STUDIES ON THE SUBCELLULAR LOCALIZATION OF HUMAN ... · methods are routinel y use tod demonstrate enzymic b activity light microscopyy . They show tha thte reaction produc is cytoplasmit

J. Cell Sci. 36, 401-412 (1979) 401Printed in Great Britain (C) Company of Biologists Limited 1 979

STUDIES ON THE SUBCELLULAR

LOCALIZATION OF HUMAN NEUTROPHIL

ALKALINE PHOSPHATASE

G. J. S. RUSTIN, P. D. WILSON AND T. J. PETERSDepartment of Medicine, Royal Postgraduate Medical School, andImperial Cancer Research Fund Laboratories, London, England

SUMMARYThe intxacellular localization of alkaline phosphatase has been determined in human neutro-

phils with analytical subcellular fractionation by density gradient centrifugation and EMcytochemistry.

Centrifugation on sucrose gradients containing 1 mM EDTA and 5 units/ml of heparinshowed that alkaline phosphatase was associated with a membranous component distinct fromplasma membrane, mitochondria, specific granules and azurophil granules. There was noresolution from the endoplasmic reticulum. Density gradient centrifugation on a sucrose—imidazole-heparin gradient showed a clear resolution of the alkaline phosphatase-containingmembranes from the Golgi and endoplasmic reticulum. Density gradient centrifugation ofneutrophils that had been disrupted in the presence of 012 mmol/1. digitonin clearly separatedalkaline phosphatase-containing membranes from the endoplasmic reticulum. Part of they-glutamyl transferase has a similar localization to that of alkaline phosphatase.

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

INTRODUCTION

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

Subcellular fractionation studies of rabbit neutrophils clearly demonstrated alkalinephosphatase activity in the specific granule (Baggiolini, Hirsch & De Duve, 1969).Similar studies on human neutrophils have however shown the enzyme to be localized

Correspondence to: Dr T. J. Peters, Dept. Medicine, Royal Post-Graduate Medical School,DuCane Road, London W12 oHS, England.

4O2 G. J. S. Rustin, P. D. Wilson and T. J. Peters

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 centri-

fugation, 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.

METHODS

Cell isolation, analytical subcellular fractionation on sucrose gradients and enzyme assayswere performed as previously described (Segal & Peters, 1977; Rustin & Peters, 1978). Inbrief, neutrophils from 40 ml blood, collected from healthy laboratory personnel, were purifiedby Dextran sedimentation, centrifugation through Ficoll - sodium metrizoate and hypotonicerythrocyte lysis. For control gradients the cells (97-99 % pure neutrophils) were suspendedin o-2 M sucrose containing i mM Na2 EDTA, pH 7 2 and 5 i.u./ml heparin. The cells weredisrupted in a small Dounce homogenizer by 25 strokes of a tight-fitting B pestle. The homo-genate was centrifuged at 800 g for 10 min to sediment nuclei and undisrupted cells and thesupernatant was layered onto a sucrose gradient extending linearly with respect] to volumefrom density 105 to 1-28 with a cushion of density 1-32 g cm""'. After centrifugation at 35000rev/min for 1 h, fractions were collected and assayed to determine the distribution of markerand unknown enzymes.

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

Unsaturated vitamin Bjj-binding capacity was determined by the charcoal radioassay ofKane, Hoffbrand & Neale (1974). Galactosyl transferase was assayed according to the methodof Beaufay et al. (1974). Inhibitor studies were performed with 1 mM Levamisole (JanssenPharmaceuticals Ltd, Marlow, U.K.).

EM studies were performed on the purified neutrophil preparations, on homogenates andon density gradient fractions. The fractions were centrifuged at 40000 rev/min (100 000 g)for 1 h in a 10 x 10 titanium angle head rotor in an MSE super 75 ultracentrifuge. All specimenswere fixed for 10 min at 4 °C in 0-5% glutaraldehyde in 0 1 M sodium cacodylate bufferpH 74 . A further 10 ml of cacodylate buffer was added and following centrifugation at 400 gfor 10 min the supernatant was removed. The pellet was resuspended in cacodylate buffer andstored at 4 °C for up to 10 days. For alkaline phosphatase cytochemistry the pellets wereincubated 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 /? glycerophosphate (SigmaGrade 1), 3-9 mM magnesium sulphate and 0-5 % of saturated alkaline lead citrate. The final

Alkaline phosphatase in human neutrophils 403

solution was adjusted to pH 9-4. Inhibitors: Levamisole (1 ITIM), L-phenylalanine (50 mil) orglycine (100 ITIM) were dissolved in the complete incubation medium. Following a brief dis-tilled water rinse, the pellets were postfixed in 1 % OsO4, dehydrated in ethanol and embeddedin Arnldite using epoxypropane as transitional solvent. Ultrathin sections were stained with25 % uranyl acetate in methanol for 10 min and viewed in a Philips 301 electron microscope.

0 J

r—1—r1 0 5 1-15 1-25 1-05 1-15 1-25

Density

Fig. i. Effect ot digitonin on organelle equilibrium density. Isopycnic centrifugationof postnuclear supernatant from control ( ) and digitonin-treated ( + ) granulo-cyte homogenate8. Frequency (± S.D. of 3 experiments) is denned as the fractionof total recovered activity present in the gradient fraction divided by density spancovered. The activity present over the density span 1-05-1 10 g cm"1 represents,over an arbitrary abscissa interval, enzyme remaining in the sample layer and is pre-sumed to reflect soluble activity. The percentages of recovered activity are: A, neutrala-glucosidase, 83; B, y-glutamyl transferase, 92; C, alkaline phosphatase, 86; D, 5'-nucleotidase, 118; E, acid phosphatase, 112; F, malate dehydrogenase, 89; G, vitaminBls-binding protein, 89; H, myeloperoxidase, 99. Control data (8 experiments) takenfrom Rustin & Peters (1978).

RESULTS

Subcellular fractionation studies. The averaged distributions of the principal markerenzymes are shown in Fig. i. The control experiments, performed in the absence ofdigitonin, showed that the distribution of alkaline phosphatase (AP) was similar tothe light peaks of neutral a-glucosidase and y-glutamyl transferase, with a modaldensity of 1-13. The plasma membrane marker 5'-nucleotidase had a significantlylighter modal density of 1-12. The dense peak of the bimodally distributed y-glutamyltransferase and neutral a-glucosidase was similar to the specific granule markervitamin B12-binding protein. Acid phosphatase also had a broad distribution over-lapping the specific granule and the azurophil (myeloperoxidase) marker enzymes.

Homogenization of the neutrophils in 0-2 mol/1. sucrose containing 0-12 mM/1.digitonin had a highly selective effect on the various organelles when the post-nuclear

404 G. J. S. Rustin, P. D. Wilson and T. J. Peters

supernatant was subjected to density gradient centrifugation. Neutral a-glucosidasewas relatively unaffected, the less dense peak decreased in density and the denserpeak showed a small increase in density. y-Glutamyl transferase showed a unimodalpeak with a density of 1-20 and some skewing towards the low density region of thegradient. The membranous elements associated with AP and with 5'-nucleotidaseshowed a marked increase in equilibrium density to 1-19 although the distributionin the gradient differed with 5'-nucleotidase showing a broader distribution. Acidphosphatase showed a greater proportion of soluble activity in the digitonin-treatedhomogenate.

(H1-05 1-15 1-25 105

Density1 15 1-25

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 72 and 5 units/ml heparin. For further details see Fig. 1.The percentages of recovered activity are: A, alkaline phosphatase, 114; D, galactosyltransferase, 96; C, y-glutamyl transferase, 101; D, neutral a-glucosidase, 112.

