RESUMO
Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.
Assuntos
Proteínas de Bactérias/biossíntese , Endotélio Vascular/microbiologia , Regulação Bacteriana da Expressão Gênica , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Ácido Aspártico/metabolismo , Elementos de DNA Transponíveis , Endotélio Vascular/ultraestrutura , Humanos , Lisina/biossíntese , Microscopia Eletrônica de Varredura , Staphylococcus aureus/ultraestrutura , Veias Umbilicais/microbiologia , Veias Umbilicais/ultraestrutura , Regulação para CimaRESUMO
Bacterial infection coincides with migration of leucocytes from the circulation into the bacterium-infected tissue. Recently, we have shown that endothelial cells, upon binding and ingestion of Staphylococcus aureus, exhibit proinflammatory properties including procoagulant activity and increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression on the cell surface, resulting in hyperadhesiveness, mainly for monocytes. The enhanced extravasation of monocytes to bacterium-infected sites is facilitated by the local production of chemotactic factors. From another study we concluded that the locally produced chemokine MCP-1 is important in the recruitment of monocytes to the peritoneal cavity in a model of bacterial peritonitis. In the present study we investigated whether cultured human endothelial cells after infection with bacteria produce and release MCP-1, which in turn stimulates monocyte chemotaxis. We observed that endothelial cells released significant amounts of MCP-1 within 48 h after ingestion of S. aureus. This was dependent on the number and the virulence of the bacteria used to infect the endothelial cells. The kinetics as well as the amount of MCP-1 released by S. aureus-infected endothelial cells differed markedly from that released by endothelial cells upon stimulation with IL-1beta. Supernatant from S. aureus-infected or IL-1beta-stimulated cells promoted monocyte chemotaxis which was almost entirely abrogated in the presence of neutralizing anti-MCP-1 antibody, indicating that most of the chemotactic activity was due to the release of MCP-1 into the supernatant. Our findings support the notion that endothelial cells can actively initiate and sustain an inflammatory response after an encounter with pathogenic microorganisms, without the intervention of macrophage-derived proinflammatory cytokines.
Assuntos
Quimiocina CCL2/biossíntese , Quimiotaxia de Leucócito , Endotélio Vascular/microbiologia , Monócitos/fisiologia , Staphylococcus aureus/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Interleucina-1/imunologia , Monócitos/imunologia , Monócitos/microbiologia , Staphylococcus aureus/imunologiaAssuntos
Moléculas de Adesão Celular/fisiologia , Endotélio Vascular/imunologia , Inflamação/imunologia , Traumatismo por Reperfusão/imunologia , Animais , Adesão Celular , Moléculas de Adesão Celular/genética , Endotélio Vascular/fisiopatologia , Regulação da Expressão Gênica , Humanos , Inflamação/fisiopatologia , Leucócitos/fisiologia , Modelos Cardiovasculares , Traumatismo por Reperfusão/fisiopatologiaRESUMO
The consequences of internalization of Staphylococcus aureus by HUVEC with respect to their adhesiveness for human monocytes and granulocytes were investigated. Viable and UV-killed, but not heat-killed, S. aureus were internalized by HUVEC, which required participation of the endothelial cytoskeleton. S. aureus-infected HUVEC displayed increased surface expression of CD106 (VCAM-1), CD54 (ICAM-1), and MHC I molecules. Expression of CD62P (P-selectin), CD62E (E-selectin), CD31 (PECAM-1), and CD102 (ICAM-2) was not affected. Concomitantly, these HUVEC expressed a time- and inoculum size-dependent hyperadhesiveness for monocytes and granulocytes. Monocyte adhesion reached maximal levels (approximately 60% adhesion) 23 h after the initial 1 h period of infection of HUVEC with about 50 bacteria per single HUVEC. To induce maximal (approximately 20%) adhesion of granulocytes, five times higher concentrations of HUVEC-infecting bacteria were required. Using the appropriate mAb, granulocyte adhesion to S. aureus-infected HUVEC was shown to be entirely mediated by the beta2 (CD11/CD18) integrins. Monocyte adhesion to these HUVEC was largely (approximately 70%) dependent on both CD11a/CD18 (LFA-1) and CD49d/CD29 (VLA-4). This demonstrates that infection of HUVEC with S. aureus potentiates CD11/CD18-mediated granulocyte adhesion and shifts the mechanism of monocyte adhesion from being completely CD11/CD18 dependent to one that also utilizes the VLA-4/VCAM-1 dependent pathway. Together, these findings indicate that in response to internalization of S. aureus, vascular endothelial cells may initiate recruitment of monocytes and granulocytes, which may be an important initial event in the pathogenesis of endovascular diseases.
Assuntos
Endotélio Vascular/metabolismo , Endotélio Vascular/microbiologia , Granulócitos/metabolismo , Monócitos/metabolismo , Staphylococcus aureus/patogenicidade , Adesão Celular/fisiologia , Moléculas de Adesão Celular/biossíntese , Células Cultivadas , Humanos , Fagocitose/fisiologia , Veias Umbilicais/citologia , Veias Umbilicais/microbiologiaRESUMO
During primary infection of mice with Listeria monocytogenes, the bacteria proliferate extensively in the liver resulting in the development of inflammatory lesions in this organ. In the present study, the effect of interferon-gamma (IFN-gamma) on the development of these lesions, and the involvement of endogenous tumour necrosis factor-alpha (TNF-alpha) in the IFN-gamma-induced effects were evaluated. During an infection of naive mice with L. monocytogenes, two types of inflammatory lesions in the liver could be distinguished: large necrotic lesions consisting of granulocytes and/or exudate macrophages and small lesions containing mainly mature macrophages, i.e. BM8-expressing cells. Necrotic lesions were characterized by the presence of CD11b-expressing cells and consisted mainly of granulocytes during days 1 and 2 of infection and thereafter of exudate macrophages. The lesions consisting of mature macrophages and lymphocytes were not associated with necrosis and were called granulomatous lesions. Some of the granulomatous lesions contained many cells that expressed Ia antigen, i.e. activated cells. Treatment of mice with recombinant (r)IFN-gamma before injection of L. monocytogenes resulted in a decrease in the number of necrotic lesions and an increase in the number of granulomatous lesions in the liver, which was accompanied by a reduced bacterial proliferation in the liver. The effect of rIFN-gamma on the development of the various types of inflammatory lesions in the liver during infection with L. monocytogenes was abrogated by anti-TNF-alpha antibody and this antibody abrogated the rIFN-gamma-induced reduction of bacterial proliferation in the liver as well. Together, the results demonstrate that endogenous TNF-alpha plays a key role in the effects of rIFN-gamma on the inflammatory response in the liver during an infection with L. monocytogenes.
Assuntos
Interferon gama/uso terapêutico , Listeriose/patologia , Fígado/patologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Feminino , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/imunologia , Listeriose/terapia , Fígado/imunologia , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos CBA , Proteínas RecombinantesRESUMO
Activated macrophages have various characteristics in common with exudate and resident macrophages, but the ability to inhibit intracellular proliferation of the protozoa Toxoplasma gondii, the expression of Ia antigen and the capacity to produce H2O2 varies among these cells. Assessment of these features of macrophages, which are generally used as criteria for macrophage activation, has certain drawbacks. Since activated murine macrophages, but not exudate or resident macrophages, produce considerable amounts of NO2-, assessment of NO2- production by these cells might serve as a measure of macrophage activation. The aim of the present study was to find out whether NO2- production by murine peritoneal macrophages correlates with the three generally accepted criteria for macrophage activation. Quantitative data on resident, exudate and activated macrophages revealed that the production of NO2- stimulated by a calcium-ionophore correlates best with the ability to inhibit the proliferation of T. gondii, Ia antigen expression, and capacity to produce H2O2. Because it is rapid and easy to perform, measurement of the amount of NO2- produced by murine macrophages stimulated with a calcium-ionophore offers the most practical criterion for distinction between activated macrophages and exudate and resident macrophages.
Assuntos
Antígenos de Histocompatibilidade Classe II/metabolismo , Peróxido de Hidrogênio/metabolismo , Ativação de Macrófagos , Macrófagos/parasitologia , Nitritos/metabolismo , Toxoplasma/fisiologia , Animais , Antígenos de Superfície/imunologia , Líquido Ascítico/citologia , Calcimicina/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Toxoplasma/imunologiaRESUMO
Supernatants of human peripheral blood mononuclear cells cultured in the presence of B. gingivalis, showed a strong osteoclast stimulating activity as measured by 45Ca release from fetal mouse long bones in vitro. These supernatants also contained a high concentration of bioactive and immunoreactive interleukin-1 (IL-1), but tumor necrosis factor (TNFa), another osteoclast-activating cytokine, was not detected. Osteoclast activation by the supernatants was inhibited by an antibody against IL-1, whereas ultrapure human IL-1 mimicked the effect of the supernatant. The ability of B. gingivalis to induce IL-1 and OAF production was heat sensitive, as 20 min heating of the bacteria at 120 degrees C caused a 50% loss of activity. In addition, purified B. gingivalis lipopolysaccharide (LPS) had little IL-1 inducing capacity, compared with LPS of Escherichia coli. These data suggest that human peripheral blood cells confronted with B. gingivalis produce large amounts of IL-1 which has strong osteoclast stimulating activity. However, in contrast with E. coli LPS, B. gingivalis LPS does not seem to be the major inducing agent. Thus other bacterial components must be responsible for the observed IL-1 and OAF induction.
Assuntos
Bacteroides/fisiologia , Reabsorção Óssea/fisiopatologia , Endotoxinas/farmacologia , Interleucina-1/fisiologia , Leucócitos Mononucleares/metabolismo , Animais , Reabsorção Óssea/metabolismo , Cálcio/farmacocinética , Escherichia coli/fisiologia , Humanos , Interleucina-1/análise , Interleucina-1/biossíntese , Leucócitos Mononucleares/fisiologia , Lipopolissacarídeos/farmacologia , Linfócitos/metabolismo , Linfócitos/fisiologia , Linfocinas/análise , Linfocinas/fisiologia , Camundongos , Monócitos/metabolismo , Monócitos/fisiologia , Osteoclastos/fisiologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
The present study was performed in order to establish whether a cell-ELISA could be used to determine the expression of antigens by adherent murine peritoneal macrophages and also quantify the numbers of such macrophages. Accurate determination of the number of adherent macrophages proved to be possible with a cell-ELISA designed to assess complement receptor type III (CRIII) expression. Expression of CRIII was considerably more sensitive than determination of the cell-protein or DNA content as a measure of the number of adherent macrophages. For the calculation of the expression of CRIII, Ia antigen, and antigen F4/80 by resident and activated macrophages, use was made of the linear part of the curve obtained when the numbers of macrophages were plotted against the absorbance values for each of the antigens. The values for CRIII expression did not differ significantly between resident macrophages, macrophages activated with recombinant interferon-gamma (rIFN-gamma) and macrophages activated with BCG/PPD. IFN-gamma-activated and BCG/PPD-activated macrophages expressed Ia antigen significantly more intensely than did resident peritoneal macrophages. In contrast the activated macrophages expressed F4/80 significantly less intensely than resident peritoneal macrophages.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Ativação de Macrófagos , Macrófagos/imunologia , Animais , Antígenos de Superfície/análise , Adesão Celular , Feminino , Antígenos de Histocompatibilidade Classe II/análise , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Complemento/análiseRESUMO
Microbiologic methods were used to assess the in vitro phagocytosis and intracellular killing of various species of bacteria by freshly isolated murine peritoneal and alveolar macrophages. Peritoneal macrophages showed effective phagocytosis of opsonized Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Listeria monocytogenes, and moderate ingestion of Staphylococcus aureus and Escherichia coli. Alveolar macrophages were poor in phagocytosing opsonized S. pyogenes, S. aureus, and E. coli; ingestion of S. pneumoniae, P. aeruginosa, and S. epidermidis was moderate. Peritoneal macrophages killed 40 to 80% of these bacteria intracellularly, but alveolar macrophages showed almost no intracellular killing of bacteria. To find out whether there is a correlation between the poor bactericidal activity of alveolar macrophages and the oxygen-dependent microbicidal mechanisms of these cells, we determined the uptake of oxygen and the release of superoxide anion and hydrogen peroxide by macrophages at rest and after stimulation with phorbol myristate acetate (PMA) or opsonized S. aureus. Upon exposure to these stimuli, peritoneal macrophages, but not alveolar macrophages, showed an increased uptake of oxygen and release of superoxide anion and hydrogen peroxide. Because alveolar macrophages contain surface active material (SAM), we investigated the phagocytosis and intracellular killing of bacteria and the release of hydrogen peroxide by peritoneal macrophages pretreated with SAM. The results showed reduced phagocytosis and impaired intracellular killing of S. epidermidis by these macrophages. The release of hydrogen peroxide by SAM-pretreated peritoneal macrophages upon stimulation with PMA or opsonized S. aureus was equal to that of the control peritoneal macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bactérias/imunologia , Macrófagos/imunologia , Fagocitose , Alvéolos Pulmonares/imunologia , Animais , Peróxido de Hidrogênio/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Consumo de Oxigênio , Cavidade Peritoneal/microbiologia , Alvéolos Pulmonares/microbiologia , Receptores de Complemento/imunologia , Receptores de Complemento 3b , Receptores Fc/imunologiaRESUMO
Peritoneal and alveolar macrophages differ in phenotype, endocytic activities, and oxidative metabolism.
Assuntos
Bactérias , Ativação de Macrófagos , Fagocitose , Alvéolos Pulmonares/citologia , Animais , Camundongos , Consumo de OxigênioRESUMO
In a child with Down's syndrome (DS) and her sibling, host immune responses were evaluated under experimental gingivitis conditions. The children live in the same environment under identical conditions. In the DS child an earlier and more extensive gingival inflammation than in her sibling had been observed. Investigation of nonspecific host defense mechanisms revealed identical results in both children for the phagocytosis and intracellular killing of Candida albicans by polymorphonuclear leukocytes in crevicular washings (CR-PMNs), in blood (PB-PMNs) and blood monocytes. Furthermore, CR- and PB-PMNs were able to secrete identical amounts of hydrogen peroxide upon stimulation. The chemotactic response of PB-PMNs in the DS child was impaired, however. The results of the studies performed on parameters of specific host defense mechanisms showed low blastogenic responses to phytohemagglutinin (PHA) and pokeweed (PWM) by lymphocytes of the DS child as compared with her sibling. Also a lack of immune regulation leading to prolonged helper/inducer cell activation on a local (gingival) and circulation level and a less pronounced T-cell depression in PB were shown. Together, these differences observed in specific and nonspecific host response mechanisms may be responsible for the earlier and more extensive gingival inflammation found in the DS child.
Assuntos
Síndrome de Down/genética , Líquido do Sulco Gengival/imunologia , Gengivite/imunologia , Neutrófilos/imunologia , Quimiotaxia de Leucócito , Criança , Síndrome de Down/imunologia , Feminino , Humanos , Ativação Linfocitária , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Reliable assessment of IL-1 production by human monocytes is critically dependent on the methods for isolation and culture of these cells. In the present study, the quality of pipettes and the preparation of Ficoll-Isopaque appear to be crucial for IL-1 production from both LPS-stimulated and unstimulated monocytes. Different brands and lots of polystyrene culture wells give rise to great variation in IL-1 production. When carefully prepared, hydrophobic teflon membranes, to which mononuclear phagocytes poorly adhere, are used as the culture substrate, stimulation of IL-1 production is observed. The HLA DR3 haplotype of the monocyte donors did not influence IL-1 production. The addition of normal human AB serum to the cultures usually increases IL-1 production, although strong inhibition of both unstimulated and LPS-stimulated IL-1 production was also observed after addition of a diet-induced hyperlipemic AB serum. This inhibition was not due to cholesterol, chylomicrons, high- or low-density lipoproteins. When monocytes were cultured at different temperatures, the only abnormality found was a decrease of cell-associated IL-1 at 41 degrees C.
Assuntos
Interleucina-1/biossíntese , Lipídeos/sangue , Monócitos/metabolismo , Animais , Separação Celular/métodos , Células Cultivadas , Meios de Cultura , Antígenos HLA-DR , Humanos , Hiperlipidemias/sangue , Macrófagos/metabolismo , Masculino , Camundongos , Monócitos/classificação , Fenótipo , TemperaturaRESUMO
An instrument combining scanning electron microscopy (SEM) and light microscopy (LM) was used to study the cell surface characteristics and DNA content of macrophages in murine bone marrow cultures. After a quantitative Feulgen DNA staining, the DNA content of the individual macrophages was measured and their cell surface morphology was studied immediately thereafter with the SEM part of the instrument. The cells were divided into six groups according to the number of microvilli and/or microridges present on their surface. A proportion of macrophages showed a DNA content more than occurs in diploid cells, which could indicate a future division. No special surface morphology could be detected in this cell type.
Assuntos
Células da Medula Óssea , DNA/análise , Macrófagos/ultraestrutura , Animais , Membrana Celular/ultraestrutura , Células Cultivadas , Macrófagos/análise , Camundongos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Microvilosidades/ultraestruturaRESUMO
The effect of hydrocortisone and dexamethasone both in vivo and in vitro was studied in mouse bone marrow cultures in methylcellulose and in two liquid culture systems, one using Leighton tubes with a flying coverslip to grow adherent colonies and the other using Teflon culture bags to obtain suspension cultures. Although the total number of nucleated bone marrow cells was not greatly influenced by glucocorticosteroid treatment of the mice, a marked decrease in the number of colony-forming units and of mononuclear phagocytes was observed. Inhibition of colony growth in methylcellulose and of growth of mononuclear phagocytes in Teflon culture bags also occurred when glucocorticosteroids were added to in vitro cultures. Both drugs caused an almost complete inhibition of the growth of adherent colonies, and 3H-thymidine labeling of the cells was correspondingly low. When the glucocorticosteroids were added to cultures pre-incubated for 5 days in the presence of conditioned medium, the 3H-thymidine labeling of macrophages and promonocytes was markedly reduced, whereas there was no change in the labeling of monoblasts.
Assuntos
Dexametasona/farmacologia , Hidrocortisona/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Medula Óssea/efeitos dos fármacos , CamundongosRESUMO
The question as to whether the various types of mononuclear phagocyte found in bone marrow cultures and recognized by specific peroxidatic (PO) activity patterns differ in the expression of binding sites for the lectin wheat-germ agglutinin (WGA) and the activity of the ectoenzyme 5'nucleotidase (5'N) was investigated. Monoblasts, promonocytes, monocytes, and/or exudate macrophages, and exudate-resident macrophages generally showed a high level of WGA binding, and a considerably lower level was found in the PO-negative cells and in resident macrophages. 5'N activity was absent in monoblasts, promonocytes, and in the great majority of the monocytes and/or exudate macrophages, but was demonstrable in exudate-resident macrophages and resident macrophages, as well as in PO-negative macrophages after 4 days of culture. On the basis of the successive occurrence of the above-mentioned phenotypes in cultures, the possibility that this diversity in WGA binding and 5'N activity is related to modulation during cell differentiation is discussed. The present findings led to the conclusion that the PO-negative macrophages, whose origin was previously not entirely certain, are precursors of resident macrophages.
Assuntos
Lectinas/metabolismo , Nucleotidases/metabolismo , Fagócitos/metabolismo , 5'-Nucleotidase , Animais , Sítios de Ligação , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Masculino , Camundongos , Fagócitos/enzimologia , Aglutininas do Germe de TrigoRESUMO
The characteristics of murine bone marrow mononuclear phagocytes in long-term cultures with embryonic fibroblast-conditioned medium were studied to determine the stage of development and state of activation of these cells. Two liquid culture systems were used: for studies on the morphology, cytochemistry, and functional characteristics at the cellular level, the cells were cultured adherent to a glass surface; and for experiments where the cells were needed in suspension (replating experiments, and studies on locomotion, intracellular killing, and cytotoxicity) use was made of Teflon culture systems. Three developmental stages of mononuclear phagocytes could be recognized easily in these cultures: monoblasts, promonocytes, and macrophages. In cultures on a glass surface, these cells grow in colonies separate from granulocytic colonies. When incubation is prolonged beyond 7-9 days, the granulocytes die, leaving pure mononuclear phagocyte cultures. Primary cultures, in which monoblasts, promonocytes, and some macrophages proliferate, can be maintained for 3-4 weeks. Calculation showed that one monoblast present on day 0 gives rise to a progeny of more than 7 X 10(3) mononuclear phagocytes by day 14; after that, the rate of proliferation declines despite the addition of fresh media. Regular replating of the cells cultured on Teflon made it possible to maintain proliferation over a period of almost 200 days. The cells in culture have the typical characteristics of mononuclear phagocytes, as judged by light microscopy, alpha-naphthyl butyrate esterase activity, lysozyme activity, presence of receptors for Fc and C3, and endocytic, microbicidal, and cytotoxic activity. The 5'nucleotidase activity, ingestion of erythrocytes via C3-receptor, locomotion, and antibody-dependent cytotoxicity indicate that the cultured bone marrow mononuclear phagocytes are more active than resident macrophages, and as active as or even more active than thioglycollate-induced macrophages. In conclusion, the population of mononuclear phagocytes in the liquid cultures of bone marrow is heterogenous with respect to developmental stage and state of activation.
Assuntos
Células da Medula Óssea , Ativação de Macrófagos , Macrófagos/imunologia , Monócitos/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Líquido Ascítico , Medula Óssea/imunologia , Contagem de Células , Divisão Celular , Movimento Celular , Células Cultivadas , Meios de Cultura , Vida Livre de Germes , Macrófagos/enzimologia , Masculino , Camundongos , Monócitos/enzimologia , Consumo de Oxigênio , FagocitoseRESUMO
Blood monocytes from healthy volunteers were isolated by Ficoll-Isopaque centrifugation and cultured (together with lymphocytes) in medium 199 with 20% heat-inactivated newborn calf serum in a Teflon culture bag. Quantifiable data on survival showed that up to 21 days of culture, approximately 40% of the initial number of monocytes were still viable. Such cultures could be maintained for more than 8 weeks without refeeding. The monocytes exhibited the morphology of macrophages after 5-7 days of culture, and increased in size during culture. Less than 1% of the cells became giant cells even after long culture periods. Almost all cultured monocytes were positive for alpha-naphthyl butyrate esterase, whereas the peroxidase-positive granules disappeared during the first week of culture. After long culture times increasing amounts of lysozyme and angiotensin-converting enzyme were detected in the culture supernatants. Phagocytosis of staphylococci did not decrease appreciably during culture, and the same holds for intracellular killing of these bacteria. Chemotactic activity decreased during culture, whereas the chemokinetic response of the monocytes persisted.
Assuntos
Técnicas Citológicas , Monócitos/fisiologia , Movimento Celular , Separação Celular , Sobrevivência Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Humanos , Monócitos/citologia , Monócitos/enzimologia , Fagocitose , PolitetrafluoretilenoRESUMO
The origin of osteoclasts was studied in an in vitro model using organ cultures of periosteum-free embryonic mouse long-bone primordia, which were co-cultured with various cell populations. The bone rudiments were freed of their periosteum-perichondrium by collagenase treatment in a stage before cartilage erosion and osteoclast formation, and co-cultured for 7 d with either embryonic liver or mononuclear phagocytes from various sources. Light and electron microscopic examination of the cultures showed that mineralized matrix-resorbing osteoclasts developed only in bones co-cultured with embryonic liver or with cultured bone marrow mononuclear phagocytes but not when co-cultured with blood monocytes or resident or exudate peritoneal macrophages. Osteoclasts developed from the weakly adherent, but not from the strongly adherent cells of bone marrow cultures, whereas 1,000 rad irradiation destroyed the capacity of such cultures to form osteoclasts. In bone cultures to which no other cells were added, osteoclasts were virtually absent. Bone-resorbing activity of in vitro formed osteoclasts was demonstrated by 45Ca release studies. These studies demonstrate that osteoclasts develop from cells present in cultures of proliferating mononuclear phagocytes and that, at least in our system, monocytes and macrophages are unable to form osteoclasts. The most likely candidates for osteoclast precursor cells seem to be monoblasts and promonocytes.
Assuntos
Células da Medula Óssea , Macrófagos/citologia , Monócitos/citologia , Osteoclastos/citologia , Animais , Medula Óssea/efeitos da radiação , Osso e Ossos , Radioisótopos de Cálcio/metabolismo , Adesão Celular , Diferenciação Celular/efeitos da radiação , Técnicas de Cultura , Embrião de Mamíferos , Feminino , Fígado , Masculino , Camundongos , Osteoclastos/fisiologia , Gravidez , Fatores de TempoRESUMO
The proliferation of human bone marrow cells was studied in a liquid culture system without colony-stimulating factor. Bone marrow cells suspended in medium containing horse serum and fetal calf serum were incubated in the Teflon culture bag. During the first week there was an increase in the number of blast cells and early cells of the granulocytic series, both of which showed a high 3H-thymidine labeling index. The total number of mononuclear phagocytes increased during the first two weeks of culture. A number of characteristics of the cultured cells (alpha-naphthyl butyrate esterase, N-acetyl-DL-alanyl alpha-naphthyl esterase, Fc receptors, and phagocytosis) were determined. It was not feasible to recognize promonocytes and monoblasts with light microscopy, but with electron microscopy and the use of peroxidatic activity as marker, monoblasts and promonocytes were identified. The monoblast is a round cell with a few surface microextensions, a large nucleus, and few cytoplasmic granules. The nuclear envelope, the rough endoplasmic reticulum, and the granules show peroxidatic activity.