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Department of Medicine,1 Pathology, University of California, San Diego, La Jolla, California 92093,2 Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021393
Received 7 March 2001/ Returned for modification 2 May 2001/ Accepted 9 October 2001
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ABSTRACT |
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INTRODUCTION |
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Infections with Giardia spp. are usually self-limited in immunocompetent individuals, indicating the presence of effective host defense mechanisms against this strictly luminal parasite, although chronic giardiasis with continued cyst excretion occurs in some individuals with no apparent immunodeficiency (15, 25). Secretory antibodies against Giardia spp. are presumed to play a central role in clearance of this parasite from the intestinal tract (1, 14). However, direct evidence is lacking, and very little is known about the physiologic functions of specific isotypes in clearing Giardia infection. Even the overall role of B cells in antigiardial defense has not been defined unequivocally, as suggested by the conflicting reports on their importance in immunity against Giardia spp.
Several studies suggest an important role for B cells in clearing Giardia infection. For example, infections of humans with Giardia lamblia or of mice with Giardia muris result in the production of antigiardial antibodies of the immunoglobulin A (IgA), IgM, and IgG isotypes in mucosal secretions and serum, and specific antibody production correlates with giardial clearance (11, 14, 18, 25, 38). Such antibodies reach their targets in vivo, since antigiardial IgA and IgG antibodies coat trophozoites in Giardia-infected mice (18). Mice depleted of B cells by treatment with anti-IgM antibodies and mice with X-linked immunodeficiency, which have a defect in B-cell development and function, are unable to clear G. muris infection (36, 37). B-cell-deficient mice generated by gene targeting appear to be unable to completely clear infections with the human pathogen, G. lamblia (41).
In contrast, other data suggest that B cells have only a limited role in antigiardial immunity. For example, mice with X-linked immunodeficiency can develop acquired immunity against secondary challenge with G. muris (35). Moreover, a recent study reported little difference in parasite clearance between wild-type littermates and B-cell-deficient mice infected with either G. lamblia or G. muris (33). Patients deficient for the production of IgA, the major immunoglobulin isotype in mucosal secretions, appear to have an only slightly increased incidence of Giardia infections (22). These data suggest that B-cell-independent host defenses against Giardia may play an important role in controlling and clearing infection.
Potential candidates for such defenses have been identified by in vitro studies. For example, defensins, small antimicrobial peptides produced by Paneth cells in the small intestine, can kill G. lamblia trophozoites in vitro (2). Nitric oxide, which can be produced by intestinal epithelial cells, inhibits proliferation and differentiation of G. lamblia trophozoites in vitro (13). However, the role of these potential host defenses against Giardia infection in vivo is not known.
Taken together, the preponderance of evidence suggests that B cells are needed for effective Giardia clearance (36, 37, 41). However, no direct evidence has been reported on the physiologic role of specific immunoglobulin isotypes in clearing Giardia infection, particularly of IgA and IgM, which are normally secreted into the intestinal lumen. To determine the physiologic importance of secretory IgA and IgM antibodies in antigiardial host defense, we used gene-targeted mice lacking IgA-expressing B cells, IgM-secreting B cells, or all B cells as controls and challenged the mice with G. muris or G. lamblia. The studies show that IgA is the major host defense mechanism against Giardia infection. B-cell-dependent but IgA-independent and B-cell-independent antigiardial defenses exist but play less important roles in controlling Giardia infection. In contrast, secreted IgM antibodies play no unique role in clearing Giardia infection.
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MATERIALS AND METHODS |
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exon, the S switch region, exon 1, and part of exon 2 of the immunoglobulin chain gene (17). These mice are completely deficient in IgA production but have modestly increased serum levels of IgM and IgG (17). Mice deficient in secreted IgM (secreted IgM KO mice) were generated by deleting the µs exon and its three downstream poly(A) sites in the immunoglobulin µ chain gene and replacing it with a cDNA fragment encoding the µm exons already spliced to the Cµ4 exon (6). These mice do not secrete IgM but still express surface IgM and IgD and undergo class switching to express other isotypes (6). As controls for B-cell KO mice, we used C57BL/6J mice (C57 mice). As controls for IgA KO mice and secreted IgM KO mice, we used wild-type littermate controls with a similar B6 x 129 genetic background as the KO mice, as well as (B6129F1/J)F2 mice, which are F2 hybrids of B6129F1/J mice derived by mating C57BL/6 and 129/J mice. No significant differences in clearing G. muris infection were observed between these two strains of mice, and the data from these mice are reported together (under the designation B6129 mice). Mice were bred and maintained at the University of California-San Diego (UCSD) animal facilities under specific-pathogen-free conditions. All animal studies were approved by the UCSD Animal Subjects Committee.
