Fish & Shellfish Immunology 19 (2005) 441e455

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Phylogeny and ontogeny of fish leucocytes

J.H.W.M. Rombout a,*, H.B.T. Huttenhuis a, S. Picchietti b, G. Scapigliati b

a Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University,

AH Wageningen 6709, The Netherlandsb Animal Biotechnology Lab, Department of Environmental Sciences, Tuscia University, Viterbo, Italy

Received 23 January 2005; accepted 2 March 2005

Available online 10 May 2005

Abstract

In contrast to higher vertebrates, most fish species hatch at the embryonic stage of life. Consequently, they have todefend against a variety of micro-organisms living in their aquatic environment. This paper is focussed on the

development of leucocytes functioning within this early innate system and later on in the acquired immune system (B andT cells). Most of the data are derived from cyprinid fish (zebrafish, carp), which are excellent models to study earlyontogeny. Attention is also paid to the phylogeny of leucocytes, with special attention to early chordates. It is clear that

young fish use innate mechanisms during the first weeks/months of their development. In zebrafish, a variety ofhematopoietic genes have been sequenced which allow a detailed picture of the development of the distinct leucocytes andtheir precursors. In cyprinids and sea bass, the thymus is the first lymphoid organ and T cells appear to be selected theremuch earlier than the first detection of T cell-dependent antibody responses. The first B cells are most probably generated

in head kidney. Although T cells are selected earlier than B cells, T cell independent responses occur earlier than the T cell-dependent responses. The very early (pre-thymic) appearance of T-like cells in gut of sea bass and carp suggests an extra-thymic origin of these cells. However, B cells populate theGALTmuch later than spleen or kidney, indicating a rather late

appearance of mucosal immunity. The first plasma cells are found long after the intake of food in cyprinids, but in manymarine fish they appear around the first food intake. In general, acquired immunity is not correlated to food intake.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Teleost; Development; Hematopoiesis; Myelopoiesis; Lymphopoiesis; Innate immunity; Adaptive immunity

* Corresponding author. Wageningen Agricultural University, Cell Biology and Immunology Group, Wageningen Inst of Animal

Sciences, P.O. Box 338, AH Wageningen 6709, The Netherlands. Tel.: C31 317 48 27 29; fax: C31 317 48 39 55.

E-mail address: jan.rombout@wur.nl (J.H.W.M. Rombout).

1050-4648/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.fsi.2005.03.007

442 J.H.W.M. Rombout et al. / Fish & Shellfish Immunology 19 (2005) 441e455

1. Introduction

In contrast to higher vertebrates, most fish species are free-living organisms already at the embryonicstage of life. Living in an aquatic environment, they must have defence mechanisms to protect themselvesagainst a variety of micro-organisms. Consequently, during a rather long period fish are dependent on theirinnate immune system, and it is expected that this develops at a very early embryonic age. This paperdescribes the development of leucocytes of the innate immune system and the acquired immune system infish. Most of the data are derived from zebrafish, which is an excellent model to study early ontogeny. Inaddition, attention is paid to the phylogeny of leucocytes, with special attention to early chordates (i.e.tunicates, cyclostomes and cartilaginous fish).

For aquaculture, the appearance of acquired immunity is important to estimate the correct age foreffective vaccination. Before that period immuno-stimulation of innate defence systems possibly offersa better protection.

2. Phylogeny of leucocytes

Data on the biology of immune systems in protostomes accumulated in recent years with the discoveryof cellular reactions and, more interestingly, with the cloning of many defence-related genes. Froma comparative immunology view, these genes and their products were classified using terms from vertebrateimmunology, such as immunological memory, lymphocytes and allograft reaction. In most casesa functional similarity with vertebrates was speculated upon, without considering that these reactionsmay have arisen independently during evolution. This consideration is supported by experimental datashowing that key molecules of the immune system i.e. the major histocompatibility complex (MHC), T cellreceptor (TcR) and immunoglobulin (Ig) have no counterparts in protostomes. As a consequence, it isinconsistent to mention lymphocytes, immunological memory and other components of the specificimmune system in the invertebrate context [1]. However, invertebrates possess an efficient defence systembased on innate humoral and cellular activities that function in a complex fashion, and these defencereactions can be classified as ‘‘inflammatory-like’’. Cellular activities of invertebrates are performed by cellscirculating in body fluids, which are collectively called hemocytes, and can mainly be classified into cellswith granules (granulocytes) and without granules (plasmatocytes) [2]. Once again it is difficult to relateinvertebrate circulating cells with vertebrate leucocytes, because similarities may derive from analogousrather than homologous responses.

Early molecules resembling Ig or TcR variable domains are present in invertebrates, although they donot perform somatic rearrangement [3e7]. It was suggested that these variable region gene segments inprimitive vertebrates served as targets for the rearranging machinery when it was introduced [8]. Recently,attention was paid to lower chordates (tunicates) with studies of the whole genome of Ciona intestinalis [9].In this species no evidence of acquired immunity genes like MHC, TcR, and Ig were found, thus indicatingthe origin of acquired immunity in vertebrates [10]. It was a matter of considerable debate whether the mostancient living vertebrates, represented today by cyclostomes, developed an acquired immune system withlymphocytes and memory. In an early study [11], the hagfish Eptatretus stoutii was found to be incapable ofproducing specific antibodies against various injected antigens and responded poorly to an allogeneic graft.On the contrary, others described the mounting of a humoral response [12,13], the presence of Ig-bearingcells [14], and T-like cells proliferating in a mixed leucocyte reaction (MLR) at 20 �C after 5 days [15]. Inanother species (Myxine glutinosa), plasma cells were identified based on morphological criteria [16]. Theputative Ig molecule of Eptatretus burgeri was purified from serum. It was heat-sensitive and displayed twomain bands at approximately 22 and 68 kDa [17]. In E. stoutii a putative Ig molecule was immuno-purified

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from serum with a mAb, and had a molecular weight (MW) of 30 and 77 kDa [18]. Although Ig, TcR, andMHC genes were not identified in jawless vertebrates, hagfish rejected skin allografts [19,23] and displayeda delayed hypersensitivity reaction [12]. These activities were performed by circulating blood cells,comparable to lymphocytes of jawed vertebrates [20e23]. Although acquired immunity genes and truelymphocytes were not identified, hagfish expressed an ikaros gene [24]. Ikaros encodes a number of zinc-finger transcription factors that play key roles in vertebrate hematopoietic stem cell differentiation and thegeneration of lymphocytes and NK cells [25,26]. These results showed that the appearance of ikarospreceded the emergence of jawed vertebrates and thus acquired immunity. Several antigens, that areimmunologically relevant in other vertebrates, were identified in lamprey leucocytes: CD45, a member ofthe receptor-type protein tyrosine phosphatase family; BCAP protein, known as B cell adaptor forphosphoinositide 3-kinase; CD33-associated signal transducer; and CD9 [27]. A recent investigation hasshed light on how lampreys, without lymphocytes as they are known in jawed vertebrates, may respond toan antigen through somatic rearrangement of a germ line gene not present in other vertebrates [28]. In thisfish species, a new type of variable lymphocyte receptor (VLR) was identified, which was composed ofhighly diverse leucine-rich repeats with an extraordinary mechanism to generate diversity from germ linesequences. Individual lymphocytes from immuno-stimulated larvae carried a unique somatic rearrangementof the VLR, thus showing the ‘‘clonality’’ of the response in lampreys. Further research will elucidatewhether VLRs are paralogous to the vertebrate lymphocyte-associated antigen receptor genes.

Cartilaginous fish and teleosts (bony fish) are the earliest vertebrate groups that have an immune systemcomparable to higher vertebrates (Table 1), with an acquired immune system consisting of B- andT lymphocytes, MHC and memory formation. The dramatic change in immune capabilities between jawlessand jawed vertebrates was attributed to a genome duplication in several studies [29,30]. It remains to beconsidered whether the ability of swallowing virtually any kind of food by early jawed vertebrates increasedthe capability of the immune system to distinguish between self and non-self. The new ability to chew andswallow food possibly increased physical injuries in the intestinal epithelium, eventually inducing newpotential antigens into the body [31]. This theory is supported by the observation that in the teleostseahorse, which has a siphon-like mouth, gut-associated lymphoid-tissue (GALT) is absent or reduced,while lymphocytes in other locations of the body are retained [32].

The most ancient living jawed vertebrates are cartilaginous fish (sharks and skates), and a considerableamount of work has been performed on the characterisation of their immune system. Genes were identifiedencoding for MHC class I and II [33,34], rag-1 [35e37], Ig [38], a surface Ig novel antigen receptor (sIg-NAR) [36,40], and for TcRa and -b [39].

