A Histochemical Study of Polyphosphate Hydrolases in Epidermal Dendritic Cells*

摘要: The epidermal dendritic cell population of mammals consists two types which differ in their ability to oxidise dihydroxyphenyla-lanine (DOPA). Thus, melanocytes are recognizable by the melanin product of. DOPA reaction, while non-melanogenic branched cells require gold impregnation or other technics visualize them. Melanocytes evenly distributed at dermoepidermal junction, closely applied basal layer epidermis, and processes best seen separated sheet preparations. In vertical sections normal epidermis (Langerhans cells) observed suprabasally with ramifying dendrites extending between keratinocytes, often terminating transitional zone. There is compelling evidence believe that a close relationship exists Langerhans manner proposed Billingham Medawar (1) considered be melanogeni-cally effete division products melanocyte population. A model has been explain certain features melano-cyte-Langerhans terms different migratory properties it suggested actively move outwards through (2).Electron micrographs suprabasal (3, 4) suggest these may migrate presumably disrupting desmosomal attachments, resembling transepithelial diapedesis leukocytes (5). Active movement also implied re-orientation from predominantly horizontal distribution ramifications cells. view putative involvement ofactomyosin-like contractile proteins cellular (6) demonstration an enzyme hydrolyzing adenosine triphosphate (ATP) (7-11) considerable significance since suggests possible association motility. present study was undertaken test this possibility attempting define some histochemical characteristics ATPase.MATERIALS AND METHODSBiopsy specimens skin Rhesus monkey, red cavy, albino mice were examined addition human obtained operation. Specimens wrapped gauze placed Ringer's solution container surrounded crushed ice. As soon as after collection, material mounted 5% aqueous carboxymethyl cellulose frozen for use cryostat -20°.Sections cut 7 fi thick picked up on coverslips. Initially comparison made air-dried, unfixed fixed ice-cold formol-calcium (12) periods varying one thirty minutes. result experience found most satisfactory results ten-minute period fixation therefore employed during study. Substitution glutaraldehyde formaldehyde procedure tried, but proved too strongly inhibitory. After fixation, washed changes distilled water transferred incubating media.Solutions prepared 0.05 M acetate buffer pH 5.4 6.5 containing substrate lead nitrate (final molarity 2.5 X 102<). At 72 0.02 tris-buffered medium 3.6 10s< molar used. 9.4 (0.05 molar) barbital-buffered calcium chloride (2 10 2< M) employed.The substrates investigated sodium 0-glycerophosphate, glucose-6-phosphate, soluble ribonucleic acid (from yeast), mono-, di-, triphosphates adenosine, cytidine, guanosine, uridine, inosine, thiamine diphosphate. Sufficient added media give final concentrations 1 3 108< M, exception RNA 10'4< concentration. Control case incubated ATP same molarity.The effects following substances in-412MELANOCYTE POLYPHOSPHATASE413vestigated adding them concentration shown :Cations: potassium 103< 5 101< lithium 10_1< magnesium 101<, 10'2<, 1.5 M.Sulfhydryl reagents: iodoacetate 4< N-ethyl maleimide (NEM) 2<, 103<, 104< p-chloromercuribenzoate (PCMB) lO3<,103<,104< M.Other agents: amobarbital azide 102< Antimycin 0.02%, cyanide 2,4 dinitrophenol dextrose 0.5 flavine adenine dinucleotide Gramicidin 0.1%, insulin (soluble) 8 units per ml, ouabain (strophanthin G) 102<, M.Pre-incubations phosphate buffered (0.1 7.4) solutions promethazine hydrochloride 10_1<, somatotrophin ml unstable when included 9.4.After incubation 37° hydrolysis visualized metal sulfide procedure. Sections lead-containing 15 seconds into dilute yellow ammonium sulphide, tapwater slides water-mounting medium. Material Padykula Herman (13) precipitated treated 30 2% cobalt prior being developed inspection.RESULTSHuman SkinLocalization reaction substrates. When monophosphate alkaline range three histologically distinct structures became stained product; inner root sheath hair follicles, dermal blood vessels, zone epidermis. positive whole showed punctate 5.4. activity cells.With diphosphate di- tri-phosphate tested similar localization. calcium-containing slight fibroblasts. neutral Except all sites displayed greatly increased medium, thispH frequently cells.Thiamine diphosphatase vessels range. fibroblasts visualized. Activity confined large arterioles, TPPase less widely cell. This particularly evident where degree manifested basally sited much greater than reverse appearances polyphosphate employed. With nucleoside di-and throughout cells, well outlining slender upwards relation finding agreement those previous workers who have phosphatase activities ATPase (8) (14). GTP GDP dense remainder pattern No formed used whereas control equimolar positive.Fixation. Unfixed 7.2 uniformly density compared sections, progressively time prolonged. 40 minutes no detected 2 hours 9.4. Ten-minues diminished activity, gave clearer localization product. Ten glutaraldehyde-cal-cium (glutaraldehyde = 6.5%) under conditions totally inhibitory except mouse liver material.Effect cations ATPase. me-414THE JOURNAL OF INVESTIGATIVE DERMATOLOGY(ft , te *dclFig. 1. Normal (9.4) showing fine demonstrated : left picture process triangular “button. ( 1200)Fig. 2. Section specimen Fig. 4