IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.

Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS.

Optimal T cell activation requires the interactions of co-stimulatory molecules, such as those in the CD28 and B7 protein families. Recently, we described the co-stimulatory properties of the murine ligand to ICOS, which we designated as B7RP-1. Here, we report the co-stimulation of human T cells through the human B7RP-1 and ICOS interaction. This ligand-receptor pair interacts with a K:(D) approximately 33 nM and an off-rate with a t((1/2)) > 10 min. Interestingly, tumor necrosis factor (TNF)-alpha differentially regulates the expression of human B7RP-1 on B cells, monocytes and dendritic cells (DC). TNF-alpha enhances B7RP-1 expression on B cells and monocytes, while it inhibits it on DC. The human B7RP-1-Fc protein or cells that express membrane-bound B7RP-1 co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN-gamma and IL-10, are induced by B7RP-1 co-stimulation. Although IL-2 levels are not significantly increased, B7RP-1 co-stimulation is dependent on IL-2. These experiments define the human ortholog to murine B7RP-1 and characterize its interaction with human ICOS.

T-cell co-stimulation through B7RP-1 and ICOS.

T-cell activation requires co-stimulation through receptors such as CD28 and antigen-specific signalling through the T-cell antigen receptor. Here we describe a new murine costimulatory receptor-ligand pair. The receptor, which is related to CD28 and is the homologue of the human protein ICOS, is expressed on activated T cells and resting memory T cells. The ligand, which has homology to B7 molecules and is called B7-related protein-1 (B7RP-1), is expressed on B cells and macrophages. ICOS and B7RP-I do not interact with proteins in the CD28-B7 pathway, and B7RP-1 co-stimulates T cells in vitro independently of CD28. Transgenic mice expressing a B7RP-1-Fc fusion protein show lymphoid hyperplasia in the spleen, lymph nodes and Peyer's patches. Presensitized mice treated with B7RP-1-Fc during antigen challenge show enhanced hypersensitivity. Therefore, B7RP-1 exhibits co-stimulatory activities in vitro and in vivo. ICOS and B7RP-1 define a new and distinct receptor-ligand pair that is structurally related to CD28-B7 and is involved in the adaptive immune response.

Human keratinocyte growth factor recombinantly expressed in Chinese hamster ovary cells: isolation of isoforms and characterization of post-translational modifications.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium conditioned by Chinese hamster ovary cells. On an SDS-PAGE gel, KGF-a migrates as two bands near 25-29 kDa and contains both N- and O-linked sugar moieties attached near the N-terminus. Detailed structural characterization confirms that KGF-a contains a single amino acid sequence predicted from cDNA sequence and the molecule has two intramolecular disulfide bridges, Cys1-Cys15 and Cys102-Cys106. An additional Cys at position 40 is free and resides in a solvent-inaccessible environment. Mass spectrometric analyses of KGF-a peptides verify the occurrence of several post-translational modifications in the molecule, including partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 and Thr22. The Asn-linked carbohydrate structures are heterogeneous, which include biantennary, triantennary, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates contain typical mucin-type structures. KGF-b is an N-terminally truncated form of KGF-a posttranslationally processed at Arg23 and is not glycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes.

Regulation of tie receptor expression on human endothelial cells by protein kinase C-mediated release of soluble tie.

The expression and activity of receptor tyrosine kinases (RTK) at the cell surface can be modulated by several different pathways including the proteolytic release of the extracellular domain as a soluble receptor. We investigated the regulation of tie receptor expression, an orphan RTK restricted to cells of hematopoietic and endothelial lineages, on primary human endothelial cells and a stably transfected Chinese hamster ovary (CHO) cell line. Tie was expressed in cells as a doublet of 135 and 125 kD; the 135-kD band represented mature cell surface receptor containing sialic acid and N-linked oligosaccharide residues, whereas the 125-kD band represented an intracellular pool of immature receptor. Phorbol 12-myristate 13-acetate (PMA) had dramatic effects on tie expression at the cell surface. Within 15 minutes of PMA treatment, the 135-kD band disappeared from the cell surface and was accompanied by the appearance of a 100-kD band in cell supernatants. The 100-kD band continued to accumulate in the media throughout the duration of PMA treatment during which mature tie receptor was undetectable on the cell surface by fluorescence-activated cell sorting (FACS) or in cell lysates by immunoblot analysis. Using specific antibodies, this 100-kD species was shown to be a soluble form of the tie receptor containing the extracellular domain. PMA-dependent release of soluble tie was mediated through the activation of protein kinase C (PKC); soluble tie was not released in the presence of PKC inhibitors, an inactive PMA analog, or following the downregulation of PKC through chronic PMA treatment. These results indicate that tie receptor expression on endothelial cells is regulated by the release of a soluble extracellular fragment following activation of PKC. Parallel pathways regulating c-kit, tumor necrosis factor (TNF), and colony-stimulating factor (CSF) receptor expression suggest that the release of extracellular receptor fragments represents an alternative mechanism through which cells modulate responses to growth factors and cytokines.

ErbB receptor activation, cell morphology changes, and apoptosis induced by anti-Her2 monoclonal antibodies.

A panel of mAbs were generated against the purified soluble form of erbB2/Her2 receptor, corresponding to the extracellular region of the receptor, and examined for their ability to mimic the receptor ligand. Some of the mAbs strongly induced tyrosine phosphorylation of 180-185 kDa proteins, including not only Her2 but also Her3 and Her4 receptors, when they were expressed on the surface of breast cancer cells. These mAbs do not cross-react with Her3 or Her4 as demonstrated by competition study. Receptor phosphorylation was also observed with the cell lines transfected with Her2 or a chimeric receptor consisting of the extracellular domain of Her2 and the transmembrane and cytoplasmic domains of epidermal growth factor receptor. Selected mAbs were tested for their ability to change cell morphology, and one specific mAb, mAb74, induced cell morphology changes and apoptosis.

Binding of Neu differentiation factor with the extracellular domain of Her2 and Her3.

The interaction of neu differentiation factor (NDF) with the extracellular domains of Her2 (sHer2) and Her3 (sHer3) have been studied using native gels, light scattering, and sedimentation equilibrium. The full-length NDF beta 2 was shown to bind sHer3 with a dissociation constant of 26 +/- 9 nM, while it showed a 1000-fold weaker binding to sHer2. Taken together, these results demonstrate that NDF is a high affinity ligand for Her3, but not for Her2. No increase in affinity of the NDF beta 2 for sHer3 was observed upon addition of sHer2 to the NDF beta 2-sHer3 mixture. Binding of NDF beta 2 to sHer3 did not induce receptor dimerization or oligomerization, the stoichiometry being one sHer3 per one NDF molecule. This finding suggests that transmembrane and/or intracellular domains of receptor family members or perhaps additional unidentified components may be involved in NDF induced dimerization and autophosphorylation, or alternatively, that dimerization is not the mechanism for Her3 autophosphorylation and signal transduction.

Biosynthetic processing of neu differentiation factor. Glycosylation trafficking, and regulated cleavage from the cell surface.

new differentiation factor (NDF), also known as heregulin, is structurally related to the epidermal growth factor family of growth factors; it stimulates tyrosine phosphorylation of the neu/HER-2 oncogene and causes differentiation of certain human breast cancer cell lines. Alternative splicing of a single gene gives rise to multiple isoforms of NDF/heregulin, as well as the neuronal homologues, designated ARIA (acetylcholine receptor inducing activity) and GGF (glial growth factor); at least 15 structural variants are known. All but two of the NDF/heregulin cDNAs are predicted to encode transmembrane, glycosylated precursors of soluble NDF. In this report we characterized the biosynthetic processing of different NDF isoforms in stably transfected Chinese hamster ovary cells expressing individual NDF isoforms, and in the native cell line Rat 1-EJ, which expresses at least six different NDF isoforms. We found that the precursors for NDF undergo typical glycosylation and trafficking. A portion of the molecules are proteolytically cleaved intracellularly leading to the constitutive secretion of soluble, mature NDF into the culture media. However, a significant portion of the newly synthesized NDF precursor molecules escape intracellular cleavage and are transported to the cell surface of both transfected and native cells, where they reside as full-length, transmembrane proteins. Finally we show that these full-length, transmembrane NDF molecules can undergo phorbol ester regulated cleavage from the membrane, releasing the soluble growth factor into the medium.

Detection of neu differentiation factor with a biospecific affinity sensor during chromatography.

A technique using a biospecific affinity sensor, BIAcore, was applied to monitor and determine mammalian cell-derived neu differentiation factor (NDF) in column fractions during chromatography. Specific purified polyclonal antibody against Escherichia coli-derived NDF was chemically bound to the surface of BIAcore sensor chips and the derivatized sensor chips were used to detect the specific binding of NDF. The measurement of NDF at very low levels can be assessed by injecting small volumes of the crude media or column fractions into the BIAcore sensor containing antibody-bound sensor chips. This automated procedure performed under computer programming control allows direct measurement of multiple NDF samples in a short period of time and provides excellent quantitative data, which is not possible using other related methods such as Western blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stimulatory activity assay on receptor autophosphorylation.

Post-translational processing of membrane-associated neu differentiation factor proisoforms expressed in mammalian cells.

Expression vectors constructed from human and rat pro-neu differentiation factor (NDF) cDNAs were transfected in Chinese hamster ovary cells for expression of recombinant NDF molecules. Soluble NDF forms were released into culture medium after post-translational processing of the membrane-bound pro-NDF forms. Different human and rat NDF isoforms, after being purified from the culture medium, were subjected to structural and biochemical characterizations. The isolated human and rat NDF isoforms have been proteolytically processed at a specific site at the N terminus, which is different from that observed for the processing of rat or human NDF molecule prepared from natural origins. The processing of each recombinant NDF isoform at its C terminus was heterogeneous but consistently occurred at nearby peptide bonds. Specific N- and C-terminal processing by Chinese hamster ovary cells has resulted in the production of two types (alpha and beta) of recombinant NDFs containing 222-225 amino acid residues. Both human and rat NDF molecules are heavily glycosylated at two of the three potential Asn-linked glycosylation sites and contain O-linked sugars at 11 of the Thr/Ser sites. Glycosylation occurs at a short, Ser/Thr-rich spacer region that connects the N-terminal immunoglobulin homology unit to the epidermal growth factor domain. Cellular phosphorylation assay indicated that these secreted forms contain similar biological activity in receptor tyrosine autophosphorylation of mammary tumor cells.

NDF/heregulin stimulates the phosphorylation of Her3/erbB3.

Her3/erbB3 has been identified as a third member of the epidermal growth factor receptor (EGFR) family [(1989) Proc. Natl. Acad. Sci. USA 86, 9193-9197; (1990) Proc. Natl. Acad. Sci. USA 87, 4905-4909]. The natural ligand for Her3 has not been identified. Although recently NDF has been proposed as a specific ligand for Her4 [(1993) Nature 366, 473-475; (1993) J. Biol. Chem. 268, 18407-18410], we report here that Her3 was phosphorylated on tyrosine not only in three breast carcinoma cell lines, MDAMB453, MDAMB468 and SKBR3, but also in Her3-transfected CHO cells in response to NDF stimulation. In further studies, cells were reacted with 125I-labeled NDF and then chemically crosslinked. Immunoprecipitation with anti-Her3 revealed a dense high Mw band, greater than 400 kDa. The results suggest that NDF may be a ligand of Her3 and induces receptor hetero-oligomerization.

Post-translational processing of membrane-associated recombinant human stem cell factor expressed in Chinese hamster ovary cells.

This report describes the structure of soluble human stem cell factor isolated from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with stem cell factor (SCF) cDNA, which encodes a leader sequence plus 248 additional amino acids. The 248 amino acids include a hydrophobic transmembrane region at positions 190-212. The isolated material is glycosylated and three bands (apparent M(r) 28,000, M(r) 35,000, and M(r) 40,000) are evident by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. After complete deglycosylation, the molecular weight by SDS-polyacrylamide gel electrophoresis is 18,000-19,000. Structural analyses of the intact SCF, the deglycosylated SCF, and a deglycosylated C-terminal peptide were performed by laser desorption, fast atom bombardment, or electrospray mass spectrometry. Pulse-labeling of cells with 35S-labeled Met and Cys resulted in cell-associated glycosylated SCF of M(r) 33,000-45,000 which was converted to M(r) 33,000 by in vitro treatment with glycosidases. During a chase with unlabeled Met and Cys, labeled SCF of M(r) 28,000, M(r) 35,000, and M(r) 40,000 appeared in the medium; it was converted to M(r) 18,000-19,000 by glycosidase treatment. SCF at the surface of the transfected CHO cells could be demonstrated by immunofluorescence. The data obtained indicate that the recombinant human stem cell factor, as isolated, represents proteolytically processed forms containing amino acids 1-165, derived from the initially synthesized membrane-bound form of 248 amino acids. Further characterization indicated that the M(r) 28,000 form is glycosylated at Asn120, the M(r) 35,000 form at Asn120 and Asn65, and the M(r) 40,000 form at Asn120, Asn93, and Asn65. Each form also contains O-linked carbohydrate. The N-linked glycosylation, particularly that at Asn93 and at Asn65, adversely affects in vitro biological activity and receptor binding.

Support of human hematopoiesis in long-term bone marrow cultures by murine stromal cells selectively expressing the membrane-bound and secreted forms of the human homolog of the steel gene product, stem cell factor.

The maintenance and differentiation of hematopoietic stem cells is influenced by cells making up the hematopoietic microenvironment (HM), including bone marrow-derived stromal cells. We and several other investigators have recently demonstrated the molecular basis of abnormal HM observed in the steel mutant mouse and cloned the normal cDNA products of this gene (termed SCF, KL, or MCF). In this report, we focus on the human counterpart of the mouse Steel (Sl) gene. Alternative splicing of the human SCF pre-mRNA transcript results in secreted and membrane-bound forms of the protein. To investigate the role of these two forms of human SCF, we targeted an immortalized stromal cell line derived from fetal murine homozygous (Sl/Sl) SCF-deficient embryos for gene transfer of various human cDNAs encoding SCF. We report that stable stromal cell transfectants can differentially process the two forms of human SCF protein product. We also demonstrate that both soluble SCF and membrane-bound SCF are active in increasing the number of human progenitor cells in the context of stromal cell cultures, although in a qualitatively different manner. Hence, the membrane-bound form of SCF may play an important role in the cell-cell interactions observed between stromal and hematopoietic cells both in vitro and in vivo.

A reassessment of methylcholanthrene transformation in the C3H10T1/2 cell culture system.

We previously demonstrated that four tumorigenic methylcholanthrene (MCA) transformed cell lines derived from C3H10T1/2 cells each contain a common G34----T nucleotide alteration in the c-Ki-ras gene. In contrast, a non-tumorigenic MCA transformant does not contain this mutation. We have now examined 75 newly isolated MCA transformants of C3H10T1/2 cells for their degree of morphological transformation, the presence of the c-Ki-ras G34----T mutation, colony formation in soft agar, and tumorigenicity in nude mice. Although many of these new MCA transformants exhibit morphological characteristics indistinguishable from previously isolated tumorigenic MCA transformants, none contain the G34----T mutation in the c-Ki-ras gene. Only one newly isolated MCA transformant can grow in soft agar. Of 14 tested, none of the new MCA C3H10T1/2 transformants are tumorigenic in nude mice.

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid differ in toxicity, mitogenic activity and induction of gene expression.

Okadaic acid (OA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are both potent tumor promoters in a mouse skin carcinogenesis experiment. OA was much more toxic than TPA for murine embryo cell lines such as Swiss 3T3 cells or C3H10T1/2 cells. TPA is a potent mitogen for 3T3 cells; in contrast OA was unable to stimulate DNA synthesis in these cells. TPA induces a family of primary response genes, the TPA induced sequence (TIS) genes, in a wide variety of cells. Although OA induced modest levels of TIS mRNA expression, the time course of the induction of TIS1 and TIS8 mRNA was delayed when compared to induction by TPA or peptide mitogens such as fibroblast growth factor (FGF). In addition TPA-mediated down-regulation of protein kinase C attenuated TIS gene induction by OA, but not by FGF.

Colony size, cell density and nature of the tumor promoter are critical variables in expression of a transformed phenotype (focus formation) in co-cultures of UV-TDTx and C3H10T1/2 cells.

UV-TDTx cells are cloned from foci arising after C3H10T1/2 cells are sequentially exposed to u.v. irradiation followed by tetradecanoylphorbol acetate (TPA). When grown in pure culture, UV-TDTx cells appear transformed. Co-culture with C3H10T1/2 cells suppresses focus formation by the UV-TDTx cells. In the presence of TPA, however, focus formation by UV-TDTx cells occurs in C3H10T1/2 co-cultures. We now demonstrate that only tumor promoters that activate protein kinase C (TPA, teleocidin) can reverse C3H10T1/2 suppression of UV-TDTx focus formation in co-culture; other promoters (diethyl stilbestrol, dioxin, saccharin, cadmium) are inactive. Retinoic acid, a potent inhibitor of many biological effects of TPA, blocks the action of TPA in UV-TDTx:C3H10T1/2 co-cultures. Focus formation by UV-TDTx cells in co-culture is dependent on the size of the UV-TDTx colony at confluence; if the UV-TDTx colony is below a minimal size when the co-cultures reach density-dependent growth arrest, suppression of focus formation by C3H10T1/2 cells occurs even in the presence of TPA. Finally, TPA must be present prior to confluence to relieve suppression of focus formation. If TPA is added to co-cultures after density arrest, UV-TDTx cells will not subsequently form foci.

Ultraviolet irradiation transforms C3H10T1/2 cells to a unique, suppressible phenotype.

Transformation of C3H10T1/2 cells by exposure to ultraviolet (UV) irradiation followed by tetradecanoyl phorbol acetate (TPA) has been used as a model of two-stage carcinogenesis. However, cells cloned from UV-TPA-induced foci (UV-TDTx cells) had a unique phenotype. Cloned UV-TDTx cells appeared transformed in pure culture but were unable to form foci when cocultured with C3H10T1/2 cells. However, in the presence of TPA, UV-TDTx cells form foci in mixed culture with C3H10T1/2 cells. This phenotype was the only one observed for UV-TPA transformants. These data suggest that communal suppression of cell division is a discrete phenomenon that must be overcome as one step in the multistage process of transformation, and this protocol permits the routine isolation of transformed cells responsive to density-dependent growth suppression.

Interaction of epidermal growth factor with initiators and complete carcinogens in the C3H10T1/2 cell culture system.

Unlike 12-O-tetradecanoylphorbol-13-acetate, epidermal growth factor (EGF) could not promote the appearance of type III foci from initiated C3H10T1/2 cells. At appropriate concentrations, EGF induced the formation of type II colonies in the absence of any initiator. At higher concentrations, EGF suppressed the induction of both type II and type III colonies elicited by methylcholanthrene.

Enhancement of oncogenesis in C3H/10T1/2 mouse embryo cell cultures by saccharin.

Impure and pure samples of saccharin (2 milligrams per milliliter) did not produce oncogenic transformation of C3H/10T1/2, clone 8, mouse embryo fibroblasts. However, after treatment of the cells with a nontransforming initiating dose (0.1 microgram per milliliter) of 3-methylcholanthrene, continuous treatment with either sample of saccharin (100 micrograms per milliliter) led to significant transformation. It is concluded that in this system saccharin is a cocarginogen, probably functioning as a promoting agent that is 1000-fold less active than the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate.

Effects of promoters on DNA synthesis in C3H/10T1/2 mouse fibroblasts.

The synthesis of DNA has been studied by autoradiography and by measurements of tritiated thymidine ([3H]TdR) incorporation in cultured C3H/10T1/2 mouse embryo fibroblasts. The cells were first treated with 3-methylcholanthrene as an initiator and then with promoters according to schedules that produce oncogenic transformation. The levels of 3-methylcholanthrene used did not affect the growth or [3H]TdR incorporation of the cells. Treatment during the log phase of growth with 12-O-tetradecanoyl-phorbol-13-acetate, phorbol didecanoate, or 4alpha-phorbol didecanoate produced a transient inhibition of [3H]TdR incorporation with the maximum at 12 hr after treatment. This resulted in a temporary delay of growth followed by recovery of the normal cell-doubling time. Phorbol did not produce these effects, suggesting that the inhibition of DNA synthesis is associated with the process of promotion. Although treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate during stationary phase resulted in a 2- to 3-fold stimulation of [3H]TdR incorporation, multiple treatments spanning log and stationary phases were found to be necessary for promotion.