cytochalasin-b and Arthritis--Rheumatoid

The targeted delivery of therapeutics to sites of rheumatoid arthritis (RA) has been a long-standing challenge. Inspired by the intrinsic inflammation-targeting capacity of macrophages, a macrophage-derived microvesicle (MMV)-coated nanoparticle (MNP) was developed for targeting RA. The MMV was efficiently produced through a novel method. Cytochalasin B (CB) was applied to relax the interaction between the cytoskeleton and membrane of macrophages, thus stimulating MMV secretion. The proteomic profile of the MMV was analyzed by iTRAQ (isobaric tags for relative and absolute quantitation). The MMV membrane proteins were similar to those of macrophages, indicating that the MMV could exhibit bioactivity similar to that of RA-targeting macrophages. A poly(lactic- co-glycolic acid) (PLGA) nanoparticle was subsequently coated with MMV, and the inflammation-mediated targeting capacity of the MNP was evaluated both in vitro and in vivo. The in vitro binding of MNP to inflamed HUVECs was significantly stronger than that of the red blood cell membrane-coated nanoparticle (RNP). Compared with bare NP and RNP, MNP showed a significantly enhanced targeting effect in vivo in a collagen-induced arthritis (CIA) mouse model. The targeting mechanism was subsequently revealed according to the proteomic analysis, indicating that Mac-1 and CD44 contributed to the outstanding targeting effect of the MNP. A model drug, tacrolimus, was encapsulated in MNP (T-RNP) and significantly suppressed the progression of RA in mice. The present study demonstrates MMV as a promising and rich material, with which to mimic macrophages, and demonstrates that MNP is an efficient biomimetic vehicle for RA targeting and treatment.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cytochalasin B; Disease Models, Animal; Erythrocytes; Gene Expression Regulation; Human Umbilical Vein Endothelial Cells; Humans; Hyaluronan Receptors; Macrophage-1 Antigen; Macrophages; Mice; Nanoparticles; Polyesters; Polylactic Acid-Polyglycolic Acid Copolymer; Proteomics; Tacrolimus

Topics: Arthritis, Rheumatoid; Blotting, Western; CD13 Antigens; Cell Communication; Cell Movement; Coculture Techniques; Collagen; Cytochalasin B; Fibroblasts; Fluorescent Antibody Technique, Indirect; Gels; Gene Expression Profiling; Humans; Ionomycin; Jurkat Cells; Lymphocyte Activation; MAP Kinase Signaling System; Microscopy, Confocal; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stromal Cells; Synovial Fluid; T-Lymphocytes; Tetradecanoylphorbol Acetate

Although neutrophilic granulocytes clearly contribute to cartilage degradation in rheumatic diseases, it is unclear if reactive oxygen species (ROS) or proteolytic enzymes are the most important components in cartilage degradation and how they interact.. Neutrophils were stimulated by chemicals conferring a different degree of ROS formation and enzyme release. Supernatants of neutrophils were incubated with thin slices of pig articular cartilage. Supernatants of cartilage were assayed by NMR spectroscopy, MALDI-TOF mass spectrometry and relevant biochemical methods. Stimulation conditions of neutrophils correlated well with the extent of cartilage degradation. Due to the release of different enzymes, cartilage degradation could be best monitored by NMR since mainly low-mass degradation products were formed. Astonishingly, the suppression of the formation of ROS resulted in decreased cartilage degradation.. ROS formed by neutrophils are not directly involved in cartilage degradation but influence the activity of proteolytic enzymes, which are the main effectors of cartilage degradation.

Topics: Animals; Arthritis, Rheumatoid; Biphenyl Compounds; Cartilage, Articular; Culture Media, Conditioned; Cytochalasin B; Endopeptidases; Humans; In Vitro Techniques; Luminescent Measurements; Magnetic Resonance Spectroscopy; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophils; Onium Compounds; Proteoglycans; Reactive Oxygen Species; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine; Tetradecanoylphorbol Acetate

The adhesive function of integrins is regulated through cytoplasmic signaling. The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines. Adhesion of T cells induced by chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta was inhibited by pertussis toxin, wortmannin, and cytochalasin B, suggesting that both G protein-sensitive phosphatidylinositol (PI) 3-kinase activation and cytoskeletal assemblies are involved. The chemokine-induced T cell adhesion could be mimicked by expression of small G proteins, fully activated H-RasV12, or H-RasV12Y40C mutant, which selectively binds to PI 3-kinase, in T cells, inducing activated form of LFA-1alpha and LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced F-actin polymerization which colocalized with profilin in T cells. Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1, which depended on endogenous MIP-1alpha and MIP-1beta through activation of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI 3-kinase activation, also induced active configuration of LFA-1 and LFA-1-mediated adhesion of ATL cells, whereas expression of a dominant-negative H-Ras mutant failed to do. Profilin-dependent spontaneous polymerization of F-actin in ATL cells was reduced by PI 3-kinase inhibitors. In this paper we propose that H-Ras-mediated activation of PI 3-kinase can be involved in induction of LFA-1-dependent adhesion of T cells, which is relevant to chemokine-mediated signaling, and that profilin may form an important link between chemokine- and/or H-Ras-mediated signals and F-actin polymerization, which results in triggering of LFA-1 on T cells or leukemic T cells.

Topics: Actins; Adult; Androstadienes; Arthritis, Rheumatoid; Cell Adhesion; Chemokine CCL3; Chemokine CCL4; Contractile Proteins; Cytochalasin B; Cytoskeleton; Endothelium, Vascular; Genes, ras; GTP-Binding Proteins; Humans; Integrins; Intercellular Adhesion Molecule-1; Leukemia, T-Cell; Lymphocyte Function-Associated Antigen-1; Macrophage Inflammatory Proteins; Microfilament Proteins; Phosphatidylinositol 3-Kinases; Profilins; Signal Transduction; T-Lymphocytes; Wortmannin

The oxidative metabolism of neutrophils isolated from the bloodstream and synovial fluid of 16 patients with rheumatoid arthritis was compared by measuring the ability of neutrophils to generate luminol dependent chemiluminescence and to secrete O2-. Measurements of receptor mediated--that is, N-formyl-methionyl-leucyl-phenylalanine stimulated--activation or receptor and second message independent--that is phorbol myristate acetate stimulated--activation showed that synovial fluid neutrophils had biochemical characteristics to suggest that they had been either up-regulated (primed) or down-regulated (activated) in vivo. These conclusions were confirmed by comparison of these responses with the changes in oxidative metabolism observed during in vitro priming and activation of control neutrophils: synovial fluid neutrophils possessed lower levels of myeloperoxidase than paired bloodstream cells, and unlike bloodstream cells could not be primed in vitro. These data thus suggest that synovial fluid neutrophils have been exposed to both priming and activating agents within rheumatoid joints.

Topics: Adult; Aged; Aged, 80 and over; Arthritis, Rheumatoid; Cytochalasin B; Humans; Luminescent Measurements; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Oxygen; Peroxidase; Stimulation, Chemical; Synovial Fluid; Tetradecanoylphorbol Acetate

Only a minority (7/35, 20%) of synovial fluids from patients with rheumatoid arthritis (RA) and none from patients with other arthritides stimulated the oxidative response of polymorphonuclear leucocytes (PMNs). Superoxide anion generation was measured by superoxide dismutase inhibitable reduction of cytochrome c. The same synovial fluids stimulated superoxide release by PMNs regardless of their source, though they elicited a greater response from RA synovial fluid PMNs than from either RA blood PMNs or blood PMNs from normal subjects. The remaining synovial fluids failed to stimulate any of the PMNs, though some (2/10) stimulated PMNs pretreated with cytochalasin B. The stimulatory activity was removed from RA synovial fluids by protein A-Sepharose and eluted with the void volume on gel chromatography. It is considered that immunoglobulin aggregates in some RA synovial fluids may stimulate the oxidative response of PMNs in the joint space but that most do not because these fluids contain either a specific inhibitor or immunoglobulin aggregates of an inappropriate type, or both.

Topics: Arthritis, Rheumatoid; Cytochalasin B; Cytochrome c Group; Female; Humans; Immunoglobulin G; Lymphocyte Activation; Male; Middle Aged; Neutrophils; Stimulation, Chemical; Superoxides; Synovial Fluid

The production of three kinds of oxygen radicals (superoxide, hydrogen peroxide, and hydroxyl radicals) by neutrophils from patients with bacterial infection or rheumatoid arthritis was measured. The stimulators used in this study were opsonized zymosan (1 mg/ml), phorbol myristate acetate (20 ng/ml), A23187 (1 microM), and platelet activating factor (1 microM). Oxygen radical production by neutrophils from patients with rheumatoid arthritis was not significantly different from that of the control group. Hydrogen peroxide production by the neutrophils from patients with bacterial infection was significantly enhanced by only opsonized zymosan, but the production of the other kinds of oxygen radicals was not. Cytochalasin B reduced the production of hydrogen peroxide induced by opsonized zymosan more markedly than that of any other kind of oxygen radical. The measurement of hydrogen peroxide is suggested to be the most accurate indicator of the enhancement of intracellular production of oxygen radicals by neutrophils during infection.

Topics: Arthritis, Rheumatoid; Bacterial Infections; C-Reactive Protein; Calcimycin; Cytochalasin B; Free Radicals; Humans; Hydrogen Peroxide; Leukocyte Count; Neutrophils; Oxygen; Platelet Activating Factor; Superoxides; Tetradecanoylphorbol Acetate; Zymosan

Adherent rheumatoid synovial cells in culture produce large amounts of prostaglandin E2 (PGE2) and collagenase. When exposed to a monocyte-derived factor, such cells exhibit marked increases in PGE2 and collagenase production. In addition, cellular morphology becomes more stellate. In the presence of this factor, indomethacin inhibits both PGE2 production and the stellate changes, whereas collagenase production usually continues at a high rate. Addition of PGE2 to cultures reproduces the stellate change as does the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP. Colchicine inhibits morphologic transformation induced by the monocyte-derived factor, whereas cytochalasin B has no effect. It appears that the stellate morphology is dependent upon PGE2-induced cAMP stimulation and is not related to collagenase production per se.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Arthritis, Rheumatoid; Blood Proteins; Cells, Cultured; Colchicine; Cyclic AMP; Cytochalasin B; Dinoprostone; Humans; In Vitro Techniques; Indomethacin; Microbial Collagenase; Monocytes; Prostaglandins E; Synovial Membrane

The effects of various chemotactic factor generation conditions and several chemotherapeutic agents on neutrophil migration were determined using in vitro assay systems designed as models for inflammatory processes occurring in the synovial cavities of patients with rheumatoid arthritis. The microtubule-promoting agent concanavalin A and the microfilament-disrupting agent cytochalasin B were shown in these systems to inhibit neutrophil migration towards zymosan-activated serum-derived chemotactic factors. Neutrophils, immunoglobulin G aggregates, and serum were required for maximum generation of comparable chemotactic factors. Insoluble immunoglobulin G aggregates with or without rheumatoid factor produced more chemotactic factor activity on interaction with neutrophils than soluble immunoglobulin G aggregates. Exposure of neutrophils to supratherapeutic levels of the nonsteroidal antiinflammatory agent aspirin decreased neutrophil response to chemotactic factors while exposure to the slow-acting or immunomodulating agents gold, D-penicillamine, or azathioprine had no effect on this neutrophil function. In vitro systems employing neutrophils, insoluble aggregates, and serum may offer useful means for assaying drug effects on important functional components of the rheumatoid inflammatory process.

Topics: Antigen-Antibody Complex; Arthritis, Rheumatoid; Blood Physiological Phenomena; Chemotaxis, Leukocyte; Concanavalin A; Cytochalasin B; Humans; Immunoglobulin G; Inflammation; Macromolecular Substances; Neutrophils

A competitive radiobioassay method for soluble immune complexes in sera has been used to study sera from patients with rheumatoid arthritis. Twenty-eight out of sixty-two sera from selected seropositive rheumatoid patients were found to produce enhanced uptake of radio-iodinated human aggregated IgG by guinea-pig macrophages in contrast with the inhibition of uptake seen with SLE sera. Twenty-two out of the twenty-eight enhancing sera belonged to patients with cutaneous vasculitis. None of the twenty-two sera from seronegative rheumatoid patients possessed enhancing properties and none of these patients had clinical evidence of vasculitis. Three patients with seropositive SLE had sera with enhancing properties and had cutaneous vasculitis. We have shown that the enhancement is probably due to the presence in sera of immune complexes or altered IgG bound to rheumatoid factor. The possible mechanism has been investigated and it appears that there is increased binding of radio-iodinated aggregated IgG to free valencies on rheumatoid factor already bound to immune complexes.

Topics: Animals; Antibodies, Antinuclear; Antigen-Antibody Complex; Arthritis, Rheumatoid; Binding Sites; Complement C3; Cytochalasin B; Guinea Pigs; Humans; Immunoglobulin Fc Fragments; Immunoglobulin G; Immunoglobulin M; In Vitro Techniques; Lupus Erythematosus, Systemic; Macrophages; Phagocytosis; Precipitin Tests; Skin; Vascular Diseases

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (beta-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10(-3)-10(-5) M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10(-3) M cyclic nucleotides and 2.8 x 10(-4)-2.8 x 10(-6) M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 microg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE(1) also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules.

Topics: Arthritis, Rheumatoid; Carbon Isotopes; Colchicine; Cyclic AMP; Cytochalasin B; Glucose; Glucuronidase; Humans; Immunoglobulin G; Immunoglobulin M; Leukocytes; Lysosomes; Male; Microtubules; Phagocytosis; Prostaglandins; Spectrophotometry, Ultraviolet; Staining and Labeling; Theophylline; Time Factors; Uric Acid; Vinblastine; Zymosan