Thioglycollate-elicited Macrophages Research Articles

Abstract 105: Timely Removal of Lipid Oxidation Products is Mediated by Macrophages.

Reciprocity of inflammation and oxidative stress is now significantly considered as an important mechanism underlying numerous diseases and cancers. As a mechanistic link connecting them, we have shown that one of the end products of lipid oxidation, 2-(ω-carboxyethyl)pyrrole (CEP) is generated during inflammation and wound healing and recognized by Toll-like receptor 2 (TLR2) on endothelial cells. It leads to angiogenesis, which is independent of vascular endothelial growth factor. Importantly, the levels of CEP are reduced back to normal when the wound is healed, but remain constitutively high in pathological conditions, including aging and cancers. Thus, for most tissue repair processes, it appears critical not only to generate CEP, but also to provide its timely removal. Accordingly, our hypothesis is that in order to avoid tissue damage by inflammation-induced oxidative products, CEP needs to be cleared from injured tissues when its role is complete. We have sought to determine a cellular and molecular mechanism of CEP clearance from injured tissues. At first, through CEP binding and internalization assay, comparing with other cell types, we verified that macrophage is the responsible cell type of CEP clearance. Specifically, resident, F4/80 hi , and M2 macrophages are much more efficient for CEP clearance rather than thioglycolate-elicited, F4/80 lo , and M1 macrophages, respectively. Secondly, by using several knockout mouse lines, we identified that both TLR2 and CD36 are largely involved in CEP clearance. To examine the in vivo relevance of CEP clearance besides skin injury model, we used ApoE deficient mice as an atherosclerotic model because atherosclerosis is known to be intimately associated with inflammation and oxidative stress. Using a CEP-specific antibody, we have been screening the CEP appearance and disappearance in early-to-late stages of atherosclerosis. Recently, we examined that CEP is strongly stained only in and adjacent to the atherosclerotic lesion of ApoE deficient mice fed with western diet for 20 weeks, but barely stained in 30 weeks. Moreover, in the plaque, macrophage-enriched region exhibits weak signals of CEP staining. Therefore, we suggest that timely removal of CEP is mediated by macrophages ex vivo and in vivo .

Action of two phospholipases A2 purified from Bothrops alternatus snake venom on macrophages

The in vitro effects of BaltTX-I, a catalytically inactive Lys49 variant of phospholipase A2 (PLA2), and BaltTX-II, an Asp49 catalytically active PLA2 isolated from Bothrops alternatus snake venom, on thioglycollate-elicited macrophages (TG-macrophages) were investigated. At non-cytotoxic concentrations, the secretory PLA2 BaltTX-I but not BaltTX-II stimulated complement receptor-mediated phagocytosis. Pharmacological treatment of TG-macrophages with staurosporine, a protein kinase C (PKC) inhibitor, showed that this kinase is involved in the increase of serum-opsonized zymosan phagocytosis induced by BaltTX-I but not BaltTX-II secretory PLA2, suggesting that PKC may be involved in the stimulatory effect of this toxin in serum-opsonized zymosan phagocytosis. Moreover, BaltTX-I and -II induced superoxide production by TG-macrophages. This superoxide production stimulated by both PLA2s was abolished after treatment of cells with staurosporine, indicating that PKC is an important signaling pathway for the production of this radical. Our experiments showed that, at non-cytotoxic concentrations, BaltTX-I may upregulate phagocytosis via complement receptors, and that both toxins upregulated the respiratory burst in TG-macrophages.

Abstract 19046: RBC-Macrophage Inflammatory Axis: Potential Role in Atherosclerosis

Background: Red blood cells (RBC) modulate vasoreactivity, serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC), and contribute to the pathology of atherosclerotic plaque rupture, suggesting a broad role in vascular pathophysiology. High fat diet (HFD) is known to negatively impact vascular function and modify RBC membrane composition, but how this affects RBC chemokine binding, and interaction with macrophages and blood vessels, is unknown. Methods and Results: RBC from C57BL/6J mice fed chow diet (chow RBC) or 60% HFD (HFD RBC) for 16 weeks were collected and studied in vitro and in vivo. Reactive oxygen species (DCFH fluorescence) and phosphatidylserine externalization (annexin V binding), a potential signal for RBC phagocytosis, were increased ~1.5 fold (p<0.05) in HFD RBC compared to chow RBC. HFD RBC released ~1.5 fold more monocyte chemoattractant protein-1 (MCP-1) (p<0.05) compared to chow RBC upon addition of heparin to disrupt DARC binding. In a transwell system, HFD RBC, as compared with chow RBC, produced a significant increase in the migration of thioglycollate-elicited macrophages through an endothelial monolayer (p<0.05). Incubation of HFD RBC with murine macrophages demonstrated a ~1.5 fold (p<0.05) increase in phagocytosis compared to chow RBC; upon their internalization by macrophages, HFD RBC more potently upregulated transcription of major pro-inflammatory chemokines Il-1b, Ccl3, and Cxcl2. As compared with chow RBC, exposure of murine aortic segments to HFD RBC under orbital shear conditions led to enhanced endothelial activation as evidenced by increased adhesion of labeled murine macrophages to the aortic endothelium (p<0.05). Finally, when labeled and injected in vivo, HFD RBC exhibited a ~3 fold (p<0.05) increase in splenic phagocytosis as compared with chow RBC. Conclusion: HFD profoundly impact RBC biology, inducing oxidative stress, altering membrane phospholipid display, and increasing chemokine release, phagocytosis by macrophages, and macrophage-blood vessel interactions. These novel findings suggest that RBC may play an important role in transducing vascular inflammation, and thus contributing to atherosclerosis, during HFD.

Proteinase 3, the Autoantigen in Granulomatosis with Polyangiitis, Associates with Calreticulin on Apoptotic Neutrophils, Impairs Macrophage Phagocytosis, and Promotes Inflammation

Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on macrophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflammatory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory "reprogramming" of macrophages.

Eosinophil contamination of thioglycollate-elicited peritoneal macrophage cultures skews the functional readouts of in vitro assays

Thioglycollate-elicited peritoneal cells are a common source of macrophages for various in vitro assays, including stimulation with TLR ligands, cell signaling assays, phagocytosis, toxicology studies, and cytokine/chemokine production. The most common method for enrichment of cultured thioglycollate-elicited peritoneal cells is adherence. However, the presence of other cell types in freshly isolated and cultured thioglycollate-elicited peritoneal cells has not been examined. Here, we demonstrate that thioglycollate-elicited peritoneal cavity contains 55-60% nonmacrophage cells, and even after adherence, there are still 12-20% nonmacrophage cells remaining. Excluding macrophages, eosinophils are the major cell type in the freshly elicited cavity (30-40%). Eosinophils are also the major cell type contaminating in vitro cultures of thioglycollate-elicited peritoneal macrophages. Moreover, the contamination of macrophage cultures by eosinophils significantly diminishes activation of p38 MAPK and the serine threonine kinase Akt and production of proinflammatory cytokines in response to LPS stimulation. Taken together, these data suggest that thioglycollate-elicited peritoneal cells are far more heterogeneous than reported previously. Further, a failure to remove contaminating eosinophils may greatly affect the interpretation of results obtained with cultured thioglycollate-elicited macrophages. Thus, our data indicate that future studies intent on accurately assessing cultured macrophage phenotype and activation require depletion of all cocontaminating cells, especially eosinophils.

Synergistic effect of Tim4 and MFG-E8 null mutations on the development of autoimmunity

Phagocytes, including macrophages, recognize phosphatidylserine exposed on apoptotic cells as an "eat me" signal. Milk Fat Globule EGF Factor VIII (MFG-E8) is secreted by one subset of macrophages, whereas Tim4, a type I membrane protein, is expressed by another. These proteins bind tightly to phosphatidylserine on apoptotic cells and enhance their engulfment by macrophages. To study the contribution of these proteins to the engulfment of apoptotic cells, we established a mouse line that was deficient in the genes encoding MFG-E8 and Tim4. The null mutation of Tim4 impaired the ability of resident peritoneal macrophages, but not thioglycollate-elicited macrophages, to engulf apoptotic cells. Mice deficient in either MFG-E8 or Tim4 on the C57BL/6 background developed hardly any autoantibodies, but aged female mice deficient in both MFG-E8 and Tim4 developed autoantibodies in an age-dependent manner. Tumour necrosis factor (TNF) α is known to protect against systemic lupus erythematosus-type autoimmunity, whereas type I IFN accelerates the disease. Indeed, the administration of an anti-TNFα antibody or a reagent that stimulates the IFN-α production [2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane)] enhanced the production of autoantibodies in the MFG-E8- and Tim4-double-deficient mice. These results suggest that the double deficiency of Tim4 and MFG-E8, phosphatidylserine-binding proteins, can trigger autoimmunity and that TNFα and type I IFN regulate reciprocally the development of autoimmune disease.

P47phox Directs Murine Macrophage Cell Fate Decisions

Macrophage differentiation and function are pivotal for cell survival from infection and involve the processing of microenvironmental signals that determine macrophage cell fate decisions to establish appropriate inflammatory balance. NADPH oxidase 2 (Nox2)-deficient chronic granulomatous disease (CGD) mice that lack the gp91(phox) (gp91(phox-/-)) catalytic subunit show high mortality rates compared with wild-type mice when challenged by infection with Listeria monocytogenes (Lm), whereas p47(phox)-deficient (p47(phox-/-)) CGD mice show survival rates that are similar to those of wild-type mice. We demonstrate that such survival results from a skewed macrophage differentiation program in p47(phox-/-) mice that favors the production of higher levels of alternatively activated macrophages (AAMacs) compared with levels of either wild-type or gp91(phox-/-) mice. Furthermore, the adoptive transfer of AAMacs from p47(phox-/-) mice can rescue gp91(phox-/-) mice during primary Lm infection. Key features of the protective function provided by p47(phox-/-) AAMacs against Lm infection are enhanced production of IL-1α and killing of Lm. Molecular analysis of this process indicates that p47(phox-/-) macrophages are hyperresponsive to IL-4 and show higher Stat6 phosphorylation levels and signaling coupled to downstream activation of AAMac transcripts in response to IL-4 stimulation. Notably, restoring p47(phox) protein expression levels reverts the p47(phox)-dependent AAMac phenotype. Our results indicate that p47(phox) is a previously unrecognized regulator for IL-4 signaling pathways that are important for macrophage cell fate choice.

Adipose-selective overexpression of ABHD5/CGI-58 does not increase lipolysis or protect against diet-induced obesity

Adipose triglyceride lipase (ATGL) catalyzes the first step of triacylglycerol hydrolysis in adipocytes. Abhydrolase domain 5 (ABHD5) increases ATGL activity by an unknown mechanism. Prior studies have suggested that the expression of ABHD5 is limiting for lipolysis in adipocytes, as addition of recombinant ABHD5 increases in vitro TAG hydrolase activity of adipocyte lysates. To test this hypothesis in vivo, we generated transgenic mice that express 6-fold higher ABHD5 in adipose tissue relative to wild-type (WT) mice. In vivo lipolysis increased to a similar extent in ABHD5 transgenic and WT mice following an overnight fast or injection of either a β-adrenergic receptor agonist or lipopolysaccharide. Similarly, basal and β-adrenergic-stimulated lipolysis was comparable in adipocytes isolated from ABHD5 transgenic and WT mice. Although ABHD5 expression was elevated in thioglycolate-elicited macrophages from ABHD5 transgenic mice, Toll-like receptor 4 (TLR4) signaling was comparable in macrophages isolated from ABHD5 transgenic and WT mice. Overexpression of ABHD5 did not prevent the development of obesity in mice fed a high-fat diet, as shown by comparison of body weight, body fat percentage, and adipocyte hypertrophy of ABHD5 transgenic to WT mice. The expression of ABHD5 in mouse adipose tissue is not limiting for either basal or stimulated lipolysis.

Galectin-9 Attenuates Acute Lung Injury by Expanding CD14–Plasmacytoid Dendritic Cell–like Macrophages

Galectin (Gal)-9 plays a crucial role in the modulation of innate and adaptive immunity. To investigate whether Gal-9 plays a role in a murine acute lung injury (ALI) model. C57BL/6 mice were pretreated with Gal-9 by subcutaneous injection 24 and 48 hours before intranasal LPS inoculation. Gal-9 suppressed pathological changes of ALI induced by LPS. Gal-9 reduced levels of proinflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-α, IL-1β, IL-6, and keratinocyte-derived cytokine; decreased neutrophils; and increased IL-10 and CD11b(+)Gr-1(+) macrophages in the bronchoalveolar lavage fluid of ALI mice. In Gal-9-deficient mice, pathological changes of ALI were exaggerated, and the number of neutrophils and the TNF-α level were increased. CD11b(+)Gr-1(+) cells were increased in the spleen of both Gal-9-treated and phosphate-buffered saline (PBS)-treated ALI mice, but only Gal-9 increased the ability of CCR2-expressing macrophages to migrate toward monocyte chemoattractant protein-1. Transfer of CD11b(+)Gr-1(+) macrophages obtained from Gal-9-treated mice ameliorated ALI. CD11b(+)Gr-1(+) macrophages obtained from Gal-9-treated but not PBS-treated mice suppressed TNF-α and keratinocyte-derived cytokine production from LPS-stimulated macrophages, and down-regulated Toll-like receptor-4 (TLR4) and TLR2 expression on thioglycollate-elicited macrophages. Fluorescence-activated cell-sorting analysis revealed that CD14 is negligible on CD11b(+)Gr-1(+) macrophages obtained from Gal-9-treated mice, although those from both groups resembled plasmacytoid dendritic cells (pDCs). Gal-9 down-regulated CD14 on pDC-like macrophages from PBS-treated mice independently of Gal-9/Tim-3 (T-cell immunoglobulin- and mucin domain-containing molecule-3) interaction, resulting in the acquisition of suppressive function, suggesting that the loss of CD14 by Gal-9 is critical for the suppression of pDC-like macrophages. Gal-9 attenuates ALI by expanding CD14(-)CD11b(+)Gr-1(+) pDC-like macrophages by preferentially suppressing macrophage functions to release proinflammatory cytokines through TLR4 and TLR2 down-regulation.

Association of liposome-entrapped [3H]methotrexate with thioglycollate-elicited macrophages in-vitro.

The association of free or liposome-entrapped [3H]methotrexate [( 3H]MTX) with thioglycollate-elicited macrophages was investigated in-vitro. [14C]Cholesteryl oleate was incorporated into the liposomes as a lipid marker. [3H]MTX association with the macrophages was 5 to 9-fold higher with liposome-entrapped [3H]MTX than with the free drug. Macrophage-liposome association was biphasic, temperature-dependent and saturable at high liposomal lipid concentration. A high liposome cholesterol (CH) content or the presence of 2,4-dinitrophenol or colchicine also reduced macrophage-liposome association.

The Immunoproteasomes Regulate LPS-Induced TRIF/TRAM Signaling Pathway in Murine Macrophages

We have proposed the novel concept that the macrophage ubiquitin-proteasome pathway functions as a key regulator of Lipopolysaccharide (LPS)-induced inflammation signaling. These findings suggest that proteasome-associated protease subunits X, Y, and Z are replaced by LMP subunits after LPS treatment of RAW 264.7 cells. The objective here was to determine the contribution of selective LMP proteasomal subunits to LPS-induced nitric oxide (NO) and TNF-α production in primary murine macrophages. Accordingly, thioglycollate-elicited macrophages from LMP7, LMP2, LMP10 (MECL-1), and LMP7/MECL-1 double knockout mice were stimulated in vitro with LPS, and were found to generate markedly reduced NO levels compared to wild-type (WT) mice, whereas TNF-α levels responses were essentially unaltered relative to wild-type responses. The recent studies suggest that the TRIF/TRAM pathway is defective in LMP knockouts which may explain why iNOS/NO are not robustly induced in LPS-treated macrophages from knockouts. Treating these macrophages with IFN-γ and LPS, however, reverses this defect, leading to robust NO induction. TNF-α is induced by LPS in the LMP knockout macrophages because IκB and IRAK are degraded normally via the MyD88 pathway. Collectively, these findings strongly support the concept that LMP7/MECL-1 proteasomes subunits actively function to regulate LPS-induced NO production by affecting the TRIF/TRAM pathway.

Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid-aminopterin conjugate

IntroductionFolate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin.MethodsUsing a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans.ResultsEC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems.ConclusionsEC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity.

IFITM6 expression is increased in macrophages of tumor-bearing mice

The family of interferon-induced transmembrane protein (IFITM) genes consists of IFITM1, 2, 3, 5, and 6. They encode cell surface proteins that modulate cell-cell adhesion and cell differentiation. In a previous study, we showed that IFITM1 is involved in the immune escape and metastasis of gastric cancer cells. In this study, we determined the difference in expression of IFITM family genes in tumor-bearing mice. IFITM1 and 6 were found to be significantly increased. IFITM6 gene expression was increased only in the spleen of tumor-bearing mice but not in the bone marrow, lymph node, or thymus. IFITM6 expression was induced in various macrophages, including splenic, thioglycollate-elicited, and bone marrow-derived macrophages, but not in T cells. Lipopolysaccharides (LPS) also increased IFITM6 expression 24 h after administration, and Toll-like receptor 1, 2, 3, 4, and 9 agonists stimulated IFITM6 expression. These findings imply that the increase in IFITM6 expression may be involved in macrophage functions of tumor-bearing mice.

Cell-Specific Determinants of Peroxisome Proliferator-Activated Receptor γ Function in Adipocytes and Macrophages

The nuclear receptor peroxisome proliferator activator receptor gamma (PPARgamma) is the target of antidiabetic thiazolidinedione drugs, which improve insulin resistance but have side effects that limit widespread use. PPARgamma is required for adipocyte differentiation, but it is also expressed in other cell types, notably macrophages, where it influences atherosclerosis, insulin resistance, and inflammation. A central question is whether PPARgamma binding in macrophages occurs at genomic locations the same as or different from those in adipocytes. Here, utilizing chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we demonstrate that PPARgamma cistromes in mouse adipocytes and macrophages are predominantly cell type specific. In thioglycolate-elicited macrophages, PPARgamma colocalizes with the hematopoietic transcription factor PU.1 in areas of open chromatin and histone acetylation, near a distinct set of immune genes in addition to a number of metabolic genes shared with adipocytes. In adipocytes, the macrophage-unique binding regions are marked with repressive histone modifications, typically associated with local chromatin compaction and gene silencing. PPARgamma, when introduced into preadipocytes, bound only to regions depleted of repressive histone modifications, where it increased DNA accessibility, enhanced histone acetylation, and induced gene expression. Thus, the cell specificity of PPARgamma function is regulated by cell-specific transcription factors, chromatin accessibility, and histone marks. Our data support the existence of an epigenomic hierarchy in which PPARgamma binding to cell-specific sites not marked by repressive marks opens chromatin and leads to local activation marks, including histone acetylation.

Peroxiredoxin 1 Stimulates Secretion of Proinflammatory Cytokines by Binding to TLR4

Peroxiredoxin 1 (Prx1) is an antioxidant and molecular chaperone that can be secreted from tumor cells. Prx1 is overexpressed in many cancers, and elevation of Prx1 is associated with poor clinical outcome. In the current study, we demonstrate that incubation of Prx1 with thioglycollate-elicited murine macrophages or immature bone marrow-derived dendritic cells resulted in TLR4-dependent secretion of TNF-alpha and IL-6 and dendritic cell maturation. Optimal secretion of cytokines in response to Prx1 was dependent upon serum and required CD14 and MD2. Binding of Prx1 to thioglycollate macrophages occurred within minutes and resulted in TLR4 endocytosis. Prx1 interaction with TLR4 was independent of its peroxidase activity and appeared to be dependent on its chaperone activity and ability to form decamers. Cytokine expression occurred via the TLR-MyD88 signaling pathway, which resulted in nuclear translocation and activation of NF-kappaB. These findings suggest that Prx1 may act as danger signal similar to other TLR4-binding chaperone molecules such as HSP72.

NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.

Knockout Mice Reveal a Role for P2Y6 Receptor in Macrophages, Endothelial Cells, and Vascular Smooth Muscle Cells

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y(6) receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y(6)-null mice by gene targeting. The P2Y(6) knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y(6) is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-alpha, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y(6) KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y(6) mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y(6)-null mice. In conclusion, we generated P2Y(6)-deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

Tumoral and macrophage uPAR and MMP-9 contribute to the invasiveness of B16 murine melanoma cells

The aim of this study was to investigate whether tumor cells as well as tumor-associated macrophages (TAMs) contribute to the generation of protease activities essential to tumor cell invasiveness, such as matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9), and the urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR). We found that the enhanced invasiveness through Matrigel-coated filters of B16 murine melanoma cells stimulated with IFNgamma was associated with an higher expression of uPAR and MMP-9 in these cells. Moreover, treatment with anti-MMP-9 or anti-uPAR monoclonal antibodies abrogated the increase of invasiveness in IFNgamma-stimulated melanoma cells, suggesting a cooperation of uPA system and MMP-9 in cytokine-stimulated invasiveness. Invasiveness through Matrigel was also enhanced in B16 melanoma cells exposed to a medium conditioned by TAMs, represented in our experimental model by thioglycollate-elicited macrophages co-cultivated with melanoma cells. Macrophages isolated from these co-cultures were found to express higher levels of uPAR and MMP-9 compared to macrophage cultures alone, and the pro-invasive activity of the co-culture-conditioned medium was abrogated by anti-MMP-9 monoclonal antibodies, but not anti-uPAR monoclonal antibodies. Furthermore, the enhanced uPAR and MMP-9 expression in macrophages co-cultivated with tumor cells seems a rather specific phenomenon, generated through a cell-to-cell contact mechanism. On the whole, our data point to a cooperation between tumor cells and macrophages elicited by tumor cells themselves in generating key enzymes essential in the promotion of tumor invasiveness, such as uPAR and MMP-9.

Neutrophil haptotaxis induced by mouse MNCF: interactions with extracellular matrix glycoproteins probably contribute to overcoming the anti-inflammatory action of dexamethasone

The Macrophage-derived Neutrophil Chemotactic Factor (MNCF) has been characterized as a dexamethasone-resistant neutrophil chemotactic lectin produced by rat macrophages. This study was undertaken to evaluate different MNCF cellular sources and investigate the mechanisms by which MNCF overcomes the anti-inflammatory actions of dexamethasone. The mouse macrophage-like cell line P388D1 and thioglycollate-elicited mouse macrophages were studied regarding their capacity to release MNCF. Neutrophil migration assays were performed in vivo and in vitro, in either the presence or absence of extracellular matrix glycoproteins (ECM). Mouse and P388D1 macrophages release a lectin that reproduces the activities of rat MNCF. The ability of MNCF to induce neutrophil adhesion and haptotaxis is enhanced through its interaction with laminin and fibronectin. These properties are not inhibited by dexamethasone. Together, our results suggest that dexamethasone-resistant neutrophil migration induced by MNCF occurs probably because of its interactions with ECM.