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Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 Macrophages
200
Biochimica et Biophysicu Acta 960 (1988) 200-209 EkVier
BBA 52818
Secretion of the lysosomal acid triacylglycerol hydrolase precursor by 5774 Macrophages Dennis I. Goldberg
* and John C. Khoo
Division of Endocrinology and Metabolism, Departmen of Medicine, University of California, San Diego, LA JoNa, CA (U.S.A.) (Received 23 September 1987) (Revised manuscript received 3 February 1988) Key words: Triacylglycerol hydrolase; Triacylglycerol lipase; Lysosomal enzyme; Protein transport; Mannose 6-phosphate receptor; (Macrophage)
5774, thioglycollate-elicited mouse peritoneal and BCGinduced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The 5774 macrophages, a cell line deficient in the calcium-independent mannOSe 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH&I induced a dfold increase in TGase secretion by alveolar macrophages, while 50 mM NH&l induced a 1Zfold increase in TGase secretion. NH&l had no effect on TGase secretion by 5774 macrophages. The TGase secreted by 5774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase lo-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose bphosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase. Introduction We have recently reported the presence of a highly active neutral triacylglycerol hydrolase
* Present address: Department of Metabolic Diseases, Central Research Division, Pfiir, Inc., Groton, CT, U.S.A. Abbreviations: TGase, triacylglycerol hydrolase (EC 3.1.1.3); a-MEM, u- minimal essential medium; BCG, Bacillus Calmette Guerin; Hepes, 4-(2-hydroxyethyl>l-piperazineethanesulfonic acid; BSA, bovine serum albumin; INT, 2-(p-iodophenyl)-3( p-nitrophenyl>5-phenyltetrazolium chloride. Correspondence: J.C. Khoo, Division of Endocrinology and Metabolism, Department of Medicine, M-013D, University of California, San Diego, La Jolla, CA 92093-0613, U.S.A. 0005-2760/88/$03.50
(TGase) in several types of macrophage [l]. This enzyme displayed a number of characteristics which distinguished it from classical acid TGase. These traits included a nearly neutral pH optimum (pH 6.4), remarkable thermal stability, the absence of activity towards monooleoylglycerol, and inhibition by 1 mg/ml bovine serum albumin (BSA). It has been proposed that this enzyme may be responsible for the preferential mobilization of triacylglycerols stored in macrophages, resulting in the enriched cholesteryl ester content of the lipid inclusions found in macrophage-derived foam cells of the atherosclerotic lesion [l]. In the present study, we report the spontaneous secretion of large quantities of neutral TGase by
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
201
5774 cells, a macrophage-like cell line deficient in the 215 kDa, bourn-~dependent mannose 6phosphate receptor (mannose 6-phosphate receptorcl) [2,3]. This receptor is involved in targeting newly synthesized enzymes to the lysosomes, where the mannose 6-phosphate recognition marker is removed and the enzyme is processed to the mature form [4]. The marmose 6-phosphate receptorc’ is located both intracellularly and on the cell surface, and the cell surface receptors are believed to mediate the endocytotic recapture of lysosomal enzyme precursors which have escaped the intracellular packaging system [4]. The ‘secretion-recapture’ pathway has been hypothesized to constitute a principal mechanism of transport for lysosomal enzymes in fibroblasts [S], although more recent theories consider the contribution to lysosomal enzyme targeting to be much more limited [6,7]. Evidence of a similar pathway in macrophages has been presented f8], but the significance of the ‘secretion-recapture’ pathway contribution to lysosomal enzyme processing in macrophages is also questionable [4]. Cells which are mannose 6-phosphate receptor ‘i-deficient, such as the 5774 or P388 Dl macrophage cell lines, are unable to effectively target or recapture lysosomal enzyme precursors, resulting in the secretion of large quantities of these enzymes [3,9]. More recently, a second mamrose 6-phosphate receptor has been identified in the P388 Dl macrophage [lO,ll]. Although the function of this 46 kDa, calcium-dependent mannose 6-phosphate receptor (mannose 6-phosphate receptor D, has not been clearly delineated, it appears to be involved in targeting of newly synthesized lysosomal enzymes, and may be responsible for the retention of large amounts of lysosomal enzymes in the P388 Dl macrophage [lo]. Although all of the lysosomal enzymes examined to date have been found to be synthesized via larger precursors, and the secretion of many lysosomal enzyme precursors has been reported [12], neither the precursor nor a mature form of the acid TGase has previously been found to be secreted. In the present study, the neutral TGase secreted from J774 cells is internalized by I-cell disease fibroblasts through their cell surface mannose 6-phosphate receptorcl, indicating the presence of phosphorylated mannose on the enzyme.
The internalized TGase was then converted to an enzyme with a pH optimum of 5.1, suggesting that the neutral TGase is a precursor of the mature lysosomal acid TGase. In contrast, macrophages which are mannose 6-phosphate receptor “-cornpetent localized the enzyme in the lysosomes and secreted much lower amounts of the neutral TGase. Materials and Methods New Zealand White rabbits (2-3 kg) were obtained from Holbert’s Rabbitry, and female Swiss-Webster mice (25-30 g) from Simonsen Laboratories. Tri[l- l4 Cloleoylglycerol and cholesteryl [l-‘4C]oleate were obtained from Amersham-Searle. Cholesteryl oleate, trioleoylglycerol, free fatty acid-poor BSA, D-mannose 6phosphate, D-mannose, glucose 6-phosphate, ammonium chloride, Freund’s complete adjuvant and NADPH were obtained from Sigma; pnitrophenyl 2-acetamido-2-deoxy-&i&pyranoside and 2-( p-iodophenyl)-3-( p-nitrophenyl)-5-phenyltetrazolium chloride (INT) from Calbiochem; OLminimal essential medium ((r-MEM) and Ham’s F-10 medium from Gibco; t~o~yco~ate medium from Difco; fetal calf serum from Irvine Scientific; and heparin-Sepharose from Pharmacia. All other chemicals were of analytical grade. The 5774 macrophage-me cell line (clone G8) was a gift of Dr. Jay Unkeless of Rockefeller University. P388 Dl cells were obtained from Dr. Hillel Koren of Duke University Medical Center, and human skin fibroblasts, from a patient with mucolipidosis II (I-cell disease), were a gift of Dr. Arnold Miller of the University of California, San Diego. Cell preparations Cultures of J774 cells were maintained in 1% mm Petri dishes containing 15 ml of a-MEM supplemented with gentamycin sulfate (50 pg/ml), fungizone (0.25 pg/ml) and 5% heat-inactivated fetal calf serum. Cells were washed with serum-free at-MEM prior to each experiment, and rn~nt~~ in serum-free cr-MEM for the duration of each ,incubation. Primary cultures of mouse peritoneal macroiphages were prepared as previously described [13]. ;Macrophages were elicited by intraperitoneal injection of thioglycollate medium 3 days prior to
202
harvest. The macrophages were plated at a density of 1 . lo7 cells per 60-mm Petri dish in a-MEM containing 10% fetal calf serum. After 3-4 h, nonadherent cells were removed by washing three times in serum-free c~-MEM. The cells were maintained in 2 ml of serum-free medium for the duration of the incubation. Rabbit alveolar macrophages were harvested in serum-free (r-MEM and cultured in 60-mm Petridishes [14]. The cells were washed and maintained as described for mouse peritoneal macrophages. I-cell disease fibroblasts were cultured in Ham’s F-10 medium containing gentamycin sulfate (50 pg/ml) and 15% fetal calf serum. The cells were washed with serum-free F-10 medium prior to use, and maintained on F-10 medium with 5% fetal calf serum during the incubation. After each incubation, the media were removed and the cells were washed with cold phosphatebuffered saline (pH 7.4), and harvested in 1 ml of 25 mM Tris-HCl/l mM EDTA/ZO% glycerol (pH 7.4) at 4°C with a rubber policeman. The media were centrifuged at 1000 x g for 10 min to precipitate any nonadherent cells which had been included in the cell harvest. Isolation of lysosomes Rabbit alveolar macrophages were induced by ear vein injection of 0.5 ml complete Freund’s adjuvant (containing heat-killed Bacillus Calmette Guerin (BCG)) diluted with 0.5 ml of sterile saline. After 2-3 weeks, the alveolar macrophages were harvested by bronchial lavage with serum-free 01MEM as described [14]. This procedure yielded (2-3). 10’ macrophages/rabbit. The cell pellet was homogenized in 25 mM Tris-HCl/l mM EDTA/250 mM sucrose (pH 7.4), according to Cohn and Wiener [15]. 3 ml of homogenate, containing (5-6). lo7 cells/ml, were centrifuged at 1000 x g for 12 min. The post-nuclear supematant fraction was layered over 30 ml of a 30-70% linear sucrose gradient, and centrifuged for 6-7 h at 53 000 x g in a Beckman SW-27 rotor at 4’ C. The gradient was harvested in 1 ml fractions with a Buchler Auto Density Flow II C fractionator. The density of each fraction was measured by weight of a known volume relative to the weight of water. Each fraction was sonicated for 12-15 s and assayed for neutral TGase, N-acetylglucosamini-
dase, and succinate tein was determined
INT reductase by the method
activities. Proof Lowry et al.
