Invertebrate Cytokines: Tunicate Cell Proliferation Stimulated by an Interleukin 1-Like Molecule (Urochrdata/Lymo Ne/Lm Mundoog/Mitogenes) DAVID A

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Proc. Natl. Acad. Sci. USA Vol. 88, pp. 9518-9522, November 1991 Cell Biology Invertebrate cytokines: Tunicate cell proliferation stimulated by an interleukin 1-like molecule (Urochrdata/lymo ne/lm munDoog/mitogeneS) DAVID A. RAFTOS*t, EDWIN L. COOPER**, GAIL S. HABICHT§, AND GREGORY BECK§ *Department of Anatomy and Cell Biology, University of California, Los Angeles, CA 90024; and §Department of Pathology, State University of New York, Stony Brook, NY 11794 Communicated by Charles H. Sawyer, July 1, 1991 (receivedfor review April 16, 1991)

ABSTRACT Tunicte pharyngeal cells include lympho- cate hemolymph and the 20-kDa tunicate IL-i-like proteins cyte-like cells and granular amoebocytes. They are Involved in are inhibited by preincubation with anti-mammalian IL-1 the specific lgeneic and phagocytic reactions of tunites. antibodies (2). Little is known about their regulation or control. A tunicate These data suggest that cytokine-like molecules have been Interleukin 1 (IL-1)-Hlke fraction is shown to snulate the conserved during evolution to the degree that functional prliferation of these cels in vitro. This fraction, designated cross-reactivity occurs over large phylogenetic distances (1, tunicate IL-f1, was isolated from tunicate hemolymph by gel 3, 6). In addition to the action of invertebrate cytokine-like fltration and chromatofociusing chromatography. Mitogenic molecules on mammalian cells, we have shown that human responses to tuncte L1 were dose dependent and could be recombinant IL-2 can stimulate the proliferation of tunicate eliminated rapidly by removing tunicate IL-1L from culture cells in vitro (14). Perhaps such cross-reactivities reflect the medium. A second tun hte molymph fraction had no effect existence of invertebrate cytokine-based regulatory systems on tunicate cell proliferation even though it exhibited IL-1-like for host defense that are homologous to those of mammals. activity in a mouse thymocyte proliferation assay. Phytohe- In this study, we investigate the function of invertebrate mawglutinin did not act synergistically with either fraction. IL-1-like molecules in their native species. An IL-i-like These data are diused in terms ofthe function and evolution fraction from tunicates, tunicate IL-1p3, is shown to stimulate Of IL-i-like molecules in invertebrates. the proliferation of tunicate cells in vitro. Interleukin 1 (IL-1)-like activities have been identified in a number of invertebrate species (1-3). Beck and Habicht (1) MATERIALS AND METHODS have shown that fractionated coelomic fluid and cell extracts Reagents, Culture Plates, and Filters. Unless stated other- from the echinoderm (starfish) Asteriasforbesi stimulate the wise, all reagents were purchased from Sigma. [methyl- proliferation of murine thymocytes in a lymphocyte prolif- 3H]Thymidine (6.7 or 20 Ci/mmol; 1 Ci = 37 GBq) and eration assay for IL-1. Stimulatory activity resides in an Ecolite scintillation cocktail were supplied by ICN. Tissue -20-kDa fraction of the coelomic fluid (3). In addition to its culture plasticware was purchased from Costar. Filter ster- thymocyte stimulatory activity, this fraction induces mam- ilization units (0.22 ,um) were purchased from either Millipore malian fibroblast proliferation, enhances fibroblast protein or Costar. synthesis, increases the production ofprostaglandin E2, and Tunicates. Solitary tunicates (Styela clava) were purchased is cytotoxic for the human cell line A375 (1, 3). All of these from Marinus (Long Beach, CA). They were maintained in an functions are characteristic of mammalian IL-1 (4-7). The aerated aquarium filled with 180 liters of artificial seawater 20-kDa coelomic fluid fraction comprises three protein spe- [ASW; 3.4% (wt/vol), Instant Ocean, Aquarium Systems, cies with isoelectric points (pI values 7.5, 5.4, and 4.8) that Mentor, OH] at 15'C. are similar to predominant forms of mammalian IL-1 (pI Harvesting of Tunicate Hemolymph. Hemolymph was har- values 7.2 and 5.0) (1, 8, 9). Moreover, polyclonal antibodies the specific to human IL-1 inhibit the stimulatory effects of the vested by severing the stolons oftunicates and collecting 20-kDa echinoderm proteins on mouse thymocytes (1). exuding fluid in chilled polyethylene centrifuge tubes. Hemo- Similar IL-i-like activities have been identified in annelids cytes were then removed by centrifugation (1000 x g for 15 and tunicates (G.B., G.S.H., and E.L.C., unpublished data; min at 40C). The resulting cell-free hemolymph was filter see also refs. 2 and 4). The detection of tunicate IL-i-like sterilized (0.22-pum filters; Costar) and stored at -20°C for up molecules is ofparticular interest. Tunicates are invertebrate to 6 months before use. members ofthe phylum Chordata and so are likely to exhibit Gel-Filtration Chromatography. Tunicate IL-1 molecules significant molecular and functional homologies with verte- were purified from 100 ml of tunicate cell-free hemolymph. brates (10). Hemolymph or cell extracts from eight species of The hemolymph was cleared by centrifugation (10,000 x g for tunicates can stimulate mouse thymocyte proliferation (2). 30 min at 40C) and concentrated =10:1 by ultrafiltration using Stimulation has been attributed to two molecular mass frac- a PM 10 membrane (Amicon). The concentrate was applied tions (:20 and >50 kDa), the smaller of which is within the to an Ultrogel AcA 54 gel-filtration column (2.6 x 85 cm; molecular mass range ofechinoderm IL-i-like molecules and Pharmacia). This column was calibrated with bovine serum mammalian IL-is (2, 5, 11, 12). The smaller tunicate IL-i-like albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A fraction segregates into neutral and acidic species with pI (25 kDa), and RNase A (13.7 kDa) (Pharmacia) as size values that correspond closely to those of echinoderm IL-1- standards. The column was eluted (1 ml/min; 40C) with like molecules and mammalian IL-is (2, 7, 13). As with echinoderms, the stimulatory effects of concentrated tuni- Abbreviations: IL-1, interleukin 1; tunicate IL-1, tunicate IL-1-like fraction; ASW, artificial seawater; PHA, phytohemagglutinin. tPresent address: c/o Prof. R. L. Raison, School of Biological and The publication costs of this article were defrayed in part by page charge Biomedical Sciences, University of Technology, P.O. Box 123, payment. This article must therefore be hereby marked "advertisement" Broadway, Sydney, N.S.W., 2007, Australia. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9518 Downloaded by guest on September 30, 2021 Cell Biology: Raftos et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9519 phosphate-buffered saline containing gentamicin (10 j.g/ml). Incubation of Tunicate Explants with IL-i-like Fractions. Three-milliliter fractions were collected and the effluent was Explants were equilibrated to culture conditions for 3 days monitored at 280 nm. Fractions were diluted 1:1 with RPMI before hemolymph fractions that contained IL-1-like activity 1640 medium and sterilized by filtration. Aliquots of the were added. After the equilibration period, explants were diluted fractions were tested for IL-1-like activity in a murine transferred to either fresh T-RPMI or to T-RPMI containing thymocyte proliferation assay using thymocytes from 4- to 20% (vol/vol) tunicate hemolymph, concentrated tunicate 8-week-old BALB/c mice or by cytotoxicity ofA375 cells as hemolymph, IL-1-like fractions, phytohemagglutinin (PHA), described (1, 3). Recombinant human IL-la (a kind gift from or combinations of the above. Subsequently, tissues were Dainippon Pharmaceutical, Osaka) was used to construct transferred to fresh medium every 3 days. standard curves to determine the number of units of IL-1 in Quantification of [3H]Thymidine Uptake. To quantify each experimental sample as described (1, 3). Specific activ- [3H]thymidine uptake by cultured tissue, explants were re- ities of highly purified tunicate IL-1 fractions were deter- moved from tissue culture plates and pooled in 2 ml of the mined as units per mg of protein. same medium in which they had been cultured. Tissues were Chromatofocusing. Gel-filtration fractions that incorpo- then incubated at 150C with 2.5 ,Ci of [3H]thymidine per ml. rated peaks of IL-1-like activity were pooled and concen- After 18 hr, excess [3H]thymidine was removed by washing trated to =5 ml (PM 10 ultrafiltration). These samples were explants four times (1 hr per wash) in 4 ml of ASW on a dialyzed against 25 mM imidazole hydrochloride (pH 8.4) hematology mixer. Washed explants were transferred to before being applied to a PBE 94 chromatofocusing column scintillation vials (1 explant per vial) containing 200 pl of (Pharmacia), which was equilibrated in 25 mM imidazole trypsin [2% (wt/vol) in ASW] and digested overnight at 3TC. hydrochloride (pH 8.4) at 40C. The chromatofocusing column Two milliliters of scintillation cocktail were then added to was eluted with Polybuffer 74 HCl (pH 4.0) (Pharmacia) to each vial so that the incorporated radioactivity could be give a linear pH gradient from pH 8.4 to pH 4.0 as per the quantified in a Beckman LS 3150P scintillation counter. manufacturer's instructions. After elution, fractions were Statistical Analysis. The significance of differences be- brought to a pH of -7.2 by adding 4 M Tris base (pH 11). The tween treatments was determined by two-tailed Student's t effluent was monitored at 280 nm, and the column eluates tests (16). Differences were deemed to be significant if P were sterilized by filtration before being assayed for IL-1 values were <0.05. activity as described above. Tunicate Tissue Culture Medium. Tunicate tissue culture medium (T-RPMI) was prepared in sterile-filtered ASW and RESULTS contained, per liter, 454 mg of RPMI 1640 powder (with Effect of T-RPMI and Unfractionated Hemolymph on L-glutamine, without sodium bicarbonate), 105 units of pen- pHlThymidine Uptake. [3H]Thymidine uptake by explants icillin sulfate, and 100 mg of streptomycin sulfate (15). This cultured in T-RPMI without added hemolymph declined over medium was sterilized by filtration and stored at 4°C. time (Fig. 1). After 9 days in vitro, [3H]thymidine uptake by Tunicate Tissue Culture Protocol. The stimulatory effects of tissues cultured in T-RPMI alone was only 38% of that tunicate hemolymph fractions were tested in cultures of incorporated 1 day after explantation. This decline in pharyngeal tissue from S. clava. The procedure used to [3H]thymidine incorporation could be slowed by supplement- culture pharyngeal explants has been described in detail ing T-RPMI with 20% (vol/vol) S. clava hemolymph (Fig. 1). elsewhere (15). Pharyngeal tissue was diced into explants (3 Cultures containing plasma maintained [3H~thymidine uptake x 3 x 1 mm) and rinsed three times in T-RPMI. Rinsed such that 93% of the original (day 1) activity was retained explants were transferred to 96-well flat-bottomed culture after 9 days in vitro. plates (1 explant per well) containing T-RPMI alone or pHlThymidine Incorporation Is Stimulated by Hemolymph T-RPMI and 20Wo (vol/vol) tunicate hemolymph (200 td per Concentrates. Fig. 2 shows that hemolymph concentrated on well). Explants were cultured at 15°C in a normal atmo- sphere. 121 131 0 10 - C.)ca 7- x 11 - 6.x E 0 CL e 0 0I- 9. 6- L- 0._CL / ._ 8 Cc 7, 'D 4.

c E CO± 5.- 2-

1:25 1:50 1:100 1:200 1:500 Control 0 2 4 6 8 10 Dilution Time (days) FIG. 2. Uptake of[3H]thymidine by pharyngeal explants cultured FIG. 1. Uptake of [3H]thymidine by tunicate pharyngeal explants for 3 days in T-RPMI containing various dilutions of hemolymph cultured for various periods in either T-RPMI alone (n) or T-RPMI ultrafiltration concentrates (>10 kDa). Bars = +1 SE; minimum of with 20% (vol/vol) tunicate hemolymph (o). Bars = ±1 SE; mini- nine replicates per point. No hemolymph concentrate was added to mum of eight replicates per point. the control cultures. Downloaded by guest on September 30, 2021 9520 Cell Biology: Raftos-et al. Proc. Natl. Acad. Sci. USA 88 (1991) PM 10 membranes (10-kDa exclusion limit) stimulated 301 [3H]thymidine uptake by cultured explants. Dilutions of up to C., 1:100 of the >10-kDa concentrate induced significantly (P < 0.05) greater [3H]thymidine uptake than that of controls x cultured in T-RPMI alone. Enhanced [3H]thymidine uptake E was not evident at greater dilutions. Effluents from PM 10 CS 20 - ultrafiltration (<10 kDa) did not significantly enhance 0.

[3H]thymidine incorporation (data not shown). 0

Characterization of IL-l-like Fractions. Isolation of tuni- 02- cate IL-1 activity was performed essentially as described for 0 data; see echinoderm IL-1 (G.B. and G.S.H., unpublished ao0) also ref. 3). Tunicate IL-1 activity was isolated from he- 10 - t1 molymph by a combination of gel-sieve and chromatofocus- E ing chromatography. This resulted in three fractions of ac- co0i. tivity, which were designated tunicate IL-1l3 (pI 7.4), tunicate IL-la (pI 5.4), and tunicate IL-la2 (pI 4.8). All fractions had activity in the murine thymocyte proliferation and A375 melanoma cytotoxicity assays. The respective specific ac- - . , , . 300 150 60 30 15 0 tivities in the two assays for the purified tunicate IL-1 fractions were the following: for tunicate IL-1/3, 5.2 x 105 tun IL1-B (ng/ml) units/mg and 5.0 x 1i0 units/mg; for tunicate IL-la, 2.0 x 106 units/mg and 2.8 x 10' units/mg; for tunicate IL-la2, 5.0 FIG. 4. Uptake of [3H]thymidine by pharyngeal explants after 3 x 105 units/mg and 1.2 x 106 units/mg. Since the tunicate days of culture in T-RPMI containing various concentrations of IL-1,B and tunicate IL-la proteins were the major constitu- tunicate IL-1p (tun IL-1,B). Bars = +1 SE; minimum of eight ents, they were tested for their ability to stimulate tunicate replicates per point. explants. pharyngeal could be detected between tissues incubated with tunicate Effect of IL-i-like Fractions on [3H]Thymidine Uptake. The tunicate IL-1f3 fraction produced significantly enhanced IL-la and controls in T-RPMI alone. [3H]thymidine uptake by explants (Fig. 3). After 3 days of Failure of PHA to Synergize Effects of Tunicate IL-1 Frac- incubation with tunicate IL-113 (300 ng/ml), explants incor- tions. The effects of both tunicate IL-la and -13 on tunicate explants were not altered by coincubation with PHA (Table porated 2.5 times (P < 0.05) the level of [3H]thymidine in cultured in T-RPMI alone. This significant 1). PHA is known to be mitogenic for tunicate explants at evident controls for difference was transient, beginning after 1 day of exposure to doses of .5 pg/ml (14). The dose of PHA (2 ,ug/ml) used tunicate IL-113 and subsiding by day 9. Dose-response anal- coincubation with tunicate IL-1 is submitogenic. There was ysis oftunicate IL-1l3 (Fig. 4) revealed significantly (P < 0.05) no significant (P > 0.05) stimulation of[3H]thymidine uptake, enhanced [3H]thymidine uptake for tunicate IL-1A3 concen- relative to controls cultured in T-RPMI, when explants were trations of -150 ng/ml. cocultured with submitogenic doses of tunicate IL-1j3 (75 The second isoelectric species isolated from hemolymph ng/ml) and PHA (2 ,ug/ml). Combination of PHA with a proteins (tunicate IL-la) did not stimulate pharyngeal ex- mitogenic concentration oftunicate IL-1,B (300 ng/ml) did not plants, although it had significant IL-1-like activity in the enhance 13H]thymidine uptake beyond that evident for cul- murine thymocyte proliferation assay (Fig. 3; see above). No tures containing 300 ng of tunicate IL-1(8 per ml alone (P > significant (P > 0.05) differences in [3H]thymidine uptake 0.05). Similarly, no significant (P > 0.05) stimulation was evident when PHA was cocultured with either 300 or 75 ng of 30 tunicate IL-la per ml (data not shown). Removal of Tunicate IL-113. The transfer of cultured explants to T-RPMI without tunicate IL-1j3 after a brief(3 days) 0 to led to a rapid x period ofexposure tunicate IL-1,B (300 ng/ml) E decline in [PH]thymidine uptake (Fig. 5). Three days after their removal from medium containing tunicate IL-13, Csc. 20 [3H]thymidine uptake by explants was equivalent to that of I.°-0 controls that had not been exposed to tunicate IL-1,3 (P > 0 0.05). 2- Restoration of [3H]Thymidine Uptake by Tunicate IL-HP. 8 Tunicate IL-1P had the capacity to restore [3H]thymidine C extended .c incorporation to explants that had been cultured for 10- in the absence of or IL-l-like fractions E periods hemolymph Table 1. [3H]Thymidine uptake by pharyngeal explants cultured I co for 3 days in T-RPMI containing various concentrations of tunicate IL-1f3 with or without PHA (2 ,ug/ml)

u-i [3H]Thymidine incorporation, 2 4 6 8 10 cpm x 10-3 ± SE Time (days) Medium - PHA + PHA T-RPMI alone 12.7 ± 1.3 11.3 ± 1.6 FIG. 3. Incorporation of [3H]thymidine by pharyngeal explants Tunicate IL-1p that had been cultured for various periods in either T-RPMI alone (o), 300 ng/ml 21.7 ± 0.9 20.7 + 0.9 T-RPMI with 300 ng oftunicate IL-la per ml (s), or T-RPMI with 300 75 ng/ml 11.8 ± 0.8 12.7 ± 1.2 ng of tunicate IL-1,B per ml (A). Bars = -+-1 SE, minimum of nine replicates per point. n 28. Downloaded by guest on September 30, 2021 Cell Biology: Raftos et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9521 20- turned to a level slightly lower than that of explants that had been cultured for only 4 days in T-RPMI alone. This level of [3H]thymidine uptake was still significantly (P < 0.05) higher x than that evident in starved tissue to which tunicate IL-1,8 0. had not been added. ci DISCUSSION We have shown that a tunicate hemolymph fraction (tunicate IL-1,8), which exhibits IL-l-like activity when tested in 10 - o.' mammalian assay systems (2), also stimulates the proliferation of tunicate cells. The methods used to quantify this .) stimulatory activity are well established. Pharyngeal explants from tunicates retain both cell viability and proliferative activity for up to 70 days in vitro (15). These cultures are I amenable to experimental manipulation. For example, proliferative activity can be modulated by incubation with lectins or mammalian interleukins (14). Variations in cell proliferation are readily quantified by the uptake of [3H]thymidine. 0 1 2 3 4 5 6 7 [3H]Thymidine incorporation by cultured explants is inhibited by irradiation and competition with unlabeled thymidine, Time (days) suggesting that there is a direct relationship between cell proliferation and [3H]thymidine uptake (14). Proliferative FIG. 5. Uptake of [3H]thymidine by pharyngeal explants that had activity in pharyngeal explants is attributed to two discrete been cultured either continuously in T-RPMI alone (o), in T-RPMI cell types (lymphocyte-like cells and granular amoebocytes), with 150 ng of tunicate IL-1p8 per ml (A), or that had been cultured both of which proliferate in vitro in the absence of for 3 days in T-RPMI with 150 ng of tunicate IL-1P per ml and then added transferred to T-RPMI alone after three 1-hr washes in T-RPMI (U). stimuli (14, 15). These cells appear to have a constitutive Bars = +1 SE; minimum of 10 replicates per point. Arrow indicates hematopoietic function (15, 17, 18). They are also involved in the day at which the indicated explants were transferred to tunicate specific allogeneic and nonspecific phagocytic reactions (19, IL-i-free medium. 20). An initial indication that cell proliferation in tunicates can (Fig. 6). Explants that had been cultured for 17 days in be stimulated by endogenous hemolymph factors is provided T-RPMI without added hemolymph components exhibited by the effect ofunfractionated hemolymph on cultured tissue. only one-third the [3H]thymidine incorporation that was Previously, we showed that proliferation is maintained at evident after 4 days of culture in T-RPMI. One day after higher levels when explants are cultured in media that contain adding tunicate IL-1f3 (300 ng/ml) to these "starved" ex- autologous hemolymph (15). This observation has been re- plants, [3HJthymidine uptake increased by 6.9 times relative peated in the current study. Such results suggest either (i) to explants cultured for 17 days in the absence of tunicate that tunicate hemolymph contains nutrients or anabolic pre- IL-1,3 and by 2 times when compared to tissue cultured for 4 cursors that are required for proliferation but are not present days in T-RPMI alone. Within 3 days of adding tunicate IL-1p in artificial (RPMI 1640 medium) culture medium or (ii) that the includes to starved explants, had re- hemolymph constitutive, mitogenic factors that [3Hjthymidine incorporation are necessary to stimulate proliferative activity. Results of the studies of fractionated tunicate hemolymph 80 support the latter alternative. The capacity to enhance [3H]thymidine uptake by explants resides in hemolymph C) concentrates (>10 kDa) that are unlikely to have retained x small nutritive (e.g., mono- and oligosaccharide) or anabolic 60 (e.g., amino acid) molecules. Stimulatory activity was asso- ciated with a chromatofocusing fraction, tunicate IL-1f3, from >10-kDa concentrates. This fraction was identified and isolated on the basis of its ability to stimulate the proliferation 40 ofmouse thymocytes (2). When tested in tunicate cell culture 0 C) systems tunicate IL-1lp also stimulated tunicate cell proliferation. The level of tunicate cell proliferation yielded by -C I:±0 incubation with tunicate IL-1,8 is similar to that of cultures maintained in medium incorporating concentrated, unfrac- T1 tionated hemolymph. Indeed, tunicate IL-1,B is capable of restoring proliferative activity to cells that have been starved of tunicate hemolymph for considerable periods. This suggests that tunicate IL-1,3 contributes substantially to the 0 stimulatory activity of tunicate hemolymph. T T B cx B Cx In contrast to tunicate IL-1f3, a second isoelectric species -4 17 18 20 isolated during gel filtration and chromatofocusing (tunicate IL-la) does not enhance tunicate cell proliferation even Time (days) though it activates mouse thymocytes. It is reassuring that not all hemolymph proteins will enhance tunicate cell FIG. 6. Uptake of [3H]thymidine by explants that had been prolif- cultured for either 4 or 17 days in T-RPMI alone (T) and by those to eration. The lack of response by tunicate cells to tunicate which 300 ng of tunicate IL-la (a) or tunicate IL-1l3 (;6) per ml were IL-la indicates that there is a degree of specificity in the added after 17 days in T-RPMI. [3H]Thymidine uptake by explants action of tunicate IL-1p. to which tunicate IL-1 had been added was tested 1 and 3 days after Given the capacity of tunicate IL-la to stimulate mouse their addition. Bars = -1 SE; minimum of 10 replicates per point. thymocytes, the lack of response to this fraction by tunicate Downloaded by guest on September 30, 2021 9522 Cell Biology: Raftos et al. Proc. Natl. Acad. Sci. USA 88 (1991) tissue may be explained in several ways. First, the culture was supported by grants from the U.S. National Science Foundation system used may not have incorporated costimulatory mol- (DCB 90 05061 to E.L.C. and DCB 88 10488 to G.B. and G.S.H.). ecules necessary for the expression of tunicate IL-la activity. When tested against murine thymocytes both tunicate 1. Beck, G. & Habicht, G. S. (1986) Proc. Nat!. Acad. Sci. USA 83, 7429-7433. IL-la and -p require costimulation with mitogenic lectins (2). 2. Beck, G., Vasta, G. R., Marchalonis, J. J. & Habicht, G. S. Here we showed that PHA, which is known to be a mitogen (1989) Comp. Biochem. Physiol. B 92, 93-98. for tunicate cells (14), does not enhance the responsiveness 3. Beck, G. & Habicht, G. S. (1991) Mol. Immunol. 28, 577-584. of tunicate explants to tunicate IL-1 proteins. However, 4. Beck, G., O'Brien, R. F. & Habicht, G. S. (1989) BioEssays 11, unknown costimulators could be required for the expression 62-67. oftunicate IL-la activity. Second, the functional spectrum of 5. Durum, S. K., Schmidt, J. A. & Oppenheim, J. J. (1985) Annu. tunicate IL-la may not include the mitogenesis of tunicate Rev. Immunol. 3, 363-387. cells even though tunicate IL-la enhances the proliferation of 6. Gery, I. & Lepe-Zuniga, J. L. (1984) Lymphokines 9, 109-125. murine thymocytes. 7. Oppenheim, J. J., Kovacs, E. J., Matsushima, K. & Durum, S. K. (1986) Immunol. Today 7, 45-56. Indeed, the physiological activities of endogenous IL-1- 8. Luger, T. A. & Oppenheim, J. J. (1983) in Advances in Inflam- like fractions in tunicates are conjectural. Our evidence mation Research, ed. Weissmann, G. (Raven, New York), Vol. suggests that one role of tunicate IL-1p may be to regulate 5, pp. 1-25. cell proliferation. We have shown that tunicate cells respond 9. Schmidt, J. A., Mizel, S. B., Cohen, D. & Green, I. (1982) J. quickly to the removal oftunicate IL-1p8 and that proliferative Immunol. 128, 2177-2182. capacity is restored upon its return. However, the physio- 10. Berrill, N. J. (1955) The Origin of Vertebrates (Oxford Univ. logical relevance of such modulation is unclear in that tuni- Press, New York). 11. Dinarello, C. A., Clowes, G. H. A., Gordon, A. H., Saravis, cate IL-1l3 is apparently constitutively present in the he- C. A. & Wolff, S. M. (1984) J. Immunol. 133, 1332-1338. molymph. Its expression and action may not be restricted to 12. Mizel, S. B. (1979) J. Immunol. 122, 2167-2172. the inflammatory functions that are characteristic of mam- 13. Simon, P. L. & Willoughby, W. F. (1981) J. Immunol. 126, malian IL-1 (6). The proliferation oftunicate pharyngeal cells 1534-1541. contributes substantially to hematopoiesis as well as to 14. Raftos, D. A., Stillman, D. L. & Cooper, E. L. (1991) Immu- immunological reactivity (15, 18, 19). Investigations of the nol. Cell Biol., in press. adaptive and defensive roles ofinvertebrate IL-1, and similar 15. Raftos, D. A., Stillman, D. L. & Cooper, E. L. (1990) In Vitro proteins, in response to antigenic challenge and of their 26, 962-970. 16. Sokal, R. R. & Rohlf, F. J. (1981) Biometry (Freeman, New effects on nonspecific defensive reactions such as phagocy- York). tosis and hemocyte migration are necessary to expand our 17. Ermak, T. H. (1975) Experientia 31, 837-839. understanding of these critical proteins. 18. Ermak, T. H. (1982) Am. Zool. 22, 795-805. 19. Raftos, D. A. & Cooper, E. L. (1991) J. Exp. Zool., in press. D.A.R. is a Fulbright Postdoctoral Research Fellow. This study 20. Wright, R. K. & Cooper, E. L. (1983) Am. Zool. 23, 205-211. Downloaded by guest on September 30, 2021