bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct Visualization of Formylpeptide Receptor Binding on Rounded and Polarized Human Neutrophils: Cellular and Receptor Heterogeneity
Authors: Walter Robert J , Marasco Wayne A
Published: January 2018

We have used light microscope autoradiography to visualize binding of the formylhexa-peptide, N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-(125l)tyrosyl-lysine to rounded and spontaneously polarized human polymorphonuclear leukocytes. These cells possess receptors known to bind with high specificity and great avidity to the chemotactic formylpeptides. Cells adherent to glass slides were exposed to (125I)-hexapeptide at 4°, fixed, and autoradiographed. Hexapeptide binding was studied over the biologically active range of peptide concentrations varying from 0.63 nM to 10 nM and autoradiographic silver grains counted on 200 rounded or 50 polarized cells at each concentration. Examination of histograms plotted from these data revealed for rounded cells: 1) two major peaks at each concentration indicating the existence of two neutrophil subpopulations, the predominant subpopulation binding one-half as much formylpeptide (peak I) as the other (peak II); 2) progressively increasing proportions of cells in peak II as the free hexapeptide concentration increased. Accordingly, at 0.63 nM hexapeptide, peak II comprised only 8% of the total cell number, whereas at 10 nM, this peak represented 35% of the total cells. This suggested that different types of receptors may exist in the two cell subpopulations (high/low affinity or high/low negative cooperativity) and that these receptor types were expressed differentially on these subpopulations. Thus, cellular heterogeneity within the neutrophil population and receptor heterogeneity among hexapeptide receptors on an individual cell were both observed here. Each of these may significantly affect neutrophil functional responses to the chemotactic formylpeptides and may explain, at least in part, the curvilinearity in the Scatchard plots of formylpeptide receptor binding that has recently been reported.

At higher concentrations of peptide (5 nM), spontaneously polarized PMN bound hexapeptide more or less uniformly over the entire cell surface. However, at lower concentrations, hexapeptide binding was markedly shifted toward the cell anterior. As a group, polarized PMN bound similar total quantities of hexapeptide, as did rounded PMN at each peptide concentration tested. Receptors displaying high- and low-affinity characteristics were, however, distributed asymmetrically over the cell surface, with the high-affinity type receptors predominantly on the anterior one-half of the cell. Such an asymmetric distribution may serve to initiate or perpetuate cell locomotion.
Quantitative Studies of Human Monokine-Induced Endothelial Cell Elongation
Authors: FitzGerald Oliver M , Hess Evelyn V , Chance Arlene , Highsmith Robert F
Published: January 2018

Leucocyte-endothelial cell interactions are important in the inflammatory response. In this study, the effect of peripheral blood mononuclear cell (PBMC) products on endothelial cell (EC) shape was examined and quantified. PBMC were obtained from normal donors by Ficoll-Hypaque separation of heparinized whole blood and cultured for 72 hr in media containing 10% fetal calf serum with and without concanavalin A (Con A). Media conditioned by PBMC or control, nonconditioned media were then added to preconfluent, first passage EC cultures derived from human umbilical veins. Conditioned media from Con A-stimulated PBMC resulted in a dose-dependent, marked elongation and whorling of cultured EC. The minimum effective concentration found to elicit a response was 1.25%, with a maximum response occurring at 10%. Quantitative morphometric analyses of treated EC indicated that the elongation was highly significant (p < 0.001) when compared to EC incubated with control, nonconditioned media. In addition, EC elongation was accompanied by a highly significant (p < 0.001) increase in cell area. Although less dramatic, conditioned media from unstimulated PBMC also elicited a similar, significant dose-dependent change in EC shape. Significant changes in EC shape were evident within 6 hr and continued over the time course of the experiment (40 h). Cell shape changes were partially reversible at 18 h after removal of the PBMC-conditioned media and replacement with control, nonconditioned media. The change in EC morphology induced by a PBMC-derived factor(s) suggests a mechanism by which activated leucocytes may modulate cellular traffic at the blood-vessel wall interface.
Increased Adhesiveness of Complement-Stimulated Neonatal Calf Neutrophils and Its Pharmacologic Inhibition
Authors: Zwahlen Roland D , Slauson David O , Neilsen Nancy R , Clifford Charles B
Published: January 2018

Several in vitro functions of neonatal neutrophils (N-PMN) have been reported to be deficient and may be functionally related to the increased susceptibility of the newborn to infection. To evaluate an in vitro event corresponding to one of the early steps in the sequence of inflammation, we used zymosan-activated plasma as a source of activated complement fragments (Cf) and measured adherence of normal and Cf-stimulated bovine N-PMN to columns of Sephadex G-25. Adherence of control N-PMN and adult PMN (A-PMN) was comparable. When N-PMN and A-PMN were stimulated with a subaggregating dose of Cf, both responded with similar increases in adhesiveness. The stimulatory effect of Cf on N-PMN adhesiveness could be inhibited by pre-incubation of the N-PMN with either steroidal (0.05 mM dexamethasone) or non-steroidal (32 mM phenylbutazone) anti-inflammatory drugs. Ultrastructural observations correlated well with the results of the adhesiveness assays, and morphometric evaluation revealed an increase in the sectional circumference of Cf-stimulated N-PMN. Control cells were round with few short cytoplasmic projections, whereas Cf-stimulated cells exhibited marked shape irregularity, polarity, and prominent organelle-free lamellipodia development. There was a highly significant (P < 0.001) increase in the measured circumference of Cf-stimulated cells. Thus, N-PMN were highly responsive to Cf stimulation, developed morphologic and functional changes indistinguishable from Cf-stimulated A-PMN, and were sensitive to pharmacologic inhibition.
Characterization of Surface Markers of Continuously Growing Murine Resident Macrophages
Authors: Muller Christian D , Lombard Yves , Bartholeyns Jacques , Poindron Philippe , Schuber Francis
Published: January 2018