1161. Enzyme assays Neutral TGase activity was assayed in an incubation mixture of 0.8 ml containing 50 mM Hepes (pH 6.4), 0.3 mg/ml BSA, 0.0625 mM tri[l-‘4C]oleoylglycerol in an ethanolic emulsion and the enzyme preparation (either cell sonicate or medium), as described previously [l]. Assays were incubated at 30’ C for 30 min. Neutral cholesteryl ester hydrolase was assayed in 0.8 ml of an incubation mixture containing 0.032 mM cholesteryl [l-‘4C]oleate in ethanol, 1.25 mg/ml BSA, 50 mM Hepes (pH 6.4) and either cell sonicate or medium. The assays were incubated for 30 min at 30°C [17]. Acid TGase was assayed in an incubation mixture containing 0.8 ml of 0.0625 mM tri[lI4 Cloleoylglycerol, 50 mM sodium acetate/ Hepes buffer (pH 5.1) 0.6 mg/ml BSA and enzyme preparation for 30 min at 30 o C. Acid cholesteryl ester hydrolase was assayed under the same conditions substituting 0.032 mM cholesteryl [li4C]oleate for the substrate All assays were terminated by the addition of 3 ml of chloroform/ methanol/ benzene (1: 2.4 : 2, v/v). [‘4C]Oleic acid was extracted after the addition of 0.1 ml of 1 M NaOH, and quantified as previously described [ 181. N-Acetylglucosaminidase was assayed according to Lewy and Conchie [19], using p-nitrophenyl 2-acetamido-2-deoxy-B-D-pyranoside as substrate. Succinate INT reductase was assayed by the method of Pennington [20]. Lactic dehydrogenase in macrophage-conditioned media was measured according to Bergmeyer et al. [21]. Results and Discussion The 5774 macrophage-like cell line expresses three TGase activities: (1) lipoprotein lipase [22]; (2) a classical acid TGase [23]; and (3) a nearly neutral TGase [l]. As shown in Fig. 1, the acid TGase and the neutral TGase activities in broken cell preparations of 5774 cells exhibited distinct pH optima, with peak activities at pH 5.5 and 6.4, respectively. Similar pH profiles were also
203
h
40
44
46
52
56
60
64
66
7.2
PH
Fig. 1. The influence of pH on TGase activity in 5774 macrophages. The 5774 cell sonicate was assayed in 50 mM Hepes/ acetate buffer as described under Materials and Methods. The activity was expressed relative to the maximal activity at its optimal pH (equal to 100%).
observed in broken cell preparations of rabbit alveolar macrophages and thioglycollate-elicited mouse peritoneal macrophages. Optimal activity for the acid TGase in the latter two cell types was observed at pH 5.1, while the more neutral TGase displayed optimal activity at pH 6.3 (not shown). It has been shown that substrate presentation as well as buffer selection can significantly alter the pH optimum observed for the lysosomal acid TGase activity of human skin fibroblasts [24]. These factors similarly affected the observed pH profile for neutral TGase. The optimal activity shifted from pH 6.4 to pH 5.4 when the enzyme was assayed with a sonicated emulsion consisting of tri[l-‘4C]oleoylglycerol, sodium taurocholate and phosphatidylcholine [25] (not shown). Since the ethanolic substrate emulsion allowed us to distinguish between the neutral TGase and acid TGase, it was used in subsequent studies. 5774 cell-conditioned culture medium contained a high level of TGase activity, with a pH optimum at 6.5 (Fig. 6A). This enzyme was distinguished from lipoprotein lipase activity in the medium by several criteria.. Lipoprotein lipase binds to heparin-Sepharose affinity gel, its activity is increased by apoprotein C-II, and the enzyme is heat-labile, losing all of its activity when heated at 5O“C for 4 h. In contrast, the neutral TGase did
not bind to heparin-Sepharose gel, its activity was not stimulated by apoprotein C-II when assayed at either pH 6.4 or 8.2, and the enzyme was extremely heat-stable, retaining all its activity after 4 h of heating at 50°C. In addition, the TGase bound weakly to DEAE-cellulose, but tightly to CM-cellulose, consistent with the observed p1 of 9, as determined by isoelectric focusing. The neutral TGase hydrolyzed trioleoylglycerol and dioleoylglycerol at equal rates, but did not display hydrolytic activity towards monooleoylglycerol. The enzyme in the medium also displayed activity against cholesteryl[l- “C]oleate, which was similar to the TGase activity in pH profile and susceptibility to BSA inhibition (60% inhibition by 1 mg/ml BSA). The secretion of neutral TGase by 5774 cells was linear with time (Fig. 2). About 50% of the total activity (cells plus medium) was recovered in the medium after 9 h of incubation (cell content of 823 f 56 mnol free fatty acids/mg protein per h). The activity in the cells, however, remained constant during the entire period of incubation. There was no detectable lactic dehydrogenase activity in the 9 h conditioned medium, ruling out cell damage as the cause for release of neutral TGase into the culture medium. r
Hours
in
Culture
Fig. 2. The release of neutral TGase activity from 5774 cells, mouse peritoneal macrophages, and rabbit alveolar macrophages. Cells were maintained in serum-free a-MEM. Media were collected and replenished with fresh cr-MEM every 3 h for 5774 cells (0) and thioglycollate-elicited mouse peritoneal macrophages (A) or every 2 h for rabbit alveolar macrophages (m). Aliquots of the media from each cell type were assayed for neutral TGase activity. The TGase activity in cell sonicates of 5774 cells, mouse peritoneal and rabbit alveolar macrophages at the end of the experiment were 823 f 56, 1227k69, and 3322k23 nmol free fatty acids (FFA)/mg protein per h, respectively.
204
Since the cellular levels of neutral TGase activity vary significantly among different types of macrophages [l], we compared the release of the enzyme from thioglycollate-elicited mouse peritoneal macrophages and from BCG-induced rabbit alveolar macrophages with that from 5774 cells. Enzyme secretion was also linear from these cells for up to 8-9 h (Fig. 2). However, the amounts of enzyme secreted relative to the cell content of enzyme varied greatly. Thioglycollate-elicited mouse peritoneal macrophages released 21% of the total activity in 9 h (cell content of 1227 f 69 nmol free fatty acids/mg protein per h), and rabbit alveolar macrophages released only 3% of the total activity in 8 h (cell content of 3322 f 23 nmol free fatty acids/mg protein per h). The levels of neutral TGase released by the three macrophage cell types were in agreement with previous reports of spontaneous secretion of P-glucuronidase by thioglycollate-elicited mouse peritoneal macrophages [26], and spontaneous secretion of P-hexosaminidase and &#.rcuronidase by 5774 cells [3]. The secretion of large quantities of lysosomal enzymes by 5774 cells has been attributed to a deficiency in the marmose 6-phosphate receptorc’, which is involved in targeting newly synthesized enzymes to the lysosomes [27]. Since the amount of enzyme released by the three cell types examined in the present study was inversely proportional to their reported expression of marmosyl-glycoprotein receptor activity [28], we examined the enzyme for the presence of the mannose 6-phosphate marker. I-cell fibroblasts, which are mannose 6-phosphate receptor-competent cells that fail to incorporate the mannose 6-phosphate marker into newly synthesized enzymes [29], were utilized in these studies. These cells possess small amounts of endogenous lysosomal enzymes, but readily internalize exogenous enzymes which contain the mannose 6-phosphate marker. The conditioned medium of 5774 cells was first concentrated lo-fold by Amicon Ultrafiltration, and then diluted 5-fold with fresh F-10 medium. This medium was then added to cultured I-cell fibroblasts. As shown in Fig. 3, intracellular neutral TGase activity increased lo-fold in 8 h. The addition of 2 mM mannose or 2 mM glucose 6-phosphate had no effect on the internalization
Fig. 3. The uptake of neutral TGase from 5774 cell-conditioned medium by I-cell fibroblasts. The 24-h 5774 cell-conditioned medium was concentrated lo-fold. The concentrated medium was diluted 5-fold with F-10 medium containing 2.5% fetal calf serum which was then added to I-cell fibroblasts (0); I-cell fibroblasts plus 2 mM mannose (A); I-cell fibroblasts plus 2 mM glucose 6-phosphate (m); and I-cell fibroblasts plus 2 mM mannose B-phosphate (A). For control, I-cell fibroblasts were incubated in F-10 medium containing 2.5% fetal calf serum (0). After incubation at 37OC for 4 h or 8 h, cells were washed, harvested, sonicated in buffer, and assayed for neutral TGase activity in 50 mM Hepes buffer at pH 6.4. TGase activity was calculated as nmol of free fatty acids released per mg cellular protein per h, and then expressed relative to the activity in the sonicate of I-cell fibroblasts incubated with 5774 cell-conditioned medium for 8 h. Each point represents the mean of duplicate assays from two 60-mm Petri dishes.
