Phagocytosis Is the Main CR3-Mediated Function Affected by the Lupus-Associated Variant of CD11b in Human Myeloid Cells

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis.     

Neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues

Antigen-transporting cells take up pathogens, and then migrate from sites of inflammation to secondary lymphoid tissues to induce an immune response. Among antigen-transporting cells, dendritic cells (DCs) are believed to be the most potent and professional antigen-presenting cells that can stimulate naïve T cells. However, the cells that transport antigens, tumor cell antigens in particular, have not been clearly identified. In this study we have analyzed what types of cells transport tumor cell antigens to secondary lymphoid tissues. We show that neutrophils, monocytes and macrophages but not DCs engulf X-irradiated P388 leukemic cells after their injection into the peritoneal cavity, and that neutrophils and monocytes but not macrophages migrate to the parathymic lymph nodes (pLN), the blood, and then the spleen. The monocytes in the pLN comprise Gr-1⁻ and Gr-1⁺ ones, and some of these cells express CD11c. Overall, this study demonstrates that neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.     

A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues

Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b^- DC, and CD11b⁺ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.     

Phagocytosis and Respiratory Burst Activity in Lumpsucker (Cyclopterus lumpus L.) Leucocytes Analysed by Flow Cytometry

In the present study, we have isolated leucocytes from peripheral blood, head kidney and spleen from lumpsucker (Cyclopterus lumpus L.), and performed functional studies like phagocytosis and respiratory burst, as well as morphological and cytochemical analyses. Different leucocytes were identified, such as lymphocytes, monocytes/macrophages and polymorphonuclear cells with bean shaped or bilobed nuclei. In addition, cells with similar morphology as described for dendritic cells in trout were abundant among the isolated leucocytes. Flow cytometry was successfully used for measuring phagocytosis and respiratory burst activity. The phagocytic capacity and ability were very high, and cells with different morphology in all three leucocyte preparations phagocytised beads rapidly. Due to lack of available cell markers, the identity of the phagocytic cells could not be determined. The potent non-specific phagocytosis was in accordance with a high number of cells positive for myeloperoxidase, an enzyme involved in oxygen-dependent killing mechanism present in phagocytic cells. Further, high respiratory burst activity was present in the leucocytes samples, verifying a potent oxygen- dependent degradation. At present, the specific antibody immune response could not be measured, as immunoglobulin or B-cells have not yet been isolated. Therefore, analyses of the specific immune response in this fish species await further clarification. The present study presents the first analyses of lumpsucker immunity and also the first within the order Scopaeniformes.     

Further characterization of low density mononuclear cells: FACS-assisted analysis of human MLR stimulators

Previously, we reported that all of the potent stimulators of the allogeneic mixed leukocyte reaction (MLR) are contained in a heterogeneous low density fraction of human peripheral blood mononuclear (PBM) cells. We have further characterized human MLR stimulators by staining them with highly specific monoclonal antibodies, and then analyzing and separating them with the fluorescence-activated cell sorter. These studies revealed two populations of low density cells with potent allogeneic stimulatory activity. One population is a monocyte subset that reacts with anti-OKM1, MO.2, and expresses C3b as well as Fc-IgG receptors. The second population exhibits even greater stimulatory capacity and does not express any of these monocyte markers. Moreover, these cells are not phagocytic and do not react with alpha-naphthyl esterase. They comprise approximately 10% of the low density fraction or 0.5% of PBM. These cells are most likely lymphoid dendritic cells, described in various species as potent MLR stimulators. In contrast to the allogeneic MLR, only the low density cell type exhibiting dendritic cell characteristics induced a potent autologous MLR.     

Thymic Dendritic Cells Are Primary Targets for the Oncogenic Virus SL3-3

The murine retrovirus SL3-3 causes malignant transformation of thymocytes and thymic lymphoma in mice of the AKR and NFS strains when they are inoculated neonatally. The objective of the present study was to identify the primary target cells for the virus in the thymuses of these mice. Immunohistochemical studies of the thymus after neonatal inoculation of the SL3-3 virus showed that cells expressing the viral envelope glycoprotein (gp70⁺ cells) were first seen at 2 weeks of age. These virus-expressing cells were found in the cortex and at the corticomedullary junction in both mouse strains. The gp70⁺ cells had the morphology and immunophenotype of dendritic cells. They lacked macrophage-specific antigens. Cell separation studies showed that bright gp70⁺ cells were detected in a fraction enriched for dendritic cells. At 3 weeks of age, macrophages also expressed gp70. At that time, both gp70⁺ dendritic cells and macrophages were found at the corticomedullary junction and in foci in the thymic cortex. At no time during this 3-week period was the virus expressed in cortical and medullary epithelial cells or in thymic lymphoid cells. Infectious cell center assays indicated that cells expressing infectious virus were present in small numbers at 2 weeks after inoculation but increased at 5 weeks of age by several orders of magnitude, indicating virus spread to the thymic lymphoid cells. Thus, at 2 weeks after neonatal inoculation of SL3-3, thymic dendritic cells are the first cells to express the virus. At 3 weeks of age, macrophages also express the virus. In subsequent weeks, the virus spreads to the thymocytes. This pathway of virus expression in the thymus allows the inevitable provirus integration in a thymocyte that results in a clonal lymphoma.     

The intracellular lifestyle of Francisella noatunensis in Atlantic cod (Gadus morhua L.) leucocytes

Francisella noatunensis causes the systemic granulomatous inflammatory disease, francisellosis in cod. Little is known about the lifestyle of this facultative intracellular bacterium within cod leucocytes. We have examined the interaction of this bacterium with phagocytic cells isolated from cod with emphasis on monocytes, macrophages, neutrophils and phagocytic B-cells. It is clear from confocal microscopy sections through adherent cell preparations that numerous bacteria were located intracellularly following in vitro infection in monocytes and macrophages. In these sections bacteria were immunostained and cell actin was stained using Alexa Fluor^® 488 phalloidin. Bacteria were observed in close association with neutrophils and intracellularly (low numbers) in B-cells. Bacteria were observed more frequently in head kidney- than in peripheral blood- and spleen- leucocytes. Following infection, bacteria were initially observed grouped together and located close to the nucleus. Later they were found spread within the cytoplasm. This indicates egression of F. noatunensis from the phagosome to the cytoplasm where replication possibly takes place. It may be hypothesised that the bacteria may alter maturation of the phagosome and thus, avoid the potent intracellular killing mechanisms of phagocytic cells. The intracellular lifestyle involving escape to cytoplasm prior to fusion with the lysosome may have consequences for vaccine development as well as antibiotic treatment of infected cod.     

Neutrophils process exogenous bacteria via an alternate class I MHC processing pathway for presentation of peptides to T lymphocytes

Peptides that are presented by class I MHC (MHC-I) molecules derive from cytosolic Ags processed via the conventional MHC-I pathway or exogenous Ags processed via alternate MHC-I processing mechanisms. Alternate MHC-I processing by macrophages and dendritic cells allows presentation of peptides from particulate Ags, including bacteria. Despite the established phagocytic activity of neutrophils, MHC-I processing and presentation of phagocytosed Ags by neutrophils has not been investigated. Murine neutrophils from peritoneal exudates were shown to express MHC-I molecules and tested for the ability to process HB101.Crl-OVA, Escherichia coli transfected to express a fusion protein containing the 257-264 epitope of OVA. Neutrophils were found to process HB101.Crl-OVA and present OVA(257-264)-K(b) complexes to CD8OVA T hybridoma cells via a pathway that was resistant to brefeldin A, an inhibitor of anterograde endoplasmic reticulum-Golgi transport, and lactacystin, a proteasome inhibitor. These results suggest that neutrophils process phagocytosed bacteria via a vacuolar alternate MHC-I pathway that does not involve cytosolic processing. In addition, neutrophils were found to secrete or "regurgitate" processed peptide that was subsequently presented by neighboring prefixed macrophages or dendritic cells. Thus, neutrophils may influence T cell responses to bacteria, either by directly presenting peptide-MHC-I complexes or by delivering peptides to other APCs for presentation. Hypothetically, neutrophils may directly present peptide to effector T cells in vivo at sites of inflammation, inducing cytokine production, whereas dendritic cells in receipt of neutrophil-derived antigenic peptides may migrate to lymphoid organs to initiate T cell responses.     

LncRNAs and immunity: watchdogs for host pathogen interactions

Immune responses combat various infectious agents by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. The polygenic responses to these external stimuli are temporally and coordinately regulated. Specific lncRNAs are induced to modulate innate and adaptive immune responses which can function through various target interactions like RNA-DNA, RNA-RNA, and RNA-protein interaction and hence affect the immunogenic regulation at various stages of gene expression. LncRNA are found to be present in various immune cells like monocytes, macrophages, dendritic cells, neutrophils, T cells and B cells. They have been shown to be involved in many biological processes, including the regulation of the expression of genes, the dosage compensation and genomics imprinting, but the knowledge how lncRNAs are regulated and how they alter cell differentiation/function is still obscure. Further dysregulation of lncRNA has been seen in many diseases, but as yet very less research has been carried out to understand the role of lncRNAs in regulation during host-pathogens interactions. In this review, we summarize the functional developments and mechanism of action of lncRNAs, in immunity and defense of host against pathogens.     

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN- dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.     

Human splenic sinusoidal lining cells express antigens associated with monocytes, macrophages, endothelial cells, and T lymphocytes

The antigenic and functional properties of splenic sinusoidal lining cells (SLC) have not been studied extensively. Some investigators have suggested that SLC are actively phagocytic, and thus part of the mononuclear phagocyte system. Others dispute this and assign the functions of endothelial cells to the SLC. During studies in situ of phenotypic subpopulations of human splenic macrophages (M phi), we found that SLC share membrane antigens (HLA-DR and OKM5), an enzyme (lysozyme), and histochemical properties (nonspecific esterases) with monocytes and M phi. In addition, we showed that LSC, like endothelial cells, synthesize factor VIII of the clotting system and also bear the receptor for transferrin. Our previous studies found that SLC express the antigens found on helper/inducer (OKT4, Leu-3a,b) and suppressor/cytotoxic (OKT8, Leu-2a) T lymphocyte subsets. We have confirmed these observations, and have shown by means of preincubation with soluble complexes of anti-human IgG-human IgG that the detection of T cell and other antigens on SLC is not due to nonspecific binding of antibodies by Fc receptors. By using techniques designed to isolate and purify splenic M phi, we were able to obtain SLC in suspension and to demonstrate that they retain the antigens detected in situ. Thus, the human splenic SLC expresses a unique combination of antigens, histochemical properties, and cell products in common with monocytes, M phi, and T lymphocytes.     

Circulating CD2+ monocytes are dendritic cells

Low levels of CD2 have been described on subsets of monocytes, macrophages, and dendritic cells. CD2 is expressed on about one-third of circulating monocytes, at levels one-half log lower than on T or NK cells, representing 2-4% of PBMC. FACS analysis of CD2+ and CD2- monocytes revealed no significant difference in the expression of adhesion molecules (CD11a/b/c), class II Ags (HLA-DR, -DQ, -DP), myeloid Ags (CD13, CD14, CD33), or costimulatory molecules (CD80, CD86). Freshly isolated CD2+ and CD2- monocytes were morphologically indistinguishable by phase contrast microscopy. However, scanning electron microscopy revealed large prominent ruffles on CD2+ monocytes in contrast to small knob-like projections on CD2- monocytes. After 2 days of culture, the CD2+ monocytes largely lost CD14 expression and developed distinct dendrites, whereas the CD2- monocytes retained surface CD14 and remained round or oval. Freshly isolated CD2+ monocytes were more potent inducers of the allogeneic MLR and more efficiently induced proliferation of naive T cells in the presence of HIV-1 gp120 than did CD2- monocytes. After culture in the presence of GM/CSF and IL-4, CD2+ monocytes were up to 40-fold more potent than monocyte-derived dendritic cells or CD2- monocytes at inducing allogeneic T cell proliferation. These findings suggest that circulating CD2+ and CD2- monocytes are dendritic cells and the precursors of macrophages, respectively. Thus, dendritic cells are far more abundant in the blood than previously thought, and they and precursors of macrophages exist in the circulation as phenotypically, morphologically, and functionally distinct monocyte populations.     

Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions

Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×10⁶±0.3×10⁶ floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.     

Relationships between Levels of Serum IgE,Cell-Bound IgE,and IgE-Receptors on Peripheral Blood Cells in a Pediatric Population

Background

Elevated serum immunoglobulin (Ig) E is a diagnostic marker of immediate-type allergic reactions. We hypothesize that serum IgE does not necessarily reflect total body IgE because in vivo IgE can be bound to cell surface receptors such as FcεRI and FcεRII (CD23). The aim of this study was to analyze the relationships between levels of serum IgE, cell-bound IgE, and IgE-receptors on peripheral blood cells in a pediatric population.

Methodology

Whole blood samples from 48 children (26 boys, 22 girls, mean age 10,3±5,4 years) were analyzed by flow cytometry for FcεRI, CD23, and cell-bound IgE on dendritic cells (CD11c+MHC class II+), monocytes (CD14+), basophils (CD123+MHC class II-) and neutrophils (myeloperoxidase+). Total serum IgE was measured by ELISA and converted into z-units to account for age-dependent normal ranges. Correlations were calculated using Spearman rank correlation test.

Principal Findings

Dendritic cells, monocytes, basophils, and neutrophils expressed the high affinity IgE-receptor FcεRI. Dendritic cells and monocytes also expressed the low affinity receptor CD23. The majority of IgE-receptor positive cells carried IgE on their surface. Expression of both IgE receptors was tightly correlated with cell-bound IgE. In general, cell-bound IgE on FcεRI+ cells correlated well with serum IgE. However, some patients carried high amounts of cell-bound IgE despite low total serum IgE levels.

Conclusion/Significance

In pediatric patients, levels of age-adjusted serum IgE, cell-bound IgE, and FcεRI correlate. Even in the absence of elevated levels of serum IgE, cell-bound IgE can be detected on peripheral blood cells in a subgroup of patients.     

Disparities in TLR5 expression and responsiveness to flagellin in equine neutrophils and mononuclear phagocytes

As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.     

Direct visualization of peptide/MHC complexes at the surface and in the intracellular compartments of cells infected in vivo by Leishmania major

Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of cells that were infected in vivo.     

F2L, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating Fpr2, the low-affinity N-formylpeptide receptor

F2L (formylpeptide receptor (FPR)-like (FPRL)-2 ligand), a highly conserved acetylated peptide derived from the amino-terminal cleavage of heme-binding protein, is a potent chemoattractant for human monocytes and dendritic cells, and inhibits LPS-induced human dendritic cell maturation. We recently reported that F2L is able to activate the human receptors FPRL-1 and FPRL2, two members of the FPR family, with highest selectivity and affinity for FPRL2. To facilitate delineation of mechanisms of F2L action in vivo, we have now attempted to define its mouse receptors. This is complicated by the nonequivalence of the human and mouse FPR gene families (three vs at least eight members, respectively). When cell lines were transfected with plasmids encoding the eight mouse receptors, only the one expressing the receptor Fpr2 responded to F2L (EC(50) approximately 400 nM for both human and mouse F2L in both calcium flux and cAMP inhibition assays). This value is similar to F2L potency at human FPRL1. Consistent with this, mouse neutrophils, which like macrophages and dendritic cells express Fpr2, responded to human and mouse F2L in both calcium flux and chemotaxis assays with EC(50) values similar to those found for Fpr2-expressing cell lines ( approximately 500 nM). Moreover, neutrophils from mice genetically deficient in Fpr2 failed to respond to F2L. Thus, Fpr2 is a mouse receptor for F2L, and can be targeted for the study of F2L action in mouse models.