Rejection response in Botryllus oozooids
1.a.
Lauren Nagashima
The rejectionreaction following tip-to-side contact between two
vascular ampullae of two incompatible Botryllus oozooids differs in the
two ampullae. The ampulla that initiated contact (aggressor) forms
a distal blood clot and responds in one of three ways that differ
in their degree of injury to the aggressing ampulla: (1) loss or
injury to the ampullar pad cells (2) loss of distal end of ampulla
by constriction (3) rapid ampullar disintegration. The receiving
ampullacshowed a more subtle rejection response, as did some other
ampullae of the oozooid pair not involved in an ampullar contact.
Rejection response in Botryllus oozooids
Lauren Nagashima
INTRODUCTION
Contact between the outgrowing vascular ampullae at the margins
of different Botryllus colonies results in either a fusion of the
colonies or a rejection and subsequent formation of a clear boundary
between the colonies (eg. Bancroft 1903, Tanaka 1973, Katow and
Watanabe 1980). Current research in this phenomenon stems from the
recognition of the similarity between this allogeneic inhibition and
mammalian rejection of foreign tissues (eg. Scofield and Weissman, in
press, Burnet 1971). The most recent description of the rejection
response by Tanaka and Watanabe (1973) divides the process into six
stages. Stage 1: The vascular ampullae extend with the subsequent
contact of the test matrices. Stage 2: The ampullae migrate into the
test of the opposite colony and reciprocal tip-to-side contact of
the ampullae is established. Stage 3: Test cells surrounding the
ampullar contact become dark and opaque. Stage 4: Test cells aggregate
at the tips of the ampullae. Stage 5: Blood flow is decreased to the
ampullae involved in the contact; test material surrounding these
contacts becomes dark and opaque. Stage 6: The vascular ampullae in
the contact area are completely separated from the test circulatory
system.
In all previous published literature, pieces of grown colonies
were used to observe the rejection reaction. Because of the many
reciprocal tip-to-side contacts of the ampullae, a clear picture of
the sequence of events in one ampullar contact was not obtained. In
the method developed by Scofield (submitted 1981) Botryllus oozooids,
Rejection response in Botryllus oozooids
Lauren Nagashima
3.
which have only eight vascular ampullae, were used. Pairs of oozooids
were observed to make a singular contact:ie,oneampulla of an oozooid
initiated contact by turning its tip to touch the side of a neighboring
oozooid's ampulla. The former ampulla shall be referred to as the
"aggressor" the latter being the "receiver". The sequence of events
that follows was seen to be much more complicated than that earlier
described by Tanaka and Watanabe (pers. comm. Scofield 1981), but
was not documented. It was the purpose of the present study to
observe the rejection process in detail and to document it with
photographs.
MATERIALS AND METHODS
Living colonies of Botryllus were collected from docks in the
Monterey Marina. Each colony was kept separate and at room temperature
in a 1L beaker, using the method of Scofield (submitted). The
developing larvae were brooded within the colony, and were allowed to
hatch as tadpoles. The beakers had been lined with glass slides on
which the tadpoles settled in a 0.5 cm wide band at the water line.
Twelve hours after the tadpoles had metamorphosed into oozooids, the
glass slides were removed for observation. Ampullar interactions were
observed and photographed (using a Nikon inversion microscope no.41368
and a 35 mm Olympus OM-2 camera) for a period of 48 hours.
Lauren Nagashima
Rejection response in Botryllus oozooids
RESULTS
In these studies, it became clear that contact between incompatible
colonies can be considered at two levels: The local level, involving
only responses of the two ampullae that came into contact; and the
systemic level, involving responses of other ampullae (on the same
individuals), which had not experienced contact.
The first level concerns the individual ampullae involved in
the tip-to-side contact (see fig.1). The reponse of the ampulla which
initiated contact (aggressor) differs from the response of the

receiving ampulla (receiver). In the aggressing ampulla, three degrees
of response are seen. Common to all three degrees of response is an
abnormally largeamount of blood which is static despite contractions
and expansions of the surrounding ampullae of the aggressing oozooid.
The first and
least drastic degree of response is an injury to, or loss of, the
columnar epithelial cells or "pad" cells at the tip of the ampulla
within 24 hours after contact; bleeding may or may not occur (see figs,
2 and 3, and table 1). The second degree of response seen in the
aggressing ampulla is an amputation of the tip of the ampulla involved
in the contact. The process of amputation consists of a constriction
in, and subsequent pinching off of, a portion of the ampullar bulb. (see table 1.
fig. 4). This response occurs between 12 and 24 hours after ampullar
contact. The third degree of response is a disintegration or
"explosion" of the ampullar wall, releasing a mass of blood cells into
the matrix of the tunic. By contrast to the other two degrees of
response, this is a quick and complete rejection response occuring
within 6 hours after tip-to-side contact had been established (see figs.
Lauren Nagashima
Rejection response in Botryllus oozooids
5 and 6, and table 1).
In the receiving ampulla, the responses to the incompatible
contact are much more subtle. In most cases, the receiving ampulla
did not respond in any visible way (see figs. 5 and 8, and table 1).
In other cases, the receiving ampulla withdrew from the ampullar
contact or became atrophied and clogged with static blood cells (see
figs. 3,4 and 6).
The second level of response to contact between incompatible
oozooids is a systemic reaction seen in the rest of the vascular
system and in ampullae uninvolved in a contact. Most commonly, the
uninvolved ampullae of either oozooid did not respond to the rejection
in progress between contacting ampullae (see figs 3,5,8). However,
in some cases, uninvolved ampullae of the aggressing or receiving
oozooid withdrew from the test cuticle, became clogged with static
blood cells and atrophied. (see figs. 4 and 6) or the adjacent
ampullae "leaked" blood cells from their ampullar wall before a
rejection response was seen in the contacting ampullar pair (see
fig. 7 and table 1).
Lauren Nagashima
Rejection responses in Botryllus oozooids
DISCUSSION
Contact between the vascular ampullae of two incompatible
oozooids leads to a rejection response. The rejection response is
both local,involving only the ampullae in contact, and systemic,
affecting the entire circulatory system. There are varying degrees
of responses in the ampulla that initiated contact (the "aggressor")
The least invasive response,seen in 50% of the rejecting pairs, was a
loss of, or injury to the ampullar pad cells. In other pairs (4.23).
amputation of a portion of the ampullar bulb was seen. In the remaining
45.83, however, a violent rejection response occured, leading to the
disintegration of the wall of the aggressing ampulla. Thus, there
seems to be a heirarchy of rejection responses.
The genetic locus which controls histocompatibility in Botryllus
is polymorphic, with an 'estimated 40 codominant alleles (Scofield and
Schlumberger, unpublished). Therefore, each combination of histo-
compatibility alleles acting in an incompatible oozooid pair is
different. In the following discussion, I am assuming that: (1)
histocompatibility alleles may be structurallydissimilar (2) these
dissimilarities may lead to the rejection reaction (3) the structural
differerences in the histocompatibility alleles may result in the
differing rejection responses (4) the greater the dissimilarity
between alleles, the greater the degree of rejection between the
contacting ampullae. Certain combinations of histocompatibility
alleles may be more structurally dissimilar and may lead to a brisk
and complete rejection response (that of the "exploding" ampulla).
Likewise, other combinations whose alleles are different, but closely
in structur
related, may ultimately result in rejection, but may allow long-term
Lauren Nagashima
Rejection response in Botryllus oozooids
contact beforehand (eg. loss of ampullar pad cells). The severity
of the rejection response seems to be inversely related to the time of
allowed contact and therefore may be directly related to the dissimilarity
between the histocompatibility alleles of the interacting oozooids.
There are two stages seen in the rejection response: The first
occurs during tip-to-side contact and is an aggregation of static
blood cells in the tip of the aggressing ampulla which then turns dark,
signaling cell death. The second stage is the local and systemic
rejection responses occuring after the tip-to-side contact has begun.
Chemotaxic factors may control both stages: (1) blood cell aggregation
and subsequent death and (2) the actual local and systemic responses
earlier described. On the other hand, the same factors may only
cause the aggregation of blood cells at the site of tip-to-side contact.
These blood cells may then release a second chemical factor as they
die, mediating the next stage of the rejection responses. Therefore,
the two parts of the rejection reaction may be coincidental or causal.
Possible sources of such chemotaxic factors are (1) blood cells or
humoral factors exchanged between ampullae at the tip-to-side junction
(2) mediators produced by-thesampullar pad cells involved in the
tip-to-side interaction.
Observation of damage to the receiving ampulla and evidence of
changes in vascular permeability during and after a rejection, reopens
the possibility of a role for blood cell or humoral factor exchange.
these sorts
Exchange of incompatible elements of, may provide the chemical.
factor which draws in blood cells into the aggressing ampulla and/or
which causes the rejection responses.
Lauren Nagashima
Rejection responses in Botryllus oozooids
The second source of the chemical factors may be the ampullar
pad cells. The pad cells involved in a "disagreeable" contact may
release a chemical signal that attracts blood cells and/or mediates
the local and systemic rejection responses,
Continued studies to determine the source of chemotaxic factors
and their role in the rejection response and the roles of necrotic
blood cells at the tip-to-side junction are in progress,
Lauren Nagashima
Rejection response in Botryllus oozooids
ACKNOW LEDGEMENTS
The author expresses her sincere gratitude to Drs. Virginia L.
gcofield and Donald P. Abbott for introducing the author to the study
of the present work, for the advice given and for the critical readings
of the manuscript and to Jay Schkumpberger for his helpful words of
encouragement.
Lauren Nagashima
Rejection response in Botryllus oozooids
10.
TABLE 1
Number of times observed
local respons
Aggressing ampulla
number percent
1. loss or injury to ampullar
pad cells
50%
2. amputation of ampullar
bulb
4.23
3."explosion" of ampulla
45.88
22
Receiving ampulla
1. no response
10
34.5%
2. withdrawal of ampulla
22
65.5%
systemic responses
Aggressing oozooid
1. no response
13
599
7
2. withdrawal of ampullae
31.88
3. bleeding from ampullar walls
9.2%
Receiving oozooid
1. no response
23
82%
2. withdrawal of ampullae
193
Lauren Nagashima
Rejection response in Botryllus oozooids
11.
LITERATURE CITED
1. Bancroft, F. W., 1903. Variation and fusion in compound ascidians.
Proc. Calif. Acad. Sci., series 3, 3: 137-186.
2. Burnet, F. M., 1971. "Self-recognition" in colonial marine forms
and flowerøing plants in relation to the evolution of immunity,
Nature., 232; 230-235.
3. Katow, H., and H. Watanabe, 1980. Fine structure of fusion reaction
in compound ascidian Botryllus primigenus OKA. Develop. Biol. 76: 1-14.
1981.
4. Scofield, V.,, Protochordate allorecognition controlled by an MC-like
gene system. Nature (submitted).
5. Scofield, V., 1981. personal communication.
6. Scofield, V., and J. Schlumpberger, 1981. umnpublished.
7. Scofield, V., and I. Weissman, 1981. personal communication,
8. Tanaka, K., 1973. Allogeneic inhibition in a compuond ascidian.
Botryllus primigenus OKA. II. cellular and humoral responses in the
"hon-fusion" reaction. Cell. Immunol. 7: 427-441.
9. Tanaka, K., and H. Watanabe, 1973. Allogeneic inhibition in a
compound ascidian, Botryllus primigenus OKA. I. processes and
features of "non-fusion" reaction. Cell.Immunol. 7: 410-126.
Rejection response in Botryllus oozooids
Lauren Nagashima
FIGURE LEGENDS: explanation of numbers placed on photograghs
1-ampulla
2-gelatinous test or tunic
3-pad cells
4-blood cells
5-necrotic zone
6-aggressing ampulla
7-test cells
8-test cuticle
9-receiving ampulla
12.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE CAPTIONS
Figure 1. A typical tip-to-side contact. The test cuticle fused
allowing the aggressing ampulla to enter into the test of
the neighboring oozooid and to form contact on the side of
a receiver ampulla. Note the static blood cells in the
aggressor's tip,
Figure 2.
Aggressing ampulla showing the first degree of response.
involving a loss of pad cells; no bleeding has occured. Note
the static blood cells in the aggressing ampullar tip. The
receiving ampulla remains uninjured and shows no obvious
response to the contact.
Figure 3. Aggressing ampulla showing a variation on the first degree
of response, involving damage to its pad cells and subsequent
bleeding. A necrotic area of dead blood and test cells is
present ar the site of injury.
Figure 4. Aggressing ampulla showing the second degree of response.
involving an amputation of a prtion of its ampullar bulb.
The aggressing ampulla, receiving ampulla and adjacent ampullae
have withdrawn from the test cuticle, are clogged with static
blood cells, and are atrophied.
Figure 5.
Aggressing ampulla showing the third and most violent rejection
response, involving a complete disintegration of its ampullar.
wall; note large amount of bleeding and necrotic zone. The
receiver and the other uninvolved ampullae show no response
to this quick and complete rejection.
13.
Lauren Nagashima
Rejeciton response in Botryllus oozooids
Figure 6 A-D. A sequence of photographs showing the third degree of
rejection response, involving an "explosion" of the am
ampullar of the aggressing ampulla.
A.6:00 a,m. The aggressor has begun a rejection response, with an injury
and bleeding from the ampullar tip. The receiver and the other
uninvolved ampullae show no response to the rejection in progress,
B.6:25 a.m. The aggressor continues to bleed profusely; no marked change
with the other ampullae.
C.6:45 a.m. The ampullar wall of the aggressor seems to have lost
its integrity; bleeding continues.
D.9:30 a.m. What is left of the aggressing ampulla has withdrawn under
the body of the oozooid, as did the other ampulla connected to the
same oozooid. A necrotic zone marks the site of the recent ampullar
contact. The other ampullae of the receiving oozooid seem to be
recovering from their response.
Figure 7. A response in the ampullae adjacent to the aggressing ampulla
on the same oozooid; involving a "leaking" of blood cells.
from their ampullar walls. This type of sysemic rejection
response is seen only in the uninvolved ampullae of the
aggressing oozooid. This phenomenon was seen after tip-to-
side contact was established, but before bleeding was seen
in the aggressor.
Figure 8.
The aggressor has "jerked" back from the ampullar contact
leaving a small necrotic zone behind. The receiving ampulla
and adjacent uninvolved ampullae on the receiving oozooid are
showing no effects fo the recent incompatible contact.
14.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 1

(for all figures)
15.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 2
16.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 3
17.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 4

.2
18.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 5


19.
Lauren Nagashima Rejection response in Botryllus oozooids
FIGURE 6-A
20.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 6-B
21.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 6-C
22.
Lauren Nagashima
Rejection reponse in Botryllus oozooids
FIGURE 6-D
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Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 7
24.
Lauren Nagashima
Rejection response in Botryllus oozooids
FIGURE 8
25.
5