bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Human Neutrophil and Pseudomonas Elastases by the Amyloid P-Component: A Constituent of Elastic Fibers and Amyloid Deposits
Authors: Vachino G , Heck L W , Gelfand J A , Kaplan M M , Burke J F , Berninger R W , McAdam K P W J
Published: January 2018

The amyloid P-component (AP), a ubiquitous component of amyloid fibrils, is also a plasma protein and a connective tissue constitutent. Its proximity to elastin, in particular, suggested that AP might serve to protect elastic tissue from hydrolytic enzymes. The inhibition of pancreatic elastase by AP has been reported. In the present study, the effects of AP on human neutrophil elastase and Pseudomonas elastase were investigated, and AP was shown to interfere with the cleavage of soluble elastin. As indicated by Michaelis-Menten analysis, AP is acting as a noncompetetlve inhibitor. C-reactive protein, which is structurally similar to AP, had no effect on either elastase. AP was also found to inhibit the degradation of secondary amyloid fibrils by neutrophil elastase when these structures were first partially purified and then reexposed to AP.

AP’s ability to inhibit elastase was compared with alpha-1 antitrypsin in the presence and absence of oxidizing agents. These substances, which are released by inflammatory cells, are known to abrogate alpha-1 antitrypsin’s anti-protease capacity. This contributes to elevated levels of free proteases in the circulation and extravascular spaces during severe inflammation. AP is not susceptible to oxidation and remains a functional inhibitor under these conditions. The potential role of AP as an elastase inhibitor is discussed.
Isolation and Characterization of Interleukin-1 From Bovine Polymorphonuclear Leukocytes
Authors: Canning Peter C , Neill John D
Published: January 2018

Interleukin-1 (IL-1α and IL-1β collectively) has been shown to be produced by a wide variety of cell types. The purpose of this study was to evaluate the ability of bovine polymorphonuclear leukocytes (PMNs) to synthesize and release IL-1-like cytokines and characterize the active molecule(s). Purified peripheral blood PMNs were cultured for various periods of time in the presence of opsonized zymosan particles. The resulting culture supernatants exhibited IL-1 activity as determined by enhanced mitogen-induced proliferation of the D10 G4.1 murine T-helper cell line. Supernatants from nonstimulated PMNs or PMNs stimulated for less than 6 h did not enhance D10 G4.1 proliferation. The active molecule (PMNIL-1) was isolated by using gel filtration high-performance liquid chromatography (HPLC). Further characterization of the HPLC-purtfied molecule by SDS-PAGE and isoelectric focusing indicates bovine PMNIL-1 has a molecular weight of 17.6 kd and a pl of 4.1.
Development, Differentiation, and Maturation of Fetal Mouse Yolk Sac Macrophages in Cultures
Authors: Naito Makoto , Yamamura Fumie , Nishikawa Shin-lchi , Takahashi Kiyoshi
Published: January 2018

The development and differentiation of macrophages in the fetal mouse yolk sac were studied morphologically in four different culture experiments. In the culture of mouse embryos with yolk sac, the development of fetal macrophages was demonstrated to precede that of promonocytes and monocytes in the yolk sac. In vitro differentiation of the fetal macrophages was consistent with the results of our previous in vivo observation indicating that fetal macrophages were differentiated from primitive macrophages, but not from the monocytic cell series. Differentiation of primitive macrophages into fetal macrophages, before the development of promonocytes and monocytes, was reproduced in the culture of cell suspensions from the fetal mouse yolk sacs, with a mouse bone marrow stromal cell clone (ST2) particularly with those at 8 days of gestation. In the soft agar or liquid culture of yolk sac cells with LP3-conditioned medium, monocyte-macrophage colonies were effectively induced, but not fetal macrophage colonies. The results provide evidence for the existence, in yolk sac hematopoiesis, of two distinct macrophage populations: a fetal macrophage population and a monocyte-derived macrophage population. The data indicate an obvious difference in development and differentiation between the two populations and the temporal precedence of fetal macrophages appearing before monocyte-macrophages.
High-Resolution Localization of Lactoferrin in Human Neutrophils: Labeling of Secondary Granules and Cell Heterogeneity
Authors: Esaguy Nair , águas Artur P, , Silva Manuel T
Published: January 2018

We studied by electron microscopy the topography of the iron-binding protein lactoferrin in human peripheral blood neutrophils by immunogold labeling of thin-sectioned cells. We show that fixation in 0.3% glutararaldehyde, supplemented by 2% formaldehyde, and combined with embedding in the hydrophilic resin LR White results in excellent preservation of the lactoferrin antigenicity and of the neutrophil microanatomy. Most of the immunogold marking was seen on the specific (secondary) granules of polymorphonuclear leukocytes, thus strengthening prevailing views on the subcellular distribution of lactoferrin in neutrophils. We document that a minority (8-10%) of neutrophils showed significant amounts of lactoferrin associated with a flocculent, protein-like material seen inside large cytoplasmic vacuoles. We show that circulating neutrophils comprise 6-10% of cells that are virtually devoid of lactoferrin labeling. This finding provides specific cytochemical support for the concept that human polimorphonuclear leukocytes are made up of a heterogeneous population of cells.
MH-S, a Murine Alveolar Macrophage Cell Line: Morphological, Cytochemical, and Functional Characteristics
Authors: Mbawuike Innocent N , Herscowitz Herbert B
Published: January 2018

A continuous cell line of murine alveolar macrophages (AM), designated MH-S, has been established following transformation of cells obtained by bronohoalvaolar lavage from Balb/cJ mice with simian virus 40 (SV40). Thirty days after infection of the AM cultures, foci of rapidly proliferating cells were recovered and these have been propagated continuously for more than 36 mo. Following its initial isolation in Fischer’s medium supplemented with L-cell-conditioned medium and horse and fetal bovine serum, the cell line is now routinely grown in RPMI-1640 medium containing 10% fetal bovine serum in the absence of conditioned medium. MH-S cells were adherent, lacked contact inhibition, and were trypsin-sensitive. They expressed intracellular T-antigen and incorporated 3H- thymidine (DNA synthesis) with a doubling time of approximately 48 h but doubled in number in 96 h. MH-S exhibited typical macrophage morphology, was >98% esterase-positive, negative for peroxidase, and expressed cell surface la and Mac-1 antigens. The cells were Fc receptor-positive as demonstrated by rosette formation with, and phagocytosis of, antibody-coated sheep erythrocytes. Constitutive IL-1 secretion was significantly increased following stimulation of the cells with lipopolysaccharide. Like freshly isolated AM, MH-S cells suppressed the in vitro plaque-forming cell (PFC) response in a dose-dependent manner when cultured with splenic lymphocytes. This cell line should facilitate studies where homogeneous populations of AM are desirable, especially those involved in determining the immunological functions of AM and their potential role in lung pathology.
Functions of Purified Mouse Neutrophils Isolated From Gelatin Sponges
Authors: Middleton Marjorie M , Campbell Priscilla A
Published: January 2018

When sterile gelatin sponges are implanted under the skin of a mouse and retrieved 6 hr later, approximately 106 neutrophils per mouse, in a 98–99% pure population, may be retrieved by simply squeezing and rinsing the sponges. These neutrophils behave similarly to peritoneal exudate neutrophils in chemotaxis and phagocytosis assays, but are bacteriostatic rather than bactericidal. The sponge method yields substantially more neutrophils than could be obtained by exsanguinating the mouse and isolating blood neutrophils, and a 5-fold purer population than is normally obtained by passage of peritoneal exudate cells over Ficoll. In addition, sponge-elicited neutrophils may be ready for use within a half hour after removal from the mouse, without being exposed to osmotic shock or Ficoll.
Heterogeneity of Macrophages in the Rat Evidenced by Variability in Determinants: Two New Anti-Rat Macrophage Antibodies Against a Heterodimer of 160 and 95 kd (CD11/CD18)
Authors: Damoiseaux J G M C , Döpp E A , Neefjes J J , Beelen R H J , Dijkstra C D
Published: January 2018

A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno- and enzyme-histochemistry on cryostat sections. The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of Mr 160,000 and 95,000.
Control of Lipoprotein Lipase Secretion by Macrophages: Effect of Macrophage Differentiation Agents
Authors: Goldman Rachel
Published: January 2018

The effect of macrophage differentiation agents on lipoprotein lipase (LPL) secretion by macrophages at different stages of differentiation/maturation was investigated. Phorbol myristate acetate (TPA) had an augmenting effect on LPL secretion by in vitro-derived bone marrow macrophages (BMMs), thioglycollate-elicited peritoneal macrophages (Tg-M⊘), and resident macrophages. Augmentation was time dependent and reached ~ twofold and ~ threefold increase over control cells within 16 and 96 hr, respectively. TPA did not affect LPL secretion from J774.1 cells treated with the agent for 16–72 hr. L-cell conditioned medium (L-CM), a source of macrophage colony-stimulating activity, augmented LPL secretion by BMMs and Tg-M⊘, and when added together with TPA had an additive augmenting effect on LPL secretion in these cells. Retinoic acid (RA) exerted a time-dependent suppressive effect on LPL secretion by BMMs (46% within 16 hr and 83% within 6 d), had a relatively small effect on secretion from J774.1 cells (~ 20% in 72 hr) and had no effect on LPL secretion by Tg-M⊘. Dexamethasone suppressed LPL secretion by BMMs, Tg-M⊘, and J774.1 cells. Optimal suppression of LPL secretion by BMMs required more than 24 hr. Thus, TPA and L-CM, agents that exert a mitogenic effect on BMMs and Tg-M⊘, augmented the secretion of LPL in these cell types, and RA and dexamethasone, agents which induce differentiation patterns in myeloid cells, suppressed LPL secretion.
Inhibition of Alveolar Macrophage Cytotoxicity by Asbestos: Possible Role of Prostaglandins
Authors: Bissonnette Elyse , Carré Brigitte , Dubois Claire , Rola-Pleszczynski Marek
Published: January 2018

Asbestosis and silicosis are chronic, fibrosing lung diseases due to prolonged inhalation of asbestos fibers or silica particles. However, little is known about the implication of these toxic dusts on cell-mediated cytotoxicity. Among the first types of ceils that are in contact with the dusts are the alveolar macrophages (AM). We studied the effect of different concentrations of UICC chrysotile asbestos and silica on 18-h cytotoxicity of AM against tumor necrosis factor (TNF)-resistant P815 target ceils or TNF-sensitive L929 target cells. Rat AM, obtained by bronchoalveolar lavage, were incubated for 2 h with 20, 50, or 100 μg/ml chrysotile or silica before the addition of target cells. AM cytotoxicity was significantly inhibited at > 20 μg/ml of chrysotile. In contrast, silica did not inhibit AM-mediated cytotoxicity at any concentration used. Asbestos, but not silica, caused significant production of PGE2 by macrophages and target cells. Addition of the cyclo-oxygenase inhibitor indomethacin to our system abolished all inhibition by asbestos. These results suggest that the inhibition of AM-mediated cytotoxicity by chrysotile was caused by prostaglandins, and that fibrogenic particles differ in their capacity to modulate AM function.
Recombinant Human Interleukin-1 Beta Primes Human Polymorphonuclear Leukocytes for Stimulus-Induced Myeloperoxidase Release
Authors: Dularay B , Elson C J , Clements-Jewery S , Damais C , Lando D
Published: January 2018

Recombinant human Interleukin-1 (rhlL-1) β was found to enhance stimulus-induced granule exocytosis from human polymorphonuclear leukocytes (PMNs). PMNs were incubated with rhlL-1β and then stimulated with either heat-aggregated IgG (Hagg) or N-formyhmethionyl leucylphenylalanine (FMLP). The release of the azurophil enzyme myeloperoxidase (MPO) was measured. Low concentrations of stimuli (10 μg/ml Hagg, 2.5 x 10-9 M FMLP) did not stimulate degranulation in the absence of rhlL-1β. However, such concentrations elicited marked degranulation from PMNs preincubated with rhlL-1β (0.2-100 ng/ml). The enhancement of degranulation was dependent on the concentration of rhIL-1β employed and on the period of incubation. In other experiments, the effect of rhlL-1β on the PMN oxidative response was determined. rhlL-1β did not directly stimulate the production of superoxide anions or enhance the oxidative response to Hagg or FMLP. It is suggested that in rheumatoid joints, IL-1β may potentiate PMN degranulation, but not their oxidative response.
Glucocorticoids Fail to Inhibit Arachidonic Acid Metabolism Stimulated by Hydrogen Peroxide in the Alveolar Macrophage
Authors: Sporn Peter H S , Murphy Teresa M , Peters-Golden Marc
Published: January 2018

We have previously demonstrated that the biologically important oxidant hydrogen peroxide (H2O2) triggers release and metabolism of arachidonic acid (AA) in the alveolar macrophage (AM). In this study, we evaluated the ability of glucocorticoids to inhibit rat AM AA metabolism stimulated by H2O2, as compared to the particulate zymosan. Methylprednisolone and other glucocorticoids failed to significantly inhibit release of AA stimulated by H2O2. while markedly reducing AA release in response to zymosan. Similarly, methylprednisolone only weakly inhibited synthesis of thromboxane (Tx)B2 stimulated by H2O2, while inhibiting zymosan-induced eicosanoid synthesis to a marked degree. On the other hand, the phospholipase inhibitor mepacrine strongly inhibited AA release and TxB2 formation stimulated by both H2O2 and zymosan, indicating that H2O2-induced AA metabolism is indeed susceptible to pharmacologic inhibition. The failure of glucocorticoids to inhibit AA metabolism stimulated by H2O2 in the AM may in part explain their inability to ameliorate oxidant-mediated lung inflammation and injury.
Constitutive Expression of Interleukin-7 mRNA and Production of IL-7 by a Cloned Murine Thymic Stromal Cell Line
Authors: Sakata Tsuneaki , Iwagami Shoji , Tsuruta Yuji , Teraoka Hiroshi , Tatsumi Yoichi , Kita Yasumichi , Nishikawa Shin-Ichi , Takai Yasuyuki , Fujiwara Hiromi
Published: January 2018

Mouse interleukin-7 (IL-7) cDNA was cloned from mouse thymic stromal cell clone MRL104.8a using a polymerase chain reaction (PCR) technique and expressed in COS-7 cells. The resulting recombinant interleukin-7 (rlL-7) supported the proliferation of mouse antigen-specific helper T cell (Th) clone 9–16 in the absence of IL-2 and antigen as well as mouse pre-B cell line DW34. It was also found that high levels of the mRNA for IL-7 were constitutively expressed in the MRL104.8a cells, and a potent amount of IL-7 was produced in its culture supernatant. These results provide the evidence for constitutive expression of IL-7 mRNA and for production of IL-7 by thymic stromal cells that have a critical role in intrathymic T cell development. The results are discussed in the context of the functional and molecular relationship between IL-7 and the previously described cytokines produced by thymic stromal cells.
Effects of Adenosine on Inositol 1,4,5-Trisphosphate Formation and Intracellular Calcium Changes in Formyl-Met-Leu-Phe-Stimulated Human Neutrophils
Authors: Walker Blair A M , Hagenlocker Brian E , Douglas Vonda K , Ward Peter A
Published: January 2018

