Eur. J. Immunol. 1987.17: 1665-1668 Interleukin 1 secretion and tumoricidal activity by macrophages 1665

Short paper

Robert Keller and Ruth Keist

Immunobiology Research Group, Institute for Immunology and Virology, University of Zurich, Zurich

Induction of interleukin 1 secretion and of tumoricidal activity in macrophages are not closely related phenomena*

Rat bone marrow-derived mononuclear phagocytes (BMMQ) , induced to differenti- ate in vitro and homogeneous with respect to the cell lineage, were interacted with various macrophage-activating agents. The effect of these agents on the secretion of interleukin 1 (IL1) activity and on the development of tumoricidal capacity was comparatively assessed. The findings show that IL 1 secretion is considerably enhanced by macrophage-activating factor and by recombinant interferon-y but remained unaffected or was even suppressed by heat-killed C. parvum or Listeria monocytogenes. In contrast, lymphokines as well as C. parvum and Listeria were similarly potent in inducing in BMMQ a tumoricidal state. The findings indicate that induction of ILl secretion and of tumoricidal activity are not necessarily closely linked phenomena.

1 Introduction

Mononuclear phagocytes, under physiological conditions in a resting state, can be functionally activated by a number of stimuli, singly or in combination [l-31. Such activated mac- rophages are critically involved in inflammatory and immune responses. While the complex processes involved in mac- rophage activation are still poorly understood, it is now widely agreed that physiologic and biochemical properties such as the expression of surface receptors, the plasma membrane pro- teins, the secretion of immunomodulatory molecules, etc., can be up- or down-regulated. Macrophage (MQ) activation may finally lead to the expression of tumoricidal activity, and this development is accompanied by the synthesis of a variety of biologically active molecules, by the increased expression of major histocompatibility complex (MHC) class I1 (Ia) molecules [4] and/or the secretion of soluble products such as complement components or interleukin 1 (IL 1). IL 1 release from macrophages can be induced by numerous bacterial products and by various irritants. IL 1 is a polypeptide exhibit- ing an array of biologic activities including stimulation of T cell growth, B cell differentiation, induction of fever, increased synthesis of actue phase proteins, stimulation of fibroblast pro- liferation and/or regulation of interactions between the central nervous system and the immune system [5, 61. More recent .work suggested that IL 1 may even be cytostatic and/or cytoci- dal for some tumors and may thus play a role in host defense

[I 62431

* This study was supported by the Swiss National Science Foundation (grants 3.396.83 and 3.336.86) and the Canton Zurich

Correspondence: Robert Keller, Immunobiology Research Group, University of Zurich, Birchstrasse 95, CH-8050 Zurich, Switzerland

Abbreviations: IL 1: Interleukin 1 BMMcP: Bone marrow-derived mononuclear phagocytes PBS: Phosphate-buffered saline IMDM: Iscove’s modified Dulbecco medium FCS: Fetal calf serum M e Macrophage@) MAF: Macropohage-activated lymphokines rIFN- y: Recombinant rat interferon-y PHA: Phytohemagglutinin PF Paraformaldehyde

against tumors [7-lo]. These findings are in obvious contrast to the findings that bacterial products (e.g. lipopolysac- charides, exotoxins) usually induce in MQ IL 1 release without the parallel induction of tumoricidal activity. By comparing the ability of various MQ-activating agents to affect the secre- tion of IL 1 and to induce tumoricidal activity in bone marrow (BM)-derived mononuclear phagocytes (BMMQ) in vitro, the present work re-evaluated this issue.

2 Materials and methods

2.1 Effector cells

Male inbred DA and colony-bred ZBZ-Cara rats (120-180 g), raised locally and maintained under conventional conditions, were the source of bone marrow cells. Cultures of BMMQ were obtained as described previously [ l l , 121. Briefly, BM cells separated from femurs by perfusion with ice-cold phos- phate-buffered saline (PBS) were suspended in Iscove’s mod- ified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum (FCS; Gibco, Grand Island, NY), 5% horse serum (Gibco) and antibiotics, and conditioned with supernat- ant (final concentration 10%) from strain L clone 929 cells (CCL1; American Type Culture Collection, ATCC). The medium was tested routinely for endotoxin contamination, and only batches containing endotoxin less than 0.05 ng/ml (as assessed by the Limulus amebocyte lysate assay; Sigma Chem- ical Co., St. Louis, MO) were used. Adherent cells from day 6-7 liquid cultures were removed with ice-cold Ca2+-, Mg2+-free PBS, washed in PBS and resuspended in IMDM supplemented with 5% FCS and antibiotics. The cells were seeded into 16-mm wells (tissue culture plates, Petra Plastic, Chur, Switzerland) and cultured for 120 min at 37°C in a humid atmosphere of 5% C02 before nonadherent cells were removed by washing with IMDM. Some of the MQ cultures were fixed with 1% paraformaldehyde for 15 min and washed; IL 1 secretion and tumoricidal activity were assessed either directly or after a further 24-h culture in medium. To enhance their functional activity, adherent BMMcP were treated for the interval specified with M@-activating agents. The extent of MQ activation was assessed by measuring tumoricidal activity.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 oO14-2980/87/1111-1665$02.50/0

1666 R. Keller and R. Keist Eur. J . Immunol. 1987.17: 1665-1668

Table 1. Induction of IL 1 secretion in BMMQ, by various MQ,-activating agents"'

Incubation medium ['HIdThd incorporation (cpm f SD) by murine thymocytes Exp. no. 1 2 3 4 5 6

Control (without PHA) 183 (f 55) 227 (k 81) 196.(4 46) 2W(? 88) 180 (f 71) 124(f 45) Control (with PHA) 645 (f 127) 592 (k 112) 725 ( 2 206) 890 (+ 1%) 988 (f 312) 521 (k 147) MAF 20% 2151 (It:468) 2795 (k341) 4188 (k369) 3529 (f254) 3030 (k421) 3371 (k612) rIFN-y 100 IU 1967 (+ 376) 2835 (+ 428) 3686 (? 417) 2971 (f 321) 2709 (f 286) 31 14 (f 580) C. parvum 300 pg 228 (f. 76) 492 (It: 125) 405 (+ 132) 413 (+ 126) 313 (+ 71) 245 (k109) Lister& 300 pg 218 (2 114) 292 (+ 98) 454 (+ 156) 401 (+ 67) 220 (It: 96) 269 (f151)

a) IL 1 activity was assayed in supernatants harvested after 24 h incubation. Results correspond to 1/4 dilutions

2.2 Ma-activating agents

MQ-activating lymphokines (MAF) were cell-free super- natants from 72-h cultures of rat spleen cells suspended in serum-free IMDM supplemented with Sepharose-bound con- canavalin A and 5 x M 2-mercaptoethanol [13]. Purified recombinant rat interferon-y (rIFN-y) was obtained from a Chinese hamster ovary (CHO) cell line as previously described [12]. Corynebacterium parvum organisms were obtained as described [12]. Briefly, C. parvum organisms (ATCC 6916) were cultured for 3-4 days in liquid brain-heart infusion (Difco Laboratories, Detroit, MI), then harvested by centrifu- gation, washed twice in PBS, and incubated for 120 min at 60 "C. Listeria monocytogenes organisms were cultured for 3 days in tryptic soy broth (Difco), then harvested by centrifu- gation, washed twice in PBS and heat killed (240 min at 60 "C) .

2.3 IL1 assay

IL 1 activity was assessed by the ability of serial logz dilutions of MQ culture supernatants to induce a proliferative response in mouse thymocytes, essentially as described by Arenzana et al. [14]. Thymocytes suspended in IMDM supplemented with 5% FCS, 2.5 x M 2-mercaptoethanol, antibiotics and sub- mitogenic concentrations of phytohemagglutinin (PHA P, Difco; final concentration 1 : 2000) were cultured for 72 h in 96-well microplates (Petra Plastic). Eighteen hours before har- vest, cultures were pulsed with 0.2 yCi = 7.4 kBq tritiated thy- midine ([meth~l-~HIdThd, New England Nuclear, Boston, MA). Inbred C3WHeJ mice raised locally and maintained under conventional conditions were the source of thymocytes.

were interacted for 36 h at 37°C with increments of effector cells to result in initial effectorltarget cell ratios of 0.5 : 1,l: 1, 2.5 : 1 and 5 : 1. Thereafter, radioactivity in the sediment and the supernatant of each reaction mixture was measured in a liquid scintillation counter, and net cytotoxicity calculated.

3 Results

3.1 IL 1 secretion by mononuclear phagocytes is differently affected by MAF andor rIFN-y vs. C. parvum andlor Listeria monocytogenes

When adherent BMMQ, obtained by day 6-7 after initiation of their in vitru culture, were incubated for a further 24 h in medium supplemented with PHA, the supernatants exhibited very little mitogenic activity. In the additional presence of MAF or IFN-y in the final 24 h BMMQ incubate, supernatants were clearly and similarly effective in enhancing thymocyte proliferation (Table 1); when directly tested on thymocytes, rIFN-y did not manifest such activity (not shown). In supernat- ants from BMMQ which were first incubated for 24 h with MAE, washed, and then incubated for a further 24 h in medium only, IL1 secretion had returned to control levels (not shown).

In contrast to lymphokines, heat-killed C. parvum and Listeria organsisms did not induce mitogenic activity in these 24-h BMMQ supernatants; IL1 activity was usually even below control levels (Table 1). Supernatants from BMMQ which had been incubated with C. parvum or Listeria for 48 or 72 h, showed similarly low IL 1 activity (not shown). However, when BMMQ were first interacted for 24 h with C. parvurn or

2.4 Ma-mediated tumoricidal activity

This was determined in a [14C]dThd release assay [15]; 5 X lo4 prelabeled P815 murine mastocytoma cells per 16-mm well

Table 2. Induction of tumoricidal activity in BMMQ, by various M@-activating agents")

Incubation medium Percent net [''CldThd release Exp. no. 1 2 3 4 5 6

Control (no stimulant) O(k3) 1 ( + 3 ) 0 ( + 2 ) 1(It:3) 0 ( & 1 ) 0 ( + 2 ) MAF 20% 49(f.5) 54(f .3) 6 0 ( f 7 ) 4 7 ( f 5 ) 58(It:7) 49(f.6) rIFN-y 100 IU 37 (It:5) 41 ( 2 4 ) 42 ( 2 5 ) 36 (24) 35 ( + 6 ) 42 ( 5 6 ) C. parvum 300 pg 61(f .7) 6 4 ( 2 8 ) 6 9 ( f 5 ) 5 9 ( * 5 ) 67(+8) 71 ( " 5 ) Listeria 300 pg 64(It:6) h l ( f . 6 ) 75 (+8) 7 1 ( + 8 ) h 5 ( + 6 ) 6 4 ( t 8 )

BMMQ, were first cultured for 24 h in medium alone (control) or in medium supplemented with MQ-ac- tivating agents in the amount indi- cated before their 36-h interaction with labeled P815 murine mastocyto- ma cells; initial effectodtarget cell ratio was 1 : 1. Effects on target cell viability are expressed as net percent [I4]dThd release. Each value repre- sents the mean of a single experi- ment, each performed in triplicate. Within a given experiment, the ef- fector cells utilized in the respective experiments in Tables 1 and 2 were derived from the same pool.

Eur. J. Immunol. 1987.17: 1665-1668 Interleukin 1 secretion and tumoricidal activity by macrophages 1667

Table 3. Fixation with PF prevents induction of tumoricidal activity in BMM@ by M@-activating agentsa)

Tumoricidal IL 1 secretion" activityb'

Control (no stimulant) 0 (f 3) 597 (f 131) PF control O(f 4) 3092 (f 321)

BMMO pretreated with MAF 79 (f 11) 2856 (f 416) PF MAF 3 ( f 5 ) 6026 (f 592) C. parvum 7 6 ( f 9) 325 (+ 88) PF C. parvum 2 ( + 6 ) 2232 (+ 312) Listeria 84 (* 12) 217 (+ 58) PF Listeria 7 ( f 9) 1992 (f 426)

a) Untreated or PF-fixed (1% for 15 min) BMM@ were cultured in medium alone (control) or in medium supplemented with one of the M@-activating agents.

b) For the assessment of tumoricidal activity, untreated or PF-fixed BMM@ were first cultured for 6 h in medium (control) or in medium supplemented with one of the M@-activating agents and then interacted for a further 36 h with labeled P815 mastocytoma cells (initial effectorkarget cell ratio 1 : 1). Effects on target cell viability are expressed as net percent [I4C]dThd release. Each value represents the mean of five experiments, each performed in triplicate.

c) IL 1 secretory activity was determined in these 24-h supernatants of BMM@, cultured in medium alone or in medium supplemented with one of the M@-activating agents. Results correspond to 114 dilutions and are expressed as ['HIdThd incorporation (cpm k SD). Effector cells utilized for the determinations of IL 1 secretory and tumoricidal activities were derived from the same pool.

Listeria, washed and incubated for a further 24 h in medium supplemented with submitogenic concentrations of PHA, the latter supernatants exhibited PHA control or, more fre- quently, moderately increased mitogenic activity.

3.2 Lymphokmes, C. parvum and Listeria are similarly potent of inducing tumoricidal activity in BMMQ,

In parallel experiments with BMMQ, derived from the same pool as utilized for the determination of IL 1 secretion, their ability to develop tumoricidal activity during in vitro interac- tion with M@-activating agents was assessed. Effector cells were first cultured for 24 h in medium alone (controls) or in medium supplemented with one of the M@-activating agents before their 36-h interaction with prelabeled tumor targets. The results in Table 2 show that each of the Ma-activating agents under study was effective of eliciting a tumoricidal state in BMMQ,; conversely, nonstimulated BMMa did not mani- fest such activity.

3.3 Paraformaldehyde-fixed B M M a are no longer able to evolve tumoricidal activity

BMM@ which had first been incubated for 15 min with 1% PF, then thoroughly washed and, either directly or after a further 24 h in medium, interacted with Ma-activating agents, were no longer able to develop tumoricidal activity (Table 3). However, such pre-fixed metabolically inert BMMQ still released considerable IL1 activity within the first 24 h after

fixation, and secreted IL 1 activity was particularly high upon interaction with IFN-y and/or MAF (Table 3).

4 Discussion

IL 1, a small polypeptide, is secreted in response to a variety of stimuli by various cell types, in particular by mononuclear phagocytes. The molecule has many potent biological proper- ties, including the stimulation of T cell growth, B cell differ- entiation, fibroblast proliferation, production of fever, synthe- sis of proteins (such as acute phase proteins) and regulation of interactions of the central nervous system with the immune system [5,6, 161. M@ activation associated with inflammatory and immune reactions is accompanied by increased expression of membrane proteins (e.g. MHC class I1 molecules), increased synthesis and secretion of enzymes and other biolog- ically active, soluble products including IL 1. In the course of the complex process of activation, M@ may evolve tumoricidal activity. Appropriately activated M@ selectively and effi- ciently kill a wide array of tumor targets in vitro and are thought to play an important role in host defense against neo- plasia (3, 171. However, the mechanism by which activated MQ, kill tumor cells is still controversial. Various effector molecules have been considered as mediators of the M@ cytocidal effects including lysosomal enzymes, oxygen inter- mediates, neutral proteinases, tumor necrosis factor, and lym- photoxin [3, 171. Recent work has shown that many agents that elicit the release of IL 1 also induce a tumoricidal state in M@ [S, 9, 181. Moreover, purified natural or recombinat IL1 was found to be cytostatic and/or cytocidal for various tumor cell lines [7, 8, 101, thus suggesting that this monokine might have a fundamental role in MQ,-mediated tumor cytotoxicity .

Recent work from this laboratory has indicated that induction of the M a tumoricidal state by lymphokines (MAF, IFN-y) vs. C. parvum follows different pathways [12, 131. In the present work, the ability of lymphokines and of heat-killed C. parvum and/or Lkteria to elicit the secretion of IL 1 and/or the tumoric- idal state in BMM@ has been comparatively examined. In keeping with earlier work [5, 12, 191, lymphokines (MAF, IFN-y) were found to induce in parallel the tumoricidal state and the secretion of IL1. Both tumoricidal activity [12, 131 and enhanced IL 1 secretion induced by MAF and/or rIFN-y rapidly decayed as soon as these M@-activating agents had been washed off. In sharp contrast, C. parvum and Listeria induce marked tumoricidal activity in BMMQ, without eliciting concomitant IL 1 production and/or secretion. It is well estab- lished that a variety of microbial agents, including endotoxins and exotoxins, are highly potent inducers of IL 1 [5]. But it has also been realized that not all stimulators of mononuclear phagocyte function elicit IL 1 production. For example, adher- ence, entry and multiplication of live Leishmania tropica in human monocytes, processes associated with MQ, activation, do not result in IL1 production [20]. However, in situations where the M@ population is contaminated with other cells including T lymphocytes the possibility has to be considered that stimulation of T cells with concomitant release of lym- phokines might induce IL 1 secretion in MQ,. Such a mechan- ism may be responsible for the recent finding that Lkteria stimulated IL 1 secretion in peritoneal cells [21]. The present and earlier [12] findings show that M@ tumoricidal activity as well as suppression of IL 1 secretion are maintained as long as C. p a w u m and Listeriu are present in the extracellular space. Additional experiments did not provide support for the thesis

1668 R. Keller and R. Keist Eur. J . Immunol. 1987.17: 1665-1668

that the secreted IL 1 might be bound and/or neutralized by the microorganisms. After washing off the extracellular organ- isms, tumoricidal activity is rapidly decaying, even though BMM@ have engulfed and are degrading a large number of organisms [12]; in contrast, IL1 secretion is resumed after removal of extracellular organisms. Recent work has indicated that IL1 may also exist in a membrane-associated form on M@; whereas rIFN-y did not affect its expression, dead Lis- feriu were effective in triggering membrane IL1 [21, 221. The present work shows that PF-fixed mononuclear phagocytes which are metabolically inert but still dispose of membrane- associated IL 1 with potent thymocyte and T cell stimulatory activity [21] are no longer able to evolve the tumoricidal state. In addition to the present findings, there are various other observations suggesting that expression of membrane-associ- ated or secretion of soluble IL 1 and ability to manifest M@- mediated tumoricidal activity need not necessarily represent concomitant phenomena. In fact, type I interferons which have been reported to augment IL 1 release [14] were not able to induce tumoricidal activity [12]; and membrane-associated IL1 was not expressed at the time of maximal activation of M@ by Lkteriu [22] . Together with these findings, the results of the present work indicate that production and/or secretion of IL 1 and development and/or manifestation of tumoricidal activity by M@ are not necessarily closely linked. It thus appears unlikely that IL1 is a major or even the universal tumoricidal agent; it may, however, represent one among many other tumor-toxic agents derived from M a .

We thank Dr. P . H. v. d . Meide for recombinant rat IFN-y, Dr. M. Aguet for determinations of antiviral activity and Ms. E. Aebli for excellent technical assistance.

Received July 1, 1987; in revised form September 5, 1987.

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