The systemic reaction during inflammation: the acute-phase proteins

The acute-phase response consists in a large number of behavioural, physiologic, biochemical, and nutritional changes involving many organ systems distant from the site, or sites, of inflammation. One of the most investigated, but still not well understood, characteristic of the acute phase is the up-regulation, or down- regulation, of many plasma proteins, known as the acute-phase proteins. The changes in the concentrations of these positive acute-phase proteins and negative acute-phase proteins are due to changes in their liver production. Their increase may vary from 25 percent to 1000 fold, as in the case of C-reactive protein and serum amyloid A. This review summarises the recent advances that have been acquired on the acute-phase proteins, in particular their function in pathologies such as infections or inflammatory lesions.     

Mechanism of enhanced RNA synthesis in acute-phase rat liver and its relationship to chromatin structure.

Nuclei isolated from the liver of rats undergoing an acute inflammatory reaction induced by turpentine treatment show increased RNA synthesis. This increase is essentially determined by a faster polyribonucleotide-elongation rate while the number of transcribing polymerase molecules is unchanged. The sensitivity of chromatin to micrococcal-nuclease digestion and the composition of chromosomal proteins are not affected by the acute-phase process. Therefore the increased RNA synthesis by liver nuclei from acutely inflamed rats does not seem to correlate with major changes in chromatin structure.     

Plasma protein and liver mRNA levels of two closely related murine alpha 1-protease inhibitors during the acute phase reaction

Plasma levels of alpha 1-PI(T) and alpha 1-PI(E), two closely related murine alpha 1-protease inhibitors, having affinities for trypsin and elastase, respectively, were compared to changes in specific liver mRNA levels after induction of the acute-phase reaction by subcutaneous injection of turpentine. In earlier, qualitative experiments an increase in plasma levels of alpha 1-PI(E), but not alpha 1-PI(T), during the acute-phase reaction had been shown. It is now shown that stimulation of plasma alpha 1-PI(E) levels reaches a maximum of 35-50% above baseline 12 h after induction of the acute-phase response using either a functional or immunological assay to measure protease inhibitor activity. Consistent with earlier observations, little or no change in plasma levels of alpha 1-PI(T) is seen. Determination of mRNA levels in the mouse liver specific for alpha 1-PI(E) and alpha 1-PI(T) was accomplished using a cell-free translation system followed by immunoprecipitation of the 35S-labeled protease inhibitors. The apparent Mr's of alpha 1-PI(E) and alpha 1-PI(T) synthesized in vitro are 42K and 46K, respectively. Apparent Mr's of the native proteins in plasma are 55K and 65K. Unexpectedly, mRNA levels for both alpha 1-PI(E) and alpha 1-PI(T) were found to increase after induction of the acute-phase reaction. Maximal stimulation for both mRNAs was approximately 300% and occurred 9 h after turpentine administration. Under these conditions, levels of translatable albumin mRNA in the mouse liver decreased to 40% of baseline in 6-9 h.     

Acute-phase protein gene expression in rat liver following whole body X-irradiation or partial hepatectomy

We studied the effect of total body X-irradiation and partial hepatectomy on the acute phase protein gene expression in rat liver. Male rats of AO strain were irradiated with high X-ray doses, without any visible tissue damage. In contrast, partial hepatectomy consisted of surgical removal of 40% liver tissue. The changes in liver mRNA concentrations for positive acute-phase reactants including cysteine protease inhibitor, alpha(1)-acid glycoprotein, fibrinogen and haptoglobin, and albumin as a negative reactant were monitored by Northern blot and slot-blot hybridizations using corresponding [32P]dCTP labeled cDNA probes. While in the first 24 h after the partial hepatectomy, liver mRNA levels for the positive acute-phase reactants increased, briefly followed by an immediate decrease, the duration and timing of the acute-phase responses to the whole body X-irradiation were slightly different and lasted for as long as 72 h. Although both treatments induced the mRNA expression of acute-phase reactants in rat liver, the observed variations in the duration and intensity of the changes in mRNA levels for the acute-phase proteins in these two types of tissue damage suggest the involvement of specific mechanisms in a fine tuning of the non-specific acute-phase responses to meet the unique requirements of the particular injury.     

Comparison of changes in gene expression of transferrin receptor-1 and other iron-regulatory proteins in rat liver and brain during acute-phase response

The “acute phase” is clinically characterized by homeostatic alterations such as somnolence, adinamia, fever, muscular weakness, and leukocytosis. Dramatic changes in iron metabolism are observed under acute-phase conditions. Rats were administered turpentine oil (TO) intramuscularly to induce a sterile abscess and killed at various time points. Tissue iron content in the liver and brain increased progressively after TO administration. Immunohistology revealed an abundant expression of transferrin receptor-1 (TfR1) in the membrane and cytoplasm of the liver cells, in contrast to almost only nuclear expression of TfR1 in brain tissue. The expression of TfR1 increased at the protein and RNA levels in both organs. Gene expression of hepcidin, ferritin-H, iron-regulatory protein-1, and heme oxygenase-1 was also upregulated, whereas that of hemojuvelin, ferroportin-1, and the hemochromatosis gene was significantly downregulated at the same time points in both the brain and the liver at the RNA level. However, in contrast to observations in the liver, gene expression of the main acute-phase cytokine (interleukin-6) in the brain was significantly upregulated. In vitro experiments revealed TfR1 membranous protein expression in the liver cells, whereas nuclear and cytoplasmic TfR1 protein was detectable in brain cells. During the non-bacterial acute phase, iron content in the liver and brain increased together with the expression of TfR1. The iron metabolism proteins were regulated in a way similar to that observed in the liver, possibly by locally produced acute-phase cytokines. The significance of the presence of TfR1 in the nucleus of the brain cells has to be clarified.     

Stimulation of the acute-phase response in simian virus 40-hepatocyte cell lines.

Seven simian virus 40 (SV40)-hepatocyte cell lines were characterized with respect to the ability to express eight liver acute-phase genes. cDNA clones corresponding to albumin, serum amyloid A, alpha 1-acid glycoprotein, haptoglobin, alpha-, beta-, and gamma-fibrinogen, and alpha 1-major-acute-phase protein mRNAs were used in Northern (RNA) or slot blot analyses. In the noninduced state, six of the seven cell lines showed significant (i.e., liverlike) levels of constitutive expression of all genes examined except that expression of haptoglobin mRNA was considerable lower than in the normal liver. To examine whether these immortalized liver cells can respond appropriately to inflammatory mediators, cells were treated with conditioned medium from activated human monocytes or mixed lymphocyte cultures. Results showed that these SV40-hepatocyte cell lines responded to the conditioned media in culture by down-regulating albumin gene expression and up-regulating other acute-phase genes in a time- and dose-dependent manner. These results indicate that the SV40-hepatocytes retained not only the ability to express a number of acute-phase genes but also the ability to respond to external stimuli. The usefulness of these cell lines for analysis of the molecular mechanisms involved in the regulation of these acute-phase genes is discussed.     

Biosynthesis and secretion of alpha 1 acute-phase globulin in primary cultures of rat hepatocytes

Experimental inflammation in rats led to a sevenfold increase in serum levels of alpha 1 acute-phase globulin. This increase is correlated with elevated levels of translatable mRNA for alpha 1 acute-phase globulin in the liver. Biosynthesis and secretion of alpha 1 acute-phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of alpha 1 acute-phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of alpha 1 acute-phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. The secreted form of alpha 1 acute-phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side-chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type alpha 1 acute-phase globulin. In the hepatocyte medium newly synthesized alpha 1 acute-phase globulin was detected 30 min after the pulse. Unglycosylated alpha 1 acute-phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in alpha 1 acute-phase globulin secretion.     

Hepcidin, interleukin-6 and hematological iron markers in males before and after heart surgery

Anemia of inflammation in patients with acute or chronic acute-phase activation is a common clinical problem. Hepcidin is a peptide shown to be the principal regulator of the absorption and systemic distribution of iron. Main inducers of hepcidin are iron overload, hypoxia and inflammation, where the latter has been linked to hepcidin via increased interleukin-6 (IL-6). This article addresses the impact and time course of postoperative acute-phase reaction in humans following heart surgery on prohepcidin, hepcidin, hematological markers and IL-6 concentrations. Serum concentrations of prohepcidin, hepcidin, IL-6 and hematological iron parameters were studied in five male patients without infection before and after heart surgery. This study, which is the first to report the impact on serum hepcidin and serum prohepcidin concentrations in patients following surgery, clearly demonstrates the induction of hypoferremia due to the postoperative acute-phase reaction. Significant changes were seen for serum iron concentration, transferrin saturation, total iron binding capacity and hemoglobin concentration. A significant increase in ferritin concentration was seen 96-144 h postoperatively. Additionally, there were significant alterations in both serum hepcidin after 96-144 h and serum prohepcidin after 48 h compared with preoperative values. Serum prohepcidin decreased, whereas serum hepcidin increased. In conclusion, changes in serum prohepcidin were followed by an increase in serum hepcidin. This speaks in favor of a chain of action where proteolytic trimming of serum prohepcidin results in increased serum hepcidin. However, hypoferremia appeared prior to the changes in serum prohepcidin and serum hepcidin.     

Effects of protein malnutrition on IL-6-mediated signaling in the liver and the systemic acute-phase response in rats

This study examines the effects of malnutrition on IL-6 signaling pathways of rats fed 2% vs. 20% casein diets for 14 days. Effects of malnutrition on the abundance and IL-6-stimulated phosphorylation of signaling proteins in the JAK-STAT and MAP kinase pathways were examined in the liver. Changes of the acute-phase response as reflected by serum alpha(1)-acid glycoprotein (AG), TNF-alpha (TNF), and IL-1beta (IL-1) were compared in the two dietary groups at 0, 4, 8, 16, and 24 h after IL-6 administration. Under basal conditions, the abundance of the IL-6 receptor, gp130, JAK1, STAT1, and STAT3 proteins and levels of phosphorylation of ERK1/2 and p38 were significantly increased in the liver in the 2% casein group compared with the 20% casein group. With IL-6 stimulation, the increased phosphorylation per unit of protein of these signaling proteins was not different in the liver between the two groups. Before IL-6 stimulation, serum levels of TNF, IL-1, IL-6, and AG were significantly higher in the 2% casein group than in the 20% casein group. After bolus injection of IL-6, changes in IL-1 and AG were similar in the two dietary groups, although a slight decline in AG level was noted after 8 h of IL-6 administration in the 2% protein group. These data demonstrate that protein malnutrition produces changes in inflammation-related proteins characteristic of a low-grade systemic inflammatory response and, thus, can serve as an inflammatory stimulus. The capacity for response to IL-6 is preserved, suggesting adaptive preservation of acute-phase responsiveness during malnutrition.     

Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein.

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.     

The role of leukocytes in the serum acute-phase reaction accompanying inflammatory reactions

The effects of whole-body X-irradiation with a dose of (7 Gray) were studied on the time course of systemic responses (fever, leukocytosis, serum coeruloplasmin concentration) in turpentine-induced inflammation and on the acute-phase reaction (coeruloplasmin, haptoglobin and seromucoid) produced by various stressful conditions (turpentine-abscess, immobilization, restraint conditions). Results showed that in the irradiated animals the lack of the leukocytic and febrile response to turpentine administration was accompanied by a normal coeruloplasmin response. Therefore, we have concluded to the involvement of more than one mediator in the development of systemic reactions accompanying inflammation. The leukopenia induced by whole-body irradiation could not prevent the development or reduce the magnitude of the acute-phase response in any of the models analysed. This observation appears to make doubtful the exclusive role of leukocytes in the mediation of the acute-phase reaction.     

Mouse C-reactive protein. Generation of cDNA clones, structural analysis, and induction of mRNA during inflammation.

A full-length C-reactive protein (CRP) cDNA clone has been isolated from a CBA/J-strain-mouse acute-phase liver library. The 1614-nucleotide cDNA specifies mRNA 5' and 3' untranslated regions of 81 and 858 bases respectively that flank 675 bases encoding mouse pre-CRP. The derived amino acid sequence predicts a 19-residue leader peptide followed by a 206-residue mature mouse CRP that shows considerable sequence identity with both human and rabbit CRP. Northern-blot analysis of mouse liver CRP mRNA concentrations after inflammatory stimuli and comparison with hepatic induction of mRNA for the major mouse acute-phase protein serum amyloid P component established that CRP, a major acute-phase reactant in human and rabbit, is a minor acute-phase reactant in mouse. The size and organization of the mouse CRP mRNA 5' and 3' untranslated regions are significantly different from those of human and rabbit CRP mRNA and may have implications for its anomalous minimal induction during acute inflammation.     

Expression of the cDNA for alpha 1-acid glycoprotein, a rat plasma protein, in Escherichia coli

Polyadenylated RNA was isolated from acute-phase liver and transcribed into cDNA. After homopolymer tailing this was cloned into the PstI site of pBR322 which was used to transform E. coli RR1 cells. Clones containing cDNA corresponding to acute-phase proteins were identified by differential hybridization with 32P-labelled normal and acute-phase cDNA. A clone synthesizing about 10 ng of alpha 1-acid glycoprotein per E. coli colony was detected using a solid-phase based immunochemical procedure. The identification of the clone was verified by competition assay with authentic alpha 1-acid glycoprotein isolated from serum and by restriction analysis of the cDNA inserted into pBR322.     

Impact of serum amyloid A on high density lipoprotein composition and levels

Serum amyloid A (SAA) is an acute-phase protein mainly associated with HDL. To study the role of SAA in mediating changes in HDL composition and metabolism during inflammation, we generated mice in which the two major acute-phase SAA isoforms, SAA1.1 and SAA2.1, were deleted [SAA knockout (SAAKO) mice], and induced an acute phase to compare lipid and apolipoprotein parameters between wild-type (WT) and SAAKO mice. Our data indicate that SAA does not affect apolipoprotein A-I (apoA-I) levels or clearance under steady-state conditions. HDL and plasma triglyceride levels following lipopolysaccharide administration, as well as the decline in liver expression of apoA-I and apoA-II, did not differ between both groups of mice. The expected size increase of WT acute-phase HDL was surprisingly also seen in SAAKO acute-phase HDL despite the absence of SAA. HDLs from both mice showed increased phospholipid and unesterified cholesterol content during the acute phase. We therefore conclude that in the mouse, SAA does not impact HDL levels, apoA-I clearance, or HDL size during the acute phase and that the increased size of acute-phase HDL in mice is associated with an increased content of surface lipids, particularly phospholipids, and not surface proteins. These data need to be transferred to humans with caution due to differences in apoA-I structure and remodeling functions.     

Glycosylation of four acute-phase glycoproteins secreted by rat liver cells in vivo and in vitro. Effects of inflammation and dexamethasone

We have studied the role of the liver in the relative increase of Concanavalin A (Con A)-reactive molecular forms of various positive rat acute-phase glycoproteins (APGPs) occurring in serum during inflammation. Secretion media of hepatocytes isolated from inflamed rats showed a 2 to 5-fold increase of the total amounts of four APGPs studied in comparison to secretion media of control hepatocytes. These changes were in analogy with those observed for corresponding sera, except for alpha 1-antitrypsin. All the different Con A-reactive molecular forms were present in the media, with exception of the most reactive form of ceruloplasmin. In vitro and in vivo, dexamethasone augmented the secretion of three APGPs, and especially of the Con A-most reactive forms. The in vitro effect of dexamethasone--augmented secretion of Con A-reactive molecular forms of alpha 1-acid glycoprotein and haptoglobin--was comparable with the results obtained for hepatocytes isolated from inflamed rats. In vivo, dexamethasone treatment resulted in an even higher increase of the serum concentration of the Con A-most reactive forms of both APGPs than experimental inflammation did. Although an extrahepatic contribution cannot be excluded, these results suggest that alterations in the Con A reactivity of APGPs as observed during the acute-phase of inflammation have their origin in the liver. A change in the Con A reactivity of glycoprotein indicates a modulation of its glycosylation. Since dexamethasone can affect these changes in vivo and in vitro, glucocorticoids most probably are involved in the regulation of the glycosylation of the APGPs during biosynthesis in the liver.