Human polymorphonuclear leukocytes of the bone marrow, circulation, and marginated pool: function and granule protein content

Polymorphonuclear leukocytes (PMN) demonstrate altered function during acute infections and after administration of corticosteroids. We questioned whether or not such changes are due to population shifts from functionally different compartments of the granulocyte pool. Volunteers were given epinephrine to induce demargination or hydrocortisone (HC) to promote egress of PMN from the bone marrow. PMN obtained before and after drug administration were compared for adherence, chemotaxis, luminol-enhanced chemiluminescence, and total content and release of lactoferrin (LF), myeloperoxidase (MPO), and beta-glucuronidase (beta-glu). Epinephrine induced a significant neutrophilia of mature PMN (segmented neutrophils), but there were no changes in function or granule protein content. HC induced a significant neutrophilia with segmented neutrophils and immature PMN (bands). Circulating PMN obtained 4 hr after HC administration demonstrated less adherence, increased chemiluminescence, increased MPO release, and decreased MPO content. Band neutrophils, however, were more adherent than segmented PMN and showed a similar decrease in adherence following HC in vivo. Thus alteration of PMN adherence following intravenous corticosteroids is not due to an influx of immature neutrophils. On the other hand, it is possible that MPO content and release and capacity for oxidative metabolism change as PMN mature.     

Quantification of the distribution of the marginating granulocyte pool in man

The kinetics of autologous granulocytes, separated from whole blood and labelled with 111In-tropolonate with continuous maintenance in plasma, have been studied in man, using a gamma camera and computer, with the aim of quantifying the distribution of the marginating granulocyte pool (MGP). We have used 3 approaches: dynamic gamma camera imaging immediately following i.v. injection of labelled cells, comparison of the activity signal from 111In-granulocytes with that from previously injected 111In-labelled red cells and absolute quantification of 111In present in liver, spleen and blood. Deconvolution analysis of the hepatic and peripheral blood time activity curves indicated that hepatic granulocyte transit time was 2.5 +/- SE 0.14 min. By comparison with 111In red cells, hepatic transit time was calculated to be 7.4 +/- SE 0.82 that of red cells, which, assuming an hepatic red cell content of 6% that of the total red cell mass, is equivalent to a transit time of 1.8 min. By comparison with 111In red cells, lung granulocyte transit time as a factor of red cel transit time was 5.4 +/- SE 0.7 at 5 min and 2.5 +/- SE 0.13 at 40 min after granulocyte injection. Using these kinetic data, in combination with previously published values for splenic granulocyte transit time, it was calculated that, 5 min after injection, the MGP accounted for 54% of the total blood granulocyte pool (TBGP), was 90% filled, and was distributed between spleen (19%), liver (26%), lung (33%) and the remainder of the body (22%). At 40 min, the MGP accounted for 60% of the TBGP, had equilibrated with the circulating granulocyte pool (CGP), and was distributed between the spleen, liver, lung and remainder of the body according to the following respective percentages: 35, 25, 10 and 30. The total granulocyte contents of the spleen and liver calculated on the basis of the kinetic data were 21% and 22% respectively and in broad agreement with the values, 34 and 23% respectively, calculated from quantitative scanning. It was concluded that about 70% of the body's MGP was present in the spleen, liver and lung. If the MGP is itself 60% of the TBGP then only about 18% of the TBGP marginates in extra-hepatosplenopulmonary sites.     

Antibiotic proteins of human polymorphonuclear leukocytes.

Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.     

Deferoxamine enhances phagocytic function of human polymorphonuclear leukocytes

Inhibition of the iron-mediated generation of toxic oxygen species by polymorphonuclear leukocytes (PMN) might prevent oxidative damage and thus enhance phagocytic function of PMN. To investigate this point, we studied the effect of the specific iron chelator, deferoxamine, on the antibacterial function of PMN. PMN were incubated for 20 hr with various concentrations of deferoxamine at 37 degrees C in medium containing 0.54 microM endogenous iron. The cells were then washed, and the phagocytic cell function was assessed. The results were compared with those for control PMN preincubated for 20 hr without deferoxamine, and those of nonincubated PMN. Compared with that of control PMN, the uptake of radiolabeled Staphylococcus aureus by PMN treated with 1 microM-1 mM deferoxamine was, on average, 10%-20% higher. This effect was not observed when iron-saturated deferoxamine (DFO) was used. Bacterial uptake was similarly increased in nonpreincubated PMN or PMN preincubated for 20 hr at 4 degrees C instead of 37 degrees C. The intracellular killing capacity of both deferoxamine-treated and control PMN exceeded 90%. PMN incubated for 20 hr at 37 degrees C with DFO not only phagocytosed more bacteria than control cells, but were also capable of killing the greater number of bacteria ingested. This increased activity of deferoxamine-treated PMN was accompanied by enhanced generation of chemiluminescence and production of superoxide during phagocytosis of S. aureus. These findings indicate that deferoxamine may enhance the antibacterial activity of PMN by protecting the cells against damage by iron-mediated generation of toxic oxygen metabolites in resting PMN.     

Functional and metabolic studies of polymorphonuclear leukocytes in the congenital Pelger-Huet anomaly

Polymorphonuclear leukocytes (PMNL) from two individuals with congenital Pelger-Huet anomaly (PHA) were examined to determine whether functional or metabolic defects accompanied the known morphological abnormality. No abnormalities of the PHA cells, as compared to normal control cells, were found when tested for quantitative leukocyte enzyme activities, nitroblue tetrazolium reduction, hexose monophosphate shunt activity, superoxide production, generation of chemiluminescence, or iodination. The PHA cells, as compared to normal PMNL, demonstrated normal chemotaxis and random migration, as well as bactericidal activity.     

Hyposmolarity stimulates myeloperoxidase exocytosis from human polymorphonuclear leukocytes

Medium hyposmolarity induced in human polymorphonuclear leukocytes treated with cytochalasin B a rapid exocytosis of the lysosomal enzyme, myeloperoxidase (MPO), which was linearly proportional to the degree of hyposmolarity between a 5 and 30% decrease (r = 0.92, p less than 0.001). Cell viability was unaffected by the hyposmolarity. The kinetics of MPO exocytosis induced by opsonized zymosan (OZ) and hyposmolarity were indistinguishable; the combination of hyposmolarity and OZ was additive. Since hyposmolarity similarly stimulates a burst of hormone secretion by perifused adenohypophyseal and pancreatic islet cells, the authors suggest that hyposmolarity-induced exocytosis is a general cellular phenomenon.     

Release of tumor necrosis factor by human polymorphonuclear leukocytes

Evidence is presented that human polymorphonuclear neutrophils (PMN) can be induced to produce tumor necrosis factor (TNF). Other investigators have previously reported that TNF has been induced from macrophages by bacteria and, more recently, from natural killer cells by certain tumor cells. Our laboratory has reported that the opportunistic fungi, Candida albicans, can induce TNF, not only from human monocytes, but also from Percoll-fractionated large granular lymphocytes. We now report that incubation of PMN with C albicans for 3 hours was sufficient for detection of TNF release, and peak induction was observed at 8 to 18 hours. This release was inhibitable by actinomycin D, an inhibitor of RNA synthesis, as well as by emetine and cycloheximide, which block protein synthesis. The TNF produced by PMN was neutralized by specific monoclonal antibodies against human TNF. These results represent an important finding that TNF production is a normal response of PMN to stimulation by fungi such as C albicans and suggest that the release of TNF may be related to autocrine activation of PMN effector function to control Candida growth.     

Phagocytosis and killing of Brucella by human polymorphonuclear leukocytes

Although cellular immunity involving activated macrophages is important in resistance to Brucella, serum factors and polymorphonuclear leukocytes (PMNLs) play some role in the initial response to infection. The interaction between human PMNLs and virulent and attenuated strains of Brucella abortus and Brucella melitensis was studied by in vitro techniques. Virulent and attenuated strains of both species were rapidly phagocytosed after opsonization with normal human serum (NHS); nonopsonized bacteria were not phagocytosed. In contrast, NHS devoid of detectable antibodies was bactericidal for strains of B. abortus but not of B. melitensis. In addition, intracellular killing of ingested bacteria was shown for virulent B. abortus but not for B. melitensis. Ultrastructural studies revealed morphological alterations in about one-half of phagocytosed B. abortus and B. melitensis after incubation for 10 min; thereafter, nearly 100% of B. abortus showed some degree of degeneration, whereas B. melitensis remained intact during 120 min of observation.     

