Ticlopidine administration to patients does not inhibit the ability of their monocytes to form rosettes and to phagocytize erythrocytes in vitro

Inhibition of monocyte function is known to have important clinical consequences and to be present in a variety of diseases: Accordingly we evaluated the effect of Ticlopidine, a powerful and widely employed inhibitor of platelet aggregation, on the capacity of peripheral blood monocytes to ingest IgG-coated erythrocytes. Our results show that in vivo administration of this drug has no effect on monocyte phagocytosis, when tested with an in vitro assay. This finding suggests that Ticlopidine can be safely used in patients with depressed monocyte function and in those in whom an impairment of monocyte phagocytosis could lead to a deterioration of the clinical picture.     

Teichoic acids are not required for Streptococcus pneumoniae and Staphylococcus aureus cell walls to trigger the release of tumor necrosis factor by peripheral blood monocytes

In gram-negative bacteria, the outer membrane lipopolysaccharide is the main component triggering cytokine release from peripheral blood mononuclear cells (PBMCs). In gram-positive bacteria, purified walls also induce cytokine release, but stimulation requires 100 times more material. Gram-positive walls are complex megamolecules reassembling distinct structures. Only some of them might be inflammatory, whereas others are not. Teichoic acids (TA) are an important portion (> or =50%) of gram-positive walls. TA directly interact with C3b of complement and the cellular receptor for platelet-activating factor. However, their contribution to wall-induced cytokine-release by PBMCs has not been studied in much detail. In contrast, their membrane-bound lipoteichoic acids (LTA) counterparts were shown to trigger inflammation and synergize with peptidoglycan (PGN) for releasing nitric oxide (NO). This raised the question as to whether TA are also inflammatory. We determined the release of tumor necrosis factor (TNF) by PBMCs exposed to a variety of TA-rich and TA-free wall fragments from Streptococcus pneumoniae and Staphylococcus aureus. TA-rich walls from both organisms induced measurable TNF release at concentrations of 1 microg/ml. Removal of wall-attached TA did not alter this activity. Moreover, purified pneumococcal and staphylococcal TA did not trigger TNF release at concentrations as high as > or =100 microg/ml. In contrast, purified LTA triggered TNF release at 1 microg/ml. PGN-stem peptide oligomers lacking TA or amino-sugars were highly active and triggered TNF release at concentrations as low as 0.01 microg/ml (P. A. Majcherczyk, H. Langen, et al., J. Biol. Chem. 274:12537-12543,1999). Thus, although TA is an important part of gram-positive walls, it did not participate to the TNF-releasing activity of PGN.     

Effects of preincubating human peripheral blood lymphocytes on their ability to bind sheep red blood cells

The effect of preincubating human peripheral lymphocytes at 37°C or 4°C for various lengths of time on their subsequent ability to form rosettes with sheep red blood cells (SRBC) was investigated. Lymphocytes suspended in either medium or medium containing fetal calf serum (FCS) and preincubated at 37°C for 30 min exhibited marked decrease in rosette formation with a return to normal values by 120 min. However, neither lymphocytes suspended in medium and preincubated at 4°C nor lymphocytes suspended in medium containing normal human serum (HS) and preincubated at 37°C exhibited this phenomenon.     

HIV-1 does not alter in vitro and in vivo IL-10 production by human monocytes and macrophages

The present study analyses the ability of HIV-1 to modulate IL-10 production in cells of monocyte-macrophage lineage cultured in the presence of macrophage colony-stimulating factor (M-CSF). Both monocytes and macrophages spontaneously produced low amount of IL-10. Lipopolysaccharide (LPS) induced a strong IL-10 response in fresh monocytes and in M-CSF-treated macrophages. In contrast, macrophages cultured in the absence of M-CSF exhibited a marked decrease in their susceptibility to LPS stimulation. M-CSF increased the IL-10 response of macrophages to LPS by enhancing both the expression of membrane-bound CD14, the protein that serves as LPS receptor, and the sensibility of CD14-expressing cells to LPS stimulation. Neither spontaneous nor LPS-induced expression of IL-10 was modulated in monocytes and macrophages by infection with eight monocytotropic strains, as demonstrated by ELISA and cytofluorimetric analysis. In contrast, all the HIV-1 strains primed macrophages for an increased IL-6 response to LPS stimulation. To determine whether IL-10 production was associated with in vivo infection, monocytes from AIDS individuals were analysed for IL-10 production. We found that neither spontaneous nor LPS-induced IL-10 production were different between healthy controls and HIV-infected patients. Taken together, these data strongly suggest that HIV-1 infection of monocytes-macrophages does not play a significant role in the regulation of IL-10 in infected patients. This study also emphasizes the role of M-CSF activation in the regulation of the cytokine response in macrophages.     

Activation of human peripheral blood monocytes by lipoproteins

Activation of human peripheral blood monocytes could enhance their attachment and or migration into the arterial intima and their various secretory and other functions, thus influencing the pathogenesis of atherosclerosis. In these experiments the authors have explored the role of lipoproteins in the activation of human blood monocytes. Monocytes were purified from citrated blood by Histopaque density gradient centrifugation and countercurrent centrifugal elutriation and cultured in DMEM in the presence of 20% acid-treated autologous serum or 100 micrograms/ml each of VLDL, LDL, Ac-LDL, and HDL. Secretion of beta-glucuronidase activity into the media was measured as a marker of activation. All of the lipoprotein density classes as well as serum stimulated secretion of beta-glucuronidase activity, with LDL and Ac-LDL having a greater influence than serum, VLDL, or HDL. Serum and LDL also stimulated secretion of prostaglandin E into the culture medium. Incubation of monocytes with serum or LDL in the presence of inhibitors of arachidonate metabolism (NDGA and indomethacin) resulted in a significant decrease in secreted and intracellular beta-glucuronidase activity, indicating a role for products of arachidonate metabolism in the activation of monocytes by lipoproteins.     

Flavivirus entry into cultured mosquito cells and human peripheral blood monocytes

Summary The entry modes of Japanese encephalitis (JE) and dengue-2 (DEN-2) viruses into C6/36 mosquito cells and of DEN-2 virus into human peripheral blood monocytes in vitro were studied. Inoculation of either JE or DEN-2 virions into C6/36 cells resulted in direct penetration of the virions into the cytoplasm at the cell surface in 3 stages. At stage 1, virions attached to the plasma membrane of host cells by their envelope spikes; at stage 2, the virion envelopes approximated to and eventually overlapped the host plasma membrane, and in the process the plasma membrane at the attachment sites dissolved; and, at stage 3, virions penetrated into the cytoplasm through the plasma-membrane disruptions created at the adsorption sites. Virions themselves apparently disintegrated at or near the penetration sites, for no virions were seen in the deeper cytoplasm. Coated pits did not form at the virion attachment sites, and virion-containing vesicles were not found in the cytoplasm. In the entry of DEN-2 virus into human peripheral blood monocytes, virions were found, adsorbed onto the external surface of the plasma membrane and attached to the luminal surface of macropinocytic vacuolar membranes. The latter apparently occurred as the result of ruffling and macropinocytic activities of the cells. At both sites virions penetrated into the cytoplasm through the plasma or vacuolar membrane in the same manner as they did through the plasma membrane of C6/36 cells. No evidence of viral entry by receptor-mediated endocytosis was observed. Implications of the entry mode of the mosquito cell-generated DEN-2 virus into human peripheral blood monocytes to an early process of natural, mosquito-transmitted infection is discussed.     

The binding of SRBC by human peripheral blood monocytes

In a population of E-rosette forming human peripheral blood cells, about 20% of the rosettes were formed by monocytes. About 60% of the peripheral blood monocytes bound SRBC in a standard E-rosette test.     

Anaerobic growth of gonococci does not alter their Opa-mediated interactions with human neutrophils.

Gonococci grown anaerobically (anaerobic gonococci) in the presence of nitrite induce the expression of at least three novel outer membrane proteins (PANs 1 to 3). Although PAN 1 is expressed by gonococci during gonorrhea, the function of the PAN proteins remains unknown. In the absence of serum, gonococci possessing opacity-associated (Opa, formerly PII) outer membrane proteins adhere to, stimulate, and are phagocytically killed by human neutrophils. Gonococci lacking Opa proteins demonstrate none of these activities. We investigated whether the PAN proteins, or any other characteristics of anaerobic gonococci, altered the ability of nonpiliated, Opa+ or Opa- gonococci to adhere to, stimulate, or be phagocytically killed by neutrophils. The expression of Opa4 by strain F62, as determined by its relative mobility on sodium dodecyl sulfate-polyacrylamide gels, appeared to be unaltered by anaerobic growth, as seen previously (V. L. Clark, L. A. Campbell, D. A. Palermo, T. M. Evans, and K. W. Klimpel, Infect Immun. 55:1359-1364, 1987). Anaerobic and aerobic Opa+ gonococci adhered to and stimulated neutrophils to the same extent. Similarly, anaerobic and aerobic Opa- gonococci adhered to and stimulated neutrophils equally poorly. Finally, anaerobic and aerobic Opa+ gonococci were equally sensitive to phagocytic killing by neutrophils, while anaerobic and aerobic Opa- gonococci were equally resistant to killing. Thus, the role of Opa proteins in mediating the interactions of gonococci with human neutrophils appears unaltered by anaerobic growth, and the PAN proteins, or other cryptic properties of anaerobic gonococci, do not seem to modulate or mediate these phenomena.     

Clinical isolates of Staphylococcus aureus vary in ability to stimulate cytokine expression in human endothelial cells

Human umbilical vein endothelial cells (HUVEC) were infected for 24 h with 18 well-characterized Staphylococcus aureus isolates, and the supernatants from infected HUVEC were analysed for interleukin (IL)-1beta, tumour necrosis factor-alpha, IL-6, IL-8, IL-10, IL-12p70, growth-related oncogene (GRO)-alpha, granulocyte macrophage colony-stimulating factor (GM-CSF) and regulated upon activation, normal T cell expressed and secreted (RANTES) by immunoassay. All staphylococcal isolates induced the expression of IL-6, IL-8, GRO-alpha, GM-CSF and RANTES. The magnitude of cytokine expression varied between isolates. Staphylococcus aureus inducing high expression of one of these cytokines also showed simultaneous high expression of the other four, indicating a common mechanism for the ability of individual S. aureus to induce expression of these cytokines. No direct correlation between cytokine expression and adhesion of S. aureus to HUVEC was observed, indicating that bacterial properties besides adhesion contribute to the activation of HUVEC.     

Human peripheral blood lymphocyte activation by protein A from Staphylococcus aureus.

Mitogenesis and polyclonal immunoglobulin production in peripheral blood lymphocyte cultures activated with Formalin-fixed or autoclaved protein A-containing Staphylococcus aureus were studied. Direct evidence for a dissociation between cell proliferation and polyclonal immunoglobulin production was found, in that S. aureus was not mitogenic after being autoclaved but retained the ability to stimulate B cells to produce immunoglobulin. Trypsin-treated S. aureus lost its binding site for immunoglobulin G, but its mitogenicity was not altered; thus, the protein A binding site for immunoglobulin G on the bacterial cell wall is not required for the stimulation of lymphocyte proliferation. Our data also show a dissociation between cell proliferation and polyclonal immunoglobulin production induced by protein A coupled to Sepharose CL-4B. These results suggest the presence of three distinct active sites on the protein A molecule: one that binds immunoglobulin G molecules, one that stimulates cell proliferation, and one that stimulates polyclonal immunoglobulin production.     

Serum capacity to neutralize superantigens does not affect the outcome of Staphylococcus aureus bacteremia

Staphylococcal superantigens (SAg) could play an important role in sepsis by activating numerous T cells. We investigated whether serum capacity to neutralize SAgs can be a prognostic factor in Staphylococcus aureus bacteremia (SAB). In a university hospital, 105 consecutive SAB patients were enrolled during a 12-month period. The earliest serum samples prior to SAB onset were stored for a later T cell proliferation assay. Multiplex polymerase chain reaction (PCR) for 19 SAg genes was performed for S. aureus blood isolates. To determine the serum capacity to neutralize SAgs, T cell proliferation by the culture supernatant of each S. aureus isolate was measured in the presence and absence of the corresponding patient’s serum. Twenty-six (24.8%) patients died within 4?weeks from SAB onset. Vascular catheter-related infection was associated with survival for ≥4?weeks. Unknown primary focus, Simplified Acute Physiology Score-II (SAPS-II), and specific SAg genes (tst, sec, sel, or sep) were associated with the 4-week mortality. No variables related to T cell proliferation assay showed statistical significance. In the multivariate analysis, SAPS-II ≥33 and tst were independently associated with the 4-week mortality. Serum capacity to neutralize SAg does not significantly affect SAB outcome. SAPS-II ≥33 and tst are independent predictors of the 4-week mortality.     

The Tween 80 reaction does not correlate to triglyceride lipase production of Staphylococcus aureus

This comparison of Tween 80 reaction with triglyceride lipase production comprises 357 Staphylococcus aureus strains from various clinical sources. Tween 80 is a water-soluble substrate, and thus estimates esterase activity. Lipase production was assayed using water-insoluble substrates (radiolabelled triglyceride emulsions). Only a minor proportion of Tween 80-positive strains had significant triglyceride lipase production (greater than 5 mU/10(9) bacteria). The discrepancy between the two methods was most pronounced in subgroups of samples with low frequency of lipase production, e.g. from isolates from superficial locations such as nasal mucosa and impetigo. Owing to its low specificity, the Tween 80 reaction therefore seems unsuitable as a marker for lipase activity in clinical isolates of Staphylococcus aureus.     

Arachidonic acid, but not its metabolites, is essential for FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes

Since arachidonic acid (AA) production by phospholipase A2 (PLA2) is essential for the Fcgamma receptor (FcgammaR)-mediated respiratory burst and phagocytosis of opsonized erythrocytes by monocytes and macrophages, we focused in this study on the role of AA and its metabolites in the FcgammaR-stimulated intracellular killing of Staphylococcus aureus by human monocytes. The results revealed that the PLA2 inhibitors, but not inhibitors of cyclo-oxygenase and lipoxygenase, markedly suppressed the FcgammaR-mediated killing process. The production of O-2 by monocytes upon FcgammaR cross-linking was inhibited by 4-bromophenacyl bromide in a dose-dependent fashion, indicating that inhibition of PLA2 activity impairs the oxygen-dependent bactericidal mechanisms of monocytes, which could be partially restored by addition of exogenous AA and docosahexaenoic acid, but not myristic acid. These polyunsaturated fatty acids, but not myristic acid, stimulated the intracellular killing of S. aureus by monocytes, although not as effectively as FcgammaR cross-linking. Furthermore, FcgammaR cross-linking stimulated the release of AA from monocytes. Studies with selective inhibitors revealed that the FcgammaR-mediated activation of PLA2 is dependent on Ca2+ and tyrosine kinase activity. Together these results indicate a key role for PLA2/AA, but not its major metabolites, in mediating the FcgammaR-stimulated intracellular killing of S. aureus by monocytes.     

Differential MHC class II expression on human peripheral blood monocytes and dendritic cells

Both monocytes (MO) and dendritic cells (DC) in human peripheral blood are of a plastic-adherent nature. The expression of the MHC class II sublocus products HLA-DP, -DQ and -DR on human peripheral blood transiently adherent cells (TA) was examined by an immunocytochemical staining technique. While most TA showed strong expression of molecules of the HLA-DR subtype, only a small proportion of cells (2-6%) showed strong HLA-DP or -DQ positivity. This strong expression of the HLA-DP and HLA-DQ sublocus products by a subset of TA was seen only after short-term culture; freshly isolated cells expressed comparatively low levels of these molecules. Enrichment for Fc receptor-negative or low-density cells from TA produced populations with strong HLA-DQ and -DP expression. Such co-enrichment of the strongly HLA-DQ+ and strongly HLA-DP+ cells suggests that the same cells express high levels of both types of MHC class II molecule. Immunocytochemical analysis of TA indicated that the strongly HLA-DQ+ cells, at least, were only weakly or non-reactive with the MO-specific monoclonal antibodies OKM1, UCHM1, MO2 and EB11. In addition, strongly HLA-DQ- or -DP-positive cells were poorly phagocytic in comparison with the majority of adherent cells. The apparent FcR-negative, low-density and weakly phagocytic nature of the strongly HLA-DQ/DP+ cells, combined with their lack of reactivity with several MO-specific antibodies, suggests that they may represent the DC component of TA. Such strong HLA-DQ/DP expression by DC may aid their positive identification in human peripheral blood and may be of relevance to DC function in antigen presentation.     

Streptococcus pneumoniae and Staphylococcus aureus surface properties in relation to their adherence to human buccal epithelial cells

Adherence to host cells by pathogenic bacteria is achieved through both specific and non-specific mechanisms. The former involve bacterial adhesin and corresponding cell receptors (Gibbons and Van Houte, 1980), while the second include electric charges and hydrophobicity of bacterial cell walls. In a previous study (Beck et al., 1988), we showed that these two cell surface characteristics vary during growth of Staphylococcus aureus in a manner which should promote adherence to host cells. The aims of the current study were to assess: (1) whether the same growth-related variations in surface properties were present in another bacterial species, Streptococcus pneumoniae; (2) whether the adherence of the two types of bacteria to epithelial cells was in fact different at different growth times; and (3) whether such differences were consistent with the observed surface properties.     

Morphological characteristics and immunophenotype of dendritic cells generated from monocytes of human peripheral blood

Morphological features and immunophenotype of mature dendritic cells (DC) generated from the monocytes of healthy donor blood after culture with GM-CSF and IL-4 with the addition of TNF, were studied using light and electron microscopy, as well as flow-cytometry. It was shown that DC were characterized by a number of morphological features such as: large size, eccentrically located nucleus, highly developed system of extensions, large vacuolated cytoplasm, prominent and activated Golgi complex and ribosomal apparatus. Mature DC are characterized by active surface expression of MHC and co-stimulatory molecules (MHC I, MHC II, CD40, CD80, CD86).     

Spontaneous cytotoxicity by human peripheral blood monocytes: inhibition by monosaccharides and oligosaccharides

The mechanism by which nonimmune cytotoxic effector cells recognize "foreign" targets for cytotoxic attack was investigated utilizing a model system in which cultured human monocytes become cytotoxic to a broad variety of xenogeneic erythrocyte target cells. Such spontaneously cytotoxic human monocytes lyse targets such as chicken (CRBC), horse (HRBC), and rat (RRBC) erythrocytes rapidly and without the addition of exogeneous lectin or antibody. It was found that a variety of simple sugars were capable of blocking the expression of cytotoxicity by precultured human monocytes, and that different oligosaccharides blocked the killing of different targets. For example, cellobiose, a beta 1-4 dimer of D (+) glucose, blocked, CRBC and HRBC lysis in vitro, but had no effect on RRBC lysis. Arabinogalactan (a complex polysaccharide with a galactose beta 1-3 galactose backbone and galactose beta 1-6 galactose side chains with terminal arabinoses), however, blocked HRBC killing, but exhibited only minor inhibition of CRBC killing. Other aspects of cell-mediated function, including lymphocyte transformation to PHA, cell viability as assessed by trypan blue exclusion, monocyte phagocytosis of opsonized CRBC's, and PHA-induced cellular cytotoxicity of CRBC targets, were essentially intact in the presence of identical concentrations of oligosaccharides. Such target-specific inhibition is consistent with the hypothesis that cytotoxic human monocytes recognize various targets through surface receptors which interact with specific monosacchardies, disaccharide, and oligosaccharide sequences present on the target surface.     

明胶法富集外周血单核细胞并刺激其成熟为树突状细胞

背景：如何简易高效分离纯化人外周血单核细胞并刺激成熟为树突状细胞,未见标准化操作流程。 目的：观察明胶法分离外周血单核细胞的效率以及将分离出的单核细胞刺激成熟为树突状细胞的表型特征,并与普通塑料黏附法对比。 方法：使用人淋巴细胞分离液分离人外周血得到单个核细胞,根据培养瓶是否进行明胶包被分为明胶包被组和普通塑料组。均分单个核细胞,按组别分离获得单核细胞并诱导刺激成熟为树突状细胞。计数各组所得单核细胞数,使用流式细胞仪检测2组单核细胞的CD14阳性率、T、B淋巴细胞污染率、树突状细胞非成熟期和成熟期CD1a,CD83的表达情况,锥虫蓝拒染法计算细胞活率,观察对比2组血小板污染情况。 结果与结论：明胶包被组单核细胞数及CD14阳性率显著高于普通塑料组(P < 0.05),普通塑料组淋巴细胞污染率显著高于明胶包被组(P < 0.05)。2组细胞活率及树突状细胞表型差异无显著性意义(P > 0.05)。明胶包被组血小板污染率低于普通塑料组。提示明胶法可以简单高效分离出单核细胞并成功刺激成熟为树突状细胞。 关键词：明胶;单核细胞;树突状细胞;人外周血;单个核细胞 doi:10.3969/j.issn.1673-8225.2012.10.037     

Differential capability for phagocytosis of apoptotic and necrotic leukemia cells by human peripheral blood dendritic cell subsets

CD11c+ dendritic cells (DC) and plasmacytoid DC (PDC) are the two major DC subsets in human peripheral blood. For the purpose of immunotherapy with DC, it is important to investigate the phagocytosis of killed tumor cells by different DC subsets. Using immature monocyte-derived DC (iMoDC) as reference, we have compared the ability of CD11c+ DC and PDC to phagocytose apoptotic and necrotic K562 leukemia cells. Freshly isolated CD11c+ DC phagocytosed apoptotic and necrotic K562 cells, whereas PDC did not show any evidence of uptake of dead cells. Blocking studies showed that CD36 is importantly involved in uptake of apoptotic and necrotic material. CD91 and CD11c were also involved. In addition, we found that beta5 integrin was expressed on CD11c+ DC but not in its classical association with alphaV. Uptake of apoptotic K562 cells by CD11c+ DC was increased following incubation with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4, alone or in combination with transforming growth factor-beta1, to levels comparable with those observed for iMoDC. Phagocytosis of dead cellular material by the GM-CSF/IL-4-treated CD11c+ DC was largely restricted to a subset expressing low levels of human leukocyte antigen-DR and CD83. Thus, the relationship between phagocytosis of antigenic material and expression of maturation-related cell-surface molecules is similar for CD11c+ DC and MoDC. We conclude that CD11c+ DC in peripheral blood are precursor cells, which under the influence of cytokines, differentiate to cells with DC phenotype and function.