Glycoconjugate with terminal galactose. A selective property of macrophages in developing rat lung

Pulmonary macrophages in pre- and postnatal rats were examined histochemically with a battery of peroxidase labeled lectins. Among them, Griffonia simplicifolia agglutinin I-B4 (GSA I-B4) which binds specifically to terminal alpha-galactose showed selective affinity in lung for the monocyte-macrophage line. These cells were demonstrable with GSA I-B4 from the 14th day of gestation through the adult. Extension to the ultrastructural level showed strong selective binding of this lectin to the surface of the plasmalemma and inner face of membranes limiting phagosomes in macrophages. At day 14 of gestation, monocyte-like cells positive with GSA I-B4 were scattered in various organs including lung. The lectin reactive cells in lung increased in number and size with development, infiltrating the interstitium through day 20 of gestation and then also entering the alveolar space. These findings suggest that GSA I-B4 recognizes a surface glycoconjugate characteristic of the pulmonary monocyte-macrophage line. Such selective lectin affinity offers a marker for detecting the pulmonary macrophages and examining their kinetics by light and electron microscopy.     

Glycoconjugate with terminal α galactose

Summary Glycoconjugates associated with the basal cell layer of various types of epithelia in the mouse and rat were examined histochemically with a battery of lectinhorseradish peroxidase (HRP) conjugates of differing sugar binding specificities. Basal cells in paraffin sections of composite tissue blocks stained with an isolectin from Griffonia simplicifolia (GSA I-B₄) specific for terminal -galactose residues but failed to react with the other lectins. Basal cells in epithelium lining striated and excretory ducts of salivary and lacrimal glands, tongue, esophagus, trachea, renal calyx, ureter, urinary bladder, urethra, epididymis and vas deferens stained selectively and intensely for content of a glycoconjugate with terminal -galactose. This galactoconjugate appeared associated with the plasmalemma of basal cells. Basal cells with a galactocalyx formed an intermittent to continuous layer generally increasing in prevalence distally in glandular duct systems. A minor population of pyramido-columnar cells with cytosolic GSA I-B₄ reactivity occurred in striated ducts and appeared less numerous in intralobular excretory ducts and more prevalent in extraglandular ducts. In trachea and renal pelvis, the GSA I-B₄ positive cell profiles ranged from low cuboidal to tall pyramidal in contour, but the latter appeared not to reach the lumen. In contrast, no GSA I-B₄ positive basal cells were seen in any segment of the pancreatic or bile ducts or in the epithelium of the gastrointestinal tract. These findings suggest that the basal cells found in similar sites in different epithelia and possessing in common a unique -galactoconjugate may function in a manner common to all and not simply in providing progenitor cells for epithelial renewal. The location and distribution of GSA I-B₄ reactive basal cells in diverse epithelia suggests that through their -galactocalyx they serve in maintaining the established composition of luminal fluid perhaps by impeding the transepithelial movement of fluid and ions.In honour of Prof. P. van DuijnThis research was supported by National Institutes of Health Grants HL-29775 and AM-10956     

α-d-Galactosylation of surface fucoglycoconjugate(s) upon stimulation/activation of murine peritoneal macrophages

Murine resident macrophages express, on their surface, carbohydrate epitopes which undergo changes during their stimulation/activation as monitored by binding of¹²⁵I labelledEvonymus europaea andGriffonia simplicifolia I-B₄ lectins. Treatment of the stimulated macrophages with coffee bean -galactosidase abolished binding of the GS I-B₄ isolectin and changed the binding pattern of theEvonymus lectin. The affinity (K _a) ofEvonymus lectin for -galactosidase-treated macrophages decreased approximately 23-fold, from 1.25×10⁸ M^–1 to 5.5×10⁶ M^–1. Subsequent digestion of -galactosidase-treated macrophages with -l-fucosidase fromTrichomonas foetus, further reduced binding ofEvonymus lectin. Resident macrophages showed the same pattern ofEvonymus lectin binding, with the same affinity, as -galactosidase-treated, stimulated macrophages. These results, together with a consideration of the carbohydrate binding specificity of theEvonymus lectin which, in the absence of -d-galactosyl groups, requires -l-fucosyl groups for binding, indicate the presence, on resident macrophages, of glycoconjugates with terminal -l-fucosyl residues. It is also concluded that during macrophage stimulation/activation -d-galactosyl residues are added to this glycoconjugate and that they form part of the receptor forEvonymus lectin. The same glycoconjugate(s) is/are also expressed on the activated macrophage IC-21 cell line which exhibits the same characteristics as that of stimulated peritoneal macrophages, i.e., it contains -d-galactosyl end groups and is resistant to the action of trypsin. Both lectins were also specifically bound toCorynaebacterium parvum activated macrophages.Abbreviations BSA bovine serum albumin - GS I-B₄ Griffonia simplicifolia I-B₄ isolectin - PBS 0.01m phosphate buffer (pH 7.1) with 0.15m NaCl (unless stated otherwise this buffer contained 3mm azide and was free of divalent cations) - PMSF phenyl methane sulfonyl fluoride - TG thioglycollate brewers medium.     

