Immunoreactive Granulocyte-macrophage Colony-stimulating Factor Research Articles

Regulation of endometrial granulocyte macrophage-colony stimulating factor (GM-CSF) in the ewe

Granulocyte macrophage-colony stimulating factor (GM-CSF) increases ovine interferon-tau (oIFNτ) secretion by ovine conceptuses, but endometrial production of GM-CSF has not been characterized. Endometrial GM-CSF expression was evaluated in ovariectomized ewes implanted with estradiol-17β (E 2) and/or progesterone (P 4) for 14 days, in day 14 cyclic and day 14 pregnant ewes. Relative levels of endometrial GM-CSF mRNA were 3-fold higher in E 2- and E 2/P 4-treated ewes than that of control or P 4-treated ovariectomized ewes. Levels of endometrial GM-CSF mRNA for cyclic ewes were similar to E 2- and E 2/P 4-treated ewes, but amounts of GM-CSF mRNA in pregnant ewes were 2-fold higher. GM-CSF concentrations in endometrial culture media, determined by GM-CSF bioassay, for cyclic and E 2/P 4-treated ovariectomized ewes were 3-fold higher than those of control, E 2- and P 4-treated ovariectomized ewes; however, amounts of GM-CSF in pregnant ewes were 2-fold higher. Immunoreactive GM-CSF, examined by western blot, was detected in the culture medium from E 2/P 4-treated ovariectomized, cyclic and pregnant ewes. Luminal and glandular epithelia and stromal regions were determined to be sites of GM-CSF expression by immunohistochemistry and in situ hybridization techniques. Data indicate that combined E 2 and P 4 treatment of ovariectomized ewes is sufficient to restore GM-CSF expression to the level found in cyclic ewes; however, GM-CSF mRNA and protein in pregnant ewes is 2-fold greater than in ovariectomized or cyclic ewes. These data suggest that the conceptus, in addition to steroids, may play a role in the regulation of endometrial production of GM-CSF.

Detection of granulocyte-macrophage colony-stimulating factor in patients with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is an idiopathic lung disease in which the alveolar spaces are filled with surfactant. Recently, it has been proposed that PAP is caused by deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) because GM-CSF-knockout mice develop the disease. To examine this possibility, we tested the two hypotheses that lung GM-CSF levels are low and that alveolar macrophages (AM) do not respond to GM-CSF in patients with PAP. Data from 10 adult patients with PAP who underwent therapeutic whole-lung lavage were compared with those of 10 healthy volunteers who underwent bronchoalveolar lavage (BAL) by fiberoptic bronchoscopy. Bronchoalveolar lavage fluid (BALF) and plasma were collected and analyzed for total protein and levels of GM-CSF, interleukin-3, and tumor necrosis factor (TNF)-alpha. Isolated AM were cultured with or without lipopolysaccharide (LPS) or GM-CSF, and production of GM-CSF and TNF-alpha was measured after 24 h. GM-CSF in BALF and plasma was higher in PAP than in control subjects (p </= 0.05), and was detectable under both reducing and nonreducing conditions as a 28-kD protein in BALF from the PAP patients. GM-CSF release by unstimulated AM from PAP patients was higher than in cells from control subjects, but the responses to LPS were similar. Mean TNF-alpha release by AM in response to GM-CSF was higher in control subjects than in PAP patients due to a low response in three patients. In conclusion, unbound immunoreactive GM-CSF is detectable in BALF and plasma of PAP patients. Most PAP patients also had intact AM responses to GM-CSF, although some may have had defects in GM-CSF receptor or signal-transduction mechanisms. Although these data exclude lack of GM-CSF production as a common etiology of human PAP, defects in GM-CSF function in PAP are under investigation.

Localization of granulocyte-macrophage colony-stimulating factor in the bovine reproductive tract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can increase embryo development to the blastocyst stage in cattle. The objective of the present study was to determine whether GM-CSF is present in the reproductive tract. Using Western blotting, immunoreactive GM-CSF was detected in uterine flushings from cows at days 0, 7, and 14 of the estrous cycle and from cows at days 14–17 of pregnancy. Also, GM-CSF was localized immunohistochemically to endometrium and oviduct. Patterns of immunohistochemical localization and intensity of reaction product were similar for all days of the estrous cycle. While present in several cell types, immunoreactive product in the endometrium was greatest in epithelium (especially luminal epithelium). Immunoreactive GM-CSF was also localized to epithelium in ampullary and isthmic regions of the oviduct, with intensity greater in ampulla. Staining was observed for both ciliated and non-ciliated cells. In conclusion, the bovine oviduct and endometrium contain immunoreactive GM-CSF and this molecule is present in uterine secretions. Thus, this cytokine is a potentially important intracellular regulator of endometrial, oviductal and embryonic function during early pregnancy in the cow.

