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Langerhans cell histiocytosis (LCH) is a rare disorder characterized by tissue accumulation of CD1a+CD207+ LCH cells. In LCH, somatic mutations of the BRAFV600E gene have been detected in tissue LCH cells, bone marrow CD34+ hematopoietic stem cells, circulating CD14+ monocytes, and BDCA1+ myeloid dendritic cells (DC). Targeting BRAFV600E in clonal Langerhans cells (LC) and their precursors is a potential treatment option for patients whose tumors have the mutation. The development of mouse macrophages and LCs is regulated by the CSF1 receptor (CSF1R). In patients with diffuse-type tenosynovial giant cell tumors, CSF1R inhibition depletes tumor-associated macrophages (TAM) with therapeutic efficacy; however, CSF1R signaling in LCs and LCH has not been investigated. We found through IHC and flow cytometry that CSF1R is normally expressed on human CD1a+CD207+ LCs in the epidermis and stratified epithelia. LCs that were differentiated from CD14+ monocytes, BDCA1+ DCs, and CD34+ cord blood progenitors expressed CSF1R that was downregulated upon maturation. Immature LCs migrated toward CSF1, but not IL34. Administration of the c-FMS/CSF1R kinase inhibitors GW2580 and BLZ945 significantly reduced human LC migration. In LCH clinical samples, LCH cells (including BRAFV600E cells) and TAMs retained high expression of CSF1R. We also detected the presence of transcripts for its ligand, CSF1, but not IL34, in all tested LCH cases. CSF1R and CSF1 expression in LCH, and their role in LC migration and differentiation, suggests CSF1R signaling blockade as a candidate rational approach for treatment of LCH, including the BRAFV600E and wild-type forms of the disease.

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Traditionally viewed as poorly plastic, neutrophils are now recognized as functionally diverse; however, the extent and determinants of neutrophil heterogeneity in humans remain unclear. We performed a comprehensive immunophenotypic and transcriptome analysis, at a bulk and single-cell level, of neutrophils from healthy donors and patients undergoing stress myelopoiesis upon exposure to growth factors, transplantation of hematopoietic stem cells (HSC-T), development of pancreatic cancer and viral infection. We uncover an extreme diversity of human neutrophils in vivo, reflecting the rates of cell mobilization, differentiation and exposure to environmental signals. Integrated control of developmental and inducible transcriptional programs linked flexible granulopoietic outputs with elicitation of stimulus-specific functional responses. In this context, we detected an acute interferon (IFN) response in the blood of patients receiving HSC-T that was mirrored by marked upregulation of IFN-stimulated genes in neutrophils but not in monocytes. Systematic characterization of human neutrophil plasticity may uncover clinically relevant biomarkers and support the development of diagnostic and therapeutic tools. We thank S. Gregori and G. Amodio for help with neutrophil isolation and culture experiments; F. Di Salvo, F. Porzio and M. Tassara for patient recruitment and data management; the Center for Omics Sciences, the Flow cytometry Resource, Advanced Cytometry Technical Applications Laboratory, Centro Risorse Biologiche at Ospedale San Raffaele; and the Centro Universitario di Statistica per le Scienze Biomediche at Vita-Salute San Raffaele University. Figures were created with Adobe Illustrator and BioRender.com. V.B. and F.V.M. conducted this study as partial fulfillment of a PhD in Molecular Medicine (Basic and Applied Immunology and Oncology program) at Vita-Salute San Raffaele University. R.D.M. is a New York Stem Cell Foundation – Robertson Investigator. M.A.C. and P.S. are supported by grants from the Italian Association for Cancer Research (AIRC) (IG 20339) and the Italian Ministry of University and Research (PRIN 20177J4E75_004). A.A. is supported by the Italian Telethon Foundation (SR-Tiget grant award B02). E.M. and N.C. are supported by fellowships from Fondazione Umberto Veronesi. This study was supported by grants from the Italian Telethon Foundation (SR-Tiget grant award F04 to R.O.) and the Italian Ministry of Health (GR-201602362156 to R.O. and S.C.). Research in the R.O. laboratory is supported by the European Research Council (starting grant 759532, X-TAM) and by AIRC (MFAG 20247 and AIRC 5×1000 special program 22737) Sí

Monocytic cells perform crucial homeostatic and defensive functions. However, their fate and characterization at the transcriptomic level in human tissues are partially understood, often as a consequence of the lack of specific markers allowing their unequivocal identification. The 6-sulfo LacNAc (slan) antigen identifies a subset of non-classical (NC) monocytes in the bloodstream, namely the slan

Increasing data from the literature point to a neutrophil-mediated role via cytokine production in several aspects of mammalian biology, including angiogenesis. In such regard, neutrophils have been shown to synthetize and release a number of molecules able to promote, directly or indirectly, the growth and migration of endothelial cells, in turn inducing the formation of new blood vessels from preexisting ones. Interestingly, neutrophil-derived cytokines can be involved either in physiological or in pathological angiogenesis, depending on either the functioning or dysregulation of sophisticated interplays among different cell types, extracellular matrix and soluble mediators within the microenvironment. Our review resumes the most interesting studies elucidating the role of neutrophil-derived cytokines in human physiological and pathological angiogenesis. When appropriate, supporting observations generated in animal models will be also mentioned. Particular emphasis will be given to VEGF and PK2/Bv8, rather than CXCL8/IL-8 and OSM. We will also discuss the potential role of neutrophil-derived cytokines such as FGF2, Ang1 and IL-17, whose roles in angiogenesis - albeit anticipated - remain to be elucidated.

Since IL-10 has been shown to up-regulate the expression of the high affinity receptor for IgG (FcγRI/CD64) in human monocytes, we examined whether the cytokine exerts a similar action toward polymorphonuclear neutrophils (PMN). Unexpectedly, we found that in neutrophils, IL-10 failed to induce either the mRNA accumulation or the surface expression of FcγRI. Consistent with these findings, stimulation of PMN with IFN-γ, but not with IL-10, resulted in the induction of specific DNA-binding activities to the IFN-γ response region (GRR), a regulatory element located in the FcγRI gene promoter, required for transcriptional activation. In electrophoretic mobility shift assays (EMSAs), we confirmed that in PBMC, IL-10 induces the binding to the GRR of both STAT1 and STAT3, two members of the STAT family. In neutrophils, however, these activators did not bind to the GRR in response to IL-10, despite the fact that both STAT1 and STAT3 are expressed in these cells. On the other hand, IFN-γ was an efficient inducer of STAT1 binding to the GRR in both PMN and PBMC. The lack of inducible GRR-binding activity in IL-10-treated PMN could not be ascribed to a lack of IL-10R, and did not appear to reflect an inhibitory effect of the cytokine. Taken together, our data suggest that IL-10 is unable to induce FcγRI gene expression in neutrophils because the intracellular signaling pathway triggered by the cytokine is impaired at the level of, or upstream of, STAT1 and/or STAT3 activation.

The neuropeptide substance P (SP), a main mediator of neurogenic inflammation, has been shown to have direct and modulatory effects on functional responses of polymorphonuclear leucocytes (PMNL). In this study, we further investigated the effects exerted by SP on human PMNL functions. Pretreatment of PMNL with SP resulted in an increase of superoxide anion (O2-) production in response to formyl-methionyl-leucyl-phenylalanine (FMLP), concanavalin A (Con A) and opsonized zymosan (STZ). In contrast, the O2- production induced by tumour necrosis factor (TNF) was strongly inhibited by pretreatment with SP. Both enhancement and inhibition of O2- response were exerted by SP in a dose-dependent manner and at concentrations which did not directly stimulate O2- production. These effects were rapid in onset, and occurred after 5 min of preincubation of cells with the neuropeptide. At concentrations that modulated O2- production by PMNL, SP also directly stimulated release of the chemotactic cytokine interleukin-8 (IL-8). Induction of IL-8 release required a longer incubation time (1 hr) with SP and was preceded by an increase of IL-8 mRNA steady-state levels. Furthermore, as well as directly stimulating IL-8 production, SP was also able to enhance the IL-8 release induced by other stimuli such as FMLP and TNF. The results of this study indicate that, in addition to the rapid and differential modulation of O2- production, SP also induces long-term changes such as IL-8 synthesis and release, and can thus amplify the process of PMNL recruitment to the inflammatory site.

