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Biosynthesis of the fifth component of complement (C5) by human fetal tissues
CLINICAL
IMMUNOLOC:Y
Biosynthesis
AND
1MMUNOPATHOLOGY
1,
346-352
(1973)
of the Fifth Component of Complement by Human Fetal Tissues1
(C5)
Tissues isolated from human fetuses as early as 9 wk gestation synthesized the fifth component of complement (C5). Production of biologically active C5 was temperature dependent, reversibly inhibited by cycloheximide, and was accompanied by incorporation of ‘C-labeled amino acids into C5 protein. Significant C5 production was detected in cultures of liver, lung, and small intestine.
In an early study of the ontogeny of serum complement (C), Rice and Silverstein found that, in fetal sheep, a functionally intact complement system did not appear until shortly before term (1). More recently, it has been shown that, of the complement components studied thus far, each is synthesized in a biologically active form early in gestation by human and/or animal fetuses. The fetus is capable of synthesizing the first (Cl) (2,3), second (C2) (4), fourth (C4) (4,5), third (C3) (4,6), sixth (C6) (7), and eighth (C8) (8) components of complement. The ontogeny of the fifth, seventh, and ninth components has not been studied. In this report, evidence is presented that human fetal tissues synthesize biologically active C5 in vitro and that this capacity is present at 9 wk gestation. Production of biologically active C5 by fetal tissues was temperature dependent, reversibly inhibited by cycloheximide, and accompanied by the incorporation of 14C-labeled amino acids into C5 protein. Significant in vitro production of C5 was detected in fetal and postnatal liver, in fetal lung and intestine, but not in stomach, muscle, kidney, or spleen. MATERIALS
AND
METHODS
Prepuwtiorl of ti.ysztes. Human fetal tissues were obtained at therapeutic abortion from fetuses delivered by hysterotomy’ Gestational ages were estimated by comparing crown-rump measurements with published standards (9). The tissues were placed immediately into ice-cold medium I99 under sterile conditions, then minced and washed three times in Neuman and Tytell’s serumless medium to reduce baseline levels of performed C5. Measured portions (approx 50-100 mg wet wt) of the washed tissue fragments were ’ Supported by USPHS Grants 2 Tissues kindly made available Division, Boston, Mass.
AI-05877 and HD-05916. by Dr. Shirley Driscoll, 346
Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reservrd.
Boston
Hospital
for Women,
Lying-In
HUMAN
FETAL
SYNTHESIS
OF
c5
347
transferred to 35 x 10 mm plastic Petri dishes (Falcon Plastics, Oxford, Calif.) and then incubated in a specially prepared minimal medium (see below). This medium supported biosynthesis of C5 for 72-96 hr and provided optimal conditions for stability of biologically active C5 in culture. Postnatal tissue specimens obtained at surgery were handled in the same way as the fetal material. Mediu. Medium 199 was purchased from Microbiological Associates (Bethesda, Md.); and Neuman and Tytell’s serumless medium, from Grand Island Biological Company (Grand Island, N. Y.). A special minimal medium designed for studies of biosynthesis of the late-acting complement components was prepared from stock reagents to contain NaClO.147 M, dextrose 0.012 M, KC1 0.4 mg/ml, NaHCO, 3.96 mg/ml, MgCI, lo-:’ M, CaCl, 1.5 X lo-” M, NaH2P0, 0.94 mg/ml, essential and nonessential amino acids (Microbiological Associates), and stock vitamins (Microbiological Associates). These reagents were dissolved in sterile pyrogen-free water; the solution was filtered (Millipore 0.22 pm) and then stored at -20°C. Immediately before use, glutamine lo-“M, hydrocortisone (Solucortef, Upjohn) 2 pglml, heated fetal calf serum (Associated Biomedical Systems) l%, penicillin 50 U/ml, and streptomycin 50 pg were added. Nernol~& Asscly of CS. Sheep erythrocytes (E) were purchased from Microbiological Associates (Bethesda, MD). Rabbit anti-Forssman antibody (A), the cell intermediate EAC14, and appropriate buffers were prepared as described in (10). Functionally purified human C2, C3, C6, C7, C8, and C9 were purchased from Cordis Corporation (Miami, Fla.). Oxidized C2 (C20xy) was prepared as described (11). C5 activity was determined by mixing 0.2 ml of a suspension of EAC14 (7.5 x 10i/ml) with 0.2 ml of a dilution of the sample and 0.4 ml of a mixture containing functionally purified human C2”““, C3, C6, and C7. The mixtures were incubated first at 30°C for 30 min, then at 37°C for 60 min after adding 0.4 ml of a mixture of C8 and C9. The cells were removed by centrifugation and the optical density of the supernatant fluids were read at 412 nm. The number of hemolytically effective C5 molecules in each sample was calculated from the extent of lysis (10). Veronal-buffered saline dextrose buffer (p = 0.075) (VBS-D) was used as the diluent throughout. This hemolytic assay for C5 is capable of detecting as few as 2 x 10’j effective molecules per milliliter. Ir~c.orl,ortrtio,b o.f14C-labeled clnrir~o (lcid.s. Tissue fragments were incubated in Ml99 with 10% fetal calf serum in which ‘Z-labeled leucine, lysine, isoleucine, and valine (New England Nuclear Corporation) at a final concentration of 1 &i/ml were substituted for the corresponding unlabeled amino acids. After 72 hr incubation, the media were harvested and dialyzed for 3 days against veronal-buffered saline containing 0.001 M NaN,. Each sample was concentrated lo- to 15-fold. A portion of each of the concentrated samples was then mixed with normal human serum (to provide carrier protein) and placed in wells cut in 1% agarose opposite rabbit antiserum specific for human C5.” After 48 hr, the immunodi&sion plates were washed first in ,: Antisera
kindly
supplied
by Dr.
Chester
A. Alper,
Children’s
Hospital,
Boston,
Mass.
348
COLTEN
0.15 M NaCI, then in distilled water; The plates were dried and then exposed to Kodak x-ray film for 6 wk. Cycloheximide purchased from Nutritional Biochemicals Corporation (Cleveland, Ohio) was used at a concentration of l-2 lg/ml in experiments designed to test the effect of a known inhibitor of protein synthesis on C5 production. RESULTS Production ofC5 by individuul tissues. Measured portions (SO-100 mg wet wt) of minced fetal lung, liver, kidney, muscle, large and small bowel, and spleen were incubated in minimal medium at 37°C in a humidified atmosphere (95% air:5% CO,). At timed intervals, O.l-ml aliquots of the media were removed, diluted in VBS-D buffer, and assayed for biologically active C5. From these data, the average rates of C5 production per 100 mg tissue were calculated. The results in Table 1 show that fetal liver, lung, and intestine were capable of producing biologically active C5 in vitro. The rate of C5 production by liver ranged between 4 x 10’ and 1 x lo* hemolytically effective C5 molecules per 100 mg tissue per hr. The rate of C5 production by fetal liver at 9 wk gestation was not significantly different from that in later fetal or postnatal liver. Significantly, one liver obtained from a patient with alpha-l antitrypsin deficiency and profound cirrhosis produced significantly less C5 than other liver specimens. Lung and intestine each produced about 1 X 10M effective molecules per 100 mg per hr. No significant C5 production was detected in cultures of fetal spleen, stomach, kidney, or muscle. However, a small amount of C5 production was detected in cultures of spleen obtained from a 15-yr-old patient with thalessemia.
In Source of tissue Fetus Fetus Fetus Fetus Fetus Fetus Fetus
Estimated gestational age
Liver
9.0 9.5 9.5 10.5 12.0 13.0 14.5
0.50 0.73 0.96 0.89 0.55 0.40 0.67
Vitro
TABLE 1 BIOSYNTHESIS
C5 effective
molecules
(X lo-“)/lo0
mg tissue/hr __
Lung
Spleen
Intestine
Kidney
---~~ MUS&
1.2 1.6 0.90 1.10
-: 0.01 1.8
0.86 1.00
CO.01
Postnatal Cirrhosis Alpha-l antitrypsin deficiency Goodpasture’s disease Chronic nephritis Chronic nephritis Thalassemia
OF C5
0.15 0.29 0.70 1.00 0.35 0.15
HUMAN
FIG. 1. In vitro production 37°C (0) and at 4°C (0).
FETAL
of C5 by fetal
SYNTHESIS
intestine:
Effect
OF
c5
of temperature.
349
Tissue
incubated
at
Effect of temper&we on C.5 production in vitro. Washed fragments of small bowel obtained from a 9-wk-old fetus were incubated at 37°C and at 4°C in minimal medium. At timed intervals, aliquots of the media were removed and assayed for C5 content. The results in Fig. 1 show that incubation of small intestine at 37°C resulted in more than a 1Sfold increase in C5; whereas, at 4°C no significant C5 production was detected. In other experiments, a similar effect of temperature on production of C5 liver and lung was observed. Effect of cycloheximide on C5 production in vitro. Fragments of jejunum and liver from a 10.Swk fetus were each incubated at 37°C in minimal medium or in minimal medium containing cycloheximide 2 pg/ml. At timed intervals, aliquots of the media were removed, diluted, and assayed for hemolytically active C5. The results in Fig. 2 show that cycloheximide inhibited C5 production by jejunum 66% and by liver about 84%. In other experiments, cycloheximide inhibited C5 production by fetal lung and postnatal liver cultures; the inhibitory effect of cycloheximide was reversed by
25 TIME (HOURS) FIG.
Triangles symbols
50
2. Effect of cycloheximide on C5 production in n&o: Circles = C5 production = C5 production by liver. Closed symbols = tissues in medium alone = tissues in medium containing cycloheximide (2 pg/ml).
by jejunum. at 37°C. Open
350
FIG. 3. Radioimmunodiffision analysis of incorporatiorl of “C-labcbled amino arids irlttr ( :.5. (Z;. and properdin factor B (CBG). Center well contains dialyzed concentrated medium harvc~~tr~d from liver culture at 72 hr. Well number (1) anti-W; (2) anti-CS; aud (3) anti-C BG.
washing the tissues. The assay and stability of C5 were unaffected by (:ycl~heximide (2 Fglml). Incorporation of ‘~-labeled umino acids into CT.5protein. Figure 3 shop c the results of radioimmunodiffusion analysis of medium harvested front L,~IItures of adult liver incubated at 37°C in the presence of “C-labeled antilt
HUMAN
FETAL
‘SYNTHESIS
OF
C5
351
of anaphylatoxic and chemotactic factors, and production of ultrastructural lesions in cell membranes do not require the action of C6-C9. Accordingly, assignment of a potential role of complement in fetal life depends on a demonstration that the developing fetus is capable of synthesizing at least the first five components in their biologically active forms. Fetal biosynthesis would be required, since for some components there is evidence against matemalfetal transplacental passage of complement (7,12,13). On the basis of evidence presented in this report, as well as others (4-6, 14), it appears that in the first trimester of gestation the human fetus is capable of synthesizing each of the first five components of complement. Additional studies are now required to determine whether the conventional and/or alternate pathways of complement activation are essential for protection against intrauterine infection. In 1962, it was shown that certain strains of mice were deficient in C5 (15). Phillips et ~1. (16) then found that transplantation of bone marrow from normal co-isogenic or allogeneic donors to C5-deficient mice resulted in a transient appearance of hemolytic complement in the recipients’ sera. Moreover, cells isolated from spleens of the recipients were capable of synthesizing C5 protein ill t;it~o. In the present study, several human tissues, including fetal lung, liver, postnatal liver, and spleen were found to synthesize C5. In these studies, the cell type that synthesized C5 has not been identified. Significant production of hemolytically active C5 was detected in cultures of fetal intestine. This production was temperature dependent and reversibly inhibited by cycloheximide, but incorporation of ‘“C-labeled amino acid into C5 protein was not detectable on three attempts. The possibility must be considered, therefore, that the intestine may not be a site of de t~oco synthesis of C5. It should be noted that in the present study tissues were incubated in a specially prepared minimal medium to detect biosynthesis of biologically active C5. Preliminary experiments indicate that this medium is also suitable for studies on the biosynthesis of biologically active C6 and C7. ACKNOWLEDGMENTS I thank Drs. Shirely Driscoll and Raphael Levey for supplying tissue samples, Dr. Chester Alper for antisera, and Miss Nobuko Sugimoto for technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
RICE, C. E., UD SILVERSTEIN, A. M. Cnti. J. Cotnp. Met/. Vet. Sci. 28, 34, 1964. COLTEN, H. R., SILVERSTEIN, A. hf., HOROS, T., AM RAPP, H. J. Irnm~~no/ogy 15,459, DAY, N. C., GEVVURZ, H., PICKERING, R. J., AND Goou, R. A. J. Zrnmrrno/. 104, 1316, COLTEN, H. R. J. C2in. Inwst. 51, 725, 1972. ADINOLFI, hf., GARDNER, B., .~ND WOOD, C.B.S. h’utuw (Londm) 219, 189, 1968. GITLIN, II., ANI BIASUCXX, A. J. Clia. Inuest. 48, 1433, 1969. BOI
1968. 1970.
1968.
352
COLTEN
11. POLLEY, M. J., AND MULLER-EBERHARD, H. J. 1. Erp. Med. 126,1013, 1967. 12. PROPP, R. P., AND ALPER, C. A. Science 162,672, 1968. 13. BACH, S., RUDDY, S., MACLAREN, J. A., AND AUSTEN, K. F. Immunology 21, 869, 1971. 14. COLTEN, H. R., GORDON, J. M., BORSOS, T., AND UPP, H. J. J. &)I. Med. 128, 595, 1968. 15. ROSENBERG, L. T., AND TACHIBANA, D. K. J. Immunol. 89,861, 1962. 16. PHILLIPS,M.E.,ROTHER,U.A.,ROTHER,K.O.,ANDTHORBECKE,G. J. Immwsology 17,315, 1969.
IMMUNOLOC:Y
Biosynthesis
AND
1MMUNOPATHOLOGY
1,
346-352
(1973)
of the Fifth Component of Complement by Human Fetal Tissues1
(C5)
Tissues isolated from human fetuses as early as 9 wk gestation synthesized the fifth component of complement (C5). Production of biologically active C5 was temperature dependent, reversibly inhibited by cycloheximide, and was accompanied by incorporation of ‘C-labeled amino acids into C5 protein. Significant C5 production was detected in cultures of liver, lung, and small intestine.
In an early study of the ontogeny of serum complement (C), Rice and Silverstein found that, in fetal sheep, a functionally intact complement system did not appear until shortly before term (1). More recently, it has been shown that, of the complement components studied thus far, each is synthesized in a biologically active form early in gestation by human and/or animal fetuses. The fetus is capable of synthesizing the first (Cl) (2,3), second (C2) (4), fourth (C4) (4,5), third (C3) (4,6), sixth (C6) (7), and eighth (C8) (8) components of complement. The ontogeny of the fifth, seventh, and ninth components has not been studied. In this report, evidence is presented that human fetal tissues synthesize biologically active C5 in vitro and that this capacity is present at 9 wk gestation. Production of biologically active C5 by fetal tissues was temperature dependent, reversibly inhibited by cycloheximide, and accompanied by the incorporation of 14C-labeled amino acids into C5 protein. Significant in vitro production of C5 was detected in fetal and postnatal liver, in fetal lung and intestine, but not in stomach, muscle, kidney, or spleen. MATERIALS
AND
METHODS
Prepuwtiorl of ti.ysztes. Human fetal tissues were obtained at therapeutic abortion from fetuses delivered by hysterotomy’ Gestational ages were estimated by comparing crown-rump measurements with published standards (9). The tissues were placed immediately into ice-cold medium I99 under sterile conditions, then minced and washed three times in Neuman and Tytell’s serumless medium to reduce baseline levels of performed C5. Measured portions (approx 50-100 mg wet wt) of the washed tissue fragments were ’ Supported by USPHS Grants 2 Tissues kindly made available Division, Boston, Mass.
AI-05877 and HD-05916. by Dr. Shirley Driscoll, 346
Copyright All rights
@ 1973 by Academic Press, Inc. of reproduction in any form reservrd.
Boston
Hospital
for Women,
Lying-In
HUMAN
FETAL
SYNTHESIS
OF
c5
347
transferred to 35 x 10 mm plastic Petri dishes (Falcon Plastics, Oxford, Calif.) and then incubated in a specially prepared minimal medium (see below). This medium supported biosynthesis of C5 for 72-96 hr and provided optimal conditions for stability of biologically active C5 in culture. Postnatal tissue specimens obtained at surgery were handled in the same way as the fetal material. Mediu. Medium 199 was purchased from Microbiological Associates (Bethesda, Md.); and Neuman and Tytell’s serumless medium, from Grand Island Biological Company (Grand Island, N. Y.). A special minimal medium designed for studies of biosynthesis of the late-acting complement components was prepared from stock reagents to contain NaClO.147 M, dextrose 0.012 M, KC1 0.4 mg/ml, NaHCO, 3.96 mg/ml, MgCI, lo-:’ M, CaCl, 1.5 X lo-” M, NaH2P0, 0.94 mg/ml, essential and nonessential amino acids (Microbiological Associates), and stock vitamins (Microbiological Associates). These reagents were dissolved in sterile pyrogen-free water; the solution was filtered (Millipore 0.22 pm) and then stored at -20°C. Immediately before use, glutamine lo-“M, hydrocortisone (Solucortef, Upjohn) 2 pglml, heated fetal calf serum (Associated Biomedical Systems) l%, penicillin 50 U/ml, and streptomycin 50 pg were added. Nernol~& Asscly of CS. Sheep erythrocytes (E) were purchased from Microbiological Associates (Bethesda, MD). Rabbit anti-Forssman antibody (A), the cell intermediate EAC14, and appropriate buffers were prepared as described in (10). Functionally purified human C2, C3, C6, C7, C8, and C9 were purchased from Cordis Corporation (Miami, Fla.). Oxidized C2 (C20xy) was prepared as described (11). C5 activity was determined by mixing 0.2 ml of a suspension of EAC14 (7.5 x 10i/ml) with 0.2 ml of a dilution of the sample and 0.4 ml of a mixture containing functionally purified human C2”““, C3, C6, and C7. The mixtures were incubated first at 30°C for 30 min, then at 37°C for 60 min after adding 0.4 ml of a mixture of C8 and C9. The cells were removed by centrifugation and the optical density of the supernatant fluids were read at 412 nm. The number of hemolytically effective C5 molecules in each sample was calculated from the extent of lysis (10). Veronal-buffered saline dextrose buffer (p = 0.075) (VBS-D) was used as the diluent throughout. This hemolytic assay for C5 is capable of detecting as few as 2 x 10’j effective molecules per milliliter. Ir~c.orl,ortrtio,b o.f14C-labeled clnrir~o (lcid.s. Tissue fragments were incubated in Ml99 with 10% fetal calf serum in which ‘Z-labeled leucine, lysine, isoleucine, and valine (New England Nuclear Corporation) at a final concentration of 1 &i/ml were substituted for the corresponding unlabeled amino acids. After 72 hr incubation, the media were harvested and dialyzed for 3 days against veronal-buffered saline containing 0.001 M NaN,. Each sample was concentrated lo- to 15-fold. A portion of each of the concentrated samples was then mixed with normal human serum (to provide carrier protein) and placed in wells cut in 1% agarose opposite rabbit antiserum specific for human C5.” After 48 hr, the immunodi&sion plates were washed first in ,: Antisera
kindly
supplied
by Dr.
Chester
A. Alper,
Children’s
Hospital,
Boston,
Mass.
348
COLTEN
0.15 M NaCI, then in distilled water; The plates were dried and then exposed to Kodak x-ray film for 6 wk. Cycloheximide purchased from Nutritional Biochemicals Corporation (Cleveland, Ohio) was used at a concentration of l-2 lg/ml in experiments designed to test the effect of a known inhibitor of protein synthesis on C5 production. RESULTS Production ofC5 by individuul tissues. Measured portions (SO-100 mg wet wt) of minced fetal lung, liver, kidney, muscle, large and small bowel, and spleen were incubated in minimal medium at 37°C in a humidified atmosphere (95% air:5% CO,). At timed intervals, O.l-ml aliquots of the media were removed, diluted in VBS-D buffer, and assayed for biologically active C5. From these data, the average rates of C5 production per 100 mg tissue were calculated. The results in Table 1 show that fetal liver, lung, and intestine were capable of producing biologically active C5 in vitro. The rate of C5 production by liver ranged between 4 x 10’ and 1 x lo* hemolytically effective C5 molecules per 100 mg tissue per hr. The rate of C5 production by fetal liver at 9 wk gestation was not significantly different from that in later fetal or postnatal liver. Significantly, one liver obtained from a patient with alpha-l antitrypsin deficiency and profound cirrhosis produced significantly less C5 than other liver specimens. Lung and intestine each produced about 1 X 10M effective molecules per 100 mg per hr. No significant C5 production was detected in cultures of fetal spleen, stomach, kidney, or muscle. However, a small amount of C5 production was detected in cultures of spleen obtained from a 15-yr-old patient with thalessemia.
In Source of tissue Fetus Fetus Fetus Fetus Fetus Fetus Fetus
Estimated gestational age
Liver
9.0 9.5 9.5 10.5 12.0 13.0 14.5
0.50 0.73 0.96 0.89 0.55 0.40 0.67
Vitro
TABLE 1 BIOSYNTHESIS
C5 effective
molecules
(X lo-“)/lo0
mg tissue/hr __
Lung
Spleen
Intestine
Kidney
---~~ MUS&
1.2 1.6 0.90 1.10
-: 0.01 1.8
0.86 1.00
CO.01
Postnatal Cirrhosis Alpha-l antitrypsin deficiency Goodpasture’s disease Chronic nephritis Chronic nephritis Thalassemia
OF C5
0.15 0.29 0.70 1.00 0.35 0.15
HUMAN
FIG. 1. In vitro production 37°C (0) and at 4°C (0).
FETAL
of C5 by fetal
SYNTHESIS
intestine:
Effect
OF
c5
of temperature.
349
Tissue
incubated
at
Effect of temper&we on C.5 production in vitro. Washed fragments of small bowel obtained from a 9-wk-old fetus were incubated at 37°C and at 4°C in minimal medium. At timed intervals, aliquots of the media were removed and assayed for C5 content. The results in Fig. 1 show that incubation of small intestine at 37°C resulted in more than a 1Sfold increase in C5; whereas, at 4°C no significant C5 production was detected. In other experiments, a similar effect of temperature on production of C5 liver and lung was observed. Effect of cycloheximide on C5 production in vitro. Fragments of jejunum and liver from a 10.Swk fetus were each incubated at 37°C in minimal medium or in minimal medium containing cycloheximide 2 pg/ml. At timed intervals, aliquots of the media were removed, diluted, and assayed for hemolytically active C5. The results in Fig. 2 show that cycloheximide inhibited C5 production by jejunum 66% and by liver about 84%. In other experiments, cycloheximide inhibited C5 production by fetal lung and postnatal liver cultures; the inhibitory effect of cycloheximide was reversed by
25 TIME (HOURS) FIG.
Triangles symbols
50
2. Effect of cycloheximide on C5 production in n&o: Circles = C5 production = C5 production by liver. Closed symbols = tissues in medium alone = tissues in medium containing cycloheximide (2 pg/ml).
by jejunum. at 37°C. Open
350
FIG. 3. Radioimmunodiffision analysis of incorporatiorl of “C-labcbled amino arids irlttr ( :.5. (Z;. and properdin factor B (CBG). Center well contains dialyzed concentrated medium harvc~~tr~d from liver culture at 72 hr. Well number (1) anti-W; (2) anti-CS; aud (3) anti-C BG.
washing the tissues. The assay and stability of C5 were unaffected by (:ycl~heximide (2 Fglml). Incorporation of ‘~-labeled umino acids into CT.5protein. Figure 3 shop c the results of radioimmunodiffusion analysis of medium harvested front L,~IItures of adult liver incubated at 37°C in the presence of “C-labeled antilt
HUMAN
FETAL
‘SYNTHESIS
OF
C5
351
of anaphylatoxic and chemotactic factors, and production of ultrastructural lesions in cell membranes do not require the action of C6-C9. Accordingly, assignment of a potential role of complement in fetal life depends on a demonstration that the developing fetus is capable of synthesizing at least the first five components in their biologically active forms. Fetal biosynthesis would be required, since for some components there is evidence against matemalfetal transplacental passage of complement (7,12,13). On the basis of evidence presented in this report, as well as others (4-6, 14), it appears that in the first trimester of gestation the human fetus is capable of synthesizing each of the first five components of complement. Additional studies are now required to determine whether the conventional and/or alternate pathways of complement activation are essential for protection against intrauterine infection. In 1962, it was shown that certain strains of mice were deficient in C5 (15). Phillips et ~1. (16) then found that transplantation of bone marrow from normal co-isogenic or allogeneic donors to C5-deficient mice resulted in a transient appearance of hemolytic complement in the recipients’ sera. Moreover, cells isolated from spleens of the recipients were capable of synthesizing C5 protein ill t;it~o. In the present study, several human tissues, including fetal lung, liver, postnatal liver, and spleen were found to synthesize C5. In these studies, the cell type that synthesized C5 has not been identified. Significant production of hemolytically active C5 was detected in cultures of fetal intestine. This production was temperature dependent and reversibly inhibited by cycloheximide, but incorporation of ‘“C-labeled amino acid into C5 protein was not detectable on three attempts. The possibility must be considered, therefore, that the intestine may not be a site of de t~oco synthesis of C5. It should be noted that in the present study tissues were incubated in a specially prepared minimal medium to detect biosynthesis of biologically active C5. Preliminary experiments indicate that this medium is also suitable for studies on the biosynthesis of biologically active C6 and C7. ACKNOWLEDGMENTS I thank Drs. Shirely Driscoll and Raphael Levey for supplying tissue samples, Dr. Chester Alper for antisera, and Miss Nobuko Sugimoto for technical assistance. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
RICE, C. E., UD SILVERSTEIN, A. M. Cnti. J. Cotnp. Met/. Vet. Sci. 28, 34, 1964. COLTEN, H. R., SILVERSTEIN, A. hf., HOROS, T., AM RAPP, H. J. Irnm~~no/ogy 15,459, DAY, N. C., GEVVURZ, H., PICKERING, R. J., AND Goou, R. A. J. Zrnmrrno/. 104, 1316, COLTEN, H. R. J. C2in. Inwst. 51, 725, 1972. ADINOLFI, hf., GARDNER, B., .~ND WOOD, C.B.S. h’utuw (Londm) 219, 189, 1968. GITLIN, II., ANI BIASUCXX, A. J. Clia. Inuest. 48, 1433, 1969. BOI
1968. 1970.
1968.
352
COLTEN
11. POLLEY, M. J., AND MULLER-EBERHARD, H. J. 1. Erp. Med. 126,1013, 1967. 12. PROPP, R. P., AND ALPER, C. A. Science 162,672, 1968. 13. BACH, S., RUDDY, S., MACLAREN, J. A., AND AUSTEN, K. F. Immunology 21, 869, 1971. 14. COLTEN, H. R., GORDON, J. M., BORSOS, T., AND UPP, H. J. J. &)I. Med. 128, 595, 1968. 15. ROSENBERG, L. T., AND TACHIBANA, D. K. J. Immunol. 89,861, 1962. 16. PHILLIPS,M.E.,ROTHER,U.A.,ROTHER,K.O.,ANDTHORBECKE,G. J. Immwsology 17,315, 1969.