Mouse alpha 1-protease inhibitor is not an acute phase reactant

Mouse plasma contains two major protease inhibitors, alpha 1-protease inhibitor (alpha 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of alpha 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic alpha 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J.M. Frazer, S.A. Nathoo, J. Katz, T.L. Genetta, and T.H. Finley [1985) Arch. Biochem. Biophys. 239, 112-119) have reported a threefold increase of mRNA for the elastase specific alpha 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, alpha 1-PI is not an acute phase reactant.     

Immunological and chemical properties of mouse alpha 1-protease inhibitors

We previously described the isolation and purification of two similar alpha 1-protease inhibitors from mouse plasma termed alpha 1-PI(E) and alpha 1-PI(T) because of their respective affinities for elastase and trypsin. Some of the biochemical and immunological properties of these proteins are reported. Both are acidic glycoproteins with pI's of 4.1-4.2. The plasma half-life of each inhibitor, determined after administration of the 125I-protein, is approximately 4 h both in normal mice and in mice after induction of the acute phase reaction. The two proteins have almost identical amino acid compositions and similar CNBr peptide maps. Tryptic maps, however, are considerably different. Reverse-phase chromatography separated alpha 1-PI(E) into three distinct isoforms, each eluting with approximately 60% acetonitrile. Under these conditions alpha 1-PI(T) shows a single peak, clearly different from those of alpha 1-PI(E). The three alpha 1-PI(E) isoforms have the same molecular weights on sodium dodecyl sulfate-gel electrophoresis and the same tripeptide sequence at their N-terminus, and appear to be immunologically identical. Polyclonal, monospecific antibodies to each native inhibitor, prepared in rabbits, showed no cross-reactivity when tested by functional assay or crossed immunoelectrophoresis. Interestingly, each antibody recognized epitopes on the C-terminal portion of its respective antigen. These studies confirm that alpha 1-PI(E) and alpha 1-PI(T), although highly similar, are products of different genes. Like human alpha 1-PI, the two mouse inhibitors are partially inactivated by mild oxidation with chloramine-T, losing all elastase inhibitor and lesser amounts of antichymotryptic and antitryptic activity. However, unlike the human protein, neither alpha 1-PI(E) nor alpha 1-PI(T) was found to have a methionine residue at its P1 site.     

Partial hepatectomy and mediators of inflammation decrease the expression of liver alpha 2-HS glycoprotein gene in rats

Liver mRNA levels of two acute phase reactant (APR) proteins, alpha 2-HS glycoprotein (a major negative APR) and alpha 1-acid glycoprotein (a major positive APR) were measured in male rats at different times after the administration of turpentine, of tumor necrosis factor, or following partial hepatectomy. In every case, a marked decrease in mRNA levels of alpha 2-HS glycoprotein was observed which reached a maximum at 24 h. A concomitant increase of alpha 1-acid glycoprotein mRNA levels was observed under the same conditions. These results indicate that the decreased levels of alpha 2-HS glycoprotein induced by the acute-phase response following inflammatory mediators and partial hepatectomy are due to a down-regulation of the gene expression of this protein in rat liver.     

In vivo cleavage of alpha2,6-sialyltransferase by Alzheimer beta-secretase

beta-Site amyloid precursor protein-cleaving enzyme 1 (BACE1) is a membrane-bound aspartic protease that cleaves amyloid precursor protein to produce a neurotoxic peptide, Abeta, and is implicated in triggering the pathogenesis of Alzheimer disease. We previously reported that BACE1 cleaved rat beta-galactoside alpha2,6-sialyltransferase (ST6Gal I) that was overexpressed in COS cells and that the NH(2) terminus of ST6Gal I secreted from the cells (E41 form) was Glu(41). Here we report that BACE1 gene knock-out mice have one third as much plasma ST6Gal I as control mice, indicating that BACE1 is a major protease which is responsible for cleaving ST6Gal I in vivo. We also found that BACE1-transgenic mice have increased level of ST6Gal I in plasma. Secretion of ST6Gal I from the liver into the plasma is known to be up-regulated during the acute-phase response. To investigate the role of BACE1 in ST6Gal I secretion in vivo, we analyzed the levels of BACE1 mRNA in the liver, as well as the plasma levels of ST6Gal I, in a hepatopathological model, i.e. Long-Evans Cinnamon (LEC) rats. This rat is a mutant that spontaneously accumulates copper in the liver and incurs hepatic damage. LEC rats exhibited simultaneous increases in BACE1 mRNA in the liver and in the E41 form of the ST6Gal I protein, the BACE1 product, in plasma as early as 6 weeks of age, again suggesting that BACE1 cleaves ST6Gal I in vivo and controls the secretion of the E41 form.     

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.     

Influence of chronic inflammation on the level of mRNA for acute-phase reactants in the mouse liver

Systemic injuries provoke a coordinate change in the hepatic synthesis and circulating concentration of plasma proteins. To assess the effect of a transition from acute to chronic inflammation on the expression of the plasma protein genes, the qualitative and quantitative changes of liver mRNA were measured in males of the inbred strain C57BL/6 of Mus musculus during a 14-day period of inflammation. Inflammation was induced and maintained by repeated injections of the irritants, turpentine, lipopolysaccharides, and celite. At various times, total RNA was extracted from the liver. The concentration of specific mRNA was quantitated by cellfree translation and by blot hybridization with cDNA. The mRNA for the increased plasma proteins serum amyloid A, alpha 1-acid glycoprotein, haptoglobin, and alpha-fibrinogen showed essentially the same time course of changes. The concentrations were maximal 24 hr after initiation of inflammation and then declined gradually during the following days. During the progression from acute to chronic inflammation, there was a switch from a predominant expression of the gene for alpha 1-acid glycoprotein-1 to one of alpha 1-acid glycoprotein-2. The prolonged inflammation had an inverse effect on the two major negative acute-phase reactants, albumin and major urinary proteins. The concentration of albumin mRNA was reduced to 50% after 24 hr of inflammation but returned to the control level within 5 days. The mRNA for major urinary proteins, however, were lowered within 2 to 5 days of inflammation to 10 to 20% and were maintained at that level. These results suggest that different regulatory factors, humoral and/or cellular, are involved in controlling the expression of the genes for positive and negative acute-phase plasma proteins during acute and chronic inflammation.     

Purification and properties of two different α1-protease inhibitors from mouse plasma

Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent M_r for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent m_r of 65,000 while the apparent m_r of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes.     

Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Human alpha-1-proteinase inhibitor(1) (alpha(1)-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. alpha(1)-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) alpha(1)-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-alpha(1)-PI), proposed as alternatives to pd-alpha(1)-PI products, have been under intensive investigation. For accurate determination of alpha(1)-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for alpha(1)-PI quantification and potency measurements. As a part of our multi-step research focused on alpha(1)-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-alpha(1)-PI and r-alpha(1)-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1-50 ng/ml) established on the bases of the following parameters: linearity (3-100 ng/ml, r(2)=0.995); accuracy (87.3-114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the alpha(1)-PI ELISA indicates that this assay can be used for monitoring concentration levels of alpha(1)-PI in multi-component biological matrices, based on the following: (a) quantification of r-alpha(1)-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of alpha(1)-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized alpha(1)-PI; (d) quantification of alpha(1)-PI in human serum; and (e) comparative quantification of alpha(1)-PI in commercially available products.     

Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.     

Molecular cloning of DNA complementary to rat alpha 2-macroglobulin mRNA

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein synthesized in the liver. Using an in vitro translation system coupled with solid-phase radioimmunoassay, alpha 2M mRNA activity was found to rise to a maximum level in 16-24 h after turpentine injection. Poly(A)+ RNA from turpentine-injected rat liver was converted to cDNA by the method of Okayama-Berg, and about 50,000 transformants were obtained. From these transformants, clones containing alpha 2M cDNA were selected using the following criteria: 1) alpha 2M cDNA should hybridize with synthetic oligonucleotides encoding portions of the alpha 2M amino acid sequence, 2) alpha 2M cDNA should hybridize preferentially with RNA which increases during inflammation, 3) mRNA which hybridizes with alpha 2M cDNA should encode a polypeptide which specifically reacts with antibody against alpha 2M, and 4) the cDNA should contain the nucleotide sequences encoding the amino acid sequences of alpha 2M. We found clones which fulfilled these criteria. Using the cDNA clone as a probe, we demonstrated that the level of alpha 2M mRNA in the liver of inflamed animal markedly increased up to 1000-fold. The size of the alpha 2M mRNA was about 4800 nucleotides in length by Northern analysis.     

Characterization and hepatic expression of rat alpha 1-inhibitor III mRNA

A 2.5-kilobase cDNA clone (AF7), encoding 785 amino acids, was isolated from a rat liver cDNA library constructed in the expression vector lambda gt11. M13 vector sequence analysis yielded a deduced protein primary structure that was 89% homologous to the prototype alpha 1-inhibitor III (alpha 1I3) sequence presented in the preceding paper by Braciak et al. (Braciak, T. A., Northemann, W., Hudson, G. O., Shields, B. R., Gehring, M. R., and Fey, G. H. (1988) J. Biol. Chem. 263, 3999-4012) with regard to exact matches and 92% homologous when considering chemically conserved residues. The clone also possessed 100% homology to the putative bait region of a variant clone (pRLA1I3/27J) of alpha 1I3. Such sequence data demonstrates that the AF7 clone corresponds to a member of the family of variant alpha 1I3 mRNAs. Furthermore, this report presents the entire mRNA sequence corresponding to the 3'-half of alpha 1I3 variant 27J. We have utilized AF7 cDNA to study the expression of alpha 1I3 messenger RNA encoding this liver-specific glycoprotein under conditions known to alter hepatic gene expression. Our data reveal that alpha 1I3 mRNA expression is not only regulated during the acute-phase response but is also modulated in response to a variety of changing physiological conditions, most notably liver development. Steady state levels of mRNA were quantified using Northern blot techniques and laser densitometry. During acute phase response initiated by turpentine injection, the relative abundance of alpha 1I3 mRNA decreased 4-5-fold over a period of 24 h. Following partial hepatectomy, the regenerating liver expressed six-fold less alpha 1I3 mRNA than untreated liver after 24 h. This reduced level was maintained over a 2-day period. We have also demonstrated that alpha 1I3 mRNA expression is developmentally regulated. Fetal rat liver did not contain detectable concentrations of rat alpha 1I3 mRNA even as late as 4 days prior to birth. However, trace amounts were observed from birth until approximately 20 days of age when alpha 1I3 mRNA levels increased 10-fold to maximal adult quantities over the following 2 or 3 weeks. During the course of pregnancy, alpha 1I3 mRNA remained essentially constant until approximately 4 days prior to birth when a precipitous decline to 40% of the original level was noted. Subsequently, normal values were gradually restored over a 30-day postpartum period.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.     

Interleukin-6 regulates hepatic transporters during acute-phase response

Cholestasis develops during inflammatory conditions characterized by the release of cytokines like interleukin-6 (IL-6), which is the major player in the hepatic acute-phase response. However, the exact contribution of IL-6 to transporter down-regulation is unclear. Therefore, we compared wild-type and IL-6-deficient mice after IL-6-injection and induction of an aseptic (turpentine-injection) or septic (LPS-injection) acute-phase response. Down-regulation of basolateral (Ntcp, Oatp1, and Mrp3) and canalicular (Mrp2, Bsep) transporter mRNA occurred after treatment with IL-6, turpentine, and LPS. In IL-6-deficient mice, turpentine failed to decrease mRNA-levels of basolateral and canalicular transporters, whereas LPS-mediated down-regulation of Ntcp, Mrp3, and Mrp2 was abolished at later time points (24 h). In conclusion, induction of an aseptic and septic acute-phase response leads to the down-regulation of basolateral and canalicular organic anion transporters. IL-6 is required for transporter down-regulation during aseptic inflammation. Furthermore, IL-6 also contributes to transporter regulation during LPS-induced cholestasis at more delayed time points.     

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.     

Induction of C-reactive protein, serum amyloid P component, and kininogens in the submandibular and lacrimal glands of rats with experimentally induced inflammation.

The mRNAs for acute-phase proteins and kininogens were found to be increased in the submandibular gland (SMG) and extraorbital and intraorbital lacrimal gland (ELG and ILG) in response to experimentally induced inflammation in rats; i.e., 24 hours after subcutaneous injection of turpentine oil, mRNAs for C-reactive protein (CRP), serum amyloid P component (SAP), and H- and T-kininogens were induced in the SMG, ELG, and ILG of rats, whereas these mRNAs were not detected in the same tissues of normal control rats. The induction of mRNAs for these inflammatory proteins by turpentine oil was preceded by a transient increase in the level of mRNA for tumor necrosis factor-alpha (TNF-alpha) at 6 hours after subcutaneous injection of the oil. This was confirmed by injection of another inflammation inducer, lipopolysaccharide (LPS), which induced the TNF-alpha mRNA in the same way at 6 hours as turpentine oil did. The up-regulation of acute-phase proteins including kininogens in the SMG, ELG, and ILG suggest the existence of a strict defense system in the exocrine glands.     

Cell-free synthesis of rat alpha 2-macroglobulin and induction of its mRNA during experimental inflammation

Poly(A)-rich RNA was isolated from the livers of acutely inflamed rats by extraction with guanidinium HCl and oligo(dT)-cellulose chromatography. After translation in a recticulocyte lysate and immunoprecipitation with a specific antiserum to alpha 2-macroglobulin a polypeptide with an apparent molecular weight of 162000 could be detected. The cell-free synthesis of alpha 2-macroglobulin was stimulated 8-fold by the addition of RNase inhibitor. Full-length alpha 2-macroglobulin polypeptide chains appeared after 35 min in the presence of 1.85 mM Mg2+ and 100 mM K+. A nucleotide number of about 5100 was estimated for alpha 2-macroglobulin by means of sucrose gradient centrifugation of poly(A)-rich RNA followed by translation in vitro and immunoprecipitation of alpha 2-macroglobulin. In normal liver alpha 2-macroglobulin mRNA represented about 0.0007% of total translatable RNA. Acute inflammation generated by intramuscular injection of turpentine led to a 66-fold increase in translatable alpha 2-macroglobulin mRNA after 18 h, followed by a rapid decrease. In accordance to the induction of alpha 2-macroglobulin mRNA serum concentrations of alpha 2-macroglobulin increased to about 2 mg/ml. Unlike alpha 2-macroglobulin mRNA serum alpha 2-macroglobulin levels remained unchanged up to 60 h.