Myeloid Monocytes Research Articles

Infiltration by monocytes of the central nervous system and its role in multiple sclerosis: reflections on therapeutic strategies.

Mononuclear macrophage infiltration in the central nervous system is a prominent feature of neuroinflammation. Recent studies on the pathogenesis and progression of multiple sclerosis have highlighted the multiple roles of mononuclear macrophages in the neuroinflammatory process. Monocytes play a significant role in neuroinflammation, and managing neuroinflammation by manipulating peripheral monocytes stands out as an effective strategy for the treatment of multiple sclerosis, leading to improved patient outcomes. This review outlines the steps involved in the entry of myeloid monocytes into the central nervous system that are targets for effective intervention: the activation of bone marrow hematopoiesis, migration of monocytes in the blood, and penetration of the blood-brain barrier by monocytes. Finally, we summarize the different monocyte subpopulations and their effects on the central nervous system based on phenotypic differences. As activated microglia resemble monocyte-derived macrophages, it is important to accurately identify the role of monocyte-derived macrophages in disease. Depending on the roles played by monocyte-derived macrophages at different stages of the disease, several of these processes can be interrupted to limit neuroinflammation and improve patient prognosis. Here, we discuss possible strategies to target monocytes in neurological diseases, focusing on three key aspects of monocyte infiltration into the central nervous system, to provide new ideas for the treatment of neurodegenerative diseases.

A Single Center Retrospective Study on the Efficacy of Haploidentical Hematopoietic Stem Cell Transplantation in Patients with Myeloid Neoplasms over 50 Years Old

Objective: This study aims to evaluate the efficacy and safety of haploidentical hematopoietic stem cell transplantation in patients with myeloid neoplasms over 50 years old, and to compare the differences in survival, recurrence, and adverse reactions between patients aged 50-55 and over 55 years old. Methods: The patients with myeloid neoplasms over 50 years old who received haploidentical hematopoietic stem cell transplantation from November 2016 to December 2021 in the Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University, School of Medicine were enrolled. According to the age at the time of transplantation, patients were divided into Group A (50-55 years old) and Group B (≥ 55 years old). The differences in Overall survival (OS), progression free survival (PFS), non-relapse mortality (NRM), cumulative incidence of relapse (CIR), incidence of graft versus host disease (GVHD), and infection rate between the two groups were analyzed. Results: A total of 163 patients (group A, n=77; group B, n=86) were enrolled, including 112 cases of acute myeloid leukemia (AML), 39 cases of myelodysplastic neoplasms (MDS), 4 cases of chronic myeloid monocyte leukemia (CMML), 3 cases of primary myelofibrosis (MF), and 5 cases of chronic myeloid leukemia (CML). There were no statistically significant differences in baseline data between the two groups, including follow-up time, disease categories, HCT-CI score, remission status of AML patients, minimal residual lesion (MRD) level of AML patients, and IPSS-R score of MDS patients. However, more patients in Group A received myeloablative (MAC) conditioning than patients in Group B (92.2% vs 50%, P=0.000). The median follow-up time was 741 (7-2377) days. The overall 2-year OS is 73.0% ± 3.5%, 2-year PFS is 71.3% ± 3.6%, 2-year NRM is12.23%（7.65%-17.93%）, and 2-year CIR is 17.20%（11.64%-23.69%）. All patients in group A achieved hematopoietic reconstitution except one patient died of graft failure, while 5 patients of group B died of graft failure(P=0.126). Neutrophils and platelet reconstruction in Group B was slightly longer than in Group A. The neutrophils reconstruction occurred at a median of 12 (8-20) day and 13 (9-25) day, respectively (P=0.007), and platelet reconstruction occurred at a median of 12 (9-28) day and 13 (8-37) day, respectively (P=0.055). The 2-year OS of Group A and Group B was 76.6% ± 4.8% and 69.0 ± 5.1%, respectively (P=0.199); The 2-year PFS was 76.2% ± 4.9% and 66.9 ± 5.1%, respectively (P=0.236); The 2-year NRM was 9.16% (4.00% -16.96%) and 18.61% (11.18% -27.51%), respectively (P=0.094); The 2-year CIR was similar, with 14.66% (7.74% -23.67%) and 19.16% (11.48% -28.33%), respectively (P=0.782). Interestingly, there was a difference in NRM between patients receiving reduced-intensity (RIC) and MAC conditioning in Group B, which was 10.2% (3.69% -20.61%) and 25.58% (13.64% -39.34%), respectively (P=0.043), while there was no difference in CIR, which was 17.31% (7.88% -29.80%) and 18.90% (8.71% -32.06%), respectively (P=0.933). It is suggested that in older patients, the strength of conditioning regimen has a greater impact on NMR. The incidence of grade 3-4 aGVHD within 100 days in all patients was 11.84% (7.31% -17.56%), and the incidence of 2-year intensive cGVHD was 8.19% (4.58% -13.14%). The incidence of grade 3-4 aGVHD within 100 days was similar between the two groups, with 9.46% (4.13% -17.46%) and 14.10% (7.46% -22.81%) (P=0.368), respectively. The 2-year intensive cGVHD was 8.07% (3.26% -15.72%) and 8.28% (3.62% -15.42%) (P=0.890), with similar incidence rates. The incidence of bacterial septicemia, pneumonia, invasive fungal infection, herpes virus and other infections were similar in both groups. Univariate and multivariate analysis both showed that 50-55 years old was an independent protective factor of OS (2.384 (1070-5.312), P=0.034) and GRFS (2.683 (1.181-6.094), P=0.018) in AML patients. Conclusion: Haploidentical hematopoietic stem cell transplantation is an effective and safe treatment for myeloid neoplasms over the age of 50. Compared with patients aged 50 to 55 years old, patients with myeloid neoplasms aged over 55 years old had similar efficacy and safety. The impact of MAC and RIC pretreatment on the prognosis of patients at different ages may be different. This retrospective study is limited in number and needs more cases and prospective cohort study to further verify.

Unraveling the mechanisms behind joint damage

A subtype of myeloid monocyte mediates the transition from autoimmunity to joint destruction in rheumatoid arthritis.

RANK+TLR2+ myeloid subpopulation converts autoimmune to joint destruction in rheumatoid arthritis.

Joint destruction is the major clinic burden in patients with rheumatoid arthritis (RA). It is unclear, though, how this autoimmune disease progresses to the point of deterioration of the joint. Here, we report that in a mouse model of RA the upregulation of TLR2 expression and its α(2,3) sialylation in RANK+ myeloid monocytes mediate the transition from autoimmunity to osteoclast fusion and bone resorption, resulting in joint destruction. The expression of α(2,3) sialyltransferases was significantly increased in RANK+TLR2+ myeloid monocytes, and their inhibition or treatment with a TLR2 inhibitor blocked osteoclast fusion. Notably, analysis of our single-cell RNA-sequencing (scRNA-seq) libraries generated from RA mice revealed a novel RANK+TLR2- a subset that negatively regulated osteoclast fusion. Importantly, the RANK+TLR2+ subset was significantly diminished with the treatments, whereas the RANK+TLR2- subset was expanded. Moreover, the RANK+TLR2- subset could differentiate into a TRAP+ osteoclast lineage, but the resulting cells did not fuse to form osteoclasts. Our scRNA-seq data showed that Maf is highly expressed in the RANK+TLR2- subset, and the α(2,3) sialyltransferase inhibitor-induced Maf expression in the RANK+TLR2+ subset. The identification of a RANK+TLR2- subset provides a potential explanation for TRAP+ mononuclear cells in bone and their anabolic activity. Further, TLR2 expression and its α(2,3) sialylation in the RANK+ myeloid monocytes could be effective targets to prevent autoimmune-mediated joint destruction.

Wasabi Component 6-(Methylsulfinyl)hexly Isothiocyanate and Derivatives Improve the Survival of Skin Allografts.

We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14+ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.

Necroptosis Contributes to Persistent Inflammation During Acute Leptospirosis.

