A carbohydrate neoepitope that is up-regulated on human mononuclear leucocytes by neuraminidase treatment or by cellular activation.

The expression of cell-surface antigens can delineate specific leucocyte developmental or functional stages. For example, certain membrane glycoproteins are expressed selectively on leucocyte subsets only after activation. Leucocyte activation can also induce changes in carbohydrate epitopes expressed on surface antigens. In the present studies, we report on a novel monoclonal immunoglobulin M antibody (mAb 13.22) that recognizes a unique carbohydrate epitope expressed on human leucocyte membrane proteins. Characterization of mAb 13.22 specificity by immunoblotting showed that it recognized proteins of MW approximately 95 000 and 150 000, including both CD18 and CD11b. The mAb 13.22 epitope was removed by N-glycosidase F but not by endoglycosidase H or fucosidase, demonstrating that it is an N-linked carbohydrate antigen. Interestingly, immunoblot staining was enhanced after neuraminidase treatment, suggesting that the antibody epitope might also be partially masked by sialic acid. In resting leucocytes, the mAb 13.22 antigen was expressed strongly on neutrophils, while dull staining was present on monocytes, and no lymphocyte staining was observed. In marked contrast, treatment of leucocytes with neuraminidase resulted in exposure of a mAb 13.22 neoepitope on a subset of lymphocytes (primarily T lymphocytes and natural killer cells) as well as up-regulated staining more than 18-fold on monocytes. Activation of lymphocytes in culture with phytohaemagglutinin or concanavalin A also unmasked the mAb 13.22 neoepitope on approximately 37% of the CD45RO+ lymphocytes. Furthermore, analysis of leucocytes collected from the synovial fluid of patients with rheumatoid arthritis showed that approximately 18% of the lymphocytes present expressed the mAb 13.22 neoepitope. Taken together, our results suggest that the mAb 13.22 carbohydrate neoepitope could represent a physiologically relevant marker that is up-regulated on leucocyte subsets during the inflammatory response.

Cell-surface lactoferrin as a marker for degranulation of specific granules in bovine neutrophils.

OBJECTIVE: To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils. SAMPLE POPULATION: Blood samples obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8). RESULTS: Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF. CONCLUSION AND CLINICAL RELEVANCE: Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood.

Priming of human neutrophils by peroxynitrite: potential role in enhancement of the local inflammatory response.

Peroxynitrite is a potent oxidant generated from the reaction of nitric oxide (NO) and superoxide anion (O2-), both of which can be produced in inflammatory tissues. In these studies, we analyzed what direct effect peroxynitrite had on neutrophil (PMN) function. We found that peroxynitrite was an effective priming agent for PMNs, as demonstrated by enhanced O2- production on subsequent activation with low doses of PMA or N-formyl-methionine-leucine-phenylalanine (fMLF), changes in the expression of PMN surface markers (L-selectin, Mac-1, flavocytochrome b, and fMLF receptor), and increased intracellular calcium levels. Analysis of the mechanism of PMN priming by peroxynitrite demonstrated that peroxynitrite resulted in minimal oxidation of protein sulfhydryl groups and subsequent protein cross-linking. In contrast, treatment of PMNs with peroxynitrite resulted in significant nitration of tyrosine residues on neutrophil proteins. In addition, inhibition of tyrosine nitration with a pyrrolopyrimidine antioxidant blocked the majority of peroxynitrite-induced priming effects, further suggesting that PMN priming was mediated primarily by nitration of tyrosine residues on PMN proteins. The ability of peroxynitrite to serve as an effective priming agent for PMNs at sites of inflammation may play a key role in modulating the host-defense process.

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils.

We characterized the dose response of bovine neutrophils to platelet-activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10(-10) and 10(-9) M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (> or = 10(-7) M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10(-9) M) caused secretory granule degranulation, moderate doses (> or = 10(-7) M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10(-5) M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations > or = 10(-7) M also caused up-regulation of the adhesion molecules Mac-l and L-selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10(-6) M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10(-5) M) caused an increased rate of PMA-stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA-induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up-regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus.

Assembly of the human neutrophil NADPH oxidase involves binding of p67phox and flavocytochrome b to a common functional domain in p47phox.

The human neutrophil NADPH oxidase is a multi-component complex composed of membrane-bound and cytosolic proteins. During activation, cytosolic proteins p47(phox), p67(phox), Rac2, and possibly p40(phox) translocate to the plasma membrane and associate with flavocytochrome b to form the active superoxide-generating system. To further investigate the role of p67(phox) in this complex assembly process, experiments were performed to identify possible regions of interaction between p67(phox) and other NADPH oxidase proteins. Using random sequence peptide phage-display library analysis of p67(phox), we identified a novel region in p47(phox) encompassing residues 323-332 and a previously identified SH3 binding domain encompassing p47(phox) residues 361-370 as p67(phox) binding sites. Synthetic peptides mimicking p47(phox) residues 323-332 inhibited the p47(phox)-p67(phox) binding interaction in an affinity binding assay; however, peptides mimicking flanking regions were inactive. Surprisingly, this same region of p47(phox) was found previously to represent a site of binding interaction for flavocytochrome b (DeLeo, F. R., Nauseef, W. M., Jesaitis, A. J., Burritt, J. B., Clark, R. A., and Quinn, M. T.(1995) J. Biol. Chem. 270, 26246-26251), and this observation was confirmed in the present report using two different in vitro assays that were not evaluated previously. Using affinity binding assays, we also found that p67(phox) and flavocytochrome b competed for binding to p47(phox)after activation, suggesting that prior to full NADPH oxidase assembly the 323-332 region of p47(phox) is associated with p67(phox) and at some point in the activation process is transferred to flavocytochrome b. Thus, taken together our data demonstrate that both p67(phox) and flavocytochrome b utilize a common binding site in p47(phox), presumably at distinct stages during the activation process, and this p47(phox) region plays a key role in regulating NADPH oxidase assembly.

Ly-6C is a monocyte/macrophage and endothelial cell differentiation antigen regulated by interferon-gamma.

Using a new Ly-6C-specific antibody (Monts-1) we show that this class of antigens are differentially expressed on monocytes/macrophages and endothelial cells. Recently elicited peritoneal exudate Mac-1+ mononuclear cells, as well as Mac-1+ mononuclear cells in the bone marrow and in the peripheral blood, express high levels of Ly-6C. Ly-6C+ mononuclear Mac-1+ cells are absent in normal uninflamed skin, but are present in high numbers in skin lesions 3 days after the s.c. injection of lipopolysaccharide, concanavalin A or complete Freund's adjuvant. In addition, large Ly-6C+ mononuclear cells are predominant in chronic granulomas induced by complete Freund's adjuvant. Resident macrophages in a variety of tissues express low levels or in many cases do not express Ly-6C. Two out of three monocyte-like cell lines are Ly-6C+, whereas macrophage-like cell lines are negative. Ly-6C+ monocytes/macrophages lose the Ly-6C antigen within 24 h after in vitro culture. Ly-6C- cultured monocytes and Ly-6C- monocyte-like cell lines, but not fully differentiated macrophages and macrophage-like cell lines, can be induced to express the Ly-6C antigen by interferon-gamma. A population of small vessel endothelial cells in diverse tissues also express high levels of Ly-6C. The present findings suggest that the Ly-6C antigen family, shown by others to be involved in T cell activation, may have more general importance in immune responses and cellular differentiation than previously appreciated.