Tissue and Cell 37 (2005) 193–202

Cytochemical and functional characterization of blood and inflammatorycells from the lizardAmeiva ameiva

Sanny O. Alberioa, Jose A. Dinizb, Edilene O. Silvac, Wanderley de Souzad,Renato A. DaMattae, ∗

a Departamento de Fisiologia, Universidade Estadual do Par´a, 66887-670 Bel´em, PA, Brazilb Unidade de Microscopia Eletrˆonica, Instituto Evandro Chagas, 66001-000 Bel´em, PA, Brazil

c Laboratorio de Parasitologia, Centro de Ciˆencias Biologicas, Universidade Federal do Par´a, 66075-110 Bel´em, PA, Brazild Laboratorio de Ultraestrutura Celular Hertha Meyer, Instituto de Biof´ısica Carlos Chagas Filho, Universidade Federal

do Rio de Janeiro, 21949-900 Rio de Janeiro, RJ, Brazile Laboratorio de Biologia Celular e Tecidual, Centro de Biociˆencias e Biotecnologia, Universidade Estadual do Norte Fluminense,

28015-620, Av. Alberto Lamego 2000, Horto, 28013-600 Campos dos Goytacazes, RJ, Brazil

Received 21 May 2004; received in revised form 22 December 2004; accepted 30 December 2004Available online 16 March 2005

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The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify graromAmeiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cusoelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, throymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Typeranulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobund higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granuleitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relatacrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the n

ype III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the mnd cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential bloodounts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyteV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/nd type II to the eosinophil granulocytes.2005 Elsevier Ltd. All rights reserved.

eywords: Ameiva ameiva; Reptilian; Leukocytes; Granulocytes; Neutrophils/heterophils; Coelomic exudate cells

. Introduction

Vertebrate blood cells are divided into erythrocytes andeukocytes. Non-mammalian erythrocytes are nucleated andasily recognized morphologically. Thrombocytes, the func-

ional analog to mammalian platelets, are also nucleated and,ike monocytes and lymphocytes, are more accurately clas-ified by transmission electron microscopy. However, there

∗ Corresponding author. Tel.: +55 22 2726 1694; fax: +55 22 2726 1514.E-mail address:renato@uenf.br (R.A. DaMatta).

is marked ambiguity in the classification of non-mammagranulocytes mainly due to the diverse terminology appthe lack of functional studies (Meseguer et al., 1994) andmorphological similarities to the mammalian granulocy(Ryerson, 1943; Sypek and Borysenko, 1988). Lately, thisterminology has been tentatively standardized and lecyte counts of some reptile species determined bymicroscopy (Egami and Sasso, 1988; Work et al., 19Alleman et al., 1992, 1999; Stacy and Whitaker, 20Harr et al., 2001). Nevertheless, some ambiguity still eists consequent to species differences (Cannon et al., 1996

040-8166/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.tice.2004.12.006

194 S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202

Bounous et al., 1996; Alleman et al., 1999). Ultrastructuraland functional studies may help to clarify this classificationproblem.

The chelonian is the reptilian clade whose leukocyte isbest characterized at the ultrastructural level. In two speciesof turtles (Ryerson, 1943) two granulocytes named heterophil(round granules) and eosinophil (ellipsoid granules) werefound.Taylor et al. (1963)also found eosinophils (one gran-ule type) and heterophils (three granule types). Recently, itwas demonstrated by electron microscopy cytochemistry thateosinophils and heterophils are two distinct cell lineages inthe blood of a turtle species (Azevedo et al., 2002; Azevedoand Lunardi, 2003). Studies carried out with theCaimancrocodilusshowed that eosinophils and heterophils are alsoseparate cell lineages (Moura et al., 1997; Oliveira et al.,1998a, 1998b). Alleman et al. (1999)found only heterophilsin the blood of the snakeCrotalus adamanteus. In the squa-mata,Kelenyi and Nemeth (1969)suggest that the two de-scribed granulocytes correspond to the same cell at differentstages of maturation.Zapata et al. (1981)characterized twogranulocytes with different types of granules and classifiedthem as heterophils. It seems clear that the controversy onthe different granulocytes is mainly due to the small numberof reptilian species examined so far, and some species mayhave only certain cell types. Furthermore, there is a lack offunctional studies on lizard leukocytes.

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methanol and stained with Giemsa for leukocyte morpho-logical classification and counting. Blood leukocytes wereseparated by adapting a previously described method usedfor chicken blood (DaMatta et al., 1998, 1999). Briefly, bloodfrom four lizards was diluted (1:3, v/v) with Dulbecco’s Mod-ified Eagle’s Medium (DMEM, Sigma), layered on top of a60% Percoll (Pharmacia) solution and centrifuged at 780×gfor 15 min at 22◦C. The diluted plasma was discarded, thebuffy coat collected and washed twice with DMEM (300×g,10 min, 4◦C).

2.3. Lizard coelomic normal and exudate cells

To determine the blood leukocyte type that migrates ex-travascularly during inflammation, 1 ml of thioglycollate(3% of sterile Brewer’s thioglycollate medium, Sigma) wasinjected intracoelomic (Baron and Proctor, 1982) into 10lizards. Twenty-four hours later and after ether euthana-sia, a coelomic wash was performed by following the stan-dard technique used for mice (Handel-Fernandez and Lopez,

Fig. 1. (a and g) Light microscopy of Giemsa-stained blood cell smear fromtheAmeiva ameivalizard. (a) Monocyte (arrow) showing kidney-shaped nu-cleus. (b) A clump of thrombocytes (arrow). (c) Lymphocyte (arrow). Notethe high nucleus/cytoplasm ratio. (d) Type I granulocyte (arrow) presentingan eccentrically located nucleus. (e) Type II granulocyte (arrow) display-ing fusiform granules. (f) Type III granulocyte (arrow) showing bilobulatednucleus. (g) Type IV granulocyte (arrow). Note the small cell size with cy-toplasm full of granules. Erythrocyte (arrowhead) presenting an ellipticalshape with a centrally placed nucleus. Bar 10�m.

The aim of this study was to characterize bloodoelomic exudate leukocytes of theAmeiva ameivabecausf the wide geographic distribution of this lizard (Vitt andolli, 1994) and its adaptability in captivity favors its usen experimental model, and intracellular parasites haveescribed infecting blood leukocytes of this lizard (Lainsont al., 2003). However, no clear identification of the leukytes exists due to a lack of morphological and functitudies.

. Materials and methods

.1. Lizards

Thirty adults (±18 cm in length) ofA. ameiva(Lepi-osauria, Teiidae) were hand-captured in Belem, PA, Brazilhey were maintained in the Instituto Evandro Chaelem animal facility, kept separately in standard moages maintained at a 10◦ angle with clean water at the boom providing 60% of dry floor, at 27–32◦C. Lizards wereed with larvae ofTenebrio molitor.

.2. Blood harvesting and leukocyte separation

Lizards were restrained manually and blood (500± 100�ler individual) collected by cardiac puncture into heparinml syringes (100 U/ml) with a 27 G needle. Lizards wereled after 2 months in captivity only for ultrastructural st

es. Blood smears from all lizards were fixed with abso

S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202 195

2000). Briefly, in 10 cm body size animals, without pullingback the skin, 5 ml of DMEM were injected into the coelomiccavity on the left of the first one-fourth (cloacae to head) ofthe lizard ventral side. The cell suspension, 4 ml, was col-lected in the same syringe and dispersed in tubes hold at

4◦C. If less than 90% of the injected medium was not re-covered, the lavage was discarded. Cells were centrifuged at500×g for 10 min at 4◦C and resuspended in DMEM. Ascontrol washes were also performed on noninjected (normal)lizards.

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ig. 2. (a and e) Transmission electron microscopy of mononuclear blood ceolgi apparatus (G), mitochondria (long arrow) and an indented nucleus (asf the upper cell in (b) showing small slender sacs (arrowhead) localized nexnd mitochondria (arrow). (e) Plasma cell showing similar lymphocyte morphar 1�m, except (c). Bar 0.25�m.

lls from theAmeiva ameivalizard. (a) Monocyte showing small vacuoles (arrow),terisk). (b) The thrombocyte has typical vacuoles (V). (c) A higher magnificationt to the plasma membrane. (d) Lymphocyte containing indented nucleus (asterisk)ology, with a large number of rough endoplasmic reticulum profiles (arrowhead).

196 S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202

S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202 197

2.4. Light microscopy

Giemsa stained blood smears were examined and pho-tographed under a Zeiss Axiophote microscope using the im-mersion 100× objective. Differential leukocyte counts weremade for the blood smears of each lizard, 200 leukocytes werecounted and the percentage of each cell type calculated. Per-centage means± standard deviation was calculated for eachcell type from the five lizards examined. Normal and exu-date cells from the lizard coelomic cavity were submitted toa Cytospin centrifuge, Giemsa stained, examined under a mi-croscope and the different cell types (200 cells) scored andrelative percentage calculated.

2.5. Transmission electron microscopy

For routine observations, blood and exudate leukocyteswere 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 ferri-cyanide in cacodylate buffer containing 5 mM calcium chlo-ride for 30 min. Cells were washed with the same buffer,dehydrated in acetone and embedded in epoxy resin. Thinsections were stained with uranyl acetate and lead citrateand observed in a Zeiss EM900 transmission electron mi-c

dK de( atr con-t ris-m atedf mMc teb xedw d ina re ob-s ranyla

a de( atr werei gstica ande thin

sections were observed in the transmission electron micro-scope.

For the detection of peroxidase at neutral pH (Roels etal., 1975) blood leukocytes were fixed in 1% glutaraldehyde(grade I) in 0.1 M cacodylate buffer, pH 7.2 for 30 min atroom temperature and washed with the same buffer contain-ing 5% sucrose. Subsequently they were incubated at roomtemperature for 30 min in cacodylate buffer, pH 7.0 contain-ing 0.5 mg/ml of diaminobenzidine, 5% sucrose and 0.003%hydrogen peroxide. Cells were washed, re-fixed in 2.5% glu-taraldehyde in cacodylate buffer, washed again, post-fixedwith 1% osmium tetroxide in cacodylate buffer, dehydratedin acetone and embedded in epoxy resin. Thin sections wereobserved in the transmission electron microscope withouturanyl acetate and lead citrate staining.

3. Results

A. ameivaperipheral blood smear cells were classifiedmorphologically by light microscopy. Erythrocytes, mono-cytes, clumped thrombocytes and lymphocytes presented adistinct morphology. Monocytes were larger than erythro-cytes, had a light pale-blue cytoplasm with a few violetpatches, a central kidney shaped nucleus that occupied about5 ane ticalw nseb sm( ad ap leuseF ran-u ad ab hilicg d nu-cI ad ab les.T welld izea lob-ug ith af andh asm

F d cells eus,v ndria ( ad)ft cation whm m nucl ta as in (a rrow), sr culum ntricb m (lon and the nuc tral nuca . Bar 0.

roscope, operated at 80 kV.For the detection of alkaline phosphatase (Robinson an

arnovsky, 1983) cells were fixed with 1% glutaraldehygrade I) in 0.1 M cacodylate buffer, pH 7.2 for 30 minoom temperature and washed with the same bufferaining 5% sucrose. Cells were washed twice in 0.1 M Taleate buffer, pH 9.0 containing 5% sucrose and incub

or 30 min at room temperature in a medium containing 4erium chloride, 10 mM�-gycerophosphate in Tris-maleauffer, pH 9.0 containing 5% sucrose. Cells were post-fiith 1% osmium tetroxide in cacodylate buffer, dehydratecetone and embedded in epoxy resin. Thin sections weerved in a transmission electron microscope without ucetate and lead citrate staining.

