FISDIRIES RESEARCH BOARD OF CANADA Translation Series No. 2776
• ; General Reyiew.2 Mat-cellà (Labr6CYtes) -
iyL. ArVy
„
Original title: Les labrocytes (Mastzellen)
From: Revue d'Hematologie (Review of Haematology), 10
Translated by the TranSlation .Section Department of the Environment
Department of. the Environment Fisheries Research Board of Canada Halifax Laboratory Halifax , N. S.
197:3
84 pages typescript ' eS30896 MT 55/1
GENERAL REVIEW
MAST-CELLS (LABROCYTES) (1)
by L. ARVY
!.1 Historical
"The entire group of mast-cell is a waiting group." ;1 1 .1- JOLLY, 1923.
In 1875, WALDEYER noted large cells containing refringent granules among the bundles of connective tissue, especially in the adrenal glands and testicular interstitial tissue, in addition to the characteristic components. Although located at various points, these tissue cells
were principally found along the blood vessels, which led WALDEYER to baptise them "plasma nellq",
In 1877 EHRLICH observed that, linder the name "plasma cells", WALDEYER had lumped together some elements which were not the same: staining connective tissue with aniline derivatives, he distinguished, among the granular cells described by WALDEYER, elements which took a
(1) Some French haematologists use the term "mastzelles" (JOLLY, 1923) or else "mastocytes". The first of these terms has the disadvantage, as a foreign word, of having to be fitted , with a French plural. The second, fairly widely used by clinicians, means literally mammary cells;
it seems to me inappropriate in relation to mammals and absurd in relation to birds, reptiles, batrachians or fish; Some histologists designate these elements as tissue "his tes with basophilic granules". HENNEGUY, BETANCES, etc. have proposed the term sitistoc tes to designate Ehrlich's "Mastzellen". •
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deep stain with the various methyl violets, their cytoplasmic granules thereby turning a metachromatic reddish colour. EHRLICH had the impression that these elementÀ were particularly abundant in tissue that was thickened, edematous, "inflamed", neoplastic or "overnourished" and, taking them to be fattened reserve elements of connective tissue, he called them "Mastzellen", from the German verb "masten": to nourish or to fatten.
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Anglo-Saxon writers use the English equivalent "mast-cells" whereas French writers call them Ehrlich's cells, "food-cells" or, still better, labrocytes (BRUMPT,.LANGERON, etc.).
JORPES (1946) suggests that the term "heparinocytes", which
EHRSTROM (1943) applied (with little justification) to leukocytes with basophilic granules, should, in the light of our-very imperfect knowledge of these relativ4.y rare cells, be reserved for mast-cells. • This viewpoint has been adopted by the Swedish, Norwegian and Finnish researchers (HOLMGREN, WILANDER, SUOMALAINEN, HARMA, LEHTO (1946-52).
Finally, RILEY (1953) has mustered arguments to show that mast- cells may very reasonably be called "histaminocytes".
NUCLEAR AND CYTOPLASMIC CHARACTERISTICS
Examined by the hanging-drop method, mast-cells generally appear perfectly round.
In smears of pleural or peritoneal fluid, they appear more or. less ovoid and rarely perfectly round or very elongated.
In slide-mounted preparations of serous membrane, they are usually ovoid, with a major diameter of 10 to 20 microns.
• In section, mast-cells are very often seen to be polyhedral, deformed by pressure exerted by neighbouring cells; in this case they are smaller, their major diameter not exceeding .8 to 15 microns. •Nevertheless, when well spread out, they may attain a size of 25 microns. They appear: to be very plastic: among the bundles of connective tissue fibres, I sometimes found almost thread-like mast-cells drawn out parallel to the fibres; in this case, in a normal rat, for example, they may attain a length of 30 microns. Pathologists (CAZAL, 1954) have observed what can without exaggeration •be described as giant mast-cells, since they measured as much as
103 microns long by 6 to 9 microns wide. 330896 MT 56/2
The mast-cell niembrane is always extremely fine if at all visible; nevertheless, it undeniably exists: it stains pure blue with toluidine blue. It has been studied under the electron . microscope by ASBOE-HANSEN (1954).
The mast-cell nucleus shows the various age-dependent aspects common to all cells. In the initial stages of its development, the cell contains a relatively large, nucleolated nucleus with a dense, delicate, rather hazy chromatin network. The nucleus is single and round or oval, its major diameter varying between 5 and 8 microns. After staining with toluidine blue, the chromatin is. barely chromophilic, homogeneous or fibrillar and pale blue. In slide-mounted preparations of . mesentery, after fixation in 10% formaldehyde, the nuclear chromatin is seen to be arranged like the spokeà of a wheel. In the final stage of its evolution, the mast-cell's nucleus has become small, round and pyknotic.
The cytoplasm appears homogeneous both before and after fixation, in ordinary and phase-contrast microscopy. It stains pale blue with toluidine blue. Nevertheless, in some mast-cells,
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the cyLoplasm contains chromophobic vacuoles Which have been interpreted either as the onset of cytoplasmic synthesis or as the vestiges of 1 excreted mast granules. Under electron microscopy, after ultracentrifugation, th è cytoplasm appears to be composed of 10 millimicron-sized particles (JULEN, 1950).
The cytoplasm of immature mast-cells contains occasional small, basophilic granules. As the cells develop, these granules increase in number and size; the particles may become sufficiently coarse and abundant to hide the nuclèus altogether. The granules vary in diameter from 0.1 to 0.4 microns. After staining with an aqueous solution of toluidine blue, the particles are swollen and often indistinct. • With thionine, they swell very little and remain clearly separated . from one another. After freeze-drying, the granules in mesenteric mast-cells from the rat have a diameter of 0.1 to 0.3 microns (SYLVEN, 1950). The giant mast-cells observed by CAZAL (1954) in the bone-marrow of a subject suffering from medullary aplasia, contained larger than normal granules with a diameter range of 1 to 1.5 microns.
Earlier writers (RAUDNITZ, 1883, WESTPHAL, 1891) noted mast granules in the vicinity of mast-cells and BALLOWITZ (1891) believed that, in reality, these granules were not extracellular but were contained in very slender projections of the cell. In fact, both cases can occur.
It seems that mast-cells are endowed with a certain chemotaxis; BAECKELAND (1950) observed that they can be attracted by the fibrinous portion of a blood clot and undergo nuclear and cytoplasmic changes culminating in the excretion of metachromatic particles without the cells having shown the slightest sign of phagocytosis. 330 896 MT 57/2
Under the influence of mechanical irritation, mast granules lose their individuality and merge into an amorphous mass, their cytoplasm . becoming hydrated and vacuolated and their membrane torn (DRENNAN, . 1951).
The abundance of the particles makes it difficult to demonstrate the mast-cell chondriome: DUBREUIL (1913), studying rabbits and . dogs, and COWDRY (1918), KIRKMAN (1950) and ZOLLINGER (1950), studying mice, did not observe any chondriome. COMPTON (1952) noted no mitochondria in the mast-cells of hamsters examined under the phase-contrast microscope either before fixation or after staining with janus green or again after fixation in Champy's solution or in osmium dichromate-acetic acid and staining with aniline fuchsine. KIRKMAN (1950) and COMPTON ( 1952) have associated the absence of chondriome with that of M or G nadi-reagent. ZOLLINGER (1950) regards mast granules as heparin-saturated mitochondria.
Nevertheless, NAKAJIMA (1928) has described a chondriome in the mast-cells of man, rhesus monkey, macaque, dog, guinea-pig, rat and mouse: one has only to fix the mitochondria and to stain them by the Altmann-Gabe method to observe perfectly clear red
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chondrioconts among the green mast granules (ARVY, previously . unpublished).
Fixation by the Da Fano or Kolatchev-Nassonov method brings out a Golgi system in the mast-cells (KIRKMAN, COMPTON, loc. cit; . previously unpublished). . ARVY,
Trial ultracentrifugation at 50,000 rpm for two hours did not result in the separation of the particles in the mast-cells from their cytoplasm (FRIBERG et al, 1951); however, differential centrifugation of suspensions of bovine hepatic capsule has been shown to separate two fractions, one of mast granules varying from 0.2 to 0.3 microns, the other of submicroscopic granules (JULEN et al, • 1950).
TECHNIQUES FOR SHOWING UP MAST-CELLS 1. - Vital and Postvital Stains.
The simplest way to become acquainted with mast-cells is to examine, by ordinary or phase-contrast microscopy, a drop of untreated pleural or peritoneal fluid from the rat, between two slides. Besides various types of leukocytes, this examination brings out, in the normal rat, elements decidedly larger than leukocytes. These elements usually have a single round or oval, sometimes notched nucleus; their cytoplasm is filled with coarse refringent and non-birefringent subequal granules, numerous to the point of concealing the . nucleus completely. Observed in ultraviolet light, the granules are not autofluorescent. Distilled water, or even saline that is isotonic to blood swells the particles which then lose their refringency,
The staining reaction of the mast granules may be followed if a drop of neutral red or janus green solution in 9% saline is introduced under one of the edges of the upper slide (COMPTON). 330896 MT 58/2
When the procedure is repeated using 1% aqueous solutions of ' methyl violet or toluidine blue, a strongly acid or weakly alkaline solution of thionine or .1% safranin in a 1% acetic solution, the mast-cells quickly die and their cytoplasmic granules take on a. metachromatic stain: red violet with methyl violet or toluidine blue, red with thionine and orange with safranin.
Another very simple method of showing up mast-cells is to take a sample of rat mesentery and stain it using the dyes already mentioned. Dahlia violet, used by EHRLICH, and polychrome blue, used by UNNA, with or without the differential action of glyceric ether, orcein or alum, bring ihem out equally well.
