ami Cell
(1991) 69. 225-234
Interleukin-2 and phytohaemagglutinin stimulat!
proliferation of tunicate cells
DAVID A. RAFTOS, DAN L. STILLMAN AND EDWIN L. COOPER
Deparimenl oj Anatomy and Cell Biology. School ofMedicini'. Cnivcrsity of California, at Los
Angck'.s. Los Angeles. California 90024-1763. USA
(Submiilcc/ 14 Jamtary 1991. Accepted for publtcation II June 199L)
Summary Profilcralivc responses of cells in lunicato pharyngca! cxplants to human intcrlcukins and
niiiogcnic Icflins were icstcd. Increased lritiaied-thymidine (pH]-TdR) uptake was detected among
pharyiigeal cells incubated with recombinanl human interleukin-2 (IL-2). and phytohaeniagglutinin-P
(PHA-P). Responses to IL-2 were dose-depcndenl and allected !yniphocytc-like cells. Enhanced
proliferation was stimulated by IL-2 in the absence of co-sliniu!ants and was not synergi/cd by
eo-incubaiion with human intcricukin-l (IL-1) or PHA-P. Anti-IL-2 polyclonakiniibody inhibited the
stimulatory activity of recombinant human interleukin-2 {rhlL-2). Of three lectms tested
(concanavalin-A [Con-A]. pokcwecd mitogen [PWM] and PHA-P). only PHA-P proved to be
mitogcnic. Con-A and PWM did not significantly increase proliferative activity even though both
lectins were capable o!" binding pharyngeai cells as revealed by How c\tometry and lluorcscence
microscopy. Similarly, human IL-I had no etil'eci on ['H]-TdR uptake either alone or in combination
with IL-2 and PHA-P. These data suggest thai the functions of some interleukin-likc c\lokines have
been conserved durmg evnlution.
INTRODUCTION
Cytokine-likeactivities have been identified in a
number of eclotherniic vertebrates and invertebrates (1-7). Prendergast and Liu have purified a protein Trom sea stars (sea star factor)
whieh stimulates delayed inflammatory skin
reaetivity. monoeyte ehemotaxis. macrophage
migration inhibition and tnacrophage activation
in mammalian assay systems (1). In its native
species, sea star factor initiates innammatory
reactions charaeteri7cd by coelomocyte adhesion and spreading (2). Similarly. Beck and
Habicht have isolated an interieukin-l (IL-1)like molecule from sea stars (3). This 29.5 kDa
protein initiates the proliferation of mammalian
tfiymoeytes and fibrobiasts (3). The stimulation
of thymocytes by sea star IL-like laetor is inhibited by anti-human-IL-1 antibody, which
suggests an antigenic relatedness between mamalian IL-1 and its echinoderm counterpart (3).
Interleukin-like molecules have also been
identified in tunicates. the most phylogenetieally pritnitive members of the phylum Chordata (4). Plasma and cell extracts from eight
tunicate species can stimulate the proliferation
of mammalian thymocytes. Partially purified
lymphocyte activating factors from tunieatcs
also induce the proliferation of mammalian
eytotoxic T cells (4). .As with echinoderm lL-1likc proteins, the stimulatory effects of tunicate
molecules on mammalian thymocytes are inhibited by anti-human-IL-l antibody (4).
The identification of interleukin-like activity
in invertebrates suggests that cytokines. which
may be related to mammalian interlcukins.
regulate the proliferation of invertebrate cells in
vivo. However the mitogenic effects of invertebrate interieukin-like proteins have only been
demonstrated in assays of mamtnalian cells
(3.4). Thete is no information regarding the effect of interleukins on invertebrate cells. Here
Correspondence: Dr D. A. Raftos. Department of Anatomy and Cell Biology. School of Medicine. University of
California at Los Angeles. Los Angeles. CA 90024-1763. USA.
Abbreviations used in this paper: ASW. artifieial seawater: BrDu. bromodeoxyuridine; Con-A. concanavalin-A;
GA. granular anioebocyte: hlL-1. human interleukin-l: ['HJ-TdR. tritiated-thymidine; Igti, immunoglobiilin G:
IL-I. interieukin-l: IL-2. interlcukin-2: LLC. lymphocyte-like cell: PBS. phosphale buffered saline: PHA. phytohaemagglutinin: PHA-P. phytohaemaggiulinin-P: PWM. pokewced mitogen: rhlL-lu. reeombinant human interleukin-l H: rhlL-2. reeombinant human interleukiii-2: SI. stimulation index; TTCM. tunicate tissue culture
medium.
