Sensory and autonomic innervation of non-hairy and hairy human skin

Summary Non-hairy and hairy human skin were investigated with the use of the indirect immunohistochemical technique employing antisera to different neuronal and non-neuronal structural proteins and neurotransmitter candidates. Fibers immunoreactive to antisera against neurofilaments, neuron-specific enolase, myelin basic protein, protein S-100, substance P, neurokinin A, neuropeptide Y, tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) were detected in the skin with specific distributional patterns. Neurofilament-, neuron-specific enolase-, myelin basic protein-, protein S-100-, substance P-, neurokinin A-and vasoactive intestinal polypeptide (VIP)-like immunoreactivities were found in or in association with sensory nerves; moreover, neuron-specific enolase-, myelin basic protein-, protein S-100, neuropeptide Y-, tyrosine hydroxylase- and vasoactive intestinal polypeptide (VIP)-like immunoreactivities occurred in or in association with autonomic nerves. It was concluded that antiserum against neurofilaments labels sensory nerve fibers exclusively, whereas neuron-specific enolase-, myelin basic protein- and protein S-100-like immunoreactivities are found in or in association with both sensory and autonomic nerves. Substance P- and neurokinin A-like immunoreactivities were observed only in sensory nerve fibers, and neuropeptide Y- and tyrosine hydroxylase-like immunoreactivities occurred only in autonomic nerve fibers, whereas vasoactive intestinal polypeptide (VIP)-like immunoreactivity was seen predominantly in autonomic nerves, but also in some sensory nerve fibers.     

Immunoreactivities of gastrin (G-) cells. I. dilution-dependent staining of G-cells by antisera and non-immune sera

Results of immunocytochemical studies reported by several laboratories suggest that gastrin (G-) cells of the stomach show immunoreactivities for various pituitary hormones (ACTH, met-enkephalin, beta-endorphin and growth hormone) in addition to gastrin. By reinvestigating the immunocytochemistry of G-cells we found that these cells exhibited reactivities towards a variety of antisera against enteric, pancreatic and hypophyseal hormones. Gastrin cells can also be "immunostained" by antisera towards proteins unrelated to any peptide hormones (e.g. alpha-fetoprotein antiserum) and by nonimmune sera. Thus the specificity of immunocytochemical findings in G-cells seems to be uncertain. According to our findings the polyvalent immunoreactivities of G-cells may be caused by a distinct binding capacity for IgG molecules. This binding of IgG to G-cells seems to be mediated by the Fab fragments of the IgG molecules which may behave like a basic dye and therefore "immunostain" anionic components within G-cells. Thus the significance of the immunocytochemical proof of peptide hormones within G-cells is limited unless extended specificity controls have been performed. The results of specificity controls performed in this study (adsorption controls, use of ascending dilutions of the primary and secondary antisera, comparison of crude antisera and affinity chromatographically purified antibodies) suggest that corticotropin-lipotropin related peptides are not contained in G-cells.     

Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig

The guinea pig lateral superior olive was examined immunocytochemically using antisera against enkephalin and choline acetyltransferase sequentially on the same sections. A colocalization of choline acetyltransferase-like and enkephalin-like immunoreactivities was found in cells of the lateral superior olive that give rise to the lateral system of olivocochlear efferents. Only choline acetyltransferase-like immunoreactivity was observed in the group of olivary nuclei that give rise to the medial group of olivocochlear fibers.     

Prostaglandin synthesis by primary cultures of mouse embryo palate mesenchyme cells

Evidence is presented for a concomitant storage of α-Neo-endorphin and dynorphin immunoreactivities in neurons of the rat brain. Antisera were raised against the structurally related opioid peptides dynorphin(1–17) and α-Neo-endorphin. Both antisera were highly specific for their respective antigen. Thus, the α-Neo-endorphin antisera did not crossreact with dynorphin and the dynorphin antisera did not crossreact with α-Neo-endorphin. Both antisera were also not cross-reactive with leu-enkephalin which is contained within the sequence of both dynorphin and α-Neo-endorphin. The antisera were used for immunofluorescent staining of frozen sections through brains from rats which had been treated with colchicine 48 hours prior to death. Both antisera revealed strong and specific immunoreactivities of magnocellular neurons in the supraoptic, retrochiasmatic supraoptic and paraventricular nuclei. Neuronal fiber systems in various areas of the brain were also labeled by the two antisera. Consecutive immunostaining of the same sections, first with dynorphin antisera and — after electrophoretic elution of the antibodies — with α-Neo-endorphin antisera or vice versa, showed that immunoreactivities for the two peptides are contained within the same hypothalamic magnocellular neurons. The neuronal fiber systems for α-Neo-endorphin and dynorphin also showed a close overlap. These studies demonstrating colocalization raise the question as to whether the two peptides have a common origin from a single precursor molecule.     

