摘要:

We demonstrate that 5′‐nucleotidase (5′NT), an ectoenzyme of guinea pig polymorphonuclear leukocytes, is largely excluded from phagosomal membrane, rather than internalized randomly during phagocytosis of heat‐killed bacteria, latex microbeads, or zymosan particles. Cells were fixed in 0.25% glutaraldehyde (pH 6.3) at 4°C for 10 min and incubated in a cytochemical medium for the demonstration of 5′NT. In the nonphagocytosing cells, 5′NT was evenly distributed on the external side of the plasma membrane. In cells phagocytosing bacteria, 5′NT appeared to be cleared from the nascent phagosomal membrane; after 5 min of phagocytosis, most of the phagocytic vacuoles containing bacteria, latex, or zymosan particles were devoid of reaction product. When phagosomes containing latex particles were isolated and biochemically assayed, they contained less than 3% of the total cellular 5′NT activity even after 60 min of phagocytosis, and at that time the total cellular 5′NT activity had not declined. When the diazonium salt of sulfanilic acid (DASA), a nonpermeable ectoenzyme inhibitor, was used to determine the distribution of extracellular and intracellular 5′NT activity, no increase in DASA‐ insensitive intracellular 5′NT was found after phagocytosis of latex or opsonized zymosan. Cytochemical and biochemical evidence led us to conclude that 5′NT is excluded from phagosomal membrane, and that the exclusion is due to redistribution rather than to inactivation by granule enzymes.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.463

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

The phagocytic process is a combination of a sequence of events which includes a recognition attachment phase and a subsequent internalization phase. The present study was designed to investigate the effect of plasma fibronectin on the attachment and ingestion of gelatinized sheep erythrocytes to isolated rat Kupffer cells in a monolayer assay. Kupffer cells were isolated by sequential collagenase‐pronase digestion followed by metrizamide density gradient centrifugation and subsequent adherence to plastic. Classification as Kupffer cells was confirmed by the presence of functional Fc receptors, a positive peroxidase reaction, and phagocytic activity. Purified plasma fibronectin as well as rat serum containing fibronectin promoted attachment of gelatinized fixed sheep erythrocytes to Kupffer cells in a dose‐dependent manner, whereas fibronectin‐deficient serum did not. Heparin did not enhance the fibronectin‐mediated attachment or ingestion of gelatinized sheep erythrocytes at lower particle doses, whereas at higher particle doses heparin augmented the response. These results indicate that fibronectin can enhance the binding and ingestion of foreign gelatin‐coated particulates by Kupffer cells.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.477

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

The detergent lysate of plastic adherent cell population of thioglycollate‐elicited peritoneal exudate cells from 100 individual Swiss mice was subjected to affinity chromatography on two different media, Sepharose coupled to heat‐ aggregated human IgG (IgG‐Sepharose) and Sepharose coupled to the phosphatidylcholine analog, rac‐1 ‐(9‐carboxyl)nonyl‐2‐hexadecy1glycero‐3‐phos‐ phorylcholine (PC‐Sepharose). Both IgG‐ and PC‐binding proteins were further purified by Sephadex G‐100 gel filtration and isoelectric focusing in the presence of 6M urea. Both IgG‐ and PC‐binding proteins thus purified appear to be homogeneous in size as well as charge properties. The IgG‐binding proteins of a molecular weight of 25,000 had an isoelectric point of 4.8, whereas the PC‐binding proteins of a molecular weight of 42,000 were more basic and had an isoelectric point of 6.0. Both materials retained their IgG‐binding capabilities as judged by their inhibitory capacity of murineEAγ rosetting systems. The subclass specificities of the IgG‐ and the PC‐binding proteins were for lgG2aand lgG2b, respectively. The PC‐binding proteins possessed a typical phospholipase A2activity, which was maximal at pH 9.5, depended on Ca++, and was specific for cleavage of fatty acid from thesn‐2position of phosphatidylcholine. The bindiong of aggregated lgG2bto the PC‐binding proteins caused the ninefold increase in noted enzymatic activity in the presence of, but not in the absence of, Ca+ +(5mM). The IgG‐binding proteins on the other hand, lacked any detectable phospholipase A2activity. Thus, the biochemical and biological properties of the PC‐ and IgG‐binding proteins isolated from murine peritoneal macrophages are essentially identical to those homologous proteins previously isolated from P388D1cells [20].

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.493

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

The interaction of elicited murine polymorphonuclear neutrophils (PMN) and the thermally dimorphic fungal pathogen Blastomyces dermatitidis in vitro was studied. The PMN elicited intraperitonealy with thioglycollate, in normal mice or mice immune to B dermatitidis, failed to reduce colony forming units (CFU) of B dermatitidis in the inoculum in a 4‐hr in vitro assay, even in the presence of 10% fresh immune serum. In contrast, PMN elicited intraperitonealy in immune mice by injection of nonviable B dermatitidis cells significantly reduced inoculum CFU (60 ± 5%) under the same conditions. Furthermore, nonviable B dermatitidis intraperitoneally (i.p.) in normal mice or nonviable Candida albicans i.p. in immune mice failed to elicit peritoneal exudate cells that reduced inoculum CFU in this system. These results support the concept that PMN, elicited in a site by means of an immunological reaction, acquired enhanced microbicidal activity. The fungicidal activity of immunologically elicited PMN was shown to be most effective at high effector to target cell ratios (1,000:1), maximal within 2 hr of coculture, and significantly enhanced in the presence of fresh immune serum compared to heat‐inactivated immune serum, normal mouse serum, or fetal bovine serum. Such PMN also had significantly enhanced fungicidal activity against C albicans compared to normal PMN. Fungicidal activity was abrogated in the presence of catalase, implicating hydrogen peroxide generation as the killing mechanism in the activated cells.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.505

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

The ability to use highly purified cryopreserved human monocytes in various in vitro assays has a number of practical and theoretical advantages, including convenience and the potential for enhanced reproducibility. Large numbers (up to 1 × 109) human monocytes can be isolated from a single donor in a purified suspension state, by a combination of leukapheresis and counter‐ current centrifugal elutriation (CCE) technologies. Following short‐ and long‐ term periods of cryopreservation, the viability and phagocytic function of these CCE‐purified monocytes was unimpaired. Cryopreserved monocytes were similar to fresh cells in their ability to release superoxide anion (O2), although unstimulated and stimulated Of release values tended to increase slightly following weeks to months of cryopreservation. In contrast, even short‐term cryopreservation diminished both the random migration and chemotactic responses of human monocytes; however, cryopreserved monocytes could be employed in this assay provided the calculated chemotactic ratio (chemotactic migration/spontaneous migration) was used. Cryopreserved monocytes demonstrate 70% of fresh monocyte accessory cell function in pokeweed mitogen‐ induced lymphocyte proliferation assays. When the binding of OKT3, OKM1, anti‐DR, and fluoresceinated pokeweed mitogen to monocytes was analyzed in the fluorescence activator cell sorter (FACS), cryopreserved and fresh monocytes displayed a similar pattern of membrane reactivity.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.521

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

Previous reports from this laboratory have revealed that macrophages obtained from 7‐day Listeria‐immune mice elicited 15 h before harvest with heat‐ killed homologous microorganisms were able to kill Listeria monocytogenes while resident or elicited cells were not [14,16]. In the present study, experiments were conducted to determine if phagocytosis‐associated oxidative metabolic activity participates in the enhanced destruction of Listeria by activated macrophages. Investigations into production of oxygen radicals by zymosan‐ stimulated macrophages revealed that Listeria‐immune antigen‐elicited (LIAE) cells produced significantly more superoxide and hydrogen peroxide than did resident, thioglycolate, or Listeria antigen‐elicited macrophages. Additionally, the percentage of nitroblue tetrazolium (NBT) dye postitive cells following exposure to zymosan was maximal in the immune‐elicited population. Utilizing a luminol‐dependent assay, a short‐term chemiluminescent (CL) burst was noted in phagocytizing macrophages. This response was greatest in the LIAE population that exhibited a tenfold increase in peak chemiluminescence over other cell types. Prolonged in vitro culture of these cells diminishes their capacity to generate oxygen radicals. Additionally, LIAE macrophages cultured in excess of 38 h exhibited a significant decrease in zymosan‐stimulated hydrogen peroxide release while the decline in superoxide generation was minimal. A substantial diminution in the Listeria‐stimulated CL response was also noted during this time period. However, phagocytosis of Listeria by LIAE cells failed to induce the level of oxygen metabolites seen when zymosan was used as the particulate stimulant. In addition, scavengers of oxygen radicals were found to be relatively ineffective in reducing the killing of L monocytogenes by immunologically activated macrophages in culture. It therefore appears that toxic oxygen species do not play a major role in the heightened killing of Listeria by activated macrophages.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.533

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

A series of experiments show the potency of a newly described microbicidal system, involving iron, H2O2, and halide, in killing a fungus (Blastomyces dermatitidis). B dermatitidis has previously been shown susceptible to the myeloperoxidase‐H2O2halide system. The present studies show killing of either of two strains in 1 hour if Fe+ +at 5 × 10−5M, H2O2at 5 × 10−M and Kl at 5 × 10−4M are all present (P<0.001). EDTA, a Fe++chelator, abrogates killing. The mechanism presumably utilizes hydroxyl radical, since an inhibitor, ethanol, also neutralizes the system. The bactericidal and fungicidal system is of great potential importance in vivo.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.545

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

Endemic Chediak‐Higashi Syndrome occurs in a restricted geographic area (Pregonero, State of Táchira, Venezuela). Neutrophils from these patients were unable to digest Candida albicans in vitro, but showed normal or increased metabolic activities. This finding supports the view that the endemic syndrome is bona fide Chediak‐Higashi Sydrome.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.549

出版商:Wiley    

年代:1984

数据来源: WILEY

摘要:

Interleukin 1 (IL‐1) is generally regarded as a major regulator of T lymphocyte proliferation. Macrophages from animals and cloned tumor cell lines have been shown to produce this monokine in response to a variety of stimuli. The ability of human monocytes and macrophages to generate IL‐1 is much less well characterized. We previously demonstrated that human monocytes cultured for 1–6 days transformed to macrophages but retained their capacity to support concanavalin A‐driven T cell proliferation. However, cultured macrophage capacity to support antigen‐driven T cell proliferation began to decline after 3 days of culture and was markedly deficient by 6 days of culture. To determine if this loss of accessory cell function was due to the inability to secrete IL‐1, we measured the monokine produced by normal fresh human monocytes and macrophages cultured in vitro from monocytes. IL‐1 was assayed by the mouse thymocyte proliferation method. Fresh monocytes secreted IL‐1 readily in response to lipopolysaccaride and latex particles. Macrophages cultured from fresh monocytes, however, lost this ability after ≥2 days in culture. Mixing experiments failed to demonstrate an inhibitor present in the macrophage supernatants that would suppress thymocyte proliferation. Stimulated T cells incubated with monocytes and 3‐day cultured macrophages failed to prolong or promote IL‐1 secretion.

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.551

出版商:Wiley    

年代:1984

数据来源: WILEY

ISSN:0741-5400    

DOI:10.1002/jlb.36.4.559

出版商:Wiley    

年代:1984

数据来源: WILEY