MCP/CCR2 Signaling Is Essential for Recruitment of Mesenchymal Progenitor Cells during the Early Phase of Fracture Healing

Objective

The purpose of this study was to investigate chemokine profiles and their functional roles in the early phase of fracture healing in mouse models.

Methods

The expression profiles of chemokines were examined during fracture healing in wild-type (WT) mice using a polymerase chain reaction array and histological staining. The functional effect of monocyte chemotactic protein-1 (MCP-1) on primary mouse bone marrow stromal cells (mBMSCs) was evaluated using an in vitro migration assay. MCP-1^−/− and C-C chemokine receptor 2 (CCR2)^−/− mice were fractured and evaluated by histological staining and micro-computed tomography (micro-CT). RS102895, an antagonist of CCR2, was continuously administered in WT mice before or after rib fracture and evaluated by histological staining and micro-CT. Bone graft exchange models were created in WT and MCP-1^−/− mice and were evaluated by histological staining and micro-CT.

Results

MCP-1 and MCP-3 expression in the early phase of fracture healing were up-regulated, and high levels of MCP-1 and MCP-3 protein expression observed in the periosteum and endosteum in the same period. MCP-1, but not MCP-3, increased migration of mBMSCs in a dose-dependent manner. Fracture healing in MCP-1^−/− and CCR2^−/− mice was delayed compared with WT mice on day 21. Administration of RS102895 in the early, but not in the late phase, caused delayed fracture healing. Transplantation of WT-derived graft into host MCP-1^−/− mice significantly increased new bone formation in the bone graft exchange models. Furthermore, marked induction of MCP-1 expression in the periosteum and endosteum was observed around the WT-derived graft in the host MCP-1^−/− mouse. Conversely, transplantation of MCP-1^−/− mouse-derived grafts into host WT mice markedly decreased new bone formation.

Conclusions

MCP-1/CCR2 signaling in the periosteum and endosteum is essential for the recruitment of mesenchymal progenitor cells in the early phase of fracture healing.     

The CCL2/CCR2 Axis Enhances Vascular Cell Adhesion Molecule-1 Expression in Human Synovial Fibroblasts

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of osteoarthritis (OA). Here, we investigated the intracellular signaling pathways involved in CCL2-induced vascular cell adhesion molecule-1 (VCAM-1) expression in human OA synovial fibroblasts (OASFs).

Methodology/Principal Findings

Stimulation of OASFs with CCL2 induced VCAM-1 expression. CCL2-mediated VCAM-1 expression was attenuated by CCR2 inhibitor (RS102895), PKCδ inhibitor (rottlerin), p38MAPK inhibitor (SB203580), and AP-1 inhibitors (curcumin and tanshinone IIA). Stimulation of cells with CCL2 increased PKCδ and p38MAPK activation. Treatment of OASFs with CCL2 also increased the c-Jun phosphorylation and c-Jun binding to the AP-1 element on the VCAM-1 promoter. Moreover, CCL2-mediated CCR2, PKCδ, p38MAPK, and AP-1 pathway promoted the adhesion of monocytes to the OASFs monolayer.

Conclusions/Significance

Our results suggest that CCL2 increases VCAM-1 expression in human OASFs via the CCR2, PKCδ, p38MAPK, c-Jun, and AP-1 signaling pathway. The CCL2-induced VCAM-1 expression promoted monocytes adhesion to human OASFs.     

Tumor-associated macrophages recruit CCR6+ regulatory T cells and promote the development of colorectal cancer via enhancing CCL20 production in mice

Background

Tumor-associated macrophages (TAMs) remodel the colorectal cancer (CRC) microenvironment. Yet, findings on the role of TAMs in CRC seem to be contradictory compared with other cancers. FoxP3⁺ regulatory T (Treg)-cells dominantly infiltrate CRC. However, the underlying molecular mechanism in which TAMs may contribute to the trafficking of Treg-cells to the tumor mass remains unknown.

Methodology/Principal Findings

CRC was either induced by N-methyl-N-nitrosourea (MNU) and H. pylori or established by subcutaneous injection of mouse colorectal tumor cell line (CMT93) in mice. CMT93 cells were co-cultured with primary macrophages in a transwell apparatus. Recruitment of FoxP3 green fluorescence protein positive (FoxP3^GFP+) Treg-cells was assessed using the IVIS Imaging System or immunofluorescence staining. A role for macrophages in trafficking of Treg-cells and in the development of CRC was investigated in CD11b diphtheria toxin receptor (CD11b-DTR) transgenic C57BL/6J mice in which macrophages can be selectively depleted. Treg-cells remarkably infiltrated solid tumor, and predominantly expressed the homing chemokine receptor (CCR) 6 in the induced CRC model. Both CMT93 cancer cells and macrophages produced a large amount of CCL20, the sole ligand of CCR6 in vitro and in vivo. Injection of recombinant mouse CCL20 into tumor sites promoted its development with a marked recruitment of Treg-cells in the graft CRC model. Conditional macrophage ablation decreased CCL20 levels, blocked Treg-cell recruitment and inhibited tumor growth in CD11b-DTR mice grafted with CMT93.

Conclusions/Significance

TAMs recruit CCR6⁺ Treg-cells to tumor mass and promote its development via enhancing the production of CCL20 in a CRC mouse model.     

Activation of Olfactory Receptors on Mouse Pulmonary Macrophages Promotes Monocyte Chemotactic Protein-1 Production

Background

Emerging evidence suggests that non-olfactory tissues and cells can express olfactory receptors (ORs), however, the exact function of ectopic OR expression remains unknown. We have previously shown in mouse models that a unique cooperation between interferon-γ (IFN-γ) and lipopolysaccharide (LPS) drives the activation of pulmonary macrophages and leads to the induction of pathogenic responses in the respiratory tract. Further, through gene array studies, we have shown that activation of macrophages by these molecules results in the selective expression of a number of ORs. In this study, we validated the expression of these ORs in mouse airway and pulmonary macrophages in response to IFN-γ and LPS (γ/LPS) stimulation, and further explored the effect of odorant stimulation on macrophage function.

Methodology/Principal Findings

OR expression in airway and pulmonary macrophages in response to IFN-γ, LPS or γ/LPS treatments was assessed by microarray and validated by q-PCR. OR expression (e.g. OR622) on macrophages was confirmed by visualization in immunofluoresence assays. Functional responses to odorants were assessed by quantifying inflammatory cytokine and chemokine expression using q-PCR and cell migration was assessed by a modified Boyden chamber migration assay. Our results demonstrate that eight ORs are expressed at basal levels in both airway and pulmonary macrophages, and that γ/LPS stimulation cooperatively increased this expression. Pulmonary macrophages exposed to the combined treatment of γ/LPS+octanal (an odorant) exhibited a 3-fold increase in MCP-1 protein production, compared to cells treated with γ/LPS alone. Supernatants from γ/LPS+octanal exposed macrophages also increased macrophage migration in vitro.

Conclusions/Significance

Eight different ORs are expressed at basal levels in pulmonary macrophages and expression is upregulated by the synergistic action of γ/LPS. Octanal stimulation further increased MCP-1 production and the motility of macrophages. Our results suggest that ORs may mediate macrophage function by regulating MCP-1 production and cell migration.     

Bone Marrow-Derived Microglia Infiltrate into the Paraventricular Nucleus of Chronic Psychological Stress-Loaded Mice

Background

Microglia of the central nervous system act as sentinels and rapidly react to infection or inflammation. The pathophysiological role of bone marrow-derived microglia is of particular interest because they affect neurodegenerative disorders and neuropathic pain. The hypothesis of the current study is that chronic psychological stress (chronic PS) induces the infiltration of bone marrow-derived microglia into hypothalamus by means of chemokine axes in brain and bone marrow.

Methods and Findings

Here we show that bone marrow-derived microglia specifically infiltrate the paraventricular nucleus (PVN) of mice that received chronic PS. Bone marrow derived-microglia are CX₃CR1^lowCCR2⁺CXCR4^high, as distinct from CX₃CR1^highCCR2^-CXCR4^low resident microglia, and express higher levels of interleukin-1β (IL-1β) but lower levels of tumor necrosis factor-α (TNF-α). Chronic PS stimulates the expression of monocyte chemotactic protein-1 (MCP-1) in PVN neurons, reduces stromal cell-derived factor-1 (SDF-1) in the bone marrow and increases the frequency of CXCR4⁺ monocytes in peripheral circulation. And then a chemokine (C-C motif) receptor 2 (CCR2) or a β₃-adrenoceptor blockade prevents infiltration of bone marrow-derived microglia in the PVN.

Conclusion

Chronic PS induces the infiltration of bone marrow-derived microglia into PVN, and it is conceivable that the MCP-1/CCR2 axis in PVN and the SDF-1/CXCR4 axis in bone marrow are involved in this mechanism.     

Hydrazinocurcumin Encapsuled Nanoparticles “Re-Educate” Tumor-Associated Macrophages and Exhibit Anti-Tumor Effects on Breast Cancer Following STAT3 Suppression

Tumor-associated macrophages (TAMs) are essential cellular components within tumor microenvironment (TME). TAMs are educated by TME to transform to M2 polarized population, showing a M2-like phenotype, IL-10^high, IL-12^low, TGF-β^high. STAT3 signaling triggers crosstalk between tumor cells and TAMs, and is crucial for the regulation of malignant progression. In our study, legumain-targeting liposomal nanoparticles (NPs) encapsulating HC were employed to suppress STAT3 activity and “re-educate” TAMs, and to investigate the effects of suppression of tumor progression in vivo. The results showed that TAMs treated by HC encapsuled NPs could switch to M1-like phenotype, IL-10^low, IL-12^high, TGF-β^low, and the “re-educated” macrophages (M1-like macrophages) considerably demonstrated opposite effect of M2-like macrophages, especially the induction of 4T1 cells migration and invasion in vitro, and suppression of tumor growth, angiogenesis and metastasis in vivo. These data indicated that inhibition of STAT3 activity of TAMs by HC-NPs was able to reverse their phenotype and could regulate their crosstalk between tumor cells and TAMs in order to suppress tumor progression.     

The Features of Inflammation Factors Concentrations in Aqueous Humor of Polypoidal Choroidal Vasculopathy

Purpose

To investigate the cytokine concentrations in the aqueous humor of patients with refractory polypoidal choroidal vasculopathy (PCV).

Methods

Three separate groups of patients were studied–refractory PCV (Group A, 41 eyes), stable PCV (Group B, 39 eyes) and senile cataract (Group C, 44 eyes). Aqueous humor samples were collected at two time points for Groups A and B–before the first intravitreal ranibizumab injection and before the last injection. Aqueous humor samples were collected prior to phacoemulsification in Group C. The cytokine concentrations of interleukin 2, 6, and 8 (IL-2, IL-6, and IL-8), tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and vascular endothelial growth factor (VEGF) were measured by cytometric bead array and flow cytometry.

Results

Before the first treatment, the MCP-1, VEGF, and TNF-α levels in Group A were significantly higher than those in Group C (P < 0.05), and the MCP-1 and VEGF levels in Group A were significantly higher than those in Group B (P < 0.05). Significantly higher MCP-1 and VEGF levels were seen in Group B compared to Group C (P < 0.05). Before the final treatment, the MCP-1, VEGF, and TNF-α concentrations in Group A were significantly higher than those in Group B (P < 0.05) and Group C (P < 0.05). IL-2 levels were significantly lower in Group A compared to Group B (P < 0.05) and Group C (P < 0.05).

Conclusion

Inflammatory cytokines such as MCP-1, VEGF, and TNF-α may be associated with the pathogenesis of both stable and refractory PCV.     

Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats

Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells but not Kat1⁻c-fms⁺ cells. Kat1⁻c-fms⁺ cells appeared in early stages of culture, but Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1⁺CD11b⁺ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1⁺c-fms⁺ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.     

BVT.2733, a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor, attenuates obesity and inflammation in diet-induced obese mice

Background

Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is being pursued as a new therapeutic approach for the treatment of obesity and metabolic syndrome. Therefore, there is an urgent need to determine the effect of 11β-HSD1 inhibitor, which suppresses glucocorticoid action, on adipose tissue inflammation. The purpose of the present study was to examine the effect of BVT.2733, a selective 11β-HSD1 inhibitor, on expression of pro-inflammatory mediators and macrophage infiltration in adipose tissue in C57BL/6J mice.

Methodology/Principal Findings

C57BL/6J mice were fed with a normal chow diet (NC) or high fat diet (HFD). HFD treated mice were then administrated with BVT.2733 (HFD+BVT) or vehicle (HFD) for four weeks. Mice receiving BVT.2733 treatment exhibited decreased body weight and enhanced glucose tolerance and insulin sensitivity compared to control mice. BVT.2733 also down-regulated the expression of inflammation-related genes including monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α) and the number of infiltrated macrophages within the adipose tissue in vivo. Pharmacological inhibition of 11β-HSD1 and RNA interference against 11β-HSD1 reduced the mRNA levels of MCP-1 and interleukin-6 (IL-6) in cultured J774A.1 macrophages and 3T3-L1 preadipocyte in vitro.

Conclusions/Significance

These results suggest that BVT.2733 treatment could not only decrease body weight and improve metabolic homeostasis, but also suppress the inflammation of adipose tissue in diet-induced obese mice. 11β-HSD1 may be a very promising therapeutic target for obesity and associated disease.     

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion,Transendothelial Migration and Phagocytosis

Background

Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β₂ integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis.

Methodology/Principal Findings

THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β₂ integrin cell surface expression and β₂ integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β₂ integrins, in particular CD11b/CD18. SIRPα overexpression reduced β₂ integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β₂ integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression.

Conclusions/Significance

SIRPα negatively regulates β₂ integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis.     

Moderate Increase of Indoxyl Sulfate Promotes Monocyte Transition into Profibrotic Macrophages

Objective

The uremic toxin Indoxyl-3-sulphate (IS), a ligand of Aryl hydrocarbon Receptor (AhR), raises in blood during early renal dysfunction as a consequence of tubular damage, which may be present even when eGFR is normal or only moderately reduced, and promotes cardiovascular damage and monocyte-macrophage activation. We previously found that patients with abdominal aortic aneurysms (AAAs) have higher CD14⁺CD16⁺ monocyte frequency and prevalence of moderate chronic kidney disease (CKD) than age-matched control subjects. Here we aimed to evaluate the IS levels in plasma from AAA patients and to investigate in vitro the effects of IS concentrations corresponding to mild-to-moderate CKD on monocyte polarization and macrophage differentiation.

Methods

Free IS plasma levels, monocyte subsets and laboratory parameters were evaluated on blood from AAA patients and eGFR-matched controls. THP-1 monocytes, treated with IS 1, 10, 20 μM were evaluated for CD163 expression, AhR signaling and then induced to differentiate into macrophages by PMA. Their phenotype was evaluated both at the stage of semi-differentiated and fully differentiated macrophages. AAA and control sera were similarly used to treat THP-1 monocytes and the resulting macrophage phenotype was analyzed.

Results

IS plasma concentration correlated positively with CD14⁺CD16⁺ monocytes and was increased in AAA patients. In THP-1 cells, IS promoted CD163 expression and transition to macrophages with hallmarks of classical (IL-6, CCL2, COX2) and alternative phenotype (IL-10, PPARγ, TGF-β, TIMP-1), via AhR/Nrf2 activation. Analogously, AAA sera induced differentiation of macrophages with enhanced IL-6, MCP1, TGF-β, PPARγ and TIMP-1 expression.

Conclusion

IS skews monocyte differentiation toward low-inflammatory, profibrotic macrophages and may contribute to sustain chronic inflammation and maladaptive vascular remodeling.     

A Pseudopterane Diterpene Isolated From the Octocoral Pseudopterogorgia acerosa Inhibits the Inflammatory Response Mediated by TLR-Ligands and TNF-Alpha in Macrophages

Several diterpenoids isolated from terrestrial and marine environments have been identified as important anti-inflammatory agents. Although considerable progress has been made in the area of anti-inflammatory treatment, the search for more effective and safer compounds is a very active field of research. In this study we investigated the anti-inflammatory effects of a known pseudopterane diterpene (referred here as compound 1) isolated from the octocoral Pseudopterogorgia acerosa on the tumor necrosis factor- alpha (TNF-α) and TLRs- induced response in macrophages. Compound 1 inhibited the expression and secretion of the inflammatory mediators TNF-α, interleukin (IL)-6, IL-1β, nitric oxide (NO), interferon gamma-induced protein 10 (IP-10), ciclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) induced by LPS in primary murine macrophages. This effect was associated with the inhibition of IκBα degradation and subsequent activation of NFκB. Compound 1 also inhibited the expression of the co-stimulatory molecules CD80 and CD86, which is a hallmark of macrophage activation and consequent initiation of an adaptive immune response. The anti-inflammatory effect was not exclusive to LPS because compound 1 also inhibited the response of macrophages to TNF-α and TLR2 and TLR3 ligands. Taken together, these results indicate that compound 1 is an anti-inflammatory molecule, which modulates a variety of processes occurring in macrophage activation.     

Novel Biochemical Markers of Psychosocial Stress in Women

Background

Prolonged psychosocial stress is a condition assessed through self-reports. Here we aimed to identify biochemical markers for screening and early intervention in women.

Methods

Plasma concentrations of interleukin (IL) 1-α, IL1-β, IL-2, IL-4, IL-6, IL-8, IL-10, interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), thyroid stimulating hormone (TSH), total tri-iodothyronine (TT3), total thyroxine (TT4), prolactin, and testosterone were measured in: 195 women on long-term sick-leave for a stress-related affective disorder, 45 women at risk for professional burnout, and 84 healthy women.

Results

We found significantly increased levels of MCP-1, VEGF and EGF in women exposed to prolonged psychosocial stress. Statistical analysis indicates that they independently associate with a significant risk for being classified as ill.

Conclusions

MCP-1, EGF, and VEGF are potential markers for screening and early intervention in women under prolonged psychosocial stress.     

Alcohol ingestion disrupts alveolar epithelial barrier function by activation of macrophage-derived transforming growth factor beta1

Background

Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Experimentally, alcohol-induced oxidative stress increases the expression of transforming growth factor β1 (TGFβ1) in the lung; however, we do not know the precise contribution of various alveolar cells in this process. In the present study, we focused on cell-cell interactions between alveolar macrophages and epithelial cells and the potential mechanisms by which TGFβ1 may become activated in the alveolar space of the alcoholic lung.

Methods

Primary alveolar macrophages and epithelial cells were isolated from control- and alcohol-fed Sprague–Dawley rats. Expression of TGFβ1 and the epithelial integrin αvβ6 were examined by real time PCR and either immunocytochemistry or flow cytometry. Alveolar epithelial cells were cultured on transwell supports in the presence of macrophage cell lysate from control- or alcohol-fed rats or in the presence of viable macrophages ± alcohol. Epithelial barrier function was assessed by transepithelial resistance (TER) and paracellular flux of Texas Red dextran.

Results

TGFβ1 expression was increased in alveolar macrophages from alcohol-fed rats, and TGFβ1 protein was predominantly membrane-bound. Importantly, alveolar macrophage cellular lysate from alcohol-fed rats decreased TER and increased paracellular dextran flux in primary alveolar epithelial cell monolayers as compared to the lysates from control-fed rats. Alcohol-induced epithelial barrier dysfunction was prevented by anti-TGFβ1 antibody treatment, indicating the presence of bioactive TGFβ1 in the macrophage lysate. In addition, co-culturing macrophages and epithelial cells in the presence of alcohol decreased epithelial barrier function, which also was prevented by anti-TGFβ1 and anti-αvβ6 treatment. In parallel, chronic alcohol ingestion in vivo, or direct treatment with active TGFβ1 in vitro, increased the expression of αvβ6 integrin, which is known to activate TGFβ1, in alveolar epithelial cells.

Conclusions

Taken together, these data suggest that interactions between alveolar epithelial cells and macrophages contribute to the alcohol-mediated disruption of epithelial barrier function via the expression and activation of TGFβ1 at points of cell-cell contact.     

Nicotine Attenuates Activation of Tissue Resident Macrophages in the Mouse Stomach through the β2 Nicotinic Acetylcholine Receptor

Background

The cholinergic anti-inflammatory pathway is an endogenous mechanism by which the autonomic nervous system attenuates macrophage activation via nicotinic acetylcholine receptors (nAChR). This concept has however not been demonstrated at a cellular level in intact tissue. To this end, we have studied the effect of nicotine on the activation of resident macrophages in a mouse stomach preparation by means of calcium imaging.

Methods

Calcium transients ([Ca²⁺]_i) in resident macrophages were recorded in a mouse stomach preparation containing myenteric plexus and muscle layers by Fluo-4. Activation of macrophages was achieved by focal puff administration of ATP. The effects of nicotine on activation of macrophages were evaluated and the nAChR involved was pharmacologically characterized. The proximity of cholinergic nerves to macrophages was quantified by confocal microscopy. Expression of β2 and α7 nAChR was evaluated by β2 immunohistochemistry and fluorophore-tagged α-bungarotoxin.

Results

In 83% of macrophages cholinergic varicose nerve fibers were detected at distances <900nm. The ATP induced [Ca²⁺]_i increase was significantly inhibited in 65% or 55% of macrophages by 100µM or 10µM nicotine, respectively. This inhibitory effect was reversed by the β2 nAChR preferring antagonist dihydro-β-eryhtroidine but not by hexamethonium (non-selective nAChR-antagonist), mecamylamine (α3β4 nAChR-preferring antagonist), α-bungarotoxin or methyllycaconitine (both α7 nAChR-preferring antagonist). Macrophages in the stomach express β2 but not α7 nAChR at protein level, while those in the intestine express both receptor subunits.

Conclusion

This study is the first in situ demonstration of an inhibition of macrophage activation by nicotine suggesting functional signaling between cholinergic neurons and macrophages in the stomach. The data suggest that the β2 subunit of the nAChR is critically involved in the nicotine-induced inhibition of these resident macrophages.     

Effects of Benzalkonium Chloride on THP-1 Differentiated Macrophages In Vitro

Purpose

To characterize the effects of benzalkonium chloride (BAK) in THP-1 differentiated cells in vitro.

Methods

Macrophages were obtained after differentiation of THP-1 cells, a human monocytic leukemia cell line. Macrophages were exposed for 24 h to 33 nM (10⁻⁵%) benzalkonium chloride (BAK), 10 nM dinitrochlorobenzene (DNCB), 100 ng/mL lipopolysaccharide (LPS), 5 ng/mL tumor necrosis factor alpha (TNF-α) or phosphate buffered saline (PBS) as controls. The expression of CD11b, CD11c, CD33 and CD54 was evaluated using immunohistochemistry and flow cytometry (FCM). Phagocytosis function was analyzed using carboxylate-modified fluorescent microspheres and quantified by FCM. Migration was evaluated in cocultures with conjunctival epithelial cells. Cytokine production was detected and quantified in culture supernatants using a human cytokine array.

Results

Stimulation of THP-1-derived macrophages with a low concentration of BAK increased CD11b and CD11c expression and decreased CD33. Macrophages exposed to BAK, LPS and TNF-α had increased phagocytosis. In contrast to LPS, BAK and TNF-α increased macrophage migration. Cytokines in supernatants of macrophages exposed to BAK revealed an increased release of CCL1, CCL4/MIP-1β, TNF-α, soluble CD54/ICAM-1 and IL-1β.

Conclusion

In vitro, BAK has a direct stimulating effect on macrophages, increasing phagocytosis, cytokine release, migration and expression of CD11b and CD11c. Long-term exposure to low concentrations of BAK should be considered as a stimulating factor responsible for inflammation through macrophage activation.     

The Role of M2 Macrophages in the Progression of Chronic Kidney Disease following Acute Kidney Injury

Introduction

Acute kidney injury (AKI) is a major risk factor in the development of chronic kidney disease (CKD). However, the mechanisms linking AKI to CKD remain unclear. We examined the alteration of macrophage phenotypes during an extended recovery period following ischemia/reperfusion injury (IRI) and determine their roles in the development of fibrosis.

Methods

The left renal pedicle of mice was clamped for 40 min. To deplete monocyte/macrophage, liposome clodronate was injected or CD11b-DTR and CD11c-DTR transgenic mice were used.

Results

Throughout the phase of IRI recovery, M2-phenotype macrophages made up the predominant macrophage subset. On day 28, renal fibrosis was clearly shown with increased type IV collagen and TGF-β. The depletion of macrophages induced by the liposome clodronate injection improved renal fibrosis with a reduction of kidney IL-6, type IV collagen, and TGF-β levels. Additionally, the adoptive transfer of the M2c macrophages partially reversed the beneficial effect of macrophage depletion, whereas the adoptive transfer of the M1 macrophages did not. M2 macrophages isolated from the kidneys during the recovery phase expressed 2.5 fold higher levels of TGF-β than the M1 macrophages. The injection of the diphtheria toxin into CD11b or CD11c-DTR transgenic mice resulted in lesser depletion or no change in M2 macrophages and had little impact on renal fibrosis.

Conclusion

Although M2 macrophages are known to be indispensible for short-term recovery, they are thought to be main culprit in the development of renal fibrosis following IRI.     

Suppression of Experimental Choroidal Neovascularization by Curcumin in Mice

Purpose

To investigate the effects of curcumin on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms.

Methods

C57BL/6N mice were pretreated with intraperitoneal injections of curcumin daily for 3 days prior to laser-induced CNV, and the drug treatments were continued until the end of the study. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day 7 and 14, and CNV leakage was evaluated by fluorescein angiography (FA) on day 14 after laser photocoagulation. The infiltration of F4/80 positive macrophages and GR-1 positive granulocytes were evaluated by immunohistochemistry on RPE-choroid flat mounts on day 3. Their expression in RPE-choroid complex was quantified by real-time PCR (F4/80) and Western blotting (GR-1) on day 3. RPE-choroid levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 were examined by ELISA on day 3. Double immunostaining of F4/80 and VEGF was performed on cryo-sections of CNV lesions on day 3. The expression of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)−1α in the RPE-choroid was determined by Western blotting.

Results

Curcumin-treated mice had significantly less CNV area (P<0.05) and CNV leakage (P<0.001) than vehicle-treated mice. Curcumin treatment led to significant inhibition of F4/80 positive macrophages (P<0.05) and GR-1 positive granulocytes infiltration (P<0.05). VEGF mainly expressed in F4/80 positive macrophages in laser injury sites, which was suppressed by curcumin treatment (P<0.01). Curcumin inhibited the RPE-choroid levels of TNF-α (P<0.05), MCP-1 (P<0.05) and ICAM-1 (P<0.05), and suppressed the activation of NF-κB in nuclear extracts (P<0.05) and the activation of HIF−1α (P<0.05).

Conclusion

Curcumin treatment led to the suppression of CNV development together with inflammatory and angiogenic processes including NF-κB and HIF−1α activation, the up-regulation of inflammatory and angiogenic cytokines, and infiltrating macrophages and granulocytes. This provides molecular and cellular evidence of the validity of curcumin supplementation as a therapeutic strategy for the suppression of age-related macular degeneration (AMD)-associated CNV.