Reference:BinI Hull 191: 159-167.(October. Acute Cytotoxic Allogeneic Histoincompatibility Reactions Involving Gray Cells in the Marine Sponge, Callyspongia diffusa CHAOQUAN YIN AND TOM HUMPHREYS KewaloMarineLaboratory, PacificBiomedicalResearch Center, UniversityofHawaii, 41 AhuiStreet, Honolulu. Hawaii 96813 Abstract. A variety ofprocedureswere used in a study tion of signals that suppress cell aggregation and cell of the histoincompatibility reactions of Callyspongia movement and attractgray cellstotheboundaryofcon- diffusa. Rejection reactions as traditionally tested be- tact; (3) acceleration ofthe sponge immune response tween laterally apposed intact fingers cut from two including the responsiveness ofgray cells to accumulate different sponges require about a week ofcontact to ex- atthe boundary ofallogeneiccontact bytissue trauma hibit cytotoxicity. In a miniaturized assay involving re- produced when the tissue iscut or dissociated it into in- actions between small pieces of tissue snipped from dividual cells; (4) arrival ofgray cellsatthe boundary of sponges with scissors and pushed together on an insect allogeneic contact: and (5) initiation of cytotoxic pro- pin, cytotoxicity is evident within 48 hours ofcontact. cesses. Reactionsofcellsdissociatedbydivalentcation removal andallowedto reaggregatein seawaterwerealsostudied. Introduction Aggregates produced from allogeneic mixtures of cells from two individuals were killed by internal cytotoxic The invertebrate immune system must protect the in- reactionswithin 36 hoursoftheinitiationofaggregation. dividual animal from a full range ofparasiticand patho- Afteronlyonehourofaggregation, aggregates from allo- genic threats (Ratcliffe el ai. 1985). For sessile species, geneic mixtures were significantly smaller than aggre- individual-specific recognition mayalso preventcellular gatesofcellsfrom asingle individual. Thisrapid slowing parasitism between contacting individuals of the same ofaggregation is the earliest response to allogeneic con- species(Buss, 1987). Very specific, immune-likehistoin- tact that we noted and does not appear to reflect early compatibility reactions occur in every invertebrate phy- cytotoxic processes. Apposition ofan aggregate contain- lum (Coopereta/.. 1992) includingsponges,thesimplest ingcellsfromonespongetoanaggregatecontainingcells metazoans(Hildemann etai. 1979). Becausethese reac- from a second individual leads to mutual destruction. tionsareusuallyveryslow,and notwelldefinedhistolog- Aggregates harvested and apposed 4 hours after initia- ically,cellsfunctioninglikevertebrateTorBcellsarenot tion ofaggregation begin to show mutual cytotoxicityat evident in any invertebrate. The idea that evolutionary 36 hours of contact. Aggregates placed in contact precedents for specific recognition events characteristic 48 hours after the initiation ofaggregation exhibit cyto- ofthese vertebrate immune cells cannot be found in in- toxicity within 8 hours. These rapidly reacting 48-hour vertebrates is often expressed (Klein, 1989; Janeway, aggregates exhibit a pronounced accumulation of gray 1992; Smithand Davidson, 1992). cells at the boundary ofallogeneic contact by 8 hours. Thislaboratoryhasobserved veryrapidallogeneicrec- These results are interpreted as indicating at least five ognition reactions involving the intimate participation steps in the histoincompatibility reactions ofC. diffusa: ofa specific cell type gray cells in the marine sponge (1) recognition soon afterallogeneic contact; (2) genera- Microciona prolifera (Humphreys, 1994). The rapidity and selectivity of these reactions have led us to focus again on the possibility ofspecificcellularrecognition in Received 16October 1995;accepted24May 1996. the immune reactions ofinvertebrates (Humphreysand 159 160 C. YIN AND T. HUMPHREYS Reinherz, 1994). FollowingBurnet's( 1971 )original