Cristina Mazzon

Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial‐monocyte association, but a NadA‐positive strain induced a significantly higher amount of TNF‐α and IL‐8 compared with the parental NadA‐negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadAΔ351–405, a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL‐8, IL‐6, MCP‐1, and MIP‐1α and low levels of the main vasoactive mediators TNF‐α and IL‐1. NadAΔ351–405 also inhibited monocyte apoptosis and determined its differentiation into a macrophage‐like phenotype.

IFN-γ and R-848 Dependent Activation of Human Monocyte-Derived Dendritic Cells by Neisseria meningitidis Adhesin A

A soluble recombinant form of Neisseria meningitidis adhesin A (NadAΔ351–405), proposed as a constituent of anti-meningococcal B vaccines, is here shown to specifically interact with and immune-modulate human monocyte-derived dendritic cells (mo-DCs). After priming with IFN-γ and stimulation with NadAΔ351–405, mo-DCs strongly up-regulated maturation markers CD83, CD86, CD80, and HLA-DR, secreted moderate quantities of TNF-α, IL-6, and IL-8, and produced a slight, although significant, amount of IL-12p70. Costimulation of mo-DCs with NadAΔ351–405 and the imidoazoquinoline drug R-848, believed to mimic bacterial RNA, increased CD86 in an additive way, but strongly synergized the secretion of IL-12p70, IL-1, IL-6, TNF-α, and MIP-1α, especially after IFN-γ priming. CD86/CD80 overexpression correlated with the occupation of high-(kd ∼ 80 nM) and low-(kd ∼ 4 μM) affinity binding sites for NadAΔ351–405. Alternatively, secretion of IL-12p70 and TNF-α, IL-6, and IL-8 corresponded to the occupation of high- or low-affinity receptors, respectively. Mo-DCs matured by IFN-γ and NadAΔ351–405 supported the proliferation of naive CD4+ T lymphocytes, inducing the differentiation of both IFN-γ and IL-4 producing phenotypes. Our data show that NadA not only is a good immunogen but is as well endowed with a proimmune, self-adjuvating, activity.

Agrin is required for survival and function of monocytic cells

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.

The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

The adhesin NadA favors cell adhesion/invasion by hypervirulent Neisseria meningitidis B (MenB). Its recombinant form NadAΔ351–405, devoid of the outer membrane domain, is an immunogenic candidate for an anti-MenB vaccine able to stimulate monocytes, macrophages and dendritic cells. In this study we investigated the molecular mechanism of NadAΔ351–405 cellular effects in monocytes. We show that NadAΔ351–405 (against which we obtained polyclonal antibodies in rabbits), binds to hsp90, but not to other extracellular homologous heat shock proteins grp94 and hsp70, in vitro and on the surface of monocytes, in a temperature dependent way. Pre-incubation of monocytes with the MenB soluble adhesin interfered with the binding of anti-hsp90 and anti-hsp70 antibodies to hsp90 and hsp70 at 37°C, a condition in which specific cell-binding occurs, but not at 0°C, a condition in which specific cell-binding is very diminished. Conversely, pre-incubation of monocytes with anti-hsp90 and anti-hsp70 antibodies did not affected NadAΔ351–405 cell binding in any temperature condition, indicating that it associates to another receptor on their plasma membrane and then laterally diffuses to encounter hsp90. Consistently, polymixin B interfered with NadAΔ351–405 /hsp90 association, abrogated the decrease of anti-hsp90 antibodies binding to the cell surface due to NadAΔ351–405 and inhibited adhesin-induced cytokine/chemokine secretion without affecting monocyte-adhesin binding. Co-stimulation of monocytes with anti-hsp90 antibodies and NadAΔ351–405 determined a stronger but polymixin B insensitive cell activation. This indicated that the formation of a recombinant NadA/hsp90/hsp70 complex, although essential for full monocyte stimulation, can be replaced by anti-hsp90 antibody/hsp90 binding. Finally, the activation of monocytes by NadAΔ351–405 alone or in the presence of anti-hsp90 antibodies were both inhibited by neutralizing anti-TLR4 antibodies, but not by anti-TLR2 antibodies. We propose that hsp90-dependent recruitment into an hsp90/hsp70/TLR4 transducing signal complex is necessary for the immune-stimulating activity of NadAΔ351–405 anti-MenB vaccine candidate.

