QUANTITÄTION OF SELECTED COMPLEMENT COMPONENTS
F. Sessions Cole David J. Gash Harvey R. Colt en
I. GENERAL INTRODUCTION
Most of the serum complement proteins are produced primari- ly in liver, but monocytes and macrophages constitute an im- portant source of extrahepatic production of many complement components. Local production of complement at tissue sites of inflammation may be of considerable importance in host defenses and immunopathological reactions. The synthesis and secretion of complement proteins by mononuclear phagocytes in culture have been measured functionally, immunochemically, and by hemo- lytic plaque assay. The hemolytic (functional) assay is used to monitor biologically active complement proteins secreted by mononuclear phagocytes in culture. The immunochemical assay is used for estimating the amount, size, and subunit structure of newly synthesized intracellular as well as secreted protein.
The hemolytic plaque assay is used to estimate the percentage of macrophages that secrete functional complement proteins, i.e., suitable for measurement of complement production by
METHODS FOR STUDYING Copyright © 1981 by Academic Press, Inc.
MONONUCLEAR PHAGOCYTES 6 5 5 All rights of reproduction in any form reserved.
ISBN 0-12-044220-5
single cells. With these techniques, several different levels of control of complement synthesis can be explored. Changes in rate of translation, posttranslational modification, and secre- tion can be defined. This approach has been applied to studies of complement biosynthesis in human, mouse, rat, and guinea pig.
Production of complement by cells from other species can be measured so long as suitable functional assays and specific an- tibodies to the individual complement proteins are available.
II. FUNCTIONAL ASSAYS
A. Reagents
The source and preparation of buffers, partially purified complement proteins, and indicator cell intermediates are des- cribed in detail by Rapp and Borsos (1).
B. Procedure
The functional assays for individual complement components are based on the one-hit theory of immune lysis. The one-hit theory states that a single effective site, resulting from the sequential action of antibody and the nine complement compo- nents, is a necessary and sufficient condition for lysis of the erythrocyte. As a consequence, from the Poisson distribution, it is possible to calculate on a molecular basis the concentra- tion of hemolytically active specific complement components present in a given sample. More detail on the theoretical and practical aspects of these assays can be found in Rapp and Borsos (1). As an example of the procedure, the hemolytic assay of the fourth component of the complement pathway (C4) will be described.
In the hemolytic assay for C4, a sample is mixed with a cell intermediate consisting of erythrocytes sensitized with anti- body and containing the first component of complement (EAC1).
After a suitable incubation period, the cells are exposed to the remaining complement components (C2, C3, C5 - 9) in excess leading to lysis of the erythrocytes. The extent of lysis (the amount of hemoglobin released) is measured spectrophotometrical- ly, and from this the absolute number of active C4 molecules in the sample can be calculated. Because of the sensitivity (pico- gram range) and specificity of these measurements, the biologi- cally active complement components can be detected in complex mixtures (e.g., tissue culture media) without fractionating or concentrating the sample (2).
Procedures for hemolytic assays of the individual components and several of the regulatory complement proteins may be found in Rapp and Borsos (1). Hemolytic assays utilizing serum defi- cient in an individual complement component have also been des- cribed for guinea pig, human, and mouse C4 (3, 4), mouse C2 (5), and C5 (6). These assays are both sensitive and specific and do not require partial purification of individual complement compo- nents. However, sera deficient in specific components are not widely available, and species incompatibilities may interfere with the assays.
C. Calculation of Data
The calculation of the percentage of lysis, the application of the Poisson distribution, and the determination of the num- ber of molecules of the active component are described in Rapp and Borsos (1).
D. Critical Comments
The hemolytic assays for individual complement components are extremely sensitive and specific (2). Less than nanogram quantities of the biologically active component can be detected in complex mixtures (2). However, the hemolytic assays for in- dividual complement components vary with respect to efficiency.
For example, whereas a single molecule of Cl is sufficient to generate a hemolytic site (7), as many as 300 C3 molecules on the red cell surface are required for one lytic site (8).
Therefore, even discounting other potential problems of stabili- ty of the component in various tissue culture media and effi- ciency of uptake of the components on the indicator cells, cer- tain components are more easily detected with this method.
Direct comparison of rates of synthesis of the individual com-
ponents must take these differences into account. Moreover,
these measurements do not detect hemolytically inactive comple-
ment proteins, nor do they permit a distinction between pre-
formed and newly synthesized protein. Other biological activi-
ties of complement components, such as immune adherence and
chemotactic activity, may be used for quantitation of biosynthe-
sis, but these have had only limited use in synthesis studies
because of lack of specificity and/or sensitivity.
I I I . HEMOLYTIC PLAQUE ASSAY
A. Reagents
Agarose (Seakem HGTP, Marine Colloids, Rockland, Maine) is dissolved in a solution consisting of equal volumes of Hank's balanced salt solution with calcium and magnesium and 0.3 M dextrose, and then maintained at approximately 50°C. Prepara- tions of buffers and other reagents are similar to those des- cribed for the hemolytic assays (1).
B. Procedure
The procedure described below may be used to estimate the number of C2 and C4 producing macrophages from guinea pig al- veolar or peritoneal lavage (9). The indicator cells are sen- sitized with a high multiplicity of Cl (400 effective molecules per cell). Eight-tenths ml of the agarose solution (final con- centration 0.5%), 0.08 ml of indicator cells (10 /ml), and 0.1 ml of macrophage cell suspension are added in rapid succes- sion to plastic tubes in a 37°C water bath. A uniform suspen- sion, prepared by quickly vortexing the mixture, is poured into plastic tissue culture dishes. After the agarose mixture solidifies, the dishes are incubated at 37°C in a humidified 5%
CC>2/95% air atmosphere. Based on preliminary kinetic experi- ments, C2 hemolytic plaques are developed after 1 hr incubation, and C4 plaques are assayed after 2 hr incubation (9). Hemo- lytic plaques are developed by successive addition of 1 ml of guinea pig C2 (3 x lO1^ effective molecules/ml) and 1 ml of rat serum in EDTA (0.01 ΛΓ) for C4, or 1 ml of rat serum in EDTA
(0.01 M) for C2.
C. Calculation of Data
Plaques are counted by visual inspection and recorded as the number of complement producing macrophages per total number of macrophages in each dish. Additional details of the pro-
cedure are given in ref. (9) .
D. Critical Cowments
Other forms of agarose such as Seaplaque (Marine Colloids, Rockland, Maine) were not suitable for these experiments be- cause of their anticomplementary effects (9).
The plaque assay allows assessment of the number of comple-
ment producing cells in a heterogeneous cell population.
Plaque formation requires the lysis of approximately 200 - 400 indicator cells (10) and is therefore a sensitive and specific indicator of C2- and C4-producing cells. Length of incubation and reagents must be individualized for mononuclear phagocytes from different species and different tissues based upon the rate of secretion, viability of the mononuclear phagocytes in agarose, and stability of indicator cell intermediates.
IV. IMMUNOCHEMICAL DETERMINATION OF COMPLEMENT PROTEINS
A. Reagents
Monospecific antisera to specific complement proteins are raised by immunization of suitable animals with purified or partially purified complement proteins (11 - 13). Other reagents used are described by Hall and Colten (14).
B
0Procedure
For each antibody-antigen combination, the zone of equiva- lence should be defined. The reagents are used in a ratio that will result in twice antibody excess and the precipitation of approximately 30 yg of protein (14).
Radiolabeled extracellular media or intracellular lysates are divided into two aliquots. One aliquot is precipitated with a test antibody-antigen combination and the other with a control antibody-antigen combination (e.g., antivalbumin- ovalalbumin) to correct for precipitation of nonspecific labeled protein. The reaction, carried out in 1.0% deoxycholate-Triton X-100 and Tris-KCl in the presence of 2 niM PMSF, is incubated for 2 hr at room temperature (14). The precipitates are then washed three times with 0.5% deoxycholate-Triton X-100, twice with 0.15 M NaCl, once with acetone, and dried under vacuum.
Total specific radioactivity is measured, or the precipitates may be subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for assessment of size and subunit structure of the newly synthesized proteins (Fig. 1).
C. Critical Comments
The choice of isotope precursor depends upon the specific
protein and question being examined. While S35 methionine is
of high specific activity, some complement proteins do not con-
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Fig. 1. SUS PAGE analysis of guinea pig Pro-C4 and native C4 synthesized by peritoneal macrophages in culture; radio-
autograph of
35S-labeled C4 antigen. Slot 1: Intracellular C4 antigen. Slot 2: C4 antigen secreted into medium.
Slot 3: Molecular weight markers. All samples electrophoresed under reducing conditions. Control immunoprecipitates con-
tained no detectable radiolabeled bands.
tain enough methionine to be adequately studied with this labeled amino acid (e.g., Clq) (15). For these proteins, other individual amino acids (e.g., proline for Clq) (15) or combina- tions of amino acids may be used. If elucidation of posttrans- lational glycosylation is being examined, labeled carbohydrates may be used (16).
Inclusion of small amounts of protease inhibitors (e.g.,
PMSF or EACA) into culture medium and intracellular lysates
may be necessary to avoid cleavage of polypeptides. Incorpora-
tion of the radioisotope into trichloroacetic acid precipitable
protein should also be assessed.
V. TISSUE CULTURE METHODS
A. Reagents
If culture conditions require serum, medium may be supple- mented with 10% heat-inactivated (56°C for 2 hr) fetal bovine serum. For radiolabeling with amino acids in the presence of serum, dialysis of heat-inactivated fetal bovine serum against 40 volumes of 0.15 M NaCl and 40 volumes of deionized water is required. Intracellular lysates are obtained by freeze-
thawing in the presence of 0.5% deoxycholate-Triton X-100 in Tris-KCl with 2 mM PMSF.
B. Procedure
After adherence, monolayers are washed with a balanced salt solution. Aliquots of medium are removed at timed intervals, the volume replenished with fresh medium, and the samples maintained at -70°C until analysis.
C. Calculation of Data
The cell number is estimated by DNA content (17).
D. Critical Comments
Adherence conditions for mononuclear phagocytes of differ- ent species and of different origins must be defined. The stability of the individual complement components under dif- ferent culture conditions must be confirmed before biosynthetic questions are examined.
VI. CONCLUDING REMARKS
The complement proteins are useful tools for elucidation of both genetic and cellular control mechanisms of the inflamma- tory process. With the methods described above, secretion of biologically active complement proteins and radiolabeled, im- munochemically detectable complement proteins may be compared.
Regulation of the rate of synthesis of precursor protein or its
posttranslational modification can be assessed. In addition,
the size and subunit structure of the precursor and functional
proteins can be examined. By using the plaque assay, the sub- population of mononuclear phagocytes that secrete functional complement components can be estimated.
With these methods, several different levels of cellular and genetic control of complement synthesis and secretion have been explored. Maturation of mononuclear phagocytes into tis- sue macrophages may be signaled or enhanced by changes in their capacity to synthesize and secrete biologically active comple- ment proteins. For example, human breast milk macrophages se- crete hemolytically active C2 and factor B immediately in vitro, while a lag of 3 to 6 days in complement production is noted in peripheral blood monocytes from the same women (18). Factor B has been shown to promote spreading of mononuclear phagocytes in serum-free medium (19). The rate of secretion of both C2 and C4 by macrophages from different tissues may be compared by simultaneous plaque assay and tissue culture (9). While al- veolar macrophages from guinea pig secrete less C2 and C4 than peritoneal macrophages in culture, the rates of C2 and C4 se- cretion per complement producing alveolar macrophage are greater than the rates per complement producing peritoneal macrophage
(9). Similarly, the response of mononuclear phagocytes to vari- ous inflammatory stimuli or pharmacologie inhibitors of inflam- mation can be assessed (9, 10, 20). The possibility that a feedback mechanism may control C4 secretion has been proposed
(.21) . The levels of control at which these mechanisms operate have not been defined.
Posttranslational modification of precursor proteins appears to be an important regulatory step in the synthesis and secre- tion of complement proteins, as it is with other proteins
(6, 22, 23). Tunicamycin, an inhibitor of posttranslational glycosylation, inhibits the secretion of C2 and C4 by guinea pig macrophages (16). Examples of genetic control mechanisms have been revealed in studies of cells from complement-deficient animals and patients. C5 deficiency in mice appears to result from a failure in secretion of C5 protein, not from a failure in biosynthesis of the precursor protein (24). This defect in posttranslational modification may be due to a primary structu- ral abnormality in the precursor protein, lack of an enzyme to perform the posttranslational modification, or lack of a spe- cific transport protein (24).
Deficiency of C4 in the guinea pig has been shown to result from a translational defect in the synthesis of C4 protein (25).
Under cell-free conditions, liver polysomes from C4-deficient guinea pigs synthesize only nascent C4 polypeptides that remain polysome bound. Thus, the mRNA appears to be present but not completely translated.
Recent studies have attempted to define the structure of the genes that code for murine C3 and C4 (26). Because the expres- sion of the Sip protein, a hemolytically nonfunctional C4 pro-
tein, is strongly dependent on testosterone, elucidation of the murine C4 gene will afford a tool to explore the role of steroid hormones in the initiation of protein synthesis
(27 - 29).
The complement proteins therefore provide tools with which to examine mechanisms of mononuclear phagocyte maturation and
"activation," which in turn govern the local inflammatory re- sponse. In addition, specific transcriptional, translational, posttranslational, and secretory control of extracellular mono- nuclear cell products can be explored.
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