RESUMO
The compartmentalization of cAMP in human neutrophils during phagocytosis of serum-opsonized zymosan suggests that cAMP is an important second messenger for regulating phagocytosis. Type 4 cAMP-specific phosphodiesterase (PDE-4), cAMP-dependent protein kinase (PKA), and adenylate cyclase are the principal effector molecules for cAMP regulation in phagocytes. Immunofluorescence microscopy demonstrated that PDE-4 isoforms (HSPDE-4A, HSPDE-4B, HSPDE-4D) were targeted to the forming phagosome in neutrophils, and were colocalized with the catalytic subunit of PKA and degranulated myeloperoxidase. Phagocytosis and accumulation of PDE-4 and PKA near adherent zymosan were inhibited by elevating cAMP levels with forskolin or rolipram. cAMP, PDE-4, and PKA were localized at sites of zymosan adherence in cells treated with cytochalasin D to inhibit phagosome formation, suggesting that zymosan engagement to Fc/CR3 receptors triggers cAMP elevations at sites of phagocytosis. HSPDE-4A, HSPDE-4B, HSPDE-4D, and PKA also were localized at the forming phagosome in monocyte-derived macrophages, and the lysosomal marker CD63 demonstrated the absence of PDE-4 around internalized phagolysosomes. These results suggest that cAMP levels are focally regulated by PDE-4 at the nascent phagosome, and that PKA may phosphorylate proteins associated with pseudopodia formation and phagosome internalization.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/análise , Compartimento Celular/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/análise , Macrófagos/enzimologia , Neutrófilos/enzimologia , Adesão Celular , Colforsina/farmacologia , AMP Cíclico/análise , GMP Cíclico/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Citocalasina D/farmacologia , Humanos , Isoenzimas/análise , Macrófagos/imunologia , Neutrófilos/imunologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , Peroxidase/análise , Fagocitose , Fagossomos/enzimologia , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Rolipram , Transdução de Sinais/imunologia , ZimosanRESUMO
The presence and physiological role of cGMP-dependent protein kinase (G-kinase) was investigated in human mononuclear phagocytes. Western blots of monocyte extracts revealed a single polypeptide band that comigrated with purified bovine lung G-kinase. G-kinase was localized by immunofluorescence microscopy in freshly isolated adherent human monocytes, monocyte-derived macrophages cultured from 4 to 14 days, and alveolar macrophages. In monocytes, G-kinase was localized in granules or vesicles in the cytoplasm, at the microtubule organizing center, on filaments, and in the nucleus. In monocyte-derived macrophages, intense staining for G-kinase was found in the vicinity of the Golgi, in vesicles throughout the cytoplasm, and diffusely in the nucleus. Dual-label confocal laser scanning microscopy demonstrated that G-kinase was colocalized with the endoplasmic reticulum. For comparison, G-kinase was localized in alveolar macrophages that were adhered from 3 to 30 min. In these cells, G-kinase was prominent within the organelle-rich area pericortical to the nucleus. However, a well-defined area of intense staining was also observed at the cell periphery at early time points during adherence and spreading. Rhodamine-labeled phalloidin showed that this peripheral area was rich in F-actin. Cytochalasin D, but not nocodazole, inhibited G-kinase targeting to the cell margin. Furthermore, the guanylate cyclase inhibitor LY83583 inhibited alveolar macrophage spreading and staining for G-kinase at the cell periphery. These data suggest that G-kinase may play an important role in cGMP-mediated regulation involved in protein processing and cell motility.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/análise , Fagócitos/enzimologia , Aminoquinolinas/farmacologia , Western Blotting , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/enzimologia , Retículo Endoplasmático/enzimologia , Guanilato Ciclase/antagonistas & inibidores , Humanos , Macrófagos Alveolares/enzimologia , Microscopia de Fluorescência , Monócitos/enzimologiaRESUMO
Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.
Assuntos
Metaloendopeptidases/antagonistas & inibidores , Fenilalanina/análogos & derivados , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Cumarínicos/farmacologia , Feminino , Humanos , Interleucinas/metabolismo , Isocumarinas , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Monócitos/metabolismo , Fenilalanina/farmacologia , Células Tumorais CultivadasRESUMO
Vitamin E is thought to enhance immunity by increasing interleukin-1 (IL-1) production and by downregulating prostaglandin E2 (PGE2) synthesis. In an effort to understand the mechanism(s) whereby the form of vitamin E known as RRR-alpha-tocopheryl succinate [also called vitamin E succinate (VES)] ameliorates retrovirus-induced immune dysfunctions, peritoneal exudate cells (PECs) derived from normal chickens and avian and murine macrophage cell lines were used as in vitro model systems to test the effects of VES treatments on PGE2 and IL-1 production. Supernatants from PECs that were exposed to avian erythroblastosis virus (AEV) for 45 minutes exhibited a 256% increase in PGE2 levels compared with supernatants from replica cultures of PECs not exposed to AEV. Pretreatment of PECs with VES before exposure to AEV maintained PGE2 levels at normal control levels. VES treatment enhanced IL-1 production by avian (HD11) and murine (P388D1) macrophage cells, respectively. Supernatants from VES-treated HD11- and P388D1-stimulated cells contained IL-1 activity 196% and 385%, respectively, greater than that observed with supernatants from untreated control cells. On the basis of these studies, downregulation of retrovirus-induced PGE2 production and/or upregulation of IL-1 production by VES are potential mechanisms for VES amelioration of retrovirus-induced immune suppression.
Assuntos
Dinoprostona/biossíntese , Interleucina-1/biossíntese , Macrófagos/efeitos dos fármacos , Vitamina E/análogos & derivados , Animais , Líquido Ascítico/metabolismo , Linhagem Celular , Galinhas , Macrófagos/metabolismo , Camundongos , Valores de Referência , Tocoferóis , Vitamina E/farmacologiaRESUMO
RRR-alpha-tocopheryl succinate (vitamin E succinate) was studied for its effects on interleukin-2 (IL-2) production by chicken splenic derived T lymphocytes and murine EL-4 thymic lymphoma cells. Supernatants from 0.1 microgram/mL vitamin E succinate-supplemented chicken splenic T cell cultures exhibited 42-72% enhanced IL-2 production over vehicle controls when tested in a chicken T cell blast bioassay. Supplementation of chicken splenic T lymphocyte cultures with butylated hydroxyanisole (BHT) and butylated hydroxytoluene (BHA) also induced elevated levels of IL-2, suggesting a role for antioxidants in IL-2 production by avian splenic T lymphocytes. Supernatants from vitamin E succinate-supplemented murine EL-4 cells (0.1 microgram/mL vitamin E succinate) induced 52-75% increased levels of IL-2 when compared to supernatants from vehicle controls when tested using a murine, IL-2-dependent CTLL-2 bioassay. IL-2 production by EL-4 cells was not enhanced by treatments with BHT, BHA, or Trolox, suggesting that vitamin E succinate-induced IL-2 production by EL-4 cells may involve a mechanism other than antioxidant effects. Vitamin E succinate plus suboptimal levels of the protein kinase C (PKC) activator phorbol myristate acetate (PMA) induced the highest levels of IL-2 by EL-4 cells. The studies provide evidence that vitamin E succinate can directly potentiate either the production or release of IL-2 from avian splenocytes and murine EL-4 cells.