Clin. exp. Immunol. (1979) 38, 300-305.
Maturation-linked expression of the Fc (IgG) receptor on developing human bone marrow and peripheral blood granulocytes C. S. SCOTT Department of Haematology, Royal United Hospital, Bath
(Accepted for publication 30 April 1979) SUMMARY
Expression of Fc(IgG) receptors, as demonstrated by IgG-coated ox cells and heat-aggregated IgG, was assessed on normal human peripheral blood and bone marrow neutrophils and their precursors. The results indicate that Fc receptor development is confined to post-mitotic granulocytes, being primarily associated with neutrophil segmentation. Fc receptors were not detected on myeloblasts or promyelocytes. A mean value of 790% and 9900 Fc positive segmented neutrophils were found in bone marrow and peripheral blood respectively. This highly significant difference (P< 0.001) appears to represent a morphologically indistinguishable terminal maturation stage. Inhibition studies with IgG show that the neutrophil Fc receptor is inhibited by aggregated but not, in contrast to the corresponding monocyte receptor, by monomeric (7S) IgG. Applications of these findings to the in vivo function of the neutrophil in health and pathology are discussed. INTRODUCTION The demonstration and characterization of specific membrane receptors on leucocytes has attracted increasing interest in recent years. Much of this work has centred on lymphocyte receptors and in particular, their relationship to in vivo function (Siegal & Good, 1977). The lymphocyte receptor for IgG is associated with B-derived cells (Dickler & Kunkel, 1972) although similar receptor activity is shown by small numbers of T lymphocytes (Ferrarini et al., 1975), particularly when activated. Whilst the function of lymphocyte Fc receptors is unclear (Nussenzweig, 1974) those found on nonlymphoid cells have a more clearly defined role. Receptors for complexed IgG present on neutrophils, monocytes and macrophages (Lo Buglio et al., 1967; Messner & Jelinek 1970) are directly associated with phagocytic function in that particle attachment though specific receptors usually precede ingestion (Rabinovitch, 1967). Although the presence of bound IgG alone may not itself lead to phagocytosis (Ehlenberger & Nussenzweig 1976) binding through the Fc receptor may trigger extracellular release of granule enzymes (Sajnani, Ranadive & Movat, 1976). As the affinity for IgG can alter with the functional state of the cell (Siegal, 1976; Rhodes, 1977) then specific changes may be induced by disease, particularly in conditions where disordered maturation is a feature. Investigation of Fc receptor activity and expression in myelopoetic malignancies should provide additional objective criteria of leukaemic cell abnormalities and, furthermore, may aid their subclassification. Interpretation of abnormal Fc receptor activity requires a prior knowledge of its expression in relation to normal maturation. Whilst this process is clearly complicated in lymphoid cells where subtle changes in morphology accompany maturation, distinct structural changes in myeloid differentiation allow easier assessment of receptor development. Correspondence: Dr C. S. Scott, Department of Haematology, Royal United Hospital, Bath, England. 0099-9141/79/1100-0300$02.00 (© 1979 Blackwell Scientific Publications
300
Expression of Fc(IgG) receptors
301
This study deals primarily with the relationship between granulocytic differentiation and receptor expression. The results clearly show that Fc receptor expression is directly related to morphological differentiation in normal human bone marrow and peripheral blood granulocytes. MATERIALS AND METHODS Preparation of EAIgG. Washed ox cells were resuspended to 107/ml in Gelatin-Veronal Buffer (GVB) pH 7-2 (Ross & Polley, 1976) and incubated at 370C for 1 hr with an equal volume of IgG rabbit anti-ox RBC. The IgG anti-ox (kindly supplied by Dr J. L. Smith) was prepared by inoculating rabbits with washed whole ox cells. The whole serum reagent obtained by bleeding was decomplemented by heat treatment and the IgG fraction purified by ammonium sulphate precipitation, followed by passage through DEAE-cellulose (DE 52 Whatman) and Sephadex G-200 (Pharmacia). The resulting IgG was tested by immunodiffusion and ouchterlony techniques for the presence of IgM, both of which were negative. The antibody concentration used for sensitization was 98 ,pg/ml and corresponded to the dilution of IgG causing least ox cell agglutination whilst still giving maximum numbers of rosettes. This in turn was determined by affinity studies with normal peripheral blood lymphocytes, monocytes and neutrophils. Preparation of immunoglobulins. Human monomeric (7S) IgG was prepared by ammonium sulphate precipitation followed by passage through DEAE-cellulose (Whatman DE52) and gel filtration on Ultrogel ACA 34 (LKB). Fractions at the 7S peak were pooled and the protein concentration measured by absorption at 280 nm. Aggregated IgG was prepared by heating 7S IgG at 630C for 20 min as previously described (Dickler, 1974). Aggregate concentration was measured by calculation of E 280 nm with corrections for light scattering. Isolation of peripheral blood and bone marrow leucocytes. Aspirated bone marrow was anti-coagulated with Heparin (25 i.u./ml) and then mixed with an equal volume of Plasmagel (Laboratoire Roger Bellon, France). Following rapid erythrocyte sedimentation at 1 g in a Wintrobe haematocrit tube the leucocyte rich supernate was washed three times in GVB. Leucocytes were then resuspended to 5 0x 106/ml in 0 05 M Tris-saline pH 8-0 for aggregate binding studies and to 2-0 x 106/ml in GVB, for the rosette assays and IgG inhibition experiments. Bone marrow was obtained by Sternal aspiration in all cases with the exception of D.L. and V.H. which were taken from the Tibia and Iliac crest respectively. Heparinized peripheral blood from normal healthy volunteers was treated in a similar way to bone marrow except that only 0-2 volumes of Plasmagel was used. Demonstration of Fc (IgG) receptors. For the rosette assay 100 ,1 leucocytes were mixed with 100 p1 EAIgG in plastic or siliconized glass tubes, incubated at 4°C for 10 min, and then centrifuged at 25 g for 5 min. Following further incubation for 60 min at 4°C the cell mixture was gently resuspended in cold GVB and cyto-centrifuged onto glass slides at 500 r.p.m. Slides were stained by May-Grunwald Giemsa and examined for rosette formation. Leucocytes were characterized by classical cytology and for this study were conventionally classified into myeloblasts, promyelocytes, myelocytes, meta-myelocytes, non-segmented and segmented neutrophils. A minimum of twenty slides were evaluated with any single marrow preparation. Demonstration of aggregate binding was achieved by mixing leucocytes with aggregated IgG to give a final IgG concentration of 0-8 mg/ml. Following incubation at 25°C for 30 min with regular gentle mixing the cells were washed in cold GVB and cytocentrifuged onto glass slides. Aggregate was visualized immunochemically by the peroxidase-antiperoxidase technique (Mason, Farrell & Taylor, 1975) and counterstained with Haematoxylin. IgG inhibition studies. The ability of monomeric or aggregated IgG to inhibit EAIgG rosette formation with peripheral blood neutrophils was assessed by incubating variable amounts of IgG with the leukocyte EAIgG mixture at 4°C for 10 min before centrifugation at 25 g.
RESULTS FAIgG Rosette Formation by Peripheral Blood Neutrophils Neutrophils from twenty-six normals were examined for their ability to form EAIgG rosettes. Binding f ox cells in most cases exceeded 10 ox cells/neutrophil whilst the mean percentage of neutrophils orming rosettes was 99%4 with a range of 94-100%. For comparison, normal peripheral blood lympho-ytes and monocytes (characterized by a-naphthyl acetate esterase activity) gave means of 24%, (range 3-43%°) and 82%, (range 72-92%) of rosette-forming cells respectively. Aggregate as revealed immunochemically was bound to virtually all neutrophils, and was seen as a lense reaction product. ,AIgG rosette formation by bone marrow neutrophils and their precursors. Neutrophils and their precursors were examined from twelve bone marrow aspirates. Relevant clinical lata including therapy and the conventional marrow report is summarized in Table 1. In all cases it H
C. S. Scott
302
FABLE 1. Clinical data relating to bone marrow aspirates examined Peripheral WBC (count x 10l1)
Marrow
Sex
Age
Clinical history
H.B.
F F F
55 50 57 52 2
Normocytic anaemia Carcinoma breast Anaemia, hepatomegaly Hodgkins disease Diamond-blackfan syndrome
Iron None None None None
9.1 * 74 74 39
74 58 63
Lymphocytic lymphoma
None Hydrallazine None
6(5
Aspirin Brufen Plaquenil None
50 * 74 9-6
V.H. F.B. I).H.
M1
D.L.
F
Il.L.
F4
l).M. R.L.
F F
H.J. M.W. E.L. A.L.
MI F F F
Therapy
Carcinoma bronchus Non-immune haemolvtic anaemia Acute R.A. R.A. nephrotic syndrome Chronic R.A. Raised plasma viscosity
56 60 67 66
*
83
6-0
l\Iarrow report Iron deficiency Normal Iron deficiencv Normal Pure ervthroblastic aplasia Normal Normal
Normoblastic hyperplasia Normal Sideroblastic ervthron Iron deficiency Normal
Results not known.
was considered that the granulocytes were present in normal numbers w hich showed normal distribution and maturation. Expression of Fc receptors as demonstrated by ox EAIgG. Table 2 shows a direct relationship with maturation, a striking increase in the number or affinity of the receptors being seen with neutrophil segmentation. Indeed there are highly significant differences between marrow nonsegmented and segmented neutrophils (P< 0001) and bone marrow and peripheral blood neutrophils (P< 0001) suggesting a rapid terminal expression of the Fc receptor in the maturation sequence. Fc receptor expression by granulocytic cells also appears to be primarily a feature of post-mitotic cells, appearing on relatively few myelocytes and not at all on promyelocytes and myeloblasts. The avidity of ox cell binding also appeared much stronger on segmented neutrophils, similar to that seen with peripheral blood neutrophils whereas the binding on earlier cells was less discrete with relatively fewer ox EAIgG being bound. TABLE 2. Fc (IgG/ox) receptor expression by neutrophils and their precursors (percentage of cells forming rosettes)
Mveloblasts Marrow
Promyelocytes
H.B.
0
V.H1. F.B. D.H.
D.L. ML. D.1M. R.L. H.J.
MW.'A. E.L. A.L. Mean Y 2 s.d.
0 0 0 * 0 * 0 0 0 0
0
1Myelocytes 3 5 25
10 5 10 0 10 6 7 0 6 5-3 72
Meta-myelocytes
Non-segmented neutrophils
15 10 17 6 18 12 12 3 7
34 18 14 22 24 28 1X 36 18 13 19 13
10-6
21-4
95
154
13 10
* Insufficient cells seen.
Peripheral
Neutrophils 88 91 74 74 85 68 89 83 73 71 81 69 78 8 165
neutrophils
26 Normals
98 9 3-0
303
Expression of Fc(IgG) receptors TABLE 3. Inhibition of neutrophil EAIgG rosette formation by IgG*
% Neutrophils forming rosettes IgG concentration (pg/ml)
Monomeric 7S IgG
Aggregated IgG
0 95 250 450 800 1100 1600
>99 >99 > 99 99 97 97 89
>99 66 45 29 26 t t
* Mean values of three experiments. t Not tested.
Phagocytosis of EAIgG was rarely seen, although capping and subsequent loss of rosettes was seen at 370C especially when the ox cells were heavily sensitized (unpublished observations).
Effect of monomeric and aggregated IgG on peripheral blood neutrophil EAIgG binding. From the results in Table 3, it can be seen that the presence of monomeric IgG had very little effect upon neutrophil rosette formation whereas when IgG was aggregated there was decreasing rosette formation with increasing aggregate concentration. Immunochemical staining of bound aggregate in the rosette test incubated with 800 ,ug/ml IgG revealed the presence of a population of cells (26%) which bound aggregate and continued to form partial ox cell rosettes. Further increases in the concentration of aggregate led to cell clumping and thus it was not possible to determine whether neutrophil Fc receptors could be completely saturated by aggregated IgG.
DISCUSSION It has been shown in this investigation that sequential expression of IgG receptors occurs in post-mitotic granulocytes. The major part of receptor synthesis and expression takes place in the bone marrow neutrophil stage and is normally complete before the appearance of the mature cell in the peripheral blood. The exact function of the Fc receptor is not completely known although in the neutrophil is clearly associated with the 'recognition' and binding of IgG-opsonized particles (Stossel, 1975). Previous estimates of the numbers of IgG-binding neutrophils in peripheral blood have shown wide variations and have not approached the levels consistently seen in this study. Messner & Jelinek (1970) using IgG coated Staph. aureus found that 5000 of neutrophils had receptors, whilst Wong & Wilson (1975) and Klempner & Gallin (1978) reported 75-85° rosette-forming neutrophils. These discrepancies are probably due to differing concentrations of indicator-bound IgG employed and reflect the higher IgG loading obtained with the ox cell system. This increased IgG concentration is thus able to detect neutrophils with relatively fewer or lower affinity receptors and is analogous to the varying Fc receptor affinities seen with lymphocyte subpopulations (Winchester et al., 1976). There are no known previous studies on expression of Fc receptors by human bone marrow neutrophils and their precursors, although studies of mouse neutrophils indicate a sequential expression of receptors during maturation (Rabellino et al., 1978). The finding that the neutrophil receptor for complexed IgG is not inhibited by monomeric IgG supports the work of Lawrence, Weigle & Spiegelberg (1975) and contrasts significantly with the monocyte/macrophage Fc receptor which is markedly inhibited by low levels of free IgG (Huber, Douglas & Fudenberg, 1969; Fleer et al., 1978). This latter finding supports in vivo studies showing that non-complement binding autoantibodies in autoimmune haemolytic anaemias are removed primarily by splenic macrophages (Mollison et al., 1965) in an environment where the relative free IgG is much lower
304
C. S. Scott
than in plasma (Van der Meulen et al., 1978) thus causing less receptor inhibition. Neutrophil IgG receptors, however, are apparently unaffected by high levels of free IgG and are presumably able to function throughout the body irrespective of external IgG concentration. Furthermore, studies by Ehlenberger & Nussenzweig (1976) show that neutrophils require large amounts of particle-complexed IgG to be stimulated for ingestion although the presence of even small amounts of bound complement appears to act synergistically with the IgG, greatly enhancing phagocytosis. These findings may be further related in the in vivo function of neutrophils, as non-phagocytic destruction of IgG-complexed particles and non-phagocytosable surfaces appears to be mediated through the Fc receptor by exocytosis of lysosomal enzymes (Hawkins, 1972; Henson & Oades, 1975). This process in pathology is clearly related to the mechanism of tissue damage seen in rheumatoid arthritis (Harris, 1976). The results of these studies provide further characteristics of neutrophil maturation not previously described. Evaluation of Fc receptor expression by neutrophils and their precursors may lead to a better understanding of neutrophil function in chronic inflammatory diseases and may further help to define abnormalities of granulocyte maturation associated with haemapoetic malignancies. I am most grateful to Dr D. Hough (University of Bath) for his guidance and advice with this project, and to Drs T. Ferguson and J. Cusworth for providing the bone marrow samples. I also wish to thank Dr R. Holman and the staff of the Haematology Department, Royal United Hospital, Bath, for their support and Mrs P. Allin for typing the manuscript. The work in this study was in part supported by a grant from the Institute of Medical Laboratory Sciences.
REFERENCES Lo BUGLIO, A.F., CoTRAN, R.S. & JANDL, J.H. (1967) Red cells coated with immunoglobulin G: binding and sphering by mononuclear cells in man. Science, 158, 1582. MASON, D.Y., FARRELL, C. & TAYLOR, C.R. (1975) The detection of intra-cellular antigens in human leucocytes by immunoperoxidase staining. Brit. J. Haemat. 31, 361. MESSNER, R.P. & JELINEK, J. (1970) Receptors for human G globulin on human neutrophils. J. Clin. Invest. 49, 2165. MOLLISON, P.L., CROME, P., HUGHES-JONES, N.C. & ROCHNA, E. (1965) Rate of removal from the circulation of red cells sensitized with different amounts of antibody. Brit. 5. Haemat. 11, 461. NUSSENZWEIG, V. (1974) Receptors for immune complexes on lymphocytes. Adv. in Immunol. 19, 217. RABELLINO, E.M., Ross, G.D., TRING, H.T.K., WILLIAMS, N. & METCALF, D. (1978) Membrane receptors of mouse leukocytes, II. Sequential expression of membrane receptors and phagocytic capacity during leukocyte differentiation. J. exp. Med. 147, 434. RABINOVITCH, M. (1967) Studies on the immunoglobulins which stimulate the ingestion of glutaraldehyde-treated red cells attached to macrophages. J. Immunol. 99, 1115. RHODES, J. (1977) Altered expression of human monocyte Fc receptors in malignant disease. Nature (Lond.) 265, 253. Ross, G.D. & POLLEY, M.J. (1976) Detection of complement receptor lymphocytes (CRL). In vitro Methods in CellMediated and Tumor Immunity. (Ed. by B.R. Bloom & J.R. David), pp. 123-136. Academic Press, New York. SAJNANI, A.N., RANADIVE, N.S. & MOVAT, H.Z. (1976) Redistribution of immunoglobulin receptors on human neutrophils and its relationship to the release of lysosomal enzymes. Lab. Invest. 35, 143. SIEGAL, F.P. (1976) IgG on infants B lymphocytes: Enpopulations. Blood, 52, 659. hanced binding of IgG by IgM bearing lymphoid cells LAWRENCE, D.A., WEIGLE, W.O. & SPIEGELBERG, H.L. in early childhood. Scand. 5. Immunol. 5, 721. (1975) Immunoglobulins cytophilic for human lymphocytes, monocytes and neutrophils. 3. Clin. Invest. 55, 368. SIEGAL, F.P. & GOOD, R.A. (1977) Human lymphocyte
DICKLER, H.B. (1974) Studies of the human lymphocyte receptor for heat aggregated or antigen complexed immunoglobulin. 3. Exp. Med. 140, 508. DIcKLu, H.B. & KUNKEL, H. (1972) Interaction of aggregated gamma-globulin with B lymphocytes. 3. Exp. Med. 136, 191. EHLENBERGER, A.G. & NussENzwEIG, V. (1976) Immunologically-mediated phagocytosis: role of C3 and Fc receptors. Clinical Evaluation ofImmune Function in Man (ed. by S.D. Litman, C.L. Christian & G.W. Siskind), pp. 47-64. Grune & Stratton, New York. FERRARINI, M., MORETTA, L., ABRILLE, R. & DURANTE, M.L. (1975) Receptors for IgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells. Eur. .7. Immunol. 5, 70. FLEER, A., VAN DER MEuLEN, F.W., LINTHOUT, E., VON DEM BORNE, A.E.G. & ENGELFRIET, C.P. (1978) Destruction of IgG-sensitized erythrocytes by human blood monocytes: Modulation of inhibition by IgG. Brit. 3. Haemat. 39, 425. HARRIS, E.D. (1976) Recent insights into the pathogenesis of the proliferative lesion in rheumatoid arthritis. Arthr. and Rheum. 19, 68. HAWKINS, D. (1972) Neutrophilic leukocytes in immunologic reactions. Evidence for the selective release of lysosomal constituents. _'. Immunol. 108, 310. HENSON, P.M. & OADEs, Z.G. (1975) Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. Secretion of granule constituents and increased oxidation of glucose. J. Clin. Invest. 56, 1053. HUBER, H., DOUGLAS, S.D. & FUDENBERG, H.H. (1969) The IgG receptor; an immunological marker for the characterization of mononuclear cells. Immunology, 17, 7. KLEMPNER, M.S. & GALLIN, J.I. (1978) Separation and functional characterization of human neutrophil sub-
Expression of Fc(IgG) receptors differentiation markers and their application to immune deficiency and lymphoproliferative diseases. Clinics in Haemat. 6.2, 355. STOSSEL, T.P. (1975) Phagocytosis: Recognition and ingestion. Neutrophil Physiology and Pathology (ed. by J.R. Humber, P.A. Miescher & E.W. Jaff6), pp. 93-126. Grune & Stratton, New York. VAN DER MEULEN, F.W., VAN DER HART, M., FLEER, A., VON DEM BORNE, A.E.G, ENGELFRIET, C.P. & VAN LOGHEM, J.J. (1978) The role of adherence to human mononuclear phagocytes in the destruction of red cells
305
sensitized with non-complement binding antibodies. Brit. a. Haemat. 38, 541. WINCHESTER, R.J., Fu, S.M., WANG, C.Y., HOFFMAN, T. & KUNKEL, H.G. (1976) Lymphocyte surface markers: Evidence for three subpopulations of Fc receptor bearing lymphocytes. Clinical Evaluation of Immune Function in Man (ed. by S.D. Litwin, C.L. Christian & G.W. Siskind), pp. 1-11. Grune & Stratton, New York. WONG, L. & WILSON, J.D. (1975) The identification of Fc and C3 receptors on human neutrophils. J. Immunol. Methods 7, 69.
Maturation-linked expression of the Fc (IgG) receptor on developing human bone marrow and peripheral blood granulocytes C. S. SCOTT Department of Haematology, Royal United Hospital, Bath
(Accepted for publication 30 April 1979) SUMMARY
Expression of Fc(IgG) receptors, as demonstrated by IgG-coated ox cells and heat-aggregated IgG, was assessed on normal human peripheral blood and bone marrow neutrophils and their precursors. The results indicate that Fc receptor development is confined to post-mitotic granulocytes, being primarily associated with neutrophil segmentation. Fc receptors were not detected on myeloblasts or promyelocytes. A mean value of 790% and 9900 Fc positive segmented neutrophils were found in bone marrow and peripheral blood respectively. This highly significant difference (P< 0.001) appears to represent a morphologically indistinguishable terminal maturation stage. Inhibition studies with IgG show that the neutrophil Fc receptor is inhibited by aggregated but not, in contrast to the corresponding monocyte receptor, by monomeric (7S) IgG. Applications of these findings to the in vivo function of the neutrophil in health and pathology are discussed. INTRODUCTION The demonstration and characterization of specific membrane receptors on leucocytes has attracted increasing interest in recent years. Much of this work has centred on lymphocyte receptors and in particular, their relationship to in vivo function (Siegal & Good, 1977). The lymphocyte receptor for IgG is associated with B-derived cells (Dickler & Kunkel, 1972) although similar receptor activity is shown by small numbers of T lymphocytes (Ferrarini et al., 1975), particularly when activated. Whilst the function of lymphocyte Fc receptors is unclear (Nussenzweig, 1974) those found on nonlymphoid cells have a more clearly defined role. Receptors for complexed IgG present on neutrophils, monocytes and macrophages (Lo Buglio et al., 1967; Messner & Jelinek 1970) are directly associated with phagocytic function in that particle attachment though specific receptors usually precede ingestion (Rabinovitch, 1967). Although the presence of bound IgG alone may not itself lead to phagocytosis (Ehlenberger & Nussenzweig 1976) binding through the Fc receptor may trigger extracellular release of granule enzymes (Sajnani, Ranadive & Movat, 1976). As the affinity for IgG can alter with the functional state of the cell (Siegal, 1976; Rhodes, 1977) then specific changes may be induced by disease, particularly in conditions where disordered maturation is a feature. Investigation of Fc receptor activity and expression in myelopoetic malignancies should provide additional objective criteria of leukaemic cell abnormalities and, furthermore, may aid their subclassification. Interpretation of abnormal Fc receptor activity requires a prior knowledge of its expression in relation to normal maturation. Whilst this process is clearly complicated in lymphoid cells where subtle changes in morphology accompany maturation, distinct structural changes in myeloid differentiation allow easier assessment of receptor development. Correspondence: Dr C. S. Scott, Department of Haematology, Royal United Hospital, Bath, England. 0099-9141/79/1100-0300$02.00 (© 1979 Blackwell Scientific Publications
300
Expression of Fc(IgG) receptors
301
This study deals primarily with the relationship between granulocytic differentiation and receptor expression. The results clearly show that Fc receptor expression is directly related to morphological differentiation in normal human bone marrow and peripheral blood granulocytes. MATERIALS AND METHODS Preparation of EAIgG. Washed ox cells were resuspended to 107/ml in Gelatin-Veronal Buffer (GVB) pH 7-2 (Ross & Polley, 1976) and incubated at 370C for 1 hr with an equal volume of IgG rabbit anti-ox RBC. The IgG anti-ox (kindly supplied by Dr J. L. Smith) was prepared by inoculating rabbits with washed whole ox cells. The whole serum reagent obtained by bleeding was decomplemented by heat treatment and the IgG fraction purified by ammonium sulphate precipitation, followed by passage through DEAE-cellulose (DE 52 Whatman) and Sephadex G-200 (Pharmacia). The resulting IgG was tested by immunodiffusion and ouchterlony techniques for the presence of IgM, both of which were negative. The antibody concentration used for sensitization was 98 ,pg/ml and corresponded to the dilution of IgG causing least ox cell agglutination whilst still giving maximum numbers of rosettes. This in turn was determined by affinity studies with normal peripheral blood lymphocytes, monocytes and neutrophils. Preparation of immunoglobulins. Human monomeric (7S) IgG was prepared by ammonium sulphate precipitation followed by passage through DEAE-cellulose (Whatman DE52) and gel filtration on Ultrogel ACA 34 (LKB). Fractions at the 7S peak were pooled and the protein concentration measured by absorption at 280 nm. Aggregated IgG was prepared by heating 7S IgG at 630C for 20 min as previously described (Dickler, 1974). Aggregate concentration was measured by calculation of E 280 nm with corrections for light scattering. Isolation of peripheral blood and bone marrow leucocytes. Aspirated bone marrow was anti-coagulated with Heparin (25 i.u./ml) and then mixed with an equal volume of Plasmagel (Laboratoire Roger Bellon, France). Following rapid erythrocyte sedimentation at 1 g in a Wintrobe haematocrit tube the leucocyte rich supernate was washed three times in GVB. Leucocytes were then resuspended to 5 0x 106/ml in 0 05 M Tris-saline pH 8-0 for aggregate binding studies and to 2-0 x 106/ml in GVB, for the rosette assays and IgG inhibition experiments. Bone marrow was obtained by Sternal aspiration in all cases with the exception of D.L. and V.H. which were taken from the Tibia and Iliac crest respectively. Heparinized peripheral blood from normal healthy volunteers was treated in a similar way to bone marrow except that only 0-2 volumes of Plasmagel was used. Demonstration of Fc (IgG) receptors. For the rosette assay 100 ,1 leucocytes were mixed with 100 p1 EAIgG in plastic or siliconized glass tubes, incubated at 4°C for 10 min, and then centrifuged at 25 g for 5 min. Following further incubation for 60 min at 4°C the cell mixture was gently resuspended in cold GVB and cyto-centrifuged onto glass slides at 500 r.p.m. Slides were stained by May-Grunwald Giemsa and examined for rosette formation. Leucocytes were characterized by classical cytology and for this study were conventionally classified into myeloblasts, promyelocytes, myelocytes, meta-myelocytes, non-segmented and segmented neutrophils. A minimum of twenty slides were evaluated with any single marrow preparation. Demonstration of aggregate binding was achieved by mixing leucocytes with aggregated IgG to give a final IgG concentration of 0-8 mg/ml. Following incubation at 25°C for 30 min with regular gentle mixing the cells were washed in cold GVB and cytocentrifuged onto glass slides. Aggregate was visualized immunochemically by the peroxidase-antiperoxidase technique (Mason, Farrell & Taylor, 1975) and counterstained with Haematoxylin. IgG inhibition studies. The ability of monomeric or aggregated IgG to inhibit EAIgG rosette formation with peripheral blood neutrophils was assessed by incubating variable amounts of IgG with the leukocyte EAIgG mixture at 4°C for 10 min before centrifugation at 25 g.
RESULTS FAIgG Rosette Formation by Peripheral Blood Neutrophils Neutrophils from twenty-six normals were examined for their ability to form EAIgG rosettes. Binding f ox cells in most cases exceeded 10 ox cells/neutrophil whilst the mean percentage of neutrophils orming rosettes was 99%4 with a range of 94-100%. For comparison, normal peripheral blood lympho-ytes and monocytes (characterized by a-naphthyl acetate esterase activity) gave means of 24%, (range 3-43%°) and 82%, (range 72-92%) of rosette-forming cells respectively. Aggregate as revealed immunochemically was bound to virtually all neutrophils, and was seen as a lense reaction product. ,AIgG rosette formation by bone marrow neutrophils and their precursors. Neutrophils and their precursors were examined from twelve bone marrow aspirates. Relevant clinical lata including therapy and the conventional marrow report is summarized in Table 1. In all cases it H
C. S. Scott
302
FABLE 1. Clinical data relating to bone marrow aspirates examined Peripheral WBC (count x 10l1)
Marrow
Sex
Age
Clinical history
H.B.
F F F
55 50 57 52 2
Normocytic anaemia Carcinoma breast Anaemia, hepatomegaly Hodgkins disease Diamond-blackfan syndrome
Iron None None None None
9.1 * 74 74 39
74 58 63
Lymphocytic lymphoma
None Hydrallazine None
6(5
Aspirin Brufen Plaquenil None
50 * 74 9-6
V.H. F.B. I).H.
M1
D.L.
F
Il.L.
F4
l).M. R.L.
F F
H.J. M.W. E.L. A.L.
MI F F F
Therapy
Carcinoma bronchus Non-immune haemolvtic anaemia Acute R.A. R.A. nephrotic syndrome Chronic R.A. Raised plasma viscosity
56 60 67 66
*
83
6-0
l\Iarrow report Iron deficiency Normal Iron deficiencv Normal Pure ervthroblastic aplasia Normal Normal
Normoblastic hyperplasia Normal Sideroblastic ervthron Iron deficiency Normal
Results not known.
was considered that the granulocytes were present in normal numbers w hich showed normal distribution and maturation. Expression of Fc receptors as demonstrated by ox EAIgG. Table 2 shows a direct relationship with maturation, a striking increase in the number or affinity of the receptors being seen with neutrophil segmentation. Indeed there are highly significant differences between marrow nonsegmented and segmented neutrophils (P< 0001) and bone marrow and peripheral blood neutrophils (P< 0001) suggesting a rapid terminal expression of the Fc receptor in the maturation sequence. Fc receptor expression by granulocytic cells also appears to be primarily a feature of post-mitotic cells, appearing on relatively few myelocytes and not at all on promyelocytes and myeloblasts. The avidity of ox cell binding also appeared much stronger on segmented neutrophils, similar to that seen with peripheral blood neutrophils whereas the binding on earlier cells was less discrete with relatively fewer ox EAIgG being bound. TABLE 2. Fc (IgG/ox) receptor expression by neutrophils and their precursors (percentage of cells forming rosettes)
Mveloblasts Marrow
Promyelocytes
H.B.
0
V.H1. F.B. D.H.
D.L. ML. D.1M. R.L. H.J.
MW.'A. E.L. A.L. Mean Y 2 s.d.
0 0 0 * 0 * 0 0 0 0
0
1Myelocytes 3 5 25
10 5 10 0 10 6 7 0 6 5-3 72
Meta-myelocytes
Non-segmented neutrophils
15 10 17 6 18 12 12 3 7
34 18 14 22 24 28 1X 36 18 13 19 13
10-6
21-4
95
154
13 10
* Insufficient cells seen.
Peripheral
Neutrophils 88 91 74 74 85 68 89 83 73 71 81 69 78 8 165
neutrophils
26 Normals
98 9 3-0
303
Expression of Fc(IgG) receptors TABLE 3. Inhibition of neutrophil EAIgG rosette formation by IgG*
% Neutrophils forming rosettes IgG concentration (pg/ml)
Monomeric 7S IgG
Aggregated IgG
0 95 250 450 800 1100 1600
>99 >99 > 99 99 97 97 89
>99 66 45 29 26 t t
* Mean values of three experiments. t Not tested.
Phagocytosis of EAIgG was rarely seen, although capping and subsequent loss of rosettes was seen at 370C especially when the ox cells were heavily sensitized (unpublished observations).
Effect of monomeric and aggregated IgG on peripheral blood neutrophil EAIgG binding. From the results in Table 3, it can be seen that the presence of monomeric IgG had very little effect upon neutrophil rosette formation whereas when IgG was aggregated there was decreasing rosette formation with increasing aggregate concentration. Immunochemical staining of bound aggregate in the rosette test incubated with 800 ,ug/ml IgG revealed the presence of a population of cells (26%) which bound aggregate and continued to form partial ox cell rosettes. Further increases in the concentration of aggregate led to cell clumping and thus it was not possible to determine whether neutrophil Fc receptors could be completely saturated by aggregated IgG.
DISCUSSION It has been shown in this investigation that sequential expression of IgG receptors occurs in post-mitotic granulocytes. The major part of receptor synthesis and expression takes place in the bone marrow neutrophil stage and is normally complete before the appearance of the mature cell in the peripheral blood. The exact function of the Fc receptor is not completely known although in the neutrophil is clearly associated with the 'recognition' and binding of IgG-opsonized particles (Stossel, 1975). Previous estimates of the numbers of IgG-binding neutrophils in peripheral blood have shown wide variations and have not approached the levels consistently seen in this study. Messner & Jelinek (1970) using IgG coated Staph. aureus found that 5000 of neutrophils had receptors, whilst Wong & Wilson (1975) and Klempner & Gallin (1978) reported 75-85° rosette-forming neutrophils. These discrepancies are probably due to differing concentrations of indicator-bound IgG employed and reflect the higher IgG loading obtained with the ox cell system. This increased IgG concentration is thus able to detect neutrophils with relatively fewer or lower affinity receptors and is analogous to the varying Fc receptor affinities seen with lymphocyte subpopulations (Winchester et al., 1976). There are no known previous studies on expression of Fc receptors by human bone marrow neutrophils and their precursors, although studies of mouse neutrophils indicate a sequential expression of receptors during maturation (Rabellino et al., 1978). The finding that the neutrophil receptor for complexed IgG is not inhibited by monomeric IgG supports the work of Lawrence, Weigle & Spiegelberg (1975) and contrasts significantly with the monocyte/macrophage Fc receptor which is markedly inhibited by low levels of free IgG (Huber, Douglas & Fudenberg, 1969; Fleer et al., 1978). This latter finding supports in vivo studies showing that non-complement binding autoantibodies in autoimmune haemolytic anaemias are removed primarily by splenic macrophages (Mollison et al., 1965) in an environment where the relative free IgG is much lower
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than in plasma (Van der Meulen et al., 1978) thus causing less receptor inhibition. Neutrophil IgG receptors, however, are apparently unaffected by high levels of free IgG and are presumably able to function throughout the body irrespective of external IgG concentration. Furthermore, studies by Ehlenberger & Nussenzweig (1976) show that neutrophils require large amounts of particle-complexed IgG to be stimulated for ingestion although the presence of even small amounts of bound complement appears to act synergistically with the IgG, greatly enhancing phagocytosis. These findings may be further related in the in vivo function of neutrophils, as non-phagocytic destruction of IgG-complexed particles and non-phagocytosable surfaces appears to be mediated through the Fc receptor by exocytosis of lysosomal enzymes (Hawkins, 1972; Henson & Oades, 1975). This process in pathology is clearly related to the mechanism of tissue damage seen in rheumatoid arthritis (Harris, 1976). The results of these studies provide further characteristics of neutrophil maturation not previously described. Evaluation of Fc receptor expression by neutrophils and their precursors may lead to a better understanding of neutrophil function in chronic inflammatory diseases and may further help to define abnormalities of granulocyte maturation associated with haemapoetic malignancies. I am most grateful to Dr D. Hough (University of Bath) for his guidance and advice with this project, and to Drs T. Ferguson and J. Cusworth for providing the bone marrow samples. I also wish to thank Dr R. Holman and the staff of the Haematology Department, Royal United Hospital, Bath, for their support and Mrs P. Allin for typing the manuscript. The work in this study was in part supported by a grant from the Institute of Medical Laboratory Sciences.
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