15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

In vitro actions on hemopoietic cells of recombinant murine GM-CSF purified after production in Escherichia coli: comparison with purified native GM-CSF

Recombinant murine GM-CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM-CSF. Both recombinant and native GM-CSF stimulated granulocyte and/or macrophage colony formation by adult and fetal mouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM-CSF (25 X 10(8) U/mg) was similar to that of the native form (15 X 10(8) U/mg). At high concentrations (greater than 200 U/ml), both forms of GM-CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (greater than 800 U/ml), megakaryocyte and some erythroid and mixed-erythroid colony formation. Recombinant GM-CSF was as effective in stimulating the proliferation of the GM-CSF-dependent cell line FD as the native molecule. Both recombinant and native GM-CSF were able to induce partial differentiation in colonies of WEHI-3B myeloid leukemic cells. Recombinant GM-CSF competed effectively for the binding of 125I-labeled native GM-CSF to hemopoietic cells, and antiserum to recombinant GM-CSF also neutralized the biological activity of native GM-CSF. The bacterially synthesized GM-CSF was a slightly more effective stimulus for megakaryocyte colony formation than the native molecule. The demonstration that purified bacterially synthesized GM-CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.     

Recombinant granulocyte-macrophage colony-stimulating factor increases adenylate cyclase activity in murine peritoneal macrophages

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotrophic cytokine which stimulates the function and proliferation of macrophage populations. Although the effects of GM-CSF are diverse and GM-CSF has entered into clinical trials, relatively little is known about signal transduction pathways utilized by GM-CSF. In view of previous studies which have suggested that some of the effects of GM-CSF on monocyte-macrophages can be mimicked by agents which increase intracellular cAMP, we investigated the effect of rGM-CSF on adenylate cyclase (AC) activity in murine peritoneal macrophages. Adenylate cyclase activity was quantitated in macrophage membrane preparations and in intact cells. In seven separate experiments, GM-CSF (50 U/ml) increased AC activity by 61(6)% relative to macrophages treated with carrier medium alone. A dose-dependent increase in AC activity was observed (10 to 100 U/ml) which peaked within 1 to 5 min after the addition of GM-CSF and returned to basal levels by 10 to 20 min. Lineweaver-Burk analysis revealed that the Vmax of macrophage AC was increased from 0.40 to 0.52 pmoles cAMP/min by GM-CSF but the Km was unchanged. Intracellular cAMP was increased by GM-CSF to 129(27)% of control values by 1 min of treatment (n = 6). Under similar experimental conditions, GM-CSF did not increase the activity of PK C (n = 14) or phospholipase A2 (n = 3) in peritoneal macrophages. These data show that macrophage adenylate cyclase activity is rapidly stimulated by GM-CSF. Moreover, these findings support further study of the role of cAMP in transmitting the intracellular signals initiated by GM-CSF in tissue macrophages.     

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.     

Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Keratinocyte-derived granulocyte/macrophage colony-stimulating factor induces DNA synthesis by peritoneal macrophages

Keratinocytes have been demonstrated to produce a number of cytokines, including growth factors such as the CSF IL-3. Circulating blood monocytes and some elicited macrophages retain a significant proliferative potential in response to colony-stimulating activity. Because a macrophage response is prominent in a variety of cutaneous immune reactions, we have studied the ability of conditioned media (CM) from a transformed murine keratinocyte cell line (PAM 212) and from normal murine keratinocytes to induce growth of peritoneal macrophages. CM from both normal and transformed keratinocyte cultures induces [3H]thymidine incorporation by thioglycollate-elicited, but not resident, peritoneal macrophages. IEF of PAM 212 CM reveals peaks of activity at pI 4.8 and less than or equal to 4.2. Analysis of CM by reversed-phase HPLC demonstrates active fractions that elute at 46 to 48% and 53 to 55% acetonitrile. The Mr of the 46 to 48% acetonitrile factor is 25 to 30 kDa by gel filtration HPLC. Polyclonal anti-granulocyte/macrophage (GM) CSF antibody blocks the induction of macrophage [3H]thymidine incorporation by factors with pI 4.8 and eluting at 46 to 48% acetonitrile but does not reduce the activity of crude CM or the factor eluting at 53 to 55% acetonitrile. Based on both physiochemical criteria and antibody neutralization, keratinocytes produce GM-CSF. Keratinocyte-derived factors, including GM-CSF, may play an important role in regulating cutaneous macrophage responses.     

