Establishment of a macrophage cell line from adherent peripheral blood mononuclear cells of Catla catla

Abstract

Catla catla, commonly known as catla, is endemic to the riverine system in northern India, Indus plain and adjoining hills of Pakistan, Bangladesh, Nepal and Myanmar. This fish grows best at water temperatures between 25°C and 32°C. Catla, an important culture species, has the highest growth rate amongst Indian major carps. A number of cell lines have been reported from catla (Ishaq Ahmad et al. 2008, 2009a, b). However, cell lines of leucocytic origin have not been reported from this species to the best of our knowledge. Such a cell line would provide useful information regarding functions of leucocytes in teleost species. Macrophages are the multipotent cells of the immune system that play a central role in innate and adaptive immune response in teleosts. In the innate immune system, macrophages are believed to be the principal phagocytic cells in fish (Blazer 1991) and are considered to be important cells in disease resistance. Fish macrophages have a scavenging function (Ganassin and Bols 1998), bactericidal activity (Honda et al. 1986), larvicidal activity (Whyte et al. 1989) and tumouricidal activity (Mulero et al. 1994). These cells also act as professional antigen presenting cells and therefore, are required for eliciting specific immune response (Guidotti and Chisari 2001). In addition, macrophages are capable of producing a large array of proinflammatory, procoagulatory and immune-regulatory products (Ellsaesser and Clem 1994). These cells are distributed in most of the organs of the body, some of which are specialised, fully differentiated cells specific for their resident organ. The macrophage cell lines provide a useful tool for studying functions of these cells. Moreover, such a cell line may contribute to the development and characterisation of macrophage cell markers and may be used as a source of macrophage signal peptides such as cytokines and other factors influencing the growth and maturation of leucocytes. These cell lines can be used for preliminary screening of immunomodulatory substances and therefore can provide an alternative to animal usage for such experiments. A number of fish macrophage cell lines have been developed namely from peripheral blood (Faisal and Ahne 1990; Vallejo et al. 1991; Weyts et al. 1997), spleen (Ganassin and Bols 1998) and kidney (Wang et al. 1995; Dannevig et al. 1997) as well as peritoneal washings (Watanabe et al. 1997). To date, no macrophage cell line is available from any of the commercially important fishes of the country. In the present study, we report the establishment and characterization of a macrophage cell line designated as C. catla macrophage (CCM) cell line from adherent blood mononuclear cells of C. catla. The heparinised blood was collected from caudal vein of a healthy catla weighing 550 g, layered over Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) and centrifuged at 400×g for 30 min. Mononuclear cells (MNCs) were collected, diluted with phosphate-buffered saline (PBS) and centrifuged at 250×g for 10 min. The pellet was suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20 % fetal bovine serum (FBS). The cells were then seeded in 25 cm flasks (Nunc, Roskilde, Denmark) at a concentration of 1×10 cells ml and the flasks were incubated at 28°C. After 24 h, the non-adherent cells were removed and fresh medium was added. The adherent cells showed aggregation and multiplication at several places in the flask (Fig. 1A, B). A D. K. Chaudhary :N. Sood (*) : P. K. Pradhan :A. Singh : P. Punia :G. Rathore National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002 Uttar Pradesh, India e-mail: sood_neeraj@rediffmail.com