Macrophage Migration Inhibitory Factor Is Involved in Ectopic Endometrial Tissue Growth and Peritoneal-Endometrial Tissue Interaction In Vivo: A Plausible Link to Endometriosis Development

Pelvic inflammation is a hallmark of endometriosis pathogenesis and a major cause of the disease''s symptoms. Abnormal immune and inflammatory changes may not only contribute to endometriosis-major symptoms, but also contribute to ectopic endometrial tissue growth and endometriosis development. A major pro-inflammatory factors found elevated in peritoneal fluid of women with endometriosis and to be overexpressed in peritoneal fluid macrophages and active, highly vascularized and early stage endometriotic lesions, macrophage migration inhibitory factor (MIF) appeared to induce angiogenic and inflammatory and estrogen producing phenotypes in endometriotic cells in vitro and to be a possible therapeutic target in vivo. Using a mouse model where MIF-knock out (KO) mice received intra-peritoneal injection of endometrial tissue from MIF-KO or syngeneic wild type (WT) mice and vice versa, our current study revealed that MIF genetic depletion resulted in a marked reduction ectopic endometrial tissue growth, a disrupted tissue structure and a significant down regulation of the expression of major inflammatory (cyclooxygenease-2), cell adhesion (αv and β3 integrins), survival (B-cell lymphoma-2) and angiogenic (vascular endothelial cell growth) factorsrelevant to endometriosis pathogenesis, whereas MIF add-back to MIF-KO mice significantly restored endometriosis-like lesions number and size. Interestingly, cross-experiments revealed that MIF presence in both endometrial and peritoneal host tissues is required for ectopic endometrial tissue growth and pointed to its involvement in endometrial-peritoneal interactions. This study provides compelling evidence for the role of MIF in endometriosis development and its possible interest for a targeted treatment of endometriosis.     

Peritoneal Fluid Reduces Angiogenesis-Related MicroRNA Expression in Cell Cultures of Endometrial and Endometriotic Tissues from Women with Endometriosis

Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis.

Methods

Endometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222) by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively.

Results

Control and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion, this “in vitro” study indicates that peritoneal fluid from women with endometriosis modulates the expression of miRNAs that could contribute to the angiogenic and proteolytic disequilibrium observed in this disease.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

Pentraxin-3 regulates the inflammatory activity of macrophages

Background and aimsPentraxin-3 (PTX3) reportedly has protective roles in atherosclerosis and myocardial infarction, and is a useful biomarker of vascular inflammation. However, the detailed functions of PTX3 in inflammation are yet to be elucidated. This study aimed to investigate the function of PTX3 in macrophages.MethodsPMA-treated THP-1 cell line (THP-1 macrophage) and monocyte-derived human primary macrophages were treated with recombinant PTX3. Cytokine and chemokine levels in the THP-1 culture medium were measured as well as monocyte chemoattractant protein (MCP-1) concentrations in the Raw 264.7 cell culture medium. PTX3-silenced apoptotic macrophages (THP-1 cell line) were generated to investigate the roles of PTX3 in phagocytosis.ResultsIn the presence of PTX3, macrophage interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and MCP-1 levels were reduced significantly (?39%, P=0.007; ?21%, P=0.008; and ?67%, P=0.0003, respectively), whilst activated transforming growth factor-β (TGF?β) was detected in the THP-1 macrophages (P=0.0004). Additionally, PTX3 induced Akt phosphorylation and reduced nuclear factor-kappa B (NF-κB) activation by 35% (P=0.002), which was induced by TNF-α in THP-1 macrophages. Furthermore, silencing of PTX3 in apoptotic cells resulted in increased macrophage binding, elevated expression rate of HLA-DR (+30%, P=0.015) and CD86 (+204%, P=0.004) positive cells, and induction of IL-1β (+36%, P=0.024) production. Conversely, adding recombinant PTX3 to macrophages reduced CD86 and HLA-DR expression in a dose-dependent manner.ConclusionsWe identified PTX3 as a novel regulator of macrophage activity, and this function suggests that PTX3 acts to resolve inflammation.     

