INTRODUCTION

Neutrophils have been recognized as a key element in host defense against microbial infections (1, 2). One of the main functions of neutrophils is the ingestion and subsequent intracellular killing of microorganisms. Intracellular killing is mediated by oxidative and/or nonoxidative mechanisms (3, 4). The former depends on oxidants (H₂O₂, hypochlorite, chloramines, and hydroxyl radicals) whose production follows the activation and assembly of NADPH oxidase on the plasma membrane. The nonoxidative mechanism reflects the actions of potent antibacterial polypeptides residing within the cytoplasmic granules. A number of antibacterial polypeptides, such as defensin, azurocidin, bactericidal/permeability-increasing protein, lysozyme, lactoferrin, cationic antibacterial proteins of 18 kDa (CAP18)¹, indolicidin, bactenecins, and protegrins, have been isolated from neutrophils of human and animal species (4-10). In general, they are highly cationic and act by perturbing the membranes of target microorganisms.

Neutrophils contain three subsets of granules, primary (azurophil) granules, secondary (specific) granules, and tertiary (gelatinase) granules (11, 12). Among antibacterial polypeptides, defensin, bactericidal/permeability-increasing protein, and azurocidin are known to be present in azurophil granules, whereas lactoferrin and CAP18 are found in specific granules, and lysozyme is present in both azurophil and specific granules (11, 12). Using makers for azurophil granules (myeloperoxidase, neutrophil elastase, and defensin), specific granules (lactoferrin) and gelatinase granules (gelatinase), mRNA expression, and protein synthesis were examined during maturation of neutrophil precursors in the bone marrow. In this context, in situ hybridization studies revealed that mRNA transcripts for myeloperoxidase, neutrophil elastase, and defensin were expressed at the promyelocyte and myelocyte stages, whereas mRNA for lactoferrin was expressed at the myelocyte and metamyelocyte stages (13, 14). In addition, biosynthesis studies using bone marrow cells sorted according to their stages of maturity showed that myeloperoxidase was mainly synthesized in the fraction containing myeloblasts and promyelocytes; lactoferrin was primarily synthesized in the fraction containing myelocytes and metamyelocytes; and gelatinase was synthesized in the fraction containing myelocytes, metamyelocytes, and band cells (15). Thus, mRNA expression and synthesis of proteins, which are characteristic of the different granule subsets, are tightly controlled at different maturation stages of neutrophil precursors.

Recently, we have purified a cationic antibacterial polypeptide of 11 kDa (CAP11) from guinea pig neutrophil granules (16). CAP11 is a homodimer of G¹LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI⁴³ joined with a disulfide bond and possesses the antimicrobial activities against Gram-negative and positive bacteria. Amino acid sequence of CAP11 shows partial homology (19-30%) to the putative active peptides of rabbit and human CAP18 (17-19), suggesting CAP11 might be a homologue of CAP18, the members of cathelin-related antibacterial proteins (cathelicidins) (20). Cathelicidin is a novel protein family with a conserved pro-region and a variable C-terminal antibacterial domain, and the pro-sequence is highly identical to the sequence of cathelin (21), a protein identified in porcine neutrophils. However, the amino acid sequence of CAP11 precursor has not been identified, and it is not known whether CAP11 is a member of cathelicidin family. Furthermore, expression of CAP11 during neutrophil maturation has not been examined, and the localization of CAP11 among granule subsets is not clear.

In this study, therefore, to deduce the amino acid sequence of CAP11 precursor, we isolated cDNA encoding CAP11 from bone marrow cells using RACE (rapid amplification of cDNA ends) technique. In addition, we evaluated the expression of CAP11 mRNA during neutrophil maturation in the bone marrow by Northern blot and in situ hybridization. We also examined the localization of CAP11 among granule subsets by extracellular release assay and subcellular fractionation experiment using Percoll density gradient centrifugation.

EXPERIMENTAL PROCEDURES

Hartley male guinea pigs weighing approximately 350 g were used as the source of cells. Blood neutrophils and mononuclear cells were isolated from heparinized blood by Ficoll-Conray centrifugation (22). Macrophages were prepared from the peritoneal cavity 4 days after the intraperitoneal injection of glycogen (22). Eosinophils were isolated from eosinophil-rich peritoneal exudates by Ficoll-Conray centrifugation (22). In some experiments, alveolar macrophages (14), lymphocytes, and monocytes purified from blood mononuclear cells (22) were also used. Isolated cells were stained with May/Grünwald/Giemsa, and the purity was >95%. Bone marrow cells were obtained from femoral bones. Cells were suspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4).

