Neutrophils as Suppressors of T Cell Proliferation: Does Age Matter?

Whereas, neutrophils have long been considered to mainly function as efficient innate immunity killers of micro-organisms at infected sites, they are now recognized to also be involved in modulation of adaptive immune responses. Immature and mature neutrophils were reported to have the capacity to suppress T cell-mediated immune responses as so-called granulocyte-myeloid-derived suppressor cells (g-MDSCs), and thereby affect the clinical outcome of cancer patients and impact the chronicity of microbial infections or rejection reactions in organ transplantation settings. These MDSCs were at first considered to be immature myeloid cells that left the bone marrow due to disease-specific signals. Current studies show that also mature neutrophils can exert suppressive activity. In this study we investigated in a robust T cell suppression assay whether immature CD11b+ myeloid cells were capable of MDSC activity comparable to mature fully differentiated neutrophils. We compared circulating neutrophils with myeloid cell fractions from the bone marrow at different differentiation stages. Our results indicate that functional MDSC activity is only becoming detectable at the final stage of differentiation, depending on the procedure of cell isolation. The MDSC activity obtained during neutrophil maturation correlated with the induction of the well-known highly mobile and toxic effector functions of the circulating neutrophil. Although immature neutrophils have been suggested to be increased in the circulation of cancer patients, we show here that immature neutrophils are not efficient in suppressing T cells. This suggests that the presence of immature neutrophils in the bloodstream of cancer patients represent a mere association or may function as a source of mature neutrophils in the tumor environment but not a direct cause of enhanced MDSC activity in cancer.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging.

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

Characterization of buffy coat-derived granulocytes for clinical use: a comparison with granulocyte colony-stimulating factor/dexamethasone-pretreated donor-derived products.

BACKGROUND AND OBJECTIVES: Buffy coat-derived granulocytes have been described as an alternative to the apheresis product from donors pretreated with dexamethasone and granulocyte colony-stimulating factor (G-CSF). The latter is - dependent on the local and national settings - obtained following a demanding and time-consuming procedure, which is undesirable in critically ill septic patients. In contrast, buffy coat-derived products have a large volume and are often heavily contaminated with red cells and platelets. We developed a new pooled buffy coat-derived product with high purity and small volume, and performed a comprehensive functional characterization of these granulocytes. MATERIALS AND METHODS: We pooled ten buffy coats following the production of platelet concentrates. Saline 0·9% was added to decrease the viscosity and the product was split into plasma, red cells and a 'super' buffy coat. Functional data of the granulocytes were compared to those obtained with granulocytes from healthy controls and G-CSF/dexamethasone-pretreated donors. RESULTS: Buffy coat-derived granulocytes showed adhesion, chemotaxis, reactive oxygen species production, degranulation, NETosis and in vitro killing of Staphylococcus aureus, Escherichia coli and Aspergillus species comparable to control and G-CSF/dexamethasone-derived granulocytes. Candida killing was superior compared to G-CSF/dexamethasone-derived granulocytes. Immunophenotyping was normal; especially no signs of activation in the buffy coat-derived granulocytes were seen. Viability was reduced. Buffy coats are readily available in the regular blood production process and would take away the concerns around the apheresis product. CONCLUSION: The product described appears a promising alternative for transfusion purposes.

Study on the protective effect of the KIR3DL1 gene in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is an autoimmune disease that mainly affects the sacroiliac joints and the spine of the lower back. The disease is strongly associated with HLA-B27. Additional genes, single-nucleotide polymorphisms, and molecular components have been identified to be associated with AS, but the exact mechanism that drives disease development remains poorly understood. The killer cell immunoglobulin-like receptors (KIRs) are regulators of cytotoxicity of natural killer cells and T cell subsets and may be relevant in binding to HLA-B27 and the development of AS. We undertook this study to identify possible associations of KIR genotype with susceptibility to AS and disease characteristics including the presence of the HLA-B27 allele, disease severity, and uveitis. METHODS: We performed complete genotyping of the KIR locus in 303 Caucasian AS patients, 119 randomly selected healthy Caucasian controls, and 50 HLA-B27-positive healthy Caucasian controls by multiplex ligation-dependent probe amplification assay for detection of gene presence and copy number. RESULTS: We did not observe a significant association of any specific KIR gene or haplotype with susceptibility to AS or any other clinical manifestation. Disease severity, as measured by fulfilling the criteria for treatment with tumor necrosis factor blocking therapy, was linked to a lower number of genes for the functional variant of KIR3DL1 (P = 0.007). CONCLUSION: Our exploratory study indicates that KIR genes are not a major risk factor for susceptibility to AS. However, the data do suggest a role for KIRs in progression of the disease, whereby KIR3DL1 has a protective effect against the more severe manifestations of AS.

Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

Novel insights in the genomic organization and hotspots of recombination in the human KIR locus through analysis of intergenic regions.

The Killer Immunoglobulin-like Receptor (KIR) proteins constitute a family of highly homologous surface receptors involved in the regulation of the innate cytotoxicity of natural killer (NK) cells. Within the human genome, 17 KIR genes are present, many of which show large variation across the population owing to the high number of allelic variants and copy number variation (CNV). KIR genotyping and CNV determination were used to map the KIR locus in a large cohort of >400 Caucasian individuals. Gene order and structure was determined by sequence-specific polymerase chain reaction of the intergenic regions. In this way, we could show that KIR3DL1 and KIR2DS4 gene variants are linked and that--contrary to current views--the gene KIR2DS5 is only present in the telomeric half of the KIR locus. Our study revealed novel insights in the highly organized distribution of KIR genes. Novel recombination hotspots were identified that contribute to the diversity of KIR gene distribution in the Caucasian population. Next-generation sequencing of the KIR intergenic regions allowed for a detailed single-nucleotide polymorphism analysis, which demonstrated several gene-specific as well as haplotype-specific nucleotides for a more accurate genotyping of this notoriously complex gene cluster.

Changes in the ornithine cycle following ionising radiation cause a cytotoxic conditioning of the culture medium of H35 hepatoma cells.

Cultured H35 hepatoma cells release a cytotoxic factor in response to irradiation with X-rays. When the conditioned medium from irradiated cells is given to nonirradiated cells, growth is inhibited and followed by cell death, possibly apoptosis, Analysis of the conditioned medium reveals a dramatic change in the ornithine (urea) cycle components after the irradiation. A strong decrease in medium arginine is accompanied with parallel increases in ornithine, citrulline and ammonia. The high level of ammonia appears to be largely responsible for the observed cytotoxicity. The development of hyperammonia by irradiated cells and the related toxicity depend on the radiation dose and the number of cells seeded thereafter for the medium conditioning. Development of cytotoxicity by irradiated cells is completely prevented with the arginase inhibitor L-norvaline, in arginine-deficient medium or when citrulline replaces arginine. These preventive measures result in subtoxic ammonia levels.

Meningeal and perivascular macrophages of the central nervous system play a protective role during bacterial meningitis.

Meningeal (MM) and perivascular macrophages (PVM) constitute major populations of resident macrophages in the CNS that can be distinguished from microglial cells. So far, there is no direct evidence that demonstrates a possible role of MM and PVM in the CNS during normal or pathologic conditions. To elucidate the role of the MM and PVM during CNS inflammation, we have developed a strategy using a single intraventricular injection of mannosylated clodronate liposomes, which results in a complete and selective depletion of the PVM and MM from the CNS. Depletion of the MM and PVM during experimental pneumococcal meningitis resulted in increased illness, which correlated with higher bacteria counts in the cerebrospinal fluid and blood. This was associated with a decreased influx of leukocytes into the cerebrospinal fluid, which occurred despite an elevated production of relevant chemokines (e.g., macrophage-inflammatory protein-2) and a higher expression of vascular adhesion molecules (e.g., VCAM-1). In contrast, the higher bacterial counts correlated with elevated production of local and systemic inflammatory mediators (e.g., IL-6) indicating enhanced local leukocyte and systemic immune activation, and this may explain the worsening of the clinical signs. These findings show that the PVM and MM play a protective role during bacterial meningitis and suggest that a primary action of these macrophages is to facilitate the influx of leukocytes at the blood-brain barrier. More in general, we demonstrate for the first time that the PVM and MM play a crucial role during inflammation in the CNS.

A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system.

