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Cellular distribution of some microsomal antigens in embryonic chicken kidneys
584
CELLULAR
DISTRIBUTION IN EMBRYONIC
OF SOME MICROSOMAL CHICKEN KIDNEYS
ANTIGENS
T. S. OKADA Laboratory
of Developmental Biology, Zoological University of Kyoto, Kyoto, Japan
Institute,
Received August 12, 1963
IT has been shown that a number of the antigenic components contained in the deoxycholate (DOC) extracts of microsomes of chicken kidney can be easily separated into three immunologically distinct sub-fractions [4, 51. The present paper will deal with a study on the cellular distribution of the microsomal antigens contained in the two different sub-fractions by means of fluorescent antibody technique. Sub-fraction K45 is the proteins precipitated from the DOC extracts of kidney microsomes between 25 and 45 per cent saturation of ammonium sulphate. It consists mainly of highly “kidney-specific” antigens [6] and consistently displays an activity of alkaline phosphatase [7]. The other sub-fraction is precipitated between 60 and 80 per cent saturation and the identical molecules are commonly found in liver microsomes too. Since the antigenic molecules contained in this sub-fraction have esterase activity [7], the abbreviation E-antigens will be adopted in the following descriptions. Crude globulins prepared from the antisera against K45 (anti-K45) and against E (anti-E) were conjugated respectively with fluorescein isothiocyanate (FITC) [2]. Conjugate of anti-E with tetramethylrhodamine (TMR) was also made for the purpose of double staining. After removal of the free dyes [2], the conjugates were treated with acetone powders prepared from adult chicken livers, those from 16-day chicken embryos devoid of kidneys and livers, and then by the homogenates of adult chicken spleens. To remove unwanted minor antibodies completely, the conjugated anti-45 was absorbed with liver microsomes and the conjugated anti-E with adult chicken serum. Before the use of the conjugates as histochemical reagents, their immunological specificities were corroborated by in vitro tests. The results demonstrate that each conjugate contains different fluorescent antibodies, which are predominently directed against different sub-fractions of microsomal extracts respectively [51. Kidneys (metanephri), livers and lungs were freshly removed from 15-16-day chicken embryos. Teased preparations and dissociated cells obtained by trypsinization were mounted on slide glasses. Fixation was made by acetone-methanol mixture (9: 1) at -20°C. It was confirmed by means of in vifro tests using isolated microsomal preparations that such a fixation did not destruct immunologically specific determinants of the microsomal antigens [8]. Staining by the conjugates lasted overnight at 4°C. For double staining, the slides were firstly stained with anti-E-TMR conjugate, washed, and then with anti-K45-FITC. The control stainings were: (1) with the conjugated globulins prepared from non-immunized rabbits, (2) Experimental
Cell Research
33
Distribution
of microsomal
antigens
Fig. I.-An intact tubule showing the brush border staining fluorescein in the lumen is not due to extracellular materials, were scattered there at the time of preparing the slide. Fig. 2.-An FITC. Fig.
3.-Four
Fig.
4.-Three
intact
tubule
cells with
consisting fluorescent
cells showing
diffuse
of the spots;
cells staining
cytoplasmic
showing with
the
585
with anti-K45-FITC. but to some positive membrane
staining
Apparent cells which with
anti-45-
anti-K45-FITC.
fluorescence;
with the conjugated antibodies wholly absorbed with (3) with the conjugated antibodies after pretreatment jugated antibodies.
staining
with
anti-E-FITC.
the homologous antigens and of the slides with the uncon-
THE STAINING WITH THE CONJUGATED ANTI-K45.-III the teased tissue preparations of kidney, fluorescence was seen only in the tubular epithelium, and no detectable staining occurred in the glomerular tissues. There are two types of tubules in staining in the respect of their fluorescent staining: i.e., the tubules with brilliant brush border as a broad intracellular band (Fig. 1) and those consisting of the cells with the staining in the cell membranes (Fig. 2). In the preparations of the dissociated cells three different cell types were recognized: (1) the cells with bright fluorescent spot and with or without the weak membrane staining (Fig. 3); (2) the cells showing only the membrane staining and (3) the cells without any fluorescence. The cells of the first type were probably derived from the tubules with the brush border staining. Cells and tissues of the heterologous organs were completely negative. In all the control stainings the cells were always dark. THE STAINING WITH THE CONJUGATED ANTI-E.-The staining in the kidney was found only in the cells of the tubular epithelium. In all the positive cells, the conjuga38 - 641819
Experimental
Cell Research
33
T. S. Okada
586
ted anti-E stained the whole cytoplasm diffusively (Fig. 4). Staining occurred also in the cytoplasm of the liver parenchyme cells. The intensity of the staining was very weak in the lung cells. DOUBLE srAiNINo.-After double staining of the kidney cells, the following four types were discernible: (1) the cells with orange cytoplasm having bright greenish spots, (2) the cells with orange cytoplasm having greenish membrane staining, (3) the cells with orange cytoplasm having no greenish fluorescence and (4) the dark cells. In some staining conditions, spots and membranes were of mixed colour. The distribution of these four types in the given cell populations are shown in Table I. When applied to the liver cells, there were many parenchyme cells with orange cytoplasm but the cells with greenish fluorescence were never observed. Remarks.-The evidence that the main antibody species contained in the present antisera were against the different microsomal molecules was previously given [5, 61. In the present immunohistological work, a complete removal of the non-specific staining from the conjugates, a choice of suitable conditions for fixation, and the negative result in the control stainings were all effectively achieved. Thus, it can be assumed that the fluorescent staining observed is due to immune reactions of the conjugates with specific cellular antigens. The similar patterns obtained by the conjugated anti-K45 were previously reported by the authors who studied the histological distribution of the so-called “kidney-specific” antigens using more complex antisera [3]. But, even anti-K45 is considered to be still of heterogeneous and may contain multiple antibody species. It seems to be probable that the antigens causing the brush border staining may be different from those showing the membrane staining. There are some reasons to believe that the brush border staining is due to the particular enzyme antigens. Anti-K45 can combine the antigenic molecules associated with alkaline phosphatase [7], and, on the other hand, conventional histochemical observations demonstrate very high activity of this enzyme along the inner lumen of the tubules [l]. The results of the double staining show that many of the tubular cells have both K45 and E. There is no cell having only K45, whereas some contain only E. As is shown in Table I, the cell population of the tubules, though morphologically homogeneous, is considered to be heterogeneous in respect of their antigenic compositions. In vitro studies using subcellular fractions isolated by differential centrifugation revealed that the main site of both K45 and E is microsomal [4, 51. But the results of immunofluorescence staining demonstrate that the patterns of the intracellular I. Distribution
TABLE
Cell populations I II
of the four different cell types in two cell populations embryonic kidneys.
of Ihe
Cell types, xa
No. of cells counted
1
2
3
4
365 358
40 36
24 23
11 14
25 27
a Cf. text.
Experimental Cell Research 33
Disfribufion
of microsomal antigens
587
distribution of K45 are conspicuously different from those of E. If the results obtained from the two different techniques were able to be correlated, differential distribution of each molecule constituting or adsorbed with endoplasmic reticulum would be surmised inside a kidney epithelial cell. The work was in part supported by the Grant-in-Aid of Education.
from the Japan Ministry
REFERENCES 1. BURSTONE, M. S., Enzyme Histochemistry and Its Application in the Study of Neoplasms. Academic Press, New York and London, 1962. 2. NAIRN, R. C., Fluorescent Protein Tracing. Livingstone, Edinburgh, 1962. 3. NAIRN, R. C., GHOSE, T., FOTHERGILL, J. B. and MCENTEGART, M. R., Nature 196, 385 (1962). 4. OKADA, T. S., Nature 94, 306 (1962). 5. OKADA, T. S. and SATO, A. G., Exptl Cell Res..31, 251 (1963). 6. __ Nature 197, 1216 (1963). 7. OZATO, K. and OKADA, T. S., ibid. 197, 1310 (1963). 8. SATO, A. G. and OKADA, T. S., Unpublished.
Experimenfal
Cell Research 33
CELLULAR
DISTRIBUTION IN EMBRYONIC
OF SOME MICROSOMAL CHICKEN KIDNEYS
ANTIGENS
T. S. OKADA Laboratory
of Developmental Biology, Zoological University of Kyoto, Kyoto, Japan
Institute,
Received August 12, 1963
IT has been shown that a number of the antigenic components contained in the deoxycholate (DOC) extracts of microsomes of chicken kidney can be easily separated into three immunologically distinct sub-fractions [4, 51. The present paper will deal with a study on the cellular distribution of the microsomal antigens contained in the two different sub-fractions by means of fluorescent antibody technique. Sub-fraction K45 is the proteins precipitated from the DOC extracts of kidney microsomes between 25 and 45 per cent saturation of ammonium sulphate. It consists mainly of highly “kidney-specific” antigens [6] and consistently displays an activity of alkaline phosphatase [7]. The other sub-fraction is precipitated between 60 and 80 per cent saturation and the identical molecules are commonly found in liver microsomes too. Since the antigenic molecules contained in this sub-fraction have esterase activity [7], the abbreviation E-antigens will be adopted in the following descriptions. Crude globulins prepared from the antisera against K45 (anti-K45) and against E (anti-E) were conjugated respectively with fluorescein isothiocyanate (FITC) [2]. Conjugate of anti-E with tetramethylrhodamine (TMR) was also made for the purpose of double staining. After removal of the free dyes [2], the conjugates were treated with acetone powders prepared from adult chicken livers, those from 16-day chicken embryos devoid of kidneys and livers, and then by the homogenates of adult chicken spleens. To remove unwanted minor antibodies completely, the conjugated anti-45 was absorbed with liver microsomes and the conjugated anti-E with adult chicken serum. Before the use of the conjugates as histochemical reagents, their immunological specificities were corroborated by in vitro tests. The results demonstrate that each conjugate contains different fluorescent antibodies, which are predominently directed against different sub-fractions of microsomal extracts respectively [51. Kidneys (metanephri), livers and lungs were freshly removed from 15-16-day chicken embryos. Teased preparations and dissociated cells obtained by trypsinization were mounted on slide glasses. Fixation was made by acetone-methanol mixture (9: 1) at -20°C. It was confirmed by means of in vifro tests using isolated microsomal preparations that such a fixation did not destruct immunologically specific determinants of the microsomal antigens [8]. Staining by the conjugates lasted overnight at 4°C. For double staining, the slides were firstly stained with anti-E-TMR conjugate, washed, and then with anti-K45-FITC. The control stainings were: (1) with the conjugated globulins prepared from non-immunized rabbits, (2) Experimental
Cell Research
33
Distribution
of microsomal
antigens
Fig. I.-An intact tubule showing the brush border staining fluorescein in the lumen is not due to extracellular materials, were scattered there at the time of preparing the slide. Fig. 2.-An FITC. Fig.
3.-Four
Fig.
4.-Three
intact
tubule
cells with
consisting fluorescent
cells showing
diffuse
of the spots;
cells staining
cytoplasmic
showing with
the
585
with anti-K45-FITC. but to some positive membrane
staining
Apparent cells which with
anti-45-
anti-K45-FITC.
fluorescence;
with the conjugated antibodies wholly absorbed with (3) with the conjugated antibodies after pretreatment jugated antibodies.
staining
with
anti-E-FITC.
the homologous antigens and of the slides with the uncon-
THE STAINING WITH THE CONJUGATED ANTI-K45.-III the teased tissue preparations of kidney, fluorescence was seen only in the tubular epithelium, and no detectable staining occurred in the glomerular tissues. There are two types of tubules in staining in the respect of their fluorescent staining: i.e., the tubules with brilliant brush border as a broad intracellular band (Fig. 1) and those consisting of the cells with the staining in the cell membranes (Fig. 2). In the preparations of the dissociated cells three different cell types were recognized: (1) the cells with bright fluorescent spot and with or without the weak membrane staining (Fig. 3); (2) the cells showing only the membrane staining and (3) the cells without any fluorescence. The cells of the first type were probably derived from the tubules with the brush border staining. Cells and tissues of the heterologous organs were completely negative. In all the control stainings the cells were always dark. THE STAINING WITH THE CONJUGATED ANTI-E.-The staining in the kidney was found only in the cells of the tubular epithelium. In all the positive cells, the conjuga38 - 641819
Experimental
Cell Research
33
T. S. Okada
586
ted anti-E stained the whole cytoplasm diffusively (Fig. 4). Staining occurred also in the cytoplasm of the liver parenchyme cells. The intensity of the staining was very weak in the lung cells. DOUBLE srAiNINo.-After double staining of the kidney cells, the following four types were discernible: (1) the cells with orange cytoplasm having bright greenish spots, (2) the cells with orange cytoplasm having greenish membrane staining, (3) the cells with orange cytoplasm having no greenish fluorescence and (4) the dark cells. In some staining conditions, spots and membranes were of mixed colour. The distribution of these four types in the given cell populations are shown in Table I. When applied to the liver cells, there were many parenchyme cells with orange cytoplasm but the cells with greenish fluorescence were never observed. Remarks.-The evidence that the main antibody species contained in the present antisera were against the different microsomal molecules was previously given [5, 61. In the present immunohistological work, a complete removal of the non-specific staining from the conjugates, a choice of suitable conditions for fixation, and the negative result in the control stainings were all effectively achieved. Thus, it can be assumed that the fluorescent staining observed is due to immune reactions of the conjugates with specific cellular antigens. The similar patterns obtained by the conjugated anti-K45 were previously reported by the authors who studied the histological distribution of the so-called “kidney-specific” antigens using more complex antisera [3]. But, even anti-K45 is considered to be still of heterogeneous and may contain multiple antibody species. It seems to be probable that the antigens causing the brush border staining may be different from those showing the membrane staining. There are some reasons to believe that the brush border staining is due to the particular enzyme antigens. Anti-K45 can combine the antigenic molecules associated with alkaline phosphatase [7], and, on the other hand, conventional histochemical observations demonstrate very high activity of this enzyme along the inner lumen of the tubules [l]. The results of the double staining show that many of the tubular cells have both K45 and E. There is no cell having only K45, whereas some contain only E. As is shown in Table I, the cell population of the tubules, though morphologically homogeneous, is considered to be heterogeneous in respect of their antigenic compositions. In vitro studies using subcellular fractions isolated by differential centrifugation revealed that the main site of both K45 and E is microsomal [4, 51. But the results of immunofluorescence staining demonstrate that the patterns of the intracellular I. Distribution
TABLE
Cell populations I II
of the four different cell types in two cell populations embryonic kidneys.
of Ihe
Cell types, xa
No. of cells counted
1
2
3
4
365 358
40 36
24 23
11 14
25 27
a Cf. text.
Experimental Cell Research 33
Disfribufion
of microsomal antigens
587
distribution of K45 are conspicuously different from those of E. If the results obtained from the two different techniques were able to be correlated, differential distribution of each molecule constituting or adsorbed with endoplasmic reticulum would be surmised inside a kidney epithelial cell. The work was in part supported by the Grant-in-Aid of Education.
from the Japan Ministry
REFERENCES 1. BURSTONE, M. S., Enzyme Histochemistry and Its Application in the Study of Neoplasms. Academic Press, New York and London, 1962. 2. NAIRN, R. C., Fluorescent Protein Tracing. Livingstone, Edinburgh, 1962. 3. NAIRN, R. C., GHOSE, T., FOTHERGILL, J. B. and MCENTEGART, M. R., Nature 196, 385 (1962). 4. OKADA, T. S., Nature 94, 306 (1962). 5. OKADA, T. S. and SATO, A. G., Exptl Cell Res..31, 251 (1963). 6. __ Nature 197, 1216 (1963). 7. OZATO, K. and OKADA, T. S., ibid. 197, 1310 (1963). 8. SATO, A. G. and OKADA, T. S., Unpublished.
Experimenfal
Cell Research 33