Production Of Granulocyte-monocyte Colony-stimulating Factor Research Articles

Testing the “Cytokine-ome” Theory: A Longitudinal Pilot Study in Renal Transplant Recipients

Cytokines function within a complex network. Their function relates to specific immunological processes that also promote autoimmunity, alloimmunity, chronic inflammation, and tissue destruction (1–6). Here, we tested the hypothesis that stable cytokine milieus (“cytokine-omes”) might define “signatures” of the cytokine network associated with pathology. To do so, we first measured the frequency of peripheral blood mononuclear cells (PBMCs) secreting the regulatory cytokine interleukin (IL)-10 in 10 healthy blood donors (7, 8). Two of 10 healthy individuals had a frequency of IL-10-secreting PBMCs of >1300/106 cells, and 8 of 10 healthy individuals had a frequency <600/106 cells (Fig. 1A). Next, the frequency of IL-10-secreting PBMCs was related to levels of homeostatic secretion of the inflammatory cytokines IL-1β, IL-8, IL-12, tumor necrosis factor (TNF)-α, and IL-2 (8). Secretion of these cytokines, with the exception of IL-8, was very low, and no co-segregation of IL-8 with the frequency of IL-10 secreting PBMCs was observed (data not shown).FIGURE 1.: A, Frequency of constitutive IL-10-secreting PBMCs. The frequency of IL-10-secreting PBMCs was polarized both in healthy donors (upper panel) and in kidney transplant recipients (lower panel), separating 2 nonoverlapping groups (Wilcoxon Sum-of-Ranks tests). After transplantation, the frequency of IL-10-secreting PBMCs decreased slightly in high-frequency individuals (as analyzed with the Student’s t-test); however, the groups remained well separated (Wilcoxon Sum-of-Ranks tests). B, Inflammatory cytokines in high vs. low IL-10-frequency kidney transplant recipients. Constitutive secretion of the cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, GM-CSF, and IL-2 was compared before and after transplantation in individuals with a high vs. a low frequency of IL-10-secreting PBMCs (Wilcoxon Sum-of-Ranks tests). Note the selective inverse correlation of IL-8 secretion and frequency of IL-10-secreting PBMCs.We then assessed the frequency of spontaneously IL-10-secreting PBMCs in a cohort of kidney transplant recipients. Patient characteristics relevant to this study are summarized in Supplementary Table 1, available for viewing online only. In these experiments, the following observations were made: (1) analogous to healthy donors, 4 of 12 patients had a pretransplant frequency of IL-10-secreting PBMCs of >1300/106 cells and 8 of 12 patients a pretransplant frequency <600/106 cells, (2) in each of the 4 transplant recipients with a high frequency of IL-10-secreting PBMCs, the frequency decreased after transplantation but remained high (posttransplant frequency: always >1300/106 cells), and (3) transplant recipients with a low frequency of IL-10-secreting PBMCs before transplantation had a stable and low frequency after transplantation (Fig. 1A). No association between the frequency of IL-10-secreting cells and total monocyte- or lymphocyte-counts was observed (Supplementary Table 1, available for viewing online only). Also, no correlation of acute-phase reaction (as reflected by levels of C-reactive protein) or frequency of IL-10 secreting cells was detected (Supplementary Table 1, available for viewing online only). At these same time points, the frequency of IL-10-secreting PBMCs was related to levels of homeostatic secretion of the inflammatory cytokines IL-1β, IL-6, IL-8, IL-12, TNF-α, granulocyte monocyte colony stimulating factor (GM-CSF), and IL-2 (8). Contrasting healthy donors, 2 patterns of cytokine secretion were observed: the group of patients with a high frequency of IL-10-producing PBMCs produced high amounts of IL-1β, IL-6, TNFα, and GM-CSF. Production of these cytokines was not influenced by immunosuppression. By contrast, patients with a low frequency of IL-10-producing PBMCs secreted large amounts of IL-8, whereas production of IL-1β, IL-6, TNF-α, and GM-CSF was low. Again, this cytokine-pattern was similar before and after transplantation. With respect to the constitutive production of IL-2 and IL-12, transplant patients did not differ (Fig. 1B). Together, these data suggest the existence of immunological milieus, exclusively observed in patients that developed end-stage renal disease, wherein inflammatory cytokines (usually thought of as a group) separate. Defining such immunological milieus (“cytokine-omes”) may prove to be an effective—and largely unexplored—approach when aiming at dissecting the “anatomy” of the complex cytokine network. Furthermore, knowledge of such differing cytokine milieus may prove relevant when interpreting cytokine levels both in physiology and pathology. ACKNOWLEDGMENTS We thank Matthias Mehling for technical assistance. O.G. is supported by the Swiss Foundation for Medical and Biological Grants and S.S. and C.H. are supported by grants from the Swiss National Science Foundation (3200B0-109302 and PP00B-114850, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Olivier Gasser Partners AIDS Research Center Massachusetts General Hospital Boston, MA Christoph Berger Gabriela Zenhaeusern Ineke Oehri Christoph Hess Immunobiology Laboratory University of Basel Basel, Switzerland Hanno Elsässer Ineke Oehri Stefan Schaub Christoph Hess Clinic for Transplantation-Immunology and Nephrology University Hospital Basel Basel, Switzerland

Wide proinflammatory effect of electronegative low-density lipoprotein on human endothelial cells assayed by a protein array

Electronegative low-density lipoprotein (LDL(−)) is a modified subfraction of LDL present in plasma able to induce the release of interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) by human umbilical vein endothelial cells (HUVEC). To ascertain whether further inflammation mediator release could be induced by LDL(−), a protein array system was used to measure 42 cytokines and related compounds. Native LDL and LDL(−) isolated from normolipemic subjects were incubated for 24 h with HUVEC and culture supernatants were used to measure inflammation mediator release. The protein array revealed that IL-6, granulocyte/monocyte colony-stimulating factor (GM-CSF) and growth-related oncogene (GRO) release were increased by cultured HUVEC in response to LDL(−). LDL(−) enhanced production of IL-6 (4-fold vs. LDL(+)), GM-CSF (4-fold), GROβ (2-fold) and GROγ (7-fold) was confirmed by ELISA. Time-course experiments revealed that IL-6 was released earlier than the other inflammation mediators, suggesting a first-wave cytokine action. However, the addition of IL-6 alone did not stimulate the production of IL-8, MCP-1 or GM-CSF. Moreover, IL-8, MCP-1 or GM-CSF alone did not promote the release of the other inflammatory molecules. Modification of LDL(+) by phospholipase A 2-mediated lipolysis or by loading with non-esterified fatty acids (NEFA) reproduced the action of LDL(−), thereby suggesting the involvement of NEFA and/or lysophosphatidylcholine in the release of these molecules. Our results indicate that LDL(−) promotes a proinflammatory phenotype in endothelial cells through the production of cytokines, chemokines and growth factors.

IL10 resistant PGS2 expression in at-risk/Type 1 diabetic human monocytes

Aberrant prostaglandin synthase 2 (PGS2/COX2) expression constitutes an antigen presenting cell (APC) dysfunction seen in monocytes of humans at risk for or with Type 1 diabetes. During endotoxin activation of PGS2 expression in healthy monocytes, granulocyte–monocyte colony stimulating factor (GM-CSF) is activated and, in turn, promotes PGS2 gene activation. GM-CSF is considered a major target the action for IL10 in its suppression of PGS2. We found that the PGS2 expression in monocytes from 47% of at-risk and diabetic humans tested were highly resistant to suppression by IL10 (maintaining ≥50% of their untreated expression), and had significantly increased GM-CSF production in vitro (1043±SD2798 pg/10 6cells, subject n=35, vs 29.7±SD91 pg/10 6cells, control n=20; P=0.0165). The PGS2 insensitivity to IL10 of these cells was not due to a lack of IL10 functionality or its suppression of GM-CSF. In contrast to its effects on PGS2, IL10 regulation of GM-CSF and other monocyte factors (i.e., DR, IL1 β, TNF α, IL12, CD54, and CD64) remained intact. These findings suggest that the inability of IL10 to properly downregulate PGS2 gene expression may contribute to its dysregulation in Type 1 diabetes.

Regulation of IL-1beta -induced GM-CSF production in human airway smooth muscle cells by carbon monoxide.

Asthma, a chronic inflammatory disease of the airways, involves the increased expression of inflammatory mediators, including granulocyte-monocyte colony-stimulating factor (GM-CSF). Heme oxygenase-1 (HO-1), a stress-response protein, confers protection against oxidative stress. We hypothesized that carbon monoxide (CO), a byproduct of HO-1-dependent heme catabolism, regulates GM-CSF synthesis in human airway smooth muscle cells (HASMC). IL-1beta treatment induced a time-dependent induction of GM-CSF in HASMC. Furthermore, IL-1beta stimulated the major MAPK pathways, including ERK1/ERK2, JNK, and p38 MAPK. Exposure of HASMC to CO at low concentration (250 ppm) markedly inhibited IL-1beta-induced GM-CSF synthesis (>90%) compared with air-treated controls. CO treatment inhibited IL-1beta-induced ERK1/2 activation but did not inhibit JNK and p38 MAPK. Furthermore, CO increased cGMP levels in HASMC. Inhibition of guanylate cyclase by IH-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-1 (ODQ) abolished the inhibitory effects of CO on GM-CSF synthesis and ERK1/2 activation. Collectively, these data demonstrate that the inhibitory effect of CO on GM-CSF synthesis depends on ERK1/2 MAPK and guanylate cyclase/cGMP-dependent pathways.

Cytokine Production by T Cells Infiltrating in the Eye of Uveitis Patients

The capacity of T cells to produce cytokines was investigated using T-cell clones (TCCs) established from infiltrating cells in the aqueous humor (AH) or peripheral blood mononuclear cells (PBMC) of patients with Vogt-Koyanagi-Harada (VKH) disease or sarcoidosis. The cytokines produced and tested in the study were interleukin (IL)-1α, IL-6, IL-8, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and granulocyte monocyte colony stimulating factor (GM-CSF). All TCCs ( n = 9) from AH of VKH patients spontaneously produced significantly larger amounts of IL-6, IL-8, and IFN-γ than TCCs from healthy donor PBMC. All TCCs ( n = 9) from AH of the sarcoidosis patient spontaneously produced significantly larger amounts of IL-1α, IL-6, and IL-8 than TCCs from healthy donor PBMC. In addition, the effects of antiinflammatory drugs on the cytokine production by the TCCs were investigated. Hydrocortisone significantly suppressed the production of IL-6, IL-8, and GM-CSF by TCCs from AH of VKH patients. Tacrolimus also significantly suppressed the production of IL-8 and GM-CSF by the TCCs. FTY720, an experimental drug, suppressed only GM-CSF production by TCCs from AH of VKH patients. Diclofenac failed to suppress the production of any cytokines by any TCCs. All tested drugs did not suppress the production of cytokines by TCCs from the sarcoidosis patient. These results thus suggest that cytokines produced by T cells infiltrating in the eye may play an important role in the pathogenesis of uveitis.

Interleukin-1α Upregulates Tumor Necrosis Factor Receptors Expressed by a Human Bone Marrow Stromal Cell Strain: Implications for Cytokine Redundancy and Synergy

To explore the biochemical and physiologic basis of the overlapping effects of interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha) on myeloid cytokine production, we have studied the dynamics of granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-stimulating factor (GM-CSF) production as well as IL-1 receptor and TNF receptor expression in a clonally derived bone marrow stromal cell strain (CDCL). IL-1 alpha and TNF alpha act in a synergistic manner to stimulate G-CSF and GM-CSF production by CDCL, resulting in an increase in CSF secretion that is 250-fold greater than that observed with either cytokine alone. This synergism in protein secretion is paralleled by synergistic increases the steady-state level of GM- and G-CSF mRNA, with supra-additive levels achieved by 24 hours. Coincident with this synergistic induction of myeloid CSFs, treatment of CDCL cells with IL-1 alpha induces a 300% increase in the expression of TNF receptors. IL-1 alpha induction of TNF receptors reaches a peak after 6 hours and gradually returns to baseline level by 24 hours. IL-1 alpha does not affect TNF receptor ligand binding affinity. A kinetic study comparing IL-1/TNF synergistic induction of growth factor secretion with IL-1 alpha induction of TNF receptors shows that these events occur in parallel. In contrast with the induction of TNF receptors by IL-1 alpha, treatment with TNF alpha has no effect on either the number of IL-1 receptors expressed by CDCL cells or IL-1 receptor ligand binding affinity. Brief treatment of IL-1 alpha/TNF alpha-stimulated CDCL cells with cycloheximide before receptor induction reduces the synergistic increase in growth factor mRNA by 40% to 60% compared with cells not treated with CHX. Taken together, these results raise the possibility that IL-1 alpha cross-induction of TNF receptors may contribute to the biochemical mechanisms underlying the synergistic stimulation of G-CSF and GM-CSF production by IL-1 alpha and TNF alpha.

Characteristics of nine newly derived mesothelioma cell lines

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies.