Immunocytological localization of dopamine in the guinea pig retina

We examined dopaminergic neurons in the guinea pig retina; antisera against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and an antiserum against gamma-aminobutyric acid (GABA) were used. In the present study, two types of amacrine cells were labeled with an anti-TH antiserum. However, no DBH and PNMT immunoreactivities were seen. The type 1 cell had a larger-sized soma located in the inner nuclear layer with processes ramifying mainly in stratum 1 of the inner plexiform layer (IPL). The type 2 cell had a smaller-sized soma and processes branching in stratum 3 of the IPL. The mean densities were 56.4 +/- 11.5/mm2 for the type 1 cell and 166.6 +/- 30.3/mm2 for the type 2 cell. Double immunocytochemistry using an antiserum against GABA revealed that while none of the type 1 cells showed GABA immunoreactivity, all of the type 2 cells displayed GABA immunoreactivity. Our results suggest that, in the guinea pig retina, the type 1 amacrine cells are pure dopaminergic and the type 2 cells are dopaminergic elements that use GABA as their second transmitter.     

Immunocytochemical demonstration of a pancreatic secretory protein of unknown function in human duodenum

We have previously isolated from human pancreatic juice a secretory glycoprotein of 19 KD (P19), devoid of known enzymatic activity. P19 gave by proteolysis a protein of 14 KD (P14), at first named protein X and also called pancreatic thread protein or pancreatic stone protein. Specific rabbit immunosera prepared against P19 and P14 were applied to localize these proteins in human small intestine. By comparison, antibodies directed against some human pancreatic enzymes (amylase, lipase, chymotrypsin, trypsinogen 1, trypsinogen 2, and trypsin 1) were also tested. Positive immunoreactivity was observed on Paneth cells with antisera directed against trypsinogens, trypsin 1, and P19-related proteins. In addition, antisera directed against P19-related proteins stained the columnar cells located in the crypts of Lieberkühn. These original findings are a further indication of the resemblance between Paneth and pancreatic acinar cells but show that their functional analogy is only partial. On the other hand, the presence of P19-related proteins on non-mature columnar cells suggests that this differential distribution is a consequence of differentiation.     

Immunohistochemical localization of polypeptide hormones in endocrine cells of the digestive tract of Branchiostoma lanceolatum

Summary The digestive tract of the cephalochordate Branchiostoma lanceolatum was investigated with regard to occurrence and distribution of endocrine cells. By the use of the peroxidase-antiperoxidase (PAP) technique, cells in the gut epithelium reacting with antisera against 8 different mammalian polypeptide hormones were localized. Positive reactions were obtained with antisera against the four mammalian islet hormones (insulin, glucagon, pancreatic polypeptide, somatostatin) and against secretin, vasoactive intestinal polypeptide, pentagastrin and neurotensin. No immunoreactivity was found with antisera against members of the lipotropin family (ACTH, met-enkephalin, -endorphin), against big-gastrin, cholecystokinin, substance P and moulin. The exact mapping of the different polypeptide immunoreactive cells throughout the digestive tract of Branchiostoma lanceolatum is presented.     

Antisera raised against eledoisin and kassinin detect immunoreactive material in rat tissue extracts: tissue distribution and chromatographic characterization

Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.     