Conditions have been described which allow an in vitro indefinite multiplication of differentiated murine macrophages (Lombard et al: Biol Cell 53, 219, 1985). R. and MAY-1 ceil lines, which were obtained, respectively, from mouse (Balb/c) spleen and resident peritoneal macrophages, have been further characterized. They present at their surface, besides the Mac-1 antigen and Fc-receptor, a mannose receptor which was characterized for its binding properties. This receptor Is responsive for a specific phagocytosis of mannosylated particles, i.e., mannosylated latex beads or oil droplets containing mannosylated bovine serum albumin. Moreover, R and MAY-1 ceils present an ectoenzyme profile (NAD+ glycohydrolase and nucleotide pyrophosphatase) similar to those of the corresponding resident macrophages.
Regulation by Interferon γ of Function in the Acute Monocytic Leukemia Cell Line, THP-1
Authors: Gaffney E V , Lingenfelter S E , Koch G A , Lisi P J , Chu C -W , Tsai S -C
Published: January 2018

THP-1 is an acute monocytic leukemia cell line which acquires phenotypic and functional monocytoid-like features following incubation with mezerein. The current study concerned the modulation of these features by rIFNγ. rIFNγ induces the time-dependent enhancement of HLA-DR expression in the presence or absence of mezerein but has no effect on the expression of Leu-M1, Leu-M2, or Leu-M3 antigens. CSF-1 production following mezerein activation was reduced by incubation in the presence of 103 and 104 units/ml rIFNγ. This was confirmed through both biological assays with mouse bone marrow cells and an indirect ELISA. In contrast, the concentration of growth inhibitory activity in conditioned medium was increased by rIFNγ. A small but significant increase in IL-1β concentration in conditioned medium was detected using a sensitive double-antibody ELISA and a radioimmunoassay. The results infer that the functional characteristics of this leukemia cell line are modulated by rIFNγ in a manner qualitatively similar to that reported for IFNγ treated normal monocytes.
Antigenic Variation and Macrophage Infiltration of Human Bladder Tumors Xenografted Into Nude Mice
Authors: Russell Pamela J , Philips Jeanette , Allan William , Hume David A
Published: January 2018

The presence of macrophages both within and around human bladder transitional cell carcinomas xenografted into nude mice has been examined using Immunocytochemical staining. Nine different xenograft lines derived from bladder tumors of eight patients were stained for F4/80, MHC II and PGP-1 labelled cells. The results revealed considerable heterogeneity both within and between tumors. All of the tumors generated some macrophage response at the tumor periphery, and this was marked in two instances, UCRU-BL-13, passage 9 and UCRU-BL-17, passage 2. In only two tumors was there substantial penetration of the tumor epithelium by macrophages, though infiltrating la+, F4/80+, PGP-1+ cells were sometimes seen spreading along the base of the epithelial sheets. Three of the tumors were characterized by the presence of L3T4+ T lymphocytes at the invasion front. The presence of macrophages or lymphocytes either at the invasion front or within the tumors did not correlate with the pathological grade of the tumors. PGP-1 monoclonal antibodies also stained granulocytes, which were present within the tumor mass in UCRU-BL-15, passage 2, possibly reflecting the production by the tumor of G-CSF. In addition, the PGP-1 antibodies stained some of the bladder tumors cells themselves, and, in some cases, the interstitial structures within the tumors. The significance of this staining is not yet understood. The xenografted tumors will provide a useful model to examine the constraints on tumor therapy using macrophage activating stimuli.
Diacylglycerols Modulate Human Polymorphonuclear Neutrophil Responsiveness: Effects on Intracellular Calcium Mobilization, Granule Exocytosis, and Superoxide Anion Production
Authors: Smith Robert J , Sam Laurel M , Justen James M
Published: January 2018

The synthetic diacylglycerols (DG), sn-1,2-dihexanoylglycerol (diC6), sn-1,2-dioctanoyl-glycerol (diC8), and 1-oleoyl-2-acetylglycerol (OAG) stimulated the release of granule constituents from and superoxide anion (Of) generation by human polymorphonuclear neutrophils (PMN). The DGs did not induce a rise in the cytosolic-free calcium concentration ([Ca2+]i), as monitored by the fluorescence of PMNs loaded with the fluorescent CA2+ indicator, Fura-2. DiC6, diC8, and OAG inhibited PMN degranulation elicited with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), acetylsn-glyceryl-3-phosphorylcholine (AGEPC), and 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4) and the calcium ionophore, A23187. In contrast to their inhibitory effects on granule exocytosis, diC6, diC8 and OAG enhanced FMLP-, AGEPC-, LTB4 and A23187-stimulated O2- production. Activation of the respiratory burst with phorbol 12-myristate 13-acetate (PMA) was unaffected by the DGs. DiC8 inhibited the rise in [Ca2+]i elicited with FMLP, LTB4, and AGEPC; this effect, as well as the DG-mediated suppression of degranulation, could be reversed with the protein kinase C (PKC) inhibitor, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7). These data indicate that in addition to possessing the intrinsic capacity to activate PMNs, DG may function in a PKC-mediated autoregulatory mode to influence PMN activation in a response-specific manner by affecting certain components of receptor-coupled and receptor-independent signal transduction systems in a stimulus-specific manner.
SHORT COMMUNICATION: Bone Marrow Progenitors Cultured in the Presence of Granulocyte-Macrophage Colony-Stimulating Factor Versus Macrophage Colony-Stimulating Factor Differentiate Into Macrophages With Distinct Tumoricidal Capacities
Authors: Falk Lydia A , Hogan M Michele , Vogel Stefanie N
Published: January 2018

Bone marrow-derived cells from C3H/HeJ mice were cultured in the presence of recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) or highly purified murine macrophage colony-stimulating factor (CSF-1) for 7 days. Following this 7-day culture period, mature macrophages were harvested and replated at precise densities in the absence of exogenous rGM-CSF or CSF-1, and assayed in a two-signal tumoricidal assay. Cultures were stimulated with medium only or with combinations of recombinant interferon-γ (rIFN-γ) as the “priming” signal, and/or butanol-extracted lipopolysaccharide (But-LPS) as the “triggering” signal for 24 hr. At this time, 51Cr-labeled, P815 tumor target cells were added, and the percent tumor cell cytotoxicity was determined after 16 hr. Macrophages derived under the influence of rGM-CSF exhibited significant tumoricidal capacity with medium alone (16 ± 5%). The addition of “priming” signal only (i.e., rIFN-γ, 10.0 U/ml) significantly increased tumoricidal capacity to 31 ± 9%. Treatment with But-LPS alone did not alter the basal tumoricidal activity of rGM-CSF-derived macrophages. Combinations of rIFN-γ (10.0 U/ml) and But-LPS (0.5–5.0 μg/ml) generated highly tumoricidal macrophages (50-60% tumor cell cytotoxicity). In contrast, medium-treated CSF-1-derived macrophages exhibited a significantly lower basal level of tumor cytotoxicity (6 ± 3%). Unlike rGM-CSF-derived macrophages, treatment of CSF-1-derived macrophages with high concentrations of rIFN-γ alone did not increase significantly the level of cytotoxicity above that of medium-treated cultures. However, CSF-1-derived macrophages responded to the highest concentrations of But-LPS (5.0 νg/ml) to increase tumoricidal activity from 6 ± 3% to 17 ± 5%. Optimal tumoricidal activity (44 ± 17%) was observed when CSF-1-derived macrophages were treated simultaneously with high concentrations of both rIFN-γ and But-LPS. Thus, macrophages derived from bone marrow progenitors in either rGM-CSF or CSF-1 exhibited tumoricidal capacities that differed in basal activity as well as in their requirements for and sensitivities to “priming” and “triggering” signals.
Suppression of Delayed-Type Hypersensitivity in Mice Pretreated With Diethylstilbesterol: Involvement of Sex Hormones in Immunomodulation
Authors: Kato Kazuyuki , Chen Yu , Nakane Akio , Minagawa Tomonori , Fujieda Kenji , Kimura Takuro , Yamamoto Ken-ichi
Published: January 2018

Delayed-type hypersensitivity (DTH) responses were suppressed in female mice treated with diethylstilbesterol (DES), a synthetic nonsteroidal compound possessing estrogenic activity, but not in male mice. Upon analysis of this DTH-suppression in DES-treated female mice by use of the macrophage migration inhibition (MI) assay, an in vitro correlate of DTH, suppressor adherent cells (i.e., macrophages) in the peritoneal cavity were found to play an important role in this DTH suppression. On the other hand, DES-induced suppression was observed in surgically castrated male mice with depressed plasma testosterone (TS) levels, but not in TS-treated female mice or in castrated male mice, which suggests that TS inhibited the DES suppression. These results provide evidence that DTH response may be modulated by sex hormones.
Platelet Activating Factor Increases Expression of Complement Receptors on Human Neutrophils
Authors: Shalit Meir , Von Allmen Carolyn , Atkins Paul C , Zweiman Burton
Published: January 2018

The phospholipid inflammatory mediator platelet activating factor (PAF) has been shown to stimulate certain functions of polymorphonuclear leukocytes (PMN). However, the effect of PAF on surface complement receptors of PMN has not been described. Using monoclonal antibodies and flow cytometry, we have assessed the effects of PAF on surface expression of membrane receptors for C3bi (CR3) and C3b (CR1) in human PMN. PAF (optimal concentration of 1 × 10-8 M) increased CR3 190% and CR1 174% compared with unstimulated cells at 37°, while the PAF analogue lyso-PAF had no stimulatory effect. Both CR3 and CR1 responses to PAF reached maximum levels at 15-30 min. PAF effects were comparable to peak effects induced by LTB4 but less than induced by FMLP. A PAF receptor antagonist, SRI 63-441, blocked the increased complement receptor expression in a dose-dependent manner with maximal inhibition of 80-95% at 5 × 10-6 M. Extracellular calcium had no effect on CR1 expression but slightly enhanced and EGTA partially inhibited the PAF-induced increase in CR3 expression. Simultaneous incubation with PAF and LTB4 enhanced CR3 and CR1 expression more than either agent alone. These findings indicate that PAF, alone and in combination with LTB4, can induce altered expression of complement receptors on the surface of PMN. This effect may enhance adhesion and phagocytosis by PMN at inflammatory reaction sites.
Flow Cytometric and Histological Analysis of Mouse Thymus in Fetal Alcohol Syndrome
Authors: Ewald Sandra J , Walden Shannon M
Published: January 2018

Reduced thymus size and fetal weight were seen in 18-day old fetuses of C57BL/6 female mice fed a complete liquid diet containing 25% ethanol-derived calories (EDC) from gestational day 0 to 18. Thymocytes from fetuses from the 25% EDC diet group and from pair-fed and ad-lib control diet groups were compared by flow cytometry for expression of thymocyte differentiation antigens. The proportions of L3T4-positive and Lyt-2-positive thymus cells were significantly reduced in alcohol-exposed fetuses compared to controls; however, the number of Thy-l-positive cells did not differ among any of the groups. Histologically, the thymus from 25% EDC fetal mice failed to show the delineation between cortex and medulla that was seen in the thymuses of control fetuses. These results indicate that thymus immaturity is one of the accompanying features of fetal alcohol syndrome.
Decrease in Pyrogenicity of Muramyl Dipeptide After Coupling With Luteinizing Hormone-Releasing Hormone
Authors: Riveau Gilles , Hosmalin Anne , Carelli Claude , Lefrancier Pierre , Chedid Louis
Published: January 2018

Muramyl dipeptide (MDP) and its adjuvant active derivative lysine-MDP (Lys-MDP) have been demonstrated to be pyrogenic and to induce endogenous pyrogen (EP) production in vivo and in vitro. It has recently been shown that immunologic castration can be achieved in mice by immunization with luteinizing hormone-releasing hormone (LHRH) directly conjugated by carbodilmide to Lys-MDP, termed LHRH-Lys-MDP (cdi), or with a linear monomeric MDP-linked molecule obtained by total synthesis, termed LHRH-Lys-MDP (s). These preparations were tested in the rabbit for their capacity to induce fever and were found to be devoid of pyrogenicity at dosage levels of Lys-MDP that induced fever. This decrease of pyrogenicity of Lys-MDP after coupling to LHRH seems to be related to the structure of the conjugate because the derivative LHRH-LysNH2-MDP exhibited the same pyrogenic activity as the free glycopeptide. Surprisingly, nonpyrogenic LHRH-Lys-MDP induced production of EP and interleukin-1 (IL-1) in vitro and increased in vivo modifications of metal levels attributed to the action of IL-1. Moreover, LHRH-Lys-MDP reduced the pyrogenic effect of an exogenous dose of EP.
Quantitation of Intracellular Mac-1 (CD11b/CD18) Pools in Human Neutrophils
Authors: Jones Douglas H , Anderson Donald C , Burr Bean L , Rudioff Helen E , Smith C Wayne , Krater Sharon S , Schmalstieg Frank C
Published: January 2018

The adhesive glycoprotein Mac-1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis-independent increase in surface Mac-1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac-1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac-1 was identified in β [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-γ (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich γ (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10−8 M, 15 min, 37°C) significantly diminished Mac-1 in pre-γ (-18% of total, P < 0.05) but not β fractions (+6% of total). Under these conditions, the content of Mac-1 in γ fractions increased 13% in association with four- to eightfold increase in surface Mac-1 expression (OKM-1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac-1 on human neutrophils by mobilizing a novel intracellular granule pool.
Membrane Oxidative Metabolism of Human Eosinophilic Cell Line EoL-1 in Response to Phorbol Diester and Formyl Peptide: Synergistic Augmentation by Interferon-γ Tumor Necrosis Factor
Authors: Yoshie Osamu , Majima Toshiro , Saito Hiroshi
Published: January 2018

Human eosinophilic cell line EoL-1 was studied using luminol-dependent chemiluminescence (CL) for the ability to produce a respiratory burst upon stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). TPA, a potent activator of protein kinase C, induced a biphasic CL response in EoL-1. Treatment of EoL-1 with interferon-γ (IFN-γ) for 5 days dramatically enhanced TPA-inducible CL, and IFN-αA had a similar effect. Neither IFN-γ nor IFN-αA strongly inhibited EoL-1 cell growth. Tumor necrosis factor (TNF) also enhanced TPA-inducible CL response of EoL-1 and, furthermore, was quite inhibitory to EoL-1 cell growth. The effects of IFN-γ and TNF were synergistic, whereas those of IFN-αA and TNF were additive. Superoxide dismutase completely abrogated TPA-induced CL, but sodium azide suppressed only the late phase of CL. EoL-1 pretreated with IFN-7, IFN-αA, or TNF also became capable of producing CL response to a chemotactic peptide (fMLP). The effects of IFN-γ and TNF were again synergistic. EoL-1 cells treated with IFN-γ, IFN-αA, or TNF had abundant cytoplasm, but only TNF increased cells having distinct eosinophilic granules. IFN-γ but not IFN-αA enhanced the cytological effect of TNF. It was further demonstrated that treatment of EoL-1 with IFN-γ and TNF strongly increased the binding sites for phorbol diesters and also dramatically induced the surface expression of fMLP receptors. IFN-γ had, however, little effect on the number or the ligand-binding affinity of TNF receptors on EoL-1. Thus, EoL-1 may provide a useful experimental model for the study of differentiation and regulation of human eosinophils.
Neutral Lipid Accumulation in Macrophages During Lipid-Induced Macrophage Growth
Authors: Yui Satoru , Yamazaki Masatoshi
Published: January 2018

We previously reported that lipids such as cholesterol esters, triglycerides, and some phospholipids that are components of cell membranes or serum lipoproteins induced the growth of mouse peritoneal macrophages in vitro. Here, we report that macrophage growth was directly induced by lipids without any soluble factors and that the continuous presence of lipids was required for their growth during culture. When macrophages were cultured with several phospholipids that stimulated macrophage growth, their triglyceride content was dramatically increased during culture, whereas their contents of phospholipids, cholesterol, and esterified cholesterols were scarcely changed. On the other hand, phosphatidylcholine and sphingomyelin, which did not stimulate macrophage growth, caused less accumulation of triglycerides in the cells. Macrophages in which triglycerides accumulated resembled foam cells, since they contained many particles that stained with oil red O. Macrophages that cultured with cholesteryl linoleate also contained many oil red O-stainable particles. These data suggest that accumulation of lipid droplets is correlated with macrophage growth induced by lipids and that foam cells in pathological states such as those in atherosclerotic or xanthoma lesions might proliferate in situ.
Differential Induction of Hematopoiesis and Immune Suppressor Cells in the Bone Marrow Versus in the Spleen by Lewis Lung Carcinoma Variants
Authors: Young M Rita , Aquino Sandy , Young Meivin E
Published: January 2018

Mice bearing large (≥ 3 g) metastatic and nonmetastatic Lewis lung carcinoma (LLC) tumors were studied to determine if the tumor variants differentially induced bone marrow versus splenic hematopoiesis and the appearance of hematopoiesis-associated immune suppressor cells. The metastatic LLC-C3 and nonmetastatic LLC-C8 tumors were equal in their stimulatory effects in vivo on both the number of bone marrow myeloid progenitor cells (CFU) and the appearance of bone marrow immune suppressor cells. In contrast, the tumor variants differed in their effects on the spleen, with the metastatic tumors causing a more pronounced increase in the number of nucleated cells and CFU, a reduced blastogenic responsiveness to concanavalin (Con-A), and an increased suppressor cell activity than nonmetastatic LLC-C8 tumors. The splenic suppressor cells of mice bearing large LLC-C3 tumors resembled the bone marrow suppressor cells which we previously described (Young et al.: Cancer Res. 47, 100, 1987) in that they were nonadherent to nylon wool, sensitive to treatment with L-leucine methyl ester, insensitive to treatment with complement and Thy-1.2, MG-1.2, asialo-GM1, or anti-IgM antibodies, and mediated their suppression through a mechanism which was only partially indomethacin sensitive. The stimulatory effects on hematopoiesis and suppressor cells by the LLC variant tumors may have been mediated by the tumor-derived colony stimulating factor (CSF) activities. Bone marrow cell proliferation and colony formation were stimulated in vitro by culture supernatants of metastatic LLC-C3 cells and, to a lesser degree, of nonmetastatic LLC-C8 cells. These colony-stimulating factor (CSF)-containing supernatants also induced normal bone marrow cells to become immune suppressive. In contrast, supernatants of only LLC-C3 cells, and not of LLC-C8 cells, stimulated in vitro growth of splenic CFU from LLC-C3-bearing mice; spleen cells from normal mice and from LLC-C8 bearers were unresponsive to supernatants of the LLC variants. These results suggest that CSF produced by either the metastatic LLC-C3 or the nonmetastatic LLC-C8 tumors could concurrently stimulate bone marrow hematopoiesis and the appearance of bone marrow suppressor cells. However, the metastatic LLC-C3 tumor cells, and not the nonmetastatic LLC-C8 cells, could also cause expansion of progenitor cells and hematopoiesis to the spleen and, consequently, induce the appearance in the spleen of hematopoiesis-associated immune suppressor cells.
Reversal of Virus-Induced Alveolar Macrophage Bactericidal Dysfunction by Cyclooxygenase Inhibition In Vitro
Authors: Laegreid W W , Liggitt H D , Silflow R M , Evermann J R , Taylor S M , Leid R W
Published: January 2018

Virus infection of alveolar macrophages (AM) both in vivo and in vitro has been associated with a decreased ability of these cells to kill bacteria, together with enhanced production of metabolites of arachidonic acid. These metabolites, especially PGE2, may be inhibitory to some phagocyte functions. Primary cultures of bovine AM obtained by bronchoalveolar lavage of normal cattle were infected in vitro with parainfluenza-3 (PI3 virus) virus. Killing of Staphylococcus epidermidis by AM was determined on days 1-4 post-infection (p.i.) PI3 virus-infected AM killed significantly fewer bacteria on day 4 p.i. compared to uninfected controls (12.1 ± 1.3% infected vs. 52.7 ± 7.2% controls, P ≤ 0.05). Bacterial killing by virus-infected AM, but not control AM, was significantly enhanced on day 4 p.i. by addition of cyclooxygenase inhibitors 1 hr prior to bactericidal assay (28.0 ± 4.5% indomethacin, 36.0 ± 4.1% mefenamic acid, 38.6 ± 7.3% piroxicam, 37.0 ± 6.4% NDGA, 44.9 ± 7.7% ETYA, P ≤ 0.05). Phagocytosis of opsonized sheep erythrocytes and superoxide generation by virus-infected AM were not significantly increased by cyclooxygenase inhibition. Phagosome-lysosome fusion was severely impaired in virus-infected AM. Pretreatment of virus-infected AM with indomethacin significantly enhanced the percentage of cell expressing fusion activity. This data suggests that in vitro bactericidal dysfunction associated with virus infection of AM is partially the result of enhanced production of prostaglandins or thromboxane by AM and/or an abnormal response to normal levels of endogenously produced cyclooxygenase metabolites. The data further indicate the presence of cyclooxygenase sensitive (phagosome-lysosome fusion) and insensitive (phagocytic) components of virus-induced bactericidal dysfunction in AM.
Formalin-Fixed Macrophages Bind Tumor Targets Similarly to Viable Macrophages
Authors: Chapes Stephen Keith
Published: January 2018

