Boar seminal plasma or hen's egg yolk decrease the in-vitro chemotactic and phagocytotic activities of neutrophils when co-incubated with boar or bull sperm

The objective was to determine the effects of boar seminal plasma and hen's egg yolk on chemotaxis and phagocytosis of porcine and bovine polymorphonuclear neutrophils (PMNs) in vitro. Chemotactic activity of PMNs was determined following culture for 90 min in a blind well chamber. Phagocytosis was assayed after co-culture of PMNs with sperm for 60 min. In the presence of ≥ 5% boar seminal plasma, chemotactic activity of PMNs was reduced (P < 0.05) in both pigs (from 1126.1 to 934.2-1009.1 cells/mm²) and in cows (from 1067.1 to 768.9-800.0 cells/mm²). Furthermore, ≥ 5% boar seminal plasma reduced (P < 0.05) leukocyte phagocytosis in pigs (26.2-32.1%) and cows (27.2-30.0%) compared to controls (41.7 and 42.1%, respectively). Although 20% hen's egg yolk increased (P < 0.05) chemotactic activity of PMNs in pigs (from 790.4 to 1006.1 cells/mm²) and cows (from 789.9 to 953.5 cells/mm²), egg yolk increased (P < 0.05) phagocytotic activity of porcine PMNs (from 24.3 to 33.8%), but not the activity of bovine PMNs (15.1 vs 15.8% in controls). Boar seminal plasma and caffeine reduced (P < 0.05) the egg yolk-induced increase in chemotaxis in both species (from 988.6 to 795.2 or 813.2 cells/mm² in pigs and from 953.5 to 779.4 or 833.8 cells/mm² in cows), and phagocytotic activities of PMN (from 33.8% to 15.2 or 13.3%) only in pigs (but not in cows; 11.2-15.1%). In conclusion, hen's egg yolk increased chemotactic activity of PMNs in both pigs and cows, whereas egg yolk increased only phagocytosis of PMNs in pigs, but not in cows. Even in the presence of egg yolk, boar seminal plasma and caffeine significantly reduced chemotactic activity of PMNs in pigs and cows, and phagocytotic activity of porcine PMNs.     

Spin Trapping of Superoxide from Glass Adherent Polymorphonuclear Leukocytes Induced by N-Formylmethionyl-Leucyl-Phenylalanine

Dahinden et al. reported that N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide release from polymorphonuclear leukocytes (PMNs) lasted more than 60 min when the cells were allowed to attach to a petri dish before induction. In contrast, it lasted only for 2.5 min when cells were in suspension (J. Clin. Invest. 72: 113-121, 1983). In spite of this report, the effect of cell adhesion has been ignored in most spin trapping studies of superoxide release from PMNs. This study shows that most PMNs in a quartz flat electron paramagnetic resonance (EPR) cuvette which was placed horizontally adhered to the wall within 3 min. In contrast, if the cuvette was placed vertically, only 20-30% of the cells became adherent in 30 min. We performed spin trapping studies using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap, and monitored the effect of cell adhesion on superoxide generation. When spin trapping was conducted on PMNs in suspension, the EPR signal of superoxide adduct (DMPO-OOH) was undetectable after stimulation with fMLP. However, PMNs which were allowed to adhere to the cuvette after stimulation generated superoxide for hours. Moreover, when PMNs were allowed to adhere prior to the stimulation, the magnitude of superoxide release was augmented three-to fourfold. Unlike fMLP, phorbol myristate acetate (PMA), which has been most commonly used in spin trapping studies, induced superoxide release which was not influenced by cell adhesion. We emphasize the importance of specifying the cell-adhesion-state in spin trapping studies.     

Effects of epoxyeicosatrienoic acids on polymorphonuclear leukocyte function

During periods of ischemia and vascular injury, factors are released which recruit monocytes and polymorphonuclear leukocytes (PMNs) to the site of injury by promoting adherence to the endothelium and transmigration across the endothelial cell (EC) layer. During coronary artery stenosis, we have shown that the endothelium-derived, cytochrome P450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs), are elevated. Therefore, we examined if the EETs could stimulate PMN adherence to cultured ECs. Pretreatment of ECs with EETs for either 30 min or 4 hr did not alter the adherence of 51Cr-labelled PMNs to ECs while phorbol myristate acetate (PMA) produced a 4-fold increase in PMN adherence. The combination of EETs and PMA did not significantly augment or diminish PMA-induced PMN adherence to ECs. When ECs and 51Cr-labelled PMNs were coincubated, treatment with EETs alone did not alter PMN adherence. However, when EETs and PMA were added together during the coincubation of ECs and 51Cr-labelled PMNs, the EETs produced a concentration-related decrease in PMN adherence. Microscopic analysis of the culture media bathing the cells revealed aggregates of the labeled PMNs. We examined the effects of the EETs on PMN aggregation. 8,9-EET (10, 50, and 100 microM) increased PMN aggregation (7 +/- 3, 35 +/- 10, and 65 +/- 11%) and intracellular calcium by 1.7 +/- 0.5, 4.7 +/- 1.4, and 6.8 +/- 2.3-fold above basal. 5,6-, 11,2- and 14,15-EETs also stimulated aggregation. FMLP stimulated the production of superoxide; however, 8,9-EET did not. These observations indicate that the decrease in PMN adherence observed in the coincubation experiment is the result of EET-induced PMN aggregation. Given the increase in EET production during coronary artery stenosis, these data may provide insight into their potential biological significance during myocardial ischemia and vascular injury.     

Concanavalin a capping of rhesus monkey polymorphonuclear leukocytes

The technique of capping, a probe designed to evaluate the fluidity and functional competence of human polymorphonuclear leukocytes (PMNs), has been successfully adapted for rhesus monkey PMNs. The capping characteristics of rhesus monkey PMNs are very similar to those of human PMNs. Utilizing this capping technique as an evaluation tool and with fetal/neonatal rhesus monkey as a functional animal model, the ontogeny of movement and chemotactic characteristics of PMNs can now be studied.     

Periodontal therapy increases neutrophil extracellular trap degradation

In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.     

Neutrophil retention during a single transit through the pulmonary circulation.

Our laboratory has previously reported that 70-80% of polymorphonuclear cells (PMNs) are delayed with respect to erythrocytes (RBCs) in a single pass through the lungs of dogs, whereas only 5-15% of PMNs are delayed in a single pass through human lungs. Because these results were obtained using a direct blood sampling method in animals and an indirect gamma camera method in humans, the reported differences could be related to differences in measurement technique. The present study was designed to settle this question by comparing both techniques in a single species. The results show that the gamma camera technique previously used in humans underestimates the retention of PMNs with respect to RBCs during a single pass through the lung. They also show that this problem can be corrected by modifying the analysis of the data obtained using the gamma camera. We conclude that the pulmonary circulation retains PMNs with respect to RBCs to a comparable degree in animals and humans.     

Modulatory effect of chiral nonsteroidal anti-inflammatory drugs on apoptosis of human neutrophils

Polymorphonuclear neutrophils (PMNs) are short-lived leukocytes that die by apoptosis. Although PMNs are crucial in the defense against infection, they have been implicated in the pathogenesis of tissue injury observed in inflammatory diseases. The induction or prevention of PMN apoptosis is currently discussed as a key event in the control of inflammation. Caspase-3 activation is the first step in the execution phase of apoptosis. In the study, effect of racemic mixtures and enantiomers of 2-arylpropionic acid derivatives: ketoprofen, flurbiprofen (FBP), and (+)-S-naproxen and 2-arylbutyric acid: indobufen on apoptosis activation via caspase-3 and phosphatidylserine (PS) translocation (annexin-V binding) in human neutrophils in vitro has been investigated. Caspase-3 activation was detected by Western blotting, fluorometric assay of DEVD-AMC cleavage, and flow cytometry with carboxyfluorescein (FAM) labeled caspase inhibitor. PMNs were isolated and cultured up to 24 h. The chiral nonsteroidal anti-inflammatory drugs (NSAIDs) were found to modulate human PMN apoptosis in a dose- and time-dependent manner. The greater activation of caspase was found at 75-150 microg/ml concentration of racemates as well enantiomers, especially for FBP, whereas NSAIDs at smaller quantities (15 microg/ml) were inactive. At concentration of 75 microg/ml, NSAIDs increased the rate of PS externalization in PMA-stimulated and non-stimulated neutrophils. Additionally, no cytotoxic effect of the NSAIDs was observed at concentration up to 75 microg/ml that induce apoptosis. Regulation of caspase activity by NSAIDs may represent a potent target to trigger apoptosis and resolve inflammatory disorders.     

Large-scale isolation of polymorphonuclear leucocytes from bovine blood

A method for the isolation of an enriched population (greater than 96%) of bovine polymorphonuclear leucocytes (PMNs) from 70 liters of blood was developed using a two-step procedure involving separation of the blood, in a packed red blood cell fraction containing the PMNs and a plasma fraction, by continuous flow blood separation. Hypotonic lysis of erythrocytes was then followed by centrifugation at 200 g sedimenting the PMNs. The yield was 93 +/- 30 g, the recovery was 62 +/- 20%, viability was greater than 95%. Since bovine blood can be obtained in unlimited amounts, the procedure described here can be applied to obtain large amounts of bovine PMNs for incubation studies and large-scale purification of intracellular enzymes suitable for biochemical characterization.     

Neutrophil caveolin-1 expression contributes to mechanism of lung inflammation and injury

Caveolin-1 present in immune cells may be involved in regulation of the inflammatory response. Here, using caveolin-1-null (Cav-1(-/-)) mice, we addressed the role of caveolin-1 in polymorphonuclear neutrophils (PMNs) in regulating PMN activation-mediated lung injury. In lungs of wild-type (Cav-1(+/+)) mice perfused at constant flow with Krebs-Henseleit solution, addition of Cav-1(+/+) PMNs (4 x 10(6) cells) into the perfusate followed by their activation with formyl-Met-Leu-Phe (fMLP, 1.0 muM) plus platelet-activating factor (1.0 nM) increased pulmonary microvessel filtration coefficient by 150% and wet-to-dry lung weight ratio by 50% as well as PMN accumulation in lungs. These responses were markedly reduced in lungs perfused with Cav-1(-/-) PMNs followed by addition of the same activating agents. fMLP-stimulated adhesion of Cav-1(-/-) PMNs to pulmonary microvascular endothelial cells and migration of Cav-1(-/-) PMNs across endothelial monolayers were also impaired compared with Cav-1(+/+) PMNs. Cav-1(-/-) PMNs showed 50-80% reduction in PMA- or fMLP-stimulated superoxide production compared with Cav-1(+/+) PMNs. In addition, Cav-1(-/-) PMNs had decreased migratory activity (50%) and adhesion to fibrinogen (40%) in response to fMLP. Rac1 and Rac2 were activated in Cav-1(+/+) PMNs after stimulation of fMLP but not in Cav-1(-/-) PMNs. Exogenous expression of caveolin-1 in COS-phox cells augmented the fMLP-induced Rac1 activation and superoxide production, indicating a direct role of caveolin-1 in the mechanism of superoxide production. Thus caveolin-1 expression in PMNs plays a key role in mediating PMN activation, adhesion, and transendothelial migration and in PMN activation-induced lung inflammation and vascular injury.     

Quantitation of tigecycline, a novel glycylcycline [corrected] by liquid chromatography

An ion-paired HPLC assay was developed to determine tigecycline (GAR-936) concentrations in Hank's balanced salts solution, tigecycline intra-cellular concentrations in human polymorphonuclear neutrophils (PMNs) and tigecycline concentrations in human serum. Minocycline was used as internal standard, 5% trichloroacetic acid was added to lyse PMNs and also precipitate proteins in PMNs and serum. The top aqueous layer was aspirated for HPLC assay. The chromatograms were performed with a reversed-phase C18 column with UV detector. The mobile phase consisted of acetonitrile, phosphate buffer (pH 3) and 1-octanesulfonic acid at a flow rate of 1 ml/min. Good linearity and recovery were achieved over the range of standard curves. The relative standard deviations of three quality controls for intra- and inter-day precision were less than 6.4%, and the relative errors of the intra- and inter-day accuracy were less than 7.0%. Tigecycline in Hank's buffer, PMNs and serum was stable under different test conditions. This new liquid chromatography assay is a simple, accurate and reproducible method for determining tigecycline in different matrix.     

Superoxide radical-scavenging effects from polymorphonuclear leukocytes and toxicity in human cell lines of newly synthesized organic selenium compounds

Synthetic organic selenium compounds such as 2-phenyl-1,2-benzisoselenazol-3(2H)-one may show glutathione peroxidase-like antioxidant activity. Recently, we synthesized new organic selenium compounds that are thought to be effective antioxidants. To study their possible applications as antioxidants, we evaluated two selenoureas, N,N-dimethylselenourea and 1-selenocarbamoylpyrrolidine, and two tertiary selenoamides, N-(phenylselenocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide, for their superoxide radical (O2-)-scavenging effects and toxicity. We measured (O2-)-scavenging effects in polymorphonuclear leukocytes (PMNs) with a specific, sensitive and real-time kinetic chemiluminescence method. Furthermore, the toxicity of these compounds was measured in some human cell lines and PMNs using the tetrazolium method. Hydrogen peroxide was measured by a scopoletin method. Finally, translocation of an NADPH oxidase component, p47 phagocyte oxidase, to the cell membrane was investigated by confocal laser scanning microscopy. N,N-Dimethylselenourea and 1-selenocarbamoylpyrrolidine effectively scavenged (O2-) released from 4beta-phorbol 12-myristate 13-acetate-stimulated PMNs, and the 50% inhibitory concentrations were 6.8 +/- 2.2 and 6.5 +/- 2.5 microm, respectively. N-(Phenylselenocarbonyl)-piperidine and N,N-diethyl-4-chloroselenobenzamide also effectively scavenged (O2-) from PMNs, and the 50% inhibitory concentrations were 11.3 +/- 4.8 and 20.3 +/- 6.4 microm, respectively. Selenoureas showed very low toxicity in human cell lines and PMNs, even at high concentrations, whereas tertiary selenoamides were cytotoxic. These compounds did not produce significant amounts of hydrogen peroxide from 4beta-phorbol 12-myristate 13-acetate-stimulated PMNs. None of the compounds significantly affected the translocation of p47 phagocyte oxidase. Selenoureas acted as effective antioxidants and showed low toxicity in some human cells. Thus, these compounds might be new candidates as antioxidative substances.     

Effect of blood serum, caffeine and heparin on in vitro phagocytosis of frozen-thawed bull sperm by neutrophils derived from the peripheral blood of cows

Although polymorphonuclear leukocytes (PMNs) are recruited into the uterine lumen to phagocytize sperm, factors controlling the phagocytotic ability of PMNs in cattle are not well documented. The objective was to determine the effects of blood serum, caffeine, and heparin on chemotaxis of PMNs for sperm and phagocytosis of sperm by PMNs in cows. Polymorphonuclear leukocytes were obtained (centrifugation) from a cow's peripheral blood. In Experiment 1, the chemotactic activity of PMNs increased (P < 0.01) when fresh serum was included in the medium (1226 cells/mm² in serum vs. 1110 cells/mm² in BSA), regardless of the presence of sperm, whereas heat-inactivated serum (1099 cells/mm²) did not affect their activity (P = 0.65). Phagocytosis of live and dead sperm by PMNs both increased (P < 0.01) in the presence of fresh serum (incidences of 54.5 and 48.0%, respectively), but stimulation was decreased (P < 0.01) by supplementation of the medium with ≥1 mM caffeine (20.6-30.3%). Serum-stimulated chemotactic activity of PMNs (1218 cells/mm²) was also decreased (P < 0.01) in the presence of caffeine (1090 cells/mm²). Furthermore, supplementation of PMNs with heparin in the presence of serum decreased (P < 0.01) both phagocytotic (from 43.8% to 21.5-31.7%) and chemotactic activities of PMNs (from 1124 to 1048-1108 cells/mm²). We inferred that opsonization in the presence of active complement stimulated phagocytotic and chemotactic activities of PMNs, and that both caffeine and heparin decreased serum-stimulated phagocytotic and chemotactic activities of PMNs.     

Effect of endocellular cryoprotectant upon polymorphonuclear neutrophil function during storage at low temperature.

The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutrophils (PMNs) during storage between 0 ° and 4 °C was investigated.Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as that of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability.Judging from the above findings, DMSO would be suitable as a crypotrotective agent although the problem on toxicity remains to be resolved.     

