Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells.

To improve the accuracy of predicting membrane protein sorting signals, we developed a general methodology for defining trafficking signal consensus sequences in the environment of the living cell. Our approach uses retroviral gene transfer to create combinatorial expression libraries of trafficking signal variants in mammalian cells, flow cytometry to sort cells based on trafficking phenotype, and quantitative trafficking assays to measure the efficacy of individual signals. Using this strategy to analyze arginine- and lysine-based endoplasmic reticulum localization signals, we demonstrate that small changes in the local sequence context dramatically alter signal strength, generating a broad spectrum of trafficking phenotypes. Finally, using sequences from our screen, we found that the potency of di-lysine, but not di-arginine, mediated endoplasmic reticulum localization was correlated with the strength of interaction with alpha-COP.

Characterization of receptors using cyanine 3-labeled neuropeptides.

We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

Leumedin NPC 15669 inhibits antigen-induced recruitment of inflammatory cells into the canine airways.

We studied the effects of NPC 15669, a member of a new class of anti-inflammatory drugs called leumedins, on chemotaxis of both human eosinophils and neutrophils and on Mac-1 receptor upregulation on stimulated eosinophils in vitro. Then, we examined the effect of NPC 15669 on antigen-induced eosinophil and neutrophil recruitment and subsequent airway hypersecretion (indicated by an increase in lysozyme concentration) in the allergic dog trachea in vivo. NPC 15669 inhibited eosinophil chemotaxis in vitro at a drug concentration of 10(-5) M (mean inhibition, 48.2%) without affecting Mac-1 receptor upregulation on stimulated eosinophils. NPC 15669 also inhibited neutrophil chemotaxis: at 10(-5) M, NPC 15669 inhibited neutrophil chemotaxis by a mean of 29.7%. In allergic dogs in vivo, NPC 15669 (10(-5) M) prevented antigen-induced recruitment of eosinophils and neutrophils and prevented the increase in elastase and lysozyme concentrations. We conclude that NPC 15669 is an effective inhibitor of antigen-induced leukocyte recruitment and elastase release and subsequent hypersecretion in airways.

Platelet microbicidal protein alone and in combination with antibiotics reduces Staphylococcus aureus adherence to platelets in vitro.

Bacterial adherence to platelets on the cardiac valve surface is believed to be critical in the induction of infective endocarditis. Recent studies have confirmed that thrombin-activated platelets secrete platelet microbicidal protein (PMP), which can both kill and exert nonlethal antiadherence effects against endovascular pathogens. In the present study, we quantified the influence of antibiotic and/or PMP exposures on in vitro platelet adherence of two Staphylococcus aureus strains, identical by DNA restriction and cell wall protein profiles, that differed in their susceptibility to PMP-induced killing (PMPs or PMPr, respectively). Adherence assays were performed by flow cytometry in the presence of sublethal PMP concentrations (1 to 2.5 micrograms/ml) alone or in combination with ampicillin (AMP) alone, sulbactam (SUL) alone, or AMP plus SUL (AMP-SUL), at levels achievable in serum. Exposure of the PMPs and PMPr S. aureus strains to antibiotics (for 2 h at 37 degrees C) prior to flow cytometry resulted in no substantive changes in the percent adherence to platelets compared with that for S. aureus cells not exposed to antibiotics, except for modestly increased adherence of both PMPs and PMPr cells exposed to AMP-SUL (18.5 and 15.8% increases, respectively). Addition of PMP to antibiotic-S. aureus mixtures (final 30 min) caused a significant decrease in S. aureus adherence to platelets, for both the PMPs and PMPr S. aureus strains, compared with antibiotic exposure alone (e.g., reduction in platelet adherence from 57.9 +/- 8.2% to 12.2 +/- 3.6% for PMPs cells exposed to AMP-SUL and PMP [P = 0.01]). Moreover, addition of PMP following exposure of the PMPs and PMPr strains to AMP-SUL reversed the enhanced bacterium-platelet adherence observed with such antibiotic exposures alone (P < or = 0.005). These data demonstrate that PMP exerts a potent antiplatelet adherence effect which is independent of its microbicidal capacity, rendering S. aureus cells less adherent to platelets in the presence or absence of antibiotics. Reduction of microbial adherence to platelets by PMP alone or with antibiotics provides further insight into the mechanism(s) that may be involved in host defense and antibiotic prophylaxis of infective endocarditis and other endovascular infections.

Fluconazole and platelet microbicidal protein inhibit Candida adherence to platelets in vitro.

Adherence to vascular endothelium is considered an essential step in the pathogenesis of hematogenously disseminated candidiasis. Platelets have been shown to promote Candida adherence to vascular endothelium in vitro. In contrast, recent studies indicate that platelets may also play a role in the primary host defense against endovascular infection by secretion of alpha granule-derived platelet microbicidal protein (PMP), which possesses both bactericidal and fungicidal activities as well as antiadherence properties. We examined the influences of PMP and the antifungal agent fluconazole on the adherence of Candida albicans to rabbit platelets, as measured by quantitative flow cytometry. In the absence of PMP and fluconazole, adherence of C. albicans to platelets was rapid (complete within 1 min), saturable, and reversible. Following 2 h of exposure to fluconazole at 10x the MIC, platelet binding of C. albicans was substantially reduced (mean reduction, 32.1%; P = 0.08). Similarly, exposure of C. albicans to PMP (range, 0.5 to 5 micrograms/ml) for 2 h (but not 30 min) significantly reduced candidal adherence to platelets 43.1 to 62.1%; (reduction range, P < 0.05). Moreover, exposure of C. albicans to PMP (5 micrograms/ml for 30 min) and then fluconazole (10x the MIC for 2 h) further decreased candidal adherence to platelets in comparison with the adherence after exposure to either agent alone (mean reduction, 57.2%; P = 0.02 and 0.05, respectively). These data demonstrate that PMP and fluconazole individually reduce the ability of C. albicans to bind to platelets in vitro and that the antiadherence activities of fluconazole are augmented by PMP.

Inhibition of platelet binding and aggregation by streptococcal exopolysaccharide.

Streptococcal exopolysaccharides are major virulence factors in the pathogenesis of endocarditis. They promote bacterial adherence to valves and subsequent vegetation formation. Since platelet binding and aggregation by streptococci are postulated mechanisms for endocardial colonization and vegetation production, the effect of exopolysaccharide on binding and aggregation was evaluated by flow cytometry and aggregometry. Streptococcus salivarius D1, a minimal exopolysaccharide producer, bound human platelets extensively (86.8% of bacteria bound by 1 min). S. Salivarius M13 and M15 and Streptococcus mitis M4 produced larger amounts of exopolysaccharide and bound platelets significantly less (52.6%, 51.2%, 52.8%, respectively). Exopolysaccharide also inhibited platelet aggregation: Strains with minimal exopolysaccharide aggregated platelets maximally, while strains with extensive exopolysaccharide failed to induce aggregation. Removal of exopolysaccharide by shearing restored aggregation by these latter strains. Thus, exopolysaccharides can inhibit the binding and aggregation of platelets by streptococci. The virulence associated with exopolysaccharide may result from the inhibition of platelet-mediated interactions that limit disease progression.

Characterization of Staphylococcus aureus-platelet binding by quantitative flow cytometric analysis.

Quantitative analyses of Staphylococcus aureus binding to platelets were done using flow cytometry after bacterial exposure to the following treatments: proteases (trypsin, protease K), antibiotics (oxacillin, gentamicin), surface carbohydrate modifiers (sodium periodate, anticapsular antibody), or platelet microbicidal protein. In separate studies, platelets were exposed to a monoclonal antibody to their Fc receptor (Fc gamma RII) before binding was quantified. The percentage of bacteria bound to platelets varied significantly among strains (22.1% +/- 3.8% to 76.4 +/- 3.2%). For all isolates, binding to platelets was rapid, saturable, and reversible, suggesting a receptor-ligand interaction. The following modifiers significantly reduced binding: platelet microbicidal protein (by 32.1% +/- 5.2%; P less than .001), homologous (but not heterologous) anticapsular antibody (by 17.7% +/- 1.9%; P less than .05), sodium periodate (by 36.3% +/- 4.3%; P less than .005), and anti-platelet Fc monoclonal antibody (by 41.5% +/- 4.4%; P less than .002). Collectively, these data suggest that the mechanism(s) involved in S. aureus-platelet binding are complex and multimodal, involving carbohydrate-rich and platelet microbicidal protein-susceptible S. aureus surface ligands as well as the platelet Fc receptor.

Agonistic activities of histamine-albumin conjugates at histamine H2 receptors on human HL-60 promyelocytic leukemia cells.

We previously identified functional histamine H2 receptors on human HL-60 promyelocytic leukemia cells [J. Biol. Chem. 264: 18356-18362 (1989)]. In the present study, we have compared the action of histamine-albumin conjugates on H2 receptor activation with that of histamine alone. Both histamine and conjugates increased intracellular levels of Ca2+ in an H2 receptor-specific manner. However, binding of fluoresceinated histamine-albumin conjugates to HL-60 cells was not dissociated by 10(-4) M unlabeled histamine, although this concentration of histamine significantly desensitized conjugate responses. These data suggest that histamine-albumin conjugates not only activate H2 receptors but also bind to HL-60 cells nonspecifically or in an H2 receptor-unrelated manner. Moreover, histamine-induced Ca2+ mobilization was transient, whereas conjugate-induced Ca2+ mobilization was sustained for more than 10 min, as a result of the influx of extracellular Ca2+. Therefore, the functional difference between histamine and conjugates may provide a good model for the further understanding of the activation mechanisms of receptor-operated Ca2+ influx.

