Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

Sulfonated human immunoglobulin enhances CD16-linked CD11b expression on human neutrophils

Intravenous human immunoglobulin therapy infrequently results in excessive inflammatory responses in vivo; these effects are not fully understood. We assessed whether sulfonated human immunoglobulin (SHIG) or polyethylene glycol-treated human immunoglobulin (PHIG) enhanced expression of inflammatory receptors on peripheral blood neutrophils in vitro, such as alphaMbeta2 (CD11b/CD18) and Fc gamma receptor type III (FcgammaRIII). CD11b and CD16 expression on neutrophils was measured by fluorescence flow cytometry. Various cytokines were assessed using a highly sensitive fluorescence microsphere system. SHIG enhanced/induced CD11b expression and partial aggregations on neutrophils, but PHIG did not. No detection of aggregation IgG was observed in SHIG and PHIG. SHIG-induced CD11b expression was inhibited by treatment of corticosteroid (dexamethasone) and by anti-CD16 monoclonal antibody. Concentrations of various cytokines such as interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, RANTES, tumor necrosis factor (TNF)-alpha, and interferon (INF)-gamma in culture supernatant were not significantly changed by SHIG or PHIG. SHIG and PHIG did not enhance CD16 on neutrophils. SHIG enhanced CD16-linked CD11b expression on neutrophils in vitro. CD11b induction was inhibited by dexamethasone and by anti-CD16 antibody. These in vitro results suggest that aggregations and enhancement of CD11b on neutrophils by SHIG may induce excessive inflammatory responses in vivo.     

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b⁻ cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b⁻ cells increased the number of AlkP⁺ cells from the plastic adherent fraction from 6.3% ± 0.8 to 56% ± 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b⁻ fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.     

CD11b表达与人工髋关节置换术后无菌性松动的相关性分析

目的:分析髋关节周围滑膜组织CD11b表达水平与人工髋关节置换术后无菌性松动的相关性。方法:以2006年5月至2016年5月于西京医院接受人工髋关节置换的患者为研究对象,对其髋关节周围滑膜组织进行CD11b免疫组化染色,并随访术后5年和10年无菌性松动的发生情况,通过单因素分析及logistic回归分析讨论CD11b表达与无菌性松动之间的相关性。结果:共300例患者纳入研究,全部获得随访,CD11b表达阳性患者163例,阳性率为54.33%;术后5年松动患者29例,术后5年无菌性松动发生率为9.67%;术后10年无菌性松动患者49例,术后10年无菌性松动发生率为16.33%;单因素分析结果表明CD11b表达阳性患者5年及10年松动率均高于CD11b表达阴性患者(P0.05);logistic回归分析结果表明CD11b过表达是髋关节置换术后无菌性松动发生的危险因素。结论:髋关节周围滑膜组织CD11b过表达是人工髋关节置换术后无菌性松动发生的危险因素,该分子或可作为无菌性松动的辅助诊断指标及潜在治疗靶点。     

URG11 promotes gastric cancer growth and invasion by activation of β‐catenin signalling pathway

Upregulated gene 11 (URG11), a new gene upregulated by Heptatitis B Virus X protein (HBx), was previously shown to activate β‐catenin and promote hepatocellular growth and tumourigenesis. Although the oncogenic role of URG11 in the development of hepatocellular carcinoma has been well documented, its relevance to other human malignancies and the underlying molecular mechanisms remain largely unknown. Here we reported a novel function of URG11 to promote gastric cancer growth and metastasis. URG11 was found to be highly expressed in gastric cancer tissues compared with adjacent nontumourous ones by immunohistochemical staining and western blot. Knockdown of URG11 expression by small interfering RNA (siRNA) effectively attenuated the proliferation, anchorage‐independent growth, invasiveness and metastatic potential of gastric cancer cells. URG11 inhibition led to decreased expression of β‐catenin and its nuclear accumulation in gastric cancer cells and extensive costaining between URG11 and β‐catenin was observed in gastric cancer tissues. Transient transfection assays with the β‐catenin promoter showed that it was inhibited by URG11‐specific small inhibitory RNA. Moreover, suppression of endogenous URG11 expression results in decreased activation of β‐catenin/TCF and its downstream effector genes, cyclinD1 and membrane type 1 matrix metallopeptidase (MT1‐MMP), which are known to be involved in cell proliferation and invasion, respectively. Taken together, our data suggest that URG11 contributes to gastric cancer growth and metastasis at least partially through activation of β‐catenin signalling pathway. These findings also propose a promising target for gene therapy in gastric cancer.     

Altered frequency of CD8+CD11c+ T cells and expression of immunosuppressive molecules in lymphoid organs of mouse model of colorectal cancer

CD11c is a member of the β2-integrin family typically used to define myeloid dendritic cells (DCs). Recent reports identify CD11c-expressing CD8⁺ T cells as a new subset of CD8⁺ regulatory T cells (Treg). Evidence exists that CD11c⁺CD8⁺ T cells may exert their effector or regulatory functions under different conditions. To date, no studies have addressed the frequency of CD11c⁺ T cells in cancer. Limited evidence exists in terms of expression of immune-checkpoint receptors, programmed cell death protein 1 (PD-1) and T-lymphocyte-associated antigen 4 (CTLA-4), as well as forkhead box P3 (Foxp3) in mouse lymphoid organs. Here, we have assessed CD11c⁺CD8⁺ and CD11c⁺CD4⁺ T cells, Foxp3, PD-1, and CTLA-4 expressing CD4⁺ T cells and CD8⁺ T cells in different tissues from three groups of male BALB/c mice—young, mature, and those with colorectal cancer (CRC). Analysis of CD3⁺CD11c⁺ T cells in the bone marrow (BM), spleen, and lymph nodes (LN) in each group showed a higher percentage of CD3⁺CD11c⁺ T cells in the BM from all groups and in the lymphoid organs of the cancer group compared with the young and mature groups. CD4^low and CD4^high cell fractions in mice BM have different expression patterns for Foxp3 and CTLA-4. We have observed a higher frequency of CD8⁺PD-1⁺ T cells in the BM, spleen, and LN of CRC mice compared with normal mice. T-cell exhaustion is associated with inhibitory receptor PD-1. According to the regulatory roles of CD11c expression in CD8⁺ T cells, we have proposed that the elevated percentage of CD11c, Foxp3, CTLA-4, and PD-1 expressing T cells were associated with immune response dysregulation in CRC.     

Gr‐1+CD11b+ myeloid cells efficiently home to site of injury after intravenous administration and enhance diabetic wound healing by neoangiogenesis

Vascularization is an important factor that affects diabetic wound healing. There is increasing evidence that myeloid cell lineages play a role in neovascularization. In this study, the efficiency of Gr‐1+CD11b+ myeloid cells to home to the site of injury and enhance diabetic wound healing by neoangiogenesis after intravenous administration was investigated. Gr‐1+CD11b+ myeloid cells were injected into tail vein after establishment of dorsal window chamber, hindlimb ischaemia and ear‐punch injury in diabetic or non‐diabetic mice. The Gr‐1+CD11b+ myeloid cells efficiently homed to the site of injury after intravenous administration and increased neoangiogenesis. The chemokine receptor type 4 (CXCR4) is robustly expressed by Gr‐1+CD11b+ myeloid cells. Inhibition of CXCR4 decreases the homing ability of Gr‐1+CD11b+ myeloid cells to the site of injury, which indicates that the CXCR4/SDF‐1 axis plays an important role in the homing of Gr‐1+CD11b+ myeloid cells to the site of injury. In addition, Gr‐1+CD11b+ myeloid cells were found to improve blood flow recovery of ischaemic limb and enhance wound healing in diabetic mice by neoangiogenesis after intravenous administration. Taken together, the results of this study suggest that Gr‐1+CD11b+ myeloid cells may serve as a potential cell therapy for diabetic wound healing.     

Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac-1)

Full-length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac-1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu-CAMs). The biosynthesis of recombinant Mac-1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac-1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac-1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC₅₀) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac-1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac-1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet-Leu-Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF-alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac-1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.     

High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.     

Increased CD11b+ Gr‐1+ cell population in the placenta after infection with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan pathogen that can cross the placenta, resulting in congenital toxoplasmosis with severe fetal brain abnormalities. The molecular mechanisms of immune responses against T. gondii infection in the placenta have largely remained unclear. An analytical method for characterizing phenotypes of immune cells in the placenta by flow cytometry was established and it was found that numbers of CD11b⁺ Gr‐1⁺ cells in the placenta increased significantly after T. gondii infection. These results suggest that innate immune responses play an important role in immunity against T. gondii infection via the feto‐maternal interface.     

Rap2b promotes proliferation,migration, and invasion of lung cancer cells

Rap2b, a member of the guanosine triphosphate-binding proteins, is widely up-regulated in many types of tumors. However, the functional role of Rap2b in tumorigenesis of lung cancer remains to be fully elucidated. In this study, we investigated the effect of Rap2b on the lung cancer malignant phenotype, such as cell proliferation and metastasis. We found that Rap2b could promote the abilities of lung cancer cell wound healing, migration, and invasion via increasing matrix metalloproteinase-2 enzyme activity. Furthermore, Rap2b overexpression could increase the phosphorylation level of extracellular signal-regulated protein kinases 1/2. In conclusion, our results suggested that Rap2b may be a potential therapeutic target for lung cancer.     

Tumor-conditioned Gr-1CD11b myeloid cells induce angiogenesis through the synergistic action of CCL2 and CXCL16 in vitro

Gr-1⁺CD11b⁺ cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1⁺CD11⁺ cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1⁺CD11b⁺ cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1⁺CD11b⁺ cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1⁺CD11b⁺ cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1⁺CD11b⁺ cells. Incubation of Gr-1⁺CD11b⁺ cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1⁺CD11b⁺ cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1⁺CD11b⁺ cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1⁺CD11b⁺ myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1⁺CD11b⁺ cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.     

CD11b promotes the differentiation of osteoclasts induced by RANKL through the spleen tyrosine kinase signalling pathway

Macrophage surface antigen‐1 (Mac‐1, CD11b/CD18) has been implicated in the regulation of osteoclastogenesis. In the synovial tissues of patients with aseptic loosening after total hip replacement, CD11b was up‐regulated, which indicated that CD11b is closely involved in osteolysis around the prosthesis. We found that CD11b, but not CD18, promoted osteoclast (OC) maturation. Here, we show CD11b up‐regulated the levels of spleen tyrosine kinase (Syk), c‐Fos and nuclear factor of activated T cells, cytoplasmic‐1 (NFATc1), as well as the activity of extracellular‐regulated kinase (Erk), and as a result, osteoclast precursors (OCPs) differentiated and became tartrate‐resistant acid phosphatase (TRAP)‐positive. In addition, increased tumour necrosis factor‐α (TNF‐α) induced by ultra‐high molecular weight polyethylene (UHMWPE) particles up‐regulated the level of CD11b. Taken together, these findings suggest that CD11b is a positive regulator of osteoclastogenesis and that it functions by activating the Syk signalling pathway, while CD18 does not have the same effect.     

Prostate cancer bone metastases promote both osteolytic and osteoblastic activity

Advanced prostate cancer is frequently accompanied by the development of metastasis to bone. In the past, prostate cancer bone metastases were characterized as being osteoblastic (i.e., increasing bone density) based on radiographs. However, emerging evidence suggests that development of prostate cancer bone metastases requires osteoclastic activity in addition to osteoblastic activity. The complexities of how prostate tumor cells influence bone remodeling are just beginning to be elucidated. Prostate cancer cells produce a variety of pro-osteoblastic factors that promote bone mineralization. For example, both bone morphogenetic proteins and endothelin-1 have well recognized pro-osteoblastic activities and are produced by prostate cancer cells. In addition to factors that enhance bone mineralization prostate cancer cells produced factors that promote osteoclast activity. Perhaps the most critical pro-osteoclastogenic factor produced by prostate cancer cells is receptor activator of NFkappaB ligand (RANKL), which has been shown to be required for the development of osteoclasts. Blocking RANKL results in inhibiting prostate cancer-induced osteoclastogenesis and inhibits development and progression of prostate tumor growth in bone. These findings suggest that targeting osteoclast activity may be of therapeutic benefit. However, it remains to be defined how prostate cancer cells synchronize the combination of osteoclastic and osteoblastic activity. We propose that as the bone microenvironment is changed by the developing cancer, this in turn influences the prostate cancer cells' balance between pro-osteoclastic and pro-osteoblastic activity. Accordingly, the determination of how the prostate cancer cells and bone microenvironment crosstalk are important to elucidate how prostate cancer cells modulate bone remodeling.     

