J. COMP.
PATH.
1977.
VOL.
43
87.
ELECTRON MICROSCOPIC INTERACTION OF CASEIN GLOBULES WITH BOVINE LEUCOCYTES DURING STAPHYLOCOCCI
OBSERVATIONS MICELLES AND POLYMORPHONUCLEAR THE PHAGOCYTOSIS IN MILK
OF MILK
THE FAT OF
BY
M. W. National
B. E.
RUSSELL,*
Institute
for
and B.
BROOKER
Research in Dairying,
Uniuersi(v
of Reading,
REITER
Reading,
England
INTRODUCTION
Bovine mastitis is characterized by an influx of polymorphonuclear leucocytes (PMN) into the milk. A pre-existing leucocytosis or one induced by endotoxin, can protect the udder against experimental challenge with Staphylococcus aureus (Newbould and Neave, 1965; Reiter and Oram, 1967) and the interaction between staphylococci and PMN may be critical for the outcome of an infection (Anderson, 1975). However, the influx of PMN in response to infection does not always eliminate the pathogens which may persist, together with large numbers of PMN, for long periods in the milk. We have shown that PMN isolated from cows’ blood and suspended in synthetic medium supplemented with normal bovine serum readily ingest and kill a mastitis strain of S. aureus, whereas PMN isolated from milk, or blood PMN suspended in separated milk, ingest and kill S. aureus much more slowly (Russell and Reiter, 1975). The cause of this impairment was attributed to casein, which inhibited phagocytosis in vitro, and reversed the bactericidal activities of PMN lysates, histones and the lactoperoxidase-hydrogen peroxide-potassium iodide system (Russell, Brooker and Reiter, 1976). We have shown, by means of immunofluorescence microscopy, that casein was associated with the surface of washed milk PMN, and with blood PMN after exposure to milk (Russell and Reiter, 1975). Furthermore, casein appeared to be ingested by PMN during the phagocytosis of staphylococci in milk. However, the limits of resolution did not permit detailed observation of how casein was taken up by PMN, or of how it probably interacted to impair the intracellular bactericidal mechanisms of PMN. The ultrastructure of PRN and the morphological events of phagocytosis are well documented (ZuckerFranklin and Hirsch, 1964; Davis and Douglas, 1972). Furthermore, the electron microscope resolves casein characteristically as electron-dense granular micelles of up to about O-3 pm in diameter (Schmidt and Buchheim, 1970; Tan, Livingstone, Gilbert and Young, 1971). It was therefore felt that this technique could be used to examine the interaction of casein with PMN during phagocytosis in milk. In this paper we present electron microscopic evidence for the ingestion of casein and fat globules by bovine PMN. and
* Present Dental
address: Schools,
Department of Oral London SEI Qrr.
Immunology
and
Microbiology,
Guy’s
Hospital
Rledical
44
M. W.
RUSSELL
MATERIALS
AND
et al. METHODS
Preparation of suspensions of PMN and staphylococci. PMN were isolated from bovine blood or milk as described previously (Russell and Reiter, 1975). Suspensions of PMN (10’ cells per ml) in milk or in synthetic milk-salts-lactose solution (MSL) plus 1 per cent normal bovine serum were mixed with 5’. aureus (10s colony-forming units/ml) in siliconized glass vials and incubated with continuous gentle agitation at 37 “C. (Russell and Reiter, 1975). When milk was used as the medium, in most experiments it was first separated to remove the cream by centrifugation at 2000 g for 10 min. No significantly different observations resulted if whole milk was used. In some experiments, after 15 min incubation, the PMN were separated from free staphylococci by centrifuging at 50g for 5 min at 4 “C and re-suspended in fresh medium for further incubation so that the post-phagocytic events could be observed without the complication of continued phagocytosis. Electron microscopy. PMN were sedimented by centrifugation at 50 g for 5 min at 4 “C and fixed by suspending in 2.5 per cent glutaraldehyde in 0.2 M cacodylate buffer pH 7.4. The cells were treated with 1 per cent osmium tetroxide, followed by 1 per cent uranyl acetate in 25 per cent ethanol, dehydrated in ethanol and embedded in Araldite. The sections were stained with uranyl acetate and lead citrate, and examined in a Hitachi HU 11E electron microscope. RESULTS
Resting PMN The structure of PMN freshly isolated from bovine blood is illustrated in Fig. 1. The cytoplasm was packed with membrane-bound granules or lysosomes (L) typical of PMN of other species. The structure of PMN isolated from milk is shown in Fig. 2. Large milk fat globules (FG), identified by the presence of
Figs 1 to 8. Electron micrographs of bovine polymorphonuclear leucocytcs (PMN). CM, Casein micelles (putative); FG, Fat globule; GB, Golgi body; L, Lysosome (both azurophil and with specific granules); M, Mitochondrion; N, Nuclear lobe; PS, Pseudopodium; PV, Phagocytic vacuole (phagosome) ; S, Staphylococcus. Fig. 1. Blood PMN, freshly isolated. x 16 000.
PHAGOCYTOSIS
OF STAPHYLOCOCCI
IiV JIILK
4.5
a characteristic membrane (Wooding, 1971) were prominent in the cytoplasm. Fig. 2 (’msttsx ) sh ows a PMN fixed in the act of ingesting a fat globule with a distinct membrane. Thr cytoplasm of milk PMN contained noticeably fewer
t ’
Fig.
)I
2. Milk PMN, freshly globule. x 10 000.
isolated.
% 11 200.
(Inset)
: milk
PMN,
freshly
isolated,
ingesting
a I~I
lysosomes than blood PMN, but there were also some small, apparently empty membrane-bound vacuoles and myelin figures. Some small dense granules of size and morphology resembling casein micelles (C:M) were seen near to
Fig.
3. Blood
PMN
after
incubation
with
S. aweus in separated
milk
for 15 min.
s 16 000.
46
M. W.
RUSSELL
et a/.
the cell membrane, sometimes in association with pseudopodia, similar granules were visible in small cytoplasmic vacuoles.
and a few
PhagoGytosis by Blood PALN The early events of phagocytosis are illustrated in Fig. 3 in which the encirclement of staphylococci (S) by pseudopodia to form phagosomes ( PV can be inferred. After prolonged incubation in the synthetic medium (M E!lL)
Fig. 4. Blood
PMN
Fig. 5. Milk
after
PMN
incubation
after
with
incubation
S. awxs
with
in MSI,
plus
1 per cent serum
S. aweus in separated
milk
for 2 h. % 12! 000.
for 2 h. x 11 200.
PHAGOCYTOSIS
plus serum, staphylococci, membranous such cells was
OF STAPHYLOCOCCI
47
IN MILK
blood PMN contained several vacuoles in which were seen mostly in various states of disintegration, together with other and granular material (Fig. 4). The vacuolated cytoplasm of usually depleted of lysosomes.
Plmgocytosis by Milk
PMJV
Milk PMN incubated with staphylococci in separated milk displayed a variety of features (Figs 5 and 6). Vacuoles containing staphyIococci were commonly
Fig. 6. Milk
PMN
after
incubation
with
S. aumw in separated
milk
for 2 h. x 12 000.
seen, and most of the organisms appeared to be structurally intact. Some staphylococci were observed to possess one complete cross-wall with a second wall at right angles and incompletely formed (Fig. 5), suggesting rapid growth and division. Other vacuoles contained granular material, some in a diffuse fornl, some in the form of dense discrete granules resembling casein micelles (C&l). Similar particles were present in association with the cell membrane of some PMD;. Vacuoles were seldom seen containing both staphylococci and casein micelles, although this feature was intentionally looked for. As in resting milk PMN, some empty vacuoles, or vacuoles containing membranous material, Some phagocytosing milk PMN appcxrcd to and myelin figures were seen. be depleted of lysosomes (Fig. 6), while others still contained a relatively large number (Fig. 5). Few milk PMN contained fat globules after 2 h incubation in separated milk. Blood PMN incubated with staphylococci in whole milk displayed features similar to those of phagocytosing milk PMN (Fig. 7). Intracellular staphylococci were usuaIly intact in structure and some appeared to be actively dividing. n’umerous vacuoles containing membranous and granular material, some of it
48
M.
W.
RUSSELL
et
al.
resembling casein micelles, were seen. Some cells possessed several large pseudopodia extending into the medium around various particles. In some instances, adjacent pseudopodia were observed encircling a particle and in intimate contact without appearing to fuse to form a typical phagosome (Fig. 8).
Fig.
7. Blood
Fig. 8. Blood
PMN
PMN
after
after
incubation
incubation
with
with
S. HUMUS in whole
S. aureus in separated
milk
milk
for 1 h. x 12 000.
for 2 h.
x
11 200.
DISCUSSION
The structural study was similar
morphology of bovine blood PMN as seen in the present to that of other species, and agreed well with the structure
PHAGOCYTOSIS
OF
STAPHYLOCOCCI
IN
MILK
49
of bovine PMN reported by Davis and Douglas (1972). The morphological events of “normal” phagocytosis, i.e. phagocytosis by blood PMN in synthetic medium plus serum, conform to the generally accepted picture of this process (Zucker-Franklin and Hirsch, 1964). Furthermore, the descriptive observations of the phagocytosis of staphylococci by blood and milk PMN in milk or synthetic medium are consistent with the quantitative results reported preLiously (Russell and Reiter, 1975). Blood PMN in MSL plus serum appeared to ingest the organisms readily, and the organisms were largely destroyed by the intracellular bactericidal systems and hydrolytic enzymes (Hegner, 1968). ‘The concomitant disappearance of the abundant lysosomal granules, presumably by fusion with the phagosomes, was an essential feature. In contrast, milk PMN did not appear to be so capable of killing ingested staphylococci, siucc many intact and dividing organisms were present in the phagosomes of milk PMN after 2 h. Since this study was inherently qualitative, it was not possible to confirm the quantitatively diminished phagocytic ingestion of staphylococci which we have already demonstrated (Russell and Reiter, 1975). The ratio of organisms to PMN was increased to 10 to 1 to facilitate observation of staphylococci in the electron microscope and this may have increased the numbers of bacteria ingested. The morphology of freshly isolated milk PMN presented some new features. Ingested milk fat globules were readily identifiable and could also be seen in suitably stained light microscopic preparations (unpublished observations). They have also been reported by Paape, Guidry, Kirk and Bolt (1975). The small dense granules seen in vacuoles and near to the surface membrane in milk PMN and blood PMN after incubation in milk had the morphology expected of casein micelles (Schmidt and Buchheim, 1970; Tan, Livingstone, Gilbert and Young, 1971), ahhough they have not been positively identified in the present investigation. The distribution of these granules agreed with the distribution of casein in similar preparations of PhSN examined by immunofluorescence microscopy, using casein-specific antiserum (Russell and Reiter, 1975). However, up to about IO per cent of the casein in cows’ milk may be present, not as particulate micelles, but in solution (Rose, 1968). Immunofluorescence cannot distinguish between the two forms, but electron microscopy resolves only the micelles. It may therefore be that molecular casein was present in association with PMN in addition to the observed micelles and also in other locations where micelles were not observed. The phagocytosis of fat globules by PMN in whole milk might be expected to inhibit the phagocytosis of staphylococci. This has been demonstrated b) Paape et al. (1975) using suspensions containing up to 40 per cent milk or its equivalent in cream. However, in experiments in which PMN were suspended in whole or separated milk, only a small diminution in phagocytosis was attributable to the presence of fat globules (Russell, 1973). When cream was added to PMN in suspension in synthetic medium, a greater inhibitory effect was observed, but microscopic examination showed that the fat globules had coalesced around the PMN. Since in whole milk, inhibition due to casein was much greater than that due to fat globules, it is possibIe that the inhibitory effect of casein is lost more easily on dilution than that of fat globules. 1)
50
M.
W.
RUSSELL
et d.
The disappearance of fat globules from milk PMN after 2 h incubation in separated milk was striking. It is not clear whether this was due to exocytosis, digestion by lipase (Elsbach and Kayden, 1965) or lipid peroxidation (Stossel, Mason and Smith, 1974). Our earlier immunofluorescence studies, which indicated that casein was ingested during the phagocytosis of staphylococci by PMN suspended in separated milk, suggested the possibility that the inclusion of casein within the phagosomes containing staphylococci would result in neutralization of the intracellular bactericidal mechanisms (Russell, Brooker and Reiter, 1976). However, casein micelles were seldom seen in phagosomes containing staphylococci, although it is possible that soluble casein was present. The evidence of ingestion of casein micelles, sometimes in apparently large quantities, does, however, suggest another possibility. If lysosomes are discharged into phagosomes containing casein, an adequate number might not be available for other phagosomes containing staphylococci. Studies using blood PMN incubated in separated milk with staphylococci were consistent with the interpretation that the simultaneous phagocytosis of staphylococci and casein results in diminished killing of the organisms by competition for and neutralization of the lysosomal bactericidal agents. Milk PMN which were exposed to the effects of casein in vivo appeared, on isolation, to contain fewer lysosomes than blood PMN. The small apparently empty vacuoles and myelin figures in milk PMN might represent the remains of casein phagosomes. There is a further possibility that exposure to casein in vivo or in vitro might result in activation of the enhanced metabolism of phagocytosis (Iyer, Islam and Quastel, 1961). A consequence of this might be that the energy reserves of milk PMN become depleted (Naidu and Newbould, 1973) or that peroxide and superoxide radicals toxic products such as lactate, hydrogen accumulate. PMN are known to be so activated on contact with agents which damage the surface membrane, such as chemotactic fragments of complement (Goetzel and Austen, 1974; Tedesco, Trani, Soranzo and Patriarca, 1975), concanavalin A (Romeo, Zabucchi and Rossi, 1973) and endotoxin (Strauss and Stetson, 1960). We have previously shown the association of casein with the PMN surface by means of immunofluorescence. An effect of casein on the PMN membrane can also be inferred from its chemotactic property which, however, has been attributed rather to soluble casein monomers and the a and p components (Wilkinson, 1972). Casein micelles are apparently not chemotactic, but it may be that their particulate nature is essential for their phagocytosis by PMN, which are not known normally to engage in the pinocytosis of liquids. SUMMARY
Polymorphonuclear leucocytes (PMN) isolated from bovine blood or milk were examined in the electron microscope before and after incubation with staphylococci in either milk or a synthetic salts medium supplemented with serum. Milk PMN on isolation contained prominent milk fat globules and a few small vacuoles containing granules resembling casein micelles. Neither of these were seen in blood PMN. Milk PMN contained fewer lysosomes than
PHAGOCYTOSIS
OF STAPHYLOCOCCI
IN
MILK
51
blood PMN, and it is suggested that phagocytosis of fat globules and cascin micelles in vivo, with consequent degranulation, accounts for their previously observed deficient phagocytosis of staphylococci. On incubation in milk in vitro with staphylococci, both blood PMN and milk PMN ingested apparently large quantities of granules resembling casein micelles, forming phagocytic vacuoles separate from those containing staphylococci. The ingested staphylococci appeared largely intact, despite the loss of’ lysosomes from the PMN cytoplasm. In contrast, the staphylococci ingested by blood PMN in synthetic medium appeared to be dead after 2 h. These observations support previous findings that casein inhibits the bactericidal activities of PMiX, and suggest possible mechanisms for this inhibition.
ACKNOWLEDGMENTS
We should like to thank Dr S. D. Douglas, University School, Minneapolis, U.S.A. for his review and comments graphs.
of Minnesota Medical on the electron micro-
REFERENCES
Anderson, J. C. (1975). Pathogenesis of experimental mastitis in the mouse caused by a strain of Staphylococcus aureus of low virulence and its modification by endotoxin. Journal of Comparative Pathology, 85, 531-538. Davis, W. C., and Douglas, S. D. (1972). Defective granule formation and function in the Chediak-Higashi syndrome in man and animals. Seminars in Haematology, 9, 43 l-450. Elsbach, P., and Kayden, H. J. (1965). Chylomicron-lipid-splitting activity in homogenates of rabbit polymorphonuclear leucocytes. American -7oournai q/* Physiology, 209, 765-769. Goetzl, E. J., and Austen, K. F. (1974). Stimulation of human neutrophil aerobic glucose metabolism by purified chemotactic factors. Journal of Clinical Investigation. 53, 591-599. Hegner, D. (1968). Isolierung und Enzymbestand von Granula aus polymorphkernigen Leukozyten des peripheren Rinderblutes. Hoppe-Seyler’s zeitschrifr .ftil physiologische Chemie, 349, 544-554. Iyer, G. Y. N., Islam, M. F., and Quastel, J, H. (1961). Biochemical aspects of phagocytosis. Nature, 192, 535-541. Naidu, T. G., and Newbould, F. H. S. (1973). Glycogen in leukocytes from bovinr blood and milk. Canadian Journal of Comparative Medicine, 37, 47-55. Newbould, F. H. S., and Neave, F. K. (1965). The response of the bovine udder to an infusion of staphylococci. Journal of Dairy Research, 32, 163-170. Paape, M. J., Guidry, A. J., Kirk, S. T., and Bolt, D. J. (1975). Measurement of phagocytosis of 32P-labelled Staphylococcus aureus by bovine leukocytes : lysostaphin digestion and inhibitory effect of cream. American Journal of Veterinary Research, 36, 1737-1743. Reiter, B., and Oram, J. D. (1967). Bacterial inhibitors in milk and other biologicat fluids. Nature, 216, 328-330. Romeo, D., Zabucchi, G., and Rossi, F. (1973). Reversible metabolic stimulation of polymorphonuclear leukocytes and macrophages by concanavalin A. .Nature, New Biology, 243, 11 l-l 12. Rose, D. (1968). Relation between micellar and serum casein in bovine milk. JorrrnaI qf Dairy Science, 51, 1897-1902. Russtall, M. W. (1973). Ph.D. Thesis, University of Reading.
52
M. w. RUSSELL et al.
Russell, M. W., Brooker, B. E., and Reiter, B. (1976). Inhibition of the bactericidal activity of bovine polymorphonuclear leucocytes and related systems by casein. Research in Veterinary Science, 30, 30-35. Russell, M. W., and Reiter, B. (1975). Phagocytic deficiency of bovine milk leucocytes: an effect of casein. Journal of the Reticuloendothelial Society, 18, 1-13. Schmidt, D. G., and Buchheim, W. (1970). Elektronmikroskopische Untersuchung der Feinstruktur von Caseinmicellen in Kuhmilch. Milchwissenschaft, 25, 596-600. Stossel, T. P., Mason, R. J., and Smith, A. L. (1974). Lipid peroxidation by human blood phagocytes. Journal of Clinical Investigation, 54, 638-645. Strauss, B. S., and Stetson, C. A. (1960). Studies on the effect of certain macromolecular substances on the respiratory activity of the leucocytes of peripheral blood. Journal of Experimental Medicine, 112, 653-669. Tan, W. C., Livingstone, D. C., Gilbert, J. D., and Young, S. (1971). Localisation in the electron microscope by acid phosphatase staining of casein particles in lactating mammary glands of rats. Journal of Microscopy, 94, 157-165. Tedesco, F., Trani, S., Soranzo, M. R., and Patriarca, P. (1975). Stimulation of glucose oxidation in human polymorphonuclear leucocytes by C3-Sepharose and soluble C567. FEBS Letters, 51, 232-236. Wilkinson, P. C. (1972). Characterisation of the chemotactic activity of casein for neutrophil leucocytes and macrophages. Experientia, 28, 1051-1052. Wooding, F. B. P. (1971). The structure of the milk fat globule membrane. Journal of Ultrastructural Research, 37, 388-400. Zticker-Franklin, D., and Hirsch, J. G. (1964). Electron microscope studies on the degranulation of rabbit peritoneal leucocytes during phagocytosis. Journal of Experimental Medicine, 120, 569-576. [Received for publication,
March
19th, 19761
PATH.
1977.
VOL.
43
87.
ELECTRON MICROSCOPIC INTERACTION OF CASEIN GLOBULES WITH BOVINE LEUCOCYTES DURING STAPHYLOCOCCI
OBSERVATIONS MICELLES AND POLYMORPHONUCLEAR THE PHAGOCYTOSIS IN MILK
OF MILK
THE FAT OF
BY
M. W. National
B. E.
RUSSELL,*
Institute
for
and B.
BROOKER
Research in Dairying,
Uniuersi(v
of Reading,
REITER
Reading,
England
INTRODUCTION
Bovine mastitis is characterized by an influx of polymorphonuclear leucocytes (PMN) into the milk. A pre-existing leucocytosis or one induced by endotoxin, can protect the udder against experimental challenge with Staphylococcus aureus (Newbould and Neave, 1965; Reiter and Oram, 1967) and the interaction between staphylococci and PMN may be critical for the outcome of an infection (Anderson, 1975). However, the influx of PMN in response to infection does not always eliminate the pathogens which may persist, together with large numbers of PMN, for long periods in the milk. We have shown that PMN isolated from cows’ blood and suspended in synthetic medium supplemented with normal bovine serum readily ingest and kill a mastitis strain of S. aureus, whereas PMN isolated from milk, or blood PMN suspended in separated milk, ingest and kill S. aureus much more slowly (Russell and Reiter, 1975). The cause of this impairment was attributed to casein, which inhibited phagocytosis in vitro, and reversed the bactericidal activities of PMN lysates, histones and the lactoperoxidase-hydrogen peroxide-potassium iodide system (Russell, Brooker and Reiter, 1976). We have shown, by means of immunofluorescence microscopy, that casein was associated with the surface of washed milk PMN, and with blood PMN after exposure to milk (Russell and Reiter, 1975). Furthermore, casein appeared to be ingested by PMN during the phagocytosis of staphylococci in milk. However, the limits of resolution did not permit detailed observation of how casein was taken up by PMN, or of how it probably interacted to impair the intracellular bactericidal mechanisms of PMN. The ultrastructure of PRN and the morphological events of phagocytosis are well documented (ZuckerFranklin and Hirsch, 1964; Davis and Douglas, 1972). Furthermore, the electron microscope resolves casein characteristically as electron-dense granular micelles of up to about O-3 pm in diameter (Schmidt and Buchheim, 1970; Tan, Livingstone, Gilbert and Young, 1971). It was therefore felt that this technique could be used to examine the interaction of casein with PMN during phagocytosis in milk. In this paper we present electron microscopic evidence for the ingestion of casein and fat globules by bovine PMN. and
* Present Dental
address: Schools,
Department of Oral London SEI Qrr.
Immunology
and
Microbiology,
Guy’s
Hospital
Rledical
44
M. W.
RUSSELL
MATERIALS
AND
et al. METHODS
Preparation of suspensions of PMN and staphylococci. PMN were isolated from bovine blood or milk as described previously (Russell and Reiter, 1975). Suspensions of PMN (10’ cells per ml) in milk or in synthetic milk-salts-lactose solution (MSL) plus 1 per cent normal bovine serum were mixed with 5’. aureus (10s colony-forming units/ml) in siliconized glass vials and incubated with continuous gentle agitation at 37 “C. (Russell and Reiter, 1975). When milk was used as the medium, in most experiments it was first separated to remove the cream by centrifugation at 2000 g for 10 min. No significantly different observations resulted if whole milk was used. In some experiments, after 15 min incubation, the PMN were separated from free staphylococci by centrifuging at 50g for 5 min at 4 “C and re-suspended in fresh medium for further incubation so that the post-phagocytic events could be observed without the complication of continued phagocytosis. Electron microscopy. PMN were sedimented by centrifugation at 50 g for 5 min at 4 “C and fixed by suspending in 2.5 per cent glutaraldehyde in 0.2 M cacodylate buffer pH 7.4. The cells were treated with 1 per cent osmium tetroxide, followed by 1 per cent uranyl acetate in 25 per cent ethanol, dehydrated in ethanol and embedded in Araldite. The sections were stained with uranyl acetate and lead citrate, and examined in a Hitachi HU 11E electron microscope. RESULTS
Resting PMN The structure of PMN freshly isolated from bovine blood is illustrated in Fig. 1. The cytoplasm was packed with membrane-bound granules or lysosomes (L) typical of PMN of other species. The structure of PMN isolated from milk is shown in Fig. 2. Large milk fat globules (FG), identified by the presence of
Figs 1 to 8. Electron micrographs of bovine polymorphonuclear leucocytcs (PMN). CM, Casein micelles (putative); FG, Fat globule; GB, Golgi body; L, Lysosome (both azurophil and with specific granules); M, Mitochondrion; N, Nuclear lobe; PS, Pseudopodium; PV, Phagocytic vacuole (phagosome) ; S, Staphylococcus. Fig. 1. Blood PMN, freshly isolated. x 16 000.
PHAGOCYTOSIS
OF STAPHYLOCOCCI
IiV JIILK
4.5
a characteristic membrane (Wooding, 1971) were prominent in the cytoplasm. Fig. 2 (’msttsx ) sh ows a PMN fixed in the act of ingesting a fat globule with a distinct membrane. Thr cytoplasm of milk PMN contained noticeably fewer
t ’
Fig.
)I
2. Milk PMN, freshly globule. x 10 000.
isolated.
% 11 200.
(Inset)
: milk
PMN,
freshly
isolated,
ingesting
a I~I
lysosomes than blood PMN, but there were also some small, apparently empty membrane-bound vacuoles and myelin figures. Some small dense granules of size and morphology resembling casein micelles (C:M) were seen near to
Fig.
3. Blood
PMN
after
incubation
with
S. aweus in separated
milk
for 15 min.
s 16 000.
46
M. W.
RUSSELL
et a/.
the cell membrane, sometimes in association with pseudopodia, similar granules were visible in small cytoplasmic vacuoles.
and a few
PhagoGytosis by Blood PALN The early events of phagocytosis are illustrated in Fig. 3 in which the encirclement of staphylococci (S) by pseudopodia to form phagosomes ( PV can be inferred. After prolonged incubation in the synthetic medium (M E!lL)
Fig. 4. Blood
PMN
Fig. 5. Milk
after
PMN
incubation
after
with
incubation
S. awxs
with
in MSI,
plus
1 per cent serum
S. aweus in separated
milk
for 2 h. % 12! 000.
for 2 h. x 11 200.
PHAGOCYTOSIS
plus serum, staphylococci, membranous such cells was
OF STAPHYLOCOCCI
47
IN MILK
blood PMN contained several vacuoles in which were seen mostly in various states of disintegration, together with other and granular material (Fig. 4). The vacuolated cytoplasm of usually depleted of lysosomes.
Plmgocytosis by Milk
PMJV
Milk PMN incubated with staphylococci in separated milk displayed a variety of features (Figs 5 and 6). Vacuoles containing staphyIococci were commonly
Fig. 6. Milk
PMN
after
incubation
with
S. aumw in separated
milk
for 2 h. x 12 000.
seen, and most of the organisms appeared to be structurally intact. Some staphylococci were observed to possess one complete cross-wall with a second wall at right angles and incompletely formed (Fig. 5), suggesting rapid growth and division. Other vacuoles contained granular material, some in a diffuse fornl, some in the form of dense discrete granules resembling casein micelles (C&l). Similar particles were present in association with the cell membrane of some PMD;. Vacuoles were seldom seen containing both staphylococci and casein micelles, although this feature was intentionally looked for. As in resting milk PMN, some empty vacuoles, or vacuoles containing membranous material, Some phagocytosing milk PMN appcxrcd to and myelin figures were seen. be depleted of lysosomes (Fig. 6), while others still contained a relatively large number (Fig. 5). Few milk PMN contained fat globules after 2 h incubation in separated milk. Blood PMN incubated with staphylococci in whole milk displayed features similar to those of phagocytosing milk PMN (Fig. 7). Intracellular staphylococci were usuaIly intact in structure and some appeared to be actively dividing. n’umerous vacuoles containing membranous and granular material, some of it
48
M.
W.
RUSSELL
et
al.
resembling casein micelles, were seen. Some cells possessed several large pseudopodia extending into the medium around various particles. In some instances, adjacent pseudopodia were observed encircling a particle and in intimate contact without appearing to fuse to form a typical phagosome (Fig. 8).
Fig.
7. Blood
Fig. 8. Blood
PMN
PMN
after
after
incubation
incubation
with
with
S. HUMUS in whole
S. aureus in separated
milk
milk
for 1 h. x 12 000.
for 2 h.
x
11 200.
DISCUSSION
The structural study was similar
morphology of bovine blood PMN as seen in the present to that of other species, and agreed well with the structure
PHAGOCYTOSIS
OF
STAPHYLOCOCCI
IN
MILK
49
of bovine PMN reported by Davis and Douglas (1972). The morphological events of “normal” phagocytosis, i.e. phagocytosis by blood PMN in synthetic medium plus serum, conform to the generally accepted picture of this process (Zucker-Franklin and Hirsch, 1964). Furthermore, the descriptive observations of the phagocytosis of staphylococci by blood and milk PMN in milk or synthetic medium are consistent with the quantitative results reported preLiously (Russell and Reiter, 1975). Blood PMN in MSL plus serum appeared to ingest the organisms readily, and the organisms were largely destroyed by the intracellular bactericidal systems and hydrolytic enzymes (Hegner, 1968). ‘The concomitant disappearance of the abundant lysosomal granules, presumably by fusion with the phagosomes, was an essential feature. In contrast, milk PMN did not appear to be so capable of killing ingested staphylococci, siucc many intact and dividing organisms were present in the phagosomes of milk PMN after 2 h. Since this study was inherently qualitative, it was not possible to confirm the quantitatively diminished phagocytic ingestion of staphylococci which we have already demonstrated (Russell and Reiter, 1975). The ratio of organisms to PMN was increased to 10 to 1 to facilitate observation of staphylococci in the electron microscope and this may have increased the numbers of bacteria ingested. The morphology of freshly isolated milk PMN presented some new features. Ingested milk fat globules were readily identifiable and could also be seen in suitably stained light microscopic preparations (unpublished observations). They have also been reported by Paape, Guidry, Kirk and Bolt (1975). The small dense granules seen in vacuoles and near to the surface membrane in milk PMN and blood PMN after incubation in milk had the morphology expected of casein micelles (Schmidt and Buchheim, 1970; Tan, Livingstone, Gilbert and Young, 1971), ahhough they have not been positively identified in the present investigation. The distribution of these granules agreed with the distribution of casein in similar preparations of PhSN examined by immunofluorescence microscopy, using casein-specific antiserum (Russell and Reiter, 1975). However, up to about IO per cent of the casein in cows’ milk may be present, not as particulate micelles, but in solution (Rose, 1968). Immunofluorescence cannot distinguish between the two forms, but electron microscopy resolves only the micelles. It may therefore be that molecular casein was present in association with PMN in addition to the observed micelles and also in other locations where micelles were not observed. The phagocytosis of fat globules by PMN in whole milk might be expected to inhibit the phagocytosis of staphylococci. This has been demonstrated b) Paape et al. (1975) using suspensions containing up to 40 per cent milk or its equivalent in cream. However, in experiments in which PMN were suspended in whole or separated milk, only a small diminution in phagocytosis was attributable to the presence of fat globules (Russell, 1973). When cream was added to PMN in suspension in synthetic medium, a greater inhibitory effect was observed, but microscopic examination showed that the fat globules had coalesced around the PMN. Since in whole milk, inhibition due to casein was much greater than that due to fat globules, it is possibIe that the inhibitory effect of casein is lost more easily on dilution than that of fat globules. 1)
50
M.
W.
RUSSELL
et d.
The disappearance of fat globules from milk PMN after 2 h incubation in separated milk was striking. It is not clear whether this was due to exocytosis, digestion by lipase (Elsbach and Kayden, 1965) or lipid peroxidation (Stossel, Mason and Smith, 1974). Our earlier immunofluorescence studies, which indicated that casein was ingested during the phagocytosis of staphylococci by PMN suspended in separated milk, suggested the possibility that the inclusion of casein within the phagosomes containing staphylococci would result in neutralization of the intracellular bactericidal mechanisms (Russell, Brooker and Reiter, 1976). However, casein micelles were seldom seen in phagosomes containing staphylococci, although it is possible that soluble casein was present. The evidence of ingestion of casein micelles, sometimes in apparently large quantities, does, however, suggest another possibility. If lysosomes are discharged into phagosomes containing casein, an adequate number might not be available for other phagosomes containing staphylococci. Studies using blood PMN incubated in separated milk with staphylococci were consistent with the interpretation that the simultaneous phagocytosis of staphylococci and casein results in diminished killing of the organisms by competition for and neutralization of the lysosomal bactericidal agents. Milk PMN which were exposed to the effects of casein in vivo appeared, on isolation, to contain fewer lysosomes than blood PMN. The small apparently empty vacuoles and myelin figures in milk PMN might represent the remains of casein phagosomes. There is a further possibility that exposure to casein in vivo or in vitro might result in activation of the enhanced metabolism of phagocytosis (Iyer, Islam and Quastel, 1961). A consequence of this might be that the energy reserves of milk PMN become depleted (Naidu and Newbould, 1973) or that peroxide and superoxide radicals toxic products such as lactate, hydrogen accumulate. PMN are known to be so activated on contact with agents which damage the surface membrane, such as chemotactic fragments of complement (Goetzel and Austen, 1974; Tedesco, Trani, Soranzo and Patriarca, 1975), concanavalin A (Romeo, Zabucchi and Rossi, 1973) and endotoxin (Strauss and Stetson, 1960). We have previously shown the association of casein with the PMN surface by means of immunofluorescence. An effect of casein on the PMN membrane can also be inferred from its chemotactic property which, however, has been attributed rather to soluble casein monomers and the a and p components (Wilkinson, 1972). Casein micelles are apparently not chemotactic, but it may be that their particulate nature is essential for their phagocytosis by PMN, which are not known normally to engage in the pinocytosis of liquids. SUMMARY
Polymorphonuclear leucocytes (PMN) isolated from bovine blood or milk were examined in the electron microscope before and after incubation with staphylococci in either milk or a synthetic salts medium supplemented with serum. Milk PMN on isolation contained prominent milk fat globules and a few small vacuoles containing granules resembling casein micelles. Neither of these were seen in blood PMN. Milk PMN contained fewer lysosomes than
PHAGOCYTOSIS
OF STAPHYLOCOCCI
IN
MILK
51
blood PMN, and it is suggested that phagocytosis of fat globules and cascin micelles in vivo, with consequent degranulation, accounts for their previously observed deficient phagocytosis of staphylococci. On incubation in milk in vitro with staphylococci, both blood PMN and milk PMN ingested apparently large quantities of granules resembling casein micelles, forming phagocytic vacuoles separate from those containing staphylococci. The ingested staphylococci appeared largely intact, despite the loss of’ lysosomes from the PMN cytoplasm. In contrast, the staphylococci ingested by blood PMN in synthetic medium appeared to be dead after 2 h. These observations support previous findings that casein inhibits the bactericidal activities of PMiX, and suggest possible mechanisms for this inhibition.
ACKNOWLEDGMENTS
We should like to thank Dr S. D. Douglas, University School, Minneapolis, U.S.A. for his review and comments graphs.
of Minnesota Medical on the electron micro-
REFERENCES
Anderson, J. C. (1975). Pathogenesis of experimental mastitis in the mouse caused by a strain of Staphylococcus aureus of low virulence and its modification by endotoxin. Journal of Comparative Pathology, 85, 531-538. Davis, W. C., and Douglas, S. D. (1972). Defective granule formation and function in the Chediak-Higashi syndrome in man and animals. Seminars in Haematology, 9, 43 l-450. Elsbach, P., and Kayden, H. J. (1965). Chylomicron-lipid-splitting activity in homogenates of rabbit polymorphonuclear leucocytes. American -7oournai q/* Physiology, 209, 765-769. Goetzl, E. J., and Austen, K. F. (1974). Stimulation of human neutrophil aerobic glucose metabolism by purified chemotactic factors. Journal of Clinical Investigation. 53, 591-599. Hegner, D. (1968). Isolierung und Enzymbestand von Granula aus polymorphkernigen Leukozyten des peripheren Rinderblutes. Hoppe-Seyler’s zeitschrifr .ftil physiologische Chemie, 349, 544-554. Iyer, G. Y. N., Islam, M. F., and Quastel, J, H. (1961). Biochemical aspects of phagocytosis. Nature, 192, 535-541. Naidu, T. G., and Newbould, F. H. S. (1973). Glycogen in leukocytes from bovinr blood and milk. Canadian Journal of Comparative Medicine, 37, 47-55. Newbould, F. H. S., and Neave, F. K. (1965). The response of the bovine udder to an infusion of staphylococci. Journal of Dairy Research, 32, 163-170. Paape, M. J., Guidry, A. J., Kirk, S. T., and Bolt, D. J. (1975). Measurement of phagocytosis of 32P-labelled Staphylococcus aureus by bovine leukocytes : lysostaphin digestion and inhibitory effect of cream. American Journal of Veterinary Research, 36, 1737-1743. Reiter, B., and Oram, J. D. (1967). Bacterial inhibitors in milk and other biologicat fluids. Nature, 216, 328-330. Romeo, D., Zabucchi, G., and Rossi, F. (1973). Reversible metabolic stimulation of polymorphonuclear leukocytes and macrophages by concanavalin A. .Nature, New Biology, 243, 11 l-l 12. Rose, D. (1968). Relation between micellar and serum casein in bovine milk. JorrrnaI qf Dairy Science, 51, 1897-1902. Russtall, M. W. (1973). Ph.D. Thesis, University of Reading.
52
M. w. RUSSELL et al.
Russell, M. W., Brooker, B. E., and Reiter, B. (1976). Inhibition of the bactericidal activity of bovine polymorphonuclear leucocytes and related systems by casein. Research in Veterinary Science, 30, 30-35. Russell, M. W., and Reiter, B. (1975). Phagocytic deficiency of bovine milk leucocytes: an effect of casein. Journal of the Reticuloendothelial Society, 18, 1-13. Schmidt, D. G., and Buchheim, W. (1970). Elektronmikroskopische Untersuchung der Feinstruktur von Caseinmicellen in Kuhmilch. Milchwissenschaft, 25, 596-600. Stossel, T. P., Mason, R. J., and Smith, A. L. (1974). Lipid peroxidation by human blood phagocytes. Journal of Clinical Investigation, 54, 638-645. Strauss, B. S., and Stetson, C. A. (1960). Studies on the effect of certain macromolecular substances on the respiratory activity of the leucocytes of peripheral blood. Journal of Experimental Medicine, 112, 653-669. Tan, W. C., Livingstone, D. C., Gilbert, J. D., and Young, S. (1971). Localisation in the electron microscope by acid phosphatase staining of casein particles in lactating mammary glands of rats. Journal of Microscopy, 94, 157-165. Tedesco, F., Trani, S., Soranzo, M. R., and Patriarca, P. (1975). Stimulation of glucose oxidation in human polymorphonuclear leucocytes by C3-Sepharose and soluble C567. FEBS Letters, 51, 232-236. Wilkinson, P. C. (1972). Characterisation of the chemotactic activity of casein for neutrophil leucocytes and macrophages. Experientia, 28, 1051-1052. Wooding, F. B. P. (1971). The structure of the milk fat globule membrane. Journal of Ultrastructural Research, 37, 388-400. Zticker-Franklin, D., and Hirsch, J. G. (1964). Electron microscope studies on the degranulation of rabbit peritoneal leucocytes during phagocytosis. Journal of Experimental Medicine, 120, 569-576. [Received for publication,
March
19th, 19761