The perivascular (PVM) and meningeal (MM) macrophages form a distinct population of resident CNS cells, selectively expressing the mature macrophage marker ED2 in the rat. In order to elucidate the role of the PVM and MM in rats during normal functioning of the brain and pathology, we have developed a strategy employing a single intraventricular injection of clodronate liposomes. This resulted in a complete depletion of the PVM and MM. Clodronate liposomes did not deplete the microglial cells. In other parts of the body, a temporal and mild depletion effect was observed, which was restored within 1 week. Detailed analysis of the elimination and repopulation kinetics of the PVM and MM revealed a slow repopulation of the CNS, starting at 14 days post depletion. This selective depletion method of the PVM and MM will enable us to get direct insight in their functions during normal and pathologic conditions of the CNS.

Cutting edge: CD43 functions as a T cell counterreceptor for the macrophage adhesion receptor sialoadhesin (Siglec-1).

Sialoadhesin (Siglec-1) is a macrophage-restricted sialic acid-binding receptor that mediates interactions with hemopoietic cells, including lymphocytes. In this study, we identify sialoadhesin counterreceptors on T lymphocytes. Several major glycoproteins (85, 130, 240 kDa) were precipitated by sialoadhesin-Fc fusion proteins from a murine T cell line (TK-1). Binding of sialoadhesin to these glycoproteins was sialic acid dependent and was abolished by mutation of a critical residue (R97A) of the sialic acid binding site in the membrane distal Ig-like domain of sialoadhesin. The 130- and 240-kDa sialoadhesin-binding glycoproteins were identified as the sialomucins CD43 and P-selectin glycoprotein ligand 1 (CD162), respectively. CD43 expressed in COS cells supported increased binding to immobilized sialoadhesin. Finally, sialoadhesin bound different glycoforms of CD43 expressed in Chinese hamster ovary cells, including unbranched (core 1) and branched (core 2) O:-linked glycans, that are normally found on CD43 in resting and activated T cells, respectively. These results identify CD43 as a T cell counterreceptor for sialoadhesin and suggest that in addition to its anti-adhesive role CD43 may promote cell-cell interactions.

Differential galactosylation of neuronal and haematopoietic signal regulatory protein-alpha determines its cellular binding-specificity.

Signal regulatory protein-alpha (SIRP alpha) is a member of the Ig superfamily selectively expressed by neuronal and myeloid cells. The molecule mediates functional interactions with CD47/integrin-associated protein. Here we provide evidence for the tissue-specific glycosylation of neuronal and haematopoietic SIRP alpha. We demonstrate a major difference in the galactosylation of N-linked glycans isolated from neuronal (i.e. brain-derived) SIRP alpha as compared to myeloid (i.e. spleen-derived) SIRP alpha, with neuronal SIRP alpha almost completely lacking galactose. beta 4-galactosyltransferase assays demonstrated that this is most likely due to a low galactosylation capacity of the brain. In order to investigate the role of galactosylation of SIRP alpha in cellular interactions, soluble recombinant SIRP alpha glycoforms containing galactose (SIRP alpha-Fc) or lacking galactose (SIRP alpha(Delta Gal)-Fc) were produced. Binding studies demonstrated superior binding of SIRP alpha(Delta Gal)-Fc to cerebellar neurons and isolated lymphocytes. In contrast, SIRP alpha-Fc bound relatively strong to macrophages. These data show that the galactosylation of SIRP alpha determines its cellular binding specificity.

Rat macrophages: membrane glycoproteins in differentiation and function.

Macrophages (mphi) play a crucial role in the immune system. The rat offers unique advantages for studying the biology of mphi. Firstly, monoclonal antibodies (mAb) against many rat mphi surface glycoproteins have become available. These have not only demonstrated a considerable heterogeneity among mphi, but have also allowed the characterization of various mphi surface molecules in terms of structure, expression regulation and function. Furthermore, substantial numbers of rat mphi can be isolated from various sites (e.g. blood, peritoneal cavity, lung and other tissues), enabling proper molecular and functional analysis of these mphi populations. Finally, a number of (unique) experimental models for human diseases have been developed in the rat, making possible the evaluation of the involvement of mphi in pathogenesis. For this, a method for the selective elimination of mphi from various tissues in vivo has proven very useful. Here, we will review the contribution that the rat has made to understanding the immunobiology of mphi. In particular, we will discuss the surface (glyco)proteins on rat mphi in differentiation and function, and the involvement of mphi in rat models of disease.

Monoclonal antibodies against rat leukocyte surface antigens.

Monoclonal antibodies have proven to be powerful tools for studying the properties of leukocyte surface antigens and the cells that express them. In the past decades many monoclonal antibodies (mAb) for identifying the different rat leukocyte surface antigens have been described. A list of mAb is provided in Table I below. The rat leukocyte surface antigens are divided into different sections, including rat CD antigens (a), rat leukocyte surface antigens without CD designation (b), rat major histocompatibility complex (MHC) antigens (c), rat T-cell receptors (d) and rat immunoglobulins (e). The molecular and functional characteristics of rat leukocyte surface antigens are discussed in more detail in some of the other chapters of this issue (e.g. Van den Berg et al., p. 45). A more extensive overview of the properties of leukocyte surface antigens is provided by Barclay et al.

Ultrastructural localisation of sialoadhesin (siglec-1) on macrophages in rodent lymphoid tissues.

In previous studies it has been demonstrated that sialoadhesin is a macrophage-restricted adhesion receptor for lymphocytes and myeloid cells. It is under normal circumstances expressed by subpopulations of macrophages in lymphoid and haemopoietic tissues. In this study different immunoelectronmicroscopical techniques are used to investigate the ultrastructural localisation of sialoadhesin within the lymph node and spleen of rodents. The results show that sialoadhesin is selectively expressed by a subset of macrophages in peripheral lymphoid tissues. Sialoadhesin was localised predominantly on the plasma membrane and in particular in areas of intimate contact with lymphocytes, thereby visualizing putative local interaction between these cells. Interestingly, sialoadhesin was also detected in intracellular vesicles that were apparently taken up by macrophages. These findings are consistent with the putative role of sialoadhesin in local cell-cell interactions in lymphoid tissues. Surprisingly, sialoadhesin was also found at contact points of macrophages with other macrophages, sinus-lining cells and reticulum cells, suggesting that sialoadhesin also mediates interactions with these cell types.

Dexamethasone promotes phagocytosis and bacterial killing by human monocytes/macrophages in vitro.

One of the actions of glucocorticoids (GC) in multiple sclerosis (MS) is an inhibitory effect on demyelination. This can be caused by a reduction in the number of infiltrating macrophages and/or by an effect on the phagocytosis of myelin. Here we investigate the effect of GC on the phagocytosis of myelin. Contrary to what was expected, we found that incubation of human monocytes with dexamethasone (DEX) for 48 h augmented (approximately threefold) the phagocytosis of myelin. This enhancement of phagocytosis by human monocytes was not restricted to myelin. Phagocytosis of various particles mediated by different macrophage receptors was increased by DEX. We found that not only the phagocytosis of Staphylococcus aureus bacteria was augmented, but also the killing of these bacteria was at least twice as effective after culture with DEX. Tumor necrosis factor alpha production of human monocyte-derived macrophages induced by lipopolysaccharide and S. aureus was suppressed by DEX. Together our results show that DEX promotes the phagocytosis of particles by human monocytes and thereby may contribute to tissue repair after immune-mediated tissue damage or infection. These data shed a new light on the clinical application of GC.

Synaptic assembly of the brain in the absence of neurotransmitter secretion.

Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.

Systemic anti-IFN-gamma treatment and role of macrophage subsets in the foreign body reaction to dermal sheep collagen in rats.

The application of a biomaterial induces a foreign body reaction. By controlling this reaction, biocompatibility could be improved. We previously demonstrated that impregnation of a biodegradable biomaterial with antibodies against interferon-gamma (IFN-gamma) inhibits the foreign body reaction. In this study we investigate whether systemic administration of the antibody can induce similar reactions. Several parameters are compared between control and anti-IFN-gamma-treated rats: cellular ingrowth; degradation of the biomaterial; ingrowth of macrophage (MO) subsets, T cells, B cells, NK cells, and granulocytes; and expression of the major histocompatibility complex class II (MHC class II) molecule on antigen presenting cells. Treatment with anti-IFN-gamma results in increased cellular ingrowth and biomaterial degradation and a decreased expression of MHC class II. Overall, systemic treatment with anti-IFN-gamma is insufficient to modulate the foreign body reaction. This suggests an alternative mechanism for MO activation besides IFN-gamma. The role of T cells and MO subsets in the foreign body reaction is discussed.

Core alpha1-->3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep.

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAcbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-Asn (core alpha1-->3-Fuc) and Xylbeta1-->2Manbeta1-->4GlcNAcbeta1-R (core beta1-->2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha1-->3-Fuc epitope. Evidence is presented that core alpha1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core alpha1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core alpha1-->3-Fuc and beta1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.