摘要:

AbstractAn important new method for phagocytosis research, confocal scanning fluorescence light microscopy (CSFM), is demonstrated using fluorescent microspheres ingested by murine macrophages. CSFM, in combination with Nomarski differential interference contrast microscopy (DIC). can resolve microspheres inside cells from microspheres attached to the surface of cells. Further, combined CSFM and DIC images can quantitate phagocytosis by individual cells aggregated together. No other method offers these capabilities. A comparison of CSFM and conventional epifluorescence light microscopy (EFM) images shows that CSFM produces significantly higher‐resolution images of microspheres than EFM, primarily because CSFM excludes the out‐of‐focus light artifacts of EFM.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.277

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractVirus infection of alveolar macrophages both in vivo and in vitro has been associated with a variety of changes in cellular function. Some of these changes are identical to the effects that arachidonate‐derived mediators, prostaglandins, leukotrienes, and hydroxyeicosatetraenoic acids, have on macrophage function. Virus infection of macrophages has been previously shown to increase the output of some arachidonate metabolites, most notably PGE2. However, the effect of virus infection on arachidonate metabolism in general has not been well described. In our experiments, primary cultures of alveolar macrophages obtained from normal cattle by bronchoalveolar lavage, were infected in vitro with parainfluenza type 3 virus. At days 0 to 4 post‐infection (p.i.) these cells were labelled with3H‐arachidonic acid and stimulated with either serum‐coated zymosan, the calcium ionophore A23187, or phorbol myristate acetate. The complete spectrum of arachidonate‐derived metabolites was determined by reverse‐phase high performance liquid chromatography with UV and on‐line radiometric monitoring of column eluant. The total output of metabolites of arachidonic acid by virus‐infected alveolar macrophages was increased over that of noninfected controls (with all stimuli tested) by day 4 p.i. (P≤ 0.05). The production of metabolites by the cyclooxygenase, 12‐and 5‐lipoxygenase enzyme systems was significantly increased, as was the release of3H‐arachidonate. The lack of stimulus specificity and the increases in arachidonate release suggest that greater substrate availability, due either to increased phosphollpase activity or direct virus‐membrane interaction, may be responsible for the virus‐induced enhancement of metabolite output.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.283

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractVirus infection of alveolar macrophages (AM) both in vivo and in vitro has been associated with a decreased ability of these cells to kill bacteria, together with enhanced production of metabolites of arachidonic acid. These metabolites, especially PGE2, may be inhibitory to some phagocyte functions. Primary cultures of bovine AM obtained by bronchoalveolar lavage of normal cattle were infected in vitro with parainfluenza‐3 (PI3 virus) virus. Killing ofStaphylococcus epidermidisby AM was determined on days 1‐4 post‐infection (p.i.) PI3 virus‐infected AM killed significantly fewer bacteria on day 4 p.i. compared to uninfected controls (12.1 ± 1.3% infected vs. 52.7 ± 7.2% controls,P≤ 0.05). Bacterial killing by virus‐infected AM, but not control AM, was significantly enhanced on day 4 p.i. by addition of cyclooxygenase inhibitors 1 hr prior to bactericidal assay (28.0 ± 4.5% indomethacin, 36.0 ± 4.1% mefenamic acid, 38.6 ± 7.3% piroxicam, 37.0 ± 6.4% NDGA, 44.9 ± 7.7% ETYA,P≤ 0.05). Phagocytosis of opsonized sheep erythrocytes and superoxide generation by virus‐infected AM were not significantly increased by cyclooxygenase inhibition. Phagosome‐lysosome fusion was severely impaired in virus‐infected AM. Pretreatment of virus‐infected AM with indomethacin significantly enhanced the percentage of cell expressing fusion activity. This data suggests that in vitro bactericidal dysfunction associated with virus infection of AM is partially the result of enhanced production of prostaglandins or thromboxane by AM and/or an abnormal response to normal levels of endogenously produced cyclooxygenase metabolites. The data further indicate the presence of cyclooxygenase sensitive (phagosome‐lysosome fusion) and insensitive (phagocytic) components of virus‐induced bactericidal dysfunction in AM.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.293

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractLeukocyte function was evaluated in five dogs with Pelger‐Huët (P‐H) anomaly and in five control dogs. No significant differences were found between groups in neutrophil adherence, random movement, chemotaxis, phagocytosis, or bacterial killing ofStaphylococcus intermedius.Neutrophils migrated rapidly into inflammatory sites where progressive nuclear lobulation was noted over time. Antibody titers to exogenous antigens were similar in the P‐H and control groups indicating B‐ and T‐lymphocyte cooperation in humoral immunity. Lymphocyte blastogenesis to phytohemagglutinin also was similar in both groups suggesting the presence of a responsive T‐lymphocyte population. These findings indicate that no apparent predisposition to infection or immunodeficiency exists in dogs with P‐H anomaly.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.301

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractMybridoma cell lines were produced by fusion of SP2/0 murine myeloma cell line with the spleen cells of Wister rats which were immunized with IgG2a‐rbinding protein isolated from the detergent lysate of a murine macrophagelike cell line, P388D1, by affinity chromatography on IgG‐Sepharose 4B. A monoclonal clone (designated as 3A2) out of a total of 13 different antibody‐secreting cell lines was found to secrete IgG1class antibodies, which inhibited more than 70% of the binding of radio‐iodinated myeloma IgG2aprotein to P38SD1cells. The 3A2 Fab fragments bound specifically to P3S8D1cells at 4° with a KDof 1.9 × 10‐8M and Bmax of 2.9 × 105per cell. This Fab fragment also specifically bound to Fcγ 2a receptor (R)‐positive T cell line (S49) with a KDof 4.4 × 10‐9M and a Bmax of 1.0 × 104but did not bind to Fcγ 2a‐negative S49 variant cell line, cyc‐. The flow cytometric analysis with the use of fluorescein‐isothiocyanate‐tagged 3A2 F(ab')2also showed that this antibody binds to Fcγ 2aR‐positive cells, P388D1and S49, but not to Fcγ 2aR‐negatlve cells, cyc‐. Monomeric and heat‐aggregated IgG2a(13‐fold molar excess) inhibited the binding of the radioiodinated 3A2 F(ab')2to P388D1cells by 70 and 49%, respectively, whereas the inhibition by monomelic and heat‐aggregated IgG2bwas 17 and 39%, respectively; 3A2 F(ab')2(100‐fold molar excess) inhibited the binding of IgG2aand IgG2bto P38SD1cells by 90 and 24%, respectively, whereas the inhibition of binding of these IgG to S49 cells was 79 and 49%, respectively. Western blotting analysis showed that 3A2 antibody recognizes a major protein (Mr= 100,000) and a minor component (Mr= 80,000) separated by SDS‐PAGE of P388D1or S49 cell lysates under nonreducing condition, whereas under reducing condition, this antibody recognized a major protein (Mr= 50,000) and two additional minor components (Mr= 40,000 and 35,000). Fcγ 2aR may thus exist at the cell surface as a disulfide linked dimer of a subunit of Mrof 50,000, which could be partially degraded during the isolation to smaller fragments of 40,000 and 35,000 Mrpeptides which are still held together by interchain disulfide bond. Furthermore, 3A2 antibody‐binding protein isolated from P388D1cell membrane lysate was found to exhibit casein kinase activity, which was previously found to be associated with IgG2a‐ but not with lgG2b‐binding protein isolated from a similar cell lysate.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.311

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractMacrophages (MPs) fixed with 1% formalin in PBS bound targets similarly to viable MPs. Like binding between viable MPs and tumor cells, the process was temperature and calcium dependent. Fixed MPs discriminated targets similarly to viable MPs. Targets not bound by viable MPs were not bound by fixed MPs. The lectinBandeiraea simplicifolia(ASI‐B4) was able to enhance MP binding to tumor cells regardless of whether MPs were fixed or viable. However, it did not appear that ASI‐B4–like molecules were involved in the direct recognition of F5b tumor cells by MPs. Target cells could not be fixed in 1% formalin for binding to occur. These data suggest that the receptor on the MP for tumor cell binding is functional in the absence of active physiological processes. In contrast, tumor cell processes that are dependent upon target cell viability are required for binding.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.322

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractThe effects of interleukin 1 (IL‐1) on induction and maintenance of immune tolerance in vitro have been studied by using splenocytes from C57BL/6 male mice. B cell tolerance to the hapten trinitrophenol (TNP) was induced with TNP‐human gamma globulin (HGG) and the cells were challenged with TNP‐Ficoll. To determine the tolerance (or immunity), antibody concentrations in the supernatant fluids were measured by using a TNP‐specific ELISA assay. Partially purified murine IL‐1 abrogated tolerance induction, and when it was added at challenge phase, it also abrogated tolerance. In addition, partially purified IL‐1 converted TNP‐HGG from a tolerogen into an immunogen without any additional exposure to antigen. Similar results were obtained when recombinant human IL‐1 α was used in place of partially purified natural IL‐1. IL‐1 is most likely acting directly on B cells rather than through the agency of T cells because purified B cells failed to become tolerant in the presence of IL‐1. Studies of IL‐1 production by antigen‐ or tolerogenstimulated splenocytes or purified B cells showed that only antigen could elicit IL‐1 production in these cells. That tolerance abrogation is unique to IL‐1 is suggested by studies which show that TNF, IL‐2, and INF γ, alone, in combination with each other, or in combination with subeffective concentrations of IL‐1 failed to effect tolerance abrogation.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.329

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractWe have previously demonstrated that there is a subpopulation of loosely adherent pulmonary mononuclear cells that can be isolated from minced and enzyme‐digested lung tissue with a potent capacity to stimulate allogeneic T lymphocyte proliferation. We now demonstrate that these cells are also capable of stimulating an autologous mixed leukocyte reaction (AMLR) and presenting antigen to autologous T lymphocytes. These loosely adherent mononuclear cells (LAM) were more effective than either alveolar macrophages or monocytes as antigen‐presenting cells. Depletion of phagocytic or Fc receptor‐positive cells from the LAM population enhanced the stimulation of an reaction AMLR while preserving antigen‐induced T lymphocyte proliferation. These results indicate that there are nonphagocytic, Fc receptor‐negative accessory cells in human lung parenchyma capable of activating resting T cells in an AMLR and supporting antigen‐specific T lymphocyte proliferation. The identity of these cells is uncertain, but the data strongly suggest that the cell is not a classical monocyte‐derived macrophage. These antigen‐presenting cells may be critical in the initiation of immune responses within the lung.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.336

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractBone‐marrow‐culture‐derived macrophages killed virally infected cells but not uninfected cells. This activity could be enhanced by preexposure of the macrophages to lipopolysaccharide (LPS), and/or purified interferons. The ability to kill virally infected targets was not restricted to a single cell type or virus. Comparing the ability of activators to induce activity against virally infected targets or tumor (P815) targets, it was found that much lower levels of LPS or α/β‐interferon were able to induce cytolytic activity for virally infected cells than were needed for tumor targets. Further, while the antitumor activity did not change significantly with an increase in the time of exposure to activating stimuli from 4 to 24 h, the activity against virally infected cells decreased dramatically with the longer exposure to stimuli.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.345

出版商:Wiley    

年代:1989

数据来源: WILEY

摘要:

AbstractHuman alveolar macrophages (AMO) have been investigated for their ability to produce three monokines, tumor necrosis factor‐alpha (TNF), macrophage colony stimulating factor (CSF‐1), and interleukin 1‐beta (IL‐1). No TNF activity was found in supematants of unstimulated AMO cultured for 20 h, although TNF mRNA was detected in the cells by Northern blot analysis. Stimulation of the cells with lipopolysaccharide (LPS) Induced production and release of high levels of TNF into the culture supernatant. Increased levels of TNF mRNA were detectable at 90 min after LPS stimulation by dot blot analysis, reaching maximum expression between 4 and 8 h and declining thereafter. TNF activity peaked at approximately 8 h in the AMO supematants. After 24 h TNF production had ended. Compared to autologous monocytes the AMO produced 5.7 times more TNF on a per cell basis (activity accumulated in 20 h supematants). Uncultured AMO expressed CSF‐1 mRNA which was translated into active protein recovered in supematants upon culture in the absence of stimulus. The addition of LPS to AMO slightly reduced both mRNA levels and amount of factor in the supematants. In contrast to the AMO, monocyte production of CSF‐1 was enhanced by LPS. CSF‐1 production by AMO continued for at least 48 h of culture. Spontaneous production of low amounts of IL‐1 was found in one‐third of the AMO samples, while low levels of IL‐1 mRNA were present in all tested preparations. LPS stimulation induced increase in IL‐1 mRNA within 90 min; mRNA levels peaked between 12 and 20 h and stayed high for at least 42 h. However, while all LPS‐stimulated AMO expressed high levels of IL‐1 mRNA biologically active IL‐1 was again detected only in a fraction of the AMO supematants. These results show that the production of monokines CSF‐1, TNF, and IL‐1 is differentially regulated by LPS in alveolar macrophages and has different responses as compared to monocytes. The longevity of the messages for each of the factors is possible indicators of the relative contribution of these factors to the response to endotoxin‐induced injury and repair processes in the lung.

ISSN:0741-5400    

DOI:10.1002/jlb.45.4.353

出版商:Wiley    

年代:1989

数据来源: WILEY