Impaired ability of burn patient neutrophils to stimulate β-defensin production by keratinocytes

Immunosuppressive neutrophils (PMN-II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN-II on the production of human β-defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual-chamber transwells. Culture fluids harvested 24?h after cultivation were assayed for HBDs using enzyme-linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony-counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN-II, because these cells produced CC-chemokine ligand 2 (CCL2), but not interleukin (IL)-12 and CC-chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell-cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell-cultured with burn-patient PMNs (36% inhibition). IL-12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL-12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn-patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN-II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.     

Effects of endotoxin on oxidative metabolism of polymorphonuclear leukocytes

The influence of endotoxin on rat polymorphonuclear leucocytes (PMN) ability to generate oxygen free radicals (OFR) has been studied by chemiluminescence method. PMNs derived from intact animals were used as a control. PMNs derived from animals with 1.5 h endotoxemia increased OFR production after stimulation by latex. In contrast, PMNs derived from intact animals and preincubated with endotoxin for 1.5 h decreased OFR production after stimulation bw latex. It was proposed that stimulating effect of endotoxin on PMNs in vivo was mediated by plasma components.     

The effects of platelet activating factor and retinoic acid on the expression of ELAM-1 and ICAM-1 and the functions of neutrophils

Preincubation of pulmonary microvascular endothelial cells (PMVECs) with platelet-activating factor (PAF) for 3.5 h increased the adhesion rate of polymorphonuclear leukocytes (PMNs) to PMVECs from 57.3% to 72.8% (p < 0.01). Preincubation of PMNs with PAF also increased PMN-PMVEC adhesion rate. All-trans retinoic acid (RA) blocked the adherence of untreated PMNs to PAF-pretreated PMVECs but not the adherence of PAF-pretreated PMNs to untreated PMVECs. PAF increased the expression of intercellular adhesion molecule-1 (ICAM-1) and E-selection (ELAM-1) on PMVECs, PMN chemotaxis to zymosan-activated serum and histamine, and PMN aggregation and the release of acid phosphatase from PMNs. Co-incubation of RA inhibited PAF-induced PMN aggregation, the release of acid phosphatase from PMNs, and PMN chemotaxis to zymosan-activated serum and histamine while the expression of ICAM-1 and ELAM-1 did not change. Our results suggest that RA can be used to ameliorate PMN-mediated inflammation.     

Intracellular Salmonella typhimurium induce lysis of human polymorphonuclear leukocytes which is not associated with the Salmonella virulence plasmid

The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro. Three S. typhimurium strains, the wild-type strain OU5043, its isogenic virulence plasmid-cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study. There was no correlation between the recovery of intracellular S. typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level. When the oxygen-dependent response of PMNs upon phagocytosis of S. typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12-myristate 13-acetate and 15% with a negative control. Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs. Microscopic analysis of Salmonella-infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs. Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24 hr of infection and this effect was not associated with the presence or absence of the virulence plasmid. On the other hand, lysis of PMNs was related to the intracellular survival of Salmnonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella-induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism. The ability to induce lysis of PMNs by either wild-type or plasmid-cured strains of S. typhimurium may play a crucial role in the pathogenesis of non-typhoid Salmonella. The contribution of pSTV to human salmonellosis is likely to be limited. Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.     

Marginated pool of neutrophils in rabbit lungs

The size and location of the marginated pool of neutrophils (PMNs) in rabbit lungs were evaluated, and the rate of exchange of the PMNs with the circulating pool was determined. 99mTc-labeled erythrocytes (99mTc-RBCs) and 125I-labeled macroaggregated albumin (125I-MAA) were used to determine RBC transit times in the pulmonary circulation. Radiolabeled PMNs were studied on their first passage through the lungs. After 10 min of circulation, the lungs were fixed, gamma counted, and prepared for morphometric and autoradiographic studies; 74 +/- 3% of the PMNs was retained in the lungs on the first passage, and 23 +/- 2% was within the pulmonary marginated pool 10 min later. The regional PMN retention and the rate of exchange between the marginated and circulating PMN pools in the lung were directly related to RBC transit time. The radiolabeled PMNs distributed similarly to the unlabeled cells within the microvasculature and had a similar exchange rate between the marginated and circulating pools (1.4 +/- 0.2%/s using labeled cells and 1.5 +/- 0.5%/s using unlabeled cells). The marginated pool was located primarily within alveolar capillaries and contained two to three times as many PMNs as the total circulating pool.