Cytochemistry of Basophil Granulocyte in Carp and Puffer
Transcript of Cytochemistry of Basophil Granulocyte in Carp and Puffer
Bulletin of the Japanese Society of Scientific Fisheries 52(11), 1895-1899 (1986)
Cytochemistry of Basophil Granulocyte in Carp and Puffer
Yuzuru Suzuki*
(Accepted April 15, 1986)
Basophil granulocytes of carp and puffer were studied cytochemically by light microscope. The basoghil cells were round with an eccentric nucleus and many coarse granules. These
granules were found to be extremely water soluble, aud fixed with basic dyes or fourth amines. They were stained by basophilic procedures and showed metachromasia with basic dyes. These findings indicate that the granules contain acid mucopolysaccharide, and"basophil"is a rational name for the granulocyte in fishes. However, these granules did not stain by astra blue or alcian blue procedures. Furthermore, metachromasia was not seen at pH 1.5 in puffer, and 3.0 in carp. In addition, basophilic material combined with basic dye was extruded easily even in extremely diluted electrolyte solution. These results indicate that the rnucapolysacchazide in fishes does not contain many suffate radicals in the molecule. This indication is in contrast to heparin in mam-malian basophils.
In mammals, basophil granulocytes are the
fe west cellular elements in the blood, and their functional features are not fully understood.
However, in some fishes such as carp and puffers, basophils are rather abundant, even though a few
studies were carried out. This inadequate study
could be due to unsettled nomenclature and dassification of fish leucocytes,1) and even some
investigators believe that the basophils do notexist in fishes. These converse results might be
due to the fact that the granules in fish basophils
are extremely water soluble. Therefore, they have been overlooked in most of the past studies.
In the present investigations, the morphological,
and cytochemical characteristics of the basophils were carefully studied.
Materials and Methods
Blood Preparation
Puffer Takifugu vermicularis, weighing 130-180g, were collected at Misaki, Kanagawa and
carp Cyprinus carpio, 170-350g, were purchased
from a local fish dealer. A toad Bufo vulgaris,
and a rabbit were also used for comparison.Blood was taken from dorsal aorta in carp and
from caudal vessels in puffer, by heparinized
syringes. Blood smears were made for cyto-
chemical observation.
Observations
For general observation, May-Grunwald
Giemsa (MGG) method of staining was employed.
Techniques for histochemical study of poly-
saccharide in the basophilic granules were as
follows: 1) fixation by basic dyes or fourth
amines.2) 2) PAS reaction, and 3) metachromatic
reaction. For metachromasia study, smears were
fixed and stained for 5 min with 1% methanolic
solutions of azure A or toluidin blue, then rinsed
in aquaous solution of hydrochloric acid of pH
1.5, 3.0 and 4.5.
Following enzymes were analyzed histochemical-
l y; peroxidase,3) acid phosphatase,4) alkalin phos-
phatase,5) β-gulucuronidase6) and esterase.7) For
analysis of esterase, two methods were adopted:
a-naphthyl butyrate and naphthol AS-D chloro-
acetate.
Solubility of the granules in electrolyte solution
was estimated by revised method of Scott and
Dorling.8) Smear preparations were fixed and
stained with 1% methylene blue methanolic solu-
tion for 5 min, then rinsed in various concentra-
tions of MgCl2 solutions. Maximum MgCl2
concentrations at which the basophilic granules
did not extruded out were determined.
Living basophils were also observed by a
Nomarsky differential interference contrast sys-
tem.
Results
General Observations
The basophil granulocytes were found to be
* Department of Fisheries, Faculty of Agriculture, The University of Tokyo, Bunkyo, Tokyo 113, Japan
(鈴 木 譲:東 京 大 学 農 学 部 水 産 学 科).
round with an eccentric nucleus. Their dia-
meters were 10 to 12μm in puffer and 13 to 15μm
in carp. In both species, coarse granules were
stained reddish purple (Fig. 1-a, b), which were
similar to rabbit (Fig. 1-c) and frog basophils.
Cytoplasm was stained weakly, and it was grayish
white colour. These coarse granules were highly
soluble in water. Therefore, they were extruded
out by Giemsa stain (fixed with methanol) or by
MGG stain with M/15 phosphate buffer. But
fine reticular structure with vacuoles remained in
the cytoplasm (Fig. 1-d, e). Dilution of dyes with
distilled water was essential for retention of the
granules in MGG stain. In carp, sometimes
degranulation was incomplete and the granules
became eosinophilic after extrusion of the baso-
philic material (Fig. 1-f).
1896 Suzuki
In addition, neutrophil granulocytes were also found in these species. The nuclei of these cell swere in different shapes, i.e. kidney shaped or seg-mented. Their unstained cytoplasm was filled
with fine and slightly eosinophilic granules. Eosinophils were not observed in both species.
CytochemistryThe granules were fixed, clearly stained, and
became water insoluble by 1% methandic solu-tion of basic dyes, such as methylen blue and safranin O. But the granules could not be stained with astra blue or alcian blue, and further de-granulation occured. The granules were also fixed well by methanolic solutions of fourth amines such as cetyl pyridinium chloride and cetyl tri-methyl ammonium bromide. The granules could be stained by Giemsa staining procedure.
Routine methods for metachromatic reactionswere not suitable for this leucocyte, because ofhighly solubility. of the granules. By revised
method, metachromasia was recognized as red-dish purple granules by azure A or toluidin blue, at pH 3.0 and 4.5 in puffer (Fig. 1-g), and at pH 4.5 in carp (Fig. 1-h).
The granules in carp basophils were clearly PAS positive, and the reaction was diastase re-sistant. However, degranulation occured in puffer basophils when methanol, Carnoy's fluid, methylenblue-methanol, and fburth amines-methanol were used for fixation. Therefore, PAS reaction was not recognized. The cytoplasm was weakly PAS positive.
The results of enzymatic cytochemical observa-tions are shown in Table 1. Due to degranula-tion, basophils reactions occured only in the
cytoplasm. In contrast to neutrophils which have active peroxidase and many hydrolases, the baso-
phils showed weak enzymatic activities except- with acid phosphatase.
Degranulation by ElectrolyteDegranulation occured at high MgCl2 con-
centration. Maximum concentration at which the granules did not extruded out was 0.01M in
puffer, 0.001M in carp 0.5M in toada and rabbit basophils.
Observation in the Living StateBasophii granulocytes of both carp.and puffer
had very active amoeboid movements (Fig. 2-a, b). They crawled on glass with lamellipods, com-
parable to those of neutrophils.
Discussion
Cytochemically, granules of basophils in carp, and puffer are characterized as follows: 1) ex-
tremely water soluble,2) insoluble in water after fixation with basic dyes or fourth amines, 3) basophilic, and 4) stain metachnomaticallq. These results indicate that the granules contain acid mucopolysaccharide. Similar results were
ported in basophils in mammals.9) Therefore, "basophil" is a rational name for that granulocyte in fishes.
In carp, the basophil granules were PAS posi-tive, but they do not contain glycogen, because
their reaction was diastase resistance. This find-ing indicates that the granules contain neutral
mucopolysaccharide or glycoprotein. These sub-stances can stain with eosin, when partial de-
granulation occurs. In contrast, the granules were not PAS positive in puffer. And the de-granulation was always complete. Therefore,
they have neither neutral mucopolysaccharide nor glycoprotein.
The basophilic granules showed following natures.1) They did not stain with astra bluc or alcian blue.2) Metachromasia was not seen at
pH 1.5 in puffer and 3.0 in carp.3) Basophilic material combined with basic dye was extruded out easily even in extremely diluted electrolyte solution. These findings indicate that acid muscopolysaccharide in fishes does not have maany sulfate radicals in the molecule. This is in con-trast to heparin in mammalian basopils.9)
"Basophils" have been seen in many species of fish, but they are called under different names.
Basophil in Carp and Puffer 1897
Fig. 1. Light micrographs of basophils. ×1700. a) Basophil of puffer, MGG stain. b)
Basophil of carp, MGG stain. c) Basophil of rabbit, MGG stain. d) Degranulated basophil
of puffer, methanol fixed Giemsa stain. e) Degranulated basophil of carp, methanol fixed
Giemsa stain. f) Partially degranulated basophil of carp, MGG stain. Granules are stained
eosinophilic. g) Basophils of puffer. The granules show methachromasia with azure A
(pH 3.0). h) Basophils of carp. The granules show methachromasia with azure A (pH 4.5).
That is because many workers did not pay enough attention to the nature of the cell as the granules are highly soluble in water. Kelenyi and Nemeth,10) who worked with carp, river bleak andtench, called them neutrophil. Chlebeck andphillips11) called the cell "secretory granulocyte" in two buffalofishes. They thought it was the advanced stage of neutrophil with vacuoles con-taining undissolved particles of the coalesced
granules. On the contrary, Yasuda et al.12) reported that the cells in carp were young neutro-
phils which stayed in the peripheral blood for shortperiod. Hines and Spira13) observed the ce
ll without granules, in carp. Therefore they called it finely reticuiar ceil. Lester and Daniels14) agreed to them and thought it is analogous to eosinophil as were observed in tissue preparations in white sucker. Sabins and Ramgneker25) and others 16-18) called it eosinophil. So far, no one
gave concrete reasons for naming this cell. How-ever in my study, I found that the granules in carp basophils become eosinophilic by partial de-
granulation. Incorrect use of the name "eosino-
Phil" is probably due to this phenomenon.
1898 Suzuki
Table 1. Enzymatic histochemical reactions of basophil (Baso) and neutrophil (Neu) of puffer
and carp
Puffer CarpEnzyme
Baso Neu Baso Neu
Peroxidase - + - +
Acid Phosphatase + + + +
Alkaline Phosphatase - - - -
β-Glucuronidase ± + ± +
Esterase
α-Naphthyl butyrate - + 士 +
Naphthol AS-D chloroacetate 士 - 士 +
Fig. 2. Living basophils of puffer (a) and carp (b) viewed by Nomarsky differential interference
contrast system. They crawl on glass with lamellipods (arrows). ×1800.
Barber and Westerman19,20) observed PAS
positive reaction of the granules of this type of cell in white sucker and named PAS positive
granular leukocyte. They concluded that this cell had no acid mucopolysaccharide from its negative reactions for metachromasia, and had no histamin because it was not affected by histamine liberator 48/80. They thought it may represent an evolutionally precursor of basophil. But most of the figures of light rnicroscopy presented in their reports showed degranulated forms. There-fore, I think reexamination is needed about cyto-chemical reactions.On the other hand, the term of basophil had
been used by Weinreb,21) and Watson et al,22) in
goldfish, and by Fey23) in many species. Fey studied cytochemically in detail and mentioned that the cells were positive for metachromasia, PAS positive, peroxidase negative, and esterase
positive. These results are almost equal to my findings. But in puffer I could not see PAS posi-tive reaction. The basophil of puffer may be
exceptional in fishes.With the exception of acid phosphatase, the
enzymatic activities of basophils were weak. Therefore they have weak ability in sterilization
and digestion. In contrast to neutrophils, the basophils of carp and puffer are not the scanvenger
cells, even both have enough motility.
Basophil in Carp and Puffer 1899
Acknowledgement
I wish to thank Professor I. Hanyu and Asso-ciate Professor K. Aida, Faculty of Agriculture,
The University of Tokyo, for reading the manu-script. This work was supported in part by
Grant-in-Aid for Encouragement of Young Scientist from the Ministry of Education, Science and Clilture.
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