Biomedical subjects

J A Hamilton

Publications and source records attributed to J A Hamilton.

At least 55 records · Page 3Linked to original sources

PubMed2000

The colony-stimulating factors and collagen-induced arthritis: exacerbation of disease by M-CSF and G-CSF and requirement for endogenous M-CSF.

There is increasing evidence that the colony-stimulating factors (CSFs) may play a part in chronic inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). We examined the involvement of macrophage CSF (M-CSF or CSF-1) and granulocyte CSF (G-CSF) in collagen-induced arthritis (CIA), a murine model of RA. Daily injections of M-CSF or G-CSF, 20-24 days postprimary immunization with type II collagen, exacerbated disease symptoms in suboptimally immunized DBA/1 mice. Support for the involvement of endogenous M-CSF in CIA was obtained by studies in which neutralizing monoclonal antibody reduced the severity of established CIA and also by studies showing the resistance of M-CSF-deficient op/op mice to CIA induction. These studies show that M-CSF and G-CSF can be proinflammatory in CIA and provide evidence that macrophage- and granulocyte-lineage cells can exacerbate CIA. Our results also show that M-CSF-dependent cells are essential for CIA development, suggesting M-CSF may be a suitable target for therapeutic intervention in RA.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.

Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A.

Animals↗

PubMed1999

Characterization of a CSF-induced proliferating subpopulation of human peripheral blood monocytes by surface marker expression and cytokine production.

The phenotype of a subpopulation(s) of human monocytes which has been shown to proliferate in vitro in response to macrophage colony-stimulating factor (M-CSF or CSF-1) and granulocyte-macrophage CSF (GM-CSF) is as yet unknown. To identify this proliferating subpopulation(s) we demonstrated first that DNA synthesis was occurring under culture conditions suitable for flow cytometric evaluation. Flow cytometric analysis of surface antigen expression identified that after 5 days of culture the proliferating subpopulation of monocytes expressed CD14, CD13, CD33, CD11b, CD11c, CD87, HLA-DR, CD45RO, and did not express CD86, CD34, CD80, CD4, CD16, and CD56. In addition, these proliferating monocytes (representing approximately 5% of total monocytes) were shown to produce the proinflammatory cytokines interleukin-6 and tumor necrosis factor alpha in response to lipopolysaccharide stimulation. Further characterization and subsequent isolation of this subpopulation of monocytes may provide new and important information necessary to understand inflammatory diseases such as rheumatoid arthritis, where local proliferation at the site of inflammation may be a key factor contributing to the chronicity of the disease.

Antigens, CD↗

PubMed1999

Expression of a Y559F mutant CSF-1 receptor in M1 myeloid cells: a role for Src kinases in CSF-1 receptor-mediated differentiation.

We have established two M1 myeloid cell lines, M1/WT cells overexpressing the wild-type CSF-1 receptor and M1/Y559F cells expressing a specific tyrosine mutant. M1/WT cells differentiated in response to CSF-1, with a reduction in their proliferative capacity. CSF-1-mediated differentiation was partially abrogated in the M1/Y559F cells, with a less marked reduction in proliferative capacity. The Src tyrosine kinases c-Src, c-Yes, c-Fyn, and c-Hck were tyrosine phosphorylated in the M1/WT cells in response to CSF-1 and bound to the WT CSF-1R through their SH2 domains. Binding of the Src kinases to the CSF-1 receptor was greatly reduced in the M1/Y559F cells. CSF-1-mediated activation of STAT3 was also abrogated in the M1/Y559F cell line. Treatment of M1/WT cells with the Src family inhibitor PP2 resulted in an inhibition of CSF-1-mediated differentiation, equivalent to that observed in the M1/Y559F cells. These data suggest that the reduced Src binding observed in the M1/Y559F cells may contribute to their reduced ability to differentiate.

Animals↗

PubMed1999

Incorporation of [1-13C]oleate into cellular triglycerides in differentiating 3T3L1 cells.

Oleate is one of the most abundant dietary fatty acids, and much remains to be learned about its metabolism in fat cells. We studied the incorporation of exogenous [1-13C]oleate into triglycerides (TG) in differentiating 3T3L1 preadipocytes using 13C NMR spectroscopy. The quantity of oleate incorporated into TG was found to increase as preadipocytes differentiated into fat cells. The ratio of unesterified [1-13C]oleate to total stored fatty acids was higher in less differentiated cells, and declined at later stages of differentiation as cells accumulated fatty acids through de novo synthesis. When added as the only exogenous fatty acid, oleate was largely esterified at the sn-2 position. When equimolar unlabeled linoleate was co-provided at the same time, the ratio of [1-13C]oleate esterified at the sn-1,3 position increased, implying competition between linoleate and oleate for esterification, especially at the sn-2 position. When cells pre-enriched with [1-13C]oleate (esterified to TG) were treated with isoproterenol, a lipolytic agent, most of the [1-13C]oleate was still found in TG, despite a high rate of lipolysis determined by measuring glycerol release. This implies extensive re-esterification of the oleate released by lipolysis.

3T3 Cells↗

PubMed1999

Direct comparison of the effects of CSF-1 (M-CSF) and GM-CSF on human monocyte DNA synthesis and CSF receptor expression.

There is evidence that a proportion of human monocytes can proliferate in vitro in response to colony-stimulating factor-1 (CSF-1, also known as M-CSF) and granulocyte-macrophage CSF (GM-CSF). To determine whether there are differences in DNA synthesis responses to these CSF, a large study using purified human peripheral blood monocytes from 45 donors was performed under optimized culture conditions. In contrast to the consistent response to CSF-1, approximately 20% of donors have monocytes that do not respond or have a minimal DNA synthesis response to GM-CSF stimulation. However, analysis demonstrated that no statistically significant differences exist in the levels of CSF-1 and GM-CSF-stimulated proliferation in monocytes. In addition, CSF-1 receptor (CSF-1R) blocking experiments indicated that a proportion of the GM-CSF-induced DNA synthesis is due to endogenous levels of CSF-1. As a further comparison of the actions of the two CSFs, CSF-1R and GM-CSFR levels were measured by flow cytometry, and it was shown that GM-CSFR levels decreased within 5 days of culture, independent of the conditions examined. In contrast, CSF-1R levels at day 5 approximated those measured in uncultured monocytes. Whether the proliferating subpopulation(s) express one or both CSF receptors at the beginning or at the end of culture is as yet unknown. The information obtained in this study will be useful for the design of strategies to enrich for the subpopulation in question based on CSF receptor expression.

Antigen-Antibody Reactions↗

PubMed1999

Separation and characterization of the activated pool of colony-stimulating factor 1 receptor forming distinct multimeric complexes with signalling molecules in macrophages.

Colony-stimulating factor 1 (CSF-1) triggers the activation of intracellular proteins in macrophages through selective assembly of signalling complexes. The separation of multimeric complexes of the CSF-1 receptor (CSF-1R) by anion-exchange chromatography enabled the enrichment of low-stoichiometry complexes. A significant proportion of the receptor in CSF-1-stimulated cells that neither possessed detectable tyrosine kinase activity nor formed complexes was separated from the receptor pool displaying autokinase activity that formed chromatographically distinct multimeric complexes. A small pool of CSF-1R formed a multimeric complex with phosphatidylinositol-3 kinase (PI-3 kinase), SHP-1, Grb2, Shc, c-Src, Cbl, and a significant number of tyrosine-phosphorylated proteins in CSF-1-stimulated cells. The complex showed a considerable amount of CSF-1R complex-associated kinase activity. A detectable level of the complex was also present in untreated cells. PI-3 kinase in the multimeric complex displayed low lipid kinase activity despite the association with several proteins. The major pool of activated CSF-1R formed transient multimeric complexes with distinctly different tyrosine-phosphorylated proteins, which included STAT3 but also PI-3 kinase, Shc, SHP-1, and Grb2. A significant level of lipid kinase activity was detected in PI-3 kinase in the latter complexes. The different specific enzyme activities of PI-3 kinase in these complexes support the notion that the activity of PI-3 kinase is modulated by its association with CSF-1R and other associated cellular proteins. Specific structural proteins associated with the separate CSF-1R multimeric complexes upon CSF-1 stimulation and the presence of the distinct pools of the CSF-1R were dependent on the integrity of the microtubular network.

Animals↗

PubMed1999

Oxidized LDL can induce macrophage survival, DNA synthesis, and enhanced proliferative response to CSF-1 and GM-CSF.

Modification of low density lipoprotein (LDL), eg, by oxidation, has been proposed as being important for the formation of foam cells and therefore for the development of atherosclerotic plaques. There are a number of reports showing that macrophage-derived foam cells can proliferate in both human and animal lesions, particularly in the early phase of the disease and possibly involving macrophage-colony stimulating factor (M-CSF, or CSF-1). We studied the in vitro effects of oxidized LDL (ox-LDL) on murine bone marrow-derived macrophages (BMMs), a cell population with a high proliferative capacity in vitro in response to CSF-1 and a dependence for survival on the presence of this growth factor. We report here that treatment of BMMs with low doses of ox-LDL, but not with native LDL, led to cell survival, DNA synthesis, and an enhanced response to the proliferative actions of CSF-1 and granulocyte macrophage-CSF (GM-CSF); the effects were dependent on the degree of LDL oxidation. For CSF-1, a synergistic effect was noticeable at suboptimal doses. The effect of ox-LDL occurred even in the absence of endogenous CSF-1 or GM-CSF. Our findings suggest that ox-LDL, and possibly other modified forms of LDL, could maintain macrophage (and foam cell) survival and therefore lengthen their tenure in a plaque; the modified LDL could also cause local macrophage proliferation or "prime" them so that they could proliferate better in response to CSF-1 (and GM-CSF) concentrations that may be present in the atheroma.

Animals↗

PubMed1999

How are free fatty acids transported in membranes? Is it by proteins or by free diffusion through the lipids?

Although transport of long-chain free fatty acids (FFAs) into cells is often analyzed in the same way as glucose transport, we argue that the transport of the lipid-soluble amphipathic FFA molecule must be viewed differently. The partitioning of FFAs into phospholipid bilayers and their interfacial ionization are particularly relevant to transport. We summarize new data supporting the diffusion hypothesis in simple lipid bilayers and in plasma membranes of cells. Along with previous supporting data, the new data indicate that transport of FFAs through membranes could occur rapidly by flip-flop of the un-ionized form of the FFA. It appears that, at least for the adipocyte, passive diffusion guarantees fast entry and exit of FFAs at both low and high concentrations. Although there are several candidate proteins for the membrane transport of FFAs, most of these proteins have other established functions. Thus, unlike the glucose transporters, these proteins would not be single-function proteins. Definitive proof of their function as FFA transporters awaits their reconstitution into simple model systems.

Animals↗

PubMed1999

A comparative study of the backbone dynamics of two closely related lipid binding proteins: bovine heart fatty acid binding protein and porcine ileal lipid binding protein.

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N[1H] NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the 'model-free' approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the microsec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.

Animals↗

PubMed1998

MRL/lpr and MRL+/+ macrophage DNA synthesis in the absence and the presence of colony-stimulating factor-1 and granulocyte-macrophage colony-stimulating factor.

Macrophage accumulation and proliferation as well as altered macrophage properties have been observed in autoimmune MRL mice. To determine whether there might be innate differences in the proliferative responses, we examined the DNA synthesis responses of peritoneal macrophages and macrophages derived in vitro from bone marrow precursors (bone marrow-derived macrophages (BMM)). Murine peritoneal exudate macrophages normally require the addition of macrophage CSF (CSF-1) to enter cell cycle in vitro. In contrast, we have found that many thioglycollate-induced adherent peritoneal macrophages, but not resident peritoneal macrophages, from both MRL/lpr and MRL+/+ mice atypically underwent DNA synthesis even in the absence of added CSF-1. They also responded very well to granulocyte-macrophage CSF. These findings may help to explain the appearance of increased macrophage numbers in MRL lesions. In contrast to a previous report, it was found that MRL/lpr and MRL+/+ BMM did not have an enhanced response to CSF-1 and that modulation of CSF-1 receptor expression was not more rapid in MRL BMM. We also found no evidence for abnormal CSF-1 internalization and degradation or for the lpr mutation to have any enhanced effect on BMM survival in the absence of CSF-1. TNF-alpha lowered the DNA synthesis response to CSF-1 of MRL/lpr BMM rather than enhanced it, as has been reported. Our data suggest that the enhanced accumulation of macrophages in the MRL/lpr kidney cannot be explained by a proposed model of enhanced responsiveness of MRL/lpr BMM to CSF-1, including a contribution by TNF-alpha.

Animals↗

PubMed1998

Direct binding of Shc, Grb2, SHP-2 and p40 to the murine granulocyte colony-stimulating factor receptor.

Granulocyte colony-stimulating factor (G-CSF) mediates the proliferation, differentiation and activation of cells in the granulocytic lineage. However, knowledge about the specific signaling pathways utilized by the G-CSF receptor (G-CSF-R) upon ligand binding remains limited. In this report, we show rapid phosphorylation of Shc upon stimulation of NFS-60 cells with G-CSF, and inducible association of Shc and Grb2 with the G-CSF-R in these cells. Using a tyrosine-phosphorylated GST-G-CSF-R fusion we demonstrate that Shc, Grb2 and SHP-2 directly bind the receptor via their respective SH2 domains, suggesting multiple routes of MAPK activation from the G-CSF-R are possible. In addition, we have identified an unknown p40 molecule which is associated with the G-CSF-R transiently following G-CSF stimulation, and a constitutively-associated p37 molecule.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Protection from collagen-induced arthritis in granulocyte-macrophage colony-stimulating factor-deficient mice.

The involvement of granulocyte-macrophage CSF (GM-CSF) in collagen-induced arthritis (CIA) was examined using GM-CSF-deficient mice. Although CIA is generally considered to be restricted to mice of the H-2q or H-2r haplotypes, we examined the role of GM-CSF in the CIA model using GM-CSF-deficient (-/-) and wild-type (+/+) mice on a C57BL/6 (H-2b) background. Mice were immunized by intradermal injection at the base of the tail with chick type II collagen followed by a repeat injection 21 days later. We found, based on both clinical and histologic assessments, that wild-type mice on this background developed severe CIA, while the GM-CSF-deficient mice had virtually no disease. Mice that were heterozygous for the GM-CSF gene (+/-) collectively displayed an intermediate response between those of the GM-CSF(+/+) and GM-CSF(-/-) groups, suggesting a gene dosage effect. GM-CSF(+/+) and GM-CSF(+/-) mice exhibited CIA responses ranging from mild (single digits) to severe swelling of all four paws, while in the few GM-CSF(-/-) mice that developed CIA the disease was confined to single digits. Despite the putative role of GM-CSF in dendritic cell development, GM-CSF-deficient mice exhibited both humoral and cellular (delayed-type hypersensitivity) responses to type II collagen; however, the cellular response was significantly reduced in the GM-CSF-deficient mice compared with the wild-type controls. These findings suggest that GM-CSF is required for CIA development in mice and support the idea that GM-CSF is a key cytokine in inflammatory joint disease.

Animals↗

PubMed1998

Proliferation-independent induction of macrophage cyclin D2, and repression of cyclin D1, by lipopolysaccharide.

D-type cyclins are induced in response to mitogens and are essential and rate-limiting for G1 phase progression in normal mammalian cells. Macrophages proliferating in response to colony-stimulating factor-1 (CSF-1) express cyclin D1 and to a lesser extent cyclin D2 but not cyclin D3. Previously we showed that the macrophage-activating agent lipopolysaccharide (LPS) blocks CSF-1-induced proliferation and cyclin D1 expression in macrophages. Here we report upon the effect of LPS on expression of cyclin D2 in normal mouse bone marrow-derived macrophages (BMM). Unexpectedly we found that this anti-mitogen raised levels of CSF-1-stimulated cyclin D2 mRNA and protein. Furthermore, LPS alone induced cyclin D2 but not cyclin D1. Inhibition of the MEK/ERK (MAPK/ERK kinase/extracellular signal-regulated kinase) mitogen-activated protein kinase pathway repressed LPS-induced cyclin D2 mRNA, whereas inhibition of the p38 mitogen-activated protein kinase enhanced expression. However, in contrast to cyclin D1, cyclin D2 in bone marrow-derived macrophages did not appear to be regulated by protein kinase A pathways. The present data (a) show elevation of a D-type cyclin in the absence of proliferation, (b) demonstrate inverse regulation of two distinct D-type cyclins under identical conditions, and (c) suggest that cyclin D2 plays a role in macrophage activation by LPS.

Animals↗

PubMed1998

Macrophage lineage cells in inflammation: characterization by colony-stimulating factor-1 (CSF-1) receptor (c-Fms), ER-MP58, and ER-MP20 (Ly-6C) expression.

Macrophage populations resident in tissues and at sites of inflammation are heterogeneous and with local proliferation sometimes evident. Using the convenient murine peritoneal cavity as an inflammation model, the appearance of macrophage lineage cells was followed with time in both thioglycollate- and sodium periodate-induced exudates. The cells were characterized by their proliferative response in vitro in response to colony-stimulating factor-1 (CSF-1) (or macrophage colony-stimulating factor [M-CSF]), particularly by their ability to form colonies in agar, in combination with flow cytometry (surface marker expression and forward and side scatter characteristics). We propose that c-Fms (CSF-1 receptor), unlike other markers, is a uniformly expressed and specific marker suitable for the detection of macrophage-lineage cells in tissues, both in the steady state and after the initiation of an inflammatory reaction. It was shown that the bone marrow myeloid precursor markers, ER-MP58 and ER-MP20 (Ly-6C), but not ER-MP12 (PECAM-1), are expressed by a high proportion of macrophage-lineage cells in the inflamed peritoneum. The macrophage colony-forming cells (M-CFCs) in a 16-hour thioglycollate-induced exudate were phenotyped as c-Fms+ERMP12-20+58+, properties consistent with their being more mature than bone marrow M-CFCs. It is proposed that ER-MP58, as well as ER-MP20, may be a useful marker for distinguishing inflammatory macrophage-lineage cells from the majority of those residing normally in tissues.

Animals↗

PubMed1998

Platelet-activating factor receptor activation. An initiator step in HIV-1 neuropathogenesis.

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system results in neuronal apoptosis. Activated HIV-1-infected monocytes secrete high levels of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the phospholipid mediator platelet-activating factor (PAF). TNF-alpha and PAF are elevated in the central nervous system of patients with HIV-1-associated dementia. We now demonstrate that conditioned media from activated HIV-1-infected monocytes induces neuronal apoptosis, which can be prevented by co-incubation with PAF acetylhydrolase, the enzyme that catabolizes PAF in the central nervous system. Preceding apoptosis is a TNF-alpha-induced increase in neuronal ceramide levels. TNF-alpha-mediated neuronal apoptosis can also be blocked by co-incubation with PAF acetylhydrolase, or a PAF receptor antagonist. Blocking pathologic activation of PAF receptors may therefore be a pivotal step in the treatment of HIV-1-associated dementia.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed1998

cAMP enhances CSF-1-induced ERK activity and c-fos mRNA expression via a MEK-dependent and Ras-independent mechanism in macrophages.

Inhibition of MAPK by elevated intracellular cAMP has often been correlated with suppression of growth factor-induced proliferation. However, in murine bone marrow-derived macrophages (BMM) we show that the cAMP analogue, 8-bromo cAMP (8BrcAMP) (1mM), despite being a dramatic G1 phase proliferation inhibitor, increased ERK activity both in the absence and presence of CSF-1; these increases were blocked by PD98059 (100 microM) suggesting MEK dependence. In contrast, CSF-1-stimulated p21Ras activity was blocked by 8BrcAMP thus correlating with the inhibition of proliferation. This is the first report to indicate that elevated intracellular cAMP can activate ERK activity while inhibiting proliferation and the data support the concept in CSF-1-treated macrophages of Ras-independent activation of ERK activity. It was also found that the acute but not the sustained elevation of c-fos mRNA expression due to 8BrcAMP was also MEK dependent indicating that there are separate pathways controlling c-fos mRNA expression in BMM.

8-Bromo Cyclic Adenosine Monophosphate↗