Total cellular RNA was isolated from bone marrow cells by the acid guanidinium thiocyanate/phenol/chloroform extraction method (23). Poly(A)⁺ RNA was purified through oligo(dT)-cellulose chromatography (Collaborative Biomedical Products), and the first strand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) using oligo(dT)₁₈ (U. S. Biochemical Corp.) as a primer. To obtain the CAP11 cDNA fragment, 5- and 3-primers corresponding to Gly¹-Lys⁸, and Trp²⁸-Tyr³⁴ of the published amino acid sequence of CAP11 (16), respectively, were used (Fig. 1): 5-primer, 5-gcgaattc-GGCCTGCGGAAGAAGTTCCGGAA-3; 3-primer, 5-gcgaattcGTACTCCCGCCAGGCC-TTCCA-3, where the lowercase letters represent EcoRI recognition sequence included to facilitate the subcloning of PCR products. Amplification was performed with 50 pmol of each primer in a final reaction mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 3 mM MgCl₂, 200 µM dNTPs, and 2.5 units of Taq DNA polymerase (TaKaRa) using the following thermal cycling parameters: 94 °C for 2 min; then 30 cycles at 94 °C for 15 s; 55 °C for 15 s; 72 °C for 30 s; followed by 72 °C for 5 min. The amplified cDNA fragment (approximately 120 base pairs was digested with EcoRI and subcloned into pBluescript SK() (Stratagene). DNA sequencing (see below) revealed that the cDNA fragment encoded the Gly¹-Tyr³⁴ sequence of CAP11. Based on the nucleotide sequence, 5RT (reverse transcription)-primer, and 5- and 3-amplification (amp) primers were synthesized and used for the RACE protocol (Fig. 1A) (24).

To amplify the 3 end (RACE3), poly(A)⁺ RNA was reverse-transcribed with oligo(dT)₁₈-adapter primer containing SalI, BamHI, and HindIII recognition sites in the adapter sequence. After purification with a spin column (Quick Spin^TM Column G-50, Boehringer Mannheim), the first strand cDNA was amplified by PCR using the upstream 3-amplification primer 5-gcgaattcACAGGAAAAGGATCCAG-3 (corresponding to nt 441-458 shown in Fig. 1B with EcoRI recognition sequence at 5 end) and the downstream adapter primer. To amplify the 5 end (RACE5), poly(A)⁺ RNA was reverse-transcribed with 5RT-primer 5-AGACTTTCCGGCCAGTTTTC-3 (complementary to nt 479-498), and the single-stranded cDNA was tailed with terminal deoxynucleotidyltransferase (TOYOBO) using dATP. Then tailed cDNA was amplified with an upstream oligo(dT)₁₈-adapter primer and a downstream 5-amplification primer 5-gcgaattcCAGTTTTCCCGATTTTCCG-3 (complementary to nt 468-486 with EcoRI recognition sequence at 5 end) located 12 nucleotides upstream of RT-primer. To assemble the full-length cDNA, the first strand cDNA synthesized with oligo(dT)₁₈ primer was amplified using the 5 end primer 5-gcgaattcGAGTGAGGACCATGGGCA-3 (corresponding to nt 1-18 with EcoRI recognition sequence at 5 end) and the 3 end primer 5-gcgtcgacCAAGAACACACTAGGTAGTC-3 (corresponding to nt 651-670 with SalI recognition sequence at 5 end). The amplified cDNA fragments were digested with EcoRI and SalI and subcloned into pBluescript SK().

The sequences of cDNAs subcloned into pBluescript SK() were determined by the dideoxy chain termination method using the ABI PRISM^TM Dye Terminator Cycle Sequencing Core Kit (with AmpliTaq® DNA Polymerase, FS) and the model 373A DNA Sequencer (Applied Biosystems). Both strands were sequenced using T3, T7 primers (Promega), and synthetic oligonucleotide primers, which were deduced from the cDNA sequence. Analyses of nucleotide and amino acid sequences were performed with the GeneWorks and PC/GENE software (IntelliGenetics), and homology search was carried out on the SWISS-PROT data base.

Total RNA (5 µg) was separated by electrophoresis on 1% agarose/formaldehyde gel and transferred by capillary blotting onto nylon membranes (Biodyne® A Membrane; Pall) (23). RNA was cross-linked with Funa®-UV-Linker (Funakoshi Co. Ltd.), and the blots were hybridized with cDNA probes labeled with Multiprime DNA labeling systems (Amersham Corp.) using [³²P]dCTP (3000 Ci/mmol; ICN Biomedicals Inc.) (23). Probes used for Northern blot analysis were the 0.67-kb CAP11 cDNA (encompassing nt 1-670; Fig. 1B), the 2.3-kb -actin cDNA (pHFA-1; graciously provided by P. Gunning and L. Kedes, Stanford University) (25), the 0.76-kb myeloperoxidase cDNA (PstI-HincII fragment of cDNA clone pMPO62) (26), the 0.47-kb defensin cDNA isolated from guinea pig bone marrow cell cDNA library (27), the 0.59-kb lactoferrin cDNA (encompassing nt 472-1060 of lactoferrin cDNA) (28) obtained by the PCR amplification of HL60 cell cDNA, and the 0.46-kb gelatinase cDNA (encompassing nt 1567-2024 of gelatinase cDNA) (29) obtained by the PCR amplification of cDNA from phorbol myristate acetate-treated U937 cells.

