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O -linked α- N -acetylgalactosamine (α-GalNAc) in the Gc protein is essential for macrophage activation; thus, the GalNAc-attached form of Gc protein is called Gc macrophage activating factor (GcMAF). O -linked glycans in Gc proteins from human plasma mainly consist of trisaccharides. GcMAF is produced when glycans on the Gc protein are hydrolyzed by α-Sia-ase and β-Gal-ase, leaving an α-GalNAc. Upon hydrolysis of α-GalNAc present on GcMAF, the protein loses the macrophage-activating effect. In contrast, our synthesized pyrrolidine-type iminocyclitol possessed strong in vitro α-GalNAc-ase inhibitory activity. In this study, we examined the protective effects of iminocyclitol against GcMAF via inhibition of α-GalNAc-ase activity. Detailed mass spectrometric analyses revealed the protective effect of the inhibitor on GcMAF. Furthermore, structural information regarding the glycosylation site and glycan structure was obtained using tandem mass spectrometric (MS/MS) analysis of the glycosylated peptides after tryptic digestion., Competing Interests: The authors declare no conflict of interest.

Human group-specific component protein (Gc protein) is a multifunctional serum protein which has three common allelic variants, Gc1F, Gc1S and Gc2 in humans. Gc1 contains an O-linked trisaccharide [sialic acid-galactose-N-acetylgalactosamine (GalNAc)] on the threonine 420 (Thr 420 ) residue and can be converted to a potent macrophage activating factor (GcMAF) by selective removal of sialic acid and galactose, leaving GalNAc at Thr 420 . In contrast, Gc2 is not glycosylated. GcMAF is considered a promising candidate for immunotherapy and antiangiogenic therapy of cancers and has attracted great interest, but it remains difficult to compare findings among research groups because different procedures have been used to prepare GcMAF. Here, we present a simple, practical method to prepare high-quality GcMAF by overexpressing Gc-protein in a serum-free suspension culture of ExpiCHO-S cells, without the need for a de-glycosylation step. We believe this protocol is suitable for large-scale production of GcMAF for functional analysis and clinical testing.

Background/aim: Serum-derived macrophage activating factor (serum-MAF) can rapidly activate macrophage phagocytic activity by inducing characteristic membrane ruffles designated as Frill-like structures. Serum-MAF contains γ-globulin, an activator of phagocytosis. This study examined whether serum-MAF and γ-globulin activate macrophages similarly., Materials and Methods: Morphological changes in macrophages were observed by time-lapse imaging and the efficiency of engulfment was analysed quantitatively. Immunological staining of talin-1 and a calpain inhibitor were performed., Results: The engulfment efficiency of serum-MAF- and γ-globulin-activated macrophages was significantly different. Talin-1 showed weak co-localisation with the Frill-like structures. Treatment with a calpain inhibitor similarly down-regulated phagocytosis irrespective of the activation factor., Conclusion: There was a difference between macrophage activation mechanisms by γ-globulin and serum-MAF. Talin may slightly contribute to serum-MAF activation. It is possible to distinguish between the calpain-dependent fundamental 'mechanism of phagocytosis' and the activating factor-dependent rapid 'activation mechanism'., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Serum-derived macrophage activating factor (serum MAF) is known to increase the phagocytic activity of macrophages and potentially plays a role in activating cancer immunity. In order to reveal the contributing factors for phagocytic activation, the migratory activity and the efficiency of engulfment was analyzed., Materials and Methods: THP-1 macrophages were induced by 12-O-tetradecanoyl-13-acetate (TPA). The migratory activity and efficiency of engulfment were analyzed by time-lapse imaging and suspension assay, respectively., Results: While the distance of migration did not change before and after activation with serum MAF, the efficiency of beads internalisation was significantly increased., Conclusion: Phagocytic activation of serum-MAF-treated macrophages was caused by increasing the efficiency of engulfment. This study contributes to the knowledge about the activation of the immune system through phagocytic activation of macrophages., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

In the constant battle against cancer cells, macrophages are of great importance. Their activation is achieved through various mechanisms such as Vitamin D binding protein (VDBP or Gc). After undergoing modifications via enzymes secreted by stimulated lymphocytes, VDBP is modified into Macrophages Activator Form/Factor (Gc-MAF). Some studies (particularly those focusing on cancer) have reported that an enzyme known as α-N-acetylgalactosaminidase (nagalase) facilitates the deglycosylation of Gc-MAF, which in turn inhibits the activation of macrophages. The aim of this review was to evaluate studies associated with nagalase and its escalation in various diseases and to propose hypothetical solutions in order to neutralize the effects of nagalase in cancer patients.

Background/aim: Macrophages are important components of human defense systems and consequently key to antitumor immunity. Human-serum macrophage activation factor (serum MAF) can activate macrophages, making it a promising reagent for anticancer therapy., Materials and Methods: We established four different macrophage subtypes through introduction of different culture conditions to THP-1- and U937-derived macrophages. We assessed phagocytic activity to understand subtype responses to typical macrophage activation factors (MAFs) and the activation mechanisms of serum MAF., Results: All four macrophage subtypes differed in their response to all MAFs. Moreover, serum MAF had two different activation mechanisms: N-acetylgalactosamine (GalNAc)-dependent and GalNAc-independent., Conclusion: Macrophage activation states and mechanisms are heterogeneous., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background/aim: Phagocytic activity is affected by a number of different stress and age-dependent factors. An easy measurement of phagocytic activity is thought to allow an indicator of an individual's health. In this study, we investigated conditions of measurement to easily evaluate the activity of phagocytosis of phagocytic cells (macrophages and neutrophils) using an easy-to-use prototype, which was improved from the device by Hamamatsu Photonics K.K., to detect neutrophil activity using subtle fluorescence., Materials and Methods: pH-sensitive fluorescent particles (pHrodo-Green E. coli Bio particles, GE particles) were added to mouse-derived macrophage cell lines (J774.1) and then incubated for 2 h at 37°C. For negative control, the phagocytosis inhibitor cytochalasin D (CyD), was added prior to culture. Next, fluorescence intensity was measured by the Prototype to evaluate the phagocytic activity of macrophages and neutrophils. Phagocytosis was also confirmed by flow cytometry., Results: The Prototype detected a steady fluorescence increase in 5 sec in J774.1 after phagocytosis, using GE particles as a negative control in the presence of CyD. Furthermore, detection was possible at 10(4) cells/test, a concentration where the flow cytometer had difficulty for detection., Conclusion: The Prototype enables measurement of the phagocytic activity within a short period of time, even with a small sample amount, thus establishing the basic conditions of measurements of phagocytosis., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background/aim: Gc protein-derived macrophage-activating factor (GcMAF) has various functions as an immune modulator, such as macrophage activation, anti-angiogenic activity and anti-tumor activity. Clinical trials of second-generation GcMAF demonstrated remarkable clinical effects in several types of cancers. Thus, GcMAF-based immunotherapy has a wide application for use in the treatment of many diseases via macrophage activation that can be used as a supportive therapy. Multiple sclerosis (MS) is considered to be an autoimmune disorder that affects the myelinated axons in the central nervous system (CNS). This study was undertaken to examine the effects of second-generation GcMAF in a patient with MS., Results: This case study demonstrated that treatments of GcMAF in a patient with MS have potent therapeutic actions with early beneficial responses, especially improvement of motor dysfunction., Conclusion: GcMAF shows therapeutic potency in the treatment of MS., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia., (© 2015 Wiley Periodicals, Inc.)

Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce the dual effect of reduction of oxaliplatin-induced neurotoxicity, together with possible synergism in the overall anticancer effect.

Lung diseases, including pneumonia and asthma, are among the most prevalent human disorders, and murine models have been established to investigate their pathobiology and develop novel treatment approaches. Whereas bronchoscopy is valuable for diagnostic and therapeutic procedures in patients, no equivalent for small rodents has been established. Here, we introduce a miniaturized video-bronchoscopy system offering new opportunities in experimental lung research. With an outer diameter of 0.75 mm, it is possible to advance the optics into the main bronchi of mice. An irrigation channel allows bronchoalveolar lavage and unilateral application of substances to one lung. Even a unilateral infection is possible, enabling researchers to use the contralateral lung as internal control.

Background: Macrophages play important roles in antitumor immunity, and immunotherapy with the group-specific component protein-derived macrophage-activating factor (GcMAF) has been reported to be effective in patients with various types of cancers. However, in macrophage research, it is important to properly evaluate macrophage activity., Materials and Methods: U937 macrophages were induced by 12-O-tetradecanoyl-13-phorbolacetate (TPA). The phagocytic activity of macrophages was evaluated as the internalized beads ratio. The MAF activity was assessed at 30 min after MAF addition as the activation ratio., Results: We established a novel assay for phagocytic activities using differentiated U937 macrophages., Conclusion: The novel protocol was simple and rapid and was sensitive for GcMAF. This protocol should be useful not only for basic studies, such as those on molecular mechanisms underlying macrophage activation, but also for clinical studies, such as assessment of GcMAF activity prior to clinical use., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Immune system dysregulation is well-recognized in autism and thought to be part of the etiology of this disorder. The endocannabinoid system is a key regulator of the immune system via the cannabinoid receptor type 2 (CB2R) which is highly expressed on macrophages and microglial cells. We have previously published significant differences in peripheral blood mononuclear cell CB2R gene expression in the autism population. The use of the Gc protein-derived Macrophage Activating Factor (GcMAF), an endogenous glycosylated vitamin D binding protein responsible for macrophage cell activation has demonstrated positive effects in the treatment of autistic children. In this current study, we investigated the in vitro effects of GcMAF treatment on the endocannabinoid system gene expression, as well as cellular activation in blood monocyte-derived macrophages (BMDMs) from autistic patients compared to age-matched healthy developing controls., Methods: To achieve these goals, we used biomolecular, biochemical and immunocytochemical methods., Results: GcMAF treatment was able to normalize the observed differences in dysregulated gene expression of the endocannabinoid system of the autism group. GcMAF also down-regulated the over-activation of BMDMs from autistic children., Conclusions: This study presents the first observations of GcMAF effects on the transcriptionomics of the endocannabinoid system and expression of CB2R protein. These data point to a potential nexus between endocannabinoids, vitamin D and its transporter proteins, and the immune dysregulations observed with autism.

Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1β, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.

Background: Group-specific component (Gc)-globulin-derived macrophage-activating factor (GcMAF) generated by a cascade of catalytic reactions with deglycosidase enzymes exerts antitumor activity. We hypothesized that degalactosyl Gc-globulin (DG3), a precursor of GcMAF, also plays a role in recovery from cancer as well as GcMAF due to progression of deglycosylation by generally resident sialidases and mannosidases., Materials and Methods: We prepared the subtypes of DG3, such as 1f1f and 1s1s and its 22 homodimers, by using vitamin D3-binding Sepharose CL-6B and examined their antitumor activity in mice bearing Lewis lung carcinoma cells, by counting the number of nodules formed in their lungs., Results: Antitumor activity of DG3 was observed regardless of its subtype, being equivalent to that of GcMAF. The injection route of DG3 affected its antitumor activity, with subcutaneous and intramuscular administration being more favorable than the intraperitoneal or intravenous route. In order to obtain significant antitumor activity, more than 160 ng/kg of DG3 were required., Conclusion: DG3 proved to be promising as an antitumor agent, similarly to GcMAF.

Background: The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity., Results: We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors., Conclusion: We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

Background: In addition to its role in calcium homeostasis and bone mineralization, vitamin D is involved in immune defence, cardiovascular function, inflammation and angiogenesis, and these pleiotropic effects are of interested in the treatment of chronic kidney disease. Here we investigated the effects of paricalcitol, a nonhypercalcemic vitamin D analogue, on human peripheral blood mononuclear cell proliferation and signaling, and on angiogenesis. These effects were compared with those of a known inhibitor of angiogenesis pertaining to the vitamin D axis, the vitamin D-binding protein-derived Gc-macrophage activating factor (GcMAF)., Methods: Since the effects of vitamin D receptor agonists are associated with polymorphisms of the gene coding for the receptor, we measured the effects of both compounds on mononuclear cells harvested from subjects harboring different BsmI polymorphisms., Results: Paricalcitol inhibited mononuclear cell viability with the bb genotype showing the highest effect. GcMAF, on the contrary, stimulated cell proliferation, with the bb genotype showing the highest stimulatory effect. Both compounds stimulated 3'-5'-cyclic adenosine monophosphate formation in mononuclear cells with the highest effect on the bb genotype. Paricalcitol and GcMAF inhibited the angiogenesis induced by proinflammatory prostaglandin E1., Conclusions: Polymorphisms of the vitamin D receptor gene, known to be associated with the highest responses to vitamin D receptor agonists, are also associated with the highest responses to GcMAF. These results highlight the role of the vitamin D axis in chronic kidney disease, an axis which includes vitamin D, its receptor and vitamin D-binding protein-derived GcMAF.

Background: A high incidence of recurrence after treatment is the most serious problem in hepatocellular carcinoma (HCC). Therefore, a new strategy for the treatment of the disease is needed. The aim of the present study was to investigate whether vitamin D binding protein-macrophage activating factor (DBP-maf) is able to inhibit the growth of HCC., Methods: The effects of DBP-maf on endothelial cells and macrophage were evaluated by WST-1 assay and phagocytosis assay, respectively. Human HCC cells (HepG2) were implanted into the dorsum of severe combined immunodeficiency (SCID) mice. These mice were divided into control and DBP-maf treatment groups (n = 10/group). The mice in the treatment group received 40 ng/kg/d of DBP-maf for 21 d., Results: DBP-maf showed anti-proliferative activity against endothelial cells and also activated phagocytosis by macrophages. DBP-maf inhibited the growth of HCC cells (treatment group: 126 ± 18mm(3), untreated group: 1691.5 ± 546.9mm(3), P = 0.0077). Histologic examinations of the tumors revealed the microvessel density was reduced and more macrophage infiltration was demonstrated in the tumor of mice in the treatment group., Conclusion: DBP-maf has at least two novel functions, namely, an anti-angiogenic activity and tumor killing activity through the activation of macrophages. DBP-maf may therefore represent a new strategy for the treatment of HCC., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The 1f1f subtype of the Gc protein (Gc(1f1f) protein) was converted into Gc-derived macrophage-activating factor (GcMAF) by enzymatic processing in the presence of β-galactosidase of an activated B-cell and sialidase of a T-cell. We hypothesized that preGc(1f1f)MAF, the only Gc(1f1f) protein lacking galactose, can be converted to GcMAF in vivo because sialic acid is cleaved by residual sialidase. Hence, we investigated the effect of preGc(1f1f)MAF on the phagocytic activation of mouse peritoneal macrophages., Results: We examined the sugar moiety of preGc(1f1f)MAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We also found that preGc(1f1f)MAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; the level of phagocytic activity was the same as that observed for GcMAF., Conclusion: PreGc(1f1f)MAF can be used as an effective macrophage activator in vivo.