Table 1. Effect of Triton, Levamisole and freezing and thawing on neutrophilhomogenate alkaline phosphatase activity

Fresh Frozen nnd thawed

Activity, m units/mg protein

Latent activity, %

Inhibition by o-i mmol/1.Levamisole, %

Mean of 4 experiments ± S.E. I m unit of enzyme activity corresponds to the hydrolysis of1 nmol of substrate i/min.

4-34 ±°-64 ±2

81 ±1

53 2 6

73

±0-46

± 3

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


Fig. 2 compares the distribution of the 'microsomal' enzymes in the sucrose-imidazole density gradients. The distribution of AP is distinct from that of galactosyltransferase. y-Glutamyl transferase shows a bimodal distribution with the less-densecomponent corresponding to that of alkaline phosphatase and the heavier componentcorresponding to the denser component of neutral a-glucosidase. The less-densecomponent of this enzyme is distinct from the AP-containing membranes.

1 05 1-15 1-25 105Density

1-15 1-25

Fig. 3. Alkaline phosphatase homogeneity in sucrose gradient fractions. Isopycniccentrifugation of postnuclear supernatant from granulocyte homogenate. For furtherdetails see Fig. 1. The percentages of recovered activity from the gradient fractionsare: A, free AP, 95; B, AP after freezing and thawing, 93; c, Triton-activated AP, 82;D, Levamisole-inhibited AP, 103.

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

Fig. 3 shows the AP activity in the gradient fractions assayed fresh, in the presenceof o-i mmol/1. Levamisole, or o-i% Triton and after freezing and thawing. Thedistribution of the alkaline phosphatase is identical in all experiments. This is strongevidence in favour of homogeneity of the AP-containing membranes in the neutrophiland is consistent with a single intracellular localization of this enzyme.

Ultrastructural studies. Fig. 4 shows that the principle site of localization of APwas in a characteristic cytoplasmic organelle. Heavy deposits of lead outlined avesicle 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 onother cytoplasmic organelles. There is no activity on the plasma membrane. Fig. 5shows a region of the cytoplasm at high magnification with irregularly shaped AP-containing structures: the contents of these organelles were heterogeneous andincluded dense mural deposits. Inhibitor studies showed that 1 mmol/1. Levamisoleand 100 mmol/1. glycine blocked the formation of reaction product.

4Q6 G. J. S. Rustin, P. D. Wilson and T. J. Peters

Fig. 6 shows AP reaction in a gently homogenized (5 strokes) neutrophil prepara-tion. Various granules and linear AP reaction production can be seen. Fig. 7 showsAP in neutrophil homogenate disrupted with 25 strokes of the tight-fitting pestle:many vesicles containing reaction product are present. Fig. 8 shows similar vesiclesin the gradient fraction when this homogenate was subjected to sucrose gradientcentrifugation. This fraction has a density of 1-13 gem" 3 and corresponds to the

r

Fig. 4. Alkaline phosphatase cytochemistry. Polymorphonuclear leucocyte incubated60 min at 37 CC for alkaline phosphatase activity. Heavy deposits of lead reactionproduct can be seen lining the inside of some granules (arrows). Lighter reaction depositsare seen in the nucleus. The plasma membrane, endoplasmic reticulum and othergranules are lacking in alkaline phosphatase. x 20250.

biochemically assayed peak of AP activity. Most of these vesicles and the membranousfragments contain dense reaction product. Fractions containing plasma membrane,specific and azurophil granules did not form alkaline phosphatase reaction productwhen EM cytochemistry was performed on them.


Fig. 5. Alkaline phosphatase cytochemistry. High-power view of the alkaline phos-phatase-containing granules (apg). Also in the field are azurophil (az) and specific (s)granules which contain no reaction product, x 66000.

Fig. 6. Alkaline phosphatase cytochemistry. Results of 4s min incubation at 37 °Cin a gently homogenized preparation (5 strokes). Heavy deposits of reaction productcan be seen in some granular (apg) and linear (/) configurations. Other profilesresembling azurophils (02) and specific granules (J) can be seen to be devoid of alkalinephosphatase reaction product, x 18000.


Fig. 7. Alkaline phosphatase cytochemistry. Reaction product in a homogenate after25 strokes incubated for 45 min at 37 °C. Deposits can be seen lining many membranevesicles and in a few linear arrangements, x 51 300.

DISCUSSIONThe alkaline phosphatase (AP)-containing membranes are clearly separated from

the plasma membrane fragments by isopycnic gradient centrifugation. This lattermembrane was characterized by its content of 5'-nucleotidase (Solyom & Trams,1972) and by the use of the proximity labels, fluorescamine and Bolton-Hunterreagent (Segal & Peters, 1977). Following digitonin treatment both the plasma andthe AP membrane show increases in equilibrium density although the increase differsfor each membrane.

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

Alkaline phosphatase in human neutrophils

8Fig. 8. Alkaline phosphatase cytochemistry. Alkaline phosphatase reaction productsurrounding vesicles from a sucrose density gradient. Incubation was for 45 min at37 °C. x 43 200.

More striking, however, is the effect of digitonin on these 2 membranes. The APmembranes show an increase in density of 0-06 g cm~3 whereas the endoplasmicreticulum shows a density decrease of 0-02 g cm"3. A similar resolution of these 2components has been demonstrated for rat liver (Tilleray & Peters, 1976), and humangut (Peters, 1976) and liver (Peters & Seymour, 1978).

The electron micrographs show that the AP membranes are derived from structureswhich bear a morphological similarity to the Golgi. However, this organelle is veryinfrequently seen in mature neutrophils, the level of the marker enzyme for thisorganelle is low and the density gradient experiments clearly resolve this organellefrom the AP membrane. Note that the glactosyl transferase shows a bimodal distri-bution with a low-density component that has a similar distribution to that of theplasma membrane. Localization of glucosyl transferases to plasma membrane (Roth,McGuire & Roseman, 1971; Roth & White, 1972; Weiser, 1973) as well as to theGolgi (Morre, Merlin & Keenan, 1969; Schachter et al. 1970; Amar-Costesec et al.1974) is well recognized.

On the basis of these experiments we conclude that AP has a unique localizationin the human neutrophil. Avila (1977) after experiments with membrane-solubilizing


agents has also tentatively suggested that AP is localized to a second light, sub-population of specific granules. The function of this organelle remains to be deter-mined. The role of AP and y-glutamyl transferase in the neutrophil, as in other tissuesremain speculative, but cytochemical studies (Bainton, 1973; Jenson & Bainton, 1973)indicate an early involvement of this organelle during phagocytosis. In view of therole 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 areinvestigating the possible localization of cAMP-degrading enzymes to this organelle.

A controversial area in the discussion of the types of neutrophil granules is thequestion of tertiary granules. Previous subcellular fractionation studies (West et al.1974; Kane & Peters, 1974) have indicated that certain acid hydrolases have a dis-tinctive distribution and are localized to a tertiary or C granule as has been demon-strated by certain morphological studies (Scott & Horn, 1970) but not by others(Farquhar, Bainton, Baggiolini & De Duve, 1972; Bainton & Farquhar, 1966). Thedensity gradient experiments on digitonin-disrupted neutrophils provides furtherevidence in favour of this hydrolase-containing granule: the distribution and solu-bilization of acid phosphatase differs from the specific and azurophil granule markerenzymes.

The precise role of these various structures in the microbicidal role of neutrophilsremain to be elucidated. Combination of subcellular fractionation techniques withcytochemical studies particularly in actively phagocytic cells should prove profitableas has been shown in the present study.

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

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{Received 29 June 1978)

Documents

STUDIES ON THE SUBCELLULAR LOCALIZATION OF HUMAN ... · methods are routinel y use tod demonstrate enzymic b activity light microscopyy . They show tha thte reaction produc is cytoplasmit