Binding of viridans group streptococci to human platelets: a quantitative analysis.

The binding of viridans group streptococci with human platelets was analyzed by two-color flow cytometry. Binding was detected within 15 s of mixing bacteria and platelets. At ratios of bacteria to platelets of 1:1, 10:1, 100:1, and 1,000:1, the percentages of bound streptococci (mean +/- standard deviation) were 93.2% +/- 5.4%, 80.0% +/- 8.6%, 39.8% +/- 11.1%, and 12.5% +/- 2.0%, respectively. Binding of labeled bacteria was reversed by adding a 500-fold excess of unlabeled streptococci. These results demonstrate that streptococcus-platelet binding is rapid, reversible, and saturable, which suggests a specific receptor-ligand interaction.

At least two non-antigen-binding molecules are required for signal transduction by the T-cell antigen receptor.

In the T-cell somatic mutant J.CaM1, the T-cell antigen receptor complex is poorly coupled to the inositolphospholipid second messenger system; some antibodies against the invariant CD3 subunit of the receptor retain their agonist function in J.CaM1. Here we show by a combination of complementation assays that the mutation in J.CaM1 affects a molecule other than the antigen-binding Ti subunit, suggesting that Ti is coupled indirectly to the signal transduction apparatus through a pathway involving the CD3 complex. We also describe another mutant, J.CaM2, in which the receptor complex is completely uncoupled from inositolphospholipid hydrolysis. J.CaM2 defines an additional complementation group, suggesting that signal transduction by the antigen receptor depends on at least two molecules distinct from Ti.

Functional competency of T cell antigen receptors in human thymus.

The T cell antigen receptor is likely to play a role in both positive and negative selection in the thymus. Three populations of thymocytes can be distinguished by the level of expression of the CD3-alpha/beta-chain heterodimer of the T cell antigen receptor (CD3/Ti alpha/beta) complex. Cells which fail to express these receptors or express low levels of receptors are contained in a population of thymocytes which express low levels of the CD5 antigen and are predominantly CD4+/CD8+. Thus, these cells appear to be relatively immature phenotypically. In contrast, the cells which express high levels of CD3/Ti alpha/beta co-express high levels of CD5 and are predominantly contained in the more mature single positive cells which express either CD4 or CD8. With the calcium-sensitive dye, Indo-1, and immunofluorescence, we demonstrated that, despite the relative phenotypic immaturity of cells which express low levels of CD3/Ti alpha/beta, these antigen receptors are able to mediate transmembrane signaling when stimulated with CD3 monoclonal antibodies. Although increases in calcium were observed in these CD3/Ti alpha/beta-low expressing cells in response to anti-CD3, no proliferative response was observed, even in the presence of phorbol myristate acetate. Proliferative responses were observed in the more mature cells which express high levels of CD3/Ti alpha/beta. These results suggest that, rather than a defect in the functional capability of the antigen receptor complex to mediate transmembrane signaling events, cellular responses to signals generated by the antigen receptor may differ at various stages of thymocyte development.

Design and application of a versatile triple-laser cell and chromosome sorter.

A high-resolution triple-laser sorter was designed and constructed to provide flexible switchover and high-resolution sorting of cells or chromosomes with any combination of one, two, or three lasers. These features provide a central facility instrument that currently serves multiple users and analyzes different stain combinations with minimal switchover effort between experiments. Improved optics and mounts that focus the three laser beams independently are able to resolve beads and chromosomes better than our previously reported dual-laser sorter. An improved signal collection unit with electronically controlled reference positions can be focused more quickly and precisely for any signal combination. A removable dye laser extends the range of usable fluorochrome labels. A rapid sheath switchover permits sorting of sterile cells and sterile chromosomes sequentially without additional sterilization or reservoir sheath change. Improved dual-laser chromosome resolution is at least as good, analyzing 8,000 chromosomes/s, compared to the previous dual-laser bench at 2,000/s. Stimulated and unstimulated peripheral blood lymphocytes were analyzed according to simultaneous measurements of cell surface receptors labeled with a fluoresceinated neuropeptide and a Texas red-labeled antibody as well as DNA content during the cell cycle. These results demonstrate the broad range of potential applications of this triple-laser system.