To confirm the phenotype of the different KO mice, we determined plasma immunoglobulin levels by enzyme-linked immunosorbent assay (ELISA). The data from male and female mice did not differ significantly and were combined for these studies. As expected, the B-cell KO mice had undetectable levels (<5 µg/ml) of plasma IgM, IgG, or IgA, while the C57 control mice had 271 ± 55 µg of IgM, 1,476 ± 382 µg of IgG, and 73 ± 11 µg of IgA per ml in the plasma (all values are means ± standard error of the mean [SEM], n 
6). The IgA KO mice had 137 ± 14 µg of IgM, 3,802 ± 437 µg of IgG, and undetectable levels (<5 µg/ml) of plasma IgA per ml. The secreted IgM KO mice had undetectable levels (<5 µg) of IgM, 2,673 ± 473 µg of IgG, and 229 ± 31 µg of IgA per ml. The B6129 control mice had 66 ± 17 µg of IgM, 913 ± 204 µg of IgG, and 122 ± 50 µg of IgA per ml of plasma. These data show that the plasma levels of IgM and IgG were 2.1- and 4.2-fold higher in IgA KO mice compared to B6129 controls (P < 0.001 by Student’s t test), which confirms previous observations in IgA KO mice (17), whereas the plasma levels of IgG and IgA were 2.9- and 1.9-fold higher in secreted IgM KO mice compared to B6129 controls (P < 0.001 by Student’s t test).
Infections. G. muris cysts were obtained from M. Belosevic (University of Alberta, Edmonton, Canada), and maintained by passage through mice every 4 to 8 weeks. Prior to infection, cysts were purified from fresh stool samples collected no more than 2 days before infection. Mice were infected orally by gavage with 104 G. muris cysts in 0.2 ml of water. Before secondary infections, all mice were treated orally with 8 mg/mouse/day of metronidazole for three consecutive days to clear any G. muris infection remaining from the primary challenge. Clearance was confirmed by the absence of G. muris cysts in fecal cyst counts. Mice had free access to food and water during the experiments.
G. lamblia GS/M-83-H7 was obtained from the American Type Culture Collection (ATCC 50581) and grown in TYI-S-33 medium as described (2, 10). Prior to infection, trophozoites from confluent cultures in tissue culture flasks were detached by chilling on ice for 10 min, washed, and resuspended in TYI-S-33 medium at 5 x 107/ml. Mice were starved overnight, infected orally by gavage with 107 G. lamblia trophozoites in a 0.2-ml volume, and returned to solid foods 2 to 3 h later. To render the different mouse strains equally infectible with G. lamblia (34), mice were given 1.5 mg of neomycin per ml in the drinking water for 24 h before infection and throughout the entire infection period.
G. muris cyst preparation and counts. Feces were collected from individual mice over a 2- to 3-h period, weighed, and homogenized in 20 ml of phosphate-buffered saline (PBS). G. muris cysts were enriched from homogenized stool samples by centrifugation over a 1 M sucrose gradient (3, 32). Briefly, 20 ml of homogenized stool was layered on top of a 10-ml cushion of a 1 M sucrose solution, and samples were centrifuged at 500 x g for 15 min at 4°C. The interface containing the G. muris cysts was collected in a 10-ml volume, diluted with 25 ml of PBS, and centrifuged at 500 x g for 15 min at 4°C. The pellet was resuspended in 1 ml of PBS, and cysts were counted in a counting chamber using a phase-contrast microscope. The detection limit of the G. muris cyst assay was 2 x 103 cysts/g of feces.
Trophozoite counts. For trophozoite counts in G. muris-infected mice, the small intestine was removed and cut into seven equal-sized pieces, numbered 1 to 7 from orad to caudad. Each segment was opened longitudinally in 4 ml of PBS, placed on ice for 10 min, and agitated on a Vortex mixer to detach trophozoites from the mucosa. Counts of viable G. muris trophozoites were performed without further purification in a hemacytometer using a phase-contrast microscope. Viability was apparent as tumbling whole-cell movements for unattached trophozoites or flagellar movements for attached trophozoites. Total trophozoite load in the small intestine was determined by adding the numbers of trophozoites in each of the seven individually counted segments. The detection limit of the assay was 2 x 103 G. muris trophozoites/small intestine.
To evaluate G. muris trophozoite distribution along the orad-caudad axis of the small intestine, we first expressed the number of trophozoites in each of the seven equally spaced segments along the small intestine as a percentage of the total trophozoite load in the small intestine. We then calculated, for each segment, the cumulative percentage of trophozoite load, i.e., the sum of the percentages of the specific segment and all the segments orad of that segment. Subsequently, the midpoint of the infection was determined mathematically, i.e., the segment number (or fraction thereof) for which 50% of all trophozoites were on the orad side and 50% were on the caudad side of the small intestine. To calculate this, we fit a curve to the data points representing segment numbers versus cumulative trophozoite percentages by iterative approximation using the three-parameter sigmoid function y = a/{1 + exp[-([x - xo]/b)]} and the Fit Curve function of the computer program Sigmaplot 4.0 (SPSS Inc., Chicago, Ill.). Based on the sigmoid function describing the best curve fit for a specific set of data points, we determined the x value (i.e., the segment number representing the midpoint of infection) for which y = 50% (i.e., a cumulative G. muris trophozoite distribution of 50%).
For G. lamblia counts, the entire small intestine was removed and opened longitudinally in 10 ml of PBS, placed on ice for 10 min, and agitated on a Vortex mixer to detach trophozoites from the mucosa. The intestinal contents were allowed to settle for 5 min on ice, and the supernatant (without the intestine and large pieces of debris) was removed and centrifuged at 1,000 x g for 15 min at 4°C. The pellet was resuspended in 1 ml of PBS, and G. lamblia trophozoites were counted in a hemacytometer using a phase-contrast microscope. The detection limit of the assay was 5 x 102 G. lamblia trophozoites/small intestine.
Data analysis. Cyst and trophozoite counts were log10 transformed, and means and SEM were calculated from the log values. Samples without detectable cysts or trophozoites were assigned a log value equivalent to half of the detection limit for each assay. Differences between groups of mice were compared by the Mann-Whitney rank sum test or Student’s t test, as appropriate. Differences with a P value of <0.05 were considered significant. Results from males and females were combined for all experiments, since no significant differences were found in stool cyst output (G. muris) or trophozoite numbers in the small intestine (G. muris and G. lamblia) at 1 and 7 weeks after infection.
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RESULTS |
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Efficient clearance of primary G. muris infection depends on IgA.
Several immunoglobulin isotypes could mediate the role of B cells in host defense against Giardia, since antigiardial antibodies of the IgA, IgM, and IgG isotypes are detectable in Giardia-infected mice (11, 18, 38). Since IgA is the most abundant immunoglobulin isotype in mucosal secretions in the intestine, we tested the role of IgA in clearing G. muris infection, using IgA KO mice (17) and B6129 littermate controls. Like C57 controls, B6129 control mice had peak stool cyst output and trophozoite numbers at 1 to 2 weeks after infection and began to clear the infection thereafter (Fig. 2). By 5 to 7 weeks after infection, cysts were undetectable in the stool, which represented a 
100-fold decrease compared with maximal levels. Mean trophozoite numbers in the small intestine 7 weeks after infection were >1,000-fold lower than the maximal levels at 1 week, and a substantial proportion (5 of 12, 42%) of B6129 mice had no detectable trophozoites.
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100-fold-higher trophozoite numbers and 10-fold-higher stool cyst output compared to B6129 controls (Fig. 2) and continued to shed cysts in the stool over at least 4 months (e.g., log10 cyst number/g of feces of 5.59 by 17 weeks after infection). Nonetheless, mean trophozoite numbers were 21-fold lower in IgA KO mice at 7 weeks compared to maximal numbers (P < 0.001 by rank sum test). This indicates that although IgA KO mice could not eradicate G. muris infection, they had some ability to control the infection, which was significantly greater than the residual ability of B-cell KO mice to control infection (log10 trophozoite numbers of 6.59 ± 0.12 in B-cell KO mice versus 5.96 ± 0.24 in IgA KO mice at week 7; P < 0.05 by rank sum test). Secreted IgM is not required for clearing primary G. muris infection. Since IgA KO mice showed a significantly greater ability to control G. muris infection than B-cell KO mice, we investigated whether another immunoglobulin, secreted IgM, may play a role in controlling Giardia infection in mice. Secreted IgM, like IgA, is transported into the intestinal lumen by the polymeric immunoglobulin receptor on intestinal epithelial cells. The course of G. muris infection in mice deficient in secreted IgM but not membrane IgM or other isotypes (6) did not differ significantly from that in B6129 littermate controls (Fig. 3), indicating that secreted IgM has no unique role in clearing G. muris infection.
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At 1 week after infection, immunized C57 controls were almost completely protected from secondary challenge, since trophozoite numbers in the small intestine (Fig. 4, top left panel) and stool cyst output (data not shown) were >100-fold lower in immunized relative to nonimmunized mice. Immune protection in C57 mice was efficient but not complete, as small numbers of cysts in the stool (data not shown) and trophozoites in the small intestine (Fig. 4) were observed occasionally in some of the immunized mice throughout the course of the infection. In contrast to control mice, immunized B-cell KO mice showed only a minor (2.0-fold) difference in the trophozoite numbers in the small intestine after 1 week compared with nonimmunized mice (Fig. 4, bottom left panel). These data demonstrate that B cells are absolutely required for developing acquired immune protection against secondary challenge with G. muris.
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B-cell-dependent host defenses affect G. muris trophozoite distribution along orad-caudad axis of small intestine. Further analysis of the trophozoite counts showed that host immune defenses influenced the distribution of G. muris trophozoites along the orad-caudad axis of the small intestine. For both groups of control mice, C57 (Fig. 5) and B6129 (data not shown), the calculated midpoint of infection was in segment 2.7 to 2.9 early in the infection (week 1) and subsequently moved progressively orad. In contrast, in B-cell KO mice, the midpoint of the infection was significantly more caudad compared to C57 controls at 1 week after infection and moved further caudad at 3 weeks after infection (Fig. 5). The orad-caudad trophozoite distribution in IgA KO mice followed a pattern similar to that in B-cell KO mice, whereas the distribution in secreted IgM KO mice was comparable to that in the B6129 control mice (data not shown). Taken together, these data show that B-cell-dependent immune defenses not only control total G. muris trophozoite load, but also significantly affect the trophozoite distribution along the orad-caudad axis of the small intestine.
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DISCUSSION |
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The data reported here demonstrate that antibodies of the IgA isotype are required for effective clearance of G. muris and G. lamblia from the murine host. This role of IgA may be expected, given that it is the most abundant immunoglobulin in mucosal secretions and antigiardial IgA antibodies are elicited by infection (18, 38). IgA is likely to exert its antigiardial functions through "immune exclusion" (e.g., immobilization or detachment of trophozoites from epithelium) rather than direct killing, since antigiardial IgA antibodies do not kill G. muris trophozoites in the presence or absence of complement (19), although one study suggested that antigiardial IgA may exert cytotoxic effects on G. lamblia (39).
Clinical reports on the incidence of Giardia infections in patients with IgA deficiency have provided conflicting results, as some studies found an increased incidence of infections, while others found that such patients are not more susceptible (9, 22, 42). Patients with IgA deficiency are usually defined by serum IgA levels of <0.05 g/liter (normal levels are 0.5 to 3.5 g/liter), but many, if not most, IgA-deficient patients have low but detectable levels of serum IgA antibodies (9, 30) and can produce IgA antibodies at near-normal levels in vitro (8, 23). This is in contrast to the complete lack of IgA production in IgA-deficient mice, which lack crucial gene segments for expressing IgA heavy chains (17). The simplest interpretation of both clinical and experimental data would be that although IgA is required for clearing Giardia infection, IgA levels well below normal are sufficient. Alternative interpretations are possible, e.g., host defenses against Giardia in mice and G. lamblia in humans differ in regard to IgA dependency, or IgA-deficient patients but not mice can develop compensatory host defenses against Giardia infection.
We found that secreted IgM (in contrast to membrane IgM, which is required for normal B-cell development and, therefore, antigiardial defense) had no unique role in clearing G. muris infection. This is in contrast to intraperitoneal infection with enteric bacteria after ligation and puncture of the cecum, where secreted IgM plays a critical role in host defense against infection (7). Antibodies of the IgM isotype, particularly natural antibodies, often recognize phylogenetically conserved structures, such as carbohydrates, phospholipids, or nucleic acids (7), suggesting that recognition of such structures may not be important for controlling giardial infection in the intestinal lumen. It is also possible that levels of secreted IgM in the intestinal lumen are too low to be important in the presence of normal IgA levels. Nonetheless, IgM may be able to cooperate with, or possibly compensate for, other host defenses, such as IgA, in antigiardial host defense. This could be particularly important if crucial antigiardial host defenses are not operative, as in IgA-deficient patients. Such a compensatory role of secreted IgM could explain why IgA KO mice have a greater residual capacity to control Giardia infection than B-cell KO mice. However, the latter finding could also be explained by a role of IgG antibodies, since IgG can be transported from the basolateral to the apical side of polarized intestinal epithelial cells in vitro (12) and can be detected in the intestinal lumen in vivo (18, 29, 31).