Regarding leucocytes of cartilaginous fish, several papers showed the presence and activity of theprincipal class of leucocytes: the macrophages. These cells performed ‘‘in vitro’’ pinocytosis andphagocytosis, and were cytotoxic against foreign cells [40,41]. In addition, cell-mediated cytotoxicity insharks [42] was performed by leucocytes purified by conventional Percoll density gradient centrifugation[43]. In the same report, purified leucocytes exhibiting spontaneous cytotoxicity were classified as non-adherent, non-phagocytic, and Ig receptor negative. Shark leucocytes responded to chemotactic stimuli andcomplement fractions [44]. B-lymphocytes produced Ig, which increased opsonisation of particles andcytotoxicity [42]. At least two populations of ‘‘in vitro’’ Ig-forming cells were identified in a skate [45].

Table 1

Presence of main immune features in the phylum of chordates

Chordate group Lymphocyte receptor MHC RAG Ikaros Memory

Tunicates e e e yes eCyclostomes VLR e ? yes e

Cartilaginous fish Ig, Ig-NARs, TcR yes yes yes likely

Teleosts Ig, TcR yes yes yes yes

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A significant amount of work was done on the immune system of bony fish in recent years: from theevolutionary point of view and for the application of results to fish farming. With respect to teleosts, bonemarrow, lymph nodes and Peyer’s patches are absent. The cephalic portion of the kidney (pronephros orhead kidney) is considered analogous to mammalian bone marrow regarding hematopoiesis [46,47].The trunk kidney (opisthonephros) is also hematopoietic, although it mainly consists of renal tissue. Inthese organs the homing and differentiation of hematopoietic stem cells occurs [48]. The spleen is involvedin hematopoiesis, although its role is generally limited to erythropoiesis and thrombopoiesis [49].

3. Ontogeny of leucocytes

3.1. Ontogeny of leucocytes in cartilaginous fish

At present, knowledge on the development of shark leucocytes and hematopoietic tissues is meagre. Inthe nurse shark (Ginglymostoma cirratum), the development of primary and secondary lymphoid tissues wasstudied [50]. After hatching, white pulp of the spleen contained B cells and small areas with T cells andantigen-presenting cells, whereas the adult spleen contained a well-defined and vascularized white pulp withT cells, antigen-presenting cells, and a low number of Ig-secreting cells. The epigonal organ performedB cell lymphopoiesis after hatching, as deduced by the presence of these cells and the expression of rag-1and TdT, which persisted into adult life. In contrast, the nurse shark spleen had a negligible lymphopoieticactivity [50]. The expression and rearrangement of sIg-NAR was recently observed in the epigonal organsof neonatal shark [51], although no information is available on the leucocytes that carry this novel Ig.Another study [52] on clearnose skate (Raja eglanteria) showed complex expression patterns of lymphocytespecific genes (Ig, TcR, rag-1 and TdT) during the development, implicating unique lymphoid tissues ingenerating the immune repertoire. For instance, IgM was first expressed in spleen, whereas IgX transcriptsfirst appeared in the gonads, the liver, the Leydig organ and even the thymus. In all organs rag-1 expressionwas found, indicating that B cells develop at different sites. In embryonic thymus, the expression of all fourclasses of TcR were described together with rag-1 and TdT expression, which indicate a less complexdevelopment of T cells compared to B cells.

Ontogenesis of GALT was investigated in Scyliorhinus canicula [53], showing that GALT was firstrepresented by individual lymphocyte-like and macrophage-like cells in the lamina propria accumulatingprior to hatching. These cells increasingly infiltrated the lamina propria and epithelium when fish startedfeeding. An important observation in this studywas thatGALTdeveloped after the thymus and the lymphoidtissue in the kidney, and approximately at the same time as the spleen, epigonal andLeydig organ. Plasma cellsand granulocytes were not observed in developing dogfish until 6 months post-hatch [53].

3.2. Ontogeny of leucocytes in teleosts

3.2.1. Zebrafish as a vertebrate model for hematopoiesisAn as yet poorly elucidated web of interactions regulates vertebrate embryonic development. In fish,

most data come from zebrafish (Danio rerio). This animal is considered as the fish model for the study ofembryonic development because of: the relative ease to study embryos, the powerful genetics which can beapplied for the generation of mutants and transgenic animals, the availability of many molecular markers,and the knowledge of the genomic sequence [54e57]. However, the zebrafish model should not beconsidered as the ontogenetic model for all fish, as it is well known that early development [58] andorganogenesis [59] differ considerably between species.

