Immunological activities of RU-41740, a glycoproteic extract from klebsiella pneumoniae

©

Ann. Inst. Pasteurllmmunol. 1987, 138, 571-584

ELSEVIER

Paris

1987

IMMUNOWGICAL ACTIVITIES OF RU-41740, A GLYCOPROfEIC EXTRACT FROM KLEBSIELLA PNEUMONIAE II. -

ACTIVATION OF MACROPHAGE CYTOTOXICITY

AGAINST TUMOUR CELLS AND PRODUCTION OF A CYTOTOXIC FACTOR

F. Vacheron (I), P. Smets (3), C. Nauciel (1) and M. Guenounou (1,2) (I) Laboratoire de Microbiologie, UFR de Medecine Paris Ouest,

92380 Ga..rches (France),

(2) UA-CNRS 40622, Universite Paris Sud, 92290 Chfitenay Malabry (France), and (3) CRI Roussel-UclaJ, 95520 Osny (France)

SUMMARY

RU-41740, a glycoprotein extract from Klebsiella pneumoniae, is an immunomodulating agent with a broad spectrum of activities. It enhances several macrophage functions, including interleukin-l (IL-l) secretion. Present data show that RU-41740 was able to promote murine macrophage cytotoxicity against tumour cells. A soluble cytotoxic factor (CF) was found in supernatants from macrophage cultures stimulated with RU-41740. These supernatants were shown to contain IL-l. CF was detected on L-929 cells sensitized by actinomycin D. CF was analysed on the basis of MW and pHi: after gel filtration on «Ultrogel Aca54 », a single peak of CF was found in the range of 50-60 Kd and was then distinct from IL-l, which was eluted at 15 Kd. After chromatofocusing, CF was found in a narrow peak of pH 4.8. CF was detected in supernatants 2 h after macrophage stimulation by RU-41740, and its release was abolished by pretreatment of macrophages with cycloheximide (2 fLg/ml). CF described here shares several properties with tumour necrosis factor (TNF). KEY·WORDS: Immunomodulation, Klebsiella pneumoniae, Glycoprotein, Macrophage; RU-41740, Cytotoxicity, Mastocytoma, L-929 cells.

Submitted January 21, 1987, accepted May 29, 1987.

572

F. VACHERON AND COLL. INTRODUCTION

RU-41740 (trade name Biostim) is a glycoprotein extract from Klebsiella pneumoniae. By the oral route, it has been used as an immunotherapeutic

agent in chronic bronchitis patients to reduce the number and length of infectious episodes [30]. In cancer patients, RU-41740 has been shown to restore cutaneous delayed-type hypersensitivity [13]. Experimental studies have shown that RU-41740 enhances both humoral and cell-mediated immune responses and protects against bacterial and viral infections [6, 26]. At the cellular level, RU-41740 acts as a selective B-cell mitogen [7,32] and is a potent inducer of interleukin-l (IL-l) production by mouse macrophages [8], human monocytes [8] and large granular leukocytes (LGL) [9]. Macrophages express tumoricidal activity after treatment in vitro by a variety of stimulants including lymphokines [4, 12, 17], interferon i [23, 14] and bacterial products such as lipopolysaccharide (LPS) [1, 31], peptidoglycans [19, 29] and muramyl dipeptide [11, 27, 28].

In this work, we investigated whether RU-41740 was able to promote murine macrophage cytotoxicity in vitro. We found that macrophages activated by RU-41740 exhibited cytotoxic activity against tumour cells. Moreover, RU-41740 was able to induce the secretion by macrophages of a soluble cytotoxic factor (CF). Such a factor was distinguishable from IL-l also released in the same conditions into the culture medium. MATERIALS AND METHODS

Animals. Female DBAI2 and C3H/HeJ mice, 7 to 10 weeks of age, were obtained from the «Ferme Experimentale de l'Institut Pasteur» (Villepreux, France). Macrophages. Four days after intraperitoneal injection of 2 ml of thioglycolate broth, peritoneal exudate cells (PEC) were harvested by washing the peritoneal cavity with 5 ml Hanks' balanced salt solution (HBSS) containing 1 0,10 heat-inactivated foetal calf serum (FCS).

abs

BSA

CF ConA CPM FCS

HBSS

IL-l Kd

absorbance. bovine serum albumin. cytotoxic factor. concanavalin A. count per minute. foetal calf serum. Hank's balanced salt solution. interleukin-l. kilodalton.

LGL

LPS

MW

PEC

PBS

TCM

TNF U

large granular leukocyte. lipopolysaccharide. molecular weight. peritoneal exudate cell. phosphate-buffered saline. tissue culture medium. tumour necrosis factor. unit.

IMMUNOLOGICAL ACTIVITIES OF RU-41740

573

Activating agents. RU-41740 (Cassenne-Roussel Uclaf, Paris) was prepared for use by making up a stock solution of 1 mg/mllyophilized powder in 0.15 M phosphate-buffered saline pH 7.2 (PBS; Gibco). The same batch (n° RP 23) of RU-41740 was used throughout. Lipopolysaccharide (LPS) from Escherichia coli was purchased from Difco (Detroit, MI) and concanavalin A (ConA) from Pharmacia (Uppsala, Sweeden). Other reagents. Actinomycin D, indomethacin and cycloheximide were purchased from Sigma (St. Louis, MO). Target cells. P815 mastocytoma cells were maintained by serial passages in the peritoneal cavity of DBAI2 mice. L-929 cells, a transformed murine fibroblast cell line, were grown at 37°C in 5 070 CO2 , in tissue culture medium (TCM) consisting of RPMI-1640 (Gibco) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 fLg/ml streptomycin and 10 % FCS. Macrophage-mediated cytostasis assay. PEC were adjusted at 2 x 106 /ml in TCM and plated under a volume of 0.1 ml into the wells of 96-well microtitre plates (Falcon, Microtest II). Two hours later, non-adherent cells were removed by washing the culture wells three times with HBSS. Adherent cells were either used immediately in a cytotoxic assay with P815 cells and bacterial stimulant, or were incubated for 18 h in the presence of bacterial stimulant before the addition of P815 cells. In both cases, bacterial stimulant was added at different concentrations under a volume of 20 fL!' P815 mastocytoma cells were harvested from the peritoneal cavity of DBAI2 mice. They were then washed, adjusted at 2 x 10 5 /ml and added (0.1 ml) to the macrophage monolayers preincubated or not for 18 h. The cultures were incubated for 24 h in an atmosphere of 5 % COz-95 % air at 37°C. They were pulsed for the final4-h culture period with 0.2 fLCi of 3H-thymidine (specific activity 1 fLCi/mM, CEA France). Cells were then harvested onto glass fibre filters with an automatic harvester (Dinatech Laboratories, Alexandria, VA). Filter disks were dried and the radioactivity was counted by liquid scintillation spectrophotometry. Results were expressed as the mean counts per minute (CPM) of triplicate samples ± the standard error. The percentage of growth inhibition (% GI) was defined as the relative radioactivity incorporated by P815 cells in the presence of macrophages (CPM P815 + macrophages) versus the mean counts incorporated in the absence of macrophages (CPM P815 alone), and was obtained by the following formula: CPM P815 + macrophages ) x 100. %01=(1CPM P815 alone Macrophage supernatant. PEC were adjusted at 0.5 x 10 6 /ml in TCM. Cells were plated into 16-mm diameter wells of multiwell plates (Falcon) under a volume of 0.5 ml per well and

574

F. VACHERON AND COLL.

incubated for 2 h at 37°C in an atmosphere of 5 % COr 95 % air. Non-adherent cells were removed by washing the cells 3 times with HBSS. The adherent cells consisted of more than 95 % macrophages, as shown by esterase staining [33]. The adherent cells were cultured in serum-free RPMI-1640 containing 25 mM HEPES (Gibco) unless otherwise stated. RU-41740 was added for various lengths of time. Supetnatants were then collected, rendered cellfree by centrifugation and tested for IL-l and cytotoxic activity. Culture supernatants designed for chromatographic studies were prepared by culturing adherent PEC in plastic flasks of 75 cm 2 (Falcon) in 15-ml medium. Thymocyte proliferation assay. IL-l activity in culture supernatants was assayed by measuring the proliferative response of mouse thymocytes. Thymocytes from C3H/HeJ mice were suspended in RPMI-HEPES containing 10 % FCS and antibiotics to a density of 1.5 x 10 7 cells/m!. The cell suspension was seeded under a volume of 0.1 ml into the wells of 96-well microtitre plates, and an equal volume of various dilutions of macrophage culture supernatants was added. Cultures were carried out with the addition of a submitogenic concentration of ConA (1 fLg/ml) and incubated in a humidified CO2 incubator for 72 h. They were pulsed for the final 6-h culture period with 0.5 fLCi 3H-thymidine. Cells were then harvested onto glass filters. Filter disks were dried and the radioactivity was counted by liquid scintillation spectrophotometry. All the samples tested were compared to a batch of partially purified murine IL-l used as a standard, arbitrarily assigned an activity of 100 V/m!. Results were expressed as units per ml as defined elsewhere [18]. Cytotoxic assay of macrophage supernatants. The method described by Fish and Gifford [5] was used. L-929 were seeded in 96-well microtitre plates at 5 X 10 4 cells/well in 0.1 ml TCM. Sample dilutions (0.1 ml) were added in the presence of actinomycin D (1 fLg/ml). The cells were incubated at 37°C for 18 h. Then supernatants were decanted and the cells were washed in HBSS. The remaining adherent cells were stained for 10 min with crystal violet (0.2 % in 2 % ethanol), washed with water and 0.1 ml of 1 % sodium dodecyl sulphate was added to each well to solubilize the stained cells. The absorbance of each well was read at 545 nm with a microELISA autoreader (LOV, Bio-Merieux, Marcy l'Etoile, France). Percent cytotoxicity was defined as the relative absorbance (abs) or sample versus control (medium only) wells: . . abs sample ) X 100. Percent CytOtOXICIty = (1abs control Cytotoxic activity units were obtained by determining the reciprocal of the dilution inducing 50 % cytotoxicity. Supernatants were also tested in a growth assay on L-929 cells or P815 cells. Target cells were incubated with macrophage supernatants for 24 h at 37°C. They were pulsed for the final 4-h culture period with 0.2 fLCi of 3H-thymidine. Percent growth inhibition was calculated as described above. Lymphokine supernatant. Lymphokine-containing supernatants were produced by incubating spleen cells from DBAI2 mice at a density of 5 X 106/ml for 24 h in TCM containing 2.5 fLg/ml ConA. The cells were removed by centrifugation and the supernatants were sterilized by filtration.

IMMUNOLOGICAL ACTIVITIES OF RU-41740

575

Gel filtration. One-hundred-fifty ml of culture supernatants from adherent PEC stimulated with

to Ilg/ml of RU-41740 were precipited with ammonium sulphate at a saturation of

70 %. After stirring for 20 h at 4°C, the precipitate was dissolved in 5 ml of 0.1 M Tris-HCI buffer (pH 7.3), dialysed against the same buffer for 48 h, applied to a column of «Ultrogel Aca54 » (Pharmacia) (2.6 x 90 em) and eluted with Tris-HCI buffer at a flow rate of 20 ml/h. Fractions of 7.5 ml were collected and lyophilized. The lyophilized fractions were resuspended in 1 ml of bidistilled water, sterilized through a 0.22-{Lm Millipore filter and tested for cytotoxic activity on L-929 cells. Bovine serum albumin (69 Kd), ovalbumin (45 Kd), chymotrypsinogen A (25 Kd) and ribonuclease A (13.5 Kd) purchased from Pharmacia, were used as standards. Void volume was calculated by the elution volume of blue dextran. Chromatofocusing. The ammonium sulphate precipitate from macrophage supernatant was dissolved in 0.025 M imidazole-HCI buffer (pH 7.4), dialysed overnight against the same buffer and fractioned by chromatofocusing on a «PBE 94» column (Pharmacia) (l x 35 cm) with 300 ml of polybuffer 74 pH 4 (Pharmacia). Fractions of 7.5 ml were collected, dialysed for 48 h against RPMI-1640 medium and tested for cytotoxic activity on L-929 cells (and for IL-1 on monocytes). RESULTS

RU-41740 can render macrophages cytostatic for tumour cells. Thioglycolate-elicited macrophages were stimulated for 18 h with RU-41740 at different concentrations; then they were washed and P815 cells were added. P815 cell growth was assessed by measuring JH-thymidine incorporation. We found that RU-41740-stimulated macrophages were able to inhibit the growth of P81S mastocytoma cells. Cytostatic activity occurred when macrophages were treated with 1 to 10 fLg/ml RU-41740 (fig. 1). Non-stimulated macrophages were weakly cytostatic « 10 % G.I) against P81S cells. Moreover, when target cells were added to macrophage monolayers at the same time as RU-41740, a more pronounced cytostasis towards P81S cells was observed. RU-41740 was active at lower concentrations (10- 3 fLg/ml to 10- 2 fLg/ml) and had no direct cytostatic activity on PSIS cells in the absence of macrophages (fig. 1). As RU-41740 was present during the entire culture time, we checked whether this cytostatic activity was due to a direct effect of RU-41740 on P815 cells. P815 cells were incubated with RU-41740 for 24 h at 37°C, then washed three times in TeM and added to non-stimulated macrophage monolayers. In the latter case, we did not observe a cytostatic effect of macrophages. RU-41740 therefore could not sensitize P815 cells to the cytostatic effect of macrophages. As previously shown for LPS [10], we examined the effect of lymphokinecontaining supernatant on RU-41740-induced cytotoxicity. Macrophages were incubated with RU-41740 and ConA-induced lymphokine-containing supernatant for 24 h. They were then extensively washed and P815 cells were added

576

F. VACHERON AND COLL.

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FIG. 1. - Macrophage monolayers were preincubated for 24 h with different concentrations of RU-41740 alone (e) or in the presence of lymphokine supernatant (0) before adding P8I5 cells.

The curve designated by open squares (0) shows the effect of RU-41740 alone when added to the macrophage monolayers at the same time as P815 cells. The figure represents the results of one representative experiment out of five. RU-41740 had no direct cytotoxic activity on P815 cells in the absence of macrophages ( • ).

to macrophages monolayers. Figure 1 shows that the addition of lymphokinecontaining supernatant alone had no effect on the macrophage cytostatic effect, which was spontaneously very low (5 to 10 0,10). The addition to such lymphokine-containing medium of minimal doses (10- 3 to 10- 2 (Jog/ml) of RU-41740 resulted in a marked increase in the cytostatic effect (55 to 90 % growth inhibition). Production of a cytotoxic factor by macrophages after activation by RU-41740.

Supernatants from macrophage cultures stimulated with RU-41740, (10 (Jog/ml) were examined for the presence of soluble cytotoxic activity against tumour cell lines. Using P815 mastocytoma cells, 30 % growth inhibition was observed with RU-41740 supernatants at a 1/4 dilution. With L-929 cells, 40 %

IMMUNOLOGICAL ACTIVITIES OF RU-41740

577

growth inhibition occurred. Supernatants from non-stimulated cultures did not inhibit the growth of the tumour cells used. When L-929 cells treated with actinomycin D (l fLg/ml) were used as targets, they were found to be more sensitive to cytotoxic activity, as shown in table I. Table I also shows that supernatants contained IL-l, as described previously [7]. Moreover, RU-41740 was able to induce the production of cytotoxic activity by macrophages from the LPS non-responder C3H/HeJ mice. An activity of 50 U was found in supernatant from C3H/HeJ macrophages stimulated with RU-41740 (10 fLg/ml). RU-41740 had no direct cytotoxicity on actinomycin-treated L-929 cells.

1. - IL-1 and cytotoxic activity in supernatants from DBA/2 and C3H/HeJ macrophages stimulated by RU·41740 and LPS.

TABLE

Stimulant RU-41740

LPS

IL-I II 12

DBAI2 Cytotoxic activity 128 80

C3H/HeJ

IL-I

6

< 2

Cytotoxix activity 50

< 2

Thioglycolate-ehcited macrophages from DBA/2 or C3H/HeJ mice were incubated for 24 h with RU-4l740 (10 I-'g/ml) or with LPS (10 I-'g/ml). Supernatants were harvested and tested for IL-l on thymocytes and cytotoxicity on L-929 cells, as described in «Material and Methods ». Results are expressed as units/m!. RU-41740 alone showed no cytotoxicity on actinomycin-treated L-929 cells.

In additional experiments, macrophages were stimulated for 2 h with RU-41740 (10 fLg/ml), washed and then incubated in medium without stimulant. Supernatants were harvested at different times and tested for cytotoxic and for IL-l activities. Figure 2 shows that cytotoxic activity was produced within 2 h in the medium following stimulation of macrophages by RU-41740. The amount of cytotoxic activity was stable for 24 h after the pulse. IL-l was released later and a maximum of secretion was reached at 24 h. It is to be noted that cytotoxic activity, detected 24 h after the 2-h pulse by RU-41740, was lower than that observed with a continuous pulse (10 times less). This was not related to the absence of RU-41740 in these supernatants, since addition of RU-41740 (10 fLg/ml) to such supernatants did not modify their cytotoxic activity. The production of both cytotoxic and IL-l activities was inhibited after treatment of macrophages by cycloheximide (2 fLg/ml) during the adherence period. Addition of indomethacin to macrophage cultures enhanced IL-l activity, but did not affect cytotoxic activity (table II).

578

F. VACHERON AND COLL. TABLE

II. - Influence of cycloheximide and indomethacin on IL-l and cytotoxic activity secretion. Treatment

Experiment 1

IL-I

Cytotoxic activity

10

80 4 190 190

(Vlml)

None Cycloheximide None Indomethacin

Experiment 2

(Vlml)

5 17 37

In experiment I, macrophages were allowed to adhere in the presence or not of cycloheximide (2 !J.g/ml), then washed and cultured 24 h in the presence of RU-41740 (10 !J.g/ml). Supernatants were harvested and tested for IL-I and cytotoxic activity. Cells assayed for viability were 90 % viable in the presence or absence of cycloheximide. In experiment 2, macrophages were cultured in the presence of RU-41740 (10 !J.g/ml) with or without indomethacin (I !J.g/ml) for 24 h. Supernatants were harvested and tested for IL-I and cytotoxic activity.

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Macrophage monolayers were incubated for 2 h with 10 fLg/ml of RU-41740. They were then washed and cultured with serum-free medium. Supernatants were collected at the time indicated and assayed for cytotoxic activity and IL-l, as described in «Materials and Methods».

Fractionation of culture supernatants. After gel filtration on «Ultrogel Aca 54», a major peak of cytotoxic activity was observed in the range of 50-60 Kd, whereas IL-l eluted at 15 Kd (fig. 3).

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Fractions of 7.5 ml were collected, lyophilized, dissolved in 1 ml water and dialysed against RPMI-1640. The fractions were tested for cytotoxic activity on L-929 cells and for IL-1 on thymocytes at 1120 dilution. BSA = bovine serum albumin ; OVA = ovalbumin; CHYM = chymotrypsinogen A; RNase = ribonucleotidase.

After chromatofocusing, cytotoxic activity was found in a narrow peak at 4.8 and IL-l in a larger zone from pH 4.6 to pH 5.4 (fig. 4). DISCUSSION

In this work, we showed that RU-41740 was able to induce non-specific cytotoxic activity in thioglycolate-elicited macrophages. When macrophages were stimulated for 18 h by RU-41740, then washed and used in a growth inhibition assay, we observed that RU-41740 moderately but significantly stimulated macrophage cytostatic activity towards tumour cells. The addition of lymphokine-containing supernatant during the 18-h pre-incubation period in the presence of RU-41740 markedly increased the cytotoxic potential of macrophages. The synergistic activation of macrophages has been

580

F. VACHERON AND COLL.

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previously reported for different combinations of immunomodulators such as lymphokines and LPS [10], lymphokines and MDP [25], interferon-I and LPS [24], and interferon-I and MDP [22]. The mechanism by which RU-41740 activates macrophage cytotoxicity was of interest, and we searched for a lytic factor in macrophage culture supernatants. Elsewhere, we described the fact that IL-l activity was detected in supernatants from macrophage cultures stimulated with RU-41740 [7]. Data presented here show that such supernatants were found to be cytotoxic towards tumour cells. A high level of cytotoxic activity was detected on L-929 cells sensitized by actinomycin D. Cytotoxicity was also detected, at a lower level, on P815 cells or L-929 in a growth assay. Fractionation of supernatants by gel chromatography showed a peak of cytotoxic activity in the range of MW from 50 to 60 Kd, and was distinct from IL-l activity. IL-l was detected principally at 15 Kd (fig. 3). In con-

IMMUNOLOGICAL ACTIVITIES OF RU-41740

581

sidering the chromatofocusing of supernatants, we observed that CF was present in a very narrow peak of pHi 4.8, whereas IL-l, which is apparently more heterogenous, was found in a large zone of pHi 4.6 to 5.2. As shown for IL-l [7], RU-41740 induces CF secretion by macrophages from LPS-non-responder C3H/HeJ mice. Indeed, RU-4l740 is a heterogenous compound mainly composed of two macromolecular fractions: a glycoprotein of 95 Kd and an LPS-like molecule bound to a protein portion (350 Kd) [7]. Experiments presently under investigation indicate that the effect on C3H/HeJ macrophages could be associated with the lipid-free 95-Kd glycoprotein present in RU-4l740. Whether the cytotoxic activity of RU-41740-activated macrophages was exclusively related to the secretion of such CF is difficult to establish. IL-l has also been shown to be cytocidal against several human and murine tumour cell lines [20, 15]. Present data show that IL-l-containing fractions were not cytotoxic against L-929 cells and P8l5 cells. This is in agreement with other works showing that IL-l is active neither on P8l5 [20] nor on L-929 cells, even in the presence of actinomycin D [15]. The CF was produced only by thioglycolate-elicited macrophages, whereas IL-l was produced by both resident and thioglycolate-elicited macrophages (data not shown). Cytotoxic activity was released more rapidly than IL-l by macrophages after addition of RU-4l740. Cytotoxic activity and IL-l production were under active synthesis, since pretreatment by cycloheximide diminished the secretion of both activities. CF was resistant to heating for 30 min at 56°C. CF activity was not inhibited by protease inhibitors such as phenylmethylsulphonyl fluoride or soybean trypsin inhibitor, nor by high concentrations of serum (20 070) (data not shown). From these properties, CF described here can be assumed to share several properties with tumour necrosis-related factor(s) [16, 3] and distinct from factors with protease activities [1, 21]. These observations further extend the range of RU-4l740 actions. Moreover, RU-4l740 has been shown to promote NK cell cytotoxicity [9] among human leukocytes. RESUME

PROPRIETES IMMUNOLOGlQUES D'UNE OLYCOPROTEINE EXTRAITE DE KLEBSIELLA PNEUMONIAE, LE RU-4l740 II. - ACTIVAnON DE LA CYTOTOXICITE DES MACROPHAGES ENVERS DES CELLULES TUMORALES ET PRODUCTION D'UN FACTEUR CYTOTOXIQUE

Le RU-4l740 est un extrait glycoproteique de Klebsiella pneumoniae doue d'activites immunostimulantes in vivo et in vitro. In vitro, il stimule de nom-

582

F. VACHERON AND COLL.

breuses fonctions du macrophage, en particulier la production d'interleukine-l (lL-l). Dans ce travail, nous montrons que les macrophages peritoneaux de souris stimules par Ie RU-41740 sont capables d'exercer une activite cytostatique envers des cellules tumorales. De plus, l'etude des surnageants de culture de macrophages actives par Ie RU-41740 montre qu'ils contiennent un facteur cytotoxique pour les cellules tumorales. L'activite cytotoxique est detectee sur les cellules L-929 sensibilisees par l'actinomycine D. Le fractionnement des surnageants de culture de macrophages par chromatographie d'exclusion en gel Aca54 montre que Ie facteur cytotoxique possede une masse moleculaire de 50-60 Kd, ce qui Ie distingue de l'IL-l (15 Kd). Par chromatofocalisation, on obtient un pic etroit d'activite cytotoxique a pH 4,8. Le facteur cytotoxique est detecte dans les surnageants 2 h apres stimulation des macrophages par Ie RU-41740, et sa secretion est inhibee par Ie cycloheximide (2 (.Lg/ml). Le facteur cytotoxique decrit ici partage de nombreuses proprietes avec Ie «tumour necrosis factor». MOTs-cLES: Immunomodulation, Klebsiella pneumoniae, Glycoproteine, Macrophage; RU-41740, Cytotoxicite, Mastocytome, Lignee L-929.

References.

[1] ADAMS, D.O., !(Ao, K., FARB, R. & PIZZO, S.V., Effector mechanisms of

[2] [3] [4] [5]

[6]

[7]

[8]

cytolytically activated macrophages. - II. Secretion of cytotoxic factor by activated macrophages and its relationship to secreted neutral proteases. J. Immunol., 1980, 124, 293-300. ALEXANDER, P. & EVANS, R., Endotoxin and double-stranded RNA render macrophages cytotoxic. Nature (Lond.), 1971, 232, 76. DRYSDALE, B., ZACHARCHUCK, C.M. & SHIN, H.S., Mechanism of macrophagemediated cytotoxicity: production of a soluble cytotoxic factor. J. Immunol., 1983, 131, 2362-2367. FIDLER, I.J., DARNELL, J.H. & BUDMEN, M.B., In vitro activation of mouse macrophages by rat lymphocyte mediators. J. Immunol., 1976,117,666-673. FISH, H. & GIFFORD, G.E., A photometric and plaque assay for macrophagemediated tumor cell cytotoxicity. J. Immunol. Methods, 1983,57,311-325. GRISCELLI, C., GROSPIERRE, B., MONTREUIL, J., FOURNET, B., BRUVIER, G., LANG, J.M., MARCHIANI, C., ZALISZ, R. & EDELSTEIN, R., Immunomodulation by glycoprotein fractions isolated from Klebsiella pneumoniae, in « Immunomodulation by microbial products and related synthetic compounds» (Yamamura, Y. & Kotani, S.) (pp. 261-265). Excerpta Medica, Amsterdam, 1982. GUENOUNOU, M., VACHERON, F., ZALISZ, R., SMETS, P. & AGNERAY, J., Immunological activities of RU-41740, a glycoproteic extract from Klebsiella pneumoniae. - I. Activation of murine B cells and induction of interleukin-l production by macrophages. Ann. Immunol. (Inst. Pasteur), 1984, 135 D, 59-69. GUENOUNOU, M., VACHERON, F., NAUCIEL, C. & AGNERAY, J., Induction of interleukin-l secretion by murine macrophages and human monocytes after stimulation by RU-41740, a bacterial immunomodulator. Int. J.Immunopharmacol, 1985, 7, 287-290.

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