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AMER. ZOOL., 15:93-106 (1975).
Ontogeny, Phytogeny, and Cellular Cooperation
LAURENS N. RUBEN
Department of Biology, Reed College, Portland, Oregon 97202
SYNOPSIS. The capacity of adult newt (Triturus viridescens) spleen cells to secrete antibody at 4
C allows simultaneous visualization and quantification of non-secretory (S~) and secretory
(S+) rosette forming cells (RFC). Visualization of mammalian S+ RFC requires 37 C, a
temperature at which S~ RFC appear to be fragile. RFC can be distinguished as S~ or S+ due
to whether one or more layers of erythrocytes adhere to the surface of sensitized spleen cells.
Different doses of horse erythrocytes (HRBC) affect newt S" RFC and S+ RFC differentially.
By varying the time between injections of different concentrations of chicken erythrocytes
(CRBC, the "carrier") and a constant dosage of CRBC-TNP (trinitrophenyl, the hapten) it is
possible to measure "helper" activity that correlates with the population of S" RFC and is
both dose and time dependent. By varying assay time after "helper" activity has been
maximized, one can determine the cytodynamics of anti-TNP antibody producing cell
(APC) activity. For the first time these morphologically separable RFC can be related to their
suspected physiologic behavior. A shift from S~ RFC to S+ RFC takes place during the
immune response. That similar dose-dependent response curves can be shown in adultRana
pipiens suggests that the newt responses represent a fundamental vertebrate pattern.
1968; Nossal et al., 1968; Claman and
Chaperon, 1969; Rajewski et al., 1969;
It should not be at all surprising to find Mitchisonetal., 1970). Excellent reviews on
papers dealing with phylogenetic aspects in this subject are available (Roitt et al., 1969;
a symposium on the development of im- Miller, 1970, 1972; Miller et al., 1971;
munity. Phylogeny and ontogeny were in- Playfair, 1971).
extricably entwined in our thinking even
T cells are antigen-recognizing cells
before the early part of the 19th century
(ARC)
with "helper" function; they rewhen von Baer first contributed his concluspond
to
antigen but do not produce antisions concerning embryos and their anceset al., 1967; Falkoff and
body
(Davies
tors. This presentation summarizes some
Kettman,
1972).
B cells, aided by sensitized
recent work relative to the evolution of celas
precursors
of plasma cells
T
cells,
serve
lular interaction in primary humoral imwhich
produce
specific
antibody
(Mitchell
mune responses. Certain immunogens,
et
al.,
1968).
Antiand
Miller,
1968;
Nossal
e.g., heterologous erythrocytes, require cellular cooperation, i.e., the interaction of at gens which are known to stimulate thymusleast two subpopulations of lymphocytes, in independent cells directly appear to be
order to elicit an immune response in ro- highly polymerized (Miller, 1971; Wilson
dents (Claman et al., 1966). One subpopu- and Feldmann, 1972) and are mitogenic for
lation is thymus-dependent (T cells), the B cells (Coutinho and Moller, 1973). The
other thymus-independent and generally mechanism of cooperation has been a subreferred to as B cells because of their deri- ject for considerable speculation (Bretscher
vation from the bursa of Fabricius in birds and Cohn, 1970; Mitchison, 1971; Dutton
(Warner et al., 1962; Cooper et al., 1966; et al., 197la,b; Feldmann and Nossal,
Rouse and Warner, 1972) or from bone 1972).
Members of all the modern vertebrate
marrow in mammals (Miller and Mitchell,
classes show the capacity to respond to
heterologous erythrocytes (Abramoff and
These studies were supported in part by a grant
La Via, 1970). Thymus tissue has been de(GB-38480) from the National Science Foundation, scribed for all vertebrate classes except that
Washington, D. C , U. S. A. Gratitude is offered Mss.
Sheryl Swink and Judith Ruben for their technical of the most primitive Agnathan, the marine
hagfish (Good et al., 1966; see also Riviere
assistance.
INTRODUCTION
93
94
LAURENS N. RUBEN
et al., 1975; Linna et al., 1975), but little is
known of thymus dependence in the antired cell response in species other than birds
and mammals. Three reports are suggestive of thymus dependence in amphibia.
DuPasquier (1970), Moticka et al. (1973),
and Manning and Turner (personal communication) all suggest thymus involve-
r
ment in Alytes obstetricians, Rana catesbiana,
and Xenopus laevis when sheep red blood
cells (SRBC) are used as immunogen.
