RESUMO
Pneumocystis carinii pneumonia remains a serious complication for immunocompromised patients. In the present study, P. carinii organisms interacted with gamma interferon (IFN-gamma)-stimulated alveolar macrophages (AMs) to activate the L-arginine-dependent cytocidal pathway involving reactive nitrogen intermediates (RNI) that were assayed as nitrite (NO2-). Unstimulated cultures of AMs produced negligible quantities of RNI. Addition of P. carinii organisms to IFN-gamma-primed AMs resulted in greatly enhanced production of RNI. NO2- levels increased from 0.8 +/- 0.4 to 11.1 +/- 3.8 microM as early as 6 h after P. carinii organisms were incubated with IFN-gamma-stimulated AMs and to 35.1 +/- 8.9 microM after a 24-h incubation, a near-maximum level. High levels of NO2- were produced by AMs primed with as little as 10 U of IFN-gamma per ml in the presence of P. carinii, and a 20-fold increase in IFN-gamma concentration resulted in only a further 65% increase in NO2- production. RNI-dependent killing of P. carinii was demonstrated by both a 51Cr release assay and a [35S]methionine pulse immunoprecipitation assay. Addition of either monoclonal tumor necrosis factor alpha (TNF-alpha) neutralizing antibody or 200 microM NG-monomethyl-L-arginine (L-NGMMA), a competitive inhibitor of the L-arginine-dependent pathway, significantly decreased NO2- production and reduced P. carinii killing. TNF-alpha alone had no effect on P. carinii viability. These results suggest that (i) the specific interaction of P. carinii organisms with IFN-gamma-primed AMs triggers the production of RNI, (ii) RNI are toxic to P. carinii, and (iii) TNF-alpha likely plays a central role in mediating P. carinii killing by IFN-gamma-stimulated AMs.
Assuntos
Arginina/fisiologia , Interferon gama/farmacologia , Macrófagos Alveolares/imunologia , Pneumocystis/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Feminino , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , ômega-N-Metilarginina/farmacologiaRESUMO
Attachment of Mycobacterium tuberculosis organisms to alveolar macrophages (AMs) is an essential early event in primary pulmonary tuberculosis. Surfactant protein A (SP-A) is a nonimmune opsonin present in the alveolar spaces that binds carbohydrate residues such as mannose. It was hypothesized that SP-A attaches to M. tuberculosis and serves as a ligand between M. tuberculosis and AMs. [125I]SP-A was found to bind to M. tuberculosis in a time- and [Ca2+]-dependent manner with a Kd of 1.9 x 10(-9) M and an apparent number of 6.3 x 10(2) SP-A binding sites/organism. Further, deglycosylated SP-A had minimal binding to M. tuberculosis, indicating that sugar moieties are important in this interaction. SP-A specifically binds to a 60-kD cell-wall protein from M. tuberculosis. SP-A-mediated attachment of 51Cr-labeled M. tuberculosis organisms to AMs is dependent on time, SP-A concentration, and Ca2+. M. tuberculosis attachment to murine AMs in the absence of SP-A was 12.8 +/- 0.9%; however, in the presence of 5 microg/ml SP-A the attachment increased to 38.6 +/- 2.9% (P < 0.001). SP-A-mediated attachment was significantly decreased from 38.6 +/- 2.9% to 18.7 +/- 3.3% (P < 0.05) in the presence of antihuman SP-A antibodies. When the attachment assay was repeated in the presence of alpha-methylene-D-mannosepyranosidase (mannosyl-BSA) and type V collagen, SP-A-mediated attachment decreased from 38.6 +/- 2.9% to 16.6 +/- 1.5% (P < 0.001) and 19.1 +/- 1.4% (P < 0.05), respectively. Further, deglycosylated SP-A had only a minimal effect on M. tuberculosis attachment to AMs. These data indicate that SP-A can mediate M. tuberculosis attachment to AMs, and suggest possible underlying mechanisms for this.
Assuntos
Aderência Bacteriana/fisiologia , Macrófagos Alveolares/microbiologia , Mycobacterium tuberculosis/fisiologia , Proteolipídeos/fisiologia , Surfactantes Pulmonares/fisiologia , Animais , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos BALB C , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , RatosRESUMO
Human immunodeficiency virus (HIV)-infected subjects are at increased risk for tuberculosis even before there is a significant loss of CD4 lymphocytes. A factor was found to be present in the bronchoalveolar lavage (BAL) of HIV-infected subjects that promoted the attachment of M. tuberculosis (MTB) organisms to alveolar macrophages (AMs). Using 51Cr-labeled MTB organisms, BAL from control subjects resulted in MTB attachment to AMs at 11.6% +/- 1.0%; in contrast, BAL from HIV-infected subjects increased attachment to 33.1% +/- 3.8% (P < 0.001). Surfactant protein A (SP-A) levels in BAL of normal controls was 1.9 +/- 0.3 micrograms/ml and was 5.5 +/- 0.4 micrograms/ml in the BAL of HIV-infected subjects (P < 0.01). When SP-A was removed by immunoprecipitation from the BAL of HIV-infected subjects, MTB attachment decreased from 33.1% +/- 3.8% to 11.3% +/- 0.4% (P < 0.001), a value identical to control levels. Exogenous human SP-A (5 micrograms/ml) was added back to the immunoprecipitated BAL and the enhanced attachment of MTB was restored. These data suggest that BAL from HIV-infected subjects contain a factor that facilitates MTB attachment to AMs, the first critical step in the establishment of infection. This factor appears to be SP-A.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Infecções por HIV/complicações , Proteolipídeos/farmacologia , Surfactantes Pulmonares/farmacologia , Tuberculose Pulmonar/etiologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Humanos , Macrófagos Alveolares/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/patogenicidade , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes PulmonaresRESUMO
Histoplasmosis, an increasingly important opportunistic infection in immunosuppressed subjects, is characterized by hematogenous dissemination of the yeast from the lung. The mechanism of this dissemination is not fully understood. Laminin, the major glycoprotein of the extracellular matrix, is known to mediate the attachment of various invasive pathogens to host tissues. In the current study, laminin is demonstrated to bind to Histoplasma capsulatum in a rapid, specific, and saturable manner. Scatchard analysis with 125I-labeled laminin revealed an estimated 3.0 x 10(4) binding sites per yeast with an apparent Kd for laminin binding of 1.6 x 10(-9) M. Laminin binding to H. capsulatum was decreased from 62 +/- 1 to 17 +/- 1 ng (P < 0.001) in the presence of 3,000 nM of Ile-Lys-Val-Ala-Val, a pentapeptide within one major cell attachment site of laminin. A 50-kD H. capsulatum laminin-binding protein was demonstrated using an 125I-Ln blot of H. capsulatum cell wall proteins. The 50-kD protein is also recognized by antibodies directed at the 67-kD laminin receptor, suggesting they are related. This study proposes a possible mechanism for H. capsulatum attachment to laminin, an important first step required for the yeast to recognize and traverse the basement membrane.
Assuntos
Histoplasma/metabolismo , Laminina/metabolismo , Sequência de Aminoácidos , Amino Açúcares/farmacologia , Animais , Anticorpos Antifúngicos/imunologia , Anticorpos Antifúngicos/farmacologia , Membrana Basal/metabolismo , Linfócitos T CD4-Positivos , Adesão Celular/efeitos dos fármacos , Parede Celular/metabolismo , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Histoplasma/imunologia , Histoplasma/patogenicidade , Laminina/química , Laminina/farmacologia , Depleção Linfocítica , Macrófagos/microbiologia , Proteínas de Membrana/metabolismo , Metilmanosídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacosRESUMO
The incidence of tuberculosis is increasing on a global scale, in part due to its strong association with human immunodeficiency virus (HIV) infection. Attachment of Mycobacterium tuberculosis to its host cell, the alveolar macrophage (AM), is an important early step in the pathogenesis of infection. Bronchoalveolar lavage of HIV-infected individuals demonstrated the presence of a factor which significantly enhances the attachment of tubercle bacilli to AMs 3-fold relative to a normal control population. This factor is surfactant protein A (SP-A). SP-A levels are increased in the lungs of HIV-infected individuals. SP-A levels and attachment of M. tuberculosis to AMs inversely correlate with peripheral blood CD4 lymphocyte counts. Elevated concentrations of SP-A during the progression of HIV infection may represent an important nonimmune risk factor for acquiring tuberculosis, even before significant depletion of CD4 lymphocytes in the peripheral blood occurs.
Assuntos
Aderência Bacteriana , HIV/fisiologia , Macrófagos Alveolares/fisiologia , Mycobacterium tuberculosis/fisiologia , Proteolipídeos/fisiologia , Surfactantes Pulmonares/fisiologia , Infecções Oportunistas Relacionadas com a AIDS/fisiopatologia , Animais , Líquido da Lavagem Broncoalveolar , Glicoproteínas/fisiologia , Humanos , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteolipídeos/análise , Proteolipídeos/biossíntese , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Surfactantes Pulmonares/análise , Surfactantes Pulmonares/biossíntese , Valores de Referência , Tuberculose/fisiopatologiaRESUMO
We previously showed that hyperthermia induced in rhesus monkeys (Macaca mulatta) by forced passive heating "primes" the peripheral lymphocyte population for increased synthesis of interferon-gamma (IFN-gamma). It was not clear whether these data could be extrapolated to the physiological response in naturally occurring fever. Therefore, in the current experiments, the temperature of rhesus monkeys was raised either by systemic injection of killed Escherichia coli or by intrahypothalamic administration of prostaglandin E2. Mononuclear cells collected subsequently from such monkeys produced more IFN-gamma in response to stimulation with mitogens than cells from control monkeys. Direct administration of IFN-alpha, -beta, or -gamma to the hypothalamus did not affect the body temperature of rhesus monkeys.
Assuntos
Dinoprostona/farmacologia , Infecções por Escherichia coli/metabolismo , Febre/metabolismo , Interferon gama/biossíntese , Animais , Antivirais/sangue , Febre/etiologia , Hipotálamo , Injeções , Macaca mulattaRESUMO
Induction of hyperthermia (39 degrees C) in human volunteers by immersion in warm water (41-45 degrees C) rapidly alters the cell populations in the peripheral blood. In addition to granulocytosis, there is an alteration of the normal ratios among T-lymphocyte subsets. Following in vitro mitogen stimulation, lymphocytes from hyperthermic individuals produce as much as 10-fold more interferon-gamma (IFN-gamma) than cells withdrawn at basal core temperatures from the same individuals. A temperature threshold of 39 degrees C for this response suggests potential relevance to fever. No change was noted in the activity of the macrophage population. The possible involvement of interleukin-2 (IL-2) in this enhanced production is discussed. No changes were noted in the circulating levels of IFN-gamma.
Assuntos
Hipertermia Induzida , Interferon gama/biossíntese , Adulto , Temperatura Corporal , Humanos , Interleucina-2/metabolismo , Contagem de Leucócitos , Linfócitos/citologia , Linfócitos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismoRESUMO
The effect of in vivo hyperthermia on plasma interferon (IFN) activity and on the induction of IFN-gamma by phytohemagglutinin (PHA) or staphylococcal enterotoxin B (SEB) in isolated leukocyte cultures was investigated. Adult rhesus monkeys (Macaca mulatta) were placed in a climatic chamber maintained at 45 degrees C until their core body temperatures increased 2 degrees C above control levels. Peripheral blood samples were withdrawn both prior to core temperature elevation and at the time of peak body temperature. Plasma IFN-alpha increased slightly from a control value of 12 U/ml to 16 U/ml at the elevated core temperature. However, this alteration of plasma IFN levels appears to be a complex process that includes the loss of certain circulating IFN-alpha subtypes and the influx of acid-labile (Type II) IFN-alpha. Additionally, a non-IFN antiviral factor present in the plasma was elevated 10-fold at the higher body temperature. When mononuclear cells were isolated and cultured at 37 degrees C in the presence of PHA or SEB, those cells isolated from animals at the peak of body temperature showed a 4- to 16-fold increase in IFN-gamma activity relative to cells isolated from the same animal before the temperature increase. Similar results were obtained with cells isolated when fever was induced by the systemic injection of nonviable Escherichia coli. These results demonstrate that increased body temperature results in a circulating lymphocyte pool which is "primed" for the production of elevated levels of IFN-gamma activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hipertermia Induzida , Interferon Tipo I/sangue , Interferon gama/biossíntese , Ativação Linfocitária , Linfócitos/imunologia , Animais , Temperatura Corporal , Células Cultivadas , Leucócitos/imunologia , Macaca mulatta , Monócitos/citologiaRESUMO
We have previously demonstrated that artificially induced hyperthermia in man enhances the subsequent production of interferon gamma (IFN-gamma) in isolated leukocyte cultures. The mechanism(s) responsible for this response may involve changes in the circulating lymphocyte populations and may also reflect activation processes occurring in vivo due to hormonal influences. In order to determine whether hyperthermia was associated with other immunostimulatory effects, we measured lymphocyte activation, natural killer cell activity, interleukin-2 (IL-2) production and endotoxin-induced tumor necrosis factor (TNF) activity in blood samples obtained from normothermic (37 degrees C) and hyperthermic (39 degrees C) individuals. Lymphocyte transformation was significantly depressed in post-hyperthermic cultures compared to pre-hyperthermic control cultures. Pre-hyperthermic autologous human plasma was less effective than fetal calf serum in promoting DNA synthesis, while post-hyperthermic plasma suppressed mitogen-induced activation. Natural killer (NK) cell activity was increased by the elevation of core body temperature. Interleukin-2 (IL-2) production in phytohemagglutinin-stimulated mononuclear cell cultures was also elevated when cells were isolated from hyperthermic individuals relative to a paired normothermic control sample. Lipopolysaccharide-induced tumor necrosis factor (TNF) synthesis in monocyte cultures was unchanged by elevation of the core body temperature. This study indicates that in vivo hyperthermia can produce an immunostimulatory effect, an immunosuppressive effect, or no effect on different parameters of the immune system.
Assuntos
Febre/imunologia , Linfocinas/biossíntese , Glicoproteínas/biossíntese , Humanos , Técnicas In Vitro , Interleucina-2/biossíntese , Células Matadoras Naturais/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Monócitos/imunologia , Fator de Necrose Tumoral alfaRESUMO
Determinations of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity in haploid strains and diploid hybrids of wild-type Saccharomyces cerevisiae revealed that a genetic basis exists for control of this key regulatory enzyme in which low enzyme activity is phenotypically dominant to high enzyme activity. These observations suggested the existence of an inhibitor of reductase activity or a suppressor of enzyme synthesis. Feeding studies using an early sterol intermediate (mevalonolactone) and end-product sterol (ergosterol) indicated that a secondary regulatory site in this pathway operates to decrease the activity of HMG-CoA reductase. This diminution of activity was paralleled by increases in the accumulation of squalene, suggesting that this intermediate (or another isoprenoid derivative) may also play a significant role in the in vivo regulation of sterol biosynthesis. Lastly, feedback inhibition of HMG-CoA reductase by ergosterol was demonstrated in a yeast mutant which is permeable to this sterol. These studies showed that yeast can serve as a eukaryotic model system for a combined biochemical and genetic investigation into the factors which control the activity of HMG-CoA reductase.