- Email: [email protected]
Dextran hypersensitivity
132
Immur~otogyT~May,rot. 3, No. 5, 1(.;82
Dextran hypersensitivity A. W. Richter and H. I. H e d i n Department of Biomedical Research, Pharmacia AB, Uppsala, Sweden Dextran, a common plasma substitute, sometimes induces life-threatening hypersensitivity reactions, in this article Wolfgang Richter and Harriet Hedin discuss recent evidence that these Type III anap@lactic reactions, caused by natural antibodies, can be abolished by pretreatment of patients with monovalent h@ten dextran. The search for more efficient and safer plasma volume expanders led to the introduction of partially hydrolyzed and purified dextrans into medicine in 1947 (Ref. 1). Clinical dextrans have a molecular size range comparable to that of plasma proteins and are prepared from native dextrans which are polysaccharides of tool. wt 10-100 millions. The latter are produced ff'om sucrose by enzymes, mainly derived from bacteria of Leuconostoc spp (for review see Ref. 2). The dextran molecules consist of more or less branched chains of glucose units connected by ct 1-6 bonds. The first generation of clinical dextrans had a moderate degree of branching and frequently caused immediate-type allergic reactions involving the skin, respiratory tract and circulation. However, clinical dextrans from European and American manufacturers had varying incidence of reactions and it was found that the conditions of manufacture i.e. the Leuconostoc strain, degree of hydrolysis, and extent of removal of high molecular weight constituents were important in diminishing reactions 3. Since 1955, B 512 d e x t r a n - a linear dextran causing a minimum of allergic reactions and produced by Leuconostoc mesenteroides strain N R R L B 512 - has been in use worldwide. Until 1968 only a few cases of dextran-induced anaphylactoid/anaphylactic reactions (ARs) were reported. However, the more widespread use of clinical dextrans, e.g. for improvement of blood flow ~ and prophylaxis of postoperative pulmonary embolism 4 led to an increase in the number of ARs reported 5. Since some of these reactions are lite-threatening, efforts to eliminate them were desirable. A collaborative study was carried out during 1968-1981 with the aim of elucidating the mechanisms underlying ARs and finding ways to prevent them. This included research on the immunogenicity of dextran, serological studies on normal individuals, and on those people who reacted to dextran, development of animal models of dextran anaphylaxis, hapten inhibition studies, and clinical multicenter trials to determine the incidence of ARs and the efficacy of hapten prophylaxis. This work was done by W. Richter, H. Hedin, K Granath, B. Ingelman and II. Renck (Research Division, Pharmacia AB, Uppsala, Sweden), K. Messmer, J. Ring, C. Mendler and H. Laubenthal (Institute for Surgical Research Univere Elseviex Biomedical PEess 1982
0167-4919/82/(1000-0000/$2,75
sity of Munich, FRG), K.-G. Ljungstr6m (1)anderyds Hospital, Danderyd, Sweden), K. Peter U. Gruber (Kantonspital, Basel, Switzerland, Klinikum Grosshadern, University of Munich, FRG), D. Kraft, O. Scheiner and H. Rumpold (Institute for General and Experimental Pathology, University of Vienna, Austria), M. Devey (Institute of Tropical Medicine, University of London), G. St~lenheim and J. Sj~Sqvist (Institute of Medical Chemistry, University of Uppsala, Sweden), H. J. M~ller-Eberhard (Scripps Clinic and Research Foundation, LaJolla, California) and others.
Immunology of d e x t r a n The antigenicity of dextran was disclosed by its serological reactivity with antibodies to Leuconostoc and pneumococci of types lI and X X (for review see Ref. 6). Later the immunogenicity of native dextrans of varying structure was demonstrated in man 3. The immunogenicity of B 512 dextran has been shown to be dependent on its molecular weight, in man 7 and mouse s. The choice of a non-immunogenic mol. wt range of 40,000-75,000 for clinical dextrans has been influenced by these findings. It must he pointed out here that the amount of clinical dextran given to patients per infusion (30-100 grams) represents an 'overwhelming' dose, which most probably leads to immunological unresponsiveness as it is known to be induced by large doses of purified polysaccharides (cited in Ref. 9). Like other polysaccharides of repetitive structure, dextran induces a thymus-independent humoral IgM class response in mice 1° and is a polyclonal B-cell activator ~. However, dextran can be converted to a thymus-dependent antigen by covalent coupling to protein 9. Such conjugates elicit a strong IgG antidextran response upon immunization. Clinical dextran does not induce mitogenic stimulation of human lymphocytes in vitro ~2,13. The antigenic determinants of different dextrans reflect their simple structure. They are constituted by oligosaccharides containing ctl-6, 1-2, 1-3 and 1-4 linked glucosyl residues in varying proportions 2. The clinically used B 512 dextran contains about 95% a l - 6 linkages 1. Inhibition studies indicated that combining sites of antidextrans are complementary to sequences
I,~,~u,oh;gy T~Jday,wd. d, .M~.5, 1~82
of 2-7 glucose residues, also suggesting the size range of its antigenic determinants t4. Antidextran combining sites may react with terminal or non-terminal determinants% The minimal size of the imrnun<;genic determinants' of B 512 dextran corresponds to two, a n d exceptionally single, d - 6 linked glucosyl residuesL In mice, the i m m u n e responses to the a l - 3 and cd-6 determinants of dextran ~(',lv have been shown to be under separate genetic control. Thus, high responder strains to the former may be low responders to the latter, and vice versa. In man, high and low responders to B 512 dextran can also be distinguished 18 but the genetic background has not been studied. Antidextrans in h u m a n s may belong to the lgG, lgA and lgM classes. Within the IgG class, the IgG 2 subgroup has been found to be p r e d o m i n a n t 19 as an expression of a restricted response. Most people have natural dextran-reactive antibodies (DRA) 3,1u,2°,21,22,23. They may be induced by dextran itself or by cross-reactive microbial polysaccharides..Native dextran is ingested as a regular
t33 c o n t a m i n a n t of sucrose <'. Dextran is also a componenl of dental plaque, may occur in food, and is produced by microorganisms of the gastrointestinal tract (cited in Ref. 29). Recently, a ubiquitous antigen found in the tissues and sera of h u m a n beings was identified as native dextran. In patients with gastrointestinal disease, high levels of 40-260 n g / m l serum were demonstrable 23. It is also of interest that in guinea pigs the permeability of the gastric mucosa to ingested dextran is increased by acetylsalicylic acid in therapeutic doses; the influx of dextran into the circulation was sufficient to induce passive cutaneous anaphylaxis 24. Thus, ample opportunity exists for dextran to induce I)RA in r a n d o m h u m a n populations. O n the other hand, h y p e r i m m u n e animal sera to pneumococci, streptococci, Salmonella, and teichoic acids crossreact with dextran (cited in Ref. 20), so theoretically 1)RA could be induced by cross-immunization. In the case of cross-reactive pneumococcal polysaccharides, results of absorption tests on h u m a n sera did not support such a view 22 (Table I).
Immunology Today, yd. 3, .Nb.5, 1982
134
TABLE I. Micrograms antibody N precipitated per ml of' serum from medical volunteer's receiving subcutaneous injections of 1 mg of various native dextrans. AntiSubject No.
