Ontogenic development of the secretory immune system in human fetal salivary glands.
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Immunopathological analysis was made of phenotypic change in a recurrent parotid gland adenocarcinoma occurring in a patient with a long clinical course of 30 years or more. At the first and second operations, in 1959 and 1978, the resected parotid gland tumors were diagnosed histopathologically as acinic cell carcinoma. However, 11 years after the second operation, in 1989, the resected recurrent tumor showed a microscopically phenotypic change towards adenocarcinoma with typical tubular arrangement. At the last operation in 1991, histopathological examination of the tumor revealed adenocarcinoma with diffuse oncocytic change in association with cervical lymph node metastasis. These findings suggest that phenotypic change may occur in vivo among human neoplasms during a long period, which may be related to the cytodifferentiation in the salivary gland tumor.
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Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. Chromogranin A is the major soluble core component in secretory vesicles. Since chromogranin A is secreted along with catecholamines, we asked whether PACAP regulates expression of the chromogranin A gene in PC12 rat chromaffin cells, so as to resynthesize the just-secreted protein, and whether such biosynthetic regulation is coupled mechanistically to catecholamine secretion. PACAP activated the endogenous chromogranin A gene by four- to fivefold. Proportional results (seven- to eightfold activation) were obtained with a transfected 1,200-bp mouse chromogranin A promoter/luciferase reporter construct. A series of chromogranin A promoter 5' deletion mutant/luciferase reporter constructs narrowed down the PACAP response element to a proximal region containing the cAMP response element (CRE box), at (-71 bp)5'-TGACGTAA-3'(-64 bp). Site-directed point mutations of the CRE site suppressed PACAP-induced trans-activation of the promoter. Thus, the proximal CRE box is entirely necessary for the chromogranin A promoter response to PACAP. Transfer of the CRE box to a neutral, heterologous promoter also conferred activation by PACAP, suggesting that the CRE domain is also sufficient to mediate the transcriptional response to PACAP. Expression of a dominant-negative mutant (KCREB) of the CRE-binding factor CREB markedly diminished trans-activation of the chromogranin A promoter by PACAP. Cotransfection of expression plasmids encoding the protein kinase A inhibitor, or an inactive protein kinase A (PKA) catalytic beta subunit, inhibited both forskolin and PACAP activation of chromogranin A transcription, revealing that PACAP-induced trans-activation is highly dependent on PKA. By contrast, inhibition of protein kinase C (by chronic exposure to phorbol ester) had no effect on transcriptional activation by PACAP. The potent PACAP/vasoactive intestinal peptide (VIP) type I receptor antagonist PACAP6-38 impaired both chromogranin A transcription or catecholamine secretion triggered by PACAP38, while the PACAP/VIP type II receptor antagonist (p-Chloro-D-Phe6, Leu17)-VIP had little or no ability to antagonize the PACAP38 effect. The agonist VIP was approximately 100- to 1,000-fold less potent than PACAP in stimulating either secretion or transcription. Thus, PACAP-evoked chromogranin A transcription and catecholamine secretion are likely mediated by the PACAP/VIP type I receptor isoform. Although the calcium channel antagonists Zn2+ (100 microM), nifedipine (10 microM), or ruthenium red (10 microM), or the cytosolic calcium chelator BAPTA-AM (50 microM) each strongly impaired PACAP-induced secretion, transcriptional activation of chromogranin A remained unaltered. Therefore, we propose that PACAP signals to chromogranin A transcription through the CRE in cis, and through PKA and CREB in trans. By contrast, a pathway involving cytosolic calcium entry through L-type voltage-dependent channels is required for PACAP to evoke catecholamine secretion.
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The ontogeny of the mucosal immune system as it relates to the development of lymphoid tissue in the respiratory tract and the gastrointestinal tract has been studied quite extensively over the past few years. It is apparent now that the bronchus-associated lymphoid tissue and gut-associated lymphoid tissue are the major sources of immunocompetent precursor B lymphocytes. After the induction of antigens in the respiratory tract or the gastrointestinal tract, precursor lymphoid cells in these sites are preferentially activated to undergo significant proliferation. Such antigen-sensitized cells eventually migrate to other mucosa sites, such as mammary glands, genital tract, conjuctiva, etc. Recent evidence has suggested that the immunocompetent tissue observed in the middle ear cleft during otitis media with effusion may function as an extension of the mucosal immune system in the upper respiratory tract. The implications of these observations relative to middle ear disease are discussed.
Cloning technologies have established unambiguously that amelogenins always seem larger in molecular weight (Mr) by gel electrophoresis (SDS-PAGE) than by mass spectrometry (MS). This has caused many problems relating cloned versions of amelogenin to proteins actually secreted by ameloblasts in vivo. In this study, discrete protein fractions at 31-20 kDa (Mr(SDS)) were prepared from freeze-dried rat incisor enamel by techniques optimized for preserving protein integrity. N-terminal sequence and amino acid compositional analyses indicated that the major protein forming these fractions was amelogenin. As expected, the molecular weights estimated by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS were significantly less than their apparent molecular weights estimated by SDS-PAGE. Plots of Mr(SDS) vs. Mr(MS) for all fractions showed high linear correlation (r = 0.992). Analysis of MS data further indicated that the major protein in the 27-kDa fraction corresponded to the R180 secretory isoform of rat amelogenin, whereas some minor proteins in the 23-kDa fraction likely corresponded to a R156 secretory isoform. This was in contrast to major proteins forming the 25-, 24-, and 23-kDa fractions (Mr(SDS)), which seemed to represent proteolytic fragments of R180 progressively altered at the P169-A170, P164-L165, and F151-S152 C-terminal cleavage sites, respectively. Proteins in the 20-kDa fraction (Mr(SDS)) most closely matched by ESI-MS fragments of the R156 secretory isoform that were C-terminally-modified at the equivalent P164-L165 site.
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The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which thrombocytopenia is associated with increased platelet size and decreased alpha-granule content. This report describes 3 new pediatric cases presenting with the classical platelet abnormalities of GPS within one family with normal parents. Examination of blood smears of the 3 patients demonstrated not only gray platelets, but also gray polymorphonuclear neutrophils (PMNs) with decreased or abnormally distributed components of secretory compartments (alkaline phosphatase, CD35, CD11b/CD18). Secondary granules were also decreased in number as assayed by immunoelectron microscopy. These data confirm that the secretory compartments in neutrophils were also deficient in this family. Megakaryocytes (MKs) were cultured from the peripheral blood CD34+ cells of the 3 patients for 14 days, in the presence of thrombopoietin and processed for immunoelectron microscopy. Although von Willebrand factor (vWF) was virtually undetectable in platelets, vWF immunolabeling was conspicuous in cultured maturing MKs, particularly within Golgi saccules, but instead of being packaged in alpha-granules, it was released into the demarcation membrane system. In contrast, P-selectin followed a more classical pathway. Double-labeling experiments confirmed that vWF was following an intracellular pathway distinct from the one of P-selectin. In these 3 new cases of GPS, the MKs appeared to abnormally process vWF, with secretion into the extracellular space instead of normal alpha-granule packaging. Furthermore, the secretory compartment of another blood cell line, the neutrophil, was also affected in this family of GPS.
Salivary antibodies were studied in 112 infants between 1 day and 8 yr of life. SIgA anticasein was present from birth in breast-fed and bottle-fed infants. Bottle-feeding resulted in significantly higher concentrations of SIgA anticasein at 3 wk to 3 months of life as compared to breast-feeding. Salivary anticasein declined toward the end of the 1st yr and was present in less than half of the children older than 1 yr. Salivary anti-beta-lactoglobulin was also present at birth in some infants. Levels increased slightly over the following 3 months but remained low. Only a minority of older children had this antibody in their saliva.
Anterior pituitary cells and other endocrine cells which synthesize and release protein hormones share the cell surface ganglioside antigens A2B5 and 3G5. In addition, these peptide hormone-producing cells contain chromogranin, a major protein component of secretory vesicles. Since human decidual cells synthesize and release a PRL that has chemical and biological properties indistinguishable from those of pituitary PRL, we examined by immunochemical techniques whether decidual cells also contain cell surface antigens A2B5, 3G5, and chromogranin. In these studies, human decidual tissue from three pregnancies and human and rat pituitary tissues were processed and stained with mouse monoclonal antibodies to A2B5, 3G5, and chromogranin by indirect immunofluorescence using fluorescein-conjugated sheep antimouse gamma-globulin. In all instances, the pituitary and pancreatic tissue stained strongly with the monoclonal antibodies to the cell surface antigens, but the decidual tissues from the three pregnancies did not. The pituitary and pancreatic cells also stained strongly with the monoclonal antibody (LK 2H10) to chromogranin, but the decidual tissues had no reactivity. In control experiments, the decidual tissues stained strongly with a monoclonal antibody to HLA antigens, and none of the tissues stained with nonimmune gamma-globulin. These results strongly suggest that the PRL-producing cells of human decidua and pituitary have different cell surface antigens. The observation that decidual cells do not contain chromogranin is consistent with previous biochemical studies suggesting that decidual PRL is not stored in secretory granules. The differences in the subcellular localization of PRL in decidual and pituitary tissues may explain, in part, the differences in the regulation of PRL release from these tissues.
As a model system to study protein traffic in polarized epithelial cells, we have used the polymeric immunoglobulin receptor. This receptor travels first to the basolateral surface, where it can bind polymeric IgA or IgM. The receptor is then endocytosed and delivered to endosomes. The receptor is sorted into transcytotic vesicles, which are exocytosed at the apical surface. The 103-amino acid cytoplasmic domain of the receptor contains several sorting signals. The 17 residues closest to the membrane are an autonomous signal that is necessary and sufficient for basolateral sorting. For rapid endocytosis there are two independent signals, both of which contain critical tyrosine residues. Finally, transcytosis is signaled by phosphorylation of a particular serine.
Proteins of human cervical mucus were quantified on polyacrylamide gels and their concentrations relative to the mucous glycoprotein were calculated. In contrast with the cyclic variation to be expected in the ratio between albumin and the mucous glycoprotein, we found a constant ratio between IgA-related secretory piece and mucous glycoprotein. Secretory piece appeared to be an important protein in the mucus with respect to its concentration. No significant differences in the amount of mucus, its cyclic variation, protein concentration and composition could be found when normal mucus was compared from women sterilized by tubal ligation.