| Neuropeptide S is a 20-amino acid neuropeptide identified with the reverse pharmacology approach by Sato S et al. in 2002. Reinscheid RK et al named this peptide neuropeptide S (NPS) for the amino-terminal residue of the mature peptede in all species is a conserved serine (S). NPS acts by activating its cognate receptor (NPSR). NPS and its receptor construct a novel neuropeptide system, and are widely distributed in central nervous system (CNS) and peripheral tissues. NPS/NPSR system has been shown to regulate a variety of physiological functions, including wakefulness, anxiety, stress, food intake, analgesia, learning and memory, and so on. At present, the studies on the effect of NPS on the immune function was focused on human and murine, as yet we have not found any reports about the gene and functions of NPS and NPSR in pigs. Therefore, small Meishan pig was used to study the distribution and immunoregulatory functions of NPS and its receptor by gene cloning, semiquantitative RT-PCR, immunohistochemistry (IHC), radioimmunoassay (RIA), immunosorbent assay (ELISA), and so on, in order to provide morphological and physiological data for the further studies on the effect of pig immue function by NPS. The results are as follows:1 Gene cloning and expression of NPS and NPSRThe coding sequence (CDs) fragment of NPS and NPSR were first cloned from the brainstem of pigs. The nucleotide sequences and the deduced amino acid sequences of NPS and NPSR were performed with the analyses of bioinformatics, including sequence alignment, homology analysis, chromosomal location, phylogenetic analysis, structure prediction, prediction of transmembrance and phosphorylation site of NPSR. We investigated the expression of NPS and NPSR in pigs by semiquantitative RT-PCR technique. The results are as follows:(1) The CDs fragment length of NPS and NPSR are 183 bp and 443 bp, encoded 59 and 147 animo acids, respectively. The sequences were submitted to the database of GenBank (accession numbers:FJ024707 and FJ444795).(2) With sequence analysis of NPS and NPSR, we found the nucleotide sequences and the encoded amino acid sequences of NPS and NPSR were of high conservation. The CDs nucleotide fragment sequences of the pig were found to be 90.0%,98.3%,88.3% and 86.7% homologous to the sequences of the previously reported human, rabbit, cattle and rat, respectively. The N-terminal amino acid sequence of pig NPS was completely conserved with the known sequences of other species. The gene of NPS and NPSR were located on chromosome 4 and 18, respectively (4q/18q), which was different from the chromosomal location of human NPS and NPSR (10q25/7p14-15). The structure of NPSR is similar to that of the family of G-protein coupled receptors, containing 7 transmembrane domains. The amino acid sequence of NPS contains 2 kinds of phosphorylation sites of protein kinase. The amino acid sequence of NPSR contains 6 kinds of phosphorylation sites of protein kinase.(3) NPS and NPSR mRNA were widely expressed in the CNS and peripheral organs of the pig. High expression level of NPS mRNA was found in the brainstem (medulla oblongata and pons), hypophysis, spleen and lung. Moderate expression of NPS mRNA was seen in the ovary, cerebral cortex, parotid gland, mammary gland, midbrain, thyroid gland, soft palatine tonsil, testis, transverse colon, cerebellum, pancreas, olfactory bulb, duodenum, hippocampus, trachea, heart, esophagus, jejunal lymph node, epididymis, jejunum, thymus, stomach, cecum, hypothalamus, adrenal gland and ileum. Weakly expression level was found in the kidney. High expression level of NPSR mRNA was detected in the hypophysis, spleen and parotid gland. Moderate levels of NPSR mRNA were found in the thyroid gland, testis, trachea, hippocampus, lung, esophagus, duodenum, thymus, heart, kidney, transverse colon, mesenteric lymph node, ileum, mammary gland, cerebral cortex, olfactory bulb, cerebellum, medulla oblongata and ovary. Furthermore, NPSR mRNA was expressed weakly in the midbrain, stomach, pancreas, epididymis and adrenal gland. There was no expression of NPSR mRNA detected in the hypothalamus, pons, jejunum, cecum and soft palatine tonsil. Generally speaking, tissues of high expression level of NPS mRNA was mainly located in CNS, while that of NPSR mRNA was mainly located in peripheral tissues and organs in pigs.2 Distribution of NPS in the pigWe studied the distribution of NPS immunoreactive cells and fibres in the pig by immunohistochemistry. The results are as follows:(1) NPS immunoreactive cells were widely located in the CNS of pigs, and immunoreactive cells typically appeared ovoid, spindle and multipolar with different size. NPS immunoreactive cells were mainly found in the principal sensory trigeminal nucleus (Pr5), locus coeruleus (LC), anterior part of the cuneate nucleus (CUR), area postrema (AP), the ventromedial nucleus (MV), and pulvinar nucleus (PUL) of the thalamus, hypothalamic paraventricular nucleus (PVN), posterior hypothalamic area (HPA), adenohypophysis, granular layer of the cerebellum and subependymal layer (SEL) of olfactory bulb. Moreover, smooth or varicose NPS immunoreactive fibres were widely distributed in the telencephalon, diencephalon, pons and olfactory bulb of the pig, with wide and dense distibution in the hypothalamus.(2) The NPS immunoreactive cells were widely expressed in various tissues and organs of the pig, including the respiratory tract (trachea and lung), alimentary tract(esophagus, stomach and intestines), endocrine organs (thyroid gland, adrenal gland and adenohypophysis), genitourinary tract (kidney, testis and ovary), lymphatic organs and tissues (jejunal lymph node, spleen and diffuse lymphoid tissues), muscles (pectoral muscle and smooth muscle in some organs), mammary gland, et al.(3) There existed both conservatism and partial interspecific difference in the distribution of NPS.3 Effects of NPS on immune function of splenic lymphocytes and pulmonary alveolar macrophages of the pig in vitroIn this study, the expression of NPS and NPSR mRNAs in splenic lymphocytes (SPLs) and pulmonary alveolar macrophages (PAMs) of pigs was detected by RT-PCR. Then the specificity for NPS immunolabeling was confirmed in SPLs and PAMs by immunocytochemistry. We further investigated the effects of NPS (0.01,0.1,1,10,100 and 1000 nM) on the proliferation of SPLs and the phagocytosis of PAMs. Moreover, the effect of NPS on the production of pro-inflammatory and anti-inflammatory cytokines was examined in pig PAMs. The results are as follows:(1) NPS and NPSR mRNA were expressed in SPLs and PAMs of the pig. The results of immunohistochemistry indicated that NPS was detected in the membrane and cytoplasm of SPLs and PAMs.(2) The proliferation of SPLs could be promoted by NPS (0.01-100 nM) treatment in vitro. The proliferation reached the maximum level, when the concentration of NPS was 1 nM. The proliferation of SPLs was also promoted by lowe dose of NPS in combination with PHA or LPS.(3) Incubation with NPS (0.01-1000 nM) resulted in dose-dependent increase in phagocytosis of the fluorescein-labeled E. coli NPS (1-10 nM) could induce maximum phagocytosis.(4) The cytokine production of PAMs could be effected by NPS. It was related with the incubation time, dose of NPS and the synergism of LPS. The pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) secreted by PAMs could be promoted by NPS (0.01-100 nM) in the presence of LPS; The anti-inflammatory cytokines (IL-4 and IL-10) secreted by PAMs could be effected by NPS (0.01-1000 nM). The production of IL-4 could be inhibited by low dose of NPS (0.01-0.1 nM), while promoted by high dose of NPS (1-1000 nM). The opposite effects were found in the production of IL-10. The production of IL-4 (10-1000 nM NPS) and IL-10 (1 nM NPS) could be promoted by NPS in the presence of LPS. The production of pro-inflammatory cytokines could be promoted by NPS in the presence of LPS in a incubation time (4-12 h). The production of IL-1β(incubation for 4 h), IL-6 (incubation for 8-12 h) and TNF-a (incubation for 4-12 