bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of IL-1α, IL-1β, and 25K Mr Non-IL-1 Activity Released by Human Mononuclear Cells
Authors: Sandborg Christy I , Berman Monique A , Imfeld Karen L , Zaldivar Frank , Masada Marvin P , Kenney John S
Published: January 2018

To determine if the release of IL-1α and IL-1β by cultured PBMC could be independently modulated by different exogenous stimuli, we examined the effect of LPS, IFNγ, latex beads, and indomethacin on the release of IL-1α and IL-1β. PBMC culture supernatants were fractionated by Sephacryl-S-200 column chromatography or HPLC (TSK G3000SW), and each fraction was tested for thymocyte mitogenic activity in the presence or absence of preincubation with anti-IL-1α or anti IL-1β monoclonal antibody (mAb) and for the presence of IL-1α or IL-1β protein by ELISA. In all experiments, thymocyte mitogenic activity not neutralizable by anti-IL-1α or anti-IL-1β mAb was detected in the 25K Mr range, which ranged from 12 to 50% of the total thymocyte mitogenic activity released, depending on the stimuli. Cultured PBMC from 95% of individuals release thymocyte mitogenic activity in the absence of exogenous stimuli, which was increased 1.3- to 7-fold by lipopolysaccharide (LPS) (25–50 üg/ml). All of this increased activity was due to increased release of IL-1β and non-IL-1 thymocyte mitogenic activity, with no change in the total amount of IL-1α released. Indomethacin (0.1 μg/ml) induced release of increased thymocyte mitogenic activity of 1.3- to 1.4-fold over unstimulated cultures. All of this increased activity was due to increased release of IL-1α and non-IL-1 activity with a concomitant decrease in IL-1β release. Interferonγ (40–100 U/ml) increased the amount of IL-1α and decreased IL-1β and non-IL-1 activity released, resulting in no overall change in the total amount of thymocyte mitogenic activity.

Molecular weight fractionation of the PBMC culture supernatants revealed that thymocyte mitogenic activity eluting in the 25K Mr range was not due to IL-1α or IL-1β. With certain culture conditions, thymocyte mitogenic activity was detected in the 30–40K Mr range. PBMC cultured with LPS and latex beads in the absence of serum released 30–40K Mr IL-1α, as well as 17K Mr IL-1α and 17K Mr IL-1β. PBMC cultured in 2% fetal calf serum (FCS) alone from some donors released only 30–40K Mr thymocyte mitogenic activity. Both IL-1α and IL-1β protein was detected by ELISA in this Mr range but only the IL-1α was bioactive. This study shows that release of IL-1α, IL-1β, and non-IL-1 thymocyte mitogenic activity can be modulated by different stimuli in consistent patterns, and suggests that the release of these factors is independently regulated.
Differential Expression of Estrogen-Regulated CD4 and Ia Positive Cells in the Immature Rat Uterus
Authors: Zheng Yi , Sundstrom Susan A , Lyttle C Richard , Teuscher Cory
Published: January 2018

Estradiol treatment of sexually immature Sprague-Dawley, Cr:NIH-nu/+ and Cr:NIH-nu/ nu rats for 2 days results in a significant increase in the number of uterine eosinophils and OX-42 positive macrophages. However, only Sprague-Dawley rats exhibit an estrogen regulated increase in the number of cells expressing immunoreactive CD4 and la, suggesting that the differential responsiveness to the effects of estradiol between the two strains of rats may be genetically controlled. Nevertheless, Cr:NIH-nu/nu rats, which are deficient in T-cells, possess virtually identical numbers of CD4 positive staining uterine cells as compared to their nu/+ littermate controls, indicating that the expression of immunoreactive CD4 is associated with a resident non-T-cell population. In addition, a message for rat CD4 was observed in Northern blots of uterine mRNA, suggesting that the immunoreactive CD4 expressed by uterine cells is probably a fully functional cell surface antigen/receptor rather than a truncated form which only expresses the antigenic determinant recognized by the W3/25 monoclonal antibody.
Alterations of Host Resistance to Mouse Typhoid Infection by Sex Hormones
Authors: Kita Eiji , Yagyu Yoshihiko , Nishikawa Fumiko , Hamuro Akiko , Oku Daisuke , Emoto Masashi , Katsui Noriaki , Kashiba Shuzo
Published: January 2018

The effect on mouse typhoid infection of a 3-day treatment of female virgin mice with 1 mg/day of female sex hormones (estrogen or progesterone), maintaining the same hormonal levels observed in pregnant mice for 30 days, was investigated in order to clarify the mechanisms of altered resistance during pregnancy. Estrogen-exposed mice were more susceptible to the intraperitoneal challenge with Salmonella typhimurium as compared with the vehicle control mice, while progesterone treatment increased the survival times of mice. Estrogen exposure increased the number of peritoneal cells after treatment, but the inflammatory cellular response after infection was significantly suppressed. Although the estrogen-treated and vehicle control mice had the same degrees of peritoneal cellular responses after infection, the death rates in the estrogen-treated mice were higher than those in the vehicle control mice against challenge with 1 LD50 of S. typhimurium. On the other hand, progesterone treatment resulted in the marked influx of peritoneal cells after treatment was terminated, and also it induced a significant increase in the number of peritoneal cells after infection. Although survival times in the progesterone group were higher than those in other groups, all progesterone-treated mice died after a challenge with 1,000 LD50 of S. typhimurium. These results suggest that progesterone enhances nonspecific resistance by increasing the influx of peritoneal cells after infection, while estrogen affects the acute inflammatory responses.
Cooperation Between Platelets and Neutrophils for Paf-Acether (Platelet-Activating Factor) Formation
Authors: Coëffier Eliane , Delautier Danièle , Le Couedic Jean-Pierre , Chignard Michel , Denizot Yves , Benveniste Jacques
Published: January 2018

Association of platelets and neutrophils is frequently observed within thrombi or inflammatory sites. Interactions between these two cell populations have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether, which is synthesized by activated platelets and neutrophils. Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelet and neutrophil cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 x 106/ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 ± 2.5 ng/ml paf-acether. Human washed platelets (3 x 108/ml) stimulated with thrombin (1 lU/ml) formed 0.60 ± 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as did platelets and neutrophils separately (10.90 ± 4.25 ng/ml, n = 6, P < .001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled (10.24 ± 3.81 ng/ml paf-acether vs. 5.30 ± 2.23, P < .05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to an increase of biosynthesis of paf-acether by neutrophils (13.86 ± 2.26 ng/ml paf-acether vs. 5.76 ± 0.38, P < .05, n = 3).

These results indicate that a cooperation between platelets and neutrophils exists for paf-acether formation. The phenomenon depends on a platelet-derived soluble factor, possibly lyso paf-acether. This cell-to-cell interaction is of interest since paf-acether is formed by and acting on platelets and neutrophils and represents a molecular basis for potent amplification of inflammatory reactions.
Stimulus-Specific Effects of Pentoxifylline on Neutrophil CR3 Expression, Degranulation, and Superoxide Production
Authors: Currie Mark S , Rao K Murali K , Padmanabhan Jaya , Jones Adrie , Crawford Jeffrey , Cohen Harvey J
Published: January 2018

The effects of pentoxifylline (Trental) on human neutrophil CR3 up-modulation, degranulation, and superoxide production were studied. We used the chemotactic peptide fMLP and the phorbol ester PMA as soluble stimuli, and β-glucan particles as a CR3-specific solid phase stimulus of neutrophil superoxide production. Since neutrophils have adenosine A2 receptors, we compared effects of pentoxifylline to effects of adenosine, and we also looked at the effect of cytochalasin B, which breaks up actin filaments. Pentoxifylline inhibited both CR3 up-modulation and degranulation of myeloperoxidase and lysozyme. Pentoxifylline is a more potent inhibitor of fMLP- compared to PMA-induced degranulation, and is especially potent against superoxide production. While pentoxifylline is less potent than adenosine in its inhibiton of fMLP-induced superoxide production, it is more potent in its inhibition of PMA- and β-glucan particle-stimulated superoxide production. Cytochalasin B, which enhances degranulation and fMLP-stimulated superoxide production, was found to inhibit β-glucan particle-stimulated superoxide production. These findings are consistent with the hypothesis that pentoxifylline can affect both the cytoskeletal architecture of unstimulated neutrophils and the activation and responses of neutrophils which involve actin polymerization and receptor-cytoskeletal interactions.
Physiologic Oxygen Tensions Limit Oxidant-Mediated Killing of Schistosome Eggs by Inflammatory Cells and Isolated Granulomas
Authors: Feldman Gerald M , Dannenberg Arthur M , Seed John L
Published: January 2018

Explanted hepatic granulomas, eosinophils obtained from the peritoneal cavity of schistosome-infected mice, schistosome egg granuloma macrophages, alveolar macrophages, and activated peritoneal macrophages obtained from Listeria-infected mice were miracidicidal when cultured at 21% oxygen. This activity was markedly attenuated at physiologic oxygen concentrations (1–15%). Catalase and superoxide dismutase blocked the miracidicidal activity of inflammatory cells but did not prevent granuloma-mediated egg killing. However, the biomimetic superoxide dismutase, copper (II) [diisopropyl salicylate]2, inhibited granuloma-mediated egg killing in a dose-dependent, apparently nontoxic manner. Thioglycollate-elicited macrophages did not kill schistosome egg miracidia even when cultured in 21% oxygen, unless pretreated with lipopolysaccharide. Isolated schistosome eggs initiated an oxidative burst in macrophages, as measured by superoxide anion production. This burst was suppressed at reduced oxygen concentrations. Thus schistosome egg miracidia can be killed nonspecifically by macrophages through the release of cytotoxic reactive oxygen intermediates triggered by the egg. This activity is not supported by the oxygen concentrations found in most tissues, with the possible exception of the lung. Schistosoma mansoni eggs, injected intraveneously and lodged in the pulmonary vasculature of mice, were killed rapidly, with a half life of 3.5 days. Eggs, injected into the mesenteric veins and lodged in the liver, remained fully viable for several weeks. The data suggest that the high oxygen tension of the lung allows for the increased production of reactive oxygen intermediates (ROI) by local inflammatory cells, which in turn increases their miracidicidal efficiency. Conversely, the relatively hypoxic environment of the liver decreases ROI production by local inflammatory cells and decreases their miracidicidal efficiency.
Immunogold Labeling of the Low-Affinity (55 kd) IL2 Receptor on the Surface of IL2 Receptor-Bearing Cultured Cells and Mitogen-Activated Peripheral Blood Lymphocytes
Authors: McGroarty Roy J , Mills Gordon B , de Harven Etienne
Published: January 2018