THE ASSAY
The quantitative assay is immunocytoadherence (Zaalberg, 1964;
Biozzi et al., 1966). The animals are immunized in vivo by intraperitoneal injections with foreign red blood cells, e.g.,
sheep (SRBC), rat (RRBC), horse (HRBC),
or chicken (CRBC). After a period of time,
the organ to be tested, e.g., the spleen, is
mechanically dissociated leaving a cellular
suspension. Aliquots of these cells are then
mixed with test or control red blood cells
and incubated overnight in the cold (3 to 5
C). When specific receptor sites occur on
the surfaces of sensitized spleen cells, erythrocytes will adhere and form "rosettes"
(Fig. 1). The rosette forming cells (RFC) are
counted visually in a hemacytometer, quantitated and expressed as RFC/106 spleen
cells. For further details see Ruben et al.
(1973).
FIG. 1. An amphibian "rosette" showing adherent
mammalian red cells bound to the sensitized spleen
cell surface. When erythrocytes adhere in several
layers of thickness, even if the entire spleen cell surface
is not covered, the rosette is considered to represent an
S+ or APC RFC (see the text), x 300.
8 days when anti-SRBC RFC activity was
100 times background. Background levels
were restored by 16 days. The
cytodynamics of this primary response in
Xenopus were similar to those reported previously for Bufo marinus (Diener and Marchalonis, 1970) and Alytes obstetricans
(DuPasquier, 1970), using the rosette assay.
While adult responses are interesting, we
especially wanted to know, instead, when
this humoral response developed and
whether any morphologic events were associated with its appearance. Analysis of
larval stages of Xenopus traced the development of this response to 1 week later
(at 25 C) than the time of allograft response
initiation (Horton, 1969; Ruben et al.,
1972). Allograft response initiation correTHE ANTI-RED CELL RESPONSE IN XENOPUS
lated with thymus maturation, but the
anti-red cell response began when lymphocyte
differentiation could first by observed
In vitro studies (Auerbach and Ruben,
in
the
spleen. This was expected since
1970) with explants of young adult Xenopus
laevis, the South African clawed toad, cell-mediated responses in amphibians insuggested that the liver and kidney failed to volve thymus-dependent cells (Cooper and
produce strong hemagglutinin responses Hildemann, 1965; Curtis and Volpe, 1971;
to SRBC; the spleen appeared to be the Horton and Manning, 1972; Tournefier,
1973; Cooper, 1974) as they do in mamprimary site. Since the spleen was engaged
mals.
Anti-red cell responses in amphibia,
in at least this type of immunologic activity,
however,
might require at least two
attention was concentrated on it during our
initial immunocytoadherence studies (Kid- cooperating lymphoid cell populations
der et al., 1973). The cellular response to from separate embryonic origins, hence
SRBC in young adults began by 4 days after dependence on maturation of the primary
challenge and the activity reached a peak at peripheral lymphoid organ, the spleen.
CELLULAR COOPERATION IN AMPHIBIA
THE ANTI-RED CELL RESPONSE IN THE NEWT
At the beginning of our studies with the
American common newt, Triturus viridescens, a urodele more primitive than anurans, no information was available on the
organs that generate the humoral responses. Since liver, spleen, and kidney are
involved in hematopoiesis we tried assaying
all three (Ruben et al., 1973). All three organs showed a substantial increase in RFC
activity after HRBC immunization, although the spleen (51 times background)
was clearly more involved than the liver (24
times background) or kidney (19 times
background). Specificity of rosette formation was demonstrated by testing with
SRBC. Red blood cell binding to HRBC was
not significantly altered when SRBC was
used as the immunogen, (0.2 ml of 25% red
cells in Alsevers solution) for the challenge
dosage to study the cytodynamics of the
newt's primary response. All three organs
produced response curves that agreed with
those for other amphibia using similar effective dosages.
CELLULAR COOPERATION
One method of demonstrating cellular
cooperation in vivo involves the use of haptens, e.g. trinitrophenyl (TNP), (Rajewski
et al., 1969; Mitchison et al., 1970). TNP
can be conjugated to erythrocytes that serve
as carrier (Rittenberg and Pratt, 1969;
Kettman and Dutton, 1970) capable of initiating anti-hapten antibody synthesis.
Preimmunization with the erythrocyte carrier will enhance the anti-hapten response.
Since there is no addition of TNP with carrier preimmunization, the concentration of
TNP available is not increased. Therefore,
enhancement occurs as a consequence of
cooperative activity of the "helper" cells
sensitized to carrier in conjunction with
APC. Thus, the degree of enhancement of
the anti-hapten response serves as a measure of "helper" activity. The population
which is carrier specific is thymusdependent in rodents (Kettman and Dutton, 1971; Falkoff and Kettman, 1972) but
the antibody producing cells (APC) are B
95
cells (Mitchell and Miller, 1968). Antihapten responses are normally assayed in
agar by showing hemolytic plaque forming
cells (PFC) (Jerne and Nordin, 1963). Control experiments for this carrier-specific
enhancement involve preimmunization
with heterologous red blood cells different
from the kind used as carrier and/or presentation of carrier-TNP without preimmunization.
Information from the newt, combining
immunocytoadherence and carrier-hapten
immunization, clearly shows enhancement
of anti-hapten response after preimmunization with the same type of erythrocyte used
as carrier, but not with a different priming
erythrocyte. CRBC and Bufo marinus
(TRBC) erythrocytes were used in the four
possible combinations of priming erythrocyte type and TNP-conjugate. HRBC and
HRBC-TNP assays were performed in all
cases. The HRBC assay provides a measure
of cross reactivity between the erythrocyte
injected in vivo and HRBC. When this
value is subtracted from the number of
RFC/106 counted in the HRBC-TNP assay,
we have a measure of anti-hapten activity.
The enhancement of anti-hapten activity
occurs in the newt following carrier-specific
preimmunization; thus, at least two interacting subpopulations of cells may be
contributing to specific adaptive immunity
in the newt.
Newts are ideal for studying the
phylogeny of cellular cooperation. Although they are primitive amphibians, they
are the most advanced vertebrate with no
bone marrow as a major hematopoietic site
(Cowden and Dyer, 1971). Clearly, cellular
cooperation appeared in evolution independent of the development of a bursa of
Fabricius or an active bone marrow. However, without additional information, it is
impossible to know if the newt's condition is
a unique consequence of parallel evolution
or whether it represents a fundamental vertebrate pattern. No information is available
on the nature of the B cell equivalent in the
newt nor is it known whether the "helper"
cells of the newt are thymus dependent.
The three studies cited earlier suggest
thymus dependence in the amphibian
anti-SRBC responses.
96
LAURENS N. RUBEN
THE EFFECT OF IMMUNOGEN DOSAGE
The intial report on the newt response
also presented some preliminary information that the intensity of the antierythrocyte response is inversely proportional to the concentration of the immunogen (HRBC). While this might be one interpretation of the result, another possibility is that different challenge dosages might
initiate antigen-binding responses with differing rate curves. Thus, whether primary
response intensity is directly or indirectly
proportional to immunogen dose would
depend on when the assays are performed.
The issue has now been explored in depth
and we can distinguish between these two
alternatives (Fig. 2; Table 1). The time
course curves of primary responses are
clearly translocated when differing challenge dosages are used. These data and all
that follow will be considered in greater
detail elsewhere (Ruben, 1975). The lower
the immunogen dose, the more rapidly is
the response peak achieved. The peak RFC
levels, however, nearly increase in a linear
fashion with each higher dosage used. It is
clear that when the assays are done at 2
days, rather than the 4-day intervals used in
our original report on the newt, a new
"shoulder" (or period of decreasing rate of
increase in numbers of antigen binding
cells within the spleen) appears between 2
and 4 days after challenge with the two
higher dosages of HRBC.
Studies of mammalian systems demonstrate that high immunogen doses obviate the necessity for cellular cooperation
(Sinclair and Elliott, 1968; Claman and
Chaperon, 1969; Taylor, 1971). These results are in agreement with those of Mitchison (1964, 1971) who suggested that T cells
are triggered by lower doses of antigen
than are B cells; "helper" activity might be
viewed as crucial to focusing relatively low
doses of antigen onto the B cells. A variety
of experiments revealed that low immunogen doses yield proportionally greater
stimulation of "helper" and memory cells
than APC (Salvin and Smith, 1964;
McDevitt et al., 1966; Hanna et al., 1967;
Greaves et al., 1970; Greaves and Moller,
1970; Kettman and Dutton, 1971; Playfair,
1971; Falkoff and Kettman, 1972). Higher
doses of antigen stimulate increasing
amounts of antibody which are generated
in shorter periods of time (Sterzl and
Trnka, 1957; Winebright and Fitch, 1962;
IMMUNOGEN (HRBC) DOSE
25%
0.25%
0.0025% •—
PRIMARY RESPONSE
T. viridescens
SPLEEN
^6000
y
5000
z
54000
3000
•
DAYS
FIG. 2. The effect of immunogen (HRBC) dose on
the primary immune response in the spleen of the
10
adult newt as assayed by immunocytoadherence.
5521 ± 11
4459 ± 131
3706 ± 759
2533 ± 161
1972 ± 234
1031 ± 318
2
4
6
8
12
16
HRBC
0
718 ±
45
313 ± 363
576 ± 285
749 ± 330
1784 ± 171
1396 ± 285
496 ± 343
1103 ± 557
95 ± 11
328 ± 242
S+
3211 ± 1103
3367 ± 440
5426 ±
550 ± 314
S- b
0.0025%
4
1235 ± 26
2172 ± 50
5622 ± 335
7173 ± 94
3337 ±
2957 ± 318
877 ±533
Total
RFC/106
19
3686 ± 226
1668 ± 258
375 ± 284
860 ± 257
9151 ± 840
2810 ± 138
1990 ± 35
1652 ±311
877 ± 553
Total
RFC/106
513 ± 509
1321 ± 1131
825 ± 561
943 ± 70
873 ±
328 ± 242
S+
1659 ± 111
4301 ± 796
6349 ± 466
2389 ± 82
2284 ± 55
550 ± 314
S—
0.25% HRBC
•13
o
1303 ±431
•V
2
599 ± 60
3087 ± 527
361 ± 165
z
3512 ± 103
5640 ± 943
2438 ± 173
O
472 ± 22
42 ± 59
328 ± 242
S+
372 ± 35
1518 ± 35
1610 ± 371
550 ± 314
S-
25% HRBC
RAT
a
An average of 16 hemacytonieter chamber counts from 4 incubations involving 2 different cellular pools, each of which is comprised of spleen cells
from 4 newts, is used for all data presented in this table.
b
S— (non-secretor) refers to RFC with adherent cells attached only to the spleen cell itself, while S+ (secretor) refers to RFC where additional
erythrocyte layers have attached to sensitized cells.
877 ± 553
Total
RFC/10 6a
0
(Background)
Day
TABLE 1. The effect of immunogen dosage (% HRBC) on the primary immune response of the newt (spleen).
CELL ULAR Co
HIBI
98
LAURENS N. RUBEN
Uhr and Finkelstein, 1963; Svehag and
Mandel, 1964; Campbell and Kind, 1969).
It appears, then, that the generation of
"helper" cells is enhanced over APC by low
immunogen doses, while the reverse is true
when higher dosages are used. Most studies
using mammalian systems utilize assays for
antibody production and this alone can account for the conclusion that higher concentrations of immunogen stimulate earlier
and stronger immune responses.
Immunocytoadherence, however, allows
the visualization of ARC activity ("helper")
as well as APC; it measures the total
number of cells capable of specific antigen
binding (Greaves and Moller, 1970;
Greaves et al., 1970; Bach and Dardenne,
1972; Haskilletal., 1972). The present data
fostered the reverse conclusion of that reported for rodents, at least with regard to
the rate of the response. Therefore, the
beginning assumption is that the earlier
shift in time courses with decreasing immunogen dose is a consequence of introducing information about "helper" or ARC
activity. This assumption, in turn, leads to
the speculation that antigen-binding cell activity, measured between days 0 and 2,
probably involve ARC mitogenesis or recruitment. ARC mitogenesis has been demonstrated (Paul et al., 1968; Marchalonis,
1971; Segal et al., 1971; Nakamura et al.,
1972) and recruitment or "homing" of
ARC into the spleen has been suggested
(Sprent et al., 1971). As in the mammalian
system, one might expect this activity to be
favored by lower immunogen doses in the
newt. The reduction in the rate of increase
in the number of antigen binding cells from
the spleen between days 2 and 4, may reflect the period of "helper" activity, when
information transfer occurs so that APC
may be triggered. Once finished, these
ARC cells may leave the spleen to serve as
"memory" cells in the circulation. Finally,
the steep increase in cell numbers between
days 4 and 8 may represent APC
mitogenesis. Neither the "shoulder" nor
the rapid increase in APC is detectable
when the lowest dose is used. It would appear that either ARC remove this small
amount of immunogen effectively enough
without the involvement of an additional
substantial cellular population, or that the
disappearance of ARC from the spleen is
partially matched by APC generation, so as
to provide for a gradual replacement of
ARC by APC. Haskill and Axelrad (1971)
have visualized this type of time-dependent
progressive shift during the immune response in the rodent.
"HELPER" AND ANTIBODY PRODUCING CELL
ROSETTES
Greaves et al. (1970) have categorized
RFC as to whether or not they appear to be
secreting antibody. Secretory (S+) or APC
RFC are sensitized spleen cells which bind
on to their surfaces more than one layer of
adherent red blood cells. Non-secretory
(S~) or ARC RFC bind only one layer of red
blood cells on to the surface. Others, e.g.,
Haskill et al. (1972), have classified
gluteraldehyde-fixed RFC according to the
number of adherent red blood cells; maximally binding cells have more than one
adherent layer and minimally binding cells
are specifically affected by adult thymectomy and anti-theta serum. Maximally binding RFC can be sorted out in gradients
along with PFC (McConnell, 1971), therefore at least some RFC are clearly related to
APC. The earlier demonstrations that some
RFC in rodents are T cells rested on less
firm ground, usually dependent on the use
of anti-theta serum and complement which
suppresses the number of antigen binding
cells (Greaves and Moller, 1970; Greaves
and Hogg, 1971; Raff, 1971). However,
Greaves and Raff (1971) and Takashashi et
al. (1971) found that some anti-theta preparations are contaminated with antibodies
directed against surface receptor sites other
than theta. At least one site is present on
thymus-independent (B) cells. However, in
support of the view that at least some RFC
are thymus dependent cells in rodents,
Bach and Dardenne (1972), using immunocytoadherence, reported antigenbinding cells from a hydrocortisone resistant pool of thymocytes. Furthermore,
about 75% of sensitized spleen cells binding
SRBC are theta positive and sensitive to
azothioprine. Modabber et al. (1970) found
specific antigen binding cells in mouse
99
CELLULAR COOPERATION IN AMPHIBIA
thymus which could be inhibited by cross
reactive materials. Recently, rosette formation by human lymphocytes has been inhibited by anti-T cell serum (Words et al.,
1973).
The situation in the chicken seems
somewhat clearer with regard to RFC origins, since bursectomy of chicken embyros
(Hemmingsson and Aim, 1972) suppresses
both RFC and PFC formation (cf. Crone et
al., 1972). Rodent RFC development is an
active secretory process at 37 C, while at 4 C
RFC formation seems related only to the
presence of antibody pre-existent on the
spleen cell surface. No secretory activity can
be discerned (Elson et al., 1972).
By adopting the S~, S+ RFC classification
of Greaves etal. (1970), I have attempted to
visualize the events previously speculated
about from the dose response curves. Figure 3 shows the time course curves after
the number of S" (ARC) RFC have been
sorted out from S+ (APC) RFC. The data
appear in Table 1. The newt rosettes were
not fixed and the minimal number of adherent red blood cells counted as a S~ rosette
is three. S+ RFC must have several layers of
bound erythrocytes, but these need not
adhere to the entire spleen cell surface. The
rosette illustrated in Figure 1 is typical of
many S+ RFC and the distribution of adherent erythrocytes suggests localization of
determinants on the sensitized cell surface.
Amphibian spleen cells are four times
larger than the mammalian cells and are
therefore useful for visualizing these local
areas of attachment. Amphibian rosettes
are also relatively stable when compared to
mammalian RFC.
The S~ (ARC) RFC response curves correspond closely to those involving total
RFC/106 counts except for two important
differences. According to the first, the peak
number of ARC of the 25% HRBC dosage
curve is 1000 RFC/106 lower than that
stimulated by the next lower dose (0.25%
HRBC). This agrees with information already considered from rodent studies;
higher doses may affect APC appearance
without as much ARC or "helper" activity.
That the 25% HRBC challenge was the only
one which initiated a relatively high
number of S+ (APC) RFC by 8 days is also in
IMMUNOGEN (HRBC) DOSE
25X — —
O.25X - - - - 0.0025*
PRIMARY RESPONSE
T. viridescens
o
SPLEEN
U
S
2000
0
T7 4ooo
6
DAYS
FIG. 3. The response curves for ARC (S") RFC and
APC (S+) RFC following challenge by three different
immunogen (HRBC) doses in the spleen of the newt,
T. viridescens.
agreement with this thesis. Another difference is the flattening of the "shoulder" between 2 and 4 days with the two higher dose
challenges. This feature of the results offers some support to the earlier speculation
that "helper" activity may be accompanied
by a loss of ARC (S~, RFC) from the spleen.
The increase in total number of RFC after 4
days can clearly be accounted for, at least in
part, by an increase in S+ (APC) RFCInformation about both S~ and S+ RFC
seems less reliable than the data on total
RFC. The standard deviations, even between assays using the same pools of cells are
large. This unreliability suggests an instability of outer layer adherent red blood cells
such that even minor differences in shearing forces generated during resuspension,
by gentle rotation of the incubation mixtures, may be sufficient to loosen them.
RBC adherent on the surface of a spleen
cell would appear to be more firmly held.
Unlike the mammalian situation where at
37 C the ARC is particularly fragile and the
APC is visualized (Haskill et al., 1972), the
100
LAURENS N. RUBEN
amphibian cells, when incubated at 4 C,
show stability of ARC but some instability of
APC.
That some antibody secretion can occur
at 4 C from poikilotherm spleen cells is an
important advantage, making it possible to
visualize both ARC and APC at the same
incubation temperature. This distinction
can be enhanced by warming the mixture
of sensitized newt spleen cells with appropriate erythrocytes for 2 hr prior to incubation in the cold. These data clarify the RFC
designations suggested above (Table 2).
There is no alteration in the proportion of
S~ to S + RFC at 2 days, but the percentage
of S+ RFC does increase at 8 days. This is to
be expected if antigen binding cells generated early in the response are ARC, while
those appearing later are APC. It is interesting that the proportion of S" and S+ antiHRBC RFC observed under these conditions (pre-warming) agrees with the proportion of anti-SRBC Tand B RFC reported by Bach and Dardenne (1972) for the
rodent.
"HELPER" ACTIVITY: A FUNCTION OF S" RFC?
It is essential to demonstrate that the
morphological criteria regarding whether
RFC are nonsecretory or secretory are real.
In other words, can one relate these two
types of RFC (S~ and S+) to their suspected
physiological behaviours, "helper" activity
and antibody production? While combining immunocytoadherence with carrierhapten immunization, it is possible to vary
by 2-day intervals the time between preimmunization and challenge by TNPconjugate. This variation provides a time
course response curve of the degree of
"helper" activity (i.e., anti-TNP RFC) as a
consequence of preimmunization. All assays are performed 8 days after presentation of the hapten. Variation in the concentration of the priming dose can provide
further information concerning the effect
of immunogen dosage on "helper" activity.
The data appear in Table 3. The red cell
used for preimmunization is CRBC at low
(0.0025%) and high (25%) concentrations.
As before, the hapten-conjugate is 10%
CRBC-TNP in all cases and the assays are
against 1% HRBC and 1% HRBC-TNP.
The volume of erythrocytes injected ip into
the newts is always 0.2 ml. Control experiments use SRBC as the primer red cell and
presentation of CRBC-TNP without any
preimmunization. No anti-TNP RFC are
formed in the absence of CRBC preimmunization. The response curves showing
dose and time dependence appear in Fig-
Incubations of both immunized and unimmunized newt spleen cells with high
hemagglutinin titer (1:512) newt antiHRBC sera and sera taken at the times of
peak anti-HRBC cellular responses (Kapp
and Benacerraf, 1972) failed to alter either
the number of RFC or the proportions of
S~ and S+ RFC. It would appear, then, that
cytophilic antibody is not generating false
RFC or converting S" to S + RFC.
I ABI.K 2. Effect ofpre-rvarming'on newt spleen cell multiple layer (S+RFC) antigen-binding of HRBC: (primary response).
HRBC dosage
0.0025%
Cold
%S-
b
25%
%S +
% S-
% S+
% incr.
S+
% S-
9£ S +
%S-
%S +
% incr.
S+
9
25
90
57
10
33
1
8
91
79
9
21
89
52
11
38
2
19
Assay day
2
8
91 C
75
Warm
Cold
Warm
a
Pre-warming entails placing cellular mixtures at 25 C for 2 hr prior to the usual overnight incubation in the
cold.
b
% S - refers to the % non-secretory RFC/10 6 spleen cells, % S+ refers to the % multilayered or secretory
RFC/10 6 spleen cells.
c
These figures are averages of two different experimental series. They represent 16 chamber counts as
described for Table 1.
101
CELLULAR COOPERATION IN AMPHIBIA
TABLE 3. Measurement of "helper" activity in newt spleen cells by varying the time between challenges of carrier (CRBC) and
carrier-hapten (CRBC-TNP) and carrier dose (CRBC-TNP = 10% in all cases).
