Effects of hyperosmolarity on annexin A1 on ocular surface epithelium in vitro.

Osmotic stress is an important challenge to cell function. Dry eye pathology is characterized by elevated tear film osmolarity as consequence of decreased tear secretion and/or increased evaporation. Dry eye pathogenesis is not completely clarified. However, it is known that tear hyperosmolarity induces NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain-cointaining 3) inflammasome activation and inflammatory mediators release that leads to ocular surface damage. Annexin A1 is a protein involved in anti-inflammatory or pro-resolution actions in different tissues while its presence and biological role on ocular surface has been scarcely examined. In this study, potential changes in annexin A1 protein expression and secretion on the ocular surface after exposure to hyperosmolar conditions were evaluated. In addition, considering the significant role of inflammation in dry eye pathology, the potential anti-inflammatory activity of Ac2-26, an annexin A1 peptide mimicking its N-terminus, was assessed. Cytosolic and membrane staining was detected for annexin A1 in corneal and conjunctival epithelial cells. A native form of annexin A1 together with a truncated form were detected by western blot analysis. Under hyperosmotic conditions increased protein levels of intracellular and secreted annexin A1 as well as higher expression of its receptor Fpr2 (formyl peptide receptor type 2) were found. Treatment with mimetic peptide Ac2-26 ameliorated NLRP3 activation and interleukin 1ß (IL-1ß) release triggered by elevated osmolarity in corneal and conjunctival epithelial cells. These findings suggest a potential role of annexin A1 and its mimetic peptide modulating key inflammatory events associated to dry eye.

Deficiency in Fpr2 results in reduced numbers of Lin-cKit+Sca1+ myeloid progenitor cells.

The Lin-c-Kit+ Sca-1+ cell population in the bone marrow (BM) serves as the direct precursor for differentiation of myeloid cells. In this study, we report that deficiency in Fpr2, a G protein-coupled chemoattractant receptor in mice, is associated with reduced BM nucleated cells, including CD31+Ly6C+ (granulocytes and monocytes), CD31-/Ly6Cint (granuloid cells), and CD31-/Ly6Chigh (predominantly monocytes) cells. In particular, the number of Lin-c-Kit+Sca-1+ (LKS) cells was reduced in Fpr2-/- mouse BM. This was supported by observations of the reduced incorporation of intraperitoneally injected bromodeoxyuridine by cells in the c-Kit+ population from Fpr2-/- mouse BM. Purified c-Kit+ cells from Fpr2-/- mice showed reduced expansion when cultured in vitro with stem cell factor (SCF). SCF/c-Kit-mediated phosphorylation of P38, STAT1, Akt (Thr-308), and Akt (Ser-473) was also significantly reduced in c-Kit+ cells from Fpr2-/- mice. Furthermore, Fpr2 agonists enhanced SCF-induced proliferation of c-Kit+ cells. Colony-forming unit assays revealed that CFU-granulocyte-macrophage formation of BM cells from Fpr2-/- mice was significantly reduced. After heat-inactivated bacterial stimulation in the airway, the expansion of c-kit+ Sca-1+ cells in BM and recruitment of Ly6G+ cells to the lungs and CD11b+Ly6C+TNFα+ cells to the spleen of Fpr2-/- mice was significantly reduced. These results demonstrate an important role for Fpr2 in the development of myeloid lineage precursors in mouse BM.

Targeting formyl peptide receptor 1 of activated macrophages to monitor inflammation of experimental osteoarthritis in rat.

Macrophages play a crucial role in the pathogenesis of osteoarthritis (OA). In this study, the feasibility of a formyl peptide receptor 1 (Fpr1)-targeting peptide probe cFLFLF-PEG-(64) Cu via positron emission tomography (PET) imaging was investigated for detection of macrophage activity during development of OA. Monoiodoacetate (MIA) was intraarticularly injected into the knee joint of Sprague-Dawley rats to induce OA. Five days later, cFLFLF-PEG-(64) Cu (∼7,400 kBq/rat) was injected into the tail vein and microPET/CT imaging was performed to assess the OA inflammation by detecting infiltration of macrophages by Fpr1 expression. In addition, a murine macrophage cell line RAW264.7 and two fluorescent probes cFLFLF-PEG-cyanine 7 (cFLFLF-PEG-Cy7) and cFLFLF-PEG-cyanine 5 (cFLFLF-PEG-Cy5) were used to define the binding specificity of the peptide to macrophages. It was found with the MIA model that the maximal standard uptake values (SUVmax ) for right (MIA treated) and left (control) knees were 17.96 ± 5.45 and 3.00 ± 1.40, respectively. Histological evaluation of cryomicrotome sections showed that Fpr1 expression, cFLFLF-PEG-Cy5 binding, and tartrate-resistant acid phosphatase activity were elevated in the injured synovial membranes. The in vitro experiments demonstrated that both fluorescent peptide probes could bind specifically to RAW264.7 cells, which was blocked by cFLFLF but not by the scramble peptide. The findings highlighted the use of cFLFLF-PEG-(64) Cu/PET as an effective method potentially applied for detection and treatment evaluation of OA. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1529-1538, 2016.

In vivo monitoring of activated macrophages and neutrophils in response to ischemic osteonecrosis in a mouse model.

Ischemic osteonecrosis (IO) is caused by disruption of the blood supply to bone. It is a debilitating condition with pathological healing characterized by excessive bone resorption and delayed osteogenesis. Although the majority of research has focused on the role of osteoblasts and osteoclasts in the disease progression, we hypothesize that innate immune cells, macrophages and neutrophils, play a significant role. With the recent development of real-time imaging probes for neutrophils and macrophages, the purpose of this study was to investigate the kinetic immune cell response in a mouse model of IO. Our results show that induction of IO leads to a significant accumulation of activated neutrophils and macrophages at the affected tissue by 48 h after surgery. Additionally, the accumulation of these immune cells remained elevated in comparison to sham controls for up to 6 weeks, indicative of chronic inflammation. Immunohistochemistry confirmed the immune cell infiltration into the necrotic bone marrow and the increased presence of TNFα-positive cells, demonstrating, for the first time, a direct response of these cells to ischemia induced necrotic bone. These new findings support a hypothesis that IO is an osteoimmunologic condition where innate immune cells play a significant role in the chronic inflammation.

Resolution of inflammation is altered in Alzheimer's disease.

BACKGROUND: Resolution is the final stage of the inflammatory response, when restoration of tissue occurs. Failure may lead to chronic inflammation, which is known as part of the pathology in the brain of individuals with Alzheimer's disease (AD). METHODS: Specialized pro-resolving mediators (SPMs), receptors, biosynthetic enzyme, and downstream effectors involved in resolution were analyzed in postmortem hippocampal tissue from AD patients and non-AD subjects. SPMs were analyzed in cerebrospinal fluid (CSF). RESULTS: SPMs and SPM receptors were detected in the human brain. Levels of the SPM lipoxin A4 (LXA4) were reduced in AD, both in the CSF and hippocampus. An enzyme involved in LXA4 synthesis and two SPM receptors were elevated in AD brains. LXA4 and RvD1 levels in CSF correlated with Mini-Mental State Examination (MMSE) scores. CONCLUSIONS: A resolution pathway exists in the brain and the alterations described herein strongly suggest a dysfunction of this pathway in AD. MMSE correlations suggest a connection with cognitive function in AD.

Differential expression of the G-protein-coupled formyl Peptide receptor in melanoma associates with aggressive phenotype.

Melanoma, due to its metastatic rate, is among the most aggressive forms of skin cancer. Human formyl peptide receptor (FPR) and its variant FPR-like 1 (FPRL1) have been associated with cell migration and invasiveness in neoplasms. We have studied the in situ expression of these receptors in a large series of melanocytic lesions and correlated the expression with clinicopathological features and prognosis. Tissue microarray blocks of 141 cases including nevi (31 cases), primary (84 cases), and metastatic melanomas (26 cases) were semiquantitatively evaluated by immunohistochemistry for the expression of FPR and FPRL1 proteins. A significant association was observed regarding diagnosis and percentage of cells showing expression of FPR (P = 0.0311) and FPRL1 (P = 0.0053). A gain of FPR immunoreactivity was observed in the lesions having ulceration (P = 0.0194) and Breslow thickness (P = 0.044). Also, high FPRL1 cytoplasmic immunoreactivity was seen in lesions without tumor regression (P = 0.04). In addition, in patients with increased cytoplasmic staining for FPR, the probability of disease-specific survival was significantly lower (log rank test, P = 0.0089). Our findings reveal that FPR and FPRL1 are overexpressed in primary melanoma and correlate with aggressive tumor characteristics, underscoring them as potential therapeutic targets.

Human spermatozoa antigens in unexplained infertility.

OBJECTIVE: To determine and compare the immunolocalization of functionally important antigens in human spermatozoa in an unexplained infertility (UI) group. MATERIALS AND METHODS: In this study, the sperm samples of 20 patients undergoing evaluation belonging to normozoospermic group, whose primary reason of infertility was under investigation for this purpose, were screened. CD46, CD55 and CD52, CD69, CD98, fMLP, HI307, and 80280 were stained on the spermatozoa through indirect immunofluorescence technique. RESULTS: In addition to CD46, CD55, and CD52 antigens, which are known to be localized on human spermatozoa, significant immunolocalization of several novel antigens including: CD52, CD69, CD98, fMLP, HI307, and 80280 were determined on the spermatozoa of the unexplained infertility group, possibly reflecting important roles in the pathophysiology of such unresolved clinical situations. CONCLUSION: Identification and characterization of antigens present on sperm cells is crucial for understanding of the diagnosis and treatment of unexplained infertility. Further studies were conducted to evaluate a possible correlation between the expression of these antigens and clinical outcomes in different well-defined infertility groups.

TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.

Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ~0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase.

Annexin 1 exerts anti-nociceptive effects after peripheral inflammatory pain through formyl-peptide-receptor-like 1 in rat dorsal root ganglion.

BACKGROUND: Annexin 1 (ANXA1) has analgesic effects in inflammatory pain. We aimed to investigate the anti-nociceptive role of ANXA1, at the dorsal root ganglion (DRG) level, through an interaction with formyl-peptide-receptor-like 1 (FPR2/ALX). METHODS: Inflammatory pain was evoked by injecting complete Freund's adjuvant (CFA, 50 µl) into the hindpaw of male Sprague-Dawley rats. The distribution of ANXA1 and FPR2/ALX in L4/5 DRGs was evaluated by immunofluorescence. The expression of ANXA1 was measured by western blot. The involvement of FPR2/ALX in the anti-nociception of ANXA1 was investigated by thermal (irradiant heat) and mechanical (von Frey filament) pain tests with intrathecal (i.t.) ANXA1-derived peptide (Anxa1(2-26)), FPR2/ALX agonist 5(S)-6(R)-7-trihydroxyheptanoic-acid-methyl-ester (BML-111), and antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc1). RESULTS: ANXA1 and FPR2/ALX localized in the satellite glial cells and neurones in L4/5 DRGs. CFA treatment (n=20) increased ANXA1 expression in L4/5 DRGs within 7 days (P<0.01). I.T. Anxa1(2-26) (20 and 100 µg µl(-1)) and BML-111 (10 and 100 nmol) reduced CFA-induced thermal and mechanical nociception within 48 h (n=40) (P<0.05). However, i.t. Boc1 10 µg intensified inflammatory pain (P<0.05) and reversed the anti-nociceptive effect of Anxa1(2-26) (n=25) (P<0.05). Moreover, ANXA1 expression increased in L4/5 DRGs after i.t. Anxa1(2-26) (20 µg µl(-1)) (P<0.05) and BML-111 (10 nmol) (P<0.01) but decreased after i.t. Boc1 (10 and 100 µg) alone (P<0.01) or Boc1 (10 µg) co-injection with Anxa1(2-26) (20 µg µl(-1)) (P<0.05). CONCLUSIONS: Endogenous ANXA1 expression at the DRG level is involved in CFA-induced inflammatory pain, and i.t. ANXA1 20 µg µl(-1) produces its anti-nociceptive effect through FPR2/ALX.

Theranostic systems assembled in situ on demand by host-guest chemistry.

Theranostic systems have been explored extensively for a diagnostic therapy in the forms of polymer conjugates, implantable devices, and inorganic nanoparticles. In this work, we report theranostic systems in situ assembled by host-guest chemistry responding to a request. As a model theranostic system on demand, cucurbit[6]uril-conjugated hyaluronate (CB[6]-HA) was synthesized and decorated with FITC-spermidine (spmd) and/or formyl peptide receptor like 1 (FPRL1) specific peptide-spmd by simple mixing in aqueous solution. The resulting (FITC-spmd and/or peptide-spmd)@CB[6]-HA was successfully applied to the bioimaging of its target-specific delivery to B16F1 cells with HA receptors and its therapeutic signal transduction with elevated Ca(2+) and phosphor-extracellular signal-regulated kinase (pERK) levels in FPRL1-expressing human breast adenocarcinoma (FPRL1/MCF-7) cells. Finally, we could confirm in vitro and in vivo stability of the highly specific host-guest interaction. The on-demand theranostic platform technology using host-guest chemistry can be exploited for various bioimaging, biosensing, drug delivery, and tissue engineering applications.

Interactions of neutrophils with silver-coated vascular polyester grafts.

BACKGROUND: In reconstructive vascular surgery, infection is one of the most feared complications because of the high mortality. While the antimicrobial effect of a silver-coated endoprosthesis has been proven in experimental trials, there are no reports on its interactions with granulocytes, the first effector cells in general inflammation and in infection. MATERIALS AND METHODS: Therefore, we investigated whether silver coating of vascular polyester grafts affects receptor expression, mediator release, and functions of human neutrophils relevant for microbicidal activity and the wound-healing process. Naïve neutrophils were analyzed for their cellular receptors such as cluster of differentiation (CD)62L, CD11b, CXCR2, and fMLP-R, the mediators interleukin 8, granulocyte elastase (human neutrophil elastase), and leukotriene B4 (LTB4) as well as for microbicidal capacity (oxidative burst) in vitro. In addition, the role of plasma coating for receptor expression was addressed. RESULTS: There was both a decrease of CD62L and CXCR2 expression and an increase of CD11b, fMLP-R expression, elastase release, and LTB4 generation, which were statistically significant (p = 0.04; p = 0.01; p = 0.0; p = 0.0; p = 0.01; p = 0.02, respectively) in the presence of the silver-coated graft compared with non-silver-coated vascular grafts. In addition, microbicidal activity was significantly (p = 0.0) impaired by the silver-coated graft. Coating of the vascular grafts with plasma did not alter the former observations significantly. CONCLUSION: The results may indicate that silver-coated vascular polyester grafts activate neutrophils chronically which may favor tissue destruction and impaired antimicrobial effects.

Glioblastoma stem cells produce vascular endothelial growth factor by activation of a G-protein coupled formylpeptide receptor FPR.

Glioma stem cells (GSCs), or stem cell-like glioma cells, isolated from malignant glioma cell lines, were capable of producing vascular endothelial growth factor (VEGF). However, the exact role of such tumour cells in angiogenesis remains unknown. In this study, we isolated a small proportion of CD133+ GSCs from the human glioblastoma cell line U87 and found that these GSCs possessed multipotent differentiation potential and released high levels of VEGF as compared with CD133(-) tumour cells. The CD133+ GSCs also formed larger xenograft tumours that contained higher VEGF immunoreactivity and denser microvessels. Moreover, GSCs expressed a functional G protein-coupled formylpeptide receptor FPR, which was activated by a chemotactic peptide ligand, N-formylmethionyl-leucyl-phenylalanine (fMLF), to mediate calcium flux and the production of VEGF by GSCs. Our results indicate that FPR expressed by human GSCs may play an important role in glioma angiogenesis.

Formyl peptide receptor-1 activation enhances intestinal epithelial cell restitution through phosphatidylinositol 3-kinase-dependent activation of Rac1 and Cdc42.

Inflammatory disorders of the gastrointestinal tract result in the breakdown of the intestinal epithelial barrier in the form of erosion and ulceration. To reestablish the epithelial barrier, the epithelium must efficiently migrate to reseal wounds. Numerous signaling cascades are involved in the induction and regulation of this complex process. N-formyl peptide receptors comprise a group of Gi-coupled receptors that regulate innate immune responses. Previously, we identified the expression of functional N-formyl peptide receptors in model SK-CO15 intestinal epithelial cells and observed a role for activation of these receptors in regulating cellular invasive behavior. In these studies, we performed formyl peptide receptor-1 (FPR) localization and evaluated its role in regulating intestinal epithelial cell wound closure. Immunolocalization studies using a recently developed specific monoclonal anti-FPR Ab demonstrated its localization along the lateral membrane of crypt epithelial cells in normal human colonic epithelium. In vitro studies using the classical FPR agonist fMLF showed that FPR activation significantly enhances model intestinal epithelial cell restitution and that FPR localized along actin filaments in lamellipodial and filopodial extrusions. The increase in cell migration was associated with activation of PI3K, Rac1, and Cdc42. Pharmacologic inhibition of PI3K activity abrogated the fMLF-induced increase in wound closure and activation of both Rac1 and Cdc42. Inhibition of Rac1 and Cdc42 using pharmacologic inhibitors and dominant negative mutants also inhibited the fMLF-induced increase in cell migration. Taken together, theses results support a novel role for FPR stimulation in enhancing intestinal epithelial cell restitution through PI3K-dependent activation of Rac1 and Cdc42.

Evidence for the presence of N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor ligands in human amniotic fluid and fMLP receptor modulation by physiological labour.

OBJECTIVES: The presence of amniotic binding sites for N-formyl-methionyl-leucyl-phenylalanine (fMLP), an inflammatory peptide, and its ability to induce prostaglandin E2 synthesis in the human amnion prompted us to investigate for: (1) the presence of fMLP receptor ligands (fMLPRL) in the amniotic fluid; (2) expression of the fMLP receptor in amniotic tissue; (3) the effect of amniotic fMLPRL on neutrophil cyclic AMP (cAMP) level and calcium concentration ([Ca2+]i) during physiological pregnancy and labour. METHODS: Binding assays were performed on neutrophils to determine the presence of fMLRL in the amniotic fluid at the 17th week of pregnancy, as well as at term, before and after the onset of labour. The expression of fMLP receptor mRNA was evaluated by RT-PCR, the cAMP level by a radiochemical assay, and the calcium concentration by Fura-2 AM fluorescence measurement. RESULTS: fMLPRLs were detectable in amniotic fluid throughout pregnancy, and their levels did not vary during gestation. Labour significantly increased both the amniotic fMLPRL level and the expression of fMLP receptor in amnion tissue. The increased amniotic fMLPRL concentration noted during labour significantly increased neutrophil cAMP level and [Ca2+]i. CONCLUSIONS: These findings demonstrate for the first time the presence of fMLP receptor ligands in amniotic fluid, and indicate a modulation of the fMLP system by the events of physiological labour.

Modification of membrane markers on THP-1 cells after ozone exposure in the presence or absence of fMLP.

The impacts of ozone and N-formyl-methionyl-leucyl-phenylalanine (fMLP) on the detection of membrane markers on non-differentiated THP-1 cells were evaluated for in vitro exposures. Several markers, specific for monocytes and macrophages, were identified on the THP-1 cells, allowing their use as a model for alveolar macrophages. Ozone exposure modified not only the detection of membrane markers, especially CD13 and CD14, monocyte and macrophage markers, but also the detection of the specific receptor for fMLP, formyl peptide receptor (FPR). Activation by fMLP also reduced the detection of the CD antigens, and a combined exposure to ozone and fMLP amplified this decrease, probably due to an additive effect of these chemicals. Overall, these results suggest important membrane rearrangements for short-term treatments to ozone and/or fMLP.