Two distinct chloride ion requirements in the constitutive protein secretory pathway

The role of chloride ions in regulated secretion is well described but remains poorly characterised in the constitutive system. In the liver, newly synthesised proalbumin is transported to the trans Golgi network where it is converted to albumin by a furin protease and then immediately secreted. We used this acid-dependent hydrolysis and the measurement of specific protein secretion rates to examine the H+ and Cl- ion dependence of albumin synthesis and secretion, a major constitutive protein secretory event in all mammals. Using permeabilised primary rat hepatocytes we show that ordinarily chloride ions are essential for the processing of proalbumin to albumin. However Cl- is not required for transport which continues but releases solely proalbumin. Prior treatment of the cells with Tris (used as a membrane-permeable weak base to neutralise Golgi luminal pH) both eliminated the formation of albumin and very greatly reduced secretion. After washing out Tris, both authentic secretion and processing could be restarted if Cl-, ATP, GTP, cAMP, Ca2+ and cytosolic proteins were added. Hence a requirement for chloride in transport, in addition to processing, can be uncovered by first neutralising pH gradients. Furthermore, the chloride channel blocker DIDS (4,4-diisothiocyanostilbene 2,2-disulphonic acid) reversibly inhibited the constitutive secretory pathway. However, the total mass of proalbumin detectable in DIDS-treated cells fell to 36% of control while the fraction processed to albumin remained almost constant. This clearly dissociates a large part of the Cl- requirement of the constitutive protein secretory pathway from the function of known liver Golgi Cl- channels.     

Partition of whey milk proteins in aqueous two-phase systems of polyethylene glycol-phosphate as a starting point to isolate proteins expressed in transgenic milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha lactoalbumin and beta lactoglobulin) and alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000; 1500 and 3350)-potassium phosphate was analysed. Bovine serum albumin and alpha lactoalbumin concentrated in the polyethyleneglycol rich phase with a partition coefficient of 10.0 and 27.0, respectively, while beta lactoglubulin and alpha-1 antitrypsin showed affinity for the phosphate-rich phase with a partition coefficient of 0.07 and 0.01, respectively. An increase of medium pH induced an increase of the partition coefficient of these proteins while the increase in polyethyleneglycol molecular mass induced the opposite behaviour. The system polyethyleneglycol 1500-pH 6.3 showed the best capacity for recovering the alpha-1 antitrypsin with a yield of 80% and a purification factor between 1.5 and 1.8 from an artificial mixture of the milk whey proteins and alpha-1 antitrypsin. The method appears to be suitable as a starting point to isolate proteins expressed in transgenic milk.     

Effects of canavanine on the secretion of plasma proteins by Hep G2 cells

Many secretory proteins contain an amino-terminal propeptide extension which is removed prior to secretion. The point of cleavage is usually marked by a basic pair of amino acids containing arginine. Canavanine, an analogue of arginine, is incorporated into protein and has been shown to inhibit the proteolytic processing of several of these prosecretory proteins. The addition of 3 mM canavanine to Hep G2 cells incubated with L-[35S]methionine inhibited the secretion of 11 plasma proteins studied. Of the secretory proteins studied only albumin is thought to contain a propeptide, which is marked by a pair of arginine residues at its point of proteolytic processing. Canavanine had varying effects on the secretion of plasma proteins; ranging from a 43-53% inhibition of secretion of alpha 1 antitrypsin and alpha 1 anti-chrymotrypsin to nearly abolishing (93% inhibition) secretion of transferrin. Canavanine also caused most of the proteins studied to migrate slower on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two of the canavanine-treated proteins (albumin and transferrin) which underwent marked changes in electrophoretic mobility were more sensitive than untreated proteins to proteolysis by Staphylococcus Aureus V8 proteinase. The slower electrophoretic migration and the greater sensitivity to proteolysis of these proteins may be attributed to marked structural changes caused by the incorporation of canavanine. This suggests that the inhibition of plasma protein secretion by canavanine is not only due to an inhibition of the processing of proteins but may be caused by structural distortions of the secretory proteins.     

Features of the milk whey protein partitioning in polyethyleneglycol-sodium citrate aqueous two-phase systems with the goal of isolating human alpha-1 antitrypsin expressed in bovine milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha-lactoalbumin and beta-lactoglobulin) and human alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000, 1450 and 3350)-sodium citrate was analysed at pH 5.2, 6.2 and 8.2. Alpha lactoalbumin concentrated in the polyethyleneglycol rich-phase, while beta-lactoglobulin, bovine serum albumin and alpha-1 antitrypsin showed affinity for the citrate rich-phase. In aqueous two-phase systems of high medium pH and high polyethyleneglycol molecular mass the protein partitioning equilibrium is displaced to the citrate rich-phase. The polyethyleneglycol 1450-pH 5.2 system with a top/bottom phase-volume ratio of 3 showed to have the best capability of recovering the alpha-1 antitrypsin from a mixture prepared with natural milk whey and human alpha-1 antitrypsin. The recovery of this protein in the bottom phase was of 90% and the purity of the obtained product was of 98%. The method appears to be suitable as a starting point to isolate other human proteins expressed in transgenic bovine milk.     

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex

1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse- chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1- antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1- antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha- mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N- linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.     

Synthesis of antithrombin III and alpha-1-antitrypsin by the perfused rat liver.

Livers isolated from control or turpentine-injected rats were perfused for 3 h with human red cells suspended in Krebs-Henseleit solution containing bovine serum albumin, dextran, glucose, heparin, cortisol, insulin, a mixture of 20 amino acids and [3H]leucine. Changes in the concentrations of antithrombin III and alpha-1-antitrypsin were evaluated by rocket immunoelectrophoresis using specific antisera, and incorporation of the 3H radioactivity into the total protein, albumin, antitrhombin III and alpha-1-antitrypsin in the perfusate was measured. The results indicate that both antithrombin III and alpha-1-antitrypsin are synthesized in the liver. Local inflammation induced in the liver donors moderately stimulated the synthesis of alpha-1-antitrypsin but it affected only marginally that of antithrombin III.     

Disparate effects of monensin and colchicine on intracellular processing of secretory proteins in cultured rat hepatocytes

We have studied the biosynthesis and intracellular processing of three major secretory proteins, albumin, alpha 1-protease inhibitor and alpha 2u-globulin, in cultured rat hepatocytes. The effect of secretion-blocking agents, monensin, a monovalent ionophore, and the microtubule-affecting agents colchicine and taxol was determined. In the control cells, alpha 1-protease inhibitor, a glycoprotein, was first synthesized as an endoglycosidase-H-sensitive form with Mr 51 000, and then processed to two endoglycosidase-H-resistant forms having Mr 51 000 and 56 000, the latter of which was secreted into the medium. Initially synthesized proalbumin was converted with chase to serum-type albumin, while no pro-type precursor was identified for alpha 2u-globulin. In the cells treated with colchicine or taxol, in which secretion was greatly inhibited, the fully processed alpha 1-protease inhibitor and albumin accumulated and were finally secreted into the medium. In the monensin-treated cells, however, most of the newly synthesized alpha 1-protease inhibitor and albumin were not processed to the final mature forms, resulting in accumulation of two 51 000-Mr forms and proalbumin, respectively. Moreover in treated cells, proalbumin and the endoglycosidase-H-resistant alpha 1-protease inhibitor were finally secreted into the medium. Such an effect was not caused by NH4Cl which also inhibited the secretion and is known to exert the similar effect as monensin on the receptor-mediated endocytosis pathway. Based on these results, the use of monensin may prove valuable for more detailed analysis of intracellular processing of various proteins.     

The disulphide mapping,folding and characterisation of recombinant Ber e 1, an allergenic protein,and SFA8, two sulphur-rich 2S plant albumins

We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.     

Demonstration of a calcium requirement for secretory protein processing and export. Differential effects of calcium and dithiothreitol.

HepG2 cells were employed as model system to investigate potential relationships between early protein processing and Ca2+ storage by the endoplasmic reticulum. Ca2+ was required for glycoprotein processing and export by intact cells. The processing and export of alpha 1-antitrypsin and the secretion of complement factor 3, which are glycosylated proteins, were inhibited by the Ca2+ ionophore ionomycin whereas the export of albumin, a non-glycoprotein, was little affected. Ionomycin blocked processing of alpha 1-antitrypsin at the conversion from the high mannose to the complex glycosylated form without affecting ATP or GTP contents. Pre-existing inhibition of intracellular processing of alpha 1-antitrypsin by ionomycin was fully reversible upon removal of the ionophore with fatty acid-free bovine serum albumin. This reversal required Ca2+. After reversal the arrested form of alpha 1-antitrypsin was fully converted to the mature form and exported to the medium. Inhibitions of alpha 1-antitrypsin processing and complement factor 3 secretion by the metalloendoprotease antagonist Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) were strongest at low extracellular Ca2+ but were reduced or prevented by high extracellular Ca2+. Processing and secretion of alpha 1-antitrypsin were reduced upon incubation in low Ca2+ medium. Exposure to dithiothreitol reduced albumin export while affecting alpha 1-antitrypsin export minimally. Suppression of amino acid incorporation into total cellular proteins of HepG2 cells accompanied inhibitions of protein processing by agents depleting sequestered Ca2+ stores or by dithiothreitol. Putative control of rates of translational initiation by the endoplasmic reticulum through linkage to rates of early protein processing is discussed.     

Tris inhibits both proteolytic and oligosaccharide processing occurring in the Golgi complex in primary cultured rat hepatocytes

Tris caused the distention of the Golgi cisternae in primary cultured rat hepatocytes and perturbed the functions occurring there. Proteolytic cleavage of precursors of both albumin and complement C3 was inhibited, whereas that of prohaptoglobin was not affected by Tris. These effects on the proteolytic cleavages resemble those of acidotropic amines (Oda, K., and Ikehara, Y. (1985) Eur. J. Biochem. 152, 605-609; Oda, K., Koriyama, Y., Yamada, E., and Ikehara, Y. (1986) Biochem. J. 240, 739-745). However, the effects of Tris significantly differed from acidotropic amines on the basis of its effects on the processing of N-linked oligosaccharides of glycoproteins. Both alpha 1-protease inhibitor and haptoglobin secreted from the Tris-treated cells were found to contain almost equal amounts of endo-beta-N-acetylglucosaminidase H-sensitive and -resistant oligosaccharides, whereas the glycoproteins from both the control and methylamine-treated cells were resistant to the enzyme. The endo-beta-N-acetylglucosaminidase-sensitive oligosaccharides were analyzed to be Man8-5GlcNAc by high resolution gel permeation chromatography, suggesting that trimming of alpha-mannose residues from the precursor Man9GlcNAc2 is incomplete in the Tris-treated cells. On the other hand, Tris did not significantly inhibit incorporation of radioactive monosaccharides (N-acetylglucosamine, galactose, and fucose) into the glycoproteins. However, two-dimensional gel electrophoresis in combination with neuraminidase digestion demonstrated that sialylation was markedly inhibited by Tris. Taken together, our results reveal that Tris inhibits not only the sialic acid addition which takes place in the trans Golgi region, but also the trimming step of high mannose-type oligosaccharides, which is thought to occur before glycoproteins reach the trans Golgi region.     

Profibrillin-1 maturation by human dermal fibroblasts: proteolytic processing and molecular chaperones

Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A(1) and incubation of dermal fibroblasts at 22 degrees C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, alpha-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway.     

A novel model and molecular therapy for Z alpha-1 antitrypsin deficiency

Animal models that closely resemble human disease can present a challenge. Particularly so in alpha-1 antitrypsin deficiency (α(1)ATD), as the mouse alpha-1 antitrypsin (α(1)AT) cluster encodes five highly related genes compared with the one in humans. The mouse PI2 homologue is closest to the α(1)AT human gene. We have changed the equivalent mouse site that results in the Z variant in man (Glu342Lys) and made both the "M" and "Z" mouse PI2 α(1)AT proteins. We have tested the ability of a small-molecular-weight compound CG to alleviate polymerisation of these mouse α(1)AT proteins as it has been shown to reduce aggregates of Z α(1)AT in man. We found that (1) CG specifically reduces the formation of polymers of recombinant mouse "Z" protein but not "M" protein; (2) whereas there is significantly more α(1)AT secreted from Chinese Hamster Ovary cells transfected with the mouse "M" α(1)AT gene than with the "Z" (20.8?±?3.9 and 6.7?±?3.6, respectively; P?相似文献   

A foetal alpha-1 antitrypsin which resembles PI*I

Summary A transient form of alpha-1 antitrypsin (PI) with an isoelectric point resembling that of the inherited PI*I allele has been detected in ten premature infants up to 35/40 weeks' gestation. It is suggested that this may be a foetal form of alpha-1 antitrypsin which is specially adapted to the uterine evironment.     

The role of bronchial epithelial cells in the pathogenesis of COPD in Z-alpha-1 antitrypsin deficiency

Background

Alpha-1 antitrypsin is the main inhibitor of neutrophil elastase in the lung. Although it is principally synthesized by hepatocytes, alpha-1 antitrypsin is also secreted by bronchial epithelial cells. Gene mutations can lead to alpha-1 antitrypsin deficiency, with the Z variant being the most clinically relevant due to its propensity to polymerize. The ability of bronchial epithelial cells to produce Z-variant protein and its polymers is unknown.We investigated the expression, accumulation, and secretion of Z-alpha-1 antitrypsin and its polymers in cultures of transfected cells and in cells originating from alpha-1 antitrypsin-deficient patients.

Methods

Experiments using a conformation-specific antibody were carried out on M- and Z-variant–transfected 16HBE cells and on bronchial biopsies and ex vivo bronchial epithelial cells from Z and M homozygous patients. In addition, the effect of an inflammatory stimulus on Z-variant polymer formation, elicited by Oncostatin M, was investigated. Comparisons of groups were performed using t-test or ANOVA. Non-normally distributed data were assessed by Mann–Whitney U test or the Kruskal-Wallis test, where appropriate. A P value of < 0.05 was considered to be significant.

Results

Alpha-1 antitrypsin polymers were found at a higher concentration in the culture medium of ex vivo bronchial epithelial cells from Z-variant homozygotes, compared with M-variant homozygotes (P < 0.01), and detected in the bronchial epithelial cells and submucosa of patient biopsies. Oncostatin M significantly increased the expression of alpha-1 antitrypsin mRNA and protein (P < 0.05), and the presence of Z-variant polymers in ex vivo cells (P < 0.01).

Conclusions

Polymers of Z-alpha-1 antitrypsin form in bronchial epithelial cells, suggesting that these cells may be involved in the pathogenesis of lung emphysema and in bronchial epithelial cell dysfunction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0112-3) contains supplementary material, which is available to authorized users.     

Regulation of sialyltransferase activity in intestinal segments of rats

A differential distribution of sialyltransferase (ST) in different regions of intestine has been shown. Jejunum and ileum homogenates from rats showed almost exclusive presence of alpha-2-3 ST (to Gal in Gal beta-1-4GlcNAc and/or to Gal in Gal beta-1-3GalNAc). In contrast, colon homogenates showed the presence of both alpha-2-3 ST (as above) and alpha-2-6 ST. Incubation of intestinal slices in presence of heat-inactivated horse serum (HHS) showed a time- and temperature-dependent secretion of soluble ST into the medium. Both jejunum and ileum slices showed high rates of secretion of alpha-2-3 ST. Colon slices, though rich in alpha-2-6 ST, secreted only alpha-2-3 ST. Colchicine, an anti-mitotic drug, injected into rats caused about 10-fold increase of the serum ST level. Jejunum slices from colchicine-treated rats showed an increased secretion of alpha-2-6 ST, suggesting that intestine undergoes a change in the expression of normal secretion of alpha-2-3 ST to a secretion of alpha-2-6 ST. The secretion of ST from incubated intestinal slices was inhibited by heparin. Certain protein factors (anti-proteases) in HHS bind to heparin-sepharose column and these protein factors are responsible for causing the secretion of ST into the medium. It has also been found that a supernatant fraction of the colon homogenate activated ST. Gel chromatography on HPLC produced 3-4 protein fractions from the colon cytosol and one of this fraction bearing high molecular weight proteins produced the maximum activation of ST.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymorphism of alpha-1 antitrypsin in dogs

• 1.1. A genetically determined polymorphism of alpha-1 antitrypsin is demonstrated in dog serum by isoelectric focusing in a pH range of 3.5–5.0, followed by direct immunoblotting using a specific antiserum.
• 2.2. Alpha 1 antitrypsin focuses as two major bands at isoelectric points of 4.60 and 4.64 or 4.67 and 4.7 in presumed homozygous animals. Heterozygotes show both sets of bands.
• 3.3. The results of seven crosses with 33 offspring are best explained by two codominant alleles, Pi^M and Pi^S at a single locus designated as Pi for proteinase inhibitor.
• 4.4. The concentration of alpha-1 antitrypsin in serum of healthy dogs was 2.65 ± 0.42 mg/ml and 2.19 ± 0.38 mg/ml in females andv males respectively.
• 5.5. The higher concentration in female dogs suggests that estrogens may influence the serum level of alpha-1 antitrypsin.

     

Secretion of proalbumin by canavanine-treated Hep-G2 cells

The two processing sites in the conversion of preproalbumin to albumin are marked by arginine residues. Therefore, to study the mechanisms of albumin processing and secretion, the arginine residues of nascent albumin were replaced with canavanine by the incubation of Hep-G2 cells with this arginine analog. During a 4-h interval, canavanine inhibited (67%) the secretion of nascent albumin and increased the intracellular transit time of albumin secretion from 24 to 39 min. At 1 h, canavanine inhibited total protein synthesis by 19% and albumin synthesis by about 40%. Both the intracellular and secreted albumin produced by canavanine-treated cells were analyzed by DEAE-cellulose chromatography and were found to be more acidic than normal proalbumin and albumin. Further analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the albumin produced and secreted by canavanine-treated cells appeared to have a larger molecular weight (by 4000) than serum albumin. The canavanine-treated cells were incubated with L-[3H]leucine and L-[3H]phenylalanine and the location of radioactive L-leucine and L-phenylalanine in the 30 NH2-terminal amino acid residues of secreted albumin was determined. The results indicated that canavanine-treated cells secreted proalbumin (79%) and also some fully processed albumin (21%). Preproalbumin was not secreted. Untreated Hep-G2 cells mostly secreted fully processed serum albumin (93%) with only traces of proalbumin (7%).     

Active Trafficking of Alpha 1 Antitrypsin across the Lung Endothelium

The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.     

Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines.

We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL-6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression.