Mechanisms underlying Retinoic acid-induced chemoattraction in molluscan neurons

Retinoic acid, a derivative of vitamin A, is known to play diverse roles in development and regeneration. Previous research in the mollusc Lymnaea stagnalis has shown that a gradient of all-trans retinoic acid attracts the growth cones of cultured neurons. The present study investigates the sub-cellular mechanisms within the growth cones of Lymnaea pedal A neurons which mediate the attractive response to a gradient of alltrans retinoic acid. In this study, the mechanism of growth cone turning is shown to be local, as neurites mechanically isolated from their cell body retain the capacity to turn towards an exogenous gradient of all-trans retinoic acid. The turning response is dependent on the initiation of protein synthesis and calcium influx, but does not appear to involve signaling through protein kinase C (PKC). The retinoid X receptor (RXR), which classically functions as a transcription factor, was also shown to be involved in the turning response, functioning locally through a non-genomic pathway. These data show, for the first time in any species, that all-trans retinoic acid's chemotropic action involves a local mechanism involving non-genomic signaling through the RXR. As retinoic acid is known to playa role in regeneration, understanding the mechanisms underlying retinoic acid signaling may lead to further advances in regenerative neuroscience.

CCR5 Mediates Pro-osteoclastic and Osteoclastogenic Leukocyte Chemoattraction

Periodontal disease (PD) progression involves the selective leukocyte infiltration into periodontium, supposedly mediated by the chemokine/chemokine receptor system. In this study, we investigated the role of chemokine receptor CCR5 in the immunoregulation of experimental PD in C57BL/6 (WT) and CCR5KO mice. Aggregatibacter actinomycetem comitans infection triggered the chemoattraction of distinct CCR5+ leukocyte subpopulations (determined by flow cytometry): CCR5+F4/80+ leukocytes, which co-express CD14, CCR2, TNF-alpha, and IL-1 beta, indicative of activated macrophages; and CCR5+CD4+ cells, which co-express CXCR3, IFN-gamma, and RANKL, indicative of Th1 lymphocytes, therefore comprising pro-osteoclastic and osteoclastogenic cell subsets, respectively. CCR5KO mice presented a lower PD severity (lower inflammation and alveolar bone loss) when compared with the WT strain, since the migration of F4/80+, TNF-alpha+, CD4+, and RANKL+ cells specifically decreased due to the lack of CCR5. Also, ELISA analysis demonstrated that the production of TNF-alpha, IL-1 beta, IL-6, IFN-gamma, and RANKL in periodontal tissues was significantly decreased in the CCR5KO strain. The periodontal bacterial load and antimicrobial patterns were unaltered in CCR5KO mice. Our results demonstrate that the chemokine receptor is involved in the migration of distinct leukocyte subpopulations throughout experimental PD, being a potential target for therapeutic intervention in PD.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in cell disassembly and the exposure of ‘flags’ at the dying cell surface that permit recognition and removal by viable cells (phagocytes). Efficient phagocytic removal of dying cells is essential to prevent inflammatory and autoimmune disorders. Relatively little is known of the molecular mechanisms underlying changes at the apoptotic cell surface. We have previously shown that ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) undergoes a change of function as cells die so that it acts as a molecular ‘flag’ to mediate corpse removal. Our work seeks to characterise apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. Here we extend earlier studies to show that apoptotic cell-associated ICAM-3 functions, at least minimally, to tether apoptotic leukocytes to macrophages via an undefined receptor. Whilst CD14 has been suggested as a possible innate immune receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Our data additionally indicate, that during apoptosis, leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates with a reduction in cell volume. This reduction in ICAM-3 is explained by cell surface shedding of microparticles (‘apoptotic bodies’) that contain ICAM-3. Such microparticles, released from apoptotic leukocytes, are strongly chemoattractive for macrophages. In addition, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. Taken together these data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of leukocyte apoptosis.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in cell disassembly and the exposure of ‘flags’ at the dying cell surface that permit recognition and removal by viable cells (phagocytes). Efficient phagocytic removal of dying cells is essential to prevent inflammatory and autoimmune disorders. Relatively little is known of the molecular mechanisms underlying changes at the apoptotic cell surface. We have previously shown that ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) undergoes a change of function as cells die so that it acts as a molecular ‘flag’ to mediate corpse removal. Our work seeks to characterise apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. Here we extend earlier studies to show that apoptotic cell-associated ICAM-3 functions, at least minimally, to tether apoptotic leukocytes to macrophages via an undefined receptor. Whilst CD14 has been suggested as a possible innate immune receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Our data additionally indicate, that during apoptosis, leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates with a reduction in cell volume. This reduction in ICAM-3 is explained by cell surface shedding of microparticles (‘apoptotic bodies’) that contain ICAM-3. Such microparticles, released from apoptotic leukocytes, are strongly chemoattractive for macrophages. In addition, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. Taken together these data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of leukocyte apoptosis.

