Siglec-9 modulated IL-4 responses in the macrophage cell line RAW264

Siglecs, an immunoglobulin-like lectin family that recognizes the sialic acid moiety, regulate various aspects of immune responses. In the present study, we investigated the effects of Siglecs on the macrophage cell line RAW264, which was stimulated with interleukin-4 (IL-4). The induction of arginase-1 (Arg1) by IL-4 was stronger in Siglec-9-expressing cells than in mock cells. Mutations in the cytoplasmic tyrosine-based inhibitory motifs in Siglec-9 markedly reduced the expression of Arg1. The phosphorylation of Akt by IL-4 and extracellular signal-regulated kinase (ERK) without IL-4 was stronger in Siglec-9-expressing cells, indicating the enhanced activation of the phosphatidylinositol 3 kinase (PI-3K) and mitogen-activated protein kinase kinase (MEK)/ERK pathways, respectively. The enhanced expression of Arg1 was inhibited by MEK inhibitors, but not by PI-3K inhibitor. These results indicate that Siglec-9 affects several different signaling pathways in IL-4-stimulated macrophages, which resulted in enhanced induction of Arg1 in Siglec-9-expressing RAW264 cells.     

Siglec-9 enhances IL-10 production in macrophages via tyrosine-based motifs

We examined whether Siglec-9 modulates cytokine production in the macrophage cell line RAW264. Cells expressing Siglec-9 produced low levels of tumor necrosis factor (TNF)-α upon stimulation with lipopolysaccharide, peptidoglycan, unmethylated CpG DNA, and double-stranded RNA. On the other hand, interleukin (IL)-10 production was strongly enhanced in Siglec-9-expressing cells. Similar activities were also exhibited by Siglec-5. However, the up-regulation of IL-10 as well as the down-regulation of TNF-α was abrogated when two tyrosine residues in the cytoplasmic tail of Siglec-9 were mutated to phenylalanine. A membrane proximal ITIM mutant of Siglec-9 did not enhance IL-10 production but partly inhibited TNF-α production, indicating diverse regulation mechanisms of TNF-α and IL-10. Siglec-9 also enhanced the production of IL-10 in the human macrophage cell line THP-1. These results demonstrate that Siglec-9 enhances the production of the anti-inflammatory cytokine IL-10 in macrophages.     

Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-kB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expres-sion of IL-12 p40 and p35 mRNA, and the degradation of IkBα induced by LPS or LPS/IFN-γ. EM-SA showed that LPS could augment the NF-kB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-kB activity nor promote the degradation of IkBα. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided with the high-level secretion of IL-12 p70 induced by IFN-γ/LPS; (ii) NF-kB signal pathway is essential for IFN-γ/LPS to induce IL-12 mRNA expression; (iii) by blocking the degradation of IkB, the PSI sup-presses the IL-12 p40/p35 mRNA expression induced by LPS and IFN-γ/LPS; (iv) NF-kB signal may not be involved in the mechanism by which IFN-γ enhanced the expression of the LPS-induced IL-12 p40/p35 mRNA.     

Uncarinic acid C plus IFN-γ generates monocyte-derived dendritic cells and induces a potent Th1 polarization with capacity to migrate

Uncarinic acid C (URC) is triterpene isolated from Uncaria rhynchophylla and modulates human DC function in a fashion that favors Th1 cell polarization depending on TLR4 signaling. The induction of dendritic cells (DC) is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Monocyte-derived DC used as adjuvant cells in cancer immunotherapy and have shown promising results. We studied the effect of interferon’s (IFN-α and IFN-γ) and TNF-α on phenotypic and functional maturation, and cytokine production of URC-primed DC in vitro. Human monocytes were exposed to either URC alone, or in combination with TNF-α, IFN-α or IFN-γ, and thereafter co-cultured with naïve T cells. We found that the expression levels of CD1a, CD83 and HLA-DR on URC-primed DC were influenced by IFN-γ and IFN-γ augmented the T cell stimulatory capacity in allo MLR to URC-primed DC. Moreover, the production of IL-12p70 by URC-primed DC was enhanced by IFN-γ. IL-12p70 production by URC-primed DC alone was influenced following treatment with anti-TLR4 mAb, but not DC differentiated with URC plus IFN-γ. URC plus IFN-γ-primed DC induced a substantial increase in the secretion of IFN-γ by T cells, which is dependent on IL-12 secretion. DC maturated with URC plus IFN-γ had an intermediate migratory capacity towards CCL19 and CCL21. In addition, the expression levels of CCR7 on URC-primed DC were enhanced by IFN-γ. In contrast, surface molecule up-regulation and function of URC-primed DC were slightly enhanced by TNF-α, and IFN-α. These results suggest that the enhancement of Th1 cells polarization to URC-primed DC induced by IFN-γ depends on the activation of IL-12p70 and independent on TLR4. DC differentiated with URC in combination with IFN-γ might be used on DC-based vaccine for cancer immunotherapy.     

