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Lack of acid phosphatase release during in vitro phagocytosis by coelomocytes of the earthworm, Lumbricus terrestris
JOURNAL
OF INVERTEBRATE
PATHOLOGY
39,
116-
118 (1982)
Lack of Acid Phosphatase Release during In Vitro Phagocytosis Coelomocytes of the Earthworm, Lumbricus terrestris The extracellular release of lysosomal and other granule-contained hydrolytic enzymes is associated with phagocytosis by vertebrate monocytes, macrophages, and polymorphonuclear leukocytes (P. M. Henson, J. Ztntnunol. 107, 1535- 1556, 1971; J. Schnyder and M. Baggliolini, J. Exp. Med. 148. 1449-1456, 1978) and by leukocytes of the invertebrate species Biomphalaria glabrcrra and Mercenaricr tnercetzaria (T. C. Cheng, G. E. Rodrick, D. A. Foley, and S. A. Koehler, J. Itzvertebr. Pathol. 25, 261-265, 1975; D. A. Foley and T. C. Cheng. J. Ztzvertebr. Puthol. 29, 321-325, 1977; T. C. Cheng, V. G. Guida, and P. L. Gerhart, J. Itzvertebr. Parhol. 32, 297-302, 1978; T. C. Cheng and M. S. Butler, J. Itzljertebr. Pathol. 34. 119- 124, 1979). Hydrolytic enzyme release by vertebrate phagocytes is believed to be instrumental in the pathogenesis of inflammation (M. Baggiolini, J. Schnyder, and U. Bretz, Advarz. Injlam. Res. 1, 263-272, 1979) while in invertebrates it may be associated with defense against invading pathogens (T. C. Cheng, V. G. Guida, and P. L. Gerhart, lot. cit., 1978). Coelomocytes of the earthworm, Lurnbricus ten’estris, are, with only minor exceptions, phagocytic (E. A. Stein, R. R. Avtalion, and E. L. Cooper, J. Morph&. 153, 467-477, 1977) and contain the lysosomal enzymes acid phosphatase and pglucuronidase (E. A. Stein and E. L. Cooper, Histochetn. J. 10, 657-678, 1978). Since phagocytosis is an important defense mechanism in Lutnbricus, and lysosomal enzyme release may also be involved in defense processes, we investigated the possible release of acid phosphatase (AcPase), a lysosomal enzyme marker, from L14172116 0022-2011/82/010116-03$01.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
by
coelomocytes during in vitro phagocytosis of yeast. We concomitantly measured the release of lactic dehydrogenase (LDH). a cytoplasmic enzyme, to monitor any enzyme release resulting from cell injury or death. Coelomic fluid, containing coelomocytes, was removed from adult Lutnbricus ferrestris by intracoelomic puncture with a sharpened Pasteur pipette. Coelomocytes were washed twice, centrifuging at 15Og for 5 min, then resuspended in sufficient Lumbrims balanced salt solution (LBSS) (E. A. Stein and E. L. Cooper, Dev. Camp. Itw murd. 5, 415-425, 1981) to give a coelomocyte concentration of -3 x 10” cells/ml. Cell viability, as determined by trypan blue dye exclusion, was 90-97%. The composition of LBSS, in millimoles per liter, is: NaCl, 71.5; KCl, 4.8; CaCl,, 3.8; MgS04, 1.1; KH,POI, 0.4; Na,HPO,, 0.4; NaHCO,, 4.2. The pH is adjusted to 7.3. The cell suspension was divided into three aliquots of 0.3 ml each. The first aliquot was processed for intracellular AcPase and LDH. Cells were separated from suspending fluid, lysed with 0.2% Triton X-100 (final concentration), centrifuged, and the particulate-free lysate analyzed for enzymatic activity. The suspending fluid was also analyzed for AcPase. To the second aliquot was added washed, heat-killed yeast (Saccharotnyces cerevisiae) in a yeast: coelomocyte ratio of 30: 1. The coelomocyteyeast suspension, along with the third aliquot of coelomocytes alone in LBSS (to provide an adherent, nonphagocytic control), was then placed in 16-mm-diameter wells of 24-well tissue culture plates and the plates set into humidified boxes at 15°C. After 1 hr of incubation, the supernatant fluid was pipetted bricus
117
NOTES
from each of the chambers, centrifuged to remove particulate matter, and analyzed for enzymatic activity. AcPase was assayed at 34”C, pH 5.8, by the method of H. U. Bergmeyer (“Methods of Enzymatic Analysis,” Vol. 2, pp. 856-859, Academic Press, New York, 1974) using p-nitrophenylphosphate as substrate. LDH was assayed by the method of L. Berger and D. Broida (Technical Bulletin No. 500, Sigma Chemical Co.. St. Louis, MO.). A series of parallel yeast-containing cultures were concurrently incubated to determine phagocytic rate and coelomocyte viability. After 1 hr, 84-92% of coelomocytes were viable by trypan blue dye exclusion, while Wright’s staining showed that 61-70% of coelomocytes contained phagocytosed yeast with an overall average of 2.6-3.8 yeast per cell. Phagocytosed yeast were distinguished from those adhering to the cell surface by the presence of a thin, nonstaining rim around all internalized yeast. Acid phosphatase was not released in detectable amounts by Lumhricus coelomocytes during phagocytosis. Although small amounts of AcPase were present in supernatants from phagocytosing cells, they were not significantly greater (P G 0.05) than supernatants from adherent, nonphagocytosing coelomocytes, nor from preadherent supernatants (Table 1). AcPase
percentages were essentially equal to those of LDH, suggesting that the small amounts (
TABLE
1 Enzyme
Acid phosphatase Enzyme assayed from: Lysed coelomocytes Preadherent supernatant Adherent, nonphagocytic supernatant Phagocytic supernatant
Lactic dehydrogenase
International enzyme units
Percentage of total enzyme”
Sigma enzyme units
Percentage of total enzyme”
27.9 k 3.8 (19)” 1.4 + 0.2 (6) 2.2 f 0.5 (19)
95.2 (19) 4.8 (6) 7.5 (19)
1629 t 477 (8) ND’ 105 + 62 (8)
93.9 (8) ND 6.1 (8)
2.6 k 0.5 (19)
8.9 (19)
148 + 65 (8)
8.5 (8)
” Total enzyme calculated as: AcPase-lysed coelomocytes plus preadherent supernatant; LDH-lysed coelomocytes plus adherent supernatant. ’ All values adjusted to 1 X 106 cells, or supernatants from 1 x 106:cells. and given as mean k SEM (n). c Not done.
118
NOTES
ing such constant phagocytic activity, damage to self tissues might result. Whether Lumbricus and Crassostrea are unusual cases, or whether lack of lysosomal enzyme release is more widespread among invertebrates than hitherto realized, requires further investigation. KEY WORDS: Lumbricus terrestris; earthworm; coelomocytes; lysosomal enzymes; acid phosphatase; lactic dehydrogenase; phagocytosis.
Supported by NIH A11597601.
Grants
1ROl
HD09333-05
and
E. A. STEIN D. H. MARKS E. L. COOPER Depurtment of Anatomy School of Medicine University of California Los Angeles, California 90024 Received July 11, 1980
OF INVERTEBRATE
PATHOLOGY
39,
116-
118 (1982)
Lack of Acid Phosphatase Release during In Vitro Phagocytosis Coelomocytes of the Earthworm, Lumbricus terrestris The extracellular release of lysosomal and other granule-contained hydrolytic enzymes is associated with phagocytosis by vertebrate monocytes, macrophages, and polymorphonuclear leukocytes (P. M. Henson, J. Ztntnunol. 107, 1535- 1556, 1971; J. Schnyder and M. Baggliolini, J. Exp. Med. 148. 1449-1456, 1978) and by leukocytes of the invertebrate species Biomphalaria glabrcrra and Mercenaricr tnercetzaria (T. C. Cheng, G. E. Rodrick, D. A. Foley, and S. A. Koehler, J. Itzvertebr. Pathol. 25, 261-265, 1975; D. A. Foley and T. C. Cheng. J. Ztzvertebr. Puthol. 29, 321-325, 1977; T. C. Cheng, V. G. Guida, and P. L. Gerhart, J. Itzvertebr. Parhol. 32, 297-302, 1978; T. C. Cheng and M. S. Butler, J. Itzljertebr. Pathol. 34. 119- 124, 1979). Hydrolytic enzyme release by vertebrate phagocytes is believed to be instrumental in the pathogenesis of inflammation (M. Baggiolini, J. Schnyder, and U. Bretz, Advarz. Injlam. Res. 1, 263-272, 1979) while in invertebrates it may be associated with defense against invading pathogens (T. C. Cheng, V. G. Guida, and P. L. Gerhart, lot. cit., 1978). Coelomocytes of the earthworm, Lurnbricus ten’estris, are, with only minor exceptions, phagocytic (E. A. Stein, R. R. Avtalion, and E. L. Cooper, J. Morph&. 153, 467-477, 1977) and contain the lysosomal enzymes acid phosphatase and pglucuronidase (E. A. Stein and E. L. Cooper, Histochetn. J. 10, 657-678, 1978). Since phagocytosis is an important defense mechanism in Lutnbricus, and lysosomal enzyme release may also be involved in defense processes, we investigated the possible release of acid phosphatase (AcPase), a lysosomal enzyme marker, from L14172116 0022-2011/82/010116-03$01.00/O Copyright All rights
Q 1982 by Academic Press, Inc. of reproduction in any form reserved.
by
coelomocytes during in vitro phagocytosis of yeast. We concomitantly measured the release of lactic dehydrogenase (LDH). a cytoplasmic enzyme, to monitor any enzyme release resulting from cell injury or death. Coelomic fluid, containing coelomocytes, was removed from adult Lutnbricus ferrestris by intracoelomic puncture with a sharpened Pasteur pipette. Coelomocytes were washed twice, centrifuging at 15Og for 5 min, then resuspended in sufficient Lumbrims balanced salt solution (LBSS) (E. A. Stein and E. L. Cooper, Dev. Camp. Itw murd. 5, 415-425, 1981) to give a coelomocyte concentration of -3 x 10” cells/ml. Cell viability, as determined by trypan blue dye exclusion, was 90-97%. The composition of LBSS, in millimoles per liter, is: NaCl, 71.5; KCl, 4.8; CaCl,, 3.8; MgS04, 1.1; KH,POI, 0.4; Na,HPO,, 0.4; NaHCO,, 4.2. The pH is adjusted to 7.3. The cell suspension was divided into three aliquots of 0.3 ml each. The first aliquot was processed for intracellular AcPase and LDH. Cells were separated from suspending fluid, lysed with 0.2% Triton X-100 (final concentration), centrifuged, and the particulate-free lysate analyzed for enzymatic activity. The suspending fluid was also analyzed for AcPase. To the second aliquot was added washed, heat-killed yeast (Saccharotnyces cerevisiae) in a yeast: coelomocyte ratio of 30: 1. The coelomocyteyeast suspension, along with the third aliquot of coelomocytes alone in LBSS (to provide an adherent, nonphagocytic control), was then placed in 16-mm-diameter wells of 24-well tissue culture plates and the plates set into humidified boxes at 15°C. After 1 hr of incubation, the supernatant fluid was pipetted bricus
117
NOTES
from each of the chambers, centrifuged to remove particulate matter, and analyzed for enzymatic activity. AcPase was assayed at 34”C, pH 5.8, by the method of H. U. Bergmeyer (“Methods of Enzymatic Analysis,” Vol. 2, pp. 856-859, Academic Press, New York, 1974) using p-nitrophenylphosphate as substrate. LDH was assayed by the method of L. Berger and D. Broida (Technical Bulletin No. 500, Sigma Chemical Co.. St. Louis, MO.). A series of parallel yeast-containing cultures were concurrently incubated to determine phagocytic rate and coelomocyte viability. After 1 hr, 84-92% of coelomocytes were viable by trypan blue dye exclusion, while Wright’s staining showed that 61-70% of coelomocytes contained phagocytosed yeast with an overall average of 2.6-3.8 yeast per cell. Phagocytosed yeast were distinguished from those adhering to the cell surface by the presence of a thin, nonstaining rim around all internalized yeast. Acid phosphatase was not released in detectable amounts by Lumhricus coelomocytes during phagocytosis. Although small amounts of AcPase were present in supernatants from phagocytosing cells, they were not significantly greater (P G 0.05) than supernatants from adherent, nonphagocytosing coelomocytes, nor from preadherent supernatants (Table 1). AcPase
percentages were essentially equal to those of LDH, suggesting that the small amounts (
TABLE
1 Enzyme
Acid phosphatase Enzyme assayed from: Lysed coelomocytes Preadherent supernatant Adherent, nonphagocytic supernatant Phagocytic supernatant
Lactic dehydrogenase
International enzyme units
Percentage of total enzyme”
Sigma enzyme units
Percentage of total enzyme”
27.9 k 3.8 (19)” 1.4 + 0.2 (6) 2.2 f 0.5 (19)
95.2 (19) 4.8 (6) 7.5 (19)
1629 t 477 (8) ND’ 105 + 62 (8)
93.9 (8) ND 6.1 (8)
2.6 k 0.5 (19)
8.9 (19)
148 + 65 (8)
8.5 (8)
” Total enzyme calculated as: AcPase-lysed coelomocytes plus preadherent supernatant; LDH-lysed coelomocytes plus adherent supernatant. ’ All values adjusted to 1 X 106 cells, or supernatants from 1 x 106:cells. and given as mean k SEM (n). c Not done.
118
NOTES
ing such constant phagocytic activity, damage to self tissues might result. Whether Lumbricus and Crassostrea are unusual cases, or whether lack of lysosomal enzyme release is more widespread among invertebrates than hitherto realized, requires further investigation. KEY WORDS: Lumbricus terrestris; earthworm; coelomocytes; lysosomal enzymes; acid phosphatase; lactic dehydrogenase; phagocytosis.
Supported by NIH A11597601.
Grants
1ROl
HD09333-05
and
E. A. STEIN D. H. MARKS E. L. COOPER Depurtment of Anatomy School of Medicine University of California Los Angeles, California 90024 Received July 11, 1980