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Keywords:

  • leucocyte scintigraphy;
  • eosinophil;
  • neutrophil;
  • ECP;
  • MPO

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

99mTc-HMPAO (Ceretec) labelling of leucocytes is used clinically for the detection of inflammatory processes in the body. This study investigated the mechanisms by which 99mTc-HMPAO is taken up by eosinophils and neutrophils. Blood cells were labelled with 99mTc-HMPAO and the cells separated by means of their densities in Percoll gradients. For other purposes, eosinophils and neutrophils were purified by means of the MACS system and, after labelling these pure cellular preparations, the cells were ultrasonicated and the organelles separated on sucrose density gradients by means of ultracentrifugation. Organelles were characterized by their morphology on electron microscopy. Granulocytes were stimulated to secrete their granule constituents by means of exposure to complement-coated particles. ECP (eosinophil cationic protein) and MPO (myeloperoxidase) were measured using specific immunoassays. The uptake of 99mTc-HMPAO was 15–25-fold higher in eosinophils than in other leucocytes. 99mTc-HMPAO was predominantly stored in the secretory granules of eosinophils and released from the eosinophil, upon activation, together with ECP. A second storage compartment was a very light density organelle of unknown nature. These results indicated that, among leucocytes, 99mTc-HMPAO is preferentially taken up by eosinophils and stored in the secretory granules, which has to be taken into consideration when evaluating images based on this technique. Our findings suggest that 99mTc-HMPAO (Ceretec) may be used as a tool to follow eosinophil turnover and activity in disease.

Labelling of blood leucocytes with 99mTc-HMPAO (Ceretec) and reinjection of the cells to patients has been used extensively in the search for inflammatory processes (Peters et al, 1986; Roddie et al, 1988). The most common clinical uses have been the search for abscesses and the demonstration and estimation of inflammatory activity of the intestine in patients with inflammatory bowel diseases (IBD) and, in this respect, the method has a very high sensitivity and specificity. The major cell attracted to abscesses is the neutrophil, whereas in inflammatory bowel diseases other cells, such as the eosinophil, are a common finding (Hällgren et al, 1989; Dvorak et al, 1993; Levy et al, 1997). Other diseases in which eosinophils are prominent are allergic diseases and, in particular, asthma (Venge et al, 1996). The role of the eosinophil in disease is still unclear, although the cell is armed with an impressive array of cytotoxic principles, such as cytotoxic proteins, e.g. ECP (eosinophil cationic protein), and the capacity to produce large amounts of oxygen free radicals, which may cause injury to tissues when released to the environment. One obvious example of the potential harmfulness of the eosinophil is the hyper-eosinophilic syndrome in which any organ may be affected.

In pioneering work, it was shown that 99mTc-HMPAO would preferentially bind to granulocytes when added to blood in vitro and that the binding to the granulocytes was more stable than to mononuclear cells (Peters et al, 1986; Roddie et al, 1988). It has also been shown that 99mTc-HMPAO binds more efficiently to eosinophils than to neutrophils, but it is still unclear to what compartment the binding occurs (Puncher & Blower, 1994). A preferential binding to eosinophils may suggest that the accumulation of leucocytes labelled with 99mTc-HMPAO preferentially reflects the accumulation of eosinophils, a notion that may explain the high sensitivity of this method in the detection of eosinophil-involving diseases such as inflammatory bowel disease. However, to substantiate this it seemed of importance to further study the relationship of 99mTc-HMPAO with eosinophils. In this report therefore, we have defined the intracellular compartments in which 99mTc-HMPAO is concentrated in eosinophils and suggested a mechanism by which this takes place.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

99mTc-HMPAO (Ceretec) was obtained from Amersham. Labelling of cells or purified granules with 99mTc-HMPAO was performed at room temperature for 15 min or as otherwise indicated in results or figure legends, according to the instruction of the manufacturer. In some experiments, cells were preincubated for 30 min with inhibitors such as cytochalasin B, iodide acetate (IAc) (Sigma Chemical, USA) or vinblastin (Eli Lilly, USA).

Venous blood was obtained from apparently healthy non-smoking subjects. Heparin was used as the anticoagulant. For some experiments, blood cells were separated on discontinuous Percoll gradients and, for other experiments, ‘pure’ eosinophils and neutrophils were obtained using the MACS system (Miltenyi, Germany) (Hansel et al, 1991).

Separation of leucocytes on Percoll gradients was performed as follows: 20 ml of blood was sedimented by means of the addition of an equal volume of 2% Dextran T 500 (Pharmacia & Upjohn AB, Sweden). The plasma containing the leucocytes was harvested. The remaining red cells were haemolysed by H2O and the leucocyte suspension washed in phosphate-buffered saline (PBS) by centrifugation. The leucocytes were finally suspended in 2 ml of PBS and the number of cells counted by means of a Bürker chamber. After labelling the cells, the cell suspension was applied on a discontinuous Percoll gradient with densities of 60%, 65%, 70%, 75%, 80% and 90% Percoll (Pharmacia & Upjohn AB). After centrifugation at 600 g for 35 min at 20°C, without the brake, the fractions were obtained. The fractions were washed twice in PBS after which the cell numbers and differentials were performed. The cell numbers were adjusted to 50 000 per vial and the radioactivity counted in each fraction.

Purification by means of the MACS system was performed as follows: 60–80 ml of blood was mixed with equal volumes of PBS. A Percoll solution of 67% was applied at the bottom of the tube. After this the tubes were centrifuged for 30 min at 1000 g at 20°C without the use of the brake. The cells above the Percoll solution containing the mononuclear cells were discarded and the remaining cells below the Percoll solution were haemolysed with water 2–3 times and washed with PBS containing 2% newborn calf serum. The cell numbers were counted and 15 μl of CD16-coated microbeads were added per 10 × 106 cells. The suspension was incubated for 1 h at +4°C. After this, the cell suspension was diluted with 0·5 ml of PBS containing 2% newborn calf serum per 10 × 106 cells and applied to the magnetic column. The purity and number of the eosinophils running through were measured. The purity of eosinophils was generally > 98% with neutrophils as the only contaminating cells and the viability was > 95%, as judged by trypan blue exclusion. The neutrophils remaining in the column were eluted after removal of the magnet and the purity and number were measured. The cell numbers were adjusted as indicated under results and the cells labelled using 99mTc-HMPAO.

Separation of subcellular organelles was performed by means of ultracentrifugation of the organelles on a continuous sucrose gradient with densities from 20% to 60%, as described in detail previously (Garcia et al, 1985). Briefly, the cell preparations were ultrasonicated for 30 s on ice. After this, the preparations were centrifuged at 1000 g to eliminate whole cells, nuclei and debris. The supernatants thus obtained were applied on the sucrose density gradient and ultracentrifuged for 18 h at 100 000 g in a swing-out rotor. The material in the gradient was harvested in 40 fractions, and the radioactivity and the ECP and myeloperoxidase (MPO) concentrations measured in each fraction. Density in the fractions was measured using refractometry and expressed as g/ml.

For the purpose of morphological characterization of the granules using electron microscopy, the dense fractions containing ECP were pooled in Beckman tubes and diluted with 10 mmol/l Pipe's buffer to 8·5 ml. The fixative solution (8% glutaraldehyde, 1% paraformaldehyde in cacodylate buffer, pH 7·4) was added up to 17 ml and samples were left on ice for 2 h. After this, the granules were pelleted by centrifugation in a Beckman ultracentrifuge at 100 000 g for 25 min at +4°C. The samples were washed in buffer and post-fixed with osmium tetroxide. Dehydration was performed in ethanol (in steps from 75%, 90% and 95% and twice in absolute ethanol) and embedded in Spurr's resin (Agar Scientific, UK) with a diamond knife (Diatome, Switzerland) and stained with uranyl acetate and lead.

The release of 99mTc-HMPAO and ECP from eosinophils was performed by incubation of a mixture of granulocytes (3 × 106/ml) with and without serum-treated G15 Sephadex particles (Pharmacia & Upjohn AB) for various times at 37°C (Winqvist et al, 1984), after which the material in the supernatants was measured and expressed as percentage release of the total material in the cells. The choice of opsonized Sephadex particles as secretagogues is based on the assumption that eosinophils are secretory cells and the knowledge that ligands coupled to large surfaces are superior stimulants of eosinophil secretion.

ECP and MPO were measured using specific immunoassays according to the instructions by the manufacturer (Pharmacia Diagnostics AB).

Gel filtration of 99mTc-HMPAO and purified ECP (a gift of Dr Agneta Trulson) was performed using a NAP-10 column (Amersham Pharmacia Biotech, Uppsala, Sweden) using a phosphate buffer, 0·05 mol/l, pH 7·4, containing 0·9% NaCl.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

The labelling of blood leucocytes was studied by the addition of Ceretec to a mixture of cells after which they were separated on discontinuous gradients of 60–90% Percoll. After separation of the cells, the cells at each density step were harvested separately. The cell and differential counts were performed on each fraction and the radioactivity measured. Two experiments were performed and presented in Table I. It is seen that the radioactivity is increased downwards in the gradient together with the proportion of eosinophils. In Fig 1, a correlation between the radioactivity in the gradient and the proportion of eosinophils is shown, showing a significant linear relationship (r = 0·92, P < 0·0001). At densities with few or no eosinophils present, the binding in these particular experiments was about 100 000 cpm compared with the activity in the last fractions with about 15% eosinophils, which was approximately fourfold higher. Extrapolating to 100% eosinophils would give a 20–25-fold higher binding of 99mTc-HMPAO to eosinophils than to neutrophils.

Table I.   The distribution of 99mTc-HMPAO (Ceretec) to various leucocyte populations after separation in a Percoll density gradient.
 Eosinophils (%)Neutrophils (%)Mononuclear cells (%)Radioactivity, cpm
Percoll fraction (%)Exp 1Exp 2Exp 1Exp 2Exp 1Exp 2Exp 1Exp 2
1 (60)0·63·26·49·090·884·2159618119408
2 (65)03·636·087·262·28·896638169557
3 (70)0·23·497·895·21·81·490555162310
4 (75)0·49·699·289·80·40·6108621215808
5 (80)4·215·295·084·20·80·6167555575525
6 (90)14·016·885·881·00·22·2427699384333
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Figure 1.  Correlation between the proportion of eosinophils in a density gradient separation of blood leucocytes and the activity of 99mTc-HMPAO (Ceretec). The coefficient of correlation (r) and the P-value are indicated.

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In order to identify the cellular compartments in eosinophils and neutrophils that bind 99mTc-HMPAO, we purified these cells using the MACS system. In one of three similar experiments shown in Figs 2 and 3, the purities of eosinophils and neutrophils were 98·7% and 99·6%, respectively, with the other cell as the major contaminating cell. In particular, no basophils were found in these preparations. After purification, the cellular concentrations were adjusted to 3·8 × 106/ml each. To 1 ml of each of the preparations the same amount of 99mTc-HMPAO was added and allowed to incubate for 15 min at room temperature. After extensive washing four times in PBS, the cell preparations were ultrasonicated and fractionated as described in Materials and methods. As shown in Fig 2, the distribution of radioactivity in the eosinophil gradient was found predominantly in one peak on top of the gradient and in another region at densities of 1·25–1·27 g/ml. This latter peak was mostly divided in two peaks but, regardless of this, the distribution of activity within this region coincided exactly with that of ECP. In Fig 3, the distribution of radioactivity in the neutrophil gradient is shown. In this gradient, the major peak is on top of the gradient with a small but distinct peak at 1·23 g/ml. This latter peak goes together with the second peak of MPO. The activity in the granule-associated region of the eosinophil preparations was about 15-fold higher than in the granule-associated peaks of the neutrophil preparations. After incubation on ice, the uptake of 99mTc-HMPAO in eosinophils and neutrophils was reduced by about 80%, suggesting the dependence on metabolically active cells for the uptake. However, the incubation of eosinophils with cytochalasin B, 10 μg/ml, vinblastin (0·1–10 μmol/l) or iodide acetate (IAc), 0·1 μmol/l, did not affect the uptake.

image

Figure 2.  The subcellular distribution of 99mTc-HMPAO (Ceretec) and ECP among eosinophil organelles after separation on a sucrose density gradient. One of three similar experiments.

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image

Figure 3.  The subcellular distribution of 99mTc-HMPAO (Ceretec) and MPO among neutrophil organelles after separation on a sucrose density gradient. One of three similar experiments.

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The organelles at densities of 1·25 to 1·27 g/ml were morphologically characterized by electron microscopy. The majority of the organelles had the typical appearance of the crystalloid-containing granules of human eosinophils.

The above experiments suggested that the storage of 99mTc-HMPAO in the eosinophils predominantly occurred in the secretory granules. In order to study this further, we labelled eosinophils with 99mTc-HMPAO after which they were exposed to a secretagogue, in this case serum-treated Sephadex particles. As shown in Fig 4, 99mTc-HMPAO was released to the extracellular environment in a time-dependent fashion together with the granule protein ECP. These results indicate that 99mTc-HMPAO was stored in the same compartment as ECP and released by similar mechanisms.

image

Figure 4.  Release of 99mTc-HMPAO (Ceretec) (top) and ECP (bottom) from granulocytes exposed to either complement-coated particles (stimulated) or buffer (unstimulated). The release is expressed as a percentage of the total amount in cellular extracts of the cells before exposure. The results of three different experiments with cells from different donors are shown.

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To further study the mechanisms by which 99mTc-HMPAO is accumulated in eosinophil granules, granules were isolated from either eosinophils or neutrophils, after which 99mTc-HMPAO was added to preparations and the uptake measured as a function of time and a function of concentration. A shown in Fig 5 (top), there was an increased accumulation of 99mTc-HMPAO in eosinophil granules as a function of incubation time. In an additional experiment we showed that, after 15 min of incubation, about 8% of 99mTc-HMPAO was taken up by the eosinophil granules, whereas about 1% of 99mTc-HMPAO was taken up by neutrophil granules. Figure 5 (bottom) shows that the uptake in eosinophils was proportional to the dose given to the granules. These data indicate that 99mTc-HMPAO diffuses into the granules freely and is concentrated in the granules by some mechanism.

image

Figure 5.  The upper panel shows the uptake of 99mTc-HMPAO (Ceretec) in purified eosinophil granules as a function of time. The lower panel shows the uptake of 99mTc-HMPAO (Ceretec) in purified eosinophil granules as a function of dose compared with the 99mTc-HMPAO (Ceretec) left in the supernatant. The different dilutions of 99mTc-HMPAO (Ceretec) were incubated with the granules for 15 min, after which the granule bound radioactivity and the activity remaining in the supernatant were separated by ultracentrifugation at 10 000 g. One of two similar experiments.

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In order to investigate whether 99mTc-HMPAO binds to ECP, 99mTc-HMPAO was chromatographed by gel filtration on a NAP-10 column in the absence and presence of ECP. The elution of 99mTc-HMPAO was identical in both experiments, excluding the possibility that 99mTc-HMPAO binds to ECP.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

In this study, we have confirmed the high selectivity of 99mTc-HMPAO (Ceretec) for eosinophils compared with neutrophils and other blood leucocytes. Our results indicated a 15–25-fold higher uptake by the eosinophils than by the neutrophils, which is somewhat higher, but similar to the results of others (Puncher & Blower, 1994). We have also shown that 99mTc-HMPAO is stored in the eosinophils mainly in two compartments. One of these is most probably the secretory granules, as suggested by the co-localization with the granule protein ECP and the localization of typical eosinophil granules in this compartment. The localization of 99mTc-HMPAO to these secretory granules is also suggested by functional data in which we have found that 99mTc-HMPAO is released in parallel with granule proteins upon activation of the cells with complement-coated particles.

The mechanisms involved in the cellular uptake of 99mTc-HMPAO in eosinophils is not known, but the fact that the activity is distinctly localized in unique compartments indicates some specificity and not just a general distribution among lipid-containing structures. According to our data, no 99mTc-HMPAO was bound to plasma membranes. It is interesting that the second largest peak of activity in eosinophils was localized to the top of the density gradient. Other data (unpublished observations) suggest that the structures found at this localization are mainly small secretory vesicles, presumably with a high lipid content. The major activity associated with neutrophils was at this localization, with only a minor fraction found in the granule compartment, i.e. the primary, MPO-containing granules. Thus, the major difference between eosinophils and neutrophils with respect to uptake of 99mTc-HMPAO is the additional storage of 99mTc-HMPAO in secretory granules of the eosinophils. The uptake of 99mTc-HMPAO does not seem to be active and involve the cell machinery, but rather is dependent on some concentration mechanism in the eosinophil granules, which has still to be defined.

The demonstration of a high selectivity of 99mTc-HMPAO for eosinophils may be important in the clinical interpretation of images based on this technique and may actually be one reason for the successful use of the Ceretec method in the detection of inflammatory processes in the intestine, such as inflammatory bowel diseases (Peters et al, 1986; Roddie et al, 1988). In IBD, the eosinophil is one of the prominent cells (Hällgren et al, 1989; Berstad et al, 1993; Makiyama et al, 1995; Bischoff et al, 1996, 1997; Levy et al, 1997). Our findings also suggest that 99mTc-HMPAO (Ceretec) could be used to study the turnover and accumulation of eosinophils in patients with eosinophilia. In patients with hyper-eosinophilic syndrome a great variety of symptoms are seen, for some of which the underlying mechanisms are obscure (Spry, 1988). Studying the accumulation of eosinophils in these cases by means of the labelling of their blood cells with 99mTc-HMPAO could give some further insight into these mechanisms and be helpful in the clinical management of these patients.

We conclude that 99mTc-HMPAO is selectively taken up by eosinophils by specific mechanisms and that 99mTc-HMPAO is stored in the secretory granules of these cells and actively secreted from this storage place upon activation of the eosinophil. The eosinophil selectivity of 99mTc-HMPAO labelling has to be kept in mind when evaluating images based on this technique. Our findings also suggest that 99mTc-HMPAO (Ceretec) may be used as a tool to follow eosinophil turnover and activity in disease.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

This study was supported by grants from the Swedish Medical Research Council.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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