[481 Electrophoretic analysis of acute-phase plasma proteins

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[48] E l e c t r o p h o r e t i c A n a l y s i s o f A c u t e - P h a s e Plasma Proteins By

HEINZ BAUMANN

Introduction Acute systemic injury causes an important change in the qualitative and quantitative composition of plasma. The concentration of a subset of proteins, the acute phase reactants, is increased. ~ These proteins are produced in the liver, and, based on their biological functions, are considered to be essential in counteracting harmful consequences of injury. 2 However, quantitative analysis of plasma composition has revealed that not only are there proteins which are enhanced, but there are also other proteins, e.g., albumin, apolipoprotein A-I, inter a-trypsin inhibitor, a2HS-glycoprotein, and transferrin, which are substantially reduced during acute phase. 3 A great wealth of information is available which addresses the identification, characterization, and regulation of several major acutephase proteins in different vertebrate species. 4 In order to understand the physiological significance of the acute-phase reactants, the entire complexity of the hepatic response must be taken into consideration, not only individual plasma components. For example, three major questions concerning acute-phase research, which are currently the focus of attention, demand complete characterization of the liver reaction: (1) what influence does a specific type of injury (i.e., inflammation, burn or parasite infection) have on the spectrum of modulated plasma proteins, (2) which factors mediate the liver response, and (3) what are the molecular mechanisms leading to the coordinated change in hepatocellular synthesis of all the acute-plasma proteins. 4 Analyses of this kind are dependent upon a simple but still sensitive and quantitative assay for acute-phase proteins. The method of choice to detect, identify, and quantitate acute-phase reactants has proved to be crossed immunoelectrophoresis based on the principle of Laurell. 5 Modification of this basic technique has facilitated and A. Koj, in "Structure and Functionof PlasmaProteins" (A. C. Allison,ed.), Vol. 1, p. 73. Plenum, London, 1974. 2 I. Kushner,Ann. N.Y. Acad. Sci. 389, 39 (1982). 3A. Koj, in "Pathophysiologyof Plasma Protein Metabolism"(G. Mariami, ed.), p. 221. Macmillan, London, 1984. 4 A. Koj, in "The Acute Phase Response to Injury and Infection" (A. H. Gordon and A. Koj, eds.), p. 181. Elsevier, Amsterdam,The Netherlands, 1985. 5C.-B. Laurell,Anal. Biochem. 10, 358 (1965). METHODS IN ENZYMOLOGY, VOL. 163

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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perfected the qualitative and quantitative analysis of multicomponent samples such as plasma or serum. 6-~° In the following sections, examples will be presented which will illustrate how the immunoelectrophoresis technique can be applied: (1) to assess the broad spectrum of inflammation-induced changes in serum proteins, (2) to identify structural and functional properties of acute-phase reactants, and (3) to measure the regulated expression of specific proteins in cultured hepatic cells. Determination of the Inflammation-Induced Changes in Serum Composition by Crossed Immunoelectrophoresis Procedure

Crossed immunoelectrophoresis separates antigenic proteins present in complex mixtures, such as plasma or serum, with high resolution. J~,lh This technique is, therefore, attractive for assessing the effect of acute phase, since it allows a very reproducible comparison of a large number of blood proteins (Fig. 1). All theoretical and practical aspects of immunoelectrophoresis in general, and crossed immunoelectrophoresis in particular, have been described in detail elsewhere. 9A°,12-14The application of this method to the analysis of acute-phase reactants is as follows. All immunoelectrophoretic steps are carried out in 1% agarose gels. Agarose, either type I: low EEO from Sigma, St. Louis, MO, or low-Mr agarose from Bio-Rad, Richmond, CA, are used and are equally satisfactory. The electrophoresis buffer for crossed immunoelectrophoresis of serum or plasma is 45 mM Tris/90 mM glycine/30 mM, barbital, pH 8.5. ~5 Comparable, but not identical, separation patterns are achieved with the less expensive buffer, 80 mM Tris/25 mM tricine, pH 8.6. ~6The electro6 B. Weeke, Scand. J. Clin. Lab. Invest. 25, 269 (1970). 7 R. P. Ganrot, Scand. J. Clin. Lab. Invest. 29 (Suppl. 124), 39 (1972). s B. Weeke, Scand. J. lmmunol. 2 (Suppl. 1), 47 (1973). 9 A. O. Grubb, Scand. J. Immunol. 17 (Suppl. 10), 113 (1983). 10 N. Hoiby, and N. H. Axelsen, Scand. J. Imrnunol. 17 (Suppl. 10), 125 (1983). II H. G. M. Clarke and T. Freeman, Clin. Sci. 35, 403 (1968). tZa M. Emmett, A. McCracken, J. L. Brown, and A. J. Crowle, J. Immunol. Methods 67, 279 (1984). ~2 C.-B. Laurell and E. J. McKay, this series, Vol. 73, p. 339. t3 j. p. Svendsen, B. Weeke, and B.-G. Johnsson, Scand. J. lmmunol. 17 (Suppl. 10), 3 (1983). 14 O. J. Bjerrum, Scand. J. lmmunol. 17 (Suppl. 10), 333 (1983). ~5 O. J. Bjerrum and P. Lundahl, Biophys. Acta 342, 69 (1974). t6 J. F. Monthony, E. G. Wallace, and P. M. Allen, Clin. Chem. 24, 1825 (1978).

Protein Stain

Autorodiocjram

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o

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r-

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r-~ "o

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(~ =

First Dimension

=

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FIG. 1. Identification of rat acute-phase reactants by crossed immunoelectrophoresls. Serum was collected from a 6-month-old male Buffalo rat prior to (Control) and 24 hr following an experimentally induced inflammation (subcutaneous injection of 250/~l turpentine) (Inflamed). An aliquot of each serum was diluted 100-fold with electrophoresis buffer (45 mM Tris/90 mM glycine/30 mM barbital, pH 8.5), and 10 #1 of the diluted sample (equivalent of 0.1/.d serum) was separated by crossed immunoelectrophoresis. First-dimension electrophoresis (from left to right) was carried out for 210 rain at 5 V/cm and 15°. The second-dimension gel was cast next to the first-dimension gel strip, and contained a mixture of goat immunoglobulins against 24 hr acute-phase rat serum (2.5 mg y-globulin added to 12 ml agarose or 33/zg/cm 2) and 2.5/.Lg of ]25I-labeled (1.2 x 106 cpm) rabbit immunoglohulin against rat arcysteine protease inhibitor. Second-dimension electrophoresis (from bottom to top) was performed for 20 hr at 2 V/cm. The immunoprecipitation pattern was visualized by staining with Coomassie Brilliant Blue R-250 and by 24 hr autoradiography. The exact alignment of the autoradiogram with the stained patterns is facilitated by using the background spots (insoluble impurities in agarose preparation) as guides. The following antigens are indicated: ALB, albumin; AGP, aracid glycoprotein; CPI, cq-cysteine protease inhibitor; t~rMG and a2-MG, al- or a2-macroglobulin; HP, haptoglobin; HPX, hemopexin; PI, cqprotease inhibitor.

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phoresis gels (17 ml) are cast onto glass plates (10 × 10 cm) and are 1.5 mm thick. The volume of agarose solution is changed proportionally to the area if other plate dimensions are used. Glass support is preferred over plastic because it allows better handling of the gel (e.g., applying intermediate gels or antiserum-containing second-dimension gel), and easy removal after electrophoresis. The latter feature is important for gels which are subsequently processed for overlay with radioactive ligands or which contain radioactive proteins and therefore require exhaustive washing. Depending on sample volume, the sample well in the first-dimension gel is either a single round hole of 4 mm diameter (for 10/~l) 1° or a oblong well (4 × 15 mm), perpendicular to the direction of electrophoresis (holding up to 80 p~l). In most cases, a single sample is applied to one gel plate. However, when identical separation of several samples is required, up to four sample wells are fit onto one gel. Electrophoresis is performed on a horizontal gel electrophoresis unit equipped with temperature-controlled (15 °) gel supports (LKB, Bio-Rad, or Pharmacia). Nonadherent dressings from Kendall (Boston, MA) serve as buffer wicks. First-dimension electrophoresis is carried out at 5 V/cm for 3.5 hr. To monitor the extent of electrophoretic migrating tracking dye, such as phenol red or bromphenol blue (10/~g/ml), can be included into the sample. After electrophoresis, the section of agarose layer not utilized for separation of the sample (normally 7.5 × l0 cm) is cut with a razor blade from the lane containing the protein sample and is removed (see Fig. 1). However, in cases where an intermediate gel is planned as part for the subsequent electrophoresis, a gel strip adjacent to the sample-containing lane is removed first from the gel plate. The width of this strip depends upon the gel with which it will be replaced and can range from 0.5 cm (for gels containing antigen or labeled ligands, see Figs. 4 and 5 below) to 4 cm (for gels containing monospecific antiserum). The resulting gap serves then as a mold for casting the intermediate gel. After completion of that, the remainder of the original and not utilized first-dimension gel is removed. If the first-dimensional gel plate contains more than one sample, the gel is cut into 2.5 cm wide strips each containing the separated proteins. ~0 These strips are placed along the edge of a new glass plate. The second-dimension gel is prepared in a tube by mixing agarose solution at 45 ° with the required amount of antiserum or immunoglobulin fraction. The total volume for a standard crossed immunoelectrophoresis (area 7.5 × l0 cm) is 12 ml. The antibody-containing agarose solution is cast next to the first-dimension gel lane.

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Electrophoresis into antibody-containing second-dimension gel (perpendicular to the first dimension) is performed at 2-4 V/cm for 16-20 hr. After the run, the individual gels are washed at room temperature with shaking in 250 ml phosphate-buffered saline for 2 hr (nonradioactive samples) or 24 hr (radioactive samples). After rinsing with distilled water for 15 min, the gel is placed onto GelBond (FMC Corp., Rockland, ME), blotted by pressing it under a stack of paper towels, and then air dried.13 The protein pattern is visualized by staining for 10 min with 0.25% Coomassie Brilliant Blue R-250 in 40% methanol/10% acetic acid followed by destaining for 1-2 min in the same methanol/acetic acid mixture. If the gel contains 3H-labeled proteins or a low amount of 35S or 1251,it is processed for fluorography prior to drying. According to the manufacturer's recommendation, the wet gel is incubated for 1 hr in EN3HANCE (New England Nuclear, Boston, MA), washed with water for 2 hr, and then dried onto GelBond. The gels are then exposed to X-ray film (XAR5, Kodak) at - 7 0 °. Optimal conditions for electrophoretic separation in the first dimension 17 and for antibody-antigen reaction in the second dimension 12,18have to be determined empirically. Technical Considerations

Detection of acute-phase-induced changes in plasma proteins relies mainly on comparison of immunoelectrophoretic patterns. Therefore, high reproducibility of the analysis is essential. To avoid variations due to technical inaccuracies, special attention must be paid to the following points: (1) Individual animals show variation in the concentrations of plasma proteins. In order to randomize these differences, equal amounts of plasma or serum from several, identically treated animals, should be pooled. (2) Within an experimental series of electrophoresis separation, all samples should be identical with respect to volume (which can vary between 5 to 60 /zl) and buffer composition. (3) Prolonged storage of frozen samples can lead to an increase in concentration due to evaporation. This is especially true when small volumes of samples are placed in large tubes and kept in a frost-free freezer. (4) Conditions for immunoelectrophoresis should be kept constant. 1z,13 Most important are composition and pH of the electrophoresis buffer, and temperature and voltage during electrophoresis, especially during the first-dimension sepa17 J.-O. Jeppsson, C.-B. Laurell, and B. Franzen, Clin. Chem. 25, 629 (1979). 18 N. H. Axelsen, E. Bock, P. Larsen, S. Blirup-Jensen, P. Just Svendsen, K. J. Pluzek, O. J. Bjerrum, T. C. Bog-Hansen, and J. Ramlau, Scand. J. Immunol. 17 (Suppl. 10), 87 (1983).

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ration. (5) Quantitative analysis of a large number of samples demands an appropriate supply of specific antiserum. To avoid use of different antiserum preparations, which would always require new calibrations and determination of optimal antigen-antibody ratios, a pool of several batches of antisera should be made at the onset of the studies. (6) If relative mobilities of antigens are of importance in interpretation of complex crossed immunoelectrophoresis patterns, a sufficient quantity of one batch of agarose should be procured. Although the currently available agarose preparations are highly purified, there are still noticeable differences in the electrophoresis behavior of proteins with different batches of agarose. 13

Results In this section, the analytical techniques are discussed using rat serum as an example, but these techniques are equally applicable to serum from other species or to plasma. The detection of acute-phase-induced changes in blood proteins by crossed immunoelectrophoresis is dependent on the type of antiserum used. Antiserum raised against acute-phase serum is strongly recommended for analysis, since it is directed mainly toward proteins that are induced by acute phase, such as a~-acid glycoprotein and aE-macroglobulin in the rat (Fig. 1), II s e r u m amyloid A in the mouse, 19or C-reactive protein in man. 2° Such antiserum is essential for identification of inflammation-specific proteins (see Fig. 3 below). On the other hand, antiserum against acute-phase plasma is more appropriate for studies which include acute-phase protein involved in coagulation, especially fibrinogens. Antiserum prepared against normal serum is also well suited for assessing the changes in the concentration of most serum proteins. It is, of course, superior in revealing those abundant proteins which are reduced by acute phase, e.g., albumin or lipoprotein (Fig. 2). However, depending upon titer and avidity of antibodies, difficulties arise in the quantitation of major acute-phase proteins whose concentration in normal serum is low, e.g., a r a c i d glycoprotein or a2-macroglobulin (Fig. 2). Both goats and rabbits may be used for immunization. Since these animals show in all instances an excellent immunogenic reaction to ratderived proteins, prime consideration for their selection is not so much the specificity but rather the quantity of antiserum produced. Sufficient supply of antisera is an important element in these studies. Immunizations t9 j. D. Sipe, Br. J. Exp. Pathol. 59, 305 (1978). z0 1. Kushner, H. Gewurz, and M. D. Benson, J. Lab. Clin. Med. 97, 739 (1981).

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FIG. 2. Analysis of serum by crossed radioimmunoelectrophoresis. Freshly prepared hepatocytes from a control rat and a rat 24 hr after turpentine-induced inflammation were labeled in tissue culture for 6 hr with [35S]methionine. Labeled medium, 25 p.l, from control and treated animals, containing 150,000 and 250,000 acid-insoluble cpm, respectively, were combined with 0.1/xl equivalents of normal or acute-phase rat serum and then separated by crossed immunoelectrophoresis as in Fig. 1. The second-dimension gel contained 600/xl of rabbit antiserum against normal rat serum (Pel-Freez, Rogers, AR). The autoradiographs were exposed for 48 hr. See Fig. 1 for definitions of the abbreviations.

with antigens follow a standard protocol. The first injections of antigen is in Freund's complete adjuvant (e.g., 150/zl acute-phase rat serum in 350 /xl phosphate-buffered saline and 500/~1 adjuvant). Three to five injections of antigen in Freund's incomplete adjuvant follow at 2-week intervals. Test bleeding (5 ml) is carried out 10 days after each injection and the titer and specificity of antibodies is determined by crossed immunoelec-

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trophoresis. Collection of 25-30 ml of rabbit immune serum or 100-200 ml goat immune serum (every 2 to 3 weeks) begins after the final antigen injection and lasts usually for 2-5 months. After a 2-month period, booster injection of antigens are performed. The preparations of immune sera showing satisfactory titer and specificity are combined and stored in aliquots at -20 °. The immunoglobulin fraction is prepared by precipitating twice with 40% saturated ammonium sulfate followed by anion-exchange chromatography on a DEAE-cellulose column. 2°a Serum proteins not only differ greatly in their concentration 2j but also in their immunogenic properties. These differences are manifested both qualitatively and quantitatively, in the wide range of precipitin lines on crossed immunoelectrophoresis plates (Fig. 1). Although a proportional increase of both antigen and antibody intensifies staining of all bands, 9,~° the resolution of minor antigen peaks in the crowded section of the pattern would be difficult. An alternate method which allows retention of high resolution and visualization of both minor and major peaks is to use radioactively labeled samples. However, since any manipulation for labeling of serum protein will change the protein concentration in the sample, an accurate radioimmunoelectrophoretic quantitation of total serum is achieved by adding to the serum sample trace amounts of serum proteins labeled to high specific activity. In order to avoid distortion in measurements, it is important that (1) the composition of the tracer is representative of the sample, (2) the amount of added protein is relatively low (below 5% of the total), and (3) the proteins are not significantly modified in structure and function by the labeling procedure, so that electrophoretic and antigenic properties are not affected. One method of generating suitable probes with high specific radioactivity (1-10 x 106 cpm//zg) is by iodination of 1- to 5-/.d aliquots of serum with 200/zCi 125I in Iodogencoated reagent tubes. 22 The nonincorporated 1251 is removed from the proteins by chromatography on a column (0.5 × 15 cm) of Sephadex G-25 in phosphate-buffered saline. Another source of radioactive plasma proteins is primary cultures of rat hepatocytes. Rat hepatocytes are prepared from 2- to 3-month-old animals, either untreated or 24 hr after an experimentally induced inflammation. 23 This is achieved by a subcutaneous injection of I00/zl turpentine per 100 g body weight. The animals are anesthesized with an injection of 100/.d of Nembutal together with 300 USP units of heparin. The liver is :0a L. Hudson and F. C. Hay. "Practical Immunology." Blackwell, Oxford, England, 1976. 2i A. Koj, H. Kasperczyk, J. Bereta, and A. H. Gordon, Biochem. J. 206, 545 (1982). 22 p. R. P. Salacinski, C. McLean, J. E. C. Sykes, V. V. Clement-Jones, and P. J. Lowry, Anal. Biochem. 117, 136 (1981). 23 p. O. Seglen, Methods Cell Biol. 13, 29 (1976).

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perfused i n s i t u from the inferior v e n a c a v a to the portal vein, first for 8 min with 300 ml of sterile perfusion buffer [25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 140 mM NaCl, 3.7 mM KCI, 4.6 mM NaCO3, pH 7.4] then for 6 min with 200 ml of Dulbecco's modified Eagle's medium (DMEM) containing 0.1 g collagenase (type I, Sigma, St. Louis, MO). The entire procedure is carried out at 37°. The liver is excised and transferred into a Petri dish with 50 ml of DMEM containing 10% heat-inactivated fetal calf serum. Each lobe is ripped open with forceps, and the cells are washed out by gentle shaking of the liver. The released cells are filtered through four layers of gauze and collected by centrifugation for 2 min at 25 g. The large-sized hepatocytes are freed from all other cell types by three rounds of centrifugation in 50 ml of serum containing DMEM for 2 min at 7 g. The parenchymal cells are enriched to more than 95% homogeneity, and the cell viability is usually above 90%. The hepatocytes are placed on collagen-coated dishes since collagenous culture substratum promotes adhesion and spreading of the liver cells. Coating of dishes is accomplished by incubating the surface of the dishes for 1 min at room temperature with Vitrogen 100 (sterile collagen solution from Collagen Corporation, Palo Alto, CA). The excess collagen solution is removed, and the dishes are air dried under a sterile hood. Prior to the plating of the cells, the collagen-coated dishes are preincubated for 30 min at room temperature with culture medium containing 10% fetal calf serum. After the hepatocytes have been plated and cultured for 30 min, nonadherent cells are removed, and adherent parenchymal cells are incubated for 6 hr with methionine- and serum-free DMEM (1 ml per 1 × 106 ceils) containing [35S]methionine (1200 Ci/ mmol, 0.5-2 mCi/ml). Under these conditions, the cells synthesize and secrete labeled plasma proteins into the culture medium with specific radioactivities ranging between 2 and l0 x l05 cpm//xg. Aliquots of the culture medium can be used for immunoelectrophoresis (Fig. 2) or polyacrylamide electrophoresis (Fig. 5 below) without further treatment. In most instances, there is no need to remove the excess nonincorporated [35S]methionine, still present in the medium, since it does not interfere with any electrophoretic analysis. If necessary however, the separation of the excess label from the labeled proteins is readily accomplished by dialysis against phosphate-buffered saline. In order to achieve a sufficient incorporation of radioactivity into the precipitin lines of serum proteins on crossed immunoelectrophoresis, aliquots of 125I- or 35S-labeled proteins with 0.1-5 x 105 cpm are normally added to serum samples (Fig. 2). The amount of radioactive antigens relative to the total can be measured by immunoelectrophoresis of the tracer proteins alone. If similar conditions are used as indicated above or

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in Fig. 2, the radioactive antigens should not contribute more than 5% to the total amount of antigen. Using radiocrossed immunoelectrophoresis, the distinction between major and minor bands depends on the length of autoradiographic exposure. A fluorograph of the gel pattern is recommended to enhance low radioactive signals. Metabolically labeled proteins in hepatocyte cultures are not only useful to increase the sensitivity of antigen-detection on crossed immunoelectrophoresis, but also allow identification of serum proteins of hepatic origin. All precipitin lines which have incorporated [35S]methionine-labeled secretory proteins of hepatocytes (Fig. 2) represent liver proteins. Additional serum proteins, but of non-hepatic origin (e.g., immunoglobulins), can be detected by comparing of the 35S-labeled pattern with that stained for proteins with Coomassie Brilliant Blue. Crossed immunoelectrophoresis patterns of normal and acute-phase serum show increases and decreases of several peaks (Figs. 1 and 2). The position of those peaks relative to albumin or other prominent peaks may help to indicate which specific protein is actually increased or decreased. However, this approach allows assignment only for a few major acutephase proteins, e.g., haptoglobin and hemopexin. In general, the high complexity of unfractionated serum prevents an unambiguous identification of the corresponding bands present on crossed immunoelectrophoresis patterns of normal and acute-phase serum. The relationship between specific precipitin peaks is readily established by employing tandemcrossed immunoelectrophoresis. 24The principle of this technique consists of electrophoresis of two different samples, i.e., equal amounts of normal and acute phase serum, in tandem in the first dimension. To do so, the samples are loaded into two separate wells 1 cm apart in the direction of the first dimension. The antigens present in the two tandem-separated samples will form a fused, double-peaked precipitin line during migration into the second-dimension gel. The area under the two peaks are indicative for the degree of the acute-phase-induced change in concentration. In order to assess unambiguously basal and acute-phase levels of specific proteins, two alternative sample arrangements (i.e., acute-phase serum ahead and behind control serum) should be tried. Using this method, the relationship of most major positive and negative acute-phase protein bands can be determined. However, this method has its limitations in that the duplication of all peaks increases complexity of the pattern and renders an analysis of weak precipitin lines impossible. Immunoelectrophoresis is also an excellent tool for detecting proteins 24 j. Kroll. Scand. J. Immunol. 17 (Suppl. 10), 135 (1983).

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Antiserum Containing Second Dimension Gel

Intermediate Gel

t

First Dimension Gel

FIG. 3. Detection of inflammation-specific serum proteins. A 0.1/xl equivalent of acutephase rat serum was separated in the first dimension. A l-cm-wide strip of 2 ml agarose with 200/zl normal rat serum was cast between the first-dimension gel lane and the seconddimension gel which contained goat immunoglobulin against acute-phase rat serum (see Fig. 1). After immunoelectrophoresis, the pattern was stained with Coomassie Blue.

which are present only in acute-phase serum (Fig. 3). The basic concept of this approach is to deplete the antiserum against acute-phase serum (used for the second-dimension gel) of all antibodies against normal serum proteins. In order to do so, an intermediate gel containing an excess of normal serum is placed between the first- and second-dimension gels. During immunoelectrophoresis, only the inflammation-specific proteins will form typical precipitin peaks and will appear at the same position as on the regular crossed immunoelectrophoresis pattern (compare Figs. 3 and 1). The antigen in Fig. 3 has been identified as a2-macroglobulin. Identification of Acute-Phase Proteins The presence of acute-phase reactants in serum can be demonstrated by crossed immunoelectrophoresis (Figs. 1 and 2). The change in concentration is evident from the area under the precipitin line) ° However, in many instances, the biochemical and functional identity of a particular protein is unknown and has to be determined separately. The acute-phase proteins for which purification schemes and/or antiserum are available (e.g., for the rat al-acid glycoprotein, haptoglobin, hemopexin, a2macroglobulin, complement component 3, fibrinogen, and al-cysteine

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protease inhibitor, 2L25,26) can be identified by their crossed immunoelectrophoretic pattern with the aid of purified protein or monospecific antibody.

Identification with Purified Acute-Phase Proteins Crossed immunoelectrophoresis of purified acute-phase proteins, carried out under the same conditions as for serum, will permit the identification of the position of a particular protein relative to other marker proteins. In order to identify the corresponding precipitin peak within the total serum protein pattern, one of the following two approaches can be applied. 1. Purified protein (e.g., 0.1-10 ng) can be added to the serum sample and then subjected to the crossed immunoelectrophoretic analysis. The increase of a specific precipitin peak above the level observed for nonsupplemented serum indicates the identity of the antigen. 27 If an excess (e.g., 0.01-1 /zg) of the purified protein is included in the serum sample, the specific precipitin peak will not be formed due to in situ depletion of the corresponding antibodies during the second-dimension electrophoresis. In this case, the removal of a precipitin line from the total serum pattern is indicative of the identity of the antigen. Alternatively, a trace amount of ~25I-labeled purified protein may be included in the sample to be analyzed. Thus, the specifically labeled precipitin band is recognized by comparing the protein stained pattern with its autoradiographic image. A modified version of this method for identification consists of separating the purified protein and a serum sample by tandem-crossed immunoelectrophoresis. 24 The purified protein (1-I00 ng) is applied to the sample well behind the well containing the acute-phase serum (0.1-0.5/zl). After separation in both dimensions, the corresponding antigens in both samples will appear as fused double peaks. The amount of purified protein and serum that is needed to achieve comparable heights of the precipitin peaks is determined empirically. 2. The second technique for identification is to apply the purified protein to crossed-line immunoelectrophoresis. 28 After separating serum in the first dimension, purified acute-phase protein (0.1-10/.tg) is incorporated into a 5- to 10-mm wide strip of agarose that is placed between the first- and second-dimension gel. During electrophoresis into the antibody25 A. Koj, E. Regoeczi, P. A. Chindemi, and J. Gauldie, Br. J. Exp. Pathol. 65, 691 (19841. 26 G. Fey, H. Doindey, K. Wiebauer, A. S. Whitehead, and K. Odink, Springer Semin. lmmunopathol. 6, 119 (1983). 27 N. H. Axelsen and E. Bock, Scand. J. lmmunol 17 (Suppl. 10), 177 (1983). 28 j. Kroll, Scand. J. lmrnunol. 17 (Suppl. 10), 171 (1983).

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containing second-dimension gel, the purified protein forms a precipitin line that fuses into the corresponding peak produced by the same protein present in the serum. Especially important for the identification of minor antigen peaks is that optimal amounts of purified protein and serum are used. This is determined empirically.

Identification with Monospecific Antibodies Monospecific antibodies against acute-phase proteins are obtained by immunization with the purified proteins, z~ High-titer antisera can be raised in rabbits and in goats by applying the standard immunization protocol: 5 to 30 tzg of purified acute-phase protein (e.g., rat a~-acid glycoprotein, oq-cysteine protease inhibitor, haptoglobin, and a2-macroglobulin, or human arantichymotrypsin) is dissolved in 500 tzl phosphatebuffered saline, suspended in 500 /.d Freund's complete adjuvant, and injected subcutaneously at multiple sites. Three additional injections of the same amounts of purified proteins but in Freund's incomplete adjuvant follow at 2-week intervals. Immune serum is collected 10 days after the last injection and again every 2 to 3 weeks. Specificity of the antiserum is determined by crossed immunoelectrophoresis using nonfractionated serum. Careful monitoring of the specificity is needed in cases where the booster injections and serum collection are carried out over prolonged periods of time (several mouths). Antibodies against traces of contaminating proteins potentially present in the original antigen preparation might appear and become relatively abundant. Limited amounts of antibodies against specific acute-phase proteins can be purified from polyspecific antisera against acute-phase serum by affinity chromatography, 2°a Monospecific antibodies against rat and human plasma proteins are commercially available [e.g., antiserum against rat albumin, transferrin, complement C3, and fibrinogen from Cooper Biomedical, Malvern, PA, or purified immunoglobulin (DAKO Immunochemicals) against human al-acid glycoprotein, albumin, arfetoprotein, OLl-antichymotrypsin, complement component 3C, C-reactive protein, ceruloplasmin, fibrinogen, haptoglobulin, hemopexin, inter-a-trypsin inhibitor, serum amyloid A, and transferrin from Accurate Chemical and Scientific Corp., Westbury, NY]. Specific antibodies help to identify the acute-phase proteins on crossed immunoelectrophoresis plates by applying one of the following two techniques. 1. Intermediate gel immunoelectrophoresis.29 In this technique, the specific antiserum against an acute-phase protein is incorporated into a 259 N. H. A x e l s e n , Scand. J. lmmunol. 17 (Suppl. 10), 141 (1983).

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to 4-cm wide gel strip that is interposed between the first-dimension gel containing the separated serum proteins and the second-dimension gel containing the polyspecific antiserum. The concentration of the specific antiserum is selected such that the antigen is precipitated entirely within the intermediate gel and may range between 0.1 and 5%. The location of the specific acute-phase protein peak within the total serum pattern is recognized by the peak's removal from the second-dimension gel and appearance in the intermediate gel. Although intermediate gel immunoelectrophoresis is most useful for identifying the position of specific protein peaks, it inevitably alters the total pattern. If the overall crossed immunoelectrophoretic pattern has to be maintained, the second technique is applied. 2. Crossed radioimmunoelectrophoresis. The principle of this technique is the identification of proteins by inclusion of radiolabeled monospecific antibodies into the second-dimension gel (Fig. 1). Polyclonal or monoclonal antibodies against purified acute-phase reactants are labeled with 125I to a high specific activity. 22 It is advantageous to use purified immunoglobulin fractions rather than serum for labeling in order to prevent nonspecific interaction and precipitation of labeled serum proteins other than immunoglobulins during subsequent immunoelectrophoresis. An aliquot of labeled antibodies (0.1-1 x 106 cpm) is mixed with the polyspecific antiserum and used in the preparation of the second-dimension gel. The radioactivity incorporated into immunoprecipitin bands is visualized by autoradiography. The same analysis will also reveal the degree of monospecificity of the antibody preparations (Fig. 1).

Identification of Acute-Phase Proteins by Function Several acute-phase proteins (e.g., haptoglobin, hemopexin, C-reactive protein, and the antiproteases) play an important role in neutralizing and removing harmful components from the circulation. Of special interest are protease inhibitors because it has been recently discovered that families of structurally related, but functionally divergent, inhibitory forms exist 3° and that the expression of these specific forms within the families is regulated differently during the acute-phase response. 31 The ability of these acute-phase proteins to specifically bind certain ligands is utilized to identify their position on crossed immunoelectrophoretic patterns. Furthermore, this technique has the potential to uncover new functional forms of acute-phase reactants and to assess the substrate specificity of known forms. The principle of the technique consists of labeling the 30 R. W. Carrell and J. Travis, Trends Biochem. Sci. 10, 20 (1985). 3~ R. E. Hill, P. H. Shaw, R. K. Barth, and N. D. Hastie, Mol. CellBiol. 5, 2114 (1985).

580

STUDY OF ACUTE-PHASE REACTANTS

[48]

c

o

(,3

mediate Gel Dimension Gel

"o

Q

E D

o

Intermediate Gel First Dimension Gel Protein Stain

Autoradiogram

FIG. 4. Identification of protease inhibitor as acute-phase reactant. The samples, consisting of 0.2/xl control and acute-phase rat serum and diluted with 7/,d electrophoresis buffer, were separated by electrophoresis. Adjacent to the first-dimension lane, an intermediate l-cm wide, agarose gel containing 50/zg ~25I-labeled bovine t~-chymotrypsin was cast (1.4 × 105 cpm/~g). This radioactive lane was separated by a 5-mm wide agarose strip from the second-dimension gel containing goat immunoglobulin against acute-phase rat serum (70 /~g/cmZ). This arrangement was necessary to identify more accurately the radioactively labeled peaks formed at the border of the second-dimension gel, and to reduce interference caused by the rather high background present in the intermediate gel. It was unavoidable that the incorporation of the chymotrypsin into the agarose gel caused some precipitation of radioactivity. The crossed immunoelectrophoretic patterns were visualized by Coomassie Blue staining and by autoradiography (24 hr exposure). See Fig. 1 for definitions of the abbreviations.

immunoprecipitated, but still active protein with radioactive ligands. One approach utilizes the following steps: Crossed immunoelectrophoresis plates of normal and acute-phase serum are prepared. After exhaustive washing, the gels are placed into a moist chamber and covered with a small volume (1-2 ml) of Tris or phosphate-buffered saline containing 1251-

[48]

ANALYSIS OF ACUTE-PHASE PLASMA PROTEINS

581

labeled (1-50/~g, 1-10 x 10 6 cpm) trypsin, chymotrypsin, papain, human neutrophil elastase, or rat hemoglobin. After incubation for 1 hr at 4° or room temperature, the nonbound radioactivity is removed by washing the gel in several changes of buffer for at least 24 hr at room temperature. The labeled bands are visualized by autoradiography or fluorography. Unfortunately, in many cases, nonspecific binding occurs with immunoprecipitates, especially with albumin. In order to minimize these nonspecific interactions, the affinity labeling is carried out during the second-dimension electrophoresis (Fig. 4). To this end, the 125I-labeled ligand (0.1-10 × 106 cpm, 0.01-1 ~g) is incorporated into an intermediate gel strip. Depending upon the electrophoretic mobility of the ligand, the intermediate gel lane is positioned either between the first and second dimension (for cathodically migrating ligands such as bovine trypsin and chymotrypsin) or at the cathodic side, behind the first-dimension gel (for anodically migrating ligand such as hemoglobin). Although the complex of protease-protease inhibitor is formed prior to immunoprecipitation in the former case, the separation of the antigens in the second dimension is not significantly modified (compare stained pattern of Fig. 4 with Fig. I). Moreover, the presence of the protease does not result in substantial loss of immunoprecipitation due to degradation. An exception to this observation is the al-acid glycoprotein peak in the example of Fig. 4. By labeling serum protein with a-chymotrypsin (Fig. 4), immunoprecipitin peaks of inhibitor of bovine chymotrypsin (i.e., al-protease inhibitor, a r and a2-macroglobulin) are predominantly obtained. Nonspecific binding to major precipitin bands, e.g., albumin, oq-cysteine protease inhibitor, or haptoglobin is not present. The same gel pattern shows the inflammation-dependent change in protease inhibitor concentration, and the appearance in acute-phase serum of additional, unidentified proteins which interact with chymotrypsin. Although all serum proteins have encountered 125I-labeled chymotrypsin during electrophoresis through the intermediate gel, this interaction must have been transient, whether with or without proteolytic action. A similarly low to nondetectable labeling of serum protein other than protease inhibitors is observed when different proteases are used. Therefore, immunoelectrophoresis of protease-labeled serum proteins is a convenient tool for identifying acute-phase reactants having protease inhibitor properties.

Identification of Acute-Phase Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis Two-dimensional polyacrylamide gel electrophoresis is characterized by high resolution. Proteins are separated in the first dimension based

582

STUDY OF ACUTE-PHASE REACTANTS

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on charge, and in the second on size. 32 This type of analysis provides fairly accurate values for isoelectric point and minimal molecular weight for many plasma proteins, 33 including major acute-phase plasma proteins. 34,35 Therefore, knowing the size and charge of a protein that has been identified as an acute-phase reactant by crossed immunoelectrophoresis is an important element in establishing its identity. The physicochemical properties of a protein present in a specific precipitin peak can be assessed a s f o l l o w s . 36 In order to facilitate detection, radioactive serum proteins are used. For example, an aliquot of serum is iodinated with ~25I, or medium proteins of primary rat hepatocytes are metabolically labeled with [35S]methionine (Fig, 2 or Fig. 6, below). Samples containing labeled serum proteins (I-50 × 106 cpm) and sufficient carrier proteins to ensure optimal separation are run on crossed immunoelectrophoresis. The gels are washed thoroughly, rinsed with water, and dried onto GelBond. According to its autoradiographic image, the precipitin line of interest, together with its plastic backing, is cut out with a razor blade. Any significant bands interfering with the peak of interest are removed. However, trace amounts of contaminants are inevitable, especially when a peak is recovered from a crowded gel section. Contaminants may serve later as useful markers for accurately aligning autoradiograms and for identifying the exact position of the protein within the total serum protein pattern. The gel fragment cut from the immunoelectrophoresis plate is placed into a conical glass tube, and 150/xl of l0 mM Tris-HCl, pH 6.8, containing 0.1% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol is added. The agarose is melted off the plastic pieces by boiling for 5 min. The presence of SDS prevents precipitation of the proteins. When cooled to 70 °, 20/zl of 10% NP-40 and 2/A of ampholine, pH 3.5-10 (LKB) are included, and the entire mixture is immediately applied with a prewarmed pipet onto the first-dimension polyacrylamide gel. After solidification of the agarose, the proteins are separated by following the standard protocol) 2 The final polyacrylamide gel is then processed for fluorography.37 The high reproducibility of two-dimensional gel electrophoresis easily allows the identification of the protein spots corresponding to the antigen within the pattern produced by serum proteins (Fig. 5).

32 p. H. O'Farrell, J. Biol. Chem. 250, 4007 (1975). 33 L. Anderson and N. G. Anderson, Proc. Natl. Acad. Sci. U.S.A. 74, 5421 (1977). 34 H. Baumann, G. P. Jahreis, and K. C. Gaines, J. Cell Biol. 97, 866 (1983). 35 H. Baumann, 1NSERM. Syrup. 126, 213 (1984). 36 H. Baumann and D. Eldridge, Cancer Res. 42, 2398 (1982). 37 W. M. Bonner and R. A. Laskey, Eur. J. Biochem. 46, 83 (1974).

[48]

583

ANALYSIS OF ACUTE-PHASE PLASMA PROTEINS

pH 7.0

6.5

6.0

5.5

5.0

4.0

5.5

MW:

I_.

_

t60 93

B

68-43--

25--

BPB FIG. 5. Identification of acute-phase proteins by two-dimensional polyacrylam~de gel electrophoresis. Hepatocytes from 24-hr turpentine-treated rats were labeled for 6 hr in tissue culture. A 50-txl aliquot of labeled medium proteins was subjected to two-dimensional polyacrylamide gel electrophoresis (A) and 500 ~1 to crossed immunoelectrophoresis (see Fig. 6 for details). The precipitin line corresponding to az-macroglobulin was cut out, and the solubilized immunocomplex also separated by two-dimensional polyacrylamide gel electrophoresis (B). First-dimension equilibrium isoelectrofocusing was carried out for 6400 Vh in a 4% polyacrylamide tube gel containing a mixture of 2% ampholine, pH 5-7, and 0.8% ampholine 3.5-10 (LKB). The second-dimension electrophoresis was performed in a uniform 10% polyacrylamide gel. The fluorographic images of the gels after 24 hr exposure are shown. The following proteins are indicated: AGP, a~-acid glycoprotein; ALB, albumins; a2u-G, azu-globulin; cq-MG, t~2-MG, al- and az-macroglobulin; CP1, c~-cysteine protease inhibitor; CT, contrapsin; Pro-HP, prohaptoglobin; fl-HB, fl-haptoglobin; TF, transferrin.

584

STUDY OF ACUTE-PHASE REACTANTS

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Regulation of Acute-Phase Proteins in Tissue Culture Cells Analysis of Tissue Culture Medium Proteins

A major aspect of the current research on the hepatic acute-phase response focuses on the cellular and molecular mechanism by which the expression of acute-phase proteins is regulated in liver cells. Most studies in this direction rely on tissue culture experiments, since regulatory processes can be assessed under more defined conditions in vitro than in vivo. Considering the pleiotropic response of liver cells to acute phase, the analysis of regulation in tissue culture cells should include a set of plasma proteins rather than being limited to a single marker protein. 38,39 The spectrum of plasma proteins produced by nontransformed liver cells in culture is identified by crossed immunoelectrophoresis. The same technique also indicates the effect of inflammation on plasma protein expression (Fig. 6). Primary cultures of adult rat hepatocytes are prepared from control liver and liver 24 hr after in vivo induced inflammation (see above). The cells have to be thoroughly washed to ensure complete removal of rat plasma proteins. The hepatocytes are metabolically labeled for 6 hr with [ 35S]methionine)4 The medium is collected and concentrated 10-fold to achieve a protein concentration sufficient for crossed immunoelectrophoresis. 4° A 500-/xl aliquot of culture medium is dialyzed for 6 hr against 25 mM ammonium bicarbonate and then lyophilized. The proteins are redissolved in electrophoresis buffer and separated by crossed immunoelectrophoresis as described for serum proteins (see Figs. I and 2). The sample volume can range between 10 and 50/zl without significant influence on separation or resolution (Fig. 6). The spectrum of proteins produced by liver cells differs remarkably from that of serum. This difference is most likely a reflection of the differences between rates of synthesis of the plasma proteins and half-lives of the same proteins in the circulation. 2~,38,4~Despite the lack of similarity in composition, the inflammation-induced changes at the cellular synthesis level are qualitatively the same as for serum protein analysis. The crossed immunoelectrophoresis of plasma proteins derived from labeled hepatocytes yields amounts of radioactivity in individual precipitin bands which are sufficient for subse38 A. Koj, J. Gauldie, E. Regoeczi, D. N. Sauder, and G. D. Sweeney, Biochem. J. 224, 505 (1984). 39 H. Baumann, R. E. Hill, D. N. Sauder, and G. P. Jahreis, J. Cell Biol. 102, 370 (1986). 4o H. Baumann, G. P. Jahreis, D. N. Sauder, and A. Koj, J. Biol. Chem. 259, 7331 (1984). 4oa B. B. Knowles, C. C. Howe, and D. P. Aden, Nature (London) 209, 497 (1980). 4~ A. Koj, in "The Acute Phase Response to Injury and Infection" (A. H. Gordon and A. Koj, eds.), p. 221. Elsevier, Amsterdam, The Netherlands, 1985.

FIG. 6. Synthesis of acute-phase proteins by hepatocytes in tissue culture. Hepatocytes were prepared from 3-month-old male rat liver (control or 24-hr turpentine-treated) by collagenase perfusion and freed from nonparenchymal cells by differential centrifugation. Aliquots of cells (8 x 105) were placed onto collagen l-coated culture plates (10 cm 2) in DMEM containing 10% heat-inactivated fetal calf serum and 1 USP unit heparin/ml. After 30 min, the adherent cells were washed once with serum-free DMEM and labeled for 6 hr in 1 ml serum free DMEM containing heparin and 200 p.Ci [35S]methionine (1200 Ci/mmol, Amersham). The medium was collected and the few suspended cells removed by centrifugation. Five hundred microliters of medium was dialyzed for 6 hr against 25 mM ammonium bicarbonate, pH 8.3, and then lyophilized. The proteins, containing 0.8 × 106 (control) and 1.2 x 106 cpm (turpentine-treated), were redissolved in 50/zl Tris/glycine/barbital buffer and separated by crossed immunoelectrophoresis. The second-dimension gel contained 33/xg/ cm 2 goat immunoglobulins against acute-phase rat serum. The autoradiogram after 16 hr exposure is shown. The precipitin peak of a2-macroglobulin (az-MG) was used for separation on two-dimensional polyacrylamide gel in Fig. 5. See Fig. 1 for definitions of abbreviations.

586

STUDY OF ACUTE-PHASE REACTANTS

[48]

quent detection and identification on two-dimensional polyacrylamide gels (Fig. 5). In order to quantitate the amount of plasma proteins produced by hepatocytes, a less complex immunoelectrophoretic pattern than that obtained by polyspecific antiserum is desirable. Therefore, a crossed immunoelectrophoretic analysis of labeled medium proteins is performed by using a mixture of monospecific antibodies (see above for their preparation) against a few marker acute-phase proteins (e.g., a2-macroglobulin, oq-cysteine protease inhibitor, al-acid glycoprotein) and control proteins (albumin, transferrin, or antithrombin I I I ) . 39 A simpler pattern is especially needed when the rate of secretion is determined by measuring the actual radioactivity incorporated into the proteins. A limited number of peaks diminishes the chance for overlapping precipitin lines. In order to measure the amount of radioactivity, the precipitin lines are cut out from the crossed immunoelectrophoresis plate, the sections are placed into a scintillation vial, and boiled in 0.5 ml water for 5 min. The still-warm agarose solution is diluted with 10 ml of water-miscible scintillation cocktail. An equal area of gel containing no detectable radioactive precipitin lines is used as a control for background. The radioactivity is expressed as percentage of total protein-bound radioactivity analyzed or as counts per minute per microgram of antigen. The amount of protein is calculated from the peak area and by comparison with a reference standard. In order to ensure linearity between the peak area and amount of protein, the crossed immunoelectrophoresis system is calibrated with various concentrations of purified standard proteins. ~0 [Rate of synthesis of acute-phase proteins is, however, more appropriately determined by short-term (15 to 30 min) labeling of hepatocytes with radioactive amino acids (e.g., [35S]methionine) followed by precipitation of the individual proteins from the cell extracts with monospecific antibodies. 4~a] The specific radioactivity incorporated into proteins in primary cultures of rat hepatocytes is variable 38-4° and depends on the uptake rates and intracellular pool size of labeled precursor amino acids. The uptake of amino acids by hepatocytes increases following inflammation. 4~,43 At the peak of hepatic acute-phase response, the uptake of [35S]methionine by rat hepatocytes is between 150 and 200% of control liver. 4° Specific labeling as well as the absolute amount of the plasma proteins synthesized and secreted by hepatocytes (nanograms per 1 × 106 cells per hour) varies 41, T. Andus, V. Gross, T.-A. Tran-Thi, G. Schreiber, M, Nagashima, and P. C. Heinrich, Eur. J. Biochem. 133, 561 (1983). 4z M. C. Powanda, G. L. Cockerell, and R. S. Pekarek, Am. J. Physiol. 225, 399 (1973). 43 R. W. Wannemacher, R. S. Pekarek, W. L. Thompson, R. T. Curnow, F. A. Beall, T. V. Zenser, F. R. de Rubertis, and W. R. Beisel, Endocrinology 96, 651 (1975).

[48]

ANALYSIS OF ACUTE-PHASE PLASMA PROTEINS

587

considerably from one cell preparation to another. 39This variability probably reflects the combined results of genetic, physiological, and technical factors. Therefore, in order to make conclusive statements about the quantitative effect of acute phase on cellular plasma protein synthesis, several independent analyses have to be performed (Fig. 6). If only an estimate of the magnitude of inflammation-induced changes in specific plasma proteins is sought, it is sufficient to express the protein production in relative values, i.e., the radioactivity incorporated into specific proteins as a fraction of either the total radioactivity in the samp i e 34,39,40 o r another plasma protein, such as albumin. 38 This approach will also facilitate comparison of data between different cell preparations and cell treatments. An equally, if not more sensitive analysis of metabolically labeled plasma proteins than that obtained by crossed immunoelectrophoresis is achieved by two-dimensional polyacrylamide gel electrophoresis (Fig. 5). This technique is extremely useful for detecting inflammation-dependent changes in the composition of medium proteins 34,35 and for measuring relative incorporation of radioactivity into specific proteins. 39.4° A major advantage of polyacrylamide gel electrophoresis over crossed immunoelectrophoresis is that only one-tenth to one-twentieth of the amount of sample (1 to 5 x 105 cpm) is needed. The quantitation is somewhat affected by the fact that between 5 and 10% of the proteins is lost during equilibration of the first dimension isoelectrofocusing gel (unpublished).44 In order to minimize the loss of radioactivity, the equilibration is limited to 30 min with two changes of SDS buffer. 32 The high resolution of proteins based on charge and size offers the additional feature of assessing the effect of acute phase on proteolytic processing (e.g., haptoglobin 45) or glycosylation of plasma proteins. 34 An inflammation-induced impairment of glycosylation results in reduced sialylation. 35,46 Undersialylated proteins are readily recognized on twodimensional gels as spot series with a less acidic mobility than that of the normal plasma proteins. 34 Use of Acute-Phase Proteins to Measure the Activity of Factors Mediating the Liver Response The major factors mediating the hepatic acute phase response are epidermal cell-derived hepatocyte-stimulating factor 39,4° (or HSF), inter44 R. P. Tracy, R. M. Currie, and D. S. Young, Clin. Chem. 28, 890 and 908 (1982). 45 j. M. Hanley, T. H. Haugen, and E. C. Heath, J. Biol. Chem. 258, 7858 (1983). 4~ N. Serbource-Goguel, M. Corbic, S. Erlanger, G. Durand, J. Agneray, and J. Feger, Hepatology 3, 356 (1983).

588

STUDY OF ACUTE-PHASE REACTANTS

[48]

leukin-6 (or monocytic H S F 4 7 ) , interleukin-1, tumor necrosis factor, and glucocorticoids. 48-5~ To properly assess the biological activity of these factors, the regulation of several proteins has to be monitored. 49,5°,5zHowever, to simplify the characterization, the analyses have often been limited to a single acute-phase protein such as rat fibrinogen, 47,53 or mouse serum amyloid p,54 o r serum amyloid A. 55 The noncorticosteroid factors acting as mediator of hepatic response have been identified in conditioned medium of peripheral blood monocytes, 47,53tissue macrophages: 6 epidermal c e l l s , 39'4° fibroblasts, 48 and transformed T cells. 57The biological activity is characterized based on the ability of these factors to stimulate in vitro the expression of specific sets of acute-phase proteins. 5°,5zThe regulation of the most prominent acute-phase plasma proteins was selected as a base for determining the factors specific activities, e.g., the increased production of al-acid glycoprotein, haptoglobin, complement C3, fibrinogen, a2-macroglobulin, and cysteine protease inhibitor in rat hepatocyte and hepatoma ce11s38,48,52,53,58; s e r u m amyloid A and p,58,59 haptoglobin, and al-acid glycoprotein in mouse hepatocytes34,54,55; and al-antichymotrypsin, 39'4° al-acid glycoprotein, haptoglobin, complement C3, 5° and Creactive protein 5°,57 in human hepatoma cells. Hepatocyte-stimulating activities, needed for experimental work, can be prepared from conditioned medium of either primary cultures of human epidermal cells, 4° human and mouse squamous carcinoma c e l l s , 39'52 lipopolysaccharide- (or Staphyloccous aureus toxin-)activated human and rodent peripheral blood monocytes4°,47,53,6° or mouse peritoneal macro47 D. G. Ritchie and G. M. Fuller, Ann. N.Y. Acad. Sci. 408, 491 (1983). 48 j. Gauldie, C. Richards, D. Harnish, P. Lansdorp, and H. Baumann, Proc. Natl. Acad. Sci. U.S.A. 84, 7251 (1987). 49 H. Baumann, C. Richards, and J. Gauldie, J. lmmunol. 139, 4122 (1987). 50 G. J. Darlington, D. R. Wilson, and L, R. Lackman, J. Cell Biol. 103, 787 (1986). 5~ D. H. Perlmutter, C. A. Dinarello, P. I. Punsat, and H. R. Colten, J. Clin. Invest. 78, 1349 (1986). 52 H. Baumann, V. Onorato, J. Gauldie, and G. P. Jahreis, J. Biol. Chem. 262, 9756 (1987). 53 D. G. Ritchie and G. M. Fuller, Inflammation 5, 275 (1981). 54 p. T. Le, M. T. Muller, and R. F, Mortensen, J. lmmunol. 129, 665 (1982). 55 M. J. Selinger, K. P. W. J. McAdam, M. M. Kaplan, J. D. Sipe, S. N. Vogel, and D. L. Rosenstreich, Nature (London) 2 ~ , 498 (1980). 56 R. F. Kampschmidt, in "Infection: The Physiologic and Metabolic Responses of the Host" (M. C. Powanda and P. G. Canonico, eds.), p. 55. Elsevier, Amsterdam, The Netherlands, 1981. 57 N. D. Goldman and T.-Y. Liu, J. Biol. Chem. 262, 2363 (1987). 5s j. D. Sipe, T. F. Ignaczak, P. S. Pollock, and G. G. Glenner, J. Immunol. 116, 1151 (1976). 59 E. Tatsuta, J. D. Sipe, T. Shirahama, M. Skinner, and A. S. Cohen, J. Biol. Chem. 258, 5414 (1983). 6o B. M. R. N. J. Woloski and G. M. Fuller, Proc. Natl. Acad. Sci. U.S,A. 82, 1443 (1985).

[48]

ANALYSIS OF ACUTE-PHASE PLASMA PROTEINS

589

phages, 4° or platelet-derived growth factor-treated fibroblasts. 5° In most cases, gel filtration is used as a principal purification step for enriching the major activity. 4°,47,53,57,6°Alternatively, purified preparations of recombinant cytokines, i.e., tumor necrosis factor, IL-1, and/or IL-6 can be used (contact biotechnology companies such as Genentech, Palo Alto, CA; Immunex Corp., Seattle, WA; or Genetics Institute, Cambridge, MA, for availability). The hormonally induced change in the production of acute-phase proteins by hepatic cells is quantitated with the highest sensitivity and accuracy (ng/ml range) by radioimmunoassay (RIA) 61 (mouse serum amyloid A and p,58 rabbit and human C-reactive protein, 6z rat a2-macroglobulin 63) or by enzyme-linked immunosorbent assay (ELISA) 64 (rat fibrinogen and albumin53.65). Although RIA and ELISA are the methods of choice for routine assay of large number of samples, immunoelectrophoresis is, in most cases, sufficient for quantitative determination of acute-phase proteins. 66 Immunoelectrophoresis is especially of value when the regulation of several plasma proteins by liver-regulating hormones is studied. 31,38Quantitation of plasma proteins is carried out by rocket immunoelectrophores i s . 8'12'18'67-'69 Aliquots of tissue culture medium are electrophoresed into an agarose gel containing monospecific antibodies against acute-phase proteins. The area under the precipitin peak is proportional to the antigen concentration in the analyzed sample. Rocket immunoelectrophoresis, whose practical application is described in detail in this series, ~2is attractive not only because of its technical simplicity, but also because it possesses high sensitivity (nanogram range) and is adaptable for routine bioassay of liver-regulating activities. 38-'4°,5°,52 Two highly specific assay systems for measuring hepatocyte-stimulating activities are described which depend on immunoelectrophoretic separation of acute-phase proteins. One system, using HepG2 cells, is ideally suited for quantitating human HSF activity, while the other, using H-35 Reuber rat hepatoma cells, 7° is preferred for characterizing the spectrum 6t C. N. Hales and J. S. Woodhead, this series, Vol. 70, p. 334. 6., S. S. Macintyre, D. Schultz, and I. Kushner, Biochem. J. 210, 707 (1983). 63 D. E. Panrucker and F. L. Lorscheider, Biochim. Biophys. Acta 705, 184 (1982). 64 E. Engvall, this series, Vol. 70, p. 419. 65 S. W. Kwan, G. M. Fuller, M. A. Krautter, J. H. van Bavel, and R. M. Goldblum, Anal. Biochem. 83, 589 (1977). 66 A. Koj, in "The Acute Phase Response to Injury and Infection" (A. H. Gordon and A. Koj, eds.), p. 310. Elsevier, Amsterdam, The Netherlands, 1985. 67 C.-B. Laurell, Anal. Biochem. 15, 45 (1966). 68 C.-B. Laurell, Scand. J. Clin. Lab. Invest. 29 (Suppl. 124), 71 (1972). 69 B. Weeke, Scand. J. lmmunol. 2 (Suppl. 1), 37 (1973). 70 M. D. Reuber, J. Natl. Cancer Inst. 26, 891 (1961).

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STUDY OF ACUTE-PHASE REACTANTS

[48]

of acute-phase proteins regulated by a given human or rodent factor. 52 The routine test of human H S F activity relies on stimulation of oq-antichymotrypsin 39,4° or fibrinogen 4s,49 synthesis in HepG2 cells. The human hepatoma cell line HepG2 (negative for hepatitis B virus antigen) has retained a wide spectrum of liver-specific functions, including the synthesis of many plasma proteins. 4°~ The cells are maintained in monolayer culture in minimal essential medium (MEM) containing 10% heat-inactivated fetal calf serum under an atmosphere of 95% air-5% CO2 and are passaged every 2 weeks. For measuring H S F activity based on antichymotrypsin regulation (Fig. 7), monolayers of HepG2 ceils are established in 24-well tissue culture plates (5 × 104 cells in 1 ml medium per well). When the cells have reached confluency (1-2 weeks after plating), the medium is replaced by 0.5 ml of serum-free MEM containing the factor to be tested (0.1-100 units/ml). Following an incubation period of 16 hr at 37 °, the medium is replaced again by 0.2 ml fresh medium. After an additional 6 hr of incubation, 50/zl of culture medium is applied into the sample well of rocket immunoelectrophoresis plates. The assay of HSF activity, based on the stimulation of fibrinogen synthesis (e.g., for IL-6 or keratinocytic HSF-II and -III49), is also carried out on HepG2 cells in 24well culture plates. To achieve optimal expression of fibrinogen, the cells are treated for 16 hr at 37 ° in 0.5 ml MEM containing 1% fetal calf serum, 1/xM dexamethasone, and the factor to be tested. The medium is replaced by 0.4 ml fresh medium, and after an additional 24-hr incubation period, 50/zl of culture medium is subjected to rocket immunoelectrophoresis. The amount of protein is calculated from the precipitin peaks based on calibration curves with purified proteins) s The number of cells in the culture well at the time of medium harvest is established by counting the cells or by measuring protein or DNA. In the example of Fig. 7, alantichymotrypsin represents a positive, and albumin a negative, acutephase reactant, aFProtease inhibitor (oq-antitrypsin) serves as a control plasma protein that is not significantly affected by HSF-I. 4°,52The control protein is a useful indicator in cases when inhibitory reactions occur due to excess amounts of HSF. Under those conditions, the production of all secreted proteins is reduced. The activity of H S F is characterized by the degree to which the factor stimulates protein production. The definition of one unit of HSF is the concentration needed to stimulate the synthesis of a major acute-phase protein, such as fibrinogen, to 50% maximal level 53 or Otl-antichymotrypsin to 33% level. 39 In order to enhance the sensitivity of the HSF activity assay, a unit definition has been proposed that is based on the ratio between a positive and a negative acute-phase reactant (e.g., a2-macroglobulin and albumin

[48]

ANALYSIS OF ACUTE-PHASE PLASMA PROTEINS

591

=i-Antichymotrypsin

Albumin

a~Antitrypsin

tO0 30

10

3

!

0.3 0.1

0

Concentrotion of HSF(Units/mL) FIG. 7. Regulation of plasma proteins by HSF in HepG2 cells. HSF-I with M~ = 30,000 and p l of 5.5 was purified from conditioned medium of the human squamous carcinoma (COLO-16) cells as described. 52The final preparation with a specific activity of 4 × 105 units/ mg and concentration of 50,000 units/ml) was tested on HepG2 cells. Confluent monolayers of HepG2 cells in 24-well culture plates (3-4 × 10~ cells per well) were treated with 0.5 ml of MEM serial dilutions of HSF-I yielding a final concentration as indicated. After 16 hr the medium was removed, and 200/xl of fresh medium was added. After 6 hr, 50-ttl aliquots were applied into the sample wells of three rocket immunoelectrophoresis gels. The gels contained monospecific goat immunoglobulins against human a~-antichymotrypsin (25/.tg/ cm2), 0.35% rabbit antiserum against human albumin (Miles Scientific, Naperville, IL), and an 0.3% rabbit antiserum against human al-protease inhibitor (Accurate Chemical and Scientific Corp., Westbury, NY). Immunoelectrophoresis was carried out in Tris-tricine buffer, pH 8.6, at 4 V/cm for 16 hr. The Coomassie Blue-stained protein patterns are reproduced.

592

STUDY OF ACUTE-PHASE REACTANTS

[48]

in rat hepatocytes). 3s In this case, the assumption is made that the increased and decreased production of the two proteins is controlled by the factors via the same cellular mechanism. If the amount of medium available from the assay cultures does not accommodate the electrophoretic determinations of all the regulated acute-phase plasma proteins (e.g., otl-acid glycoprotein, arantichymotrypsin, arantitrypsin, complement C3, haptoglobin, fibrinogen, albumin, arfetoprotein, and transferrin in HepG2 cells), the rocket immunoelectrophoresis is modified. One modification is to include two or three antisera in the agarose gel. 38 The concentration of each antiserum has to be chosen in such a way that a clear separation of the precipitin peaks is achieved. Alternatively, the agarose gel is divided into two or three sections, each containing one antiserum preparation (see Fig. 8). 48It is advisable that the gel furthest away from the sample well contains antiserum against plasma proteins with good anodal mobility (e.g., arproteins or albumin) and that the gel closest to the sample well contains antiserum against proteins with poor mobility (e.g., complement C3, hemopexin, or fibrinogen). The specific pattern of acute-phase plasma proteins modulated by a liver-regulating factor is determined in H-35 rat hepatoma cells. A clonal cell line has been established (available from my laboratory) that not only regulates all major positive rat acute-phase proteins, but also responds to human and rodent factors by stimulating the synthesis of specific sets of these proteins. 52 The assay protocol is as follows. Confluent cultures of H-35 ceils are released by trypsin and suspended in MEM containing 10% heat-inactivated fetal calf serum at a concentration of 1 × 10 4 cells per milliliter. One-milliliter aliquots of the cell suspension are placed into wells of 24-well culture plates and incubated at 37° under an atmosphere of 95% air-5% CO2. Within 3 to 4 days, the cells have grown to 80% confluent monolayers. The cells are washed once with 1 ml serum-free MEM and then overlayed with 0.5 ml serum-free MEM containing 1/zM dexamethasone and the factors to be tested. Following an incubation for 24 hr at 37 °, the medium is replaced by 0.4 ml fresh stimulatory medium. After an additional 24-hr culture period, the medium is removed, and aliquots ranging between 10 to 70 p.l are subjected to rocket immunoelectrophoresis for quantitating the amounts of secreted plasma proteins. Since the relative proportion of the acute-phase proteins is indicative of the factor's specificity,52 it is advantageous to analyze individual samples simultaneously for at least two diagnostic acute-phase proteins. Optimal is the paired immunoelectrophoretic determination of a type I acute-phase protein (aracid glycoprotein, complement C3, or haptoglobin) and a type II acute-phase protein (arantichymotrypsin, arantitrypsin, c~2-macro-

[48]

ANALYSIS OF ACUTE-PHASEPLASMA PROTEINS

68

593

25 15

CP[

C5

11

14 13

16 15

18 17

20 19

22 21

24 23

28 26

32 30

36 34

41 47 ;38 4 4

Fractions FIG. 8. Identification of HSF activities by stimulation of acute-phase proteins in H-35 cells. Two milliliters of 10x concentrated, 4d-conditioned medium of the human squamous carcinoma (COLO-16) cells were chromatographed on a column (2.5 × 95 cm) of Sephadex G-100 in phosphate-buffered saline. The eluate was collected in 3.5 ml fractions. Fifty microliters from the fractions indicated and 5/~1 of the starting sample (total) were mixed with 1500 ~1 serum-free MEM containing 1 /.tM dexamethasone. After sterile filtration, 0.5 ml of the media was added to 80% confluent H-35 cell monolayers in 24-well cluster plates. After 24 hr incubation at 37°, the media were replaced by 0.4 ml fresh stimulatory media. Following an additional 24-hr incubation period, 70/xl of the culture media was withdrawn and applied to an immunoelectrophoresis plate. The gel (13 × 20.5 cm) was cast in two sections. The lower section contained goat antiserum against rat complement C3 (C3) (Cooper Biochemical) (15/xl in 25 ml agarose solution), while the upper section contained rabbit antiserum against rat al-cysteine protease inhibitor (CPI) (50 /~1 in 25 ml agarose solution). Immunoelectrophoresis was carried out in Tris-tricine buffer at 4 V/cm for 20 hr. The Coomassie Blue-stained protein pattern is reproduced. The appearance of the precipitin peaks of c~rcysteine protease inhibitor within the lower section is due to the cathodal migration of the antibodies. The elution position of molecular weight marker proteins (in thousands) is indicated at the top. V0, void volume; Vt, total volume of the Sephadex G-100 column. globulin, a~-cysteine protease inhibitor, h e m o p e x i n , or fibrinogen)J2 The s y n t h e s i s o f a n t i t h r o m b i n I I I is n o t significantly affected b y h o r m o n a l t r e a t m e n t , w h i c h a l l o w s u s e o f the v a l u e for a n t i t h r o m b i n I I I c o n c e n t r a t i o n in the a s s a y m e d i u m as a n i n t e r n a l m a r k e r for relative c o m p a r i s o n b e t w e e n d i f f e r e n t t r e a t m e n t s . F i g u r e 8 illustrates the a c t u a l a p p l i c a t i o n of the H-35 cell a s s a y . I n this e x a m p l e , the a s s a y n o t o n l y i n d i c a t e s the

594

STUDY OF ACUTE-PHASEREACTANTS

[49]

chromatographic elution of HSF activity present in conditioned medium of human squamous carcinoma cells, but also the differentiation between the activity of HSF-III/HSF-II and HSF-I/IL-1. Virtually identical information would have been obtained if cysteine protease inhibitor was substituted, for instance, by a2-macroglobulin or hemopexin and complement C3 by haptoglobin. The same H-35 cell assay system is also applicable for (1) quantitating HSF activity of various cytokines, either present in crude mixtures or in homogeneous preparations, (2) comparison of factors from different cell origins or different species, and (3) comparison of recombinant and natural products. Acknowledgments The studies on which the above procedures are based were supported by NIADDKD grant DK33886 and NCI grant CA26122. I am a recipient of an Established Investigatorship Award from the American Heart Association. I am indebted to Drs. Aleksander Koj, JagielIonian University, Krakow, Poland, and Jack Gauldie, McMaster Univesity, Hamilton, Ontario, Canada, for providing monospecific antisera, Victoria Onorato and Gerald P. Jahreis for technical help, Karen P. Prowse for correcting the manuscript, and Marcia Held for secretarial assistance.

[49] H e p a t o c y t e - S t i m u l a t i n g Factor

By BARRY M.

R. N. J. WOLOSK! and GERALD M. FULLER

Introduction

It has been established from in vivo studies, as well as studies using isolated hepatocytes, that the synthesis of the acute-phase reactant group of plasma proteins is regulated by monocyte-derived factors by direct action on the liver. 1-5 Hepatocyte-stimulating factor (HSF) is a major A. Koj, Blut 51, 267 (1985). 2 G. M. Fuller, J. M. Otto, B. M. R. N. J. Woloski, C. T. McGary, and M. A. Adams, J. Cell Biol. 101, 1481 (1985). 3 B. M. R. N. J. Woloski and G, M. Fuller, Proc. Natl. Acad. Sci. U.S.A. 82, 1443 (1985). 4 G. Ramadori, J. D. Sipe, C. A. Dinarello, S. B. Mizel, and H. R. Colten, J. Exp. Med. 162, 930 (1985). B. M. R. N. J. Woloski, E. Gospodarek, and J. C. Jamieson, Biochem. Biophys. Res. Commun. 13tl, 30 (1985).

METHODS IN ENZYMOLOGY, VOL. 163

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.