In the presence of adenosine, formyl-Met-Leu-Phe-stimulated human neutrophils show a greatly diminished production of superoxide anion. Analysis of changes in levels of intracellular calcium revealed that the immediate increase (occurring within seconds) in intracellular calcium following addition of formyl-Met-Leu-Phe is not affected by the presence of adenosine, although there are significantly lower intracellular calcium levels during the late phase (occurring 1–4 min after addition of formyl-Met-Leu-Phe). Consistent with these findings is the fact that adenosine does not affect the production of inositol 1,4,5-trisphosphate in formyl-Met-Leu-Phe-stimulated neutrophils. These data suggest that the profound inhibitory effects of adenosine on superoxide responses in formyl-Met-Leu-Phe-stimulated neutrophils may be related to an action of adenosine occurring late in the sequence of events of signal transduction.
Influence of Extracellular Calcium on the Metabolism of Arachidonic Acid in Alveolar Macrophages
Authors: Taylor Stephen M , Laegreid William W , Englen Mark D , Dani Ginger M , Silflow Ron M , Liggitt H Denny , Leid R Wes
Published: January 2018

Bovine alveolar macrophage metabolism of arachidonic acid (AA) via cyclooxygenase (CO) and lipoxygenase (LO) pathways is stimulus specific. Under standard conditions with RPMI-1640 media containing 0.4 mM Ca2+, the calcium ionophore A23187 stimulates the release of both CO and LO products, whereas opsonized zymosan and phorbol myristate acetate (PMA) selectively stimulate the CO pathway in these cells. We have examined the effect of varying the extracellular concentration of calcium (0–2.4 mM) on the profiles of AA metabolites secreted with differing stimuli. All stimuli caused the release of CO products, even in the absence of calcium in the media. The magnitude of release was correlated with increasing extracellular calcium concentrations, indicating some dependence of phospholipase activation on extracellular calcium. However, there were notable differences between stimuli regarding the magnitude of CO product formation and dependence on extracellular Ca2+. No 5-LO products were demonstrable with either zymosan or PMA at any concentration of extracellular calcium tested, and inhibition of CO by indomethacin did not result in 5-LO product formation for these stimuli. The production of 5-LO products in bovine alveolar macrophages by A23187 required extracellular calcium, demonstrating an absolute dependence for activation of the 5-LO pathway on an influx of extracellular calcium. Our results indicate that intracellular and extracellular Ca2+ have differing roles in the metabolism of AA down CO and LO pathways in bovine alveolar macrophages depending on the stimulus used. This regulation suggests that the pools of calcium required for activation of phospholipase A2 (PLA2) are not necessarily available for the 5-LO enzyme.
Stimulation of a Histone H4 Protein Kinase in Triton X-100 Lysates of Rabbit Peritoneal Neutrophils Pretreated With Chemotactic Factors: Effect of Leukotriene B4 and Cytochalasin B
Authors: Huang Chi-Kuang , Laramee Gary R
Published: January 2018

The effects of leukotriene B4 on the stimulation of a histone H4 kinase activity in rabbit peritoneal neutrophils have been studied. LTB4 activated the kinase within 10 sec with the increasing activity decaying to near basal level after 20 sec of stimulation. The effect was not inhibited by removal of extracellular calcium or loading the cells with quin-2/AM. Pretreatment of the cells with cytochalasin B greatly enhanced and prolonged the activation of the kinase. Pretreatment of the cells with LTB4 or fMet-Leu-Phe inhibited the effect of the later addition of LTB4. Since similar characteristics of LTB4 and cytochalasin B have also been observed in LTB4-stimulated responses such as secretion and superoxide generation, the results suggest a potential role of H4 kinase in LTB4-stimulated responses.
Immune Regulation by Platelet-Activating Factor: II. Mediation of Suppression by Cytokine-Stimulated Endothelial Cells In Vitro
Authors: Lacasse Chantal , Rola-Pleszczynski Marek
Published: January 2018

Human umbilical vein endothelial cells (EC) can respond to endotoxin or to the inflammatory cytokines tumor necrosis factor (TNF) and interleukin 1 (IL-1) by producing platelet-activating factor (PAF). When EC were preexposed to TNF-α (25 U/ml) for 1 h, and then washed, their subsequent coculture with peripheral blood mononuclear cells (PBMC) resulted in suppressed proliferative response of the latter to the mitogen Con A (P < 0.05). This effect was completely reversed by the concomitant use of the PAF receptor antagonist BN 52021 (0.1 mM). Preexposure of EC to IL-1β (0.5 U/ml) induced similar effects, but IL-1 and TNF were not additive. Removal of monocytes from the PBMC population abolished the effects. On the other hand, coculture of monocytes with cytokine-preexposed EC resulted in significant induction of suppressor activity on lymphocyte proliferation. Our data indicate that EC, preexposed to inflammatory cytokines, can modulate lymphocyte functions via the production of PAF and its action on monocytes.
Chemotactic Activity and Receptor Binding of Neutrophil Attractant/Activation Protein-1 (NAP-1) and Structurally Related Host Defense Cytokines: Interaction of NAP-2 With the NAP-1 Receptor
Authors: Leonard Edward J , Yoshimura Teizo , Rot Antal , Noer Kathleen , Walz Alfred , Baggiolini Marco , Walz Daniel A , Goetzl Edward J , Castor C William
Published: January 2018

Neutrophil attractant/activation protein-1 (NAP-1) has sequence similarity to platelet factor-4 (PF-4) and to NAP-2 (a truncated form of connective tissue activating protein-Ill [CTAP-III(des 1–15)]. We compared chemotactic activity for neutrophils of these related proteins. We also included for comparison CTAP-III, CTAP-III(des 1–13), the C-terminal dodecapeptide of PF-4 [PF-4(59–70)], and C5a. Chemotactic potency (EC50) was highest for NAP-1 and C5a. Although chemotactic efficacy (peak percentage of neutrophils migrating) was comparable for C5a, NAP-1, and NAP-2, the NAP-2 response occurred only at concentrations 100-fold higher than the NAP-1 EC50 of 10-8 M. Data for the CTAP-III proteins confirmed that CTAP-III is not an attractant and that chemotactic activity appears as a result of cleavage of residues at the N-terminus to make CTAP-III(des 1–13) or NAP-2 [CTAP-III(des 1–15)]. Chemotactic activity of PF-4 was low and variable, with no significant response by neutrophils from six of nine subjects. In contrast, PF-4(59–70) regularly induced high chemotactic responses, although the EC50 of 1.6 × 10-5 M was 1,000-fold greater than that of NAP-1. The binding of fluoresceinated NAP-1 to neutrophils was inhibited by unlabeled NAP-1 or NAP-2 but not by PF-4 or PF-4 (59–70). This suggests that NAP-2 interacts with the neutrophil NAP-1 receptor. Despite the low chemotactic potency of NAP-2, it is a potential attractant at sites of injury because of the relatively large amounts of the parent CTAP-III released from platelets, as indicated by a serum concentration of approximately 10-6 M.
In Vitro Modulating Effect of Human Very-Low-Density Lipoproteins on Human Polymorphonuclear Leukocyte Oxidative Metabolism and Migration
Authors: Idohou N , Couderc R , Roch-Arveiller M , Bonneau C , Giroud J P , Raichvarg D
Published: January 2018

In this work, the in vitro effects of very-low-density lipoproteins (VLDL) on human polymorphonuclear leukocyte (PMN) oxidative metabolism and migration were studied.

VLDL stimulated PMN superoxide generation in absence of other stimulating agents. The effect of VLDL from normotriglyceridemic subjects was more marked than with VLDL from hypertriglyceridemia subjects.

VLDL reduced in a dose-dependent manner the luminol-dependent chemiluminescence of PMN stimulated by phorbol myristate acetate (PMA) and, to a lesser degree, by opsonized zymosan. This effect was observed with VLDL concentrations found in healthy and hypertriglyceridemic patients. Superoxide anion generation was also reduced by preincubation of PMN with VLDL before stimulation with PMA but not opsonized zymosan. VLDL were not cytotoxic for PMN. The above effects appear to be an intrinsic property of VLDL and might lead to reduced PMN-mediated non-specific host defences in hypertriglyceridemic subjects.
Proteolysis of Gelatin-Bound Fibronectin by Activated Leukocytes: A Role for Leukocyte Elastase
Authors: Daudi Imbesat , Gudewicz Paul W , Saba Thomas M , Cho Eshin , Vincent Peter
Published: January 2018

Fragmentation of subendothelial matrix-bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125l-labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I-fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose-dependent manner by the addition of both methoxysuccinyl-alanine-alanine-valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non-specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60–200 kD) were not labelled, but the smaller molecular weight fragments (<45 kD), derived from the artifical matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.
Analysis of the Number and Distribution of Macrophages, Lymphocytes, and Granulocytes in the Mouse Uterus From Implantation Through Parturition
Authors: De Mamata , Wood Gary W
Published: January 2018

The metrial gland and decidua basalis are uterine structures which, during pregnancy in mice and rats, contain bone marrow derived cells. The current study demonstrates that large numbers of bone marrow derived cells, identified by common leukocyte antigen (CLA) positivity, accumulate in the mesometrial uterus between days 6 and 10 and contribute significantly to the cellularity of both the metrial gland and the decidua basalis. The distribution of F4/80+ cells (macrophages) is similar, suggesting that CLA+ cells in the metrial gland and decidua basalis are macrophages. Disappearance of luminal epithelium in the mesometrial uterus between days 11 and 12 leads to regression of metrial gland and decidua basalis and coincident disappearance of CLA+ and F4/80+ cells. A second population of CLA+ and F4/80+ cells appears in association with the development of new uterine luminal epithelium which surrounds the fetus and placenta during the final week of pregnancy. These very large accumulations of macrophages invariably are related to presence of epithelium.
1,25-Dihydroxyvitamin D3 Induces Monocytic Differentiation of the PLB-985 Leukemic Line and Promotes c-fgr mRNA Expression
Authors: Perkins Sherrie L , Link Daniel C , Kling Stephen , Ley Timothy J , Teitelbaum Steven L
Published: January 2018

1,25-dihydroxyvitamin D3 [1,25(OH)2D] is known to stimulate maturation of the human promyelocytic line HL-60 and murine bone marrow precursor cells along a monocyte/macrophage pathway. In this study, the steroid's effects on the PLB-985 leukemic line were examined. We found that 1,25(OH)2D induces monocytic differentiation of PLB-985 as manifested by morphological appearance, histochemical staining, and changes in cell surface antigen expression. Additionally, there was acquisition of functional monocyte characteristics including phagocytic activity and superoxide anion production via the respiratory burst pathway. Steady state levels of mRNA derived from the leukocyte-specific proto-oncogene c-fgr were also increased by the steroid. Thus, 1,25(OH2)D effectively differentiates PLB-985 cells along a monocytic pathway, providing a useful model of macrophage differentiation.
A Cytosolic Inhibitor of Human Neutrophil Elastase and Cathepsin G
Authors: Thomas Raymond M , Nauseef William M , Iyer Shankar S , Peterson Michael W , Stone Phillip J , Clark Robert A
Published: January 2018

The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is α1-proteinase inhibitor, a member of a superfamily of homologous proteins known as serpins. Neutrophil cytosol inhibited the activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 125l-elastase or 125l-cathepsin G with neutrophil cytosol or α1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of α1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100° but were dissociated by hydrolysis in ammonium hydroxide (1.5 mol/L) at 37°. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either α1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5–5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpin-like protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.
Absence of FMLP Receptors on Rat Macrophages
Authors: Walker Blair AM , Seiler Andrew J , Owens Catherine A , Hagenlocker Brian E , Ward Peter A
Published: January 2018

Although rat peritoneal neutrophils in the presence of cytochalasin B demonstrate superoxide (O2-) responses to the chemotactic peptide N'-formyl-met-leu-phe (FMLP), neither elicited rat peritoneal macrophages nor rat alveolar macrophages show an O2-response to FMLP (in the presence or absence of cytochalasin B), although a good O2-response to opsonized zymosan is demonstrated by both types of macrophages. Using Fura-2 loaded cells, peritoneal macrophages failed to show an increase in intracellular calcium after exposure to FMLP, f-nor-leu-phe, F-met-met-met, or F-norleu-leu-phe-norleu-lys. FMLP also failed to induce elevations in intracellular calcium in alveolar macrophages. In 3H-FMLP binding studies, the lack of responsiveness of peritoneal and alveolar macrophages was associated with the lack of FMLP receptors on these cell types, in striking contrast to the presence of functional receptors on rat neutrophils.
Absence of interleukin 1α radioprotection in tumor-bearing animals: elevated plasma levels of prostaglandin E versus a preexisting primed marrow
Authors: Kovacs C J , Harrell J P , Evans M J , Johnke R M
Published: January 2018

Recombinant human interleukin 1 (IL-1) administered as a “priming” agent 24 h prior to hematopoietically lethal doses of total body irradiation (TBI) confers radioprotection to normal G57B1/6 (B6) mice, but not to B6 tumor-bearing animals (TBAs) known to have altered hematopoietic steady states. Using the Lewis lung tumor (LLca) in the B6 mouse, studies were carried out to determine whether the failure of IL-1 to radioprotect the LLca TBA was related to a preexisting “primed” hematopoietic state in the TBA or resulted from inhibition of myelopoietic activity associated with the production of prostaglandin E (PGE) by, or in response to, the tumor. Both normal B6 and LLca B6 TBAs were injected (every 24 h × 1–5) with 100 μg of indomethacin (IND) prior to the administration of IL-1. A single treatment with IND was sufficient to reduce the elevated levels of PGE found in the plasma of the TBAs. After five treatments, IND reduced the PGE level to below that of controls. Neither the acute nor the protracted IND treatment, however, affected the expansion of the stem and progenitor cell compartments of the marrow in the LLca TBA. Furthermore, no evidence of restoration of the radioprotective properties of IL-1 was observed in TBAs pretreated with IND. Collectively, these data suggest that the failure of IL-1 to provide radioprotection to the LLca TBA is not a direct result of the elevated plasma PGE levels associated with growth of the LLca tumor. In addition, these studies provide insight into the importance of examining in vivo effects of biological molecules in altered, as well as normal, physiological states.
Production of tumor necrosis factor α by resident and activated murine macrophages
Authors: Tachibana Katsuyasu , Chen Guan-jie , Huang Dennis S , Scuderi Philip , Watson Ronald Ross
Published: January 2018

Alveolar macrophages (Am⊘s), resident peritoneal macrophages (RPm⊘s), and thioglycolate-elicited peritoneal macrophages (TGPm⊘s) were isolated from C57BL/6 mice and incubated with lipopolysaccharide (LPS), stimulated cell supernatant, or recombinant interferon γ (IFN-γ) for 24 h. Tumor necrosis factor (TNF) in cell-free supernatants was measured by enzyme-linked immunosorbent assay. Anio⊘s incubated with 103 ng/ml LPS produced 50 times more TNF than RPm⊘s and 5 times more than TGPm⊘s, and LPS alone induced maximum TNF production by Am⊘s. Stimulated cell supernatant or recombinant IFN-γ alone did not induce TNF production. A combination of LPS with stimulated cell supernatant or IFN-γ had only a limited synergistic effect on TNF production by Am⊘s. However, both LPS and stimulated cell supernatant or recombinant IFN-γ induced maximum TNF production by RPm⊘s and TGPm⊘s. TGPm⊘s showed greater sensitivity to LPS and stimulated cell supernatant or IFN-γ with regard to TNF production than the other macrophage populations investigated.
Characterization of a direct effect of phorbol myristate acetate on human neutrophil cell membrane using 31D8 monoclonal antibody
Authors: Woronick Charles L , Maderazo Eufronio G , Anthony Monique N , Krause Peter J , Sha'afi Ramadan I
Published: January 2018

We found that 4-β-phorbol 12-myristate 13-acetate (PMA) caused decreased expression of the polymorphonuclear neutrophil (PMN) surface antigen 31D8. In contrast to the rapid initiation of the oxidative burst caused by PMA, the effect was slow to start but increased during incubation periods up to 50 min. To study this apparent protein kinase C-independent late effect of PMA, we measured 31D8 expression in PMNs after incubation with various concentrations of PMA. The maximum PMA-induced inhibition was 76 ± 2%, with an ID50 of 3.9 ± 0.4 ng/ml. Oxidants and prooxidants (hydrogen peroxide, hypochlorite, taurine-chloramine, and ferrous iron, with or without H2O2) had no direct effect on 31D8 antigen expression. The following substances were not protective against the inhibitory affect of PMA: (1) antioxidants (superoxide dismutase, catalase, azide, dimethyl sulfoxide, Desferal, and ascorbate, with the exception of α-tocopherol), (2) inhibitors of protein kinase C (H7 and W7), (3) inhibitors of 5-lipoxygenase (A-63162, MK886, and high-dose indomethacin) and (4) inhibitors of cyclooxygenase (low-dose indomethacin). Myeloperoxidase-deficient PMNs had normal 31D8 antigen expression and a decrease of 31D8 antigen expression by PMA, as did normal PMNs. The inactive analog of PMA, 4-α-phorbol didecanoate, had no effect on 31D8 antigen expression. α-Tocopherol (50 μg/ml) and betamethasone (150 μg/ml) protected against the PMA effect by 30.5 ± 7.3 (P < .0005) and 52 ± 15 (P < 0.004) channels, respectively. These results indicate that PMA has a protein kinase C-independent late effect on human neutrophils, which can be prevented by pretreatment with α-tocopherol or the steroid betamethasone. These compounds probably exert their protective effect by membrane stabilization.
The role of oxidative stress in disease progression in individuals infected by the human immunodeficiency virus
Authors: Baruchel Sylvain , Wainberg Mark A
Published: January 2018

This review describes the potential role of oxidative stress as a cofactor of disease progression from asymptomatic human immunodeficiency virus (HIV) infection to the acquired immunodeficiency syndrome (AIDS). Oxidative stress is a known activator of HIV replication in vitro through the activation of a factor that binds to a DNA-binding protein, NF-B, which in turn stimulates HIV gene expression by acting on the promoter region of the viral long terminal repeat. Tumor necrosis factor α (TNF-α), an essential cytokine produced by activated macrophages, is also involved in the activation of HIV infection through similar mechanisms. TNF-mediated cytotoxicity of cells exposed to this substance is related to the generation of intracellular hydroxyl radicals. An indirect argument in favor of the role of oxidative stress in HIV-associated disease progression is the consumption of glutathione (GSH), a major intracellular antioxidant, during HIV infection and progression. GSH is known to play a major role in regulation of T cell immune functions. Oxidative stress may also play an important role in the genesis of cellular DNA damage and, in this context, may be related to HIV-associated malignancies and disease progression. Finally, the role of antioxidants as components of therapeutic strategies to combat HIV disease progression is discussed.
Assessment of neutrophil leukocyte secretory response to fMLP in whole blood in vitro
Authors: Chadwick Vinton S , Ferry Dianne M , Butt Terence J
Published: January 2018

A simple, precise method has been developed for assessing neutrophil secretory responses (release of vitamin B12 binding protein from specific granules) to challenge of aliquots of whole blood with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Dose-response studies performed on blood from normal healthy volunteers showed higher maximal secretory responses in males than females (33.3 ± SEM 2.2 vs. 27.4 ± 2.5, P<.005) a left shift in dose-response curves after feeding compared to fasting (P<.005), spontaneous up-regulation of responses in blood incubated at 37° for 1 h, and marked up-regulation in response to preincubation with endotoxin. This whole blood challenge method may be used to study neutrophil responses in groups of individuals or patients without the confounding effects of changes in cell responses resulting from cell isolation procedures. The method may also be used as a bioassay for neutrophil-activating factors.
Nitric oxide production by tumor targets in response to TNF: paradoxical correlation with susceptibility to TNF-mediated cytotoxicity without direct involvement in the cytotoxic mechanism
Authors: Fast David J , Lynch Robert C , Leu Richard W
Published: January 2018

