Pierre Garrone

A type I interferon autocrine–paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells

Dendritic cells (DC) produce interleukin-12 (IL-12) in response to Toll-like receptor (TLR) activation. Two major TLR signaling pathways participate in the response to pathogens: the nuclear factor-κB (NF-κB)–dependent pathway leading to inflammatory cytokine secretion including IL-12 and the interferon (IFN)-dependent pathway inducing type I IFN and IFN-regulated genes. Here we show that the two pathways cooperate and are likely both necessary for inducing an optimal response to pathogens. R-848/Resiquimod (TLR7 ligand in the mouse and TLR7/8 ligand in human) synergized with poly(I:C) (TLR3 ligand) or lipopolysaccharide (LPS; TLR4 ligand) in inducing high levels of bioactive IL-12p70 secretion and IFN-β mRNA accumulation by mouse bone marrow–derived DC (BM-DC). Strikingly, IL-12p70 but not IL-12p40 secretion was strongly reduced in BM-DC from STAT1−/− and IFNAR−/− mice. STAT1 tyrosine-phosphorylation, IL-12p35, and IFN-β mRNA accumulation were strongly inhibited in IFNAR−/− BM-DC activated with the TLR ligand combinations. Similar observation were obtained in human TLR8-expressing monocyte-derived DC (moDC) using neutralizing anti-IFNAR2 antibodies, although results also pointed to a possible involvement of IFN-λ1 (also known as IL-29). This suggests that TLR engagement on DC induces endogenous IFNs that further synergize with the NF-κB pathway for optimal IL-12p70 secretion. Moreover, analysis of interferon regulatory factors (IRF) regulation in moDC suggests a role for IRF7/8 in mediating IRF3-independent type I IFN and possibly IL-12p35 synthesis in response to TLR7/8.

FDF03, a Novel Inhibitory Receptor of the Immunoglobulin Superfamily, Is Expressed by Human Dendritic and Myeloid Cells

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a− DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c− DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcγRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-γ. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-γ production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-γ production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-γ production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I–specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1α specific inhibitor

Interleukin-1 (IL-1) defines two polypeptides, IL-1α and IL-1β, that possess a wide spectrum of biological effects. Two natural antagonists of IL-1 action have been characterized: the IL-1 receptor antagonist (IL-1Ra) and a soluble form of the type II IL-1 receptor. Neutralizing autoantibodies to IL-1α have also been detected in sera of healthy individuals and patients with autoimmune or inflammatory diseases. To characterize such antibodies molecularly, we attempted to generate B cell clones producing anti-IL-1α human monoclonal antibody (HuMAb) by combining Epstein-Barr virus-immortalization and CD40-activation of B lymphocytes from individuals with circulating anti-IL-1α. We describe herein the generation and properties of a natural IgG4κ anti-IL-1α monoclonal autoantibody, HuMAb X3, that bound specifically to human IL-1α, but not to IL-1β and IL-1Ra, with a high affinity (Kd = 1.2 × 10−10 M). HuMAb X3 inhibited IL-1α binding to IL-1 receptors and neutralized biological activities of both recombinant and natural forms of IL-1α. A recombinant form of HuMAb X3 was found to display identical specific IL-1α antagonism. The presence of somatic mutations within X3 variable regions suggests an antigen-driven affinity maturation. This study extends the demonstration of the presence of high affinity neutralizing anti-IL-1α autoantibodies that can function as a third type of IL-1 antagonist.

Virus overrides the propensity of human CD40L‐activated plasmacytoid dendritic cells to produce Th2 mediators through synergistic induction of IFN‐γ and Th1 chemokine production

Depending on the activation status, plasmacytoid dendritic cells (PDC) and myeloid DC have the ability to induce CD4 T cell development toward T helper cell type 1 (Th1) or Th2 pathways. Thus, we tested whether different activation signals could also have an impact on the profile of chemokines produced by human PDC. Signals that induce human PDC to promote a type 1 response (i.e., viruses) and a type 2 response [i.e., CD40 ligand (CD40L)] also induced PDC isolated from tonsils to secrete chemokines preferentially attracting Th1 cells [such as interferon‐γ (IFN‐γ)‐inducible protein (IP)‐10/CXC chemokine ligand 10 (CXCL10) and macrophage inflammatory protein‐1β/CC chemokine ligand 4 (CCL4)] or Th2 cells (such as thymus and activation‐regulated chemokine/CCL17 and monocyte‐derived chemokine/CCL22), respectively. Activated natural killer cells were preferentially recruited by supernatants of virus‐activated PDC, and supernatants of CD40L‐activated PDC attracted memory CD4+ T cells, particularly the CD4+CD45RO+CD25+ T cells described for their regulatory activities. It is striking that CD40L and virus synergized to trigger the production of IFN‐γ by PDC, which induces another Th1‐attracting chemokine monokine‐induced by IFN‐γ/CXCL9 and cooperates with endogenous type I IFN for IP‐10/CXCL10 production. In conclusion, our studies reveal that PDC participate in the selective recruitment of effector cells of innate and adaptive immune responses and that virus converts the CD40L‐induced Th2 chemokine patterns of PDC into a potent Th1 mediator profile through an autocrine loop of IFN‐γ.

Increased incidence of neutralizing autoantibodies against interleukin-1α (IL-1α) in nondestructive chronic polyarthritis

Cytokines such as IL-1 and tumor necrosis factor a (TNFa) play a critical role in chronic joint inflammation and destruction. To study their regulation, we looked for circulating antiproinflammatory cytokine autoantibodies in 318 patients with chronic arthritis by immunoprecipitation with protein G. Anti-IL-1α but not anti-IL-1β or anti-TNFα IgG antibodies were detected in 9% of blood donors and 18.9% of chronic arthritis patients. These antibodies were found more commonly and at a higher level in patients with nondestructive arthritis. Negative correlations were observed between the antibody levels and indices of disease activity and joint destruction. There was a negative association between the presence of anti-IL-1α antibodies and that of HLA-DR4. These circulating anti-IL-1α antibodies were not complexed with IL-1α and could block specifically the biological activity of IL-1α and its binding to membrane IL-1 receptors. These results indicate that these antibodies are beneficial, suggesting their contribution in the clinical presentation.

Immunosuppressive effects of Pseudomonas aeruginosa exotoxin A on human B-lymphocytes

In this study we investigated the effects of exotoxin A on proliferation and differentiation of human B-cells in vitro. Exotoxin A at a concentration of 1 microgram/ml inhibited the proliferation of B-cells preactivated by insolubilized anti-IgM antibody or by formalinized Staphylococcus aureus particles, plus IL-2 or IL-4. B-cell blasts obtained after preactivation of tonsillar B-cells produce IgG and IgM in culture supernatants, and this Ig production is enhanced by IL-2 or IL-4. Exotoxin A inhibited the production of IgG and IgM by the B-blasts at the concentration of 1 microgram/ml.

Cross‐linking of antigen receptor via Ig‐β (B29, CD79b) can induce both positive and negative signals in CD40‐activated human B cells

Antigen‐dependent activation of B lymphocytes is mediated through surface immunoglobulins and their associated molecules Ig‐α (CD79a, Mb1) and Ig‐β (CD79b, B29). Here we show that an antibody directed against the extracellular part of human Ig‐β can, when cross‐linked by CD32‐transfected L cells, induce an IL‐2‐dependent proliferation of tonsil B cells. With the use of L cells stably transfected with both CD32 and CD40L, anti‐Ig‐β activation of B cells was combined with CD40 triggering, an important component of the T cell‐dependent B cell activation. This dual cellular activation resulted in two different phases, with initially synergistic proliferative effects, both without and with IL‐2 or IL‐10. Then, after 5–6 days of culture, cells stimulated with both anti‐Ig‐β and CD40L underwent massive cell death, in contrast to B cells activated with CD40L alone. Cell death was not prevented by the addition of IL‐2 or IL‐10, but was prevented by the addition of IL‐4. These results are discussed in the context of positive and negative selection of mature B cells.

Regulation of Peripheral B-Cell Growth and Differentiation

The life histories of B lymphocytes and T lymphocytes share many similar features. They derive from immature precursor cells and use similar genes and strategy to encode, assemble, and select their surface antigen receptors. T-cell development in the thymus involves a series of stringent positive and negative selection processes that ensure (1) that the generated T-cell receptors distinguish self and nonself antigen and (2) the dependence on the recognition of self-MHC to respond to nonself antigens. B-cell development within the bone marrow also involves a series of selection processes that ensure that the generated B-cell antigen receptors directly recognize nonself antigen without the involvement of selfMHC. The current views of primary T-cell and B-cell lymphopoiesis in the thymus and bone marrow, respectively, are generated by two considerations: (1) each stage of gene rearrangement and the selection of the host cells correspond to a particular stage of cellular differentiation characterized by surface molecules that collectively define a cell subset; (2) a particular differentiation stage/cell subset is localized within a particular anatomic compartment of the thymus or bone marrow. Thus, the whole differentiation pathway seems to follow a specific migration pattern through different functional compartments of thymus or bone marrow, where they interact with different stromal cells/accessory cells. These two experimental approaches, together with gene cloning, should ultimately permit the isolation of the regulatory molecules that drive the cells from one differentiation stage to the next during the primary B-cell and T-cell developments.