bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Neutrophil Degranulation Stimulated by Aspergillus fumigatus
Authors: Levitz Stuart M , Farrell Timothy P
Published: January 2018

Previous studies have established that human neutrophils (PMN) are unable to kill resting conidia (RC) of Aspergillus fumigatus but can kill conidia that have been preincubated in culture medium until swollen but not yet germinated. Compared with swollen conidia (SC), RC stimulate a relatively weak PMN respiratory burst In the present study, we further examined the mechanisms of resistance of RC to neutrophil killing by comparing neutrophil degranulation and phagocytosis following stimulation by RC and SC opsonized in pooled human serum. RC, compared with SC, stimulated significantly less release of both the primary granule marker β-glucuronidase and the secondary granule marker lactoferrin. PMN also phagocytosed significantly greater numbers of SC, although the differences in phagocytosis were not great enough to account for the differences in degranulation. Suboptimal stimulation of degranulation and phagocytosis may thus contribute to the inability of neutrophils to kill RC. Moreover, reagent lactoferrin bound avidly to both RC and SC, raising the possibility that PMN-released lactoferrin may contribute to antifungal activity at the conidial surface by competing for iron or catalyzing the formation of oxygen radicals.
Kupffer Cell Proliferation and Glucan-Induced Granuloma Formation in Mice Depleted of Blood Monocytes by Strontium-89
Authors: Yamada Masahiko , Naito Makoto , Takahashi Kiyoshi
Published: January 2018

In mice with prolonged severe monocytopenia induced by selective irradiation of the bone marrow with the bone-seeking isotope 89Sr, the proliferative capacity of Kupffer cells was studied by immunohistochemistry with an anti-mouse macrophage monoclonal antibody, F4/80, ultrastructural peroxidase (PO) cytochemistry, and tritiated thymidine (3HTdR) autoradiography. The number and 3HTdR uptake of Kupffer cells were significantly increased in the splenectomized mice after severe monocytopenia had continued for more than 4 wk, and almost all the Kupffer cells showed a localization pattern of PO activity similar to that of resident macrophages in the liver of normal mice. In the glucan-induced granuloma formation in similar mortocytopenic mice, Kupffer cells proliferated. conglomerated, and transformed into epithelioid cells, which fused together to become multinuclear giant cells. These results suggest that Kupffer cells are a self-renewing population by their own cell division and can participate actively in granulomatous inflammations in severely monocytopenic and intact mice.
Differential Expression of M-CSF, G-CSF, and GM-CSF by Human Monocytes
Authors: Lee Mei-Ting , Kaushansky Kenneth , Ralph Peter , Ladner Martha B
Published: January 2018

The colony-stimulating factors (CSF) belong to a group of proteins which regulate blood cell production. Human monocytes allowed to adhere express high levels of M-CSF transcripts and secreted protein at 24 h in the presence but not in the absence of indomethacin (Indo), an inhibitor of prostaglandin E (PGE) production. When induced with lipopolysaccharide (LPS), adherent monocytes express M-CSF, G-CSF, and GM-CSF transcripts and secrete these proteins and TNF. M-CSF and GM-CSF messages increase in LPS-induced monocytes by the addition of Indo, while G-CSF mRNA appears to decrease. Exogenous addition of PGE-2 to LPS-induced monocytes down-modulates the expression of M-CSF and GM-CSF transcripts. G-CSF message is elevated, suggesting an alternate pathway to G-CSF regulation. PGE-2 inhibits the secretion of CSFs and TNF. In contrast, LPS-induced monocytes held 24 h in nonadherent culture express G- and GM-CSF but not M-CSF. Monocytes that are adhered for 24 h and then treated with LPS for an additional 24 h express only M-CSF message and secrete M-CSF and TNF. PGE-2 added with LPS during the 24–48 h induction blocks M-CSF and TNF production, but appears to enhance M-CSF message expression, in contrast to its effect on 0 h inductions. These results suggest that adherence alone induces M-CSF gene expression, but low levels of PGE or other arachidonic acid metabolites limit this expression. Other events in 1 d-cultured monocytes block the ability to induce G-CSF and GM-CSF expression with LPS, and block the suppressive effect of PGE-2 on M-CSF expression at the RNA level.
Adenosine and Its Derivatives Control Human Monocyte Differentiation Into Highly Accessory Cells Versus Macrophages
Authors: Najar Hossain Motieian , Ruhl Stephan , Bru-Capdeville Anne Christine , Peters Johann Hinrich
Published: January 2018

Human peripheral blood monocytes have been found to undergo a transitory state of high accessory activity before they fully become macrophages. Time kinetics were done to follow this accessory potential. Studying the regulation of accessory activity, we have found that monocyte-derived accessory cells (m-AC) pass through two phases of development, which both are adversely controlled by cyclic nucleotides. Phase I is positively correlated by intracellular cAMP increase and can be arrested by adenosine 3′;5′ cyclic monophosphate (cAMP) and synergystic agents. In addition to cAMP, non-cyclic adenine nucleotides and adenosine also mimic all cAMP effects. This behavior is explained by the known presence of surface 5′ nucleotidase and adenosine receptor, which in turn leads to activation of adenylate cyclase. At phase II serum is required to convert m-AC into macrophages. In the absence of serum, cells were arrested in the m-AC state. Adenine nucleotides effectively counteract the serum induction leading to the development of m-AC even in the presence of serum. Monocyte/macrophage markers such as Fc receptors and non-specific esterase strictly correlate negatively with the expression of accessory activity. Morphologically, the appearance of veils positively correlates with all experimental situations of high accessory activity. Therefore, it is evident that serum contains regulatory factors that strongly modify the accessory potency of the m-AC via the cyclic nucleotide system, thus presenting a potent immunoregulatory principle at the beginning of the immune cascade.
Recombinant Granulocyte–Macrophage Colony-Stimulating Factor Activates Human Macrophages to Inhibit Growth or Kill Mycobacterium avium Complex
Authors: Bermudez Luiz Eduardo M , Young Lowell S
Published: January 2018

