RESUMEN

IgA has traditionally been regarded a non-inflammatory antibody. This might indeed be true for secretory IgA (SIgA), which exerts its function at mucosal surfaces where commensal microorganisms and dietary antigens prevail. Serum IgA, however, potently triggers (pro)-inflammatory activity upon binding to the myeloid IgA receptor, FcalphaRI. Here, new insights in the roles of IgA and FcalphaRI are addressed and a model integrating the various functions of IgA in immunity is discussed.

Asunto(s)

Antígenos CD/inmunología , Inmunoglobulina A/inmunología , Receptores Fc/inmunología , Animales , Antígenos CD/química , Humanos , Inmunoglobulina A/química , Conformación Proteica , Receptores Fc/química

RESUMEN

Despite the well-recognized involvement of immunoglobulin (Ig) A in mucosal immunity, the function of its receptor, FcalphaRI (CD89), is poorly understood. The ability of FcalphaRI to activate leukocytes seems to conflict with the proposed anti-inflammatory activity of secretory IgA. We show here that in a transgenic mouse model, inflammatory mediators induced expression of FcalphaRI on Kupffer cells, which enabled efficient phagocytosis in vivo of bacteria coated with serum IgA. Secretory IgA did not initiate phagocytosis. Therefore, interactions between serum IgA and FcalphaRI on Kupffer cells may provide a 'second line of defense' in mucosal immunity, by eliminating invasive bacteria entering through the portal circulation and thus preventing disease.

Asunto(s)

Antígenos CD/inmunología , Inmunoglobulina A/inmunología , Macrófagos del Hígado/inmunología , Receptores Fc/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Escherichia coli/inmunología , Expresión Génica , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina A Secretora/inmunología , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/microbiología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Transgénicos , Fagocitosis/inmunología , Receptores Fc/biosíntesis , Receptores Fc/genética

RESUMEN

We constructed an immunotoxin, composed of an antibody directed against the high-affinity IgG receptor CD64 and Ricin-A, with the aim of resolving chronic inflammation through elimination of activated macrophages. In vitro, this immunotoxin proved very efficient in inducing apoptosis in activated macrophages, leaving resting and low CD64-expressing macrophages unaffected. We examined the activity of our immunotoxin in a sodium lauryl sulfate (SLS)-induced cutaneous inflammation model, using transgenic mice expressing human CD64. Upon intradermal injection of the immunotoxin (IT), cutaneous inflammation resolved in 24 h. This was demonstrated histologically by clearance of all CD64-expressing macrophages, followed by clearance of other inflammatory cells. Clinical parameters associated with inflammation, such as local skin temperature and vasodilation, also decreased.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Dermatitis/tratamiento farmacológico , Dermatitis/inmunología , Inmunotoxinas/toxicidad , Macrófagos/inmunología , Piel/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Apoptosis/efectos de los fármacos , Temperatura Corporal/efectos de los fármacos , Enfermedad Crónica , Dermatitis/patología , Dermatitis/fisiopatología , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Humanos , Inmunotoxinas/administración & dosificación , Inmunotoxinas/inmunología , Inmunotoxinas/metabolismo , Inyecciones Intradérmicas , Interferón gamma/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Transgénicos , Receptores Fc/metabolismo , Receptores de IgG/inmunología , Ricina/administración & dosificación , Ricina/metabolismo , Ricina/toxicidad , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Piel/patología , Dodecil Sulfato de Sodio/farmacología , Factores de Tiempo , Células U937 , Vasodilatación/efectos de los fármacos

RESUMEN

Interferons are able to enhance the expression of carcinoembryonic antigen (CEA) on tumour cells, allowing improved tumour targeting. In this report the hypothesis is tested that combinations of cytokines may further increase the tumour antigen expression. The combination of both IFN-gamma and IFN-alpha with interleukin-6 demonstrated a significant additive effect on the CEA-expression. This was found by quantitatively analysing the CEA-expression on human colorectal tumour cells by flow cytometry. It is concluded that combinations of cytokines show the potential of inducing tumour antigen expression for improved tumour targeting.

Asunto(s)

Antígeno Carcinoembrionario/metabolismo , Citocinas/farmacología , Regulación hacia Arriba , Neoplasias Colorrectales/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Técnicas In Vitro , Interferón gamma/farmacología , Interleucina-6/farmacología , Factores de Tiempo , Células Tumorales Cultivadas

RESUMEN

We report here that tumor angiogenesis-mediated endothelial cell (EC) anergy can be overcome by inhibitors of angiogenesis. We found previously that tumor growth, known to be dependent on angiogenesis, results in down-regulation of endothelial adhesion molecules and tumor EC anergy to inflammatory signals. We hypothesized that counteracting angiogenesis induces re-expression of adhesion molecules and normalizes responses to inflammatory cytokines. Here, we present data to show that the angiogenesis inhibitor platelet factor-4 (PF4) is able to prevent basic fibroblast growth factor (bFGF)-induced down-regulation of intercellular adhesion molecule-1 (ICAM-1). Furthermore, PF4 restores ICAM-1 expression following bFGF-induced down-regulation of ICAM-1. This PF4 effect occurs at the protein level and the RNA level and it has functional impact on leukocyte adhesion. In addition, PF4 overcomes the tumor-induced EC anergy to inflammatory signals such as tumor necrosis factor alpha (TNF alpha). Our findings may be the basis of new cancer therapies by combining anti-angiogenic therapy and immunotherapy to decrease blood vessel formation and to increase the effectiveness of inflammatory reactions against tumors.

Asunto(s)

Endotelio Vascular/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Factor Plaquetario 4/farmacología , Animales , Bovinos , Adhesión Celular , Anergia Clonal , Regulación hacia Abajo , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/citología

RESUMEN

The in vitro culture of endothelial cells (EC) is dependent on the presence of a coated surface and the availability of growth factors in the medium. The aim of the present research is to investigate whether in vitro EC culture conditions, such as serum source and surface coating, determine the growth characteristics of EC. The phenotype of EC was studied at the level of adhesion molecule expression and down-regulation by angiogenic factors. We found that human umbilical vein EC adhere well to and stretch well with plastic coated with fibronectin, collagen, gelatin and hyaluronan in contrast to non-coated plastic. While low in hyaluronan-coated wells, the spontaneous proliferation of EC was enhanced in fibronectin-collagen and gelatin-coated wells as compared to non-coated wells. Basic fibroblast growth factor bFGF-induced proliferation, however, was best on hyaluronan-coated plastic. A markedly up-regulated proliferation was measured on fibronectin and collagen while EC on gelatin-coated plastic only showed moderate bFGF-induced proliferation. On non-coated plastic EC were not inducible with bFGF. The induction of apoptosis by serum deprivation on these different matrices was most efficient when no coat was available or when wells were coated with hyaluronan, and bFGF inhibited apoptosis induction under all conditions. The use of different culture media demonstrated that human and bovine serum both can be used for human EC assays. The synthetic medium Utroser G prevented both spontaneous and growth factor-induced proliferation. We found that apart from some magnitude differences, the down-regulation of intercellular adhesion molecule-1 (ICAM-1) by angiogenic factors such as bFGF is not dependent on specific culture conditions.

Asunto(s)

Endotelio Vascular/citología , Matriz Extracelular/fisiología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Proteínas Sanguíneas/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Colágeno/farmacología , Medios de Cultivo/farmacología , Endotelio Vascular/química , Matriz Extracelular/química , Factor 2 de Crecimiento de Fibroblastos/fisiología , Fibronectinas/farmacología , Gelatina/farmacología , Humanos , Receptores de Hialuranos/análisis , Ácido Hialurónico/farmacología , Molécula 1 de Adhesión Intercelular/análisis , Neovascularización Fisiológica/fisiología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Venas Umbilicales/citología

RESUMEN

Recirculation of leukocytes is mediated by the intricately regulated expression of adhesion molecules on both the vessel wall and leukocyte membranes. In the present paper it is demonstrated that tumor angiogenesis factors impair leukocyte rolling and adhesion under flow conditions. Three lines of evidence presented in this paper support this finding; (i) treatment of cultured endothelial cells (EC) with the angiogenic factor basic fibroblast growth factor (bFGF) results in decreased ICAM-1 expression and decreased numbers of adhering leukocytes under flow conditions. (ii) flow induced upregulation of endothelial ICAM-1 in the presence of bFGF does not yield ICAM-1 levels higher than on resting EC. (iii) bFGF decreases the TNFalpha mediated induction of E-selectin and ICAM-1 expression, resulting in decreased rolling and firm adhesion of leukocytes on the endothelial surface. For ICAM-1 it is demonstrated that bFGF inhibits TNFalpha induced levels of mRNA, and that this effects is transcriptionally regulated. These findings support our earlier described hypothesis that angiogenic factors are involved in the tumor derived escape mechanism from immune surveillance, since we demonstrate here that these mechanisms are operative under physiologic flow conditions.

RESUMEN

CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.

Asunto(s)

Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/patología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Receptores de Hialuranos/fisiología , Linfocinas/farmacología , N-Glicosil Hidrolasas , Neovascularización Patológica/fisiopatología , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/fisiología , Carcinoma de Células Renales/irrigación sanguínea , División Celular , Células Cultivadas , Inhibición de Contacto , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/inmunología , Inmunotoxinas/farmacología , Neoplasias Renales/irrigación sanguínea , Proteínas de Plantas/farmacología , Reacción en Cadena de la Polimerasa , Empalme del ARN , Proteínas Inactivadoras de Ribosomas Tipo 1 , Saporinas , Piel/irrigación sanguínea , Estimulación Química , Células Tumorales Cultivadas , Venas Umbilicales/citología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

We report the suppressed vascular CD34 expression in renal cell carcinoma. This was found by quantitatively analyzing CD34 expression on normal and tumor derived EC by flow cytometry. In vitro studies revealed that culture of umbilical cord or dermis derived microvascular EC with angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor induced downregulation of CD34. This angiogenesis-induced downregulated expression of CD34 adhesion molecule may contribute to the tumor mediated escape mechanism from immune surveillance. It is concluded that there are quantitative differences in expression of endothelial CD34 in different compartments of the vasculature, that angiogenic factors affect this expression and that subpopulations of EC exist with differences in EAM expression.

Asunto(s)

Antígenos CD34/metabolismo , Carcinoma de Células Renales/metabolismo , Endotelio Vascular/metabolismo , Neoplasias Renales/metabolismo , Anticuerpos/metabolismo , Bromodesoxiuridina/análisis , División Celular/efectos de los fármacos , Selectina E/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/química , Endotelio Vascular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Citometría de Flujo , Humanos , Receptores de Hialuranos/metabolismo , Molécula 1 de Adhesión Intercelular/inmunología , Riñón/metabolismo , Linfocinas/farmacología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

We previously showed that endothelial cells (EC) from the vasculature of human solid tumors have a decreased expression of intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 as compared with normal tissue EC. This effect is explained by EC exposure to angiogenic factors. It is known that upregulation of endothelial adhesion molecules (EAM) is a sign of EC activation in inflammatory responses. We therefore tested the effect of angiogenic factors on upregulation of EAM on tumor EC and human umbilical vein EC (HUVEC) by proinflammatory cytokines. Incubation of tumor-derived EC in tumor necrosis factor alpha (TNF alpha) did result in expression levels of only 20% of the level of similarly treated normal tissue-derived EC. Pretreatment of HUVEC with 10 ng/ml basic fibroblast growth factor (bFGF) for 3 days, before TNF alpha- or interleukin-1 alpha (IL-1 alpha) stimulation, resulted in ICAM-1 levels of only 30% to 60% of cells without pretreatment. Also, the induction of vascular EC adhesion molecule-1 (VCAM-1) and E-selectin by TNF alpha was significantly inhibited by prior exposure to bFGF. Vascular endothelial growth factor had similar but less prominent effects. The effect of transforming growth factor-beta and IL-8 was studied as well. The functional relevance of the finding of a decreased EC inflammatory response was confirmed by adhesion assays. Our results show that tumor angiogenesis induces EC anergy. This may serve as a tumor-protecting mechanism by impairing the development of an efficient leukocyte infiltrate in tumors.

Asunto(s)

Citocinas/farmacología , Selectina E/biosíntesis , Endotelio Vascular/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neovascularización Patológica/patología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Selectina E/genética , Factores de Crecimiento Endotelial/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Inflamación , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/farmacología , Interleucina-1/farmacología , Interleucina-8/farmacología , Linfocinas/farmacología , Proteínas de Neoplasias/genética , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales , Molécula 1 de Adhesión Celular Vascular/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Conflicting data have been published on whether low-dose methotrexate (MTX) treatment of rheumatoid arthritis (RA) is able to slow down radiological joint damage, i.e. retard the destruction of articular cartilage and (subchondral) bone. We studied the effects of MTX on proteoglycan (PG) turnover and interleukin-1 (IL-1)- and RA mononuclear cell (RA-MNC)-induced cartilage damage in human articular cartilage tissue cultures, and the effects of MTX on basal and RA-MNC-influenced proliferation and differentiation of osteoblasts in cultures of human bone-derived osteoblasts. MTX exerted no direct effect on cartilage nor did MTX influence IL-1- or RA-MNC-induced cartilage damage, despite strong suppression of basal as well as mitogen- and antigen-induced RA-MNC proliferation. MTX induced strong inhibition of osteoblast proliferation, but did not significantly interfere with osteoblast differentiation (i.e. alkaline phosphatase activity). RA-MNC-enhanced proliferation and differentiation of osteoblasts were abolished by MTX. These results suggest that if MTX is able to induce retardation of radiological progression in RA, this is not based on an initial direct effect of MTX on cartilage as measured by PG turnover, nor on an initial inhibition of IL-1- or RA-MNC-induced cartilage damage. However, longstanding MTX-induced inhibition of RA-MNC proliferation may lead to reduction of the catabolic activity involved in cartilage destruction. On the other hand, long-term inhibition of osteoblast proliferation may eventually lead to decreased bone formation and osteopenia. Whether this will turn out to be a problem of clinical importance in the treatment of RA has to be established.

Asunto(s)

Antirreumáticos/farmacología , Artritis Reumatoide/patología , Cartílago Articular/efectos de los fármacos , Metotrexato/farmacología , Osteoblastos/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Cartílago Articular/metabolismo , División Celular/efectos de los fármacos , Técnicas de Cultivo , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad , Osteoblastos/citología , Proteoglicanos/biosíntesis , Proteoglicanos/efectos de los fármacos

RESUMEN

Intercellular adhesion molecule 1 (ICAM-1) is involved in the recirculation of blood leukocytes and, presumably, in the infiltration of cytolytic effector leukocytes into tumors. The present report describes a down-regulated expression of vascular ICAM-1 on tumor-infiltrating endothelial cells (EC) in renal cell carcinoma. This finding was obtained by flow cytometric analysis of tumor EC compared to EC obtained from healthy tissue. Since growth of solid tumors is dependent on the formation of new blood vessels (angiogenesis), we hypothesized that angiogenic factors are responsible for the down-regulation of ICAM-1. This hypothesis was investigated in vitro using human umbilical vein- and dermis-derived EC. Using flow cytometry, we found a biphasic regulation of ICAM-1 during stimulation of cultured EC with the angiogenic agent basic fibroblast growth factor (bFGF). Although 16-24 h after activation a marked up-regulation of ICAM-1 was observed, expression was significantly decreased after 48h. The longevity of this down-regulation was at least 7 days. Northern blot analysis revealed down-regulation of the steady-state mRNA level of the gene. ICAM-2 showed similar results of intial up- and later down-regulation. Functional relevance for the changes in ICAM-1 expression was demonstrated by a corresponding biphasic regulation of EC-leukocyte adhesion after EC activation by bFGF. The described effects are specific for bFGF since other angiogenic factors (such as vascular endothelial growth factor, transforming growth factor beta, and interleukin 8) did not affect adhesion molecule expression. Subsequent experiments showed that angiogenic factors decrease the sensitivity of EC to activation with tumor necrosis factor-alpha in regard to adhesion molecule expression. The present results reveal a tumor-derived escape mechanism from cytolytic effector leukocytes by down-regulation of vascular adhesion molecules in vivo and in vitro and decreased responsiveness to proinflammatory cytokines.

Asunto(s)

Carcinoma de Células Renales/metabolismo , Endotelio Vascular/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Neoplasias Renales/metabolismo , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/patología , Adhesión Celular , Células Cultivadas , Regulación hacia Abajo , Endotelio Vascular/patología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/patología , Neovascularización Patológica

Asunto(s)

Carcinoma de Células Renales/irrigación sanguínea , Endotelio Vascular/efectos de los fármacos , Neoplasias Renales/irrigación sanguínea , Molécula 1 de Adhesión Celular Vascular/uso terapéutico , Animales , Antígenos CD , Carcinoma de Células Renales/tratamiento farmacológico , Endoglina , Endotelio Vascular/inmunología , Femenino , Humanos , Inmunotoxinas/farmacología , Neoplasias Renales/tratamiento farmacológico , Ratones , Receptores de Superficie Celular , Molécula 1 de Adhesión Celular Vascular/efectos adversos , Molécula 1 de Adhesión Celular Vascular/biosíntesis

RESUMEN

This study was designed to investigate whether methotrexate (MTX), used in the treatment of rheumatoid arthritis (RA), affects proliferation and differentiation of human osteoblasts in culture. The effects of MTX were assessed by analyzing markers of proliferation and differentiation of human trabecular bone-derived osteoblast-like cells cultured in the presence or absence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). Treatment of the osteoblastic cells with MTX resulted in a strong dose-dependent inhibition of cell proliferation with half maximal response at a dose of 30 nM. MTX did not interfere with cellular alkaline phosphatase (AP) activity, the number of cells expressing cytochemical AP, or basal osteocalcin production. Addition of 1,25(OH)2D3 to the cultures caused an enhanced AP expression and osteocalcin production coinciding with a decreased osteoblast proliferation. Coincubation of 1,25(OH)2D3 with MTX in doses > or = 100 nM further inhibited osteoblast growth and induced a significant stimulation of AP expression and activity, and production of osteocalcin above the values reached in the 1,25(OH)2D3 cultures. In conclusion, MTX proved to be a potent inhibitor of osteoblast proliferation but did not affect basal osteoblastic phenotypic expression. In the presence of the osteoblast differentiation-promoter, 1,25(OH)2D3, MTX further inhibited cell growth which was associated with enhanced AP activity and osteocalcin production. Thus, MTX may have profound effects on bone metabolism and remodeling by interfering with bone cell turnover.

Asunto(s)

Antirreumáticos/toxicidad , Calcitriol/farmacología , Metotrexato/toxicidad , Osteoblastos/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Análisis de Varianza , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Remodelación Ósea/efectos de los fármacos , Remodelación Ósea/fisiología , Huesos/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Cabeza Femoral/citología , Cabeza Femoral/efectos de los fármacos , Humanos , Inmunohistoquímica , Metotrexato/uso terapéutico , Osteoblastos/citología , Osteoblastos/enzimología , Osteocalcina/biosíntesis , Radioinmunoensayo

RESUMEN

Endogenous growth factors may be involved in the prevention of bone loss by estrogen and progestins in postmenopausal women. The present study was performed to compare the action of estrogen/progestins on bone-derived cells with the effects of exogenously added purified growth factors. Human osteoblast-like (HOB) cells were incubated with 17 beta-estradiol (E2), progesterone (P), dydrogesterone (DD), 20 alpha-dihydroxydydrogesterone (DHD), with and without the growth factors, insulin-like growth factors-I/-II (IGF-I/-II) or transforming growth factor-beta type 1 (TGF-beta 1) for 24 h under serum-free conditions. Cell growth and DNA synthesis were assessed by spectophotometrical analysis of total cell number and immunochemical detection of BrdU incorporation, respectively. Compared with the sex steroids, incubation of the cells with IGF-I or TGF-beta 1 resulted in at least a two-fold increase of total HOB cell numbers. No difference in stimulating HOB growth was observed between IGF-II and the female sex steroids E2 and P. Combining IGF-I/-II or TGF-beta 1 with either E2 or P did not result in a significantly further increase in the human osteoblast-like cell growth. In conclusion, the bone anabolic growth factors, IGF-I and TGF-beta 1, may be more important regulators of osteoblast proliferation than the female sex steroids. An interaction of estrogen/gestagens with the growth factors IGF-I/-II or TGF-beta 1 was not evident from the growth of human bone-forming cells in short-term cultures.

Asunto(s)

Estradiol/farmacología , Osteoblastos/citología , Progestinas/farmacología , Somatomedinas/farmacología , Factor de Crecimiento Transformador beta/farmacología , Anciano , División Celular/efectos de los fármacos , Células Cultivadas , Didrogesterona/análogos & derivados , Didrogesterona/farmacología , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor II del Crecimiento Similar a la Insulina/farmacología , Osteoblastos/efectos de los fármacos , Progesterona/farmacología

RESUMEN

Estrogen/gestagen replacement therapy prevents excess bone loss in postmenopausal women. The mode of action by which these sex steroids exert their anabolic effects on bone has not been completely clarified yet. In this study, 17 beta-estradiol (E2), as well as progestins progesterone (P), dydrogesterone (DD), 20 alpha-dihydroxydydrogesterone (DHD), medroxyprogesterone acetate (MPA), and cyproterone acetate (CPA) were able to stimulate the mitogenesis and differentiation of normal adult human osteoblast-like (HOB) cells harvested from female trabecular bone explants. The different progestins exerted a more pronounced stimulatory effect on HOB proliferation than E2 did. The combination of E2 with P, DD, or DHD did not result in a statistically significant further increase of HOB proliferation, as compared with the progestins alone. In general, E2 showed a stronger differentiation-inducing effect than the progestins, as measured by histochemical staining of the HOB cells for alkaline phosphatase activity. Combining E2 and the progestins did not result in a further increase of the number of alkaline phosphatase positive cells, compared with E2 alone. The different progestins proved to be equally potent in stimulating HOB proliferation and differentiation. In conclusion, progestins as well as E2 exerted anabolic but differential effects on normal adult human osteoblasts in vitro.

Asunto(s)

Estrógenos/farmacología , Osteoblastos/citología , Progestinas/farmacología , Anciano , Fosfatasa Alcalina/análisis , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Terapia de Reemplazo de Estrógeno , Femenino , Humanos , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Osteoporosis Posmenopáusica/prevención & control

RESUMEN

Here we report that osteoblast-like cells derived from female and male adult human trabecular bone are able to directly respond to 17 beta-estradiol (E2) and progesterone (P). In short-term (1 day) cultures using serum-free and phenol red-free medium, both steroid hormones were found to stimulate DNA synthesis and growth of the human osteoblast-like cells. P was more potent in stimulating osteoblast growth compared to E2. On the other hand, E2 showed a stronger differentiation-inducing effect as determined by analysis of the number of cells displaying cytochemical alkaline phosphatase (AP) activity, a marker for the mature osteoblast phenotype. Combination of E2 and P resulted in a further increase in DNA synthesis, but did not further affect the number of cells expressing AP activity. In conclusion, female sex steroids may be involved in regulating bone mass in human adults via a direct anabolic action on the bone forming cells.

Asunto(s)

Estradiol/farmacología , Osteoblastos/citología , Progesterona/farmacología , Fosfatasa Alcalina/metabolismo , Bromodesoxiuridina , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Replicación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Cinética , Masculino , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología