The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kerepesi, L. A.
Right arrow Articles by Abraham, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kerepesi, L. A.
Right arrow Articles by Abraham, D.
The Journal of Immunology, 2006, 176: 4315-4322.
Copyright © 2006 by The American Association of Immunologists

Complement Component C3 Is Required for Protective Innate and Adaptive Immunity to Larval Strongyloides stercoralis in Mice1

Laura A. Kerepesi*, Jessica A. Hess*, Thomas J. Nolan{dagger}, Gerhard A. Schad{dagger} and David Abraham2,*

* Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107; and {dagger} Department of Pathobiology, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA 19107


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
This study examines the role of complement components C3 and C5 in innate and adaptive protective immunity to larval Strongyloides stercoralis in mice. Larval survival in naive C3–/– mice was increased as compared with survival in wild-type mice, whereas C3aR–/– and wild-type mice had equivalent levels of larval killing. Larval killing in naive mice was shown to be a coordinated effort between effector cells and C3. There was no difference between survival in wild-type and naive C5–/– mice, indicating that C5 was not required during the innate immune response. Naive B cell-deficient and wild-type mice killed larvae at comparable levels, suggesting that activation of the classical complement pathway was not required for innate immunity. Adaptive immunity was equivalent in wild-type and C5–/– mice; thus, C5 was also not required during the adaptive immune response. Larval killing was completely ablated in immunized C3–/– mice, even though the protective parasite-specific IgM response developed and effector cells were recruited. Protective immunity was restored to immunized C3–/– mice by transferring untreated naive serum, but not C3-depleted heat-inactivated serum to the location of the parasites. Finally, immunized C3aR–/– mice killed larvae during the adaptive immune response as efficiently as wild-type mice. Therefore, C3 was not required for the development of adaptive immunity, but was required for the larval killing process during both protective innate and adaptive immune responses in mice against larval S. stercoralis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The complement system is an integral component of both the innate and adaptive immune responses in the defense against invading pathogens (1, 2, 3). Complement must be activated through either the classical, alternative, or lectin pathways, which results in a complex multicomponent cascade of proteins with wide-ranging functions in the control of pathogens. One of these proteins, C3, is required in all three activation pathways, and has been shown to function in both innate and adaptive protective immunity to pathogens (1, 2, 3). C3 also acts as a bridge between innate and adaptive immunity, as mice deficient in C3 have an impaired ability to mount humoral immune responses to T-dependent Ags (4, 5), especially when Ag dose is limiting (4, 6). Additionally, C3 has been shown to influence the development of the Th2 immune response (7, 8). Two molecules result from the activation and cleavage of C3: C3a, which acts as a chemoattractant, and C3b, which acts as an opsonin (9). One of the key downstream proteins resulting from complement activation is C5. Upon activation, this molecule is converted into C5a, which acts as a chemoattractant for cells including neutrophils (2, 10), and C5b, which initiates the formation of the membrane attack complex (11).

Infection with Strongyloides stercoralis, an intestinal nematode of humans, is initiated by the penetration of the infective third-stage larvae (L3).3 The infection induces Th2 immune responses in humans (12, 13), and protective immunity to L3 in humans operates through an Ab-dependent mechanism (14). In mice, the immune mechanisms capable of killing larval S. stercoralis have been studied on a variety of mouse backgrounds, including C57BL/6J, BALB/cJ, BALB/cByJ, and CBA/J (15, 16, 17). Innate protective immunity, evaluated 3 days after infection of naive mice, was shown to be IL-5 and eosinophil dependent (18). Adaptive protective immunity developed after immunization of mice with live larvae and resulted in ~90% of challenge larvae killed within 1 day postchallenge (15). Several immune components have been identified to be required for the development and functioning of the protective adaptive immune response, including CD4+ Th2 cells (17) and B cells (16) producing IgM (14, 19, 20). The rapid killing of larvae by the adaptive immune response apparently also required neutrophils as effector cells (14, 20).

Previous studies have also shown complement to be required in both innate and adaptive protective immune responses to S. stercoralis. Complement enhanced the ability of naive neutrophils from humans to bind to the surface of S. stercoralis L3 in vitro, and cellular attachment was prevented when activation of the complement pathways was inhibited (21). Complement was also required for larval killing by the adaptive immune response in mice; protective immunity was eliminated from immunized mice by treatment with cobra venom factor to eliminate C3 at the time of the challenge infection (14, 19, 20). The objectives of this study were to: 1) evaluate the relative roles of the complement components C3 and C5 in larval killing during protective innate and adaptive immune responses against larval S. stercoralis; 2) determine whether C3 or C5 was required as a bridge between the innate and the adaptive immune response; 3) identify the functional subcomponents of C3 and/or C5 that effectuate larval killing, and thereby identify the mechanism through which complement functions in protective immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and parasites

C57BL/6J, BALB/cJ, C3–/– (B6.129S4-C3tm1Crr/J on the C57BL/6 background), µMT (B6.129S2-Igh4m1Cgn/J, B cell deficient on the C57BL/6 background), B10.D2/oSnJ (C5 deficient), and B10.D2/nSnJ (C5 sufficient) mice were purchased from The Jackson Laboratory. C3aR–/– mice (22) on the BALB/c background were provided by C. Gerard and A. Humbles (Children’s Hospital, Boston, MA). C3–/– and C3aR–/– mice were bred at Thomas Jefferson University. All mice were housed in filter-top microisolator boxes under light- and temperature-controlled conditions. S. stercoralis L3 were obtained from the cultures of fresh stools from a laboratory dog infected with the parasite, according to methods previously described (15). Larvae were collected from 7-day charcoal cultures, washed, and resuspended in culture medium.

