Neutrophil recruitment as a factor limiting injury or promoting recovery from acute lung injury.

We studied 1) neutrophil mobilization in sheep given endotoxin (10 ng.kg-1.min-1, n = 5) for 4 h, 2) surviving (n = 17) and nonsurviving sheep (n = 8) during a 12-h infusion of endotoxin, and 3) adult sheep (n = 8) or lambs (n = 8) infused with endotoxin for 12 h. Bone marrow cells of sheep declined from a baseline value of 10,533 +/- 1,784 to 5,966 +/- 1,980 cells/microliter (P < 0.05) 4 h after endotoxin. After 12 h of endotoxin infusion, circulating neutrophils remained reduced from baseline values of 2,000-4,000 cells/microliter to 343 +/- 70 in lambs and 484 +/- 236 in nonsurviving sheep, while beginning to recover in surviving sheep to 1,838 +/- 467 cells/microliter. In lambs and nonsurviving sheep, a 12-h infusion of endotoxin increased lung lymph protein clearance tenfold compared with a fivefold increase in surviving sheep. Neutrophils cultured from sheep bone marrow exposed to lamb postendotoxin plasma failed to increase in cell number (609 +/- 229 to 610 +/- 182 cells/microliter), whereas similar cells exposed to adult sheep postendotoxic plasma showed a significant increase in cell number (1,069 +/- 101 to 2,293 +/- 448 cells/microliter, P < 0.05). Our results are consistent with the hypothesis that the ability to recruit neutrophils to the circulation during periods of inflammation is important in limiting the severity of acute lung injury.

In vivo labeling of neutrophils using a fluorescent cell linker.

To study neutrophil circulation time and trafficking in vivo requires labeling the cells so that their movement can be followed temporally and spatially. Labeling procedures used to date, however, have relied on ex vivo separation and labeling methods, an undesired consequence of which may be neutrophil activation. Moreover, the labeled cells preferentially stick in the pulmonary circulation for several hours upon reinjection into the host. Therefore, we devised an alternate labeling procedure that relied on intra-arterial infusion of the fluorescent phagocytic cell linker PKH26. We infused PKH26 (5.5 x 10(-6) M for 1 h) into six awake sheep and measured systemic and pulmonary hemodynamics and lung lymph dynamics continuously for 3-4 h after the infusion. We collected simultaneous arterial and venous blood samples hourly for 4 h, and again 24 h after the infusion ended, for white blood cell and neutrophil differential counts and to identify labeled cells in fresh smears by fluorescence and differential interference contrast (DIC) microscopy. Infusion of PKH26 had minimal and transient physiologic effects on systemic and pulmonary artery pressure, lung lymph flow, and leukocyte counts. Labeled cells were present in venous blood after the infusion for at least 24 h. DIC microscopy observation of the blood smears indicated that the fluorescently labeled cells were exclusively neutrophils. Unstimulated superoxide anion release from neutrophils isolated 2 and 24 h after PKH26 infusion was not different from baseline release. Phorbol myristate acetate-stimulated release was not affected either. The labeled neutrophils responded to chemoattractants by migrating to extravascular sites. Our results indicate that neutrophils can be selectively labeled in vivo, after which trafficking of the labeled neutrophils can be determined.

TNF-alpha and the pathophysiology of endotoxin-induced acute respiratory failure in sheep.

We studied changes in cardiovascular and pulmonary function during prolonged endotoxemia in conscious sheep. Sheep with chronic lung lymph fistulas received a 12-h infusion of Escherichia coli endotoxin (10 ng x kg-1 x min-1) and allowed to recover for 12 h. Supportive therapies were withheld. Prolonged endotoxemia without volume support caused systemic hypotension associated with reduced cardiac output and increased systemic vascular resistance, pulmonary hypertension, and acute lung injury with progressive respiratory failure. Plasma tumor necrosis factor-alpha (TNF-alpha) concentrations increased transiently. Sustained pulmonary hypertension and increased pulmonary and systemic vascular resistances contributed to a poor outcome in 9 of 34 sheep (26%). Plasma TNF-alpha concentrations were significantly greater in survivors with sustained pulmonary hypertension and in nonsurviving sheep than in surviving sheep without pulmonary hypertension. Endotoxin (1 ng/ml) increased neutrophil expression of TNF-alpha in vitro. Addition of interleukin-6 prevented this response. Synthesis and release of TNF-alpha may be an important proximal event influencing the development of sustained pulmonary hypertension and progressive respiratory failure with endotoxemia. Interleukin-6 may contribute to the phasic nature of the TNF-alpha response.

