Amyloid-ß aggregates activate peripheral monocytes in mild cognitive impairment.

The peripheral immune system is important in neurodegenerative diseases, both in protecting and inflaming the brain, but the underlying mechanisms remain elusive. Alzheimer's Disease is commonly preceded by a prodromal period. Here, we report the presence of large Aß aggregates in plasma from patients with mild cognitive impairment (n = 38). The aggregates are associated with low level Alzheimer's Disease-like brain pathology as observed by 11C-PiB PET and 18F-FTP PET and lowered CD18-rich monocytes. We characterize complement receptor 4 as a strong binder of amyloids and show Aß aggregates are preferentially phagocytosed and stimulate lysosomal activity through this receptor in stem cell-derived microglia. KIM127 integrin activation in monocytes promotes size selective phagocytosis of Aß. Hydrodynamic calculations suggest Aß aggregates associate with vessel walls of the cortical capillaries. In turn, we hypothesize aggregates may provide an adhesion substrate for recruiting CD18-rich monocytes into the cortex. Our results support a role for complement receptor 4 in regulating amyloid homeostasis.

Expression of the phagocytic receptors αMß2 and αXß2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells.

Activation of the integrin phagocytic receptors CR3 (αMß2, CD11b/CD18) and CR4 (αXß2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to ß2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (ß2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMß2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMß2 and αXß2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXß2.

Allosteric activation and silencing of leukocyte ß2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXß2 ligand-binding domain-the αX I-domain-corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH- and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXß2. This work highlights how intrinsically flexible pH- and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Complement Potentiates Immune Sensing of HIV-1 and Early Type I Interferon Responses.

Complement-opsonized HIV-1 triggers efficient antiviral type I interferon (IFN) responses in dendritic cells (DCs), which play an important role in protective responses at the earliest stages in retroviral infection. In contrast, HIV-1 suppresses or escapes sensing by STING- and MAVS-associated sensors. Here, we identified a complement receptor-mediated sensing pathway, where DCs are activated in CCR5/RLR (RIG-I/MDA5)/MAVS/TBK1-dependent fashion. Increased fusion of complement-opsonized HIV-1 via complement receptor 4 and CCR5 leads to increased incoming HIV-1 RNA in the cytoplasm, sensed by a nonredundant cooperative effect of RIG-I and MDA5. Moreover, complement-opsonized HIV-1 down-modulated the MAVS-suppressive Raf-1/PLK1 pathway, thereby opening the antiviral recognition pathway via MAVS. This in turn was followed by MAVS aggregation and subsequent TBK1/IRF3/NF-κB activation in DCs exposed to complement- but not non-opsonized HIV-1. Our data strongly suggest that complement is important in the induction of efficient antiviral immune responses by preventing HIV-1 suppressive mechanisms as well as inducing specific cytosolic sensors. IMPORTANCE Importantly, our study highlights an unusual target on DCs-the α chain of complement receptor 4 (CR4) (CD11c)-for therapeutic interventions in HIV-1 treatment. Targeting CD11c on DCs mediated a potent antiviral immune response via clustering of CR4 and CCR5 and subsequent opening of an antiviral recognition pathway in DCs via MAVS. This novel finding might provide novel tools for specifically boosting endogenous antiviral immunity via CR4, abundantly expressed on multiple DC subsets.

Biologia Futura: stories about the functions of ß2-integrins in human phagocytes.

Integrins are essential membrane proteins that provide a tightly regulated link between the extracellular matrix and the intracellular cytoskeletal network. These cell surface proteins are composed of a non-covalently bound α chain and ß chain. The leukocyte-specific complement receptor 3 (CR3, αMß2, CD11b/CD18) and complement receptor 4 (CR4, αXß2, CD11c/CD18) belong to the family of ß2-integrins. These receptors bind multiple ligands like iC3b, ICAMs, fibrinogen or LPS, thus allowing them to partake in phagocytosis, cellular adhesion, extracellular matrix rearrangement and migration. CR3 and CR4 were generally expected to mediate identical functions due to their structural homology, overlapping ligand specificity and parallel expression on human phagocytes. Despite their similarities, the expression level and function of these receptors differ in a cell-type-specific manner, both under physiological and inflammatory conditions.We investigated comprehensively the individual role of CR3 and CR4 in various functions of human phagocytes, and we proved that there is a "division of labour" between these two receptors. In this review, I will summarize our current knowledge about this area.

Extracellular matrix protein-1 secretory isoform promotes ovarian cancer through increasing alternative mRNA splicing and stemness.

Extracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXß2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXß2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXß2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXß2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.

BCR activated CLL B cells use both CR3 (CD11b/CD18) and CR4 (CD11c/CD18) for adhesion while CR4 has a dominant role in migration towards SDF-1.

Chronic lymphocytic leukaemia (CLL) is the most common leukaemia in the western world. In previous studies, various proportion of patients was found to carry CD11b+ or CD11c+ B cells whose presence was an unfavourable prognostic factor. The exact mechanism however, how these receptors contribute to the pathogenesis of CLL has not been revealed so far. Here we analysed the role of CD11b and CD11c on B cells of CLL patients in the adhesion to fibrinogen and in the migration towards stromal cell derived factor-1 (SDF-1) and studied the role of CR4 in the adherence of the CD11c+ B cell line BJAB. We observed that both CR3 and CR4 mediate adhesion of the malignant B cells. Moreover, we found, that CR4 was strongly involved in the migration of the leukemic cells towards the chemoattractant SDF-1. Our data suggest that CR3 and CR4 are not only passive markers on CLL B cells, but they might contribute to the progression of the disease. Since the role of SDF-1 is prominent in the migration of CLL cells into the bone marrow where their survival is supported, our findings help to understand how the presence of CD11c on leukemic B cells can worsen the prognosis of chronic lymphocytic leukaemia.

HIV-1 subverts the complement system in semen to enhance viral transmission.

Semen is important in determining HIV-1 susceptibility but it is unclear how it affects virus transmission during sexual contact. Mucosal Langerhans cells (LCs) are the first immune cells to encounter HIV-1 during sexual contact and have a barrier function as LCs are restrictive to HIV-1. As semen from people living with HIV-1 contains complement-opsonized HIV-1, we investigated the effect of complement on HIV-1 dissemination by human LCs in vitro and ex vivo. Notably, pre-treatment of HIV-1 with semen enhanced LC infection compared to untreated HIV-1 in the ex vivo explant model. Infection of LCs and transmission to target cells by opsonized HIV-1 was efficiently inhibited by blocking complement receptors CR3 and CR4. Complement opsonization of HIV-1 enhanced uptake, fusion, and integration by LCs leading to an increased transmission of HIV-1 to target cells. However, in the absence of both CR3 and CR4, C-type lectin receptor langerin was able to restrict infection of complement-opsonized HIV-1. These data suggest that complement enhances HIV-1 infection of LCs by binding CR3 and CR4, thereby bypassing langerin and changing the restrictive nature of LCs into virus-disseminating cells. Targeting complement factors might be effective in preventing HIV-1 transmission.

Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia: a genetic and functional study.

OBJECTIVE: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. DESIGN: A case-control study. SETTING: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. POPULATION: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. METHODS: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. MAIN OUTCOME MEASURES: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. RESULTS: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. CONCLUSIONS: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. TWEETABLE ABSTRACT: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXß2.

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMß2 and αXß2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective ß2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α'- chain (α'NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α'NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.

Role of Complement Receptors (CRs) on DCs in Anti-HIV-1 Immunity.

Upon entry of human immunodeficiency virus 1 (HIV-1) into the host, innate immune mechanisms are acting as a first line of defense, that considerably also modify adaptive immunity by the provision of specific signals. Innate and adaptive immune responses are intimately linked and dendritic cells (DCs) together with complement (C) play an important role in regulation of adaptive immunity. Initially, the role of complement was considered to primarily support - or COMPLEMENT - cytolytic actions of antibodies or antibody-complexed antigens (immune complexes, ICs) or directly kill the pathogens by complement-mediated lysis. Recently, the role of complement was revised and found to significantly augmenting and modulating adaptive immunity, in particular against viruses. Complement and DCs are therefore predestined to open novel avenues for antiviral research and potential therapeutic interventions. Recent studies on interactions of complement-opsonized HIV-1 with DCs demonstrated a high potential of such primed DCs to initiate efficient antiviral and cytotoxic anti-HIV-1 immunity and complement-coated viral particles shift DCs functions via CR3 and CR4 in an antithetic manner. This review will focus on our current knowledge of CR3 and CR4 actions on DCs during HIV-1 binding and the outcome of infection influenced by entry and signaling pathways.

CR4 Signaling Contributes to a DC-Driven Enhanced Immune Response Against Complement-Opsonized HIV-1.

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.

Differential Interactions of Serum and Bronchoalveolar Lavage Fluid Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.

Phagocytic Integrins: Activation and Signaling.

Phagocytic integrins are endowed with the ability to engulf and dispose of particles of different natures. Evolutionarily conserved from worms to humans, they are involved in pathogen elimination and apoptotic and tumoral cell clearance. Research in the field of integrin-mediated phagocytosis has shed light on the molecular events controlling integrin activation and their effector functions. However, there are still some aspects of the regulation of the phagocytic process that need to be clarified. Here, we have revised the molecular events controlling phagocytic integrin activation and the downstream signaling driving particle engulfment, and we have focused particularly on αMß2/CR3, αXß2/CR4, and a brief mention of αVß5/αVß3integrins.

The differential role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in the adherence, migration and podosome formation of human macrophages and dendritic cells under inflammatory conditions.

CR3 and CR4, the leukocyte specific ß2-integrins, involved in cellular adherence, migration and phagocytosis, are often assumed to have similar functions. Previously however, we proved that under physiological conditions CR4 is dominant in the adhesion to fibrinogen of human monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs). Here, using inflammatory conditions, we provide further evidence that the expression and function of CR3 and CR4 are not identical in these cell types. We found that LPS treatment changes their expression differently on MDMs and MDDCs, suggesting a cell type specific regulation. Using mAb24, specific for the high affinity conformation of CD18, we proved that the activation and recycling of ß2-integrins is significantly enhanced upon LPS treatment. Adherence to fibrinogen was assessed by two fundamentally different approaches: a classical adhesion assay and a computer-controlled micropipette, capable of measuring adhesion strength. While both receptors participated in adhesion, we demonstrated that CR4 exerts a dominant role in the strong attachment of MDDCs. Studying the formation of podosomes we found that MDMs retain podosome formation after LPS activation, whereas MDDCs lose this ability, resulting in a significantly reduced adhesion force and an altered cellular distribution of CR3 and CR4. Our results suggest that inflammatory conditions reshape differentially the expression and role of CR3 and CR4 in macrophages and dendritic cells.

Size-Selective Phagocytic Clearance of Fibrillar α-Synuclein through Conformational Activation of Complement Receptor 4.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

An acidic loop within the human soluble CD23 protein may direct the interaction between sCD23 and the αXß2 integrin.

CD23 is involved in a myriad of immune reactions. It is not only a receptor for IgE, but also functions in the regulation of IgE synthesis, isotype switching in B cells, and induction of the inflammatory response. These effector functions of CD23 arise through its interaction with another leukocyte-specific cell surface receptor - the ß2 integrin subfamily. It has been shown that CD23 is also capable of interacting with the ß3 and ß5 integrin ß-subunit of integrins via a basic RKC motif in a metal cation-independent fashion. In this study the interaction was probed for whether or not the RKC motif governs the interaction between CD23 and the αXß2 integrin as well. This was done by performing bioinformatic docking predictions between CD23 and αXß2 integrin αI domain and SPR spectroscopy analysis of the interaction. This revealed that in the absence of cations, the RKC motif is involved in interaction with the integrin αI domain. However, in the presence of divalent metal cations the interaction showed the involvement of a novel acidic motif within the CD23 protein. This same pattern of interaction was seen in docking predictions between CD23 and the ß3I-like domain. This study thus presents an alternative site as a possible contributor to the CD23-integrin interaction exhibiting cation-dependence.

Structural Immunology of Complement Receptors 3 and 4.

Complement receptors (CR) 3 and 4 belong to the family of beta-2 (CD18) integrins. CR3 and CR4 are often co-expressed in the myeloid subsets of leukocytes, but they are also found in NK cells and activated T and B lymphocytes. The heterodimeric ectodomain undergoes considerable conformational change in order to switch the receptor from a structurally bent, ligand-binding in-active state into an extended, ligand-binding active state. CR3 binds the C3d fragment of C3 in a way permitting CR2 also to bind concomitantly. This enables a hand-over of complement-opsonized antigens from the cell surface of CR3-expressing macrophages to the CR2-expressing B lymphocytes, in consequence acting as an antigen presentation mechanism. As a more enigmatic part of their functions, both CR3 and CR4 bind several structurally unrelated proteins, engineered peptides, and glycosaminoglycans. No consensus motif in the proteinaceous ligands has been established. Yet, the experimental evidence clearly suggest that the ligands are primarily, if not entirely, recognized by a single site within the receptors, namely the metal-ion dependent adhesion site (MIDAS). Comparison of some recent identified ligands points to CR3 as inclined to bind positively charged species, while CR4, by contrast, binds strongly negative-charged species, in both cases with the critical involvement of deprotonated, acidic groups as ligands for the Mg2+ ion in the MIDAS. These properties place CR3 and CR4 firmly within the realm of modern molecular medicine in several ways. The expression of CR3 and CR4 in NK cells was recently demonstrated to enable complement-dependent cell cytotoxicity toward antibody-coated cancer cells as part of biological therapy, constituting a significant part of the efficacy of such treatment. With the flexible principles of ligand recognition, it is also possible to propose a response of CR3 and CR4 to existing medicines thereby opening a possibility of drug repurposing to influence the function of these receptors. Here, from advances in the structural and cellular immunology of CR3 and CR4, we review insights on their biochemistry and functions in the immune system.

Facilitating THP-1 macrophage studies by differentiating and investigating cell functions in polystyrene test tubes.

Macrophage cell lines are a useful model to explore the properties of primary macrophages. However, a major limitation in the use of these cells is that when they are differentiated, they become adherent and hence present with the same limitation as natural macrophages. The cells need to be detached and are often subjected to detachment techniques such as detachment buffers containing proteolytic enzymes or scraping with a rubber 'policeman'. These steps are time-consuming, reduce cell yields as well as cell viability and function. We have therefore investigated the possibility of differentiating the human macrophage THP-1 cell line in polystyrene FACS tubes to enable cells to be directly used for investigations by flow cytometry. Here we demonstrate that when the human macrophage cell line THP-1 are cultured in FACS tubes with phorbol myristate acetate added, they undergo differentiation into macrophages, assessed morphologically and by autofluorescence expression, in a similar manner to those cultured in tissue culture dishes. The cells can be readily washed and adjusted in concentration by centrifugation in the same tubes and can be directly tested for expression of cell surface markers and function by flow cytometry. This avoids the use of either detachment reagents or physical cell scraping. Consequently, we showed that the tube culture method results in increased cell yield and viability compared to those subjected to detachment procedures. The tube method generated functional macrophages which expressed the complement receptors, CR3 and CR4, and effectively phagocytosed complement opsonised Staphylococcus aureus via these receptors.

Functional studies of chronic lymphocytic leukemia B cells expressing ß2-integrin type complement receptors CR3 and CR4.

The expression and role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in B cells are not yet explored in contrast to myeloid cells, where these ß2-integrin type receptors are known to participate in various cellular functions, including phagocytosis, adherence and migration. Here we aimed to reveal the expression and role of CR3 and CR4 in human B cells. In B cells of healthy donors CR3 and CR4 are scarcely expressed. However, two patients with chronic lymphocytic leukemia (CLL) characterized by a peculiar immune-phenotype containing both CD5-positive and CD5-negative B cell populations made possible to study these molecules in distinct B cell subsets. We found that CD11b and CD11c were expressed on both CD5-positive and CD5-negative B cells, albeit to different extents. Our data suggest that these receptors are involved in spreading, since this activity of CpG-activated B cells on fibrinogen could be partially blocked by monoclonal antibodies specific for CD11b or CD11c. CpG-stimulation lead to proliferation of both CD5-positive and CD5-negative B cells of the patients with a less pronounced effect on the CD5-positive cells. In contrast to normal B cells, CLL B cells of both patients reacted to CpG-stimulation with robust IL-10 production. The concomitant, suboptimal stimulus via the BCR and TLR9 exerted either a synergistic enhancing effect or resulted in inhibition of proliferation and IL-10 production of patients' B cells. Our data obtained studying B cells of leukemic patients point to the role of CR3 and probably CR4 in the interaction of tumor cells with the microenvironment and suggest the involvement of IL-10 producing B cells in the pathologic process.