A Photoreactive Fluorescent Marker for Identifying Eosinophils and Their Cytoplasmic Granules in Tissues

Abstract

Here we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photoreactive complexes that produce stable fluorescent emissions. This method worked readily with histological tissue sections 6–60 μm thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed. Deep red (>605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510–590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm). The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable (>10 min). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore. This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers. Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.

The detection of cytoplasmic components, such as granules and granule products, is central to the study of eosinophils, whose prominent specific granules are central to their action in the many pathological conditions in which they are involved. In particular, there is a need to understand the movement and release (“degranulation”) of the eosinophil contents in these conditions. However, aside from basic histochemical stains and some limited subcomponent antibody markers (Keeping and Lyttle 1984; Hallgren et al. 1989), more specific approaches, such as receptor localization and other immunocytochemical and molecular studies, are often hindered by the inherent fluorescent properties of the eosinophil granules or their endogenous enzymes that can interfere or confuse secondary enzyme markers, among other things.

Commonly used histochemical approaches for light microscopy include Luna's stain for eosinophil granules (Luna 1968), Lendrum's chromotrope 2R, and the standard hemotoxylin-eosin or Giemsa stains, all of which highlight the eosinophil cytoplasmic granule because of its basic nature (Spicer and Lillie 1961). The fluorescing compound aniline blue has also been used to identify eosinophils (McCrone et al. 1988).

Luna's protocol utilizes Biebrich scarlet, a chromophore with high specificity for basic proteins. This water-soluble, deep-red anionic dye is synthesized by coupling diazotized 4-amino-1, 1′-azobenzene-3, 4′-disulfonic acid to 2-naphthol and is also known as acid red 66. The four aromatic hydrocarbon ring structures linked by double-bond (azo) nitrogens indicates a strong capacity for electron resonance in this molecule. Spectral absorbance data on this compound showed a broad (450–575-nm) peak with a maxima at 505 nm (Green 1990). These data support the hypothesis that this compound might possess fluorescent capabilities when bound to appropriate substrates that would immobilize the molecule on each side of the azo nitrogens. Here we show that this is indeed so and that this property can be used to study eosinophil biology.

Materials and Methods

Peritoneal Lavage Cell Suspensions

August (AUG) strain of dilute hooded rats (Harlan Olac; Bicester, UK) infected with Nippostrongylus brasiliensis were sacrificed by CO₂ inhalation and peritoneal cells were isolated by creating a small ventral incision in the abdomen. Then 30 ml cold (wet ice) Ca⁺⁺/Mg⁺⁺-free Hank's balanced salt solution (HBSS) (Sigma Chemical; St Louis, MO) was placed in the animal's peritoneal cavity. After gently massaging the animal for 1 min, the HBSS and suspended cells were removed with a disposable syringe. Cells were then centrifuged at 200 X g for 10 min at 4C and the supernatant discarded. The resulting pellet was resuspended in 5 ml HBSS and returned to wet ice.

Intestinal Tissue Specimens

Animals (see above) were sacrificed by CO₂ inhalation before tissue collection. After this, 8 cm of the proximal jejunum, beginning at a point just distal from the duodenal loop adjacent to the pancreatic rests, was ligated into 2–4-cm compartments. The segments were injected with cold (held on wet ice) 10% neutral buffered formalin (NBF), excised, and placed in NBF for 15 minutes at room temperature (RT), with the NBF then replaced with fresh fixative solution and the samples stored at RT until paraffin embedment.

Biebrich Scarlet Staining

Deparaffinized histological sections (6 and 60 μm) of proximal jejunum fixed in 10% NBF, as well as Cytospin3 (Shandon; Pittsburg, PA) slide preparations of peritoneal lavage cells (see above) similarly fixed and unfixed, were used. Preparations were rehydrated in distilled H₂O at RT for 5 min. The slides were then stained in an aqueous solution (pH 6.8) of 1% Biebrich scarlet (Sigma Chemical) for 10 sec (10 min for 60-μm sections) and cleared in running H₂O for 5 min. Slides were dehydrated in 100% ethanol (2 X) and washed twice in xylene before being mounted in Permount (Fisher Scientific; Fair Lawn, NJ) and allowed to dry overnight.

Fluorescence Microscopy

Microscopy was performed on a Nikon FXA epifluorescent research microscope (Nikon; Tokyo, Japan) illuminated by a 100-W mercury arc lamp with a G-2A filter cassette (excitation filter EX510–590, dichroic mirror DM580, barrier filter BA590). Fluorescence intensity measurements using a gray scale of 0 (black) to 255 (white) were recorded by a Hamamatsu XC-77 CCD camera (Hamamatsu Photonics; Kyoto, Japan) and analyzed on a Metamorph Imaging system (Universal Imaging; West Chester, PA). The emission intensities from equal areas (approximately 50 μm²/field) of connective tissue and eosinophil granules were averaged from 10 fields. Images were recorded on Kodak Ektachrome 50 slide film (Eastman Kodak; Rochester, NY) and transferred to Polaroid 59 prints (Polaroid; Cambridge, MA) using a Polaroid Daylab II.

Confocal Laser Scanning Microscopy (CLSM)

The AUG rat samples were observed on an Ultima-Z-312 (Meridian Instruments; Okemos, MI) CLSM with an excitation line at 514 nm and emission filters of 575 nm (short) and 605 nm (long). These images were recorded as TIFF image files on floppy discs and converted to dye sublimation prints on a color digital printer (Tektronix; Wilsonville, OR).

Results

Fluorescent Microscopy

A deep-red fluorescent label was observed in eosinophilspecific granules, red blood cells, some macrophage phagolysosomes, and the granules of large granular lymphocytes (LGLs) (Figures 1A–1D) in the tissues examined. These fluorophore-tissue complexes were the source of emission spectra (>605 nm) on photoexcitation and proved to be stable, producing increased quantum emission for the first minute of continuous photoexcitation and then stabilizing (Figure 2). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in this time frame, including areas of background Biebrich scarlet binding in which photoreactive complexes were not formed (e.g., collagen). The remaining deep-red fluorescent emissions were from the various cell constituents described above, in strong contrast to the dark background.

Confocal Laser Scanning Microscopy

The CLSM provided exceptional resolution of individual eosinophil-specific granules (Figure 1C) through optical sectioning. The Biebrich scarlet-labeled granules were clearly visualized. In addition, three-dimensional reconstructions of successive optical sections resolved specific granule profiles and dispersion patterns previously unobtainable from whole eosinophils in histological sections (Figure 1C). The use of narrow excitation spectra and emission filtration showed that the emission spectra varied in wavelength with some of the Biebrich scarlet complexes (i.e., eosinophil-specific granules vs macrophage phagolysosomes and LGL granules) and were distinctly separable (Figure 1D).

Figure 1

Micrographs of the red fluorescent emissions from the BS-protein complexes in NBF-fixed jejunal lamina propria secondarily labeled with Hoechst 33258 in blue (A) and unfixed cytospin preparations (B). Computer-aided 3D reconstruction of the BS-eosinophil granule complex fluorescence in the tissues of the lamina propria (green) as detected by CLSM (C). The label includes eosinophil-specific granules (^∗) and red blood cells (arrows). Variation in fluorescence emission by different BS-protein complexes as detected by the CLSM (D). Eosinophils (^∗) are red, while macrophage phagolysomes and LGL granules were differentiated by emission (yellow). Red blood cells (arrows) emitted at a similar wavelength as the eosinophil granules. Bars = 10 μm.

Figure 2

The fluorescence intensity profiles of the Biebrich scarlet-eosinophil granule complexes (A) and autofluorescence in background (B) of Nippostrongylus-infected gut sections.

Discussion

Analysis of microscopy specimens by fluorescent markers is not limited to the interference properties of transmitted light. Fluorescent markers provide point sources of narrow-band emission spectra as opposed to the transmission of full-spectrum light through the entire thickness of a sample under brightfield microscopy. Tissue often has a high degree of variability with respect to optical properties such as refractive index and absorption. Therefore, fluorescent markers often provide better 3D imaging than brightfield chromophores and produce exceptional clarity for labeled objects nearest the objective. Moreover, if a given fluorophore provides enough quantum yield at wavelengths sufficiently separate from the excitation wavelength, then a brighter image on a darker field will result. This improvement of signal-to-noise can provide better resolution when imaging in thick biological specimens and/or tissue sections is performed (Inoue 1990). Lastly, the use of CLSM usually requires fluorescent markers. The primary virtue of this technology is high-resolution optical sectioning of biological specimens. Optical sectioning eliminates the structural artifacts and invasive nature of mechanical sectioning and allows the visualization of both living and fixed cells. The shallow depth of field (0.1–0.5 μm) of better CLSMs limits the information gathered to a small section of the entire sample. This eliminates the background and scattered fluorescence produced by the rest of the specimen and improves contrast, clarity, and detection (Wright et al. 1993).

Several properties are necessary for a dye to be useful in marking cells for fluorescent detection. These include spectral properties, chemical properties, and specificity (Stewart 1978). The spectral properties of the Biebrich scarlet complexes described here are exemplary. A good quantum yield was observed at a longer wavelength, minimizing the interference with tissue autofluorescence that is common at shorter spectra. The emission increased over time and then stabilized. This is in sharp contrast to the vast majority of fluorochromes, which tend to photobleach under continuous photoexcitation. Finally, a wide separation of excitation and emission maxima of BS, coupled with the photoreactivity, provides a brighter image against a dark field with a standard filter set.

Biebrich scarlet has a known structure and synthesis, making it readily available in high purity. Solubility of Biebrich scarlet in water at neutral pH is essential for its use in preparation protocols for both fixed and living cells and probably played a role in the fluorophore penetration of thick tissue sections observed in this study. The ability to form covalent bonds with cell constituents prevents dyes from redistributing during tissue preparation and analysis. Biebrich scarlet has two separate sulfonated benzene ring structures. These sulfonic acid groups bind covalently with proteins and other compounds containing amino or sulfhydryl groups (Stewart 1978).

Spicer and Lillie (1961) have described the compound's affinity for basic proteins. Nevertheless, the difference in photoreactivity observed between Biebrich scarlet bound to eosinophil-specific granules and that bound to collagen cannot be a simple matter of fluorophore binding concentration. The fluorophore-eosinophil granule complexes described in this study were clearly photoreactive, increasing their quantum emission and then stabilizing with continuous photo-excitation, whereas background Biebrich scarlet staining photobleached. This characteristic is particularly useful in eosinophils, which possess diffuse autofluo-rescence (520 nm) due to a granule-associated flavin adenine dinucleotide (Fuerst and Jannach 1965; Mayeno et al. 1992). The masking of eosinophil autofluo-rescence by Biebrich scarlet not only provides definitive specific granule profiles not seen by autofluorescence but also allows dual labels with fluorescent markers that emit at this wavelength. Finally, the fluorescent complexes observed in eosinophils were separable from those in the macrophages and LGLs by virtue of their different emission spectra under CLSM.

The specific nature of these fluorescent complexes is unknown. A possible scenario may be a complex between the two sulfonic acid groups of Biebrich scarlet and the amine group and the guanidinium function of arginine, known to be in high concentration in eosinophil-specific granule proteins (Yokota et al. 1984; Egesten et al. 1986; Pimenta et al. 1987). Of interest is that major basic protein, a primary constituent of eosinophil-specific granules, is packaged as an acidic proprotein (pI 6.2) (Barker et al. 1988). However, because most aspects of the eosinophil-specific granule-packaging scheme are unknown, this may have no bearing on the availability of amine groups for binding of Biebrich scarlet. Nevertheless, the amount of complex formation or the saturation of binding by the sulfonic acid groups in Biebrich scarlet may affect emission spectra from the observed cell constituents. One relevant point is the fact that the fluorescent complexes were formed in both fixed and unfixed eosinophils. This indicates the stability of these binding sites under aldehyde fixation.

This investigation has described a new fluorophore for the study of eosinophil-specific granules in individual cells and tissues. Although many investigations are needed to fully characterize the nature of Biebrich scarlet-tissue complexes, a careful analysis of emission spectra and chemical binding influences on this fluorophore may provide new data on the nature of packaging and release of these granule proteins from their cell compartments. The need for fluorescent markers for CLSM and the properties of Biebrich scarlet-eosinophil granule complexes described here demonstrate a strong potential for the use of Biebrich scarlet in future eosinophil-related research.