Immunology 1977 32 955
The role of Fc and C3b receptors in phagocytosis by inflammatory polymorphonuclear leucocytes in man
J. M. A. WILTON, H. H. RENGGLI * & T. LEHNER Department of Oral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London and *Department of Periodontology, University of Nijmegen, The Netherlands
Received 1 October 1976; acceptedfor publication 5 November 1976
Summary. Polymorphonuclear leucocytes from the gingival crevice (CREV-PMN) in man have a defective capacity to phagocytose Candida albicans blastospores. Phagocytosis of zymosan particles, which detect C3b receptors, is also impaired but ingestion of latex beads coated with heat-aggregated IgG, which detects Fc receptors, is normal compared to peripheral blood polymorphonuclear leucocytes (PB-PMN). If phagocytosis is inhibited by Cytochalasin B, fewer CREV-PMN bind Candida and zymosan but the binding of IgG-coated latex beads remains unchanged. CREV-PMN have IgG (88%) IgM (45 ) and C3 (48 Y.) on their cell membrane, whilst less than 5%Y. of PB-PMN have any of these components. Incubation of PB-PMN in fluid from the gingival crevice confers surface IgG and C3 to the cells. Such treatment also inhibits the subsequent binding of IgG coated latex beads. The results suggest that the deficiency of phagocytosis by CREVPMN is due to decreased binding of particles to the C3b receptor of PMN, whilst the Fc receptor system remains intact.
large numbers through the mucosa of the intestina and respiratory tracts, but the functional ability of these cells has not been investigated. A convenient source of PMN which have migrated through epithelium are those found in the mouth at the gingival crevice (Attstrom, 1970; Skapski & Lehner, 1976). We have recently shown in man that the gingival crevicular polymorphonuclear leucocytes (CREVPMN) are capable of phagocytosing and killing Candida albicans (CA) blastospores (Wilton, Renggli & Lehner 1977). However the CREV-PMN are defective when compared with the peripheral blood polymorphonuclear leucocytes (PB-PMN) from the same subject, in that a smaller number of cells phagocytose CA blastospores and fewer particles are taken up by CREV-PMN than PB-PMN. Nevertheless, the cells from both sources kill CA with equal efficiency (Wilton et al., 1977). In patients with rheumatoid arthritis, Turner, Shumacher & Myers (1973) found that PMN from synovial fluid take up fewer bakers' yeast particles than blood PMN from the same subject. They speculated that this deficiency could be due to previous endocytosis in vivo of IgG rheumatoid factor complexes; such complexes when taken up by normal blood PMN would inhibit subsequent phagocytosis of yeast (Turner et al., 1973). Our own data (Wilton et al., 1977) could be interpreted as an impaired function of either the
INTRODUCTION
Polymorphonuclear leucocytes (PMN) migrate in Correspondence: Dr J. M. A. Wilton, Department of Oral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London SEI 9RT.
I
955
956
J. M. A. Wilton, H. H. Renggli & T. Lehner
PMN receptor for the activated third component of complement (Henson, 1969) or the Fc portion of immunoglobulin (Henson, 1969; Messner & Jelinek, 1970) on the surface of the CREV-PMN, leading to diminished phagocytosis of the CA blastospores. There is now evidence that the two receptors may have different functions in both murine (Mantovani, 1975) and human PMN (Scribner & Fahrney, 1976). The C3b receptor principally mediates particle binding whilst the Fc receptor mediates both particle binding and initiates endocytosis. The purpose of this investigation was to compare both the binding and phagocytosis of particles by CREV-PMN and PB-PMN, in an attempt to determine whether the phagocytic defect observed in the CREV-PMN is due to an Fc or C3b receptor deficiency. We have used serum-treated zymosan particles (Gewurz, 1972) to detect the C3b receptor (Goldstein, Roos, Kaplan & Weissman, 1975) and heat-aggregated human IgG bound to latex particles to detect the Fc receptor.
MATERIALS AND METHODS
Isolation of glass-adherent CREV-PMN Crevicular washings were collected from twelve healthy human subjects of both sexes, aged 20-44 years, by the method of Skapski & Lehner (1976). The individual washings in Hanks's balanced salt solution (HBSS) were buffered to 7-2 with sodium bicarbonate and were then pooled and washed once in HBSS by centrifugation. After the removal of a sample for viable counting using trypan blue exclusion, the specimen was adjusted to a final volume of 200 p1 with a final concentration of 30%. autologous serum in HBSS. The mean number of PMN recovered from the washings was 92,648 ± s.d. (21658) with a mean viability of 84 6 ±4 7%. Fifty-microlitre aliquots were then placed onto the glass 'wells' of a glass slide coated with Hilflow (Jencons, Hemel Hempstead), which had been covered by four templates each of 5 mm diameter. Thus each slide contained four separate 50-.ul drops. After incubation at 370 for 60 min in a moist chamber, the slides were washed first by gentle aspiration and the replacement of fresh HBSS several times and then by immersing the slide in a beaker of warmed HBSS. Each well contained only glass-adherent cells and differential counts of
Giemsa-stained preparations showed greater than 98%Y. PMN. Isolation of glass-adherent PB-PMN Blood was obtained by venepuncture and the erythrocytes were separated by the addition of Dextran (T250 Pharmacia, Uxbridge) to a final concentration of 3 % (v/v). The leucocyte-rich plasma was aspirated after 60 min at 370 and layered onto a Ficol-Hypaque gradient. After centrifugation, the pellet was suspended in a large excess of buffered isotonic ammonium chloride for 10 min at 4°. The cells were washed twice in HBSS, adjusted to the concentration of the CREV-PMN in HBSS containing 30%o autologous serum and then attached to glass as outlined for the CREV-PMN.
Phagocytosis assay (1) Candida albicans. The phagocytosis of live Candida albicans blastospores was assayed by the method of Schmid & Brune (1974), modified for the slides used in this study. Viable CA blastospores were added to each well in a total volume of 50 u1 in HBSS, containing 30%. fresh autologous serum, at a particle ratio of 5 CA: 1 PMN and incubated for 30 min in a moist chamber at 37°. The preparation was washed in HBSS to remove free CA and mounted in HBSS for viewing. A minimum of 200 cells was counted and the number of PMN phagocytosing one or more CA expressed as a percentage of the total cell count. (2) Zymosan. Zymosan A (Sigma, London) was heated at 1000 for 1 h and suspended in HBSS at 1 mg/ml. One milligram samples of zymosan were incubated with 0 5 ml of fresh human serum for 30 min at 370 and washed three times in HBSS. After adjusting the concentration to 0 5 mg/ml the zymosan was stored in small aliquots at -70°. The particles were then used in the phagocytosis assay as outlined for CA, by adding 50 ,ul of the suspension to each well. (3) IgG-coated latex beads. Five millilitres of IgGcoated latex beads (Rheuma-Wellcotest, Wellcome Reagents, Beckenham) were washed three times in HBSS and resuspended in 10 ml of HBSS. The beads were stored at 40 and used in the phagocytosis assay at 50 p1 per well.
Particle-binding assays. The binding of CA blasto-
957
Fc and C3b receptors in phagocytosis
spores, zymosan and IgG-coated latex beads was estimated after inhibiting phagocytosis with Cytochalasin B (Zurier, Hofstein & Weissmann 1973). Cytochalasin B (Calbiochem, Hereford) was dissolved in 0-1 % dimethyl sulphoxide at a concentration of 50 ,ug/ml. The adherent cell monolayers were pretreated with Cytochalasin B at a final concentration of 5 ug/ml for 15 min, before the particles were overlaid and also during the period of time allowed for binding.
Immunofluorescent staining of PMN. Cytochalasin B treated monolayers of CREV-PMN or PB-PMN were overlaid with 50 pl of optimally diluted, aggregate free (140,000 g, 90 min) fluorescein-
conjugated sheep anti-human IgG, IgM or IgA (Wellcome Reagents Beckenham), anti-C3 (Hoechst Pharmaceuticals, Hounslow) or anti-albumin (Wellcome Reagents, Beckenham) for 30 min at 4°. The anti-albumin serum was conjugated with fluorescein isothiocyanate (BDH, Poole, Dorset) by the method of Johnson & Holborow (1973) and had a final fluorescein to protein ratio of 0-85. After washing with phosphate-buffered saline, the preparations were mounted in saline and membrane immunofluorescence assessed using a Leitz Ortholux II microscope fitted with a Ploem vertical illuminator. In some tests PMN were preincubated with heataggregated human IgG for 15 min at 630, at a concentration of 1 mg/ml for 30 min before staining with anti-IgG. The effect of crevicular fluid on PB-PMN Adherent cell monolayers were pre-treated with the supernatant fluid collected from the gingival washings. The pre-treatment was carried out at 370 for 30 min and control monolayers were treated with HBSS. Particle binding assays and immunofluorescent staining were then performed as described above. Antibody absorption and complement reconstitution One-millilitre volumes of serum were absorbed with 109 killed CA blastospores for 1 h at 370 with constant agitation, and then overnight at 4°. After centrifuging to remove blastospores, the serum was stored at - 20° until used. Pre- and postabsorption sera were assayed for antibodies to Candida using the indirect fluorescent antibody test (Wilton et al., 1977). Guinea-pig complement was obtained from Wellcome Reagents, Beckenham.
Statistical analysis The results were analysed using Student's t-test for dependent means. RESULTS
Binding and phagocytosis of CA blastospores Significantly greater numbers of PB-PMN (85-3 ± 4-2) phagocytosed these particles than CREV-PMN (52-3 ± 166; P
The role of Fc and C3b receptors in phagocytosis by inflammatory polymorphonuclear leucocytes in man
J. M. A. WILTON, H. H. RENGGLI * & T. LEHNER Department of Oral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London and *Department of Periodontology, University of Nijmegen, The Netherlands
Received 1 October 1976; acceptedfor publication 5 November 1976
Summary. Polymorphonuclear leucocytes from the gingival crevice (CREV-PMN) in man have a defective capacity to phagocytose Candida albicans blastospores. Phagocytosis of zymosan particles, which detect C3b receptors, is also impaired but ingestion of latex beads coated with heat-aggregated IgG, which detects Fc receptors, is normal compared to peripheral blood polymorphonuclear leucocytes (PB-PMN). If phagocytosis is inhibited by Cytochalasin B, fewer CREV-PMN bind Candida and zymosan but the binding of IgG-coated latex beads remains unchanged. CREV-PMN have IgG (88%) IgM (45 ) and C3 (48 Y.) on their cell membrane, whilst less than 5%Y. of PB-PMN have any of these components. Incubation of PB-PMN in fluid from the gingival crevice confers surface IgG and C3 to the cells. Such treatment also inhibits the subsequent binding of IgG coated latex beads. The results suggest that the deficiency of phagocytosis by CREVPMN is due to decreased binding of particles to the C3b receptor of PMN, whilst the Fc receptor system remains intact.
large numbers through the mucosa of the intestina and respiratory tracts, but the functional ability of these cells has not been investigated. A convenient source of PMN which have migrated through epithelium are those found in the mouth at the gingival crevice (Attstrom, 1970; Skapski & Lehner, 1976). We have recently shown in man that the gingival crevicular polymorphonuclear leucocytes (CREVPMN) are capable of phagocytosing and killing Candida albicans (CA) blastospores (Wilton, Renggli & Lehner 1977). However the CREV-PMN are defective when compared with the peripheral blood polymorphonuclear leucocytes (PB-PMN) from the same subject, in that a smaller number of cells phagocytose CA blastospores and fewer particles are taken up by CREV-PMN than PB-PMN. Nevertheless, the cells from both sources kill CA with equal efficiency (Wilton et al., 1977). In patients with rheumatoid arthritis, Turner, Shumacher & Myers (1973) found that PMN from synovial fluid take up fewer bakers' yeast particles than blood PMN from the same subject. They speculated that this deficiency could be due to previous endocytosis in vivo of IgG rheumatoid factor complexes; such complexes when taken up by normal blood PMN would inhibit subsequent phagocytosis of yeast (Turner et al., 1973). Our own data (Wilton et al., 1977) could be interpreted as an impaired function of either the
INTRODUCTION
Polymorphonuclear leucocytes (PMN) migrate in Correspondence: Dr J. M. A. Wilton, Department of Oral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London SEI 9RT.
I
955
956
J. M. A. Wilton, H. H. Renggli & T. Lehner
PMN receptor for the activated third component of complement (Henson, 1969) or the Fc portion of immunoglobulin (Henson, 1969; Messner & Jelinek, 1970) on the surface of the CREV-PMN, leading to diminished phagocytosis of the CA blastospores. There is now evidence that the two receptors may have different functions in both murine (Mantovani, 1975) and human PMN (Scribner & Fahrney, 1976). The C3b receptor principally mediates particle binding whilst the Fc receptor mediates both particle binding and initiates endocytosis. The purpose of this investigation was to compare both the binding and phagocytosis of particles by CREV-PMN and PB-PMN, in an attempt to determine whether the phagocytic defect observed in the CREV-PMN is due to an Fc or C3b receptor deficiency. We have used serum-treated zymosan particles (Gewurz, 1972) to detect the C3b receptor (Goldstein, Roos, Kaplan & Weissman, 1975) and heat-aggregated human IgG bound to latex particles to detect the Fc receptor.
MATERIALS AND METHODS
Isolation of glass-adherent CREV-PMN Crevicular washings were collected from twelve healthy human subjects of both sexes, aged 20-44 years, by the method of Skapski & Lehner (1976). The individual washings in Hanks's balanced salt solution (HBSS) were buffered to 7-2 with sodium bicarbonate and were then pooled and washed once in HBSS by centrifugation. After the removal of a sample for viable counting using trypan blue exclusion, the specimen was adjusted to a final volume of 200 p1 with a final concentration of 30%. autologous serum in HBSS. The mean number of PMN recovered from the washings was 92,648 ± s.d. (21658) with a mean viability of 84 6 ±4 7%. Fifty-microlitre aliquots were then placed onto the glass 'wells' of a glass slide coated with Hilflow (Jencons, Hemel Hempstead), which had been covered by four templates each of 5 mm diameter. Thus each slide contained four separate 50-.ul drops. After incubation at 370 for 60 min in a moist chamber, the slides were washed first by gentle aspiration and the replacement of fresh HBSS several times and then by immersing the slide in a beaker of warmed HBSS. Each well contained only glass-adherent cells and differential counts of
Giemsa-stained preparations showed greater than 98%Y. PMN. Isolation of glass-adherent PB-PMN Blood was obtained by venepuncture and the erythrocytes were separated by the addition of Dextran (T250 Pharmacia, Uxbridge) to a final concentration of 3 % (v/v). The leucocyte-rich plasma was aspirated after 60 min at 370 and layered onto a Ficol-Hypaque gradient. After centrifugation, the pellet was suspended in a large excess of buffered isotonic ammonium chloride for 10 min at 4°. The cells were washed twice in HBSS, adjusted to the concentration of the CREV-PMN in HBSS containing 30%o autologous serum and then attached to glass as outlined for the CREV-PMN.
Phagocytosis assay (1) Candida albicans. The phagocytosis of live Candida albicans blastospores was assayed by the method of Schmid & Brune (1974), modified for the slides used in this study. Viable CA blastospores were added to each well in a total volume of 50 u1 in HBSS, containing 30%. fresh autologous serum, at a particle ratio of 5 CA: 1 PMN and incubated for 30 min in a moist chamber at 37°. The preparation was washed in HBSS to remove free CA and mounted in HBSS for viewing. A minimum of 200 cells was counted and the number of PMN phagocytosing one or more CA expressed as a percentage of the total cell count. (2) Zymosan. Zymosan A (Sigma, London) was heated at 1000 for 1 h and suspended in HBSS at 1 mg/ml. One milligram samples of zymosan were incubated with 0 5 ml of fresh human serum for 30 min at 370 and washed three times in HBSS. After adjusting the concentration to 0 5 mg/ml the zymosan was stored in small aliquots at -70°. The particles were then used in the phagocytosis assay as outlined for CA, by adding 50 ,ul of the suspension to each well. (3) IgG-coated latex beads. Five millilitres of IgGcoated latex beads (Rheuma-Wellcotest, Wellcome Reagents, Beckenham) were washed three times in HBSS and resuspended in 10 ml of HBSS. The beads were stored at 40 and used in the phagocytosis assay at 50 p1 per well.
Particle-binding assays. The binding of CA blasto-
957
Fc and C3b receptors in phagocytosis
spores, zymosan and IgG-coated latex beads was estimated after inhibiting phagocytosis with Cytochalasin B (Zurier, Hofstein & Weissmann 1973). Cytochalasin B (Calbiochem, Hereford) was dissolved in 0-1 % dimethyl sulphoxide at a concentration of 50 ,ug/ml. The adherent cell monolayers were pretreated with Cytochalasin B at a final concentration of 5 ug/ml for 15 min, before the particles were overlaid and also during the period of time allowed for binding.
Immunofluorescent staining of PMN. Cytochalasin B treated monolayers of CREV-PMN or PB-PMN were overlaid with 50 pl of optimally diluted, aggregate free (140,000 g, 90 min) fluorescein-
conjugated sheep anti-human IgG, IgM or IgA (Wellcome Reagents Beckenham), anti-C3 (Hoechst Pharmaceuticals, Hounslow) or anti-albumin (Wellcome Reagents, Beckenham) for 30 min at 4°. The anti-albumin serum was conjugated with fluorescein isothiocyanate (BDH, Poole, Dorset) by the method of Johnson & Holborow (1973) and had a final fluorescein to protein ratio of 0-85. After washing with phosphate-buffered saline, the preparations were mounted in saline and membrane immunofluorescence assessed using a Leitz Ortholux II microscope fitted with a Ploem vertical illuminator. In some tests PMN were preincubated with heataggregated human IgG for 15 min at 630, at a concentration of 1 mg/ml for 30 min before staining with anti-IgG. The effect of crevicular fluid on PB-PMN Adherent cell monolayers were pre-treated with the supernatant fluid collected from the gingival washings. The pre-treatment was carried out at 370 for 30 min and control monolayers were treated with HBSS. Particle binding assays and immunofluorescent staining were then performed as described above. Antibody absorption and complement reconstitution One-millilitre volumes of serum were absorbed with 109 killed CA blastospores for 1 h at 370 with constant agitation, and then overnight at 4°. After centrifuging to remove blastospores, the serum was stored at - 20° until used. Pre- and postabsorption sera were assayed for antibodies to Candida using the indirect fluorescent antibody test (Wilton et al., 1977). Guinea-pig complement was obtained from Wellcome Reagents, Beckenham.
Statistical analysis The results were analysed using Student's t-test for dependent means. RESULTS
Binding and phagocytosis of CA blastospores Significantly greater numbers of PB-PMN (85-3 ± 4-2) phagocytosed these particles than CREV-PMN (52-3 ± 166; P
