Production Of Colony-stimulating Factor Research Articles

Co-expression of proteinase inhibitor enhances recombinant human granulocyte–macrophage colony stimulating factor production in transgenic rice cell suspension culture

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte–macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.

Hematopoietic response of rats exposed to the impact of an acute psychophysiological stressor on responsiveness to an in vivo challenge with Listeria monocytogenes: Modulation by Chlorella vulgaris prophylactic treatment

In this study, we investigated the hematopoietic response of rats pretreated with CV and exposed to the impact of acute escapable, inescapable or psychogenical stress on responsiveness to an in vivo challenge with Listeria monocytogenes. No consistent changes were observed after exposure to escapable footshock. Conversely, the impact of uncontrollable stress (inescapable and psychogenical) was manifested by an early onset and increased severity and duration of myelossuppression produced by the infection. Small size CFU-GM colonies and increased numbers of clusters were observed, concurrently to a greater expansion in the more mature population of bone marrow granulocytes. No differences were observed between the responses of both uncontrollable stress regimens. CV prevented the myelossuppression caused by stress/infection due to increased numbers of CFU-GM in the bone marrow. Colonies of cells tightly packed, with a very condensed nucleus; in association with a greater expansion in the more immature population of bone marrow granulocytes were observed. Investigation of the production of colony-stimulating factors revealed increased colony-stimulating activity (CSA) in the serum of normal and infected/stressed rats treated with the algae. CV treatment restored/enhanced the changes produced by stress/infection in total and differential bone marrow and peripheral cells counts. Further studies demonstrated that INF-γ is significantly reduced, whereas IL-10 is significantly increased after exposure to uncontrollable stress. Treatment with CV significantly increased INF-γ levels and diminished the levels of IL-10. Uncontrollable stress reduced the protection afforded by CV to a lethal dose of L. monocytogenes, with survival rates being reduced from (50%) in infected rats to 20% in infected/stressed rats. All together, our results suggest Chlorella treatment as an effective tool for the prophylaxis of post-stress myelossupression, including the detrimental effect of stress on the course and outcome of infections.

Human Bone Marrow Adipocytes Block Granulopoiesis Through Neuropilin-1-Induced Granulocyte Colony-Stimulating Factor Inhibition

Adipocytes are part of hematopoietic microenvironment, even though up to now in humans, their role in hematopoiesis is still questioned. We have previously shown that accumulation of fat cells in femoral bone marrow (BM) coincides with increased expression of neuropilin-1 (NP-1), while it is weakly expressed in hematopoietic iliac crest BM. Starting from this observation, we postulated that adipocytes might exert a negative effect on hematopoiesis mediated through NP-1. To test this hypothesis, we set up BM adipocytes differentiated into fibroblast-like fat cells (FLFC), which share the major characteristics of primitive unilocular fat cells, as an experimental model. As expected, FLFCs constitutively produced macrophage colony stimulating factor and induced CD34(+) differentiation into macrophages independently of cell-to-cell contact. By contrast, granulopoiesis was hampered by cell-to-cell contact but could be restored in transwell culture conditions, together with granulocyte colony stimulating factor production. Both functions were also recovered when FLFCs cultured in contact with CD34(+) cells were treated with an antibody neutralizing NP-1, which proved its critical implication in contact inhibition. An inflammatory cytokine such as interleukin-1 beta or dexamethasone modulates FLFC properties to restore granulopoiesis. Our data provide the first evidence that primary adipocytes exert regulatory functions during hematopoiesis that might be implicated in some pathological processes. Disclosure of potential conflicts of interest is found at the end of this article.

Propofol pretreatment attenuates LPS-induced granulocyte–macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-κB translocation

Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 μM after 48 h incubation. Pretreatment with 100 μM PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-α, and granulocyte–macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and IκBα, as well as the nuclear translocation of NF-κB primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-κB nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.

IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia

Emerging evidence supports the concept that T helper type 17 (T(H)17) cells, in addition to mediating autoimmunity, have key roles in mucosal immunity against extracellular pathogens. Interleukin-22 (IL-22) and IL-17A are both effector cytokines produced by the T(H)17 lineage, and both were crucial for maintaining local control of the Gram-negative pulmonary pathogen, Klebsiella pneumoniae. Although both cytokines regulated CXC chemokines and granulocyte colony-stimulating factor production in the lung, only IL-22 increased lung epithelial cell proliferation and increased transepithelial resistance to injury. These data support the concept that the T(H)17 cell lineage and its effector molecules have evolved to effect host defense against extracellular pathogens at mucosal sites.

Inhibition of Interleukin-12 Production by Trypanosoma brucei in Rat Macrophages

The immune response of a host infected with Trypanosoma brucei is modulated by trypomastigotes. We examined the changes in cytokine production in T. brucei gambiense (Wellcome strain; WS) infected rats and the influence on production of interleukin (IL)-12 by macrophages. The blood concentration of interferon-gamma, tumor necrosis factor-alpha, and IL-10 increased beginning the second day after infection. However, an increase in IL-12p40 was not observed until 4 days after infection. When spleen macrophages and Kupffer cells harvested from uninfected rats and HS-P cells (a rat macrophagelike cell line) were cocultured with WS, IL-12p40 production did not change. When HS-P cells were cultured with WS, transport of nuclear factor-kappaB into the nucleus increased. Levels of macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony-stimulating factor mRNA in the spleens and livers of WS-infected rats were high in comparison with uninfected rats, suggesting that the WS promotes macrophage proliferation. The level of IL-12p40 mRNA in HS-P cells cocultured with WS increased in response to transfection with a small interfering RNA against M-CSF or addition of anti-M-CSF antibody. These results suggest that the WS inhibits IL-12p40 mRNA production by promoting production of macrophage colony-stimulating factor by macrophages.

Ingested (Oral) SIRS Peptide 1-21 Suppresses Type 1 Diabetes in NOD Mice

Type 1 diabetes (T1D) is a chronic disorder that results from autoimmune destruction of the insulin-producing pancreatic beta cell. The nonobese diabetic (NOD) mouse is a model of the human autoimmune disease T1D. Soluble immune response suppressor (SIRS) is a nonspecific protein suppressor of immune response produced by immunomodulatory T cells stimulated by type I interferon (IFN). SIRS inhibits antibody responses in vivo, lipopolysaccharide (LPS)-induced fever, and delayed-type hypersensitivity (DTH) responses. Previous investigators have isolated the N-terminal sequence of SIRS protein consisting of 21 amino acids. Mice ingesting 1 microg SIRS peptide 1-21 showed significant delayed onset of T1D and a decreased frequency of T1D compared with mock-fed and 10-microg-fed mice and a significant decrease in islet inflammation. There were significant decreases in islet lymphocyte chemokine production of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein-1 gamma (MIP-1 gamma), regulated upon activation, normal T cell-expressed, and presumably secreted (RANTES), and stromal cell-derived factor-1 (SDF-1) in the SIRS-fed mice, factors important in migration of inflammatory cell into the islets. Ingested (oral) SIRS peptide inhibits clinical T1D by decreasing target organ cellular migration of islet destructive populations by suppression of islet lymphocyte chemokine secretion.

Increased hGM-CSF production and secretion with Pluronic F-68 in transgenicNicotiana tabacum suspension cell cultures

The effects of Pluronic F-68, a nonionic surfactant, on the production and secretion of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) in a transgenicNicotiana tabacum cell suspension culture were investigated in this study. The addition of Pluronic F-68 was shown to extend cell survival in the stationary phase, but had no influence on effective initial cell growth. With regard to production, it increased the level of extracellular hGM-CSF two-fold. This may be attributable not only to the enhanced expression level, but also to the improved permeability of the cell membrane due to the interaction between Pluronic F-68 and the cell membrane and cell wall. The effect of Pluronic F-68 on the production and secretion of hGM-CSF in a bioreactor was also evaluated. hGM-CSF production in the bioreactor after the addition of Pluronic F-68 proved more effective than in flask cultures.

Granulocyte Colony-stimulating Factor Production and Rapid Progression of Gastric Cancer after Histological Change in the Tumor

Granulocyte colony-stimulating factor (G-CSF)-producing malignancies are thought to be rare and associated with advanced disease and poor prognosis. Here, we report on a 77-year-old patient with G-CSF-producing gastric cancer. We observed this patient from the stage prior to the diagnosis of gastric cancer when leukocyte count was normal to the stage of advanced disease associated with remarkable leukocytosis. Immunohistochemical analysis demonstrated G-CSF expression in the advanced-stage, poorly differentiated adenocarcinoma, but not in the early-stage, well-differentiated adenocarcinoma. G-CSF receptor was not detected to be expressed in the advanced-stage tumor. Based on these results it appears that a histological change in the tumor may influence G-CSF production and the concomitant rapid progression in gastric cancer.

Development of efficient method for purified recombinant bovine granulocyte-macrophage colony-stimulating factor production with baculovirus-silkworm gene expression system

Recombinant bovine granulocyte-macrophage colony-stimulating factor (rboGM-CSF) was produced by the baculovirus-silkworm expression system. It was purified to 98% by (NH(4))(2)SO(4), followed by a three-step column chromatography with silica gel, ion exchange resin and a metal chelate column. The specific activity of purified rboGM-CSF was 1.6-6.3 x 10(6) ED(50) mg(-1). By this method, the specific activity was raised 160-fold and 21% of the expressed rboGM-CSF was recovered.

Immunological Consequences of Apoptotic Cell Phagocytosis

Cells undergo apoptosis in development, tissue homeostasis, and disease and are subsequently cleared by professional and nonprofessional phagocytes. There is now overwhelming evidence that phagocyte function is profoundly altered following apoptotic cell uptake, with consequences for the ensuing innate and adaptive immune response. Pathogens and tumors exploit the changes in macrophage function following apoptotic cell uptake. Here, we will outline the consequences of apoptotic cell phagocytosis and illustrate how apoptotic cells could be used to manipulate the immune response for therapeutic gain.

Differential regulation of lipopolysaccharide and Gram‐positive bacteria induced cytokine and chemokine production in macrophages by Gαi proteins

Heterotrimeric G(i) proteins play a role in signalling activated by lipopolysaccharide (LPS), Staphylococcus aureus (SA) and group B streptococci (GBS), leading to production of inflammatory mediators. We hypothesized that genetic deletion of G(i) proteins would alter cytokine and chemokine production induced by LPS, SA and GBS stimulation. LPS-induced, heat-killed SA-induced and heat-killed GBS-induced cytokine and chemokine production in peritoneal macrophages from wild-type (WT), Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice were investigated. LPS induced production of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), IL-10 and interferon-gamma-inducible protein-10 (IP-10); SA induced TNF-alpha, and IL-1beta production; and GBS induced TNF-alpha, IL-6, IL-1beta, macrophage inflammatory protein-1alpha (MIP-1alpha) and keratinocyte chemoattract (KC) production were all decreased (P < 0.05) in Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. In contrast to the role of G(i) proteins as a positive regulator of mediators, LPS-induced production of MIP-1alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in macrophages from Galpha(i1/3) (-/-) mice, and SA-induced MIP-1alpha production was increased in both groups of Galpha(i) protein-depleted mice. LPS-induced production of KC and IL-1beta, SA-induced production of GM-CSF, KC and IP-10, and GBS-induced production of IL-10, GM-CSF and IP-10 were unchanged in macrophages from Galpha(i2) (-/-) or Galpha(i1/3) (-/-) mice compared with WT mice. These data suggest that G(i2) and G(i1/3) proteins are both involved and differentially regulate murine inflammatory cytokine and chemokine production in response to both LPS and Gram-positive microbial stimuli.

Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy

Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy

Theoretical Basis for Herbal Medicines, Tokishakuyaku‐San and Sairei‐To, in the Treatment of Recurrent Abortion: Enhancing the Production of Granulocyte–Macrophage Colony‐Stimulating Factor in Decidual Stromal Cells

To get insight into the basis for the empirical usage of herbal medicines, such as Tokishakuyaku-san (Toki) and Sairei-to (Sai) in the treatment of recurrent abortion and intrauterine growth restriction, we examined whether these medicines modulate the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine working as an important mediator for intercellular communication in the embryonic development, in decidual stromal cells (DSCs). Human DSCs were cultured with either Toki or Sai at several different concentrations. The effect on cell proliferation was assessed by WST-8 assay. GM-CSF released into culture medium was analyzed using enzyme-linked immunosorbent assay, and semi-quantitative polymerase chain reaction was carried out to see GM-CSF mRNA expression in DSCs. Sai inhibited the proliferation of cultured DSCs, while no interference was observed in the presence of Toki. Both Toki and Sai enhanced the release of GM-CSF into culture medium. The amount of GM-CSF mRNA in cultured DSCs was as well increased by either Toki or Sai. Considering the significance of GM-CSF in embryonic development, clinical benefit of these herbal medicines in the treatment of recurrent abortion might be based on the shown pharmacological reaction related to GM-CSF.

Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum

The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium.

Granulocyte-Macrophage Colony-Stimulating Factor Production by Human Keratinocytes in the Presence of Pimecrolimus

Granulocyte-Macrophage Colony-Stimulating Factor Production by Human Keratinocytes in the Presence of Pimecrolimus

Human peritoneal mesothelial cells isolated from spent dialysate fluid maintain contaminating macrophages via production of macrophage colony stimulating factor

Human peritoneal mesothelial cells (HPMC) are useful for the analysis of peritoneal reactions to various insults and to peritoneal dialysate. HPMC can be readily obtained from spent dialysis fluid, but leucocyte contamination is a major problem when using these cells for in vitro experiments. Therefore, we examined the persistence of leucocyte contamination in HPMC cultures obtained from spent dialysate. Cells were obtained from spent patient dialysate bags by centrifugation and analysed for specific cell phenotypes by flow cytometry at the initial collection and during sequential passages in cell culture. Cell proliferation was assessed by either bromodeoxyuridine incorporation or a dehydrogenase assay. Cytokine secretion was analysed by enzyme-linked immunosorbent assay. Spent dialysate bags contained two major cell populations: CD45+ leucocytes and cytokeratin-8/18+ cells. Initially, most collected cells were CD45+, but their numbers decreased rapidly during the first week of culture. However, a persistent contamination of CD45+ leucocytes, approximately 20% of cells, was evident during the next three passages. This persistent CD45+ contamination was identified as CD68+ macrophages and contained bromodeoxyuridine + proliferating cells. These macrophages could be removed by fluorescence-activated cell sorting using anti-CD45 antibody, resulting in highly purified HPMC which expressed cytokeratin-8/18 and calretinin. Supernatant obtained from these purified HPMC contained macrophage colony stimulating factor and induced proliferation of bone marrow-derived macrophages. Spent dialysate contains macrophages which persist in culture and are associated with HPMC secretion of macrophage colony stimulating factor and macrophage proliferation. Therefore, contaminating macrophages should be specifically removed from HPMC preparations before performing in vitro studies.

Promotion of recombinant macrophage colony stimulating factor production by dimethyl sulfoxide addition in Chinese hamster ovary cells

A Chinese hamster ovary (CHO) cell line is the primary choice for the production of recombinant protein drugs with glycosylation modification. Recombinant protein productivity was enhanced by the addition of dimethyl sulfoxide (DMSO) in CHO cells. However, DMSO induced G0/G1 phase growth arrest and reduced cell growth rate. A two-stage process was developed to utilize the stimulatory effect of DMSO on recombinant protein production and mitigate the problem of growth inhibition in this study. In the first stage, cells are cultured without DMSO for a certain period in order to obtain a high cell density. Sequentially, DMSO is added to achieve a high specific productivity in the second stage. Using this approach, we found that by adding 1% DMSO after 24 h of growth, macrophage colony-stimulating factor (M-CSF) volumetric productivity is increased by 57% compared with the value obtained without the addition of DMSO.

MEKK3 is essential for lipopolysaccharide‐induced interleukin‐6 and granulocyte–macrophage colony‐stimulating factor production in macrophages

Mitogen-activated protein/ERK kinase kinase 3 (MEKK3) is a Ser/Thr protein kinase belonging to the MEKK/STE11 subgroup of the MAP3K family. Recently, we found that MEKK3 plays a critical role in interleukin-1 (IL-1) receptor and Toll-like receptor 4 signalling using established primary mouse embryonic fibroblast (MEF) cell lines. However, the function of MEKK3 in immune cells has not been studied because germ-line MEKK3 knockout mice are embryonically lethal between embryonic days 10 and 11. In this study, we used small interference RNA to the mouse Mekk3 gene to specifically knock down MEKK3 expression in the macrophage line Raw264.7. We found that the lipopolysaccharide-induced IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production was dramatically decreased in MEKK3 knockdown cells whereas the tumour necrosis factor-alpha and IL-1beta production were not affected. We also observed that the ERK1/2, p38 and JNK MAPK induction in MEKK3 knockdown cells were moderately inhibited within the first 60 min of stimulation, while the ERK and p38 were more severely inhibited after 2-4 hr of stimulation. Degradation of IkappaBalpha was also partially blocked in MEKK3 knockdown cells. Notably, the impairment in IL-6 and GM-CSF production in the MEKK3 knockdown cells was restored by reintroducing a human Mekk3 cDNA that could not be targeted by mouse Mekk3-siRNAs. In conclusion, this study showed that MEKK3 is a crucial and specific regulator of the proinflammatory cytokines IL-6 and GM-CSF in macrophages and provided a novel method for investigating MEKK3 function in other immune cells.

Nicotine Treatment Induces Expression of Matrix Metalloproteinases in Human Osteoblastic Saos-2 Cells

Tobacco smoking is an important risk factor for the development of severe periodontitis. Recently, we showed that nicotine affected mineralized nodule formation, and that nicotine and lipopolysaccharide stimulated the formation of osteoclast-like cells by increasing production of macrophage colony-stimulating factor (M-CSF) and prostaglandin E2 (PGE2) by human osteoblastic Saos-2 cells. In the present study, we examined the effects of nicotine on the expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), the plasminogen activation system including the component of tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PA inhibitor type 1 (PAI-1), alpha7 nicotine receptor, and c-fos. We also examined the effect of the nicotine antagonist D-tubocurarine on nicotine-induced expression of MMP-1. Gene expression was examined using real-time polymerase chain reaction (PCR) to estimate mRNA levels. In addition, expression of the MMP, TIMP, uPA, tPA, and PAI-1 proteins was determined by Western blotting analysis. Nicotine treatment caused expression of MMP-1, 2, 3, and 13, but not MMP-14, to increase significantly after 5 or 10 d of culture; MMP-14 expression did not change through day 14. Enhancement of MMP-1 expression by nicotine treatment was eliminated by simultaneous treatment with D-tubocurarine. In the presence of nicotine, expression of uPA, PAI-1, or TIMP-1, 2, 3, or 4 did not change over 14 d of culture, whereas expression of tPA increased significantly by day 7. Nicotine also increased expression of the alpha7 nicotine receptor and c-fos genes. These results suggest that nicotine stimulates bone matrix turnover by increasing production of tPA and MMP-1, 2, 3, and 13, thereby tipping the balance between bone matrix formation and resorption toward the latter process.