Proc. Nati. Acad. Sci. USA Vol. 91, pp. 999-1003, February 1994 Immunology Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide ISABELLE P. OSWALD*t, ISAM ELTOUM*, THOMAS A. WYNN*, BERTRAND SCHWARTZt, PATRICIA CASPAR*, DENISE PAULIN§, ALAN SHER*, AND STEPHANIE L. JAMES* *Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; tLaboratoire de G6ndtique Moldculaire, Ecole Nationale Vdtdrinaire, 94704 Maison-Alfort, France; and 1Laboratoire de Biologie Mol6culaire et de la Differentiation, Institut Pasteur, 75754 Paris, France Communicated by Richard M. Krause, September 27, 1993

ABSTRACT Like many pathogens that undergo an intra- macrophages, produce toxic metabolites that kill these patho- vascular stage of development, larvae of the helminth parasite gens directly. Schistosoma mansoni migrate through the blood vessels, where Schistosoma mansoni is a helminth parasite that spends they are in close contact with endothelial cells. In vito exposure most of its life in the vertebrate host in an intravascular of murine endothelial cells to various cytokines (interferon y, environment. Its developmental pathway involves penetra- tumor necrosis factor a, and interleukin la or 113) resulted in tion of the skin, followed by rapid intravascularization and their activation to kill schistosomula through an arginine- transit through the lungs to the liver and hepatic portal dependent mechanism involving production of nitric oxide system. Exposure to radiation-attenuated schistosome larvae (NO). Cytokine-treated endothelial cells showed increased ex- confers a high level ofresistance against subsequent infection pression of mRNA for the inducible form of the NO synthase, in several mammalian species (4) and thus offers an ideal and both NO production and larval killing were suppressed by model in which to study both the targets and the mechanisms treatment with competitive inhibitors. The effector function of of protective immunity. In mice receiving a single exposure cytokine-treated endothelial cells was similar to that of acti- to irradiated schistosome larvae, protective immunity is vated inflammatory tissue macrophages, although activation largely dependent on the induction of a Th, helper T-cell appeared to be differentially regulated in these two cell types. immune response, correlates with the development of larvi- Activated endothelial cells killed older (18-day) forms of the cidal activated macrophages, and is accompanied by an parasite, such as those currently thought to be a primary target increase of Ia+, oxidatively reactive macrophages within the of immune elimination in the lungs of mice previously vacci- lungs (4, 5). Analyses of the migration of challenge parasites nated with radiation-attenuated cercariae, as well as newly in mice vaccinated with irradiated larvae indicate that the transformed larvae. In C57BL/6 mice, which become resistant majority are eliminated within a few weeks ofinfection, after to S. mansoni infection as a result ofvaccination with irradiated they reach the lungs and before they develop into adult cercariae, endothelial cell morphology characteristic of acti- worms (5). vation was observed in the lung by 1-2 weeks after challenge In vitro, interferon fy (IFN-y)-activated murine macro- infection. Similar endothelial cell changes were absent in phages kill larval schistosomula through an arginine- P-strain mice, which do not become resistant as a result of dependent mechanism involving production of reactive ni- trogen oxides (6). NO has likewise been implicated as an vaccination. Together, these observations indicate that endo- effector molecule of macrophage-mediated cytotoxicity thelial cells, not traditionally considered to be part of the against tumor cells and multiple protist targets, where it has immune system, may play an important role in immunity to S. been shown to interfere with DNA replication, the citric acid mansoni and, by means ofNO-dependent killing, could serve as cycle, and mitochondrial respiration (7). Two- to 3-week-old effectors of resistance to other intravascular pathogens. parasites as well as early skin-stage larvae are susceptible to killing by activated macrophages (5), suggesting that this Cell-mediated immunity (CMI) is traditionally thought to be mechanism might play a role in immune elimination of mediated by T lymphocytes plus accessory cells, notably schistosomula in the lungs of vaccinated mice. However, macrophages, operating against pathogens within tissue parasites migrating through the lungs are initially located sites. Vascular endothelium plays a major role in this process intravascularly where they are in intimate contact with ECs. by localizing leukocytes to the site of the infection, a re- Since cytokine-treated ECs can produce NO (8), we consid- sponse dependent on the cytokine-induced expression of ered the possibility that they could serve as effector cells in adhesion molecules on endothelial cells (ECs) (1, 2). Cyto- schistosomulum killing and thus contribute to the mechanism kines also upregulate the expression of major histocompati- of immune elimination of parasites in vaccinated mice. bility complex molecules on ECs, allowing them to function as antigen-presenting cells for T-cell induction (2, 3). These MATERIALS AND METHODS findings indicate that ECs are cytokine-activatable cells that play an important role in the afferent limb of CMI. Animals and Parasites. Female C57BL/6 (Division of Can- In the case of blood-borne infections, the target pathogen cer Treatment, National Cancer Institute, Frederick, MD) or often residues intravascularly. The effector mechanism by which CMI contributes to control or elimination of a disease Abbreviations: IL, interleukin; IFN, interferon; TGF, transforming agent in the blood is poorly defined. One possibility is that growth factor; TNF, tumor necrosis factor; L-NMMA, N"- cytokine-stimulated ECs in analogy with cytokine-activated monomethyl-L-arginine; LNNA, N"-nitro-L-arginine; CMI, cell- mediated immunity; iNOS, inducible NO synthase; EC, endothelial cell. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Laboratory of payment. This article must therefore be hereby marked "advertisement" Parasitic Diseases, Building 4, Room 126, National Institutes of in accordance with 18 U.S.C. §1734 solely to indicate this fact. Health, Bethesda, MD 20892. 999 Downloaded by guest on September 28, 2021 1000 Immunology: Oswald et al. Proc. Nati. Acad. Sci. USA 91 (1994)

P/J (The Jackson Laboratory) mice were used at 7-8 weeks. RESULTS S. mansoni cercariae (NMRI strain) were provided by F. Lewis (Biomedical Research Institute, Rockville, MD). In Vitro Larvicidal Activity of Cytokine-Treated ECs. The For histological analysis of the lung tissues, mice were ability of ECs to kill schistosomula following exposure to immunized with 500 irradiated (50 krad; 1 rad = 0.01 Gy) various cytokines was compared with that of inflammatory cercaria by tail immersion in water containing the larvae. peritoneal macrophages (Table 1). No single agent was able Immunized mice or control unimmunized animals were in- to induce EC larvicidal activity, although inflammatory mac- fected 5 weeks later by exposure of shaved abdominal skin to rophages were effectively activated by either IFN-y or LPS 500 unattenuated cercariae. alone. However, several combinations of cytokines (IFN-y For in vitro larvicidal assays, both newly transformed (3 hr) plus TNF-a, either IFN-,y or TNF-a plus IL-la or IL-1lP, and and 2.5-week-old schistosomula were used. Three-hour either IFN--y or TNF-a plus LPS) were able to stimulate schistosomula were mechanically transformed from cercar- significant cytotoxicity by ECs. iae (6). Older parasites were recovered by perfusion from the Interestingly, the cytokine combinations that were effec- hepatic portal system of mice that were infected 18 days tive for activation differed between ECs and macrophages before by percutaneous exposure to 3 x 103 cercaria (5). (Table 1). Thus, the combination ofTNF-a with IL-la or -1(, Reagents. Recombinant murine IFN-y and tumor necrosis which activated ECs, had little effect on macrophages. Con- factor a (TNF-a) were gifts from Genentech, recombinant versely, IFN-y plus IL-2 or IL-6 activated macrophages but human interleukin 2 (IL-2) was a gift from Cetus, and purified was ineffective on ECs. IL-10 was a gift from R. Coffman (DNAX). Recombinant Arginine Dependence of EC Larviddal Activity. As shown murine IL-la, IL-1,8, IL-4, and IL-6 were purchased from in Table 1, the cytokine combinations which induced schis- Genzyme, recombinant human transforming growth factor tosomulum killing by ECs also induced nitrite production. (31 (TGF-(31) was from R & D Systems, bacterial lipopoly- Four hours after treatment with IFN-y plus TNF-a, mRNA saccharide (LPS, Escherichia coli 0128:B12 phenol extract) for iNOS was also upregulated as measured by a semiquan- and Nw-nitro-L-arginine (L-NNA) were from Sigma, and titative reverse transcription-PCR (Fig. 1). N"'-monomethyl-L-arginine (L-NMMA) monoacetate was To further examine the role of NO in larval killing, ana- from Calbiochem. logues of L-arginine with N-guanidino substitutions that Histological Analysis. The left lobe of the lung was pro- specifically inhibit NO synthesis (L-NMMA and L-NNA) cessed as described (9). Arteritis was scored by using an were added to the cultures. At 1 mM, L-NMMA completely optical micrometer to measure the thickness of the wall and inhibited the ability of ECs to kill schistosomula (Table 2). the surrounding of the inflammatory infiltrate ofjuxtabron- L-NNA also effectively inhibited schistosomula killing by chial arteries. cytokine-activated cells (83% reduction of larval killing and Effector Cells. Brain ECs from 129/J mice were immortal- 95% reduction of nitrite production in the presence of 2 mM ized by microinjection of the cell with the HuVim 830-T/t L-NNA with IFN-y-plus TNF-a-activated ECs). recombinant gene (10). The cells were maintained in a Downregulation ofEC Activation by Cytokies. IL4, IL-10, mixture of RPMI 1640 and Dulbecco's modified Eagle's and TGF-( downregulate IFN-y-induced macrophage acti- medium supplemented with 10% fetal bovine serum (com- vation (11, 12). In contrast, neither IL-10 nor TGF-,B inhibited plete medium) (11, 12). Another murine EC line, LE-II, derived from lung capillaries, was also used in some exper- Table 1. Comparison of the activation of murine ECs or iments (gift from T. Maciag, Holland Laboratory, American inflammatory macrophages (MO) following cytokine treatment Red Cross, Rockville, MD) (13). Alternatively, nontrans- Schistosomulum Nitrite formed murine ECs isolated from brain microvessels, hepatic klilling* productiont sinusoids, or lung (gift from R. Kilbourn and G. Nicolson, University ofTexas, Houston) were continuously maintained Cytokine treatment ECs M4o ECs MO with endothelial mitogen (Biomedical Technologies, Stough- None 10 ± 5 10 ± 2 <4 <2 ton, MA) (14) at 100 pg/ml. Inflammatory macrophages were IFN-'y(l00 units/ml) 10 ± 3 52 ± 6 7 ± 2 44 ± 4 harvested from the peritoneal cavities of C57BL/6 mice 5 TNF-a (1000 units/ml) 9 ± 1 25 ± 7 5 ± 3 <2 days after intraperitoneal injection of 1.5 ml of sterile 2.3% LPS (250 ng/ml) 17 ± 3 74 ± 7 7 ± 4 45 ± 8 thioglycollate broth (Sigma). IL-la (6.25 ng/ml) 18 ± 5 18 ± 2 10 ± 3 <2 Larvicidal Assay. For larvicidal assays (6, 11), ECs or IL-1,B (6.25 ng/ml) 20 ± 4 17 ± 1 11 ± 5 <2 macrophages were cultured with newly transformed schisto- IL-2 (500 units/ml) 12 ± 1 18 ± 3 <4 <2 somula or 2.5-week-old parasites for48 or 72 hr, respectively, IL-6 (10 ng/nl) 12 ± 3 24 ± 7 <4 <2 at effector/target ratios