The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

Transgenic Leishmania expressing fluorescent reporter proteins such as green fluorescent protein (GFP) have opened the way for a flow cytometry (FACS)-based method to assess the killing of Leishmania parasites inside their macrophage host. Compared with counting parasites in microscopic preparations, the assessment of anti-leishmanial effects by FACS analysis promises both strict objectivity and significant reduction of labour-per-sample while scanning thousands of cells within seconds. Compared with other semi-automated methods based on host cell lysis and biochemical quantification of released parasites, the procedure is more direct and simple, reducing handling artefacts. An assay system is described using highly pure murine bone marrow-derived macrophages infected in vitro as a suspension culture with GFP-transfected Leishmania major promastigotes. The cells were rested for 24 h, allowing intracellular promastigotes to transform into amastigotes, and then exposed to macrophage-activating agents (IFN-gamma, LPS) or standard anti-leishmanial therapeutics. Within 48 h, the GFP signal from parasitized macrophages became indiscernible by FACS analysis, both in activated host cells and in cultures treated with the anti-leishmanials. In cultures activated with rIFN-gamma+LPS this coincided with the release of nitric oxides, but this was not the case in cultures treated with anti-leishmanials. Furthermore, by adding propidium iodide immediately before FACS analysis, the effect of treatment on the viability of the host cell was assessed at the same time. The combination of FACS analysis, and PI and NO detection offers a rapid and objective means of testing for intracellular anti-leishmanial effects and general cytotoxicity and gives a first indication of whether the former is due to direct leishmanicidal activity or indirect functions via macrophage activation.

Rational protein design has been successfully used to create mimics of natural proteins that retain native activity. In the present work, de novo protein engineering is explored to develop a mini-protein analogue of Gc-MAF, a glycoprotein involved in the immune system activation that has shown anticancer activity in mice. Gc-MAF is derived in vivo from vitamin D binding protein (VDBP) via enzymatic processing of its glycosaccharide to leave a single GalNAc residue located on an exposed loop. We used molecular modeling tools in conjunction with structural analysis to splice the glycosylated loop onto a stable three-helix bundle (alpha3W, PDB entry 1LQ7). The resulting 69-residue model peptide, MM1, has been successfully synthesized by solid-phase synthesis both in the aglycosylated and the glycosylated (GalNAc-MM1) form. Circular dichroism spectroscopy confirmed the expected alpha-helical secondary structure. The thermodynamic stability as evaluated from chemical and thermal denaturation is comparable with that of the scaffold protein, alpha3W, indicating that the insertion of the exogenous loop of Gc-MAF did not significantly perturb the overall structure. GalNAc-MM1 retains the macrophage stimulation activity of natural Gc-MAF; in vitro tests show an identical enhancement of Fc-receptor-mediated phagocytosis in primary macrophages. GalNAc-MM1 provides a framework for the development of mutants with increased activity that could be used in place of Gc-MAF as an immunomodulatory agent in therapy.

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Rainbow trout (Oncorhynchus mykiss) cells of a monocyte-macrophage lineage (rtMOCs) were used to characterize the ability of the trout innate immune system to recognize and respond to different pathogen associated molecular pattern (PAMP) bearing substances. Compared to what has been reported for mammalian macrophages, rtMOCs responded with lower sensitivity to lipopolysaccharide (LPS) from Escherichia coli (EC-LPS) and Pseudomonas aeruginosa (PA-LPS). The sensitivity of rtMOCs to LPS was not influenced by the presence of serum which suggests that the resistance to endotoxic shock in fish may be due to the lack of serum-borne factors that confer sensitivity to LPS in mammals. The time course of the response to PAMPs could be separated into two patterns. EC-LPS induced stable cytokine expression whereas PA-LPS, zymosan and muramyl dipeptide induced transient TNF2 expression. By analogy to the type of stimulation observed in mammals it can be hypothesized that different signaling pathways, possibly initiated by different receptors, may be involved in the recognition of these PAMPs by rtMOCs.

Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor. More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip. In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor.

Background: Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition., Materials and Methods: We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release., Results: The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected., Conclusion: The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.

Vitamin D-binding protein-macrophage activating factor (DBP-MAF) has previously been shown to stimulate bone resorption and correct the skeletal defects associated with osteopetrosis in two nonallelic mutations in rats. This same protein and a small fragment of the protein have now been shown to demonstrate an anabolic effect on the skeleton of both newborn and young adult, intact rats. The novel peptide fragment was synthetically produced based on the human amino acid sequence at the site of glycosylation in the third domain of the native protein (DBP). The peptide tested is 14 amino acids in length and demonstrates no homologies other than to that region of DBP. Newborn rats were injected i.p. with saline, peptide (0.4 ng/g body wt.) or DBP-MAF (2 ng/g body wt.) every other day from birth to 14 days of age. On day 16 the rats were euthanized and the long bones collected for bone densitometry by pQCT. After 2 weeks of treatment with either the whole protein (DBP-MAF) or the small peptide, bone density was significantly increased in the treated animals compared to the saline controls. Young adult female rats (180 grams) were given s.c. injections of saline or peptide (0.4 ng/g body wt. or 5 ng/g body wt.) every other day for 2 weeks; 2 days after the final injections, the rats were euthanized and the femurs and tibias collected for bone densitometry. Both doses of the peptide resulted in significant increases in bone density as determined by pQCT. Young adult rats were injected locally with a single dose of the peptide (1 microg) or saline into the marrow cavity of the distal femur. One week after the single injection, the bones were collected for radiographic and histological evaluation. The saline controls showed no evidence of new bone formation, whereas the peptide-treated animals demonstrated osteoinduction in the marrow cavity and osteogenesis of surrounding cortical and metaphyseal bone. These data suggest that DBP-MAF and the synthetic peptide represent therapeutic opportunities for the treatment of a number of bone diseases and skeletal disorders. Systemic administration could be used to treat osteoporosis and a number of other osteopenias, and local administration could be effective in fractures, bony defect repairs, spinal surgery, and joint replacement.

Background: Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF., Materials and Methods: Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay., Results: We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity., Conclusion: Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.

The effect of viral hemorrhagic septicemia virus (VHSV) in vitro infection on the nitric oxide (NO) production by turbot Scophthalmus maximus kidney macrophages has been addressed in the past. Previously, we had determined that only a small fraction of turbot possess head kidney macrophages that respond to a single exposure of lipopolysaccharide (LPS) with NO production (LPS-responsive macrophages), whereas macrophage cultures from other individuals were not activated by LPS alone and needed a combination of stimuli to respond (LPS-non-responsive macrophages). In the current work, we examined the effect of VHSV on NO production by macrophages characterized as LPS-responsive macrophages or LPS-non-responsive macrophages. Combinations of LPS and tumor necrosis factor alpha (TNF-alpha) and macrophage-activating factor (MAF) were also used to stimulate the cells for NO production. The effect of VHSV on NO production depends on the response to LPS alone. When a low multiplicity of infection was used (1.78 x 10(-3)), the NO production in response to LPS in LPS-responsive macrophages was significantly decreased. However, LPS-non-responsive macrophage cultures produced NO when a combination of LPS and VHSV was used. In the case of a higher VHSV multiplicity of infection (1.78), no significant change was observed in LPS-non-responsive animals. Combinations of LPS with TNF-alpha, LPS with MAF, and TNF-alpha with MAF were used to induce NO production in LPS-non-responsive macrophages. In all these cases, VHSV suppressed NO production, although at a significant level only when a combination of TNF-alpha and MAF was used for the induction of NO.

The ability of Japanese encephalitis virus (JEV) and JEV-induced macrophage-derived factor (MDF) to modulate nitric oxide synthase (NOS) activity in brain and tumor necrosis factor-alpha (TNF-alpha) and the possible antiviral role of NOS during JEV infection were investigated. NOS activity and particularly that of the inducible form of NOS (iNOS) was significantly enhanced in JEV or JEV-induced MDF-treated mice. Following JEV infection, total NOS activity in brain was gradually increased from Day 3 and reached a peak on Day 6. MDF-induced NOS activity and iNOS activity were dose dependent and maximum activity was observed at 1 h after treatment. The response was sensitive to anti-MDF antibody treatment and N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS. Pretreatment of JEV-infected mice with L-NMMA increased the mortality as evident from reduced mean survival time (MST, 11.8 days) compared to placebo treated JEV-infected mice (MST, 17 days). The enhanced level of TNF-alpha observed in the early phase of JEV infection correlated well with the enhanced activity of iNOS. These observations thus provide evidence of the protective role of iNOS during JEV infection and indicate that iNOS may be a key mediator in host innate immune response to infection., (Copyright 2001 Academic Press.)

Phagocytic functions of rat alveolar macrophages (AM) following intraperitoneal injection of conagenin (CNG) and of AM sub-populations fractionated by Percoll discontinuous gradient centrifugation were investigated. Phagocytosis of opsonized-sheep red blood cells (SRBC) following in vitro incubation with CNG showed a significant increase in a higher density of AM (fraction IV). In addition, phagocytosis was also increased in lower density ones (fractions I and II) by macrophage-activating factor (MAF) co-cultivation. CNG-injected rats for 5 consecutive days showed a dose-dependent increase in phagocytosis of AM compared to the control rats. Although the distribution of AM sub-population in rats injected CNG was not significantly different compared to the control rats, phagocytosis was significantly increased in AM of a lower density fraction (fraction II). These results suggest that CNG directly increases phagocytosis of AM in a higher density fraction, and indirectly enhances phagocytosis in AM of a lower density fraction via increasing MAF-like material production.

Serum amyloid A (A-SAA) has previously been reported to be an acute-phase protein in salmonids. Hepatocytes represent a major source of A-SAA in salmonids, but nothing is known about hepatocyte SAA synthesis in fish. In the present work, the expression of A-SAA transcripts in primary cultures of Atlantic salmon hepatocytes in response to macrophage derived cytokines, human recombinant cytokines and bacterial lipopolysaccharide (LPS) was studied by Northern blot analysis. The macrophage supernatants were prepared by stimulating Atlantic salmon head kidney macrophages with LPS, yeast glucan or a leukocyte derived macrophage activating factor (MAF). The supernatants from glucan- or MAF-stimulated macrophages had no effect on A-SAA expression of the hepatocytes, while supernatants from LPS-stimulated macrophages gave about a 2-fold increase in expression. The combination of either glucan and MAF, or LPS and MAF were more effective and these supernatants gave a 3.4- and 5.2-fold increase in A-SAA expression, respectively. The hepatocytes were also treated with the human recombinant cytokines TNFalpha, IL-1beta and IL-6, alone or in combination. The A-SAA response to each of them alone was modest, but TNFalpha and IL-6 or IL-1beta and IL-6 in combination gave a higher response than each cytokine alone. These data suggest that the expression of A-SAA by hepatocytes from Atlantic salmon is induced by cytokine-like molecules. Interestingly, hepatocytes treated directly with LPS gave a more than 10-fold increase in SAA mRNA expression, but it is not known if this is a direct effect of LPS on the hepatocytes or if it is mediated by other contaminating cell types.

In contrast to the studies that describes the effects of conagenin (CNG) on the cellular immunity of lymphocytes (references), we investigated the in vitro effect of CNG on macrophage function. Phagocytosis of alveolar macrophages (AM) against opsonized-sheep red blood cells (SRBC) was significantly enhanced following in vitro incubation with CNG for 12 hours at 37 degrees C, which was closely associated with increased expression of Fc-receptor in AM membranes. Macrophage-activating factor (MAF), prepared from splenic lymphocytes in vitro stimulated with concanavarin A (Con A) for 48 hours at 37 degrees C, had also the enhancing effect on phagocytosis of AM against opsonized-SRBC. Preincubation with CNG (0.1 microg/ml) and MAF (1/100 dilution) induced the additional effect on phagocytosis of AM, which was associated with the increased expression of Fc-receptor in AM membranes. These results suggest that CNG enhances AM phagocytosis by increasing the expression of Fc-receptor on their membranes via either effecting different sub-populations of AM cells or by activating independent mechanism on the same AM cell population.

In mammals, the increased generation of prostaglandins (PG) during the onset of inflammatory responses and activation of immune cell types has been attributed to the induction of a novel cyclo-oxygenase (COX) isoform, termed COX-2, which is distinct from the well-characterized constitutive activity (COX-1). Goldfish (Carassius auratus) macrophages exposed to bacterial lipopolysaccharide and leucocyte-derived macrophage-activating factor(s) showed a significant increase in the generation of the major COX product, PGE2, within the first 6 h of stimulation. The selective COX-2 inhibitor, NS398, inhibited this elevated generation of PGE, whereas the basal level of this product synthesized by unstimulated macrophages was unaffected by such exposure. PGE generation by goldfish macrophages was similarly inhibited by the glucocorticoid, dexamethasone, and an inhibitor of protein synthesis, cycloheximide, suggesting that this stimulation may be due to an inducible enzyme equivalent to mammalian COX-2. The complete coding sequence of rainbow trout (Oncorhynchus mykiss) COX-2 was obtained by PCR. The gene contains a 61 bp 5'-untranslated region (UTR), a 1821 bp open reading frame and a 771 bp 3'UTR containing multiple copies of an mRNA instability motif (ATTTA). The predicted translation product had high homology to known mammalian and chicken COX-2 (83-84%) and COX-1 (77%) sequences. Reverse-transcriptase PCR with cDNA from control and bacterially challenged fish revealed that trout COX-2 expression was not constitutive but could be induced. Overall, these studies show for the first time that the inducible isoform of COX has a long evolutionary history, probably dating back to the evolution of fish over 500 million years ago.

Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.

Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized beta-galactosidase or treatment of human Gc protein with immobilized beta-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum alpha-N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 10(5) cells) showed a mean survival time of 35 +/- 4 days, whereas tumor-bearing controls had a mean survival time of 16 +/- 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 10(5) tumor cells, survived up to 32 +/- 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 10(5) cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 10(5) tumor cells, also survived up to 32 +/- 4 days, while control mice that received the 5 x 10(5) ascites tumor cells only survived for 14 +/- 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 10(5) Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90.

Murine peritoneal macrophages, activated by BCG vaccine, and human peripheral blood monocytes, activated by lipopolysaccharides, exerted neurite stimulating or neurite inhibiting effects in various periods of activation. The supernatants of these preparations were active in organotypic culture of chick embryo dorsal root ganglia. The inhibition of neurite growth on the 1st day of cultivation was followed by the neurite-stimulating effect. The fluctuation of neurite-inhibition and neurite-stimulation effect of macrophage supernatants suggest the availability of certain changes in cytokine composition in different periods of macrophage activation.

Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein-macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the macrophage/osteoclast lineage can be functionally upregulated with the subsequent addition of DBP-MAF to perform the activities of phagocytosis and bone resorption. The in vitro data also showed that DBP-MAF did not support colony development as in CSF-1 or the combination treatment. The recruitment and activation of cells into the macrophage/ osteoclast lineage may help to correct the bone and immune defects found in diseases demonstrating a significant lack of myeloid cells, as well as neutrophilia disorders and the disease, osteopetrosis.

