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The Journal of Immunology, 1999, 163: 4976-4984.
Copyright © 1999 by The American Association of Immunologists

Nitric Oxide and the Th2 Response Combine to Prevent Severe Hepatic Damage During Schistosoma mansoni Infection1

L. Rosa Brunet2,*,{dagger}, M. Beall*, D. W. Dunne{dagger} and E. J. Pearce3,*

* Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and {dagger} Department of Pathology, Cambridge University, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During infection with Schistosoma mansoni, NO production increases following the deposition of parasite eggs in the liver. In wild-type C57BL/6 mice, NO levels peak during the sixth week of infection and are subsequently down-regulated. Inducible NO synthase (iNOS) mRNA was found in diseased liver tissue along with TNF-{alpha} and IFN-{gamma}, which are known promoters of iNOS expression. Mice treated with aminoguanidine, a selective inhibitor of iNOS, exhibited cachexia and exacerbated liver pathology, suggesting that NO limits hepatocyte damage when the liver is first exposed to eggs. Hepatic iNOS is up-regulated in SCID mice, indicating that NO production is part of an innate response. Studies with infected highly susceptible IL-4-/- mice revealed that prolonged NO production is in itself deleterious and that a major function of the Th2 response, which is severely compromised in the absence of IL-4, is to regulate NO production. In these animals, plasma NO levels are high compared with those in infected wild-type mice and remain elevated until death. Nevertheless, the underlying importance of NO is illustrated by the finding that aminoguanidine treatment leads to more severe liver disease and reduced time to death in infected IL-4-/- mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pathogenic helminths of the genus Schistosoma infect an estimated 200 million people worldwide (1). Disease is caused by the host reaction to tissue-trapped parasite eggs, which induce granulomatous lesions composed primarily of macrophages, eosinophils, lymphocytes, and fibroblasts (1, 2). Granulomatous pathology is orchestrated by CD4+ cells, and the most severely affected organ during Schistosoma mansoni infection is the liver. During infection, the CD4+ cell response to parasite eggs is strongly type 2 in nature and characterized by the production of high levels of IL-4, IL-5, and IL-10 (3, 4, 5). We recently reported that during murine infection the production of Th2 cytokines is essential for host-survival (6). In our hands, type 2 response-defective IL-4-/- C57BL/6 mice develop severe morbidity and succumb during the acute phase of the infection (6), which is defined as the 2- to 3-wk period following the onset of egg production. In these animals, increased NO production by cultured spleen cells correlated tightly with increased disease severity (6), suggesting a deleterious role for this mediator. Defining the role of NO during schistosomiasis has become particularly relevant in light of recent findings showing that peripheral blood mononuclear cells from infected patients are capable of in vitro NO production following exposure to S. mansoni Ags (7).

NO, an endogenously secreted short-lived lipophilic free radical, is the byproduct of the conversion of arginine and oxygen into citrulline in an enzymatic reaction mediated by NO synthase (NOS)4 (8, 9). Three NOS isoforms have been described to date, each encoded by separate genes and under distinct induction requirements. The constitutive forms of NOS, present in neuronal (nNOS, NOS-1) and endothelial (eNOS, NOS-3) cells, are constitutively expressed at basal levels (10, 11) and produce only low amounts of NO that act in physiological processes including neurotransmission and vasorelaxation (9, 12, 13). The third isoform, the inducible NOS (iNOS, NOS-2), is present in virtually all cells (14), is expressed in response to proinflammatory cytokines (such as IFN-{gamma}, TNF-{alpha}, and IL-1ß) and/or microbial products (such as LPS), and results in the prolonged production of large amounts of NO (15, 16). The function of NO in the immune response varies depending on the biological milieu (13, 16, 17, 18). NO may be host-protective, acting as an effector molecule of macrophage cytotoxicity against tumor cells and invading pathogens (8, 19, 20, 21) and as a regulator of inflammatory responses (13, 22). Despite these beneficial roles, NO has been found to mediate disease processes by inducing cell apoptosis in tissues and causing cell damage through the formation of toxic radicals (23, 24, 25, 26, 27). In light of these distinct observations, the exact role of NO in many pathological settings remains to be defined.

The role of NO during schistosomiasis has been investigated previously only in the context of its function as an effector molecule of the protective type 1 immune response induced by an experimental radiation-attenuated vaccine (28, 29). In this study, we examined the role of NO during natural infection with S. mansoni. We found that in contrast to wild-type (WT) mice in which NO production is under strict regulation, systemic NO levels are elevated in IL-4-/- mice throughout the course of acute disease. In the hepatic tissues of WT and IL-4-/--infected mice, iNOS, TNF-{alpha}, and IFN-{gamma} mRNA are up-regulated at the onset of egg deposition. In an effort to characterize the role of NO during acute schistosomiasis, we treated mice with aminoguanidine (AMG), which prevents NO production by selectively inhibiting iNOS (30, 31, 32, 33, 34). Preventing NO production at the onset of egg deposition had a profound deleterious effect. Following iNOS inhibition, both WT and IL-4-/- mice developed severe cachexia and hepatosplenomegaly was significantly reduced. Most strikingly, infected WT mice developed more severe liver damage characterized by elevated hepatocellular enzyme release and increased hepatocyte necrosis and apoptosis. Under the same conditions, IL-4-/- mice developed severe hepatotoxicity and succumbed prematurely. We interpret these data as suggestive of a protective role for NO during the early stages of acute schistosomiasis and hypothesize that to prevent NO-induced tissue damage associated with elevated NO levels, NO production in infected hosts requires tight regulation by egg-induced type 2 responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental infections

