The Role of Cytokines in Neutrophil Development, Tissue Homing, Function and Plasticity in Health and Disease.

Neutrophils are crucial innate immune cells and comprise 50-70% of the white blood cell population under homeostatic conditions. Upon infection and in cancer, blood neutrophil numbers significantly increase because of the secretion of various chemo- and cytokines by, e.g., leukocytes, pericytes, fibroblasts and endothelial cells present in the inflamed tissue or in the tumor microenvironment (TME). The function of neutrophils in cancer has recently gained considerable attention, as they can exert both pro- and anti-tumorigenic functions, dependent on the cytokine milieu present in the TME. Here, we review the effect of cytokines on neutrophil development, tissue homing, function and plasticity in cancer and autoimmune diseases as well as under physiological conditions in the bone marrow, bloodstream and various organs like the spleen, kidney, liver, lung and lymph nodes. In addition, we address several promising therapeutic options, such as cytokine therapy, immunocytokines and immunotherapy, which aim to exploit the anti-tumorigenic potential of neutrophils in cancer treatment or block excessive neutrophil-mediated inflammation in autoimmune diseases.

Characterization of human Fc alpha receptor transgenic mice: comparison of CD89 expression and antibody-dependent tumor killing between mouse strains.

Since mice do not express a homologue of the human Fc alpha receptor (FcαRI or CD89), a transgenic mouse model was generated in four different backgrounds (C57BL/6, BALB/c, SCID and NXG) expressing the FcαRI under the endogenous human promoter. In this study, we describe previously unknown characteristics of this model, such as the integration site of the FCAR gene, the CD89 expression pattern in healthy male and female mice and in tumor-bearing mice, expression of myeloid activation markers and FcγRs and IgA/CD89-mediated tumor killing capacity. In all mouse strains, CD89 expression is highest in neutrophils, intermediate on other myeloid cells such as eosinophils and DC subsets and inducible on, among others, monocytes, macrophages and Kupffer cells. CD89 expression levels are highest in BALB/c and SCID, lower in C57BL/6 and lowest in NXG mice. Additionally, CD89 expression on myeloid cells is increased in tumor-bearing mice across all mouse strains. Using Targeted Locus Amplification, we determined that the hCD89 transgene has integrated in chromosome 4. Furthermore, we established that wildtype and hCD89 transgenic mice have a similar composition and phenotype of immune cells. Finally, IgA-mediated killing of tumor cells is most potent with neutrophils from BALB/c and C57BL/6 and less with neutrophils from SCID and NXG mice. However, when effector cells from whole blood are used, SCID and BALB/c are most efficient, since these strains have a much higher number of neutrophils. Overall, hCD89 transgenic mice provide a very powerful model to test the efficacy of IgA immunotherapy against infectious diseases and cancer.

Effective, Long-Term, Neutrophil Depletion Using a Murinized Anti-Ly-6G 1A8 Antibody.

Neutrophils are crucial innate immune cells but also play key roles in various diseases, such as cancer, where they can perform both pro- and anti-tumorigenic functions. To study the function of neutrophils in vivo, these cells are often depleted using Ly-6G or Gr-1 depleting antibodies or genetic "knockout" models. However, these methods have several limitations, being only partially effective, effective for a short term, and lacking specificity or the ability to conditionally deplete neutrophils. Here, we describe the use of a novel murinized Ly-6G (1A8) antibody. The murinized Ly-6G antibody is of the mouse IgG2a isotype, which is the only isotype that can bind all murine Fcγ receptors and C1q and is, therefore, able to activate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) pathways. We show that this mouse-Ly-6G antibody shows efficient, long-term, and near-complete (>90%) neutrophil depletion in the peripheral blood of C57Bl6/J, Balb/c, NXG and SCID mice for up to at least four weeks, using a standardized neutrophil depletion strategy. In addition, we show that neutrophils are efficiently depleted in the blood and tumor tissue of IMR32 tumor-bearing SCID mice, analyzed six weeks after the start of the treatment.

Deubiquitinating enzymes USP4 and USP17 finetune the trafficking of PDGFRß and affect PDGF-BB-induced STAT3 signalling.

Interaction of platelet-derived growth factor (PDGF) isoforms with their receptors results in activation and internalization of receptors, with a concomitant activation of downstream signalling pathways. Ubiquitination of PDGFRs serves as a mark to direct the internalization and sorting of the receptors. By overexpressing a panel of deubiquitinating enzymes (DUBs), we found that USP17 and USP4 efficiently deubiquitinate PDGF receptor ß (PDGFRß) and are able to remove both Lys63 and Lys48-linked polyubiquitin chains from the receptor. Deubiquitination of PDGFRß did not affect its stability, but regulated the timing of its trafficking, whereby USP17 prolonged the presence of the receptor at the cell surface, while USP4 affected the speed of trafficking towards early endosomes. Induction of each of the DUBs in BJhTERT fibroblasts and U2OS osteosarcoma cells led to prolonged and/or shifted activation of STAT3 in response to PDGF-BB stimulation, which in turn led to increased transcriptional activity of STAT3. Induction of USP17 promoted acute upregulation of the mRNA expression of STAT3-inducible genes STAT3, CSF1, junB and c-myc, while causing long-term changes in the expression of myc and CDKN1A. Deletion of USP17 was lethal to fibroblasts, while deletion of USP4 led to a decreased proliferative response to stimulation by PDGF-BB. Thus, USP17- and USP4-mediated changes in ubiquitination of PDFGRß lead to dysregulated signalling and transcription downstream of STAT3, resulting in defects in the control of cell proliferation.

Erratum: Specific targeting of PDGFRß in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression: Erratum.

[This corrects the article DOI: 10.7150/thno.37851.].

Specific targeting of PDGFRß in the stroma inhibits growth and angiogenesis in tumors with high PDGF-BB expression.

PDGF-BB/PDGFRß signaling plays an important role during vascularization by mediating pericyte recruitment to the vasculature, promoting the integrity and function of vessels. Until now it has not been possible to assess the specific role of PDGFRß signaling in tumor progression and angiogenesis due to lack of appropriate animal models and molecular tools. Methods: In the present study, we used a transgenic knock-in mouse strain carrying a silent mutation in the PDGFRß ATP binding site that allows specific targeting of PDGFRß using the compound 1-NaPP1. To evaluate the impact of selective PDGFRß inhibition of stromal cells on tumor growth we investigated four tumor cell lines with no or low PDGFRß expression, i.e. Lewis lung carcinoma (LLC), EO771 breast carcinoma, B16 melanoma and a version of B16 that had been engineered to overexpress PDGF-BB (B16/PDGF-BB). Results: We found that specific impairment of PDGFRß kinase activity by 1-NaPP1 treatment efficiently suppressed growth in tumors with high expression of PDGF-BB, i.e. LLC and B16/PDGF-BB, while the clinically used PDGFRß kinase inhibitor imatinib did not suppress tumor growth. Notably, tumors with low levels of PDGF-BB, i.e. EO771 and B16, neither responded to 1-NaPP1 nor to imatinib treatment. Inhibition of PDGFRß by either drug impaired tumor vascularization and also affected pericyte coverage; however, specific targeting of PDGFRß by 1-NaPP1 resulted in a more pronounced decrease in vessel function with increased vessel apoptosis in high PDGF-BB expressing tumors, compared to treatment with imatinib. In vitro analysis of PDGFRß ASKA mouse embryo fibroblasts and the mesenchymal progenitor cell line 10T1/2 revealed that PDGF-BB induced NG2 expression, consistent with the in vivo data. Conclusion: Specific targeting of PDGFRß signaling significantly inhibits tumor progression and angiogenesis depending on PDGF-BB expression. Our data suggest that targeting PDGFRß in the tumor stroma could have therapeutic value in patients with high tumor PDGF-BB expression.

PDGF-BB enhances collagen gel contraction through a PI3K-PLCγ-PKC-cofilin pathway.

Cell-mediated contraction of collagenous matrices is modulated by various growth factors and cytokines, such as platelet-derived growth factor-BB (PDGF-BB). Here we used a genetic cell model to delineate defined signaling pathways that enhance collagen gel contraction downstream of ligand-stimulated platelet-derived growth factor receptor-ß (PDGF-Rß). Our data show that PDGF BB-enhanced activations of phosphatidylinositol 3'-kinase (PI3K) and phospholipase Cγ (PLCγ) were necessary for PDGF-enhanced collagen gel contraction. Importantly, other defined signaling pathways down-stream of PDGF-Rß were, however, dispensable. The decisive roles for PI3K and PLCγ were corroborated by experiments using selective inhibitors. Furthermore, we show that de-phosphorylation and thereby activation of cofilin that is important for the turnover of actin filaments, is depended on PI3K and PLCγ down-stream of PDGF-Rß. Moreover, inhibition of protein kinase C (PKC) by GÖ6976 and bisindolylmaleimide-II abolished cofilin de-phosphorylation, as well as PDGF-enhanced contraction. In contrast, activation of the PKC protein family by 4ß-phorbol 12-myristate 13-acetate (PMA) did not accelerate collagen gel contraction although it induced long-term cofilin de-phosphorylation, showing the need of a dynamic control of cofilin de-phosphorylation for PDGF-enhanced collagen gel contraction. Taken together, our data point to the involvement of a PI3K/PLCγ-PKC-cofilin pathway in both PDGF-enhanced cofilin de-phosphorylation and PDGF-enhanced collagen gel contraction.

Platelet-derived growth factor (PDGF)-induced activation of Erk5 MAP-kinase is dependent on Mekk2, Mek1/2, PKC and PI3-kinase, and affects BMP signaling.

Platelet-derived growth factor-BB (PDGF-BB) binds to its tyrosine kinase receptors (PDGFRs) and stimulates mitogenicity and survival of cells of mesenchymal origin. Activation of PDGFRs initiates a number of downstream signaling pathways, including phosphatidyl 3'-inositol kinase (PI3-kinase), phospholipase Cγ and MAP kinase pathways. In this report, we show that Erk5 MAP kinase is activated in response to PDGF-BB in the smooth muscle cell line MOVAS in a manner dependent on Mekk2, Mek1/2, Mek5, PI3-kinase and protein kinase C (PKC). The co-operation of Mek1/2 and Mekk2 in the activation of Erk5, suggests a close co-regulation between the Erk1/2 and Erk5 MAP kinase pathways. Furthermore, we found that classical PKCs are important for Erk5 activation. In addition, we found that PKCζ interacts with Erk5 and may exert a negative feed-back effect. We observed no nuclear accumulation of Erk5 in response to PDGF-BB stimulation, however, we identified a mechanism by which cytoplasmic Erk5 influences gene expression; Erk5 was essential for PDGF-BB-mediated Smad1/5/8 signaling by stimulating release and/or activation of bone morphogenetic protein(s) (BMPs). Thus, PDGF-BB-induced Erk5 activation involves parallel stimulatory and inhibitory pathways and promotes Smad1/5/8 signaling.

The Ubiquitin Ligases c-Cbl and Cbl-b Negatively Regulate Platelet-derived Growth Factor (PDGF) BB-induced Chemotaxis by Affecting PDGF Receptor ß (PDGFRß) Internalization and Signaling.

Protein ubiquitination controls protein stability and subcellular localization of tyrosine kinase receptors, hence affecting signaling both quantitatively and qualitatively. In this report, we demonstrate that, after ligand stimulation, the PDGF ß receptor (PDGFRß) becomes ubiquitinated in a manner requiring both the c-Cbl and Cbl-b ubiquitin ligases. Simultaneous depletion of c-Cbl and Cbl-b resulted in reduced ligand-induced PDGFRß clearance from the cell surface because of reduced endocytosis of the receptor. Cbl-b formed a complex with c-Cbl, as well as with the PDGFRß, in response to PDGF-BB stimulation. We were unable to find a direct interaction between the receptor and c-Cbl, raising the possibility that Cbl-b is necessary for c-Cbl to interact with PDGFRß. Phosphorylated Tyr-1021 in PDGFRß was the primary interaction site for Cbl-b, with some contribution from Tyr-1009. Depletion of c-Cbl and Cbl-b led to an increased ligand-induced tyrosine phosphorylation of the receptor. Several tyrosine residues with elevated phosphorylation (i.e. Tyr-579, Tyr-581, Tyr-1009, and Tyr-1021) have previously been shown to interact with Src kinases and PLCγ. Indeed, in cells depleted of c-Cbl and Cbl-b, both Src and PLCγ phosphorylation were enhanced, whereas activation of other pathways, such as Erk1/2 MAP kinase and Akt, were not affected. In addition, Stat3 phosphorylation, which has been connected to Src activity, was also elevated in cells lacking c-Cbl and Cbl-b. Functionally, we found that cells depleted of c-Cbl and Cbl-b were more prone to migrate toward PDGF-BB, whereas no reproducible effect on cell proliferation could be observed. In conclusion, internalization as well as signaling via PDGFRß are controlled by ubiquitination.

AtHESPERIN: a novel regulator of circadian rhythms with poly(A)-degrading activity in plants.

We report the identification and characterization of a novel gene, AtHesperin (AtHESP) that codes for a deadenylase in Arabidopsis thaliana. The gene is under circadian clock-gene regulation and has similarity to the mammalian Nocturnin. AtHESP can efficiently degrade poly(A) substrates exhibiting allosteric kinetics. Size exclusion chromatography and native electrophoresis coupled with kinetic analysis support that the native enzyme is oligomeric with at least 3 binding sites. Knockdown and overexpression of AtHESP in plant lines affects the expression and rhythmicity of the clock core oscillator genes TOC1 and CCA1. This study demonstrates an evolutionary conserved poly(A)-degrading activity in plants and suggests deadenylation as a mechanism involved in the regulation of the circadian clock. A role of AtHESP in stress response in plants is also depicted.