Subcellular distribution of terminal α-D- and β-D-galactosyl residues in Ehrlich tumour cells studied by lectin-gold techniques

We have studied by high resolutionin situ light and electron microscopic lectin-gold techniques the subcellular distribution of -d-Gal residues using theGriffonia simplicifolia I-B₄ isolectin and compared it with that of -d-Gal residues as detected with theDatura stramonium lectin in Ehrlich tumour cells grown as ascites or monolayer. The microvillar but not the smooth plasma membrane regions were labelled with theGriffonia simplicifolia I-B₄ isolectin whereas both plasma membrane regions were equally well labelled with theDatura stramonium lectin. Elements of the endocytotic/lysosomal system such as coated membrane invaginations and vesicles, early and late endosomes and secondary lysosomes were positive for both -d-Gal and -d-Gal residues. A particular feature of Ehrlich tumour cells is an elaborate tubular membrane system located in the pericentriolar region which is labelled throughout by both lectins and represents part of the endosomal system. In the Golgi apparatus labelling with both lectins was observed to commence in trans cisternae which is indirect evidence for a joint distribution of the sequentially acting 1,4 and 1,3-galactosyl-transferases.     

Origin of the central cells of erythroblastic islands in fetal mouse liver: ultrahistochemical studies of membrane-bound glycoconjugates

 To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells. Accepted: 22 January 1997     

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells

Background

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity.

Methods

MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting.

Results

BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α₁, α₆ and β₁ integrin subunit expression, and increases α₅ subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21.

Conclusion

BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells.

General significance

Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer.     

Role of the tachykinin NK1 receptor in a murine model of cigarette smoke-induced pulmonary inflammation

Background

The tachykinins, substance P and neurokinin A, present in sensory nerves and inflammatory cells such as macrophages and dendritic cells, are considered as pro-inflammatory agents. Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and increased tachykinin levels are recovered from the airways of COPD patients. The aim of our study was to clarify the involvement of the tachykinin NK₁ receptor, the preferential receptor for substance P, in cigarette smoke (CS)-induced pulmonary inflammation and emphysema in a mouse model of COPD.

Methods

Tachykinin NK₁ receptor knockout (NK₁-R^-/-) mice and their wild type controls (all in a mixed 129/sv-C57BL/6 background) were subjected to sub acute (4 weeks) or chronic (24 weeks) exposure to air or CS. 24 hours after the last exposure, pulmonary inflammation and development of emphysema were evaluated.

Results

Sub acute and chronic exposure to CS resulted in a substantial accumulation of inflammatory cells in the airways of both WT and NK₁-R^-/- mice. However, the CS-induced increase in macrophages and dendritic cells was significantly impaired in NK₁-R^-/- mice, compared to WT controls, and correlated with an attenuated release of MIP-3α/CCL20 and TGF-β1. Chronic exposure to CS resulted in development of pulmonary emphysema in WT mice. NK₁-R^-/- mice showed already enlarged airspaces upon air-exposure. Upon CS-exposure, the NK₁-R^-/- mice did not develop additional destruction of the lung parenchyma. Moreover, an impaired production of MMP-12 by alveolar macrophages upon CS-exposure was observed in these KO mice. In a pharmacological validation experiment using the NK₁ receptor antagonist RP 67580, we confirmed the protective effect of absence of the NK₁ receptor on CS-induced pulmonary inflammation.

Conclusion

These data suggest that the tachykinin NK₁ receptor is involved in the accumulation of macrophages and dendritic cells in the airways upon CS-exposure and in the development of smoking-induced emphysema. As both inflammation of the airways and parenchymal destruction are important characteristics of COPD, these findings may have implications in the future treatment of this devastating disease.     

Macrophage derived chemokine (CCL22), thymus and activation-regulated chemokine (CCL17), and CCR4 in idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a chronically progressive interstitial lung disease of unknown etiology. Previously, we have demonstrated the selective upregulation of the macrophage-derived chemokine CCL22 and the thymus activation-regulated chemokine CCL17 among chemokines, in a rat model of radiation pneumonitis/pulmonary fibrosis and preliminarily observed an increase in bronchoalveolar (BAL) fluid CCL22 levels of IPF patients.

Methods

We examined the expression of CCR4, a specific receptor for CCL22 and CCL17, in bronchoalveolar lavage (BAL) fluid cells, as well as the levels of CCL22 and CCL17, to elucidate their pathophysiological roles in pulmonary fibrosis. We also studied their immunohistochemical localization.

Results

BAL fluid CCL22 and CCL17 levels were significantly higher in patients with IPF than those with collagen vascular diseases and healthy volunteers, and there was a significant correlation between the levels of CCL22 and CCL17 in patients with IPF. CCL22 levels in the BAL fluid did not correlate with the total cell numbers, alveolar lymphocytes, or macrophages in BAL fluid. However, the CCL22 levels significantly correlated with the numbers of CCR4-expressing alveolar macrophages. By immunohistochemical and immunofluorescence analysis, localization of CCL22 and CCR4 to CD68-positive alveolar macrophages as well as that of CCL17 to hyperplastic epithelial cells were shown. Clinically, CCL22 BAL fluid levels inversely correlated with DLco/VA values in IPF patients.

Conclusion

We speculated that locally overexpressed CCL22 may induce lung dysfunction through recruitment and activation of CCR4-positive alveolar macrophages.     

Characterization of specific receptors for leukotriene D4 on human alveolar macrophages

Using ³H-leukotriene D₄, a specific receptor assay has been developed for human alveolar macrophages, obtained by broncho-alveolar lavage of patients undergoing fiberoptic bronchoscopy because of suspected bronchial carcinomaa. Lavage was performed in a carcinoma-free lobe of the lung and alveolar macrophages were subsequently isolated and incubated for binding studies. ³H-Leukotriene D₄ was found to bind specifically with high affinity (K_d = 3.8 nM), in a saturable manner (B_max = 90 fmol/10⁶ cells), reversible and selective. Specific binding was linear with protein concentration and equilibrium binding at 4°C was reached at 50 min. Scatchard and Hill analysis revealed a single class of binding sites with no cooperativity among the sites. displacement studies with LTD₄, the selective SRS-A antagonist FPL 55712 and with leukotriene C₄ revealed respective K_i values of 3.4; 16; and 110 nM. The data suggest that human alveolar macrophages may contain a specific receptor type for LTD₄, which has a relatively low affinity for LTC₄, and are discussed in relation to modulatory processes in the lung, apart from direct actions of LTD₄ on smooth muscle receptors. From the data here acquired, it may be apparent that the study of characteristics of receptors specific for a broncho-active substance like LTD₄ on huma alveolar macrophages, which play an important role in immuno-inflammatory processes seen in many chronic lung diseases, may yield major insights into the pathogenesis and therapy decisions involved in these diseases.     

Characterization of glycoconjugates in developing rat respiratory system by means of conventional and lectin histochemistry

Summary The glycoconjugates of the respiratory system of rats from 15 days of gestation through the adult period have been characterized by means of both conventional and lectin histochemistry. The main changes occurred at 20–21 days of gestation immediately before birth. An increase of acidic groups in the glycoproteins of the lung and airway epithelium was observed by conventional mucin histochemistry. The combined use of neuraminidase digestion and lectin histochemistry demonstrated an increase of sialic acid residues at the terminal position of the glucidic moieties of the glycoproteins. The sialic acid residues were linked (2–3, 6) to d-galactose (1–3)-N-acetylgalactosamine, thus masking the PNA-reactivity detected on the luminal surface of Clara cells and pneumonocytes before birth. In the adult period, -l-fucose residues, detected by UEA-I, were localized in the glycoproteins contained in goblet cells and periciliary layer of the rat airway epithelium. The modifications observed in the lung of developing rats are similar to those previously described in human fetal and neonatal lungs. This suggests that the rat represents a useful model to study the glycoprotein synthesis during lung development.     

M2 macrophages promote myofibroblast differentiation of LR-MSCs and are associated with pulmonary fibrogenesis

Background

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the histopathological pattern of usual interstitial pneumonia and is associated with a high mortality rate. Recently, lung resident mesenchymal stem cells (LR-MSCs) have been identified as an important contributor to myofibroblast activation in pulmonary fibrosis. Macrophages are also believed to play a critical role in pulmonary fibrosis. However, the underlying connections between LR-MSCs and macrophages in the pathogenesis of pulmonary fibrosis are still elusive.

Methods

In this study, we investigated the interaction between LR-MSCs and macrophages using a bleomycin-induced mouse pulmonary fibrosis model and a coculture system.

Results

Here, we show that blocking pulmonary macrophage infiltration attenuated bleomycin-induced pulmonary fibrosis. In addition, as determined by flow cytometry, we discovered that the recruited macrophages in fibrotic lungs of bleomycin-treated mice were mainly M2 macrophages. In particular, we found that M2, rather than M1 macrophages, promoted myofibroblast differentiation of LR-MSCs. Moreover, we demonstrated that suppression of the Wnt/β-catenin signaling pathway could attenuate myofibroblast differentiation of LR-MSCs induced by M2 macrophages and bleomycin-induced pulmonary fibrosis. Tissue samples from IPF patients confirmed the infiltration of M2 macrophages and activation of Wnt/β-catenin signaling pathway.

Conclusion

In summary, this study furthered our understanding of the pulmonary fibrosis pathogenesis and highlighted M2 macrophages as a critical target for treating pulmonary fibrosis.

     

Enzyme-linked lectin assay (ELLA): Use of alkaline phosphatase-conjugated Griffonia simplicifolia B4 isolectin for the detection of α- -galactopyranosyl end groups

Alkaline phosphatase has been coupled to Griffonia simplicifolia I B₄ isolectin using a one-step glutaraldehyde conjugation procedure. This enzyme-lectin conjugate (AP-GS I-B₄) has been used to specifically detect plastic-bound natural and synthetic glycoproteins bearing α- -galactopyranosyl end groups. The extent of reactivity of the AP-GS I-B₄ with the glycoproteins appears to be proportional to the number of terminal galactosyl residues present. Furthermore, this assay, termed ELLA (enzyme-linked lectin assay), is specifically inhibitable by low-molecular-weight sugars containing terminal α- -galactosyl groups. The ELLA reactions may be assayed rapidly and objectively by the use of commercially available ELISA-plate readers using standard filters.     

Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung

High affinity (Kd=0.2 nM), low capacity (48 fmoles per mg protein), stereospecific binding sites, with properties characteristic of the β₁-subtype of β-adrenergic receptors, have been detected in fetal rabbit lung membranes as early as the 22nd day of gestation. The concentration of the receptor did not change significantly between the 22nd and 26th day of gestation, but increased 3-fold between the 26th and 29th day, reaching a level of 198 fmoles per mg protein. A further increase (from 198 to 315 fmoles per mg protein) in receptor concentration was observed in adult female rabbits. The increase in the levels of pulmonary β-adrenergic receptors between the 26th and 29th day of gestation is temporally related to the increase in surfactant release into the alveolar spaces of the fetal lung. Thus β-adrenergic agonists may act directly on the fetal lung to regulate surfactant secretion.     

Histochemical demonstration of O-glycosidically linked,type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Summary Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo-and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with -N-acetylgalactosaminidase and -L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with -galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B₄ reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo--N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from nonsecretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(1-3)-N-acetyl-D-galactosamine1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.     

Temporal relationship between immune cell influx and the expression of inducible nitric oxide synthase, interleukin-4 and interferon-gamma in pancreatic islets of NOD mice following adoptive transfer of diabetic spleen cells

Beta cell destruction in NOD mice can be accelerated by adoptive transfer of diabetic spleen cells into irradiated adult NOD mice. Here mice receiving diabetic spleen cells were examined at days 0, 7, 14, 21 and at onset of diabetes for the resulting insulitis and the number of intra-islet CD4 and CD8 cells and macrophages. The progression of insulitis and the number of intra-islet CD4 and CD8 cells and macrophages were correlated with the expression and co-localization of inducible nitric oxide synthase, interferon- and interleukin-4 by dual-label light and confocal immunofluorescence microscopy. Diabetes developed in 7/8 mice by 27 days following cell transfer. The insulitis score increased slightly by day 7 but rose sharply at day 14 (p=0.001) and was maintained until diabetes. The mean number of intra-islet CD4 and CD8 cells and macrophages showed a similar trend to the insulitis scores and were present in almost equal numbers within the islets. Immunolabelling for inducible nitric oxide synthase was observed at day 7 in only some cells of a few islets but increased sharply from day 14. It was restricted to islets with insulitis and was co-localized in selective macrophages. Weak intra-islet interleukin-4 labelling was observed at days 7 and 14 but became more pronounced at day 21 and at onset of diabetes, being present in selective CD4 cells. Intra-islet labelling for interferon- was first observed at day 21, but became more intense at onset of diabetes and was co-localized in a proportion of macrophages. Both cytokines were expressed in islets with advanced insulitis. Interferon- staining was also observed within endothelial cells located in the exocrine pancreas. We conclude that transfer of diabetic spleen cells results in a rapid influx of CD4 and CD8 cells and macrophages within the pancreas of recipient mice. During the period of heightened insulitis, selective immune cells begin to express inducible nitric oxide synthase and the opposing cytokines, interferon- and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.     

Isolation of a melibiose-binding protein from human spleen

A melibiose-binding protein was isolated from human spleen by serial affinity chromatography on lactose-, mannose-, and melibiose-Sepharose. The purified protein agglutinated rabbit erythrocytes and re-bound to melibiose, but did not bind to murine nor human laminin. The protein was composed of 58 kDA, 32 kDa and 26 kDa polypeptides. The polypeptides were detected in buffy coat cell extracts and they were synthesizedin vitro by B lymphoblastoid cells. The polypeptides did not react with anti-galaptin, anti-C-reactive protein, anti-amyloid P, anti-keratin, and anti-rat lung lectin 29 sera. The 58 kDa polypeptide reacted very weakly with anti-core-specific lectin serum and reacted with anti-IgG serum. The data suggest that the major protein isolated is an anti-Gall 6 immunoglobulin.Abbreviations ME mercaptoethanol - PMSF phenylmethylsufonyl fluoride - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanosulfonate - PBS 0.01m PO₄, 0.12m NaCl, pH 7.3 - TBS 0.1m NaCl, 0.05m Tris, 0.05% NaN₃, 0.01m CaCl₂, 0.001m MgCl₂, pH 7.3 - BSA bovine serum albumin - GSI Griffonia simplicifolia I - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macrophages and regulated as a function of the Toll-like receptor immune response. Here, we investigated the intracellular impact of the down-regulation of the proprotein convertase 1/3 in NR8383 macrophages and confirmed the results on macrophages from PC1/3 deficient mice. A complete proteomic study of secretomes and intracellular proteins was undertaken and revealed that inhibition of proprotein convertase 1/3 orient macrophages toward an M1 activated phenotype. This phenotype is characterized by filopodial extensions, Toll-like receptor 4 MyD88-dependent signaling, calcium entry augmentation and the secretion of pro-inflammatory factors. In response to endotoxin/lipopolysaccharide, these intracellular modifications increased, and the secreted factors attracted naïve T helper lymphocytes to promote the cytotoxic response. Importantly, the application of these factors onto breast and ovarian cancer cells resulted in a decrease viability or resistance. Under inhibitory conditions using interleukin 10, PC1/3-knockdown macrophages continued to secrete inflammatory factors. These data indicate that targeted inhibition of proprotein convertase 1/3 could represent a novel type of immune therapy to reactivate intra-tumoral macrophages.Innate immunity is the first line of immune defense and is common to all metazoans (1, 2). In this immune system, macrophages play a crucial role in the maintenance of tissue homeostasis. These cells are involved in almost every disease through their immunological and wound-healing functions (1, 2). During a pathogenic infection, trauma or neurodegeneration, macrophages are recruited and activated contributing to the phagocytosis of pathogens and the secretion of cytokines and chemokines activating other immune cells. Macrophages can develop into classically pro-inflammatory (M1) or alternatively (M2) activated macrophages. M1 macrophages are characterized by the secretion of pro-inflammatory cytokines whereas M2 macrophages secrete anti-inflammatory cytokines (3). Stimulation of macrophages with LPS activates TLR4 signaling leading to the nucleus translocation of NF-κB or IRF3 which activate genes encoding proteins involved in innate immune response (4). Many of these proteins are secreted (cytokines, chemokines…) to attract and activate other immune cells like T lymphocytes. In tumors, macrophages are oriented toward the M2 phenotype and promote cancer growth by suppressing immune cells function (5). Current research in the therapeutic field focus on ways to reactivate macrophages.Surprisingly, we have shown that during immune responses, macrophages secrete typical neuroendocrine molecules (6–8), such as neuropeptides (9) or the proprotein convertases (PC)¹ PC2 and PC1/3 and that PC1/3 is an important regulator of innate immune responses (10–12). Proprotein convertases cleave precursor proteins which can lead to the activation, inactivation or functional changes. PC2 and PC1/3 operate within the regulated secretory pathway. Their expression is not restricted to neuroendocrine tissues, they are also expressed in macrophages and lymphocytes (12). In a previous study from our group, PC1/3 knockout (KO) in mice challenged with LPS caused innate immune defects and uncontrolled cytokine secretion (10). Th1 pathway is enhanced in PC1/3 KO mice. Following LPS treatment, PC1/3 colocalized with TLR4 in the endosomal compartment (11). We concluded that PC1/3 contributes to the regulation of TLR4 signaling and the resulting cytokine secretion.The NR8383 rat pulmonary macrophage cell line was previously shown as a good model to study the role of PC1/3 in the macrophage innate immune response (13). In the present study, we developed a PC1/3-knockdown (K_D) NR8383 cell line using lentiviral-delivered shRNAs. Our aim is to understand the cellular impact of PC1/3 inhibition in macrophages and the consequences on their activation. Proteomic analysis of secreted proteins allowed us to identify pro-inflammatory cytokines and alarmins already at 24h of LPS stimulation in PC1/3-K_D secretomes which was confirmed by cytokines array. Proteomic studies of PC1/3-K_D NR8383 cellular extracts revealed an important perturbation in the intracellular trafficking machinery through the disorganization of cytoskeletal protein expression. These results were confirmed on macrophages from PC1/3 KO mice. Cytokines secretion and cytoskeleton reorganization can be linked to intracellular calcium increase in PC1/3-K_D cells. Moreover, we showed that MyD88-dependant TLR4 signaling was sustained when PC1/3 is down-regulated. We describe here that inhibition of PC1/3 induced classically activated phenotype (M1) in macrophages. The chemotactic and anti-tumor properties of the PC1/3-K_D macrophage secretome promoted the cytotoxic immune response and inhibited cancer cell viability. The down-regulation of PC1/3 could be used in cancer immunotherapy to reactivate macrophages.     

Lectin-binding sites in isolated equine cumulus-oocyte complexes: Differential expression of glycosidic residues in complexes recovered with compact or expanded cumulus

Equine cumulus-oocyte complexes (COCs) were analyzed by means of 13 lectins to evaluate their glycoconjugate patterns and to verify differences between COCs recovered with compact (Cp) and expanded (Exp) cumulus. Cumulus cells showed a similar staining pattern in both Cp and Exp COCs with all lectins used, except for a higher reactivity with SNA and GSA II in Cp COCs and SBA in Exp COCs. The zona pellucida (ZP) showed (1) uniform staining with MAL II, RCA₁₂₀, and SBA in both Cp and Exp COCs, (2) trilaminar binding pattern with WGA as well as higher Con A reactivity in the outer region of both types of COCs, (3) uniform staining with PNA only in Exp COCs, (4) uniform and trilaminar binding pattern with SNA in Cp and Exp COCs, respectively, and (5) major reactivity with GSA II in Exp COCs. Ooplasm showed similar staining intensity with Con A, HPA, GSA I-B₄, and WGA in both Cp and Exp COCs, with stronger reactivity to GSA II in Exp COCs, whereas SNA, UEA I, and LTA binding sites were present only in Cp COCs. Oocyte cortical granules of both Cp and Exp COCs reacted with Con A and WGA. These results suggest that, in the mare, viable (Cp) and atretic (Exp) COCs display different glycoconjugate staining pattern, which may account for the different maturation and developmental competence of COCs.