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Alcchol-based fixatives have the disadvantage of vacuolating the cytoplasm and swelling the mast granules. •
The Swedish researchers use, as mast-cell fixatives, quinine and basic lead acetate solutions; both are good preservatives of the granules, but have the disadvantage of being very poor histological fixatives. This disadvantage may, however, be attenuated by the addition of' alcohol Or formaldehyde.
Fixatives based on formaldehyde, picro-formaldehyde or dichromate (BOUIN, HELLY, MAXIMOW, etc.) are good preservatives of mast-cells. •
III. - General Stains.
The procedures currently used in histology: picro-indigo- carmine hemalum*, eosin hemalum, azo dye, the triple stains of Masson, Mallory, Prenant, Flemming, etc. do not bring out mast-cells.
After fixation in 10% formaldehyde and paraffin sectioning, mast granules from the mouse are Gram-pcitive and appear grey or black with Heidenhain's hematoxylin.
Mouse mast-granules in frozen or paraffin sections fixed in 10% formaldehyde stain blue with Nile blue sulphate and brillant cresyl blue.
In frozen sections, hematin acid stains the mast particles from dog, man, rat and mouse, but not after pyridine treatment, whether the fixative used is 10% formaldehyde or calcium formal- dehyde (MONTAGNA, 1948; MONTAGNA and PARKS, 1948; KIRKMAN, 1950). 3 30896 MT .• 59/2
After fixation in Helly's solution and paraffin or freeze sectioning, the granules in mast-cells from the mouse stain violet . with azure II and a very intense green with Altmann-Gabe. ,
After fixation by all current histologic methods, the mast granules stain with Gomori's chromic hematoxylin, fuchsine. paraldehyde and pyronine (pyroninophilia is resistant to ribonuclease).
In the course of my investigation of the variable factors of mast-celis from the rat or mouse, I used almost exclusively staining with methyl or cresyl violets or toluidine blue or, to produce deep colouring, picro-fuchsine or picro-indigo-carmine. These stains have the advantage of acting rapidly; moreover, the violet and blue stains colour the mast granules a dark red and picro-fuchsine and picro-indigo-carmine turn the dark red to black whereas the tissues stain yellow, pink or green; finally, the mast-cells stand out clearly against the background of the other tissue components, thus facilitating counting and photographing.
Some tissues are so rich in metachromatic ground substance that the mast-ceils are scarcely visible; this is true with the umbilical cord.
* hemalum: alum hematoxylin. transi.
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PRAKKEN and WOERDEMANN (1952) were able to observe mast-cells by treating sections with a hyaluronidase which lyses the metachromatic substance which is the essential constituent of Wharton's jelly.
IV. - IDENTIFYING STAINS:
The granules in the mast-cells of man and the rhesus monkey do not restain leuco-fuchsine* unless they are first treated with a chromium-based fixative, but they do restain leuco-fuchsine after periodate oxidation. Oxidation by periodic acid selectively breaks the bonds in 1-2 glycol, thus causing the appearance of aldehyde intermediates capable of reacting with Schiff reagent to yield a purple red compound. Oxidized in this way glycogen, chitin, the mucins and mucoprotides, hyaluronic acid, the glyco-protides, reticulin, the mucopolysaccharides, the cerebrosides, the lipid monosaccharides, the oleic or cinnamic acids, the ketosteroids and heparin monosulphate restain Schiff's leuco-fuchsine. The most potent heparin known is composed of one completely esterified molecule; since all its radicals are blockeà by sulphuric radicals, it cannot react with periodic acid (JORPES et al, 1948); heparin monosulphate which has two adjacent hydroxyls is oxidized by this acid and restains Schiff reagent, - not without prior oxidation, however. From these findings, JORPES and GARDELL (1948) have concluded that mast granules are made up of heparin monosulphate or a derivative of this compound.
Under the same conditions, chondroitin sulphuric acid does not stain with Shiff - reagent; for this reason, cartilage remains colourless when treated with periodic acid and fuchsine sulphurous acid. This
• fact .indicates a difference between chondroitin sillphuric acid and heparin monosulphuric acid, not only as regards the internal bonding of the two carbohydrate molecules, but also the position of the sulphate groups. 330896 MT 60/2
Glycogen and hyaluronic acid behave like heparin monosulphate; nevertheless, the mast granules probably have no connection with these compounds, since the specific reactions of glycogen and hyaluronic acid on the granules are negative: subjected to the prior action of an amylase (LEBLOND, 1950), a glycogenase or a hyaluronidase, the granules still restain leuco-fuchsine after periodate oxidation.
This interpretation of the character of mast-cells is supported by numerous facts. Assaying the sulphuric esters from a variety of viscera specimens by hydrolysis, which liberates the sulphates, followed by precipitation of the sulphates by barium chloride, shows that the sulphuric ester content increases as the number of mast-cells present (HOLMGREN and WILANDER, 1937). HOLMGREN noted, for example, that the pig aorta, which contains virtually no heparin compounds, has very few mast-cells, whereas the aorta or vena cava of the cow or the horse, both rich in mast-cells, are also
* fuchsine decolourized with sulphurous acid (or sulphur, dioxide) transi.. .
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rich in heparin compounds. In the sanie way WILANDER (1937) observed
that the rat liver, which contains no mast-cells, contains no heparin conv- pourds, whereas the subcutanaxis tissue of thesane animal, rich in mast-cells, assays 65 mg/kg of heparin. EHRLICH et al (1949) were able to extract 126 mg of purified heparin from an elephantiasic scrotum rich in mast-cells. In addition, the number of mast-cells substantial in the case of certain tumours (BLOOM, 1942); OLIVER et al (1947) noted that mastocytomas in the dog yield about 50 times more heparin per unit oe weight than the richest normal sources of high activity heparin.
Nevertheless, the intensity of staining in mast-cells treated with Schiff reagent after periodate oxidation varies according to species: the mast granules of the pig stain poorly and those of the rat yield only a pink tint (LEBLOND, 1950). Moreover, for a given species (the observation has been made in man, mouse and rat), after treatment with a variety of fixatives and paraffin sectioning some mast-cells do not restain Schiff reagent after periodate oxidation (LILLIE, 1950). This fact may be related either to the absence of heparin compound or to the presence of heparin alone
which, as a carrier compound for three sulphate groups, blocks three of the four hydroxyls of the molecule which thus cannot further undergo periodate oxidation.
Thus for a single species and even for a given tissue, after treatment with SPA (1.) , some of the stained mast-cells appear orange while the rest appear purple. Apart from possible differences in staining reaction arising either . from differences in the thickness of the sections, in the depth of cut into the mast-cell made by the -microtome (whether a tangential or equatorial cut) or from the vanishing of the granules or the most soluble parts of them, one may imagine that at any given moment the essential compound in the granules has not arrived at the same stage of synthesis in all the mast-cells or in all the granules of a given mast-cell. The experiments of JORPES and GARDELL (1948) strongly suggest that some
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granules might contain strongly esterified heparin which is inactive under SPA, while other granules contain only heparin monosulphate. This suggestion contains a very important and irrefutable element of truth: to prove it, one has only to repeat the experiments of
LILLIE (1950) or DRENNAN (1951). If the sections are stained with thionine, after identification of the mast-cells and decolourizing with acid-alcohol, SPA brings out only some of the mast-cells revealed by thionine. In the same way, if the sections are stained first with SPA and then with toluidine blue, the latter stain brings out a number of mast-cells which were not revealed by the former
(DRENNAN, 1951):
Tongue • SPA Toluidine Blue Man Oesophagus 43 155 65 127 Tongue 210 319 Woman Oesophagus 73 194 • Tongue 28 60 • Woman Oesophagus 86 211
(1) Schiff reagent: periodic acid.
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A final argument in favour of the richness in heparin compouàs of mast granules is furnished by their metachromasia. Heparin compounds carry an exceptionally high electric charge, which is perhaps responsible for their inhibiting the formation of chrombin from prothrombin (ARVY, 1947); excessively rich in sulphuric acid, these compounds mark an unusual kind of synthesis in a livinij organism. Their acidity and high molecular weight confer on them the ability to yield with some stains, tautomeric molecules giving different colour effects. LISON (1935) demonstrated that metachromasia is a sign of sulphuric esters of high molecular weight: all the known sulphuric esters of high molecular weiet are metachromatic and lose their metachromasia if deprived of their sulphuric radical. The phosphoric esters or cerebroside sulphuric acid do not bring out the purpletautomeric form of toluidine blue. Nevertheless, the vitreous humour, synovial fluid and a purified preparation of hyaluronic acid all stain metachromatically and, according to ASBOE-HANSEN (1954) contain no sulphur.
Of course, metachromasia is fairly common among vertebrate tissues: the intercellular matrix of cartilage, the arterial wall in mucoid degeneration, the basic substance of the heart valves, sciera and tendons, secretion from the epithelial mucous glands, Wharton's jelly, the mucoid substance in eggs, certain nucleoprotides, etc. However, the aniline stains related to toluidine blue, have, in vitro, a special affinity for heparin, so that even a diluted toluidine blue solution instantly precipitates the heparin from uncoagulable heparinized blood which thereupon recovers its normal coagulability. This reaction is so reliable that it is used for adjusting the dose of heparin compounds.
Mast granules stained with a . 0.1% solution of toluidine blue either in water or in various strengths of alcohol are seen to be metachromatic in aqueous and weak alcohol.solutions up to 600 (Baumé);
After staining with a toluidine blue solution in 70 0 alcohol, the granules are blue and after treatment with toluidine blue in 80 ° alcohol;
-1 330896 MT 62/2 they are no longer coloured. Even with weak alcohol solutions, the metachromasia of the mast-cells is still less marked than with aqueous solutions.
Pretreatment of the slides with periodic acid for 45 minutes does not modify the metachromatic reaction of the granules to toluidine blue or change their shape.
The question has been raised whether the metachromatic substance in mast-cells is actually in the granules and not in the cytoplasm. Under ordinary microscopy, the granules in sharp focus appear violet blue and the underlying ones pinkish; in cells with abundant granulation, this colour effect may lead to the belief that the intergranular cytoplasm is metachromatic (FRIBERG et al, 1951).