Does iron overload in metabolic syndrome affect macrophage profile? A case control study

AimsDysmetabolic iron overload syndrome (DIOS) is common but the clinical relevance of iron overload is not understood. Macrophages are central cells in iron homeostasis and inflammation. We hypothesized that iron overload in DIOS could affect the phenotype of monocytes and impair macrophage gene expression.MethodsThis study compared 20 subjects with DIOS to 20 subjects with metabolic syndrome (MetS) without iron overload, and 20 healthy controls. Monocytes were phenotyped by Fluorescence-Activated Cell Sorting (FACS) and differentiated into anti-inflammatory M2 macrophages in the presence of IL-4. The expression of 38 genes related to inflammation, iron metabolism and M2 phenotype was assessed by real-time PCR.ResultsFACS showed no difference between monocytes across the three groups. The macrophagic response to IL-4-driven differentiation was altered in four of the five genes of M2 phenotype (MRC1, F13A1, ABCA1, TGM2 but not FABP4), in DIOS vs Mets and controls demonstrating an impaired M2 polarization. The expression profile of inflammatory genes was not different in DIOS vs MetS. Several genes of iron metabolism presented a higher expression in DIOS vs MetS: SCL11A2 (a free iron transporter, +76 %, p = 0.04), SOD1 (an antioxidant enzyme, +27 %, p = 0.02), and TFRC (the receptor 1 of transferrin, +59 %, p = 0.003).ConclusionsIn DIOS, macrophage polarization toward the M2 alternative phenotype is impaired but not associated with a pro-inflammatory profile. The up regulation of transferrin receptor 1 (TFRC) in DIOS macrophages suggests an adaptive role that may limit iron toxicity in DIOS.     

Pigment Epithelium Derived Factor Inhibits the Growth of Human Endometrial Implants in Nude Mice and of Ovarian Endometriotic Stromal Cells In Vitro

Angiogenesis is a prerequisite for the formation and development of endometriosis. Pigment epithelium derived factor (PEDF) is a natural inhibitor of angiogenesis. We previously demonstrated a reduction of PEDF in the peritoneal fluid, serum and endometriotic lesions from women with endometriosis compared with women without endometriosis. Here, we aim to investigate the inhibitory effect of PEDF on human endometriotic cells in vivo and in vitro. We found that PEDF markedly inhibited the growth of human endometrial implants in nude mice and of ovarian endometriotic stromal cells in vitro by up-regulating PEDF expression and down-regulating vascular endothelial growth factor (VEGF) expression. Moreover, apoptotic index was significantly increased in endometriotic lesions in vivo and endometriotic stromal cells in vitro when treated with PEDF. In mice treated with PEDF, decreased microvessel density labeled by Von Willebrand factor but not by α-Smooth Muscle Actin was observed in endometriotic lesions. And it showed no increase in PEDF expression of the ovary and uterus tissues. These findings suggest that PEDF gene therapy may be a new treatment for endometriosis.     

Oxygenated phosphatidylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2

During cancer therapy, phagocytic clearance of dead cells plays a vital role in immune homeostasis. The nonapoptotic form of cell death, ferroptosis, exhibits extraordinary potential in tumor treatment. However, the phagocytosis mechanism that regulates the engulfment of ferroptotic cells remains unclear. Here, we establish a novel pathway for phagocytic clearance of ferroptotic cells that is different from canonical mechanisms by using diverse ferroptosis models evoked by GPX4 dysfunction/deficiency. We identified the oxidized phospholipid, 1-steaoryl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH), as a key eat-me signal on the ferroptotic cell surface. Enriching the plasma membrane with SAPE-OOH increased the efficiency of phagocytosis of ferroptotic cells by macrophage, a process that was suppressed by lipoprotein-associated phospholipase A₂. Ligand fishing, lipid blotting, and cellular thermal shift assay screened and identified TLR2 as a membrane receptor that directly recognized SAPE-OOH, which was further confirmed by TLR2 inhibitors and gene silencing studies. A mouse mammary tumor model of ferroptosis verified SAPE-OOH and TLR2 as critical players in the clearance of ferroptotic cells in vivo. Taken together, this work demonstrates that SAPE-OOH on ferroptotic cell surface acts as an eat-me signal and navigates phagocytosis by targeting TLR2 on macrophages.Subject terms: Cancer, Cancer microenvironment     

Effect of Fonsecaea monophora on the Polarization of THP-1 Cells to Macrophages

Background

Chromoblastomycosis is a chronic, progressive fungal disease of the skin and subcutaneous tissue caused by a unique group of dematiaceous fungi. Fonsecaea monophora, a new species distinct from Fonsecaea pedrosoi strains, is the main pathogen responsible for chromoblastomycosis in south China. Macrophages can be polarized into two categories: classically activated and alternatively activated.

Objectives

Little is known about the relationship between F. monophora and macrophage polarization. This study aimed to study the effect of F. monophora on the polarization of THP-1 cells to macrophages.

Methods

We established coculture systems of F. monophora and THP-1-derived macrophages in different activation states.

Results

F. monophora enhanced the phagocytosis by macrophages in the initially activated state and weakened the phagocytosis by classically activated macrophages without affecting that by alternatively activated macrophages. Classically activated macrophages had the strongest killing effect on F. monophora, while the initially activated macrophages had the weakest. The pathogen could not be rapidly cleared by any type of macrophage. F. monophora promoted the expression of proinflammatory cytokines and inhibited that of anti-inflammatory cytokines.

Conclusions

F. monophora promoted the polarization of THP-1 cells to classically activated macrophages and inhibited that of THP-1 cells to alternatively activated macrophages.

     

Potential Link between the Sphingosine-1-Phosphate (S1P) System and Defective Alveolar Macrophage Phagocytic Function in Chronic Obstructive Pulmonary Disease (COPD)

Introduction

We previously reported that alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) are defective in their ability to phagocytose apoptotic cells, with a similar defect in response to cigarette smoke. The exact mechanisms for this defect are unknown. Sphingolipids including ceramide, sphingosine and sphingosine-1-phosphate (S1P) are involved in diverse cellular processes and we hypothesised that a comprehensive analysis of this system in alveolar macrophages in COPD may help to delineate the reasons for defective phagocytic function.

Methods

We compared mRNA expression of sphingosine kinases (SPHK1/2), S1P receptors (S1PR1-5) and S1P-degrading enzymes (SGPP1, SGPP2, SGPL1) in bronchoalveolar lavage-derived alveolar macrophages from 10 healthy controls, 7 healthy smokers and 20 COPD patients (10 current- and 10 ex-smokers) using Real-Time PCR. Phagocytosis of apoptotic cells was investigated using flow cytometry. Functional associations were assessed between sphingosine signalling system components and alveolar macrophage phagocytic ability in COPD. To elucidate functional effects of increased S1PR5 on macrophage phagocytic ability, we performed the phagocytosis assay in the presence of varying concentrations of suramin, an antagonist of S1PR3 and S1PR5. The effects of cigarette smoking on the S1P system were investigated using a THP-1 macrophage cell line model.

Results

We found significant increases in SPHK1/2 (3.4- and 2.1-fold increases respectively), S1PR2 and 5 (4.3- and 14.6-fold increases respectively), and SGPL1 (4.5-fold increase) in COPD vs. controls. S1PR5 and SGPL1 expression was unaffected by smoking status, suggesting a COPD “disease effect” rather than smoke effect per se. Significant associations were noted between S1PR5 and both lung function and phagocytosis. Cigarette smoke extract significantly increased mRNA expression of SPHK1, SPHK2, S1PR2 and S1PR5 by THP-1 macrophages, confirming the results in patient-derived macrophages. Antagonising SIPR5 significantly improved phagocytosis.

Conclusion

Our results suggest a potential link between the S1P signalling system and defective macrophage phagocytic function in COPD and advise therapeutic targets.     

氧化低密度脂蛋白对THP-1巨噬细胞吞噬和分泌功能的影响

目的:探讨氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)对巨噬细胞源性泡沫细胞吞噬功能和炎症相关因子分泌功能的影响。方法:利用佛波酯(phorbol ester,PMA)诱导THP-1细胞分化形成巨噬细胞,之后采用ox-LDL处理48小时后,诱导其形成泡沫细胞。利用中性红吞噬实验,分析泡沫细胞形成前后吞噬功能的变化;通过ELISA法,检测细胞培养上清中肿瘤坏死因子α(tumor necrosis factorα,TNF-α)含量,观察ox-LDL对THP-1巨噬细胞功能的影响。结果:细胞形态学结果表明,我们成功利用ox-LDL诱导THP-1巨噬细胞形成泡沫细胞;进一步发现ox-LDL诱导THP-1巨噬细胞表面的清道夫受体CD36表达升高,并促进细胞吞噬功能增加,进一步促进细胞内胆固醇含量显著升高(P0.05);同时,ox-LDL能够刺激巨噬细胞大量分泌TNF-α(P0.05)。结论:ox-LDL通过增强清道夫受体CD36表达,提高巨噬细胞的吞噬功能,引起大量胆固醇聚集,产生细胞毒性损伤,并促进TNF-α炎性因子的大量分泌。     

Interaction between macrophages and ferroptosis

AbstractFerroptosis, a newly discovered iron-dependent cell death pathway, is characterized by lipid peroxidation and GSH depletion mediated by iron metabolism and is morphologically, biologically and genetically different from other programmed cell deaths. Besides, ferroptosis is usually found accompanied by inflammatory reactions. So far, it has been found participating in the development of many kinds of diseases. Macrophages are a group of immune cells that widely exist in our body for host defense and play an important role in tissue homeostasis by mediating inflammation and regulating iron, lipid and amino acid metabolisms through their unique functions like phagocytosis and efferocytosis, cytokines secretion and ROS production under different polarization. According to these common points in ferroptosis characteristics and macrophages functions, it’s obvious that there must be relationship between macrophages and ferroptosis. Therefore, our review aims at revealing the interaction between macrophages and ferroptosis concerning three metabolisms and integrating the application of certain relationship in curing diseases, mostly cancer. Finally, we also provide inspirations for further studies in therapy for some diseases by targeting certain resident macrophages in distinct tissues to regulate ferroptosis.Facts

• Ferroptosis is considered as a newly discovered form characterized by its nonapoptotic and iron-dependent lipid hydroperoxide, concerning iron, lipid and amino acid metabolisms.
• Ferroptosis has been widely found playing a crucial part in various diseases, including hepatic diseases, neurological diseases, cancer, etc.
• Macrophages are phagocytic immune cells, widely existing and owning various functions such as phagocytosis and efferocytosis, cytokines secretion and ROS production.
• Macrophages are proved to participate in mediating metabolisms and initiating immune reactions to maintain balance in our body.
• Recent studies try to treat cancer by altering macrophages’ polarization which damages tumor microenvironment and induces ferroptosis of cancer cells.

Open questions

• How do macrophages regulate ferroptosis of other tissue cells specifically?
• Can we use the interaction between macrophages and ferroptosis in treating diseases other than cancer?
• What can we do to treat diseases related to ferroptosis by targeting macrophages?
• Is the use of the relationship between macrophages and ferroptosis more effective than other therapies when treating diseases?

Subject terms: Cell death and immune response, Cytokines, Cancer immunotherapy     

Inulin stimulates phagocytosis of PMA-treated THP-1 macrophages by involvement of PI3-kinases and MAP kinases

Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation.     

