Clin. exp. Immunol. (1978) 33, 79-87.
Sex steroid hormones and antibodies to Candida albicans S. MATHUR, R. S. MATHUR, H. DOWDA*, H. 0. WILLIAMSON, W. P. FAULK & H. H. FUDENBERG Department of Basic and Clinical Immunology and Microbiology, and Reproductive Endocrinology Section, Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, South Carolina, and * Diagnostic Microbiology Laboratory, South Carolina Health and Environmental Department, Columbia, South Carolina, USA
(Received 2 February 1978)
SUMMARY
The effect of changes in progesterone (P) and estradiol (E2) on titres of antibodies to Candida albicans was studied by measurement of these three parameters in the following endocrinologically diverse human groups: normal females, gonadal dysgenetics, users of a sequential oral contraceptive (Oracon) and normal males. In females, C. albicans titres (mean+ s.e.m.) were significantly higher (P< 005) in the luteal (74+ 14) than in the follicular phase (34+ 19) of the cycle, and there were similar significant increases in P and E2. In the gonadal dysgenetic group (n = 29), with E2 levels comparable with males, the antibody titres were also equivalent to those in normal males (40+ 0 5), but were significantly lower than those of normal females in the follicular phase (P< 0-05). In contrast, Oracon users, with high blood progestin levels, had C. albicans titres (118+ 15) significantly higher (P< 0001) than those of control subjects during the follicular phase. A significant correlation (P< 0.05) was observed between P and C. albicans titres (mainly IgA) in randomly selected samples (n = 112) from normal females during the follicular and luteal phases, and in two subjects from whom blood samples were drawn daily for the entire cycle. In the latter, an increase in E2 but not P in the late follicular phase was accompanied by a marked decrease in C. albicans titres. No changes were observed in total immunoglobulin levels or antibodies to SRBC or Herpes virus in response to the marked changes in hormones. These results indicate that the production of antibodies to C. albicans may be specifically influenced by sex steroid hormones, being enhanced by P and E2 at low levels but depressed by E2 at high levels.
INTRODUCTION Chronic infection with Candida albicans is common in women, especially during pregnancy (Buck & Hasenclever, 1963). Studies correlating the use of oral contraceptives and altered immunity to viral infections (Royal College of General Practitioners, 1974) indicate the need for defining the role of sex hormones in immune mechanisms. It has been suggested that factors associated with the various stages of the menstrual cycle may influence local immune responses (Schumacher, 1973a; Hulka & Omran, 1969). Chipperfield & Evans (1975), however, reported that levels of IgA and IgG in cervical secretions did alter during the menstrual cycle. Although some attempts have been made to study the immunological effects of synthetic hormone uptake during the menstrual cycle in rats (Wira & Sandoe, 1977), few studies on the possible role of sex steroid hormones in human immune mechanisms, in general, have been proposed. Correspondence: Dr S. Mathur, Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina 29401, USA. 0099-9104/78/0700-0079$02.00 (0 1978 Blackwell Scientific Publications
79
S. Mathur et al.
80
The present study was designed to evaluate the relationship of the sex hormones progesterone and estradiol with the titres of antibodies to C. albicans. Levels of the sex hormones and of anti-Candida antibodies were measured in the plasma of normal cycling females, gonadal dysgenetics, long-term users of the sequential oral contraceptive Oracon (with persistently high synthetic steroid hormones in the blood) and normal males. Estimations of serum immunoglobulins as well as antibodies to sheep red blood cells and Herpes virus were also carried out. MATERIALS AND METHODS Subjects Plasma samples were obtained from normal females, gonadal dysgenetics (ovarian dysfunction) and long-term users of Oracon (Mead Johnson Co.; 0-1 mg of ethinyl estradiol daily for 16 days, followed by 25 mg of dimethisterone, a synthetic progestin, plus 0-1 mg ethinyl estradiol for 6 days) attending the Obstetrics and Gynecology Clinics, Medical University of South Carolina. Samples from normal males and daily samples through a complete menstrual cycle from two normal females were obtained from volunteers. All the subjects were free from clinical symptoms of candidiasis. Antigen. Cultures of C. albicans (courtesy Dr. W. Kaplan, Center for Disease Control, Atlanta, Georgia 30333) were grown in yeast nitrogen base (BBL, Cockeysville, Maryland), supplemented with 30%4 glucose. Blastospores were harvested after 48 hr, washed three times in PBS (pH 7.2) and lyophilized. Whole blastospores and germ tubes were used in the indirect fluorescent antibody assay, whereas a cytoplasmic extract obtained by sonicating the cells and extracting with PBS was used for the passive haemagglutination test. Antisera. Goat fluorescein-conjugated antisera against IgG, IgA and IgM were obtained commercially from BurroughsWellcome Co. (Research Triangle Park, North Carolina). Assays. Passive haemagglutination (Gold & Fudenberg, 1967) was used for evaluation of plasma antibodies to C. albicans and sheep red blood cells. Antibodies to Herpes virus were estimated using complement fixation test (Lennette, Melrick & Magoffin, 1974). Antibody titres were expressed as arithmetic means of reciprocals, as in previous reports (Lehner, 1965; Dolan & Stried, 1973; Mathur et al., 1977a,c). Chromic chloride was used as the coupling agent to bind the antigens from C. albicans to O+, Rh+ red blood cells. The indirect fluorescent antibody assay was carried out as described elsewhere (Mathur et al., 1977c). Quantification of plasma immunoglobulins was carried out by radial immunodiffusion using monospecific antisera to IgG, IgA and IgM, as described previously (Mancini, Carbonara & Heremans, 1965). Progesterone and estradiol were estimated by the radioimmunoassay methods of De Villa et al. (1972) and Parker, Ellegood & Mahesh (1975) respectively, as detailed elsewhere (Mathur et al., 1977b). Statistical analysis. The data were analysed by the Student's t-test and by linear regression analysis (r test).
TABLE 1. Plasma levels of progesterone and estradiol and plasma antibody titres to C. albicans in subjects with various levels of circulating sex steroid hormones
Subjects Normal females Follicular phase Luteal phase
Oraconusers* Gonadal dysgenetics Normal males
Number studied
Progesterone
Estradiol
C. albicans
(ng/100 ml)
(ng/100 ml)
reciprocal
46 66 24 29 36
149+ 15 (a) 1348+ 38 (b) 60+5 (c) 100+5 (d) 101+ 8 (e)
4 7+ 0 4 (f) 8-0+ 0-8 (g) 2-5+0-3 (h) 27± 0-2 (i)
34+ 19 (k) 74+ 14 (1) 118+ 15 (m) 3-9+0 5 (n) 7.4+ 1.5 (o)
3-0±0.2 (j)
Data shown are mean+ s.e.m. Significance levels by Student's t-test: (a) vs (b) P< 0-001; (f) vs (g) P< 0-001; (k) vs (1) P< 0-025; (a) vs (c) P< 0-001; (f) vs (h) not significant; (k) vs (m) P< 0-001; (a) vs (d) not significant; (f) vs (i) P< 0 005; (k) vs (n) P< 0-025; (a) vs (e) not significant; (f) vs (j) P< 0 025; (k) vs (o) P< 0-001; (d) vs (e), (i) vs (j) and (n) vs (o) not significant. Correlation coefficient (r) for (a) vs (k) and (b) vs (1): P< 0-05. * Synthetic sex hormone levels are not measured by specific progesterone radioimmunoassay used.
81
Sex hormones and antibodies to C. albicans TABLE 2. Anti-Candida antibody titres and total immunoglobulins in sera from two volunteers (C.C. and C.D.) through one menstrual cycle
Measurement
C.C. Progesterone (ng/100 ml) Estradiol (ng/100 ml) Anti-Candida antibodies (reciprocal) Passive haemagglutination Immunofluorescence IgG IgA IgM Total immunoglobulins (mg/100 ml) IgG IgA IgM C.D. Progesterone (ng/100 ml) Estradiol (ng/100 ml) Anti-Candida antibodies (reciprocal) Passive haemagglutination Immunofluorescence IgG IgA IgM Total immunoglobulins (mg/100 ml) IgG IgA IgM
Follicular (mean+ s.e.m.)
Luteal (mean+ s.e.m.)
Significance* (P)
72+ 5 5 5+ 1-3
664+ 131 8-4+ 1-8
< 0 001 < 0 05
6 5+ 8-6
89+ 71
< 0 001
1-8+0.2 3-2+0-8 1-4+ 0-1
10-1+3-7 67-6+ 14-1 4-5+ 1.4
n.s. < 0 001 < 0-05
992+ 230 226+ 90 130+ 26
987+ 240 249+ 80 138+47
n.s. n.s. n.s.
146+ 12 13-3+ 1-5
1362+ 204 14-7+ 1-3
< 0 001 n.s.
7-5+ 9-3
30+ 23
< 0-005
39+ 15 13-2+ 3-6 1-8+0-8
13-9±40 69-8+ 16.9 6-1+1-5
< 0 01
999+ 140 250+ 57 188+ 32
772+ 304 260+ 67 194+ 31
< 0 05 n.s. n.s.
Sex steroid hormones and antibodies to Candida albicans S. MATHUR, R. S. MATHUR, H. DOWDA*, H. 0. WILLIAMSON, W. P. FAULK & H. H. FUDENBERG Department of Basic and Clinical Immunology and Microbiology, and Reproductive Endocrinology Section, Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, South Carolina, and * Diagnostic Microbiology Laboratory, South Carolina Health and Environmental Department, Columbia, South Carolina, USA
(Received 2 February 1978)
SUMMARY
The effect of changes in progesterone (P) and estradiol (E2) on titres of antibodies to Candida albicans was studied by measurement of these three parameters in the following endocrinologically diverse human groups: normal females, gonadal dysgenetics, users of a sequential oral contraceptive (Oracon) and normal males. In females, C. albicans titres (mean+ s.e.m.) were significantly higher (P< 005) in the luteal (74+ 14) than in the follicular phase (34+ 19) of the cycle, and there were similar significant increases in P and E2. In the gonadal dysgenetic group (n = 29), with E2 levels comparable with males, the antibody titres were also equivalent to those in normal males (40+ 0 5), but were significantly lower than those of normal females in the follicular phase (P< 0-05). In contrast, Oracon users, with high blood progestin levels, had C. albicans titres (118+ 15) significantly higher (P< 0001) than those of control subjects during the follicular phase. A significant correlation (P< 0.05) was observed between P and C. albicans titres (mainly IgA) in randomly selected samples (n = 112) from normal females during the follicular and luteal phases, and in two subjects from whom blood samples were drawn daily for the entire cycle. In the latter, an increase in E2 but not P in the late follicular phase was accompanied by a marked decrease in C. albicans titres. No changes were observed in total immunoglobulin levels or antibodies to SRBC or Herpes virus in response to the marked changes in hormones. These results indicate that the production of antibodies to C. albicans may be specifically influenced by sex steroid hormones, being enhanced by P and E2 at low levels but depressed by E2 at high levels.
INTRODUCTION Chronic infection with Candida albicans is common in women, especially during pregnancy (Buck & Hasenclever, 1963). Studies correlating the use of oral contraceptives and altered immunity to viral infections (Royal College of General Practitioners, 1974) indicate the need for defining the role of sex hormones in immune mechanisms. It has been suggested that factors associated with the various stages of the menstrual cycle may influence local immune responses (Schumacher, 1973a; Hulka & Omran, 1969). Chipperfield & Evans (1975), however, reported that levels of IgA and IgG in cervical secretions did alter during the menstrual cycle. Although some attempts have been made to study the immunological effects of synthetic hormone uptake during the menstrual cycle in rats (Wira & Sandoe, 1977), few studies on the possible role of sex steroid hormones in human immune mechanisms, in general, have been proposed. Correspondence: Dr S. Mathur, Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina 29401, USA. 0099-9104/78/0700-0079$02.00 (0 1978 Blackwell Scientific Publications
79
S. Mathur et al.
80
The present study was designed to evaluate the relationship of the sex hormones progesterone and estradiol with the titres of antibodies to C. albicans. Levels of the sex hormones and of anti-Candida antibodies were measured in the plasma of normal cycling females, gonadal dysgenetics, long-term users of the sequential oral contraceptive Oracon (with persistently high synthetic steroid hormones in the blood) and normal males. Estimations of serum immunoglobulins as well as antibodies to sheep red blood cells and Herpes virus were also carried out. MATERIALS AND METHODS Subjects Plasma samples were obtained from normal females, gonadal dysgenetics (ovarian dysfunction) and long-term users of Oracon (Mead Johnson Co.; 0-1 mg of ethinyl estradiol daily for 16 days, followed by 25 mg of dimethisterone, a synthetic progestin, plus 0-1 mg ethinyl estradiol for 6 days) attending the Obstetrics and Gynecology Clinics, Medical University of South Carolina. Samples from normal males and daily samples through a complete menstrual cycle from two normal females were obtained from volunteers. All the subjects were free from clinical symptoms of candidiasis. Antigen. Cultures of C. albicans (courtesy Dr. W. Kaplan, Center for Disease Control, Atlanta, Georgia 30333) were grown in yeast nitrogen base (BBL, Cockeysville, Maryland), supplemented with 30%4 glucose. Blastospores were harvested after 48 hr, washed three times in PBS (pH 7.2) and lyophilized. Whole blastospores and germ tubes were used in the indirect fluorescent antibody assay, whereas a cytoplasmic extract obtained by sonicating the cells and extracting with PBS was used for the passive haemagglutination test. Antisera. Goat fluorescein-conjugated antisera against IgG, IgA and IgM were obtained commercially from BurroughsWellcome Co. (Research Triangle Park, North Carolina). Assays. Passive haemagglutination (Gold & Fudenberg, 1967) was used for evaluation of plasma antibodies to C. albicans and sheep red blood cells. Antibodies to Herpes virus were estimated using complement fixation test (Lennette, Melrick & Magoffin, 1974). Antibody titres were expressed as arithmetic means of reciprocals, as in previous reports (Lehner, 1965; Dolan & Stried, 1973; Mathur et al., 1977a,c). Chromic chloride was used as the coupling agent to bind the antigens from C. albicans to O+, Rh+ red blood cells. The indirect fluorescent antibody assay was carried out as described elsewhere (Mathur et al., 1977c). Quantification of plasma immunoglobulins was carried out by radial immunodiffusion using monospecific antisera to IgG, IgA and IgM, as described previously (Mancini, Carbonara & Heremans, 1965). Progesterone and estradiol were estimated by the radioimmunoassay methods of De Villa et al. (1972) and Parker, Ellegood & Mahesh (1975) respectively, as detailed elsewhere (Mathur et al., 1977b). Statistical analysis. The data were analysed by the Student's t-test and by linear regression analysis (r test).
TABLE 1. Plasma levels of progesterone and estradiol and plasma antibody titres to C. albicans in subjects with various levels of circulating sex steroid hormones
Subjects Normal females Follicular phase Luteal phase
Oraconusers* Gonadal dysgenetics Normal males
Number studied
Progesterone
Estradiol
C. albicans
(ng/100 ml)
(ng/100 ml)
reciprocal
46 66 24 29 36
149+ 15 (a) 1348+ 38 (b) 60+5 (c) 100+5 (d) 101+ 8 (e)
4 7+ 0 4 (f) 8-0+ 0-8 (g) 2-5+0-3 (h) 27± 0-2 (i)
34+ 19 (k) 74+ 14 (1) 118+ 15 (m) 3-9+0 5 (n) 7.4+ 1.5 (o)
3-0±0.2 (j)
Data shown are mean+ s.e.m. Significance levels by Student's t-test: (a) vs (b) P< 0-001; (f) vs (g) P< 0-001; (k) vs (1) P< 0-025; (a) vs (c) P< 0-001; (f) vs (h) not significant; (k) vs (m) P< 0-001; (a) vs (d) not significant; (f) vs (i) P< 0 005; (k) vs (n) P< 0-025; (a) vs (e) not significant; (f) vs (j) P< 0 025; (k) vs (o) P< 0-001; (d) vs (e), (i) vs (j) and (n) vs (o) not significant. Correlation coefficient (r) for (a) vs (k) and (b) vs (1): P< 0-05. * Synthetic sex hormone levels are not measured by specific progesterone radioimmunoassay used.
81
Sex hormones and antibodies to C. albicans TABLE 2. Anti-Candida antibody titres and total immunoglobulins in sera from two volunteers (C.C. and C.D.) through one menstrual cycle
Measurement
C.C. Progesterone (ng/100 ml) Estradiol (ng/100 ml) Anti-Candida antibodies (reciprocal) Passive haemagglutination Immunofluorescence IgG IgA IgM Total immunoglobulins (mg/100 ml) IgG IgA IgM C.D. Progesterone (ng/100 ml) Estradiol (ng/100 ml) Anti-Candida antibodies (reciprocal) Passive haemagglutination Immunofluorescence IgG IgA IgM Total immunoglobulins (mg/100 ml) IgG IgA IgM
Follicular (mean+ s.e.m.)
Luteal (mean+ s.e.m.)
Significance* (P)
72+ 5 5 5+ 1-3
664+ 131 8-4+ 1-8
< 0 001 < 0 05
6 5+ 8-6
89+ 71
< 0 001
1-8+0.2 3-2+0-8 1-4+ 0-1
10-1+3-7 67-6+ 14-1 4-5+ 1.4
n.s. < 0 001 < 0-05
992+ 230 226+ 90 130+ 26
987+ 240 249+ 80 138+47
n.s. n.s. n.s.
146+ 12 13-3+ 1-5
1362+ 204 14-7+ 1-3
< 0 001 n.s.
7-5+ 9-3
30+ 23
< 0-005
39+ 15 13-2+ 3-6 1-8+0-8
13-9±40 69-8+ 16.9 6-1+1-5
< 0 01
999+ 140 250+ 57 188+ 32
772+ 304 260+ 67 194+ 31
< 0 05 n.s. n.s.
