/ . Embryol. exp. Morph., Vol. 16, 3, pp. 519-530, December 1966
Printed in Great Britain
519
The release of soluble
H-2 alloantigens during disaggregation of mouse
embryo tissue by a chelating agent
By MICHAEL EDIDIN 1
From the Section of Cell Biology, The Weizmann Institute of Science,
Rehovoth, and Department of Bacteriology and Immunology, Harvard
Medical School, Boston, Massachusetts
Treatment with chelating agents binding divalent cations has been found to
effect the dissociation of a variety of tissues of both embryo and adult animals
(reviewed in Steinberg, 1958). In the course of dissociation it appears that
materials are released from cell surfaces which play a part in their specific
adhesion, and which may be shown experimentally to promote selectively the
re-aggregation of dissociated cells (Humphreys, 1963; Moscona, 1963). The
extracted materials appear to be glycoprotein complexes (Humphreys, 1965),
made up of fairly small subunits, estimated to be of 13000-20000 molecular
weight (Margoliash et al. 1965). Units of about the same size appear to be the
antigenic sites involved in the blocking of sponge cell aggregation by rabbit
anti-sponge serum, specific for a given sponge species (MacLennan, 1963). I
shall here present evidence that materials of similar molecular weight bearing
immunological specificities of the H-2 alloantigen system are released from the
tissues of certain mouse embryos during the course of their dissociation by the
chelating agent Versene (ethylenediaminetetraacetic acid).
Antigens of the H-2 system function as transplantation antigens determining
the fate of allogeneic homografts, as immunogens stimulating the production of
circulating antibody, and as antigens of erythrocytes and leucocytes reacting
with antibody (Stimpfling & Snell, 1961; Snell, Hoecker, Amos & Stimpfling,
1964). The antigens extracted by Versene are most conveniently detected by
their ability to inhibit the cytotoxic action of antibody produced by tissue
allografts made between mouse strains differing at the H-2 locus. The potency
of the antigens extracted is thus expressed in terms of their inhibition of a
standard cytotoxic alloantiserum. This technique allows determination of very
small amounts of material and it avoids the problems of immunization and
quantitative scoring of skin graft survival, which are encountered when H-2
1
Author's address: Department of Bacteriology and Immunology, Harvard Medical School,
25 Shattuck Street, Boston, Massachusetts 02115, U.S.A.
520
M. EDIDIN
antigens are tested in terms of their ability to sensitize animals to subsequent skin
grafts. All the results presented here concern H-2 antigens rather than H-2
immunogens. These antigens, which might be considered H-2 haptens, are of
interest both because of the manner of their extraction, and because they are
the smallest forms of H-2 antigens yet reported and characterized (Herzenberg &
Herzenberg, 1962; Lejeune-Ledant, 1962; Brent, Medawar & Ruskiewicz, 1962;
Davies, 1962; Kandutsch & Stimpfling, 1963; Manson, Foschi, Dougherty &
Palm, 1964; Haughton, 1965; Heberman & Stetson, 1965; Kahan, 1965;
Nathenson & Davies, 1966).
MATERIALS AND METHODS
Mice. Animals of inbred strains A (H-2a) C57B1/6 (H-2b), Balb/c (H-2d),
C3H (H-2a) and (DBA/2 x B a l b / c ^ hybrids (H-2 d /H-2 d ), referred to as D 2 C,
were used in the experiments reported. These animals came either from the
colony maintained by the Weizmann Institute of Science, Rehovoth, Israel, or
from the Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine. No
difference between embryos from the two colonies, or in the materials extracted
from them, was noted; hence, no distinction of mouse origin will be made in
reporting the results. The principal H-2 antigens of the strains used are listed
in Table 1.
Table 1. H-2 classification of various mouse strains*
Strain
H-2
Antigens
A
C57B1/6
Balb/c
(DBA/2 x Baltyc^
C3H
a
b
d
d
k
1,3,4,5,6,8,10,11,13,14,25,27,28,29
2,5,6, 14,22, 27,28,29, 33
3,4, 6, 8, 10, 13, 14, 27, 28, 29, 31
1, 3, 5, 8, 11,25, 32
* Antigens not listed are either absent, or as yet undetermined in the strain.
Data from Snell et al. 1964.
Matings for embryos were made between D 2 C males and C57B1/6 various
females. This combination gave the greatest frequency of successful matings of
those tested. Females were brought to oestrus by injection of hormones, after
McLaren & Michie (1959), and pregnancies were timed from the day of
observation of a copulation plug.
Alloimmune sera were prepared by injecting appropriate mice with spleen
and lymph node cell suspensions at 1- to 2-week intervals. Approximately
25 x 106 nucleated cells were given per injection for a total of four to six injections. Animals were bled on days 4-6 after the last injection; the sera were
pooled and stored at - 50° C.
The cytotoxic test employed measures cell death in terms of permeability to
dye; it has been previously described in detail (Gorer & O'Gorman, 1956;
Soluble H-2 antigens from mouse embryos
521
Boyse, Old & Thomas, 1961; Edidin, 1964). In the present series of tests the diluent
for cells and sera was 0-1 % crystalline bovine serum albumin (Armour, Fraction V),
in tris-buffered Hanks's solution, rather than protein-free Hanks's solution.
Target mesenteric lymph node cells were suspended at a concentration of
1 x 106 cells per ml. Ten microliters of this suspension was mixed with 10 /A of
antiserum on a slide, and incubated in a moist-box at 37 °C. After 20 min,
complement (normal guinea-pig serum) was added and the slides were incubated
for a further 40 min. Nigrosin, 0-6 % in heated normal mouse or calf serum,
was then added and after a few minutes the cells were examined at x 200-250
magnification. One hundred to two hundred cells per drop were counted and
classified as stained or unstained. Five replicate tests were placed on a single
slide.
RESULTS
Preparation of antigenic extracts
Soluble H-2 antigens, SAg, were prepared from batches of 9 to 10-day-old
embryos. After removal from the uterus and washing in Hanks's solution, the
embryos were staged according to number of somites and transferred to 0-02 %
Versene in phosphate-buffered NaCl (PBS). The embryos were minced with fine
forceps and incubated for 2-3 h at 37 °C with constant swirling or stirring. After
incubation the suspension was made 5 % in trichloroacetic acid, held at 4 °C for
10 min and centrifuged at 10,000 g for 30 min, removing a variety of precipitated
proteins and nucleic acids, as well as the embryo tissue fragments, but leaving
antigenic activity in solution. The TCA supernatant was dialysed overnight in
the cold against one change of distilled water. This brief dialysis removed most
of the TCA without appreciable loss of antigen activity. The dialysed material
was lyophilized and either stored at — 20 °C, or immediately made up in PBS,
at 2-5 x concentration, for testing with antiserum.
Activity of the extracts
The dialysed, concentrated, TCA supernatant was an active inhibitor of
alloimmune sera tested as cytotoxins against mouse lymphoid cells. A typical
assay with the serum tested at two dilutions is shown in Fig. 1. This figure also
indicates that the extracted activity is strain-specific, since Balb/c anti-A antiserum, as expected, did not react with the (D2C x C57B1/6)F1 embryo extract.
The specifically inhibiting extracts appear to contain a full range of H-2 antigens. This is shown in Table 2, in which antigens peculiar to H-2 b and H-2 d ,
numbers 2, 33 and 31 (Snell et al. 1964), are detected by appropriate antisera
and, in addition, antigens common to a number of H-2 loci are also detected
by means of the appropriate cross-reactive serum.
It appears that Versene is necessary for the extraction of SAg, and that its
action may be blocked by the presence of excess calcium and magnesium ions.
Table 3 presents experiments in which embryos were extracted by a variety of
522
M. EDIDIN
salt solutions either containing an excess of calcium ion, or lacking Versene, or
containing another disaggregating agent, tetraphenylboron (Rappaport &
Howze, 1964), which binds potassium. Of all these solutions, only that containing Versene in the absence of excess calcium was effective in extracting the
antigens.
170
340
Serum dilution
Fig. 1. Equal amounts of SAg extracted from (DBA/2 x Balb/c)F 1 x Balb/c embryos
(H-2d/H-2d) were incubated with equivalent amounts of A anti-Balb/c and Balb/c
anti-A sera. In each case the serum plus SAg is compared to serum plus a phosphatebuffered NaCl blank (PBS).
= A anti-Balb/c plus SAg;
= A antiBalb/c plus PBS;
= Balb/c anti-A plus SAg; - - = Balb/c Anti-A plus PBS.
Table 2. Antigenic range of SAg
SAg from (D2C x C57B1/6)F 1 embryos*
Fraction of cells stained
Antiserum
+ SAg
-SAg
R
C3H anti-C57Bl/6
A anti-Balb/c
C57B1/6 anti-A
0-27
0-43
0-75
0-55
052
0-88
049
O80t
O75t
* Data are for two different batches of embryos.
t Measurements are for a single column fraction of the material, not for intact SAg.
Equivalent amounts of antiserum used for each test.
The experiments also suggest that Versene does not act by changing the permeability of cell membranes, allowing contents to leak out, but rather that it
removes SAg from cell surfaces; ineffective agents gave at least the same percentage of damaged cells as shown by staining with nigrosin (Kaltenbach,
Kaltenbach & Lyons, 1958) as did Versene. Further evidence for the surface
Soluble H-2 antigens from mouse embryos
523
action of Versene is derived from the data on re-extraction of once extracted
broken cells; this treatment did not yield more SAg activity.
Another parameter in the extraction of soluble H-2 antigens was embryo age.
SAg could not be obtained from batches of embryos with an average age greater
than 40-44 somites. While it is difficult to establish the exact cut-off point for
extraction it appears to be no later than the 11th day of gestation. Certainly
much older embryos, as well as new born and adult tissues, do not yield active
SAg.
Table 3. Extraction of embryos with a variety of salt solutions and
chelating agents
Fraction of cells damaged
by antiserum
Extracting solution
Tris-buffered NaCl
Tris-buffered Hanks's solution
0 0 2 % Versene-PBS
0 0 2 % Versene-PBS 2 x 10~ 3 M Ca 2+
0 0 2 % Versene-PBS
0 0 2 % Versene-PBS 2 x 10~ 3 M Ca 2+
10~2 M tetraphenylboron
0 0 2 % Versene-PBS
10~2 M tetraphenylboron
0 0 2 % Versene-PBS second extraction
(serum tested at two dilutions)
+ extract
0-68
0-67
0-32
0-47
0-44
0-63
0-65
0-39
0-63
J0-31
10-71
— extract
Fraction of
cells staining
with supravital dye
0-69
0-69
0-49
049
0-57
0-57
0-57
0-60
060
0-40)
0-66J
—
0-38
0-50
—
—
—
0-80
100
—
0-99
Note. Approximately equal amounts of embryo tissues were used in each experiment.
Characteristics of the extracts
The molecular size of the soluble materials extracted from 10- to 11-day
embryos was determined by chromatography on Sephadex. Whole extracts,
shown to be active inhibitors, separated into two peaks on Sephadex G-25 (Fig. 2).
A slow-eluting peak, estimated to be material of approximately 2000 M.W.
was variably present, depending on the degree of dialysis of the preparation.
A faster-eluting peak, running in the excluded volume of the column, was rerun on Sephadex G-50. Here the activity peak eluted just after the excluded
volume of the column (determined by simultaneously running blue dextran,
with a molecular weight of approximately 2000000); this leads to an estimate
of about 8000 for the molecular weight of the peak material (Fig. 3).
Table 4 gives data on the chemical composition of whole extracts, before
column fractionation. Fig. 4 shows various determinations, including amino
sugars, on a series of G-25 fractions. In the latter case, no correlation is apparent
between any of the chemical entities measured and serum-inhibiting activity.
The effect of proteolytic enzymes on SAg was next examined. SAg was treated
with a crude pancreatic protease mixture, Difco Trypsin 1:300, acidified with
524
M. EDIDIN
TCA to remove enzyme protein and then dialysed to remove TCA appeared to
lose all activity. The same result was also obtained when highly purified trypsin
(Worthington, 5 x crystalline) was substituted for the crude enzyme mixture.
0-10 _
0-20
Void volume
r
i
0-40
'A
0-80
1-2
i
i
i
i
i
i
i
i
i
11
13
15
17
Fraction
Fig. 2. Whole SAg extracted from DBA/2 x Balb/c)F 1 x Balb/c embryos was
loaded on a Sephadex G-25 column, and eluted with phosphate-buffered NaCl.
Antigen activity of each fraction was determined with A anti-Balb/c serum. Values
of R are values for degree of serum inhibition by each fraction.