The Expression of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Its Regulation by Ovarian Steroids in Rat Uterine Stromal Cells

The effects of ovarian steroids on the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in rat uterus were examined. Intense expression of GM-CSF mRNA was dispersedly located in the endometrial-myometrial junction and stroma of the uterus. GM-CSF was immuno-histochemically localized in stromal cells and luminal epithelium. Ovariectomy significantly reduced the appearance of GM-CSF-mRNA-positive cells and the levels of expression for GM-CSF mRNA in the whole uterus, whereas treatment with 17β-estradiol (E2) or a combination of E2 and progesterone (P4) for 5 days on ovariectomized animals recruited GM-CSF-mRNA-positive cells and stimulated its expression. The combined treatment with E2 and P4 also stimulates the expression for GM-CSF mRNA and the production of immunoreactive GM-CSF in the stromal tissues. These data suggest that the expression of GM-CSF in uterine stromal cells is partially regulated by ovarian steroids.

Human fallopian tube expresses granulocyte-macrophage colony stimulating factor (GM-CSF) and GM-CSF alpha and beta receptors and contain immunoreactive GM-CSF protein

Human fallopian tube expresses granulocyte-macrophage colony stimulating factor (GM-CSF) and GM-CSF alpha and beta receptors and contain immunoreactive GM-CSF protein

Human fallopian tube expresses granulocyte-macrophage colony stimulating factor (GM-CSF) and GM-CSF alpha and beta receptors and contain immunoreactive GM-CSF protein.

Using specific primers, 35S-40mer oligonucleotide probe and monoclonal antibody, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical observations respectively revealed that human fallopian tube expresses granulocyte macrophage colony stimulating factor (GM-CSF) mRNA and protein, as well as GM-CSF alpha and beta receptors mRNA. The RT-PCR products revealed the predicted 286, 546 and 380 bp fragments for GM-CSF, GM-CSF alpha receptor and GM-CSF beta receptor respectively, which were further verified by restriction enzyme digestion analysis. Tubal epithelial cells in the ampullary and isthmus regions (ciliated and nonciliated) are the primary site of GM-CSF mRNA expression and its immunoreactive gene product, and present to a lesser extent in tubal stromal, smooth muscle, and arterial endothelial and smooth muscle cells. The in situ hybridization and immunostaining observations indicated that the expression of GM-CSF mRNA and protein appeared to be cycle dependent and considerably higher during mid-late proliferative and early-mid secretory, than early proliferative and reduced at late secretory phases of the menstrual cycle and postmenopausal period. The results demonstrate for the first time that human fallopian tube expresses GM-CSF and its alpha and beta receptors mRNA and contains the immunoreactive gene product for GM-CSF, and may be potentially regulated by ovarian steroids. The results imply the importance of GM-CSF in a variety of tubal functions and possibly the early embryonic development.

Specific Repression of Granulocyte-Macrophage and Granulocyte Colony-Stimulating Factor Gene Expression in lnterleukin-1-Stimulated Endothelial Cells With Antisense Oligodeoxynucleotides

Abstract Antisense oligodeoxynucleotides (ODNs) have been used to effect the specific inhibition of cellular gene expression. We have evaluated the application of this approach to the inhibition of interleukin-1 (IL-1)- induced granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in cultured human umbilical vein endothelial cells. Antisense ODNs or control ODNs (sense ODNs or missense ODNs containing random base substitutions) were added to cultures of endothelial cells, the cells were induced with IL- 1 alpha, and the conditioned media were assayed for GM-CSF and G-CSF by quantitative bioassays and for immunoreactive GM-CSF by enzyme immunoassay. Antisense ODNs complementary to the first 15 or 18 bases of the translation start sites of GM-CSF or G-CSF mRNAs inhibited, in a concentration-dependent fashion, the IL-1-stimulated expression of the corresponding factor, but did not affect expression of the other factor. Control ODNs did not affect GM-CSF or G-CSF expression. Exposure to a GM-CSF antisense ODN, but not a control ODN, substantially reduced cytoplasmic GM-CSF mRNA levels in IL-1-stimulated endothelial cells. Neither ODN affected levels of endothelial leukocyte adhesion molecule (ELAM)1 or glyceraldehyde-3-phosphate dehydrogenase mRNAs. We conclude that antisense ODNs complementary to the translation start sites of GM-CSF or G-CSF mRNAs inhibit expression of the corresponding factor in a sequence-specific fashion and this effect is mediated, at least in part, by reduction in the cytoplasmic level of the targeted mRNA. Moreover, IL-1-induced GM-CSF or G-CSF expression does not depend on expression of the other factor.