sug- ductivecellularlevelanalysisofallogeneicrecognition in gestion, we have emphasized direct, specific, selfrecog- thisspecies. nition as the guiding principle for invertebrate immu- nity. Conceptually, ifinvertebrate immunocytes recog- Materialsand Methods nize individual self histocompatibility marker proteins, Sponges they can immediately, upon contact, recognize as non- selfall cells that do not display the identical self-marker Thebrilliant purplesponge Callyspongiadiffiisa(Rid- proteins. Theabilityofinvertebrateimmunocytestorec- ley)wascollected fromtheshallowreefsaroundCoconut ognizeselfcouldbelikenedtotheabilityofvertebrateT- Island, Kaneohe Bay, Oahu, Hawaii, and maintained in cells to recognize selfmajor histocompatibility complex the running seawatertanksat the Kewalo Marine Labo- (MHC)proteins. JustasvertebrateT-cellslearn to recog- ratory. Sponges of 10 to 50 grams or more survive and MHC nize self proteins through the process ofpositive continue to grow slowly for six months to one year in selection,onecanimaginethatduringdifferentiation, in- these tanks in spite ofthe excision, from timetotime, of vertebrate immunocytes learn to recognize selfproteins small portionsoftheirtissue forexperiments. encoded by the specificallelesthat the individual animal has inherited at a polymorphic locus (or loci) that en- Ilistticonipatibilin assays codes self marker proteins (Humphreys and Reinherz, In addition to the original grafting system described 1994). The tropical marine sponge Callyspongia diffiisa was for C. diffiisa (Hildemann el a/.. 1979) in which two in- used in some ofthe first experimentson histoincompati- ttaocgtetfhienrgeorsn,at2y"piXca3l"lymiscerveorsaclopceenstliimdeetmearisntlaonign,edarien steiae-d bility in sponges(Hildemann clai. 1979). Subsequently, water, we have developed a miniaturized assay. Two graft rejection reactions have been described in a variety small pieces oftissue a few mm in dimensions, snipped oofccsupron(gVeasnpecdieesVaynvdertwoanddistBinucstceremjae,ctio1n988m)o.dalSiotimees fromthespongewithscissors, arepushedtogetheron the end of a #0 stainless steel insect pin that is incubated sponges for example, C. diffiisa produce a nonspe- standingin 3 ml offiltered seawaterinawell ofa 24-well cificcytotoxicreactionthat killsallcellswithinafewmil- tissueculture plate(Falcontype 3047)shakingat 50 rpm cllimae/t..ers19o8f0)t.heOtbhoeurndsaprecyieosf,fsoruecihgnascoMn.tapcrtol(iHfierlad,ecmaarnrny onWaerotatory shaker. The seawaterischanged daily. also assayed the histoincompatibility reactions out an encapsulation-like processand producea barrier, thatoccurwithinaggregatesofdissociatedcellsproduced oftencontainingcollagen,betweenthecontactingtissues (Humphreys, 1994). Our observations on M. prolifera with mixturesofcellsfrom twodifferent sponges, aswell csougngteasctt iwimtmhedtihaetreelceealsleuloafrarcehcoegmnoiktiionneuthpaotnatatlrlaocgtesntehiec gasretghaeterseaapctpioosnesdtihnatalolcocguernebicetpwaeiresn. Tfuhlelyddeteaviellsoopfetdheasge- gray cellsto accumulate in the zoneofcontact and orga- assays follow a description ofcell dissociation and reag- gregation. nize the formation of the collagen layer (Humphreys, 1994). Although reactions of other species of sponges Celldissociation andreaggregation have been examined in considerable histological detail mm (Van de Vyver and Buscema, 1988: Smith and Hilde- Sponge tissue (5 to 10 ofa finger ofthe sponge, mann, 1986a, b), few othercluesconcerningthecellular about 0.5 gram), is washed four times, 10 minutes per and mechanistic bases ofrecognition or rejection reac- wash, with 10 ml pergram oftissueofcalcium and mag- tions haveemerged. nesium free seawater (CMF-SW) (Humphreys, 1963) at Encouraged by a report that mixed aggregates ofdis- room temperature, 22-25C (lowering the temperature sociated cells from two individualsofC. diffiisa undergo to 4C kills C. diffiisa cells). The washed tissue is then a rapid rejection reaction which causes selfannihilation squeezed repeatedly in a bag of 100 ^m Nitex mesh to ofthe aggregates within two days (Johnston. 