Our findings of a central role of B cells and IgA in antigiardial host defense differ from those in a recent report by Singer and Nash, in which B-cell KO mice were shown to control G. lamblia infection as well as normal littermate controls after 4 weeks, suggesting that B cells played a limited role, if any, in controlling acute G. lamblia infection in those studies (33). Although it is difficult at this point to reconcile the data, differences in the experimental design may be responsible for the apparent discrepancy. For example, we used higher G. lamblia inocula, which yielded 10- to 100-fold-higher peak infectious loads 1 week after infection (33). It is possible that B cells are required for controlling higher infectious burdens, while they play a less important role in controlling lower initial and peak infectious loads of Giardia. Furthermore, we treated mice with a nonabsorbable oral antibiotic, neomycin, throughout the infections, which minimizes differences in G. lamblia susceptibility of different strains of mice but has no direct effects on G. lamblia, as a previous study had suggested that such susceptibility differences are largely due to differences in the intestinal microbiota (34). The antibiotic treatment protocol in our studies might have helped to reveal host defenses directed against Giardia itself rather than those that might affect Giardia indirectly by controlling the composition or density of the intestinal microbiota. In any case, the data from Singer and Nash support the presence of B-cell-independent antigiardial host defenses, which is consistent with our finding that B-cell KO mice had some, albeit limited, capacity to control G. muris infection. The relative importance of B-cell-dependent and -independent host defenses for controlling and ultimately clearing Giardia infection is likely to depend on multiple parasite factors (e.g., peak infectious burden) and host factors (e.g., intestinal microbiota), whose specific roles in antigiardial host defense remain to be defined.
Giardia spp. mostly colonize the duodenum and jejunum in humans and mice, although trophozoites can occasionally be found in the ileum, colon, stomach, and bile ducts (3, 26). The characteristic distribution of trophozoites along the orad-caudad axis of the intestine is likely to result from a complex interplay of host and parasite factors. On the parasite side, motility, the ability to attach and detach from the intestinal epithelium and mucus, proliferative capacity, the ability to differentiate into cysts, and the need for optimal nutrient access are important factors in determining their localization in the small intestine (16, 43). On the host side, physiologic functions (e.g., intestinal motility and secretory responses) and anatomic factors are likely to be important in controlling giardial numbers in different regions of the small intestine. For example, few trophozoites are present upstream of the entry point of the common bile duct into the duodenum, supporting an important role for bile and/or pancreatic secretions in giardial survival in the host (16). Our data show that specific antigiardial host defenses, i.e., antibody production, also play a significant role in determining trophozoite distribution along the orad-caudad axis of the small intestine. The mechanisms underlying this effect are not clear but could relate to direct effects (e.g., stronger B-cell defenses in the caudad portions of the small intestine) or indirect effects (e.g., a role of B cells in intestinal motility or in controlling the intestinal microbiota).
Our data and other experimental and clinical studies indicate that effective acquired immune defenses exist against Giardia which can attenuate, if not prevent, reinfections of the same host. These defenses may not be complete, since reinfections are relatively common in areas with endemic giardiasis, which may be related to differences in infecting giardial strains or antigenic variation (24). Nonetheless, antigiardial immunity elicited by infection with a specific Giardia strain or exposure to Giardia extracts can reduce severity and duration of repeated infections with the same or a different strain (25, 28), which constitutes the rationale for developing antigiardial vaccines. Successful vaccination trials have been reported in dogs and cats, validating the feasibility of antigiardial vaccination (27, 28), although the underlying immune mechanisms are not well understood. The findings reported here indicate that IgA-dependent host defenses can overcome antigenic variation as an immune evasion strategy of Giardia spp. in vivo (5, 24), possibly by targeting nonvariable antigens. Therefore, our data suggest that vaccination efforts should be targeted mostly towards stimulating antigiardial IgA.
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ACKNOWLEDGMENTS |
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We are grateful to Tom Blaze for invaluable help with the animal experiments and Jennifer Smith for excellent technical support.
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FOOTNOTES |
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) and wild-type littermate C57 control mice (•) were infected orally with 104 G. muris cysts. Infection intensity was assessed at the indicated times after infection by determining stool cyst output (top panel) and total trophozoite numbers in the small intestine (bottom panel). All data are means ± SEM from nine or more mice for each data point. The detection limits of the assays are indicated by the gray line in each panel. Asterisks indicate values of B-cell KO mice that are significantly different from controls at the same time point (P < 0.01 by rank sum test).
