Immunology 1990 69 312-315

Production of tumour necrosis factor by mastocytoma P815 I. OHNO, Y. TANNO, K. YAMAUCHI & T. TAKISHIMA First Department School of Medicine, Sendai, Japan

cells

of Internal Medicine, Tohoku University

Acceptedfor publication 20 September 1989

SUMMARY P815, a transformed mouse mastocytoma cell line, produced and released cytotoxic factors after stimulation with phorbol 12-myristate 13-acetate (PMA), but not with lipopolysaccharide (LPS), calcium ionophore A23187 and IgE receptor triggering. The cytotoxic activity was reduced 60% by antibodies to mouse tumour necrosis factor (TNF). In addition, we demonstrated that TNF mRNA was already expressed under normal culture conditions and that it increased after stimulation with PMA. Although it was unknown whether factors other than TNF were lymphotoxin or some other unknown factors, it has been suggested that mast cells have cytotoxic activity, and that they contribute to inflammatory response through the release of TNF, which has a wide range of biological activity.

INTRODUCTION Tumour necrosis factor (TNF) was originally identified in the serum of mice infected with Mycobacterium bovis strain BCG (Bacillus Calmette-Guerin) and subsequently injected with endotoxin (Carswell et al., 1975). It could elicit haemorrhagic necrosis of some tumours in recipient animals (Carswell et al., 1975) and was cytotoxic to tumour cells in vitro without killing normal cells (Carswell et al., 1975). Activated monocytes/ macrophages are known to be the major cellular source of TNF (Carswell et al., 1975; Matthews & Watkins, 1978). Recently, the expression of TNF mRNA by other cells has been detected. For example, it was known that human peripheral T lymphocytes produce TNF with IL-2 or anti-T3 and express its gene detected by Northern analysis using human TNF (hTNF) cDNA (Steffen, Ottmann & Moore, 1988). B lymphoblastoid cells (Williamson et al., 1983) and human endothelial tumour cell line (Spriggs et al., 1988) were also reported to produce TNF. In addition, basophils/mast cells, which have been recognized to play a major role in immediate allergic reaction by releasing chemical mediators such as histamine (reviewed by Wasserman, 1980), were observed to have a potential for releasing cytotoxic factors related to TNF. Previously, we reported that rat basophilic leukaemia (RBL) cells could produce and release the cytotoxic factor related to TNF by triggering IgE receptors (Ohno et al., 1988). Moreover, Young et al. reported that murine mast cells produced a TNF-like factor after stimulation with a combination of phorbol 12myristate 13-acetate (PMA)/concanavalin A (ConA) or lipo-

polysaccharide (LPS) (Young et al., 1987), and other investigators demonstrated the presence of TNF mRNA and TNF protein in bone marrow-derived mast cells (Steffen et al., 1989). On the other hand, P815, which is a mouse mastocytoma cell line (Dunn & Potter, 1957), showed no cytotoxicity without stimulation (Jadus et al., 1986; Liu et al., 1989). However, gene expression and production of TNF by P815 cells with stimulation has not yet been evaluated. Therefore, to determine whether P815 cells produced TNF after stimulation, we assayed cytotoxicity in culture supernatants and cell lysates and performed Northern analysis of TNF mRNA with mouse TNF (mTNF) cDNA.

Abbreviation: TNF, tumour necrosis factor. Correspondence: Dr I. Ohno, First Dept. of Internal Medicine, Tohoku University School of Medicine, Sendai 980, Japan.

Stimulation of cells 106 cells each of J774A. 1 scraped from adherent culture or P815 harvested from floating culture were incubated with 1 ml of

MATERIALS AND METHODS

Cells TNF sensitive cell line L929 was provided by Dr 0. Yoshie, Department of Bacteriology, Tohoku University School of Medicine, Sendai, Japan (Yoshie, Tada & Ishida, 1986). Mouse monocyte/macrophage cell line, J774A.1 and mouse mastocytoma cell line, P815 were obtained from Japanese Cancer Research Resources Bank, Tokyo, Japan. Cells were grown in RPMI-1640 (Gibco Laboratories, Grand Island, NY), except for J774A. 1 grown in NYSF404 (Kohjin Co., Tokyo, Japan), supplemented with 100 U/ml penicillin (Meiji Seika Kaisha Ltd., Tokyo, Japan), 100 Mg/ml streptomycin (Meiji) and 10% heat-inactivated fetal bovine serum (Flow Laboratories Inc., Mclean, VA) at 370 in a humidified atmosphere of 5% C02:95% air.

312

Production of TNF by mastocytoma cells stimulant in each well of a 12-well tissue culture plate (Coaster, Cambridge, MA). As stimulants, LPS (E. coli; Sigma Chemical Co., St. Louis, MO) was used at a final concentration of 10 Mg/ ml, PMA (Sigma) at 5 ng/ml, calcium ionophore A23187 (Sigma) at 5 Mg/ml, in RPMI-1640 containing 2% of fetal bovine serum, and medium alone as control. These experiments were performed in duplicate in culture plates. After incubation for various periods, the culture supernatants were harvested. The cell lysates of P815 were obtained by freezing and thawing, sonication and centrifugation (Young et al., 1987). IgE receptor triggering Anti-ovalbumin (OVA) mouse serum was obtained from 6- to 8-week-old female C3H/HeN mice, which were bred in the Institute for Experimental Animals, Tohoku University School of Medicine, Sendai, Japan, injected twice at a 3-week interval with 5 pg of OVA (Sigma) in 0-5 mg of aluminium hydroxide (Wako Pure Chemical Industries Ltd, Osaka, Japan) (Ida, Siraganian & Notkins, 1983). Passive sensitization and antigen challenge of P815 cells were performed as described previously (Ohno et al., 1988). Briefly, the cells were passively sensitized at 3 x 106/ml with RPMI-1640 containing 5% of anti-OVA mouse serum at 370 for 1 hr. After sufficient washing with RPMI-1640, 106 cells were incubated with 1 ml of OVA at 10-' to I03 ng/ml and harvested as described above. It has been reported that these procedures induced histamine release from basophilic cells through a IgE receptor-mediated mechanism (Segal et al., 1981).

Histamine assay At 30 min after stimulation, the cell supernatants were harvested and assayed for histamine content with an automated fluorometric technique. The total histamine content of each well was determined by adding 6% of perchloric acid. Spontaneous histamine obtained from the cells stimulated with medium alone was less than 10% of total histamine content. The experimental results were expressed as the percentage of histamine release calculated from the following formula:

histamine release (%) = (E-S) x 100/(T-S) where E was the histamine content of experimental sample, T was the total histamine content and S was the spontaneously released histamine content (Siraganian & Hook, 1980).

Assayfor cytotoxic activity The cytotoxic activity of the culture supernatants and the cell lysates was measured on L929 cells with actinomycin D (Sigma) (Warren & Ralph, 1986). Briefly, L929 cells were seeded at a density of 3 x 104 cells per well in 96-well plastic tissue culture plates (Corning Glass Works, Corning, NY) in 100 p1 of RPMI1640 containing 10% of fetal bovine serum. After incubation for 24 hr, the medium of the L929 culture was discarded and replaced with 2-fold serial dilutions of the test samples containing 1 pg/ml of actinomycin D in RPMI-1640 containing 2% of fetal bovine serum. After incubation for 18 hr, the culture medium was discarded and the L929 cells were further incubated with RPMI-1640 containing 5% fetal bovine serum and 0-006% Neutral Red (Gibco) for 75 min. After washing with 0 1 M phosphate-buffered saline, pH 7-4, Neutral Red in the viable L929 cells was eluted into 100 p1 of 0-01 N HCI containing 30% of ethanol, and the absorbance of each well was read at 540 nm

313

with Titertek Multiskan (Flow Laboratories, McLean, VA). A total of 100% lysis of cells was achieved with 100 u1 of RPMI1640 containing 5% of Triton X-100 (Sigma) and 0% lysis was determined to have occurred in wells incubated only with reagents used for stimulation of cells. Percent cytotoxicity was calculated from the following formula: cytotoxicity (%) = [A(0)-A(S)] x 100/[A(0)-A(100)] where A(0), A(l00) and A(S) were A54o of each well of 0% lysis, 100% lysis and samples. Data were averaged from 4 wells. In addition, recombinant hTNF (5 x 105 U/ml on L929 cells, providied from Asahi Chemical Industry Co. Ltd, Tokyo, Japan), recombinant mTNF (>99% pure, 4 x 107 U/mg on L929 cells, Genzyme Corporation, Boston, MA) and culture supernatants were also assayed after incubation for 30 min at 370 with a polyvalent rabbit antiserum against recombinant mTNF-a (approximately 106 neutralizing units per ml, Gemzyme) or normal rabbit serum (TAGO Inc., Burlingame, CA). Effect of a protein synthesis inhibitor on the induction of cytotoxicity In some experiments, cells were incubated with stimulants in the presence of cycloheximide (Sigma) at 2 ug/ml. The cytotoxic activity was assayed after 24 hr as described above. RNA preparation and Northern blot analysis Extraction of cellular RNA and evaluation of TNF mRNA were performed as described previously (Chirgwin et al., 1979; Yamauchi, Martinet & Crystal, 1987). 107 to 108 of cells were incubated with a stimulant, which was either 10 yg/ml of LPS for J774A.1 or 5 ng/ml of PMA for P815, in RPMI-1640 containing 2% of fetal bovine serum. After stimulation for 4 hr in tissue culture dishes (150 x 25 mm style; Becton-Dickinson Labware, Lincoln Park, NJ), the cells were lysed in 5-2 M guanidine isothiocyanate (Wako) solution. The lysates were mixed with caesium chloride (Mitsuwa's Pure Chemicals, Osaka, Japan; (1 g/2-5 ml), layered on a cushion of 5-6 M caesium chloride, 0-1 M EDTA (Wako), pH 7-6, in a polyallomer tube (Hitachi Koki Co. Ltd, Tokyo, Japan) and centrifuged at 200 in a RPS50-2 roter (Hitachi) at 36,000 r.p.m. for 12 hr. The pellet was dissolved in 100 mm NaCl, 10 mm Tris-HCl, pH 8-6, 1 mm EDTA, 1% (w/v) SDS (Sigma), precipitated with ethanol, redissolved in autoclaved water, and stored in REVCO freezer (Rheem Manufacturing Company, Asheville, NC) at -800. TNF mRNA in the cellular RNA was evaluated using Northern analysis. Twenty micrograms of each RNA were fractionated in 1 % agarose gel (International Biotechnologies Inc., New Haven, CT) under denaturing conditions with 6% of formaldehyde, and transferred to a nitrocellulose filter (Bethesda Research Laboratories Life Technologies Inc, Gaithersburg, MA) which was stored at 40 overnight. The nitrocellulose filter was hybridized with mTNF cDNA (provided from Asahi Chemical Industry Co. Ltd) (Shirai et al., 1988) labelled with multiprime DNA labelling system (Amersham International plc., Buckinghamshire, U.K.) using 32P-deoxycytidine 5'-triphosphate (New England Nuclear, Boston, MA). Hybridization was performed at 420 for 18 hr followed by washing the filter four times (20,5 min each) in 2 x SSC, 0-05% SDS and four times (600, 30 min each) in 0-1 x SSC, 0 05% SDS. Autoradiogram was performed by exposure to RX X-ray film (Fuji Photo Film Co. Ltd, Tokyo, Japan).

I. Ohno et al.

314

Table 1. Cytotoxic activity in culture supernatants of P815 cells incubated with various stimulants

Stimulants*

J774A.I P815

A23187

OVA

18-1+5-3 (4)

ND:

26-1±+45 (6)

0-2+0 2 (4)

ND 0 2+0 2 (4)

Medium

LPS

PMA

00+00 (4)t 2-6+1-0 (6)

79-1+8-5 (4) 4-2+2-6 (4)

Cells

* One ml of the stimulants was added to 106 ofthe cells at the concentration as described in Materials and Methods. t After incubation for 24 hr, cytotoxic activity was assayed and expressed as mean + SE (n). t ND, not done.

I

30

,

2 3

28S 18S

20

10,

0.

5

8

24

Incubation time (hr) Figure 1. Kinetics of cytotoxic activity from P815 cells. P815 cells were incubated with PMA at S ng/ml. After incubation for various periods, the culture supernatants (open circle) and cell lysates (closed circle) were assayed for cytotoxic activity and expressed as mean + SE of four samples.

RESULTS

Cytotoxic activity in culture supernatants of P815 with various stimulants One million of the cells were incubated with various stimulants, and the cytotoxic activity of the supernatants was assayed after incubation for 24 hr (Table 1). The cytotoxic activity of the supernatants from J774A. 1 cells, which were reported to produce TNF (Pennica et al., 1985), was 79-1+85% when stimulated with LPS, and 18-1 +5-3% with PMA. For stimulation of P815, in addition to PMA and LPS, calcium ionophore A23187 and the combination of anti-OVA mouse serum and OVA were used. Regardless of stimulant, no cytotoxicity could be detected after 24 hr, except for the supernatant with PMA which showed 27-9 + 7-1 % of cytotoxicity significant to medium (2-6+ 1-0%, P

Production of tumour necrosis factor by mastocytoma P815 cells.

P815, a transformed mouse mastocytoma cell line, produced and released cytotoxic factors after stimulation with phorbol 12-myristate 13-acetate (PMA),...
828KB Sizes 0 Downloads 0 Views