varying from 0.5 x 104:1 to 2 x 104:1 IFN-y + TNF-a 100 ±0 91 ± 7 70 ± 3 72 ± 4 in complete medium. Larval killing was determined micro- IFN-y + IL-la 88 ± 5 52 ± 6 106 ± 8 43 ± 3 scopically by the criteria of internal granularity and loss of IFN-y + IL-1, 91 ± 6 55 ± 9 110 ± 9 38 ± 6 motility (6). Initial experiments verified that neither cyto- IFN-y+ LPS 50±4 87 ± 3 87 ± 10 83 ± 5 kines (IFN-y, TNF-a, IL-1, IL-2, IL-6) nor reagents (LPS, IFN-y + IL-2 15 ± 1 70 ± 12 7 2 51 7 L-NMMA, L-NNA) were directly toxic to the larvae at the IFN-y + IL-6 8 2 56 ± 16 9 3 35 5 concentrations used. TNF-a + IL-la 49 8 26 7 82 12 <2 Measurement of Nitrite Production. ECs were cultured on TNF-a + IL-1, 43 9 26 2 108 10 <2 six-well plates at 2.5 x 105 cells per ml in 2 ml of culture TNF-a + LPS 36 8 68 0 41 6 53 + 7 medium supplemented with the stated concentration of cy- IL-2 + IL-la 25 4 17 4 12 4 <2 tokines. After 48 hr, nitrite production was determined by a IL-2 + IL-l,8 23 4 19 7 13 3 <2 standard Griess reaction adapted to microplates (11, 12). IL-2 + IL-6 8 3 20 7 <4 <2 Quantitation ofmRNA. Relative amounts ofmRNA for the IL-6 + IL-la 9 1 14 6 7 + 3 <2 inducible NO synthase (iNOS) were determined by a quan- IL-6 + IL-1 9 2 19 5 <4 <2 titative reverse transcription-PCR protocol (9). Primer and *ECs (0.5 x 106) or inflammatory macrophages (1.0 x 106) were probe sequences for iNOS were as follows: sense primer, cultured with 100 parasites for 48 hr. Results indicate percent dead 5'-CTGGAGGAGCTCCTGCCTCAT-3'; antisense primer, parasites (mean ± SEM) of three to six experiments. 5'-GCAGCATCCCCTCTGATGGTG-3'; probe, 5'-CTG- tResults indicate nmol of nitrite per culture (mean ± SEM) of three GATGAGCTCATCTTTGCC-3'. to six experiments. Downloaded by guest on September 28, 2021 Immunology: Oswald et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1001

cytokine Schistosomulum killing (% dead) M0 IENDOTHELIAL addition 20 40 60 80 100 CELLS none ...... y b U. UL z 2 I-. TGF-P 26 + 2 + U U IU L L F IL-10 _ INDUCIBLE NO so SYNTHASE IL-4

_ _mu .HPRT FIG. 2. Effect of IL-10, IL-4, and TGF-,3 on the inhibition of EC activation for schistosomulum killing. Results pooled from three experiments are presented as mean percent larval killing ± SEM. FIG. 1. Cytokine activation ofthe production of iNOS mRNA by Endothelial cells were activated with either IFN-y (20 units/ml) and ECs and macrophages. ECs and macrophages (MO) were stimulated TNF-a (200 units/ml) (solid bars) or IFN-y (20 units/ml) and IL-la for 4 hr with IFN--y (100 units/ml) or TNF-a (1000 units/ml) alone or (1.25 ng/ml) (stippled bars) and treated with TGF-,3, IL-10 or IL-4 at in combination. Unstimulated cells were used as controls. After 4 hr, 500 pg/ml, 250 units/ml, or 250 units/ml, respectively. Untreated total RNA was isolated, reversed-transcribed, and then amplified by cytokine-activated cells were used as positive control. Significance PCR with specific primers for iNOS or hypoxanthine phosphoribo- ofinhibition was assessed by a paired t test comparing the larvicidal syltransferase (HPRT). activity of positive control cells to that of cells treated with modu- latory cytokines: **, P < 0.01. EC activation by combinations of either of IFN-y plus IL-la or IFN-y plus TNF-a as measured by cytotoxicity (Fig. 2) or nitrite production (data not shown). IL-4, however, was able lungs of mice vaccinated with radiation-attenuated parasites to completely block the ability of IFN-y-plus TNF-a- after challenge infection. In addition to the presence of activated or IFN-y plus IL-la-activated ECs to kill schisto- inflammatory foci in the lung parenchyma previously de- somula (Fig. 2). Likewise, IL-4 treatment substantially de- scribed (5, 15), remarkable endarteritis and periarteritis were creased iNOS mRNA (54% inhibition in IFN-y-plus TNF-a- observed, with mononuclear cell and eosinophil infiltration. activated cells). This inhibitory effect of IL4 could be In the early lesion the ECs appeared swollen, with globoid overcome by activation of ECs with higher (>5-fold) con- vesicular nuclei and abundant eosinophilic cytoplasm (Fig. centrations of upregulatory cytokines (7% inhibition of par- 3B), compared with the flat ECs with dark nuclei seen in the asite killing in the presence of IL-4 at 250 units/ml). normal vessels (Fig. 3A). Later, there was evidence ofintimal Cytotoxic Effects ofECs from Various Tissues. Because ECs thickening that sometimes completely occluded the blood have been shown to exhibit different properties depending on vessels (Fig. 3C). These reactions increased gradually over a the organ from which they were derived (14), the function of 2-week period following the challenge infection (Fig. 4). ECs of different origins was compared. Similar results were Similar arteritis was not observed in control animals that had obtained with either transformed EC lines derived from brain been likewise infected. or lung cells or primary ECs isolated from brain microves- Interestingly, only minimal endothelial changes were ob- sels, hepatic sinusoids, or lungs (Table 3). served in similar experiments with P mice, a strain that fails Susceptibility of Post-Lung-Stage Schistosomula to Acti- to develop a significant level of protective immunity upon vated ECs. Because schistosome larvae exhibit a biphasic vaccination with irradiated cercaria (5) (Fig. 3D). Thus, in age-dependent susceptibility to killing by activated macro- these animals, there was no difference in the arteritis between phages (5), EC killing of older parasites was examined. As observed with newly transformed larvae, cytokine-activated ECs killed a substantial proportion of 2.5-week-old parasites Table 3. Larvicidal activity and nitrite production by ECs from (Table 3). different sources Evidence for in Vivo EC Activation in Vaccinated Mice. To evaluate the relevance of these in vitro observations to Schistosomulum Nitrite killingt % dead production, immunity in vivo, we examined morphologic changes in the Activation nmol per 5 x cells Table 2. Arginine dependence of larval killing by EC type status* 3 hr 2.5 weeks 106 cytokine-activated ECs Transformed 9 ± 1 15 ± 4 >4 Schistosomulum Nitrite productiont Brain Null Activated 100 ± 0 52 ± 7 53 ± 4 % dead nmol 5 x 106 cells killing,t per Lung Null 9 ± 2 8 ± 3 >4 Cytokine - + - + Activated 74 ± 8 45 ± 2 89 ± 4 treatment* L-NMMA L-NMMA L-NMMA L-NMMA Primary None 7 3 ND <4 ND Brain Null 14 ± 3 13 7 >4 ± 40 ± 5 IFN-y + TNF-a 100 0 7 ± 3 68 ± 5 8 ± 2 Activated 79 7 47 6 8 ± 2 2 >4 IFN-y + LPS 70 5 3 ± 1 65 ± 5 8 ± 3 Liver Null 13 ± 5 43 6 38 ± 6 IFN-y + IL-1-a 90 5 5 ± 3 120 ± 4 18 ± 2 Activated 69 Lung Null 20 ± 5 10 1 >4 IFN-y + IL-1-. 98 5 9 4 125 ± 9 20 2 65 ± 55 1 85 ± 4 TNF-a + IL-1-a 77 7 7 3 85 ± 1 11 ±2 Activated 8 TNF-a + IL-1-P 80 4 10 3 90 ± 5 13 2 *Cells were activated with a combination of IFN-y (100 units/ml) ND, not determined. plus TNF-a (1000 units/ml) and, for primary lung ECs, IL-la (6.25 *IFN-{y, 100 units/ml; TNF-a, 1000 units/ml; IL-la or -1,, 6.25 ng/ml). ng/ml; LPS, 250 ng/ml. tThree-hour-old schistosomula were cultured for 48 hr with 0.5 x 106 ECs; 2.5-week-old parasites were cultured for 72 hr with 2 x 106 tMean ± SEM (n = 4) in the presence or absence of 1 mM L-NMMA. ECs (half of the cells were added at 24 hr of culture). Results show mean ± SEM of three to six experiments. Downloaded by guest on September 28, 2021 1002 Immunology: Oswald et A Proc. Nadl. Acad. Sci. USA 91 (1994) DISCUSSION 9§ Previous work on the immunologic functions ofthe endothe- lium has revealed that ECs play a central role in both the induction and orchestration of cell-mediated inflammatory responses. The present study establishes that these cells can I'.: also operate in the effector arm of host defense by directly £ killing