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

GM-CSF and the impaired pulmonary innate immune response following hyperoxic stress

We have previously demonstrated that mice exposed to sublethal hyperoxia (an atmosphere of >95% oxygen for 4 days, followed by return to room air) have significantly impaired pulmonary innate immune response. Alveolar macrophages (AM) from hyperoxia-exposed mice exhibit significantly diminished antimicrobial activity and markedly reduced production of inflammatory cytokines in response to stimulation with LPS compared with AM from control mice in normoxia. As a consequence of these defects, mice exposed to sublethal hyperoxia are more susceptible to lethal pneumonia with Klebsiella pneumoniae than control mice. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a growth factor produced by normal pulmonary alveolar epithelial cells that is critically involved in maintenance of normal AM function. We now report that sublethal hyperoxia in vivo leads to greatly reduced alveolar epithelial cell GM-CSF expression. Systemic treatment of mice with recombinant murine GM-CSF during hyperoxia exposure preserved AM function, as indicated by cell surface Toll-like receptor 4 expression and by inflammatory cytokine secretion following stimulation with LPS ex vivo. Treatment of hyperoxic mice with GM-CSF significantly reduced lung bacterial burden following intratracheal inoculation with K. pneumoniae, returning lung bacterial colony-forming units to the level of normoxic controls. These data point to a critical role for continuous GM-CSF activity in the lung in maintenance of normal AM function and demonstrate that lung injury due to hyperoxic stress results in significant impairment in pulmonary innate immunity through suppression of alveolar epithelial cell GM-CSF expression.     

Control of macrophage lineage populations by CSF-1 receptor and GM-CSF in homeostasis and inflammation

There is recent interest in the role of monocyte/macrophage subpopulations in pathology. How the hemopoietic growth factors, macrophage-colony stimulating factor (M-CSF or CSF-1) and granulocyte macrophage (GM)-CSF, regulate their in vivo development and function is unclear. A comparison is made here on the effect of CSF-1 receptor (CSF-1R) and GM-CSF blockade/depletion on such subpopulations, both in the steady state and during inflammation. In the steady state, administration of neutralizing anti-CSF-1R monoclonal antibody (mAb) rapidly (within 3-4 days) lowered, specifically, the number of the more mature Ly6C(lo) peripheral blood murine monocyte population and resident peritoneal macrophages; it also reduced the accumulation of murine exudate (Ly6C(lo)) macrophages in two peritonitis models and alveolar macrophages in lung inflammation, consistent with a non-redundant role for CSF-1 (or interleukin-34) in certain inflammatory reactions. A neutralizing mAb to GM-CSF also reduced inflammatory macrophage numbers during antigen-induced peritonitis and lung inflammation. In GM-CSF gene-deficient mice, a detailed kinetic analysis of monocyte/macrophage and neutrophil dynamics in antigen-induced peritonitis suggested that GM-CSF was acting, in part, systemically to maintain the inflammatory reaction. A model is proposed in which CSF-1R signaling controls the development of the macrophage lineage at a relatively late stage under steady state conditions and during certain inflammatory reactions, whereas in inflammation, GM-CSF can be required to maintain the response by contributing to the prolonged extravasation of immature monocytes and neutrophils. A correlation has been observed between macrophage numbers and the severity of certain inflammatory conditions, and it could be that CSF-1 and GM-CSF contribute to the control of these numbers in the ways proposed.     

The Interaction of Naegleria fowleri Amoebae with Murine Macrophage Cell Lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D₁, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowler bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

毛喉萜对小鼠骨髓细胞和腹腔巨噬细胞表面胰岛素受体和GM－CSF受体的下调作用

放射性标记受体分析表明毛喉萜（ＦＳＫ）可以降低小鼠骨央细胞和腹腔巨噬细胞表面的胰岛素（ＩＮＳ）和粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）受体数目,而且对ＧＭ－ＣＳＦ的作用有剂量和时间依赖性,经ＦＳＫ处理的巨噬细胞酸性磷酸酶活性增加,微丝更加舒展。     

毛喉萜对小鼠骨髓细胞和腹腔巨噬细胞表面胰岛素受体和GM－CSF受体的下调作用

放射性标记受体分析表明毛喉萜可以降低小鼠骨央细胞和腹腔巨噬细胞表面的胰岛素和粒细胞－巨噬细胞集落刺激因子受体数目,而且对ＧＭ－ＣＳＦ的作用有剂量和时间依赖性,经ＦＳＫ处理的巨噬细胞酸性磷酸酶活性增加,微丝更加舒展。     

Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage

Transgenic mice carrying the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene expressed from a retroviral promoter exhibit elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. The eyes of transgenic mice are opaque, contain accumulations of macrophages, and develop retinal damage. Similarly, lesions containing macrophages develop in striated muscle. The mice also display an accumulation of large, often multinucleate, activated macrophages in the peritoneal and pleural cavities. The transgene is transcribed in peritoneal cells, as well as in eyes and infiltrated striated muscle. A high proportion of transgenic mice die with muscle wasting when aged 2-4 months, possibly because of macrophage activation resulting from the high levels of GM-CSF.     

Interleukin-10 contributes development of macrophage suppressor activities by macrophage colony-stimulating factor, but not by granulocyte-macrophage colony-stimulating factor

Macrophages are known to possess suppressor activities in immune responses. To determine the effects of GM-CSF and M-CSF on the expression of macrophage suppressor activities, monocyte-derived macrophages cultured with GM-CSF (GM-Mphis) were compared with those cultured with M-CSF (M-Mphis) for antigen-specific proliferation and interferon-gamma (IFN-gamma) production by lymphocytes. Both GM-Mphis and M-Mphis equally suppressed lymphocyte proliferation, but only M-Mphis suppressed IFN-gamma production in response to purified protein derivative (PPD). M-Mphis, but not GM-Mphis, released IL-10 not only in the course of macrophage differentiation but also in response to PPD after maturation to macrophages. From the results that (i) exogenous IL-10 suppressed IFN-gamma production, but not proliferation of lymphocytes, and that (ii) neutralizing antibody to IL-10 reversed suppressor activities of M-Mphis on IFN-gamma production, but not lymphocyte proliferation, it appeared that IL-10 was the major factor responsible for suppression of IFN-gamma production. Thus, these results suggest that only M-CSF augments IL-10-dependent suppressor activity of macrophages on IFN-gamma production and that both GM-CSF and M-CSF induce IL-10-independent macrophage suppressor activity on lymphocyte proliferation.     

Both granulocyte-macrophage CSF and macrophage CSF control the proliferation and survival of the same subset of alveolar macrophages

The effect of granulocyte-macrophage (GM)-CSF on the proliferation of murine pulmonary alveolar macrophages in vitro was investigated. About 20% of freshly isolated alveolar macrophages formed colonies in both liquid and soft agar cultures in the presence of GM-CSF. GM-CSF was also found to be capable of maintaining the survival of these colony-forming cells in vitro. Moreover, GM-CSF could substitute for CSF-1 in maintaining the survival of CSF-1-responding pulmonary alveolar macrophage colony-forming cells in the absence of CSF-1. The concentration of GM-CSF required for maintaining the survival of colony-forming cells without proliferation was much lower than that required for the proliferation of these cells in vitro. It also enhanced the CSF-1-dependent clonal growth of alveolar macrophages. These data suggest that the colony-forming cells that respond to GM-CSF are the same subset of macrophages that form colonies in the presence of CSF-1. GM-CSF did not inhibit the binding of 125I-CSF-1 to alveolar macrophages at 0 degrees C. However, the preincubation of macrophages with GM-CSF at 37 degrees C resulted in a transient down-regulation of CSF-1 binding activity.     

Growth inhibitory factor of human alveolar macrophage

The effect of human lung conditioned medium (HLCM) on the DNA synthesis in cultured human alveolar macrophages (HAM) was evaluated. The medium from human lung cultured for 3 days (3d-HLCM) promoted the DNA synthesis as well as the recombinant human GM-CSF does. On the other hand, that cultured for six days (6d-HLCM) did not promote the DNA synthesis but strongly suppressed GM-CSF induced DNA synthesis in HAM. This growth inhibitory effect was also observed when several macrophage like cell lines were cultured with 6d-HLCM. Partial purification of an inhibitory factor on gel permeation HPLC revealed two distinct peaks of activity with molecular weights of 38 kd and 110 kd.     

Interferon-gamma synergizes with tumor necrosis factor and with interleukin 1 and requires the presence of both monokines to induce antitumor cytotoxic activity in macrophages

Small concentrations of recombinant murine interferon-gamma (MuIFN-gamma), recombinant human interleukin 1 (HuIL-1), and recombinant murine tumor necrosis factor (MuTNF), added separately to cultures of thioglycolate-elicited peritoneal macrophages, produced no cytotoxic activity against L5178Y cells, a tumor cell line which is resistant to the direct toxic effects of these cytokines, either alone or in combination. However, small concentrations of MuIFN-gamma when combined with small concentrations of either HuIL-1 or MuTNF activated these macrophages to produce cytotoxic effects against L5178Y cells; small concentrations of HuIL-1 and MuTNF in combination had no macrophage activating activity. Specific antibody to MuTNF blocked the macrophage-activating synergistic effects of MuIFN-gamma + HuIL-1, and specific antibody to HuIL-1 blocked the macrophage-activating activity of MuIFN-gamma + MuTNF, indicating that MuTNF was induced in macrophage cultures treated with MuIFN-gamma + HuIL-1, and that murine IL-1 was induced in macrophage cultures treated with MuIFN-gamma + MuTNF. These results indicate that all three cytokines are required for induction of antitumor cytotoxic activation of macrophages. Experiments with a concentration of MuIFN-gamma which alone could activate macrophages revealed that both MuTNF and murine IL-1 were required for this activation. The demonstration that small concentrations of these three cytokines can act synergistically, but not separately, to activate macrophages indicates the importance of cytokine combinations in immunoregulation and in anti-tumor cell-mediated immune responses.