RESUMEN

Antiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards beta2-glycoprotein 1 (beta2GP1)phospholipid complexes, we investigated the localisation of beta2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti-beta2GP1 antibodies, we detected beta2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti-beta2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti-beta2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti-beta2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

Asunto(s)

Endosomas/química , Endotelio Vascular/citología , Glicoproteínas/metabolismo , Anticuerpos/metabolismo , Anticuerpos/farmacología , Anticuerpos Antifosfolípidos , Anticoagulantes/inmunología , Anticoagulantes/metabolismo , Síndrome Antifosfolípido/etiología , Endotelio Vascular/ultraestructura , Glicoproteínas/inmunología , Humanos , Microscopía Fluorescente , Receptor IGF Tipo 2/efectos de los fármacos , Receptor IGF Tipo 2/metabolismo , Venas Umbilicales/citología , beta 2 Glicoproteína I

RESUMEN

Anti-phospholipid antibodies (APLAs) are associated with thrombosis and/or recurrent pregnancy loss. APLAs bind to anionic phospholipids directly or indirectly via a cofactor such as beta(2)-glycoprotein 1 (beta(2)GPI). The lipid target of APLA is not yet established. Recently, we observed that APLAs in vitro can bind lysobisphosphatidic acid (LBPA). The internal membranes of late endosomes are enriched in this phospholipid. The current study was undertaken to determine to what extent binding of APLA to LBPA is correlated with binding to cardiolipin and to beta(2)GPI and to determine whether patient antibodies interact with late endosomes of human umbilical vein endothelial cells (HUVECs) and thus modify the intracellular trafficking of proteins. Binding of patient immunoglobulin G (n=37) to LBPA was correlated significantly with binding to cardiolipin. Although LBPA binding was correlated to a lesser extent with beta(2)GPI binding, we observed that beta(2)GPI binds with high affinity to LBPA. Immunofluorescence studies showed that late endosomes of HUVECs contain LBPA. Patient but not control antibodies recognized late endosomes, but not cardiolipin-rich mitochondria, even when we used antibodies that were immunopurified on cardiolipin. Incubation of HUVECs with patient plasma samples immunoreactive toward LBPA resulted in an accumulation of the antibodies in late endosomes and led to a redistribution of the insulinlike growth factor 2/mannose-6-phosphate receptor from the Golgi apparatus to late endosomes. Our results suggest that LBPA is an important lipid target of APLA in HUVECs. These antibodies are internalized by the cells and accumulate in late endosomes. By modifying the intracellular trafficking of proteins, APLA could contribute to several of the proposed pathogenic mechanisms leading to the antiphospholipid syndrome.

Asunto(s)

Anticuerpos Antifosfolípidos/inmunología , Endosomas/inmunología , Endotelio Vascular/inmunología , Anticuerpos Monoclonales/inmunología , Cardiolipinas/inmunología , Células Cultivadas , Endosomas/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Glicoproteínas/inmunología , Humanos , Inmunoglobulina G/inmunología , Lisofosfolípidos/inmunología , Lisofosfolípidos/metabolismo , Monoglicéridos , beta 2 Glicoproteína I

RESUMEN

By releasing interleukin (IL)-12 in the lung, alveolar macrophages (AM) may profoundly modify an immune response. The autocrine regulation of the heterodimeric, biologically active form of IL-12 (IL-12 p70) by IL-10 was studied, as well as the expression of its subunits of 35 kD (p35) and 40 kD (p40). AM cultured in medium alone expressed only p35 mRNA. Both p35 and p40 mRNA levels were induced by lipopolysaccharide (LPS) and were further increased by interferon-gamma (IFN-gamma). LPS alone induced IL-12 p40 but not IL-12 p70 production in monocytes and in AM. However, IL-12 p70 was released when the autocrine production of IL-10 was neutralized by IL-10 blocking antibody, and IL-12 p40 production increased. Although IFN-gamma markedly decreased LPS-induced IL-10 production in AM, neutralizing IL-10 further enhanced the level of LPS and IFN-gamma-induced IL-12 p70 in AM. In contrast, neutralizing the trace amount of IL-10 released by AM stimulated by CD40 crosslinking and IFN-gamma did not increase IL-12 p70. Thus, IL-12 p70 production by AM appears to be tightly controlled by the autocrine release of IL-10 when stimulated by LPS, or by LPS and IFN-gamma, whereas CD40 crosslinking triggered IL-12 p70 production in the absence of autocrine regulation by IL-10.