Leptospirosis is an emerging infectious disease. Recently, canine and human leptospirosis outbreaks were reported in California and New York, respectively. In this study we evaluated the role that cell death processes play in the inflammatory response to Leptospira. Groups of male C3H/HeJ mice were infected with pathogenic L. interrogans and non-pathogenic L. biflexa for 24 and 72 hours; inflammatory processes were characterized for apoptosis and necroptosis by flowcytometry of spleen cells and were further assessed for expression of biomarkers of necroptosis by western blot. We found that pathogenic L. interrogans promotes apoptosis in myeloid neutrophils and monocytes at 24h and 72h post-infection, whereas L. biflexa promotes apoptosis of myeloid monocytes only at 24h post-infection. It is interesting that the immune cells undergoing the common programmed cell death pathway (apoptosis) are the cell types which were not increased in frequency in spleen of mice infected with L. interrogans (neutrophils) and L. biflexa (monocytes) in our previous study. The same trend was observed with pathogenic L. interrogans inducing necroptosis of myeloid neutrophils in addition to monocytes and macrophages at 24h and/or 72h post-infection, whereas L. biflexa promoted this pro-inflammatory cell death process in monocytes and macrophages only at 24h post-infection. Thus, early apoptosis and necroptosis of these cell types may explain its absence in frequency in spleen. Furthermore, at 24h and 72h, expression of the necroptosis molecular biomarkers p-MLKL, p-RIP1 and p-RIP3 was increased post infection with pathogenic L. interrogans. These data suggest that the underlying cell death processes involved in immune responses to pathogenic Leptospira contribute directly to persistent inflammation during the early stages of leptospirosis.

Interfering with alternatively activated macrophages by CSF-1R inhibition exerts therapeutic capacity on allergic airway inflammation

PurposeAllergic asthma is a chronic inflammatory disorder with airway hyperresponsiveness and tissue remodeling as the main pathological characteristics. The etiology of asthma is relatively complicated, involving genetic susceptibility, epigenetic regulation, environmental factors, and immune imbalance. Colony stimulating factor 1 receptor (CSF-1R), highly expressed in myeloid monocytes, plays an important role in regulating inflammation. However, the pathological role of CSF-1R and the therapeutic effects of CSF-1R inhibitor in allergic airway inflammation remain indistinct. MethodsThe house dust mite (HDM)-triggered allergic airway inflammation model was conducted to fully uncover the efficacies of CSF-1R inhibition, as illustrated by histopathological examinations, biochemical analysis, ELISA, RT-PCR, Western blotting assay, immunofluorescence, and flow cytometry. Furthermore, bone marrow-derived macrophages (BMDMs) were differentiated and polarized upon IL-4/IL-13 induction to clarify the underlying mechanisms of CSF-1R inhibition. ResultsHerein, we presented that the expression of CSF-1R was increased in HDM-induced experimental asthma and inhibition of CSF-1R displayed dramatic effects on the disease severity of asthma, referring to suppressing the secretion of allergic mediators, dysfunction of airway epithelium, and infiltration of inflammatory cells. Furthermore, CSF-1R inhibitor could markedly restrain the polarization and expression of transcriptional factors of alternatively activated macrophages (AAMs) in the presence of IL-4/IL-13 and reduce the recruitment of CSF-1R-dominant macrophages, both in acute and chronic allergic airway inflammation model. ConclusionCollectively, our findings demonstrated the molecular pathological mechanism of CSF-1R in allergic airway diseases and suggested that targeting CSF-1R might be an alternative intervention strategy on the homeostasis of airway immune microenvironment in asthma.

Is There Any Evidence of Monocytes Involvement in Alzheimer's Disease? A Pilot Study on Human Postmortem Brain.

Background:The role of neuroinflammation has become more evident in the pathogenesis of neurodegenerative diseases. Increased expression of microglial markers is widely reported in Alzheimer’s disease (AD), but much less is known about the role of monocytes in AD pathogenesis. In AD animal models, bone marrow-derived monocytes appear to infiltrate the parenchyma and contribute to the phagocytosis of amyloid-β depositions, but this infiltration has not been established in systematic studies of the human brain postmortem.Objective:In addition to assessing the distribution of different subtypes of microglia by immunostaining for CD68, HLA-DR, CD163, and CD206, we focused on the involvement of C-chemokine receptor type2 (CCR2) positive monocytes during the AD course.Methods:We used formalin-fixed and paraffin-embedded tissue from four vulnerable brain regions (hippocampus, occipital lobe, brainstem, and cerebellum) from neuropathologically characterized AD cases at different Braak stages and age-matched controls.Results:Only singular migrated CCR2-positive cells were found in all brain regions and stages. The brainstem showed the highest number of positive cells overall, followed by the hippocampus. This mechanism of recruitment seems to work less efficiently in the human brain at an advanced age, and the ingress of monocytes obviously takes place in much reduced numbers or not at all.Conclusion:In contrast to studies on animal models, we observed only a quite low level of myeloid monocytes associated with AD pathology. Furthermore, we provide evidence associating early microglial reactions carried out in particular by pro-inflammatory cells with early effects on tangle- and plaque-positive vulnerable brain regions.

PRT4165 nanocomposite promoting epigenetic retardation through proteasomal depletion of polycomb in acute myeloid leukemia

The polycomb repressive complex 1 (PRC1) protein, Bmi1 is overexpressed in acute myeloid leukemia and mediates histone H2A monoubiquitination through its ubiquitin (E3) ligase activity. The 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) inhibits PRC1-mediated H2A monoubiquitination but the poor aqueous solubility, and physiological instability limit its application. In current study, the encapsulation of PRT4165 in human serum albumin nanoparticles (HSANPs) improved its therapeutic efficacy as evident through the enhanced apoptosis, sub-G1 cell cycle arrest, depolarized mitochondrial membrane, reactive oxygen species generation and caspase 3 activation. The nanoformulation repressed Bmi1 through ubiquitin-proteasome pathway (UPP) and regulated H2AK119 ubiquitination in AML cells. Co-immunoprecipitation studies revealed the direct interaction of Bmi1 and C-Myb, a crucial regulator of AML pathogenesis. Moreover, PRT4165 encapsulated HSANPs showed improved in vivo biodistribution, better dispersibility and biocompatibility, and exhibited enhanced suppression of leukemia stem cell marker, CD45+ and activation of myeloid monocytes differentiation marker, CD11b+ in AML xenograft mouse model.

Synergy Between Peroxisome Proliferator-Activated Receptor γ Agonist and Radiation Therapy in Cancer

Approximately 60% of newly diagnosed cancer patients receive radiotherapy to accomplish neoadjuvant, definitive, adjuvant or palliative purpose as required. However, radiotherapy may trigger host-mediated proangiogenic and proinflammatory effects that lead to tumor recurrence. Angiogenesis and inflammation are crucial processes, through which tumor microenvironment (TME) influences tumor growth, invasiveness and metastasis. Peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone has anti-inflammatory, antiangiogenic and antineoplastic activities from preclinical data. We hypothesize that rosiglitazone and radiation therapy synergistically amplify the antitumor effect, which is based on the evidence that rosiglitazone remodels tumor vasculature and limits tumor-associated macrophages (TAMs) infiltration. The aim of this study is to evaluate the synergistic effect of PPARγ agonist rosiglitazone combined with radiation therapy. Each female Balb/c mouse was inoculated with 5X105 murine colon carcinoma cell line CT26 or breast cancer 4T1. When tumor size approximately reached 250-300 mm3, 12Gy in a fraction as hypofractionated irradiation therapy (HFRT) or 12Gy in 4 daily fractions of 3Gy as conventional fractionated irradiation therapy (CFRT) was delivered to the tumor, meanwhile administration of 100mg/kg rosiglitazone was started daily for 2 weeks. Tumors were harvested according to the schedule. Flow cytometry, ELISA, immunofluorescence, western blot, and so forth were performed to test the synergistic mechanisms. We presented that rosiglitazone has antiangiogenic effect by inhibiting VEGF-VEGFR2 signaling pathways. Immunofluorescence work identified that rosiglitazone can remodel tumor vasculature contributing to decrease of microvessel density, promotion of pericyte coverage, reduction of TAMs infiltration, and even further accompanied improvement of intratumoral hypoxia and vessel perfusion compared with the control (P<0.05). 4T1 or CT26 tumor-bearing mice treated with rosiglitazone in combination with HFRT or CFRT showed a significant reduction in lesion size and lung metastases compared with the control (P<0.001), rosiglitazone(P<0.001) or radiotherapy group (P<0.05). Analysis of ELISA, immunofluorescence and flow cytometry showed that tumor-derived chemokine CCL2 in reaction to HFRT facilitates the recruitment of migrating CD11b+ myeloid monocytes and TAMs into irradiated sites that initiates vasculogenesis and allows tumor recurrence after HFRT. We found that rosiglitazone decreased CCL2 and suppressed CD11b+ myeloid monocytes and TAMs infiltrations after HFRT, which delayed tumor recurrence. Rosiglitazone may elicit normalization of tumor vasculature and limit TAMs infiltration, and thereby delay tumor progression with a fresh perspective. Rosiglitazone, as an adjuvant therapy to HFRT, inhibits CCL2 mediating tumor recurrence.

Synergy between peroxisome proliferator-activated receptor γ agonist and radiotherapy in cancer.

Angiogenesis and inflammation are crucial processes through which the tumor microenvironment (TME) influences tumor progression. In this study, we showed that peroxisome proliferator‐activated receptor γ (PPARγ) is not only expressed in CT26 and 4T1 tumor cell lines but also in cells of TME, including endothelial cells and tumor‐associated macrophages (TAM). In addition, we showed that rosiglitazone may induce tumor vessel normalization and reduce TAM infiltration. Additionally, 4T1 and CT26 tumor‐bearing mice treated with rosiglitazone in combination with radiotherapy showed a significant reduction in lesion size and lung metastasis. We reported that a single dose of 12 Gy irradiation strongly inhibits local tumor angiogenesis. Secretion of C‐C motif chemokine ligand 2 (CCL2) in response to local irradiation facilitates the recruitment of migrating CD11b+ myeloid monocytes and TAM to irradiated sites that initiate vasculogenesis and enable tumor recurrence after radiotherapy. We found that rosiglitazone partially decreases CCL2 secretion by tumor cells and reduces the infiltration of CD11b+ myeloid monocytes and TAM to irradiated tumors, thereby delaying tumor regrowth after radiotherapy. Therefore, combination of the PPARγ agonist rosiglitazone with radiotherapy enhances the effectiveness of radiotherapy to improve local tumor control, decrease distant metastasis risks and delay tumor recurrence.

Targeted disruption of adenosine kinase in myeloid monocyte cells increases osteoclastogenesis and bone resorption in mice.

Adenosine kinase (ADK) serves an important role in intracellular adenosine clearance via phosphorylating adenosine to AMP. The role of adenosine and its receptors in the maintenance of bone homeostasis is well studied, particularly in osteoclastogenesis and bone resorption; however, the function of ADK in bone metabolism is still unclear. In the present study, utilizing the cre/floxp recombination system, mice with conditional loss of ADK function in myeloid monocyte cells were used to assess the effect of ADK deficiency on bone metabolism. Mice were evaluated by means of gross observation and bone histomorphometric analysis. Exvivo osteoclast differentiation and bone resorption were also examined using genetic deletion and pharmacologic inhibition of ADK in osteoclasts. Compared with control mice, the results of the present study demonstrate that adult mice lacking ADK in the myeloid monocyte cells had reduced body weight and nasoanal length. The results of bone histomorphometric analysis revealed that bone mass was significantly decreased and osteoclastic parameters were increased in the study mice. Furthermore, invitro cell culture revealed that inhibition of ADK function promoted osteoclast differentiation and bone resorption. Osteoclast‑associated gene expression, including tartrate‑resistant acid phosphatase, nuclear factor of activated T‑cells, cytoplasmic 1, matrix metalloproteinase 9, Cathepsin K and calcitonin receptor, was also significantly increased. These results suggest that mice with ADK deficiency have reduced bone formation due to increased osteoclastogenesis and bone resorption. The present study provides further insight into the mechanism by which ADK serves a key role in bone metabolism.

Central role of myeloid MCPIP1 in protecting against LPS-induced inflammation and lung injury

Although systemic inflammatory responses attributable to infection may lead to significant lung injury, the precise molecular mechanisms leading to lung damage are poorly understood and therapeutic options remain limited. Here, we show that myeloid monocyte chemotactic protein-inducible protein 1 (MCPIP1) plays a central role in protecting against LPS-induced inflammation and lung injury. Myeloid-specific MCPIP1 knockout mice developed spontaneous inflammatory syndromes, but at a late age compared to global MCPIP1 knockout mice. Moreover, mice with a myeloid-specific deletion of MCPIP1 were extremely sensitive to LPS-induced lung injury due to overproduction of proinflammatory cytokines and chemokines. We identified C/EBPβ and C/EBPδ, two critical transcriptional factors that drive cytokine production and lung injury, as targets of MCPIP1 RNase. LPS administration caused MCPIP1 protein degradation in the lungs. Pharmacological inhibition of MALT1, a paracaspase that cleaves MCPIP1, by MI-2 selectively increased the MCPIP1 protein levels in macrophages and in the lungs. Meanwhile, administration of MI-2 protected mice from LPS-induced inflammation, lung injury and death. Collectively, these results indicate that myeloid MCPIP1 is central in controlling LPS-induced inflammation and lung injury. Pharmacological inhibition of MALT1 protease activity may be a good strategy to treat inflammatory diseases by enhancing MCPIP1 expression in myeloid cells.

Differential control of hypoxia-inducible factor 1 activity during pro-inflammatory reactions of human haematopoietic cells of myeloid lineage

The biological responses of human haematopoietic cells of myeloid lineage following malignant transformation depends crucially on their adaptation to signalling stress. This adaptation is dependent on the activation of the hypoxia-inducible factor 1 (HIF-1) transcription complex, although the mechanisms underlying its control remain unclear. Here, we report that mammalian target of rapamycin (mTOR) kinase, a central regulator of myeloid cell growth and metabolism, is crucially involved in HIF-1α accumulation/HIF-1 activation in human THP-1 myeloid monocytes, LAD2 mast cells and primary basophils in a variety of different settings. In THP-1 cells, mTOR was a major contributor to HIF-1 activation induced by ligands of both membrane-associated and endosomal toll-like receptors that recognise molecular patterns shared by pathogens. In LAD2 mast cells and primary human basophils mTOR was vital for HIF-1α accumulation/HIF-1 activity during immunoglobulin E (IgE)-dependent pro-allergic responses as well as in maintaining constitutive HIF-1α levels in mast cells. Furthermore, we observed that nitric oxide synthase activity was dependent on mTOR in these myeloid cells and involved in HIF-1α accumulation mediated by endosomal toll-like receptor 7/8 triggering in THP-1 cells as well as IgE-dependent basophil, but not mast cell, responses. In both cases the process is likely to be associated with an impact of reactive nitrogen species on HIF-1α-degrading prolyl hydroxylation. These studies underline the importance of mTOR as a fundamental regulator of HIF-1 signalling in myeloid cells.

CCL2 and Interleukin-6 Promote Survival of Human CD11b+ Peripheral Blood Mononuclear Cells and Induce M2-type Macrophage Polarization

CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.

Culture and identification of mouse myeloid semimature dendritic cells

To investigate the methods of culturing and identifying mouse myeloid semimature dendritic cell (smDC) in vitro. Myeloid monocytes derived from 6-week-old C57 BL/6 mice were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 2 ng/ml recombinant murine granulocyte macrophage-colony stimulating factor (GM-CSF), and 20 ng/ml recombinant murine interleukin (IL)-4 for 9 days. Then cells were incubated with 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) for 24 hours to obtain smDC. Meanwhile, smDC was differentiated into mature dendritic cell (mDC) or immature dendritic cell (iDC) by treatment with 1 micro/m1 lipopolysaccharide (LPS) or without LPS. The morphological features of smDC were assayed by inverted microscopy and scanning electron microscopy. Surface markers such as CD11c, CD4O, CD8O, CD86, and MHC-II were tested by flow cytometry. IL-1beta, IL-6, IL-12, and IL-10 in the supernatant were tested by ELISA. The activation of allogene lymphocyte (BALB/c mice) stimulated by C57BL/6 myeloid smDC in mixed lymphocyte reaction was examined by Cell Counting Kit-8 in vitro. The shape of smDC was round or oval-shaped, and the diameter of smDC was about 15 microm. The length of smDC dendrite was between 5 to 10 microm. smDC, iDC, and mDC all expressed high level of CD11 c. The expressions of MHC-II, CD40, CD80, and CD86 on smDC were higher than those of iDC and lower than those of mDC. IL-1beta, IL-6, and IL-12 secretion of smDC was significantly lower than that of mDC (P < 0.01), and IL-12 was significantly lower than that of iDC (P < 0.05), while no significant difference of IL-1beta and IL-6 secretion was found between smDC and iDC (P > 0.05). Furthermore, IL-10 secretion was not significantly different among these three kinds of DCs (P > 0.05). The effect of allogene lymphocytes activation on smDC was significantly lower than that of mDC and positive control (P < 0.01), but had no significant difference when compared with that of iDC and negative control (P > 0.05). smDC may be a relatively independent dendritic cell sub-population in terms of function and morphology. It is a feasible way to induce myeloid monocytes to differentiate into smDC using GM-CSF, IL-4, and TNF-alpha in vitro.

Impaired Inflammatory Response to Glial Cell Death in Genetically Metallothionein-I- and -II-Deficient Mice

Metallothionein I+II (MT-I+II) are acute-phase proteins which are upregulated during pathological conditions in the brain. To elucidate the neuropathological importance of MT-I+II, we have examined MT-I+II-deficient mice following ip injection with 6-aminonicotinamide (6-AN). 6-AN is antimetabolic and toxic for bone marrow cells and grey matter astrocytes. In MT+/+ mice, injection with 6-AN resulted in breakdown of the blood–brain barrier (BBB) and absence of GFAP-positive astrocytes in specific grey matter areas of the brain stem. Reactive astrocytosis encircled the damaged grey matter areas, which were heavily infiltrated by microglia/macrophages. The recruitment of hematogenous macrophages was accompanied by leakage of the BBB. The immunoreactivity (ir) of granulocyte–macrophage-colony-stimulating factor (GM-CSF) and the receptor for GM-CSF (GM-CSFrec) was significantly upregulated in astrocytes and microglia/macrophages, respectively. MT-I+IIir was also clearly increased in astrocytes surrounding the damaged areas, while that of the CNS-specific MT isoform, MT-III, was mildly increased in both astrocytes and microglia/macrophages. In MT−/− mice injected with 6-AN, the BBB remained almost intact. The damage to specific grey matter areas was similar to that observed in MT+/+ mice, but reactive astrocytosis, microglia/macrophages infiltration, and GM-CSFir and GM-CSFrecir were clearly reduced in MT−/− mice. In contrast, MT-IIIir was dramatically increased in MT−/− mice. Total zinc decreased and histochemically detectable zinc increased in the brain stem after 6-AN similarly in MT+/+ and MT−/− mice. Bone marrow myeloid monocytes and macrophages were increased as a reaction to 6-AN only in MT+/+ mice. The results demonstrate that the capability of MT−/− mice to mount a normal inflammatory response in the brain is severely attenuated, at least in part because of 6-AN-induced bone marrow affectation, involving MT-I+II for the first time as major factors during CNS tissue damage.

Multiple effects of human recombinant interleukin 4 on human myeloid monocyte cell lines

The effects of recombinant human interleukin 4 (rIL-4) on proliferation and differentiation on human myeloid/monocytic leukemia cell lines were examined. At high concentrations, rIL-4 had a slight enhancing effect on [3H]thymidine incorporation by U937 cells. rIL-4 markedly induced expression of the Fc epsilon receptor (CD23) and the Leu-M3 antigen (CD14) on U937 cells. HL60 and THP-1 cells treated with rIL-4 also showed increased CD23 expression, but little change of CD14 antigen expression. CD23 induction required lower amounts of IL-4 than needed for T cell growth, indicating that CD23 induction on U937 will serve as a sensitive assay for human IL-4. rIL-4 reduced the steady state level of IL-1 beta mRNA in U937.

Human breast cancer cells share antigens with the myeloid monocyte lineage.

We have examined the expression of several myeloid cell associated antigens, some of which are involved in myelomonocyte adhesion, in seven well characterized human breast cancer cell lines, since common properties of adhesiveness and migration are found in haemopoietic cells and epithelial cancer cells. Five of these cell lines were of metastatic origin and two were derived from primary breast carcinoma. Antigenic expression was evaluated by immunofluorescence (IF), flow cytometry (FCM), radioimmunoassay on live cells (RIA) and immunoperoxidase staining. None of these cell lines expressed T or B lymphoid specific antigens. Myeloid antigens My4, MO1, and MOF11 (derived from the hybridization of mouse X63 - Ag8 cells with spleen cells from Balb/c mice immunized with purified human monocytes) were expressed in the 7 cell lines. Leu M1, Leu M3, My9, and MO2 antigens were expressed in some of the cell lines. Leu M2 and My7 antigens were not expressed or at very low levels. The expression of these myeloid antigens was also tested by immunoperoxidase staining, and found on frozen sections of normal mammary gland, fibroadenoma of the breast, primary breast cancer, and lymph node and skin metastases of breast tumours. This common expression in epithelial breast cells and in myeloid cells might be related to common biological functions such as interaction with extracellular matrix which precedes cell migration, a normal function of macrophages and an abnormal function expressed or amplified in human cancer epithelial cells.

Rabbit antisera to cell lines RC2a and U937: antigens expressed on human leukaemic cells of myeloid, monocyte and T-lymphocyte lineage.

Rabbit antisera have been produced to an acute myelomonocytic leukaemia (AMML)-derived cell line (RC2a) and a histiocytic lymphoma derived cell line (U937) having macrophage characteristics. The antisera were screened by complement-mediated cytotoxicity and immunofluorescence (cytofluorograph analysis) against separated leukaemic (122 patients plus 13 cell lines) and normal haematologic cell populations (60 preparations from 20 donors plus 10 B-lymphoblastoid cell lines). The sera were absorbed with pooled B-lymphoblastoid cell lines including the autologous B-lymphoblastoid cell line to RC2a (CESS-B) or alternatively with B-CLL and T-CLL cells. All leukaemic cell populations were confirmed using the markers SIg, E-rosette receptor, cALL antigen, alpha-naphthyl butyrate esterase and myeloperoxidase. Rabbit anti-RC2a (Adherent cells) (RARC2a(Ad) ) and rabbit anti-U937 (RAU937) recognised antigens common to immature myeloid monocyte and T-lymphocyte lineage but did not react by cytotoxicity, absorption or cytofluorographic analysis with cells of B-lymphocyte lineage (B-lymphoblastoid or B-CLL) and reacted only occasionally with cALL patients' cells (includes pre B phenotype). These sera reacted with peripheral blood monocytes but not with other mature blood leucocytes. RAU937 reacted with a major mononuclear population from normal marrow and with more differentiated myeloid leukaemia cells. RARC2a(Ad) and RAU937 detected overlapping subgroups of myeloid leukaemia (AMoL, AMML, AML and CML) patients and Null-ALL and T-ALL patients. These subgroups are now being examined for prognostic significance.