CD166 promotes the cancer stem-like properties of primary epithelial ovarian cancer cells

Cancer stem cells (CSCs) or tumor-initiating cells are thought to play critical roles in tumorigenesis, metastasis, drug resistance, and tumor recurrence. For the diagnosis and targeted therapy of CSCs, the molecular identity of biomarkers or therapeutic targets for CSCs needs to be clarified. In this study, we identified CD166 as a novel marker expressed in the sphere-forming CSC population of A2780 epithelial ovarian cancer cells and primary ovarian cancer cells. The CD166⁺ cells isolated from A2780 cells and primary ovarian cancer cells highly expressed CSC markers, including ALDH1a1, OCT4, and SOX2, and ABC transporters, which are implicated in the drug resistance of CSCs. The CD166⁺ cells exhibited enhanced CSC-like properties, such as increased sphere-forming ability, cell migration and adhesion abilities, resistance to conventional anti-cancer drugs, and high tumorigenic potential in a xenograft mouse model. Knockdown of CD166 expression in the sphere-forming ovarian CSCs abrogated their CSC-like properties. Moreover, silencing of CD166 expression in the sphere-forming CSCs suppressed the phosphorylation of focal adhesion kinase, paxillin, and SRC. These results suggest that CD166 plays a key role in the regulation of CSC-like properties and focal adhesion kinase signaling in ovarian cancer.     

Myeloperoxidase deficiency enhances zymosan phagocytosis associated with up-regulation of surface expression of CD11b in mouse neutrophils

Myeloperoxidase (MPO), a major component of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion. Phagocytosis is a critical event induced by neutrophils for host defense and inflammation. Interestingly, we found that MPO-deficient (MPO^?/?) neutrophils engulfed larger amounts of zymosan than wild-type neutrophils. Blocking of the CD11b subunit of complement receptor 3 (CR3) as well as inhibition of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) dramatically reduced zymosan phagocytosis. In contrast, blocking of dectin-1, toll-like receptor 2 (TLR2), or spleen tyrosine kinase (Syk) had no significant effects on phagocytosis. Western blotting analysis showed that inhibition of FAK decreased the phosphorylation of ERK1/2, indicating that ERK1/2 is a downstream regulator of FAK in neutrophils. Importantly, we found that cell surface expression of CD11b and phosphorylation of ERK1/2 was significantly higher in zymosan-stimulated MPO^?/? neutrophils than in zymosan-stimulated wild-type neutrophils. Pretreatment with the MPO inhibitor 4-aminobenzoic acid hydrazide dramatically enhanced both zymosan phagocytosis and the surface expression of CD11b in wild-type neutrophils, but not in MPO^?/? neutrophils. Collectively, these results strongly suggest that up-regulation of the CD11b/FAK/ERK signaling pathway due to absence of MPO enhances the zymosan phagocytic activity of mouse neutrophils.     

CD11b在小鼠急性结肠炎模型中的表达及鼠李糖乳杆菌的调节作用

目的 研究CD11b在炎症性肠病模型小鼠结肠组织中的表达及定位,探讨鼠李糖乳杆菌(LGG)对CD11b的调节作用.方法 将28只SPF级小鼠随机分为3组:Ctrl组8只、DSS组10只、DSS+LGG组10只,给予3％ DSS自由饮用建立急性结肠炎模型,DSS+ LGG组小鼠提前1周管饲LGG共14 d;模型建立期间观...