h) could also be promoted by NPS in coordination with LPS; The production of IL-4 (4 h) and IL-10 (2 h and 8 h) could be promoted by NPS. The production of NPS IL-4 could be promoted (4 h) or inhibited (8-12 h) by NPS in coordination with LPS.4 Effect of intracerebroventricular injection of NPS on the level of pro-inflammatory and anti-inflammatory cytokines in the serum and immune organs of pigsIntracerebroventricular injection (i.c.v.), RIA, ELISA and semiquantitative RT-PCR were used to study the effect of high dose (30 nmol) and low dose (10 nmol) of NPS on the serum level of pro-inflammatory (IL-1β, IL-6 and TNF-α) and anti-inflammatory cytokines (IL-4 and IL-10) of pigs. The mRNA and protein level of the cytokines in the immune organs (thymus, spleen, jejunal lymph node and soft palatine tonsil) was also detected. The results are as follows:(1) The level of serum pro-inflammatory cytokines could be effected by i.c.v. of NPS. The content of serum IL-6 and TNF-a could be up-regulated by high and low doses of NPS. The content of serum IL-1βcould be up-regulated by high dose of NPS. It could reach the peak level within a short time (10-70 min) after the injection. High level of the pro-inflammatory cytokines maintained a certain time (20-110 min). IL-1βand TNF-αwhich had both returned to the level of control group in 2 h, while IL-6 was kept at a higher level. The features of these effects (acting time, maintain time, peak time and peak level) were correlated with the doses of NPS. This also might be related to the characteristic secretion of cytokines in vivo.(2) The level of serum anti-inflammatory cytokines could be effected by i.c.v. of NPS. The effect of NPS on the serum IL-4 and IL-10 was different from that of pro-inflammatory. The content of serum IL-4 could be down-regulated by low dose of NPS, while being up-regulated by high dose of NPS. The opposite effects were found in the production of serum IL-10. It’s similar to the effect of NPS on the production of anti-inflammatory cytokines of PAMs in vitro. The level of serum IL-4 and IL-10 could return to the level of control group in 2 h. The features of these effects might be correlated with the doses of NPS and the characteristic secretion of cytokines in vivo.(3) The level of pro-inflammatory cytokines in immune organs of pigs could be effected by i.c.v. of NPS. The content of IL-1βand IL-6 in the soft palatine tonsil and spleen could be up-regulated by high dose of NPS, while the content of TNF-a in the jejunal lymph node could be down-regulated. The content of IL-1β, IL-6 and TNF-a in the soft palatine tonsil coule be up-regulated by low dose of NPS. The content of IL-1βand IL-6 in the spleen could also be up-regulated by low dosse of NPS, while being down-regulated in the thymus gland and jejunal lymph node. The effect of NPS on the expression level of pro-inflammatory cytokine mRNA was similar to that of the same protein. Besides that, TNF-a in the soft palatine tonsil was up-regulated by low dose of NPS.(4) The level of anti-inflammatory cytokines in immune organs of pigs could be effected by i.c.v. of NPS. The content of IL-4 in the thymus gland and spleen and IL-10 in the soft palatine tonsil and thymus gland) could be up-regulated by high dose of NPS, while the content of IL-4 in the soft palatine tonsil and IL-10 in the spleen could be down-regulated. The content of IL-4 in the thymus gland could be up-regulated by low dose of NPS, while down-regulated in the jejunal lymph node. The effect of NPS on the expression level of anti-inflammatory cytokine mRNA was similar to that of the same protein. Besides that, the mRNA level of IL-10 in the jejunal lymph node was down-regulated by high and low dose of NPS, and was up-regulated in the soft palatine tonsil by high dose of NPS.5 Effect of intravenous injection of NPS on the level of pro-inflammatory and anti-inflammatory cytokines in the serum and immune organs of pigs We also used the methods mentioned above to investigate the effect of high dose (6μg/kg weight) and low dose (3μg/kg weight) of peripheral NPS on the level of pro-inflammatory (IL-1β, IL-6 and TNF-α) and anti-inflammatory cytokines (IL-4 and IL-10) in the serum and immune organs of pigs. The results are as follows:(1) The level of serum cytokines could be effected by intravenous injection (i.v.) of NPS. The content of serum pro-inflammatory (IL-1βand IL-6) and anti-inflammatory cytokines (IL-4 and IL-10) could be up-regulated by high and low doses of NPS. The content of serum TNF-a could also be up-regulated by high dose of NPS. It could reach peak level within a short time (30-80 min) after the injection. The level of the cytokines could all return to that of control group in 2 h. The effect of i.v. of NPS was similar to that of the i.c.v. of NPS.(2) The level of pro-inflammatory cytokines in the immune organs of pigs could be effected by i.v. of NPS. The effect of NPS on the expression level of pro-inflammatory cytokine mRNA was similar to that of the same protein. The content of IL-1βin the jejunal lymph node could be down-regulated by high dose of NPS. The expression level of IL-6 mRNA in thymus gland and spleen could be down-regulated by high dose of NPS. There was no effect on the content of TNF-a in the jejunal lymph node. The content of IL-1βin the jejunal lymph node could be down-regulated by high dose of NPS. The content of pro-inflammatory cytokines in other immune organs could be up-regulated by high dose of NPS; The content of IL-1βin the soft palatine tonsil and spleen and IL-6 in the jejunal lymph node could be up-regulated by low dose of NPS, while the content of IL-1βand IL-6 in the thymus gland and IL-1βin the jejunal lymph node could be down-regulated. Only the protein level of IL-6 in the oft palatine tonsil and TNF-a in the soft palatine tonsil and spleen could be down-regulated by low dose of NPS.(3) The level of anti-inflammatory cytokines in immune organs of pigs could be effected by i.v. of NPS. The effect of NPS on the expression level of anti-inflammatory cytokine mRNA was not completely consistent with that of the same protein. The content of IL-4 in the thymus gland (protein level) and spleen could be up-regulated by high dose of NPS. The content of IL-4 in the soft palatine tonsil and the expression level of IL-4 mRNA in the jejunal lymph node mRNA could be down-regulated by high dose of NPS. The content of IL-10 in the jejunal lymph node could be up-regulated by high dose of NPS. The content of IL-4 in the jejunal lymph node and IL-10 in the thymus gland could be up-regulated by low dose of NPS. The expression level of IL-4 in the jejunal lymph node and IL-10 in the thymus gland could be up-regulated by low dose of NPS, while the expression level of IL-4 in the soft palatine tonsil and IL-10 in the soft palatine tonsil and spleen could be up-regulated by low dose of NPS.In conclusion, the CDs fragment of NPS and NPSR was first cloned from pig. With the analyses of bioinformatics, the nucleotide sequences and the deduced amino acid sequences of NPS and NPSR were of high conservation. NPS and its receptor are extensive mainly located in lymphatic organs, immune-related tissues and immune cells (SPLs and PAMs). These studies provided morphological data for NPS in regulating immune function of pigs. The in vitro test indicated that the proliferation of SPLs could be effected by NPS, as well as the phagocytosis and cytokine production of PAMs. The in vivo test indicated that the level of cytokines in the serum and immune organs could be effected by the central and peripheral administration of NPS in pigs. These findings have great significance to the further study on the immunoregulatory role of NPS. As an immunomodulatory factor, NPS plays an important role in the cellular and humoral immune. All these studies above provided morphological and physiological data for NPS role in regulating immune function, from mRNA level to protein level, morphology and distribution to function, in vitro to in vivo. |
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