Monocyte Suspensions Research Articles

Regulation of monocyte migration by amphoterin (HMGB1)

AbstractAmphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-γ (IFN-γ)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5′-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium. (Blood. 2004;104:1174-1182)

Initial interaction of citrate-coated iron oxide nanoparticles with the glycocalyx of THP-1 monocytes assessed by real-time magnetic particle spectroscopy and electron microscopy

Interaction with biological material can alter physicochemical parameters of magnetic nanoparticles and might thereby change their magnetic behavior with potentially important implications for various nanoparticle applications. Little is known about changes of the magnetic behavior that occur during the initial phase of cell binding and uptake. We investigate the magnetic behavior of very small superparamagnetic iron-oxide nanoparticles (VSOP) during initial contact with THP-1 monocytes. We combine real-time magnetic particle spectroscopy (MPS), a fast and sensitive method for specific detection of magnetic nanoparticles in biological specimen with high-pressure-freezing/freeze-substitution transmission electron microscopy (HPF/FS-TEM), enabling us to generate snapshots of the interaction of VSOP with the cellular glycocalyx. MPS reveals significant changes of the dynamic magnetic behavior within seconds after VSOP injection into monocyte suspensions that correlate with the formation of nanoparticle clusters in the glycocalyx. The combination of real-time MPS and HPF/FS-TEM provides an ideal platform to analyze magnetic behaviors of nanoparticles upon interaction with cells and tissues.

P61: DNA-vaccines against melanoma: Design and investigation of antigenic properties

Melanoma is the most aggressive and dangerous skin cancer. In 2013 about 23.8% of patients developed III–IV stages of a disease in Russia ( http://clinical-pharmacy.ru ). The most common approaches to control melanoma are surgical resection, chemotherapy, radiotherapy, and immunotherapy (Wildemore et al., 2001). In the case of immunotherapy interferon preparations are the most commonly used, however, it is impossible to increase overall survival rate. Vaccine therapy is more promising method including engineered DNA-vaccines. The goal of this work is to design and study candidate DNA-vaccines against melanoma pMEL-TCI-A0201 and pMEL-TCI in vitro and ex vivo. Using original software we designed two T-cell immunogens MEL-TCI-A0201 and MEL-TCI which are candidate DNA-vaccines against melanoma. The first allele-specific immunogen MEL-TCI-A0201 comprises CTL-epitopes restricted by only one allelic variant HLA-A∗0201 that is prevailing in global population. The second universal immunogen MEL-TCI comprises CTL-epitopes restricted by 12 major allelic variants of HLA class I molecules. Furthermore, DNA-construct pcDNA-mart1 carrying full-length variant of antigen MART-1 was designed. This construct was used as a positive control. Genes encoding target immunogens have been synthesized and cloned into vector plasmid DNA3.1 (Antonets et al., 2010, Bazhan et al., 2010). Evidence of the target genes expression in vitro after transfection of 293T-cells with obtained DNA-vaccine constructs p MEL-TCI and pMEL-TCI-A0201 were evaluated using three methods: reverse transcription-PCR to determine specific polyepitope immunogen mRNA; western immunoblotting assay with 29F2 monoclonal antibodies to “marker” epitope at the C-terminus of the polyepitope constructs; flow cytometry assay of stained 293T cells transfected with obtained plasmids using FITC-labeled 29F2 monoclonal antibodies. It was shown that all obtained plasmids were provided expression of target genes in eukaryotic cells. Plasmids pMEL-TCI and pMEL-TCI-A0201 were used for transfection of human monocyte derived dendritic cells to estimate capacity of polyepitope constructs to induce specific T-cell immune response by IFN γ ELISpot analysis ex vivo. The study was carried out in collaboration with researchers from laboratory of molecular immunology at Scientific Research Institute of Clinical immunology, Siberian Branch, Russian Academy of Medical Sciences. License for conducting the study was approved by the local ethic committee at this institute. Cell culture of human melanoma Mel Is (HLA-A∗0201+, MART-1+) was kindly provided by N. N. Blokhin Russian Cancer Research Center (RAMS) and was used as target cells. Monocyte suspension and suspension of co-incubated monocytes with dendritic cells transfected with vector plasmid DNA3.1 were used as negative controls. Co-incubated monocytes with dendritic cells transfected with plasmid pcDNA-mart1, encoding full-length melanoma antigen MART-1, were used as positive control. It was shown that in the presence of melanoma cell lysates co-incubated with monocytes and dendritic cells transfected with plasmids pMEL-TCI-A0201 and pcDNA-mart1 (positive control) reliably more IFN γ -producing cells are detected compared with negative control samples in the presence of lysate of melanoma cells. Thus, it was shown that obtained plasmids pMEL-TCI-A0201, pMEL-TCI, and pcDNA-mart1 are able to provide expression of target proteins in eukaryotic cells. Moreover using IFN γ ELISpot assay we demonstrated that DNA vaccine pMEL-TCI-A0201 induced T-cell immune response against human melanoma antigens and its efficiency is equal to full-length melanoma antigen MART-1. This study was supported by Federal Program “Research and development on priority directions of scientific-technological complex of Russia for 2007–2012”, State contrast No. 16.512.11.2186.

Dopamine increases CD14+CD16+ monocyte migration and adhesion in the context of substance abuse and HIV neuropathogenesis.

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes, a mature subpopulation of peripheral blood monocytes, are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy, continued influx of CD14+CD16+ monocytes, both infected and uninfected, contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain, CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study, we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R, D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist, SKF38393, increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion, live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay, dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition, adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain regions.

Extra domain A of fibronectin primes leukotriene biosynthesis and stimulates neutrophil migration through activation of Toll-like receptor 4

There is increasing evidence of a role for Toll-like receptors (TLRs) in inflammatory arthritis. The extra domain A (ED-A)-containing isoform of fibronectin is generated under pathologic conditions such as rheumatoid arthritis, and ED-A has been identified as an endogenous TLR-4 ligand. Leukotriene B4 (LTB4) and polymorphonuclear neutrophils (PMNs) play a critical role in murine models of inflammatory arthritis. The aim of this study was therefore to investigate the putative effects of ED-A on leukotriene biosynthesis and PMN migration through TLR signaling. The effect of recombinant human ED-A (rhED-A) on leukotriene biosynthesis was evaluated in isolated human blood PMNs and monocytes by high-performance liquid chromatography. The capacity of rhED-A to stimulate PMN migration was evaluated using a transendothelial/matrix migration assay in vitro and the mouse air-pouch model in vivo. Recombinant human ED-A efficiently primed the biosynthesis of LTB4 in PMN and monocyte suspensions. This priming effect was dependent on TLR-4 activation, since the TLR-4-signaling inhibitor CLI-095 completely blocked the effect of rhED-A but not that of other TLR ligands (R-848, Pam2 CSK4) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB4 receptor 1 (BLT1) antagonist CP105,696 or the cytosolic phospholipase A2 α inhibitor pyrrophenone. In vivo, rhED-A induced a significant PMN recruitment into the air pouch of C3H/HeOuJ mice (expressing functional TLR-4), but not in C3H/HeJ mice (expressing nonsignaling TLR-4). These results demonstrate the ability of rhED-A to promote LTB4 biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB4 biosynthesis and PMN infiltration in inflammatory joint diseases.

NHE-1 and β1 integrin dependent monocyte adhesion and migration after glucose, insulin or PPARγ stimulation

In the present study the effect of high glucose concentrations, insulin, PPARγ activators (rosiglitazone) and NHE-1 inhibitors (cariporide) in atherosclerosis-related functions of human monocytes was investigated. Monocyte adhesion to laminin-1, collagen type IV and endothelial cells, as well as monocyte migration through the same substrates were studied. Incubation of the monocyte suspension with high glucose concentrations, insulin and rosiglitazone induced all the studied atherosclerosis-related functions of the monocytes. In all these functions the addition of cariporide counteracted the activity of glucose, insulin and rosiglitazone. The use of antigen for β1 integrin also counteracted the activity of the above in monocyte adhesion in all three substrates. The data of the present study suggests that PPARγ activation in monocytes induces atherosclerosis, and that NHE-1 and β1 integrin play an important role in the beginning of atherosclerosis.p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Lucida Grande'}

Native LDL-Cholesterol Mediated Monocyte Adhesion Molecule Overexpression is Blocked by Simvastatin

This study sought to evaluate the effect of nLDL concentrations on monocyte adhesion molecule expression in hypercholesterolemic patients with stable coronary artery disease (CAD) and to determine whether lipid-lowering therapy with simvastatin would change this effect. Blood samples from patients with hypercholesterolemia (mean LDL 152 mg/dL) and CAD (HC, n = 23) were collected before and after a 12-week treatment with 40 mg of simvastatin. Healthy individuals (mean LDL 111 mg/dL) were used as controls (CT, n = 15). Isolated nLDL, at a fixed concentration of 100 mg/dL, was added to monocyte suspensions obtained before and after the simvastatin treatment. Monocyte activation was determined by changes in cellular adhesion molecule expression. In response to nLDL, CD11b and CD14 adhesion molecule expression was higher in HC patients than in CT patients before treatment (174.2 +/- 8.4 vs 102.2 +/- 6.3, P < 0.03 and 140.4 +/- 5.0 vs 90.4 +/- 6.7, P < 0.04). After simvastatin treatment, CD11b expression decreased to 116.9 +/- 12.5 (P < 0.03) and CD14 expression to 107.5 +/- 6.2 (P < 0.04). Alternatively, L-selectin expression was lower in HC patients than in CT patients before therapy (46.0 +/- 3.5 vs 62.1 +/- 5.5, P < 0.04), and it increased markedly after lipid reduction to 58.7 +/- 5.0 (P < 0.04 vs baseline). After simvastatin treatment, LDL was reduced to mean 101.5 mg/dL. These data demonstrate that monocytes from HC patients are more prone to marked nLDL-mediated changes of adhesion molecule expression than monocytes from controls. Simvastatin is capable of inhibiting such nLDL effects. This proinflammatory response to nLDL may have a role in the early onset of atherosclerosis.

Human recombinant IL-1 receptor antagonist (IL-1Ra) inhibits leukotriene B4 generation from human monocyte suspensions stimulated by lipopolysaccharide (LPS).

The effect of human recombinant IL-1 receptor antagonist (hrIL-1Ra) on leukotriene B4 (LTB4) release was investigated in activated human monocyte cultures. To stimulate LTB4 generation, LPS was used as an agonist. Detection was performed with the highly sensitive radioimmunoassay method. The cells were treated with scalar concentrations using LPS at 1-1000 ng/ml for different periods of time. The greater LTB4 stimulation was found at LPS 100 ng/ml for 18 h incubation time. Preincubation of monocytes with cytochalasin B (CB) (5 micrograms/ml) for 15 min augmented the release of LTB4 when LPS was used. A dose-dependent inhibition was found when human monocytes were pretreated for 10 min with hrIL-1Ra at different concentrations (0.25-250 ng/ml) and then treated with LPS 100 ng/ml for 18 h. Maximum inhibition was observed at the highest concentration of hrIL-1Ra (250 ng/ml). Macrophages treated with a non-selective 5-lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), used at 10 microM, added 15 min before LPS 100 ng/ml, produce a dose-dependent inhibition of LTB4. Cells pretreated with arachidonic acid, at various concentrations (10(-9)-10(-5) M) for 10 min and then treated with LPS 100 ng/ml for 18 h, were also inhibited in a dose-dependent manner by hrIL-1Ra in their production of LTB4. The inhibition of LTB4 release by hrIL-1Ra, in LPS-stimulated human monocytes, may suggest an important modulatory role for this new cytokine (monokine) in inflammation and immunity and may hold future therapeutic implications for diseases involving LTB4 as a mediator.

Blockade by ruthenium red of tissue factor-initiated coagulation.

The ability of ruthenium red (RuR) to inhibit tissue factor (TF)-initiated blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma. In a single-stage clotting assay, RuR concentration-dependently inhibited rabbit brain thromboplastin (rbTF)-induced coagulation and offset bacterial endotoxin (LPS)-induced monocytic TF (mTF) hypercoagulation; the IC(50)s were estimated at 7.5 and 12.3 microM, respectively. A 15-min preincubation of RuR with rbTF or monocyte suspension resulted in the pronounced inhibition with a significantly lowered IC(50) at 1.8 or 7.7 microM for rbTF or mTF procoagulation, respectively. The differences in IC(50)s between rbTF and mTF without or with the preincubation indicated that TF was a primary target for RuR action. The effect of RuR on the physiological function of TF in FVII activation was demonstrated by the proteolytic cleavage of FVII zymogen to its active forms of serine protease on Western blotting analyses. RuR readily blocked TF-catalyzed FVII activation (diminished FVIIa formation), thus down regulating the initiation of blood coagulation. Inclusion of RuR into human plasma samples in vitro significantly prolonged prothrombin time, indicating the depressed coagulation. FVII activity was inhibited by 30 - 60% depending on the dose; as a result, FX activity also decreased. However, RuR showed no effect on thrombin time. Thus, RuR inhibited FVII activation to block the initiation of coagulation.

Enhanced low shear stress induced platelet aggregation by Shiga-like toxin 1 purified from Escherichia coli O157.

The effect of Shiga-like toxin 1 (Stx1) produced by Escherichia coli O157 on platelets was studied with an argon laser light-assisted shear-induced platelet aggregometer and with binding assays. Stx1 markedly enhanced the platelet aggregation under low shear stress but did not affect it under high shear stress. Minimal concentration of Stx1 required for the enhancement was 0.25 ng/ml, and almost maximal enhancement was observed at a final concentration of > or =2.5 ng/ml. This enhanced platelet aggregation disappeared after leukocyte depletion from normal platelet-rich plasma with a specific filter. In contrast, a standard platelet aggregometer was unable to detect this enhanced platelet aggregation in either the presence or the absence of ADP. 125I-labeled purified Stx1 did not specifically bind to normal washed platelets depleted of leukocytes, and thin-layer chromatographic analysis of glycolipids extracted from normal platelet lysates also confirmed that leukocyte-depleted normal platelets lack Stx1-specific receptor globotriaosylceramide (Gb3). Supernatant from the monocyte suspension stimulated with Stx1 exhibited the enhanced low shear stress induced platelet aggregation, but that from the polymorphonuclear cell suspension did not. Several cytokines produced from monocytes reproduced this event in vitro. Further, plasmas from six out of seven patients with hemolytic uremic syndrome (HUS) had activity similar to the purified Stx1. This activity was almost totally impaired after treatment of HUS plasmas with Gb3 in accord with reduction of plasma Stx1 levels. Taken together, these results indicate that platelets lack Gb3, and Stx1 appears to modulate platelet aggregation in an indirect fashion, presumably by the release of cytokines or chemical compounds from the target tissues.

Specific antibody to Cryptococcus neoformans alters human leukocyte cytokine synthesis and promotes T-cell proliferation.

Addition of a monoclonal antibody which binds the Cryptococcus neoformans capsule to suspensions of human monocytes, T lymphocytes, and cryptococcal cells (i) enhances interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-2 production; (ii) reduces IL-10 secretion; and (iii) promotes T-cell proliferation. The ability of specific antibody to influence cytokine production and lymphoproliferation suggests a mechanism by which humoral immunity can influence cell-mediated immunity.

Insulin induces tyrosine dephosphorylation of a 92 kDa protein in suspended monocytes

Monocytes bear insulin receptors similar to those expressed in other tissues, but insulin action in these cells remains unclear. There is evidence that adhesion, by generating a complex array of irreversible transformations, may modify the response of cells to other stimuli, such as hormones. The present study aimed to characterise the pattern of insulin induced tyrosine phosphorylation of monocytes in suspension. Monocytes in suspension were obtained by sequential gradient centrifugation and the tyrosine phosphoproteins were analyzed by immunoblot with antiphosphotyrosine antibodies. The major result of the study is that in suspended monocytes insulin induced a dose and time dependent dephosphorylation of a protein with a molecular mass of about 92 kDa without stimulating the tyrosine phosphorylation of the Insulin Receptor Substrat-1 (IRS-1). In conclusion, we showed that in monocytes in suspension insulin seems to activate a tyrosine phosphatase, which, in turn, dephosphorylates a protein with an apparent molecular weight of 92 kDa.

Cytokine induction of the ability of human monocyte CD44 to bind hyaluronan is mediated primarily by TNF-alpha and is inhibited by IL-4 and IL-13.

Ligation of CD44 by hyaluronan (HA) is a key proinflammatory event that regulates lymphocyte and monocyte adhesion and cytokine production. While most immune cells express CD44, few immune cells constitutively bind HA. We have previously shown that monocyte CD44 acquires the ability to bind HA after in vitro culture of PBMC in human serum and, therefore, we have investigated a series of human cytokines and bacterial LPS for their ability to induce and/or inhibit monocyte CD44 to bind HA. We found that IL-1alpha, IL-1beta, IL-3, granulocyte macrophage (GM)-CSF, and TNF-alpha, as well as bacterial LPS, all directly induced peripheral blood monocytes to bind HA. In contrast, IL-2 and IL-15 up-regulated monocyte CD44 HA-binding in PBMC suspensions, but not in purified monocyte suspensions. An anti-TNF-alpha-neutralizing Ab inhibited IL-1alpha-, IL-1beta-, IL-3-, and GM-CSF-mediated monocyte HA binding. In addition, treatment of IL-2- and IL-15-stimulated PBMC cultures with an anti-TNF-alpha Ab prevented IL-2- and IL-15-induced monocyte HA binding, thus identifying TNF-alpha as a lymphocyte-derived factor that acted on monocytes to induce HA binding. In contrast, IL-4 and IL-13 were potent inhibitors of monocyte CD44-HA binding induced by either human serum or by IL-1alpha, IL-1beta, IL-3, GM-CSF, or TNF-alpha. IL-10 had dual effects on monocyte CD44-HA binding. Alone, IL-10 induced HA binding to PBMC monocyte CD44, while in contrast, IL-10 inhibited IL-1-induced monocyte CD44 binding to HA. Taken together, these studies identify a network of T cell and monocyte-derived cytokines that regulate HA binding to peripheral blood monocyte CD44, primarily through TNF-alpha.

Human monocyte binding to fibronectin enhances IFN-gamma-induced early signaling events.

Leukocyte integrins are fundamentally important in modulating adhesion to extracellular matrix components and to other cells. This integrin-mediated adhesion controls leukocyte arrest and extravasation during the onset of inflammatory responses. Moreover, integrin-ligand interactions trigger signaling pathways that may influence leukocyte phenotype and function at sites of inflammation. In the current studies, we evaluated the combinatorial effects of monocyte adhesion and IFN-gamma on intracellular signaling pathways. IFN-gamma triggers a well-defined signal transduction pathway, which although not directly stimulated by monocyte adherence to fibronectin or arginine-glycine-aspartate (RGD)-coated substrata, was enhanced significantly in these matrix-adherent cells. Compared with monocytes in suspension or adherent on plastic surfaces, monocytes adherent to fibronectin or RGD exhibited a greater than threefold increase in steady state levels of IFN-gamma-induced mRNA for the high affinity Fc gammaRI receptor. By electrophoretic mobility shift assays, this increase in mRNA was associated with a 5- to 10-fold increase in the STAT1-containing DNA-binding complex that binds to Fc gammaRI promoter elements. Furthermore, the tyrosine phosphorylation of STAT1 and the tyrosine kinases JAK1 and JAK2 was enhanced significantly in RGD-adherent monocytes compared with control cells. These results suggest a novel mechanism by which integrin-mediated cell adhesion can modulate the magnitude of cytokine-induced signal transduction pathways, thereby amplifying cellular events leading to monocyte activation and inflammation.

Human recombinant interleukin-1 beta induces thromboxane A2 release in polymorphonuclear leukocytes, macrophages and platelets: effect of IL-1 receptor antagonist.

Prostaglandins and thromboxanes (Txs) are produced by polymorphonuclears (PMNs) and macrophages (Mphis) in response to various stimuli. PMNs were separated from other human blood cells and Mphis were separated from rat peritoneal lavage. In this paper we show that human recombinant interleukin-1 (hrIL-1) can stimulate the release of thromboxane B2 (TxB2) by PMNs and Mphis. In addition, we have shown that aggregation of PMNs may occur when calcium ions (7 mM) and hrIL-1 (100 ng/ml) are added to the cell preparation, but not when Ca2+ alone, hrIL-1 alone, or first hrIL-1 then calcium are added to the cell preparation. The treatment of human platelets with hrIL-1 shows that after 15 min incubation TxB2 is released. In addition, we compared the aggregation of platelets caused by ADP with that caused by hrIL-1. Human recombinant IL-1 at a concentration of 100 ng/ml also causes little aggregation of platelets, in this case the aggregation is reversible. In conclusion, hrIL-1 beta stimulates TxB2 release in PMNs, Mphis and platelets and this effect increases with addition of Ca2+ ions. The mixture of hrIL-1 and Ca2+ causes little aggregation of PMNs. In monocyte suspensions, pretreated with human recombinant IL-1 receptor antagonist (IL-1ra) 500 ng/ml for 10 min and then treated with LPS or hrIL-1 beta 10 micrograms/ml, the release of TxB2 was partially inhibited. IL-1ra may play a significant role in the control of IL-1 and LPS induction in the release of TxB2.

Rhinovirus enters but does not replicate inside monocytes and airway macrophages.

Potential interactions between rhinovirus (RV) and both the airway macrophage and its precursor cell, the blood monocyte, were investigated in terms of direct binding, intracellular replication, cell survival, and cytokine production. When HeLa cell suspensions are inoculated with RV as a positive control, virus titer increases by 100-fold in the first 24 h, confirming intracellular replication. In contrast, RV titer in monocyte and macrophage suspensions steadily decreased. Despite a lack of productive RV replication, cell-associated RV RNA was detectable using a biotin-labeled cDNA probe as early as 6 h after inoculation. Direct binding of RV16 to macrophages was confirmed using radiolabeled virus, although preincubation with anti-ICAM-1 mAb did not block this interaction. Synthesis of RV RNA, as indicated by [3H]uridine incorporation in actinomycin D-treated cells, was detected in HeLa cells but not macrophages, suggesting that the viral RNA detected inside macrophages was from input virus and was not newly synthesized. RV inoculation did not adversely affect monocyte or macrophage viability. Finally, RV caused macrophage activation, as indicated by the induction of TNF-alpha secretion. These in vitro findings suggest that macrophages interact with major group RV in vivo, and raise the possibility that there is a second cellular receptor for these viruses. Furthermore, macrophages do not serve as permissive host cells during in vivo RV infection, but instead may be active participants in anti-RV immunity and RV-induced airway inflammation.

High-yield purification of rat monocytes by combined density gradient and immunomagnetic separation

Satisfactory purification of rodent monocytes in suspension has not been achieved up to now because in rats and mice these cells occur as a minor population of peripheral blood leukocytes overlapping with lymphocytes in size and density. We describe a two-step procedure for the isolation of monocytes from rat blood with high yield and purity. This method permits the recovery of more than 90% of monocytes collected by perfusion of the vasculature and avoids loss of major subpopulations. Percoll density gradient centrifugation of perfusate cells is combined with subsequent indirect immunomagnetic depletion of lymphocytes using an antibody cocktail. The method described produces more than 90% pure rat monocytes.

Interactions of endotoxin with human blood cells and serum proteins.

Endotoxin interacts with several plasma protein systems and blood cells, causing release of a multitude of endogenous mediators that contribute to the pathophysiological process of sepsis. Binding of 125I-labelled lipopolysaccharide, LPS, to human blood in vitro showed that the major part of the 125I-LPS was recovered in plasma, whereas only small amounts were retained in washed suspensions of granulocytes, erythrocytes, monocytes and lymphocytes, respectively. Whole leukocyte preparations or isolated subpopulations incubated with 125I-LPS or fluorescein-conjugated LPS followed by autoradiography, flow cytometry or immunofluorescence microscopy showed unequivocally that monocytes bound much more LPS than did granulocytes and lymphocytes. Lipoprotein electrophoresis followed by autoradiography showed that 125I-LPS bound to all the purified lipoprotein fractions, which was also confirmed by gel filtration chromatography. These findings demonstrate that monocytes represent the most important blood cell for LPS binding and that radiolabelled LPS is able to bind to lipoproteins as well as to other serum constituents.

Cu/Zn superoxide dismutase in patients with non-familial Alzheimer’s disease

Chromosome 21 contains genes whose altered expression has long been associated with Down's syndrome and whose altered structure with some cases of Alzheimer's disease (AD). The gene for the Cu/Zn superoxide dismutase enzyme (SOD-1), a key enzyme in the metabolism of oxygen free radicals, is located on the distal portion of chromosome 21. Due to the triplication of the SOD-1 gene, patients with Down's syndrome have an almost 50% increase in their SOD activity. On the other hand, almost 25% of the patients with Down's syndrome over 40 years of age develop progressive dementia, with clinical symptoms of AD. Therefore, we decided to evaluate whether abnormalities in the production of free radicals could be detected in blood cells from AD patients, and whether they correlated with molecular variations in the Cu/Zn SOD-1 gene. Superoxide anion production was evaluated spectrophotometrically in suspensions of monocytes from 9 sporadic AD patients, and from 9 aged-matched apparently normal controls. After stimulation with increasing concentrations of n-formyl-methionyl-leucyl-phenylalanine (fMLP) or Ca ionophore A23187, monocyte free radical generation was quantitatively and qualitatively normal. Furthermore, restriction fragment length polymorphism (RFLP) analysis of leukocyte DNA digested with a variety of enzymes, gave comparable results in patients and controls. Our data support the possibility that in addition to the generation of free radicals, other directions should be explored to elucidate the mechanisms of dementia in AD.

Monocyte adhesion to fibronectin in psoriasis.

After vascular extravasation, mononuclear cells (MNC) undergo chemotaxis and adhesion to extracellular matrix proteins, resulting in their differentiation into macrophages. Although endothelial adhesion and chemotaxis are altered in psoriasis, MNC adhesion to extracellular matrix proteins has not been previously studied in the disease. Since MNC adhesion to endothelial cells is abnormally regulated in psoriasis by TGF-beta, we tested they hypothesis that in psoriasis substance P also regulates the adhesion of monocytes to the extracellular matrix protein fibronectin. Monocytes from 16 normal controls and 11 psoriatic individuals were isolated and purified using a two-step gradient centrifugation procedure. Adhesion to fibronectin was studied by plating monocyte suspensions onto fibronectin-precoated microtiter plates. The number of adherent cells was quantified by measuring their hexosaminidase activity. Although statistically significant differences in the basal (unstimulated) adhesion or in the substance P-stimulated adhesion between normal control monocytes and those obtained from psoriatic individuals were not observed, a subpopulation of psoriatics was identified who responded to substance P. Furthermore, this in vitro response to substance P was correlated with the clinical status of the subpopulation which was characterized by unstable psoriasis triggered by stressful life events. The results of this study indicate that priming of monocytes by the extracellular matrix protein fibronectin or by elevated levels of substance P are not critical steps in the pathogenesis of stable, chronic psoriasis. Substance P may contribute to the appearance of new lesions in some individuals with unstable psoriasis.