Macrophages (MPs) fixed with 1% formalin in PBS bound targets similarly to viable MPs. Like binding between viable MPs and tumor cells, the process was temperature and calcium dependent. Fixed MPs discriminated targets similarly to viable MPs. Targets not bound by viable MPs were not bound by fixed MPs. The lectin Bandeiraea simplicifolia (ASI-B4) was able to enhance MP binding to tumor cells regardless of whether MPs were fixed or viable. However, it did not appear that ASI-B4–like molecules were involved in the direct recognition of F5b tumor cells by MPs. Target cells could not be fixed in 1% formalin for binding to occur. These data suggest that the receptor on the MP for tumor cell binding is functional in the absence of active physiological processes. In contrast, tumor cell processes that are dependent upon target cell viability are required for binding.
Differential Production of Tumor Necrosis Factor, Macrophage Colony Stimulating Factor, and Interleukin 1 by Human Alveolar Macrophages
Authors: Becker Susanne , Devlin Robert B , Haskill J Stephen
Published: January 2018

Human alveolar macrophages (AMO) have been investigated for their ability to produce three monokines, tumor necrosis factor-alpha (TNF), macrophage colony stimulating factor (CSF-1), and interleukin 1-beta (IL-1). No TNF activity was found in supematants of unstimulated AMO cultured for 20 h, although TNF mRNA was detected in the cells by Northern blot analysis. Stimulation of the cells with lipopolysaccharide (LPS) Induced production and release of high levels of TNF into the culture supernatant. Increased levels of TNF mRNA were detectable at 90 min after LPS stimulation by dot blot analysis, reaching maximum expression between 4 and 8 h and declining thereafter. TNF activity peaked at approximately 8 h in the AMO supematants. After 24 h TNF production had ended. Compared to autologous monocytes the AMO produced 5.7 times more TNF on a per cell basis (activity accumulated in 20 h supematants). Uncultured AMO expressed CSF-1 mRNA which was translated into active protein recovered in supematants upon culture in the absence of stimulus. The addition of LPS to AMO slightly reduced both mRNA levels and amount of factor in the supematants. In contrast to the AMO, monocyte production of CSF-1 was enhanced by LPS. CSF-1 production by AMO continued for at least 48 h of culture. Spontaneous production of low amounts of IL-1 was found in one-third of the AMO samples, while low levels of IL-1 mRNA were present in all tested preparations. LPS stimulation induced increase in IL-1 mRNA within 90 min; mRNA levels peaked between 12 and 20 h and stayed high for at least 42 h. However, while all LPS-stimulated AMO expressed high levels of IL-1 mRNA biologically active IL-1 was again detected only in a fraction of the AMO supematants. These results show that the production of monokines CSF-1, TNF, and IL-1 is differentially regulated by LPS in alveolar macrophages and has different responses as compared to monocytes. The longevity of the messages for each of the factors is possible indicators of the relative contribution of these factors to the response to endotoxin-induced injury and repair processes in the lung.
Thrombin-Treated Endothelium Primes Neutrophil Functions: Inhibition by Platelet-Activating Factor Receptor Antagonists
Authors: Vercellotti G M , Wickham N W R , Gustafson K S , Yin H Q , Hebert Mary , Jacob H S
Published: January 2018

We have shown previously that fluid phase platelet-activating factor (PAF) can enhance or “prime” polymorphonuclear (PMN) responses to subsequent stimulation with agonists such as formyl-methionine-leucine-phenylalanine (FMLP). Since thrombin induces PAF production in endothelial cells, we tested whether this thrombin-provoked endothelial PAF primes responses of marginateci PMNs. Monolayers of human umbilical vein endothelial cells were exposed to either thrombin (0.5-5.0 units/ml) or buffer for up to 5 min and then PMNs were layered on top of the endothelial cells. After a further 5 min incubation, the PMNs were stimulated with a suboptimal concentration of FMLP (10-7 M), and their superoxide production, elastase release, adhesion to endothelium, and capacity to cause endothelial cell lysis and detachment were assessed. Thrombin pretreatment significantly enhanced each of these FMLP-stimulated neutrophil responses. The extent of this enhancement correlated with both the dose and duration of thrombin treatment of endothelial cells and also the duration of PMN incubation with thrombin-exposed endothelium. Evidence that the augmentation was due to endothelial-derived PAF was obtained as follows: (1) thrombin induced [3H]acetate incorporation into endothelial PAF (assayed in lipid extracts); (2) antithrombin III conjointly inhibited this [3H]acetate uptake and prevented the priming effect of thrombin-treated endothelium on PMN responses; and (3) the PAF receptor antagonist BN52021. when preincubated with PMNs, also effectively blocked the enhancement of PMN responses. We conclude that thrombin stimulation of endothelial cells initiates a sequence of events culminating in the production of PAF—a membrane phospholipid capable of priming marginated PMNs. We suggest that this coagulation-fostered endothelial PMN interaction may underlie a paracrine response that may potentiate PMN-mediated endothelial injury during sepsis and other thrombin-generating disorders.
Monokine Secretion in Aging and Protein Malnutrition
Authors: Bradley Suzanne F , Vibhagool Asda , Kunkel Steven L , Kauffman Carol A
Published: January 2018

Aged and protein-malnourished hosta have diminished febrile responses and increased morbidity and mortality from infection that could be due to deficiencies in the production of certain monokines. In this study, the ability of peritoneal macrophages from aged and protein-malnourished rats to produce IL-1 and TNF was explored. Aged rats fed a standard diet produced less IL-1 and TNF, as measured by the thymocyte proliferation and L929 cytotoxicity assays, than young and middle-aged rats. Monokine production was not diminished by protein malnutrition in any age group. No synergistic decline in IL-1 or TNF production was seen with increasing age in malnourished rats. Diminished IL-1 and TNF production may partially explain the severity of infection seen in the elderly patient, but not the malnourished host. The role of other cytokines such as IL-6 and cytokine inhibitors in aging and malnutrition should be explored.
Selective Potentiation of Lymphocyte-Derived Macrophage Chemotactic Factor Release in Complete Freund’s Adjuvant-Treated Guinea Pigs
Authors: Sakata Ken-mei , Muramoto Kazuhiro , Hirashima Mitsuomi , Hayashi Hideo
Published: January 2018

Dinitrophenol (DNP)-ovalbumin(OA)-induced tissue macrophage reaction in sensitized guinea pigs is enhanced by treatment with complete Freund’s adjuvant (CFA). The enhancement of the reaction may be due to the increased production of a T-lymphocyte-derived macrophage chemotactic factor (LDMCF) because treatment of animals with CFA potentiates antigen- and concanavalin A(ConA)-induced release of LDMCF activity from spleen cells of the CFA-treated animals in vitro. This potentiating effect by CFA seems to be ascribed to the release of an adherent-cell-derived soluble factor from the CFA-treated animals. The adherent cell-derived factor, LDMCF-potentiating factor (LDMCF-PF), preferentially potentiates the release of LDMCF activity but not of eosinophil chemotactic activity from antigen- or Con-A-stimulated T lymphocytes. Protein synthesis is required for release of LDMCF-PF. Molecular weight of LDMCF-PF activity is assumed to be about 10,000-20,000. LDMCF-PF activity is sensitive to trypsin, to neuraminidase, and also to alkalinity at pH 11, suggesting that LDMCF-PF is a glycoprotein. The present study provides one explanation for the enhanced macrophage reaction in delayed-type hypersensitivity reactions.
Human Neutrophils Activated by Interferon-γ and Tumour Necrosis Factor-α Kill Entamoeba histolytica Trophozoites In Vitro
Authors: Denis Michel , Chadee Kris
Published: January 2018

The interaction between cytokine-activated human neutrophils and Entamoeba histolytica trophozoites was studied as well as the mechanism(s) involved. Treatment of neutrophils with rlFN-γ alone allowed them to kill 30% of E. histolytica trophozoites; however, rlFN-γ and rTNF-α pretreatments in combination increased neutrophil killing to 70%. In the absence of direct contact between neutrophils and amebae, rlFN-γ-treated neutrophils were shown to kill 70% of amebae, and rlFN-γ- and rTNF-α-treated neutrophils killed 97% of amebae. Neutrophil enhancement of amebicidal activity following cytokine treatments was correlated with Increased neutrophil resistance to amebic contact-dependent killing and was shown to be 73% H2O2 dependent.
Gamma Interferon Induces Monocyte Killing of Listeria monocytogenes by an Oxygen-Dependent Pathway; Alpha- or Beta-Interferons by Oxygen-Independent Pathways
Authors: Peck Richard
Published: January 2018

Human peripheral blood monocytes purified by counterflow centrifugal elutriation were treated with recombinant interferons–-gamma (IFN-γ), alpha (IFN-α), or beta (IFN-β)–-and tested for their capacity to kill Listeria monocytogenes. All three IFNs increased the monocyte bactericidal activity in a dose-dependent fashion. Exogenous catalase, an inhibitor of monocyte-generated hydrogen peroxide, did not affect bactericidal activity. However, exogenous superoxide dismutase inhibited killing by IFN-γ-activated monocytes, but not by IFN-α- or IFN-β-activated monocytes. By contrast, exogenous soybean trypsin inhibitor inhibited killing by IFN-α- or IFN-β-activated monocytes, but not by IFN-γ-activated monocytes. Combinations of IFN-γ and IFN-α resulted in no increase in bactericidal activity. Finally, treatment with IFN-γ resulted in different receptiveness of the cell to subsequent oxidative burst-stimulating signals than did treatment with either IFN-α or IFN-β. These results suggest that monocytes treated with IFN-γ kill L monocytogenes by an oxygen-dependent mechanism, but treatment with IFN-α or IFN-β elicits principally oxygen-independent mechanisms.
Three-Dimensional Reconstruction of Anomalous Beige Mouse Macrophage Lysosomes
Authors: Strausbauch Paul , Sehgal Nanda
Published: January 2018

Cells of the beige mouse, the murine counterpart of the Chediak-Higashi Syndrome, contain enlarged anomalous lysosomes. The three-dimensional structure of these lysosomes from peritoneal macrophage was ascertained by study of electron micrographs taken in a series of serial sections combined with computer-assisted analysis. The most common basic structural units identified were biconcave discs and hollow cup/ovoid-shaped structures. Other more bizarre forms were represented by the fusion of these basic lysosomal variants. Anomalous lysosomal forms were found in both beige resident and exudate peritoneal macrophage. A similar study of lysosomes in animals not carrying the beige mutation revealed the presence of smaller but structurally similar forms of the basic lysosomal variants. This latter finding indicates that the presence of lysosomal variants per se in the beige animal is not a finding unique to these animals but rather that their distinctiveness derives from their increased size and propensity to undergo fusion with each other. The usefulness of computer-assisted three-dimensional reconstruction is demonstrated.
Quantitation of Conjugate Formation Between Human Polymorphonuclear Leukocytes and Antibody-Coated Target Cells by Flow Cytometry: The Role of Fc Receptor and LFA-1 Antigen
Authors: van Kessel Kok P M , van Strijp Jos A G , van der Tol Marijke E , van Kats-Renaud Henny J , Thijssen Rik M W M , Fluit Ad C , Verhoef Jan
Published: January 2018

The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common β-chain of the adhesion molecule family and a combination of antibodies against the α-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the β-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.
Influenza A Virus and the Neutrophil: A Model of Natural Immunity
Authors: Hartshorn Kevan L , Karnad Anand B , Tauber Alfred I
Published: January 2018

Natural immune reactions are mediated by lymphocytes, macrophages/monocytes, and neutrophils. The latter have been implicated in a variety of self-surveillance models, i.e., activity against malignant host cells, participation in wound repair, and infliction of damage in postischemic perfusion injury. Better characterized are the interactions with unopsonized pathogens through lectinophagocytosis mechanisms, where the lectin resides either on the phagocyte or on the microorganism. This review examines the infection by influenza A virus (IAV) of the human neutrophil, which results in the vigorous metabolic response of the cell to generate toxic oxygen species. This response is not necessarily characteristic of response to unopsonized particles, as the neutrophil exhibits no such activity to unopsonized zymosan or chlamydia. The virus elicits calcium mobilization from intracellular stores through a pertussis toxin-insensitive mechanism, and in its particulars the activation cascade is unique in comparison to any other characterized agonist. The putative receptor for the IAV binding protein, hemagglutinin (HA), contains the sialic acid residues; identification of specifically linked protein receptors will allow characterization of this stimulation pathway and will define the molecular biology of this activation sequence. Insight into this particular pathway may allow definition of a primitive recognition system that represents a fundamental basis for discernment of self and nonself entities.
In Vivo and In Vitro Assessment of Porcine Neutrophil Activation Responses to Chemoattractants: Flow Cytometric Evidence for the Selective Absence of Formyl Peptide Receptors
Authors: Fletcher Mark P , Stahl Gregory L , Longhurst John C
Published: January 2018

Interest in the role that activated granulocytes play in C5a-induced myocardial ischemia prompted us to investigate and compare activation responses of pig and human neutrophils. The responses of Hypaque-Ficoll purified porcine (P-PMN) and human neutrophils (H-PMN) to stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, phorbol myristate acetate (PMA), and calcium ionophore A23187 (A23187) were compared by flow cytometrically measured changes in the cells' forward (FWD-SC) (a measure of shape/volume change) and right angle (90°-SC) light scatter (a measure of secretion), and in the distribution of the membrane potential sensitive fluorescent probe di-O-C(5) (3). FMLP, C5a, and Zymosan-activated serum (ZAS) stimulated chemotaxis and FMLP vs. PMA-stimulated adherence to plastic were also compared. Unstimulated P-PMN had lower FWD-SC and 90°-SC than H-PMN (39.4 ± 1.4 vs. 48.4 ± 2.0 P < 0.05, and 32.7 ± 2.7 vs. 52.4 ± 1.5 units, P < 0.005, for FWD-SC and 90°-SC of P-PMN vs. H-PMN, respectively). P-PMN selectively failed to increase their FWD-SC upon stimulation with FMLP (0.0 ± 0.5% vs. 26.1 ± 6.8%, P-PMN vs. H-PMN), or decrease their 90°-SC when treated with cytochalasin B + FMLP (secretion) (2.4 ± 0.1% vs. -35.8 ± 4.6% change in 90°-SC, P-PMN vs. H-PMN), while responding comparably to C5a, PMA, and A23187. P-PMN failed to depolarize in response to FMLP but responded similarly to H-PMN when activated by C5a, A23187, and PMA. P-PMN's chemotactic response to FMLP was selectively absent since the cells responded well to purified pig C5a. FMLP stimulated significant increases in H-PMN adherence to bovine serum albumin-coated plastic (44.1 ± 6.7% vs. 12.6 ± 3.7%, FMLP vs. buffer, P < 0.025), but failed to increase adherence of P-PMN above baseline 0.68 ± 0.20% vs. 2.12 ± 1.90%, FMLP vs. buffer, P > 0.05. PMA (100 ng/ml) stimulated comparable increases in adherence in both PMN types (48.6 ± 5.2% vs. 58.7 ± 4.9%, P-PMN vs. H-PMN, P < 0.025). Binding studies using the fluoresceinated N-formyl peptide f-met-leu-phe-lysine-fluorescein-isothiocyanate (FMLPL-FITC) in the absence and presence of excess non-fluoresceinated FMLPL indicated that P-PMN lack specific binding sites for the N-formyl peptides. Intracoronary (LAD) infusion of FMLP in the instrumented intact pig produced no change in neutrophil extraction, LAD regional blood flow, or myocardial contractility while infusion of purified porcine C5a induced a rapid and marked increase in myocardial neutrophil extraction, a decrease in LAD coronary blood flow, and diminished contractility. It is concluded that P-PMN and H-PMN respond comparably to C5a, PMA, and A23187, but P-PMN are selectively unresponsive to activation by FMLP both in vitro and in vivo due to the absence of FMLP binding.