Contact-induced neutrophil activation by platelets in human cell suspensions and whole blood.

Platelet-dependent activation of polymorphonuclear neutrophils (PMNL) was investigated with a lumi-aggregometer in heparinized whole blood and platelet-PMNL suspensions. The lumi-aggregometer allowed us to simultaneously monitor increases in impedance or light transmission as consequences of platelet aggregation and luminol-enhanced chemiluminescence (CL) as a measure of the oxidative burst in PMNL. Aggregation and platelet-PMNL contacts were also checked by light and electron microscopy. In whole blood, adenosine diphosphate (ADP) and the thromboxane A2 mimetic U 46619 induced the aggregation (increase in impedance) and the CL, which were both suppressed by EDTA, arginyl-glycyl-aspartyl-serine (RGDS) peptide, and the absence of stirring. In contrast, FMLP caused only CL that was unaffected by EDTA, RGDS peptide, and nonstirring. Similar observations were obtained with mixed suspensions containing washed platelets and PMNL at their physiologic concentrations. ADP, U 46619, and thrombin induced both aggregation (increase in light transmission) and CL, whereas FMLP caused CL but only very weak aggregation. Exogenous fibrinogen strongly enhanced the effects of ADP and U 46619. Iloprost, EDTA, RGDS peptide, red blood cell (RBC) ghosts, and nonstirring inhibited the effects induced by the platelet agonists, but were ineffective on the CL induced by FMLP. Treatment of platelets with aspirin did not affect the CL of PMNL induced by platelets. Microscopic examination, the requirements of stirring, Ca2+, and fibrinogen, and the inhibitory effects of RGDS peptide and RBC ghosts show that stimulated platelets activate PMNL in a contact-dependent manner that depends on fibrinogen binding. This was confirmed by the immunochemical demonstration of fibrinogen (but not of fibronectin) in the contact spaces between activated platelets and PMNL. Because supernatants and lysates of resting or thrombin-stimulated platelets did not induce the CL of PMNL, soluble agonists did not appear to be involved. Nonstimulated washed platelets also caused CL of PMNL that required stirring and Ca2+ and was inhibited by RBC ghosts. No CL occurred in unstimulated stirred whole blood, suggesting that a preactivation of platelets during the preparation may be responsible for the effects of unstimulated washed platelets. The results show that platelets provide a strong stimulus for PMNL that requires intercellular contact. Fibrinogen exposure on the platelet surface seems to be necessary for the activation of PMNL by stimulated platelets.     

Transient pulmonary platelet sequestration during endotoxemia in dogs

We evaluated the time-course of regional platelet sequestration, following a bolus dose of endotoxin in anesthetized dogs. Autologous indium 111 labeled platelets, representing less than 1% of the circulating platelets, were injected 35-60 min prior to administering endotoxin intravenously to dogs. A gamma camera was used to monitor the distribution of these platelets within the thorax and abdomen. Alterations in the circulating blood platelet count paralleled the changes in blood radioactivity, enabling us to use external imaging to evaluate platelet kinetics. Marked hypotension and thrombocytopenia occurred within 6 min after administering endotoxin. The platelet pool in the lungs peaked at 9 min and was temporally related to the decrease in circulating platelet count, hypotension and increase in liver size. Translocation of platelets from the lungs to the circulating platelet pool occurred during the subsequent hour with sequestration occurring in the liver and possibly other organs. During this phase there was a recovery in platelet count to 35% of baseline levels but without significant recovery in mean arterial pressure. Based on these results we propose that endotoxin-induced thrombocytopenia results from pulmonary and hepatic sequestration of platelets, but that sequestration of platelets in the lungs is only transient. The mechanism and significance of subsequent translocation of platelets from the lungs to other sites, particularly the liver and the circulating platelet pool, remain to be investigated.     

Physiologic responses to small emboli and hemodynamic effects of changes in deformability of polymorphonuclear leukocytes in isolated rabbit lung.

We hypothesized that polymorphonuclear leukocytes (PMNs) exposed to lipopolysaccharide (LPS) or chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) would alter the pulmonary hemodynamics of buffer-perfused rabbit lung. Pulmonary arterial pressure (Ppa) was measured at baseline, at peak response, and at 30 min after PMN infusion in the perfusate (Ppa x time, PT product). Infusion of peritoneal-harvested PMNs resulted in a transient increase in both pulmonary vascular resistance (PVR) and lung weight. PVR also increased when glutaraldehyde-treated rabbit PMNs (GPMNs) or beads were infused. Upstream PVR (Pao-Pdo) remained high with the infusion of GPMNs and beads and returned to baseline only when PMNs were infused 30 min thereafter. FMLP-exposed PMNs increased the peak Ppa and PT product. Pretreatment with 3-isobutyl-1-methylxanthine (IBMX) blocked this increase in pressure, suggesting the release of vasoconstrictor(s) or a direct effect of FMLP. PMNs exposed to LPS increased peak Ppa and PT product with and without the addition of IBMX. Cytochalasin D treatment of PMNs prevented the increase in PT product, suggesting that actin polymerization of PMNs is involved. The effects of these agents on PMN rigidity were verified by means of 6.5-microm polycarbonate filters. PMN suspension treated with FMLP or LPS increased filter perfusion pressure and PT product. Cytochalasin D prevented these increases. These results suggest that, initially after injection, PMNs behave like small beads embolizing primarily the small arteries in the lung and that they then move distally through the vasculature. Exposure to FMLP or LPS alters PMN deformability and the ability of PMNs to pass through the pulmonary vasculature, resulting in increased pulmonary vascular resistance.     

Platelet survival and turnover: important factors in predicting response to splenectomy in immune thrombocytopenic purpura

Autologous indium-111 platelet sequestration and survival studies were performed on 59 immune thrombocytopenic purpura (ITP) patients, 21 of whom underwent splenectomy shortly thereafter. Sequestration patterns were primarily splenic in 46 patients, primarily hepatic in 6 patients, and both splenic and hepatic in 8 patients. The mean platelet survival ranged from 15 to 211 hr (normal, 180-220 hr), and mean platelet turnover (a measure of platelet production rate) varied from 99 platelets/microliters/hr to 7,585 platelets/microliters/hr (normal 1,200-1,600 platelets/microliters/hr). Among splenectomy patients, 13 had an excellent response, and 8 had a fair or poor response. Neither the pattern of platelet sequestration nor the quantity of platelet-associated IgG was useful in predicting response to splenectomy. There was, however, a striking correlation between platelet studies showing short survival/high turnover and subsequent excellent response to splenectomy. Conversely, patients with only moderately decreased survival and low turnover had an unpredictable response to splenectomy. This investigation demonstrates that ITP patients are a heterogeneous population and include a significant subset whose thrombocytopenia results primarily from decreased turnover. Platelet kinetic studies appear useful in predicting beneficial response to splenectomy.     

Abnormal neutrophil-pulmonary interaction in the adult respiratory distress syndrome. Qualitative and quantitative assessment of pulmonary neutrophil kinetics in humans with in vivo 111indium neutrophil scintigraphy

In the absence of direct toxins, the majority of evidence from animal models suggests that neutrophils (PMN) are necessary for the full expression of the abnormal pulmonary permeability accompanying acute microvascular lung injury. We therefore studied the role of the PMN in the human correlate of this disease, the adult respiratory distress syndrome (ARDS), by assessing the pulmonary retention of infused autologous 111Indium-labeled PMN (PMN-In). We evaluated 79 patients, prospectively categorized as: "active" ARDS (Aa; n = 30), "active" ARDS and concurrent corticosteroid therapy (As; n = 11), "resolving" ARDS (Ar; n = 13), sepsis without pulmonary edema (S; n = 7), and cardiac pulmonary edema (C; n = 18). This clinical separation was confirmed by retrospective analysis of associated measures of hemodynamic and respiratory dysfunction. We found that both analog scintigrams (positive/negative for diffuse pulmonary PMN-In sequestration) and computer-assisted quantitative analysis in 46 patients (T 1/2 of first hour demargination and percentage of peak activity/pixel/second remaining at 17 to 20 h) showed a significant rank order decrease in the pulmonary retention of labeled PMN-In through the Groups Aa----As----S----Ar----C. Our findings recognized aspects of in vivo PMN-In behavior that implied pathophysiologic differences between groups of critically ill patients in either the PMN themselves or in PMN-pulmonary endothelial interaction. This demonstrates the possibility of abnormal in vivo PMN-endothelial interaction in ARDS by virtue of the greater pulmonary localization of PMN in active ARDS versus resolving disease, septic non-ARDS states, and cardiac pulmonary edema.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of platelet glycoprotein Ib by elastase released from primed neutrophils

Summary Mutual interactions between neutrophils (PMN) and platelets are recognized to be important in modulating the respective functions of these two cell types. Here we show that primary granule secretion from appropriately-stimulated PMN can lead to complete proteolytic removal of GPIb from the platelet surface.
Thus, when the PMN in PMN/platelet mixtures were stimulated by FMLP, platelets lost GPIb as measured by ristocetin-induced aggregation, flow cytometry and SDS-PAGE analysis. This loss was most marked when PMN were primed by GM-CSF, and could be inhibited by a specific elastase inhibitor. As expected, the al-antiproteinase in plasma inhibited GPIb loss, but when PMN were strongly stimulated by FMLP and GM-CSF in the presence of platelets this inhibition was incomplete or absent.
It is concluded that joint priming of PMN with GM-CSF and platelets can cause a previously unrecognized degree of primary granule secretion which, via elastase, leads to platelet GPIb loss. We suggest that this loss is likely to be of physiological and pathophysiological importance.     

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.     

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.     

Inhaled platelet-activating factor causes pulmonary neutrophil sequestration in normal humans.

Inhaled platelet-activating factor (PAF) causes bronchoconstriction and transient peripheral neutropenia in humans. We studied eight normal subjects to investigate whether inhaled PAF caused pulmonary neutrophil sequestration. All subjects received autologous 99mTc-red cells as a blood pool marker, seven received 111In-neutrophils, and one received 111In-platelets. Six subjects inhaled 48 micrograms of PAF. There was immediate pulmonary sequestration of 111In-neutrophils, maximal (218% baseline) at 6 min (p less than 0.001), returning to normal by 3 h. There was no change in circulating platelet count or pulmonary 111In-platelet transit. Methacholine inhalation caused equivalent bronchoconstriction to PAF, but it had no effect on neutrophil count or pulmonary 111In-neutrophil activity. We have demonstrated pulmonary neutrophil, but not platelet, sequestration after PAF. This supports a role for PAF as an inflammatory mediator in humans. This may be a useful model for exploring pulmonary neutrophil kinetics and preinflammatory processes.     

Platelets express functional Toll-like receptor-4

Profound thrombocytopenia occurs in humans with sepsis and in mice administered lipopolysaccharide (LPS). Growing evidence indicates that platelets may contribute to these abnormalities, but whether that is a direct result of LPS activation of platelets or an indirect result of other inflammatory mechanisms remains unclear. Here we demonstrate that although platelets do not increase P-selectin expression in response to LPS, platelets bind more avidly to fibrinogen under flow conditions in a Toll-like receptor-4 (TLR4)-dependent manner. In addition, we find that CD41+ megakaryocytes grown from fetal livers and adult circulating platelets express significant amounts of TLR4. LPS induced thrombocytopenia in wild-type mice but not in TLR4-deficient (TLR4def) mice. Wild-type platelets accumulated in the lungs of wild-type mice in response to LPS; TLR4def platelets did not. However, wild-type platelets did not accumulate in the lungs of LPS-treated TLR4def mice. Neutrophils also accumulated in the lungs, and this preceded platelet accumulation. Neutrophil depletion completely abolished LPS-induced platelet sequestration into the lungs, but platelet depletion did not affect neutrophil accumulation. Thus, our data show for the first time that platelets do express functional levels of TLR4, which contribute to thrombocytopenia through neutrophil-dependent pulmonary sequestration in response to LPS.     

Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous 111In-labeled platelets and homologous 51Cr- labeled platelets differ

Mean platelet survival and turnover were simultaneously determined with autologous 111In-labeled platelets (111In-AP) and homologous 51Cr- labeled platelets (51Cr-HP) in ten patients with chronic immune thrombocytopenic purpura (ITP). In vivo redistribution of the 111In-AP was quantitated with a scintillation camera and computer-assisted image analysis. The patients were divided into two groups: those with splenic platelet sequestration (spleen-liver 111In activity ratio greater than 1.4), and those with diffuse sequestration in the reticuloendothelial system. The latter patients had more severe ITP reflected by pronounced thrombocytopenia, decreased platelet turnover, and prominent early hepatic platelet sequestration. Mean platelet life span estimated with 51Cr-HP was consistently shorter than that of 111In-AP. Platelet turnover determined with 51Cr-HP was thus over-estimated. The difference in results with the two isotope labels was apparently due to greater in vivo elution of 51Cr. Although the limitations of the techniques should be taken into account, these findings indicate that platelet turnover is not always normal or increased in ITP, but is low in severe disease. We suggest that this may be ascribed to damage to megakaryocytes by antiplatelet antibody. The physical characteristics in 111In clearly make this radionuclide superior to 51Cr for the study of platelet kinetics in ITP.     

Acute Complement-mediated Haemolysis in the Rabbit: Relationship between the Thrombocytopenia and Neutropenia and Complement Consumption

S ummary . There appears to be a direct relationship between complement consumption in the rabbit (demonstrated by a fall in CH₅₀ and plasma C3 concentration) and the temporary disappearance of platelets and neutrophils from the circulation during IgM cold antibody induced intravascular haemolysis. Studies with ⁵¹Cr-labelled platelets showed that although the platelet counts returned to initial values following the thrombocytopenia, 18–38% of the labelled platelets failed to return, suggesting platelet loss at the site of sequestration (mainly the lungs) and dilution of the recirculating platelets with unlabelled platelets from another pool. There is no evidence that these events trigger intravascular coagulation. Complement-depleted rabbits are totally refractory to further attempts to produce lysis, thrombocytopenia and neutropenia by cold antibody, though, even when complement activity in the plasma is allowed to recover, the lytic response to doses of cold antibody similar to those originally given, is reduced. The surviving circulating red cells are coated with C₃ following an acute lytic episode and the immune-adherence of neutrophils and platelets to fixed-C3 on red-cell membranes in the pulmonary circulation provides one possible mechanism for the thrombocytopenia and neutropenia.     

Kinetics, in vivo redistribution and sites of sequestration of indium-111-labelled platelets in giant platelet syndromes

Six patients with giant platelet syndrome were examined: four with Bernard-Soulier syndrome (two were asplenic); one with hereditary thrombopathic thrombocytopenia; and one with May-Hegglin anomaly. Autologous platelets were labelled with In-111-oxine and in vivo redistribution and sites of sequestration measured with quantitative imaging. In Bernard-Soulier syndrome platelet survival was normal or moderately shortened; platelet turnover was decreased only in the two patients with thrombocytopenia. In the patients with thrombopathia or May-Hegglin anomaly, platelet survival and turnover was moderately decreased. In those patients with normal-sized spleens, the mean splenic platelet pool consisted of 35.5% of the platelet mass, i.e. normal. The intrasplenic transmit time of the megathrombocytes was prolonged. Splenic blood flow was within normal limits. There was a marked accumulation of platelets in the liver at equilibrium: 15.5-58.8% of whole body radioactivity (normal 9.6 +/- 1.2%). This finding is unexplained. The final sites of sequestration of platelets were mainly in the liver and spleen, similar to that seen in normal subjects. We conclude that there is no inverse relationship between cell size and splenic platelet transit time. Platelet size therefore does not determine the size of the splenic platelet pool. The size of the platelets also does not seem to affect the sites of sequestration at the end of their life span.     

Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium

The CD11/CD18 family of glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to inflammatory responses. Using a monoclonal antibody (MoAb) against CD18 (TS1/18), the role of these molecules in polymorphonuclear neutrophil (PMNL) adhesion to cultured primary human umbilical vein endothelial cells (HUVEC) was examined under venous flow conditions. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to interleukin-1 (IL-1)-treated HUVEC at 2.0 dynes/cm2 (TS1/18-treated 305 +/- 58 PMNL/mm2 v 334 +/- 63 PMNL/mm2 on control). Furthermore, incubation of HUVEC with R6.5.D6, an MoAb against intercellular adhesion molecule-1 (ICAM-1) did not significantly inhibit PMNL adhesion to IL-1-treated HUVEC at 2.0 dynes/cm2 (P greater than .3). In contrast to the lack of inhibition of adhesion under conditions of flow, incubation of PMNL with TS1/18 reduced PMNL adherence in static adhesion assays. PMNL migration beneath HUVEC monolayers has been shown to be stimulated by 4-hour IL-1 treatment. TS1/18 and R6.5.D6 significantly inhibited migration of PMNL beneath IL-1-treated HUVEC monolayers under flow conditions by slightly more than 80% (P less than .005). In flow experiments with CD18-deficient PMNL, virtually no transendothelial migration was observed. The effect of FMLP (10(-8) mol/L) on PMNL adhesion to untreated HUVEC at wall shear stresses ranging from 0.25 to 2.0 dynes/cm2 was also investigated. FMLP had little effect on PMNL adherence at shear stresses above 0.5 dynes/cm2 (P greater than .45). In response to FMLP exposure at lower wall shear stresses, PMNL adherence to untreated HUVEC increased 6.9-fold at 0.5 dynes/cm2 (P less than .001). At 0.25 dynes/cm2, FMLP stimulation increased PMNL adherence to untreated HUVEC 6.5-fold compared with controls (P less than .005), and FMLP failed to make CD18-deficient PMNL more adherent. In experiments with PMNL pretreated with TS1/18 (anti-CD18), there was a 67% inhibition of FMLP-stimulated adhesion at 0.5 dynes/cm2 (P less than .025). The upper threshold of CD18-mediated PMNL adhesion appears to be between 0.5 and 1.0 dyne/cm2. Above these wall shear stresses, the initial attachment of PMNL to cultured endothelium was mediated almost exclusively by CD18-independent mechanisms. By simulating some of the flow parameters in the microcirculation with well-characterized shear forces, PMNL adhesion by CD18-independent and dependent mechanisms can be differentiated. These data also indicate that CD18 is an important mediator of transendothelial migration by PMNL, which have attached to the endothelium by a CD18-independent mechanism.     

Myeloperoxidase-mediated modulation of chemotactic peptide binding to human neutrophils

Methionine-containing chemotactic peptides, such as formyl-methionyl- leucyl-phenylalanine (FMLP), are inactivated via a neutrophil-derived, myeloperoxidase-mediated oxidation of the methionine residue. We report that extracellular inactivation of FMLP by myeloperoxidase modulates the apparent binding of methionine-containing chemotactic peptides to their surface receptors. Inhibitors of myeloperoxidase enhanced FMLP binding. At subsaturating concentrations of 3H-FMLP (20 nM), 1 mM cyanide (KCN) increased the binding of 3H-FMLP to human neutrophils (PMN) by 51% +/- 12%. Similar increases occurred with 0.1 mM azide and 10 mM aminotriazole (ATZ). KCN had little effect on maximal 3H-FMLP binding to PMN at saturation (control-17,040 +/- 910 receptors/PMN; KCN- 16,820 +/- 1,940 receptors/PMN), but decreased the concentration of 3H- FMLP required to half-saturate the PMN receptors (control-39 +/- 3 nM; KCN-17 +/- 1 nM). ATZ gave similar results. The binding to PMN of the non-methionine-containing chemotactic peptide 125I-formyl-norleucyl- leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (125I-FNLPNTL) was unaltered by KCN. Also, the binding of 3H-FMLP to myeloperoxidase- deficient PMN was unaltered by KCN. Both KCN and ATZ decreased the oxidation of FMLP by PMN. Finally, ATZ (but not KCN) enhanced the chemotactic migration of PMN in response to submaximal concentrations of FMLP. These studies show that intact PMN inactivate methionine- containing chemotactic peptides by a pathway that is sensitive to myeloperoxidase inhibitors and is absent in myeloperoxidase-deficient PMN. This action results in an apparent decrease in the affinity of the chemotactic peptide receptor for methionine-containing chemotactic peptides, which may modulate chemotatic events in inflammatory loci.     

Effect of thoracotomy and cardiopulmonary bypass on activated platelet and neutrophil dynamics and platelet emboli in a pig model

The effects of thoracotomy and components of extracorporeal circuits on dynamics of platelets and neutrophils were quantified with autologous In-111-labeled platelets (INPLT) and neutrophils (INN) during cardiopulmonary bypass (CPB) operations in Yorkshire pigs. Cardiopulmonary bypass was carried out with a hollow-fiber oxygenator and an arterial filter in 48 pigs (30–35 kg); 12 unoperated controls for platelets and neutrophils; 12 sham operated controls; 12 with 180 minutes of CPB with platelets and neutrophils; 12 with 90 minutes of CPB and 90 minutes of reperfusion at 2.5–3.5 one/min. Platelets and neutrophils were labeled with In-111 tropolone and were injected intravenously: platelets at 24 hours and neutrophils at 15 minutes before CPB. All pigs were systemically heparinized [activated coagulation time (ACT) > 400 seconds]; CPB was instituted with a roller pump, oxygenator (OX; Bentley Univox, 1.8 m²), and arterial filter (AF; 0.025 m²) for durations of 180 minutes and 90 minutes of bypass, followed by 90 minutes of reperfusion. The kinetics and pooling of platelets and neutrophils were monitored by a Geiger probe. The adherent thrombi and neutrophils in the OX, AF, viscera, and brain were imaged with a gamma camera and were measured with an ion chamber and a gamma counter. The percentile distribution of labeled platelets and neutrophils expressed as the mean ± standard deviation of injected dose in eight groups was calculated and statistical analyses were performed (ANOVA and paired t-test). Sham operation alone increased platelet retention in the lung, heart, and brain significantly (p < 0.001) over that of unoperated pigs. Neutrophil margination to lung immediately after injection was high; CPB and reperfusion altered the distribution in blood, viscera, and connective tissues. During CPB, an equilibrium among single platelets, platelet thrombi, and emboli was reached in the blood, oxygenator, arterial filter, perfused organs, and tissues. After CPB, the pulmonary neutrophil retention increased significantly (p < 0.001). Reperfusion of 90 minutes following 90 minutes of CPB decreased the level of neutrophils and increased the level of platelets in the lung. Only a small amount of platelets and neutrophils was retained in the oxygenator and arterial filter. Neutrophil retention in the OX and AF was higher than that of platelets. The small amount of retained neutrophils in the heart, kidneys, and brain suggested that cytokines, rather than marginated neutrophils alone, may play a major role in inflammatory insult to these organs during and after CPB. OX thrombi increased with the time of CPB; AF thrombus in both groups was almost similar. During CPB, AF functioned minimally as a thrombus trap with a small percent of retained thrombi; reperfusion post-CPB did not change the amount. Thoracotomy alone has a significant effect on platelet and neutrophil kinetics, and on the subsequent effect of thrombus formation, embolization, and neutrophil margination in organs during the CPB procedure.     

Vaso-occlusive crisis-associated neutrophil dysfunction in patients with sickle-cell disease

Polymorphonuclear leukocyte (PMN) aggregation and chemotaxis were studied in 27 patients with sickle cell disease (SCD). Pain-free patients with SCD had a significantly impaired aggregation response to stimulation with n-formylmethionyl-leucyl-phenylalanine (FMLP) with or without cytochalasin B (CB), compared with normal volunteers (p less than 0.001). Patients with SCD in vaso-occlusive crisis had PMN aggregation induced by FMLP with or without CB that was significantly increased compared with the cohort of pain-free SCD patients (p less than 0.001). PMN from pain-free patients had normal chemotaxis, whereas patients in vaso-occlusive crisis had a significant impairment in PMN chemotaxis. PMN chemotaxis was inversely related to the PMN aggregation response to FMLP with CB (r = -0.75). Thus, the PMN from pain-free patients with SCD appears to have normal or decreased "stickiness" and to develop increased stickiness during vaso-occlusive crisis. The mechanisms responsible for these changes need further elucidation. Alterations in PMN function may be responsible, in part, for the increased risk of infection noted in individuals with SCD and may play a role in the development of the acute chest syndrome.     

111In-oxine platelet survivals in thrombocytopenic infants

Thrombocytopenia is a common occurrence (20%) in sick neonates, but the causes have not been well studied. In this report we demonstrate that thrombocytopenia in the neonate is characterized by increased platelet destruction as shown by shortened homologous 111In-oxine-labeled platelet life spans. Thirty-one prospectively studied thrombocytopenic neonates were investigated by measuring the 111In-labeled platelet life span, platelet-associated IgG (PAIgG), and coagulation screening tests. In every infant, the thrombocytopenia was shown to have a destructive component since the mean platelet life span was significantly shortened to 65 +/- 6 (mean +/- SEM) hours with a range of one to 128 hours compared with adult values (212 +/- 8; range, 140 to 260; gamma function analysis). The platelet survival was directly related to the lowest platelet count and inversely related to both the highest mean platelet volume and duration of the thrombocytopenia. In 22 infants the percent recovery of the radiolabeled platelets was less than 50%, which suggested that increased sequestration also contributed to the thrombocytopenia. Infants with laboratory evidence of disseminated intravascular coagulation (n = 8) or immune platelet destruction evidenced by elevated levels of PAIgG (n = 13) had even shorter platelet survivals and a more severe thrombocytopenia compared with the ten infants in whom an underlying cause for the thrombocytopenia was not apparent. Full-body scintigraphic images obtained in 11 infants showed an increased uptake in the spleen and liver, with a spleen-to- liver ratio of 3:1. This study indicates that thrombocytopenia in sick neonates is primarily destructive, with a subgroup having evidence of increased platelet sequestration.     

Second malignancies in hairy cell leukemia (leukemic reticuloendotheliosis)

Kinetics andw quantification of the sites of destruction of ¹¹¹-Indium-oxine-labeled autologous platelets were investigated in eight patients with idiopathic thrombocytopenic purpura. The mean platelet count was 17 ± 9 × 10⁹/liter; platelets were separated by differential centrifugation and labeled with 5.6 ± 2.5 MBq ¹¹¹In. Whole body and organ ¹¹¹In-platelet distribution was quantitated with a scintillation camera and a computer-assisted imaging system acquisition matrix. Areas of interest were selected with the computer and organ ¹¹¹In-radioactivity expressed as a percentage of whole body activity. Mean platelet survival was 49.5 ± 29.6 hr and the survival curves were exponential. Equilibrium percentage organ ¹¹¹In-radioactivity was (normal values in parentheses): spleen 33.7 ± 8.8(31.1 ± 10.2); liver 16.1 ± 9.5(13.1 ± 1.3); thorax 22.8 ± 3.7(28.2 ± 5.6). Percentage organ ¹¹¹In-activity at the time when labeled platelets had disappeared from the circulation was: spleen 44.5 ± 16.4 (40 ± 16); liver 16.0 ± 11.5 (32.4 ± 7.2); thorax 19.7 ± 6.0 (17.7 ± 10.3). Thorax activity corresponds to bone marrow radioactivity. Three patterns of platelet sequestration were evident. Three patients had mainly splenic sequestration, two mainly hepatic sequestration, and three diffuse reticuloendothelial system sequestration with a major component of platelets destroyed in the bone marrow. Splenectomy was performed in two patients. The pattern of ¹¹¹In-platelet sequestration was not predictive of response of glucocorticoid therapy or indicative of the necessity for splenectomy. Quantitative ¹¹¹In-labeled autologous platelet kinetic studies provide a new tool for the investigation of platelet disorders.     

Neutrophil activation by Helicobacter pylori.

Helicobacter pylori infection of the stomach is accompanied by a persistent polymorphonuclear leukocyte (PMNL) infiltrate of the mucosa. The aim of this work was to study the activation of human PMNL by substances produced by H pylori. Filtered H pylori conditioned media stimulated a significant PMNL oxidative burst (p less than 0.002). This was equal to 26% of the maximal response stimulated by the PMNL chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 1 mumol/l). The response to FMLP was prolonged by the combined presence of complement inactivated human anti-H pylori plasma and conditioned medium (p less than 0.002). High pressure liquid chromatography of an extract of conditioned medium showed a fraction that stimulated PMNL, eluted, and antigenically cross reacted with FMLP. Washed H pylori cells, and those opsonised with complement inactivated human anti-H pylori plasma, did not induce a significant oxidative burst. Opsonized H pylori, however, prolonged the oxidative burst induced by FMLP (p less than 0.02). In conclusion, H pylori synthesizes and secretes a substance, probably FMLP, that may account for the PMNL accumulation that accompanies H pylori infections. Immune complexes composed of H pylori antigen and specific antibody potentiate the PMNL oxidative burst. This combination of H pylori derived products, and host PMNL and antibodies, may be involved in the mucosal damage observed in H pylori associated gastritis.