Lectin-dependent localization of cell surface sialic acid-binding lectin Siglec-9

Siglecs are immunoglobulin lectin group proteins that recognize the sialic acid moiety. We previously reported that the expression of Siglec-9 on the macrophage cell line RAW264 markedly enhanced Toll-like receptor (TLR)-induced interleukin (IL)-10 production and inhibited the production of proinflammatory cytokines. In this study, we examined the lectin-dependent anti-inflammatory activities of Siglec-9. IL-10 production was modestly reduced by a mutation that disrupted the lectin activity of Siglec-9, while the reduction in tumor necrosis factor-α was not affected. Membrane fractionation experiments revealed that a part of Siglec-9 resided in the detergent-insoluble microdomain, the so-called lipid raft fraction. The amount of Siglec-9 in the lipid raft fraction rapidly increased following TLR2 stimulation by peptidoglycan and peaked after 3–10 min. This time course was similar to that of TLR2. The double tyrosine mutant in immunoreceptor tyrosine-based inhibitory motifs moved to lipid rafts in a similar manner, while lectin-defective Siglec-9 was not detected in the lipid raft fraction. The production of IL-10 was partially reduced by cholesterol oxidase that disturbed lipid raft organization. Taken together, these results suggest that Siglecs exhibit lectin-dependent changes in cellular localization, which may be partly linked to its control mechanism that increases the production of IL-10.     

不同诱导因子对人外周血单个核细胞P2X7受体表达的作用

ATP激活P2X7受体可产生一系列的白细胞功能反应,因此P2X7受体的表达调控引起我们的兴趣。然而P2X7受体在正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)、单核细胞中的表达调控机制尚未阐明。本文用半定量RT-PCR方法检测多种细胞因子、细菌抗原、丝裂原对P2X7受体表达的诱导作用,探索P2X7受体的诱导表达模式。结果表明,单个核细胞和单核细胞可检出P2X7受体的表达;白细胞介素2、4、6(interleukin-2、-4、-6,IL-2、IL-4、IL-6)、肿瘤坏死因子仪(tumour necrosis factor-α,TNF-α)等细胞因子和金黄色葡萄球菌CowanⅠ株(Staphylococcus aureus Cowan strainⅠ,SAC)、脂多糖(lipopolysaccharide,LPS)能上调PBMC的P2X7受体表达,而γ干扰素(interferon-γ,IFN-γ)、粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GM-CSF)、巨噬细胞集落刺激因子(macmphage colony-stimulating factor,M-CSF)和植物血凝素(phytohemagglutinin-M,PHA-M)等则没有作用;LPS和M-CSF可以提高单核细胞的P2X7受体表达,IFN-γ、TNF-α、GM-CSF作用较弱,但是这些因子的预处理并不能增强LPS对P2X7受体表达的诱导。炎症因子促进P2X7受体的表达,提示P2X7受体可能在对抗细菌感染的免疫反应中起一定作用,这有待于进一步研究。     

Cytokine Regulation of Human Immunodeficiency Virus Type 1 Entry and Replication in Human Monocytes/Macrophages through Modulation of CCR5 Expression

Human macrophages express chemokine receptors that act as coreceptors for human immunodeficiency virus type 1 (HIV-1) and are major targets for HIV-1 infection in vivo. The effects of cytokines on HIV-1 infection of macrophages and on the expression of CCR5, the principal coreceptor for macrophage-tropic viruses, have now been investigated. Expression of CCR5 on the surface of freshly isolated human monocytes was virtually undetectable by flow cytometry with the monoclonal antibody 5C7. However, after culture of monocytes for 48 h in serum-free medium, approximately 30% of the resulting macrophages expressed CCR5 and the cells were susceptible to infection by macrophage-tropic HIV-1. Addition of either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cultures markedly increased both the extent of HIV-1 entry and replication as well as surface expression of CCR5. In contrast, addition of the T-helper 2 (Th2) cell-derived cytokine interleukin-4 (IL-4) or IL-13 prevented the expression of CCR5 induced by culture in medium alone, and IL-4 inhibited virus entry, replication, and cytopathicity under these conditions. IL-4 or IL-13 also prevented the stimulatory effects of M-CSF or GM-CSF on CCR5 expression as well as HIV-1 entry and replication. In addition, IL-4 reversed the increase in CCR5 expression induced by pretreatment of cells with M-CSF. Although IL-10 also inhibits HIV-1 replication in macrophages, it did not suppress surface CCR5 expression induced by colony-stimulating factors. These results indicate that the cytokine environment determines the susceptibility of macrophages to HIV-1 infection by various mechanisms, one of which is the regulation of HIV-1 coreceptor expression.     

Integrating macrophages into organotypic co-cultures: a 3D in vitro model to study tumor-associated macrophages

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.     

Cloning, characterization, and phylogenetic analysis of siglec-9, a new member of the CD33-related group of siglecs. Evidence for co-evolution with sialic acid synthesis pathways

The Siglecs are a subfamily of I-type lectins (immunoglobulin superfamily proteins that bind sugars) that specifically recognize sialic acids. We report the cloning and characterization of human Siglec-9. The cDNA encodes a type 1 transmembrane protein with three extracellular immunoglobulin-like domains and a cytosolic tail containing two tyrosines, one within a typical immunoreceptor tyrosine-based inhibitory motif (ITIM). The N-terminal V-set Ig domain has most amino acid residues typical of Siglecs. Siglec-9 is expressed on granulocytes and monocytes. Expression of the full-length cDNA in COS cells induces sialic-acid dependent erythrocyte binding. A recombinant soluble form of the extracellular domain binds to alpha2-3 and alpha2-6-linked sialic acids. Typical of Siglecs, the carboxyl group and side chain of sialic acid are essential for recognition, and mutation of a critical arginine residue in domain 1 abrogates binding. The underlying glycan structure also affects binding, with Galbeta1-4Glc[NAc] being preferred. Siglec-9 shows closest homology to Siglec-7 and both belong to a Siglec-3/CD33-related subset of Siglecs (with Siglecs-5, -6, and -8). The Siglec-9 gene is on chromosome 19q13.3-13.4, in a cluster with all Siglec-3/CD33-related Siglec genes, suggesting their origin by gene duplications. A homology search of the Drosophila melanogaster and Caenorhabditis elegans genomes suggests that Siglec expression may be limited to animals of deuterostome lineage, coincident with the appearance of the genes of the sialic acid biosynthetic pathway.     

TGF-β1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3 days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium⁺ uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium⁺ uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium⁺ uptake in a concentration-dependent fashion with a maximum effect at 5 ng/ml and with an IC₅₀ of ~ 0.4 ng/ml. Moreover, ATP-induced YO-PRO-1²⁺ uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity.     

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

Interferon-γ, tumor necrosis factor, and interleukin-18 cooperate to control growth of Mycobacterium tuberculosis in human macrophages

Mycobacterium tuberculosis (MTB) remains a leading infectious threat to human health. Macrophages are the cells targeted for infection by the bacterium as well as key effector cells for clearance of the pathogen. Interleukin (IL)-27 opposes macrophage-mediated control of MTB because supplying IL-12 and blocking the activity of IL-27 limits bacterial growth in primary human macrophages. The purpose of this study was to determine the immunological regulators of this macrophage mechanism to restrict MTB growth. Interferon (IFN)-γ, TNF-α, and IL-18 were all demonstrated to be important to the environment that limits bacterial growth when IL-12 is supplied and IL-27 is neutralized. We find IL-18 works in conjunction with IL-12 to achieve optimal IFN-γ production in this system. We also demonstrate novel interactions between these cytokines to influence the expression or responsiveness to one another. Quantitative assays show that IFN-γ enhances expression of the IL-18 receptor signaling chain, as well as TNF expression and secretion. In turn, TNF-α augments expression of the receptor for IFN-γ, the amount at the cell surface, and the extent of IFN-γ -induced signaling. We further define how the cytokine environment supports an enhanced state of classical macrophage activation. Collectively, these results describe novel immunological mechanisms that provide additional insights into the effects of IL-12 and IL-27 on macrophage regulation during MTB infection.     

Production of IL-1 receptor antagonist by human in vitro-derived macrophages. Effects of lipopolysaccharide and granulocyte-macrophage colony-stimulating factor.

The objective of these experiments was to evaluate the production of IL-1ra, a specific receptor antagonist of IL-1, by human in vitro-derived macrophages, a model for differentiated macrophages. IL-1ra protein levels in supernatants and lysates of cultured cells were determined by a specific ELISA. Relative steady-state IL-1ra mRNA levels were measured using a specific cDNA probe. Human monocytes were differentiated by 6 days culture in either medium or granulocyte-macrophage colony-stimulating factor (GM-CSF), after which the effects of subsequent LPS and/or GM-CSF on the production of IL-1ra were evaluated. In vitro-derived macrophages cultured in medium for 6 days constitutively produced IL-1ra protein during the 24-h period of the 7th day in culture. The constitutive production of IL-1ra by medium-aged cells correlated with low steady-state IL-1ra mRNA levels determined over this same time period. In contrast, cells cultured for 6 days in GM-CSF synthesized significantly increased levels of IL-1ra protein during the 7th day in culture but the secreted levels remained unchanged. Cells differentiated in GM-CSF displayed enhanced steady-state levels of IL-1ra mRNA in comparison with cells aged in medium. Stimulation of in vitro-derived macrophages aged for 6 days in medium or in GM-CSF, with LPS or adherent IgG, did not result in increased levels of IL-1ra protein production in comparison with non-LPS stimulated cells. The IL-1ra protein detected in the supernatants of cells differentiated in GM-CSF was biologically active in the IL-1-augmented murine thymocyte proliferation assay. By Western blot analysis, the IL-1ra protein in the in vitro-derived macrophage supernatants was predominantly the 22- to 24-kDa glycosylated species, whereas the lysates contained additional lower molecular weight forms. These results suggest that as monocytes differentiate in vitro into macrophages, they constitutively produce IL-1ra protein and that this production is enhanced by the continuous presence of GM-CSF.     

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.     

Gender difference in tumor necrosis factor-α production in human neutrophils stimulated by lipopolysaccharide and interferon-γ

The gender difference in tumor necrosis factor-α (TNF-α) production in human neutrophils stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was explored by using peripheral blood neutrophils from young men and women. As compared with female neutrophils, male neutrophils released greater amounts of TNF-α, and exhibited stronger activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase in response to LPS stimulation. LPS-induced TNF-α production was markedly enhanced by pretreatment of cells with IFN-γ, and IFN-γ-mediated priming in male neutrophils was significantly greater than that in female neutrophils. Male neutrophils showed higher expression of TLR4, but not IFN-γ receptors, than female neutrophils, and its expression was increased by stimulation with IFN-γ or IFN-γ plus LPS. These findings indicate that male neutrophils show higher responsiveness to stimulation with LPS and IFN-γ than female neutrophils, and suggest that the gender difference in neutrophil responsiveness to LPS and IFN-γ is partly responsible for that in the outcome of sepsis, in which premenopausal women show a favorable prognosis as compared with men.     

M2 macrophages induced by prostaglandin E2 and IL-6 from cervical carcinoma are switched to activated M1 macrophages by CD4+ Th1 cells

Monocytes attracted by tumor-induced chronic inflammation differentiate to APCs, the type of which depends on cues in the local tumor milieu. In this work, we studied the influence of human cervical cancer cells on monocyte differentiation and showed that the majority of cancer cells either hampered monocyte to dendritic cell differentiation or skewed their differentiation toward M2-like macrophages. Blocking studies revealed that M2 differentiation was caused by tumor-produced PGE(2) and IL-6. TGF-β, IL-10, VEGF, and macrophage colony-stimulating factor did not play a role. Notably, these CD14(+)CD163(+) M2 macrophages were also detected in situ. Activation of cancer cell-induced M2-like macrophages by several TLR-agonists revealed that compared with dendritic cells, these M2 macrophages displayed a tolerogenic phenotype reflected by a lower expression of costimulatory molecules, an altered balance in IL-12p70 and IL-10 production, and a poor capacity to stimulate T cell proliferation and IFN-γ production. Notably, upon cognate interaction with Th1 cells, these tumor-induced M2 macrophages could be switched to activated M1-like macrophages that expressed high levels of costimulatory molecules, produced high amounts of IL-12 and low amounts of IL-10, and acquired the lymphoid homing marker CCR7. The effects of the interaction between M2 macrophages and Th1 cells could partially be mimicked by activation of these APCs via CD40 in the presence of IFN-γ. Our data on the presence, induction, and plasticity of tumor-induced tolerogenic APCs in cervical cancer suggest that tumor-infiltrated Th1 cells can stimulate a tumor-rejecting environment by switching M2 macrophages to classical proinflammatory M1 macrophages.     

Induction of hepatocyte growth factor/scatter factor by interferon-γ in human leukemia cells

Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-γ remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-α and IFN-β were as effective as IFN-γ in RPMI-8226 cells, but less than IFN-γ in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-γ. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1β, and tumor necrosis factor (TNF)-α, the former three were as effective as IFN-γ in KG-1 cells, but only TNF-α stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-α, IFN-β, and IFN-γ. The effect of IFN-γ in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-γ in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-γ is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-γ is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis. J. Cell. Physiol. 174:107–114, 1998. © 1998 Wiley-Liss, Inc.