Protein aggregation mediated by cysteine oxidation during the stacking phase of discontinuous buffer SDS-PAGE.

The resolution of complex protein mixtures by discontinuous buffer SDS-PAGE is accomplished by their concentration into thin bands in the stacking gel, followed by their separation during migration through the resolving gel. Recombinant human interferon-inducible protein-10 (IP-10), a 10-kDa C-X-C chemokine with four cysteines, aggregated during the stacking phase of SDS-PAGE and generated a band with an apparent molecular mass of 18 kDa. This aggregation depended on the presence of reduced sulfhydryl residues on IP-10, on the amount of loaded protein, and on the concentration of the ammonium persulfate used to polymerize the stacking gel. The aggregation of IP-10 could be prevented by reduction of its sulfhydryls with dithiothreitol followed by irreversible blockade with iodoacetamide. These methods may be useful in the prevention of aggregation of sulfhydryl-containing proteins during SDS-PAGE, especially when large quantities are analyzed to assess their purity.

Molecular characterization of a granulocyte macrophage-colony-stimulating factor receptor alpha subunit-associated protein, GRAP.

The granulocyte macrophage-colony-stimulating factor receptor (GM-CSF-R) is a heterodimer composed of 2 subunits, alpha and beta, and ligand binding to the high-affinity receptor leads to signalling for the multiple actions of GM-CSF on target cells. In order to explore the role of the alpha subunit in signalling, we used a yeast-2-hybrid system to identify proteins interacting with the intracellular domain of the GMR-alpha. A cDNA encoding a predicted protein of 198 amino acids, designated GRAP (GM-CSF receptor alpha subunit-associated protein), was isolated in experiments using the intracellular portion of GMR-alpha as bait. The interaction between GRAP and GMR-alpha was confirmed by coimmunoprecipitation in mammalian cells. GRAP mRNA is widely expressed in normal human and mouse tissues and in neoplastic human cell lines, but it is not restricted to cells or tissues that express GM-CSF receptors. Three discrete GRAP mRNA species were detected in human tissues and cells, with estimated sizes of 3.3, 3.1, and 1.3 kb. GRAP is highly conserved throughout evolution, and homologues are found in yeast. The GRAP locus in Saccharomyces cerevisiae was disrupted, and mutant yeast cells showed an inappropriate stress response under normal culture conditions, manifested by early accumulation of glycogen during the logarithmic growth phase. GRAP is, therefore, a highly conserved and widely expressed protein that binds to the intracellular domain of GMR-alpha, and it appears to play an important role in cellular metabolism.

Relationships of apoptotic signaling mediated by ceramide and TNF-alpha in U937 cells.

It is commonly assumed that ceramide is a second messenger that transduces signaling leading to apoptosis. We tested this hypothesis by investigating the role of ceramide in TNF-alpha-initiated apoptotic signaling using the histiocytic lymphoma cell line U937. We found considerable differences between cell killing by TNF-alpha and by ceramide. U937 cells treated with TNF-alpha are committed early and irreversibly to the apoptotic pathway and start to die 90 min after treatment. U937 cells treated with ceramide start to die 12 h after the initial treatment. The cell death signaling initiated by TNF-alpha is transduced within minutes of exposure to TNF-alpha and it is irreversible. Exogenous ceramide increases the intracellular level of ceramide rapidly, significantly, and well above the physiological levels, within minutes, but cellular commitment to death does not occur until after the first 6 h of incubation. Furthermore, the endogenous ceramide in U937 cells treated with TNF-alpha increases well after the commitment to the apoptotic pathway. The differences between ceramide and TNF-alpha in the kinetics and the commitment to the apoptotic pathway suggest that, (a) ceramide is not a second messenger in the apoptotic signaling of TNF-alpha, (b) ceramide elevations, in TNF-alpha treated cells, are a consequence rather than a cause of apoptosis and (c) exogenously added ceramide and TNF-alpha kill cells via different pathways.

Interferon-inducible protein 10 as a possible factor in the pathogenesis of cutaneous T-cell lymphomas.

Human IFN-gamma-inducible protein 10 (IP-10), a C-X-C chemokine secreted by IFN-gamma-stimulated keratinocytes, is chemotactic for normal CD4-positive lymphocytes and inhibits the proliferation of early subsets of normal and of leukemic hemopoietic progenitors. Cutaneous T-cell lymphoma (CTCL) is an indolent lymphoproliferative disorder of CD4-positive lymphocytes that remain confined to the skin for many years before visceral dissemination. Because IFN-gamma mRNA was detected in the epidermis of CTCL lesions, we decided to investigate the role of IP-10 in the epidermotropism of CTCL by determining its expression in normal skin and in CTCL lesions. Using purified recombinant IP-10 (rIP-10) or a recombinant fusion protein between IP-10 and the straight phi10 protein of phage T7, we generated rabbit antisera that recognized and neutralized rIP-10. Immunoperoxidase staining of normal epidermis demonstrated that IP-10 was expressed by basal keratinocytes but not by the more differentiated cells. In the often hyperplastic epidermis overlying CTCL lesions, IP-10 immunostaining was enhanced compared to normal skin and extended to the suprabasal keratinocytes in 28 of 29 patients for a frequency of 97% and a 95% confidence interval of 82-100%. However, IP-10 was detectable in the dermal or epidermal lymphoid infiltrates in only 3 of 29 patients (10%; 95% confidence interval, 2-29%). Skin clinically free of CTCL demonstrated normal IP-10 immunostaining. In one patient who had matching biopsies performed before and after treatment, IP-10 was overexpressed before treatment but was normally expressed at remission. The in vitro proliferation of primary normal human keratinocytes was inhibited in a dose-dependent manner by rIP-10. These results suggest that IP-10 plays a role in the epidermotropism of CTCL. Additional work is needed to determine whether IP-10 stimulates or inhibits CTCL proliferation. A better understanding of the growth controls operating in CTCL may be useful in the development of curative strategies for this disorder.

Stereospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis.

We investigated the ability of N-octanoyl-sphingosine (C8-Cer) stereoisomers, N-octanoyl-DL-erythro-dihydrosphingosine (DL-e-DHC8-Cer), and a new ceramide derivative, N-octyl-D-erythro-sphingosine (D-e-C8-Ceramine), to induce apoptosis in U937 cells. We found the C8-Cer stereoisomers to be stereospecific with the D- and L-threo stereoisomers being severalfold more potent than the erythro in inducing nucleosomal fragmentation. The order of potency was: D-t-C8-Cer = L-t-C8-Cer > L-e-C8-Cer > D-e-C8-Cer > DL-e-DHC8-Cer. The importance of the carbonyl group in apoptosis was investigated by using a new ceramide derivative, D-e-C8-Ceramine, in which the carbonyl group was replaced by a methylene group. The carbonyl group was not necessary for triggering apoptosis. In fact, replacement of the carbonyl group decreased substantially the time required for cells to die, with maximum DNA fragmentation occurring at 6 h as opposed to the 18 h required by D-e-C8-Cer. To explore possible mechanisms by which these compounds trigger the apoptotic pathway, we tested their ability to increase the endogenous levels of cellular ceramide and to differentially activate a ceramide-activated protein kinase (CAPK). While the potent DNA fragmentation-inducing compounds D-e-C8-Ceramine and L-t-C8-Cer failed to increase the cellular ceramide levels, D-e-C8-Cer, D-t-C8-Cer and D-e-C8-Ceramine activated the CAPK equally. These studies suggest that the DNA fragmentation-inducing ability of the threo stereoisomers and D-e-C8-Ceramine cannot be attributed either to an increase in the activity of CAPK, or, as illustrated by D-e-C8-Ceramine and L-t-C8-Cer, to the differential elevation of endogenous ceramide. The phosphatase inhibitor okadaic acid failed to protect U937 cells from apoptosis induced by D-e-C8-Cer.

Cytokine loops involving interferon-gamma and IP-10, a cytokine chemotactic for CD4+ lymphocytes: an explanation for the epidermotropism of cutaneous T-cell lymphoma?

Human interferon-gamma (IFN-gamma)-inducible protein 10 (IP-10), a C-X-C chemokine, is secreted by IFN-gamma-stimulated keratinocytes and is chemotactic for CD4+ lymphocytes. We therefore investigated its role in the epidermotropism of cutaneous T-cell lymphoma (CTCL) that is known to express IFN-gamma mRNA in the epidermis and is characterized by an indolent course with multiple relapses that remain confined to the skin for many years. By injecting purified recombinant (r) IP-10 we generated a polyclonal rabbit antiserum that specifically recognized and neutralized rIP-10. With immunoperoxidase staining, IP-10 expression was limited to the basal epidermal keratinocytes of normal skin. In biopsies of CTCL lesions the expression of IP-10 was markedly increased and it extended to the suprabasal keratinocytes in 17 of 18 patients, but it was detectable only faintly in the dermal or epidermal lymphoid infiltrates in 2 of these 18 patients. In 1 patient who had matching biopsies performed before and after treatment, IP-10 was overexpressed before treatment, but was normally expressed in the posttreatment biopsy that showed resolution of the CTCL. Increased IP-10 expression was not detected in any of 4 patients with B-cell lymphoma involving the dermis. On the basis of these findings and a review of the literature, we propose that secretion of IFN-gamma by the lymphoid infiltrate in CTCL induces the epidermal keratinocytes to secrete IP-10 that, in turn, is chemotactic for CTCL, accounting for its epidermotropism. This model may be used as a basis for future investigations of the pathogenesis of CTCL.

Human interferon-inducible protein 10: expression and purification of recombinant protein demonstrate inhibition of early human hematopoietic progenitors.

Human interferon-inducible protein 10 (IP-10), a member of the family of the small secreted proteins called intercrine cytokines or chemokines, is secreted by interferon gamma-stimulated T cells, monocytes, endothelial cells, and keratinocytes. We have begun to explore the biological properties of IP-10 by cloning and overexpression in baculovirus and in bacterial protein expression systems. A 9.9-kD protein was secreted by infected insect cells, which on sodium dodecyl sulfate-polyacrilamide gel electrophoresis comigrated with keratinocyte IP-10 and with f(22-98), a bacterial recombinant fragment lacking the signal sequence but containing all other residues of IP-10. All three reacted with antibodies recognizing residues 10-98 (alpha IP-10) and 77-98 of IP-10 (alpha 22), demonstrating that it is secreted by keratinocytes and insect cells after removal of the signal sequence but without proteolysis of the COOH-terminal end. Purified rIP-10 suppresses in vitro colony formation by early human bone marrow progenitor cells which need r-steel factor (rSLF) and rGM-CSF or rSLF and r-erythropoeitin (rEPO). The inhibition is dose dependent, is complete at concentrations > or = 50 ng/ml, is prevented by preincubation of rIP-10 with alpha IP-10, but not by alpha 22, and is seen with highly purified CD34+ cells, suggesting direct effect of rIP-10 on the progenitors. Combination of rIP-10 and other chemokines at inactive concentrations inhibited colony formation in a synergistic manner. rIP-10 did not affect colony formation in the absence of any growth factors or in the presence of rEPO or rGM-CSF but in absence of rSLF. The effects of IP-10 may be relevant to normal marrow function and might be harnessed to protect human hematopoietic progenitors from the cytotoxic effects of chemotherapy.