Organisms belonging to the Mycobacterium avium complex (MAC) are associated with life-threatening bacteremia in patients with the acquired immunodeficiency syndrome (AIDS). As these organisms survive within macrophages, we examined the ability of recombinant human granulocyte–monocyte colony-stimulating factor (GM-CSF) to activate human monocyte-derived macrophages to inhibit the intracellular growth or kill the most mouse-virulent MAC strain In our collection that belongs to serotype 1. While unstimulated cells did not inhibit intracellular growth of MAC, macrophages activated by GM-CSF (10–104 U/ml) inhibited or killed up to 58 ± 5% of the initial inoculum. This activation was dose-dependent, with maximal change occurring with a dose of 100 U/ml after 72 hr exposure. Inhibition or killing was demonstrated if GM-CSF was given both before or after establishment of infection. The combination of GM-CSF (102 U/ml) plus TNF (102 U/ml) augmented macrophage killing (range, 31 ± 4%) compared with GM-CSF (102 U/ml) alone, but the combination of recombinant human interferon-gamma (IFNγ) plus GM-CSF resulted in a significant decrease in intracellular inhibition of growth or killing (13.3 ± 2%) compared with 57.7 ± 5% obtained with GM-CSF alone. These results indicate that: 1) GM-CSF can activate macrophages to inhibit intracellular growth or kill MAC; 2) killing may be augmented by TNF; and 3) IFNγ may impair GM-CSF-dependent macrophage activation.
Characteristics of the Chemotactic Activity of Heparin Cofactor II Proteolysis Products
Authors: Hoffman Maureane , Pratt Charlotte W , Corbin Lisa W , Church Frank C
Published: January 2018

The physiological function of the serpin (serine proteinase inhibitor) heparin cofactor II (HCII) is not well understood. A role for HCII as an inhibitor of thrombin in the presence of dermatan sulfate and heparin has been proposed. Neutrophils (PMN) are the major cellular component of acute inflammation. HCII can be proteolytically inactivated by cathepsin G (CG) and elastase (LE), which are released by stimulated PMN. We have recently shown that reaction products of HCII with CG and LE are potent chemotaxins for PMN. Monocytes (monos) appear later in the course of inflammation than do PMN. They differentiate into macrophages in the tissues and participate in healing of damaged tissue and initiating immune responses. We found that the proteolysis products of HCII were chemotactic for monocytes in a fashion similar to their effects on PMN. At 10-8 to 10-9 M, the chemotactic activity of HCII proteolysis products was comparable to that of 10-8 M N-formyl-Met-Leu-Phe (fMLP). The chemotactic activity of HCII-proteinase reaction products is mediated by a different mechanism than that of α1 proteinase inhibitor (α1PI)-LE complexes or fMLP. Our data suggest that chemotactic activity generated by proteolysis of HCII is not due to the conformational change induced by cleavage of the exposed loop near the reactive site nor by release of the reactive site peptide. We also compared the effects of HCII reaction products and fMLP on expression of Mac-1 and p150,95 adhesive proteins. Mac-1 has been implicated in mono adhesion and chemotaxis and as a potential initiator of coagulation. The surface expression of Mac-1 was not increased above control levels by incubation of leukocytes with HCII digests, even though fMLP did increase surface Mac-1. Proteolysis products of HCII could play a role in the initial influx of PMN into a thrombus, and in the transition from acute to chronic inflammation, or to granulation and healing.
Eicosanoid Production in Nonparenchymal Liver Cells Isolated From Rats Infused With E. coli Endotoxin
Authors: de Turco E B Rodriguez , Spitzer J A
Published: January 2018

Continuous i.v. infusion of a nonlethal dose of Escherichia coll endotoxin induced an early (3-h) accumulation of neutrophils in the rat liver followed by a later (30-h) greater extravasation of mononuclear phagocytes (MNP). These inflammatory cells, recovered together by centrifugal elutriation, were analyzed for their potential capacity to metabolize [1-14C]-AA. Ca2+ ionophore A23187 (5 μM) stimulated the release of [1-14C]-AA from PC and PI both in cells from saline- and ET-infused rats, the latter showing a higher capacity to further metabolize AA to eicosanoids. LTB4 and 5-HETE were the major metabolites accumulated in cells from rats infused with ET for 3 h, while PGD2 played the main role in cells from saline-infused rats. This could reflect [1-14C]-AA metabolism by PMNP and Kupffer cells, respectively. By 30 h of ET-infusion, a shift from PGD2 to PGE2 release was observed. These results suggest that eicosanoids released by nonparenchymal cells (i.e., Kupffer and endothelial cells) and PMNP in the liver of ET-infused rats may alter the normal intercellular information flow between parenchymal and nonparenchymal cells, contributing to the severe impairment in liver function and metabolism during endotoxicosis and sepsis.
Oxidative Metabolism of Polymorphonuclear Leukocytes: Modulation by Adhesive Stimuli
Authors: Laurent F , Benoliel A M , Capo C , Bongrand P
Published: January 2018

Different agents such as phorbol myristate acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), or opsonized zymosan induced an oxidative burst in rat peritoneal polymorphonuclear leukocytes (PMNs) elicited by casein. Plastic adhesion of PMNs down-regulated superoxide (O2) release stimulated by PMA or fMet-Leu-Phe but had no effect on zymosan-induced O2 generation, indicating that the O2 forming enzyme, the NADPH oxidase, was not affected by modulation and that a common step of the transductional events induced by PMA or fMet-Leu-Phe might be involved in this regulation. We demonstrated that a differential translocation of protein kinase C (PKC) was not responsible for that modulation. PMA-induced secretion of granule content (vitamin B12-binding protein) was not susceptible to modulation, suggesting that the transductional pathways leading to O2 generation and granule secretion are partly separated. The adhesion of PMNs to different substrates (glass, plastic, albumin-, laminin-, fibronectin-, poly-lysine-, or concanavalin A-coated plastic) down-regulated to different extent superoxide release. Whether the nature of the biochemical signal induced by the diverse adhesive stimuli or a physical parameter such as binding strength was involved in this differential behavior remains to be elucidated. Since adhesiveness was dependent on the state of the cytoskeleton and O2 inducers were reported to stimulate actin polymerization, we studied the F-actin content and distribution of PMNs by using the specific fluorescent probe NBD-phallacidin and an original methodology allowing a quantitative analysis of fluorescence on both adherent and suspended cells. PMA induced a polarization of F-actin on suspended PMNs but had no effect on the intracellular distribution of F-actin in adherent PMNs. Thus, we suggest that the adhesion of PMNs induced an immobilization of F-actin, possibly correlated to the down-regulation of one of the transductional pathways involved in the NADPH oxidase activation.
Tumor Necrosis Factor and Granulocyte Macrophage-Colony Stimulating Factor Stimulate Human Macrophages to Restrict Growth of Virulent Mycobacterium avium and to Kill Avirulent M. avium: Killing Effector Mechanism Depends on the Generation of Reactive Nitrogen Intermediates
Authors: Denis Michel
Published: January 2018

An avirulent and a virulent strain of Mycobacterium avium were selected on the basis of their growth patterns in human monocyte-derived macrophages. The virulent 7497 M. avium grew progressively in untreated macrophages, whereas the avirulent LR/149 M. avium was killed to a moderate extent by untreated human macrophages (50% of the original infectious inoculum killed 7 days after infection). We set out to investigate the possibility of modulating these growth patterns by cytokine treatment. Application of tumor necrosis factor (TNF) (100 U/ml) led to macrophages restricting significantly the growth of virulent M. avium 7497 (tenfold decrease at 7 days). TNF was also effective at modulating positively the interaction between avirulent LR/149 M. avium and macrophages inasmuch as TNF-treated cells killed 99% of infecting mycobacteria at 7 days. Granulocyte macrophage-colony stimulating factor (GM-CSF) (100–10,000 U/ml) treatment led to macrophages being as mycobacteriostatic for virulent 7497 M. avium as TNF-α-treated cells (i.e., tenfold reduction in growth). Treatment of macrophages with both GM-CSF and TNF-α was shown to have additive effects on bacteriostatic activity on M. avium. The mechanism of killing of avirulent M.  avium by TNF-α was shown to be dependent on the generation of reactive nitrogen intermediates, as seen by inhibition of effector mechanisms by NG-monomethyl-arginine and arginase. Moreover, there was a correlation between NO2– generation and mycobactericidal activity of macrophages. Addition of superoxide dismutase reversed the killing of avirulent M. avium by untreated or TNF-treated macrophages. This abrogation was also apparent in chronic granulomatous disease (CGD) macrophages, which were inefficient at generating reactive oxygen intermediates. Moreover, macrophages from CGD patients killed avirulent M. avium as efficiently as cells from normal individuals. We conclude from these results that 1) GM-CSF and TNF-α, alone or in combination, increase effector functions of macrophages against virulent and avirulent strains of M. avium; 2) reactive nitrogen intermediates seem to be involved in this effector mechanism; and 3) superoxide dismutase protected M. avium against macrophage effector function, seemingly by protecting the bacteria against endogenous superoxide anion. The implications of these findings for host resistance to atypical mycobacteria are discussed.
Cellular Binding Mechanism on Rat Macrophages for Sialylated Glycoconjugates, Inhibited by the Monoclonal Antibody ED3
Authors: Damoiseaux J G M C , Döpp E A , Dijkstra C D
Published: January 2018

The mAb ED3 recognizes a subpopulation of rat macrophages, with a highly restricted tissue distribution. The tissue distribution as well as the in vitro expression of the ED3 antigen and of the sheep erythrocyte receptor (SER), binding unopsonized erythrocytes in the mouse, are very similar. This receptor has almost the same binding characteristics, although a different tissue distribution, as the sialic acid binding receptor (SAR), binding ganglioside-coated erythrocytes in the rat In this study we summarize the available literature concerning these sialic acid binding receptors (SER and SAR). Furthermore we have identified ED3 as SER by inhibition studies of erythrocyte binding with mAb ED3, as well as by the newly developed equivalents ED16 and ED17. We also show that light trypsin treatment of alveolar macrophages, expressing SAR, results in SER-like activity. This obtained SER-like activity could not be blocked by the mAb ED3, indicating that SER and SAR are different receptors. It appears that rat macrophages can express two receptors for sialylated glycoconjugates, a high-affinity receptor SER, recognized by mAb ED3, and a low-affinity receptor SAR, not recognized by mAb ED3.
The Human Monocyte-Like Cell Line THP-1 Expresses FcγRI and FCγRII
Authors: Fleit Howard B , Kobasiuk Catherine D
Published: January 2018

