No abstract

We have analyzed peripheral blood mononuclear cell preparations from a rat model of combined injury (CI) [whole-body irradiation (500 cGy 60Co) followed by a thermal injury (20% body surface area, dorsal, scald burn)] for the expression of OX8 antigens. Ficoll-separated mononuclear fractions were labeled with monoclonal antibodies MRC OX8, MRC OX19, W3/13 HLK, or W3/25 for flow cytometric analysis. Combined-injury trauma resulted in decreased mononuclear cells to 6% of normal. This effect was due to the rapid decrease in radiosensitive lymphocytes from 83% to 10%. The relative numbers of monocytes increased from a normal 13% to 70% at day 4 after CI. Labeling of cells with OX8 after CI shifted to a population which was significantly larger in volume than normal lymphocytes. At the same time the mean fluorescence intensity of OX8-positive cells was considerably reduced. With the use of a F(ab) fragment of OX8 as a probe, these results could be partially explained as unspecific binding of the whole molecule of OX8 to Fc receptors expressed by activated monocytes. But double-labeling and cell-sorting experiments also revealed the expression of OX8 antigens by a subset of OX8+/OX19- monocytes after CI.

No abstract

Analysis of cellular effector function(s) often requires their isolation from other cellular types. Cell separatory techniques could mask, or select out, clinically important functional lesions. We examined differences in canine peripheral blood neutrophil functions, i.e. migration and H202 production, following two commonly used cell separation techniques: isotonic lysis or density gradient (Percoll) centrifugation. Separation methodology was observed to have a significant impact on both metabolic and mobility functions. In comparison to isotonic lysis, Percoll separation caused near 100% increases in random migration, near 40% decreases in chemotaxis and 70% increases in H202 production.

Neutrophils were isolated from the peripheral blood of clinically normal canines by hypotonic lysis or density gradient (Percoll) centrifugation techniques. As a function of preparative technique, separated neutrophils were examined in vitro for alterations in membrane lipid integrity, and both granular and cytosolic specific enzymes. Membrane lipid disorder was assessed by merocyanine 540 (MC540), a fluorescent bioprobe, which intercalates into disrupted cellular lipid membranes. Evidence of membrane lipid disorder was based upon comparisons of mean cellular fluorescence exhibited by unstimulated cells. Based upon comparisons of mean cellular fluorescence the isolation methodologies did not appear significantly different. Significant levels of membrane disruption, due to preparative technique, were evident upon neutrophil stimulation with phorbol myristate acetate (PMA). Neutrophils which were Percoll-separated and PMA-stimulated demonstrated significant levels of membrane disruption not apparent in lysis-separated stimulated aliquots of cells. Cellular isolation methods did not significantly alter cellular myeloperoxidase (MPO) levels based on comparisons of cellular totals. Extracellular lactate dehydrogenase (LDH) levels of lysis-separated cells were four-times those of Percoll-separated neutrophils.