An association between lung lymph endothelin concentration and survival during endotoxemia in awake sheep.

Endotoxin stimulates synthesis of endothelin which can cause pulmonary and systemic vasoconstriction and bronchoconstriction. Prolonged endotoxemia in sheep results in dramatic increases in pulmonary and systemic vascular resistances in nonsurvivors compared with survivors. Experiments were conducted in 12 conscious sheep (seven survivors, five nonsurvivors) to determine if synthesis of endothelin might contribute to the pathophysiology in nonsurvivors. Endotoxin was infused at 10 ng/min/kg for 12 h followed by a 4 h recovery in survivors. Lung lymph endothelin concentration peaked at 38.7 +/- 5.8 pg/mL during the endotoxin infusion in survivors compared with a peak of 128.7 +/- 33.0 pg/mL (p < .05) in nonsurvivors. Cardiac output was lower, and systemic and pulmonary vascular resistances and the Aa gradient in PO2 were significantly greater in nonsurvivors compared with survivors. These variables are those likely to be affected by increased circulating endothelin concentrations which suggests that endothelin contributes to early mortality during prolonged endotoxemia.

Microplate reader assay for measurement of opsonized zymosan-stimulated superoxide anion production by neutrophils.

We report a microplate reader assay for the measurement of superoxide anion (O2-) production from opsonized zymosan-stimulated sheep polymorphonuclear leukocytes (PMNs). This method, for use with the particulate neutrophil activator, is a modification of the cytochrome c reduction system that measures O2- release from adherent neutrophils stimulated with soluble mediators such as phorbol-myristate-acetate (PMA). We measured O2- release from PMNs (250,000 per well) stimulated with opsonized zymosan over a dose range from 0.0625 mg/mL to 0.5 mg/mL. A dose of 0.125 mg/mL was shown to stimulate maximal superoxide production while having little effect on the optical density of the reagent blank. O2- values obtained using this method were not statistically different from values measured using cell-free supernatants. This system was useful in demonstrating PMN priming, as evidenced by the 2.7 times increase in O2- production above baseline from neutrophils isolated from sheep given a 12-hour, low-dose endotoxin infusion. This is a convenient and sensitive method for measurement of neutrophil O2- production in response to a receptor-mediated, particulate stimulus.

Endotoxin infusion in anesthetized sheep is associated with intrapulmonary sequestration of leukocytes that immunohistochemically express tumor necrosis factor-alpha.

Plasma levels of tumor necrosis factor-alpha (TNF-alpha) peak between 2 and 4 h during a 12-h continuous infusion of endotoxin in awake sheep. We hypothesized that a source of this TNF-alpha is the pool of leukocytes that accumulate in the pulmonary circulation. To test this hypothesis, we physiologically monitored six anesthetized sheep during baseline and 4-h endotoxin infusion periods (10 ng/kg x min). We obtained open-lung biopsies at baseline and at 20 min and 2 and 4 h during the endotoxin infusion period for immunohistochemical localization of TNF-alpha. The plasma concentration of TNF-alpha increased from an average baseline concentration of 0.06 +/- 0.03 ng/ml (mean +/- SD) to a peak of 1.40 +/- 0.28 ng/ml at 2 h of the endotoxin infusion. We observed increased cytoplasmic TNF-alpha immunoreactivity in situ among neutrophils and intravascular mononuclear phagocytes during the endotoxin infusion compared with baseline. Also, the number of immunopositive leukocytes increased in the pulmonary circulation during the continuous infusion of endotoxin. We conclude that TNF-alpha-producing leukocytes accumulate in the pulmonary circulation during endotoxemia. These cells probably contribute to both the rise in the circulating levels of TNF-alpha and the development of acute lung injury.

In vivo endotoxin exposure increases neutrophil counts without maintenance of primed superoxide anion release in short-term cultures of sheep bone marrow.

A short-term bone marrow culture was established to determine the functional and proliferative responses of neutrophils sampled from sheep exposed to endotoxin in vivo. Iliac crest bone marrow was drawn from surgical control animals and from sheep given a 12-h infusion of endotoxin. In these bone marrow aspirates, neutrophils from endotoxin-treated sheep showed primed superoxide anion release compared with controls when stimulated with phorbol myristate acetate. Priming was no longer present in neutrophils tested after 2 days in culture. The neutrophil cell count, however, was higher after 2 days in culture than in controls. Increased neutrophil production/viability was reproduced in naive bone marrow cultures by replacing part of the normal serum medium with plasma taken from sheep several hours after endotoxin infusion. These data suggest that a dissociation exists between priming and increased neutrophil production/viability in response to endotoxin.

Loss of endothelium-dependent vasodilation in the pulmonary vessels of sheep after prolonged endotoxin.

We examined the hemodynamic response of awake sheep to prolonged endotoxin infusion (10 ng.kg-1 x min-1 for 12 h) and the in vitro endothelium-dependent relaxation of pulmonary arterial vessels excised 12 h after the end of endotoxin infusion to determine whether the development of pulmonary hypertension after endotoxin is associated with loss of endothelium-dependent relaxation. In seven of nine sheep, there was a maintained increase (4-68% of baseline) in pulmonary arterial pressure 24 h after the beginning of endotoxin infusion. The greater the increase in pulmonary arterial pressure in vivo, the greater was the in vitro deficit in endothelium-dependent relaxation of the pulmonary vessels. The maximum in vitro vessel dilation was 59% for pulmonary artery rings isolated from sheep without a sustained increase in pulmonary arterial pressure 24 h after endotoxin. Prolonged endotoxin infusion did not alter the in vitro response of pulmonary arterial vessels to KCl or 10(-5) M norepinephrine. Force development, response to 10(-5) M norepinephrine, and vasodilation in response to acetylcholine were also not altered in pulmonary vessels taken from control sheep and exposed in vitro to tumor necrosis factor-alpha (400 U/ml). Our results suggest that loss of endothelium-dependent relaxation in pulmonary vessels supports the sustained pulmonary hypertension that develops after prolonged exposure to endotoxin.

Physical and cytochemical properties of neutrophils activated in situ in the lung during ZAP infusion in sheep.

Increased retention of activated neutrophils in the lungs contributes to endothelial cell injury. However, characterization of the morphological changes that occur in neutrophils during activation in the pulmonary microcirculation has not been fully determined in vivo. Therefore, the present study was designed to determine structural and cytochemical properties of neutrophils in situ in pulmonary arterioles and alveolar capillaries during the infusion of zymosan-activated plasma (ZAP) or plasma (control) in anesthetized sheep. Quantitative morphological methods showed that ZAP infusion caused significant retention of neutrophils in alveolar capillaries [2.19 +/- 0.40 (SD) x 10(8) neutrophils/ml of capillary blood volume] and pulmonary arterioles (1.02 +/- 0.46 x 10(8) neutrophils/ml of arterial blood volume) compared with plasma infusion (1.03 +/- 0.15 and 0.30 +/- 0.10 x 10(8) neutrophils/ml, respectively; P < 0.05). Harmonic mean diameter of ZAP-activated neutrophils in situ (7.19 +/- 0.44 microns) was significantly greater than the diameter of neutrophils in plasma-treated sheep (6.29 +/- 0.17 microns; P < 0.05). Neutrophil cross-sectional area (54 +/- 3 microns2) and volume (248 +/- 27 microns3) in situ in alveolar capillaries were also significantly greater in ZAP-treated sheep than in control sheep (41 +/- 4 microns2 and 184 +/- 9 microns3, respectively; P < 0.05). Similarly, microvascular neutrophils in ZAP-treated sheep were vacuolated and elongated, filamentous actin was redistributed peripherally, and the cells were degranulated. We conclude that during ZAP infusion, neutrophils become enlarged and degranulated in pulmonary microvessels, especially in alveolar capillaries. The structural and cytochemical changes that occur are consistent with the hypothesis that neutrophil activation is accompanied by alterations in neutrophil physical properties, alterations that may facilitate retention and contribute to endothelial cell injury.

Neutrophil-endothelial cell interactions in the lung.

