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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 4026-4027
CORRESPONDENCE
Evidence for Continuous Basal Generation of Gc-MAF: Absence in
Infantile Osteopetrosis and Restoration After Bone Marrow Transplant
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LETTER |
To the Editor:
Recent studies by Yamamoto et al1 and Benis and
Schneider2 in Blood suggest that Gc-globulin (also
known as vitamin D-binding protein) may have an important role in
macrophage activation and osteoclast differentiation from monocytes and
so may control bone morphogenesis and remodeling.1-5
Deglycosylation of Gc-globulin (removal of galactose and sialic acid
from the trisaccharide leaving N-acetyl-galactosamine [GalNAc])
produces a potent macrophage-activating factor (Gc-MAF).1
In osteopetrotic rat and mice models4,5 and in a single
human study,1 indirect data suggest a defect in
lysophospholipid-inducible Gc-MAF, although direct estimation of this
factor in normal and disease state has never been performed.
In this report, we extend earlier observations by showing a basal level
of Gc-MAF generation in normal healthy human subjects, its absence in a
patient with infantile autosomal recessive osteopetrosis (IOP), and
supression in IOP carriers. The patient, a female infant of related
(second cousin) South Asian parents, presented with seizures on the
eighth day of life. Investigation showed severe hypocalcemia (plasma
calcium, 1.25 mmol/L; albumin 36 g/L), with elevated parathyroid
hormone level (208 pg/L reference, 10 to 54 pg/mL). Bone marrow
aspirate and biopsy were consistent with a diagnosis of osteopetrosis.
The extended family was highly consanguineous, with several
marriages between cousins, and a history of osteopetrosis in
two distant cousins. A skeletal survey demonstrated uniformly dense
bones with associated metaphyseal lucent bands in keeping with
osteopetrosis. The father was a genotypical HLA match, and the patient
underwent a conditioned unmanipulated bone marrow transplant at the age
of 10 months. By day 28 post bone marrow transplant, she had an
increasing lymphocyte count with serum Ca2+ 2.29 mmol/L,
phosphate 1.27 mmol/L, and albumin 36 g/L. Molecular DNA studies
confirmed engrafting.
The measurement of exposed N-acetyl-galactosamine (GalNac) component of
Gc-MAF was performed by lectin-based immunoassay using a combination of
antibody monospecific to Gc-globulin protein (Diasorin, Berkshire, UK),
and Helix pomatia lectin conjugated to horseradish peroxidase
(HRP) (Sigma, Dorset, UK), which specifically binds to
GalNac. The specificity and reproducibility of the assay used was
confirmed using appropriate positive and negative controls.
The rates of generation of the factor for the patient, her parents, and
for male control subjects (n = 9) are shown in Fig 1. All control subjects showed basal
noninduced generation of Gc-MAF when lymphocytes were incubated with
the plasma Gc-globulin ( OD620 range, 84 to 180;
mean ± SD, 95 ± 18). The patient showed complete lack of
generation of Gc-MAF pretransplantation, despite prolonged incubation
of lymphocytes and plasma, lasting 16 hours. After
transplantation, Gc-MAF generation was shown. The patient's parents showed only 60% to 80% of the Gc-MAF generation of controls, findings in keeping with this recessive trait. The generation of
activating factor was constant over the first 8 hours; thereafter, there was slight decline in the rate of production. The plasma Gc-globulin concentrations in the control subjects (range, 25 to 47;
mean ± SD, 35 ± 5.3 mg/100 mL), the patient (37 mg/100 mL), and
the carriers (24 and 27 mg/100 mL for mother and father, respectively)
were quite similar. All levels were found to be within the reference
range (male, 24 to 47 mg/100 mL and female 20 to 47 mg/100 mL).
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| Fig 1.
Noninducible basal Gc-MAF generation from plasma
Gc-globulin by lymphocytes in male control subjects, osteopetrotic
patient, and her parents. The patient showed complete lack of
generation of Gc-MAF before transplantation, but after transplantation
Gc-MAF generation was evident. ( ), Controls; ( ), father; ( ),
mother; ( ), op/op; ( ), op/op (RX).
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Previous studies had assumed that Gc-MAF generation and, therefore, the
enzymes involved in its production are only induced as part of
an inflammatory response.4,5 In these investigations only lysophospholipid-inducible enzymes (B-lymphocyte
 -galactosidase and T-lymphocyte sialidase) and Gc-MAF
generation were measured and inducible Gc-MAF generation was thought to
be defective. The results of these studies suggest that lymphocytes
play an important role in modulating osteoclast activity. This is not
altogether surprising because osteoclasts are members of the
macrophage/monocyte lineage. The modulation of macrophage function by T
lymphocytes is well recognized, and a defect in T lymphocytes is
responsible for uncontrolled macrophage proliferation in Griscelli and
Chediak-Hegashi syndromes.6 Our own experience has shown
that osteopetrotic patients have a surprisingly higher incidence of pre
and post bone marrow transplant viral infections than other patients
(unpublished observations, March 1997). Further studies of
T- and B-cell function in IOP are needed as well as work on the precise
contribution of Gc-MAF in the activation of other cells of
macrophage/monocyte and osteoclast lineages. It is also of note that
infants born with absent T cells (T-negative severe combined
immunodeficiency) do not show features of osteopetrosis, so other cell
lineages must also generate Gc-MAF. These observations imply that
Gc-MAF may have a hitherto unrecognized role in normal bone
morphogenesis and remodeling. A recent unpublished observation suggests
that lymphocytes may have a prominent role in osteoclastogenesis from monocytes (R.M. Kanan and H.K. Datta, February 1999). The
putative role of lymphocytes in osteoclast activation and
differentiation may in fact be mediated by Gc-MAF acting in concert
with recently described cytokine TRANCE/RANKL (tumor necrosis
factor-related activated-induced cytokine).7 The results
of this study and other investigations in animal models suggest that
the phenotypic manifestation of osteopetrosis may arise from defective
Gc-MAF generation. Infusion of Gc-MAF into affected individuals may
mitigate the life-threatening effect of IOP.
Harish K. Datta
David B. Cook
Raed M. Kanan
Department of Clinical
Biochemistry
The Medical School
Newcastle upon Tyne, UK
Andrew R. Gennery
Andrew J. Cant
Department of
Paediatrics
Newcastle General Hospital
Newcastle upon Tyne,
UK
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REFERENCES |
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Yamamoto N, Naraparaju VR, Orchard PJ:
Defective lymphocyte glycosidases in the macrophage activation cascade of juvenile osteopetrosis.
Blood
88:1473, 1996[Abstract/Free Full Text]
2.
Benis KA, Schneider GB:
The effects of vitamin D binding protein-macrophage activating factor and colony stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats.
Blood
88:2898, 1996[Abstract/Free Full Text]
3.
Schneider GB, Benis KA, Flay NW, Ireland RA, Popoff SN:
Effects of vitamin D binding protein-macrophage activating factor (DBP-MAF) infusion on bone resorption in two osteopetrotic mutations.
Bone
16:657, 1995[Medline]
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Yamamoto N, Naraparaju VR:
A defect in inducible -galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse.
Immunology
88:604, 1996[Medline]
[Order article via Infotrieve]
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Yamamoto N, Lindsay DD, Naraparaju VR, Ireland RA, Popoff SN:
A defect in the inflammation-primed macrophage activation cascade in osteopetrotic rat.
J Immunol
152:5100, 1994[Abstract]
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Faigle W, Raposo G, Tenza D, Pinet V, Vogt AB, Kropshofer H, Fischer A, de Saint-Basile G, Amigorena S:
Deficient peptide loading and MHC class II endosomal sorting in a human genetic immunodeficiency disease: the Chediak-Higashi syndrome.
J Cell Biol
141:1121, 1998[Abstract/Free Full Text]
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Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T:
Osteoclast differentiation factor is ligand for osteoprotegerin/osteoclastogenesis inhibitory factor and is identical to TRANCE/RANKL.
Proc Natl Acad Sci USA
95:3597, 1998[Abstract/Free Full Text]
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