Asunto(s)

Interleucina-10/biosíntesis , Interleucina-12/biosíntesis , Macrófagos Alveolares/metabolismo , Secuencia de Bases , Antígenos CD40/farmacología , Cartilla de ADN , Humanos , Interferón gamma/farmacología , Interleucina-10/antagonistas & inhibidores , Cinética , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos Alveolares/efectos de los fármacos

RESUMEN

Tumor necrosis factor alpha(TNF alpha), a proinflammatory cytokine secreted predominantly by monocytemacrophages, interacts with two cell-surface receptors: TNF-R55 and TNF-R75. Few studies have been devoted to their modulation on human alveolar macrophages (AM). Both source and target of TNF(alpha), AM also release its inhibitors, the soluble receptors, following the cleavage of the extracellular domain of TNF-R55 and TNF-R75. Because in vivo AM are subject to activation by exogenous or endogenous stimuli, we analyzed the release of both receptors into the cell culture supernatant in response to lipopolysaccharide (LPS), phorbol myristate acetate (PMA), and cytokines such as interleukin 2(IL-2), IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon gamma (IFN-gamma). Results were compared with those obtained on peripheral blood monocytes (Mo), and the role of receptor recycling was investigated using inhibitors such as monensin and chloroquine. In our culture conditions, basal release by unstimulated AM amounted to 0.3 +/- 0.1 and 0.5 +/- 0.1 ng/ml for TNF-sR55 and TNF-sR75, respectively. In the same conditions, Mo released 1.2 +/- 1.2 ng/ml of TNF-sR55 and 5.1 +/- 0.1 ng/ml of TNF-sR75. PMA slightly increased mRNA expression and release of TNF-sR55, but those of TNF-sR75 were enhanced approximately 4-fold. After 24 h of culture, the release of TNF-sR75 was 2.5-fold higher on Mo than on AM. Of the cytokines tested on AM, IFN-gamma increased the release of TNF-sR75 3-fold, but that of TNF-sR55 only between 1.5- and 2-fold. GM-CSF enhanced them to a lower extent (approximately 1.5-fold). Shedding occurred despite the presence of chloroquine, monensin and colchicine, suggesting that cleavage takes place on the cell surface rather than after internalization. Addition of colchicine increased the release of TNF-sR75 induced by LPS and IFN-gamma, but not by PMA. In conclusion, Mo and AM differ in their ability to release TNF(alpha) and TNF-sR. On AM the release of each receptor appears to be regulated separately. Finally, IFN-gamma was among the most efficacious cytokines to induce the release of both receptors, with TNF-sR75 being more liable to shedding. Thus, the two TNF-R seem to be ruled by separate mechanisms and to differ in terms of release sensitivity.

Asunto(s)

Antígenos CD/metabolismo , Interferón gamma/farmacología , Macrófagos Alveolares/metabolismo , Monocitos/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Animales , Antígenos CD/análisis , Células Cultivadas , Cloroquina/farmacología , Colchicina/farmacología , Medios de Cultivo , Sustancias de Crecimiento/farmacología , Humanos , Células L , Lipopolisacáridos/farmacología , Activación de Macrófagos , Ratones , Microtúbulos/efectos de los fármacos , Monensina/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/biosíntesis , Receptores del Factor de Necrosis Tumoral/análisis , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo

Asunto(s)

Macrófagos Alveolares/metabolismo , Monocitos/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Sialoglicoproteínas/metabolismo , Animales , Citocinas/fisiología , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Modelos Biológicos , Receptores de Interleucina-1/antagonistas & inhibidores

RESUMEN

The concentration of C-reactive protein (CRP) increases in human plasma up to a thousandfold during inflammatory states. Because tissue macrophages have been shown to have receptors for CRP, the question arises of whether these cells may respond to increased local concentrations of CRP by producing cytokines capable of participating in the inflammatory response. Accordingly, we examined the capacity of alveolar macrophages--relatively accessible human macrophages--to produce interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) in response to CRP. We found that production of IL-1 alpha, IL-1 beta, and TNF-alpha, as measured by bioassay and immunoassay, increased in a dose-dependent manner after stimulation by CRP and that the levels of the respective mRNAs analyzed by Northern blot increased proportionally. These findings suggest that one of the functions of CRP may be to stimulate the production of IL-1 and TNF by macrophages at inflammatory sites where alterations of capillary permeability combined with an increased serum level lead to enhanced local concentrations of this acute-phase protein.

Asunto(s)

Proteína C-Reactiva/farmacología , Interleucina-1/biosíntesis , Macrófagos Alveolares/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Actinas/biosíntesis , Actinas/genética , Bioensayo , Northern Blotting , Líquido del Lavado Bronquioalveolar , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Interleucina-1/genética , Cinética , Neoplasias Pulmonares/metabolismo , Macrófagos Alveolares/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Factor de Necrosis Tumoral alfa/genética

RESUMEN

Human alveolar macrophages (AM) are antigen-presenting cells that have an important immune effector function in the lung. We have previously shown that AM produce a specific interleukin-1 (IL-1) inhibitor of 20 to 25 kD that blocks biologic activities of IL-1 alpha and IL-1 beta such as prostaglandin E2 production by fibroblasts. This inhibitor acts as a receptor antagonist (IL-1ra) by binding to the IL-1 receptor. We are now presenting evidence that the natural AM-derived IL-1ra is immunologically identical to IL-1ra cloned from human peripheral blood monocytes and shows a band at 20 kD compatible with the natural glycosylated IL-1ra. No constitutive expression of IL-1 mRNA was detected when analyzed by Northern blot immediately after bronchoalveolar lavage from six control patients. Comparison of in vitro kinetics of IL-1ra, IL-1 alpha, and IL-1 beta analyzed during culture in the presence or absence of phorbol myristate acetate revealed that their mRNA expression was asynchronous. IL-1 alpha and IL-1 beta mRNA were expressed after as little as 15 min, whereas IL-1ra mRNA was detectable only after 3 h in culture. The production of IL-1ra was measured by enzyme-linked immunosorbent assay and compared with that of IL-1 alpha and IL-1 beta. In freshly isolated AM (10(6)/ml), cell-associated IL-1ra was present in an average amount of 2.0 +/- 0.5 ng/ml, i.e., 25 and 100 times more than IL-1 alpha and IL-1 beta, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Interleucina-1/biosíntesis , Interleucina-4/farmacología , Lipopolisacáridos/farmacología , Macrófagos Alveolares/metabolismo , Receptores de Interleucina-1/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Western Blotting , Células Cultivadas , Humanos , Interleucina-1/genética , Cinética , Macrófagos Alveolares/efectos de los fármacos , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

To study the interaction of lymphocytes and macrophages in the control of extracellular matrix turnover, we determined the effects of several soluble T cell products on mononuclear phagocyte production of metalloproteinases. Cytokines including IL-2, IL-4, IL-6, tumor necrosis factor alpha (TNF alpha), GM-CSF, and IFN-gamma were each tested for capacity to modulate macrophage metalloproteinase and tissue inhibitor of metalloproteinases (TIMP) expression. The addition of IL-4 to cells cultured under basal conditions caused a dose-dependent suppression in the release of 92-kD type IV collagenase without affecting TIMP production. 92-kD enzyme secretion was inhibited by 50% with 1-2 ng/ml of IL-4 and by 90% with 10 ng/ml of IL-4. When cells were first exposed to killed Staphylococcus aureus to induce metalloproteinase production, IL-4 potently blocked the stimulated release of both interstitial collagenase and 92-kD type IV collagenase, again without effect upon TIMP. Metabolic labeling experiments and Northern hybridizations demonstrated that IL-4 exerted its action at a pretranslational level. Furthermore, IL-4 possessed the capacity to inhibit metalloproteinase expression even in the relatively immature peripheral blood monocyte. As reported previously (Shapiro, S. D., E. J. Campbell, D. K. Kobayashi, and H. G. Welgus. 1990. J. Clin. Invest. 86:1204), IFN-gamma suppressed constitutive macrophage production of 92-kD type IV collagenase. Despite the frequent antagonism observed between IL-4 and IFN-gamma in other systems, the combination of these two agents lowered metalloproteinase biosynthesis dramatically, whereas IL-4 opposed the IFN-gamma-stimulated production of cytokines (IL-1 and TNF alpha). IL-6 had only minimal effect upon metalloproteinase production, but appeared to specifically augment TIMP release. In summary, cytokines released by activated T cells may profoundly reduce the capacity of the macrophage to mediate extracellular matrix degradation.

Asunto(s)

Interleucina-4/farmacología , Macrófagos Alveolares/enzimología , Metaloendopeptidasas/biosíntesis , Colagenasa Microbiana/metabolismo , Expresión Génica/efectos de los fármacos , Glicoproteínas/metabolismo , Humanos , Técnicas In Vitro , Interferón gamma/farmacología , Interleucina-1/biosíntesis , Activación de Macrófagos , Metaloendopeptidasas/genética , ARN Mensajero/genética , Inhibidores Tisulares de Metaloproteinasas , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

Alveolar macrophages have the ability to downregulate immune processes in vitro. We have recently suggested the presence of interleukin-1 (IL-1) inhibitors in the supernatants of human bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis or sarcoidosis. In the present study, we further analyze the cellular origin and the biologic properties of a 20- to 25-kD IL-1 inhibitor spontaneously produced by cultured human alveolar macrophages (AM). The inhibitor blocks IL-1-induced prostaglandin E2 production by human fibroblasts and the IL-1-related increase of phytohemagglutinin-induced murine thymocyte proliferation. After rigorous IL-1 alpha and IL-1 beta depletion, supernatants of lung macrophages specifically block the binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1 in a dose-dependent manner. These results indicate that AM from both normal donors and patients produce a specific IL-1 inhibitor that may be of importance in protecting the alveolar environment from the deleterious effects of excessive IL-1 production.

Asunto(s)

Interleucina-1/antagonistas & inhibidores , Macrófagos/fisiología , Alveolos Pulmonares/citología , Bioensayo , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Dinoprostona/biosíntesis , Humanos , Técnicas In Vitro , Interleucina-1/metabolismo , Activación de Linfocitos , Peso Molecular , Receptores Inmunológicos/fisiología , Receptores de Interleucina-1

RESUMEN

A small portion of human lung mononuclear cells are very potent stimulators of allogeneic resting T cells. Although several-fold more effective than phagocytic alveolar macrophages (AM) and blood monocytes (Mo), they do not produce more of the lymphocyte co-stimulators interleukin-1-alpha (IL-1 alpha), interleukin-1-beta (IL-1 beta), or tumor necrosis factor-alpha (TNF-alpha) than did Mo. Blocking antibodies against IL-1 alpha, IL-1 beta, TNF-alpha, and IL-6 did not reduce T cell proliferation. These potent antigen-presenting cells (APC) are loosely adherent and do not have phagocytic inclusions. Most of them have the marker RFD1 of dendritic cells (DC) rarely present on Mo or AM and have a strong tendency to form clusters with T cells like murine DC. Thus, we demonstrate an example in the human system of a dissociation between T cell activation and IL-1 or TNF-alpha production by DC or Mo, implying a major role for other "co-stimulating signals" by lung APC with dendritic features. The presence of different APC with various co-stimulating signals may be of importance for T cell subsets modulation.

Asunto(s)

Interleucina-1/biosíntesis , Pulmón/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Anticuerpos/inmunología , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Unión Competitiva , Humanos , Técnicas In Vitro , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/efectos de los fármacos

RESUMEN

Interleukin 1 (IL-1) possesses multiple biological activities that may be blocked selectively by different inhibitors. Some known inhibitors block the lymphocyte activating factor (LAF/IL-1) but not the mononuclear cell factor (MCF/IL-1) measured by its capacity to stimulate prostaglandin E2 (PGE2) and collagenase production. The presence of IL-1 in vivo may be difficult to detect due to the presence of inhibitor(s) and the level of the inhibitor(s) may vary depending upon pathological conditions. We have found that urine from three patients with monocytic leukemia (M5) contained high levels of inhibitor(s) of MCF/IL-1, whereas urine of normal subjects did not contain significant amounts. Urine from two patients with other blood neoplasic diseases also contained little inhibitory activity. The MCF/IL-1 inhibitor(s), which also acts on human recombinant IL-1 beta, is approximately 25-35 kD, is not retained on concanavalin A-Sepharose column and can be partially destroyed with urea and boiling. At this stage of the purification the fraction containing the MCF/IL-1 inhibitor(s) also inhibits the LAF/IL-1 assay. However, this inhibitor(s) is probably distinct from other inhibitors already described.

Asunto(s)

Fibroblastos/metabolismo , Interleucina-1/orina , Linfocinas/orina , Colagenasa Microbiana/biosíntesis , Prostaglandinas E/biosíntesis , Membrana Sinovial/metabolismo , Adulto , Anciano , Dinoprostona , Femenino , Calor , Humanos , Masculino , Membrana Sinovial/citología

RESUMEN

Normal human epidermal cells produce, in primary culture, activities which stimulate the release of PGE2 and collagenase by dermal fibroblasts; this factor(s) might play an important role in epidermal-dermal interactions. Since these activities were mainly found in the cell lysates with only little being detected in the conditioned media, we investigated further the problem of cell-associated versus released activity in the model of the human epidermoid carcinoma cell line A431. The activities were consistently found in the cell lysate and in the conditioned media only when the cells were leaky. No membrane-associated activities were identified. Purification of the cytosolic activities were identified. Purification of the cytosolic activities yielded two differently charged species both with a MW of approximately 17K. The copurification of PGE2- and collagenase-stimulating activities with thymocyte comitogenic activity suggests a close physiochemical relation to IL-1. The activities described here might therefore correspond to the intracellular counterpart of epidermal IL-1 formerly described as epidermal cell-derived thymocyte activating factor (ETAF) and identified in the conditioned medium of cultured epidermal cells. These observations are of importance when studying the modulation of these activities.

Asunto(s)

Carcinoma de Células Escamosas/análisis , Interleucina-1/análisis , Carcinoma de Células Escamosas/inmunología , Línea Celular , Cromatografía/métodos , Citosol/análisis , Dinoprostona , Humanos , Colagenasa Microbiana/metabolismo , Peso Molecular , Prostaglandinas E/metabolismo

RESUMEN

The pathogenesis and progression of rheumatoid arthritis involves the production of biologically active lymphokines and monokines. Of these, interleukin 1 (IL-1) has been somewhat of a controversial molecule because it seems to evoke various biological responses in several different tissues. In these studies we demonstrate that three biological properties of human monocyte-derived IL-1 (T-lymphocyte activation and human synovial cell prostaglandin E2 and collagenase production) co-purify. The complementary DNA for the prominent pI 7 form of human IL-1 was expressed, purified, and tested. Any controversy now appears resolved since homogeneous recombinant human IL-1 stimulates prostaglandin E2 and collagenase from human synovial cells as well as activates T cells in vitro.

Asunto(s)

Artritis Reumatoide/metabolismo , Interleucina-1/fisiología , Colagenasa Microbiana/biosíntesis , Prostaglandinas E/biosíntesis , Membrana Sinovial/metabolismo , Animales , Células Cultivadas , Cromatografía , ADN Recombinante , Dinoprostona , Escherichia coli/genética , Humanos , Interleucina-1/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C3H , Monocitos/metabolismo , Proteínas Recombinantes

RESUMEN

Cultured human alveolar macrophages from smokers with lung cancer produced spontaneously variable amounts of factors stimulating fibroblast proliferation and production of prostaglandin E2 and collagenase by fibroblasts. These biological activities belong to molecules similar or identical to interleukin 1. Exogenous leukotriene B4 added to alveolar macrophage cultures increased the production of these factors. The Ca++ ionophore A23187 was found to have similar effects. By the control of monokine production, leukotriene B4 locally released by inflammatory cells may modulate lung fibroblast functions.

Asunto(s)

Calcimicina/farmacología , Fibroblastos/efectos de los fármacos , Leucotrieno B4/farmacología , Proteínas/metabolismo , Alveolos Pulmonares/efectos de los fármacos , División Celular/efectos de los fármacos , Dinoprostona , Femenino , Fibroblastos/fisiología , Humanos , Técnicas In Vitro , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Colagenasa Microbiana/biosíntesis , Persona de Mediana Edad , Monocinas , Prostaglandinas E/biosíntesis , Alveolos Pulmonares/metabolismo