- Email: [email protected]
Distribution and levels of properdin in human body fluids
CLINICAL
IMMI‘NOLOGY
ASD
Distribution
IMM”N”PATHOLOGY
and Levels
J. 0. MINTA*, Department
Department Immunology,
5, 84-90
(1976)
of Properdin Fluids’
in Human
Body
P. D. JEZYK, AND I. H. LEPOW
of Medicine,
The University of Connecticut Connecticut 06032, und of Pathology, Division of Experimental University of Toronto, Medicul Sciences Canada
Health
Pathology Building.
Center,
Farmington,
and institute oJ’ Toronto. M5S IA8,
Received July 14, 1975 Radioimmunoassay of71 normal adult human sera gave a mean properdin concentration of 19.3 + 5.4 &ml. Similar values were found in sera from patients with sickle cell anemia and in maternal serum at the time of full-term delivery. A limited ontogenetic study revealed that the concentration of properdin in fetal serum increased progressively during gestation, reaching one-half to two-thirds of adult levels at parturition and full adult levels about 1 yr later. Properdin was not present in significant concentrations in amniotic fluid, milk, colostrum, parotid saliva, nasal washings, sweat, gastric juice, bile, urine, seminal fluid, and cerebrospinal fluid indicating the absence of an intact properdin pathway in these normal fluids.
INTRODUCTION
The properdin system was described in 1954 (1) as a group of normal serum proteins and magnesium ions constituting a pathway to the terminal components of complement and implicated in killing of susceptible bacteria, neutralization of certain viruses, and lysis of abnormal erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (l-4). In addition to properdin, the constituents of this pathway included proteins which resembled but were apparently different from the early acting components (C 1, C4, C2) of the classical complement sequence (1,5,6). More recently, multiple independent lines of evidence that have more definitively established the properdin system as a distinct alternative pathway for the activation of terminal components of complement (C3, CS-C9), have been reported (7-16). In accordance with current conventions, the properdin system is now designated as the properdin or alternative pathway, the rigorously defined components of which are properdin, Factor B, Factor D, and C3.3 The biologic importance of the properdin pathway for the activation of the terminal complement components has been reemphasized in a case report of a patient with increased susceptibility to infections due to impaired complement * This investigation was supported by NIH Grant Al-08251 and the Medical Research Council of Canada (MA-5063). 2 Research Scholar of the Canadian Heart Foundation. 3 Factor B is synonymous with C3 proactivator (C3PA), glycine-rich P-glycoprotein (GBG) and heat-labile factor; Factor D with C3PA convertase and GBGase; C3 with Factor A. 84 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROPERDIN
LEVELS
IN
BODY
FLUIDS
85
mediated functions (17). This patient was shown to be deficient in C3b inactivator that destroys a product (C3b) shown to initiate a positive feedback amplification mechanism involving Factor B and Factor D for further cleavage of C3 and activation of the terminal C5-C9 components of complement (9,18,19). Properdin has been reported to be deposited together with other immune reactants in the renal glomeruli of patients with glomerulonephritis (20), lupus nephritis (20,21), and in the dermal-epidermal junction of certain skin diseases (21,22). The properdin concentrations in the sera of these patients were also correspondingly depressed (20-25). Highly purified properdin has been shown to interact in serum to initiate the activation of the constituents of the properdin system and the late acting complement components (26,27). We have recently devised a sensitive solid phase radioimmunoassay for measuring properdin concentration in the nanogram range (28). In this report, we have used this assay to study the distribution and levels of properdin in normal and pathological body fluids in order to gain additional biological and pathobiological information on the potential role of the properdin pathway in humoral homeostasis. METHODS
AND MATERIALS
Solid phase radioimmunoassay was performed in polystyrene tubes (Falcon Plastics, Oxnard, California) as previously described (28). In this assay, a fixed area of the interior of polystyrene tubes was coated with an optimal dilution of monospecific rabbit antihuman properdin antiserum (l/1000) in 0.06 M sodium barbital buffer, pH 9.6, for 2 hr at 23” C. The competitive binding of increasing concentrations of unlabeled properdin (lo-60 ng) and a constant amount of rzsI properdin (40 ng) to the antibody-coated tubes was a linear function of the concentration of the unlabeled protein. Properdin concentrations in unknown samples were then estimated from the standard curve. Properdin was purified from human serum according to the method of Pensky et al. (7) and was trace-iodinated with 12sI by the ICI method of Helmkamp et al. (29), as described by Minta et al. (28). Rabbit antihuman properdin antiserum was prepared by injection of purified properdin (100 pg) in complete Freund’s adjuvant at multiple intradermal sites, followed by booster injections 4 weeks later using the same amount of properdin in incomplete Freund’s adjuvant. Antisera were harvested beginning 7-10 days after the booster injections. Normal adult human blood. This was obtained from laboratory personnel and a commercial blood donor service(Knickerbocker, New York). Serum from patients with sickle cell anemia was kindly supplied by Dr. Fred S. Rosen, Childrens Hospital, Boston. Maternal and cord blood. Blood was obtained from pregnant mothers by venipuncture at the time of delivery. Blood from newborn infants from normal full-term pregnancies was obtained from the umbilical cord immediately after delivery. Maternal and cord blood were collected by Dr. Richard Osborne, University of Connecticut. Fetal cord blood. This was obtained from conceptuses that were aborted for
86
MIN’I’A,
J”““”
AND
IXPOM
medical or psychiatric indications. Gestational age was estimated from Streeter’s data for embryos with a crown-rump length of 30 mm or less and Patten’s data were used for crown-rump length of 30 mm or more (30,311. Amniotic fluid. Fluid was collected by Dr. Saul Rosenberg, New Britain General Hospital, New Britain, from normal term pregnancies by aspiration with needle and syringe directly from the amniotic sac. Colostrum clnd milk. These were collected by Dr. Martha Lepow. Department of Pediatrics, University of Connecticut. Parotid saliva. Saliva from laboratory personnel was collected into Curby cups by stimulating with citric acid solution on the opposite side of the tongue after the cup was sealed on the buccal mucosa. The pH of the parotid saliva was checked to make sure that there was no leakage of the citric acid into the saliva collected. Dr. Irving Goldschneider, Department of Pathology, University of Connecticut, kindly performed these collections. Nasul washings. These were collected by gently instilling 5 ml of saline with a syringe into the anterior nares doing one site at a time and holding the head backward for few seconds before expelling into a beaker. Nasal washings were collected from laboratory personnel without any upper respiratory infection. The assistance of Dr. Irving Goldschneider is gratefully acknowledged. Sweat. Sweat was collected from three volunteers by the technique of iontophoresis by Dr. Robert Greenstein, Department of Pediatrics, University of Connecticut. Bile and gastric juices. Dr. James Foster, Hartford Hospital, Hartford, collected these from patients on the surgical service. Urine. Samples were collected from laboratory personnel without known renal disease. Semen. Dr. Eugene Sigman, Department of Surgery, Division of Urology, University of Connecticut, collected semen samples by prostatic massage of normal individuals who had undergone vasectomy. Cerebrospinal jluid. This was collected by Dr. Melville Roberts, Division of Neurosurgery. Newington Veterans Administration Hospital, by lumbar puncture on patients undergoing myelography for possible laminectomy. None of these patients had spinal cord tumors. Infant blood. Samples were obtained by heel puncture at 4- 12 months of age and were kindly provided from an independent study by Dr. Martha Lepow and Dr. Irving Goldschneider. The infants were all born by normal full-term deliveries and had satisfactory general state of development. All body fluids were ceritrifuged and stored at -70°C prior to radioimmunoassay. RESULTS
AND DISCUSSION
The results of measurements of properdin concentration in several different human body fluids are presented in Table 1. The range of properdin concentration in 71 normal adult sera varied between 10.5 and 36.5 pg/ml, with the mean and the standard deviation of the mean at 19.3 +- 5.4 &ml. It has been shown by Johnston et al. (32) that sera from patients with sickle
PROPERDIN
LEVELS
IN
TABLE PROPERDIN
BODY
87
FLUIDS
1
CONCENTRATION
IN BODY
FLUIDS
Properdin concentration Sample
Number tested
Range
71 27 20 46 5 8 8 10 9 3 5 4 12 3 4
10.5-36.5 11.6-23.9 14.6-33.3 3.1-23.4 O-O.32 0 0 O-O.26 O-O.26 O-O.68 o-0.40 0 o-o. 17 o-o.51 0
Normal adult human serum Sickle cell serum Maternal serum Cord serum Amniotic fluid Milk Colostrum Parotid saliva Nasal washings Sweat Gastric juice Bile Urine Seminal fluid Cerebrospinal fluid
(CLg/ml)
Mean -C SD 19.3 17.0 24.8 11.2 0.1 0 0 0.09 0.06 0.39 0.08 0 0.03 0.37 0
+ 2 2 ?