of neutral TGase activity. However, when 2 mM mannose 6-phosphate was added to the culture medium, internalization of the neutral TGase was blocked by 70%, indicating the presence of the mannose 6-phosphate recognition marker on the enzyme. Since enzymes bearing the marmose 6-phosphate recognition marker are targeted to the lysosomes by receptor-competent cells, secretion of the enzymes by these cells should be increased by weak bases, such as NH,Cl [18]. Weak bases raise the lysosomal pH and interfere with the recycling of the mannose 6-phosphate receptors back to the Golgi apparatus or cell surface [30]. We attempted to increase the secretion of neutral TGase by incubating alveolar macrophages with NH,CI. As
205
TABLE I THE EFFECT OF NH&l ON THE SECRETION OF NEUTRAL TRIACYLGLYCEROL HYDROLASE BY RABBIT ALVEOLAR MACROPHAGES Rabbit alveolar macrophages were incubated at 37 o C for 4 h or 8 h in serum-free a-MEM, or in cu-MEM containing 25 mM NH,Cl or 50 mM NH,Cl. The media were collected, centrifuged at 1000 x g to remove nonadherent cells, and assayed for neutral TGase activity in 50 mM Hepes buffer at pH 6.4. The nonadherent cells were added to the cells recovered from each Petri dish, which were washed, sonicated in buffer, and assayed for neutral TGase activity. Activity is expressed as nmol free fatty acids released/mg cell protein per h. Each value represents the mean of duplicate assays on two 60-mm Petri dishes. The numbers in brackets indicate the range of the different plates as a percent of the mean. Addition to incubation
None
25 mM NH&l
50 mM NH&l
Triacylglycerol hydrolase activity (mnol free fatty acids/mg cell protein per h) media (4 h)
media (8 h)
20 (17.8)
$.3)
963
60 (28.3)
181
116
345
(4.3)
cells (8 h)
(3.9)
(3.5)
(1.7) 878 (0.3) 658 (3.8)
shown in Table I, the secretion of neutral TGase activity was low in cells incubated for 4 or 8 h in serum-free LX-MEM.Addition of 25 mM NH,Cl caused a 3-fold increase in the amount of enzyme secreted at 4 h, and a 6-fold increase at 8 h. Furthermore, addition of 50 mM NH,Cl resulted in a 6-fold increase in the secretion of the enzyme in 4 h, and a 1Zfold increase in 8 h. The neutral TGase activity in the cells decreased reciprocally during the 8-h incubation periods. There was no appreciable accumulation of lactic dehydrogenase activity in the media, indicating that the increase in neutral TGase released into the culture media was not attributable to cell breakage. In contrast, the addition of NH,Cl to 5774 cells in culture had no effect on the rate of the secretion of neutral TGase, consistent with the spontaneous release of the precursor forms of lysosomal enzymes from a mannose 6-phosphate receptor-deficient cell. These results are similar to previous reports concerning effects of NH,Cl or particle stimulation on the spontaneous secretion of fi-hexosaminidase by P388 Dl macrophages [8,9].
These data suggested that the neutral TGase might be a lysosomal enzyme. We made an attempt to localize this enzyme in the lysosomes of 5774 cells. However, we were unable to define the intracellular location of either the neutral TGase or N-acetylglucosaminidase, a lysosomal marker enzyme. This was presumably due to the inefficient sequestration of lysosomal enzymes in 5774 cells [3]. Rabbit alveolar macrophages, induced by heat-killed BCG, have been shown to contain very dense lysosomes which are readily separated from other cellular organelles [15]. In addition, rabbit alveolar macrophages contain a very high level of neutral TGase activity [l], and are mannose 6phosphate receptor-competent cells [28]. We therefore utilized BCG-induced rabbit alveolar macrophages to isolate lysosomes. As shown in Fig. 4, the post-nuclear supematant fraction which entered a 30-708 isopycnic sucrose gradient (fractions 6-30) was separated into two major protein peaks. The less dense peak (fractions 10-17) contained the mitochondrial marker enzyme, succinate INT reductase. The more dense peak (fractions 18-26) contained the lysosomal markq enzyme, N-acetylglucosaminidase. The location of neutral TGase paralleled the lysosomal fractions, providing evidence that it is a lysosomal enzyme. To verify that the neutral TGase was distinct from the acid TGase, fractions 19-24 were pooled and the pH profile was examined. As shown in Fig. 5, the acid TGase exhibited a pH optimum of 5.5, while the neutral TGase exhibited a pH optimum of 6.7. These activity peaks were consistent with those observed in whole cell homogenates of 5774 cells, rabbit alveolar macrophages, and thioglycollate-elicited mouse peritoneal macrophages. The lysosomal fraction also contained two peaks of cholesteryl ester hydrolase activity. The acid cholesteryl ester hydrolase displayed a pH optimum of 5.1, and was only slightly inhibited by 1 mg/rnl of BSA during assay. Activity of the neutral peak (optimal activity at pH 6.7) was inhibited 61% by the addition of 1 mg/ml BSA, similar to the inhibition of neutral TGase activity in 5774 cell-conditioned medium (data not shown). Although it was clear that the neutral TGase was distinct from the acid TGase, and that the neutral TGase was a lysosomal enzyme, the rela-
206
_
_
0
2
_
4
6
_
f3 10
12
14
16
Fraction
18
20
22
24
26
28
30J
Number
,
1
I
1
I
I
1
I
1.10
1.12
1.14
1.16
1.18
I
1.20
122
1.24
1.26
Density
(g/ml)
Fig. 4. Isopycmc sucrose density gradient of the post-nuclear supernatant fraction prepared from BCG-induced rabbit alveolar macrophages. Rabbit alveolar macrophages were harvested and homogenized as described under Materials and Methods. The post-nuclear supematant fraction was layered over a 30-708 sucrose gradient and centrifuged at 53000x g for 7 h. 1 ml fractions were collected and each fraction was assayed for protein (A), neutral TGase activity (A), N-acetylglucosamidase activity (o), and succinate INT reductase activity (0). The enzymatic activity in each fraction was expressed as percentage of the total activity in all fractions.
‘1: 40
44
48
52
56
60
64
68
72
PH
Fig. 5. pH profile of the TGase activity in the lysosomes of BCG-induced rabbit alveolar macrophages. Lysosomal fractions from the isopycnic sucrose density gradient of Fig. 4 were pooled and sonicated for 10 s. The sonicated fraction was assayed for TGase activity in 50 mM Hepes/acetate buffer at various pH values. The activity was expressed relative to that at pH 6.7.
tionship between the two enzymes was not clear. It has been shown that the precursors of the lysosomal enzymes, which are secreted by I-cell fibroblasts, are not subjected to the normal proteolytic processing in the lysosome 1121. Similarly, lysosomal enzyme secretion mediated by NH,Cl will include the precursor enzyme form [31]. Since the neutral TGase was secreted from 5774 cells as a result of a defect which bypasses normal processing, we considered the possibility that the neutral TGase might be a precursor of the acid TGase, rather than a different enzyme. We examined, therefore, the pH profiles of TGase activity in I-cell fibroblasts after incubation for 8 h in the presence of 5774 cell-conditioned medium, and in I-cell fibroblasts incubated in the same manner, but after a 4 h chase in fresh F-10 medium. As shown in Fig. 6A, the acid TGase in the homogenate of I-cell fibroblasts had optimal activity at pH 5.1, but activity levels were very low (16 nmol free fatty acids/mg cell protein per h). The TGase in the conditioned medium of 5774 cells displayed
207
TABLE
(4
II
THE EFFECTS OF THIOGLYCOLLATE AND MINERAL OIL ELICITATION ON THE CELL CONTENT AND SECRETION OF NEUTRAL TRIACYLGLYCEROL HYDROLASE BY MOUSE PERITONEAL MACROPHAGES Resident, thioglycollate-elicited, and mineral oil-elicited macrophages were cultured in serum-free (Y-MEM. After 24 h, cells and media were assayed for neutral TGase activity at pH 6.4 in 50 mM Hepes buffer. Triacylglycerol lipase activity is expressed as nmol free fatty acids/mg cell protein per hkS.E. (number of observations). Macrophages
Triacylglycerol hydrolase (nmol free fatty acids/ mg cell protein per h) cells
0l.I 4.0
’ 4.8
’
5.2
’
5.6
’
60
’
6.4
’ 68
PH
media (6)
176 f 11.7 (6)
Thioglycollate-elicited macrophages
1442 f 22.7 (4)
368 f 24.6 (6)
Mineral-oil-elicited macrophages
1314 f 30.5 (4)
352 f 29.4 (6)
Resident ’ 4.4
activity
macrophages
504k20.3
160-
8060-
Fig. 6. The effect of pH on TGase activity in 5774 cell-conditioned medium and homogenate of I-cell fibroblasts. (A) The 24-h 5774 cell-conditioned medium (0) and the sonicate of I-cell fibroblasts (A) were assayed for TGase activity in 50 mM Hepes/acetate buffer at the various pH values indicated. The specific activity of the TGase in I-cell fibroblasts at pH 5.1 was 16 nmol free fatty acids/mg protein per h. Activities were normalized and expressed relative to their activities at their optimal pH values (equal to 100%). (B) I-cell fibroblasts incubated with 5774 cell-conditioned medium for 8 h, and in I-cell fibroblasts incubated with 5774 cell-conditioned medium for 8 h, followed by a 4 h chase. The conditioned medium was concentrated lo-fold by Amicon Ultrafiltration. The concentrated medium was diluted 5-fold with F-10 medium containing 2.5% fetal calf serum and added to cultured I-cell
a pH optimum of 6.5. As shown in Fig. 6B, I-cell fibroblasts that had been incubated in 5774 cell-conditioned medium for 8 h exhibited optimal activity at pH 5.3. After a chase for an additional 4 h to allow all the surface membrane-bound enzyme to reach the lysosomes for processing, a predominant peak of activity was observed at pH 5.1. The activity at pH 5.1 remained lo-times higher than the activity in control I-cell fibroblasts. These data indicate that the neutral TGase was converted to the mature acid form of lysosomal TGase after reaching the lysosomes. Increased synthesis and secretion of a number of acid hydrolases, among other alterations in cellular physiology, have been reported in inflammatory macrophages [32] and also in particle-stimulated macrophages [33]. Since thioglycollate-elicited mouse peritoneal macro-
fibroblasts. After 8 h, the cells were washed, harvested, and sonicated in buffer and then assayed for TGase activity (A). A second series of I-cell fibroblasts was incubated in the same manner for 8 h, and then incubated for an additional 4 h in fresh F-10 medium containing 2.5% fetal calf serum. The cell sonicate was assayed for TGase activity (0). FFA, free fatty acids.