C57BL/6 WT mice (Taconic, Germantown, NY), C57BL/6 SCID mice (Taconic), and C57BL/6 IL-4-/- mice (35), bred at Cornell University, were infected percutaneously with S. mansoni (Puerto Rican strain, Naval Medical Research Institute) cercariae shed from infected Biomphalaria glabrata snails. Mice were individually identified and weighed regularly (6). We used weight loss as an indicator of morbidity and terminated experiments once we detected significant differences in weight changes in treated and untreated animals. At necropsy, mice were bled by cardiac puncture and plasma samples were used to determine systemic NO, TNF-{alpha}, and IFN-{gamma} levels (see below). Whole livers were weighed and tissue samples collected for RT-PCR analysis and to quantitate organ egg-burden (36). The hepatic frontal lobes were fixed in neutral buffered formalin and used for histological examination. Hepatic granuloma volumes and areas of hepatic necrosis were measured using an ocular micrometer on Masson’s trichrome and hematoxylin and eosin stained sections, respectively. Sections were also stained with periodic acid schiffs (PAS) to evaluate changes in hepatic glycogen.

AMG treatment

Mice were treated orally with 100 mM AMG (hemisulfate salt; Sigma, St. Louis, MO) by adding it to their sole source of drinking water. To confirm that this dose was effective in inhibiting iNOS, we injected mice with 10 µg of LPS (Escherichia coli 0111:B4; Difco, Detroit, MI) and compared NO levels (as measured by the accumulation of stable nitrite, see below) in the plasma of mice that received AMG treatment or that were left untreated.

Immune response measurements

Spleens were aseptically removed, weighed, and single-cells suspensions prepared. Splenocytes (2 x 106cells/well) were incubated in vitro with mAb anti-CD3 (0.5 µg/well plate-bound) to polyclonally stimulate T cells. Inhibition of NO production was maintained in vitro by the addition of N{omega}-monomethyl-L-arginine monoacetate salt (L-NMMA, 25 µM final; Calbiochem, La Jolla, CA). IL-4, IL-5, IL-10, TNF-{alpha}, and IFN-{gamma} levels in culture supernatants and/or plasma were determined by ELISA (6, 37). NO production was estimated by measuring accumulated nitrite by the Greiss reaction as previously described (6). Plasma nitrite levels were measured following a reduction step using E. coli for 1 h at 37°C (38). Proliferation assays were conducted to determine the effect of AMG treatment on splenic proliferative responses. Splenocytes (2.5 x 105cells/well) were cultured in triplicate on plate-bound anti-CD3 in 96-well U-bottom microtiter plates, and proliferation was measured after 120 h. Cells were pulsed with 1 µCi of [3H]TdR/well (Amersham, Arlington Heights, IL) during the last 12 h of culture, and thymidine incorporation into DNA was determined by liquid scintillation counting. Throughout these experiments, spleens from appropriate groups of uninfected animals treated with AMG or left untreated were used as controls.

For semiquantitative competitive RT-PCR analysis, groups of infected WT, SCID, and IL-4-/- mice were euthanized on days 41–43 postinfection, soon after the onset of egg deposition. Total RNA, extracted from liver samples using RNAzol (Tel-Test, Friendswood, TX) as per the manufacturer’s instructions, was reverse transcribed using Superscript II (Life Technologies, Gaithersburg, MD) and random hexamer primers (Pharmacia Biotech, Piscataway, NJ) as described (39). Following reverse transcription, cDNA aliquots were used for semiquantitative PCR amplification (40). Briefly, cDNA obtained from experimental animals was amplified using oligonucleotide primers for the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) and for iNOS, TNF-{alpha}, and IFN-{gamma} in the presence of a polycompetitor construct containing mutated cDNA sequences for these genes (39). Amplifications (37 cycles for HPRT and 41 cycles for iNOS, TNF-{alpha}, and IFN-{gamma}) were conducted under the following conditions: 94°C for 45 s, 66°C for 15 s, and 72°C for 45 s. Concentrations of cDNAs were individually adjusted using HPRT before assaying for iNOS, TNF-{alpha}, and IFN-{gamma}. Final PCR products were resolved by electrophoresis on 2.5% agarose gels followed by ethidium bromide staining and quantitation by densitometry (EAMGle-Eye; Becton Dickinson, Sunnyvale, CA). The larger m.w. of the competitor band was used as an internal standard to determine the relative amounts of the experimental cDNA with a lower m.w.

Assessment of hepatic cellular damage

To assess the extent of hepatic cellular damage, we measured the circulating levels of the hepatocellular enzyme aspartate aminotransferase (AST) using a commercially available kit (Sigma). An increase in AST levels is a clinically accepted indicator of hepatic injury (41). Baseline plasma AST levels (SF units (SFU)) were determined in uninfected control animals treated with AMG or left untreated. Increases in AST levels during infection were expressed as fold increase over the baseline.

Immunohistochemical staining

Formalin-fixed, paraffin-embedded tissue sections were microwaved and stained with a polyclonal anti-mouse iNOS Ab (Transduction Laboratories, Lexington, KY) (42). Bound Ab was detected using the avidin-biotin complex-based method according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). Control slides were stained with an anti-ß-galactosidase Ab.

Cellular apoptosis was detected immunohistochemically by TUNEL as per the manufacturer’s instructions (Boehringer Mannheim, Indianapolis, IN). The fluorescent-labeled DNA fragments were analyzed using immunofluorescence microscopy.

Statistics

Significant differences in organ weights, liver egg burdens, granuloma volumes, AST levels, and NO and cytokine production between AMG-treated and untreated mice were determined by Student’s t test. When required, nonparametric statistical analysis was conducted using the Wilcoxon signed rank test or Mann-Whitney test. Probability values (2) 0.05 were considered significant. Unless otherwise stated, data are presented as mean ± SE for individual experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Systemic NO production during infection

Our recent work with infected IL-4-/- mice suggested that, in the absence of a Th2 response, the severity of cachexia during acute schistosomiasis correlates positively with NO production (6). In light of this observation we investigated the kinetics of systemic in vivo NO production in infected WT and IL-4-/- mice as measured by nitrite levels in the plasma of infected animals. Soon after the onset of egg deposition, NO levels appeared slightly elevated in both WT and IL-4-/- mice (Fig. 1Go). However, in contrast to WT animals, which effectively reduce NO levels as infection progresses, IL-4-/- mice maintain elevated systemic NO levels until the time of death (Fig. 1Go and not shown). Because NO is the end product of a complex inflammatory response, we determined whether we could detect the presence of other inflammatory mediators in the plasma of infected animals. However, we found that in both infected WT and IL-4-/- animals, TNF-{alpha} and IFN-{gamma} levels in plasma were below detection levels (not shown).



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  		FIGURE 1. NO levels in the plasma of infected WT and IL-4-/- mice during the early stages of disease. Mice were each exposed to ~60 cercariae and bled at three different time points following the onset of egg deposition. Plasma levels of accumulated nitrite, used as an indicator of NO production, were measured. Levels in the plasma of uninfected animals were below detection. There were four animals per group. Nitrite levels are expressed as means ± SE. Results are representative of three separate experiments. (*, p <= 0.05).

 
We further investigated the kinetics of NO production in infected WT mice using an in vitro assay (Fig. 2Go) and found that in these animals production of NO by Ag-stimulated splenocytes peaked between days 37 and 45 of infection, coincident with the deposition of eggs in host tissues. NO levels sharply declined thereafter such that by day 70 of infection they had returned to the levels found in naive uninfected mice. NO remained low for the remainder of the infection.



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  		FIGURE 2. In vitro NO production by infected WT mice. Mice were each infected with ~30 cercariae. Spleen cells collected at various times postinfection were stimulated in vitro with ({circ}) PBS or a combination of (•) SEA (50 µg/ml) and SWAP (50 µg/ml). There were four animals per group. Nitrite levels are expressed as means ± SE. Results are representative of three separate experiments.

 
Effects of iNOS inhibition on morbidity during infection

Beginning at days 35–37 postinfection, we treated IL-4-/- and WT mice with AMG, an inhibitor of iNOS, and compared disease progression with that observed in similarly infected but untreated animals. We confirmed the effectiveness of AMG in preventing NO production by injecting uninfected AMG-treated or untreated WT animals with LPS (10 µg, i.p.) and measuring plasma NO levels; NO production in treated animals was significantly reduced (8 ± 1.6 µM) compared with levels in LPS-injected untreated mice (100.5 ± 1 µM).

Contrary to our expectations, iNOS inhibition led to an exacerbation of symptoms in IL-4-/- mice, which suffered increased weight loss compared with untreated infected mice (Fig. 3Go). Infected WT mice treated with AMG also suffered significant weight loss compared with untreated infected animals (Fig. 3Go). Weight loss was not due to any inherent toxicity of AMG as uninfected AMG-treated IL-4-/- and WT mice were indistinguishable from untreated uninfected mice in this regard (not shown, p = 0.56). Interestingly, iNOS inhibition by AMG appeared to have a deleterious effect only when treatment was initiated at the onset of egg deposition. When chronically infected WT mice (32 wk postinfection) were treated for a 10-day period, we found no exacerbation of morbidity, as measured by weight change, compared with similarly infected untreated mice (not shown, p = 0.99). Even when AMG treatment was continued for 3 wk in chronically infected WT mice (52 wk postinfection) we detected no worsening of disease compared with similarly infected untreated controls (not shown, p = 0.58). Our data suggest that NO is effective in preventing the severe morbidity that may result from the host’s first exposure to parasite eggs.



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  		FIGURE 3. Morbidity in infected WT and IL-4-/- mice is exacerbated in the absence of NO. The time course of cumulative weight change in AMG-treated WT and IL-4-/- mice that had each been exposed to ~70 cercariae was followed. AMG treatment was started at day 35 postinfection. Animals similarly infected but left untreated were followed for comparison. There were five animals per group. Changes in body weight are expressed as means ± SE. Results are representative of at least three separate experiments. Compared with untreated infected animals of the appropriate genotype, cumulative weight changes in WT and IL-4-/- mice treated with AMG are significantly different (*, p <= 0.05).

 
Because the host immune response can limit egg production by adult worms (43), we compared liver egg loads in AMG-treated and untreated mice to determine whether the lack of NO promotes parasite egg production, thereby causing severe morbidity. We found that AMG-treated WT and IL-4-/--infected mice had liver egg loads comparable to those of untreated mice (Table IGo). On the basis of these results, we conclude that the increased morbidity apparent in AMG-treated infected mice is not due to increased burden of infection but rather reflects the absence of some host-beneficial process mediated by NO.


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  		Table I. Effects of AMG treatment on WT and IL-4-/- micea

 
Effects of iNOS inhibition on organomegaly

A consistent feature of schistosomiasis in immunocompetent hosts is the development of liver and spleen organomegaly. However, the spleens and livers of infected mice treated with AMG were significantly less enlarged than those of control infected animals (Table IGo). While a comparison of body weight relative to organ weight at the time of death (not shown) reveals that this in part reflects the cachectic state of the AMG-treated infected mice, it may also indicate that during S. mansoni infection NO plays a more direct role in the development of organomegaly. To ensure that AMG treatment did not affect organ size per se in normal animals, we compared liver and spleen weights in treated and untreated age-matched uninfected WT and IL-4-/- mice. As anticipated, the organ weights of AMG-treated uninfected mice were similar to those of untreated age-matched controls (Table IGo).

Effects of iNOS inhibition on liver pathology

The liver, which is the major target organ of pathology during schistosomiasis, is clearly affected by the absence of NO (Table IGo). One implication of these observations is that iNOS is expressed in the liver and that NO is produced locally in this organ. To assess whether or not this is the case, we used RT-PCR to examine whether iNOS is expressed in the liver during the natural course of infection. At the onset of egg deposition in infected animals, expression of the iNOS gene is markedly elevated (Fig. 4Go); hepatic tissue from uninfected mice does not contain iNOS mRNA (Fig. 4Go). Consistent with our findings of increased systemic NO production in the absence of IL-4, we observed that compared with WT mice, hepatic iNOS mRNA is up-regulated to a greater extent in IL4-/- animals (Fig. 4Go; transcript:competitor ratio of 0.33 ± 0.1 vs 0.59 ± 0.4, respectively). We found that similarly to WT mice, iNOS expression is up-regulated early during infection in SCID mice (Fig. 4Go). Taken together our data suggest that the production of NO at the onset of egg deposition is at least in part regulated by IL-4 and part of an innate immune response to parasite eggs in the liver.



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  		FIGURE 4. Up-regulation of hepatic iNOS, TNF-{alpha}, and IFN-{gamma} mRNA during infection in WT, IL-4-/-, and SCID mice. Liver samples from mice infected with ~70 cercariae each were collected at day 41 of infection and used as a source of mRNA for competitive RT-PCR analysis of gene transcripts. The upper bands represent amplicons from the competitor plasmid while the lower bands are from the cDNAs. C, control uninfected mice (lanes 1, 2, 6, 7, and 11); I, infected mice (lanes 3–5, 8–10, 12, and 13).

 
Increased NO production is often associated with elevations in other inflammatory mediators such as TNF-{alpha} and IFN-{gamma}, which ultimately may synergize with NO and in combination further potentiate its effects (13). Indeed, liver TNF-{alpha} and IFN-{gamma} transcripts are both increased as a result of infection (Fig. 4Go). Unlike the case of iNOS, TNF-{alpha} transcripts were not increased in infected SCID mice (Fig. 4Go).

In S. mansoni-infected hosts, normal hepatic tissue architecture is altered by the development of granulomatous lesions around tissue-trapped eggs (Fig. 5GoA). In light of the observed increase in iNOS mRNA levels in hepatic tissue at the onset of egg deposition, we wished to establish whether iNOS was being expressed within the granulomas and/or by resident liver cells. Using immunohistochemistry, iNOS was localized in the developing granulomas of infected WT (Fig. 5GoJ) and IL-4-/- mice (Fig. 5GoN). The intensity of staining in samples from IL-4-/- mice was invariably greater than that observed in those from WT mice (compare Fig. 5GoN to Fig. 5GoJ). As time progressed, staining in granulomas of WT mice became less apparent (Fig. 5GoK).



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  		FIGURE 5. Histological evidence for increased hepatic damage in infected mice following inhibition of iNOS. Liver samples from AMG-treated and control untreated infected WT and IL-4-/- mice were collected at day 47 of infection, fixed, sectioned, and stained. Arrows indicate eggs, arrowheads indicate points of interest. A, WT, hematoxylin and eosin stain, x100. B, WT treated with AMG, hematoxylin and eosin stain, x100. Note area of coagulative necrosis, outlined with a black line. C, WT treated with AMG, hematoxylin and eosin stain, x400. Areas of coagulative necrosis adjacent to eggs are visible. Dead cell in an area of necrosis is indicated with an arrowhead. D, WT treated with AMG, TUNEL staining by immunofluorescence, x200. Area similar to that outlined in B stains positively with TUNEL. Note highly fluorescent nuclei of cells and complete lack of staining in the unaffected tissue surrounding the damaged region. E, IL-4-/-, hematoxylin and eosin stain, x100. F, IL-4-/- treated with AMG, hematoxylin and eosin stain, x100. Lightly staining areas contain vacuolated hepatocytes. G, IL-4-/- treated with AMG, hematoxylin and eosin stain, x400. Hepatocytes around granuloma are highly vacuolated. H, WT, PAS stain, x100. Most hepatocytes are filled with glycogen. An example of a positively staining cell is indicated with an arrowhead. I, WT treated with AMG, PAS stain, x200. Hepatocytes contain less glycogen than those in H. J, WT, immunostained with anti-iNOS, x200. Cells reacting with the Ab are restricted to areas of leukocyte infiltration. K, WT, immunostained with anti-iNOS, x200. This liver tissue was taken from a chronically infected mouse at wk 20 of infection. Note the lack of reactivity around the eggs compared with that seen in J. L, IL-4-/-, PAS stain, x200. Arrowhead indicates a heavily stained cell. M, IL-4-/- treated with AMG, PAS stain, x200. Lightly staining areas contain vacuolated hepatocytes that contain no detectable glycogen (arrowhead). Large tracts of the livers of these animals were glycogen depleted. N, IL-4-/-, immunostained with anti-iNOS, x200. Reactive cells are within the granuloma. More cells per granuloma than for the WT mice appear to stain positively.

 
During the early stages (wk 5–7) of acute disease, we and others (S. J. Davies and J. H. McKerrow, unpublished observations) have found that the liver of infected WT mice develops areas of coagulative necrosis, which histologically appear as strongly eosinophilic areas in which hepatocellular detail is lost. These areas are associated with some but not all parasite eggs and developing granulomas. In infected WT mice, iNOS inhibition resulted in increased hepatic tissue damage reflected histologically by more numerous and larger coagulative necrotic areas (Fig. 5Go, B and C). Because throughout our studies we always used the hepatic frontal lobes for histological examination, we felt that comparison of the numbers of these necrotic regions, and of their total surface area, was appropriate. We found that WT mice treated with AMG had a significantly (p < 0.05) higher number of necrotic areas per section compared with untreated mice (6.5 ± 1.5 vs 2.7 ± 0.8). Moreover the total area of necrosis per liver section was significantly (p < 0.05) larger in AMG-treated animals (247.9 ± 44.9 µm (2) compared with untreated mice (93.1 ± 38.2 µm (2). Because the size of the liver lobes examined reflected the size of the liver in general, being smaller in AMG-treated infected mice than in untreated infected animals (Table IGo), our data suggest that the extent of tissue necrosis in the absence of NO is greater than in control animals.

Compared both to AMG-treated infected WT and to untreated infected IL-4-/- mice, hepatic damage was substantially more pronounced in AMG-treated infected IL-4-/- mice. In these animals, iNOS inhibition led to gross hepatotoxicity characterized macroscopically by a yellow/white discoloration in two of three AMG-treated infected IL-4-/- mice. On tissue sections, hepatocytes from these hepatotoxic animals appeared swollen, with evidence of microvescicular cytoplasmic damage and nuclear alterations (Fig. 5Go, F and G). In contrast, none of the untreated infected IL-4-/- mice showed any evidence of hepatotoxicity at the times examined (Fig. 5GoE).

Cachexia resulting from iNOS inhibition in infected mice was reflected in changes in hepatocyte glycogen levels. At day 47 of infection, PAS staining of tissue sections from infected WT and IL-4-/- animals revealed extensive glycogen stores (Fig. 5Go, H and L). The absence of NO led to severe depletion of glycogen in both genotypes (Fig. 5Go, I and M), with glycogen loss being particularly severe and extensive in the IL-4-/- animals (Fig. 5GoM).

The development of hepatotoxicity has been associated with impaired granuloma formation, which may allow for the escape of egg hepatotoxins into surrounding hepatic tissue (44). To determine whether granulomas are smaller in AMG-treated mice, we measured their volume. We found that the absence of NO in infected WT and IL-4-/- mice resulted in a trend toward decreased granuloma volume compared with untreated animals (Table IGo). Aside from volume, granulomatous lesions were otherwise unaffected by the absence of NO. Both the eosinophil cellular composition of the granuloma infiltrate and the extent of hepatic collagen deposition were similar in treated and untreated mice of either genotype (not shown).

iNOS inhibition is associated with hepatocyte apoptosis

Studies have implicated NO in protection against TNF-{alpha}-induced hepatocyte apoptosis (45). Hence, we investigated whether the observed liver damage in the absence of NO is due to apoptosis mediated by TNF-{alpha}, the hepatic expression of which is up-regulated during infection (Fig. 4Go and Refs. 46, 47, 48). By TUNEL staining, we found evidence for hepatocytes undergoing apoptosis in the areas of coagulative necrosis observed in AMG-treated WT mice (Fig. 5GoD). In contrast, apoptotic hepatocytes were seldom seen in untreated infected WT mice (not shown).

Hepatocyte damage in AMG-treated infected IL-4-/- mice was not limited to specific necrotic areas, nor was it associated with increased TUNEL staining (not shown). Rather it appeared to affect most liver tissue, interfering with appropriate hepatic functions. For example, PAS staining of liver sections, used as an indicator of glycogen stores, revealed dramatic depletion of glycogen in AMG-treated infected IL-4-/- mice (Fig. 5GoM). We conclude that during schistosome infection, the absence of both IL-4 and NO leads to extensive hepatic tissue damage.

iNOS inhibition is associated with exacerbated hepatocellular damage

We assessed the extent of cellular hepatic damage in AMG-treated infected animals by measuring plasma levels of AST, an hepatocellular enzyme. In all infected mice regardless of genotype, AST levels were significantly elevated over normal background levels, consistent with hepatic involvement typical of schistosomiasis (Fig. 6Go). However, in both infected WT and IL-4-/- mice, AST levels increased further as a result of iNOS inhibition (Fig. 6Go). Consistent with the liver damage observed macro and microscopically, AST levels were highest in AMG-treated IL-4-/- animals. AMG treatment did not affect plasma AST levels in uninfected mice: AST levels in uninfected WT mice treated with AMG were comparable to those of untreated mice (22.2 ± 2 SFU vs 24.9 ± 2.3 SFU, p = 0.36, respectively); similarly, AST levels in uninfected IL-4-/- mice treated with AMG were comparable to those of untreated animals (26.3 ± 3.3 SFU vs 24.2 ± 2.3 SFU, p = 0.64, respectively).



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  		FIGURE 6. Increased hepatocyte damage in infected WT and IL-4-/- mice following inhibition of iNOS. Plasma levels of AST, an hepatocellular enzyme, of mice exposed to ~70 cercariae 46 days earlier, were measured. AST levels in animals that had received AMG treatment (started on day 37 of infection) were compared with those from untreated infected animals. Fold increases in AST levels are expressed as means ± SE. There were five animals per group. Results are representative of three separate experiments. Compared with untreated infected animals of the appropriate genotype AST levels were significantly higher in infected WT and IL-4-/- mice in the absence of NO (*, p <= 0.05).

 
T cell responses in the absence of NO

Because under certain conditions NO has been reported to down-regulate production of IFN-{gamma} and TNF-{alpha} (49), we hypothesized that the increased morbidity observed during AMG-treatment may be dependent on increased production of these proinflammatory cytokines. Elevation in TNF-{alpha} production is an attractive explanation for the cachexia observed with iNOS inhibition (Fig. 3Go). To determine whether AMG treatment for a 10-day period broadly affected the immune response of infected WT and IL-4-/- mice, we measured the in vitro production of key cytokines including TNF-{alpha}, IFN-{gamma}, IL-4, IL-5, and IL-10. We found that, for the most part, iNOS inhibition did not significantly alter subsequent in vitro cytokine production by anti-CD3-stimulated spleen cells (Fig. 7Go, IL-4 not shown). Thus the increased morbidity observed in the absence of NO cannot simply be ascribed to increased production of proinflammatory cytokines such as IFN-{gamma} or TNF-{alpha} or to decreased production of anti-inflammatory cytokines such as IL-4 or IL-10.



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  		FIGURE 7. Inhibition of iNOS does not affect in vitro production of TNF-{alpha}, IL-5, or IFN-{gamma} by T cells. Spleen cells were from AMG-treated WT and IL-4-/- mice that had each been exposed to ~70 cercariae 46 days earlier. AMG treatment was started at day 37 of infection. Spleens were assayed individually and stimulated in vitro with anti-CD3 mAb. The absence of NO was maintained in vitro by the addition of L-NMMA. Cytokine levels were measured by ELISAs in 24 h (TNF-{alpha}) and 72 h (IFN-{gamma}, IL-5, and IL-10) supernatants. There were five animals per group. Cytokine levels are expressed as means ± SE. Results are representative of three separate experiments. Aside from reduced IL-10 production by spleen cells from AMG-treated infected IL-4-/- mice, we detected no difference between the in vitro cytokine production of splenocytes from AMG-treated or untreated animals of the appropriate genotype. Note, however, that regardless of treatment splenocytes from infected IL-4-/- show significant impairment in cytokine production compared with infected WT mice following in vitro anti-CD3 stimulation (*, p <= 0.05).

 
Because AMG treatment prevented the development of splenomegaly, we investigated whether the ability of splenocytes to proliferate in vitro was impaired in the absence of NO. We found proliferation of splenocytes from WT animals not to be affected by iNOS inhibition (Fig. 8Go). In contrast, the absence of NO significantly impaired proliferative responses by spleen cells from IL-4-/- mice, which at the time of acute disease already exhibit impaired proliferation compared with similarly infected WT animals. It remains to be defined whether this proliferative defect is due to NO or rather is a result of the severe disease in these animals. Regardless, it seems unlikely that decreased T cell proliferation accounts for reduced splenomegaly in the absence of NO.



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  		FIGURE 8. Proliferative responses of infected WT and IL-4-/- mice in the absence of NO. Proliferative responses of splenocytes from WT and IL-4-/- mice that had each been exposed to ~70 cercariae 46 days earlier were assayed using [3H]thymidine incorporation into DNA. AMG treatments was started at day 37 postinfection. The absence of NO was maintained in vitro by the addition of L-NMMA. There were five animals per group. Proliferative responses are expressed as means ± SE. Results are representative of two separate experiments. The absence of NO significantly affected proliferative responses only in infected IL-4-/- mice (*, p <= 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we have attempted to define the role of NO during the acute stage of schistosomiasis. Our study was prompted by the finding of a correlation in infected IL-4-/- mice between elevated NO production by cultured spleen cells and increased severity of disease (6). Based on this finding, we hypothesized that a major function of the Th2 response that develops in WT mice during infection is to regulate NO production and thus prevent toxic effects mediated by this gas (6). We anticipated that inhibition of NO production would have no effect in infected WT mice and decrease morbidity and increase life-expectancy in infected IL-4-/- mice. However, our data show that the inhibition of iNOS has severe detrimental effects in both infected WT and IL-4-/- animals such that at the time the liver first becomes exposed to parasite eggs, the absence of NO leads to severe morbidity and increased hepatic damage. These observations have led us to propose a novel function for NO during schistosomiasis. At the onset of egg deposition when the host immune response is characterized by the production of Th1 cytokines (3, 4), NO may act in limiting the hepatocyte damage caused by the arrival of eggs in the liver. With the induction of the Th2 response stimulated by the parasite eggs themselves, the Th1 response is down-regulated and NO is no longer required to protect hepatocytes. Rather, tight regulation of NO production is required to prevent the harmful effects associated with the excess production of this inflammatory mediator.

NO has been the focus of many investigations attempting to define its role in the immune response. Unfortunately, such studies, which have used a variety of experimental models ranging from endotoxemia to cirrhosis, have produced conflicting results. Under different conditions, NO can have cytoprotective or cytotoxic effects depending not only on the initial insult but also on the source, rate of production, and concentration of NO in the biological milieu (18, 19, 23, 45, 50, 51, 52, 53). Hence, it is the balance between NO and other inflammatory cytokines and mediators (such as TNF-{alpha}, IL-1ß, IFN-{gamma}, reactive oxygen species, to name a few) that is crucial in determining its effect on the host. To further complicate the issue, it has now become apparent that cells are differentially susceptible to the effects of NO. In contrast to cells in the gut that are quite susceptible to damage induced by high levels of NO (54), hepatocytes appear quite resistant to its effects (55). Data accumulated in recent studies have suggested that NO plays a unique role in the liver, and its involvement in a variety of hepatic processes is supported by the finding that the majority of cells found in the liver, such as hepatocytes, Kupffer cells, fat-storing cells, and endothelial cells, are able to produce NO under adequate stimulation (14, 56, 57, 58). However, whether NO is cytoprotective or cytotoxic in this organ is still a matter of controversy (53, 59).

In the course of our work, we found evidence for elevated production of NO in both WT and IL-4-/- mice during infection. Plasma NO levels in WT mice and in vitro NO production from splenocytes of WT and IL-4-/- mice increase during the early stages of acute infection (Fig. 3Go and Ref. 6) but in WT animals diminish at later time points following the development of a strong Th2 response. In contrast, systemic levels of NO remain elevated in infected IL-4-/- mice up until the time that these animals die. Systemic NO could presumably be coming either from the diseased tissue, such as the liver or intestine, or from reactive lymphoid organs. The finding of iNOS mRNA in hepatic samples plus iNOS protein in granulomas supports the view that NO is being made in quantity within the liver itself. It is possible that exposure to parasite eggs induces the production of NO in an environment in which cells have been primed by adult worm Ags to produce IFN-{gamma} and TNF-{alpha} (3, 4, 46); mRNA for both of these cytokines were found in diseased liver.

We observed that the absence of NO during acute schistosomiasis leads to increased hepatic damage characterized by hepatocyte apoptosis and increased hepatocellular enzyme release. Our findings are supportive of the proposed protective role for NO in preventing hepatocyte damage induced by TNF-{alpha} and oxygen radicals (45, 53, 60, 61, 62, 63, 64, 65, 66). In several models of hepatic injury, tissue damage is exacerbated in the absence of NO and ameliorated following TNF-{alpha} neutralization. Our study, using a parasitic infection to induce hepatic injury, also suggests a cytoprotective effect of NO. During schistosomiasis, hepatic TNF-{alpha} mRNA transcripts (Fig. 4Go; Refs. 47 and 48) and protein levels (L.R.B. and E.J.P., unpublished observations) are significantly increased on arrival of eggs in the liver. NO production is also elevated at this time; hepatic iNOS mRNA transcripts are up-regulated and cells recruited to form granulomas around trapped eggs stain positively with anti-iNOS Abs. Therefore, we postulate that the observed up-regulation of iNOS mRNA is necessary to prevent severe hepatic damage at the time when eggs first arrive in the liver where they induce the production of potentially hepatotoxic cytokines such as TNF-{alpha}. At this time the infected host has yet to mount a T cell response against egg Ags. Thus NO production may represent an innate protective response to the egg. This view is supported by the observation that hepatic iNOS expression is up-regulated in SCID mice. Taken together our data argue for a host protective role of NO during the innate immune response to parasite eggs in the liver.

Why a lack of NO is more detrimental to liver health in infected IL-4-/- mice than in infected WT mice is unclear. The severe morbidity that develops in the absence of IL-4 is not associated with the significant changes in liver function and pathology detected using the techniques described here. Rather, in IL-4-/- mice the intestine is the site of altered pathologic changes that differ from those seen in infected WT mice (6). The pathologic changes that develop in the hepatocytes of AMG-treated infected IL-4-/- mice are highly reminiscent of those that occur in infected nude or T cell-depleted mice. T cell responses are clearly compromised in infected IL-4-/- mice (Figs. 7Go and 8Go, and J. Pedras, L. Rosa, and E. Pearce, manuscript in preparation), raising the possibility that in the absence of a robust egg-specific T cell response, NO produced as part of an innate response assumes a role of central importance in protecting the liver.

The absence of NO had no significant effects on the in vitro cytokine production by splenocytes of infected WT mice following anti-CD3 stimulation. This lack of effect on the immune response is in contrast to previous reports suggesting that NO down-regulates IFN-{gamma} and TNF-{alpha} in a negative feedback mechanism (28, 49). Similarly, when we compared cytokine production by splenocytes from infected IL-4-/- mice following AMG treatment, we detected no significant differences in cytokine levels aside from a further impairment of IL-10 production in the absence of NO; presumably lower production of the antiinflammatory cytokine IL-10 could lead to exacerbated disease and account for some of the accentuated cachexia observed following AMG treatment in these mice.

The functions of NO in various disease processes have been studied experimentally using inhibitors of NOS. Unfortunately most inhibitors are nonselective, inhibiting all three isoforms and ultimately interfering with normal physiological functions. In our study we used AMG, which is regarded as 10- to 100-fold more selective for iNOS compared with constitutively expressed nNOS and eNOS (30, 31, 32, 33, 34). AMG is the most commonly used iNOS inhibitor and despite other biochemical actions has been found not to be toxic per se (67, 68, 69). In our hands, uninfected animals treated with AMG show no sign of morbidity and no histological evidence of hepatic damage. Similarly, AST levels, used to indicate hepatocellular damage, are not increased in these animals corroborating further the lack of a direct hepatotoxic effect of AMG treatment alone. Therefore, we conclude that the severe hepatic damage associated with AMG treatment in this study is due to the absence of inducible NO during acute schistosomiasis, and not to an inherent hepatotoxicity of AMG. We hypothesize that NO may be playing a host protective role in the liver at the time this organ cytokine milieu is still type 1 as shown by elevated levels of IFN-{gamma}, TNF-{alpha}, and IL-1ß (Fig. 4Go, and L.R.B. and E.J.P., unpublished observations). This view is further supported by the finding that AMG treatment had no measurable impact on chronically infected WT mice, which do not have elevated systemic NO levels, but which have severely damaged livers. Taken together the data support the view that the effects of AMG treatment are due to the inhibition of NO production and not to any direct hepatotoxic effect.

We hypothesize three potentially protective roles for NO at the time the liver is first exposed to eggs: 1) by inhibiting TNF-{alpha}-dependent activation of caspase 3, a protease mediator of TNF-{alpha}-induced death (70), NO may prevent hepatocyte apoptosis (45, 63, 64, 66) induced by the increased levels of TNF-{alpha} during infection (47, 48); 2) NO may protect against damage mediated by reactive oxygen species (61, 62, 65), which may become elevated during infection (71); and 3) NO may be maintaining hepatocellular viability by limiting intrahepatic thrombosis, causing vasodilation and thereby promoting organ perfusion (61). We are in the process of addressing these issues to determine which if any is correct.


   Acknowledgments
 
Dr. A. W. Cheever performed the measurements of circumova granulomas in infected mice presented in this study. Dr. A. Alcaraz helped with histological examinations of tissues. We thank Tony Marshall and Drs. Eric Denkers, Padraic Fallon, Anne LaFLamme, and Phil Scott for helpful discussion.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI32573 (to E.J.P.). L.R.B. was supported by National Research Service Award AI-09512 while performing the work described in this manuscript and is now a recipient of a Burroughs Wellcome Hitchings Elion Fellowship. Schistosome life stages for this work were supplied through National Institutes of Health-National Institute of Allergy and Infectious Diseases Contract N01-AI-55270. Back

2 Current address: Department of Pathology, Cambridge University, Cambridge, U.K. Back

3 Address correspondence and reprint requests to Dr. Edward J. Pearce, Department of Microbiology and Immunology, C5-165 VMC, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. E-mail address: Back

4 Abbreviations used in this paper: NOS, NO synthase; nNOS, neuronal NOS; eNOS, endothelial NOS; WT, wild type; AMG, aminoguanidine; PAS, periodic acid schiffs; L-NMMA, N{omega}-monomethyl-L-arginine monoacetate salt; HPRT, hypoxanthine-guanine phosphoribosyl transferase; AST, aspartate aminotransferase. Back

Received for publication May 18, 1999. Accepted for publication August 16, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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