Immunology 1976 30 825

Lymphocyte receptors I. RECEPTORS FOR Fc OF IgG AND COMPLEMENT (C3b) ON IMMUNOGLOBULINBEARING, ANTIGEN-BINDING AND ANTIBODY-SECRETING CELLS

I.

McCONNELL & C. M. HURD Department of Immunology, Royal Postgraduate Medical School, London

Received 3 November 1975; acceptedfor publication 20 November 1975

Summary. Immunoglobulin (Ig) bearing, antigenbinding and antibody-secreting cells were investigated for the presence of membrane receptors for the Fc part of IgG and for C3b on their surface. Fc and C3b receptors were detected on the surface of most Ig-bearing and of most antigen-binding cells. Fc receptors were also detected on IgM antibodysecreting cells but not on IgG-secreting cells. C3b receptors were not detected on any antibodysecreting cells. These receptors are either lost from the B cell surface as they differentiate into antibodysecreting cells or are blocked in vivo by immune complexes or C3b. INTRODUCTION Lymphocytes possess a variety of surface receptors. B lymphocytes express membrane receptors for antigen (see review by Warner, 1974), the Fc part of IgG (Basten, Miller, Sprent and Pye, 1972), the third component of complement, C3b (Bianco and Nussenzweig, 1970) and certain viruses such as Epstein-Barr virus (Jondal and Klein, 1973). Apart from the antigen receptor which is structurally related to immunoglobulin molecules (McConnell, Correspondence: Dr I. McConnell, MRC Group on Mechanisms in Tumour Immunity, MRC Laboratory of Molecular Biology, University Postgraduate Medical School, Hills Road, Cambridge CB2 20H.

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Munro, Gurner and Coombs, 1969; Raff, Sternberg and Taylor, 1970; Warner, 1974) little is known about the structural or chemical nature of the other 'receptors'. Recent evidence suggests that the Fc receptor on B lymphocytes may be associated with the Ia antigens determined by the I region of the major histocompatibility complex (Dickler, Cone, Kubicek and Sachs, 1975). T lymphocytes similarly express an array of surface receptors. These include antigen receptors (Basten, Miller, Warner and Pye, 1971); 'receptors' for the Fc part of IgG mainly found on subpopulations of T lymphocytes; (Yoshida and Andersson, 1972; Soteriades-Vlachos, Gyongybssy and Playfair, 1974; Stout and Herzenberg, 1975), receptors for heterologous erythrocytes (Brain, Gordon and Willets, 1970; Coombs, Gurner, Wilson, Holm and Lindgren, 1970) and measles virus (Valdimarsson, Agnarsdottir and Lachmann, 1975). Activated T lymphocytes may also express a membrane receptor for C3b (ArnaizVillena, Gydngy6ssy and Playfair, 1974) although this has been disputed by Parish (1975). More recently it has been reported that a proportion of human T lymphocytes possess a membrane receptor for IgM (Moretta, Ferrarini, Durante and Mingari, 1975) and this has been confirmed by McConnell and Hurd (1976). The expression of surface antigens and receptors by differentiating lymphocytes shows considerable

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I. McConnell & C. M. Hurd

heterogeneity. Both quantitative and qualitative changes in the expression of alloantigens, surface receptors and surface immunoglobulin during immunocompetent cell differentiation have been described (Aoki, Hammerling, de Harven, Boyse and Old, 1969; Takahashi, Old, McIntire and Boyse, 1971). It is from this aspect that we have investigated the distribution of Fc and C3b receptors amongst immunoglobulin-bearing, antigen-binding and antibody-secreting cells. MATERIALS AND METHODS

Materials Animals. Six to 8-week-old male and female BALB/c mice were used throughout. Lymphocytes. Spleen cell suspensions were prepared by gently teasing out spleens into minimum essential medium (MEM) (Flow-Laboratories) buffered to pH 7 2 with 20 mm HEPES (Sigma). Lymph node cell suspensions were prepared from axillary and precrural lymph nodes. All cell suspensions were washed three times at 40 in MEMHEPES before use. Antigens. Erythrocytes. Freshly drawn sheep (SRBC) and ox erythrocytes (Ox RBC) were stored at 40 in ACD and used within 10 days. Prior to use they were washed four times in phosphate-buffered saline (PBS). Bovine serum albumin. 5 x crystallized BSA (Fraction V, Armour & Company) in PBS was dialysed against two changes of PBS (200 volumes) and centrifuged before use. Ficoll-Hypaque. 14 per cent Ficoll (Pharmacia, Sweden) in distilled water and 34 per cent Hypaque (Winthrop, Surbiton, England) in distilled water mixed in ratio of 24 volumes Ficoll to 10 volumes Hypaque. Antisera. (a) JgM and IgG antibody to ox RBC. Anti-ox RBC was raised in rabbits by a primary injection of 1 ml 10 per cent ox RBC emulsified in Freund's complete adjuvant and given in two different intramuscular sites. Three weeks later boosters of 0 5 ml 10 per cent ox RBC were given at intervals of 2 weeks. IgM and IgG fractions were prepared from early and late bleeds respectively by chromatography on Sephadex G-200. The IgM and IgG fractions were concentrated by 50 per cent ammonium sulphate precipitation. After dialysis against PBS the fractions were directly agglutinating

to 1/40 and had a haemolytic titre (with guinea-pig complement) of 1/1000-1/2000. The poor agglutinability of certain ox erythrocytes (Uhlenbruck, Seaman and Coombs, 1967) provides ideal particles which can be sensitized with IgG antibody (or IgM antibody and complement) for the detection of Fc and C3b receptors by rosette formation (Hallberg, Gurner and Coombs, 1973). (b) Anti-immunoglobulin sera. Himalayan strain rabbits were primed intra-articularly with 200 ug alum-precipitated, mouse IgG2a myeloma protein together with 4 x 109 heat-killed B. pertussis and similarly boosted at 3-weekly intervals with 50 #g IgG2a in saline. (The IgG2a was a gift from Dr A. Munro, Department of Pathology, Cambridge.) Pig anti-rabbit IgM (W.444) and goat antirabbit IgG (A58) (kindly donated by Dr A. Feinstein, Institute of Animal Physiology, Cambridge) were made class specific by adsorption with a rabbit Fab'-Sepharose 4B immunoadsorbent. (c) Fluorescent anti-rabbit IgG. IgG purified from the goat anti-rabbit IgG serum by DEAE chromatography was conjugated to fluorescein isothiocyanate (FITC) by standard methods. The fluorescein/protein ratio of the conjugate was 2 3. Indicator particles for rosette assays. (a) EAISM and EAIG.Ox erythrocytes sensitized with 1/60 rabbit IgM and 1/100 rabbit IgG anti-ox RBC antibody respectively. Anti-rabbit ,u agglutinated EAI3M but not EA1.G. Anti-y gave the reverse agglutination pattern. (b) EAC. Maximum sensitization with complement for C3b rosette formation was given by a 5 min incubation of 1 ml, 2 per cent EAIM in fresh mouse serum from AKR (C5-deficient) strains diluted 1/2 in complement fixation test (CFT) diluent (Oxoid, London). We have consistently found that 20-30 min incubation (Bianco and Nussenzweig, 1970) yields poor EAC for detecting C3b receptors on lymphocytes. EAC is presumably KAF reacted during the longer incubation period (Lachmann, Hobart and Aston, 1973). The EAC were checked for C3b by agglutination with specific rabbit anti-mouse C3. (c) Fluoresceinated indicator erythrocytes. Sheep (or ox) erythrocytes were directly fluoresceinated as described (Matuhashi, 1964; Wolf, Janeway, Coombs, Catty, Gell and Kelus, 1971). FITC-ox erythrocytes were conjugated to BSA to give BSAFITC-ox RBC by carbodiimide (Golub, Mishell, Weigle and Dutton, 1968) using 1-ethyl-3-(3-dimeth-

Fc and C3b receptors on lymphocytes

aminoproply) carbodiimide HCI (ECDI-Sigma, London). Methods Rosetting assays for Fc and C3b receptors. A suspension-centrifugation technique was used (McConnell et al., 1969) in which 50 p1 of lymphocytes at 2 x 106 ml were mixed with 50 p1 of 2 per cent EA1R (for Fc receptors) or 2 per cent EAC (for C3b receptors) in a 50 mm x 5 mm siliconized glass tube. After centrifugation (1000 r.p.m., 4° for 4 min) the Fc rosettes were counted. For C3b rosettes the tubes were incubated for 30 min at 37°. For counting, the rosettes were gently resuspended and 20 p1 of fluorescein diacetate (diluted 2 x 10-4) added to label live cells (Rotman and Papermaster, 1966) before being loaded on the counting chamber (Neubauer, 0-2 mm deep). Within the rosettes the live cells were strongly fluorescent under incident u.v. illumination. The percentage of resetting lymphocytes was determined from counts on three replicate preparations, 300 lymphocytes per count. Mean figures were expressed as a percentage, 1 s.d. usually being within 6 per cent of the total rosette count. Mixed rosette assay. Different receptors on the one lymphocyte were detected by a mixed fluorescentrosetting assay (Wolf et al., 1971). A mix of fluorescent and non-fluorescent indicator particles each at a concentration suitable for a given receptor, were used in the suspension-centrifugation test. By phase contrast and u.v. illumination the adherent erythrocytes in a single rosette were scored either as being all of the one type, i.e. either all fluorescent or all non-fluorescent (one receptor type) or a mixture of two indicator particles, i.e. fluorescent and nonfluorescent (two receptor types per cell). Antigen-binding rosettes. These were detected as previously described (McConnell et al., 1969). Antigen receptors for BSA were detected on lymphocytes using BSA-conjugated SRBC. Rosette-depletion technique. Rosetted cells were separated from non-rosetted cells by one step gradient fractionation on Ficoll-Hypaque (Bach, Muller and Dardenne, 1970; Parish and Hayward, 1974a, b). In our assay the rosettes were formed in bulk by centrifuging 2 ml of 2 per cent indicator erythrocytes (either EAIG or EAC) with 2 ml of lymphocytes at 4 x 106/ml in 75 mm x 12-5 mm siliconized glass tubes. Control preparations were rosetted with E(ox RBC) alone.

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The cells were resuspended in 0 8 ml MEMHEPES with 10 per cent FCS and a pool (2 ml) overlayed on 3 ml Ficoll-Hypaque gradients in siliconized glass tubes. After centrifugation at 3000 r.p.m. (about 1000 g) for 25 min at 200 the rosettes and red cells were present in the bottom layers of the gradient. Non-rosetted lymphocytes were recovered from the interface and upper parts of the gradient, diluted with medium, washed three times and assayed as described in the text. In the control (E rosetted) this procedure gives a non-specific loss of cells (15-20 per cent) there being additional specific loss of Fc and C3b rosettes in the test (EA- or EAC-rosetted) fractions. In the results the reacting cells are given as a percentage of the recovered cells. Indirect immunofluorescence. Surface immunoglobulin on mouse lymphocytes was detected by indirect immunofluorescence using rabbit antimouse IgG (adsorbed mouse red cells and thymus cells) followed by fluorescent anti-rabbit IgG. Haemolytic, plaque-forming cells (PFC). Direct PFC to SRBC were detected by the Jerne plaque assay, indirect plaques being developed with a rabbit anti-mouse IgG.

RESULTS Relationship between lymphocytes expressing surface immunoglobulin (Ig+), Fc receptors (Fc+) and C3b receptors (C3b+) The tissue distribution of cells expressing surface immunoglobulin, Fc and C3b receptors is summarized in Table 1. Less than 1 per cent of thymus cells were positive and the percentage of Fc+ and C3b+ cells in the spleen was greater than in lymph node. Any comparison, however, between Ig+, Fc+ and C3b+ cells is difficult with spleen cells since they contain a large number of macrophages and monocytes which also express Fc and C3b receptors. Thus in this study most experiments were made on lymph node cell suspensions which contain fewer cells (

Lymphocyte receptors. I. Receptors for Fc of IgG and complement (C3b) on immunoglobulin-bearing, antigen-binding and antibody-secreting cells.

Immunology 1976 30 825 Lymphocyte receptors I. RECEPTORS FOR Fc OF IgG AND COMPLEMENT (C3b) ON IMMUNOGLOBULINBEARING, ANTIGEN-BINDING AND ANTIBODY-SE...
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