Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.

The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.     

Production of hemopoietic colony-stimulating factors by astrocytes.

Astrocytes may play a central role in regulation of immune mediated processes in the central nervous system. By their inducible expression of MHC class II Ag and secretion of cytokines they may propagate expansion and activation of T and B lymphocytes invading the brain tissue. Here we report that astrocytes may also contribute to the macrophage response observed in inflammatory and degenerative diseases of the brain. Murine astrocytes secrete granulocyte-macrophage CSF (GM-CSF) as evidenced by induction of colony formation in bone marrow cells and growth of FDC-P1 cells. Both effects are neutralized with anti-GM-CSF- but not with anti-IL-3-antibodies. Some residual activity detected in the bone marrow assay after antibody treatment can be explained by concomitant production of granulocyte CSF (G-CSF). The mRNA of both G- and GM-CSF are identified by Northern blots in astrocytes. Furthermore, mRNA for IL-1 alpha and IL-1 beta are detected in comparable amounts in astrocytes and brain macrophages, the latter, however, comprising much more potent sources of TNF-alpha.     

Influence of IL-1 alpha and -1 beta on the survival of human bone marrow cells responding to hematopoietic colony-stimulating factors

Purified recombinant human (rhu) IL-1 alpha and IL-1 beta were evaluated for their effects on the proliferation and survival of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells from normal human bone marrow (BM). Using nonadherent low density T lymphocyte depleted (NALT-) BM cells cultured in the presence or absence of IL-1, CSF-deprivation studies demonstrated that IL-1 alpha or IL-1 beta by itself did not enhance the proliferation of CFU-GM or BFU-E. They did, however, promote the survival of progenitors responding to the delayed addition of media conditioned by the 5637 cell line (5637 conditioned medium), rhu GM-CSF and erythropoietin. The survival promoting effects of IL-1 alpha on CFU-GM and BFU-E were neutralized by anti-IL-1 alpha mAb added to the cultures. The survival promoting effect of IL-1 alpha did not appear to be mediated by CSF, because neither CSF nor erythroid burst promoting activity were detectable in cultures in which NALT- cells were incubated with rhuIL-1 alpha. In addition, suboptimal concentrations of rhu macrophage CSF (CSF-1), G-CSF, GM-CSF, and IL-3, which were just below the levels that would stimulate colony formation, did not enhance progenitor cell survival. Survival of CFU-GM and BFU-E in low density (LD) bone marrow cells did not decrease as drastically as that in NALT- BM cells, and exogenously added IL-1 did not enhance progenitor cell survival of CFU-GM and BFU-E in LD BM cells. However, addition of anti-IL-1 beta decreased survival of CFU-GM and BFU-E in LD BM cells. These results implicate IL-1 in the prolonged survival of human CFU-GM and BFU-E.     

Prostaglandin E2-dependent induction of granulocyte-macrophage colony-stimulating factor secretion by cloned murine helper T cells

PG are known to inhibit T cell proliferation, at least in part by suppressing IL-2 production, but effects of PG on the production of other lymphokines have not been well studied. We have found that PGE2 and PGE1, but not PGF2 alpha, inhibit both proliferation and production of granulocyte-macrophage (GM)-CSF by murine TH clones stimulated with Ag or anti-CD3 antibody. Thus, signals generated via the Ag receptor:CD3 complex were inhibited by PGE. Most interesting, however, was the finding that PGE2 and PGE1 could act synergistically with IL-2 for the induction of GM-CSF in some TH1 clones. Dependence on PGE2 for this response was not found in all clones, as some TH1 cells could produce GM-CSF after IL-2 alone, and some cells did not produce GM-CSF even in the presence of PGE2 and IL-2. These observations indicate that there is a subset of TH1 cells receptive to a stimulating activity of PGE2 in the presence of IL-2. PGE2 is known to elevate cAMP levels in T cells. Therefore, we tested whether other agents known to increase cAMP, such as forskolin and cholera toxin, could act in conjunction with IL-2 to induce GM-CSF secretion. As was found with PGE2, these compounds also induced GM-CSF activity in the presence of IL-2, suggesting a critical role for cAMP in this process. Overall these data indicate that the requirements for activation of GM-CSF secretion vary among individual T cells. Most importantly they provide the first evidence that E-series PG are positive signals for lymphokine induction in certain T cells, whereas simultaneously acting as negative signals limiting proliferation. This result also suggests that treatment with anti-inflammatory drugs that decrease PGE2 concentrations may inhibit lymphokine secretion normally stimulated by this pathway.     

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.     

Interdependence of the radioprotective effects of human recombinant interleukin 1 alpha, tumor necrosis factor alpha, granulocyte colony-stimulating factor, and murine recombinant granulocyte-macrophage colony-stimulating factor

Interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are molecularly distinct cytokines acting on separate receptors. The release of these cytokines can be concomitantly induced by the same signal and from the same cellular source, suggesting that they may cooperate. Administered alone, human recombinant (hr)IL-1 alpha and hrTNF alpha protect lethally irradiated mice from death, whereas murine recombinant GM-CSF and hrG-CSF do not confer similar protection. On a dose basis, IL-1 alpha is a more efficient radioprotector than TNF alpha. At optimal doses, IL-1 alpha is a more radioprotective cytokine than TNF alpha in C57BL/6 and B6D2F1 mice and less effective than TNF alpha in C3H/HeN mice, suggesting that the relative effectiveness of TNF alpha and IL-1 alpha depends on the genetic makeup of the host. Administration of the two cytokines in combination results in additive radioprotection in all three strains. This suggests that the two cytokines act through different radioprotective pathways and argues against their apparent redundancy. Suboptimal, nonradioprotective doses of IL-1 alpha also synergize with GM-CSF or G-CSF to confer optimal radioprotection, suggesting that such an interaction may be necessary for radioprotection of hemopoietic progenitor cells.     

