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Developmental and Comparative Immunology 34 (2010) 791–796

Contents lists available at ScienceDirect

Developmental and Comparative Immunology

journa l homepage: www.e lsev ier .com/ locate /dc i

hort communication

he primitive immune system of amphioxus provides insights into the ancestraltructure of the vertebrate immune system

an Han1 , Gonghua Huang1 , Qinfen Zhang, Shaochun Yuan, Jianzhong Liu, Tingting Zheng, Lifei Fan,hangwu Chen, Anlong Xu ∗

tate Key Laboratory of Biocontrol, Guangdong Key Laboratory of Pharmaceutical Functional Genes, College of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People’sepublic of China

r t i c l e i n f o

rticle history:eceived 11 December 2009eceived in revised form 27 March 2010ccepted 29 March 2010vailable online 9 April 2010

a b s t r a c t

Amphioxus is considered to be the basal chordate. However, the structural and anatomical features of theamphioxus immune system are still elusive. Here we report a profile of structural studies of the amphioxusgill and gut, the first line of defending against microbes, through optical and electron microscopy. Theamphioxus gut and gill are characterized by the following morphological criteria compared with verte-brates: primary and secondary lymphoid-like tissue clustered in the gill, a thicker basement membrane

eywords:mphioxusertebrate

mmune systemtructureill

with a large villus channel and lack of muscular layer in the gut, along with blood vessels that fill withphagocytes following microbial challenge. The phenomena of tissue repair after microbial invasion wasobserved, though no phagocytes were observed in the region of tissue necrosis. The epithelium cells ofamphioxus gut showed active phagocytosis after the microbial challenge. A small number of free andfixed macrophage-like cells were also found in the amphioxus gut. The current results described thestructure of the immune system and cellular defense against infection in a protochordate, which may

the s

ut help us in understanding

. Introduction

Amphioxus (the lancelet, subphylum Cephalochordata), whichs the contemporary representative of an ancient chordate lin-age with a fossil record dating back to the Cambrian period,s considered to be the basal chordate (Putnam et al., 2008;olland et al., 2008). Together with vertebrates and urochordates,mphioxus descended from a common ancestor that lived around50 million years ago (Putnam et al., 2008). In many respects,mphioxus reflects the primitive vertebrate condition, but alsoxhibits uniquely specialized features that arose in the half bil-ion years since its divergence from the main chordate lineageHolland et al., 2008). Recent studies placed urochordates as the

ister group of vertebrates (Blair and Hedges, 2005; Delsuc et al.,006), and revealed that amphioxus was not a close relative ofertebrates as had previously been thought (Putnam et al., 2008;uang et al., 2008), which only increased its importance in our

Abbreviations: TNFR, tumor necrosis factor receptor; S.c., Staphylococcus aureus;.p., Vibrio parahaemolyticus.∗ Corresponding author at: Department of Biochemistry, College of Life Sciences,

un Yat-sen University, 135 Xingangxi Road, Guangzhou 510275, People’s Republicf China. Tel.: +86 20 39332990; fax: +86 20 39332950.

E-mail address: lssxal@mail.sysu.edu.cn (A. Xu).1 These authors contributed equally to this work.

145-305X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.dci.2010.03.009

tructural origin of the vertebrate immune system.© 2010 Elsevier Ltd. All rights reserved.

understanding of fundamental features of the chordate ancestralcondition.

Amphioxus is widely used as a model for studying evolution anddevelopment and there are extensive observations on its locomo-tory, sensory, and nervous systems (Holland et al., 2004). However,very few studies of its immune system have been conducted, espe-cially of the structure and evolutionary significance of immunetissues and organs.

In Elie Metchnikoff’s late 19 century study of amphioxus, whichhe called the last survivor of the lower vertebrates, he stated “allattempts therefore to provoke inflammatory phenomena in it havegiven only negative results” (Metchnikoff, 1891). For many years,it was not known how amphioxus could survive microbial infec-tion. However, two decades ago, Rhodes et al. (1982) reported asmall number of free and fixed phagocytes in the coelomic cav-ity of amphioxus. These cells typically possessed a cleft nucleus,lysosome-like bodies, and, often, cilia and rootlet structures. Theyconcluded that the cells might play a vital role in defending againstcertain infection. However, Silva et al. (1995) failed to observephagocytes by light or electron microscopy as they examined a

wound in the distal portion of amphioxus. The existence of phago-cytes in amphioxus therefore remains controversial.

The amphioxus gut is constantly exposed to abundant commen-sal bacteria and has regular if not frequent contact with pathogenicmicrobes in contaminated food and water. The gut plays an indis-

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ensable role in immune defense against microbial challenge (Liu etl., 2009). Gerzeli (1961) reported the presence of enterochromaf-n cells in the gut of amphioxus but did not identify their functionr the basis for their role in defending against certain infection.

We have been striving to study the immune system of this ani-al at the molecular level for many years. This has led to some

mportant findings such as the galectin (Yu et al., 2007a) and the-type lectin system (Yu et al., 2007b), the tumor necrosis factorsystem (Yuan et al., 2007), the V and C domain-bearing protein (Yut al., 2005), toll like receptors (Yuan et al., 2009a), tumor necro-is receptor associated factors (Yuan et al., 2009b), and especiallyymphocyte-like cells (Huang et al., 2007a). These results contributeo a better understanding of the amphioxus immune system. A sys-ematic comparative analysis of the amphioxus genome databaseas also carried out in our laboratory (Huang et al., 2008), which

howed that it has an innate immune diversity and complexitynparalleled among the vertebrates. However, there has been noystematic study of the anatomy of its immune system.

In this study, we try to reveal the structure of amphioxus gut andill and offer some cellular evidence of its immune defense system.e also want to further understand the relationship between the

tructure and function of this primitive immune system and to seehether amphioxus has the same inflammatory response as that

n vertebrates, and then to compare the structural similarities andifferences between amphioxus and vertebrates.

. Materials and methods

.1. Amphioxus care and maintenance

Mature adults of Chinese amphioxus, Branchiostoma belcherisingtaunese, were obtained from Kioachow Bay near Qingdao,hina, and cultured in our laboratory in aquaria supplied with cir-ulating filtered seawater maintained at 20–25 ◦C.

.2. Immune challenge of animals

For light microscopy, live Staphylococcus aureus (S.c.) was sus-ended in PBS and 0.5 �l (108 cells/ml) was microinjected intouscle that located at one-third of anterior back of each individual

mphioxus. Amphioxus which were microinjected with 0.5 �l PBSere used as controls. We use 30 amphioxus for experiment group

nd 30 for control group. The animals were kept in the aquaria fordays.

For electron microscopy, a total of 5 �l (108 cells/ml) of liveibrio parahaemolyticus (V.p.) in PBS was microinjected into theosition of one-third of hindgut from the anus of each individualmphioxus. Amphioxus which were microinjected with 5 �l PBSere used as controls. We use 24 amphioxus for experiment group

nd 24 for the control group. The animals were kept in the aquariaor 2 days.

.3. Microscopy

For light microscopy, the whole body of S.c. injected amphioxusere fixed in 4% formaldehyde. Fixed tissue was washed with PBS,ehydrated in a graded series of ethanols, embedded in paraf-n, cut in 5 �m sections and stained with hematoxylin and eosin.mphioxus injected with PBS only was used as controls.

For electron microscopy, at the time point of 6, 12, 24 and 48 hfter the V.p. injection, the amphioxus guts were dissected under

he microscope and cut into small pieces. The cut samples werexed in 2.5% glutaraldehyde and 1% osmium tetroxide, and washedith PBS. After dehydrating in a graded series of ethanols and ace-

one, the samples were embedded in fresh Spurr’s Resin (Spurrmbedding kit, EMS). Ultrathin sections were stained with uranyl

tive Immunology 34 (2010) 791–796

acetate and lead citrate for examination. Amphioxus injected withPBS only were used as controls. Images were collected on JEOL100CX-II at 100 kV.

2.4. In situ analysis of amphioxus tumor necrosis factor receptor1 (named as BbtTNFR1)

Adults of the Chinese amphioxus Branchiostoma belcheri tsing-taunese, were collected and kept in filtered seawater for 2 days.The animals were sacrificed and cut at 1-cm intervals. The tissueblocks obtained were fixed in 4% paraformaldehyde in PBS andembedded in paraffin. Tissue blocks were cut transversally andmounted on glass slides coated with poly-lysine. The digoxigenin-labeled probes were prepared by using the plasmids that containthe sequences of the BbtTNFR1 with the SP6 promoter sequenceat the 3′ end of the sequences as template and the antisenseprobes were synthesized with the SP6 RNA polymerase accordingto the protocol of the digoxigenin DIG RNA labeling kit (Roche).In situ hybridization was performed according to Li et al. (2004)with a little modification. That is before hybridization, the sec-tions were de-waxed, re-hydrated, absterged, bleached with 3%H2O2 to inhibit endogenous enzyme activity, and digested byproteinase K. The probes were added to the sections at a concen-tration of 1 �g/ml. After overnight hybridization at 42 ◦C, sectionswere treated with a series of high stringency washes followed byimmunodetection.

