Marion Kleijer

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.

The Fc gamma RIIIA-158F allele is a risk factor for systemic lupus erythematosus

Objective. To study whether the Fc gamma RIIIA-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 gamma R 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 gamma RIIIA-158F allele was 0.74, versus 0.57 in healthy controls (P = 0.003), The genotype distribution of the Fc gamma RIIIA-158V/F polymorphism was also significantly different from that of the control population (P = 0.004). The distribution of the other Fc gamma R polymorphisms-Fc gamma RIIA-131R/H, Fc gamma RIIIB-NA(1/2), and Fc gamma RIIIA-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 gamma RIII4-158V/F polymorphism nas significantly different from that in the control group. This might indicate that macrophage expression of the Fc gamma RIIIA-158F isoform is involved in the disturbed clearance of immune complexes in patients with SLE.

The composition of the mesenchymal stromal cell compartment in human bone marrow changes during development and aging

Life-long hematopoiesis depends on the support of mesenchymal stromal cells within the bone marrow. Therefore, changes in the hematopoietic compartment that occur during development and aging probably correlate with variation in the composition of the stromal cell microenvironment. Mesenchymal stromal cells are a heterogeneous cell population and various subtypes may have different functions. In accordance with others, we show that CD271 and CD146 define distinct colony-forming-unit-fibroblast containing mesenchymal stromal cell subpopulations. In addition, analysis of 86 bone marrow samples revealed that the distribution of CD271brightCD146− and CD271brightCD146+ subsets correlates with donor age. The main subset in adults was CD271brightCD146−, whereas the CD271brightCD146+ population was dominant in pediatric and fetal bone marrow. A third subpopulation of CD271−CD146+ cells contained colony-forming-unit-fibroblasts in fetal samples only. These changes in composition of the mesenchymal stromal cell compartment during development and aging suggest a dynamic system, in which these subpopulations may have different functions.

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

Summary. FcγRIII (the CD16‐antigen), a low‐affinity receptor for IgG, is expressed by neutrophils, natural killer lymphocytes and macrophages. A soluble form of FcγRIII has been identified in human plasma. This soluble form of FcγRIII (sFcγRIII) originates from release by neutrophils. In the present study we show by transfusions of plasma that contains sFcγRIII of one allotype (NA1‐FcγRIII) in recipients homozygous for the other allotype (NA2‐FcγRIII) that the clearance of sFcγRIII is about 0.7 ml/min. Because the concentration of sFcγRIII was found to be constant in a small cohort of donors followed for about 1‐5 years, the half‐life of NA1‐sFcγRIII is about 1.8 d, assuming a one‐compartment model.

NA‐phenotype‐dependent differences in neutrophil FcγRIIIb expression cause differences in plasma levels of soluble FcγRIII

Soluble FcγRIII 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 FcγRIII in plasma and released FcγRIII in cell supernatants. Both sFcγRIIIa, derived from NK cells and sFcγRIIIb, derived from neutrophils are detected in the assay. However, plasma analysis of FcγRIIIB gene‐deficient donors suggested that sFcγRIIIa 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 sFcγRIII than plasma of homozygous NA2‐positive donors. Heterozygous donors were found to have intermediate levels of sFcγRIII in their plasma. Hemizygous FcγRIIIB gene‐deficient donors were found to have half the amount of sFcγRIII in their plasma compared to donors with two FcγRIIIB alleles. These NA phenotype‐dependent differences in plasma sFcγRIII could not be contributed to either an assay artefact or NA‐dependent differences in shedding of FcγRIIIb upon neutrophil activation. Calibration curves constructed with plasma of homozygous donors did not reveal NA‐dependent differences in antibody affinity. Measurement of released FcγRIIIb 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 FcγRIIIb expression were present, comparable to the differences found in plasma levels of sFcγRIII. Differences in the amounts of released FcγRIII in supernatants of NA‐typed apoptotic neutrophils were similar to initial differences in FcγRIIIb expression, again being lower in NA1‐positive than in heterozygous and NA2‐positive donors. In conclusion, NA‐dependent differences in plasma levels of soluble FcγRIII seem to be caused by differences in expression of the receptor on the neutrophil membrane.