Native dextran Code
21
CSC-N 279
34
Swed. 3079
44
NRRL-B 742
49
NRRL-B 1254
51
NRRL-B 512 F
a CI SII
Bleeding
CI a
SII
SXII
SXX
Antidextran after absorption with pneumococcal polysaccharides
Pre-immunization PostPrePostPrePostPrePostPrePost-
0.3 1.2 0.9 1.0 1.5 1.3 0.7 0.5 1.6 1.2
1.1 1.4 0.5 1.3 0.0 0.0 2.8 2.9 0.0 0.0
0.9 0.7 0.9 0.4 0.6 1.6 0.0 0.4 1.2 0.0
1.0 1.0 0.5 0.6 0.6 1.2 0.0 0.7 0.3 0.5
0.2 5.9 1.1 14.9 3.0 6.5 0.8 4.1 2.7 19.4
C polysaccharide from type I pneumococcus = pneumococcal polysaccharide specific for type 1I
Antidextran before absorption with pneumococcal polysaccharides 1.8 6.0 1.5 16.2 5.1 6.3 0.5 4.1 3.1 17.1
SXII - pneumococcal polysaccharide speeific for type Xll SXX = pneumococcal polysaccharide specific for type XX From Ref. 22
Pathogenetic mechanisms Impurities In adverse drug reactions, the drug itself, its metabolites, or impurities m a y be elicitors. W i t h regard to dextran, the molecule itself was i n c r i m i n a t e d in severe A R s 3.20,30 rather than impurities 31-33 but the role of m a c r o m o l e c u l a r c o n t a m i n a n t s in provoking mild reactions c a n n o t be ruled out since traces of such material are d e m o n s t r a b l e in the majority of clinical d e x t r a n p r e p a r a t i o n s 33. T h e removal of impurities is therefore desirable a n d o n e m a n u f a c t u r e r has recently achieved this 33. Benefits m a y be e x p e c t e d but d e m o n s trating t h e m will be difficult, for technical a n d biometric reasons. Dextran reactive antibodies ( D R A ) T h e t h e o r e t i c a l risks of d e x t r a n i n f u s i o n in individuals with high titers of circulating D R A were p o i n t e d out in 1950 (Ref. 34). A few years later, K a b a t el al) d e m o n s t r a t e d a positive relationship b e t w e e n skin tests to native dextran, high spec!fic precipitin levels, a n d a p r o p e n s i t y to systemic allergic reactions u p o n infusion of non-B 512 a n d B 512 clinical dextrans into 101 volunteers. T h e results indicated that D R A participate in elicitation of such reactions. However, J a c o b s s o n 2~, c o n c l u d e d that D R A have no p a t h o g e n i c importance, since the great majority of volunteers with high D R A levels tolerate B 512 d e x t r a n infusion. Despite the m a n y published case reports of A R s immunological observations are rare a n d give n o c l e a r p i c t u r e of t h e u n d e r l y i n g m e c h a n i s m s . W e therefore studied the levels a n d c o m p o s i t i o n of D R A in sera from m a n y patients who h a d e x p e r i e n c e d A R s of varying severity and from normal individuals. Sera were collected over 12 years from different countries. In a n a p h y l a c t i c shock in h u m a n s , specific IgE-class antibodies are often considered responsible for the
d e v e l o p m e n t of clinical signs, but I g G - m e d i a t e d anaphylaxis has also been d o c u m e n t e d 35. N o D R A of IgEclass could be found before or after A R s by passive c u t a n e o u s a n a p h y l a x i s in monkeys, the radioallergos o r b e n t technique, a n d the modified radioactive redcell-linked a n t i g e n - a n t i g l o b u l i n reaction 2°,3°. Thus, A R s do not conform to the concept of I g E - m e d i a t e d anaphylaxis. Sera of d e x t r a n reactors were also e x a m i n e d for D R A by passive h e m a g g l u t i n a t i o n 2°. In this way, total activity of the Ig-classes G, A, M a n d D is d e t e r m i n e d . It is a p p a r e n t from Fig. 1 that the titer of D R A is positively related to the degree of severity of ARs. All patients with severe reactions have high titers. It should be e m p h a s i z e d that these results have been o b t a i n e d from s e r u m samples fortuitously collected from patients shortly before A R s occurred. If sera d r a w n soon after A R s are examined, erroneously low titers are found, due to neutralization of D R A by the infused dextran. A c o m p a r i s o n b e t w e e n D R A - t i t e r distributions in patients with severe A R s a n d in a r a n d o m p o p u l a t i o n shows that 76% of the d e x t r a n reactors a n d only 4% of n o r m a l s had high titers of 512 or more (Fig. 2). Clearly, the group at risk is a small s u b p o p u l a t i o n of high r e s p o n d e r s to dextran. However, c o m p a r i s o n of the incidence of severe A R s (0.05%) a n d the frequency of high titers of I ) R A in n o r m a l people shows that only a small p r o p o r t i o n of those with high titers develop ARs. This is in accord with J a c o b s s o n ' s results 2~ a n d m a y be explained by several sets of factors TM. Some of these are: Ig-class a n d s u b g r o u p composition, affinity a n d c o n c e n t r a t i o n of D R A , platelet fragility a n d responsiveness of the p a t i e n t ' s vascular system. For further analysis of D R A , the modified red-celllinked antigen-antiglobulin reaction was e m p l o y e d to d e t e r m i n e Ig-class and s u b g r o u p c o m p o s i t i o n 3°.
135
Immunology Today, vol. 3, No. 5, 1982
262144. 1310726553632768163848102409620481024< 512256128'~ 64~
it lit
mm
ill
•
•
mm
mmmm
n•
•
•magi
mn
•m•nmn
mum
mum
emiR
iit
lib
lllll
E -~ ~8
3216842-
ll
~-
0
Ill
•
•
mnmu
Hi •
•••••t
n, •
decrease in tile levels of Clq in severe ARs ~t. Concentrations of the other complement proteins and of serum carboxypeptidase B were normal. These results indicate that the classical pathway is being activated in dextran reactors by immune complexes and accord with the presence of high titers of DRA in patients with severe reactions. Available evidence does not favour the activation of the alternative pathway by infusion of clinical dextran.
Lung histopathology Histological examination of sections of lung tissue from patients with fatal ARs showed occlusion of pulmonary vessels (Ref. 36 and unpublished data). The occluding material consisted of platelets, leukocytes and hyaline globuli with the staining properties of fibrin. These findings are interpreted to suggest that
ii
% 222018Ill
I
16-
II
ill
G r a d e o f severity o f A R
Fig. ! Relationship between serum titers of hemagglutlnating DRA and grade of severity in patients with ARs. Samples were collectedprior to the dextran infusion (n = 84). ] = fatal outcome. (From Hedin and Richter, in press) Dextran-reactive IgG antibodies of high titer were regularly found in patients with severe reactions. IgGtiter strongly correlated with grade of severity; four lethal cases all belonged to the highest titer range of 16,384 to 32,768. This corresponds to 0.4 mg DRA per ml serum. In addition, high IgA-titers were sometimes found, whereas IgM levels were low and IgD antibodies were absent. All four subgroups of IgG were demonstrable but the contribution of IgG 2 was considered the most important. Skin tests with clinical B 512 dextran were positive in 32% of dextran reactors and often correlated with high titers of hemagglutinating DRA 2°. The best predictive diagnostic test for dextran hypersensitivity is determination of dextran reactive IgG. However, such a test can only delineate a risk group of a few per cent in a random population, and cannot predict individual predisposition.
1412108-
,-]20 %J 222018161412-
lO. 8.
6"k-
CompLement Since native dextran can activate the alternative pathway of complement in vitro, it was suggested that ARs might be triggered in a similar way (for review see Ref. 31). Complement profiles were established for dextran reactors to study this possibility. In addition, serum carboxypeptidase B levels were determined, since low levels might lead to high concentrations of a n a p h y l a t o x i n with potential shock p r o d u c i n g activity. The most important finding was a significant
20
16 8
64 32
256 1024 4086 16384 65536 262144 128 512 2848 8192 32768 131072
Titer of hemagglutinating
DRA
Fig. 2 Distribution of serum titers of hemagglutinating DRA in individuals with no history of ARs, n = 1,408 (upper graph) and in patients with ARs of grades III to IV, n - 46 (lower graph). (From Hedin and Richter, in prcss)
136
Immunology Today, ~o{. ,3, ,No..5, 7~)~2
insoluble a n t i g e n - a n t i b o d y complexes have been deposited in the lungs and b o u n d ieukocytes and platelets by a n interaction between Fc-receptors on cells and Fc-pieces of DRA. T h e fibrin deposits appearing as globular microthrombi are thought to be due to activation of the coagulation system via its connection with other plasma enzyme systems, e.g. the complement system. Conclusion All these findings taken together strongly support i m m u n e complex (Type III) anaphylaxis as the mechanism of severe ARs. Mild reactions may be antibody-dependent or not. The sequence of events leading to antibody-dependent reactions may be envisaged as follows. In a small subpnpulation of individuals with high titers of preformed D R A of IgGclass, infusion of clinical dextran generates harmful i m m u n e complexes. They activate complement and aggregation of leukocytes and platelets occurs. The aggregated material is sequestered in the lung and release of vasoactive mediators leads to the anaphylaetic symptoms. Since a n t i g e n - a n t i b o d y complexes are of crucial pathogenic importance, interference with their formation by h a p t e n inhibition was proposed as a means of preventing ARs 2°.
,.... e
S ~*'°~
." d~
•
•
i
•o
7°*j
i
Ioe•ea
eeeeee
/ Z ee eo eo
5....
/
% eo%~
%.z
°eee°*~
""
• •
"-.......-......t
Fig. 3 Illustration of the hapten inhibition principle Monovalent haptendextran, i.e. Dextran 1 (3) binds competitively to combining sites (4) of DRA (I) thus preventing formation of large complexes between polyvalent clinical dextran (2) and DRA (1).
Hapten inhibition A h a p t e n is a substance capable of binding to antibodies with corresponding specificity but which cannot induce antibody tbrmation. Haptens may be polyvalent or monovalent with regard to the n u m b e r of antigenic determinants. A polyvalent h a p t e n may form complexes with antibodies, as a n antigen does, but a monovalent h a p t e n can bind only to individual combining sites of antibodies. Fig. 3 illustrates the i n h i b i t i o n of i m m u n e complex f o r m a t i o n by a monovalent hapten. ln-vilro studies H a p t e n inhibition of precipitation in the d e x t r a n antidextran system has been extensively investigated by K a b a t and co-workers (for review see Ref. 14). Only inhibition studies with B 512 dextran are considered here. Isomalto-oligosaccharides consisting of a l - 6 linked glucosyl residues were found most effective. T h e i r inhibitory power increased strikingly from isomaltose to isomaltopentaose with little further increase for isomalto-hexaose and -heptaose 3v (Fig. 4). These results were confirmed by the technique of 'indirect' single radial immunodiffusion ~*. In this way a dextran fragment of 6 glucose units (mol. wt 990) was found suitable as a monovalent h a p t e n preparation for in-vivo experiments. Animal models H a p t e n inhibition has been shown to prevent lethal cytotropic d e x t r a n a n a p b y l a x i s in g u i n e a pigs. Dextran cross-reactive rabbit antipneumococcal type II sera 3v and rabbit antisera to B 512 dextran 4° were used for sensitization. The following information on the molecular mechanism was obtained, using B 512 dextran fractions of varying size tot challenge. The amount of sensitizing antibody is critical: polyvalent dextran may elicit anaphylaxis in strongly sensitized animals, but not in weakly sensitized ones. Noneliciting dextran fractions always exerted inhibition, when given together with eliciting fractions. The molecular weight of such protective isomalto-oligosaccharides or dextran fi'actions ranged from about 1,000-10,000 (Ref. 40,41). Inhibition of anaphylaxis was achieved by a moderate molar excess of noneliciting/eliciting fractions (1.9-7.6). W i t h maximal sensitization, the smallest dextran fragment with elicitor action in passive cutaneous anaphylaxis proved to be isomaltodecaose (mol. w t 1,600) 41, see Table I1. In contrast, isomaltohexaose (mol. wt 990) never elicited anaphylaxis, showing that it was truly a monovalent hapten. It was also considered important to study immune complex anaphylaxis in animals, since ARs in man conform to this type. For this purpose, a special low molecular dextran product (Dextran 1, i5% solution) c o m p r i s i n g a s e l e c t e d m i x t u r e of i s o m a l t o oligosaccharides of mol. wt (~w) 1,000 was developed (Ingelman el al., unpublished); factors other t h a n inhibitory power were also taken into consideration. A model was established in dogs made hypersensitive to
137
Immunology To&O,, voL 3, No..5, 1982
so
A' ¢/'~.
,o 0
/ ~ ~'/"~ / ,
i
i ~l,nT
"
J
i
/
0.5ml
i i i,,g°
,
.......
i, o ~ '4 ,o1.......
1
i
D4
J
TABLE 11. Monovalency of dextran fragments ot six glucose residues indicated by incapability to elicit passive cutaneous anaphylaxis (PCA) in guinea pigs. gg antidextran Challenging Mo[. wt PCA-lesion per skin site agent (X'iw) area (mm 2)
i ,
0.3 0.3 0.3 0.3 0.3 0.9 0.9 0.9 0.9
1.0ml20D,o
/S /
,1/./
/7/
30
/P"
...-
. . . . . . . ,,,o , , , , MICRflMOLES OLIGOSACCHAHIOE AODED
ISOMALTOHEIAOSE
, METR~'L-a-ISOMALTOSIDE
• ISOMALTOPENTAQSE
• ISOMALTOSE
o tSOMALTOTETRAOSE
* PAAOSE
• METHYL-a-ISDMALTOTRIOSIDE
•
IM-6 IM-10 Dx-fr. Dx-fr. Dx-fr. IM-6 [M-10 I)x-fr. Dx-fr.
990 1,638 3,100 10,500 71,000 990 1,638 3,100 71,000
0 0 121 121 289 0 225 196 361 From Ref. 41
4~ISOMALTOTRIOSYL-D-GLUCOSE
• ISOMALTOTfllOSE
Fig. 4 Inhibition by various mono- and oligo-saccharides of precipitation of antidextran by dextran.
(From Ref. 3) B512 d e x t r a n by i m m u n i z a t i o n with a proteind e x t r a n corljugate 42. U p o n challenge of these dogs u n d e r anaesthesia with 1 ml of ' M a c r o d e x ' (mol. wt 60,000) 59% of animals s h o w e d a n a p h y l a c t i c reactions of varying severity. A m o n g reactors, severe reactions occurred in 37%. T h e y were characterized by decrease in cardiac output a n d of m e a n arterial pressure, w h e r e a s p u l m o n a r y arterial pressure a n d p u l m o n a r y resistance increased. F u r t h e r , the n u m b e r of circulating leukocytes and platelets fell, a n d a decrease in the titer of D R A was noted. T h e a d m i n i s t r a t i o n of D e x t r a n 1 either as preinjection or as a d m i x t u r e to the challenging a n a p h y l a c t o g e n i c dextran, significantly r e d u c e d b o t h the incidence and the severity of anaphylaxis. In the c o m b i n e d h a p t e n - t r e a t e d groups, only 16% of the dogs s h o w e d reactions. A m o n g the reactors, 7% developed severe anaphylaxis4L Similar results were o b t a i n e d in n o n - a n a e s t h e t i z e d dogs by preinjection of D e x t r a n l (Ref. 43). Preventior7 of a&erse reaclions z'r~man
Clinical trials with D e x t r a n 1 (Promit ®, 15°70, Pharmacia AB) were now considered. Theoretically, a given dose should be more effective w h e n a d m i n i s t e r e d as preinjection t h a n w h e n m i x e d with clinical dextran. Suitable doses, r e p r e s e n t i n g a m o l a r excess of 50-200 times, were calculated from the n u m b e r of a n t i b o d y c o m b i n i n g sites of D R A of lgG-class in sera of patients with severe D I A R . Clinical studies were initiated in S w e d e n and G e r m a n y in 1978. In each patient, a preinjection of 10 or 20 ml of D e x t r a n 1 or an a d m i x t u r e of 20 ml of D e x t r a n 1-500 ml of clinical d e x t r a n is given. For each patient, a protocol c o n t a i n i n g information on sex, age, m a i n diagnosis, type of operation p e r f o r m e d etc., is completed. W h e n an adverse reaction is observed or suspected, details about the reaction a n d the p a t i e n t ' s history are obtained. Further, s e r u m samples d r a w n before a n d after the reaction are analyzed for D R A . T o achieve a u n i f o r m classification of reactions, clinical a n d immunological data are c o n t i n u o u s l y
