RESUMO
A young man suffering from recurrent Neisseria infections was shown to lack detectable serum complement factor D hemolytic activity. Addition to the patient's serum of purified factor D to a final concentration of 1 microgram/ml resulted in full restoration of the activity of the alternative pathway. Using an enzyme-linked immunosorbent assay, it was shown that the patient's serum did not contain measurable amounts of factor D antigen either. The sister, the father, as well as the parents of the mother had factor D levels within the normal range, and the factor D level of the mother was decreased. The capacity of the patient's serum, at concentrations up to 5%, to promote phagocytosis of Escherichia coli by normal human granulocytes was low when compared to normal serum. Substitution of the patient's serum with purified factor D resulted in a full restoration of opsonic activity. This study describes the first complete deficiency of factor D, and demonstrates its possible relation to recurrent Neisseria infections.
Assuntos
Enzimas Ativadoras do Complemento/deficiência , Fator D do Complemento/deficiência , Gonorreia/etiologia , Meningite Meningocócica/etiologia , Adulto , Fator D do Complemento/genética , Fator D do Complemento/uso terapêutico , Ensaio de Atividade Hemolítica de Complemento , Via Alternativa do Complemento , Escherichia coli , Humanos , Masculino , Linhagem , Fagocitose , Sepse/etiologiaRESUMO
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
In a previous study we have demonstrated that heat-killed Staphylococcus aureus opsonized with either purified human serum IgA or secretory IgA (sIgA) can induce a respiratory burst (measured as H2O2 release) in human polymorphonuclear leucocytes (PMN; Gorter et al., 1987). In the present study we have investigated whether opsonization of IgA-coated staphylococci with complement has an additional effect on the H2O2 release of PMN. It was demonstrated that staphylococci coated with IgA (or sIgA) and subsequently opsonized with complement induced at least a two-fold increase in the specific H2O2 release compared with bacteria coated with IgA (or sIgA) alone (P less than 0.05 and P less than 0.02, respectively). The co-operative effect of IgA and complement was also observed in the presence of 10 mM ethyleneglycoltetraacetic acid containing 5 mM MgCl2 (MgEGTA), suggesting that activation of the alternative pathway of complement is sufficient to exert this effect. Using D-deficient serum as a source of complement we could demonstrate that activation of the alternative pathway is essential for the co-operative effect of complement and IgA. The increase in specific H2O2 release caused by complement was found to be dependent on the amount of IgA initially used to opsonize the bacteria. Finally the co-operative effect of IgA and complement was not restricted to one IgA subclass, because an additional opsonization of S. aureus coated with sIgA1 or sIgA2 with complement resulted in both cases in a statistically significant enhanced specific H2O2 release by PMN (P less than 0.05).
Assuntos
Proteínas do Sistema Complemento/fisiologia , Peróxido de Hidrogênio/metabolismo , Imunoglobulina A/fisiologia , Neutrófilos/metabolismo , Complemento C3/fisiologia , Via Alternativa do Complemento , Humanos , Consumo de OxigênioRESUMO
In order to develop a suitable model for studying the role of granulocytes and monocytes in resistance against pathogenic microorganisms, we investigated the effect of irradiation and cytostatic treatment (cyclophosphamide and VP-16) on the number of both peripheral blood and peritoneal leukocytes in male Swiss mice. Irradiation and cyclophosphamide treatment severely decreased the number of both granulocytes and monocytes in peripheral blood, whereas VP-16 only lowered the number of blood monocytes to a significant degree and had little effect on the number of blood granulocytes or lymphocytes. When normal mice were injected intraperitoneally with newborn calf serum (NBCS) the number of peritoneal granulocytes rose about 100-fold within 6 h. In irradiated and cyclophosphamide-treated mice, this influx of granulocytes into the peritoneal cavity was virtually eliminated, as was the concomitant increase in the number of blood granulocytes; in VP-16-treated mice, on the other hand, the number of peripheral blood and peritoneal granulocytes increased to the same degree as in normal mice. An increase in the number of peripheral blood monocytes and peritoneal macrophages occurred 24-48 h after injection of NBCS in normal mice. This increase was significantly impaired by irradiation as well as by treatment with cyclophosphamide or VP-16.
Assuntos
Inflamação/sangue , Leucócitos/efeitos da radiação , Animais , Ciclofosfamida/farmacologia , Modelos Animais de Doenças , Etoposídeo/farmacologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Granulócitos/efeitos da radiação , Inflamação/imunologia , Inflamação/patologia , Contagem de Leucócitos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/efeitos da radiação , Masculino , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/efeitos da radiação , Cavidade Peritoneal/citologiaRESUMO
The aim of the present study was to investigate the contribution of granulocytes and monocytes to resistance against an acute systemic candidal infection in mice. To this end granulocytopenia and monocytopenia were induced by irradiation or treatment with cyclophosphamide, and monocytopenia was obtained by treatment with VP-16. After intravenous injection of 1 X 10(4) Candida albicans into mice irradiated with 8 GY, the number of Candida albicans cultured from the kidneys, expressed as the geometric mean of the number of CFU/g tissue, was 5.4 X 10(4), 7.1 X 10(6) and 5.8 X 10(7) on days 1, 3 and 5 of infection respectively (p less than 0.001 compared to normal mice). The number of Candida albicans cultured from the liver and spleen was also significantly higher for irradiated animals than for normal mice (p less than 0.001). For cyclophosphamide-treated mice the number of organisms in the kidney (1.7 X 10(4) CFU/g on day 1, 1.9 X 10(6) on day 3 and 3.8 X 10(6) on day 5 of infection) and spleen was significantly higher (p at least less than 0.02) than for normal mice after injection of 1 X 10(3) Candida albicans. Monocytopenia induced by VP-16 did not result in an increase in the number of Candida albicans cultured from the kidney or spleen after infection. From these studies it is concluded that granulocytes and not monocytes or exudate macrophages play an important role in resistance against Candida albicans during the first five days of a systemic infection.
Assuntos
Candidíase/imunologia , Granulócitos/imunologia , Monócitos/imunologia , Animais , Ciclofosfamida/farmacologia , Etoposídeo/farmacologia , Imunidade Inata , Contagem de Leucócitos , Masculino , Camundongos , Irradiação Corporal TotalRESUMO
Extracellular ATP in its tetra-anionic form (ATP4-) induces ion fluxes and membrane depolarization in the mouse macrophage-like cell line J774.2 and in resident mouse macrophages. We analyzed the effects of extracellular ATP4- by both patch-clamp and intracellular microelectrode techniques. Whole-cell patch-configuration membrane potential measurements on J774.2 cells revealed that ATP4- -induced depolarization occurred within 40 ms of pulsed application of ATP and was completely reversible. The depolarizations were accompanied by a dramatic increase in membrane conductance and showed no sign of adaptation to ATP over a period of 30 min. At 5 mM total ATP (ATPt) the whole-cell conductance was approximately 10 nS, and an upper limit of 20 pS for a single-channel conductance has been established. The reversal potential associated with the ATP-induced depolarization at asymmetric K+, Na+, Ca2+, and Cl- concentrations across the membrane was 0 mV. In patch-clamped cells depolarization was complete at 20 microM ATP4-, and repolarization from full depolarization occurred in approximately 5 s. In contrast, in intact cells measured by microelectrode impalement, complete depolarization occurred at approximately 2 mM ATP4- and repolarization was much slower (approximately 100 min). These findings indicate that the changes in intracellular ionic composition that occur after ATP treatment affect the rate of cell repolarization. At lower concentrations of ATP, potassium conductances modulated the depolarizing effect of ATP. ATP also depolarized mouse peritoneal macrophages, but a variant cell line (ATPR B2), derived from J774.2 cells by prolonged exposure to ATP, was insensitive to ATP. Our results provide a membrane electrophysiological description and analysis of a large nonselective plasma membrane conductance of macrophages induced by extracellular ATP.
Assuntos
Trifosfato de Adenosina/farmacologia , Macrófagos/efeitos dos fármacos , Animais , Cálcio/metabolismo , Membrana Celular , Cloretos/metabolismo , Condutividade Elétrica , Eletrofisiologia , Macrófagos/fisiologia , Camundongos , Microeletrodos , Potássio/metabolismo , Sódio/metabolismoRESUMO
Nosocomial infections with Legionella pneumophila serogroups 1 and 10 in the Leiden University Hospital and infections with L. pneumophila serogroup 6 in neighboring hospitals gave us an opportunity to study the development of opsonizing antibodies against L. pneumophila serogroups 1, 6, and 10 in the serum of 13 patients. Seven of these patients were infected with L. pneumophila serogroup 1, two were infected with serogroup 6, and four were infected with serogroup 10. The opsonic cross-reactivity of antibodies against these serogroups of L. pneumophila and complement involvement in opsonization were also investigated. Convalescent-phase sera from patients infected with L. pneumophila serogroup 1 or 6 were able to promote ingestion of these serogroups by polymorphonuclear leukocytes, whereas ingestion of L. pneumophila serogroup 10 was enhanced only in the presence of convalescent-phase sera from patients infected with this serogroup. Opsonization of L. pneumophila serogroups 1, 6, and 10 was complement dependent.
Assuntos
Anticorpos Antibacterianos/isolamento & purificação , Legionella/imunologia , Doença dos Legionários/imunologia , Humanos , Legionella/classificação , Doença dos Legionários/sangue , Neutrófilos/fisiologia , Fagocitose , SorotipagemRESUMO
The present study has shown that changes in ionic channel currents accompany the phagocytosis of particles by mononuclear phagocytes. The patch-clamp technique in the cell-attached configuration was applied to human monocyte-derived macrophages to measure the activity of single transmembrane ionic channels in intact cells. During such measurements, IgG-opsonized and non-opsonized latex particles were offered for phagocytosis under continuous video-microscopical observation. Single particles were presented to the phagocytes at a membrane location some distance from that of the patch electrode. After a lag period following particle attachment, enhanced inward and outward time-variant single channel currents coinciding with particle engulfment were observed. On the basis of current-voltage characteristics and membrane potential measurements, the outward-directed channels were identified as K+ channels. Phagocytosis was also accompanied by slow transient changes in background membrane currents, probably due to changes in the membrane potential of the phagocytosing cell. Phagocytosis of IgG-coated latex particles differed from phagocytosis of uncoated or albumin-coated particles by a shorter lag time between particle attachment and the onset of enhanced ionic channel activity.
Assuntos
Canais Iônicos/fisiologia , Macrófagos/fisiologia , Fagocitose , Condutividade Elétrica , Eletrofisiologia , Humanos , Técnicas In Vitro , Proteínas Opsonizantes , Receptores Fc/fisiologiaRESUMO
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
The relationship between the resting membrane potential and the intracellular ionic concentrations in human monocytes was investigated. Cell volume, cell water content, and amount of intracellular K+, Na+, and Cl- were measured to determine the intracellular concentrations of K+ (Ki), Na+ (Nai) and Cl- (Cli) of monocytes, and of lymphocytes and neutrophils. Values found for monocytes were similar to those for neutrophils, i.e., cell volumes were 346 and 345 micron3, respectively, cell water content 78%, and Ki, 128 and 125, Nai, 24 and 26, and Cli, 102 and 103 mmol/l cell water, respectively. Lymphocytes, however, had different values: 181 micron3 cell volume, 77% cell water content, and for Ki, Nai, and Cli, 165, 37, and 91 mmol/l cell water, respectively. The resting membrane potential of cultured human monocytes (range -30 to -40 mV), determined by measurement of the peak potential occurring within the first milliseconds after microelectrode entry, was most dependent on extracellular K+, followed by Cl-, and Na+. The membrane permeability ratio of Cl- to K+ was estimated by use of the constant field equation to be 0.23 (range 0.22 to 0.30).
Assuntos
Líquidos Corporais/análise , Cloretos/farmacologia , Líquido Intracelular/análise , Leucócitos Mononucleares/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potássio/farmacologia , Sódio/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Concentração OsmolarRESUMO
The purpose of the present study was to compare the monoclonal antibody (Mab) binding patterns of various tissue macrophages with each other and with blood monocytes. To allow recovery from the effects of the isolation procedure, or to obtain purified populations, macrophages were cultured for 24 hr and 48 hr. For comparison, blood monocytes were also cultured for 24 hr and 48 hr. Mab binding to individual cells, detected by the biotin avidin immunoperoxidase method, was quantified cytophotometrically and the results expressed as the median of the specific mean absorbance per 0.25 micron2 cell surface area or as specific integrated absorbance per cell. Analysis of the quantitative data in relation to the results of subjective evaluation of the peroxidase reaction product, demonstrating Mab binding to cells, yielded three classes for description of the intensity of antigen expression by cells: weak (specific mean absorbance per unit cell surface less than 0.07), moderate (values between 0.07 and 0.14), and intense (values more than 0.14). No matter how the results were expressed, comparison of the Mab binding patterns of macrophages with those of blood monocytes showed that spleen macrophages bound significantly less F4/80 and more M5/114 (Ia antigen). Kupffer cells and skin macrophages bound either approximately the same amount or considerably less of the various Mabs than monocytes did. Pulmonary tissue and alveolar macrophages bound significantly more 30.G.12 (leucocyte antigen), M3/38 (Mac-2 antigen), and M3/84 (Mac-3 antigen) and comparable amounts or considerably less of the other Mabs than the monocytes did. Peritoneal macrophages bound significantly more F4/80, M1/70 (complement receptor III), and 2.4.G.2. (Fc receptor II) and comparable amounts or considerably less of the other Mabs than monocytes did. It is concluded that macrophages from different organs and different anatomical sites within one organ differ from one another, for example, peritoneal macrophages do not resemble any other population of macrophages and alveolar macrophages do not resemble pulmonary tissue macrophages, and differentiation of blood monocytes into tissue macrophages does not show a distinct pattern.
Assuntos
Antígenos de Superfície/análise , Macrófagos/imunologia , Monócitos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Separação Celular , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos , Cavidade Peritoneal/imunologia , Alvéolos Pulmonares/imunologia , Pele/imunologia , Baço/imunologiaRESUMO
The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.
Assuntos
Interferon gama/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Animais , Suscetibilidade a Doenças , Feminino , Listeria monocytogenes , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Cavidade Peritoneal/citologia , Fagocitose , Proteínas Recombinantes/farmacologia , Salmonella typhimurium , Organismos Livres de Patógenos Específicos , Baço/microbiologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
To find out whether activated macrophages display a nonspecific enhancement of antibacterial activity, we determined the intracellular killing of bacteria by peritoneal macrophages from CBA and C57BL/10 mice infected with BCG and challenged with mycobacterial antigens (purified protein derivative (PPD]. After in vivo phagocytosis, the rate of in vitro intracellular killing of Listeria monocytogenes by bacillus Calmette-Guérin (BCG)-PPD-activated macrophages from CBA mice increased by a factor of 1.7 and that of those from C57BL/10 mice by a factor of 2.0, relative to the rate in normal resident macrophages. The increased listericidal activity of BCG-PPD-activated macrophages could not have been due to an increased number of peroxidase-positive macrophages because exudate macrophages obtained after i.p. injection of proteose peptone into BCG-infected mice or PPD into control mice, killed ingested Listeria about as efficiently as normal resident macrophages did. In contrast, BCG-PPD-activated macrophages from both mouse strains killed Salmonella somewhat less efficiently and Escherichia coli and Staphylococcus aureus with the same efficiency as normal resident macrophages did. These cells, however, inhibited the intracellular replication of Toxoplasma gondii. Activated peritoneal macrophages from listeria-infected mice showed a similar increase of the rate of intracellular killing of Listeria and absence of change in rate of intracellular killing of Salmonella. Consistent with the in vitro findings, the number of viable L. monocytogenes in the spleen and liver of BCG-infected CBA and C57BL/10 mice decreased during the first 2 days after i.v. injection, whereas Salmonella typhimurium proliferated in these organs of both mouse strains. Checking the state of activation of BCG-PPD-activated macrophages showed that these cells displayed enhanced O2-consumption and H2O2 release after stimulation with phorbol myristate acetate compared with resident macrophages. The present findings show that the antimicrobial activity of immunologically activated macrophages is not uniformly increased: for certain microorganisms (L. monocytogenes, T. gondii), this effector function is enhanced, whereas for others (S. typhimurium, S. aureus, E. coli), it is not.
Assuntos
Ativação de Macrófagos , Macrófagos/fisiologia , Animais , Bacteriólise , Atividade Bactericida do Sangue , Escherichia coli , Feminino , Listeria monocytogenes , Fígado/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL/imunologia , Camundongos Endogâmicos CBA/imunologia , Salmonella typhimurium , Organismos Livres de Patógenos Específicos , Baço/microbiologia , Staphylococcus aureusRESUMO
The aim of the present study was to investigate whether corpuscular immune complexes containing human IgA were able to interact with human polymorphonuclear leucocytes (PMN). As a model for corpuscular IgA immune complexes (IgA IC), heat-killed Staphylococcus aureus (S. aureus) opsonized with either purified human serum IgA or purified secretory IgA (sIgA) isolated from human colostrum was used. In order to determine the capacity of IgA and sIgA to opsonize S. aureus the phagocytosis of these IgA IC by PMN was measured. S. aureus opsonized with IgA, sIgA, IgG, heat-inactivated serum or fresh serum was ingested by 23 +/- 8%; 28 +/- 9%; 39 +/- 7%; 31 +/- 10% and 78 +/- 10% of the PMN (S. aureus:PMN = 10:1, n = 4), respectively. These results were significantly different (P less than 0.05) from the percentage obtained with unopsonized S. aureus (9 +/- 3%), indicating that IgA and sIgA induce ingestion of S. aureus. The phagocytic index for PMN incubated with S. aureus opsonized with sIgA (231) was higher than for S. aureus opsonized with IgA (119), indicating a better uptake of S. aureus opsonized with sIgA in our system. Bacteria opsonized with either IgA or sIgA were also capable of triggering H2O2 release of PMN in a dose-dependent manner. The H2O2 release by PMN triggered with S. aureus opsonized with IgA could not be inhibited with a F(ab')2 anti-Fe gamma receptor monoclonal antibody, whereas the H2O2 release triggered with S. aureus opsonized with IgG was fully inhibited. Soluble heat-aggregated IgA (AIgA) also induced H2O2 release of PMN, suggesting that the IgA itself is essential for the induction of a respiratory burst.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Imunoglobulina A/imunologia , Neutrófilos/imunologia , Fagocitose , Antígenos de Bactérias/imunologia , Humanos , Peróxido de Hidrogênio/metabolismo , Imunoglobulina A Secretora/imunologia , Neutrófilos/metabolismo , Staphylococcus aureus/imunologiaRESUMO
The aim of the present study was to determine whether the difference between the rate of intracellular killing of Salmonella typhimurium by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 mice also holds for other salmonellae and other bacteria species. After in vivo phagocytosis, the initial rate of in vitro intracellular killing of S. typhimurium phagetype 505, S. typhimurium phagetype 510, and S. typhimurium M206 by macrophages of CBA mice amounted always to approximately 1.7 times the value found for macrophages of C57BL/10 mice (p less than 0.001), indicating that the difference in killing efficiency between CBA and C57BL/10 macrophages holds for various strains of S. typhimurium. However, some other salmonella species, i.e., S. dublin and S. heidelberg, as well as E. coli 054 and 02K1+, Listeria monocytogenes EGD and L347, and Staphylococcus aureus were killed equally efficiently by macrophages of both mouse strains. These findings indicate that the difference between the rates of intracellular killing by macrophages of salmonella-resistant CBA and salmonella-susceptible C57BL/10 does not hold for several other bacteria species and thus might be specific for S. typhimurium. Subsequent experiments showed that the in vivo proliferation of S. typhimurium 510 in the first 2 days after i.v. injection was 2.0-fold to 3.0-fold higher in the spleens and livers of C57BL/10 mice than in those of CBA mice, whereas the in vivo proliferation of S. dublin and S. heidelberg was between 1.0-fold to 1.4-fold higher in the C57BL/10 mice. These findings suggest that the differences between the rate of in vitro intracellular killing of salmonella by CBA and C57BL/10 macrophages are reflected in differences in the rate of in vivo proliferation of these microorganisms in CBA and C57BL/10 mice. To gain insight into the involvement of the oxidative metabolism of CBA and C57BL/10 macrophages in the difference in the rate of intracellular killing of S. typhimurium, the O2 consumption and H2O2 release by resident peritoneal macrophages was determined. The amplitudes of the respiratory burst and the release of H2O2 was identical in macrophages of the two mouse strains after triggering by either preopsonized heat-killed S. typhimurium or phorbol myristic acetate. These findings indicate that the mouse species-associated difference in the intracellular killing of S. typhimurium is not caused by a difference in the oxidative metabolism of CBA and C57BL/10 macrophages.
Assuntos
Macrófagos/fisiologia , Fagocitose , Salmonelose Animal/patologia , Salmonella typhimurium/fisiologia , Animais , Feminino , Peróxido de Hidrogênio/metabolismo , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Consumo de Oxigênio , Cavidade Peritoneal/patologia , Especificidade da EspécieRESUMO
The purpose of the present study was to compare the phenotype of tissue macrophages with that of their precursors in the bone marrow and blood. The phenotype was determined on the basis of the quantitative binding of monoclonal antibodies to cell-surface antigens (antigen F4/80, complement receptor III, Fc receptor II, Ia antigen, common leukocyte antigen, and Mac-2 and Mac-3 antigens) on individual mononuclear phagocytes. Monoclonal antibody binding to cells, detected by the biotin-avidin immunoperoxidase procedure, was quantitated by cytophotometric determination of the amount of enzyme reaction product on cells. The results of this quantitation are expressed as the median of the specific absorbance per unit of cell-surface area (0.25 micron2) and per cell. Shortly after collection of the mononuclear phagocytes, binding of all monoclonal antibodies except those directed against the common leukocyte and Mac-2 antigens to peritoneal macrophages was enhanced compared with binding to blood monocytes; for alveolar macrophages we found reduced binding of monoclonal antibodies F4/80 and M1/70 (complement receptor III) and enhanced binding of monoclonal antibodies with specificity for the common leukocyte antigen and Mac-2 and Mac-3 antigens. The results obtained with cultured mononuclear phagocytes show that during the development from monoblast to tissue macrophages, monoclonal antibody binding to the various types of mononuclear phagocyte, expressed per unit of cell-surface area, was not significantly altered except that of M3/38 (Mac-2 antigen) to peritoneal macrophages and that of F4/80 and M1/70 (complement receptor III) to alveolar macrophages. Expressed on a per cell basis, the results show an increase in the binding of all monoclonal antibodies except those directed against the Fc receptor II and Mac-3 antigen during the development from promonocytes to peritoneal macrophages; binding of most monoclonal antibodies to alveolar macrophages was considerably lower than that to blood monocytes. It is concluded that the expression of the various cell-surface antigens alters during mononuclear phagocyte differentiation. The expression changed also during culture, although distinct patterns of alteration could not be distinguished.
Assuntos
Antígenos de Superfície/análise , Macrófagos/citologia , Monócitos/citologia , Cavidade Peritoneal/citologia , Fagócitos/citologia , Animais , Anticorpos Monoclonais , Células Sanguíneas/citologia , Células da Medula Óssea , Diferenciação Celular , Células Cultivadas , Histocitoquímica , Imunoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Monócitos/classificação , Fagócitos/imunologia , Fenótipo , Alvéolos Pulmonares/citologiaAssuntos
Antígenos CD , Imunoglobulina A Secretora/imunologia , Imunoglobulina A/imunologia , Neutrófilos/imunologia , Complexo Antígeno-Anticorpo/imunologia , Humanos , Peróxido de Hidrogênio/metabolismo , Fragmentos Fab das Imunoglobulinas/imunologia , Técnicas In Vitro , Neutrófilos/metabolismo , Proteínas Opsonizantes/imunologia , Fagocitose , Receptores Fc/imunologia , Receptores de IgG , Receptores Imunológicos/imunologia , Staphylococcus aureus/imunologiaRESUMO
The composition and opsonizing activity of five commercially available immunoglobulin preparations for intravenous use (Venoglobulin I, Venilon, Gammagard, Polyglobin, and Sandoglobulin) were studied. The composition of these preparations does not differ very much as far as total protein, immunoglobulin class and IgG subclass concentrations are concerned. The only exceptions were that Veniglobulin I, Gammagard and Sandoglobulin contain IgA, which might cause side effects in patients with anti-IgA antibodies, Gammagard contains very little IgG4, and Venilon and Polyglobin contain no and almost no IgG3, respectively, which might explain their very low opsonic activity. It was found that Venilon and Gammagard activate complement in the ready-for-infusion state. The opsonic activity of Venoglobulin I, Sandoglobulin and Gammagard is about equal to that of inactivated serum: Staphylococcus aureus, Escherichia coli with K antigen, Streptococcus pyogenes and Streptococcus group B are well opsonized and E. coli without K antigen and Streptococcus pneumoniae are poorly opsonized.