Biomedical subjects

J A Hamilton

Publications and source records attributed to J A Hamilton.

At least 19 recordsLinked to original sources

PubMed2006

Therapeutic potential of treating chronic obstructive pulmonary disease (COPD) by neutralising granulocyte macrophage-colony stimulating factor (GM-CSF).

Chronic obstructive pulmonary disease (COPD) is a major incurable global health burden and will become the third largest cause of death in the world by 2020. It is currently believed that an exaggerated inflammatory response to inhaled irritants, in particular cigarette smoke, causes progressive airflow limitation. This inflammation, where macrophages and neutrophils are prominent, leads to oxidative stress, emphysema (loss of lung structure), small airways fibrosis and mucus hypersecretion. However, COPD responds poorly to current anti-inflammatory treatments including potent glucocorticosteroids, which produce little or no benefit. In this review we consider the therapeutic potential of targeting granulocyte macrophage-colony stimulating factor (GM-CSF) for the treatment of COPD. GM-CSF is a major regulator of both macrophage and neutrophil activation and survival in the lung-these cells are intimately linked to COPD. Animal data indicates that neutralisation of GM-CSF ameliorates experimental COPD and predicts therapeutic utility in treating stable COPD and treating exacerbations. As such, GM-CSF represents an attractive therapeutic target for the treatment of COPD.

Animals↗

PubMed2005

Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy.

Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [(13)C]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA-HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [(13)C]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [(13)C]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites.

Binding Sites↗

PubMed2005

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens.

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.

Adult↗

PubMed2003

Energy translocation across cell membranes and membrane models.

Fatty acid transport is an important process in cellular energy distribution and storage in both normal and pathological states, especially obesity-linked type 2 diabetes mellitus. Fatty acid transport has been studied by the complementary approaches of cell biology and biophysics. According to the latter approach, specific proteins that enhance the uptake and storage of fatty acids are posited as fatty acid translocases, which facilitate fatty acid movement from the outer to inner leaflets of the plasma membrane. According to biophysical studies conducted in vitro, fatty acid translocation occurs by a rapid diffusive process that does not require a protein. Herein, we critically review these two mechanisms and their importance in the regulation of fatty acid uptake in vivo.

Adipose Tissue↗

PubMed2002

The generation of highly enriched osteoclast-lineage cell populations.

Osteoclasts form when hematopoietic cells are stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) or tumor necrosis factor-alpha (TNFalpha). Osteoclast precursors derive from M-CSF-dependent proliferating hematopoietic cells but cannot yet be purified from mixed populations. M-CSF stimulation of bone marrow cells results in large numbers of nonadherent, proliferating macrophage precursors. These rapidly form adherent bone marrow macrophages (BMM). BMM and their precursors can be isolated free from mesenchymal and lymphocytic cells. BMM precursors derived from CBA-strain mouse bone marrow, when cocultured with ST2 cells (which express RANKL and M-CSF), formed numerous mononuclear osteoclasts, which resorbed bone and expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors (CTR). Addition of approximately 10 BMM precursors to ST2 cultures resulted in over 80% of these cocultures forming functional osteoclasts, suggesting that they are a highly enriched source of osteoclast progenitors. Supporting this, recombinant RANKL/M-CSF-stimulated BMM precursors formed populations in which all cells expressed TRAP. While only a small proportion of these cells (8.6%) expressed CTR, with transforming growth factor-beta (TGFbeta) present RANKL/M-CSF-stimulated BMM precursors formed almost pure (98.4%) CTR-positive osteoclasts after 7 days. This suggests that TGFbeta stimulated the maturation rate of these cells. Passaged or viably frozen BMM precursors gave rise to BMM that also all formed osteoclasts lineage cells after RANKL/M-CSF stimulation. These data suggest that BMM precursors derived from CBA mice are an expanded pool of osteoclast progenitors. These can be employed to generate osteoclast populations of high purity and in large numbers when stimulated by TGFbeta, which greatly augments the osteoclastogenic effects of RANKL.

Animals↗

PubMed2002

High concentrations of CA 125 in uterine flushings: influence of cause of infertility and menstrual cycle day.

Uterine flushings were obtained under transvaginal ultrasonographic control from 132 women presenting for investigation and treatment of infertility. Levels of CA 125 were measured by radioimmunoassay and results expressed in relation to the total protein concentration of the same flushings. CA 125 was detected in uterine fluid at levels higher than those previously reported in peripheral blood. Uterine fluid CA 125 concentrations varied throughout the menstrual cycle, being highest in the mid-follicular phase (days 6 to 10). Uterine fluid CA 125 concentrations may reflect endometrial secretion of this protein more directly than serum levels. CA 125 concentrations did not vary according to the cause of infertility but further work in larger numbers of women is required.

Adult↗

PubMed2001

Granulocyte macrophage colony-stimulating factor: a new putative therapeutic target in multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, can be induced by immunization with a number of myelin antigens. In particular, myelin oligodendrocyte glycoprotein, a central nervous system (CNS)-specific antigen expressed on the myelin surface, is able to induce a paralytic MS-like disease with extensive CNS inflammation and demyelination in several strains of animals. Although not well understood, the egress of immune cells into the CNS in EAE is governed by a complex interplay between pro and antiinflammatory cytokines and chemokines. The hematopoietic growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), is considered to play a central role in maintaining chronic inflammation. The present study was designed to investigate the previously unexplored role of GM-CSF in autoimmune-mediated demyelination. GM-CSF(-/)- mice are resistant to EAE, display decreased antigen-specific proliferation of splenocytes, and fail to sustain immune cell infiltrates in the CNS, thus revealing key activities for GM-CSF in the development of inflammatory demyelinating lesions and control of migration and/or proliferation of leukocytes within the CNS. These results hold implications for the pathogenesis of inflammatory and demyelinating diseases and may provide the basis for more effective therapies for inflammatory diseases, and more specifically for multiple sclerosis.

Animals↗

PubMed2001

Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity.

M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells.

Animals↗

PubMed2001

Structure of pyruvate dehydrogenase kinase. Novel folding pattern for a serine protein kinase.

The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases. Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases. The 2.5-A crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size. The N-terminal half is dominated by a bundle of four amphipathic alpha-helices, whereas the C-terminal half is folded into an alpha/beta sandwich that contains the nucleotide-binding site. Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotide-binding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.

Adenosine Triphosphate↗

PubMed2001

Tissue-type plasminogen activator deficiency exacerbates arthritis.

Fibrin deposition, cell migration, and tissue remodeling are key components in the lesions of inflammatory joint diseases, such as rheumatoid arthritis. The plasminogen activators (PAs), namely, tissue-type PA (t-PA) and urokinase PA, are implicated in these aspects of an inflammatory response, although their precise roles are yet to be defined. We therefore used gene-deficient mice to explore their role in a two-stage arthritis model involving intraarticular methylated BSA injection, followed by systemic IL-1 treatment. We report in this study that both t-PA and urokinase PA are protective for the mild arthritis induced by intraarticular methylated BSA injection alone, since absence of either of them exacerbates the response; following s.c. IL-1 injection, t-PA(-/-) mice had particularly severe disease. Fibrin deposition appeared to parallel disease severity under the various conditions, suggesting that PA-mediated fibrinolysis may be normally playing a protective role in inflammatory joint disease.

Animals↗

PubMed2001

Blockade of collagen-induced arthritis post-onset by antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in the effector phase of disease.

There is mounting evidence for a role of the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) in inflammatory disease, including arthritis. In the present study, we examined the effectiveness of treatment of collagen-induced arthritis (CIA) with a neutralizing mAb to GM-CSF. DBA/1 mice were immunized for the development of CIA and treated at different times, and with different doses, with neutralizing mAb to GM-CSF or isotype control mAb. Anti-GM-CSF mAb treatment prior to the onset of arthritis, at the time of antigen challenge, was effective at ameliorating the ensuing disease. Modulation of arthritis was seen predominantly as a reduction in overall disease severity, both in terms of the number of limbs affected per mouse and the clinical score of affected limbs. Importantly, anti-GM-CSF mAb treatment ameliorated existing disease, seen both as a reduction in the number of initially affected limbs progressing and lower numbers of additional limbs becoming affected. By histology, both inflammation and cartilage destruction were reduced in anti-GM-CSF-treated mice, and the levels of tumor necrosis factor-a and IL-1beta were also reduced in joint tissue washouts of these mice. Neither humoral nor cellular immunity to type II collagen, however, was affected by anti-GM-CSF mAb treatment. These results suggest that the major effect of GM-CSF in CIA is on mediating the effector phase of the inflammatory reaction to type II collagen. The results also highlight the essential role of GM-CSF in the ongoing development of inflammation and arthritis in CIA, with possible therapeutic implications for rheumatoid arthritis.

Animals↗

PubMed2001

Proteomic analysis of macrophage differentiation. p46/52(Shc) Tyrosine phosphorylation is required for CSF-1-mediated macrophage differentiation.

Macrophage colony stimulating factor (M-CSF or CSF-1) acts to regulate the development and function of cells of the macrophage lineage. Murine myeloid FDC-P1 cells transfected with the CSF-1 receptor (FD/WT) adopt a macrophage-like morphology when cultured in CSF-1. This process is abrogated in FDC-P1 cells transfected with the CSF-1 receptor with a tyrosine to phenyalanine substitution at position 807 (FD/807), suggesting that a molecular interaction critical to differentiation signaling is lost (Bourette, R. P., Myles, G. M., Carlberg, K., Chen, A. R., and Rohrschneider, L. R. (1995) Cell Growth Differ. 6, 631--645). A detailed examination of lysates of CSF-1-treated FD/807 cells by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) revealed a number of proteins whose degree of tyrosine phosphorylation was modulated by the Y807F mutation. Included in this category were three phosphorylated proteins that co-migrated with p46/52(Shc). Immunoprecipitation, Western blotting, and in vitro binding studies suggest that they are indeed p46/52(Shc). A key regulator of differentiation in a number of cell systems, ERK was observed to exhibit an activity that correlated with the relative degree of differentiation induced by CSF-1 in the two cell types. Transfection of cells with a non-tyrosine-phosphorylatable form of p46/52(Shc) prevented the normally observed CSF-1-mediated macrophage differentiation as determined by adoption of macrophage-like morphology and expression of the monocyte/macrophage lineage cell surface marker, Mac-1. These results are the first to suggest that p46/52(Shc) may play a role in CSF-1-induced macrophage differentiation. Additionally, a number of proteins were identified by two-dimensional SDS-PAGE whose degree of tyrosine phosphorylation is also modulated by the Y807F substitution. This group of molecules may contain novel signaling molecules important in macrophage differentiation.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Enhancement of macrophage survival and DNA synthesis by oxidized-low-density-lipoprotein (LDL)-derived lipids and by aggregates of lightly oxidized LDL.

Human atherosclerotic plaque contains a partially characterized range of normal and oxidized lipids formed mainly from free and esterified cholesterol and phospholipids, some of which can be located in macrophage-derived "foam" cells. Oxidation of low-density lipoprotein (LDL) is often considered as an important event leading to subsequent foam-cell development, which may also include enhanced cell survival and/or proliferation. The active component(s) in oxidized LDL (ox.LDL) causing macrophage proliferation is debated. We report here that the lipid component of ox.LDL can promote macrophage survival and DNA synthesis, the latter response showing a synergistic effect in the presence of low concentrations of macrophage colony-stimulating factor. 7-Ketocholesterol showed some stimulation of macrophage DNA synthesis whereas hypochlorite-oxidized (i.e. apolipoprotein B-oxidized) LDL did not. Plaque-derived lipids could enhance macrophage survival. It has not been proven that LDL in lesions is oxidized sufficiently to be the dominant source of sterols in vivo or to be able to induce macrophage growth in vitro or in vivo; it has been suggested that aggregation of modified LDL in vivo is an important step in the deposition of intracellular lipid. We found that aggregation of lightly oxidized LDL potentiated dramatically its ability to stimulate macrophage DNA synthesis, indicating that extensive oxidation of LDL is not required for this response in vitro and perhaps in vivo.

Animals↗

PubMed2001

Inflammatory microcrystals induce murine macrophage survival and DNA synthesis.

The interaction of particulates with resident macrophages is a consistent feature in certain forms of crystal-induced inflammation, for example, in synovial tissues, lung, and the peritoneum. The mitogenic activity of basic calcium phosphate (BCP) crystals and calcium pyrophosphate dihydrate (CPPD) crystals on synovial fibroblasts has been considered relevant to the synovial hyperplasia observed in crystal-induced arthritis. The aim of the study was to determine whether microcrystals such as these could enhance macrophage survival and induce DNA synthesis, thus indicating that they may contribute to the tissue hyperplasia. Murine bone-marrow-derived macrophages were treated in vitro with microcrystals, the cell numbers were monitored over time, and DNA synthesis was measured as the incorporation of [methyl-(3)H]thymidine (TdR). We report here that BCP, monosodium urate, talc, and, to a lesser extent, CPPD crystals promote macrophage survival and DNA synthesis; the latter response is particularly striking in the presence of low concentrations of macrophage-colony stimulating factor (M-CSF, CSF-1). Enhanced macrophage survival or proliferation may contribute to the synovial hyperplasia noted in crystal-associated arthropathies, as well as to talc-induced inflammation and granuloma formation. The crystals studied join the list of particulates having these effects on macrophages, indicating the generality of this type of response.

Animals↗

PubMed2001

Comparison of macrophage responses to oxidized low-density lipoprotein and macrophage colony-stimulating factor (M-CSF or CSF-1).

Modification of low-density lipoprotein (LDL), for example by oxidation, could be involved in foam cell formation and proliferation observed in atherosclerotic lesions. Macrophage colony-stimulating factor (CSF-1 or M-CSF) has been implicated in foam cell development. It has been reported previously that oxidized LDL (ox.LDL) and CSF-1 synergistically stimulate DNA synthesis in murine bone-marrow-derived macrophages (BMM). The critical signal-transduction cascades responsible for the proliferative response to ox.LDL, as well as their relationship to those mediating CSF-1 action, are unknown. We report here that ox.LDL stimulated extracellular signal-regulated protein kinase (ERK)-1, ERK-2 and phosphoinositide 3-kinase activities in BMM but to a weaker extent than optimal CSF-1 concentrations at the time points examined. Inhibitor studies suggested at least a partial role for these kinases, as well as p70 S6-kinase, in ox.LDL-induced macrophage survival and DNA synthesis. For the DNA synthesis response to CSF-1, the degree of inhibition by PD98059, wortmannin and rapamycin was significant at low CSF-1 concentrations but was reduced as the CSF-1 dose increased. Using BMM from CSF-1-deficient mice (op/op) and a neutralizing antibody approach, we found no evidence for an essential role for endogenous CSF-1 in ox.LDL-mediated survival or DNA synthesis; likewise, with the same approaches, no evidence was obtained for an essential role for endogenous granulocyte/macrophage-CSF in ox.LDL-mediated macrophage survival and, in contrast with the literature, ox.LDL-induced macrophage DNA synthesis.

Androstadienes↗

PubMed2001

Binding of 13-HODE and 15-HETE to phospholipid bilayers, albumin, and intracellular fatty acid binding proteins. implications for transmembrane and intracellular transport and for protection from lipid peroxidation.

Transport and utilization of fatty acids (FA) in cells is a multistep process that includes adsorption to and movement across the plasma membrane and binding to intracellular fatty acid binding proteins (FABP) in the cytosol. We monitored the transbilayer movement of several polyunsaturated FA and oxidation products (13-hydroxy octadecadienoic acid (HODE) and 15-hydroxytetraenoic acid (HETE)) in unilamellar protein-free phospholipid vesicles containing a fluorescent pH probe. All FA diffused rapidly by the flip-flop mechanism across the model membrane, as revealed by pH changes inside the vesicle. This result suggests that FA oxidation products generated in the cell could cross the plasma or nuclear membrane spontaneously without a membrane transporter. To illuminate features of extra- and intracellular transport, the partitioning of unsaturated FA and oxidized FA between phospholipid vesicles and albumin or FABP was studied by the pyranin assay. These experiments showed that all polyunsaturated FA and oxidized FA (13-HODE and 15-HETE) desorbed rapidly from the phospholipid bilayer to bind to bovine serum albumin, which showed a slight preference for the unsaturated FA over the oxidized FA. FABP rapidly bound FA in the presence of phospholipid bilayers, with a preference of 13-HODE over the unsaturated FA and with a specificity depending on the type of FABP. Liver FABP was significantly more effective than intestinal FABP in binding 13-HODE in the presence of vesicles. The more effective binding of the FA metabolite, 13-HODE, than its precursor 18:2 by FABP may help protect cellular membranes from potential damage by monohydroxy fatty acids and may contribute a pathway for entry of 13-HODE into the nucleus.

Animals↗

PubMed2001

Dependence of interleukin-1-induced arthritis on granulocyte-macrophage colony-stimulating factor.

OBJECTIVE: To determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF or CSF-1) are involved in the methylated bovine serum albumin/interleukin-1 (mBSA/IL-1)-induced arthritis model. METHODS: Following systemic injection, IL-1 has been shown to augment a weak inflammatory response to mBSA in murine joints and to induce an acute erosive arthritis. GM-CSF and M-CSF have been implicated in inflammatory reactions, including those in joints, and have recently been shown to exacerbate murine arthritis. Since in vitro studies have found that IL-1 can enhance GM-CSF and M-CSF production, we reasoned that they might be playing a part in IL-1-mediated arthritis. GM-CSF-deficient (GM-CSF-/-) and M-CSF-deficient (op/op) mice were injected intraarticularly with mBSA and subcutaneously with IL-1. Arthritis was monitored histologically on day 7. Normal mice were also treated intraperitoneally with blocking monoclonal antibodies to GM-CSF and M-CSF, and to the M-CSF receptor. Numbers of macrophages (Mac-2 and F4/80 staining) were monitored, as was the number of cycling (bromodeoxyuridine-positive) cells. RESULTS: GM-CSF-/- mice and normal mice treated with anti-GM-CSF antibody did not show IL-1-induced arthritis progression. There was a dramatic reduction in synovial cellularity, including reduced numbers of macrophages and cycling cells. The op/op mice did not develop mBSA/IL-1-induced disease, but blocking antibody to M-CSF or to the M-CSF receptor failed to diminish disease in normal mice. CONCLUSION: GM-CSF is involved in the IL-1-induced arthritis that follows mBSA injection; M-CSF involvement in the model is also suggested, since op/op mice did not develop arthritis. These studies provide the first in vivo evidence for a role of GM-CSF, and possibly M-CSF, in the proinflammatory actions of IL-1.

Animals↗

PubMed2001

Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells.

Using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) of 32P-labeled cytosolic and membrane extracts, we identified a 21.5 kDa phosphoprotein with an isoelectric point of 6.0 in NFS-60 cells that was phosphorylated maximally at 15 min by treatment with granulocyte-colony stimulating factor (G-CSF) but not with interlevkin-3 (IL-3) or colony-stimulating factor-1 (macrophage-colony stimulating factor (CSF-1 (M-CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaffected by a series of antiproliferative agents [32]. These findings suggested that the 21.5/6.0 phosphoprotein may be involved in specific G-CSF-mediated biological responses such as activation and/or differentiation. We sought to characterize this 21.5/6.0 by a novel combination of 2-D SDS-PAGE and hydroxyapatite (HTP)-chromatography. Amino acid sequence determination of 21.5/6.0 revealed it to share a high level of homology with copper/zinc superoxide dismutase (Cu/Zn-SOD), indicating that a Cu/Zn-SOD is phosphorylated following treatment with G-CSF. This is the first report of the phosphorylation and possible involvement of Cu/Zn-SOD protein in granulocyte activation/differentiation events. In addition, Cu/Zn-SOD levels and activity were diminished by G-CSF but not IL-3 treatment. This new protocol combining 2-D SDS-PAGE and HTP-chromatography allows the characterization of low abundance phosphoproteins involved in the cellular responses to G-CSF and presumably to other cytokines/growth factors.

Animals↗