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Biomedical subjects

J A Hamilton

Publications and source records attributed to J A Hamilton.

At least 163 records · Page 9Linked to original sources
PubMed1993

Regulation of macrophage colony-stimulating factor (M-CSF) production in cultured human synovial fibroblasts.

OBJECTIVE: To study the regulation of macrophage-colony stimulating factor (M-CSF) formation in vitro by human synovial fibroblast-like cells. METHODS: Human synovial cell explant cultures were established using cells from non-rheumatoid donors. M-CSF antigen was measured by immunoassay, and messenger RNA (mRNA) levels were determined by Northern blot. RESULTS: The cytokines, interleukin-1 (IL-1), tumor necrosis factor alpha (TNF alpha), interferon-gamma (IFN-gamma) and IL-4, increased production of M-CSF above constitutive levels. The presence of the cyclooxygenase inhibitor, indomethacin, potentiated the action of IL-1 on M-CSF synthesis, suggesting that an endogenous cyclooxygenase product(s) can down-regulate M-CSF formation. Changes in M-CSF mRNA levels paralleled those in protein levels. The glucocorticoid, dexamethasone, and the retinoid, all-trans retinoic acid, stimulated M-CSF formation. The control of M-CSF synthesis in the synovial fibroblasts differs from that for granulocyte macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF). CONCLUSION: These results suggest that cytokine-stimulated synovial fibroblasts may be a source of M-CSF production in the joints of patients with inflammatory arthritis; as a result, monocyte/macrophages may be activated, leading to perpetuation of the inflammation and destructive events occurring in these lesions.

Cells, Cultured↗
PubMed1993

Regulation of M-CSF production by cultured human thymic epithelial cells.

We have studied the regulation of M-CSF production by human thymic epithelial cells (TEC) in a continuing effort to better understand the contribution of TEC to the intrathymic cytokine network. The levels of M-CSF were measured by radioimmunoassay. Five different TEC cultures were studied and we found that all cells examined produced M-CSF constitutively. We also studied the effects of cytokines on the regulation of the M-CSF secretion profile. IL-1, which strongly induces the secretion of a number of cytokines in TEC, was found to up-regulate M-CSF levels. The effects of IL-1 were dose and time dependent suggesting a direct effect on TEC. IFN-gamma is known to up-regulate cell surface antigens, and to modulate the IL-1-induced cytokine response in TEC. IFN-gamma could induce M-CSF by itself, but the effects were less pronounced than those of IL-1. IFN-gamma did not modify the IL-1-induced M-CSF. IL-4, which has been shown to partially modulate the IL-1-induced GM-CSF in TEC, had no effect on constitutive or induced M-CSF levels. These data are discussed in the context of the regulation of myelopoietic growth factor production by thymic stromal cells.

Abortion, Induced↗
PubMed1993

Contrasting effects of interleukin-4 on colony-stimulating factor and interleukin-6 synthesis by vascular endothelial cells.

Endothelial cells (EC) may regulate both local and systemic aspects of inflammation through the synthesis of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin-6 (IL-6). EC are known to synthesize these cytokines in response to interleukin-1 (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide (LPS). In this paper, we illustrate the effect of interleukin-4 (IL-4) in reducing the synthesis of GM-CSF by EC stimulated with IL-1 alpha, TNF-alpha, or LPS. This is compared with the previously reported strong synergy between IL-4 and IL-1 alpha, TNF-alpha, or LPS in the synthesis of IL-6 by EC. No clear effect of IL-4 was seen in the synthesis of G-CSF or M-CSF. The range of concentrations of IL-4 at which these effects were seen was identical for both reduced GM-CSF synthesis and increased IL-6 synthesis. The effect of IL-4 on IL-6 synthesis was seen by 4 h of treatment, while that on GM-CSF was apparent between 4 and 8 h. It is suggested that these contrasting effects of IL-4 may reflect a biological role for this cytokine in the regulation of leukocytosis and the acute phase response.

Acute-Phase Reaction↗
PubMed1993

Expression and amplification of cyclin genes in human breast cancer.

Cyclins, the regulatory subunits of cyclin-dependent kinases, play an important role in the control of cellular proliferation. Since dysregulated expression of these genes may contribute to the malignant phenotype the expression and amplification of cyclin A, B1, C, D1, D2, D3 and E genes were studied in 20 breast cancer cell lines. Increased expression of one or more of the cyclin A, B1, D1 or E genes was found in seven cell lines (35%); of these five (25%) showed increased expression of cyclin D1. Overexpression occurred in both the presence and absence of gene amplification. Conversely, amplification did not invariably lead to overexpression. Cyclin D2 expression was lower in breast cancer cell lines than in cultured normal breast epithelial cells. Cyclin D1 expression was further investigated in breast tumour biopsies: 56 of 124 specimens (45%) expressed higher levels of cyclin D1 mRNA than normal breast tissue. These data implicate dysregulated expression of several cyclin genes, particularly cyclin D1, as a potential factor in the pathogenesis of breast cancer.

Base Sequence↗
PubMed1993

Evidence against a requirement for Na+/H+ exchange activity for proliferation or differentiation of HL60 leukemic cells.

Although increased Na+/H+ exchange (antiport) activity has been shown to precede both granulocytic and monocytic differentiation of HL60 cells, its requirement for this process remains controversial. We show here that, in the absence of HCO3-, the Na+/H+ exchanger is the predominant mechanism involved in rapid recovery of pHi from cytoplasmic acidosis; however, in the presence of HCO3-, other mechanisms also contribute to pHi homeostasis. In the absence of HCO3-, dimethyl sulfoxide and 4 beta-phorbol 12-myristate 13 alpha-acetate result in an acute activation of antiport activity, whereas all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, gamma-interferon, and tumor necrosis factor alpha do not. Furthermore, whether or not HCO3- is present, the amiloride analogue, 5-N,N-dimethylamiloride, a potent and more specific antiport inhibitor than the parent compound, failed to suppress HL60 cell proliferation or the decreased proliferation in response to dimethyl sulfoxide, all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, gamma-interferon, or tumor necrosis factor alpha. In the absence of HCO3-, 5-N,N-dimethylamiloride also had little effect on the ability of these agents to induce functional maturation as assessed by acquisition of respiratory burst activity. Inhibition of antiport activity also did not prevent 4 beta-phorbol 12-myristate 13 alpha-acetate-induced expression of c-fos mRNA or cell adherence. The results suggest that, even under conditions in which the Na+/H+ exchanger is the predominant operable pHi regulator, antiport activity is not required for HL60 proliferation or differentiation.

Amiloride↗
PubMed1992

Zymosan-triggered tyrosine phosphorylation in mouse bone-marrow-derived macrophages is enhanced by respiratory-burst priming agents.

We have investigated the relationship between tyrosine phosphorylation and respiratory-burst activity in mouse bone-marrow-derived macrophages (BMM). We demonstrate that zymosan, an agent known to trigger the macrophage respiratory burst, also triggers the activation of tyrosine kinase activity, resulting in rapid tyrosine phosphorylation on numerous proteins, and provide evidence for the role of tyrosine phosphorylation in the triggering of the BMM respiratory burst. Agents, such as tumour necrosis factor alpha (TNF alpha), interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS), which prime the macrophage for an enhanced zymosan-triggered respiratory burst, increase tyrosine phosphorylation triggered by zymosan. The zymosan-triggered tyrosine phosphorylation and respiratory-burst activity were partially suppressed by the tyrosine kinase inhibitors alpha-cyano-3-ethoxy-4-hydroxy-5-phenylmethylcinnamide (ST638) and herbimycin A. In addition, pre-exposure of BMM to vanadate, a phosphotyrosine phosphatase inhibitor, greatly enhanced the ability of zymosan to induce tyrosine phosphorylation and trigger the respiratory burst. These data highlight the importance of the balance between tyrosine kinase and phosphotyrosine phosphatase activity in determining the ultimate level of tyrosine phosphorylation in BMM and suggest that zymosan-triggered tyrosine phosphorylation is an important biochemical signal for triggering of the respiratory burst.

Animals↗
PubMed1992

pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.

A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release of protons from approximately 50% of these fatty acid molecules (apparent pKa in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagram of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t1/2 < or = 2 sec) whereas ionized fatty acid flip-flops slowly (t1/2 of minutes). Since fatty acids move across phosphatidylcholine bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid flip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia.

Biological Transport↗
PubMed1992

Human arterial smooth muscle cells synthesize granulocyte colony-stimulating factor in response to interleukin-1 alpha and tumor necrosis factor-alpha.