THP-1 cells are a monocyte-like cell line derived from a patient with acute monocytic leukemia and unlike other leukemic cell lines has a normal diploid karyotype. We have characterized FcγR expression on this cell line by flow cytometry, radiolabeled lgG1 and monoclonal antibody (mAb) binding assays, and biochemical analysis. Flow cytometric analysis of THP-1 cells with anti-FcγRI, II, and III mAb, and a rabbit anti-FcγRIII F(ab')2 demonstrated that only FcγRI and FCγRII are expressed by these cells. A panel of anti-FcγRIII mAb (anti-CD16) failed to bind to THP-1 cells. Biochemical studies identified polypeptides of 64 to 78 kDa (FcγRI) and of 42 to 53 kDa (FcγRII). FcγR expression was determined by binding of radioiodinated human lgG1 (to detect FcγRI), mAb IV.3 (to detect FcγRII), or rabbit IgG immune complexes. Thirty-five thousand high affinity binding sites (dissociation constant [KD] = 4.22 × 10-9M) for lgG1 were found on THP-1 cells. Interferon-γ (IFNγ) upregulated FcγRI expression by THP-1 cells 2.8-fold, whereas FcγRI on U937 cells was increased six- to eight-fold by this cytokine. Phorbol myristate acetate (PMA), tumor necrosis factor-α (TNFα), and vitamin D3 had no effect on lgG1 binding by THP-1 cells. Fifty thousand IgG molecules in immune complexes bound to THP-1 cells. IFNγ treatment increased this binding by four-fold, PMA treatment resulted in a 50% increase in the number of IgG immune complexes bound, whereas vitamin D3 treated THP-1 cells bound half as many IgG immune complexes as control cells. Binding assays utilizing mAb IV.3 identified 50,000 sites per cell. Treatment of THP-1 cells with IFNγ, TNFα, PMA, or vitamin D3 had no effect on FcγRII expression. That FcγRI plays a predominant role in immune complex binding was demonstrated by inhibition studies. Human lgG1 as well as mouse lgG2a mAb to FcγRII inhibited immune complex binding by 76 to 84%, whereas mouse lgG1 mAb to FcγRII had minimal effect on immune complex binding. FcγR expression may not be linked to differentiation of THP-1 cells since only 1,25 vitamin D3 was able to induce the expression of CD14, a marker of mature monocytic phenotype.
Effect of Dietary Fish Oil on Development and Selected Functions of Murine Inflammatory Macrophages
Authors: Hubbard Neil E , Somers Scott D , Erickson Kent L
Published: January 2018

Inflammatory macrophages from mice fed diets containing menhaden fish oil (MFO) have a reduced capacity for cytotoxicity of mastocytoma cells upon activation with interferon-γ (IFNγ) and lipopolysaccharide due to an altered responsiveness to IFNγ. In an effort to elucidate further how dietary MFO effects macrophage function, we have studied the maturation of inflammatory macrophages from mice fed MFO compared with mice fed safflower oil (SFO) using several processes that serve as markers of the activational state. No significant differences in the recruitment or percentage of peritoneal exudate cells as macrophages after thioglycollate injection and no differences in spreading, binding, or phagocytosis of sheep erythrocytes or phagocytosis of yeast by inflammatory macrophages were observed when the dietary groups were compared. However, MFO macrophages had an altered capacity for peroxide release when stimulated with unopsonized zymosan (10–200 μg/ml). Furthermore, to elucidate how MFO feeding could alter IFNγ-induced responses of inflammatory macrophages, we assessed phorbol-12-myristate-13-acetate-induced hydrogen peroxide production and expression of class II MHC determinants (la). There were no differences between macrophages from mice fed the two diets with respect to the production of peroxide when they were preincubated with 0.1–10 U/ml of IFNγ. However, MFO macrophages had greater peroxide production after enhancement with 100 U/ml of IFNγ. With respect to la induction, the percentage of macrophages responding to IFNγ was not altered by diet, and there were no differences in expression of la induced by 24 hr exposure to IFNγ. Thus the differential effect of MFO compared with SFO is probably mediated not by an alteration in the maturation of inflammatory macrophages but rather through the alteration of IFNγ-induced functions such as peroxide production.
Activated Macrophage Conditioned Medium: Identification of the Soluble Factors Inducing Cytotoxicity and the L-Arginine Dependent Effector Mechanism
Authors: Amber Ina J , Hibbs John B , Parker Charles J , Johnson Barbara B , Taintor Read R , Vavrin Zdenek
Published: January 2018

Conditioned medium (CM) from cultures of cytotoxic activated macrophages causes inhibition of mitochondrial respiration, DNA synthesis, and aconitase activity in murine EMT-6 mammary adenocarcinoma cells by an l-arginine dependent effector mechanism. CM induces cytotoxicity and nitrite synthesis in EMT-6 cells in a dose dependent manner. We have identified the soluble factors in CM that induce cytotoxicity and synthesis of inorganic nitrogen oxides from l-arginine by EMT-6 cells. Using functional inhibition experiments, the activity of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) in CM was investigated. The LPS inhibitor polymyxin B and TNFα antibody produced a modest decrease in nitrite production, while IFNγ antibody markedly inhibited both nitrite production and cytostasis. Simultaneous treatment with polymyxin B, TNFα antibody, and IFNγ antibody reduced EMT-6 cell nitrite production by 81%, and cytostasis by 74%. By Western blot, IFNγ and TNFα were shown to be present in CM. When CM was subjected to hydrophobic interaction chromatography, a single peak of activity was eluted, and Western blot showed that the active fractions contained IFNγ. Furthermore, IFNγ antibody neutralized the activity in these chromatographic fractions. We conclude that induction of inorganic nitrogen oxide synthesis from l-arginine by the synergistic combination of IFNγ, TNFα, and LPS accounts for most of the biologic activity of CM, and that IFNγ is the major priming factor.
Developmental Regulation of the Cytokine Repertoire in Human Macrophages: IL-1, IL-6, TNF-α, and M-CSF
Authors: Scheibenbogen Carmen , Andreesen Reinhard
Published: January 2018

We were interested in the dependence of constitutive and stimulated cytokine secretion on the stage of macrophage (MAC) differentiation in vitro. Elutriation-purified blood MO were cultured up to 28 days and their secretory repertoire was analyzed under adherence conditions at various culture stages. For each of the cytokines tested, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage colony-stimulating factor (M-CSF), a different pattern of regulation was observed. During the initial phase of maturation (up to day 7 in culture) within which the characteristics of normal MO to MAC transformation are achieved, M-CSF was the only cytokine to be secreted constitutively. From the LPS-dependent cytokines, IL-1β and IL-6 were downregulated whereas TNF-α levels increased severalfold. For the release of IL-1β, IL-6, and TNF-α a synergistic effect of interferon-gamma (IFN-g) and lipopolysaccharide (LPS) was observed. M-CSF release increased until day 7 in culture with LPS being stimulatory for this particular cytokine only during the first days of differentiation. Upon further cultivation of MAC up to 28 days, LPS-induced IL-1β levels remained very low, but IL-6 levels increased again reaching that of blood MO, and TNF-α continued to rise reaching levels up to 30-fold higher than in blood MO. M-CSF secretion stayed high with LPS even suppressing constitutive secretion. Long-term cultured MAC started to release IL-6 and TNF-α also in the absence of a stimulus and, furthermore, became responsive to IFN-g alone. Our data show that the release of each cytokine investigated is differently regulated during maturation. These results document the functional plasticity of human MAC and emphasize the impact that MO to MAC differentiation may have in vivo.
Chronic Treatment With P2-Purinergic Receptor Agonists Induces Phenotypic Modulation of the HL-60 and U937 Human Myelogenous Leukemia Cell Lines
Authors: Cowen Daniel S , Berger Melvin , Nuttle Louise , Dubyak George R
Published: January 2018

In previous studies we have demonstrated that extracellular ATP (and UTP), acting through P2-purinergic receptors, can stimulate the inositol phospholipid signaling system in neutrophils and monocytes, as well as in neutrophil monocyte progenitor cells. In this study we have examined the ability of extracellular nucleotides to modulate the phenotype of myelomonocytic progenitor cells. As model systems, we utilized the established HL-60 promyelocytic and U937 promonocytic human cell lines which were cultured in the continuous presence of nucleotides known to be potent agonists for P2-purinergic receptors. When cultured for 5 days with ATPγS (a phosphatase resistant analog of ATP) plus 10% fetal bovine serum, both HL-60 cells and U937 cells expressed several (but not all) phenotypic characteristics of differentiated phagocytes. In HL-60 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides, (2) a reduction in cell size with a decreased nuclear cytoplasmic ratio, (3) a sharply reduced rate of proliferation, (4) a reduction in the percentage of cells expressing surface transferrin receptors, and (5) an increase in the percentage of cells expressing the type 1 complement receptor (CR1). In U937 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides and platelet activating factor, (2) a reduced rate of proliferation, (3) a reduction in the percentage of cells expressing surface transferrin receptors, and (4) increases in the percentage of cells expressing both type 1 (CR1) and type 3 (CR3) complement receptors. During the first 12-24 hr after exposure to ATPγS, HL-60 cells showed no obvious changes in morphology, viability, or the levels of β-actin mRNA, but did show (1) a 4-fold increase in chemotactic peptide-induced Ca2+ mobilization, and (2) a >90% decrease in c-myc mRNA levels. Significantly, when HL-60 cells were treated under serum-free conditions, the ability of ATP to enhance expression of functional FMLP receptors could be dissociated from the inhibitory effects of adenine nucleotides on cell proliferation observed in serum containing media. Moreover, treatment of serum-free HL-60 cultures with UTP, another P2-purinergic receptor agonist, also resulted in enhanced expression of functional FMLP receptors.
Effect of Tumor Necrosis Factor on the Generation of Chlorinated Oxidants by Adherent Human Neutrophils
Authors: Test Samuel T
Published: January 2018

Human neutrophils adherent to simulated biologic surfaces undergo significant activation of the respiratory burst over prolonged periods of time in response to stimulation with the cytokines tumor necrosis factor-alpha (TNFα) or tumor necrosis factor-beta (TNFβ) or with the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP). In this study, neutrophils were examined for their ability to generate the highly reactive and powerful oxidant hypochlorous acid (HOCI) and the longer-lived, less reactive endogenous nitrogen-chlorine (N-CI) derivatives in response to these stimuli either alone or when exposed to recombinant human TNFα (rTNFα) or β (rTNFβ) prior to addition of FMLP. Neutrophils adherent to fetal bovine serum-coated polystyrene tissue culture wells were able to generate only small quantities of HOCI when incubated with rTNFα, rTNFβ, or FMLP individually. However, when neutrophils were first incubated with either rTNFα or rTNFβ prior to addition of FMLP, there was a marked increase in HOCI generation. Neutrophils stimulated in such a manner consumed ~18% of the HOCI generated in the formation of N-CI derivatives. Further scrutiny of the response to the combination of rTNFα and FMLP revealed that HOCI release was rapid, with 80% of total HOCI accumulation occurring within 15 min after FMLP addition. The amount of HOCI generated was dependent on the number of cells added and on the concentration of both rTNFα and FMLP. Comparison of HOCI generation with superoxide anion and myeloperoxidase release showed that the amount of HOCI generated was limited primarily by the amount of myeloperoxidase released rather than by the degree of respiratory burst activation. These results demonstrate that human neutrophils stimulated with FMLP after a brief incubation with rTNFα or rTNFβ can generate cytotoxic and microbicidal concentrations of chlorinated oxidants.
Phagocytosis of Opsonized Yeast Induces Tumor Necrosis Factor-α mRNA Accumulation and Protein Release by Human Polymorphonuclear Leukocytes
Authors: Bazzoni Flavia , Cassatella Marco A , Laudanna Carlo , Rossi Filippo
Published: January 2018

In this report we show that phagocytosis of yeast particles opsonized with IgG (Y-IgG) by human polymorphonuclear cells (PMN) results in the selective induction of tumor necrosis factor (TNF-α) messenger RNA (mRNA) and release of its mature protein. Lipopolysaccharide (LPS) was also found able to induce TNF-α secretion by PMN, but was a less potent stimulus compared with Y-lgG. There was no evidence of interleukin-6 (IL-6) gene expression in PMN after phagocytosis of Y-lgG or in response to LPS, whereas IL-6 mRNA expression and secretion were induced by either stimulus in monocytes. These findings demonstrate that a physiological function such as phagocytosis modulates the gene expression for a cytokine in PMN and shed new light on the understanding of the pathogenesis of the inflammatory process.
Leukocyte-Endothelial Cell Adhesive Interactions: Role of Xanthine Oxidase-Derived Oxidants
Authors: Suzuki Masayuki , Grisham Matthew B , Granger D Neil
Published: January 2018

The objective of this study was to determine whether agents that either scavenge or inhibit the production of oxygen radicals can alter the adhesive interactions between leukocytes and venular endothelium elicited by ischemia-reperfusion. Cat mesenteric and intestinal blood flows were reduced to 20% of baseline for 1 hr, followed by 1 hr of reperfusion. Sixty minutes after reperfusion, red blood cell velocity (Vr), leukocyte rolling velocity (Vw), and the number of adherent leukocytes were measured in mesenteric venules. Then, either manganese-superoxide dismutase (Mn-SOD), catalase, desferrioxamine, or oxypurinol was administered intravascularly. Ten minutes later, repeat measurements were obtained and compared with pretreatment values. Catalase, Mn-SOD, and oxypurinol significantly attenuated neutrophil adherence while neither inactivated-catalase nor desferrioxamine altered the reperfusion-induced leukocyte adhesion. The ratio of Vw to erythrocyte velocity, an index of the fracture stress between rolling leukocytes and venular endothelium, was not altered by any of the agents studied. These results and data in the literature indicate that many of the agents that are commonly used to either scavenge or inhibit the production of oxygen radicals in postischemic tissues exert a significant inhibitory influence on leukocyte adhesion to microvascular endothelium in vivo. Our results are also consistent with the view that xanthine oxidase-derived oxidants contribute to the leukocyte-endothelial cell adhesive Interactions associated with reperfusion of ischemic tissues.
Antibodies Against Granule Proteins Activate Neutrophils In Vitro
Authors: Charles Linda A , Caldas Maria Lucia R , Falk Ronald J , Terrell Regina S , Jennette J Charles
Published: January 2018

Polymorphonuclear leukocyte (PMN) respiratory burst was stimulated by heterologous antibodies against PMN granule proteins but not by control antibodies. Fluorescence-activated cell sorter (FACS) analysis of activated PMN demonstrated the presence of two primary granule proteins, proteinase 3 (PR-3) and cationic protein 57 (CAP-57) at the membrane surface. The presence of myeloperoxidase (MPO) at the cell surface of primed and unprimed PMN was confirmed by immunoelectron microscopy. Priming doses of recombinant tumor necrosis alpha (rTNFα) enhanced the rate of superoxide (O2-) production by these antibodies and increased the amount of surface protein accessible to these antibodies. Anti-neutrophil cytoplasmic autoantibodies (ANCA) with specificities for PMN granule proteins are present in patients with Wegener's granulomatosis, polyarteritis nodosa, and idiopathic and crescentic glomerulonephritis. The demonstration that antibodies against granule proteins activate PMN supports the hypothesis that the vasculitis seen in these diseases is due in part to PMN mediated oxidative injury following PMN stimulation by ANCA.
Histochemical and Immunochemical Study of the Fate of Candida albicans Inside Human Neutrophil Phagolysosomes
Authors: Marquis Gabriel , Garzon Simon , Montplaisir Serge , Strykowski Henri , Benhamou Nicole
Published: January 2018

To further define the urtrastructural events associated with the killing of Candida albicans by human neutrophils, four methods were used: (1) the periodate-thiocarbohydrazidesilver proteinate (PA-TCH-SP) staining of vicinal-glycol-containing complex carbohydrates; (2) the localization of thermostable immunodeterminants of the yeast cell wall, mannans or mannoproteins, using monospecific antibodies and a protein A-gold complex (monAb-gold); (3) the localization of mannose residues with concanavalin A labeled with gold particles (Con A-gold); (4) the localization of chitin oligomers using wheat germ agglutinin and ovomucoid labeled with gold particles (WGA-gold). The mannan-rich cell wall layers were progressively lost as shown by altered PA-TCH-SP reactivity and a diffuse pattern of staining with Con A-gold and monAb-gold. The de novo appearance of conspicuous amounts of glycogen-like particles near the plasmalemma and in the cell wall was interpreted as evidence of a reparative process of the yeast cell wall. Chitin was seemingly unaltered and readily demonstrated by the WGA-gold in the wall remnants of ghost cells.
C-reactive protein inhibits intracellular calcium mobilization and superoxide production by guinea pig alveolar macrophages
Authors: Földes-Filep Éva , Filep János G , Sirois Pierre
Published: January 2018

C-reactive protein (CRP) is a prototypical acute-phase reactant, the humoral and plasma concentrations of which rise dramatically after tissue injury or inflammation. The effects of CRP on superoxide production and intracellular calcium mobilization by guinea pig alveolar macrophages challenged with platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and phorbol 12-myristate 13-acetate (PMA) were studied. CRP by itself did not activate alveolar macrophages up to a concentration of 100 μg/ml, whereas it inhibited superoxide production in a time- and dose-dependent manner with median inhibitory concentration (IC50) values of 4.2 ± 0.3,3.0 ± 0.2, and 3.2 ± 0.3 μg/ml for PAF (10-7 M), fMLP (10-7 M), and PMA (10-9 M), respectively. When CRP was incubated with the agonists before addition to cells, it inhibited PMA-, PAF-, and to a lesser extent fMLP-induced superoxide production. CRP also attenuated the rise in intracellular free calcium levels evoked by fMLP or PAF in a dose-dependent manner. These findings suggest that CRP may play a role in attenuating tissue damage secondary to activation of alveolar macrophages by inhibiting superoxide generation and mobilization of intracellular free calcium.
Macrophage cytoskeleton association with CR3 and CR4 regulates receptor mobility and phagocytosis of iC3b-opsonized erythrocytes
Authors: Ross Gordon D , Reed William , Dalzell Juanita G , Becker Susanne E , Hogg Nancy
Published: January 2018

Alveolar macrophages (AMø) were examined for CR1 (C3b receptor, CD35), CR3 (iC3b receptor; CD11b/CD18), and CR4 (iC3b receptor; CD11c/CD18) by assays for binding of C3-opsonized sheep erythrocytes (EC3b or EC3bi) and uptake of specific monoclonal antibodies (mAbs). In AMø isolates from nine normal volunteers, 49% of cells bound EC3b and 71% bound EC3bi. Quantitation of receptors per cell with [125I]mAbs showed 8.5 × 104 CR4, 5.1 × 104 CR3, and 2.6 × 104 CR1. With most AMø preparations, CR3 was the major receptor mediating attachment of EC3bi, despite the predominance of CR4 antigens. Anti-CR3 inhibited EC3bi rosettes by ≥50%, whereas anti-CR4 blocked rosettes by ≤18%. U937 cells differentiated with phorbol myristate acetate resembled AMø in receptor expression but exhibited almost no CR4-dependent rosetting. Despite the relative inability of CR4 to mediate EC3bi attachment, AMø ingestion of [51Cr]EC3bi was blocked by either anti-CR3 or anti-CR4. Two lines of evidence indicated that CR3 were more mobile within the membrane than were CR4. Immunofluorescence staining demonstrated patching and occasional capping of CR3, whereas CR4 remained uniformly distributed. This patching and capping of CR3 required the actin cytoskeleton, as it was inhibited by cytochalasin D. Modulation experiments using surfaces coated with anti-CR3 or anti-CR4 also showed that CR3 was more mobile than was CR4. However, there was some variation among AMø isolates from different donors. In seven isolates, no CR4 modulation was produced with anti-CR4, whereas in six other isolates, CR4 was modulated by 66%. Incubation of cells in cytochalasin D increased modulation of both CR3 and CR4 on mAb-coated surfaces. Cells exhibiting increased mobility of CR4 showed an increased ability to form CR4-dependent EC3bi rosettes. The data are consistent with the hypothesis that CR3 and CR4 exhibit a variable association with the cytoskeleton that regulates their mobility and function. A relatively mobile subset of CR3 and/or CR4 mediates EC3bi attachment, whereas a relatively immobile subset of CR3 and/or CR4 fails to mediate EC3bi attachment but functions to promote ingestion of EC3bi.
Functional alterations of swine peripheral blood mononuclear cells by methadone
Authors: Molitor Thomas W , Murtaugh Michael P , Click Robert E , Gekker Genya , Chao Chun , Peterson Phillip K
Published: January 2018

In vitro exposure of the synthetic opiate drug methadone allowed evaluation of putative immunomodulatory activities of swine peripheral blood mononuclear cells. Respiratory burst, an index of microbicidal activity, was suppressed by methadone in a dose-dependent manner following exposure for 48 h. The suppression was blocked by the opiate antagonist naloxone. Another macrophage function phagosome-lysosome fusion was impaired by exposure to methadone. A primary lymphocyte-mediated function natural killer cell activity was also affected. In contrast, the macrophage function antibody-mediated phagocytosis was not affected. Because the functions affected by methadone are critical to host defenses against pathogenic organisms, our findings suggest that opiate-mediated immunomodulation merits further study. Moreover, our studies suggest that swine may provide an ideal model for the investigation of opiate-mediated suppression of immune cell functions.
Increase in the rat blood leukocyte counts induced by PAF-acether is suppressed by general anesthesia
Authors: Bozza Patricia T , Silva Patricia M R , Castro-Faria-Neto Hugo C , Martins Marco A , Cordeiro Renato S B
Published: January 2018

Blood leukocyte count alterations induced by PAF-acether in anesthetized and nonanesthetized rats were investigated. Intravenous injection of increasing amounts of PAF-acether (1.5-8 μg/kg) in nonanesthetized animals induced dose-dependent hemoconcentration and leukocytosis. The former was apparent within 10 min, peaked from 30 min to 1 h, and diminished thereafter. The leukocytosis was noted within 30 min, was maximal at 1 h, and was over 4 h after injection of PAF-acether (4 μg/kg). It was characterized by a marked increase in the blood neutrophil counts under conditions in which the number of lymphocytes, monocytes, and eosinophils remained unchanged. PAF-acether–induced leukocytosis occurred in parallel with a marked decrease in the number of bone marrow nucleated cells, suggesting that the latter phenomenon may determine the former one. Leukocytosis by PAF-acether was inhibited dose-dependently by specific PAF-acether antagonists including BN 52021 (median effective dose ED50 = 4.99 mg/kg), WEB 2086 (ED50 = 4.59 mg/kg), and 48740 RP (ED50 = 9.02 mg/kg). General anesthesia by either pentobarbital, urethane, or ether inhalation, but not by ketamine, also impaired the PAF-acether–induced blood leukocytosis under conditions in which the hemoconcentration was not modified. In addition, pentobarbital-anesthetized rats did not have reduced bone marrow nucleated cell counts after PAF-acether stimulation. These findings are consistent with the assessment that PAF-acether–induced rat leukocytosis is accounted for by a bone marrow neutrophil mobilization process that is clearly suppressed in animals anesthetized by pentobarbital.
The up- and down-modulation of immunoglobulin G Fc receptors and complement receptors on activated human neutrophils depends on the nature of activator
Authors: Leino Lasse , Lilius Esa-Matti
Published: January 2018

Human neutrophils were activated with soluble stimuli, fonnyl-methionyl-leucyl-phenylalanine (fMLP) or ionophore A23187, and with opsonized particles, zymosan or Streptococcus pneumoniae bacteria. Monoclonal antibodies and flow cytometry were used to assess the expression of Fc-γ receptors (FcRI, FcRII, FcRIII) and complement receptors (CR1, CR3). The role of extracellular calcium and magnesium in the modulation of receptor expression was also examined. The low-level expression of FcRI was not affected by any activator tested. fMLP and A23187 did not alter the expression of FcRII, whereas a significant, Ca2+- and Mg2+-independent down-modulation was observed upon activation with opsonized particles. All activators clearly decreased the surface expression of FcRIII in the presence of Ca2+ and Mg2+, probably as a consequence of shedding of the phosphatidylinositol-glycan–anchored receptor protein. The removal of calcium and magnesium blocked the shedding of FcRIII caused by soluble stimuli, whereas it retarded but did not abolish the fall in FcRIII expression when cells were incubated with opsonized particles. This fall was likely due to internalization of the receptor molecules while the shedding was blocked. A rapid increase in CR1 and CR3 expression was seen upon activation with soluble stimuli. The change in CR1 expression was independent of extracellular Ca2+ and Mg2+. The increase in CR3 number required an influx of divalent cations. No total up-modulation of complement receptors occurred when neutrophils were activated with opsonized particles. However, the kinetic analysis revealed a temporary up-modulation that was followed by a down-modulation. The results indicate that the expression of both Fc-γ and complement receptors on human neutrophils is changed upon activation and that the up- and down-modulation of these receptors depends on the nature of activator. We also suggest that in neutrophils the FcRIII down-modulation is the result of both receptor shedding and internalization, while FcRII is down-modulated by receptor internalization.
High deformability and motility of lymphokine-activated killer cells in vitro and in vivo
Authors: Bouwens Luc , Narayani Inna , Wisse Eddie
Published: January 2018

The motility and deformability of lymphokine-activated killer cells, purified by their adherence to plastic (A-LAK cells), was investigated in vivo and in vitro. In vitro, A-LAK cells were observed as intrinsically motile cells. They continuously changed their shape while forming protopods or pseudopods and crawled over the culture surface. A-LAK cells were able to migrate across micropores of 3 μm diameter, which was three times smaller than the average diameter of an A-LAK cell. Even in the absence of serum factors and of interleukin-2 (IL-2), more than half of the inoculated cells migrated across such micropore membranes within 18 h. Electron microscopic examination of these micropore membranes showed that the A-LAK cells were highly deformable. A-LAK cells also migrated across a confluent monolayer of endothelium-like 10T1/2 cells. After 24 h incubation on the monolayers, about 20% of the A-LAK cells were found underneath the monolayer. There, they actively moved in the narrow space between the monolayer and the bottom of the culture dish. In vivo IL-2-activated cells showing the large granular lymphocyte morphology accumulated in the hepatic microvasculature. Electron microscopic observations indicate that these cells did not accumulate by mechanical entrapment due to rigidity and size but rather by adherence to the endothelial lining of the sinusoidal capillaries. This also appeared to be the case for adoptively transferred A-LAK cells, which were seen to be attached to the capillary wall. These observations stress the importance of adhesive entrapment to explain the accumulation of intravenously transferred LAK cells in the vascular beds.
Synergism of interleukin 6 and 1α,25-dihydroxyvitamin D3 in induction of myeloid differentiation of human leukemic cell lines
Authors: Duits Ashley J , Dimjati Wati , van de Winkel Jan GJ , Capel Peter JA
Published: January 2018

Interleukin 6 (IL-6) is a multifunctional cytokine with an important role in immunity. We analyzed the effect of recombinant human IL-6 in combination with 1α,25-dihydroxyvitamin D3 (Vit. D3) on differentiation of the human myeloid leukemic cell lines U937 and HL-60 with respect to alterations in antigen expression and functional activity. Of a panel of antigens analyzed, only CD11b (the α chain of CR3), and GD14 (a cell surface protein recognizing the lipopolysaccharide-binding protein-lipopolysaccharide complex) had significantly increased expression. Expression of ICAM-1 (CD54), a ligand for LFA-1, was also found to be enhanced with a concomitant increase of ICAM-1 mRNA levels. Enhanced nonspecific esterase levels and induction of respiratory burst activity confirmed that cell differentiation was induced. Furthermore, IL-6 and Vit. D3 had a profound effect on functional activities, as shown by enhancement of rosetting between sheep erythrocytes, sensitized with C3bi (EAC), and either U937 or HL-60 cells. Also, phorbol myristate acetate–induced homotypic adhesion of U937, which is ICAM-1 dependent, was markedly induced by these agents. These results indicate an important role of IL-6 and Vit. D3 in myeloid cell Function and development.