Læknablaðið : fylgirit - 01.05.1978, Page 150

L. Thorsteinsson, S.S. Frjáland and J.B. Natvig.
Institute of Immunology and Rheumatology, Riks-
hospitalet, University Hospital, Oslo, Norway
Correspondence: Leifur Thorsteinsson, Institute
of Immunology and Rheumatology, Fr. Qvamsgt.
1, Oslo 1, Norway.
Abstract:
Staining with peroxidase, EA-rosette formation
and determination of cytotoxic activity were
used to characterize the third lymphocyte-like
cell population and to see whether these cells
were of monocytic origin. The cell suspensions
studied contained about 2Ö/o peroxidase positive
celis as well as 207o cells bearing Fc-receptors
(EA-RFC). Direct peroxidase staining of the
EA-rosette preparations showed, however, that
only a small proportion of the EA-RFC were
peroxidase positive. Furthermore, after frac-
tionation on nylon fibre columns, the cell
suspensions were practically depleted of
peroxidase positive cells, while the cytotoxic
activity was increased and the percentage of
EA-RFC only moderately reduced. Conversely,
EA-rosette depletion experiments almost totally
removed EA-RFC while, the cytotoxic activity
and the percentage of peroxidase positive cells
remained, although clearly reduced. In addi-
tion, mitogen stimulation depleted the cell
suspensions of peroxidase positive cells. It is
therefore concluded, that our data do not support
the theory that the cells in question are of
monocytic origin because they lack one of the
most reliable marker for monocytes.
Intr oduc tion:
Lymphoid cells isolated from peripheral blood
by Isopaque-Ficoll gradient centrifugation can be
classified in at least 3 different populations (3,
10). T-Iymphocytes with receptors for sheep
erythrocytes, B-lymphocytes with membrane-
bound immunoglobulin and the so-called "third
lymphocyte-like cell population" with receptors
for the Fc-part of IgG (10,11,12,16). The last
mentioned cells can be detected by using human
ORH+ erythrocytes sensitized with strong anti-CD
antibodies (10,11,12,16). The effector cells
recently termed K-cells responsible for lymphocyte
mediated cytotoxicity against antibody coated target
cells are found among these cells (7,10,11,24,25).
Whether these cells represent a separate popula-
tion of lymphoid cells or some kind of immature
FURTHER CHARACTERIZATION
OF THE nTHIRD LYMPHOCYTE-
LIKE CELL POPULATION’*
cells belonging to the monocyte line or the B- or
T-cell lines is unknown. Thus, it has been
suggested that they might be precursor cells of
B-lymphocytes (5,6) or that they might be
developmentally related to monocytes (13).
Most of the monocytes contain the enzyme
peroxidase which has gained acceptance as one
of the most reliable monocyte markers. The
peroxidase activity shows atendencyto decrease
during differentiation from the monocyte stem
cell in the bone-marrow to tissue macrophages '
(17,18,23). If the third lymphocyte-like cell
population belongs to some stage of the monocyte
cell-line, they would be expected to be peroxidase
positive. The purpose of this work was to inves-
tigate these cells for peroxidase activity with
several of the available peroxidase staining
techniques.
Materials and methods:
Mononuclear leukocytes were isolated
from peripheral blood of healthy donors (10 IU
heparin per ml) either from a blood bank or
laboratory personell by the Isopaque/Ficoll
gradient centrifugation technique (4). After being
washed 3 times in Hanks BSS the cells were
suspended in the same Hanks BSS containing 10%
normal human AB serum (N.H.S.) with 1 mg
EDTA per ml. This suspension, (3x10^ cells on
each slide) was used to make cytocentrifuge
preparation and stain the cells for peroxidase.
Peroxidase staining methods. Three
different methods were used for peraxidase
staining.
A. A modification of the Graham
Knoll technique (2 0). Air dried cjrtocen-
trifuge preparations were fixed for 30 seconds in
a solution of 407o formalin and concentrated (96f%)
ethanol 1:9 washed with tap water and dried. The
preparations were incubated for 7 minutes in a
mixture of 250 mg O-tolidin, 6 ml etanol (96%),
4 ml dest. water and 0.02 ml 3f% H2O2, washed
several times with tap water and counterstained
(May-Grunwald Giemsa). These reactions give
blue cell nuclei and peroxidase positive structures
show green to brownish hue.
B. The Kaplow technique (14). Air
dried cytocentrifuge preparations were fixed for 60
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