Federica Calzetti

The neutrophil as a cellular source of chemokines.

Neutrophils are known to play an important role in inflammatory responses by virtue of their ability to perform a series of effector functions that collectively represent a major mechanism of innate immunity against injury and infection. In recent years, however, it has become obvious that the contribution of neutrophils to host defence and natural immunity extends well beyond their traditional role as professional phagocytes. Indeed, neutrophils can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses. These include not only Fc receptors, complement components, cationic antimicrobial and NADPH oxidase proteins, but also a variety of cytokines (including tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-1R alpha, IL-12 and vascular endothelial growth factor), and chemokines such as IL-8, growth-related gene product, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interferon-gamma-inducible protein of 10 kDa and monokine induced by interferon-gamma. Because these chemokines are primarily chemotactic for neutrophils, monocytes, immature dendritic cells and T-lymphocyte subsets, a potential role for neutrophils in orchestrating the sequential recruitment of distinct leukocyte types to the inflamed tissue is likely to occur. The purpose of this review is to summarize the essential features of the production of chemokines by polymorphonuclear neutrophil leukocytes and the contribution that we have made to characterize some aspects of this newly discovered crucial function of neutrophils.

Phosphatidic acid and not diacylglycerol generated by phospholipase D is functionally linked to the activation of the NADPH oxidase by FMLP in human neutrophils

It is widely accepted that the activation of the NADPH oxidase of phagocytes is linked to the stimulation of protein kinase C by diacylglycerol formed by hydrolysis of phospholipids. The main source would be choline containing phospholipid via phospholipase D and phosphatidate phosphohydrolase. This paper presents a condition where the activation of the respiratory burst by FMLP correlates with the formation of phosphatidic acid, via phospholipase D, and not with that of diacylglycerol. In fact: 1) in neutrophils treated with propranolol, an inhibitor of phosphatidate phosphohydrolase, FMLP plus cytochalasin B induces a respiratory burst associated with a stimulation of phospholipase D, formation of phosphatidic acid and complete inhibition of that of diacylglycerol. 2) The respiratory burst by FMLP plus cytochalasin B lasts a few minutes and may be restimulated by propranolol which induces an accumulation of phosphatidic acid. 3) In neutrophils stimulated by FMLP in the absence of cytochalasin B propranolol causes an accumulation of phosphatidic acid and a marked enhancement of the respiratory burst without formation of diacylglycerol. 4) The inhibition of the formation of phosphatidic acid via phospholipase D by butanol inhibits the respiratory burst by FMLP.

Toll‐Like Receptor‐3‐Activated Human Mesenchymal Stromal Cells Significantly Prolong the Survival and Function of Neutrophils

Bone marrow‐derived mesenchymal stromal cells (BM‐MSCs) are stromal precursors endowed with extensive immunomodulative properties. In this study, we aimed to assess whether Toll‐like receptor‐3 (TLR3)‐ and TLR4‐activated BM‐MSC influence human neutrophil (PMN) responses under coculture conditions. We show that TLR3 triggering by polyinosinic:polycytidylic acid dramatically amplifies, in a more significant manner than TLR4 triggering by lipopolysaccharide, the antiapoptotic effects that resting BM‐MSC constitutively exert on PMN under coculture conditions, preserving a significant fraction of viable and functional PMN up to 72 hours. In addition, TLR3‐ and TLR4‐activated BM‐MSC enhance respiratory burst ability and CD11b expression by PMN. The coculture in the absence of cell contact and the incubation of PMN in supernatants harvested from TLR3‐ and TLR4‐activated BM‐MSC yield comparable results in terms of increased survival and immunophenotypic changes, thus suggesting the involvement of endogenous soluble factors. Neutralizing experiments reveal that the biological effects exerted on PMN by TLR3‐activated BM‐MSC are mediated by the combined action of interleukin 6, interferon‐β (IFN‐β), and granulocyte macrophage colony‐stimulating factor (GM‐CSF), while those exerted by TLR4‐activated BM‐MSC mostly depend on GM‐CSF. MSC isolated from thymus, spleen, and subcutaneous adipose tissue behaves similarly. Finally, the effects exerted by TLR3‐ or TLR4‐stimulated BM‐MSC on PMN are conserved even after the previous priming of BM‐MSC with IFN‐γ and tumor necrosis factor‐α. Our data highlight a novel mechanism by which MSC sustain and amplify the functions of PMN in response to TLR3‐ and TLR4‐triggering and may consequently contribute to inflammatory disorders. STEM CELLS 2011;29:1001–1011

Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF−/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-γ by CD4+ T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.

Up-regulation of IL-10R1 expression is required to render human neutrophils fully responsive to IL-10.