Tumor necrosis factor (TNF) is selectively cytotoxic for some tumor cells in vivo and in vitro. We determined whether TNF-mediated cytotoxicity for TNF-sensitive tumor targets was related to TNF-stimulated production of NO by the tumor cell itself. We found that a cell line that was sensitive to TNF-mediated cytotoxicity produced NO in response to TNF as measured by the accumulation of nitrite in the supernatants of TNF-stimulated cells. Production of NO in response to TNF was inhibited by the competitive substrate inhibitor, NG-monomethyl-l-arginine (NMMA). The kinetics of NO production in response to TNF indicated that most of the NO was produced during the first 24 h and peaked after 48 h of culture and that TNF-stimulated NO production was dose dependent. TNF-resistant cell lines produced less NO than a TNF-sensitive cell line, and the amount of nitrite produced correlated with the relative sensitivity of each cell line to TNF-mediated cytotoxicity. In addition, recombinant interferon-γ augmented the amount of NO produced in response to TNF by both sensitive and resistant cells and correspondingly enhanced the susceptibility of resistant cells to TNF cytotoxicity. Both sensitive and resistant cells were sensitive to NO, however, in that NO generated exogenously by culture in the presence of sodium nitroprusside was cytotoxic for both sensitive and resistant cells in a dose-dependent manner. We were unable, however, to demonstrate directly a role for NO in TNF-mediated cytotoxicity as NMMA- and arginine-free media provided little protection from TNF-mediated cytotoxicity. We tentatively conclude that the ability of adherent murine tumor cells to produce nitric oxide in response to TNF correlates directly with their level of sensitivity to TNF-mediated cytotoxicity, although NO thus produced appears not to be directly involved in the cytotoxic mechanism.
Relationship of large and small CD3- CD56+ lymphocytes mediating NK-associated activities
Authors: Ortaldo John R , Winkler-Pickett Robin , Kopp William , Kawasaki Akemi , Nagashima Kunio , Okumura Ko , Yagita Hideo , Bach Fritz H
Published: January 2018

We have defined a population of CD3-, CD56+ small lymphocytes (SLs) that exhibit the same phenotype and lytic capacity as natural killer (NK) cells. NK cells characteristically express the surface markers CD16 and CD56, mediate non–major histocompatibility complex (MHC)–restricted lysis, and have been equated with CD3- large granular lymphocytes (LGLs). In the present study we extended the observation that CD3-, CD56+ SLs can mediate NK- and antibody-dependent cellular cytotoxicity activity by studying the activation signals and lytic mechanisms that might be utilized by CD3-, CD56+ SLs in comparison to CD3- CD56+ LGLs. Our results show that CD3- SLs, similar to CD3- LGLs, exhibited activated killing in response to interleukin-2 (IL-2). In addition, after IL-2 activation, the CD3- SLs exhibited morphologic changes, including increases in size and granularity, and both morphologically and phenotypically became virtually indistinguishable from CD3- LGLs. Similar to CD3- LGLs, CD3- SLs could be directly activated by IL-2 alone to secrete significant quantities of interferon-γ (IFN-γ) and to express IL-2 receptor (IL-2R) p55. Examination of serine esterases and pore-forming protein (PFP) demonstrated that these cells exhibited a cytoplasmic distribution of perforin, which, unlike that of CD3- LGLs, was not associated with dense cytoplasmic azurophilic granules. Serine esterase levels were similar. However, after IL-2 activation PFP was concentrated in dense cytoplasmic granules, similar or identical to the situation in CD3-, CD56+ LGLs. These CD3-, CD56+ subsets appear to represent a continuum of activated cells that might represent various states of maturation of NK cells.
A simple and rapid method for isolation of eosinophilic granulocytes from human blood
Authors: Cramer Rita , Dri Pietro , Zabucchi Giuliano , Patriarca Pierluigi
Published: January 2018

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic nor-modense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.
Inhibition site of dexamethasone on extravasation of polymorphonuclear leukocytes in the hamster cheek pouch microcirculation
Authors: Oda Takaharu , Katori Makoto
Published: January 2018

A video system was used to investigate the inhibitory effect(s) of the glucocorticoid dexamethasone (DEX) on polymorphonuclear leukocyte (PMN) extravasation in the microcirculation of hamster cheek pouch. DEX was given intraperitoneally at 0.1 or 0.3 mg/kg body weight 2 h before induction of extravasation by topical application of leukotriene B4 (LTB4) or formyl-methionyl-leucyl-phenylalanine (fMLP) on the microvasculature. The number, time course, and behavior of PMNs were examined in the following five steps of extravasation: (1) rolling on the venular endothelium, (2) adhesion on the endothelium, (3) passage between the endothelial cells, (4) staying in the venular wall, and (5) migration from the venular wall into the interstitial space. In either the presence or absence of DEX, topical application of LTB4 (15 pmol/50 μl) or fMLP (10 nmol/50 μl) caused an increase in the number of PMNs that adhered to the venules. Thus, DEX did not inhibit the adhesion of PMNs on the endothelial cells. The adhered PMNs induced by these chemoattractants became gradually smaller, finally disappearing in the vascular lumen, as observed on the monitor screen. The whole process took about 10 min. This passage of PMNs between the endothelial cells was also not inhibited by DEX, as over 90% of the adhered PMNs passed through the endothelial cells and advanced to the next step of extravasation in the presence or absence of DEX. When these chemoattractants were applied to DEX-untreated animals, the PMNs that passed through the endothelial cells stayed for about 30 min in the venular wall. Thereafter, PMNs migrated into the interstitial space. The numbers of PMNs in the interstitial space were counted at 30, 60, and 90 min after the application of chemoattractants. In the DEX-untreated animals, PMNs in the interstitial space started to appear by 30 min, and thereafter the number further increased. However, in the DEX-treated animals, the number of PMNs at 60 and 90 min was significantly suppressed. Since DEX may inhibit the synthesis and/or release of a proteinase(s) in the PMN granules, which would normally degrade the basement membrane, this inhibition may be due to the inability of PMNs to penetrate the basement membrane.