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359.
Immunology, 1965, 9,
Complement in Skin Grafting in Mice
LINDA D. CAREN AND L. T. ROSENBERG
Department ofMedical Microbiology, Stanford University,
Stanford, California
(Received 25th January 1965)
Summary. The role of haemolytic complement activity in the rejection of skin
homografts in mice was studied. Two co-isogenic lines of mice were used, which
are only known to differ in one respect. One line (BIO.D2 new) possesses haemolytic
complement activity, while the other line (BIO.D2 old) lacks such activity. Both
lines of mice rejected CF-l and C57B1/6J skin grafts at essentially the same rate.
It was therefore concluded that the presence or absence ofhaemolytic complement
activity does not affect the rejection of skin homografts in mice. To determine if
BIO.D2 new and BlO.D2 old line are histocompatible, skin grafts were exchanged
between members ofboth lines. Although BIO.D2 new line mice accepted BlO.D2
old line skin grafts without exception, the reverse situation did not always obtain.
The possible role of Hc, as well as alternative reasons for the observed histo-
incompatibility, are discussed.
INTRODUCTION
The role of 'complement' in immunologic reactions is known with considerable
uncertainty. Experiments designed to test the importance of complement are beset with
the hazards attendant upon study ofcomplex reactions ofill-defined largely uncharacter-
ized proteins in biologic systems. The existence of lines of mice that differ with respect
to haemolytic complement activity has recently been reported (Rosenberg and Tachibana,
1962). Ofparticular interest are the strains C57Bl/10-H-2d (Hc'/Hc') (BIO.D2 new line)
which possess haemolytic complement activity, and C57BI/10-H-2d (Hc0/Hc') (BIO.D2
old line) mice which lack such activity, (Herzenberg, Tachibana, Herzenberg and
Rosenberg, 1963). It has been shown that this difference is due to two alleles, Hc' and
Hco, acting at a single locus (Herzenberg et al., 1963; Tachibana, Ulrich and Rosenberg,
1963). The dominant allele is Hc', which, when present, results in the synthesis of a
P-migrating serum protein. This serum protein, designated hc', functions as a component
of complement, while Hc' produces no detectable product (Tachibana and Rosenberg,
1964). This serum protein system has recently been reported under another name
(Cinader and Dubiski, 1963, 1964).
In the present study the rates of rejection of foreign skin grafts placed on members of
each line are compared. Also reported are experiments to determine if BlO.D2 new and
BlO.D2 old line are histocompatible, i.e. if the gene Hc' could function as a histocom-
patibility gene, or ifthe two strains differed from each other at any loci other than the Hc
locus.
359
360 D. Caren and L. T. Rosenberg
Linda
MATERIALS AND METHODS
Animals used. The following inbred and random bred strains ofmice were obtained from
stocks maintained in this department: two sub-lines of C57Bl/l0-H-2d (BlO.D2),
BlO.D2/Sn new line (G12F3G6F2G4F12) and B10.D2/Sn old line (G12F33); C57B1/6J;
and CF-1 (non-inbred). All donors and recipients were matched as to sex.
Grafting procedure. Sterile 4 per cent chloral hydrate (0-1 ml/g body weight), injected
i.p., was used as anaesthetic whenever necessary. Since each donor contributed many
grafts, the skin was removed from the trunk of the body in one entire piece. Thereafter
the grafting techniques were essentially as described by Billingham et al., (Billingham,
1954; Billingham and Medawar, 1951; Billingham and Silvers, 1961). When four grafts
were placed on a recipient, two fitted grafts in one bed were placed on each side. The
grafts were covered with a sterile gauze patch, a 35 x 75 mm strip of cohesive latex
bandage (Bryant and Bernard, 1955) (The Sealtex Company, Chicago, Illinois), and two
strips ofsimilar size surgical adhesive tape. The cephalic halfofthe bandage was covered
with the same size strip ofplaster ofParis impregnated gauze. set reactions were pre-
Spleen cell suspensions to be used in attempts to elicit second i.p. (1/2 spleen
paredinHanks'sBalanced Salt Solutionimmediately prior to beinginjected new line
equivalent/injection). The serum isoantigen, hc', which distinguishes Bi0.D2
from B1O.D2 old line, was prepared as described previously (Tachibana and Rosenberg,
1964). Animals were shown to be effectively immunized against the isoantigen by the
demonstration of precipitating serum antibody to hc' by double diffusion in agar
(Tachibana and Rosenberg, 1964). and examinedonthetenthpost-operative
Evaluation ofgrafts. The grafts were unwrapped described by Billingham (1954).
day, and evaluated visually according to the standards
The grafts were checked daily until they were 1 month old, then at weekly intervals.
RESULTS
EXPERIMENT I
Four skin grafts (CF-1, C57B1/6J, B1O.D2 new, BlO.D2 old) were placed on each
BlO.D2 new and BlO.D2 old line recipient. In order to control position effects, all com-
binations ofpositions and contiguity were equally represented. The median survival time
(MST), the day by which 50 per cent of the grafts were rejected, was determined when
applicable (Billingham, Brent, Medawar and Sparrow, 1954). In addition, thirty-seven
mice were subjected to autografts. There were no graft failures among all thirty-seven
mice, thirty-three ofwhich lived at least 17 weeks. in the first three columns ofTable 1.
The fate ofthe transplanted CF-1 grafts is shown of B10.D2 new and BlO.D2 old line
The second and third columns show the proportionthe shown in the
recipients which rejected their CF-1 skin grafts on post-operative day ofeach line
first column. The MST values of CF-1 skin grafts transplanted to recipients
are identical, 11 days in each case. All twenty-one B10.D2 old line recipients and all
thirty-seven BlO.D2 new line recipients rejected their CF-1 skin grafts by the twenty-third
post-operative day. grafts transplanted to BlO.D2 new and B1O.D2 old line
The fate of the C57B1/6J skin the fourth and fifth columns of Table 1. The MST
recipients is similarly shown in B1O.D2 new and B1O.D2 old mice do not
values of C57B1/6J skin grafts transplanted to
in Mice 361
Skin Grafting
TABLE 1
THE REJECTION OF CF-1 AND C57B1/6J SKIN GRAFTS BY BMW.D2 NEW AND B10.D2
OLD LINE RECIPIENTS
Post- CF-I grafts placed on C57B1/6J grafts placed on
operative BMM.D2 BlM.D2 B10.D2 BMM.D2
day new line old line new line old line
recipients recipients recipients recipients
10 32/32 21/21 16/16 34/34
11 2/32 5/21 10/16 11/34
12 0/32 5/21 7/16 8/34
13 0/32 3/21 5/16 6/34
14 0/32 0/21 4/16 5/34
15-23 0/32 0/21 0/16 0/34
Fractions indicate proportion ofgrafts surviving to day indicated.
differ significantly, being 12 and 11 days respectively. All thirty-four B1O.D2 old line
recipients and all sixteen B1O.D2 new line recipients rejected their C57B1/6J grafts by the
twentieth post-operative day. line mice their and skin
Since BlO.D2 old and BlO.D2 new rejected CF-1 C57B1/6J
grafts at essentially the same rate, it was concluded that the presence or absence ofhaemo-
lytic complement activity does not detectably affect the rejection of homologous skin
grafts in mice.
EXPERIMENT II
The purpose of the second experiment was to determine if BlO.D2 new and BlO.D2
old line mice were histocompatible. Table 2 summarizes the behaviour of BlO.D2 new
and BlO.D2 old line skin grafts when transplanted to BlO.D2 new and BlO.D2 old line
recipients. The BlO.D2 new line mice freely accepted all the BlO.D2 new and BlO.D2
oldline skin grafts applied. Although the BIO.D2 old line recipients accepted all the BIO.D2
old line skin grafts, three BlO.D2 new line skin grafts were rejected during the seventeenth
post-operative week. There were, however, other BlO.D2 new line skin grafts which were
not rejected by BlO.D2 old line recipients, including several which have persisted on
surviving mice for up to 6 months. Since there were no rejections seen when BlO.D2 old
line skin grafts were placed on BlO.D2 new line recipients, it seemed possible that the Hc'
gene might be functioning as a histocompatibility gene. Therefore, three additional
TABLE 2
HOMOGRAFTS AND ISOGRAFTS IN B1O.D2 NEW AND B1O.D2 OLD LINE
RECIPIENTS
Type of Type ofskin Fraction of
recipient graft applied successful grafts
BMM.D2 new B10.D2 new 11/11
B10.D2 new B1O.D2 old 13/13
B10.D2 old B10.D2 old 11/11
B10.D2 old B10.D2 new 7/10*
* The three rejections observed in this group occurred in the fourth
month after grafting.
D. Caren and L. T. Rosenberg
362 Linda
experiments were designed to clarify the importance of the Hc' gene in the three rejec-
tions of BlO.D2 new line grafts by BIO.D2 old line recipients. The purpose of all three
experiments was to determine if second set (accelerated) rejections of BIO.D2 new line
grafts by BIO.D2 old line recipients could be elicited. BlO.D2 new line skin graft to each
The first experiment consisted of applying another new line skin
ofthe three BIO.D2 old line recipients which had rejected their first BIO.D2 weakens
grafts. It has been shown that active immunity elicited by a single skin homograft
perceptibly but is still strong 4 months after grafting (Medawar, Billingham and Sparrow,
1953). The second grafts have not been rejected during the period of observation. This is
shown in Table 3.
TABLE 3
FAILURE TO DEMONSTRATE ACCELERATED REJECTION OF BlO.D2 NEW LINE SKIN GRAFTS
BY BlO.D2 OLD LINE RECIPIENTS
Method of presensitization No. of No. of mice Duration of
ofBlO.D2 old line recipients recipients which rejected surviving grafts
their grafts (weeks)
Rejection ofprevious BlO.D2 3 0 >5
new line skin graft
Injection of BlO.D2 new line 8 0 >13
spleen cell suspension i.p.,
1-3 weeks prior to
grafting*
Immunization with BlO.D2 4 0 >11
new line serumt
*Used 1/2 spleen equivalent per recipient. See Materials and Methods. See
tRecipients were shown to be producing anti-hc' antibody at time ofgrafting.
Materials and Methods.
In the second experiment, eight BIO.D2 old line mice, which had been injected with
BlO.D2 new line spleen cells 1-3 weeks previously, were grafted with BlO.D2 new line
skin grafts. Spleen cells have been shown to function as effective immunizers when the H-2
locus or H-Y locus is involved, but less effectively when the H-3 locus is involved (Berrian
and McKhann, 1960; Sachs and Heller, 1958). As is shown in Table 3, all eight B1O.D2
old line mice have retained their grafts. No rejections have occurred during the 13 weeks of
observation. four B1O.D2 old line mice immunized with BlO.D2 new line
In the third experiment, of new line skin They were
serum euglobulin were used as recipients B10.D2 grafts.
given a booster shot 5-7 days prior to grafting, and were shown to be producing pre-
cipitating anti-hc' antibody on the day grafted. As is shown in Table 3, there have been
no rejections during the period ofobservation.
DISCUSSION
The first question which these experiments were designed to answer was: does the
presence or absence of haemolytic complement activity play a role in the rejection of
homologous skin grafts? Since B1O.D2 new and B1O.D2 old line mice rejected CF-1 and
C57B1/6J skin grafts with essentially equal alacrity, it may be concluded that haemolytic
complement activity is not necessary for skin graft rejection in mice.
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