Nuclear Receptors Nur77 and Nurr1 Modulate Mesenchymal Stromal Cell Migration

Detailed understanding of mesenchymal stromal cells (MSC) migration is imperative for future cellular therapies. To identify genes involved in the process of MSC migration, we generated gene expression profiles of migrating and nonmigrating fetal bone marrow MSC (FBMSC). Only 12 genes showed differential expression in migrating versus nonmigrating FBMSC. The nuclear receptors Nur77 and Nurr1 showed the highest expression in migratory MSC. Nur77 and Nurr1 are members of NR4A nuclear orphan receptor family, and we found that their expression is rapidly increased upon exposure of FBMSC to the migratory stimuli stromal-derived factor-1α (SDF-1α) and platelet-derived growth factor-BB. Lentiviral expression of Nur77 or Nurr1 resulted in enhanced migration of FBMSC toward SDF-1α compared with mock-transduced FBMSC. Analysis of the cell cycle, known to be involved in MSC migration, revealed that expression of Nur77 and Nurr1 decreases the proportion of cells in S-phase compared with control cells. Further, gain-of-function experiments showed increased hepatocyte growth factor expression and interleukin (IL)-6 and IL-8 production in MSC. Despite the altered cytokine profile, FBMSC expressing Nur77 or Nurr1 maintained the capacity to inhibit T-cell proliferation in a mixed lymphocyte reaction. Our results demonstrate that Nur77 and Nurr1 promote FBMSC migration. Modulation of Nur77 and Nurr1 activity may therefore offer perspectives to enhance the migratory potential of FBMSC which may specifically regulate the local immune response.

The Presence of Activated CD4+ T Cells Is Essential for the Formation of Colony-Forming Unit-Endothelial Cells by CD14+ Cells

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study, the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also, however, CD4+ T cells were found to be indispensable for the induction of CFU-EC colonies, mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4+ T cells or blocking MHC class II molecules on monocytes, it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes, the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14+ cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization.

Cell cycle and tissue of origin contribute to the migratory behaviour of human fetal and adult mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are potential cells for cellular therapies, in which the recruitment and migration of MSC towards injured tissue is crucial. Our data show that culture‐expanded MSC from fetal lung and bone marrow, adult bone marrow and adipose tissue contained a small percentage of migrating cells in vitro, but the optimal stimulus was different. Overall, fetal lung‐MSC had the highest migratory capacity. As fetal bone marrow‐MSC had lower migratory potential than fetal lung‐MSC, the tissue of origin may determine the migratory capacity of MSC. No additive effect in migration towards combined stimuli was observed, which suggests only one migratory MSC fraction. Interestingly, actin rearrangement and increased paxillin phosphorylation were observed in most MSC upon stromal cell‐derived factor‐1α or platelet‐derived growth factor‐BB stimulation, indicating that this mechanism involved in responding to migratory cues is not restricted to migratory MSC. Migratory MSC maintained differentiation and migration potential, and contained significantly less cells in S‐ and G2/M‐phase than their non‐migrating counterpart. In conclusion, our results suggest that MSC from various sources have different migratory capacities, depending on the tissue of origin. Similar to haematopoietic stem cells, cell cycle contributes to MSC migration, which offers perspectives for modulation of MSC to enhance efficacy of future cellular therapies.

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

ABSTRACT: We have previously described reduced expression of the Fcγ receptor type III on the cell membrane (M-FcRIH) of neutrophils from very preterm neo- nates. 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.

Mesenchymal Stromal Cells Stimulate the Proliferation and IL-22 Production of Group 3 Innate Lymphoid Cells

Infusion of mesenchymal stromal cells (MSCs) is a promising and increasingly applied therapy for patients who suffer from a variety of inflammatory diseases, including graft-versus-host disease (GvHD), a common and life-threatening complication after allogeneic hematopoietic stem cell transplantation. The therapeutic effect of MSCs is mainly ascribed to their ability to suppress T cells and to support tissue repair. However, clinical response rates in patients with GvHD are limited to 50%, and the determinants for MSC responsiveness are unknown. We recently reported that high frequencies of activated group 3 innate lymphoid cells (ILC3s) before and after allogeneic hematopoietic stem cell transplantation were associated with a lower risk of GvHD. This may be related to IL-22 production by ILC3s, a cytokine important for intestinal epithelial cell homeostasis. In this study, we investigated whether ILC3s may contribute to the therapeutic effect of MSCs by studying the interaction between MSCs and ILC3s in vitro. ILC3s isolated from human tonsils were cocultured with human bone marrow–derived MSCs for 5 d in the presence of IL-2. Coculture with MSCs enhanced the proliferation and IL-22 production of ILC3s. Reciprocally, ILC3s promoted ICAM-1 and VCAM-1 expression on MSCs. For both directions, the activation was mainly mediated by cell–cell contact and by MSC-derived IL-7 and likely by aryl hydrocarbon receptor ligands. Thus, in addition to inhibiting the proliferation of alloreactive T cells, MSCs also promote the expansion and IL-22 production of ILC3s, which may contribute to healthy homeostasis and wound repair in the treatment of various inflammatory conditions in the intestine, including GvHD.