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)

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.
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PMID:Adhesion of subsets of human blood mononuclear cells to endothelial cells in vitro, as quantified by flow cytometry. 136 Oct 77

Primitive clonogenic progenitor cells in human bone marrow bind to preformed marrow-derived stromal layers in vitro and generate colonies of blast cells. The binding interaction does not require calcium or magnesium ions and occurs equally well in serum-free and serum-supplemented culture medium. It does not appear to involve known cell adhesion molecules (CAMs) for which monoclonal antibodies are available (integrins, N-CAM, LFA-1, and ICAM-1), and we were unable to demonstrate a role for the progenitor cell antigen CD34 in progenitor cell adhesion to cultured stroma. The CAM expressed by the blast colony-forming cells may exist in transmembrane or phosphatidylinositol (PI)-linked forms because it is only partially degraded by exposure to trypsin or to PI-specific phospholipase C. However, binding of these cells to stroma is not prevented in the presence of monoclonal antibodies reacting with known PI-linked structures (Thy-1, CD14, and CD16). It is either masked by neuraminidase-sensitive residues or is no longer expressed as cells mature, respectively, along the granulocytic or erythroid lineages. The properties of the hemopoietic progenitor CAM are discussed with reference to the properties of other CAMs and of hemopoietic progenitor cell markers.
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PMID:Hemopoietic progenitor cell binding to the stromal microenvironment in vitro. 237 49

The lymphocyte function-associated (LFA)-1 molecule is expressed on certain populations of macrophages that have an augmented capacity to capture tumor cells. Accordingly, we analyzed the role of LFA-1 in the establishment of such cell-cell interactions. F(ab')2 fragments of the M17/4, anti-LFA-1 monoclonal antibody (MAb) inhibited the interaction between activated macrophages and tumor cells by up to 80% in a dose-dependent manner. The anti-LFA-1 MAb reduced (between 55 to 79%) the number of P815, LSTRA, or EL-4 tumor cells bound to trypsin-sensitive structures on bacillus Calmette Guerin activated macrophages. The inhibition appeared selective, because a F(ab')2 fragment of anti-Mac-1 did not inhibit such binding. Inhibition of tumor cell capture could be observed as soon as 15 min after the onset of the cell-cell interaction between activated macrophages and tumor cells. Optimal inhibition occurred when both tumor targets and macrophages were precoated with the MAb. Although P815, LSTRA, EL-4, and BW5147 tumor cells all expressed LFA-1, only the first three but not BW5147 cells were bound by activated macrophages. Furthermore, endotoxin-pulsed macrophages elicited by thioglycollate broth expressed the LFA-1 antigen but did not exhibit selective tumor cell capture. Finally, anti-LFA-1 inhibited the development of weak into strong binding. Taken together, the results suggest that LFA-1 molecules can participate in the interaction between activated macrophages and neoplastic cells.
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PMID:Mechanisms of tumor cell capture by activated macrophages: evidence for involvement of lymphocyte function-associated (LFA)-1 antigen. 242 81

We asked whether we could distinguish the roles of the human lymphocyte membrane proteins LFA-1, LFA-2, and LFA-3 in the function of CTL-mediated killing. Little is known about the functions of these molecularly distinct proteins beyond the facts that i) binding of a monoclonal antibody (MAb) to any one of them is sufficient to inhibit killing, ii) that in each case inhibition involves prevention of CTL-target cell conjugate formation, and iii) that MAb to LFA-1 and LFA-2 inhibit best when bound to the CTL, whereas anti-LFA-3 inhibits only when bound to the target cell. This latter is despite the fact that (in our test system) LFA-1 and LFA-3 are expressed both on the CTL and on the target. When the target cells were pretreated with trypsin, the sensitivity of CTL-mediated killing was affected in a different way for each site. Inhibition of anti-LFA-1 was increased by approximately 20-fold. Inhibition by anti-LFA-2 was unaffected. Inhibition by anti-LFA-3 was abolished. Trypsin did not remove the specific antigens recognized by the various CTL, HLA-A,B,C or HLA-DR. Nor did it remove LFA-1 from the target cell. It did, however, selectively remove LFA-3 from the target cell. These results indicate, for the first time, that LFA-1 and LFA-2 have functionally distinct roles. They suggest that an unidentified trypsin-sensitive target cell molecule, operationally designated the "trypsin-sensitive counter blocker" (TSCB), plays an important role in the function of LFA-1, possibly by providing a target cell binding site for LFA-1 on the CTL. The hypothesis that this TSCB is identical to LFA-3 (and the related possibility that LFA-1 and LFA-3 are mutual ligands) is not favored by our data, but is not excluded. Finally, the data indicate that the mechanisms by which MAb inhibit killing differ at the LFA-1 and LFA-3 sites. They are consistent with LFA-1 providing adhesion strengthening by binding to another site (the TSCB?) and with LFA-3 delivering an inhibitory signal when provoked with MAb.
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PMID:Functional distinctions between the LFA-1, LFA-2, and LFA-3 membrane proteins on human CTL are revealed with trypsin-pretreated target cells. 387 Nov 2

We sought additional evidence for an inverse relationship between functional CTL-target cell affinity on the one hand, and susceptibility of the CTL-mediated killing to inhibition by alpha LFA-1 and alpha Lyt-2,3 monoclonal antibodies on the other hand. Previously, we experimentally reduced affinity by pretreating the target cells with papain. This removed most of the class I H-2 antigens, had little effect on the ability of allospecific CTL to recognize and kill these targets, but dramatically reduced the initial strength of CTL-target cell adhesion, and increased by more than 10-fold the susceptibility of the killing to inhibition by alpha Lyt-2,3 and alpha LFA-1 MAb. In the present report, we find that pretreating the target cells with trypsin, like papain, does not significantly change the susceptibility of the target cells to killing by allospecific CTL in a 2-hr assay, and increases by about 10-fold susceptibility of the killing to inhibition by alpha LFA-1. Unlike papain, however, trypsin does not consistently increase blocking by alpha Lyt-2,3, does not remove class I H-2 antigens from the target cell, and does not substantially reduce the strength of initial CTL-target adhesion formation (estimated by post dispersion lysis after a 5-min conjugate-forming incubation). These results show a functional difference between LFA-1 and Lyt-2,3. Both papain and trypsin produced similar 10-fold increases in susceptibility to blocking by alpha LFA-1. In contrast, susceptibility to inhibition by alpha Lyt-2,3 was increased nearly 100-fold by papain, but was not consistently affected by trypsin. Thus, the above-mentioned inverse relationship holds for alpha Lyt-2,3 but not for alpha LFA-1. Our results are consistent with the hypothesis that Lyt-2,3 but not LFA-1 participates in recognition of class I H-2 antigens. Possibly LFA-1 participates in an adhesion-strengthening process that follows T cell recognition, and which may also be used by other LFA-1 expressing leucocytes in intercellular interactions. Finally, our results suggest (for the first time in the mouse system) that an unidentified non-H-2 "trypsin-sensitive counter blocking" molecule on the target cell plays an important role in CTL-target cell interaction.
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PMID:Blocking of CTL-mediated killing by monoclonal antibodies to LFA-1 and Lyt-2, 3. II. Evidence that trypsin pretreatment of target cells removes a non-H-2 molecule important in killing. 388 May 79

Antibodies to most cytolytic T lymphocyte (CTL) external membrane antigens have no effect on CTL-mediated killing in the absence of complement. However, antibodies which do inhibit killing have now been identified for 7 distinct molecular sites. Antibodies to 6 of these "lymphocyte function-associated antigens" (LFAs, also called "blocking sites") inhibit when bound to the CTL, and to the 7th, when bound to the target cell. Mouse homologs have been identified for only 4 of the 7 human LFAs. 5 (probably 6) of the blocking sites inhibit by interfering with adhesion formation between the CTL and the target cell; the exception is T3. None of the presently identified blocking sites are believed to be lethal hit structures (CTL "toxin"). Reduction of target cell H-2 alloantigen density by pretreatment with papain reduces CTL-target functional "affinity", and increases susceptibility to inhibition 100-fold for anti-Lyt-2,3 and 10-fold for anti-LFA-1. This is consistent with the hypothesis that Lyt-2,3 aids in recognition of class 1 MHC antigens, perhaps by strengthening intercellular adhesion. On the other hand, LFA-1 appears to function differently. Trypsin pretreatment of target cells has little effect on MHC antigens or CTL-target affinity, yet still increases by 10-fold susceptibility to inhibition by anti-LFA-1. This is seen in both human and mouse CTL systems. These results suggest the existence of a non-MHC target structure which participates in the adhesion-strengthening function of LFA-1, and which is trypsin (and papain) sensitive: the "trypsin-sensitive counter blocker" (TSCB). LFA-3 may be the human TSCB. The roles of these LFAs in intercellular adhesion extend to more general cell adhesions. Anti-LFA-1 and anti-LFA-3 weaken the spontaneous adhesions which form between cells of the human B cell line JY. These homotypic adhesions are not initiated by immunologic recognition. Anti-LFA-1 is more potent at prolonging allograft survival in vivo than are anti-Lyt-2,3, anti-T200, anti-Thy-1, or anti-I-A. Thus, the potent anti-adhesion properties of LFA-1 seen in vitro may lead to useful immunotherapy in the clinic.
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PMID:Lymphocyte function-associated antigens: regulation of lymphocyte adhesions in vitro and immunity in vivo. 389 52

In many immunoinflammatory diseases, macrophages, by producing interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha), stimulate protease secretion in fibroblasts, thus contributing to tissue destruction. Monocyte/macrophage activation is prompted by soluble factors released by activated T cells as well as by cell-cell contact. Indeed, previous studies have shown that monocytes exposed to paraformaldehyde (PFA)-fixed, activated T cells produced high amounts of IL-1 beta. In this report, we used the T cell line HUT-78 to further characterize the T cell factor(s) responsible for monocyte activation by cell-cell contact. After subcellular fractionation, most of the activity was found in the cellular membrane fraction of PHA/PMA-stimulated HUT-78 cells, and proved to be due to glycoproteins, following trypsin digestion and tunicamycin treatment. HUT-78 cells acquired the capacity to stimulate monocytic cells after as little as 1h of stimulation. De novo protein synthesis was required for the expression of the IL-1 beta inducing factor, as shown by cycloheximide treatment. When membrane proteins of PHA/PMA-stimulated HUT-78 cells were separated on SDS-polyacrylamide gel, a peak of stimulatory activity was observed at Mr--25-35 x 10(3). By using specific cytokine inhibitors or blocking mAbs, we ascertained that cell-associated cytokines (IL-1, IL-2, IFN gamma and GM-CSF) were not involved in monocyte activation by cell contact. Anti-CD2 and -CD11a (LFA-1) mAbs partially blocked IL-1 beta production by -25% and -35%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cell surface glycoproteins expressed on activated human T cells induce production of interleukin-1 beta by monocytic cells: a possible role of CD69. 849 Jan 1

The adhesion molecule L-selectin is proteolytically cleaved from the surface of lymphocytes and neutrophils within minutes after stimulation by phorbol ester or calcium ionophores. In contrast to neutrophils, soluble factors have not been shown to induce down-regulation of L-selectin on lymphocytes. We therefore examined whether signals generated by interaction with cell surface receptors could deliver physiological stimuli inducing this regulatory mechanism. While cross-linking of several adhesion molecules (CD2, CD44, alpha 4-integrin, LFA-1) by antibody did not result in a significant reduction of the expression of L-selectin, antibodies against CD45 and Thy-1.2, both involved in the regulation of lymphocyte activation, induced loss of cell surface L-selectin within minutes, even at 4 degrees C, by shedding into the supernatant. Cross-linking of these molecules was shown to be essential, but Fc interactions or adherent cells were not required. A similar response, albeit less effective, was found after cross-linking of CD3. Interestingly, initiation of shedding only occurred in the presence of cell-cell contact, pointing to a second, as yet unknown, signal required. Loss of L-selectin induced by CD45 cross-linking is followed by a rapid re-expression of the molecule upon incubation at 37 degrees C. This reaction is also dependent on specific triggering signals as rapid re-expression was not observed after removal of L-selectin by trypsin. The data indicate that the protein phosphatase CD45 as well as the TCR complex itself in combination with a further, as yet unknown, cell-cell contact-dependent stimulus have a regulatory role in the dynamic control of L-selectin expression in lymphocytes.
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PMID:CD45-mediated signals can trigger shedding of lymphocyte L-selectin. 913 16

Clinical trials that test the efficacy of Phlogenzym (consisting of the hydrolytic enzymes bromelain and trypsin and the anti-oxidant rutosid) as a treatment for T cell-mediated autoimmune diseases including multiple sclerosis (MS), type 1 diabetes and rheumatoid arthritis are presently ongoing. We tested the effects of Phlogenzym treatment in the murine model for MS, experimental allergic encephalomyelitis (EAE), a disease induced in SJL mice by immunization with proteolipid protein (PLP) peptide 139-151. Oral administration of Phlogenzym resulted in complete protection from EAE. In Phlogenzym-treated mice, the dose response curve of the PLP:139-151-specific T cell response was shifted to the right, that is, the primed T cells required higher peptide concentrations to become activated. Additionally, the T cell response to this peptide was shifted towards the T helper 2 cytokine profile. Both effects are consistent with an increased T cell activation threshold. In support of this interpretation, we found that the accessory molecules CD4, CD44, and B7-1 (all of which are involved in T cell co-stimulation) were cleaved by Phlogenzym, while CD3 and MHC class II molecules (which are involved in the recognition of antigens by T cells) and LFA-1 were unaffected. These data show the efficacy of oral Phlogenzym treatment in an animal model of T cell-mediated autoimmune disease and suggest that the protective effect might be the result of an increase in the activation threshold of the autoreactive T lymphocytes brought about by the cleavage of accessory molecules involved in the interaction of T cells and antigen presenting cells.
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PMID:Prevention of murine EAE by oral hydrolytic enzyme treatment. 1022 28

Type 1, or cellular, immune response is characterized by overproduction of TNF-alpha, IFN-gamma, IL-1, IL-2 and IL-8 and is the underlying immune mechanism of psoriasis, alopecia areata, rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin-dependent diabetes mellitus and experimental autoimmune uveitis (EAU). Type 2 immune response is seen in antibody-mediated autoimmune diseases. Based on the pharmacokinetic effects of cetirizine and allopurinol, this paper introduces these two safe and inexpensive drugs as novel potential agents against cell-mediated autoimmune disorders. Cetirizine, supposed to inhibit DNA binding activity of NF-kappa B, inhibits the expression of adhesion molecules on immunocytes and endothelial cells and the production of IL-8 and LTB4, two potent chemoattractants, by immune cells. It induces the release of PGE2, a suppressor of antigen presentation and MHC class II expression, from monocyte/macrophages and reduces the number of tryptase positive mast cells in inflammation sites. Tryptase is a chemoattractant, generates kinins from kininogen, activates mast cells, triggers maturation of dendritic cells and stimulates the release of IL-8 from endothelial cells and the production of Th1 lymphokines by mononuclear immunocytes. Allopurinol is a free radical scavenger, suppresses the production of TNF-alpha and downregulates the expression of ICAM-1 and P2X(7) receptors on monocyte/macrophages. ICAM-1 serves as a ligand for LFA-1 (on T lymphocytes), allowing proper antigen presentation. P2X(7) receptors are thought to be involved in IL-1beta release, mitogenic stimulation of T lymphocytes and the probable cytoplasmic communication between macrophages and lymphocytes at inflammation sites. Allopurinol was markedly more effective than prednisolone in treating experimental autoimmune uveitis and in combination with cyclosporine suppressed the inflammatory reaction of this condition more effectively than either agent alone. As allopurinol is a competitive inhibitor of xanthine oxidase and decreases serum levels of uric acid, which is protective against multiple sclerosis, it should preferably be coadministered with uric acid precursors in the treatment of this condition. Cetirizine and allopurinol may prove of benefit in the treatment of various cellular autoimmune disorders.
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PMID:Cetirizine and allopurinol as novel weapons against cellular autoimmune disorders. 1503 12


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