The cells were treated with 5 mM diisopropyl fluorophosphate (DFP) and disrupted in ice by sonication (Tomy Ultrasonic Disruptor UD-201, Tominaga Works Ltd.). The sonicates were centrifuged at 200 × g for 10 min to sediment nuclei, and the supernatants (10⁶ cell equivalents) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 7.5-22% linear gradient of polyacrylamide (14). The resolved polypeptides were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell). The membrane was blocked in 3% normal goat serum/PBS and probed with affinity purified rabbit anti-CAP11 antibody (100 ng/ml; see below). The membrane was further probed with a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Organon Teknika Corp.), and CAP11 was finally detected with ECL^TM Western blotting detection reagents (Amersham Corp.).

Anti-CAP11 serum was raised in rabbits using the synthetic peptide (corresponding to L²RKKFRKTRKR¹² of CAP11) (16) covalently coupled to keyhole limpet hemocyanin, and anti-CAP11 antibody was purified by affinity chromatography using the synthetic peptide-conjugated epoxy-activated Sepharose 6B (Pharmacia Biotech Inc.), according to the manufacturer's instruction.

Bone marrow cells were mixed with 1% dextran T-500 (Pharmacia), and erythrocytes were sedimented. The resulting supernatant was centrifuged at 200 × g for 10 min, and the pelleted cells were suspended in PBS at 10⁷ cells/ml. The cells (15 ml) were applied on top of a 50-ml centrifuge tube containing 9 ml of Percoll (Pharmacia), density 1.080 g/ml, layered below 9 ml of Percoll, density 1.065 g/ml (15). The desired densities of Percoll were obtained by mixing 10 × PBS with precalculated amounts of Percoll and distilled H₂O, as described by the manufacturer. The gradient was centrifuged at 1000 × g for 20 min at 4 °C. This resulted in the fractionation of bone marrow cells into three groups: band 1, a well defined band on top of the gradient; band 2, all the cells between band 1 and a cell pellet at the bottom, band 3. Band 3 was contaminated with erythrocytes and therefore subjected to a hypotonic lysis. Cytocentrifuge preparations were made using Cytospin 2 (10⁵ cells/slide, 340 rpm, 10 min; Shandon Instruments) and stained with May/Grünwald/ Giemsa. Differential counting of stained myeloid cell preparations revealed that band 1 contained 18 ± 6% myeloblasts, 56 ± 4% promyelocytes, 16 ± 4% myelocytes, and 10 ± 4% metamyelocytes; band 2 contained 5 ± 3% promyelocytes, 36 ± 7% myelocytes, 44 ± 8% metamyelocytes, 10 ± 5% band cells, and 5 ± 3% segmented cells; band 3 contained 4 ± 3% myelocytes, 12 ± 4% metamyelocytes, 49 ± 5% band cells, and 35 ± 7% segmented cells (mean ± S.D., n = 3), as previously reported (15). Total cellular RNA was isolated from the three groups of cells and subjected to Northern blot analysis (described above). To measure the relative amounts of mRNA, the detected bands were quantified using MasterScan^TM System (Scanalytics).

In situ hybridization was performed essentially based on the methods previously described (14, 30). Bone marrow cells were cytocentrifuged (see above) onto siliconized RNase-free glass slides, fixed in 4% paraformaldehyde/PBS, and treated with 0.25% acetic anhydride, 0.1 M triethanolamine buffer, pH 8.0. The cytocentrifuge preparations were hybridized at 45 °C for 14-16 h with ³⁵S-labeled antisense or sense cRNA probe (10⁷ cpm/ml) and then treated with RNase. For autoradiography, the slides were coated with Konica NR-M2 emulsion (Konica Corp.) diluted 1:1 with 0.3 M ammonium acetate at 42 °C, air-dried, and stored in the dark at 4 °C. After 3-10 days of exposure, the slides were developed in Konicador Super developer, fixed in Fujifix (Fuji Photo Film Co., Ltd.), and stained with May/Grünwald/Giemsa. Silver grain counts above individual cells or in their close vicinity were microscopically evaluated, and bone marrow cells hybridized with antisense cRNA probes were considered as positive if they contained more than four grains per cell; this was based on the results that the cells hybridized with all five sense probes (see below) contained less than four grains per cell. A minimum of 500 cells was evaluated in each preparation using the morphologic criteria described before (13, 14, 31), and the data were expressed as the percentage of positive cells within each maturation stage. To generate ³⁵S-labeled cRNA probes, the CAP11, myeloperoxidase, defensin, lactoferrin, and gelatinase cDNAs (described above) were subcloned into the transcription vector pBluescript SK (), and after appropriate linearization, labeled antisense and sense cRNA probes were synthesized by transcription of cDNAs with T3 (Stratagene) or T7 (TOYOBO) RNA polymerase using ³⁵S-UTP (1000 Ci/mmol; ICN Biomedicals Inc.).