intravascular pathogens through the production of toxic nitrogen metabolites. This potential effector function was demonstrated in vitro with S. mansoni schistosomula as the target. During their developmental cycle, schistosome larvae are in intimate contact with endothelium for a pro- longed period and might be expected to be highly sensitive to EC-mediated damage. Studies tracking the migration pattern of radiolabeled S. mansonilarvae in mice previously immunized with irradiated parasites suggest that the majority of the parasites are elim- inated in the lungs several weeks after challenge infection(5). On day 21 after infection, most ofthe larvae detectable in the lungs of vaccinated mice are located within blood vessels, although -25% are found within alveolar spaces. About half of the identifiable larvae in either site are surrounded by moderate to intense inflammatory reactions consisting mostly of mononuclear cells (15, 16). While the question of what becomes ofchallenge parasites in the lungs of immunized animals currently remains unan- swered, there is now consensus among many investigators that the protection conferred by a single vaccination with attenuated cercaria results from CMI (4,5). It is apparent that f a',$ > A larvae which have broken out into the alveolar spaces may sJ '-jv*allg~ - come into direct contact with activated macrophages present * 4*' , in these inflammatory reactions (17). However, intravascular larvae are in more intimate connection with the ECs lining the blood vessels of the lung. It is doubtful that labile effector molecules such as NO produced by macrophages within the FIG. 3. Lung sections taken 14 days after challenge infection of perivascular infiltrate might reach these larval targets. Cy- unvaccinated (A) or vaccinated (B and C) C57BL/6 mice or from a tokines produced within the inflammatory cell infiltrate sur- vaccinated mouse ofthe P/J strain (which fails to develop protection rounding the vessel might, however, affect EC function, and after immunization) (D). Note normal EC size (arrow) and lack of vice versa (18). arteritis in A, gradual increase of EC size from the normal (arrow) to Indeed, ECs were found to be activated for larval kig by the inflammated part of the artery (double arrow) in B, obliteration stimulation with combinations of and as of the lumen by cells adherng to the activated endothelium in C, and IFN-'y, TNF-a, IL-1, the minimal periarteritis in D. (Hematoxylin and eosin staining; well as endotoxin (Table 1). The cytotoxic mechanism was x400.) arginine-dependent (Table 2), and correlated with production of toxic nitrogen oxides. Two categories of enzymes are vaccinated and control animals when measured 2-3 weeks responsible for the production of NO from L-arginine (19). after challenge infection (mean ± SD of arteritis, 8.3 ± 1.0 The first type of enzyme, constitutive NOS, is constitutively mm in vaccinated animals, and 7.4 ± 0.2 mm in controls). expressed, generates a low output of NO, and is known to be expressed in ECs as well as in the brain. The second category includes the iNOS which is inducible by cytokines and/or E 16 microbial products, generates high NO output, and was first E described in macrophages. Our results show that ECs are likewise able to generate high levels of NO (Table 1) and to 6 12 express the iNOS gene after stimulation by cytokines (Fig. 1). a Thus, these data confirm at the gene level previous indica- C tions that ECs express the cytokine-inducible NOS, based on U 8 -C observations of cofactor requirement (20). ECs appear to be capable of mediating larval killing in a 0 r.a 4 manner analogous to that observed with tissue macrophages IC (6, 11). However, conditions for the participation of ECs in 4c the effector mechanism of CMI to S. mansoni may be more 0o stringent than those stimulating macrophage function, since o 2 4 6 8 10 12 14 16 multiple