Type I interferon regulates cytokine-delayed neutrophil apoptosis, reactive oxygen species production and chemokine expression.

Interferons (IFNs) are key regulators of a number of inflammatory conditions in which neutrophils play an important role in pathology, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), where type I IFNs are implicated in disease pathology. However, IFNs are usually generated in vivo together with other cytokines that also have immunoregulatory functions, but such interactions are poorly defined experimentally. We measured the effects of type I (IFN-α) IFN, elevated in both RA and SLE, on the functions of healthy neutrophils incubated in vitro in the absence and presence of proinflammatory cytokines typically elevated in inflammatory diseases [tumour necrosis factor (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF)]. IFN-α alone had no effect on neutrophil apoptosis; however, it abrogated the anti-apoptotic effect of GM-CSF (18 h, P < 0·01). The enhanced stability of the anti-apoptotic protein myeloid cell leukaemia 1 (Mcl-1) and delayed activation of caspase activation normally regulated by GM-CSF were blocked by IFN-α: this effect was mediated, in part, by activation of p38 mitogen-activated protein kinase (MAPK). IFN-α alone also primed reactive oxygen species (ROS) production and maintained the transient priming effect of TNF-α for up to 4 h: it also down-regulated GM-CSF- and TNF-α-activated expression of chemokine (C-X-C motif) ligand (CXCL)1, CXCL2, CXCL3, CXCL8, CCL3 and CCL4 but, in contrast, increased the expression of CXCL10. These novel data identify complex regulatory signalling networks in which type I IFNs profoundly alter the response of neutrophils to inflammatory cytokines. This is likely to have important consequences in vivo and may explain the complexity and heterogeneity of inflammatory diseases such as RA, in which multiple cytokine cascades have been activated.

APPA (apocynin and paeonol) modulates pathological aspects of human neutrophil function, without supressing antimicrobial ability, and inhibits TNFα expression and signalling.

Neutrophils are key players in the pathophysiological process underlying inflammatory conditions not only by release of tissue-damaging cytotoxic enzymes, reactive oxygen species (ROS) but also by secretion of important immunomodulatory chemokines and cytokines. Here, we report the effects of the novel agent APPA, undergoing formal clinical development for treatment of osteoarthritis, and its constituent components, apocynin (AP) and paeonol (PA) on a number of neutrophil functions, including effects on TNFα- expression and signalling. Neutrophils were treated with APPA (10-1000 µg/mL) prior to the measurement of cell functions, including ROS production, chemotaxis, apoptosis and surface receptor expression. Expression levels of several key genes and proteins were measured after incubation with APPA and the chromatin re-modelling agent, R848. APPA did not significantly affect phagocytosis, bacterial killing or expression of surface receptors, while chemotactic migration was affected only at the highest concentrations. However, APPA down-regulated neutrophil degranulation and ROS levels, and decreased the formation of neutrophil extracellular traps. APPA also decreased cytokine-stimulated gene expression, inhibiting both TNFα- and GM-CSF-induced cell signalling. APPA was as effective as infliximab in down-regulating chemokine and IL-6 expression following incubation with R848. Whilst APPA does not interfere with neutrophil host defence against infections, it does inhibit neutrophil degranulation, and cytokine-driven signalling pathways (e.g. autocrine signalling and NF-κB activation), processes that are associated with inflammation. These observations may explain the mechanisms by which APPA exerts anti-inflammatory effects and suggests a potential therapeutic role in inflammatory diseases in which neutrophils and TNFα signalling are important in pathology, such as rheumatoid arthritis.

The CDK inhibitor purvalanol A induces neutrophil apoptosis and increases the turnover rate of Mcl-1: potential role of p38-MAPK in regulation of Mcl-1 turnover.

Human neutrophils are terminally differentiated cells that do not replicate and yet express a number of enzymes, notably cell cycle-dependent kinases (CDKs), that are associated normally with control of DNA synthesis and cell cycle progression. In neutrophils, CDKs appear to function mainly to regulate apoptosis, although the mechanisms by which they regulate this process are largely unknown. Here we show that the CDK2 inhibitor, purvalanol A, induces a rapid decrease in myeloid cell leukaemia factor-1 (Mcl-1) levels in human neutrophils and peripheral blood mononuclear cells (PBMCs), but only induces apoptosis in neutrophils which are dependent upon expression on this protein for survival. This rapid decrease in cellular Mcl-1 protein levels was due to a purvalanol A-induced decrease in stability, with the half-life of the protein decreasing from approximately 2 h in control cells to just over 1 h after addition of the CDK2 inhibitor: it also blocked the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent stabilization of Mcl-1. Purvanalol A blocked GM-CSF-stimulated activation of extracellular-regulated kinase (Erk) and signal transducer and activator of transcription (STAT)-3, and stimulated an additive activation of protein kinase B (Akt) with GM-CSF. Purvalanol A alone stimulated a rapid and sustained activation of p38-mitogen-activated protein kinase (MAPK) and the pan p38-MAPK inhibitor, BIRB796, partly blocked the purvalanol A-induced apoptosis and Mcl-1 loss. These novel effects of purvalanol A may result, at least in part, from blocking GM-CSF-mediated Erk activation. In addition, we propose that purvalanol A-induced activation of p38-MAPK is, at least in part, responsible for its rapid effects on Mcl-1 turnover and acceleration of neutrophil apoptosis.

Janus kinase inhibitors prevent migration of rheumatoid arthritis neutrophils towards interleukin-8, but do not inhibit priming of the respiratory burst or reactive oxygen species production.

Neutrophils play a crucial role in the pathophysiology of rheumatoid arthritis (RA) via the release of reactive oxygen species (ROS), proteases and cytokines. Orally active Janus kinase (JAK) inhibitors (JAKi), e.g. baricitinib and tofacitinib, have high clinical efficacy in RA but are linked with neutropenia and increased infections. Our aim was to determine the effect of JAK inhibition with baricitinib and tofacitinib on healthy control and RA neutrophil lifespan and function. RA (n = 7) and healthy control (n = 7) neutrophils were treated with baricitinib or tofacitinib for 30 min, prior to incubation in the absence or presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon (IFN)-γ. JAKi prevented GM-CSF- and IFN-γ-induced apoptosis delay in RA and healthy control neutrophils in a dose-dependent manner. Baricitinib decreased the rate of chemotaxis towards interleukin (IL)-8, but not f-Met-Leu-Phe (fMLP) in RA neutrophils. While healthy control neutrophils incubated with GM-CSF became primed to produce ROS in response to stimulation with fMLP and phorbol-12-myristate-12-acetate (PMA), RA neutrophils produced increased levels of ROS without the need for priming. JAKi prevented ROS release from primed healthy control neutrophils in response to fMLP, but had no effect on ROS production by RA neutrophils. Baricitinib reversed GM-CSF priming of ROS production in response to fMLP in healthy control, but not RA, neutrophils. We conclude that incubation with JAKi prevents chemotaxis of RA neutrophils towards IL-8, but does not prevent the production of ROS or increase the level of apoptosis. This may be due to the in-vivo exposure of RA neutrophils to priming agents other than those that activate JAK/signal transducer and activator of transcription (STAT) signalling.

Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism.

Disease recurrence and rejection following liver transplantation for autoimmune chronic active liver disease.

Autoimmune chronic active liver disease (ACALD), a major indication for liver transplantation, is associated strongly with antigenic determinants HLA-B8 and DR3. A retrospective analysis of 43 patients who underwent OLTx for putative ACALD and who, as well as their tissue organ donors, were typed, was performed. Disease recurrence and graft rejection episodes were determined by chart review and histopathological review of all material available. Disease recurrence was histologically documented in 11 (25.6%) of these 43 cases. Graft rejection episodes occurred in 24 (55.8%). All recurrences were in recipients of HLA-DR3-negative grafts. Nine of the recurrences were in HLA-DR3-positive recipients (odds ratio: 6.14, P less than 0.03). Two of 11 cases of disease recurrence were in recipients who were HLA-DR3-negative. Nine of these 11 had received HLA-DR3-negative grafts. Rejection occurred in 13 HLA-B8-positive recipients, 12 of whom received HLA-B8-negative grafts. Eleven HLA-B8-negative recipients experienced at least one rejection episode and 9 of these had received HLA-B8-negative grafts. Based upon these data we conclude: 1) that recurrence of putative ACALD is more likely to occur in HLA-DR3-positive recipients of HLA-DR3-negative grafts; (2) that recurrences were not seen in recipients of HLA-DR3-positive grafts; (3) that HLA-B8 status does not affect disease recurrence; and (4) that neither the HLA-B8 nor the DR3 status of the graft or recipient has an effect on the observed frequency of rejection.

A study of the effectiveness of a specific treatment for elective mutism.

Elective mutism represents a relatively rare clinical syndrome in which children with normal verbal capabilities totally refuse to speak in select settings for prolonged periods of time. It is often described as particularly treatment resistant. This study describes 20 strictly diagnosed elective mutes treated, using the Hawthorn Center approach. As found in other studies, most of the elective mutes in this sample (90%) were described as controlling, negative, or oppositional, and a high rate (50%) of maternal-child overenmeshment was found. Treatment outcome using the Hawthorn Center approach was found to be at least fair for all cases and excellent for 85%.

RANK, RANKL and osteoprotegerin in bone biology and disease.

Upon the discovery of RANK, RANKL and OPG in the late 1990s, their importance in the maintenance of the skeletal structure and their dramatic role in bone disease were largely unexpected. In recent years the understanding of these proteins, in particular their regulation, has greatly increased. This review aims to bring the interested reader up to date with the latest news and views on the mechanisms controlling bone resorption in normal and pathological conditions.