~.
226
D. A. KAFTOSt/.-li.
we show that mammalian interleukins and other
mitogens can stitnulate the proliferation of invertebrate (tunicate) cells. This study has been
facilitated by the development of a method for
the in mw culture of cells from the solitary tunicate. 5M't'/fl(7rtri;. Circulatory haemoeytesand
single cell suspensions from solid tissues of S.
clava do not proliferate in viiro (8). However we
have established a technique for culturing
pharyngeai explants whieh maintains both ceil
viability and proliferation for extended periods
(8).
MATERIALS AND METHODS
Tunicates
S. clava were purchased from Marinus Inc.
(Long Beach. CA. USA). They were maintained
in a refrigerated {I 5T) aquarium filled with 180
L artificial seawater (3.4% w/v. Instant Ocean.
Aquarium Systems. Mentor, OH. USA).
Chemicals and reagents
KeLotnbinant human interleukin-la (rhIL-la:
1.3 X 10' U/mg). purified human interleukin-l
(hIL-I; 1.15 U/mL) and recombinant human
interleukin-2 (rhIL-2: 6.2X10*' U/mg) were
purchased from Collaborative Research Inc.
(Bedford. MA. USA). Tritiated thymidine([^H]TdR; 6.7 Ci/mmol. 1 jtCi/^iL) and Eeolite
scintillation cocktail were obtained from ICN
Biomedicals (Costa Mesa. CA. USA). Mouse
anti-broniodeoxyuridine monoclonal antibody
(clone B44) was produced by the Beeton Dickinson Monoclonal (^cnter (Mountain View. CA.
US.A,). Rabbit anti-human IL-2 polycional antibody was purchased from Genzyme (Los
Angeles. CA. USA). Immunohistoehemical
reagents were from Vector Laboratories (Burlingame. CA. USA). All other chemicals and reagents were supplied by Sigma Chemicals (St
Louis, MO. USA).
Media
All media were stored at 4 T and sterilized by
filtration (0.2 ^m filters. Costar. Cambridge.
MA. USA) prior to use. Artificial seawater
(ASW) that had been removed from the aquarium in which tunicates were tnaintained was
used to wash explants and to prepare culture media. //( viiro cultures of pharyngeai
tissue were established in tunicate tissue cul-
ture medium (TTC M) which was ptepared in
ASW and contained powdered RPMI-1640
(454 mg/L). streptomycin sulfate (40 mg/L).
penicillin sulfate (10-* U/L) and .V. clava plasma
(20% v/v; 8). Phosphate bufiered saline (PBS:
120 mmoI/L) incorporating heat-inactixated
normal horse serum (0.2"!'o v/v) was used to wash
tissue sections and to dilute reagents during
immunohistoehemical procedures.
Tissue culture protocols
The effects of interleukins and leetins on cell
proliferation were tested in cultures of pharyngeai tissue from S. clava. The culture system has
been described in detail previously (8|. Cultures
were established by dicing pharyngeai tissue dissected from live tunicates into 2 X 2 X I mm
explants. Explants were washed by orbital rotation in ASW and then transferred (I explant
per well) to 96-well sterile culture plates (0.32
em- growth area, tlat bottomed. C ostar, Cambridge. MA. USA)containing200|.iL TTCM per
well. Cultures were maintained in normal atmosphere at 15°C. Explants were transferred to
plates containing fresh media every 3 days. Interleukins and iectins were added to TTCM
immediately prior to the commencement of
culture and with each change of medium. The
concentration of interleukins was calculated in
half-maximal units/niL (U/mL) based on the
manufacturer's data for the stimulation of mammalian cells. According to this information.
the dose o'i rhIL-2 yielding half-maximal proliferation of murine eytotoxic T cells was
1.8 U/mL.
Immuno-absorption of rhIL-2
The spjeeificity of responses to rhIL-2 was confirmed by absorbing rhlL-2. prior to its addition
to culture media, with anti-IL-2 antiserum.
Twenty-five microlitrcs undiluted rhlL-2 was
incubated (3 h. room temperature) with 200 ^L
undiluted anti-IL-2. or with non-specific rabbit
immunoglobulin G (IgG) as a control. Insoluble
protein A (100 )jg in 180 ^L TTCM) was then
added and the incubation continued for a
further 1 h. The incubate was centrifuged
(UOOOXtr. 5 min) and the supernatant added
to culture medium as described abo\e.
Quantification ofpHJ-thymidine incorporation
Explants were incubated with
(5MCi/mL) for 18 h (15°C). Exeess thymidinc
TUNICATE CELL PROLIFERATION
was removed by 4 washes in ASW (I h/wash
with rotation). Indi\idual cxplants were then
transferred to scintillation vials and digested in
250 ML trypsin (2%w/v in ASW. 24 h. 36T)
prior to the addition of 2 niL Ecolite scintillation cocktail. Radioactivity was counted in a
Beckman LS 3I5OP liquid scintillation counter.
Proliferative activities were calculated as
stimulation indexes (SI) by the formula:
SI=[^H]-TdR cl/min incorporated inio cxplanis
ciillurcd with interleukins and lectins i- ct/min in
control tissues cultured in TTCM vvithout additives.
The specificity of [^H]-TdR uptake was confirmed by competitively inhibiting incorporation with non-isotopic thymidine (1 mg/mL)
that was added to incubates 30 min prior to [-'H]TdR. Additional cxplants were exposed to
gamma radiation (5000 rad) irom a ^"Co source
and then washed extensively in ASW prior to the
commencement of culture. This dose of radiation has been shown previously to inhibit proliferative activity in pharyngeal cultures (8).
Immanobiswchemical analysis of
bromodeoxyuridine uptake
Cultured pharyngeal tissue was incubated with
bromodeoxyuridine (BrDu: 10 |.imol/L in
TTCM) for' 4h at 15°C. Explants were then
washed three times in ASW and fixed overnight
in formaldehyde (4"/() v/v in ASW). Fixed tissues
were embedded in paraffin and sectioned
(7nm). After hydrolysis in HCl (3N. 30 min)
and digestion in pepsin (0.5% w/v. pH 1.5. 30
min), sections were ineubated with mouse-antiBrDu monoclonal antibody (1:400 dilution in
PBS. 30 min). Bromodeoxyuridine-positive
cells were then stained using an avidin-biotinalkaiine phosphatase immunohistochemical kit
(Veetastain ABC-AP. Veetor Laboratories) according to the manufacturer's instruetions. Differential counts of BrDu-positive cells were
made using the criteria of Wright (9) to discern
morphologically discrete eell types.
Binding of lectins to pharyngeal cells
The ability of lectins to bind pharyngeal cells
was tested by fluorescent labelling. Single cell
suspensions were prepared by macerating
pharyngeal explants with scissors, repeated passage through 18.5 gauge syringe needles and filtration through nylon mesh (20 ^im pore size).
227
Suspensions {2X lOf" cells in 1 mL ASW) were
then ineubated (1 h. room temperature) with
lOtig/mL of FITC-conjugated Con-A. PHA-P.
PWM, PHA-P combined with A-acetylgalactosamine (100 |.ig/mL). or PH.A-P eombined with
A'-acetyiglucosamine (lOOjig/mL). After three
washes in ASW, the binding of tluorochromeconjugated lectins was analysed with a FACScan
flow cytometer (Becton Dickinson, Mountain
View. CA. USA). Debris and eell aggregates were
eliminated by gating in forward v.v 90° light scatter. The frequency of labelled cells relative to the
gated population was determined in red v.vgreen
fluorescence plots. Propidium iodide (1 ng/mL)
was added to samples immediately prior to
analysis so that dead eells eould be eliminated
from calculations. Aliquots of lectin-incubated
suspensions were also inspected with a fluorescence microscope to determine the morphology
of labelled eells. Differential counts of labelled
eells were also based on the morphological criteria of Wright (9) with eells being allotted to
diseretegroups(lymphocyte-like. amoeboid and
vesieulated) by phase microscopy.
Cell viability assay
The viability of pharyngeal cells was tested by
eosin Y dye exclusion. One hundred microlitre
aliquots of single eeil suspensions prepared from
eultured explants (2 X [Q^ eells/niL) were mixed
for 5 min with IO0|.iL eosin Y (0.5% w/v in
ASW) containing trypsin (1% w/v). The relative
frequeney of eosin Y-positi\e cells was then determined with a haemocvtometer.