activating stimuli are apparently required (Table 1 Day post challenge and ref. 21). Nevertheless, analyses of mRNA levels in the FIG. 4. Effect of vaccination on arteritis in the lungs of mice lungs of vaccinated mice suggest that the cytokines partici- infected with S. mansoni. Arteritis was determined (mean of four pating in EC activation-IFN-'y, TNF-a, and IL-1-might all animals ± SEM) as thickness of the wall of juxtabronchial arteries be expected to be produced within the inflammatory reac- and surrounding inflammatory reaction in the lungs of mice percu- tions to challenge schistosomula in vivo (T.A.W., I.E., taneously infected with 500 irradiated cercariae. o, Vaccinated; u, I.P.O., S.L.J., and A.S., unpublished work). Interestingly, nonvaccinated. conditions for downregulation of EC activity appear also to Downloaded by guest on September 28, 2021 Immunology: Oswald et al. Proc. Natl. Acad. Sci. USA 91 (1994) 1003 be stringent (Fig. 2), since two of the three macrophage activation and downregulation by cytokines of this effector modulatory cytokines tested here exerted little effect on ECs. function. Together, these observations suggest that ECs To fully define the ability of ECs to function as immune should be considered as potential effector cells in the mech- effector cells in vaccinated mice, it is also necessary to anism of resistance to any pathogen that is intravascular at determine their ability to kill older migrating forms of the some developmental stage. parasite, since these are the presumed targets of protective immunity. Schistosomula go through at least two stages of Note Added in Proof. A study (26) has been published on induction susceptibility to killing by lymphokine-activated macro- of the NOS gene in rat ECs. phages: within the first few days after transformation from the infective cercarial stage and 2-4 weeks thereafter (5). We thank Drs. R. Kilbourn, G. Nicholson, and T. Maciag for Cytokine-activated ECs were likewise able to kill a signifi- providing the endothelial cells used in this study; R. Coffman for the cant portion of older parasites (Table 3). These results purified IL-10; G. Nicholson for his valuable advice; and Sara Heiny indicate that S. mansoni larvae are vulnerable to EC- for expert technical assistance. This investigation received financial support from the United Nations Development Program/World mediated damage at a time when the majority of the larvae Bank/World Health Organization Special Program for Research and would still be located in the lungs of vaccinated mice (17). Training in Tropical Diseases. Immunologic activation of ECs at the time when challenge larvae are found in the lungs is suggested by the observation 1. Cavender, D. E. (1991) Int. Rev. Exp. Pathol. 32, 57-94. of hypertrophy and possible hyperplasia of the endothelium 2. Shimizu, Y., Newman, W., Tanaka, Y. & Shaw, S. (1992) ofpulmonary vessels in regions ofinflammatory activity (Fig. Immunol. Today 13, 106-111. 3). The observation of EC alterations upon challenge infec- 3. Bosse, D., George, V., Candal, F. J., Lawley, T. J. & Ades, tion of a resistant mouse strain, but not in the case of a strain E. W. (1993) Pathobiology 61, 236-238. (P) that fails to develop protective immunity as a result of 4. James, S. L. & Sher, A. (1990) Contemp. Topics Microbiol. vaccination (Fig. 3D), is also very suggestive that these Immunol. 155, 21-31. responses play a role in resistance. 5. James, S. L. & Boros, D. L. (1993) in Macrophages-Patho- Several ways in which activated ECs could be expected to gen Interactions, eds. Zwilling, B. & Eisenstein, T. (Dekker, participate in vaccine-induced immunity have been docu- New York), pp. 461-473. 6. James, S. L. & Glaven, J. (1989) J. Immunol. 143, 4208-4212. mented in the current study. First, occlusion of the pulmo- 7. James, S. L. (1991) Exp. Parasitol. 