Maturation of the Human Complement System

I. ONSETTIME ANDSITES OF FETAL Clq, C4, C3,

ANDC5 SYNTHESIS

PmEE F. Kocm

From the Division of Clinical Immunology, Department of Medicine,University of Colorado Medical Center, Denver, Colorado 80220

A B S T R A C T The onset times and sites of humanClq, C4, C3, and C5 synthesis were determined byculturing tissues from 23 fetuses, 8-25 wk old, in thepresence of [14C]lysine and isoleucine. In parallel, IgGand IgM production was followed. Liver, spleen, pla-centa, peritoneal and bone marrow cells, thymus, andcolon were cultured for 48 h and the concentratedmedia studied by immunoelectrophoresis and subsequentautoradiography using adult human serum as car-rier and specific antisera. The quantitative synthesiswas approximated by scoring the intensity of thelabeled precipitin lines using uniform conditions. C5production was detected earliest at 8 wk gestation andby 11 wk and thereafter, C3, C4, and C5 synthesis wasuniformly present in multiple tissues. Clq synthesis,however, was limited almost exclusively to the spleen,began at 14 wk, and was not uniformly present. Incontrast IgG and IgM production did not occur inthree fetuses synthesizing complement and while de-tected as early as 11 wk, was inconstant, occurredpredominantly in the spleen, and was quantitativelymuch less compared to C3, C4, and C5. These findingssuggest that developmentally the complement system isa more primative biological defense mechanism thanantibody.

INTRODUCTIONThe fundamental importance of the complement systemas an effector mechanism modulating the diverse bio-

Presented in part at the Seventh International Congressof Allergy, Florence, Italy, October 1970 (Excerpta Med.Int. Congr. Ser. 211: 28).

Dr. Kohler is a recipient of v Research Career Develop-ment Award, HD 42404 from the National Institutes ofHealth.

Received for publication 30 March 1972 and in revisedform 23 June 1972.

logical consequences of antigen-antibody interactionshas been delineated over the past decade (reviewedin reference 1). Recently, evidence for complementactivation independent of antibody (2, 3) and for its-participation in blood coagulation (4) have also beenreported.

Relatively limited data are available on the develop-mental aspects and tissue origins of the proteins whichcomprise the human complement system (5-8). Suchinformation has potential relevance to human patho-physiologic processes in general.

The objective of this study was to determine whenfetal synthesis of complement proteins participating ininitial, mid-, and terminal stages of the reaction se-quence, e.g., Clq, C4, C3, and C5, begins. In parallel,the sites and onset time for synthesis of these proteinswere compared and the relative synthesis of each pro-tein was estimated. In addition, the temporal relation-ship between immunoglobulins G and M and comple-ment synthesis was determined. To these ends, thetechnique of in vitro tissue culture in the presence ofcarbon-"'-labeled amino acids followed by immunoelec-trophoresis (IEP) 1 using specific antisera and subse-quent autoradiography, was employed (9).

The results indicate that complement synthesis pre-cedes immunoglobulin synthesis in the human fetussuggesting that it constitutes a more primitive, albeitless specific, defense system. In addition, complementproduction occurs in a wider range of tissues and isquantitatively greater than immunoglobulin synthesis.

METHODSFetal tissues. Tissues were cultured from 37 fetuses ob-

tained after spontaneous or therapeutic abortion at the Uni-versity of Colorado Medical Center and the General Rose

"Abbreviations used in this paper: autoradiograph, ARG;C3 PA, C3 proactivator; IEP, immunoelectrophoresis; PBS,phosphate-buffered saline; WHS, goat antiwhole human sera.

The Journal of Clinical Investigation Volume 52 March 1973 671

Hospital, Denver. The fetuses were aborted spontaneously intwo instances, by amniocentesis in 12 and by hysterotomy orhysterectomy in the remainder. Fetal gestational age rangedfrom 8 to 25 wk as estimated from measurement of crown-heel length according to Scammon and Calkins (10).

Cultures. In vitro culture of various tissues was carriedout in media containing carbon-14 amino acids. This wasfollowed by cellulose acetate electrophoresis and IEP of theconcentrated culture fluid with subsequent autoradiographyas modified by van Furth (11) from the original techniqueof Hochwald, Thorbecke, and Asof sky (9). From 60 to325 mg (wet weight) of tissue from (a) liver, (b) spleen,(c) placenta, (d) thymus, and (e) colon, were minced witha scalpel blade and washed at 40C in 2 ml special McCoy'smedia 5-A lacking lysine and isoleucine (MicrobiologicalAssociates, Inc., Bethesda, Md.), then cultured in 2-ml por-tions of media to which high specific activity ["4C] lysine andisoleucine (302-312 mCi/mmol, Amersham/Searle Corp.,Arlington Heights, Ill.) were added at concentrations of1 iuCi/ml each. In addition, suspensions of (f) peritonealcells obtained by injecting 2-4 ml of the special media intothe peritoneal cavity followed by gentle kneading and' re-covery of the fluid, and (g) bone marrow cells, recoveredfrom a single femur by injecting 2 ml of special mediathrough the marrow cavity, were similarly studied. The 8 wkfetus which measured 22 mmwas cultured in toto.

Glass culture tubes (125-X-15 mm) were gassed with amixture of 5% C02 and 95%o air, capped, and incubated at370C in a rotating drum for 48 h. The contents were frozenat -70'C, then thawed and dialyzed against 2 liter volumesof 0.015 M phosphate-buffered saline (PBS) X6 for 72 h.After centrifugation at 40C to remove insoluble constituents,the supernatant media were lyophilized and reconstituted with0.3 ml distilled H20, and thereafter stored at -70°C. Cellu-lose acetate electrophoresis of the concentrated culture fluidswithout carrier serum was done with a Beckman MicrozoneCell (Beckman Instruments, Inc., Fullerton, Calif.) using a25 ul sample applicator. Electrophoresis was carried out for20 min at 25 V/cm in 0.075 MVeronal buffer pH 8.6. Serumfrom the same fetus, when available, was included for com-parison with the protein and autoradiograph (ARG) patternsof the various tissues.

Antisera. Monospecific rabbit antisera to the complementcomponents, Clq, C4, C3, and C5 were either gifts from Dr.H. J. Muller-Eberhard (12) or prepared in this laboratory.The rabbit antiserum to IgG and subgroup G3 was raised byimmunization with DEAE-cellulose-purified IgG from a pa-tient with IgG3 myeloma. This antiserum did not react withpurified kappa or lambda light chains. In addition, goat anti-whole human serum (WHS) and anti-IgM (Hyland Div.,Travenol Labs., Inc., Costa Mesa, Calif.) were used. Thespecificity of each antiserum was verified by Ouchterlonyanalysis and IEP.

Immunoelectrophoresis. The micromethod of Scheidegger(13) was used with 2%o Difco purified agar (Difco Labora-tories, Detroit, Mich.) in 0.04 M Veronal buffer pH 8.2 ata potential of 5 V/cm for 45 min. The antigen wells, capac-ity 4 ,d, were first filled with fresh carrier WHSfollowed bythree applications of culture fluid. The precipitin patternswere developed over 48 h; the slides were washed first in0.15 M NaCl, then distilled H20 with frequent changes for72 h, dried under filter paper, and stained with amido black.For the analysis of Clq, 1% agarose in 0.05 M PBS wasused and the carrier was the H-chain disease plasma Matt.,which contains an elevated Clq concentration of 0.410 mg/ml(14). Electrophoresis was for 90 min at 5 V/cm and the

washing and staining as described above. To prevent com-plexing of Clq to Clr and Cls, 0.01 Mtrisodium EDTAwasincluded in the buffer.

Autoradiography and synthesis scoring. The stainedslides were exposed in sealed boxes for 6 wk to Kodak KKor No Screen X-ray film (Eastman Kodak Co., Rochester,N.Y.) using foam rubber to compress the film firmly to theslides and then developed with Kodak Rapid X-Ray De-veloper. The intensity of the ARG precipitin lines wasscored: +, questionable; 1+, weak but definite; 2+, mod-erate; and 3+, intense. Only 1+ or greater ARG lines areconsidered in the results. To verify that the ARGlines werea direct consequence of in vitro protein synthesis, liver, pla-centa, and colon from a 25 wk fetus were cultured as shownin the presence of 12.5, 25.0, and 50 /Ag/ml of puromycin.

