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A 4-kilobase complementary DNA (cDNA) encoding human macrophage-specific colony-stimulating factor (CSF-1) was isolated. When introduced into mammalian cells, this cDNA directs the expression of CSF-1 that is structurally and functionally indistinguishable from the natural human urinary CSF-1. Direct structural analysis of both the recombinant CSF-1 and the purified human urinary protein revealed that these species contain a sequence of at least 40 amino acids at their carboxyl termini which are not found in the coding region of a 1.6-kilobase CSF-1 cDNA that was previously described. These results demonstrate that the human CSF-1 gene can be expressed to yield at least two different messenger RNA species that encode distinct but related forms of CSF-1.

A well-established model of bowel inflammation is the HLA-B27 transgenic rat that exhibits a spontaneous disease phenotype resulting in chronic diarrhea caused by immune cell activation. Estrogens have previously been shown to modulate the immune system, and both estrogen receptors (ERalpha and ERbeta) are present in the intestine and cells of the immune system. Therefore, the ability of estrogen to ameliorate disease progression in the HLA-B27 transgenic rat was determined. HLA-B27 transgenic rats with chronic diarrhea were treated with 17alpha-ethynyl-17beta-estradiol (EE) for 5 days. EE treatment dramatically improved stool scores after only 3 days. Histological scores of the degree of ulceration, inflammatory cell infiltration, fibrosis, and lesion depth of the colon were also improved by EE treatment. Because neutrophil infiltration into the colon is involved in the development and propagation of disease, myeloperoxidase (MPO) activity was measured. MPO levels were reduced by 80% by EE treatment. Cotreatment with the pure ER antagonist ICI-182780 (ICI) blocked the effects of EE on stool character, MPO activity, and histology scores, strongly suggesting that the activity of EE is mediated through ER. Mast cell proteases can promote neutrophil infiltration, and gene expression analysis demonstrated that mast cell protease 1, 3, and 4 mRNA were all decreased in colons from estrogen-treated rats. In addition, a direct effect of estrogen on bone marrow-derived mast cell activity was demonstrated, suggesting that ER-mediated inactivation of mast cells may contribute to the improvement in the clinical sign and histological scores in this model.

We have isolated a subline of the M-07 human megakaryoblastic leukemia cell line, designated M-07e, that requires either interleukin-3 (IL-3) or granulocyte macrophage colony-stimulating factor (GM-CSF) for growth, even in the presence of fetal calf serum. This cell line will not grow long term in any other cytokine although it responds slightly to IL-2, IL-4, IL-6, IL-9, and interferon-gamma. We have used the M-07e subline to develop a quantitative bioassay for the measurement of levels of either GM-CSF or IL-3. This assay is as sensitive to either factor as the human bone marrow colony assay (CFU-GM) or the chronic myelogeneous leukemic (CML) blast cell proliferation assay for these factors and is much more convenient and reliable than either. With this assay, as little as 25-50 pg/ml of either IL-3 or GM-CSF can be detected, a level that should render the assay useful for analysis of these molecules in samples from patients undergoing colony-stimulating factor therapy and from conditioned media from natural sources of the factors. In these cases, neutralizing antisera to each cytokine are required to demonstrate the specificity of the assay. This assay, in combination with quantitative immunoassays, should greatly facilitate the analysis of the roles of IL-3 and GM-CSF in regulating hematopoiesis both in patients and in natural sources of the cytokines.

Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based anity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murinē t4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptidelike sequence that is removed to generate the mature Nterminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21 000 M r polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.Keywords: Flt4; Flt4 ligand; VEGF-C; BIAcoreTo identify a source of the Flt4 ligand, we used a Flt4-Fc fusion protein. This consists of the extracellular region of murine Flt4 (Finnerty et al., 1993) fused to the hinge-C H 2-C H 3 domains of human immunoglobulin g1. The conditioned media (CM) of over 100 dierent cell lines were screened for binding to Flt4-Fc using a BIAcore 2000 instrument. The media conditioned by the murine embryonic liver cell line, BNL 1NG A.2, human bladder carcinoma 5637, and bualo rat BRL 3A each showed binding to Flt4-Fc, but not human IgG1 (Figure 1). The binding was speci®c because when the CM was coinjected with excess Flt4-Fc, but not human IgG1 or KIT-Fc, the signal was inhibited (Figure 1). The presence of Flt4 ligand (Flt4-L) in BNL 1NG A.2 CM was con®rmed by demonstrating that it activated the Flt4 receptor protein tyrosine kinase. CHO cells stably transfected with a murine Flt4 cDNA expression vector were treated with 50-fold concentrated BNL 1NG A.2 CM for 10 min at 378C. This resulted in a marked increase in tyrosyl phosphorylation of Flt4 (Figure 2a, lane 1) relative to mock stimulated samples (Figure 2a, lane 2).Six micrograms of Flt4-L were puri®ed from 15L of BNL 1NG A.2 CM using Flt4-Fc anity chromatography. Flt4-Fc (100 mg/ml) or KIT-Fc (200 mg/ml) were directly immobilized to 1 mL Pharmacia HiTrap NHS columns. A volume of 50 ml of concentrated BNL 1NG A.2 CM was loaded ®rst onto the KIT-Fc column to adsorb nonspeci®c binding, then onto the Flt4-Fc column. The columns were washed and separately eluted with 24 mM hydrochloric acid. As expected, only fractions from the Flt4-Fc anity column had binding to Flt4-Fc on the BIAcore assay. The speci®c activity in response units (RU)/mg was determined using RU that were in the linear range of RU versus concentration of Flt4-L puri®cation fractions. The speci®c activity of Flt4-L from BNL 1NG A.2 was 4.2610 6 RU/mg, an increase of 98 455-fold after t...