The intracellular localization of alkaline phosphatase has been determined in human neutrophils with analytical subcellular fractionation by density gradient centrifugation and EM cytochemistry.

Centrifugation on sucrose gradients containing 1 mM EDTA and 5 units/ml of heparin showed that alkaline phosphatase was associated with a membranous component distinct from plasma membrane, mitochondria, specific granules and azurophil granules. There was no resolution from the endoplasmic reticulum. Density gradient centrifugation on a sucrose— imidazole-heparin gradient showed a clear resolution of the alkaline phosphatase-containing membranes from the Golgi and endoplasmic reticulum. Density gradient centrifugation of neutrophils that had been disrupted in the presence of 0·12 mmol/1. digitonin clearly separated alkaline phosphatase-containing membranes from the endoplasmic reticulum. Part of the γ-glutamyl transferase has a similar localization to that of alkaline phosphatase.

EM cytochemistry of neutrophils, neutrophil homogenates and of the density gradient fractions identified alkaline phosphatase-containing granules as irregular-shaped, often tubular, structures. It is suggested that alkaline phosphatase and part of the γ-glutamyl transferase activity are localized to a unique organelle in the human neutrophil.

The intracellular localization of neutrophil alkaline phosphatase (AP) has remained unclear despite many biochemical and cytochemical studies. Several histochemical methods are routinely used to demonstrate enzymic activity by light microscopy. They show that the reaction product is cytoplasmic and that the amount can vary greatly in different clinical situations (Kaplan, 1968). Using different EM cytochemical techniques Nakatsui (1969), Geddes, Kirchen & Marshall (1975) and Borgers, Thone, De Cree & De Cork (1978) demonstrated activity in different neutrophil organelles including plasma membrane, Golgi and various unspecified vesicular structures, whereas Bainton, Ullyot & Farquhar (1971) could only detect activity in certain specific granules at the myelocytic stage of maturation.

Subcellular fractionation studies of rabbit neutrophils clearly demonstrated alkaline phosphatase activity in the specific granule (Baggiolini, Hirsch & De Duve, 1969). Similar studies on human neutrophils have however shown the enzyme to be localized to unidentified membranes (microsomes) and not in the specific granules (West, Rosenthal, Gelb & Kimball, 1974; Bretz & Baggiolini, 1974; Spitznagel et al. 1975). Microsomes is an operational term for the heterogenous collection of membranes, derived from plasma membrane, mitochondrial outer membranes, Golgi and the endoplasmic reticulum, obtained by high-speed centrifugation of tissue homogenates. These components can be resolved by analytical sucrose density gradient centrifugation, particularly if combined with selective membrane perturbants such as digitonin (Amar-Costesec et al. 1974; Mitropoulos, Verkatesan, Balasubramanian & Peters, 1978; Tilleray & Peters, 1976). These techniques have been used to investigate the intracellular localization of alkaline phosphatase in human neutrophils. The fractionation procedures have been complemented by EM cytochemical studies on intact cells, homogenates and density gradient fractions. Special attention has been paid to the use of procedures which minimize enzyme inactivation and non-specific lead precipitation (Wilson, 1973). The results of this combined approach have been to demonstrate the localization of AP to a previously unrecognized organelle.

Cell isolation, analytical subcellular fractionation on sucrose gradients and enzyme assays were performed as previously described (Segal & Peters, 1977; Rustin & Peters, 1978). In brief, neutrophils from 40 ml blood, collected from healthy laboratory personnel, were purified by Dextran sedimentation, centrifugation through Ficoll -sodium metrizoate and hypotonic erythrocyte lysis. For control gradients the cells (97–99% pure neutrophils) were suspended in 0·2 M sucrose containing 1 mM Na2 EDTA, pH 7·2 and 5 i.u./ml heparin. The cells were disrupted in a small Dounce homogenizer by 25 strokes of a tight-fitting B pestle. The homogenate was centrifuged at 800 g for to min to sediment nuclei and undisrupted cells and the supernatant was layered onto a sucrose gradient extending linearly with respect] to volume from density 1·05 to 1·28 with a cushion of density 1·32 g cm−3. After centrifugation at 35000 rev/min for 1 h, fractions were collected and assayed to determine the distribution of marker and unknown enzymes.

Following kinetic studies, certain modifications were made to the enzyme assays described by Kane & Peters (1975). Alkaline phosphatase was assayed at pH 9·3 and acid phosphatase at pH 4·6, both with 4 methylumbelliferyl phosphate substrates. Neutral α-glucosidase was assayed at pH 6·5 whilst lysozyme, malate dehydrogenase, lactate dehydrogenase, 5’-nucleo-tidase, γ-glutamyl transferase and myeloperoxidase were assayed as described by Segal & Peters (1977).

Unsaturated vitamin B12-binding capacity was determined by the charcoal radioassay of Kane, Hoffbrand & Neale (1974). Galactosyl transferase was assayed according to the method of Beaufay et al. (1974). Inhibitor studies were performed with 1 mM Levamisole (Janssen Pharmaceuticals Ltd, Marlow, U.K.).

EM studies were performed on the purified neutrophil preparations, on homogenates and on density gradient fractions. The fractions were centrifuged at 40000 rev/min (100000 g) for 1 h in a 10×10 titanium angle head rotor in an MSE super 75 ultracentrifuge. All specimens were fixed for to min at 4 °C in 0·5% glutaraldehyde in 0·1 M sodium cacodylate buffer pH 7·4. A further 10 ml of cacodylate buffer was added and following centrifugation at 400 g for 10 min the supernatant was removed. The pellet was resuspended in cacodylate buffer and stored at 4 °C for up to 10 days. For alkaline phosphatase cytochemistry the pellets were incubated for 45–60 min at 37 °C in the substrate media of Mayahara, Hirano, Saito & Ogawa (1967).

This contained 28 mM Tris-HCl buffer pH 8·5, 20 mM sodium β glycerophosphate (Sigma Grade 1), 3·9 mM magnesium sulphate and 0·5 % of saturated alkaline lead citrate. The final solution was adjusted to pH 9·4. Inhibitors: Levamisole (1 HIM), L-phenylalanine (50 mM) or glycine (100 mM) were dissolved in the complete incubation medium. Following a brief distilled water rinse, the pellets were postfixed in 1 % OsO4, dehydrated in ethanol and embedded in Araldite using epoxypropane as transitional solvent. Ultrathin sections were stained with 25 % uranyl acetate in methanol for 10 min and viewed in a Philips 301 electron microscope.

Subcellular fractionation studies

The averaged distributions of the principal marker enzymes are shown in Fig. 1. The control experiments, performed in the absence of digitonin, showed that the distribution of alkaline phosphatase (AP) was similar to the light peaks of neutral α-glucosidase and γ-glutamyl transferase, with a modal density of 1·13. The plasma membrane marker 5’-nucotidase had a significantly lighter modal density of 1·12. The dense peak of the bimodally distributed γ-glutamyl transferase and neutral a-glucosidase was similar to the specific granule marker vitamin B12-binding protein. Acid phosphatase also had a broad distribution overlapping the specific granule and the azurophil (myeloperoxidase) marker enzymes.

Fig. 1.

Effect of digitonin on organelle equilibrium density. Isopycnic centrifugation of postnuclear supernatant from control (……) and digitonin-treated (+) granulocyte homogenates. Frequency (±S.D. of 3 experiments) is defined as the fraction of total recovered activity present in the gradient fraction divided by density span covered. The activity present over the density span 1·05-1·10 g cm−3 represents, over an arbitrary abscissa interval, enzyme remaining in the sample layer and is presumed to reflect soluble activity. The percentages of recovered activity are: A, neutral α-glucosidase, 83; B, γ-glutamyl transferase, 92; C, alkaline phosphatase, 86; D, 5’-nucleotidase, 118; E, acid phosphatase, 112; F, malate dehydrogenase, 89; G, vitamin B12-binding protein, 89; H, myeloperoxidase, 99. Control data (8 experiments) taken from Rustin & Peters (1978).

Fig. 1.

Effect of digitonin on organelle equilibrium density. Isopycnic centrifugation of postnuclear supernatant from control (……) and digitonin-treated (+) granulocyte homogenates. Frequency (±S.D. of 3 experiments) is defined as the fraction of total recovered activity present in the gradient fraction divided by density span covered. The activity present over the density span 1·05-1·10 g cm−3 represents, over an arbitrary abscissa interval, enzyme remaining in the sample layer and is presumed to reflect soluble activity. The percentages of recovered activity are: A, neutral α-glucosidase, 83; B, γ-glutamyl transferase, 92; C, alkaline phosphatase, 86; D, 5’-nucleotidase, 118; E, acid phosphatase, 112; F, malate dehydrogenase, 89; G, vitamin B12-binding protein, 89; H, myeloperoxidase, 99. Control data (8 experiments) taken from Rustin & Peters (1978).

Homogenization of the neutrophils in 0·2 mol/l. sucrose containing 0·12 mM/l. digitonin had a highly selective effect on the various organelles when the post-nuclear supernatant was subjected to density gradient centrifugation. Neutral a-glucosidase was relatively unaffected, the less dense peak decreased in density and the denser peak showed a small increase in density. γ-Glutamyl transferase showed a unimodal peak with a density of 1·20 and some skewing towards the low density region of the gradient. The membranous elements associated with AP and with 5’-nucleotidase showed a marked increase in equilibrium density to 1·19 although the distribution in the gradient differed with 5’-nucleotidase showing a broader distribution. Acid phosphatase showed a greater proportion of soluble activity in the digitonin-treated homogenate.

Vitamin B12-binding capacity also shows a greater portion of soluble activity with the particulate component having a greater equilibrium density. Myeloperoxidase shows no significant change in the proportion of soluble activity but the particulate component shows a marked increase in density. The distribution of malate dehydrogenase was identical in the control and digitonin-treated neutrophils.

Fig. 2 compares the distribution of the ‘microsomal’ enzymes in the sucroseimidazole density gradients. The distribution of AP is distinct from that of galactosyl transferase. γ-Glutamyl transferase shows a bimodal distribution with the less-dense component corresponding to that of alkaline phosphatase and the heavier component corresponding to the denser component of neutral α-glucosidase. The less-dense component of this enzyme is distinct from the AP-containing membranes.

Fig. 2.

Distribution of alkaline phosphatase, endoplasmic reticulum and Golgi markers. Isopycnic centrifugation of postnuclear supernatant from granulocyte homogenates (3 experiments). Cells were homogenized in 0·2 mol/1. sucrose containing 3 mmol/1. imidazole -HC1 buffer pH 7·2 and 5 units/ml heparin. For further details see Fig. 1. The percentages of recovered activity are: A, alkaline phosphatase, 114; B, galactosyl transferase, 96; C, γ-glutamyl transferase, 101; D, neutral α-glucosidase, 112.

Fig. 2.

Distribution of alkaline phosphatase, endoplasmic reticulum and Golgi markers. Isopycnic centrifugation of postnuclear supernatant from granulocyte homogenates (3 experiments). Cells were homogenized in 0·2 mol/1. sucrose containing 3 mmol/1. imidazole -HC1 buffer pH 7·2 and 5 units/ml heparin. For further details see Fig. 1. The percentages of recovered activity are: A, alkaline phosphatase, 114; B, galactosyl transferase, 96; C, γ-glutamyl transferase, 101; D, neutral α-glucosidase, 112.

Table 1 shows the effect of Triton, of freezing and thawing, and of Levamisole on the AP activity of neutrophil homogenates. In freshly prepared cells two thirds of the activity is latent. After freezing and thawing the free activity was reduced to a greater extent than the total activity so that the percent latent activity is greater.

Table 1.

Effect of Triton, Levamisole and freezing and thawing on neutrophil homogenate alkaline phosphatase activity

Effect of Triton, Levamisole and freezing and thawing on neutrophil homogenate alkaline phosphatase activity
Effect of Triton, Levamisole and freezing and thawing on neutrophil homogenate alkaline phosphatase activity

Fig. 3 shows the AP activity in the gradient fractions assayed fresh, in the presence of 0·1 mmol/1. Levamisole, or 0·1% Triton and after freezing and thawing. The distribution of the alkaline phosphatase is identical in all experiments. This is strong evidence in favour of homogeneity of the AP-containing membranes in the neutrophil and is consistent with a single intracellular localization of this enzyme.

Fig. 3.

Alkaline phosphatase homogeneity in sucrose gradient fractions. Isopycnic centrifugation of postnuclear supernatant from granulocyte homogenate. For further details see Fig. 1. The percentages of recovered activity from the gradient fractions are: A, free AP, 95; B, AP after freezing and thawing, 93; c, Triton-activated AP, 82; D, Levamisole-inhibited AP, 103.

Fig. 3.

Alkaline phosphatase homogeneity in sucrose gradient fractions. Isopycnic centrifugation of postnuclear supernatant from granulocyte homogenate. For further details see Fig. 1. The percentages of recovered activity from the gradient fractions are: A, free AP, 95; B, AP after freezing and thawing, 93; c, Triton-activated AP, 82; D, Levamisole-inhibited AP, 103.

Ultrastructural studies

Fig. 4 shows that the principle site of localization of AP was in a characteristic cytoplasmic organelle. Heavy deposits of lead outlined a vesicle that had a variety of profiles, mostly regular and irregular spheres and rods. Scattered reaction-product of low density is seen over the nuclei and occasionally on other cytoplasmic organelles. There is no activity on the plasma membrane. Fig. 5 shows a region of the cytoplasm at high magnification with irregularly shaped AP-containing structures: the contents of these organelles were heterogeneous and included dense mural deposits. Inhibitor studies showed that 1 mmol/1. Levamisole and 100 mmol/1. glycine blocked the formation of reaction product.

Fig. 4.

Alkaline phosphatase cytochemistry. Polymorphonuclear leucocyte incubated 60 min at 37 °C for alkaline phosphatase activity. Heavy deposits of lead reaction product can be seen lining the inside of some granules (arrows). Lighter reaction deposits are seen in the nucleus. The plasma membrane, endoplasmic reticulum and other granules are lacking in alkaline phosphatase, × 20250.

Fig. 4.

Alkaline phosphatase cytochemistry. Polymorphonuclear leucocyte incubated 60 min at 37 °C for alkaline phosphatase activity. Heavy deposits of lead reaction product can be seen lining the inside of some granules (arrows). Lighter reaction deposits are seen in the nucleus. The plasma membrane, endoplasmic reticulum and other granules are lacking in alkaline phosphatase, × 20250.

Fig. 5.

Alkaline phosphatase cytochemistry. High-power view of the alkaline phosphatase-containing granules (apg). Also in the field are azurophil (az) and specific (s) granules which contain no reaction product, × 66000.

Fig. 5.

Alkaline phosphatase cytochemistry. High-power view of the alkaline phosphatase-containing granules (apg). Also in the field are azurophil (az) and specific (s) granules which contain no reaction product, × 66000.

Fig. 6 shows AP reaction in a gently homogenized (5 strokes) neutrophil preparation. Various granules and linear AP reaction production can be seen. Fig. 7 shows AP in neutrophil homogenate disrupted with 25 strokes of the tight-fitting pestle: many vesicles containing reaction product are present. Fig. 8 shows similar vesicles in the gradient fraction when this homogenate was subjected to sucrose gradient centrifugation. This fraction has a density of 1·13 g cm−3 and corresponds to the biochemically assayed peak of AP activity. Most of these vesicles and the membranous fragments contain dense reaction product. Fractions containing plasma membrane, specific and azurophil granules did not form alkaline phosphatase reaction product when EM cytochemistry was performed on them.

Fig. 6.

Alkaline phosphatase cytochemistry. Results of 45 min incubation at 37 °C in a gently homogenized preparation (5 strokes). Heavy deposits of reaction product can be seen in some granular (apg) and linear (l) configurations. Other profiles resembling azurophils (az) and specific granules (S) can be seen to be devoid of alkaline phosphatase reaction product, × 18000.

Fig. 6.

Alkaline phosphatase cytochemistry. Results of 45 min incubation at 37 °C in a gently homogenized preparation (5 strokes). Heavy deposits of reaction product can be seen in some granular (apg) and linear (l) configurations. Other profiles resembling azurophils (az) and specific granules (S) can be seen to be devoid of alkaline phosphatase reaction product, × 18000.

Fig. 7.

Alkaline phosphatase cytochemistry. Reaction product in a homogenate after 25 strokes incubated for 45 min at 37 °C. Deposits can be seen lining many membrane vesicles and in a few linear arrangements, × 51 300.

Fig. 7.

Alkaline phosphatase cytochemistry. Reaction product in a homogenate after 25 strokes incubated for 45 min at 37 °C. Deposits can be seen lining many membrane vesicles and in a few linear arrangements, × 51 300.

Fig. 8.

Alkaline phosphatase cytochemistry. Alkaline phosphatase reaction product surrounding vesicles from a sucrose density gradient. Incubation was for 45 min at 37 °C. × 43200.

Fig. 8.

Alkaline phosphatase cytochemistry. Alkaline phosphatase reaction product surrounding vesicles from a sucrose density gradient. Incubation was for 45 min at 37 °C. × 43200.

The alkaline phosphatase (AP)-containing membranes are clearly separated from the plasma membrane fragments by isopycnic gradient centrifugation. This latter membrane was characterized by its content of 5’-nucleotidase (Solyom & Trams, 1972) and by the use of the proximity labels, fluorescamine and Bolton-Hunter reagent (Segal & Peters, 1977). Following digitonin treatment both the plasma and the AP membrane show increases in equilibrium density although the increase differs for each membrane.

Although the endoplasmic reticulum marker enzyme neutral α-glucosidase has a similar distribution in sucrose-EDTA-heparin density gradients, there is clear resolution of this component from the alkaline phosphatase membrane in sucrose-imidazole-heparin gradients.

More striking, however, is the effect of digitonin on these 2 membranes. The AP membranes show an increase in density of 0·06 g cm−3 whereas the endoplasmic reticulum shows a density decrease of 0·02 g cm−3. A similar resolution of these 2 components has been demonstrated for rat liver (Tilleray & Peters, 1976), and human gut (Peters, 1976) and liver (Peters & Seymour, 1978).

The electron micrographs show that the AP membranes are derived from structures which bear a morphological similarity to the Golgi. However, this organelle is very infrequently seen in mature neutrophils, the level of the marker enzyme for this organelle is low and the density gradient experiments clearly resolve this organelle from the AP membrane. Note that the glactosyl transferase shows a bimodal distribution with a low-density component that has a similar distribution to that of the plasma membrane. Localization of glucosyl transferases to plasma membrane (Roth, McGuire & Roseman, 1971; Roth & White, 1972; Weiser, 1973) as well as to the Golgi (Morré, Merlin & Keenan, 1969; Schachter et al. 1970; Amar-Costesec et al. içf]4) is well recognized.

On the basis of these experiments we conclude that AP has a unique localization in the human neutrophil. Avila (1977) after experiments with membrane-solubilizing agents has also tentatively suggested that AP is localized to a second light, subpopulation of specific granules. The function of this organelle remains to be determined. The role of AP and y-glutamyl transferase in the neutrophil, as in other tissues remain speculative, but cytochemical studies (Bainton, 1973; Jenson & Bainton, 1973) indicate an early involvement of this organelle during phagocytosis. In view of the role of cyclic AMP in phagosome-lysosome fusion (Bourne, Lehrer, Cline & Melmon, 1971; Zurier et al. 1974; Lowrie, Jackett & Ratcliffe, 1975; Boxer et al. 1976), we are investigating the possible localization of cAMP-degrading enzymes to this organelle.

A controversial area in the discussion of the types of neutrophil granules is the question of tertiary granules. Previous subcellular fractionation studies (West et al. 1974; Kane & Peters, 1974) have indicated that certain acid hydrolases have a distinctive distribution and are localized to a tertiary or C granule as has been demonstrated by certain morphological studies (Scott & Horn, 1970) but not by others (Farquhar, Bainton, Baggiolini & De Duve, 1972; Bainton & Farquhar, 1966). The density gradient experiments on digitonin-disrupted neutrophils provides further evidence in favour of this hydrolase-containing granule: the distribution and solubilization of acid phosphatase differs from the specific and azurophil granule marker enzymes.

The precise role of these various structures in the microbicidal role of neutrophils remain to be elucidated. Combination of subcellular fractionation techniques with cytochemical studies particularly in actively phagocytic cells should prove profitable as has been shown in the present study.

We thank Mr P. White and Mrs R. Watson for expert technical assistance and Ms Jean de Luca for typing the manuscript. G.J.S.R. and T.J.P. receive support from the Leukaemia Research Fund.

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