Interleukin 1 production by human lung tissue. II. Inhibition by anti-inflammatory steroids

Glucocorticoids block the localized accumulation of leukocytes as sites of inflammation by preventing their adherence to vascular endothelium. This implies that glucocorticoids are acting either on the leukocytes, endothelium, or cells which produce adherence-promoting factors (such as interleukin 1 (IL-1)). Previous studies have shown that dexamethasone (DEX) treatment of either polymorphonuclear leukocytes (PMN) or human umbilical vein vascular endothelial cells (VEC) or both in vitro does not prevent adherence induced by thrombin or formylmethionyl-leucyl-phenylalanine (f-met peptide). We now show that pretreatment of PMN and/or VEC for 24 hr with 0.1 microM DEX had no effect on adherence of PMN to VEC activated with IL-1 (2 U/ml), lipopolysaccharide (1 microgram/ml), or 12-O-tetradecanoylphorbol-13-acetate (30 ng/ml) suggesting that glucocorticoids may inhibit adherence in vivo by blocking formation of IL-1 and other adherence-inducing stimuli. We have recently established that cultured human lung fragments produce IL-1 in vitro. To investigate whether glucocorticoids could inhibit the production of adherence-inducing factors, we examined the effect of glucocorticoids on IL-1 production from human lung tissue. Treatment of human lung fragments in vitro for 18 hr with glucocorticoids such as DEX and hydrocortisone resulted in dose dependent inhibition of IL-1 production; these and other glucocorticoids, at concentrations ranging between 0.1 and 1 microM, produced greater than 50% inhibition of IL-1 release. Nonglucocorticoid steroids including testosterone and beta-estradiol (1 microM) had no effect. Inhibition of IL-1 production occurred after a lag period 5 of 16 hr, and the relative glucocorticoid potencies agreed with their known anti-inflammatory potencies in vivo (beta-methasone approximately triamcinolone acetonide greater than DEX greater than fludrocortisone greater than prednisolone greater than hydrocortisone). Inhibition of IL-1 production in vivo may, in part, explain the remarkable ability of glucocorticoids to prevent the adherence of leukocytes to endothelium and their accumulation at an inflammatory site.     

Interleukin 1 production by human polymorphonuclear neutrophils

The purpose of this study was to determine whether human polymorphonuclear neutrophils (PMN), which share a common cell lineage with macrophages, could produce factors such as IL 1. Other properties which these two cell types share are their phagocytic nature and the common receptor and antigens on their cell surfaces. IL 1, in many of its physical, biochemical, and functional characteristics, is found to resemble endogenous pyrogen (EP). PMN have been cited as a possible cell source of EP, but there have also been reports in which the capacity of PMN to produce EP has been questioned. This study shows that normal human PMN can be stimulated by particulate agents such as zymosan and soluble agents such as phorbol myristic acetate to produce a factor(s) which induces proliferation of mouse thymocytes, i.e., PMN IL 1. This PMN IL 1 was released from PMN in a dose- and time-dependent fashion. PMN IL 1 was nondialyzable, was heat-labile, and was inactivated at pH below 5 and above 8. PMN IL 1 stimulated the proliferation of normal human synovial fibroblasts and caused release of a neutral protease (plasminogen activator) from synovial cells. The synovial and thymocyte-proliferating capacity of PMN IL 1 was not affected by the protease inhibitor aprotinin or by soybean trypsin inhibitor. Gel filtration studies estimate the m.w. of PMN IL 1 to be approximately 13,000 to 17,000.     

Interleukin 1 production by a human acute monocytic leukemia cell line

Human interleukin 1 (IL-1) was produced under serum-free conditions by stimulating a human monocytic leukemia cell line (THP-1) with silica or lipopolysaccharide (LPS). The IL-1 from THP-1 cells has a molecular weight of 12,000-20,000, consistent with the low-molecular-weight form of IL-1 from human peripheral blood monocytes. Further characterization by isoelectrofocusing showed one major peak of activity at pI 7 for the THP-1 cell-derived IL-1. In contrast, the low-molecular-weight form of IL-1 from human monocytes has two major species, pI 5 and pI 7. This cloned THP-1 cell line produces levels of IL-1 activity comparable to those obtainable from peripheral blood monocytes. Thus THP-1 cells can serve as a valuable source of relatively homogeneous human IL-1 for further purification and molecular characterization of its role in regulating immune functions.     

Interleukin 1 induces interleukin 1. II. Recombinant human interleukin 1 induces interleukin 1 production by adult human vascular endothelial cells

Interleukin 1 (IL-1) alters several potentially pathogenic endothelial cell (EC) functions. The authors report here that recombinant human IL-1 (rIL-1) alpha (0.1 to 10 ng/ml) or IL-1-beta (1 to 100 ng/ml) induce concentration- and time-dependent increases in IL-1-beta mRNA levels in EC derived from adult human saphenous vein. rIL-1 induced IL-1-alpha mRNA only in EC treated concomitantly with cycloheximide (2 micrograms/ml). IL-1-beta mRNA production began within 1 hr of exposure to rIL-1, peaked after 24 hr, and declined thereafter. Actinomycin D prevented the appearance of IL-1 mRNA in rIL-1-treated EC. rIL-1 also induced the release of biologically active IL-1 from EC, which was inhibited by cycloheximide (1 microgram/ml). When compared on the basis of their activity in the thymocyte costimulation assay, rIL-1-alpha and rIL-1-beta were equipotent as inducers of IL-1 production by EC. EC stimulated with rIL-1 produced prostaglandin E2, which inhibits IL-1 production by other cell types and also decreases the responsiveness of thymocytes to IL-1. When EC were exposed to rIL-1 in the presence of indomethacin (1 microgram/ml), which blocked prostaglandin E2 production, greater amounts of rIL-1-induced IL-1 release were detected, although the inhibitor did not affect IL-1-beta mRNA levels. IL-1-induced IL-1 production was unlikely to be caused by endotoxin contamination of tissue culture media or IL-1 preparations, because the lipopolysaccharide (LPS) antagonist polymyxin B (10 micrograms/ml) blocked LPS-induced IL-1 production by EC but did not affect IL-1 release in response to rIL-1-beta (100 ng/ml). The IL-1-inducing property of rIL-1-beta was heat-labile, whereas heated LPS stimulated EC IL-1 production. The source of IL-1 in our cultures was not monocyte/macrophages, as treatment of EC with monoclonal antibody to the monocyte antigen Mo2 under conditions that lysed adherent peripheral blood monocytes did not affect production of IL-1 by EC in response to LPS (1 microgram/ml) or rIL-1-beta (100 ng/ml). IL-1 elicits a coordinated program of altered endothelial function that increases adhesiveness for leukocytes and coagulability. IL-1-induced IL-1 gene expression in human adult EC could thus provide a positive feedback mechanism in the pathogenesis of vascular disease including atherosclerosis, vasculitis, and allograft rejection.     

Interleukin 15 stimulates production of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by human peripheral blood mononuclear cells

In peripheral blood mononuclear cells (PBMC), matrix metalloproteinase (MMP)-9 mediates the extravasation of immune cells and may be involved in tissue destruction during inflammation. We investigated the effect of the pro-inflammatory cytokines interleukin (IL-)12 and 15 on the secretion of MMP-9 in PBMC. IL-15, but not IL-12, induces MMP-9 in PBMC and in T cells. Moreover, the combination of IL-15 and IL-2 had an additive effect. In contrast, both IL-12 and IL-15 induced the release of tissue inhibitor of metalloproteinases (TIMP)-1. IL-15 led to a dose-dependent increase of the MMP-9/TIMP-1 ratio as a measure for increased proteolytic capacity. We conclude that IL-15 mediates its effects in inflammation in part through MMP-9.     

Interleukin 1 and the glomerular mesangium. I. Purification and characterization of a mesangial cell-derived autogrowth factor

The proteins expressing interleukin 1 (IL 1) activity from rat peritoneal macrophages and cultured glomerular mesangial cells were compared after purification to apparent homogeneity. The purified IL 1 shared a number of biochemical features including m.w., charge, and specific activity. These findings were extended by the results of proteolytic peptide mapping, which revealed similar breakdown oligopeptides, confirming the close resemblance of these two IL 1 species produced by macrophages and mesangial cells. The purified mesangial cell IL 1 acts as an autocrine or paracrine growth factor. The local release of this cytokine may be an important factor in glomerular diseases characterized by mesangial proliferation and matrix expansion.     

Interleukin 1 (IL 1)-dependent lymphokine production by human leukemic T cell line HSB.2 subclones

Cloning of a human T cell leukemic cell line, HSB.2, was performed by a limiting dilution method to obtain clones with high levels of IL 2 production. None of the subclones that were obtained produced IL 2 constitutively, and only a low level of IL 2 was produced by the stimulation of these subclones with phytohemagglutinin (PHA) alone. High levels of IL 2 production (greater than 300 U/ml) were observed in several clones when stimulated with a cocktail of PHA and IL 1. Among them, HSB.2-A7-D2, A7-D9, or C5-B2 subclones, which were selected after cloning twice, were most effective in IL 1-dependent IL 2 production. HSB.2 subclones exhibited IL 1-dependent production of a variety of lymphokines other than IL 2, e.g., interferon-gamma (IFN-gamma), B cell growth factor (BCGF), and colony-stimulating factor (CSF). We observed that subclones with high IL 2-producing capacity tended to produce high levels of IFN-gamma or BCGF as well, while the capacity of CSF production was not parallel to these properties. Although several subclones were found to produce IFN-gamma and BCGF simultaneously with minimal IL 2 activity, no subclones with an exclusive BCGF production were obtained. Furthermore, when supernatants from the stimulated A7-D9 subclone were applied to an Ultro-gel AcA54 gel chromatography, it was revealed that IL 2 activity (m.w. 17K to 18K) and IFN-gamma (40K to 45K) were clearly separated, whereas two peaks of BCGF activity coincided with each peak of IL 2 and IFN-gamma, respectively. On the other hand, CSF activity was eluted at a different peak (30K to 35K). These data indicate that IL 2, IFN-gamma, and CSF activities are based on distinct molecules, whereas BCGF activities are indistinguishable from IL 2 and IFN-gamma. The HSB.2 subclones thus selected will provide a useful model for delineating the mechanism of IL 1-dependent lymphokine(s) production, and are a promising candidate for better lymphokine(s) producers.     

Identification and characterization of human ethanolaminephosphotransferase1

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EPT) catalyzes the transfer of phosphoethanolamine from CDP-ethanolamine to diacylglycerol to produce phosphatidylethanolamine (PE). To date, the dual specificity of choline/ethanolaminephosphotransferase (CEPT) has been recognized as the total activity responsible for the synthesis of PE via the CDP-ethanolamine pathway in human. We report here the identification and characterization of another human cDNA that encodes CDP-ethanolamine-specific human EPT (hEPT1). Through homology search, we found that human selenoprotein I contained the CDP-alcohol phosphatidyltransferase signature, a common motif conserved in phospholipid synthases. Bacterial expression of the cDNA in Escherichia coli demonstrated that the product specifically used CDP-ethanolamine as the phosphobase donor to produce PE with the activation by both Mn(2+) and Mg(2+). RT-PCR and Northern blot analysis revealed that hEPT1 was ubiquitously expressed in multiple tissues, but in brain it was highly expressed in cerebellum. Here, we propose that in addition to previously identified CEPT, hEPT1 is involved in the biosynthesis of PE via the Kennedy pathway.     

Interleukin 1 preferentially stimulates the production of tissue-type plasminogen activator by human articular chondrocytes

Interleukin 1, derived from human placenta, stimulates plasminogen activator activity in human articular chondrocytes. The stimulation of plasminogen activator activity can be abolished by preincubation of placental interleukin 1 with an antiserum to homogeneous 22K factor, a species of interleukin 1 beta, indicating that the stimulation of plasminogen activator activity is due to interleukin 1 and not contaminating factors. Chondrocytes produce three species of plasminogen activator, with apparent Mr approximately 50,000, 65,000 and 100,000 as determined after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis with gels containing casein and plasminogen. Both placental interleukin 1 and 22K factor enhance the production of the species of Mr approximately 65,000 and 100,000. Comparison of the mobility of the plasminogen activator species on SDS-polyacrylamide gel electrophoresis with human urokinase (u-PA) and human melanoma tissue-type plasminogen activator (t-PA) and studies with antibodies to these enzymes indicate that the Mr approximately 50,000 species is a u-PA and the Mr approximately 65,000 a t-PA. The Mr approximately 100,000 species is possibly an enzyme-inhibitor complex. Interleukin 1 therefore appears to enhance the production of t-PA and a putative enzyme-inhibitor complex. Abolition of plasminogen activator activity in the fibrin plate assay with antibodies to t-PA and u-PA also confirms enhanced t-PA production on interleukin 1 stimulation, though there is also evidence for increased cell-associated production of u-PA.     

Identification and characterization of human Fas associated factor 1, hFAF1.

We have identified and characterized a cDNA encoding human Fas associated factor 1 (hFAF1) cDNA and a shorter form of hFAF1 cDNA [hFAF1(s)] with a 456 bp internal in-frame deletion from a human HeLa cDNA library. The nucleotide sequences of hFAF1 and hFAF1(s) were identical except for the deletion. GST-hFAF1 fusion protein bound to the in vitro translation product of Fas. The N-terminal region (amino acid 1 approximately 201) including the upstream ubiquitin homology domain of hFAF1 could bind with the death domain of Fas unlike that of qFAF1 whose binding region with Fas could not be determined. However hFAF1 did not bind to the death domain of Fas mutant, lpr(cg). hFAF1 was expressed abundantly in testis, skeletal muscle, and heart as 2.8 kb mRNA. Polyclonal antibody against hFAF1 detected 74 kD protein, a deduced protein size from the ORF and 40 kD protein in some cell lines.     

Interleukin 1 regulates human metallothionein gene expression.

Incubation of cultured human cells with interleukin 1 leads to increased expression of the human metallothionein-IIA gene. Recently, metallothionein has been shown to be an efficient free radical scavenger, and induction by interleukin 1 may be part of a protective response to minimize damage by hydroxyl radicals.     

Testosterone metabolism by human lung tissue

The metabolism of [7-³H(N)]-testosterone by slices of human lung tissue obtained from an adult male at surgery was studied in a time course experiment. The 17β-hydroxysteroid oxidoreductase activity of human lung was expressed in the synthesis of isotope-labeled 4-androstene-3,17-dione, 5α-androstane-3,17-dione, androsterone and isoandrosterone. The rate of formation of 4-androstene-3, 17-dione followed a linear course for about five min (160 pmol/lOOmg protein/min), while the formation of the other 17-oxosteroid metabolites followed a linear course for about 1 h (5α-androstane-3, 17-dione, 80 pmol/100mg protein/h; androsterone, 2.4pmol/100mg protein/h; isoandrosterone, ∼28 pmol/lOOmg protein/h). Thus, human lung tissue, in vitro, expresses significant 17β-hydroxysteroid oxidoreductase activity and, notably, a cofactor milieu that favors the 17-oxo over the 17β-hydroxysteroid oxidoreduetion state. This obtains since 5α-dihydrotestosterone formation constituted a very small fraction of total 17-oxosteroid metabolites (∼4.6 pmol/100mg protein/h vs. 2,832 pmol/100mg protein/h). In addition to 17β-hydroxysteroid oxidoreductase activity, the human lung has demonstrable 5α-reductase, 3β-hydroxysteroid oxidoreductase and 3α-hydroxysteroid oxidoreductase activities, findings which support the concept that the lung is a site of metabolism of plasma C₁₉-steroids.     

Identification and characterization of adenosine 5'-tetraphosphate in human myocardial tissue

Endocrine functions of the human heart have been studied extensively. Only recently, nucleotidergic mechanisms have been studied in detail. Therefore, an isolation strategy was developed to isolate novel nucleotide compounds from human myocardium. The human myocardial tissue was fractionated by several chromatographic studies. A substance purified to homogeneity was identified as adenosine 5'-tetraphosphate (Ap(4)) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), post-source decay MALDI MS, and enzymatic cleavage analysis. Furthermore, Ap(4) was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap(4) elicited dose-dependent vasodilations. Vasodilator responses were abolished in the presence of the P(2Y1) receptor antagonist MRS 2179 (1 microm) or the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (50 microm). After removal of the endothelium by Triton X-100, Ap(4) induced dose-dependent vasoconstrictions. Inhibition of P(2X) receptors by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (30 microm) or desensitization of P(2X) receptors by alpha,beta-methylene ATP (alpha,beta-meATP, 1 microm) diminished these vasoconstrictor responses completely. In the present study Ap(4) has been isolated from human tissue. Ap(4) was shown to exist in human myocardial tissue and was identified in ventricular specific granules. In coronary vasculature the nucleotide exerted vasodilation via endothelial P(2Y1) receptors and vasoconstriction via P(2X) receptors on vascular smooth muscle cells. Ap(4) acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion.     

Interleukin 1 increases the production of endothelin-1 by cultured endothelial cells

We examined the effect of human recombinant interleukin 1 (IL-1) on the production of endothelin-1 by cultured porcine endothelial cells. The induction of endothelin-1 mRNA began within 1 hr of exposure to IL-1, showed twin peaks at 4 and 24 hr, and declined thereafter. Enzyme-linked immunosorbent assay revealed that the amount of endothelin-1 peptide in conditioned media was also increased by IL-1 in a dose- and time-dependent manner. Our results suggested that IL-1, a macrophage-derived cytokine, may affect the contraction and proliferation of vascular smooth muscle cells by stimulating the production of endothelin by endothelial cells.     

Human tissue kallikrein. I. Isolation and characterization of human pancreatic kallikrein from duodenal juice

Human pancreatic kallikrein was purified from duodenal juice by ion exchange chromatography on DEAE-Sepharose and immunoaffinity chromatography. Thus, an enzyme preparation with a specific activity (using Ac-Phe-Arg-OEt as substrate) of 1 000 U/mg protein was obtained. A specific biological activity of 1310 KE/mg protein was measured in the dog blood pressure assay and of 0.361 HMW kininogen-U/mg, corresponding to the liberation of 383 micrograms bradykinin-equivalents per mg enzyme per min from HMW kininogen in the rat uterus assay. In dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 27 kDa was obtained. Using gel filtration on Ultrogel AcA-44 a molecular mass of 40 kDa was measured. The amino-acid composition was determined and isoleucine and alanine were identified as the only N-terminal amino-acid residues. On isoelectric focusing four protein bands with isoelectric points of 5.60, 5.65, 5.70 and 5.85 were separated. The bimolecular velocity constant for the inhibition by diisopropyl fluoro phosphate was determined as 10.5 l x mol-1 x min-1. The dissociation constant Ki of the human pancreatic kallikrein-aprotinin complex was calculated to be 1.5 x 10(-10)M. The kinetic constants for the kallikrein-catalysed hydrolysis of Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. Immunological studies showed a close relationship between the human pancreatic kallikrein and other human tissue kallikreins, especially with human urinary kallikrein. Detergents such as Triton X-100, Tween 20 and lysolecithin, as well as human serum albumin, activated the human pancreatic kallikrein preparation.     

Identification and characterization of the human Pgp-1 glycoprotein

Two monoclonal antibodies have been raised against human Pgp-1 by the immunization of mice with human fibroblasts. The human molecule, like the previously identified mouse counterpart, is an abundant membrane protein (M_r approximately 95 000) with a broad tissue distribution. Pgp-1 is phosphorylated, and phosphoamino acid analysis demonstrates that this occurs exclusively on serine residues. A major difference between the mouse and the human is that 50–60% of human thymocytes are Pgp-1⁺ compared to 5–10% of mouse thymocytes at an equivalent stage in development. Immunofluorescence studies of cryostat sections showed that the majority of human medullary thymocytes are strongly stained with Pgp-1-specific antibody, whereas the expression of Pgp-1 on cortical thymocytes is much more heterogenous.     

Identification and characterization of the human MOG1 gene

Many of the proteins that mediate transport into and out of the nucleus have been structurally and functionally conserved throughout evolution. Here we describe the sequence and characterization of the human MOG1 gene. The MOG1 gene was originally identified in Saccharomyces cerevisiae as a multi-copy suppressor of conditional alleles of the yeast nuclear transport factor, GSP1 (scRan) (Oki and Nishimoto (1998) Proc. Natl. Acad. Sci. USA 95, 15388-15393). A search of the expressed sequence tag database identified a putative human protein that is 29% identical and 47% similar to the yeast protein. Our experiments demonstrate that the human MOG1 message is expressed in a variety of tissue samples. Several experiments indicate that the human MOG1 protein binds to both yeast and human Ran suggesting functional conservation between the yeast and human MOG1 proteins. Furthermore, hMOG1a, like scMOG1, is localized throughout the cell but is concentrated within the nucleus. Consistent with these findings, hMOG1a can partially complement the growth defect present in yeast MOG1 deletion cells. Taken together, our findings suggest that MOG1 is an evolutionarily conserved Ran binding protein that could play a role in regulating nuclear protein trafficking.