Peroxidase activity within circulating neutrophils correlates with pulmonary phenotype in cystic fibrosis

Excess neutrophils are present in the airways of patients with cystic fibrosis (CF). Myeloperoxidase (MPO) activity of acid extracts of sputum is directly correlated with airflow obstruction in CF patients. We hypothesized that the sputum MPO was derived from the MPO of neutrophils that entered the airways from the circulation. Active MPO without protease activity injures airways. If MPO activity from circulating neutrophils that emigrate into the airways of these patients causes increased airway epithelial permeability and mucus-gland secretion, then (1) those patients with greater MPO activity per circulating neutrophil would be more likely to produce sputum and (2) the MPO activity per circulating neutrophil would positively correlate with airflow obstruction. We determined the MPO activity for both circulating and sputum neutrophils. Spirometry and respiratory cultures were obtained simultaneously with blood and sputum samples. CF patients with more MPO activity within their circulating neutrophils were more likely to produce sputum ( P =.001, chi 2 test), and the MPO activity per circulating neutrophil was positively correlated with airflow obstruction as measured on the basis of the ratio of 1-second forced expiratory volume to forced vital capacity ( P <. 03, Kruskal-Wallace test). These associations were independent of age, sex, the results of respiratory-tract culture, or protease activity in the circulating neutrophils. MPO activity in circulating neutrophils from CF patients homozygotic for the deletion of phenylalanine at position 508 in the CF transmembrane regulator protein is directly related to the severity of these patients' pulmonary disease. Our results are consistent with the hypothesis that circulating neutrophils deliver active MPO to the airway, producing airway injury and airflow obstruction in homozygotic delF508 CF patients.     

Neutrophil kinetics in man.

A method has been developed for measuring neutrophil cellularity in normal human bone marrow, in which the neutrophil-erythroid ratio was determined from marrow sections and marrow normoblasts were estimated by the erythron iron turnover. Neutrophil maturational categories, defined by morphologic criteria, were supported by autoradiographs of marrow flashed-labeled with 3H-thymidine. Correction for multiple counting error was empirically derived by counting serial sections through cells of each maturational category. The normal neutrophil-erythroid ratio in 13 normal human subjects was 1.5 +/- 0.07. The mean number of normoblasts in the same subjects was estimated to be 5.07 +/- 0.84 X 10(9) cells/kg. Total marrow neutrophils (X 10(9) cells/kg) were 7.70 +/- 1.20, the postmitotic pool (metamyelocytes, bands, and segmented forms) was 5.59 +/- 0.90 and the mitotic pool (promyelocytes + myelocytes) was 2.11 +/- 0.36. Marrow neutrophil ("total") production has been determined from the number of neutrophils comprising the postmitotic marrow pool divided by their transit time Transit time was derived from the appearance in circulating neutrophils of injected 3H-thymidine. The postmitotic pool comprised 5.59 +/- 0.90 X 10(9) neutrophils/kg, and the transit time was 6.60 +/- 0.03 days. From these data marrow neutrophil production was calculated to be 0.85 X 10(9) cells/kg per day. Effective production, measured as the turnover of circulating neutrophils labeled with 3H-thymidine, was 0.87 +/- 0.13 X 10(9) cells/kg per day. This value correlated well with the calculation of marrow neutrophil production. A larger turnover of 1.62 +/- 0.46 X 10(9) cells/kg per day was obtained when diisopropylfluorophosphate-32P was used to label circulating neutrophils. Studies using isologous cells doubly labeled with 3H-thymidine and diisopropylfluorophosphate-32P demonstrated a lower recovery and shorter t1/2 of the 32P label.     

Neutrophil kinetics in acute infection

Neutrophil kinetics of acute experimental infection were studied with diisopropylfluorophosphate-³²P labeling in 31 dogs inoculated intrabronchially with pneumococci. In vitro neutrophil labeling indicated a rapid transit time through the blood in early infections, with an elevated marginal granulocyte pool sometimes preceding an elevation of the circulating granulocyte pool. 13 hr after infection, the circulating and total blood granulocyte pools were increased but the rate of neutrophil transit through the blood was normal. During the recovery from infection there was a marked prolongation of neutrophil blood transit time, suggesting virtually complete cessation of bone marrow release of neutrophils into the blood. Labeling of neutrophils in vivo indicated an increased rate of emptying of the bone marrow storage pool proportional to the severity of infection as measured by the fever index. The change in the blood ratio of nonsegmented to segmented neutrophils was a much more accurate index of the severity of infection than the blood granulocyte concentration, correlating significantly with the fever index.     

5-Hydroxyeicosanoids selectively stimulate the human neutrophil 15- lipoxygenase to use endogenous substrate

When human neutrophils, prelabeled with [3H]arachidonic acid, were incubated with 5S,15S-dihydroxyeicosatetraenoic acid (5,15-diHETE), a dose-dependent increase in the 15-lipoxygenase product [3H]-15-HETE was observed relative to untreated cells. Typically, a fivefold increase in [3H]-15-HETE formation was obtained upon exposure of these cells to 3 muM 5,15-diHETE. There was no appreciable enhancement of the 5-lipoxygenase metabolite [3H]-5-HETE. Product identities were confirmed by comparing retention times on straight- and reversed-phase HPLC with authentic standards, and RIA. Other 5-hydroxyeicosanoids, such as 5-HETE, 5-HETE methyl ester, and leukotriene B4(5S,12R-diHETE), were equally effective in stimulating the formation of [3H]-15-HETE, but exogenously added lipoxin A4, lipoxin B4, 15-HETE, and 12-HETE were much less potent, whereas stearic acid was ineffective. The diHETEs also showed a greater selectivity in activating the 15-lipoxygenase relative to the 5-lipoxygenase. A likely source of substrate for the 15- and 5-lipoxygenases is a pool of cell-associated but noncovalently bound arachidonic acid. In [3H]arachidonic acid-prelabeled neutrophils, the amount of free [3H]arachidonic acid ranged between 50 and 700 fmol/10(7) cells, whereas unlabeled neutrophils contained 100-2,200 pmol/10(7) cells of nonesterified arachidonic acid. The exogenously added hydroxyeicosanoids induce a 0.5-3% conversion of this substrate pool to product. These findings indicate that the 15-lipoxygenase in human neutrophils is a cryptic enzyme that needs to be stimulated in order to metabolize endogenous substrate. It is possible that 5-hydroxyeicosanoids may mimic an as yet unidentified physiological activator of the 15-lipoxygenase.     

Eosinophil kinetics in the hypereosinophilic syndrome.

The kinetics of 51-chromium-labeled eosinophils were studied in 6 patients with the idiopathic hypereosinophilic syndrome. The kinetics of these cells were compared with 51-chromium-labeled neutrophils of 9 normal subjects. The patient studies consistently showed that autologous labeled eosinophils transiently left the circulating cell pool in the first 3 hours after infusion, re-entered the circulating pool, and then disappeared from the circulation with a mean blood half-life of 44 +/- 2.0 hours. In contrast, the neutrophils of normal subjects left the circulation progressively with an estimated blood half-life of 12.4 +/- 2.0 hours. These data suggest that the leukocytosis of the hypereosinophilic syndrome may be due to the presence in the blood of an increased number of cells with a relatively long blood half-life.     

Blood lactoferrin release induced by running exercise in normal volunteers: antibacterial activity

BACKGROUND: The aim of this study was to determine serum lactoferrin concentrations and serum antibacterial activity before and after running exercise. METHODS: Twenty-four healthy young men were randomly assigned to high, middle, or low intensity of exercise groups (5000 steps running at 180, 130, and 80 steps/min, respectively). Blood samples were collected at baseline and immediately, 1 and 4 h after exercise. Concentrations of circulating neutrophils, serum lactoferrin, iron in whole blood, and serum iron were measured. Antibacterial activity of serum was evaluated using live Micrococcus luteus. RESULTS: The numbers of circulating neutrophils were increased by 20.0% and 15.5% 1 h after exercise in high and middle groups (both P<0.01), respectively. Serum lactoferrin concentrations were significantly increased immediately after exercise by 48.3% and 33.0% in the high and middle groups (both P<0.01), respectively. No significant changes in total iron or serum iron concentrations were observed during the study. Antibacterial activities of serum collected immediately after exercise in the high and middle groups were significantly stronger than those before exercise, by 31.2% and 25.4% (both P<0.05), respectively. CONCLUSIONS: Serum lactoferrin concentrations are increased immediately after running exercise and may play an antibacterial role in host defenses before mobilization of neutrophils into the circulating pool.     

Neutropenia, Inflammation, and the Kinetics of Transfused Neutrophils in Rabbits

A rabbit model was used to study the effects of neutropenia and inflammation on the intravascular distribution, survival, and tissue accumulation of transfused neutrophils. Donor blood labeled with [(3)H]thymidine was infused into normal or neutropenic (vinblastine treated) animals. Inflammation was created by subcutaneous implantation of polyvinyl sponges, some with added endotoxin. Initial circulating neutrophil pool recovery, survival, and inflammatory site accumulation of labeled neutrophils were measured.Neutropenia was associated with a relative increase in the marginal pool size, manifested by a diminished initial circulating pool (CNP) recovery of transfused cells. The CNP recovery was directly proportional to recipient neutrophil count. Neutropenia had no effect on the intravascular survival of transfused cells and was accompanied by only a modest decrease in the inflammatory site recovery of the transfused neutrophils (10.4+/-5.4 vs. 14.4+/-4.0% in normals).Inflammation in the form of subcutaneous polyvinyl sponges was accompanied by an increase in margination with initial CNP recoveries of 24.3+/-4.7 and 27.6+/-8.8% at zero and 4 h after implantation respectively (normal, 38.2+/-9.9%). Transit through the CNP was hastened by inflammation with a t((1/2)) of 2.02+/-0.72 h (normal, 3.2+/-1.0 h).Addition of endotoxin to the sponges further perturbed cell kinetics. CNP recoveries were considerably lower and half-lifes were initially shorter and subsequently uninterpretable in studies done after endotoxin sponge insertion. Inflammatory site accumulation was markedly diminished to 7.4+/-1.9% of injected neutrophil label in the endotoxin sponge animals, suggesting that many of the transfused cells were functionally unavailable rather than marginated. These studies demonstrate that neutropenia and inflammation with or without endotoxin markedly alter the kinetics of transfused neutrophils and that CNP recovery of transfused cells is not necessarily predictive of their inflammatory site accumulation.     

Neutrophil modulation of the pulmonary chemokine response to lipopolysaccharide

When cells within the intrapulmonary compartment are exposed to pathogens or their products such as lipopolysaccharide, they produce CXC chemokines in order to attract circulating neutrophils into the lower respiratory tract. Previous studies have shown that as neutrophils (PMNs) enter the lung, bronchoalveolar lavage (BAL) chemokine levels are decreased. In this study, we determined the intrapulmonary and systemic responses to two important rat chemokines, cytokine-induced neutrophil chemoattractant (CINC) and macrophage inflammatory protein-2 (MIP-2), to intratracheal (i.t.) LPS (100 microg in 0.5 mL of phosphate-buffered saline) under neutropenic (cyclophosphamide [CPA]) and neutrophilic (G-CSF) conditions. By 4 h after i.t. LPS, CPA pretreatment decreased PMN recruitment 83% and G-CSF increased PMN recruitment 91% compared with recruitment into the lung in vehicle-pretreated rats (42.7 +/- 19.3 million PMNs). Neutropenic rats had increased CINC and MIP-2 concentrations in BAL fluid 4 h after i.t. LPS when compared with levels seen in vehicle controls (P < 0.05). In vitro LPS-stimulated chemokine production by alveolar macrophages obtained from CPA- and vehicle-pretreated animals did not differ. The increase in BAL fluid chemokine levels in neutropenic rats corresponded to increased chemotaxis of neutrophils to BAL fluid from CPA-pretreated rats as compared with the chemotaxis response of PMN to BAL fluid from vehicle-pretreated rats. In contrast, G-CSF enhancement of neutrophil recruitment decreased chemotactic activity of BAL fluid collected 4 h after i.t. LPS. These data show that as neutrophils are recruited into the lung, they alter chemokine levels, which most likely serves to down-regulate the inflammatory response.     

A Comparative Evaluation of Inpatient Rehabilitation for Older Adults With Debility, Hip Fracture, and Myopathy

Kortebein P, Granger CV, Sullivan DH. A comparative evaluation of inpatient rehabilitation for older adults with debility, hip fracture, and myopathy.

Objective

To compare the functional outcomes and discharge location of older adults admitted to inpatient rehabilitation for debility, hip fracture, and myopathy.

Design

Retrospective cohort study from 2002 to 2003 with information from the Uniform Data System for Medical Rehabilitation (UDSMR).

Setting

United States inpatient rehabilitation facilities subscribing to the UDSMR.

Participants

Patients 65 years or older (N=84.701) with primary diagnoses of debility (n=14,835), hip fracture (n=68,915), and myopathy (n=951).

Interventions

Not applicable.

Main Outcome Measures

Change in functional status, including efficiency (change in functional status divided by length of stay in days) and discharge setting.

Results

The efficiency of the patients with debility (1.7±2.1) was significantly lower than that of the patients with hip fracture (1.9±1.6; P<.001), but not different from the patients with myopathy (1.6±1.4; P=.3). Significantly more patients with debility (68%) were discharged home than the hip fracture and myopathy groups (66% and 65%, respectively; P<.001).

Conclusions

Although statistical differences exist, the functional recovery and rate of discharge home of older adult patients admitted to inpatient rehabilitation with a primary debility diagnosis are essentially the same clinically as those of patients with a diagnosis of either hip fracture or myopathy. Given these findings, and given that hip fracture and myopathy are approved medical conditions according to the Centers for Medicare and Medicaid Services 75% rule, the medical condition debility warrants consideration for inclusion as a qualifying medical diagnosis under this rule. However, further research is needed to develop relatively objective criteria for the debility diagnosis, and to identify those patients with debility who are most likely to benefit from inpatient rehabilitation.     

Proteolysis by Neutrophils: RELATIVE IMPORTANCE OF CELL-SUBSTRATE CONTACT AND OXIDATIVE INACTIVATION OF PROTEINASE INHIBITORS IN VITRO

Polymorphonuclear leukocytes have been implicated in connective tissue injury in a variety of disease processes. To gain insight into mechanisms by which neutrophils might degrade connective tissue macromolecules in the presence of proteinase inhibitors, we have used a model system that allows neutrophils to be held in vitro under physiologic conditions in close proximity to a very proteinase-sensitive substrate, (125)I-labeled fibronectin. We have found: (a) neutrophils spread rapidly on the fibronectin substrate; (b) fibronectin proteolysis by neutrophils is largely attributable to released elastase, and is linearly related to cell number over the range of 2,000 to 30,000 cells per assay; (c) oxidants released from neutrophils stimulated by opsonized zymosan or phorbol myristate acetate do not protect released elastase from inhibition by alpha(1)-proteinase inhibitor or alpha(2)-macroglobulin; (d) neutrophil myeloperoxidase and enzymatically generated superoxide anion render alpha(1)-proteinase inhibitor ineffective against fibronectin proteolysis when neutrophils are added 30 min later; and (e) alpha(1)-proteinase inhibitor and alpha(2)-macroglobulin incompletely inhibit fibronectin proteolysis by neutrophils (79.8+/-6.3 and 73.5+/-12.0%, respectively.) The data suggested that proteolysis due to neutrophils that are in contact with susceptible macromolecules may occur due to partial exclusion of inhibitors from the cell-substrate interface. Although confirming that alpha(1)-proteinase inhibitor is ineffective against neutrophil-derived proteolysis after exposure to oxidants, these studies did not support the hypothesis that oxidants released from stimulated neutrophils enhance activity of proteinases they release in the presence of alpha(1)-proteinase inhibitor. We anticipate that further studies with this test system will be helpful in defining conditions that modulate inflammatory connective tissue injury in diseases such as pulmonary emphysema and rheumatoid arthritis.     

A highly sensitive fluorimetric assay for pyridoxal phosphate phosphatase

A highly sensitive fluorimetric assay for pyridoxal phosphate phosphatase is described. The assay involves separation of the substrate and product by ion-exchange chromatography followed by treatment of pyridoxal with potassium cyanide under slightly alkaline conditions to form 4-pyridoxolactone, a highly fluorescent compound. Certain kinetic properties of the enzyme activities in human neutrophils are described.     

Analysis of the expression of NB1 antigen using two monoclonal antibodies

BACKGROUND: Neutrophil-specific antigen NB1 is expressed on neutrophil subpopulations in 97 percent of healthy individuals and is located on 56- to 64-kDa glycoprotein. While the molecule carrying NB1 has been identified, the nature of the NB1 epitope has not been well characterized. STUDY DESIGN and METHODS: Two monoclonal antibodies (MoAbs), 1B5 and the recently produced 7D8, and four alloantibodies, all specific for NB1, were used to investigate the expression of NB1 on neutrophils from several donors. RESULTS: MoAb 7D8 was shown to be specific for NB1. It reacted with NB1-positive neutrophils from 52 donors in the granulocyte immunofluorescence assay and did not react with NB1-negative neutrophils from 8 donors. MoAb 7D8 immunoblotted a 56- to 64-kDa molecule on neutrophils from eight NB1-positive donors and did not react with this molecule on NB1-negative neutrophils from two donors. When 7D8 was tested in the monoclonal antibody immobilization of granulocyte antigens assay, it reacted with two NB1 alloantibodies, but not with NA1 or NA2 alloantibodies. To determine if MoAbs 7D8 and 1B5 recognized the same epitope, both were tested against the same NB1-positive neutrophils and the cells were analyzed by two- color flow cytometry. Both antibodies bound independently to neutrophils, which indicated that the antibodies recognized different epitopes. When similar studies were performed with MoAb 7D8 and three NB1 alloantibodies, 7D8 partially inhibited the binding of two of the alloantibodies. The size of the NB1-positive subpopulation was analyzed in 25 people using flow cytometry with both MoAbs and three alloantibodies. The subpopulation of antigen-positive cells was similar in all donors when 7D8 and the three NB1 alloantibodies were tested; however, the subpopulation recognized by MoAb 1B5 was smaller in two of the donors. Neutrophils from one of these people were analyzed by immunoblotting, and no differences were detected in the molecule carrying NB1 in those neutrophils and that molecule in control neutrophils. CONCLUSION: NB1 specificity is made up of at least two separate epitopes. The expression of NB1 varied among antigen-positive individuals. While NB1 is expressed by a 56- to 64-kDa glycoprotein, the structure of this protein on antigen-negative cells has not been determined.     

The expression of the NB1 antigen on myeloid precursors and neutrophils from children and umbilical cords

The neutrophil-specific antigen NB1 is expressed by neutrophils from 97% of healthy adults. However, membrane expression of this molecule is unique in that it is found on only a subpopulation of neutrophils present in NB1-positive adults. We have investigated the ontogeny of NB1 antigen expression by haematopoietic progenitor cells to determine the stage and pattern of antigen expression during granulocytic cell differentiation. In addition, we examined whether the ontogeny and frequency of granulocytic cells expressing the NBl antigen might vary in subjects according to age. A monoclonal antibody (MoAb) specific for NB1 (1B5) and flow cytometry was used to assess the frequency and characteristics of the NB1-positive cells found in umbilical cord blood (n = 11), children (n = 37), healthy adults (n = 46) and patients with chronic myelogenous leukaemia (n = 8). We also used flow cytometry to isolate NB1-positive and NB1-negative bone marrow and peripheral blood cells from various tissue sources. The separated subpopulations were then analysed by Wright stain and light microscopy. The size of the NB1-positive neutrophil subpopulation in 46 healthy adults (56 ± 19%) was identical to that found for neutrophils from 36 children ranging in age from 8 months to 18 years (56 ± 11%). In contrast, expression of the NB1 antigen by the neutrophils present in umbilical cord blood (91 ± 3%, n = 11) was significantly greater than that in adults (P < 0.002) or children (P < 0.002). We also examined the size of the NB1-positive subpopulation among neutrophils from eight patients with chronic myelogenous leukaemia (CML). The NB1-positive subset in CML subjects (29.5 ± 22.4%) was significantly less that in healthy adults (P < 0.02) or children (P < 0.02). Marrow cells from eight adults were similarly separated and analysed. We found that 69 ± 17% of segmented and band forms of neutrophils, 70 ± 2% of metamyelocytes and 61 ± 23% of myelocytes were NB1-positive. In fetal bone marrow, 86 ± 9% of the segmented and band forms, 82 ± 10% of the metamyelocytes and 3 ± 4% of myelocytes were NB1-positive. In conclusion, neutrophil-specific antigen NB1 is first expressed at the myelocyte stage of myeloid differentitation. In adult bone marrow, the percentages of myelocytes, metamyelocytes and segmented or band cells that expressed this antigen were similar and comparable in magnitude to the frequency of NB1-positive neutrophils found in the circulation. Although the size of the NBl-positive neutrophil subpopulation was the same in healthy adults and children, it was significantly increased in umbilical cord blood, and in fetal marrow cells.     

Effects of adrenaline on glucose and glutamine metabolism and superoxide production by rat neutrophils.

Despite the large body of information on the role of corticosteroids in regulating lymphocyte and phagocyte function, the role of the hormone adrenaline in immunoregulation is an under-investigated topic. The present study has addressed the effects of adrenaline on the rates of utilization and oxidation of glucose and glutamine, the phagocytic capacity and the rate of superoxide production by rat neutrophils. Incubation of rat neutrophils in the presence of 50 microM adrenaline caused a marked elevation in glucose metabolism, an effect that could be blocked by propranolol. Adrenaline caused a partial inhibition of glutamine utilization by neutrophils, an effect that was also blocked by propranolol. These effects of adrenaline could be mimicked by 100 microM dibutyryl cAMP. Phosphate-dependent glutaminase activity was significantly elevated in neutrophils incubated in the presence of 50 microM adrenaline or 100 microM dibutyryl cAMP for 1 h, whereas glutamine oxidation was significantly depressed (P<0.05) under these conditions. The elevation in enzyme activity was only partially blocked by propranolol. The phagocytic activity of rat neutrophils was not altered by adrenaline in the presence of either glucose or glutamine. The rate of phorbol 12-myristate 13-acetate-induced superoxide production in the presence of glucose was potently reduced by the addition of 5 nM or 50 microM adrenaline. This effect could be mimicked by dibutyryl cAMP. However, when rat neutrophils were incubated in the presence of glutamine plus adrenaline (5 nM or 50 microM), the rate of superoxide production was only marginally reduced. These findings support the proposition that adrenaline may deviate the flux of glucose from the NADPH-producing pentose phosphate pathway, thus reducing substrate availability for the superoxide-generating NADPH oxidase. However, glutamine metabolism may still give rise to substantial quantities of NADPH from the glutaminolysis pathway. We postulate that glutamine metabolism may thus provide a protective mechanism against the inhibitory effect of adrenaline on superoxide production by neutrophils.     

Circulating anti-neutrophil cytoplasmic autoantibodies in subglottic stenosis: a useful aid in diagnosing vasculitis in this condition?

Five patients with subglottic stenosis, occurring either as a presenting symptom or as a manifestation in the course of a systemic disease, are described. Indirect immunofluorescence revealed the presence of circulating autoantibodies against both cytoplasmic and perinuclear constituents of neutrophils in all five. Antibodies directed against a 29 kDa antigen of the azurophilic granules (two patients), against myeloperoxidase (one patient), and against both the 29 kDa antigen and myeloperoxidase (one patient) were found by enzyme-linked immunosorbent assay. These autoantibodies have previously been found in patients with Wegener's granulomatosis, microscopic polyarteritis, (idiopathic) glomerulonephritis and Churg-Strauss syndrome. However, only one of these five patients fulfilled the criteria for these conditions. Since these autoantibodies are seldom observed in other conditions, and other diseases had been excluded by careful evaluation, we suggest that their presence places subglottic stenosis within the spectrum of necrotizing (granulomatous) vasculitis. Whether immunosuppressive therapy is always warranted in patients with subglottic stenosis and circulating anti-neutrophil cytoplasmic antibodies is a matter of debate.     

Motility and Adhesiveness in Human Neutrophils: EFFECTS OF CHEMOTACTIC FACTORS

Human peripheral blood neutrophils (PMN) obtained from healthy adults were examined in vitro with techniques adapted to assess the effects of chemotactic factors (CF) on cellular configuration and adhesiveness. The results were compared with those that use certain conventional techniques for assessing chemotaxis and chemokinesis. Exposure of PMN to N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe), zymosan-activated serum, bacterial chemotactic factor, or a low molecular weight chemotactic factor from activated serum (C5a) in the absence of a gradient resulted in a change in cellular shape from a spherical to a polarized configuration in a high percentage of cells. This occurred rapidly in suspension, under conditions designed to exclude a role for cell adhesiveness, and was reversible upon removal of the CF. Restimulation of cells with the CF resulted in reappearance of the polarized configuration to the same extent as on initial stimulation with one exception: f-Met-Phe pretreated cells failed to respond to f-Met-Phe, though they responded fully to the other CF. Each CF caused a significant increase in PMN attachment to protein-coated glass. This enhanced adhesiveness was not reversible upon removal of the CF when the cells were treated under conditions shown to produce chemotactic deactivation. Cells treated under these conditions also exhibited significantly reduced motility on glass and in micropore filters in the absence of a gradient of CF. Bacterial chemotactic factor, even at high concentrations, failed to produce deactivation and did not cause a sustained enhancement of adhesiveness.     

In-vivo Myocardial Substrate Alteration during Perfused Ventricular Fibrillation

OBJECTIVES: Earlier work suggests the in-vivo heart alters its substrate utilization as a function of cardiac work. Previous work has also demonstrated the high oxygen requirements of the heart during ventricular fibrillation (VF). The authors hypothesized that myocardial substrate utilization during VF with perfusion is similar to the normal beating heart under conditions of increased workload. METHODS: Myocardial substrate selection was studied in the in-vivo porcine myocardium using 13carbon nuclear magnetic resonance (13C NMR) under conditions of increased cardiac work (dobutamine group) and VF with extracorporeal perfusion (VF group). Once the animal preparation was completed, metabolic steady state was achieved with the infusion of unlabeled acetate into the left anterior descending (LAD) coronary artery. The infused substrate was then changed to [2-13C] acetate and glutamate pool labeling was monitored by 13C NMR. The glutamate C4 resonance areas at baseline and after intervention of either increased workload (dobutamine group) or perfused VF (VF group) were compared within groups using paired t-tests. RESULTS: Baseline aortic and great cardiac vein lactates, glucose levels, blood gases, hemoglobin levels, and temperatures were similar between groups. In both groups, there was a significant decrease from baseline in the labeling of C4 glutamate peaks (dobutamine group: 20.2+/-14.9 vs 84.7+/-32.7, p = 0.002; and VF group: 49.8+/-24.4 vs 83.9+/-24.4, p = 0.02), indicating selection against acetate oxidation in favor of other endogenous substrates. CONCLUSIONS: In the in-vivo heart, despite the absence of functional contractions, changes in substrate utilization during perfused VF are similar to changes that occur with increased workload in the normal beating heart.     

Marrow erythroid and neutrophil cellularity in the dog.

This paper describes a method for determining the number of marrow erythroid and neutrophil cells in which the cellularity of marrow sections was related to that of the total marrow by radioiron dilution. Tissue sections were prepared from methacrylate-embedded dog marrow biopsies, and neutrophils were identified by staining of their primary granules. After correction of direct section counts for multiple counting error, accurate neutrophil-erythroid ratios were established with a coefficient of variation of less than 10 percent when 10-4 cells were examined. An average neutrophil-erythroid ratio of 1.2 was found in six normal dogs. The total number of nucleated red cells in the dog was 5.48 plus or minus 0.78 times 10-9/kg (plus or minus 1 SD), and the corresponding erythron iron turnover was 0.90 plus or minus 0.11 mg Fe/100 ml whole blood/day. The total number of marrow neutrophils, derived from the neutrophil-erythroid ratio, was 6.6 plus or minus 0.59 times 10-9 cells/kg, of which 1.4 were promyelocytes and myelocytes, 2.3 were metamyelocytes and bands, and 3.0 were segmented neutrophils. Leukopheresis studies were carried out in six dogs to confirm the accuracy of these cellular measurements. Marrow counts showed a mean decrease of 22.7 times 10-9 cells or 35 percent of the postmitotic neutrophil pool, and it was calculated that 10.2 times 10-9 additional cells had been taken from already circulating blood. This estimated deficit of 32.9 times 10-9 was almost identical to the 33 times 10-9 cells actually counted in the removed blood.     

Neutropenia induced by systemic infusion of 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid: correlation with its in vitro effects upon neutrophils.

5(S), 12(S)-Dihydroxy-cis-14,trans-6,8,10-eicosatetraenoate (compound I), 5(S),12(R)-dihydroxy-cis-14,trans-6,8,10-eicosatetraenoate (compound II), and 5(S),12(R)-dihydroxy-cis-6,14,trans-8,10-eicosatetraenoate (compound III) were prepared from rabbit peritoneal neutrophils challenged with arachidonic acid plus ionophore A23187. Each arachidonate metabolite caused rabbit neutrophils to aggregate and, in cells treated with cytochalasin B, release granule-bound enzymes. Compound III was 10- to 100-fold more potent than compounds II and I. When intravenously infused into rabbits at doses of 100--1,000 ng/kg, compound III induced abrupt, profound, transient neutropenia associated with a rapidly reversing accumulation of neutrophils in the pulmonary circulation. This in vivo action correlated closely with the ability of the fatty acid to activate neutrophils in vitro: neutropenia, aggregation, and degranulation occurred at similar doses of stimulus and the rapid, reversing kinetics of the neutropenic response paralleled the equally rapid, reversing formation of aggregates. The fatty acid did not alter the circulating levels of lymphocytes or platelets and did not aggregate platelets in vitro. At comparable doses (i.e., 100--1,000 ng/kg), compounds I and II did not cause neutropenia. Thus, compound III possesses a high degree of structural and target-cell specificity in stimulating neutrophils in vitro and in vivo. Clinical and experimental syndromes associating neutropenia with increased levels of circulating arachidonate metabolites may involve compound III as a mediator of neutrophil sequestration in lung.