Quantitative flow cytometric analysis of expression of tumor necrosis factor receptor types I and II on mononuclear cells

Abstract

Background: Tumor necrosis factor (TNF)-α is an inflammatory cytokine, the biological effects of which are mediated by the interaction with specific membrane-bound receptors. To assess TNF-α receptor (TNFR) expression, it is important to estimate both the number of cells that carry these receptors and the number of receptors per cell, because the cell fate depends on the balance between TNFRI and TNFRII signaling. Objective: The aim of the present study was to develop an optimized protocol to estimate the level of expression of membrane-bound TNFRI and TNFRII, using QuantiBRITE PE calibration beads. Materials and methods: The percentage of cells that expressed membrane-bound TNFRI and TNFRII and the mean number of receptors per cell were determined by flow cytometry using PE-labeled antibodies against TNFR. To create a calibration curve and convert cell fluorescence intensity values to absolute numbers of receptors, we used QuantiBRITE PE beads. Results: CD19⁺ B lymphocytes had the least percentage of cells expressing TNFRI and the greatest number of receptor molecules per cell, whereas CD3⁺ T lymphocytes had the greatest percentage of cells expressing TNFRII and the lowest density of these receptors. We also established that stimulation of peripheral blood mononuclear cells (PBMCs) with the lipopolysaccharide (LPS) significantly increased the number of TNFRI and TNFRII on CD14⁺ monocytes. Conclusion: Application of the protocol-identified differences in the percentage of cells that expressed TNFRs, as well as the absolute number of receptors per cell, among different subpopulations of PBMCs, and between PBMCs cultured with and without LPS.     

“Decoding the Dots”: The ImageStream system (ISS) as a novel and powerful tool for flow cytometric analysis

The aim of this article is to provide a brief review of the ImageStream system (ISS). The ISS technology was developed as a novel method for multiparameter cell analysis and subsequently as a supportive tool for flow cytometry (FC). ISS integrates the features of FC and fluorescent microscopy collecting images of acquired cells for offline digital image analysis. The article presents an overview of the main characteristics of ISS and a comparison between ISS, FC and the laser scanning cytometer (LSC). We reviewed ISS applications focusing on those involved in cellular phenotyping and provide our own experience with using ISS as a supportive tool to classical FC and demonstrate the compatibility between FC and ISS photometric analysis as well as the advantages of using ISS to confirm FC results.     

Detection of surface asialoglycoprotein receptor expression in hepatic and extra-hepatic cells using a novel monoclonal antibody

The asialoglycoprotein receptor (ASGPR) is a heterodimeric membrane protein which is involved in the internalization of desialylated glycoproteins and also in the binding and uptake of various pathogenic viruses. To facilitate the analysis of ASGPR expression, we generated a monoclonal antibody, termed ASSA-1, that is specific to the ASGPR H1 subunit based on ELISA and Western blots analysis. ASSA-1 also reacted to surface-displayed ASGPR in live cells thus enabling analysis of ASGPR expression by immunofluorescence flow cytometry, which we used to analyze established human liver cell lines previously confirmed to be positive for ASGPR mRNA expression. In agreement with previous reports, surface ASGPR was also detected in extra-hepatic cells and, surprisingly, even in human T cell lines, which was then further confirmed in activated, but not in resting, primary human peripheral blood lymphocytes. These observations suggest that ASGPR has a broad pattern of expression that even extends into cells from the immune system, which biological meanings still have to be analyzed. We expect that monoclonal antibody ASSA-1 will serve as a new powerful tool in analyzing the biological role of ASGPR in hepatic and extra-hepatic cells.     

The roles of interleukin-1 and interleukin-1 receptor antagonist in antigen-specific immune responses

Despite evidence that interleukin (IL)-1 promotes the proliferation of some T helper 2 (Th2) cell clones in vitro, the physiological role of IL-1 in the regulation of antigen-specific immune responses remains undefined. Using a liposome-DNA delivery system, we transiently expressed IL-1 receptor antagonist (IL-1Ra) to suppress IL-1 functions at the site of the antigen-specific primary immune response. Our data indicate, for the first time, that IL-1Ra downregulates antigen-specific IL-4 and IgE responses, with concomitant enhancement of interferon- and IgG2a responses in vivo. In addition, IL-1 can promote Th2 development in an IL-4-independent manner in vitro. Thus, the balance between endogenous IL-1 and IL-1Ra during the primary immune response can be an important factor in determining the antigen-specific effector function of T cells.     

