bluuebunny/crossref_metadata_2025 huggingface dataset:
Culture of ovine bone marrow–derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro
Authors: Francey T , Jungi TW , Rey O , Peterhans E
Published: January 2018

An in vitro system allowing the culture of ovine bone marrow–derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4-to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to fivefold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)–containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1β (IL-1β) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1β) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.
Ultrastructural identification of human mast cells resembling skin mast cells stimulated to develop in long-term human cord blood mononuclear cells cultured with 3T3 murine skin fibroblasts
Authors: Dvorak Ann M , Furitsu Takuma , Kissell-Rainville Susan , Lshizaka Teruko
Published: January 2018

Human mast cells developed in vitro when cord blood mononuclear cells were cocultured for 3 months with 3T3 embryonic mouse skin fibroblasts. The metachromatic cells that arose in these cultures contained histamine, a functional Fcε receptor and granule proteases (tryptase, chymase), and they were definitively identified by the ultrastructural demonstration of crystal granules. We present a detailed ultrastructural analysis of this newly available system for the reliable development of human mast cells in vitro and provide criteria for definitive identification of the mast cell and basophil lineages in humans.
Defective lipopolysaccharide-induced production of both interleukin 1α and interleukin 1β by A/J mouse macrophages is posttranscriptionally regulated
Authors: Brandwein Sydney R , Maenz Lynn
Published: January 2018

Interleukin 1 (IL-1) production by A/J (A) and C57BL/6J (B6) mouse peritoneal macrophages stimulated with lipopolysaccharide (LPS) was determined. Strain A macrophages produced low levels of soluble IL-1 bioactivity compared with B6 macrophages. This defect was not reversed by indomethacin, interferon-γ, phorbol myristate acetate, or calcium ionophore A23187. In contrast, cytosolic IL-1 bioactivity was similar in LPS-stimulated A and B6 macrophages. Western blotting revealed that A macrophage supernatants contained lower levels of both 17-kd IL-1α and 17-kd IL-1β but similar levels of tumor necrosis factor α compared with B6 macrophages. Cytosolic levels of 31-kd Pro-IL-1α and also 31-kd pro-IL-1β were similar in A and 6 macrophages. Oligonucleotide probing indicated that A and B6 macrophages contained similar levels of IL-1α and also IL-1β mRNA. These findings indicate that LPS-stimulated A macrophage culture supernatants contain low levels of both IL-1α and IL-1β compared with B6 macrophages and that these defects in IL-1 production are posttranscriptionally regulated.
Cytokine secretion by immune cells in space
Authors: Chapes Stephen K , Morrison Dennis R , Guikema James A , Lewis Marian L , Spooner Brian S
Published: January 2018

Cultured, bone marrow–derived macrophages, murine spleen and lymph node cells, and human lymphocytes were tested for their ability to secrete cytokines in space. Lipopolysaccharide-activated bone marrow macrophages were found to secrete significantly more interleukin-1 and tumor necrosis factor when stimulated in space than when stimulated on earth. Murine spleen cells stimulated with poly I:C in space released significantly more interferon-α at 1 and 14 hours after stimulation than cells stimulated on earth. Similarly, murine lymph node T cells and human peripheral blood lymphocytes, stimulated with concanavalin A in space, secreted significantly more interferon-γ than ground controls. These data suggest that space flight has a significant enhancing effect on immune cell release of cytokines in vitro.
Interleukin-8 induces changes in human neutrophil actin conformation and distribution: relationship to inhibition of adhesion to cytokine-activated endothelium
Authors: Westlin William F , Kiely Jeanne-Marie , Gimbrone Michael A
Published: January 2018

Interleukin-8 (IL-8) induces diverse biological responses in neutrophils, including inhibition of adhesion to cytokine-activated endothelium, which we have termed the leukocyte adhesion inhibition (LAI) effect. Pretreatment of neutrophils with cytochalasin B abolished the LAI effect of IL-8, suggesting a microfilament-dependent mechanism. Interleukin-8 induced a rapid increase (≤ 15 s) in the polymerization of actin filaments in human neutrophils that was blocked by pretreatment with cytochalasin B. F-actin depolymerization occurred gradually at a rate inversely proportional to IL-8 concentration. This temporal pattern of actin polymerization-depolymerization was similar to that induced by other chemotactic factors such as C5a and N-formylmethionyl-leucyl-phenylalanine, which also exhibit a marked LAI effect, but the lipid mediators, leu-kotriene B4 and platelet-activating factor, lack any significant LAI effect. Scanning confocal microscopy demonstrated that neutrophil actin microfilaments undergo a dramatic rearrangement prior to detachment of the neutrophil from a surface. We suggest that the ability of IL-8 and certain other leukocyte agonists to regulate the actin polymer network of neutrophils may play an important role in adhesive interactions with the vascular endothelium.
Low response of BALB/c macrophages to priming and activating signals
Authors: Oswald Isabelle P , Afroun Samia , Bray Dorothy , Petit Jean-François , Lemaire Genevieve
Published: January 2018

Trehalose dimycolate (TDM), a mycobacterial glycolipid, is a powerful macrophage-priming agent. However, its efficiency seems limited in the case of BALB/c mice. Peritoneal macrophages harvested from TDM-treated BALB/c mice did not control BCG growth in vitro as efficiently as similar macrophages from two other mouse strains, (B6 × D2)F1 and C57BL/6, which are respectively Bcgr and Bcgs. BALB/c macrophages elicited by TDM also exhibited a low capacity to produce hydrogen peroxide and, after activation by lipopolysaccharide (LPS), weak cytostatic activity against P815 mastocytoma cells. Finally, alkaline phosphodiesterase, a marker of resident and inflammatory macrophages, was still expressed at a high level in macrophages of BALB/c mice treated with TDM. Low responsiveness of BALB/c macrophages to stimuli was not observed with TDM only; activation for tumor cytotoxicity of thioglycolateelicited macrophages from BALB/c mice required also higher doses of interferon-γ, and LPS. l-Arginine–dependent production of nitric oxide was inducible in macrophages from BALB/c mice, but the conditions required for its induction were more stringent. Thus, the reduced antiproliferative effects of BALB/c macrophages may be due to uncomplete induction of NO synthase after suboptimal stimulation.
Activation of bovine neutrophils by Pasteurella haemolytica leukotoxin is calcium dependent
Authors: Ortiz-Carranza O , Czuprynski CJ
Published: January 2018

In this study, we used the fluorescent probe Fluo-3 to show that an increase in cytosolic free calcium, [Ca2+]i, occurred when suspensions of bovine neutrophils were incubated with sublethal concentrations of P. haemolytica leukotoxin. This increase in [Ca2+]i was dependent on the concentration of leukotoxin present in the medium and, at a given concentration of leukotoxin, dependent on the external calcium concentration. The calcium channel blocker verapamil and the β-adrenergic antagonist propranolol inhibited leukotoxin-stimulated Ca2+ gain, as did a neutralizing antileukotoxin monoclonal antibody. As reported previously, incubation of bovine neutrophils with partially purified leukotoxin stimulated a vigorous luminol-dependent chemiluminescence response (LDCL). The present study shows that LDCL stimulation was dependent on the presence of extracellular calcium and was inhibited by the addition of verapamil and propranolol. These data indicate that bovine neutrophils exhibit a considerable increase in cytoplasmic free calcium when they are incubated with P. haemolytica leukotoxin in the presence of external calcium. They also provide evidence that an increased [Ca2+]i is required for functional activation of the bovine neutrophil oxidative burst by P. haemolytica leukotoxin.
Cytokines and suppressor macrophages cause tumor-bearing host CD8+ T cells to suppress recognition of allogeneic and syngeneic MHC class II molecules
Authors: Walker Thomas M , Yurochko Andrew D , Burger Carol J , Elgert Klaus D
Published: January 2018

Quantitative and qualitative tumor-associated changes in T cell phenotype and function were identified in CD8+ T cells. Tumor growth changed splenic CD4+/ CD8+ T cell ratios and induced the appearance of more cells with the CD8+ phenotype. In comparison to equal concentrations of normal host (NH) counterparts, tumor- bearing host (TBH) CD8+ T cells were highly suppressive to allorecognition and autorecognition. Suppression was not due to quantitative reductions in CD4+ T cells, although minor qualitative differences were observed. Suppression appeared to be mediated partly by prostaglandin E2 (PGE2). Interferon-γ (IFN-γ) and interleukin-4 (IL-4) contributed to TBH CD8+ T cell-mediated suppression. Blocking studies using monoclonal antibodies (mAb) in conjunction with indomethacin suggested that cytokine networks involving IFN-7, IL-4, and PGE2 were disrupted during tumor growth and promoted TBH CD8′+' T cell suppression. Alloresponses and autoresponses were significantly suppressed when TBH CD8+ T cells mediated these reactions simultaneously with TBH la” macrophages. Inhibition of PGE2 production was unable to reverse the additive suppression caused by these two cell types. These results collectively suggest that tumor- induced changes in CD8+ T cells lead to suppressed allorecognition and autorecognition through both soluble mediator molecules and cellular interactions.
Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils
Authors: Smith Duane M , Waite Moseley
Published: January 2018

We describe here and partially characterize a Ca2+-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophos- phatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachi- donic acid from exogenous [l-14C]stearoyl-2-[3H]arachi- donyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/ cyclooxygenase inhibitors eicosatetrayenoic acid and nor- dihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, mono- glyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl- methionyl-leucyl-phenylalanine, and then subfraction- ated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane- enriched fractions. These data suggest that this Ca2+- independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.
Three distinct cell phenotypes of induced-TNF cytotoxicity and their relationship to apoptosis
Authors: Woods Keith M , Chapes Stephen K
Published: January 2018

