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Allograft rejection and alloimmune memory in the solitary urochordate, Styela plicata
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. ii, pp. 343-351, 1987. 0145-305X87 $3.00 + .00 Printed in the USA. Copyright (c) 1987 Pergamon Press Ltd. All rights reserved.
ALLOGRAFT REJECTION AND ALLOIMMUNE MEMORY IN THE SOLITARY UROCHORDATE, STYELA PLICATA
D.A. Raftos, N.N. Talt and D.A. Briscoe School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2109, Australia.
ABSTRACT The responses of the solitary urochordate, St_£ela ~iicata, to firstand second-set tunic grafts c o n f i r m the existence of a sensitive histocompatibility system. Most first-set allografts were eliminated (median rejection time, RT50, of 38.2 ± 5.9 days) w h e r e a s the majority of autografts remained viable. Allograft rejection exhibited s i g n i f i c a n t m e m o r y with second-set a l l o g r a f t s being lost far more r a p i d l y (RT50 = 22.0 Z 2.6 days) than first-set allografts. This alloimmune memory was shown to survive for up to 50 days after firstset rejection. Furthermore, 3rd party grafting indicated that memory was specific to the p r e s e n s i t i z i n g tissue type. These results are discussed in terms of the evolution of vertebrate immunity.
INTRODUCTION Many i n v e r t e b r a t e s possess histocompatibility systems which are capable of recognizing and eliminating allogeneic tissue (HJldemann, 1979). In the p h y l a A n n e l i d a ( C o o p e r , 1969; H o s t e t t e r a n d C o o p e r , 1973), C n i d a r i a ( H i l d e m a n n et al., 1980), E c h i n o d e r m a t a ( K a r p a n d H i l d e m a n n , 1976) a n d P o r i f e r a (Bigger e t a l . , 1982) t h e s e h i s t o c o m p a t i b i l i t y reactions have a number of fundamental characteristics i n common w i t h t h e v e r t e b r a t e immune r e s p o n s e . I n p a r t i c u l a r they exhibit alloimmune memory, which is specific to the presensitizing tissue type. It has been postulated that such similarity reflects evolutionary homology between invertebrate histocompatibility and the vertebrate immune s y s t e m ( H i l d e m a n n , 1974, 1979; C o o p e r , 1969, 1982). In light of this hypothesis studies of histocompatibility in the phylum Urochordata are of considerable interest. It is widely believed that urochordates are the closest extant relatives of the vertebrates and thus have the greatest chance of yielding identifiable homology (Berrill, 1955). I n many colonial urochordate species allogeneic discrimination takes the form of rapid non-fusion reactions, which prevent the development of incompatible chimeric c o l o n i e s ( M u k a i a n d W a t a n a b e , 1974). N o n - f u s i o n r e a c t i o n s are controlled by a s i n g l e g e n e ( S a b b a d i n , 1982) w h i c h S c o f i e l d e t a l . (1982) s u g g e s t i s a n c e s t r a l
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to t h e vertebrate Major Histocompatibility Complex. Histocompatibility has also been investigated in the s o l i t a r y urochordate, Ciona intestinalis. Reddy et al_., (1975) have found that allografts of tunic tissue from this species underwent chronic rejection which appeared to be mediated by lymphocyte-like cells. A l t h o u g h such studies have identified a sensitive a l l o d i s c r i m i n a t i v e capacity amongst urochordates, none have been amenable to the investigation of memory and specificity. However, these characteristics remain fundamental to comparisons with the vertebrate immune system (Hildemann, 1984). Thus it is the a i m of the current study to investigate both the first- and second-set histocompatibility responses of the solitary urochordate, Styela plicata.
METHODS AND MATERIALS Animals: Specimens of S_= Plicata were collected from two sites on Sydney Harbour (Balmoral Beach and Clifton Gardens) s e p a r a t e d by a p p r o x i m a t e l y 5 km of coastline. Individuals with wet weights ranging from 27 to 35 g were collected from shark netting and cleaned of adherent material. Animals were held in the laboratory for not more than 3 days in 3Oi polyethylene aquaria under constant filtration and aeration. Following grafting, recipients and donors were returned to Balmoral Beach where they were secured in nylon mesh cages to shark netting at the 0.1m tide mark. Graft Implantation: Grafts r e p r e s e n t e d lcm long cores of tunic tissue r e m o v e d from the stolonic margin of individuals by inserting a length of 3mm diameter syringe tubing. They were implanted into holes prepared in the same manner, which had been filled with warm 3O% w/v gelatin; in tunlcate saline (Warr et al., 1977) c o n t a i n i n g 105i.u/] p e n i c i l l i n sulphate and 250 mg/l streptomycin sulphate. After i m p l a n t a t i o n , holes r e m a i n i n g at the end of the graft bed w e r e sealed with petroleum gel. The various grafting regimes employed are depicted in Figure 1. All graft recipients were taken from the Balmoral Beach p o p u l a t i o n w h e r e a s a l l o g r a f t donors were collected from Clifton Gardens. Each donor provided allografts for 4 recipients. First-set Grafting Experiments: 220 hosts in 3 i n d e p e n d e n t trials w e r e used for first-set g r a f t i n g experiments. All first-set hosts received a single autograft implanted 1 cm a n t e r i o r of its original p o s i t i o n on the dorsal stolonic margin. A single allograft was embedded 1 cm anterior of the autograft. The viability of these grafts w a s a s s e s s e d at various times over a 100 day e x p e r i m e n t a l period to provide a sequential analysis of rejection. In each trial, groups of at least 10 hosts/tlme point were scored for rejection and then discarded. Secondary Grafting Experiments: 321 individuals, comprising 2 independent trials, were used for secondary grafting. Secondary grafts were implanted 65 days after the hosts had received first-set grafts. Each host was implanted with a single second-set autograft on the.ventral stolonic margin and either a flrst-set allograft taken from the s a m e donor as used in the flrst-set or a third party graft from an a n i m a l u n r e l a t e d to the f i r s t - s e t donor. S e c o n d a r y graft r e j e c t i o n was scored at a number of time points up to 60 days after implantation with a minimum of 10 hosts/point assessed in each trial.
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All secondary grafting experiments were accompanied by first-set controls to negate the effect of variable water temperature on rejection kinetics• Relationship Between Third Party and First-set Donors: 42 of the donors of 3rd party graft tissue used in secondary grafting experiments received a single allograft from the animal which had acted as the donor of first-set grafts. R e j e c t i o n was assessed in these a n i m a l s 65 days after implantation. This interval is in excess of that required for n o r m a l first-set allograft rejection (see Results). Duration of Allolmmune Memory: S e c o n d - s e t a l l o g r a f t s w e r e i m p l a n t e d into individuals at intervals of either; 55, 67, 75, 85, 95 or 115 days after flrst-set g r a f t i n g (16 individuals per interval). Rejection was assessed 30 days after implantation. This time point provides an accurate discrimination between "rapid" (memory based) and "normal" rejection sequences (see Results).
A n a l y s i s of G r a f t R e j e c t i o n : Graft rejection was a s s e s s e d by r e m o v i n g b l o c k s o f t i s s u e c o n t a i n i n g g r a f t s and s e c t i o n i n g them by hand. R e j e c t i o n was d e f i n e d as t h e t o t a l l o s s of g r a f t t i s s u e in t h e s e s e c t i o n s . In f i r s t - s e t and s e c o n d a r y g r a f t i n g e x p e r i m e n t s d a t a from i n d e p e n d e n t trials were pooled. The resulting total frequencies for rejection at various t i m e s after g r a f t i n g w e r e t r a n s f o r m e d into logit units and subjected to tentative weighted least squares regression analysis to provide estimates of median rejection times (RT50) for incompatible grafts (Flnney, 1971).
2A.._ /
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.
.
.
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Fig.l.
Schematic representation of grafting regimes for autografts (H); f i r s t - s e t a l l o g r a f t s (1A), s e c o n d - s e t a l l o g r a f t s (2A), t h i r d - p a r t y allografts (3A) and a l l o g r a f t s between f i r s t - s e t d o n o r s and t h i r d p a r t y d o n o r s (B)•
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RESULTS First-set Responses: F i r s t - s e t auto- and a l l o - g r a f t s rapidly healed into their graft beds. Points of fusion b e t w e e n the tunic m a t r i x e s of graft and host w e r e evident within 5 days of implantation. Figure 2 reveals that, after fusing with the host, the vast majority of f i r s t - s e t a u t o g r a f t s r e m a i n e d fully viable over the 100 day e x p e r i m e n t a l period. Only 6.1~ of autografts were eventually lost with all such cases being a t t r i b u t a b l e to technical failure of the t r a n s p l a n t a t i o n procedure. In contrast, most f i r s t - s e t a l l o g r a f t s w e r e e v e n t u a l l y e l i m i n a t e d f r o m their hosts. Allograft rejection was first detected 35 days after implantation. This rejection, as d e f i n e d by the total loss of intact tissue, w a s preceded by necrotic activity and discolouration within grafts . The frequency of graft loss increased rapidly to a maximum of approximately 75~ of a11ografts within 50 days. There was no further increase in rejection frequency throughout the remainder of the experimental 100 day period indicating that 25~ of allografts were compatible with their hosts.
100 90 80
/
7c
e
.~ 4o
2c 1(:
I
lO
Days after grafting
Fig. 2. Frequency of rejection (~, n ~ 1 0 g r a f t s per point) for first-set autografts (1H,$) and first-set allografts (1A, • ) a t v a r i o u s t i m e s a f t e r i m p l a n t a t i o n i n t o S__ p l i c a t a .
Second-set Prior
Responses: grafting had no a p p a r e n t
effect
upon t h e
viability
of
autografts.
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The total f r e q u e n c y of s e c o n d - s e t a u t o g r a f t loss was 5.1~, w h i c h did not differ s i g n i f i c a n t l y (p>0.05) from that for first-set autografts. H o w e v e r repeated g r a f t i n g did affect a l l o g r a f t survival. Figure 3 reveals that the onset of second-set a l l o g r a f t r e j e c t i o n was far more rapid than that for first-set allografts. Correspondingly, the RTs0 value for s e c o n d - s e t a l l o g r a f t s was 22.0 ~ 2.6 days as opposed to 38.2 3 5.9 days for first-set allografts.
1:F 80
25-
7C
0
=40 O
2G
i
10
i
I
20 30 Days after
I
40
I
50
I
60
grafting
Fig. 3. F r e q u e n c y of rejection, (~, n ~ 10 grafts per point) for first-set allografts (1A, • ), second-set allografts ( 2 A , • ) and third-party allografts (3A, A ) at various times after implantation into S_~.plicata. (Secondary grafts were implanted 65 days after the first-set).
This enhancement of allograft rejection provided a clear distinction between firstand second-set responses. Logit regressions for firstand second-set rejection did not overlap significantly (p
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when a further 50~ of 3rd party grafts were eliminated. This second phase of r e j e c t i o n o c c u r r e d on a t i m e scale s i m i l a r to that for f i r s t - s e t a l l o g r a f t rejection. Table 1 reveals that 80~ of 3rd party donors in the rapidly rejected group failed to eliminate grafts from individuals used for the firstset g r a f t i n g of their r e s p e c t i v e hosts. Conversely, only 10~ of n o r m a l l y rejected 3rd party animals retained grafts f r o m their r e s p e c t i v e f i r s t - s e t donors. These levels of compatibility between 3rd party and first-set donors differed significantly (p
TABLE 1. Relationship Between "Rapid" (<30 days) Third Party Allograft Rejection by Second-Set Hosts and Compatibility of Third Party Donor with Respective Presensitizing (First-set) Donors. Speed of Third Party Rejection by Second-Set Host
Compatibility of Third-Party Donor with Presensitizing Donor I
1 -
Compatible Incompatible
Rapid 21t 5~
Grafts assessed 65 days after implantation,
Normal 7~ 67~
n=42
Duration of A11oimmune Memory: The f r e q u e n c y of "rapid" (<30 days past grafting) s e c o n d - s e t a l l o g r a f t r e j e c t i o n was d e p e n d e n t upon the interval b e t w e e n first- and second-set transplantation. Figure 4 reveals that the level of "rapid" rejection remained relatively constant at approximately 60~ for grafts implanted within 85 days of the first-set. H o w e v e r longer periods b e t w e e n first- and second- set grafting led to a decline in "rapid" rejection frequencies. An interval of 115 days yielded a level of "rapid" response which was indistinguishable from that for technical autograft loss (11.1~).
DISCUSSION The data presented here confirm that urochordates possess hlstocompatibility s y s t e m s capable of d i s c r i m i n a t i n g b e t w e e n self and a l l o g e n e i c tissue. 75~ of f i r s t - s e t a l l o g r a f t s in S_~.~ l i c a t a were rejected whilst autografts remained viable. However such first-set analysis does little to d e l i n e a t e the nature of a l l o g r a f t recognition. At this level it is impossible to determine whether rejection is derived from; (a) the passive loss of a l l o g r a f t s due to general b i o c h e m i c a l i n c o m p a t i b i l i t i e s or, (b) specific h i s t o c o m p a t i b i l i t y s y s t e m s d i r e c t e d against foreign tissue types. Indeed, the high levels of first-set allograft compatibility (25~) identified
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in this study tend histocompatibility variations t h a n by a integrity, w h i c h Bay
349
to support the former option. Limited poiymorphism in can be more easily explained by random biochemical s p e c i f i c s y s t e m d e v o t e d to the m a i n t e n a n c e of i n d i v i d u a l r e l y upon h i g h l e v e l s of p o l y m o r p h i s m f o r i t s e f f i c i e n c y .
80 70 6O
-i
== " 40 C .o •~ 30 re
20 10
5'5 Time
I
I
65 of implantation(days
Fig. Frequency of rejection (~, grafting for second-set allografts transplantation.
I
85
95 after
I
I
115
1st set grafting)
4.
n ~ 10 g r a f t s per point) implanted at various times
30 d a y s a f t e r after first-set
This conflict h a s b e e n r e s o l v e d by s e c o n d a r y g r a f t a n a l y s i s . Second-set allografts i n S__~.p l i c a t a w e r e r e j e c t e d f a r more r a p i d l y t h a n f i r s t - s e t grafts indicating the presence of an adaptive system rather than one based upon p a s s i v e d e g e n e r a t i o n . F u r t h e r m o r e , t h e p r o l o n g a t i o n o f a l l o - i m m u n e memory f o r periods up t o 50 d a y s a f t e r first-set rejection is indicative of a specifically initiated response as opposed to a general physiological e n h a n c e m e n t s t i m u l a t e d by f i r s t - s e t allografts ( H t l d e m a n n , 1984). B u r n e t (1974) h a s s u g g e s t e d t h a t , i n u r o c h o r d a t e s , allorecognition differs f u n d a m e n t a l l y from t h a t of v e r t e b r a t e s . With t h e s u p p o r t of g e n e t i c e v i d e n c e from the colonial genus, ~KX!~, he p o s t u l a t e s that histocompatibility responses are essentially n o n - s p e c i f i c a n d a r i s e from a f a i l u r e t o d e t e c t s e l f determinants on a l l o g e n e i c c e l l s ( B u r n e r ; 1971, 1 9 7 4 ) . By d e f i n i t i o n this s y s t e m w o u l d be i n c a p a b l e of discriminating between various types of allogenetc t i s s u e and t h u s c o u l d n o t p r o v i d e s p e c i f i c i t y toward particular tissue types in memory responses. However, such specificity has been identified in the current study. Nost 3rd party allografts i n S_~. ~ l i c a t a a r e r e j e c t e d on a t i m e s c a l e s i m i l a r t o t h a t f o r f i r s t - s e t allografts a n d do n o t
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invoke memory responses.. This suggests that alloimmune memory is developed specifically against the presensitlzing tissue and is not cross reactive to other a l l o t y p e s w i t h i n the population. However, given the high levels of allograft compatibility mentioned previously, some 3rd party grafts should bear the s a m e tissue type as that of the p r e s e n s i t i z i n g donor. These grafts would be expected to undergo "rapid" rejection mediated by memory responses developed toward the original donor. Such cross reactivity is exemplified by the small group of 3rd party grafts (25~) in S. plicata w h i c h w e r e rapidly rejected and showed hlgh levels of compatibility to their first-set donors. The identification of memory and specificity in this study reflects an obvious s i m i l a r i t y b e t w e e n u r o c h o r d a t e and v e r t e b r a t e h i s t o c o m p a t i b i l i t y systems. This may be derived from an evolutinary confluence in the functional components of h i s t o c o m p a t i b i l i t y mechanisms. We p o s t u l a t e that, in urochordates, graft rejection involves the r e c o g n i t i o n of foreign a l l o t y p i c cell surface markers which induce histocompatibility reactions. This system may be specifically involved in the prevention of fusion between conspecifics, w h i c h r e p r e s e n t s a frequent threat to individual integrity for sessile aggregative animals such as urochordates (Schmidt, 1982). In vertebrates the role of histocompatibility systems is altered. Since the threat of fusion between con-specifics is absent, the previous functional basis of histocompatibility would be lost. Hence the system may have adapted to a new function involving the e l i m i n a t i o n of infective pathogens (Klein, 1977). This change may have been initiated by the diversification of allotype markers and receptors ultimtely y i e l d i n g the v e r t e b r a t e MHC and a s s o c i a t e d r e c o g n i t i o n molecules. Such d i v e r s i f i c a t i o n w o u l d have a l l o w e d p r e v i o u s l y existing cytotoxicity mechanisms to recognize not only allogeneic tissue but also viral d e t e r m i n a n t s r e s t r i c t e d w i t h self MHC markers. In light of this hypothesis many controversial aspects of the vertebrate MHC may be reconciled w i t h its e v o l u t i o n a r y h i s t o r y rather than its i m m e d i a t e function. However, confirmation of this theory awaits comparison between urochordate cell surface determinants and MHC glycoproteins.
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ALLOGRAFT REJECTION AND ALLOIMMUNE MEMORY IN THE SOLITARY UROCHORDATE, STYELA PLICATA
D.A. Raftos, N.N. Talt and D.A. Briscoe School of Biological Sciences, Macquarie University, North Ryde, N.S.W. 2109, Australia.
ABSTRACT The responses of the solitary urochordate, St_£ela ~iicata, to firstand second-set tunic grafts c o n f i r m the existence of a sensitive histocompatibility system. Most first-set allografts were eliminated (median rejection time, RT50, of 38.2 ± 5.9 days) w h e r e a s the majority of autografts remained viable. Allograft rejection exhibited s i g n i f i c a n t m e m o r y with second-set a l l o g r a f t s being lost far more r a p i d l y (RT50 = 22.0 Z 2.6 days) than first-set allografts. This alloimmune memory was shown to survive for up to 50 days after firstset rejection. Furthermore, 3rd party grafting indicated that memory was specific to the p r e s e n s i t i z i n g tissue type. These results are discussed in terms of the evolution of vertebrate immunity.
INTRODUCTION Many i n v e r t e b r a t e s possess histocompatibility systems which are capable of recognizing and eliminating allogeneic tissue (HJldemann, 1979). In the p h y l a A n n e l i d a ( C o o p e r , 1969; H o s t e t t e r a n d C o o p e r , 1973), C n i d a r i a ( H i l d e m a n n et al., 1980), E c h i n o d e r m a t a ( K a r p a n d H i l d e m a n n , 1976) a n d P o r i f e r a (Bigger e t a l . , 1982) t h e s e h i s t o c o m p a t i b i l i t y reactions have a number of fundamental characteristics i n common w i t h t h e v e r t e b r a t e immune r e s p o n s e . I n p a r t i c u l a r they exhibit alloimmune memory, which is specific to the presensitizing tissue type. It has been postulated that such similarity reflects evolutionary homology between invertebrate histocompatibility and the vertebrate immune s y s t e m ( H i l d e m a n n , 1974, 1979; C o o p e r , 1969, 1982). In light of this hypothesis studies of histocompatibility in the phylum Urochordata are of considerable interest. It is widely believed that urochordates are the closest extant relatives of the vertebrates and thus have the greatest chance of yielding identifiable homology (Berrill, 1955). I n many colonial urochordate species allogeneic discrimination takes the form of rapid non-fusion reactions, which prevent the development of incompatible chimeric c o l o n i e s ( M u k a i a n d W a t a n a b e , 1974). N o n - f u s i o n r e a c t i o n s are controlled by a s i n g l e g e n e ( S a b b a d i n , 1982) w h i c h S c o f i e l d e t a l . (1982) s u g g e s t i s a n c e s t r a l
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to t h e vertebrate Major Histocompatibility Complex. Histocompatibility has also been investigated in the s o l i t a r y urochordate, Ciona intestinalis. Reddy et al_., (1975) have found that allografts of tunic tissue from this species underwent chronic rejection which appeared to be mediated by lymphocyte-like cells. A l t h o u g h such studies have identified a sensitive a l l o d i s c r i m i n a t i v e capacity amongst urochordates, none have been amenable to the investigation of memory and specificity. However, these characteristics remain fundamental to comparisons with the vertebrate immune system (Hildemann, 1984). Thus it is the a i m of the current study to investigate both the first- and second-set histocompatibility responses of the solitary urochordate, Styela plicata.
METHODS AND MATERIALS Animals: Specimens of S_= Plicata were collected from two sites on Sydney Harbour (Balmoral Beach and Clifton Gardens) s e p a r a t e d by a p p r o x i m a t e l y 5 km of coastline. Individuals with wet weights ranging from 27 to 35 g were collected from shark netting and cleaned of adherent material. Animals were held in the laboratory for not more than 3 days in 3Oi polyethylene aquaria under constant filtration and aeration. Following grafting, recipients and donors were returned to Balmoral Beach where they were secured in nylon mesh cages to shark netting at the 0.1m tide mark. Graft Implantation: Grafts r e p r e s e n t e d lcm long cores of tunic tissue r e m o v e d from the stolonic margin of individuals by inserting a length of 3mm diameter syringe tubing. They were implanted into holes prepared in the same manner, which had been filled with warm 3O% w/v gelatin; in tunlcate saline (Warr et al., 1977) c o n t a i n i n g 105i.u/] p e n i c i l l i n sulphate and 250 mg/l streptomycin sulphate. After i m p l a n t a t i o n , holes r e m a i n i n g at the end of the graft bed w e r e sealed with petroleum gel. The various grafting regimes employed are depicted in Figure 1. All graft recipients were taken from the Balmoral Beach p o p u l a t i o n w h e r e a s a l l o g r a f t donors were collected from Clifton Gardens. Each donor provided allografts for 4 recipients. First-set Grafting Experiments: 220 hosts in 3 i n d e p e n d e n t trials w e r e used for first-set g r a f t i n g experiments. All first-set hosts received a single autograft implanted 1 cm a n t e r i o r of its original p o s i t i o n on the dorsal stolonic margin. A single allograft was embedded 1 cm anterior of the autograft. The viability of these grafts w a s a s s e s s e d at various times over a 100 day e x p e r i m e n t a l period to provide a sequential analysis of rejection. In each trial, groups of at least 10 hosts/tlme point were scored for rejection and then discarded. Secondary Grafting Experiments: 321 individuals, comprising 2 independent trials, were used for secondary grafting. Secondary grafts were implanted 65 days after the hosts had received first-set grafts. Each host was implanted with a single second-set autograft on the.ventral stolonic margin and either a flrst-set allograft taken from the s a m e donor as used in the flrst-set or a third party graft from an a n i m a l u n r e l a t e d to the f i r s t - s e t donor. S e c o n d a r y graft r e j e c t i o n was scored at a number of time points up to 60 days after implantation with a minimum of 10 hosts/point assessed in each trial.
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All secondary grafting experiments were accompanied by first-set controls to negate the effect of variable water temperature on rejection kinetics• Relationship Between Third Party and First-set Donors: 42 of the donors of 3rd party graft tissue used in secondary grafting experiments received a single allograft from the animal which had acted as the donor of first-set grafts. R e j e c t i o n was assessed in these a n i m a l s 65 days after implantation. This interval is in excess of that required for n o r m a l first-set allograft rejection (see Results). Duration of Allolmmune Memory: S e c o n d - s e t a l l o g r a f t s w e r e i m p l a n t e d into individuals at intervals of either; 55, 67, 75, 85, 95 or 115 days after flrst-set g r a f t i n g (16 individuals per interval). Rejection was assessed 30 days after implantation. This time point provides an accurate discrimination between "rapid" (memory based) and "normal" rejection sequences (see Results).
A n a l y s i s of G r a f t R e j e c t i o n : Graft rejection was a s s e s s e d by r e m o v i n g b l o c k s o f t i s s u e c o n t a i n i n g g r a f t s and s e c t i o n i n g them by hand. R e j e c t i o n was d e f i n e d as t h e t o t a l l o s s of g r a f t t i s s u e in t h e s e s e c t i o n s . In f i r s t - s e t and s e c o n d a r y g r a f t i n g e x p e r i m e n t s d a t a from i n d e p e n d e n t trials were pooled. The resulting total frequencies for rejection at various t i m e s after g r a f t i n g w e r e t r a n s f o r m e d into logit units and subjected to tentative weighted least squares regression analysis to provide estimates of median rejection times (RT50) for incompatible grafts (Flnney, 1971).
2A.._ /
.I r, /
3A ..... " ~m o
OQ ~ # ~
°
•
j .
.
.
.
Q .
.
.
.
.
.
.
.
O~
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Fig.l.
Schematic representation of grafting regimes for autografts (H); f i r s t - s e t a l l o g r a f t s (1A), s e c o n d - s e t a l l o g r a f t s (2A), t h i r d - p a r t y allografts (3A) and a l l o g r a f t s between f i r s t - s e t d o n o r s and t h i r d p a r t y d o n o r s (B)•
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RESULTS First-set Responses: F i r s t - s e t auto- and a l l o - g r a f t s rapidly healed into their graft beds. Points of fusion b e t w e e n the tunic m a t r i x e s of graft and host w e r e evident within 5 days of implantation. Figure 2 reveals that, after fusing with the host, the vast majority of f i r s t - s e t a u t o g r a f t s r e m a i n e d fully viable over the 100 day e x p e r i m e n t a l period. Only 6.1~ of autografts were eventually lost with all such cases being a t t r i b u t a b l e to technical failure of the t r a n s p l a n t a t i o n procedure. In contrast, most f i r s t - s e t a l l o g r a f t s w e r e e v e n t u a l l y e l i m i n a t e d f r o m their hosts. Allograft rejection was first detected 35 days after implantation. This rejection, as d e f i n e d by the total loss of intact tissue, w a s preceded by necrotic activity and discolouration within grafts . The frequency of graft loss increased rapidly to a maximum of approximately 75~ of a11ografts within 50 days. There was no further increase in rejection frequency throughout the remainder of the experimental 100 day period indicating that 25~ of allografts were compatible with their hosts.
100 90 80
/
7c
e
.~ 4o
2c 1(:
I
lO
Days after grafting
Fig. 2. Frequency of rejection (~, n ~ 1 0 g r a f t s per point) for first-set autografts (1H,$) and first-set allografts (1A, • ) a t v a r i o u s t i m e s a f t e r i m p l a n t a t i o n i n t o S__ p l i c a t a .
Second-set Prior
Responses: grafting had no a p p a r e n t
effect
upon t h e
viability
of
autografts.
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The total f r e q u e n c y of s e c o n d - s e t a u t o g r a f t loss was 5.1~, w h i c h did not differ s i g n i f i c a n t l y (p>0.05) from that for first-set autografts. H o w e v e r repeated g r a f t i n g did affect a l l o g r a f t survival. Figure 3 reveals that the onset of second-set a l l o g r a f t r e j e c t i o n was far more rapid than that for first-set allografts. Correspondingly, the RTs0 value for s e c o n d - s e t a l l o g r a f t s was 22.0 ~ 2.6 days as opposed to 38.2 3 5.9 days for first-set allografts.
1:F 80
25-
7C
0
=40 O
2G
i
10
i
I
20 30 Days after
I
40
I
50
I
60
grafting
Fig. 3. F r e q u e n c y of rejection, (~, n ~ 10 grafts per point) for first-set allografts (1A, • ), second-set allografts ( 2 A , • ) and third-party allografts (3A, A ) at various times after implantation into S_~.plicata. (Secondary grafts were implanted 65 days after the first-set).
This enhancement of allograft rejection provided a clear distinction between firstand second-set responses. Logit regressions for firstand second-set rejection did not overlap significantly (p
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when a further 50~ of 3rd party grafts were eliminated. This second phase of r e j e c t i o n o c c u r r e d on a t i m e scale s i m i l a r to that for f i r s t - s e t a l l o g r a f t rejection. Table 1 reveals that 80~ of 3rd party donors in the rapidly rejected group failed to eliminate grafts from individuals used for the firstset g r a f t i n g of their r e s p e c t i v e hosts. Conversely, only 10~ of n o r m a l l y rejected 3rd party animals retained grafts f r o m their r e s p e c t i v e f i r s t - s e t donors. These levels of compatibility between 3rd party and first-set donors differed significantly (p
TABLE 1. Relationship Between "Rapid" (<30 days) Third Party Allograft Rejection by Second-Set Hosts and Compatibility of Third Party Donor with Respective Presensitizing (First-set) Donors. Speed of Third Party Rejection by Second-Set Host
Compatibility of Third-Party Donor with Presensitizing Donor I
1 -
Compatible Incompatible
Rapid 21t 5~
Grafts assessed 65 days after implantation,
Normal 7~ 67~
n=42
Duration of A11oimmune Memory: The f r e q u e n c y of "rapid" (<30 days past grafting) s e c o n d - s e t a l l o g r a f t r e j e c t i o n was d e p e n d e n t upon the interval b e t w e e n first- and second-set transplantation. Figure 4 reveals that the level of "rapid" rejection remained relatively constant at approximately 60~ for grafts implanted within 85 days of the first-set. H o w e v e r longer periods b e t w e e n first- and second- set grafting led to a decline in "rapid" rejection frequencies. An interval of 115 days yielded a level of "rapid" response which was indistinguishable from that for technical autograft loss (11.1~).
DISCUSSION The data presented here confirm that urochordates possess hlstocompatibility s y s t e m s capable of d i s c r i m i n a t i n g b e t w e e n self and a l l o g e n e i c tissue. 75~ of f i r s t - s e t a l l o g r a f t s in S_~.~ l i c a t a were rejected whilst autografts remained viable. However such first-set analysis does little to d e l i n e a t e the nature of a l l o g r a f t recognition. At this level it is impossible to determine whether rejection is derived from; (a) the passive loss of a l l o g r a f t s due to general b i o c h e m i c a l i n c o m p a t i b i l i t i e s or, (b) specific h i s t o c o m p a t i b i l i t y s y s t e m s d i r e c t e d against foreign tissue types. Indeed, the high levels of first-set allograft compatibility (25~) identified
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ALLOIMMUNE MEMORY IN UROCHORDATES
in this study tend histocompatibility variations t h a n by a integrity, w h i c h Bay
349
to support the former option. Limited poiymorphism in can be more easily explained by random biochemical s p e c i f i c s y s t e m d e v o t e d to the m a i n t e n a n c e of i n d i v i d u a l r e l y upon h i g h l e v e l s of p o l y m o r p h i s m f o r i t s e f f i c i e n c y .
80 70 6O
-i
== " 40 C .o •~ 30 re
20 10
5'5 Time
I
I
65 of implantation(days
Fig. Frequency of rejection (~, grafting for second-set allografts transplantation.
I
85
95 after
I
I
115
1st set grafting)
4.
n ~ 10 g r a f t s per point) implanted at various times
30 d a y s a f t e r after first-set
This conflict h a s b e e n r e s o l v e d by s e c o n d a r y g r a f t a n a l y s i s . Second-set allografts i n S__~.p l i c a t a w e r e r e j e c t e d f a r more r a p i d l y t h a n f i r s t - s e t grafts indicating the presence of an adaptive system rather than one based upon p a s s i v e d e g e n e r a t i o n . F u r t h e r m o r e , t h e p r o l o n g a t i o n o f a l l o - i m m u n e memory f o r periods up t o 50 d a y s a f t e r first-set rejection is indicative of a specifically initiated response as opposed to a general physiological e n h a n c e m e n t s t i m u l a t e d by f i r s t - s e t allografts ( H t l d e m a n n , 1984). B u r n e t (1974) h a s s u g g e s t e d t h a t , i n u r o c h o r d a t e s , allorecognition differs f u n d a m e n t a l l y from t h a t of v e r t e b r a t e s . With t h e s u p p o r t of g e n e t i c e v i d e n c e from the colonial genus, ~KX!~, he p o s t u l a t e s that histocompatibility responses are essentially n o n - s p e c i f i c a n d a r i s e from a f a i l u r e t o d e t e c t s e l f determinants on a l l o g e n e i c c e l l s ( B u r n e r ; 1971, 1 9 7 4 ) . By d e f i n i t i o n this s y s t e m w o u l d be i n c a p a b l e of discriminating between various types of allogenetc t i s s u e and t h u s c o u l d n o t p r o v i d e s p e c i f i c i t y toward particular tissue types in memory responses. However, such specificity has been identified in the current study. Nost 3rd party allografts i n S_~. ~ l i c a t a a r e r e j e c t e d on a t i m e s c a l e s i m i l a r t o t h a t f o r f i r s t - s e t allografts a n d do n o t
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invoke memory responses.. This suggests that alloimmune memory is developed specifically against the presensitlzing tissue and is not cross reactive to other a l l o t y p e s w i t h i n the population. However, given the high levels of allograft compatibility mentioned previously, some 3rd party grafts should bear the s a m e tissue type as that of the p r e s e n s i t i z i n g donor. These grafts would be expected to undergo "rapid" rejection mediated by memory responses developed toward the original donor. Such cross reactivity is exemplified by the small group of 3rd party grafts (25~) in S. plicata w h i c h w e r e rapidly rejected and showed hlgh levels of compatibility to their first-set donors. The identification of memory and specificity in this study reflects an obvious s i m i l a r i t y b e t w e e n u r o c h o r d a t e and v e r t e b r a t e h i s t o c o m p a t i b i l i t y systems. This may be derived from an evolutinary confluence in the functional components of h i s t o c o m p a t i b i l i t y mechanisms. We p o s t u l a t e that, in urochordates, graft rejection involves the r e c o g n i t i o n of foreign a l l o t y p i c cell surface markers which induce histocompatibility reactions. This system may be specifically involved in the prevention of fusion between conspecifics, w h i c h r e p r e s e n t s a frequent threat to individual integrity for sessile aggregative animals such as urochordates (Schmidt, 1982). In vertebrates the role of histocompatibility systems is altered. Since the threat of fusion between con-specifics is absent, the previous functional basis of histocompatibility would be lost. Hence the system may have adapted to a new function involving the e l i m i n a t i o n of infective pathogens (Klein, 1977). This change may have been initiated by the diversification of allotype markers and receptors ultimtely y i e l d i n g the v e r t e b r a t e MHC and a s s o c i a t e d r e c o g n i t i o n molecules. Such d i v e r s i f i c a t i o n w o u l d have a l l o w e d p r e v i o u s l y existing cytotoxicity mechanisms to recognize not only allogeneic tissue but also viral d e t e r m i n a n t s r e s t r i c t e d w i t h self MHC markers. In light of this hypothesis many controversial aspects of the vertebrate MHC may be reconciled w i t h its e v o l u t i o n a r y h i s t o r y rather than its i m m e d i a t e function. However, confirmation of this theory awaits comparison between urochordate cell surface determinants and MHC glycoproteins.
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HILDEMANN, W.H. JOKIEL, P.L., BIGGER, C.H., a n d JOHNSTON, I.S. Allogenelc polymorphism and alloimmune memory in the coral, Monti~o_Kr~ verrucosa. Transplantation 30, 297, 1980.
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Received: Accepted:
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W.H. A question
A. Formal
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a
J.N., WEST, L.A. and W E I S S M A N N , I.L. is c o n t r o l l e d by an M H C - l i k e gene