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

Enhancement of beta 2-microglobulin formation induced by phytohemagglutinin and mercuric ion in cultured human leucocytes.

Vol. 91, No. 2, 1979 November BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 569-574 28, 1979 ENHANCEMENT OF !32-MICROGLOBULIN FORMATIO...
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