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Originally published online as doi:10.1189/jlb.0203063 on October 23, 2003

Published online before print October 23, 2003
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(Journal of Leukocyte Biology. 2004;75:190-197.)
© 2004 by Society for Leukocyte Biology

Phenotypic and functional alterations of peripheral blood monocytes in neutrophil-specific granule deficiency

Masaaki Shiohara*,1, Adrian F. Gombart{dagger}, Yukio Sekiguchi*, Eiko Hidaka{ddagger}, Susumu Ito§, Takashi Yamazaki*, H. Phillip Koeffler{dagger} and Atsushi Komiyama*

Departments of
* Pediatrics and
{ddagger} Laboratory Medicine and
§ Division of Blood Transfusion, Shinshu University School of Medicine, Matsumoto, Japan; and
{dagger} Division of Hematology/Oncology, Cedars-Sinai Medical Center, Los Angeles, California

1 Correspondence: Department of Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390-8621 Japan. E-mail: mjshio{at}poa.matsumoto.ne.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil-specific granule deficiency (SGD) is a rare, congenital disease characterized by atypical neutrophil structure and function, resulting in recurrent bacterial infections from early infancy. Homozygous recessive mutations in the CCAAT/enhancer-binding protein {varepsilon} (C/EBP{varepsilon}) gene were described in two of five SGD patients, indicating loss of C/EBP{varepsilon} function as the primary genetic defect in this disease. C/EBP{varepsilon} is expressed in murine and human macrophages. Macrophages from the C/EBP{varepsilon}-deficient mice show impaired differentiation, phagocytic activity, and transcription of macrophage-specific genes. To determine if monocyte/macrophage cells are impacted in SGD, we analyzed phenotypic features of peripheral blood (PB) monocytes in a SGD individual lacking functional C/EBP{varepsilon}. Flow cytometric analysis of PB leukocytes revealed aberrant expression of CD45, CD11b, CD14, CD15, and CD16 on cells from the SGD individual. Also, the PB CD14+ cells from this individual, weakly stained for the monocyte-specific enzyme, nonspecific esterase, and electron microscopic examination, indicated morphologic differences between the SGD cells and those from normal controls. Serum interleukin (IL)-6 levels in the SGD individual during a severe bacterial infection were lower compared with levels in other non-SGD individuals with sepsis. In contrast, serum IL-8 levels were markedly elevated in the SGD individual compared with those of non-SGD individuals in sepsis. PB CD14+ cells from the SGD individual expressed higher IL-8 mRNA levels compared with normal controls in response to lipopolysaccharide and interferon-{gamma}. These phenotypic and functional alterations of PB monocytes in the SGD individual suggest that C/EBP{varepsilon} plays a critical role in monocyte/macrophage development of humans and is consistent with observations in the murine system. This study implicates abnormalities in monocytes/macrophages and neutrophils in the onset and development of SGD.

Key Words: immunodeficiency • C/EBP{varepsilon} • CD14 • esterase • IL-8


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil-specific granule deficiency (SGD) is a disorder characterized by a lifetime of recurrent, pyogenic infections [1 , 2 ]. Neutrophils from these individuals have abnormalities in nuclear morphology and lack primary, specific, and tertiary granule proteins including lactoferrin, collagenase, and defensins [1 2 3 4 5 ]. Also, they are defective in chemotaxis and killing of bacteria. Additionally, eosinophil-specific granule content, including eosinophil cationic protein, eosinophil-derived neurotoxin, and major basic protein (MBP), is deficient in SGD [6 ]. CCAAT/enhancer-binding protein {varepsilon} (C/EBP{varepsilon}) is a transcription factor that is primarily expressed in the myeloid and T cell lineages [7 , 8 ]. Neutrophils from C/EBP{varepsilon}-deficient (-/-) mice have distinctive morphological features including bilobed nuclei and absence of specific and tertiary granule proteins, similar to neutrophils of individuals with SGD [9 , 10 ]. Also, the mRNAs encoding eosinophilic granule proteins, eosinophil peroxidase (EPX) and MBP, are not expressed [11 ]. In addition, C/EBP{varepsilon} (-/-) neutrophils are defective in chemotaxis and bactericidal activity [12 ]. These phenotypic similarities between human SGD and C/EBP{varepsilon} (-/-) mice suggested a loss of a functional C/EBP{varepsilon} in SGD (Table 1 ). Indeed, germ-line C/EBP{varepsilon} mutations in two SGD patients were reported, explaining the genetic defect responsible for this disease [13 , 14 , 16 ].


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  		Table 1. Comparison of C/EBP{varepsilon} Knockout (KO) Mouse and SGD

 
C/EBP{varepsilon} is highly expressed at the promyelocyte and later stages of granulocytic development but is detectable in monocytes/macrophages and related cell lines in mice as well [15 ]. Additional studies suggested a role in monocyte/macrophage differentiation, gene expression, and/or function. Overexpression of C/EBP{varepsilon} in a pre-B cell acute lymphoblastic leukemia cell line induced expression of the macrophage-colony stimulating factor receptor as well as chemokines, including macrophage inflammatory protein (MIP)-1{alpha} and MIP-1ß [17 ]. Representational difference analysis using neutrophils and macrophages from wild-type and C/EBP{varepsilon} (-/-) mice identified a set of differentially expressed genes, including cathepsin L, MIP-1{gamma}, and macrophage chemoattractant protein-3, which are specific to myelomonocytic cells [18 ]. More recently, Tavor et al. [15 ] demonstrated that macrophages from C/EBP{varepsilon}-deficient mice displayed signs of immaturity, as well as impaired phagocytosis and gene expression. These findings indicate that C/EBP{varepsilon} regulates myelomonocytic target genes and that loss of normal C/EBP{varepsilon} function affects monocyte/macrophage function in the mouse. Altered monocyte/macrophage function has not been reported in SGD; therefore, we examined the peripheral blood (PB) monocytes of a SGD patient, lacking functional C/EBP{varepsilon}, for abnormalities of morphology, protein expression, cell-surface markers, and enzymatic activity. Our results revealed differences between normal and SGD monocyte/macrophages, indicating that loss of C/EBP{varepsilon} impacts monocytic cells in humans and mice. The parallels between the human and murine conditions indicate that the C/EBP{varepsilon}-deficient murine model will serve as an extremely useful tool in further characterizing this rare human disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient
Materials from a female patient described previously, lacking neutrophil-specific granules, were studied after receiving her informed consent [2 ]. This patient has a homozygous germ-line alteration involving a single A-nucleotide insertion at nucleotide 1113 in exon 2 of C/EBP{varepsilon} [14 ]. Samples from non-SGD patients with sepsis and normal healthy volunteers were obtained after receiving their informed consent. We defined sepsis as follows: those patients presenting with high fever, high value of C-reactive protein (>15.0 mg/ml), and bacterial growth from blood culture with infection foci. Patient profiles are listed in Table 2 .


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  		Table 2. Serum Cytokine Levels of the SGD Patient

 
Reagents
Human interferon-{gamma} (IFN-{gamma}) was purchased from Gibco-BRL (Rockville, MD). Lipopolysaccharide (LPS), purified from Esherichia coli O55:B5, was obtained from Sigma-Aldrich (St. Louis, MO). LPS was trichloroacetic acid-precipitated and purified by gel filtration chromatography and contained 0.8% of protein (manufacturer’s instruction).

Flow cytometric analysis and sorting
Monoclonal antibodies (mAb) for CD45, CD11b, CD14, CD15, CD16, and CD34 were purchased from BD Immunocytometry Systems (Mountain View, CA). For the analysis of surface markers on the PB leukocytes, 1–2 x 106 buffy coat cells were collected in polystyrene tubes and were incubated with appropriately diluted fluorescein isothiocyanate (FITC)- or phocoerythrin (PE)-mAb, as described previously [19 ]. The cells were washed twice, after which their surface markers were analyzed with the FACScan flow cytometer, using the Lysis II software program (BD Immunocytometry Systems). Viable cells were gated according to their forward light-scatter characteristics (FSC) and side-scatter characteristics (SSC). In some experiments, CD14+ cells were sorted by FACScan flow cytometer. PB cells were stained with mAb for CD14 and CD15, and the cells that have high intensities of CD14 and have less CD15 expression were gated and sorted. Flow cytometric analysis of the cells sorted by this system revealed that less than 5% of control CD14+ cells expressed CD15. In contrast, ~15% of CD14+ cells of the SGD patient expressed CD15 (data not shown).

Analysis of nonspecific esterase (NSE) activity
PB CD14+ cells were isolated using the magnetic cell sorting separation system (Miltenyi Biotec, Auburn, CA). NSE activity was examined on cytocentrifuge preparations as described by the manufacturer (esterase stain kit, Muto Pure Chemicals Co., Ltd., Tokyo, Japan) [20 ].

Ultrastructural analysis by electron microscopy (EM)
For the ultrastructural examination, PB CD14+ cells were fixed with 1.25% gluteraldehyde in 0.1 mol/l phosphate buffer (pH 7.2) for 2 h and were postfixed in 1% osmium tetroxide. The specimens were dehydrated in alcohol and embedded in Araldite (Nissin Co., Tokyo, Japan). Immunostaining using antilysozyme antibody was performed as described previously [21 ]. Ultrathin sections were examined in a Hitachi H-300 electron microscope.

Assay of serum cytokine levels
Serum Th1 cytokine (IFN-{gamma}, TNF-{alpha}, and IL-2) and Th2 cytokine (IL-10, IL-6, and IL-4) levels were measured by the BD CBA kit (BD Biosciences, San Diego, CA), following the manufacturer’s instructions. Serum concentrations of IL-8 protein were measured by a human IL-8 ELISA kit (Fujirebio, Tokyo, Japan).

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the PB-derived CD14+ cells of the patient and the healthy volunteer, by Isogen reagent (Nippon Gene, Tokyo, Japan), following the manufacturer’s instruction. The RNA (200 ng) was reverse-transcribed using random-hexamer primers and avian myeloblastosis virus–RT (Takara, Tokyo, Japan). One-fifth of the RT reaction was used directly in a PCR reaction. The PCR reaction conditions were 67 mM Tris-HCl (pH 8.8), 400 nM each primer, and 0.5 unit Taq polymerase in a 25-µl reaction. Twenty-five cycles of PCR (94ºC, 30 s; 55–60ºC, 30 s; and 72ºC, 2 min) were performed. A sense primer 5'-ATGACTTCCAAGCTGGCCGTG-3' (nt 1–21) and an antisense primer 5'-TTATGAATTCTCAGCCCTCTTCAAAAACTTCTC-3' (nt 302–269) were used for amplification of the IL-8 cDNA (GenBank/EMBL/DDBJ accession no. Z11686). The ß-actin primers were 5'-CTGGCCGGGACCTGACTGACTACCTCATGA-3' (nt 617–646, sense) and 5'-ACTCTAACCGTACCGAAATAAACAAA-3' (nt 1256–1231, antisense). Reaction products were separated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide, and photographed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of cell-surface markers of PB leukocytes
The expression of CD45, CD11b, CD14, CD15, and CD16 on PB leukocytes was examined by flow-cytometric analysis (Fig. 1 ). Viable PB leukocytes were gated according to the FSC and SSC. R1 (red) and R2 (green) correspond to normal monocyte and neutrophil populations, respectively (Fig. 1A) . Distribution of cell populations developed by SSC and CD45 showed that the SGD neutrophilic granulocytes (green) were much lower in SSC height compared with the normal control, consistent with the low granularity and immaturity of these cells (Fig. 1B) . In the SGD sample, monocytes were not separated but combined with the neutrophil population. The cell populations expressing CD11b were clearly divided into neutrophils (green) and monocytes (red) in the normal control (Fig. 1C) . In contrast, the SGD cells expressing CD11b were not clearly separated into two subpopulations. Normal PB leukocytes divided into two subpopulations on the basis of CD14 and CD15 expression: CD14-/CD15+ (neutrophils) and CD14+/CD15- (monocytes) (Fig. 1D) . In the SGD individual’s PB, however, these two subpopulations were not clearly separated. The expression of CD16, which is a low-affinity Fc receptor for immunoglobulin G (IgG; Fc{gamma}R) and mainly expressed on neutrophils, was markedly decreased on the SGD patient’s leukocytes, compared with the normal control (Fig. 1E) . In contrast, CD16 was expressed at similar levels on natural killer (NK) cells from the SGD patient and the normal control (Fig. 1E , circles). The SGD patient’s eosinophils, detected by CD45 and CD9, were reduced in number compared with the normal control (data not shown). Expression of CD11c, CD13, CD18, CD21, CD32, CD33, CD34, CD35, CD41b, CD42b, CD61, CD71, CD117, and glycophorin A on PB leukocytes was unchanged in the SGD patient as compared with the normal controls (data not shown).



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  		Figure 1. Flow cytometric analysis of PB leukocytes, which were gated according to the FSC and SSC. R1 and R2 correspond to normal monocytes and neutrophil populations, respectively (A). Cells colored by red (R1) and green (R2) in dot-blot analysis represent monocyte and neutrophil populations on the basis of FSC and SSC, respectively (B). The surface expression of CD45 (B), CD11b (C), CD14 (D), CD15 (D), and CD16 (E) was determined by flow cytometric analysis. PER CP, peridinin chlorophyll protein.

 
NSE activity of PB monocytes
Monocytes, but not neutrophils, possess NSE activity. Intensities of NSE staining of CD14+ PB cells were compared between the normal and the SGD patient, by counting 100 cells in each field and calculating average percentages of strongly or weakly NSE-positive cells in a total of five independent fields (Fig. 2 ). Strongly positive cells in normal (Fig. 2B and 2D) versus the SGD (Fig. 2A and 2C) sample were 85% versus 21%, and weakly positive cells were 15% versus 79% (Fig. 2E) , respectively. These findings indicate that the NSE activity of the SGD patient’s monocytes was notably much weaker than those of the normal control, suggesting a functional impairment of the patient’s PB monocytes.



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  		Figure 2. Nonspecific esterase activity of PB monocytes. (A–D) PB CD14+ cells from a normal volunteer (A and C) and the SGD patient (B and D) were purified by anti-CD14 immunomagnetic beads. Cytochemical staining was performed on cytocentrifuged samples. Original magnification, x100 (A and B); x400 (C and D). (E) The ratio of strongly and weakly positive cells for nonspecific esterase activities in normal and SGD CD14+ cells. Data shown represent mean ± SD of five independent counts of the cells.

 
EM analysis of monocytes in SGD
The EM findings showed that CD14+ monocytes from PB of a normal control have small, round or rod-shaped, cytoplasmic granules. In contrast, CD14+ monocytes from the SGD individual have large cytoplasmic granules, many of which contained electron-dense cores. Rod-shaped granules observed in the normal control were not seen in the SGD cells (Fig. 3A 3B 3C 3D ). These granules are thought to contain lysosomal enzymes including lysozyme, which degrades the sugar backbone of peptidoglycan by hydrolyzing the bond between N-acetylglucosamine and N-acetylmuramic acid. This enzyme is present in primary and secondary granules of neutrophils and monocytes [22 , 23 ]. Immunostaining with gold particles bound with antilysozyme Ab revealed that the SGD monocytes as well as the control monocytes stained positively (Fig. 3E and 3F) .



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  		Figure 3. Ultrastructural appearance of PB monocytes. Electron micrographs from the normal control (A, C, and E) and from the SGD patient (B, D, and F) were prepared from CD14+ PB cells. (E and F) Immunogold staining using antilysozyme Ab. Arrowheads indicate gold particles bound with antilysozyme Ab. Mitochondria are in close proximity to the granules (E). Original magnifications were 5000x (A and B) and 25,000x (C–F).

 
Serum cytokine levels of the patient
Frequent and severe infections occur in individuals with SGD. The manifestations of infectious states result from the secretion of inflammatory cytokines and/or chemokines in the course of immune-defensive responses against bacterial infections. In SGD, the serum cytokine or chemokine levels in steady-state or inflammatory conditions have not been analyzed. CD4+ Th cells were shown to differentiate and polarize into two populations according to their cytokine production pattern (i.e., Th1 cells producing IFN-{gamma}, TNF-{alpha}, and IL-2 and Th2 cells producing IL-10, IL-6, and IL-4) [24 , 25 ]. We measured the levels of these cytokines and one of the CXC chemokines, IL-8, in the serum of the SGD patient and compared them with levels found in non-SGD patients with sepsis. Serum concentrations of seven cytokines were <20 pg/ml in three normal volunteers without any obvious infections (Table 2) . This included serum IL-8 concentrations of 19 and 20 pg/ml in these normal controls. Concentrations of IFN-{gamma}, TNF-{alpha}, IL-2, and IL-4 in the serum of the SGD patient with necrotizing fasciitis and S. aureus sepsis were also <20 pg/ml. The same is observed in the normal and the SGD patient after antibiotic therapy and no longer suffering from sepsis. In contrast, serum IL-8 levels were markedly elevated (17,252 pg/ml) in the SGD patient with sepsis as compared with the non-SGD controls with sepsis (ranged from 111 to 7680 pg/ml). IL-6 levels were slightly elevated (94 pg/ml) but were well below the levels found in three non-SGD septic cases. Concentrations of IL-10 in the serum of the SGD patient with sepsis were <20 pg/ml. In contrast, serum IL-10 levels were elevated in two of three non-SGD controls with sepsis (ranged from 72 to 413 pg/ml; Table 2 ). These results suggest that expression of certain cytokines (IL-6 and IL-8) was increased during bacterial infection of the SGD individual, and the other cytokines were not. Clearly, a clearer picture of the capacity to make cytokines will require additional septic and nonseptic SGD individuals.

RT-PCR analysis of IL-8 mRNA expression in CD14+ cells
Analysis of serum cytokine levels showed that the IL-8 concentration was markedly elevated in the serum of the SGD patient with severe bacterial infection. We analyzed IL-8 mRNA expression in CD14+ cells by RT-PCR (Fig. 4 ). PB CD14+ cells were isolated and incubated with/without 5 ng/ml LPS and 50 U/ml IFN-{gamma} for 4 h. After incubation, the cells were harvested, RNA was extracted, and RT-PCR was performed. CD14+ cells from normal controls did not express IL-8 mRNA, but their expression of IL-8 increased upon incubation with LPS and IFN-{gamma}. CD14+ cells from the SGD patient expressed IL-8 weakly, and their expression markedly increased by incubation with LPS and IFN-{gamma} (Fig. 4) .



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  		Figure 4. Expression of IL-8 mRNA in PB CD14+ cells, which were incubated without or with 5 ng/ml LPS and 50 U/ml IFN-{gamma} for 4 h. Cells were harvested, and RNA was extracted. RT-PCR was performed using primers specific for IL-8 and ß-actin. PCR products were subjected to electrophoresis through 1.5% agarose gels, stained with ethidium bromide, and photographed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
SGD involves morphological and functional abnormalities in neutrophils and eosinophils. In addition, C/EBP{varepsilon}-deficient murine macrophages showed signs of immaturity, impaired phagocytic function, and a decreased or absence of cytokine production including IL-6, IL-10, IL-12, and IL-18, demonstrating the importance of C/EBP{varepsilon} in macrophage development and function in the mouse [15 ]. To determine if monocyte/macrophage defects existed in SGD, we analyzed PB monocytes from a SGD individual lacking functional C/EBP{varepsilon} [14 ]. Phenotypical (EM and surface-receptor molecules) and functional (NSE staining and gene expression) abnormalities in PB monocytes were observed in SGD.

Flow cytometry is a powerful tool in the diagnosis of hematologic diseases involving myeloid and lymphoid leukemias by analyzing the patterns and intensity of antigen expression [26 ]. CD45 is a tyrosine phosphatase expressed on the cell membrane of leukocytes, and its expression increases as they mature [27 ]. Blood is readily separated into its cellular constituents by flow cytometry when CD45 is combined with side-scatter [26 ]. The SSC, FSC, and CD45 patterns of the PB cell distribution of SGD, especially granulocyte and macrophage populations, were quite different from the normal control. Immature myeloid populations were increased in SGD, similar to that observed in acute myeloid leukemia. Besides regulating the synthesis of secondary granule proteins in granulocytes, C/EBP{varepsilon} probably also influences the granulocyte-macrophage differentiation program.

The CD11b (ß2 integrin complement receptor 3), CD14 (glycosylphosphatidylinositol-anchored receptor expressed on the surface of the monocytes/macrophages, which binds to the complex of LPS and LPS-binding protein), CD15 (glycoprotein Lewisx), and CD16 (IgG low-affinity receptor, Fc{gamma}III) are important molecules required for innate immunity to invading microbes. In SGD, aberrant expression of CD11b, CD14, and CD15 in PB monocytes was revealed by flow cytometry. This suggests that monocytes in SGD may be defective in their innate-immune response, thus contributing to susceptibility to bacterial infections in these individuals. The expression of CD14, monocyte chemotactic protein-3 (MCP-3), and plasminogen-activator inhibitor type 2, which participate in the regulation of the inflammatory response, was decreased in the murine C/EBP{varepsilon}-deficient macrophages, indicating that C/EBP{varepsilon} is involved in regulation of these molecules in the mouse [15 ]. Our data suggest that in vivo, C/EBP{varepsilon} is involved in regulation of CD11b, CD14, and CD15 levels. Prior studies showed that CD11b expression was significantly decreased on murine C/EBP{varepsilon}-deficient neutrophils early after thioglycollate challenge, but then expression became abundant by 24 h after challenge [12 ]. CD16, which is mainly expressed on granulocytes and NK cells, was severely reduced on granulocytes but normally expressed on NK cells in the SGD patient (Fig. 1E) . These data suggest that CD16 is regulated by C/EBP{varepsilon} in granulocytes and is controlled by a different mechanism in NK cells.

We have shown that T cell receptor-mediated proliferation of T cells was impaired in C/EBP{varepsilon} (-/-) mice [28 ]. Spleen cells from C/EBP{varepsilon} (-/-) mice expressed lower levels of mRNA encoding IFN-{gamma}, IL-4, IL-12p40, and IL-2 compared with the wild-type mice, indicating a defect in the expression of macrophage-specific genes important in the control of T cell function [9 ]. In addition, the expression of IL-10, IL-12, and IL-18, which are key cytokines in the development of Th1 and Th2 cell responses, was decreased in C/EBP{varepsilon} (-/-) macrophages [15 ]. In this study, serum IL-6 levels in SGD and normal controls were undetectable in the absence of sepsis. However, during sepsis, levels markedly rose in non-SGD patients but not in the SGD individual. The promoter for the IL-6 gene contains functional C/EBP-binding sites in its promoter region [29 , 30 ], and C/EBP{varepsilon} is important for the basal expression of IL-6 in murine macrophages [15 ]. The observed defect in IL-6 production in SGD suggests that IL-6 is a target gene of C/EBP{varepsilon} in humans as well.

In contrast to IL-6, the serum concentration of IL-8 was slightly elevated in the uninfected SGD patient and markedly increased by severe infection as compared with the septic, non-SGD patients. Furthermore, IL-8 mRNA in PB CD14+ cells of SGD increased dramatically after stimulation with LPS and IFN-{gamma} compared with the normal control. IL-8 is one of the CXC chemokines that induces the chemotaxis of neutrophils, monocytes, T lymphocytes, and eosinophils [31 , 32 ]. Chemokines, including IL-8, are produced by monocytes and regulate T cell differentiation and function through chemokine receptors expressed on the T cells [33 , 34 ]. The IL-8 promoter is positively regulated by transcription factors including nuclear factor-{kappa}B, activated protein-1, and/or C/EBP in a cell line-specific manner [35 , 36 ]. In contrast, the POU (founding members Pit-1, Oct-1/2, and Unc-86) homeodomain transcription factor, Oct-1, strongly represses transcriptional activity of the IL-8 promoter by binding to an element overlapping the C/EBP site [37 ]. Alterations of C/EBP{varepsilon} may change the interactions of the transcription factor Oct-1 with the C/EBP element. Aberrant IL-8 production induced by severe bacterial infections may modify immunological responses and clinical manifestations of individuals with SGD. Serum IL-10 levels were <20 pg/ml in the uninfected SGD patient and were not increased by sepsis. In contrast, serum concentrations of IL-10 were elevated in two of three non-SGD controls. C/EBPß and -{delta} were shown to activate LPS-induced IL-10 gene expression in mouse macrophages [38 ]. In addition, the expression of IL-10 was impaired in C/EBP{varepsilon} (-/-) macrophages [15 ]. C/EBP{varepsilon} may be involved in the expression of IL-10 in humans as well as in mice. Expression of other chemokines, including MIP-1{alpha}, MIP-1ß, and MCP-1 in PB CD14+ cells stimulated with IFN-{gamma} and LPS, was not impaired in SGD compared with normal controls, as analyzed by RT-PCR (data not shown).

These phenotypic and functional alterations of PB monocytes of the SGD patient may not necessarily be a result of the inherent defect of the monocytes/macrophages lineage but could be the consequences of abberrant stimulation of monocyte/macrophage differentiation. For example, abberrant proliferation of T cells and macrophage-specific cytokine production of spleen cells of C/EBP{varepsilon}-deficient mice [9 , 28 ] might partially affect the differentiation and function of immune-response cells including monocytes/macrophages. Further studies are needed to elucidate the indirect effects on monocyte/macrophage differentiation in SGD.

SGD is an extremely rare disease. The individual in this study is one of two with reported germ-line mutations in the C/EBP{varepsilon} locus. Five SGD cases have been reported worldwide. Material for study from these patients is extremely difficult to obtain. The existence of a murine model for SGD helps circumvent this obstacle; however, it is important to determine if observations in the murine model are consistent with the human disease. The loss of C/EBP{varepsilon} activity on granulocyte differentiation and function is strikingly similar in the murine model. This indicates a strong conservation of C/EBP{varepsilon} function in humans and mice. The discovery of monocyte/macrophage defects in the murine model raised the question of how these cells are affected in the human disease. The current study indicates that the transcription factor C/EBP{varepsilon} plays a critical role in monocyte/macrophage function and development in humans as well. Such defects have not been described previously. Impairment of the granulocytic and monocytic lineages likely contributes to the profound defects in the innate-immune response, which result in the severe and frequent bacterial infections that occur in these patients.


   ACKNOWLEDGEMENTS
 
H. P. K. was supported by NIH grants as well as The Parker Hughes fund. He holds the endowed Mark Goodson Chair in Oncology Research and is a member of the Jonsson Cancer Center and the Molecular Biology Institute at UCLA. We thank Prof. Mitsuoki Eguchi (Dokkyo Medical College, Japan) for his assistance in the EM analysis.

Received February 9, 2003; revised September 8, 2003; accepted September 18, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Strauss, R. G., Bove, K. E., Jones, J. F., Mauer, A. M., Fulginiti, V. A. (1974) An anomaly of neutrophil morphology with impaired function N. Engl. J. Med. 290,478-484
  2. Komiyama, A., Morosawa, H., Nakahata, T., Miyagawa, Y., Akabane, T. (1979) Abnormal neutrophil maturation in a neutrophil defect with morphologic abnormality and impaired function J. Pediatr. 94,19-25[CrossRef][Medline]
  3. Ambruso, D. R., Sasada, M., Nishiyama, H., Kubo, A., Komiyama, A., Allen, R. H. (1984) Defective bactericidal activity and absence of specific granules in neutrophils from a patient with recurrent bacterial infections J. Clin. Immunol. 4,23-30[CrossRef][Medline]
  4. Tamura, A., Agematsu, K., Mori, T., Kawai, H., Kuratsuji, T., Shimane, M., Tani, K., Asano, S., Komiyama, A. (1994) A marked decrease in defensins mRNA in the only case of congenital neutrophil-specific granule deficiency reported in Japan Int. J. Hematol. 59,137-142[Medline]
  5. Johnston, J. J., Boxer, L. A., Berliner, N. (1992) Correlation of messenger RNA levels with protein defects in specific granule deficiency Blood 80,2088-2091[Abstract/Free Full Text]
  6. Rosenberg, H. F., Gallin, J. I. (1993) Neutrophil-specific granule deficiency includes eosinophils Blood 82,268-273[Abstract/Free Full Text]
  7. Antonson, P., Stellan, B., Yamanaka, R., Xanthopoulos, K. G. (1996) A novel human CCAAT/enhancer binding protein gene, C/EBP{varepsilon}, is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 close to the T-cell receptor {alpha}/{delta} locus Genomics 35,30-38[CrossRef][Medline]
  8. Chumakov, A. M., Grillier, I., Chumakova, E., Chi, D., Slater, J., Koeffler, H. P. (1997) Cloning of the novel human myeloid-cell-specific C/EBP{varepsilon} transcription factor Mol. Cell. Biol. 17,1375-1386[Abstract]
  9. Yamanaka, R., Barlow, C., Lekstrom-Himes, J., Castilia, L., Liu, P., Eckhaus, M., Decker, T., Wynshaw-Boris, A., Xanthopoulos, K. G. (1997) Impaired granulopoiesis, myelodysplasia, and early lethality in C/EBP{varepsilon} deficient mice Proc. Natl. Acad. Sci. USA 94,13187-13192[Abstract/Free Full Text]
  10. Verbeek, W., Lekstrom-Himes, J., Park, D. J., Dang, P. M., Vuong, P. T., Kawano, S., Babior, B. M., Xanthopoulos, K., Koeffler, H. P. (1999) Myeloid transcription factor C/EBP{varepsilon} is involved in the positive regulation of lactoferrin gene expression in neutrophils Blood 94,3141-3150[Abstract/Free Full Text]
  11. Gombart, A. F., Kwok, S. H., Anderson, K. L., Yamaguchi, Y., Torbett, B. E., Koeffler, H. P. (2003) Regulation of neutrophil eosinophil secondary granule gene expression by transcription factors C/EBP{varepsilon} and PU.1 Blood 101,3265-3273[Abstract/Free Full Text]
  12. Lekstrom-Himes, J., Xanthopoulos, K. G. (1999) CCAAT/enhancer binding protein epsilon is critical for effective neutrophil-mediated response to inflammatory challenge Blood 93,3096-3105[Abstract/Free Full Text]
  13. Lekstrom-Himes, J., Dorman, S. E., Kopar, P., Holland, S. M., Gallin, J. I. (1999) Neutrophil-specific granule deficiency results from a novel mutation with loss of function of the transcription factor CCAAT/enhancer binding protein {varepsilon} J. Exp. Med. 189,1847-1852[Abstract/Free Full Text]
  14. Gombart, A. F., Shiohara, M., Kwok, S. H., Agematsu, K., Komiyama, A., Koeffler, H. P. (2001) Neutrophil-specific granule deficiency: homozygous recessive inheritance of a frameshift mutation in the gene encoding transcription factor CCAAT/enhancer binding protein-{varepsilon} Blood 97,2561-2567[Abstract/Free Full Text]
  15. Tavor, S., Vuong, P. T., Park, D. J., Gombart, A. F., Cohen, A. H., Koeffler, H. P. (2002) Macrophage functional maturation and cytokine production are impaired in C/EBP{varepsilon}-deficient mice Blood 99,1794-1801[Abstract/Free Full Text]
  16. Gombart, A. F., Koeffler, H. P. (2002) Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP{varepsilon} Curr. Opin. Hematol. 9,36-42[CrossRef][Medline]
  17. Williams, S. C., Du, Y., Schwartz, R. C., Weiler, S. R., Ortiz, M., Keller, J. R., Johnson, P. F. (1998) C/EBP{varepsilon} is a myeloid-specific activator of cytokine, chemokine and macrophage-colony-stimulating factor receptor genes J. Biol. Chem. 273,13493-13501[Abstract/Free Full Text]
  18. Kubota, T., Kawano, S., Chi, D. Y., Hisatake, Y., Chumakov, A. M., Taguchi, H., Koeffler, H. P. (2000) Representational difference analysis using myeloid cells from C/EBP{varepsilon} deletional mice Blood 96,3953-3957[Abstract/Free Full Text]
  19. Shiohara, M., Uskokovic, M., Hisatake, J., Hisatake, Y., Koike, K., Komiyama, A., Koeffler, H. P. (2001) 24-Oxo metabolites of vitamin D3 analogues: disassociation of their prominent antileukemic effects from their lack of calcium modulation Cancer Res. 61,3361-3368[Abstract/Free Full Text]
  20. Shiohara, M., Dawson, M. I., Hobbs, P. D., Sawai, N., Higuchi, T., Koike, K., Komiyama, A., Koeffler, H. P. (1999) Effects of novel RAR- and RXR-selective retinoids on myeloid leukemic proliferation and differentiation in vitro Blood 93,2057-2066[Abstract/Free Full Text]
  21. Hidaka, E., Ota, H., Katsuyama, T., Nakayama, J., Momose, M., Hidaka, H., Ishii, K., Murata, F., Tsuyama, S., Kurihara, M., Ishihara, K., Hotta, K. (2000) Coexistance of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells Histochem. Cell Biol. 113,91-98[CrossRef][Medline]
  22. Mutasa, H. C. F. (1989) Combination of diaminobenzidine staining and immunogold labeling: a novel technical approach to identify lysozyme in human neutrophils Eur. J. Cell Biol. 49,319-325[Medline]
  23. Miyauchi, J., Sasadaira, H., Watanabe, K., Watanabe, Y. (1985) Ultrastructural immunocytochemical localization of lysozyme in human monocytes and macrophages Cell Tissue Res. 242,269-277[Medline]
  24. Mosmann, T. R., Coffman, R. L. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties Annu. Rev. Immunol. 7,145-173[CrossRef][Medline]
  25. Romagnani, S. (1992) Induction of TH1 and TH2 responses: a key role for the ‘natural’ immune response? Immunol. Today 13,379-381[CrossRef][Medline]
  26. Jennings, C. D., Foon, K. A. (1997) Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy Blood 90,2863-2892[Free Full Text]
  27. Altin, J. G., Sloan, E. K. (1997) The role of CD45 and CD45-associated molecules in T cell activation Immunol. Cell Biol. 75,430-445[Medline]
  28. Kawano, S., Gombart, A. F., Hisatake, Y., Verbeek, W., Lekstrom-Himes, J., Xanthopoulos, K. G., Koeffler, H. P. (1999) TCR-mediated proliferation of T-cells is severely impaired in C/EBP{varepsilon} knockout mice Blood 94,370a(abstract).
  29. Bretz, J. D., Williams, S. C., Baer, M., Johnson, P. F., Schwartz, R. C. (1994) C/EBP-related protein 2 confers lipopolysaccharide-inducible expression of interleukin 6 and monocyte chemoattractant protein 1 to a lymphoblastic cell line Proc. Natl. Acad. Sci. USA 91,7306-7310[Abstract/Free Full Text]
  30. Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., Kishimoto, T. (1990) A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family EMBO J. 9,1897-1906[Medline]
  31. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E. A., Appella, E., Oppenheim, J. J., Leonard, E. J. (1987) Purification of a human monocyte-derived neutrophil chemotactic factor that has sequence similarity to other host defense cytokines Proc. Natl. Acad. Sci. USA 84,9233-9237[Abstract/Free Full Text]
  32. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E., Kung, H. F., Leonard, E. J., Oppenheim, J. J. (1988) Molecular cloning of a human monocyte-derived neutrophil chemotactic (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor J. Exp. Med. 167,1883-1893[Abstract/Free Full Text]
  33. Ward, S. G., Bacon, K., Westwick, J. (1998) Chemokines and T lymphocytes: more than an attraction Immunity 9,1-11[CrossRef][Medline]
  34. Sallusto, F., Lanzavecchia, A., Mackay, C. R. (1998) Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses Immunol. Today 19,568-574[CrossRef][Medline]
  35. Yasumoto, K., Okamoto, S., Mukaida, N., Murakami, S., Mai, M., Matsushima, K. (1992) Tumor necrosis factor {alpha} and interferon {gamma} synergisticallyinduce interleukin 8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-{kappa}B-like binding sites of the interleukin 8 gene J. Biol. Chem. 267,22506-22511[Abstract/Free Full Text]
  36. Mukaida, N., Mahe, Y., Matsushima, K. (1990) Cooperative interaction of nuclear factor-{kappa}B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines J. Biol. Chem. 265,21128-21133[Abstract/Free Full Text]
  37. Wu, G. D., Lai, E. J., Huang, N., Wen, X. (1997) Oct-1 and CCAAT/enhancer-binding protein (C/EBP) bind to overlapping elements within the interleukin-8 promoter J. Biol. Chem. 272,2396-2403[Abstract/Free Full Text]
  38. Liu, Y. W., Tseng, H. P., Chen, L. C., Chen, B. K., Chang, W. C. (2003) Functional cooperation of simian virus 40 promoter factor and CCAAT/enhancer-binding protein ß and {delta} in lipopolysaccharide-induced gene activation of IL-10 in mouse macrophages J. Immunol. 171,821-828[Abstract/Free Full Text]



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