We have recently shown that IL-10 fails to trigger Stat3 and Stat1 tyrosine phosphorylation in freshly isolated human neutrophils. In this study, we report that IL-10 can nonetheless induce Stat3 tyrosine phosphorylation and the binding of Stat1 and Stat3 to the IFN-γ response region or the high-affinity synthetic derivative of the c-sis-inducible element in neutrophils that have been cultured for at least 3 h with LPS. Similarly, the ability of IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was dramatically enhanced in cultured neutrophils and, as a result, translated into the SOCS-3 protein. Since neutrophils’ acquisition of responsiveness to IL-10 required de novo protein synthesis, we assessed whether expression of IL-10R1 or IL-10R2 was modulated in cultured neutrophils. We detected constitutive IL-10R1 mRNA and protein expression in circulating neutrophils, at levels which were much lower than those observed in autologous monocytes or lymphocytes. In contrast, IL-10R2 expression was comparable in both cell types. However, IL-10R1 (but not IL-10R2) mRNA and protein expression was substantially increased in neutrophils stimulated by LPS. The ability of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3 synthesis and to regulate IL-1 receptor antagonist and macrophage-inflammatory protein 1β release in LPS-treated neutrophils correlated with this increased IL-10R1 expression, and was abolished by neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results demonstrate that the capacity of neutrophils to respond to IL-10, as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and modulation of cytokine production, is very dependent on the level of expression of IL-10R1.

Activation of an immunoregulatory and antiviral gene expression program in poly(I:C)-transfected human neutrophils.

Neutrophils, historically known for their involvement in acute inflammation, are also targets for infection by many different DNA and RNA viruses. However, the mechanisms by which they recognize and respond to viral components are poorly understood. Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic mimetic of viral dsRNA that is known to interact either with endosomal TLR3 (not expressed by human neutrophils) or with cytoplasmic RNA helicases such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). In this study, we report that intracellularly administered poly(I:C) stimulates human neutrophils to specifically express elevated mRNA levels encoding type I IFNs, immunoregulatory cytokines, and chemokines, such as TNF-α, IL-12p40, CXCL10, CXCL8, CCL4, and CCL20, as well as classical IFN-responsive genes (IRG), including IFIT1 (IFN-induced protein with tetratricopeptide repeats 1)/IFN-stimulated gene (ISG)56, G1P2/ISG15, PKR (dsRNA-dependent protein kinase), and IFN-regulatory factor (IRF)7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the MAPK-, PKR-, NF-κB-, and TANK (TNF receptor-associated NF-κB kinase)-binding kinase (TBK1)/IRF3-signaling transduction pathways, as illustrated by the use of specific pharmacological inhibitors. Consistent with the requirement of the cytoplasmic dsRNA pathway for antiviral signaling, human neutrophils were found to constitutively express significant levels of both MDA5 and RIG-I, but not TLR3. Accordingly, neutrophils isolated from MDA5-deficient mice had a partial impairment in the production of IFN-β and TNF-α upon infection with encephalomyocarditis virus. Taken together, our data demonstrate that neutrophils are able to activate antiviral responses via helicase recognition, thus acting at the frontline of immunity against viruses.

The MYD88-Independent Pathway Is Not Mobilized in Human Neutrophils Stimulated via TLR4

LPS activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. This prompted us to revisit the intriguing issue of CXCL10 production, which we previously showed to be inducible in neutrophils stimulated with LPS and IFN-γ but not with either stimulus alone, contrary to other myeloid cells. We now report that in neutrophils the MyD88-independent pathway is not activated by LPS. Indeed, microarray and real-time PCR experiments showed that neither IFNβ nor IFNβ-dependent genes (including CXCL10) are inducible in LPS-treated neutrophils, in contrast to monocytes. Further investigation into the inability of LPS to promote IFNβ expression in neutrophils revealed that the transcription factors regulating the IFNβ enhanceosome, such as IFN-regulatory factor-3 and AP-1, are not activated in LPS-treated neutrophils as revealed by lack of dimerization, nuclear translocation, confocal microscopy, and inducible binding to DNA. Moreover, we show that the upstream TANK-binding kinase-1 is not activated by LPS in neutrophils. A lack of IFNβ/CXCL10 mRNA expression and IFN-regulatory factor 3 activation was also observed in myeloid leukemia HL60 cells differentiated to granulocytes and then stimulated with LPS, indicating that the inability of neutrophils to activate the MyD88-independent pathway represents a feature of their terminal maturation. These results identify a disconnected activation of the two signaling pathways downstream of TLR4 in key cellular components of the inflammatory and immune responses and help us to better understand the primordial role of neutrophils in host defense against nonviral infections.

Gene Expression and Production of Tumor Necrosis Factor Alpha, Interleukin-1β (IL-1β), IL-8, Macrophage Inflammatory Protein 1α (MIP-1α), MIP-1β, and Gamma Interferon-Inducible Protein 10 by Human Neutrophils Stimulated with Group B Meningococcal Outer Membrane Vesicles

ABSTRACT Accumulation of polymorphonuclear neutrophils (PMN) into the subarachnoidal space is one of the hallmarks of Neisseria meningitidis infection. In this study, we evaluated the ability of outer membrane vesicles (OMV) from N. meningitidis B to stimulate cytokine production by neutrophils. We found that PMN stimulated in vitro by OMV produce proinflammatory cytokines and chemokines including tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-8, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β. A considerable induction of gamma interferon (IFN-γ)-inducible protein 10 (IP-10) mRNA transcripts, as well as extracellular IP-10 release, was also observed when neutrophils were stimulated by OMV in combination with IFN-γ. Furthermore, PMN stimulated by OMV in the presence of IFN-γ demonstrated an enhanced capacity to release TNF-α, IL-1β, IL-8, and MIP-1β compared to stimulation with OMV alone. In line with its downregulatory effects on neutrophil-derived proinflammatory cytokines, IL-10 potently inhibited TNF-α, IL-1β, IL-8, and MIP-1β production triggered by OMV. Finally, a neutralizing anti-TNF-α monoclonal antibody (MAb) did not influence the release of IL-8 and MIP-1β induced by OMV, therefore excluding a role for endogenous TNF-α in mediating the induction of chemokine release by OMV. In contrast, the ability of lipopolysaccharide fromN. meningitidis B to induce the production of IL-8 and MIP-1β was significantly inhibited by anti-TNF-α MAb. Our results establish that, in response to OMV, neutrophils produce a proinflammatory profile of cytokines and chemokines which may not only play a role in the pathogenesis of meningitis but may also contribute to the development of protective immunity to serogroup B meningococci.

Neutrophil activation and survival are modulated by interaction with NK cells

It is increasingly evident that neutrophils are able to cross-talk with other leukocytes to shape ongoing inflammatory and immune responses. In this study, we analyzed whether human NK cells may influence the survival and activation of neutrophils under co-culture conditions. We report that NK cells exposed to either IL-15 or IL-18 alone strongly protect the survival of neutrophils via the release of IFNγ and granulocyte macrophage colony-stimulating factor (GM-CSF) plus IFNγ, respectively, and cause a slight up-regulation of neutrophil CD64 and CD11b expression. In comparison, NK cells exposed to both IL-15 and IL-18 show a lesser ability to increase the survival of neutrophils but can more potently up-regulate CD64 and CD11b expression, as well as induce the de novo surface expression of CD69, in neutrophils. Analysis of the events occurring in neutrophil/NK co-cultures exposed to IL-15 plus IL-18 revealed that (i) neutrophil survival is positively affected by NK-derived GM-CSF but negatively influenced by a CD18-dependent neutrophil/NK contact, (ii) NK-derived IFNγ is almost entirely responsible for the induction of CD64, (iii) both soluble factors (primarily GM-CSF) and direct cell-cell contact up-regulate CD11b and CD69 and (iv) NK-derived GM-CSF induces the expression of biologically active heparin-binding EGF-like growth factor (HB-EGF) in neutrophils. Finally, we demonstrate that NK cells can also express HB-EGF when stimulated with either IL-2 or IL-15, yet independently of endogenous GM-CSF. Altogether, our results define a novel interaction within the innate immune system whereby NK cells, by directly modulating neutrophil functions, might contribute to the pathogenesis of inflammatory diseases.

Human neutrophils interact with both 6-sulfo LacNAc + DC and NK cells to amplify NK-derived IFNγ: role of CD18, ICAM-1, and ICAM-3

The role of neutrophils as key players in the regulation of innate and adaptive immune responses is increasingly being recognized. We report that human neutrophils establish a network with both natural killer (NK) cells and 6-sulfo LacNAc(+) dendritic cells (slanDCs), which ultimately serves to up-regulate NK-derived interferonγ (IFNγ). This network involves direct reciprocal interactions and positive amplification loops mediated by cell-derived cytokines. Accordingly, we show that after lipopolysaccharide + interleukin-2 (IL-2) or IL-15/IL-18 stimulation, neutrophils directly interact with and potentiate the activity of both slanDCs and NK cells. On the one hand, neutrophils augment the release of IL-12p70 by slanDCs via a CD18/ intercellular adhesion molecule-1 (ICAM-1) interaction that stimulates activated NK cells to produce IFNγ. IFNγ further potentiates the interaction between neutrophils and slanDCs and the release of slanDC-derived IL-12p70, thus creating a positive feedback loop. On the other hand, neutrophils directly co-stimulate NK cells via CD18/ICAM-3, leading to the production of IFNγ. Colocalization of neutrophils, NK cells, and slanDCs, as well as of IL-12p70 and IFNγ, in inflamed tissues of Crohn disease and psoriasis provides strong evidence for a novel cellular and cytokine cooperation within the innate immune system in which neutrophils act as amplifiers of NK cell/slanDC-mediated responses.