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Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 320-327
PHAGOCYTES
From the Departments of Medicine, Pediatrics, and Pathology,
University of Washington School of Medicine, Seattle, WA.
Myelokathexis is a congenital disorder that causes severe chronic
leukopenia and neutropenia. Characteristic findings include degenerative changes and hypersegmentation of mature neutrophils and
hyperplasia of bone marrow myeloid cells. The associated neutropenia can be partially corrected by treatment with granulocyte
colony-stimulating factor (G-CSF) or granulocyte-macrophage
colony-stimulating factor (GM-CSF). These features led us to propose
that accelerated apoptosis of neutrophil precursors might account for
the neutropenic phenotype. Blood and bone marrow aspirates were
obtained from 4 patients (2 unrelated families) with myelokathexis
before G-CSF therapy and from 2 of the affected persons after
G-CSF therapy (1 µg/kg per day subcutaneously for 3 weeks). Bone
marrow was fractionated using immunomagnetic bead cell sorting into
CD34+, CD33+/CD34
Myelokathexis is a rare cause of severe
chronic neutropenia characterized by degenerative changes and
hypersegmentation in mature neutrophils, first described by Zuelzer in
1964.1 Subsequently, additional congenital and acquired
cases have been reported.2-13 Affected persons have
recurrent bacterial infections attributed to neutropenia and to
depressed functional activity of their neutrophils.5,8 The
pathophysiology underlying myelokathexis has been attributed to
prolonged retention of neutrophils in the bone marrow
compartment.1,2,14 Administration of either granulocyte
colony-stimulating factor (G-CSF) or granulocyte-macrophage
colony-stimulating factor (GM-CSF) to persons with myelokathexis
reportedly increases the number of neutrophils in circulation and leads
to clinical improvement during episodes of bacterial
infection.8,10-12
Tissue homeostasis during development and the host immune
response are regulated by apoptosis, or programmed cell
death.15,16 The apoptotic pathway involves a series of
sequential morphologic and biochemical changes in affected cells,
including early membrane blebbing (zeiosis) and redistribution of
phospholipids in the plasma membrane, followed later by cytoplasmic
shrinkage, chromatin condensation, and, ultimately, internucleosomal
DNA fragmentation. Senescence cells are then removed by resident
scavenger phagocytes.15-17
It is now clear that the apoptotic program in a particular cell or
tissue can be regulated by either pro-apoptotic factors or
anti-apoptotic factors.18,19 Recent evidence indicates that the Fas (APO-1; CD95)/Fas-ligand system is an important cellular pathway regulating the apoptotic program in diverse
tissues,20-22 including the "professional" bone
marrow-derived phagocytes.17,23-26 Among anti-apoptotic
factors, the protein products of the protooncogenes bcl-2 and
bcl-x have received considerable attention.27-31
Based originally on morphologic examination of neutrophils and their precursors, we hypothesized that accelerated apoptosis of myeloid progenitor cells may account for the lack of peripheral neutrophils in
myelokathexis. Therefore, we examined spontaneous apoptosis of myeloid
progenitor cells from patients with myelokathexis and analyzed the
expression pattern of genes implicated in apoptotic cell death in these
respective cell populations. These studies showed that the accelerated
apoptosis of bone marrow myeloid progenitors and the aberrant
expression of bcl-x are central features of this disorder.
Furthermore, these abnormalities can be abrogated in vivo by treatment
with G-CSF.
Clinical histories of patients with myelokathexis
Patient 1 (family 1).
This 40-year-old woman was recognized as severely leukopenic and
neutropenic when she was admitted to the hospital at age 5 with
pneumonia. From early childhood through adulthood, numerous white blood
cell counts of <1.0 × 109/L have been documented,
with absolute neutrophil counts of 0.1 to
0.3 × 109/L and normal hemoglobin levels,
hematocrits, and platelet counts. Her clinical history is remarkable
for gingivitis and episodic cutaneous and sinopulmonary infections.
Examination of bone marrow aspirates obtained from childhood through
adulthood has revealed a hypercellular marrow with marked granulocytic
hyperplasia. Bone marrow neutrophils in these aspirates contain
hypersegmented nuclei with highly condensed chromatin. The nuclear
lobes are often separated by long, thin strands of chromatin,
consistent with the diagnosis of myelokathexis (Figure
1). Many reticuloendothelial cells within the bone marrow contain basophilic material. Cytogenetic examination results on multiple occasions have been normal. At age 12, she had mild
hypogammaglobulinemia (IgG, 525 mg/dL). The neutropenia was attributed
to the ineffective production of neutrophils, based on a quantitative
bone marrow biopsy and ferrokinetic studies performed in 1971.
Patient 2 (family 1).
This 20-year-old son of patient 1 was recognized to have neutrophil
counts of 0.1 to 0.5 × 109/L and
hypogammaglobulinemia early in childhood. Like his mother, he had
recurrent infections, including otitis media and otitis externa, severe
chicken pox, gingivitis, and an episode of pneumonia. When he was 4 to
8 years of age, he was treated with intramuscular pooled IgG
injections, which were discontinued because of allergic reactions.
Cellulitis has often developed after relatively minor cuts or
scratches, but serious infections have been infrequent during early
adulthood. Hematologic data have been identical to those of his mother,
including granulocytic hyperplasia in bone marrow aspirates and normal
cytogenetics. Hematologic evaluation results of all other immediate
family members were normal.
Patient 3 (family 2).
This 20-year-old woman has experienced recurrent ear and sinopulmonary
infections since early childhood. At 2 years of age, she was found to
have a white blood cell count of 0.3 to 0.7 × 109/L
and to be severely neutropenic. In addition to recurrent bacterial infections, multiple warts have developed, particularly on her hands.
The diagnosis of myelokathexis was based on the presence of
granulocytic hyperplasia in bone marrow aspirates and on the observation of extremely pyknotic nuclei and vacuolated cytoplasms in
both blood and bone marrow neutrophils. Hematocrit and platelet counts
have been normal. Treatment with G-CSF at 3 µg/kg per day subcutaneously for 3 days increased blood neutrophils from
0.1 × 109/L to 0.5 × 109/L, but
therapy was discontinued at the patient's request.
Patient 4 (family 2).
This 12-month-old daughter of patient 3 had severe leukopenia and
neutropenia from birth. She has experienced recurrent sinopulmonary infections. The morphology of her neutrophils is identical to that of
her mother. Because of recurrent infections she was treated with G-CSF
(3 µg/kg per day), which increased the blood neutrophil count from
0.05 × 109/L to 1.0 × 109/L.
G-CSF treatment was associated with the development of
thrombocytopenia, which necessitated discontinuation of G-CSF. No other
affected family members have been identified.
Blood and bone marrow samples
Preparation of purified neutrophils
Purification of bone marrow progenitor cells The monocytoid cell fraction was isolated by modification of previously described methods.33 Bone marrow mononuclear cells from patients and healthy donors were fractionated into CD34+ early progenitors, CD33+/CD34
myeloid progenitors, and
CD15+/CD33-/CD34 bone marrow
granulocyte precursor subpopulations using specific monoclonal
antibodies and immunomagnetic beads (Miltenyi, Auburn, CA) according to
the manufacturer's recommendations. Purity of each bone marrow
hematopoietic subpopulation was >96% as tested by FACS analysis.
Culture conditions for bone marrow and neutrophils Apoptosis assays. Apoptosis of bone marrow cells and peripheral blood neutrophils was assessed by 2 methods, analysis of apoptotic (hypodiploid) nuclei by flow cytometry and annexin-V binding. For analysis of apoptotic nuclei, 5 × 106 phosphate-buffered saline (PBS)-washed neutrophils were gently resuspended in 0.5 mL hypotonic fluorochrome solution 50 µg/mL propidium iodide in 0.1% sodium citrate and 0.1% Triton X-100 and analyzed by flow cytometry. Propidium iodide fluorescence of individual nuclei was filtered through a 585/42-nm band-pass filter and measured on a logarithmic scale by a FACScan cytometer (Becton Dickinson, Mountain View, CA) while gating on physical parameters to exclude cell debris. Data were analyzed using CellFIT Cell-Cycle Analysis software (Becton Dickinson). At least 10,000 events per sample were counted. Results are reported as the percentage of cells with hypodiploid nuclei, which reflects the relative proportion of apoptotic cells.34-36 Annexin-V binding to neutrophils and bone marrow progenitor cells was performed using an apoptosis detection kit (R&D Systems, Minneapolis, MN). Briefly, 5 to 20 × 104 freshly isolated cells or cells stored overnight in RPMI (BioWhittaker, Walkersville, MD) in the presence of 10% autologous serum were labeled with annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide for 20 minutes at room temperature, washed twice, and analyzed by 2-color flow cytometry using CellQuest Analysis software (Becton Dickinson). Results are reported as a percentage of annexin-V-positive cells in early and late stages of apoptosis. Colony-forming assays Purified bone marrow CD34+ cells (3000 per plate) were plated in soft agar (Difco, Detroit, MI) in Iscove's modified Dulbecco's medium (GIBCO BRL, Grand Island, NY) containing 5 × 10-5 mol/L 2 -mercaptoethanol (Sigma) and
penicillin-streptomycin, supplemented with human recombinant
hematopoietic growth factor mix (20 ng/mL SCF, 50 ng/mL Flt3 ligand, 10 ng/mL IL-3, 10 ng/mL G-CSF, and 10 ng/mL GM-CSF) in triplicate 1-mL
plates at 3 × 103 cells/plate as
described.37 The plates were incubated at 37°C in a
humidified atmosphere containing 5% CO2. On day 15, the
resultant colonies were evaluated based on morphology, density, and
number of cells, and they were grouped into CFU-high proliferative
potential primitive progenitor cells (>1000 cells/colony), early
myeloid progenitors CFU-GM (>100 cells/colony), and late myeloid
precursor CFU-GM clusters (<50 cells/colony). The results are
presented as a percentage of primitive, early, and late myeloid
compartments in the bone marrow, representing the mean number of
morphologically distinct colonies in triplicate plates.
Electron and light microscopy Bone marrow samples were fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for 2 hours, washed in the same buffer, and postfixed for 4 hours at room temperature in 2% osmium tetroxide in distilled water to which a few drops of 2% aqueous potassium-ferrocyanide were added. The tissue and cells were rinsed in distilled water, then block-stained with 0.5% aqueous uranyl acetate for 20 minutes and again rinsed in distilled water. Cells were embedded in 2% agar in 0.1 mol/L sodium cacodylate buffer. The tip of the agar blocks containing the cell pellet was cut off, dehydrated in a graded series of ethanol, and embedded in Eponate 12 resin (Ted Pella, Redding, CA). Samples of bone marrow tissue were directly dehydrated and embedded in resin without agar embedding. Thin sections were cut with a diamond knife on an LKB Nova ultramicrotome (LKB, Bromma, Sweden) and collected on parlodion-coated 200 mesh copper grids (Ted Pella, Redding, CA). Sections were stained with uranyl acetate and lead citrate and examined with a JEOL JEM 100B electron microscope (JEOL, Tokyo, Japan).Immunohistochemical staining for bcl-2 and bcl-x Immunohistochemical staining of CD34+, CD33+/CD34, and
CD34/CD33 cell populations from
patients with myelokathexis and healthy control volunteers was
performed on cytocentrifuge preparations. Cells were fixed in 100%
ethanol at 4°C for 10 minutes. Specimens were blocked with 5%
normal goat serum diluted in Tris-buffered saline, pH 8, for 20 minutes
at room temperature. The hamster-antihuman bcl-2 monoclonal
antibody 6C8 or a hamster monoclonal antibody of irrelevant specificity
TN3 19.12 (as a control IgG), adjusted to equal concentrations, was
incubated with the specimens, followed by biotinylated goat-antihamster
IgG (United States Biochemical, Medford Lakes, NJ) at a 1:40 dilution
and ABC alkaline phosphatase reagent (Vector Laboratories, Burlingame,
CA). Alternatively, rabbit polyclonal antihuman bcl-x antibody
(gift of Dr. Craig Thompson, University of Chicago, Chicago,
IL)38 or dilution buffer was incubated with the specimens,
followed by biotinylated goat-antirabbit IgG (Vector Laboratories) at a
dilution of 1:40 and ABC alkaline phosphatase reagent. All incubations
were performed at 4°C for 1 hour. The primary and secondary
antibodies were diluted in Tris-buffered saline supplemented with 5%
normal goat serum. Staining was developed using bromochlorindoyl
phosphate and nitroblue tetrazolium substrate. Specimens were
counterstained using 0.1% acridine orange and 0.1% safranin O. The
RL7 cell line was used as a positive control for bcl-2
expression.39 Unseparated normal mononuclear bone marrow
cells were used as a positive control for bcl-x expression.
Immunofluorescence flow cytometry for detection of Fas expression Cell surface expression of Fas on purified bone marrow precursor cells was assayed by direct immunofluorescence flow cytometry using saturating concentrations of FITC-conjugated Fas-specific UB2 monoclonal antibody. In brief, 106 freshly isolated cells were incubated in the presence of UB2-FITC monoclonal antibody for 45 minutes at 4°C, washed once with PBS containing 0.1% sodium azide, then fixed with 1% paraformaldehyde in PBS. Simultaneous negative control staining reactions were performed with a saturating concentration of irrelevant murine IgG1-FITC in place of UB2-FITC. The plates were kept at 4°C until the stained cells were analyzed by flow cytometry using a FACScan and BDIS Consort software (Becton Dickinson).Immunofluorescence flow cytometry for detection of FasL expression Cell surface expression of FasL on cells of interest was assayed by indirect immunofluorescence flow cytometry using Fas-Ig for primary staining and FITC-conjugated affinity-purified F(ab')2 goat-antihuman IgG for secondary staining. In brief, primary staining was performed with 106 cells incubated for 45 minutes on ice in the presence of Fas-Ig (20 µg/mL) in PBS containing 10% normal human serum. The cells were washed once with PBS containing 0.1% sodium azide and 0.1% bovine serum albumin, and a secondary staining was performed using FITC-conjugated affinity purified F(ab')2 goat-antihuman IgG (10 µg/mL) in PBS containing 10% goat serum. After a single PBS wash, the cells were fixed with 1% paraformaldehyde in PBS. Simultaneous negative control staining reactions were performed by omitting Fas-Ig from the primary staining step. In separate neutrophil assays, B7-Ig was also substituted for Fas-Ig as a negative control. The plates were kept at 4°C until the stained cells were analyzed by flow cytometry. Mean fluorescence intensity was calculated by subtraction of the mean fluorescence channel of the appropriate negative control.
Survival characteristics of peripheral blood neutrophils and bone marrow hematopoietic progenitors from patients with myelokathexis To investigate whether the neutropenia in myelokathexis is caused by accelerated apoptosis, we first compared the viability and rate of apoptotic cell death of peripheral blood neutrophils and CD15+ bone marrow neutrophil precursors in vitro from patient 2 and a healthy control. No substantial differences in viability or in the level of apoptosis were present in freshly isolated neutrophils and in CD15+ cells from the myelokathexis patient and the healthy volunteer (Table 1). However, examination of the cell populations after overnight storage revealed a substantially larger proportion of cells undergoing apoptosis in neutrophils and in CD15+/CD34/CD33 cells
from the patient with myelokathexis compared with the healthy volunteer. On overnight storage approximately 55% to 60% of
peripheral blood neutrophils from the patient with myelokathexis
developed features of apoptosis compared to only 10% to 20% of
neutrophils from the healthy donor. Similarly, approximately 90% of
bone marrow CD15+ cells from the patient with myelokathexis
were apoptotic after overnight storage. In contrast, only 20% of the
comparable cells from the healthy volunteer underwent apoptosis during
this time period.
Characteristics of bone marrow progenitor cells in myelokathexis
Electron microscopy of bone marrow cells in myelokathexis
Expression of pro-apoptotic and anti-apoptotic factors in bone
marrow precursor cells in myelokathexis
Effect of G-CSF treatment
Effect of G-CSF treatment on survival characteristics of neutrophils
and bone marrow-derived cell populations
Severe neutropenia is a well-recognized risk factor for the
development of severe bacterial and fungal infections.41
Patients with severe chronic neutropenia experience recurrent
infections usually caused by surface organisms of the oropharynx, skin,
and gastrointestinal tract. These infections are often associated with
substantial morbidity. Several forms of severe chronic neutropenia have
been described, including congenital, cyclic, autoimmune, idiopathic
neutropenia, and myelokathexis.42,43
The authors thank Drs M. Kang, C. Hatlestad, A. Kattamis, J. Kwiatkowski, M. Wener, G. Segal, and M. Brouns, and they thank Audrey Anna Boyard of the Severe Chronic Neutropenia International Registry for the referral of patients with myelokathexis. They also
thank Linda Slane for help with manuscript preparation.
Submitted June 8, 1999; accepted September 2, 1999.
Supported by grants from the National Institutes of Health
(2RO1-DK-18951) and Amgen, Inc. (Thousand Oaks, CA).
Reprints: David C. Dale, Department of Medicine, Box 356422, University of Washington, Seattle, WA 98195-6422; e-mail: dcd{at}u.washington.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
