Granulocyte Colony-stimulating Factor mRNA Expression Research Articles

Kidney injury enhances renal G-CSF expression and modulates granulopoiesis and human neutrophil CD177 in vivo.

SummaryKidney injury significantly increases overall mortality. Neutrophilic granulocytes (neutrophils) are the most abundant human blood leukocytes. They are characterized by a high turnover rate, chiefly controlled by granulocyte colony stimulating factor (G‐CSF). The role of kidney injury and uremia in regulation of granulopoiesis has not been reported. Kidney transplantation, which inherently causes ischemia–reperfusion injury of the graft, elevated human neutrophil expression of the surface glycoprotein CD177. CD177 is among the most G‐CSF‐responsive neutrophil genes and reversibly increased on neutrophils of healthy donors who received recombinant G‐CSF. In kidney graft recipients, a transient rise in neutrophil CD177 correlated with renal tubular epithelial G‐CSF expression. In contrast, CD177 was unaltered in patients with chronic renal impairment and independent of renal replacement therapy. Under controlled conditions of experimental ischemia–reperfusion and unilateral ureteral obstruction injuries in mice, renal G‐CSF mRNA and protein expression significantly increased and systemic neutrophilia developed. Human renal tubular epithelial cell G‐CSF expression was promoted by hypoxia and proinflammatory cytokine interleukin 17A in vitro. Clinically, recipients of ABO blood group‐incompatible kidney grafts developed a larger rise in neutrophil CD177. Their grafts are characterized by complement C4d deposition on the renal endothelium, even in the absence of rejection. Indeed, complement activation, but not hypoxia, induced primary human endothelial cell G‐CSF expression. Our data demonstrate that kidney injury induces renal G‐CSF expression and modulates granulopoiesis. They delineate differential G‐CSF regulation in renal epithelium and endothelium. Altered granulopoiesis may contribute to the systemic impact of kidney injury.

Ureaplasma species modulate cell adhesion molecules and growth factors in human brain microvascular endothelial cells

Ureaplasma species (spp.) are considered commensals of the adult urogenital tract, but may cause chorioamnionitis and preterm birth as well as sepsis and meningitis in neonates. Pathomechanisms in Ureaplasma-driven neuroinflammation are largely unknown. This study addressed mRNA and protein expression of intercellular and vascular cell adhesion molecules (ICAM-1, VCAM-1), granulocyte-colony stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF) in native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) exposed to Ureaplasma (U.) urealyticum or U. parvum. Ureaplasma spp. reduced G-CSF mRNA (p < 0.05) and protein expression (p < 0.01) and increased VEGF mRNA levels (p < 0.01) in native HBMEC. Upon co-stimulation, Ureaplasma isolates enhanced LPS-evoked VEGF and ICAM-1 mRNA expression (p < 0.05), but mitigated G-CSF and VCAM-1 mRNA responses (p < 0.05). In line with previous findings, our results indicate an ability of Ureaplasma spp. to compromise blood-brain barrier integrity, mitigate immune defense, and subdue neuroprotective mechanisms. This may facilitate intracerebral inflammation, allow chronic infections, and promote brain injury. More pronounced effects in co-stimulated cells may indicate an immunomodulatory capacity of Ureaplasma spp.

LPS-Induced G-CSF Expression in Macrophages Is Mediated by ERK2, but Not ERK1.

Granulocyte colony-stimulating factor (G-CSF) selectively stimulates proliferation and differentiation of neutrophil progenitors which play important roles in host defense against infectious agents. However, persistent G-CSF production often leads to neutrophilia and excessive inflammatory reactions. There is therefore a need to understand the mechanism regulating G-CSF expression. In this study, we showed that U0126, a MEK1/2 inhibitor, decreases lipopolysaccharide (LPS)-stimulated G-CSF promoter activity, mRNA expression and protein secretion. Using short hairpin RNA knockdown, we demonstrated that ERK2, and not ERK1, involves in LPS-induced G-CSF expression, but not LPS-regulated expression of TNF-α. Reporter assays showed that ERK2 and C/EBPβ synergistically activate G-CSF promoter activity. Further chromatin immunoprecipitation (ChIP) assays revealed that U0126 inhibits LPS-induced binding of NF-κB (p50/p65) and C/EBPβ to the G-CSF promoter, but not their nuclear protein levels. Knockdown of ERK2 inhibits LPS-induced accessibility of the G-CSF promoter region to DNase I, suggesting that chromatin remodeling may occur. These findings clarify that ERK2, rather than ERK1, mediates LPS-induced G-CSF expression in macrophages by remodeling chromatin, and stimulates C/EBPβ-dependent activation of the G-CSF promoter. This study provides a potential target for regulating G-CSF expression.