We have identified three distinct cell phenotypes with respect to the conditions under which cells became susceptible to TNF-mediated lysis. These conditions include: 1) treatment with the protein synthesis inhibitor, cycloheximide; 2) contact with activated macrophages, and 3) infection with vaccinia virus. Whereas vaccinia virus-infected 3T3 cells became sensitive to soluble TNF, F5b cells required contact with activated macrophages. We showed that the “macrophage-resistant” F5m cells did not become sensitive to TNF or to killing by activated macrophages after infection with vaccinia virus. Therefore, vaccinia infection does not sensitize all cells to TNF. We also determined the pathways of lysis for cells after sensitization. Whereas 3T3, LM929, and F5b cells were killed by the process of necrosis, F5m cells lysis was characterized by the release of low mol wt DNA fragments (apoptosis). J. Leukoc. Biol. 53: 37–44; 1993.
Selective refractoriness of macrophages to endotoxin-induced production of tumor necrosis factor, elicited by an autocrine mechanism
Authors: Fahmi Hassan , Chaby Richard
Published: January 2018

Tolerance of macrophages to endotoxin (lipopolysaccharide, LPS) can be induced in vitro by LPS itself. We show here that one of the mechanisms of tolerance to LPS can be mediated via an autocrine process. Continuous exposure to LPS is not required to induce macrophage desensitization. Refractoriness to production of tumor necrosis factor (TNF) in response to LPS can be transferred from tolerant to naive macrophage populations by incubation of the latter with the culture supernatant of the former, in the absence of endotoxin. The active factor present in this macrophage-desensitizing culture supernatant (MD-Sup) is more efficiently removed by incubation with tolerant macrophages than by incubation with naive macrophages. The refractoriness elicited by treatment with MD-Sup is restricted to a decreased TNF response to LPS; interleukin-1 (IL-1) and IL-6 responses are not affected. J. Leukoc. Biol. 53: 45-52; 1993.
Inhibition of Corynebacterium parvum–primed and lipopolysaccharide-induced hepatic necrosis in rats by selective depletion of neutrophils using a monoclonal antibody
Authors: Sato Tsukasa , Shinzawa Haruhide , Abe Yoshihiro , Takahashi Tsuneo , Arai Shigeru , Sendo Fujiro
Published: January 2018

To examine whether neutrophils are involved in the pathogenesis of experimental liver dysfunction, we observed the effect of selective in vivo neutrophil depletion by a monoclonal antibody (RP-3) on the pathogenesis of acute experimental hepatic necrosis in rats induced by a preparative injection of heat-killed Corynebacterium parvum and a challenging dose of lipopolysaccharide (LPS) (positive control group). The serum transaminase titer 8 h after LPS injection was reduced by selective depletion of peripheral blood neutrophils as a result of RP-3 treatment (RP-3 group). A kinetic study showed that the serum transaminase titer of the RP-3 group was significantly lower than that of the positive control group from 4 to 24 h after LPS injection. The transaminase level was significantly lower in the group with less than 400/mm3 peripheral blood neutrophils than in the group with a greater number. The effect of RP-3 on the transaminase level was due neither to the injection of cancer ascites nor to RP-3 injection with the accompanying decrease in complement titer. These results suggest that neutrophils play an important role in the induction of liver dysfunction in this system. J. Leukoc. Biol. 53: 144–150; 1993.
Isolation of a human endothelial cell C1q receptor (C1qR)
Authors: Peerschke Ellinor IB , Malhotra Rajneesh , Ghebrehiwet Berhane , Reid Kenneth BM , Willis Anthony C , Sim Robert B
Published: January 2018

Clq binding to endothelial cells has been described previously, but the putative cell surface receptor (s) has not been identified. In the present study, modifications of a reported purification of lymphocyte Clq receptor (ClqR) were used to isolate Clq binding sites from human umbilical vein endothelial cells. Cells were harvested, without protease treatment, at passage 10-17 and lysed with 1% Triton X-100. The lysate was fractionated on Fast-performance liquid chromatography (FPLC) Mono-Q using a linear NaCl gradient, followed by high-performance liquid chromatography (HPLC) ion exchange (TSKgel DEAE-NPR). A major protein was eluted that had the same mobility on sodium dodecyl sulfate-polyacrylamide gels and the same NHHerminal sequence as lymphocyte ClqR. This protein was expressed on the surface, as judged by surface radioiodination, bound to Clq-coated surfaces, and was recognized by polyclonal antilymphocyte ClqR antibodies. Thus, endothelial cells express a Clq receptor that appears identical to lymphocyte ClqR. The data further support the hypothesis that cell surface ClqRs identified on a variety of somatic and cultured cells are either identical or constitute a family of closely related molecules. J. Leukoc. Biol. 53: 179–184; 1993.
Interleukin-1-dependent mitogenic responses induced by protoscoleces of Echinococcus multilocularis in mur ine lymphocytes
Authors: Kizaki Takako , Ishige Masamichi , Bingyan Wang , Kumagai Mitsuru , Day Noorbibi K , Good Robert A , Onoé Kazunori
Published: January 2018

