Blood and inflammatory cells of the lungfish Lepidosiren paradoxa

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1 Fish & Shellfish Immunology 23 (2007) 178e187 Blood and inflammatory cells of the lungfish Lepidosiren paradoxa Maria Lucia da S. Ribeiro a,d, Renato A. DaMatta b, *, José A.P. Diniz c, Wanderley de Souza d, Jose Luiz M. do Nascimento e, Tecia Maria U. de Carvalho d a Departamento de Farmácia, Centro de Ciências da Saúde, Universidade Federal do Pará, Av. Augusto Corrêa 1, Bairro Guamá, , Belém, Pará, Brazil b Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Avenida Alberto Lamego 2000, , Parque Califórnia, Campos dos Goytacazes, Rio de Janeiro, RJ, Brazil c Unidade de Microscopia Eletrônica, Instituto Evandro Chagas, Av. Almirante Barroso 492, Bairro Marco, , Belém, Pará, Brazil d Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fund~ao, Rio de Janeiro, RJ, Brazil e Laboratório de Neuroquímica, Departamento de Fisiologia, Centro de Ciências Biológicas, Universidade Federal do Pará, Av. Augusto Corrêa 1, Bairro Guamá, , Belém, Pará, Brazil Received 13 July 2006; revised 27 September 2006; accepted 17 October 2006 Available online 25 October 2006 Abstract A special interest exists concerning lungfish because they may possess characteristics of the common ancestor of land vertebrates. However, little is known about their blood and inflammatory cells; thus the fine structure, cytochemistry and differential cell counts of coelomic exudate and blood leucocytes were studied in Lepidosiren paradoxa. Blood smear analyses revealed erythrocytes, lymphocytes, monocytes, polymorphonuclear agranulocytes, thrombocytes and three different granulocytes. Blood monocytes and lymphocytes had typical vertebrate morphology. Thrombocytes had large vacuoles filled with a myelin rich structure. The polymorphonuclear agranulocyte had a nucleus morphologically similar to the human neutrophil with no apparent granules. Types I and II granulocytes had eosinophilic granules. Type I granulocytes had round or elongated granules heterogeneous in size, while type II had granules with an electron dense core. Type III granulocyte had many basophilic granules. The order of frequency was: type I granulocyte, followed by lymphocyte, type II granulocyte, monocyte, polymorphonuclear agranulocyte and type III granulocyte. Peroxidase localized mainly at the periphery of the granules from type II granulocytes, while no peroxidase expression was detected in type I granulocytes. Alkaline phosphatase was localized in the granules of type II granulocyte and acid phosphatase cytochemistry also labelled a few vacuoles of polymorphonuclear agranulocyte. About 85% of the coelomic inflammatory exudate cell population was type II granulocyte, 10% polymorphonuclear agranulocyte and 5% macrophages as judged by the nucleus and granule morphology. These results indicate that this lungfish utilises type II granulocytes as its main inflammatory granulocytes and that the polymorphonuclear agranulocyte may also be involved in the inflammatory response. The other two granulocytes appear similar to the mammalian eosinophil and basophil. In * Corresponding author. Tel./fax: þ address: renato@uenf.br (R.A. DaMatta) /$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi: /j.fsi

2 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e summary, this lungfish appears to possess the typical inflammatory granulocytes of teleosts, however, further functional studies are necessary to better understand the polymorphonuclear agranulocyte. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Lungfish; Lepidosiren paradoxa; Blood leucocytes; Granulocytes; Inflammatory cells 1. Introduction Lungfish are from the Osteichthyes class and Sarcopterygii subclass, a survivor of a once dominant group [1]. These fishes are considered living fossils and may still possess characteristics of the common ancestor of land vertebrates [2]. Thus, particular interest exists concerning the lungfish and its relatives with regard to vertebrate evolution [1e3]. Little is known about the blood cells of the Sarcopterygii. Of the few articles published, only one has characterised, at the ultrastructural level, blood cells of the Australian lungfish (Neoceratodus forsteri) [4]. The fine structure [4] and histochemistry [5] of monocytes, thrombocytes, lymphocytes, neutrophils, eosinophils, heterophils and basophils have been described and there is a previous attempt to explore the inflammatory response of N. forsteri which found no difference in the coelomic cell population after lipopolysaccharide injection [5]. Finally, there is an article characterising granulocytes of the South American lungfish (Lepidosiren paradoxa), which is mainly related to haematopoietic tissue [6], however, in this report (contrary to the work of Hine et al. [4,5]), only three types of granulocytes are described [6]. Here the morphology, fine structure, and peroxidase and phosphatase localisation of blood leucocytes from L. paradoxa and the inflammatory granulocyte population after thioglycollate coelomic stimulation are described. A better understanding of these cells may shed light on fish granulocyte heterogeneity [7] and on the evolution of tetrapod leucocytes. 2. Material and methods 2.1. Lungfish, blood harvesting and leucocyte separation Lepidosiren paradoxa was captured by local fishermen in the flooded regions surrounding the city of Belém, PA, Brazil. Ten animals, varying from 30 to 70 cm in length, were maintained in water tanks ( cm) on a local farm. Lungfish were maintained in good health by feeding with small chunks of raw fish once a week with no animals dying over a two-year period. A week before blood harvesting, animals were transferred to separate tanks ( cm) and kept at the animal facility in the Instituto Evandro Chagas, Belém. Lungfish were restrained manually; 1 ml of blood was collected by cardiac puncture into 1 ml syringes and added to a tube containing EDTA as anticoagulant (0.5% final concentration). Blood smears from all lungfishes were fixed with absolute methanol and stained with Giemsa for leucocyte morphological classification and counting. Animals were re-bled after 15 days only for ultrastructural studies where blood leucocytes from each individual fish were separated from erythrocytes as described [8] Fish coelomic exudate leucocytes In order to determine the inflammatory blood granulocyte, 1 ml of thioglycollate (3% aqueous solution of Brewer s thioglycollate medium (Sigma), was injected intracoelomically into three fish. These were killed by a blow to the head after 6, 36 and 48 h and a coelomic wash was performed with Dulbecco s Modified Eagle s Medium (DMEM). Briefly, in 60 cm body size animals, without pulling back the skin, 10 ml of DMEM was injected into the coelomic cavity of the fish. The cell suspension was collected in the same syringe, dispersed in tubes disposed on ice and smears performed. The cell suspension was centrifuged at 500 g for 10 min at 4 C and fixed for transmission electron microscopy Light microscopy Giemsa stained blood smears were examined and photographed under a Zeiss Axiophote microscope using the immersion 100 objective. After leucocyte classification, differential counts were made for each blood smear of 10 fish

3 180 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e187 examined; 100 leucocytes were counted and the mean percentages standard deviation were calculated for each cell type. The diameter of the different cells was measured using a standard pre-calibrated stage micrometer. Smears from exudate coelomic washes of the three fishes were Giemsa stained, observed and the percentage of the different cell types evaluated Transmission electron microscopy Blood and exudate leucocytes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, ph 7.2 for 2 h at room temperature, washed twice and post-fixed with 1% osmium tetroxide and 0.8% potassium ferricyanide in cacodylate buffer containing 5 mm calcium chloride for 30 min. Cells were washed with the same buffer, dehydrated in acetone and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate and observed in a Zeiss EM900 transmission electron microscope, operated at 80 kv. For alkaline phosphatase localisation [9], blood leucocytes were fixed in 1% glutaraldehyde (grade I) in 0.1 M cacodylate buffer, ph 7.2 for 30 min at room temperature and washed with the same buffer containing 5% sucrose. Cells were washed twice in 0.1 M Trisemaleate buffer, ph 9.0 containing 5% sucrose and incubated for 60 min at room temperature in 4 mm cerium chloride, 10 mm b-glycerophosphate in Trisemaleate buffer, ph 9.0 containing 5% sucrose. Cells were post-fixed with 1% osmium tetroxide in cacodylate buffer, dehydrated in acetone and embedded in epoxy resin. Thin sections were observed without uranyl acetate and lead citrate staining. For the detection of acid phosphatase, acetate buffer of ph 4.6, was used in the protocol above. For control of the specificity of the reaction, the cells were incubated in the absence of b-glycerophosphate. For peroxidase localisation [10], blood leucocytes were fixed in 1% glutaraldehyde (grade I) in 0.1 M cacodylate buffer, ph 7.2 for 30 min at room temperature and washed with the same buffer containing 5% sucrose. Fixed cells were divided in two tubes and incubated for 10 min with 2.5 mm of DAB in cacodylate buffer, ph 7.2 (neutral) or ph 9.5 (alkaline peroxidase). Subsequently, cells were incubated at room temperature for 30 min in cacodylate buffer, ph 7.2 or 9.5 containing 0.5 mg ml 1 of diaminobenzidine, 5% sucrose and 0.003% hydrogen peroxide. Cells were washed, re-fixed in 2.5% glutaraldehyde in cacodylate buffer, washed, post-fixed with 1% osmium tetroxide in cacodylate buffer, dehydrated in acetone and embedded in epoxy resin. Thin sections were observed in the transmission electron microscope without uranyl acetate and lead citrate staining. For control of the specificity of the reaction, the cells were incubated in the absence of diaminobenzidine. 3. Results Lepidosiren paradoxa peripheral blood smear cells were classified morphologically by light microscopy into eight cell types. Erythrocytes were elliptically shaped, about 53 mm in its largest diameter, with cytoplasmic inclusions and a centrally located oval nucleus that contained condensed chromatin (Fig. 1A right). Lymphocytes were about 23 mm in length, had a thin rim of light purple cytoplasm surrounding a centrally located basophilic nucleus, which occupied most of the cell (Fig. 1A left). Monocytes were about 30 mm in length, had a light purple cytoplasm with a few eosinophilic inclusions and a kidney shaped nucleus that occupied about 60% of the cell (Fig. 1B). Thrombocytes were about 37 mm in its largest diameter, were often found in clumps with a dark pink cytoplasm and a basophilic nucleus that occupied most of the cell (Fig. 1C). Polymorphonuclear agranulocytes were also observed, they were about 30 mm in length, had a light basophilic cytoplasm with rare granules seen only in some cells. They possessed an eccentric polymorphonucleus with purple chromatin; some of these cells had up to five nuclear lobes connected by a fine rim of chromatin (Fig. 1D). Three different granulocyte types were observed. Type I granulocytes were about 40 mm in length, had a faint pink cytoplasm with round eosinophilic granules homogeneously distributed. These cells had an eccentrically located bi to trilobulated nucleus with condensed chromatin (Fig. 1E). Type II granulocytes were about 55 mm in diameter, had a colourless cytoplasm with oval and round, highly eosinophilic granules heterogeneous in size, a few possessed a more stained core. They contained an eccentric nucleus that stained lightly blue with a few patches of condensed purple chromatin (Fig. 1F). Type III granulocytes were about 50 mm in length, had a light blue cytoplasm containing many highly basophilic, round granules that covered some areas of the nucleus. These cells had an eccentrically located, highly basophilic nucleus (Fig. 1G). Based on the histo-morphological classification above, differential blood leucocyte counts were performed. Type I granulocyte was the largest leucocyte population found ( %), followed by lymphocyte ( %),

4 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e A B C D E F G Fig. 1. Light microscopy of Giemsa stained blood cell smear from the Lepidosiren paradoxa. (A) Lymphocyte (left) with a high nucleus/cytoplasm ratio and erythrocyte (right) with cytoplasmic inclusions. (B) Monocyte showing kidney shaped nucleus. (C) Thrombocytes containing small vacuoles (arrow). (D) Polymorphonuclear agranulocyte with an eccentrically polymorphonucleus. Note thin rims of chromatin connecting the lobes (arrow) and no apparent granules. (E) Type I granulocyte displaying trilobulated nucleus and evident granules. (F) Type II granulocyte showing an eccentrical nucleus and evident granules. Some granules with a denser stained core can be seen (arrow, inset). (G) Type III granulocyte with evident basophilic granules. Bar 30 mm. Same bar for A, B, E, F, G, inset 6 mm. type II granulocyte ( %), monocyte ( %), polymorphonuclear agranulocyte ( %) and type III granulocyte ( %). Blood thrombocyte, monocyte and lymphocyte were recognised by ultrastructural observations. The lymphocyte had an oval form, the nucleus occupied more cytoplasm than for the thrombocyte, a fine cytoplasm line surrounding

5 182 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e187 the nucleus, with few mitochondria and filiform projections at the surface were also observed (Fig. 2A). The monocyte had a circular form, a nucleus with condensed chromatin, mitochondria, endoplasmic reticulum and filiform projections at the surface (Fig. 2B). The thrombocytes were irregular in form, their nucleus occupied most of the cell and they contained many small vacuoles next to the nuclear border and larger ones filled with an electron dense myelin rich structure (Fig. 2C). The polymorphonuclear agranulocyte had a circular form, a polylobulated nucleus with marginal heterochromatin and cytoplasm containing almost no granules, scattered mitochondria, and some vesicles; filiform surface projections were evident (Fig. 2D). Three types of granulocytes were observed by transmission electron microscopy. The type I granulocyte had an irregular form, with a polylobulated nucleus (Fig. 3A), and many round or elongated granules heterogeneous in size (Fig. 3B). The type II granulocyte also had an irregular form, a polymorphic nucleus with no more than three lobes and a cytoplasm full of an exclusive granule type possessing an electron dense core (Fig. 3C, D). The only type III granulocyte observed had round heterogeneously sized granules (Fig. 3E). Localisation of peroxidase activity to the whole or periphery of the granules of the type II granulocyte was ph independent (Fig. 4A, B), but activity was not detected in type I granulocytes (not shown). Alkaline phosphatase was found in the core and in areas around a few granules of type II granulocyte (Fig. 4C). Acid phosphatase cytochemistry labelled a few vacuoles of the polymorphonuclear agranulocyte (Fig. 4D). Cell and nucleus morphology, and staining properties on smears of the coelomic thioglycollate exudate cell revealed that about 85% of the cell population was composed of type II granulocytes, 10% was of polymorphonuclear agranulocytes and 5% were macrophages. No difference in the composition of the exudate cell population was found A B * C * D Fig. 2. Transmission electron microscopy of agranulocyte blood cells from the Lepidosiren paradoxa. (A) Lymphocyte has a few mitochondria and a fine cytoplasm surrounding the nucleus. (B) Monocyte with mitochondria and endoplasmic reticulum can be seen. A higher magnification of mitochondria (arrow, inset) and endoplasmic reticulum (asterisk, inset) can be seen. (C) Thrombocyte has big and small vacuoles. The bigger vacuoles (arrow) show myelin structures (asterisk, lower inset). Small vacuoles next to the nuclear borders can be seen (arrow, upper inset). (D) Polymorphonuclear agranulocyte. Note the polylobulated nucleus, vesicles, mitochondria (arrow, inset) and a few granules (arrowhead, inset). Bar 5 mm, inset 1 mm.

6 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e A B C D E Fig. 3. Transmission electron microscopy of granulocyte blood cells from Lepidosiren paradoxa. (A) Type I granulocyte displays a polylobulated nucleus and granules heterogeneous in size and form. (B) A higher magnification of the granules of type I granulocyte; note elongated and round granules and a few mitochondrion (arrowhead). (C) Type II granulocyte showing a polymorphic nucleus, and granules heterogeneous in size with an electron dense core. (D) A higher magnification of the granules of type II granulocyte; note the electron dense core. (E) Type III granulocyte with round granules heterogeneous in size. Bar 5 mm, except C, D and E Bar 1 mm. at 6, 36 and 48 h after stimulation. Ultrastructural observations of the exudate leucocytes revealed type II granulocytes with an eccentrically located nucleus, higher amounts of heterochromatin, granules with an electron dense core and membrane projections (Fig. 5A, B). Macrophages with many membrane projections, a large cytoplasm to nucleus ratio and vacuoles, were also observed (Fig. 5C). The polymorphonuclear agranulocyte was not observed in ultrastructural preparations.

7 184 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e187 A B C * * * D Fig. 4. Transmission electron microscopy of blood type II granulocyte and polymorphonuclear agranulocyte of Lepidosiren paradoxa after cytochemistry staining. (A) Cytochemistry for peroxidase in type II granulocyte. Note reaction product in the periphery (arrow) or at the whole granules (arrowhead). (B) Higher magnification of type II granulocyte; note labelling pattern mostly at the periphery of the granules. (C) Alkaline phosphatase localization in type II granulocyte. Reaction product can be seen in some granules, mainly at its core (arrow). (D) Cytochemistry for acid phosphatase in polymorphonuclear agranulocyte. Note nucleus morphology (asterisk) and reaction product at some cytoplasmic structures (arrow). Bar 5 mm.

8 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e B A C Fig. 5. Transmission electron microscopy of coelomic exudate cells obtained after 36 h of thioglycollate stimulation. (A) Type II granulocyte with an eccentrically located nucleus, granules with an electron dense core and membrane projections. (B) A higher magnification of the granules of type II granulocyte; note the electron dense core. (C) Macrophages with many membrane projections and vacuoles. Bar 5 mm, except B Bar 1 mm. 4. Discussion A striking fact about L. paradoxa blood was the large size of its cells. It has been reported that erythrocyte size decreases in the following group sequence: rays, sharks and teleosts [11]. This is probably related to the metabolic activity, where more active fishes need extra oxygen and thus, have smaller erythrocytes [11e13]. L. paradoxa is not an active fish and is capable of estivation [14]. Thus, large erythrocytes are probably suitable for its survival. Large blood cells have also been related to fishes that have evolved first in the evolutionary scale [13,15], this agrees with the early appearance of lungfishes on earth (180 million years ago) [1]. In the Australian lungfish, Hine et al. [4,5] described circulating blood cells as heterophils, neutrophils, eosinophils, basophils, lymphocytes, monocytes, thrombocytes and erythrocytes. Erythrocytes, lymphocytes and monocytes of L. paradoxa were all similar to those found in other vertebrate species. Thrombocytes contained large vacuoles containing myelin structures reported before in the African lungfish [16] and also in shark thrombocytes [17], but not common in other thrombocytes. Four types of granulocytes (basophil, two eosinophils and neutrophils) have also been described in the Australian lungfish [18]. In L. paradoxa, Bielek and Strauss [6] have observed the presence of only three granulocyte types. The present work also confirmed the existence of three granulocytes. In addition, a polymorphonuclear cell with nucleus morphology analogous to the human blood neutrophil was found. However, similar to monocytes, rare granules were observed by light and electron microscopy. Thus, the polymorphonuclear cell was