Specific repression of granulocyte-macrophage and granulocyte colony- stimulating factor gene expression in interleukin-1-stimulated endothelial cells with antisense oligodeoxynucleotides

Antisense oligodeoxynucleotides (ODNs) have been used to effect the specific inhibition of cellular gene expression. We have evaluated the application of this approach to the inhibition of interleukin-1 (IL-1)-induced granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in cultured human umbilical vein endothelial cells. Antisense ODNs or control ODNs (sense ODNs or missense ODNs containing random base substitutions) were added to cultures of endothelial cells, the cells were induced with IL-1 alpha, and the conditioned media were assayed for GM-CSF and G-CSF by quantitative bioassays and for immunoreactive GM-CSF by enzyme immunoassay. Antisense ODNs complementary to the first 15 or 18 bases of the translation start sites of GM-CSF or G-CSF mRNAs inhibited, in a concentration-dependent fashion, the IL-1-stimulated expression of the corresponding factor, but did not affect expression of the other factor. Control ODNs did not affect GM-CSF or G-CSF expression. Exposure to a GM-CSF antisense ODN, but not a control ODN, substantially reduced cytoplasmic GM-CSF mRNA levels in IL-1-stimulated endothelial cells. Neither ODN affected levels of endothelial leukocyte adhesion molecule (ELAM)1 or glyceraldehyde-3-phosphate dehydrogenase mRNAs. We conclude that antisense ODNs complementary to the translation start sites of GM-CSF or G-CSF mRNAs inhibit expression of the corresponding factor in a sequence-specific fashion and this effect is mediated, at least in part, by reduction in the cytoplasmic level of the targeted mRNA. Moreover, IL-1-induced GM-CSF or G-CSF expression does not depend on expression of the other factor.

Granulocyte/Macrophage Colony-stimulating Factor (GM-CSF) Gene Expression by Eosinophils in Nasal Polyposis

Nasal polyposis is a chronic inflammatory condition of the upper airways characterized by infiltration of activated inflammatory cells, particularly eosinophils. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a cytokine with powerful biologic effects including the regulation of survival, proliferation, and activation of granulocytes as well as differentiation of hemopoietic cells. To examine the potential role of GM-CSF in the pathogenesis of this condition, we investigated gene expression and production of GM-CSF in nasal polyp tissues as well as in the normal nasal mucosa. Immunoreactive GM-CSF was detected by enzyme-linked immunosorbent assay in the 24-h supernatant of nasal polyp tissues placed in culture. By Northern blot analysis and Southern blot analysis following a reverse-transcription polymerase chain reaction using a human GM-CSF cDNA probe, we detected GM-CSF mRNA in nasal polyp tissues, as well as in the tissue from a patient with allergic rhinitis, but not in the normal nasal mucosa. By in situ hybridization using the same probe, cells expressing mRNA specific for GM-CSF were observed in nasal polyp tissues and in the allergic nasal mucosa. In addition, by the combination of in situ hybridization and counterstaining with chromotrope 2R, we demonstrated that approximately 30% of eosinophils infiltrating the polyp tissue express the GM-CSF gene. These results suggest a novel mechanism by which eosinophils may contribute to the pathogenesis of chronic inflammatory diseases such as nasal polyposis, allergic rhinitis, and, by implication, asthma.

Endobronchial allergen challenge in asthma. Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization.

Airway inflammation is thought to play an important role in the pathogenesis of asthma. We have used in situ hybridization and an immunoassay to determine whether granulocyte macrophage colony-stimulating factor (GM-CSF) (a cytokine capable of eosinophil activation) is present in the airway of asthmatics (n = 6) who have 37.0 +/- 15.1% airway eosinophilia after endobronchial allergen challenge. Levels of immunoreactive GM-CSF (less than 4 pg/ml pre-allergen versus 180.5 +/- 46.9 pg/ml post-allergen) increased significantly 24 h after endobronchial allergen stimulation. The cellular source of bronchoalveolar lavage (BAL) GM-CSF, as determined by in situ hybridization and immunoperoxidase staining, was derived predominantly from UCHL-1 positive BAL lymphocytes, as well as from a smaller population of alveolar macrophages. Before local endobronchial allergen challenge, less than 1% of lymphocytes and alveolar macrophages recovered by BAL expressed GM-CSF mRNA, whereas after allergen stimulation 92.6 +/- 3.4% of lymphocytes and 17.5 +/- 22.7% of alveolar macrophages expressed GM-CSF mRNA. This study provides evidence that in an experimental model of allergen-induced asthma, activation of the immune and inflammatory response (BAL lymphocyte and alveolar macrophage production of GM-CSF) is temporally associated with an inflammatory cell influx of eosinophils into the airway.

