Acute-phase Proteins Alpha1-antitrypsin Research Articles

Clinical Significance of SERPINA1 Gene and Its Encoded Alpha1-antitrypsin Protein in NSCLC.

High expression of SERPINA1 gene encoding acute phase protein, alpha1-antitrypsin (AAT), is associated with various tumors. We sought to examine the significance of SERPINA1 and AAT protein in non-small-cell lung cancer (NSCLC) patients and NSCLC cell lines. Tumor and adjacent non-tumor lung tissues and serum samples from 351 NSCLC patients were analyzed for SERPINA1 expression and AAT protein levels. We also studied the impact of SERPINA1 expression and AAT protein on H1975 and H661 cell behavior, in vitro. Lower SERPINA1 expression in tumor but higher in adjacent non-tumor lung tissues (n = 351, p = 0.016) as well as higher serum levels of AAT protein (n = 170, p = 0.033) were associated with worse survival rates. Specifically, in NSCLC stage III patients, higher blood AAT levels (>2.66 mg/mL) correlated with a poor survival (p = 0.002). Intriguingly, levels of serum AAT do not correlate with levels of C-reactive protein, neutrophils-to-leukocyte ratio, and do not correlate with SERPINA1 expression or AAT staining in the tumor tissue. Additional experiments in vitro revealed that external AAT and/or overexpressed SERPINA1 gene significantly improve cancer cell migration, colony formation and resistance to apoptosis. SERPINA1 gene and AAT protein play an active role in the pathogenesis of lung cancer and not just reflect inflammatory reaction related to cancer development.

Genotyping diagnosis of alpha-1 antitrypsin deficiency in Saudi adults with liver cirrhosis.

The acute phase protein alpha-1 antitrypsin (AAT) is mainly produced in liver cells. AAT deficiency affects the lungs and liver. We conducted a case-control study to define a valuable method for the proper diagnosis of alpha-1 antitrypsin deficiency (AATD), as well as the association of liver cirrhosis with AATD in Saudi adults.Blood samples from 300 liver cirrhosis patients and 400 controls were analyzed according to serum AAT concentration, phenotyping, and genotyping. Nephelometry was used for AAT quantification, isoelectric focusing electrophoresis was used for phenotyping detection, and real-time PCR was used for genotyping to determine the Z and S deficiency alleles.This study highlights the accuracy of using genotyping in addition to AAT quantification, since this technique has proven to be successful in the diagnosis of AATD for 100% of our cases. A significant deviation in AAT genotypes frequencies from the Hardy–Weinberg equilibrium in the adult cirrhosis group occurred due to a higher observed frequency than expected for the Pi ZZ homozygous genotype.Pi ZZ in adults may be considered as the risk factor for liver cirrhosis. However, we could not establish this relationship for heterozygous AATD genotypes (such as Pi MZ and Pi SZ).

Novel anti-inflammatory effects of alpha-1-antitrypsin on lung microvascular endothelial cells

The acute phase protein alpha-1-antitrypsin (AAT) is released into the circulation at high concentrations (1.3–2mg/ml). The knowledge that an inherited AAT-deficiency increases susceptibility to chronic inflammatory conditions and that AAT is one of the major inhibitors of neutrophil proteases, suggests an important role for AAT in controlling acute phase inflammation and preventing development of chronic inflammation and tissue damage. Recent studies have also shown that exogenous AAT can be internalized by endothelial cells and suppresses cell apoptosis by inhibiting caspases-1 and 3. To further investigate the effects of AAT on endothelial cells, we first examined its influence on release of pro-inflammatory cytokine, TNFα. Primary lung microvascular endothelial cells were stimulated for 4 to 8h with TNFα in the presence or absence of human plasma derived preparation of AAT (Prolastin®, 2mg/ml). Under these experimental conditions, AAT clearly reduced TNFα concentrations in cell supernatants (61±11%) as compared to cells stimulated with TNFα alone. We next focused on the intracellular calcium-dependent calpains which are known to enhance leukocyte-endothelium interactions and endothelial dysfunction. Pre-incubation of endothelial cells for 1 hour with AAT (2mg/ml), efficiently reduced calpain activity (up to 53±8% of basal levels). Thus, our results give further evidence for anti-inflammatory activities of AAT and reveal a previously unknown function for AAT, namely to inhibit calpains in endothelial cells. Calpain activity is implicated in numerous pathological conditions including Alzheimer's disease, demyelination events of multiple sclerosis, neuronal damage after spinal cord injury and hypoxic/ischaemic injury to brain, kidney and heart organs. Therefore, the property of AAT to regulate calpain activity may be important in a wide array of pathological states.

Induction of alpha1-antitrypsin synthesis in human articular chondrocytes by interleukin-6-type cytokines: evidence for a local acute-phase response in the joint.

We have previously shown that human articular chondrocytes synthesize large amounts of interleukin-6 (IL-6) upon stimulation with proinflammatory cytokines and that they express the IL-6 receptor. The present study was undertaken to analyze whether different IL-6-type cytokines can induce synthesis of the acute-phase protein alpha1-antitrypsin in human articular chondrocytes. Chondrocytes from human articular cartilage, cultured in agarose, were stimulated with IL-6-type cytokines. Total RNA was isolated and analyzed by Northern blotting. Levels of alpha1-antitrypsin protein were determined by enzyme immunoassay. Stimulation of chondrocytes with oncostatin M (OSM) and IL-6 led to a 5-10-fold increase in alpha1-antitrypsin synthesis. This increase was dose and time dependent. Furthermore, OSM and IL-6 induced IL-6 synthesis in chondrocytes, resulting in an autocrine amplification loop. Our data strongly suggest the existence of a local acute-phase response in the joint. Synthesis of the acute-phase protein alpha1-antitrypsin, a major inhibitor of serine proteinases, may be an important protective mechanism of articular chondrocytes to prevent cartilage damage in inflammatory joint diseases.

Translational enhancement of H-ferritin mRNA by interleukin-1 beta acts through 5' leader sequences distinct from the iron responsive element.

Interleukin-1 beta (Il-1 beta), a key cytokine in the acute phase response, elevates hepatic expression of both the heavy (H) and light (L) ferritin subunits without influencing the steady-state levels of either ferritin transcript. Transfection experiments with human hepatoma cells reveal that sequences within the 5' untranslated region (5'UTR) of H-ferritin mRNA confer translational regulation to chimaeric chloramphenicol acetyl transferase (CAT) mRNAs in response to Il-1 beta in the absence of marked changes in CAT mRNA levels. Il-1 beta dependent translational enhancement is mediated by a distinct G + C rich RNA sequence within 70 nucleotides (nt) of the start codon. The upstream Iron Responsive Element RNA stemloop does not confer increased expression to CAT mRNA in Il-1 beta stimulated hepatoma transfectants. A 38 nucleotide consensus sequence within the 5'UTRs of the mRNAs encoding the hepatic acute phase proteins alpha 1-antitrypsin (alpha 1AT), alpha 1-acid glycoprotein (AGP) and haptoglobin (Dente et al., 1985) is similar to sequences in the G + C rich H-ferritin mRNA translational regulatory element. Deletion of three nucleotides from this region of the 61 nt G + C rich element in the H-ferritin mRNA 5' leader eliminates Il-1 beta translational enhancement of the CAT reporter transcripts.

The acute-phase protein alpha 1-antitrypsin inhibits growth and proliferation of human early erythroid progenitor cells (burst-forming units-erythroid) and of human erythroleukemic cells (K562) in vitro by interfering with transferrin iron uptake

We have previously shown that the hepatic acute-phase protein alpha 1- antitrypsin (alpha 1-AT) inhibits transferrin (tf) binding to its receptor (tfR) of human placental membranes. To evaluate the possibility that this interaction can explain the pathophysiology of the changes in iron metabolism in the course of chronic disease, subsequently leading to anemia in chronic disease (ACD), we examined the effect of alpha 1-AT on cells of the erythroid cell line. alpha 1- AT completely prevented tf binding to tfR on K562 human erythroleukemic cells and on reticulocytes. This inhibitory potency was dose-dependent and competitive, as proved in equilibrium saturation and kinetic studies. The cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha showed no such effect. Internalization of the tf-tfR complex was inhibited with alpha 1-AT in a dose-dependent manner. Furthermore, alpha 1-AT profoundly reduced the growth of K562 cells as well as their proliferation, albeit to a lesser degree. Growth of early erythroid progenitor cells (burst-forming units-erythroid) was significantly suppressed by alpha 1-AT, but no effect on the growth of late erythroid progenitor cells (colony-forming units-erythroid) was detected. These inhibitions of alpha 1-AT were seen in high physiologic concentrations attained in the course of acute-phase situations. These data show that alpha 1-AT might be a mediator of the changes in iron metabolism that are characteristic of clinical findings in the course of ACD.

The hepatic acute-phase proteins alpha 1-antitrypsin and alpha 2-macroglobulin inhibit binding of transferrin to its receptor.

Transferrin binding to human placental sites was inhibited by the acute-phase proteins alpha 1-antitrypsin (alpha 1-AT) and alpha 2-macroglobulin (alpha 2-MG), whereas haptoglobin, C-reactive protein and ferritin displayed no such effect. In equilibrium saturation binding assays, the effective acute-phase proteins decreased the apparent affinity of the binding sites for transferrin, but the transferrin binding-site density Bmax. was not significantly changed. For instance, the addition of 30 microM alpha 1-AT increased the KD of transferrin from 8.46 +/- 1.51 nM to 21.6 +/- 3.04 nM; the Bmax. values were 1.17 +/- 0.18 pmol/mg of protein and 1.04 +/- 0.25 pmol/mg of protein respectively. In kinetic studies, alpha 1-AT decreased the association rate constant k+1 of the 125I-transferrin-binding-site complex from 2.18(+/- 0.21) x 10(7) M-1.min-1 to 3.99(+/- 0.18) x 10(6) M-1.min-1. In contrast, the dissociation rate constant k-1 was not changed (0.0948 +/- 0.002 min-1, 0.089 +/- 0.0017 min-1). On isoelectric focusing, no alteration in transferrin protein pattern or shift in isoelectric point was detected in the presence of alpha 1-AT. Inhibition of transferrin binding by the acute-phase proteins alpha 1-AT and alpha 2-MG is competitive. Interestingly, inhibition is already present at physiological concentrations. However, full inhibition is only achieved at concentrations above the normal range, which are attained in acute-phase reactions.

Interferon beta 2/interleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear phagocytes and in human hepatoma cells.

The cytokine IFN beta 2/IL-6 has recently been shown to regulate the expression of genes encoding hepatic acute phase plasma proteins. INF beta 2/IL-6 has also been shown to be identical to MGI-2, a protein that induces differentiation of bone marrow precursor cells toward mature granulocytes and monocytes. Accordingly, we have examined the effect of IFN beta 2/IL-6 on expression of the IL-1- and tumor necrosis factor-unresponsive acute phase protein alpha 1-antitrypsin (alpha 1 AT) in human hepatoma-derived hepatocytes and in human mononuclear phagocytes. Purified human fibroblast and recombinant IFN beta 2/IL-6 each mediate a specific increase in steady-state levels of alpha 1 AT mRNA and a corresponding increase in net synthesis of alpha 1 AT in primary cultures of human peripheral blood monocytes as well as in HepG2 and Hep3B cells. Thus, the effect of IFN beta 2/IL-6 on alpha 1 AT gene expression in these cells is primarily due to an increase in accumulation of alpha 1 AT mRNA and can be distinguished from the direct, predominantly translational effect of bacterial lipopolysaccharide on expression of this gene in monocytes and macrophages. The results indicate that IFN beta 2/IL-6 regulates acute phase gene expression, specifically alpha 1 AT gene expression, in extrahepatic as well as hepatic cell types.

Angiotensinogen is an acute-phase protein in man

Accumulating information concerning the structure of angiotensinogen suggests a resemblance of this component of the renin-angiotensin system to the acute-phase protein alpha 1-antitrypsin. Compared to a group of age- and sex-matched controls without signs of infection, markedly elevated levels of angiotensinogen (increase in median value: 70%), alpha 1-antitrypsin (102%), caeruloplasmin (76%), haptoglobin (261%), and orosomucoid (162%) were found in plasma from 14 patients with acute inflammatory disease. This finding indicates that angiotensinogen should be included in the list of acute-phase proteins in man, and raises the question whether angiotensinogen is involved in the regulation of the renin-angiotensin system during inflammation and tissue injury.

Stress-metabolism after myocardial infarction-demonstrated by means of the behaviour of plasma proteins with short half-life (author's transl)

The kinetics of plasma proteins with short half-life during stress-metabolism in patients after myocardial infarction with and without clinical complications and after angina pectoris were compared. The acute-phase proteins alpha1-antitrypsin, C-reactive protein (CRP), fibrinogen, haptoglobin, and the transport proteins prealbumin and transferrin were analyzed with the method of radial immunodiffusion. Whereas angina pectoris doesn't influence the protein kinetics, one can recognize after myocardial infarction a continuous increase of the acute-phase proteins to maxima between the 3rd and 5th day after the attack. Parallel to these changes, the transport proteins decrease with subsequent increase. The changes, which are similar to those seen after surgical trauma, are dependent on the severity of illness, and can be used as prognostic parameters. During stress metabolism, the concentrations of the proteins depending on nutrition, prealbumin and transferrin, are modified by the type and severity of stress, and by nutritional influences. The mechanisms of these changes and the consequences for their use as diagnostic parameters are discussed.