Carrier
Carrier
dose
CRBC
0.0025%
CRBC
25%
SRBC
SRBC
0.0025%
b
25%
—
Days between
injections
Anti-TNPa RFC/10"
("helper" activity)
% Increase
in S + RFC
2
7113 ± 613
6642 ± 1775
2551 ± 424
246 ± 346
1636+ 114
1791 ± 1215
2404 ± 620
0±0
0±0
0±0
0±0
79
85
95
100
66
69
100
—
4
6
8
2
4
6
8
2-8
2-8
—
a
Anti-TNP RFC/106 are determined by subtracting the RFC/106 assayed with HRBC from the number
assayed with HRBC-TNP. All assays were done 8 days after CRBC-TNP injection.
b
Control with 10% CRBC-TNP injected at day 0 and assayed 8 days later.
ure 4, and the data show S , S+, and total
RFC/106.
The results clearly support the previous
conclusions that low concentrations of immunogen which generate high numbers of
S~ RFC are particularly effective in
stimulating "helper" activity, i.e. enhancement of anti-TNP RFC. This enhancement
provided by low but not high dose priming
correlates with the excess of S~ RFC generated in response to low but not high dosage
challenge. Furthermore, most of the enhancement in the number of anti-TNP
RFC can be accounted for by S+ RFC, expected if anti-TNP activity is a function of
APC and not of ARC which should respond
to preimmunization by carrier erythrocytes. "Helper" activity is maximized in the
2- to 4-day period following preimmunization.
ANTIBODY PRODUCTION: A FUNCTION OF S+ RFC
"Helper" activity can now be maximized
by using low dose (0.0025% CRBC) priming followed by CRBC-TNP 4 days later. If
the times of assay are varied by 2-day intervals thereafter, information can be derived
concerning the kinetics of specific (antiTNP) antibody production by RFC. Figure
5 clearly demonstrates that anti-TNP RFC
activity appears first after 6 days and
quickly rises to a peak at 8 days after TNP
presentation. This activity disappears from
the newt spleen just as quickly, i.e., 2 days
later. This result is confirmatory of peak S+
RFC formation at 8 days in primary responses. Further, it supports the notion
that multilayered rosettes are APC, since
nearly all of the anti-TNP activity can be
accounted for by increase in this type of
rosette (S+ RFC).
These results employing a combination
of poikilotherm spleen cells, imTHE KINETICS OF HELPER ACTIVITY
T. viridescens
SPLEEN
Priming Dose - Day 0
9000
25% CRBC
0.0025% CRBC
SOOO
|
CRBC- TNP = 10%
1.
7000
Assays 8 days post-TNP injection
1
***^ I\
-> 6 0 0 0
\
\
£
5000
Z
4000
\
\
\\\
\
\\
< 3000
V
^
T
LU
'
2000
^
1
\
T
\ \
\ \
1000
0
1
2
3
4
5
6
7
1
DAYS BETWEEN PRIMING AND TNP PRESENTATION
FIG. 4. Anti-TNP RFC enhancement, i.e., "helper"
activity is both dose and time dependent in the newt
spleen.
102
LAURENS N. RUBEN
THE KINETICS OF ANTIBODY PRODUCING
CELLS (APC) IN THE NEWT SPLEEN
0.0025X CRBC - 0 DAY
10*CRBC-TNP-DAY4
4
6
•
10
12
14
16
ASSAY DAYS AFTER TNP PRESENTATION
FIG. 5. The kinetics of anti-TNP RFC activity in the
spleen of the newt.
munocytoadherence and carrier-hapten
immunization then, suggest that both
"helper" and antibody producing cell activities are being visualized when one observes the rosette population shift from S~
to S+ RFC as the immune response progresses. A remote possibility that ARC and
"helper" cells may be separate populations
remains, since "helper" cells could be stimulated proportionately but may not, like
ARC, bind antigen.
THE ANTI-RED CELL RESPONSE IN RANA PIPIENS
Adult Rana pipiens (the leopard frog) are
members of the most advanced order (Anura) of Amphibia (Noble, 1931). Recently,
Levin (personal communication) tested the
effect of different dosages of SRBC (by
using immunocytoadherence) on the antired cell response of spleen and thymus
from adult Rana pipiens. The volume of the
challenge dose was increased to 0.5 ml injected ip, since frogs are substantially larger
than newts. The SRBC concentrations were
equivalent to those used for HRBC in the
newt studies (0.0025%, 0.25%, and 25%
SRBC). All the other experimental conditions were identical except for the dissociation medium for frog spleen and thymus
cells. The medium used for newt cell
maintenance is 7 parts L-15 (LeibowitzGIBCO):2 parts twice glass distilled water;
the medium for frog cells is 5 parts L-15
and 4 parts water (Balls and Ruben, 1966);
in the past Kidder et al. (1973) used one
part decomplemented fetal calf serum.
The data appear in Tables 4 and 5 and in
Figures 5, 6, and 7. The three dose response curves for frog spleen cells are
clearly similar to the newt's. The same time
translocations appear with low dose priming and generate the most rapid cellular
response. Each more concentrated dose
stimulates a higher activity level at the time
of peak response. The standard deviations,
however, of the frog data represent variations among individuals since each adult
frog spleen is sufficiently large to assay
separately. Newt spleens, on the other
hand, must be pooled (four) and therefore
standard deviations illustrate variations between different cellular pools taken from
different groups of animals.
An additional advantage in using the
frogs is that their paired thymus is, like the
spleen, large and can be pooled for immunocytoadherence assays. Information
derived from thymus cells is particularly
useful because all thymus antigen binding
TABLE 4. Primary immune response in Rana pipiens adults (total RFC/106 viable spleen cells counted).
Assay
day
2
4
6
8
10
12
16
H R B C dose
0.0025%
0.25%
a
3875 ± 619
1552 ± 171
*
1140 ± 481
*
1011 ± 112
615 ± 58
1790 ±
1386 ±
4874 ±
2727 ±
275
455
662
822
*
1925 ± 682
944 ±414
25%
1035 ± 265
1022 it
3246 dt
7550 dt
3246 it
2546 dt
1420 ±
282
156
1737
716
265
187
' Since individual spleens were assayed, standard deviations refer to variation among individuals (4).
No assays were performed on these days.
!
103
CELLULAR COOPERATION IN AMPHIBIA
TABLE 5. Primary immune response in Rana pipiens adults (total RFC/10e viable thymus cells counted).
Assay
day
2
4
6
8
10
12
16
0.0025%
0.25%
25%
2615 ± 624"
1839 ± 104
2493 ± 121
2407 ± 786
2344 ± 122
1905 ± 449
3016 ± 1092
3120 ± 1088
1508b
1348 ± 476
1250"
1158 ± 330
775 ± 268
896b
*
*
337"
296"
1425 ± 229
518 ± 8
a
Since thymii from 4 individuals were pooled for each assay, standard deviations refer to differences between
2 different cellular pools.
b
These figures are from only one cellular pool of thymocytes.
* No assay was performed on this day.
cells are S RFC regardless of challenge
dose and time. This is supportive of the
likelihood that splenic S" RFC or "helper"
RFC are thymus-dependent cells in the
Amphibia, as they are in mammals. In fact,
the embryonic thymus of Rana pipiens may
produce most adult lymphocytes (Turpen
et al., 1973). Larval thymectomy ofTriturus
alpestris appears to suppress RFC formation
(Tournefier, personal communication).
CONCLUSION
There is similarity in the dose-dependent
IMMUNOGEN (SRBC) DOSE
25*
0.25S
response patterns between newts and frogs.
Furthermore, there is agreement in the
cytodynamics of the primary antierythrocyte responses among Triturus viridescens (the newt); Alytes obstetricans,
Xenopus laevis (both primitive Anura), Bufo
marinus, and Rana pipiens (both advanced
Anura). This suggests that visualizations of
the primary immune response and cellular
cooperation are more likely to be patterns
fundamental to the vertebrate group than a
phenomenon which evolved uniquely
within the newts. These common features
support the view that ancestral amphibians
may have possessed these characteristics
which evolved in all modern amphibian
groups. These may also have evolved in
ancestral reptiles which eventually gave rise
to modern reptiles, birds, and mammals.
PRIMARY RESPONSE
Rana pipiens
SPtEEN
IMMUNOGEN
(SRBC) DOSE
25*
0.25%
0.0025%
PRIMARY RESPONSE
Rono pipiens
THYMUS
DAYS
FIG. 6. The effect of immunogen (SRBC) dose on
the primary immune response in the spleen of the
adult frog Rana pipiens.
>
DAYS
12
14
16
FIG. 7. The effect of immunogen (SRBC) dose on
antigen binding in the thymus of the hogRana pipiens.
104
LAURENS N. RUBEN
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