Cellular metachromasia in cystic fibrosis

  • Published on
    06-Jul-2016

  • View
    213

  • Download
    0

Transcript

  • CELLULAR METACHROMASIA I N CYSTIC FIBROSIS

    G. B. REED, A. D. BAIN, W. M. MCCRAE AND F. M. SCOTT Department of Pathology, Royal Hospital for Sick Children, Universiiy of Edinburgh

    CYSTIC fibrosis of the pancreas has been defined as an inherited disease in which there is a dysfunction of all or most of the exocrine glands (di Sant Agnese, 1964 ; Lobeck, 1966). The fundamental cause remains unknown and it has been difficult to suggest a pathogenesis recessively inherited yet leading to a general involvement of exocrine glands, especially when there is a disorder of ionic secretion in some glands and in others a disorder of seromucous secretion.

    The demonstration of metachromasia in cultured fibroblasts from patients with this disease has directed interest to its study at a cellular level. Although the mechanisms by which this metachromasia is produced remain obscure, it has been reported to occur only when the fibroblasts were grown in media supplemented with newborn calf serum (Danes and Bearn, 1969). With this tissue-culture medium, no difference has been observed in the amount of metachromasia between those homozygous and those heterozygous for cystic fibrosis.

    As cellular metachromasia has been observed in tissue cultures from normal individuals and in disorders other than cystic fibrosis the necessity for accurate definition of the in-vitro culture conditions must now assume considerable importance. There are apparently no reports of trials of fibroblast cultures from patients with cystic fibrosis in tissue-culture media supplemented with pooled human serum and relating the incidence of the metachromasia so obtained to the period in culture. Consequently the present investigation was undertaken to obtain information on this point and to see whether, by using pooled human serum, we could show any difference in the incidence of metachromasia between the homozygous patients and the heterozygous parents.

    MATERIALS AND METHODS Skin biopsies from the forearm were set up as explant cultures in stoppered Pyrex baby

    feeding bottles, with chicken plasma and thrombin, and gassed with 5 per cent. COz in air. The growth medium at this stage consisted of Eagles minimum essential medium (Burroughs Wellcome, based on Earles solution) together with chick embryo extract and 20 per cent. inactivated pooled human serum. Outgrowth on the average after 3-4 wk permitted sub- culture into further baby bottles and stock cultures were maintained in this way. Serial subcultures were taken at varying intervals into stoppered 12 x 150-mm test-tubes containing 2 ml of medium, 10 per cent. of serum and a flying coverslip (Paul, 1965). Coverslip pre- parations were examined after 24 hr and some at intervals of 4 and 6 days after subculture. Other subcultures were also made with medium containing human serum in varying con- centrations, with and without embryo extract and at intervals of 3-7 days. Before staining, the coverslips were washed twice in warm phosphate-buffered saline, fixed for 5 min. in methanol, air-dried and then stained with toluidine blue at pH 4.5 for 10 s (Culling, 1963). They were

    Received 3 Dec. 1969; accepted 15 Feb. 1970. 8 . PATH.-VOL. 101 (1970) 251

  • 252

    then briefly rinsed in distilled water and mounted in DePex (Gurr). Each coverslip was examined in the same manner; ten fields with approximately 100 cells were examined along the long axis of the slide; alternate fields were chosen and the entire slide surveyed. All cells with metachromatic cytoplasm within the ten fields were counted, the total being expressed as the number of cells with metachromasia per thousand cells surveyed on each coverslip.

    G. B. REED, A. D. BAIN, W. M. McRAE AND F. M. SCOTT

    200 U

    175 Id E 150

    125

    .-

    V 5 100- s 2 7 5 - c,

    a) I 50- - 2 25- 8 20- zi 15

    - U

    e 10- u m 5 - r

    0 -

    L W

    - 11 patients with cystic fibrosis of the pancreas -

    - - 0

    0 a 0

    0 0 -

    8 :: 0 :*.. 0 - 0 o e

    0

    0 0 o e 00 -

    0 0 . 0

    em 0 . 8 0 0 . 0 - 8 o ' b f p ' & o e e o a m

    O . 0

    * - 0 0 * e

    0 e.o.:o

    1 1 1 1 1 l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

    f

    20 15

    9 normal patients

    5 'I....... e.0 a o e a + e s + s o - r o e o o 0

    5 10 15 20 25 30 35 40 15 50 55 60 65 70 75 80 85 90 a

    95 100 105 -I 110

    Days in culture

    FIG. 1.-Incidence of metachromatic cells in relation to time in culture (normal subjects).

    RESULTS Our findings are based on cultures obtained from 9 normal children,

    8 parents of children with cystic fibrosis and 11 children with cystic fibrosis ranging in age from 1 to 7 yr. Of the latter children, 6 were male and 5 female and all presented the characteristic triad of chronic pulmonary disease, pan- creatic deficiency and elevated sweat electrolytes. The time from primary explant to first subculture varied from 20 to 53 days, The number of subcultures

  • METACHROMASIA IN CYSTIC FIBROSIS 253

    ranged from 5 to 25 and the total time in culture from 52 to 108 days. The morphological changes in the cells consist of cytoplasmic metachromasia similar to that previously described (Danes and Bearn, 1969). These authors separated the staining pattern of their cultures into two metachromatic cate- gories: type I , vesicular metachromasia (A with few vesicles, B with many vesicles) and type 2, diffuse vesicular and granular metachromasia. In our series type-3 cases are those without metachromasia.

    In the present series of cultures from 19 individuals an attempt was made to follow this classification and, on a broad basis, 4 were classified as type 1, 12 as type 2 and 3 as type 3. Difficulties arose in this classification, however, as there was some overlap between type-I and type-2 metachromasia, depending on the coverslip field examined and the passages selected. Nevertheless, as

    8 parents

    L L I I I I I I I I 1 1 I I I I ' I I I I I l ~

    5 10 15 20 25 30 35 10 15 50 55 60 65 70 75 80 85 90 95 100 105 110

    Days in culture FIG. 3.-hcidence of metachromatic cells in relation to time in culture (parents of subjects with

    cystic fibrosis).

    already noted by Danes and Bearn (1969), cultures from individuals in the same family appeared to be of the same classified type.

    In the cultures from normal children, the percentage of cells showing metachromasia ranged from 0 to 2 per cent. (average 0.2 per cent.). In the cultures from children with cystic fibrosis, the metachromasia ranged from 0 to 19.3 per cent. (average 2.2 per cent.)-a ten-fold difference in the mean, This does not, however, reflect the actual distribution, which is better illustrated by the scattergrams (figs. 1 and 2) which, although emphasising the difference between normal individuals and patients with cystic fibrosis, show a wide variation in the amount of metachromasia observed in the latter. The amount of cellular metachromasia observed in a group of parents of children with cystic fibrosis (fig. 3) was similar to that observed in normal children. Variations in the amount of metachromasia were also found in the consecutive subcultures from the same patients with cystic fibrosis and it is of interest that on two occasions the amount of metachromasia in the particular case illustrated in fig. 4 was 0 and near 0. This is in contrast with the observations of Danes and Bearn (1968), who noted a progressive rise with subculture in the percentage of metachromasia to a constant 50-100 per cent.

    J . PATH.-VOL. 101 (1970) R

  • 254 G. B. REED, A. D. BAIN, W. M. McRAE AND F. M. SCOTT

    100

    z 9 0 - g a o - I= .- E

    0)

    z 7 0 - z 2 6 0 - E 5 0 -

    4 0 -

    3 0 -

    3

    +-' L

    VI - - U .-

    2 5 20-

    p 10- (D c)

    0 -

    DISCUSSION Using reinforced Eagle's medium with 10 per cent. newborn calf serum,

    Danes and Bearn (1968) have demonstrated that 50-100 per cent. of fibroblasts cultured from the skin in cases of cystic fibrosis exhibit cellular metachromasia. Cellular metachromasia has also been described in fibroblast cultures taken

    - 1201 110

    t 1 I I 1

    1 I I I I I I I I I I

    I I I I I I l

    0 I

    II u a '

