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