The interaction between Treponema pallidum and human polymorphonuclear leukocytes

The interaction between polymorphonuclear leukocytes (PMNLs) and Treponema pallidum was studied. Intradermal injection of greater than or equal to 10(6) T. pallidum into rabbits caused a rapid accumulation of PMNLs. Human serum released chemotaxigenic factor (C5a) during incubation in vitro with T. pallidum. Incubation of T. pallidum with human PMNLs in vitro (ratio, 100:1) stimulated chemiluminescence. These responses were dependent upon the presence of both antibody and complement and were greatest when serum from a patient with late secondary syphilis was used as a chemotaxigenic source or for opsonization. Electron microscopic studies documented the rapid uptake of T. pallidum into membrane-bound vacuoles in the human PMNLs in vitro after incubation for as little as 5 min, with leukocyte degranulation and loss of treponemal integrity observed after 4 hr. T. pallidum were found within PMNLs 3 hr after intradermal inoculation of rabbits. These data show that PMNLs are attracted to, and appear to ingest, T. pallidum, but they fail to explain why inoculation of these organisms is not followed by eradication.     

Diminished release of lactoferrin from polymorphonuclear leukocytes of human neonates

In neonatal and adult polymorphonuclear leukocytes (PMN) we determined the content and the release of beta-glucuronidase, myeloperoxidase, lysozyme and lactoferrin. We found an equal total content of these proteins in adult and neonatal PMN, except for a lower lysozyme concentration in neonatal PMN. In the presence of opsonized zymosan myeloperoxidase, lysozyme and beta-glucuronidase were released in equal amounts; lactoferrin, however, was released to a lower rate from neonatal than from adult PMN (p less than 0.0005).     

Superoxide anion generation of human polymorphonuclear leukocytes in urine]

In order to clarify the role of polymorphonuclear leukocytes (PMNs) in the defence mechanism of the urinary tract, we investigated the superoxide anion (O2-) generation of PMNs in the urine. We tested urines mixing concentrated urine with diluted ones in various ratios. Peripheral blood derived PMNs were stimulated in tested urines or phosphate buffer solution (1M, pH 7.0) with phorbol-myristate acetate and O2- generation of PMNs was measured by means of chemiluminescence (CL) method. The CL values of PMNs in tested urines were 1/30 that of phosphate buffer solution. However, PMNs generated O2- at maximum levels when the urine osmolarity was 300-400 mOsm/kg. Furthermore, PMNs did not generate O2- when the urine osmolarity was more than 600 mOsm/kg, or less than 200 mOsm/kg. We also investigated the influence of urea and creatinine on O2- generation of PMNs. The O2- generation of PMNs decreased when the urea concentration rose. However, there was no influence of creatinine on O2- generation of PMNs. From these results, it was elucidated that PMNs generated O2- when the urine osmolarity was 300-400 mOsm/kg. Therefore, urine osmolarity of the patients with urinary tract infection should be kept at 300-400 mOsm/kg, and diuresis might be important for the patients with urinary tract infection.     

Regulatory mechanisms of a chemoattractant receptor on human polymorphonuclear leukocytes

The oligopeptide chemotactic factor receptor in leukocyte membranes exists in two affinity states that are in part interconvertible. Convertibility is regulated by guanine nucleotides, which suggests that a nucleotide regulatory unit allosterically modifies receptor affinity and participates in its transduction mechanisms. Approximately one-third of the high-affinity receptors in polymorphonuclear leukocyte (PMN) membranes are not subject to guanine nucleotide regulation. This fraction can be increased by agonist preincubation and could represent an intermediate form of the receptor prior to signal transduction and/or internalization. Pharmacologic manipulation of viable PMNs demonstrates that the affinity and functional activity of the chemoattractant receptor can be altered divergently by aliphatic alcohols and polyene antibiotics. The alcohols raise the receptors' affinity, enhance chemotaxis, but markedly depress chemoattractant-induced secretory mechanisms. In contrast, polyene antibiotics lower the receptors' affinity, depress chemotaxis, but enhance specific granule secretion. Thus, a relationship exists between the chemoattractant receptors' affinity and their ability to transduce signals for either chemotaxis or secretion.     

Effect of cyclic adenosine monophosphate elevations on functional responses of polymorphonuclear leukocytes from patients with cystic fibrosis

Recently, it has been suggested that the primary metabolic defect of cystic fibrosis (CF) is in the metabolic steps between an elevation of cyclic adenosine monophosphate (cAMP) and the opening of the Cl- ion channel in airway epithelial cells. Results from other studies have indicated that the primary defect may also be present in polymorphonuclear leukocytes (PMNL). In PMNL, cAMP is a potent inhibitor of cell function. We therefore used three compounds (epinephrine, theophylline, and forskolin) known to elevate cAMP in PMNL by three different mechanisms, to study the effect of an elevation of cAMP on various cell functions, such as superoxide production, membrane depolarization, and degranulation of CF patient PMNL and PMNL from healthy individuals. The baseline rates of superoxide production, degranulation, and membrane depolarization were identical for CF and control PMNL. After exposure to epinephrine as well as theophylline plus forskolin, the cell function of CF PMNL was inhibited to the same extent as that of control PMNL. Our results argue against an abnormal response of PMNL from patients with CF to an elevation of cAMP.     

Antibiotic proteins of polymorphonuclear leukocytes

Abstract: The polymorphonuclear leukocyte (PMN) plays an essential role in the innate defense of the mammalian host against bacterial invaders. Responding chemotactically, the PMN delivers a complex antibiotic arsenal to sites of infection. Among these cytotoxic systems is an array of antimicrobial proteins and peptides that the PMN directs at microorganisms both before (i.e. extracellularly) and after sequestration into a phagocytic vacuole. In addition to their microbicidal capacity, several of these proteins bind to and neutralize the endotoxic activity of Gram-negative bacterial lipopolysaccharides (LPS). In this review the principle features of these antibiotic proteins are briefly summarized with emphasis on their possible actions in biological settings. In many instances, additional functions independent of cytotoxicity have been described raising the possibility that some of these proteins subserve multiple roles in inflammation.     

Action of collagenase and elastase from human polymorphonuclear leukocytes on human articular cartilage

Summary Collagenase from human polymorphonuclear leukocytes (neutrophil collagenase) attacks collagen type II in solution at a rate intermediate to those of type I and III collagens. This enzyme alone is not able to initiate degradation of native human articular cartilage. If the cartilage is first treated with leukocyte elastase, collagenase slowly degrades collagen.Confirming earlier findings by other investigators, elastase has a dual action on cartilage: The enzyme removes proteoglycans, thus demasking collagen fibers and giving collagenase access to them, and solubilizes collagen at a sizable rate.Although neutrophil collagenase cleaves collagen type II in solution at a high rate, the native, cross-linked status of collagen in cartilage makes it a relatively poor substrate for this enzyme. On a weight by weight scale, elastase and collagenase display about the same collagenolytic potential on human articular cartilage.The elastase/collagenase system from human polymorphonuclear leukocytes could represent a cooperative proteolytic complex in the destruction of cartilage in rheumatoid arthritis.     

Oxidative inactivation of leukotriene C4 by stimulated human polymorphonuclear leukocytes

Leukotriene C₄ (LTC₄) was metabolized by human polymorphonuclear leukocytes (PMNs) stimulated with phorbol myristate acetate (PMA) into three sets of products. These products differed in mobility on reverse-phase high-performance liquid chromatography (RP HPLC) from LTC₄ and also from leukotriene D₄ (LTD₄) and leukotriene E₄ (LTE₄), the sequential products of peptide cleavage of LTC₄. Products I, II, and III were eluted as doublets with an average retention time for each doublet of 7.5 ± 0.3, 10.5 ± 0.6, and 16.3 ± 1.1 min (mean ± SD), respectively, as compared with 13.8 min for LTC₄. Doublet I material was biologically inactive and showed <5% of the immunoreactivity of LTC₄, doublet II material had 1% of the spasmogenic activity of LTC₄ on the guinea pig ileum and was equally immunoreactive, and doublet III material was neither biologically active nor immunoreactive. When [14,15-³H]LTC₄ and [³⁵S]LTC₄ were metabolized, all three doublet products retained the ³H label, whereas only the doublet I and doublet II products retained the ³⁵S label. The UV absorbance spectra of the three sets of metabolites were as follows: doublet I, maximum at 280 nm with shoulders at about 270 and 290 nm; doublet II, maximum at 284.5 nm with shoulders at about 275 and 295 nm; and doublet III, maximum at 269 nm with shoulders at about 259 and 279 nm. The metabolism of LTC₄ to the three classes of functionally inactive products by stimulated PMNs was completely blocked by catalase and azide, indicating a requirement for H₂O₂ and myeloperoxidase. When hypochlorous acid (HOCl)—considered to be a natural product of the interaction of myeloperoxidase, H₂O₂, and chloride ion—was formed chemically and allowed to react with LTC₄, the resulting products were indistinguishable by UV and HPLC analyses from the doublet II and doublet III metabolites of LTC₄. The doublet II products were identified as the two diastereoisomeric sulfoxides of LTC₄ by comparison with synthetic reference compounds. The doublet III products were shown to be identical with synthetic samples of (5S, 12S)- and (5S, 12R)-6-trans-LTB₄. The formation of two diastereoisomeric LTC₄ sulfoxides and 6-trans-LTB₄ can be explained in terms of an S-chlorosulfonium ion as the initial reactive intermediate, which subsequently undergoes conversion to product II by hydrolysis and product III by carbocation formation.     

Correlation of the biologic effects and binding of cytochalasins to human polymorphonuclear leukocytes

Treatment of human PMNs with cytochalasins (CE, CD, CB, and H2CB) results in alteration of cell morphology and inhibition of cell motility. Morphological changes are similar to those reported for nonamoeboid fibroblasts--rounding, zeiosis, and arborization. Mean cell velocity of PMNs, as measured by quantitative analysis of time-lapse videotape recordings, was reduced to 0.1 micron/min (control, 7.3 +/- 4.2 micron/min). Phagocytosis by PMNs, as measured by phagocytosis of latex beads, was inhibited by 75%. The relative potency of the cytochalasins for inducing morphological change or for inhibiting locomotion and phagocytosis is similar to their relative potencies for affecting non-amoeboid cells: CE greater than CD greater than CB greater than or equal to H2CB. Quantitative binding of 3H-CB to purified PMNs under equilibrium conditions reveal two types of specific CB binding sites: high-affinity sites (KD approximately 3 x 10(-7) M, 3 x 10(6) sites/cell) and low affinity sites (KD approximately 2 x 10(-6) M). The relative affinities of the cytochalasins for the high-affinity and low-affinity CB binding sites parallel their relative potencies for inducing biologic effects (i.e. CE greater than CD greater than CB greater than or equal to H2CB).     

A quantitative colorimetric method to evaluate the functional state of human polymorphonuclear leukocytes

Summary The colorimetric assay previously described by Mosmann [11] for the measurement of cell viability and proliferation has been modified for the assessment of the functional state of human polymorphnuclear cells (PMNs). The ability of PMNs to reduce the tetrazolium salt MTT to formazan reflects directly the degree of stimulation induced by various agents. The underlying mechanism of MTT-reduction to formazan seems to be similar to that of nitroblue tetrazolium (NBT)-reduction. In contrast to the NBT-reduction assay, the formazan produced from MTT can easily be measured by an ELISA reader. Parallel experiments revealed a qualitative correlation between the concentration of formazan produced from MTT and the concentration of cytochrome C reduced by PMNs. Although oxidative burst may not be the actual lytic mechanism in cellular cytotoxicity of PMN, we also observed an association between MTT-reduction capacity and the cytotoxic activity of PMNs from normal donors in antibody dependent cellular cytotoxicity. Our results indicate that the MTT-reduction assay can be employed to estimate the functional state of polymorphnuclear granulocytes.Supported in part by grant no. CA33484 from the NIH