5.4 3.5 3.6 3.6
cell anemia are defective in opsonic activity. The properdin content of sera from 27 patients with sickle cell anemia assayed in this study was within the normal range. Thus, the defective opsonic activity that has been observed in sickle cell anemia patients does not appear to be related to properdin concentration. However, it is recognized that the radioimmunoassay measures total antigen concentration and does not distinguish between functionally active protein molecules and inactive species that still retain antigenic determinants. The properdin concentration in 20 maternal sera collected at the onset of parturition was 24.8 ? 3.6 pg/ml and was in good agreement with normal adult values. However, the properdin concentration in 46 cord sera collected from full-term babies was comparatively low, ranging from 3.1 to 23.4 pg/ml with the mean at 11.2 ? 3.6 pg/ml. The properdin concentrations in 11 paired maternal and cord sera are presented in Table 2. The maternal serum levels of properdin ranged from 14.6 to 33.3 pg/rnl and averaged 23.1 ? 6.8 pg/ml. The properdin concentration in patient cord sera was 8.1-23.4 pg/ml, with the mean at 15.6 ? 5.7 pg/ml. In all but one case, the concentration of properdin in the maternal serum was higher than in the corresponding cord serum, confirming an earlier report that the hemolytic titers of properdin in cord blood TABLE PROPERDIN
CONCENTRATION
2
IN PAIRED
MATERNAL
AND
CORD
SERA
Properdin concentration (&ml) Sample
Number tested
Range
Mean 2 SD
Maternal serum Cord serum
11 11
14.6-33.3 8.1-23.4
23.lk6.8 15.6k5.7
88
MINTA,
J”“YK
AND
LEPOW
were lower than in maternal blood (33). In the one exception, titers of properdin in cord and maternal sera were the same. Statistically, the properdin concentration in maternal sera was significantly higher than in cord sera (P < 0.0005). A limited number of fetal sera have also been analyzed for properdin concentration. The ages of the fetuses studied ranged from 9 to 22 weeks. Properdin was barely detectable (1.5 pgiml) in the cord blood of a 9-week-old fetus. The properdin concentrations in a 15 and a 17-week-old fetus were 2.9 and 5.6 pg/ml, respectively. Cord blood from two 22-week-old fetuses contained 9.4 and 11.7 pg/rnl respectively. These results of a limited number of fetal sera indicated a progressive increase in serum properdin levels in the conceptuses as the period of gestation increased. Properdin was found to be present in low concentrations in fetal sera during early embryonic life and increased progressively with gestational time, although adult values were not obtained at full-term. In a limited number of samples examined. properdin was completely undetectable or was present only in trace amounts (less than 1 pg/ml) in amniotic fluid, milk, colostrum, parotid saliva, nasal washings, sweat, gastric juice. bile, urine, seminal fluid, and cerebrospinal fluid (Table 1). The traces of properdin found in some of these samples could have been due to contamination with blood. These results in part are in agreement with those of Pillemer et ul. (I) who found by less sensitive hemolytic assays that properdin activity was undetectable in amniotic fluid, colostrum, and mature milk. The absence of significant amounts of properdin in these body fluids indicate that an intact properdin pathway is not present and is therefore not participating in normal homeostasis at these extravascular sites. However, these findings do not rule out the possibility that properdin might be present in these body fluids as a result of an inflammatory process and increased vascular permeability. Properdin in fetal sera may be derived from the maternal circulation via the allantoic placenta or synthesized by the fetus or both. It is also possible that properdin may be transferred from the mother to the fetus through the amniotic fluid and then by absorption through the fetal gastrointestinal tract or through the fetal lungs. However, this is an unlikely route of transfer since amniotic fluids from five conceptuses at full term did not contain properdin or properdin was present in barely detectable traces much lower than the concentrations in the maternal or cord blood. Although transplacental passage cannot be completely excluded at this point. the possibility that properdin in cord blood originated in the fetus is indirectly indicated by its absence in amniotic fluid and low concentration in fetal and cord blood compared to maternal blood. The definition of the sites and the exact time of onset of biosynthesis of properdin must await in ritrt~ studies with fetal tissues. The concetration of properdin in sera of randomly selected infants of various postnatal ages (4,8, and 12 months) are presented in Table 3. It can be seen that the properdin concentration in the 4-month-old infant (15.3 + 3.1 pg/ml) was approximately the same as in the newborn. However, by 8 months of age the properdin levels had rise to a mean of 17.5 -+ 4.4 /&ml, a significant increase over the previous value (P < 0.05). By 12 months of age, adult values were reached and the increase in serum properdin levels over
PROPERDIN
LEVELS
IN
TABLE SERUM
PROPERDIN
LEVELS
BODY
89
FLUIDS
3 OF NORMAL
INFANTS
Properdin concentration (&ml) Serum sample 4-month-old infants g-month-old infants 12-month-old infants
Number tested
Range
Mean * SD
11 11 11
9.0- 19.6 8.6-24.5 13.9-26.6
15.3 2 3.1 17.5 k 4.4 20.3 f 4.6
those at 8 months of age was also statistically significant (P < 0.025). The difference in serum properdin levels at 12 and 4 months of age was highly significant (P < 0.005). These results indicate that the properdin concentration in the neonatal serum was not depressed below the levels at the time of delivery but increased progressively with the attainment of adult values at about 12 months of age. REFERENCES 1. Pillemer, L., Blum, L., Lepow, I. H., Ross, 0. A., Todd, E. W., and Wardlaw, A. C., Science 120, 279, 1954. 2. Wardlaw, A. C., and Pillemer, L., J. Exp. Med. 103, 553, 1956. 3. Wedgwood, R. J., Ginsberg, H. S., and Pillemer, L., J. Exp. Med. 104, 707, 1956. 4. Hinz, C. F., Jr., Jordan, W. S., Jr., and Pillemer, L., J. Clin. Znvesr. 35, 453, 1956. 5. Pensky, J., Wurz, L., Pillemer, L., and Lepow, I. H., Z. Zmmunifatsforsch. 118, 329, 1959. 6. Blum, L., Pillemer. L., and Lepow, I. H., Z. Immuni&rsforsch. 118, 349, 1959. 7. Pensky, J., Hinz, C. F., Jr., Todd, E. W., Wedgood, R. J., Boyer, J. T., and Lepow, 1. H., J. Immunol. 100, 142, 1968. 8. Gotze, O., and Muller-Eberhard, H.-J., J. Exp. Med. 134, 190s 1971. 9. Muller-Eberhard, H.-J., and Gotze, O., J. Exp. Med. 135, 1003, 1972. 10. Goodkofsky, I. and Lepow, I. H., J. Immunol. 107, 1200, 1971. 11. Alper, C. A., Goodkofsky, I., and Lepow, I. H., J. Exp. Med. 137, 424, 1973. 12. Goodkofsky, I., Stewart, A., and Lepow, I. H., J. Immunol. 111, 287, 1973. 13. Sandberg, A. L., Osler, A. G., Shin, H. S., and Oliveira, B., J. Immunol. 104, 329, 1970. 14. Marcus, R. L., Shin, H. S., and Mayer, M. M., Proc. Nat. Acad. Sci. USA 68, 1351, 1971. 15. Frank, M. M., May, J., Gaither, T., and Ellman, L., J. Exp. Med. 134, 176, 1971. 16. Hunsicker, L. G., Ruddy, S., and Austen, K. F., J. Immunol. 110, 128, 1973. 17. Alper, C. A., Abramson, N., Johnston, R. B., Jr., Jandl, J. H., and Rosen, F. S., N. Engl. .I. Med. 282, 349, 1970. 18. Alper, C. A., Rosen, F. S., and Lachmann, P. J., Proc. Nat. Acad. Sci. USA 69, 2910, 1972. 19. Nicol, P., and Lachmann, P. J., Immunology 24, 259, 1973. 20. Westberg, N. G., Naff, G. B., and Boyer, J. T., J. Clin. Invesr. 50, 642, 1971. 21. Rothtield, N. F., Ross, 0. A., Minta, J. 0.. and Lepow, I. H., N. Engl. J. Med. 287, 681, 1972. 22. Provost, T. T. and Tomasi, J. B., Jr., J. Cfin. Znvesr. 52, 1779, 1973. 23. Jordan, R. E., Schroeter, A. L., Good, R. A., and Day, N. K., Clin. Immunol. Immunopathol. 3, 307, 1975. 24. McLean, R. H., and Michael, A. T., J. Clin. Invest. 52, 634, 1973. 25. Perrin, L. H., Lambert, P. H., and Miescher, P. A., Clin. Exp. Immunol. 16, 575, 1974. 26. Go&e, O., and Muller-Eberhard, H.-J., J. Immunol. 111, 189, 1973. 27. Minta, J. O., and Lepow, I. H., Zmmunochem, 11, 261, 1974. 28. Minta, J. O., Goodkofsky, I., and Lepow, I. H., Immunochemistry 10, 341, 1973. 29. Helmkamp, R. W., Goodland, R. I., Bale, W. F., Spar, I. L., and Mutschler, L. W., Cancer Res. 20, 1495, 1960.
90 30. 31. 32. 33.
MINTA,
JEZYK
AN11
LEPOM
Streeter. G. L.. Cuvnegie Contrib. Embryo/. 32, 133, 194X. Patten. B. M., “Human Embryology.” Blackiston, New York. 1948. Johnston, R. B.. Jr., Newman, S. L.. and Struth, A. G.. N. EngI. J. Med. 288, 803, 1973. Yoshioka, H.. Matsumoto, S., Itagaki, M., Ogata. R.. and Shirashita. H.. Aun. Pueditrt. 187. 1964.
203
IMMI‘NOLOGY
ASD
Distribution
IMM”N”PATHOLOGY
and Levels
J. 0. MINTA*, Department
Department Immunology,
5, 84-90
(1976)
of Properdin Fluids’
in Human
Body
P. D. JEZYK, AND I. H. LEPOW
of Medicine,
The University of Connecticut Connecticut 06032, und of Pathology, Division of Experimental University of Toronto, Medicul Sciences Canada
Health
Pathology Building.
Center,
Farmington,
and institute oJ’ Toronto. M5S IA8,
Received July 14, 1975 Radioimmunoassay of71 normal adult human sera gave a mean properdin concentration of 19.3 + 5.4 &ml. Similar values were found in sera from patients with sickle cell anemia and in maternal serum at the time of full-term delivery. A limited ontogenetic study revealed that the concentration of properdin in fetal serum increased progressively during gestation, reaching one-half to two-thirds of adult levels at parturition and full adult levels about 1 yr later. Properdin was not present in significant concentrations in amniotic fluid, milk, colostrum, parotid saliva, nasal washings, sweat, gastric juice, bile, urine, seminal fluid, and cerebrospinal fluid indicating the absence of an intact properdin pathway in these normal fluids.
INTRODUCTION
The properdin system was described in 1954 (1) as a group of normal serum proteins and magnesium ions constituting a pathway to the terminal components of complement and implicated in killing of susceptible bacteria, neutralization of certain viruses, and lysis of abnormal erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (l-4). In addition to properdin, the constituents of this pathway included proteins which resembled but were apparently different from the early acting components (C 1, C4, C2) of the classical complement sequence (1,5,6). More recently, multiple independent lines of evidence that have more definitively established the properdin system as a distinct alternative pathway for the activation of terminal components of complement (C3, CS-C9), have been reported (7-16). In accordance with current conventions, the properdin system is now designated as the properdin or alternative pathway, the rigorously defined components of which are properdin, Factor B, Factor D, and C3.3 The biologic importance of the properdin pathway for the activation of the terminal complement components has been reemphasized in a case report of a patient with increased susceptibility to infections due to impaired complement * This investigation was supported by NIH Grant Al-08251 and the Medical Research Council of Canada (MA-5063). 2 Research Scholar of the Canadian Heart Foundation. 3 Factor B is synonymous with C3 proactivator (C3PA), glycine-rich P-glycoprotein (GBG) and heat-labile factor; Factor D with C3PA convertase and GBGase; C3 with Factor A. 84 Copyright @ 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROPERDIN
LEVELS
IN
BODY
FLUIDS
85
mediated functions (17). This patient was shown to be deficient in C3b inactivator that destroys a product (C3b) shown to initiate a positive feedback amplification mechanism involving Factor B and Factor D for further cleavage of C3 and activation of the terminal C5-C9 components of complement (9,18,19). Properdin has been reported to be deposited together with other immune reactants in the renal glomeruli of patients with glomerulonephritis (20), lupus nephritis (20,21), and in the dermal-epidermal junction of certain skin diseases (21,22). The properdin concentrations in the sera of these patients were also correspondingly depressed (20-25). Highly purified properdin has been shown to interact in serum to initiate the activation of the constituents of the properdin system and the late acting complement components (26,27). We have recently devised a sensitive solid phase radioimmunoassay for measuring properdin concentration in the nanogram range (28). In this report, we have used this assay to study the distribution and levels of properdin in normal and pathological body fluids in order to gain additional biological and pathobiological information on the potential role of the properdin pathway in humoral homeostasis. METHODS
AND MATERIALS
Solid phase radioimmunoassay was performed in polystyrene tubes (Falcon Plastics, Oxnard, California) as previously described (28). In this assay, a fixed area of the interior of polystyrene tubes was coated with an optimal dilution of monospecific rabbit antihuman properdin antiserum (l/1000) in 0.06 M sodium barbital buffer, pH 9.6, for 2 hr at 23” C. The competitive binding of increasing concentrations of unlabeled properdin (lo-60 ng) and a constant amount of rzsI properdin (40 ng) to the antibody-coated tubes was a linear function of the concentration of the unlabeled protein. Properdin concentrations in unknown samples were then estimated from the standard curve. Properdin was purified from human serum according to the method of Pensky et al. (7) and was trace-iodinated with 12sI by the ICI method of Helmkamp et al. (29), as described by Minta et al. (28). Rabbit antihuman properdin antiserum was prepared by injection of purified properdin (100 pg) in complete Freund’s adjuvant at multiple intradermal sites, followed by booster injections 4 weeks later using the same amount of properdin in incomplete Freund’s adjuvant. Antisera were harvested beginning 7-10 days after the booster injections. Normal adult human blood. This was obtained from laboratory personnel and a commercial blood donor service(Knickerbocker, New York). Serum from patients with sickle cell anemia was kindly supplied by Dr. Fred S. Rosen, Childrens Hospital, Boston. Maternal and cord blood. Blood was obtained from pregnant mothers by venipuncture at the time of delivery. Blood from newborn infants from normal full-term pregnancies was obtained from the umbilical cord immediately after delivery. Maternal and cord blood were collected by Dr. Richard Osborne, University of Connecticut. Fetal cord blood. This was obtained from conceptuses that were aborted for
86
MIN’I’A,
J”““”
AND
IXPOM
medical or psychiatric indications. Gestational age was estimated from Streeter’s data for embryos with a crown-rump length of 30 mm or less and Patten’s data were used for crown-rump length of 30 mm or more (30,311. Amniotic fluid. Fluid was collected by Dr. Saul Rosenberg, New Britain General Hospital, New Britain, from normal term pregnancies by aspiration with needle and syringe directly from the amniotic sac. Colostrum clnd milk. These were collected by Dr. Martha Lepow. Department of Pediatrics, University of Connecticut. Parotid saliva. Saliva from laboratory personnel was collected into Curby cups by stimulating with citric acid solution on the opposite side of the tongue after the cup was sealed on the buccal mucosa. The pH of the parotid saliva was checked to make sure that there was no leakage of the citric acid into the saliva collected. Dr. Irving Goldschneider, Department of Pathology, University of Connecticut, kindly performed these collections. Nasul washings. These were collected by gently instilling 5 ml of saline with a syringe into the anterior nares doing one site at a time and holding the head backward for few seconds before expelling into a beaker. Nasal washings were collected from laboratory personnel without any upper respiratory infection. The assistance of Dr. Irving Goldschneider is gratefully acknowledged. Sweat. Sweat was collected from three volunteers by the technique of iontophoresis by Dr. Robert Greenstein, Department of Pediatrics, University of Connecticut. Bile and gastric juices. Dr. James Foster, Hartford Hospital, Hartford, collected these from patients on the surgical service. Urine. Samples were collected from laboratory personnel without known renal disease. Semen. Dr. Eugene Sigman, Department of Surgery, Division of Urology, University of Connecticut, collected semen samples by prostatic massage of normal individuals who had undergone vasectomy. Cerebrospinal jluid. This was collected by Dr. Melville Roberts, Division of Neurosurgery. Newington Veterans Administration Hospital, by lumbar puncture on patients undergoing myelography for possible laminectomy. None of these patients had spinal cord tumors. Infant blood. Samples were obtained by heel puncture at 4- 12 months of age and were kindly provided from an independent study by Dr. Martha Lepow and Dr. Irving Goldschneider. The infants were all born by normal full-term deliveries and had satisfactory general state of development. All body fluids were ceritrifuged and stored at -70°C prior to radioimmunoassay. RESULTS
AND DISCUSSION
The results of measurements of properdin concentration in several different human body fluids are presented in Table 1. The range of properdin concentration in 71 normal adult sera varied between 10.5 and 36.5 pg/ml, with the mean and the standard deviation of the mean at 19.3 +- 5.4 &ml. It has been shown by Johnston et al. (32) that sera from patients with sickle
PROPERDIN
LEVELS
IN
TABLE PROPERDIN
BODY
87
FLUIDS
1
CONCENTRATION
IN BODY
FLUIDS
Properdin concentration Sample
Number tested
Range
71 27 20 46 5 8 8 10 9 3 5 4 12 3 4
10.5-36.5 11.6-23.9 14.6-33.3 3.1-23.4 O-O.32 0 0 O-O.26 O-O.26 O-O.68 o-0.40 0 o-o. 17 o-o.51 0
Normal adult human serum Sickle cell serum Maternal serum Cord serum Amniotic fluid Milk Colostrum Parotid saliva Nasal washings Sweat Gastric juice Bile Urine Seminal fluid Cerebrospinal fluid
(CLg/ml)
Mean -C SD 19.3 17.0 24.8 11.2 0.1 0 0 0.09 0.06 0.39 0.08 0 0.03 0.37 0
+ 2 2 ?
5.4 3.5 3.6 3.6
cell anemia are defective in opsonic activity. The properdin content of sera from 27 patients with sickle cell anemia assayed in this study was within the normal range. Thus, the defective opsonic activity that has been observed in sickle cell anemia patients does not appear to be related to properdin concentration. However, it is recognized that the radioimmunoassay measures total antigen concentration and does not distinguish between functionally active protein molecules and inactive species that still retain antigenic determinants. The properdin concentration in 20 maternal sera collected at the onset of parturition was 24.8 ? 3.6 pg/ml and was in good agreement with normal adult values. However, the properdin concentration in 46 cord sera collected from full-term babies was comparatively low, ranging from 3.1 to 23.4 pg/ml with the mean at 11.2 ? 3.6 pg/ml. The properdin concentrations in 11 paired maternal and cord sera are presented in Table 2. The maternal serum levels of properdin ranged from 14.6 to 33.3 pg/rnl and averaged 23.1 ? 6.8 pg/ml. The properdin concentration in patient cord sera was 8.1-23.4 pg/ml, with the mean at 15.6 ? 5.7 pg/ml. In all but one case, the concentration of properdin in the maternal serum was higher than in the corresponding cord serum, confirming an earlier report that the hemolytic titers of properdin in cord blood TABLE PROPERDIN
CONCENTRATION
2
IN PAIRED
MATERNAL
AND
CORD
SERA
Properdin concentration (&ml) Sample
Number tested
Range
Mean 2 SD
Maternal serum Cord serum
11 11
14.6-33.3 8.1-23.4
23.lk6.8 15.6k5.7
88
MINTA,
J”“YK
AND
LEPOW
were lower than in maternal blood (33). In the one exception, titers of properdin in cord and maternal sera were the same. Statistically, the properdin concentration in maternal sera was significantly higher than in cord sera (P < 0.0005). A limited number of fetal sera have also been analyzed for properdin concentration. The ages of the fetuses studied ranged from 9 to 22 weeks. Properdin was barely detectable (1.5 pgiml) in the cord blood of a 9-week-old fetus. The properdin concentrations in a 15 and a 17-week-old fetus were 2.9 and 5.6 pg/ml, respectively. Cord blood from two 22-week-old fetuses contained 9.4 and 11.7 pg/rnl respectively. These results of a limited number of fetal sera indicated a progressive increase in serum properdin levels in the conceptuses as the period of gestation increased. Properdin was found to be present in low concentrations in fetal sera during early embryonic life and increased progressively with gestational time, although adult values were not obtained at full-term. In a limited number of samples examined. properdin was completely undetectable or was present only in trace amounts (less than 1 pg/ml) in amniotic fluid, milk, colostrum, parotid saliva, nasal washings, sweat, gastric juice. bile, urine, seminal fluid, and cerebrospinal fluid (Table 1). The traces of properdin found in some of these samples could have been due to contamination with blood. These results in part are in agreement with those of Pillemer et ul. (I) who found by less sensitive hemolytic assays that properdin activity was undetectable in amniotic fluid, colostrum, and mature milk. The absence of significant amounts of properdin in these body fluids indicate that an intact properdin pathway is not present and is therefore not participating in normal homeostasis at these extravascular sites. However, these findings do not rule out the possibility that properdin might be present in these body fluids as a result of an inflammatory process and increased vascular permeability. Properdin in fetal sera may be derived from the maternal circulation via the allantoic placenta or synthesized by the fetus or both. It is also possible that properdin may be transferred from the mother to the fetus through the amniotic fluid and then by absorption through the fetal gastrointestinal tract or through the fetal lungs. However, this is an unlikely route of transfer since amniotic fluids from five conceptuses at full term did not contain properdin or properdin was present in barely detectable traces much lower than the concentrations in the maternal or cord blood. Although transplacental passage cannot be completely excluded at this point. the possibility that properdin in cord blood originated in the fetus is indirectly indicated by its absence in amniotic fluid and low concentration in fetal and cord blood compared to maternal blood. The definition of the sites and the exact time of onset of biosynthesis of properdin must await in ritrt~ studies with fetal tissues. The concetration of properdin in sera of randomly selected infants of various postnatal ages (4,8, and 12 months) are presented in Table 3. It can be seen that the properdin concentration in the 4-month-old infant (15.3 + 3.1 pg/ml) was approximately the same as in the newborn. However, by 8 months of age the properdin levels had rise to a mean of 17.5 -+ 4.4 /&ml, a significant increase over the previous value (P < 0.05). By 12 months of age, adult values were reached and the increase in serum properdin levels over
PROPERDIN
LEVELS
IN
TABLE SERUM
PROPERDIN
LEVELS
BODY
89
FLUIDS
3 OF NORMAL
INFANTS
Properdin concentration (&ml) Serum sample 4-month-old infants g-month-old infants 12-month-old infants
Number tested
Range
Mean * SD
11 11 11
9.0- 19.6 8.6-24.5 13.9-26.6
15.3 2 3.1 17.5 k 4.4 20.3 f 4.6
those at 8 months of age was also statistically significant (P < 0.025). The difference in serum properdin levels at 12 and 4 months of age was highly significant (P < 0.005). These results indicate that the properdin concentration in the neonatal serum was not depressed below the levels at the time of delivery but increased progressively with the attainment of adult values at about 12 months of age. REFERENCES 1. Pillemer, L., Blum, L., Lepow, I. H., Ross, 0. A., Todd, E. W., and Wardlaw, A. C., Science 120, 279, 1954. 2. Wardlaw, A. C., and Pillemer, L., J. Exp. Med. 103, 553, 1956. 3. Wedgwood, R. J., Ginsberg, H. S., and Pillemer, L., J. Exp. Med. 104, 707, 1956. 4. Hinz, C. F., Jr., Jordan, W. S., Jr., and Pillemer, L., J. Clin. Znvesr. 35, 453, 1956. 5. Pensky, J., Wurz, L., Pillemer, L., and Lepow, I. H., Z. Zmmunifatsforsch. 118, 329, 1959. 6. Blum, L., Pillemer. L., and Lepow, I. H., Z. Immuni&rsforsch. 118, 349, 1959. 7. Pensky, J., Hinz, C. F., Jr., Todd, E. W., Wedgood, R. J., Boyer, J. T., and Lepow, 1. H., J. Immunol. 100, 142, 1968. 8. Gotze, O., and Muller-Eberhard, H.-J., J. Exp. Med. 134, 190s 1971. 9. Muller-Eberhard, H.-J., and Gotze, O., J. Exp. Med. 135, 1003, 1972. 10. Goodkofsky, I. and Lepow, I. H., J. Immunol. 107, 1200, 1971. 11. Alper, C. A., Goodkofsky, I., and Lepow, I. H., J. Exp. Med. 137, 424, 1973. 12. Goodkofsky, I., Stewart, A., and Lepow, I. H., J. Immunol. 111, 287, 1973. 13. Sandberg, A. L., Osler, A. G., Shin, H. S., and Oliveira, B., J. Immunol. 104, 329, 1970. 14. Marcus, R. L., Shin, H. S., and Mayer, M. M., Proc. Nat. Acad. Sci. USA 68, 1351, 1971. 15. Frank, M. M., May, J., Gaither, T., and Ellman, L., J. Exp. Med. 134, 176, 1971. 16. Hunsicker, L. G., Ruddy, S., and Austen, K. F., J. Immunol. 110, 128, 1973. 17. Alper, C. A., Abramson, N., Johnston, R. B., Jr., Jandl, J. H., and Rosen, F. S., N. Engl. .I. Med. 282, 349, 1970. 18. Alper, C. A., Rosen, F. S., and Lachmann, P. J., Proc. Nat. Acad. Sci. USA 69, 2910, 1972. 19. Nicol, P., and Lachmann, P. J., Immunology 24, 259, 1973. 20. Westberg, N. G., Naff, G. B., and Boyer, J. T., J. Clin. Invesr. 50, 642, 1971. 21. Rothtield, N. F., Ross, 0. A., Minta, J. 0.. and Lepow, I. H., N. Engl. J. Med. 287, 681, 1972. 22. Provost, T. T. and Tomasi, J. B., Jr., J. Cfin. Znvesr. 52, 1779, 1973. 23. Jordan, R. E., Schroeter, A. L., Good, R. A., and Day, N. K., Clin. Immunol. Immunopathol. 3, 307, 1975. 24. McLean, R. H., and Michael, A. T., J. Clin. Invest. 52, 634, 1973. 25. Perrin, L. H., Lambert, P. H., and Miescher, P. A., Clin. Exp. Immunol. 16, 575, 1974. 26. Go&e, O., and Muller-Eberhard, H.-J., J. Immunol. 111, 189, 1973. 27. Minta, J. O., and Lepow, I. H., Zmmunochem, 11, 261, 1974. 28. Minta, J. O., Goodkofsky, I., and Lepow, I. H., Immunochemistry 10, 341, 1973. 29. Helmkamp, R. W., Goodland, R. I., Bale, W. F., Spar, I. L., and Mutschler, L. W., Cancer Res. 20, 1495, 1960.
90 30. 31. 32. 33.
MINTA,
JEZYK
AN11
LEPOM
Streeter. G. L.. Cuvnegie Contrib. Embryo/. 32, 133, 194X. Patten. B. M., “Human Embryology.” Blackiston, New York. 1948. Johnston, R. B.. Jr., Newman, S. L.. and Struth, A. G.. N. EngI. J. Med. 288, 803, 1973. Yoshioka, H.. Matsumoto, S., Itagaki, M., Ogata. R.. and Shirashita. H.. Aun. Pueditrt. 187. 1964.
203