73, 223-226. nary microvessels might impede migration ofthe larvae in the 8. Li, L., Kilbourn, R. G.,Adams, J. & Fidler, I. J. (1991) Cancer lungs. Trapped larvae would thus be forced into long-term Res. 51, 2531-2535. contact with activated ECs producing toxic mediator sub- 9. Wynn, T. A., Eltoum, I., Cheever, A. W., Lewis, F. A., stances, such as NO and TNF-a. Over time, especially at the Gause, W. C. & Sher, A. (1993) J. Immunol. 151, 1430-1440. high concentrations expected to be generated at the contact 10. Schwartz, B., Vicart, P., Delouis, C. & Paulin, D. (1991) Biol. points between cells and larvae, these toxic effector mole- Cell. 73, 7-14. cules could inhibit various metabolic functions dependent on 11. Oswald, I. P., Gazzinelli, R. T., Sher, A. & James, S. L. (1992) FeS-containing enzymes (7), eventually resulting in death of J. Immunol. 148, 3578-3582. the parasites. Alternatively, such hostile conditions may 12. Oswald, I. P., Wynn, T. A., Sher, A. & James, S. L. (1992) make it attractive for the parasite to attempt to escape from Proc. Natl. Acad. Sci. USA 89, 8676-8680. its intravascular location. Cytokine treatment has been 13. Schreiber, A. B., Kenney, J., Kowalski, W. J., Freisel, R., Mehlman, T. & Maciag, T. (1985) Proc. Natl. Acad. Sci. USA shown to increase EC "leakiness" (22), a fact that may not 82, 6138-6142. only contribute to perivascular inflammation but also pro- 14. Belloni, P. N., Carney, D. H. & Nicholson, G. L. (1992) Mi- mote the exodus of some larvae. In addition, because NO crovasc. Res. 43, 20-45. may also be toxic to the cells which produce it (23), self- 15. Crabtree, J. E. & Wilson, R. A. (1986) Parasite Immunol. 8, destruction of activated ECs could facilitate the escape of 265-285. some larvae into the alveolar spaces, from which they cannot 16. Olakunkle, 0. K., Dean, D. A., Mangold, B. L. & von Lich- return to the bloodstream and where they may become the tenberg, F. (1992) Am. J. Trop. Med. Hyg. 47, 231-237. target of inflammatory reactions with activated macro- 17. Coulson, P. S. & Wilson, R. A. (1988) Am. J. Trop. Med. Hyg. phages. Thus, the results reported here suggest a potential 38, 529-534. 18. Mantovani, A., Bussolno, F. & Dejana, E. (1992) FASEB J. 6, role for cells that are not traditionally considered to be part 2591-2599. of the immune system in mediating protective immunity 19. Marletta, M. A. (1993) J. Biol. Chem. 268, 12231-12234. against S. mansoni or any pathogen that is intravascular at 20. Gross, S. S., Jaffe, E. A., Levi, R. & Kilbourn, R. G. (1991) some developmental stage. Preliminary studies in our labo- Biochem. Biophys. Res. Commun. 178, 823-829. ratory indicate that human ECs can also kill schistosomula 21. Aarden, L., Helle, M., Boeije, L., Pascual-Salcedo, D. & de upon cytokine activation, although as previously observed Groot, E. (1991) Eur. Cytokine Netw. 2, 115-120. with human macrophages (24), cytotoxicity appears indepen- 22. Queluz, T. T., Brunda, M., Vladutiu, A. O., Brentjens, J. R. & dent of NO production. Andres, G. (1991) Exp. Lung Res. 17, 1095-1108. Cytokine-activated ECs have been shown to kill tumor 23. Estrada, C., Gomez, C., Martin, C., Moncada, S. & Gonzalez, and C. (1992) Biochem. Biophys. Res. Commun. 186, 475-482. cells in vitro by means of an NO-dependent reaction (8) 24. James, S. L., Woods-Cook, K. & Lazdins, J. K. (1990) J. to inhibit intracellular growth of the protozoan parasite Immunol. 145, 2686-2690. Toxoplasma gondii by an unidentified mechanism (25). The 25. Woodman, J. P., Dimier, I. H. & Bout, D. T. (1991) J. Immu- results of the current study with an extracellular helminth nol. 147, 2019-2023. establish a role for reactive nitrogen oxides in the mechanism 26. Suschek, C., Roth, H., Fehsel, K., Enezmann, J. & Kolb- ofparasite killing by murine ECs and define the conditions for Bachofen, V. (1993) J. Immunol. 151, 3283-3291. Downloaded by guest on September 28, 2021