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes. © 2012 Macmillan Publishers Limited All rights reserved.

Chemosensory response of marine flagellate towards L- and D- dissolved free amino acids generated during heavy grazing on bacteria

This study investigated the generation of dissolved free amino acids (DFAA) by the bacterivorous flagellate Rhynchomonas nasuta when feeding on abundant prey. Specifically, it examined whether this flagellate protist exhibits a chemosensory response towards those amino acids. The concentrations of glycine and the and D-enantiomers of glutamate, serine, threonine, alanine, and leucine were determined in co-cultures of the flagellate and bacteria. Glycine, L- and D-alanine, and L-serine were found to accumulate under these conditions in amounts that correlated positively with flagellate abundance, suggesting that protists are involved in their generation. Investigations of the chemotactic response of young and old foraging protists to the same amino acids, offered in concentrations similar to those previously generated, showed that glycine elicited the strongest attraction in both age groups. Young protists were strongly attracted to all the assayed amino acids, whereas older protists maintained a high level of attraction only for glycine. These results suggest that glycine generated by protists actively grazing in bacterially enriched patches functions as an infochemical, signaling to foraging protists the presence of available prey in the aquatic environment.

Elucidation of the Retinoid Signalling Pathway Involved in Axon Guidance in Lymnaea stagnalis and Xenopus laevis

The vitamin A metabolite, retinoic acid (RA), is known to play a crucial role in several developmental processes including axial patterning and differentiation. More recently, RA has been implicated in the regenerative process acting through its classical signaling pathway, the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), to mediate gene transcription. Moreover, RA has been shown to act as a guidance molecule for growth cones of regenerating motorneurons of the pond snail, Lymnaea stagnalis. Our lab has recently shown that RA can induce this morphological response independent of nuclear transcription, however, the role of the retinoid receptors in RA-induced chemoattraction is still unknown. Here, I show that the retinoid receptors, RXR and RAR, may mediate the growth cones response to the metabolically active retinoic acid isomers, all-trans and 9-cis RA, in Lymnaea stagnalis. Data presented here show that both an RXR and RAR antagonist can block growth cone turning in response to application of both isomers. Because no prior investigations have shown growth cone turning of individual vertebrate neurons, I aimed to show that both retinoic acid isomers were capable of inducing growth cone turning of embryonic spinal cord neurons in the frog, Xenopus laevis. For the first time in Xenopus, I showed that both all-trans and 9-cis RA were able to induce significantly more neurite outgrowth from cultured embryonic spinal cord neurons and induce positive growth cone turning of individual growth cones. In addition, I showed that the presence of the RXR antagonist, HX531, blocked 9-cis RA-induced growth cone turning and the RARβ antagonist, LE135, blocked all-trans RA-induced growth cone turning in this species. Evidence provided here shows for the first time, conservation of retinoic acid-induced growth cone turning in a vertebrate model system. In addition, these data show that the receptors involved in this morphological response may be the same in vertebrates and invertebrates.

Investigating the role of retinoic acid in the vertebrate and invertebrate nervous system

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999.5 B56 D64 2007

The protein LJM 111 from Lutzomyia longipalpis Salivary Gland Extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model

Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF-alpha and IFN-gamma released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry. TNF-alpha and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF-alpha and IFN-gamma. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF-alpha whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils. (C) 2012 Elsevier B.V. All rights reserved.

Gene Expression Profiling in Limb-Girdle Muscular Dystrophy 2A

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in calpain 3 (CAPN3). Calpain 3 plays different roles in muscular cells, but little is known about its functions or in vivo substrates. The aim of this study was to identify the genes showing an altered expression in LGMD2A patients and the possible pathways they are implicated in. Ten muscle samples from LGMD2A patients with in which molecular diagnosis was ascertained were investigated using array technology to analyze gene expression profiling as compared to ten normal muscle samples. Upregulated genes were mostly those related to extracellular matrix (different collagens), cell adhesion (fibronectin), muscle development (myosins and melusin) and signal transduction. It is therefore suggested that different proteins located or participating in the costameric region are implicated in processes regulated by calpain 3 during skeletal muscle development. Genes participating in the ubiquitin proteasome degradation pathway were found to be deregulated in LGMD2A patients, suggesting that regulation of this pathway may be under the control of calpain 3 activity. As frizzled-related protein (FRZB) is upregulated in LGMD2A muscle samples, it could be hypothesized that β-catenin regulation is also altered at the Wnt signaling pathway, leading to an incorrect myogenesis. Conversely, expression of most transcription factor genes was downregulated (MYC, FOS and EGR1). Finally, the upregulation of IL-32 and immunoglobulin genes may induce the eosinophil chemoattraction explaining the inflammatory findings observed in presymptomatic stages. The obtained results try to shed some light on identification of novel therapeutic targets for limb-girdle muscular dystrophies

Sperm capacitation in humans is transient and correlates with chemotactic responsiveness to follicular factors.

In humans, only a small fraction (2-12%) of a sperm population can respond by chemoattraction to follicular factors. This recent finding led to the hypothesis that chemotaxis provides a mechanism for selective recruitment of functionally mature spermatozoa (i.e., of capacitated spermatozoa, which possess the potential to undergo the acrosome reaction and fertilize the egg). This study aimed to examine this possibility. Capacitated spermatozoa were identified by their ability to undergo the acrosome reaction upon stimulation with phorbol 12-myristate 13-acetate. Under capacitating conditions, only a small portion (2-14%) of the spermatozoa were found to be capacitated. The spermatozoa were then separated according to their chemotactic activity, which resulted in a subpopulation enriched with chemotactically responsive spermatozoa and a subpopulation depleted of such spermatozoa. The level of capacitated spermatozoa in the former was approximately 13-fold higher than that in the latter. The capacitated state was temporary (50 min < life span < 240 min), and it was synchronous with the chemotactic activity. A continuous process of replacement of capacitated/chemotactic spermatozoa within a sperm population was observed. Spermatozoa that had stopped being capacitated did not become capacitated again, which indicates that the capacitated state is acquired only once in a sperm's lifetime. A total sperm population depleted of capacitated spermatozoa stopped being chemotactic. When capacitated spermatozoa reappeared, chemotactic activity was restored. These observations suggest that spermatozoa acquire their chemotactic responsiveness as part of the capacitation process and lose this responsiveness when the capacitated state is terminated. We suggest that the role of sperm chemotaxis in sperm-egg interaction in vivo may indeed be selective recruitment of capacitated spermatozoa for fertilizing the egg.

Neogenin interacts with RGMa and Netrin-1 to guide axons within the embryonic vertebrate forebrain

In the embryonic forebrain, pioneer axons establish a simple topography of dorsoventral and longitudinal tracts. The cues used by these axons during the initial formation of the axon scaffold remain largely unknown. We have investigated the axon guidance role of Neogenin, a member of the immunoglobulin (Ig) superfamily that binds to the chemoattractive ligand Netrin-1, as well as to the chemorepulsive ligand repulsive guidance molecule (RGMa). Here, we show strong expression of Neogenin and both of its putative ligands in the developing Xenopus forebrain. Neogenin loss-of-function mutants revealed that this receptor was essential for axon guidance in an early forming dorsoventral brain pathway. Similar mutant phenotypes were also observed following loss of either RGMa or Netrin-1. Simultaneous partial knock downs of these molecules revealed dosage-sensitive interactions and confirmed that these receptors and ligands were acting in the same pathway. The results provide the first evidence that Neogenin acts as an axon guidance molecule in vivo and support a model whereby Neogenin-expressing axons respond to a combination of attractive and repulsive cues as they navigate their ventral trajectory. (c) 2006 Elsevier Inc. All rights reserved.

Characterisation of ICAM-3 and extracellular vesicles in the clearance of apoptotic cells

Apoptotic cell clearance by phagocytes is a vital part of programmed cell death that prevents dying cells from undergoing necrosis which may lead to inflammatory and autoimmune disorders. Apoptotic cells (AC) are removed by phagocytes, in a process that involves 'find me' and 'eat me' signals that facilitate the synapsing and engulfment of cell corpses. Extracellular vesicles (EV) are shed during apoptosis and promote phagocyte recruitment. Binding of AC is achieved by multiple ligand-receptor interactions. One interesting AC associated ligand is ICAM-3, a highly glycosylated adhesion molecule of the IgSF family, expressed on human leukocytes. On viable cells ICAM-3 participates in initiating immune responses, whereas on AC we show it attracts phagocytes through EV and aids in the binding of AC to the phagocytes. This project aims to characterize the role of ICAM-3 and EV in the clearance of AC and to identify the mechanisms that underlie their function in apoptotic cell clearance. Human B cells induced to apoptosis by UV irradiation were observed during their progression from viable to apoptotic via flow cytometry. The involvement of ICAM-3 in mediating interaction between AC and MØ was assessed. The ability of ICAM3 on EV to mediate chemoattraction was observed using chemotaxis assays. Additionally the anti-inflammatory effect was assessed using LPS-induced TNF-α production that suggested it may have anti-inflammatory effects. Future work in this project will assess the role of ICAM3 on EV from different phases of apoptosis to exert functional effects both in vitro and in vivo.