BIOCHEMICAL

Vol. 168, No. 2, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 396-401

April 30. 1990

ImmnoreactiveGrowthHonmne

(GH) Secretion byEIunranLyr@ocybes:

AugrssntedRe1eas.e Naoki

Hattori,

Akira

bymenous

Shimatsu,

Masahiko

GH

Sugita,

Shunichi

Kumagai,

andHirooImura Second

Received

Division,

Kyoto

University

February

26,

Department Faculty

of Internal

of Medicine,

Medicine,

Kyoto

606,

Japan

1990

Peripheral blood mononuclear cells (PBMCs) from normal adults secreted small amounts of human growth hormone (GH;0.2-0.6 pg/105 cells/7 days culture) as measured by a highly sensitive enzyme immunoassay. Stimulation of PBMCs with phytohemagglutinin (PHA) consistently showed a 4-6 fold increase in GH secretion. Transformed B-lymphocytes by Epstein-Barr virus also secreted GH (O-8-4.8 pg/5 x lo* cells/7 days culture). GH secreted by lymphocytes comigrated with pituitary GH on an Ultrogel AcA44 column. Addition of GH during the culture augmented endogenous GH secretion from PHA-stimulated PBMCs. GH-releasing hormone and a somatostatin analogue, SMS 201-995, did not affect GH secretion from non-stimulated and PHAstimulated PBMCs. These findings suggest that both T and B lymphocytes secrete immunoreactive GH in a different manner from that in the anterior pituitary. o 1990 Academic Pres*, Inc. Recent tween have It

studies

the

immunoregulatory

has been

ACTH,

shown

endorphin

(GH)-related phocytes that

Very

by

bioactivity, describe

TSH (3)

a highly

were

recently,

quantitative sensitive

communication

systems

cytokines produce

also

is

et

similar

and molecular analysis enzyme

hormones

have

neuroendocrine

effects.

(4).

immunoassay

have secrete

pituitary

weight.

of GH secretion (EIA)

such as hormone

mitogen-stimulated

(6) and

to

peptides

Prolactin/growth in

al.

synthesize

be-

Neuroendocrine

detected

Weigent can

exists

(1).

neuroendocrine

and prolactin

leukocytes

antigenecity, the

and

lymphocytes

lymphocytes

bidirectional

immune

effects

mRNA species (5).

that and

that (2),

mononuclear

produced

using

indicate

neuroendocrine

provided GH in GH in

In the present

lymevidence vitro.

GH

terms

of

study,

we

by T and B lymphocytes for

GH (7).

Venous blood was taken after an overnight fast from normal adult volunteers. Plasma GH levels determined by EIA at the time of blood withdrawal ranged from 0.18-l-5 ug/L. Peripheral blood mononuclear cells (PBMCS) were isolated by standard Ficoll-Hypaque gradient centrifugation (8) and suspended in culture medium (RPM1 1640) containing 10% fetal calf serum (Filtron, Australia) and antibiotics. PBMCs (1 x 10'cells in 200 pL) ooo6-291x.90 $1.50 Copyright 6 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

396

Vol.

168, No. 2, 1990

BlOCHEMlCAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

were incubated in triplicate at 37OC in 5% COz with the following additives: 0.2% phytohemagglutinin (PHA;Difco, Detroit, MI), 1% pokeweed mitogen (PWM;Sigma, St. Louis, MO), 20 mg/L lipopolysaccharide (LPS; Difco, Detroit, MI), human pituitary GH (Sumitomo Pharmaceuticals, Tokyo, Japan), GH-releasing hormone (GHRH;Peptide Institute, Osaka, Japan), a somatostatin analogue (SMS 201-995;Sandos Pharmaceuticals, Bazel, Switzerland). After incubation, the cells were removed by centrifugation and the culture supernatant was used for the assay. Epstein Barr (EB) virus-transformed B cell lines were established from normal subjects, as previously described (9). Gel chromatography The culture supernatant of PHA-stimulated T cells or EB virustransformed B cells was concentrated lo-30 fold with polyethylene glycol. The concentrated samples (0.5-1.0 mL) or GH standards in culture medium were applied onto an Ultrogel AcA44 column (0.9 x 70 cm) and eluted with 0.01 mol,/L phosphate buffer containing 0.1 mol/L NaCl, 0.1% NaNa, and 0.1% bovine serum albumin at 4'C. The fractions of 0.8 mL were collected and assayed for GH. The column was calibrated by markers with various molecular weights. Assays GH was measured by a highly sensitive enzyme immunoassay (EIA) as previously described (6). In brief, samples (100 pL) were incubated with anti-GH IgG-coated polystyrene balls at 37OC for 6h. After washing with saline, the polystyrene balls were incubated with anti-GH Fab' peroxidase conjugate at 4 'C or 16h and at 20°C for 6h. After washing with saline, peroxidase activity bound to the balls was assayed using a fluorescence method. A spectrofluorophotometer (Shimadzu RF-540, Kyoto, Japan) was used to measure the fluorescence intensity. The detection limit was 1 x lO"ug/L. The intraand inter-assay coefficients of variation were 6.0% and 8.6%, respectively. Statistical analysis The data were expressed as means?SEM. Student's t-test analysis of variance in combination with Duncan's new multiple were used for statistical analysis as appropriate.

and one way range test

RESULTS PBMCs (1 x 10' of

immunoreactive

Stimulation

of PBMCs with

increased not

pg/well)

7 days

into

the

two fold, EB virus

for

7 days

a T-cell

the

time-course

while

small

as shown mitogen,

a B-cell

addition

transformed

secreted

secreted

media,

PWM cl%),

in GH secretion. about

GH secretion.

cultured

for

PHA (0.0125-0.2%),

increase

GH secretion

stimulate

cultured

GH (0.2-0.6

a dose-related

/well)

cells/well)

in

Fig.1.

resulted mitogen,

in also

of LPS (20 mg/L)

B cell

variable

amounts

lines

amounts

did

(5 x 10'cells

of

GH (0.8-4.8

pg/well). Fig.2a

shows

PHA-stimulated incubation. incubation

lymphocytes. Small for

amounts from

day

to

and continued

stimulated

GH secretion

GH secretion not

unchanged

PBMCs was increase

was dependent

the

were

during

9 days

9th

on the

397

non-stimulated

immediately

pg/well)

stimulated

till

from

detected

of GH (0.2-0.6

6h and remained

PHA. GH secretion

of GH was

secreted

day.

As shown

number

the

after

the

of culture

by PHA after

the

and

after

without 3rd

in

Fig.Zb,

of cultured

cells.

to

5th PHA-

Vol. 168, No. 2, 1990

BIOCHEMICAL

PHA

stimulated

PWM LPS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EB-V transformed B cell lines

Time

xl05cellshel

Figure 1. Secretion of immunoreactive GH from non-stimulated, mitogens lymphocytes, and three EB virus-transformed B (PHA, PWM, LPSJ-stimulated cell lines during the culture for 7 days. Figure 2. (a) Time course of GH secretion from non-stimulated (triangle) and PHA-stimulated (circle) lymphocytes in 2 normal subjects. The number of cells was 1 x lo5 /well. (b) The relationship of GH secretion from PHAstimulated lymphocytes with the cell numbers in the 7 days culture. Mean?SE of GH levels in triplicate wells are shown. To investigate 100

x lo-'

with

pq/L)

GH secretion

was added

PBMCs. GH was stable

calculated

the

GH from

the

creased

recovered

(Table

exogenous affect

1).

in

not

curve

curve

position

of were

GH (lo-

and incubated

in media

without

cells

(mean recovery:99.3%).

of secreted

GH by subtracting

GH levels.

The

from

addition

PHA-stimulated numbers

of

not

in

changed

We

of added

(10“

in-

a dose-related

by the

SMS 201-995

non-stimulated

amounts

I days

GH significantly

lymphocytes

were

M) and/or

the

for

addition

of

-10s6 M) did

and PHA-stimulated

not

lymphocytes

shown).

A dilution standard

exogenous

media

cell

GH secretion

regulated,

culture

from The

was

the

GR. GHRH (lo- 'O-10*'

the

(data

amounts

GH secretion

manner

forms

whether

of

of

GH produced

GH (Fiq.3).

22K GH on gel also

Table

observed

1. Effects

by lymphocytes

Immunoreactive chromatography

as in the of exogenous

0 10

(Fiq.4).

Minor

case of pituitary

GH.

Secreted non-stimulated 0.2 kO.1

50

100 Values show mean rSE of secreted * PCO.02 vs. secreted GH from in the absence of exogenous ** PCO.01 vs. secreted GH from in the absence of exogenous ***p