Interleukin-6 and transforming growth factor-beta regulate the expression of monocyte chemoattractant protein-1 and colony-stimulating factors in human thyroid follicular cells.

Monocytes and T-lymphocytes, both of which play a pivotal role in immune/inflammatory responses, can be attracted from the circulation into tissues by monocyte chemoattractant protein-1 (MCP-1), and monocytes can be further activated by colony-stimulating factors (CSFs), granulocyte/macrophage CSF (GM-CSF) or macrophage CSF (M-CSF). We examined whether either interleukin-6 (IL-6) or transforming growth factor-beta (TGF-beta), both of which are produced by thyroid follicular cells (TFC), can regulate the production of MCP-1 or CSF(s) in human TFC. IL-6, being effective only in the presence of soluble IL-6 receptor (sIL-6R), stimulated the expression of both MCP-1 and M-CSF, but was inhibitory on GM-CSF expression. On the other hand, TGF-beta stimulated the expression of both MCP-I and GM-CSF, but suppressed M-CSF expression. These results suggest a possible role of IL-6 or TGF-beta on the initiation and/or modulation of thyroid immune/inflammatory responses via MCP-1 production and differential production of GM-CSF or M-CSF by TFC.     

Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation

GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from murine bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-kappaB and AP-1 activities. GM-BMM preferentially produced TNF-alpha, IL-6, IL-12p70, and IL-23 whereas, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12p70 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IkappaBalpha degradation, RelA nuclear translocation, and NF-kappaB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pretreated with the other CSF before LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-kappaB activity. Thus, GM-CSF and M-CSF demonstrate, at the level of macrophage cytokine production, different and even competing responses with implications for their respective roles in inflammation, including a possible dampening or suppressive role for M-CSF in certain circumstances.     

The role of lymphocytes and monocytes in hematopoietic growth factor production by peripheral blood mononuclear cells

Stimulated peripheral blood mononuclear cells (MNC) are one of the richest described physiologic sources of colony-stimulating activity. To understand the molecular basis for, and the cellular sources of, this MNC activity, we cultured purified human lymphocytes and monocytes for 2 hr to 6 days and examined colony-stimulating factor (CSF) gene activity by Northern blot analysis. We show that MNC are capable of expressing messenger RNA for macrophage (M)-CSF, granulocyte (G)-CSF, GM-CSF, and multi-CSF when stimulated with mitogens. The time courses of induction of these genes differ, with G-CSF induction preceding that of the other CSFs. In addition, the spectra of CSFs produced by cell populations enriched for lymphocytes, monocytes, or macrophages differ. The implications of these findings for the selective activation of hematopoiesis are discussed.     

Radiosensitivity increases with differentiation status of murine hemopoietic progenitor cells selected using enriched marrow subpopulations and recombinant growth factors

The radiosensitivity of populations of colony-forming cells (CFC) in murine bone marrow was investigated using different recombinant colony-stimulating factors (CSFs; murine IL-3 and granulocyte-macrophage CSF and human granulocyte CSF), or purified murine macrophage CSF. With unfractionated normal bone marrow the CFC increased in radiosensitivity as they progressed through the granulocyte lineage. The D0 values ranged from 129 +/- 12 cGy for CFC stimulated with GM-CSF down to 42 +/- 2 cGy after stimulation with G-CSF. IL-3 stimulated a CFC population which gave the only survival curve with a shoulder (n = 1.9 +/- 0.3). With semipurified populations of primitive or bipotential CFC, D0 values were generally lower with respect to the equivalent values for unpurified bone marrow (range 62 +/- 7 cGy to 135 +/- 7 cGy). Changes in cluster/colony ratio and colony morphology together possibly with products of accessory cells influence the interpretation of the radiosensitivity parameters.     

Human macrophage colony-stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone

Colony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)-CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M-CSF did not alter cytoplasmic motility in time-lapse recordings, suggesting that M-CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte-macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M-CSF production may be a mechanism by which osteoblastic cells, which produce M-CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M-CSF induces a diversion of lineage resources away from osteoclastic function.     