Vascular smooth muscle cells (SMC) are a major cell type comprising the walls of blood vessels. We report the synthesis of granulocyte colony-stimulating factor (G-CSF) by cultured human SMC obtained from the internal mammary artery and thoracic aorta. Interleukin-1 alpha (IL-1 alpha) greatly increased in a dose-dependent manner the amount of this cytokine produced by the SMC, with tumor necrosis factor-alpha (TNF-alpha) being less effective. Newly formed G-CSF could be detected in culture supernatants within 6 hours after IL-1 alpha or TNF-alpha treatment. Northern blot analysis of SMC stimulated with IL-1 alpha and TNF-alpha showed an increase in the amount of mRNA for G-CSF as compared with control cells. Enhanced G-CSF mRNA levels were observed when SMC were treated with cycloheximide in the absence or presence of added cytokine. In vasculitis, the walls of blood vessels become inflamed as evidenced by a leucocytic infiltrate usually dominated by polymorphonuclear neutrophil leukocytes (PMNs). G-CSF is known to stimulate PMNs, and our findings raise the possibility that G-CSF made by SMC contributes to the development of vasculitis lesions.

Aorta↗
PubMed1992

Plasminogen activator in granulocyte-macrophage-CSF transgenic mice.

The pattern of expression of urokinase type plasminogen activator (PA) in granulocyte-macrophage-CSF transgenic mice and their normal littermates was studied using RNAse protection assays and a plasminogen-dependent fibrinolytic assay for PA. Urokinase type PA mRNA was expressed at a high level in transgenic peritoneal cells and at a lower level in transgenic eye tissue and spleen, but not in equivalent tissue from the normal mice. Enzymically active PA was detectable in protein extracts from peritoneal cells taken from transgenic mice of less than 8 wk of age (young mice) but not from normals. Paradoxically, extracts from transgenic mice of more than 12 wk of age (old mice) showed little detectable PA activity despite continuing transcription in some mice of this age. The production of PA by peritoneal cells may be responsible for the spontaneous i.p. bleeding which is a feature of the transgenic mice and production in other tissues may help explain the local pathologic changes.

Age Factors↗
PubMed1992

Interfacial conformation of dipalmitoylglycerol and dipalmitoylphosphatidylcholine in phospholipid bilayers.

Diacylglycerols are minor constituents of membrane lipids, yet are essential in the activation and membrane association of protein kinase C. Solid-state 13C NMR experiments have been used to characterize the orientation of the glycerol backbone of dipalmitoylglycerol (DPG) and dipalmitoylphosphatidylcholine (DPPC) in egg phosphatidylcholine (PC) bilayers. The 13C NMR spectra of both DPG and DPPC specifically 13C-labeled at the sn-2 chain carbonyl exhibit a single narrow resonance (approximately 2 ppm) in liquid-crystalline egg PC bilayers. In contrast, specific 13C-labeling of both the sn-1 and sn-2 chain carbonyls results in an additional broad component (24-32 ppm) with an axially symmetric line shape. These data reveal that DPG has a distinct motionally-averaged structure in PC bilayers that is similar to that of DPPC and is not significantly affected by the absence of the large polar PC headgroup. The NMR line shapes are roughly consistent with the results of previous FTIR and NMR studies that indicate the sn-1 chain extends from the C1 carbon of the glycerol backbone into the hydrophobic interior of the bilayer, while the sn-2 chain first extends parallel to the bilayer surface and incorporates a bend at the ester linkage in order to keep the sn-1 and sn-2 chains parallel. However, the data suggest that the time-averaged orientation of the glycerol backbone is tilted from the bilayer normal, in contrast to the nearly parallel orientation observed in the crystal structures of phosphatidylcholines and phosphatidylethanolamines or the perpendicular orientation observed in the crystal structures of diacylglycerols.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Gamma-interferon counteracts interleukin-1 alpha stimulated expression of urokinase-type plasminogen activator in human endothelial cells in vitro.

The effect of gamma-interferon (gamma-IFN) on the interleukin-1 alpha (IL-1 alpha) induced stimulation of urokinase-type plasminogen activator (u-PA) expression in human foreskin microvascular endothelial cells (HFMEC) and in human umbilical vein endothelial cells (HUVEC) was investigated. When gamma-IFN and IL-1 alpha were added to the cells simultaneously, gamma-IFN inhibited the IL-1 alpha induced increase in u-PA antigen production in both HFMEC and HUVEC in a dose dependent fashion, with a maximum inhibitory effect achieved between 2.0 and 20.0 U/ml of gamma-IFN. Pretreatment of HFMEC with gamma-IFN for 1 hour before addition of IL-1 alpha resulted in a significant reduction in u-PA synthesis. However, when HFMEC were pretreated for 8 hours with gamma-IFN before the addition of IL-1 alpha the reduction in u-PA production was even more significant. When gamma-IFN was added to HFMEC 1 hour after IL-1 alpha, a significant inhibition in u-PA synthesis was seen. In contrast only a slight inhibition in IL-1 alpha induced u-PA production was seen when gamma-IFN was added to the cells 8 hours after IL-1 alpha. gamma-IFN also inhibited significantly the IL-1 alpha induced increase in u-PA specific mRNA in HUVEC and HFMEC.

Blotting, Northern↗
PubMed1992

The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B.

The action of three matrix metalloproteinases (MMPs), 72- and 95-kDa gelatinases (MMP-2 and MMP-9) and PUMP (MMP-7), and a cysteine proteinase, cathepsin B, were investigated on aggrecan the major proteoglycan of cartilage. All the enzymes cleaved aggrecan although the activity of the 95-kDa gelatinase was very low. Specific cleavage sites were investigated following incubation with a purified aggrecan G1-G2 domain fragment (150 kDa). Both gelatinases produced 110-kDa G2 and 56-kDa G1 products by a single cleavage at an Asn-Phe bond within the interglobular domain close to the G1 domain. This was similar to the action of stromelysin (MMP-3) (Fosang, A. J., Neame, P. J., Hardingham, T. E., Murphy, G., and Hamilton, J. A. (1991) J. Biol. Chem. 266, 15579-15582). Cathepsin B also produced two fragments from a single cleavage at a Gly-Val bond only three amino acids C-terminal to the metalloproteinase cleavage site. PUMP cleaved at the metalloproteinase Asn-Phe site, but in addition produced a low yield of a smaller G2 fragment (56 kDa) corresponding to cleavage between Asp441 and Leu442 (human sequence), within the interglobular domain, close to the G2 domain. The apparent difference in size between the two G2 fragments released by PUMP (110 and 56 kDa) was much greater than predicted from the peptide length between the cleavage sites (100 amino acids). However, keratanase digestion greatly reduced the size of the 110-kDa G2 fragment, while producing only a small reduction in size of the 56-kDa product, showing that there was approximately 30-40 kDa of keratan sulfate attached to the interglobular domain between the PUMP cleavage sites. This new structural information on aggrecan may account for the previously observed stiffness of the interglobular domains when viewed by rotary shadowing electron microscopy (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D. G., Hardingham, T. E., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). These results show that in spite of a high keratan sulfate content the interglobular domain provides important sites for cleavage by different proteinases, including several members of the matrix metalloproteinase family.

Aggrecans↗
PubMed1992

Selective suppression of growth factor-induced cell cycle gene expression by Na+/H+ antiport inhibitors.

Activation of Na+/H+ exchange activity is a ubiquitous response to growth factors and has been implicated in the mitogenic response. Little is known of how the antiport influences events in the nucleus which ultimately control the cell cycle. Using potent Na+/H+ exchange inhibitors we show for normal mouse bone marrow-derived macrophages that this activity is required for the colony-stimulating factor-1-induced gene expression of the M1 and M2 subunits of ribonucleotide reductase, an enzyme critical for DNA synthesis. Suppression of M1 and M2 mRNA levels occurred when the inhibitors were added up to 8 h after the growth factor, mirroring their ability to prevent entry into S phase at similar times. Antiport activity was not required for the induction of other genes associated with cell cycle progression including proliferating cell nuclear antigen and the G1 cyclin, CYL1. These results highlight the differential expression of various cell cycle-associated genes and demonstrates that non-coordinate regulation of CYL1 cyclin and DNA synthesis gene expression can occur. The selective dependence of ribonucleotide reductase subunit gene expression on Na+/H+ exchange activity may provide a biochemical basis for the requirement of persistent antiporter activity during G1 for subsequent entry into S phase.

Amiloride↗
PubMed1992

Priming of the respiratory burst of bone marrow-derived macrophages is associated with an increase in protein kinase C content.

The biochemical mechanism(s) underlying the priming of the macrophage for an enhanced PMA-induced respiratory burst is not understood. Because the cellular receptor for PMA is thought to be protein kinase C (PKC), we have investigated the effects of priming agents on cellular PKC levels. Sonicates from unprimed bone marrow-derived macrophages (BMM) were found to contain PKC activity (309 +/- 51 pmol 32P-incorporated/mg/min; mean +/- SE, n = 17) as measured by the phospholipid-, diacylglycerol-, and calcium-dependent phosphorylation of histone. Exposure of BMM to priming agents such as TNF-alpha, LPS, and granulocyte/macrophage-CSF resulted in a significant increase in both histone-phosphorylating activity and levels of immunoreactive PKC protein in these cells. A minimum of 6-h exposure, with an increasing effect up to 48 h, was required for a detectable increase in PKC level. The activity from primed BMM, like that of the untreated cells, was predominantly cytosolic. The kinetics and concentration dependence of the priming agent-induced increase in the PKC content of BMM closely paralleled the enhancing effects of these agents on the PMA-stimulated respiratory burst. Furthermore, CSF-1, a cytokine that does not prime BMM, failed to increase PKC activity. We propose that the exposure of BMM to priming agents leads to an increase in the expression of a stimulatory isozyme(s) of PKC, resulting in an enhanced ability to mount a respiratory burst in response to stimulation with PMA.

Animals↗
PubMed1992

Interleukin-4 suppresses plasminogen activator inhibitor-2 formation in stimulated human monocytes.

Using a specific enzyme-linked immunosorbent assay, plasminogen activator inhibitor-2 (PAI-2) was quantitated in cultures of human monocytes. Lipopolysaccharide (LPS) increased both extracellular and cell-associated PAI-2 levels, as well as PAI-2 mRNA measured by Northern analysis. Both the lymphokine, interleukin-4 (IL-4) (greater than or equal to 10 pmol/L), and the glucocorticoid, dexamethasone (100 nmol/L), inhibited PAI-2 formation and PAI-2 mRNA induction. Another lymphokine, interferon-gamma (IFN-gamma) (100 U/mL), as for IL-4 alone, did not stimulate PAI-2 formation; however, in contrast to IL-4, IFN-gamma did not reverse the LPS effect but could potentiate it. The suppression of PAI-2 formation by IL-4 and glucocorticoid in stimulated human monocytes extends the list of monocyte products whose synthesis can be downregulated in these cells by the two agents. The findings could have relevance to the control by monocytes/macrophages of connective tissue resorption, including that of fibrin, at sites of inflammation.

Cells, Cultured↗
PubMed1992

Inhibition of S-phase progression in macrophages is linked to G1/S-phase suppression of DNA synthesis genes.

Some of the important controlling events regulating eukaryotic S-phase progression are considered to occur late in the G1 stage of the cell cycle. We show here that stimulation of DNA synthesis in bone marrow-derived macrophages (BMM) by macrophage CSF-1 is preceded by G1 expression of three genes which encode proteins associated with the DNA synthesis machinery--the M1 and M2 subunits of ribonucleotide reductase and proliferating cell nuclear Ag (PCNA). Increased expression for these genes correlated well with the mitogenic response and sustained expression required de novo RNA and protein synthesis and also the presence of CSF-1 for at least most of G1. Inhibitors of BMM proliferation (LPS, TNF-alpha, IFN-gamma, and cAMP elevating agents) suppressed CSF-1-induced expression of M1, M2, and PCNA mRNA measured at 22 h. This suppression occurred even when added up to 12 h after the CSF-1, a period coinciding with the G1/S-phase boundary. The delayed kinetics of this effect parallels the ability of these agents to maximally inhibit CSF-1-induced BMM DNA synthesis when added at similar times. Decreased expression of M1, M2, and PCNA was not merely a consequence of DNA synthesis inhibition because the S-phase inhibitor, hydroxyurea, did not suppress CSF-1-induced gene expression. These results suggest that inhibition of DNA synthesis by antiproliferative agents involves inhibition of expression of several genes associated with the DNA synthesis machinery.

8-Bromo Cyclic Adenosine Monophosphate↗