The large yellow–brown cytoplasmic phagosomes seen in the Kupffer cells of the juvenile Nile crocodile were positive for both melanin and hemosiderin, a feature also described in other reptiles (Jacobson, 2007). Hemosiderin is usually seen in cells responsible for the breakdown of effete red blood cells and consists, among others, of ferritin and glycoproteins (Smith and Laurie, 2010). Ceroid pigment, lipofuscin pigment and glycoproteins are all PAS positive (Schaffner, 1998, Smith and Laurie, 2010) and may account for the third pink-staining element observed in the phagosomes. Ceroid and lipofuscin are both positive with the PAS-D reaction while glycoproteins are negative, excluding it as the third element. The rate of accumulation of ceroid and lipofuscin differs, lipofuscin accumulates slowly with age, whereas the formation of ceroid pigment may occur rapidly at any age (Ghadially, 1988; Yin, 1996; Porta, 2002). We, therefore, suggest that the third element consists of ceroid pigment as the livers studied were obtained from juvenile crocodiles. Ceroid pigment occurs as residual bodies due to lysosomal activity (Ghadially, 1988) and represents degraded cellular debris (Schaffner, 1998). Ceroid is also described as a type of lipofuscin (Smith and Laurie, 2010), closely related to lipofuscin or even consisting of the same material in different oxidative stages (Ghadially, 1988).
Transmission Electron Microscopy
The location and positioning of Kupffer cells has been noted in several publications (Junqueira et al., 1975; Wisse and Knook, 1977; Phillips et al., 1987; Wisse et al., 1996; Ross et al., 2003; Ghoddusi and Kelly, 2004) including being found inside the sinusoidal lumen, forming part of the sinusoidal lining, embedded in the endothelial lining, located as intraluminal macrophages or existing as fixed cells. Elias (1955) mentioned that the sinusoids of vertebrates were lined by potential phagocytes that never bridge the lumen and Naito et al. (2004) described Kupffer cells in mammals as being attached to the surfaces of fenestrated sinusoidal endothelial cells. In reptilian sauropsids, the Kupffer cells were attached to the endothelial surfaces of the sinusoids (Schaffner, 1998). They were present in the sinusoidal lumen and in the space of Disse of the West African crocodile (Storch et al., 1989). The Kupffer cells of the juvenile Nile crocodile liver did not reflect these findings as they were found in several positions, not only in the sinusoidal spaces but also bridging the sinusoidal lumen regularly, as well as occupying perisinusoidal locations. In the Nile crocodile, these cells can, therefore, be regarded as being highly mobile and not fixed cells.
Some authors differentiate between Kupffer cells and melanomacrophages which have also been referred to as “pigment cells” or “specialized Kupffer cells” when they contain melanosomes (Storch et al., 1989; Schaffner, 1998; McClellan-Green et al., 2006; Jacobson, 2007). The juvenile Nile crocodile liver contained numerous but isolated pigmented Kupffer cells as opposed to the collections of specialized Kupffer cells and pigment cells that had been noted in other reptiles (Hack and Helmy, 1964; Henninger, 1982; Henninger and Beresford, 1990; McClellan-Green et al., 2006; Jacobson, 2007). Storch (1989) distinguished between Kupffer cells and pigment cells in the West African crocodile describing them as two separate cell populations. Schaffner (1998) mentioned a second pigmented macrophage in addition to Kupffer cells in a perisinusoidal location in reptiles and Jacobson (2007) termed similar cells melanomacrophages. The present study identified matching ultrastructural features, including the tubular structures (tubulosomes), in the “pigmented” cells in all the mentioned locations, that is, sinusoidal, perisinusoidal and among hepatocytes, and concluded that these cells are all Kupffer cells. This concurs with findings in amphibians that the pigment cells in livers are Kupffer cells (Sichel et al., 1997).
The phagocytosis of apoptotic cells by Kupffer cells forms part of the regulatory processes of inflammation and immunity (Dini et al., 2002). Two pigments, namely melanin and hemosiderin, coexisted in the phagosomes of the Kupffer cells of the Nile crocodile. Hemosiderin is derived from the breakdown of haemoglobin and catalyses the formation of free superoxide radicals that are trapped by melanin (Beresford, 1987; Sichel et al., 2002; McClellan-Green et al., 2006). Kupffer cells of reptiles and amphibians are able to synthesize melanin (Henninger and Beresford, 1990; Kalashnikova 1992; Corsaro et al., 2000; Sichel et al., 2002) which is deposited in melanosomes. Melanosomes are specialised lysosomes (Orlow, 1995) and the production of melanin acts as a defence mechanism and as protection in response to the possibly harmful hemosiderin (Scalia et al., 1988, 1990; Henninger and Beresford, 1990). The phagocytic process in the Kupffer cells of the Nile crocodile is consequently similar to that taking place in other reptiles and amphibians. The ultrastructural morphology of lipofuscin differs from that of the ceroid pigment present in the phagosomes of the Kupffer cells of the Nile crocodile. Lipofuscin consists of membrane-bound structures containing lipid droplets intermingled with electron-dense material (Ghadially, 1988), whereas the ceroid in the current study consisted of nonmembrane bound, pleomorphic spherical structures of medium electron density without the characteristic lipid droplets. This finding concurs with the light microscopical conclusion that the third element present in the phagosomes is ceroid pigment. Myelinosomes are additionally found in cells that have phagocytosed erythrocytes (Ghadially, 1988).
No structures comparable to the conspicuous tubular cytoplasmic organelles (tubulosomes) with filamentous or crystalline interiors found in the Kupffer cells could be traced in the literature. Storch et al. 1989 mentioned the presence of small membrane-bound vesicles of unknown origin in the Kupffer cells of the West African crocodile, however, no images were published to illustrate their structure. Boler (1969) described the ultrastructure of cytosomes in canine Kupffer cells, but these were larger than the tubulosomes, spherical in form and contained tubular structures. It is hypothesised that these organelles (tubulosomes), given their close relationship with the phagosomes, are specialised lysosomes involved in melanin synthesis and dedicated to the breakdown of phagosomal contents. Mitochondria are often closely associated with other organelles that require adenosine triphosphate (Lehninger, 1965, in Ghadially, 1988) for energy transfer, which may explain the close relationship between the tubulosomes and the mitochondria in the Kupffer cell cytoplasm of the Nile crocodile.
Lipid droplets were present in the Kupffer cells in this study, as well as in Kupffer cells of the lizard (Taira and Mutoh, 1981), but were absent in mammalian studies (McCuskey and McCuskey, 1990). Several of the mentioned publications refer to vermiform processes (invaginations of the cell membrane) formed by Kupffer cell membranes in mammals, but none were found in the liver of the juvenile Nile crocodile.