Hemopexin is a developmentally regulated, acute-phase plasma protein in the chicken.

Identity has been established between chicken hemopexin and alpha 1-globulin "M," a plasma known for the hormone responsiveness of its synthesis in monolayer cultures of embryonic chicken hepatocytes (Grieninger, G., Plant, P. W., Liang, T. J., Kalb, R. G., Amrani, D., Mosesson, M. W., Hertzberg, K. M., and Pindyk, J. (1983) Ann. N. Y. Acad. Sci. 408, 469-489). Identification was based on immunological cross-reactivity, electrophoretic behavior on sodium dodecyl sulfate-polyacrylamide gels, heme-binding capacity, and pattern of cleavage by proteolytic enzymes. Electroimmunoassays were used to investigate plasma protein levels, particularly those of hemopexin, in the acute-phase response and embryonic development. Acute-phase plasma protein production, elicited by injection of chickens with turpentine, bore many similarities to the pattern of hepatocellular plasma protein synthesis produced in response to the addition of specific hormones in culture. The response of the stressed chickens included elevated levels of hemopexin and fibrinogen (5- and 2-fold, respectively) accompanied by a 50% drop in albumin. Hemopexin levels of developing chick embryos were measured for several days before and after hatching. Onset of hemopexin production occurred around the time of hatching, and was followed by a steep increase (more than 1000-fold over 4 days). Similarly, it was not until the 12th h of culture that hepatocytes isolated from both early and late stage chicken embryos began to produce hemopexin, although, from their initiation in culture, they secreted a number of other plasma proteins in quantity. After 12 h, hepatocellular output of hemopexin rapidly accelerated. This precocious induction ex vivo required no hormonal or macromolecular medium supplements. These observations indicate that the embryonic chicken hepatocyte culture system will provide a useful model for studying the regulation of hemopexin biosynthesis in hepatic development and the acute-phase response.


489).
Identification was based on immunological crossreactivity, electrophoretic behavior on sodium dodecyl sulfate-polyacrylamide gels, heme-binding capacity, and pattern of cleavage by proteolytic enzymes. Electroimmunoassays were used to investigate plasma protein levels, particularly those of hemopexin, in the acute-phase response and embryonic development. Acute-phase plasma protein production, elicited by injection of chickens with turpentine, bore many similarities to the pattern of hepatocellular plasma protein synthesis produced in response to the addition of specific hormones in culture. The response of the stressed chickens included elevated levels of hemopexin and fibrinogen (5-and 2-fold, respectively) accompanied by a 50% drop in albumin. Hemopexin levels of developing chick embryos were measured for several days before and after hatching. Onset of hemopexin production occurred around the time of hatching, and was followed by a steep increase (more than 1000-fold over 4 days). Similarly, it was not until the 12th h of culture that hepatocytes isolated from both early and late stage chicken embryos began to produce hemopexin, although, from their initiation in culture, they secreted a number of other plasma proteins in quantity. After 12 h, hepatocellular output of hemopexin rapidly accelerated. This precocious induction ex vivo required no hormonal or macromolecular medium supplements. These observations indicate that the embryonic chicken hepatocyte culture system will provide a useful model for studying the regulation of hemopexin biosynthesis in hepatic development and the acute-phase response.
Expression of a variety of hepatic genes is confined to specific phases of development, but only in certain cases are *This research was supported by National Institutes of Health Grants HL-09011 and AM-30203. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the factors that affect this timing understood (1)(2)(3)(4)(5). Studies with hepatocytes derived from chicken embryos suggest that they may provide an ex vivo system in which to clarify t.he critical factors involved in the bransition from fetal to adult hepatic protein synthesis.
Once established in monolayer culture, these embryonic cells begin to develop certain characteristics typical of adult hepatocytes, including a lack of cell division (6, 7) and adult levels of glycogen deposition (8), UDP-glucuronyi transferase activity (9), and cytochrome P-450 (IO). Moreover, synthesis of several plasma proteins, not produced by the embryo, is induced. Five such plasma proteins were demonstrated by crossed immunoelectrophoresis of spent culture medium using anti-adult chicken serum absorbed with embryonic serum (11). The most prominent of these is an a,-globulin which we termed "M." Chicken embryo hepatocyte cultures have also been shown to express the adult form of antithrombin-111 (12).
The fortuitous presence of anti" antibodies in rabbit an& albumin (Cohn Fraction V) enabled us to devise a reagent for monitoring secreted M in the hepatocyte cultures (13). It was found that accelerated production of this protein began at about 12 h of culture and continued thereafter (7,11).
An intriguing feature of this culture system is that viable monolayers, which become induced for M synthesis, are established even in the absence of serum or hormonal supplementation of the medium (7). It has also been found that M production is highly responsive t.o a number of hormones, including insulin (71, glucocorticoids (14), and thyroid hormones (15). The stimulatory effect was even greater when the individual hormones were combined (14), with the secretion of M accounting for 5-10% of total plasma protein output. under such conditions (6,14).
T o identify the function of N ] -globulin M, we have purified it and compared it with known chicken plasma prot.eins. In this study, we demonstrate that M is the heme transport protein, hemopexin, and t.hat, in addition to its being a developmentally regulated protein, it is a major participant in the acute-phase response of the chicken. Our findings suggest that, by experimental manipulation of the conditions of culture, it may be possible to discern the factors responsible for triggering major changes in hemopexin production in pathological states and during hepatic development.

E X P E R l M E N T A L PROCEDURES
Reagents--3,3',5-Triiodo-~-thyronine (thyroid hormone or, simply, Ta)' (sodium salt) was obtained from Sigma as were insulin The abbreviations used are: Ta, 3,3',5-triiodo-~-thyronine (thyroid hormone); HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate; Hx, hemopexin.  Methods-Primary monolayer cultures of hepatocytes from 16day-old chicken embryos were prepared as described (7,16). They were plated and maintained in modified Ham's F-12 medium without hormones or serum supplement so that the cells were cultured from the onset in a chemically defined medium, free of added macromolecules. Cultures derived from younger (12-day) or older (19-day) embryos were prepared in a similar fashion with the aim of obtaining similar cell densities. Culture medium was replaced with an equal volume of fresh medium every 24 h unless otherwise indicated.
Hemopexin was affinity-purified from pooled adult chicken plasma by t.he method of Tsutsui and Mueller (18) using a heme-agarose column, adapted as described (19). Serum was obtained from hatched chickens by cardiac puncture. Serum was obtained from chicken embryos by drawing blood from a large vein near the air space. For this procedure, the top of the shell was removed without nicking the membrane, and a drop of mineral oil placed on the membrane to facilitate visualization of underlying blood vessels.
Methods employed in analysis of plasma proteins by electroimmunoassay (Is), crossed immunoelectrophoresis (I]), and immunoprecipitation of plasma proteins (17, 20) have been described previously. Hemopexin was quantified by electroimmunoassay with either anti-hemopexin or anti" antibodies (see below) using purified hemopexin as standards. Immunoprecipitation of secreted hemopexin in spent culture medium using anti-hemopexin IgG and processing of immunoprecipitates for SDS-polyacrylamide gel electrophoresis under disulfide-reducing conditions followed described procedures (17, 20). SDS-polyacrylamide gel electrophoresis under disulfide-reducing conditions and two-dimensional peptide mapping of proteins were performed, if not otherwise stated, as described (20,21). For analysis of unreduced proteins, our standard sample buffer (70 mM dithiotbreitol, 70 mM Na2C03, 0.53 M sucrose, 2.7% SDS, 1 mM EDTA, and 0.05% bromphenol blue) was replaced by 6 M urea, 40 mM Tris-HCI, pH 6.8,4% SDS, and 0.05% bromphenol blue, and the alkylation step omitted.
Antibodies to chicken al-globulin M were obtained from rabbit anti-albumin (Cohn Fraction V) antiserum by repeated absorption with embryonic chicken (16-day-old) serum, which contains no M, to remove antibodies against albumin and transthyretin' (prealbumin "C") (13). The IgG-enriched fraction recovered after precipitating the absorbed antiserum with 35% ammonium sulfate was reabsorbed with embryonic chicken serum before being passed over DEAE-cellulose.
The resulting IgGs recognized only al-globulin M when tested against adult chicken serum proteins with electroimmunoassays and with crossed immunoelectrophoresis. Antiserum against affinity-purified chicken hemopexin was raised in rabbits and a monospecific IgG fraction immunopurified (19). The specific antisera recognizing chicken fibrinogen and albumin have been described previously (13,17, 20). Other purified chicken plasma proteins and/or their respective antisera were the generous gifts of several of our colleagues: al-acid glycoprotein from E. Regoeczi (22), antithrombin III from D. Amrani (12), haptoglobin from F. Delers Spent culture medium for isolation of al-globulin M was obtained from hepatocyte monolayers that were given fresh medium containing 'The plasma protein previously named "C" (11,42) has recently been identified as transthyretin ("prealbumin") (S. Lee and G. Grieninger, unpublished observation). ~ _ _ _~ insulin (35 nM) a t 24 and again a t 48 h of culture to boost M production (7). At 72 h, spent medium was harvested and concentrated 50-fold by ultrafiltration with a supported YM-IO membrane (Amicon). Routinely, 0.7 ml of concentrated medium, containing approximately 0.2 mg of M was applied to the anti" IgG column.
Thirty minutes were allowed for binding of M to the coupled antibody. One milliliter of sodium borate buffer (0.1 M sodium borate, p H 7.5, 0.5 M NaCI, 0.1 mM PMSF, 0.02% sodium azide) was added and allowed to equilibrate with the gel for another 30 min, this step being repeated once. Unbound proteins were washed off the column with sodium borate buffer. Bound al-globulin M was eluted with 3 M potassium thiocyanate, 0.1 mM PMSF, 0.02% sodium azide, and promptly subjected to diafiltration (Amicon, YM-10 membrane) using 0.01 M sodium phosphate, p H 8.0, 0.15 M NaCI, 0.1 mM PMSF, and 0.02% sodium azide. The entire procedure was performed a t 4 "C.
The recovery of M, as evaluated by electroimmunoassay, averaged 50-60%. Before use in the experiments described, the protein samples from different runs were combined, subjected to diafiltration using the above phosphate buffer, but without PMSF or sodium azide, and further concentrated t o 0.64 mg/ml.

RESULTS AND DISCUSSION
The protein known as a,-globulin M (1 1) was isolated from spent medium of chicken embryo hepatocyte cultures. Purification was accomplished by immunoadsorbent column chromatography, using bound anti" IgG prepared from rabbit anti-albumin (Cohn Fraction V) antiserum that had been previously made monospecific by absorption with embryonic chicken serum (see "Experimental Procedures"). In analysis of the eluted protein by crossed immunoelectrophoresis with antiserum against most chicken plasma proteins, only one peak was observed (not shown). The homogeneity of the preparation was corroborated by electrophoresis on SDSpolyacrylamide gels, where a single band ( M , 49,500) was obtained (Fig. 1, lane 2). When the protein was reduced with dithiothreitol and alkylated prior to electrophoresis, its mobility decreased, resulting in an apparent molecular weight of 62,000 (lane 4 ) . This dramatic shift suggests that M may be a tightly folded protein constrained by internal disulfide bonds.
When purified al-globulin M and its antiserum were tested against a number of known chicken plasma proteins-including al-acid glycoprotein, antithrombin 111, haptoglobin, hemopexin, transthyretin, and retinol-binding protein-and/or their corresponding antisera (not shown), cross-reaction was found only with hemopexin. A single band, co-migrating with M on SDS-polyacrylamide gels, both before and after treatment with dithiothreitol, was immunoprecipitated when antihemopexin IgG was incubated with metabolically labeled plasma proteins from spent hepatocyte culture medium (Fig.  1, compare lanes 5 and 7 with Lanes 4 and 2, respectively).
The parallel electrophoretic behavior of the two proteins synthesized in culture-one immunopurified with anti", the other immunoprecipitated with anti-hemopexin-strongly supports the contention that M is hemopexin. Of note, the mobility of hemopexin purified by passing adult chicken plasma over a heme-affinity column was slightly greater than that of the hepatocyte culture-derived molecules (Fig. 1, compare lanes 1 and 3 with 2 and 4 ) , possibly as a result of either a reduction in carbohydrate content or a slight degree of proteolysis. That hepatocyte hemopexin, like plasma hemopexin, is highly glycosylated is indicated by the greater mobility of the polypeptide immunoprecipitated from the medium of tunicamycin-treated hepatocytes (Fig. 1, lunes 6 and  8; see also Ref. 26).
Identification of al-globulin M as hemopexin was extended by two-dimensional peptide mapping in which the single band corresponding to M/hemopexin in the first-dimension gel was subjected to limited proteolysis with Staphylococcus aureus V8 protease; the fragments thus generated were then sepa- To obtain the labeled proteins, hepatocyte monolayers were exposed at 3 and 24 h of culture to fresh medium containing 10 nM TB, 1 nM dexamethasone, and 35 nM insulin. Treatment of monolayers with tunicamycin was initiated 3 h before radioactive labeling by adding the inhibitor to the medium (5 pg/ml) and maintaining it during the labeling period. Under these conditions, N-glycosylation of fibrinogen is completely inhibited (21). The cells were labeled at 48 h in the presence and absence of tunicamycin with 1.5 and 1.0 mCi of [35S] methionine, respectively, per 0.4 ml of medium. Two hours later, spent media were collected, and 0.01 ml was immunoprecipitated with anti-hemopexin IgG and electrophoresed. Samples separated in lanes 3,4, 5, and 6 were reduced and alkylated prior to electrophoresis and are marked with an asterisk (*). Lanes 1 and 3*, hemopexin (2 pg) affinity-purified from chicken plasma; lanes 2 and 4*, a,-globulin M (2 pg) immunoadsorbed from hepatocyte cultures (see "Experimental Procedures"); lanes 5* and 7, immunoprecipitated hemopexin from untreated cultures; lanes 6* and 8, immunoprecipitated hemopexin from tunicamycin-treated cultures. The numbers on the left and right side of the panels indicate molecular mass in kilodaltons. rated in the second-dimension gel. Immunoadsorbed M and immunoprecipitated hemopexin from the cultures gave rise, by this procedure, to peptide maps which were virtually indistinguishable from each other or from that of hemopexin affinity-purified from adult chicken plasma (Fig. 2, compare  trucks 1-3). Nonglycosylated hemopexin from tunicamycintreated cells had a much different partial peptide map (track 4 ) , except in the lowest region of the gel, suggesting that normally all but the smallest fragments bear carbohydrate residues.
The function of hemopexin in the blood stream is that of a heme transport molecule, preventing the urinary excretion of heme and facilitating the conservation of iron (27)(28)(29). Chicken plasma-derived hemopexin binds heme in an equimolar ratio as does hemopexin from other species (19). Titration of the heme-binding capacity of the immunoadsorbed hepatocyte M preparation also indicated a molar ratio of heme to protein of 1:1 (Fig. 3). Intersection of the titration curve with the ordinate (0 addition of heme) further suggests that 62% of the M molecules purified from spent culture medium contain heme.
The binding of heme, it has been found, protects plasma hemopexin to a degree from proteolytic attack by trypsin (19, (Tu) 31,32). Under our assay conditions, for example, hemehemopexin ( i e . plasma-derived protein, saturated with heme) remained largely intact, with about 20-30% of the molecules suffering only a small loss of approximately 7,000 daltons (Fig. 4, compare lunes 2 and 3 ) . The apoprotein, in contrast, was proteolyzed to at least half a dozen lower molecular weight fragments, ranging in size from 40,000 to 14,000 (lanes 4 and 5 ) . Similar patterns were generated with al-globulin M isolated from culture. Treatment of fully loaded heme" produced two high molecular weight bands (lunes 6 and 7 ) , comigrating with those obtained with heme-hemopexin. However, due to the partially (62%) loaded nature of the M preparation as isolated (see Fig. 3), its treatment generated a profile that was a composite of the higher and lower molecular weight bands (lunes 8 and 9 ) .
Thus, on the basis of immunological cross-reactivity, electrophoretic behavior on SDS-polyacrylamide gels, heme-binding capacity, and pattern of cleavage by proteolytic enzymes, we have concluded that al-globulin M is hemopexin. Henceforth, we will drop the term al-globulin M in referring to this protein.
Clinical studies indicate that serum hemopexin increases 10-100-fold in humans from the fetal to the adult level (33). In mice, it has been shown that hemopexin synthesis increases as part of the acute-phase response to injury or stress (34,35). However the agents and mechanisms involved in the regulation of hemopexin synthesis in vivo have yet to be defined. Because of the demonstrated hormone sensitivity of hemopexin production in chicken embryo hepatocyte cultures (14), we have begun to investigate this system's potential as a model for studying the regulation of hemopexin synthesis. In this context, we have characterized the acute-phase and developmentally altered hemopexin levels that occur in the phosphate, 100 mM NaCl, pH 7.4. The concentration of heme was measured spectrophotometrically in 40% dimethyl sulfoxide using a millimolar extinction coefficient of 180 at 400 nm (30). The binding of heme to M/hemopexin was determined by adding increasing amounts of heme to two cuvettes, one containing phosphate buffer alone and the other containing the protein solution (44.7 pg in 1.0 ml), and recording the difference in absorption at 414 nm. For determining the molar ratio of binding, a molecular weight of 62,000 has been used for M/hemopexin as derived from Fig. 1, which compares well with the one estimated by gel filtration (19). After extrapolation to 0 AOD411, it follows that when saturation is reached, 0.70 nmol of heme is bound to 0.72 nmol of protein.
chicken and compared them with the changes in hemopexin production elicited in culture. Prior to this report, the acutephase response of the chicken had been characterized only in terms of changes in the electrophoretic profile of serum proteins (36). T o assess the acute-phase response, blood samples were withdrawn from chickens (3.5 months) for several days following subcutaneous injection with turpentine. This standard method of experimentally eliciting the acute-phase response brought about dramatic differences in the plasma protein profile, as illustrated by crossed immunoelectrophoresis (Fig.  5). Secretion of a few plasma proteins (e.g. "Z") was not affected. Hepatocellular production of the remainder evidenced either a temporary enhancement (e.g. hemopexin,peak 6; fibrinogen, peak 21 ) or a temporary reduction (e.g. albumin, peak 4; transthyretin, peak 2). The &fold increase in hemopexin was accompanied by a nearly 2-fold elevation of fibrinogen and a more than 50% drop in albumin (Fig. 6). These apparently compensating changes, which have also been noted in the acute-phase response of other animal species (37)(38)(39), reached their maximum 2-3 days following injection of the irritant.
It has been shown for other species that many of the changes originate in altered rates of hepatic biosynthesis of these proteins (34,35,(38)(39)(40)(41); however, no single agent has yet been shown to mimic completely the pattern of acutephase plasma protein production by its action on liver cells in culture. Using as a base line the low level output of plasma proteins by the chicken embryo hepatocytes cultured in the absence of hormones, serum, or other macromolecular supplement, we have measured the effects of the controlled addition of physiological concentrations of several hormones, including thyroid hormones, glucocorticoids, and insulin, on plasma protein production. In each case, production of hemopexin (M) was as much or more responsive to stimulation than that of the other plasma proteins assayed, whether the hormones were added individually or in combination (14). All three together, for example, elicited a 7.2-fold increase in hemopexin secretion as compared with 2.7-, 2.6-, and 1.4-fold stimulation for that of fibrinogen, albumin, and transferrin, respectively. Interestingly, the pattern of production elicited by the combination of only glucocorticoids and thyroid hormones most closely resembled that seen in acute-phase chickens: 5.3-and 4.8-fold enhancements of hemopexin and fibrinogen, respectively, with no increase in albumin. A direct link between these culture conditions and the hormonal status of the traumatized animal has not been established; however, further studies along these lines may ultimately resolve the critical balance of factors upon which the hormonal milieu in vivo is built in both normal and pathological states.
Our previous studies have shown that although hemopexin is present in normal adult chicken serum, it is absent from the serum of 17-day-old chicken embryos (11). T o pinpoint the induction of hemopexin in uiuo, serum hemopexin levels of developing chicks were evaluated daily before and after hatching (Fig. 7A). No hemopexin was detected by electroimmunoassay in samples of undiluted chicken embryo serum taken throughout the 5 days prior to hatching. An abrupt change occurred on the day of hatching, however, initiating a steady rise in the level of hemopexin over the 4 subsequent days to achieve a concentration of 150 pg/ml or half the level FIG. 5. Crossed immunoelectrophoresis of proteins in chicken plasma before and during the acutephase response. Four chickens (3.5 months old) were injected subcutaneously with turpentine (0.5 ml per kg of body weight) in the scapular area. Blood samples were drawn from the wing vein and collected in heparin, and plasma was prepared. Plasma samples, drawn from one representative chicken on day 0, day 3, and day 10 following turpentine injection, were analyzed by crossed immunoelectrophoresis. Samples (3 pl) were placed in the appropriate well in the lower left corner of each panel. Electrophoresis in the first dimension was performed from left to right and in the second dimension from bottom to top. The second-dimension gel (antibody-containing) contained a mixture of antisera, similar to that used previously (42), which recognizes most chicken plasma proteins. Immunoplates were stained with Coomassie Blue. In this assay, the amount of each plasma protein is reflected by the intensity and area of its respective peak (11). Several peaks have been identified with the use of specific antisera or purified antigens and are numbered according to Ref. 42. Peak 2, transthyretin (previously termed "prealbumin C"); peak 4, albumin; peak 6, hemopexin; peak 17, al-antitrypsin; peak 19, transferrin; peak 21, fibrinogen; peak 25, chicken immunoglobulins.  found in fully grown chickens (3.5 months old). Based on the limit of detection of our electroimmunoassay (0.05 pg/ml), the serum hemopexin level of the 4-day-old chicks represents a more than 1000-fold increase over embryonic levels. During the period of most dramatic change for hemopexin, albumin levels increased by only a factor of three (Fig. 7 B ) , in accord with the findings of others (43).
Developmental induction of hemopexin production at hatching bears some similarities to the changes observed in rat liver enzyme activities around the time of birth (reviewed in Ref. 1). Based on rapidity and magnitude, the elevated levels of hemopexin most likely reflect an increase in hemopexin mRNA. In this context, we note that there is a rapid accumulation of mRNA for contrapsin (a plasma protein of the anti-chymotrypsin family) on a comparable scale in the developing mouse embryo liver just prior to birth (3,4).