Regulated endocrine-specific protein 18 (RESP18) is localized to and regulated in A-like cells and G-cells in rat stomach

The regulated endocrine-specific protein 18 (RESP18) has previously been localized to different endocrine cells and neurons, in particular the pituitary gland and hypothalamus. It is found in the lumen of the endoplasmic reticulum and is degraded at the post-ER pre-Golgi compartment, and a role in processing of secreted peptides has been hypothesized. The present study examines localization of RESP18 in the gastrointestinal mucosa of rats by immunohistochemistry, and expression and regulation in response to hypergastrinemia induced by acid inhibition (pantoprazole), gastrin antagonism (YF476), fasting-refeeding and octreotide by mRNA measurements. RESP18 was mainly found in the gastric mucosa, but could also be detected in a few, scattered cells in the lower small intestine and in colon. In the antral mucosa, all RESP18 immunoreactivity was localized to ghrelin-producing A-like cells and gastrin-producing G-cells. In the corpus mucosa, a significant fraction, but not all of the RESP18 immunoreactive cells, were A-like cells. In both antrum and corpus, Resp18 mRNA seemed to vary similarly with the activation of the A-like cells, and in the antrum also with stimulation of the G-cells. This study demonstrates, for the first time, the localization of RESP18 to specific neuroendocrine cells of the gastrointestinal mucosa and that it seems to be regulated synchronously with the peptides secreted from these cells. This suggests that Resp18 may indeed have a functional role in the synthesis or storage of these gastrointestinal peptides.     

Constitutive expression of IL-18 binding protein in murine testicular tissues and cells

In the present study, we examined the cellular origin and the expression levels of interleukin-18 binding protein (IL-18 BP), during normal maturation of murine testis. Immunohistochemical staining of testicular tissues from sexually mature mice, shows that testicular germ cells, at different stages of differentiation, express IL-18 BP. Leydig cells/interstitial cells and peritubular cells express higher levels of IL-18 BP, as compared to other testicular cells. The levels of IL-18 BP were similar in testicular tissues and homogenates from sexually immature and mature mice, as examined by western blot, ELISA and real time PCR analysis. Our results demonstrate, for the first time, the expression of IL-18 BP in murine testicular tissues and cells. Together with our previous studies, which showed over-expression of IL-18 in testicular tissues of immature mice as compared with mature mice, these results may indicate a role for IL-18 in testicular development, and also in the regulation of testicular functions under physiological conditions.     

Over-expression of IL-18, ICE and IL-18 R in testicular tissue from sexually immature as compared to mature mice

In this study we examined the cellular origin and the expression levels of interleukin-18 (IL-18), IL-18 receptor (IL-18R) and IL-1beta-converting enzyme (ICE), which activates pro-IL-18, during normal maturation of murine testis. The levels of IL-18, IL-18R and ICE were significantly higher in testicular tissues and homogenates (but not in the spleen or liver) from sexually immature than mature mice. Immunohistochemical staining of testicular tissues from sexually immature and mature mice shows that testicular germ cells and Leydig cells/interstitial cells express higher levels of IL-18, as compared to other testicular cells. Peritubular cells of sexually immature and mature mice also expressed IL-18. Our results demonstrate, for the first time, over-expression of the IL-18 family in testicular tissues of sexually immature mice, as compared to mature mice, as well as the expression of IL-18 in the different stages of differentiation of testicular germ cells. Thus, our results may indicate involvement of the endocrine system (gonadotropins and testosterone) in the regulation of the testicular IL-18 family, which could be involved in the regulation of testicular functions, development and spermatogenesis under physiological conditions.     

Splice variants of the OB receptor gene are differentially expressed in brain and peripheral tissues of mice

A high affinity receptor for OB protein was recently cloned from the choroid plexus of mice. At least six alternatively spliced forms of the OB receptor (OB-R) gene have been described, all of which encode proteins containing the OB-R extracellular domain. One splice variant encodes a receptor with a long intracellular domain, OB-RL, that has been implicated in OB-R signaling. Here, we have used in situ hybridization to examine the localization of OB-R splice variants in brain and peripheral tissues of adult and newborn mice. Using a probe hybridizing with all known splice variants, we confirmed that OB-R mRNA was widely distributed in the adult tissues. In the CNS, choroid plexus was the major site of expression. We now demonstrate that OB-R mRNA is expressed in peripheral tissues; primarily associated with connective tissues. In addition, OB-R mRNA was detected at higher levels in peripheral tissues of newborn mice than in adult mice. With a probe specific for OB-RL, we confirmed that high mRNA expression was detected in hypothalamic nuclei, while low levels were observed in choroid plexus. We now report that in peripheral tissues of adult mice, OB-RL mRNA expression was either very low or undetectable. In newborn mice, the pattern of OB-RL message expression in the CNS was similar to that of adult mice, while bone was the site of highest OB-RL message expression in the peripheral tissue. These data suggest different biological roles for OB-R splice variants encoding the short and long forms of OB-R. The localization of OB-RL to hypothalamic nuclei supports the idea that OB-RL is the brain receptor that mediates OB protein signaling and actions. In addition, the expression of OB-R message in newborn mice also suggests a biological role of OB-R during development in mice.     

Dual specificity phosphatases 18 and 21 target to opposing sides of the mitochondrial inner membrane

Although large-scale approaches have identified numerous mitochondrial phosphoproteins, little is known about the mitochondrial kinases and phosphatases that regulate these phosphoproteins. Here, we identify two members of the atypical dual specificity phosphatases (DSP), DSP18 and DSP21, that are localized in mitochondria. Although DSP18 is widely expressed in several mammalian tissues, DSP21 is selectively expressed in the testes. We demonstrate that DSP18 and DSP21 are targeted to mitochondria by cryptic internal localization signals. Subfractionation of mitochondria demonstrated that DSP18 is located in the intermembrane space as a peripheral membrane protein of the inner membrane. In contrast, subfractionation of rat testis mitochondria revealed DSP21 is localized to the matrix as a peripheral membrane protein of the inner membrane. Moreover, we demonstrate that a previously reported substrate for DSP18, the stress-activated protein kinase, does not localize to mitochondria in several different tissues, making it an unlikely substrate for DSP18. Finally, we show that induction of apoptosis by treatment with staurosporine causes translocation of DSP18 from the intermembrane space into the cytosol similar to other apoptogenic factors such as cytochrome c. This work rigorously demonstrates the unique location of two highly similar DSPs on opposing sides of the mitochondrial inner membrane.     

Up-regulation of IL-18BP,but not IL-18 mRNA in rat liver by LPS

Interleukin (IL)-18 is a pro-inflammatory cytokine that plays a critical role in inflammation leading to liver damage, through promotion of Fas-mediated apoptosis. Inhibition of IL-18 activity protects against LPS-induced lethality in mice and against liver damage induced by LPS after sensitisation of mice with Proprionibacterium acnes. A specific, potent, endogenous inhibitor of IL-18 (IL-18BP) has been identified in mice and humans, and IL-18BP mRNA is expressed constitutively in liver. The objectives of this study were to compare changes in IL-1beta and IL-18 mRNA expression in the liver of rats in response to peripheral injection of LPS, using real-time PCR, and also to investigate whether IL-18BP mRNA expression is affected by this treatment. LPS rapidly up-regulated IL-1beta mRNA expression, but IL-18 mRNA expression was unaffected by LPS treatment. Unlike IL-18, IL-18BP mRNA was up-regulated dramatically by approximately 12-fold above nai;ve levels, peaking 3 h after LPS injection. This ability of LPS to up-regulate expression of the endogenous IL-18 inhibitor may indicate a mechanism by which the inflammatory response to LPS is regulated.     

Altered interleukin-1 receptor antagonist and interleukin-18 mRNA expression in myocardial tissues of patients with dilatated cardiomyopathy

Interleukin-1 (IL-1) is a potent regulator of cell proliferation, inflammation, and contraction of cardiovascular cells. It has been proposed that the IL-1/IL-1ra (IL-1 receptor antagonist) ratio influences these functions. Other members of the IL-1 family and the related caspase-1 also contribute to regulation of IL-1-mediated functions. We determined the mRNA expression of caspase-1, caspase-3, IL-1alpha , IL-1beta , IL-18, IL-1 receptor type I (IL-1-RI), and IL-1ra in left ventricle tissue of hearts from patients with ischemic or dilated cardiomyopathy (ICM or DCM) and in control tissues from unused donor transplant hearts in RT-PCR experiments. We show that the expression of caspase-1, caspase-3, IL-1beta , and IL-1-RI mRNA was not different between patients and control tissues. Furthermore, we did not find detectable amounts of IL-1alpha mRNA in any of these adult myocardial tissues. On the other hand, expression of IL-18 RNA was lower in myocardium of both patient groups compared with control hearts. Furthermore, IL-1ra mRNA expression was significantly lower in tissues of DCM patients compared with ICM patients and controls. This was in line with a trend towards lower IL-1ra protein levels in myocardial tissues of DCM patients. In contrast with the adult tissues discussed above, which did not express IL-1alpha mRNA, commercially available human fetal tissue expressed IL-1alpha mRNA. On the other hand IL-1beta mRNA was present in fetal and in adult human heart tissue. Our data provide evidence for an altered ratio of IL-1/IL-1ra in DCM patients. This dysregulation may contribute to pathogenesis and/or progression of heart disease by modulating the otherwise balanced IL-1-mediated functions in cardiovascular cells.     

Interleukin-18 (IL-18) mRNA expression and localization of IL-18 mRNA-expressing cells in the mouse uterus

Interleukin-18 (IL-18) belongs to the interleukin-1 family and was identified as an interferon-gamma inducing factor. We investigated IL-18 mRNA-expressing cells in the mouse uterus. By RNase protection assay, IL-18 mRNA and alpha subunit of IL-18 receptor mRNA were detected in the uterus. In the uterus, IL-18 mRNA levels increased during sexual maturation. In situ hybridization analysis demonstrated IL-18 mRNA-expressing cells in the mouse uterus of different ages. At 21 days of age, IL-18 mRNA-expressing cells were detected in the luminal epithelial cells and stromal cells although the IL-18 mRNA signal was weak. At 42 days of age, IL-18 mRNA signal was mainly detected in the stromal cells located near the myometrium, and in some of the luminal and glandular epithelial cells. In the uterus of 63-day-old adult mice, a strong hybridization signal for IL-18 mRNA was detected at estrus, but was weak at diestrus. IL-18 mRNA was mainly detected in the glandular epithelial cells and stromal cells. The effect of estradiol-17beta (E(2)) on IL-18 mRNA-expressing cells in the uterus was examined in ovariectomized mice. In oil-treated mice IL-18 mRNA signal was localized in luminal epithelial cells and stromal cells, while in E(2)-treated mice IL-18 mRNA signal was localized in stromal cells alone. These results suggest that the mouse uterus has an IL-18 system, and IL-18 exerts a physiological role within the uterus in a paracrine manner, and that IL-18 gene expression is regulated by estrogen.     

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.     

Contribution of Langerhans cell-derived IL-18 to contact hypersensitivity

The epidermal Langerhans cells (LC), a member of the dendritic cell family, and the LC-derived cytokine IL-12 play a pivotal role in the initiation of contact hypersensitivity (CHS), a Th1 immune response in the skin. Because IL-18, another LC-derived cytokine, shares functional and biological properties with IL-12, we examined a potential role for IL-18 in CHS initiation. Our studies demonstrated that during the induction phase of murine CHS, IL-18 mRNA was significantly up-regulated in the skin-draining lymph nodes (LN). Migratory hapten-modified LC in LN expressed high levels of IL-18 mRNA and secreted functional IL-18 protein. LN cells produced significant amounts of IFN-gamma following in vitro IL-12 stimulation, which could be partially blocked by anti-IL-18 Ab, suggesting a synergistic role for endogenous IL-18 in IFN-gamma production by LN cells. Because mature IL-18 requires cleavage of immature precursors by caspase-1, we further examined IL-12-induced IFN-gamma production in caspase-1(-/-) LN cells. An impaired IFN-gamma production was seen in caspase-1(-/-) LN cells, which could be restored by addition of exogenous IL-18, supporting a role for caspase-1-cleaved, mature IL-18 in IFN-gamma production. Finally, in vivo studies showed that CHS responses were significantly inhibited in mice treated with neutralizing IL-18 Ab as well as in caspase-1(-/-) mice deficient in mature IL-18, indicating functional relevance for IL-18 in CHS. Taken together, our studies demonstrate that LC-derived IL-18 significantly contributes to CHS initiation.     

Interferon-gamma mediates gene expression of IL-18 binding protein in nonleukocytic cells

Interleukin-18 (IL-18) binding protein is a soluble decoy receptor for IL-18 which efficiently antagonizes biological functions of IL-18 in vitro and in vivo. Since regulation of IL-18 activity likely contributes to the pathogenesis of inflammatory diseases as well as malignancies, we investigated gene expression of IL-18 binding protein (IL-18BP) in different human cell systems, namely in the keratinocyte cell line HaCaT, in the colon carcinoma cell line DLD-1, and in primary renal mesangial cells. In unstimulated cells only minute amounts of mRNA coding for IL-18 binding protein were detectable. However, in all three cell types gene expression was markedly upregulated by interferon-gamma (IFN-gamma). IL-18 is recognized as a pivotal mediator of IFN-gamma production. Therefore, the present data imply that activity of IL-18 is modulated by a negative feedback mechanism which is mediated by IFN-gamma-induced IL-18 binding protein.     

IL-18 inhibits diabetes development in nonobese diabetic mice by counterregulation of Th1-dependent destructive insulitis.

The development of type 1 diabetes in animal models is T cell and macrophage dependent. Islet inflammation begins as peripheral benign Th2 type insulitis and progresses to destructive Th1 type insulitis, which is driven by the innate immune system via secretion of IL-12 and IL-18. We now report that daily application of IL-18 to diabetes-prone female nonobese diabetic mice, starting at 10 wk of age, suppresses diabetes development (p < 0.001, 65% in sham-treated animals vs 33% in IL-18-treated animals by 140 days of age). In IL-18-treated animals, we detected significantly lower intraislet infiltration (p < 0.05) and concomitantly an impaired progression from Th2 insulitis to Th1-dependent insulitis, as evidenced from IFN-gamma and IL-10 mRNA levels in tissue. The deficient progression was probably due to lesser mRNA expression of the Th1 driving cytokines IL-12 and IL-18 by the innate immune system (p < 0.05). Furthermore, the mRNA expression of inducible NO synthase, a marker of destructive insulitis, was also not up-regulated in the IL-18-treated group. IL-18 did not exert its effect at the levels of islet cells. Cultivation of islets with IL-18 affected NO production or mitochondrial activity and did not protect from the toxicity mediated by IL-1beta, TNF-alpha, and IFN-gamma. In conclusion, we show for the first time that administration of IL-18, a mediator of the innate immune system, suppresses autoimmune diabetes in nonobese diabetic mice by targeting the Th1/Th2 balance of inflammatory immune reactivity in the pancreas.     

Systemic administration of IL-18 promotes diabetes development in young nonobese diabetic mice

IL-18 is now identified as a pleiotropic cytokine that acts as a cofactor for both Th1 and Th2 cell development. Type 1 diabetes is considered a Th1-type autoimmune disease, and to date, the suppressive effect of exogenous IL-18 on the development of diabetes has been reported in 10-wk-old nonobese diabetic (NOD) mice. In the present study we administered exogenous IL-18 systemically in 4-wk-old NOD mice using i.m. injection of the IL-18 expression plasmid DNA (pCAGGS-IL-18) with electroporation. Contrary to previous reports, the incidence of diabetes development was significantly increased in NOD mice injected with pCAGGS-IL-18 compared with that in control mice. Systemic and pancreatic cytokine profiles deviated to a Th1-dominant state, and the the frequency of glutamic acid decarboxylase-reactive IFN-gamma-producing CD4(+) cells was also high in the IL-18 group. Moreover, it was suggested that the promoting effect of IL-18 might be associated with increased peripheral IL-12, CD86, and pancreatic IFN-inducible protein-10 mRNA expression levels. In conclusion, we demonstrate here that IL-18 plays a promoting role as an enhancer of Th1-type immune responses in diabetes development early in the spontaneous disease process, which may contribute to elucidating the pathogenesis of type 1 diabetes.