Reference: Biol. Bull. 196: 26-33. (February. 1999) ddification of the Phagosome in Crassostrea virginica Hemocytes Following Engulfment of Zymosan AMY E. BEAVEN 1 AND KENNEDY T. PAYNTER* ^Department of Biology and Chesapeake Biological Laboratoiy, University of Maryland, College Park, Mainland 20742 Abstract. Phagocytic hemocytes are responsible for en-gulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reac-tive oxygen intermediate (ROI) producing enzymes in ver-tebrate cells, we measured phagosomal pH in oyster hemo-cytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), con-jugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocytosis of zymo-san. The average pH of 1079 phagosomes within 277 he-mocytes 1 h after phagocytosis of zymosan was 3.9 0.03. Observations of 141 hemocytes with internalized zymosan by light microscopy revealed that, over a 60-min time period, 51% of highly granular hemocytes became partially granular, and 29% became agranular. In addition, 83% of partially granular hemocytes containing zymosan at time = became agranular within 60 min. A comparison revealed that the phagosomes of agranular hemocytes were much more acidic (pH 3.1 0.02) than those of highly granular hemocytes (4.9 0.02: P < 0.05). These values are sig-nificantly lower than most reported in the literature for blood cells from metazoan organisms. Introduction Phagocytic hemocytes are the primary cells involved in the oyster's internal defense response against invading or-ganisms (Alvarez et at, 1989; McCormick-Ray and Howard, 1991). Oyster hemocytes morphologically resem-ble vertebrate monocytes and macrophages (Anderson, Received 9 July 1998; accepted 25 November 1998. * To whom correspondence should be addressed. E-mail: paynter(S' mees.umd.edu 1981; Adema et ul.. 1991) and, like these immune cells, have the ability to recognize, engulf, and internally degrade a variety of particles within the hemolymph and tissue (Foley and Cheng. 1975). The phagocytic process begins with recognition of a foreign particle and its engulfment into a phagosome. Once sequestered within the phagosome, the particle is subjected to various hydrolytic enzymes and reactive oxygen intermediates (ROIs), which aid in destroy-ing the particle (Adema et al, 1991; Anderson et at, 1995). Many of the enzymes contained within the hemocytes of bivalves, including lysozyme (Rodrick and Cheng, 1974), /3-glucuronidase (Cheng and Rodrick, 1975). and myeloper-oxidase (MPO) (Wojcik and Paynter. 1995), have acidic pH optima ranging from pH 4.5-5.5. In addition, phagocytosis of foreign organisms by vertebrate macrophages and poly-morphonuclear leukocytes (PMNs), as well as by hemo-cytes of Mytilus edulis, is accompanied by rapid acidifica-tion of the phagosome lumen (Jensen and Bainton, 1973; Rathman et at. 1996; Kroschinski and Renwrantz, 1988). This suggests that, as in vertebrate phagocytes, phagosomal acidification may be required for activation of hydrolytic and ROI-producing enzymes in bivalve hemocytes, and may therefore be essential for the hemocyte antimicrobial defense response in molluscs. Over the past few decades, the protozoan parasite Perk-insus marinus has caused widespread oyster mortalities in the Atlantic coast region. Although oyster hemocytes readily engulf P. marinus, it appears that the parasite may be able to survive within the hemocyte (Chu and La Peyre. 1993). In fact, many microorganisms can escape intracellu-lar destruction by blocking phagosome-lysosome fusion (Jones and Hirsch. 1972; Horwitz. 1983; Mauel, 1984) or phagosomal acidification (Horwitz and Maxrield, 1984; Black etui., 1986; Sibley et al., 1985: Crowle et at, 1991), or by adapting to the acidic environment of the phagosome 26
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