Immunology 1979 37 111

Subceliular localization of the eosinophil chemotactic factor (ECF) and its inactivator in human polymorphonuclear leucocytes (PMN)

N. FRICKHOFEN* & W. KONIG Institute of Medical Microbiology, Johannes Gutenberg-University, D-6500 Mainz, W. Germany

Received 13 July 1978; acceptedfor publication 17 October 1978

vator can be recovered from the peroxidase positive (azurophilic) granules and has a mol. wt of 60,000 and less. We suggest that low mol. wt ECF is derived from the plasma membrane of PMN which can be inactivated by components of the azurophilic granules. The mechanism of inactivation is still unresolved.

Summary. An eosinophil chemotactic factor (ECF) of low MW can be released from human polymorphonuclear leucocytes (PMN) on stimulation with the Ca-ionophore, arachidonic acid and during phagocytosis. After a rapid rise of ECF activity in the supernatant a steep fall of in its activity occurred at the later times of secretion suggesting a mechanism of ECF inactivation. ECF obtained at the later times of secretion represents a stable biological activity and does not decrease on further incubation. In addition, intact PMN and ECF combined do not lead to its inactivation, while incubation of homogenized PMN with ECF decreased its activity. These data suggest the presence of an inactivator for ECF within human PMN. The purpose of the study was to localize ECF and its inactivator within human PMN. After cell disruption, differential and equilibrium gradient centrifugation, subcellular components of human PMN can be obtained which reveal eosinophilotactic (ECF) or ECF-inactivating activity. ECF activity can be recovered (in a structurally bound state) from the microsomal fraction of unstimulated and stimulated PMNs, while another portion is obtainable as a soluble, low mol. wt ECF. The PMN-derived ECF inacti-

INTRODUCTION We recently demonstrated the generation of eosinophilotactic activity from human PMN by the Ca-ionophore (Czarnetzki, Konig & Lichtenstein, 1976), during phagocytosis (Konig, Czarnetzki & Lichtenstein, 1976) and by stimulation with arachidonic acid (Konig, Tesch & Frickhofen, 1978; Tesch, Konig & Frickhofen, 1979). With each stimulus, the ECF activities obtained are of low molecular weight (about 500) and not preformed within PMN. The molecular analysis of the ECF is currently under investigation. Since ECF-A is associated with the granules of mast cells, it was of interest to localize ECF (synthesis) within PMN by subcellular fractionation techniques. Furthermore, it was demonstrated that, on stimulation, PMN showed a rapid rise of ECF activity in the supernatant, followed by a steep fall-off in activity at later times of secretion (Konig et al., 1976; Konig, Czarnetzki & Lichtenstein, 1978a). Since it was shown that ECF represents a stable biological activity, we suggested that the decrease of ECF activity is due to

* In partial fulfilment of N.F.'s M.D. thesis. Correspondence: Dr. W. Konig, Johannes-GutenbergUniversitait, Institat fur Medizinische Mikrobiologie, 65 Mainz, Obere Zahlbacher Strasse 67, W. Germany. 0019-2805/79/0500-011 1$02.00 © 1979 Blackwell Scientific Publications

111

112

N. Frickhofen & W. Konig

the effect of (a) cell-derived inactivator(s). This assumption was confirmed experimentally when supernatants of stimulated cells at later times of secretion were dialysed and incubated with a stock amount of ECF. In these experiments, ECF activity was reduced suggesting that the inactivator might be secreted under physiological conditions. The inactivator obtained from the supernatant of stimulated cells, however, was too low in its activity and concentration in order to be analysed satisfactorily (Czarnetzki, K6nig, & Lichtenstein, 1978). Since homogenates of unstimulated PMN, incubated with ECF also led to a decrease in eosinophilotactic activity, it appeared reasonable to characterize the inactivator by subcellular fractionation studies. The mechanism of inactivation is still unresolved. Inactivation may either be due to binding or to enzymatic cleavage. MATERIAL AND METHODS

Methods for the preparation of human and guinea-pig PMN, of human eosinophils and buffers used for mediator release are the same as described previously (Konig et al., 1978c). Surface iodination of human PMN was performed as has been described using lactoperoxidase and 1251 (Konig & Ishizaka, 1976). Commercial source of reagents See Konig, Frickhofen & Tesch, 1979: succinic acid, INT 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride, D-glucose-6-phosphate, ,B-nicotinamide adenine dinucleotide reduced form (NADH), stachyose, maltotriose (Sigma, Munchen, W. Germany). Reagents not further listed were purchased from E. Merck, Darmstadt, W. Germany.

Preparation of ECF PMN (1 x 107/ml) were incubated with either the ionophore A 23187 at 2-5 x 10-6 M (a gift of Dr R. Hamill of the Eli Lilly Research Laboratories, Indianapolis, Indiana, U.S.A.) or zymosan coated with complement (Zx) at a concentration of 3 mg/ml. The incubation time for ionophore stimulation amounted to 12 min, while with (Zx) a 15 min incubation was found to be

optimal for mediator release (Czarnetzki et al., 1976; Konig et al., 1976). The incubation was stopped by centrifugation in the cold for 5 min at 400 g. The supernatant containing ECF activity was recovered and the cell pellet was washed in Tris buffer without

CaC12 and MgCl2 and resuspended in the homogenization medium.

Cell breakage Three methods were used: (a) sonication was carried out for 10-30 s at 40 (Mullard equipment Ltd, England); (b) nitrogen cavitation was performed using a nitrogen bomb (Artisan Industries, Mass., U.S.A.) applying a pressure of 60 atmospheric units at 40 for 30 min; (c) homogenization was performed in a PotterElvehjem teflon glass homogenizer (Braun, Melsungen, 30 ml, clearance 0-095-0 115 mm) driven by a drilling machine (Janke & Kunkel, Staufen, W. Germany) at 2000 r.p.m. Approximately 40-80 strokes were applied. In each case the final cell concentration was 5 x 107/ml. Four different media, each buffered with 0-005 M Tris-HCl, pH 7 4, were assayed with regard to their property for cell disruption: (a) hypotonic NaCl (022%), adjusted to isotonicity after homogenization by addition of NaCl (1 .6%); (b) isotonic sucrose (0 34 M); (c) isotonic sucrose with heparin (50 u/ml); (d) hypotonic sucrose (0-1 M) adjusted to isotonicity after homogenization by addition of the same volume of 0-58 M sucrose. During homogenization the cells were kept in ice and the degree of cell breakage was followed by microscopy using toluidine blue staining of the cells. Subcellular fractionation Subcellular fractionation was performed by differential and equilibrium gradient centrifugation. (a) Differential centrifugation was carried out in refrigerated centrifuges (H. Christ, Osterode, W. Germany and Beckman Instruments). Five fractions were obtained by centrifuging the whole homogenate at 400, 3000, 20,000 g for 15 min and at 200,000 g for 60 min, leading to four pellets ('P 1-4') and a final supernatant fraction (P 5'). The pellets were adjusted to the same volume and medium as present in the final supernatant. In some experiments the pellets were washed to obtain a better separation. The wash-supernatant was then analysed separately. (b) Equilibrium gradient centrifugation was performed in a Beckman Spinco L2-65B ultracentrifuge with the following rotors: SW27, SW40, SW65. Trisbuffered (0 005 M, pH 7-4) sucrose gradients were prepared using a continuous gradient former. The density range and shape of the gradients varied depending on the material to be separated (see Results). After centrifugation at 100,000 g for 8-12 h,

ECF and its inactivator in human PMN

113

were obtained by piercing the bottom of the polycarbonate tubes. The density of the fractions was measured with a Zeiss Refractometer. Each fraction was adjusted to the same sucrose concentration and volume, in order to obtain comparable conditions; sonicated fractions were used for the determination of chemotactic and enzyme activities.

tions of 2 7 ml each were collected. Blue dextran, lysozyme (mol. wt 15,000) and sucrose (mol. wt 342) were used as molecular weight markers. Blue dextran was determined spectrophotometrically (260 nm) as well as vitamin B 12 (360 nm); lysozyme was measured as described; sucrose was determined by the method of Dubois et al. (Dubois, Gilles, Hamilton, Rebers & Smith 1956) and ['4C]-histamine was analysed in a scintillation counter.

Analytical techniques Chemotaxis. The method for measuring the eosinophil migration has been described in detail (Czarnetzki et al., 1976). The subcellular fractions (100-500 1) were assayed for eosinophilotactic activity and for their property to inactivate a stock amount of ECF. Biochemical assays. Protein, DNA and enzymes were measured as outlined previously (Konig et al., 1978c). Additional enzymes were assayed as has been described: 3.1.3.9 glucose-6-phosphatase (Chen, Toribara & Warner, 1956), 3.1.3.5 5'nucleotidase (Bergmeyer, 1970). Unit definition. One unit is defined as the enzyme activity which splits 1 gM of substrate per minute at the described conditions. The specific activities (mu/mg protein) of the enzymes measured routinely within PMN are summarized in Table 1. Chromatography. Gel filtration analysis of the eosinophilotactic factor was performed on a Sephadex G-25 column (1 -6 x 25 cm) as has been described previously. The inactivator was characterized by gel filtration analysis on a Sephadex G-200 column (2-5 x 37 cm). Elution was performed in TCM buffer and frac-

RESULTS Homogenization The purpose of our studies was to analyse the subcellular distribution of ECF and its inactivator. Human PMN were suspended in medium and then subjected to either sonication, homogenization or nitrogen cavitation to obtain intact subcellular components. Microscopic examination was performed to calculate the percentage of homogenized cells. It was found that sonication and nitrogen cavitation led to an almost complete disruption of the cells in either medium, whereas with homogenization the degree of cell breakage strongly depends on the medium. Experiments, in which the cells were first suspended in a hypotonic medium (0 1 M suCr'ose in Tris-HCI, 0-005 M, pH 7-4), homogenized and then readjusted to isotonicity by addition of 0 58 M sucrose led to a satisfactory degree of cell disruption. Our experiments were performed under hypotonic conditions without heparin.

seventeen to twenty-one fractions of equal volume

Table 1. Calculation of specific enzyme activities in human PMN. SA, specific activity; SD, standard deviation; n, number of independent cell preparations and independent determinations. Recovery during differential and equilibrium gradient centrifugation was always 80-110%. The latter value (110%) is due to the fact that separated components express higher enzyme activities as compared to an unseparated homogenate.

Enzyme Peroxidase Lysozyme

P-glucuronidase ,B-glycerophosphatase Acid p-Np-phosphatase Alkaline p-Np-phosphatase Mg2 +-ATPase LDH

SA (mu/mg)

± SD

n

1640 115 2-00 26-0 132 4-43 13-6 998

+ 398 + 31 + 0-69 + 58 + 31 + 184 + 3-4

17 17 17 13 17 17 13 13

+215

Subcellular fractionation Subcellular fractions were obtained by differential and equilibrium gradient centrifugation. Differential centrifugation. Unstimulated PMN were homogenized and fractions were obtained at 400, 3000, 20,000 and 200,000 g with a final 200,000 g supernatant fraction (see Materials and Methods). It is apparent (Fig. 1) that ECF activity can be recovered from the 400 and 200,000 g precipitate and supernatant fraction. These data were confirmed by eight separate experiments. On stimulation, PMN showed a three to five-fold increase in ECF activity affecting mainly the 400 g precipitate and 200,000g supernatant fraction. Each fraction was then assayed with regard to its inactivating property for ECF. An aliquot of 200-500 ,l was incubated with a stock amount of ECF for 30 min at 37°. As a control, ECF was diluted in the suspension medium without the addition of subcellular fractions and kept for 30 minutes at 37°. Figure I