For the ethanolic phosphotungstic acid method (Gordonnd Bensch, 1968), cells were fixed in 1% glutaraldehygrade I) in 0.1 M cacodylate buffer, pH 7.2 for 30 minoom temperature and dehydrated in ethanol. Then cellsncubated for 2 h at room temperature in 2% phosphotuncid in absolute ethanol, washed with propylenum oxidembedded in epoxy resin. Unstained and lead stained

ig. 3. (a and f) Transmission electron microscopy of granulocyte blooesiculated rough endoplasmic reticulum (long arrow), small mitochohe granules contain amorphous material (inset). (b) A higher magnifiitochondria (arrow). (c) Granulocyte type II displays eccentric disforrrow). The granules are surrounded by membrane (inset). Bar sameound or fusiform granules (arrowhead) and rough endoplasmic retiilobulated nucleus, small mitochondria (arrow), endoplasmic reticuluensity. Note higher amounts of heterochromatin when compared tond granules homogenous in size (arrow). Bar 1�m, except inset and (b)

0% of the cell (Fig. 1a). Thrombocytes were smaller thrythrocytes and often found in clumps. They were ellipith a thin rim of clear cytoplasm surrounding an inteasophilic nucleus occupying most of the cell cytoplaFig. 1b). Lymphocytes were larger than thrombocytes, hale-purple cytoplasm, with a round and pale-violet nucccentrically located, occupying most of the cell (Fig. 1c).our different granulocyte types were observed. Type I glocytes was typically the largest cell observed. They hlue cytoplasm with round, eosinophilic and some basopranules. These cells had an oval, eccentrical, unlobulateleus and a small nucleus-to-cytoplasm ratio (Fig. 1d). TypeI granulocytes were similar in size to monocytes and hlue cytoplasm with more elongated eosinophilic granuhey had a centrally located nucleus with borders notefined (Fig. 1e). Type III granulocytes had a similar snd cytoplasm color in relation to type I. They had a bilated nucleus and basophilic granules (Fig. 1f). Type IVranulocytes were slightly larger than thrombocytes, w

aint-blue cytoplasm, an eccentrically located nucleusighly basophilic granules occupying most of the cytopl

from theAmeiva ameivalizard. (a) Granulocyte type I. Note the eccentric nuclarrow), granules heterogeneous in size, shape and content (arrowhe. Some oof (a) showing vesiculated rough endoplasmic reticulum profiles (arroead) andeus, normal rough endoplasmic reticulum (arrow) and surface projecions (long). (d) A higher magnification of (c) showing long mitochondria (long amall

profiles (arrow).Bar same as in (b). (e) Granulocyte type III showing ecceg arrow) and granules (arrowhead) very heterogeneous in size, formd electronleus of type I granulocyte (a). (f) Granulocyte type IV presents a cenleus,25�m.

198 S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202

(Fig. 1g). Erythrocytes were elliptically shaped with a cen-trally located oval nucleus. Cytoplasmic inclusions were rarlyseen (Fig. 1g).

Based on the above histo-morphological classification,differential blood leukocyte counts of lizards were per-

formed. Type III granulocytes were the largest leukocytepopulation found (44.2± 3.41), followed by lymphocytes(24.8± 2.49), type I granulocytes (12.5± 1.29), type II gran-ulocytes (10.2± 0.69), monocytes (7.3± 0.69) and type IVgranulocytes (1.0± 0.18).

Ffpds

ig. 4. (a and d) Transmission electron microscopy of granulocyte blood celor alkaline phosphatase in type I (upper cell) and type II granulocytes. Typositive reaction in the small granules (arrow). (b) Basic proteins could be de) Peroxidase cytochemistry at neutral pH. Type I granulocyte had a positivhows positive reaction in small and fusiform granules (d). Bar 1�m.

ls from theAmeiva ameivalizard after cytochemistry staining. (a) Cytochemistrye I granulocyte contains large unlabeled granules; the type II granulocyte has atected in medium and small sized granules found in the type I granulocyte. (c ande reaction in all granules and endoplasmic reticulum (c) and type II granulocyte

S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202 199

Fig. 5. (a and e) Transmission electron microscopy of coelomic exudate cells obtained after 24 h of thioglycollate stimulation. (a) Macrophage presentingmembrane projections, cisternae (long arrow) and normal (arrowhead) vacuoles, granules (small arrow) and mitochondria (big arrow) were observed.(b) TypeIII granulocyte presenting bilobulated nucleus with higher amounts of heterochromatin, small mitochondria (arrow) and heterogeneous granules. (c) Basicproteins were detected basically at the nucleus of macrophages. (d) Basic proteins were detected at the nucleus and granules of type III granulocyte.(e) Alkalinephosphatase was detected at macrophages (M) and granulocyte type III (G) at the plasma membrane. (a–d) Same bar. Bar 1�m.

200 S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202

Blood leukocytes were characterized by transmissionelectron microscopy following separation from erythro-cytes by centrifugation over a Percoll cushion. The adaptedmethodology was satisfactory because a greater concentra-tion of leukocytes over erythrocytes was obtained. Attemptsto achieve higher leukocyte purity by increasing the densityof the Percoll cushion resulted in less erythrocyte contami-nation; however, granulocytes loss increased.

Blood monocytes, thrombocytes and lymphocytes wereeasily recognized at the ultrastructural level. Monocytes pre-sented small vacuoles, Golgi apparatus, mitochondria and anindented nucleus (Fig. 2a). Thrombocytes had typical vac-uoles that appeared empty (Fig. 2b). Thrombocytes also hadcanalicular systems next to the plasma membrane easily seenat higher magnification (Fig. 2c). Lymphocytes were roundwith an indented nucleus, scattered mitochondria and a cyto-plasm with few membrane bound organelles (Fig. 2d). Plasmacells were rarely found and were characterized by the prolificrough endoplasmic reticulum (Fig. 2e).

The four different blood granulocyte types seen by lightmicroscopy were also observed by transmission electron mi-croscopy. Type I granulocytes presented an eccentric nucleuswith heterochromatin mainly arround the borders. These cellshad a large number of intracytoplasmic granules heteroge-neous in size, shape and electron density (Fig. 3a). Someof the granule had amorphous internal structures and werem to-c scat-tT phicn ders( rlym drT bu-l atin.G elec-t thec smicr s-p atinm us ins

ion,c ba-s oundi fews -u d pe-r sw ctedi cifica nulesw edm ran-u ac-

tivity was localized in granules and endoplasmic reticulumfound in type I granulocytes (Fig. 4c), in small and fusiformgranules in type II granulocytes (Fig. 4d), type III was notobserved.

Cytospin analyses of the normal coelomic cell populationof nonstimulated lizards revealed that theA.ameivapresentedmainly macrophages and a few type III granulocytes (lessthan 5%). After 24 h of thioglycollate stimulation, a largeincrease in the granulocyte population, primarily type III, wasdetected (above 50%), less than 5% of type I was observed.

Ultrastructural observations of the 24 h thioglycollate ex-udate cells from the coelomic cavity revealed macrophageswith membrane projections, cisternae, vacuoles, granules andmitochondria (Fig. 5a), and type III granulocytes presentingbilobulated nucleus with higher amounts of heterochromatin,small mitochondria and heterogeneous granules (Fig. 5b).Basic proteins were detected in macrophages and type IIIgranulocyte at the nucleus and granules (Fig. 5c and d). Al-kaline phosphatase labeled both cells at the plasma membraneonly (Fig. 5e).

4. Discussion

Erythrocytes, thrombocytes, monocytes, lymphocytes,plasma cells, and four types of granulocytes were observed int ac-t om-b hoses rke ker,2 -b stem.L ocyte( esl r sys-tm ruc-t theyw con-t cytec ka hec lles.T s af-t cellsc stemw hasa .,1

mi-c TypeI st tot isticw ytesh orm,

embrane bound (Fig. 3a, inset). Small and scattered mihondria, and vesiculated rough endoplasmic reticulumered throughout the cytoplasm was also observed (Fig. 3b).ype II granulocytes displayed an eccentric pleomorucleus with heterochromatin mainly arround the borFig. 3c). Small, round or fusiform granules were cleaembrane bound (Fig. 3c, inset). Long mitochondria an

ough endoplasmic reticulum were also present (Fig. 3d).ype III granulocytes were round, with an eccentric bilo

ated nucleus containing higher amounts of heterochromranules were very heterogeneous in size, form and

ron density, small mitochondria were scattered throughytoplasm and very few structures resembling endoplaeticulum were observed (Fig. 3e). Type IV granulocytes dilayed a round form, a central nucleus with heterochromainly around the borders, large granules homogeno

ize and a few mitochondria (Fig. 3f).In order to improve blood granulocyte characterizat

ytochemistry for the localization of two enzymes andic proteins were used. Alkaline phosphatase was not fn the larger granules of type I granulocytes, although,mall granules were positive (Fig. 4a, top cell). Type II granlocytes presented a positive reaction in the small rounipheral granules (Fig. 4a, bottom cell); fusiform granuleere also positive (not shown). Basic proteins were dete

n type I granulocytes at the cell cytoplasm and in spereas of the small and medium size granules; larger graere not labeled (Fig. 4b). Type III granulocytes presentedium granules positive for basic proteins and large gles were not labeled (not shown). Neutral peroxidase

heA. ameivaperipheral blood. The morphological chareristics obtained by light microscopy of erythrocytes, throcytes, monocytes and lymphocytes were similar to teen in other reptilian species (Egami and Sasso, 1988; Wot al., 1998; Alleman et al., 1992, 1999; Stacy and Whita000; Harr et al., 2001). At the ultrastructural level thromocytes had large vacuoles and a typical canalicular syarge vacuoles are also present in the chicken thrombDaimon et al., 1977). However, the tortoise thrombocytack large vacules and has a very extensive canaliculaem (Daimon et al., 1987). Thus, theA. ameivaresembleorphologicaly more the chicken thrombocytes. Ultrast

urally, monocytes had the usual organelles, however,ere scarcely distribute through the cytoplasm. This in

rast to the description of other reptiles where the monoytoplasm is full of organelles (Taylor et al., 1963; Sypend Borysenko, 1988). This cell type also contrasts with toelomic macrophage that has a cytoplams full of organehis difference may be related to the maturation proces

er monocytes migration to inflammed tissue. Plasmaharacterized by its profound endoplasmic reticular syere rarely found in the blood of this lizard. This findinglso been described in other squamata species (Zapata et al981; Sypek and Borysenko, 1988).

The four different blood granulocytes seen by lightroscopy were also observed by electron microscopy.granulocytes had large, round-oval granules in contra

ype II that had small fusiform granules. This characteras confirmed at the ultrastructure level. Type II granulocad a similar morphology, such as granule size and f

S.O. Alberio et al. / Tissue and Cell 37 (2005) 193–202 201

to granulocytes of other turtle species described as the ma-malian eosinophils (Ryerson, 1943; Wood and Ebanks, 1984;Azevedo et al., 2002; Azevedo and Lunardi, 2003). Type Igranulocytes contained round granules, similar to those foundin type III cells. The essential differences between these twocells were the bilobulated nucleus, amount of heterochro-matin and the more heterogeneous granules present in typeIII. Type I granulocytes also contained a vesiculated roughendoplasmic reticulum not present in type III cells. Duringmaturation of erythrocytes there is an increase in heterochro-matin (Grasso et al., 1962). The increase in heterochromatinfound in type III granulocyte, and the other differences inrelation to type I granulocyte, suggest that type III may be amore mature state of type I granulocyte. Thus, both cells mayconstitute the same cell lineage.

Type II granulocytes had plasma membrane projectionsand a low number of granules (round and elongated). Thesecharacteristics strongly indicate that this type II is a differentcell lineage when compared to types I and III granulocytes.Furthermore, alkaline phosphatase was not found in the largegranules of type I granulocytes. However, it was detectedin granules of type II granulocytes. Differences in the cy-tochemical localization of enzymes between two cells havebeen used to distinguish cell lineages (Azevedo et al., 2002;Azevedo and Lunardi, 2003). Thus, the cytochemical resultsfurther support the differentiation of type II granulocyte fromt rvedi tal-l cytese or-p l andh acteri l.,2

tiono ofu agesa ani ypeI ulo-c thece xo IIIg nale mamm t them edc re-i tures cellm

pho-t s oft erea tes.

The resemblance of the morphology and basic proteins lo-calization between blood and coelomic granulocytes furtherindicates that type III (and to a less extents, type I) is theblood granulocyte that migrates to the inflamed tissue. It hasbeen shown that activation of the rat neutrophils increasesalkaline phosphatase activity (Jiang et al., 1998). Alkalinephosphatase labeling in the granulocytes of thecoelomicex-udate was higher than in the blood granulocyte. This suggeststhat during migration to the inflamed tissue the lizard granu-locyte is activated as in the rat system (Mathison et al., 2003).This activation might also explain the differences in the typeof labeling of basic proteins between blood andcoelomicgranulocytes.

The percentage of types I and III granulocytes in the bloodwas higher than the other cell types. The lymphocyte popu-lation was followed by the monocyte, type II granulocyte,and basophils had a very low number, as expected. A 44%of the total leukocyte population is achieved if the relativepercentage means of types I and III are combined. This highpercentage suggests that this population is functionally analo-gous to the mammalian neutrophil, since the latter cell type isalso present in higher numbers in the blood of mammals dueto its function as an essential first line of defense (Zychlinskyet al., 2003).

In conclusion, the morphological, cytochemical and func-tional data presented in this study indicates that types I andI atesa phil.T ulo-c phil.

A

a , Dr.R andM aesa witht on-s(S -p RJ),F Fi-n Na-c ad er-f ws(

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A l andtoise

ypes I and III. Although amorphous structures were obsen some granules of blood type III granulocyte, no crysoid structure was seen in the granules of all the granuloxamined. The type IV granulocytes were similar in mhology and relative number to the mammalian basophiave been classified as such by other authors that char

zed reptilian blood cells (Alleman et al., 1999; Harr et a001).

Functional analyses provided additional characterizaf A. ameivagranulocytes. The coelomic cell populationninjected animals was mainly composed of macrophnd type III granulocytes. After thioglycollate injection,

ncrease in the type III, and a small number of the t, granulocytes were observed. However, type II granytes were not found. An inflammatory response inoelomiccavity of mice (Baron and Proctor, 1982) and chick-ns (Trembicki et al., 1984) is characterized by an influf neutrophils/heterophils. The migration of types I andranulocytes to the inflamed coelomic cavity is a functiovidence that these granulocytes are analogous to thealian neutrophils and avian heterophils. The fact thaajority of the granulocyte population found in the inflam

oelomic cavity was mainly composed of type III cell,nforces the suggestion that this cell type is a more matate of type I, because migration is usually favored byaturation.Basic proteins, detected using the ethanolic phos

ungstic acid technique, were localized in the granuleypes I and III granulocytes of the blood. These proteins wlso localized in the coelomic exudate type III granulocy

-

-

II granulocytes may correspond to different maturation stnd are equivalent to the mammalian neutrophil/heteroype II granulocytes may represent the eosinophil granyte, while type IV corresponds to the mammalian baso

cknowledgments

The authors would like to thank Andrea Carvalho Cesarnd Dr. Thereza Kipnis for proof-reading the manuscriptalph Lainson for permission to use the animal facilityarcia A. Dutra, Arthur Rodrigues, Giovana A. de Mornd Beatriz F. Ribeiro for their invaluable assistance

he photographic work. This work was supported by Celho Nacional de Desenvolvimento Cientıfico e TecnologicoMCT-CNPq), Fundac¸ao de Coordenac¸ao de Pessoal de Nıveluperior (CAPES), Fundac¸ao Carlos Chagas Filho de Amaro a Pesquisa do Estado do Rio de Janeiro (FAPEundac¸ao Estadual do Norte Fluminense (FENORTE),anciadora de Estudos e Projetos (FINEP), Programaional de Cooperac¸ao Academica (PROCAD) and Programe Nucleos de Excelencia (PRONEX). The experiments p

ormed in this work comply with the current Brazilian laIBAMA 02018.000301/02-13).

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