Immunohistochemical evidence of gastro-entero-pancreatic neurohormonal peptides of vertebrate type in the nervous system of the larva of a dipteran insect, the hoverfly, Eristalis aeneus

Using rabbit and guinea-pig antisera, raised against GEP neurohormonal peptides of mammalian origin, cells were observed in the brain and/or in the fused ventral ganglia of the last (fifth) larval instar of the hoverfly, Eristalis aeneus, being immunoreactive with antisera against insulin, somatostatin, glucagon, PP, secretin, gastrin/CCK/caerulein; substance P, enkephalin and endorphin. Most of these GEP neurohormonal peptides also occurred in nerve fibers. No immunoreactive cells or nerve fibers could be detected with antisera against GIP, VIP, (the central fragments of) CCK, bombesin or neurotensin. The antisera tested failed to reveal any immunoreactive cells or nerves in Weismann's ring (fused corpus allatum/corpus cardiacum and thoracic gland) or in different parts of the alimentary tract. The observations support the hypothesis that neuronal GEP hormonal peptide production in the brain is a genuinely original mechanism and the appearance of endocrine cells in the gut a later feature in evolution.     

Presence of sauvagine-like epitopes in the interrenal gland of the bullfrog Rana catesbeiana

Immunocytochemistry was used to investigate the presence of corticotropin-releasing factor-like peptides in the interrenal (adrenal) glands of the bullfrog Rana catesbeiana by using specific antisera raised against synthetic nonconjugated rat/human corticotropin-releasing factor, urotensin I, and sauvagine. From these three antisera, covering a broad range of corticotropin-releasing factor-like immunoreactivities, only the sauvagine antiserum gave positive immunoreactivity. Sauvagine immunoreactivity was found in cortical cells grouped into cords in the renal zone of the interrenal gland. The central and subcapsular cords were less stained. Tyrosine hydroxylase-positive chromaffin cells were not sauvagine-immunoreactive. The immunoreactivity was abolished, in all cases, by previous immunoabsorption of the sauvagine antiserum with synthetic sauvagine (0.1 7M), but it was not eliminated by sucker (Catostomus commersoni) urotensin I, sole (Hippoglossoides elassodon) urotensin I, sucker corticotropin-releasing factor, rat/human corticotropin-releasing factor, or ovine corticotropin-releasing factor (0.1-10 7M). In a sauvagine radioimmunoassay, interrenal extracts displaced ¹²⁵I-sauvagine from antiserum only partially, and not in parallel with the sauvagine standard curve. The results suggest that the sauvagine immunoreactivity in the R. catesbeiana interrenal gland may represent a novel sauvagine-like peptide.     

Molecular characterization of the human VIP prohormone

Two distinct cell groups contain α-melanocyte-stimulating hormone-like immunoreactivity in rat hypothalamus. Only one group, located in the arcuate nucleus, contains other opiocortin peptide immunoreactivity. Combined immunocytochemistry, radioimmunoassay and high performance liquid chromatography, using two different antisera, were used in an attempt to characterise the immunoreactive material present in each cell group. The results thus obtained from normal rats, using an antiserum against α-melanocyte-stimulating hormone and one against COOH-terminal adrenocorticotropin, were compared with those obtained from rats treated neonatally with monosodium glutamate, which destroys the arcuate nucleus.In animals treated with monosodium glutamate, cells of the arcuate nucleus, staining with both antisera, were reduced in number. Cells containing only α-melanocyte-stimulating hormone-like immunoreactivity in the lateral hypothalamus were unaffected. Peptide levels detected by radioimmunoassay with both antisera were reduced in parallel. Chromatographed extracts showed parallel reductions in α-melanocyte-stimulating hormone-like and COOH-terminal adrenocorticotropin-like immunoreactivities.These results suggest that if the immunostained cells of the lateral hypothalamus contain conventional α-melanocyte-stimulating hormone, it constitutes only a very small proportion of the total hypothalamic concentration. However, the possibilities that the antiserum is crossreacting with a different molecular species, or with a similar compound synthesised by a different pathway cannot be excluded.     

Multiple peptide immunoreactivities in the nervous system of Aeschna cyanea (Insecta, Odonata). An immunohistochemical study using antisera to cholecystokinin octapeptide, somatoliberin, vasoactive intestinal peptide, motilin and proctolin

By use of the indirect immunoperoxidase method, the brain, the suboesophageal ganglion and the corpora cardiaca of the dragonfly Aeschna cyanea have been shown to be immunoreactive to proctolin antiserum and to several mammalian peptide antisera including unsulfated cholecystokinin octapeptide (CCK-8 NS) (Andriès et al. 1989), vasoactive intestinal peptide (VIP), human somatoliberin (hGRF) (Andriès et al. 1984) and motilin antisera. Immunohistochemical studies have been performed on material fixed in a solution of picricacid paraformaldehyde or in Bouin Hollande's sublimate solution. Antisera were applied on alternate sections or, according to the elution-restaining method of Tramu et al. (1978), one after another on the same section. Multiple peptide immunoreactivities appear expressed in the brain and the suboesophageal ganglion. Cells reactive to both hGRF and VIP antisera show also gastrin/CCK-like immunoreactivity and some of them are also detected by motilin antiserum. Besides, some cells immunopositive to CCK-8 NS and motilin antisera do not show hGRF or VIP immunoreactivity. At least, two pairs of protocerebral cells appear immunoreactive to both CCK-8 NS and proctolin antisera. Therefore, the present observations support our previously developed idea (Andriès et al. 1989) that the population of CCK-like cells is heterogenous.     

Electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of the calf fundic glands

The electron immunocytochemical co-localization of prochymosin and pepsinogen in chief cells, mucous neck cells and transitional mucous neck/chief cells of calf fundic glands was studied using specific antisera for prochymosin and pepsinogen with a protein A-gold method. Prochymosin and pepsinogen immunoreactivities were detected in the same secretory granules of the chief, mucous neck and transitional cells, simultaneously using small and large colloidal gold particles. In chief cells, both immunoreactivities were distributed uniformly over the same zymogen granules showing a round, large, homogeneous and electron-dense appearance. In mucous neck cells, both immunoreactivities were found exclusively on the same electron-dense core located eccentrically in the mucous granule showing light or moderate electron density. In transitional mucous neck/chief cells, electron-dense cores became larger in size and some granules were occupied by the electron-dense core without a halo between the core and the limiting membrane. Both immunoreactivities were found uniformly over the electron-dense core. The granules having no halo in the transitional cells could not be distinguished from the typical zymogen granules in the chief cells.     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.     

Ultrastructural and immunohistochemical study of endocrine cells in the midgut of the cockroach Blaberus craniifer (Insecta,Dictyoptera)

Summary The midgut of Blaberus craniifer is principally made up of columnar epithelial cells which are derived from small regenerative cells found grouped in nidi. Between them, small sparsely granulated cells with clear cytoplasm can be observed lying on the basal lamina. Mainly based on the size, shape and texture of their secretory granules, at least ten types of such endocrine cells have been identified. Five cell types contain a uniform population of dense granules: (1) medium-sized, round to oval granules; (2) small elongated granules; (3) large irregular granules; (4) oval granules with a highly osmiophilic core; (5) oval, haloed granules. Five others are characterized by a heterogeneous population of granules: (6) small, round to oval, variably electron-dense granules; (7) oval medium-sized granules of variable electron density; (8) large irregular granules of variable electron density; (9) small dense granules and large vesicles with filamentous material; (10) small dense granules and very large pale vesicles.In addition, near the regenerative cells, large cells characterized by very large, irregular, dense granules (up to 4 m), lack contact with the lumen, and reach the basal lamina only by slender cytoplasmic processes.Several antisera raised against mammalian peptides and amine were used to reveal axonal fibers and endocrine cells. Serotonin-like immunoreactivity is localized in a profuse innervation of the muscle layers that surround the epithelium, whereas cholecystokinin and methionine-enkephalin antisera stain a more moderate number of axonal fibers. Cholecystokinin-, methionine-enkephalin-, substance P-, vasoactive intestinal peptide-, somatoliberin-, and gonadoliberin-like immunoreactivities were detected in endocrine cells of the epithelium. While most of the cells appear pyramidal, oval, fusiform or bowl-shaped, and seem to lack contact with the lumen, cells reaching it have been detected reacting with antisera to cholecystokinin, substance P, vasoactive intestinal peptide, somatoliberin and gonadoliberin.     

Polyclonal Antisera To Distinguish Strains and Form Variants of Photorhabdus (Xenorhabdus) luminescens

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode strain NLH-E87.3) and against the primary and secondary forms of P. luminescens PSH (obtained from nematode strain DH-SH1). In double-diffusion tests all form variants of strain PE reacted with the antiserum against the primary form, but each variant produced a different diffusion pattern. The primary and secondary forms of strain PSH were also serologically different. Antiserum 9226 reacted with almost all P. luminescens strains tested, but it reacted differently with each strain in the double-diffusion test, showing that the strains were serologically different. The specificity of the antisera was increased by cross-absorption. After cross-absorption the antiserum against the strain PSH primary or secondary form was specific for that form and did not react with the other form. Using the cross-absorbed antisera in immunofluorescence cell-staining tests, we could distinguish primary and secondary form cells in a mixed strain PSH culture.     

Substance P-, neurotensin- and bombesin-like immunoreactivities in the gill epithelium of Ciona intestinalis L.

Summary Substance P-, neurotensin- and bombesin-like immunoreactivities were localised in some gill epithelial cells in the pharynx of Ciona intestinalis L. No immunoreactivity was obtained with antisera to gastrin, glucagon, insulin, pancreatic polypeptide or calcitonin. Some of the epithelial cells of the gills were shown to be argyrophilic with the Grimelius technique.     

Is helodermin-like immunoreactivity in human thyroid C cells due to a salmon calcitonin-like substance?

Helodermin-like and salmon calcitonin (sCT)-like immunoreactivities co-existed in a subset of human calcitonin (hCT)-containing cells in normal human thyroid tissue and medullary thyroid carcinomas. Helodermin/sCT-immunoreactive cells were mostly different from calcitonin gene-related peptide (CGRP)-positive cells. Helodermin and sCT immunoreactivities were not identified in pulmonary and pancreatic hCT-positive neuroendocrine tumors, except for a few lung tumor cells showing positive staining with one of two sCT antisera used. Helodermin immunoreactivity demonstrated by rabbit antiserum R0086 was completely abolished in the presence of synthetic sCT, while sCT immunoreactivity was not absorbed by synthetic helodermin. The carboxyl terminal Arg30-Thr31 sequence (and Pro35 amide structure) of helodermin would be the epitopic site recognized by this antiserum, since a similar amino acid sequence is present in sCT molecules but absent from hCT and CGRP.     

Multiple peptide immunoreactivities in the nervous system of Aeschna cyanea (Insecta,Odonata)

Summary By use of the indirect immunoperoxidase method, the brain, the suboesophageal ganglion and the corpora cardiaca of the dragonfly Aeschna cyanea have been shown to be immunoreactive to proctolin antiserum and to several mammalian peptide antisera including unsulfated cholecystokinin octapeptide (CCK-8 NS) (Andriès et al. 1989), vasoactive intestinal peptide (VIP), human somatoliberin (hGRF) (Andriès et al. 1984) and motilin antisera. Immunohistochemical studies have been performed on material fixed in a solution of picricacid paraformaldehyde or in Bouin Hollande's sublimate solution. Antisera were applied on alternate sections or, according to the elution-restaining method of Tramu et al. (1978), one after another on the same section. Multiple peptide immunoreactivities appear expressed in the brain and the suboesophageal ganglion. Cells reactive to both hGRF and VIP antisera show also gastrin/CCK-like immunoreactivity and some of them are also detected by motilin antiserum. Besides, some cells immunopositive to CCK-8 NS and motilin antisera do not show hGRF or VIP immunoreactivity. At last, two pairs of protocerebral cells appear immunoreactive to both CCK-8 NS and proctolin antisera. Therefore, the present observations support our previously developed idea (Andriès et al. 1989) that the population of CCK-like cells is heterogenous.     

Caspase-12 processing and fragment translocation into nuclei of tunicamycin-treated cells

Excess endoplasmic reticulum (ER) stress induces processing of caspase-12, which is located in the ER, and cell death. However, little is known about the relationship between caspase-12 processing and cell death. We prepared antisera against putative caspase-12 cleavage sites (anti-m12D318 and anti-m12D341) and showed that overexpression of caspase-12 induced autoprocessing at D(318) but did not induce cell death. Mutation analysis confirmed that D(318) was a unique autoprocessing site. In contrast, tunicamycin, one of the ER stress stimuli, induced caspase-12 processing at the N-terminal region and the C-terminal region (both at D(318) and D(341)) and cell death. Anti-m12D318 and anti-m12D341 immunoreactivities were located in the ER of the tunicamycin-treated cells, and some immunoreactivities were located around and in the nuclei of the apoptotic cells. Thus, processing at the N-terminal region may be necessary for the translocation of processed caspase-12 into nuclei and cell death induced by ER stress. Some of the caspase-12 processed at the N-terminal and C-terminal regions may directly participate in the apoptotic events in nuclei.