Challenge infections

Mice were challenged with 50 S. stercoralis L3 contained within diffusion chambers that were implanted s.c. on the dorsal flank of the mice. Construction of diffusion chambers covered with either 0.1- or 2.0-µm Isopore membranes (Millipore) followed previously described methods (15). Diffusion chambers were sterilized by exposure to 100% ethylene oxide and aerated for 12 h. Implanted diffusion chambers were removed from the mice 1 day after challenge in experiments studying adaptive immunity or 3 days after challenge in experiments studying innate immunity. Larval viability was determined based on motility and morphology. Cells recovered from the diffusion chambers were quantitated, centrifuged onto slides through the use of a Cytospin 3 centrifuge (Thermo Shandon), and stained for differential counts with DiffQuik (Baxter Healthcare).

Complement depletion was accomplished by i.p. injection of cobra venom anti-complementary protein factor (Sigma-Aldrich) into mice 3 days before challenge infection and on the day of challenge, as previously described (19). The amount of cobra venom factor given to the mice was 400 µg/kg body weight based on published protocols (23).

Serum transfer and serum reconstitution

For serum transfer experiments, serum from immunized and naive mice was injected into mice at the time of challenge into the s.c. pocket surrounding the implanted diffusion chamber. Serum from immunized wild-type and C3–/– mice was pooled from each mouse in the group so each mouse was equally represented in the serum pool. Each serum recipient mouse was given 100 µl of serum brought up to 200 µl with 100 µl of PBS. For C3 reconstitution experiments, serum from naive mice was untreated or heat inactivated (56°C for 30 min), and 100 µl of serum was placed into the diffusion chamber with the larvae at the time of challenge infection.

Ag preparation

Deoxycholate (DOC)-soluble larval proteins were prepared by previously described methods (24). Briefly, L3 were washed in PBS supplemented with antibiotics and stored at –80°C. L3 were thawed and homogenized for 1 h in the presence of a protease inhibitor mixture (Sigma-Aldrich) and sonicated. Homogenized L3 were incubated in PBS at 4°C overnight with continuous mixing. PBS-soluble proteins were removed, and the PBS-insoluble proteins were resuspended in 20 mM Tris-HCl/0.5% DOC (Sigma-Aldrich) and mixed overnight at 4°C. The DOC-soluble proteins were then dialyzed against PBS overnight, concentrated, and filter sterilized. Protein concentration was quantitated by a Micro BCA Protein Assay kit (Pierce) and stored at –80°C.

Spleen cell stimulation

Spleens from naive and immunized mice were aseptically removed 1 wk after challenge and made into single cell suspensions. Cells were cultured in a 96-well plate at 2 x 106/well. Spleen cells were stimulated with anti-CD3 mAb (BD Pharmingen), coated with anti-CD3 mAb at 0.5 µg/ml for 2 h at 37°C, and washed with PBS. Cells were incubated at 37°C for 3 days, and supernatants were collected and frozen at –20°C.

Ab and cytokine ELISA

Nunc Maxisorp 96-well plates (Nunc) were coated overnight at 4°C with 50 µl of S. stercoralis-soluble L3 Ags at 10 µg/ml. Plates were blocked with borate blocking buffer solution (0.17 M boric acid, 0.12 M NaCl, 0.05% Tween 20, 0.25% BSA, 1 mM EDTA (pH 8.5)) at 37°C for 1 h. Wells were washed with distilled water, and test samples, diluted in borate blocking buffer solution, were placed in duplicate wells at serial dilutions and incubated at 37°C for 2 h. Appropriately matched biotinylated goat anti-mouse IgM (Vector Laboratories), IgG1, and IgG2a (BD Pharmingen) Abs were added, and plates were incubated at 37°C for 2 h. Plates were washed, and ExtrAvidin peroxidase (Sigma-Aldrich) was added for 30 min at room temperature, followed by the peroxidase substrate ABTS (Kirkegaard & Perry Laboratories); color reaction was measured at 405 nm.

Cytokine ELISA for IL-5 and IL-4 was performed using appropriately matched anti-IL-5 and anti-IL-4 mAbs for coating and capture Ab (BD Pharmingen), and the protocol was as above. An IFN-{gamma} kit (AN-18 monoclonal; BD Pharmingen) was used following manufacturer’s provided protocol. The 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) was used as the substrate; the reaction was stopped using 0.5 M H2SO4, and color reaction was measured at 450 nm.