Dexamethasone regulation of the expression of cytokine mRNAs induced by interleukin-1 in the astrocytoma cell line U373MG

BSF-2/IL-6, GM-CSF and IL-1 beta mRNAs were induced by recombinant IL-1 in human astrocytoma cell line U373MG. The induction of BSF-2/IL-6 and IL-1 beta mRNAs did not require de novo protein synthesis while that of GM-CSF mRNA required a newly synthesized protein. Dexamethasone inhibited the induction of these cytokine mRNAs by IL-1. This process seems to require continued protein synthesis. These results suggest that the production of these cytokines are positively and negatively controlled by IL-1 and glucocorticoids, respectively, in astrocytes.     

Regulation of granulocyte-macrophage colony-stimulating factor in human retinal pigment epithelial cells by IL-1beta and IFN-gamma.

GM-CSF production by RPE cells, which form part of the blood-retina barrier, is upregulated by IL-1beta and this increase can be reversed by IFN-gamma. IL-1beta up-regulation is not dependent on PKC but the PKC activator PMA induces low levels of GM-CSF production and acts synergistically with IL-1beta to further increase GM-CSF. Although A23187 and ionomycin stimulated low levels of GM-CSF production, the IL-1beta pathway was cyclosporin A insensitive and did not interact with the calcium pathway. IL-1beta-stimulated GM-CSF mRNA expression and production was strongly dependent on NF-kappaB. IFN-gamma inhibition of the GM-CSF response to IL-1beta acted via NF-kappaB, reducing the translocation of NF-kappaB to the nuclei of RPE cells treated with IL-1beta and IFN-gamma. The results show that IFN-gamma down-regulation acts either directly on NF-kappaB or its activation or by blockade of a pathway upstream of NF-kappaB. However, any such blockade does not involve PKC or intracellular calcium.     

Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell.

32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes. The acquired competence to respond to GM-CSF remains irreversibly encoded in the primed cells, although the GM-CSF receptor can be down regulated by interleukin-3. This phenomenon suggests a mechanism by which hematopoietic precursors may obtain additional receptors, thereby increasing their differentiative potential.     

Interleukin 1 stimulates human endothelial cells to produce granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Endothelial cells are a potent source of hematopoietic growth factors when stimulated by soluble products of monocytes. Interleukin 1 (IL 1) is released by activated monocytes and is a mediator of the inflammatory response. We determined whether purified recombinant human IL 1 could stimulate cultured human umbilical vein endothelial cells to release hematopoietic growth factors. As little as 1 U/ml of IL 1 stimulated growth factor production by the endothelial cells, and increasing amounts of IL 1 enhanced growth factor production in a dose-dependent manner. Growth factor production increased within 2 to 4 hr and remained elevated for more than 48 hr. To investigate the molecular basis for these findings, oligonucleotide probes for granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and multi-CSF were hybridized to poly(A)-containing RNA prepared from unstimulated and IL 1-stimulated endothelial cells. Significant levels of GM-CSF and G-CSF, but not M-CSF or multi-CSF, mRNA were detected in the IL 1-stimulated endothelial cells. Biological assays performed on the IL 1-stimulated endothelial cell-conditioned medium confirmed the presence of both GM- and G-CSF. These results demonstrate that human recombinant IL 1 can stimulate endothelial cells to release GM-CSF and G-CSF, and provide a mechanism by which IL 1 could modulate both granulocyte production and function during the course of an inflammatory response.     

Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.     

Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors

Highly purified mouse colony-stimulating factors (CSF) were tested for their effect on neutrophil cytotoxic function in a homologous antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which TNP-coupled mouse thymoma cells coated with mouse anti-TNP antibodies were used as targets, and purified normal mouse bone marrow neutrophils or induced peritoneal neutrophils were used as effector cells. Biochemically pure granulocyte-macrophage (GM)- and granulocyte (G)-CSF enhanced the cytotoxic activity of neutrophils obtained from both sources, allowing them to kill target cells at low antibody concentrations. Furthermore, GM- and G-CSF showed an additive effect, suggesting either the presence of separate receptors for GM- and G-CSF or of separate subsets of neutrophils. Induced peritoneal neutrophils showed a higher level of basal cytotoxic activity than did bone marrow neutrophils, suggesting neutrophil activation in vivo, but both reached similar levels of cytotoxicity upon maximal stimulation with CSF. In addition, CSF was found to be cross-reactive between mouse and human species in their enhancement of neutrophil cytotoxicity. By testing purified mouse CSF on human neutrophils, it could be shown that G-CSF and GM-CSF are functionally distinct molecules, because only G-CSF enhanced ADCC by human neutrophils. These experiments show that the purified factors that control the production of neutrophils by progenitor cells in vitro also activate differentiated neutrophils to carry out their cytotoxic activity in a more effective manner.