9 186 M.L.daS. Ribeiro et al. / Fish & Shellfish Immunology 23 (2007) 178e187 considered an agranulocyte cell type. The neutrophils described by Hine et al. [4,5] have the same nuclear morphology observed in the polymorphonuclear agranulocyte, though, granules were observed. However, some agranular neutrophils were also reported [5]. It is reasonable to correlate the neutrophil described by Hine et al. [4,5] with the polymorphonuclear agranulocyte seen in our preparation. The rare granules observed by us, may be related to species differences. Although this cell type was not the major population in the coelomic inflamed cavity, it was also present, indicating that it may be involved in the inflammatory response (further discussed). The type II granulocyte is probably related to the eosinophilic type I granulocyte described by Bielek and Strauss [6] as judged by the morphological (electrondense core) and cytochemical (peroxidase) characteristics of its granules. Type III granulocyte was the only cell with extremely basophilic granules and it also had distinct granule morphology in comparison to the other granulocytes. Furthermore, its relative abundance was below 1.5%. These results suggest that this cell type is a separate lineage and might be related to the mammalian basophil. The type I granulocyte was similar to what Bielek and Strauss [6] named the eosinophilic II granulocyte. No direct morphological correlation of this cell type could be found in Hine et al. s [4,5] work. By exclusion, it is possible that this cell type may be related to the mammalian eosinophil. The type II granulocyte and the polymorphonuclear agranulocyte were found in the inflamed coelomic cavity. However, the type II granulocyte was much more abundant. In addition, the granules of this cell type were positive for peroxidase and for alkaline phosphatase, both markers of neutrophils/heterophils [7,19,20]. These results indicate that this cell type is the major inflammatory granulocyte of L. paradoxa and, thus, is related to the avian and reptile heterophil. Inflammatory granulocytes with eosinophilic granules have also been described in a reptile [21], and in fishes [22] as the major inflammatory leucocyte. However, it cannot be ruled out that the polymorphonuclear agranulocyte is also involved in the migration to inflamed tissue. Considering that this cell type has few granules, that subpopulations of monocytes have been described in the blood of a teleost [23], and that blood monocytes migrate to inflamed tissue, it is not unreasonable to relate the polymorphonuclear agranulocyte to a monocyte subpopulation. Because lungfish may still possess characteristics of the common ancestor of teleosts and land vertebrates, it also seems reasonable to suggest that during the evolution of the former, the type II granulocyte became the primordial inflammatory granulocyte. Further experiments to determine the phagocytic ability and its capacity to release oxygen radicals are necessary to better characterise the function of the lungfish polymorphonuclear agranulocyte. Macrophages were also found in the exudate coelomic cell population. These cells had typical vertebrate macrophage morphology with membrane projections and vacuoles similar to the cell described in the Australian lungfish [5]. Although in their work Hine et al. [5] injected lipopolysaccharide in the coelomic cavity, no further characterization of exudate cell population was described because no change in the cell number was found. Although a change in the composition of the inflammatory population after 48 h in fish would normally be expected [24], no fluctuations were detected in the relative number of granulocytes and macrophages in the 48 h period examined after thioglycollate injection. This might reflect a less efficient transmigration response of lungfish. Further studies examining longer periods of thioglycollate injection will address this question. Acknowledgments The authors would like to thank Dr. Kevin Tyler and Andrèa Carvalho César for proof-reading the manuscript, Dr. Ralph Lainson for allowing us to use the animal facility and Marcia A. Dutra, Giovana A. de Moraes, and Beatriz F. Ribeiro for their invaluable assistance with the photographic work. This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT-CNPq), Fundaç~ao de Coordenaç~ao de Pessoal de Nível Superior (CAPES), Fundaç~ao Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Financiadora de Estudos e Projetos (FINEP), Programa Nacional de Cooperaç~ao Acadêmica (PROCAD) and Programa de Núcleos de Excelência (PRONEX). The experiments performed in this work comply with the current Brazilian laws. References [1] Brinkmann H, Denk A, Zitzler J, Joss JJ, Meyer A. 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