A bronchial epithelial cell-derived factor in asthma that promotes eosinophil activation and survival as GM-CSF.

We have examined the in vitro interaction between bronchial epithelial cells and eosinophils derived from five asthmatics by determining the effect of epithelial cell-conditioned medium on the survival and activation of peripheral blood eosinophils. The supernatants of epithelial cells from six normal donors were used as control. The asthmatic epithelial cell-conditioned medium significantly increased the survival of eosinophils cultured for 3 (P less than 0.025) and 6 (P less than 0.05) days. The incubation of eosinophils with the supernatants of asthmatic epithelial cells for 1 h also increased the generation of superoxide anion and the release of leukotriene C4, triggered by phorbol myristate acetate and calcium ionophore, by more than twofold. The preincubation of asthmatic epithelial cell-conditioned media with saturating concentrations of a mono-specific antiserum against granulocyte-macrophage colony-stimulating factor (GM-CSF) completely abolished their activity, whereas the addition of recombinant human GM-CSF restored it. The supernatants of asthmatic epithelial cells contained 0.88 +/- 0.09 (SD) ng/5 X 10(5) cells immunoreactive GM-CSF, and this amount was significantly greater than that measured in the supernatants of normal epithelial cells (0.21 +/- 0.105, P less than 0.025). Bronchial epithelial cells from asthmatics also expressed increased levels of GM-CSF mRNA when compared with normal epithelial cells. Thus both the synthesis and release of GM-CSF by bronchial epithelial cells are upregulated in asthma, and this may contribute to the persistence of eosinophil infiltration and activation in asthmatic airways.

Interleukin-1 Binds to Specific Receptors on Human Bronchial Epithelial Cells and Upregulates Granulocyte/Macrophage Colony-stimulating Factor Synthesis and Release

Cultured human bronchial epithelial cells constitutively produce granulocyte/macrophage colony-stimulating factor (GM-CSF). An upregulation of the synthesis and release of GM-CSF from those cells might contribute to the persistence of infiltration and local activation of inflammatory cells in some inflammatory diseases of the airways, such as asthma. Increased levels of immunoreactive and biologically active interleukin-1 (IL-1) have been identified in the airway secretions of asthmatic patients, together with an increase in GM-CSF contents. As IL-1 is known to upregulate GM-CSF production in many cell populations, in this study we investigated the ability of IL-1 to bind to specific receptors on bronchial epithelial cells and promote GM-CSF synthesis and release. Bronchial epithelial cells possessed specific single-class surface receptors for recombinant IL-1. The addition of exogenous IL-1 led to a dose-dependent increase in the accumulation of GM-CSF mRNA and release of immunoreactive GM-CSF to the culture medium. Release of IL-1 in the bronchial mucosa during allergic and nonallergic responses may lead to enhanced GM-CSF synthesis and release by epithelial cells, thus promoting airway inflammation.

The effects of dose and route of the administration on the pharmacokinetics of granulocyte-macrophage colony-stimulating factor

The pharmacokinetics of granulocyte-macrophage colony stimulating factor (GM-CSF) (0.3–30 μg/kg) were studied after subcutaneous bolus ( n = 16) or intravenous bolus ( n = 5) injection or 2 h intravenous infusion ( n = 12). Each method of administration gave a different GM-CSF concentration—time profile. Highest peak serum concentrations ( C max) followed the intravenous bolus, and the time GM-CSF persisted at a concentration greater than 1 ng/ml ( t > 1 ng/ml) was longer after a subcutaneous than after an intravenous injection. Area under the concentration—time curve (AUC), C max and t > 1 ng/ml all increased with dose for each method of administration. After intravenous administration, there was a two-phase decline in concentration. The half-life ( t 1 2 ) of the terminal phase following an intravenous bolus ranged from 0.24 to 1.18 h and, following intravenous infusion, from 0.62 to 9.07 h and appeared to increase with dose. The apparent clearance was greatest following subcutaneous injection at doses below 3 μg/kg, suggesting a saturable mechanism or different bioavailability. Only 0.001%–0.2% of the injected dose appeared in the urine as immunoreactive GM-CSF.