Blood outgrowth endothelial cells from cord blood and peripheral blood: angiogenesis-related characteristics in vitro.

BACKGROUND: Blood outgrowth endothelial cells (BOEC) are good candidates for vascular (re-) generating cell therapy. Although cord blood (CB) BOEC have been reported as more proliferative than peripheral blood (PB) BOEC, not much is known about their functional properties. OBJECTIVES: We have studied the following determinants in BOEC expanded from CB and PB: endothelial phenotype, in vitro adhesion, migration, proliferation, and angiogenic tube forming capacity. METHODS/RESULTS: Endothelial phenotype of BOEC was evaluated by fluorescence activated cell sorting (FACS) analysis and confirmed the presence of endothelial markers including CD31, CD105, CD144, CD146, KDR/VEGFR-2, Tie-2, and TNF-alpha-induced VCAM-1 and ICAM-1. Evaluation of cell proliferation revealed a higher basal proliferation of CB-BOEC, which increased after exposure to bFGF but not VEGF. The lower basal proliferation of PB-BOEC increased with VEGF or bFGF addition. Array analysis of angiogenic genes showed many comparable expressions in both BOEC, and a slightly more pronounced pro-angiogenic profile in CB-BOEC than PB-BOEC. Both BOEC were able to form tubular structures in a three-dimensional fibrin matrix. Tube formation in CB-BOEC was markedly induced by TNF-alpha only and inhibited by anti-urokinase antibodies. It was comparable to that induced by combined addition of TNF-alpha and VEGF or bFGF, while maximal tube formation in PB-BOEC required simultaneous exposure to TNF-alpha/VEGF or TNF-alpha/bFGF. CONCLUSIONS: The endothelial phenotype and characteristics for homing, adhesion, migration, inflammation, and angiogenic tube formation are almost equal for BOEC from CB and PB. A slightly more angiogenic phenotype favors CB-BOEC. However, addition of VEGF to PB-BOEC induces equal proliferation and tube formation.

Neutrophil antibody specificity in different types of childhood autoimmune neutropenia.

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 microL(-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-FcgammaRIIIb, 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-FcgammaRIIIb 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-FcgammaRIIIb antibodies. Thus, characterization of the antibodies in sera from children with AIN discriminates patients with primary AIN from those with secondary AIN.

Actin polymerization induces shedding of FcgammaRIIIb (CD16) from human neutrophils.

FcgammaRIIIb (CD16) is a glycosyl phosphatidylinositol (GPI)-anchored low-affinity IgG receptor, exclusively expressed on human neutrophils. FcgammaRIIIb associates with complement receptor 3 (CR3, Mac-1, CD11b/CD18), which may indirectly link FcgammaRIIIb to the actin cytoskeleton. Upon neutrophil activation, apoptosis, or chemotaxis, FcgammaRIIIb is shed from the cell surface. In all of these events, actin rearrangements play an important role. To establish a role for the actin cytoskeleton in the control of FcgammaRIIIb shedding, we treated human neutrophils with jasplakinolide, an actin-polymerizing peptide. We show that enhanced actin polymerization induces time- and dose-dependent shedding of FcgammaRIIIb. This effect was not restricted to FcgammaRIIIb, because the cell surface expression of CD43, CD44, and L-selectin was also downregulated after induction of actin polymerization. This actin-dependent pathway is staurosporine sensitive but does not appear to involve activation of PKC or CR3. These data show that the actin cytoskeleton can regulate protein ectodomain shedding from human neutrophils.

The Fc gammaRIIIA-158F allele is a risk factor for systemic lupus erythematosus.

OBJECTIVE: To study whether the Fc gammaRIIIA-158V/F polymorphism, which affects IgG binding affinity, is a risk factor for systemic lupus erythematosus (SLE). METHODS: We genotyped a group of 70 Caucasian SLE patients for all known Fc gammaR polymorphisms. Of this group, 45 patients (64%) had nephritis. In 35 patients, this diagnosis was confirmed by renal biopsy. RESULTS: In the total group of 70 SLE patients, the frequency of the Fc gammaRIIIA-158F allele was 0.74, versus 0.57 in healthy controls (P = 0.003). The genotype distribution of the Fc gammaRIIIA-158V/F polymorphism was also significantly different from that of the control population (P = 0.004). The distribution of the other Fc gammaR polymorphisms--Fc gammaRIIA-131R/H, Fc gammaRIIIB-NA(1,2), and Fc gammaRIIIA-48L/R/H--was similar in SLE patients and controls. CONCLUSION: In our group of SLE patients, only the distribution of the alleles of the Fc gammaRIIIA-158V/F polymorphism was significantly different from that in the control group. This might indicate that macrophage expression of the Fc gammaRIIIA-158F isoform is involved in the disturbed clearance of immune complexes in patients with SLE.

Clinical value of soluble IgG Fc receptor type III in plasma from patients with chronic idiopathic neutropenia.

Previous studies have shown that the plasma level of soluble IgG Fc receptor type III (sFcgammaRIII) is a measure of the total body neutrophil mass. The aim of this study was to determine whether the plasma level sFcgammaRIII is associated with the risk of contracting bacterial infections in patients with neutropenia. We collected blood from 66 patients suffering from acquired idiopathic neutropenia, whose blood was sent to our laboratory for diagnostic evaluation of neutropenia (neutrophil count <1,500 cells/microL). Soluble FcgammaRIII levels were measured in plasma. Genotype distibutions of FcgammaR polymorphisms were determined. Clinical data were obtained from the patient files. Patients were assessed as to whether or not they had suffered from a bacterial infection 3 months before to 3 months after a single sFcgammaRIII measurement. In addition, longitudinal data were obtained from 21 patients. Of the 66 neutropenic patients who were included, 15 had suffered from a bacterial infection in the period 3 months before to 3 months after sFcgammaRIII measurement. The age and sex distribution was equal among the groups with and without infections, as were the genotype frequencies of neutrophil FcgammaR polymorphisms. Both neutrophil count and plasma level sFcgammaRIII were significantly lower in the patient group with infections, compared with the noninfected group (P = .03 and P < .0001, respectively). No infections were reported for patients who had plasma sFcgammaRIII levels above 100 arbitrary units (AU; normal value, 30 to 200). After matching each infected patient with two noninfected patients having the same neutrophil count, sFcgammaRIII plasma levels remained significantly lower in the group with infections (P = . 0001). For the patients who were followed in time, no infections were reported when sFcgammaRIII levels were above 100 AU. In conclusion, our population of patients with chronic idiopathic neutropenia with plasma sFcgammaRIII levels above 100 AU did not show an increased risk of contracting bacterial infections.

Fc gamma RIIIB gene duplication: evidence for presence and expression of three distinct Fc gamma RIIIB genes in NA(1+,2+)SH(+) individuals.

Recently, a new alloantigen on IgG Fc receptor type IIIb (Fc gamma RIIIb), SH, was described (Bux et al, Blood 89:1027, 1997). We identified three healthy individuals whose neutrophils reacted positively with the SH antiserum. The neutrophil antigen (NA) phenotype of all three donors was NA(1+,2+). Analysis of genomic DNA showed that the three donors were positive for the described SH-encoding mutation in the NA2-Fc gamma RIIIB gene, 266C-->A. However, NA(1,2) genotyping and nucleotide sequencing of an NA2-specific fragment amplified from the genomic DNA fragment showed that these individuals carried three Fc gamma RIIIB genes, namely, NA1-Fc gamma RIIIB, NA2-Fc gamma RIIIB, and SH-Fc gamma RIIIB, encoding NA1-Fc gamma RIIIb, NA2-Fc gamma RIIIb, and SH-Fc gamma RIIIb, respectively. Southern blot analysis confirmed these findings. Furthermore, all three transcripts were isolated from neutrophil mRNA. To investigate whether the presence of three Fc gamma RIIIB genes resulted in a higher membrane expression of Fc gamma RIIIb, we measured the reactivity of neutrophils from NA(1+,2+)SH(+) individuals with a panel of CD16 monoclonal antibodies (MoAbs) in comparison with neutrophils from NA(1+,2+)SH(-) controls. Reactivity of four different anti-pan-Fc gamma RIII MoAbs and NA2-specific MoAb GRM1 was higher with SH(+) neutrophils compared with controls, whereas that of NA1-specific MoAbs was similar, which is in concordance with the results from the genomic analysis. We observed that reactivity with NA2-specific CD16 MoAb PEN1 was sixfold higher in SH(+) individuals compared with controls. Apparently, the 60Ala-->Asp substitution in SH-Fc gamma RIIIb influences the epitope recognized by PEN1. In conclusion, we identified three NA(1+,2+)SH(+) individuals carrying three Fc gamma RIIIB genes and observed a clear gene-dosage effect on the level of expression of neutrophil Fc gamma RIIIb.

Fc gammaRIIIa-158V/F polymorphism influences the binding of IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype.

We analyzed a genetic polymorphism of Fc gamma receptor IIIa (CD16) that is present on position 158 (Phe or Val) in the membrane-proximal, IgG-binding domain. With a polymerase chain reaction-based allele-specific restriction analysis assay we genotyped 87 donors and found gene frequencies of 0.57 and 0.43 for Fc gammaRIIIA-158F and -158V, respectively. A clear linkage was observed between the Fc gammaRIIIA-158F and -48L genotypes on the one hand and the Fc gammaRIIIA-158V and -48H or -48R genotypes on the other hand (chi2 test; P < .001). To determine the functional consequences of this Fc gammaRIIIa-158V/F polymorphism, we performed IgG binding experiments with natural killer (NK) cells from genotyped donors. All donors were also typed for the recently described triallelic Fc gammaRIIIa-48L/R/H polymorphism. NK cells were treated with lactic acid to remove cell-associated IgG. Fc gammaRIIIa(NK)-158F bound significantly less IgG1, IgG3, and IgG4 than did Fc gammaRIIIa(NK)-158V, irrespective of the Fc gammaRIIIa-48 phenotype. Moreover, freshly isolated NK cells from Fc gammaRIIIa-158VV individuals carried significantly more cytophilic IgG than did NK cells from Fc gammaRIIIa-158FF individuals. In addition, CD16 monoclonal antibody (MoAb) MEM154 bound more strongly to Fc gammaRIIIa-158V, compared with -158F, again independently of the Fc gammaRIIIa-48 phenotype. The binding of MoAb B73.1 was not influenced by the Fc gammaRIIIa-158V/F polymorphism, but proved to depend solely on the amino acid present at position 48 of Fc gammaRIIIa. In conclusion, the previously reported differences in IgG binding among the three Fc gammaRIIIa-48L/R/H isoforms are a consequence of the linked, biallelic Fc gammaRIIIa-158V/F polymorphism at amino-acid position 158.

NA-phenotype-dependent differences in neutrophil Fc gamma RIIIb expression cause differences in plasma levels of soluble Fc gamma RIII.

Soluble Fc gamma RIII in plasma is primarily derived from neutrophils and is a measure of the total body neutrophil mass. We have developed a new, sensitive 'sandwich' ELISA to measure soluble Fc gamma RIII in plasma and released Fc gamma RIII in cell supernatants. Both sFc gamma RIIIa, derived from NK cells and sFc gamma RIIIb, derived from neutrophils are detected in the assay. However, plasma analysis of Fc gamma RIIIB gene-deficient donors suggested that sFc gamma RIIIa contributes only marginally to the total amount measured in healthy individuals. Furthermore, we observed that plasma of homozygous NA1-positive donors contained lower amounts of sFc gamma RIII than plasma of homozygous NA2-positive donors. Heterozygous donors were found to have intermediate levels of sFc gamma RIII in their plasma. Hemizygous Fc gamma RIIIB gene-deficient donors were found to have half the amount of sFc gamma RIII in their plasma compared to donors with two Fc gamma RIIIB alleles. These NA phenotype-dependent differences in plasma sFc gamma RIII could not be contributed to either an assay artefact or NA-dependent differences in shedding of Fc gamma RIIIb upon neutrophil activation. Calibration curves constructed with plasma of homozygous donors did nor reveal NA-dependent differences in antibody affinity. Measurement of released Fc gamma RIIIb in supernatants of neutrophils stimulated with PMA, and inhibition of this signal with human IgG revealed no NA-dependent differences. However, NA-dependent differences in neutrophil Fc gamma RIIIb expression were present, comparable to the differences found in plasma levels of sFc gamma RIII. Differences in the amounts of released Fc gamma RIII in supernatants of NA-typed apoptotic neutrophils were similar to initial differences in Fc gamma RIIIb expression, again being lower in NA1-positive than in heterozygous and NA2-positive donors. In conclusion, NA-dependent differences in plasma levels of soluble Fc gamma RIII seem to be caused by differences in expression of the receptor on the neutrophil membrane.

A triallelic Fc gamma receptor type IIIA polymorphism influences the binding of human IgG by NK cell Fc gamma RIIIa.

A donor-dependent difference in electrophoretic mobility of deglycosylated released Fc gamma RIIIa derived from NK cells and macrophages was observed. We investigated whether this was based on a polymorphism of the Fc gamma RIIIA gene. Cloning and sequencing of Fc gamma RIIIa-encoding cDNA derived from an apparently heterozygous donor showed one single nucleotide substitution at position 230 (T-->G), which was responsible for a leucine (L)-->arginine (R) substitution at position 48 in the first extracellular Ig-like domain (EC1) of Fc-gamma RIIIa and caused a higher electrophoretic mobility of Fc gamma RIIIa. An allele-specific primer annealing PCR assay was developed to amplify specifically an Fc gamma RIIIA gene-derived fragment, which was digested with AciI (recognizing G230) or MnlI (recognizing T230). MnlI restriction analysis revealed the presence of a third Fc gamma RIIIa allele with a T230-->A substitution, which predicts a change of 48-leucine into 48-histidine (H). A gene frequency of 86% for the T230 (48-L) allele, 6% for G230 (48-R), and 8% for A230 (48-H) was found. A significantly different genotype distribution was found among 12 unrelated Caucasian Fc gamma RIIIB gene-deficient donors. Fc gamma RIIIa-48R and Fc gamma RIIIa-48H showed a higher binding capacity of human (h)IgG1, hIgG3, and hIgG4 compared with Fc gamma RIIIa-48L. Finally, the CD16 mAbs 1D3 and MEM154 bound more strongly and Leu11c (B73.1) bound less to the newly identified Fc gamma RIIIa isoforms.

Abnormal neutrophil phenotype and neutrophil FcRIII deficiency corrected by bone marrow transplantation.

BACKGROUND: Neutrophils from a patient in first remission of acute myeloid leukemia were found to lack NA1 and NA2 alloantigens. This NA null phenotype was converted to the normal phenotype of NA1, NB2 by the transplantation of bone marrow from an HLA-identical sibling. To investigate the inherited or acquired nature of this rare phenotype, a combination of conventional neutrophil serology and recently developed restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) assays was used. STUDY DESIGN AND METHODS: Diagnosis, remission, and posttransplant patient peripheral blood samples were used for neutrophil phenotyping by granulocyte agglutination and immunofluorescence tests. The presence and dose of the gene for neutrophil Fc gamma RIIIb (Fc gamma RIIIB) were tested for with RFLP and Southern analysis and PCR-based RFLP tests. Plasma levels of circulating soluble Fc gamma RIII (sFc gamma RIII) were measured with radioimmunoassay. The sibling bone marrow donor and the patient's parents were also studied. RESULTS: RFLP analysis of DNA obtained from the patient at the time of diagnosis showed that she lacked the Fc gamma RIIIB gene for neutrophil Fc gamma RIII (i.e., Fc gamma RIIIb), but that, in DNA prepared from posttransplant samples, the Fc gamma RIIIB gene was present. Quantitation of plasma levels of soluble FcRIII (sFcRIII) demonstrated a complete absence of sFcRIII in the patient's pretransplant plasma. However, 20 units of sFcRIII were detected in the patient's plasma by 160 days after graft. Hair samples from the patient provided sufficient nonhematopoietic, genomic DNA to confirm that her genotype was NA0NA0. DNA prepared from lymphocytes of both parents and the sibling marrow donor was used to quantitate their Fc gamma RIIIB gene dose. The mother and brother had only one Fc gamma RIIIB gene each, while the father apparently had a normal complement of two Fc gamma RIIIB genes. CONCLUSION: In this case, an inherited absence of Fc gamma RIIIB gene in a patient with acute myeloid leukemia was unintentionally corrected by the transplantation of bone marrow from a sibling donor who himself carried only one Fc gamma RIIIB gene.

Neutrophil Fc gamma RIIIb deficiency, nature, and clinical consequences: a study of 21 individuals from 14 families.

Several individuals have been described whose neutrophils lack the normally abundantly expressed IgG Fc gamma receptor IIIb (Fc gamma RIIIb). We now studied the responsible genomic defect and analyzed the medical history in detail of 21 Fc gamma RIIIb-negative donors identified in 14 unrelated families. We developed a polymerase chain reaction allele-specific-primer annealing assay to genotype for the NA polymorphism of the Fc gamma RIIIB gene. All Fc gamma RIIIb-deficient individuals were negative for both the NA1 and the NA2 allele. In all cases the complete absence of the Fc gamma RIIIB alleles was confirmed using a Southern blot-based restriction fragment length polymorphism assay. Furthermore, an additional deletion of the next more telomeric located Fc gamma RIIC gene was found. Family studies showed that at least one Fc gamma RIIIB allele was absent in both parents in 6 families, whereas in 2 families the father had a normal phenotype. Two individuals suffered from an autoimmune thyroiditis. Four individuals had had multiple episodes of infection, 3 had only incidental infections, and 14 never had any serious infection. Genotyping showed a normal Fc gamma RIIa phenotype distribution among the Fc gamma RIIIb-negative individuals, thus excluding the possibility that the presence of the favorable IgG2-binding low-responder isoform of Fc gamma RIIa (131-H) contributed to the overall absence of recurrent bacterial infections.

Isoimmune neonatal neutropenia caused by Fc gamma RIIIb antibodies in a Spanish child.

BACKGROUND: Fc gamma RIIIb deficiency is a rare defect in which neutrophils do not express Fc gamma RIIIb and therefore the individuals with this defect have an NA null phenotype. Soluble Fc gamma RIII in plasma is severely decreased and almost undetectable. During pregnancy, Fc gamma RIIIb deficiency may cause the formation of maternal Fc gamma RIIIb antibodies, which leads to an isoimmune neonatal neutropenia. The first known case of isoimmune neonatal neutropenia caused by these antibodies in a Spanish child was identified. CASE REPORT: A newborn infant was severely affected by omphalitis; analysis of his blood showed an absolute neutropenia, but he responded well on intravenous immunoglobulin therapy. The maternal antiserum reacted strongly with all tested Fc gamma RIIIb-positive neutrophils. A family study showed that the infant's mother, one of the mother's sisters, and her mother were Fc gamma RIIIb deficient. No neutrophil antibodies were found in the plasma from these other Fc gamma RIIIb-negative women, although both had had numerous pregnancies. The three women were healthy, but one had recurrent otitis. DNA analysis of the family showed the absence of both Fc gamma RIIIB genes in the three Fc gamma RIIIb-negative women. The father of the child and all the children of the Fc gamma RIIIB gene-deficient women were shown to lack one of the Fc gamma RIIIB genes. CONCLUSION: A new case of isoimmune neonatal neutropenia caused by anti-Fc gamma RIIIb is identified. The family study indicates that the Fc gamma RIIIb deficiency is a hereditary genetic defect. In accordance with the location of Fc gamma RIIIB on chromosome 1, an autosomal pattern of inheritance of the Fc gamma RIIIB-deficient allele was observed.

Status epilepticus during vigabatrin treatment: a report of three cases.

Vigabatrin (gamma-vinyl-GABA or GVG) is an irreversible inhibitor of gamma-aminobutyric acid transaminase (GABA-T), which is an enzyme responsible for gamma-aminobutyric acid (GABA) catabolism. Inhibition of GABA catabolism increases brain concentration of GABA, a neural inhibitor. GVG has been found to be a potent new anti-epileptic drug, especially in the treatment of refractory epilepsy, in particular of complex partial seizures. Three patients who developed a severe status epilepitus while on GVG treatment are reported. A possible proconvulsive effect of GVG is hypothesized, which might result from disinhibition in the nigro-collicular pathway due to increased GABA-levels.

The plasma concentration of soluble Fc-gamma RIII is related to production of neutrophils.

Fc gamma RIII (the CD16-antigen), a low-affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes and macrophages. A soluble form of Fc gamma RIII has been identified in human plasma. This soluble form of Fc gamma RIII (sFc gamma RIII) originates from release by neutrophils. In the present study we show by transfusions of plasma that contains sFc gamma RIII of one allotype (NA1-Fc gamma RIII) in recipients homozygous for the other allotype (NA2-Fc gamma RIII) that the clearance of sFc gamma RIII is about 0.7 ml/min. Because the concentration of sFc gamma RIII was found to be constant in a small cohort of donors followed for about 1.5 years, the half-life of NA1-sFc gamma RIII is about 1.8 d, assuming a one-compartment model. The plasma concentration of sFc gamma RIII depended mainly on the production of neutrophils in the bone marrow, and was not influenced by shifts of neutrophils from one pool to another (storage, marginating or circulating pool). Because Fc gamma RIII is only expressed on mature neutrophils, this implies that the concentration of sFc gamma RIII depends on production of mature neutrophils.

Soluble Fc gamma RIIIa is present in plasma and is derived from natural killer cells.

Fc gamma RIII (CD16), a receptor for complexed IgG, is encoded by two very homologous genes: Fc gamma RIIIA and Fc gamma RIIIB. NK cells and macrophages express Fc gamma RIIIa, whereas only neutrophils constitutively express Fc gamma RIIIb. In a previous study we found that soluble (s)Fc gamma RIII in plasma seemed to originate only from neutrophils. However, CD16 mAb, directed against different epitopes of Fc gamma RIII, precipitated a glycoprotein from plasma of homozygous Fc gamma RIIIB gene-deficient donors. This glycoprotein migrated in a similar way as did released Fc gamma RIIIa derived from NK cells, whereas Fc gamma RIIIa released by cultured monocytes migrated differently and appeared to be more heavily glycosylated on SDS-PAGE. After deglycosylation, the M(r) of the plasma sFc gamma RIIIa was similar to that of released Fc gamma RIIIa. Moreover, V8-protease maps were identical. Therefore, we conclude that sFc gamma RIIIa is also present in plasma and is derived from NK cells. Because sFc gamma RIII levels are hardly detectable in the plasma of most homozygous Fc gamma RIIIB gene-deficient donors, we suspect that the sFc gamma RIIIa level is negligible compared with the level of sFc gamma RIIIb in plasma of healthy donors. Two patients with an NK cell lymphocytosis had a high plasma level of sFc gamma RIIIaNK. Furthermore, high levels of sFc gamma RIIIaNK were found in plasma of two patients with rheumatoid arthritis. Thus, the level of sFc gamma RIIIaNK might reflect either an increase in circulating NK cells or an enhanced release of Fc gamma RIIIaNK in certain diseases. This study shows that an assay that discriminates between sFc gamma RIIIa and sFc gamma RIIIb is necessary for the interpretation of sFc gamma RIII levels in patients.

Recombinant granulocyte colony-stimulating factor administration to healthy volunteers: induction of immunophenotypically and functionally altered neutrophils via an effect on myeloid progenitor cells.

We performed a detailed kinetic study on the in vivo effect of a single subcutaneous dose of granulocyte colony-stimulating factor (G-CSF; 300 micrograms) in four healthy individuals on the expression and function of neutrophil Fc gamma receptors (Fc gamma R). G-CSF did not induce Fc gamma RI (CD64) on circulating neutrophils. However, neutrophils newly formed in response to G-CSF were Fc gamma RI positive and were able to perform antibody-dependent cellular cytotoxicity in an Fc gamma RI-dependent way. Fc gamma RII (CD32) expression was not changed significantly. Fc gamma RIII (CD16, phosphatidylinositol-linked) expression, slightly increased immediately (30 minutes) postinjection, was found to be strongly decreased on the newly formed population. For comparison, we studied the expression of the PI-linked proteins leukocyte alkaline phosphatase (LAP) and CD14. Intracellular levels of LAP mirrored the biphasic expression pattern as membrane-bound Fc gamma RIII. In contrast, CD14 expression on neutrophils was initially constant, followed by high levels on the newly formed neutrophils. Soluble CD14 levels were found to be elevated transiently, whereas peak levels of soluble Fc gamma III were observed as late as 6 days postinjection. In conclusion, we have shown that G-CSF results in an immunophenotypically and functionally altered neutrophil population for an important part as a result of its effect on myeloid precursor cells.

Changes in plasma FcRIII demonstrate increasing receptor production during late pregnancy and after preterm birth.

We have previously described reduced expression of the Fc gamma receptor type III on the cell membrane (M-FcRIII) of neutrophils from very preterm neonates. To investigate the mechanism underlying this reduced receptor expression, we have measured neutrophil-derived soluble FcRIII (S-FcRIII) in the plasma of fetuses and neonates from 19 wk gestation. S-FcRIII in fetal plasma and in preterm neonates born before 32 wk gestation was consistently low [mean 13.6 +/- 1.2% (mean adult S-FcRIII = 100%, range 30-240%)]. In utero, S-FcRIII starts to rise from 33 wk and increases more than 4-fold to reach adult levels by term. S-FcRIII measured sequentially in preterm infants born as early as 24 wk of gestation showed a rapid postnatal increase to reach adult levels within 3 wk of birth. The changes in S-FcRIII paralleled changes in M-FcRIII expression on the cell surface. These observations point to a reduced rate of FcRIII production by fetal neutrophils as opposed to an increase in receptor release. The parallel increase in S-FcRIII and M-FcRIII suggests that there may be a programmed activation of FcRIII synthesis within individual cells late in the 3rd trimester of fetal development. In addition, FcRIII production may be switched on early by preterm birth.

Maternal genomic neutrophil FcRIII deficiency leading to neonatal isoimmune neutropenia.

The healthy mother of a child with transient immune neutropenia was found to be "NA-null." The mother's neutrophils did not react with anti-NA1 and anti-NA2 antibodies (polyclonal human alloantibodies and mouse monoclonal antibodies). A healthy donor was discovered during routine neutrophil antigen typing whose neutrophils were also "NA-null." This NA-phenotype was due to the absence of FcRIII (CD16 antigen) on neutrophils as demonstrated with anti-FcRIII monoclonal antibodies. The neutrophils of these two individuals were not able to bind dimeric immunoglobulin G. However, their cells had a normal expression of other phosphatidylinositol (PI)-linked membrane glycoprotein (CD24, CD67, and CLB gran/5 antigens), ruling out the existence of a PI-linkage defect, such as paroxysmal nocturnal hemoglobinuria. The mother (propsitus) had isoantibodies in her blood against neutrophil-FcRIII without allospecificity, apparently produced during pregnancy and responsible for the neutropenia of her child. The expression of FcRIII on natural killer lymphocytes of both individuals was normal. FcRIII is encoded by two separate genes, one (FcRIII-1) for the neutrophil-PI-linked receptor, another (FcRIII-2) for the natural killer cell and macrophage-transmembrane receptor. By messenger RNA and DNA analysis (with an FcRIII-cDNA probe and restriction endonucleases) the neutrophil-FcRIII deficiency appeared to be due to deletion of the FcRIII-1 gene in both individuals, while the FcRIII-2 gene was normally present. The parents of the propositus were found to be heterozygous for this defect. Thus, FcRIII-1 gene deficiency of the mother may be a cause of (iso)immune neutropenia of the newborn. Whether this deficiency may have other clinical consequences has to be studied.

Soluble Fc gamma receptor III in human plasma originates from release by neutrophils.

FcRIII (the CD16-antigen), a low affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes, and macrophages. We have developed a sensitive radioimmunoassay to quantify FcRIII. A soluble form of FcRIII was identified in human plasma. Immunoprecipitation of FcRIII from plasma showed that the plasma form of FcRIII has an identical electrophoretic mobility as the FcRIII expressed by neutrophils. Moreover, the plasma form of FcRIII exhibited the same polymorphism as does the neutrophil FcRIII. The neutrophil expresses the phosphatidylinositol-linked form of FcRIII, encoded by the gene FcRIII-1. Because it is not known whether this gene is also active in nonhematopoietic cells, we analyzed patients with an acquired clonal disorder of their hematopoietic cells, paroxysmal nocturnal hemoglobinuria (PNH). PNH patients appeared to have a strongly reduced expression of FcRIII on their neutrophils. The concentration of FcRIII in the plasma of these patients was also reduced, indicating that plasma FcRIII originates from neutrophils. A patient deficient in FcRIII-1 but with a normal expression of FcRIII-2 had no soluble FcRIII in her plasma, also indicating that plasma FcRIII originates from neutrophils. The electrophoretic mobility of the protein backbone of plasma FcRIII and FcRIII released by activated neutrophils was identical, whereas deglycosylated FcRIII obtained from a lysate of neutrophils migrated slower. This indicates that plasma FcRIII originates from activation-induced release by neutrophils. Stimulation of neutrophils or neutrophil cytoplasts (closed membrane vesicles filled with cytoplasm) with low concentrations of FMLP (10(-9)-10(-8) M) or phorbol myristate acetate (1-10 ng/ml) induced a dose-dependent release of FcRIII. The plasma concentration of FcRIII was relatively constant (range 40-280% of the mean). Soluble FcRIII was also detected in inflamed joint fluids of arthritis patients, suggesting that FcRIII is also released by activated neutrophils in vivo.