TABLE Ill. Statistical comparison (Fisher's one-sided test) of the incidences of mild and severe ARs in patients receiving 10 or 20 ml of Dextran 1 before infusion of clinical dextran. Status: September, 1981. Incidence of Dose
Mild reactions (Grades I + 11)
Severe reactions (Grades Ill + IV)
10ml 20ml
65/35346=0.184% 92/65048=0.141%
9/35346 = 0.025% 3/65048=0.005%
p = 0.06 p = 0.006 Note: The reported incidencc per patient of severe ARs without hapten prophylaxis is 0.037%-0.080% (Ljungstr6m, Renck el at., to be published). discussed b e t w e e n the physicians responsible arm the study co-ordination centre. At present, trials are going on in six countries a n d more t h a n 100,000 patients have been i~westigated. I n t e r i m results from Scandinavia (Co-ordinators: H. Renck and K.-G. L j u n g s t r 6 m ) and Bavaria (Coordinators: K. M e s s m e r , K. Peter a n d H. L a u b e n t h a [ ) are r e p o r t e d in Ref. 44 a n d in "Fable II1. A dosed e p e n d e n t prophylactic effect of preinjection of D e x t r a n 1 is evident. T h e incidence of severe A R s is decreased by 30-50% with the ]0 ml dose and by 90% with the 20 ml dose in c o m p a r i s o n with controls (I~jungstr/Sm, Renck et aL, to be published). T h e prophylactic effect of a d m i x t u r e of D e x t r a n 1 to clinical d e x t r a n on severe A R s is insignificant. T h e clinical results are s u p p o r t e d by immunological findings ( D R A titer analysis), indicating a t t e n u a t i o n of severe reactions into mild ones. T h e incidence of mild reactions is affected only slightly by h a p t e n prophylaxis. T h e series of investigations described here d e m o n strates the applicability of h a p t e n inhibition to the elimination of adverse drug reactions - a principle that has also been applied to penicillin allergy 4s. References
1 lngelman, B., Gr6nwall, A., Gelin, L. E. and Eliasson, R. (1969) Properties a~d App/icalion oj De.~harT.~, Ahnqvist and Wiksell, Stockholm 2 Jeanes, A., Haynes, W. C., Wilham, C. A. e/a/. (1954)J. Am Chem. Soc. 76, 5041-5052
Immurmlogy Today, vol. 3, No. 5, 1982
138 3 Kabat, E. A., Turino, G. M., Tarrow, A. B. and Maurer, P. H. (t957) J. Clin. lnvest. 36, 1160-1170 4 Bergentz, S. E. (1978) WorldJ. Surg. 2, 19-24 5 Furhoff, A. K. (1977) Acla Anaesthesinl. Seand. 21,161-167 6 Hehre, E. J., Sugg, J. Y. and Neill, J. M. (1952) Ann. N. Y. Acad. &'i. 55,467-470 7 Kabat, E. A. and Bezer, A. E. (1958) Arch. Biochem. Biophys. 78, 306-318 8 Howard, J. G., Vicari, G. and Courtenay, B. (1975) immunology 29, 585-597 9 Richter, W. (1981) in Clin. Immunol. (Steften, C. and Ludwig, H., eds) Vol. 14, pp. 235-246, Elsevier/North Holland Biomedical Press, Amsterdam 10 Chen, J. C. and Leon, M. A. (1976) J. lmmuuol. 116,416-422 II Coutinho, A., M611er, G. and Richter, W. (1974) Scan& J. Immunol. 3, 321-338 12 Ring, J. (1978) Anaphylaktoide Reaklior~en (Diss) Anaeslhe.siologie und Intenriv-medizin Vol. tit, Springer Verlag, Berlin 13 Cunnington, P. G., Blackshaw, R. M. and Sykes, 1. K. (1980) Int. Arch. Allergy Appl. Immunol. 63, 195-200 14 Kabat, E. A. (1961) in Experimental Irumunochemistry (Kabat, E. A. and Mayer, M. M., eds) 2nd edn, pp. 241-267, Thomas, U.S.A. 15 Cisar, J., Kabat, E. A., Dorner, M. E. and Liao, J. (1975) J. Exp. Med. 142, 435-459 16 Blomberg, B. S., Geckeler, W. R. and Weigert, M. (1972) &ience 177, 178-180 17 Fernandez, C., Liebermann, R. and M611er, G. (1979) &and. J. Immunol. 10, 77-80 t8 Hedin, H. and Richter, W. (1982) [rTt. Arch. Allergy Appl. Immunol. in press 19 Yount, W.J., Dorner, M. M., Kunkel, H. G. and Kabat, E. A. (1968) J. Exp. Med. i27,633-646 20 Hedin, H., Richter, W. and Ring, J. (1976) Int. Arch. Allergy Appl. Immunol. 52, 145-159 2l Jacobsson, L. (1959) &udies on Partially ttydrolyzed Dextran with Special Reference to its use fi)r Plasma Vdume Determination in Man (Diss), Uppsala, Sweden
22 Maurer, P. H. (1953) Proc. Soc. t'2xp. Biol. Med. 83,879-884 23 Palosuo, T. and Milgrom, F. (1980) Int. Arch. Alle*igy Appl. Immunol. 65, 153-161 24 Flemstr6m, G., Marsden, N. V. B. and Richter, W. (1976) Int. Arch. Allergy Appl. ImmunoI. 51,627-636 25 B6ttiger, L. E. (1979) Acla Med. &'and. 205,451-456 26 Ring, J. and Messmer, K. (I977) Lancet i, 466-469 27 Bauer, ,~. and Ostling, G (I970) Acta Anaestheszol. Stand. Suppl. 37, 182 185 28 Schaning, B. and Koch, H. (1975) Anaesthesirt 24, 507-516 29 Gruber, U. F., Saldeen, T., Brokop, T., Ekl6f, B. E T AL. (1980) Br. Med. J. 280, 69-77 30 Hedin, H., Kraft, D., Richter, W., Scheiner, O. and Devey, M. (1979) lmmunobiology 156,289 31 Hedin, H. (1977) Dextran-InducedAr~aphylactoidReactim~s in Man. Immunological in vitro and in vivo Studies (Diss), Uppsala, Abstract Ups. Diss. Fac. Sci. No. 432 32 Richter, W. (1973) Immunological in vivo and in vitro Studies q[ the 1)extran Antidexlran System (Diss), Uppsala 33 Richter, W. (1980) Int. Arch. Allergy Appl. lmmanol. 61,457-466 34 Hehre, E.J. and Sugg, J. Y. (1950) Fed. Proc. 9,383 35 Leikola, J., Koistinen, J., Lehtinen, M. and Virolainen, M. (I973) Blood42, 111-119 36 Ziegler, H. K. (1978) Med. Klin. 73, 1089-1090 37 Kabat, E. A. (1957) J. CellComp. Phy.~iol. 50, 79-102 38 Richter, W. (1971)J. Immunology 107, 948 952 39 Hoene, R., Swineford, O. and Quelch, S. (1961)J. Allergy 32, 381-391 40 Richter, W. (1971) Int. Azch. Allezgy Appl. lmmunol. 41,826-844 41 Richter, W. (1972) Inl. Arch. Allergy Appl. fmmunology 43, 252-268 42 Mendler, C. (1980) Hapten-Hemmung der Dexlran-lrdnzierlen Anaphylaklischen Reaktion beim Hund (Diss), University Munich 43 Schwarz, J. A. and Raschak, M. (1978) Allergologie 1,184 44 Hedin, H., Richter, W., Messmer, K., Renck, H., Ljungstr6m, K. G. and Laubenthal, H. (1981) Dev. Biol. &and. 48, 179-189 45 Week, de, A. L. and Girard, J. P. (1972) Int. Arch. AllergyAppl. lmmunol. 42, 798-815
T-cell membrane antigens associated with cytotoxic function Benjamin Bonavida, John Fan and John C. Hiserodt Department of Microbiology and Immunology, U C L A School of Medicine, Los Angeles, CA 90024, U.S.A. In the pas! .few years the.first steps have been taken towards an understanding of the molecular basis o[ %ce//medialed eyloloxicity. Parlicular alten~ion has been paid to the par! played in cyloloxicily by idiotype-bearing antigen-receptors arid other molecules on lhe T-eel! .r1~rface. This arlicle reveiws lhese .rludies and.focuses on lhe information obtained from studies on the inhibition (~f cylotoxic T cells with anlisera or monoclonal antibodies directed against ran/codes on their szlr[ace.
C y t o t o x i c T l y m p h o c y t e s ( C T L s ) are involved in allograft rejection in vivo I a n d t h e lysis of allogeneic 2 a n d virus- or h a p t e n - m o d i f i e d s y n g e n e i c target cells in /3itro3,4. L y s i s takes place in t h r e e discrete stepsS: (i) the b i n d i n g of C T L s to target cells (a process p r o b a b l y involving T-ceil r e c e p t o r a n d target cell a n t i g e n s ) ; (ii) ' p r o g r a m m i n g for lysis' (a Ca2+ a n d t e m p e r a t u r e d e p e n d e n t process) a n d ; (iii) t h e d i s i n t e g r a t i o n of the target cell (a step i n d e p e n d e n t of t h e effector cell). In t h e initial event of C T L - m e d i a t e d target cell lysis, eflector l y m p h o c y t e s interact with a n d b i n d to t a r g e t cells. Elsevier Biomedical Piess 1982 0167 4919/82/0000 0000/$275
D e s p i t e the recent a d v a n c e s in our" u n d e r s t a n d i n g of t h e physiological r e q u i r e m e n t s a n d cellular p r o c e s s e s in C T L - m e d i a t e d cytolysis, the m o l e c u l a r m e c h a n i s m involved in target cell lysis r e m a i n s to be resolved. Several m o d e l s have b e e n p r o p o s e d . T h e s e i n c l u d e (l) i n t e r a c t i o n s involving m e m b r a n e - a s s o c i a t e d m o l e c u l e s of C T L o t h e r t h a n t h e a n t i g e n - b i n d i n g receptor; (2) t h e release from C T I . s of soluble m e d i a t o r s a n d ; (3) t h e role of t h e p e r t u r b a t i o n of t h e t a r g e t cell m e m b r a n e following a n i n t e r a c t i o n b e t w e e n t h e C T L r e c e p t o r a n d target-cell a n t i g e n - w h i c h p e r t u r b s the target cell's m e m b r a n e a n d so results in its lysis. T h e r e is
Immur~otogyT~May,rot. 3, No. 5, 1(.;82
Dextran hypersensitivity A. W. Richter and H. I. H e d i n Department of Biomedical Research, Pharmacia AB, Uppsala, Sweden Dextran, a common plasma substitute, sometimes induces life-threatening hypersensitivity reactions, in this article Wolfgang Richter and Harriet Hedin discuss recent evidence that these Type III anap@lactic reactions, caused by natural antibodies, can be abolished by pretreatment of patients with monovalent h@ten dextran. The search for more efficient and safer plasma volume expanders led to the introduction of partially hydrolyzed and purified dextrans into medicine in 1947 (Ref. 1). Clinical dextrans have a molecular size range comparable to that of plasma proteins and are prepared from native dextrans which are polysaccharides of tool. wt 10-100 millions. The latter are produced ff'om sucrose by enzymes, mainly derived from bacteria of Leuconostoc spp (for review see Ref. 2). The dextran molecules consist of more or less branched chains of glucose units connected by ct 1-6 bonds. The first generation of clinical dextrans had a moderate degree of branching and frequently caused immediate-type allergic reactions involving the skin, respiratory tract and circulation. However, clinical dextrans from European and American manufacturers had varying incidence of reactions and it was found that the conditions of manufacture i.e. the Leuconostoc strain, degree of hydrolysis, and extent of removal of high molecular weight constituents were important in diminishing reactions 3. Since 1955, B 512 d e x t r a n - a linear dextran causing a minimum of allergic reactions and produced by Leuconostoc mesenteroides strain N R R L B 512 - has been in use worldwide. Until 1968 only a few cases of dextran-induced anaphylactoid/anaphylactic reactions (ARs) were reported. However, the more widespread use of clinical dextrans, e.g. for improvement of blood flow ~ and prophylaxis of postoperative pulmonary embolism 4 led to an increase in the number of ARs reported 5. Since some of these reactions are lite-threatening, efforts to eliminate them were desirable. A collaborative study was carried out during 1968-1981 with the aim of elucidating the mechanisms underlying ARs and finding ways to prevent them. This included research on the immunogenicity of dextran, serological studies on normal individuals, and on those people who reacted to dextran, development of animal models of dextran anaphylaxis, hapten inhibition studies, and clinical multicenter trials to determine the incidence of ARs and the efficacy of hapten prophylaxis. This work was done by W. Richter, H. Hedin, K Granath, B. Ingelman and II. Renck (Research Division, Pharmacia AB, Uppsala, Sweden), K. Messmer, J. Ring, C. Mendler and H. Laubenthal (Institute for Surgical Research Univere Elseviex Biomedical PEess 1982
0167-4919/82/(1000-0000/$2,75
sity of Munich, FRG), K.-G. Ljungstr6m (1)anderyds Hospital, Danderyd, Sweden), K. Peter U. Gruber (Kantonspital, Basel, Switzerland, Klinikum Grosshadern, University of Munich, FRG), D. Kraft, O. Scheiner and H. Rumpold (Institute for General and Experimental Pathology, University of Vienna, Austria), M. Devey (Institute of Tropical Medicine, University of London), G. St~lenheim and J. Sj~Sqvist (Institute of Medical Chemistry, University of Uppsala, Sweden), H. J. M~ller-Eberhard (Scripps Clinic and Research Foundation, LaJolla, California) and others.
Immunology of d e x t r a n The antigenicity of dextran was disclosed by its serological reactivity with antibodies to Leuconostoc and pneumococci of types lI and X X (for review see Ref. 6). Later the immunogenicity of native dextrans of varying structure was demonstrated in man 3. The immunogenicity of B 512 dextran has been shown to be dependent on its molecular weight, in man 7 and mouse s. The choice of a non-immunogenic mol. wt range of 40,000-75,000 for clinical dextrans has been influenced by these findings. It must he pointed out here that the amount of clinical dextran given to patients per infusion (30-100 grams) represents an 'overwhelming' dose, which most probably leads to immunological unresponsiveness as it is known to be induced by large doses of purified polysaccharides (cited in Ref. 9). Like other polysaccharides of repetitive structure, dextran induces a thymus-independent humoral IgM class response in mice 1° and is a polyclonal B-cell activator ~. However, dextran can be converted to a thymus-dependent antigen by covalent coupling to protein 9. Such conjugates elicit a strong IgG antidextran response upon immunization. Clinical dextran does not induce mitogenic stimulation of human lymphocytes in vitro ~2,13. The antigenic determinants of different dextrans reflect their simple structure. They are constituted by oligosaccharides containing ctl-6, 1-2, 1-3 and 1-4 linked glucosyl residues in varying proportions 2. The clinically used B 512 dextran contains about 95% a l - 6 linkages 1. Inhibition studies indicated that combining sites of antidextrans are complementary to sequences
I,~,~u,oh;gy T~Jday,wd. d, .M~.5, 1~82
of 2-7 glucose residues, also suggesting the size range of its antigenic determinants t4. Antidextran combining sites may react with terminal or non-terminal determinants% The minimal size of the imrnun<;genic determinants' of B 512 dextran corresponds to two, a n d exceptionally single, d - 6 linked glucosyl residuesL In mice, the i m m u n e responses to the a l - 3 and cd-6 determinants of dextran ~(',lv have been shown to be under separate genetic control. Thus, high responder strains to the former may be low responders to the latter, and vice versa. In man, high and low responders to B 512 dextran can also be distinguished 18 but the genetic background has not been studied. Antidextrans in h u m a n s may belong to the lgG, lgA and lgM classes. Within the IgG class, the IgG 2 subgroup has been found to be p r e d o m i n a n t 19 as an expression of a restricted response. Most people have natural dextran-reactive antibodies (DRA) 3,1u,2°,21,22,23. They may be induced by dextran itself or by cross-reactive microbial polysaccharides..Native dextran is ingested as a regular
t33 c o n t a m i n a n t of sucrose <'. Dextran is also a componenl of dental plaque, may occur in food, and is produced by microorganisms of the gastrointestinal tract (cited in Ref. 29). Recently, a ubiquitous antigen found in the tissues and sera of h u m a n beings was identified as native dextran. In patients with gastrointestinal disease, high levels of 40-260 n g / m l serum were demonstrable 23. It is also of interest that in guinea pigs the permeability of the gastric mucosa to ingested dextran is increased by acetylsalicylic acid in therapeutic doses; the influx of dextran into the circulation was sufficient to induce passive cutaneous anaphylaxis 24. Thus, ample opportunity exists for dextran to induce I)RA in r a n d o m h u m a n populations. O n the other hand, h y p e r i m m u n e animal sera to pneumococci, streptococci, Salmonella, and teichoic acids crossreact with dextran (cited in Ref. 20), so theoretically 1)RA could be induced by cross-immunization. In the case of cross-reactive pneumococcal polysaccharides, results of absorption tests on h u m a n sera did not support such a view 22 (Table I).
Immunology Today, yd. 3, .Nb.5, 1982
134
TABLE I. Micrograms antibody N precipitated per ml of' serum from medical volunteer's receiving subcutaneous injections of 1 mg of various native dextrans. AntiSubject No.
Native dextran Code
21
CSC-N 279
34
Swed. 3079
44
NRRL-B 742
49
NRRL-B 1254
51
NRRL-B 512 F
a CI SII
Bleeding
CI a
SII
SXII
SXX
Antidextran after absorption with pneumococcal polysaccharides
Pre-immunization PostPrePostPrePostPrePostPrePost-
0.3 1.2 0.9 1.0 1.5 1.3 0.7 0.5 1.6 1.2
1.1 1.4 0.5 1.3 0.0 0.0 2.8 2.9 0.0 0.0
0.9 0.7 0.9 0.4 0.6 1.6 0.0 0.4 1.2 0.0
1.0 1.0 0.5 0.6 0.6 1.2 0.0 0.7 0.3 0.5
0.2 5.9 1.1 14.9 3.0 6.5 0.8 4.1 2.7 19.4
C polysaccharide from type I pneumococcus = pneumococcal polysaccharide specific for type 1I
Antidextran before absorption with pneumococcal polysaccharides 1.8 6.0 1.5 16.2 5.1 6.3 0.5 4.1 3.1 17.1
SXII - pneumococcal polysaccharide speeific for type Xll SXX = pneumococcal polysaccharide specific for type XX From Ref. 22
Pathogenetic mechanisms Impurities In adverse drug reactions, the drug itself, its metabolites, or impurities m a y be elicitors. W i t h regard to dextran, the molecule itself was i n c r i m i n a t e d in severe A R s 3.20,30 rather than impurities 31-33 but the role of m a c r o m o l e c u l a r c o n t a m i n a n t s in provoking mild reactions c a n n o t be ruled out since traces of such material are d e m o n s t r a b l e in the majority of clinical d e x t r a n p r e p a r a t i o n s 33. T h e removal of impurities is therefore desirable a n d o n e m a n u f a c t u r e r has recently achieved this 33. Benefits m a y be e x p e c t e d but d e m o n s trating t h e m will be difficult, for technical a n d biometric reasons. Dextran reactive antibodies ( D R A ) T h e t h e o r e t i c a l risks of d e x t r a n i n f u s i o n in individuals with high titers of circulating D R A were p o i n t e d out in 1950 (Ref. 34). A few years later, K a b a t el al) d e m o n s t r a t e d a positive relationship b e t w e e n skin tests to native dextran, high spec!fic precipitin levels, a n d a p r o p e n s i t y to systemic allergic reactions u p o n infusion of non-B 512 a n d B 512 clinical dextrans into 101 volunteers. T h e results indicated that D R A participate in elicitation of such reactions. However, J a c o b s s o n 2~, c o n c l u d e d that D R A have no p a t h o g e n i c importance, since the great majority of volunteers with high D R A levels tolerate B 512 d e x t r a n infusion. Despite the m a n y published case reports of A R s immunological observations are rare a n d give n o c l e a r p i c t u r e of t h e u n d e r l y i n g m e c h a n i s m s . W e therefore studied the levels a n d c o m p o s i t i o n of D R A in sera from m a n y patients who h a d e x p e r i e n c e d A R s of varying severity and from normal individuals. Sera were collected over 12 years from different countries. In a n a p h y l a c t i c shock in h u m a n s , specific IgE-class antibodies are often considered responsible for the
d e v e l o p m e n t of clinical signs, but I g G - m e d i a t e d anaphylaxis has also been d o c u m e n t e d 35. N o D R A of IgEclass could be found before or after A R s by passive c u t a n e o u s a n a p h y l a x i s in monkeys, the radioallergos o r b e n t technique, a n d the modified radioactive redcell-linked a n t i g e n - a n t i g l o b u l i n reaction 2°,3°. Thus, A R s do not conform to the concept of I g E - m e d i a t e d anaphylaxis. Sera of d e x t r a n reactors were also e x a m i n e d for D R A by passive h e m a g g l u t i n a t i o n 2°. In this way, total activity of the Ig-classes G, A, M a n d D is d e t e r m i n e d . It is a p p a r e n t from Fig. 1 that the titer of D R A is positively related to the degree of severity of ARs. All patients with severe reactions have high titers. It should be e m p h a s i z e d that these results have been o b t a i n e d from s e r u m samples fortuitously collected from patients shortly before A R s occurred. If sera d r a w n soon after A R s are examined, erroneously low titers are found, due to neutralization of D R A by the infused dextran. A c o m p a r i s o n b e t w e e n D R A - t i t e r distributions in patients with severe A R s a n d in a r a n d o m p o p u l a t i o n shows that 76% of the d e x t r a n reactors a n d only 4% of n o r m a l s had high titers of 512 or more (Fig. 2). Clearly, the group at risk is a small s u b p o p u l a t i o n of high r e s p o n d e r s to dextran. However, c o m p a r i s o n of the incidence of severe A R s (0.05%) a n d the frequency of high titers of I ) R A in n o r m a l people shows that only a small p r o p o r t i o n of those with high titers develop ARs. This is in accord with J a c o b s s o n ' s results 2~ a n d m a y be explained by several sets of factors TM. Some of these are: Ig-class a n d s u b g r o u p composition, affinity a n d c o n c e n t r a t i o n of D R A , platelet fragility a n d responsiveness of the p a t i e n t ' s vascular system. For further analysis of D R A , the modified red-celllinked antigen-antiglobulin reaction was e m p l o y e d to d e t e r m i n e Ig-class and s u b g r o u p c o m p o s i t i o n 3°.
135
Immunology Today, vol. 3, No. 5, 1982
262144. 1310726553632768163848102409620481024< 512256128'~ 64~
it lit
mm
ill
•
•
mm
mmmm
n•
•
•magi
mn
•m•nmn
mum
mum
emiR
iit
lib
lllll
E -~ ~8
3216842-
ll
~-
0
Ill
•
•
mnmu
Hi •
•••••t
n, •
decrease in tile levels of Clq in severe ARs ~t. Concentrations of the other complement proteins and of serum carboxypeptidase B were normal. These results indicate that the classical pathway is being activated in dextran reactors by immune complexes and accord with the presence of high titers of DRA in patients with severe reactions. Available evidence does not favour the activation of the alternative pathway by infusion of clinical dextran.
Lung histopathology Histological examination of sections of lung tissue from patients with fatal ARs showed occlusion of pulmonary vessels (Ref. 36 and unpublished data). The occluding material consisted of platelets, leukocytes and hyaline globuli with the staining properties of fibrin. These findings are interpreted to suggest that
ii
% 222018Ill
I
16-
II
ill
G r a d e o f severity o f A R
Fig. ! Relationship between serum titers of hemagglutlnating DRA and grade of severity in patients with ARs. Samples were collectedprior to the dextran infusion (n = 84). ] = fatal outcome. (From Hedin and Richter, in press) Dextran-reactive IgG antibodies of high titer were regularly found in patients with severe reactions. IgGtiter strongly correlated with grade of severity; four lethal cases all belonged to the highest titer range of 16,384 to 32,768. This corresponds to 0.4 mg DRA per ml serum. In addition, high IgA-titers were sometimes found, whereas IgM levels were low and IgD antibodies were absent. All four subgroups of IgG were demonstrable but the contribution of IgG 2 was considered the most important. Skin tests with clinical B 512 dextran were positive in 32% of dextran reactors and often correlated with high titers of hemagglutinating DRA 2°. The best predictive diagnostic test for dextran hypersensitivity is determination of dextran reactive IgG. However, such a test can only delineate a risk group of a few per cent in a random population, and cannot predict individual predisposition.
1412108-
,-]20 %J 222018161412-
lO. 8.
6"k-
CompLement Since native dextran can activate the alternative pathway of complement in vitro, it was suggested that ARs might be triggered in a similar way (for review see Ref. 31). Complement profiles were established for dextran reactors to study this possibility. In addition, serum carboxypeptidase B levels were determined, since low levels might lead to high concentrations of a n a p h y l a t o x i n with potential shock p r o d u c i n g activity. The most important finding was a significant
20
16 8
64 32
256 1024 4086 16384 65536 262144 128 512 2848 8192 32768 131072
Titer of hemagglutinating
DRA
Fig. 2 Distribution of serum titers of hemagglutinating DRA in individuals with no history of ARs, n = 1,408 (upper graph) and in patients with ARs of grades III to IV, n - 46 (lower graph). (From Hedin and Richter, in prcss)
136
Immunology Today, ~o{. ,3, ,No..5, 7~)~2
insoluble a n t i g e n - a n t i b o d y complexes have been deposited in the lungs and b o u n d ieukocytes and platelets by a n interaction between Fc-receptors on cells and Fc-pieces of DRA. T h e fibrin deposits appearing as globular microthrombi are thought to be due to activation of the coagulation system via its connection with other plasma enzyme systems, e.g. the complement system. Conclusion All these findings taken together strongly support i m m u n e complex (Type III) anaphylaxis as the mechanism of severe ARs. Mild reactions may be antibody-dependent or not. The sequence of events leading to antibody-dependent reactions may be envisaged as follows. In a small subpnpulation of individuals with high titers of preformed D R A of IgGclass, infusion of clinical dextran generates harmful i m m u n e complexes. They activate complement and aggregation of leukocytes and platelets occurs. The aggregated material is sequestered in the lung and release of vasoactive mediators leads to the anaphylaetic symptoms. Since a n t i g e n - a n t i b o d y complexes are of crucial pathogenic importance, interference with their formation by h a p t e n inhibition was proposed as a means of preventing ARs 2°.
,.... e
S ~*'°~
." d~
•
•
i
•o
7°*j
i
Ioe•ea
eeeeee
/ Z ee eo eo
5....
/
% eo%~
%.z
°eee°*~
""
• •
"-.......-......t
Fig. 3 Illustration of the hapten inhibition principle Monovalent haptendextran, i.e. Dextran 1 (3) binds competitively to combining sites (4) of DRA (I) thus preventing formation of large complexes between polyvalent clinical dextran (2) and DRA (1).
Hapten inhibition A h a p t e n is a substance capable of binding to antibodies with corresponding specificity but which cannot induce antibody tbrmation. Haptens may be polyvalent or monovalent with regard to the n u m b e r of antigenic determinants. A polyvalent h a p t e n may form complexes with antibodies, as a n antigen does, but a monovalent h a p t e n can bind only to individual combining sites of antibodies. Fig. 3 illustrates the i n h i b i t i o n of i m m u n e complex f o r m a t i o n by a monovalent hapten. ln-vilro studies H a p t e n inhibition of precipitation in the d e x t r a n antidextran system has been extensively investigated by K a b a t and co-workers (for review see Ref. 14). Only inhibition studies with B 512 dextran are considered here. Isomalto-oligosaccharides consisting of a l - 6 linked glucosyl residues were found most effective. T h e i r inhibitory power increased strikingly from isomaltose to isomaltopentaose with little further increase for isomalto-hexaose and -heptaose 3v (Fig. 4). These results were confirmed by the technique of 'indirect' single radial immunodiffusion ~*. In this way a dextran fragment of 6 glucose units (mol. wt 990) was found suitable as a monovalent h a p t e n preparation for in-vivo experiments. Animal models H a p t e n inhibition has been shown to prevent lethal cytotropic d e x t r a n a n a p b y l a x i s in g u i n e a pigs. Dextran cross-reactive rabbit antipneumococcal type II sera 3v and rabbit antisera to B 512 dextran 4° were used for sensitization. The following information on the molecular mechanism was obtained, using B 512 dextran fractions of varying size tot challenge. The amount of sensitizing antibody is critical: polyvalent dextran may elicit anaphylaxis in strongly sensitized animals, but not in weakly sensitized ones. Noneliciting dextran fractions always exerted inhibition, when given together with eliciting fractions. The molecular weight of such protective isomalto-oligosaccharides or dextran fi'actions ranged from about 1,000-10,000 (Ref. 40,41). Inhibition of anaphylaxis was achieved by a moderate molar excess of noneliciting/eliciting fractions (1.9-7.6). W i t h maximal sensitization, the smallest dextran fragment with elicitor action in passive cutaneous anaphylaxis proved to be isomaltodecaose (mol. w t 1,600) 41, see Table I1. In contrast, isomaltohexaose (mol. wt 990) never elicited anaphylaxis, showing that it was truly a monovalent hapten. It was also considered important to study immune complex anaphylaxis in animals, since ARs in man conform to this type. For this purpose, a special low molecular dextran product (Dextran 1, i5% solution) c o m p r i s i n g a s e l e c t e d m i x t u r e of i s o m a l t o oligosaccharides of mol. wt (~w) 1,000 was developed (Ingelman el al., unpublished); factors other t h a n inhibitory power were also taken into consideration. A model was established in dogs made hypersensitive to
137
Immunology To&O,, voL 3, No..5, 1982
so
A' ¢/'~.
,o 0
/ ~ ~'/"~ / ,
i
i ~l,nT
"
J
i
/
0.5ml
i i i,,g°
,
.......
i, o ~ '4 ,o1.......
1
i
D4
J
TABLE 11. Monovalency of dextran fragments ot six glucose residues indicated by incapability to elicit passive cutaneous anaphylaxis (PCA) in guinea pigs. gg antidextran Challenging Mo[. wt PCA-lesion per skin site agent (X'iw) area (mm 2)
i ,
0.3 0.3 0.3 0.3 0.3 0.9 0.9 0.9 0.9
1.0ml20D,o
/S /
,1/./
/7/
30
/P"
...-
. . . . . . . ,,,o , , , , MICRflMOLES OLIGOSACCHAHIOE AODED
ISOMALTOHEIAOSE
, METR~'L-a-ISOMALTOSIDE
• ISOMALTOPENTAQSE
• ISOMALTOSE
o tSOMALTOTETRAOSE
* PAAOSE
• METHYL-a-ISDMALTOTRIOSIDE
•
IM-6 IM-10 Dx-fr. Dx-fr. Dx-fr. IM-6 [M-10 I)x-fr. Dx-fr.
990 1,638 3,100 10,500 71,000 990 1,638 3,100 71,000
0 0 121 121 289 0 225 196 361 From Ref. 41
4~ISOMALTOTRIOSYL-D-GLUCOSE
• ISOMALTOTfllOSE
Fig. 4 Inhibition by various mono- and oligo-saccharides of precipitation of antidextran by dextran.
(From Ref. 3) B512 d e x t r a n by i m m u n i z a t i o n with a proteind e x t r a n corljugate 42. U p o n challenge of these dogs u n d e r anaesthesia with 1 ml of ' M a c r o d e x ' (mol. wt 60,000) 59% of animals s h o w e d a n a p h y l a c t i c reactions of varying severity. A m o n g reactors, severe reactions occurred in 37%. T h e y were characterized by decrease in cardiac output a n d of m e a n arterial pressure, w h e r e a s p u l m o n a r y arterial pressure a n d p u l m o n a r y resistance increased. F u r t h e r , the n u m b e r of circulating leukocytes and platelets fell, a n d a decrease in the titer of D R A was noted. T h e a d m i n i s t r a t i o n of D e x t r a n 1 either as preinjection or as a d m i x t u r e to the challenging a n a p h y l a c t o g e n i c dextran, significantly r e d u c e d b o t h the incidence and the severity of anaphylaxis. In the c o m b i n e d h a p t e n - t r e a t e d groups, only 16% of the dogs s h o w e d reactions. A m o n g the reactors, 7% developed severe anaphylaxis4L Similar results were o b t a i n e d in n o n - a n a e s t h e t i z e d dogs by preinjection of D e x t r a n l (Ref. 43). Preventior7 of a&erse reaclions z'r~man
Clinical trials with D e x t r a n 1 (Promit ®, 15°70, Pharmacia AB) were now considered. Theoretically, a given dose should be more effective w h e n a d m i n i s t e r e d as preinjection t h a n w h e n m i x e d with clinical dextran. Suitable doses, r e p r e s e n t i n g a m o l a r excess of 50-200 times, were calculated from the n u m b e r of a n t i b o d y c o m b i n i n g sites of D R A of lgG-class in sera of patients with severe D I A R . Clinical studies were initiated in S w e d e n and G e r m a n y in 1978. In each patient, a preinjection of 10 or 20 ml of D e x t r a n 1 or an a d m i x t u r e of 20 ml of D e x t r a n 1-500 ml of clinical d e x t r a n is given. For each patient, a protocol c o n t a i n i n g information on sex, age, m a i n diagnosis, type of operation p e r f o r m e d etc., is completed. W h e n an adverse reaction is observed or suspected, details about the reaction a n d the p a t i e n t ' s history are obtained. Further, s e r u m samples d r a w n before a n d after the reaction are analyzed for D R A . T o achieve a u n i f o r m classification of reactions, clinical a n d immunological data are c o n t i n u o u s l y
TABLE Ill. Statistical comparison (Fisher's one-sided test) of the incidences of mild and severe ARs in patients receiving 10 or 20 ml of Dextran 1 before infusion of clinical dextran. Status: September, 1981. Incidence of Dose
Mild reactions (Grades I + 11)
Severe reactions (Grades Ill + IV)
10ml 20ml
65/35346=0.184% 92/65048=0.141%
9/35346 = 0.025% 3/65048=0.005%
