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Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles following severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1β (IL-1β) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1α and IL-1β, we show that IL-1 signaling contributes to NHO development following SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor or C-C chemokine ligand-2 did not promote NHO development. Finally using NHO biopsies from SCI and TBI patients, we show that IL-1β is expressed by CD68+ macrophages. IL-1α and IL-1β produced by activated human monocytes promote calcium mineralization and RUNX2 expression in fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether these data suggest that interleukin-1 promotes NHO development in both humans and mice. This article is protected by copyright. All rights reserved.

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.

Skeletal metastases present a major clinical challenge for prostate cancer patient care, inflicting distinctive mixed osteoblastic and osteolytic lesions that cause morbidity and refractory skeletal complications. Macrophages are abundant in bone and bone marrow and can influence both osteoblast and osteoclast function in physiology and pathology. Herein, we examined the role of macrophages in prostate cancer bone lesions, particularly the osteoblastic response. First, macrophage and lymphocyte distributions were qualitatively assessed in patient's prostate cancer skeletal lesions by immunohistochemistry. Second, macrophage functional contributions to prostate tumour growth in bone were explored using an immune-competent mouse model combined with two independent approaches to achieve in vivo macrophage depletion: liposome encapsulated clodronate that depletes phagocytic cells (including macrophages and osteoclasts); and targeted depletion of CD169(+) macrophages using a suicide gene knock-in model. Immunohistochemistry and histomorphometric analysis were performed to quantitatively assess cancer-induced bone changes. In human bone metastasis specimens, CD68(+) macrophages were consistently located within the tumour mass. Osteal macrophages (osteomacs) were associated with pathological woven bone within the metastatic lesions. In contrast, lymphocytes were inconsistently present in prostate cancer skeletal lesions and when detected, had varied distributions. In the immune-competent mouse model, CD169(+) macrophage ablation significantly inhibited prostate cancer-induced woven bone formation, suggesting that CD169(+) macrophages within pathological woven bone are integral to tumour-induced bone formation. In contrast, pan-phagocytic cell, but not targeted CD169(+) macrophage depletion resulted in increased tumour mass, indicating that CD169(-) macrophage subset(s) and/or osteoclasts influenced tumour growth. In summary, these observations indicate a prominent role for macrophages in prostate cancer bone metastasis that may be therapeutically targetable to reduce the negative skeletal impacts of this malignancy, including tumour-induced bone modelling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.

Stem cells and their malignant counterparts require the support of a specific microenvironment or "niche". While various anti-cancer therapies have been broadly successful, there are growing opportunities to target the environment in which these cells reside to further improve therapeutic efficacy and outcome. This is particularly true when the aim is to target normal or malignant stem cells. The field aiming to target or use the niches that harbor, protect, and support stem cells could be designated as "nichotherapy". In this essay, we provide a few examples of nichotherapies. Some have been employed for decades, such as hematopoietic stem cell mobilization, whereas others are emerging, such as chemosensitization of leukemia stem cells by targeting their niche.

Mobilization and collection of haemopoietic stem and progenitor cells (HSPC) is the cornerstone of autologous and allogeneic stem cell transplantation for a wide variety of haematological and some non-haematological malignancies. Centres providing this service face the challenge of optimizing the likelihood of successful collection of transplantable doses of cells, while maximizing the efficiency of the apheresis unit and minimizing the risk of toxicity as well as mobilization failure. Recent developments in the understanding of the molecular mechanisms of mobilization have led to the emergence of novel strategies for HSPC mobilization, which may assist in meeting these imperatives. The task for clinicians is how to incorporate the use of these strategies into practice, in the light of emerging evidence for efficacy and safety of these agents. Herein, the literature is reviewed, and a proposed algorithm for HSPC mobilization is presented.

Endolyn (CD164) is a sialomucin that functions as an adhesion molecule and a negative regulator of CD34+ CD38- human haematopoietic precursor cell proliferation. The 105A5 and 103B2/9E10 CD164 monoclonal antibodies (mAbs), which act as surrogate ligands, recognize distinct glycosylation-dependent classes I and II epitopes located on domain I of the native and recombinant CD164 proteins. Here, we document five new CD164 mAbs, the 96 series, that rely on conformational integrity, but not glycosylation, of exons 2- and 3-encoded CD164 domains, thereby resembling the class III mAbs, N6B6 and 67D2. Although all the 96 series class III mAbs labelled both the 105A5+ and 103B2/9E10+ cells, cross-competition and immunoblotting studies allow them to be categorized into two distinct class III subgroups, i.e. the N6B6-like subgroup that only recognizes 80-100 kDa proteins and the 67D2-like subgroup that also recognizes a higher molecular weight (>220 kDa) form. To more closely define the reactivity patterns of mAbs to the classes I and II epitopes, the global glycosylation patterns of the soluble human (h) CD164 proteins were determined using lectin binding, high-performance liquid chromatography (HPLC) and mass spectrometry. hCD164 recombinant proteins bound to the lectins, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin and peanut agglutinin, indicating the presence of high mannose and complex N-glycans, in addition to core 1 O-glycans (the Tn antigen) and alpha2-3 and alpha2-6 sialic acid moieties. Our HPLC and mass spectrometry results revealed both high mannose and complex N-glycosylation with various numbers of branches increasing the complexity of the glycosylation pattern. Most O-glycans were small, core 1 or 2 based. High levels of sialylation in alpha2-3 and alpha2-6 linkages, without sialyl-Lewis X, indicate that the majority of these hCD164 recombinant proteins are unable to bind to selectins in our assay system, but may interact with Siglec molecules.

We have previously shown (Zhou et al: Blood, 72:1870, 1988) that IL3, added with low concentrations of CSF-1 (1 ng/ml) to normal human CD34+ enriched cells, promoted the development of various types of colonies including those containing immature monocytes. However, when high concentrations of CSF-1 (20 ng/ml) were added alone or together with IL3, smaller colonies with mature macrophages were found. Here we show by in situ hybridization that IL3 allows the development, from CD34+ cells, of a subpopulation of immature progenitors which express the CSF-1 receptor (c-fms) mRNA. The expression of c-FMS protein was also substantiated by immunocytochemical studies using anti-c-fms antibody. The percentage of c-fms positive cells peaked at day 7 and began to decrease thereafter. When anti-CSF-1 antibodies were included in the culture, the decrease in c-fms mRNA after day 7 was abrogated. This indicated that endogenous CSF-1 was produced as CD34+ cells developed into monocytes or progenitors of monocytes and that CSF-1 modulates c-fms expression. We further demonstrated that when a high dose of CSF-1 (20 ng/ml) was added at day 7 to IL3-stimulated CD34+ cells, a rapid down-regulation of c-fms mRNA and protein was seen. No down-regulation was observed with low concentration of CSF-1 (1 ng/ml). The possibility that different concentrations of CSF-1 could modulate the development of monocytic progenitors is discussed.

The use of mobilised blood to effect haematological rescue now exceeds that of bone marrow. Despite the now broad application of blood stem cells (BSC) in a variety of clinical settings little is known of the precise mechanisms responsible for their release into the peripheral circulation. Under steady state conditions, primitive haemopoietic progenitor cells (HPC) are restricted to the bone marrow and circulate at low to undetectable levels. Although the basis for this specific retention of HPC within the BM remains to be defined, adhesive interactions between HPC and their surrounding stromal cell and ECM environment are hypothesised to play a major role. Mobilisation occurs in response to a variety of perturbations including physical exercise, following myelosupressive chemotherapy and following administration of a number of different cytokines. The kinetics of release may vary from as little as a few minutes to several weeks. Such observations suggest that a single unifying mechanism is therefore unlikely to be responsible for mobilisation in all cases and in all likelihood the predominant mechanism(s) will vary according to the mobilising agent employed. Nevertheless, mobilising agents are proposed to act to a greater or lesser extent by perturbing the function of the various cell adhesion molecules (CAM) or ligands which restrict HPC to the BM. In accord with this notion, studies by other have shown that antibody induced perturbation of VLA-4 function results in mobilisation of HPC in primates. How therefore do cytokines, the most commonly mobilising agents, result in HPC release? The most frequently employed agent to induce HPC release is granulocyte colony-stimulating factor (G-CSF). We have previously demonstrated that in vitro exposure of human CD34+ cells (HPC) to G-CSF, in common with several cytokines including IL-3, GM-CSF and SCF, results in transient increases in the affinity of the β 1 integrins VLA-4 and VLA-5 for their ligands fibronectin (FN) and VCAM-I. Thus in addition to the well documented mitogenic effects of cytokines these molecules also regulate the adhesive properties of HPC. Data will therefore be presented which examine the regulation of integrin function on human HPC and its relevance to the phenomenon of mobilisation. In addition we will also review recent observations in man and in a murine model which suggest an unanticipated link between bone turnover and BSC release. Collectively these studies provide some novel insights into the phenomenon of mobilisation but also suggest that the actual mechanisms involved are more complex than is currently appreciated.