Statistical analysis

Experiments consisted of five mice per group, unless otherwise noted. All experiments were performed at least twice, results were reproducible, and data shown are from one representative experiment. Statistical analysis of the data was performed using multivariate general linear hypothesis multifactorial ANOVA with Systat version 11 software (Systat). Fisher’s least significant difference test was performed for post hoc analyses. Values of p <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Role of C3 and C5 during the innate immune response

Naive C57BL/6 mice, treated with cobra venom factor to deplete C3, were infected with S. stercoralis L3. The larvae were implanted in cobra venom factor-treated and untreated mice for 1 or 3 days in diffusion chambers covered with 2.0-µm pore-size membranes that allow cellular ingress and egress. Larval survival in untreated mice decreased by 44% from day 1 to day 3, thereby demonstrating innate immune control of the infection. Survival of larvae in cobra venom factor-treated mice was equal to control mice on day 1 and was significantly greater than in controls on day 3, demonstrating a role for C3 in innate protective immunity (Fig. 1A). The requirement for C3 in the innate immune response was further examined using C3–/– mice. Naive wild-type and C3–/– mice were infected for 3 days with L3 contained within diffusion chambers constructed with either 2.0-µm pore-size membranes, which allowed cell entry, or 0.1-µm pore-size membranes, which prevented entry of cells into the larval microenvironment. Naive C3–/– mice had a diminished ability to kill L3 compared with wild-type mice when the larvae were contained within diffusion chambers covered with 2.0-µm pore-size membranes (Fig. 1B). The number of cells migrating into the diffusion chambers implanted in wild-type and C3–/– mice was equivalent (Fig. 1C), thus indicating that there was no cellular recruitment defect in the C3–/– mice in response to S. stercoralis L3 during the innate immune response. Preventing cells from entering the diffusion chambers through the use of 0.1-µm pore-size membranes resulted in equivalent larval survival in naive wild-type and C3–/– mice. Furthermore, larval survival in C3–/– mice challenged with L3 in 2.0- and 0.1-µm pore-size membrane-covered diffusion chambers was equal (Fig. 1B), suggesting that cells and complement function cooperatively during the innate immune response. In the absence of either C3 or cell contact, larval killing by the innate immune response was significantly decreased.


Figure 1
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 1. Innate immune response to larval S. stercoralis in the absence of C3. Larval survival during the innate immune response was evaluated in naive mice treated with cobra venom factor (CVF) on days 1 and 3 (A). *, Statistical significance (p ≤ 0.05) in parasite survival in untreated wild-type mice between days 1 and 3. **, Statistical significance (p ≤ 0.05) in parasite survival between cobra venom factor-treated and untreated wild-type mice on day 3. Larval survival in diffusion chambers covered with either 0.1- or 2.0-µm membranes implanted in naive C57BL/6 and C3–/– mice (B). *, Statistical significance (p ≤ 0.05) in parasite survival between C57BL/6 and C3–/– mice when L3 were implanted in diffusion chambers covered with 2.0-µm membranes. Cellular recruitment into diffusion chambers containing L3 implanted in naive C57BL/6 and C3–/– mice (C). Each experiment was performed at least twice; data shown represent the means and SDs of a minimum of five mice per group.

 
Natural Ab has been shown to be involved in the recognition of invading pathogens and in the activation of the classical complement pathway (25). Naive µMT mice, which intrinsically lack Ab, were challenged with L3 for 3 days to determine whether natural Ab was required in the innate immune response. Percentage of larval survival in C57BL/6J (49 ± 21) and µMT (51 ± 22) mice was equivalent. Activation of C3 results in the cleavage of the molecule into the functional components C3a and C3b. To test whether C3a was required for innate immunity, naive C3aR–/– mice were challenged with L3 within diffusion chambers covered with 2.0-µm pore-size membranes for 3 days. There was no difference in larval survival in BALB/cJ (52 ± 17) and C3aR–/– (52 ± 26) mice, nor was a difference seen in cell recruitment.

Naive C5-deficient mice were infected for 3 days with larval S. stercoralis to determine whether byproducts of the complement cascade downstream of the cleavage of C3 were required for larval killing during the innate immune response. C5-deficient mice eliminated larvae at the same rate as C5-sufficient wild-type control mice, indicating that the molecular cascade beyond C3 was not required for larval killing in the innate immune response (Fig. 2A).


Figure 2
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 2. C5 is not required for innate or adaptive protective immunity. Naive C5-sufficient wild-type and C5-deficient mice were challenged, and larval survival was ascertained (A). C5-sufficient and -deficient mice were immunized against infection and then challenged. Larval survival (B) and the total number of cells recruited into the diffusion chamber were analyzed after challenge infections (C). *, Statistical significance (p ≤ 0.05) between naive and immune. Each experiment was performed at least twice; data shown represent the means and SDs of a minimum of five mice per group.

 
Role of C3 and C5 during the adaptive immune response

C5-deficient mice were immunized with L3 of S. stercoralis to determine whether adaptive protective immunity would develop in these mice. Both immunized wild-type C5-sufficient and C5-deficient mice killed challenge infections within 24 h (Fig. 2B), indicating that C5 was not required for larval killing in the adaptive immune response. Additionally, there was no difference in cell numbers found in the diffusion chambers between immunized wild-type C5-sufficient and C5-deficient mice (Fig. 2C), indicating that C5a is not required for cell recruitment to the larval microenvironment in the adaptive immune response.

The role of C3 in the adaptive immune response to S. stercoralis was studied by immunizing C3–/– mice; protective immunity did not develop in the immunized C3–/– mice (Fig. 3A). Parasite-specific IgM and IgG1 levels were equivalent between immunized wild-type and C3–/– mice, whereas parasite-specific IgG2a responses were absent in the immunized mice (Fig. 3B). Therefore, there was no defect in the ability of B cells to produce Ab in C3–/– mice following immunization with S. stercoralis L3. To determine whether there was a qualitative change in the Ab response, serum collected from immunized wild-type and C3–/– mice was passively transferred to naive wild-type mice at the time and location of the challenge infection. Serum from immunized C3–/– mice transferred protective immunity to naive mice at levels equivalent to that seen with serum from immunized wild-type mice (Fig. 3C). Therefore, C3–/– mice do not have a quantitative or qualitative defect in their ability to mount a protective Ab response against S. stercoralis L3.


Figure 3
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. Adaptive protective immunity to larval S. stercoralis was absent in immunized C3–/– mice. Larval survival was assessed in immunized and naive C57BL/6 and C3–/– mice (A). Parasite-specific IgG2a, IgG1, and IgM responses were measured in immunized C57BL/6 and C3–/– mice (B). Larval survival was assessed to determine the ability of serum from C3–/– mice to passively transfer immunity to naive C57BL/6 mice (C). *, Statistical significance (p ≤ 0.05) between naive and immune. Each experiment was performed at least twice; data shown represent the means and SDs of a minimum of five mice per group.

 
The number and type of cells recruited into the diffusion chambers were evaluated to ascertain whether there was a defect in the effector cell response of immunized C3–/– mice. There was no difference in the total number of cells recruited into the diffusion chambers between immunized wild-type or C3–/– mice (Table I). Furthermore, there was no significant change in the number of neutrophils, eosinophils, or macrophages present in the diffusion chambers (Table I).


View this table:
[in this window]
[in a new window]
  		Table I. Total number of cells and number of neutrophils, eosinophils, and macrophages (x104/ml) recruited to diffusion chambers in immunized wild-type C57BL/6 and C3–/– micea

 
To confirm that C3 was only required during the effector stage of the adaptive immune response, normal or heat-inactivated naive serum was transferred to immunized C3–/– mice at the time and location of the challenge infection. Transfer of normal naive serum to immunized C3–/– mice restored the ability of these mice to kill the challenge larvae (Fig. 4A), whereas transfer of heat-inactivated serum did not (Fig. 4B). Therefore, the heat-labile C3 was required only at the time of challenge infection and not for the development of the protective adaptive immune response.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 4. Untreated naive serum, but not heat-inactivated serum, restores protective immunity to immunized C3–/– mice. At the time of challenge, either untreated serum (A) or heat-inactivated serum (B) was transferred to naive and immunized wild-type and C3–/– immune mice, and larval survival was determined. *, Statistical significance (p ≤ 0.05) between naive and immune. Each experiment was performed at least twice; data shown represent the means and SDs of a minimum of five mice per group.

 
To determine whether C3a was required for the larval killing mechanism in the adaptive response, C3aR–/– mice were immunized and challenged with S. stercoralis larvae. Protective immunity developed in both immunized wild-type and C3aR–/– at equivalent levels (Fig. 5A), indicating that C3a was not required for the protective immune response to S. stercoralis. There was no difference in the number of cells recruited into the diffusion chambers implanted in immunized C3aR–/– as compared with wild-type mice (Fig. 5B), indicating that C3a was not required for cell recruitment. Parasite-specific IgM and IgG1 levels were equivalent between immunized wild-type and C3aR–/– mice, whereas parasite-specific IgG2a responses were absent in the immunized mice (Fig. 5C). Finally, IL-4, IL-5, and IFN-{gamma} were measured in spleen cell cultures derived from naive and immunized mice. After stimulation with anti-CD3 mAb, IL-4 and IL-5 levels were equivalently elevated in immunized wild-type and C3aR–/– mice. IFN-{gamma} was not elevated in the immunized mice regardless of strain (Fig. 6).


Figure 5
View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 5. Adaptive protective immunity is unaffected in C3aR–/– mice. BALB/c and C3aR–/– mice were immunized. The following parameters were measured in the diffusion chambers implanted in the naive and immune mice: A, larval survival; B, cellular recruitment; and C, quantity of parasite-specific IgM (1:2000), IgG1, and IgG2a (1:50). *, Statistical significance (p ≤ 0.05) between naive and immune. Each experiment was performed at least twice; data shown represent the means and SDs of a minimum of five mice per group.

 

Figure 6
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 6. Cytokine responses are unaffected in C3aR–/– mice. BALB/c and C3aR–/– mice were immunized, and IL-4 (A), IL-5 (B), and IFN-{gamma} (C) production by splenocytes following stimulation with anti-CD3 mAb was measured. *, Statistical significance (p < 0.05) between naive and immune. Each experiment was performed at least twice; data shown represent the means and SDs of a minimum of five mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The objective of this study was to determine the role of C3 and C5 in innate and adaptive protective immunity to larval S. stercoralis. It was demonstrated that C3 functioned in both the innate and the adaptive protective immune responses, whereas C5 was not required in either. Naive C5-deficient mice killed larvae at levels comparable to C5-sufficient wild-type mice. Furthermore, immunized C5-deficient mice killed larvae in challenge infections and recruited cells at levels that were comparable to immunized C5-sufficient mice. Previous studies have shown C5-deficient mice to be more susceptible to infection with the cestode Echinococcus granulosus (26). The role of C5 in the innate immune response to helminths is therefore parasite species dependent. Based on the observation that protective immunity functioned without C5, three potential mechanisms of complement-mediated larval killing were eliminated. First, the pore-forming membrane attack complex does not function to "punch holes" into the surface of larval S. stercoralis. Second, C5 is not required for the initiation of the parasite-specific Ab response, confirming a previous study that showed that C5-deficient mice did not have an impaired ability to mount humoral immune responses (27). Third, C5a is not required as a chemoattractant or as an activator of effector cells responding to the worm. C5a is a highly potent chemoattractant and stimulator of degranulation for neutrophils (28, 29). Therefore, neutrophils, required for killing larval S. stercoralis during the adaptive immune response (14, 20), function in a C5a-independent manner.

In the current study, naive C3–/– mice demonstrated that in the absence of C3, larval survival increased, thereby demonstrating a role for C3 in the protective innate immune response. When cells were prevented from entering the parasite microenvironment in naive wild-type mice, there was also an increase in larval survival. However, restricting cell contact with the larvae in C3–/– mice did not further reduce larval killing, suggesting that cells and C3 are concurrently required to affect larval killing. Previous in vitro studies have shown that complement promotes the binding of monocytes and neutrophils to surface of S. stercoralis L3 (21) and the adherence of cells to L3 of Strongyloides ratti (30). Complement not only promotes cellular adherence, but is also required for in vitro neutrophil-mediated killing of Angiostrongylus cantonensis L3 and eosinophil-mediated killing of Schistosoma mansoni schistosomula (31, 32). It is therefore possible that C3 functions as an attachment site for cells on the larval surface during the innate response to S. stercoralis in mice.

The classical, alternative, and lectin complement pathways can all be activated during the innate immune response. Activation of the classical complement pathway during the innate immune response is initiated by natural Abs that recognize a variety of pathogen-associated molecules (25). In naive µMT mice, which lack circulating natural IgM, larval killing was equivalent to that seen in wild-type mice, demonstrating that complement activation via the classical pathway was not required for killing L3 in innate immunity. Adherence of canine cells to Ancylostoma caninum and human cells S. stercoralis in vitro is mediated by the alternative complement pathway (21, 33). Therefore, activation of the murine complement system by S. stercoralis L3 during the innate immune response may involve interaction between the surface of the worm and the lectin or alternative complement activation pathways.

C3–/– mice failed to kill larvae via the adaptive immune response, and experiments were performed to identify the deficiency that would explain the absence of protective immunity. C3 is important for the development of the T-dependent B cell responses and enhances the humoral immune response especially when Ag dose is a limiting factor (4, 34). Mice lacking complement receptors CR1 and CR2 have impaired humoral responses to T-dependent Ags, as well as a reduction in B-1 cells (35). B-1 cells (16) and IgM are required for protective immunity against S. stercoralis L3 in mice (19). In addition, C3–/– mice infected with S. mansoni had decreased production of Th2-associated cytokines and enhanced Th1 cytokines. The cytokine results were reflected in the Ab responses, in that IgG1 responses in C3–/– mice were significantly impaired, IgG2a responses were unaffected, and infected C3–/– mice actually had increased production of parasite-specific IgM (8). In the current study, C3–/– mice developed IgM, IgG1, and IgG2a responses comparable to that seen in wild-type mice, indicating that there was no significant defect in the Th2-mediated B cell response. Furthermore, transfer of serum from immunized C3–/– mice to naive wild-type mice passively transferred protective immunity, thereby providing confirmation that C3 was not required for B cell function. Therefore, it was concluded that the development of protective IgM to S. stercoralis is not C3 dependent.

There appears to be an inconsistency between the observation that C3 functions during the innate response, yet is not required for the development of the adaptive response. Larvae must die and be processed by APCs to initiate the adaptive Ab response, and this is apparently occurring in the absence of C3. Complement enhanced the rate of eosinophil-mediated killing of Hemonchus contortus larvae, but was not required for the killing process to occur (36). Therefore, it is possible that in the present study C3 increased the rate, but was not essential for cell-mediated killing of larval S. stercoralis during the innate immune response. Alternative C3-independent killing mechanisms apparently compensate for the absence of C3, killing larvae and thereby promoting the development of adaptive immunity.

It was hypothesized that the absence of adaptive protective immunity in C3–/– mice might have been caused by a defect in cell requirement. Total cell and granulocyte numbers recruited to the larval microenvironment were equal in immunized wild-type and C3–/– mice. It was then postulated that C3 might be required for the actual larval killing process. This hypothesis was confirmed by experiments in which it was shown that normal serum transferred at the time of the challenge infection could reconstitute the killing capacity of immunized C3–/– mice, whereas heat-inactivated serum could not. It has been reported previously that complement is activated during the adaptive protective immune response to S. stercoralis in mice by the Ab-dependent classical activation pathway (19). Thus, the production of IgM during the adaptive immune response may amplify the amount of C3 fixed on the surface of the larvae, thereby increasing the efficiency of larval killing. Therefore, C3 is required as an integral component during the adaptive immune response in collaboration with IgM and neutrophils for killing S. stercoralis larvae.

The importance of C3a as an immune mediator has been observed in mouse models of allergy (22) and endotoxic shock (37). C3a activates mast cells (38) and acts as chemotaxic factor for eosinophils, but not for neutrophils (39). In addition, in studies on allergic inflammation, it was observed that C3aR–/– mice have enhanced secretion of Th2 cytokines by splenocytes and a concomitant elevation in Ag-specific IgG1 (40). Data presented in the current study using C3aR–/– mice indicate that C3a is not required for larval killing during either the innate or adaptive immune response. Immunized C3aR–/– mice developed IgM, IgG1, and IgG2a responses comparable to that seen in wild-type mice, and splenocytes from immunized C3aR–/– and wild-type mice secreted equal quantities of IL-4, IL-5, and IFN-{gamma}. These data indicate that there was no significant change in the Th2 or the B cell response in the immunized C3aR–/– mice as was reported in the allergy model (40). One possible explanation for this discrepancy is that in the allergy model the enhanced secretion of Th2 cytokines and elevation in Ag-specific IgG1 were observed if there was an epicutaneous sensitization, but not if there was i.p. sensitization (40). Therefore, the route of immunization and the type of immunization may control whether C3aR–/– mice develop enhanced Th2 responses.

Because C3 is cleaved into C3a and C3b, and C3aR–/– mice have no apparent defects in immunity against S. stercoralis, it may be concluded that C3b is the active component of C3 that is required to mediate larval killing. There are several possible mechanisms through which C3b may function in the larval killing process. C3b acts as an adherence molecule for neutrophils (41), and neutrophils are required for larval killing during the adaptive immune response (14, 20). Significantly higher levels of fixed C3b have been found on the surface of S. stercoralis L3 recovered from immune mice 6 h after infection as compared with larvae recovered from naive mice (19). Therefore, C3b may serve as an anchor for neutrophils to adhere to on the surface of larvae. Alternatively, C3b may facilitate neutrophil activation and/or degranulation, although it has been shown that neutrophil degranulation does not occur after binding to C3b alone, but requires secondary signals (42) such as beta-glucans (43, 44). These secondary signals may be either immune system- or worm-derived molecules that may function with neutrophils and C3b to facilitate the killing of the larvae.

The current study has shown that C3 functions during the innate immune response and is required during the adaptive protective immune response of mice to larval S. stercoralis. C3b appears to be the active component of C3 and possibly cooperates with eosinophils, facilitating eosinophil degranulation and larval death during the innate immune response (18, 45). C3b also appears to be the active component of C3 during the adaptive response, and it functions at the terminal end of the killing process. Killing of larval S. stercoralis by immunized mice is dependent on the interaction among IgM (19, 20), neutrophils (18, 20), and C3. It is hypothesized that the presence of IgM, generated during the adaptive immune response, increases the amount of fixed C3b present on the surface of the larvae, thereby increasing the efficiency by which neutrophils eliminate larval S. stercoralis.


   Acknowledgments
 
We thank Ann Marie Galioto, Juergen Landmann, Udai Padigel, and Gilberto Santiago for expert technical and analytical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants RO1 AI47189 (to D.A.) and RO1 AI22662 (to G.A.S.). Back

2 Address correspondence and reprint requests to Dr. David Abraham, Department of Microbiology and Immunology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107. E-mail address: David.Abraham{at}jefferson.edu Back

3 Abbreviations used in this paper: L3, third stage infective larvae; DOC, deoxycholate. Back

Received for publication June 7, 2005. Accepted for publication January 4, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Carroll, M. C.. 2004. The complement system in regulation of adaptive immunity. Nat. Immunol. 5: 981-986. [Medline]
  2. Gasque, P.. 2004. Complement: a unique innate immune sensor for danger signals. Mol. Immunol. 41: 1089-1098. [Medline]
  3. Volanakis, J. E.. 2002. The role of complement in innate and adaptive immunity. Curr. Top. Microbiol. Immunol. 266: 41-56. [Medline]
  4. Fischer, M. B., M. Ma, S. Goerg, X. Zhou, J. Xia, O. Finco, S. Han, G. Kelsoe, R. G. Howard, T. L. Rothstein, et al 1996. Regulation of the B cell response to T-dependent antigens by classical pathway complement. J. Immunol. 157: 549-556. [Abstract]
  5. Pepys, M. B.. 1974. Role of complement in induction of antibody production in vivo: effect of cobra factor and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J. Exp. Med. 140: 126-145. [Abstract]
  6. Bottger, E. C., T. Hoffmann, U. Hadding, D. Bitter-Suermann. 1985. Influence of genetically inherited complement deficiencies on humoral immune response in guinea pigs. J. Immunol. 135: 4100-4107. [Abstract]
  7. Drouin, S. M., D. B. Corry, J. Kildsgaard, R. A. Wetsel. 2001. Cutting edge: the absence of C3 demonstrates a role for complement in Th2 effector functions in a murine model of pulmonary allergy. J. Immunol. 167: 4141-4145. [Abstract/Free Full Text]
  8. La Flamme, A. C., A. S. MacDonald, C. R. Huxtable, M. Carroll, E. J. Pearce. 2003. Lack of C3 affects Th2 response development and the sequelae of chemotherapy in schistosomiasis. J. Immunol. 170: 470-476. [Abstract/Free Full Text]
  9. Frank, M. M., L. F. Fries. 1991. The role of complement in inflammation and phagocytosis. Immunol. Today 12: 322-326. [Medline]
  10. Hopken, U. E., B. Lu, N. P. Gerard, C. Gerard. 1996. The C5a chemoattractant receptor mediates mucosal defense to infection. Nature 383: 86-89. [Medline]
  11. Rus, H. G., F. I. Niculescu, M. L. Shin. 2001. Role of the C5b-9 complement complex in cell cycle and apoptosis. Immunol. Rev. 180: 49-55. [Medline]
  12. Porto, A. F., F. A. Neva, H. Bittencourt, W. Lisboa, R. Thompson, L. Alcantara, E. M. Carvalho. 2001. HTLV-1 decreases Th2 type of immune response in patients with strongyloidiasis. Parasite Immunol. 23: 503-507. [Medline]
  13. Neva, F. A., J. O. Filho, A. A. Gam, R. Thompson, V. Freitas, A. Melo, E. M. Carvalho. 1998. Interferon-{gamma} and interleukin-4 responses in relation to serum IgE levels in persons infected with human T lymphotropic virus type I and Strongyloides stercoralis. J. Infect. Dis. 178: 1856-1859. [Medline]
  14. Kerepesi, L. A., T. J. Nolan, G. A. Schad, S. Lustigman, D. R. Herbert, P. B. Keiser, T. B. Nutman, A. J. Krolewiecki, D. Abraham. 2004. Human immunoglobulin G mediates protective immunity and identifies protective antigens against larval Strongyloides stercoralis in mice. J. Infect. Dis. 189: 1282-1290. [Medline]
  15. Abraham, D., H. L. Rotman, H. F. Haberstroh, W. Yutanawiboonchai, R. A. Brigandi, O. Leon, T. J. Nolan, G. A. Schad. 1995. Strongyloides stercoralis: protective immunity to third-stage larvae in BALB/cByJ mice. Exp. Parasitol. 80: 297-307. [Medline]
  16. Herbert, D. R., T. J. Nolan, G. A. Schad, D. Abraham. 2002. The role of B cells in immunity against larval Strongyloides stercoralis in mice. Parasite Immunol. 24: 95-101. [Medline]
  17. Rotman, H. L., S. Schnyder-Candrian, P. Scott, T. J. Nolan, G. A. Schad, D. Abraham. 1997. IL-12 eliminates the Th-2 dependent protective immune response of mice to larval Strongyloides stercoralis. Parasite Immunol. 19: 29-39. [Medline]
  18. Herbert, D. R., J. J. Lee, N. A. Lee, T. J. Nolan, G. A. Schad, D. Abraham. 2000. Role of IL-5 in innate and adaptive immunity to larval Strongyloides stercoralis in mice. J. Immunol. 165: 4544-4551. [Abstract/Free Full Text]
  19. Brigandi, R. A., H. L. Rotman, W. Yutanawiboonchai, O. Leon, T. J. Nolan, G. A. Schad, D. Abraham. 1996. Strongyloides stercoralis: role of antibody and complement in immunity to the third stage of larvae in BALB/cByJ mice. Exp. Parasitol. 82: 279-289. [Medline]
  20. Ligas, J. A., L. A. Kerepesi, A. M. Galioto, S. Lustigman, T. J. Nolan, G. A. Schad, D. Abraham. 2003. Specificity and mechanism of immunoglobulin M (IgM)- and IgG-dependent protective immunity to larval Strongyloides stercoralis in mice. Infect. Immun. 71: 6835-6843. [Abstract/Free Full Text]
  21. De Messias, I. J., R. M. Genta, W. D. Mohren. 1994. Adherence of monocytes and polymorphonuclear cells to infective larvae of Strongyloides stercoralis after complement activation. J. Parasitol. 80: 267-274. [Medline]
  22. Humbles, A. A., B. Lu, C. A. Nilsson, C. Lilly, E. Israel, Y. Fujiwara, N. P. Gerard, C. Gerard. 2000. A role for the C3a anaphylatoxin receptor in the effector phase of asthma. Nature 406: 998-1001. [Medline]
  23. Hanski, C., M. Naumann, A. Grutzkau, G. Pluschke, B. Friedrich, H. Hahn, E. O. Riecken. 1991. Humoral and cellular defense against intestinal murine infection with Yersinia enterocolitica. Infect. Immun. 59: 1106-1111. [Abstract/Free Full Text]
  24. Herbert, D. R., T. J. Nolan, G. A. Schad, S. Lustigman, D. Abraham. 2002. Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice. Exp. Parasitol. 100: 112-120. [Medline]
  25. Boes, M.. 2000. Role of natural and immune IgM antibodies in immune responses. Mol. Immunol. 37: 1141-1149. [Medline]
  26. Ferreira, A. M., M. Breijo, R. B. Sim, A. Nieto. 2000. Contribution of C5-mediated mechanisms to host defense against Echinococcus granulosus hydatid infection. Parasite Immunol. 22: 445-453. [Medline]
  27. Heyman, B., L. Pilstrom, M. J. Shulman. 1988. Complement activation is required for IgM-mediated enhancement of the antibody response. J. Exp. Med. 167: 1999-2004. [Abstract/Free Full Text]
  28. Ward, P. A., L. J. Newman. 1969. A neutrophil chemotactic factor from human C'5. J. Immunol. 102: 93-99. [Abstract/Free Full Text]
  29. Henson, P. M.. 1971. The immunologic release of constituents from neutrophil leukocytes. I. The role of antibody and complement on nonphagocytosable surfaces or phagocytosable particles. J. Immunol. 107: 1535-1546. [Abstract/Free Full Text]
  30. Grove, D. I., C. Northern, H. J. Dawkins. 1985. Interactions of Strongyloides ratti free-living and skin-penetrated infective larvae and parasitic adults with serum and cells in vitro. Aust. J. Exp. Biol. Med. Sci. 63: 521-529.
  31. Shaio, M. F., S. C. Hou, J. G. Chen, C. C. Wu, K. D. Yang, F. Y. Chang. 1990. Immunoglobulin G-dependent classical complement pathway activation in neutrophil-mediated cytotoxicity to infective larvae of Angiostrongylus cantonensis. Ann. Trop. Med. Parasitol. 84: 185-191. [Medline]
  32. Anwar, A. R., S. R. Smithers, A. B. Kay. 1979. Killing of schistosomula of Schistosoma mansoni coated with antibody and/or complement by human leukocytes in vitro: requirement for complement in preferential killing by eosinophils. J. Immunol. 122: 628-637. [Abstract/Free Full Text]
  33. Klaver-Wesseling, J. C., J. C. Vetter, E. N. Schoeman. 1982. The in-vitro interaction between several components of the canine immune system and infective larvae of Ancylostoma caninum. Parasite Immunol. 4: 227-232. [Medline]
  34. Dempsey, P. W., M. E. Allison, S. Akkaraju, C. C. Goodnow, D. T. Fearon. 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271: 348-350. [Abstract]
  35. Ahearn, J. M., M. B. Fischer, D. Croix, S. Goerg, M. Ma, J. Xia, X. Zhou, R. G. Howard, T. L. Rothstein, M. C. Carroll. 1996. Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen. Immunity 4: 251-262. [Medline]
  36. Rainbird, M. A., D. Macmillan, E. N. Meeusen. 1998. Eosinophil-mediated killing of Haemonchus contortus larvae: effect of eosinophil activation and role of antibody, complement and interleukin-5. Parasite Immunol. 20: 93-103. [Medline]
  37. Kildsgaard, J., T. J. Hollmann, K. W. Matthews, K. Bian, F. Murad, R. A. Wetsel. 2000. Cutting edge: targeted disruption of the C3a receptor gene demonstrates a novel protective anti-inflammatory role for C3a in endotoxin-shock. J. Immunol. 165: 5406-5409. [Abstract/Free Full Text]
  38. Legler, D. F., M. Loetscher, S. A. Jones, C. A. Dahinden, M. Arock, B. Moser. 1996. Expression of high- and low-affinity receptors for C3a on the human mast cell line, HMC-1. Eur. J. Immunol. 26: 753-758. [Medline]
  39. Daffern, P. J., P. H. Pfeifer, J. A. Ember, T. E. Hugli. 1995. C3a is a chemotaxin for human eosinophils but not for neutrophils. I. C3a stimulation of neutrophils is secondary to eosinophil activation. J. Exp. Med. 181: 2119-2127. [Abstract/Free Full Text]
  40. Kawamoto, S., A. Yalcindag, D. Laouini, S. Brodeur, P. Bryce, B. Lu, A. A. Humbles, H. Oettgen, C. Gerard, R. S. Geha. 2004. The anaphylatoxin C3a down-regulates the Th2 response to epicutaneously introduced antigen. J. Clin. Invest. 114: 399-407. [Medline]
  41. Scribner, D. J., D. Fahrney. 1976. Neutrophil receptors for IgG and complement: their roles in the attachment and ingestion phases of phagocytosis. J. Immunol. 116: 892-897. [Abstract/Free Full Text]
  42. Goldstein, I. M., H. B. Kaplan, A. Radin, M. Frosch. 1976. Independent effects of IgG and complement upon human polymorphonuclear leukocyte function. J. Immunol. 117: 1282-1287. [Abstract/Free Full Text]
  43. Xia, Y., V. Vetvicka, J. Yan, M. Hanikyrova, T. Mayadas, G. D. Ross. 1999. The beta-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells. J. Immunol. 162: 2281-2290. [Abstract/Free Full Text]
  44. Vetvicka, V., B. P. Thornton, G. D. Ross. 1996. Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J. Clin. Invest. 98: 50-61. [Medline]
  45. Rotman, H. L., W. Yutanawiboonchai, R. A. Brigandi, O. Leon, G. J. Gleich, T. J. Nolan, G. A. Schad, D. Abraham. 1996. Strongyloides stercoralis: eosinophil-dependent immune-mediated killing of third stage larvae in BALB/cByJ mice. Exp. Parasitol. 82: 267-278. [Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
U. M. Padigel, L. Stein, K. Redding, J. J. Lee, T. J. Nolan, G. A. Schad, L. Birnbaumer, and D. Abraham
Signaling through G{alpha}i2 protein is required for recruitment of neutrophils for antibody-mediated elimination of larval Strongyloides stercoralis in mice
J. Leukoc. Biol., April 1, 2007; 81(4): 1120 - 1126.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
A. M. Galioto, J. A. Hess, T. J. Nolan, G. A. Schad, J. J. Lee, and D. Abraham
Role of Eosinophils and Neutrophils in Innate and Adaptive Protective Immunity to Larval Strongyloides stercoralis in Mice.
Infect. Immun., October 1, 2006; 74(10): 5730 - 5738.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kerepesi, L. A.
Right arrow Articles by Abraham, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kerepesi, L. A.
Right arrow Articles by Abraham, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS