Semaphorins and neuropilins expression in salivary gland tumors.

BACKGROUND: Salivary gland tumors (SGT) account for 3-10% of all head and neck neoplasms, and little is known about their angiogenic properties. Despite semaphorins and neuropilins have been demonstrated to be prognostic determinants in many human cancers, they remain to be investigated in SGT. Therefore, the objective of this study was to analyze the clinical significance of the expression of class 3 semaphorins A (Sema3A) and B (Sema3B) and neuropilins-1 (Np-1) and neuropilins-2 (Np-2), in SGT. METHODS: Two hundred and forty-eight SGT were organized in tissue microarray paraffin blocks and expression of CD34, Sema3A, Sema3B, Np-1, and Np-2 was determined through immunohistochemistry. The immunoreactions were quantified using digital algorithms and the results correlated with clinicopathological parameters. RESULTS: Malignant tumors had an increased vascular density than their benign counterparts and their increased vascular area significantly correlated with recurrences (P < 0.05). Patients older than 40 years and the presence of recurrences determined an inferior survival rate (P = 0.0057 and P = 0.0303, respectively). In normal salivary glands, Np-1 and Np-2 expression was restricted to ductal cells, whereas Sema3A and Sema3B were positive in the serous acinar compartment. Tumors were positive for all markers and the co-expression of Np-1/Np-2 significantly correlated with the presence of paresthesia and advanced stages of the tumors (P = 0.01 and P = 0.04, respectively). CONCLUSION: Sema3A, Sema3B, Np-1, and Np-2 may be involved in the pathogenesis of SGT, but their expression did not present a statistically significant prognostic potential in this study.

Induction of regulatory T cells by a murine ß-defensin.

ß-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine ß-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system.

Expression patterns of Sema3F, PlexinA4, -A3, Neuropilin1 and -2 in the postnatal mouse molar suggest roles in tooth innervation and organogenesis.

OBJECTIVE: Semaphorins form a family of axon wiring molecules but still little is known about their role in tooth formation. A class 3 semaphorin, Semaphorin3F (Sema3F), besides acting as a chemorepellant for different types of axons, controls a variety of non-neuronal developmental processes. MATERIALS AND METHODS: Cellular mRNA expression patterns of Sema3F as well as neuropilin 1 (Npn1), neuropilin 2 (Npn2), plexinA3 and plexinA4 receptors were analyzed by sectional in situ hybridization in the mouse molar tooth during postnatal days 0-7. The expression of the receptors was studied in PN5 trigeminal ganglia. RESULTS: Sema3F, Npn1, -2 and plexinA4 exhibited distinct, spatiotemporally changing expression patterns, whereas plexinA3 was not observed in the tooth germs. Besides being expressed in the base of the dental mesenchyme Sema3F, like plexinA4, Npn1 and -2, was present in the ameloblast cell lineage. Npn1 and Npn2 were additionally seen in the pulp horns and endothelial cells and like PlexinA4 in the developing alveolar bone. Npn1, plexinA3 and -A4 were observed in trigeminal ganglion neurons. CONCLUSIONS: Sema3F may act as a tooth target-derived axonal chemorepellant controlling establishment of the tooth nerve supply. Furthermore, Sema3F, like Npn1, -2 and plexinA4 may serve non-neuronal functions by controlling the development of the ameloblast cell lineage. Moreover, Npn1 and Npn2 may regulate dental vasculogenesis and, together with PlexinA4, alveolar bone formation. Further analyses such as investigation of transgenic mouse models will be required to elucidate in vivo signaling functions of Sema3F and the receptors in odontogenesis.

A role for class 3 semaphorins in prostate cancer.

BACKGROUND: Class 3 semaphorins are secreted proteins that act as guidance cues for migrating cells via their transmembrane receptors plexins and neuropilins. Semaphorins have a role in cancer affecting tumor progression both directly, and indirectly by affecting angiogenesis. METHODS: The expression of semaphorins and their receptors in prostate cancer cell lines and tissue was determined by RT-PCR, Western blotting and immunohistochemistry. The effect of Sema3E on prostate cancer cell lines was determined by adhesion assays and transwell migration assays. RESULTS: Semaphorins and their receptors, plexins and neuropilins, are widely co-expressed in prostate cancer cell lines and tissue with a significant overexpression of Sema3E in tumor tissue. Sema3E affected integrin-mediated adhesion to fibronectin of prostate cancer cells, and inhibited their motility. Expression of Sema3C was upregulated and Sema3A and Sema3E were down regulated in prostate cells by hypoxia, consistent with an additional role for Sema3A and 3E as anti-angiogenic factors in prostate cancer. CONCLUSIONS: Semaphorin 3E is aberrantly expressed in prostate cancer and affects adhesion and motility of prostate cancer cells, indicating a role for the Sema3E/PlexinD1 signaling pathway in prostate cancer and identifying a new possible target for therapy.