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Nevertheless, JULEN et al (1950) believe the metachromatic substance to be contained in an intergranular ultramicroscopic fraction. In fact, comparative studies of the duration of staining reactions, such as those conducted by ZOLLINGER (1950), bring the realization that toluidine blue stains granules suspended in an orthochromatic medium metachromatically, but as the reaction progresses, the granules are seen to swell up while the intergranular cytoplasm turns violet. About two hours later, the granules are dark blue and the cytoplasm in the mast-cells metachromatic. This seems to indicate that the metachromatic, perhaps heparin-based sUbstance in the particles is a precipitate on their surface which, under the effect of hydration, diffuses into the intergranular cytoplasm which thus gains in metachromasia at the expense of the granules.
V. - SPECIFIC STAINING REACTIONS.
a) Glycogen. - ARNOLD (1906) noted that -mast-cells react positively to Best's carmine and concluded that they contained glycogen; in fact, LEHNER (1924), WISLOCKI and DEMPSEY (1946) and KIRKMAN (1950) have demonstrated that they contain none.
Bauer reagent gives a weak stain to the mast granules of man, rat, mouse, rhesus monkey and macaque in paraffin sections, and this reaction persists after application of saliva or amylase. Moreover, • tissues fixed in formaldehyde vapour and incubated in a saliva 0 medium for 24 or even 72 hours at 37 C stain with Schiff reagent after periodate oxidation, in the same way as tissues not treated with saliva (WISLOCKI et al, 1949; FADEM, 1951).
• h) Mucoid Substances. - Early .histochemists - HOYER (1880), HARRIS (1900) and STAEMMLER (1921)'- believed that mast granules were rich in mucin since they were stainable by muci-carmine and muci-haematin. o In fact, after fixation in 10% formaldehyde or 80 alcohol and either freeze or paraffin sectioning, the granules in mast-cells yield only a weak colour with Mayer's muci-carmine.
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c) Hyaluronic Acide - According to the research of ASBOF-HANSEN . it would seem that the metachromasia of human mast-cells (1950-51), disappears after treatment with hyaluronidase; that author claims to have observed changes in the number and character of mast-cells and mast granules (in particular, loss of their metachromasia), using as study material a biopsy specimen of human skin previously injected with a quantity of enzyme. .
Nevertheless, whether smeared directly on the slide or prepared
as a paraffin section, after fixation in either , 4% basic lead acetate or 10% formaldehyde, the granules of mast-cells from rhesus monkey,
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macaque, mouse, rat and hamster keep their metachromasia (DAVIES, 1942, WISLOCKI et al, 1946 and 1947; COMPTON, 1952; PRAKKEN and WOERDEMANN, 1952), whereas, under the same conditions, the vitreous humour, the synovial membrane and Wharton's jelly lose their metachromasia.
In 1952, COMPTON reported that a subcutaneous injection of 5 mg/cc of a saline solution of hyaluronidase does not provoke any change in the mast-cells in a tissue fragment taken from the inguinal region of the adult Cricetus auratus examined both before and after fixation in neutral formaldehyde and safranin staining, whether examinations were made 10 to 30 minutes or 1, 2, 3, 4 or 6 hours after injection with the enzyme.
It seems indisputable that the granules in normal mast-cells do not contain hyaluronic acid: introduction of hyaluronidase into a preparation of rat peritoneal juice smeared between slide and cover-plate in 9% sodium chloride and observed for variable lengths of time up to 8 hours in a warm room does not provoke any noteworthy change in the particles.
d) Lipids. - In 1908, PAPPENHEIM asserted that mast-cells contained
a mucolecithide, but CIACCIO affirmed in 1913 that normal mast-cells do not contain any lipids, although the latter can sometimes appear in pathological states. Nevertheless, the mast granules of man
- and rat, fixed either in 10% formaldehyde . or in Baker's calcium- cadmium formaldehyde, in frozen sections, take Sudan black (MONTAGNA and NOBACK, 1947; WISLOCKI et al, 1947); this colour is shown even if a prior acetone extraction is tried on the mast-cells of man, rat and mouse. •
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PAFF et al (1947) detected lipids that were stainable by Sudan B black in dog mast-cells, and SNELLMAN et al. (1951) have affirmed that mast granules contain lecithin, cholesterol and neutral fats. Nevertheless, COMPTON (1952) did not discover any lip,ids in mast granules.
e) Protides. - In 1904 MEYER believed that the particles in mast-cells contained nucleic acid, but recent investigation has been able to demonstrate neither ribonucleic nor desoxyribonucleic acid in the granules and ribonuclease affects them no more than desoxyribonuclease (WISLOCKI and DEMPSEY, 1946; ZOLLINGER, 1950).
f) Enzymes. - In man, macaque, dog, guinea-pig, rat and mouse, the granules of mast-cells give the characteristic pei'oxidase reaction IAKAJIMO, 1928) and in frozen thyroid sections, the reaction of rat mast granules to benzidine peroxide is positive (DE ROBERTIS and GRASSO, 1946). Nevertheless, in both cases it is a matter of pseudo-peroxidase since the ensyme is thermostable (MONTAGNA and NOBACK, 1948).
Study of the mesentery in toto, before or after fixation in 10% formaldehyde and after freeze sectioning, shows that the granules i
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rat, dog, mouse and hamster mast-cells give the characteristic nadi-reaction indicative of a cytochrome oxidase (MONTAGNA and NOBACK, 1946 and 1947; MONTAGNA, 1948; PAFF et al, 1947; KIRKMAN, 1950; COMPTON, 1952). Nevertheless, after fixation in formaldehyde vapour or a formaldehyde-alcohol mixture, FADEM (1951) was not able to detect cytochrome oxidase in man.
Mast granules show an alkaline and acid phosphatase activity (whether the test methods of Gomori, Newman et al, 1950, or Wolf et a1,.1943, are used). This fact is now well established in man, dog, rat, mouse and hamster (NOBACK and MONTAGNA, 1946 and 1948; WISLOCKI et al, 1946 and 1949; DAHLGAARD, 1948; PAFF, MONTAGNA and BLOOM, 1947; RILEY and DRENNAN, 1949; FADEM, 1951; COMPTON, 1952).
After acetone fixation either on the slide or in paraffin sections, the granules of man, dog, rat, hamster and mouse react positively to Gomori's lipase test (NOBACK and MONTAGNA, 1946; MONTAGNA et al, 1948; KIRKMAN, 1950; COMPTON, 1952).
g) Various Substances. - After fixation in Bouin's solution and paraf fin trapping, the granules in mouse mast-.cells are not fin in Cowdry's test; therefore, they contain neither poly- argentaf . phenol nor aminophenol (KIRKMAN, 1950).
In Baker's reaction on rat tissues fixed in Bouin's solution and paraffin-trapped, the granules in mast-cells do not contain arginine, but their cytoplasm does give a positive reaction (KIRKMAN, 1952).
After fixation in Bouin's solution and paraffin sectioning, the granules in rat mast-cells appear variably black or red in Copeland's (1948) chloride test (KIRKMAN, 1952).
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JORPES et al (1953) and ASBOE-HANSEN (1953) studied mast-cells by autoradiographic techniques. The latter author demonstrated a radioactive sulphate in the mast-cells of a skin tumour in a mouse injected with S35 tagged sodium sulphate; he noticed that some mast- cells, in every other respect like the rest, did not contain radioactive sulphur.
VI. - Mast-Cells in Tissue Culture.
PAFF et al (1947) observed that most mast-cells originating in mastocytoma fragments from the dog proliferate when cultivated in vitro unlike other types of cells which very quickly disappear from the cultures This finding should be compared with the observations made by FISHER (1927 and 1936), GOERNER (1930) and ZAKRZEi/SXI (1932 and 1933) on the inhibiting effects of heparin on tissue growth and differentiation.
PAFF and BLOOM (1949) observed mast granules both dissolving and being expelled in tissue cultures; they interpret these results in terms of heparin release by the cell.
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ZITGER et al (1953) made some interesting observations on human mast-cells taken by biopsy from the neck of a child suffering from urticaria pigmentosa (1), and cultivated for 13 months. The mast-cells multiply best in the semi-anaerobic state; the number and size of the granules increase as the cell develops; the fine granules found in early-stage cultures contrast with the coarse granules of migratory mast-cells or mast-cells f6und in old cultures; some mast-cells, however, contain both fine and coarse granules.
BIOCHEMICAL PECULIARITY HISTAMINE ABUNDANCE
WILANDER observed that the blood of dogs in peptonic or anaphylatic shock increases in both histamine and heparin content. This finding, confirmed by ROCHA and SILVA (1952), remained unexplained until the research of RILEY and WEST (1952). These authors noted a connection not only between heparin and mast-cells, as reported by the Swedish investigators, but also between mast-cells and histamine:
Histamine Mast-Cells Rat liver 0.3 0 - Pig Aorta 0.7 0 Bovine Aorta 10 Rat Subcutaneous - Connective Tissue 16 . 4-4
Bovine Inferior Vena Cava 20 +4- Bovine Glisson's Capsule 40 +1L+
Bovine pleura is richer in mast-cells than Glisson t s capsule from the same animal; in fact, it is the histamine-richest tissue known to date (200 to 280 microg/g). Mastocytomas in the child and the dog are extremely rich in histamine; in one case, RILEY and WEST (1953) were able to assay 1,000 microg of histamine per gram of tumour tissue.
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Finally, RILEY derived some interesting observations from the. use of potent histamine liberators. After rapid intravenous injection of a lethal dose of stilbamidine or 2-hydroxystilbamidine, the fluorescent diamidine can generally be observed in the cytoplasm of peritoneal mast-cells. If the injection is given slowly, the mast-cells are vacuolated or they burst. Analogous changes occur in rat mas t-cells after injection with Bordet's anaphylatoxin. In both cases, however, such changes can be prevented by administration of antihistamines.
Nettleship's disease, characterized by proliferation of the connective tissue elements predominant around the blood vessels and sweat glands and having melanocytes, plasmocytes and lymphocytes dispersed among them. Recent studies include those of SUTTON (1939), ELLIS (1949) and DRENNAN (1951 ),.
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This increase in the number of mast-cells, followed by the dispersal of their granules during anaphylactic shock (STUART, 1952), may consequently be interpreted in various ways. It may be regarded as representing a defense reaction of the organism, since heparin protects sensitized pigeons against anaphylactic shock (KEYS et al, 1926), in the same way that it protects guinea-pigs (WILLIAMS et al, 1927) or rabbits (SILFERSKIOLD, 1940), but this increase may also arise from a release of histamine since it is inhibited by antihistamines.
ZOOLOGICAL DISTRIBUTION
I. - Invert'èbrates.
In invertebrates, mast-cells appear to be entirely lacking.
I personally have never encountered, either in passing or in the course of research of another kind, any elements resembling mast-cells in sponges, coelenterons, echinoderms, worms, mollusks, arthropods o2 tunicates. Moreover, no systematic research using the most appropriate detection techniques has, to my knowledge, been undertaken in this direction.
Nevertheless, ZOTTA (1922) described "mastocytes" in the blood of the imago of Carausius morosus, Philium sp. and Cyphocranio gigas; he also reported them in Truxalis nasuta and in various locusts; moreover, ZOTTA is of the opinion that the amphophilic elements in Mantis religiosà described by KOLLMANN (1908) could be placed in the same category. In fact, working either alone or with my collaborators (1944-54), I have never seen any similar elements in the numerous normal and abnormal insects that I have examined either in the course of blood studies or in searching for leukopoietie centres.
md J.
_ a 330896 MT 6 7/ 2
II. - Lower Vertebrates.
Although mast-cells in lOwer vertebrates were described as early
as 1878-80 by EHRLICH and his student WESTPHAL, comparative histology and haematology alike provide very few data on these elements. Most of the observations made on them are old and sometimes contradictory. Clearly a methodical inventory of the mast-cells in lower vertebrates still remains to be made.
A) Fish. - "As far as present investigation has gone the mast granules in fish are by far the most soluble in the animal kingdom." (MICHELS, 1923).
RAWITZ (1899) and GRUN BERG (1902) were not able to find any mast-cells in Scyllium catalus, nor was MEINERTZ (1902) in Perca, so that, as late as 1909, PAPPENHEIM believed that fish did not possess any: he himself had not been -
md 330896 MT 68/1
able to find them in the hagfish, nor in Esox lucius, Petromyzon or -Gadas. At the same period, WEIDENREICH shared this opinion.
DRZEWINA (1911), in her study of 68 teleost species, did not report the existence of mast-cells, although she was familiar with these elements, having studied them in Didelphis lanigera (1903).
Nevertheless MICHELS (1923), using Maximow's technique, found numerous mast-cells in the swim-bladder of Leuciscus sp.; they are particularly dense around the blood vessels, which they sometimes cover completely. They are also abundant in the mesentery, the peritoneum and especially in peritoneal fatty tissue. Absent from paraffin sections of the alimentary canal, they appear numerous in frozen sections of the lamina propria. They are not found in paraffin sections of the liver and kidneys, but can be seen in kidney accretions.
The swim-bladder of Cyprinus çarpio contains mast-cells arranged parallel to the muscular fasciculi and the vessels. Their granules stain a dark red with thionine, with slight variations in tint not only from one cell to the other, but also within a given cell. The mast granules of this teleûst are particularly soluble, and specimens mounted in toto stained by Dominici's method show no trace of mast-cells. The same result,is obtained with paraffin sections of swim-bladder, muscle, liver, kidney or gill (MICHELS,.1923).
Anguilla vulgaris (according to MICHELS, loc. cit.) is entirely lacking in mast-cells; MICHELS did not find any in mesentery specimens mounted in toto or freeze sectioned, and concluded: "Hence whilst mast"cells are present in some types of fish, they are lacking •in others." Nevertheless, research in another direction gave me an oppdrtunity to observe very numerous mast-cells in Anguilla vulgaris both in the alimentary canal and the meninges, and the saine result was obtained with Callionymus lyra and Ammodytes lanceolatus.
Ind 330896 MT 68/2
Cvprinus carpi° possesses mast-cells capable of very definite amoeboid locomotion and grouped in a crown formation around the melanophores of the scales (GABE and VEIL, 1948); in a normal carp wei5Iling about 20 g, the count made at a linear magnification of 350 diameters showed a hundred odd mast-cells per microscopic field in the pigmented part of the scales (VEIL and GABE, 1950). It is possible to obtain from the scales of this teleost an extract which retards the coagulation of dog plasma in much the same way as heparin (VEIL and QUIVY, 1950). •
In summary, all that seems necessary for observing numerous mast-cells in fish is to prepare a mesenteric smear or a slide-mounted scale sample and stain it with a solution of toluidine blue, methyl violet or cresyl violet in 9% saline. Piscine mast-cells are generally smaller than those of mammals (7 to 12 microns) and contain fewer granules so that their nucleus is usually visible.
md 330896 MT 69/1
B) Batrachians - JOLLY (1901) stated, without further argument, that mast-cells are distributed throughout connective tissue both in anurans and urodelans. According to him, they are very irregular, dendritic elements with multiple extremities, hence the naine RANVIER (1890-1900). "clasmatocytes" given them by
MAXIMOW (1906) reported the existence of mast-cells in tadpoles, - young fogs and axolotib. He found the mast-cells of the latter to be both very irregular and very large, reaching as much as mm in length; . • they extend pseudopods which do not anastomose with those of adjacent mast-cells. These elements are particularly numerous in subcutaneous connective tissue, the perimysia and adventitia of the vessels. They can multiply by mitosis if stimulated by the introduction of a foreign body.
ARNOLD (1906) described elements very reminiscent of the preceding in thefrog. He noted that the mast-cells were sometimes binucleate, that their chromatin was filamentous and usually arranged like the spokes of a wheel,_and finally that after vital staining the swollen granules tended to cluster together.
PARDI (1909), examining . anurans and urodelans described mast-cells as much as 1 mm in length in Triton cristatus.
PAPPENHEIM' (1909) confirmed the existence of mast-cells in Rafla temp'oraria and WERZBERG (1911) in R.'esculenta.
Finally, MICHELS (1923) made an inventory of the mast-cells of
Rana esculenta and R. pipiens. Thesubcutaneous connective tissue contains numerous mast-cells and the body wall is thin enough to permit their examination in specimens mounted in toto; the mesentery, peritoneum, spleen, intermuscular connective tissue and lungs are also rich in mast-dells; however, the latter are rare in the liver and kidneys.
md 330896 MT 69/2
In Ambystoma, mast-cells abound in the mesentery and the digestive mucosa Where they are preferentially located under the villi; extremely variable in appearance, they are always very large.and difficult to . distinguish from pigment cells; the lung and liver contain only rare- mast-cells.
Cryptobranchus alleghaniensis is not believed to posses any mast-cells in the mesentery, alimentary canal, lung: or liver.
In fact, the same criticism is applicable to all the preceding investigations: they all used Maximow's method or variations of it. The specimens to be stained had first to stand for several days in 0 over 70 alcohol (since water tends to spoil the iparticles even after treatment With absolute alcohol), before being stained for 10 to 20
minutes in 70 g alcohol (or absolute alcohol) saturated with thionine under addition of 11 drops of 2% sodium carbonate per 10 cc of thionine in alcohol solution.
Ind 330896 MT 70/1
In reality, vital and postvital mesentery preparations from anurous batrachians slide-mounted and stained with solutions of
toluidine blue, methyl violet or cresyl violet in 6% saline reveal mast-cells very similar to those found in the rat mesentery; if the preparation is left unsealed for as little as 24 hours, however the round, oval or slightly dendritic mast-cells of the day before are replaced by very elongated elements in all respects similar to those described by the preceding authors.
• Mast-cells are found in the choroid plexuses of Iriturus cristatus carnifex, Triturus vulgaris and Hydromantes italicus germani (MAZZI, 1954).
C) Reptiles. - a) Chelonians: EBERHARDT (1907) reported the . existence of mast-cells in the turtle and noted that its spleen was. . . remarkably rich in them.
Using Maximow's method, MICHELS (1923) studied 4 turtles: Chrysemys picta, Emys europea, Clemys leprosa and Testudo mauritanica. Whereas numerous mast-cells are found in the subcutaneous connective tissue, mesentery, spleen, mucosa and
submucosa of the alimentary canal, liver and lung of the first two species, Clemys appears to have no mast-cells in its alimentary • canal, either in paraffin or frozen sections. In lestudo mauritanica, the spleen contains only a few mast-cells and the peritoneum, muscle —tissue, alimentary canal, lung and kidney contain none at all. The author finds only one possible explanation for this difference; namely, that Testudo was examined at the end of hibernation, exactly two days after its reawakening, while the other three species were examined before hibernation.
md IL
33089 6 MT 70/2
b) Lacertilians: It has been known for some time that Agama inermis (PAPPENHEIM, 1909), Hemidactylus and Gongylus (WERZBERG, 1911)
possess mast-cells. In Gongylus ocellatus, numerous mast-cells are found in connective tissue, spleen, peritoneum, muscular aponeurose, lung and liver, whereas they are scarcer in the alimentary canal (MICHELS, 1923).
In Phrynosoma coronatum, the mast-cells are extremely numerous
in the mesentery and always of very large size in the alimentary canal, tongue, lung and liver; they are rare in the spleen (MICHELS, 1923).
The choroid plexuses of Lacerta muralis nigriventris and L. sicula campestriS contain mast-cells (MAZZI, 1954)
Paraffin sections of Tarentola mauritanica exhibit numerous mast- cells in the meninges and, after postvital.toluidine blue staining, the mesentery from the same animal shows numerous mast-cells with their usual predominance around the vessels (ARVY, previously unpublished).
D) Birds. - The mesentery of birds possesses mast-cells; "they are generally few, small, irregularly rounded or elongated and are predominant around the vessels" (JOLLY, 1923).
md 330896 MT 71/1
•;
Mast-cells are found in the connective tissue of Meleagris gallopavo,
Gallus domesticus, Columba palumbus, Passer domesticus and various
Corvides (Corvus frugilegus, C. Corone and C. monedula) (ARVY, previously unpublished).
In the spinal ganglia and their posterior roots, the mast-cells of the immature domestic chicken are, according to BRUSA, more numerous than in the guinea-pig or rat; in the peripheral nerves of the chicken, they are smaller and more streamlined than in the latter two species and their granules are less nuMerous (BRUSA, 1954).
E) Mammals. - With use of the appropriate techniques, mast-cells have been invariably found in man, rhesus monkey, macaque, cow, horse, dog, cat, goat, sheep, pig, hare, rabbit, guinea-pig, hamster, bat, rat,. and mouse
Their abundance varies according to the species: Jolly found them to be scarce in rabbit, hare and guinea-pig and abundant in bat, '
goat and calf (JOLLY, 1923):
HISTOLOGICAL DISTRIBUTION
Mast-cells are invariably found in the skin of healthy adult mammals; they hug the collagenous fibres or are arranged along the dermal vessels, around the sweat glands or the hair follicles. They are relatively abundant in the aorta wall of some animals (cows, horse)
and rare in the pig aorta (HOLMGREN and WILANDER, 1937; RILEY and WEST, 1953). They are also numerous in the cornea (JORPES et al, 1937). Mast-cells have been found to be remarkably abundant in the tongue
of the bat (WESTPHAL, 1880; WILLIAMS, 1900) and entirely lacking in the tongue of the rabbit. They are also found in the connective tissue of the thyroid and variouà salivary glands; they are rare in the capsules of the thymus and spleen and not abundant in the connective tissue of the epididymus, prostate and bladder.
mci 330896 MT • 71/2
SUOMALAINEN, HARMA and LETHO (1951-52) observed numerous mast-cells
-in the lung of Erinaceus around the bronchi.
HAMMAR (1894), PETERSEN (1930), DAVIES (1942) McDONALD (1948) and ASBOE-HANSEN (1950) consider mast-cells to be the predominant elements in the synovial membranes of man.
ROSENHEIM (1886), RAKMANOW (1912), PALEARI (135), ROSSI and
GASTALDI (1935) and CARGNALLO (1940) observed mast-cells within the sheaths of Schwann in the peripheral nerves of normal man; they were found to increase during certain infections (syphilis, poliomyelitis,
meningoencephalitis and toxic neutris (MEDEA, 1906);
md 330896 MT 72/1
ROSSI, 1929; BIELCHOWSKY, 1935). ZIMMERMANN (1908) observed mast-cells in the peripheral nervous system of the guinea-pig; ZALLA (1909) reported them in the dog and BOSSI (1935) in the cow.
MEYER (1906) and TOBINAGA (1936) described mast-cells in the gasserian ganglion in man.
McKibben (1914) observed mast-cells in the meninges of Necturus and MAZZI (1954) and ARMY (previously unpublished) have found them in the choroid plexuses of various Lacertidae.
The presence of mast-cells in the hypophysis and infundibulum were reported by GRAY (1935); . it has recently been confirmed in the hypophysis of man (CASTIGLIONI, 1947; TINEL and VINEUX, 1952).
MONTAGNA and NOBACK (1946) noted that the preputial gland (1) of the rat is particularly rich in mast-cells; they are also abundant in that of the mouse (ARMY, BOIFFARD and GABE, 1954).
The rat and mouse uterus contains mast-cells in the myometrium, at the base of the endometrium and around the uterine glands. There. are only a few in the medial plain of the uterus, the number increasing from the middle of the horn cf the uterus toward the ovarian sac and the vagina; it is rare not to find any within the ovarian capsule (ARVY, previously unpublished). MacKAY (1950) reported the presence of mast-cells in the endometrium, myometrium and the uterine mucosa of woman. TINEL and VIMEUX (1952) observed numerous mast-cells in the basal layer of the vaginal epithelium of woman, cow and ewe; they noted that, while the ovarian stroma of.young woman is rich in mast-cells, that of post-menopausal woman contains none. According to them, the ovaries of the doe rabbit and the ewe contain no mast-cells. BRUSA
(1954) noted an increase in the number of mast-cells in the uterine wall in the course of post-gravid return to menstruation.
md 330896 MT 72/2 4
JANES and MacDONALD (1945) reported numerous mast-cells around the glandular acini of the breast in woman; mast-cells are also numerous in the cow's udder (TINEL and VIMEUX, 1952).
There are numerous mast-cells in the unbilical cord (PRAKKEN and WOERDEMANN, 1952) but the placenta of wcaman does not contain any, at least during the third month of pregnancy (TINNEL and VINEUX, 1952).
In the normal condition, the parenchymas of the kidneys, liver, lungs, spleen, tendons, adrenal glands, testicles and epiphyses have not been found to contain any mast-cells..
The mesentery and omentum, the hepatic capsule and the pleura are. always rich in mast-cells.
(1) And not the prepuce of the female rat . (!), as DRENNAN (1951) maintains41
md 330896 11 r 73/1
ORIGIN OF MAST-CELLS
THEIR APPEARANCE IN THE COURSE OF ONTOGENESIS
Some authors believed that mast-cells originated in the blood and were formed from lymphocytes, large mononuclears or plasmocytes (WEIDENREICH, 1908; DANTSCHAKOFF, 1910; SABRAZES and
LAFON, 1908; DOWNEY, 1911; MICHELS, 1922). CHEVREMONT (1950) still maintains that "created in the bone marrow, mast-ceils pass into the blood" and can leave the vessels to "take up residence in the various connective tissues" (loc. cit., p. 274).
Others believed that mast-cells were formed from migratory white corpuscles (VAN DER STRICHT, 1901) and represented only degenerating leukocytes, their granules being only by-products of mucoid degeneration (WEIDENREICH, 1908-10, PAPPENHEIM, 1904-08)..
EBERHARDT (1907) induced an aseptic inflammation in Emys, lutaria by introduction of a foreign body in its tissues, and observed "the mobilization of the eosinophilic leukocytes and the lymphocytes from the vessels"; next "polyblasts" appeared which phagocytosed the tissue mast-cells while basophilic leukocytes migrated toward the affected tissue, became hypertrophied and chamged into mast-cells which finalli surrounded the capsule of connective tissue enveloping the foreign body.
In birds, EBERHARDT (loc. cit.) and SOLUM (1908) assume that basophilic leukocytes can emigrate from the blood to the tissues and be transformed into mast-cells.
In fish, MICHELS (loc. cit.) makes the assumption that mast-cells appear in the mesentery, swim-bladder and kidney at the expense of parenchymal cells, whereas in reptiles they are differentiated from "lymphocytes" in the spleen. However, some of these "lymphocytes" have a lobed nucleus and others are even polynucleated, so that it is impossille to know what MICHELS means by the term "lymphocyte". Finally, the mast-cells of amphibians are allegedly differentiated from "polyblasts" (MICHELS, 1923). fs 330896 MT 73/2
jOLLY (1923) seems to have been the first to refuse this connection between mast-cells and leukocytes: . "Lymphoid mast-cells are distinct cells; they do not result from the degeneration of other types of medullary cells." He demonstrated that, during the second half of a mouse or rat embryo's life, the connective tissue has a fibrillated texture and it is possible to observe mast-cells arising by differentiation of ordinary connective tissue cells (JOLLY, 1901). 330896 Ma 74/1
Studying various foetuses (human, rabbit, cat or rat), HOLMGREN (1946) was able to analyse the differentiation of mast-cells at the expense of mesenchymal cells. According to COMPTON (1952), however, hamsters less than a day old do not exhibit any mast-cells; it is only around their third or fourth day that any are observed; ' ROSENHEIM (1886) did not observe . mast-cells in the sheaths of Schwann of man until after the fifth year, but noted that they were numerous in the aged.
HELLSTROM and HOLMGREN (1947) beleive that mast-cells are less numerous in subcutaneous tissue, that their particles become more disperse and less metachromatic as the subject ages and, still' according to the authors, women over-;0. invariably' passess , more mast-cells than men.
MULTIPLICATION AND FATE OF THE MAST-CELLS
In the adult mammal, it is exceptional for mast-cells to,- undergo mitosis (MAXIMOW, 1906-11;-.DOEYÏ 1913). .71à72.-the mammalian - embryo, however, mitosis is not a rarze,,a(7=rr.enceLEEZEW, 1924; LEHNER, 1924; NAGAYO, 1928; DERINGER and DUNN, 1947).
A number of authors have reported mitosis of the labrocytes- in lower vertebrates; MAXIMOW (1906 ).served that the introducti, ,, • of a foreign body provoked proliferatiOn of the 'màst-cells in the axolotl, and that it was not unusual for them to undergo mitosis. EBERHARDT (1907) observed mitosis of the mast-cells in the turtle spleen; ARNOLD (1906) saw mast-cells undergoing mitosis in the frog; DANTSCHAKOFF (1910) observed numerous occurrences of mitosis in the mast-cells of the Tropidonotus natrix embryo, and MICHELS (1922) reported it in mast-cells of the sp_1.t of the Gongylus lizard.
Some authors have regarded mast-cells with double or dumbbell-shaped nuclei as undergoing amitotic division (SABRAZES and
LAFON,.1908; LEHNER, 1924; STOCKINGER, 1927; BLOOM, 1942). fs .330 896 trr 74/2
In its final stage of development when the cytoplasm is •
packed with particles and the nucleus is pyknotic, the mast-cell
appears to undergo a.kind of holocrine dissolution; the cell membrane
tears and the granules scatter while the karyorrhectic nucleus is
phagocytosed. In tissue cultures, PAFF and BLOOM (1949) observeà granules being expelled from the cell and dissolving; they interpreted • this finding as the release of a heparin compound by the cell and were of the opinion that the secretory cycle implies a degenerative
process which ends in the cell's death; MAXIMOW, (1904), STAEMMLER (1925)
and QUENSEL (1933) had earlier described mast-cells as "one-celled
. glands of the connective tissue", a concept already put forward by RANVIER (1870) in explanation of blood leukocytes. 330896 VC: 75/1
RELATIONSHIP OF MAST-CELLS TO OTHER CELLS
• I. - J4ekoc tes. A number of authors assume that there is a close relationship between mast-cells and the leukocytes with polymorphous nuclei and basophilic granules found in blood and bone marrow; some Anglo-Saxon authors do not differentiate between "mast-leukocytes", "blood mast-cells" or "basicytes" i5çeferring to leukocytes with basophilic
granules, whereas others make a distinction between these elements, as found in blood or bone marrow, and "tissue mast-cells".
The situation in poikilotherms is thought to be quite different from that in homeotherms. According to WEIDENREICH, DANTSCHAKOFF, PARDI and PAPPENHEIM (loc. cit.), cells with basophilic granules are ubiquitous in poikilotherms and can function equally well in the blood and the connective tissues. MAXIMOW (loc, cit.) opposed this theory; he believed that, even in lower vertebrates, there were two ontogenetically and morphologically distinct types of basophilic cells peculiar to tissue and blood respectively; their only common characteristici he stated, was the presence of metachromatic basophilic granules in both types. However, mast granules are fine, register a pale colour with basic stains and are very abundant in the cytoplasm, whereas the leukocytes granules are larger, bulkier, less numerous and stain so intensely that they appear black.
• Indubitably, there is a wide gap in our knowledge of this subject, owing in part to the fact that haematologists do not study connective tissue and histologists do not study blood and bone marrow.
Some authors have assumed a complementary correlation between mast-cells and basophilic leukocytes. MAXIMOW (1906) reported that the rabbit possessed only a few mast-cells, whereas its basophilic leukocytes were relatively numerous; on the other hand, the cat, rat and mouse which possess numerous mast-cells are poor in basophilic leukocytes. Moreover, the same is true of Ambystoma, Clemys, Leuciscus and Cyprinus. Nevertheless, Gongylus, p_h_r_y_nauma, Chrysemis and Emys, are well supplied with both mast-cells and basophilic leukocytes and
fs 7r31.:91i-M'eZei.ere.TerMe-7.1:7'e."-?7''',''' 330896 •-Ur 75/2
çryptobranchus which has basophilic leukocytes appears to be entirely
lacking in mast-cells (MICHELS, 1923). •
In the light of our durrent knowledge, we can do little more than take note of the various observations, even if they appear strange or inexplicable. It has been reported that hyaluronidase is one of the factors resulting in the excretion of granules by basophilic leukocytes of the blood; the effect of this enzyme is allegedly to cause excretion of the granules so that the leukocyte-, once deprived
fs :330896 76/1 of them, reveals its chondriome and centrosome. In this way, all , that is needed to follow the creation of new cytoplasmic inclusions is to apply a neutral red vital stain (BEAUZAMY-MODAY, 1954). It should be noted, however, that this action of hyaluronidase was detected in basophilic leukocytes taken from a subject suffering from basophilic myelogenous leukemia and it is not certain whether the basophilic leukocytes of a normal subject would behave in the same way. If not, it should be concluded that basophïlic leukocytes of the blood and mast-cells are essentially different, since parallel trials, both in vivo and in vitro, using either bacterial or testicular hyaluronidases, did not bring out any discernible change in the mast granules on any occasion on which the experiment was carried out on mast-cells of apparently normal connective tissue (DAVIES, 1942); WISLOCKI et al, 1946; COMPTON, 1952), or on mast-cells of the peritoneal fluid from the adult white rat (AM7Y, previously unpublished).
In a recent studv (1) , TURCHINI and KHAU VAN KIEN investigated the histochemical properties of basophilic leukocytes of the blood in the course of infections involving an increase in the number of these elements (cirrhosis of the liver and chronic myeloid leukosis). In the authors' opinion, "it seems that the basophils, during their normal progression from marrow to peripheral blood and depending on the ambient medium, secrete a whole series of substances, the end product of which is heparin. This normal metabolic process may be disturbed during insults, mild pathological infections and chronic illnesses. The basophilic elements, as carriers of the necessary material, are capable of degrading or breaking down the complex products to provide, as needed, the means of eliminating or stemming the propagation of toxic substances, as is demonstrated by phase-contrast microscopy of living tissue. Under the effect of foreign substances such as hyaluronidase, the basophil.s excrete their granules. In the course of chronic myeloid leukosis, the granules in immature cells in the circulating blood are rich in hyaluronic acid. By contrast, the more advanced elements exhibit granules containing heparin derivatives which do not go beyond the complexity of heparin monosulphuric fs 330896 0 1 76/2
acid and are probably accompanied by the formation of intermediate derivatives such as chondroitin sulphuric acid". Thus the research of TURCHINI and KHAU VAN KIEN establishes a lin k between normal and deviant or arrested metabolism during heparin synthesis. It suggests that "the basophils of the blood perform a defence function implemented by degradation of the granular elements of the cell or
1 - destruction of the latter acdompanied by the release of products necessary for the elimination of toxic substances. The two phenomena of normal and deviant or arrested synthesis may take place simultaneously in the peripheral blood. In non-leukemic blood, the synthesis resulting only in hyaluronic acid gives way to noimal or paranoimal heparin s mthesis, followed by degradation of the heparin derivatives as far down the scale as hyaluronic acid and
th (1) Communication to the 5 International Congres!1, Paris, September 7-12, 1954.
E s 330896 MY 77/1
even below." During their investigations, however, TURCHINI and MEN found that, in general, the reactions of the various constituents of basophilic leukocyte granules are always less well-defined and more difficult to obtain than with mast granules.
Nevertheless, it seems necessary, despite the very similar reactions of their granules, to separate leukocytes with polymorphous nuclei and basophilic granules from mast-cells.
Like leukocytes, mast-cells are differentiated mesenchymal cells, but, in the normal animal, they undergo differentiation in extramedullary connectives tissue while leukocytes are differentiated in bone marrow.
Leukocytes witli basophilic granulation are the same size as neutrophils or eosinophils, that is, they are generally smaller than mast-cells: their granules are usually coarser and less numerous- and their nucleus is generally polymorphous whereas mast-cells have •a single, occasionally slightly indented nucleus.
In bone marrow, stem cells are found, promyelocytes and myelocytes with basophilic granules, which evolve into basophilic leukocytes. Their nuclear morphology presents no analogy with that of mast-cells. The evolutionary line of leukocytes with basophilic granulation is homologous fo that of leukocytes with neutrophilic or eosinophilic granulation.
One argument for the absence of a connection between leukocytes • with basophilic particles and mast-cells is furnished by pathology. Some cases of basophilic leukemia have been encountered and, in the rare instances in which a search for mast-cells has been made, the distribution and abundance of the latter presented no anomalies. Moreover, cases of mastocytosis and mastocytoma have been encountered which were not associated with any change in the evolutionary line of the leukocytes with basophilic granulation or in the percentage
Ind 330896 mr 77/2 of the latter among the other blood leukocytes: the mast-cell proliferation by which Nettleship's disease is characterized, for example, is not accompanied by any increase in the number of blood leukocytes with basophilic granules.
Nevertheless, it is conceivable that, like other types of leukocytes, those with basophilic granules may fortuitously pass into the tissues and that, inversely, *mast-cells may pass into the bone marrow or bloodstream, since they are migratory connective tissue cells.
In addition, it is quite certain that mast-cells may appear in the bone marrow in the course of various myelopathies. Their presence has been associated with lymphoid leukemias, plasmocytoses, agranulocytoses, splenopathic medullary inhibition, Hegglin's (1) anomaly , haemolytic anaemia, Marchiafava-Micheli's anaemia and aplastic anaemia, and in every case, the presence of mast-cells suggests a poor prognosis (UNDRITZ, 1946; LEITNER, 1949; ROHR, 1949; BREMY, 1950; FADEM, 1951). •
(1) Polyphyletic disorder common in adolescence characterized by lasting basophilic deposits in the cytoplasm known as Doehle's inclusion bodies.
md 330896 HT 78/1
FADEM (1951) observed mast-cells in human bone marrow in 7 out of 2,800 cases examined; in 4 cases, the mast-cells represented less than 1% of all cells. In one case of aplastic anaemia accompanied by leukopenia and thrombocytopenia, however, the only cellular elements of the medulla appearing intact were plasmocytes, lymphocytes and binucleate erythroblasts. There were purpura and haemorrhaging. Over a period of 7 months, the mast-cells increased - from 2.7% to 7%; at the time of death, the marrow no longer contained cells other than lymphocytes, plasmocytes and mast-cells. FADEM is of the opinion that, when the preportion of mast-cells reaches a considerable level, the quantity of heparin released is sufficient to inhibit multiplication of the other cell categories and medullary aplasia becomes irreducible; according to FADEM, then, anaemia results from mastocytosis. •
11. Ranvierl, s Clasmatocytes.
Using the methods of Ehrlich (acetified dahlia violet) and Ranvier (violet 5B), JOLLY (1900) made a comparative study of the clasmatocytes and mast-cells of rabbit, guinea-pig, rat, dog, man, frog and triton. "In the triton where the clasmocytes are easily recognized by their enormous dendritic and fragmented protuberances", these elements may be considered as identical to mast-cells: thy are dendritic mast-cells which undergo clasmatosis. -"Morphologically speaking, the dendritic and fragmented form shown by the clasmatocytes of Batrachians is also the one in which mast-cells most frequently appear in the connective tissue of these animals" (JOLLY, loc, cit.).
On the other hand (JOLLY dixit), the epiploon of rabbit, guinea-pig, rat, dog or man, treated by Ranvier's method, exhibits numerous clasmatocytes with often moniliform, sometimes very long protuberances and having rounded, flared extremities. Staining the same epiploon by Ehrlich's method reveals mast-cells which are
md 330896 PU- 78/2
large and numerous in the rat, small in the dog, less numerous in man, scarce in the guinea-pig and exceptional in the rabbit. The clasmatocytes in mammals described by RANVIER differ from mast-cells" (JOLLY, loc. cit.).
FUNCTIONAL CHANGES IN MAST-CELLS
I. - Variations in the Normal Subject: Fasting and Hibernation.
Mast-cells have been found to increase in the frog when, after a long period of fasting, it is offered abundant food (KORYBUTT- DACZKIEWICZ, 1878; STASSANO, 1900); however, BALLOWITZ (1891) observed no change in the mast-cells of these hibernators, either in autumn when the animals are well-fed or at the spring reawakening when they are emaciated by several months of fasting.
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: • • • ••• ' • • • - 4.-- ' . • .• 0 • • .„ •. • r • • ' 4•••'.- • •• • • go • ci.. ' . . . -• - - . •• • ,.. • ._._.'.1,
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FIG. 1. - Rat thymus after treatment with cortisone, Bouin's solution, cresyl violet and pidro-fuchsine.
FIG, 2. - Juxta-pancreatic rat mesentery after treatment with thiourea, Bouin's solution and Dominici's stain.
330896 HY 80/i
BALLOWITZ (1891) starved yesnEtilo bats for 6 months, killing them at various times during winter sleep; in autumn, the mast-cells were found to be very numerous in the various organs; during the winter, they decreased and their granules were smaller and less . chromophilic.
ROSSI and VILIANI (1935) observed an increase in the mast-cells of the intestinal lamina propria in mice and guinea-pigs subjected • to a long fast.
SUOMALAINEN and H.F:RMA (1951) observed considerable hibernation-dependent variations in the number of mast-cells in
Erinaceus europeus. Normal Hibernating Awaking Hedgehog Hedgehog Hedgehog 4.3 24.3 14.1 Mast-cells in the small intestine... 5.9 18.2 7.9 4.2 11.9 - 27 75.5 69.7 Peribronchial mast-cells 39.2, 70.2 74.5 25.4 92.3
SUOMALAINEN and his students (1951-53) have established a connection between the increase in the number of mast-cells during hibernation and winter bradycardia and hypocoagulability of the blood.
Not only does the number of mast-cells increase greatly in hibernating hedgehogs, but their appearance is greatly modified; during the summer, the mast-cells are very rich in granules, only a small number being poorly supplied with them. During the winter, however, the•proportiOn of mast-cells analogous •to those previously observe'd in the dog during peptonic shock (WILANDER, 1937), that is mast-cells containing only a few granules distributed mainly, if not exclusively, around the cell periphery, becomes enormous.
md 330896 NT 80/2
' Since in the hibernating Erinaceus (SUOMALAINEN and SARJAS, 1951), the heart beat is weak and the circulation slows down, the danger of' thrombosis would be great if the blood were not hypocoagulable
(SUOMALAINEN and LEY1TO, 1952). For this reason the mast-cells. increase and excre"hs their particles lvishly (SUOMALAINEN and
HARMA, 1951).
Citellus, the American squirrel, shows a longer , blood coagulation. time during its summer sleep and it would be interesting to know whether corresponding changes take place in the mast-cells.
II. Experimentally Produced Variations in Mast-Cells.
Cortisone has been found to inhibit the activity of mast-cells in embryo skin or spleen cultures in vitro (PAFF and STEWART, 1953). In man, rabbit, guinea-pig and mouse, ASBOE-HANSEN (1950-52) has reported a decrease in the number of mast-cells accompanied by morphological
d
330896 ti
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- .... • . •:.. '....•--"..i•a•-•,i.,_••;---•-",'•-t."'"-• - . • - .. • " 1.C:a. ... 1Y. • ..: ..• • -...:..-'• ..• . . . —...... .— p •• . - "'« - = 4 / -e, . • le - - • '47 «, •''' C.` ' I 1" sf."- 7 :,• • • . - • : . •-•-;""i-•-•• e; • • ' le-,,esr„F; • ce. • - 6 "- - • •-•. 1;1 • *- : 4 -t r -.0 •„. f •,. - .• , - es :•-e•••ser * . . e e . .1 a • • -----;••;*1 : '! • • ,F • - •••• „,• •-• ...t.....„›.40• • r - • • • .,.-h•• ••••-• • ' t • " • • , • • • 4 • • -. • •.' ' • - • •••• • 1•• • lt • -• • j.-••• • • • • ./ .4. 14J
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FIG. 3. - Mouse . lymphatic ganglion after X-ray treatment, Bouin's solution, toluidine blue and picro-fuchsine.
FIG. 4. - Mouse vagina after folliculin treatment, Bouin's solution and Dominici's stain.
md
, • • 330896 Ni 82/1
changes consisting in vacuolation of the cytoplasm, coalescence of the granules and irregularity of the cell contour after cortisone or corticotrophic hormone administration. CAVALURO et al (1951), STUART (1951), FULTON and MAYNARD (1953) and BAKER (1953) reported
similar variations in the mast-cells after cortisone injection.. On the other hand, SCHOCK and CLICK (1953) did not observe this • effect and ARVY et al (1952) invariably observed an increase in the . number of mast-cells in the thymus and the lymphatic ganglia of the mouse after cortisone administration. Under the effect of thyroxine treatment, mast-cells have been
. found to dwindle and lose their granules (ASBOE-HANSEN, 1950), On the other hand, an injection of thyrotropin caused an increase in the number of mast-cells in the guinea-pig (ASBOE-HANSEN and INVERSEN, 1951). Thiourea provoked their increase in the rat (ARVY and GABE, 1950) and folliculin in the mouse (ARVY, in press).
ASBOE-HANSEN (1951) believes that various hormones cause mast-cells to secrete hyaluronic acid and sees a connection between
the absence of this acid in subcutaneous tissue during thyrotoxicosis ' and the scarcity of mast-cells and their dearth of granules.'
SYVEN (1940) at first saw mast-cells lose their granules and decrease in number following X-ray irradiation, then become abundant in the irradiated tissues. SMITH et al (1953) noted coalescence of the mast granules and the rupture of the cellular membrane after X-ray irradiation. ARVY et al. (1951-54) invariably noted an increase in the number of mast-cells as a result of X-ray exposure.
FRICK (1950) saw an increase in the number of mast-cells after heparin injection whereas they decrease after injection with thrombokinase while hepar±nemia becomes more pronounced.
md 330896 NI" 82/2
GIANNICO (1950) observed a drop in the number of mast-cells in . the rat after dicumarol administration.
Nevertheless, DEWITT et al (1954), from their study of mast-cells using injections of thyroxine, desoxycorticosterone, cortisone acetate, heparin, hyaluronidase or a potent histamine liberator resulting from condensation of p-methyloxy-phenyl-ethyl-methylamine with formaldehyde, concluded that the morphology of mast-cells is so variable and artefacts so frequent as to render a valid interpretation of the action of these substances impossible: "in fact, the experimental animals and their controls yielded extremely variable results".
• After section of the peripheral nerve ends of the branchial plexus in the guinea-pig, rat and the young Gallus domesticus; proliferation of the mast-cells around the tigrolyzing and atrophying cells in the corresponding spinal ganglia takes place; whereas, in the normal state, 8 to 10 mast-cells can be counted per section in the root of a noimal spinal ganglion, there are 40 to
50 after resection of the nerve (BRUSA, 1954).
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PIG. 5 - Rat lymphatic ganglion after alloxan treatment, Bouin's solution and Dominici's stain.
FIG. 6 - Rat submaxillary gland after thiourea treatment, toluidine blue and picro-fuchsine.
Ind 330896 MT 84/1
In Cyprinus carpio, an initial injection of horse serum produces a drop in the number-of mast-cells in the pigmented area of the : scales, whereas a second injection given three weeks after the first. results in a substantial although short-lived increase in these . elements; this increase is the only stigma of the anaphylactoid reaction experienced by the carp as a result of horse serum administration (VEIL and GABE, 1950).
Finally, it appears that mechanical or chemical irritation may
provoke the disappearance of granules from cutaneous mast-cells; DRENNAN (1951) observed the same reaction after scraping of urticaria pigmentosa lesions, as did SYLVEN and LARSSON (1948) after daubing of the skin with . phenol, benzene or naphthalene.
III. - Pathological Variations in Mast-Cells.
Man may contract Nettleship's disease characterized by diffuse proliferation of the mast-cells in the subcutaneous connective tissue; this mastocytosis is accompanied by melanocytosis and pruritis.
Dermatologists have noted an unusual abundance of mast-cells in subcutaneous connective tissue as a feature of numerous dermatites: neurodermatitis, lichen planus, urticaria, Besnier's pruritis, pityriasis, chronic eczema, lupus erythematosus, scleroderma, vitiligo, scarlet fever rash, and atrophying poikiloderma (SMIRNOVA-ZAMBOWA, 1926; BURKL and LEONHARTSBERGER, 1951; ASBOE-HANSEN, 1951 and KEINING and BRAUN-FALCO, 1952).
Amyloidosis lesions are rich in mast - cells (CAZAL, 1942).
Perivascular mast-cells are particularly numerous in the course of periarteritis nodosa and myxedema (ALTSCHULER and ANGEVINE, 1949; - ASBOE-HANSEN, 1950..
ind ' 330896 Nï 84/2
Cutaneous neoplasms, sarcomas, myofibromas and lipomas are always accompanied by mastocytosis which is often localized at the periphery of the abnormal tissue. As early as 1883, ACKERMANN reported the existence of numerous mast-cells around sarcomas and remarked that these elements did not eXist in the tumour tissue itself; this observation has been confirmed by many authors in man and other animals both in spontaneous and induced tumours (FROMME, 1906; STAEMMLER, 1921; HIGUCHI, 1930; CRAMER and SIMPSON, 1944).
An unusual abundance of mast-cells has been reported in chronic lesions of varied aetiology: tuberculoma, syphiloma, brown induration of the lungs, myocarditis, cirrhosis of the liver, joint or heart lesions of rheumatic origin, glaucomatous iridocyclitis, etc. (NEUMANN, 1885; HOLMGREN and STENBECK, 1940; ZOLLINGER, 1949; ALTSCHULER and ANGEVINE, 1949; HAMILTON and SYVERTON, 1950; SAPHIR, 1950).
Mastocytomas associated with massive idiopathic proliferation of mast-cells have been observed in man, horse, dog and mouse (SABRAZES and LAFON, 1908; BLOOM, 1948; DERINGER and DUNN, 1947 and OLIVER et al, 1947).
md 330896 NI 85/1
THE ROLE OF MAST-CELLS
"The tremendous number of mast-cells encountered, especially along the blood vessels, is sufficient cytological evidence to warrant the adscription of some important role to these cells" (MICHELS, 1923).
The most varied hypotheses have been advanced in explanation of the role of mast-cells.
Many authors (EHRLICH, 1878; CAJAL, 1896; CALLEJA, 1896) have considered mast-cells to be wandering cells endowed with trophic properties. UNNA (1925) 'regarded mast-cells as one-celled connective tissue glands secreting a substance which disaggregated, dissolved and became . mixed with the interstitial plasma of the organs so as to transmit to them nutritive and perhaps bactericidal properties.
STAEMMLER (1921) claimed that mast-cells elaborated a mucin necessary in the formation of intercellular cement. •
SCHUMACHER (1925) believed that the role of the granules was to transform molecular oxygen into active oxygen and MICHELS (1923) was convinced that mast-cells were oxygen-carriers. These affirmations, supported only b.y its differences in distribution and particularly the predominance of mast-cells near the blood vessels, have found some confirmation in-recent research. In fact, there is a cytochrome C-cytochrome oxidase system in mast granules. Since the cytochrome oxidase catalyzes the oxidation of cytochrome C by activating the oxygen, the mast granules could well be the agents
.of an energy transfer and thus the sites at which oxidation takes place (NOBACK and MONTAGNA, 1946).
md 330896 Hf 85/2
A number of histologists have attached great importance to a "secretory atmosphere", "pericellular metachromatic areas" and extracellular, metachromatic and nongrannular "hazes". Drawing attention to these aspects, PITTALUGA (1922) interpreted them as stigmata of a diastase secretory activity of the mast-cells.. It is now generally assumed that they are fixation artefacts due to the hydrosolubility of the mast granules.
ARNOLD (1906) and WEIDENREICH (1908) believed that mast-cells were phagocytes: since the research of MICHELS (1923), this • possibility is no longer contemplated: "the sole valid conclusion that can be drawn from the present studies is a negative one: mast-cells are not correlated with the process of phagocytosis, for on no occasion was a phagocytosed material encountered in the mast cytoplasm".
RHEINDORF (1905), STAFFEL (1907) and MEIROWSKY (1908) believed mast-cells to be associated in some way with the metabolism of pigment. This hypothesis hàs been refuted by LEHNER (1924) and has not been seriously entertained since.
md 330896 MT' 86/1
ASBOE-HANSEN (1950) put forward the theory that mast-cells play a defensive role against infection through their production of ' hyaluronic acid, a factor controlling cell permeability. That. author claims to have noted a correlation between the abundance, of mast-cells in tissues and their hyaluronic acid content as detectable by chemical and histochemical procedures.
MacCUTCHEON and COMAN (1947) are of the opinion that the sulphuric mucopolysaccharide contained in the mast granules acts as a hyaluronidase inhibitor and can oppose the growth of hyaluronidase-producing tumours.
SYLVEN and LARSSON (1948) have put forward the theory that mast-cells contribute to the detoxification of the tissues and, since 1952, TURCHINI and his students have maintained that mast-cells play a role in the elimination of toxic substances by "acetylation, glycuro-conjugation and sulpho-conjugation" through the action of the heparin compounds contained in . their granules.
GEREBTZOFF has observed the invariable presence of large numbers of mast-cells in the cerebral haemorrhagic centres and BAECKELAND (1950) noted that, in contact with blood and fibrin clots, mast-cells undergo nuclear and cytoplasmic transformations ending in the excretion of mast granules within the clot and the lysis of the fibrin. The author concluded that mast-cells must be a factor, not only in maintenance of the fluidity of the interstitial fluids, but also in the organism's reactions to pathological coagulations of the blood.
The presence of mast-cells in the cornea suggested to JORPES et al (1937) that the'se elements were responsible for the fact that small corneal haemorrhages never produce a clot. In the same way,. it is.not unreasonable to associate the extreme abundance of maàt-cells in the synovial membranes with tho . failure of haemarthroses to coagulate.
md 330896 mr 86/2
Mast-cells may well in some way be a factor in the metabolism of lipoproteines, since there is a correlation among their abundance, the magnitude of alimentary lipemia and atherosclerosis. A comparative study of mast-cell abundance for the same surface area of myocardium showed 193 mast-cells in an atherosclerotic older woman,
276 mast-cells in a non-atherosclerotic woman of the same age and 351 mast-cells in a healthy young woman (CAIRNS et al, 1954). Moreover, it is known from experiments that heparin increases the dispersion of the lipoproteins of high molecular weight as well as the electrophoretic mobility of beta-ceobulins (GOFMAN et al, 1949; KAUFMANN et al., 1953).
CAZAL (1942), PARROT and LABORDE (1951) and DEMPSEY (1952) have conjectured that mast-cells are a source not only of heparin' but also of histamine. The research of RILEY and WEST (1952-53) has' actually demonstrated that mast-cells contain two principles. Yet- nothing is known of the ability of mast-cells to synthesize histamine. All that is known at present is that the activity of heparin is reduced by histamine (GERENDAS, 1948) and that
md 330896 nT 87/1 histamine can be tied by heparin (PARROT and LABORDE, 1951), and that, the histamine-heparin bond being unstable, another amine competing for the heparin can break it. This peculiarity seems worth remembering since, under certain conditions, 1 mg of heparin can tie 1 microg of histamine dichlorhydrate.
CONCLUSIONS
Since EHRLICH's discovery of mast-cells, our conception of these connective tissue cells'has evolved a great deal with respect to their nature, origin, distribution and role. It is no exaggeration to say that, of all the connective tissue cells, mast-cells are now the best known.
The first findings were purely morphological, then the metachromasia of mast granules gave rise to histochemical research and recent pharmacodynamic work suggests that the term "heparinocyte" fits only one of of the cells' characteristics and the term "histaminocyte"_is equally justified.
The various aspects of mast-cells under normal, experimental or pathological conditions are by now well established. They are free connective tissue cells capable of amoeboid locomotion, incapable of phagocytosis, multiplying by mitosis and deriving from the mesenchyma. In passing, or under experimental or pathological conditions, it is possible to find mast-cells in the bone marrow of vertebrates; in this case, however, they are so different from basophilic leukocytes that the possibility is excluded of their being in the saine cellular line of descent. The reticular cells of the bone marrow show none of the characteristic reactions of mast-cells and their nucleus is very different with respect both to morphology and stainability.
md 330896 ;1 1 87/2
The essential characteristic of mast-cells is the richness of their cytoplasm in granules of a diameter not exceeding 0.5 microns; these particles are so abundant as to conceal in most cases the various cell constituent: nucleus, nucleolus, Golgi systems and chondriome; of remarkably uniform size, these mast granules are metachromatic owing to their high content of sulphuric esters, and'their ability to restain fuchsine sulphurous acid points to a metachromasia arising from a heparin monosulphate. This staining reaction, moreover, brings out differences (from species to species, from mast-cell to mast-cell, or even from granule to granule), forcing the conclusion that not all mast granules contain heparin monosulphate; heparin monosulphate saturated in sulphuric acid could yield a heparin bi- or trisulphate, the latter constituent, by far the most active physiologically, being inactive with respect to fuchsine sulphurous acid.
Histochemistry confirms the richness of mast granules in pseudo-perioxidase, cytochrome-oxidase, alkaline or acid phosphatase
and lipase.
md 330896 Mi 88/1
Through experiments on the ileum isolated from the guinea-pig, students of pharmacodynamics have been able to discover relatively large quantities of histamine in mast-cells (1,000 microg of histamine per g of tissue) and to state that, under experimental conditions, in vitro, 1 mg of heparin can tie 1 microg of histamine dichlorhydrate.
Mast-cells have been found in every vertebrate when the appropriate detection methods were applied; their existence in invertebrates is still problematical, if they are to be studied in invertebrates, it would be des'irable to identify both their biochemical and pharmacodynamic properties.
Mast-cells are found wherever there is connective tissue, which means in virtually every organ in vertebrates; However, they exhibit preferential locations which seem to vary from species to species.
The number of mast-cells in a given tissue depends on the physiological state (fasting, hibernation, endocrine balance, various infections).
.In conclusion, mast-cells appear to be polyvalent cells with a particular connection with heparin and histamine metabolism.
(Laboratoire d'Anatomie et Histologie comparées, Sorbonne, Paris.)
(Laboratory for Comparative Anatomy and Histology, Sorbonne, Paris.) • • (Manuscript submitted September 15, 1954.)
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