114

N. Frickhofen & W. Konig %

Eos/5HPF

100I

Inactivation

1502iX

510

ErhncrementUll

500

1 r 100

300 b)stim. cells,

E

100

5'-Nucleotidase* 0.6 -

_ze

102 _ ~ _x_

0,2

mU/ml Alk. p-Nitrophenyi

mU/ml Alk. Phospho-

Phasphatase

5

diesterase I

0.2 3-

1~~~~~~0

mU/ml Acid p-Nitraphenyi- mU/ml

Phosphose

100

Glucose-6-

Phosphatose

0n3

60 mU/ml

2.0 1.2

0.4

-2 mU/ml

fl-Glucuronidase

400, 3000 and 20,000 g with their major amount in fractions at 3000 and 20,000 g. This distribution pattern differed from that of fi-glycerophosphatase, which was found throughout all fractions with major peaks at 3000, 20,000 g and in the 200,000 g supernatant fraction (see discussion). When the fractions were analysed for microsomal enzymes, it was apparent that Mg2 I-ATPase activity was almost completely confined to the 200,000 g precipitate. Alkaline p-Npphosphatase showed a major peak in the 200,000 g fraction, while acid p-Np-phosphatase was distributed throughout all fractions with the major peak in the 200,000 g supernatant. 5'-nucleotidase and glucose-6phosphatase showed low activity as well as alkaline phosphodiesterase I. In addition, the activities of the latter enzymes were strongly inhibited in the presence of Na-tartrate. These data indicate that the lysosomal phosphatase may induce the release of inorganic phosphorus from the substrate by non-specific hydrolysis. The results so far obtained, were then calculated in terms of relative specific activity (RSA), i.e. percentage activity to percentage of protein (Fig. 2). Our data show that ECF activity is distributed in a similar pattern as the microsomal enzyme markers, which are enriched by three- to five-fold in a fraction of low protein content. Inactivator activity is present in granular fractions which are concentrated by two- to three-fold and which contain the lysosomal enzyme markers, lysozyme, fl-glucuronidase, peroxidase and acid 13-glycerophosphatase. The presence of fl-glycerophosphatase activity in the microsomal fraction is partly due to the fact that acid p-Np-phosphatase cleaves the same substrate. Equilibrium density centrifugation. Further characterization of ECF and inactivator-containing fracwas obtained by equilibrium density centrifugation in a Beckman ultracentrifuge. The data presented here are representative of twelve separate experiments. It became apparent that a better resolution was achieved with continuous as compared to discontinuous gradients. Furthermore, the separation of granules from a postnuclear supernatant fraction led to better results as compared to fractions which were analysed after differential centrifugation at 3000 and 20,000 g. In these experiments, the clumping of granules impaired the separation significantly. In the following experiment (Fig. 3), a postnuclear supernatant (P2-P4 2 ml; 4-6 mg protein) was layered on a continuous sucrose gradient (36 ml; 19-54% w/w sucrose). Centrifugation was carried out in a SW27 rotor for 10 h at 100,000 g. After centrifugation, fracat

ECF

2000

LDH

1200

400-

Figure 1. Subcellular fractionation of unstimulated (a) and ionophore stimulated (b) human PMN. Cells were homogenized in Tris-(0 01) buffered sucrose (0 1 M) and sucrose (0 58 M) was added to obtain a final concentration of 0 34 M. Mean values of the five fractions obtained by differential centrifugation during eight separate experiments are shown. The variation coefficient (calculated on the basis of eight separate experiments) varied between 11 and 380', which is due to differences in the degree of homogenization. The distribution pattern of biological activities and enzymes was, however, similar in each experiment. Steps indicate the five fractions (400, 3000, 20,000, 200,000g precipitates and supernatant), obtained after differential centrifugation. Broken lines represent enzyme activity in the presence of sodium tartrate (10 mM).

demonstrates that ECF was inactivated by 50% with the fractions at 3000 and 20,000 g, while the 400 and 200,000 g precipitate and supernatant fraction led to enhanced ECF activity, which is partly due to the presence of endogenous ECF. As to the granular markers, it is found that peroxidase, lysozyme and f,-glucuronidase are distributed in fractions obtained

ECF and its inactivator in human PMN Mg++-ATPase RSA 4 32

INA Peroxidose

ECF ( unstim. cells )

RSA

RSA

115

4 3

811 611

4

2-

2

0

0

50 100 % Protein

50 100 % Protein

0

Alk. p-Np-

Lysozyme

RSA 4 3 2

Phosphotose

RSA

100

ECF l stim. cells )

RSA

4

4

3

3-

2]-

2-

L

50

% Protein

1 0

50

0

100

°/0 Protein

50 100 % Protein

Acid p-NpPhosphotose

p-Glycero Phosphatose

RSA

0

50 100 % Protein

RSA

3l

3 2

2

0

50 100 % Protein

0

50 100 % Protein

Alk.

-Glucuronidase

RSA

RSA

Phospho-*

ldiesterase

I

411 311 2]

4-

3-

1

0

50 100 % Protein

0

RSA 4 3'21I

50 100 % Protein

% Protein

Figure 2. Biological and enzyme activities in fractions after differential centrifugation plotted against the protein content as 'relative specific activities (RSA)'. Results were obtained from the experiment depicted in Fig. 1; abscissa: Total protein content in the five fractions is expressed as 100%; width of the steps represents the percentage of protein in each fraction; ordinate: RSA 1 indicates the same concentration as is present in the total homogenate; RSA 2 indicates twice the concentration, i.e. a twofold purification; shaded areas represent the degree of purification. =

=

tions were obtained, adjusted to the same sucrose concentration and final volume. The fractions were sonicated, assayed for protein content, enzyme and chemotactic activity. It can be seen that the major amount of protein remained on top of the gradient with two minor peaks at a density range of [-17-1 24 g/cm3 and 1 12 g/cm3. The ECF inactivator eluted preferentially in fraction B and ECF activity was present in fraction containing microsomal enzymes (M). Activities on top of the gradient (fractions 17-21) represent soluble and not structurally bound components.

When the

enzyme

markers

are

correlated to the

protein pattern, it can be seen that the granular such as lysozyme, fl-glycerophosphatase, peroxidase and ,B-glucuronidase correspond to the denser protein band. While lysozyme and fl-glycerophosphatase showed two distinct peaks in fractions A (1[23 g/cm3) and C (1 19 g/cm3), the major portion of peroxidase and fl-glucuronidase was present in between the two peaks in fraction B (1 21 g/cm3). These results suggest that the PMN granules consist of at least three types with different densities (A,B,C). The inactivator apparently corresponds to the peroxidase-positive subpopulation (B). The protein band of lesser density (Fig. 3) contained enzymes,

116

N. Frickhofen & W. Konig D*nssity

Protein

Ig/c i)

g/nl 1

M

EosO/SHPF 400 Enhancement

1 30

1.20-

-I

I

i

1000

5000

1.10

1.00

5

mU/Iml 30

10

15

100 .jInacti

m

on

ECF

20

Lysozyme

5

,

.-1

~~~~~~~50

_

_

>

200

10

15

mU

Acid p-Nitrophenyl-

80

Phosphotase

20

60

20-

40 10-

20 5

10

15

/3-Glycero-

mU/ ,ml 3I

5

4

1 10

15

Alk p-Nitrophenyl-

200

10 5

20

(i-Glucuronidose

10

15

20

Mg"-ATPase

mU 1ml

20

mU/ml

20

Phosphatose

5

400-

15

15

2-

30

10

10

3

600

5

I

20

Peroxidase

mUI/ml

5 mU /ml

Phosphatase

M

_~~~~~-

20

10

15

20

Alk. Phospho-* diestercse I

mU/r nl

0.02 0.01 0.1 5

10

15

20

5

10

15

20

Figure 3. Subcellular separation of a post-nuclear supernatant on a linear sucrose gradient after equilibrium gradient centrifugation. Correlation of biological activity to enzyme markers (pm, distance of migration into the filter). Abscissa: fractions obtained after ultracentrifugation. Inactivation is expressed as decrease in ECF activity after its incubation with the fractions; stock ECF shows in this experiment an eosinophilotactic activity of 250 eosinophils per five highpower fields; A,B,C represent different types of granules; M indicates the pattern of microsomes.

the microsomal markers Mg2 + -ATPase, alkaline p-Np-phosphatase and a minor portion of acid p-Npphosphatase (1 125 g/cm3). In agreement with our results obtained by differential centrifugation, it was

demonstrated that acid p-Np-phosphatase seems to be loosely attached to subcellular structures and/or consists of a particle bound and a soluble (cytoplasmic) enzyme. As to alkaline phosphodiesterase I its major

ECF and its inactivator in human PMN

peak equilibrated at a density of 1-145 g/cm3 with a shoulder at 1 125 g/cm3. The overall ECF activity is low, since unstimulated cells were used in this experiment. Furthermore, because of the high sucrose concentration and its interference with eosinophil chemotaxis only 100-200 p1 of each fraction could be assayed in the Boyden chamber. Experiments were carried out to determine the localization of the ECF-containing or producing particles more precisely (Fig. 4). The 200,000 g precipitate (1 ml, 0 5-0 7 mg) of ionophore-stimulated cells was layered on top of a shallow sucrose gradient (19-45% w/w sucrose). Centrifugation was performed in a SW40 rotor for 10 h at 100,000 g. It is apparent that the protein was present in almost every fraction with minor fluctuations. A distinct peak of ECF activity appeared at a density of 1-13 g/cm3 with a smaller peak Protein Ipg/ml I

Density

Ig/cm'|

M2

Ml

117

at 1-17 g/cm3. The elution pattern of the microsomal enzymes Mg2 +-ATPase, alkaline and acidp-Np-phosphatase (and in part alkaline phosphodiesterase I) showed a similar distribution as ECF. Furthermore, ECF co-sediments with radiolabeled structures of the PMNs after surface iodination. Since the peroxidase positive granules, which are also iodinated by this method, have been removed in this experiment, it may be assumed that the iodinated components most likely represent the plasma membrane of the PMN. These data combined suggest that ECF is derived from microsomal components of the cells and perhaps from the plasma membrane. From the previous experiment (Fig. 3) it appeared that the inactivator was present within a distinct subpopulation of PMN granules. To analyse these data more precisely, experiments were carried out using a

Am ECF

1.20

200 -150 100

200 -l 150 100

1.00 j-50

50-

1.10

_ 5

mU/ml

10

15

mU/ml

Acid p-NitrophenylPhosphotose

I

605

5

10

15

A]

20

Alk. p-NitrophenylPhosphatose

432-

40

20

5

20

1-1 10

15

20

5 mU)l/ml

Alk.

10

15

20

Phospho-*

diesterase I

0.03 -

0.02 0.015

10

15

20

5

10

15

20

5

10

15

20

cpm

800600400-

200-

Figure 4. Localization of structurally bound ECF after ultracentrifugation of the 200,000 g precipitate of stimulated PMN on a linear sucrose gradient; Ml, M2, separation of microsomal components; pm, distance of migration into the filter.

N. Frickhofen & W. Kdnig

118

granules. Since the postnuclear supernatant was analysed on a gradient of high density, cell constituents of lesser density, such as microsomes, cytoplasma and cell structures, which are solubilized by the homogenisation procedure, equilibrated on top of the gradient. When this latter fraction is incubated with a stock amount of ECF, a marked enhancement in ECF activity can be observed, which cannot be explained by the presence of microsomal ECF activity in this fraction alone. At the present, this problem is under investigation.

shallow gradient of high density, thus allowing a better separation of granules with different densities. A postnuclear supernatant fraction of unstimulated cells was obtained and applied to a linear sucrose gradient (43-53% sucrose w/w). Ultracentrifugation was then carried out for 10 h at 100,000 g in a SW40 rotor. It is apparent from Fig. 5 that the granules, as assessed by enzyme markers, were clearly separated. The heavy portion of the granules, containing lysozyme and fl-glycerophosphatase activity, equilibrates at a density of 1 23 g/cm3, while the less dense component (1-19 g/cm3) coincides with the soluble portion of the enzyme. On a shallow gradient, the peroxidase and f-glucuronidase containing granules were separated into two distinct peaks eluting at 1 21 and 1-22 g/cm3. The inactivator eluted at a position which corresponds to the less dense peak of the peroxidase positive Density 19/cm31 1 30

Gel filtration analysis Since by subcellular fractionation studies, ECF activity can be recovered from the 200,000 g precipitate and supernatant fractions, it was of interest to deter-

Protein

AB2 B,

-

c[/sg/mIJ -

Subcellular localization of the eosinophil chemotactic factor (ECF) and its inactivator in human polymorphonuclear leucocytes (PMN).

Immunology 1979 37 111 Subceliular localization of the eosinophil chemotactic factor (ECF) and its inactivator in human polymorphonuclear leucocytes...
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