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Originally published online as doi:10.1189/jlb.0607437 on August 14, 2008

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(Journal of Leukocyte Biology. 2008;84:1279-1286.)
© 2008 by Society for Leukocyte Biology

Vitamin D3 induces pro-LL-37 expression in myeloid precursors from patients with severe congenital neutropenia

Jenny Karlsson*,1, Göran Carlsson{dagger}, Olivia Larne*, Mats Andersson* and Katrin Pütsep*

* Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden; and
{dagger} Childhood Cancer Research Unit, Department of Woman and Child Health, Karolinska University Hospital Solna, Stockholm, Sweden

1 Correspondence: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail: jenny.karlsson{at}ki.se

ABSTRACT

The innate immune system produces a number of effector molecules that are important for protection against bacterial infections. Neutrophils and antimicrobial peptides are major components of innate defense with the capacity of rapid bacterial killing. Patients with severe congenital neutropenia (SCN) experience recurrent and chronic infections despite recombinant G-CSF-mobilized neutrophils. We have shown previously that these neutrophils are deficient in that they lack the antimicrobial peptide LL-37. Here, we show that pro-LL-37 mRNA is not expressed in neutrophil precursors from patients with SCN, although the gene and promoter region for pro-LL-37, CAMP, does not display any mutations. The hormonal form of vitamin D3 [1,25(OH)2D3] induced the expression of pro-LL-37 in isolated neutrophil progenitors and in EBV-transformed B cells from patients with SCN, whereas all-trans retinoic acid only induced expression in transformed B cells. These results demonstrate that myeloid cells of patients with SCN can produce pro-LL-37, suggesting that other pathways are impaired.

Key Words: 1.25(OH)2D3 • Kostmann syndrome • hCAP18 • all-trans retinoic acid

INTRODUCTION

Patients with the disease severe congenital neutropenia (SCN or Kostmann syndrome) have a bone marrow neutrophil maturation failure that results in neutropenia and life-threatening infections [1 ]. Maintenance therapy using recombinant human (rh)G-CSF overcomes this maturation arrest and mobilizes the neutrophils into the circulation [2 ]. The mechanisms causing the pathology of SCN have not been resolved completely, but gene defects in neutrophil elastase 2 (ELA2) or HCLS1 associated protein X-1 (1) (HAX1) confer similar phenotypes [3 , 4 ]. Nonetheless, a number of patients exhibit neither mutation [5 ], underscoring the prevailing uncertainty concerning the underlying molecular mechanisms of this disease.

Neutrophils are pivotal in preventing bacterial infections, and most patients with SCN respond to G-CSF with restored blood neutrophil numbers [2 ]. Despite G-CSF treatment, severe periodontal disease and residual susceptibility to infection often persist [6 7 8 ], indicating that their neutrophils may be malfunctioning. We have demonstrated previously that the neutrophils from patients with SCN are deficient with respect to expression of the antimicrobial peptide LL-37 and its pro-form pro-LL-37 (human cathelicidin antimicrobial peptide 18, hCAP18 alternatively and cathelin-LL-37) [9 , 10 ]. Pro-LL-37 is normally localized to neutrophil secondary granules, and its processed form, LL-37, is known primarily for its importance as an innate defense molecule with broad and highly efficient antibiotic activity [11 12 13 14 ]. Additional reported activities of LL-37 include chemoattractant function [15 ] and inhibition of neutrophil apoptosis [16 ]. The deficiency of neutrophil pro-LL-37 is a common denominator for patients with SCN, irrespective of genetic mutation or inheritance, and affected individuals may be identified through significantly decreased pro-LL-37 plasma levels [5 , 9 ].

Neutrophil maturation from myelocytic progenitors occurs in the bone marrow and follows a strictly coordinated program in which the transcription factors C/EBP{alpha} and PU.1 are critical regulators of early differentiation [17 , 18 ]. Various neutrophil granule proteins are produced at different stages of maturation [19 ], and the lineage-specific transcription factor C/EBP{epsilon} is essential for synthesis of secondary granule proteins [20 ]. It was demonstrated recently that the hormonal form of vitamin D3, [1,25(OH)2D3], is a strong inducer of pro-LL-37 expression in monocytes/macrophages [21 , 22 ]. A major function of 1,25(OH)2D3 is to control calcium and phosphorus homeostasis and thereby skeletal development [23 , 24 ]. The observation that a number of patients with SCN present with osteoporosis/osteopenia [8 , 25 ] and that the gene for LL-37 has vitamin D-responsive elements in the promoter region [21 , 22 , 26 ] urged us to investigate whether 1,25(OH)2D3 may play a role in defective pro-LL-37 synthesis.

In this study, we report that the gene encoding pro-LL-37, including the three vitamin-responsive elements, is intact in patients with SCN. However, the pro-LL-37 transcript levels were low in patient neutrophil progenitors, whereas the major neutrophil differentiation transcription factors were at similar levels to that of healthy subjects. Interestingly, 25(OH)D3 and 1,25(OH)2D3 could induce pro-LL-37 expression in isolated neutrophil precursors from these patients in vitro, demonstrating that their capacity to produce pro-LL-37 is not hampered.

MATERIALS AND METHODS

Patients and control individuals
Patients with SCN of various inheritances were included (Table 1 ). Patient identity code numbers are consistent with those previously reported [5 , 10 ]. Bone marrow and blood from control subjects included two pediatric, post-acute lymphoblastic leukemia (ALL) patients with normal white blood cell counts and bone marrow appearance. Informed consent was obtained from patients, parents, and control individuals. The ethical committees of the Universities of Umeå, Göteborg, Swedish Uppsala and Karolinska Institutet (Stolkholm) approved this study.


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  		Table 1. Patients with SCN

Sequencing of the CAMP gene
DNA was isolated from the lymphocyte fraction of blood using the GeneElute mammalian genomic DNA miniprep kit (Sigma-Aldrich, St. Louis, MO, USA) in accordance with manufacturer’s recommendations. The CAMP gene (cathelicidin antimicrobial peptide, available from GenBank/European Molecular Biology Laboratory/DNA DataBank of Japan under Gene ID 820), and 800 bp upstream of the first exon, was sequenced. Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA), including 1x GC buffer, 0.2 mM each dNTP, and 0.5 µM each primer (5'-gaa aag atg ggg tca aga agc-3', 5'-ctg ggg gtt cca ctg aga atg-3'), was used to amplify the gene. The PCR cycle was as follows: 60 s of initial denaturation at 98°C, followed by 35 cycles of 64°C for 30 s, 72°C for 120 s, and 98°C for 15 s. The primers used for sequencing are available upon request.

Neutrophil precursor preparation
Bone marrow (0.5–2 ml) was aspirated with 0.9% sodium chloride with heparin as an anticoagulant, and RBC contamination was reduced through Dextran T500 sedimentation (Amersham Biosciences, Piscataway, NJ, USA). The upper, leukocyte-rich layer was used for enrichment of myeloid precursors by density centrifugation using a two-layer discontinuous Percoll (Amersham Biosciences) gradient as described previously [27 , 28 ]. The more mature cells (mainly myelocytes and metamyelocytes) floating above the pellet and the early progenitor cells (mainly myeloblasts and promyelocytes) present in the interface between the different Percoll layers were pooled, representing a neutrophil precursor cell fraction. The pellet, which mainly contains band cells and mature neutrophils, was not used in these experiments.

Isolation of neutrophil precursors by flow cytometry
The neutrophil precursor cells derived from 10 patients and two control individuals were resuspended in ice-cold buffer (PBS with 1% BSA and 0.1% sodium azide) and labeled with FITC-conjugated mouse anti-human CD13 and R-phycoerythrin-conjugated monoclonal mouse anti-human CD11b (Dako A/S, Glostrup, Denmark). Alternatively, dextran-sedimented bone marrow cells from two patients were labeled as above but without prior Percoll density centrifugation. Cells were gated for by granularity and size (P1) and subsequently sorted into CD11blow and CD11bhigh differentiation stage populations using a FACScan Vantage SE sorter (Becton Dickinson, Franklin Lakes, NJ, USA). The expression of CD11b and CD13 antigens varies with neutrophil precursor maturation [29 30 31 ] in the following order of increasing maturity: Promyelocytes are CD13highCD11blow, myelocytes are CD13lowCD11blow, metamyelocytes are CD13lowCD11bhigh, and band cells/segmented cells are CD13highCD11bhigh. No differences in transcript expression of selected targets could be noted between the two methods used for neutrophil progenitor enrichment (see Results).

RNA isolation, RT-PCR, and transcript analysis
Total RNA was prepared from 0.3–1 x 106 flow cytometer-sorted cell populations using the SV Total RNA isolation system (Promega, Madison, WI, USA) or alternatively, the RNeasy min kit (Qiagen, Hilden, Germany). The synthesis of first-strand cDNA was accomplished using the ImProm-IITM RT system (Promega), both steps in accordance with the manufacturers’ instructions.

Power SYBR Green PCR master mix and ABI 7900FAST machine (both from Applied Biosystems, Foster City, CA, USA) were used to determine pro-LL-37 (5'-aacctctaccgcctcctggacctg-3', 5'-caatcctctggtgactgctgtgtcg-3'), C/EBP{alpha} (5'-accttgtgccttggaaatgc-3', 5'-ggggtctgctgtagcctcg-3'), and GAPDH (5'-tgccatcactgccacccagaag-3', 5'-atgaccttgcccacagccttgg-3') transcript levels. The quantitative PCR (qPCR) cycle was as follows: 50°C for 2 min and 95°C for 10 min of initial denaturation, followed by 35 cycles of 95°C for 15 s and 60°C for 30 s. TaqMan fast universal PCR master mix was used to determine C/EBP{epsilon} (hs00357657m1), PU.1 (hs02786711m1), and GAPDH (Hs99999905m1) transcript levels (all from Applied Biosystems) using standard conditions from the manufacturer. Target gene mRNA expression levels were assessed with the {Delta} comparative threshold method for quantification and given as percent of GAPDH. Transcripts of human neutrophil peptide 1–3 (HNP1–3) were assessed using PCR Core System II (Promega) with sequence-specific primers as described in ref. [32 ] but with 35 cycles and double-time for annealing, extension, and denaturation. β-Actin was amplified as in ref. [33 ], except for running 31 cycles. The reaction products were analyzed using 1% agarose gel. Band intensity was determined with Image J for HNP1–3 and β-actin (NIH Image program, http://rsb.info.nih.gov/nih-image), given as percent of β-actin.

Stimulation of EBV-transformed B cells
EBV-transformed B lymphoblastoid cell lines from one patient with SCN as well as from one healthy sibling were a generous gift from Hans G. Boman and Ingemar Ernberg (Karolinska Institutet). Transformed cells were stimulated at a density of 0.8 x 109 cells/L in RPMI including 15 mM Hepes for 3–4 days at 37°C supplemented with 100 nM 1,25(OH)2D3 or different concentrations of all-trans retinoic acid (ATRA; both from Sigma-Aldrich). Cell culture medium and cells were harvested by centrifugation (200 g). The cell culture medium was concentrated using Sep-Pak light tC18 cartridges (Waters), according to the manufacturer’s recommendation with 0.1% trifluoroacetic acid (TFA) in water as conditioner and wash solvent. The cartridges were washed further with 0.1% TFA in 30% acetonitrile, and bound proteins were eluted with 0.1% TFA in 60% acetonitrile. The eluted proteins were frozen, lyophilized, and resuspended in NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Carlsbad, CA, USA), thus obtaining a 50x concentrate of the original cell culture medium. Equal numbers of seeded cells from each well were snap-frozen and stored at –80°C until use.

Neutrophil precursor stimulation
Neutrophil precursor cells were seeded at a density of 0.8–1 x 109 cells/L and maintained in Stem-Pro-34 SFM basal liquid medium (Invitrogen) or in RPMI medium with 15 mM Hepes. The medium was supplemented with 2% autologous bone marrow plasma and 5 ng/ml rhG-CSF (Sigma-Aldrich). Cells were stimulated for 12 h–5 days at 37°C with 12–120 nM 1,25(OH)2D3, 1 µM ATRA, 100 nM 25(OH)D3, 1 µM cholecalciferol, or 1 µM 9-cis retinoic acid. The cell culture medium from each experiment was collected following centrifugation (200 g). Equal numbers of seeded cells from each well were snap-frozen and stored at –80°C until resuspension in immunoblot sample buffer, or alternatively, RNA was isolated using the RNeasy min kit (Qiagen).

SDS-PAGE and immunoblotting
Samples for immunoblots were dissolved in NuPAGE LDS sample buffer (Invitrogen) including 10% β-ME and heated at 70°C before loading. Proteins were separated in 1.0 mm 4–12% NuPAGE Bis-Tris gel (Invitrogen), and immunoblotting was performed as described [10 ] using the following antibodies: goat anti-rabbit IgG (H+L; BioRad Laboratories, Hercules, CA, USA), rabbit anti-LL-37 (Innovagen, Lund, Sweden [9 ]), and rabbit anti-actin (directed against residues 20–33; Sigma-Aldrich). The pro-LL-37 signal from the cell culture medium was normalized to the number of cells in each well, and equal numbers of seeded cells from each well were dissolved in loading buffer.

RESULTS

Patients with SCN display low pro-LL-37 transcript levels in neutrophil precursors
We analyzed pro-LL-37 and the {alpha}-defensins HNP1–3 transcript levels in bone marrow neutrophil progenitors representing early and late stages of maturation. One typical example of the CD13/CD11b expression profile of cells at different maturation stages during normal neutrophil maturation is displayed in Figure 1B and a representative plot from one patient with SCN in Figure 1C . In patients with SCN, the G-CSF-driven myeloid maturation generated a higher proportion of the more mature cell population, although the earlier myeloid maturation stages were represented but at lower numbers compared with the control (Fig. 1C , and data not included).


Figure 1
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  		Figure 1. Analysis of transcript levels in subsets of bone marrow-derived neutrophil precursors. (A–C) Cell sorting by flow cytometry of neutrophil precursors from control and patients with SCN. (A) Gate is displayed. SSC and FSC, Side-scatter and forward-scatter, respectively. (B) Cells were sorted into one early (CD11blow) and one late (CD11bhigh) maturation-stage population of bone marrow neutrophil precursors, based on CD13/CD11b expression as described [29 30 31 ]. The different maturation stages are indicated: P, promyelocytes; M, myelocytes; MM, metamyelocytes; BS, band cells/segmented cells. The BMT patient (#1) served as a control. An example of a patient (#7) neutrophil maturation profile is depicted in C. Transcript levels of pro-LL-37 (D), C/EBP{alpha} (F), C/EBP{epsilon} (G), and PU.1 (H) as compared with GAPDH for six patients (#2, #6, #9, #13, #27, and #28) by qRT-PCR. (E) HNP1–3 transcript levels for four patients (#2, #3, #6, and #7) as compared with β-actin by PCR. (D, F–H) Controls are post-ALL individuals. (1) E Control is the BMT patient (#1). (D–H) Results are depicted as means and ranges. (I) Organization of the CAMP gene (Chr3p21.3), coding for pro-LL-37, including 800 bp of the 5'-untranslated (UTR) domain. The three vitamin D- and vitamin A-responsive elements of DR3 and DR5 type are marked. The gene region displayed was amplified and sequenced for four patients (#3, #6, #7, and #8) according to standard procedures.

Pro-LL-37 synthesis commences at the myelocyte-to-metamyelocyte stage of neutrophil maturation in the bone marrow, whereas HNP1–3 synthesis begins earlier at the myeloblast-to-promyelocyte stage [19 , 34 ]. From Figure 1D , it is clear that pro-LL-37 transcripts were deficient in the neutrophil progenitors of all patients tested, irrespective of the presence or absence or type of disease-associated mutations (Table 1) , whereas the control individuals, as expected, exhibited high levels of the transcript in the neutrophil precursors of late maturation stage (CD11bhigh). Conversely, the transcript levels for HNP1–3 did not differ between patients and control and were readily detectable in early- and late-stage populations (Fig. 1E) . The patients’ deficiency of pro-LL-37 transcripts is not likely to be caused by the G-CSF therapy, as stem-cell donors receiving G-CSF displayed neutrophil pro-LL-37 protein levels almost as high as prior to G-CSF treatment (data not included). The slight reduction may be a result of increased release from neutrophil granules or a function of G-CSF-accelerated bone marrow transit.

Deficiency of pro-LL-37 transcripts is neither a result of mutations in the gene coding for pro-LL-37 (CAMP) nor lack of key transcription factors for neutrophil differentiation
We next analyzed the CAMP gene for mutations by sequencing the entire gene as well as 800 bp of the 5'-UTR region that contains binding sites for vitamin D-response elements, PU.1, C/EBPs, and other potential transcription factors [22 ] (Fig. 1I) . Neither mutations in the CAMP gene nor the upstream region were detected in the four patients analyzed. These patients harbor one of two different ELA2 mutations or one HAX1 mutation or display no known disease-associated mutation, respectively (Table 1) .

Major transcription factors governing neutrophil differentiation include C/EBP{alpha}, C/EBP{epsilon}, and PU.1. Their transcripts were measured by qRT-PCR (Fig. 1 F-H) in early (CD11blow) and late (CD11bhigh) differentiation stage populations. Overall, the transcript levels of C/EBP{alpha}, C/EBP{epsilon}, and PU.1 displayed no major difference between controls and SCN individuals. For C/EBP{epsilon}, a slight reduction in patient neutrophil progenitors at the early stage of maturation was noted, but the difference, less than twofold, was not conclusive in view of the variation in patient sample range (Fig. 1G) .

Lymphoblastoid cells from patients with SCN produce pro-LL-37 in response to vitamin D3
Lymphocytes are known to produce low levels of endogenous pro-LL-37, and its production can be elevated by different inducers [32 ]. We therefore investigated whether EBV-transformed B cells from a patient with SCN had the capacity to produce pro-LL-37. Cells from a patient and a sibling produced pro-LL-37 following stimulation with 1,25(OH)2D3 (Fig. 2A ). This induction was time- and concentration-dependent using physiological concentrations of 1,25(OH)2D3 ranging from 0.01 to 10 nM (data not included). We also investigated the effect of ATRA, and Figure 2B illustrates that EBV-transformed B cells produce pro-LL-37 in a concentration-dependent manner similar to that induced by 1,25(OH)2D3. The stimulation was time-dependent, with increased protein concentrations in the cell culture medium following 3 days of stimulation (data not included). These experiments demonstrate that cells of the lymphoid lineage from a patient with SCN have the ability to transcribe and translate pro-LL-37.


Figure 2
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  		Figure 2. Immunoblot of EBV-transformed B cell pro-LL-37 response to 1,25(OH)2D3 or ATRA stimulation. (A) Transformed cells from a patient with SCN (#3) and a healthy sibling were stimulated with 100 nM 1,25(OH)2D3 for 3 days. Cells and cell culture medium (Sup) were assayed for pro-LL-37 response (16 kD). Two gels were used with the same standard on both gels, the cuts marked by *. (B) EBV-transformed B cells from the same patient and control were stimulated for 4 days with different concentrations of ATRA. The cell culture medium from B cells was concentrated 50x prior to analysis. Patient gene mutation is denoted inside parentheses (see Table 1 ).

Neutrophil precursors from patients with SCN have the capacity to produce pro-LL-37 protein in vitro
We next evaluated the responsiveness of patient myeloid cells to 1,25(OH)2D3, ATRA, or a combination of these, respectively. A pronounced induction of pro-LL-37 was noted when neutrophil precursors from patients as well as from control individuals were incubated with 1,25(OH)2D3 (Fig. 3 A and B ). The induction was time-dependent, and at 3.5 days pro-LL-37 was detected in collected cells as well as in the cell culture medium (Fig. 3B) , whereas after 5 days, pro-LL-37 was mainly noticeable in the culture medium (Fig. 3A) . In contrast to the bone marrow cells, blood-derived mature neutrophils from controls and patients did not respond with pro-LL-37 production following 1,25(OH)2D3 stimulation (data not included).


Figure 3
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  		Figure 3. Immunoblot of bone marrow-derived neutrophil precursor pro-LL-37 response (16 kD) to vitamin stimuli. (A) Cell culture medium after 5 days of stimulation with 1,25(OH)2D3, ATRA, or a combination thereof. Samples from a healthy individual and a patient (#6) were analyzed in the same gel. (B) Cell culture medium (Sup) and cell content of pro-LL-37 were measured after 3.5 days of stimulation of neutrophil precursors from Patient #9. Cell content was analyzed for β-actin (40 kD) as loading control. (C) Cell culture medium from a healthy individual and a patient (#8) following 5 days of stimulation with 1,25(OH)2D3, ATRA, the previtamins 25(OH)D3, and cholecalciferol and 9-cis retinoic acid, respectively. All samples from the patient were analyzed in the same gel but with lanes removed in between, indicated with *. Patient gene mutations are denoted inside parentheses (see Table 1 ). n.d., Not determined.

Unlike for the transformed B cells, neither ATRA nor the geometric isomer of ATRA, 9-cis retinoic acid, induced pro-LL-37 synthesis in neutrophil precursors (Fig. 3 A-C) . ATRA and 1,25(OH)2D3, in combination, counteracted the pro-LL-37 synthesis obtained with vitamin D3 alone (Fig. 3 A and B) , and the reduction of pro-LL-37 was evident in cells and supernatants (Fig. 3B) . Incubation with ATRA alone resulted in increased clumping and colony formation, indicating differentiation toward the neutrophil arm of the myeloid lineage. This phenomenon did not occur following 1,25(OH)2D3 stimulation (data not included).

Circulating vitamin D3 levels are within the normal range in patients with SCN, and vitamin D3 metabolism is not hampered
Many patients with SCN present with osteopenia/osteoporosis (Table 2 and refs. [8 , 25 ]), and a major function of 1,25(OH)2D3 is to control calcium and phosphorus homeostasis, as well as bone formation. Three patients were analyzed for 25(OH)D3 and 1,25(OH)2D3 levels in blood serum, and the laboratory findings displaying bone formation biochemical markers are presented in Table 2 . Both molecules were within reference values, suggesting that the patients have no major defect in vitamin D3 metabolism.


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  		Table 2. Biochemical Bone Markers and Vitamin D3 Major Metabolites in Serum from Patients with SCN

One patient was administered a low dose of 1,25(OH)2D3 daily in an effort to prevent osteopenia (Table 1) . Plasma levels of pro-LL-37 were measured before, during, and after therapy, but no induction of pro-LL-37 protein was observed (data not included).

Active 1,25(OH)2D3 is produced from 25(OH)D3 through the action of the processing enzyme 25-hydroxyvitamin-D3-1{alpha}-hydroxylase [23 , 35 ] (transcribed from the CYP27B1 gene). 25(OH)D3 is in turn produced from cholecalciferol. We analyzed vitamin D3 metabolism in neutrophil precursor cells from patients with SCN using pro-LL-37 synthesis as a read-out. Stimulation of neutrophil precursor cells with 25(OH)D3 or cholecalciferol resulted in an increased level of pro-LL-37 in the cell culture medium (Fig. 3C) , indicating that the vitamin D3-converting enzymes of the patients are functional.

1,25(OH)2D3-Induced pro-LL-37 transcription does not correlate with increased C/EBP{epsilon} transcription
C/EBP{epsilon} is a major transcription factor for the genes encoding the secondary granule proteins during neutrophil maturation in the bone marrow [20 ]. Pro-LL-37 and C/EBP{epsilon} transcripts were therefore monitored following 1,25(OH)2D3 and ATRA stimulation (Fig. 4 A and B ). No concomitant induction of C/EBP{epsilon} could be noted, indicating that C/EBP{epsilon} is not involved in 1,25(OH)2D3-induced pro-LL-37 synthesis. Figure 4A also demonstrates that pro-LL-37 transcripts are generated following combined ATRA/1,25(OH)2D3 stimulation, suggesting that the vitamin D3-induced transcription of the pro-LL-37 gene is subject to further regulation post-transcriptionally, as the combination reduced pro-LL-37 at a protein level (Fig. 3 A and B) .


Figure 4
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  		Figure 4. Analysis of transcript levels in stimulated bone marrow-derived neutrophil precursors from patients with SCN. Neutrophil precursors were isolated, stimulated with 1,25(OH)2D3, ATRA, or a combination thereof, and harvested at different time-points: 12 h, 2.5 days, or 5 days. Transcript analysis was conducted for pro-LL-37 (A) and C/EBP{epsilon} (B) compared with GAPDH for two patients (#2 and #27) by qRT-PCR.

DISCUSSION

We have reported recently that the antimicrobial propeptide pro-LL-37 is deficient in neutrophils of patients with SCN, which is contrary to patients with autoimmune- and idiopathic neutropenia and may therefore serve as a phenotypic biomarker for SCN [5 , 9 ]. In the current study, we indicate that the defect in pro-LL-37 protein synthesis most likely arises as a function of inadequate transcription. This conclusion is based on the findings that isolated neutrophil precursor cells from patients with SCN were almost devoid of pro-LL-37 mRNA, and there were no mutation(s) in the gene coding for pro-LL-37 (CAMP) or in the CAMP promoter region. The 1,25(OH)2D3 and its precursor forms, however, increased the synthesis of pro-LL-37 in neutrophil precursor cells in vitro, demonstrating that the bone marrow neutrophil precursors indeed have the capacity to produce pro-LL-37.

The differentiation of cells within the myeloid lineage is influenced by extracellular signals provided by cytokines and the nuclear receptor ligands 1,25(OH)2D3 and ATRA [36 ]. There are three putative vitamin D- and vitamin A-responsive elements in the promoter of CAMP (Fig. 1I) of the DR3 and DR5 type [26 ], indicating that ATRA and 1,25(OH)2D3 are potential regulators of the gene. ATRA and 1,25(OH)2D3 were herein used to evaluate the inducibility of the CAMP gene in cells from patients with SCN. ATRA did not induce any pro-LL-37 production in bone marrow-derived neutrophil precursors but did so in patient-derived, EBV-transformed B cells, thus demonstrating that lymphocytes from patients with SCN have the capacity to produce pro-LL-37. The unresponsiveness of patient myeloid cells to ATRA concords with the unchanged pro-LL-37 expression following ATRA stimulation in bone marrow mononuclear cells and the myeloid cell lines NB4 and HL-60, reported by An et al. [37 ]. In contrast to ATRA, 1,25(OH)2D3 stimulation of neutrophil precursors from three SCN patients (with ELA2, HAX1, or no known disease-associated mutation, respectively) all responded by pro-LL-37 production, as did the EBV-transformed B cells. These results further underscore the intricate regulation of pro-LL-37 in cells of different lineages [38 ].

We have no indication that the pro-LL-37 dysregulation is a result of aberrant vitamin D3 metabolism, as the systemic levels of 1,25(OH)2D3 and 25(OH)D3 in patients with SCN are within the range of healthy individuals. Furthermore, neutrophil precursors from patients were able to respond in vitro to the provitamins cholecalciferol and 25(OH)D3, respectively, by pro-LL-37 production, indicating that the local conversion enzymes to 1,25(OH)2D3 are functional. Hence, the failed induction of pro-LL-37 in one patient following oral 1,25(OH)2D3 administration (see Table 1 ) may have been a result of too low of a dose rather than malfunctioning vitamin D3 metabolism. We cannot exclude, however, that the proportion of minor, more polar metabolites of 25(OH)D3 exhibiting lower biological activity compared with 1,25(OH)2D3 is different in patients compared with in healthy individuals [39 ].

The basal levels of the instrumental transcription factors C/EBP{alpha} and PU.1, which govern early myeloid differentiation in patient bone marrow precursors, did not differ from that of healthy individuals. Gene-inactivating mutations in the neutrophil lineage-specific transcription factor C/EBP{epsilon} have been identified in patients with specific granule deficiency [20 ], a rare neutrophil disorder, but unlike those patients [40 ], the neutrophils derived from patients with SCN contained lactoferrin and gelatinase [10 ]. A major defect in C/EBP{epsilon}-driven transcription is therefore unlikely. In addition, the level of C/EBP{epsilon} transcripts in neutrophil progenitors was similar to that of controls. Mutations in the transcription regulator growth factor-independent 1 (Gfi1) have been recorded in a few patients with SCN [41 ], and transfer of one gene-inactivating mutation to a mouse model rendered the mouse neutropenic [42 ]. PU.1 and C/EBP{epsilon} are negatively regulated by GFI1 during myelopoiesis [43 ]. However, the transcript levels of PU.1 and C/EBP{epsilon} were similar to control individuals in the present group of patients with SCN, suggesting that GFI1 is functional. It is therefore unlikely that GFI1 is involved in the pro-LL-37 deficiency.

The CAMP gene is transcribed during neutrophil differentiation in the bone marrow, commencing at the myelocyte stage of maturation [44 ]. ATRA and 1,25(OH)2D3 have opposing effects on early myeloid precursors in such a way that 1,25(OH)2D3 preferentially directs myeloid precursors to the monocytic lineage, whereas ATRA guides them toward the neutrophil lineage [45 ]. The early neutrophil precursors, the promyelocytes, may redirect toward monocytes/macrophages in vitro in response to 1,25(OH)2D3, regardless of the presence of G-CSF [46 ]. It may be argued that the pro-LL-37-inducing effect of 1,25(OH)2D3 reflects production from cells differentiating toward the monocytic lineage. The reported level of pro-LL-37 produced by monocytes is, however, low, and therefore, the high levels in patient progenitors most likely derive from cells of the neutrophil lineage [47 ]. When a combination of 1,25(OH)2D3 and ATRA was used for stimulation, pro-LL-37 protein levels were reduced. The induction at the mRNA level, however, was not compromised by coincubation with ATRA, suggesting that pro-LL-37 may, in addition, be post-trancriptionally regulated. Interestingly, 1,25(OH)2D3-induced pro-LL-37 transcription correlated inversely with C/EBP{epsilon} transcription levels, suggesting that transcription factor(s) other than C/EBP{epsilon} are active in 1,25(OH)2D3-induced pro-LL-37 synthesis.

In summary, myeloid precursors of patients with SCN responded to 1,25(OH)2D3 with pro-LL-37 transcription and protein synthesis. We could not detect any alterations in the vitamin D3 synthesis pathways that could explain the prevailing neutrophil pro-LL-37 deficiency. We conclude that the deficiency is most likely a result of improper transcriptional regulation during myelopoiesis, as the pro-LL-37 gene was functionally accessible by 1,25(OH)2D3 in EBV-transformed B cells as well as in neutrophil precursor cells in vitro. Neutrophil differentiation can be induced through more than one pathway, as demonstrated by others [48 ]. It may be that the forced myelopoiesis induced by pharmacological doses of G-CSF fails to recruit transcription factors essential for pro-LL-37 synthesis or alternatively, that there is an inherent defect in specific transcription regulator(s) necessary for pro-LL-37 production. Further identification of components involved in the defective transcription of the CAMP gene may provide tools for elevating pro-LL-37 levels, which in turn, may therapeutically benefit patients with SCN.

NOTE ADDED IN PROOF

Details on patient #28 have been published recently and refer to patient #8 in Carlsson, G., et al. (May 29, 2008) J. Intern. Med. PMID: 18513342 [Epub ahead of print].

ACKNOWLEDGEMENTS

This work was supported by the Swedish Research Council (06X-12634 to M. A. and 06XD-14653 to K. P.), the Swedish Society for Medical Research (to K. P.), the King Oscar II Jubilee Foundation (to M. A.), the Magnus Bergwall Foundation (to M. A.), the Swedish Medical Society (to K. P.), the Åke Wiberg foundation (to K. P.), the Swedish Childhood Cancer Foundation (to G. C.), and the Söderbergska stiftelsen. We are grateful to all patients and their families and physicians. We acknowledge Hans G. Boman and Ingemar Ernberg for their generous gift of cell lines and for valuable discussions. We are grateful to Anita Vestman for cell culture assistance, Birgitta Wester for FACS analyses, and Robert A. Harris for linguistic advice. We also acknowledge Charles L. Bevins and Wilhelm Paulander for PCR assistance and Ramona Petersson for immunoblot analyses.

Received June 27, 2007; revised May 25, 2008; accepted July 9, 2008.

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