Granulocyte Pool Research Articles

Radiation-induced effects on bone marrow of bank voles inhabiting the Chornobyl exclusion zone

Purpose To investigate the effects of chronic exposure to low-dose radiation on bone marrow hematopoiesis of bank voles inhabiting the radioactively contaminated territory of the Chornobyl exclusion zone. Materials and methods Animals were collected within the highly radioactive area of the so-called Red Forest located close to the destroyed 4th reactor of the Chornobyl Nuclear Power Plant. Radioecological investigations included evaluation of radiocontamination of soil samples by 90Sr and 137Cs, levels of incorporated radionuclides in animals’ bodies and organs, as well as the absorbed dose rates. The study of peripheral blood and bone marrow parameters combined with cytogenetic analysis of bone marrow micronucleated polychromatic erythrocytes and standard metaphase test was carried out. Results The blood system of the exposed animals manifested significant changes in peripheral blood parameters (anaemia and leucocyte formula left shift), ineffective differentiation and maturation of bone marrow cells, particularly relevant to the erythroid and granulocyte pools. Increased yields of bone marrow micronucleated polychromatic erythrocytes and chromosomal aberrations, including dicentrics and Robertsonian fusion-like configurations, were revealed. Conclusions Observed disturbances in the bone marrow and peripheral blood suggest functional instability and inefficient compensatory and recovery reactions of the blood system of the bank voles from the contaminated areas of the Chornobyl exclusion zone. We assume that they are the consequences both of direct radiation exposure and hereditary pathological changes that have formed in a number of generations inhabiting radioactively contaminated areas.

Relationship of granulocytic inflammation of bronches with the level of asthma control in patients with cold airway hyperresponsiveness under inhalation therapy of extrafine beclomethasone/formoterol

Introduction. Obstruction of small airways in asthma is accompanied by greater hypersensitivity to nonspecific irritants and inflammation activity, which is associated with a worsening of the clinical course of the disease. The combination of beclomethasone dipropionate/formoterol fumarate (BDP/FF), used in the form of an extrafine metered dose aerosol, is capable of having an anti-inflammatory effect on the small airways. Dynamic changes in the structure and function of granulocytes of inflammatory bronchial infiltrate after exposure to cold air in patients with asthma treated with BDP/FF have been little studied.Aim. To study the nature of changes in eosinophil and neutrophil pools of bronchial granulocytes in asthma patients with cold airway hyperresponsiveness (CAHR) during long-term therapy with extrafine BDP/FF.Materials and methods. The clinical study involved 25 asthma patients with CAHR. The design of the work included a questionnaire survey of patients using the Asthma Control Test (ACT) questionnaire, spirometry (Easy on PC, ndd Medizintechnik AG, Switzerland), an isocapnic hyperventilation by cold air (IHCA), collection and cytological examination of induced and spontaneously produced sputum. The examination was carried out at the beginning and after 12 weeks of BDP/FF therapy (100/6 μg, twice a day).Results. 12-week treatment with extrafine BDP/FF led to an improvement in asthma control (ACT increased from 17.1±1.1 to 22.5±0.5 points, p˂0.001) and patency of small bronchi (increase in МEF50 was 0.47±0.21 L/s, МEF25-75 – 0.42±0.17 L/s). BDP/FF therapy had a positive effect on cellular inflammation, leading to a decrease in the number of eosinophils in sputum from 9.5 (3.0; 19.5) to 2.2 (1.3; 4.7)% (p˂0,05). A decrease in the number of eosinophils was also observed after cold bronchoprovocation: from 9.0 (2.8; 15.4) to 4.7 (2.8; 7.8)% (p˂0.05). There was no dynamics in neutrophils after BDP/FF treatment before and after IHCA (59.9±1.3 and 57.1±2.0%, respectively, p>0.05).Conclusion. Improvement of disease control in asthma patients with CAHR after 12 weeks of therapy with extrafine BDP/FF is associated with the regulation of eosinophilic inflammation, a decrease in the number of eosinophils in the airways and suppression of the activity of the neutrophilic pool of bronchial granulocytes in response to cold bronchoprovocation.

Radiolabelled leucocytes in human pulmonary disease.

Radionuclides for leucocyte kinetic studies have progressed from non-gamma emitting cell-labelling radionuclides through gamma emitting nuclides that allow imaging of leucocyte kinetics, to the next goal of positron emission tomography (PET). Mostly the authors' own studies, following on from studies of the early pioneers. From early imaging studies, it appeared that the majority of the marginated granulocyte pool was located in the lungs. However, later work disputed this by demonstrating the exquisite sensitivity of granulocytes to ex vivo isolation and labelling, and that excessive lung activity is artefactual. Following refinement of labelling techniques, it was shown that the majority of marginated granulocytes are located in the spleen and bone marrow. The majority of leucocytes have a pulmonary vascular transit time only a few seconds longer than erythrocytes. The minority showing slow transit, ~5% in healthy persons, is increased in systemic inflammatory disorders that cause neutrophil priming and loss of deformability. Using a range of imaging techniques, including gamma camera imaging, whole-body counting and single photon-emission computerized tomography, labelled granulocytes were subsequently used to image pulmonary trafficking in lobar pneumonia, bronchiectasis, chronic obstructive pulmonary disease and adult respiratory distress syndrome. More recently, eosinophils have been separated in pure form using magnetic bead technology for the study of eosinophil trafficking in asthma. These include advancement of eosinophil imaging, development of monocyte labelling, development of cell labelling with PET tracers and the tracking of lymphocytes.

Substantial changes in hemocyte parameters of Manila clam Ruditapes philippinarum two years after the Hebei Spirit oil spill off the west coast of Korea

Two years after the Hebei Spirit oil spill occurred off the west coast of Korea, we determined sub-lethal effects of the spilled oil on hemocyte parameters of Ruditapes philippinarum in the damaged areas. Clams in the spilled sites displayed unusually high proportion of granulocytes, which may result in higher phagocytosis capacity and reactive oxygen species production. Hemocytes in clams from the polluted sites also displayed less DNA damage and mortality than in the control site, possibly due to a faster phagocytosis of the impaired cells. Glycogen, the major energetic reserve, was depleted in clams from the spilled sites, potentially due to energetic consumption for maintenance of a large pool of granulocytes, detoxification processes and oxidative stress. Modified hemocyte parameters in clams in the spilled area, may reflect sub-lethal physiological stresses caused by the residual oils in the sediment, in conjunction with environmental modifications such as food availability and pathogens pattern.

Neutrophil Phenotypic Characteristics in Children with Congenital Asplenia and Splenectomized for Hereditary Spherocytosis

The spleen plays an important role in the granulocyte homeostasis due to such mechanisms as pooling, elimination of senescent cells and regulatory effects on granulocyte renewal in the bone marrow. The expression profile of granulocyte receptors was tested in children with congenital asplenia, and splenectomized for spherocytosis. Receptors tested included those appearing with maturation (CD16, CD11b, CD11c, TREM-1), disappearing (CD54, CD49d, CD64) and maintained during maturation (CD11a, CD45). In general, we found that the circulating granulocyte pool in the asplenic patients had phenotypical features of highly matured but not apoptotic neutrophils with a significantly elevated expression of CD16 (CD16high), tendency to a lower expression of CD45 (CD45low) and an unchanged expression of CD64 (and other markers indicating systemic inflammatory reactions). The high fluorescence intensity of CD11b,c, and TREM-1 in the congenital asplenia may indicate a potentially elevated pro-inflammatory status of granulocytes, possibly due to the low activity of vagus nerve and spleen-dependent cholinergic anti-inflammatory pathway.

Discovery of Mouse Spleen Signaling Responses to Anthrax using Label-Free Quantitative Phosphoproteomics via Mass Spectrometry

Inhalational anthrax is caused by spores of the bacterium Bacillus anthracis (B. anthracis), and is an extremely dangerous disease that can kill unvaccinated victims within 2 weeks. Modern antibiotic-based therapy can increase the survival rate to ∼50%, but only if administered presymptomatically (within 24-48 h of exposure). To discover host signaling responses to presymptomatic anthrax, label-free quantitative phosphoproteomics via liquid chromatography coupled to mass spectrometry was used to compare spleens from uninfected and spore-challenged mice over a 72 h time-course. Spleen proteins were denatured using urea, reduced using dithiothreitol, alkylated using iodoacetamide, and digested into peptides using trypsin, and the resulting phosphopeptides were enriched using titanium dioxide solid-phase extraction and analyzed by nano-liquid chromatography-Linear Trap Quadrupole-Orbitrap-MS(/MS). The fragment ion spectra were processed using DeconMSn and searched using both Mascot and SEQUEST resulting in 252,626 confident identifications of 6248 phosphopeptides (corresponding to 5782 phosphorylation sites). The precursor ion spectra were deisotoped using Decon2LS and aligned using MultiAlign resulting in the confident quantitation of 3265 of the identified phosphopeptides. ANOVAs were used to produce a q-value ranked list of host signaling responses. Late-stage (48-72 h postchallenge) Sterne strain (lethal) infections resulted in global alterations to the spleen phosphoproteome. In contrast, ΔSterne strain (asymptomatic; missing the anthrax toxin) infections resulted in 188 (5.8%) significantly altered (q<0.05) phosphopeptides. Twenty-six highly tentative phosphorylation responses to early-stage (24 h postchallenge) anthrax were discovered (q<0.5), and ten of these originated from eight proteins that have known roles in the host immune response. These tentative early-anthrax host response signaling events within mouse spleens may translate into presymptomatic diagnostic biomarkers of human anthrax detectable within circulating immune cells, and could aid in the identification of pathogenic mechanisms and therapeutic targets.

The chemokine Bv8/prokineticin 2 is up-regulated in inflammatory granulocytes and modulates inflammatory pain

Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freund's adjuvant-induced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced up-regulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain.

The evaluation of neutropenia: the use of the granulocyte mobilization test

Ten individuals with idiopathic neutropenia and similar numbers of normal and abnormal controls were tested for mobilization of their marginal granulocyte pools and bone marrow reserve by using epinephrine and hydrocortisone intravenously. Individuals with 'benign' idiopathic neutropenia appeared to have a normal response while half the abnormal controls responded poorly. It is suggested that granulocyte mobilization tests are valuable in the assessment of individuals with neutropenia.

Modelling Perspectives on Aging: Can Mathematics Help us Stay Young?

We survey several types of mathematical models that keep track of age distributions in a population, or follow some aspects of aging, such as loss of replicative potential of stem cells. The properties of a class of linear models of this type are discussed and compared. We illustrate the applicability of such models with a simple example based on hypothetical stem cell dynamics developed to address age-related telomere loss in the human granulocyte pool. We then describe the contrasting behaviour of nonlinear systems. Examples are drawn from the class of “dynamical diseases” to illustrate some of the aspects of nonlinear systems. Applications of these, and other models to the problems of aging and replicative aging are discussed.

Investigation of Ethnic Neutropenia by Assessment of Bone Marrow Colony-Forming Cells

Healthy individuals with African ancestry have lower neutrophil counts than Caucasians. It has previously been postulated that this was consequent on either a diminished bone marrow granulocyte reserve or an altered distribution of neutrophils between the circulating and marginated granulocyte pools. Recent indirect evidence supports the former hypothesis. In this study we have compared the number of granulocyte plus granulocyte-macrophage colony-forming units (CFUs) in the bone marrow of healthy African and Afro-Caribbean subjects with the number of CFUs in the bone marrow of healthy age and sex-matched Caucasians. We found the group with African ancestry to have significantly fewer CFUs than the Caucasian group. There was no evidence of any qualitative difference between the CFUs of the two ethnic groups: they showed similar sensitivity to granulocyte-monocyte colony stimulating factor and similar enhancement of growth when cultured with a larger range of cytokines. These observations suggest that ethnic neutropenia observed in those with African ancestry is likely to result from reduced numbers of bone marrow progenitor cells in comparison with numbers present in Caucasians.

Effects of treatment of rheumatoid arthritis patients with an antibody against tumour necrosis factor α on reticuloendothelial and intrapulmonary granulocyte traffic

The therapeutic effects of a monoclonal antibody against tumour necrosis factor alpha (TNFalpha) were evaluated objectively in 10 patients with rheumatoid arthritis by 111In-labelled granulocyte imaging before and after treatment, and compared with changes in granulocyte kinetics with respect to the liver, spleen and lungs. Anti-TNFalpha resulted in a decrease in the size of the whole-body pool of marginating granulocytes, as reflected by a significant increase in the 30 min intravascular recovery of labelled granulocytes from 40% (S.D. 10) to 47% (S.D. 16) of injected activity (P<0.02). The 111In contents of the spleen, liver and lungs were unchanged, so the origin of the increment in recovery was presumed to be a reduction in granulocyte margination in inflamed synovium, although this was not quantifiable. The sizes of the granulocyte pools in the liver and lungs, expressed as the 111In content of the organ per unit of circulating 111In-labelled cells, were not significantly different after treatment, but the splenic granulocyte pool decreased by 16% (S.D. 19) (P<0.05). Individual changes in the size of the splenic pool showed no significant correlation with corresponding changes in 30 min recovery or with corresponding indices of inflammation (24 h 111In-granulocyte joint activity and C-reactive protein). We conclude that anti-TNFalpha produces an obvious resolution in inflammatory joint activity that is accompanied by an increased circulating component of the total blood granulocyte pool, as a result of decreased margination at sites of inflammation. Anti-TNFalpha may also produce a specific decrease in splenic granulocyte pooling, independent of any anti-inflammatory effects, although a similar decrease in the lungs, which might be anticipated as a result of reduced cytokine-induced granulocyte activation, could not be detected.

Pulmonary granulocyte kinetics in relation to endothelial and granulocyte activation

The aim of the study was to measure the peripheral blood levels of soluble E-selectin in patients with systemic inflammation and compare them with in vivo granulocyte activation, pulmonary intravascular granulocyte pooling, pulmonary extravascular granulocyte migration and 99mTc-diethylenetriaminepenta-acetic acid (DTPA) aerosol clearance, an index of lung injury. The level of soluble E-selectin was measured by capture ELISA. Granulocytes were labelled with 111In and 99mTc for quantification of pulmonary granulocyte kinetics. The pulmonary vascular granulocyte pool (PGP) was expressed as a fraction of the total blood granulocyte pool. Pulmonary granulocyte migration was quantified on 24-h images using the 111In signal. Granulocyte activation was quantified as the percentage of circulating cells showing shape change ('primed'). Lung injury was assessed from the clearance rate of inhaled 99mTc-DTPA aerosol. Eighteen patients with systemic inflammation were studied: five with inflammatory bowel disease, eight with systemic vasculitis, four with graft versus host disease and one with a recent renal transplant. The peripheral blood levels of soluble E-selectin were significantly elevated in patients with systemic inflammation. The level of soluble E-selectin showed a significant association with granulocyte migration (Spearman rank correlation coefficient, Rs=0.53; P<0.05) but not with PGP or with the percentage of cells showing shape change (P>0.05 for both). Granulocyte migration was bimodal: patients were therefore subdivided into 'migrators' and 'non-migrators'. Soluble E-selectin level, 99mTc-DTPA clearance and PGP, but not the percentage of cells showing shape change, were significantly higher in migrators than in non-migrators. We conclude that pulmonary intravascular granulocyte pooling is increased in the presence of increased numbers of circulating primed granulocytes but increased pooling does not by itself promote granulocyte migration into the lung interstitium. Insofar as an elevated level of E-selectin in peripheral blood reflects vascular endothelial activation, the data are consistent with the notion that pulmonary endothelial activation is required, in addition to granulocyte activation and an expanded PGP, for granulocyte migration into lung parenchyma and, therefore, for lung injury to occur.

Bimodal granulocyte transit time through the human lung demonstrated by deconvolution analysis

The lungs are an important site of granulocyte pooling. The aim of the study is to quantify pulmonary vascular granulocyte transit time using deconvolution analysis, as has previously been performed to measure pulmonary red cell transit time. Granulocyte and red cell studies were performed in separate groups of patients. Both cell types were labelled with Tc-99m, which for granulocyte labelling was complexed with hexamethylpropyleneamine oxime (HMPAO). The red cell impulse response function (IRF) was monoexponential with a median transit time of 4·3 s. The granulocyte IRF was biexponential in 19 of 22 subjects, 18 of whom had systemic inflammation (inflammatory bowel disease, systemic vasculitis or graft- vs-host disease) and four were controls without inflammatory disease. The median transit time of the fast component ranged from 20 to 25 s and of the slow component 120–138 s in the four patient groups. The fraction of cells undergoing slow transit correlated significantly with (a) mean granulocyte transit time and (b) the fraction showing shape change in vitro. We conclude that granulocyte transit time through the pulmonary circulation is bimodal and that shape-changed (activated) cells transit more slowly that non-activated cells. The size of the fraction undergoing slow transit is closely related to mean granulocyte transit time and is an important determinant of the size of the pulmonary vascular granulocyte pool.

Mice Deficient in Fas Ligand (gld) or Fas (lpr) Show Few Alterations in Granulopoiesis

Evidence exists that the life span of mature, circulating neutrophils is influenced by apoptosis induced by interactions between Fas ligand (FasL) and Fas (CD95). However, the role of FasL/Fas-mediated apoptosis in granulopoiesis has not been explored. The present study assessed differences in granulopoiesis between normal (B6) mice and mice carrying mutations in the genes for FasL (B6/gld) or Fas (B6/lpr). Granulopoiesis was examined by quantitating mature granulocytes in the blood, committed myeloid progenitor cells (or colony-forming units; CFU) in the bone marrow (BM), and granulocyte lineage cells in the BM. The present study also characterized through flow cytometry the ability of GR-1+granulocyte lineage cells from B6, B6/gld,and B6/lprmice to undergo spontaneous apoptosisin vitro. In comparison to B6 mice, B6/gldmice (but not B6/lprmice) showed small, but significant increases in the number and percentage of blood granulocytes and in the number of myeloid CFU. However, the number and percentage of GR-1+granulocyte lineage cells in the BM were similar in the three strains. The rate of spontaneous apoptosis of GR-1+granulocyte lineage cells also did not differ between B6, B6/gld,and B6/lprmice. In B6 and B6/gldmice, Fas expression on granulocyte lineage cells was downregulated in conjunction with a decrease in forward-angle light scatter (fsc) and externalization of phosphatidylserine (PS), two measures of apoptosis. These results suggest that FasL–Fas interactions play only a minor role in modulating numbers of committed myeloid progenitor cells and the size of the peripheral pool of mature granulocytes. Interactions between FasL and Fas do not influence the size of the BM pool of granulocyte lineage cells or the ability of those cells to undergo spontaneous apoptosis.

Relationship between granulocyte activation, pulmonary granulocyte kinetics and alveolar permeability in extrapulmonary inflammatory disease.

1. The aim of the study was to examine the relationship between granulocyte activation, pulmonary intravascular granulocyte transit, pulmonary extravascular granulocyte migration and lung injury in patients with systemic conditions (bone marrow transplant recipients, inflammatory bowel disease and systemic vasculitis) in which abnormalities of pulmonary granulocyte traffic have previously been reported. 2. A double 111In-99mTc granulocyte labelling technique was used for quantification of granulocyte kinetics in 23 patients, of whom five were control patients. The pulmonary vascular granulocyte pool was measured from dynamic data centred on the 99mTc signal and expressed as a percentage of the total blood granulocyte pool. Granulocyte migration was quantified on 24 h images using the 111In signal. Granulocyte activation was measured as the percentage of cells showing a change in shape. The clearance rate of an inhaled aerosol of 99mTc-diethylenetriaminepenta-acetic acid (DTPA) was used as a marker of lung injury. 3. Pulmonary granulocyte pool, migration, activation and aerosol clearance, although highly variable in the patient groups, were, in general, elevated compared with the controls. 4. Granulocyte activation correlated with pulmonary granulocyte pool (Rs = 0.72, n = 22, P < 0.01), while the t1/2 of DTPA clearance correlated with migration (Rs = -0.84, n = 17, P < 0.01). Fifteen patients had an expanded pulmonary granulocyte pool, of whom six with no evidence of migration, had a normal DTPA clearance, while nine, who had an abnormal migration signal, had an accelerated DTPA clearance. The pulmonary granulocyte pool in these nine was significantly higher than in the six without a migration signal. 5. Activation of granulocytes results in delayed transit through the lung vasculature. With increasing margination, granulocytes migrate into the lung interstitium and injure the lung. An increased intravascular pool does not by itself lead to lung injury.

Leukocyte response to toxic injury.

Exposure to a variety of drugs and toxins can induce hematopoietic damage. These agents exert their effects through several distinct mechanisms including destruction or suppression of hematopoietic stem cells, cytotoxic destruction of rapidly proliferating precursor cells, immune-mediated hematotoxicity, altered hematopoietic microenvironment, genetic mutation, and microvascular injury. Some toxins consistently produce suppression of granulopoiesis in a dose-dependent manner, whereas others produce idiosyncratic reactions. Although all types of injury tend to result in granulocytopenia, the time course of the changes varies with the type of injury. With acute destruction of the proliferative pool of granulocytes, neutropenia develops within 7 days and recovery occurs within days after discontinuing treatment. With stem cell injury, the onset of hematotoxicity is variable and damage is often permanent. Immune-mediated reactions may occur acutely or after months or years of treatment with a drug. Drugs or toxins that act as mutagens can induce a variety of hematopoietic disorders including aplastic anemia, myelodysplasia, and leukemia. Therefore, the time course of onset of leukopenia after drug or chemical exposure and the rapidity of hematopoietic recovery give clues to the mechanism by which granulopoiesis is suppressed.

Transfer and expression of the human multiple drug resistance gene into live mice.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

Granulocyte turnover in the feline intestine.

The objective of this study was to determine the turnover rate of the extravascular pool of granulocytes in different regions of the feline gastrointestinal tract. Leukocyte emigration from the vasculature was prevented over a 48-h period by repeated intravenous injections of a monoclonal antibody (MAb IB4) directed against the leukocyte adhesion glycoprotein complex CD11/CD18. Tissue-associated myeloperoxidase (MPO) activity was used to monitor the total tissue granulocyte pool at 0.5, 12, 24, and 48 h after MAb IB4 administration. The mucosal layer of the duodenum, jejunum, ileum, and colon exhibited different kinetics of granulocyte clearance, with average life-spans (t1/2) ranging between 6.9 (colon) and 10.4 h (duodenum). Granulocyte clearance rates of 0.5 x 10(6) and 2.4 x 10(6) cells/h/g tissue were estimated (from measured values of t1/2 and tissue granulocyte pool) for the small bowel and colonic mucosae, respectively. The submucosal layer of the intestine exhibited a biphasic reduction in tissue MPO activity following immunoneutralization of CD11/CD18, with an initial t1/2 < or = 0.5 h followed by a t1/2 of 36-60 h. The initial rapid decline in tissue MPO suggests that a significant fraction of granulocytes in the submucosa is localized in a readily exchangeable pool (e.g., marginated cells within the vasculature). The results of this study indicate that the average life-span of resident granulocytes varies significantly between different regions of the gastrointestinal tract, with the intestinal mucosa exhibiting a t1/2 comparable to that previously reported for circulating feline neutrophils (approximately 8 h).

Increased Neutrophil Mobilization and Decreased Chemotaxis During Cortisol and Epinephrine Infusions

Although hormones are putative mediators of neutrophil changes after injury, the effects of trauma-induced levels of plasma cortisol and epinephrine on circulating neutrophils have not been reported in humans. The dynamics of PMN mobilization and chemotaxis were evaluated during 19 infusions of epinephrine or cortisol or a combined infusion of both hormones in ten normal volunteers. Basal levels of epinephrine and cortisol increased during infusions to levels consistent with those reported following severe injury. Circulating neutrophil counts increased in parallel with plasma cortisol levels. Epinephrine mobilized the entire marginated pool of neutrophils, and the neutrophil half-life was extended from a normal of 6.6 hours to 10.4 hours by cortisol. Chemotaxis after six hours of epinephrine infusion was reduced compared with baseline chemotaxis. In four volunteers who had a second infusion of cortisol, chemotaxis was significantly depressed ten days after the infusion compared with baseline. From these data we conclude that stress levels of epinephrine mobilize the marginated pool of granulocytes into the circulating pool in a linear fashion, and cortisol raises the half-life of circulating neutrophils. Reduced neutrophil chemotaxis seen as a consequence of these infusions could account for some of the increased susceptibility to infection that occurs after major trauma.

Inhibition of leukocyte rolling in venules by protamine and sulfated polysaccharides

Intravital video microscopy was used to investigate leukocyte margination in 80 mesenteric venules (19 to 54 microns) of 50 anesthetized rabbits. After intravenous (IV) bolus injection, sulfated polysaccharides reduced in a reversible and dose-dependent way the number of leukocytes rolling slowly along the venular wall. The presence of sulfate groups is essential because other negatively charged or neutral polysaccharides had no effect. It was not caused by an increase in RBC velocity or chelation of divalent cations. Inhibition by sulfated dextrans (n = 7) was independent of molecular weight (mol wt 13,000 to 500,000) but was influenced by the average number of sulfate groups per monosaccharide. With substitution 0.13, the 90%-inhibition dose was 104 mg/kg, with 0.7 it was 56 mg/kg, and between substitution 1 and 2 it ranged from 20 to 23 mg/kg. At 100 mg/kg, plasma concentration was 0.6 to 0.7 mg/mL. Xylan sulfate (mol wt 6,000, substitution 1.8) gave 90% inhibition at 11 mg/kg, and heparin gave 90% inhibition at 97 mg/kg. Duration of inhibition (0.5 to 2 hours) depended on mol wt and appeared to be related to plasma clearance. Because protamine also inhibited rolling (12 mg/kg; less than 10 minutes), we propose that repetitive formation and breakup of ionic bonds between sulfate groups and positively charged amino acids is involved in leukocyte rolling. During inhibition of rolling, systemic lymphocyte/monocyte levels appeared to increase. Granulocyte counts did not change, indicating that rolling is not the main mechanism responsible for the marginal granulocyte pool.