Journal of Immunological Methods, 23 (1978) 69--78

69

© Elsevier/North-Holland Biomedical Press

PHAGOCYTOSIS OF TECHNETIUM-99m SULFUR COLLOID BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES *

PRADIP K. R US T A G I and ALBERT F. LoBUGLIO * *

Ohio State University, Division of Hematology and Oncology, Columbus, 0H43210, U.S.A. (Received 27 December 1977, accepted 10 March 1978)

This study characterizes a new phagocytic assay system utilizing technetium-99m sulfur colloid as the phagocytic particle. Uptake of sulfur colloid by human polymorphonuclear leukocytes is a time and temperature dependent process that requires glucose for optimal uptake. In contrast to many other systems, sulfur colloid phagocytosis appears to be serum and divalent cation independent. An attractive feature of this system is the 10-fold increase in particle uptake with phagocytosis as compared to that at zero time.

INTRODUCTION

Technetium-99m sulfur colloid (TcSC) is used exl~ensively in clinical practice as an imaging agent of the reticuloendothelial system (RES). The virtually exclusive in vivo uptake of these and other particles by the RES has been attributed to the efficiency with which fixed macrophages ingest foreign colloidal materials (Saba, 1970). Little is known regarding the characteristics of ingestion of these particles by phagocytic leukocytes in vitro (English and Andersen, 1975; McAfee and Thakur, 1976). Several characteristics of this material suggested that it might be of interest to study in terms of its use as a phagocytic particle. First, this colloidal suspension does not sediment readily and should be easily separated from leukocytes during centrifugation. Second, the isotope is a strong gamma emitter which simplifies radioactive counting and procedures have been established for the easy production of relatively standardized and stable colloidal suspensions. Quantitative data on leukocyte TcSC uptake kinetics and energy requirements for TcSC uptake by phagocytic cells are not presently available. This study was undertaken to characterize an in vitro phagocytic system utilizing TcSC as the particle and the human granulocyte as the phagocyte.

* This work was supported by the National Cancer Institute Contract N01-CB-53936. ** Reprint requests to: Albert F. LoBuglio, M.D., University Hospital N-1022, 410 W. 10th Ave, Columbus, OH 43210, U.S.A.

70 METHODS

Cells

PMNs were obtained from normal human volunteers by dextran sedimentation of peripheral venous blood with subsequent shock lysis to remove contaminating red cells as previously described (Rinehart et al., 1974). Cell suspensions were washed with Selegman's balanced salt solution (SBSS), counted (Coulter Electronics, Inc.), and adjusted to a concentration of 107 granulocytes/ml SBSS. L e u k o c y t e preparations were 70--90% granulocytes, 10--20% l y m p h o c y t e s and 3--8% m o n o c y t e s by supravital staining and 95--100% viable by trypan blue exclusion. Mononuclear cell preparations were obtained by Ficoll--Hypaque separation of heparinized blood (Rinehart et al., 1974) and were 60---70% lymphocytes, 25--35% m o n o c y t e s and 98%) were obtained by resuspending the donor cell b u t t o n from the Ficoll--Hypaque separation followed by dextran sedimentation and shock lysis to remove contaminating red blood cells. Purified l y m p h o c y t e preparations (>98% lymphocytes) were obtained by preincubation of blood with iron particles in 4% dextran followed by Ficoll--Hypaque density centrifugation (King et al., 1975). Particles

TcSC was prepared from a Mallinckrodt kit. Four to 6 mCi of 99m Tc-sodium pertechnetate eluted from a 500 mCi 99 Mo-99m Tc fission generator (Mallinckrodt, Inc.) was used to label the sulfur colloid. The TcSC thus prepared was centrifuged at 6785 X g for 10 min in a Sorvall RC-5 superspeed refrigerated centrifuge to remove larger particles. This procedure consistently resulted in sedimentation of 75--80% of the radioactivity. The supernatant was aspirated and served as the source of 'small' particles in the phagocytic assay. There was no further loss of activity from the supernatant when the small particles were centrifuged at 200 X g, indicating that those sulfur colloid particles capable of being separated o u t at cell-sedimenting forces had been removed by the centrifugation procedure. Centrifugation at 100,000 X g sedimented >95% of the radioactivity in this small particle suspension indicating that the pertechnetate was attached to particulate colloid. The TcSC particles were sized by a polycarbonate film filter radioactivity retention technique (Davis et al., 1974). Prior to centrifugation, 50% of the TcSC was retained by a 0.4 pm Nuclepore membrane. (This correlated with a particle diameter range of 0.1--0.7 t~m as measured by scanning electron microscopy). The small particle preparation was n o t retained by the 0.4 pm filter, b u t 30% was retained b y a 0.1 pm Nuclepore filter. The 'small particle' preparation was used for all subsequent experiments and will be simply referred to as TcSC.

71

Serum Autologous serum was obtained from clotted whole blood and used fresh in the complement-active state.

TcSC uptake After establishing the characteristics of particle uptake, a standard assay was designed. Siliconized glass tubes (100 mm X 16 mm) containing 0.4 ml autologous serum, 1.0 ml Hank's balanced salt solution (HBSS), and 0.1 ml TcSC were preincubated at 37°C for 30 min. 0.5 ml of granulocyte suspension (5 × 106 granulocytes) was warmed to 37°C and added to the assay tubes for a total reaction volume of 2 ml. The tubes were plugged with tightfitting siliconized rubber stoppers and incubated at 37°C for 30 min on a rotating mixer. The viability of cells following this 30 min incubation was 92--98%. The reaction was terminated by the addition of 9 ml ice-cold normal saline containing 1 mM N-ethyl maleimide (NEM) followed by p r o m p t centrifugation of the tubes at 200 X g for 10 min. Zero time values were obtained by adding cells at 37°C to assay tubes that had been cooled to 4°C, immediately adding the NEM wash, and sedimenting the cells. After the supernatant had been decanted, the cell pelle.ts were resuspended in fresh NEM in saline as before and washed a second time. The final cell b u t t o n was resuspended in 1 ml HBSS and transferred carefully to counting tubes for determination of cell-associated radioactivity. Radioactive counts were obtained in a Packard model 3003 Auto gamma scintillation spectrometer. Each determination was performed in duplicate and the mean calculated; individual values were within 5% of the mean. The uptake of TcSC was expressed in terms of counts per minute (cpm) or converted to grams of elemental sulfur X 10-s/5 × 106 granulocytes/30 min.

Electron microscopy Scanning electron microscopy (SEM) was performed on a Cambridge Stereoscan Mark IIa microscope. Specimens from a standard assay were fixed in 2% phosphate-buffered glutaraldehyde, sedimented onto aluminum foil in a cytocentrifuge (Shandon-Elliot, Inc.), dried and gold-coated for SEM studies as previously described (Rinehart et al., 1971). Transmission electron microscopy (TEM) was performed on a Philips 300 electron microscope. Specimens were taken from a standard assay using purified granulocyte cell preparations. Control preparations were made by utilizing TcSC which had been made particle free by centrifugation at 100,000 X g and then used in the standard assay. Cell pellets were fixed in 2% phosphatebuffered glutaraldehyde, p H 7 . 4 , post-fixed in 1% buffered osmium tetroxide and e m b e d d e d in Araldite 502. Thin sections were routinely

72

stained with lead citrate and uranyl acetate as previously described (Rinehart et al., 1975). RESULTS

Fig. 1 illustrates the uptake of TcSC with time by two normal individuals with differing uptake capacities. The reaction was terminated at each time interval by the addition of 4.5 vol of cold 1 mM NEM in saline. The uptake in both cases increased linearly for 30 min and then remained constant up to 45 min. Zero time and coincubation of particles and cells for short intervals at 37°C (2 min) produced low uptake values. Fig. 1 also illustrates the ability of cold NEM to prevent particle uptake in that its addition at zero time inhibited uptake over the subsequent 45 min time period. The efficient separation of particles from the leukocytes is obvious from the NEM incubation system. These results provided the rationale for using a 30 min incubation period for subsequent studies and for terminating the reaction by diluting the system with NEM at 4°C. Thirty minute uptake of TcSC increased linearly with increasing dosage of sulfur up to 21 pg. At doses >21 pg, no further increase was noted and at higher doses, inhibition of uptake was seen suggesting toxic effects of the

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I~) 30 4; INCUBATION TIME (MINUTES) Fig. 1. T i m e course o f T c S C u p t a k e b y t h e l e u k o c y t e s o f t w o n o r m a l individuals. Results o b t a i n e d u n d e r t w o d i f f e r e n t c o n d i t i o n s of i n c u b a t i o n are s h o w n . Specific activities o f t h e T c S C used in each e x p e r i m e n t were 2.0 X 103 c p m / 1 0 -8 g sulfur (solid lines) a n d 3.8 × 103 c p m / 1 0 -s g sulfur ( d o t t e d lines).

73

colloidal suspension at high concentrations. Uptake at zero time was negligible at all doses and there was no dose effect on zero time uptake. For subsequent experiments, 21 ~g of sulfur (0.1 ml TcSC) were used. The uptake of TcSC was found to be linear with increasing granulocyte number while zero time uptake remained at approximately 10% of the 30 min uptake (Fig. 2). A cell number of 5 × 106 were used in the standard assay to insure that particles were present in excess. We then used the standardized assay to examine the characteristics of TcSC uptake by granulocytes. The effect of temperature on TcSC uptake by PMNs is depicted in Fig. 3. Uptake was only 38 _+ 7% of control at 25°C and 13 + 1% of control at 4°C. To examine the role of serum factors, 5% human albumin (Travenol Laboratories, Inc.} were substituted for fresh serum and resulted in no impairment of particle uptake. In fact, uptake of TcSC in protein-free media was identical to that in the serum system. Thus, uptake of TcSC did not appear to be dependent on serum factors (opsonins). Uptake of TcSC in the serum-free system was inhibited 85% by 4°C temperature similar to the temperature dependence noted in the standard assay. To examine the role of Ca 2÷ and Mg 2÷, TcSC uptake was examined using Ca 2÷- and Mg2÷-free SBSS and 5% albumin. No impairment of uptake was noted. Addition of 4 mM EDTA did not adversely affect TcSC uptake by granulocytes. To examine the metabolic requirements for TcSC hptake, granulocyte preparations were preincubated for 10 min with metabolic inhibitors and

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Fig, 2. E f f e c t o f cell n u m b e r on TcSC uptake, Results o b t a i n e d at zero t i m e and following 30 rain i n c u b a t i o n are s h o w n . T h e data are from 3 experiments. Fig. 3. E f f e c t of temperature o n TcSC u p t a k e . U p t a k e achieved at each i n c u b a t i o n temperature is expressed as the per cent o f u p t a k e achieved at 37°C. The bars represent the m e a n -+ S.E. o f 3 experiments.

74

then used in the assay. Under these conditions, 1 mM NEM produced 57 ± 17% (n = 3) inhibition of particle uptake. 1 mM 2,4-dinitrophenol, an inhibitor of oxidative phosphorylation and 0.1 mM p-chloromercurobenzene sulfonic acid, a membrane sulfhydryl blocking agent, produced no significant inhibition of TcSC uptake in 3 experiments. These observations suggested that TcSC uptake may be dependent on glycolysis. To examine" this more directly, TcSC uptake was examined in glucose-free media and found to be 45 _+ 19% (n = 3) of control values. Fig. 4 illustrates the role of glucose in particle uptake. Cells isolated in glucose free media and placed in the assay with no glucose had 56% inhibition of TcSC uptake. The uptake was completely restored to control values by addition of glucose in physiologic concentrations just prior to particle contact. To assure ourselves that the assay was not simply reflecting pinocytosis (cell ingestion of soluble media), free Tc-99m pertechnetate was substituted for TcSC in the assay system. No cell associated radioactivity was noted over a 30 min period of incubation. The uptake of TcSC by m o n o c y t e s and lymphocytes was compared to that of granulocyte preparations. As can be

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Fig. 4. E f f e c t o f glucose o n TcSC uptake. U p t a k e achieved in the standard assay s y s t e m served as the c o n t r o l value. U p t a k e achieved b y the same individual's cells, isolated in glucose-free m e d i a and i n c u b a t e d w i t h particles in the absence and presence o f glucose (1 m g / m l ) is e x p r e s s e d as per cent o f the control value. Fig. 5. U p t a k e o f TcSC b y m o n o n u c l e a r cells and a purified l y m p h o c y t e preparation. Results obtained at zero time and f o l l o w i n g 30 m i n i n c u b a t i o n are s h o w n . T h e bars represent the u p t a k e b y s u s p e n s i o n s c o n t a i n i n g 5 × 106 m o n o c y t e s or l y m p h o c y t e s . T h e data are from s i m u l t a n e o u s e x p e r i m e n t s on one donor.

75

1 J

Fig. 6. Transmission electron photomicrographs (original magnification: 10,260 diameters). A: granulocyte following 0 min incubation with TcSC. Few phagocytic vacuoles are seen. B : granulocyte following 30 min incubation with TcSC. Numerous large phagocytic vacuoles can be seen. C: granulocytes following 30 min incubation with a particle-free preparation. Phagocytic vacuoles are absent.

seen in Fig. 5, m o n o c y t e uptake appears similar to that established for granulocytes while purified l y m p h o c y t e preparations had negligible uptake. Uptake by purified granulocytes (not shown) was identical to m o n o c y t e uptake in 3 experiments. Examination of granulocyte preparations following 30 min incubation with TcSC by scanning electron microscopy failed to reveal surface bound particles on 50 cells examined although the particles themselves could be visualized when placed on membrane filters and examined by SEM. Transmission electron microscopy (TEM) of granulocytes incubated with TcSC or particle-free preparations (100,000 X g supernate) are illustrated in Fig. 6. Numerous phagocytic vacuoles are present in the TcSC preparation. These vacuoles are considerably larger than the TcSC particles and probably represent fusion of smaller phagosomes. The particles themselves are n o t visualized since they are dissolved by the fixation and staining procedures. Finally, the uptake of TcSC by the granulocytes of 15 normal individuals

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was determined (Fig. 7). 2.2 _+0.9 × 10 -8 g sulfur was taken up by PMNs at zero time, compared with 23 _+3.9 × 10 -8 g after 30 min. DISCUSSION

The major hurdle in the interpretation of leukocyte--particle interaction is the differentiation between cell surface adherence versus actual ingestion (phagocytosis). Stossel has recently reviewed criteria characterizing cell surface adherence and phagocytic systems (Stossel, 1975). Using these criteria, the interaction of TcSC with human granulocytes appears to reflect almost exclusively a phagocytic system. The zero time values are low with a linear uptake with time, uptake is severely restricted at 4°C and is energy dependent (56% inhibition in the absence of glucose) and uptake can be saturated by excess particles. In addition, SEM failed to reveal surface bound particles and TEM studies revealed phagocytic vacuoles. Finally, phagocyte deficient l y m p h o c y t e preparations failed to demonstrate uptake of TcSC as would occur with non-specific surface adherence to cell surfaces. A good example of the use of these criteria can be seen in a study by Davies that indicates that the interaction between latex particles and leukocytes in serum-free media reflects surface adherence while in the presence of fresh sera reflects phagocytosis (Davies et al., 1975). A less difficult differentiation between phagocytosis and pinocytosis is readily made in cell systems of this type. Since pinocytosis is the nonspecific ingestion of media at the cell surface, it usually requires hours of incubation rather than reaching a m a x i m u m plateau at 30 min. In addition,

77 labeling of the surrounding media with radioactive non-particulate technetium 99m failed to produce any radioactive uptake under conditions identical to that used for uptake of TcSC. The characteristics of TcSC uptake by granulocytes are similar to some other phagocytic particles b u t unique in several ways. The linear uptake over a 30 min time period is similar to that seen with latex particles b u t considerably longer than the 4--6 min noted using albumin coated parafin oil emulsions. The lack of serum dependence in terms of uptake is quite unusual and suggests that TcSC phagocytosis is independent of gamma-globulin and complement receptors on the cell surface. This is in contrast to the commonly used particles (red cells (Greendyke et al., 1963), bacteria ( R o o t et al., 1972), latex particles (Davies et al., 1975), parafin oil emulsions (Stossel, 1973), etc.) where serum factors play an important role in phagocytosis. The lack of dependence on divalent cations is also unusual although carbon particle ingestion has been reported to occur in their absence (Metzger and Casarett, 1967). It is unclear what properties of the TcSC account for these differences although particle size, electrostatic charge and chemical characteristics are potential factors. However, gold colloid particles of a similar order of magnitude in size as the sulfur colloid particles used in our studies have been noted to be taken up poorly by PMNs (Gosselin, 1967). When we examined the TcSC uptake by various blood leukocytes, we were somewhat surprised to find that m o n o c y t e uptake of TcSC was comparable to that of granulocytes. It has been reported that human monocytes have 'sluggish' phagocytic capacity as compared to granulocytes (Territo and Cline, 1977). Whether this finding is related to characteristics of TcSC which make it more palatable to cells of the reticuloendothelial system or differences in technical aspects of m o n o c y t e preparation is unclear. TcSC uptake by m o n o c y t e s has similar characteristics to that of granulocytes with respect to kinetics, serum dependence, and divalent cation dependence (manuscript in preparation). Several practical aspects of TcSC make it a useful particle for studies of phagocytosis. The material is readily available and inexpensive in that most Nuclear Medicine departments prepare and utilize large amounts daily. The short half-life and gamma emission of Tc make it a relatively safe isotope with little or no need for sample preparation in order to measure radioactivity. The ability to stop the reaction readily with cold NEM allows multiple samples to be studied concurrently. Finally, studies in 15 normal individuals revealed approximately a 10-fold increase between zero time and 30 min uptake. Thus, TcSC appears to be a useful particle for studies of leukocyte phagocytosis. Since it does n o t depend on serum factors, its utilization can allow evaluation of cell phagocytic capacity independent of complement and immunoglobulin receptor activity.

78 ACKNOWLEDGEMENTS We w o u l d like t o a c k n o w l e d g e t h e R o e s s l e r F o u n d a t i o n f o r s u p p o r t o f P r a d i p K. Rustagi; t h e e x p e r t e l e c t r o n m i c r o s c o p y o f Dr. G. A d o l p h A c k e r m a n , D e p a r t m e n t o f A n a t o m y , a n d Dr. J o h n Scheu, Division o f N u c l e a r Medicine; a n d H e l e n Ilc f o r h e r aid in m a n u s c r i p t p r e p a r a t i o n . REFERENCES Davies, W., M. Thomas, P. Linkson and R. Penny, 1975, J. Reticuloendothel. Soc. 18, 136. Davis, M.A., A.G. Jones and H. Trindade, 1974, J. Nuc. Med. 15,923. English, D. and B.R. Andersen, 1975, J. Nuc. Med. 16, 5. Gosselin, R.E., 1967, Fed. Proc. 26, 987. Greendyke, R.M., R.E. Brierty and S.N. Swisher, 1963, Blood 22,295. King, G.W., G. Bain and A.F. LoBuglio, 1975, Cell. Immunol. 16,389. McAfee, J.G. and M.L. Thakur, 1976, J. Nuc. Med. 17,488. Metzger, G.V. and L.J. Casarett, 1967, Adv. Exp. Med. Biol. 1, 163. Rinehart, J.J., S.P. Balcerzak and A.F. LoBuglio, 1971, J. Lab. Clin. Med. 78,167. Rinehart, J.J., S.P. Balcerzak, A.L. Sagone and A.F. LoBlugio, 1974, J. Clin. Invest. 54, 1337. Rinehart, J.J., A.L. Sagone, G.A. Ackerman and A.F. LoBulgio, 1975, New Engl. J. Med. 292, 83. Root, R.K., L. Ellman and M.M. Frank, 1972, J. Immunol. 109,477. Saba, T.M., 1970, Arch. Intern. Med. 126, 1031. Stossel, T.P., 1973, J.Celt Biol. 58,346. Stossel, T.P., 1975, Sere. Hematol. 12, 83. Territo, M.C. and M.J. Cline, 1977, J. Immunol. 118,187.