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Bacterial endotoxin (LPS) modulation of CSF-1, granulocyte-macrophage (GM)-CSF, G-CSF, IL-1, TNF, and Kit Ligand receptors on murine bone marrow cells (BMC) in vivo and in vitro was investigated. In vivo LPS reduced the binding of hrIL-1 (> 90%), mrTNF (> 62%), mrGM-CSF (> 42%) and hrG-CSF (> 91%) to BMC within 2 h, but elevated IL-1 binding (> 8.4-fold) between 8 to 48 h. In vitro, LPS decreased G-CSF and IL-1 binding after 8 h yet increased TNF binding in a dose-dependent manner, suggesting that in vivo, the LPS up-regulation of IL-1 binding to BMC was indirect. Because in vivo LPS elevated the levels of TNF, IL-6, GM-CSF, and glucocorticoids, we further examined GM-CSF and TNF modulation of cytokine receptors on BMC in vivo. In vivo, TNF decreased the binding of TNF (> 88%), G-CSF (> 89%), and IL-1 (> 73%) within 30 min, but increased IL-1 binding (> 4.8-fold) after 10 h. In contrast, in vitro TNF decreased IL-1 binding after 8 h, implying that in vivo TNF up-regulation of Il-1 binding to BMC was also due to an indirect mechanism. However, GM-CSF increased IL-1 binding to BMC both in vivo and in vitro after 8 h. Further studies showed that in vitro GM-CSF and dexamethasone synergistically increased IL-1 binding to BMC in a time- and dose-dependent manner. This synergistic modulation depended on synthesis of protein and mRNA, and was due to an increase in receptor number rather than an increase in receptor affinity. Because in vivo, LPS and LPS-induced cytokines (IL-1 and TNF) elicited the secretion of glucocorticoid and CSF activities, our results revealed a mechanism for LPS up-modulation of IL-1R on BMC in vivo.

Modulation of IL-1R on human neutrophils was investigated after in vitro treatment of cells with human recombinant (hr) granulocyte (G)-CSF, hrgranulocyte-macrophage (GM)-CSF, hrCSF-1, hrIL-1 alpha, hrIL-2, hrIL-3, hrIL-4, hrIL-5, hrIL-6, hrIL-7, transforming growth factor-beta 1, or hrTNF-alpha. At 4 degrees C, 125I-IL-1 binding was competed by IL-1 but not by other cytokines tested. At 37 degrees C, GM-CSF, TNF-alpha, and IL-1 decreased 125I-IL-1 binding in a dose-dependent manner. Kinetic studies showed that GM-CSF reduced > 45% IL-1 binding within 15 min but later (8 h) produced a > 2-fold increase. In contrast, TNF decreased > 85% IL-1 binding within 15 min with a recovery of > 80% relative to that of control after 24 h. Scatchard analysis revealed that TNF or GM-CSF down-modulation of IL-1 binding was due to a decrease of IL-1R number. Further studies showed that dexamethasone and GM-CSF (or G-CSF) synergistically increased IL-1 binding after 8 h. This synergistic modulation was a cytokine dose- and time-dependent process, and was due to an increase in IL-1R numbers rather than a change in binding affinity. In addition, human bone marrow neutrophils, cord blood neutrophils, and several human hematopoietic cell lines (HL-60, U-937, and AML-193) responded to dexamethasone and GM-CSF (or G-CSF) with a superadditive increase in IL-1 binding. Because mammalian systems respond to bacterial endotoxins with secretion of TNF, IL-1, glucocorticoids, G-CSF and GM-CSF, our results shed additional light on this highly regulated cytokine network and revealed a novel role for GM-CSF.

Human recombinant granulocyte CSF (G-CSF) modulation of cytokine receptors on murine bone marrow cells (BMC) in vivo and in vitro was investigated. In vivo, G-CSF reduced 125I-G-CSF binding to BMC by greater than 95% within 30 min, with return to base line after 48 h. Human rCSF-1 binding was reduced greater than 85% after 30 min and failed to recover even after 48 h. Murine rTNF-alpha or recombinant granulocyte/macrophage CSF binding was not significantly altered. However, human rIL-1 alpha binding increased greater than 1.5-fold after 3 h, was elevated greater than 5-fold between 6 and 12 h, and declined to base line after 48 h. In vitro, G-CSF induced a greater than 1.5-fold increase in IL-1 binding to BMC after 8 h, suggesting that up-modulation of IL-1 binding in vivo required G-CSF and other influences. Further studies indicated that BMC responded to glucocorticoids and G-CSF with a synergistic increase of IL-1 binding. This synergistic IL-1R modulation was a time- and dose-dependent process and was inhibited by cycloheximide or actinomycin D in a dose-dependent manner. Binding studies further revealed that the synergistic stimulation of IL-1R expression on BMC was probably due to increased receptor number, rather than increased receptor affinity. In addition, this phenomenon was also observed in other hematopoietic cells. Our results demonstrated that G-CSF was capable of stimulating IL-1R expression on BMC both in vivo and in vitro and G-CSF in combination with glucocorticoids synergistically up-modulated IL-1 binding to BMC in vitro. Inasmuch as IL-1 induces the secretion of G-CSF and glucocorticoids in vivo, this synergistic induction may play an important, as yet unknown, role in the inflammatory cascade.

We here investigated IL-1 modulation of cytokine receptors on murine bone marrow cells (BMC). In vivo, IL-1 treatment reduced greater than 88% of granulocyte-CSF, greater than 35% of TNF, and greater than 51% of granulocyte/macrophage-CSF binding to BMC after 3 h, and returned to base line after 48 h. However, IL-1 binding to BMC decreased greater than 30% after 30 min, dramatically increased greater than 9-fold between 6 and 10 h, and declined to base line after 48 h. In vitro incubation of BMC with IL-1 did not markedly alter IL-1 and granulocyte-CSF binding, suggesting that modulation of granulocyte-CSF and IL-1 binding to BMC by IL-1 in vivo is due to an indirect mechanism. Further in vitro studies showed that IL-1 binding to BMC was specifically induced by glucocorticoids rather than other steroids, and is a time- and dose-dependent process. IL-1 induced IL-1R up-regulation was suppressed by ketoconazole, cycloheximide, and actinomycin D in a dose-dependent manner. In addition, in vivo dexamethasone imitated the action of IL-1 in stimulating IL-1 binding to BMC and in inducing neutrophilia. Furthermore, IL-1 binding to BMC from sham mice 8 h after IL-1 administration was 2.5 times higher than that observed in adrenalectomized mice. Our results demonstrate, for the first time, that in vivo IL-1-induced increased IL-1 binding to BMC was due to an indirect mechanism, and glucocorticoids stimulated IL-1 binding to BMC in vivo and in vitro. Inasmuch as serum glucocorticoid levels can be elevated by IL-1 in vivo, these results reveal a novel mechanism by which IL-1 modulates its own receptors in vivo.

Modulation of granulocyte CSF (G-CSF) receptors on murine peritoneal exudate macrophages (PEM) by various cytokines was investigated. At 4 degrees C, 125I-G-CSF receptor binding on PEM reached a plateau after 6 h and was specifically competed by unlabeled human rG-CSF but not by other cytokines, including human rG-CSF-1, murine recombinant granulocyte-macrophage CSF, murine rIFN-gamma, human rIL-1 beta, and murine rTNF-alpha. 125I-G-CSF bound to PEM has a half-life of 30 min at 37 degrees C. Preincubation of PEM with murine rTNF, murine recombinant granulocyte-macrophage CSF, CSF-1, or G-CSF for 30 min at 37 degrees C resulted in partial reduction of 125I-G-CSF binding capacity, whereas IL-1 or IFN-gamma did not inhibit G-CSF binding. Further studies indicated that reduction of G-CSF binding caused by TNF was a dose- and time-dependent process and did not require FCS. The reduction was transient, and receptor binding was recovered by incubation at 37 degrees C for 8 h. The recovery of G-CSF binding was inhibited in the presence of cycloheximide. In addition, G-CSF binding studies suggested that the TNF-induced decrease in G-CSF binding to PEM was probably due to a reduction in receptor number rather than receptor affinity. Modulation of G-CSFR by TNF was also observed on nonelicited macrophages from various strains of mice. Our results demonstrate a physiologic response of G-CSFR on macrophages that is modulated by TNF. This phenomenon may play an important, as yet unknown, role in the macrophage inflammatory response.

The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis.