bluuebunny/crossref_metadata_2025 huggingface dataset:
rTNFα Facilitates Human Polymorphonuclear Leukocyte Adherence to Fibrinogen Matrices With Mobilization of Specific and Tertiary but Not Azurophilic Granule Markers
Authors: Hanlon W A , Stolk J , Davies P , Humes J L , Mumford R , Bonney R J
Published: January 2018

rTNFα facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen-coated surfaces in a concentration- and time-dependent manner. The adhesion was maximal with 1.0 nM rTNFα within 40–50 min at 37°C. A monoclonal antibody (1B4) directed toward the β2-chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNFα induced adhesion. TNFα caused a time-dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of Aα(1–21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNFα could be further stimulated with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and Aα(1–21) production over time. Thus the rTNFα-facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.
Effect of Chronic Endotoxemia on Concanavalin A-Stimulated Inositol Lipid Metabolism in Rat Splenocytes
Authors: Hagar Arthur F , de Turco Elena B. Rodriguez , Spitzer Judy A
Published: January 2018

The effect of a chronic, noniethal infusion of endotoxin (ET) on the phosphatidylinositol (PI) cycle in rat splenocytes was evaluated. Rats were infused intravenously with sterile saline or Escherichia coli ET (0.1 mg/ 100 g body weight/ 24 hr) for 30 hr via subcutaneously implanted osmotic pumps. The splenocytes were then labeled in vitro for 90 min with [32P]PO4 or for 3 hr with [3H]myoinositol to assess the status of the resynthesis and degradative parts of the PI cycle, respectively. PI cycle activity was depressed in splenocytes of ET-infused rats as evidenced by a 25% reduction in the incorporation of [32P]PO4 into phosphatidic acid (PA), PI, and the polyphosphoinositides and by a similar decrease in the production of [3H]inositol phosphates. Stimulation of splenocytes with concanavalin A (Con A) resulted in dose-dependent increases in the incorporation of [32P]PO4 into PA and PI and in the production of [3H]inositol phosphates, indicating that Con A stimulates the PI cycle in these cells. The Con A-stimulated increase in inositol phosphate production was higher in splenocytes from ET-infused rats. We have previously shown that splenocytes from rats infused for 30 hr with ET exhibit a decreased blastogenic responsiveness to Con A and lipopolysaccharide [Spitzer et al., Proc. Soc. Exp. Biol. Med. 186,27,1987]. The present data do not support the notion that inositol lipid-mediated signaling mechanisms are solely responsible for the expression of the appropriate functional response and suggest that in ET-infused rats there is an uncoupling of the initial response to Con A (i.e., the production of inositol lipid-derived second messengers) and the long-term (i.e., mitogenic) response.
A Method for the Quantitative Analysis and Standardization of Interleukin-1 Bioactivity
Authors: Krakauer Teresa , Krakauer Henry
Published: January 2018

A method is presented for the reproducible quantitation of the biological activity of interleukin 1 (IL-1). This method provides diagnostic tools which give insights into the qualitative aspects of the binding of IL-1 and of the resulting activation of the responder thymocytes; for example, whether the lymphokine and/or the responder population is heterogeneous, or whether a threshold level exists. It establishes under what circumstances the assumptions on which it is based are reasonably adhered to and, consequently, quantitative estimation in the manner it prescribes is justified. It also gives a simple way to calculate both the maximal response attainable for each preparation in an assay and the dilution of a particular preparation that would produce a half-maximal response, the accepted unit of activity of IL-1. This empirical technique provides an improved means of comparing the activities of various preparations of IL-1 in bioassays using various stocks of responder cells and reagents. It should also be applicable to the evaluation of the biological activity of lymphokines in general.
Tumor-Associated Macrophages Share In Vitro Growth Characteristics With Resident but not Elicited Macrophages
Authors: Reed Doug , Burnham Kim
Published: January 2018

Recent studies have shown that tissue macrophages are capable of proliferation and that this capability is enhanced by various cytokines, including macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumor-associated macrophages (TAM) have been demonstrated to proliferate in vitro, but no information is currently available on the ability of M-CSF and GM-CSF to enhance this response. To address this problem, limiting dilution analysis was utilized to examine the proliferative ability of macrophages isolated from two murine tumors of distinct origin following growth in secondary hosts. As a means of comparison, resident peritoneal macrophages (RPM) and thioglycolate-elicited macrophages (TEM) were also analyzed. Results indicate that a rare subset of TAM and RPM is capable of proliferation and that M-CSF and GM-CSF enhance the frequency of TAM and RPM which proliferate, but do not enhance the growth of TEM.
Genetic Resistance/Susceptibility to Mycobacteria: Phenotypic Expression in Bone Marrow Derived Macrophage Lines
Authors: Radzioch D , Hudson T , Boulé M , Barrera L , Urbance J W , Varesio L , Skamene E
Published: January 2018

Congenic strains of mice susceptible (B10A.Begs) or resistant (B10A.Bcgr) to BCG were established. Here we describe the model system which has been established to analyze the functional activities of macrophages in the two strains. We have immortalized bone marrow macrophages from B10A.Bcgs and B10A.Bcgr congenic strains of mice and derived cloned macrophage lines designated B10S and B10R, respectively. B10R and B10S cell lines exhibited surface markers and morphology typical of macrophages. B10S and B10R were similar in their phagocytic activity, in their level of c-fms, in their transforming growth factor β (TGFβ) mRNAs expression, and in their expression of tumoricidal activity in response to interferon-gamma (IFNγ) plus lipopolysaccharides (LPS). However, B10R macrophages expressed a higher level of la mRNA when activated with IFNγ compared with B10S macrophages. Analysis of the bacteriostatic activity of the two cell lines revealed that B10R macrophages were much more active in inhibiting Mycobacterium smegmatis replication than B10S. To measure the intracellular destruction of bacilli, a bactericidal assay based on hybridization with an oligonucleotide probe specific for mycobacterial ribosomal RNA was designed. The results demonstrated that B10R macrophages were endowed with enhanced constitutive bactericidal activity as compared with B10S.

In conclusion we have obtained macrophage lines from bone marrow of B10A.Bcgs and B10A.Bcgr mice that express to a similar extent functional and phenotypic characteristics of macrophages. However, we demonstrate that relative to B10S macrophages, the B10R macrophages have higher expression of la mRNA and that they are constitutively more active in expressing mycobactericidal activity.
Dynamics of Human Neutrophil Aggregation Evaluated by Flow Cytometry
Authors: Rochon Yvan P , Frojmovic Mony M
Published: January 2018

In order to directly monitor neutrophil aggregation, we have developed a simple particle counting technique using flow cytometry. Flow times were used to determine aggregation from changes in the total number of particles per unit volume (%PAT), while fluorescent signals emitted from glutaraldehyde-fixed neutrophils were used to measure changes in the total number of singlet neutrophils (%PAgs). Flow cytometrically-determined %PA values were found to be virtually identical to values determined from microscopy. We show that aggregation parameters can be evaluated and compared simply from measures of changes in total particle count without a need for distinguishing singlets from murtiplets. A number of aggregation parameters are introduced and related to the initial cell concentration (No) and stir speed (shear). Aggregation of neutrophils, following stimulation with 0.5 μM FMLP (N-formyl-methionyl-leucyl-phenylalanine), showed a latent time (ti) of 4 ± 1.5 sec independent of No or stir speed; had a forward rate of aggregation (v,) which was proportional to the initial cell concentration and the stir speed; plateaued for ≥ 60 s; and showed partial to complete reversal by 7.5 min. Above a critical stir speed, the extent and duration of aggregation varied inversely with the stir speed. The stir and No dependence of the aggregation parameters studied suggest the existence of subpopulations of neutrophils with distinct efficiencies of aggregation.
Regulation of Complement 5a Receptor Expression in U937 Cells by Phorbol Ester
Authors: Rubin Janet , Titus Louisa , Nanes Mark S
Published: January 2018

Receptors for the anaphylactic portion of complement, C5a, are not initially expressed in the monoblastic U937 cell line, but appear as the cell is induced to differentiate by the synergistic actions of 1,25(OH)2D and cyclic adenosine monophosphate (cAMP). Phorbol myristate acetate (PMA), which activates the protein kinase C pathway (PKC), does not cause C5a receptor (C5aR) expression when used as a single agent. The induction of C5aR by the synergistic actions of 1,25(OH)2D and cAMP, however, can be augmented as much as 180% by the addition of PMA. C5aR arising in cells exposed to 1,25(OH)2D and 8,4-chlorophenylthio-cAMP have an affinity constant of about 0.4 nM as assessed by cold competition analysis. We show here that when phorbol augmentation of receptor number occurs, the affinity constant is increased by 3.6-fold. In an effort to ascertain whether the change in C5aR Kd involved a PKC-dependent event we examined whether 5–60 min exposure of C5aR-positive cells to PMA would change C5aR Kd. Acutely, PMA caused a downregulation of receptor binding with decreases in apparent receptor number out of proportion to changes in Kd. One hundred nanomolar PMA, which effects nearly complete translocation of PKC to the membrane, consistently caused a 70–90% decrease in C5a surface binding. This downregulation was proportional to PMA dose and exposure time. Micromolar concentrations of the microtubule depolymerizing agents colchicine and vinblastine caused a less drastic downregulation, about 50% of the maximal phorbol effect. Our data suggest that activation of the PKC system might acutely limit the macrophage's ability to respond to C5a; chronically, phorbols upregulate receptor expression, most likely through positive effects on C5aR gene expression.
Inhibition of neutrophil chemotaxis and chemokinesis associated with a plasma protein in aging rats: selective depression of cell responses mediated by complement-derived chemoattractants
Authors: Mello S B V , Farsky S H P , Sannomiya P , Garcia-Leme J
Published: January 2018

The influence of aging on neutrophil chemotaxis, chemokinesis, and superoxide production was investigated in rats. Animals of two age groups, 3 to 4 months and 20 to 21 months, were used. Equivalent neutrophil chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP), leukotriene B4 (LTB4), and bacterial lipopolysaccharide (LPS)-activated plasma were observed in both groups of animals, with cells suspended in Hanks' balanced salt solution (HBSS). However, cross-incubation studies in which cells from young adult rats were exposed to plasma from aged donors, then resuspended in HBSS for testing, showed marked changes in the ability of the cells to respond to the chemoattractants. The response to LPS-activated plasma was reduced, whereas responses to fMLP and LTB4 remained unaltered. Previous incubation of the cells with homologous plasma from young donors produced no effect. The inhibitory activity developing with advancing age affected not only chemotaxis but also random movement stimulated by LPS-activated plasma. The inhibitory activity of chemotaxis and chemokinesis in plasma of aged animals was heat labile (56°), vanished in the presence of a proteolytic enzyme like trypsin, and was maintained after dialysis with 12,000-Mr retention dialysis tubing. The material did not influence superoxide production by stimulated neutrophils. It is suggested that inhibition of neutrophil locomotion with advancing age is associated with a plasma protein capable of interacting with neutrophil receptors for complement-derived chemoattractants. The inhibitory substance might influence neutrophil responses to infection and inflammation in the elderly.
Macrophage and lymphocyte functions are down-regulated in newborn rabbits
Authors: Tomai Mark A , Fitzgerald Thomas J , Froberg M Kent
Published: January 2018

We have developed an animal model for congenital syphilis. Treponema pallidum is injected intravenously into pregnant rabbits and fetuses are infected in utero. As a prelude to characterizing the immunologic consequences of fetal infection, it was necessary to expand on the baseline information about newborn rabbit immune capabilities. Studies were undertaken to determine splenic macrophage and T lymphocyte functions with emphasis on newer immunologic parameters. Newborns aged 2 weeks were compared to adults. Macrophage capabilities in newborn rabbits differed from those of their adult counterparts. These cells produced similar basal levels of interleukin 1 (IL-1) but failed to respond to the IL-1 stimulants of lipopolysaccharide (LPS) or T. pallidum. Macrophages also exhibited diminished levels of la expression and increased levels of prostaglandin E2 (PGE2) secretion. T lymphocyte functions were altered in newborn spleen preparations. Following concanavalin A (Con A) stimulation, interferon γ production was half that of adults; in direct contrast, IL-2 production was twice that of adults. Con A–induced lymphocyte proliferation was markedly decreased in newborn preparations. This diminished response resulted from down-regulation rather than immaturity. When newborn splenic cells were stimulated with Con A in the presence of indomethacin, anti-transforming growth factor (anti-TGF), or exogenous IL-1/IL-2, better proliferation resulted. PGE2, which is well established as a down-regulator of newborn immune functions in human and mouse systems, also appears to play a role in suppressing newborn rabbit functions. TGF is a potent suppressor of a number of adult immunologic reactions. This is the first documentation of the potential role of this factor in down-regulating newborn immune capabilities. These findings provide a framework for future investigations of our congenital syphilis model.
Carcinoembryonic antigen binding proteins on elicited peritoneal macrophages
Authors: Ann Toth Carol
Published: January 2018

Carcinoembryonic antigen (CEA), a member of the immunoglobulin supergene family, is recognized by a specific 80-kd receptor on Kupffer cells and alveolar macrophages. Peritoneal macrophages were harvested from male Sprague-Dawley rats 72 h after intraperitoneal injection with thioglycolate broth and from unstimulated animals. The elicited peritoneal macrophages had a much greater capacity to endocytosed CEA than the cells isolated from unstimulated animals. They took up CEA in a concentration- and time-dependent manner. Cellular uptake was temperature and colchicine sensitive indicating internalization of CEA. The process was saturable and could be specifically inhibited by CEA and nonspecific crossreacting antigen (NCA), a member of the CEA supergene family. Two binding proteins of ~ 35 and 80 kd were isolated from the surface of the peritoneal macrophages by affinity chromatography on a CEA-Sepharose column. Using CEA coupled to sulfo-succininidyl-2-[p-azidosalicyl amide]-1,3′-dithiopropionate (SASD), a photoactivatable crosslinker, two binding proteins of 55 and 80 kd were identified on the surface of live peritoneal macrophages. The specificity of CEA endocytosis and binding proteins exhibited by the elicited peritoneal cells are similar to that previously reported for Kupffer cells and alveolar macrophages.
Monoclonal antibody NIMP-R10 directed against the CD11b chain of the type 3 complement receptor can substitute for monoclonal antibody 5C6 to exacerbate listeriosis by preventing the focusing of myelomonocytic cells at infectious foci in the liver
Authors: Conlan J Wayne , North Robert J
Published: January 2018

Treatment of mice with a monoclonal antibody (mAb) designated NIMP-R10, directed against the CD11b polypeptide of the CD18/CD11b heterodimeric type 3 complement receptor (CR3), exacerbates listeriosis by preventing myelomonocytic cells from focusing at sites of infected hepatocytes in the liver. Under these conditions an otherwise sublethal Listeria inoculum grows unrestrictedly within hepatocytes and causes death in 3 days. The results obtained with NIMP-R10 are similar to those previously obtained with a different anti-CD11b mAb (5C6), although mAb NIMP-R10 is more effective at enhancing infection. Therefore, both mAbs can be used to analyze host antibacterial defenses in vivo.
Postreceptor events associated with human neutrophil activation by interleukin-8
Authors: Smith Robert J , Sam Laurel M , Leach Karen L , Justen James M
Published: January 2018

Recombinant human monocyte–derived interleukin-8 (IL-8M), recombinant human endotheliumderived IL-8 (IL-8E), and a recombinant human truncated form of IL-8 (IL-8T) stimulated a time-dependent (t1/2 ~ 2–3 s) and concentration-dependent (0.1–100 nM) release of azurophil (myeloperoxidase) and specific (vitamin B12 binding protein, gelatinase) granule constituents from cytochalasin B–treated human neutrophils (HNs) wherein IL-8T = IL-8M > IL-8E. An increase in the cytosolic free calcium concentration ([Ca2+]i) was greater in IL-8T– than in IL-8M– or IL-8E–activated HNs, and IL-8T was more potent than either IL-8M or IL-8E in sequentially desensitizing the HNs to the effects of the other IL-8 forms. IL-8 induced a time- and concentration-dependent (0.1–100 nM) increase in the production of inositol 1,4,5-trisphosphate (IP3) in HNs. U-73122 (1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a potent inhibitor of phospholipase C–catalyzed events in HNs, suppressed IL-8–triggered IP3 production, increased [Ca2+]i and granule exocytosis in HNs. The membrane-associated activity of the α and β subtypes of protein kinase C was significantly enhanced in IL-8–activated cells.
Wheat germ agglutinin inhibits the C5a receptor interaction: implications for receptor microheterogeneity and ligand binding site
Authors: Johnson Richard J , Simpson Stacy , van Epps Dennis E , Chenoweth Dennis E
Published: January 2018

Wheat germ agglutinin (WGA) has been shown to inhibit the interaction of C5a with the C5a receptor on both polymorphonuclear neutrophils (PMNs) and the histiocytic cell line U937. The level of inhibition with isolated receptor preparations is 100%, and on intact cells 10 to 20% of the receptor population appear to retain their ability to bind C5a in the presence of WGA. In contrast, this lectin completely inhibits the C5a-mediated degranulation of PMN primary and secondary granules, suggesting that the population of C5a receptors responsible for mediating degranulation is also recognized by WGA. More than 50% of the receptors appear to be blocked before an effect on degranulation occurs. This inhibition by WGA does not appear to be due to down-regulation of C5a receptors from the cell surface, excessive aggregation of receptor sites, or interaction of WGA with the carbohydrate portion of the C5a molecule. The inhibition is reversed by N-acetylglucosamine but not by sialic acid. This effect appears to be specific for WGA because various other lectins do not inhibit the C5a receptor interaction. That the inhibition by WGA is due to direct binding of the lectin to N-acetylglucosamine residues on the C5a receptor is strongly supported by the ability of the cross-linked C5a-receptor complex to bind to and be specifically eluted from a WGA-Affigel affinity matrix. These observations are consistent with hypothesis that the population of C5a receptors on leukocytes exhibits microheterogeneity with respect to structure (carbohydrate content) and/or function. J. Leukoc. Biol. 52: 3–10; 1992.
Direct interaction between an antigen-specific B cell clone and an MHC class II–reactive helper T cell clone
Authors: Hamano Teruaki , Asano Yoshihiro , Iwasaki Tsuyoshi , Yamasaki Tadakuni , Hase Kazunobu , Kakishita Eizo
Published: January 2018

TP67.14 established by somatic hybridization is a 2,4,6-trinitrobenzenesulfonic acid (trinitrophenyl, TNP)-specific B cell clone with a receptor molecule for TNP on the cell membrane, and MS202 is an interleukin-2 (IL-2)–dependent T helper (Th) cell clone reactive to auto-MHC class II antigens (IAk and IEk) as previously reported. In the present study it was shown that MS202 considerably induced the maturation of TP67.14 into anti-TNP plaque-forming cells (PFCs), and this response was markedly augmented by the addition of TNP–keyhole limpet hemocyanin (KLH). Recombinant cytokines and the culture supernatant of MS202 with TP67.14 did not affect the generation of anti-TNP antibodies by TP67.14. Also, neither anti–IL-4 nor anti–IL-5 monoclonal antibody (mAb) inhibited the maturation of TP67.14 mediated by MS202. The differentiative effect of MS202 on TP67.14 was completely lost when each cell was separately cultured using a semipermeable membrane. Monoclonal antibodies against LFA-1β molecules significantly blocked the development of anti-TNP PFCs induced by MS202, as well as anti-IAk and anti-IEk ImAbs. Interestingly, the plasma membrane–enriched fraction (PM) derived from MS202 exhibited much more differentiative effects on TP67.14 treated with TNP-KLH than PM from other T cell lines and concanavalin A–induced T lymphoblasts. In addition, TNP-conjugated PM from MS202 by itself induced a great number of anti–TNP PFCs. The present findings indicate that MS202 is capable of inducing the maturation of TP67.14, which is considered to represent a population of B cells with antigen specificity in a late lineage of B cell maturation, through direct cell contact but not soluble factors. This suggests that B cells with antigen specificity, in the presence of antigen, can be induced to mature into antibody-secreting cells through direct contact with Th cells; in this process surface major histocompatibility complex class II and lymphocyte function-associated antigen 1 (LFA-1) molecules are directly involved and the cell membrane derived from Th cells provides a transductional signal for maturation of B cells with antigen specificity in the presence of antigen.
Differences in the onset of the inflammatory response to cutaneous leishmaniasis in resistant and susceptible mice
Authors: Beil Waltraud J , Meinardus-Hager Georg , Neugebauer Dorothea-Ch , Sorg Clemens
Published: January 2018

Sites of cutaneous infection with Leishmania major in genetically susceptible (BALB/c) and resistant (C57B1/6) mice were investigated for the early inflammatory response (6 h to 12 days) by electron microscopy combined with enzyme-histochemical methods. Susceptible BALB/c mice spontaneously recruited only polymorphonuclear leukocytes (PMNs) at the site of infection. Infiltrating mononuclear phagocytes (and eosinophils) were first observed at day 1 in a ratio equal to the influx of PMNs (about 40%). This pattern persisted during the following 11 days of infection. In the resistant C57/B16 mice, the first cellular infiltrate at the infected site contained mononuclear phagocytes (25%) and eosinophils (15%) besides PMNs (60%). Within 3 days after infection, mononuclear phagocytes became the dominant population of cells in cutaneous lesions (up to 80%). It was found in situ that L. major accumulated and replicated in immature macrophages, that is, intermediate stages between monocytes and resident macrophages, which were found in lesions of both strains. The burden of parasites was, however, degraded more rapidly by the infiltrating cells of the resistant mice than by those of the susceptible ones. Within the first 4 days of infection, the parasites were found in PMNs, mononuclear phagocytes, and extracellular spaces in both strains. In susceptible mice this distribution pattern persisted up to 12 days after infection; in resistant C57B1/6 mice parasites accumulated inside mononuclear phagocytes within this period. It is concluded that the features of acute inflammation during leishmaniasis in BALB/c mice are sustained over a prolonged period that is ineffective in the elimination of L. major.
Macrophage antimicrobial functions in a chicken MHC chromosome dosage model
Authors: Lin H-K , Bloom SE , Dietert RR
Published: January 2018

Macrophages respond to certain inflammatory signals with a marked increase in respiratory burst and the production of reactive oxygen intermediates; these metabolites play an essential role in the destruction of invading microorganisms. In this study, macrophage antibacterial inflammatory responses were compared among chickens having two (disomic), three (trisomic), or four (tetrasomic) copies of the major histocompatibility complex (MHC)–encoding microchromosome (B15 haplotype). Phorbol myristate acetate (PMA)–stimulated superoxide anion (O2-) production by cross-linked dextran (Sephadex)–elicited peritoneal macrophages was measured at early (4 h), intermediate (24 h), and late (42 h) stages of the inflammatory response using ferrocytochrome c reduction. Significantly elevated O2- production was observed for trisomic versus disomic macrophages during both early and intermediate stages of the inflammatory response. Late in the response, tetrasomic macrophages produced a significantly higher level of O2- than disomic cells. When PMA was used to trigger hydrogen peroxide (H2O2) production, no significant genotype difference was found for any stage of the inflammatory response. Phagocytosis of heat-killed Salmonella enteritidis by macrophages differed among the three genotypes: trisomic macrophages were superior to disomic cells during early inflammation, no genotypic difference was observed at the intermediate stage, and disomic cells had greater phagocytic capacity than aneuploid macrophages late in the response. Likewise, when S. enteritidis was cultured with macrophages to induce oxygen intermediate secretion, H2O2 production followed a kinetic pattern among the genotypes similar to that observed for bacterial phagocytosis. Endogenous superoxide dismutase (SOD), catalase, and glutathione peroxidase (GP) activities were determined for the macrophages during intermediate and late inflammatory stages. Tetrasomic macrophages had reduced SOD activity at the late stage, no significant difference was observed in catalase activity among genotypes at either time point, and trisomic macrophages had enhanced GP activity compared to disomic cells at both time points. These results indicate that differences in MHC gene dosage are associated with differences in chicken macrophage activation for the acquisition of selected antibacterial functions.
Substrate-specific proteases (BLT-esterase) are localized predominantly in the natural killer cells of unprimed mice
Authors: Savers Thomas J , Mason Llewellyn H , Pilaro Anne , Wiltrout Theresa A , Komschlies Kristin , Munger William L , Wiltrou Robert H
Published: January 2018

In leukocytes isolated from unprimed mice, the levels of extractable Nα-Cbz-Lys-thiobenzylester- esterase (BLT-esterase) closely correlated with the number of natural killer (NK) cells. The spleens of mice that exhibit severe combined immunodeficiency (SCID) contained much higher levels of this enzyme than other mouse strains. Treatments that resulted in a local accumulation of NK cells (as assessed by lytic activity) produced a concomitant increase in BLT-esterase activity. However, short-term in vitro treatment of spleen cells with interferon (IFN)-α/β indicated that BLT-esterase levels correlated more closely with absolute numbers of NK cells than with their lytic capacity. There was a very good correlation between the numbers of cells bearing the NK phenotype (NK-1. 1+) and BLT-esterase levels. Cells positively sorted using the NK-specific antibodies NK-1. 1 and LGL-1 had high enzymatic activity. The BLT- esterase levels were high in both the NK-1. 1+LGL-1” and NK-l. r/LGL-r subsets. Highly purified CD4+ and CD8+ T cells and slg+ B cells demonstrated negligible enzyme, as did populations of cells highly enriched for macrophages or neutrophils. However, it should be stressed that the inbred mice used on this study have been maintained in a pathogen-free facility. It would be anticipated that mice maintained under less stringent conditions could exhibit appreciable levels of BLT-esterase activity in their T cells. Nonetheless, BLT-esterase is present at high levels in NK cells and cannot be regarded as a T cell-specific enzyme. J. Leukoc. Biol. 53: 454–461; 1993.
Endocytosis of ß2 integrins by stimulated human neutrophils analyzed by flow cytometry
Authors: Chambers J David , Simon Scott I , Berger Elaine M , Sklar Larry A , Arfors Karl-E
Published: January 2018

Flow cytometry and fluorescently labeled monoclonal antibodies were used to investigate endocytosis of human neutrophil ß2 integrins following cellular activation. GDI 8 initially present on the cell surface cycled in two phases after exposure to formyl peptide or plateletactivating factor. The first phase lasted 3 min at 37°C; after a lag, CD18 was specifically internalized at approximately 20%/min. Subsequently a second phase was detectable consisting of exponential reduction of internal fluorescence with a half-time of approximately 2 min, representing probe reexpression. At peak endocytosis approximately 40% of CD18 was internalized. All of the internalized CD18 was associated with αM (CR3); no endocytosis of αL (LFA-1) was observed. When neutrophils were stimulated with phorbol esters or calcium iono- phore, CD18 was internalized much more slowly (t1/2 = 5 min) and probe was not reexpressed. Endocytosis of CD18 may participate in regulating neutrophil adhesiveness, removing activated receptors, or permitting receptor recycling. J. Leukoc. Biol. 53: 462–469; 1993.
In vivo immortalization of murine peritoneal macrophages: a new rapid and efficient method for obtaining macrophage cell lines
Authors: Adami Cecilia , Brunda Michael J , Palleroni Alicia V
Published: January 2018

Although several murine macrophage (mφ) cell lines from different sites have previously been obtained by in vitro infection with the J2 murine retrovirus, which carries the v-raf and v-myc oncogenes, it was not possible to immortalize thioglycolate-elicited peritoneal macrophages (Pmφs) by this in vitro procedure. A technique utilizing in vivo injection of the J2 virus has been developed to overcome this problem. The J2 virus immortalized Pm φs in a very efficient manner in vivo because no exogenous growth factors were required for the in vitro proliferation of these cells and numerous continuous cloned cell lines were readily established. In contrast, Pmφs obtained from uninfected mice or Pmφs infected in vitro with the J2 virus did not proliferate. The in vivo immortalized cells had many of the morphological and functional characteristics of mφs. Analysis of two of the clones, PMJ2-PC and PMJ2-R, demonstrated intracellular expression of the product of the v-raf gene, presence of mφ-associated cell surface antigens, interleukin-6 secretion induced by lipopolysaccharide, and biological response modifier-induced cytotoxic activity against tumor cells. In addition, one of the clones, PMJ2-PC, constitutively expressed major histocompatibility complex (MHC) class II antigens, and in the other clone, PMJ2-R, MHC class II antigen expression was induced by recombinant murine interferon-γ. This method of utilizing the J2 virus in vivo represents a novel technique for obtaining hematopoietic cell lines from cells that are difficult to immortalize in vitro. J. Leukoc. Biol. 53: 475–478; 1993.
Monoclonal antibodies to the carbohydrate structure Lewisx stimulate the adhesive activity of leukocyte integrin CD11b/CD18 (CR3, Mac-1, αmβ2) on human granulocytes
Authors: Stöckl Johannes , Magdic Otto , Rosenkranz Alexander , Fiebiger Edda , Kniep J Bernhard , Stockinged Hannes , Knapp Walter
Published: January 2018

Several carbohydrate structures on human granulocytes have been discussed as potential ligands for C- type lectins (selectins) on endothelial cells. Among them are the lacto-series type II chain antigens sialyl-Lewisx (SLex), Lewisx (Lex), and VIM2. We demonstrated in this study that monoclonal antibodies (mAbs) to Lex and to SLex, but not other anticarbohydrate mAbs (VIM2, CDwl7, CD24), can stimulate granulocytes to form homotypic aggregates. This effect was particularly noticeable with three distinct anti-Lex mAbs (3C6, 4D1, 6C7). Much less impressive effects were also seen with nine other anti-Lex mAbs and with the anti-SLex mAb CSLEX1. Aggregation was shown to be an active process. It is temperature and energy dependent, requires divalent cations, and is selective in terms of mAb specificity. Anti- Lex-induced homoaggregate formation could be inhibited with CDllb mAb JML-H11 and CD54 (ICAM1) mAb LB-2 and thus seems to be associated with activation of the β2-integrin cytoadhesion pathway. J. Leukoc. Biol. 53: 541–549; 1993.
Mitogenic signal transduction in T lymphocytes in microgravity
Authors: Cogoli Augusto , Bechler Birgitt , Cogoli-Greuter Marianne , Criswell Sue B , Joller Helen , Joller Peter , Hunzinger Elisabeth , Muller Otfried
Published: January 2018

The activation by concanavalin A Con A of human peripheral blood lymphocytes (PBLs) in the presence of monocytes as accessory cells was investigated in cultures exposed to microgravity conditions in Spacelab. Activation of T cells was measured as incorporation of [3H] thymidine into DNA, secretion of interleukin-2 (IL-2), and interferon-7, and expression of IL-2 receptors. Whereas, as discovered in earlier experiments, the activation of resuspended T cells is strongly inhibited, activation of cells attached to microcarrier beads is more than doubled in microgravity. The results suggest that the depression of the activation in resuspended cells may be attributed to a malfunction of monocytes acting as accessory cells. In fact, although the ultrastructure of resuspended monocytes is not altered in microgravity, the secretion of IL-1 is strongly inhibited. Our data suggest that (1) IL-2 is produced independently of IL-1, (2) IL-1 production is triggered only when monocytes (and lymphocytes?) adhere to microcarriers, (3) the expression of IL-2 receptors depends on IL-1, and (4) provided sufficient IL-1 is available, activation is enhanced in microgravity. Finally, cultures of resuspended PBLs and monocytes in microgravity constitute a complete and natural system in which monocytes are not operational. This may be useful for studies of the role of accessory cells and cell-cell interactions in T lymphocyte activation. J. Leukoc. Biol. 53: 569–575; 1993.
Granulocyte activation induced by intense interval running
Authors: Gray AB , Telford RD , Collins M , Baker MS , Weidemann MJ
Published: January 2018

Activation of granulocytes has been associated with normal immune function, inflammation, and exercise-induced muscle damage. The effect of intense interval running on granulocyte activation was examined by use of flow cytometry, monoclonal antibodies, and spectrophotometric techniques. Eight trained males [maximal oxygen uptake Vo2max, mean (SD) = 64.4 (3.6) ml/kg/min; age 30.1 (4.8) years] undertook an intense interval exercise (treadmill running) protocol to exhaustion. Subjects completed an average of 16.5 one- minute runs. Granulocyte expression of CR3 (GDI lb), receptor for complement component C3bi (6 and 24 h post-test), and FcγRIII (GD16) (24 h post-test) and the plasma concentration of elastase-inhibitor complex (1 h post-test) increased significantly (all P < .05). Subjects (8 of 8) exhibited a post-test decrease at either 1 or 6 h (P < .01) and a 24-h post-test significant increase (7 of 8; P < .05) in granulocyte 90° light scattering (LS). Plasma lactoferrin (Lf) concentration, although increased by 17% at 6 h post-test, was not significantly different from resting values at any sampling point. Changes in plasma Lf and median channel 90° LS were significantly correlated (r = - .43, P = .04), raising the possibility of monitoring exercise-induced granulocyte activation (degranulation) by flow cytometry. Intense interval exercise appears to induce granulocyte activation, as manifested by release of granule proteins and changes in 90° LS and expression of both Fc and complement receptors. J. Leukoc. Biol. 53: 591–597; 1993.
Characterization of a 64-kd protein phosphorylated during chemotactic activation with IL-8 and fMLP of human polymorphonuclear leukocytes. II. Purification and amino acid analysis of phosphorylated 64-kd protein
Authors: Shibata Michio , Yamakawa Yoshio , Ohoka Tadakazu , Mizuno Satoshi , Suzuki Kazuo
Published: January 2018

Lung giant cell carcinoma-derived chemotactic protein (LUCT)/IL-8 and fMet-Leu-Phe stimulate phosphorylation of a 64-kd protein (p64) in 32P-labeled human polymorphonuclear leukocytes (PMNs). The p64 was purified from cytosol of human PMNs (1.8 × 109 cells) by DEAE-Sepharose CL6B column chromatography, hydroxyapatite HPLC, and reverse-phase HPLC. By hydroxyapatite HPLC, p64s were separated and produced two peaks containing both nonphosphorylated and phosphorylated p64. Amino acid composition of each p64 was determined. Each p64 was directly subjected to amino acid sequencing, but the amino acid residue in the amino-terminal of the proteins could not be detected. From the results of amino acid composition and other characters of p64, it is suggested that p64 is identical to I-plastin, which is a leukocyte-specific protein.
Taurine chloramine inhibits the synthesis of nitric oxide and the release of tumor necrosis factor in activated RAW 264.7 cells
Authors: Park Eunkyue , Quinn Michael R , Wright Charles E , Schuller-Levis Georgia
Published: January 2018

Taurine is present in high concentrations in most mammalian tissues, including those that prodigiously produce oxidants. Taurine protects against bronchiolar damage induced by NO2, ozone, bleomycin, and amio- darone. Taurine is chlorinated to form taurine chloramine (Tau-Cl) by the halide-dependent myeloperoxidase system and, under physiological conditions, reduces HOC1 toxicity. Although NO and its metabolites, ΝO2− and NOs−, are thought to be major mediators of tissue damage resulting from oxidant exposure, cytokines, including tumor necrosis factor (TNF), are also involved. We examined the effects of Tau-Cl on NO production and TNF release by using RAW 264.7 cells activated with recombinant interferon-γ (rIFN-γ; 50 U/ml) and lipopoly- saccharide (LPS; 10 μg/ml). NO was measured spec- trophotometrically as ΝO2− after reaction with Griess reagent and TNF was measured by FLISA. Tau-Cl (0.5 mM) inhibits NO and TNF released into the medium by 47% and 43%, respectively. Tau-Cl is actively transported into RAW 264.7 cells by an uptake system that is energy, temperature, and Na+ dependent. Competition experiments demonstrate that the uptake system for Tau- Cl is distinct from that for taurine. In addition, the NO synthase activity of cytosolic preparations from activated RAW 264.7 cells is irreversibly inhibited by pretreatment with Tau-Cl. We demonstrate that Tau-Cl inhibits production of NO and TNF by activated macrophages and suggest a mechanism through which taurine supplementation may protect against oxidant-induced tissue damage.
Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators
Authors: Lee Alison , Whyte Moira K B , Haslett Christopher
Published: January 2018

Neutrophil apoptosis leads to macrophage ingestion of intact senescent neutrophils. This may represent a neutrophil removal mechanism that is important both in the control of inflammatory tissue injury and for the normal resolution processes of inflammation. Because apoptosis is likely to be a key control process in cell and tissue homeostasis, a number of inflammatory mediators were tested for their ability to modulate the rate of apoptosis in populations of neutrophils aging in culture. Endotoxic lipopolysaccharide, human recombinant complement factor 5a, and human recombinant granulocyte- macrophage colony-stimulating factor all markedly inhibited the rate of neutrophil apoptosis in a concentration-dependent fashion, without inducing necrosis (as assessed by trypan blue exclusion). This inhibitory effect on the rate of neutrophil apoptosis was shown by morphological criteria and confirmed by gel electrophoresis of extracted DNA. Inhibition of apoptosis of aging neutrophil populations was associated with prolongation of the functional life span of the population as assessed by the ability of neutrophils to spread on glass surfaces, to polarize in response to deliberate stimulation with N-formyl-Met-Leu-Phe (fMLP), and to release the granule enzyme marker myeloperoxidase on fMLP stimulation. These observations show that inflammatory mediators prolong the functional life span of neutrophils through modulation of apoptosis. Further elucidation of these mechanisms will lead to a better understanding of the processes controlling neutrophil residence and function in inflamed tissues and may provide further insights into the molecular mechanisms of apoptosis, which is of widespread importance in tissue biology.
The in vivo effects of rhIL-lα therapy on human monocyte activity
Authors: Lee Anna M , Vadhan-Raj Saroj , Hamilton Raymond F , Scheule Ronald K , Holian Andrij
Published: January 2018

Pleiotropic cytokines such as interleukin-lα (IL-lα) have multiple effects on peripheral blood monocytes (PBMs). This study examined the ability of in vivo recombinant human IL-lα (rhIL-lα) therapy to enhance clinically important monocyte functions in ovarian cancer patients prior to chemotherapy. After 4 days of continuous infusion, in vivo rhIL-lα therapy amplified both the number and activity of PBMs. Therapy with rhIL-lα increased the number of PBMs sixfold. These monocytes had a significantly increased ability to produce superoxide anion in response to phorbol 12,13- dibutyrate stimulation. Their ability to secrete spontaneously the immunomodulatory cytokines IL-lα and IL-1β was significantly increased, but their ability to secrete tumor necrosis factor a (TNF-α) was not significantly elevated. These effects of rhIL-lα infusion on cytokine secretion by PBMs appear to be related to rhlL-lα-in- duced increases in the mRNA levels for these cytokines. In contrast, rhIL-lα therapy did not significantly alter PBM response to lipopolysaccharide (10 μg/ml). In summary, infused rhIL-lα, in addition to its use as a myelo- protective agent, has enhancing effects on the number and activity of PBMs. The effects of rhIL-lα infusion on PBM function demonstrated here should at least transiently increase the ability of monocytes to combat infection and enhance host immune response.
Monocyte adherence to human vascular endothelium
Authors: Beekhuizen Henry , van Furth Ralph
Published: January 2018

An acute inflammatory response requires that circulating monocytes bind to and migrate across the endothelium of the vessel wall to gain access to inflamed sites. Various mediators of inflammation - cytokines and chemoattractants - have been shown to initiate and regulate the margination and extravasation of monocytes. This review summarizes evidence that the mechanism underlying the initial adhesion of monocytes to normal and cytokine-stimulated endothelial cells and their subsequent transendothelial migration are successive events in monocyte-endothelial cell interaction. Special emphasis is given to the current knowledge of the contribution of adhesion molecules belonging to the family of 13i- and 132-integrins, the immunoglobulin supergene family, and the selectins to such cellular interaction. The sequence of events that allow monocytes to attach to, migrate over and finally pass the endothelium is discussed in detail.
NADPH-oxidase activity: the probable source of reactive oxygen intermediate generation in hemocytes of the gastropod Lymnaea stagnalis
Authors: Adema C M , van Deutekom-Mulder E C , van der Knaap W P W , Sminia T
Published: January 2018

Macrophage-like defense cells (hemocytes) of the pond snail Lymnaea stagnalis generate reactive oxygen intermediates (ROIs) upon contact with non-self, following kinetics similar to those of ROI production by mammalian leukocytes during respiratory burst. In this study, several inhibitors of NADPH-oxidase, the key enzyme of the respiratory burst in mammalian phagocytes, were tested for their effect on oxidative activities [as demonstrated by nitroblue tetrazolium (NBT) reduction and luminol-dependent chemiluminescence (LDCL)] of phagocytosing snail hemocytes. In the presence of di- phenylenc iodonium, zymosan-stimulated hemocytes of L. stagnalis failed to reduce NBT and showed a markedly reduced LDCL response. Also, compounds that prevent assembly of functional NADPH-oxidase complexes in activated mammalian cells were effective; preincubation of hemocytes with 1,4-naphthoquinone inhibited the LDCL response and NBT reduction upon phagocytic stimulation. Furthermore, coincubation but not preincubation with five different catechol-like phenols inhibited oxidative activities of zymosan-stimulated hemocytes. These results imply similarities in composition and regulation of the ROI-generating mechanisms of both mammalian and snail defense cells. It is postulated that in L. stagnalishemocytes, (1) NADPH-oxidase activity is responsible for ROI production, (2) an active NADPH-oxidase enzyme complex has to be assembled from putative cytosolic and membrane-associated components, and (3) continuous replacement of active NADPH-oxidase enzyme complexes is necessary to sustain respiratory burst-like oxidative activities during interactions with non-self.