Search results for: Mouse Anti Human IL_1 beta FITC
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The immune reactivity role of HCV-induced liver infiltrating lymphocytes in hepatocellular damage.
Liver infiltrating lymphocytes (LIL) were isolated from HCV-positive (+) and HCV-negative (-) end-stage livers. Phenotypic analysis and functional studies using proliferative and lymphocytotoxic assays were performed with the isolated LIL. Two CD3+ lymphocyte populations were found in LIL using FITC anti-CD3 monoclonal antibodies (mAb). One was a bright fluorescence intensity population (as in PBL), and the other dim. We calculated the number of FITC-anti-CD3 mAbs bound per lymphocyte on PBL and LIL and found 80,040 +/- 4628 and 39,615 +/- 3932, respectively. Therefore, HCV+ and HCV- patient PBL contained approximately twice the number of CD3 molecules per cell than patient CD3+ LIL. LIL also contained approximately a threefold higher concentration of TCR alpha beta +, CD4-CD8-, and CD56,16 (NK) cells than the patient PBL. Thus, a major subset of LIL is phenotypically similar to mouse NK1.1+ "intermediate" T cells. LIL freshly isolated from HCV+ livers exhibited weak CTL activity against EBV- or Con A-transformed lymphoblast targets infected with vaccinia-HCV recombinant virus (rHCV) or primary hepatocyte cultured cells. However, after in vitro coculture of LIL with rHCV, these cells developed a strong cytotoxicity for the above targets. In contrast, LIL from HCV- livers were not cytotoxic against the same targets. Histochemical studies (in situ) demonstrated that these hepatocytes express CD95, and stains demonstrated apoptosis. The HCV+ hepatocytes also express class I MHC molecules and ICAM-1. The addition of mAb specific for these adhesion molecules inhibited CML activity. Short-term cultured hepatocytes (targets) from HCV+ and HCV- patients produced low levels of cytokines IL-1 beta, IL-2, IL-6, TNF alpha, and IFN-gamma but a high level of IL-8. It is speculated that LIL expressing reduced numbers of CD3 molecules may even function as immune regulators as proposed for intermediate T cells in mice.Y Jin, L Fuller, M Carreno, K Zucker, D Roth, V Esquenazi, T Karatzas, S J Swanson, A G Tzakis, J Miller
1305 related Products with: The immune reactivity role of HCV-induced liver infiltrating lymphocytes in hepatocellular damage.
96 assays1 Product tipe: Instrumen1 mg
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Combinations of low concentrations of cytokines and acute agonists synergize in increasing the permeability of endothelial monolayers.
The deposition of circulating immune reactants in blood vessels, an important event in the pathogenesis of certain types of vasculitis, requires an increase in permeability in the endothelial monolayer. An in vitro model to examine the integrity of endothelial cell monolayers and their response to inflammatory mediators has been developed. Human umbilical vein endothelial cells were grown to confluence on an FITC-labelled matrix and monolayer integrity was assessed by the exclusion of a 125I-anti-FITC antibody. Alteration in endothelial monolayer permeability was associated with an increase in uptake of 125I-anti-FITC antibody, expressed as a percentage of the maximal uptake of antibody on to FITC-matrix from which endothelial cells had been stripped. We determined the effects on endothelial monolayer permeability of acute agonists (thrombin and histamine), cytokines (tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), IL-1 and IL-4) and combinations of acute agonists and cytokines. Addition of thrombin in concentrations ranging from 0.5 to 15 U/ml led to an increased uptake of 125I-anti-FITC antibody from 2% to 15% relative to unstimulated endothelium. For other agonists and cytokines the increases in permeability were: (i) histamine (50-400 pmol/ml) increased uptake 5-22%; (ii) TNF (12.5-100 ng/ml) increased uptake 2-12%; (iii) IFN-gamma (125-250 U/ml) increased uptake 1.5-3%. IL-1 beta (50-100 U/ml) and IL-4 (50-100 U/ml) had no effect. Synergistic interactions on endothelial monolayer permeability were seen with the following combinations: (i) IL-4 (100 U/ml) and TNF (12.5 ng/ml) uptake 11%; (ii) IL-4 (100 U/ml) and IFN-gamma (125 U/ml) uptake 6.5%; (iii) TNF (12.5 ng/ml) and IFN-gamma (125 ng/ml) uptake 7%; (iv) thrombin (0.5 U/ml) and histamine (50 pmol/ml) uptake 13.5%; and (v) TNF (12.5 ng/ml) and thrombin (0.5 U/ml) uptake 8.5%. These observations suggest that interactions between cytokines and acute inflammatory mediators such as thrombin and histamine may be important in determining whether immune complexes are deposited in vessel walls. This model system may now be useful for the further investigation in vitro of the mechanisms involved in the pathogenesis of immune complex-mediated vascular damage.H L Beynon, D O Haskard, K A Davies, R Haroutunian, M J Walport
1903 related Products with: Combinations of low concentrations of cytokines and acute agonists synergize in increasing the permeability of endothelial monolayers.
5 G16 Arrays/Slide1.00 flask11.00 flask1.00 flask
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The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.
Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.M P de Caestecker, B A Telfer, I V Hutchinson, F W Ballardie