* Department of Hematology, Lund University, Sweden; and
The Netherlands Cancer Institute, Amsterdam
Correspondence: Elinor Bülow, Dept. of Hematology, BMC, C14, S-221 84 Lund, Sweden. E-mail: elinor.bulow{at}hematologi.lu.se
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Key Words: secretory pathway • storage • LL-37 • lysosome-related organelle • azurophil granule
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Azurophil granules are considered lysosome-like [14 ]. Although poorly understood, their biogenesis may, however, differ from that of the typical lysosome-related organelles of many other hematopoietic cells that involve transfer of vesicles from the trans Golgi network (TGN) to late endosomes, often with the appearance of multivesicular bodies, followed by mature granule formation [15 ]. In contrast, biogenesis of azurophil granules may have certain features in common with that of granules for regulated secretion in endocrine, neuroendocrine, and exocrine cells. During maturation of regulated secretory granules of the latter cells, the composition of granules is refined by removal of mistakenly sorted, soluble and membrane proteins before maturation to dense-core granules [16 ]. The production of specific granules of neutrophils has not yet been thoroughly investigated, but acidification mechanisms are important in transfer of lactoferrin for normal storage in specific granules [17 ].
The aim of this work is to investigate whether hCAP-18, a normal constituent of specific granules, can also be sorted for storage in lysosome-related organelles of myeloid cells and remain stable. Therefore, we characterize sorting and processing of hCAP-18 after expression in the rat basophilic leukemia (RBL) and the mouse promyelocytic (MPRO) cell lines. Thus, our experiments represent an attempt of mistargeting hCAP-18 for storage in lysosome-related organelles where it does not normally belong. The rationale for doing this is that many azurophil granule proteins have been expressed in RBL cells, and in other myeloid cell lines before, and were shown to be processed correctly under the same conditions used here for the hCAP-18 experiments. For instance, hematopoietic serine proteases, MPO, and BPI have been investigated extensively and have demonstrated as being targeted, processed, and stored in lysosome-related organelles of RBL cells [2 ]. The latter organelles are regarded as equivalent to azurophil granules of neutrophils, although the latter, in contrast to the RBL organelles, lack some lysosome-associated membrane proteins [18 , 19 ]. In any case, lysosome-related organelles of RBL cells should represent a rather natural environment for storage of azurophil granule proteins but not for hCAP-18. The present results may therefore shed light on mechanisms of retrieval for storage in myeloid granules, the stability of proteins in this environment, and the consequences of timing of expression of genes for granule proteins. Widening the window for biogenesis through constitutive expression in HL-60 cells of neutrophil gelatinase-associated lipocalin (NGAL), like hCAP-18, a normal constituent of specific granules, resulted in targeting to azurophil granules, followed, however, by slow degradation in this proteolytic environment [20 ]. We demonstrate that hCAP-18 can be mistargeted for storage in lysosome-related granules of RBL and MPRO cells. The antibiotic C-terminal peptide of hCAP-18 was, however, released prematurely, more strongly in RBL granules than in MPRO granules. In conclusion, our results illustrate that mistargeting specific granule proteins to lysosome-related organelles may furnish premature processing to mature forms and potential instability depending on protein conformation.
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Construction of expression vectors
A cDNA for hCAP-18 was generously provided by Dr. Hans G. Boman (Stockholm University, Sweden) [9
]. The pBluescript KS vector was cleaved with Pvu1 and was used as a template in a 20-cycle polymerase chain reaction to create the insert hCAP-18 containing the Kozak consensus leader sequence for maximal translational efficiency [21
] and the flanking restriction enzyme sites for HindIII and Not1, added by the primers. The upstream primer 5'-GACTTCAGAAGCTTCCGCCACCATGAAGACCCAAAGGAATGGCCAC(primer no. 1) and downstream primer 5'-GACTTCAGGCGGCCGCCTAGGACTCTGTCCTGGGTACAAGA (primer no. 2; start and stop codons in boldface and restriction enzyme sites underlined) were used. The resulting product was cloned into the plasmid pcDNA3 to create the expression vector pcDNA3/hCAP-18.
cDNA constructs were investigated by in vitro translation, and the expected size was verified in each case.
Cell culture
The rat basophilic/mast cell line RBL-1 [22
] was cultured as described previously [23
]. The mouse promyelocyte cell line MPRO (MPRO.C1, monoclonal subclone) [24
] was grown in DMEM supplemented with 7% FCS, 20% HM-5 conditioned medium as a source of granulocyte macrophage-colony stimulating factor, and 1% L-glutamine [24
, 25
]. The HM-5 cells, kindly provided by Dr. Jack Cowland (Copenhagen, Denmark), were maintained in RPMI 1640 supplemented with 10% FCS. The conditioned medium was obtained by growing the cells to 80% confluence, changing to fresh medium, and harvesting conditioned medium after 48 h [25
].
Transfection procedures
RBL and MPRO cells were transfected with pcDNA3 constructs using the Bio-Rad Gene PulserTM (Bio-Rad, Hercules, CA), with electrical settings of 260 V and 960 µF as described previously [26
]. After electrophoresis, geneticin (2 and 1 mg/ml, respectively) was added to select for recombinant clones containing the geneticin resistance of pcDNA3. Individual antibiotic-resistant RBL cell clones (monoclonal) [23
] or antibiotic-resistant MPRO cell pools (polyclonal) were selected and expanded in suspension cultures and were screened for expression of the transfected protein.
Biosynthetic radiolabeling
Biosynthetic radiolabeling of the newly synthesized proteins by stably transfected cell lines was carried out as described previously [26
]. Cells were starved for 30 min followed by incubation with [35S]methionine/[35S]cysteine or [14C]leucine (pulse-labeling). For chase of the radiolabeled proteins, the cells were resuspended in complete nonradioactive medium after radiolabeling. At various time intervals, cells were withdrawn and subjected to analysis.
Subcellular fractionation
Subcellular fractionation was performed as described [26
]. Thus, the postnuclear cell homogenate was centrifuged in a Percoll density gradient followed by collection of nine fractions, fraction 9 containing the cytosol. The distribution of lysosome-related organelles and Golgi elements in the gradient was determined by assaying ß-hexosaminidase and galactosyl transferase as described elsewhere [27
, 28
]. The peak activities of ß-hexosaminidase and galactosyl transferase in subcellular fractions from RBL cells were localized in fractions 1–2 (containing the dense cytoplasmic organelles referred to in this work) and 5–8, respectively [23
].
Immunoprecipitation
Immunoprecipitation was performed as described previously [29
].
Western blotting
The enhanced chemiluminescence Western blotting kit was used according to the manufacturer’s instructions. Briefly, 5 x 106 cells were washed once in phosphate-buffered saline and were then frozen at -80°C for at least 20 min. The cell pellet was diluted in 100 µl lysis buffer [86 mM Tris (pH 6.8), 11% (v/v) glycerol, 2.3% (w/v) sodium dodecyl sulfate (SDS), 1.2% (v/v) ß-mercaptoethanol, 0.005% (v/v) bromphenol blue] containing protease inhibitors (CompleteTM), after which the cells were lysed by sonication for 
10 s with a Dr. Hielsher sonicator (B. Braun Biotech International, Melsungen, Germany). Samples were boiled for 5 min and collected by centrifugation at 14,000 g at 4°C for 5 min. A lysate from 0.5 x 106 cells was loaded in each lane of a precast 10–20% Tris-glycine gel. Proteins were transferred electrophoretically to Hybond-P nitrocellulose membrane in blotting buffer. Detection was performed according to the manufacturer’s instructions, and the membranes were exposed to HyperfilmTM ECLTM for 60 s.
Immunoelectron microscopy
RBL cells stably transfected with hCAP-18 were fixed for 24 h in 4% paraformaldehyde in 0.1 M PHEM (PIPES-HEPES-EGTA-Mg2+) buffer (pH 6.9) and were then processed for ultrathin cryosectioning as described before [30
]. Cryosections (45 nm) were cut at -125°C using diamond knives (Drukker Cuijk, The Netherlands) in an ultracryomicrotome (Leica Aktiengesellschaft, Vienna, Austria) and were transferred with a mixture of sucrose and methyl cellulose onto formvar-coated copper grids [31
]. The grids were placed on 35-mm petri dishes containing 2% gelatin. Double-immunolabeling was performed using a previously described procedure [32
] with 10- and 15-nm protein-A-conjugated colloidal gold probes (EM Lab., Utrecht University, The Netherlands). After immunolabeling, the cryosections were embedded in a mixture of methylcellulose and uranyl acetate and examined with a Philips CM 10 electron microscope (Eindhoven, The Netherlands). For the controls, the primary antibody was replaced by a nonrelevant rabbit antiserum.
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Figure 1. Expression of hCAP-18 in RBL cells. Cells transfected with cDNA of hCAP-18 were incubated for 30 min with [35S]methionine/[35S]cysteine (A and D) or [14C]leucine (B), followed by chase of the label for indicated time periods. (D) As indicated, NH4Cl (10 mM) or chloroquine (1 µM) was present during starvation, labeling, and chase. [14C]Leucine was used to get radiolabeling of LL-37 that lacks methionine/cysteine residues. At depicted time points, cells were removed and after lysis, were subjected to immunoprecipitation with anti-hCAP-18 (A) or anti-LL-37 (B and D). In addition, hCAP-18 was precipitated from the incubation medium at each chase time point. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by fluorography. The fluorograms were exposed for 7 days (B) or 9 days (D). (C) Whole cell lysates of RBL/wild (wild) or RBL/hCAP-18 (hCAP-18) were subjected to Western blotting using anti-hCAP-18 or anti-LL-37 antibodies. For immunoprecipitation, the antibodies have the following specificity: Anti-LL-37 detects 17-kDa proform and mature 18-kDa hCAP-18. Anti-hCAP-18 detects the 17-kDa proform, mature 18-kDa hCAP-18, and the 15- and 16-kDa cathelin-processing forms. In addition, anti-LL-37 detects LL-37 by Western blotting but not when used in immunoprecipitation of hCAP-18-transfected cells.
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To determine if the processing took place in a lysosomal compartment, cells were incubated with NH4Cl or chloroquine, thus blocking lysosomal proteolysis by increasing the pH. Results from pulse-chase labeling experiments carried out with either agent and using anti-LL-37 for immunoprecipitation showed a slight protection against proteolysis of hCAP-18 (Fig. 1D) , indicating that degradation may take place in a lysosomal/granule compartment. The constitutive secretion was not affected by either agent.
The promyelocytic MPRO cell line was also stably transfected with hCAP-18 cDNA. MPRO can be induced to differentiate into neutrophils with synthesis of specific granule transcripts by supraphysiological concentrations of ATRA [24 , 25 ]. hCAP-18 and its 17-kDa proform were observed after biosynthetic radiolabeling (Fig. 2 , top panel). Only hCAP-18, which dominated during chase of the radiolabel, was secreted. In contrast to RBL cells, only small amounts of cathelin-like processing forms were observed in MPRO cells, indicating less premature processing in MPRO granules than in RBL granules (compare with Fig. 1A ). After induction of differentiation with ATRA toward neutrophils, MPRO cells showed a slightly different processing of hCAP-18 in this experiment detected with anti-LL-37, which also detects hCAP-18. The conversion of the 17-kDa proform into hCAP-18 was slower, and secretion of proform was higher, but generation of visible LL-37 could not be observed (Fig. 2 , lower panel); the anti-LL-37 antibody is not able to detect LL-37 by immunoprecipitation (see below).
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Figure 2. Expression of hCAP-18 in MPRO cells. Cells transfected with cDNA of hCAP-18 were incubated (top panel) or differentiated for 4 days with 10-5 M ATRA before the incubation (bottom panel) with [14C]leucine for 30 min, followed by chase of the label for indicated time periods. At depicted time points, 20 x 106 cells were removed and after lysis, were subjected to immunoprecipitation with anti-hCAP-18 or anti-LL-37. In addition, hCAP-18 was precipitated from the incubation medium at each chase time. The immunoprecipitates were analyzed as described in the legend to Figure 1
. The fluorograms were exposed for 29 and 36 days, respectively.
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Figure 3. Targeting hCAP-18 to dense cytoplasmic organelles in RBL cells. Cells (100x106) expressing hCAP-18 were subjected to homogenization, followed by subcellular fractionation of the postnuclear supernatant by centrifugation in Percoll with subsequent collection of nine subcellular fractions, with fraction no. 9 containing all cytosol. The fractions were lysed, subjected to immunoprecipitation with anti-hCAP-18, and analyzed by Western blot with anti-hCAP-18. Peak activities of ß-hexosaminidase, fractions 1–2, and galactosyl transferase, fractions 5–8, indicate the positions of lysosomes and Golgi elements, respectively.
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Figure 4. Targeting hCAP-18 to dense cytoplasmic organelles in MPRO cells. Cells expressing hCAP-18, undifferentiated (A) or differentiated for 3 days with 10-5 M ATRA (B), were incubated with [35S]methionine/[35S]cysteine for 30 min followed by chase of the label for 3 h. At depicted time periods, 100 x 106 cells were withdrawn and subjected to homogenization followed by subcellular fractionation, as described in the legend to Figure 2
, before the fractions were combined as indicated and immunoprecipitated with anti-hCAP-18. The immunoprecipitates were analyzed by SDS-PAGE followed by fluorography. The fluorograms were exposed for 8 weeks.
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Figure 5. Colocalization of hCAP-18, lgp 120, and RMCP. (A and B) Ultrathin cryosections from RBL cells expressing hCAP-18 were double-labeled with rabbit anti-lgp 120 followed by protein A gold (10 nm) and with rabbit anti-hCAP-18 followed by protein A gold (15 nm). (C) Cells were double labeled with mouse anti-RMCP followed by rabbit anti-mouse and protein A gold (10 nm) and with rabbit anti-hCAP-18 followed by protein A gold (15 nm). (D) Double labeling was performed with rabbit anti-lgp 120 followed by protein A gold (10 nm) and with mouse anti-RMCP followed by rabbit anti-mouse and protein A gold (15 nm). (A) An area of a cell is shown with granules highly labeled for hCAP-18; also the TGN is labeled. (B) A higher magnification of the marked granule in A (*), and labeling is seen for lgp 120 on the membrane and for hCAP-18 on the matrix. (C) A granule double labeled for hCAP-18 and RMCP on the matrix, and (D), a granule is shown double labeled for lgp 120 on the membrane and RMCP on the matrix. Original bar, 200 nm.
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The composition of neutrophil granules is governed by coordination of expression of the genes for the granule proteins, and specific sorting mechanisms for targeting to different granule subtypes have not yet been identified [3 ]. Conversely, retrieval from the secretory pathway is necessary to allow targeting for granule storage. hCAP-18 was retrieved for storage even when expressed outside its normal biosynthetic window, here accomplished by widening the window through constitutive gene expression. We demonstrated previously that exogenous nonmyeloid, normally secretory proteins can be retained and are acceptable for storage in lysosome-related organelles [29 ]. Consequently, retention for storage may be a myeloid-specific property, allowing proteins with a compatible conformation to be sorted. However, not all soluble proteins delivered to the Golgi are sorted for storage in myeloid cells [36 ], suggesting the existence of conformation-based quality control for sorting. In contrast to endocrine and exocrine cells, many myeloid cells use the endosomal pathway for granule formation, a pathway that was involved in targeting hCAP-18, as judged by the results from immunogold labeling in RBL cells. Thus, hCAP-18 colocalized with lysosomal membrane markers in multivesicular bodies and small electron-dense cytoplasmic granules. Another specific granule protein, NGAL, has been shown to be targeted to azurophil-like granules when constitutively expressed in leukemic HL-60 cells, again emphasizing targeting mechanisms that are not necessarily protein-specific [20 ]. Moreover, HL-60 cells induced to mature into neutrophils did not retain newly synthesized NGAL, but released it to the exterior through constitutive secretion coupled to an inability of maturing HL-60 cells to form specific granules [37 ]. Similarly, induction of differentiation of MPRO cells resulted in augmented secretion of hCAP-18 in transfected cells. Accordingly, even if these cells have been shown to up-regulate transcripts for specific granule proteins such as lactoferrin and gelatinase during differentiation [25 ], the retention of constitutively expressed hCAP-18 diminished during maturation. This may result from down-regulation of the sorting machinery with lack of specific granule formation, despite gene expression for secondary granule proteins.
Lysosome-like azurophil granules may be proteolytically active at an early stage of formation in neutrophil precursors, as judged by their ability to process proforms of granule proteins to mature forms [2 ]. This has also been demonstrated for lysosome-related organelles of RBL cells in which proteolytic processing into mature granule proteins was observed as a post-sorting event [2 ]. The fully processed azurophil granule proteins are relatively stable in this proteolytic environment. However, in contrast to lack of processing in specific granules (the normal destination for hCAP-18), targeting to lysosome-related organelles of the myeloid cell lines resulted in processing with premature generation of LL-37, judging by our results from immunoprecipitation using antibodies with varying specificity. On the basis of antibody specificity, it is also concluded that the approximately 15-kDa processing forms observed in granules represent cathelin-like propeptides. The normal biosynthetic window of hCAP-18 in neutrophil precursors prevents targeting to azurophil granules that are formed prior to specific granules. It may be necessary to separate hCAP-18 from serine proteases of azurophil granules to prevent premature proteolytic release of its antibacterial activity (LL-37) and degradation. hCAP-18 is susceptible to cleavage by elastase, cathepsin G, and proteinase 3, normally stored in azurophil granules of neutrophils. However, proteinase 3 was shown to be responsible for the cleavage of hCAP-18 after exocytosis, although all the proteases may be released during degranulation [12 ].
We conclude that a specific granule protein, hCAP-18, can be targeted as a holoprotein for storage in lysosome-related organelles when constitutively expressed in myeloid cell lines, indicating the existence of cell-specific retention mechanisms for intracellular storage. Moreover, the mistargeting to lysosome-related organelles can lead to premature release of the antibiotic peptide LL-37 in the proteolytic environment of these organelles.
Received November 21, 2001; revised January 8, 2002; accepted January 25, 2002.
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