0-2
Void volume
04
- i
X
0-6
X
0-8
X
X
A
A A
X
10 I
i
A A
1
10
A L i\
A
&,
A
A
A
J& fa OX
l
1
1
12
14
16
Fraction
Fig. 3. The void volume peak of activity of SAg chromatographed on Sephadex G-25
was re-chromatographed on Sephadex G-50. x = antigen activity measured with
A-anti-Balb/c serum; A = antigen activity measured with C57B1/6 anti-A serum
(cross-reactive). The 'void volume' indicates the elution peak of high molecular
weight blue dextran.
Soluble H-2 antigens from mouse embryos
525
Table 4. Protein* and sugar content and antigenic potency of
various SAg preparations
SAg
l.vii.
Embryo
age
(somites)
33
27
28
27
Activity of
a standard
amount of
Total sugar
SAg(24/*g) Total Folin- (excluding
plus a
positive
aminosugars)
standard serum materials
as/ig
(as fi% BSA)
dose, R =
Peptide/sugar
glucose
0-50
0-50
n.d.
n.d.
248
830
220
162
92
150f
95J
88 §
2-7/1
5-5/1
2-3/1
1-8/1
* Protein by the method of Lowry et al. Tryptophane-sulphuric acid method for nonamino sugar.
t Pentose (by the cysteine-sulphuric acid method) and iV-acetylhexosamine (by the method
of Aminoff, Morgan & Watkins) were detected. About 30 ji% of each was present.
J Less than 2 /tg/ml of nucleic acids was present (by O.D. 260 m/*). However a total of 25 /tg
pentose was found.
§ Total of 28 /tg N-acetylhexosamine present.
Note. All methods were taken from Kabat & Mayer (1961).
6
8
10
Fraction
14
Fig. 4. Lower graph, antigen activity of SAg fractioned on Sephadex G-25 as
in Fig. 2. Upper graph: O = /Mg/ml peptide (0-0280); • = /*g/ml amino sugar as
glucosamine; x = /tg/ml nucleic acids (00260).
33
JEEM l6
526
M. EDIDIN
However, when the trypsin-treated materials were run through Sephadex to
remove the enzyme, and were not dialysed it was seen that trypsin acts to reduce
the molecular size of the extracts, without destroying their antigenic activity.
Fig. 5 shows the effect of crystalline trypsin on the G-25 void-volume peak
(molecular weight about 8000) of SAg. The material was treated with 1 mg %
trypsin for 1 h at 37 °C and then rechromatographed on the G-25 column. It will
be seen that after treatment antigen activity is considerably retarded in its elution
10
10
12
14
16
18
20
Fraction
Fig. 5. The void volume peak of SAg fractioned on G-25 was treated with 1 mg%
crystalline trypsin for 1 h at 37 °C and then rechromatographed on G-25. R values
measure antigen activity of each fraction, x = before trypsin treatment ; • = after
trypsin treatment.
Table 5. Trypsinization of newborn or adult mouse liver, followed
by extraction with 0-02 % Versene
Time of trypsinization (min)
Newborn
Adult
R* = 0-66
R* = 0-83
10
15
20
30
0-35
0-71
0-61
0-72
0-39
0-61
0-87
0-82
* Relative potency of treated alloantiserum corrected to constant amount of tissue extracted.
from the column, and appears to be associated with molecules of molecular
weight less than 2000. Similar results were obtained when SAg was treated with
crystalline pepsin.
The results of tryptic digestion of SAg suggested that it might be possible to
Soluble H-2 antigens from mouse embryos
527
remove antigenic fragments from tissues of older embryos, or even from adult
mouse tissues by vigorous treatment with trypsin. Such treatment is known to
remove a variety of cell surface components (Cook, Heard & Seaman, 1962;
Eylar & Madoff, 1962; Langley & Ambrose, 1964), and it appeared that the
H-2 determinants themselves are resistant to proteolytic enzymes. Table 5
indicates the activity extracted from newborn liver after digestion for various
times with crude trypsin, followed by extraction with Versene, acidification with
TCA and overnight dialysis against distilled water; the treatment also yielded
H-2 activity from older embryos and from adult spleens. All of these extracts
appear to be far more heterogeneous, in both chemical composition and fragment size, than are extracts of 9- to 10-day embryos. Work is currently in
progress on the purification of SAg extracted from adult tissues.
DISCUSSION
In the experiments presented here chelation, presumably of calcium and
magnesium ions, effects the solution of rather small alloantigenic components
of mouse embryo cells. Re-extraction of cells with fresh Versene, leading to
further damage of their membranes, or damage to their membranes in the
course of treatment with tetraphenylboron did not lead to release of soluble
H-2 antigens, indicating that the mechanism of SAg release by Versene is not
simply a change in membrane permeability, and suggesting that it releases the
antigen directly from the cell surface.