Mitogenic effects of protoscoleces (PSCs) of Echinococcus multilocularis on murine lymphocytes were studied. Spleen cells from normal BALB/c mice showed significant proliferative responses when cocultured with PSCs. Proliferative responses were observed in both the T and B cell populations. The PSCs also stimulated cells of the macrophage/monocyte lineage to secrete inter-leukin-1 (IL-1). Depletion of plastic- and Sephadex G-10-adherent cells from the spleen cell population significant-ly reduced the proliferative responses to PSCs and the low responsiveness was restored by addition of plastic-ad-herent cells to these cultures. Furthermore, addition of anti-lL-l serum to the spleen cell cultures stimulated with PSCs completely suppressed the proliferative responses. These findings demonstrate that the mitogenic effect of PSC on lymphocytes depends on EL-1 secreted by cells of macrophage/monocyte lineage. J. Leukoc. Biol. 53: 233–239; 1993.
Laminin cleavage by activated human neutrophils yields proteolytic fragments with selective migratory properties
Authors: Steadman Robert , Irwin Michael H , John Patricia L St , Blackburn Warren D , Heck. Louis W , Abrahamson Dale R
Published: January 2018

We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain- specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three-armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophos- phate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes. J. Leukoc. Biol. 53: 354–365; 1993.
Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro
Authors: Belenky Sergei N , Robbins Richard A , Rubinstein Israel
Published: January 2018

Nitric oxide synthase (NOS) inhibitors have been shown to modulate neutrophil migration. We hypothesized that the NOS inhibitors NG-monomethyl-L- arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), and L-canavanine (L-CAN) also modulate human peripheral blood monocyte chemotaxis. To test this hypothesis, monocyte chemotaxis toward formylmethionyl- leucyl-phenylalanine (fMLP) was assessed using a modified blindwell chemotaxis chamber technique. L- NMMA and L-NAME, but not D-NMMA or L-CAN, significantly attenuated fMLP-induced monocyte chemotaxis (P < .05). L-Arginine and sodium nitroprusside, but not D-arginine, reversed NOS inhibitor-induced responses. Dibutryl cyclic guanyl monophosphate (cGMP) attenuated the inhibitory effects of L-NMMA on monocyte chemotaxis (P < .05). Finally, fMLP increased cGMP generation by monocytes, which was significantly attenuated by L-NMMA (P < .05). These data indicate that the L-arginine/NO biosynthetic pathway regulates human monocyte chemotaxis in vitro. J. Leukoc. Biol. 53: 498–503; 1993.
Tumor growth increases la− macrophage synthesis of tumor necrosis factor-α and prostaglandin E2: changes in macrophage suppressor activity
Authors: Alieva David G , Burger Carol J , Elgert Klaus D
Published: January 2018

Although tumor growth enhances macrophage (mφ) cytotoxic activity by increasing their tumor necrosis factor-a (TNF-a) production, increased prostaglandin E2 (PGE2) synthesis reduces most immune responses during tumor growth. Macrophages that do not express major histocompatibility complex class II molecules (la− mφ) are the predominant suppressor and cytotoxic population and are more abundant in tumor-bearing hosts (TBHs). This study determined if TBH la’ mφs are the major population producing TNF-α and PGE2 and if these molecules affect la’ mφ-mediated suppression of alloantigen-stimulated T cell proliferation. Normal host (NH) and TBH splenic Ia+-depleted (la−) mφs synthesized more TNF-a than their respective whole populations (WPs) when cultured with lipopolysaccharide and interferon-7. TBH la’ mφs produced the most TNF-α. Northern blot analyses showed that la’ mφs had higher amounts of TNF-α mRNA expression than their respective WP, and TBH la” mφs expressed the highest amounts of TNF-α mRNA. When WP and la’ NH and TBH mφs were added to alloantigen-stimulated T cells, suppression of T cell proliferation mediated by la’ mφs was greater than by their respective WP. TBH Ia−  mφs were most suppressive. The blockage of PGE2 production reduced suppression mediated by TBH la” mφs more than by all other mφ populations. A PGE2-specific enzyme-linked immunosorbent assay showed that PGE2 production was greater in la’ mφ- than in WP mφ-containing cultures and greatest in cultures containing TBH la’ mφs. Because TNF-α enhances T cell responses, its effects on la−mφ PGE2-mediated suppression was determined. When TNF- a was added to mφ-containing T cell cultures, TNF-α directly stimulated NH, but not TBH, la’ mφs, which enhanced T cell proliferation. However, inhibiting PGE2 production allowed TNF-a to stimulate T cell proliferation in TBH la” mφ-containing cultures. Collectively, these data show that la’ mφs are the major TNF-α- and PGE2-producing cells and that these molecules are partly responsible for the tumor-induced increase in mφ- mediated cytotoxicity and suppression, respectively. TNFα not only mediates cytotoxicity but also counteracts la−  mφ PGE2-mediated suppression. Although tumor growth increases la’ mφ TNF-α production, enhanced PGE2 production blocks TNF-α’s stimulatory action on la− mφs, which favors their suppressor function during tumor growth. J. Leukoc. Biol. 53: 550–558; 1993.
Effects of inhibition of lipoxygenase and guanylate cyclase on human neutrophil responses to formyl peptide and granulocyte-macrophage colony-stimulating factor
Authors: Coffey Ronald G , Diaz Sharon , Coffey Christina S
Published: January 2018

Human neutrophils were activated by the bacterial chemotactic peptide N-formylmethionyl-leucyl- phenylalanine (fMLP) to produce superoxide (O2”) and to release the primary granule enzyme β-glucuronidase and the predominantly secondary granule enzyme lysozyme. Pretreatment with granulocyte-macrophage colony- stimulating factor (GM-CSF) increased the secretion of all three substances upon addition of fMLP. The augmentation by GM-CSF was significantly attenuated by the 5-lipoxygenase inhibitor AA861 and by the guanylate cyclase inhibitor LY83583. The secretion induced by fMLP alone was much less affected by either of the two inhibitors. AA861 inhibited leukotriene B4 production in neutrophils primed with GM-CSF and stimulated with fMLP, and LY83583 inhibited GM-CSF-evoked increases of 3′,5′-guanosine monophosphate. The data suggest that activation of lipoxygenase and guanylate cyclase is not critical to the fMLP stimulation pathway, but they may be important components of the pathway by which GM-CSF augments neutrophil responses to fMLP. However, AA861 and LY83583 may have important actions in addition to inhibition of 5-lipoxygenase and guanylate cyclase.
Overview of spaceflight immunology studies
Authors: Taylor Gerald R
Published: January 2018

The effects of spaceflight and analogues of spaceflight are discussed here and in nine accompanying articles. In this summary we present spaceflight studies with human subjects, animal subjects, and cell cultures and we review ground-based systems used to model the observed effects of spaceflight on the immune system. Human paradigms include bed rest, academic or psychological stress, physical stress, hypobaric or high altitude stress, and confinement. Animal models include antior- thostatic and orthostatic suspension, hypobarism, and confinement. The ten manuscripts in this collection were selected to provide a summary that should give the reader an overview of the various activities of spaceflight immunology researchers throughout the history of space travel. This manuscript identifies the major contributors to the study of spaceflight immunology, explains what types of studies have been conducted, and how they have changed over the years. Also presented is a discussion of the unusual limitations associated with spaceflight research and the efforts to develop appropriate ground-based surrogate model systems. Specific details, data, and mechanistic speculations will be held to a minimum, because they will be discussed in depth in the other articles in the collection.
The role of cytokines in immune changes induced by spaceflight
Authors: Sonnenfeld Gerald , Miller Edwin S
Published: January 2018

It has become apparent that spaceflight alters many immune responses. Among the regulatory components of the immune response that have been shown to be affected by spaceflight is the cytokine network. Spaceflight, as well as model systems of spaceflight, have been shown to affect the production and action of various cytokines including interferons, interleukins, colony stimulating factors, and tumor necrosis factors. These changes have been shown not to involve a general shutdown of the cytokine network but, rather, to involve selective alterations of specific cytokine functions by spaceflight. The full breadth of changes in cytokines in- duced by spaceflight, as well as mechanisms, duration, adaptation, reversibility, and significance to resistance to infection and neoplastic diseases, remains to be established.
Granulocytes enhance LPS-induced tissue factor activity in monocytes via an interaction with platelets
Authors: Halvorsen Hanne , Olsen Jan Ole , Osterud Bjarne
Published: January 2018

In the present study we have investigated the effect of platelets and granulocytes on bacterial lipopoly- saccharide (LPS)-induced tissue factor (TF) activity in monocytes. Experiments were performed on freshly isolated cells resuspended in heparinized plasma and recombined with platelet-poor or platelet-rich plasma. In a platelet-dependent reaction the granulocytes enhanced LPS-induced TF activity by an average of 100%. The effect was dose dependent with regard to the number of both granulocytes and platelets, respectively. Granulocytes and/or platelets did not affect LPS-induced tumor necrosis factor (TNF) secretion from monocytes. Phorbol myristate acetate (PMA) per se was not able to induce TF activity in our system. In contrast, the agonist caused a substantial increase in TF activity induced by LPS. The effect was totally dependent on the presence of platelets and was shown to be due to stimulation of both granulocytes and monocytes (the activity rose from 30 ± 7 to 83 ± 12 mU/106 cells in the presence of platelets and from 69 ± 8 to 143 ± 22 mU/106 cells in the presence of platelets and granulocytes). Effects similar to those observed with PMA were obtained with physiological concentrations (10 ng/ml) of TNF. A combination of these two agonists gave no further amplification of LPS-induced TF activity compared with the effect of the agonists separately. Low concentrations of a monoclonal anti- CD15 antibody abolished the stimulatory effects of platelets and granulocytes. Furthermore, the anti-GDI 5 antibody neutralized the effect of TNF, whereas the PMA effect was reduced by almost 75%. These results were confirmed in a whole-blood system. The inhibitory effect of the antibody may be associated with CD15’s role as a complementary ligand for PADGEM. Our study indicates that a close interaction between granulocytes, platelets, and monocytes is essential for optimal TF activity induced by LPS. It is hypothesized that the effect of granulocytes is related to their ability to activate platelets. We propose that upon activation granulocytes secrete a product that enhances the capacity of platelets to stimulate TF activity in monocytes.
Effect of lipoxins and other eicosanoids on phagocytosis and intracellular calcium mobilisation in rainbow trout (Oncorhynchus mykiss) leukocytes
Authors: Knight J , Lloyd-Evans R , Rowley A F , Barrow S E
Published: January 2018

Rainbow trout (Oncorhynchus mykiss) macrophages generated lipoxin (LX) A4, LXA5, leukotriene (LT) B4, LTB5 and 12-hydroxyeicosatetraenoic acid (12-HETE) during the phagocytosis of zymosan and Escherichia coli, but not of the yeast Saccharomyces cerevisiae. Prostaglandin (PG) E2 was also detected in supernatants from macrophages incubated with either zymosan or calcium ionophore A23187. LXA4 (10−8- 10−6 M) and LTB4 (10−9-10−7 M) provoked rapid and transient dose-dependent increases in intracellular calcium ([Ca]i) concentrations in leukocyte suspensions containing 40-60% macrophages. EC50 values were 14.9 and 1.2 nM, respectively. PGE2 and 12-HETE had no effect on [Ca]i at concentrations up to 30 μM. PGE2 and 12-HETE (10−5-10−10 M) enhanced the in vitro phagocytosis of yeast test particles by trout macrophages, whereas LXA4 and LTB4 had no demonstrable effect on the responses of these cells at concentrations up to 10−5 M. In conclusion, the processes involved in trout macrophage stimulation are complex but involve generation of both cyclooxygenase and lipoxygenase products. The increase in [Ca]i caused by LXA4 and LTB4 may form part of the chemotactic transduction mechanism that recruits granulocytes and macrophages to sites of inflammation. The effects of eicosanoids on phagocytosis appear to be independent of changes in [Ca]i.
Expression of human perforin in a mouse cytotoxic T lymphocyte cell line: evidence for perturbation of granule-mediated cytotoxicity
Authors: Thia Kevin Y T , Smyth Mark J , Trapani Joseph A
Published: January 2018

Expression of the pore-forming protein perforin is normally restricted to the cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Perforin, which causes cell death by osmotic lysis, has the ability to form transmembrane channels in target cell membranes. This function makes perforin crucial in the granule-exocytosis model of T cell-mediated cytotoxicity. In the present study, variants of the mouse cytotoxic T lymphocyte cell line CTLL-R8 have been produced which express human perforin. A full-length cDNA clone (HP-10) encoding human perforin was inserted in the sense orientation into the expression plasmid pCMV5neo. The resultant construct, designated pCMV5neoHP-10, was used to transfect GTLL-R8 cells. Of eight G418-resistant clones studied, four clones expressed human perforin mRNA by Northern analysis and three of these clones also expressed human perforin protein by Western blotting. The expression of human perforin protein was associated with a pronounced (55-74%) and consistent reduction in the killing of three target cell lines, P815, YAC-1, and EL4, compared with parental GTLL-R8 cells. The reduction in target cell lysis could not be attributed to nonspecific effects of the transfection, as clones transfected with neo alone showed no reduction in killing in comparison with parental GTLL-R8 cells. Clones expressing human perforin showed very similar growth characteristics, surface phenotype, and 2 N-α- benzyloxycarbonyl-L-thiobenzyl-esterase release compared with untransfected CTLL-R8 cells. The mechanism of reduction of cytolysis is unclear but may involve competition by human perforin in the handling or packaging of endogenous granule constituents (including mouse perforin) or assembly of human perforin into mouse polyperforin channels in target cell membranes. The expression of human perforin in mouse cytotoxic T cells provides a potential model for studying how cytotoxic T cells process, package, utilize, and protect themselves against the perforin molecules they produce.
Effects of macrophage colony-stimulating factor (M-CSF) on the development, differentiation, and maturation of marginal metallophilic macrophages and marginal zone macrophages in the spleen of osteopetrosis (op) mutant mice lacking functional M-CSF activity
Authors: Takahashi Kiyoshi , Umeda Syuji , Shultz Leonard D , Hayashi Shin-Ichi , Nishikawa Shin-Ichi
Published: January 2018

Immunohistochemical techniques using an anti-mouse panmacrophage monoclonal antibody and anti-mouse monoclonal antibodies specific for marginal metallophilic macrophages or marginal zone macrophages were used to detect red pulp macrophages, marginal metallophilic macrophages, and marginal zone macrophages in the spleen of op/op mice. In the mutant mice, the red pulp macrophages were reduced to about 60% of those in the normal littermates and the marginal metallophilic macrophages and marginal zone macrophages were absent. After administration of recombinant human macrophage colony-stimulating factor (rhM-CSF), numbers of red pulp macrophages increased rapidly, reaching levels found in normal littermates 1 week later. In contrast, the marginal metallophilic macrophages as well as the marginal zone macrophages appeared slowly after rhM-CSF administration and their numbers were less than half of the baseline level of normal littermates even at 12 weeks of administration. The distribution of marginal metallophilic macrophages and marginal zone macrophages appearing after M-CSF administration was irregular in the spleen of the op/op mice. These splenic macrophage subpopulations differed in their responses to rhM-CSF, suggesting that distinct mechanisms may be involved in their development and differentiation. The splenic red pulp macrophages present in unmanipulated op/op mice are an M-CSF–independent macrophage population. Although the marginal metallophilic macrophages and marginal zone macrophages are thought to be M-CSF-dependent, their development and differentiation appear to be influenced by locally produced M-CSF or other cytokines. J. Leukoc. Biol. 55: 581-588; 1994.
Effect of thioglycollate and BCG stimuli on glucose and glutamine metabolism in rat macrophages
Authors: Costa Rosa L F B P , Safi D A , Curi R
Published: January 2018

The macrophage is a differentiated cell that takes part in a wide range of physiological and pathological processes, such as inflammatory and immunological responses. The stimulation of macrophages involves the acquisition of some functional characteristics such as phagocytosis, ability to kill tumor cells, and processing and presentation of antigens. In this study we have investigated the changes in macrophage glucose and glutamine metabolism as induced by stimulation by thioglycollate (inflammatory) and bacille Calmette-Guérin (BCG) (activated). The results showed an increase in hexokinase and citrate synthase activities, due to both stimulatory processes. One difference between inflammatory and activated cells is the higher rate of glucose oxidation in the latter. The activation with BCG, however, also led to an enhancement in glutamine metabolism that is not observed in the inflammatory cell. These results suggest that glutamine metabolism might be important for macrophage function during immunological response. J. Leukoc. Biol. 56: 10–14; 1994.
Interaction of nuclear proteins with an AP-1/CRE-like promoter sequence in the human TNF-α gene
Authors: Newell Christine L , Deisseroth Albert B , Lopez-Berestein Gabriel
Published: January 2018

The tumor necrosis factor α (TNF-α) promoter contains an AP-l/CRE-like binding site, TGAGCTCA. AP-1 elements generally transduce signals involving protein kinase C; the CRE site mediates a cAMP response, involving protein kinase A. Thus, this element has the potential to receive signals through divergent signaling pathways. Nuclear protein binding studies using extracts from THP-1 monocytic cells treated with lipopolysaccharide (LPS), which stimulates, or dexamethasone (Dex) or pentoxifylline (PTX), which inhibit TNF production, respectively, suggest that two lowmobility complexes could be involved in regulation through this promoter region. PTX and Dex increase binding of both these complexes compared with untreated cells; approximately 2 hours after LPS induction, the upper complex becomes undetectable. This upper complex is composed of ATF2 (activating transcription factor 2, a cyclic AMP responsive element binding protein) homodimers; the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun/ATF2 complexes, which could be activating complexes. In this case, the simultaneous presence of both complexes, which would occur in the presence of Dex or PTX, could reduce the amount of TNF transcription through competitive binding. Through in vitro competitive binding studies comparing the binding affinities of the TNF promoter sequence and a consensus CRE, we further suggest how variation of endogenous binding sequences from consensus may be an important property for regulatory control of particular genes J. Leukoc. Biol. 56: 27–35; 1994.
Effect of the carbocyclic nucleoside analogue MDL 201,112 on inhibition of interferon-γ-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression
Authors: Edwards Carl K , Watts Lynnetta M , Parmely Michael J , Linnik Matthew D , Long Richard E , Borcherding David R
Published: January 2018

