ARTICLE |
Correspondence to: Andreas Grützkau, Virchow Klinikum Dermatologie, Augustenburger Platz 1, 13353 Berlin, Germany.
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Summary |
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The chemokine interleukin-8 (IL-8) mediates infiltration and adhesion of neutrophils during inflammatory processes. We have previously shown that this cytokine can be produced and released by normal and leukemic human mast cells (HMC-1 cells). To assess whether and to what extent this cytokine is stored intracellularly, we investigated production and localization of IL-8 at the single-cell level by combined use of flow cytometry (FACS) and immunoelectron microscopy. Conditions necessary for optimal fixation and permeabilization of HMC-1 cells were determined by measuring changes in cell-specific light scatter parameters and by estimating cellular uptake of propidiumiodide (PI). In this way, we were able to detect IL-8 with a monoclonal antibody in stimulated cells that were microwave-fixed with a combination of paraformaldehyde (4%) and glutaraldehyde (0.1%), followed by permeabilization with saponin (0.025%). FACS analysis revealed time-dependent synthesis of IL-8 with at most 50% positively stained cells at 8-12 hr after stimulation. For pre-embedding immunogold electron microscopy, cells were treated according to the protocol established by flow cytometry. IL-8 was found to be located in specific cytoplasmic, electron-dense granules of stimulated HMC-1 cells. These results confirm and extend our previous findings by demonstrating IL-8 expression in HMC-1 cells at the single-cell level. In addition, we propose that quantitative FACS can be reliably used in a timesaving manner to establish appropriate conditions for pre-embedding immunoelectron microscopy of intracellular antigens. (J Histochem Cytochem 45:935-945, 1997)
Key Words: mast cells, HMC-1 cells, interleukin-8, immunoelectron microscopy, pre-embedding immunocytochemistry, flow cytometry, colloidal gold
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Introduction |
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The chemokine interleukin-8 (IL-8), initially described as a granulocyte-directed chemotactic stimulus, is a potent proinflammatory cytokine and has been identified in a number of inflammatory diseases including psoriasis, rheumatoid arthritis, and lung diseases (
For intracellular detection of cytokines by these methods, particularly in mast cells, two major difficulties are encountered: (a) cytokines are soluble mediators that are easily eluted during specimen processing (
In addition to these applications, flow cytometry enables quantification of antibody binding under various conditions of cell fixation by use of a fluorescence-labeled secondary antibody. The successful application of flow cytometry for the detection of cell-associated cytokines has been demonstrated predominantly for mononuclear leukocytes (
In this report we present a semiquantitative flow cytometric assay to detect intracellular IL-8 in HMC-1 cells at the single-cell level. Furthermore, we offer a strategic protocol established by flow cytometry that allowed rapid optimization of conditions necessary for fixation and permeabilization of mast cells before subcellular localization of IL-8 by pre-embedding immunoelectron microscopy. We suggest that this procedure is applicable to other cells and intracellular antigens.
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Materials and Methods |
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Cell Culture
HMC-1 cells (kindly provided by Dr. Butterfield; Rochester, MN), which are immature human leukemic mast cells (
Cell Fixation
The following fixatives were compared: 1% PFA; 4% PFA; 1% PFA/0.1% GA; 4% PFA/0.1% GA; 4% PFA/2% GA; and 2% PFA/10 mM lysine periodate (PLP). PFA (Merck; Darmstadt, Germany) was freshly prepared and diluted in PBS without magnesium and calcium. PLP was also prepared just before use according to the method of
Cell Permeabilization
Cells were permeabilized with increasing concentrations of Triton X-100 (Sigma) or saponin (Sigma), as indicated. Permeabilization with Triton X-100 (0.02-0.05%) was done for 20 min at 4C before antibody staining. Saponin treatment (0.003-0.05%) was done during the entire staining and washing procedure. The efficiency of permeabilization was controlled by staining cells with propidium iodide (PI; Sigma) at a concentration of 200 µg/ml for 10 min at room temperature. Incorporation of the dye and cellular integrity were quantified by an EPICS XL flow cytometer (Coulter Electronics; Krefeld, Germany).
Antibodies
The following monoclonal antibodies (MAbs) were used for flow cytometry and pre-embedding immunoelectron microscopy: anti-tryptase [AA1; kindly provided by Dr. Walls, Southampton, UK (
Immunofluorescence Staining Protocol for Flow Cytometry
For detection of intracellular antigens, HMC-1 cells were microwave-fixed in a mixture of 4% PFA and 0.1% GA as described above. To block nonspecific FcR-mediated binding of MAbs, 5 x 105 cells were preincubated for 30 min at 4C with human heat-inactivated AB serum (Behring Werke; Marburg, Germany) containing 0.025% saponin. Saponin treatment was done during the entire staining procedure as described above. After blocking, primary MAbs were added to a final volume of 50 µl. Cells were incubated for 30 min at 4C, washed twice in PBS containing 0.5% BSA/0.025% saponin (washing buffer), and were resuspended in human AB serum. Binding of the primary MAb was visualized by indirect immunofluorescence using a DTAF-conjugated F(ab')2 fragment of goat anti-mouse IgG antibodies (Dianova). The secondary antibody was added at a final concentration of 20 µg/ml, and cells were further incubated for 30 min at 4C. Finally, cells were washed and fixed in PBS containing freshly prepared 1% PFA. At least 10,000 cells were analyzed using an EPICS XL flow cytometer (Coulter Electronics). Results were expressed as percent positive cells, taking into account the amount of nonspecific binding of the isotype-matched control antibody (negative control).
Immunoelectron Microscopy (Pre-embedding Procedure)
The protocols for cell fixation, permeabilization, and staining with the primary MAb used for immunoelectron microscopy were identical with those described for flow cytometric detection of intracellular antigens. Binding of the primary antibody was visualized by incubating cells overnight at 4C with goat anti-mouse IgG conjugated with 1-nm colloidal gold (British BioCell International; Cardiff, UK). Negative controls included the substitution of an isotype-matched control MAb for the primary antibody in the labeling scheme and omitting the primary MAb. The secondary antibody was diluted 1~200 in buffer containing 0.5% BSA/0.025% saponin and 0.1% fish gelatin (British BioCell International). After intense washing, cells were fixed for 10 min in 2.5% GA and were then washed two more times with PBS for 10 min each, followed by an incubation in 2% OsO4 for 1 hr at 4C. Finally, cells were washed in PBS and distilled water and immunolabeling was completed by silver stain amplification with a commercially available "Silver Enhancing Kit" (British BioCell International) according to the instructions of the manufacturer. Enhancement was performed at 20C for 30 min and was terminated by washing the specimens in distilled water for 5 min. Before dehydration, cells were centrifuged in molten agar (1% in distilled water at 45C) to form cell pellets that were sliced and processed as small blocks. Dehydration was done in a graded series of ethanol solutions at 4C. The dehydrated samples were transferred to propylene oxide and embedded in Araldite (Serva; Heidelberg, FRG). Polymerization was done for 48 hr at 60C. Semithin sections (0.5 µm) were mounted on glass slides and stained with toluidine blue for quick evaluation. Ultrathin sections (50 nm) were picked up on copper grids and stained with aqueous uranyl acetate and lead citrate. Specimens were examined with a Zeiss EM906 transmission electron microscope at 80 kV.
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Results |
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Permeabilization Protocol
Figure 1 shows the influence of increasing concentrations of saponin (0.003-0.05%) on the permeabilization of unfixed HMC-1 cells measured by the uptake of propidium iodide (Fl2/PI) and on the cellular integrity determined by measuring forward scatter parameter (FS). As little as 0.006% saponin caused a distinct permeabilization of nearly 50% of cells, but this effect was accompanied by cell destruction, as is evident from the decrease in the forward scatter parameter and from light microscopic evaluation. At a concentration of 0.025% all cells were stained. Triton X-100 produced similar effects, but at higher concentrations: at a threshold concentration of 0.03%, Triton X-100 caused permeabilization of 60% of cells, whereas no effect was detectable at 0.02% (data not shown).
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These results indicated that the plasma membrane of mast cells reacts extremely sensitively to the detergents used, and it was therefore concluded that permeabilization requires additional cell fixation.
Fixation Protocol
The effect of various fixatives on permeabilization and cell integrity was investigated for saponin- and Triton X-100-treated as well as for untreated cells (Figure 2). The light scatter properties of the majority of cells (>95%) that had not been treated with a detergent but were microwave-fixed with PLP or 1% PFA indicated sufficient preservation of the cell structure. However, under these conditions no cell permeabilization was achieved because these cells stained negatively for PI. The minor population of small cells that had incorporated PI probably represent damaged and dead cells.
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Cells fixed with combinations of PFA and GA showed significant PI uptake, indicating partial cell permeabilization (Figure 2). Because cells fixed in 4% PFA alone showed only minor PI uptake, it is likely that permeabilization was mainly induced by GA. To ensure that GA alone or its combination with microwave irradiation is responsible for the permeabilizing effect observed, cells were fixed without irradiation and the same effect was ascertained (data not shown). This fixation-induced permeabilization allowed diffusion of small molecules such as PI through the plasma and nuclear membrane, but was insufficient for immunoglobulins, as could be shown by an insufficient staining of these cells for intracellular antigens such as CD68 and IL-8 in comparison to cells treated with saponin or Triton X-100 after fixation (data not shown). Destruction of cell integrity by saponin could be prevented by prefixation of cells in solutions containing at least 4% PFA and 0.1% GA (Figure 3). Fixation with PFA alone or PLP did not ensure sufficient stabilization of cell structure in the presence of saponin (Figure 3). This holds also true for Triton X-100-permeabilized cells (data not shown).
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Immunolabeling for Flow Cytometry
After determination of optimal conditions for cell fixation (4% PFA/0.1% GA) and permeabilization (0.025% saponin), the binding of MAbs directed against intracellular antigens [tryptase (AA1), CD68 (KP1), and IL-8 (52E8)] was quantified by flow cytometry. CD68 and tryptase, two antigens expressed intracellularly in human mast cells (
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The histograms in Figure 5 demonstrate the expression of IL-8 in unstimulated and stimulated HMC-1 cells. After cell fixation and permeabilization, the cytokine was measurable in small amounts in unstimulated and calcium ionophore-treated cells (Figure 5A and Figure 5B). PMA treatment and, to a much greater extent, the combination of calcium ionophore and PMA induced a significant increase in the expression of cytoplasmic IL-8 (Figure 5C and Figure 5D). The plasma membrane of calcium ionophore/PMA-stimulated and unstimulated cells was negative for IL-8, as determined by immunostaining in the absence of cell fixation and permeabilization (data not shown). The time course of the accumulation of intracellular IL-8 in calcium ionophore/ PMA-stimulated cells is shown in Figure 6. Even after 2 hr of stimulation there was a measurable increase in IL-8 expression, which reached its maximum at 12 hr. Thereafter, the cytoplasmic IL-8 content declined.
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Stimulation times longer than 12 hr induced a decrease in cell viability of 10-20%, but dead cells and debris were electronically gated out on the basis of shifted forward and side-angle light scatter parameters.
Immunolabeling for Electron Microscopy
The fixation and permeabilization protocol, as established by flow cytometry, was applied for pre-embedding immunoelectron microscopic detection of IL-8 in HMC-1 cells. Under these conditions good ultrastructural preservation was noticeable, as verified by the depiction of mitochondrial membranes and secretory granules (Figure 7). HMC-1 cells exhibited the typical appearance of immature human mast cells, with large nucleoli in a monolobed round or ovoid nucleus, some immature granules, and many mitochondria. Granules of the cells exhibited an amorphous structure without the crystalline gratings frequently seen in the granules of mature tissue mast cells (Figure 7B-D). Furthermore, the majority of HMC-1 cells cultured under suspension conditions are characterized by the appearance of empty granule containers and degranulation channels, indicating spontaneous piecemeal degranulation (Figure 7A). Most importantly, the antigenicity of intracellular IL-8 was retained. In HMC-1 cells stimulated for 8 hr with 0.25 µM A23187 and 25 ng/ml PMA, silver-enhanced 1-nm colloidal gold complexes were observed as large, specific electron-dense particles that were mainly located at the periphery of cytoplasmic granules (Figure 7B and Figure 7C). Only a few granules showed a more homogeneous or even negative staining for IL-8 (Figure 7B and Figure 7C). Empty granule containers and degranulation channels exhibited only minimal or no immunostaining for IL-8 (Figure 7C). In granules of unstimulated cells, no immunoreactivity was detectable (Figure 7A). The nonspecific background labeling was minimal, and no immunoreactive structures were seen in cells processed in the presence of an isotype-matched irrelevant MAb (Figure 7D, negative control) or after omitting the primary MAb during immunostaining (data not shown).
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Discussion |
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We previously identified IL-8 expression in human mast cells under inflammatory conditions (
The successful identification of IL-8 by the pre- embedding immunogold technique described suggests that flow cytometry is a useful tool for rapid screening of multiple parameters necessary for optimal cell fixation, permeabilization, and antibody binding. We used PI as a reliable probe for the determination of the extent of cellular permeabilization, although it must be remembered that PI possesses a substantial lower molecular size than immunoglobulins. For cell fixation, we applied the microwave energy technology because this method has been described to ensure an excellent preservation of the ultrastructure of mast cells and of various mast cell-associated antigens (
Fixation with 4% PFA and 2% GA according to
Treatment with Triton X-100 and saponin induced sufficient membrane permeabilization at very low but distinct threshold concentrations (0.025% and 0.006%, respectively). This extreme sensitivity of mast cells in response to saponin indicates a high cholesterol content in the plasma membrane because it has been reported that the plant glycoside intercalates into eukaryotic cell membranes by reversibly interacting with this steroid (
Taken together, the present findings demonstrate semiquantitative IL-8 expression at the single-cell level and its ultrastructural localization in stimulated HMC-1 cells. Furthermore, this study provides a strategic protocol established by flow cytometry that allows rapid optimization of fixation and permeabilization conditions for detection of intracellular antigens in mast cells by pre-embedding immunoelectron microscopy.
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Acknowledgments |
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Supported in part by "Forschungsprojektschwerpunkt" of the Free University of Berlin.
We are grateful to Prof Schnoy from the Department of Pathology, Virchow Clinics, Berlin, for making available the facilities of transmission electron microscopy, to Ms Lajous-Petter for helpful suggestions and advice, and to Ms Pröhl for photographic assistance.
Received for publication October 25, 1996; accepted February 13, 1997.
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