Clin. exp. Immunol. (1978) 34, 423-428.
Suppression by IgA of IgG-mediated phagocytosis by human polymorphonuclear leucocytes J. M. A. W I LTO N Department ofOral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London (Received 13 June 1978) SUMMARY
IgA from normal human serum, a myeloma IgA and human colostral IgA were found to inhibit the IgG-dependent phagocytosis of Candida albicans (CA) blastospores by polymorphonuclear leucocytes (PMN) from normal human subjects. The inhibition was dependent on the Fc portion of IgA, was enhanced by heat treatment (560C for 20 min) and depended on binding to the PMN, but not to the opsonized particle. Binding of CA to the PMN IgG Fc receptor was inhibited, whilst the binding to the PMN C3b receptor was unaffected. INTRODUCTION Immunoglobulin A has been reported to have a modulating effect on some of the biological effects of immunoglobulins G and M. In the rabbit, serum IgA inhibited the complemented-mediated bacteriolytic effect of IgG and IgM antibodies against Brucella abortus (Hall, Mannion & Zimmerman, 1971). In man, serum IgA was found to inhibit the serum bactericidal activity of IgG and IgM antibodies against Nesseria meningitidis (Griffiss, 1975). The lack of a bactericidal effect against N. meningitidis in human serum was shown to be due to the inhibitory activity of IgA on IgM-mediated bacteriolysis (Griffiss & Bertram, 1977). Secretory IgA was inhibitory to the bactericidal effect of rabbit IgG and IgM against Salmonella typhimurium (Eddie, Schulkind & Robbins, 1971). Patients with alcoholic liver disease were shown to have inhibitors of polymorphonuclear leucocytes (PMN) chemotaxis in serum which were associated with IgA (Van Epps, Strickland & Williams, 1975). Myeloma IgA of both the IgA1 and IgA2 subclasses and secretory IgA from human colostrum would inhibit PMN chemotaxis in response to a variety of different chemotactic agents (Van Epps & Williams, 1976). In the present study, the effect of human IgA from serum and colostrum on the phagocytosis and killing of Candida albicans (CA) by human PMN was investigated. These functions of the PMN are optimal when both anti-Candida IgG antibodies and complement are present in the assay (Wilton, Renggli & Lehner, 1977). It would appear that the IgG-mediated phagocytosis of Candida albicans by human PMN is inhibited by both serum and secretory IgA, but that complement-mediated phagocytosis and binding are unaffected. The killing of CA by the PMN was not affected by the IgA of either type. MATERIALS AND METHODS Preparation of immunoglobiulins. IgA was prepared from two pools of normal human serum by starch block electrophoresis and DEAE cellulose chromatography according to the method of Fahey, McCoy & Goulian (1968). Colostral IgA was purified from pooled human colostrum by the method of Tomasi et al. (1965). Myeloma IgA was obtained from a single myeloma serum (IgA concentration 1100 mg per 100 ml) using, in sequence, hydrophobic affinity chromatography, DEAE cellulose chromatography and Sephadex G200 gel filtration following the method of Doellgast & Plaut (1976). None of the IgA preparations contained IgG when assayed at 5 0 mg/ml by electrophoresis and single radial diffusion. The IgA preparations were Correspondence: Dr J. M. A. Wilton. Department of Oral Immunology and Microbiology, Guy's Hospital, London SEI 9RT. 0099-9104/78/0120-0423$02.00 (D 1978 Blackwell Scientific Publications 11
423)
J. M. A. Wilton
424