VEGF receptors and neuropilins are expressed in the urothelial and neuronal cells in normal mouse urinary bladder and are upregulated in inflammation.

Recent evidence supports a role for vascular endothelium growth factor (VEGF) signaling in bladder inflammation. However, it is not clear what bladder cells are targeted by VEGF. Therefore, we determined the nature of cells responding to VEGF in normal and inflamed bladders by tagging such cells in vivo with a targeted fluorescent tracer, scVEGF/Cy, an engineered single-chain VEGF labeled with Cy5.5 dye, which identifies cells with accessible and functionally active VEGF receptors. Inflammation was induced by intravesical instillation of PAR-activating peptides or BCG. In vivo NIRF imaging with intravenously injected scVEGF/Cy revealed accumulation of the tracer in the control mouse bladder and established that inflammation increased the steady-state levels of tracer uptake. Ex vivo colocalization of Cy5.5 dye revealed that in normal and at a higher level in inflamed bladder, accumulation of scVEGF/Cy occurs in both urothelial and ganglial cells, expressing VEGF receptors VEGFR-1 and VEGFR-2, as well as VEGF coreceptors neuropilins (NRP) NRP1 and NRP2. PCR results indicate that the messages for VEGF-Rs and NRPs are present in the bladder mucosa and ChIP/QPCR analysis indicated that inflammation induced upregulation of genes encoding VEGFRs and NRPs. Our results strongly suggest new and blossoming VEGF-driven processes in bladder urothelial cells and ganglia in the course of inflammation. We expect that molecular imaging of the VEGF pathway in the urinary tract by receptor-mediated cell tagging in vivo will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of bladder-targeting drugs and treatments.

Phenotypic and functional characterization of ultraviolet radiation-induced regulatory T cells.

Sensitization through UV-exposed skin induces regulatory T cells (Treg). In contrast to the classical CD4+CD25+ Treg that act contact dependent, UV-induced Treg (UV-Treg) suppress via IL-10, indicating a distinct subtype that requires further characterization. Depletion studies revealed that UV-Treg express the glucocorticoid-induced TNF family-related receptor (GITR) and the surface molecule neuropilin-1. The injection of T cells from UV-tolerized mice after depletion of UV-Treg into naive recipients enabled a contact hypersensitivity response, indicating that tolerization also induces T effector cells. Adoptive transfer experiments using IL-10-deficient mice indicated that the IL-10 required for suppression is derived from UV-Treg and not from host-derived cells. Activation of UV-Treg is Ag specific, however, once activated suppression is nonspecific (bystander suppression). Hence, speculations exist about the therapeutic potential of Treg generated in response to Ag that are not necessarily the precise Ag driving the pathogenic process. Thus, we studied the consequences of multiple injections of 2,4-dintrofluorobenzene (DNFB)-specific Treg into ears of naive mice followed by multiple DNFB challenges. DNFB-specific Treg were injected once weekly into the left ears of naive mice and DNFB challenge was performed always 24 h later. After three injections, a challenging dose of DNFB was applied on the right ear. This resulted in pronounced ear swelling, indicating that the subsequent boosting of DNFB-specific Treg had caused sensitization of the naive mice against DNFB. These data demonstrate that UV-Treg express GITR and neuropilin-1 and act via bystander suppression. However, constant boosting of Treg with Ag doses in the challenging range results in final sensitization that might limit their therapeutic potential.

Pancreatic carcinoma cells express neuropilins and vascular endothelial growth factor, but not vascular endothelial growth factor receptors.

BACKGROUND: Neuropilins (NRPs) are characterized as coreceptors of vascular endothelial growth factor (VEGF). In the current study, the authors assessed the expression of NRPs, VEGF, and vascular endothelial growth factor receptors (VEGFRs), as well as VEGF-induced cell proliferation, in pancreatic carcinoma cell lines and tissue specimens. METHODS: Human pancreatic carcinoma cell lines (Panc-1 and MIA PaCa-2), normal human pancreatic ductal epithelial cells (HPDE), and human umbilical vein endothelial cells (HUVECs) were cultured. Human pancreatic adenocarcinoma tissue specimens were also studied. Expression levels of NRPs, VEGFRs, and VEGF were determined by real-time polymerase chain reaction analysis and immunostaining. Cell proliferation was examined using a [3H]thymidine incorporation assay. RESULTS: Both NRP-1 and NRP-2 were expressed in Panc-1 cells, HPDE cells, and HUVECs but were expressed minimally in MIA PaCa-2 cells. Panc-1 expressed 30 times more NRP-1 mRNA than NRP-2 mRNA. NRP-1 levels in Panc-1 cells were 5.3 times higher than in HPDE cells but were similar to NRP-1 levels in HUVECs. NRP-2 levels in Panc-1 cells were similar to NRP-2 levels in HPDE cells but lower than NRP-2 levels in HUVECs. Expression of all three VEGFRs was observed only in HUVECs. However, VEGF mRNA was detected in all cell types except for HUVECs. NRP-1 immunoreactivity levels were much higher than NRP-2 immunoreactivity levels in Panc-1 and human pancreatic adenocarcinoma tissue specimens, whereas VEGFRs were not detected in either of these two settings. In response to VEGF165, [3H]thymidine incorporation in Panc-1 cells increased significantly (by 61%; P < 0.01). A monoclonal antibody against human NRP-1 significantly blocked VEGF-induced cell proliferation in Panc-1 cells. CONCLUSIONS: The pancreatic carcinoma cell line Panc-1 and adenocarcinoma tissue specimens expressed high levels of NRP-1 and VEGF, but not VEGFRs, and exogenous VEGF significantly increased NRP-1-mediated, but not VEGFR-mediated, Panc-1 cell proliferation. These data suggested that NRP-1 may be involved in the pathogenesis of pancreatic carcinoma.

Repulsion and attraction of axons by semaphorin3D are mediated by different neuropilins in vivo.

Class 3 semaphorins are known to repel and/or sometimes attract axons; however, their role in guiding developing axons in the CNS in vivo is still essentially unknown. We investigated the role of Semaphorin3D (Sema3D) in the formation of the early axon pathways in the zebrafish CNS. Morpholino knock-down shows that Sema3D is essential for the correct formation of two early axon pathways. Sema3D appears to guide axons of the nucleus of the medial longitudinal fasciculus (nucMLF) by repulsion and modulation of fasciculation. In contrast, Sema3D appears to be attractive to telencephalic neurons that form the anterior commissure (AC). Knock-down of Neuropilin-1A (Npn-1A) phenocopied the effects of Sema3D knock-down on the nucMLF axons, and knock-down of either Npn-1A or Npn-2B phenocopied the defects of the AC. Furthermore, simultaneous partial knock-down experiments demonstrated genetic interactions among Sema3D, Npn-1A, and Npn-2B. Together, these data support the hypothesis that Sema3D may act as a repellent through receptors containing Npn-1A and as an attractant via receptors containing Npn-1A and Npn-2B.

Cloning and embryonic expression of zebrafish neuropilin genes.

Neuropilin (Nrp), a cell surface receptor for class 3 semaphorins and for certain heparin forms of vascular endothelial growth factors, functions in many biological processes including axon guidance, neural cell migration and angiogenesis in the development of the nervous system and the cardiovascular system. To understand the role of neuropilins in zebrafish embryogenesis, we have cloned three zebrafish neuropilin homologues, nrp1b, nrp2a and nrp2b. Based on synteny, zebrafish nrp1b and the previously cloned nrp1a are orthologous to human nrp1, and zebrafish nrp2a and 2b orthologous to human nrp2. We have characterized the expression patterns of these four zebrafish neuropilin genes in wild type embryos from the beginning of somitogenesis to 48 h post-fertilization. Zebrafish nrp1a is expressed in the neural tube including telencephalon, epithalamus, cells along the axonal trajectory of the posterior commissure and the medial longitudinal fascicle, hindbrain neurons, vagus motor neurons and spinal motoneurons. Zebrafish nrp1b is expressed in the nose, the cranial neural crest cell (NCC) derived tissue underlying the hypothalamus, endothelial precursors and the trunk and tail vasculature. Zebrafish nrp2a is expressed in telencephalon, anterior pituitary, oculomotor and trochlear motor neurons, cells along the supra-optic and posterior commissures, hindbrain rhombomere 1, hindbrain neurons, cranial NCCs and sclerotome. Zebrafish nrp2b is expressed in telencephalon, thalamus, hypothalamus, epiphysis, cells along the anterior and posterior commissures, post-optic and supra-optic commissures and the olfactory axonal trajectory, hindbrain neurons, cranial NCCs, somites and spinal cord neurons.

