Vol. 91, No. 2, 1979 November
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 569-574
28, 1979
ENHANCEMENT OF !32-MICROGLOBULIN FORMATION INDUCED BY PHYTOHEMAGGLUTININ AND MERCURIC ION IN CULTURED HUMAN LEUCOCYTES M. Ohsawa*
and M. Kimura
Department of Occupational Diseases and Department of Experimental National Institute of Industrial Health, Nagao, Tama-ku, Kawasaki Received
October
Toxicology, 213, Japan
13,1979 SUMMARY
Human leucocyte cell cultures were stimulated to initiate DNA synthesis by phytohemagglutinin and mercuric chloride. Both mitogens enhanced the accumulation of 8z-microglobulin in the medium, which was synthesized by lymphocytes. Mercuric chloride promoted the accumulation of this protein optimaly with a concentration (1 x 10m5M) to produce the maximum stimulation of Combined use of phytohemagglutinin (50 pg/ml) and mercuric DNA synthesis. chloride (1 x 10e5M) produced additive effect on both DNA synthesis and 82microglobulin accumulation. These findings suggest that mercuric ion causes the proliferative response of lymphocytes by a mechanism different from that for the stimulation by phytohemagglutinin. INTRODUCTION Transformation
of human lymphocytes
by phytohemagglutinin
(PHA)
stimulate
lymphocytes
as a non-specific
phocytes
can enhance
metal
ions
ions,
82-Microglobulin been
located
tion
by lymphocytes
lation
on the cell
in vitro
*
reaction
These
* Present Sagamiko, addressed.
facts
address: Kanagawa
(12,13) mean that Faculty 199-01,
response
(4,5)
like
PHA.
of a molecular
of lymphocytes found
antiserum
(8). (9)
human lymto heavy Among those
stimulant
(4).
weight
11,800
synthesis
and promoted
of lymphocytes
protein
Normal
(4,6,7).
Its
or antibody
and has and secre-
under
the
stimu-
of 82-microglobulin
to PHA (12),
the mixed
proliferative
may play
in vitro
shown to
in response
potent
and an antigen-induced this
have been
and zinc
the most
protein
have been
Moreover,
ions
and transformation
has been
surface
the proliferative
lymphocyte (13)
ion
mitogen
nickel
is a simple
of PHA (10,ll).
inhibits
(2-4),
mercuric
known to be induced
Some heavy metal
DNA synthesis
such as mercury
heavy metal
(1).
is well
a critical
of Pharmaceutical Sciences, Teikyo Japan. TO whom all correspondence
response role
on the
University, should be
0006-291X/79/220569-06$01.00/0 569
Copyright @ 1979 by Academic Press. Inc. AN rights of reproduction in any form reserved.
Vol. 91, No. 2, 1979
response
of lymphocytes
vestigate
whether
in vitro metal
BIOCHEMICAL
is ion
stimulate
to mitogens
or not
enhanced increases
AND BIOPHYSICAL
and antigens.
the accumulation
by mercuric
lymphocytes
It
has prompted
of $z-microglobulin
ion.
The present
the accumulation
RESEARCH COMMUNICATIONS
work
by lymphocytes has shown that
of B2-microglobulin,
by a mechanism
differnt
from
but
that
us to in-
for
that the
this
it
may
stimulation
by PBA. MATERIALS
AND METHODS
Leucocytes were obtained from the peripheral blood of a normal donor with no history of sensitivity to mercury by allowing the blood to settle for 1 hour at 37°C after adding dextran to give a final concentration of 0.4%. Aliquots of the cell suspension containing approximately lo6 cells per ml medium were cultured in 1 ml Eagle's minimum essential media containing 20% heat-inactivated fetal calf serum, 50 units/ml penicillin G and 50 ug/ml streptomycin under 5% CO2 atmosphere. At the onset of cultivation, 20~1 sterilized solution of PBA-M (Grand Island Biological Co., Grand Island, N.Y., U.S.A.) and/or mercuric chloride were added to appropriate cultures to give a final concentration of 12.5 to 200 ug per ml medium and 1 x 10B6 to 1 x 10e4M, respectively. For the mercury experiment two kinds of control cultures were set up: one with 50 ng PBA per ml medium and another one without any stimulant. Lymphocyte stimulation activity was estimated by determining [3Blthymidine incorporation into cold trichloroacetic acid-insoluble (acid-insoluble) fraction of cells. Tritiated thymidine (0.5 uC/ml culture, specific activity 150 mC/m mol) was added for the final 24 hours of the culture by use of 20 ul DNA synthesis was then arrested by the addition of 1 ml of working solution. ice-cold saline and immersing in ice bath. The cells was separated by centrifugation at 50g for 5 min from the medium which was collected for &-microglobulin assay. The precipitated cells was re-suspended in saline and was then filtered on the glass fiber filter discs (Whatman GF/C) with a sampling Those discs were washed twice with ice-cold manifold (Millipore Co., Ltd.). 5% trichloroacetic acid and once with ice-cold ethanol. Tritium radioactivity Amounts of B2on the disc was counted with a liquid scintillation system. microglobulin in the media were determined by a radioimmunoassay method (11, 14). RESULTS AND DISCUSSION Figure
1 shows
the dose-effect
and $2-microglobulin
accumulation
caused
increase
a significant
insoluble
fraction
PBA-M per
ml medium.
41 to 150 rig/ml
Amounts
Human Bz-microglobulin ring.
As cells
the increased
were
of
maintained
for
the medium
after
thymidine
leucocytes
under
to the
detected under
in
in the original
of B2-microglobulin
570
3-day
stimulation
culture.
PBA
incorporation
increased
healthy
lymphocyte
the presence
B2-microglobulin
in response was not
accumulation
in
in tritiated
of cultured
medium
relationship
condition
into
of 12.5
the medium concentration
in the medium
to 200 pg
increased of PEA.
medium before after
acid-
3-day
cultuculture,
seems to result
from
BIOCHEMICAL
Vol. 91, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
T-
10
fira 8 b