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PubMed2005

Aberrant TNF secretion by whole blood in healthy subjects with a history of reactive arthritis: time course in adherent and non-adherent cultures.

BACKGROUND: The pathogenesis of reactive arthritis (ReA) apparently involves aberrations in innate immune functions such as monocyte tumour necrosis factor (TNF) generation. OBJECTIVE: To investigate TNF production in healthy subjects with previous yersinia triggered reactive arthritis. METHODS: The study comprised HLA-B27 positive subjects with previous reactive arthritis (B27+ReA+), and B27+ReA- and B27-ReA- subjects (n = 15 each). Whole blood TNF production was induced by lipopolysaccharide (LPS), which binds to CD14/TLR4 on the monocyte surface, or by a combination of phorbol 12-myristate 13-acetate (PMA) and Ca(2+) ionophore A23187, which activates monocytes independently of cell surface receptors. To further evaluate the possible role of adhesion mediated signalling on TNF production, blood samples were incubated in adherent or non-adherent conditions. TNF levels in culture supernatants were measured using an automated immunoassay analyser. The CD14(-159)C/T genotype was determined by a cycle minisequencing method. RESULTS: B27+ReA+ supernatants had higher TNF levels than B27+ReA- supernatants in PMA/A23187 wells in two hour (p = 0.004) and four hour cultures (p = 0.001). Rapid initial TNF release took place in adherent but not in non-adherent conditions. This adhesion associated difference was greater in the B27+ReA+ group than in the B27+ReA- or B27-ReA- group in response to PMA/A23187 (p values <0.001), and greater in the B27+ReA+ group than in the B27-ReA- group in response to LPS (p = 0.021). CD14(-159)T was associated allele dose dependently with an increase in the LPS induced TNF secretion allele (p = 0.030). CONCLUSIONS SUBJECTS: who have recovered from yersinia arthritis show enhanced TNF production, which may be regulated at the level of monocyte adhesion.

Adult↗
PubMedDate not supplied

Signal transduction in adherent and non-adherent human cell lines after fibronectin stimulation.

It is shown that adherent and non-adherent human ovarian carcinoma cells (OVP 10) secrete MMPs and their production was stimulated by fibronectin as documented by gelatinise zymography. These cells also presented an increase of ERK phosphorylating activity following fibronectin stimulation, regardless of their adhesion. Contrary to OVP 10 cells, the human urothelial cells (HCV-29) are more anchorage-dependent. They only secreted the MMPs under adherent conditions and they revealed a lower level of basal and fibronectin stimulated ERK phosphorylation activity. In addition, non-adhering HCV-29 cells showed post translational down-regulation of focal adhesion kinase.

Blotting, Western↗
PubMed1988

Modulation of human neutrophilic granulocyte functions by recombinant human tumor necrosis factor and recombinant human lymphotoxin. Promotion of adherence, inhibition of chemotactic migration and superoxide anion release from adherent cells.

Recombinant human tumor necrosis factor (TNF) and recombinant human lymphotoxin (LT) were analyzed for their effects on inflammation-related functions of human polymorphonuclear neutrophilic granulocytes (PMN) in vitro, TNF at a concentration of 10 U/ml (corresponding to 10(-11) mol/l) enhanced PMN adherence to nylon fibres. It strongly inhibited the chemotactic migration of PMN in the Boyden chamber assay towards the chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, LTB4 and a monocyte-derived chemotaxin (MOC) without affecting random migration and without being chemotactic itself. It did not stimulate superoxide anion (-O2.) production of PMN in suspension. However, it induced considerable -O2. release from PMN that had become adherent on nylon fibres. All these effects were abrogated by prior incubation of the cytokine with polyclonal and monoclonal antibodies against TNF. LT concentrations of 1,000 U/ml or higher were required to observe a moderate inhibition of chemotactic migration towards the above chemotactic factors and to elicit some -O2. production from nylon fibre-adherent PMN. LT did not increase the adherence of PMN to nylon fibres and it was not chemotactic. The results indicate that TNF is a potent modulator of PMN functions.

Cell Adhesion↗
PubMed1979

Cell-mediated cytotoxicity of adherent and non-adherent mouse lymph node cells sensitized in vitro against tumour-associated antigens of syngenetic methylcholanthrene-induced sarcomas.

Cell-mediated cytotoxicity of adherent and non-adherent subpopulations of mouse lymph node cells sensitized in vitro on monolayers of syngeneic, methylcholanthrene-induced sarcoma cells was examined by inhibition of 3H-thymidine incorporation. The cell-mediated cytotoxicity of nylon wood-adherent, non-adherent and unfractionated lymph node cells was found to be equally efficient.

Animals↗
PubMed1995

Adherence of human monocytes to haemodialysis membranes: LFA 1 (CD11a/CD18) CR1 (CD35) and CR3 (CD11b/CD18) triggering promotes the biosynthesis of platelet-activating factor and adherence.

BACKGROUND: Platelet-activating factor is a mediator of inflammation involved in the blood-membrane interaction. We report that selective stimulation of complement receptors (CR1 and CR3) triggers PAF synthesis and monocyte adherence to complement-activating membranes. METHODS: The synthesis of PAF was studied after stimulation of normal human adherent monocytes with F(ab)2 and Fab fragments of monoclonal antibodies specific to CR1 and CR3. CD11a, CD11b, CD18, and CD35 was studied by flow cytometry on neutrophils and monocytes. The molecular species of PAF from stimulated monocytes were identified by reverse-phase high-performance liquid chromatography coupled with mass spectrometry. RESULTS: Anti-CR1 and anti-CR3 monoclonal antibodies induced a dose-dependent C-16 but not C-18 PAF production. The latter occurred also with monovalent Fab fragments of both anti-CR1 and anti-CR3 monoclonal antibodies, that were not internalized as seen by immunofluorescence. Adherence of monocytes to Cuprophan membranes was markedly higher (P < 0.01) in membranes pretreated with fresh than with heat-inactivated normal plasma. However, the high adherence to fresh plasma-treated membranes was completely abrogated by coincubating the cells with Web 2170, a specific PAF receptor antagonist. This was not due to downregulation of adhesion molecules expression on leukocytes. CONCLUSIONS: These studies implicate a crucial role of PAF in blood interaction with haemodialysis membranes that fix complement activated products.

Antibodies, Monoclonal↗
PubMed1997

Fibronectin increases both non-adherent cells and CFU-GM while collagen increases adherent cells in human normal long-term bone marrow cultures.

Normal haematopoietic proliferation and differentiation occur within the human bone marrow microenvironment which is comprised of stromal cells including fibroblasts, adipocytes, macrophages and endothelial cells as well as the extracellular matrix made of collagen, fibronectin, laminin, vitronectin, thrombospondin and haemonectin. All haematopoietic progenitor cells including primitive LTC-IC, multilineage CFU-mix, myeloid CFU-GM and erythroid BFU-E adhere to the heparin-binding domains of the extracellular matrix component fibronectin. Human long-term bone marrow cultures (LTHBMC) represent the best available approximation for the in vivo marrow microenvironment in which the proliferation and differentiation of haematopoietic progenitor cells depend on the presence of marrow stromal cells and their attendant matrices. Since extracellular matrix components have been shown to promote myelopoiesis in long-term murine bone marrow cultures, we have examined the effect of two main components of the extracellular matrix: fibronectin and collagen type I on myelopoiesis in LTHBMC in an effort to increase the myeloid progenitor cell production. The present study revealed different modulatory effects for these two components. Collagen significantly increased the adherent fraction of LTHBMC (p < 0.05) but always resulted in a decreased myeloid progenitor cell (CFU-GM) production throughout the whole 8 weeks of culture. On the other hand, fibronectin significantly increased the number of both non-adherent cells. CFU-GMs (p < 0.01) and to a lesser extent the number of adherent cells as well as maintaining the LTHBMC up to 14 weeks. Fibronectin has been previously shown to stimulate the development of CFU-GMs in short-term semisolid cultures and to play an active role in haematopoietic progenitor cell-microenvironment interactions. Therefore, the presence of fibronectin in LTHBMC could increase both the productivity and longevity of myelopoiesis in the system. The integration of fibronectin in the ex vivo expansion systems currently undergoing development would ensure a sustained effective cumulative production of the myeloid progenitor cells (CFU-GMs), and consequently could accelerate the rate of haematological recovery in transplanted patients.

Bone Marrow Cells↗
PubMed1983

Detection of different states of immunity to colon cancer in rats by a leukocyte adherence inhibition (LAI) assay assessing the adherence of T lymphocytes and monocytes selectively.

Peripheral blood mononuclear cells (MNC) of rats, which had received a subcutaneous inoculation of either viable or X-irradiated syngeneic DMH-W49 colon carcinoma cells, were investigated sequentially for sensitization against tumor-associated antigens by a new micro-glass-tube leukocyte adherence inhibition (LAI) assay. The number of adherent cells of MNC subpopulations was estimated by a cellular radioimmunoassay (CRIA), which utilizes anti-monocyte (MC) antiserum or anti-T-cell monoclonal antibody (McAb) and 125I-labelled protein A. LAI reactivity was demonstrated in rats sensitized with X-irradiated tumor cells when assessing the adherence of both T lymphocytes and monocytes. It was found that reactivity with the two different types of indicator cells did not usually coincide in time. The same phenomenon was also observed in tumor-bearing rats although with lower levels of LAI reactivity. In both groups the LAI response detected by anti-T-cell reagent mostly appeared earlier after tumor-cell inoculation than the response demonstrated by anti-MC serum. The T-lymphocyte-associated reactivity also disappeared more rapidly. These results suggest that different states of immune reactivity are reflected in LAI responses involving T lymphocytes and monocytes as indicator cells.

Adenocarcinoma↗
PubMed1986

New micro-glass-tube leukocyte adherence inhibition assay assessing cell adherence of mononuclear cell subpopulations defined by monoclonal antibodies.

A new micro-glass-tube leukocyte adherence inhibition (LAI) assay which is appropriate for detecting delayed type hypersensitivity in vitro has been developed for human leukocytes. Enumeration of adherent cells is replaced by a cellular radioimmunoassay determining antibody binding of the monoclonal reagents, OKT4, OKT8 and OKM1, to glass-adherent cells, fixed by glutaraldehyde or formaldehyde. An LAI reactivity to purified protein derivative of tuberculin (PPD) was detectable in donors giving a positive PPD skin test with OKT4 reagent, but not with the other two reagents.

Antibodies, Monoclonal↗
PubMed1979

Adherence, augmented adherence, and aggregation of polymorphonuclear leukocytes.

Adherence of polymorphonuclear leukocytes to nylon fiber was found to be plasma-independent and distinct from the processes of augmented adherence, leukocyte aggregation, and inhibition of random leukocyte migration, all of which are dependent on "activated" plasma. Nylon fibers were unable to "activate" plasma, in contrast to the complement-activating agent zymosan. Prior incubation of plasma with antiserum to the third component of complement (C3) inhibited aggregation of polymorphonuclear leukocytes by plasma incubated with zymosan, and heat-inactivation blocked augmentation of adherence by treatment of plasma with zymosan.

Adult↗
PubMed1976

The role of adherent cells in the immune response. Fibroblasts and products released by fibroblasts and peritoneal cells can substitute for adherent cells.

The primary immune response to sheep erythrocytes in adherent cell-depleted cultures was restored by adding a critical number of peritoneal cells. Complete substitution was achieved also with supernatants from allogeneic and syngeneic peritoneal cells. Both living fibroblasts and supernatants from fibroblast cultures were found to be highly efficient substitutes for adherent cells in both syngeneic and allogeneic systems. Supernatants from non-antigen-reated peritoneal cells and fibroblasts caused increased DNA synthesis and induction of polyclonal antibody synthesis in normal spleen cells. Thus, adherent cells need not function in the immune response by presenting antigen to the B cells via 'IgT' or by releasing signal-2 activity, which acts on lymphocytes that have already received signal 1.

Animals↗
PubMed1984

Repeated micro-monocyte adherence inhibition assay: a new technique of leukocyte adherence inhibition to detect tumor immunity in patients with hepatoma.

A microplate leukocyte adherence inhibition (micro-LAI) assay was performed with peripheral blood mononuclear cells obtained from patients with hepatoma and control subjects (including healthy donors and patients with other diseases). Cell extracts of human hepatoma cells (HCC-M) and human hepatic cells (Chang liver cell) in tissue culture were prepared by sonication followed by centrifugation. The supernatants of these two cell lines were used as a specific antigen and a nonspecific antigen, respectively. It was found that monocytes were major indicator cells and that monocytes produced an LAI reaction in the absence of lymphocytes. Therefore, a repeated microplate monocyte adherence inhibition (MAI) assay was developed, in which the monocyte population of adherent cells is increased by removing nonadherent cells after an initial assay in fetal calf serum-containing medium without test antigens, and monocytes are counted selectively as peroxidase-positive cells in a subsequent second assay with test antigens. With regard to sensitivity and reproducibility, the repeated micro-MAI assay is superior to a micro-MAI assay in which the initial assay is omitted although monocytes are selectively counted. With this simple and sensitive technique a hepatoma-associated immune response to the extract of HCC-M was detected in 16 out of 22 patients (73%) with hepatoma, whereas the false-positive rate was 7% (3/41) in all control subjects.

Carcinoma, Hepatocellular↗
PubMed1996

Differential expression of the heavy-chain ferritin gene in non-adhered and adhered oligodendrocytes.

Oligodendrocytes (OLGs) synthesize and maintain central nervous system myelin. Little is known about the molecules and pathways involved in signaling the commencement of myelination; yet myelination is spatially and temporally controlled. We are interested in deciphering the signaling events that control the on and off switch of myelination. To address these questions we are using an in vitro model system consisting of pure cultures of OLGs, isolated from postmyelination brains. We have shown that in vitro, these OLGs regenerate and reenact the ontogenic development of myelin upon adhesion to a substratum; we have also shown that when transplanted in vivo, they can form myelin. Hence, identifying the genes that are turned on upon OLG adhesion seemed a worthwhile approach to singling out those genes that are critical to the differentiation process. For this we adopted a novel technique-differential display-that permits the comparison of gene expression under two sets of conditions; in our case between non-adhered and adhered OLGs. Using the differential display method in conjunction with a set of five arbitrary primers, we have identified five cDNAs that are activated upon OLG substratum adhesion. Four of these cDNAs proved to be fragments of the heavy (H)-chain ferritin cDNA. H-chain ferritin is involved in the rapid sequestration and delivery of iron; it is also a cytoprotectant. Brain iron is localized predominantly in OLGs. Our finding that the expression of H-chain ferritin is upregulated upon OLG regeneration/differentiation agrees with reports, in other cell types, that H-chain ferritin transcription is modulated by factors that control cell growth and differentiation. The enhanced transcription results in a twofold augmentation in ferritin synthesis. This is the first demonstration of an adherence-mediated activation of the H-chain ferritin gene. The observation has interesting implications. The transcription of the H-chain ferritin gene is associated with tissue stress such as might occur during rapid cell growth. The fact that this transcription takes place upon OLG-substratum adhesion can be viewed as another manifestation of the anchorage-induced signal that drives OLGs toward a differentiation program. This entails the synthesis of all the necessary components for the assembly of large quantities of membranes, hence constituting a period of rapid growth and considerable cell stress. We have sequenced the full-length ferritin cDNA of ovine OLGs; the cDNA is 775 bp long. The coding sequence starts at residue 57 with the ATG codon and terminates at residue 602, with the stop codon TAA. The cDNA codes for 181 amino acids with a predicted size for the protein of 22 kD. The deduced amino acid sequence is 93% identical to human ferritin. We postulate that OLG H-chain ferritin may function at more than one level: 1) it provides the iron required to sustain an oxidative metabolism; and 2) it acts as a cytoprotectant against oxidant-mediated injury.

Amino Acid Sequence↗
PubMed1981

Measurement of macrophage-mediated cytotoxicity against adherent and non-adherent target cells by release of 11 indium-oxine.

This report describes the utilization of 111 indium-oxine chelate ([111In]Ox) for studies of macrophage-mediated cytotoxicity. [111In]Ox efficiently labeled both non-adherent and adherent tumor targets with no decrease in cell viability. Spontaneous release of intracellularly incorporated [111In]Ox was very slow (0.25-0.50%/h) from most targets, making isotope-release assays of at least 48 h feasible. In addition, released [111In]Ox was not reutilized. In contrast to its low spontaneous release from intact cells, incorporated [111In]Ox was rapidly released from tumor targets after interaction with activated macrophages. Levels of [111In]Ox released in response to cytolytic macrophages correlated well with those observed for the 51Cr and [3H]TdR radiolabels. Therefore, [111In]Ox can be utilized for relatively short-term (less than 20h) assays with lymphoma targets, as well as for longer-term assays with adherent cells. This should facilitate the testing, with the same radioisotope-release assay, of a wide range of tumor targets for susceptibility to macrophage-mediated cytotoxicity.

Animals↗
PubMed1989

Internalization of phosphatidylserine by adherent and non-adherent rat mononuclear cells.

Energy-dependent, protein-mediated incorporation of radiolabeled phosphatidylserine vesicles is observed in casein-elicited rat peritoneal cells. Cell fractionation and a comparison with other phospholipids demonstrate the selective interaction of phosphatidylserine with the mononuclear fraction of these cells. During 60 min of incubation, unchanged phosphatidylserine accumulates in the cells whereas lysophosphatidylserine is released in the medium. When adherence is used to fractionate the mononuclear cells, phosphatidylserine uptake is detected in the macrophage-enriched fraction (adherent cells) and in the lymphocyte-enriched fraction (non-adherent cells). Evidence of stereoselective uptake and of phosphatidylserine internalization in both cells is obtained by the use of phosphatidyl-D-serine and by digestion of the extracellular phospholipid with phospholipase A2. Only in lymphocytes is the uptake of phospholipid substantially inhibited by cytochalasin B, metabolic poisons and a low incubation temperature (17 degrees C). Phosphatidylserine deacylation-reacylation is instead detected in both cells. It is concluded that lymphocytes actively concur in the uptake of phosphatidylserine by rat mononuclear cells.

Animals↗
PubMed1990

Effects of doxorubicin on maturation of human monocytes in adherent and non-adherent cultures.

Purified human monocytes were cultured for 2 h, 88 h, and 10 days in plastic tubes (adherent) and for 10 days in Teflon foil bags (non-adherent). Monocytes were incubated with doxorubicin by two short-term exposures (750 or 1500 ng/ml) for 1 h or by continuous exposure (75 ng/ml). Maturation was monitored by measuring the intracellular activity of three metabolic enzymes and two acid hydrolases. Expression of receptors for the Fc moiety of immunoglobulin G (FcRI, FcRII, FcRIII), CD14, and HLA-DR was assayed by indirect immunofluorescence with monoclonal antibodies. In the presence of doxorubicin, the adherent capacity, the yield, and the enzyme activities reflecting growth and intermediary metabolism were similar to the control groups. However, doxorubicin reduced the expression of FcRI (32-45%), FcRII (10-26%), CD14 (20-37%), and HLA-DR (25-34%) on the monocyte-derived macrophages. Expression of FcRIII was not detectable after 10 days of culture.

Antigens, CD↗
PubMed2003

Exercise interventions for smokers with a history of alcoholism: exercise adherence rates and effect of depression on adherence.

This study examined the adherence rates and the effect of depression on adherence in two studies conducted among smokers with a past history of alcoholism. In both studies, subjects participated in a 12-session group-based exercise intervention for smoking cessation. The target quit date (TQD) was Session 8. Participants in Study 1 were 73 smokers (43% female). Exercise instructions began at Session 8 and continued through Session 12. Mean frequency and number of minutes of exercise decreased during the 4 weeks of exercise treatment (P<.001). Study 2, conducted with 18 smokers (50% female), examined the feasibility of commencing exercise at Session 1, well before the TQD. The mean number of minutes exercised increased from Sessions 1 to 12 (P=.013). In both studies, average session attendance was high (82%). Combining subjects from both studies, depressed smokers at baseline reported greater mean frequency of exercise per week than nondepressed smokers (P=.05). The results suggest that depressed smokers can be engaged in an exercise program. Further research is needed to determine if commencing exercise early during treatment, prior to the TQD, improves adherence.

Adult↗
PubMed1992

Generation of lymphokine-activated killer cells by adherent LGL phenotype cells and non-adherent T lymphocytes.

In this work we separated human blood lymphocytes (PBL) in two populations (A-LAK and NA-LAK cells) by the adherence plastic method. A maximum adherence of cells was obtained after 2 days of PBL incubation in LAK medium containing 500 U/ml rIL-2. The A-LAK cells had LGL phenotype but 40% of them had a macrophage phenotype marker and less than 20% weakly expressed a T-cell marker. This population, when reincubated in culture, produced an increasing titre of interferon. At the same time, a significant NK activity against K562 target cells was measured just after enrichment; these enriched adherent cells also developed an increased LAK activity against DAUDI cell lines, ninefold more at 6 days than when assayed just after enrichment. In contrast, 75% of the NA-LAK enriched cells expressed T-cell marker; these produced two- to threefold less interferon than A-LAK cells at all time-points. The NA-LAK lymphocytes enhanced principally LAK activity measured by 70% lysis against DAUDI target cells tested at 6 days of culture. Further studies are in progress to determine the nature of the effector cells that mediated LAK activity.

Cell Adhesion↗