Pivotal role of IL-6 in the hyperinflammatory responses to subacute ozone in adiponectin-deficient mice

Adiponectin is an adipose-derived hormone with anti-inflammatory activity. Following subacute ozone exposure (0.3 ppm for 24-72 h), neutrophilic inflammation and IL-6 are augmented in adiponectin-deficient (Adipo(-/-)) mice. The IL-17/granulocyte colony-stimulating factor (G-CSF) axis is required for this increased neutrophilia. We hypothesized that elevated IL-6 in Adipo(-/-) mice contributes to their augmented responses to ozone via effects on IL-17A expression. Therefore, we generated mice deficient in both adiponectin and IL-6 (Adipo(-/-)/IL-6(-/-)) and exposed them to ozone or air. In ozone-exposed mice, bronchoalveolar lavage (BAL) neutrophils, IL-6, and G-CSF, and pulmonary Il17a mRNA expression were greater in Adipo(-/-) vs. wild-type mice, but reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice. IL-17A(+) F4/80(+) cells and IL-17A(+) γδ T cells were also reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice exposed to ozone. Only BAL neutrophils were reduced in IL-6(-/-) vs. wild-type mice. In wild-type mice, IL-6 was expressed in Gr-1(+)F4/80(-)CD11c(-) cells, whereas in Adipo(-/-) mice F4/80(+)CD11c(+) cells also expressed IL-6, suggesting that IL-6 is regulated by adiponectin in these alveolar macrophages. Transcriptomic analysis identified serum amyloid A3 (Saa3), which promotes IL-17A expression, as the gene most differentially augmented by ozone in Adipo(-/-) vs. wild-type mice. After ozone, Saa3 mRNA expression was markedly greater in Adipo(-/-) vs. wild-type mice but reduced in Adipo(-/-)/IL-6(-/-) vs. Adipo(-/-) mice. In conclusion, our data support a pivotal role of IL-6 in the hyperinflammatory condition observed in Adipo(-/-) mice after ozone exposure and suggest that this role of IL-6 involves its ability to induce Saa3, IL-17A, and G-CSF.

Extracellular signal-regulated kinase 2 mediates the expression of granulocyte colony-stimulating factor in invasive cancer cells

Granulocyte colony-stimulating factor (G-CSF) affects granulopoiesis and is important for mobilizing neutrophils into blood circulation. Due to the hematopoietic properties of G-CSF, it has been widely used to clinically treat chemotherapy-induced neutropenia. However, G-CSF can promote tumors by inhibiting innate and adaptive immunity and enhancing angiogenesis and neoplastic growth. Most G-CSF-producing tumors are associated with a poor prognosis. This indicates that G-CSF promotes cancer progression. Thus, identifying regulatory molecules involved in tumor-derived G-CSF expression may provide therapeutic targets for cancer treatment. This study identified considerable G-CSF expression in malignant breast, lung and oral cancer cells. However, G-CSF expression was barely detectable in non-invasive cell lines. Expression of G-CSF mRNA and protein increased during exposure to tumor necrosis factor-α (TNF-α). Treatment with U0126 (a mitogen-activated protein kinase inhibitor) drastically reduced basal levels of G-CSF and TNF-α-induced G-CSF in aggressive cancer cells. This study also showed that knockdown of extracellular signal-regulated kinase (ERK)2 by shRNA was necessary and sufficient to eliminate the expression of tumor-derived G-CSF. This did not apply to ERK1. Therefore, ERK2 (but not ERK1) is responsible for the transcriptional regulation of tumor-derived G-CSF. The results indicate the pharmaceutical value of specific ERK2 inhibitors in treating patients with G-CSF-producing tumors.

IL-17A stimulates granulocyte colony-stimulating factor production via ERK1/2 but not p38 or JNK in human renal proximal tubular epithelial cells

We investigated the potential role of IL-17A in the induction of granulocyte colony-stimulating factor (G-CSF), a critical granulopoietic growth factor, in human renal proximal tubular epithelial cells. Human renal proximal tubular cells (HK-2, ATCC) were used to characterize the effects of IL-17A or IL-17F on G-CSF production, using ELISA, real-time RT-PCR, and immunoblotting. The cell surface expression of IL-17 receptors (IL-17Rs) was analyzed by flow cytometry. IL-17A stimulation of proximal tubular cells led to a dose- and time-dependent increase in secreted G-CSF. This effect was dependent on mRNA transcription and protein translation. Real-time RT-PCR demonstrated that G-CSF mRNA expression reached a maximum level at 6 h following IL-17A stimulation and that this increase was dose dependent. Both IL-17RA and IL-17RC were expressed on proximal tubular cells. IL-17A also enhanced TNF-α- or IL-1β-mediated G-CSF secretion from cells. Additionally, IL-17A induced MAPK (ERK1/2 but not p38 MAPK or JNK) activation, and pharmacological inhibitors of MEK1/2 (U0126) but not of p38 MAPK (SB203580) or JNK (SP600125), significantly blocked the IL-17A-mediated G-CSF release. We demonstrated the potential ability of IL-17A to induce G-CSF in renal proximal tubular cells. It is proposed that IL-17A may play an important role in neutrophil transmigration and activation via stimulation of G-CSF in tubular injury.

Granulocyte-colony stimulating factor is involved in low-dose LPS-induced neuroprotection

Granulocyte-colony stimulating factor (G-CSF) has recently been noted for neuroprotective function. Evidence has been given to indicate that G-CSF is naturally expressed in neurons and directly activates anti-apoptosis pathways. Finding out the agents inducing G-CSF production is of value for understanding the neuroprotection network in central nervous system. It is known that lipopolysaccharide (LPS) can induce macrophages to produce G-CSF. Here we demonstrate that hippocampal neurons exhibited the expression of toll-like receptor-4, and prove that low-dose LPS treatment increased the expression and production of G-CSF mRNA and protein in cultured neurons. We further indicate that the neutralization of G-CSF with corresponding anti-G-CSF antibodies abolished the neuroprotective effect of LPS pretreatment in N-methyl-d-aspartic acid-induced neuronal injury by MTT/CCK-8 assays and LDH release. Thus our results reveal that G-CSF may be involved in LPS-mediated neuroprotection in vivo.

Induction of granulocyte colony-stimulating factor by globular adiponectin via the MEK–ERK pathway

Adiponectin, an adipocyte-derived cytokine, affects a number of physiological processes, including immune function and inflammation. We investigated whether globular adiponectin (gAd) affects the expression of inflammation-related genes in murine macrophages (RAW264 cells). DNA microarray analysis indicated that granulocyte colony-stimulating factor (G-CSF) showed the largest increase in expression in gAd-stimulated RAW264 cells. The gAd-induced secretion of G-CSF increased in a time- and dose-dependent manner. U0126 (MEK1/2 inhibitor) and PD98059 (MEK1 inhibitor) reduced the gAd-induced G-CSF mRNA expression and G-CSF protein production. gAd induced the phosphorylation of MEK1/2 and ERK1/2 in RAW264 cells. In addition, the gAd-induced phosphorylation of MEK1/2 and ERK1/2 was dramatically reduced by PD98059 and U0126, respectively. Collectively, these results suggest that MEK1/2-ERK1/2 signaling is involved in the adiponectin-induced secretion of G-CSF.