    10 20 30 40 50 60 70 80 90 100 1 I I I I 1 ,

    Days in culture

    FIG. 4.-Variation in incidence of metachromatic cells (one subject with cystic fibrosis).

    from the skin in many other diseases (Matalon and Dorfmann, 1969). Cart- Wright, Danks and Jack (1969), however, have stressed the need for further study of the effect of growth rate and of subculturing on cellular metachromasia. Nadler et al. (1969) have also indicated that many conditions of tissue culture, including length of time in culture, relation of staining to subculture and composition of medium affect the presence and degree of metachromasia. Attention has also been drawn to the importance of the medium, and partic- ularly of its vitamin-C content (Punnett, Kistenmacher and Niederer, 1968). These observations appear relevant to the results obtained when the amount of cellular metachromasia in cystic fibrosis was plotted against the time of subculture.

    Nevertheless, our results confirm that there is a difference between the

  • METACHROMASIA IN CYSTIC FIBROSIS 255

    amount of metachromasia in cultures from patients with cystic fibrosis and cultures from normal children. The amount of metachromasia, however, was generally less than one-tenth of that previously reported (Danes and Bearn, 1968). The reasons for this are not yet apparent, but are probably related to the tissue-culture systems employed. The metachromasia, moreover, in our experience, varied in amount, not only from case to case but also in the indi- vidual subculture from each patient. Metachromasia with toluidine blue is thought to be due to the accumulation of sulphated mucopolysaccharides. We have so far been unable to establish the nature of the stored material, although we assume it to be an acid mucopolysaccharide (AMPS). The intracellular site of the metachromatic material is unknown. As has been postulated in Hurlers disease, the site may be in the lysosomes (De Mars and Leroy, 1966; Van Hoof and Hers, 1968) or in the Golgi apparatus (Matalon and Dorfmann, 1966).

    Dingle and Webb (1965) review the effects of culture conditions on the production of mucopolysaccharides, and stress the composition of the medium as being the most important factor. They also quote other work showing that the rate of polysaccharide production varied considerably in different periods of subculture and was probably related to the time of synthesis of DNA. Our experience has been that with the same basic culture medium, alteration in the percentage of serum or the presence or absence of embryo extract produced no difference in the amount of metachromasia. Likewise, cell dosage, with a range of inoculum between 2 x 104 and 1 x 105 cells per ml per tube, had no appreciable effect. From these preliminary data it is apparent that in relating cellular metachromasia to disease, culture conditions should be accurately defined.

    It is possible that the discrepancy in the amount of metachromasia in cystic fibrosis obtained by us as compared with others might be a reflection of differences in the culture medium. The most obvious difference in regard to the present investigation has been the use of pooled human serum. The signscance of this is as yet undetermined, but it may be relevant to the observa- tions of Spock et al. (1967), who have demonstrated that in patients with cystic fibrosis, there is a serological factor that may, it has been suggested, be a biologically active peptide concerned with membrane transport abnormalities (Talamo, 1969).

    Recent evidence indicates that in the mucopolysaccharidoses fibroblasts cultured from skin exhibit changes that reflect at a cellular level the probably generalised biochemical disorder. Fibroblast cultures from Hurlers disease and Marfans syndrome have shown an increase in total intracellular AMPS largely due to dermatan sulphate in Hurlers and to hyaluronic acid in Marfans syndrome. In contrast, cultures of fibroblasts from cases of cystic fibrosis have shown an increased cell content of total AMPS which, however, parallels the normal as regards the relative proportions of hyaluronic acid, dermatan sulphate and chondroitin sulphate (Matalon and Dorfmann, 1968). Thus the possibilities of characterising a disease on the biochemical level by assays of cultured cells emphasise the importance of cellular metachromasia as a screening

  • 256

    procedure. Much therefore depends on further knowledge of the chemical nature, cultural factors and mechanisms underlying the in-vitro production of cellular metachromasia, as well as the reasons for the time-lag in culture (Danes and Bearn, 1968) before its appearance. Whatever the significance of this metachromasia, an in-vitro model has been established to study a disease such as cystic fibrosis, the pathogenesis of which still remains an enigma.

    G. B. REED, A. D. BAIN, W. M. McRAE AND F. M. SCOTT

    SUMMARY

    Fibroblasts were cultured from children with cystic fibrosis of the pancreas and their parents. The same basic tissue-culture principles as other workers have used were relied on, but the tissue-culture medium was supplemented with pooled human serum instead of calf serum. The finding of increased metachromasia in fibroblasts from children with cystic fibrosis was confirmed, but the amount of this metachromasia was only one-tenth of that previously reported-a difference that would appear to be related to the in-vitro culture conditions. The amount of metachromasia in parents of children with cystic fibrosis approximated to that of the normal. In the individual cases of cystic fibrosis the various subcultures showed a considerable range in the percentage of meta- chromasia. As the technique of fibroblast culture is likely to have wide applica- tions in the study of the genetic metabolic diseases, attention is drawn to the need for further study of the culture conditions, and particularly of the sero- logical factors involved in the in-vitro production of cellular metachromasia.

    This work was supported by a grant to the University of Edinburgh from the Distillers Company Ltd and the Cystic Fibrosis Research Trust.

    REFERENCES CARTWRIGHT, E., DANKS, D. M., AND JACK, I. 1969. Metachromatic fibroblasts in pseudo-

    xanthoma elasticum and Marfans syndrome. Lancet, 1, 533. CULLING, C. F. A. 1963. In Handbook of histopathological techniques, 2nd ed., London,

    p. 232. DANES, B. S., AND BEARN, A. G. 1968. A genetic cell marker in cystic fibrosis of the pancreas.

    Lancet., 1, 1061. DANES, B. S., AND BEARN, A. G. 1969. Cystic fibrosis of the pancreas: a study in cell

    culture. J. Exp. Med., 129, 775. DE MARS, R., AND LEROY, J. G. 1966. In In vitro, Baltimore, vol. 2, p. 107. DINGLE, J. T., AND WEBB, M. 1965. In Cells and tissues in culture, ed. by E. N. Willmer,

    London, vol. 1, p. 372. DI SANTAGNESE, P. A. 1964. In Textbook of paediatrics, 8th ed., edited by W. Nelson,

    Philadelphia, p. 773. LOBECK, C. C. 1966. In Metabolic basis of inherited disease, 2nd ed., edited by J. B.

    Stanbury, J. B. Wyngaarden and D. S. Fredrickson, New York, p. 1300. MATALON, R., AND DORFMANN, A. 1966. Hurlers syndrome: biosyntheses of acid muco-

    polysaccharides in tissue culture. Proc. Natn. Acad. Sci. USA, 56, 1310. MATALON, R., AND DORFMANN, A. 1968. Acid mucopolysaccharides in cultured fibroblasts

    of cystic fibrosis of the pancreas. Biochem. Biophys. Res. Commun., 33, 954. MATALON, R., AND DORFMANN, A. 1969. Acid mucopolysaccharides in cultured human

    fibroblasts. Lancet, 2, 838. NADLER, H. L., WODMCKI, J. M., SWAE, M. A., AND OFLYNN, M. E. 1969. Cultivated

    amniotic fluid cells and fibroblasts derived from families with cystic fibrosis. Lancet, 2,84.

  • METACHROMASIA IN CYSTIC FIBROSIS 257

    PAUL, J. 1965. In Cell and tissue culture, 3rd ed., London, pp. 8 and 177. PUNNETT, HOPE H., KISTENMACHER, MILDRED, AND NIEDERER, BETTINA S. 1968. Meta-

    SPOCK, A., HEICK, H. M. C., CRESS, H., AND LOGAN, W. S. 1967. Abnormal serum factor

    TALAMO, R. C. 1969. Cystic fibrosis of the pancreas-new clues to the metabolic riddle.

    VAN HOOF, F., AND HERS, H. C. 1968. The abnormalities of lysosomal enzymes in muco-

    chromasia in fibroblasts. Lancet, l, 1433.

    in patients with cystic fibrosis of the pancreas. Pediut. Res., 1, 173.

    Calif. Med., 110, 432.

    polysaccharidoses. Eur. J. Biochem., 7 , 34.

Recommended

View more >