The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage

CCRL2 is a 7-transmembrane domain receptor that shares structural and functional similarities with the family of atypical chemokine receptors (ACKRs). CCRL2 is upregulated by inflammatory signals and, unlike other ACKRs, it is not a chemoattractant-scavenging receptor, does not activate β-arrestins, and is widely expressed by many leukocyte subsets. Therefore, the biological role of CCRL2 in immunity is still unclear. We report that CCRL2-deficient mice have a defect in neutrophil recruitment and are protected in 2 models of inflammatory arthritis. In vitro, CCRL2 was found to constitutively form homodimers and heterodimers with CXCR2, a main neutrophil chemotactic receptor. By heterodimerization, CCRL2 could regulate membrane expression and promote CXCR2 functions, including the activation of β2-integrins. Therefore, upregulation of CCRL2 observed under inflammatory conditions is functional to finely tune CXCR2-mediated neutrophil recruitment at sites of inflammation.

Flow cytometry applications for the analysis of chemokine receptor expression and function.

Chemokine receptors play an important role in leukocyte migration, both in physiological and pathological conditions, and the interest in new methodologies for their detection is increasing. In this review, we focused on chemokine receptors detection through flow cytometric approaches, including the use of specific antibodies and fluorescent chemokines, and on approaches aimed at the analysis of their functions, from intracellular trafficking to signaling activities.

Expression and function of IL-1R8 (TIR8/SIGIRR), a regulatory member of the IL-1 receptor family in platelets

AIMS Platelets express functional interleukin-1 receptor-1 (IL-1R1) as well as a repertoire of toll-like receptors (TLRs) involved in platelet activation, platelet-leucocyte reciprocal activation, and immunopathology. IL-1R8, also known as single Ig IL-1-related receptor (SIGIRR) or TIR8, is a member of the IL-1R family that negatively regulates responses to IL-1R family members and TLRs. In the present study, we addressed the expression of IL-1R8 in platelets and megakaryocytes and its role in the control of platelet activation during inflammatory conditions and thromboembolism. METHODS AND RESULTS Here, we show by flow cytometry analysis, western blot, confocal microscopy, and quantitative real-time polymerase chain reaction that IL-1R8 is expressed on human and mouse platelets at high levels and on megakaryocytes. IL-1R8-deficient mice show normal levels of circulating platelets. Homotypic and heterotypic (platelet-neutrophil) aggregation triggered by Adenosine DiPhosphate (ADP) and IL-1 or lipopolysaccharide (LPS) was increased in IL-1R8-deficient platelets. IL-1R8-deficient mice showed increased soluble P-selectin levels and increased platelet-neutrophil aggregates after systemic LPS administration. Commensal flora depletion and IL-1R1 deficiency abated platelet hyperactivity and the increased platelet/neutrophil aggregation observed in Il1r8(-/-) mice in vitro and in vivo, suggesting a key role of IL-1R8 in regulating platelet TLR and IL-1R1 function. In a mouse model of platelet-dependent pulmonary thromboembolism induced by ADP administration, IL-1R8-deficient mice showed an increased frequency of blood vessel complete obstruction. CONCLUSION These results show that platelets, which have a large repertoire of TLRs and IL-1 receptors, express high levels of IL-1R8, which plays a non-redundant function as a regulator of thrombocyte activity in vitro and in vivo.

From tango to quadrilla: current views of the immunological synapse.

All T cell functions require establishing contacts with other cells. In the last ten years, the immunological synapse, the contact-site between T cells and their partners, has been the object of numerous investigations and recent advances in imaging technologies have provided significant insights into the mechanism of immunological synapse formation and its functional outcomes. Considering all the available data, the immunological synapse can be defined as a dynamic structure, formed between a T cell and one or more antigen-presenting cells, showing lipid and protein segregation, signaling compartmentalization, and bidirectional information exchange though soluble and membrane-bound transmitters. In this review, we present the current views on the immunological synapse and discuss about some interesting unresolved questions.

From Tango to Quadrilla

All T cell functions require establishing contacts with other cells. In the last ten years, the immunological synapse, the contact-site between T cells and their partners, has been the object of numerous investigations and recent advances in imaging technologies have provided significant insights into the mechanism of immunological synapse formation and its functional outcomes. Considering all the available data, the immunological synapse can be defined as a dynamic structure, formed between a T cell and one or more antigen-presenting cells, showing lipid and protein segregation, signaling compartmentalization, and bidirectional information exchange though soluble and membrane-bound transmitters. In this review, we present the current views on the immunological synapse and discuss about some interesting unresolved questions.