208
phages and rabbit alveolar macrophages are both considered to be inflammatory macrophages [34], it was likely that the high levels of neutral and acid TGase activities might reflect the activation state of the macrophages. We compared, therefore, the activity of the acid and neutral TGase in resident, thioglycollateand mineral-oil-elicited mouse peritoneal macrophages. As shown in Table II, the cellular activity assayed at pH 6.4 incresed 2.9-fold in thioglycollate-, and 2.6-fold in mineraloil-elicited macrophages as compared to resident macrophages. The secretion of the enzyme by these cells remained constant in proportion to total activity (about 20%). In contrast, the activity of the neutral cholesteryl ester hydrolase, an enzyme believed to be responsible for the hydrolysis of cytoplasmic cholesteryl esters, did not increase in either type of elicited macrophage, indicating that the increase in precursor form of acid TGase was a specific part of the inflammatory process. Activity of the mature acid TGase, measured at pH 5.1, was increased 3.4-fold in thioglycollateelicited macrophages. The mature form of the TGase was predominant in the homogenate prepared from human skin fibroblasts, and exhibited a pH optimum of 5.1. However, when the fibroblasts were incubated with 25 mM NH,Cl, the TGase activity secreted into the culture medium was predominantly in the precursor form, and exhibited an optimal activity at pH 6.2. The pH profile of the TGase activity in the culture medium of human skin fibroblasts was similar to that of 5774 cell-conditioned medium (data not shown). This is consistent with the hypothesis that NH,Cl interferes with the recycling of mannose 6-phosphate receptors, preventing the sequestration of the newly synthesized lysosomal enzymes in human skin fibroblasts. In the absence of mannose 6-phosphate receptor recycling, the fibroblasts functioned in the same manner as receptor-deficient 5774 cells and secreted the precursor form of lysosomal acid TGase. Concluding remarks We have presented evidence that 5774 macrophages spontaneously secreted a neutral TGase which appears to be the precursor form of lysosomal acid TGase. The secretion of large quanti-
ties of this immature enzyme is apparently facilitated by the absence of the mannose 6-phosphate receptor ” . The 5774 cells retained 50% of the precursor TGase after 9 h in culture, and also exhibited the mature form of the acid TGase intracellularly. The ability to retain and process lysosomal enzymes may have been due to the presence of the mannose 6-phosphate receptorCD. Although this second receptor has not been identified in the 5774 cell line, it is present in P388 Dl macrophages, another murine tumor cell line which is mannose 6-phosphate receptor ‘t-deficient [lo]. The distinction between precursor and mature forms was facilitated by the use of an ethanolic tri[ l4 Cjoleoylglycerol emulsion substrate presentation in the enzyme assays. The precursor (neutral TGase) had a pH optimum of 6.4, whereas the mature enzyme had a pH optimum of 5.5. We were unable to detect the activity of the mature form (lysosomal acid TGase) in the conditioned medium of either 5774 or rabbit alveolar macrophages. If the neutral TGase were a second lysosomal enzyme rather than a precursor to the lysosomal acid TGase, we would expect both of the enzymes to be found in the media. In addition, while the homogenates of rabbit alveolar macrophages and human skin fibroblasts contain both the neutral and acid forms of TGase, the neutral form was secreted predominantly in response to NH,Cl. Since NH,Cl interferes with the transport of newly synthesized lysosomal enzymes to the endosomes, where they are proteolytically converted to the mature forms [35], the enzyme secreted under these conditions is primarily the precursor form. These data, combined with data concerning the uptake and conversion of the neutral TGase to the acid TGase in I-cell fibroblasts support the conclusion that the neutral TGase is the lysosomal acid TGase precursor. While there is no evidence that the neutral TGase functions in vivo, we cannot preclude the possibility that it may function intracellularly, and/or extracellularly in an appropriately neutral environment. For example, it has been shown that macrophage endosomes contain proteinases which degrade endocytosed protein ligands [36]. A second possibility is that the neutral TGase plays an extracellular role at the site of inflammation, since macrophages do secret significant amounts of
lysosomal enzymes in response to inflammatory [32] and phagocytosable stimuli [33]. Secretion of the neutral TGase was increased similarly in thioglycollate-elicited mouse peritoneal macrophages in the present study. Acknowledgements
The authors thank Elizabeth Miller, Scott Herdman and Patricia McLoughhn for their technical assistance. This work was supported by National Institutes of Health Research Grant HL34724 and Training Grant HL-07276 awarded by the National Heart, Lung and Blood Institute. References 1 Khoo, J.C., Vance, J.E., Mahoney, E.M., Jensen, D., Wancewicz, E. and Steinberg, D. (1984) Arteriosclerosis 4, 34-40. 2 Msrland, B. and Kaplan, G. (1978) Exp. Cell Res. 115, 63-72. 3 Gabel, C.A., Goldberg, D.E. and Komfeld, S. (1983) Proc. Natl. Acad. Sci. USA 80, 775-779. 4 Shepherd, V.L., Freeze, H.H., Miller, A.L. and Stahl, P.D. (1984) J. Biol. Chem. 259, 2257-2261. 5 Hickman, S. and Neufeld, E.F. (1972) B&hem. Biophys. Res. Commun. 49, 992-999. 6 Sly, W.S. and Stahl, P. (1978) in Transport of Macromolecules in Cellular Systems (Silverstein, S.C., ed.), pp. 229-244, DahIen Konferenzen, Berlin. 7 Von Figura, K. and Weber, E. (1978) Biochem. J. 176, 943-950. 8 Jessup, W. and Dean, R.T. (1982) B&hem. Biophys. Res. Commun. 105, 922-927. 9 Jessup, W. and Dean, R.T. (1980) Biochem. J. 190,847-850. 10 Hoflack, B. and Komfeld, S. (1985) Proc. Natl. Acad. Sci. USA 82,4428-4432. 11 Hoflack, B. and Komfeld, S. (1985) J. Biol. Chem. 260, 12008-12014. 12 Hasihk, A. and Von Figura, K. (1984) in Lysosomes (DingIe, J.T., Dean, R.T. and Sly, W., eds.), Vol. 7, pp. 3-16, Elsevier, Amsterdam. 13 Mahoney, E.M., HamiU, A.L., Scott, W.A. and Cohn, Z.A. (1977) Proc. Natl. Acad. Sci. USA 74, 4895-4899.
14 Mahoney, E.M., Khoo, J.C. and Steinberg, D. (1982) Proc. Natl. Acad. Sci. USA 79,1639-1642. 15 Cohn, Z.A. and Wiener, E. (1963) J. Exp. Med. 118, 991-1008. 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 17 Khoo, J.C., Mahoney, E.M. and Steinberg, D. (1981) J. Biol. Chem. 256,12659-1266X 18 Khoo, J.C. and Steinberg, D. (1975) Methods Enzymol. 35, 181-189. 19 Lewy, G.A. and Conchie, J. (1966) Methods Enzymol. 8, 571-584. 20 Pennington, R.J. (1961) B&hem. J. 80, 649-654. 21 Bergmeyer, H.U., Bemt, E. and Hess, B. (1963) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed.), pp. 736-743, Academic Press, New York. 22 Khoo, J.C., Mahoney, E.M. and Witztum, J.L. (1981) J. Biol. Chem. 256, 7105-7108. 23 Werb, Z. and Cohn, Z.A. (1972) J. Exp. Med. 135, 21-44. 24 Sando, G.N. and Rosenbaum, L.M. (1985) J. Biol. Chem. 260, 15186-15193. K., Ohkuma, S. and Takano, 25 Imanaka, T., Amanuma-Muto, T. (1984) J. B&hem. 96,1089-1101. M.D. and Swank, R.T. (1981) J. Biol. Chem. 26 Skudlarek, 256, 10137-10144. 27 Goldberg, D.E., Gabel, C.A. and Komfeld, S. (1983) J. Cell Biol. 97, 1700-1706. 28 Shepherd, V.L. and Stahl, P.D. (1984) in Lysosomes (Dingle, J.T., Dean, R.T. and Sly, W., eds.), Vol. 7, pp. 83-98, Elsevier, Amsterdam. 29 Creek, K.E. and Sly, W.S. (1984) in Lysosomes (DingIe, J.T., Dean, R.T. and Sly, W., eds.), Vol. 7, pp. 63-82, Elsevier, Amsterdam. A., Grubb, J.H., Talkad, V. and Sly, 30 Gonzalez-Noriega, W.S. (1980) J. Cell Biol. 85, 839-852. 31 Skudlarek, M.D., Novak, E.K. and Swank, R.T. (1984) in Lysosomes (DingIe, J.T., Dean, R.T. and Sly, W., eds.), Vol. 7, pp. 17-43, EIsevier, Amsterdam. 32 Schnyder, J. and Baggiohni, M. (1978) J. Exp. Med. 148, 435-450. 33 McCarthy, K., Musson, R.A. and Henson, P.M. (1982) J. Reticulendothel. Sot. 31, 131-144. 34 Adams, D.O. and Hamilton, T.A. (1984) Amm. Rev. Immunol. 2, 283-318. 35 Brown, W.J., Goodhouse, J. and Farquhar, M.G. (1986) J. Cell Biol. 103, 1235-1247. 36 Diment, S. and Stahl, P. (1985) J. Biol. Chem. 260, 15311-15317.
Biochimica et Biophysicu Acta 960 (1988) 200-209 EkVier
BBA 52818
Secretion of the lysosomal acid triacylglycerol hydrolase precursor by 5774 Macrophages Dennis I. Goldberg
* and John C. Khoo
Division of Endocrinology and Metabolism, Departmen of Medicine, University of California, San Diego, LA JoNa, CA (U.S.A.) (Received 23 September 1987) (Revised manuscript received 3 February 1988) Key words: Triacylglycerol hydrolase; Triacylglycerol lipase; Lysosomal enzyme; Protein transport; Mannose 6-phosphate receptor; (Macrophage)
5774, thioglycollate-elicited mouse peritoneal and BCGinduced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The 5774 macrophages, a cell line deficient in the calcium-independent mannOSe 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH&I induced a dfold increase in TGase secretion by alveolar macrophages, while 50 mM NH&l induced a 1Zfold increase in TGase secretion. NH&l had no effect on TGase secretion by 5774 macrophages. The TGase secreted by 5774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase lo-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose bphosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase. Introduction We have recently reported the presence of a highly active neutral triacylglycerol hydrolase
* Present address: Department of Metabolic Diseases, Central Research Division, Pfiir, Inc., Groton, CT, U.S.A. Abbreviations: TGase, triacylglycerol hydrolase (EC 3.1.1.3); a-MEM, u- minimal essential medium; BCG, Bacillus Calmette Guerin; Hepes, 4-(2-hydroxyethyl>l-piperazineethanesulfonic acid; BSA, bovine serum albumin; INT, 2-(p-iodophenyl)-3( p-nitrophenyl>5-phenyltetrazolium chloride. Correspondence: J.C. Khoo, Division of Endocrinology and Metabolism, Department of Medicine, M-013D, University of California, San Diego, La Jolla, CA 92093-0613, U.S.A. 0005-2760/88/$03.50
(TGase) in several types of macrophage [l]. This enzyme displayed a number of characteristics which distinguished it from classical acid TGase. These traits included a nearly neutral pH optimum (pH 6.4), remarkable thermal stability, the absence of activity towards monooleoylglycerol, and inhibition by 1 mg/ml bovine serum albumin (BSA). It has been proposed that this enzyme may be responsible for the preferential mobilization of triacylglycerols stored in macrophages, resulting in the enriched cholesteryl ester content of the lipid inclusions found in macrophage-derived foam cells of the atherosclerotic lesion [l]. In the present study, we report the spontaneous secretion of large quantities of neutral TGase by
0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
201
5774 cells, a macrophage-like cell line deficient in the 215 kDa, bourn-~dependent mannose 6phosphate receptor (mannose 6-phosphate receptorcl) [2,3]. This receptor is involved in targeting newly synthesized enzymes to the lysosomes, where the mannose 6-phosphate recognition marker is removed and the enzyme is processed to the mature form [4]. The marmose 6-phosphate receptorc’ is located both intracellularly and on the cell surface, and the cell surface receptors are believed to mediate the endocytotic recapture of lysosomal enzyme precursors which have escaped the intracellular packaging system [4]. The ‘secretion-recapture’ pathway has been hypothesized to constitute a principal mechanism of transport for lysosomal enzymes in fibroblasts [S], although more recent theories consider the contribution to lysosomal enzyme targeting to be much more limited [6,7]. Evidence of a similar pathway in macrophages has been presented f8], but the significance of the ‘secretion-recapture’ pathway contribution to lysosomal enzyme processing in macrophages is also questionable [4]. Cells which are mannose 6-phosphate receptor ‘i-deficient, such as the 5774 or P388 Dl macrophage cell lines, are unable to effectively target or recapture lysosomal enzyme precursors, resulting in the secretion of large quantities of these enzymes [3,9]. More recently, a second mamrose 6-phosphate receptor has been identified in the P388 Dl macrophage [lO,ll]. Although the function of this 46 kDa, calcium-dependent mannose 6-phosphate receptor (mannose 6-phosphate receptor D, has not been clearly delineated, it appears to be involved in targeting of newly synthesized lysosomal enzymes, and may be responsible for the retention of large amounts of lysosomal enzymes in the P388 Dl macrophage [lo]. Although all of the lysosomal enzymes examined to date have been found to be synthesized via larger precursors, and the secretion of many lysosomal enzyme precursors has been reported [12], neither the precursor nor a mature form of the acid TGase has previously been found to be secreted. In the present study, the neutral TGase secreted from J774 cells is internalized by I-cell disease fibroblasts through their cell surface mannose 6-phosphate receptorcl, indicating the presence of phosphorylated mannose on the enzyme.
The internalized TGase was then converted to an enzyme with a pH optimum of 5.1, suggesting that the neutral TGase is a precursor of the mature lysosomal acid TGase. In contrast, macrophages which are mannose 6-phosphate receptor “-cornpetent localized the enzyme in the lysosomes and secreted much lower amounts of the neutral TGase. Materials and Methods New Zealand White rabbits (2-3 kg) were obtained from Holbert’s Rabbitry, and female Swiss-Webster mice (25-30 g) from Simonsen Laboratories. Tri[l- l4 Cloleoylglycerol and cholesteryl [l-‘4C]oleate were obtained from Amersham-Searle. Cholesteryl oleate, trioleoylglycerol, free fatty acid-poor BSA, D-mannose 6phosphate, D-mannose, glucose 6-phosphate, ammonium chloride, Freund’s complete adjuvant and NADPH were obtained from Sigma; pnitrophenyl 2-acetamido-2-deoxy-&i&pyranoside and 2-( p-iodophenyl)-3-( p-nitrophenyl)-5-phenyltetrazolium chloride (INT) from Calbiochem; OLminimal essential medium ((r-MEM) and Ham’s F-10 medium from Gibco; t~o~yco~ate medium from Difco; fetal calf serum from Irvine Scientific; and heparin-Sepharose from Pharmacia. All other chemicals were of analytical grade. The 5774 macrophage-me cell line (clone G8) was a gift of Dr. Jay Unkeless of Rockefeller University. P388 Dl cells were obtained from Dr. Hillel Koren of Duke University Medical Center, and human skin fibroblasts, from a patient with mucolipidosis II (I-cell disease), were a gift of Dr. Arnold Miller of the University of California, San Diego. Cell preparations Cultures of J774 cells were maintained in 1% mm Petri dishes containing 15 ml of a-MEM supplemented with gentamycin sulfate (50 pg/ml), fungizone (0.25 pg/ml) and 5% heat-inactivated fetal calf serum. Cells were washed with serum-free at-MEM prior to each experiment, and rn~nt~~ in serum-free cr-MEM for the duration of each ,incubation. Primary cultures of mouse peritoneal macroiphages were prepared as previously described [13]. ;Macrophages were elicited by intraperitoneal injection of thioglycollate medium 3 days prior to
202
harvest. The macrophages were plated at a density of 1 . lo7 cells per 60-mm Petri dish in a-MEM containing 10% fetal calf serum. After 3-4 h, nonadherent cells were removed by washing three times in serum-free c~-MEM. The cells were maintained in 2 ml of serum-free medium for the duration of the incubation. Rabbit alveolar macrophages were harvested in serum-free (r-MEM and cultured in 60-mm Petridishes [14]. The cells were washed and maintained as described for mouse peritoneal macrophages. I-cell disease fibroblasts were cultured in Ham’s F-10 medium containing gentamycin sulfate (50 pg/ml) and 15% fetal calf serum. The cells were washed with serum-free F-10 medium prior to use, and maintained on F-10 medium with 5% fetal calf serum during the incubation. After each incubation, the media were removed and the cells were washed with cold phosphatebuffered saline (pH 7.4), and harvested in 1 ml of 25 mM Tris-HCl/l mM EDTA/ZO% glycerol (pH 7.4) at 4°C with a rubber policeman. The media were centrifuged at 1000 x g for 10 min to precipitate any nonadherent cells which had been included in the cell harvest. Isolation of lysosomes Rabbit alveolar macrophages were induced by ear vein injection of 0.5 ml complete Freund’s adjuvant (containing heat-killed Bacillus Calmette Guerin (BCG)) diluted with 0.5 ml of sterile saline. After 2-3 weeks, the alveolar macrophages were harvested by bronchial lavage with serum-free 01MEM as described [14]. This procedure yielded (2-3). 10’ macrophages/rabbit. The cell pellet was homogenized in 25 mM Tris-HCl/l mM EDTA/250 mM sucrose (pH 7.4), according to Cohn and Wiener [15]. 3 ml of homogenate, containing (5-6). lo7 cells/ml, were centrifuged at 1000 x g for 12 min. The post-nuclear supematant fraction was layered over 30 ml of a 30-70% linear sucrose gradient, and centrifuged for 6-7 h at 53 000 x g in a Beckman SW-27 rotor at 4’ C. The gradient was harvested in 1 ml fractions with a Buchler Auto Density Flow II C fractionator. The density of each fraction was measured by weight of a known volume relative to the weight of water. Each fraction was sonicated for 12-15 s and assayed for neutral TGase, N-acetylglucosamini-
dase, and succinate tein was determined
INT reductase by the method
activities. Proof Lowry et al.
1161. Enzyme assays Neutral TGase activity was assayed in an incubation mixture of 0.8 ml containing 50 mM Hepes (pH 6.4), 0.3 mg/ml BSA, 0.0625 mM tri[l-‘4C]oleoylglycerol in an ethanolic emulsion and the enzyme preparation (either cell sonicate or medium), as described previously [l]. Assays were incubated at 30’ C for 30 min. Neutral cholesteryl ester hydrolase was assayed in 0.8 ml of an incubation mixture containing 0.032 mM cholesteryl [l-‘4C]oleate in ethanol, 1.25 mg/ml BSA, 50 mM Hepes (pH 6.4) and either cell sonicate or medium. The assays were incubated for 30 min at 30°C [17]. Acid TGase was assayed in an incubation mixture containing 0.8 ml of 0.0625 mM tri[lI4 Cloleoylglycerol, 50 mM sodium acetate/ Hepes buffer (pH 5.1) 0.6 mg/ml BSA and enzyme preparation for 30 min at 30 o C. Acid cholesteryl ester hydrolase was assayed under the same conditions substituting 0.032 mM cholesteryl [li4C]oleate for the substrate All assays were terminated by the addition of 3 ml of chloroform/ methanol/ benzene (1: 2.4 : 2, v/v). [‘4C]Oleic acid was extracted after the addition of 0.1 ml of 1 M NaOH, and quantified as previously described [ 181. N-Acetylglucosaminidase was assayed according to Lewy and Conchie [19], using p-nitrophenyl 2-acetamido-2-deoxy-B-D-pyranoside as substrate. Succinate INT reductase was assayed by the method of Pennington [20]. Lactic dehydrogenase in macrophage-conditioned media was measured according to Bergmeyer et al. [21]. Results and Discussion The 5774 macrophage-like cell line expresses three TGase activities: (1) lipoprotein lipase [22]; (2) a classical acid TGase [23]; and (3) a nearly neutral TGase [l]. As shown in Fig. 1, the acid TGase and the neutral TGase activities in broken cell preparations of 5774 cells exhibited distinct pH optima, with peak activities at pH 5.5 and 6.4, respectively. Similar pH profiles were also
203
h
40
44
46
52
56
60
64
66
7.2
PH
Fig. 1. The influence of pH on TGase activity in 5774 macrophages. The 5774 cell sonicate was assayed in 50 mM Hepes/ acetate buffer as described under Materials and Methods. The activity was expressed relative to the maximal activity at its optimal pH (equal to 100%).
observed in broken cell preparations of rabbit alveolar macrophages and thioglycollate-elicited mouse peritoneal macrophages. Optimal activity for the acid TGase in the latter two cell types was observed at pH 5.1, while the more neutral TGase displayed optimal activity at pH 6.3 (not shown). It has been shown that substrate presentation as well as buffer selection can significantly alter the pH optimum observed for the lysosomal acid TGase activity of human skin fibroblasts [24]. These factors similarly affected the observed pH profile for neutral TGase. The optimal activity shifted from pH 6.4 to pH 5.4 when the enzyme was assayed with a sonicated emulsion consisting of tri[l-‘4C]oleoylglycerol, sodium taurocholate and phosphatidylcholine [25] (not shown). Since the ethanolic substrate emulsion allowed us to distinguish between the neutral TGase and acid TGase, it was used in subsequent studies. 5774 cell-conditioned culture medium contained a high level of TGase activity, with a pH optimum at 6.5 (Fig. 6A). This enzyme was distinguished from lipoprotein lipase activity in the medium by several criteria.. Lipoprotein lipase binds to heparin-Sepharose affinity gel, its activity is increased by apoprotein C-II, and the enzyme is heat-labile, losing all of its activity when heated at 5O“C for 4 h. In contrast, the neutral TGase did
not bind to heparin-Sepharose gel, its activity was not stimulated by apoprotein C-II when assayed at either pH 6.4 or 8.2, and the enzyme was extremely heat-stable, retaining all its activity after 4 h of heating at 50°C. In addition, the TGase bound weakly to DEAE-cellulose, but tightly to CM-cellulose, consistent with the observed p1 of 9, as determined by isoelectric focusing. The neutral TGase hydrolyzed trioleoylglycerol and dioleoylglycerol at equal rates, but did not display hydrolytic activity towards monooleoylglycerol. The enzyme in the medium also displayed activity against cholesteryl[l- “C]oleate, which was similar to the TGase activity in pH profile and susceptibility to BSA inhibition (60% inhibition by 1 mg/ml BSA). The secretion of neutral TGase by 5774 cells was linear with time (Fig. 2). About 50% of the total activity (cells plus medium) was recovered in the medium after 9 h of incubation (cell content of 823 f 56 mnol free fatty acids/mg protein per h). The activity in the cells, however, remained constant during the entire period of incubation. There was no detectable lactic dehydrogenase activity in the 9 h conditioned medium, ruling out cell damage as the cause for release of neutral TGase into the culture medium. r
Hours
in
Culture
Fig. 2. The release of neutral TGase activity from 5774 cells, mouse peritoneal macrophages, and rabbit alveolar macrophages. Cells were maintained in serum-free a-MEM. Media were collected and replenished with fresh cr-MEM every 3 h for 5774 cells (0) and thioglycollate-elicited mouse peritoneal macrophages (A) or every 2 h for rabbit alveolar macrophages (m). Aliquots of the media from each cell type were assayed for neutral TGase activity. The TGase activity in cell sonicates of 5774 cells, mouse peritoneal and rabbit alveolar macrophages at the end of the experiment were 823 f 56, 1227k69, and 3322k23 nmol free fatty acids (FFA)/mg protein per h, respectively.
204
Since the cellular levels of neutral TGase activity vary significantly among different types of macrophages [l], we compared the release of the enzyme from thioglycollate-elicited mouse peritoneal macrophages and from BCG-induced rabbit alveolar macrophages with that from 5774 cells. Enzyme secretion was also linear from these cells for up to 8-9 h (Fig. 2). However, the amounts of enzyme secreted relative to the cell content of enzyme varied greatly. Thioglycollate-elicited mouse peritoneal macrophages released 21% of the total activity in 9 h (cell content of 1227 f 69 nmol free fatty acids/mg protein per h), and rabbit alveolar macrophages released only 3% of the total activity in 8 h (cell content of 3322 f 23 nmol free fatty acids/mg protein per h). The levels of neutral TGase released by the three macrophage cell types were in agreement with previous reports of spontaneous secretion of P-glucuronidase by thioglycollate-elicited mouse peritoneal macrophages [26], and spontaneous secretion of P-hexosaminidase and &#.rcuronidase by 5774 cells [3]. The secretion of large quantities of lysosomal enzymes by 5774 cells has been attributed to a deficiency in the marmose 6-phosphate receptorc’, which is involved in targeting newly synthesized enzymes to the lysosomes [27]. Since the amount of enzyme released by the three cell types examined in the present study was inversely proportional to their reported expression of marmosyl-glycoprotein receptor activity [28], we examined the enzyme for the presence of the mannose 6-phosphate marker. I-cell fibroblasts, which are mannose 6-phosphate receptor-competent cells that fail to incorporate the mannose 6-phosphate marker into newly synthesized enzymes [29], were utilized in these studies. These cells possess small amounts of endogenous lysosomal enzymes, but readily internalize exogenous enzymes which contain the mannose 6-phosphate marker. The conditioned medium of 5774 cells was first concentrated lo-fold by Amicon Ultrafiltration, and then diluted 5-fold with fresh F-10 medium. This medium was then added to cultured I-cell fibroblasts. As shown in Fig. 3, intracellular neutral TGase activity increased lo-fold in 8 h. The addition of 2 mM mannose or 2 mM glucose 6-phosphate had no effect on the internalization
Fig. 3. The uptake of neutral TGase from 5774 cell-conditioned medium by I-cell fibroblasts. The 24-h 5774 cell-conditioned medium was concentrated lo-fold. The concentrated medium was diluted 5-fold with F-10 medium containing 2.5% fetal calf serum which was then added to I-cell fibroblasts (0); I-cell fibroblasts plus 2 mM mannose (A); I-cell fibroblasts plus 2 mM glucose 6-phosphate (m); and I-cell fibroblasts plus 2 mM mannose B-phosphate (A). For control, I-cell fibroblasts were incubated in F-10 medium containing 2.5% fetal calf serum (0). After incubation at 37OC for 4 h or 8 h, cells were washed, harvested, sonicated in buffer, and assayed for neutral TGase activity in 50 mM Hepes buffer at pH 6.4. TGase activity was calculated as nmol of free fatty acids released per mg cellular protein per h, and then expressed relative to the activity in the sonicate of I-cell fibroblasts incubated with 5774 cell-conditioned medium for 8 h. Each point represents the mean of duplicate assays from two 60-mm Petri dishes.
of neutral TGase activity. However, when 2 mM mannose 6-phosphate was added to the culture medium, internalization of the neutral TGase was blocked by 70%, indicating the presence of the mannose 6-phosphate recognition marker on the enzyme. Since enzymes bearing the marmose 6-phosphate recognition marker are targeted to the lysosomes by receptor-competent cells, secretion of the enzymes by these cells should be increased by weak bases, such as NH,Cl [18]. Weak bases raise the lysosomal pH and interfere with the recycling of the mannose 6-phosphate receptors back to the Golgi apparatus or cell surface [30]. We attempted to increase the secretion of neutral TGase by incubating alveolar macrophages with NH,CI. As
205
TABLE I THE EFFECT OF NH&l ON THE SECRETION OF NEUTRAL TRIACYLGLYCEROL HYDROLASE BY RABBIT ALVEOLAR MACROPHAGES Rabbit alveolar macrophages were incubated at 37 o C for 4 h or 8 h in serum-free a-MEM, or in cu-MEM containing 25 mM NH,Cl or 50 mM NH,Cl. The media were collected, centrifuged at 1000 x g to remove nonadherent cells, and assayed for neutral TGase activity in 50 mM Hepes buffer at pH 6.4. The nonadherent cells were added to the cells recovered from each Petri dish, which were washed, sonicated in buffer, and assayed for neutral TGase activity. Activity is expressed as nmol free fatty acids released/mg cell protein per h. Each value represents the mean of duplicate assays on two 60-mm Petri dishes. The numbers in brackets indicate the range of the different plates as a percent of the mean. Addition to incubation
None
25 mM NH&l
50 mM NH&l
Triacylglycerol hydrolase activity (mnol free fatty acids/mg cell protein per h) media (4 h)
media (8 h)
20 (17.8)
$.3)
963
60 (28.3)
181
116
345
(4.3)
cells (8 h)
(3.9)
(3.5)
(1.7) 878 (0.3) 658 (3.8)
shown in Table I, the secretion of neutral TGase activity was low in cells incubated for 4 or 8 h in serum-free LX-MEM.Addition of 25 mM NH,Cl caused a 3-fold increase in the amount of enzyme secreted at 4 h, and a 6-fold increase at 8 h. Furthermore, addition of 50 mM NH,Cl resulted in a 6-fold increase in the secretion of the enzyme in 4 h, and a 1Zfold increase in 8 h. The neutral TGase activity in the cells decreased reciprocally during the 8-h incubation periods. There was no appreciable accumulation of lactic dehydrogenase activity in the media, indicating that the increase in neutral TGase released into the culture media was not attributable to cell breakage. In contrast, the addition of NH,Cl to 5774 cells in culture had no effect on the rate of the secretion of neutral TGase, consistent with the spontaneous release of the precursor forms of lysosomal enzymes from a mannose 6-phosphate receptor-deficient cell. These results are similar to previous reports concerning effects of NH,Cl or particle stimulation on the spontaneous secretion of fi-hexosaminidase by P388 Dl macrophages [8,9].
These data suggested that the neutral TGase might be a lysosomal enzyme. We made an attempt to localize this enzyme in the lysosomes of 5774 cells. However, we were unable to define the intracellular location of either the neutral TGase or N-acetylglucosaminidase, a lysosomal marker enzyme. This was presumably due to the inefficient sequestration of lysosomal enzymes in 5774 cells [3]. Rabbit alveolar macrophages, induced by heat-killed BCG, have been shown to contain very dense lysosomes which are readily separated from other cellular organelles [15]. In addition, rabbit alveolar macrophages contain a very high level of neutral TGase activity [l], and are mannose 6phosphate receptor-competent cells [28]. We therefore utilized BCG-induced rabbit alveolar macrophages to isolate lysosomes. As shown in Fig. 4, the post-nuclear supematant fraction which entered a 30-708 isopycnic sucrose gradient (fractions 6-30) was separated into two major protein peaks. The less dense peak (fractions 10-17) contained the mitochondrial marker enzyme, succinate INT reductase. The more dense peak (fractions 18-26) contained the lysosomal markq enzyme, N-acetylglucosaminidase. The location of neutral TGase paralleled the lysosomal fractions, providing evidence that it is a lysosomal enzyme. To verify that the neutral TGase was distinct from the acid TGase, fractions 19-24 were pooled and the pH profile was examined. As shown in Fig. 5, the acid TGase exhibited a pH optimum of 5.5, while the neutral TGase exhibited a pH optimum of 6.7. These activity peaks were consistent with those observed in whole cell homogenates of 5774 cells, rabbit alveolar macrophages, and thioglycollate-elicited mouse peritoneal macrophages. The lysosomal fraction also contained two peaks of cholesteryl ester hydrolase activity. The acid cholesteryl ester hydrolase displayed a pH optimum of 5.1, and was only slightly inhibited by 1 mg/rnl of BSA during assay. Activity of the neutral peak (optimal activity at pH 6.7) was inhibited 61% by the addition of 1 mg/ml BSA, similar to the inhibition of neutral TGase activity in 5774 cell-conditioned medium (data not shown). Although it was clear that the neutral TGase was distinct from the acid TGase, and that the neutral TGase was a lysosomal enzyme, the rela-
206
_
_
0
2
_
4
6
_
f3 10
12
14
16
Fraction
18
20
22
24
26
28
30J
Number
,
1
I
1
I
I
1
I
1.10
1.12
1.14
1.16
1.18
I
1.20
122
1.24
1.26
Density
(g/ml)
Fig. 4. Isopycmc sucrose density gradient of the post-nuclear supernatant fraction prepared from BCG-induced rabbit alveolar macrophages. Rabbit alveolar macrophages were harvested and homogenized as described under Materials and Methods. The post-nuclear supematant fraction was layered over a 30-708 sucrose gradient and centrifuged at 53000x g for 7 h. 1 ml fractions were collected and each fraction was assayed for protein (A), neutral TGase activity (A), N-acetylglucosamidase activity (o), and succinate INT reductase activity (0). The enzymatic activity in each fraction was expressed as percentage of the total activity in all fractions.
‘1: 40
44
48
52
56
60
64
68
72
PH
Fig. 5. pH profile of the TGase activity in the lysosomes of BCG-induced rabbit alveolar macrophages. Lysosomal fractions from the isopycnic sucrose density gradient of Fig. 4 were pooled and sonicated for 10 s. The sonicated fraction was assayed for TGase activity in 50 mM Hepes/acetate buffer at various pH values. The activity was expressed relative to that at pH 6.7.
tionship between the two enzymes was not clear. It has been shown that the precursors of the lysosomal enzymes, which are secreted by I-cell fibroblasts, are not subjected to the normal proteolytic processing in the lysosome 1121. Similarly, lysosomal enzyme secretion mediated by NH,Cl will include the precursor enzyme form [31]. Since the neutral TGase was secreted from 5774 cells as a result of a defect which bypasses normal processing, we considered the possibility that the neutral TGase might be a precursor of the acid TGase, rather than a different enzyme. We examined, therefore, the pH profiles of TGase activity in I-cell fibroblasts after incubation for 8 h in the presence of 5774 cell-conditioned medium, and in I-cell fibroblasts incubated in the same manner, but after a 4 h chase in fresh F-10 medium. As shown in Fig. 6A, the acid TGase in the homogenate of I-cell fibroblasts had optimal activity at pH 5.1, but activity levels were very low (16 nmol free fatty acids/mg cell protein per h). The TGase in the conditioned medium of 5774 cells displayed
207
TABLE
(4
II
THE EFFECTS OF THIOGLYCOLLATE AND MINERAL OIL ELICITATION ON THE CELL CONTENT AND SECRETION OF NEUTRAL TRIACYLGLYCEROL HYDROLASE BY MOUSE PERITONEAL MACROPHAGES Resident, thioglycollate-elicited, and mineral oil-elicited macrophages were cultured in serum-free (Y-MEM. After 24 h, cells and media were assayed for neutral TGase activity at pH 6.4 in 50 mM Hepes buffer. Triacylglycerol lipase activity is expressed as nmol free fatty acids/mg cell protein per hkS.E. (number of observations). Macrophages
Triacylglycerol hydrolase (nmol free fatty acids/ mg cell protein per h) cells
0l.I 4.0
’ 4.8
’
5.2
’
5.6
’
60
’
6.4
’ 68
PH
media (6)
176 f 11.7 (6)
Thioglycollate-elicited macrophages
1442 f 22.7 (4)
368 f 24.6 (6)
Mineral-oil-elicited macrophages
1314 f 30.5 (4)
352 f 29.4 (6)
Resident ’ 4.4
activity
macrophages
504k20.3
160-
8060-
Fig. 6. The effect of pH on TGase activity in 5774 cell-conditioned medium and homogenate of I-cell fibroblasts. (A) The 24-h 5774 cell-conditioned medium (0) and the sonicate of I-cell fibroblasts (A) were assayed for TGase activity in 50 mM Hepes/acetate buffer at the various pH values indicated. The specific activity of the TGase in I-cell fibroblasts at pH 5.1 was 16 nmol free fatty acids/mg protein per h. Activities were normalized and expressed relative to their activities at their optimal pH values (equal to 100%). (B) I-cell fibroblasts incubated with 5774 cell-conditioned medium for 8 h, and in I-cell fibroblasts incubated with 5774 cell-conditioned medium for 8 h, followed by a 4 h chase. The conditioned medium was concentrated lo-fold by Amicon Ultrafiltration. The concentrated medium was diluted 5-fold with F-10 medium containing 2.5% fetal calf serum and added to cultured I-cell
a pH optimum of 6.5. As shown in Fig. 6B, I-cell fibroblasts that had been incubated in 5774 cell-conditioned medium for 8 h exhibited optimal activity at pH 5.3. After a chase for an additional 4 h to allow all the surface membrane-bound enzyme to reach the lysosomes for processing, a predominant peak of activity was observed at pH 5.1. The activity at pH 5.1 remained lo-times higher than the activity in control I-cell fibroblasts. These data indicate that the neutral TGase was converted to the mature acid form of lysosomal TGase after reaching the lysosomes. Increased synthesis and secretion of a number of acid hydrolases, among other alterations in cellular physiology, have been reported in inflammatory macrophages [32] and also in particle-stimulated macrophages [33]. Since thioglycollate-elicited mouse peritoneal macro-
fibroblasts. After 8 h, the cells were washed, harvested, and sonicated in buffer and then assayed for TGase activity (A). A second series of I-cell fibroblasts was incubated in the same manner for 8 h, and then incubated for an additional 4 h in fresh F-10 medium containing 2.5% fetal calf serum. The cell sonicate was assayed for TGase activity (0). FFA, free fatty acids.
208
phages and rabbit alveolar macrophages are both considered to be inflammatory macrophages [34], it was likely that the high levels of neutral and acid TGase activities might reflect the activation state of the macrophages. We compared, therefore, the activity of the acid and neutral TGase in resident, thioglycollateand mineral-oil-elicited mouse peritoneal macrophages. As shown in Table II, the cellular activity assayed at pH 6.4 incresed 2.9-fold in thioglycollate-, and 2.6-fold in mineraloil-elicited macrophages as compared to resident macrophages. The secretion of the enzyme by these cells remained constant in proportion to total activity (about 20%). In contrast, the activity of the neutral cholesteryl ester hydrolase, an enzyme believed to be responsible for the hydrolysis of cytoplasmic cholesteryl esters, did not increase in either type of elicited macrophage, indicating that the increase in precursor form of acid TGase was a specific part of the inflammatory process. Activity of the mature acid TGase, measured at pH 5.1, was increased 3.4-fold in thioglycollateelicited macrophages. The mature form of the TGase was predominant in the homogenate prepared from human skin fibroblasts, and exhibited a pH optimum of 5.1. However, when the fibroblasts were incubated with 25 mM NH,Cl, the TGase activity secreted into the culture medium was predominantly in the precursor form, and exhibited an optimal activity at pH 6.2. The pH profile of the TGase activity in the culture medium of human skin fibroblasts was similar to that of 5774 cell-conditioned medium (data not shown). This is consistent with the hypothesis that NH,Cl interferes with the recycling of mannose 6-phosphate receptors, preventing the sequestration of the newly synthesized lysosomal enzymes in human skin fibroblasts. In the absence of mannose 6-phosphate receptor recycling, the fibroblasts functioned in the same manner as receptor-deficient 5774 cells and secreted the precursor form of lysosomal acid TGase. Concluding remarks We have presented evidence that 5774 macrophages spontaneously secreted a neutral TGase which appears to be the precursor form of lysosomal acid TGase. The secretion of large quanti-
ties of this immature enzyme is apparently facilitated by the absence of the mannose 6-phosphate receptor ” . The 5774 cells retained 50% of the precursor TGase after 9 h in culture, and also exhibited the mature form of the acid TGase intracellularly. The ability to retain and process lysosomal enzymes may have been due to the presence of the mannose 6-phosphate receptorCD. Although this second receptor has not been identified in the 5774 cell line, it is present in P388 Dl macrophages, another murine tumor cell line which is mannose 6-phosphate receptor ‘t-deficient [lo]. The distinction between precursor and mature forms was facilitated by the use of an ethanolic tri[ l4 Cjoleoylglycerol emulsion substrate presentation in the enzyme assays. The precursor (neutral TGase) had a pH optimum of 6.4, whereas the mature enzyme had a pH optimum of 5.5. We were unable to detect the activity of the mature form (lysosomal acid TGase) in the conditioned medium of either 5774 or rabbit alveolar macrophages. If the neutral TGase were a second lysosomal enzyme rather than a precursor to the lysosomal acid TGase, we would expect both of the enzymes to be found in the media. In addition, while the homogenates of rabbit alveolar macrophages and human skin fibroblasts contain both the neutral and acid forms of TGase, the neutral form was secreted predominantly in response to NH,Cl. Since NH,Cl interferes with the transport of newly synthesized lysosomal enzymes to the endosomes, where they are proteolytically converted to the mature forms [35], the enzyme secreted under these conditions is primarily the precursor form. These data, combined with data concerning the uptake and conversion of the neutral TGase to the acid TGase in I-cell fibroblasts support the conclusion that the neutral TGase is the lysosomal acid TGase precursor. While there is no evidence that the neutral TGase functions in vivo, we cannot preclude the possibility that it may function intracellularly, and/or extracellularly in an appropriately neutral environment. For example, it has been shown that macrophage endosomes contain proteinases which degrade endocytosed protein ligands [36]. A second possibility is that the neutral TGase plays an extracellular role at the site of inflammation, since macrophages do secret significant amounts of
lysosomal enzymes in response to inflammatory [32] and phagocytosable stimuli [33]. Secretion of the neutral TGase was increased similarly in thioglycollate-elicited mouse peritoneal macrophages in the present study. Acknowledgements
The authors thank Elizabeth Miller, Scott Herdman and Patricia McLoughhn for their technical assistance. This work was supported by National Institutes of Health Research Grant HL34724 and Training Grant HL-07276 awarded by the National Heart, Lung and Blood Institute. References 1 Khoo, J.C., Vance, J.E., Mahoney, E.M., Jensen, D., Wancewicz, E. and Steinberg, D. (1984) Arteriosclerosis 4, 34-40. 2 Msrland, B. and Kaplan, G. (1978) Exp. Cell Res. 115, 63-72. 3 Gabel, C.A., Goldberg, D.E. and Komfeld, S. (1983) Proc. Natl. Acad. Sci. USA 80, 775-779. 4 Shepherd, V.L., Freeze, H.H., Miller, A.L. and Stahl, P.D. (1984) J. Biol. Chem. 259, 2257-2261. 5 Hickman, S. and Neufeld, E.F. (1972) B&hem. Biophys. Res. Commun. 49, 992-999. 6 Sly, W.S. and Stahl, P. (1978) in Transport of Macromolecules in Cellular Systems (Silverstein, S.C., ed.), pp. 229-244, DahIen Konferenzen, Berlin. 7 Von Figura, K. and Weber, E. (1978) Biochem. J. 176, 943-950. 8 Jessup, W. and Dean, R.T. (1982) B&hem. Biophys. Res. Commun. 105, 922-927. 9 Jessup, W. and Dean, R.T. (1980) Biochem. J. 190,847-850. 10 Hoflack, B. and Komfeld, S. (1985) Proc. Natl. Acad. Sci. USA 82,4428-4432. 11 Hoflack, B. and Komfeld, S. (1985) J. Biol. Chem. 260, 12008-12014. 12 Hasihk, A. and Von Figura, K. (1984) in Lysosomes (DingIe, J.T., Dean, R.T. and Sly, W., eds.), Vol. 7, pp. 3-16, Elsevier, Amsterdam. 13 Mahoney, E.M., HamiU, A.L., Scott, W.A. and Cohn, Z.A. (1977) Proc. Natl. Acad. Sci. USA 74, 4895-4899.
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