A flow cytometry approach for quantitative analysis of cellular phosphatidylserine distribution and shedding

Phospholipids are asymmetrically distributed across the membrane of all cells, including red blood cells (RBCs). Phosphatidylserine (PS) is mainly localized in the cytoplasmic membrane leaflet, but during RBC ageing it flip-flops to the external leaflet—a process that is increased in certain pathological conditions (e.g., β-thalassemia). PS externalization in RBCs mediates their phagocytosis by macrophages and removal from the circulation. PS is usually measured by flow cytometry and is reported as the percentage of cells with external PS. In the current study, we developed a novel two-step flow cytometry procedure to quantitatively measure not only the external PS but also the intracellular and shed PS. In this method, PS is first bound to fluorescent annexin V, and then the residual nonbound annexin is quantified by binding to PS exposed on apoptotic cells. Using this method, we measured 1.1 ± 0.2 and 0.12 ± 0.04 μmol inner and external PS, respectively, per 10⁷ normal RBCs. Thalassemic RBCs demonstrated increased PS externalization (1.7-fold) and shedding (11-fold) that was accompanied by lower intracellular PS (31%). These results suggest that quantitative flow cytometry of PS could have a diagnostic value in evaluating the pathology of RBCs in hemolytic anemias associated with increased PS externalization and shortening of the RBC life span.     

Interleukin-33/ST2 signaling promotes production of interleukin-6 and interleukin-8 in systemic inflammation in cigarette smoke-induced chronic obstructive pulmonary disease mice

Interleukin-33 is a newly described member of the interleukin-1 family. Recent research suggests that IL-33 is increased in lungs and plays a critical role in chronic airway inflammation in cigarette smoke-induced chronic obstructive pulmonary disease (COPD) mice. To determine the role of IL-33 in systemic inflammation, we induced COPD mice models by passive cigarette smoking and identified the IL-33 expression in bronchial endothelial cells and peripheral blood mononuclear cells (PBMCs) of them. After isolation, PBMCs were cultured and stimulated in vitro. We measured expressions of interleukin-6 and interleukin-8 in PBMCs in different groups. The expression of IL-33 in bronchial endothelial cells and PBMCs of COPD mice were highly expressed. Stimulated by cigarette smoke extract (CSE), the expression of IL-6 and IL-8 were induced and enhanced by IL-33. PBMCs of COPD mice produced more IL-6 and IL-8 stimulated by CSE and IL-33. Expression of IL-6 and IL-8 were decreased when stimulated by IL-33 together with soluble ST2. The mRNA production of ST2 in IL-33 stimulated PBMCs was increased. Being pretreated with several kinds of MAPK inhibitors, the secretions of IL-6 and IL-8 in PBMCs did not decrease except for the p38 MAPK inhibitor. We found that IL-33 could induce and enhance the expression of IL-6 and IL-8 in PBMCs of COPD mice via p38 MAPK pathway, and it is a promoter of the IL-6 and IL-8 production in systemic inflammation in COPD mice.     

Uptake and killing of Listeria monocytogenes by normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting

Cellular components of innate immunity (NK cells, monocytes and granulocytes) play an important role in early resistance to Listeria monocytogenes in the mouse model. Minimally invasive methods of measuring the bacteriocidal capacity of these cells may be useful as a biomarker of susceptibility in humans. A technique was developed whereby the uptake and survival of L. monocytogenes could be measured in human granulocytes and monocytes using small volumes of peripheral blood. This method used flow cytometry to detect the presence of PKH-2-labeled bacteria within these cells. Survival of bacteria was determined by sorting of infected cells based on a combination of fluorescence and light scattering properties. Considerable variation in bacterial recovery was seen between normal volunteers. There was consistently greater survival of a fully virulent strain of L. monocytogenes within monocytes and granulocytes compared with an isogenic strain lacking the hemolysin, listeriolysin O, when measured at baseline. There was no evidence of longer-term bacterial survival or growth at 2 or 24 h. This technique may be useful for assessment of both host resistance and pathogen virulence.     

Flow cytometric analysis of chemokine receptor expression on cerebrospinal fluid leukocytes

Collection of cerebrospinal fluid (CSF) from the lumbar subarachnoid space is a routine procedure in clinical neurology, providing an opportunity to obtain hematogenous cells from the central nervous system environment in vivo. The ability to study individual cells in samples with low cell numbers has made flow cytometry an attractive method for studies of chemokine receptor expression on such cells. Several methodological variables such as staining temperature and cell isolation techniques may, however, influence the final outcome of the staining. In addition, low numbers of cells in the normal lumbar CSF, together with a tendency of CSF cells to decay rapidly after sampling, require meticulous handling of the samples. Here, we describe the methodology used in our laboratory to study chemokine receptor expression on cells in paired samples from peripheral blood and CSF using flow cytometry.     

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Size measurements on isolated rat heart cells using coulter analysis and light scatter flow cytometry

Isolated ventricular muscle cells from the adult rat heart have been examined by both Coulter analysis and light scatter flow cytometry. The dispersed cell preparations contain two main cell types: viable, rod-shaped cells and damaged, round cells. Coulter analytical techniques provided statistical data on cell volume for both cell types. The contribution of each population to the Coulter pulse height distributions were separated by a subtraction method using data obtained from digitonin-treated preparations that contain only round cells. A shape factor for cells aligned with the flow direction was computed from light microscope measurements and the effects of cell orientation within the Coulter aperture were approximately assessed. The estimated volumes for intact myocytes compare favourably with those reported in the literature. No significant size difference was observed between fresh and fixed cells.Narrow angle, forward light scatter measurements were made on individual cells flowing across a focused laser beam. Both scatter pulse height and pulse width (pulse duration) distributions were collected. Values for myocyte length calculated from pulse width information agree well with published data and confirm that the hydrodynamic forces in the flow system produced alignment of the cells with the flow direction. Scatter pulse width distributions reveal two distinct peaks assignable to either rod or round cells. Preliminary electronic gating experiments, using pulse height signals, suggest that signals derived from round cells could be eliminated entirely using a gating regime based on pulse width. This would enable flow cytometric measurements to be made on only the intact myocytes present in heterogeneous preparations.     

Establish a flow cytometric method for quantitative detection of Beclin-1 expression

Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2 % bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004 μg/μL. Samples were incubated at room temperature (25 °C) for 30 min. The prepared samples had better to be detected immediately or to be stored at 4 °C and detected within 6 h, otherwise the samples should be fixed in 1 % paraformaldehyde storing at 4 °C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G–fluorescein isothiocyanate (IgG–FITC) for flow cytometry or with anti-rabbit IgG–peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with ¹²⁵I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1–100 ng/ml LPS; however, it was reduced by approximately 30–40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.     

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.     

Correlation of neutrophil and monocyte derived interleukin-1 receptor antagonist and interleukin-8 with colitis severity in the rabbit

Activated neutrophils and monocytes produce interleukin (IL)-8, a pro-inflammatory chemokine, but also IL-1 receptor antagonist (IL-1ra), which is an anti-inflammatory cytokine. We were interested to see the profiles of IL-8 and IL-1ra in the colonic tissue and in the peripheral blood leukocytes (PBL) during the development of immune complex induced colitis in rabbits. IL-1ra and IL-8 in PBL were measured in 26 rabbits at time 0 h, 24 h, and 48 h after induction of colitis. The colons were removed at 48 h for measuring myeloperoxidase (MPO), ulcer area, IL-1ra and IL-8. Epithelial damage, crypt abscess formation and leukocyte infiltration of the colonic tissue were major features of this colitis model. During the development of colitis, there was an increase in circulating neutrophils and monocytes (P < 0.0001), but not lymphocytes. Likewise, elevated amounts of IL-1ra (P = 0.0001) and IL-8 (P = 0.0219) production by PBL were observed following induction of colitis. Flow cytometry revealed major source of IL-1ra was monocytes, while the main sources of IL-8 were neutrophils and monocytes. There was correlation between MPO and ulcer area (R_s = 0.6327, P < 0.0001). At 24 h, PBL from MPO^High group (n = 11) showed increased IL-1ra (P = 0.027) and IL-8 (P = 0.0128) levels vs MPO^Low group (n = 15). IL-8 production by PBL showed correlation with tissue MPO (R_s = 0.4273, P = 0.0295). The colitis in this model was associated with an increase in circulating monocytes and neutrophils, which released increased amounts of IL-8 and IL-1ra. Further, IL-8 and IL-1ra showed correlation with the severity of colitis. These observations should significantly further understandings on the role of neutrophils and monocytes in the immunopathogenesis of ulcerative colitis.     

A statistical theory for flow cytometry profiles in terms of the binding of ligands to cell surface receptors and changes in gene expression

Flow cytometry analysis is a technique used for obtaining light scattering and fluorescence intensity data in order to characterise a chosen cell line. From a sample of the data obtained, it is desired to infer the distribution of cell size, cell granularity and occupancy of cell surface receptors, by constructing histograms for the variables of interest. Often an attempt is made, for instance, to account for the changes in shape of these histograms in terms of alterations in gene expression, etc. In this paper we analyse the way that changes in the sample histograms can be interpreted in three frequently encountered situations, namely (a) when there is one cell line exposed to alterations in chemical potential of ligand, (b) when there are two cell lines exposed separately to saturating concentrations of the same ligand, and (c) when two ligands are added in saturating amounts, first separately, then together, to the same cell line. We demonstrate that, under a wide range of assumptions, the change in histogram shape can be accounted for in terms of a proportionate and absolute component and examples are given to illustrate this. Finally, a computer program to analyse experimental data in terms of estimated shift and stretch parameters is described.     

A simple protocol to purify fresh nuclei from milligram amounts of meristematic pea root tissue for biochemical and flow cytometry applications

A simple protocol to purify fresh nuclei from very small amounts (mg) of Pisum sativum (cv. Lincoln) root tissue is presented. The protocol is reliable and has been repeated many times; it can be performed within a few hours and needs only a simple modification of common glassware for tissue homogenization. Similar yields of purified nuclei are obtained both with meristematic and adult root tissues using a detergent-free glyeerol buffer. Purified nuclei can be stored at –20°C for several weeks without any appreciable loss of integrity. The high yield of purified nuclei enables us to use our preparations for biochemical and cytometric investigations.     

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at −80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.