Foreign antigens or mitogens initiate activation of T lymphocytes through antigen-specific receptors. After antigen-receptor interaction, T cells release a lymphotrophic hormone, Interleukin 2 (IL2), and high-affinity IL2 receptors (IL2R) are progressively expressed on their surfaces. When evaluated under the scanning electron microscope after specific immunolabeling with colloidal gold markers, the density of expression of the 55 kd low-affinity IL2 receptor (Tac) increases in a time-dependent manner, reaching a maximum 72 hours after the onset of phytohemagglutinin (PHA) activation. In contrast, the number of cells expressing the 55 kd molecule reaches a maximum by 24 hours and remains constant through 72 hours. Immunogold labeling allows simultaneous determination of IL2 receptor density at the level of each individual cell and of the percentage of IL2 receptor-positive cells in any cell population under study. The rank order of receptor expression on the surface of established culture cell lines correlates with results reported from other laboratories with immunofluorescence and Scatchard analysis of binding of radiolabeled IL2. Although at high density of receptor expression Immunogold labeling with 40 nm gold markers detects significantly fewer sites than radiolabeling, the method appears very sensitive in identifying low levels of expression. This results from 1) the extremely low non-specific background of the immunogold labeling technique and 2) the analysis of single cells rather than cell populations. Our results indicate that 2 cell lines, reported as negative for the expression of the 55 kd IL2 receptor (YT2C2 and MLA144), express low numbers of this molecule as demonstrated by immunogold labeling.
FcγRII-Mediated Superoxide Production by Phagocytes Is Augmented by GM-CSF Without a Change in FCγRII Expression
Authors: Roberts Pamela J , Devereux Steven , Pilkington Glenn R , Linch David C
Published: January 2018

Freshly purified neutrophils and monocytes respond to multiple cross-linking of FCγRII with the lgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)I, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30–60 min at 37°C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking FcγRII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhlFN-γ (100 u/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking FcγRII, there was no concomitant change in FcγRII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of FCγRII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.
Modulation of an Adhesion-Related Surface Antigen on Equine Neutrophils by Bacterial Lipopolysaccharide and Antiinflammatory Drugs
Authors: Bochsler Philip N , Slauson David O , Neilsen Nancy R
Published: January 2018

The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common β-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the monoclonal recognized a functional epitope on equine PMN involved in adhesion-related functions. Cells pretreated only with bacterial lipopolysaccharide (LPS; 1 μg/ml) exhibited moderate increased binding of MoAb 60.3 as determined by fluorescence intensity. Preincubation of PMN with LPS resulted in a slight increase in MoAb 60.3 binding after subsequent ZAS stimulation, greater than that with either LPS or ZAS as sole stimulus. Similarly, enhanced binding of MoAb 60.3 was observed with LPS preincubation when PMA was used as a stimulus, but this effect was dose dependent and was observed at only one of three PMA concentrations tested (1 ng/ml). In other experiments, preincubation of PMN with antiinflammatory drugs inhibited 41.5–45.1% of ZAS-stimulated PMN adhesion to monolayers of equine endothelial cells. To determine whether modulation of expression of the adhesion-related antigen recognized by MoAb 60.3 correlated with these observed adhesive responses of PMN, we used immunofluorescence flow cytometry to assess expression of the antigen on drug-treated PMN. Using 10% ZAS as a stimulus, phenylbutazone (PBZ; 100 μg/ml) pretreatment of PMN reduced subsequent MoAb 60.3 binding by only 12.3%, and dexamethasone (DEX; 10-5 M) reduced binding by only 1.0%; reductions of 16.4% with PBZ and 9.3% with DEX occurred when PMA (10 ng/ml) was used as the PMN stimulant. These data suggest that equine PMN express a functional adhesion molecule similar to those found on human PMN and that LPS may enhance the expression of this surface antigen. Expression of this adhesion-related surface antigen on equine PMN does not correlate well with levels of drug-induced diminished adhesion of PMN to endothelium in vitro.
Failure of F-Met-Leu-Phe to Induce Chemotaxis in Differentiated Promyelocytic (HL-60) Leukemia Cells
Authors: Sirak Andrea A , Laskin Jeffrey D , Robertson Fredika M , Laskin Debra L
Published: January 2018

Treatment of HL-60 promyelocytic leukemia cells with dibutyryl cyclic adenosine 3′,5′-monophosphate (dbcAMP) induced these cells to differentiate toward mature neutrophilic granulocytes (PMN). DbcAMP was found to produce a rapid time- and dose-dependent inhibition of HL-60 cell proliferation, reaching a maximum after 48 hr treatment of the cells with 250–500 μM. This was associated with morphologic alterations consistent with maturing PMN. Using immunofluorescence and flow cytometry, we found that dbcAMP-treated HL-60 cells expressed some, but not all, surface markers characteristic of mature phagocytes. Thus receptors for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP); the monocyte/granulocyte markers My-8, Mo-1, and Leu-15; as well as the monocyte markers Mo-2 and My-4 were identified on the cells. In contrast, dbcAMP-treated cells did not express the PMN-specific antigen DL1.2. We also found that dbcAMP-treated HL-60 cells produced hydrogen peroxide in response to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). However, the magnitude of this response was significantly less than that observed in mature PMN. In contrast, fMLP-stimulated levels of hydrogen peroxide production were comparable in both cell types. In addition, despite the fact that dbcAMP-treated cells expressed fMLP receptors, they did not exhibit directed migration toward this chemotactic peptide. Taken together, these data suggest that dbcAMP-induced differentiation of HL-60 cells is incomplete.
Fc-γ Receptor-Ligand Interactions Enhance Macrophage GSIB4-Binding Activity
Authors: Tabor Dale R , Thompson Joseph W , Lary Cynthia H , Jacobs Richard F
Published: January 2018

Activated macrophages have an increased ability to bind the lectin Griffonia simplicifolia-IB4 (GSIB4). Since macrophages readily use the Fc-receptor (FcR) during several immunologic and inflammatory processes, it is important to determine whether interactions with this moiety affect GSIB4-binding ability. Peritoneal macrophages cultured in vitro with Fc fragments of immunoglobulin G (IgG), whole IgG, or heat-aggregated IgG demonstrate an increase in this function. Conversely, treatment of macrophages with (Fab')2fractions alone has no direct effect on this activity. Although the GSIB4-binding response is minimally expressed by normal macrophages, it is more markedly apparent on macrophages from LPS-treated animals. In both cases, however, pretrypsinization of the cells renders them refractory to IgG-mediated induction of the GSIB4-binding response. Moreover, macrophages cultured independently with IgG subclasses 1, 2a, or 3 demonstrate that the magnitude of their response to this signal is directly associated with the type of subclass used. Although each subclass enhanced the response, in this study interactions with IgG2a produced the best results.

Overall, however, the greatest GSIB4-binding activity is generated when FcRs are crosslinked by aggregated IgG rather than simply bound by independent monomeric interactions at the FcRs. This suggests that the event of appropriately interacting with the FcRs amplifies the GSIB4-binding function. Such a mechanism could play a key role in coordinating the humoral, cell-mediated, and innate responses of the immune system.
Splenic Macrophages From Tumor-Bearing Mice Co-Expressing MAC-1 and MAC-2 Antigens Exert Immunoregulatory Functions Via Two Distinct Mechanisms
Authors: Watson Gordon A , Fu Yang-Xin , Lopez Diana M
Published: January 2018

Tumor burden has been shown to induce a variety of phenotypic and functional changes in the cellular constituents of the host's immune system. These changes have been implicated as mechanisms by which tumors avoid rejection. Studies of BALB/c mice bearing a D1-DMBA-3 mammary adenocarcinoma showed alterations of the splenocyte populations. There was a five-fold increase of macrophages (Mφ) that were phenotypically and functionally analyzed to establish their role in tumor-induced modifications of the host's immune response. Monoclonal antibody staining defined a Mac-1+2+ population which comprised up to 20% of the splenocytes in tumor-bearers (TB), but is negligible in spleens from normal mice. These Mao-1+2+ Mφ were found to mediate down-regulation of both polyclonal and antigen-specific T and B cell responses in vitro and in vivo. Although B cell responses were suppressed via prostaglandin E2 (PGE2) production by the TB Mφ, T cell responses were relatively refractory to PGE2-mediated down-regulation. Instead, they were suppressed by a contact-dependent T cell-Mφ interaction. Furthermore, tumor-derived factors such as granulocyte-Mφ colony-stimulating factor (GM-CSF) seem to play an important role in the induction and expansion of the Mac-1+2+ Mφ. These cells appear to mediate down-regulation of the host immune responses by at least two distinct mechanisms: 1) PGE2 production and 2) a cell contact-dependent, but non-major-histocompatibility-complex-specific, interaction.
GM-1, a Clone of the Monoblastic Phagocyte U937 That Expresses a Large Respiratory Burst Capacity Upon Activation With Interferon-γ
Authors: Garotta Gianni , Thelen Marcus , Delia Domenico , Kamber Markus , Baggiolini Marco
Published: January 2018

The human cell line U937 was cloned and screened for the responsiveness to interferon-γ (INF-γ). The selected subclone, named GM-1, expressed a high density of IFN-γ receptors and showed HLA typing similar to that of the parental line but was devoid of the Y chromosome. GM-1 cells display a promyeloid phenotype as revealed by flow cytometry using a panel of murine antibodies. Following treatment with IFN-γ GM-1 cells differentiated to a more mature monocyte stage and acquired the capacity to mount a respiratory burst. After treatment with differentiation promoters, such as phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO), and retinoic acid, GM-1 showed a more limited respiratory burst capacity. Superoxide release in IFN-γ-activated cells was stimulated with f-Met-Leu-Phe, C5a, or PMA. The development of the respiratory burst capacity was accompanied with the expression of cytochrome b558, a component of the phagocyte NADPH-oxidase. GM-1 cells are useful for the study of the effects of IFN-γ on the respiratory burst. They are more sensitive and yield a more homogenous response to IFN-γ than U937 cells. The phenotype of GM-1 cells was stable for more than 5 years.
Recombinant Interleukin-4 Enhances the Chemiluminescent Oxidative Burst of Murine Peritoneal Macrophages
Authors: Tan Henkie P , Nehlsen-Cannarella Sandra L , Garberoglio Carlos A , Tosk Jeffrey M
Published: January 2018

We evaluated the effects of murine recombinant interleukin-4 (rIL-4) on murine peritoneal macrophages. We showed a marked, dose-dependent stimulation of respiratory oxidative burst by IL-4 in peptone-elicited murine peritoneal macrophages. This effect was abolished by a neutralizing monoclonal antibody (mAb) to rlL-4 confirming that the enhanced chemiluminescence was due to IL-4. In contrast, rlL-4 depressed the respiratory oxidative burst of a transformed murine macrophage cell line, J774, in a dose-dependent mAb-reversible manner.
Alterations in Tyrosine Protein Kinase Activities Upon Activation of Human Neutrophils
Authors: Berkow Roger L , Dodson Robert W
Published: January 2018

Human neutrophils are phagocytic cells that can be activated by a variety of agonists to undergo a group of physiologic responses. This “stimulus-response” coupling is thought to be dependent on the phosphorylation of specific proteins. We have previously shown that, in addition to the widely distributed serine and threonine protein kinases, neutrophils contain tryosine protein kinase activity in the cell cytosol and particulate fractions. When neutrophils are treated with a variety of agents, phosphorylation of both cytosolic and particulate proteins on tyrosine residues occurs. Increases in tyrosine phosphorylation may be a result of increases in the activity of tyrosine kinases or a decrease in the activity of phosphotyrosine phosphatases. In this study, we have used a novel nondenaturing polyacrylamide gel electrophoretic method to demonstrate that treatment of human neutrophils with the chemotactic peptide FMLP or the phorbol ester phorbol myristate acetate induces a time- and concentration-dependent increase in the tyrosine protein kinase activity found in the cell cytosol and cell particulate fraction. The time course and concentration range over which these changes occur are similar to those seen for activation of the neutrophil oxidative burst and phosphorylation of proteins on tyrosine residues, suggesting that these events may be related.
Expression and Capping of a Proliferation-Associated Surface Membrane p34 kDa Antigen on Different Human Hematopoietic Cell Lines
Authors: Streck Richard J , Hurley Edward L , Pauly John L
Published: January 2018

A novel surface membrane nonglycosylated acidic polypeptide (34 kDa), encoded by a structural gene on chromosome 11, has been identified using murine monoclonal antibody (MoAb) 53.6 (lgG2a). MoAb 53.6, raised against uninduced cells of a human erythroleukemia line (HEL), recognizes a surface membrane antigen that is displayed on proliferating (cell cycle phase G., S, and M + G2 phase) human leukocytes. The expression and redistribution (i.e., patching and capping) of the p34 kDa antigen on 27 different long-term human hematopoietic cell (HHC) lines was defined by fluorescence microscopy. These lines had been established from patients with leukemia or healthy donors and included phenotypically defined populations of T cells, B cells, and myelomonocytic cells. Almost all (>95%) of the leukocytes of the 27 lines reacted strongly with MoAb 53.6. The majority of the leukocytes displayed p34 kDa antigen patching (26/27 lines; patched cells, 96–100%); moreover, 20 of 27 lines exhibited p34 kDa antigen capping (capped cells, 8–96%). Presentation of the p34 kDa antigen on surface membrane ultrastructures, imaged with immunogold using an indirect antibody labeling procedure, was illustrated by scanning electron microscopy, and endocytosis of the gold-tagged antigen-antibody complex was studied by transmission electron microscopy. The HHC lines are thought to represent immortalized populations of different human leukocyte subsets that are in different stages of maturation and/or differentiation; thus these lines should prove useful as models for further characterizing this unique p34 kDa proliferation-associated antigen and for defining the mechanisms and significance of surface membrane antigen redistribution and modulation that has been associated with leukocyte activation and propagation.
[Ca2+]i Changes and Respiratory Burst in Human Neutrophils and Monocytes Induced by NAP-1/Interleukin-8, NAP-2, and gro/MGSA
Authors: Walz Alfred , Meloni Federica , Clark-Lewis Ian , von Tscharner Vinzenz , Baggiolini Marco
Published: January 2018

The stimulatory effects of neutrophil-activating peptide 1 (NAP-1), also termed interleukin 8 (IL-8), neutrophil-activating peptide 2 (NAP-2), and melanoma growth-stimulatory activity (gro/MGSA) on human neutrophils and monocytes were compared on the basis of two responses that can be assessed in real time, the changes in cytosolic free calcium and the respiratory burst. All three peptides induced a rapid and transient rise of cytosolic-free calcium and the respiratory burst in neutrophils. Both responses were also obtained in monocytes on stimulation with NAP-1/IL-8 and gro/MGSA, but not with NAP-2, which appeared to be more selective for neutrophils. Pretreatment with concanavalin A (ConA) enhanced several fold the rate and duration of the respiratory burst of neutrophils stimulated with all three peptides and of monocytes stimulated with NAP-1/IL-8 and gro/MGSA, but not with NAP-2. Sequential stimulation showed mutual cross desensitization by NAP-2 and gro/MGSA in neutrophils. In addition, desensitization of neutrophils toward NAP-2 and gro/MGSA, and of monocytes toward gro/MGSA, was obtained by prestimulation with NAP-1/IL-8. Prestimulation with either NAP-2 or gro/MGSA, however, did not desensitize the cells for NAP-1/IL-8.

These results suggest that under conditions where multiple stimulatory agents are produced, neutrophil-activating peptides may contribute to the formation of substantial amounts of oxygen-derived radicals. In addition, the study shows that NAP-1/IL-8 and gro/MGSA, but not NAP-2, have some stimulatory effects on monocytes as well.
Reactivity of antineutrophil cytoplasmic autoantibodies with mononuclear phagocytes
Authors: Charles Linda A , Falk Ronald J , Jennette J Charles
Published: January 2018

Antineutrophil cytoplasmic autoantibodies (ANCA) react with proteins found in the granules of neutrophils and the peroxidase-positive lysosomes of monocytes, including myeloperoxidase (MPO), proteinase 3 (PR-3), and elastase. ANGA-associated diseases, such as Wegener's granulomatosis and polyarteritis nodosa, are characterized by necrotizing vascular inflammation. The inflammatory lesions typically contain both neutrophils and mononuclear phagocytes, with the latter sometimes predominating, for example, in the granulomatous lesions of Wegener's granulomatosis. We investigated the presence of the ANCA target antigens PR3, MPO, and elastase in mononuclear phagocyte cytoplasm during the course of differentiation in vitro and in alveolar and peritoneal macrophages. We observed that ANCA antigens were down-regulated during mononuclear phagocyte differentiation, with the loss corresponding to that of peroxidase-positive granules. This suggests that ANCA can directly interact only with monocytes and early exudative macrophages and not with mature macrophages.
Heterogeneity in the mobilization of cytoplasmic calcium by human polymorphonuclear leukocytes in response to fMLP, C5a and IL-8/NAP-1
Authors: Eisner Jörn , Kaever Volkhard , Emmendörffer Andreas , Breidenbach Thomas , Lohmann-Matthes Marie-Luise , Roesler Joachim
Published: January 2018

The visible excitation and emission wavelengths of the recently developed fluorescent Ca2+ indicator fluo-3 permit analysis of the intracellular Ca2+ concentration, [Ca2+]i, in flow cytometry with a 488-nm argon laser. The role of [Ca2+]i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1) by flow cytometry. The [Ca2+]i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti-CD16 phycoerythrin-conjugated antibody and fluo-3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type lb. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL-8/NAP-1 (10-10 M) two subsets of neutrophils appeared; one of them showed an increase in [Ca2+]i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single-cell [Ca2+]i distribution patterns, which is important for the understanding of [Ca2+]i in neutrophil heterogeneous activation processes.
Differential production of granulocyte-macrophage colonystimulating factor by macrophages from mice susceptible and resistant to Leishmania mexicana amazonensis
Authors: Soares Luis Roberto B , Barcinski Marcello A
Published: January 2018

The present results demonstrate that macrophages from mice susceptible to infection with Leishmania mexicana amazonensis sustain a higher production of granulocyte-macrophage colony-stimulating factor (GM-GSF) throughout the in vitro infection than macrophages from a resistant strain. Resident macrophages from BALB/c and C57B1/10 mice were infected with promastigotes of L. mexicana amazonensis and the amount of biologically active GM-CSF was measured in the supernatants collected at different times of infection. Measurements were made by bone marrow and GM-CSF/interleukin-3 addicted cell proliferation. Because GM-GSF is a disease-exacerbating cytokine, its differential production by infected macrophages may be one of the mechanisms defining resistance or susceptibility to a leishmanial infection.
Synthetic hexapeptides derived from the transmembrane envelope proteins of retroviruses suppress N-formylpeptide–induced monocyte polarization
Authors: Oostendorp Robert AJ , Schaaper Wim M M , Post Jacob , Meloen Rob H , Scheper Rik J
Published: January 2018

Retroviral infections are frequently associated with immunosuppression. Retroviral transmembrane envelope proteins (TM proteins) play an important role in this phenomenon. CKS-17, a synthetic heptadecapeptide, represents the immunosuppressive site of these retroviral TM proteins. Here we report on the further delineation of this immunosuppressive site using CKS-17-derived hexapeptides. The N-formyl-methionyl-leucyl-phenylalanine-induced monocyte polarization assay was used throughout this study because this monocyte function has been shown to be highly sensitive to TM protein p15E-related immunosuppression. We found that in addition to CKS-17 one CKS-17-derived hexapeptide, LDLLFL, reversibly inhibited monocyte polarization, with 50% inhibitory concentrations of 20 and 2 μM respectively. LDLLFL-mediated inhibition was sequence specific because the reverse peptide LFLLDL and scrambled peptides were not inhibitory. Hexapeptides corresponding to LDLLFL, but derived from various retroviruses other than murine leukemia virus, also inhibited monocyte polarization. Peptides most homologous to LDLLFL –- LDILFL (feline leukemia virus) and LDLLFW (human T lymphotropic virus types I and II)–-were the most potent inhibitors. Peptides homologous to primate and human endogenous proviruses were not suppressive. LDLLFL and some of its homologues also inhibited polarization of neutrophilic granulocytes. These findings lend further support to the view that conserved retroviral TM protein–related peptides can play an important role in suppression of inflammatory cell function as encountered in retrovirus-associated immunosuppression.
In vivo metabolic response of hepatic nonparenchymal cells and leukocytes to granulocyte-macrophage colony-stimulating factor
Authors: Spolarics Zoltan , Schuler Agnes , Bagby Gregory J , Lang Charles H , Nelson Steve , Spitzer John J
Published: January 2018

This study investigates the in vivo glucose utilization of various immune-competent cells after an intra-arterial injection of a nonlethal dose (30 μg/kg body weight) of murine recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Injection of GM-CSF resulted in a rapid but transient reduction in the number of circulating neutrophils. After 20 min the number of neutrophils returned to normal values, and by 4 h it was about 80% greater than in time-matched saline-injected controls. One hour after the treatment, neutrophils were accumulated in the livers of GM-CSF-injected animals but not in control livers. In vivo glucose utilization by circulating neutrophils and mononuclear cells and various liver cell types was investigated by combining the 2-deoxyglucose tracer technique with cell isolation procedures. GM-GSF increased the in vivo glucose utilization of circulating and infiltrating neutrophils by more than 200%. Glucose utilization by circulating mononuclear cells was also doubled. After GM-GSF injection, glucose utilization by Kupffer cells was increased by 130% and by hepatic endothelial cells was increased by 60%. Indomethacin pretreatment blunted the hyperglycemia caused by GM-CSF injection; however, it did not inhibit the increased glucose utilization by immune-competent cells. This suggests that the effect of GM-GSF on glucose utilization by these cells is not mediated by prostanoids and is at least partially independent of the mass action of elevated glucose concentration. These findings indicate that GM-CSF may be an important member of the cytokine cascade that mediates the acute in vivo metabolic response of immune-competent cells in sepsis or endotoxemia.
In vitro adhesion of human epidermal Langerhans cells to laminin and fibronectin occurs through β1 integrin receptors
Authors: Le Varlet B , Staquet M J , Dezutter-Dambuyant C , Delorme P , Schmitt D
Published: January 2018

Human epidermal Langerhans cells are dendritic cells that can capture, process, and present antigens to T cells. It was previously shown that these Langerhans cells express the very late activation antigen (VLA) protein family of β1 integrins. β1 integrins mainly mediate the adhesion of cells to a number of extracellular components, such as laminin, fibronectin, and collagen, which are present in the skin. In this report, we demonstrate that a large percentage of the Langerhans cell population was able in vitro to attach to laminin and fibronectin but not to collagen. An ultrastructural study of adherent Langerhans cells showed that they were spread largely on laminin, with a loss of their round shape, and partially on fibronectin. Langerhans cell binding to laminin or fibronectin induced a decrease of the Birbeck granule number. Specific inhibitions of cell adhesion were performed, and it was demonstrated that VLA-6 was the major receptor involved in Langerhans cell adhesion to laminin. This adhesion was not RGD dependent and was slightly enhanced by Mn2+. VLA-3 and especially VLA-5 mediated Langerhans cell binding to fibronectin through the RGDS sequence of the protein. Mn2+ sharply increased the Langerhans cell adhesion to fibronectin. VLA-6 mediated in vitro Langerhans cell adhesion to laminin, which suggests that in vivo VLA-6 could permit Langerhans cells to attach and migrate through the basement membrane. Moreover, VLA-5 and VLA-3 take part in the in vitro Langerhans cell binding to fibronectin, suggesting that in vivo these fibronectin receptors could facilitate Langerhans cell passage throughout the fibronectin network of the dermis before migration to lymph nodes.
Formation of LTB4 by fMLP-stimulated alveolar macrophages accounts for eosinophil migration in vitro
Authors: Hidi Rabiâ , Coëffier Eliane , Vargaftig B Boris
Published: January 2018

Guinea pig alveolar macrophages obtained by bronchoalveolar lavage were isolated by adherence for 2 h and stimulated with 1 μM of N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) for different time intervals. The supernatants then were tested for their chemotactic effect on guinea pig peritoneal normodense eosinophils and for release of thromboxane B2, leukotriene B4 (LTB4), and platelet activating factor (PAF). The supernatant from fMLP-stimulated alveolar macrophages induced a significant eosinophil attraction (96.0 ± 11.9, number of migrating eosinophils [mean ± SEM], n = 17) as compared to unstimulated macrophages (4.8 ± 1.4, n = 15). This effect was not accounted for by fMLP carry-over to the macrophages because, in contrast to human eosinophils, fMLP has no chemotactic effect on guinea pig eosinophils. Pretreatment of eosinophils with BN 52021 (100 μM), a specific PAF antagonist, and with indomethacin (10 μM), a cyclooxygenase inhibitor, failed to inhibit migration of eosinophils induced by supernatants from either stimulated or unstimulated alveolar macrophages. In contrast, inhibition of the 5-lipoxygenase enzyme with N-(3-phenoxycinamyl)-acetohydroxamic acid (1 μM) suppressed eosinophil migration by alveolar macrophage supernatants (94.1 ± 2.6% of inhibition, n = 6). Desensitization of eosinophils by and to LTB4 (10 nM) inhibited migration induced by supernatants from stimulated alveolar macrophages (87.5 ± 5.4% of desensitization toward LTB4 and 83.1 ± 5.4% of desensitization toward supernatants, n = 5). Under the present experimental conditions, LTB4 is the only agent implicated in eosinophil migration induced by supernatants from fMLP-stimulated alveolar macrophages.
Inhibition of some human neutrophil functions by the cyclooxygenase inhibitor ketorolac tromethamine
Authors: Hyers Thomas M , Tricomi Sally M , Liao Jeng-Jong
Published: January 2018

Ketorolac tromethamine, a new nonsteroidal anti-inflammatory agent of the pyrrolo-pyrrole group, was assayed for inhibitory effects on polymorphonuclear leukocytes (PMN) in a variety of systems. Ketorolac inhibited PMN superoxide anion generation, lysozyme release, myeloperoxidase release, adherence to plastic surfaces, and chemotaxis in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) in a dose-dependent manner. Ketorolac also inhibited phorbol myristate acetate-stimulated adherence of PMN to bovine pulmonary artery endothelial cells. The drug inhibited lysozyme and myeloperoxidase release by PMN in response to C5a but failed to inhibit C5a stimulation of PMN in any of the other assays. Levels of ketorolac required to inhibit PMN function in most systems were in the range of 0.2 to 1.0 mg/ml, but chemotaxis to fMLP was inhibited by concentrations of ketorolac as low as 1 μg/ml. Ketorolac, currently the only nonsteroidal anti-inflammatory drug available in a parenteral form may have therapeutic usefulness in a variety of conditions thought to be mediated in part by PMN, including sepsis.
Tumor necrosis factor α and interleukin 1 α enhance lipopolysaccharide-mediated bovine endothelial cell injury
Authors: Sharma Smita A , Olchowy Timothy WJ , Yang Zhengang , Breider Mike A
Published: January 2018

Alveolar macrophages (AMs) are important in the host response to aerogenous pulmonary bacterial infections, such as Pasteurella haemolytica–induced pneumonia in cattle. Previous work has shown that AMs enhance P. haemolytica–mediated pulmonary endothelial cell (EC) damage in vitro. The purpose of this study was to determine the mechanism of AM-enhanced EC damage using an in vitro AM-EC coculture system consisting of AMs cultured on culture plate insert membranes and ECs in the underlying chamber. The addition of lipopolysaccharide (LPS) to the culture plate insert chamber resulted in EC damage indicated by 51Cr release, which was enhanced in the presence of AMs. To determine the role of AM-secreted cytokines, recombinant human interleukin 1 α (IL-1) or tumor necrosis factor α (TNF) was added to ECs simultaneously with varying concentrations of LPS. Although TNF and IL-1 alone had only marginal toxic effects on ECs, the simultaneous treatment of TNF or IL-1 with LPS greatly increased the LPS cytotoxic effect on ECs. In addition, IL-1 receptor antagonist eliminated the IL-1 enhancement of LPS-mediated EC toxicity. These results suggest that macrophage-secreted cytokines synergistically enhance LPS-mediated pulmonary EC damage.
Effect of divalent cations on adhesion of polymorphonuclear leukocytes to matrix molecules in vitro
Authors: Lundgren-Åkerlund Evy , Berger Elaine , Arfors Karl-E
Published: January 2018

Adhesion of N-formyl-methionyl-leucyl-phenylalanine-stimulated human polymorphonuclear leukocytes (PMNs) to dishes coated with laminin, fibronectin, or collagen types I and IV was dependent on the presence of magnesium (Mg2+) but not calcium (Ca2+). Addition of manganese (Mn2+) in combination with Ca2+ and Mg2+ further increased the number of PMNs adhering to the matrix proteins. Monoclonal antibody 60.3 (mAb 60.3) was equally effective at inhibiting adhesion of PMNs to all the matrix proteins. The presence of Mn2+ (50 μM), in addition to 1 mM Ca2+ and Mg2+, required higher concentrations of mAb 60.3 to inhibit adhesion of PMNs to collagens type I or IV, suggesting increased affinity of PMNs for these substrates. These findings suggest that the PMNs may regulate the affinity of CD11/CD18 (or multiple ligands by binding different divalent cations to the receptor.
Heterogeneity in the circulating neutrophil pool: studies on subpopulations separated by continuous flow electrophoresis
Authors: Eggleton Paul , Fisher D , Crawford Neville
Published: January 2018

Isolated blood neutrophils from healthy individuals have been separated by continuous flow electrophoresis (CFE) as a Gaussian-shaped profile extending over 12–15 fractions, on the basis of differences in cell surface electrical charge. The fractions were pooled into three or four subpopulations; the mean electrophoretic mobilities of the least and most electronegative cells were 0.96 and 1.22 μm/sec/V/cm, respectively. Each pool of neutrophils was analyzed for functional and biochemical differences. Expression of respiratory burst in terms of the rate of superoxide production by the least and most electronegative cells to fixed concentrations of N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10−7 M) or phorbol myristate acetate (PMA, 1 μg/ml) revealed that the least electronegative cells generated superoxide anion (O2−) at approximately twice the rate of the most electronegative cells. However, when lower concentrations of fMLP were used (1–5 × 10−9 M), the most electronegative cells were most active. The least electronegative cells were also the most active in terms of phagocytosis and chemotaxis. In accordance with these differences in motile function, the basal F-actin content of the least electronegative cell pool was greater than the F-actin levels found in the most electronegative cells and remained so upon stimulation with fMLP. Such neutrophil heterogeneity as detected by CFE may be important in selective marginatum and recruitment of cells to inflammatory foci and sites of infection and may in part represent subsets of cells within the circulation that are primed in vivo to respond to inflammatory stimuli.