part with the Fc surface receptors of the mast cell. This mechanism requires therefore the intervention (of a soluble antigen, foreign to the mast cell. In contradistinction DAAD needs no antigen introduction. It may therefore well be actually self-triggering, by virtue of the fact that both the activable Fc receptors and the specific antigen (Fab reactive) are borne by the mast cells (Fig. 15~). The antigenic stimulus might however conceivably be provided by histocompatibility antigens dissolved in the medium (Fig. 15a). This possibility seems to be excluded by the fact that the supernatant medium is unable by itself to trigger the degranulation of mast cells presensitized with the relevant immune serum. according to the procedure of classical anaphylactic mast cell degranulation nith a soluble antigen. be that membrane transplantation antigens of otller Another possibility would cells present in peritoneal suspensions play the triggering role. This interestingpossibility would give an example of anaphylaxis triggered by particulate, cell membrane linked transplantation antigens (Fig. 1%) as already demonstrated in a previous work (36). This cannot be formally excluded as long as one does not have highly purified mast cell populations to work with. However, it is nlade unlikely by the fact that this previous work (36) has shown that a ratio lymphoitl cells/mast cells of 500/l is necessary to obtain 500/C cell degranulation in a system where the antigen is provided by lymphocytes allogeneic to the mast cells, while the usual proportion in the peritoneal suspension cells utilized in DAAD was about 30/l, a proportion that d’K1 not bring an\- significant degree of tlegranulatiotl in those preceding experiments.

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Moreover, the instantaneity of DAAD requires the system that offers the highest opportunity for an immediate and almost simultaneous Fab and Fc fixation with immediate activation, i.e., the self-triggering mechanism, Finally the DAAD phenomenon presents both a practical and a theoretical interest. As a technique, it is specific for histocompatibility antigens and due only to anaphylactic antibodies. It is reproducible and allows a quantitation of transplantation anaphylactic antibodies hardly attainable by PCA reactions (22). Actually, DAAD has already been utilized as a technique for the identification of transplantation anaphylactic (mainly IgG1) antibodies during the rejection and facilitation reactions toward allografts and in placenta eluates (37). Biological Significance

of Transplantation

Anaphylactic

Antibodies

This phenomenon confirms the existence and biological activity of transplantation antibodies of IgGl class, that had only been previously ascertained by PCA reactions (1, 5). It also shows the existence of transplantation anaphylactic antibodies with the characteristics of reagins or IgE. Associated with two other phenomena described in this laboratory, DAAD adds further support to the concept of transplantation anaphylaxis. The first phenomenon was that of cutaneous transplantation anaphylaxis studied as a PCA reaction performed with soluble transplantation antigens in the same way as a usual PCA (1). The other phenomenon was the immunoallogeneic shock (2) in which histocompatibility alloantibodies, when injected iv to the corresponding recipients trigger a general shock with dyspnea, hypothermia, and often death. Whole sera as well as DEAE fractions were correlated in their shocking and PCA activities and the phenomenon is inhibited by anti-histaminic drugs. DAAD may further offer a cellular basis to the understanding of the mechanism of this immunoallogenic shock and provide an in vitro model for transplantation anaphylaxis. The relations between transplantation anaphylaxis and the mechanism and consequences of the homeostatic facilitation reaction (38, 39) remain to be established in view of the parallelism between transplantation IgGl (mouse and guinea pig) antibodies, transplantation anaphylaxis, and enhancement-facilitation (3-6). The answer may lie in lymphocytes possessing histamine receptors (40-42). ACKNOWLEDGMENTS This work has been realized with the help of Mrs. B. Fauchet and S. Righenzi (technicians), Mr. C. Pinet (draftsman), Mr. G. Develay and Y. IssouliC (photographers), and Mrs. A. Vioux (secretary-typist).

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Kinsky, R., Voisin, G. A., Hilgert, I., and Due, H. T., Transplantation 12, 171, 1971. Voisin, J., Kinsky, R., and Voisin, G. A., Transplantation 15, 206, 1973. Voisin, G. A., Kinsky, R., Jansen, F., and Bernard, C., Transplantation 8, 618, 1969. Voisin, G. A., Transplant. Proc. 3, 1229, 1971. Kinsky, R. G., Voisin, G. A., and Due, H. T., Transplantation 13, 452, 1972. Kinsky, R., Voisin, G. A., and Due, H. T., Advan. Exp. Med. Biol. 29, 435, 1973. Vuagnat, P., Neveu, T., and Voisin, G. A., Eur. J. Immunol. 3, 90, 1973. Vuagnat, P., Neveu, T., and Voisin, G. A., J. Exp. Med. 137, 265, 1973.

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9. IO. 11. 12. 13. 14. IS. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

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DEGKAS17LATION

15.5

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