Download inability of peripheral lymphoid cells of

Aust. J. exp. Biol. med. Sci. (1969), 47, pp. 759-761
Brief Communication:
INABILITY OF PERIPHERAL LYMPHOID CELLS OF
AGAMMAGLOBULINAEMIC PATIENTS TO BIND RADIOIODINATED
ALBUMINS
by D. NAOR," Z. BENTWICH AND C . CIVIDALLI
(From the Departments of Immunology, Internal Medicine and Pediatrics and
Child Health, Hebrew University-Hadassah Medical School, Jerusalem, Israel).
{Accepted for publication 18th July, 1969.)
In previous publications (Naor and Sulitzeanu, 1967; Sulitzeanu and Naor, 1969) we
described an attempt to test directly one of the central postulates of Burnet's clonal selection theory (Burnet, 1959), namely, that antibody-like receptors are present on the surface
of lymphoid cells. This was done by testing the ability of normal mouse spleen cells to
bind bovine serum albumin (BSA), when exposed to very small amounts of a highly radioiodinated antigen preparation in vitro, in the cold. The results, as assessed by autoradiography, suggested that spleen and lymph node cells were capable of binding BSA as well as
three other albumins. The binding ability was restricted to a small number of cells (about
1 in 1500). Many more cells became labelled after exposure to a mixture of four radioactive
albumins than after exposure to any one of the individual albumins (Sulitzeanu and Naor,
1969). This was interpreted as showing that different cells bind different antigens. Comparable observations were made by Byrt and Ada and Humphrey (personal communication).
AgammaglobuUnaemic patients are restricted in their ability to produce antibodies.
The purpose of this work was to test if this defect is accompanied by an inability of the
patients' cells to bind radioiodinated antigens. As the binding is apparently related to the
presence of cell receptors, the peripheral white cells of agammaglobulinaemic patients would
be unable to bind radioiodinated albumins if receptors were absent in their cells.
The cell donors were: (1) A male infant 14 months old, with "congenital agammaglobulinaemia" (Bruton's type); (2) a normal infant of the same age as patient No. 1;
(3) a female adult patient, 28 years old, with "primary acquired agammaglobulinaemia";
(4) a normal adult 33 years old.
Crystalline BSA was purchased from Nutritional Biochemicals Corporation, Ohio.
Cuinea-pig serum albumin (CPSA) was isolated from serum by preparative disc electrophoresis (Sulitzeanu, Slavin and Yecheskeli, 1967). Iodination was performed following the
method of Creenwood, Hunter and Clover (1963). Immunoglobulin of patients was analyzed
by immunoelectrophoresis and gel diffusion. Response of patients' lymphocytes to phytohaemagglutinin (PHA) was determined by counting the number of blasts and by measuring
the incorporation of tritiated thymidine in cell cultures. Leucocyte culture was prepared
with 108 cells in TC199 (Difco) medium containing 20% autologous serum and 0-1 ml.
PHA. The incorporation was studied by adding 10 n c of tritiated thymidine on the 4th
day of the culture for 5 hr. (patient No. 1) or 16 hr. (patient No. 3). Binding of radioiodinated antigens was performed as follows: to 10 ml. of heparinized peripheral blood,
which served as a source for the white blood cells, 2 ml. of plasma gel (Laboratoire Roger
Bellon Neuilly, France) were added. After 30 min. the plasma, containing white cells.
•Present address: University of California, Department of Bacteriology and Immunology,
Berkeley, California 94720.
760
D. NAOR, Z. BENTWICH AND G. CIVIDALLI
was centrifuged at 400 g. The cell sediment was resuspended in saline C (Hammerstrom
and Stoner, 1963), containing polyvinyl pyrrolidone (1-8%) and heparin (10 units/ml.).
50 X 106 white cells were incubated for 1 hr. at 4° with a mixture of 1125 BSA (1-5 x lO^
counts/min.; specific activity 130 x 108 counts/min./ml) and I'S.T GPSA (1-5 x 10«
counts/min.; specific activity 380 x 10" counts/min./ml.) in a final volume of 1 ml. The
use of two antigens was intended to yield an increased number of labelled cells. It was
argued that, should a particular cell be restricted to bind a single antigen, exposition
of the cell suspension to a mixture of different antigens should result in a higher yield of
labelled cells than exposition to a single antigen. The cells were washed three times, smeared
on slides and covered with Ilford K-5 nuclear emulsion for autoradiography. 5,000 to 10,000
cells were surveyed for each experiment. Cells were arbitrarily designated as labelled if
containing seven or more grains; in any case, a highly significant difference was found by
the Chi2 test (p < 0-0001) between the expected values of labelled cells (calculated on
the basis of the Poisson distribution) and the experimental values (Sulitzeanu and Naor
1969).
Immunoelectrophoretic analysis of serum of patient No. 1 showed very low levels of
IgG; IgA and IgM were absent. On incubation with PHA blast transformation of his
lymphocytes occurred in 61% of the cells (within normal limits). 4-7% of the added
tritiated thymidine was incorporated by the lymphocytes in cell culture as compared with
6-8% in a nonnal control studied at the same time and 0-37% in a patient with thymic
dysplasia.
Serum protein analysis of patient No. 3 showed a sharp decrease of IgG (90%) while
IgA and IgM were not detected. Blast transformation of lymphocytes on incubation with
PHA occurred in 79% of the cells. Upon the addition of PHA, incorporation of tritiated
thymidine into lymphocytes increased from 3% to 15-5%. A skin allograft was rejected by
the patient after 20 days (upper limit of normal).
TABLE 1.
Diilribulion
of grain counts in human peripheral white blood celln from agammaglobuUnaeinic
normal donors.
and
No. of peripheral white blood celLs
Cell donors
with grain counts:
0-3
4-6
7-10
Congenital agammaglobulinaemic
infant
9956
42
2
Normal infant cells
9852
106
28
Patient with acquired
agammaglobulinaemia
9828
152
20
Normal adult
9772
180
30
11-15 16-30
30
No. of cells
counted
10000
12
2
—
10000*
10000*
10
4
4
10000*
The cells were incubated with a mixture of '^'I-BSA and "^
*5000 cells were surveyed in this slide. The results were multiplied by 2.
Table 1 shows the distribution of silver grains in the white blood cells of agammaglobulinaemic patients and normal donors of the same age. Only 2 cells of the patient with
the congenital form of disease were labelled, as compared with 42 cells of the corresponding
control: likewise, 20 cells of the patient with acquired agammaglobulinaemia were labelled
as compared with 48 cells of the normal adult. Moreover, cells containing 11 grains or
more (medium or heavily labelled) could not be found in the smears prepared with the
LYMPHOID CELLS IN AGAMMAGLOBULINAEMIA
761
patients' cells. Over 50X of the medium and heavy labelled cells of the controls were
lymphocytes.
In our previous communications we showed that a small number of spleen and lymphnode cells of normal mice bind antigen. This has now been found to apply also to peripheral lymphoid cells of normal humans. The inability of the peripheral white blood cells
of agammaglobulinaemic patients to bind antigen strengthens our assumption that the binding
is due to specific cell receptors.
Acknowledgements. The authors are indebted to Professor D. Sulitzeanu for advice
during the preparation of the manuscript, and to Miss Judith Shtulberg for excellent technical assistance. The work was supported by the Joint Research Fund of the Hebrew
University and Hadassah.
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(1969}-