Expression of granulocyte colony-stimulating factor is induced in injured rat carotid arteries and mediates vascular smooth muscle cell migration.

Granulocyte colony-stimulating factor (G-CSF) is a lineage-restricted hematopoietic growth factor that stimulates proliferation and maturation of hematopoietic progenitors and is a known powerful mobilizer of bone marrow-derived stem cells. Very little has been reported on G-CSF expression and modulation of vascular smooth muscle cell (VSMC) activation. The purpose of this study was to characterize the expression and effects of G-CSF on primary human VSMC and balloon angioplasty-injured rat carotid arteries. In cultured human VSMC, G-CSF mRNA and protein expression are induced by several cytokines, with the most potent being fetal calf serum and T-lymphocyte-conditioned media. G-CSF is not expressed in naive rat carotid arteries but is induced in neointimal SMC in carotid arteries subject to balloon angioplasty. G-CSF is chemotactic for human VSMC. There is a significant difference between unstimulated cells and those treated with G-CSF at 100 and 1,000 pg/ml (P < 0.01 and 0.05 for 3 experiments). G-CSF also activates the GTPase Rac1, a regulator of cellular migration in VSMC. Inhibition of Rac1 inhibits G-CSF-driven VSMC migration. Important signal transduction protein kinases, including p44/42 MAPK, Akt, and S6 kinase, are also activated in response to G-CSF. This is the first report describing the expression of G-CSF in injured arteries and the multiple effects of G-CSF on VSMC activation. Together, our data suggest that G-CSF is an important mediator of inflammatory cell-VSMC communication and VSMC autocrine activation and may be an important mediator of the VSMC response to injury.

Granulocyte Colony Stimulating Factor-Producing Multiple Myeloma Associated with Neutrophilia

We report a case of IgG-kappa multiple myeloma associated with neutrophilia (WBC 31,300/microl, neutrophil 90.5%). Interestingly, the serum level of granulocyte colony stimulating factor (G-CSF) in this patient was elevated to 1,500 pg/ml (normal range: 5.78-27.5). Plasma cells were 35% in the bone marrow and were strongly stained with anti-G-CSF antibody. To directly study the production of G-CSF from plasma cells in this patient, CD138 positive plasma cells were purified from bone marrow of multiple myeloma patients by magnetic sorting. The expression of G-CSF mRNA was observed in CD138 positive plasma cells from this myeloma patient with neutrophilia by RT-PCR. In contrast, the expression of G-CSF mRNA was not detected in CD138 positive plasma cells from the other multiple myeloma patients without neutrophilia and 4 human myeloma cell lines (HS-Sultan, IM9, RPMI8226, U266) by RT-PCR. After the CD138 positive plasma cells were cultured in vitro for 48 h, the production of G-CSF protein was confirmed (71.8 pg/ml) in the supernatant by ELISA. These results indicated plasma cells of this myeloma patient directly produced G-CSF and that this was the primary cause of neutrophilia.

Establishment and Characterization of a New Lung Cancer Cell Line (MI‐4) Producing High Levels of Granulocyte Colony Stimulating Factor

We established a human lung cancer cell line, MI‐4 from the pleural effusion of a 69‐year‐old male with advanced large cell undifferentiated carcinoma of the lung complicated by leukocytosis. The culture supernatant of MI‐4 contained high levels of granulocyte colony stimulating factor (G‐CSF). The intracellular localization of the G‐CSF was identified by immunocytochemistry. Reverse transcription‐polymerase chain reaction (RT‐PCR) revealed G‐CSF mRNA expression in this cell line. The cell line was successfully transplanted into nude mice. The transplanted nude mice also showed leukocytosis with a high serum G‐CSF level. Southern blot analysis did not show amplification or rearrangement of the G‐CSF gene in MI‐4 cells. Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) analyses revealed that this cell line has an additional chromosome 17 attached to a segment of chromosome 10 besides two intact chromosomes 17, and that each of these three chromosomes 17 has a G‐CSF gene on chromosome 17q. Inflammatory cytokines, tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, significantly enhanced G‐CSF expression at both the protein and mRNA levels in MI‐4. However, these cytokines did not stimulate the growth of MI‐4 cells, regardless of abundant G‐CSF production. TNF‐α rather suppressed it, in a dose‐dependent manner. Exogenous recombinant human G‐CSF and anti‐G‐CSF antibody did not promote or inhibit the growth of MI‐4 cells at any concentration examined. In addition, RT‐PCR analysis did not show G‐CSF receptor mRNA expression. These results suggest that this cell line does not have an autocrine growth loop for G‐CSF. This cell line should be very useful for understanding the biological activity of G‐CSF in G‐CSF‐overproducing lung cancer.

Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor

Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron–exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.

Granulocyte colony-stimulating factor-producing primary pericardial mesothelioma

A 54-year-old male patient presented with a granulocyte colony-stimulating factor (G-CSF)-producing primary pericardial mesothelioma, while showing symptoms of congestive heart failure, a fever of 38 to 39°C, and marked leucocytosis of 52.7 × 10 3 cells/mm 3. The histopathologic diagnosis was established after autopsy. G-CSF production was confirmed by the expression of G-CSF mRNA in the tumor extract and the patient's high serum G-CSF concentration. The expression of G-CSF by benign and malignant mesothelial cells has already been reported. However, this is the first case report of G-CSF production in a pericardial mesothelioma.

Alveolar type II-like cells release G-CSF as neutrophil chemotactic activity.

We evaluated the potential of A549 cells, an alveolar type II epithelial cell line, to release granulocyte colony-stimulating factor (G-CSF), in addition to interleukin (IL)-8 and leukotriene B4, as neutrophil chemotactic activity (NCA). Human recombinant IL-1beta stimulated A549 cells to release NCA in a time- and dose-dependent fashion. The released NCA was blocked by mouse anti-human G-CSF polyclonal antibody. Molecular-sieve column chromatography revealed that IL-1beta induced the release of a 19- to 20-kDa chemotactic mass that was inhibited by anti-human G-CSF antibody. IL-1beta stimulated the release of G-CSF in a dose-dependent fashion, but the time-dependent profile of G-CSF showed that the concentration of G-CSF declined after 48 h. Tumor necrosis factor (TNF)-alpha, Escherichia coli lipopolysaccharide (LPS), and bradykinin (BK) stimulated A549 cells to release NCA that was inhibited by anti-G-CSF antibody. The release of G-CSF in response to TNF-alpha, LPS, and BK was significantly increased. The similar concentrations of human recombinant G-CSF (10-1,000 pg/ml) as in the supernatant fluid induced neutrophil chemotaxis. G-CSF mRNA was expressed time and dose dependently at 4 h and declined after 4 h in response to IL-1beta as evaluated by RT-PCR. The expression of G-CSF mRNA was also observed by TNF-alpha, LPS, and BK stimulation. These data suggest that type II alveolar epithelial cells may produce G-CSF as NCA and may participate in the regulation of leukocyte extravasation.

Interleukin-1β and Tumor Necrosis Factor-α Stimulate Granulocyte Colony-Stimulating Factor Production by Placental Villous Core Mesenchymal Cells

To test the hypothesis that interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) regulate granulocyte colony-stimulating factor (G-CSF) production by human placental villous core mesenchymal cells. Villous core mesenchymal cells were isolated from placentas at 14-20 weeks' gestation and cultured in vitro. Cells were treated with IL-1 beta or TNF-alpha in dose-response and time-course studies. We measured G-CSF mRNA expression by Northern blot analysis and G-CSF protein production by enzyme-linked immunosorbent assay of the conditioned media. Unstimulated mesenchymal cells expressed negligible G-CSF. Steady-state G-CSF mRNA expression was maximal 3-6 hours after IL-1 beta treatment and 6-18 hours after TNF-alpha treatment. Each cytokine induced G-CSF protein production in dose-and time-dependent manners, with IL-1 beta more potent than TNF-alpha. The G-CSF mRNA expression and G-CSF protein production induced by the combination of both cytokines exceeded that induced by either cytokine alone. Interleukin-1 beta and TNF-alpha stimulate G-CSF production by placental villous core mesenchymal cells in vitro. These results identify a potential mechanism by which villous core mesenchymal cells mediate, in part, the placental response to these two cytokines.

Granulocyte colony-stimulating factor (G-CSF) production by astrocytoma cells and its effect on tumor growth.

We investigated the expression of granulocyte colony-stimulating factor (G-CSF), G-CSF mRNA, and G-CSF receptor mRNA in astrocytoma cell lines, G-CSF in astrocytoma cyst fluid, and the effect of recombinant G-CSF on the proliferation of astrocytoma cells in vitro and in vivo. We first examined supernatants from astrocytoma cell lines for the presence of G-CSF by ELISA. G-CSF was expressed by 6 of 14 astrocytoma cell lines constitutively, and, was detected after stimulation with tumor necrosis factor-alpha (TNF-alpha) in four of eight cell lines which did not produce G-CSF constitutively. G-CSF mRNA was detected by reverse-transcriptase polymerase chain reaction (RT-PCR) in all cell lines studied, suggesting that astrocytoma cells have the potential to produce G-CSF. We also analyzed the presence of G-CSF by ELISA in five astrocytoma cyst fluids. G-CSF was detected in one case. Although, in vitro study, the growth of glioma cells was not affected by rG-CSF, in a mouse model, the administration of G-CSF significantly shortened the time to tumor appearance and accelerated tumor growth. These data suggest that G-CSF has a stimulatory effect on the proliferation of astrocytoma cells in vivo through the mediation of host factors.

Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) in Behçet's disease.

Increases in the number and activity of peripheral polymorphonuclear neutrophils (PMNs) is often found in Behçet's disease (BD), indicating that PMN may play an important role in the pathogenesis of this disorder. It has recently been reported that G-CSF and GM-CSF, a family of hematopoietic growth factors, enhance PMN activity. To explore the role of these two CSFs in BD, we first examined the chemotactic response of PMNs to these CSFs by performing a polarization assay. PMN response to G-CSF in BD patients was lower than that in controls, while PMN response to GM-CSF was similar in patients and controls. However, PMNs from BD patients showed an enhanced chemotactic response to N-formyl-L-methionyl-leucyl-phenylalanine. Thus, it is speculated that the PMNs of the patients might have already been activated in vivo by G-CSF and thus could not respond further to this agent in vitro. We examined G-CSF and GM-CSF mRNA expressions in peripheral mononuclear cells stimulated with LPS, PMA, and Con A by Northern hybridization. G-CSF mRNA expression levels in BD patients were higher than in the controls, while GM-CSF mRNA expression levels were lower than in the controls. We also examined the serum levels of the two CSFs by ELISA and EIA. However, all levels of the two CSFs in both patients and controls were not detectable, except in the case of one BD patient in the active stage of the disease, who showed high levels of G-CSF, but not of GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesion of NFS-60 myeloid leukemia cells to MC3T3-G2/PA6 stromal cells induces granulocyte colony-stimulating factor production

To examine the interaction between immature myeloid cells and stromal cells in the induction of granulocyte colony-stimulating factor (G-CSF) production, stromal cells of the MC3T3-G2/PA6 (PA6) murine cell line, which has preadipocyte characteristics and can support hematopoiesis, were cocultured with various myeloid cell lines and G-CSF mRNA expression was examined by Northern and reverse transcriptase- polymerase chain reaction analyses. A significant amount of G-CSF mRNA was induced by the culture of an interleukin-3/G-CSF-dependent murine myeloid leukemia cell line, NFS-60, on PA6 stromal cells for 16 hours. Using a G-CSF-dependent subline of DA-1 (DA-1N), the biologic activity of G-CSF was also detected in PA6/NFS-60 coculture supernatants, but not in the culture supernatant of PA6 or NFS-60 alone. Direct contact of NFS-60 cells with the PA6 stromal layer was essential for the induction of G-CSF mRNA, as indicated by the following observations: (1) NFS-60 cells efficiently adhered to PA6 cells; (2) medium conditioned by NFS-60 cells did not contain the activity to induce G- CSF mRNA in PA6 cells; and (3) induction of G-CSF mRNA was not observed when NFS-60 cells were separated from PA6 cells by a microporous membrane (0.45-microns pore size). Several other myeloid cell lines, including FDC-P2, 32Dcl3, WEHI-3, and DA-1, did not induce G-CSF mRNA expression after the coculture with PA6 cells, although significant numbers of these cells adhered to PA6 cells. Therefore, NFS-60 cells may express or overexpress a molecule that is involved in adhesion- mediated induction of G-CSF production.