The effects of the carbocyclic nucleoside MDL 201,112 and the purine nucleoside adenosine on the interferon-γ (IFN-γ)-induced priming of macrophages (mfs) for the respiratory burst and major histocompatibility class II (MHC class II) Ia+ antigen expression were compared. Priming of purified, peritoneal mφs from Lewis (LEW/N) rats for 18 h with recombinant rat IFN-γ (rRaIFN-γ) in the presence of either adenosine (100 μM) or MDL 201,112 (10 μM) resulted in a fourfold decrease in superoxide anion (02-) production after stimulation with opsonized zymosan. Both agents were effective even when added 2 or 4 h after rRaIFN-γ treatment. Peritoneal mφs from LEW/N rats stimulated with LPS/rRaIFN-γ were observed to secrete immunoreactive and bioactive TNF-α over 18 h in vitro and this cytokine could be dose-dependently inhibited by MDL 201,112. MDL 201,112 did not bind to classical A1 or A2 receptors on rat brain homogenates. Physiological levels of adenosine deaminase, or treatment with the nucleoside transport inhibitor dipyridamole, reversed the effects of adenosine; however, these agents at physiological concentrations had little or no effect on the inhibition of 02- release mediated by MDL 201,112. Furthermore, incubation of LEW/N mφs for 18 h in vitro with rRaIFN-γ resulted in significant enhancement of MHC class II Ia+ antigen expression, and these levels could be blocked by nearly 50% by either MDL 201,112 (10 μM) or adenosine (100 μM). MDL 201,112 and adenosine were also effective in decreasing mφ opsonized zymosan-stimulated 02- levels and MHC class II Ia+ antigen expression in vivo. The effects of MDL 201,112 on the down-regulation of heat-killed M. tuberculosis-activated LEW/N mφ MHC class II Ia+ antigen expression in vitro appear to be mediated by a novel pathway, because there was no rank order of potency of ADO A1 or A2 agonist/antagonists (CCPA, NECA, XAC, or CPT) in our in vitro system. In summary, our data provide compelling evidence that immunoregulatory carbocyclic nucleoside analogues such as MDL 201,112 or adenosine appear to regulate LEW/N rat mf activation through novel molecular mechanisms and may have important therapeutic implications for acute and chronic inflammatory diseases. J. Leukoc. Biol. 56: 133–144; 1994.
Glucosidase trimming inhibitors preferentially perturb T cell activation induced by CD2 mAb
Authors: van Kemenade F J , Rotteveel F T M , van den Broek L A G M , Baars P A , van Lier R A W , Miedema F
Published: January 2018

Glycosidase trimming inhibitors may be used to study contribution of N-linked glycan moieties in T cell function. We have studied the effects of castanospermine (Cas), swainsonine (Swain), 1-deoxynojirimycin (dNM), and 1-deoxymannojirimycin (dMM) on T cell activation and differentiation. Our analysis included a new dNM derivative, N-pentyl-1-deoxynojirimycin (pentyldNM). Previous reports showed inhibitory action of trimming inhibitors, such as Swain and Cas, on pokeweed mitogen-driven immunoglobulin (Ig) production. We extend these findings for pentyldNM and observed that glucosidase inhibitors, Cas and pentyldNM were effective in inhibiting CD2 and CD3 monoclonal antibody (mAb) driven Ig production. The pattern of inhibition by mannosidase and glucosidase inhibitors correlated with inhibitory action on T cell activation: only glucosidase trimming inhibitors (Cas and pentyldNM with comparable potency) perturbed mAb-induced T cell activation, in particular if induced by CD2 mAb. Expression of the early activation marker CD69 was not decreased in the presence of these inhibitors, while addition of exogenous recombinant interleukin-2 partially overcame inhibitory effects during proliferation. These findings suggest that glucosidase, but not mannosidase, trimming inhibitors interfere with a late phase of T cell activation. In addition, the enhanced sensitivity of CD2 mAb-induced proliferation for glucosidase trimming inhibitors suggests dependence on N-linked glycans during CD2-mediated adhesion and triggering functions. J. Leukoc. Biol. 56: 159–165; 1994.
Variables in the isolation and culture of human monocytes that are of particular relevance to studies of HIV
Authors: Bennett Suzanne , Breit Samuel N
Published: January 2018

Mononuclear phagocytes play an important role in human immunodeficiency virus (HIV) infection. However, knowledge of the extent of macrophage involvement in the pathogenesis of HIV infection is still incomplete. This is due in part to the technical difficulties associated with isolating, purifying, and culturing these cells. Isolation of a pure population of human monocytes is essential when studying the role of these cells in HIV infection. In addition, the differentiation, activation, and replication of human monocytes are critically affected by both isolation and culture conditions. This review examines the different monocyte isolation and purification methods and culture conditions that are currently being employed and assesses their suitability for studying the functions of monocytes in HIV infection. J. Leukoc. Biol. 56: 236–240; 1994.
HIV protease inhibitors: effects on viral maturation and physiologic function in macrophages
Authors: Bugelski Peter J , Kirsh Richard , Hart Timothy K
Published: January 2018

Human immunodeficiency virus (HIV), the retrovirus believed to be the cause of acquired immunodeficiency syndrome (AIDS), infects a variety of CD4+ cells, including lymphocytes and cells of the monocyte/macrophage lineage. Encoded in the HIV genome are several precursor proteins that must undergo proteolytic cleavage to yield functional proteins. The gag precursor protein of HIV (p55) is cleaved by a virally encoded aspartate protease (HIV protease). Because cleavage of p55 is required for viral maturation and infectivity, inhibition of HIV protease is an attractive target for therapy designed to block the progression of HIV infection. Inhibitors of HIV protease from a variety of chemical classes have been synthesized and antiviral activity has been demonstrated in lymphocytes and cells from the monocyte/macrophage lineage. A few HIV protease inhibitors have progressed to the clinic and some have shown promise in early trials. There are, however, several important issues that will affect the development of a successful HIV protease inhibitor, including cell and tissue distribution and immunotoxicity. These issues are discussed, with an emphasis on the complexities posed by cells of the monocyte/macrophage lineage. J. Leukoc. Biol. 56: 374–380; 1994.