Genetic mechanism of human neutrophil antigen 2 deficiency and expression variations.

Human neutrophil antigen 2 (HNA-2) deficiency is a common phenotype as 3-5% humans do not express HNA-2. HNA-2 is coded by CD177 gene that associates with human myeloproliferative disorders. HNA-2 deficient individuals are prone to produce HNA-2 alloantibodies that cause a number of disorders including transfusion-related acute lung injury and immune neutropenia. In addition, the percentages of HNA-2 positive neutrophils vary significantly among individuals and HNA-2 expression variations play a role in human diseases such as myelodysplastic syndrome, chronic myelogenous leukemia, and gastric cancer. The underlying genetic mechanism of HNA-2 deficiency and expression variations has remained a mystery. In this study, we identified a novel CD177 nonsense single nucleotide polymorphism (SNP 829A>T) that creates a stop codon within the CD177 coding region. We found that all 829TT homozygous individuals were HNA-2 deficient. In addition, the SNP 829A>T genotypes were significantly associated with the percentage of HNA-2 positive neutrophils. Transfection experiments confirmed that HNA-2 expression was absent on cells expressing the CD177 SNP 829T allele. Our data clearly demonstrate that the CD177 SNP 829A>T is the primary genetic determinant for HNA-2 deficiency and expression variations. The mechanistic delineation of HNA-2 genetics will enable the development of genetic tests for diagnosis and prognosis of HNA-2-related human diseases.

SPACA3 gene variants in a New Zealand cohort of infertile and fertile couples.

SPRASA (also referred to as SLLP1) is a protein identified in the acrosome of human sperm and encoded by the gene SPACA3. SPRASA is associated with sperm-oocyte recognition and binding, and may play a role in fertility. In order to determine whether variants in the SPACA3 gene are associated with human infertility, we undertook a genetic analysis of 102 infertile and 104 fertile couples. Three gene variants were identified using PCR-based DNA sequencing; 1) an insertion of TGC within a quadruple tri-nucleotide (TGC) repeat region in the 5' untranslated region (UTR) (g.-22TGC(4_5), 2) a guanine to adenosine transition at position 239 (c.239G>A) resulting in a non-synonymous amino acid substitution from cysteine to tyrosine (p.C80Y) at position 80 in the putative transmembrane region, and 3) a novel nucleotide variant (c.691G>C) located in the 3'UTR. A functional effect of the g.-22TGC (4_5) was confirmed by a luciferase expression assay, while the effects of the variants c.239G>A and c.691G>C were predicted using in silico analysis. Although the frequencies of these variants were not significantly different between the infertile and fertile populations, we present evidence that the variants could affect the expression levels or function of SPRASA, thereby affecting a couple's fertility. Larger populations, especially individuals/couples with unexplained infertility, need to be screened for these variants to validate a relationship with fertility.

Brain-derived antigens in lymphoid tissue of patients with acute stroke.

In experimental animals, the presence of brain-derived constituents in cervical lymph nodes has been associated with the activation of local lymphocytes poised to minimize the inflammatory response after acute brain injury. In this study, we assessed whether this immune crosstalk also existed in stroke patients. We studied the clinical course, neuroimaging, and immunoreactivity to neuronal derived Ags (microtubule-associated protein-2 and N-methyl d-aspartate receptor subunit NR-2A), and myelin-derived Ags (myelin basic protein and myelin oligodendrocyte glycoprotein) in palatine tonsils and cervical lymph nodes of 28 acute stroke patients and 17 individuals free of neurologic disease. Stroke patients showed greater immunoreactivity to all brain Ags assessed compared with controls, predominantly in T cell zones. Most brain immunoreactive cells were CD68(+) macrophages expressing MHC class II receptors. Increased reactivity to neuronal-derived Ags was correlated with smaller infarctions and better long-term outcome, whereas greater reactivity to myelin basic protein was correlated with stroke severity on admission, larger infarctions, and worse outcome at follow-up. Patients also had more CD69(+) T cells than controls, indicative of T cell activation. Overall, the study showed in patients with acute stroke the presence of myelin and neuronal Ags associated with lymph node macrophages located near activated T cells. Whether the outcome of acute stroke is influenced by Ag-specific activation of immune responses mediated by CD69 lymphocytes deserves further investigation.

Elevated neutrophil membrane expression of proteinase 3 is dependent upon CD177 expression.

Proteinase 3 (PR3) is a major autoantigen in anti-neutrophil cytoplasmic antibodies (ANCA)-associated systemic vasculitis (AASV), and the proportion of neutrophils expressing PR3 on their membrane (mPR3+) is increased in AASV. We have shown recently that mPR3 and CD177 are expressed on the same cells in healthy individuals. In this study we try to elucidate mechanisms behind the increased mPR3 expression in AASV and its relationship to CD177. All neutrophils in all individuals were either double-positive or double-negative for mPR3 and CD177. The proportion of double-positive neutrophils was increased significantly in AASV and systemic lupus erythematosus patients. The proportion of mPR3+/CD177+ cells was not correlated to general inflammation, renal function, age, sex, drug treatment and levels of circulating PR3. AASV patients had normal levels of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Pro-PR3 was found to constitute 10% of circulating PR3 but none of the mPR3. We found increased mRNA levels of both PR3 and CD177 in AASV, but they did not correlate with the proportion of double-positive cells. In cells sorted based on membrane expression, CD177-mRNA was several-fold higher in mPR3+ cells. When exogenous PR3 was added to CD177-transfected U937 cells, only CD177+ cells bound PR3 to their membrane. In conclusion, the increased membrane expression of PR3 found in AASV is not linked directly to circulating PR3 or PR3 gene transcription, but is dependent upon CD177 expression and correlated with the transcription of the CD177 gene.

Molecular studies reveal that A134T, G156A and G1333A SNPs in the CD177 gene are associated with atypical expression of human neutrophil antigen-2.

BACKGROUND AND OBJECTIVES: The human neutrophil antigen-2 (HNA-2) is expressed on a subpopulation of neutrophils as most subjects present a negative plus a positive HNA-2 population of neutrophils. The number of neutrophils expressing HNA-2 is variable and may increase in pregnancy, infections, myeloproliferative disorders and after G-CSF. This study investigated the presence of polymorphisms in the gene encoding HNA-2 (CD177) in individuals presenting different patterns of antigen expression and determined the association of single nucleotide polymorphisms (SNPs) with the heterogeneous HNA-2 expression. MATERIALS AND METHODS: Flow cytometry was employed to analyse the HNA-2 expression on neutrophils from 135 healthy subjects using two monoclonal antibodies (TAG4, 7D8). Sequencing reactions were performed on subjects whose antigen expression was low (< or = 50%), high (> or = 80%) or atypical (a nonreactive population plus two distinct positive cell populations). RESULTS: Five SNPs were detected, two of them (A793C, G1084A) were related to a low expression of HNA-2 (P = 0.031 and P = 0.004). Atypical antigen expression was observed in 5.9% (8/135) of the individuals, three nonpregnant women and five men. In these cases, the cDNA sequences revealed three SNPs (A134T, G156A and G1333A) strongly related to this atypical HNA-2 expression (P = 0.004, P = 0.006 and P < 0.0001, respectively). CONCLUSIONS: Our data show that polymorphisms in the CD177 are associated with variations in the HNA-2 expression and may be the cause of atypical expressions.

Activation, immune polarization, and graft-versus-leukemia activity of donor T cells are regulated by specific subsets of donor bone marrow antigen-presenting cells in allogeneic hemopoietic stem cell transplantation.

We investigated the roles of specific subsets of donor APCs purified from bone marrow in donor T cell activation and graft-vs-leukemia (GvL) activity in murine models of hemopoietic stem cell transplantation. Lineage(-)CD11c(+) APC precursors were separated from donor bone marrow based on expression of CD11b. Transplanting lineage(-)CD11c(+)CD11b(-) APC (CD11b(-) APC) in combination with c-kit(+)Sca-1(+)lineage(-) hemopoietic stem cells (HSC) and congenic donor T cells led to increased donor CD4(+) and CD8(+) T cell proliferation and higher donor T cell chimerism than with transplanting grafts containing HSC, T cells, and lineage(-)CD11c(+)CD11b(+) APCs (CD11b(+) APC), or grafts containing only HSC and T cells. Transplanting CD11b(-) APCs induced Th1/type 1 cytotoxic T lymphocyte donor T cell immune polarization and enhanced GvL activity of donor T cells without increased graft-vs-host disease in both MHC- and minor histocompatibility Ag-mismatched murine hemopoietic stem cell transplantation models, whereas CD11b(+) APCs led to Th2/type 2 cytotoxic T lymphocyte donor T cell immune polarization. Donor CD11b(-) APCs were plasmacytoid dendritic cell progenitors (>90% CD317; PDCA-1(+)) and up-regulated CD80, CD86, and IL-12 during alloantigen presentation, whereas CD11b(+) APCs expressed Gr-1 and up-regulated expression of programmed death ligands-1 and 2 after activation. These results are the first to show that manipulation of the content of donor APCs in allogeneic HSC grafts can regulate donor T cell immunity and enhance GvL without increasing graft-vs-host disease activity.

Granulocyte concentrates: prolonged functional capacity during storage in the presence of phenotypic changes.

BACKGROUND: Granulocyte transfusion has been proposed as a bridging therapy for patients with prolonged periods of chemotherapy-induced neutropenia, who suffer from severe fungal and bacterial infections. To recover, adequate numbers of granulocytes are required when the patients are refractory to standard treatment. The aim of this study was to assess the functional characteristics and efficacy of granulocyte colony-stimulating factor/dexamethasone-mobilized granulocytes used for transfusions. DESIGN AND METHODS: Granulocytes from the leukapheresis products were tested for the expression of cell-surface antigens, interactions with endothelial cells, motility, killing of microbes and survival. The granulocytes were used in vivo for transfusion in 16 severely ill children, who were--apart from a patient with a granulocyte dysfunction--all suffering from prolonged neutropenia. RESULTS: Mobilization of granulocytes with granulocyte colony-stimulating factor and dexamethasone caused phenotypic changes (decreased CD62L expression and increased levels of CD66b and CD177). The ability of the granulocytes to interact with endothelial cells (rolling, adhesion, transmigration) and to kill various types of pathogens was not affected by the mobilization, leukapheresis and irradiation procedures. The granulocytes were functionally indistinguishable from those isolated from untreated donors, even after 24 hours of storage. Granulocyte transfusion seemed to benefit 70% of patients, as 11 out of the 16 children showed clinical recovery within 1-2 weeks after beginning the transfusions. CONCLUSIONS: Although CD62L expression is downregulated on granulocytes used for granulocyte transfusions, concomitant CD177 upregulation may explain the intact interactions with endothelial cells. All other granulocyte functions tested were intact, including the ability to kill fungi. Granulocyte concentrates can be stored without loss of in vitro viability and functionality for at least 24 hours. As demonstrated in vivo, granulocyte transfusions may be an effective therapy for neutropenic pediatric patients suffering from life-threatening infections.

Increased CD177 (PRV1) expression in thalassaemia and the underlying erythropoietic activity.

CD177 (PRV1) expression is strongly related to polycythaemia vera (PV). Whilst studying CD177 expression in PV patients and controls, individuals with beta-thalassaemia minor were found to display an elevated expression of CD177. The study was expanded to include patients with thalassaemia intermedia, sickle cell/beta-thalassaemia and thalassaemia major. CD177 expression was increased in these thalassaemic groups and correlated with their erythropoietic activity, as assessed by the measurement of serum erythropoietin and soluble transferrin receptor levels. Within this context, elevated CD177 expression is not only a specific feature of PV but may be an indicator of increased erythropoietic activity in thalassaemia syndromes.

A novel high throughput immunomagnetic cell sorting system for potential clinical scale depletion of T cells for allogeneic stem cell transplantation.

OBJECTIVE: To develop an immunomagnetic cell separation system for allogeneic hematopoietic stem cell (HSC) transplantations, which can achieve a high level of T-cell depletion (at least 4.0 log(10)), high level of recovery of hematopoietic stem cells (>90%), with a high throughput (>10(6) cells/second). METHODS: Peripheral blood leukocytes (PBLs) from buffy coats were spiked with CD34-expressing cells (KG1a) to mimic a leukaphoresis product containing stimulated HSCs. T cells were labeled with anti-CD3(+) Dynabeads and separated in a quadrupole magnetic cell sorter (QMS). The performance of the system with respect to T-cell depletion and recovery of non-T cells and spiked KG1a was determined using four-color, flow cytometry analysis, with the aid of Trucount cell-concentration calibration beads. Limiting dilution assays were also performed to quantify the log(10) depletion of clonable T cells. RESULTS: While the general performance of the QMS system is governed by proven theoretical principles, significant system variability exist, not all of which can be explained by our current understanding. Consequently, a factorial design was employed, guided by JMP software, to optimize the labeling conditions and operation of the QMS focused on maximizing the depletion of T cell, and recovery of unlabeled cells including KG1a cells. From these studies, an optimized, no wash, immunomagnetic labeling protocol and optimized QMS operating conditions were developed. For an average initial cell concentration of 1.7 x 10(8) total cells, an average 3.96 +/- 0.33 log(10) depletion (range, 3.53-4.34) of CD3(+)CD45(+) cells with a mean 99.43% +/- 4.23% recovery of CD34(+)CD45(+) cells (range, 94.38-104.90%) was achieved at a sorting speed of 10(6) cells/s (n = 6). Limiting dilution assays on the T-cell depleted fractions, which gave a log(10) depletion of 3.51 for the clonable T cells. CONCLUSION: We suggest that this system will provide superior performance with respect to T-cell depletion and CD34(+) recovery for clinical allogeneic bone marrow transplants. Ongoing studies, on a clinical scale, are being conducted to demonstrate this claim.