Cranial expression of class 3 secreted semaphorins and their neuropilin receptors.

The semaphorin family of chemorepellents and their receptors the neuropilins are implicated in a variety of cellular processes, including axon guidance and cell migration. Semaphorins may bind more than one neuropilin or a heterodimer of both, thus a detailed knowledge of their expression patterns may reveal possible cases of redundancy or mutual antagonism. To assess their involvement in cranial development, we cloned fragments of the chick orthologues of Sema3B and Sema3F. We then carried out mRNA in situ hybridisation of all six class 3 semaphorins and both neuropilins in the embryonic chick head. We present evidence for spatiotemporal regulation of these molecules in the brainstem and developing head, including the eye, ear, and branchial arches. These expression patterns provide a basis for functional analysis of semaphorins and neuropilins in the development of axon projections and the morphogenesis of cranial structures.

From the discovery of neuropilin to the determination of its adhesion sites.

Neuropilin (NRP) and plexin (Plex) that are now known to be semaphorin receptors were initially identified as antigens for monoclonal antibodies (MAbs) that bound to particular neuropiles and plexiform layers of the Xenopus tadpole optic tectum, several years before the discovery of semaphorin. The extracellular segment of the NRP protein is a mosaic of 3 functionally different protein motifs that are thought to be involved in molecular and/or cellular interactions, suggesting that NRP serves in a various cell-cell interaction by binding a variety of molecules. The first identified function of NRP was the cell adhesion activity; Cell reaggregation study using NRP-expressing cell lines revealed that NRP can mediate cell adhesion via heterophilic molecular interaction. Later, NRP was shown to bind semaphorins and vascular endothelial growth factor (VEGF). It was also shown that NRP makes receptor complexes with Plex to propagate semaphorin signals.

The role of neuropilin in vascular and tumor biology.

Neuropilin-1 (NRP1) and NRP2 are related transmembrane receptors that function as mediators of neuronal guidance and angiogenesis. NRPs bind members of the class 3 semaphorin family, regulators of neuronal guidance, and of the vascular endothelial growth factor (VEGF) family of angiogenesis factors. There is substantial evidence that NRPs serve as mediators of developmental and tumor angiogenesis. NRPs are expressed in endothelial cells (EC) and bind VEGF165. NRP1 is a co-receptor for VEGF receptor-2 (VEGFR2) that enhances the binding of VEGF165 to VEGFR2 and VEGF165-mediated chemotaxis. NRP1 expression is regulated in EC by tumor necrosis factor-alpha, the transcription factors dHAND and Ets-1, and vascular injury. During avian blood vessel development NRP1 is expressed only in arteries whereas NRP2 is expressed in veins. Transgenic mouse models demonstrate that NRP1 plays a critical role in embryonic vascular development. Overexpression of NRP1 results in the formation of excess capillaries and hemorrhaging. NRP1 knockouts have defects in yolk sac, embryo and neuronal vascularization, and in development of large vessels in the heart. Tumor cells express NRPs and bind VEGF165. NRP1 upregulation is positively correlated with the progression of various tumors. Overexpression of NRPI in rat tumor cells results in enlarged tumors and substantially enhanced tumor angiogenesis. On the other hand, soluble NRP1 (sNRP1) is an antagonist of tumor angiogenesis. Semaphorin 3A binds to EC and tumor cells. It also inhibits EC motility and capillary sprouting in vitro. VEGF165 and Sema3A are competitive inhibitors for NRP1 mediated functions in EC and neurons. These results suggest that NRP1 is a novel regulator of the vascular system.