Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.     

Control of lipoprotein lipase secretion in mouse macrophages

The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells.     

Colony stimulating factor-1 stimulates diacylglycerol generation in murine bone marrow-derived macrophages, but not in resident peritoneal macrophages

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in murine bone marrow-derived macrophages (BMM); however, unlike BMM, murine resident peritoneal macrophages (RPM) undergo a poor proliferative response. It has previously been shown that phosphatidylinositol-4,5-bisphosphate hydrolysis is not associated with CSF-1 action in BMM. In this report we demonstrate that, despite a lack of inositol trisphosphate generation, CSF-1 transiently elevated both [3H]myristoyl- and [3H]arachidonyl-diacylglycerol (DAG) in BMM in a dose-dependent fashion. CSF-1 failed, however, to stimulate an increase in either species of DAG in RPM. Thus, DAG could be a second messenger for the proliferative action of CSF-1 in macrophages. Other mitogenic agents, 12-0-tetradecanoyl phorbol 13-acetate (TPA) and exogenous phospholipase C, also increased BMM levels of [3H]myristoyl- and [3H]arachidonyl-DAG. The nonmitogenic agents, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and zymosan, had different effects on the generation of either species of DAG in BMM. LPS failed to elevate either form, TNF-alpha increased only [3H]arachidonyl-DAG, while zymosan stimulated levels of both species of DAG. It therefore appears that increased diacylglycerol generation may be necessary, but perhaps not sufficient, for macrophage proliferation.     

Establishment of Self-Renewable GM-CSF-Dependent Immature Macrophages In Vitro from Murine Bone Marrow

Macrophages play a key role in the innate immune system. Macrophages are thought to originate from hematopoietic precursors or the yolk sac. Here, we describe the in vitro establishment of self-renewable GM-CSF-dependent immature macrophages (GM-IMs) from murine bone marrow (BM). GM-IMs grow continuously in vitro in conditioned medium containing GM-CSF. The immunophenotype of GM-IMs is F4/80^high CD11b^high CD11c^low Ly6C^low. By comparing gene expression in GM-IMs and BM dendritic cells, we found that GM-IMs expressed lower levels of chemokines, cytokines and their receptors. GM-IMs are round in shape, attach loosely to non-coated culture dishes and have a marked phagocytic capacity. These results indicate that GM-IMs are macrophage precursor cells. Following stimulation with LPS, monocyte-like GM-IMs converted to flat macrophage-like cells that tightly adhered to non-coated culture dishes and produced pro-inflammatory cytokines TNFα, IL-6 and IL-1β. These results indicated that GM-IMs differentiated to M1 pro-inflammatory macrophages. This was confirmed by stimulation of GM-IMs with IFNγ, an inducer of M1 markers. GM-IMs showed enhanced expression of M2 macrophage markers such as Arg1 and Retnla following stimulation by Th2 cytokines IL-4 and IL-13. When GM-IMs were injected into mice at sites of wounding, wound repair was enhanced. These results indicate that GM-IMs can differentiate to M2 macrophages. When GM-IMs were injected into clodronate-treated mice, they induced resident macrophage proliferation by producing M-CSF. In conclusion we have established self-renewable GM-CSF-dependent immature macrophages in vitro from murine BM, which differentiate to M1 or M2 macrophages.     

Functional defects of culture-grown bone marrow-derived macrophages from autoimmune MRL/MpJ-lpr/lpr mice

To investigate the primary defects and development of macrophages in MRL/MpJ-/pr/lpr (MRL/l) mice, we used a pure population of macrophages derived from bone marrow precursor cells cultured in the presence of L-cell conditioned medium (LCM) as a source of colony stimulating factor. Bone marrow-derived macrophages (BMM phi) from MRL/l mice had lower antigen presenting activity as detected by the induction of antigen-specific T cell proliferation, than age- and sex-matched control mice (CBA/J). Cell surface antigens (Ia and Mac-1) were determined quantitatively by a cell sorter as markers of macrophage differentiation. The BMM phi from MRL/l contained a much smaller number of Ia antigen-positive macrophages than those from normal mice. Treatment of BMM phi with an Ia-inducing of factor (IFN-gamma) markedly increased the expression of Ia antigens. This increase was significantly greater in BMM phi from MRL/l mice than in BMM phi from control mice. Expression of Mac-1 antigen was not different in BMM phi from the two strains. The Fc-mediated phagocytosis of IgG-coated sheep red blood cells was decreased in BMM phi from MRL/l mice compared with those from control mice. The function of nonspecific phagocytosis as measured by latex-bead incorporation was also impaired in MRL/l mice. The functional defects of MRL/l BMM phi found in these experiments are not secondary defects acquired under the influence of environmental signals during development, but are derived from the primary abnormalities which already exist in myeloid stem cells.     

Roles of the mitogen-activated protein kinase family in macrophage responses to colony stimulating factor-1 addition and withdrawal.

Colony stimulating factor-1 (CSF-1) (or macrophage CSF) is involved in the survival, proliferation, differentiation, and activation of cells of the monocyte/macrophage lineage. Because the mitogen-activated protein kinase family members extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinase are widely implicated in such cellular functions, we measured their activity in growing and growth-arrested cultures of bone marrow-derived macrophages (BMM), as well as their stimulation by saturating concentrations of CSF-1. ERK activity was approximately 2-fold higher in cycling BMM compared with growth-arrested BMM; in addition, CSF-1-stimulated BMM DNA synthesis was partially inhibited by PD98059, a specific inhibitor of MEK activation, suggesting a role for a mitogen-activated protein-ERK kinase (MEK)/ERK pathway in the control of DNA synthesis but surprisingly not in the control of cyclin D1 mRNA or c-myc mRNA expression. The suppression of BMM apoptosis by CSF-1, i.e. enhanced survival, was not reversed by PD98059, suggesting that a MEK/ERK pathway is not involved in this process. Using a quantitative kinase assay, it was found that CSF-1 gave a slight increase in BMM p38 activity, supporting prior data that CSF-1 is a relatively weak stimulator of inflammatory cytokine production in monocytes/macrophages. Relatively high concentrations of the p38 inhibitor, SKB202190, suppressed CSF-1-stimulated BMM DNA synthesis. No evidence could be obtained for the involvement of p38 activity in BMM apoptosis following CSF-1 withdrawal. We were not able to show that CSF-1 enhanced BMM JNK-1 activity to a significant extent; again, no role could be found for JNK-1 activity in the BMM apoptosis occurring after CSF-1 removal.     

CpG DNA activates survival in murine macrophages through TLR9 and the phosphatidylinositol 3-kinase-Akt pathway

Bacterial CpG-containing (CpG) DNA promotes survival of murine macrophages and triggers production of proinflammatory mediators. The CpG DNA-induced inflammatory response is mediated via TLR9, whereas a recent study reported that activation of the Akt prosurvival pathway occurs via DNA-dependent protein kinase (DNA-PK) and independently of TLR9. We show, in this study, that Akt activation and survival of murine bone marrow-derived macrophages (BMM) triggered by CpG-containing phosphodiester oligodeoxynucleotides or CpG-containing phosphorothioate oligodeoxynucleotides was completely dependent on TLR9. In addition, survival triggered by CpG-containing phosphodiester oligodeoxynucleotides was not compromised in BMM from SCID mice that express a catalytically inactive form of DNA-PK. CpG DNA-induced survival of BMM was inhibited by the PI3K inhibitor, LY294002, but not by the MEK1/2 inhibitor, PD98059. The effect of LY294002 was specific to survival, because treatment of BMM with LY294002 affected CpG DNA-induced TNF-alpha production only modestly. Therefore, CpG DNA activates macrophage survival via TLR9 and the PI3K-Akt pathway and independently of DNA-PK and MEK-ERK.     

The regulation of mononuclear phagocyte entry into S phase by the colony stimulating factor CSF-1

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. Populations of adherent bone marrow-derived macrophages (BMM) devoid of CSF-1 producing cells were used to study regulation by CSF-1 of macrophage entry into S phase. More than 95% of BMM possess the CSF-1 receptor. It was shown that 93-98% of BMM are cycling (S phase 8-9 hr, doubling time 24-28 hr) when cultured in the presence of CSF-1. BMM incubated with 15% FCS in the absence of CSF-1 or in the presence of CSF-1 concentrations inducing survival without proliferation enter a quiescent state. This state is characterized by a reduction in the synthesis of DNA (98%), total protein (35%), ribosomal protein (76%), and histone (96%) compared with the synthetic rate of these components in exponentially growing cells. Addition of CSF-1 to BMM rendered quiescent by removal of CSF-1 stimulated entry into S phase with a lag period of approximately 12 h. This lag period is reduced to 8 hr in BMM made quiescent at concentrations of CSF-1 inducing survival without proliferation, an effect which may be related to the expected higher protein content of these cells (Tushinski and Stanley, J. Cell. Physiol., 116:67-75). Neutralization of CSF-1 by antibody at different times during the lag period indicates that CSF-1 is required for almost the entire lag period for the entry of any cells into S phase. In BMM rendered quiescent by removal of both serum and CSF-1, purified CSF-1 without serum stimulated entry of cells into S phase, whereas serum alone was ineffective. The results are consistent with a primary regulatory role of CSF-1 in mononuclear phagocyte proliferation, survival, and function.     

Activation and proliferation signals in murine macrophages: stimulation of glucose uptake by hemopoietic growth factors and other agents

Purified colony-stimulating factor (CSF-1) (or macrophage colony stimulating factor [M-CSF]) stimulated the glucose uptake of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM) as measured by 3H-2-deoxyglucose (2-DOG) uptake. Similar concentrations of CSF-1 stimulated the 2-DOG uptake and DNA synthesis in BMM. Other purified hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and interleukin-3 (IL-3) (or multi-CSF), and the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though differing in their mitogenic capabilities on BMM, were also stimulators of 2-DOG uptake in BMM and RPM. The nonmitogenic agents, lipopolysaccharide (LPS) and concanavalin A (Con A), were also active. The inhibition by cytochalasin B and by high concentrations of D-glucose suggest that the basal and stimulated 2-DOG uptake occurred via a carrier-facilitated D-glucose transport system. The responses of the two macrophage populations to the hemopoietic growth factors and to the other agents were quite similar, suggesting that events that are important for the induction of DNA synthesis are not tightly coupled to the earlier rise in glucose uptake. For the BMM, the ability of a particular agent to stimulate glucose uptake did not parallel its ability to promote cell survival. However, stimulation of glucose uptake could still be a necessary but insufficient early macrophage response for cell survival and subsequent DNA synthesis.     

Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

Transforming growth factor-beta 1 inhibits scavenger receptor activity in THP-1 human macrophages.

The macrophage scavenger receptor, a 220-kDa trimeric membrane glycoprotein, mediates the internalization of modified forms of low density lipoprotein (LDL) such as acetyl-LDL and oxidized-LDL and thus is likely to play a key role in atheroma macrophage foam cell formation. In addition, recent evidence suggests that the scavenger receptor may be an important macrophage binding site for lipopolysaccharide involved in lipopolysaccharide scavenging by macrophages. However, little is known about the regulation of this important receptor. We now report that the induction of scavenger receptor activity (as measured by acetyl-LDL stimulation of intracellular cholesterol esterification) seen in phorbol ester-differentiated THP-1 human macrophages was completely suppressed to the level seen in undifferentiated THP-1 monocytes by picomolar concentrations of transforming growth factor-beta 1 (TGF-beta 1). 125I-Acetyl-LDL degradation was inhibited in a dose-dependent manner by TGF-beta 1, with maximal inhibition (approximately 70%) occurring at 24 pM TGF-beta 1. Scatchard analysis revealed that TGF-beta 1 treatment resulted in a approximately 2-fold decrease in receptor number, and Northern blot analysis of RNA isolated from differentiated THP-1 macrophages demonstrated approximately 2-fold less scavenger receptor mRNA in TGF-beta 1-treated cells compared with that in macrophages not treated with TGF-beta 1. Since TGF-beta 1 is thought to be present in both atherosclerotic and inflammatory lesions, the above findings may have physiological relevance regarding the regulation of atheroma foam cell formation and/or the regulation of lipopolysaccharide clearance by macrophages.     

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.     

Biochemical events accompanying macrophage activation and the inhibition of colony-stimulating factor-1-induced macrophage proliferation by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide.

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.     

Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function

The effects of granulocyte-macrophage (GM)-CSF on the synthesis of MHC class II molecules and on the Ag presentation capacity by bone marrow derived macrophages (BMM phi) was investigated. BMM phi obtained by in vitro culture in the presence of macrophage-CSF were negative for synthesis of I-A molecules and induced the Ag-mediated proliferation of insulin-specific T clone cells with lower efficiency than splenic accessory cells. After pulse treatment with GM-CSF for 24 to 48 h, day 12 BMM phi exhibited highly efficient Ag presentation function which was superior to that induced by IFN-gamma. Expression of membrane-bound IL-1 was augmented significantly by GM-CSF, but not by IFN-gamma. However, the T cell clone used to probe for accessory cell function of BMM phi was not dependent on IL-1 for optimal proliferation. Concomitantly, GM-CSF induced the de novo synthesis of I-A molecules, although to a lesser extent than optimal doses of IFN-gamma. Thus GM-CSF appears to elicit properties in addition to Ia molecule synthesis and membrane IL-1 expression in BMM phi being essential for efficient accessory cell function to the T clone cells. The activation of BMM phi by GM-CSF was reversible and could be repeated. These data show that GM-CSF exerts a modulatory influence on preformed BMM phi, reversibly activating cells to Ia biosynthetic potential and pronounced accessory cell capacity, thus rendering the explanation unlikely that differentiation of precursor cells into a constitutively functional state had occurred.     

Regulation of colony-stimulating factor 1-dependent macrophage precursor proliferation by type beta transforming growth factor

Transforming growth factor (TGF) type beta, a potent growth modulator, has recently been shown to inhibit the proliferation and function of several types of immune cells. This report investigates the effect of human platelet purified TGF-beta on CSF-1-induced proliferation in liquid cultures. We used two cell types to study TGF-beta effects, bone marrow precursors and a c-myc partially transformed CSF-1-dependent macrophage cell line designated BMM-8. We found that CSF-1-dependent proliferation of both cell types was strongly inhibited by TGF-beta in a dose-dependent manner. Approximately 1.6 and 8 pM TGF-beta inhibited 50% of CSF-1 proliferation of the bone marrow precursors and BMM-8, respectively. Inhibition appeared to be reversible, as bone marrow and BMM-8 cells proliferated in response to CSF-1 after preincubation of the cells in TGF-beta. Interestingly, inhibition of hematopoietic cells was observed only after a lag period of 24 to 48 h after onset of cultures. TGF-beta inhibition was partially diminished when increasing amounts of CSF-1 were added to the cultures. TGF-beta inhibition did not involve secondary inhibitory factors such as IFN or PG, both of which have been previously shown to suppress CSF responsiveness. Finally, flow cytometric analysis of the cell cycle indicated that within 48 h, TGF-beta-treated BMM-8 cells were prevented from entering S phase. These results suggest that TGF-beta may play an important role in the negative regulation of macrophage production.     

Protein kinase C has both stimulatory and suppressive effects on macrophage superoxide production.

Unlike resident peritoneal macrophages (RPM) or tumor necrosis factor alpha (TNF alpha)-primed bone marrow-derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNF alpha-primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan-induced production of superoxide by unprimed and TNF alpha-primed BMM and RPM, in spite of substantial inhibition of PMA-induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10(-7) M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan-induced response, a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt-induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpretation, the co-stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan-induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide production.     

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.