Eur. J. Irnmunol. 1988.18: 817-820 Single-cell oxygen burst reveals neutrophil heterogeneity 817

Short paper

Rainer Fritzsche and Alain L. De Week

Institute of Clinical Immunology, Inselspital, Bern

1 Introduction

Chemiluminescence microscopy reveals functional heterogeneity in single neutrophils undergoing oxygen burst

Activated polymorphonuclear leukocytes (PMN) respond to various triggers with an oxygen burst, during which the release of reactive oxygen species (ROS) plays a key role in microbial killing. The biological function of the ROS-associated light emissions is not known. However, this particularly weak cell-derived chemiluminescence (CL) may serve as a parameter for the identification of PMN activation. In this study we describe a novel technique which we termed CL microscopy. A microscope-based low-light image-processing system was applied which was sensitive enough to detect single photons, capable of two-dimensional signal accumulation, and digital image analysis. This technique permitted, for the first time, the visualization of the oxygen burst in single cells. Furthermore, quantitative evaluation of cell-derived luminol-enhanced CL revealed functional heterogeneity. Single-cell investigations of activated living PMN of normal human donors showed clear differences in kinetics and intensity of the oxygen burst related to different stimuli. The chemical agent (phorbol 12-myris- tate 13-acetate) induced CL in 83% of PMN. In contrast, the complement-mediated phagocytic stimulation by opsonized zymosan gave much higher light intensities of individual cells, but only in part of the PMN population (30%). CL microscopy presents a new and highly sensitive technique with considerable potential for single- cell analysis in immunological research.

The continuous fluorescent or chemiluminescent light emis- sions by living creatures are commonly observed phenomena, whereas the role of ultraweak luminescence in intracellular interactions or its function in the cellular metabolism still remains to be established [ 1, 21.

Chemiluminescence (CL) of activated polymorphonuclear leukocytes (PMN) during the oxidative metabolic burst is a low-light phenomenon which occurs at the single photon level. PMN activation results in the release of highly reactive oxygen species (ROS); such as H202, '02, *OH, 02-, OC1-; which lead to the emission of single photons via short-lived inter- mediate states of endogenous substrates [3].

The generation of cellular CL is commonly determined with luminometers equipped with one-dimensional photomultiplier tubes which, however, provide only numerical data, and whole cell suspensions (104-106 cells) are required [3]. The minimal number of activated neutrophils needed to detect luminol- enhanced CL was reported [2] to be about 10 to 100. This study describes a novel technique, which we have termed chemiluminescence microscopy. It permits the visualization of a single-cell oxygen burst in human PMN and provides data on their functional heterogeneity to chemical or to phagocytosis- associated stimulation.

[I 65821

Correspondence: Rainer Fritzsche, Institute of Clinical Immunology, Inselspital, CH-3010 Bern, Switzerland

Abbreviations: C L Chemiluminescence PMA: Phorbol 12-myris- taste 13-acetate PMN: Polymorphonuclear leukocytes

2 Materials and methods

2.1 Photonic-microscope system

Cellular CL was monitored with a photonic-microscope sys- tem, constructed by modification of commercially available equipment. Three functionally different parts were combined: a microscope, a camera head and an image-processing compu- ter system. The inverted microscope (IMT-2 Olympus Optical Co. Ltd., Tokyo, Japan) was modified for darkroom applica- tion, inverted heating and maximum light output. Coupled to the microscope is the camera head with a two-dimensional photon-counting tube with two-stage microchannel plates (MCP) and a low-lag vidicon detector (C 1966-20, Hamamatsu Photonics K. K., Hamamatsu City, Japan). Imaging from ordinary low-light in phase contrast down to single photon level (10' to 10' photons/mm2 x sec) was possible. For CL measurements, light signals were amplified with a maximum ratio (lo6) of the MCP, digitally processed and accumulated over defined time intervals in video frame memories (512 x 512 x 16 bit) of the image processor (C1966 AVECNIM, Photoniks K. K.). Cellular light emissions and their time- dependant accumulation were continuously monitored on TV monitors and video taped for documentation. Photographs of CL images were taken from a pseudo-color monitor.

2.2 Cells

PMN were obtained from the heparinized venous blood of normal human donors by methamizol-metrizoat sedimenta- tion, Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) centrifu- gation and hypotonic lysis. The cells were resuspended in phosphate-buffered saline, (PBS-Dulbecco's; Gibco, Grand Island, NY), and kept on ice until use. A sample, about 2 x

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheirn, 1988

818 R. Fritzsche and A. De Weck Eur. J. Immunol. 1988.18: 817-820

Figure 1. Photonic imaging of CL light released during the phagocytosis-associated respiratory burst of activated single human PMN. Mixed cell population (all cells viable, 88% PMN), (a) illumination in phase contrast, (image taken directly before the addition of opsonized zymosan; arrows indicate CL-emitting PMN as identified by comparison with Fig. 1 f ) , (b-f) Photonic imaging after addition of opsonized zymosan; (b) individual photons visualized as dots after one million-fold amplification; (c) photon counting, first period 5-15 rnin, (upper large timing indicates accumulated time of photon counting in minutes, lower small timing indicates total time elapsed since addition of stimulus; digitized images are divided into eight levels consisting of 32 grey values each and displayed in pseudocolors, respectively, b/w-printing); (d) second period, (15-25 rnin), different PMN undergo oxygen burst and subsequently emit light; (e) further screening (31-41 min) demonstrates the decrease of reactive oxygen production; (f) total accumulated image of cellular CL (1 h period photon counting).

Eur. J. Immunol. 1988.18: 817-820

lo5 cells, was transferred to a culture chamber on the inverted microscope platform. After a 10-min equilibration period at 37"C, a triggering solution was added.

Single-cell oxygen burst reveals neutrophil heterogeneity 819

2.3 Triggering agents

A phagocytic stimulus, opsonized zymosan (Sigma; opsonized with fresh human serum according to the manufacturer's instructions), was added together with the nontoxic chemiluminescence enhancer, luminol (Boehringer, Man- nheim, FRG; final conc. 250 pmoYl in PBS, diluted from 100 mmoVl stock solution in dimethylsulfoxide, DMSO). Visual control ensured that all cells in the microscopic field of view were covered with zymosan particles. Alternatively to zymo- san, PMA (Sigma, 1 pmoyl in PBS, diluted from 5 mmoyl stock solution in DMSO) was applied as a purely chemical triggering agent. Following the addition of the trigger- enhancer solution, photonic imaging started under darkroom conditions.

2.4 Image acquisition

Images of the microscopic field of view were acquired in bright light phase contrast (before and after photon counting), as CL images, or fluorescence images (after additional staining pro- cedures). Cellular light emissions were accumulated in video frame memories of the image processor over desired time intervals of partial (e.g. 10-min periods as in Fig. 1 c, d, e) or total CL observation (1 h).

2.5 Additional staining procedures

To confirm cell viability and differentiate activated PMN from other cells, including eosinophils, additional sequential incu- bations with fluorescent viability stain, (acridine orange, ethidium bromide), and chromogenic stain (eosin) were per- formed on the same group of single cells after photon counting was concluded.

2.6 Data analysis and cell identification

Superposition of the different photonic images (Fig. lc-e) resulted in an overall image of the cellular CL due to the phagocytic (Fig. If) or chemical stimulation (images not shown). Quantitative evaluation of CL emission as provided by pulse counting and image analysis features was achieved on a single-cell basis (see Fig. 2).

The comparison of the different sequentially acquired images (bright light, photon accumulation, fluorescence) led to the identification of activated light-emitting cells concerning cell type and viability.

vation (in terms of CL intensity), specific cell type and viability status can be performed. The CL images yield spatial informa- tion; and a quantitation of CL intensity can be obtained on a single-cell basis.

All light-emitting cells were identified as PMN. Furthermore, the results demonstrate that human neutrophils show heterogeneous reactions, in response to chemical phorbol 12- myristate 13-acetate (PMA), as well as phagocytic (opsonized zymosan) stimuli (Fig. 2). A variable percentage of viable PMN does not react in terms of detectable oxygen burst on either stimulus. Among the activated cells there is a wide range of measurable CL intensities between 1.4 and 45-fold background level. The maximally zymosan-activated neu- trophil of a normal donor gave about 2.3 x lo6 counts, com- pared to 5 x lo4 counts background noise, when measured close to chemiluminescent cells; and only 4.2 X 10' counts, attributed to the dark current of the image processorkamera system (all values/1200 pixel X 60 min). PMN of the same donor showed much stronger CL intensities following stimula- tion with zymosan than with PMA. But more cells reacted to the chemical (83%) than the phagocytic stimulus (30%, Table

Onset of CL emission of PMN in response to chemical activa- tion was simultaneous. In contrast, the phagocytic stimulus resulted in a heterogeneous start of the respiratory burst in the same sample with a time delay in numerous cells of more than 10 min (Fig. l c , d). The overall duration of detectable CL emission monitored in single cells lasted between 9 and 70

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3 Results

The oxygen burst of single activated cells could be visualized. The photonic microscope system allows the measurements from small sample sizes, screening of mixed cell populations, identification of single cells and long-term observation of indi- vidual PMN. In addition, a comparison between cellular acti-

rn PHA Zymosan*

Figure 2. Quantitative analysis of single-cell CL with typical distribu- tion within the neutrophil population of normal donors. Each dot represents integrated photon emission of one PMNII h photon count- ing. Dotted lines show background values.

* Data evaluation from same image as Fig. 1 f .

820 R. Fritzsche and A. De Weck Eur. J. Immunol. 1988.18: 817-820

Table 1. Percentage of PMN with detectable light emissions following stimulation

No. of No. of vital ‘?t, PMN with donors PMN/sample CL emission

Mean Range Mean Range

Opsonizedzymosan 10 109 81-122 30 18-41 ?MA 5 102 94-120 83 69-91

4 Discussion

CL microscopy thus provides a unique analytical tool to assess individual cell activation following various stimuli. There are no comparable methods for continuously monitoring low-light emissions during the single-cell oxygen burst in a two-dimen- sional (2D) fashion. Furthermore, the sensitivity of the 2D photon-counting image processing technique for luminol- enhanced CL measurement seems to be superior to other luminometers. The spectral sensitivity of the bialkali photo- cathode (&,,, 350-500 nm) fits the emission characteristics of the commonly used synthetic luminogenic compounds with blue-emitting reactions, such as the phthalazine dione, luminol and the acridinium salt, lucigenin. However, the bialkali photocathode is less sensitive for yellow and red emitters.

In contrast to dye-based determinations of reactive oxygen species, CL microscopy does not require a fluorochrome exci- tation, or any other external light source. Thus, cell-toxic and photo-bleaching effects of dyes are prevented, while the viabil- ity and reactivity of biological specimens are preserved and a stable quantitative data analysis is maintained. On the other hand, a combined single-cell analysis of CL with low-light- level fluorescence investigations on identical cells seems to be promising, e.g. for the additional study of intracellular mes- sengers or associated membrane mobility using conjugated dyes.

PMN play a crucial role in cellular defence mechanisms. Since 1972, it is known that activated human neutrophils emit light during phagocytosis [4]. Now, for the first time, a single cell respiratory burst could be visualized. Studying cellular CL emissions, we discovered large differences in intensity and kinetics of the oxygen burst as well as in the total number of activated individual cells reacting to PMA or to serum-acti-

vated zymosan. Interestingly, less than one third of the PMN reacted to zymosan, which is opsonized with serum comple- ment fractions and triggers the process of phagocytosis mainly via cellular C3b receptors. Whether these activated neu- trophils represent a specific subpopulation or whether their reaction is due to an antigenic priming or preconditioning in vivo will be the subject of further investigations. Besides PMN, various other cell types [2] are capable of CL emission by producing reactive oxygen species.

CL microscopy provides a variety of potential applications for cell biologists as well as in biomedical research and clinical laboratory; for example, the single-cell analysis of physiologic states or disorders, that are associated with an increased or inadequate oxidative product formation [5, 61; multi-label immunoassays, using a combination of fluorescent, chromogenic and chemiluminescent [7] markers or substrates; or recombinant DNA technology with chemiluminogenic probing [8] for the detection of single-cell gene expression. Whether this technique will permit the evaluation of light sig- nals coming from intracellular origin or intercellular interac- tions (following cell-cell contact or via humoral factors) will depend on the future development and availability of new luminogenic compounds [9] with improved CL efficiency.

Received January 1, 1988; in revised form February 19, 1988.

5 References

1 Slawinska, D. and Slawinska, J., in Burr, J. G. (Ed.), Chemi- and Bioluminescence, Marcel Dekker, Inc., New York, Base1 1985, p. 495.

2 Campbell, A. K., Hallett, M. B. and Weeks, I., Methodr Biochem. Anal. 1985.31: 317,

3 Allen, R. C., in Adam, W. and Cilento, G. (Eds.) Chemical and Biological Generation of Exited States, Academic Press, Inc., Orlando 1982, p. 309.

4 Allen, R. C., Stjemholm, R. C. and Steele, R. H., Biochem. Bio- phys. Res. Commun. 1972. 47: 679.

5 McCord, J. M., N . Engl. J. Med. 1985. 312: 159. 6 Weiss, S. J. and Lobuglio, A. F., Lab. Invest. 1982. 47: 5. 7 Whitehead, T. P., Thorpe, G. H. G., Carter, T. J. N., Groucutt, C.

8 Matthews, J. A., Batki, A., Hynds, C. and Kricka, L. J., Anal.

9 Roberts, P. A., Knight, J. and Campbell, A. K., Anal. Biochem.

and Kricka, L. J., Nature 1983. 305: 158.

Biochem. 1985. 151: 205.

1987. 160: 139.