Examination of survival, proliferation and cell surface antigen expression of human monocytes exposed to macrophage colony-stimulating factor (M-CSF)

The effects of macrophage colony-stimulating factor (M-CSF or CSF-1) on the survival, proliferation, maturation and activation of human blood monocytes were examined. M-CSF (100-1,000 U/ml) doubled the number of monocytes surviving after eight days in culture and accelerated the usual increase in cell volume. Antiserum to M-CSF abolished both of these effects. There was no sizable increase in 3H-thymidine incorporation in monocytes over this time period. Of various factors tested, including gamma-interferon (gamma-IFN), interleukin (IL) 1 alpha, granulocyte CSF (G-CSF), platelet-derived growth factor (PDGF), and lipopolysaccharide (LPS), only granulocyte-macrophage CSF (GM-CSF) could also enhance survival and augment cell volume. While antiserum to human M-CSF eliminated the increase in survival induced by GM-CSF, it could not ablate the GM-CSF-stimulated increase in monocyte cell volume. Monocyte cell surface markers that increase with maturation (i.e., Fc gamma RIII) or with activation (i.e., Fc gamma RI) were unaffected by incubation with M-CSF.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.     

Upregulation of HLA class II, but not intercellular adhesion molecule 1 (ICAM-1) by granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3) in synergy with dexamethasone.

In this study we have compared the effects of granulocyte macrophage colony stimulating factor (GM-CSF) on purified normal blood monocytes, with two other haemopoietic growth factors, Interleukin (IL-) 3 and Macrophage (M-)CSF on HLA class I, class II and intercellular adhesion molecule 1 (ICAM-1) expression in the presence and absence of dexamethasone (Dex). IL-3 alone, like GM-CSF, was a weak inducer of HLA class II expression but in combination with Dex markedly enhanced HLA-DR, DP and DQ expression. Similar changes were observed for HLA class I expression. The response to both IL-3 and GM-CSF was not additive in the presence of an optimal concentration of one cytokine and titrating concentrations of the other indicating that they may use common receptors and signal transduction mechanisms. Although IL-3 or GM-CSF alone also enhanced ICAM-1 expression, Dex inhibited both constitutive and the cytokine induced expression of this antigen. In contrast M-CSF, in the presence or absence of Dex, failed to enhance ICAM-1, HLA class I or II expression. These observations further highlight differences between the effects of the haemopoietic growth factors GM-CSF and IL-3 versus M-CSF in the regulation of monocyte function. Finally, the distinct effect of a combination of glucocorticoids with GM-CSF or IL-3 to induce high levels of HLA expression on human monocytes suggests they may have an important role during inflammatory conditions in vivo.     

Murine recombinant IL-4 is a bifunctional regulator of macrophage growth induced by colony-stimulating factors.

Murine peritoneal exudate macrophage (PEM) coexpress receptors for both granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) and can be induced by both factors, either alone or in combination, to undergo extensive proliferation in vitro. In this study the effect of murine rIL-4 (MurIL-4) on the proliferation of PEM was examined. MurIL-4 alone did not support macrophage proliferation but prolonged their survival in vitro. When MurIL-4 was combined with human (Hu)rM-CSF, it enhanced the proliferative response of PEM to rHuM-CSF in a dose-dependent manner, reaching a maximum at approximately 10 ng/ml. Contrarily, MurIL-4 suppressed the proliferative response of PEM to MurGM-CSF. Receptor binding assays using radiolabeled ligands showed that MurIL-4 selectively enhanced the expression of M-CSF receptors; suggesting that at least part of the synergistic effect of MurIL-4 is mediated at the receptor level. Of relevance to this effect is the finding that MurIL-4 greatly promoted the responsiveness of PEM to low concentrations of HurM-CSF. Unlike M-CSF receptors, however, MurIL-4 treatment failed to modulate the levels of GM-CSF receptors in PEM. The proliferative responses of PEM to both MurGM-CSF and HurM-CSF could be inhibited by MurIFN-gamma with similar sensitivity. This inhibitory effect of MurIFN-gamma was partially neutralized by MurIL-4 in cultures containing HurM-CSF but not those containing MurGM-CSF. This study demonstrates that IL-4 is involved directly in the regulation of macrophage production by modulating their responsiveness to various cytokines.     

