Eur. J. Immunol. 1985.15: 553-558 Parasite killing by a macrophage cell line 553

Phillip Scott, Stephanie James and Alan Sher

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda

The respiratory burst is not required for killing of intracellular and extracellular parasites by a lymphokine-activated macrophage cell line

The macrophage cell line, IC-21, was found to be incapable of producing the oxygen products associated with the respiratory burst. However, IC-21 cells were activated by lymphokine (LK) to kill intracellular (Leishmania donovani amastigotes) and extracellular (Schistosoma mansoni larvae) parasites, as well as tumor cells. In each case, the cytotoxicity exhibited by activated IC-21 cells and activated peritoneal mac- rophages was indistinguishable. However, nonactivated IC-21 cells were unable to kill L. donovani log-growth phase promastigotes, while nonactivated peritoneal mac- rophages destroyed > 90% of the initial infection. These results indicate that amasti- gotes and schistosome larvae are susceptible to killing by nonoxidative cytotoxic mechanism induced by lymphokine activation but, on the other hand, support the concept that the killing of log-growth phase promastigotes by nonactivated cells is dependent upon the respiratory burst. We propose that the IC-21 cell line may be a useful model for studying nonoxidative killing functions of activated macrophages.

1 Introduction

The activated macrophage (MQ) is a major effector cell involved in host defense against infectious agents and neo- plasms [l]. In the process of killing parasites and tumor cells, MQ undergo a respiratory burst (RB), and it has been post- ulated that toxic oxygen products released during the RB mediate the destruction of these targets [2]. In addition, oxy- gen-independent killing mechanisms have also been described, which involve the release of various cytotoxic agents, such as cytolytic factor and tumor necrosis factor [3-51. However, because of the close association of the RB with MQ-target interaction, it has been difficult to distinguish between MQ cytocidal activity mediated by oxygen metabolites, and non- oxygen-dependent killing mechanisms.

In this study, we examined the influence of the RB upon MQ killing of the protozoan parasite, Leishmania donovani, and the helminth, Schistosoma mansoni. The Leishmania life cycle includes the promastigote stage, found within the insect vec- tor, and the amastigote form, an obligate intracellular parasite of MQ. These two stages of the parasite exhibit marked differ- ences in their sensitivity to MQ-mediated killing. Thus, most promastigotes in logarithmic growth phase are killed in nonac- tivated MQ, while amastigotes survive and are only killed fol- lowing MQ activation. However, in both cases leishmanicidal activity has been attributed to toxic oxygen metabolites pro- duced during the RB [6-81. Similarly, a role for oxygen metabolites in extracellular killing by activated MQ has also been suggested [9, lo].

[I 48301

Correspondence: Phillip Scott, National Institute of Allergy and Infec- tious Diseases, National Institutes of Health, Building 5, Room 112, Bethesda, MD 20205, USA

Abbreviations: RB: Respiratory burst pM8: Peritoneal macro- phages FBS: Fetal bovine serum KRPG: Kreb’s Ringer phosphate buffer with glucose PMA: Phorbol myristate acetate OZ: Opson- ized zymosan NBT: Nitroblue tetrazolium CGD: Chronic granulo- matous disease

To examine the mechanisms involved in killing these parasites, we used the IC-21 cell line. This cell line was originally isolated from SV40-transformed C57BW6 peritoneal MQ (pMQ), and retains a variety of MQ characteristics, including normal phagocytosis, Fc and C3 receptors and the ability to present antigen [ll-131. In this study we demonstrate that in contrast to normal pMQ, nonactivated as well as lymphokine (LK)- activated IC-21 cells fail to exhibit a RB in response to para- sites or artificial stimulants. Thus, this cell line provides an excellent model with which to investigate the importance of the RB in the cytocidal activity of M a .

2 Materials and methods

2.1 Cells

Resident pMQ were derived from female C3WHeN (Division of Research Sciences, NIH, Bethesda, MD; 6-8 weeks old) by peritoneal lavage. Elicited cells were harvested from C3H/ HeN mice 4 days following i.p. injection of 1 ml of sodium caseinate. Resident p M a were used in the leishmanicidal assay, and elicited cells were used in all assays involving larval or tumor cell targets. The IC-21 cell line was maintained by weekly passage in Iscove’s modified Dulbecco’s medium (Gibco, Grand Island, NY) with 10% fetal bovine serum (FBS; HyClone “defined” sera, Sterile systems Inc., Logan, UT), 1% nonessential amino acids (Flow Labs, McLean, VA), 100 U/ml penicillin G-potassium, 100 pg/ml streptomycin and 2 mML-glutamine. To inhibit growth during killing assays, IC- 21 cells were irradiated with 3300 rds prior to use.

2.2 Parasites

Leishmania donovani (1 S , clone 2D) was obtained from Dr. Dennis Dwyer, LPD, NIH. Amastigotes were harvested from spleens of infected Syrian outbred hamsters. Amastigotes were used immediately following isolation or following storage in liquid nitrogen. Promastigotes were grown in Medium 199 (M. A. Bioproducts, Walkersville, MD) with 20% FBS at 2I0C, and used when they were in log-growth phase. S. man-

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554 P. Scott, S . James and A. Sher Eur. J. Immunol. 1985.15: 553-558

soni cercariae (NMRI strain) were kindly provided by F. A. Lewis (Biomedical Research Institute, Rockville, MD). Schis- tosomula were prepared from the cercariae by mechanical transformation and purification on Percoll [14]. In the nitro- blue tetrazolium (NBI) and chemiluminescence assays schis- tosomula were opsonized by pretreatment with 10% chronic immune serum, obtained from 12-week S. mansoni-infected C57BL/6 mice, to enhance rapid Ma-target interaction. In the larvacidal assay opsonization was not performed. LK was pre- pared by PPD challenge of spleen cells from Bacillus Cal- mette-Guerin (BCG)-immune mice as previously described. Murine recombinant interferon-y was provided by Genentech Inc. (San Francisco, CA) and used at a final concentration of 60 U/ml.

2.3 Oxygen metabolite assays

The cytochrome c assay was performed as described by John- ston [16] with the modifications outlined below. Briefly, 2 x lo6 elicited pMQ or IC-21 cells were incubated in 24-well plates (Costar, Cambridge, MA) for 5 h with 10% LK, washed 3 times with Kreb's Ringer phosphate buffer with glucose (KRPG), and phorbol myristate acetate (PMA; 200 ng) or complement-opsonized zymosan (OZ; 0.5 mg) added to the cells with 80 VM cytochrome c (ferricytochrome c, horse heart, type 111, Sigma Co., St. Louis, MO). Additional wells received the same stimulants with 40 yg/ml superoxide dismu- tase (Sigma). After 90 min incubation at 37"C, the supernate was removed, centrifuged in a microcentrifuge, and the absorbance of the supernate determined spectrophotometri- cally at 550 nm. The concentration of reduced cytochrome c was determined using the absorptivity A550nm = 2.1 X lo4 M-' . cm-l. The scopoletin assay was performed exactly as described by Nathan [17] for adherent cells observed at a single time point, with the exception that the assay was terminated after 60 min. Elicited cells were pretreated with 10% LK for 5 h before testing, and then exposed to 200 ng PMA. All reagents used in this assay were obtained from Sigma. For the qualita- tive NBT assay, pMQ or IC-21 cell monolayers were incubated with 10% LK for 5 h, and then incubated for 90 min with NBT (0.05% in KRPG with 25% FBS) and schistosomula, Leish- mania or OZ in 8-well chamber slides (LabTek, Miles Labs, Inc., Naperville, IL). Subsequently, formazan deposition was assessed microscopically on unstained slides of larvae, and stained (Diff-Quik, Harleco, Gibbsville, NJ) slides of Leish- mania and OZ. For the quantitative NBT assay, 1 X lo6 cells were plated in 24-well chamber slides (Costar), exposed to LK for 5 h, and then exposed to OZ, amastigotes, promastigotes, or schistosomula larvae in the presence of NBT. After 90 min, cells were washed with phosphate-buffered saline and removed with a rubber policeman. Duplicate wells were pooled, centrifuged and 2 ml of pyridine was added to the pellet. Tubes were boiled for 10 min to extract the dye, cen- trifuged and the supernate was read at 515 nm on a spec- trophotometer. Results are expressed as nmol NBT reduced/ lo6 cells/90 min using the absorptivity 2.62 x lo4 W1 cm-' determined in pyridine. Chemiluminescence, enhanced with 1.25 x M luminol (Sigma), was measured in a Beckman liquid scintillation counter (Model LS9000) set on single photon count following addition of OZ (0.5 mg), antibody- opsonized S. mansoni schistosomula (1.5 x lo4) or L. dono- vani amastigotes (1 X lo7) to 8 x 106 LK-pretreated pMQ, or IC-21 cells. Elicited cells were used with schistosomula, and resident pMQ, were used with amastigotes and OZ.

2.4 Leishmanicidal assay

Amastigote killing assays were performed as previously described [18]. Briefly, 5 x lo5 resident pMQ or IC-21 cells were cultured in polypropylene tubes (Falcon # 2063) with or without 10% LK for 5 h. These cells were then infected with L. donovani amastigotes at a 4 : 1 ratio for 2 h, subsequently centrifuged at 1000 rpm to remove most of the uningested parasites, resuspended in RPMI 1640 (Advanced Biotechnol- ogy Inc., Silver Spring, MD) with 10% FBS, penicillin-strep- tomycin and L-glutamine, with or without 10% LK, and cul- tured for 72 h. Samples were harvested at 2, 24 and 72 h after infection by cytocentrifuge, stained with Diff-Quik (Harleco, Gibbsville, NJ) and the percent infected MQ, and number of parasites per infected M@ determined microscopically. For promastigote killing assays p M a or IC-21 cells were plated in 8-well chamber slides (LabTek), and pretreated with LK prior to infection. Cells were exposed to promastigotes for 2 h at a 4 : 1 parasite to cell ratio. Following infection, monolayers were washed to remove excess promastigotes and fresh LK (10%) added to the appropriate wells. Samples were harvested at 2,24 and 72 h, stained and the infection assessed microscop- ically. The percent leishmanicidal activity was equal to 1- (no. of parasited100 MQ, in LK or control cultures at 72 h divided by the number of parasites per 100 M a in LK or control cul- tures at 2 h) x 100.

2.5 Extracellular killing assays

Schistosomula and tumor cytotoxicity was performed with sodium caseinate-elicited pMQ at effector to target ratios of 104 : 1 and 20 : 1, respectively. Inflammatory M@ have previ- ously been shown to be more effective than resident cells in these assays [19, 201. Larvicidal activity was determined microscopically using nonopsonized larvae after incubation of the schistosomula for 48 h with LK-treated or control cells. Percent killing is expressed as the mean percent mortality in duplicate samples. Tumor cytotoxicity was determined by measuring release of incorporated [3H]thymidine from pre- labeled L929 tumor cells at 48 h and expressed as percentage of total counts released by detergent lysis [19].

3 Results

3.1 The inability of the IC-21 cell line to produce a RB

The IC-21 cell line was tested for its ability to produce a RB in response to two potent stimulants, OZ and PMA. Two oxygen metabolites of the RB were measured: superoxide anion (02) in the cytochrome c assay, and hydrogen peroxide (H202), in the scopoletin assay (Table 1). To mimic the in vitro killing assays described below, PM and IC-21 cells were pretreated for 5 h with 10% LK. Such LK treatment has previously been demonstrated to induce optimal M@ activation for killing these targets [15, 221. In response to either stimulant, pMQ produced a RB, as evidenced by the release of oxygen metabo- lites. However, under the same conditions IC-21 cells pro- duced only negligable amounts of these metabolites. Moreover, the inability of IC-21 cells to exhibit a RB could not be overcome by treating the cells with LK for 72 h (Table 1).

Eur. J. Immunol. 1985.15: 553-558 Parasite killing by a macrophage cell line 555

Table 1. Oxygen metabolite production by pM@ and IC-21 cells")

Cells Stimulant L K ~ ) 0; H202 nmov107 nmou106

celld90 min celld60 min (h)

pMQ PMA') 5 72.9 f 1.5 9.4 OZd' 5 57.8 f 1.6 ND"

IC-21 PMA 5 2.6 f 0.4 < 0.05 oz 5 0.4 f 0.6 ND

PMA 72 0 fO ND oz 72 0 fO ND

a) 0; was measured by the cytochrome c assay and Hz02 was mea- sured by the scopoletin assay as described in Sect. 2.3.

b) Amount of time cells were treated with LK (10%) prior to expo- sure to stimulant.

c) Two hundred ng PMA. d) One half mg OZ. e) ND = Not determined.

Since our primary interest was to study the role of the RB in M@ killing of parasites, we next compared the response of pM@ and IC-21 cells to parasite targets in an NBT assay, which also measures 0;. Reduction of NBT to formazan by

05, was qualitatively assessed by microscopic observation, and quantitatively assessed by spectrophotometric determination of the amount of formazan generated. An important advan- tage of the NBT assay, when studying intracellular killing, is that unlike the cytochrome c assay, which only measures extracellular O,, the NBT assay measures 0; generated on the surface membrane, as well as within the phagosome.

pM@ or IC-21 cells were incubated for 90 min with NBT and either S. mansoni larvae (schistosomula), L. donovani amasti- gotes or OZ. As seen in Fig. 1, pMQ, produced a strong RB as measured by formazan deposition after exposure to OZ, S. mansoni schistosomula or L. donovani amastigotes. However, the IC-21 cell line did not reduce NBT to formazan under the same conditions. The lack of a RB was not due to decreased phagocytosis of OZ or amastigotes. Thus, after 1 h 59% of the pM@ and 57% of the IC-21 cells contained equivalent num- bers of L. donovani amastigotes (pM@, 2.7 2 0.1; IC-21, 2.5 k 0.2) and > 60% of the amastigotes in pMQ, had forma- zan granules associated with them. Similarly, both pM@ and IC-21 cells adhered to the surface of the schistosome larvae, but only pM@ demonstrated a RB upon contact with the schis- tosome surface.

The ability of parasite targets to induce a RB in pM@ and IC- 21 cells was also measured in a quantitative NBT assay

Figure 1. Reduction of NBT by pM@ (A, C, E) or the IC-21 M@ cell line (B, D, F) following ex- posure to antibody-opsonized Schistosoma mansoni schis- tosomula (A, B), Leishmania donovani amastigotes (C, D) or complement OZ (E, F). Adher- ent pM@ (elicited) and IC-21 cells were pretreated with 10% LK for 5 h. M@ monolayers were then incubated for 90 min with NBT (0.05% in KRPG with 25% FBS) and schistosomula, Leishmania or zymosan in 8-well chamber slides. Subsequently, formazan deposition was as- sessed microscopically on un- stained slides of larvae, and stained slides of Leishmania and zymosan. (A) Formazan was de- posited on the larvae at the site of attachment of LK-activated pM@ to the schistosomula tegu- ment. (B) Multiple LK-activated IC-21 cells are attached to the larvae with no evidence of NBT reduction. (C) Black formazan completely surrounds amasti- gotes within phagosomes of the pM@ (arrow). (D) The nucleus and the kinetoplast of the in- tracellular amastigotes (arrow) can be clearly seen, with no evi- dence of formazan deposition. (E) Intracellular zymosan parti- cles are completely surrounded by black formazan. (F) No for- mazan deposition is seen.

556 P. Scott, S. James and A. Sher Eur. J. Imrnunol. 1985.15: 553-558

Table 2. Quantitative NBT reduction by pM@ and IC-21 cells follow- ing exposure to parasite targets

nmol NBT reduce&l@ celld60 min IC-21

LK PM@

Control LK Control

oza' 23 + 2 23 + 3 2 ? I 1 + 2 hastigotes 6 + 5 8 5 7 0 .7+ 1 0.4? 1 Promastigotes 11 f l 14 + 4 2 + 3 3 + 4 Schistosomulab) 10.6 10.8 0 1.1

a) OZ, amastigotes and promastigotes were added to resident pM@ or IC-21 cells at a 20 : 1 target to cell ratio. Data shown represents the mean nmol NBT reduced k SD of 2 experiments.

b) Schistosomula (12000) were added to elicited pM@ or IC-21 cells.

(Table 2). All three parasite targets induced a significant response in both nonactivated and activated pMQ, while only minimal responses were obtained with IC-21 cells.

Finally, to further demonstrate that the IC-21 M@ cell line is incapable of producing a RB, we examined the chemiluminescence response of these cells (Fig. 2). Chemiluminescence, a measure of the light emitted during the RB, is believed to result from the interaction of RB products with luminol and target components [21]. A large increase in chemiluminescence was observed when L. donovani amasti- gotes and OZ were added to LK-pretreated resident pM@. In addition, S. mansoni larvae induced a significant increase in the chemiluminescence of LK-pretreated elicited pM@. This increase was smaller than that observed with the intracellular targets, probably because only those cells which adhered to the surface of the larvae, approximately 10% of the total

* O t d lo t i

&=--&--&-*--rsl--9r' - _ _ _ _ _ _ _ _. &::g;;g::&:;jz<$ - - - . . -. -_ -. 20 40 60

MINUTES

Figure 2. Cherniluminescence, following addition of complement-OZ (0.5 mg), antibody opsonized S. mansoni schistosomula (1.5 X lo4) or L donovani amastigotes (1 x lo') to 8 x lo6 LK pre-treated pM@ or IC-21 cells. Elicited pM@ were used with S. mansoni schistosomula, and resident pM@ were used with L. donovani amastigotes and OZ. Closed symbols represent pM@, open symbols represent IC-21 cells. (@,O---) Zymosan; (A,&--) Leishmania; (H,U--) schis-

Table 3. Extracellular killing by the IC-21 M@ cell line

Target Cells KiIIing") (%) Control LK

Schistosomula PM@ 6 + 3 96+ 6b) 89 f 15

Tumor cells PM@ 16+ 7 42+ 11 53 f 16

IC-21 112 4

IC-21 2 2 2 12

a) The percent killing was calculated as described in Sect. 2.4, and the results presented are the mean + SD of three or more experi- ments.

b) The percent killing with LK-treated cells was significantly greater than controls (p < 0.005) with both schistosomula and tumor cells.

number, would be expected to respond. In contrast, LK-pre- treated IC-21 cells demonstrated no chemiluminescence with any of the targets. Taken together, these data demonstrate that not only do IC-21 cells have a generalized impairment in oxygen metabolite production, evidenced by their response to OZ and pM@, but that these cells are unable to exhibit a RB upon contact with amastigotes, promastigotes and schis- tosomula.

3.2 Extracellular killing by IC-21 cells

To determine what role the RB plays in activated M@ killing of S. mansoni larvae, pM@ or IC-21 cells were incubated with schistosomula in the presence or absence of LK. Larvacidal activity was determined microscopically after incubation of the schistosomula for 48 h with M a . As previously observed [22], nonactivated pM@ exhibited negligible cytotoxicity against S. mansoni larvae, while significant killing was observed by acti- vated pM@ (96% larvacidal activity) (Table 3). Similarly, nonactivated IC-21 cells exhibited minimal cytotoxicity against larvae. However, once activated by LK these cells were as capable of killing schistosomula as pM@ (89% larvacidal activ- ity). No difference in the degree of IC-21 cell-mediated killing was seen between antibody-opsonized and nonopsonized targets in the larvacidal assay (data not shown).

The ability of LK-activated IC-21 cells to kill extracellular targets was confirmed using [3H]thymidine-labeled L929 tumor cells (Table 3). Following LK activation IC-21 cells demonstrated 53% tumoricidal activity, while activated pMQ exhibited 42% tumoricidal activity. With both cell types, only minimal tumoricidal activity was observed in the absence of activation.

3.3 Intracellular killing by IC-21 cells

To assay intracellular killing, resident pM@ and IC-21 cells were infected with L. donovani amastigotes. While nonacti- vated pM@ and IC-21 cells did not demonstrate any leish- manicidal activity, following LK treatment both cell popula- tions eliminated >95% of the amastigotes (Fig. 3). The per- cent of infected IC-21 cells decreased from 75% at 24 h to 1% at 72 h and a similar decrease from 60% to 2% was seen over

tosomula. the same time period with LK-activated pM@. Thus, the IC-21

Eur. J. Immunol. 1985. I S : 553-558 Parasite killing by a macrophage cell line 557

1 24 72 72 HOURS

Figure 3. Killing of L. donovani amastigotes by LK-activated pM@ (A) or IC-21 cells (B) in vitro as described in Sect. 2.4. Unbroken line, control cells; broken line, LK-treated cells.

Table 4. Survival of L. donovani promastigotes in normal and LK- activated pM@ and IC-21 cells

No. of parasited100 M@ Killing”’ 2 h 72 h (”/.I

LK 162 (60) 0 (0) 100

IC-21 Control 199 (71) 315 (67) 0 LK 159 (61) 0 (0) 100

pM@ Control 300 (79)b’ 12 ( 5 ) 96

a) The percent killing was calculated as described in Sect. 2.4. The data presented are from one representative experiment out of three. The mean % killing k SD for all three experiments was: pM@ control, 97 k 3; pM@ LK, 99 k 1; IC-21-control, 10 k 17; IC- 21-LK, 98 C 3.

b) Percent infected M a .

cells were as effective as pMQ, in killing Leishmania amasti- gotes. In addition, both cell populations were induced to kill amastigotes after exposure to murine interferon-y, a primary mediator of MQ, activation [23] (% killing k SD; pMQ,, 93 k 4; IC-21,95 k 1). This finding suggests that IC-21 cells and pMQ, may be activated by similar mechanisms. When log-growth phase promastigotes of Leishmania were tested for sensitivity to killing by pMQ, and IC-21 cells, a different set of results was obtained (Table 4). Nonactivated pMQ, infected with Leish- mania promastigotes destroyed > 90% of the initial inoculum. In contrast, IC-21 cells did not eliminate promastigotes unless they were activated by LK. Thus, while both nonactivated and activated pMQ, could destroy promastigotes, IC-21 cells required LK activation to kill promastigotes.

4 Discussion

MQ, cytotoxicity can be separated into nonactivated and acti- vated killing mechanisms. Certain pathogens, such as Leish- mania promastigotes in log-growth phase, Trypanosoma cruzi epimastigotes, as well as many bacteria, are readily killed by normal MQ, while other targets, such as Leishmania amasti- gotes, S. mansoni larvae and tumor cells, are only killed when

MQ, have been activated. A variety of mechanisms responsible for mediating both types of MQ, cytotoxicity have been described, including oxygen-dependent and oxygen-indepen- dent mechanisms. We used the M@ cell line, IC-21, to study the mechanisms responsible for activated and nonactivated M@ killing of some of these intracellular and extracellular parasites. Because this cell line was found to be defective in oxygen metabolism, it provides an excellent tool with which to dissociate these two killing mechanisms.

The importance of toxic oxygen metabolites produced during the RB in certain types of MQ, microbicidal activity, such as nonactivated MQ, killing, is well established. Monocytes from patients with chronic granulomatous disease (CGD), which are defective in oxygen metabolism, are unable to kill a variety of bacteria, fungi, as well as protozoa, and this defect is believed to be directly related to the impaired oxygen metabolism of these cells [24-261. Similarly, mutant clones selected from the J774 cells line for their inability to exhibit a RB were unable to kill Trypanosoma cruzi epimastigotes, while clones with normal oxygen metabolism killed these para- sites [27]. With Leishmania promastigotes, it was found that glucose derivation, which decreases the RB, or addition of the oxygen metabolite scavenger, catalase, inhibited killing by pMQ, [6]. Our results support these earlier findings. Thus, similar to the mutant J774 cells that were defective in oxygen metabolism and epimastigote killing, nonactivated IC-21 cells were unable to exhibit a RB or kill Leishmania log-growth phase promastigotes, while normal pM@ killed greater than 90% of these organisms.

In contrast to nonactivated MQ, killing, the role that the RB plays in activated MQ, killing of both intracellular and extracel- lular targets is less clear. A variety of mediators for the cytoci- dal effects of activated MQ, have been proposed, including oxygen metabolites, such as H202 [2-10, 281. To a large extent, the data supporting a role for oxygen metabolites have been indirect. A strong correlation between enhanced produc- tion of oxygen intermediates and enhanced MQ, killing has been observed [29]. Similarly, catalase or glucose deprivation has been reported to inhibit LK-mediated activated MQ, kil- ling of both intracellular and extracellular targets, suggesting that H202 is a primary mediator of killing [30, 311. Thus, these results would suggest that intracellular survival of Leishmania amastigotes is dependent upon the failure to stimulate a RB, resistance to oxygen metabolites, or both, and that once acti- vated by LK, MQ, kill these parasites by generating increased levels of toxic oxygen metabolites during phagocytosis. How- ever, not all of the evidence points to a primary role for oxy- gen metabolites in activated MQ, microbicidal activity. First of all, amastigotes do stimulate a significant RB in nonactivated resident and elicited pMQ,, as well as human monocytes - although less than that elicited by promastigotes - and are able to survive ([7], Table 2). Secondly, it has been shown that MQ, can be activated to kill Leishmania amastigotes after infection [15, 181. This result strongly argues that modulation by LK of the RB associated with amastigote phagocytosis is not required for MQ, microbicidal activity. Finally, it has recently been shown that monocytes from CGD patients can be acti- vated by LK to kill Leishmania promastigotes and inhibit Leishmania amastigote growth [32]. These results, taken together with the findings reported here, strongly suggest that nonoxidative killing mechanisms play an important role in activated MQ, killing of Leishmania. There are also data indi- cating that oxygen metabolites may not play a primary role in

558 P. Scott. S. James and A. Sher

activated M@ killing of extracellular targets [33]. Some inves- tigators have reported that catalase has no effect on extracellu- lar killing by activated M@ [34, 351, that tumoricidal activity can occur anerobically [36, 371, and that enhanced Hz02 release can be dissociated from tumoricidal and larvacidal activity [28, 38, 391. Moreover, cytolytic factors, obtained from activated MQ,, can kill tumor cells in the absence of any oxygen intermediates [3-51. Preliminary experiments suggest that such M@-derived factors are also effective in killing schis- tosomula (S. James, unpublished observations) .Our results demonstrate that in the absence of measurable oxygen metabolite release, LK-activated IC-21 cells can kill schis- tosomula and tumor cells as well as normal pM0. Conversely, nonactivated pMQ, exhibited a significant RB following con- tact with s. mansoni larvae, but demonstrated no cytocidal activity (Tables 2, 3). Taken together, these results suggest that oxygen metabolites may not be the primary mediators of activated M@ killing for these extracellular targets.

Nevertheless, our data do not completely rule out a role for oxygen metabolites in LK-mediated activated M@ killing. Although IC-21 cells were found to be deficient in oxygen metabolite production, it is difficult to exclude the possibility that nondetectable levels of oxygen intermediates are being produced. Low level production of oxygen metabolites could facilitate nonoxygen-dependent killing mechanisms. For example, it has been reported previously that nontoxic levels of H202 enhance the activity of a cytolytic factor in a tumorici- dal assay [40]. However, it should be stressed as discussed above, that killing of Leishmania amastigotes, schistosomula and tumor cells by LK-activated IC-21 cells cannot be primar- ily mediated by nondetectable oxygen metabolites because nonactivated pM@ produce significant levels of oxygen metabolites following interaction with these targets (Table 2 ) , and yet demonstrate no cytotoxic activity.

Our results with the IC-21 cell model suggest that the primary mechanisms of cytotoxicity used by activated and nonactivated macrophages may differ. Thus, targets that are susceptible to toxic oxygen intermediates, such as Leishmania log-growth phase promastigotes, T. cruzi epimastigotes and certain bac- teria, are killed by nonactivated MQ, through oxidative mechanisms. Activation of M@ may enhance oxygen metabo- lite production, and thus facilitate killing of oxygen metabolite susceptible targets. However, LK activation also appears to stimulate nonoxidative killing mechanisms, which can kill a larger range of targets, including Leishmania amastigotes, S. mansoni larvae and tumor cells. These nonoxidative mechan- isms are not well defined, although several potential mediators of nonoxidative killing have been described, including cytoly- tic proteases and tumor necrosis factor [3-51. Since IC-21 cells can be grown in quantity and are free of contaminating cell types, this cell line should be an excellent model for determin- ing the role of these and other potential mediators in nonox- idative killing by activated MQ.

We wish to thank Dr. J . Lazdins for helpful discussions and Dr. E. Handman for providing the IC-21 cell line. We also thank Ms. Brenda 8. Martin and Ms. Wilma Davis for editoral assistance.

Received November 6, 1984.

Eur. J. Immunol. 1985.15: 553-558

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