stored in small aliquots at -70'C until required. IgG was isolated from Cohn Fraction II gamma globulins (Sigma, London) by DEAE cellulose chromatography and stored at -70'C. Whole serum, isolated serum and IgG were heated at 560C for 30 min before being tested for inhibition. Pepsin digestion of IgA. (Fab')2 fragments of IgA were isolated from a peptic digest of whole IgA by Sephadex G200 filtration according to the method of Madsen & Rodkey (1976). The eluted protein was concentrated to the original volume of the sample; analysis by Ouchterlony immunodiffusion showed that there were no Fc antigenic determinants. Opsonisation ofCandida albicans (CA). CA (109) were added to 1-0 ml of high titre (1: 1024 by indirect fluorescent antibody test) anti-Candida serum from a patient with chronic mucocutaneous candidiasis. After incubation at 370C for 1 hr and at 4VC for 18 hr, the CA were washed three times in Hanks's buffered salt solution (HBSS) and adjusted to a concentration of 108 CA/ml in HBSS. The CA carried both IgG and C3 on their surface, as judged by direct immunofluorescence using fluorescein-conjugated sheep anti-human IgG (Wellcome Reagents, Beckenham, UK) or fluorescein-conjugated rabbit anti-human C3 (Hoechst, Hounslow, UK). The antisera were centrifuged at 10,000 g for 90 min before the PMN were stained. These CA were designated CA-IgG-C3. CA coated with only C3 were prepared according to the method of Goldstein et al. (1976). The CA-IgG-C3 were heated in 2 M NaCI for 15 min, washed twice with complement fixation test diluent (Oxoid, Basingstoke) and adjusted to a concentration of 108 CA/ml in HBSS. These CA had only C3 on their surface when stained with antisera against IgG and C3 and were designated CA-C3. Phagocytosis and killing of CA by human PMN. PMN were obtained from heparinized (10 u/ml) venous blood of clinically healthy laboratory personnel using dextran (T250 Pharmacia, Uxbridge, UK) sedimentation for 1 hr at 37°C. Leucocytes were allowed to settle on glass slides and the experimental design is shown in Fig. 1. Phagocytosis and killing was assayed using the method of Schmid & Brune (1974), as modified for microscope slides by Wilton et al. (1977). Adherence of CA was assayed using Cytochalasin B (Sigma, London) treated PMN. The binding of immunoglobulin to PMN was detected using direct immunofluorescence with the appropriate antiserum and Cytochalasin B-treated PMN (Wilton et al., 1977). Inhibition of phagocytosis was expressed as the percentage inhibition given by serum, whole IgA and IgG or (Fab')2 fragments of IgA, of the control PMN phagocytosis or binding of the CA-IgG-C3 or CA-C3 particles. Statistical treatment ofresults. The results were analysed using Student's t-test for dependent means.
RESULTS Neither of the two serum pools from which the IgA was isolated were inhibitory to the phagocytosis and binding of CA-IgG-C3 by PMN from normal subjects, whether tested before or after heating at 56°C. In contrast, the unheated isolated IgA from the first serum pool was only weakly inhibitory of PMN phagocytosis, yielding 6*4+2*1% s.d. at a concentration of 20 jg, but IgA from the second pool showed 12-0+5-2% inhibition at the same dose. After heating at 56°C for 30 min and testing at the same concentration, the first IgA was inhibitory, depressing phagocytosis by 23-4±5-1% and the second IgA showed an inhibitory activity of 21.6+6.3%. The IgA myeloma serum inhibited phagocytosis by 6 % at a concentration of 11 tg/ml and this did not increase further when heated serum was used. 25 3 3+ Dextran separated PMN 60min, 37°C ; Wash x 2 HBSS Adhere to glass 30 min, 370 C in 10% autologous serum
Incubate with HBSS aggregated IgA, IgG or secretory IgA 15 min, 37°C in 10% autologous
Pre-treat with cytochalasin B 5 Ekg/mL 15 min, 37°C
serum
Add CA-IgG-C3 or CA-C3 in HBSS 30min, 37°C
Stain with FITC anti-IgA or I gG
Count PMN phagocytosing or rosett ng
Count PMN with membrane staining
I
FIG. 1. Experimental design to test the inhibition of polymorphonuclear leucocyte phagocytosis and binding of Candida albicans by IgA or IgG.
.
IgA inhibits PMN phagocytosis
_
_
425
Unheated IgG showed 441±2.7% inhibition of phagocytosis at a concentration of 20 pg/ml which increased to 9-6+41% after heating. The dose response curves of the combined data for the two aggregated normal serum IgA preparations and the aggregated myeloma IgA are shown in Fig. 2, and also the dose response curve for aggregated IgG which was used as a control. Aggregated serum IgA significantly inhibited PMN phagocytosis at concentrations as low as 80 jig in contrast to aggregated serum IgG, where significant inhibition was not noted until a concentration of 31-25 jg and where comparable inhibition to that shown by IgA was not shown until a concentration of 62-5 pg of IgG was reached. The inhibition was not only restricted to the numbers of PMN phagocytosing CA since the numbers of Candida taken up by those PMN which had phagocytosed was also depressed compared with the control. For example, at a dose of 250 jig of IgA, the mean number of CA ingested was 1-6 CA/PMN±0-18 compared with 4-24024 of the control. A comparable degree of inhibition of the numbers taken up was noted at doses of IgA which inhibited the percentage of PMN phagocytosing CA-IgG-C3. It was expected that phagocytosis of CA-IgG-C3 would be inhibited by aggregated IgG which binds to the Fc-IgG receptor (Table 2) and prevents the attachment of the IgG opsonized particle. It is evident, however, that the concentration of IgA required to inhibit phagocytosis is four times lower than that required for IgG. 100
_
80 _ 0
60
M
0
tO40 .0
DE
20 XT
~ ~ 0
1
l 50
150 200 250 ,ug Agg IgA/ IgG per tube
100
500
1000
FIG. 2. The inhibition by aggregated serum IgA (A A) or IgG (0-0) of the phagocytosis of Candida albicans coated with antibody and complement by polymorphonuclear leucocytes.
TABLE 1. The inhibition by aggregated normal serum IgA and myeloma IgA of the binding of Candida albicans coated with antibody and complement (CA-IgG-C3) or complement alone (CA-C3) to polymorphonuclear leucocytes Percentage PMN binding
IgA (pg) 0 7-8 15-6 31-25 62-5 125-0 500 0
CA-IgG-C3
Percentage inhibition
P*
11 16 21 38 61 81
n.s. < 0 05
Suppression by IgA of IgG-mediated phagocytosis by human polymorphonuclear leucocytes J. M. A. W I LTO N Department ofOral Immunology and Microbiology, Guy's Hospital Medical and Dental Schools, London (Received 13 June 1978) SUMMARY
IgA from normal human serum, a myeloma IgA and human colostral IgA were found to inhibit the IgG-dependent phagocytosis of Candida albicans (CA) blastospores by polymorphonuclear leucocytes (PMN) from normal human subjects. The inhibition was dependent on the Fc portion of IgA, was enhanced by heat treatment (560C for 20 min) and depended on binding to the PMN, but not to the opsonized particle. Binding of CA to the PMN IgG Fc receptor was inhibited, whilst the binding to the PMN C3b receptor was unaffected. INTRODUCTION Immunoglobulin A has been reported to have a modulating effect on some of the biological effects of immunoglobulins G and M. In the rabbit, serum IgA inhibited the complemented-mediated bacteriolytic effect of IgG and IgM antibodies against Brucella abortus (Hall, Mannion & Zimmerman, 1971). In man, serum IgA was found to inhibit the serum bactericidal activity of IgG and IgM antibodies against Nesseria meningitidis (Griffiss, 1975). The lack of a bactericidal effect against N. meningitidis in human serum was shown to be due to the inhibitory activity of IgA on IgM-mediated bacteriolysis (Griffiss & Bertram, 1977). Secretory IgA was inhibitory to the bactericidal effect of rabbit IgG and IgM against Salmonella typhimurium (Eddie, Schulkind & Robbins, 1971). Patients with alcoholic liver disease were shown to have inhibitors of polymorphonuclear leucocytes (PMN) chemotaxis in serum which were associated with IgA (Van Epps, Strickland & Williams, 1975). Myeloma IgA of both the IgA1 and IgA2 subclasses and secretory IgA from human colostrum would inhibit PMN chemotaxis in response to a variety of different chemotactic agents (Van Epps & Williams, 1976). In the present study, the effect of human IgA from serum and colostrum on the phagocytosis and killing of Candida albicans (CA) by human PMN was investigated. These functions of the PMN are optimal when both anti-Candida IgG antibodies and complement are present in the assay (Wilton, Renggli & Lehner, 1977). It would appear that the IgG-mediated phagocytosis of Candida albicans by human PMN is inhibited by both serum and secretory IgA, but that complement-mediated phagocytosis and binding are unaffected. The killing of CA by the PMN was not affected by the IgA of either type. MATERIALS AND METHODS Preparation of immunoglobiulins. IgA was prepared from two pools of normal human serum by starch block electrophoresis and DEAE cellulose chromatography according to the method of Fahey, McCoy & Goulian (1968). Colostral IgA was purified from pooled human colostrum by the method of Tomasi et al. (1965). Myeloma IgA was obtained from a single myeloma serum (IgA concentration 1100 mg per 100 ml) using, in sequence, hydrophobic affinity chromatography, DEAE cellulose chromatography and Sephadex G200 gel filtration following the method of Doellgast & Plaut (1976). None of the IgA preparations contained IgG when assayed at 5 0 mg/ml by electrophoresis and single radial diffusion. The IgA preparations were Correspondence: Dr J. M. A. Wilton. Department of Oral Immunology and Microbiology, Guy's Hospital, London SEI 9RT. 0099-9104/78/0120-0423$02.00 (D 1978 Blackwell Scientific Publications 11
423)
J. M. A. Wilton
424
stored in small aliquots at -70'C until required. IgG was isolated from Cohn Fraction II gamma globulins (Sigma, London) by DEAE cellulose chromatography and stored at -70'C. Whole serum, isolated serum and IgG were heated at 560C for 30 min before being tested for inhibition. Pepsin digestion of IgA. (Fab')2 fragments of IgA were isolated from a peptic digest of whole IgA by Sephadex G200 filtration according to the method of Madsen & Rodkey (1976). The eluted protein was concentrated to the original volume of the sample; analysis by Ouchterlony immunodiffusion showed that there were no Fc antigenic determinants. Opsonisation ofCandida albicans (CA). CA (109) were added to 1-0 ml of high titre (1: 1024 by indirect fluorescent antibody test) anti-Candida serum from a patient with chronic mucocutaneous candidiasis. After incubation at 370C for 1 hr and at 4VC for 18 hr, the CA were washed three times in Hanks's buffered salt solution (HBSS) and adjusted to a concentration of 108 CA/ml in HBSS. The CA carried both IgG and C3 on their surface, as judged by direct immunofluorescence using fluorescein-conjugated sheep anti-human IgG (Wellcome Reagents, Beckenham, UK) or fluorescein-conjugated rabbit anti-human C3 (Hoechst, Hounslow, UK). The antisera were centrifuged at 10,000 g for 90 min before the PMN were stained. These CA were designated CA-IgG-C3. CA coated with only C3 were prepared according to the method of Goldstein et al. (1976). The CA-IgG-C3 were heated in 2 M NaCI for 15 min, washed twice with complement fixation test diluent (Oxoid, Basingstoke) and adjusted to a concentration of 108 CA/ml in HBSS. These CA had only C3 on their surface when stained with antisera against IgG and C3 and were designated CA-C3. Phagocytosis and killing of CA by human PMN. PMN were obtained from heparinized (10 u/ml) venous blood of clinically healthy laboratory personnel using dextran (T250 Pharmacia, Uxbridge, UK) sedimentation for 1 hr at 37°C. Leucocytes were allowed to settle on glass slides and the experimental design is shown in Fig. 1. Phagocytosis and killing was assayed using the method of Schmid & Brune (1974), as modified for microscope slides by Wilton et al. (1977). Adherence of CA was assayed using Cytochalasin B (Sigma, London) treated PMN. The binding of immunoglobulin to PMN was detected using direct immunofluorescence with the appropriate antiserum and Cytochalasin B-treated PMN (Wilton et al., 1977). Inhibition of phagocytosis was expressed as the percentage inhibition given by serum, whole IgA and IgG or (Fab')2 fragments of IgA, of the control PMN phagocytosis or binding of the CA-IgG-C3 or CA-C3 particles. Statistical treatment ofresults. The results were analysed using Student's t-test for dependent means.
RESULTS Neither of the two serum pools from which the IgA was isolated were inhibitory to the phagocytosis and binding of CA-IgG-C3 by PMN from normal subjects, whether tested before or after heating at 56°C. In contrast, the unheated isolated IgA from the first serum pool was only weakly inhibitory of PMN phagocytosis, yielding 6*4+2*1% s.d. at a concentration of 20 jg, but IgA from the second pool showed 12-0+5-2% inhibition at the same dose. After heating at 56°C for 30 min and testing at the same concentration, the first IgA was inhibitory, depressing phagocytosis by 23-4±5-1% and the second IgA showed an inhibitory activity of 21.6+6.3%. The IgA myeloma serum inhibited phagocytosis by 6 % at a concentration of 11 tg/ml and this did not increase further when heated serum was used. 25 3 3+ Dextran separated PMN 60min, 37°C ; Wash x 2 HBSS Adhere to glass 30 min, 370 C in 10% autologous serum
Incubate with HBSS aggregated IgA, IgG or secretory IgA 15 min, 37°C in 10% autologous
Pre-treat with cytochalasin B 5 Ekg/mL 15 min, 37°C
serum
Add CA-IgG-C3 or CA-C3 in HBSS 30min, 37°C
Stain with FITC anti-IgA or I gG
Count PMN phagocytosing or rosett ng
Count PMN with membrane staining
I
FIG. 1. Experimental design to test the inhibition of polymorphonuclear leucocyte phagocytosis and binding of Candida albicans by IgA or IgG.
.
IgA inhibits PMN phagocytosis
_
_
425
Unheated IgG showed 441±2.7% inhibition of phagocytosis at a concentration of 20 pg/ml which increased to 9-6+41% after heating. The dose response curves of the combined data for the two aggregated normal serum IgA preparations and the aggregated myeloma IgA are shown in Fig. 2, and also the dose response curve for aggregated IgG which was used as a control. Aggregated serum IgA significantly inhibited PMN phagocytosis at concentrations as low as 80 jig in contrast to aggregated serum IgG, where significant inhibition was not noted until a concentration of 31-25 jg and where comparable inhibition to that shown by IgA was not shown until a concentration of 62-5 pg of IgG was reached. The inhibition was not only restricted to the numbers of PMN phagocytosing CA since the numbers of Candida taken up by those PMN which had phagocytosed was also depressed compared with the control. For example, at a dose of 250 jig of IgA, the mean number of CA ingested was 1-6 CA/PMN±0-18 compared with 4-24024 of the control. A comparable degree of inhibition of the numbers taken up was noted at doses of IgA which inhibited the percentage of PMN phagocytosing CA-IgG-C3. It was expected that phagocytosis of CA-IgG-C3 would be inhibited by aggregated IgG which binds to the Fc-IgG receptor (Table 2) and prevents the attachment of the IgG opsonized particle. It is evident, however, that the concentration of IgA required to inhibit phagocytosis is four times lower than that required for IgG. 100
_
80 _ 0
60
M
0
tO40 .0
DE
20 XT
~ ~ 0
1
l 50
150 200 250 ,ug Agg IgA/ IgG per tube
100
500
1000
FIG. 2. The inhibition by aggregated serum IgA (A A) or IgG (0-0) of the phagocytosis of Candida albicans coated with antibody and complement by polymorphonuclear leucocytes.
TABLE 1. The inhibition by aggregated normal serum IgA and myeloma IgA of the binding of Candida albicans coated with antibody and complement (CA-IgG-C3) or complement alone (CA-C3) to polymorphonuclear leucocytes Percentage PMN binding
IgA (pg) 0 7-8 15-6 31-25 62-5 125-0 500 0
CA-IgG-C3
Percentage inhibition
P*
11 16 21 38 61 81
n.s. < 0 05
