Blood group antigens in urinary bladder: from A antigen to A epitopes

© ELSEVIER Paris 1987

Ann. Inst. PasteurlImmunol. 1987, 138, 885-889

BWOD GROUP ANTIGENS IN URINARY BLADDER: FROM A ANTIGEN TO A EPITOPES P. Rouger, P. Gane and C. Salmon Centre National de Reference pour les Groupes Sanguins (CNRGS), Universite Pierre et Marie Curie, Institut National de Transfusion Sanguine, 53, bid Diderot, 75012 Paris

Introduction. Several reports concerning the expression of blood group A in tumours have described the frequent disappearance of A antigen in transitional cell carcinoma of the urinary bladder. Several authors [1, 2, 3, 4] tried to correlate blood group expression with the grade and stage of vesical tumours. The discrepancy between the results of the different studies shows that the demonstration of the presence of blood group antigens in normal and tumoral tissues comes up against a number of problems, including: a) the method of storing tissues; b) the complexity of the tissue structures bearing these determinants; and now c) the reagents used in the different studies. All these works were performed with polyclonal human sera. In order to understand these anomalies, it seemed necessary to analyse in detail the distribution of blood group A in normal bladder epithelium with a series of monoclonal antibodies (mAb). Distribution of A antigen in urothelium as shown by poIyclonal reagent. In using human anti-A reagent, A antigen was found on all cell layers of normal urothelium, mainly in subjects with the AI> secretor and Lewispositive phenotype. Several studies [5, 6, 7] demonstrated that the expression of A antigen was partly controlled by the secretor status; the cells of non-secretor subjects were weakly stained or unstained. In some non-secretor

Received October 29,1987, from the Symposium «Blood group substances and associated antigens in nornial differentiation and oncogenesis» (Paris, September 25, 1987).

P. ROUGER AND COLL.

886

individuals, only the basal layer cells were stained. Cells of Le(a-b-) secretors were strongly stained. The staining of basal cells was essentially membranous and the staining of cells of intermediary or upper layers seemed to be cytoplasmic. The staining of urothelium of the A2 non-secretor phenotype was very poor.

From A antigen to A epitopes. Polyclonal reagents consist of a series of different antibodies which react with different parts of A antigen. When polyclonal reagents are used in indirect immunofluorescence assay, only global staining is observed, which is the result of different phenomena. The use of monoclonal reagents enables analysis of the epitope rather than the antigen. In order to perform correct analysis, it is essential these monoclonal reagents be very well characterized. Recently in the «First international workshop on monoclonal antibodies against human red blood cell and related antigens », 31 monoclonal anti-A antibodies were characterized by 12 laboratories using agglutination, fluorescence and biochemical approaches [8]. The conclusions of this Workshop can be applied to the understanding of the distribution of A markers in normal human urothelium.

Distribution of A epitopes in urothelium as shown by monoclonal reagents. Using monoclonal reagents, the expression of A markers appeared to depend on 4 criteria: a) chain types; b) fucosylation; c) secretor status; and d) Lewis phenotype.

Distribution according to the different chain types. Type 1 and 2 antigens are found on all cell layers. However, type 1 chain appears to be more strongly expressed in the superficial layers whereas type 2 chains are essentially present in the deepest layers. Type 3 and 4 chains were not found in the cell membrane; the supranuclear staining pattern observed in immunofluorescence suggested that these structures were expressed in the Golgi apparatus (table I).

mAb

=

monoclonal antibody.

BLOOD GROUP A ANTIGENS IN UROTHELIUM T ABLE I. -

Monofucosylated determinants A type 1

887

A and AY antigen chain types.


2

I

1



I

1
A type 3




2

I

1
Example of difucosylated determinants
I

1
I

1
Distribution oj mono- and difucosylated structures. Difucosylated A structures are found on cells of superficial layers. On the contrary, antibodies specific for monofucosylated determinants react exclusively with basal and intermediary urothelial cells (table II).

Contribution oj the secretor status. The urothelium of subjects with non-secretor status does not exhibit a significant number of type 1 difucosylated structures; only a reduced quantity of type 2 monofucosylated structures can be demonstrated (table III).

888

P. ROUGER AND COLL. TABLE

II. - Expression of 6 types of A determinants in cells of superficial and basal layers of urotbelium.

A determinant

Superficial layers

Basal Layers

+++ +

+ +++

(+)

+++

Type 1 chain Type 2 chain Type 3 chain Type 4 chain Monofucosylated chain Difucosylated chain

(1) (1)

+++

Expression ranges from - to +++ according to the intensity of immunofluorescence staining. (I) = only the Golgi apparatus stained.

The Lewis phenotype. The presence of the Lewis gene seems to have no effect on the expression of A antigen in the cells of the basal layers. On the contrary, the absence of the Lewis gene is correlated with the expression of A antigen in cells of the superficial layers. It appears that the fucosylation step in superficial layers is controlled by the Lewis gene. Conclusion. The use of mAb reveals new aspects of the distribution of blood group antigens. Polyclonal reagents demonstrated all A determinants, but now, monoclonal reagents can detect the A epitope according to the chain type and fucosylation. Most published studies on ABO distribution in urinary bladder

TABLE

Phenotype

III. - Expression of A determinants according to Lewis status in different layers of urotbelium. Le(a-b+)

Le(a+b-)

Le(a- b-) secretor

2

2

2

Layers Superficial Basal

+++

( +)

+++

++

(+)

++

++ +++

+++ +++

Le(a-b-) non-secretor 2

++

(+) ++

Expression ranges from - to +++ according to the role of the system in the expression of A markers. 1 =reaction observed with antibodies specific for monofucosylated structures; 2 = reactions observed with antibodies recognizing difucosylated structures.

BLOOD GROUP A ANTIGENS IN UROTHELIUM

889

tumours were performed with polyclonal reagents; however, the conclusions were unclear and often disputable. In the future, it seems necessary to analyse bladder tumours with a panel of selected mAb. MOTS-CLI~S:

Epitope.

Vessie, Tumeur, Epithelium, Groupe sanguin A; Antigene,

REFERENCES.

[1] ASKARl, A., COLMENARES, E., SABERI, A. & JARMAN, W.D., Red cell surface antigen and its relationships to survival of patients with transitional cell carcinoma of bladder. J. Urol., 1981, 125, 182-184. [2] CATALONA, W. J ., Practical utility of specific red cell adherence test in bladder cancer. Urology, 1981,28,113-117. [3] LANGE, P.H., LIMAS, L. & FRALEY, E.E., Tissue blood group antigens and prognosis in low stage transitional cell carcinoma of the bladder. J. Urol., 1978, 119, 52-55. [4] VALLANCIEN, G., ROUGER, Ph., LECLERC, J.P. & Kuss, R., Immunofluorescence study of the distribution of A, Band H cell surface antigens in bladder tumor. J. Urol., 1985, 130, 67-70. [5] COON, J.S. & WEINSTEIN, R.S., Variability in the expression of the O(H) antigen in human transitional epithelium. J. Urol., 1981, 125, 301-306. [6] JUHL, B.R., Methodologic and genetic influence on immunohistolochemical demonstration and semi-quantitation of blood group antigen A in human ureter urothelium. J. Histochem. Cytochem., 1985, 33, 21-26. [7] LIMAS, C. & LANGE, P., A,B,H antigen detectability in normal and neoplastic urothelium. Influence of methodologic factors. Cancer, 1982,49,2476-2484. [8] First international workshop on monoclonal antibodies against human red blood cell and related antigens - Blood transfusion and immunohematology (P. Rouger, D. Anstee & C. Salmon). Arnette, Paris, 1987 (in press).