It appears that Versene-extractable H-2 antigens are present only during early
stages of mouse embryo development, at a time when the quantity of these
antigens bound to cells is small (Edidin, 1964). Perhaps the soluble materials
represent precursors of cell bound H-2 antigens requiring further covalent bond
synthesis to be fully fixed to the surface, possibly the sort of in situ cross-linking
proposed for the mucopeptide of gram-positive bacterial cell walls (Strominger
& Tipper, 1965).
That the H-2 antigenic materials studied here contain polysaccharide determinants is suggested by the failure of proteolytic enzymes to inactivate them. It
accords well with the finding by Nathenson & Davies (1966) that the H-2 activity
in their preparations, made from adult mouse tissues, is associated with glycoprotein. On the other hand, the failure to abolish antibody-neutralizing activity
by treatment with trypsin is in marked contrast to reports of its effects in at least
partially inactivating a number of insoluble H-2 preparations (Billingham,
Brent &Medawar, 1958 ;Basch& Stetson, 1962;Kandutsch&Stimpfling, 1962;
Mann, Corson & Dammin, 1962). This difference suggests that the inactivation
observed with trypsin in the experiments cited was due to loss of small, antigenic
molecules after digestion, or to the failure of antigenic determinants on small
carriers to elicit transplantation immunity.
Finally it should be noted that two treatments which result in disaggregation
33-2
528
M. EDIDIN
of tissue cells, Versene chelation and trypsinization, yield molecular fragments
carrying H-2 specificities. Though there may well be no necessary connexion
between the two, the association of tissue disaggregation and release of H-2
antigens suggests a functional site for H-2 at the cell surface. The suggestion is
furthered by the correspondence in size of the SAg molecules, measured in
calcium-free solution, and that of the subunits of sponge aggregation factor
(Margoliash et al. 1965) which appear in the absence of calcium and magnesium
ions. This interpretation is discordant with the observation that free lymphocytes
and other leucocytes carry the highest per cell charge of H-2 (Hoecker & Pizarro,
1961; Basch & Stetson, 1962), though it does accord with the paucity of H-2 on
erythrocytes. Though many other antigenic factors are associated with the
phenomena of cell social behaviour, such as Forssman antigen (Fogel & Sachs,
1964), H-2 antigens are known to be involved in cell recognition (Moller, 1965)
and may also be part of the material basis of cell adhesion.
SUMMARY
1. Treatment of 10-day-old mouse embryos with the chelating agent Versene
releases water-soluble materials carrying H-2 alloantigen activity, as measured
by inhibition of cytotoxic sera.
2. The materials extracted (SAg) are specific for the strain of mouse embryo
used.
3. SAg extracts may be resolved into two peaks of activity by chromatography on Sephadex G-25. The peaks correspond to molecular weights of
approximately 8000 and 2000.
4. The molecular weight of SAg may be reduced to one-fourth its original
value by treatment with trypsin.
RESUME
La liberation d''alloantigenes H-2 solubles au cours de la desagregation de tissu
embryonnaire de sour is par un agent chelateur
1. Le traitement au Versene d'embryons de souris de 10 jours libere des
substances hydrosolubles portant une activite alloantigenique H-2, telle qu'on
la mesure par inhibition de serums cytotoxiques.
2. Les substances extraites (SAg) sont specifiques pour la souche de souris
utilisee.
3. Les extraits SAg peuvent etre separes en deux pointes d'activite par
chromatographie sur Sephadex G-25. Les pics correspondent a des poids
moleculaires d'environ 8000 et 2000.
4. Le poid moleculaire de SAg peut etre reduit au quart de sa valeur originelle
par traitement des antigenes a la trypsine.
Soluble H-2 antigens from mouse embryos
529
This research was performed during the tenures of a National Science Foundation Postdoctoral Fellowship and an American Heart Association Career Investigator Fellowship.
I am grateful to Professor Michael Feldman and Professor Albert H. Coons for their hospitality and their interest in, and encouragement of, my work. I would like to thank Mrs
Valerie A. Parsegian and Miss Ceyran Yilmaz for their able technical assistance.
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{Manuscript received 3 June 1966)