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

Generation of macrophage-activating factor requires a precursor protein, Gc protein (serum vitamin D3-binding protein), as well as participation of beta-galactosidase of inflammation-primed B lymphocytes and sialidase of T lymphocytes. The treatment of human peripheral blood mononuclear cells with an inflammatory lysophospholipid induced beta-galactosidase and sialidase activity of lymphocytes, leading to the generation of macrophage-activating factor and activation of monocytes/macrophages. However, lysophospholipid treatment of peripheral blood mononuclear cells from three infantile patients with osteopetrosis resulted in no significant activation of monocytes/macrophages. The lysophospholipid-inducible beta-galactosidase activity of B lymphocytes as well as that of the sialidase of T lymphocytes was found to be defective in these patients.

Modulation of the respiratory burst activity of head kidney macrophages isolated from rainbow trout (Oncorhynchus mykiss) was observed following treatment with several biologically active substances. Macrophage-activating factor (MAF) induced the highest increment if respiratory burst activity relative to treatment with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha) or beta-glucans from Saccharomyces cerevisiae. Increased responses were more evident when these molecules were combined in pairs. Negative regulation of respiratory burst activity was observed when diMePGE2 was added to the macrophages, with maximal inhibition seen using a concentration of 2.6 microM. Inhibition was also seen using stimulated macrophages, either by co-incubation of stimuli and diMePGE2 or by adding diMePGE2 to previously stimulated cells. The inhibitory effect on macrophages was detectable with 3 h of incubation with diMePGE2 and by 24 h the level of the response was even lower than that from unstimulated (control) macrophages. Of significance was the finding that the inhibitory effect of prostaglandin on macrophage function could be overcome by co-incubation with stimulatory molecules or by pre-treatment with MAF and LPS or MAF and TNF alpha Thus, the regulation of macrophage activation in fish is likely to be as complex as in mammals.

Tumor necrosis factor (TNF) is considered to be deeply involved in the hepatocyte damages in severe hepatitis. To delineate which mediators are involved in the production of TNF in vivo, we examined regulatory mechanisms of the production of TNF by rat Kupffer cells using a variety of mediators. Lipopolysaccharide (LPS) markedly induced TNF production by Kupffer cells. Kinetic studies revealed a rapid release of TNF within 3-4 hrs after the addition of LPS to the culture medium. Spleen cell derived-macrophage activating factor prepared from rat spleen cells did not by itself induce the production of TNF. However, the presence of a small amount of the factor during or before exposure to LPS induced higher levels of TNF, suggesting that macrophage activating factor had a priming effect. Recombinant human interferon-gamma and recombinant human granulocyte-macrophage colony stimulating factor, the natural types of which are components of the macrophage activating factor, displayed similar effects. Prostaglandin E2 (PGE2) and dexamethasone both inhibited LPS-induced TNF production in a dose dependent manner. Indomethacin, a cyclooxygenase inhibitor, increased LPS-induced TNF production. Interestingly, a combination of PGE2 and indomethacin inhibited TNF production more strongly than PGE2 alone, suggesting that the simultaneous treatment with PGE2 and indomethacin decreases liver damage in severe hepatitis rather than PGE2 alone. In addition, PGE2 pretreatment reduced the response to the newly added PGE2, suggesting the presence of a desensitization mechanism in the PGE2 receptor system. These findings suggest that spleen cell-derived macrophage activating factor and bowel-derived LPS take important parts in TNF production through the portal blood in the liver in vivo.

Recent studies in our laboratory demonstrated that fish macrophages produce nitric oxide. To elucidate the mechanisms which regulate nitric oxide production in teleosts, we examined whether macrophage activating factors (MAFs) secreted by mitogen stimulated leukocytes, induced nitric oxide production in a long-term cultured macrophage cell line and in primary cultures of kidney macrophages from the goldfish. The results indicate that both primary and long term cultured goldfish macrophages produce nitric oxide in response to MAF or bacterial lipopolysaccharide (LPS), and co-stimulation with both factors results in a synergistic induction of nitric oxide production. MAF that induced nitric oxide production were present in leukocyte supernatants as early as 24 h after addition of mitogens to cell cultures. The production of MAF was dependent upon the incubation temperature, presence of serum in the culture medium and duration of incubation: maximal MAF activity was detected in 72-96 h supernatants raised in media with serum at 30 degrees C. MAF-induced nitric oxide production by long term cultured macrophages was inhibited by 1000 microM NG-monomethyl-L-arginine or amino-guanidine, indicating an L-arginine-dependent metabolic pathway for the production of the reactive nitrogen intermediates in teleosts. The biochemical events of cytokine induced nitric oxide production by teleost macrophages appear to be similar to those of mammalian macrophages.

A variety of supernatants were prepared by stimulating rainbow trout Oncorhynchus mykiss head kidney macrophages with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-alpha), or a leucocyte-derived macrophage-activating factor (I-MAF), individually and in combination. If generated using a 12-h stimulation period, such supernatants were found to elevate significantly the respiratory burst activity of target macrophages; that is, they contained a macrophage-derived MAF (m-MAF), but supernatants generated using a shorter incubation period showed no significant activity. Combinations of these treatments were particularly effective in generating m-MAF-containing supernatants. The elevation of respiratory burst activity by supernatants generated using combined treatments could be partially inhibited by prior treatment of the target macrophages with anti-TNF-alpha receptor 1 (TNFR1) monoclonal antibodies (mAbs). Similarly, treatment of macrophages with combinations of 1-MAF and m-MAF generated supernatants with potent m-MAF activity and this activity was partially inhibited by prior treatment of the target cells with anti-TNFR1 mAb. In addition, the presence of anti-transforming growth factor beta 1 (TGF-beta 1) serum while generating these latter supernatants resulted in significantly increased m-MAF activity. Such data suggest that fish leukocytes secrete a variety of potent macrophage-activating (TNF-alpha) and -deactivating (TGF-beta) factors.

Osteopetrosis is a heterogeneous group of bone diseases characterized by an excess accumulation of bone and a variety of immune defects. Osteopetrosis (op) and incisors absent (ia) are two nonallelic mutations in the rat which demonstrated these skeletal defects as a result of reduced bone resorption. Osteopetrotic (op) rats have severe sclerosis as a result of reduced numbers of osteoclasts which are structurally abnormal. The sclerosis in ia rats is not as severe as in op mutants; they have elevated numbers of osteoclasts, but they are also morphologically abnormal, lacking a ruffled border. Both of these mutations have defects in the inflammation-primed activation of macrophages. They demonstrate independent defects in the cascade involved in the conversion of vitamin D binding protein (DBP) to a potent macrophage activating factor (DBP-MAF). Because this factor may also play a role in the pathogenesis of osteoclastic dysfunction, the effects of ex vivo-generated DBP-MAF were evaluated on the skeletal system of these two mutations. Newborn ia and op rats and normal littermate controls were injected with DBP-MAF or vehicle once every 4 days from birth until 2 weeks of age, at which time bone samples were collected to evaluate a number of skeletal parameters. DBP-MAF treated op rats had an increased number of osteoclasts and the majority of them exhibited normal structure. There was also reduced bone volume in the treated op animals and an associated increased cellularity of the marrow spaces. The skeletal sclerosis was also corrected in the ia rats; the bone marrow cavity size was significantly enlarged and the majority of the osteoclasts appeared normal with extensive ruffled borders.

Mycoplasmas and mycoplasma membranes have been shown to induce the production of inflammatory cytokines, including tumor necrosis factor alpha and interleukin-6, as well as nitric oxide, by mouse macrophages and rat brain astrocytes. Luminol-enhanced chemiluminescence was used as a sensitive method to show that Mycoplasma capricolum membranes induce mouse peritoneal macrophages to produce reactive oxygen radicals. Coincubation of the mycoplasma with a secondary stimulus, namely macrophage-activating factor or interferon-gamma, increased the chemiluminescence. The augmentation was abolished by the nitric oxide synthase inhibitor NG-methyl-L-arginine, indicating the involvement of nitric oxide. The coproduction of superoxide and nitric oxide by the same cell allows the formation of the powerful oxidant peroxynitrite, which could be responsible for the increased chemiluminescence. Induction of oxidizing radicals by mycoplasmas may contribute to the clinical pathology seen in mycoplasma infections.

Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to wild-type rats but not in osteopetrotic (op) mutant rats. In vitro treatment of wild-type rat peritoneal cells with lyso-Pc or DDG efficiently activated macrophages whereas treatment of op mutant rat peritoneal cells with lyso-Pc or DDG did not activate macrophages. The inflammation-primed macrophage activation cascade in rats requires participation of B lymphocytes and vitamin D binding protein (DBP). Lyso-Pc-inducible beta-galactosidase of wild-type rat B lymphocytes can convert DBP to the macrophage-activating factor (MAF), whereas B lymphocytes of the op mutant rats were shown to be deficient in lyso-Pc-inducible beta-galactosidase. DBP is conserved among mammalian species. Treatment of human DBP (Gc1 protein) with commercial glycosidases yields an extremely high titrated MAF as assayed on mouse and rat macrophages. Because the enzymatically generated MAF (GcMAF) bypasses the role of lymphocytes in macrophage activation, the op mutant rat macrophages were efficiently activated by administration of a small quantity (100 pg/rat) of GcMAF. Likewise, in vitro treatment of op rat peritoneal cells with as little as 40 pg GcMAF/ml activated macrophages.

Phagocytosis of rat alveolar macrophages (AM) was enhanced by the infusion of arginine-rich solution for 7 days. The enhancement of phagocytosis by arginine-rich solution was due to not the difference in the distribution of AM subpopulations (I to IV) but the difference in phagocytic activity of AM in fraction IV. In the process of phagocytosis, there were no significant differences in the stages of migration, attachment, and digestion between control and arginine-rich solutions, although AM from fraction IV of rats infused with arginine-rich solution showed significantly higher ingestion of opsonized sheep red blood cells (SRBC) compared to that of control group. Furthermore, the production of macrophage-activating factor (MAF) from rat splenocytes was higher in arginine-rich group than that of control group. AM from fraction IV of rats fed a stock diet had a higher arginase activity and showed a significant increase of phagocytosis following in vitro incubation with L-arginine (25 and 50 mM) for 24 h. From these results, the enhanced phagocytosis of AM by arginine-rich solution may be due to the increased phagocytosis of AM from fraction IV, in which the higher sensitivity of AM from fraction IV to arginine and the higher production of MAF from splenocytes following the infusion of arginine-rich solution participate.