Proteinase 3 and CD177 are expressed on the plasma membrane of the same subset of neutrophils.

Proteinase 3 (PR3) is found in granules of all neutrophils but also on the plasma membrane of a subset of neutrophils (mPR3). CD177, another neutrophil protein, also displays a bimodal surface expression. In this study, we have investigated the coexpression of these two molecules, as well as the effect of cell activation on their surface expression. We can show that CD177 is expressed on the same subset of neutrophils as mPR3. Experiments show that the expression of mPR3 and CD177 on the plasma membrane is increased or decreased in parallel during cell stimulation or spontaneous apoptosis. Furthermore, we observed a rapid internalization and recirculation of mPR3 and plasma membrane CD177, where all mPR3 is replaced within 30 min. Our findings suggest that the PR3 found on the plasma membrane has its origin in the same intracellular storage as CD177, i.e., secondary granules and secretory vesicles and not primary granules. PR3- and CD177-expressing neutrophils constitute a subpopulation of neutrophils with an unknown role in the innate immune system, which may play an important role in diseases such as Wegener's granulomatosis and polycythemia vera.

Reconstitution of allogeneic hemopoietic stem cells: the essential role of FcR gamma and the TCR beta-chain-FCp33 complex.

Transplantation of purified allogeneic hemopoietic stem cells (SC) alone is characterized by a decreased risk of graft-vs-host disease but increased incidence of engraftment failure. It has been established that the facilitating cell (FC) promotes allogeneic SC reconstitution and results in donor-specific transplantation tolerance across MHC disparities, without graft-vs-host disease. Although the requirements for this facilitating function are not well-characterized, it is known that facilitation is dependent on FC expression of a unique heterodimer consisting of the TCR beta-chain (TCRbeta) and a 33-kDa protein, FCp33. The current study confirms that CD3epsilon and TCRbeta expression are present on the FC at the time of transplantation and demonstrates that the majority of cells in the FC population express the TCR signaling molecule, FcRgamma, rather than the more conventional CD3zeta receptor. Of particular significance, we have now demonstrated that FC-mediated allogeneic SC reconstitution is critically dependent on FcRgamma expression and that FcRgamma coprecipitates with the TCRbeta-FCp33 heterodimer. The mandatory requirement of TCRbeta and FcRgamma for FC function provides the first evidence of a previously undescribed role for FcRgamma in the facilitation of allogeneic SC reconstitution and establishes that FcRgamma is part of the TCRbeta-FCp33 complex uniquely expressed on FC.

The effect of the JAK2 V617F mutation on PRV-1 expression.

JAK2(V617F)an acquired mutation of JAK2, is present in a majority of patients with polycythemia vera and to a lesser extent among patients with the other myeloproliferative disorders. We analyzed the effect of JAK2(V617F) on the expression of polycythemia rubra vera 1(PRV-1), using an in vitro model. Compared to wild-type JAK2, the presence of JAK2(V617F) increased both PRV-1 protein and mRNA levels in murine myeloid cells. A JAK2 inhibitor eliminated the V617F-induced increase in PRV-1 expression.

Sex-dependent up regulation of CD 177-specific mRNA expression in cord blood due to different stimuli.

BACKGROUND: The neutrophil-specific CD 177 molecule (NB1 glycoprotein/HNA-2a) has gained clinical interest because of its involvement in severe antibody-dependent diseases like transfusion-related acute lung injury and neonatal alloimmune neutropenia. Up regulation of CD 177 in response to different stimuli (granulocyte-colony-stimulating factor, N-formyl-Met-Leu-Phe, bacterial infections) has been described in adults. STUDY DESIGN AND METHODS: The regulation of CD 177 expression was evaluated on mRNA and glycoprotein levels from cord blood neutrophils of 56 neonates, 38 of them with complications during pregnancy or delivery. Real-time polymerase chain reaction and flow cytometry were used for quantification. RESULTS: White blood cells from neonates of both sexes showed significantly elevated glycoprotein and mRNA levels compared to adults. In addition, there was a significant mRNA up regulation in female newborns predominantly occurring in cases with pathologic cardiotocogram, premature rupture of the amniotic membrane, and health disorders of the mother. CONCLUSION: These findings show a significantly increased CD 177 expression in neutrophils from newborns compared to adults, which suggests the existence of additional factors being able to stimulate CD 177 expression.