Autocrine and paracrine growth control by granulocyte-monocyte colony- stimulating factor of acute lymphoblastic leukemia cells

Blast colony assays were performed on freshly obtained bone marrow samples from 19 newly diagnosed or relapsed children with acute lymphoblastic leukemia (ALL) of B lineage to determine the effect of added granulocyte-monocyte colony-stimulating factor (GM-CSF). Of the 19 marrow samples tested, 7 responded to GM-CSF with a mean increase in ALL blast colonies of 346%. Blast cells from one of the responders chosen for flow cytometric study showed expression of GM-CSF receptors on 38% of cells. These findings prompted us to establish five ALL cell lines of diverse phenotypes to examine the expression of GM-CSF and GM- CSF receptor genes in human leukemia, and to determine the role of GM- CSF in autocrine and paracrine growth control of ALL cells. One line, termed G2, manifested a GM-CSF-mediated autocrine pattern of cell growth with the following features: G2 blast colony growth in a serum- free system without added growth factor was density dependent; exogenous GM-CSF augmented G2 colony formation when the cells were seeded at low density; G2 cells constitutively expressed mRNA for GM- CSF and GM-CSF receptor; G2 cells also produced and secreted measurable amounts of GM-CSF into cell culture supernatant; and, monoclonal anti- GM-CSF antibodies abolished G2 colony growth when added to cultures with cells seeded at low density without growth factors. Of the other four ALL cell lines, three expressed mRNA for GM-CSF receptor and responded in vitro to added GM-CSF with increased blast colony growth; however, none of these four cell lines expressed mRNA for constitutive production of GM-CSF. A fifth ALL cell line lacked receptors for GM-CSF and did not respond in clonogenic assays to added GM-CSF. Thus, a bioregulator of normal hematopoiesis plays a central role in autocrine growth control of G2 ALL cells, and an important paracrine growth- promoting role in three of four other ALL cell lines.

Autocrine and Paracrine Growth Control by Granulocyte-Monocyte Colony-Stimulating Factor of Acute Lymphoblastic Leukemia Cells

Blast colony assays were performed on freshly obtained bone marrow samples from 19 newly diagnosed or relapsed children with acute lymphoblastic leukemia (ALL) of B lineage to determine the effect of added granulocyte-monocyte colony-stimulating factor (GM-CSF). Of the 19 marrow samples tested, 7 responded to GM-CSF with a mean increase in ALL blast colonies of 346%. Blast cells from one of the responders chosen for flow cytometric study showed expression of GM-CSF receptors on 38% of cells. These findings prompted us to establish five ALL cell lines of diverse phenotypes to examine the expression of GM-CSF and GM-CSF receptor genes in human leukemia, and to determine the role of GM-CSF in autocrine and paracrine growth control of ALL cells. One line, termed G2, manifested a GM-CSF-mediated autocrine pattern of cell growth with the following features: G2 blast colony growth in a serum-free system without added growth factor was density dependent; exogenous GM-CSF augmented G2 colony formation when the cells were seeded at low density; G2 cells constitutively expressed mRNA for GM-CSF and GM-CSF receptor; G2 cells also produced and secreted measurable amounts of GM-CSF into cell culture supernatant; and, monoclonal anti-GM-CSF antibodies abolished G2 colony growth when added to cultures with cells seeded at low density without growth factors. Of the other four ALL cell lines, three expressed mRNA for GM-CSF receptor and responded in vitro to added GM-CSF with increased blast colony growth; however, none of these four cell lines expressed mRNA for constitutive production of GM-CSF. A fifth ALL cell line lacked receptors for GM-CSF and did not respond in clonogenic assays to added GM-CSF. Thus, a bioregulator of normal hematopoiesis plays a central role in autocrine growth control of G2 ALL cells, and an important paracrine growth-promoting role in three of four other ALL cell lines.

Activation of the granulocyte-monocyte colony stimulating factor gene in acute myeloid leukaemia cells is not related to gene rearrangement

Several reports have documented that leukaemic blasts produce a number of cytokines among them the granulocyte-monocyte colony stimulating factor (GM-CSF). We analysed the structure of the gene that codes for GM-CSF in 44 acute myeloid leukaemia (AML) cases in an attempt to establish whether the autocrine production of GM-CSF was due to a structural gene alteration. No structural alteration was detected in the GM-CSF gene in any of the 44 cases studied. We, therefore, conclude that the autocrine production of GM-CSF by leukaemia blasts is not dependent on gene rearrangement.