Statistical analysis
Statistical calculations were made using Solo
statistical software (BMOP Statistical Software
Inc.. Los Angeles. C.A, LiSA). The significance of
differences between treatments was determined
by one-way analysis of \ariance (10), Variation
between mean values was considered to be significant if F-ratios yielded probabilities of less
than 5%. interleukins and lectins were considered to be mitogenic if they stimulated significantly (P<0.05) greater ["^H]-TdR uptake
than that detected ineontrol explants cultured in
TTCM alone. The dose of rhlL-2 predicted to
induce half-niaximal stimulation of cxplants
was determined by interpolation of a minimum
weighted least squares linear regression which
plotted the stimulation indices for explants cultured in 5-fold dilutions of rhIL-2 against log
228
D. A. R.AFTOS A;7M/..
transformations of the corresponding rhIL-2
doses (10).
Table 1. ['H]-TdR uptake hy pharyngeal cxplants.
Culture medium
RESULTS
idine uptake hy control cultures
Pharyngcal cxplants cultured in TTCM without
Icctinsor intcrleukins incorporated 8374 + 804
ct/min (/? = 26) [-^H]-TdR when tested after 3
days in riliv. Similar values were obtained after
6 and 9 day culture periods. Competitive inhibition using excess non-isotopic thymidinc reduced [-^HI-TdR incorporation to 24 ± 4%
(/j= 10) of normal levels (7?<0.05). Similarly,
explants cultured lor 9 days after irradiation exhibited only 17 ± 2.1 %(/('= 10) of the [^'<]-TdR
uptake evident in cxplants cultured in TTCM
alone (F<0.05).
Enhanced j^Hf-thymidine uptake in the
presence of IL-2
IL-2 enhanced [^H]-TdR uptake by pharyngcal
explants {Fig. I). Prolifcrativc activity increased
in a dose-dependent fashion. Alter 3 days, concentrations of I U/mL rhlL-2 were not significantly (F>0.05) stimulatory, whereas 125
U/mL yielded 2.7 X the [^H]-TdK uptake evident in explants cultured without rhIL-2
(P<0.05). Based on this dose-response, the
concentration of rhIL-2 predicted to yield halfmaximal stimulation was 4.9 ± 0.6 U/mL. The
[-^Hl-TdR uptake (ct/min)
TTCM
rhlL-2
RahlL-2 absorbed rh!L-2
RIgCi absorbed rhlL-2
6728 ±827
15936± 1346
9082± 1089
12011 ± 918
Pharyngeal explants were cultured for 3 days in
TTCM. 20 U/niL rblL-2. or 20 U/mL rhlL-2 and
pre-incubated with either rabbit anti-human IL-2
(RahlL-2) or non-specitic rabbit IgG (RIgG)
(n = 20 ± s.e.m.).
dose-dependent pattern of [^H]-TdR uptake
stimulated by rhIL-2 remained constant overa 9
day period. Stimulation indexes lor four dilutions of rhIL-2 (1-125 U/mL) did not difler
significantly (P>0.05) between explants tested
after 3. 6 and 9 days.
Absorption of IL-2 activity
The stimulatory effect of rhlL-2 on pharyngeal
expiants was inhibited by immuno-absorption
with anti-IL-2 (Table I). Explants cultured in
media prepared with anti-IL-2 absorbed rhIL-2
incorporated significantly (/'<0.05) less I'H]TdR than tissue cultured vvith untreated rhIL-2.
Pre-ineubation of rhIL-2 with non-specific rabbit IgG had no such eftect.
Failure of human IL-1 to enhance
pHJ-thymidine uptake
In contrast to IL-2. IL-I had no eftect on ['H]TdR uptake (Fig, 2). No significant {P>0.Q5)
stimulation was apparent overa 6 day period in
the presence of 1-50 U hIL-l/mL. Similarly,
incubation with 1-50 U rhII-la/mL had no significant {P>0.05) inductive effect (data not
shown).
Comhined effect of IL-1 and IL-2
Time (days)
Fig. I. Stimulation of('H]-TdR uptake (stimulation
indexes. SI) by pharyngcal c.vplanls cultured for up to 9
days in increasing concentrations of rhlL-2 ( « ^ 9 .
bars = s.e.m.).
Human IL-1 did not enhance the responsiveness
of explants to rhlL-2 (Fig. 3). ( o-culture with
hlL-l and a sub-mitogenic dilution of rhlL-2 (I
LJ/mL) did not significantly (/^>0.()5) increase
('H]-TdR uptake relative to controls cultured
without interleukins. Similarly, stimulation indexes for explants eo-incubated with hlL-1 and
stimulatory doses of rhlL-2 (>5 (U/mL) did not
vary significantly (/'>0.05) from those of tissue
cultured with the corresponding rhlL-2 concentrations alone.
