SciELO - Scientific Electronic Library Online

 
vol.25 número2Variaciones Múltiples de las Ramas de la Arteria Axilar Relevantes para la Cirugía Plástica: Reporte de CasoEstilos de Aprendizaje y su Correlación con el Rendimiento Académico en Anatomía Humana Normal índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

Compartir


International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.25 n.2 Temuco jun. 2007

http://dx.doi.org/10.4067/S0717-95022007000200021 

 

Int J. MorphoL, 25(2):363-366,2007.

 

Characterization of Blood Mononuclear Phagocytes in Phrynops hilarii (Chelonia Chelidae)

Caracterización de los Fagocitos Mononucleares en la Sangre de Phrynops hilarii (Chelonia Chelidae)

 

*Dimitrius Leonardo Pitol; **Joáo Paulo Mardegan Issa; ***Flávio Henrique Caetano & ****Laurelúcia Orive Lunardi

*Graduate student, Biosciences Institute- Molecular and Cellular Biology (UNESP), Rio Claro, Sao Paulo, Brazil.
**
Graduate student, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.
*** Professor of Cellular Biology, Biosciences Institute- Molecular and Cellular Biology (UNESP), Rio Claro, Sao Paulo, Brazil.
****Professor of Histology, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.

Dirección para correspondencia


SUMMARY: The localization of peroxidase activity in different cell regions is used as a criterion for the classification of the stage of maturation of mammalian mononuclear phagocytes with a positive peroxidase reaction indicating the presence of monoblasts, promonocytes, monocytes and macrophages. In this study it was evaluated the peroxidase activity of blood mononuclear phagocytes of this turtle detected at different stages of differentiation. The present observations suggest that, in turtles, the differentiation of mononuclear phagocytes occur in the blood circulation, in contrast to animals, where only are monocytes in circulating blood and macrophage differentiation occurs in other body compartments.

KEYWORDS: Mononuclear phagocytes; Blood; Turtle.

RESUMEN: La localization de la actividad de la peroxidasa en diversas regiones de la célula se utiliza como criterio para la clasificación de la etapa de maduración de fagocitos mononucleares. Una reacción positiva de peroxidasa indica la presencia de monoblastos, promonocitos, monocitos y macrófagos. En este estudio fue evaluada la actividad de la peroxidasa de los fagocitos mononucleares de la sangre de la tortuga Phrynops Hilarii detectada en diversas etapas de la diferenciación. Las actuales observaciones sugieren que, en tortugas, la diferenciación de fagocitos mononucleares ocurre en la circulación de la sangre, en contraste a los mamíferos, donde están solamente los monocitos en la sangre circulante y la diferenciación de los macrófagos ocurre en otras partes del cuerpo.

PALABRAS CLAVE: Fagocitos mononucleares; Sangre; Tortuga.


INTRODUCTION

Mononuclear phagocytes represent a cell lineage consisting of monoblasts, promonocytes, monocytes, and macrophages. The similarities of the morphological, cytochemical, and functional characteristics of these cells have led to the concept of a monuclear phagocyte system (Van Furth etal, 1972; Pellizzoneiet al. 2002). One of the major functions of mononuclear phagocytes is phagocytosis and the killing of microorganisms (Langermans etal, 1994; Halliwell, 2006).

In mammals, mononuclear phagocytes differentiate in bone marrow from stem cells- monoblasts, promonocytes and monocytes. Monocytes enter the circulation and migrate to body cavities and or tissue, where they differentiate into macrophages as growth factors, interleukins and others (Van Furth etal, 1972;Beelen&Fuitsma, 1982; Beelen et al., 1989; Metcalf, 1989; Metcalf & Nicola, 1992; Naito, 1993; Sudhakaran et al, 2007). Mononuclear phagocytes in the different stages of development have been well characterized in mammals on the basis of ultrastructural observation of peroxidase activity and distribution (Beelen ei et al. 1978,1979; Beelen, 1981; Beelen & Fuitsma).

Mononuclear phagocytes have not been fully characterized in turtles. The turtle {Phrynops hilarii) has special interest because of its easy breeding, rapid development, and easy access and lodging. In this study it was evaluated the peroxidase activity of blood mononuclear phagocytes of this turtle detected at different stages of differentiation.

MATERIAL AND METHOD

This study followed the requirements of the Ethics Committee on the Use of Animals in Experimentation at the University of Sao Paulo, Brazil.

The study was conducted on five adult Phrynops hilarii turtles (Chelonia, Chelidae) of both sexes weighing 600-1200g. The animals were captured around the estuary of the Guaiba River, near Porto Alegre, Brazil. All the observations were made during the spring and early summer at a temperature ranging from 20 to 30oc. Three mL of blood obtained by puncturing the jugular vein of the neck were collected into a heparinized glass tube and centrifuged for 20 min at 1000 rpm. The coat buffer was fixed for 10 min in 1% glutaraldehyde in 0.1M Na-cacodylate-HCL buffer, pH 7.4, at 4oc. This was then washed in the same buffer and pre-incubated in 0.1% 3,3-diaminobenzidine-tetrahydrochloride (Polyscience, Warrington, PA) in 0.1M Na-cacodylate-HCL buffer, pH 6.5, in the absence the H202 at room temperature for 45 min in the dark.

The coat buffer was divided into two aliquots, one of which was incubated in freshly prepared medium in the presence of 0.01% H2O2 for 30 min at room temperature, while the other (control) was incubated in fresh medium in the absence of H202 (Beelen & Fuitsma). The cells were washed in 0.1M Na-cacodylate, pH 7.5, and post-fixed in 0.1 % OS04 in the same buffer for 30 min at 4oc, then rinsed in distilled water, dehydrated and embedded in epoxy resin. Thin sections were observed with a transmission electron microscope (Jeol 100C).

RESULTS

Different stages of mononuclear phagocyte differentiation are observed in the circulating blood of turtle, as defined by ultrastructural peroxidase cytochemistry.

In this study, we identified cells with monoblast characteristics, i.e., a large nucleus with proeminent nucleoli, a nucleocytoplasmatic ratio greater than 1, and the peroxidase reaction was localized in the cytoplasmatic granules (Fig. 1 A). Promonocytes, with an indented nucleus and a nucleocytoplasmatic ratio of less than 1, clearly visible cytoplasmatic prolongations, peroxidase reaction in the nuclear envelope, rough endoplasmatic reticulum, and cytoplasmatic granules can be seen in Fig. 1B. Monocytes have a positive peroxidase reaction in the cytoplasmatic granules and peripheral nucleus (Fig. 1C). A circulating macrophage with a positive peroxidase reaction in cytoplasmatic granules, nuclear envelope, and granular endoplasmic reticulum is illustrated in Fig. 1D.


Fig. 1A. Monoblast showing positive reaction for peroxidase observed in the cytoplasmatic granules (arrow) of the rough endoplasmic reticulum.

Fig. 1B. Promonocyte with positive reaction for peroxidase in the cytoplasmatic granules (arrow), in the rough endoplasmic reticulum (arrow) and in the nuclear envelope (arrow).

Fig. 1C. Monocyte showing a few cytoplasmatic granules with positive reaction for peroxidase in the nuclear envelope (arrow).

Fig. 1D. Macrophage circulating in blood showing peroxidase activity in the rough endoplasmic reticulus (arrow), and in the nuclear envelope (arrow).

DISCUSSION

Peroxidase activity was observed ultrastructurally in the circulating blood of Phrynops hilarii, identifying monoblasts, promonocytes, monocytes and macrophages.

In mammals, the monoblast-promonocyte-monocyte differentiation occurs in bone marrow, with monoblasts and promonocytes being detected in circulating blood only in specific pathological situations (Van Furth, 1989, 1992). In Phrynops hilarii this cell differentiation was observed in circulating blood under normal conditions. The distribution of peroxidase activity in these mononuclear phagocytes was similar to that observed in the bone marrow, peritoneal cavity, and inflammatory sites of mammals (Bellen eí al, 1978,1979; Beelen, 1981; Van Furth, 1989; Pellizzon et al).

In mammals, it has been suggested that the monocytes production is controlled by different growth factors as CFU-GEMM, CFU-GM e M-CSF (Metcalf; Metcalf & Nicola) or 11 -6 (Otsuka et al., 1991). These cells in the bone marrow move to circulating blood, 24h after its differentiation. They are spread off in the circulating and endothelial vase borders, posteriorly they move into tissues and serum cavities and differentiate into exudates and resident macrophages (Van Furth, 1989; Sudhakaran et al.). In the inflammatory responses, the circulating monocytes influx are mediated by different chemotactic factors as the protein-1 (MCP-1), and in the inflammatory focus differentiate into exudates macrophage (Bodeleiet al. 1977; Beelen et al., 1978,1981; Van Furth, 1989; Leonard & Yoshimura, 1990;Issekutzei et al. 1981; Jandhl, 1991; Wiktor-Jedrzejczak et al., 1992). Monocytes, macrophages with peroxidatic activity characteristic of resident macrophages, and negative peroxidase macrophages, were observed in this study in the turtles circulating blood suggesting that these cells move to the tissues probably in a differentiated stage.

It was observed in this study macrophages exhibiting a great phagocytose in the turtles circulating blood. These cells showed two forms, one of these is the morphological form characterized as mature macrophages and others very similar as the observed in mouse during the fetal development (Takahashi et al., 1989; Naito et al., 1990).

Naito defined these primitive macrophages as "fetal macrophages", we prefer to define as primitive macrophage, considering that the studied animals were in reproductive phase, thus adult animals. The mononuclear phagocytes differentiation in mammalians is dependent of the local environment conditions and the specific conditions of the specific tissues. These conditions are mediated by CSF and cytokines (Geislier et.al., 1989;Falk&Vogel, 1990; Naito et al., 1991,1993).Naito et al, 1991 work, using bone marrow culture, showed that M-CSF presence is fundamental for monocyte differentiation into mature macrophage. The results of this study showed that mononuclear phagocytes differentiation into monoblasts-promonocytes-macrophages are not similar to mononuclear phagocytes in vertebrates because apparently they do not depend of the tissues environment conditions, and there is not necessary the monocytes migration into tissue with the objective to promote the macrophages differentiation, because this process occurs during the period that these cells are circulating. The significance of this defensive process in turtles, as well as, the necessity to understand the regulatory mechanism of the macrophages differentiation in these animals, especially the growth factors participation (CSF) in this process, must study in future researches.

 

REFERENCES

Metcalf, D.; Nicola, N. A. Blood, 79:2861-6, 1992.        [ Links ]

Beelen, R. H. J.; Fluitsma, D. M.; Kom, C. & Hoefsmit, E. C. Identification of exúdate - residente macrophages on the basis of peroxidatic activity. J. Reticuioendothel Soc., 25:103-15, 1978.        [ Links ]

Beelen, R. H. J.; Fuitsma, D. M.; Van de Meer, J.W.; Hoefsmit, E.C.M. & Hoefsmit, E.C.M. Development of different peroxidatic activity patterns in peritoneal macrophages in vivo and in vitro. J. Reticuioendothel Soc., 25:513-24, 1979.        [ Links ]

Beelen, R. H. J. Development of exudates-resident macrophages in vivo and vitro. In: Hematology and blood transfusion. 27. Disorders of the monocyte macrophage system. Smalzl D., Huhn D. and Schafe H.F. eds., Springer Verlag, Berlin, 1981. pp. 89-100        [ Links ]

Beelen, R. H. J. & Fuitsma, D.M. What is the relevance of exúdate resident. Immunobiology., 161:266-73, 1982.        [ Links ]

Beeelen, R. H. J.; Bos, H. J.; Kamperdijk, E. W. A. & Hoefsmit, E. C. M. Ultrastrucuture of monocytes and macrophages. In: Human monocytes edited by M. Zembala and G. L. Asherson, Academic Press, London, 1989. pp7-15.        [ Links ]

Bodel, R. T.; Nichois, B. A. & Baiton, D. F. Appearance of peroxidase reactivity within the rough endoplasmic reticulum of blood monocytes after surface adherence. J. Exp. Med., 145:264-74, 1977.        [ Links ]

Falk, L. A. & Vogel, S. N. Differential production of IFN-/ by CSF-1 and GM- CSF derived macrophages. J. Leukocyte. Biol, 48:43-9, 1990.        [ Links ]

Geislier, K.; Harrington, M.; Srivastava, C; Leemhuies, T.; Tricot, G. & Broxmeyer, H. E. Effects of recombinant human colony stimulating factors (CSF) (granulocyte-macrophage colony stimulating factor CSF and CSF-1) on human monocyte/macrophage differentiation. J. Immunol, 143: 140-6, 1989.        [ Links ]

Halliwell, B. Phagocyte-derived reactive species: salvation or suicide? Trends. Biochem. Sci, 31:509-15, 2006.        [ Links ]

Issekutz, T. B.; Issekutz, A. C. & Movat, H. Z. The in vivo quantitation and kinetics of monocyte migration into acute inflammatory tissue. Am. J. Pathol,103:47-55, 1981.        [ Links ]

Jandhl, J. H. Blood Pathophysiology. Blackwell Scientific Publications, Boston, 1991. Cap. 11, pp 211-227.        [ Links ]

Langermans, J. A.; Hazenbos, W. L. & Van Furth, R. Antimicrobial functions of mononuclear phagocytes. J. Immunol. Methods., 174: 185-94, 1994.        [ Links ]

Leonard, E. J. & Yoshimura, T. Human monocyte chemoattractant protein-1 (MCP-1). Immunol. Today, 11:97-101, 1990.        [ Links ]

Metcalf, D. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature, 339: 27-30, 1989.        [ Links ]

Naito, M.; Yamamura, F; Nishikawa, S. I. & Takahashi, K. Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures. J. Leukocyte Biol, 46:1-10, 1990.        [ Links ]

Naito, M.; Hayashi, S.; Yoshida, H.; Nishikawa, S.; Shultz, L. D. & Takahashi K. Abnormal differentiation of tissue macrophage populations in osteopetrosis (op) mice defective m the production of macrophage colony-stimulanting factor. Amer J. Pathol, 139: 657-67, 1991.        [ Links ]

Naito, M. Macrophage heterogeneity in development and differentiation. Arch. Histol. Cytol, 56:331-51, 1993.        [ Links ]

Otsuka, T.; Thacker, J. D. & Hogge, D. E. The effects of interleukin 6 and interleukin 3 on early hematopoietic events in long term cultures of human marrow. Exp. Hematol, 19:1024-48, 1991.        [ Links ]

Pellizzon, C. H.; Nakaghi, L. S.; Azevedo, A.; Casaletti, L. & Lunardi, L.O. Localization of peroxidase activity in blood mononuclear phagocytes in pacu fish (Piaractus mesopotamicus). J. Submicrosc. Cytol. Pathol, 34:377-9, 2002.        [ Links ]

Sudhakaran, P. R.; Radhika, A. & Jacob, S. S. Monocyte macrophage differentiation in vitro: Fibronectin-dependent upregulation of certain macrophage-specific activities. Glycoconj., 24:49-55, 2007.        [ Links ]

Takahashi, K.; Yamamura, F. & Naito, M. Differentiation, maturation and proliferaton of macrophages m the mouse yolk sac. A light microscopic, enzyme-cytochemical, immunohistochemical and ultraestructural study. J. Leukocyte Biol, 45:87-96, 1989.        [ Links ]

Van Furth, R.; Cohn, Z. A.; Hirsh, J. G.; Humpiy, J. H.; Spector, W. G.; Langevoort, H. L. The mononuclear phagocyte system. A new classification of macrophages, monocytes and their precursors. Bull WHO., 46:845-52, 1972.        [ Links ]

Van Furth, R. Origin and turnover of monocytes and macrophages. Curr Topic. Pathol, 79:125-47, 1989.        [ Links ]

Van Furth, R. Production and migration of monocytes and kinetics of macrophages, 3-12. In "Mononuclear phagocytes .Biology of monocytes and macrophages" ed. by R. van Furth.Kluwer Academic. Publishers.Boston-London, 1992.        [ Links ]

Wiktor-Jedrzejczak, W.; Ratajczak, M. Z.; Ptasznik, A.; Sell, K.W.; Ahmed-Ansari, A. & Ostertag, W. CSF-1 deficiency in the op/op mouse has differential effects on macrophage populations and differentiation stages. Exp. Hematol, 20:1004-10,1992.        [ Links ]

 

Received. 06-01-2007 Accepted: 05-04-2007

Correspondence to:

Dimitrius Leonardo Pitol
Faculdade de Odontología de Ribeirao Preto - USP
Departamento de Morfología, Estomatología e Fisiología
CEP: 14040-904
Av. Café S/N, Ribeirao Preto, SP, Brazil
Email: dimipitol@yahoo.com.br

 

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons