Changes of hepatic lactoferrin gene expression in two mouse models of the acute phase reaction

Lactoferrin (Ltf), an iron binding glycoprotein, is a pleiotropic molecule whose serum concentration increases under acute phase conditions. The physiological roles of this protein have been well elucidated, but the source and serum regulation of Ltf gene expression have not been investigated in detail as part of the acute phase reaction (APR). In the current work, the changes in hepatic Ltf-gene-expression during turpentine oil- (TO-) or LPS-induced APR were investigated. Ltf was upregulated at both the mRNA and protein levels in the liver of TO- and LPS-treated wild type (WT) mice. The pattern of induction however was different in both animal models indicating distinctive signalling patterns resulting in an acute phase reaction. Cytokines are the core regulators of APR. Among the major cytokines, IL-6 is an important signalling molecule, which also regulates iron homeostasis in response to an inflammatory situation. In this study, the administration of IL-6 induced Ltf gene expression in the liver of WT mice, in murine hepatocytes and in hepa 1-6 cells. Ltf-gene-expression was upregulated also in the liver of TO- and LPS-treated IL-6 knockout (KO) mice. The increase in serum Ltf after LPS injection was greater than after TO-injection both in WT and IL-6-KO mice. To evaluate the contribution of other acute phase cytokines in the regulation of Ltf-gene-expression in the liver, both in vitro and in vivo studies with IL-1β, TNF-α, or IFN-γ were performed. The results demonstrate that TNF-α and IFN-γ also upregulated Ltf-gene-expression, while IL-1β has no role in the regulation of Ltf-gene-expression.     

The acute phase response of Atlantic cod (Gadus morhua): humoral and cellular response

Intra-muscular injection of turpentine oil was used to induce acute phase response (APR) in Atlantic cod (Gadus morhua L.). The effects on the serum cortisol, total protein, IgM and pentraxin concentration were examined as well as the effects on natural antibody, anti-trypsin and leukocyte respiratory burst activity. The turpentine injection resulted in a 26 fold increase in the cortisol level after 72 h. Slightly reduced serum protein level in both groups was attributed to the restricted feeding during the experimental period. The IgM serum concentration was significantly reduced after 168 h in the turpentine treated fish while the natural antibody activity was not affected. The anti-trypsin activity was initially suppressed but recovered to normal levels at the end of the experiment. The turpentine injection had little effect on the serum level of the pentraxins, CRP-PI and CRP-PII. The respiratory burst activity was significantly suppressed after 72 h. It is concluded that 1) cod shows a relatively slow humoral and cellular response to APR induction, 2) the increase in serum cortisol level may be the key modulator of the mainly suppressive effects on the immune parameters and 3) pentraxins are not typical acute phase proteins in cod.     

Serum alpha2-macroglobulin and cytokine measurements in an acute inflammation model in rats

An enzyme-linked immunosorbent assay (ELISA) for rat alpha2-macroglobulin (alpha2M) using a monoclonal antibody was developed, and used to measure alpha2M in sera from rats injected intramuscularly with turpentine oil as an inflammatory agent. The mean concentration of alpha2M gradually increased and peaked 2 days after the turpentine oil injection. The peak alpha2M concentration ranged from 2362-8472 microg/ml (mean 4531 microg/ml), which was 50-290 times higher than the pre-dosing levels of 23-61 microg/ml. In addition, interleukins (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon (IFN)-gamma were measured using commercial ELISA reagent kits. IL-6 and IL-8 increased and peaked 12 h after turpentine oil injection, the levels being 5-51 times and 2-38 times the pre-dosing ones, respectively. The concentrations of IL-1, IL-2, IL-4, IL-10 and IFN-gamma in rats injected with turpentine oil did not change.     

Suppression of IL-2-induced SAA gene expression in mice by the administration of an IL-1 receptor antagonist

The hepatic acute phase response induced by the administration of interleukin (IL)-2 is most likely mediated by secondary cytokines. In this investigation, we examined the role of endogenous IL-1 in the synthesis of the hepatic acute phase protein serum amyloid A (SAA) during IL-2 treatment. The injection of IL-2 induced SAA gene expression in the liver. The concurrent administration of an IL-1 receptor antagonist (IL-1RA) markedly reduced hepatic SAA mRNA levels and, to a lesser extent, SAA protein levels in the serum. Although IL-1 is an inducer of IL-6 production, the administration of the IL-1RA had no effect on circulating IL-6 levels in IL-2-treated mice. These findings suggest that the production of IL-1 is an important factor in the induction of SAA mRNA in mice undergoing immunotherapy with IL-2.     

CCAAT enhancer-binding protein alpha is required for interleukin-6 receptor alpha signaling in newborn hepatocytes

The acute phase response is an evolutionarily conserved response of the liver to inflammatory stimuli, which aids the body in host defense and homeostasis. We have previously reported that CCAAT enhancer-binding protein alpha (C/EBPalpha) is required for the induction of acute phase protein (APP) genes in newborn mice in response to lipopolysaccharide. In this paper, we describe a mechanism by which C/EBPalpha knock-out mice are unable to induce APP gene expression in response to inflammatory stimuli. We demonstrate that the lack of acute phase response in C/EBPalpha knock-out mice is because of a hepatocyte autonomous defect. C/EBPalpha knock-out hepatocytes do not activate STAT3 in response to recombinant interleukin (IL)-6, indicating a defect in the IL-6 pathway. C/EBPalpha knock-out hepatocytes also do not show activation of other IL-6 receptor (IL-6R)-mediated Janus kinase substrates, gp130, SHP-2, and Tyk2. Further examination of the IL-6 pathway demonstrated that C/EBPalpha knock-out hepatocytes have decreased IL-6Ralpha protein levels caused, in part, by reduced protein stability. However, other components of the IL-6 pathway are intact, as demonstrated by rescue of STAT3 activation and APP gene induction with recombinant-soluble IL-6R linked to IL-6 cytokine (Hyper-IL-6) or with another gp130 signaling cytokine, Oncostatin M. In conclusion, C/EBPalpha is required for the proper regulation of IL-6Ralpha protein in hepatocytes resulting in a lack of acute phase protein gene induction in newborn C/EBPalpha null mice in response to lipopolysaccharide or cytokines.     

Involvement of the acute phase protein alpha 1-acid glycoprotein in nonspecific resistance to a lethal gram-negative infection

Resistance to gram-negative infection can be induced by pretreating animals with several agents such as turpentine and interleukin (IL)-1. Because these agents are powerful inducers of acute phase proteins, we wondered whether these proteins, more particularly alpha(1)-acid glycoprotein (alpha(1)-AGP), are involved in nonspecific resistance to infection. Turpentine and IL-1 protect completely against a lethal challenge of Klebsiella pneumoniae when given 48 and 12-48 h before the challenge, respectively. alpha(1)-AGP induction in the serum reached peak values 48 h after turpentine and 12-48 h after IL-1 injection. Administration of alpha(1)-AGP, 2 h before a challenge of K. pneumoniae, significantly increased the survival. Numbers of bacteria cultured from blood and organs were significantly lower in mice pretreated with a protective dose of turpentine, IL-1, or alpha(1)-AGP. These data suggest that alpha(1)-AGP is a possible mediator in turpentine- or IL-1-induced protection because time points of maximal induction of alpha(1)-AGP by turpentine or IL-1 and of optimal protection by alpha(1)-AGP coincide. Transgenic overexpression of rat alpha(1)-AGP protected mice from a K. pneumoniae infection. Bacterial counts in blood and organs were significantly lower in transgenic mice, and only in control mice were large necrotic areas, apoptosis, and blood clots observed in the spleen. Our data suggest that alpha(1)-AGP prevents gram-negative infections and may be an essential component in nonspecific resistance to infection.     

The acute phase response of cod (Gadus morhua L.): expression of immune response genes

An acute phase response (APR) was experimentally induced in Atlantic cod (Gadus morhua L.) by intramuscular injection of turpentine oil. The change in the expression of immune related genes was monitored in the anterior kidney and the spleen over a period of 7 days. The genes examined were two types of pentraxins, apolipoprotein A1 (ApoA-I), the complement component C3, interleukin-1β (IL-1β), transferrin, cathelicidin, and hepcidin. All genes were constitutively expressed in both organs and their expression amplified by the turpentine injection. A pattern of response was observed both with respect to the organ preference and to the timing of a maximum response. The increased gene expression of the pentraxins, ApoA-I and C3 was restricted to the anterior kidney, the gene expression of IL-1β, cathelicidin, and transferrin increased in both organs, while hepcidin gene expression was only significantly increased in the spleen. The pentraxins and ApoA-I appear to be early mediators of APR in cod, possibly stimulating C3 and IL-1β response, while the antimicrobial peptides may play a minor role. The increase in transferrin gene expression in both organs, and apparent indifference to cortisol release associated with the turpentine injection, suggests that this could be a typical acute phase protein in cod.     

Modulation of the acute phase response by altered expression of the IL-1 type 1 receptor or IL-1ra

A complete understanding of the role for endogenously produced interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) in the acute phase response to inflammation remains unknown. In the present studies, knockout mice lacking either a functional IL-1 type I receptor (IL-1RI(-/-)), a TNF type I receptor (TNFR-I(-/-)), or both IL-1 type I and TNF type I receptors (IL-1RI(-/-)/TNFR-I(-/-)) received a turpentine abscess. Additional mice deficient in IL-1ra protein (IL-1ra(-/-)) or overexpressing IL-1ra protein (IL-1ra(tg)) were similarly treated. After a turpentine abscess, IL-1 receptor knockout mice exhibited an attenuated inflammatory response compared with wild-type or animals lacking a functional TNFR-I. Mice overexpressing IL-1ra also had an attenuated hepatic acute phase protein response, whereas IL-1ra knockout mice had a significantly greater hepatic acute phase response. We conclude that the inflammatory response to a turpentine abscess is the result of a balance between IL-1ra expression and IL-1 binding to its type I receptor. Endogenously produced IL-1ra plays a central role in mitigating the magnitude of the IL-1-mediated inflammatory response and, ultimately, the outcome to a turpentine abscess.     

Acute phase protein response in the capybara (Hydrochoerus hydrochaeris)

We evaluated the acute phase protein response in capybaras (Hydrochoerus hydrochaeris). Three animal groups were used: 1) healthy animals (n=30), 2) a group in which experimental inflammation with turpentine was induced (n=6), and 3) a group affected with sarcoptic scabies (n=14) in which 10 animals were treated with ivermectin. Haptoglobin (Hp), acid-soluble glycoprotein (ASG) and albumin were analyzed in all animals. In those treated with turpentine, Hp reached its maximum value at 2 wk with a 2.7-fold increase, whereas ASG increased 1.75-fold and albumin decreased 0.87-fold 1 wk after the induction of inflammation. Capybaras affected with sarcoptic scabies presented increases in Hp and ASG of 4.98- and 3.18-fold, respectively, and a 0.87-fold decrease in albumin, compared with healthy animals. Haptoglobin and ASG can be considered as moderate, positive acute phase proteins in capybaras because they showed less than 10-fold increases after an inflammatory process and reached their peak concentrations 1 wk after the induction of inflammation. Conversely, albumin can be considered a negative acute phase protein in capybaras because it showed a reduction in concentration after inflammatory stimulus.     

Regulation of rabbit acute phase protein biosynthesis by monokines.

We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.     

Expression of the phospholipid scramblase (PLSCR) gene family during the acute phase response

Phospholipid scramblase 1 (PLSCR1) is a member of PLSCR gene family that has been implicated in multiple cellular processes including movement of phospholipids, gene regulation, immuno-activation, and cell proliferation/apoptosis. In the present study, we identified PLSCR1 as a positive intracellular acute phase protein that is upregulated by LPS in liver, heart, and adipose tissue, but not skeletal muscle. LPS administration resulted in a marked increase in PLSCR1 mRNA and protein levels in the liver. This stimulation occurred rapidly (within 2 h), and was very sensitive to LPS (half-maximal response at 0.1 microg/mouse). Moreover, two other APR-inducers, zymosan and turpentine, also produced significant increases in PLSCR1 mRNA and protein levels, indicating that PLSCR1 was stimulated in a number of models of the APR. To determine signaling pathways by which LPS stimulated PLSCR1, we examined the effect of proinflammatory cytokines in vitro and in vivo. TNFalpha, IL-1beta, and IL-6 all stimulated PLSCR1 in cultured Hep B3 hepatocytes, whereas only TNFalpha stimulated PLSCR1 in cultured 3T3-L1 adipocytes, suggesting cell type-specific effects of cytokines. Furthermore, the LPS-stimulated increase in liver PLSCR1 mRNA was greatly attenuated by 80% in TNFalpha and IL-1beta receptor null mice as compared to wild-type controls. In contrast, PLSCR1 levels in adipose tissue were induced to a similar extent in TNFalpha and IL-1beta receptor null mice and controls. These results indicate that maximal stimulation of PLSCR1 by LPS in liver required TNFalpha and/or IL-1beta, whereas the stimulation of PLSCR1 in adipose tissue is not dependent on TNFalpha and/or IL-1beta. These data provide evidence that PLSCR1 is a positive intracellular acute phase protein with a tissue-specific mechanism for up-regulation.     

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.     

Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein.

The human acute phase protein, C-reactive protein (CRP), is capable of specifically binding to and modulating the function of mononuclear phagocytes. To investigate whether CRP can also affect the capacity of these cells to produce inflammatory cytokines, enzyme immunoassays and Western blot techniques were used to quantitate interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) produced by freshly-isolated normal human monocytes. CRP induced the rapid release of each cytokine, with significantly elevated levels in culture supernatants at 4 hours and maximal levels of TNF-alpha at 8 hours, and of IL-1 beta and IL-6 at 16 hours of culture. The effects of CRP were dose-dependent; greater than 10-fold increases of each cytokine were observed following culture with greater than or equal to 50 micrograms/ml CRP, concentrations which are often found in the presence of moderate to severe inflammation or tissue injury. The induction of cytokine release by CRP was unaffected by inclusion of 25 micrograms/ml polymyxin-B in culture media, but was completely abrogated by prior boiling of the CRP, a procedure which had no effect on induction of monocyte cytokine release by lipopolysaccharide. The dose-dependent induction of inflammatory cytokines by CRP provides further support for the hypothesis that interaction with mononuclear phagocytes constitutes an important biological role for this acute phase protein.     

Effect of turpentine oil on C-reactive protein (CRP) production in rainbow trout (Oncorhynchus mykiss)

The effect of turpentine oil on C-reactive protein (CRP) production was studied in rainbow trout (Oncorhynchus mykiss). Serum CRP concentration was estimated by sandwich enzyme-linked immunosorbent assay using anti-rainbow trout CRP monoclonal antibody (mAb) AC4 and polyclonal antibody. Intracellular CRP was demonstrated by flow cytometry using anti-trout CRP mAb. Hepatocytes, head kidney macrophages, spleen lymphocytes and peripheral blood lymphocytes showed reaction against AC4, but RTG-2 fibroblastic line cells, derived from rainbow trout gonad did not. This is the first report on the detection of intracellular CRP in fish. CRP levels decreased significantly 1 day after intramuscular injection of turpentine oil and remained low for 14 days. Significant decreases in the expression of CRP in hepatocytes, head kidney macrophages and spleen lymphocytes after injection of turpentine oil were found. The reduction of serum CRP concentration after turpentine oil injection may be attributed to decreases in intracellular CRP synthesis.     

Identification of alpha 2-macroglobulin as a carrier protein for IL-6

In this report we demonstrate that alpha 2-macroglobulin (alpha 2M) is a carrier protein for IL-6. IL-6 was found to bind plasma proteins and an immunoblot analysis revealed that the complex between IL-6 and plasma proteins contains alpha 2M. Furthermore, purified alpha 2M bound IL-6. alpha 2M did not inhibit IL-6 activity or its binding to homologous receptor. IL-6 bound to alpha 2M retained its biologic activity and became resistant to treatment with proteases, although free IL-6 was easily degraded. These findings indicate that alpha 2M plays an important role as a carrier protein for IL-6 in serum and makes IL-6 produced at the local inflammatory site available to lymphocytes, hepatocytes, and hematopoietic stem cells, resulting in the induction of the coordinate systemic host defense reactions, such as immune response, acute phase reaction, and hematopoiesis.     

Implication of the hepatokine,fibrinogen-like protein 1 in liver diseases,metabolic disorders and cancer: The need to harness its full potential

Fibrinogen-like protein 1 (FGL1) is a novel hepatokine that forms part of the fibrinogen superfamily. It is predominantly expressed in the liver under normal physiological conditions. When the liver is injured by external factors, such as chemical drugs and radiation, FGL1 acts as a protective factor to promote the growth of regenerated cells. However, elevated hepatic FGL1 under high fat conditions can cause lipid accumulation and inflammation, which in turn trigger the development of non-alcoholic fatty liver disease, diabetes, and obesity. FGL1 is also involved in the regulation of insulin resistance in adipose tissues and skeletal muscles as a means of communication between the liver and other tissues. In addition, the abnormally changed FGL1 levels in the plasma of cancer patients make it a potential predictor of cancer incidence in clinical practice. FGL1 was recently identified as a major functional ligand of the immune inhibitory receptor, lymphocyte-activation gene 3 (LAG3), thus making it a promising target for cancer immunotherapy except for the classical programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis. Despite the potential of FGL1 as a new cancer biomarker and therapeutic target, there are few related studies and much of what has been reported are superficial and lack depth and particularity. Therefore, elucidating the role and underlying mechanisms of FGL1 could be crucial for the development of promising diagnostic and therapeutic strategies for related diseases. Here, we provide a comprehensive review of the cellular mechanisms and clinical prospects of FGL1 in the prevention and treatment of liver diseases, metabolic disorders and cancer, and proffer suggestions for future studies.     

Expression of c-jun and Egr-1 genes in rat liver in response to partial hepatectomy, and injection of turpentine.

In rat liver stimulated by partial hepatectomy a significant expression of Egr-1 and to less extent c-jun genes was observed within 15-30 min after the surgery. The induction is transient and vanishes within one hour. Egr-1 and c-jun were also induced in liver after subcutaneous injection of turpentine, a strong inducer of acute phase response. A maximum activation of these genes was observed within 4-9 hours after administration of turpentine. Both Egr-1 and c-jun extend the list of "immediate early" genes involved in proliferative response and constitute a group of genes inducible at the beginning of acute phase response.