AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 857-862

Vol. 168, No. 2, 1990 April 30, 1990

BIOCHEMICAL

MONONUCLEAR

CELLS ENHANCE

PROSTAGLANDIN

OF POLYMORPHONUCLEAR TUMOR NECROSIS

E2 PRODUCTION

LEUKOCYTES FACTOR a

VIA

Hideto Akama, Yoichi Ichikawa, Yasutsugu Matsushita, Taeko Shinozawa and Mitsuo Homma Department of Internal Medicine, School of Medicine, Keio University, Received

March

19,

Shinjuku-ku,

Tokyo 160, Japan

1990

SUMMARY: To clarify the interactions between mononuclear cells and polymorphonuclear leukocytes, and to identify the cytokine(s) that mediate the interaction? the effects of a culture supernatant of LPS-stimulated mononuclear cells on production of arachidonic acid metabolites of polymorphonuclear cells were studied. The culture supernatant of LPS-stimulated mononuclear cells increased production of prostaglandin Ez of polymorphonuclear cells. TNFa, but not IL-l, IL-2, IL-6, or IFNr, enhanced the prostaglandin Ez production when added in vitro. Additionally, an anti-rTNFa monoclonal antibody inhibited the stimulating activity of the culture supernatants. TNFa, produced by mononuclear cells, appears to play an important role in the development of inflammation, such as rheumatoid arthritis, by enhancing the arachidonic acid metabolism of the polymorphonuclear cells, cu1990Academic Press,bb2. Polymorphonuclear

leukocytes (PMNs) contribute to the pathogenesis of joint

damage such as occurs in rheumatoid arthritis (l-5). Activation of these PMNs with certain stimuli results in the generation of potentially toxic enzymes, oxygen metabolites, and a wide range of membrane-derived

arachidonic acid metabolites

(6,7). On the other hand, various cytokines such as interleukin-1

(IL-l)

and tumor

necrosis factor a (TNFa) produced by monocytes/macrophages or synovial lining cells appear to possessmany biologic activities

including

the resorption

of bone and

cartilage, and the production of collagenase and prostaglandins by synoviocytes (812). Also, there is growing evidences which suggest that these cytokines potentiate or modify PMN functions (13-14): oxygen radical production, chemotactic activity, and degranulation.

However,

little

yet is known about the regulation

of the

arachidonic acid metabolism of PMNs by the mononuclear cells. We thus have attempted to characterize the effect of cytokines produced in the culture supernatant of LPS-stimulated mononuclear cells on the PMN functions, especially on the production of arachidonic acid metabolites of the PMNs, and to identify the Abbreviations: PMNs, polymorphonuclear leukocytes; rIL, recombinant interleukin; TNF, tumor necrosis factor; IFN, interferon; PG, prostaglandin; HBSS, Hanks’ balanced salt solution; HEPES, N-[2-Hydroxyethyll piperazine-N’-[2ethanesulfonic acid]; LPS, lipopolysaccharide. 0006-291X/90 Copyright All rights

$1.50

0

1990 by Academic Press, Inc. of reproduction in any form reserved.

857

Vol.

168, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

cytokine(s) precisely, by using various human recombinant cytokines and an antirTNFa monoclonal antibody. This communication shows that TNFa rather than IL-1 plays an important role in the production of prostaglandin E2 (PGE2) of the PMNs. MATERIALS

AND METHODS

Materials: RPMI-1640, HBSS, HEPES, LPS (Escherichia coli 026:66), human interferon7 (IFNr) and human rIL-2 were purchased from Sigma Chemical Co. (St. Louis, MO). Human rIL-6 was kindly supplied by Dr. Hirano (Osaka Univ., Japan). Human rIL-la and rIL-1P were purchased from Otsuka Pharmaceutical Co. (Tokushima, Japan). Human rTNFa (2.3 X 106 U/mg protein) and monoclonal antibody against human rTNFa were generously provided by the Asahi Chemical Industry Co. (Tokyo, Japan). Preparation of culture supernatants of LPS-stimulated mononuclear cells: Peripheral blood mononuclear cells were isolated from heparinized blood of healthy adult donors by Ficoll-Hypaque centrifugation. The mononuclear cells from the interface were washed three times with RPMI-1640, supplemented with 10% fetal calf serum, 2 mmole/l L-glutamine, 100 pg/ml streptomycin, and 200 U/ml penicillin. For the preparation of the cytokine-containing culture supernatants, the mononuclear cells were suspended at a concentration of 2 X 106 cells/ml in a supplemented RPMI-1640 medium containing 2.5 p /ml LPS, and cultured for 24 hr at 37°C in 5% CO2 in humidified air. After centri Pugation at 150 g for 5 min, the supernatants were collected and stored at -20°C. Culture supernatants that were obtained from mononuclear cells suspended in a supplemented RPMI-1640 medium without LPS were used as a control. Activation of PMNs: The PMNs were isolated from venous blood of healthy adult donors by centrifugation on Ficoll-Hypaque, dextran sedimentation, and a hypotonic lysis of erythrocytes, as has been previously described (15). The final cell suspension consisted of more than 96% PMNs, as monitored in Giemsa-stained cytopreparations. Their viability was consistently more than 95% as determined b trypan blue exclusion. The PMNs (5 X 106 cells/ml) were suspended in HEPE B (30mM)-buffered HBSS. Aliquots of purified PMNs were warmed to 37°C for 5 min and then incubated with the same volume of the culture supernatant of LPSstimulated mononuclear cells at 37°C. Several human cytokines were added to the suspensions of these PMNs, and incubated in the same fashion as for the culture supernatant. Measurement of PGEz production of PMNs: The amount of the PGE2 concentration in the PMNs supernatant were determined by radioimmunoassay kit (New England Nuclear, Boston, MA). As PGE2 also was produced by the mononuclear cells, the amount of the PGE2 production of the PMNs was estimated by the following formula: PGE2 production of PMNs = PGE2 concentration after stimulation of PMNs - l/2 of PGE2 concentration of the mononuclear cell culture supernatants. Each experiment was done in triplicate, and all values are expressed as means f SE. RESULTS The time course of the PGEz production

of the PMNs with

the culture

supernatant of LPS-stimulated or non-stimulated mononuclear cells is shown in Figure 1. The PMNs incubated with the culture supernatants of LPS-stimulated mononuclear cells showed an enhanced production of PGE2. The culture supernatant of the non-stimulated mononuclear cells did little to augment the PGE2 production of the PMNs. 858

Vol.

168, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

200 l

: LPs(+)

0: ”

(-)

z:M+SE(n=3)

E kJ ,a $4 2 e looN

Incubation

time

(min)

Fimre 1. Effects of incubation with the culture supernatant of LPS-stimulated mononuclear cells (SUP) on the PGE2 production of the PMNs. The SUP (@) or nonstimulated culture supernatant (0) was incubated with the same volume of suspensionsof PMNs at 37°C. Values are expressedasmeans f SE of three normal donors.

To clarify the cytokine(s) which augmented the PGE2 production of the PMNs, the effects of 120 min incubation with various human cytokines were investigated. As shown in Table 1, LPS itself did not augment the PGE2 production of the PMNs. A significant increment of PGE2 in the supernatants, however, was noticed after the addition of rTNFa. The other cytokines, rIL-la, rIL-l&3, rIL-2, rIL-6, and IFNr, demonstrated no effects on the PGE2 production of the PMNs. The dose-dependent effects of rTNFct on the production of PGEz during a 120min incubation is shown in Figure 2. The maximum enhancement was observed at 100 - 1000 U/ml of rTNFa.

Table 1 A

Effects of human cytokines on the PGE2production of the PhINs PGEz * (pg/ml) Control LPS rTNFa rIL-la rIL-lfl rIL-2 rIL-6 IFNr

14 16 102 13 20 16 13

(2.5 pg/ml) (100 U/ml) (100 U/ml) (100 U/ml) (100 U/ml) (100 U/ml) (100 U/ml)

9

f 3 f 3 f 21 § f 2 f 7 f 3 f 3 f

5

*M f SE (n=3). §p

Mononuclear cells enhance prostaglandin E2 production of polymorphonuclear leukocytes via tumor necrosis factor alpha.

To clarify the interactions between mononuclear cells and polymorphonuclear leukocytes, and to identify the cytokine(s) that mediate the interaction, ...
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