Biol. Neonate 27: 318-328(1975)
Phagocytic Cells in Cord Blood1 Gregor Prindull2, Brigitte Prindull, Zvi Palti and Joseph M. Yoffey Department of Anatomy, Obstetrics, and Haematology, The Hebrew University-Hadassah Medical School, Jerusalem
Key Words. Monocytes - Neutrophils • Lymphocytoid cells • Phagocytes • Cord blood Abstract. A suspension of fine carbon particles was added to cord blood of healthy premature and full-term infants, and the mixture was incubated for 3 h, after which the uptake of carbon particles by blood leukocytes was examined. The results were compared with those from the blood of adults. A gradient of phagocytic activity was observed. The most active uptake of carbon was by the leukocytes of premature infants, the least active by leukocytes of adults. In cord blood and blood of adults, phagocytic activity was evident in both monocytes and neutrophils..In addition, two types of what have been termed ‘lympho cytoid’ phagocytes were seen. These resemble lymphocytes in their morphology. One type possesses basophilic cytoplasm, and has been found only in premature cord blood. The presence of lymphocytoid phagocytes affords a further indication of the differences between the circulating lymphocyte population in the prenatal and perinatal period as compared with the adult.
Introduction
1 This study was supported in part by a grant from the Joint Research Fund of the Hebrew University and Hadassah, and by grant Pr 75/6 from the ‘Deutsche Forschungs gemeinschaft’. 1 On leave from the Department of Paediatrics, University of Gottingen, FRG. Under the auspices of the Committee for Scientific Co-operation between German Institutions and the Weitzmann Institute.
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Fetuses after the 15th week of gestation are capable of reacting effectively by humoral and cellular antibody production when exposed to foreign antigenic material (21). Antibody production against particulate matter is preceded by phagocytosis, which in the fetus can be studied conveniently in cord blood leukocytes. In a series of such studies, attention has been directed inter alia to the identity of the phagocytic cells contained therein.
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Matoth (14), observing the ingestion of starch granules by cord blood leuko cytes from full-term infants, seems to have noted ingestion only by polymorphs. Gluck and Silverman (10) found a number of phagocytic cells in cord blood of both premature and full-term infants, which they referred to as ‘effective phago cytes’ whithout any further attempt at identification. They made only a passing reference to monocytes, which according to Knoll (12) are present in the blood from the 5th month of fetal life onwards. Playfair et al. (19) saw monocytes in the blood of all fetuses examined between the ages of 8 and 27 weeks. More recently, Prindull and Prindull (23, 24) noted an appreciable monocyte reserve during the first few days after birth in infants subjected to therapeutic exchange transfusions. In view of the known phagocytic activity of monocytes, it seemed surprising that specific reference has not been made by previous investigators to their phagocytic activity - or lack of activity —in cord blood. The present study was therefore undertaken, primarily in order to establish whether monocytes in cord blood were in fact capable of phagocytosis. As the work progressed, further observations were made on the phagocytic activity of neutrophils, and also of a group of cells, not previously described, which while possessing the morphology of lymphocytes, appeared to be the most actively phagocytic of all the cells in cord blood. Materials and Methods Cord blood from 10 full-term infants, and from 3 premature infants (gestational ages 34 weeks) was collected into heparinized tubes. Deliveries of the infants as well as their post-natal courses were uneventful. Venous blood was also withdrawn into heparinized syringes from 10 healthy volunteers. Heparin was used in amounts of 20 lU/ml of blood collected. Processing of the blood from both infants and adults was done within 2 h of collection. After allowing the blood to sediment at 37 °C for 1 h, cell suspension of 40 ± 5 million total leukocytes per millilitre in medium TC 199 were prepared from the buffy coat. Through a hypodernic needle, each sample received one drop of a 1:10 dilution of carbon ink (Gunther Wagner, West Germany; particle size 200-500 A) in medium TC 199. The ink was thoroughly mixed with the cell suspension by a vortex shaker and incubated at 37 °C for periods of 1 and 3 h. At first the cells were washed with tissue culture medium immedi ately after incubation, but subsequently this step was omitted, since it did not yield any obvious advantage. Blood smears were made after incubation and stained with Wright’s stain. Phagocytosis was assessed in terms both of the percentage of cells which were phago cytic, and of the amount of carbon ingested by individual cells.
Four morphologically distinct types of phagocytic cells have been identified in cord blood, three in both full-term and premature infants, and a fourth only
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in the latter. The three which are common to both groups are: (1) polymorpho nuclear neutrophil granulocytes; (2) monocytes, and (3) lymphocyte-like cells with abundant cytoplasm. The fourth type of cell, which so far has only been seen in the blood of premature infants, is also lymphocyte-like, but has only a small amount of basophilic cytoplasm.
Monocytes In premature infants the monocytes, like the neutrophils, had a very high phagocytic index. Even after only 1 h incubation, 8 0 -9 0 % of the monocytes had ingested ink, while after 3 h the figure increased to practically 100%. Fur thermore, the monocytes in these infants ingested far more carbon than the neutrophils, and they also segregated the particles to form larger clumps than were seen in neutrophils (fig. 5). In full-term infants, about 10% of the monocytes did not contain any ink after incubation for 3 h. Furthermore, the amount of carbon ingested by indi vidual monocytes was appreciably less than in premature infants (fig. 5). Monocytes of adults showed the lowest phagocytic index of all three groups. Even after incubation for 3 h, over 60 % of the monocytes did not contain ink. It was also noteworthy that in monocytes of adult blood the par ticle size was usually small, and there was singularly little tendency to clumping (fig. 7). Figure 2 summarizes the main findings on carbon ingestion in the three cell groups examined. From a morphological point of view, the monocytes of
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Granulocytes As far as granulocytes are concerned (fig. 3,4), three points call for empha sis. Phagocytic activity was found only in neutrophils. Furthermore, in our preparations only an occasional granulocyte in adult blood had ingested carbon particles after 1 and 3 h incubation, in marked contrast to the number of ac tively phagocytic neutrophils in cord blood. Premature infants showed the highest phagocytic index. After even 1 h incubation, 60—70 % of granulocytes had ingested carbon particles, while incubation for 3 h resulted in practically 100 % uptake. Segregation of ingested particles to form clumps occurred to a marked extent in the granulocytes of premature infants, to a lesser but still quite appre ciable extent in full-term infants, but hardly at all in the neutrophils of adults. Figure 1 summarizes the main findings concerning carbon ingestion by neutro phil granulocytes. It is based, as is figure 2, on a visual assessment of the load of carbon uptake by individual' neutrophils or monocytes, respectively, as approx imately heavy, medium, or light, from one representative subject of each of the three groups examined. The selection of subjects was made after a detailed quantitative evaluation of the three premature infants, of four full-term infants and of four adults.
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Fig. 1. Ingestion of carbon particles by neutrophil granulocytes of cord blood of pre mature and full-term infants, compared with adult blood. Carbon uptake after 3 h incuba tion, graded by simple visual assessment as 1 = heavy, 2 = medium, 3 = light, 4 = no uptake. The data represent studies on one representative subject from each of the groups studied. Fig. 2. Ingestion of carbon particles by monocytes of cord blood of premature and full-term infants, compared with adult blood.
Lymphocytoid Phagocyte Type / A completely unexpected finding was the presence of two types of phago cytic cell with lymphocyte morphology. Pending a final decision as to their identity, these cells are being termed ‘lymphocytoid’. The lymphocytoid cell most commonly seen has been designated type I. It occurs most conspicuously in the blood of full-term infants, has a nucleus which resembles that of a me dium or large lymphocyte, and possesses a relatively large amount of pale cyto plasm. These cells may also be found in adult blood, though in rather smaller numbers than in cord blood. In an ordinary blood film, when these cells are seen without carbon particles, it is difficult to regard them as anything other than lymphocytes (fig. 10, 11).
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cord blood tend to show greater irregularity in nuclear shape than those of the adult. Thus, figure 8 is a monocyte with a triradiate nucleus, while figure 9 shows more marked segmentation than one would normally expect to find.
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Lymphocytoid Phagocyte Type II The type II lymphocytoid phagocyte has so far only been found in the cord blood of premature infants. Figure 12 illustrates one of these cells containing carbon, while figure 13 illustrates the appearance without ingested carbon. In marked contrast to the type I phagocyte, this cell possesses a high N:C ratio, and a relatively small amount of basophilic cytoplasm. It is all the more striking that this relatively narrow border of cytoplasm can become so tightly packed with ingested carbon particles.
Discussion Phagocytic Gradient A comparison of the phagocytic activity of neutrophils and monocytes in premature and full-term infants and in adults brings out what may be termed a phagocytic gradient. This is indicated by the presence of phagocytic cells in each group, and by the amount of carbon ingested by the individual cells. The latter parameter can be graded approximately by simple visual assessment of carbon uptake as heavy, medium, or light. Our results show clear evidence of the pres ence of a phagocytic gradient in both monocytes and granulocytes (fig. 1, 2).
Fig. 3-13. Uptake of carbon particles after 3 h incubation by blood cells. Cells from cord blood of premature and full-term infants compared with blood from adults, x 1,200. 3 Heavily loaded neutrophil from premature newborn infant. 4 Lightly loaded neutrophil from blood of normal healthy adult. 5 Heavily loaded monocyte from blood of a prema ture infant. 6 Moderately loaded monocyte from a full-term infant. 7 Lightly loaded mono cyte from normal adult. 8 Trilobate monocyte from blood of premature infant. 9 Marked segmentation in monocyte of full-term infant. Slight carbon uptake. Type I lympho cytoid phagocyte from premature infant. Heavy carbon uptake. 11 Type 1 lymphocytoid cell from adult blood incubated without addition of carbon. Compare with figure 12. 12 Type II lymphoid phagocyte from cord blood of premature infant. Heavy carbon uptake. 13 Type II lymphocytoid cell from blood of premature infant before incubation with carbon. In its general configuration, this cell resembles that of figure 12. The cytoplasm in figures 12 and 13 is basophilic, though this docs not show in the black and white picture.
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Variation in Phagocytic Activity o f Neutrophils One of the most obvious points to emerge in the analysis of the present results is the great variability in the amount of particle uptake by individual neutrophils even of one and the same subject. The reasons for this are not clear. One possible source of difficulty may be the different types of particle em ployed, e.g. starch particles (14), Higgins’ India ink (10), and polystyrene par ticles (3).
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A further source of difficulty lies in the action of serum factors such as opsonins (5, 9, 18). Cord sera apparently possesses a low degree of opsonic activity in comparison to that of adult blood (4, 25). In some instances, opsonic activity may even be markedly deficient (25). This might conceivably have some bearing on the almost complete absence of neutrophil phagocytic activity in some of our infants. For phagocytosis of monocytes, opsonins may not be so essential, since these cells were found to display phagocytic activity in all the cord blood examined. In this respect cord blood monocytes would resemble adult blood monocytes, which can be phagocytic even in the absence of serum (3).
Lymphocytoid Cells and Monocytes The finding of phagocytic lymphoid cells raised the question whether these were a distinctive cell group belonging to the lymphoid series, or whether they might be atypical forms of monocytes.
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Metabolic Factors Apart from opsonins, metabolic factors influence the phagocytic activity of neutrophils. Even in the resting stage, oxygen consumption by neutrophils of newborn infants is significantly higher than in matched maternal leukocytes (17). Also increased is the number of neutrophils which will spontaneously reduce dye substances such as nitroblue tetrazolium (NBT) (11). The question then arises, whether cells with a high oxygen consumption already in the resting stage, would on that account be more actively phagocytic. Our own findings would be wholly consistent with such a view, through they do not prove it. Alternatively, it could be argued that these phagocytic cells activated already in the resting stage, also had altered cell membrane structures, more permeable not only to the dye NBT, but also facilitating the entry into the cell of carbon particles. The marked difference in carbon uptake between premature and full term infants observed in this study would then be interpreted to repre sent differences in cell membrane permeability between the two groups. How ever, it is difficult to imagine that carbon particles of 200-500 A in size would diffuse through a cell membrane as readily as the NBT solution. From what we know about the NBT test, it appears most likely that the greater reactivity of newborn infants’ granulocytes in this test is due to the increased rate of oxydative reduction of the dye secondary to the activated cell metabolism, rather than to an increased amount of unreduced NBT diffusing into the cell. We therefore believe that the increased number of carbon particles found inside of leukocytes particularly in premature infants, and to a lesser but still substantial degree in full-term infants — in comparison to adult leukocytes — is due to true phago cytosis and not the result of a facilitated diffusion of particles secondary to an altered membrane permeability.
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A cursory survey of the literature suffices to bring out the fact that mono cytes are a very pleomorphic group of cells (2, 13, 29). Though the nucleus is generally regarded as being bean-shaped, with a characteristic nuclear ‘h o f, it may not infrequently be oval or circular, as in the lymphoid cells. However, the internal structure of the monocyte nucleus, often described as ‘lacy’, ‘skein-like’ or ‘reticular’, appears to be regarded as more distinctive than the actual shape. The cytoplasm, furthermore, in films stained with Wright’s stain, as in the pres ent study, is described by Wintrobe (29) as ‘greyish or cloudy blue’, and possessing ‘abundant fine, lilac or reddish blue granules’. By these criteria, neither of the two types of lymphocytes can be classed as monocytes. The type II cell has a basophilic cytoplasm and can certainly not be regarded as monocytic. The type I lymphocytoid cell, with its pale cytoplasm, is also not a monocyte, if the presence of the characteristic monocytic granules is a valid criterion. Most of the pale lymphocytoid cells do not have granules. Occasion ally, they do have a few azurophilic granules, which, however, are typically lymphocytic (15). A few of the type I cells contain nuclei whose structures are not too different from that of monocytic nuclei, but for the most part these cells possess nuclei which are typically pachychromatic, as in lymphocytes. In their classical study, Downey and McKinlay (6) described a cell with an abundance of pale cytoplasm — their type III lymphocyte resembling in its general morphology the type I cell of this study. But it is difficult to press this comparison too closely, since their description was based on pathological mate rial (7). Furthermore, the authors (6, 7) make no reference to the phagocytic properties of these cells. Lymphocytes are not generally regarded as phagocytic. However, in her investigation of the phagocytosis of latex particles by blood ‘mononuclear cells’, Zucker-Franklin (33; fig. 2a, b, c) has provided evidence of phagocytic cells, which even by electron microscopy were ‘... difficult to distinguish from lym phocytes’. She came to the conclusion that ‘About 6—8 % of the cells containing particles cannot be identified with certainty ... Therefore, the possibility cannot be excluded that a very small percentage of bona fide lymphocytes are also able to take up particles...’ A fuller discussion of the phagocytic properties of lym phocytes or of lymphocyte derivatives will be found elsewhere (27, 32). The presence of the lymphocytoid cells adds one more piece of evidence to recent observations that, on both morphological and functional grounds, there are important differences between the blood lymphocyte of the fetus and neo nate, as compared with those in postnatal life: (a) Neonatal lymphocytes contain a higher proportion of medium and large cells than the blood of adults (8); (b) medium-size lymphocytes in both neonatal (8) and prenatal (28) blood possess the characteristic morphology of transitional cells, as observed by both light and electron microscopy; (c) in view of the proliferative properties of tran sitional cells, it is not therefore surprising that prenatal and neonatal lympho
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cytes contain a higher proportion of cells in spontaneous DNA synthesis than lymphocytes in the blood of adults (8, 22, 28); (d) neonatal lymphocytes have been reported to respond to PHA to a greater extent than lymphocytes in the blood of adults (20); (e) phagocytic lymphocytoid cells with basophilic cyto plasm (the type 11 cells of this study) have so far been found only in cord blood of premature infants. The question at once arises: What is the significance of the differences in the lymphocyte populations of fetal and adult blood? If it is permitted at this point to speculate, the answer to this question may be twofold, partly immunological and partly haematological. Immunologically speaking, the differences in the blood lymphocyte population may conceivably reflect to a large extent the differences in the antigenic stimulation before and after birth as compared to adult life (8, 16,26). As far as haematopoiesis is concerned, it is now well-established (31) that the blood of many mammals, and presumably of man also, contains haemato poietic stem cells. In the case of the mouse, the number of stem cells in fetal blood may be more than 100 times as great as in the adult (1). Though the identity of the stem cell is still a matter of dispute, it has been suggested that in all probability it is a member of the transitional cell group (30). In the human fetus, the majority of the lymphocytes found in the blood in mid-fetal life are transitional cells (28). As full-term approaches, the transitional cells fall, while the small lymphocytes increase. Whatever the true explanation of the differences already recognized between the lymphocyte populations of fetal and adult blood, the lymphocytoid cells now described add another component to the complex and heterogenous group of blood cells which we term ‘lymphocytes’.
A cknowledgements It is a pleasure to place on record our indebtedness to Professor G. Gitlin, for the provision of laboratory facilities in the Anatomy Department, and to Professor G. Izak, of the Department of Haematology, for generous assistance with experimental material. We are also grateful to Mrs. Eva Salomon, for her skilled photomicrographic assistance, and to sister Balya Caleb, for help in obtaining cord blood.
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Barnes, D.W.H. and Loutit, J.F.: Haemopoietic stem cells in peripheral blood. Lancet if: 1138 1141 (1967). Bloom, W.: Lymphocytes and monocytes; theories of haematopoiesis; in Handbook of haematology, vol. 2, p. 1427 (Hoeber, Ne-.v York 1938).
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References
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Cline, M.J. and Lehrer, R.I.: Phagocytosis by human monocytes. Blood 32: 423 -429 (1968) . Dosset, J.H. and Quie, P.C.: Serum bacterial opsonins and polymorphonuclear leuko cytes in mothers and newborns. J. Pediat. 74: 817-818 (1969). Dosset, J.H.; Williams, R.C., and Quie, P.G.: Studies on interaction of bacteria, serum factors, and polymorphonuclear leukocytes in mothers and newborns. Pediatrics 44: 49-57 (1969). Downey, H. and McKindlay, C.A.: Acute lymphadenosis compared with acute lym phatic leukaemia. Archs intern. Med. 32: 82-101 (1932). Drescher, J. und Diedenhofen, H.: Uber die Beziehungen zwischen basophilen und hellen, azurgranulierten Blutzellen der lymphatischen Reaktion, aufgrund von Leuko zytenkulturversuchen beim Pfeifferschen Drüsenfieber. Blut 27: 384-392 (1973). Faulk, W.P.; Goodman, J.R.; Maloney, M.A.; Fudenberg, N.H. and Yoffey, J.M.: Morphology and nucleoside incorporation of human neonatal lymphocytes. Cell Immunol. 8: 166 172 (1973). Forman, M.L. and Stiehm, E.R.: Impaired opsonic activity but normal phagocytosis in low-birth-weight infants. New Engl. J. Med. 281: 926-931 (1969). Gluck, L. and Silverman, W.A.: Phagocytosis in premature infants. Pediatrics 20: 951-957 (1957). Humbert, J.R.; Kurtz, M.L., and Hathaway, W.E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants. Pediatrics 45: 125-128 (1970). Knoll, W.: Die Blutbildung beim Embryo; in Handbuch der allgemeinen Haematologie, vol. 1. p. 553 (Urban & Schwarzenberg, Berlin 1932). Leder, L.-D.: Der Blutmonozyt. Exp. Med. Path. Klin., vol. 23 (Springer, Berlin 1967). Matoth, Y.: Phagocytic and ameboid activities of the leukocytes in the newborn infant. Pediatrics 9: 748-754 (1952). Michaelis, L. and Wolff, A.: Über Granula in Lymphozyten. Virchows Arch. 167: 151-163 (1902). Nathenson, G.; Schorr, J.B., and Litwin, S.D.: Gm factor fetomaternal gamma-globulin incompatibility. Pediat. Res. 5: 2 -9 (1971). Park, B.H.; Holmes, B., and Good, R.A.: Metabolic studies on newborn leukocytes. Pediat. Res. 3: 376 (1969). Pearson, H.A.: Phagocytosis by leukocytes of newborn infants. J. Pediat. 74: 329 - 330 (1969) . Playfair, J.H.L.; Wolfendale, M.R., and Kay, H.E.M.: The leukocytes of peripheral blood in the human foetus. Brit. J. Haemat. 9: 336-344 (1963). Prindull, G.: An in vitro quantitative study of phytohaemagglutinin (PHA) induced transformation of lymphocytes from premature newborn infants, from older prema ture infants, and from full-term newborn infants. Blut 2.?.' 7-13 (1971). Prindull, G.: Maturation of cellular and humoral immunity during human embryonic development. Acta paediat., Stockh. 63: 607-615 (1974). Prindull, G.: Spontaneous DNA synthesis of blood lymphoid cells in premature new born infants, in older premature infants, and in full-term newborn infants. Z. Kinderheilk. 118: 197-206(1974). Prindull, G. and Prindull, B.: Leukocyte reserves of newborn infants. I. Observations during exchange transfusions. Blut 21: 79-89 (1970). Prindull, G. and Prindull, B.: Leukocyte reserves of newborn infants. II. Restoration of new leukocyte circulating levels after exchange transfusion. Blut 2/.' 155-161 (1970). Stossei, T.P.; Alper, C.A., and Rosen, F.S.: Opsonic activity in the newborn; role of properdin. Pediatrics 52: 134-137 (1973).
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Steinberg, A.G.: Genetic variations in human immunoglobulins: the Gm and I nv types: in Advances in Immunogenetics, p. 75 (Lippincott, Philadelphia 1967). 27 Trowell, O.A.: Lymphocytes; in Willmer Cells and tissues in culture, pp. 95 172 (Academic Press, New York 1965). 28 Winter, C.C.B.; Byles, A.B., and Yoffey, J.M.: Blood lymphocytes in newborn and adults. Lancet ii: 932-933 (1965). 29 Wintrobe, M.M.: Clinical haematology, 6th ed. (Lea & Febiger, Philadelphia 1967). 30 Yoffey, J.M.: The stem cell problem in the foetus. Israel J. Med. Scis 7: 825-831 (1971). 31 Yoffey, J.M.: Bone marrow in hypoxia and rebound (Thomas, Springfield 1974). 32 Yoffey, J.M. and Courtice, F.C.: Lymphatic, lymph and the lymphomycloid complex (Academic Press, New York 1970). 33 Zucker-Franklin, D.: The percentage of monocytes among ‘mononuclear’ cell fractions obtained from normal human blood. J. Immunol. 112: 234-240 (1974).
Dr. G. Prindull, Assistant Professor, Univ.-Kinderklinik, Humboldtallce 38,D -3 4 Gottingen (FRG)
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Phagocytic Cells in Cord Blood1 Gregor Prindull2, Brigitte Prindull, Zvi Palti and Joseph M. Yoffey Department of Anatomy, Obstetrics, and Haematology, The Hebrew University-Hadassah Medical School, Jerusalem
Key Words. Monocytes - Neutrophils • Lymphocytoid cells • Phagocytes • Cord blood Abstract. A suspension of fine carbon particles was added to cord blood of healthy premature and full-term infants, and the mixture was incubated for 3 h, after which the uptake of carbon particles by blood leukocytes was examined. The results were compared with those from the blood of adults. A gradient of phagocytic activity was observed. The most active uptake of carbon was by the leukocytes of premature infants, the least active by leukocytes of adults. In cord blood and blood of adults, phagocytic activity was evident in both monocytes and neutrophils..In addition, two types of what have been termed ‘lympho cytoid’ phagocytes were seen. These resemble lymphocytes in their morphology. One type possesses basophilic cytoplasm, and has been found only in premature cord blood. The presence of lymphocytoid phagocytes affords a further indication of the differences between the circulating lymphocyte population in the prenatal and perinatal period as compared with the adult.
Introduction
1 This study was supported in part by a grant from the Joint Research Fund of the Hebrew University and Hadassah, and by grant Pr 75/6 from the ‘Deutsche Forschungs gemeinschaft’. 1 On leave from the Department of Paediatrics, University of Gottingen, FRG. Under the auspices of the Committee for Scientific Co-operation between German Institutions and the Weitzmann Institute.
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Fetuses after the 15th week of gestation are capable of reacting effectively by humoral and cellular antibody production when exposed to foreign antigenic material (21). Antibody production against particulate matter is preceded by phagocytosis, which in the fetus can be studied conveniently in cord blood leukocytes. In a series of such studies, attention has been directed inter alia to the identity of the phagocytic cells contained therein.
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Matoth (14), observing the ingestion of starch granules by cord blood leuko cytes from full-term infants, seems to have noted ingestion only by polymorphs. Gluck and Silverman (10) found a number of phagocytic cells in cord blood of both premature and full-term infants, which they referred to as ‘effective phago cytes’ whithout any further attempt at identification. They made only a passing reference to monocytes, which according to Knoll (12) are present in the blood from the 5th month of fetal life onwards. Playfair et al. (19) saw monocytes in the blood of all fetuses examined between the ages of 8 and 27 weeks. More recently, Prindull and Prindull (23, 24) noted an appreciable monocyte reserve during the first few days after birth in infants subjected to therapeutic exchange transfusions. In view of the known phagocytic activity of monocytes, it seemed surprising that specific reference has not been made by previous investigators to their phagocytic activity - or lack of activity —in cord blood. The present study was therefore undertaken, primarily in order to establish whether monocytes in cord blood were in fact capable of phagocytosis. As the work progressed, further observations were made on the phagocytic activity of neutrophils, and also of a group of cells, not previously described, which while possessing the morphology of lymphocytes, appeared to be the most actively phagocytic of all the cells in cord blood. Materials and Methods Cord blood from 10 full-term infants, and from 3 premature infants (gestational ages 34 weeks) was collected into heparinized tubes. Deliveries of the infants as well as their post-natal courses were uneventful. Venous blood was also withdrawn into heparinized syringes from 10 healthy volunteers. Heparin was used in amounts of 20 lU/ml of blood collected. Processing of the blood from both infants and adults was done within 2 h of collection. After allowing the blood to sediment at 37 °C for 1 h, cell suspension of 40 ± 5 million total leukocytes per millilitre in medium TC 199 were prepared from the buffy coat. Through a hypodernic needle, each sample received one drop of a 1:10 dilution of carbon ink (Gunther Wagner, West Germany; particle size 200-500 A) in medium TC 199. The ink was thoroughly mixed with the cell suspension by a vortex shaker and incubated at 37 °C for periods of 1 and 3 h. At first the cells were washed with tissue culture medium immedi ately after incubation, but subsequently this step was omitted, since it did not yield any obvious advantage. Blood smears were made after incubation and stained with Wright’s stain. Phagocytosis was assessed in terms both of the percentage of cells which were phago cytic, and of the amount of carbon ingested by individual cells.
Four morphologically distinct types of phagocytic cells have been identified in cord blood, three in both full-term and premature infants, and a fourth only
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in the latter. The three which are common to both groups are: (1) polymorpho nuclear neutrophil granulocytes; (2) monocytes, and (3) lymphocyte-like cells with abundant cytoplasm. The fourth type of cell, which so far has only been seen in the blood of premature infants, is also lymphocyte-like, but has only a small amount of basophilic cytoplasm.
Monocytes In premature infants the monocytes, like the neutrophils, had a very high phagocytic index. Even after only 1 h incubation, 8 0 -9 0 % of the monocytes had ingested ink, while after 3 h the figure increased to practically 100%. Fur thermore, the monocytes in these infants ingested far more carbon than the neutrophils, and they also segregated the particles to form larger clumps than were seen in neutrophils (fig. 5). In full-term infants, about 10% of the monocytes did not contain any ink after incubation for 3 h. Furthermore, the amount of carbon ingested by indi vidual monocytes was appreciably less than in premature infants (fig. 5). Monocytes of adults showed the lowest phagocytic index of all three groups. Even after incubation for 3 h, over 60 % of the monocytes did not contain ink. It was also noteworthy that in monocytes of adult blood the par ticle size was usually small, and there was singularly little tendency to clumping (fig. 7). Figure 2 summarizes the main findings on carbon ingestion in the three cell groups examined. From a morphological point of view, the monocytes of
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Granulocytes As far as granulocytes are concerned (fig. 3,4), three points call for empha sis. Phagocytic activity was found only in neutrophils. Furthermore, in our preparations only an occasional granulocyte in adult blood had ingested carbon particles after 1 and 3 h incubation, in marked contrast to the number of ac tively phagocytic neutrophils in cord blood. Premature infants showed the highest phagocytic index. After even 1 h incubation, 60—70 % of granulocytes had ingested carbon particles, while incubation for 3 h resulted in practically 100 % uptake. Segregation of ingested particles to form clumps occurred to a marked extent in the granulocytes of premature infants, to a lesser but still quite appre ciable extent in full-term infants, but hardly at all in the neutrophils of adults. Figure 1 summarizes the main findings concerning carbon ingestion by neutro phil granulocytes. It is based, as is figure 2, on a visual assessment of the load of carbon uptake by individual' neutrophils or monocytes, respectively, as approx imately heavy, medium, or light, from one representative subject of each of the three groups examined. The selection of subjects was made after a detailed quantitative evaluation of the three premature infants, of four full-term infants and of four adults.
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Fig. 1. Ingestion of carbon particles by neutrophil granulocytes of cord blood of pre mature and full-term infants, compared with adult blood. Carbon uptake after 3 h incuba tion, graded by simple visual assessment as 1 = heavy, 2 = medium, 3 = light, 4 = no uptake. The data represent studies on one representative subject from each of the groups studied. Fig. 2. Ingestion of carbon particles by monocytes of cord blood of premature and full-term infants, compared with adult blood.
Lymphocytoid Phagocyte Type / A completely unexpected finding was the presence of two types of phago cytic cell with lymphocyte morphology. Pending a final decision as to their identity, these cells are being termed ‘lymphocytoid’. The lymphocytoid cell most commonly seen has been designated type I. It occurs most conspicuously in the blood of full-term infants, has a nucleus which resembles that of a me dium or large lymphocyte, and possesses a relatively large amount of pale cyto plasm. These cells may also be found in adult blood, though in rather smaller numbers than in cord blood. In an ordinary blood film, when these cells are seen without carbon particles, it is difficult to regard them as anything other than lymphocytes (fig. 10, 11).
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cord blood tend to show greater irregularity in nuclear shape than those of the adult. Thus, figure 8 is a monocyte with a triradiate nucleus, while figure 9 shows more marked segmentation than one would normally expect to find.
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Lymphocytoid Phagocyte Type II The type II lymphocytoid phagocyte has so far only been found in the cord blood of premature infants. Figure 12 illustrates one of these cells containing carbon, while figure 13 illustrates the appearance without ingested carbon. In marked contrast to the type I phagocyte, this cell possesses a high N:C ratio, and a relatively small amount of basophilic cytoplasm. It is all the more striking that this relatively narrow border of cytoplasm can become so tightly packed with ingested carbon particles.
Discussion Phagocytic Gradient A comparison of the phagocytic activity of neutrophils and monocytes in premature and full-term infants and in adults brings out what may be termed a phagocytic gradient. This is indicated by the presence of phagocytic cells in each group, and by the amount of carbon ingested by the individual cells. The latter parameter can be graded approximately by simple visual assessment of carbon uptake as heavy, medium, or light. Our results show clear evidence of the pres ence of a phagocytic gradient in both monocytes and granulocytes (fig. 1, 2).
Fig. 3-13. Uptake of carbon particles after 3 h incubation by blood cells. Cells from cord blood of premature and full-term infants compared with blood from adults, x 1,200. 3 Heavily loaded neutrophil from premature newborn infant. 4 Lightly loaded neutrophil from blood of normal healthy adult. 5 Heavily loaded monocyte from blood of a prema ture infant. 6 Moderately loaded monocyte from a full-term infant. 7 Lightly loaded mono cyte from normal adult. 8 Trilobate monocyte from blood of premature infant. 9 Marked segmentation in monocyte of full-term infant. Slight carbon uptake. Type I lympho cytoid phagocyte from premature infant. Heavy carbon uptake. 11 Type 1 lymphocytoid cell from adult blood incubated without addition of carbon. Compare with figure 12. 12 Type II lymphoid phagocyte from cord blood of premature infant. Heavy carbon uptake. 13 Type II lymphocytoid cell from blood of premature infant before incubation with carbon. In its general configuration, this cell resembles that of figure 12. The cytoplasm in figures 12 and 13 is basophilic, though this docs not show in the black and white picture.
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Variation in Phagocytic Activity o f Neutrophils One of the most obvious points to emerge in the analysis of the present results is the great variability in the amount of particle uptake by individual neutrophils even of one and the same subject. The reasons for this are not clear. One possible source of difficulty may be the different types of particle em ployed, e.g. starch particles (14), Higgins’ India ink (10), and polystyrene par ticles (3).
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A further source of difficulty lies in the action of serum factors such as opsonins (5, 9, 18). Cord sera apparently possesses a low degree of opsonic activity in comparison to that of adult blood (4, 25). In some instances, opsonic activity may even be markedly deficient (25). This might conceivably have some bearing on the almost complete absence of neutrophil phagocytic activity in some of our infants. For phagocytosis of monocytes, opsonins may not be so essential, since these cells were found to display phagocytic activity in all the cord blood examined. In this respect cord blood monocytes would resemble adult blood monocytes, which can be phagocytic even in the absence of serum (3).
Lymphocytoid Cells and Monocytes The finding of phagocytic lymphoid cells raised the question whether these were a distinctive cell group belonging to the lymphoid series, or whether they might be atypical forms of monocytes.
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Metabolic Factors Apart from opsonins, metabolic factors influence the phagocytic activity of neutrophils. Even in the resting stage, oxygen consumption by neutrophils of newborn infants is significantly higher than in matched maternal leukocytes (17). Also increased is the number of neutrophils which will spontaneously reduce dye substances such as nitroblue tetrazolium (NBT) (11). The question then arises, whether cells with a high oxygen consumption already in the resting stage, would on that account be more actively phagocytic. Our own findings would be wholly consistent with such a view, through they do not prove it. Alternatively, it could be argued that these phagocytic cells activated already in the resting stage, also had altered cell membrane structures, more permeable not only to the dye NBT, but also facilitating the entry into the cell of carbon particles. The marked difference in carbon uptake between premature and full term infants observed in this study would then be interpreted to repre sent differences in cell membrane permeability between the two groups. How ever, it is difficult to imagine that carbon particles of 200-500 A in size would diffuse through a cell membrane as readily as the NBT solution. From what we know about the NBT test, it appears most likely that the greater reactivity of newborn infants’ granulocytes in this test is due to the increased rate of oxydative reduction of the dye secondary to the activated cell metabolism, rather than to an increased amount of unreduced NBT diffusing into the cell. We therefore believe that the increased number of carbon particles found inside of leukocytes particularly in premature infants, and to a lesser but still substantial degree in full-term infants — in comparison to adult leukocytes — is due to true phago cytosis and not the result of a facilitated diffusion of particles secondary to an altered membrane permeability.
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A cursory survey of the literature suffices to bring out the fact that mono cytes are a very pleomorphic group of cells (2, 13, 29). Though the nucleus is generally regarded as being bean-shaped, with a characteristic nuclear ‘h o f, it may not infrequently be oval or circular, as in the lymphoid cells. However, the internal structure of the monocyte nucleus, often described as ‘lacy’, ‘skein-like’ or ‘reticular’, appears to be regarded as more distinctive than the actual shape. The cytoplasm, furthermore, in films stained with Wright’s stain, as in the pres ent study, is described by Wintrobe (29) as ‘greyish or cloudy blue’, and possessing ‘abundant fine, lilac or reddish blue granules’. By these criteria, neither of the two types of lymphocytes can be classed as monocytes. The type II cell has a basophilic cytoplasm and can certainly not be regarded as monocytic. The type I lymphocytoid cell, with its pale cytoplasm, is also not a monocyte, if the presence of the characteristic monocytic granules is a valid criterion. Most of the pale lymphocytoid cells do not have granules. Occasion ally, they do have a few azurophilic granules, which, however, are typically lymphocytic (15). A few of the type I cells contain nuclei whose structures are not too different from that of monocytic nuclei, but for the most part these cells possess nuclei which are typically pachychromatic, as in lymphocytes. In their classical study, Downey and McKinlay (6) described a cell with an abundance of pale cytoplasm — their type III lymphocyte resembling in its general morphology the type I cell of this study. But it is difficult to press this comparison too closely, since their description was based on pathological mate rial (7). Furthermore, the authors (6, 7) make no reference to the phagocytic properties of these cells. Lymphocytes are not generally regarded as phagocytic. However, in her investigation of the phagocytosis of latex particles by blood ‘mononuclear cells’, Zucker-Franklin (33; fig. 2a, b, c) has provided evidence of phagocytic cells, which even by electron microscopy were ‘... difficult to distinguish from lym phocytes’. She came to the conclusion that ‘About 6—8 % of the cells containing particles cannot be identified with certainty ... Therefore, the possibility cannot be excluded that a very small percentage of bona fide lymphocytes are also able to take up particles...’ A fuller discussion of the phagocytic properties of lym phocytes or of lymphocyte derivatives will be found elsewhere (27, 32). The presence of the lymphocytoid cells adds one more piece of evidence to recent observations that, on both morphological and functional grounds, there are important differences between the blood lymphocyte of the fetus and neo nate, as compared with those in postnatal life: (a) Neonatal lymphocytes contain a higher proportion of medium and large cells than the blood of adults (8); (b) medium-size lymphocytes in both neonatal (8) and prenatal (28) blood possess the characteristic morphology of transitional cells, as observed by both light and electron microscopy; (c) in view of the proliferative properties of tran sitional cells, it is not therefore surprising that prenatal and neonatal lympho
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cytes contain a higher proportion of cells in spontaneous DNA synthesis than lymphocytes in the blood of adults (8, 22, 28); (d) neonatal lymphocytes have been reported to respond to PHA to a greater extent than lymphocytes in the blood of adults (20); (e) phagocytic lymphocytoid cells with basophilic cyto plasm (the type 11 cells of this study) have so far been found only in cord blood of premature infants. The question at once arises: What is the significance of the differences in the lymphocyte populations of fetal and adult blood? If it is permitted at this point to speculate, the answer to this question may be twofold, partly immunological and partly haematological. Immunologically speaking, the differences in the blood lymphocyte population may conceivably reflect to a large extent the differences in the antigenic stimulation before and after birth as compared to adult life (8, 16,26). As far as haematopoiesis is concerned, it is now well-established (31) that the blood of many mammals, and presumably of man also, contains haemato poietic stem cells. In the case of the mouse, the number of stem cells in fetal blood may be more than 100 times as great as in the adult (1). Though the identity of the stem cell is still a matter of dispute, it has been suggested that in all probability it is a member of the transitional cell group (30). In the human fetus, the majority of the lymphocytes found in the blood in mid-fetal life are transitional cells (28). As full-term approaches, the transitional cells fall, while the small lymphocytes increase. Whatever the true explanation of the differences already recognized between the lymphocyte populations of fetal and adult blood, the lymphocytoid cells now described add another component to the complex and heterogenous group of blood cells which we term ‘lymphocytes’.
A cknowledgements It is a pleasure to place on record our indebtedness to Professor G. Gitlin, for the provision of laboratory facilities in the Anatomy Department, and to Professor G. Izak, of the Department of Haematology, for generous assistance with experimental material. We are also grateful to Mrs. Eva Salomon, for her skilled photomicrographic assistance, and to sister Balya Caleb, for help in obtaining cord blood.
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Barnes, D.W.H. and Loutit, J.F.: Haemopoietic stem cells in peripheral blood. Lancet if: 1138 1141 (1967). Bloom, W.: Lymphocytes and monocytes; theories of haematopoiesis; in Handbook of haematology, vol. 2, p. 1427 (Hoeber, Ne-.v York 1938).
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Cline, M.J. and Lehrer, R.I.: Phagocytosis by human monocytes. Blood 32: 423 -429 (1968) . Dosset, J.H. and Quie, P.C.: Serum bacterial opsonins and polymorphonuclear leuko cytes in mothers and newborns. J. Pediat. 74: 817-818 (1969). Dosset, J.H.; Williams, R.C., and Quie, P.G.: Studies on interaction of bacteria, serum factors, and polymorphonuclear leukocytes in mothers and newborns. Pediatrics 44: 49-57 (1969). Downey, H. and McKindlay, C.A.: Acute lymphadenosis compared with acute lym phatic leukaemia. Archs intern. Med. 32: 82-101 (1932). Drescher, J. und Diedenhofen, H.: Uber die Beziehungen zwischen basophilen und hellen, azurgranulierten Blutzellen der lymphatischen Reaktion, aufgrund von Leuko zytenkulturversuchen beim Pfeifferschen Drüsenfieber. Blut 27: 384-392 (1973). Faulk, W.P.; Goodman, J.R.; Maloney, M.A.; Fudenberg, N.H. and Yoffey, J.M.: Morphology and nucleoside incorporation of human neonatal lymphocytes. Cell Immunol. 8: 166 172 (1973). Forman, M.L. and Stiehm, E.R.: Impaired opsonic activity but normal phagocytosis in low-birth-weight infants. New Engl. J. Med. 281: 926-931 (1969). Gluck, L. and Silverman, W.A.: Phagocytosis in premature infants. Pediatrics 20: 951-957 (1957). Humbert, J.R.; Kurtz, M.L., and Hathaway, W.E.: Increased reduction of nitroblue tetrazolium by neutrophils of newborn infants. Pediatrics 45: 125-128 (1970). Knoll, W.: Die Blutbildung beim Embryo; in Handbuch der allgemeinen Haematologie, vol. 1. p. 553 (Urban & Schwarzenberg, Berlin 1932). Leder, L.-D.: Der Blutmonozyt. Exp. Med. Path. Klin., vol. 23 (Springer, Berlin 1967). Matoth, Y.: Phagocytic and ameboid activities of the leukocytes in the newborn infant. Pediatrics 9: 748-754 (1952). Michaelis, L. and Wolff, A.: Über Granula in Lymphozyten. Virchows Arch. 167: 151-163 (1902). Nathenson, G.; Schorr, J.B., and Litwin, S.D.: Gm factor fetomaternal gamma-globulin incompatibility. Pediat. Res. 5: 2 -9 (1971). Park, B.H.; Holmes, B., and Good, R.A.: Metabolic studies on newborn leukocytes. Pediat. Res. 3: 376 (1969). Pearson, H.A.: Phagocytosis by leukocytes of newborn infants. J. Pediat. 74: 329 - 330 (1969) . Playfair, J.H.L.; Wolfendale, M.R., and Kay, H.E.M.: The leukocytes of peripheral blood in the human foetus. Brit. J. Haemat. 9: 336-344 (1963). Prindull, G.: An in vitro quantitative study of phytohaemagglutinin (PHA) induced transformation of lymphocytes from premature newborn infants, from older prema ture infants, and from full-term newborn infants. Blut 2.?.' 7-13 (1971). Prindull, G.: Maturation of cellular and humoral immunity during human embryonic development. Acta paediat., Stockh. 63: 607-615 (1974). Prindull, G.: Spontaneous DNA synthesis of blood lymphoid cells in premature new born infants, in older premature infants, and in full-term newborn infants. Z. Kinderheilk. 118: 197-206(1974). Prindull, G. and Prindull, B.: Leukocyte reserves of newborn infants. I. Observations during exchange transfusions. Blut 21: 79-89 (1970). Prindull, G. and Prindull, B.: Leukocyte reserves of newborn infants. II. Restoration of new leukocyte circulating levels after exchange transfusion. Blut 2/.' 155-161 (1970). Stossei, T.P.; Alper, C.A., and Rosen, F.S.: Opsonic activity in the newborn; role of properdin. Pediatrics 52: 134-137 (1973).
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Steinberg, A.G.: Genetic variations in human immunoglobulins: the Gm and I nv types: in Advances in Immunogenetics, p. 75 (Lippincott, Philadelphia 1967). 27 Trowell, O.A.: Lymphocytes; in Willmer Cells and tissues in culture, pp. 95 172 (Academic Press, New York 1965). 28 Winter, C.C.B.; Byles, A.B., and Yoffey, J.M.: Blood lymphocytes in newborn and adults. Lancet ii: 932-933 (1965). 29 Wintrobe, M.M.: Clinical haematology, 6th ed. (Lea & Febiger, Philadelphia 1967). 30 Yoffey, J.M.: The stem cell problem in the foetus. Israel J. Med. Scis 7: 825-831 (1971). 31 Yoffey, J.M.: Bone marrow in hypoxia and rebound (Thomas, Springfield 1974). 32 Yoffey, J.M. and Courtice, F.C.: Lymphatic, lymph and the lymphomycloid complex (Academic Press, New York 1970). 33 Zucker-Franklin, D.: The percentage of monocytes among ‘mononuclear’ cell fractions obtained from normal human blood. J. Immunol. 112: 234-240 (1974).
Dr. G. Prindull, Assistant Professor, Univ.-Kinderklinik, Humboldtallce 38,D -3 4 Gottingen (FRG)
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