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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3683-3693
By
From Services d'Hématologie Biologique et Clinique,
Hôpital Lariboisière, Paris, France; Service
d'Immunologie-Hématologie, Hôpital Raymond Poincaré,
Paris, France; INSERM U. 474, Hôpital Henri Mondor,
Créteil, INTS, Paris, France; Haemophilia Centre, St Thomas'
Hospital, London, UK; Service d'Immuno-Hématologie Clinique,
Hôpital Saint Louis, Paris, France; and Service
d'Hématologie Biologique, Hôpital Necker-Enfants Malades,
Paris, France.
Erythroblastic synartesis is a rare form of acquired
dyserythropoiesis, first described by Breton-Gorius et al in 1973. This syndrome is characterized by the presence of septate-like membrane junctions and "glove finger" invaginations between erythroblasts, which are very tightly linked together. This phenomenon, responsible for ineffective erythropoiesis, leads to an isolated severe anemia with
reticulocytopenia. In the following report, we describe 3 new cases of
erythroblastic synartesis associated with dysimmunity and monoclonal
gammapathy. In all cases, the diagnosis was suggested by characteristic
morphological appearance of bone marrow smears, and further confirmed
by electron microscopy. Ultrastructural examination of abnormal
erythroblast clusters showed that these cells were closely approximated
with characteristic intercellular membrane junctions. The pathogenesis
of the dyserythropoiesis was modeled in vitro using crossed
erythroblast cultures and immunoelectron microscopy: when cultured in
the presence of autologous serum, the erythroblasts from the patients
displayed synartesis, whereas these disappeared when cultured in normal
serum. Moreover, synartesis of normal erythroblasts were induced by the
patient IgG fraction. Immunogold labeling showed that the monoclonal
IgG were detected in, and restricted to, the synartesis. A discrete
monoclonal plasmacytosis was also found in the patient bone marrow. The
adhesion receptor CD36 appeared to be concentrated in the junctions,
suggesting that it might be involved in the synartesis. These
experiments indicated that a monoclonal serum immunoglobulin (IgG in
the present cases) directed at erythroblast membrane antigen was
responsible for the erythroblast abnormalities. Specific therapy of the
underlying lymphoproliferation was followed by complete remission of
the anemia in these cases.
HEMATOPOIETIC CELLS, unlike many other
tissues that develop specialized intercellular junctions, remain loose
and do not normally generate cytoplasmic interconnections with adjacent cells. In normal bone marrow, erythroid precursors are often observed to cluster around macrophages, forming the erythroblastic island: A
privileged relationship exists between these three cell types, in which
one cell nurtures the other. This phenomenon, in which an erythroblast
takes up ferritin from the macrophage, has been termed rhopheocytosis.
However, in areas of intimate association between a macrophage and
erythroblasts, or between 2 erythroblasts, no morphologic evidence of
surface specialization has been noted, and only ferritin has been seen
in the interstitium.1,2
Acquired cases of dyserythropoiesis may have multiple causes and the
erythroid lineage may exhibit varied morphological features on bone
marrow examination. However, alterations at the sites of contacting
membranes have rarely been reported. Abnormal linkages between
erythroblasts have been observed in hypersiderotic marrows, and a
common feature was that ferritin deposition was consistently located
along the pathological junctions.3 Some specific
morphological abnormalities were described in 1973 in a patient with an
acquired dyserythropoietic anemia, where erythroblasts were tightly
linked together.4 In that case, electron microscopy showed
that these junctions were formed by interdigitating cell processes and
that they were linked by regularly spaced septake-like structures that were devoid of ferritin deposition. This observation led to the description of a new syndrome called "erythroblastic synartesis," from the Greek words " Patients
Case 1.
A 24-year-old woman was admitted to hospital in September 1973 with
severe aplastic, slightly microcytic anemia. The blood count showed a
hemoglobin level of 6 g/dL, with normal white blood cell (WBC) and
platelet counts and a normal serum iron level; the reticulocyte count
was reduced. The patient had splenomegaly and small diffuse
lymphadenopathy, and a lymphoproliferative disease was discovered
(small lymphocytic lymphoma). Unfortunately, no record of serum
electrophoresis and immunoelectrophoresis were found in this patient's
file. There was no lymphocytosis in the peripheral blood; direct Coombs
test was positive for complement. The red blood cell (RBC) aplasia did
not respond to initial therapy with chlorambucil. The hemoglobin level
steadily decreased (3 g/dL within 1 month) and the patient continued to
be RBC transfusion dependent.
Case 2.
A 70-year-old woman was admitted to hospital in November 1992 because
of severe and progressive anemia. Nine years previously the patient had
been hospitalized for Sjögren's syndrome. Investigations on that
occasion had showed a low complement C4 level and strongly positive
antinuclear antibodies. Testing for the lupus anticoagulant and serum
monoclonal immunoglobulin was negative and there had been no evidence
of hemolysis. On this admission, the hemoglobin level was 5.5 g/dL with
a reticulocyte count of 20.109/L. RBC indices confirmed a
normocytic and normochromic anemia. Examination of the blood smears
showed normal WBC and differential counts, and a normal platelet count.
Serum iron was 35 mmol/L and serum ferritin 175 mg/L. Serum
lactate-dehydrogenase was 667 (normal <330), bilirubin was 9.3 mg/dL,
and haptoglobins were markedly reduced. The direct Coombs test was
positive for complement. The lupus anticoagulant was present.
Anti-cardiolipin antibody tests were also positive at 53 U/mL. Serum
immuno-electrophoresis showed a monoclonal kappa IgG paraprotein at a
concentration of 3 g/L. Oral corticosteroid therapy was started. This
treatment resulted in progressive disappearance of the bone marrow
erythroid dysplasia and to the disappearance of the anemia. However,
the monoclonal immunoglobulin remained unchanged. When withdrawal of
corticosteroid treatment was attempted, the anemia relapsed. Thus, the
patient continues to receive corticosteroid therapy and maintains a
normal hemoglobin level.
Case 3.
In July 1995, a 46-year-old woman was referred because of
lymphadenopathy and lymphocytosis. WBC count was 9,200 × 106/L with 38% polymorphonuclear cells and 58%
lymphocytes. The hemoglobin level was 13.2 g/dL. The platelet count was
317 × 109/L. Morphology and immunophenotyping were
consistent with the diagnosis of chronic lymphocytic leukemia.
Hypogammaglobulinemia was present (4.8 g/L) associated
with a monoclonal IgG kappa gammapathy (1.7 g/L). By February 1996, the
tumoral syndrome had progressed. The WBC was 18.7 × 109/L
with 76% lymphocytes and the hemoglobin level had decreased to 7.9 g/dL. The direct Coombs test was negative. The patient was started on
low-dose CHOP (chlorambucil, vincristine, and
prednisone) chemotherapy regimen. Because of the occurrence of
Streptococcus pneumoniae pneumonia she also
received prophylactic intravenous immunoglobulins for 6 months. In July
1997, after 6 cycles followed by 1 year of chlorambucil therapy, she
was in clinical remission. The WBC was 4.5 × 109/L with
72% neutrophils and 16% lymphocytes and the hemoglobin level was 12.7 g/dL. The monoclonal IgG kappa was still present (2 g/L). Therapy was
stopped. In December 1997, she relapsed with lymphadenopathy,
splenomegaly, and severe anemia. The hemoglobin level was 6.9 g/L, the
reticulocyte count was 42 × 109/L, the
lactico-deshydrogenase (LDH) level was 265 IU/L
(N < 190), and the direct Coombs test was negative. The monoclonal
IgG kappa level was stable (2 g/L). Polymerase chain reaction (PCR)
analysis for Parvovirus B19 was negative in both bone marrow and blood. Fludarabine therapy was associated with transient incomplete remission and the patient died in October 1998 with septic cellulitis.
Cell Preparations
Bone marrow.
Bone marrow from the patients was aspirated from the sternum and the
smears were prepared with Romanovsky staining. Control human bone
marrow was harvested from normal adult donors during hip surgery after
obtaining informed consent and in accordance with the institutional
guidelines of the Committee on Human Investigation.
Isolation of a Purified IgG Fraction
Erythroblast Cultures
Electron Microscopy Aspirated bone marrow samples and cultured cells were directly fixed on 2.5% glutaraldehyde in phosphate buffer 0.1 mol/L, pH 7.4 for 1 hour at 22°C, then washed 3 times in phosphate buffer. Alternatively, tannic acid was added into the fixative to enhance electron density in the extracellular space.Flow Cytometry The fixation of normal and pathological IgG on erythroblast and erythrocyte membranes was tested by flow cytometry (FACSalibur; Becton Dickinson, Mountain View, CA). Mean fluorescence intensity (MFI) was used to measure antibody binding.Western Blot Proteins of erythroblast stroma were separated by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate and lithium dodecyl sulfate and transferred onto nitrocellulose sheets, then incubated with purified IgG of a normal subject and of the 2 patients (nos. 2 and 3) after the technique described by Towbin et al.7 An anti-CD36 immune serum raised in the rabbit was used as a positive control. The reaction was detected with ECL detection kit (Amersham, Little Chalfont, UK).
Bone Marrow Morphology Light microscopy.
Bone marrow aspirates from the 3 patients showed marked
hypercellularity and erythroid hyperplasia (>70% for case nos. 1 and 2). Bone marrow smear from patient 3 showed diffuse lymphocytic infiltration (86%) and numerous erythroid islands (Fig
1). The main abnormalities observed in the bone marrow
smears of the 3 patients were the presence of numerous erythroid
islands, with aggregated erythroblasts and frequent binuclear forms.
Most often, adjacent cells shared the appearance of the same maturation
stage, but some cells of apparently different maturity were also in
close contact. A nonbasophilic clear area was observed at sites of
close proximity between adjacent erythroblasts. This feature allowed a
distinction to be made between this type of erythroid island and that
of sideroblastic anemia. No macrophages were found in contact with
these erythroblast clusters.
Electron microscopy.
Electron microscopy confirmed the diagnosis of erythroblastic
synartesis in the 3 patients. Plasma membranes of adjacent
erythroblasts were joined by closely interdigitating processes (Fig
2). Cytochemical demonstration of the peroxidatic
activity of hemoglobin (not shown) allowed identification of maturation
stages of the erythroblasts, because its level of intensity relates to
the hemoglobin content of each cell. This technique showed that
erythroblasts of the same maturation stage, as well as those of
different maturation stages, were linked. Ribosomes were absent at
sites of linkage via interdigitating process, corresponding with the
nonbasophilic areas observed by light microscopy (Fig
3a). At the junction sites, coated pits and
rhopheocytosis vesicles were absent, probably because a macrophage
expansion would find it impossible to penetrate this zone of tightly
linked plasma membrane. Ferritin granules were never observed within
these junctions. Junctions between erythroblasts had a characteristic
morphology: resembling gap junctions, they were formed by 2 closely
placed membranes joined by periodical structures every 150 Å, giving
rise to a zipper-like appearance (Fig 3b). Isolated normoblasts often
displayed picnotic nuclei and/or binuclearity. The 2 external leaflets
of the plasma membrane of adjacent erythroblasts were separated by a
constant space of 145 Å in patient 1, and 180 Å in patients 2 and 3.
In Vitro Experiments The erythroblastic synartesis could be reproduced in vitro: indeed, erythroblast cultures established with bone marrow progenitors from cases 2 and 3 displayed similar morphological abnormalities to those observed in vivo (Fig 4a, see page 3686 ). Crossed culture experiments using the patient erythroblasts grown in the presence of their own serum or of a control serum, or using erythroblasts of a control subject grown in the presence of the patient sera (see Table 1) were performed and gave the following results: the erythroblast abnormalities reproduced in culture in the presence of autologous serum were absent when the culture was performed with a control AB serum (Fig 4b). Electron microscopy confirmed these findings, showing that the same intercellular junctions were observed when the patient erythroblastic progenitors were cultured with autologous serum, but that they were absent when the culture was performed in the presence of a control serum. These abnormalities were not reproduced in vitro when the erythroblasts were grown in the patient sera that had been IgG depleted. This allowed us to conclude that the synartesis was caused by an IgG component. Further confirmation was given by the observation that cultured normal erythroblasts displayed authentic synartesis when grown in the presence of patient to serum. Finally, the IgG fractions from the patients were added to normal serum at a concentration of 2 g/L and were able to induce typical synartesis on control erythroblasts grown in this preparation (Fig 5).
Flow Cytometry The results of the immunofluorescence test are indicated in Table 2. Purified IgG of patient no. 2 was detected on the surface of erythroblasts (MFI: 52) whereas the binding of the same IgG preparation on erythrocytes was weak (MFI: 2.5). Background fluorescence measured after incubation with IgG purified from a normal serum shows an MFI of 2.2 whatever the tested bone marrow cells were.
Immuno-electron Microscopy (Table 3) A positive immunogold labeling for immunoglobulin gamma chains was specifically detected along the synartesis, indicating that immunoglobulin could be the responsible agent for the synartesis. Moreover, the observation of a positive reaction only for the gamma chains and not for the mu chains suggested that the immunoglobulin located within the synartesis was an IgG. Finally, immunolabeling for the immunoglobulin light chains kappa and lambda showed that only kappa chains were detected in the synartesis, whereas lambda chain labeling was consistently negative. This observation was evidence for the monoclonal nature of the IgG. Knowing that both patients had a serum monoclonal IgG kappa, the IgG detected in a synartesis seemed to be identical to the one in their serum.
Western Blot Analysis
This report describes 3 new cases of erythroblastic synartesis, whose
diagnosis was made possible by morphological examination of bone marrow
erythroblasts by light and electron microscopy. Very few cases of this
disease have been described in the literature. This report presents a
detailed clinical and morphological description of the disease. It is
important that erythroblastic synartesis, which is probably an
under-recognized disease, is not misdiagnosed as refractory anemia or
as a malignant erythroblastic leukemia since a specific therapeutical
approach should be undertaken. This report also elucidates the
pathophysiology of the formation of abnormal junctions between the
erythroblasts, and shows that this is due to serum IgG, and most
probably to the monoclonal component IgG kappa, ie, a monoclonal
immunoglobulin. Finally, successful therapeutic interventions are
derived from these findings.
The authors acknowledge Dr Jean-Philippe Rosa for helpful discussions;
Prof. Henri Rochant for useful clinical informations concerning patient
1; Dr Paul-Henri Roméo for constant support; Josette Guichard,
Martine Debbia, Pierre Gane, and Viviane Bony for their precious
technical assistance, and Dorothée Ménage for secretarial help.
Submitted January 4, 1999; accepted July 22, 1999.
Supported by "Association pour la Recherche sur le Cancer" (ARC),
and "Fondation pour la Recherche Médicale."
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 Elisabeth M. Cramer, MD, PhD, INSERM U.474,
Hôpital Henri Mondor 94010 Créteil, France; e-mail:
emcramer{at}im3.inserm.fr.
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TOP
ABSTRACT
INTRODUCTION
" (with) and
"
entire or IgG depleted, or normal serum
containing the patient's IgG (2 g/L). Cultures were set up in presence
of recombinant human (rhu)-erythropoietin (Janssen-Cilag,
Issy-les-Moulineaux, France) 2 U/mL. Dishes were
incubated at 37°C in air atmosphere supplemented with 5%
CO2 and saturated with humidity. Erythroid colony
burst-forming units erythroid (BFU-E) were harvested at day 14-15. The
methyl cellulose was dissolved in an excess of culture medium. The
cells were either cytospun or fixed for electron microscopy.