3. Results and discussion

3.1. Histopathology of amphioxus muscle following microbialchallenge

To detect whether amphioxus has the same inflamma-tory response as that in vertebrates, S.c. were injected intothe amphioxus muscle and the histopathological change wereobserved. Compared with PBS injection group, which had shown noobvious damage; after 24 h, the S.c. injected amphioxus swam errat-ically and 96.6% of amphioxus showed redness of the surroundinginjection site. The histopathological change of the injection site wasfurther studied under light microscopy. The phenomena of nero-sis in the site of injection with normal muscle structure lost werefound (Fig. 1A and B). However, no cells with the characteristicsof phagocytes were seen infiltrating the lesions or phagocytiz-ing necrotic tissue (Fig. 1C), which further confirmed the previousobservations and investigations by Metchnikoff (1891) and Silvaet al. (1995) regarding the absence of phagocytes in a woundand lack of inflammatory response in amphioxus. The potentialmolecular mechanism behind this observation may be due to theover-expressed protease cascade and protease inhibitory compo-nents (Huang et al., 2007b), which contributed to the digestion ofthe necrotic tissue after wounding or infection. This was obviouslydifferent from mechanisms seen in invertebrates which mainlyuse phagocytes to perform this function. The lesions in 90% S.c.injected amphioxus did not heal, but slowly progressed, leadingto its death, indicating that the clearance of extruded microbesby enzymatic mechanisms might not be under effective regula-tion in most amphioxus. Surprisingly, we found that about 10% ofamphioxus could survive and have tissue repair (Fig. 1D) after 5days injection compared to the control group which were injectedwith PBS. We further analyzed the process of the tissue repair and

found that during the early stages of repair, there is a granulationtissue-like substance that is later replaced by fibrosis, which fillsinto the space cleared of necrotic tissue. Thus, it seems that the tis-sue repair ability in amphioxus was limited and showed individualdifferences.

Y. Han et al. / Developmental and Comparative Immunology 34 (2010) 791–796 793

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ig. 1. The pathological changes of the amphioxus after treated with PBS and Stap00 �m. (B) The necrotic liquerfaction of muscle fibre (indicated by arrow) at theorphological characteristics of phagocytes infiltrated into the lesions or phagocyt

njected with S.c., scar repair was observed in the necrotic regions. Scale bar, 50 �m

.2. The putative lymphoid-like tissues in amphioxus gill

In our previous study (Huang et al., 2007a), we reportedhat under the epithelium near the basement membrane in theill, some immature lymphocyte-like cells with apparently lesslectron-dense nuclear could be seen. In the current study, weurther studied the lymphoid-like tissues in amphioxus gills. Weound that the gill was composed of gill bars, which were formed

f a core of muscle with an outer sheath of epithelium (Fig. 2A).n electron microscope sections, clusters of lymphocyte-like cells

ith large nuclei surrounded by a thin layer of cytoplasm were seenn the epithelium of the gill (Fig. 2B and C). The nuclei containedperipheral rim of heterochromatin adjacent to the nuclear enve-

ig. 2. Gill structure with light and electron microscopy. (A) Gill bar with epithelium overill epithelia. Scale bar for B is 5 �m and scale bar for C is 2 �m. (D) A lymph node-like sthe cells shown in (B), (C) and (D). Gill bar (GB), muscle (M), lymphocyte-like cell (L), nuc

ccus aureus (S.c.). (A) The PBS treated amphioxus muscles were normal. Scale bar,ay after injected with S.c. was found. Scale bar, 100 �m. (C) There is no cell with

he necrotic tissue in the necrotic region. Scale bar, 50 �m. (D) At the fifth day aftercle (M), granulation tissue-like substance (G), fibrosis (F).

lope. Unexpectedly, a lymph node-like structure, which may be theprimitive form of the vertebrate lymph node and is believed to beone of the important components of the lymphoid system, was alsoseen in the epithelium (Fig. 2D).

3.3. Structural similarities and differences of gut betweenamphioxus and vertebrate

In the vertebrate gut, the absorptive cell and the goblet cells inthe mucous membrane contribute to the digestion and absorptionof nutrients. Paneth’s cell and the lymph nodes in the lamina pro-pria mainly contribute to the immune defense against microbes.The structure of the amphioxus gut shares certain similarities with

laying the muscle core. Scale bar, 50 �m. (B and C) The lymphocyte-like cells in theructure in the epithelium. Scale bar, 2 �m. The box in (A) shows the localization ofleus (N).

794 Y. Han et al. / Developmental and Comparative Immunology 34 (2010) 791–796

Fig. 3. The structure of the guts under the light and transmission electron microscopy. (A) The structure of muscle and gut under the light microscopy. Scale bar, 50 �m. (B)The apical surfaces of the cells formed cell protrusions that pinched off and entered the gut lumen. Scale bar, 2 �m. (C) The mitochondria in column-like epithelium. Scalebar, 1 �m. (D) The development of cell protrusions in apical surfaces of columnar epithelium. Scale bar, 3 �m. (E) The villus channel in the basement membrane. Scale bar,3 m. (Gw ere fo( nt me(

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�m. (F) Amphioxus has an apparent and large blood vessel in the gut. Scale bar, 2 �as challenged with the V.p., many phagocytes, which contained the phagosome, w

Mi), cell protrusions (CP), column-like epithelium (*), secretory granule (S), basemeLy), phagocyte (Ph).

he vertebrate’s such as the absorptive cell and the goblet cell inhe mucous membrane, but the boundary between the mucous

embrane and the submucosa was not apparent in amphioxus.he anterior mid-gut was lined with a tall column-like epithelium.he apical surfaces of the cells, which enveloped some secretoryranules, formed cell protrusions that pinched off and entered theut lumen (Fig. 3B). Many mitochondria in column-like epitheliumould be seen, and this may be closely related to the energeticetabolism of epithelia whose metabolic process needed a large

mount of energy (Fig. 3C). The secretory granules accumulatedn the protrusion, and slowly progressed, leading to the discon-

ection with epithelium. The protrusion swelled, and dissolved,hen died with releasing the secretory granules and other contentsFig. 3D).

Unlike the urochordate colonial ascidian Botryllus schlosseriBurighel and Milanesi, 1977), the amphioxus gut lacks microvilli

) Blood vessel in the gut of PBS control group. Scale bar, 2 �m. (H) When amphioxusund in the vessels. Scale bar, 2 �m. The picture showed muscle (M), mitochondriambrane (BM), villus channel (VC), blood vessel (BV), endothelial cell (EC), lysosome

in the epithelia. Moreover, there is no muscular layer in amphioxusgut which mainly participates in the movements of the enteronwhen compared with vertebrate. The amphioxus also has a thickerbasement membrane and the villus channel in the basement mem-brane is bigger (Fig. 3E). We also found that the amphioxus hasa large visible blood vessel in the gut and there are endothelialcells which contain many lysosomes lining in the blood vessel(Fig. 3F). Compared with amphioxus of the PBS control group, whenamphioxus was challenged with the V.p., many phagocytes contain-ing a large phagosome were found in the vessels (Fig. 3G and H),which were obviously different from the vertebrates. Furthermore,

at the molecular level, amphioxus tumor necrosis factor recep-tor 1 (named as BbtTNFR1), which is just one of the importantimmune-related genes, was expressed in gut. Results of in situ anal-ysis showed that the mRNA of BbtTNFR1 could be detected in theintestines and sexual-gland (Supplementary Fig. 1). The immune-

Y. Han et al. / Developmental and Comparative Immunology 34 (2010) 791–796 795

Fig. 4. Transmission electron-microscopic structure of the amphioxus guts cells. (A) Normal epithelia cells of amphioxus gut in the PBS treated group. There were manyvacuoles but no secondary-lysosome-like bodies. Scale bar, 2 �m. (B) Many big secondary-lysosome-like bodies and vacuoles were found in the epithelia cells of amphioxusgut after treated with V.p. These cells possessed cilia. Scale bar, 3 �m. (C) The V.p. in the gut became attached to membrane of the macrophage-like cells. Scale bar, 2 �m. (D)T ith thu dies wc e-likem

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he V.p. was ingested, forming phagosome. Enzymes-contrined lysosomes fused wnder the basement membrane after treated with V.p. 4 h. Some lysosome-like boilia (C), secondary-lysosome-like bodies (L), vacuoles (V), nucleus (N), macrophagembrane (BM).

elated genes localizing in gut may indicate an important role ofhe gut in defending against pathogen infection.

Given the evolutionary significance of this animal, all theseifferences indicate that each organ in amphioxus might havearious functions, although it has more simple tissue organiza-ion than vertebrates, but the function of homologous organsecame more specific when they evolved into vertebrates duringvolution.

.4. The phagocytosis phenomenon in amphioxus gut

The appropriate distribution of immune cells among organsn the body is crucial for the immune surveillance and effectorunctions of the immune system (Moser and Loetscher, 2001).his is also true for amphioxus. When V.p. was injected into theut of amphioxus, after 48 h, many cells were seen to penetratento the gut. In order to further understand the characteristicsf those exudative cells, we used electron microscopy to observehe structural changes of the gut. Contrary to the amphioxus ofhe PBS control group which has normal epithelial cells with vac-oles and cilia of the amphioxus gut (Fig. 4A), many epithelial cellsith large secondary-lysosome-like bodies were observed after

he V.p. injection (Fig. 4B), and specially, those cells showed activehagocytosis.

A process of phagocytosis could also be seen in the gut ofhe infected amphioxus while compared with the PBS control

roup in which no obvious phagocytosis was seen. In the firsttep of phagocytosis, macrophage-like cells became attached tohe V.p. (Fig. 4C) and encapsulated the V.p. through phagocyto-is to form a phagosome. The phagosome fused with a lysosomeo form a phagolysosome (Fig. 4D), which was substantiated by

e phagosome to form phagolysosome. Scale bar, 1 �m. (E) and (F) The phagocytesere observed in the cells. Scale bar, 2 �m. The picture showed epithelial cells (*),cells (ML), phagocyte (Ph), bacteria (B), lysosome (Ly), phagosome (P), basement

the finding of the genes related to energy metabolism (such ascytochome c oxidase subunit) up-regulated by bacterial infectionin our previous study (Huang et al., 2007b). Under the basementmembrane, following infection with V.p., some phagocytes withlysosome-like bodies were also observed in the lamina propria(Fig. 4E and F).

Thus, the issue about “the existence of phagocytes in theamphioxus is a matter of debate” has been obviously resolved inthis study. The occurrence of the functional macrophage-like cellsin amphioxus suggests that a primitive form of vertebrate immunesystem is found in this animal during evolution. Moreover, theactive phagocytosis of those epithelial cells indicates that besidesthe digestive and absorptive function for all epithelia, the epithe-lia of amphioxus gut might play an even more important role inthe immune defensive function. The primitive immune cells mayhave originated from those ancestral cells which have been thoughtonly as a specific function for digestion and absorption in the diges-tion system of vertebrates, suggesting that the immune system isoriginated from digestive system.

Thus, the current paper was very helpful for us to study theancestral structure of the vertebrate immune system. Consideringthe fact that amphioxus hold so many distinct features comparedwith vertebrate, we might believe that the amphioxus will providean insight to uncover novel defense mechanisms against infec-tion, to further reveal new roles of phagocytes to understand itsphysiological and pathological roles in vertebrates. In addition,

further functional analyses of those identified tissues and struc-ture, along with the advances from the molecular biological studiesfrom this animal will put the amphioxus in a key position tounderstand the origins and characteristics of vertebrate immunesystem.

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cknowledgments

We gratefully acknowledge Dr. Jinlang Wu (Lab of Electronicroscopy, Zhong-Shan Medical College, Sun Yat-sen University)

or his critical comments on the electron microscopy.This work was supported by Project 2007CB815800 of the

ational Basic Research Program (973), Project 2006AA09Z433f the State High-Tech Development Project (863), and Project007DFA30840 of International S&T Cooperation Program from theinistry of Science and Technology of China, Key Project (0107)

rom the Ministry of Education, and Key Projects of Commissionf Science and Technology of Guangdong Province and Guangzhouity. A Xu is the recipient of “Outstanding Young Scientist Award”f National Natural Science Foundation of China.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.dci.2010.03.009.

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