Immunology 1978 34 689
A new semiquantitative radiometric opsonin assay SELECTIVE MEASUREMENT OF OPSONIZING CAPACITY OF THE ALTERNATIVE PATHWAY
M. YA MAM URA & H. VALDI MARS SON Department of Immunology, St Mary's Hospital Medical School, London W2 1PG
Received 30 June 1977; acceptedfor publication 18 August 1977
Summary. A new semiquantitative radiometric opsonin assay is described. It was found that the opsonin activity generated by incubating brewer's yeast, Saccharomyces cerevisiae, in medium containing less than 5 % human serum was exclusively complement dependent. In contrast, C. albicans was effectively opsonized in the absence of complement. Antibodies and the early classical complement pathway did not contribute to the opsonization of S. cerevisiae and neither did C5-9. The brewer's yeast assay can therefore be used for measuring selectively the opsonizing capacity of the alternative pathway. Sera from approximately 7 % of apparently healthy adult controls consistently failed to generate significant opsonin activity while 8 out of 26 patients with suspected immune deficiency of unknown cause were defective in this assay. All opsonin deficient sera so far tested had haemolytically normal alternative pathway and Factor B activity. INTRODUCTION
before specific immunity is acquired. The alternative pathway is a major source of opsonin activity, and micro-organisms capable of directly activating this pathway are therefore obvious targets for antibody independent opsonization. Assays for analysing opsonization have not been significantly improved since the phenomenon was first described (Wright & Douglas, 1903). Thus, with a few exceptions (Stossel, 1973), tests for opsonin activity still depend on counting ingested particles under the microscope. One such assay, in which heat killed baker's yeast is used, has been reported to selectively detect complement-derived opsonins (Miller & Nilsson, 1970). The difficulty of distinguishing between adherent and engulfed particles is a serious drawback inherent in all microscopical techniques. We have recently developed an objective method for measuring phagocytosis of yeast organisms, in which this problem was overcome (Yamamura, Boler & Valdimarsson, 1977). This technique has now been modified for analysing and quantifying opsonin activity.
Antibody-independent opsonization may be important for microbial defence, especially early in life
MATERIALS AND METHODS
Correspondence: Dr H. Valdimarsson, Department of Immunology, St Mary's Hospital Medical School, London W2 1PG.
3H-uridine (Specific activity 5 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, 0019-2805/78/0400-0689 $02.00 (©1978 Blackwell Scientific Publications 689
690
M. Yamamura & H. Valdimarsson
England. Peptone was purchased from Difco. Medium RPML 1640 (Flow Laboratories) was buffered with HEPES and used throughout. LP3 tubes (Luckham) were used for mixing yeast and polymorphonuclear leucocytes (PMNs) during phagocytosis, but aliquots for measuring uridine uptake were transferred into microtitre wells (Cooke).
Yeast Fast growing strain of Saccharomyces cerevisiae (Strain L) was isolated from CWE brewer's yeast, which is specially formulated for rapid fermentation. The strain was kindly identified by Mrs Barbara Kirsop, of the Brewing Industry Research Foundation, Redhill, Surrey. The yeast was stored on 2% glucose, 1 % peptone and 2 % agar slopes at 40, and transferred to a new agar slope every 1-2 months. Subcultures were made in RPMI 1640 at 280 for 48-72 h and then kept at 40 for up to 1 week without loss of number or viability. The viability was greater than 98 %.
Polymorphonuclear leucocytes (PMNs) These were obtained from heparinized blood. Erythrocytes were sedimented by adding Dextran 110 (Dextran 110, Fison Ltd.). PMNs were washed at least 5 times with RPMI 1640 and adjusted to 5 x 105/ml and 2-5 x 105/ml.
Sera Sera were separated from clotted blood samples and kept at - 1900. Human R3 reagent was prepared by treating serum with Zymosan (Sigma) (Lachmann, Hobart & Aston, 1973). It retained 40% of intact C3, tested by two dimensional immunoelectrophoresis, but no haemolytic complement was present at a dilution of 1/5 and Factor B activity was also depleted. There was no decrease in haemolytically active C2 and C4. C3 deficient serum was kindly supplied by Dr P. J. Lachmann and C5 deficient serum was obtained from B1OD2 strain mice. Assay principle We have previously demonstrated that inhibition of 3H-uridine incorporation into yeast in a log growth phase can be used as a sensitive index of phagocytic function (Yamamura et al., 1977). This is because: (1) there is a linear relationship between uridine
incorporation and yeast number; (2) yeast replicating inside phagocytic cells do not take up uridine from culture medium; and (3) phagocytic cells do not incorporate significant amounts of uridine in short term cultures. Using the S. cerevisiae (L strain) it was found that during an incubation period of 60 min, 3 x 103 organisms incorporated sufficient amounts of 3H-uridine and there was a linear relationship between uridine uptake and yeast number between 3 x 103 and 3 x 105 per culture.
Standard assay procedure Unless otherwise stated, assays were performed as follows: a mixture containing 5 x 105 yeast cells and 5 x 104 or 1 x 105 PMNs was rotated at 370 for 30 min in 0 5 ml of medium containing 2-5 and/or 1 -25 % serum. Duplicate 0-2 ml aliquots were transferred to microtitre wells to which 0-2 pCi of 3H-uridine had previously been added and the microtitre plates incubated for a further 60 min at 370. The cells were then collected onto glass fibre filter paper by a Skatron Multiple Harvester, the dried discs placed in scintillation vials, covered with 5 ml of PPO-toluene (4 g/1) scintillation fluid and counted by a Packard Tri-carb Liquid Scintillation Spectrometer, Model B2450. Presentation of data Opsonin activity has traditionally been expressed as the opsonic index which is the average number of particles ingested by each phagocyte under conditions where phagocytosis is limited by the opsonin concentration. In the assay described here the results can be expressed as per cent inhibition of uridine incorporation (I) caused by phagocytosis or c.p.m. in PMNs present 100 c.p.m. in PMNs absent) It is also possible to work out the average number of yeast ingested by each PMN leucocyte because there is a linear relationship between the number of phagocytosed yeast and the inhibition of uridine incorporation. The data in this paper will be presented as Opsonin Indices by the following formula: I
(1
IxR where I is the per cent inhibition of uridine 100 incorporation caused by phagocytosis and R the yeast/PMN ratio.
Radiometric opsonin assay RESULTS
Evaluation of assay conditions Assay conditions for opsonin activity must be adjusted so that the number of particles ingested is limited in a linear fashion by the opsonin concentration and not by the number of particles available for phagocytosis.
4
3
C2
0E
691
Fig. 1 shows that in the presence of 1 -25 and 2 5% serum, phagocytosis was dependent on the availability of yeast when there were less than 5 organisms per PMN leucocyte, but remained constant when the yeast/PMN ratio exceeded 5/1. Using yeast/PMN ratios of 5/1 and 10/1, it was further demonstrated that phagocytosis increased in a linear fashion when serum concentration was raised from 0 625 to 2 5 % while a plateau was usually reached at 5 % (Fig. 2). These observations indicate that, with yeast/PMN ratios between 5/1 and 10/1, there is a linear relationship between phagocytosis and serum concentration up to 2-5 %. As normal sera gave a wide scatter at 0 625 %, it was decided to use serum dilutions of 1 -25 and 2 5 % for measuring opsonin activity.
0
Analysis of the opsonin activity Table 1 demonstrates that C. albicans was effectively 10/5 10/2 Ratio (S. cerevisioel PM N)
Table 1. The different serum requirements for opsonization of S. cerevisiae and C. albicans*
Figure 1. Phagocytosis of S. cerevisiae using yeast/PMN ratios from 1/1 to 10/1. The ingestion was no longer limited by the availability of yeast if the ratio was 5/1 or more. (0) 1-5 % serum concentration; (0) 2-5 % serum concentration. 6 5
0
Normal serat Serum heated 560 for 30 min Zymosan treated serum C2 deficient serum C3 deficient serum Agammaglobulinaemic serum ( < 50 mg/I) Serum free medium
4 0 ±0-6 0 0 30 01
5-0 ±1-2 5-6 4-6 49 6-5
3-2 0
4-8 0-1
-0
4
.0
S. cerevisiae C. albicans
* All sera used at 25 %. t Mean ± s.d., n = 20.
3 0
0 0 Q.
0
2
-/I
0-625
25 1I25 Concentration of serum (%)
5-0
Figure 2. Four experiments with yeast/PMN ratio of 10/1. The phagocytosis was limited by the serum concentration up to 2-5 %.
opsonized by heat inactivated serum, while S. cerevisiae, was not. Similarly, zymosan treated serum had normal opsonin activity for C. albicans but none for S. cerevisiae. Sera deficient in C2 or antibodies had normal opsonin capacity for both yeast types whilst C3 deficient serum opsonized only C. albicans and not S. cerevisiae. C5 deficient mouse serum had normal opsonin activity for S. cerevisiae (Fig. 3). In order to test whether antibodies, although not
692
M. Yamamura & H. Valdimarsson Table 2. Normal ranges for opsonic activity of adult human sera against S. cerevisiae
4 X 3 '(3 ._
-i 2 0
Serum concentration 0
0x
Mean opsonin index Standard deviation Normal ranges (95 % conf. limit) 2-5 1-25 5 Concentration of serum (%)
Figure 3. Opsonin activity of C5 deficient compared with normal mouse serum (0).
mouse serum
*
125%
25%
2-7 04 1 9-3 5
4-0 0-6 2 8-5-2
n = 20; 2 healthy donors with low values were excluded.
(0)
low in opsonin activity and, in contrast to all other healthy controls, these individuals did not show any increase in opsonin activity when the serum concentration was increased from 1 25 to 2 5 %. Table 3 shows the reproducibility of the assay in a longitudinal study of 5 individuals.
essential, might nevertheless contribute to the opsonization of S. cerevisiae, the yeast was preincubated in 20 % heat inactivated sera, washed and then tested in 1 -25 and 2 5 % of the R3 reagent. No opsonization was detected in these experiments. It is therefore evident that under the assay conditions used, the opsonin activity for S. cerevisiae was exclusively derived from direct activation of the alternative complement pathway. In contrast, phagocytosis of C. albicans did not require complement activation.
Comparison with the microscopical baker's yeast opsonin assay Six sera that had been found to lack opsonin activity in the microscopical baker's yeast assay (Soothill & Harvey, 1976) were kindly supplied by Professor J. F. Soothill. They were all found to be deficient in the radiometric assay. However, 1 of the 3 control sera which were normal in the microscopical assay showed opsonin deficiency in the radiometric assay. Altogether, 3 out of 39 sera from healthy controls were found to be deficient in this assay (Fig. 4). So far, sera have been tested from 26 patients with clinically suspected immune deficiency, but without detectable immune abnormality in laboratory tests, and 8 were found to lack opsonin activity.
Normal ranges Sera from 20 healthy adults were assayed for opsonin activity towards S. cerevisiae. Table 2 shows the means and standard deviations of opsonin indices for serum concentrations of 1 -25 and 2 5 %. Normal ranges with 95 % confidence limits for 1 -25 and 2-5 % serum were 1 9-3 5 and 2-8-5-2 respectively. However, 2 healthy donors were consistently very
Table 3. Reproducibility of the assay in longitudinal studies
No. of Serum donors
Normal healthy donor (1) Normal healthy donor (2) Healthy donor with opsonin deficiency (1) Healthy donor with opsonin deficiency (2) Patient with recurrent infections
tests
4 4 3 3 3
Opsonin indices
1-25% serum
3-5; 2-7; 2-6; 3-2 2-5; 2-3; 2-0; 2-3 0-1; 0-5; 0 0; 0; 0 0-5; 0-2; 0-1
2 5 % serum
4-1; 4-8; 3 9; 5 1 4 5; 3-0; 3-1; 3-4 0-1; 0 5; 0 0; 0; 0 0 3; 0 3; 0-2
_A\~ ~ ~ Radiometric opsonin assay
i593
0
5
S
4
3 0 c
0
Vt
TO 2
*0O 0
=9I 0^
1 0 0
.3. a
I
O1
A
aa B
0 0 S
S
Y
0
_
D
C
I
Figure 4. Opsonin activity of sera from 39 control subjects, 6 samples with known opsonin defect as determined by microscopical yeast assay and 26 patients suspected of immune deficiency. All sera were tested at a concentration of 1-25 %. (A) Healthy normal subjects; (B) healthy subjects with opsonin defect; (C) known opsonin defect; (D) suspected immunodeficiency.
DISCUSSION Opsonization is of major importance for effective pathogen and antigen elimination. However, the molecular basis of this phenomenon is poorly understood and quantitative opsonin assays, suitable for general clinical use, have not been available. The radiometric assay described in this paper is semiquantitative and easy to perform. It is essential, however, to select and use a homogeneous strain of fast growing yeast organisms. Employing a fast growing strain of S. cerevisiae, we have demonstrated that the opsonin activity generated by the alternative complement pathway can be selectively measured. This assay correlates with a microscopical baker's yeast test hitherto used for complement derived opsonins. In a preliminary clinical study, sera from 8 out of 26 patients with unexplained immune deficiency have failed to generate opsonin activity in our assay. This is in good agreement with the recent report of Soothill and Harvey who found defective opsonization in 11 of 43 children with unexplained infections. H
They also observed that the majority of healthy mothers of affected children had defective opsonization, suggesting a primary abnormality rather than a depletion phenomenon (Soothill & Harvey, 1976). Moreover, and in good agreement with the findings of Soothill & Harvey (1976) approximately 7% of our control subjects, who did not have a history of abnormal infections also showed defective opsonization, suggesting perhaps a relatively common biological deviation not necessarily associated with clinical immune deficiency. Interestingly, approximately 25 % of atopic individuals have recently been found to have opsonin defects identified by the microscopical baker's yeast assay (Turner, Brostoff, Mowbray, Wells, Harvey & Soothill, 1978). It is tempting to speculate that our defective controls, the immune deficient patients and the atopic individuals all have the same type of defect which alone is not sufficient to produce clinical disease but predisposes to both immune deficiency and allergy. This would be an additional link between atopic and immune deficiency diseases (Soothill, 1976). Indeed, 4 of our 8 patients with immune deficiency and
694
M. Yamamura & H. Valdimarsson
defective opsonization were also atopic, whilst none of the remaining 18 patients with normal alternative pathway opsonin capacity had history of atopic allergy. These questions can only be answered by elucidating the biochemical basis of the opsonin defect which must clearly be located in the alternative complement pathway. Soothill & Harvey have indeed demonstrated that sera with the opsonizing defect in their assay show a subnormal consumption of C5-9 when incubated at 370 with inulin and endotoxin (Soothill & Harvey, 1977). However, all defective sera so far tested by us and by Soothill and Harvey have had normal and occasionally high levels of alternative pathway and Factor B activity as measured in haemolytic assays.
ACKNOWLEDGMENTS This work was supported by the Multiple Sclerosis Society G.B. and the Wellcome Trust. We thank Professor J. F. Soothill for providing sera from patients, Professor P. J. Lachmann and Professor J. Mowbray for complement deficient sera and Mrs G. Dasilva for excellent technical assistance.
REFERENCES LACHMANN P.J., HOBART M.J. & ASTON W.P. (1973) Complement Technology. In: Handbook of Experimental Immunology, 2nd edn (ed. by D. M. Weir), I. Chapt. 5. Blackwell Scientific Publications, Oxford. MILLER M.E. & NILSSON U.R. (1970) A familial deficiency of the phagocytosis-enhancing activity of serum related to a dysfunction of the fifth component of complement (CS). N. Eng. J. Med. 282, 354. SOOTHILL J.F. & HARVEY B.A.M. (1976) Defective opsonization. A common immunity deficiency. Arch. Dis. Childh. 51, 91. SOOTHILL J.F. (1976) Some intrinsic and extrinsic factors predisposing to allergy. President's Address. Proc. R. Soc. Med. 69, 439. SOOTHILL J.F. & HARVEY B.A.M. (1977) A defect of the alternative pathway of complement. Clin. exp. Immunol. 27, 30. STOSSEL T.P. (1973) Evaluation of opsonic and leucocyte function with a spectrophotometric test in patients with infection and phagocytic disorders. Blood, 42, 121. TURNER M.W., BROSTOFF J., MOWBRAY J., WELLS R.S., HARVEY B.A.M. & SOOTHILL J.F. (1978) Defective yeast opsonization and C2 deficiency in atopic patients. (In
preparation.) WRIGHT A.E. & DOUGLAS S.R. (1903) An experimental and investigation of the role of blood fluids in connection with phagocytosis. Proc. R. Soc. London. R. V. of Sci. 72, 357. YAMAMURA M., BOLER J. & VALDIMARSSON H. (1977) Phagocytosis measured as inhibition of uridine uptake by Candida albicans. J. Immunol. Methods, 14, 19.
A new semiquantitative radiometric opsonin assay SELECTIVE MEASUREMENT OF OPSONIZING CAPACITY OF THE ALTERNATIVE PATHWAY
M. YA MAM URA & H. VALDI MARS SON Department of Immunology, St Mary's Hospital Medical School, London W2 1PG
Received 30 June 1977; acceptedfor publication 18 August 1977
Summary. A new semiquantitative radiometric opsonin assay is described. It was found that the opsonin activity generated by incubating brewer's yeast, Saccharomyces cerevisiae, in medium containing less than 5 % human serum was exclusively complement dependent. In contrast, C. albicans was effectively opsonized in the absence of complement. Antibodies and the early classical complement pathway did not contribute to the opsonization of S. cerevisiae and neither did C5-9. The brewer's yeast assay can therefore be used for measuring selectively the opsonizing capacity of the alternative pathway. Sera from approximately 7 % of apparently healthy adult controls consistently failed to generate significant opsonin activity while 8 out of 26 patients with suspected immune deficiency of unknown cause were defective in this assay. All opsonin deficient sera so far tested had haemolytically normal alternative pathway and Factor B activity. INTRODUCTION
before specific immunity is acquired. The alternative pathway is a major source of opsonin activity, and micro-organisms capable of directly activating this pathway are therefore obvious targets for antibody independent opsonization. Assays for analysing opsonization have not been significantly improved since the phenomenon was first described (Wright & Douglas, 1903). Thus, with a few exceptions (Stossel, 1973), tests for opsonin activity still depend on counting ingested particles under the microscope. One such assay, in which heat killed baker's yeast is used, has been reported to selectively detect complement-derived opsonins (Miller & Nilsson, 1970). The difficulty of distinguishing between adherent and engulfed particles is a serious drawback inherent in all microscopical techniques. We have recently developed an objective method for measuring phagocytosis of yeast organisms, in which this problem was overcome (Yamamura, Boler & Valdimarsson, 1977). This technique has now been modified for analysing and quantifying opsonin activity.
Antibody-independent opsonization may be important for microbial defence, especially early in life
MATERIALS AND METHODS
Correspondence: Dr H. Valdimarsson, Department of Immunology, St Mary's Hospital Medical School, London W2 1PG.
3H-uridine (Specific activity 5 Ci/mmol) was obtained from the Radiochemical Centre, Amersham, 0019-2805/78/0400-0689 $02.00 (©1978 Blackwell Scientific Publications 689
690
M. Yamamura & H. Valdimarsson
England. Peptone was purchased from Difco. Medium RPML 1640 (Flow Laboratories) was buffered with HEPES and used throughout. LP3 tubes (Luckham) were used for mixing yeast and polymorphonuclear leucocytes (PMNs) during phagocytosis, but aliquots for measuring uridine uptake were transferred into microtitre wells (Cooke).
Yeast Fast growing strain of Saccharomyces cerevisiae (Strain L) was isolated from CWE brewer's yeast, which is specially formulated for rapid fermentation. The strain was kindly identified by Mrs Barbara Kirsop, of the Brewing Industry Research Foundation, Redhill, Surrey. The yeast was stored on 2% glucose, 1 % peptone and 2 % agar slopes at 40, and transferred to a new agar slope every 1-2 months. Subcultures were made in RPMI 1640 at 280 for 48-72 h and then kept at 40 for up to 1 week without loss of number or viability. The viability was greater than 98 %.
Polymorphonuclear leucocytes (PMNs) These were obtained from heparinized blood. Erythrocytes were sedimented by adding Dextran 110 (Dextran 110, Fison Ltd.). PMNs were washed at least 5 times with RPMI 1640 and adjusted to 5 x 105/ml and 2-5 x 105/ml.
Sera Sera were separated from clotted blood samples and kept at - 1900. Human R3 reagent was prepared by treating serum with Zymosan (Sigma) (Lachmann, Hobart & Aston, 1973). It retained 40% of intact C3, tested by two dimensional immunoelectrophoresis, but no haemolytic complement was present at a dilution of 1/5 and Factor B activity was also depleted. There was no decrease in haemolytically active C2 and C4. C3 deficient serum was kindly supplied by Dr P. J. Lachmann and C5 deficient serum was obtained from B1OD2 strain mice. Assay principle We have previously demonstrated that inhibition of 3H-uridine incorporation into yeast in a log growth phase can be used as a sensitive index of phagocytic function (Yamamura et al., 1977). This is because: (1) there is a linear relationship between uridine
incorporation and yeast number; (2) yeast replicating inside phagocytic cells do not take up uridine from culture medium; and (3) phagocytic cells do not incorporate significant amounts of uridine in short term cultures. Using the S. cerevisiae (L strain) it was found that during an incubation period of 60 min, 3 x 103 organisms incorporated sufficient amounts of 3H-uridine and there was a linear relationship between uridine uptake and yeast number between 3 x 103 and 3 x 105 per culture.
Standard assay procedure Unless otherwise stated, assays were performed as follows: a mixture containing 5 x 105 yeast cells and 5 x 104 or 1 x 105 PMNs was rotated at 370 for 30 min in 0 5 ml of medium containing 2-5 and/or 1 -25 % serum. Duplicate 0-2 ml aliquots were transferred to microtitre wells to which 0-2 pCi of 3H-uridine had previously been added and the microtitre plates incubated for a further 60 min at 370. The cells were then collected onto glass fibre filter paper by a Skatron Multiple Harvester, the dried discs placed in scintillation vials, covered with 5 ml of PPO-toluene (4 g/1) scintillation fluid and counted by a Packard Tri-carb Liquid Scintillation Spectrometer, Model B2450. Presentation of data Opsonin activity has traditionally been expressed as the opsonic index which is the average number of particles ingested by each phagocyte under conditions where phagocytosis is limited by the opsonin concentration. In the assay described here the results can be expressed as per cent inhibition of uridine incorporation (I) caused by phagocytosis or c.p.m. in PMNs present 100 c.p.m. in PMNs absent) It is also possible to work out the average number of yeast ingested by each PMN leucocyte because there is a linear relationship between the number of phagocytosed yeast and the inhibition of uridine incorporation. The data in this paper will be presented as Opsonin Indices by the following formula: I
(1
IxR where I is the per cent inhibition of uridine 100 incorporation caused by phagocytosis and R the yeast/PMN ratio.
Radiometric opsonin assay RESULTS
Evaluation of assay conditions Assay conditions for opsonin activity must be adjusted so that the number of particles ingested is limited in a linear fashion by the opsonin concentration and not by the number of particles available for phagocytosis.
4
3
C2
0E
691
Fig. 1 shows that in the presence of 1 -25 and 2 5% serum, phagocytosis was dependent on the availability of yeast when there were less than 5 organisms per PMN leucocyte, but remained constant when the yeast/PMN ratio exceeded 5/1. Using yeast/PMN ratios of 5/1 and 10/1, it was further demonstrated that phagocytosis increased in a linear fashion when serum concentration was raised from 0 625 to 2 5 % while a plateau was usually reached at 5 % (Fig. 2). These observations indicate that, with yeast/PMN ratios between 5/1 and 10/1, there is a linear relationship between phagocytosis and serum concentration up to 2-5 %. As normal sera gave a wide scatter at 0 625 %, it was decided to use serum dilutions of 1 -25 and 2 5 % for measuring opsonin activity.
0
Analysis of the opsonin activity Table 1 demonstrates that C. albicans was effectively 10/5 10/2 Ratio (S. cerevisioel PM N)
Table 1. The different serum requirements for opsonization of S. cerevisiae and C. albicans*
Figure 1. Phagocytosis of S. cerevisiae using yeast/PMN ratios from 1/1 to 10/1. The ingestion was no longer limited by the availability of yeast if the ratio was 5/1 or more. (0) 1-5 % serum concentration; (0) 2-5 % serum concentration. 6 5
0
Normal serat Serum heated 560 for 30 min Zymosan treated serum C2 deficient serum C3 deficient serum Agammaglobulinaemic serum ( < 50 mg/I) Serum free medium
4 0 ±0-6 0 0 30 01
5-0 ±1-2 5-6 4-6 49 6-5
3-2 0
4-8 0-1
-0
4
.0
S. cerevisiae C. albicans
* All sera used at 25 %. t Mean ± s.d., n = 20.
3 0
0 0 Q.
0
2
-/I
0-625
25 1I25 Concentration of serum (%)
5-0
Figure 2. Four experiments with yeast/PMN ratio of 10/1. The phagocytosis was limited by the serum concentration up to 2-5 %.
opsonized by heat inactivated serum, while S. cerevisiae, was not. Similarly, zymosan treated serum had normal opsonin activity for C. albicans but none for S. cerevisiae. Sera deficient in C2 or antibodies had normal opsonin capacity for both yeast types whilst C3 deficient serum opsonized only C. albicans and not S. cerevisiae. C5 deficient mouse serum had normal opsonin activity for S. cerevisiae (Fig. 3). In order to test whether antibodies, although not
692
M. Yamamura & H. Valdimarsson Table 2. Normal ranges for opsonic activity of adult human sera against S. cerevisiae
4 X 3 '(3 ._
-i 2 0
Serum concentration 0
0x
Mean opsonin index Standard deviation Normal ranges (95 % conf. limit) 2-5 1-25 5 Concentration of serum (%)
Figure 3. Opsonin activity of C5 deficient compared with normal mouse serum (0).
mouse serum
*
125%
25%
2-7 04 1 9-3 5
4-0 0-6 2 8-5-2
n = 20; 2 healthy donors with low values were excluded.
(0)
low in opsonin activity and, in contrast to all other healthy controls, these individuals did not show any increase in opsonin activity when the serum concentration was increased from 1 25 to 2 5 %. Table 3 shows the reproducibility of the assay in a longitudinal study of 5 individuals.
essential, might nevertheless contribute to the opsonization of S. cerevisiae, the yeast was preincubated in 20 % heat inactivated sera, washed and then tested in 1 -25 and 2 5 % of the R3 reagent. No opsonization was detected in these experiments. It is therefore evident that under the assay conditions used, the opsonin activity for S. cerevisiae was exclusively derived from direct activation of the alternative complement pathway. In contrast, phagocytosis of C. albicans did not require complement activation.
Comparison with the microscopical baker's yeast opsonin assay Six sera that had been found to lack opsonin activity in the microscopical baker's yeast assay (Soothill & Harvey, 1976) were kindly supplied by Professor J. F. Soothill. They were all found to be deficient in the radiometric assay. However, 1 of the 3 control sera which were normal in the microscopical assay showed opsonin deficiency in the radiometric assay. Altogether, 3 out of 39 sera from healthy controls were found to be deficient in this assay (Fig. 4). So far, sera have been tested from 26 patients with clinically suspected immune deficiency, but without detectable immune abnormality in laboratory tests, and 8 were found to lack opsonin activity.
Normal ranges Sera from 20 healthy adults were assayed for opsonin activity towards S. cerevisiae. Table 2 shows the means and standard deviations of opsonin indices for serum concentrations of 1 -25 and 2 5 %. Normal ranges with 95 % confidence limits for 1 -25 and 2-5 % serum were 1 9-3 5 and 2-8-5-2 respectively. However, 2 healthy donors were consistently very
Table 3. Reproducibility of the assay in longitudinal studies
No. of Serum donors
Normal healthy donor (1) Normal healthy donor (2) Healthy donor with opsonin deficiency (1) Healthy donor with opsonin deficiency (2) Patient with recurrent infections
tests
4 4 3 3 3
Opsonin indices
1-25% serum
3-5; 2-7; 2-6; 3-2 2-5; 2-3; 2-0; 2-3 0-1; 0-5; 0 0; 0; 0 0-5; 0-2; 0-1
2 5 % serum
4-1; 4-8; 3 9; 5 1 4 5; 3-0; 3-1; 3-4 0-1; 0 5; 0 0; 0; 0 0 3; 0 3; 0-2
_A\~ ~ ~ Radiometric opsonin assay
i593
0
5
S
4
3 0 c
0
Vt
TO 2
*0O 0
=9I 0^
1 0 0
.3. a
I
O1
A
aa B
0 0 S
S
Y
0
_
D
C
I
Figure 4. Opsonin activity of sera from 39 control subjects, 6 samples with known opsonin defect as determined by microscopical yeast assay and 26 patients suspected of immune deficiency. All sera were tested at a concentration of 1-25 %. (A) Healthy normal subjects; (B) healthy subjects with opsonin defect; (C) known opsonin defect; (D) suspected immunodeficiency.
DISCUSSION Opsonization is of major importance for effective pathogen and antigen elimination. However, the molecular basis of this phenomenon is poorly understood and quantitative opsonin assays, suitable for general clinical use, have not been available. The radiometric assay described in this paper is semiquantitative and easy to perform. It is essential, however, to select and use a homogeneous strain of fast growing yeast organisms. Employing a fast growing strain of S. cerevisiae, we have demonstrated that the opsonin activity generated by the alternative complement pathway can be selectively measured. This assay correlates with a microscopical baker's yeast test hitherto used for complement derived opsonins. In a preliminary clinical study, sera from 8 out of 26 patients with unexplained immune deficiency have failed to generate opsonin activity in our assay. This is in good agreement with the recent report of Soothill and Harvey who found defective opsonization in 11 of 43 children with unexplained infections. H
They also observed that the majority of healthy mothers of affected children had defective opsonization, suggesting a primary abnormality rather than a depletion phenomenon (Soothill & Harvey, 1976). Moreover, and in good agreement with the findings of Soothill & Harvey (1976) approximately 7% of our control subjects, who did not have a history of abnormal infections also showed defective opsonization, suggesting perhaps a relatively common biological deviation not necessarily associated with clinical immune deficiency. Interestingly, approximately 25 % of atopic individuals have recently been found to have opsonin defects identified by the microscopical baker's yeast assay (Turner, Brostoff, Mowbray, Wells, Harvey & Soothill, 1978). It is tempting to speculate that our defective controls, the immune deficient patients and the atopic individuals all have the same type of defect which alone is not sufficient to produce clinical disease but predisposes to both immune deficiency and allergy. This would be an additional link between atopic and immune deficiency diseases (Soothill, 1976). Indeed, 4 of our 8 patients with immune deficiency and
694
M. Yamamura & H. Valdimarsson
defective opsonization were also atopic, whilst none of the remaining 18 patients with normal alternative pathway opsonin capacity had history of atopic allergy. These questions can only be answered by elucidating the biochemical basis of the opsonin defect which must clearly be located in the alternative complement pathway. Soothill & Harvey have indeed demonstrated that sera with the opsonizing defect in their assay show a subnormal consumption of C5-9 when incubated at 370 with inulin and endotoxin (Soothill & Harvey, 1977). However, all defective sera so far tested by us and by Soothill and Harvey have had normal and occasionally high levels of alternative pathway and Factor B activity as measured in haemolytic assays.
ACKNOWLEDGMENTS This work was supported by the Multiple Sclerosis Society G.B. and the Wellcome Trust. We thank Professor J. F. Soothill for providing sera from patients, Professor P. J. Lachmann and Professor J. Mowbray for complement deficient sera and Mrs G. Dasilva for excellent technical assistance.
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