Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Immunocompetent cells of the epidermis can interact by the elaboration and recognition of cytokines. Although much new information has been reported concerning the cytokines secreted by keratinocytes, little is known about cytokines derived from Langerhans cells (LC). To address this deficiency, we examined cytokine mRNA profiles in different epidermal preparations from BALB/c mice, taking advantage of the sensitive technique of polymerase chain reaction (PCR), after reverse transcription of mRNA. In assays of epidermal sheets separated from dermis by ammonium thiocyanate, mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-7, tumor necrosis factor alpha (TNF alpha), TNF beta, granulocyte macrophage/colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) were unequivocally present. By contrast, faint bands were detected for IL-4, IL-5, and interferon gamma (IFN gamma), and no PCR signal was detected for IL-2. Importantly, assays of epidermal cells (EC) dissociated with trypsin revealed similar mRNA profiles. To determine the effects of cell isolation, fluorescence-activated cell sorter (FACS)-purified Ia- EC were first analyzed; all of the previously cited cytokine mRNA were present except for IL-1 beta and MIP-1 alpha. EC depleted of LC by a second technique, lysis using anti-Ia monoclonal antibody and complement, revealed similar profiles, with substantially reduced PCR signals for IL-1 beta and MIP-1 alpha. Finally, FACS-purified LC (Ia+ EC) clearly expressed IL-1 beta and MIP-1 alpha mRNA, a finding that was verified by Southern blotting using internal oligo probes. We conclude that these cell-isolation procedures did not produce substantial alterations in basal mRNA profiles and that LC are the principal source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated EC in mice.
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PMID:Langerhans cells are the major source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated mouse epidermal cells. 138 44

Interleukin-1 (IL-1) is an important mediator in inflammation and immunological processes. The findings of native IL-1 inhibitors suggest a negative feedback mechanism to down-regulate IL-1 mediated acute inflammation. IL-1 inhibitors were also found elevated in disease states associated with high IL-1 levels. We have previously described one such IL-1 inhibitor derived from the human M20 myelomonocytic cell line. In this paper we present several biological and biochemical characteristics of the M20 IL-1 inhibitor. Various in vitro activities of the inhibitor are described and its IL-1 specificity in these assays is demonstrated. Purification of the inhibitor was performed by DEAE-high performance liquid chromatography, isoelectric focusing, gel filtration and dye ligand chromatography column. This protein factor has a MW of 52 +/- 4 kDa and a pI of 4.15 +/- 0.1. The inhibitor has no cross-reactivity against a panel of known cytokines (IL-1 alpha, IL-1 beta, IL-2, sIL-2R, IL-6, tumor necrosis factor (TNF), interferon-gamma (IFN-gamma)) and is distinct from the IL-1 receptor antagonist (IL-1ra). The purified IL-1 inhibitor was destroyed by trypsin, 2-mercaptoethanol, sodium dodecyl sulfate and extremes in pH and in temperature. Only IL-1 induced (but not the IL-2, IL-6 or TNF induced) thymocyte proliferation and PGE2 production by fibroblasts were inhibited by the inhibitor, thus showing specificity to IL-1 in these assays.
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PMID:The M20 IL-1 inhibitor. II. Biological characterization. 143 Nov 47

Phytohemagglutinin (PHA) injection induces transient protease-sensitive traffic of lymphocytes in skin and other tissues in several species. Examination of the possible roles of cytokines in such reactions showed that recombinant bovine and human tumor necrosis factor (TNF)-alpha potently induce dose-dependent lymphocyte traffic in pig skin (and in other tissues including the draining lymph nodes) with early kinetics and a morphology of the inflammatory reaction similar to that of PHA (peaking 9-12 h). Recombinant human interleukin (IL)-1 alpha also induces dose-dependent lymphocyte traffic, but it peaks at 4 h. Entry of labeled lymphocytes into inflammatory sites induced by PHA, TNF-alpha and IL-1 alpha, but not into normal skin, is inhibited by approximately 80% by their pretreatment with trypsin, indicative of the induction of endothelial determinants recognized by protease-sensitive surface molecules on the lymphocytes. Even the minimal lymphocyte traffic induced by interferon-gamma and lipopolysaccharide was similarly protease sensitive. At the earliest stage (approximately 2 h) of significant induction of lymphocyte entry by TNF-alpha and IL-1 alpha the inductive signal for each appears easily saturated. Thus lymphocyte entry is little increased by increasing low cytokine doses over 100-fold: However, these reactions are additive, and this was used to confirm that they are distinct from each other and from PHA. A further distinction was revealed by the homing of lymphocytes pretreated with pertussis toxin: such lymphocytes were greater than 90% inhibited in their homing to tissues through constitutive high endothelial venules (HEV) and greater than 60% inhibited in homing to TNF-alpha and IL-1 alpha skin sites, but unaffected in homing to PHA skin sites (like most non-HEV-mediated traffic). Moreover, potent chicken anti-TNF-alpha, which prevented TNF-induced lymphocyte entry, did not affect PHA-induced traffic. Thus, these three agents which induce peripheral lymphocyte traffic appear to involve different mechanisms as shown by differences in (i) their kinetics; (ii) the effect of anti-TNF-alpha and (iii) the effect of pertussis toxin treatment of the lymphocytes and by the fact that their inductive mechanisms are additive in effect.
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PMID:Active lymphocyte traffic induced in the periphery by cytokines and phytohemagglutinin: three different mechanisms? 151 13

Mononuclear cell production of cytokines that stimulate fibroblast production of prostaglandin E (PGE) is an important mechanism by which mononuclear cells regulate fibroblast function. The objective of this investigation was to determine the effects of the cytokines interleukin 1 beta (IL-1 beta), interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma), alone or in paired combinations, on PGE production by near-confluent human periodontal ligament (PDL) fibroblasts in vitro. Premolars extracted in the course of orthodontic treatment were used for this study. Fibroblast cultures, free of epithelial cells, were obtained after the fourth subculture by the use of accurately-timed trypsin treatment. Cells in the fourth to sixth passage, incubated in DMEM supplemented with 10% equine serum, were used for these experiments. Cells (1 x 10(5)) were seeded in 12- x -75-mm tissue culture tubes and incubated with various doses of IL-1 beta, IL-1 alpha, TNF-alpha, and IFN-gamma, alone or in specific combinations, for 15 min, two, 12, 24, and 72 h. PGE concentrations in the media were measured by radio-immunoassay. The results showed that human PDL fibroblasts responded to the administration of cytokines by an elevation in the synthesis of PGE in a dose- and time-related fashion. The increase in PGE production was inhibited by the addition of indomethacin. The interactions between these cytokines varied in degree, depending on the particular combinations of cytokines. In addition, the administration of cytokine combinations was found to be additive, synergistic, subtractive, or suppressive on the production of PGE by PDL fibroblasts, depending on the duration of incubation. These experiments demonstrate the importance of the consideration of the interplay between cytokines produced by mononuclear cells on the mechanisms that regulate the functions of PDL fibroblasts.
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PMID:Interactive effects between cytokines on PGE production by human periodontal ligament fibroblasts in vitro. 211 29

Previous studies have demonstrated that paraformaldehyde-treated macrophages possess IL-1 alpha activity in a variety of bioassay systems. However, no definitive biochemical data in support of the membrane IL-1 alpha concept has been reported. The purpose of the present study was to determine if the biologic activity associated with treated cells is due to a membrane form of IL-1 alpha or alternatively, to the leakage of IL-1 alpha. If the former case was true, then the exposed membrane IL-1 alpha should bind anti-IL-1 alpha antibodies or be cleaved by mild trypsin treatment. In both instances, IL-1 alpha activity should be lost when measured in a subsequent IL-1 bioassay. Our results indicate that pulsing paraformaldehyde-treated normal or cell line macrophages with anti-IL-1 alpha antibodies or treating the cells with trypsin did not affect the ability of the treated cells to function in a murine thymocyte proliferation assay. Furthermore, the standard short term treatment of cells with paraformaldehyde (15 min) did not prevent the leakage of IL-1 alpha from the cells or the processing of the precursor forms of the protein. When cells were treated with paraformaldehyde for 2 h, they no longer released IL-1 alpha or possessed thymocyte stimulatory activity. We also found that short term glutaraldehyde treatment of macrophages completely blocked the release of IL-1 alpha from cells as well as the appearance of cell-associated IL-1 alpha activity. Our results support the conclusion that the stimulatory activity of paraformaldehyde-treated macrophages is not due to a membrane form of IL-1 alpha but is, in fact, due to the continuous release of IL-1 alpha from the cells.
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PMID:Evidence against the existence of a membrane form of murine IL-1 alpha. 278 40

Coculturing IM9 human lymphocytes and A549 human lung adenocarcinoma cells results in a 2-3-fold increase in the density of beta-adrenergic receptors in the latter, as quantified by 125I-cyanopindolol binding. Lymphocyte-conditioned medium (LCM) has the same effect, which is moderately sensitive to heat, is retained by ultrafiltration over a Mr 10,000 cut-off filter, and is reduced by trypsin treatment or by preincubation of lymphocytes with 0.3 micrograms/ml cycloheximide. Treatment of lung cells with cycloheximide also prevents the effect of LCM. Glucocorticoids, which also increase beta-receptor density in A549 cells, markedly potentiate the effect of LCM. Gel permeation high pressure liquid chromatography of LCM yields three peaks of biological activity with Mr 70,000, 35,000, and 15,000. Monocytic interleukin-1 (IL-1) mimics the effect of LCM in that it increases beta-receptor density in A549 cells (EC50 0.3 pM), and its effect is potentiated by cortisol. Recombinant IL-1 alpha is somewhat more potent than IL-1 beta, while interleukin-2 and interferon-alpha are ineffective. Tumor necrosis factor alpha causes a small increase in beta-receptors, which is not influenced by glucocorticoids. A polyclonal anti-IL-1 antibody inhibits the effect of IL-1 and the effect of the 15-kDa but not the 35- and 70-kDa fractions of LCM. The activity of the latter two fractions is also unaffected by anti-tumor necrosis factor alpha antibody. These results indicate that lymphocytes release protein factors including IL-1 that up-regulate pulmonary beta-adrenergic receptors by an action that involves protein synthesis. The possible relevance of this regulatory mechanism for the pathomechanism of certain respiratory diseases is discussed.
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PMID:Synergistic regulation of pulmonary beta-adrenergic receptors by glucocorticoids and interleukin-1. 284 27

The human IL-1 molecules (IL-1 alpha and IL-1 beta) are post-translationally cleaved from 31-kDa precursor to 18-kDa biologically active molecules. During the course of studies of post-translational modifications of human IL-1, we have observed that although LPS induced the production of both intracellular IL-1 alpha and IL-1 beta in human monocytes, [32P]orthophosphate labeling of these cells revealed that intracellular precursor of IL-1 alpha (pre-IL-1 alpha) to be phosphorylated at least 10-fold more than intracellular pre-IL-1 beta. However, no 32P-incorporation could be detected in the 18-kDa processed IL-1 alpha and IL-1 beta. Analysis by TLC revealed that the major phosphorylation site occurred at serine residue(s). The 32P was incorporated into multiply cleaved precursors of IL-1 alpha, which appeared in the absence of protease inhibitors. Since the smallest Mr pre-IL-1 alpha that was labeled with 32P was 22 kDa, the phosphorylated serine residue is presumably located adjacent to a sequence of four basic amino acids located in the 4-kDa region at the amino terminus of the 22-kDa precursor of IL-1 alpha. This serine residue might also be a major phosphorylation site for a cAMP-dependent protein kinase. This hypothesis was substantiated by the demonstration that a synthetic peptide analogue of this region (residue 84 to 112) could be similarly phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, a truncated pre-IL-1 alpha (residue 64 to 271) and a "fusion" protein containing staphylococcal protein A and an amino-terminal half-portion of pre-IL-1 alpha (residue 1 to 112), but not mature IL-1 alpha (residue 113 to 271), could also be phosphorylated by cAMP-dependent protein kinase. There is no comparable amino acid sequence in IL-1 beta which could be expected to be phosphorylated by a cAMP-dependent protein kinase. The physiologic relevance of phosphorylation of pre-IL-1 alpha was investigated. The data showed that phosphorylation of truncated pre-IL-1 alpha greatly enhanced its susceptibility to digestion by trypsin and promoted the conversion of pre-IL-1 alpha to the more biologically active IL-1. Although the precise role of the rather selective phosphorylation of pre-IL-1 alpha is not known, our findings do suggest that the phosphorylation of serine close to dibasic/tetrabasic amino acid sequence functions to facilitate the processing and/or release of IL-1 alpha.
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PMID:Phosphorylation of intracellular precursors of human IL-1. 325 35

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.
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PMID:Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop. 762 17

Human chorion, but not amnion, tissue explants produced substantial quantities of neutrophil chemoattractant in response to interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha). This suggested that chorion is one of the chemoattractant producing tissues. Therefore, the biochemical properties and the regulation of a chemoattractant in human chorionic cells were examined. IL-1 alpha and TNF alpha stimulated human chorionic cells to produce neutrophil chemotactic factor in both a dose- and time-dependent manner. This chemotactic factor was a heat-stable and trypsin-sensitive protein with an apparent molecular weight of 10000, and it was also immunologically identified as a chemotactic cytokine of the human IL-8 family. Immunohistochemical observations with IL-1 alpha- and TNF alpha-treated chorion explants indicated that trophoblasts and stromal cells, including fibroblast-like and macrophage-like cells, but not endothelial cells, were characterized as IL-8-producing cells. From these observations, it is very likely that both IL-1 and TNF alpha may participate in the production of chemotactic factor/IL-8 in pre-term parturition, accompanied by an intraamniotic infection, along with their known promotive effect on the production of matrix metalloproteinases, which is connected with the destruction of matrix components of fetal membranes.
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PMID:Stimulation of the biosynthesis of interleukin 8 by interleukin 1 and tumor necrosis factor alpha in cultured human chorionic cells. 770 64

Dendritic epidermal T cells (DETCs) are Thy-1+, CD45+, CD3+, CD4-, CD8-, and T-cell receptor-V gamma 3/V delta 1+ leukocytes that reside normally in adult mouse skin. We have demonstrated previously that keratinocytes serve as adhesion substrates for DETCs, and that interleukin 7 (IL-7), which is produced by keratinocytes, serves as a growth factor for DETCs. The present study was conducted to address the mechanisms by which DETCs migrate into the epidermis, reasoning that keratinocytes may also be a source of chemotactic activity. Short-term DETC lines were 35S-labeled and tested for migration toward Pam 212 keratinocyte culture supernatants using a modified Boyden chamber method; cell movement from upper chambers toward test samples in lower chambers was traced by counting radioactivity. DETC displayed rapid (within 60 min) and marked (> 50%) migration toward keratinocyte supernatants. The majority of cells that had migrated into keratinocyte supernatants expressed the V gamma 3 T-cell receptor, thus verifying that the migrating cells were DETCs. Addition of keratinocyte supernatants to the upper chambers completely blocked migration, suggesting its chemotactic nature. By contrast, no DETC migration was observed toward 3T3 fibroblast supernatants. Chemotactic activities were 1) produced by Pam 212 cells even in the absence of serum; 2) greater than 12 kD in size; 3) heat and pH labile; 4) trypsin sensitive; and 5) precipitated by 60-100% ammonium sulfate. Several cytokines (e.g., IL-1 alpha and IL-8) failed to mediate DETC migration when added to the lower chambers. Likewise, the same cytokines, when added to the upper chambers, failed to inhibit DETC migration toward Pam 212 supernatants. These results support our hypothesis that keratinocytes facilitate the residence of DETC in epidermis by secreting unique chemotactic factors, by providing adhesion substrates, and by elaborating specific growth factors.
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PMID:Mouse dendritic epidermal T cells exhibit chemotactic migration toward PAM 212 keratinocyte culture supernatants. 839 9


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