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3.2.2. Embryonic hematopoiesisEmbryonic and definitive hematopoiesis are separate and different processes in zebrafish development,

similar to the situation described in mammals [60]. The hematopoietic tissues of zebrafish development arepresented in Fig. 1. In the lateral plate mesoderm (LPM), the first expression of hematopoiesis-relevantgenes was observed around 12 hours post-fertilisation (hpf) [61]. During the migration of LPM cells, theintra-embryonic intermediate cell mass (ICM) is formed. This structure progressively converges in a rostralto caudal direction and ends just caudal of the future anus [62,63]. When the caudal vein is formed, theICM in the tail is called the posterior blood islet. At an early stage (18 somitesZ 20 hpf) the ICM consistsof undifferentiated hemangioblasts that still have the potential to form endothelial, erythroid, myeloid, andlymphoid precursors [56,63]. Between 22 and 30 hpf, a variety of stem cell marker genes are expressed. Theyare given in Fig. 2, which indicates the differentiation of the different hematopoietic cell lineages. Theposterior blood islet of zebrafish appears around 42 hpf [62] and remains hematopoietic till at least 96 hpf[64]. Possibly, hematopoiesis still continues in the posterior blood islet, because a high number of mpxpositive cells were detected at 6 dpf [65]. In carp, a similar development was observed, though a posteriorblood islet was observed from 36 hpf (at 25 �C), and remained hematopoietic considerably longer (till 2weeks post-fertilisation (wpf)). The posterior blood islet of the carp embryo was the main myelopoietictissue (neutrophils, basophils and monocytes/macrophages), as was observed with immuno-histochemistryusing leukocyte subpopulation-specific monoclonal antibodies and transmission electron microscopy (EM)

Fig. 1. Hematopoietic tissues during zebrafish development, identified using cell morphology and expression of genes important for

hematopoiesis. Anterior is up, dorsal is to the right. Lateral Plate (ventral) Mesoderm (LPM) [61]; Intermediate Cell Mass

(ICM) [62], the dotted line represents mainly vasculogenic tissue [63]; ventral part of dorsal aorta [61,64]; caudal

(posterior) ventral vein region (plexus) or posterior blood islet [62]; kidney; thymus. From 16 hpf, macrophages originate

from the rostral LPM [67], and from 24 hpf erythrocytes and mpx positive cells appear in the ICM [61,62,65,72]. The ICM can be

divided into separate areas based on expression patterns and developmental speed [61,72]. Between 24 and 30 hpf, the ICM disappears

as cells are taken up by the nascent circulatory system [62]. At 48 hpf hematopoiesis takes place in the ventral wall of the aorta [61,72],

and in the posterior blood islet [62]. From approximately 4 dpf, hematopoiesis starts in thymus and head kidney [62].

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[66]. Furthermore, the ICM forms the aorta and the vena cardinalis. Myelopoiesis was observed in theventral wall of the zebrafish aorta from 48 to 96 hpf, until hematopoiesis starts in the kidney [61,64].

3.3. Teleost myelopoiesis in development

Myeloid cells develop from a multipotent myeloideerythroid progenitor (MMP), which also has thepotential to differentiate into thrombocytes, as shown in Fig. 2. The MMP is considered a myeloid lineageprecursor when spi.1 or c/ebp1 is expressed [56]. Macrophages differentiate from this precursor as soon as

Embryonic SC

ICM cells

Hemangioblast

Hematopoietic SC Endothelial SC

MMP BLP

Erythrocyte LP Thrombocyte LP Myeloid LP Pro-B cell Pro-thymocyte

Granulocytes Monocytes/

Macrophages Effector B cells Effector T cells

zbmp-2

scl/lmo2/hhex

flk-1 gata-2

c-myb, cbf ikaros

gata-3

rag-1/2, TdT

rag-1/2,TdT, lck

mpxdra, fms,L-plastin

spi-1,c/ebp1

Endothelium

Fig. 2. Schematic representation of the molecular pathways involved in zebrafish hematopoiesis. This scheme should be considered

a simplified working model of the actual biological situation. SC: stem cell; ICM: intermediate cell mass; LP: lineage precursor; MMP:

multipotent myelo-erythroid progenitor; BLP, bipotent lymphoid progenitor. Genes: zbmp-2: zebrafish bone morphogenic protein 2; scl:

stem cell leukemia transcription factor; lmo-2: lim domain only-2; hhex: hematopoietically expressed homeobox; flk-1: fetal liver tyrosine

kinase-1; gata-2/3: gata-binding protein-2 and -3, respectively; c-myb: myeloblastosis oncogene; cbfb: core binding factor b; spi-1: serine

protease inhibitor-1 (Z pu.1); c/ebp1: CCAAT/enhancer binding protein 1; mpx: myelo-peroxidase; dra: draculin; L-plastin: leucocyte-

specific plastin; fms: receptor for macrophage colony-stimulating factor; rag-1/2: recombination activation gene-1 and -2, respectively;

TdT: terminal deoxynucleotidyl transferase; lck: T cell-specific tyrosine kinase gene promotor [56,61,73,88,110e112].

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they start to express fms and L-plastin [56]. This process commences between 12 and 20 hpf in zebrafishmacrophages derived from the LPM, anterior to the heart [67]. These cells, that bypassed the monocyticseries, migrated onto the yolk sac around 24 hpf. From the yolk sac many of them migrated to themesenchyme of the head and into the blood circulation [67]. Before and during migration the earlymacrophages initially expressed the dra gene, which was subsequently replaced by L-plastin. Apart fromphagocytosing apoptotic cells, these macrophages also engulfed and destroyed intravenously injected Grampositive and Gram negative bacteria, and in addition they showed activation and migration towardsinfected sites [67]. These data indicate that zebrafish macrophages operate as a first line of defence at thisage. No data are available yet on the type of chemotactic molecules that play a role in the attraction ofmacrophages. Moreover, carp IL-1b was expressed from 1 dpf onwards using real time quantitative PCR,and was up-regulated when LPS was injected in the yolk sac of carp embryos [66]. Although it is not provenwhether this molecule play a role in chemotaxis of leucocytes, it certainly indicates that inflammatorycytokines are present very early in ontogeny. From 48 to 72 hpf (in zebrafish and carp, respectively), theposterior blood islet must be considered the major producer of myeloid cells. From 4 dpf (zebrafish andcarp) onwards the kidney appears and will become the main myelopoietic tissue. In carp, ontogeny wasstudied using a macrophage-specific monoclonal antibody (WCL15 [66,68,69]). The first macrophages weredetected at 2 dpf in the kidney region and on the yolk sac. At 4 dpf they were found in thymus and gutepithelium and from 7 dpf also in kidney and spleen.

In addition to macrophages, granulocytes are derived from the myeloid lineage precursor. At least twotypes are described in each fish species studied, but their nomenclature is rather inconsistent [70,71].Ultrastructurally, both types have a similar appearance in all species described. The first type containssmaller oval granules with a crystalline structure inside, and is generally called neutrophil or heterophil. Thenomenclature of the second type, with larger round to oval electron-dense granules and a less polymorphicnucleus, is less consistent. The granules differ significantly in their electron densities. Even within one celldifferent granules were noticed, from electron dense to electron lucent with all variations in between. Thename of this second granulocyte type should depend on the staining with eosin (i.e. salmonids), but in carpit is not eosinophilic cytochemically. These cells with ultrastructural similarities with mammalian mast cellsstain well with toluidine blue and are abundant in mucosal tissues. These characteristics justify thedesignation basophil, even when these cells are eosinophilic in certain fish species. Unfortunately,knowledge about their function is limited. Both cell types were found early in carp ontogeny: neutrophilsfrom hatching onwards and basophils from 3 dpf, which indicates an early defence function for both [66].

Neutrophils in zebrafish displayed a high peroxidase-activity and were easily distinguished in tissues bya peroxidase staining. In addition, they expressed neutrophil-specific myelo-peroxidase (mpx) very early indevelopment. mpx was expressed first in the ICM of zebrafish at 18 hpf [65,72]. From 24 to 72 hpf, mpx-expressing cells were scattered throughout the embryo, with the majority in the posterior blood islet[56,65,72]. In carp, neutrophils were not clearly peroxidase-positive, but a monoclonal antibody is available(TCL-BE8). Although reactivity was found with monocytes (in adults), the combination of electronmicroscopy and immuno-cytochemistry located carp neutrophils at the same locations as in zebrafish,indicating that the posterior blood islet is an important granulopoietic tissue. Similar to macrophages,neutrophils also migrated to an infection or inflammation site (after tail transection [56,65]) indicating theirrole in innate defence. Already during the hematopoietic period of the posterior blood islet, (head) kidneydevelops as the definitive myelopoietic tissue (in carp around 5 dpf) [66,68].

3.4. Teleost T cell lymphopoiesis in development

When embryonic developmental processes are completed, the various hematopoietic processes graduallyshift towards the organs they are associated with in the juvenile and adult situation. In general, the thymusis the first of these organs to become lymphoid [73e76], although in flounder (Platichthys flesus L.) and cod

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(Gadus morhua L.) thymus is reported to appear later than spleen and kidney [59,77]. In zebrafish and carp,the thymus anlage is lymphoid at 3 and 4 dpf, respectively [62,78]. These early thymocytes probablyoriginated from the ICM as indicated by ikaros gene expression [76]. Ikaros is a zinc-finger DNA bindingprotein considered to be a master hematopoietic switch gene involved in the earliest commitment ofhematopoietic stem cells to the lymphoid lineage [25,26]. With RT-PCR (not published), carp and zebrafishboth expressed at least 5e6 isoforms of ikaros, of which 4e5 were expressed at high levels (ik-1, ik-2, ik-7and ik4/8) and 1 (ik-3) at a low level. In trout, 8 isoforms were described [25]. Fig. 3 displays the expressionof these isotypes in early zebrafish development. Assuming all isotypes are necessary to produce lymphoidprogenitor cells, we conclude this is possible from 17 hpf. Ikaros expression was first observed in the lateralmesoderm of zebrafish at 16 hpf, followed by the ICM from 19 hpf and was detected in thymus at 72 hpf[76]. In trout, developing considerably slower at lower temperatures, ikaros was also expressed early inontogeny: at 3e4 days in the yolk sac and at 5e6 days in the embryo proper [25]. At 84 hpf rag-1, gata-3and lck expression was detected in zebrafish thymus [55,57]. In carp, rag-1 expression appeared ata comparable age [66], while in trout rag-1/2 expression was found at 10 dpf and TdT expression at 20 dpf(6 days before hatching), when thymus and head kidney appeared [25].

The first detection of thymic cortex and medulla in carp and zebrafish was reported at 7 dpf [66], and ofcourse considerably later in trout (54e56 dpf) [25]. In carp, cortex and medulla were also demonstrated witha cortical thymocyte marker (mAbWCL9) that reacted with thymocytes immediately after their appearancein the thymus [78]. WCL9C cells expressed rag-1 in the thymus [66]. Interestingly, WCL9C lymphoid cellswere detected in blood, spleen, head kidney and gut in the first weeks post-fertilisation, although insignificantly lower proportions compared to thymus. However, after 5 wpf their presence was restricted to thethymus [69]. The significance of this feature still has to be investigated, but this is considerably hampered bythe fact that the function of the WCL9 immuno-reactive molecule is not known yet.

Fig. 3. Ikaros expression during the first 29 h of zebrafish development. RT-PCR with ikaros primers was carried out on RNA isolated

from pools of 10 embryos of equal age, indicated above each lane. All embryos were grown from the same clutch of eggs. A molecular

weight marker (M) is loaded on the gel on the far left and right sides. Note the presence of the 5 isoforms in zebrafish (4 strong and 1

weak) from 17 hpf onwards, before that age only 2e3 bands were detected. Embryos were monitored till 61 hpf, but showed the same

5e6 isotype pattern (unpublished results).

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The first TcRa gene expression was detected in zebrafish at 3 dpf using RT-PCR, and the expressionsteadily increased till adult levels were reached between 4 and 6 wpf [57]. In another study on zebrafish,using in situ hybridisation, the first TcRa expression was found in thymus at 4 dpf and adult levels werereported at 3 wpf [79]. In the same report, the first TcRa-positive cells outside the thymus were found in theregion of intestine and gills at 9 dpf, followed by head kidney and subsequently spleen.

MHC-I expression is intimately related to T cell function. In carp, MHC-Ia expression (using RT-PCR)was detected from 1 dpf, although b2m first appeared at 7 dpf, indicating that MHC-I molecules arefunctional from 1 wpf [80]. With antibodies against both components of MHC-I, expression on thymocyteswas observed from 3 wpf onwards, but may be earlier because younger stages were not investigated.However, b2m was expressed on the surface of a higher number of thymocytes than MHC-Ia, suggesting anassociation of b2m to another molecule, possibly a non-classical class I molecule [80]. In spleen and headkidney the expression of both molecules was similar, but after 8 wpf all cells expressed both molecules,while in thymus the difference in immuno-reactivity remained. A clear indication for effective selection ofthymocytes is the presence of apoptotic cells. The first apoptotic cells in carp thymus were described at 7 dpfand their number increased with age, especially in the cortical area [78]. At 4 wpf high numbers of apoptoticcells were found, indicating an active selection process. This is supported by the appearance (EM) ofreticular epithelial cells and nurse-like cells between 1 and 4 wpf [78].

These data fit more or less with earlier immunological experiments in carp. Immunisation in the first mpfresulted in ‘‘neonatal’’ tolerance [81,82]. Carp injected with T cell independent antigen (LPS) developedantibody responses and memory from 4 wpf, while they responded against T cell-dependent antigens(HGG) from 8 wpf [83]. Similar results were obtained for rainbow trout [84,85] and recently also forzebrafish [57], reporting T cell independent antibody responses from 4 wpf and T cell-dependent antibodyresponses at 6 wpf. So, it must be concluded that T helper cell function appears somewhere between 6 and8 wpf, dependent on the species studied, which is surprisingly late with respect to the earlier appearance ofcortical and medullary thymocytes.

The first ‘‘cytotoxic responses’’ in carp were described at 19 dpf (at 22 �C instead of the generally used25 �C) based on scale graft rejection [86]. Following the mammalian paradigm, this immunologicalresponse was caused by either specific cytotoxic/helper T cells, or by non-specific cytotoxic cells. Thepresence of specific T cells at this age was not detected in the immunisation experiments mentioned inthe preceding paragraph, so that the latter option gains credibility. Unfortunately, knowledge about theontogeny of non-specific cytotoxic cells is limited.

In carp, a mAb (WCL38) mainly reacting with mucosal Ig� lymphoid cells (putative T cells) wasproduced. It reacted with the majority (G60%) of lymphoid cells isolated from gills, gut and skin of adults[87]. WCL38C cells were detected in the gut epithelium from 3 dpf. Their number increased considerably ingut and gills in the first 5 weeks of development [66]. Surprisingly, these cells appeared in the mucosalorgans before the thymus is populated by thymocytes, and they were very abundant in mucosal organsbefore acquired immunity was detected. From 8 wpf onwards around 5e10% of thymocytes wereWCL38C, but before that age these cells do not populate the thymus. WCL38C cells are suggested to be gdT cells [87], but till now no evidence is available. For trout, it was also suggested that cells showing rag andTdT expression in the gut may be extrathymically developing gd IEL (intraepithelial lymphocytes) T cellhomologues [88]. Preliminary results showed low cytotoxic activity of these IEL of carp and hardly anyreaction with the non-specific cytotoxic cell (NCC) marker 5C6 [87]. Possibly these cells are regulatory ‘‘T’’cells in the acquired immune system at later ages, but their function in the first mpf is not defined at present.Comparable results were obtained in sea bass [89] using a mAb (DLT15) recognising most if not all T cells(DLT15C cells expressed TcRb) [90]. The first DLT15C cells were detected between 5 and 12 days post-hatching (dph) [91] using flow cytometry on macerated embryos. However, this early appearance is likelydue to cross-reaction with neural cells located in the olfactory bulbs (unpublished results). Recently, DLT15was used in an immuno-histochemical study: the appearance of B and T cells in the distinct lymphoid

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organs of sea bass and carp is given in Table 2. These results are in agreement with previous observations[92,93], and are consequently considered as definitive for sea bass. Comparison with carp data is difficultbecause of the large difference in the developmental speed (which is only partly due to temperaturedifferences), but also because a pan T cell marker is not available in carp. In both species T cells weredetected in gut and thymus before spleen and head kidney.

3.5. Teleost B cell lymphopoiesis in development

Next to thymus as primary T cell organ, head kidney is considered the primary B cell organ. Expressionof genes specific for the lymphoid cell lineage, such as rag-1, rag-2 and ikaros were described in thymus andhead kidney [55,57]. These genes are considered lymphoid cell lineage specific.

Using RT-PCR, first Igm expression in carp was found in head kidney from 7 dpf [66], which agrees withthe first expression of the membrane form of Igm in zebrafish [94]. In the latter study the secretory Igmexpression started at 2 wpf. However, another zebrafish report described the first IgLc expression inzebrafish at 3 dpf [57]. In conclusion, Igm mRNA expression precedes detectable membrane expression onB cells.

At 4 dpf, first extra-thymic rag-1 expression was observed outside the thymus in the same region whereinsulin is expressed: the pancreas. According to this study [94], B cells develop in the zebrafish pancreas,which is supported by the first detection of Igm expression at the same location at 10 dpf. In carp, neitherIgm expression nor WCI12C cells were observed in the hepato-pancreas [66], indicating this organ does notplay a role in B cell production. Another recent and elegant zebrafish study indicated the kidney as theprimary organ for B cells [95]. In this report transgenic zebrafish were used in which rag-2 or lck wascoupled to GFP expression. Fluorescence was observed in the thymus and kidney, but not in the pancreas.Within zebrafish further experiments are necessary to explain this discrepancy.

Surface IgC cells were first detected in carp head kidney at 2 wpf [66,69] (Table 2) using WCI12, a mAbdirected against serum IgM heavy (H) chain. In an earlier study [96], heterogeneity in carp IgM wasdetected using a second mAb against the carp IgM H chain (WCI4). With WCI4 an additional populationof B cells was detected, which reacted with 50% of all B cells detected in carp head kidney at 2 wpf, but theproportion decreased to around 20% in head kidney, spleen and blood from 4 weeks onwards. These dataindicate that WCI4C12� B cells play a more important role in ontogeny than WCI4C12C and WCI4�12C

cells, of which the latter is the main B cell population in older animals. Although IgD has been sequenced inseveral fish species [97e99], it is unlikely that WCI4 detects IgD, because the molecular weight of theimmuno-reactive molecule is of the IgM H chain size. A separate IgM isotype is more logical, because theuse of a distinct IgM isotype is also suggested for mucosal immunity [100].

In sea bass, B cells also appeared considerably later than T cells (45 dph vs 28 dph: Table 2). The firstIgMC cells in sea bass were cytoplasmic-positive, followed by surface IgMC cells 1e2 weeks later [91]. This

Table 2

Immunocytochemical appearance of B and T cells in lymphoid organs of sea bass (in dph, GSD; at 16 �C) and carp (in dpf at 25 �C;hatching at 2 dpf)

Tissue T cells (DLT15) sea bass B cells (DLIg3) sea bass Ig� lymphoid cells carp B cells (WCI12) carp

Thymus 28G 2 90G 5 4a negligible

Head kidney 35G 5 45G 5 7a 14

Spleen 45G 3 45G 3 7a 14

Intestine 28G 2 90G 5 3b 35

The sea bass data are based on G100 sections/organ, and in carp on immuno-histochemistry and flow cytometry.a Based on WCL9 (cortical thymocytes in adult carp).b Based on WCL38 (putative mucosal T cells).

451J.H.W.M. Rombout et al. / Fish & Shellfish Immunology 19 (2005) 441e455

sequence of IgM appearance is also reported for trout [101], and comparable with the first m-chainappearance in amphibians [102,103] and mammals [104]. The first cytoplasmic IgC plasma cells weredetected in carp head kidney from 1mpf [66,105]. In zebrafish secretory Igm expression started at 2 wpf [94],which fits more or less with the first plasma cell detection in carp. These observations on plasma cells agreewith the immunisation results mentioned earlier for carp and zebrafish, both showing an inducible antibodyresponse against a T cell independent antigen after immunisation at 4 wpf. In any case, the first appearanceof plasma cells in carp and zebrafish is not correlated to the first feeding (at 4e5 dpf), which contrasts withthe observations in some marine species [77].

Carp B cells depart from kidney and spleen at 5 wpf to populate peripheral organs, amongst which is theintestine. Consequently, B cells appear considerably later in the intestine than carp WCL38C cells,indicating that mucosal immunity does not develop before this stage in carp and sea bass. Similarconclusions were drawn for spotted wolffish, because in situ hybridisation using a probe complementary tothe secretory Igm chain revealed also a delayed appearance of plasma cells in the gut [106].

4. Conclusions

Acquired immune responses developed in jawed vertebrates. Studies on the more ancient species livingtoday (cartilaginous and bony fish) certainly shed light on the evolution of acquired immune responses.Ontogenesis is a sequence of molecular and cellular events regulated by time and space, leading to thedevelopment of a fully functional organism. This review clearly shows that young fish use innatemechanisms during the first weeks/months (dependent on the species) of their development. With respect tothis, ontogenetic studies are important to understand evolutionary pathways and to defend farmed fishagainst pathogens at early age.

It is evident that T cells develop much earlier than B cells in all species investigated. Extra-thymic originof T cells mostly of the gd phenotype was reported in mammals [107,108], and the data obtained on carp,sea bass, and zebrafish, reported in this review, indicate that a similar process takes place in bony fish. Thisis an important finding, since thymus and GALT are evolutionary related and GALT possibly precedesthymus during evolution [109].

Acknowledgements

The authors wish to thank Karin Fieten for her support on zebrafish ontogeny and the participants ofthe FISHAID group, especially Dr. Roy Dalmo (co-ordinator) for their stimulating suggestions. This workis granted by the EU 5th FP: contract no. QLK2-CT-2000-01076.

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