229
TUNICATE CELL PROLIFERATION
Binding oflectins to pharyngeal cells
Time (days)
Fig. 2. Stimulation indexes (SI) for pharyngeal explants cultured for 3 and 6 days with increasing concentrations of hlL-1 ( / O 9 , bars = s.c.m.).
All three of the leetins tested (Con-A. PWM.
PHA-P) were capable of labelling pharyngeal
cells. Flow cytometric analyses revealed that
83%. 35% and 20% of pharyngeal cells were
bound by detectable quantities of Con-A. PWM
and PHA-P. respectively (Table 2). The addition of A'-acetylgalactosamine inhibited the
binding of PHA-P to cells so that enhaneed
fluorescence, relative to unlabelled controls,
eould not be detected. However, co-ineubation
with A'-acetylglucosamine did not significantly
(P>0.05) in'hibit the binding of PHA-P.
Fluorescence microscopy revealed that all of
the cell types found in single cell suspensions of
pharyngeal explants were labelled by the three
leetins. There was no significant (F>0.05)
difference in the frequency with which the three
leetins labelled the various cell typres
(Table 2).
Effect oflectins on pHf-thymidine uptake
1
5
IL-2 (U/mL)
25
Fig. 3. EReet of co-eulture for 3 days with various
combinations of hIL-l and rhlL-2 on the uptake of
[-'H]-TdR by pharyngcal expiants (stimulation indexes. Si) ('?> 9. bars= s.e.m.).
Con-A and PWM (1-25 pg/niL) had no effect
(F>0.05)on the uptakeof ['H)-TdR by explants
after 3 days in eulture (Fig. 4). In contrast.
PHA-P increased [^HJ-TdR uptake. Although
PHA-P was not stimulatory at a dose of I |.ig/mL
(F>0.05), higher coneent rations (5 and
25 |jg/mL) signifieantly (F<0.05) enhaneed the
uptake of ['H]-TdR relative to untreated controls (Fig. 4). However this stimulatory effect
was transient (Fig. 5). Increased [-'H]-TdR uptake could not be detected at any of the concentrations tested after 6 and 9 days of incubation
with PHA-P. The failure to maintain enhaneed
[•'HJ-TdR incorporation corresponded with a
decreased viability of cells in the presence
of PHA-P. Only 62.3 + 4.8% («= 5) and
45.4 ± 6.3% (n^5) of pharyngeal cells were
Table 2. Percentages of tunicate pharyngeat eells labelled by FITC-conjugated leetins and the percentages ol three
discrete cell types comprising the labeiled celi population* (/i = 3 ± s.e.m.).
Lcctin
Ceils iabeiled (%)
LLC
Labelicd ceils (%)
Amoeboeytes
Vacuotated
None
O.i2 + O.Ol
83.1 ± 1.2
35.4 ±4.2
20.6 ±3.1
i8.3±4.3
0.56 ±0.1
i 5.4 ±2.1
21.7 ±3.4
18.9± i.4
21.4 + 2.2
69.8 ±
65.4 ±
63.3 ±
64.5 ±
14.1 ±2.4
i 3.0 ±2.2
17.8 ± i.6
14.2 ±3.0
Con-A
PWM
PHA
I'iHA + /V-acetyigiucosamine
?H.\ + ,'V-acetylgalactosamine
i.9
i.3
1.8
1.0
*Wherc no Icciin was added frequencies for ibe different cell types are based on the total (unstained) eeil
population.
230
D, A. RAFTOS ET.H.
1
Mitogen
Fig. 4. Stimulation indexes (S!) for pharyngeal expluiits cultured for 3 days will) increasing concentrations ol" Con-A. PWM and PHA-P (/j=10.
bars = s.c.m.).
PHA-P
Fig. 6. Stimulation of ['H)-TdR uptake (stimulation
indexes. SI) by pharyngeal cxplants al'lcr .1 days of coculture with various combinations ol" PHA-P and
rhIL-2 (H3=9. bars = s.e.m.).
nificantly <i'>0.05) from that induced by rhIL-2
alone. Similarly, a stimulatory dose of PHA-P
(5 jig/mL) combined with rhIL-2 yielded no
synergistic increase in [^H)-TdR uptake. Coculture with 5 pg/mL PHA-P and rhIL-2 resulted in [^H)-TdR incorporation equivalent to
that resulting from either PHA-P or rhIL-2
administered independently (/'>0.05).
PH.VP also failed to enhance [^HJ-TdR uptake in response to IL-I (Fig. 7). No significant
(/'>0.05) stimulation was evident in cultures
containing both hIL-l and a sub-mitogenic conTime (days)
Fig. 5. EflTect of duration of culture with various
doses of PHA-P on llic stimulation of [^H]-TdR uplake (stimulation indexes. SI) by pharyngeal explants
(/(= 10. bars = s.e.m.).
viable after 6 days of incubation with 5 and 25
^lg/mL PHA-P. respectively. Over the same period. 83.4 ± 3,3% {/)= 10) of cells from explants
cultured without PHA-P remained viable.
Failure of PHA-P to synergize responses to
interleukins
0
PHA-P did not synergize the stimulatory effect
of IL-2 {Fig. 6). [-^H]-TdR uptake by tissues cocultured with a sub-mitogenic concentration of
PH.A-P (I ng/mL) and rhlL-2 did not vary sig-
IL-1 (U/mL)
Fig. 7. EHect of co-culture for 3 days with PHA-P
a n d h l L - l on the stimulation indexes (SI) tor pharyngcal cxplants (/? ^ 9. bars = s.e.m.).
231
TUNICATE CELL PROLIFERATION
cenlration of PHA-P (1 Mg/mL). The ['Hl-TdR
incorporation resulting iVoin co-incubation with
hlL-1 and a mitogenic dose of PH.VP(5 ng^'mL)
did not vai7 (F>0.05) from that induced by
PHA-P alone.
Morphology of BrDu stained cells
Immunohistochemical analyses of BrDu-pulscd
explants revealed that two cell types (lymphocyte-like cells. LLC and granular amoebocytcs. GA) incorporated BrDu (Fig. 8). Other
cell populations {vacuolated ceils, epithelial cells
and hyaline amoehocytes) did not take up detectable quantities of BrDu. Lymphocyte-like
cells were small (5-7 ^im diameter), round eells
with large nuclei and limited, agranular cytoplasm (9: Fig. 8). Granular amoebocytes were
larger, round to ovoid cells (8-12 pm diameter)
with small nuclei and extensive granular cytoplasm (9: Fig. 8). Incubation with rhIL-2 stimu-
lated enhanced BrDu uptake by LLC (Fig. 9).
Twice as many BrDu-positive LLC were evident
in cultures incubated with 25 U/mL lL-2 than in
tissue that was not exposed to interleukin
(/•'<0.05). The frequency of BrDu-positive GA
increased over the range of IL-2 doses tested but
did not signifieantly exceed that of controls
which excluded \L-i (F>0.05).
DISCUSSION
Interleukin-2 and PHA-P stimulate the proliferation of tunicate pharyngeal cells. In vivo, the
pharynx is a major haematopoietic organ (1113). Lymphocyte-like stem cells undergo proliferation and differentiation to yield a variety
of morphologically distinct hacmoeyte types
(11.12). The proliferation of LLC is also stimulated by allogenic antigens indicating that these
cells have an additional immunoiogical function
t
Fig. 8. Bright field micrographs ofcells within pharyngeal explants. (A) Hematoxylin and eosin stained section of
an explant after 3 days ofculturc in TTCM (bar= 20 fim). (B) Imniunohistochcmically stained cells after BrDu
ircatmcnt of'an cxplani that had been cultured for ? days in TTCM containing 25 IJ/mL rhIL-2. BrDu-positive
cells have darkly siained nuclei (bars = 20 (im). .Abbreviations: A. amoebocyte: L. lymphocytc-likc cell; V. vesiculaled cell.
232
D. A. RAFTOS /:/ .1/
T
IL-2 (U/mL)
Kig. 9. Number of BrDu-positi\e LLC and G.-\ per
mm- (/' = 5. bars ^ s.e.m.) in sections oi' phar\ngcal
explants cultured for 3 days in increasing concenli'ations orrhiL-2.
(14). //) vilro. pharyngcal cxplants remain viable
for up to 70 days wiicn cultured in an osniotically compatible medium containing tunicate
plastna. RPMI-1640 and antibiotics (8). Cell
proliferation is maintained in these pharyngeal
cultures tor extended periods and can be quantilicd by llie incorporation of either BrDu or
['H]-TdR. The prolitcrative basis of BrDu uptake has been coniirmed previously by correlation vviih increased cell numbcrand sensitivity
to irradiation (8). Similarly. [''Hl-TdR has
freciuentK been used loanalyseccM proliferation
and dificrentiation in lunicates {11-13). In the
current study, a relationship between [-'HJ-TdR
incorporation into pharyngeal explants and
DNA replication is indicated by the inhibition
of pH]-TdR uptake after cither irradiation or
co-incubation vvith non-isotopie thymidinc. The
limited variation in ['H]-TdR uptake evident
between replicated samples (e.g. 8374 ± 804
ct/min. n^lt
for controls) confirms that this
quantification ol prolilerativcactivity isconsistent and reproducible.
Three leetins (PHA-P. Con-A and PWM) that
induce the proliferation of mammalian
lymphocytes were tested for their stimulatory
effects on tunicate cells. All three leetins bound
to tunicate pharyngcal celts as indicated by
lluorescent labelling. Differences that were
evident in the proportion of pharyngeal cells
labelled by the three leetins (Con-A. 83.1%:
PWM. 35.4%: PHA. 20.6%) probably rellect variations in the conjugation of lluorescent labels to
leetins or differences in the affinity of leetins for
target carbohydrates. None of the leetins dis-
criminated between the ditlerent pharyngeal cell
types.
Although all three leetins bound tunicate eells.
only PHA-P stimulated cell proliferation. This
induction by PH.\-P was transient. Enhanced
prolileralion could only be detected for 3 days.
Over longer periods, niitogenie doses proved
cytotoxic so that enhanced proliferation was not
evident. The pattern of inhibition resulting from
co-incubation with amino sugars suggests that,
as in mammals. PH.A-P binds to cells via A'acetylgalactosaminc.
The identification of leelin-mediated niitogencsis in tunicates could reflect a physiological
function for carbohydrate-specific proteins.
Many invertebrates produce endogenous carbohydrate-binding leetins (I 5). The conjugation
of endogenous leetins to carbohydrate moieties
on the surface of foreign particles may be a
predominant mechanism by which invading
pathogens are recognised by pliagoc\ tes (15,16).
Perhaps invertebrate leetins act not only in
this opsonic role but are also capable of stimulating proliferative responses to infection. Phagocytic cells from some invertebrates are both
sensitive to endogenous iectins and proliferate
in response to bacterial immunization (17.18).
The niitogenie activity of PHA-P in tunicates
could rcilect ihe existence of a lectin-mediated
activation pathway that is responsible lor such
adaptive proliferation. Certainly, the demonstration that Con-A and PWM bind to tunicate
cells but are not niitogenie suggests that PHA-P
initiates a specific transduction system rather
than non-specifically altering the physiological
state of resting cells.
The existence of such a transduction mechanism in tunicates need not imply the evolutionary
conservation of systems regulating cell proliferation. Endogenous leetins may be critical to recognition processes in both vertebrates and invertebrates (16.19.20). However the generalized
nature of the earbohvdrate ligands recognized by
leetins (A'-acetylgalactosamine in the case of
PHA) is such that chance cross-reactivities
should be relatively common between animal
groups. Cross-reactivily suggests only that the
cells of different taxa express the same transduetive carbohydrate moieties.
The capacity of mammalian IL-2 to stimulate
cell proliferation in tunicates is of greater phylogenetic consequence. Recombinant human IL-2
stimulates the proliferation of tunicate cells in a
dose-dependent fashion. Half-maximal stimulation of phar\'ngeal cxplants occurs at concentrations of IL-2 (4.9 U/niL)that are similar to
those required by mammalian CTL (—1.8
TUNICATE CELL PROLIFERATION
U/mL). Moreover, the ability of anti-IL-2 antibody to specifically inhibit stimulatory activity
confirms that rhlL-2. and not contaminants
within rhIL-2 preparations, was responsible for
enhanced proliferalion.
Unlike leetins. IL-2 gains its stimulatory effects upon mammalian T cells via a protein
receptor (Tac antigen) of exclusive specificity
(21-23). IL-2 dependent proliferative mechanisms that do not rely upon such a receptor have
not been identified (23). Given that the recognition of IL-2 in mammals is mediated by eoniplex and specific reactive sites, the funetional
cross-reactivity of human IL-2 in tunicates can
be explained in two ways. First, convergent evolution could have given rise to an unrelated
receptor that mimics the specificity of mammalian Tac antigens and also hastheeapaeity to
induce cell proliferation. Second, cross-reactivity may reflect the evolutionary conservation of
a receptor that has maintained sufficient fioniology to allow the recognition of lL-2 across
substantial phylogenetic distances.
By either of these explanations, a corollary to
the existence of a reactive site for human IL-2 in
tunicates is that endogenous lL-2-like ligands
also exist. Functional homologs of IL-2 have
been identified in birds, fish and amphibians,
even though cross-reactivities with mammalian
IL-2 are not always evident (5.6). Among invertebrates. Reck eta/, have found that fractionated
proteins from tunicates exhibit IL-2-1 ike activity
when tested in mammalian T cell systems (4).
Sueh endogenous IL-2-like activity either could
regulate the proliferation of tunicate cells
following antigenic stimulation or may modulate hacmatopoiesis. These potential Inunctions
are indicated by the nature of the cells which
respond to IL-2. IL-2 promotes the proliferation
of tunicate LLC. This cell type has at least two
functions. In visceral tissues, such as the
pharynx. LLC act as stem ceils which give rise to
circulatory haemocytes (11-14). The higfi level
of proliferative activity evident among pharyngeal cultures in the absence of IL-2 reflects this
haematopoietic activity. In addition. LLC act as
immunoeompetent cells. The proliferation of
LLC is enhanced by allogeneic stimuli (14). and
LLC in the periphery are associated with the recognition and destruetion of allogeneic tissue
(24).
The immunoeompetent function of LLC suggests that tfieir response to IL-2 is analogous to
that of mammalian T cells. However there are
notable differences between the reactions of
tunicate LLC and mammalian lymphocytes to
interleukins. First, the level of stimulation
233
evident in tunicate pharyngeal cultures
following IL-2 treatment (up to 3X that of control cultures) is substantially lower than that
reported for mammalian lymphoeytcs. Again.
this difference may be due to the added
haematopoietic function of tunicate LLC. Unlike mammalian T cell cultures (22). proliferation is maintained among tunicate pharyngeal
explants in the absence of lL-2. Hence diUcrenees between stimulated and unstimulated tunicate cultures should be lower than those for
mammalian cells.
A second difference in the response of
tunicate and mammalian tissues to IL-2 is tfiat
the niitogenie effect of rhIL-2 in tunieates does
not require eo-stimulation with Iectins. conjugating antibodies or antigen (21). IL-2 induced
proliferation proceeds in the absence of costimulation and is not synergized by PHA. This
independence from co-stimulation eould be explained by the nature of the tissues used to test
IL-2 activity in tunieates. Haematopoiesis is
mainlained in tunicate pharyngeal cultures
without the addition of stimulants (8). Hence
endogenous co-stimulatory factors or simply the
haematopoietic nature of these cultures may
negate a requirement for additional synergism.
Tunicate cells also differ from mammalian T
cells in their sensitivity to IL-1. Neither purified
hlL-1 nor hIL-Ia affect the proliferation of tunicate eells. The failure of IL-1, eithcraloneor in
combination with PHA. to promote division
suggests that it is incapable of inducing the production of endogenous IL-2-likc molecules.
Similarly, its failure tosynergize responsiveness
when co-cultured vvith IL-2 indicates that lL-1
does not enhance IL-2-likc receptor expression.
The most parsimonious explanation for the
failure of IL-1 to stimulate tunicate cultures is
that the human IL-1 tested here have not been
sufficiently conserved during evolution to be
recognized by tunicate eells. However the available evidence suggests that !L-l-like molecules,
and by corollary their receptors, do exist in tunieates (4). Henee human lL-1 might recognize
tunicate cells but its effects may not interact with
those of lL-2. We are currently investigating the
role of IL-1-like proteins by analysing the function of endogenous cytokines in tunicates.
ACKNOWLEDGEMENTS
The authors thank Mrs Sharon Sampogna and
her staff in the Microscopic Techniques Laboratory, UCLA School of Medicine, for tfieir help
2.14
D. A. R.AFTOS
with histological procedures. Flow cytonictric
facilities were provided by the Flow Cytotnetry
Core Laboratory. Johnson Comprehensive Cancer Center. UCLA. We gratefully acknowledge
Mrs Ingrid Schmid for her assistance with tlow
cytometric analyses. This study v^as supported
by the US National Science Foundation {Grant
no. DCB 85-19848). Flow eytometrie faeilities
were sponsored in part by a Johnson Cancer
Centre Core Grant (CA 16042). David Raftos is
a Fulbright Postdoctoral Fellow and recipient of
a Frcdcrik B. Bang Scholarship in Marine Invertebrate Immunology administered by the Ameriean Association of Immunologists. Dan Stillman was supported by an REU supplement from
the National Science Foundation.
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