p = 0.06 p = 0.006 Note: The reported incidencc per patient of severe ARs without hapten prophylaxis is 0.037%-0.080% (Ljungstr6m, Renck el at., to be published). discussed b e t w e e n the physicians responsible arm the study co-ordination centre. At present, trials are going on in six countries a n d more t h a n 100,000 patients have been i~westigated. I n t e r i m results from Scandinavia (Co-ordinators: H. Renck and K.-G. L j u n g s t r 6 m ) and Bavaria (Coordinators: K. M e s s m e r , K. Peter a n d H. L a u b e n t h a [ ) are r e p o r t e d in Ref. 44 a n d in "Fable II1. A dosed e p e n d e n t prophylactic effect of preinjection of D e x t r a n 1 is evident. T h e incidence of severe A R s is decreased by 30-50% with the ]0 ml dose and by 90% with the 20 ml dose in c o m p a r i s o n with controls (I~jungstr/Sm, Renck et aL, to be published). T h e prophylactic effect of a d m i x t u r e of D e x t r a n 1 to clinical d e x t r a n on severe A R s is insignificant. T h e clinical results are s u p p o r t e d by immunological findings ( D R A titer analysis), indicating a t t e n u a t i o n of severe reactions into mild ones. T h e incidence of mild reactions is affected only slightly by h a p t e n prophylaxis. T h e series of investigations described here d e m o n strates the applicability of h a p t e n inhibition to the elimination of adverse drug reactions - a principle that has also been applied to penicillin allergy 4s. References
1 lngelman, B., Gr6nwall, A., Gelin, L. E. and Eliasson, R. (1969) Properties a~d App/icalion oj De.~harT.~, Ahnqvist and Wiksell, Stockholm 2 Jeanes, A., Haynes, W. C., Wilham, C. A. e/a/. (1954)J. Am Chem. Soc. 76, 5041-5052
Immurmlogy Today, vol. 3, No. 5, 1982
138 3 Kabat, E. A., Turino, G. M., Tarrow, A. B. and Maurer, P. H. (t957) J. Clin. lnvest. 36, 1160-1170 4 Bergentz, S. E. (1978) WorldJ. Surg. 2, 19-24 5 Furhoff, A. K. (1977) Acla Anaesthesinl. Seand. 21,161-167 6 Hehre, E. J., Sugg, J. Y. and Neill, J. M. (1952) Ann. N. Y. Acad. &'i. 55,467-470 7 Kabat, E. A. and Bezer, A. E. (1958) Arch. Biochem. Biophys. 78, 306-318 8 Howard, J. G., Vicari, G. and Courtenay, B. (1975) immunology 29, 585-597 9 Richter, W. (1981) in Clin. Immunol. (Steften, C. and Ludwig, H., eds) Vol. 14, pp. 235-246, Elsevier/North Holland Biomedical Press, Amsterdam 10 Chen, J. C. and Leon, M. A. (1976) J. lmmuuol. 116,416-422 II Coutinho, A., M611er, G. and Richter, W. (1974) Scan& J. Immunol. 3, 321-338 12 Ring, J. (1978) Anaphylaktoide Reaklior~en (Diss) Anaeslhe.siologie und Intenriv-medizin Vol. tit, Springer Verlag, Berlin 13 Cunnington, P. G., Blackshaw, R. M. and Sykes, 1. K. (1980) Int. Arch. Allergy Appl. Immunol. 63, 195-200 14 Kabat, E. A. (1961) in Experimental Irumunochemistry (Kabat, E. A. and Mayer, M. M., eds) 2nd edn, pp. 241-267, Thomas, U.S.A. 15 Cisar, J., Kabat, E. A., Dorner, M. E. and Liao, J. (1975) J. Exp. Med. 142, 435-459 16 Blomberg, B. S., Geckeler, W. R. and Weigert, M. (1972) &ience 177, 178-180 17 Fernandez, C., Liebermann, R. and M611er, G. (1979) &and. J. Immunol. 10, 77-80 t8 Hedin, H. and Richter, W. (1982) [rTt. Arch. Allergy Appl. Immunol. in press 19 Yount, W.J., Dorner, M. M., Kunkel, H. G. and Kabat, E. A. (1968) J. Exp. Med. i27,633-646 20 Hedin, H., Richter, W. and Ring, J. (1976) Int. Arch. Allergy Appl. Immunol. 52, 145-159 2l Jacobsson, L. (1959) &udies on Partially ttydrolyzed Dextran with Special Reference to its use fi)r Plasma Vdume Determination in Man (Diss), Uppsala, Sweden
22 Maurer, P. H. (1953) Proc. Soc. t'2xp. Biol. Med. 83,879-884 23 Palosuo, T. and Milgrom, F. (1980) Int. Arch. Alle*igy Appl. Immunol. 65, 153-161 24 Flemstr6m, G., Marsden, N. V. B. and Richter, W. (1976) Int. Arch. Allergy Appl. ImmunoI. 51,627-636 25 B6ttiger, L. E. (1979) Acla Med. &'and. 205,451-456 26 Ring, J. and Messmer, K. (I977) Lancet i, 466-469 27 Bauer, ,~. and Ostling, G (I970) Acta Anaestheszol. Stand. Suppl. 37, 182 185 28 Schaning, B. and Koch, H. (1975) Anaesthesirt 24, 507-516 29 Gruber, U. F., Saldeen, T., Brokop, T., Ekl6f, B. E T AL. (1980) Br. Med. J. 280, 69-77 30 Hedin, H., Kraft, D., Richter, W., Scheiner, O. and Devey, M. (1979) lmmunobiology 156,289 31 Hedin, H. (1977) Dextran-InducedAr~aphylactoidReactim~s in Man. Immunological in vitro and in vivo Studies (Diss), Uppsala, Abstract Ups. Diss. Fac. Sci. No. 432 32 Richter, W. (1973) Immunological in vivo and in vitro Studies q[ the 1)extran Antidexlran System (Diss), Uppsala 33 Richter, W. (1980) Int. Arch. Allergy Appl. lmmanol. 61,457-466 34 Hehre, E.J. and Sugg, J. Y. (1950) Fed. Proc. 9,383 35 Leikola, J., Koistinen, J., Lehtinen, M. and Virolainen, M. (I973) Blood42, 111-119 36 Ziegler, H. K. (1978) Med. Klin. 73, 1089-1090 37 Kabat, E. A. (1957) J. CellComp. Phy.~iol. 50, 79-102 38 Richter, W. (1971)J. Immunology 107, 948 952 39 Hoene, R., Swineford, O. and Quelch, S. (1961)J. Allergy 32, 381-391 40 Richter, W. (1971) Int. Azch. Allezgy Appl. lmmunol. 41,826-844 41 Richter, W. (1972) Inl. Arch. Allergy Appl. fmmunology 43, 252-268 42 Mendler, C. (1980) Hapten-Hemmung der Dexlran-lrdnzierlen Anaphylaklischen Reaktion beim Hund (Diss), University Munich 43 Schwarz, J. A. and Raschak, M. (1978) Allergologie 1,184 44 Hedin, H., Richter, W., Messmer, K., Renck, H., Ljungstr6m, K. G. and Laubenthal, H. (1981) Dev. Biol. &and. 48, 179-189 45 Week, de, A. L. and Girard, J. P. (1972) Int. Arch. AllergyAppl. lmmunol. 42, 798-815
T-cell membrane antigens associated with cytotoxic function Benjamin Bonavida, John Fan and John C. Hiserodt Department of Microbiology and Immunology, U C L A School of Medicine, Los Angeles, CA 90024, U.S.A. In the pas! .few years the.first steps have been taken towards an understanding of the molecular basis o[ %ce//medialed eyloloxicity. Parlicular alten~ion has been paid to the par! played in cyloloxicily by idiotype-bearing antigen-receptors arid other molecules on lhe T-eel! .r1~rface. This arlicle reveiws lhese .rludies and.focuses on lhe information obtained from studies on the inhibition (~f cylotoxic T cells with anlisera or monoclonal antibodies directed against ran/codes on their szlr[ace.
C y t o t o x i c T l y m p h o c y t e s ( C T L s ) are involved in allograft rejection in vivo I a n d t h e lysis of allogeneic 2 a n d virus- or h a p t e n - m o d i f i e d s y n g e n e i c target cells in /3itro3,4. L y s i s takes place in t h r e e discrete stepsS: (i) the b i n d i n g of C T L s to target cells (a process p r o b a b l y involving T-ceil r e c e p t o r a n d target cell a n t i g e n s ) ; (ii) ' p r o g r a m m i n g for lysis' (a Ca2+ a n d t e m p e r a t u r e d e p e n d e n t process) a n d ; (iii) t h e d i s i n t e g r a t i o n of the target cell (a step i n d e p e n d e n t of t h e effector cell). In t h e initial event of C T L - m e d i a t e d target cell lysis, eflector l y m p h o c y t e s interact with a n d b i n d to t a r g e t cells. Elsevier Biomedical Piess 1982 0167 4919/82/0000 0000/$275
D e s p i t e the recent a d v a n c e s in our" u n d e r s t a n d i n g of t h e physiological r e q u i r e m e n t s a n d cellular p r o c e s s e s in C T L - m e d i a t e d cytolysis, the m o l e c u l a r m e c h a n i s m involved in target cell lysis r e m a i n s to be resolved. Several m o d e l s have b e e n p r o p o s e d . T h e s e i n c l u d e (l) i n t e r a c t i o n s involving m e m b r a n e - a s s o c i a t e d m o l e c u l e s of C T L o t h e r t h a n t h e a n t i g e n - b i n d i n g receptor; (2) t h e release from C T I . s of soluble m e d i a t o r s a n d ; (3) t h e role of t h e p e r t u r b a t i o n of t h e t a r g e t cell m e m b r a n e following a n i n t e r a c t i o n b e t w e e n t h e C T L r e c e p t o r a n d target-cell a n t i g e n - w h i c h p e r t u r b s the target cell's m e m b r a n e a n d so results in its lysis. T h e r e is