Detection of murine bone marrow granulocyte/macrophage progenitor cells (GM-CFU) in serum-free cultures stimulated with purified M-CSF or GM-CSF

Colony formation by mouse granulocyte/macrophage progenitors (GM-CFU) responding to purified colony-stimulating factors (CSF) in serum-free cultures is described. Analysis of the lipid requirements for colony growth stimulated by purified macrophage CSF (M-CSF) demonstrated that cholesterol is essential. Linoleic acid further promoted colony growth only if cholesterol was present, but phospholipid was inhibitory. More colonies were obtained in serum-free cultures, than in serum-supplemented controls. This difference could not be attributed to a change in the range of sensitivity to M-CSF. Stimulation of GM-CFU with granulocyte/macrophage CSF (GM-CSF) required further supplementation with hydrocortisone for optimal expression of colony-forming capacity in serum-free cultures. Hydrocortisone slightly inhibited colony growth stimulated with M-CSF. Under these culture conditions, the number of GM-CFU responding to GM-CSF was twice that obtained with M-CSF.     

Differential activity of granulocyte-macrophage and macrophage colony stimulating factors on bone resorption in fetal rat long bone organ cultures.

In this study, the ability of recombinant human macrophage (M) and murine granulocyte-macrophage (GM) colony stimulating factor (CSF) to affect both basal and stimulated bone resorption in fetal rat long-bone organ cultures was assessed. It was found that M-CSF does not affect basal bone resorption or bone resorption stimulated by parathyroid hormone, recombinant human interleukin 1 beta, prostaglandin E2 (PGE2), and 1,25 dihydroxy vitamin D3. Specifically, M-CSF at concentrations as high as 30 nM (1 microgram/mL) did not modulate 45Ca release from fetal rat long bones stimulated by these agents. The addition of recombinant murine GM-CSF (at equal molar concentration to M-CSF) also did not affect bone resorption stimulated by parathyroid hormone and interleukin 1 beta. On the other hand, GM-CSF stimulated basal bone resorption over a 120-h period and augmented the resorption mediated by exogenous PGE2 over a 48-h incubation. In addition, GM-CSF was shown to stimulate production of endogenous PGE2 in cultures of bone rudiments. These effects on bone resorption were blocked by the addition of prostaglandin synthesis inhibitors and specific antibodies to murine GM-CSF. These data indicate that M-CSF does not act as a regulator of bone turnover, but GM-CSF may cause bone resorption by stimulating the synthesis of PGE2 in bone.     

Differentiation of human monocytes in vitro with granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression

The cytokines macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) promote differentiation of monocytes into macrophages with distinct phenotypes and unique functional abilities. In this report, we characterize how monocytes and macrophages differentiated from monocytes with M-CSF and GM-CSF regulate their cGMP levels by controlling which phosphodiesterases (PDEs) and guanylyl cyclases (GCs) are expressed. We find that PDE1B and PDE2A are expressed at low levels in monocytes, but are the major cGMP PDEs expressed in macrophages. M-CSF differentiation triggers increased expression of PDE1B and PDE2A, while GM-CSF causes a large increase only in PDE1B. Based on PDE expression, we identified THP-1 and U937 cell lines as possible models for studying the roles of PDE1B and PDE2A in macrophage function. We additionally characterized changes in expression of GCs upon differentiation. We found that GM-CSF differentiation triggers a small decrease in soluble guanylyl cyclase (sGC) and a large increase in GC-A, while M-CSF significantly decreases sGC.     

Comparison of the effects of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity

Cultured human monocytes undergo a process of differentiation and maturation lasting 5 to 10 days that ultimately leads to the appearance of large macrophage-like cells. This differentiation is growth factor dependent: of all the cytokines tested, only macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3 proved capable of supporting the differentiation and the long term survival of the macrophage-like cells. Although all three cytokines yield cells with macrophage characteristics, cells developed in M-CSF have features distinct from those matured in either IL-3 or GM-CSF. At the morphologic level, the M-CSF-supported monocyte cultures yield elongated, spindle-shaped cells whereas those supported with IL-3 or GM-CSF yielded round cells with distinct nuclei. All three macrophage populations expressed similar levels of HLA-DR, CD11b, and CD11c, but the M-CSF-treated cultures yielded more CD14+ and CD16+ (Fc gamma RIII) cells. All three cell populations developed capacity for antibody-dependent cellular cytotoxicity (ADCC) as well as antibody-independent cytotoxicity with peak activity achieved after 8 to 12 days in culture. ADCC capacity developed earliest and the level of activity was usually greatest in the M-CSF-treated cultures, possibly correlating with the higher level of expression of CD16. Our findings indicate that any of these cytokines, but particularly M-CSF, may be useful clinically in enhancing the tumoricidal capacity of tumor-specific mAb through augmentation of macrophage capacity for ADCC.     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.