Faculty Bibliography

One approach to study the inflammatory potential of neutrophils involves in vitro methods, using either adherent or suspended cells. In order to do structure-function studies, traditional methods require that experiments be done in parallel: one experiment for structure and another for function. In this report new morphological methods for coordinated structure-function studies on the same cells are described. Isolation and biochemical analysis of sheep and human adherent neutrophils were done as described in the companion paper (Cerasoli et al., 1988). Then, at designated time points, the adherent cells were fixed and processed in the microtiter wells for high-resolution light microscopy and transmission electron microscopy. The principal obstacle to the morphological studies was chemical etching of the microtiter wells by the processing solutions and embedding media. Insertion of No. 3 BEEM capsule 'sleeves' (the cap and conical tip were removed) into the wells before processing eliminated the obstacle and provided standard-sized, polymerized blocks for microtomes. Adherent neutrophils activated in vitro with 10(-7) M phorbol myristate acetate developed prominent cytoplasmic vacuoles. Furthermore, the activated cells assumed irregular shapes and cytoplasmic processes. These changes in adherent cell morphology in vitro are similar to those seen in neutrophils which have been activated and fixed in vivo. Thus, the in vitro approach we devised retains the morphologic characteristics of cells in vivo and provides an efficient method to do integrated structure-function studies. Using these techniques, we have studied alveolar neutrophils obtained from a patient with acute respiratory failure. These cells contained conspicuous cytoplasmic vacuoles, few granules, and their border was ruffled. The same morphologic changes observed after activation of peripheral blood neutrophils with phorbol myristate acetate in vitro were seen in the alveolar neutrophils obtained from this patient. Therefore, these studies reveal that similar morphologic changes are seen in neutrophils stimulated in vitro as well as cells which have been activated pathophysiologically in vivo

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo