Comparative studies of microscopically determined aggregation, degranulation, and light transmission after chemotactic activation of adult and newborn neutrophils

Abstract

Changes in the light transmission of suspensions of activated neutrophils are widely used to measure the dynamics of neutrophil aggregation. Such studies have suggested, for example, that aggregation is irreversible for human newborn neutrophils but fully reversible for adult cells. We have evaluated aggregation directly by microscopic particle counting and compared it with changes in light transmission (delta T) and with release from three granule subsets for neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP). Maximal increases in %T in response to 0.5 micromol/L FMLP were approximately 25% larger for newborn than for adult neutrophils, and were only partially reversible by 8 minutes, while %T increases for adult neutrophils were fully reversible. However, measurements of neutrophil aggregation using light microscopy showed that both newborn and adult neutrophils fully deaggregated. A further independence of delta T from aggregation was found by pretreating adult neutrophils with cytochalasin B (5 micrograms/mL) in the presence of 0.5% gelatin, a pretreatment that blocked FMLP-induced neutrophil aggregation while allowing large increases in %T and degranulation. In response to FMLP, newborn neutrophils released more enzyme from each granule subset than did adult neutrophils. Our results suggest that cellular events associated with neutrophil activation (other than aggregation) are implicated in light transmission responses and that these differ for adults and newborns. These results also suggest that reports of neutrophil aggregation should be based on direct particle counting methods rather than on %T responses.

The Platelet Extinction Coefficient Measured by Aggregometry Is Dependent on Platelet Composition rather than Size: Implications for Studies of Platelet Heterogeneity and Abnormalities

“…including plat~lets (1, 2, 4-7), erythrocytes (2), monocytes (8) and neutrophils (9,10). The most widely used instrument for measuring changes in extinction or light transmission (% T) for aggregating blood cells is the aggregometer, which has a wide collection angle (y) for the light transmitted through the stirred cell suspension, typically y = 25 ± so and 10° respectively for Payton (2,6,10) and Scienco (9) models. For a given instrument geometry and light wave length, the mean extinction of a suspension of cells (E) will depend on the mean cell v~lume (V), shape and relative refractive index (m) (1)(2)(3).…”

Inhibition of Platelet Aggregation by an SLE-Derived Human Hybridoma Autoantibody Against an Activation-Dependent Antigen

“…Neutrophils were isolated according to the method described by Rochon ct al. (46) and contained less than 10% contaminating platelets, as detected by FACS analysis using antigpllb-llla antibody, AP2.…”