In myeloid blasts, the expression and release of the multifunctional chemokine IL-8 could be expected to be differentiation-associated.We investigated the profile of interleukin-8 (IL-8) expression and release by leukemic cells obtained at diagnosis from 42 untreated adult patients with acute myeloid leukemia of various FAB subtypes (2 M0, 7 M1, 6 M2, 6 M3, 10 M4 and 11 M5). IL-8 transcripts were evaluated by Northern blot and densitometric analysis. IL-8 release by myeloid blasts was evaluated by a specific ELISA either in sera at diagnosis or in supernatants (SN) obtained from cultured leukemic cells.In basal conditions, Northern blot analysis revealed detectable IL-8 transcripts in 15/29 cases, eleven of which were classified as M4-M5 and 4 as FAB M0-M3. Densitometric analysis of IL-8 transcript bands showed higher expression in M4-M5 than in M0-M3 cases (mean values +/- SD: 16.5 +/- 21 and 0.77 +/- 1.36 densitometric units, respectively; p = 0.012). Higher IL-8 serum levels were observed in leukemic patients as opposed to normal controls (mean values +/- SD: 0.53 +/- 0.75 vs 0.003 +/- 0.014 ng/mL, respectively; p = 0.006). Furthermore, a trend (though not of statistical significance) towards higher IL-8 serum values was observed in M4-M5 as opposed to M0-M3 subtypes. After 24 hours of culture, the majority of myeloid blasts (95%) spontaneously released detectable amounts of IL-8 into SN. However, M4-M5 released substantially higher amounts of IL-8 than M0-M3 blasts (mean +/- SD: 68 +/- 46 and 8.5 +/- 12 ng/mL, respectively; p0.001). This difference between M0-M3 and M4-M5 blasts was already observed after 6 hours of culture and increased over 72 hours.Our findings confirm and further support the preferential release of high levels of IL-8 by myeloid blasts showing monocytic differentiation.

Monokine induced by IFN-γ (MIG), IFN-inducible T cell α chemoattractant (I-TAC), and IFN-γ-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that bind to a common receptor, CXCR3, and that are produced by different cell types in response to IFN-γ. We have recently reported that human polymorphonuclear neutrophils (PMN) have the capacity to release IP-10. Herein, we show that PMN also have the ability to produce MIG and to express I-TAC mRNA in response to IFN-γ in combination with either TNF-α or LPS. While IFN-γ, alone or in association with agonists such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression, IFN-α, in combination with TNF-α, LPS, or IL-1β, resulted in a considerable induction of IP-10 release by neutrophils. Furthermore, IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10, and I-TAC mRNA and the extracellular production of MIG and IP-10 in neutrophils stimulated with IFN-γ plus either LPS or TNF-α. Finally, supernatants harvested from stimulated PMN induced migration and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes; these activities were significantly reduced by neutralizing anti-MIG and anti-IP-10 Abs, suggesting that they were mediated by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the ability of neutrophils to produce these chemokines might contribute not only to the progression and evolution of the inflammatory response, but also to the regulation of the immune response.

IL-8 is a novel chemotactic cytokine, produced by a variety of blood and tissue cells, that has marked activating effects on polymorphonuclear leukocytes (PMN). We report that IL-8 is produced and released by human PMN after stimulation with the chemotactic agonist FMLP. Release of IL-8 in response to FMLP was transient and not influenced by PMN adherence or by the absence of serum in the medium. Maximum yields were usually obtained with 10 nM FMLP within 2 h of stimulation (0.5-3.5 ng/ml/7 x 10(6) cells, range of 17 different donors). IL-8 release was dependent on FMLP-induced de novo protein synthesis because it was inhibited by cycloheximide, was paralleled by enhanced expression of IL-8 mRNA and was potentiated from two- to sixfold after preincubation of PMN with cytochalasin B. The FMLP effect was direct and not dependent on LPS or on contaminating monocytes, which showed only low responsiveness to FMLP. Pretreatment of PMN with pertussis toxin prevented FMLP-dependent IL-8 production, the effect being evident both at the level of mRNA expression and protein secretion. In addition, two other chemoattractans, platelet-activating factor and C5a, were found capable to induce release of IL-8 by PMN. The results of this study suggest that chemotactically stimulated PMN may be able to amplify the recruitment process of PMN to the inflammatory site by releasing IL-8. As a long-lived cytokine, IL-8 could markedly prolong the attractant effect.