Thrombotic and bleeding complications in four subpopulations of patients with essential thrombocythemia defined by c-Mpl protein expression and PRV-1 mRNA levels.

C-Mpl and PRV-1 expression was measured in a cohort of 48 patients with essential thrombocythemia (ET). A retrospective analysis was conducted to asses whether the presence of one or both markers correlates with a higher risk of developing thromboembolic complications. In this cohort, PRV-1 overexpression was associated with a significantly increased risk of thrombosis, whereas decreased c-Mpl expression was not. The results of this retrospective analysis must now be corroborated in a large, prospective trial of newly diagnosed patients.

Reactive oxygen species and 12/15-lipoxygenase contribute to the antiproliferative capacity of alternatively activated myeloid cells elicited during helminth infection.

Understanding the role of CD11b(+)GR-1(+) myeloid suppressor cells in the immune suppression and immunoregulation associated with a variety of diseases may provide therapeutic opportunities. In this article, we show, in a model of helminth infection, that CD11b(+)GR-1(+) myeloid suppressor cells but not CD11b(+)F4/80(high) mature macrophages expanded in the peritoneal cavity of BALB/c mice implanted with Taenia crassiceps. Peritoneal cell populations from early stage-infected animals impaired T cell proliferation by secreting NO. Yet, they lost their ability to secrete NO in the late stage of infection. Concomitantly, their capacity to exert arginase activity and to express mRNAs coding for FIZZ1 (found in inflammatory zone 1), Ym, and macrophage galactose-type C-type lectin increased. Furthermore, cells from early stage-infected mice triggered T cells to secrete IFN-gamma and IL-4, whereas in the late stage of infection, they only induced IL-4 production. These data suggest that CD11b(+)GR-1(+) myeloid suppressor cells displaying an alternative activation phenotype emerged gradually as T. crassiceps infection progressed. Corroborating the alternative activation status in the late stage of infection, the suppressive activity relied on arginase activity, which facilitated the production of reactive oxygen species including H(2)O(2) and superoxide. We also document that the suppressive activity of alternative myeloid suppressor cells depended on 12/15-lipoxygenase activation generating lipid mediators, which triggered peroxisome proliferator-activated receptor-gamma. IL-4 and IL-13 signaling contributed to the expansion of myeloid suppressor cells in the peritoneal cavity of T. crassiceps-infected animals and to their antiproliferative activity by allowing arginase and 12/15-lipoxygenase gene expression.

The effects of hydroxyurea on PRV-1 expression in patients with essential thrombocythemia and polycythemia vera.

The study was designed to investigate the influence of hydroxyurea (HU) treatment on PRV-1 expression. Eighteen newly diagnosed patients with essential thrombocythemia (ET) or polycythemia vera (PV) were included. HU significantly increased PRV-1 gene expression in the early stage of treatment.

Induction of a CD4+ T regulatory type 1 response by cyclooxygenase-2-overexpressing glioma.

PGE(2), synthesized by cyclooxygenase-2 (COX-2)-overexpressing tumor, is known to contribute to cellular immune suppression in cancer patients, but the mechanism remains unclear. We report the mechanism of a CD4(+) T regulatory type 1 (Tr1) induction by CD11c(+) mature dendritic cells (DCs) that phagocytose allogeneic and autologous COX-2-overexpressing glioma. A human glioma cell line, U-87MG, and primary cultured glioblastoma cells (MG-377) overexpressed COX-2. We did not detect IL-10Ralpha expression in these gliomas, and rIL-10 did not suppress their COX-2 expression. Exposure to COX-2-overexpressing glioma induced mature DCs to overexpress IL-10 and decreased IL-12p70 production. These DCs induced a Tr1 response, which is characterized by robust secretion of IL-10 and TGF-beta with negligible IL-4 secretion by CD4(+) T cells, and an inhibitory effect on admixed lymphocytes. Peripheral CD4(+) T cell populations isolated from an MG-377 patient also predominantly demonstrated a Tr1 response against MG-377 cells. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the time of phagocytic uptake by DCs abrogated this regulatory response and instead elicited Th1 activity. COX-2 stable transfectants in LN-18 (LN-18-COX2) also induced a Tr1 response. The effect of a COX-2 inhibition in LN-18-COX2 is reversible after administration of PGE(2). Taken together, robust levels of PGE(2) from COX-2-overexpressing glioma, which is unresponsive to IL-10 within the local microenvironment, may cause DCs to secrete high levels of IL-10. These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which is mediated by tumor-exposed, IL-10-enhanced DCs.