Cytocentrifuge preparations (see above) were immunostained as described previously (14). To inactivate the endogenous peroxidase activity, the slides were treated with 0.1 M periodic acid and 0.02% sodium borohydride. The slides were incubated with 100 ng/ml rabbit anti-CAP11 antibody or rabbit control IgG (Sigma) in Tris-buffered saline (20 mM Tris-HCl, pH 7.5, 500 mM NaCl) containing 1% gelatin and 0.05% Tween 20, and after washing, further incubated with a 1:2000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG. The slides were finally developed in 0.05% 3,3-diaminobenzidine tetrahydrochloride, 0.01% H₂0₂ and counterstained with May/Grünwald/Giemsa.

Neutrophils (5 × 10⁷ cells/ml) were incubated at 37 °C for 15 min with or without various concentrations of N-formyl-Met-Leu-Phe (fMLP; Sigma) in a total volume of 1 ml of PBS containing 1 mM CaCl₂ and 1 mM MgCl₂. After incubation, each tube was placed in ice and centrifuged at 1850 × g for 5 min. The supernatants were mixed with 5 mM DFP and used for the assay of CAP11, lactoferrin, gelatinase, -glucuronidase, and lactate dehydrogenase. To measure the total contents, neutrophils sonicates containing 5 mM DFP were used. Extracellular release was calculated as the content in the supernatant in terms of percentage of the total content. CAP11 and lactoferrin were detected by Western blot analysis using rabbit anti-CAP11 antibody and rabbit anti-lactoferrin serum (1:1000 dilution; Organon Teknika), respectively, as primary antibodies, as described above. Gelatinase was analyzed by zymography with SDS-PAGE on 10% polyacrylamide containing 1 mg/ml gelatin under nonreducing conditions (32). To measure the relative amounts, CAP11 (11 kDa) and lactoferrin (80 kDa) bands detected by Western blotting and gelatinase bands (92, 135, and 220 kDa) (33, 34) detected by zymography as clear zones of gelatin lysis were scanned with MasterScan^TM System. The densities of CAP11, lactoferrin, and defensin (see below) bands and the areas of clear zones in gelatin zymograms were quantitated in the ranges where they were proportional to the amounts of samples used as follows: CAP11, 10⁵ to 10⁶ cell equivalents; lactoferrin and defensin, 10⁴ to 10⁵ cell equivalents; gelatinase, 10³ to 10⁴ cell equivalents. -Glucuronidase and lactate dehydrogenase activities were measured as described previously (35).

Subcellular fractionation of neutrophils was performed by density centrifugation on Percoll gradients (36). After treatment with 5 mM DFP, neutrophils were suspended in disruption buffer (100 mM KCl, 3 mM NaCl, 1 mM ATP, 3.5 mM MgCl₂, 10 mM PIPES, pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluoride at 5-8 × 10⁷ cells/ml. Cells were disrupted by nitrogen cavitation (350 p.s.i., 20 min; Parr Instrument Co.), and the cavitate was collected dropwise into EGTA, pH 7.4, sufficient for a final concentration of 1.25 mM. Nuclei and unbroken cells were sedimented by centrifugation at 400 × g for 15 min. The postnuclear supernatant (S₁; 10 ml) was applied on top of a 3 × 10-ml three-layer Percoll gradient (1.05/1.09/1.12 g/ml) and centrifuged at 37,000 × g for 30 min. This resulted in a gradient with four visible bands, from the bottom designated the -band (containing azurophil granules), the ₁-band (containing specific granules), the ₂-band (containing gelatinase granules), and the -band (containing plasma membrane and secretory vesicles). The four bands were recovered and used for marker assays after Percoll was removed by spinning at 180,000 × g for 2 h. CAP11, lactoferrin, and gelatinase were assayed as described above. Defensin (3.8 kDa) was analyzed by Western blotting using rabbit anti-defensin serum (1:1,000 dilution) as a primary antibody (14). Myeloperoxidase and leucine aminopeptidase activities were spectrophotometrically measured (35). The densities of Percoll were adjusted by mixing 10 × disruption buffer with precalculated amounts of Percoll and distilled H₂O, and 1 mM ATP, 0.5 mM phenylmethylsulfonyl fluoride, and 1.25 mM EGTA were added before use.

RESULTS

Bone marrow cell cDNA was PCR-amplified using 5-primer and 3-primer corresponding to Gly¹-Lys⁸ and Trp²⁸-Tyr³⁴ of mature CAP11 sequence, respectively (Fig. 1A). DNA sequencing revealed that the amplified cDNA fragment encoded Gly¹-Tyr³⁴ sequence of CAP11. Based on the nucleotide sequence, 5RT-primer, and 5- and 3-amplification primers were synthesized and used for the RACE protocol. cDNA fragment corresponding to nt 1-486 was obtained by RACE 5, whereas cDNA fragment corresponding to nt 441-729 with poly(A) tail at 3 end was obtained by RACE 3. Finally, sequences of these cDNA fragments were confirmed by isolation and sequencing of the full-length cDNA (corresponding to nt 1-670) amplified with 5 and 3 end primers. The nucleotide sequence (nt 1-729) was deposited with the accession number D87405. Northern blot analysis using ³²P-labeled CAP11 cDNA probe demonstrated a 0.9-kb mRNA in bone marrow cells (Fig. 3A). Assuming a poly(A) tail of 200-300 nt (37), CAP11 mRNA is predicted to encode 600-700 nt. Thus, CAP11 cDNA (nt 1-729) likely represents a nearly complete transcript of CAP11 mRNA.

The complete cDNA sequence of 729 nt and its deduced amino acid sequence are shown in Fig. 1B. An open reading frame of 534 nt was preceded by a short 5-untranslated region of 11 nt. The sequence flanking the putative translation start site at position 12 showed homology to a Kosak consensus sequence (38). A stop codon was found at position 546, followed by a 3-untranslated region of 181 nt. A typical polyadenylation signal was found at position 707, 23 nt upstream of the poly(A) tail.

The cDNA encoded a polypeptide of 178 amino acid residues (prepro-CAP11) with a calculated mass of 20,132 and an overall calculated pI of 11.02. The start methionine was followed by a 29-amino acid hydrophobic region (Met¹³⁵-Ala¹⁰⁷) corresponding to a putative signal peptide, as expected for a protein stored in cytoplasmic granules. The signal peptide was followed by a portion (Trp¹⁰⁶-Val¹) including 13 positively and 11 negatively charged residues distributed over 106 residues, with a calculated pI of 8.30. The cDNA sequence corresponding to mature CAP11 sequence (Gly¹-Ile⁴³ with a calculated pI of 12.32) was present at the 3 end of the open reading frame, and its deduced sequence was identical with the amino acid sequence previously determined (16). No putative N-glycosylation site was found in the prepro-CAP11 sequence.

Homology search through the SWISS-PROT data base revealed that the deduced sequence of prepro-CAP11 was homologous to the known members of the cathelicidin family of antibacterial polypeptides (Fig. 2) (20). Alignment of these polypeptides showed that their structural similarities were confined to the N-terminal regions, whereas the C-terminal regions were very different, with the exception of short regions of high similarities among CAP11 and CAP18. The pro-region of CAP11 precursor shared 31-52% identities with the members of cathelicidins. As observed for cathelicidins, four conserved cysteine residues that could form two intramolecular disulfide bridges (20) were clustered in the C-terminal moiety of the pro-sequence of CAP11 precursor.

To evaluate the regulation of CAP11 expression, we examined mRNA expression for CAP11 in various types of leukocytes. Northern blot analysis revealed that bone marrow cells and neutrophils expressed the 0.9-kb CAP11 mRNA, but the expression was more abundant in bone marrow cells than in mature neutrophils (Fig. 3A). No CAP11 mRNA transcripts were observed in macrophages, eosinophils, and mononuclear cells. However, when the same RNA samples were analyzed with -actin cDNA probe, the 2.2-kb -actin mRNA transcripts were observed in these cells. Next, CAP11-containing cells were investigated by Western blot analysis. The 11-kDa bands corresponding to CAP11 were detected in bone marrow cells and mature neutrophils but not in other types of leukocytes (Fig. 3B). Expression of CAP11 was also evaluated using lymphocytes, monocytes, and alveolar macrophages by Northern blot and Western blot analyses. However, these cells neither expressed CAP11 mRNA nor contained CAP11 (data not shown). Thus, among cells examined, mature neutrophils and bone marrow cells expressed CAP11 mRNA and contained CAP11, suggesting that the expression of CAP11 is likely neutrophil lineage-specific.

In addition, expression of myeloperoxidase, defensin, lactoferrin, and gelatinase mRNA was examined using bone marrow cells and mature neutrophils and compared with that of CAP11 mRNA. Myeloperoxidase and defensin mRNA transcripts were detected in bone marrow cells but not in mature neutrophils (Fig. 3C). In contrast, lactoferrin and gelatinase mRNA transcripts were detected in both bone marrow cells and mature neutrophils, although the mRNA levels were much lower in mature neutrophils than in bone marrow cells, as with CAP11 mRNA.

The expression of CAP11 gene is assumed to be modulated during neutrophil maturation in the bone marrow, since the levels of CAP11 mRNA transcripts are different between mature neutrophils and bone marrow cells that consist of neutrophil precursor cells at various maturation stages. Then, CAP11 mRNA expression was evaluated using bone marrow cells separated according to their maturation stages (15). Based on the association between cell maturation and specific cell density, bone marrow cells were sorted into three groups by density gradient centrifugation as follows: band 1, containing immature cells (myeloblasts and promyelocytes); band 2, containing cells of intermediate maturity (myelocytes and metamyelocytes); band 3, containing the most mature cells (band cells and segmented neutrophils). Expression of myeloperoxidase and defensin mRNA transcripts was most pronounced in the cells from band 1 but was remarkably reduced in more mature cells, especially cells from band 3 (Fig. 4, A and B). In contrast, expression of lactoferrin mRNA was greatly increased in the cells from band 2 in comparison with cells from band 1. However, the mRNA level was strikingly decreased in the cells from band 3. Interestingly, gelatinase and CAP11 mRNA transcripts were markedly increased in the cells from band 2 compared with cells from band 1, and the mRNA levels were considerably maintained in the cells from band 3. Expression of -actin mRNA was almost the same among cells from three bands. These observations indicate that CAP11 and gelatinase mRNA transcripts are predominantly expressed at the later stages of neutrophil maturation in the bone marrow and more abundantly expressed in mature cells than lactoferrin mRNA.

To further identify mRNA-expressing cells, we analyzed bone marrow cells with in situ hybridization using ³⁵S-labeled cRNA probes (Figs. 5 and 6). Positive cells for myeloperoxidase and defensin mRNA transcripts were predominantly present in the promyelocyte (89-99%) and myelocyte (57-93%) stages with much smaller percentages of positive metamyelocytes. In contrast, lactoferrin mRNA-positive cells were mostly found at the myelocyte stage (90%), and the percentages of positive cells decreased after the metamyelocyte stage. In addition, gelatinase mRNA-positive cells were mainly observed in the myelocyte (71%) and metamyelocyte (90%) stages, and the proportions of positive cells decreased in band cells and segmented neutrophils. Interestingly, CAP11 mRNA-positive cells were primarily found in the metamyelocyte stage (96%) with much smaller percentages of positive myelocytes, band cells, and segmented neutrophils. No cells of eosinophil, erythroid, or mononuclear cell lineage showed hybridization with CAP11 cRNA antisense probe.

In addition, bone marrow cells containing CAP11 were immunohistochemically evaluated using anti-CAP11 antibody. CAP11 was detected predominantly in the cells of neutrophil lineage such as metamyelocyte, band cells, and segmented neutrophils (Fig. 6) but not detected in the cells of other cell lineage. Quantification of CAP11-positive cells at different maturation stages revealed that positive cells were less than 30% at the myelocyte stage. Interestingly, coincident with the increase of CAP11 mRNA levels, the percentage of CAP11-positive cells markedly increased at the metamyelocyte stage (>95%), and the increased levels remained almost constant (>99%) in band cells and segmented neutrophils, although the expression of CAP11 mRNA was reduced in these cells.

Together these observations indicate that CAP11 gene transcription likely occurs during a very limited stage of neutrophil maturation, mostly at the metamyelocyte stage, and results in the synthesis and cytoplasmic accumulation of CAP11, which is present in the subsequent stages of neutrophil maturation.

Gelatinase is synthesized and stored in the granules during later stages of neutrophil maturation (11, 12, 15). Thus, concomitant expression of CAP11 and gelatinase mRNA transcripts in metamyelocytes suggests that CAP11 and gelatinase might be localized in the same subset of granules. Gelatinase is localized in both specific and gelatinase granules, and a low dose of fMLP (10 nM) is reported to induce the selective release of gelatinase from gelatinase granules without release of specific and azurophil granule components (36). Then, the localization of CAP11 among neutrophil granule subsets was examined by comparing the extracellular release of granular components. At low concentrations of fMLP (0.5-10 nM), gelatinase was readily released from neutrophils without release of lactoferrin (a specific granule marker) above resting level, and the release further increased at a higher concentration of fMLP (100 nM), accompanied with the release of lactoferrin (Fig. 7). In contrast, extracellular release of -glucuronidase (an azurophil marker) and lactate dehydrogenase (a cytosol marker) was less than 10% at any concentration of fMLP. Interestingly, extracellular release of CAP11 apparently occurred at low concentrations of fMLP (0.5-10 nM) but was not augmented by 100 nM fMLP. Furthermore, localization of CAP11 was assessed using granule subset fractions prepared by Percoll density gradient centrifugation (Fig. 8) (36). The -band contained the majority of azurophil granule markers myeloperoxidase and defensin, whereas the -band contained plasma membrane identified by leucine aminopeptidase (22). The ₁-band contained the majority of specific granule marker lactoferrin, whereas almost equal amounts of gelatinase (approximately 45% of total cell content) were located in both the ₁-band (containing specific granules) and ₂-band (containing gelatinase granules), as previously observed (36). In addition, albumin (a matrix marker for secretory vesicles) (11, 12) was mostly detected in the -band by SDS-PAGE analysis (data not shown), indicating that the -band contains secretory vesicles as well as plasma membrane (36). Interestingly, CAP11 was found to be exclusively localized in the gelatinase granule-containing ₂-band. Together these observations suggest that CAP11 may be stored in the granule subset, possibly the gelatinase granule.

DISCUSSION

To understand the regulation of CAP11 expression, we have isolated and analyzed cDNA encoding CAP11. Furthermore, we have investigated the expression of CAP11 mRNA during neutrophil maturation and localization of CAP11 among neutrophil granule subsets. Sequence analysis of CAP11 cDNA isolated from guinea pig bone marrow cells using RACE technique has revealed that CAP11 is likely synthesized as a precursor comprising 178 amino acid residues, which is composed of a signal peptide (N-terminal 29 residues), a propeptide (106 residues), and a C-terminal mature peptide (43 residues). Interestingly, the predicted CAP11 precursor is homologous with the members of cathelicidins, a novel family of antibacterial polypeptides, and displays the characteristic features of cathelicidins that consist of a highly conserved prepro-region of 128-143 residues including a putative 29-30-residue signal peptide and a cathelin-like propeptide of 99-114 residues containing four invariant cysteine residues and a variable C-terminal antimicrobial region ranging in length from 12 to 100 residues (20). Based on the structures, the C-terminal peptides of cathelicidins are classified into -helical peptides (CAP18, PMAP-36) (18, 19, 39, 40), proline- and arginine-rich peptides (C12, Bac7, Bac5, PR-39) (41-44), tryptophan-rich peptides (indolicidin, PMAP-23) (45, 46), and loop-forming peptides with one or two disulfide bonds (cyclic dodecapeptide, protegrins) (47, 48). CAP11 possesses the amphipathic -helical structure in the N-terminal cationic region (Gly¹-Lys¹⁵), which is assumed to be implicated in the antibacterial activity (16). Thus, CAP11 could be regarded as a member of cathelicidins that contain an amphipathic -helical peptide at the C-terminal region. However, it is worth noting that CAP11 has a homodimeric structure joined with a disulfide bond (16), whereas other -helical peptides (CAP18, PMAP-36) contain no cysteine residue involved in the covalent dimerization. The predicted amino acid sequence of prepro-CAP11 suggests that the pro-CAP11 must be further cleaved to generate mature CAP11, following signal peptide removal. We have tried to detect the precursors of CAP11 by Western blot analysis using anti-CAP11 antibody. However, we have been unsuccessful in detecting the high molecular weight bands corresponding to CAP11 precursors in bone marrow cells and mature neutrophils (data not shown). This may be due to the transient nature of the precursor in myeloid cells and/or the fact that the antibody has been developed against the peptide of mature CAP11 and not the precursor sequence. The processing of CAP11 precursors could be elucidated in the future using antibody recognizing the propeptide sequence of CAP11 and cellular extracts prepared from metabolically labeled myeloid cells.

Neutrophils contain three subsets of granules, azurophil granules, specific granules, and gelatinase granules (11, 12). During differentiation and maturation of neutrophil precursors in the bone marrow, azurophil granules appear at the promyelocyte stage, whereas specific granules appear at the myelocyte stage, and gelatinase granules are formed later than specific granules (11, 12, 31). Consistent with this, mRNA for myeloperoxidase, neutrophil elastase, and defensin (azurophil granule constituents) is expressed at promyelocyte and myelocyte stages, whereas mRNA for lactoferrin (a specific granule constituent) is expressed at myelocyte and metamyelocyte stages (13, 14). Furthermore, biosynthesis experiments have indicated that myeloperoxidase is mainly synthesized in the bone marrow cell fraction containing myeloblasts and promyelocytes; lactoferrin is primarily synthesized in the fraction containing myelocytes and metamyelocytes; gelatinase (specific and gelatinase granule constituents) is synthesized in the fractions containing myelocytes, metamyelocytes, and band cells (15). In the present study, CAP11 has been shown to be specifically expressed in the neutrophil lineage. Furthermore, Northern blot analyses using bone marrow cells and mature neutrophils have revealed that mature neutrophils express CAP11, gelatinase, and lactoferrin mRNA but not myeloperoxidase and defensin mRNA, although bone marrow cells express all these mRNA transcripts, suggesting that CAP11 mRNA is expressed at later stages of neutrophil maturation. In fact, Northern blot analysis using bone marrow cells separated according to their maturation stages has shown that CAP11 mRNA as well as gelatinase mRNA is abundantly expressed in the cells at later stages of neutrophil maturation. Furthermore, in situ hybridization using specific antisense cDNA probes has demonstrated that CAP11 mRNA is primarily expressed in metamyelocytes and to a lesser extent in myelocytes and band cells, whereas lactoferrin mRNA is mainly detected in myelocytes with limited expression in metamyelocytes and band cells. Interestingly, gelatinase mRNA transcripts are mostly expressed in both myelocytes and metamyelocytes and to a lesser extent in band cells. Thus, CAP11 mRNA is simultaneously expressed with gelatinase mRNA at metamyelocyte and band cell stages, although substantial amounts of gelatinase mRNA are also expressed at myelocyte stage, as with lactoferrin mRNA. In addition, coincident with the expression of CAP11 mRNA, the number of CAP11-containing cells has markedly increased at metamyelocyte stage. Together these observations suggest that CAP11 is actively synthesized at the metamyelocyte stage, accompanied with its mRNA expression, and likely stored with gelatinase in the granule subset, possibly the gelatinase granule, which can be supported by the results of extracellular release assay and subcellular fractionation experiments. However, it is also possible that CAP11 and gelatinase are present in the different granule subpopulations within the ₂ fraction. This could be definitively resolved by double-labeling immunogold electron microscopy, which has been employed to distinguish gelatinase granules from specific granules in human neutrophils (11).

Several antibacterial polypeptides belonging to cathelicidin family have been purified from neutrophils (6-10, 20); however, their subcellular localization and gene expression during neutrophil maturation have not been examined in detail. Recently, CAP18, a member of cathelicidins, has been identified in the specific granules of human neutrophils (8). Colocalization of CAP18 and lactoferrin in the same granule subset (specific granules) apparently suggest that CAP18 gene transcription occurs primarily at the myelocyte stage of neutrophil maturation, like lactoferrin gene transcription. Importantly, CAP18 mRNA has been found to be predominantly expressed in myelocytes with limited mRNA expression in more immature cells (promyelocytes) and mature cells (metamyelocytes, band cells and segmented neutrophils) by in situ hybridization using CAP18-specific cRNA probe and human bone marrow cells.² Thus, expression of CAP18 and CAP11 genes seems to be differently regulated at the stages of neutrophil maturation in the bone marrow, although CAP18 and CAP11 are both the members of cathelicidin family.

For understanding the in vivo function of CAP11, it is noteworthy that CAP11 is readily released extracellularly from neutrophils accompanied with gelatinase. During stimulation of neutrophils, gelatinase granules are mobilized more extensively than specific and azurophil granules, and the preferential mobilization of gelatinase granules leads to the extracellular release of a collagenolytic enzyme, gelatinase, which may facilitate extravasation of neutrophils (11, 12). Interestingly, CAP11 has been shown to retain the antibacterial activities even in the presence of a physiological concentration of NaCl (0.15 M) (16), suggesting that CAP11 can exert the antibacterial activity in the extracellular fluid. Thus, CAP11 is assumed to play an important part in the killing of bacteria in the extracellular milieu during migration of neutrophils into the infected tissues.

In the present study, we have revealed that the genes for azurophil granule proteins (myeloperoxidase and defensin), specific granule proteins (lactoferrin and gelatinase), gelatinase granule protein (gelatinase), and CAP11 are stage-specifically expressed during differentiation and maturation of neutrophil precursors in the bone marrow. Consequently, identification of factors that control the transcription of genes coding for different granule proteins seems to be important for the understanding of mechanisms controlling differentiation and maturation of neutrophil lineage precursor cells. Based on this concept, the promoters for the myeloperoxidase, neutrophil elastase, and defensin genes have been investigated, and transcription factors such as PU.1 and CCAAT/enhancer-binding protein are assumed to be involved in the myeloid-specific expression of these genes (49-51). Isolation and characterization of the promoter for the CAP11 gene will help us to clarify the transcriptional control that is acting at the later stage of neutrophil maturation.