The large marginated pool of neutrophils normally present in the pulmonary circulation provides a unique opportunity to study the relationship between neutrophils and endothelial cells in situ. Unlike other organs in which neutrophil density in the vascular space is low, adequate numbers of cells are readily available in the lung to assess such important characteristics as distribution, density, size, volume, and immunocytochemical and cytochemical characteristics. To take advantage of such a large marginated pool, morphologic techniques should be more universally applied in studies of neutrophil-mediated tissue injury. From the data presented, it seems clear that factors governing neutrophil-endothelial cell interactions may differ in the lung compared to systemic vascular beds. We have suggested that this difference may be related to the unique characteristics of the lung with regard to anatomy and blood flow rather than site-specific differences in endothelial cells. With the discovery of specialized proteins involved in cell adhesion and the increasing availability of appropriate antibodies to adhesion proteins, unprecedented opportunities exist to address these questions. It is an exciting time to study neutrophil-endothelial cell interactions in the lung.

Downregulation of blood and bone marrow neutrophils decreases expression of acute lung injury in sheep.

We have shown that infusion of zymosan-activated plasma (ZAP) in sheep causes acute lung injury and downregulates peripheral blood neutrophils in that elicited superoxide release is reduced for at least 24 h after the infusion. The present study was designed to test the following hypotheses: 1) peripheral blood neutrophils are representative of neutrophils marginated in the pulmonary circulation, 2) blood neutrophils are downregulated because neutrophils developing in bone marrow are similarly affected, and 3) downregulated neutrophils have a reduced capacity to produce tissue injury. In a series of experiments in 21 sheep, we showed that elicited superoxide release was similar in peripheral blood neutrophils and in marginated neutrophils washed out of the pulmonary vascular bed. Measurements of superoxide release from blood and bone marrow neutrophils collected 2-24 h after ZAP infusion revealed progressive downregulation with time and greater downregulation of superoxide release in bone marrow neutrophils compared with peripheral blood neutrophils. Finally, after downregulating peripheral blood neutrophils, subsequent infusion of ZAP in conscious sheep produced sequestration of neutrophils in the pulmonary circulation but failed to produce a sustained increase in lung lymph protein clearance. The results suggest that neutrophil downregulation, as measured in vitro, is expressed in vivo as reduced ability of neutrophils to produce tissue injury when challenged by an activating agent.

Infusion of zymosan-activated plasma affects neutrophils in peripheral blood and bone marrow in sheep.

Intravenous infusion of zymosan-activated plasma (ZAP) in sheep results in acute lung injury mediated in part by free radical release from stimulated neutrophils that are retained in increased numbers in the pulmonary microcirculation. ZAP infusion is also associated with long-term reduction in elicited superoxide anion generation from segmented neutrophils in the circulating and bone marrow pools for at least 24 h after the infusion. The purpose of our study was to investigate the effect of ZAP infusion on leukocyte counts and neutrophil granule-associated enzyme activity in the circulating and bone marrow pools. Cytochemical methods were used to characterize enzyme activity in primary granules (acid phosphatase and myeloperoxidase) and secondary granules (alkaline phosphatase). During the infusion period, neutrophil differential counts decreased less in venous blood than in matched arterial blood. Release from bone marrow stores was evident as increased numbers of circulating band neutrophils during and after the infusion. Neutrophils in venous and arterial blood smears were degranulated acutely during and 1-3 h after the infusion was stopped. Band and segmented neutrophils in bone marrow also appeared slightly degranulated 1-2 h after the infusion. In vitro experiments showed that band and segmented neutrophils in bone marrow degranulated in response to ZAP incubation. Immature polymorphonuclear leukocytes did not degranulate. Five to 24 h after ZAP infusion, enzyme activity in circulating and bone marrow neutrophils was at baseline levels, suggesting that new cells were being released into the circulating pool because degranulated neutrophils do not synthesize new granules. Another indication of a long-term effect in bone marrow were slight decreases in the percentage of immature polymorphonuclear leukocytes and band neutrophils and a significant decrease in the percentage of eosinophils, all of which coincided with a significant increase in the percentage of segmented neutrophils. Our results demonstrate that circulating complement anaphylatoxins affect neutrophils in bone marrow as well as the vascular space.

Plasma tumor necrosis factor-alpha during long-term endotoxemia in awake sheep.