1988), we disperse the cells from the matrix into 10 ml CMF-SW began to explore the possibility ofmore rapid assays of pergram oftissue. The cell concentration ofthe suspen- recognition in this species. In fact, we describe cellular sion typically ranges from 10 to 20 X 106 cells per ml. assays that can detect recognition reactions within an The viability ofthe cells is checked by dye exclusion, ei- hour ofallogeneic contact. We have also discovered ac- ther with 1% trypan blue in CMF-SW or the live/dead celeration and maturation processes that are compo- viability/cytotoxicity kit of Molecular Probes, Inc. (L- nents of the reacting immune system. In addition we 3224). Freshlydissociated cells routinely have a viability have described gray cells in C. diffiisa and have shown greaterthan 95%. Thedissociatedcellsarecentrifuged at that recognition signalscause them to accumulate at the 1500 rpm for 1 '/: min at 15C (Sorvall Rt6000B Refrig- zone ofallogeneic contact in association with the cyto- erated Centrifuge), resuspended at aconcentration of 10 toxic process. These results provide a beginning for pro- X 106 cells/ml in 0.45 ^m Millipore filtered SW supple- ACUTE HISTOINCOMPATIBILITY REACTIONS IN SPONGES 161 merited with 0.01% bovine serum albumen (BSA-SW; between autogeneic and allogeneic contacts. At about the addition ofthe very dilute BSA to the SW seems to 6 days, depending somewhat on temperature (Johnston improve the viability and health ofthe cells and aggre- et ai. 1981), the organization ofthe cells along the zone gates). To produce aggregates, the cell suspension is im- ofcontactchangesvisibly, and 24to 36 hourslatercellu- mediately dispensed in 0.4-ml aliquots into 16-mm di- lar degeneration becomes apparent along the boundary ameterwellsof24-well tissueculture plates(Falcon type ofcontact. Asalready analyzed extensively (Jokiel etai, 3047). Aggregation is initiated by placing the multiwell 1982), certain allogeneic combinations ofspecific indi- plates on a rotatory platform shaker with a 3" diameter viduals reproducibly reactweakly orslowly, presumably ofrotation (Henkart and Humphreys, 1970) shaking at due to genetic similarity of individual sponges in these 50 rpm. The BSA-SW is gently replaced at 8 hours and populations. For the results presented here, reactive then every day. Fortime seriesdeterminations, replicate sponge pairswere used in all experiments. We multiwell plates are started to provide an undisturbed miniaturized the histocompatibility assay by ex- sample for each time point determination. Aggregates amining the reactions of two small pieces of tissue are photographed with direct illumination undera Wild pressed together on an insect pin. These pieces adhere M5 binocular microscope using Kodak Ektachrome rapidly in both autogeneic and allogeneic combinations film. (Fig. 1). The former heal together and remain healthy indefinitely. The latter usually exhibit degeneration Histoincompatibilityreactions inaggregates along the zone ofcontact beginning by 48 hours. Figure To examine the reactions of allogeneic mixtures of t1hsreheowaslliongseenceticpipnaiarsisnagysspoofstshibeltehrbeeetwaeuteongtehnreeieciannddivtihde- cseplolsn,geasggarreegaptreosduccoendt.aiTnwinog0c.e2l-lmslfarloimquottwsoofdidfifsesroecnit- ouafltisspsounegiessveafrtyerev7i2dehnoturasloonfgctohnteabcot,unwdhaernydoefgceonnetraacttioinn ated cell suspension from each oftwo spongesare mixed theallogeneicpairs. An interestingfeatureofthereacting in the same aggregation well ofa multiwell plate, which tissue in these insect pin assays, evident on pinsCand D imsatTnhuheaenllrpeylaaacctpeipdoonossninotgfhetpawrioortsaagtogofrreyaggasgthreaeskg,earet.aecsharmeadeexaumpinoefdcelblys nifnortoFmiogbtushreeerav1re.edaisiontfhnceyotmno-itrgoerxaaictctiiornnegaocttfiiscosenulsel..sSoAulnlctholietvhmieinggprciaentlliasownaariyes ofonly one sponge. Aggregates are selected individually usually eliminated from the original sponge matrix in frommtmhe aggregation wells using a micropipette with an these miniaturized grafts by 4 days. Under this proto- 0.5 bore under a binocular microscope. Aggregates mm col involving both wounding of the contact surfaces 0.2 to 0.3 in diameterare selected and paired in the and trauma to the sponge tissue by the crushing action bottom ofa well ofa 96-well, U-bottom Micro Test III of the scissors during the process of cutting the small flexible Assay Plate (Falcon type 391 1) with 0.1 ml of pieces allogeneic reactionsaresignificantly more rapid filtered seawater. For allogeneic reactions, an aggregate with cell degeneration evident by 48 hours. This may be from one aggregation well containing cells ofonly one contrastedwith reactionsinabout 7 daysforlaterallyap- sponge isapposed to an aggregate from adifferent aggre- posed complete fingers that have not been traumatized gationwell containingonlycellsfromadifferentsponge. bycuttingin thezoneofcontactingtissue(Hildemann el ai. 1980). Graycellfraetionation Graycellsarepurifiedbycentrifugingapreparationof Cellreaggregalion dissociated cells in CMF-SW layered over a cushion of 8% Ficoll, 16% sodium diatrizoate in CMF-SW at Following the observations ofJohnston (1988) on C. 3000 rpm in an IEC refrigerated centrifuge at 15C for diffusa, we examined histoincompatibility reactions of 15 minutes. The pellet is resuspended in CMF-SW and allogeneic mixtures ofreaggregated cells. We confirmed consistsofabout 50-70%graycells(SeeFig. 6Aand 6B). thatcells, mechanicallydissociatedinseawater(SW)and allowed to settle on glass or plastic, aggregate and differ- Results entiate into small functional sponges within a few days when they are derived from one individual. In contrast, Tissuegrafting allogeneic mixtures ofcells from two spongesdie within The original histocompatibility experiments with C. 48 hours. We also examined these reactions with cells diffusa were replicated by tying two fingers cut from that had been dissociated bythe removal ofdivalentcat- different sponge individuals side by side on a glass slide ions (Humphreys, 1963), returned to complete SW, and maintained in running seawater (Hildemann et ai. reaggregated in suspension in wells ofa multiwell plate 1979). As described previously, the fingers adhere on a rotatory shaker. Similar killing ofmixed aggregates strongly for several days without discernible differences occurs undertheseconditions. 162 C. YIN AND T. HUMPHREYS '' Figure I. Histoincompatihilityassay.Thethreeautograftandthethreeallograftcombinationspossible between three individual sponges are shown 72 hoursafter apposition ofpiecesoftissue on insect pins. Twoapposed piecesoftissuefromthesamespongeremain healthyand healtogether(a,b, f)-Twopieces oftissuefromdifferentspongeindividualsreactandcreateazoneofcelldeathexposingtheskeletalmatrix alongthezoneofallogeneiccontact(c,d,e). Barequals0.4em. Figure 2. Aggregates from dissociatedcellsfrom eachofthreeindividual sponges. Aggregatesofpure populationsofcellsfrom thethreesponges(A, B.C),andthethreepossiblepair-wiseallogeneicmixtures ofdissociatedcellsfromthethreesponges(D,E.F),2 hoursaftertheinitiationofaggregationinsuspension shakercultures.Theaggregatesofmixedcellsfromtwoindividualsin D.E.andFaresignificantlysmaller thanaggregatesfromindividualpurecellaggregatesinA, BandC. Barequals0.5 mm. Figure3. Cytoto.xic histoincompatibility reactions in aggregatescontainingan allogeneic mixture of dissociatedcellsderived from twodifferentsponges. A. A lumpy surfaceat 20hoursofaggregation isthe firstevidenceofcytotoxicreactionsin mixedaggregates. B. By 21 hourstheroughenedappearanceofthe aggregatesurface hasbecome morepronounced. C. By 26hoursthe mixedaggregateshavebegun to fall apartintosmallercellclumpswhichsoondisintegratecompletely. D.Aggregatesofcellsderivedfromone individualremainsmoothlysphericalasshownafter82 hoursofaggregation. Barequals0.2 mm. ACUTE H1STOINCOMPATIBILITY REACTIONS IN SPONGES 163 TableI Averagediameter<>/thelenlar/;t'xtaggregatesinshakerwells after2limit'solaggregation Cellsfromsponge 164 C. YIN AND T. HUMPHREYS groupings ofcellsas shown after6 or 12 hours in Figure concentrated by purification on a density gradient, are 4D and E. In Figure 4E the edges ofthe aggregates near shown in Figure 6 A and B as photographed at high the boundary ofallogeneic contact have become rough. powerwith DICoptics. Thecharacteristic featureofgray Overthe next6to8 hourstheaggregateswillbreakdown cells is the cytoplasm filled with densely packed, highly into smaller clumps ofcells that proceed to disintegrate refractive, oblategranules. The gray cellsdo not contain completely. The failureofthecellswithin thetwoadher- the purple pigment evident in Figure 6A and B in the ent aggregates to round up into one spherical aggregate variety ofcell typescontaminatingthe gray cell prepara- suggests that cell movement has been suppressed by the tion. histoincompatibility reactions. Gray cells were originally described in M. prolifera as We also discovered that mature aggregates harvested appearing gray when viewed live under Kohler bright 48 hours after initiation of reaggregation react more field microscopy (Wilson and Penney, 1930). Gray cells quickly than newly formed aggregates. Aggregates col- appeargray underKohleropticsbecause they are unpig- lected 4 to 6 hours after the start ofaggregation exhibit mented, and their cytoplasm is completely filled with cytotoxicprocessesonlyafter36 hoursofcontact. Aggre- smallgranulesofhighrefractiveindexthatcreateanaura gates 24 hours old react more quickly (Fig. 4) and 48- ofdarkness about the cell. Because the original designa- hour aggregates initiate cytotoxic processes within tion ofthe gray cells in M. prolifera was based on their 8 hours of apposition. Thus, the processes responsible microscopicappearance, werefertothesecorresponding for the cell-killing reactions mature and proceed more cells in C. diffusaasgraycells.Graycellsrepresentabout rapidly in the olderaggregates. 1 to 3% ofthe total cells released from C. diffusa tissue bydissociation in CMF-SW. In M. prolifera, gray cellsaccumulateat the boundary Graycells ofallogeneiccontact,creatingavisibleband in the react- Because gray cells are intimaMtely involved in the his- ingtissue(Humphreys. 1994). Weexamined reactingin- toincompatibility reactionsof pmlij'cra(Humphreys, tact fingers, reacting pieces oftissue on insect pins, and 1994), weexamined thegraycellsofC diffusa. Previous reacting newly formed aggregates ofC. diffusa to ascer- histologicalstudieson C. diffusa(Smith and Hildemann, tain whether similar accumulations ofgray cells occur 1986a,b)failed toidentifygraycells. However,anexam- at the boundary between reacting tissue in this species. ination of CMF-SW-dissociated, living C. diffusa cells Although there were suggestions in many experiments under Kohler ordifferential interference contrast (DIG) that gray cellswere accumulating, wewere unable tode- optics revealed a small but distinct population oflarge, vise protocols that would yield striking and consistent multigranular cells resembling gray cells. These cells. accumulation ofgray cells at the boundary of reaction Figure6. Gray Cells. A and B. Dissociated gray cells enriched by density gradient centrifugation C. Graycellsin a migratingbandscatteredamongthepurplishcellsofareacting48-houraggregate;6hours aftertheaggregateswereapposed.theywerecompressedto 1 to20/jrn underacoverslip.examined,and photographed. Gray cells (marked by arrowheads) are bright, non-pigmented cells whose cytoplasm is packed with small oblate granules. The various non-gray cell types in the preparations can be seen to exhibitadiversityinnumberandtypeofcytoplsamicinclusionsandthepurplepigmentationcharacteristic ofC'. ili/fuxa. Observedat 100Xwithdifferentialinterferencecontrastoptics. Barequals 10^m. ACUTE HISTOINCOMPATIBILITY REACTIONS IN SPONGES 165 in these experimental settings. However, when 48-hour tion signal. This regulation ofcell behavior can serve as aggregates,whichreactin 8 hours,areapposed,abandof a time- and possibly cell-proximal assay for the activa- lightercolored tissue parallel to the boundary ofcontact tion ofthe rejection reaction. It could prove useful for became detectable within four to eight hours. A pair of identifying the cells involved in recognition and signal- reacting48-hour aggregates, photographed 6 hours after ing and for screening compounds, such as sugars, pairing,exhibitssuchaband, and itseemstobeconverg- cyclosporinA,ormonoclonalantibodies,which impinge ing on the boundary ofallogeneic contact (Fig. 5A). If directly on theearly cellulareventsofrecognition. the adhering aggregates are greatly compressed under a We have also established that C. diffusa hasa popula- cover slip until they are amenable to examination by tion ofgray cells that is specifically involved in the allo- DIC optics, an accumulation ofgray cells is evident. A geneic rejection reaction. Sponges in general (Simpson, general view, taken with a 20x DICobjective, showsthe 1984),andC. diffusaspecifically(Smithand Hildemann, aggregatepairfrom Figure5A compressedtoone fifth its 1986a, b; 1988), have fewerthan adozen cell types. The original thickness (Fig. 5B). The brighter spots arrayed suggestion that gray cells, which are present in most parallel to the boundary ofallogeneiccontactamongthe sponge species (Boury-Esnault, 1977), may serve an im- otherwise lightly purple cells represent gray cells. Ifthe mune function (Humphreys, 1994) providesinsight into aggregatesarecompressed even more and observed with the natureofthiscell type, whose function hasotherwise a 100X oil DICobjective,thesebrighterspotscanbeseen been unknown (Simpson, 1984). Identifying a specific tobedefinitivegraycellswiththeirdenselypackedoblate cell type found in many species ofsponges as a possible granules (Fig. 6C). One or two hours later, when the ag- immunocyte will provide focus forcellularand molecu- gregatesfirstexhibitsignsofcytotoxicreactions, thegray larstudiesofimmune function in sponges. cells are concentrated at the boundary of contact be- The following aspects ofour results will be discussed tween the allogeneic cells as shown at low power in Fig- more fully: (1) the basis for the more rapid, 48-hour re- ure 5C. We were unable to continue to find and identify actions ofthe tissue fragments on insect pins, and ofal- thegraycellsin the aggregatesbeyond thisperiodduring logeneic mixed cells in aggregates, relative to the one- whichthecytotoxicreactionsbegin. Noaccumulation of week reactions ofintact tissues; (2) the nature ofthe re- graycellsoccursin remodelingpairsofcontactingaggre- actionsthat inhibit cell aggregation ofthe allogeneiccell gatesofcellsfrom the samesponge. mixtures; and (3) The processes occurring in newly formed, individual-specific aggregates that shorten the time to cytotoxic reactions from 36 hours ofallogeneic Discussion contact to 8 hours and allow the rapid accumulation of We have studied the histoincompatibility reactions in graycellsat theallogeneic boundary. the marine sponge C. diffusa and have developed novel modifications of this interesting experimental system 48-hourreactions that will help to elucidate the cellular and molecular mechanismsofimmunerecognitionintheinvertebrates. When intact fingers of C. diffusa are apposed, cyto- The histoincompatibility reactions in this species have toxic reactions are not detectable until about one week beenwellstudied(Hildemann eltil., 1979).Cytotoxicre- later(Hildemannelal.. 1980,andourownexperiments). actionsbetweenparabiosedintact fingersoftwodifferent Incontrast,thecytotoxicreactionsintheinsectpinassay spongesoccurafteraweekanddestroyafew millimeters orwithin aggregatesofdissociatedcellsbegin in lessthan oftissuealongtheboundaryofallogeneiccontact. These 48 hours. We propose that wounding or dissociation of reactions have been followed by extensive histological the tissue quickly releases rate-limitingstepsto the initi- examination (Smith and Hildemann 1986a, b; 1988) ation ofthe sponge immune response, a process that re- without a clear delineation ofany specifically immune quires several days when activated by allogeneic contact cells. The observations ofJohnston (1988), that alloge- signals alone. The biological purpose for rate-limiting neic mixtures ofdissociated cells begin cytotoxic reac- stepsmaybetodelayasmuch aspossiblethenonspecific tionswithin 48 hours, suggested to usthat furtherexam- cell killing, which destroy thecellsofthereactinganimal ination ofthese more rapid reactionsofdissociated cells as well as those of the foreign organism (Bigger el al, might yield further insight into possible cellular mecha- 1981). Thus, the sponge seemsto have mechanismsthat nisms. respond slowly against foreign contactswhen they mini- Ourexperiments present two new results that may be mally impinge on the sponge tissue, but can react vigor- important for understandingthe cell biology ofrecogni- ouslytoforeigncontactswhentissuedamageisinvolved. tion. We discovered an experimentally observable pa- The more rapid second-set reactions that have been de- rameter, therateofcell movementand aggregation, that scribed in C. diffusa (Hildemann el al., 1980) may be changes within one hour of actual allogeneic contact, related to thisacceleration. These authors noted that for possibly reflecting the generation ofa non-selfrecogni- about three weeks after undergoinga rejection reaction, 166 C. YIN AND T. HUMPHREYS a finger ofC. diffusa will react more rapidly to a second and that require 36 hours to react with cytotoxicity. If allogeneiccontact. these suggestions are valid, the slowness ofthe cells to carryoutallogeneic recognitionperseisnot the limiting Inhibition ofaggregation factor in the slower reactions. Rather, the delay seems The inhibition ofaggregation, which is manifested in relatedtotheabilityoftheimmuneprocesstoproceedto an allogeneic mixture ofdissociated cells within an hour the cytotoxic effector function in response to allogeneic offirst contact, appears to be a specific response to non- recognition signals. The striking observation that the selfrecognition upon allogeneiccontact. Webelieve that gray cells in 48-hour aggregates are rapidly mobilized aggregation slows because cell movement is suppressed, suggests that the responsivenessofgray cells may be one possiblyby achemokine released aspart ofthesignaling of the accelerated parameters. The acceleration in the ofnon-selfrecognition. In theexperimental setting, are- spongesofalloreactivityuponwoundingordisruption of dlaurcgteirocnellinclcuelmlpsm,otbielciatuysewoceullldmoslvoewmetnhet fisorremqautiiroend toof tvehretteibsrsauteemiamymbuenceonscyesptteumallthyatreltahteerdetaorteheaivdaeraieitnythoef stabilize nascent attachments between two cell clumps "danger" signalsthat maketheanimal more likely to re- when they happen to make contact in the shaking sus- act to a given antigen as foreign (Matzinger, 1994). In pensions. Insteadofcells movingsothattheinitial adhe- C. diffusa, these "danger" signals produced from tissue sion spreads and becomes more stable, the two clumps wounding accelerate cytotoxic reactivity from a time amdahteerley osnhleyaraetdtahpearptoiangtaionfbiynittihael mcoonttiaocntoafntdhearleiquulitdi.- couWresesoufgg1e7s0thaobuorvsetothoantetohfe8ihnohuirbsi.tion of aggregation may be a response ofcells to the release ofa chemokine wIinldleeadd,heirfeshbauktindgo insostttoepnpdedtothfeoralmloagesnienigcleagspghreergea,teas at the boundary ofallogeneic contact. We have demon- processrequiringcell movement(Steinberg, 1963). Like- strated that graycellsare induced to move tothe zone of wisethe failure oftwoaggregates in allogeneiccontact to allogeneic contact, a responseanalogous to those guided rreomuanidnuapdhienrtoenotn,ecsopnhfeirrimcsalthaagtgrceeglaltme,oveveemnenthtohuagshbteheeny bthyecvheermtoebkrianteesigmemneurnaetesdydstuermi.ngInthveerrteeabcrtaitoenss,otfhceerlelsarien many chemokines, each with a variety of functions. suppressed (Steinberg, 1963). Studies with an individual specific monoclonal anti- Thus, from this model one may suppose that one, or obfodtywo(SpmariatbhiaonseddHfiilngdeermsaonfnC,. 1d9if8f6ubs)asdhoonwotthmaitxthdeurceilnlgs mmoorvee,mecnhte,mopkrienveenstimnagythweeilnlgraecststooffionrhiebiigtn cgeelnlse,raalndcetlol the several days they are in contact before the cytotoxic attractgraycellsto move through these inhibited cellsto reaction begins. Because sponge cells usually migrate the boundary ofallogeneiccontact. constantly, and visual observation of the adhesion be- tween the cells ofthe surfaces oftwo parabiosed sponge Anoverviewoftheimmunereaction ofC. diffusa fingers establishes that they are in intimate contact, the Superficial contact ofC. diffusawith non-selftissueor failure ofthe cells ofthe two contacting sponges to mix cellsisquickly recognized and leadsto theproduction of wouldbesurprisingifcell movementwereactive.In vivo a signal that suppressesthe movement ofselfcells in the the suppression of cell movement upon contact with zone ofcontact. This suppression may reduce the entry non-selftissue may functionally reduce the potential for offoreign cells into the sponge tissue. Ifthe contact per- parasitic or pathogenic cells to enter the sponge tissue sists, or ifcontact involves significant tissue damage, an during the early phases ofallogeneic contact before the accelerationoftheimmuneprocessoccurs,andgraycells cytotoxic rejection reactionsbegin. become more responsive to the signal to move to the boundary ofallogeneic contact. As the gray cells accu- 8-hourreactions mulate at the zone of allogeneic contact, cell killing The ability of48-hour aggregates to carry out the cell effector mechanisms are initiated, and all cells in the killingreaction within 8 hoursofallogeneiccontactindi- zone of contact, both foreign and self, are killed. This catesthat tissueandcellsareable, underproperlystimu- later response, although harmful to the sponge, presum- latedconditions,toprogressfrom theprimaryallogeneic ably eliminates any organism that is attacking or trying recognition event to the cytotoxic effector activity in togain entry into the sponge tissue. The state ofacceler- only 8 hours. We havealsosuggested that suppression of ated reactivity ofthe sponge immune system may last aggregation in allogeneic mixtures ofcells reflects a sig- aboutthreeweeks. nal from primary allogeneic recognition and occurs within one hour ofallogeneic cell contact. This signal, LiteratureCited ewvheinchinsucpeplrlsestsheastcaerlel mfroevsehlmyenisto,laitseedvfidreonmtiinntaocntethisosuure BiggAefrf,erCe.ntH.,seWns.itHi.zaHtiiolndeamnadnnef,fePr.eLn.tJcoyktioetlo,xiacnidtyI.iSn.aJlolhongsetnoeinc.t1i9s8s1ue. ACUTE HISTOINCOMPATIBILITY REACTIONS IN SPONGES 167 responsesofthe marinespongeCallyspongiadiffusa. Transplanta- The influence oftemperatureon the kineticsofallograft reactions tion31:461-464. inatropicalspongeandareefcoral.Biol. Bull 160:280-291. Boury-K.snault, N. 1977. A cell type in sponges involved in the me- Jokiel, P.L., W. II. Ilildemann,andC. II. Bigger. 1982. Frequency tabolismofglycogen:thegraycells.CellTina. 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