Mechanisms of Invasion and Replication of the Intracellular Stage in

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1 NFECTON AND MMUNTY, Nov. 1984, p Vol. 46, No /84/ $02.00/0 Copyright 1984, American Society for Microbiology Mechanisms of nvasion and Replication of the ntracellular Stage in Trypanosoma cruzi ROBERT E. McCABE,1'2 JACK S. REMNGTON,12* AND FAUSTO G. ARAUJO1 Department of mmunology and nfectious Diseases, Research nstitute, Palo Alto Medical Foundation, Palo Alto, California 94301,1 and Division of nfectious Diseases, Department of Medicine, Stanford University Medical Center, Stanford, California Received 21 May 1984/Accepted 20 August 1984 Amastigotes obtained from spleens of mice infected ith different strains of Trypanosoma cruzi ere examined for their ability to invade macrophages and L929 cells and to initiate infection in mice. Both types of cells ere readily invaded by organisms of the strains Y, MR, and Tulahuen. Organisms of the CL strain ere taken up by both types of cells at a rate that as significantly loer than that for organisms of the other strains. Hoever, all strains multiplied intracellularly. Activated macrophages inhibited the replication of intracellular organisms. Treatment of normal macrophages ith cytochalasin B, trypsin, chymotrypsin, or pronase significantly inhibited phagocytosis, but the inhibitory effect as reversible. Mice injected ith spleen amastigotes developed parasitemia and died of the infection. These results demonstrate that spleen amastigotes are able to infect, survive, and replicate ithin professional and nonprofessional phagocytes and to initiate infection in vivo. nteriorization of spleen amastigotes is by phagocytosis and is dependent upon a proteasesensitive receptor(s) on the cell surfaces of host macrophages. Trypanosoma cruzi, the agent of Chagas' disease, assumes the trypomastigote and amastigote forms in vertebrate hosts. Trypomastigotes are nondividing, circulating forms that enter cells and initiate infection in vertebrates. Amastigotes divide intracellularly, induce inflammatory and immunological responses in vivo, and destroy cells in vitro (6, 8). ntracellular amastigotes are protected from immune responses and chemotherapeutic agents presently used for the treatment of Chagas' disease. t is assumed that the pathogenesis and maintenance of infection ith T. cruzi depend mostly upon the amastigote stage (11). Carvalho et al. (10) suggested that amastigotes from spleens of infected mice did not replicate after interiorization into macrophages from normal mice. On the other hand, Pan (18) demonstrated that amastigotes from cell-free cultures entered and multiplied in human skin-muscle cells. The amastigotes used by Carvalho et al. (10) and Pan (18) may not be comparable. Hoever, their observations, as ell as our previous findings (14), indicated that further ork on the interaction beteen amastigotes and phagocytic or nonphagocytic cells as arranted. We used amastigotes from spleens of mice infected ith four different strains of T. cruzi. Upon phagocytosis by normal mouse macrophages or internalization by mouse fibroblasts of the L929 cell line, the organisms replicated in both types of cells. Uptake of amastigotes by macrophages as markedly inhibited by cytochalasin B and as apparently mediated by a protease-sensitive membrane component(s). Activated macrophages markedly inhibited the replication of intracellular amastigotes in vitro. n addition, amastigotes caused lethal infection hen injected into mice. MATERALS AND METHODS Mice. Siss-Webster female mice (Simonsen Laboratories, Gilroy, Calif.), eighing 18 to 20 g, ere used to obtain blood-form trypanosomes (BFT), spleen amastigotes, and peritoneal macrophages. To determine the infectivity of * Corresponding author. 372 spleen amastigotes in vivo, e used C57BL/6N female mice (Simonsen Laboratories). T. cruzi. Strains Y, CL, MR (5, 16), and Tulahuen (22) ere used. BFT ere isolated (9) from mice infected for 7 days ith the Y or Tu strain and for 12 days ith the MR or CL strain. Spleen amastigotes ere obtained by a slight modification of the procedure described by Carvalho et al. (10). Briefly, spleens collected aseptically 7 days after infection ere homogenized, and the suspension as passed three times through a syringe packed ith gauze and to times through a syringe packed ith glass ool. Thereafter, the suspension as filtered through a polycarbonate membrane filter ith a pore diameter of 3,um (Nuclepore Corp., Pleasanton, Calif.). The filtrate as centrifuged at 1,200 x g for 15 min and then alloed to stand for 30 min at 37 C. The supernatant as removed, and the pellet as suspended in Hank balanced salt solution, layered on sodium metrizoate-ficoll (Lymphoprep; Nyegaard & Co., Oslo, Noray), and centrifuged at 800 x g for 20 min. The layer above the sodiutn metrizoate-ficoll as collected and centrifuged at 1,200 x g for 15 min, the supernatant as discarded, and the sediment as suspended, in RPM 1640 medium (GBCO Laboratories, Grand sland, N.Y.) supplemented ith 10% inactivated fetal bovine serum, 100 U of penicillin per ml,. and 100,ug of streptomycin per ml. This medium as used for in vitro experiments. All steps ere performed at room temperature. This method yielded preparations that ere at least 98% amastigotes, as determined by light microscopy (6). Each spleen yielded 5 x 106 to 10 x 106 amastigotes after infection ith 5 x 105 BFT. This yield could be increased by intraperitoneal injection of the mice ith cyclophosphamide (2 mg per mouse) 48 h before and 48 h after infection. Peritoneal macrophage and L929 cell monolayers. Peritoneal macrophage monolayers ere established as previously described (15). The viability of the cells as >95%, as determined by trypan blue exclusion. The cells ere plated on four- or eight-chamber tissue culture slides (Lab-Tek; Miles Laboratories, nc., Naperville, ll.) and incubated for 2 to 4 h at 37 C in an atmosphere of air and 5% CO2.

2 VOL. 46, 1984 Thereafter, the monolayers ere ashed to remove nonadherent cells and incubated ith medium overnight. To infect the cells, e added a suspension ith the desired number of amastigotes to the monolayers for various periods of time. Thereafter, the monolayers ere ashed, fixed ith Bouin solution, and stained overnight ith Giemsa stain. Confluent areas of to replicate monolayers ere examined for each time point to determine the percentage of infected cells. At least 500 cells ere examined. The number of organisms per macrophage as determined by counting at least 25 infected cells hen the percentage of infection as <5% and at least 50 cells hen the percentage of infection as higher. Monolayers of L929 cells ere processed similarly. Activated macrophages. Mice ere injected intravenously ith 1.4 mg of Corynebacterium parvum (lot CA763; Burroughs Wellcome Co., Research Triangle Park, N.C.). Seven days later, the peritoneal cells ere harvested, and monolayers ere established as described above. Control mice received phosphate-buffered saline (ph 7.2). Enzyme treatment. The method described by Alcantara and Brener (2) as used. Macrophage monolayers ere incubated ith trypsin (Worthington Diagnostics, Elizabeth, N.J.), chymotrypsin (Worthington Diagnostics), or pronase (Calbiochem-Behring, Los Angeles, Calif.) at a concentration of 500 jig/ml of medium. After 30 min, the monolayers ere ashed five times ith medium containing 50% inactivated fetal bovine serum, fresh medium as added, and the cells ere incubated for 1 h. Thereafter, the cells ere exposed to either BFT or amastigotes at a parasite-tomacrophage (P/M) ratio of 3:1 for either 3 or 8 h. Treatment of macrophages ith cytochalasin B. Cytochalasin B (Sigma Chemical Co., St. Louis, Mo.) as dissolved in dimethylsulfoxide and diluted in medium to a concentration of5 or 10 pg/ml (17). Macrophage monolayers ere incubated ith different concentrations of cytochalasin B or ith control medium containing 0.1% dimethyl sulfoxide for 1 h. After this period of time, a suspension of amastigotes as added to achieve a P/M ratio of 1:1. One hour later, the slides ere ashed, fixed, and stained. n some experiments, either the monolayers ith cytochalasin B and amastigotes ere ashed gently and incubated ith medium for 42 h or the amastigotes and cytochalasin B remained in the macrophage monolayers for 24 h. Zymosan coated ith human type AB serum (GBCO Laboratories) as used at a concentration of 1.3 mg/ml of medium to assess the effectiveness of cytochalasin B in preventing phagocytosis (4). RESULTS nfection of macrophages and L929 cells. The results of infection of macrophages ith amastigotes of each of the T. cruzi strains are shon in Fig. 1. For strains Y, Tu, and MR, the infection rates ere similar (Fig. 1A, B, and C, respectively). Thus, for strains Y (Fig. 1A) and Tu (Fig. 1B), ca. 50% of the cells ere infected by 24 h of incubation (P/M ratio of 3:1). For strain MR, 40% of the cells ere infected (Fig. 1C). The number of cells infected ith amastigotes of the CL strain as markedly loer than the number of cells infected ith amastigotes of the other strains (Fig. 1D). Only 2% of macrophages exposed to amastigotes of the CL strain ere infected after 24 h of incubation. NVASON OF MACROPHAGES BY T. CRUZ AMASTGOTES 373 a - U. z For all strains, the infection rates in monolayers of L929 cells ere considerably loer (<1%) than those obtained ith macrophages (data not shon). To assess the possibility that trypomastigotes that contaminated the amastigote preparation ere the organisms that infected the macrophages, e added isolated trypomasti- -z 0L 60r- (A) 40 20k v. so 440 _ A (C) c 60 40[ 201 (B) 60r(D) V- o 8p2 a HOURS NCUBATON FG. 1. nfection of normal mouse macrophages (M4) ith spleen amastigotes. Amastigotes of the Y (A), Tu (B), MR (C), and CL (D) strains of T. cruzi ere added to the cells at a P/M ratio of 0.5:1 (0), 1:1 (A), 1.5:1 (x), 2:1(EO), or 3:1 (V) and incubated for the indicated number of hours. gotes to macrophage monolayers at the same concentration that as found in the amastigote preparation. n this experiment, 3 x 106 amastigotes and 5.4 x 103 trypomastigotes ere added to separate monolayers. The results (Fig. 2) revealed that macrophages ere rarely infected ith this inoculum of trypomastigotes, hereas 50% of the macrophages exposed to the amastigote preparation ere infected after 24 h. ntracellular replication of amastigotes ithin macrophages. Macrophages ere exposed to amastigotes for 10 h. Thereafter, extracellular organisms ere removed, fresh medium as added, and the monolayers ere incubated for an additional 48 h. The number of intracellular organisms of all four strains increased markedly (Fig. 3A through D). Macrophages infected ith amastigotes of the Y strain (P/M ratio of 3:1) contained an average of 2.5 and 13 organisms after 24 and 48 h of incubation, respectively. ncubation for 72 or 96 h resulted in lysis and destruction of the monolayers Ȧlthough the infection rate in L929 cells as loer than that in macrophages, the replication of amastigotes ithin these cells as evident, as each infected cell had ca. eight intracellular organisms by 48 h of incubation (data not shon). nhibition of replication of amastigotes in activated macrophages. The replication of amastigotes ithin activated macrophages as markedly inhibited (Fig. 4). Thus, by 72 h, the average number of intracellular organisms per cell in activated macrophage monolayers as 3, hereas in controls it as 14 Ėffect of cytochalasin B on phagocytosis. The uptake of amastigotes by macrophages as completely blocked by cytochalasin B at a concentration of 5 or 10,ug/ml (Table 1). The organisms adhered to the surfaces of the macrophages, and removal of the drug resulted in their phagocytosis. Thus, monolayers treated ith cytochalasin B and amastigotes for

3 374 McCABE, REMNGTON, AND ARAUJO NFECT. MMUN. 0aJ u - Uż 6Or_ O c z U 20 _ a- nu. V. -m HOURS NCUBATON '4 FG. 2. nfection of macrophages (MO) ith a concentration of trypomastigotes similar to the concentration that contaminated the spleen amastigote preparation. Symbols: 0, spleen amastigotes (P/M ratio of 1:1; x, trypomastigotes (P/M ratio of 0.018:1). See text for details. 1 h and then ashed had numbers of infected cells similar to those in control monolayers after 42 h of incubation. Cytochalasin B had no apparent effect on these amastigotes since their replication as similar in macrophages treated ith cytochalasin B and in controls (Table 1). To confirm that the inhibition of phagocytosis as dependent upon the presence of cytochalasin B, e incubated macrophages for 24 h ith the drug and either amastigotes or zymosan. n control monolayers, 14.5% of the macrophages ere infected, hereas 0.3 and 0.2% of macrophages incubated ith 5 or 10,ug of cytochalasin B per ml ere infected, respectively. Virtually all (>90%) control cells had internal (A) 15 (C) 1o - (B) - AE cn4 2 HOURS NCUBATON FG. 4. nhibition of the replication of amastigotes in nonspecifically activated macrophages (MO). Normal (0) or C. parvumactivated (V) macrophages ere infected ith spleen amastigotes at a P/M ratio of 1:1. ized zymosan, hereas cytochalasin B-treated cells only rarely (<1%) had intracellular zymosan. Effect of proteases on phagocytosis. All three enzymes tested markedly inhibited phagocytosis of both amastigotes and BFT after 3 h of incubation (Fig. 5). The inhibitory effect as more pronounced ith trypsin and pronase than ith chymotrypsin. After 8 h of incubation, the uptake of amastigotes by protease-treated macrophages as still markedly less than that by controls. Hoever, by this time, the uptake of BFT had returned to control values for trypsin-treated macrophages and almost to control values for chymotrypsinand pronase-treated macrophages. nfectivity of amastigotes for mice. The mortality for mice injected ith 104 amastigotes as 100% by day 35 of infection. Control mice ere injected ith ca. 53 BFT, as this number of organisms as equivalent to the number of BFT that contaminated the amastigote preparation. Only 43% of the control mice had died by day 35 of infection (P = 0.03, Fisher exact test). The surviving mice ere still alive 3 months after infection. DSCUSSON Our results revealed that amastigotes isolated from 5 _ spleens of mice infected ith four different strains of T. cruzi infected and multiplied ithin normal murine macrophages i- and L929 cellsṫheinteriorization of amastigotes of the CL strain as considerably loer than that of amastigotes of the ~~Y, MR, and Tu strains. There are a number of characteristics that differentiate organisms of the CL strain of T. cruzi from ( D) organisms of other strains (7). One of these is the reduced '5 D capacity of BFT of the CL strain to infect macrophages, as o0 - The X"PV compared ith that of BFT of other strains of T. cruzi (1). reason for this is unclear. A different cell membrane receptor(s) may play a fundamental role (2). Our demonstra HOURS NCUBATON FG. 3. ntracellular replication of amastigotes in normal macrophage monolayers (MO.). Macrophages monolayers ere infected ith spleen amastigotes of the Y (A), Tu (B), MR (C), and CL (D) strains of T. cruzi at a P/M ratio of 0.5:1 (0), 2:1 (O), or 3:1 (V) and incubated for the indicated number of hours. Results for P/M ratios of 1:1 and 1.5:1 ere similar to the results shon in the figure. 5 TABLE 1. Cytochalasin B and internalization of spleen amastigotes in macrophages % Macrophages Cytochalasin B infected at (h): P/M at concn (jig/ml) 42 h 1 42" a Monolayers ere ashed extensively after 1 h of incubation and ere then incubated ith medium alone.

4 VOL. 46, 1984 NVASON OF MACROPHAGES BY T. CRUZ AMASTGOTES u z i-40 z 10 r AM BFT r v 3 HOURS AM BFT r1 v., r 8 HOURS FG. 5. Effect of the treatment of macrophages (MO4) ith various proteases on the phagocytosis of amastigotes (AM) and BFT. Macrophages ere treated for 30 min ith 500,ug of trypsin (T), chymotrypsin (C), or pronase (P) per ml. The monolayers ere ashed, and spleen amastigotes or BFT ere added and incubated for 3 or 8 h. The results are expressed as the percentage of treated macrophages infected, taking the number of control macrophages infected as 100%. tion that CL amastigotes infect macrophages at a significantly loer rate than Y, MR, and Tu amastigotes suggests that a CL BFT receptor(s) may be present in the CL amastigotes as ell. nterestingly, the rate of replication of CL amastigotes once the organisms had been phagocytized as similar to the rate noted for amastigotes of the other strains. n contrast to the results of Carvalho et al. (10), our results revealed that spleen amastigotes infected and multiplied intracellularly, resulting in the ultimate destruction of the infected cells. The method that e used to isolate amastigotes from spleens of infected mice as basically similar to the method reported by Carvalho et al. (10). Thus, the differences beteen our results and those of Carvalho et al. (10) are probably not due to differences in the protocol for the isolation of amastigotes. Amastigotes, and not trypanosomes present in the amastigote preparation, ere the agents responsible for the infection of macrophages or L929 cells. Previous reports have shon that amastigotes from cell cultures are interiorized and multiply ithin HeLa cells, L929 cells, and calf embryo fibroblasts (17). Our results revealed similar properties for spleen amastigotes. The results for cytochalasin B experiments support phagocytosis as the major means of uptake of amastigotes by macrophages. Similar results have been reported for cultured trypomastigotes (17). The active invasion of cells by amastigotes as not ruled out by our experiments. Active invasion has been reported to be one of the mechanisms by hich BFT infect cells (12). The attachment of amastigotes to the cell surface is not affected by cytochalasin B. Thus, penetration may occur given sufficient time. We tested this hypothesis and noted that cytochalasin B prevented the uptake of both amastigotes and zymosan over a 24-h period. Thus, if active penetration of cells by amastigotes occurs, it appears to be rare in an in vitro system. Nonprofessional phagocytes can be infected by amastigotes, as e shoed ith L929 cells and as Nogueira and Cohn (17) shoed ith L929 and HeLa cells and ith calf embryo fibroblasts. n these cells, a phagocytic process rather than direct invasion may occur, as the integrity of the plasma membrane is apparently preserved (17). Hoever, dissolution of the cell membrane has been reported after invasion of HeLa cells by cultured trypanosomes (19). The replication of intracellular amastigotes as markedly inhibited in activated macrophages. ntracellular killing probably occurred in these cells, as degenerating forms ere observed and the percentage of infected macrophages decreased ith time. The inhibition or killing of intracellular T. cruzi has been reported to occur in macrophages nonspecifically activated by injection of donor mice ith BCG cells (13) or by infection of mice ith Toxoplasma gondii or Besnoitia jellisoni (3, 21). The treatment of macrophages ith the enzymes trypsin, chymotrypsin, and pronase inhibited the uptake of BFT, as previously reported (1, 17). The inhibitory effect of these enzymes as greater for amastigotes than for BFT. n addition, our results revealed that for BFT the inhibitory effect of the enzymes had almost disappeared 8 h after their removal from the macrophage cultures, hereas for amastigotes the effect as still evident after this period of time. Removal of the enzymes as folloed by rapid synthesis of the receptors necessary for the uptake of BFT and, possibly, for the uptake of amastigotes by macrophages. With regard to the pathogenesis of the T. cruzi infection, our results suggest that, despite the considerable destruction of organisms in tissues during acute infection (20), amastigotes released from infected cells are able to infect and multiply ithin professional and nonprofessional phagocytes. Thus, amastigotes appear capable of maintaining and amplifying the infection and destruction of cells in vitro. Whether amastigotes are the primary agents for tissue destruction in vivo remains to be established in future studies. ACKNOWLEDGMENTS This investigation received financial support from the UNDP/ World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases and from the National nstitutes of Health (Public Health Service grant Al 18794). R.E.M. is a recipient of an Edith Milo Felloship and of National nstitutes of Health Felloship grant Al LTERATURE CTED 1. Alcantara, A., and Z. Brener The in vitro interaction of Trypanosoma cruzi blood stream forms and mouse peritoneal macrophages. Acta Trop. 35: Alcantara, A., and Z. Brener Trypanosoma cruzi: role of macrophage membrane components in the phagocytosis of bloodstream forms. Exp. Parasitol. 50: Araujo, F. G., and E. Nascimento Trypanosoma cruzi infection in mice chronically infected ith Toxoplasma gondii. J. Parasitol. 63: Axline, S. G., and E. P. 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