We used a continuous 12-h infusion of Escherichia coli endotoxin (10 ng.min-1.kg-1) in 10 awake sheep equipped with a lung lymph fistula and vascular catheters to determine the time course of increased plasma tumor necrosis factor-alpha (TNF-alpha) during the infusion and a 12-h postinfusion period. Lung lymph flow increased progressively during the infusion to a peak value averaging 8.6 +/- 2.0 times the baseline flow of 6.3 +/- 1.3 g/h. During the postinfusion period, lung lymph flow remained elevated at three to four times baseline. The lymph-to-plasma protein concentration ratio was unchanged from baseline over 24 h, indicating a dramatic increase in net protein flux across pulmonary microvessels. The TNF-alpha concentration peaked early in the infusion and then declined, despite the continuing presence of endotoxin. Plasma TNF-alpha concentration increased 10-fold (0.33 +/- 0.05 ng/ml at baseline to 3.89 +/- 0.78 ng/ml peak) 2 h into the endotoxin infusion. At the end of the endotoxin infusion, plasma TNF-alpha had decreased to 1.16 +/- 0.19 ng/ml. The circulating TNF-alpha concentration did not correlate with pathophysiology or outcome in these sheep.

A multicenter registry of patients with acute respiratory distress syndrome. Physiology and outcome.

In a multicenter registry conducted over 2 yr of patients with acute respiratory distress syndrome (ARDS), we enrolled 153 patients and collected data daily for 7 consecutive days and weekly thereafter until death or hospital discharge. The purposes of the registry were (1) to determine whether a more liberal definition of ARDS (PaO2/FIO2 < or = 250; bilateral pulmonary infiltrates within 7 days) than those commonly used would result in enrollment of patients earlier in their clinical course, and (2) to study the clinical course of the syndrome in survivors and nonsurvivors. The mortality rate was 54% and it was significantly greater in older versus younger patients (75% versus 37%) and in septic versus nonseptic patients (60% versus 43%). We found that the definition of ARDS used for the registry resulted in enrollment of patients 1 to 7 days earlier than was the case when other published definitions of ARDS were applied to the patient population. Fewer than 2% of the patients failed to meet one of the nonregistry definitions of ARDS within 7 days. The mortality rate was independent of the definition used to identify ARDS patients. Our results suggest that a more liberal definition of ARDS than those commonly used can result in identification of the same population of patients earlier in their clinical course.

PGE1 inhibited PMN attachment to air emboli in vivo during infusion of ZAP without preventing lung injury.

Prostaglandin E1 (PGE1) treatment of neutrophils inhibits their adherence to substrates in vitro, including endothelial cell monolayers. Demonstration that PGE1 inhibits neutrophil adherence in vivo in the lung, however, is complicated by PGE1 effects on cells other than neutrophils, such as endothelial cells. To determine whether PGE1 inhibits neutrophil adherence properties in vivo, we used air emboli as intravascular targets for neutrophil attachment. Four experimental conditions were studied in anesthetized and awake sheep that were treated with 1) PGE1 and air emboli, 2) saline and air emboli, 3) PGE1 and zymosan-activated plasma (ZAP) + air emboli, and 4) saline and ZAP + air emboli. PGE1 (30 ng.kg-1.min-1) or saline was infused continuously 1 h before and 1 h during the infusion of air emboli (group 1; n = 13 sheep) or ZAP + air emboli (group 2; n = 13 sheep). The number of neutrophils (PMNs) attached to air emboli in four anesthetized sheep per condition was significantly less in sheep given PGE1 and ZAP + air emboli [8 +/- 3 (SD) PMNs/mm of embolus perimeter] than in the other three conditions (14-21 PMNs/mm; P less than 0.05). Repeated experiments in five awake sheep per group showed that PGE1 treatment did not prevent increased lung lymph protein clearance in either group compared with saline treatment. We conclude that PGE1 specifically inhibited attachment of ZAP-activated neutrophils to air emboli in vivo. The lack of pathophysiological protection suggests that PGE1-induced alterations in neutrophil attachment properties were independent of other cellular activation responses.

Recurrent noncardiac pulmonary edema accompanying pregnancy-induced hypertension.

Severe pulmonary edema occurred in a patient during the third trimester of two consecutive pregnancies, 17 months apart. Noncardiac origin of the pulmonary edema was demonstrated by normal pulmonary capillary wedge pressures, normal roentgenographic cardiac dimensions with absence of effusions, normal echocardiographic ejection fraction, and elevated thermodilution cardiac outputs; moderate reduction in serum albumin levels may have contributed. In the setting of pregnancy-induced hypertension, the development of ARDS on each occasion suggests a pathophysiologic link.

Physiology of aging related to outcome in the adult respiratory distress syndrome.

Thirty-nine patients with adult respiratory distress syndrome (ARDS) were enrolled in a study to identify potential age-related changes in organ system function that may help explain the apparent association between age and poor outcome in these patients. Criteria for enrollment included an arterial PO2-to-inspired O2 concentration ratio less than or equal to 200 in a clinical setting consistent with ARDS. Patients were excluded if they were less than 18 yr old, had clinical manifestations of congestive heart failure, were seropositive for the human immunodeficiency virus, or had stage II metastatic lung cancer. Patients were divided into two groups: those less than 60 yr old (mean 42 +/- 3 yr, n = 17) and those greater than or equal to 60 yr old (73 +/- 2 yr, n = 16). A group of six patients was analyzed as a separate subset based on a body temperature less than or equal to 97.5 degrees F at enrollment (hypothermic patients, 73 +/- 4 yr old). Sepsis was present in 67% of the nonhypothermic patients and in all the hypothermic patients. Mortality rates were 12% in the patients less than 60 yr and 69% in the nonhypothermic patients greater than or equal to 60 yr. All the hypothermic patients died. Sequential data obtained over 6 days were compared within and between groups. The following results were obtained. 1) The ratio of arterial PO2 to inspired O2 fraction was greater and the positive end-expiratory pressure used was significantly less in the patients greater than or equal to 60 yr old compared with the younger group.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide anion release from blood and bone marrow neutrophils is altered by endotoxemia.

In vivo endotoxin infusion produces neutrophil-mediated acute lung injury and increases superoxide anion release from phorbol myristate acetate (PMA)-stimulated blood neutrophils collected 18-24 hours after the infusion. Because the turnover time of circulating blood neutrophils is only 6-8 hours, it was hypothesized that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with increased superoxide anion release from bone marrow neutrophils. To test this hypothesis, two doses of Escherichia coli endotoxin (5.0 and 0.5 micrograms/kg) were infused into chronically instrumented awake sheep. Blood and bone marrow neutrophils were collected 24 hours after the infusion, and superoxide anion release from unstimulated and PMA-stimulated neutrophils was measured in vitro. Endotoxin infusion produced an increase in pulmonary microvascular permeability, in intravascular activation (degranulation) of blood neutrophils, and in circulating blood neutrophils 24 hours after the infusion. High-dose endotoxin (5.0 micrograms/kg; n = 4) increased superoxide anion release from unstimulated peripheral blood neutrophils (2.25 +/- 0.38 times baseline [p less than or equal to 0.05]) and from peripheral blood neutrophils stimulated with 10(-9) M PMA in vitro (1.46 +/- 0.55 times baseline). Low-dose endotoxin (0.5 micrograms/kg; n = 5), on the other hand, did not alter superoxide anion release from peripheral blood neutrophils. Bone marrow neutrophils could not be isolated reproducibly after high-dose endotoxin because of leukoaggregation. Bone marrow neutrophils were isolated after low-dose endotoxin infusion. Stimulation of these cells with 10(-9) M PMA in vitro resulted in a two- to fourfold increase above control release (p less than or equal to 0.05). Increased superoxide anion release from both peripheral blood and bone marrow neutrophils occurred in the absence of circulating endotoxin, as measured by a Limulus assay. These results show that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with an increase in the superoxide anion release from bone marrow neutrophils. Furthermore, the recruitment of affected bone marrow neutrophils into peripheral blood may explain the increased superoxide anion release from blood neutrophils 24 hours after endotoxin infusion.

Calcium is required for PMA induced superoxide release from human neutrophils.

Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O2-) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca++ or 0 mM Ca++ Hanks' buffer for 60 min prior to activation as well as during measurement of O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 +/- 1.2 nmol O2- in 20 min (n = 4). O2- release was not significantly different for high density cells incubated and stimulated in 0 mM Ca++. In the presence of 1 mM Ca++, 0.2 million adherent neutrophils released 6.3 +/- 0.5 nmols O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 +/- 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca++ had no effect on O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- was independent of verapamil (10(-5)-10(-7) M), suggesting that voltage-dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca++ increased after addition of PMA. Unstimulated cells in 1 mM Ca++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O2- secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O2- and increasing superoxide release after a maximum stimulating dose of PMA.