Adhesion of NFS-60 Myeloid Leukemia Cells to MC3T3-G2/PA6 Stromal Cells Induces Granulocyte Colony-Stimulating Factor Production

To examine the interaction between immature myeloid cells and stromal cells in the induction of granulocyte colony-stimulating factor (G-CSF) production, stromal cells of the MC3T3-G2/PA6 (PA6) murine cell line, which has preadipocyte characteristics and can support hematopoiesis, were cocultured with various myeloid cell lines and G-CSF mRNA expression was examined by Northern and reverse transcriptase-polymerase chain reaction analyses. A significant amount of G-CSF mRNA was induced by the culture of an interleukin-3/G-CSF-dependent murine myeloid leukemia cell line, NFS-60, on PA6 stromal cells for 16 hours. Using a G-CSF-dependent subline of DA-1 (DA-1N), the biologic activity of G-CSF was also detected in PA6/NFS-60 coculture supernatants, but not in the culture supernatant of PA6 or NFS-60 alone. Direct contact of NFS-60 cells with the PA6 stromal layer was essential for the induction of G-CSF mRNA, as indicated by the following observations: (1) NFS-60 cells efficiently adhered to PA6 cells; (2) medium conditioned by NFS-60 cells did not contain the activity to induce G-CSF mRNA in PA6 cells; and (3) induction of G-CSF mRNA was not observed when NFS-60 cells were separated from PA6 cells by a microporous membrane (0.45-microns pore size). Several other myeloid cell lines, including FDC-P2, 32Dcl3, WEHI-3, and DA-1, did not induce G-CSF mRNA expression after the coculture with PA6 cells, although significant numbers of these cells adhered to PA6 cells. Therefore, NFS-60 cells may express or overexpress a molecule that is involved in adhesion-mediated induction of G-CSF production.

G(AnH)MTetra, a naturally occurring 1,6-anhydro muramyl dipeptide, induces granulocyte colony-stimulating factor expression in human monocytes: a molecular analysis.

N-Acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-isoglutam yl-m- diaminopimelyl-D-alanine [G (Anh)MTetra], a naturally occurring breakdown product of peptidoglycan from bacterial cell walls, was studied for its ability to induce granulocyte colony-stimulating factor (G-CSF) mRNA and protein expression in human adherent monocytes. Resting monocytes did not express G-CSF mRNA or secrete G-CSF protein. In contrast, monocytes exposed to G(Anh)MTetra showed a dose-dependent increase in G-CSF mRNA accumulation, which correlates with the secretion of G-CSF protein. Maximal levels of G-CSF mRNA were reached within 2 h of activation. Expression of G-CSF was mediated by an increase in the stability of G-CSF transcripts rather than by an increase in the transcription rate of the G-CSF gene. Experiments with the protein synthesis inhibitor cycloheximide revealed that G(Anh)MTetra-induced G-CSF mRNA expression was independent of new protein synthesis. Furthermore, it was shown that the effect of G(Anh)MTetra was regulated by a protein kinase C-dependent pathway, whereas protein kinase A and tyrosine kinases were not involved. Finally, it was shown that G(Anh)MTetra also induced G-CSF mRNA expression in human endothelial cells. The data indicate that, besides lipopolysaccharide, other naturally occurring bacterial cell wall components are able to induce G-CSF expression in different hematopoietic cells.