bluuebunny/crossref_metadata_2025 huggingface dataset:
Thiol Proteinase Inhibitors Reverse the Increased Protein Kinase C Down-Regulation and Concanavalin A Cap Formation in Polymorphonuclear Leukocytes From Chediak-Higashi Syndrome (Beige) Mouse
Authors: Sato Akihiko , Tanabe Fuminori , Ito Masahiko , Ishida Eiko , Shigeta Shiro
Published: January 2018

Protein kinase C (PKC) plays an essential role in intracellular signal transduction for various cell functions, including concanavalin A (Con A)-induced cap formation. This enzyme is known to be proteolysed by calpain, which is a Ca2+ -dependent thiol proteinase. As reported previously, in polymorphonuclear leukocytes (PMNs) from beige mouse, the model of Chediak-Higashi syndrome, Con A-induced cap formation significantly increased compared with that in normal mouse. However, after pretreatment of beige PMNs with the thiol proteinase inhibitors leupeptin or E-64, the capping decreased to normal levels. Meanwhile, Con A-induced the translocation of PKC from the cytosolic to membrane fraction within 5 min in both mice, which is essential to the activation of this enzyme. However, after the translocation, an abnormal rapid decline in membrane-bound PKC activity was noted in beige mouse PMNs. Both leupeptin and E-64 also corrected the rapid decline in PKC activity observed in the beige mouse. These findings suggest that the normalization of Con A cap formation in beige mouse PMNs by the thiol proteinase inhibitors is associated with the correction of abnormality in PKC activity.
Characterization of Macrophage Subsets Regulating Murine Natural Killer Cell Activity
Authors: Nelson John A S , Parhar Ranjit S , Scodras John M , Lala Peeyush K
Published: January 2018

We examined the relationship of l-A expression by normal murine macrophages to their immunoregulatory role on natural killer cell activity. Macrophages were isolated on the basis of plastic adherence; characterized on the basis of conventional markers such as phagocytic ability, cytoplasmic non-specific esterase activity, surface MAC-1 and F4/80 antigen expression; and then used for functional studies relative to their expression of surface l-A. Two functional macrophage subsets were identified: NK-stimulatory and NK-suppressive subsets. The former function was associated with splenic macrophages, which were predominantly l-A+ as identified with a radioautographic immunolabeling technique; the latter function with peritoneal macrophages which were predominantly l-A-. Loss of macrophage l-A expression in vitro was delayed in the presence of indomethacin and enhanced in the presence of PGE2, indicating that PGE2 down-regulates l-A expression on macrophages. The NK stimulatory function of l-A+ macrophages was attributable to a soluble mediator, identified as IFN-γ, since the stimulatory ability was abrogated with an anti-IFN-γ antibody. l-A expression appears to be important for the stimulatory function, since some interference with this function was noted in the presence of anti-I-A antibody. The NK-suppressor function of I-A- macrophages was attributable to the soluble mediator PGE2, since this function was abrogated with indomethacin or anti-PGE2 antibody. These results are relevant to the understanding of normal in vivo immunoregulation by macrophages.
Down-Regulation of Macrophage I-A Expression in Tumor-Bearing Mice
Authors: Nelson John A S , Parhar Ranjit S , Scodras John M , Lala Peeyush K
Published: January 2018

During the subcutaneous growth of a highly metastatic mammary adenocarcinoma line, C3-L4 in C3H/HeJ mice, there was a rapid decline in macrophage l-A expression in vivo. The incidence of l-A+ macrophage subset in the spleen declined from 90% to 10% or less within 5 days of tumor transplantation, with a parallel decline in their absolute number. l-A expression in these cells remained suppressed for a long time until tumors became necrotic and ulcerated. In spite of a low incidence (15–20%) of l-A+ macrophages in the normal peritoneal space, tumor transplantation caused a long-lasting decline in their incidence to one-fourth of the original level. Tumor-associated macrophages were predominantly l-A- throughout the tumor life span. Thus l-A- macrophages dominated in all anatomical compartments in tumor-bearing mice. Macrophage l-A expression was substantially restored (spleen) or stimulated (peritoneal space and tumor) in tumor-transplanted mice subjected to chronic indomethacin therapy in the drinking water, indicating a reversal of prostaglandin-mediated down-regulation of macrophage l-A expression in situ. Concomitantly, this therapy caused regression of the primary tumors and prevented lung metastasis. These results are relevant to the understanding of tumor–host interactions and its exploitation for immunotherapy.
High-Affinity Binding of Fibronectin to Cultured Kupffer Cells
Authors: Cardarelli Pina M , Blumenstock Frank A , McKeown-Longo Paula J , Saba Thomas M , Mazurkiewicz Joseph E , Dias James A
Published: January 2018

Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinizedcoated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 × 10-9 M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800–3,500 binding sites or putative “fibronectin receptors” per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.
Activated Neutrophils Induce Erythrocyte Immunoglobulin Binding and Membrane Protein Degradation
Authors: Weiss Douglas J , Murtaugh Michael P
Published: January 2018

Neutrophil-induced alterations in feline erythrocytes were investigated to elucidate the pathogenesis off the erythrocyte destruction associated with inflammatory disease. Incubation of erythrocytes with zymosan-activated neutrophils, at target:effector concentrations of 50:1, resulted in immunoglobulin binding to the erythrocyte membrane and degradation of membrane proteins. Vitamin E and superoxide dismutase/catalase attenuated the immunoglobulin binding. Proteolysis was not prevented by addition of antioxidants or protease inhibitors to the erythrocyte:neutrophil coculture and was not reproduced with chemical oxidation or calcium loading of erythrocytes.
Effect of Retinoids on the Release and Gene Expression of Tumor Necrosis Factor-α in Human Peripheral Blood Monocytes
Authors: Turpin Jim , Mehta Kapil , Blick Mark , Heater Jeane P , Lopez-Berestein Gabriel
Published: January 2018

The effect of retinoic acid (RA) and retinol (ROH) on the release of tumor necrosis factor (TNF) by human peripheral blood monocytes (HPBM) was determined. HPBM were cultured for various periods of time in either 5% complete (cAB) or delipidized (DLS) AB serum. TNF release (L929 cytolytic assay) in the presence of cAB occurred during the first 3 days of in vitro culture. Delipidization of AB serum completely inhibited the lipopolysaccharide (LPS)-induced release of TNF by HPBM. Addition of RA (0.5 μM) to DLS restored LPS-induced TNF release by HPBM, and supplementation with ROH (1.0 μM) resulted in release of TNF-like activity, but only after 3 days of in vitro culture. The maintenance of TNF release by the addition of exogenous RA after 3 days of in vitro culture suggested that depletion of endogenous RA was partially responsible for loss of TNF-like activity. The levels of endogenous TNF protein and mRNA were not influenced by delipidization of serum and were found to be similar to those of HPBM cultured in the presence of AB serum. TNF protein and mRNA were undetectable in HPBM ROH-treated cell lysates, although cytolytic activity was observed in culture supernatants. These results suggest that retinoids are required for the release of cytolytic factors from HPBM and that non-TNF cytolytic factors may be released by these cells at different stages of maturation.
Bleomycin Binding Sites on Alveolar Macrophages
Authors: Denholm Elizabeth M , Phan Sem H
Published: January 2018

Previous work has demonstrated that bleomycin can directly stimulate alveolar macrophage secretion of fibroblast growth factors and monocyte chemotactic factors. In this study, rat alveolar macrophages obtained by bronchoalveolar lavage were examined for the presence of bleomycin binding sites, which might mediate this response. The results indicated that alveolar macrophages have specific, saturable, and reversible binding sites. Both high- and low-affinity binding sites were found; each macrophage possessed 6.7 × 104 high-affinity sites, with a Kd, of 528 nM, and 2.2 × 106 low-affinity sites, with a Kd of 65 μM. The Kd of the high-affinity sites corresponds closely to the ED50 obtained from dose-response curves of the bleomycin-stimulated secretion of both fibroblast growth and monocyte chemotactic factors, suggesting that bleomycin stimulation of alveolar macrophage function responses may be mediated by bleomycin interaction with these sites.
Bacterial Lipopolysaccharide Induces Release of Tumor Necrosis Factor-α From Bovine Peripheral Blood Monocytes and Alveolar Macrophages In Vitro
Authors: Adams Jeffrey L , Czuprynski Charles J
Published: January 2018

In this study, we demonstrate that freshly adherent bovine monocytes release tumor necrosis factor-α (TNF-α) in response to stimulation with bacterial lipopolysaccharide (LPS). TNF-α was detected using actinomycin D-treated WEHI-164 murine fibrosarcoma cells as targets in an 18 hr cytotoxicity assay. Doses of LPS from 20 ng/ml to 20 μg/ml were capable of inducing bovine TNF-α. The kinetics of TNF-α release from bovine monocytes demonstrated peak levels of cytotoxic activity at 1–3 hr post-LPS treatment, with a subsequent decline to background levels by 18 hr post-LPS treatment. A monoclonal antibody that neutralizes recombinant human TNF-α (rHuTNF-α) significantly reduced the cytotoxicity of LPS-stimulated bovine monocyte culture supematants. Size exclusion high-performance liquid chromatography (HPLC) analysis of LPS-stimulated monocyte and alveolar macrophage culture supematants resulted in a molecular weight elution profile similar to that of recombinant human TNF-α. These elution profiles are consistent with the presence of multimers of TNF-α. This is believed to be the first report of the in vitro production of bovine TNF-α.
Modulation of Nitrogen Oxide Synthesis In Vivo: NG-Monomethyl-L-Arginine Inhibits Endotoxin-Induced Nitrate/Nitrate Biosynthesis While Promoting Hepatic Damage
Authors: Billiar T R , Curran R D , Harbrecht B G , Stuehr D J , Demetris A J , Simmons R L
Published: January 2018

Attempts were made to promote or inhibit nitric oxide (·N=O) synthesis in a murine model of hepatic damage (Corynebacterium parvum followed by lipopolysaccharide; LPS) to determine the role of N=O in the liver injury. Moderate hepatic damage and increases in circulating NO2-/NO3- levels were detectable after C. parvum alone. Administration of LPS to these mice resulted in severe hepatic damage and acute elevations in circulating nitrogen oxide levels. L-arg had no influence on the C. parvum or LPS-induced changes. NG-monomethyl-L-arginine (NMA) had no effect in the absence of LPS, but when given with LPS, a dose-dependent suppression in plasma NO2-/NO3- levels and an increase in liver injury were seen. The NMA-induced changes were partially reversed by the simultaneous administration of L-arg. These findings suggest a protective role for · N=O in this model.
Stimulus-Dependent Leukotriene Release From Human Basophils: A Comparative Study of C5a and Fmet-leu-phe
Authors: MacGlashan Donald , Warner Jane
Published: January 2018

Previous studies of human basophil mediator release have noted that the bacterial peptide fmet-leu-phe and the anaphylatoxin C5a induce comparable levels of histamine release while only fmet peptide induces leukotriene release. Since 5-lipoxygenase metabolism of arachidonic acid is calcium dependent, we examined the characteristics of the human basophil [Ca++]i response which follows its activation by either fmet peptide or C5a. While the peak [Ca++]i response was essentially identical for these two stimuli, fmet peptide induced a prolonged increase in [Ca++]i while C5a stimulated only a transient increase in [Ca++]i that was essentially over within 2 minutes of adding the stimulus. Simultaneous addition of EDTA with fmet peptide revealed the two phases of the [Ca++]i response and demonstrated that leukotriene release was dependent on an elevated [Ca++]i level in the 2–5 minutes following challenge. Enhancement of leukotriene release induced by C5a by agents such as staurosporine and interieukin-3 also produced a [Ca++]i kinetic curve which resembled fmet piptide. Single cell studies of the [Ca++]i response could detect no subpopulations of cells which responded preferentially to fmet peptide or C5a, eliminating the possibility that the ability of fmet peptide to induce leukotriene was a result of its action on a functionally distinct population of basophils.
Syngeneic B Lymphoma Cells Provide a Unique Stimulus to Natural Killer (NK) Cells in Genetically Low-NK SJL/J Mice
Authors: Lin Tao-Zhen , Ponzio Nicholas M
Published: January 2018

SJL/J mice are a genetically low-NK strain, and their cytotoxic activity cannot be augmented with conventional NK inducers. In contrast, effector cells taken from the lymphoid tissues of SJL mice bearing a syngeneic B cell lymphoma (RCS) show variable, but significant levels of cytotoxic activity against NK-susceptible targets, such as YAC-1. Previous results suggested that the RCS cells themselves contributed to this cytotoxicity. However, results presented here indicate that most, if not all of the activity present within the lymphoid tissues of RCS-bearing mice is mediated by RCS-activated, host NK cells. These results were confirmed by in vitro studies, which demonstrate that both gamma irradiated (γ-) RCS cells and γ-allogeneic spleen cells induce cytotoxic activity in SJL spleen cells against YAC targets. However, the cytotoxicity induced by γ-allogeneic cells is mediated largely by lymphokine-activated killer (LAK) cells, since these effectors also lyse NK-resistant target cells, such as L1210. In contrast, the cytotoxic effector cells that are induced by syngeneic γ-RCS cells cause lysis of YAC targets, but not L1210 target cells. These data indicate that the syngeneic B cell lymphomas of SJL mice are a unique stimulus for host NK cells in this strain. Since activated NK cells produce a variety of lymphokines, RCS stimulation of host NK cells in SJL mice may provide some of the growth-promoting lymphokines that are known to be necessary for progressive growth of these lymphoma cells.
Tumoricidal Activity of Alveolar and Peritoneal Macrophages of C57BL/6 Mice Bearing Metastatic or Nonmetastatic Variants of Lewis Lung Carcinoma
Authors: Duffie G Patrick , Young M Rita I
Published: January 2018

The spontaneous tumoricidal abilities of alveolar and peritoneal macrophages from C57B1/6 mice bearing a metastatic or a nonmetastatic cloned variant of Lewis lung carcinoma (LLC) were measured. Cytotoxicity by alveolar macrophages was enhanced during the first few weeks after subcutaneous (s.c.) or intravenous (i.v.) injection of metastatic LLC-C3 cells, but not after injection of nonmetastatic LLC-C8 cells. Alveolar macrophages from mice with s.c.-lnjected metastatic tumors, but not with nonmetastatic tumors, could be further activated in vitro, but not beyond the maximal level of spontaneous cytotoxicity. Late in tumor growth, the spontaneous cytotoxicity by alveolar macrophages of metastatic LLC-C3 tumor bearers was suppressed and could not be increased by in vitro activation. The tumoricidal abilities of peritoneal macrophages from mice bearing either LLC-C3 or LLC-C8 tumors were modulated in a similar way, as were alveolar macrophages. The reduced cytotoxicity by alveolar macrophages from mice with nonmetastatic tumors or from mice bearing large metastatic tumors was not due to suppression by macrophage-derived prostaglandins. The loss of tumoricidal capabilities by macrophages from mice with large metastatic LLC-C3 tumors was not caused by elevated systemic prostaglandin E2 (PGE2) levels. These results suggest that alveolar and peritoneal macrophages are activated to be cytotoxic during development of pulmonary metastases and do not need to be functionally depressed for successful establishment of metastases.
Monovalent Fab Fragments of D7.5 Monoclonal Antibody Activate Intracellular Ca2+ Mobilization and Secretion of Cytolytic Factors by Thymus Cells
Authors: Peacock James S , Tan Jacqueline , Guffee Judy , Stein-Streilein Joan
Published: January 2018

Previous studies have identified two mouse monoclonal antibodies, D7.5 and G1.4, that each can cause secretion of cytolytic factors from natural killer (NK) cells and resting T cells in humans, rats, and hamsters. Toward elucidation of the molecular identity and the transmembrane signaling mechanism of the antibody-defined trigger molecules, we investigated 1) their surface density, 2) the crosslinking requirement for signaling, and 3) their ability to mobilize intracellular calcium ions. Equilibrium binding studies using monovalent Fab fragments of D7.5 showed the presence of approximately 20,000 trigger molecules per thymus cell and an antibody dissociation constant of 1.8 x 10-6 M. Thymus cells incubated in the presence of either intact or Fab fragments of D7.5 acquired the ability to lyse Yac-1 cells. Furthermore, the incubation media of thymocytes that were treated with either intact or Fab fragments of D7.5 contained effector-cell derived factors that could lyse Yac-1 cells. Flow cytometry measurements of changes in intracellular free calcium concentration ([Ca2+]i) in thymocytes. A significantly elevated level of [Ca2+]i was observed at 60s after antibody addition and this response, which required the external calcium source, increased continuously during the 30 min incubation. Furthermore, dbcAMP plus theophylline did not alter the ability of D7.5 to induce calcium mobilization suggesting that the antibody-defined trigger may not use the signaling pathway involving breakdown of phosphatidylinositol.
Inhibition of Cluster Formation and Lymphocyte Proliferation by Anti-Fibronectin Antiserum
Authors: Klingemann H-G , Storb R , Deeg H J
Published: January 2018

Dendritic cells (DC) confer concanavalin A responsiveness to accessory cell-depleted canine lymphocytes. DC-lymphocyte cluster formation is followed by blast transformation and proliferation of lymphocytes. We investigated whether fibronectin, which is known to be involved in cell:cell interactions, might also be involved in the interaction between canine DC and lymphocytes. The addition of rabbit anti-dog fibronectin antiserum to concanavalin A-stimulated peripheral blood mononuclear cells decreased cluster formation and reduced 3H-thymidine incorporation by 57–70%. Similarly, when cell fractions, enriched for DC, were pretreated with anti-fibronectin antiserum before being added back to accessory cell-depleted lymphocytes, cluster formation was reduced and lymphocyte proliferation after concanavalin A stimulation decreased by 18–46% as measured by 3H-thymidine uptake. Ferritin-conjugated anti-fibronectin antibody was bound to the surface of DC, primarily at the dendritic processes. We conclude that fibronectin, located on the surface of canine DC, participates in the accessory cell function of these cells.
Monoclonal Antibodies to the Leukocyte Membrane CD18 Glycoprotein Complex and to Intercellular Adhesion Molecule-1 Inhibit Leukocyte-Endothelial Adhesion in Rabbits
Authors: Argenbright L W , Letts L G , Rothlein R
Published: January 2018

Increasing evidence indicates that leukocyte-endothelium adhesion is mediated, in part, by the CD11/CD18 family of heterodimeric glycoproteins expressed on the leukocyte plasma membrane and by intercellular adhesion molecule-1 (ICAM-1) which is expressed on endothelial cells. We have used the technique of intravital microscopy to visualize the microcirculation of the rabbit mesentery and to evaluate effects of antibodies against several adhesion glycoproteins on C5a-induced leukocyte adhesion. Addition of zymosan-activated serum (a source of C5a) to the buffer superfusing the mesenteric microvasculature induced rapid adhesion of leukocytes to the endothelium of post-capillary venules. Monoclonal antibodies R15.7 (anti-CD18), R7.1 (anti-CD11a, LFA-1), and R6.5 (anti-ICAM-1), administered intravenously before C5a exposure, strongly inhibited leukocyte adherence while antibody LM2 (anti-CD11b, Mac-1) produced significant, but weaker, inhibition. If these antibodies were administered after C5a-induced adhesion had begun, both R15.7 and R7.1 displaced adherent leukocytes and prevented further leukocyte accumulation: LM2 and R6.5 did not displace adherent leukocytes or inhibit incoming leukocytes from adhering. These data confirm earlier findings establishing a role for CD18 in leukocyte adhesion in vivo and extend those observations to implicate both CD11a and CD11b in that adhesion. In addition, we report that ICAM-1 mediates, in part, the initial leukocyte–endothelial cell adhesion following C5a exposure in vivo.
Role of Endogenously Derived Leukotrienes in the Regulation of Lysosomal Enzyme Expression in Macrophages Exposed to β 1,3-Glucan
Authors: Lew D Betty , Leslie Christina C , Henson Peter M , Riches David W H
Published: January 2018

The expression of the lysosomal enzyme hexosaminidase has been shown to be stimulated by the exposure of mouse macrophages to β 1,3-glucan, a particulate component of yeast cell walls and zymosan particles. Exposure of mouse peritoneal macrophages to particulate β 1,3-glucan (100 μg/ml) was also found to stimulate the production of eicosanoids from both the cyclooxygenase (prostaglandin E2) and 5′-lipoxygenase (leukotriene C4) pathways. The objective of this study was to determine the relationship, if any, between the production of arachidonic acid metabolites and the increased expression of lysosomal enzymes. To determine if products of the cyclooxygenase or 5′-lipoxygenase pathway were involved in the regulation of hexosaminidase expression, macrophages were exposed to β 1,3-glucan in the presence of indomethacin, an inhibitor of cyclooxygenase; 4,7,10,13 ETYA, an inhibitor of both cyclooxygenase and 5′-lipoxygenase; or AA861, a selective inhibitor of 5′-lipoxygenase. While the increased expression of hexosaminidase was not affected in macrophages stimulated with β 1,3-glucan in the presence of indomethacin, both 4,7,10,13 ETYA and AA861 completely blocked the response, suggesting a role for products of the 5′-lipoxygenase pathway in the regulation of hexosaminidase expression. To further explore the relationship between arachidonate release and the increased expression of hexosaminidase, macrophages were exposed to phospholipase A2 in an attempt to circumvent the interaction between β 1,3-glucan and the macrophage membrane. Incubation with phospholipase A2 was found both to induce the accumulation of LTC4 in the culture supernatant and to stimulate the increased expression of hexosaminidase. The mechanism of regulation of hexosaminidase expression by products of the 5′-lipoxygenase pathway was investigated by incubating macrophages with purified luekotrienes in either the presence or absence of β 1,3-glucan. Incubation of macrophages with purified LTC4 or LTB4 in the absence of β 1,3-glucan failed to stimulate the expression of hexosaminidase. However, challenging macrophage monolayers with LTC4 or LTB4 in the presence of a suboptimal concentration of β 1,3-glucan (1 μg/ml) led to a synergistic increase in the expression of hexosaminidase. Collectively these data suggest that the leukotriene products of the 5′-lipoxygenase pathway, LTC4 and LTB4, regulate the expression of lysosomal enzymes by apparently priming macrophages, thereby increasing their sensitivity to triggering agents such as β 1,3-glucan. Since macrophages produce LTC4, and the increased expression of hexosaminidase is prevented by inhibitors of the 5′-lipoxygenase pathway, the data further suggest that LTC4 may prime macrophages in an autocrine or paracrine fashion.
Neutrophil Erythrotoxicity Induced by Phorbol Myristate Acetate: Mechanisms Involved in Neutrophil Activation
Authors: Geffner Jorge R , Fontán Patricia A , Sordelli Daniel O , Isturiz Martin A
Published: January 2018

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2–0.5 ng/ml) for 18 hr at 37°C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.
Selective Radiation Resistance of Immunologically Induced T Cells as the Basis for Irradiation-Induced T-Cell-Mediated Regression of Immunogenic Tumor
Authors: Dunn Pamela L , North Robert J
Published: January 2018

Sublethal, whole-body γ-irradiation of immunocompetent, but not T cell deficient, mice bearing an established immunogenic tumor results in T-cell-mediated complete tumor regression and in long-term host survival. This striking T-cell-dependent immunotherapeutic action of irradiation was paradoxically associated with the destruction of over 90% of host T cells and with a state of severe immunodepression as evidenced by the inability of irradiated mice to reject a tumor allograft. Furthermore, whereas exposure to 500 rads caused regression of a syngeneic tumor implanted 6 days before irradiation, it caused enhanced growth of a different syngeneic tumor growing on the same animal and implanted 1 day before. This ability of irradiation to cause regression of a 6 day tumor, but accelerated growth of a 1 day tumor, was also seen when the tumors were implanted in the reverse order. This means that, between days 1 and 6 of a tumor growth, tumor-specific T cells are converted from a radiosensitive to a highly radioresistant state, almost certainly because of having been activated and inducted into the antitumor immune response. This explanation for the selective radioresistance of effector T cells is based on publications showing that activated, in contrast to resting, T cells are highly radioresistant. Thus irradiation-induced, T-cell-mediated tumor regression depends not only on the destruction of radiosensitive suppressor T cells but also on the selective sparing of radioresistant activated effector T cells that are needed to destroy the tumor in the absence of suppression.
Cyclosporine A Induces an Early and Transient Rigidification of Lymphocyte Membranes
Authors: Niebylski Charles D , Petty Howard R
Published: January 2018

The effect of cyclosporine A (CsA) on peripheral blood lymphocyte (PBL) membranes was studied using fluorescence techniques. Light scattering and intercalation experiments indicate CsA has a critical micelle concentration of 3 × 10–5 M. Doses above this critical concentration were avoided during these in vitro studies. Steady-state fluorescence polarization of the lipid probe DPH in peripheral blood lymphocyte membranes revealed that 10–5 M CsA caused a significant increase in the fluorescence polarization 1–5 min after exposure (15–16% above control DPH polarization values). This initial increase in polarization plateaued at 10–11% above control values within roughly 40 min. Fluorescence lifetime measurements of DPH during this response reveal a slight increase in DPH lifetime. The increased DPH lifetimes showed that polarization measurements alone underestimate the rigidity of DPH's microenvironment. Measurements of DPH polarization and lifetime were analyzed using the Perrin equation to calculate the apparent microviscosity. Control PBL membranes at 37°C exhibited a microviscosity (η) equal to 1.89 poise (P). Five to 10 min after CsA exposure η = 2.62–2.68 P and plateaus at η = 2.31–2.51 P 20–120 min after exposure. Dose-response studies show that prolonged (>90 min) exposure to CsA alters lymphocyte membrane fluidity. This early response in lymphocytes to CsA may correspond to other events associated with the initial binding of CsA to lymphocyte membranes such as membrane depolarization and alterations in phospholipid metabolism. Intravenous delivery of CsA in clinical studies has shown that a similar CsA dose of near 10–5 M is available in the blood during the first hour after injection. The evidence presented here suggests that peripheral blood lymphocyte membranes are rigidified when exposed to CsA doses similar to those found in patient's blood immediately following intravenous administration.
HIV Replication and Persistence in Human Gastrointestinal Cells Cultured In Vitro
Authors: Moyer Mary Pat , Gendelman Howard E
Published: January 2018

Epithelial and submucosal mesenchymal (SM) cells from normal human small intestine and colon could be directly infected by several strains of the human immunodeficiency virus (HIV). Macrophage-derived virus strains were more potent than the HTLVIIIB prototype strain. Persistent release of virus over several months implies that the human gastrointestinal tract may serve as a site for primary infection and as a reservoir for the virus. Furthermore, HIV infection of SM cells may be an in vitro model of Kaposi's sarcoma.
Inflammatory Responses to Cryptococcosis in Congenitally Athymic Mice
Authors: Salkowski Cindy A , Balish Edward
Published: January 2018

Histopathology revealed that nu/nu mice developed both acute and chronic inflammatory responses following infection with Cryptococcus neoformans. In comparison to inflammatory responses in nu/+ mice, the responses in nu/nu mice were delayed, less intense, contained predominantly more polymorphonuclear leukocytes (PMNs) than macrophages (Mφs), and did not develop into granulomas. In addition, nu/nu mice developed cryptococcal skin lesions demonstrating that C. neoformans is dermatotropic in a T-cell deficient host. Quantitative culturing of infected organs confirmed that delayed and incomplete inflammatory responses observed in nu/nu mice correlated with their enhanced susceptibility to C. neoformans.
T Helper/Inducer (CD4+) Cells Prestimulated With PPD Induce Monocytes to Produce Interleukin-1β
Authors: Dunlap Nancy E , Tilden Arabella B
Published: January 2018

We obtained peripheral blood mononuclear cells (PBMC) from four healthy, tuberculin purified protein derivative (PPD) reactive donors and cultured these cells in media containing PPD (low dose = 200 ng/ml or high dose = 1 fig/ml). Five days after the addition of PPD, T cells were isolated, washed, and added to autologous adherent cell cultures at a 1:1 ratio. Adherent cells were then cultured for 24 h in media only (baseline), media plus lipopolysaccharide (LPS, 2 μg/ml; positive control), or media containing the prestimulated T cells. After 24 h, supernatants were harvested and interleukin 1β (IL-β) levels were assayed by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The results show that T cells prestimulated with low dose PPD (200 μg/ml) did not induce IL-1 production by adherent cells (mean increase over baseline 0.2 ± 1.3 standard deviation [SD] ng/ml, P = 0.61). However, T cells prestimulated with high dose PPD (1 tig/ml) did induce adherent cells to secrete IL-1β (mean increase over baseline 1.7 ± 0.62 [SD] ng/ml, P = 0.01), but this induction was abolished when cell-to-cell contact was prevented by use of double well chambers (mean increase over baseline 0.1 ± 0.36 [SD] ng/ml, P = 0.69). Prestimulated T helper (CD4+) cells were able to induce monocytes to secrete IL-1β but prestimulated CD8+ T cells were not. These data suggest that when T helper (CD4+) cells are sufficiently activated they acquire the ability to induce monocytes to secrete IL-1β. Cell-to-cell contact between monocytes and T cells is required. This function of activated T cells may be important in the normal cellular immune response.
FcR-Independent Antibody-Mediated Cellular Cytotoxicity
Authors: Colsky Arthur S , Mendez Luis E , Peacock James S
Published: January 2018

In this study we used palmitate-derivatized antibodies (pal-Ab) to examine the minimum contribution of Fc receptors (FcR) to macrophage (Mφ)-mediated lysis and phagocytosis in antibody-dependent cellular cytotoxicity (ADCC). Pal-Ab specific for chicken erythrocytes (CE) were incorporated into the plasma membranes of Mφ by insertion of the palmitate hydrocarbon chains into the outer leaflet of the phospholipid bilayer. In this system, the palmitate anchor bypassed the requirement for FcR in antibody-dependent effector-target conjugation and provided a unique opportunity to uncouple antibody-FcR interactions in ADCC. We show that binding of CE targets by P388D1 Mφ effector cells through anti-CE pal-F(ab')2 can lead to efficient extracellular target cell lysis, but not phagocytosis. In contrast, pal-F(ab')2-mediated interactions between CE and peritoneal exudate Mφ (PEM) activated both ingestion and extracellular lysis of the targets. In normal ADCC, FcR-dependent interactions between CE and either P388D1 cells or PEM triggered both extracellular lysis and phagocytosis. Our results demonstrate that lysis and phagocytosis in CE-directed ADCC by Mφ have different minimum requirements for FcR functions. Moreover, our results suggest that FcR-independent triggers on the PEM surface are capable of triggering target cell lysis and internalization following antibody-mediated interactions.
Selective Depletion of Liver and Splenic Macrophages Using Liposomes Encapsulating the Drug Dichloromethylene Diphosphonate: Effects on Antimicrobial Resistance
Authors: Pinto Angelo J , Stewart Deneen , van Rooijen Nico , Morahan Page S
Published: January 2018

The current results provide direct evidence for a role of tissue macrophages (Mφ) in natural immunity and support the use of immunomodulators to enhance antiviral resistance in immunocompromised individuals. In this study, macrophages (Mφ) in the spleen and liver were eliminated by intravenous (i.v.) injection of the drug dichloromethylene diphosphonate (DMDP) encapsulated in liposomes. The effect of this depletion system on peritoneal Mφ, peripheral blood leukocytes, splenic natural killer (NK) activity, and natural and immunomodulator-induced host resistance was then assessed. Barrier-maintained CD-1 female mice were inoculated i.v. either with DMDP liposomes, free liposomes (containing no DMDP), or saline on day -2 or on days -3 and -1 before cell population analysis or infection. Single or double treatment with DMDP liposomes had no effect on peritoneal Mφ as indicated by no changes in total number, differential counts, or ectoenzyme patterns. Double treatment with DMDP liposomes caused a marked leukocytosis in blood, primarily of lymphocytes and polymorphonuclear leukocytes (PMN), and a transient depression of spontaneous and interferon-inducible splenic NK activity. The effects on host resistance to i.v. infection with Listeria monocytogenes or herpes simplex virus type 2 (HSV-2) indicated that i.v. treatment with DMDP liposomes significantly reduced natural resistance to these microorganisms as evidenced by increased mortality and decreased median survival time. When DMDP liposomes-treated mice were given the immunomodulator maleic anhydride divinyl ether copolymer (MVE-2) intraperitoneally the day before infection with HSV-2, the immunosuppressive effect of DMDP liposome treatment was significantly reversed.
The Role of NK Cells in the Regulation of Experimental Metastasis in a Murine Lymphoma System
Authors: Smithson Glennda , Bittick Thomas H , Chervenak Robert , Lee Shu-Jan , Wolcott R Michael
Published: January 2018

This report describes the role of natural killer (NK) cells in regulating the localization of two closely related murine lymphomas (ASL1 and ASL1w) to various organ sites. These tumors are phenotypically similar yet differ in their overall malignancy and experimental metastatic pattern. Both tumors show similar patterns of migration in kinetic studies, however, they vary in their NK susceptibility. Cold target inhibition analysis and assessment of susceptibility to cytotoxic T lymphocytes (CTL) suggested that the NK resistance of ASL1 was due to the lack of expression of a relevant target structure(s) required for NK recognition. The role of NK cells in regulating the growth of the tumors in vivo was studied by altering NK cell levels in recipient mice prior to tumor injection. Neither NK cell depletion nor augmentation affected the growth of the NK resistant ASL1 tumor but NK depletion significantly shortened the mean survival time (MST) of recipients of the NK sensitive ASL1w tumor as well as changed its metastatic pattern; conversely, NK augmentation significantly increased the MST of ASL1w recipients. These data suggest that NK cells play a critical role in determining the metastatic characteristics of the ASL1w lymphoma.
Development, Differentiation, and Maturation of Macrophages in the Chorionic Villi of Mouse Placenta With Special Reference to the Origin of Hofbauer Cells
Authors: Takahashi Kiyoshi , Naito Makoto , Katabuchi Hidetake , Higashi Kenji
Published: January 2018

In blood vessels of the chorionic villi of mouse placenta, primitive macrophages first emerged at 10 days of gestation, then differentiated and matured into fetal macrophages. After emigration into the chorionic villous mesenchymal stroma, they ingested fluid-like stromal materials and transformed into Hofbauer cells. In our observation of their differentiation and maturation, no promonocytes or monocytes were detected. In the culture of cell suspensions from the placenta with LP3-conditioned medium, CFU-GM was confirmed, but in the culture with the mouse bone marrow stromal cell line (ST2) the primitive/fetal macrophage population occurred predominantly before the development of the monocyte/macrophage population. Proliferative potential of the primitive and fetal macrophages is slight. With the progress of gestation, the monocyte/macrophage population appeared in the chorionic villous stroma, forming a heterogeneous population of placental macrophages in the late fetal stage.
Effects of Corticosterone and Microgravity on Inflammatory Cell Production of Superoxide
Authors: Fleming Sherry D , Edelman Linda S , Chapes Stephen K
Published: January 2018

In this investigation we studied the effects of corticosterone and microgravity on Propionibacterium acnes-induced inflammatory cells ability to produce superoxide (O2-). We found in vitro and in vivo exposure of murine peritoneal inflammatory cells to corticosterone did not inhibit the O2- response. We also found that in microgravity P. acnes-induced inflammatory cells were capable of producing four times as much O2- as at 1g. Therefore, neither corticosterone nor microgravity experienced during parabolic flight prevents an O2- response by inflammatory cells.
Granulated Metrial Gland Cells: Pregnancy Specific Leukocytes?
Authors: Stewart I J
Published: January 2018

Granulated metrial gland (GMG) cells and their analogous cells are a prominent feature of the maternal placenta in rodents and in primates, including the human. Evidence which relates to the origin of these cells is reviewed and shows that there is now conclusive evidence that GMG cells are a type of leukocyte. Studies to define the type of leukocyte have been inconclusive overall, although several studies have suggested that GMG cells are a type of NK cell. However, it is proposed that attempting to define GMG cells as a type of leukocyte which is already known, like NK cells, is inappropriate and GMG cells should be considered as a separate leukocyte population. Granulated metrial gland cells are closely associated with pregnancy but technically cannot be described as pregnancy specific. The function of GMG cells remains to be established, but the possibility that GMG cells and their analogous cells are controlling the invasion of trophoblast into the uterine wall and/or the removal of aberrant trophoblast is currently under consideration.
Lipopolysaccharide-Dependent and Lipopolysaccharide-Independent Pathways of Monocyte Desensitisation to Lipopolysaccharides
Authors: Annenkov Alexander Y , Baranova Flora S
Published: January 2018

The present study demonstrates that with time in culture blood monocytes (MO) lose their ability to express procoagulant activity (PCA) and secrete tumor necrosis factor-alpha (TNFα) in culture medium in response to lipopolysaccharide (LPS) stimulation. Thus, upon 10 μg/ml LPS stimulation for 4 hours 2-day-old MO produced lower levels of PCA and TNFα than fresh MO. The decrease in responsiveness was not caused by cell death, since in the case of TNFα it was fully reversible by interferon-gamma (IFN-γ). Compared with cells pre-incubated in medium alone, the responsiveness of MO pre-incubated in LPS was further decreased. Thus, in MO LPS pre-incubation was followed by an LPS refractory state. It was expected that the decrease in responsiveness induced by cultivation in medium alone was mediated by LPS contamination of culture medium. However, as we were unable to prevent this decrease by neutralizing LPS contamination of the culture medium with polymyxin B, the loss in LPS-induced activities of cultured MO is likely to be mediated by culture conditions other than LPS contamination. Taken together the present data demonstrate that LPS-dependent as well as LPS-independent pathways of MO desensitization to LPS exist.
Viscoelastic and Motile Properties of Hamster Lung and Peritoneal Macrophages
Authors: Bizal Colette L , Butler James P , Valberg Peter A
Published: January 2018

The motile and rheologic properties of hamster lung and peritoneal macrophages (LMs and PMs) were examined by following the motions of magnetizable iron oxide (γ-Fe2O3) particles contained within phagolysosomes of these cells. As a measure of intracellular motility, γ-Fe2O3 particles in cells were magnetically aligned and the decay rate of the remanent magnetic field (RMF) in the direction of initial magnetization was monitored over time. Cytoplasmic rheology was measured by twisting the intracellular particles with a magnetic field (Btw) applied perpendicularly to the direction of initial magnetization. We measured changes in the RMF associated with application and removal of Btw. Intracellular motility in LMs and PMs was not significantly different (P > 0.20); similarly, cytoplasmic viscosity was not significantly different in LMs and PMs (P > 0.12); deformation on application of torque was significantly greater (P < 0.0001) and elastic recoil on removal of torque was significantly smaller (P < 0.0001) in PMs than in LMs; and by qualitative observation, the yield stress of cytoplasm (associated with a plastic, nonrecoverable deformation) was lower in PMs than in LMs. These results show that although cytoplasmic motion and viscosity are similar in the two cell types, PM cytoplasm is less stiff than LM cytoplasm as determined by yield stress.
Determination of the Number and Distribution of Macrophages, Lymphocytes, and Granulocytes in the Mouse Uterus From Mating Through Implantation
Authors: De Mamata , Choudhuri Rajani , Wood Gary W
Published: January 2018

The concentration and distribution of F4-80 positive cells (macrophages) and common leukocyte antigen (CLA) positive (bone marrow derived) cells were assessed in mouse uterus between days 1 and 8 of pregnancy. High numbers of polymorphonuclear leukocytes and lymphocytes were present on days 1 and 2, but not thereafter. Granulocytes were found both in the endometrium and within the luminal epithelium. The percentage of total cells contributed by macrophages was high on days 1 and 2. That percentage decreased significantly on day 3, then increased again on day 5 and remained high through day 8. Macrophages always were found in myometrial stroma. Macrophages were found throughout the endometrium on days 2 through 8. High numbers of macrophages were observed near epithelia, particularly on days 1,2,4, and 5. Few F4-80+ or CLA+ cells were observed within the developing primary and secondary decidua. The results demonstrate that an inflammation-like cellular response occurs in the uterus following mating and that macrophages are a major cellular component of the uterus during early pregnancy.
Role of 5-Lipoxygenase Products of Arachidonic Acid in Cell-to-Cell Interaction Between Macrophages and Natural Killer Cells in Rat Spleen
Authors: Chang Ke-Jian , Saito Hiroyuki , Tatsuno Ichiro , Tamura Yasushi , Yoshida Sho
Published: January 2018

In the present study we performed experiments to reveal the role off 5-lipoxygenase products of arachidonic acid in cell-to-cell interaction between rat peritoneal macrophages and spleen natural killer (NK) cells. Peritoneal macrophages were found to significantly enhance NK cell activity. It was found that the enhancing activity of macrophages was significantly inhibited by the ingestion of highly purified eicosapentaenoic acid-ethyl ester (EPA-E). But direct cytotoxic action of macrophage on target cells (YAC-1) was unchanged by the ingestion of EPA-E. In addition, the depressed enhancing activity of EPA-enriched macrophages (EPA macrophages) was partially, but significantly, restored by the addition of either 10-10 M leukotriene B4 (LTB4) or 10-12 M 5-hydroperoxyeicosatetraenoic acid (5-HPETE). The partial restoring effect of 10-10 M LTB4 was further significantly enhanced by the addition of 5-HPETE. In contrast 5-hydroxyeicosatetraenoic acid (5-HETE) had no modulatory effect on the depressed activity of EPA macrophages. LTB4,5-HPETE, and 5-HETE had no direct effect on NK cell activity itself. Significantly less amounts of LTB4 and 5-HETE were produced in EPA macrophages as compared with that in control macrophages. The present study indicates that macrophage modulatory action on NK cells can be partly mediated by LTB4 and 5-HPETE, which are produced in macrophages.