Activation of p38 MAPKalpha by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure

We have previously demonstrated that constant 20 mmHg extracellular pressure increases serum-opsonized latex bead phagocytosis by phorbol 12-myristate 13-acetate (PMA)- differentiated THP-1 macrophages in part by inhibiting focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Because p38 MAPK is activated by physical forces in other cells, we hypothesized that modulation of p38 MAPK might also contribute to the stimulation of macrophage phagocytosis by pressure. We studied phagocytosis in PMA-differentiated THP-1 macrophages, primary human monocytes, and human monocyte-derived macrophages (MDM). p38 MAPK activation was inhibited using SB-203580 or by p38 MAPK small interfering RNA (siRNA). Pressure increased phagocytosis in primary monocytes and MDM as in THP-1 cells. Increased extracellular pressure for 30 min increased phosphorylated p38 MAPK by 46.4 ± 20.5% in DMSO-treated THP-1 macrophages and by 20.9 ± 9% in primary monocytes (P < 0.05 each). SB-203580 (20 µM) reduced basal p38 MAPK phosphorylation by 34.7 ± 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPK siRNA reduced total p38 MAPK protein by 50–60%. Neither SB-203580 in THP-1 cells and peripheral monocytes nor p38 MAPK siRNA in THP-1 cells affected basal phagocytosis, but each abolished pressure-stimulated phagocytosis. SB-203580 did not affect basal or pressure-reduced FAK activation in THP-1 macrophages, but significantly attenuated the reduction in ERK phosphorylation associated with pressure. p38 MAPK siRNA reduced total FAK protein by 40–50%, and total ERK by 10–15%, but increased phosphorylated ERK 1.4 ± 0.1-fold. p38 MAPK siRNA transfection did not affect the inhibition of FAK-Y397 phosphorylation by pressure but prevented inhibition of ERK phosphorylation. Changes in extracellular pressure during infection or inflammation regulate macrophage phagocytosis by a FAK-dependent inverse effect on p38 MAPK that might subsequently downregulate ERK. force; inflammation; infection; leukocyte; mechanotransduction; signal transduction     

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.     

Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK

We hypothesized that changes in extracellular pressure during inflammation or infection regulate macrophage phagocytosis through modulating the focal adhesion kinase (FAK)-ERK pathway. Undifferentiated (monocyte-like) or PMA-differentiated (macrophage-like) THP-1 cells were incubated at 37°C with serum-opsonized latex beads under ambient or 20-mmHg increased pressure. Pressure did not affect monocyte phagocytosis but significantly increased macrophage phagocytosis (29.9 ± 1.8 vs. 42.0 ± 1.6%, n = 9, P < 0.001). THP-1 macrophages constitutively expressed activated FAK, ERK, and Src. Exposure of macrophages to pressure decreased ERK and FAK-Y397 phosphorylation (77.6 ± 7.9%, n = 7, P < 0.05) but did not alter FAK-Y576 or Src phosphorylation. FAK small interfering RNA (SiRNA) reduced FAK expression by >75% and the basal amount of phosphorylated FAK by 25% and significantly increased basal macrophage phagocytosis (P < 0.05). Pressure inhibited FAK-Y397 phosphorylation in mock-transfected or scrambled SiRNA-transfected macrophages, but phosphorylated FAK was not significantly reduced further by pressure in cells transfected with FAK SiRNA. Pressure increased phagocytosis in all three groups. However, FAK-SiRNA-transfected cells exhibited only 40% of the pressure effect on phagocytosis observed in scrambled SiRNA-transfected cells so that phagocytosis inversely paralleled FAK activation. PD-98059 (50 µM), an ERK activation inhibitor, increased basal phagocytosis (26.9 ± 1.8 vs. 31.7 ± 1.1%, n = 15, P < 0.05), but pressure did not further increase phagocytosis in PD-98059-treated cells. Pressure also inhibited ERK activation after mock transfection or transfection with scrambled SiRNA, but transfection of FAK SiRNA abolished ERK inhibition by pressure. Pressure did not increase phagocytosis in MonoMac-1 cells that do not express FAK. Increased extracellular pressure during infection or inflammation enhances macrophage phagocytosis by inhibiting FAK and, consequently, decreasing ERK activation. force; inflammation; infection; leukocyte; mechanotransduction; signal transduction     

Intervertebral disc cells as competent phagocytes in vitro: implications for cell death in disc degeneration

Introduction

Apoptosis has been reported to occur in the intervertebral disc. Elsewhere in the body, apoptotic cells are cleared from the system via phagocytosis by committed phagocytes such as macrophages, reducing the chance of subsequent inflammation. These cells, however, are not normally present in the disc. We investigated whether disc cells themselves can be induced to become phagocytic and so have the ability to ingest and remove apoptotic disc cells, minimising the damage to their environment.

Method

Bovine nucleus pulposus cells from caudal intervertebral discs were grown in culture and exposed to both latex particles (which are ingested by committed phagocytes) and apoptotic cells. Their response was monitored via microscopy, including both fluorescent and video microscopy, and compared with that seen by cell lines of monocytes/macrophages (THP-1 and J774 cells), considered to be committed phagocytes, in addition to a nonmacrophage cell line (L929 fibroblasts). Immunostaining for the monocyte/macrophage marker, CD68, was also carried out.

Results

Disc cells were able to ingest latex beads at least as efficiently, if not more so, than phagocytic THP-1 and J774 cells. Disc cells ingested a greater number of beads per cell than the committed phagocytes in a similar time scale. In addition, disc cells were able to ingest apoptotic cells when cocultured in monolayer with a UV-treated population of HeLa cells. Apoptotic disc cells, in turn, were able to stimulate phagocytosis by the committed macrophages. CD68 immunostaining was strong for THP-1 cells but negligible for disc cells, even those that had ingested beads.

Conclusion

In this study, we have shown that intervertebral disc cells are capable of behaving as competent phagocytes (that is, ingesting latex beads) and apoptotic cells. In terms of number of particles, they ingest more than the monocyte/macrophage cells, possibly due to their greater size. The fact that disc cells clearly can undergo phagocytosis has implications for the intervertebral disc in vivo. Here, where cell death is reported to be common yet there is normally no easy access to a macrophage population, the endogenous disc cells may be encouraged to undergo phagocytosis (for example, of neighbouring cells within cell clusters).     

Leishmania (Viannia) peruviana (MHOM/PE/LCA08): Comparison of THP-1 cell and murine macrophage susceptibility to axenic amastigotes for the screening of leishmanicidal compounds

This study, undertaken to compare the susceptibility of THP-1 cells and murine peritoneal macrophages to Leishmania peruviana amastigotes, obtained THP-1 infection with 10 parasites/cell compared to 2 parasites/murine macrophage. The parasite burden was maximal at 72 h post-infection (h.p.i.) for THP-1 cells, while it was still increasing at 120 h.p.i. for murine macrophages. Since in both cases the infection with L. peruviana affected cell viability, we recommend evaluating any leishmanicidal activity at 72 h.p.i. Amphotericin B reduced Leishmania infection by 50% at concentrations of 0.1 μM in THP-1 and murine macrophages at 72 h.p.i.Our results demonstrate that amastigotes of L. peruviana can infect THP-1 cells and murine macrophages and indicate the suitability of this model to screen compounds for leishmanicidal activity.     

Surfactant proteins A and D enhance the phagocytosis of Chlamydia into THP-1 cells

Chlamydiae are intracellular bacterial pathogens that infect mucosal surfaces, i.e., the epithelium of the lung, genital tract, and conjunctiva of the eye, as well as alveolar macrophages. In the present study, we show that pulmonary surfactant protein A (SP-A) and surfactant protein D (SP-D), lung collectins involved in innate host defense, enhance the phagocytosis of Chlamydia pneumoniae and Chlamydia trachomatis by THP-1 cells, a human monocyte/macrophage cell line. We also show that SP-A is able to aggregate both C. trachomatis and C. pneumoniae but that SP-D only aggregates C. pneumoniae. In addition, we found that after phagocytosis in the presence of SP-A, the number of viable C. trachomatis pathogens in the THP-1 cells 48 h later was increased approximately 3.5-fold. These findings suggest that SP-A and SP-D interact with chlamydial pathogens and enhance their phagocytosis into macrophages. In addition, the chlamydial pathogens internalized in the presence of collectins are able to grow and replicate in the THP-1 cells after phagocytosis.