leucyl-leucine-methyl-ester and leucine-methyl-ester

T614 (3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-o ne) is a member of the family of methanesulfonanilide non-steroidal anti-inflammatory drugs (mNSAIDs), most of which act as cyclooxygenase (COX)-2 inhibitors. L-leucine methyl ester (Leu-OME) is a reagent which has been shown to kill phagocytes following interaction with intracellular proteases. There are two pathways whereby Leu-OME becomes cytotoxic to phagocytes. Within lysosomes, Leu-OME is converted into free Leu, which causes disruption of the lysosomes and subsequent cell necrosis. The other is the conversion of Leu-OME into (Leu-Leu)(n)-OME, which is associated with the induction of apoptosis. In the present study, we examined the action of T614 on Leu-OME mediated killing of THP-1, a human monocytic cell line. We revealed that T614 and phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, inhibited Leu-OME mediated killing of THP-1 cells. All the other mNSAIDs, including nimesulide (NIM-03), fluosulide (CGP28238), FK3311 and NS398, also rescued THP-1 from Leu-OME mediated killing, although to a lesser degree. Of the classical NSAIDs tested, a protective effect was observed with diclofenac at high concentration, but not with naproxen or indomethacin. Unlike conventional lysosomal inhibitors, such as chloroquine and ammonium chloride (NH(4)Cl), T614 and PMSF did not raise lysosomal pH, as measured by flow cytometry using fluorescein isothiocyanate dextran (FITC-dextran). Therefore, the mechanism whereby T614 and PMSF inhibit Leu-OME killing is distinct from that of chloroquine or NH(4)Cl. Based on the similarity of T614 and PMSF, we suggest that, besides their roles as COX-2 inhibitors, T614 and other mNSAIDs may act as lysosomal protease inhibitors.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Benzopyrans; Cell Death; Dipeptides; Growth Inhibitors; Humans; Hydrogen-Ion Concentration; Leucine; Lysosomes; Monocytes; Phenylmethylsulfonyl Fluoride; Sulfonamides; Tumor Cells, Cultured; U937 Cells

Porcine polymorphonuclear cells (PMN) and murine macrophages (M phi) were treated in vitro with Leu-OMe or Leu-Leu-OMe (1.5-5.0 mM) for various periods of time. It was found that the Leu-OMe and Leu-Leu-OMe entered cells rapidly, concomitantly the intracellular leucine accumulated. The methyl derivative diffused faster than Leucine due to its lipophylic character. The Leucine-O-methylesters hydrolysed rapidly as a consequence of the esterase and peptidase activities. The cells treated with Leu-OMe accumulated a high amount of Leucine and some Leu-Leu-OMe too. It was found that the formation of the didpeptide-methylester is not a spontaneous process, rather an enzymatic one. The Leu-OMe treated cells serve as a model which can be used to investigate the effect of the amino acid metabolism and the formation of dipeptides intracellularly and extracellularly.

Topics: Animals; Cells, Cultured; Chromatography, Thin Layer; Dipeptides; Hydrolysis; Immunosuppressive Agents; Leucine; Macrophages; Mice; Neutrophils; Swine

The uptake of Leu-OMe and Leu-Leu-OMe was studied in vitro in porcine PMN cells. Both methylesters are metabolized leading to the intracellular accumulation of leucine. Part of the hydrolyzed leucine gradually filtrates back into the culture medium in a time-, temperature- and methylester substrate concentration-dependent manner. Another portion of Leu-OMe is converted to Leu-Leu dipeptide. With respect to the cellular effects of Leu-OMe treatment ultrastructural studies showed the presence of large vacuoles without significant alteration of cell viability. Increased exocytosis of lysosomal enzymes did not lead to lytic events. Changes in the plasma membrane are indicated by the observation that Leu-OMe treatment causes the loss of the chemotactic activity to formyl-Met-Leu-Phe.

Topics: Animals; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dipeptides; Exocytosis; In Vitro Techniques; Leucine; Leukocytes, Mononuclear; Macrophages, Peritoneal; Neutrophils; Swine

The selective cytotoxicity of the lysosomotropic leucine methyl ester and its lysosomal condensation product leucyl-leucine methyl ester have been used to investigate their effect on a range of lymphocyte subsets and on the cellular proliferation and secretion by immunoglobulin-secreting B cells from axillary regional draining lymph nodes of breast cancer patients. CD2+, CD3+, and CD8+ lymphocyte subsets were selectively reduced by the leucyl-leucine methyl ester treatment (CD2: 84.2-67.5%; CD3: 76.1-62.3%; and CD8: 8.0-3.4%), but there was no significant reduction in the CD4+ and CD19+ subsets (CD4: 68.2-64.7%; and CD19: 22.6-33.2%). In the presence of mouse splenic macrophages as antigen-presenting cells, rIL-2, IFN-gamma, and pokeweed mitogen-stimulated lymphocyte supernatant, leucyl-leucine methyl ester-treated lymphocytes showed a significant increase in 3H-thymidine incorporation and in the number of immunoglobulin-secreting B cells following coculture with the breast tumor cell line MCF-7. In this study, we have characterized some of the cellular and cytokine factors that are necessary for in vitro immunization of human B lymphocytes. Hopefully, this will enable human MAbs to be produced in vitro against tumor-associated antigens.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antigen-Presenting Cells; Antigens, Neoplasm; Axilla; B-Lymphocyte Subsets; Breast Neoplasms; Cell Fusion; Cell Line; Dipeptides; Humans; Interferon-gamma; Interleukin-2; Leucine; Lymph Nodes; Macrophages; Mice; Pokeweed Mitogens; Recombinant Proteins

Treatment of peripheral blood mononuclear cells (PBMC) with the lysosomotropic agent leucine methyl ester (Leu-OMe) eliminates monocytes/macrophages and cytotoxic lymphocytes including CD3- CD16+ natural killer (NK) cells and a fraction of T cell receptor (TcR) alpha beta + CD8+ T cells. We report that freshly isolated peripheral blood gamma delta T cells are highly sensitive to Leu-OMe treatment as well. After incubation of PBMC with 5 mM Leu-OMe or incubation of purified T cells with 50 microM leucyl leucine methyl ester (Leu-Leu-OMe) and subsequent overnight culture, CD3-CD16+ NK cells and gamma delta T cells were no longer detectable by immunofluorescence analysis. The two major gamma delta T cell subsets V gamma 9+V delta 2+ and V gamma 9-V delta 1+ were equally susceptible to Leu-OMe and Leu-Leu-OMe treatment. The elimination of V gamma 9+ T cells by Leu-OMe treatment was confirmed in functional assays. Stimulation of peripheral blood T cells with killed mycobacteria resulted in selective expansion of V gamma 9+ T cells. In contrast, no activation of gamma delta T cells was elicited in Leu-OMe-treated responder T cells stimulated with killed mycobacteria.

Topics: Blood; Cells, Cultured; Cytotoxicity, Immunologic; Dipeptides; Flow Cytometry; Fluorescent Antibody Technique; Humans; Immunosuppressive Agents; Leucine; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocyte Subsets; T-Lymphocytes

The stimulation and immortalisation of human peripheral blood B lymphocytes specific for Candida albicans antigen were investigated. An in vitro immunisation system was employed which involved pretreatment of mononuclear cells with L-leucyl L-leucine methyl ester which removes the suppressive effects of CD8+ T cells, NK cells and monocytes. The remaining cells, CD4+ T cells, B cells and dendritic cells, were cultured with antigen and a mixture of cytokines. A mixture of IL-2, -4 and -6 was found to be optimal for antibody production as determined by an Elispot assay. Transformation of the activated B cells by Epstein Barr virus was found to be optimal after 2 days and lines secreting anti-Candida antibodies were established. These lines could form the basis for specific monoclonal antibody production by generating hybridomas, or by a newly described technique whereby cDNA encoding antibody Fab regions is transferred into phage display libraries. The overall strategy might be generally applicable for the generation of human monoclonal antibodies to infectious agents.

Topics: Antibodies, Monoclonal; B-Lymphocytes; Candida albicans; Cell Line; Dipeptides; Herpesvirus 4, Human; Humans; Immunization; Interleukin-2; Interleukin-4; Interleukin-6; Leucine; Lymphocyte Activation

We describe a simple and inexpensive chemical procedure for the selective expansion of human CD4+ lymphocytes. The method employs L-leucine methyl ester (LME) to deplete monocytes and large granular lymphocytes, as well as to inhibit growth of CD8+ lymphocytes. LME treatment eliminates granular cells, but most CD8+ lymphocytes, B-lymphocytes, and CD4+ lymphocytes remain. Peripheral blood mononuclear cells (PBMCs) from normal and HIV-positive individuals are treated with LME for 1 h at ambient temperature and cultured in the presence of IL-2 to expand the cell number. Stimulation with the T-cell mitogens concanavalin A, phytohemagglutinin, or OKT3 antibody augments lymphocyte expansion and within 1-3 weeks the culture is greatly enriched (90-100%) in CD4+ lymphocytes. LME-treated lymphocytes expand up to 10-fold during culture in the presence of IL-2 alone and up to 400-fold following treatment with T-cell mitogens. The immune function of LME-treated and expanded peripheral blood lymphocytes was examined using the response to the recall antigens tetanus toxoid and Candida albicans. Fresh PBMCs exposed to these recall antigens proliferated readily. Similarly, LME-treated lymphocytes following expansion responded to these recall antigens with good fidelity to the original PBMC response patterns in four of six donors. The expanded and LME-treated lymphocytes also exhibited good mitogen responses in three of three donors. The LME procedure allows for the simple and inexpensive generation of expanded, immunologically functional, CD4+ lymphocytes.

Topics: CD4-Positive T-Lymphocytes; Cell Division; Cells, Cultured; Dipeptides; HIV Infections; Humans; Immunophenotyping; Leucine; Lymphocyte Activation; Mitogens; T-Lymphocytes, Regulatory

Cultured Schistosoma mansoni schistosomula of various ages were exposed to several lysosomotropic agents. Weak bases such as chloroquine, ammonium chloride, and acridine orange caused gut swelling upon protonation. The latter compound fluoresced a bright orange indicating the acidic nature of the gut contents. Hydrolysis of ingested L-amino acid methyl esters also resulted in gut swelling, indicating the nonpermeant nature of the resulting L-amino acids. Neither age nor feeding status influenced these swelling effects. Treatment of schistosomula with D-amino acid esters, free L-amino acids, or methanol had no effect. Thin-layer chromatographic analysis of worms treated with radiolabeled L-leucine methyl ester provided evidence that the ester was hydrolyzed. These results support the premise that the schistosome gut is an acidic compartment and is reminiscent of a secondary lysosome in its reaction to lysosomotropic agents.

Topics: Acridine Orange; Alanine; Amino Acids; Ammonium Chloride; Animals; Chloroquine; Dipeptides; Esters; Hydrogen-Ion Concentration; Leucine; Microscopy, Electron; Microscopy, Fluorescence; Schistosoma mansoni; Tryptophan

L-Leucine methyl ester (Leu-OMe) is a lysosomotropic compound that irreversibly removes natural killer cell (NK) function from human peripheral blood mononuclear cells. This effect was dependent on the presence of mononuclear phagocytes (M phi) or polymorphonuclear leukocytes (PMN) and was prevented by lysosomal inhibitors such as chloroquine or NH4Cl. When M phi or PMN were incubated with Leu-OMe, a product was formed that eliminated all NK function from mixed lymphocyte populations. This effect did not require the presence of M phi or PMN and was not prevented by lysosomal enzyme inhibitors. Thin-layer chromatography and mass spectral analysis revealed that this NK-toxic product was L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). When human lymphocytes were exposed to greater than 50 microM Leu-Leu-OMe for 15 min, all NK function was irreversibly eliminated. Other dipeptide methyl esters containing nonpolar L amino acids caused similar effects, but substitution with amino acids containing polar or charged side chains or with D stereoisomers produced compounds that had no toxic effect on NK. These findings indicate that M phi and PMN can regulate NK function by releasing the dipeptide condensation product Leu-Leu-OMe generated from Leu-OMe via a lysosomally mediated mechanism. The data show that specific products of lysosomal enzyme activity may have potent effects on the function of adjacent cells.

Topics: Dipeptides; Humans; In Vitro Techniques; Killer Cells, Natural; Leucine; Lysosomes; Monocytes; Neutrophils