Fetal sera. Blood was obtained from 15 of the fetuses bycardiac puncture and/or from the umbilical vessels aftercareful cleansing of the cord. After clotting for up to 2 hat room temperature, the sera were separated by centrifuga-tion in the cold and stored at -70'C. Studies of total he-molytic complement and component protein concentrations inthese sera will be reported in detail later.2

RESULTSEvidence that the positive ARG's were a direct con-sequence of in vitro protein synthesis was provided bycultures of liver, placenta, and colon in the presence ofpuromycin. A progressive decrease in ARG intensity,on both cellulose acetate and IEP with anti-WHS,occurred at concentration of 12.5 and 25 /Ag/ml puro-mycin and were completely inhibited at 50 /ig/ml. Re-sults with cellulose acetate electrophoresis and IEPusing anti-WHS were concordant except for occa-sional 1 or 2+ single lines in peritoneal and bonemarrow cell cultures corresponding to haptoglobin oralpha2 macroglobulin, whereas the cellulose acetateARG's were negative.

In each experiment, the culture was judged viableif fetal tissue, other than placenta, demonstrated apositive cellulose acetate ARG and a 1 + or greaterARG precipitin line against anti-WHS on IEP. Theviability of the seven individual tissues was similarlyconsidered.

By these criteria, 23 of the 37 cultures were success-ful. In the 14 fetuses obtained from spontaneous abor-tions or amniocenteses, placental protein synthesis oc-

curred in 11 but only 2 had synthesis by another tissue,i.e., only 2 were viable. In contrast, 21 of 25 fetusesobtained by hysterotomy or hysterectomy were viablein culture. Viability was 100% for liver (22/22), spleen(21/21), and placenta (19/19) cultures, 93% for colon

(18/19), 91% for thymus (20/22), and 74% for bonemarrow (15/21) and peritoneal (15/21) cells (TableI).

2 Kohler, P. F. Maturation of the human complementsystem. II. Component levels in fetal and neonatal sera.Manuscript in preparation.

672 P. F. Kohler

TABLE IMean Synthesis Scores of Viable Tissues from 22 Fetal Cultures, Number of Tissues Synthesizing

Each Protein, and Protein's Carbon-14 Specific Activity

Viable cultures*

Tissue % Number Clq C4 C3 Cs IgG IgM

Liver 100 22 0.1 (2)1 1.2 (16) 2.7 (22) 1.8 (17) 0.2 (4) 0.2 (4)Spleen 100 21 1.3 (12) 0.9 (10) 0.7 (10) 1.2 (12) 0.8 (9) 0.6 (9)Placenta 100 19 0 0.9 (11) 0.9 (11) 1.1 (12) 0.6 (9) 0.2 (2)Peritoneal cells 74 15 0.1 (1) 0.2 (2) 0.2 (2) 0.5 (6) 0 0Bone marrow cells 74 15 0 0.6 (4) 0.2 (2) 1.1 (6) 0.1 (2) 0.1 (1)Thymus 91 20 0.1 (1) 1.4 (16) 1.9 (18) 1.6 (15) 0.5 (8) 0.4 (6)Colon 93 18 0.2 (2) 1.3 (16) 1.4 (15) 1.5 (15) 0.4 (7) 0.4 (6)

Specific activity§ 230 172 164 286 117 620Specific activity 1.38 1.03 1.00 1.61 0.70 3.72

relative to C3

* Producing positive (>.+) ARG's on cellulose acetate electrophoresis and with anti-WHS on IEP.Numbers in parentheses indicate number of cultures synthesizing protein.

§ Moles lysine and isoleucine per mole protein.

SITES OF SYNTHESISFigs. 1 and 2 illustrate typical ARGresults in the dif-ferent tissues cultured from the same 16 wk fetus. The

Immunoelectrophoresis

Clq precipitins (Fig. 2, left) are diffuse, thereby de-creasing the intensity of the ARG produced by thespleen (Fig. 2, right). This occurred frequently with

AutoradiographsAnti -%A/IWC

Liver

vvno ;;-y b;^r..SI.X._w...3+

.0

IgGM 2

1+

C3 2+

C4 2+

2+

C5 . 2+

Placenta _::

Per1tr3nea}C:elts.... .... ..

J!!vl!g!_t

....---- An..E,^b_* . . . . A.. d.... : . .... : ..... :..

F_... . .._#... . ..... _ S.w . . .

........;...>S. 6.'E,,

2+0i

0;

1+0

1+

0

1+

0

2+

0

FIGURE 1 Synthesis of C3, C4, C5, and IgG by tissues from a 16 wk fetus. Left: IEP patternsproduced by carrier serum and specific antisera. Right: ARG's resulting from in vitro synthesisof protein by liver, spleen, placenta and peritoneal cells, bone marrow, and thymus. Scoringfrom 0 to 3+ is indicated on the ARG's. Line tracings are provided for specific labeled precipitinlines which are indistinct on reproduction. The peritoneal culture is not viable.

Clq, C4, C3, and C5 Synthesis in Human Fetal Tissue 673

Immunoelectrophoresis Autoradiographs

0.. ..

0

.0.

FIGURE 2 Synthesis of Clq by the same 16 wk f etal tissuesas shown in Fig. 1. Carrier anti-Clq IEP pattern (lef t) andARG (right). Definite (2+) synthesis is present in spleenonly while a questionable (±+) ARG is present in the pla-centa. Scoring is indicated on the ARG's.

Clq; however, the other precipitins were consistentlysharp, making their ARG's more comparable. Table Iis a summary of the mean protein synthesi's scores, via-bility, and the number of each tissue producing theprotein.

Liver. The primary site of complement protein syn-thesis was in liver with 1+ or greater labeling of twoor more components in all viable cultures. In the cul-ture of the total body from an 8-wk old fetus, a ±-C3ARGoccurred while labeling of C5 was 1+. Definitesynthesis was apparent by 11 wk in the two earliestfetal livers studied; C3 synthesis occurred in 100%,C4 in 73%, and C5 in 77% of the livers (Table I).Immunoglobulin G and M synthesis (1+) occurred infour livers from fetuses 11-22 wk old. Weak Clq syn-thesis was also detected in two livers at 16 and 17 wk.

Immunoelectrophoresis AutoradiographsAnti-

WHS

IgG+G3

FIGURE 3 Restricted labeling of IEP lines by IgG synthe-sized by a 14 wk placenta. Carrier IEP patterns vs. anti-WHSand anti IgG-G3 (left) and ARG's (right). Only theanodal portion of the IgG precipitins are labeled indicatingsynthesis of an electrophoretically restricted IgG population.This finding was also frequently present in spleen, thymus,and colon.

Spleen. The splenic cultures were of particular in-terest in that Clq synthesis was limited almost exclu-sively to their tissue (Fig. 2) and was of much greatermagnitude compared to other sites (Table I). Clqsynthesis first occurred at 14 wk, considerably laterthan the other components. Splenic C5 synthesis wasessentially equal to Clq followed by C4 and C3. In addi-tion, IgG and IgM production occurred primarily inthe spleen and was first detected at 11 wk, increasingin parallel with fetal age.

Placenta. C5, C3, and C4 were the major productsof the placenta, but occurred only in 58-63% of cul-tures. Placental IgG synthesis was present in approxi-mately half of the cultures beginning at 14 wk. Inmany instances, only the anodal portion of the pre-cipitin arch was labeled as seen in Fig. 3. IgM syn-thesis was present in a single culture from a 14 wkfetus. No evidence for placental Clq production wasfound aside from a single ±ARG from one 16 wkplacenta (Table I and Fig. 2).

Peritoneal cells. C5 was the primary complementprotein synthesized but was produced in only 40% ofthe viable cultures. Labeled protein with a fast betamobility corresponding to haptoglobin was uniformlypresent with 2-3+ scores. A single culture at 20 wkdemonstrated 2+ Clq synthesis while none showed IgGor IgM synthesis.

Bone marrow cells. C5 was the major product ofthe bone marrow cells occurring in 40% of viable cul-tures followed by C4 in 27%. Minimal synthesis of C3,IgG, and IgM occurred and there was no evidence forproduction of Clq (Table I). As in the peritoneal cellcultures, labeling of haptoglobin was frequently present.

Thymus. Significant labeling of C3, C4, and C5occurred in 75-90% of the viable cultures. In addition,IgG and IgM production occurred as early as 14 wk,but was much less in comparison with the complementproteins. Thymic Clq synthesis occurred in one 16 wkfetus.

Colon. Significant synthesis of C3, C4, and C5 in83-89% of the cultures was apparent as early as 11wk and was essentially equal for all three components.Weak synthesis of both IgG and IgM was also presentat 11 wk increasing in parallel with fetal age to 2+at 17 wk. Clq synthesis (2+) occurred in a singleculture obtained from a 16 wk fetus.

DISCUSSIONThe technique of tissue culture in the presence of["C]amino acids allows identification of individual pro-tein synthesis by subsequent immunoautoradiography(6-9, 11). In theory, a positive ARGresults only fromde novo protein synthesis. Several precautions are es-

sential for valid interpretations of the ARG results,

674 P. F. Kohler

however. The use of algal mixtures of [14C]amino acidsmay result in nonspecific ARG's secondary to binding oflabeled cysteine (9, 11). Nonspecific labeling of alpha2macroglobulin has been attributed to its carrier proper-ties and labeling of haptoglobin may occur via its com-plexing with ['C]hemoglobin (11). These explanationsprobably account for the occasional appearance of labelin these precipitin lines in cultures which failed to showany other labeled lines when tested against ant-i-WHSor the various monospecific antisera.

The relative synthetic rates of individual proteinswere approximated by using standardized cultures, IEP,and ARGconditions. Scoring from 0 to 3+ was doneby the author on several occasions on all ARG's withminor variation. van Furth (11) has reported thatunder standardized experimental conditions, as little as0.0002 Ag of labeled immunoglobulins in a precipitinline is detectable by autoradiography and that quantita-tive comparisons of protein synthesis are possible.

The proportion of lysine and isoleucine (moles lysineand isoleucine per mole protein) in the complementproteins (1) is relatively low but constant: Clq, 230;C4, 172; C3, 164; and C5, 268. In contrast, the pro-portion is much higher in IgM, 620, and slightly lowerin IgG, 117 (15). The 1'C specific activities of theseproteins, relative to C3, are Clq, 1.38; C4, 1.03; C3,1.00; C5, 1.61; IgG, 0.70; and IgM, 3.72 (Table I).Therefore detection of IgM, C5, and Clq was favoredin comparison with the other proteins in these experi-ments. In addition, the fivefold variation in the wetweight of the solid tissues cultured and their greatexcess compared to the bone marrow and peritonealcells influenced the quantity of labeled protein recoveredas did several indeterminate variables such as aminoacid membrane transport and pool size, which maydiffer in various tissues. Thus, comparisons of synthe-sis scores in different tissues are gross approximations.More meaningful comparisons can be made betweenproteins synthesized by the same tissue.

Complement components. Synthesis of at least onecomponent was seen beginning with the youngest fetus.C5 synthesis was the first to occur at 8 wk. In thisinstance, the entire fetus was cultured. A questionable(±) C3 ARGwas also produced but no evidence ofClq or C4 production was found. Thereafter, everyfetus produced C5 in two or more of the seven tissuescultured. C5 synthesis was greatest, and essentiallyequal, in liver, thymus, and colon cultures but alsooccurred in spleen, placenta and peritoneal cells, andbone marrow (Table I). These are the first studies ofhuman C5 synthesis. In the mouse C5 production hasbeen reported to occur in liver and spleen (16).

Both C3 and C4 synthesis began at 11 wk gestationand were present in every culture from this age on-

wards. The liver was the main site of C3 and C4 pro-duction but thymus and colon were nearly as active(Table I).

C3 and/or C4 synthesis by human fetal tissues hasbeen studied previously by others with the techniqueused herein. In 10 fetuses from 10 to 20 wk gestationstudied by Adinolfi, Gardner, and Wood (7), hepaticC3 production first occurred in one of three cultures at10 wk and C4 at 14 wk. Thorbecke, Hochwald, vanFurth, Mfiller-Eberhard, and Jacobsen (6) had earlierobserved C3 and C4 synthesis by liver from fourfetuses 20-25 wk old. Gitlin and Biasucci (8) haveprovided evidence for C3 as well as C1 inhibitor pro-duction by fetal yolk sac cultures as early as 29 daysgestation. In 7 of their 15 fetal cultures, algal hydroly-sate mixtures of [C]amino acids were used introducingthe possibility of nonspecific binding of labeled cys-teine to carrier protein.

A distinctive finding was that Clq synthesis wasalmost exclusively limited to the spleen and beganlater in gestation than the other components whichwere produced in essentially all tissues cultured (Figs.1 and 2, Table I).

Since Clq has receptors for sites on the Fc frag-ments of IgG and IgM, the possibility of interaction ofcarrier Clq with labeled IgG and IgM produced invitro must be considered. No correlation between im-munoglobulin and Clq synthesis existed in the presentstudy. In one culture Clq synthesis occurred in theabsence of immunoglobulin and in seven cultures IgGand/or IgM synthesis occurred but not Clq (Fig. 4).Therefore, the positive Clq ARG's are not felt to bedue to binding of labeled immunoglobulins to carrierClq.

Clq synthesis is clearly not limited to the spleen inadults as evidenced by serial quantitation of serumClq concentrations in five adults before and up to 1mo after splenectomy. In no instance was there a sig-nificantly prolonged decreased Clq compared to C4,C3, or C5 after splenectomy.8

In a previous report of in vitro Clq synthesis (17),employing the same technique used in this study,splenic production occurred in two of two human fetalcultures and three of five cultures from adults. Peri-toneal and lung macrophages, however, were moreactive in Clq as well as C3 and C4 synthesis. Afterpurification of the macrophages by adherence to glass,synthesis of Clq was decreased. These investigatorsconcluded that macrophages were the primary site ofClq, C3, and C4 synthesis. Synthesis of Clq by skinfibroblasts has also been reported (18).

In sharp contrast, Colten, Gordon, Borsos, and Rapp(19) using a hemolytic assay have presented evidence

'Kohler, P. F. Unpublished observations.

Clq, C4, C3, and C5 Synthesis in Human Fetal Tissue 675

Immunogkobulins

U-

ComplementComponents

.'43

he U8 lb 12 14 16 18 20 22 24 26

Fetal Age in Weeks

FiGuRE 4 Composite summary of protein synthesis by the individual fetuses. Blackened squaresindicate synthesis of the complement protein (below) and blackened triangles synthesis of IgGand/or IgM (above). While synthesis of C3, C4, and CS was uniformly present from 11 wkgestation onward, immunoglobulin synthesis was inconstant and quantitatively much less (seetext and Table I). In three fetuses, ages 8, 11, and 16 wk, complement but not IgG or IgMsynthesis occurred.

that the entire human trimolecular first component com-plex, Clq,r,s, is formed primarily, if not exclusively,by colon and ileum in vitro. In a single experimentwith 19 wk fetal tissue, ileum and colon synthesizedhemolytically active C1 but not spleen, stomach, thy-mus, liver, lung, or kidney. These investigators hadreported previously that in the guinea pig, the intestinalepithelial cell was not responsible for biologically activeC1 production. A specific cell was not identified in thehemolytically active human colon and ileal cultures.

Immunoglobulins. Synthesis of both IgG and IgMbegan at 11 wk, similar to the findings of Gitlin andBiasucci (8), but was inconstant in comparison withthe regular synthesis of C4, C3, and C5. In threefetuses complement synthesis occurred but not IgG orIgM (Fig. 4). Spleen and colon were the main sitesof IgM production while spleen and placenta were forIgG (Table I). Often synthesis of one immunoglobulinoccurred without production of the other (Fig. 4).Quantitatively, immunoglobulin production was signifi-cantly less than that of C4, C3, and C5 (Table I). Thispoint is further strengthened by the fact that the ex-perimental conditions strongly favored the detection ofIgM, specific activity 3.72 relative to C3 (Table I).

Of interest was the frequent confinement of the ARGto the anodal end of the IgG precipitin lines (Fig. 3)suggesting synthesis of an electrophoretically distinctpopulation of gamma heavy chains by fetal tissues.Stecher and Thorbecke (20) have reported a similaroccurrence in rodent tissues but attributed it to free

light chain synthesis. In no instance was a ARGpresent in the IgG3 precipitin line except for twoplacental cultures. This indicates that IgG3 synthesis bythe fetus may begin subsequent to the other sub-group(s).

GENERALCOMMENTSThese findings indicate that during maturation of thehuman fetus, complement production precedes and isquantitatively greater than that of IgG or IgM. Relatedstudies of serum concentrations of complement proteinsin fetuses from this study' and another report (21) offetal serum levels also support this conclusion. Re-cent evidence that complement system activation canoccur independent of antibody-antigen interactions isconsonant with the hypothesis that complement may bea more primitive biologic defense system than specificantibody (2, 3, 22). This hypothesis is also supportedby the observations that cobra factor can activate thecomplement sequence from C3 onwards, via a bypassmechanism involving C3 proactivator (C3 PA), ininvertebrates which lack specific antibody protein (23).Investigation of fetal C3 PA synthesis, the protein re-sponsible for the bypass (3) would provide additionalevidence bearing on the postulate that ontogeneticallycomplement is a more primitive biologic system thanimmunoglobulin.

ACKNOWLEDGMENTSThe excellent technical assistance of Mr. Marlin Nofzigerand Miss Joyce Trembath is gratefully appreciated. I also

676 P. F. Kohler

I .

he

thank Dr. H. J. Mfiller-Eberhard for his generous gift ofantisera and the Obstetrics Department of this institutionfor providing the tissues.

This work was supported in part by grants HD03381 andAI 00013 from the National Institutes of Health.

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Clq, C4, C3, and C5 Synthesis in Human Fetal Tissue 677