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Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1797-1802
Neutrophil Antibody Specificity in Different Types of
Childhood Autoimmune Neutropenia
By
Marrie C.A. Bruin,
Albert E.G.Kr von dem Borne,
Rienk Y.J. Tamminga,
Marion Kleijer,
Lica Buddelmeijer, and
Masja de Haas
From the Department of Hematology of the University Hospital for
Children and Youth `The Wilhelmina Childrens Hospital,' Utrecht; CLB,
Sanguin Blood Supply Foundation, and Laboratory for Experimental and
Clinical Immunology, Academic Medical Centre, University of Amsterdam,
Amsterdam; the Department of Hematology, Academic Medical Centre,
University of Amsterdam, Amsterdam; and the Department of Pediatrics,
University Hospital Groningen, Groningen, The Netherlands.
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ABSTRACT |
Autoimmune neutropenia (AIN) in children can be divided into 2 forms. In primary AIN, neutropenia is the sole abnormality, and
although neutrophil counts are generally below 500 µL 1, mild bacterial infections occur. Primary AIN is
mostly seen in young children and shows a self-limited course. AIN
occurring in association with autoimmune diseases (secondary AIN) often shows more severe infectious complications. We analyzed clinical and
serological data from 28 pediatric patients with AIN to evaluate whether there is a possible relationship between specificity of the
neutrophil autoantibodies and the clinical course of the disease. Specificity of the circulating antibodies was determined with the
indirect granulocyte immunofluorescence test (GIFT) and a panel of
phenotyped donor neutrophils. The samples were further analyzed in the
monoclonal antibody immobilization of granulocyte antigens assay
(MAIGA) for neutrophil antigen (NA)1, NA2, CD11a, and
CD11b specificity. With the indirect GIFT, an antibody specificity was
deduced in 26 of the 28 analyzed samples. In all but 3 sera from
patients with primary AIN, NA1-(76%) or NA2-(10%) specific antibodies
were detected with the indirect GIFT. In 2 samples, the reactivity in
the indirect GIFT was too weak to draw conclusions, but the MAIGA
showed NA1 and/or NA2 specificity of the antibodies. One serum, from a
patient with primary AIN with a persistent neutropenia for more than 6 years, contained NA1, possibly pan-Fc RIIIb, and CD11a antibodies. In
4 sera from patients with primary AIN, weak antibodies with CD11a or
CD11b specificity were detected with the MAIGA. Sera from 7 patients
with secondary AIN contained in all cases antibodies with
pan-FcRIIIb specificity, as deduced from the indirect GIFT results
and absorbance/elution experiments performed with 2 sera. The MAIGA
confirmed this for only 1 of the 5 tested sera. Furthermore, CD11a
antibodies were detected in 1 of the 5 tested sera. In conclusion, our
results indicate that primary AIN is usually associated with
NA-specific antibodies, whereas secondary AIN seems to be associated
with pan-FcRIIIb antibodies. Thus, characterization of the
antibodies in sera from children with AIN discriminates patients with
primary AIN from those with secondary AIN.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
CHRONIC NEUTROPENIA is defined by an
absolute neutrophil count (ANC) below 1,500 cells per µL of blood,
lasting for at least 6 months. In 1975, Lalezari et al1
demonstrated that chronic neutropenia can be caused by autoantibodies.
Autoimmune neutropenia (AIN) is divided into primary and secondary
forms.2 In primary AIN, neutropenia is the sole hematologic
abnormality. Primary AIN is the most common form of neutropenia in
young children, generally diagnosed at the age of about 8 months and
disappearing around the age of 3 years. In case of secondary AIN,
neutropenia is present in association with another autoimmune disorder
or a malignant lymphoproliferative disease. Secondary AIN can occur at
any age and the clinical course is variable. Previous studies showed
that in patients with primary AIN, the neutrophil autoantibodies are
frequently directed against 1 of the alloforms of the neutrophil antigen (NA) system and, in particular, against the NA1
alloform.1-5 The specificity of autoantibodies in children
with secondary AIN has not yet been studied in detail in a large number
of patients.6
The NA antigens are located on the IgG-Fc receptor type IIIb
(FcRIIIb; CD16), which is exclusively expressed by
neutrophils.7 The NA phenotype frequency in the white
population is as follows: 11% of the population is NA(1+,
2), 45% is NA(1,
2+), and 43% is NA(1+,
2+).7 Recently, a new polymorphism of the
NA system was found to encode the so-called SH antigen, a genetic
variant involved in neonatal alloimmune neutropenia, which is expressed
by approximately 4% of the white population.8,9
We investigated the neutrophil-autoantibody specificity in 21 children
with primary AIN and in 7 children with secondary AIN. In patients with
self-limited primary AIN, the autoantibodies were in all cases directed
against 1 of the NA alloforms, whereas in patients with secondary AIN,
we found only antibodies with pan-FcRIIIb specificity.
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MATERIALS AND METHODS |
Patients.
Serum and/or EDTA-anticoagulated blood was sent to our laboratory for
diagnostic evaluation of neutropenia. Informed consent and clinical
information were obtained via the attending pediatrician. Chronic
neutropenia was defined as an absolute neutrophil count (ANC) below
1,500 neutrophils/µL of blood present for at least 6 months.
In 1992, a diagnostic procedure based on the diagnostic procedure of
the International Severe Chronic Neutropenia Registry was introduced in
The Netherlands for pediatric patients with chronic neutropenia. This
procedure starts with a 6-week period in which peripheral blood
leukocyte and neutrophil counts are measured twice weekly. When
persistent neutropenia is diagnosed and no spontaneous recovery occurs
within 3 months, a more extensive procedure is advised, including
immunologic tests, eg, immunoglobulin profile and screening for
autoantibodies, screening for metabolic diseases and viral infections,
bone marrow aspiration, neutrophil mobilization tests, and measurement
of leukocytes and ANC in the blood from the parents of the patients. In
patients with severe bacterial infections and/or clinical features of a
certain syndrome, the diagnostic procedure is accelerated and extended
to avoid delay in appropriate treatment.
The pediatric patients (n = 43) for whom the blood was sent to our
laboratory for detection of autoantibodies against neutrophils formed
the basis of the study, and only patients with positive neutrophil-autoantibody tests were included (n = 28).
Monoclonal antibodies (MoAabs) and human antisera.
Anti-pan-FcRIIIb MoAbs (CD16) used were: CLBFcRgran1, 3G8 (Medarex,
West Lebanon, NH), BW209/2, and MEM154. BW209/2 was a generous gift
from Dr R. Kurrle (Behring Werke, Marburg, Germany) and
MEM154 was a kind gift from Dr V. Horejsi (Prague, Czech Republic). CLBFcRgran1 (CD16), TB133 (CD11a), W6/32 (anti-HLA class I), and irrelevant murine MoAbs were obtained from our institute, the Central
Laboratory of the Netherlands Red Cross Blood Transfusion Service
(CLB), Amsterdam, The Netherlands. S131 (CD11b), IB4 (CD18), and J3D3
(CD35) were obtained via the Fourth and Fifth Workshop on Human
Leukocyte Differentiation Antigens.
NA1-, NA2-, NB1-, CD11a-, CD11b- and I-reactive human antisera were
obtained either from women immunized during pregnancies or from
patients immunized by blood transfusions. Sera from healthy volunteers
(AB positive) were used as controls. Fluorescein isothiocyanate (FITC)-labeled F(ab')2 fragments of goat antimurine
Ig and FITC-labeled goat anti-human Ig were from the CLB.
Isolation of the cells.
Fresh anticoagulated blood from volunteers or patients was centrifuged
over a Ficoll-Hypaque layer (Pharmacia Fine Chemicals AB, Uppsala,
Sweden) with a specific gravity of 1.076 g/mL. Mononuclear cells were
harvested from the interphase, and the pellet was treated with ice-cold
NH4Cl solution (155 mmol/L NH4Cl, 10 mmol/L
KHCO3, 0.1 mmol/L EDTA, pH 7.4) to lyse the erythrocytes.
The remaining cells were more than 95% neutrophils.
Detection of neutrophil antibodies in the direct and indirect
granulocyte immunofluorescence test (GIFT).
The isolated neutrophils were fixed with 1% (wt/vol) paraformaldehyde
(PFA). In the direct GIFT, the patients' neutrophils were incubated
with FITC-labeled F(ab') fragments of goat anti-human Ig to
detect neutrophil-bound antibodies. In all cases, the patients' sera
was tested with the indirect GIFT for circulating neutrophil antibodies
with a panel of phenotyped donor neutrophils
(Table 1).10,11 In the indirect
GIFT, donor neutrophils were incubated with serum for 30 minutes at
room temperature (RT). After washing with
phosphate-buffered saline (PBS) containing 0.2% (wt/vol), bovine serum
albumin (BSA), the neutrophils were stained with FITC-labeled
F(ab')2 fragments of goat anti-human Ig. Binding of
the antibodies was measured with a FACScan (Becton Dickinson, San Jose,
CA).
Detection of neutrophil antibodies with the MoAb-specific
immobilization of granulocyte antigens (MAIGA).
Sera were tested in the MAIGA as described previously.11
Briefly, 1 × 106 neutrophils expressing either NA1 or
NA2, or FcRIIIb-negative neutrophils were incubated with 50 µL of
serum for 30 minutes at 37°C. The cells were washed with PBS/BSA,
10 µL of diluted MoAb was added, and the incubation was continued for
30 minutes at 37°C. MoAb 3G8 and BW209/2 were used if NA1-positive
neutrophils were tested and MoAbs 3G8, MEM154, and BW209/2 were used in
assays with NA2-positive neutrophils. TB133 (CD11a), S131 (CD11b), IB4 (CD18), and J3D3 (CD35) were only used in assays with
FcRIIIb-negative neutrophils. The neutrophils were washed 3 times
with PBS/BSA and were lysed with 1% (vol/vol) Triton-X100-containing
buffer in the presence of protease inhibitors EDTA (5 mmol/L),
phenylmethylsulphonyl fluoride (PMSF, 2 mmol/L), and soybean-trypsin
inhibitor (200 ng/L) for 30 minutes at 4°C. After centrifugation of
the lysate for 30 minutes at 15,000g, 70 µL of the lysate was
diluted with 180 µL of Tris-buffered NaCl (0.9%, wt/vol) containing
1% Triton-X 100 (vol/vol), 0.05% (vol/vol) Tween-20, and 0.5 mmol/L
CaCl2. A total of 100 µL of the diluted lysate was added
to the wells of an enzyme-linked immunosorbent assay (ELISA) plate
(Maxisorp, Nunc, Roskilde, Denmark), coated with goat
anti-mouse IgG (3 µg/mL). Incubation was performed overnight at
4°C. The plates were washed, 100 µL of horseradish
peroxidase-labeled goat anti-human IgG was added, and the incubation
was continued for 2 hours. After washing, a substrate was added to
measure the amount of bound antibody. All tests were performed in
duplicate. In each assay, incubation of neutrophils with a serum
containing HLA antibodies followed by incubation with MoAb W6/32 served
as a control for the whole procedure. Sera with known NA1 and NA2
specificity and serum from an AB-positive control served as positive
and negative controls, respectively.
Evaluation of FcRIIIb or NA allospecificity.
To evaluate the presence of NA1-, NA2-, or pan-FcRIIIb-specific
antibodies in the sera, 150 µL of serum was incubated twice with 7.5 × 106 neutrophils from either NA1 or NA2 homozygous
positive donors for 45 minutes at RT. Absorbed antibodies were eluted
from the cells by incubation with 1 mol/L isotonic citrate buffer (pH
2.8) for 10 minutes at RT, and the pH of the harvested
eluates was neutralized by adding an equal volume of 1 mol/L isotonic
Tris-HCl (pH 10.6).12,13 The reactivity of
the absorbed sera and eluates was tested with NA1- and NA2-positive
neutrophils. NA1- and NA2-specific antisera and sera from AB-positive
donors served as controls. The elution procedure did not alter the
specificity of the control sera, indicating that no artificial immune
complexes were formed that could bind to the neutrophil FcRs.
Immunoprecipitation.
A total of 2 × 107 neutrophils of an
NA(1+, 2+) genotyped donor was radiolabeled
with 37 MBq 125iodide by the Iodogen method
according to the manufacturer's instructions (Pierce, Rockford, IL).
Labeled cells (2 × 106) were incubated with either 1 of the patient sera, control human serum, or CLBFcRgran1 MoAb for 1 hour at RT. The cells were washed and lysed in 0.5% (wt/vol) Nonidet
P-40 in the presence of protease inhibitors EDTA (5 mmol/L), PMSF (2 mmol/L), and soybean-trypsin inhibitor (200 ng/mL) for 30 minutes at
4°C. The lysed samples were centrifuged for 30 minutes at
12,000g. Subsequently, precipitation was performed with
protein-G-coated sepharose beads (Pharmacia). The immune precipitate
was subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under nonreducing conditions and to autoradiography.
| |
RESULTS |
Patient groups.
Blood samples from 43 pediatric patients were analyzed. These were
samples from patients with a chronic neutropenia, who were screened
according to a standard diagnostic procedure, as described in Materials
and Methods. Fifteen patients were excluded from our study. The sera of
these patients contained no detectable antibodies against neutrophils.
Four of these patients had neutropenia in association with infections,
lasting only 3 to 5 months. In 8 other patients, the following
diagnoses were made: Kostmann's disease (n = 1), familial neutropenia
(n = 1), cyclic neutropenia (n = 1), glycogen storage disease (n = 1),
lazy leukocyte syndrome (n = 1), combined immune deficiency
(n = 2), idiopathic neutropenia with limb malformations (n = 1). One
patient developed acute lymphoblastic leukemia after a 4-month period
of neutropenia. In 2 patients, no classifying diagnosis was made. The
antibody tests, performed with both GIFT and MAIGA, were negative. One
of these last 2 patients has a neutropenia persisting for more than 5 years now. In the other patient, neutropenia spontaneously resolved
after 13 months. Although this last patient was likely to have had AIN,
as a result of failure to detect antibodies, this patient was not
included in the study.
Twenty-eight patients were included in the study. Their clinical
characteristics are shown in Table 2. Based
on the clinical data, the patients were divided into 2 groups. The
first group consisted of 21 patients with the typical features of
primary AIN: neutropenia was the only clinical problem and was
diagnosed before the age of 1 year. Bacterial infections were mild and
usually affected the skin, the middle ear, the oropharynx, and the
upper respiratory tract. In 18 cases, the neutropenia resolved
spontaneously within 30 months (range, 16 to 52 months; mean, 28 months). Three patients in the primary AIN group showed persistent
neutropenia during the study period (Table 2). However, 2 of these
patients were diagnosed less than 2 years ago. One patient, who is now 6 years old, has a consistent neutrophil count of less than 500/µL without any clinical problems.
The second group consisted of 7 patients who all suffered from
secondary AIN (Table 2). The age of onset of the neutropenia was
variable, ranging from 6 months to 14 years. In 2 patients, neutropenia
was the presenting problem. One of these patients showed neutropenia at
the age of 6 months. Ten months later, immune-mediated thrombocytopenia
was diagnosed. In the other patient, neutropenia was diagnosed at the
age of 13 months followed by autoimmune hemolytic anemia 1 month
afterwards. The duration of neutropenia in these patients ranged from 1 to 6 years. Infections in this group were severe in 3 cases, and 1 patient also with combined immune deficiency died. Two patients, who
both suffered from severe skin and respiratory tract infections, were
treated with recombinant human granulocyte colony-stimulating factor
(rh-G-CSF) for more than 2 years. Subsequently, the
ANC normalized and no more bacterial infections occurred. In 3 other
patients, immune-modulating therapy, eg, corticosteroids or high-dose
intravenous immunoglobulin administration, were temporarily beneficial
with respect to the ANC. Withdrawal of therapy gave a relapse of the
neutropenia in 2 of these 3 patients, whereas 1 patient showed a normal
ANC during 1 year and was subsequently lost for follow-up. Finally, 1 of the patients, suffering from mild autoimmune hemolytic anemia and
immune thrombocytopenic purpura (ITP) (Evans
syndrome), received prophylaxis with antibiotics (cotrimoxazole) and
was clinically well.
Specificity of the neutrophil antibodies as determined with the
indirect GIFT.
Because of the neutropenia, the direct GIFT could only be performed in
4 patients, with a positive result in all cases. The presence of
circulating neutrophil antibodies was one of the inclusion criteria of
this study. The presence of HLA antibodies was excluded in all cases by
simultaneously testing the donor lymphocytes (data not shown). Table 2
shows the results of the antibody screening with a panel of phenotyped
donor granulocytes. In patients with primary AIN (n = 21), we found NA1
specificity in 16 (76%) and NA2 specificity in only 2 patients (10%).
In 3 cases, no specificity could be determined based on the results
with the panel, but they were directed against FcRIIIb epitopes
because the neutrophils of a FcRIIIb-negative donor were not stained
by these sera. The antibodies from 1 of these patients seemed to show
pan-FcRIIIb specificity in this test, although weaker binding to
homozygous NA2-positive neutrophils compared with homozygous
NA1-positive neutrophils was noted. In the other 2 patients, the
reactivity of the serum was too weak to draw conclusions. In 5 patients, serial antibody tests were performed. The antibody tests in
these patients were negative after 6 to 15 months of follow-up. In all of the patients, disappearance of the antibodies preceded the recovery
of the neutrophil counts by several months.
The sera from the patients with secondary AIN showed all pan-FcRIIIb
specificity. These sera were positive with the neutrophils from all
NA-positive donors and negative with the cells of the FcRIIIb-negative donor.
Reactivity of patients sera in the MAIGA with CD16, CD11a, and CD11b
MoAbs.
To confirm our results obtained with the screening in the indirect
GIFT, we performed the MAIGA. All available sera (n = 23) were tested
in duplicate with homozygous NA1-positive and homozygous NA2-positive
neutrophils (Table 3). Thirteen sera with
NA1 antibodies in the GIFT were tested. Confirmation of NA1 specificity
was obtained in 3 cases, whereas 6 sera showed negative results (Table
3). Four sera showed variable reactions, dependent on the MoAb used in
the MAIGA (see Table 3). The 2 sera with NA2-reactive antibodies in the
GIFT showed positive results in the MAIGA with homozygous NA2-positive
neutrophils (Table 3). The 2 sera with too weak reactivity in the
indirect GIFT to draw conclusions on antibody specificity reacted
positively in the NA1-MAIGA, whereas 1 of these sera was also weakly
positive in the NA2-MAIGA (Table 3).
All of these sera were also tested in the MAIGA with CD11a and CD11b
MoAbs. Two sera containing NA1 antibodies showed weak reactivity in the
MAIGA with the CD11a MoAb, and 1 serum containing NA2 antibodies showed
weak reactivity in the MAIGA with the CD11a MoAb and strong reactivity
in that with the CD11b MoAb (data not shown).
With the MAIGA, the presence of pan-FcRIIIb-specific antibodies in
5 tested sera from patients with secondary AIN was confirmed in 1 case,
whereas 3 sera showed negative results. One serum showed only a
positive result in the MAIGA with BW209/2 and homozygous NA1-positive
cells; with 3G8 and NA2-positive cells, all results were negative. A
weak reaction in the MAIGA with the CD11a MoAb was obtained with the
serum in which pan-FcRIIIb antibodies were confirmed with the MAIGA.
One patient, diagnosed as having primary AIN, but with persistent
neutropenia after 6 years, showed in her serum pan-FcRIIIb
specificity of the antibodies concluded from the indirect GIFT. The
serum of this patient showed the presence of both NA1 and NA2
antibodies in the MAIGA (Table 3). Furthermore, very strong
reactivity in the MAIGA with the CD11a MoAb and weak reactivity in the
CD11b MAIGA were observed. To further characterize the neutrophil
antibodies present in this serum, immune precipitation was performed
with NA(1+, 2+) phenotyped neutrophils; only
NA1-FcRIIIb was precipitated (Fig 1). As
shown in Fig 1, lane 3, NA1-Fc RIIIb was precipitated by another
serum from a patient with primary AIN and NA1 antibodies, and in 4 of
the 9 additionally tested cases, NA1-FcRIIIb was precipitated (data
not shown). All immune precipitation experiments with sera from
patients with secondary AIN, containing antibodies with pan-FcRIIIb
specificity, showed negative results (Fig 1, lane 2, in total 5 sera
were tested, data not shown).
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| Fig 1.
Immune precipitation from 125I-labelled
NA(1+,2+)-phenotyped neutrophils by
neutrophil antisera and CD16 MoAbs. Lane 1, serum from the patient with
the clinical pattern of primary AIN, but with chronic neutropenia. This
serum reacted positively in the indirect GIFT and MAIGA with both
NA(1+, 2) and NA(1,
2+)-phenotyped neutrophils. Lane 2, serum from a patient
with secondary AIN with pan-FcRIIIb antibodies as shown by the
indirect GIFT. Lane 3, serum from a patient with primary AIN and NA1
antibodies, shown in the GIFT and the MAIGA. Lane 4, control serum
containing isoantibodies with pan-FcRIIIb specificity. Lane 5, MoAb
CLB-gran11 (NA1-FcRIIIb). Lane 6, MoAb CLB-FcRgran1
(pan-FcRIIIb).
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Confirmation of pan-FcRIIIb specificity by absorption/elution
tests.
To prove that the sera from patients with secondary AIN contained
antibodies with pan-FcRIIIb specificity, absorbance and elution
experiments were performed with 2 of these sera. We observed that both
homozygous NA1-positive and homozygous NA2-positive neutrophils
absorbed all reactivity: the twice incubated sera no longer reacted
with neutrophils of the same or the opposite NA phenotype
(Fig 2). Eluates prepared from
serum-incubated NA1-positive neutrophils reacted positively with fresh
homozygous NA1-positive and with homozygous NA2-positive neutrophils
and vice versa (Fig 2). The staining of homozygous NA1-positive
neutrophils was brighter compared with that of homozygous NA2-positive
neutrophils. This explains the relatively higher binding to
NA2-positive neutrophils of the eluate prepared from NA1-positive
neutrophils.
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| Fig 2.
Analysis of the presence of allospecific antibodies in
FcRIIIb-antisera from patients with secondary AIN. The left panel
shows the reactivity of the sera or eluates with homozygous
NA1-positive cells and the right panel with homozygous NA2-positive
cells. The mean of experiments with 2 different sera is depicted.
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| |
DISCUSSION |
In this study, we analyzed the neutrophil antibody specificity in sera
from 2 pediatric patient groups with autoimmune-mediated neutropenia.
The patients were selected by the criteria of a positive neutrophil
antibody test. We used the indirect GIFT with a panel of donor
granulocytes, including a FcRIIIb-negative donor for detection of
neutrophil antibodies. In the report of the Second International
Granulocyte Serology Workshop, Bux et al14 concluded that a
combination of the GIFT and GAT (granulocyte agglutination test) is the
best means of antibody detection. From the results of the Workshop and
from our own experience, it follows that the GAT is especially
necessary for detection of antibodies with 9a and 5b specificity. In
pediatric patients and especially in patients with AIN, 5b and 9a
antibodies have never been described. Therefore, we did not use the GAT
as a second test for screening antibodies in this patient group. The
sera of the pediatric patients with neutropenia of unknown origin were
all tested in the GIFT and in the MAIGA. From the literature, it is
known that children with primary AIN have, in up to 95% of the tested
cases, circulating antibodies directed against neutrophils. Specificity
of these antibodies could be detected in about one third of the cases. In the majority of these cases, antibodies showed NA specificity. The
specificity of the neutrophil antibodies in children with secondary AIN
has not previously been studied extensively. We detected a difference
in antibody specificity between patients with primary AIN and secondary
AIN. In serum from all children with secondary AIN (n = 7), antibodies
with pan-FcRIIIb specificity were detected, whereas in all sera from
patients with primary AIN (n = 21), antibodies with NA specificity were
found. The serum of 1 patient with the clinical picture of primary AIN,
but with chronic neutropenia by now lasting for 6 years, seemed to
contain both antibodies with NA1 specificity and with pan-FcRIIIb
specificity, as concluded from the screening with the indirect GIFT,
the MAIGA, and immune precipitation results. In addition to these
antibodies, in the MAIGA performed with neutrophils from a
FcRIIIb-negative donor, strongly reactive CD11a-specific antibodies
and weakly reactive CD11b antibodies were detected in this serum. Until
now, no other autoimmune or hematological problems have evolved in this
patient. The persistence of the neutropenia for more than 6 years, as
well as the different specificity of antibodies in the serum compared
with the sera of the other 20 patients with primary AIN, suggests a
different type of neutropenia than the classical primary AIN.
Overall, weak CD11a- or CD11b-specific antibodies were detected in 3 of
the 13 tested sera from other patients with primary AIN and in 1 of the
4 tested sera from patients with secondary AIN. In the 3 patients with
primary AIN, whose sera showed both NA-specific and CD11a or CD11b
antibodies, the neutropenia resolved. Bux et al found 21% of the
samples positive in a MAIGA with CD11b and CD18 MoAbs, which is similar
to the 28% (4 of 14 tested samples) we found. Hartman and
Wright15 described the presence of antibodies with
CD11b/CD18 specificity in 14% of 50 adult patients with immune neutropenia. These investigators used an immunobead assay for detection
of antibodies. Six patients with CD11b/CD18 antibodies in this study
had multiple autoimmune problems, and 5 of the 7 patients had recurrent
infections. Thus, CD11b/CD18 antibodies are detectable both in adult
and childhood populations with immune-mediated neutropenia.
In all but 3 cases of primary AIN (86%), we defined the NA specificity
of the neutrophil antibodies by screening of the sera with a panel of
phenotyped donor granulocytes. In a recent report on analysis of
antibody specificity in 240 cases of AIN, Bux et al5 found
with the same test NA specificity in only 34% of the cases, in all
other tested cases the serum reacted both with NA1-and with
NA2-positive neutrophils. Furthermore, these investigators observed
that NA1- and NA2-reactive antibodies were detected in the MAIGA in
sera from patients with primary AIN.5 We observed that not
in all cases all tested MoAbs showed a positive result. In general, the
positive signals obtained in the MAIGA with sera from patients with AIN
were much lower compared with those obtained with sera containing allo-
or isoantibodies (data not shown). In our opinion, screening of
patients' sera with a well-defined donor granulocyte panel is to be
preferred over testing of the sera in the MAIGA, although this latter
test can be helpful to further analyze antibody specificities present
in indirect-GIFT-negative samples.
The etiology of AIN is not clear. In primary AIN, the very early onset
of the disease, together with the self-limited course, is remarkable.
In secondary AIN, the existence of other autoimmune diseases is of
special interest. In their review, Shastri and Logue16
discussed the pathophysiology of immune neutropenia. Their discussion
is based on the 2 major pathways for immune dysregulation. In the first
pathway, antibodies to foreign antigens, eg, viruses or bacteria,
cross-react with self-antigens. The antibodies found in this situation
are supposed to be polyclonal, and in the case of a normally developed
immune system, this type of immune reaction is self-limited. Hence,
primary AIN may be explained by a mechanism of immunity due to
cross-reactivity. There are, however, questions that remain. The
antibodies are present during a relatively long period (months or
years). A possible explanation for this phenomenon, suggested by other
investigators,17 is that full development of the suppressor
T-cell function occurs during the first years of life. Apparently, the
NA1 antigen forms a risk factor contracting primary AIN. Parvovirus B19
has been believed to play a role in the
etiology,18 but this was not confirmed.5 The
second mechanism in the pathway of immune dysregulation involves loss
of suppression of a clone of cells that are able to react with
self-antigens. Little is known about the clonality of the antibodies in
AIN in children. In secondary AIN, the development of other autoimmune diseases suggests an etiology in which disturbance of the
self-tolerance plays an important, but until now, incompletely
understood role.
In conclusion, our data show that children with immune-mediated chronic
neutropenia and NA-specific antibodies in their sera suffer from
primary AIN and therefore have a benign, and in most cases,
self-limited disease. On the contrary, children with chronic neutropenia and antibodies with pan-FcRIIIb specificity have a risk
of developing other autoimmune diseases and therefore should be
followed in a different way . Especially in young children with AIN,
characterization of the antibodies can be helpful in differentiating
between primary and secondary AIN.
| |
ACKNOWLEDGMENT |
The authors acknowledge all pediatricians who sent material and data
for this study. We thank the Department of Leukocyte and Thrombocyte
Serology of the CLB for serologic screening and for their help with the MAIGA.
| |
FOOTNOTES |
Submitted September 29, 1998; accepted May 6, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Masja de Haas, MD, PhD,
Central Laboratory of The Netherlands Red Cross Blood Transfusion
Service, Plesmanlaan 125, 1066 CX Amsterdam, PO Box 9190, 1006 AD
Amsterdam, The Netherlands; e-mail: m_de_haas{at}clb.nl.
| |
REFERENCES |
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Chronic autoimmune neutropenia due to anti-NA1 antibody.
N Engl J Med
293:744, 1975[Abstract]
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Autoimmune neutropenia.
Semin Hematol
29:45, 1992[Medline]
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3.
Lalezari P, Manoocherehr K, Petrosova M:
Autoimmune neutropenia of infancy.
J Pediatr
109:764, 1986[Medline]
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Autoimmune neutropenia: Clinical and laboratory studies in 143 patients.
Ann Hematol
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Bux J, Behrens G, Jaeger G, Welte K:
Diagnosis and clinical course of autoimmune neutropenia in infancy: Analysis of 240 cases.
Blood
91:181, 1998[Abstract/Free Full Text]
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The use of rh-G-CSF in chronic autoimmune neutropenia: Reversal of autoimmune phenomena, a case history.
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Huizinga TWJ, Kleijer M, Tetteroo PA, Roos D, von dem Borne AEGK:
Biallelic neutrophil NA-antigen system is associated with a polymorphism on the phospho-inositol-linked Fc gamma receptor III (CD16).
Blood
75:213, 1990[Abstract/Free Full Text]
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Bux J, Stein EL, Bierling P, Fromont P, Clay ME, Stroncek DF, Santoso S:
Characterisation of a new alloantigen (SH) on the human neutrophils FcReceptor IIIb.
Blood
89:1027, 1997[Abstract/Free Full Text]
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Koene H, Kleijer M, Roos D, De Haas M, Von dem Borne AEG Kr:
Fc RIIIB gene duplication: Evidence for presence and expression of three distinct Fc genes in NA(1+,2+)SH(+) individuals.
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Detection of granulocyte allo-antibodies by an indicrect immunofluorescence test.
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Analysis of granulocyte-reactive antibodies using an immunoassay based upon monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA).
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Elution of granulocyte and platelet antibodies.
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Report on the Second International Granulocyte Serology Workshop.
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Identification of autoantibodies specific for the neutrophil adhesion glycoproteins CD11b/CD18 in patients with autoimmune neutropenia.
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Autoimmune neutropenia.
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Autoimmunization against the neutrophil-specific NA1 antigen is associated with HLA-DR2.
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McClain K, Estrov Z, Hong Chen, Mahoney DH:
Chronic neutropenia of childhood: Frequent association with parvovirus infection and correlations with bone marrow culture studies.
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ABSTRACT
INTRODUCTION