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The Journal of Immunology, 2005, 175: 1827-1833.
Copyright © 2005 by The American Association of Immunologists

Critical Role for IgM in Host Protection in Experimental Filarial Infection

Bhargavi Rajan1, Thirumalai Ramalingam1 and Thiruchandurai V. Rajan2

Department of Pathology, University of Connecticut Health Center, Farmington, CT 06032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have previously shown that B cells (in particular B1 cells) are important in host protection against brugian infections in a murine i.p. model. In this study, we show that mice deficient in circulating IgM (secIgM–/–), but otherwise normal in their humoral responses, manifest a significant impairment in worm elimination, suggesting that one critical B cell function is the production of Ag-specific IgM. Efficient elimination of larvae is IgM dependent for both primary and challenge infections. The ability to eliminate worms is restored in secIgM–/– mice by administering sera from primed mice. We corroborated these in vivo studies with in vitro observations which show that IgM is the only isotype that reacts strongly with the surface of Brugia L3. Furthermore, activated peritoneal exudate cells adhere to L3 only in the presence of filaria-specific sera or IgM purified from them. This attachment is not reduced by heat inactivation of the serum, suggesting complement independent activity. Peritoneal exudate cells from primed mice, especially activated macrophages, carry high levels of IgM on their surfaces. Our observations suggest that an IgM-mediated reaction initiates the formation of host-protective granulomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The mechanisms involved in mammalian host protection against large, tissue-dwelling parasites such as the filarial nematodes are not well understood. In an attempt to understand host protective mechanisms, we have analyzed the kinetics of the host response to the i.p. injection of infective brugian larvae (1, 2). We have shown that infection is followed by an exuberant inflammatory response, resulting in the accumulation of ~10 times as many leukocytes as are present in naive mice. This response is followed, ~7–8 days after infection, by adherence of host leukocytes to the larvae, progressing incrementally until the larvae are completely covered with host cells. Histologically, these aggregates are composed predominately of macrophages (some of which fuse to form multinucleated giant cells) and eosinophils. These multilayered structures are referred to as granulomas in the pathology literature (1, 2). Granulomas with a striking histological resemblance to that described in the murine i.p. Brugia infection model (1), have been documented in jirds (3), hamsters (4), cats and dogs (5), and humans (6), suggesting that a phylogenetically conserved mechanism is involved in the sequestration and elimination of the parasite. We and others have demonstrated a critical requirement for adaptive immunity in achieving sterile immunity. T cells, B cells, the IL-4 signaling pathway, and IL-5 are essential for efficient elimination of a primary infection (7, 8, 9, 10, 11, 12). We have also demonstrated a crucial role for B1 cells (12, 13).

The last observation led us to examine the role of IgM in host protection in our experimental model. The predominant Ab isotype made by B1 B cells is IgM (14). We reasoned that the substantial (>90%) deficit in IgM in these mice could account for the delayed clearance of infection in Btkxid mice compared with their normal counterparts. Although IgM Abs are present in every mammalian species tested, their precise role in host defense has been difficult to elucidate. The recent description of a mouse that lacks circulating Ig has made this a more approachable problem. This mouse was generated by Boes et al. (15 by deleting the µs exon as well as the three polyadenylation sites associated with this exon. As a consequence, these mice are able to express IgM on the cell surface, but are unable to secrete it. In their first report on these mice (16), the authors showed that they have normal levels of conventional B2 B lymphocytes but, unexpectedly, a substantial increase in the number of B1 B lymphocytes in the peritoneum and other serosal cavities. Serologically, the mice had higher than normal levels of circulating IgG2a, IgG3, and IgA. The production of IgG Ab responses to suboptimal levels of a T cell-dependent Ag was impaired. There were diminished numbers of germinal centers and impairment of the affinity maturation of Abs, which might account for the deficit.

These mice have been helpful in understanding the role of IgM in host defense. Boes et al. (16) examined this question using the cecal ligation and puncture model, which results in an acute peritonitis. Approximately 20% of normal mice succumb to this procedure. In contrast, 70% of secretory IgM-deficient mice died within the first 32 h of CLP. Based on this observation, Boes et al. (16) concluded that IgM is important in systemic bacterial infection.

The role of IgM in other infections is less clearly defined. One viral infection in which the role of IgM has been examined carefully is influenza (17). Baumgarth et al. (17) showed that IgM from both B1 and B2 B lymphocytes is required for host defense. They also showed that the repertoire and affinity of Abs made is impaired in the absence of IgM. Recently, Salinas-Carmona and Perez-Rivera (18) have shown that protection against nocardial infections requires IgM, using a passive-transfer approach.

The role of IgM in host protection against larger parasitic organs, such as the nematodes, is also unclear. Abraham and his colleagues (19, 20) have correlated IgM titers with host protection against the parasitic organisms, Strongyloides stercoralis. They have further shown that the absence of IgM correlates with a poor prognosis.

We report here the course of experimental Brugia pahangi infections in mice that lack circulating IgM and document a critical role of IgM in host protection. Our results demonstrate that adherence of host immune cells to the Brugia larvae is dependent on circulating IgM.


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

C57BL/6J (hereafter wild type (wt)3) mice were obtained from The Jackson Laboratory. B6;129S4-Igh-6tm1Che/J (hereafter secIgM–/–) mice were obtained initially from The Jackson Laboratory. They were subsequently housed and bred at the American Association of Laboratory Animal Care accredited University of Connecticut Health Center Vivarium. B6.JHD mice bear a targeted mutation in the Ig J and D segments (21). These mice lack B lymphocytes and do not have detectable serum Ig.

All mice were maintained under specific pathogen-free conditions in microisolator cages. They were given lab chow and sterile water ad libitum. The integrity of our secIgM–/– colony was periodically confirmed by quantitating serum IgM levels in randomly selected mice by sandwich ELISA.

Infectious larvae

Brugia pahangi L3 were harvested at TRS Inc. (Dr. J. McCall, University of Georgia, Athens, GA) or the University of Louisiana (Dr. T. Klei) from infected Aedes aegytpi mosquitoes and transported in RPMI 1640 supplemented with antibiotics as described previously (22).

Experimental infection

Mice were injected with aliquots of ~50 B. pahangi L3 i.p. in 500 µl of RPMI 1640 using 1-ml syringes fitted with 5/8-inch 25-gauge needles. For challenge infections, 50 L3 of the same species were injected i.p. into mice previously sensitized with 20 L3 two months earlier.

Peritoneal exudate cells (PECs)

Mice were sacrificed at various time points postinfection. They were first subjected to transthoracic cardiac bleeds for serum collection and for blanching the mesenteric vasculature, thereby minimizing blood contamination of the lavage. Peritoneal lavages were performed using ice-cold RPMI 1640 medium supplemented with 5 U of heparin/ml. Lavage fluid was passed through 100-µm pore-sized nylon mesh screens and collected into 15-ml polystyrene tubes.

Worm recovery

Following peritoneal lavage, intestines were removed and soaked in PBS. The scrotal sacs were opened, and carcasses were placed in PBS for further soaking. Carcasses were then rinsed and soaked in PBS. Viable worms were enumerated in the peritoneal lavage, intestinal washes, and carcass soaks under a dissecting microscope.

Cell counting and flow cytometry

The total number of peritoneal exudate cells in each mouse was determined using the Advia 120 Hematology System (Bayer Diagnostic Division) with version 2.2.06-MS software. PECs were incubated with Fc Block (BD Pharmingen) at a dilution of 1/50 for 10 min at 4°C to minimize nonspecific labeling through Fc{gamma} receptors. Following a wash with 4 ml of FACS buffer (PBS, 0.2% BSA, and 0.1% NaN3), PECs were stained with a mixture of conjugated mAbs (CD19-PE, CD3-CyChrome, Ly6G-FITC, and CD11b-allophycocyanin). The fluorochrome-conjugated Abs were used at 1/100 dilutions (except for CD11b-allophycocyanin which was used at 1/400). Following a wash with 4 ml of FACS buffer, cells were fixed with 0.5% paraformaldehyde and acquired on a FACSCalibur (BD Biosciences). Data were subsequently analyzed using FlowJo (version 6; TreeStar). All Abs used for flow cytometry were purchased from BD Pharmingen.

Measurement of Ag-specific Ig by ELISA

To generate a B. pahangi Ag extract, a mixture of ~3000 L3s, ~300 L4s, and 30 adults were washed several times with sterile PBS. The mixture of worms was placed in 500 µl of PBS in an Eppendorf tube and sonicated with the microprobe for 10 min on ice, with 15-s pulses followed by 5-s rest utilizing a power setting of 6 (Sonic Dismembrator XL2020; Fisher Scientific). After sonication, the tube was spun at 10,000 rpm for 30 min at 4°C. The supernatant was harvested and its protein content was measured using the bicinchoninic acid assay (Pierce). ELISA plates were coated with 1 µg/ml worm Ag in pH 9.0 sodium phosphate coating buffer. Plates were blocked for 1 h at room temperature with 1% BSA-PBS solution. Sera diluted serially 3-fold from 1/100 were incubated for 2 h at room temperature. After further washes, appropriate alkaline phosphatase-labeled isotype-specific secondary Abs were added for 1 h. The plates were washed and developed with phosphatase substrate (Sigma-Aldrich). OD at 405 nm was determined using an E-Max plate reader (Molecular Devices).

In vitro cell adherence assay

PECs were recovered from secIgM–/– mice 14 days postinfection. The cells were washed with complete RPMI 1640 (RPMI 1640 containing 100 U/ml penicillin, 20 µg/ml gentamicin, 4 µg/ml ciprofloxacin, and 2 µg/ml ceftazidime) and resuspended at 20 x 106/ml. Sera were collected from naive or infected wt or secIgM–/– mice and frozen at –20°C as 20% stocks in complete RPMI 1640. They were thawed immediately before use. In some cases, freshly collected sera were used. In other instances, sera were heated at 56°C for 30 min to inactivate heat-labile complement components.

B. pahangi L3 larvae were washed extensively in complete RPMI 1640 by serial passage through fresh medium in 6-well polystyrene cluster dishes. Five cleaned L3s were added per well of a 96-well flat-bottom plate containing 50 µl of complete RPMI 1640. A total of 106 PECs were added in a volume of 50 µl. Sera from 20% stocks were added at final concentrations ranging from 2 to 10% before adjusting the final volume of the well to 200 µl. The plates were incubated at 37°C for 2–3 h before scoring. Each condition was tested in duplicate.

An independent observer, always blinded to the experimental conditions, scored the larvae for cell adherence on a scale ranging from 0 to 3: 0, if no cells were attached; 1, if few cells were attached in a single layer for even a part of the worm; 2, if several layers of cells were partially covering the worm; and 3, if the worm was completely covered by several layers of cells.

Immunofluorescence

Ten fresh washed L3s were incubated with day 14 sera from primed wt mice at a final concentration of 10% in RPMI 1640 for 1 h at 37°C in 96-well flat-bottom plates. The L3s were transferred to 48-well flat-bottom plates and excess serum was washed away with PBS and vigorous shaking on an orbital shaker. They were blocked with 1% BSA in PBS overnight at 4°C and then incubated with fluorochrome-conjugated secondary Abs against IgM, IgG1, IgG2b, IgG3, or trinitrophenyl (BD Pharmingen) for 30 min at room temperature on a shaker. Excess secondary Ab was removed by three vigorous washes in PBS. The worms were transferred into a Lab-Tek II 8 chambered cover glass (Nalge Nunc) for confocal microscopy using a Zeiss LSM 410.

IgM purification

IgM was purified from serum using commercially available peptidomimetic KAPTIV-M columns (Bio/Can Scientific) according to the manufacturer’s recommended protocol. Briefly, sera were diluted 1/5 in loading buffer (0.05 M sodium phosphate buffer, pH 7). The sample was loaded onto a packed column and washed with loading buffer until UV absorbance at 280 nm returned to baseline. The retained IgM was eluted with elution buffer (0.1 M acetic acid) and immediately neutralized with 0.1 M Tris-HCl (pH 9.0). Although the eluate was highly enriched for IgM, some IgA was also detectable by ELISA. IgG and IgE were absent. The column wash, which had higher concentrations of IgA than the eluate, was included in the cell adherence assay to control for IgA contamination in the eluate.

Graphs and statistics

Data were plotted using Microsoft Excel 10 and Prism 4.02 (GraphPad Software). Statistical comparisons between groups were performed using the Student’s t test in either of these programs. A p < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IgM is critical for worm elimination

In view of our previous data that mice bearing the Btkxid mutation manifest delayed clearance of i.p. brugian infections, we reasoned that IgM may play a role in host protection. To test our assumption, we used mice deficient in circulating IgM (secIgM–/– mice). We enumerated live worm recoveries on day 16 after i.p. infection with B. pahangi L3. secIgM–/– mice harbored significantly more parasites (18 ± 8) than their wt counterparts (1.2 ± 0.8, Fig. 1a).



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  		FIGURE 1. IgM is critical for worm elimination. a, Larval yields after primary infections: 50 B. pahangi L3 were injected i.p. into groups of 5 wt and secIgM–/– mice. Both groups were sacrificed on day 16. Worm yields were enumerated and represented as percentages of initial load. b, Cellular analyses of the peritoneal exudates were conducted by four-color flow cytometry as described in Materials and Methods. Data are representative of five experiments. c, Ten wt and secIgM–/– mice were injected with 50 B. pahangi. Eight weeks later, they were challenged with a second dose (50 L3s) of B. pahangi. Ten days after the second infection, all groups were sacrificed and worm recoveries were determined. Live worm recoveries from the challenge infection are plotted as percentages of worms injected. Any worms persisting from the priming infection could be easily distinguished from those from the challenge infection by the size difference. Data are representative of two experiments. d, Serum donor and recipient mice received 20 and 50 B. pahangi L3 i.p., respectively. On day 13 after infection, sera were collected from the donor mice and 350 µl of sera were transferred i.p into each secIgM–/– recipient mouse. Worm yields were determined on day 18 after infection. Similar results were obtained in three experiments. *, p < 0.05.

 
Previous studies from our laboratory have shown that T cell-deficient mice are impaired in leukocyte recruitment to the site of infection. This lesion is associated with a defect in host protection. secIgM–/– mice do not exhibit this defect (Fig. 1b). In all experiments we have conducted, they recruit more PECs than wt mice. Among the various components of the PEC, B1 cell numbers are consistently elevated in absolute numbers in secIgM–/– mice, in agreement with the original description (15, 16). Macrophage numbers also were significantly higher in secIgM–/– mice. Other cells, although increased in absolute numbers, were not significantly different (Fig. 1b).

We reasoned that the dependence on IgM for worm elimination during a primary infection would disappear following challenge infection if the protective specificities class switch to other isotypes, as they do in many infectious models. secIgM–/– mice were impaired in eliminating a challenge infection as well. They harbored 26 ± 4% of the larvae administered as challenge infection as compared with wt mice that had only 2.4 ± 0.8% of larvae from this infection (Fig. 1c). Although wt mice had completely cleared the priming infection as expected, secIgM–/– mice still harbored 7% of the priming infection at the time of sacrifice. This is consistent with the data shown in Fig. 1a, which showed increased worm burdens in these mice following a primary infection, but at an earlier time point.

Passive transfer of primed wt serum restores protection in secIgM–/– mice

Since genetically altered mice sometimes exhibit phenotypes that are not directly related to the gene mutation, we sought to determine whether the impairment of worm clearance in secIgM–/– mice could be corrected by reconstitution with primed wt serum that would contribute Ag-specific IgM. On day 13 postinfection, cohorts of secIgM–/– mice were passively administered 350 µl of primed wt or JHD sera. On day 18, the mice were sacrificed and worm recoveries were enumerated. secIgM–/– mice receiving wt serum had statistically significantly fewer worms (8.4 ± 3.5) than those that received JHD serum (32.5 ± 5.6) or no serum at all (25.5 ± 6.5, Fig. 1d).

IgM is essential for cell adherence to Brugia L3

Even though secIgM–/– mice recruit substantial numbers of leukocytes to the site of infection, they were significantly impaired in their ability to eliminate the infection. We reasoned that the defect in these mice, in contrast to T cell-deficient mice, was not in cell recruitment but at a later stage in host defense, perhaps in adherence of leukocytes to larvae to initiate granuloma formation. To determine whether IgM played a role in adherence of PECs to worms, we used an adaptation of an in vitro assay previously described by Chandrashekar et al. (23). We tested the adherence of peritoneal cells to Brugia L3 from naive or primed wt and secIgM–/– mice in the presence of either naive or primed sera.

The data in Fig. 2represent blinded scores of cell adherence assays performed with naive and Brugia- primed cells, respectively. The result illustrates several important points. First, naive peritoneal cells are incapable of adhering to worms in the presence of naive sera, irrespective of the source of serum. Second, in the presence of primed wt serum, but not primed secIgM–/– serum, naive PECs are able to adhere to larvae. Third, PECs recovered from primed wt mice are capable of adhering to worms even in the absence of serum. Finally, cells from secIgM–/– mice are incapable of adhering to the worms in the presence of their own sera, but they are capable of adhering to the larvae in the presence of sera from wt mice.



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  		FIGURE 2. PECs from secIgM–/– mice do not adhere to worms in vitro. Peritoneal cells harvested from naive (a) or primed (b) secIgM–/– or wt mice were incubated with no serum or a serum source as indicated below the x-axis along with five B. pahangi larvae. The cell adherence was scored by a blinded observer on a scale of 0 (no cell adherence) to 3 (maximal cell adherence) as described in detail in Materials and Methods. Similar results were obtained in other repetitions.

 
IgM is sufficient for cell adhesion

Even though the studies using secIgM–/– sera strongly suggest that IgM is the critical isotype for mediating cell adherence, we sought to demonstrate it directly. We purified IgM using a peptidomimetic column. We tested the ability of primed wt serum, the IgM-depleted flow through, and the IgM-enriched eluate to reconstitute cell adherence (Fig. 3). The data demonstrate that although primed wt serum could support cell adherence, IgM-depleted flow through could not. Moreover, the enriched IgM fraction could also restore cell adherence. The data also rule out the possibility of the presence of inhibitory factors in secIgM–/– serum, since all wells contained secIgM–/– serum and cell adherence took place even in its presence.



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  		FIGURE 3. Purified IgM can reconstitute secIgM–/– serum to support cell adherence. PECs from primed secIgM–/– mice were incubated with B. pahangi L3 in the presence of 5% primed secIgM–/– serum. Duplicate wells received 2% of secIgM–/– serum, serum source used for IgM purification, IgM affinity column flow-through, or the eluate. Cell adherence was scored by a blinded observer as described in detail in Materials and Methods. 3 = maximal cell adherence and 0 = no cell adherence. Results are representative of two similar experiments.

 
IgM Abs in immune sera recognize Brugia L3

If circulating IgM plays a critical role in host protection and if the defect in secIgM–/– mice is the lack of this isotype, then normal mice should have filarial-specific IgM in the circulation after infection. To evaluate the contribution of Ig isotypes in recognizing worm Ags, we performed ELISA on sera from wt mice on various days after Brugia infection. The results demonstrate that the predominant isotype recognizing worm Ags is IgM, beginning at day 12 and increasing significantly until day 21 after infection (Fig. 4). IgG isotypes examined displayed modest, if any, binding to the worm extract (data for IgG1 shown in Fig. 4; data not shown for other isotypes). To determine whether Ig recognition of the surface of the L3 followed a similar pattern, we performed isotype-specific immunofluorescence. The results clearly indicated that the only isotype that bound to the L3 was IgM (Fig. 5). These data are similar to those reported in the literature with Strongyloides stercoralis, suggesting that IgM may be the predominant isotype involved in recognition of parasitic nematodes (19).



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  		FIGURE 4. IgM is the only isotype that strongly reacts with worm Ag extract. Ag-specific ELISA: sera from three to five wt mice were collected at the indicated time points after B. pahangi infection. The isotype distribution of anti-filarial Abs in the sera was determined using ELISA. Data are plotted as geometric means of the OD at 405 nm, with SE (note: some error values are too low to be noticeable). Similar results were obtained in another repetition.

 


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  		FIGURE 5. IgM is the only isotype that recognizes surface of Brugia L3: Brugia L3 were incubated with 10% primed wt serum. The isotypes recognizing the surface of the worm were detected by probing with a panel of fluorescently labeled isotype-specific Abs or their corresponding isotype controls. Consistently, the IgM was the only isotype that was detected on the surface of the larvae. The left and right panels represent the differential interference microscopy and fluorescent image for each isotype examined. Similar results were obtained in two experiments. Negative results were obtained for IgG isotypes (data not shown).

 
IgM directly binds to macrophages in PECs

The previous experiment confirms that normal mice have circulating IgM capable of binding to larvae. One possible mechanism for IgM-dependent cell adherence is through activation of complement components. Cell adherence could be mediated by C3 receptors on PECs binding to activated C3 on larvae. However, heat inactivation of sera at 56°C for 30 min did not abrogate cell adherence, suggesting a complement-independent mechanism (data not shown). An alternative possibility is that IgM is responsible for forming a bridge between larvae and PECs. To test this, we examined for the presence of IgM and IgG on the surface of PECs recovered from naive and primed wt animals. The only isotype that we consistently detected in high amounts on the surface was IgM (Fig. 6). Not surprisingly, the highest expression of IgM in naive PECs was on B1 and B2 cells. However, macrophages could be stained with anti-IgM even in naive mice, albeit modestly. IgM levels on the surface of macrophages were significant increased following infection, suggesting up-regulation of an IgM receptor on activated cells.



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  		FIGURE 6. IgM binding on wt macrophages increases following Brugia infection. Naive or day 14 Brugia-primed PECs were harvested from B6 wt mice and stained for CD11b, CD19, CD3, and IgM. Different cell populations were identified as previously described (2 12 ). IgM expression on the surface of various cell types is represented as an offset histogram overlay. Similar results were obtained in another repetition. Eos, eosinophils; Mac, macrophages.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Over the past decade, we have been investigating host defense mechanisms against filarial infections using an i.p. infection model. We have used either the human parasite, Brugia malayi, or the closely related feline parasite, B. pahangi, in our studies. We have shown in previous studies that normal, immunocompetent mice eliminate the infection within a few weeks after introduction of the parasite into the peritoneal cavity, before maturation to the adult and the production of microfilariae (1). We also have shown that this efficient host protection has an absolute requirement for T lymphocytes, as indicated by the fact that mutant strains that lack T lymphocytes alone (such as nude, TCR{beta}, or TCR{beta}{delta} knockout mice) or in combination with B cells (such as SCID or RAG knockout mice) fail to reject worms and permit them to persist to maturity (9, 24). These mice become productive of microfilariae.

Our studies over the past several years have also suggested that the end product of host defense is the production of cell aggregates around the larvae, called granulomas. The process of conversion of the larva from the pristine state, devoid of adhering host leukocytes, to one fully encased in a granuloma appears to take place in a number of steps: accumulation of cells in the peritoneal cavity; adherence to larvae; formation of the granuloma; and killing of larvae within the granuloma.

In the case of T cell knockout mice, the failure in host protection takes place in the very first step. We have been able to show that mice that do not have T lymphocytes (alone or in combination with other defects) fail to recruit leukocytes, a critical first step for the formation of the granulomas (2, 12).

In previous studies, we also showed that mice that lack B lymphocytes manifest defects in host protection, with persistence to the larvae until late stages of infection when they become adults (13). Surprisingly, however, B cell knockout mice seldom, if ever, become productive of microfilariae, even when the numbers of adult worms within them are comparable to those within their T cell-deficient counterparts. The reason for the failure of host defense in B cell knockout mice is not the same as it is in T cell knockout mice. B cell knockout mice recruit cells as effectively as wt mice, even though there is a slight delay in the kinetics of cell recruitment. In this report, we suggest that the defect in B cell-deficient mice is at a later stage in host defense and that it could be due to the lack of circulating IgM.

Mice specifically lacking circulating IgM manifest defects in worm clearance in primary and challenge infections, underscoring the significance of this isotype in filarial infection. As is true of global B cell deficiencies, the impairment in worm elimination in secIgM–/– mice is not associated with deficiency in recruiting cells to the site of infection. Rather, their cellularity is consistently higher than that observed in wt mice. Transfer of primed wt sera to secIgM–/– mice on day 13 resulted in a significant decrease in worm burdens in the recipient mice, in comparison to unreconstituted secIgM–/– control mice. This experiment rules out the possibility that the observed phenotype in the mutant mice is due to some unrelated defect.

Our results with secIgM–/– mice confirm the importance of IgM in host defense. But what is the precise role of this isotype? We reasoned that IgM might be involved in adherence of peritoneal cells to the larvae to initiate granuloma formation. In vitro experiments designed to address this hypothesis confirmed our prediction and suggested that IgM is required for leukocyte adherence to L3. Adherence depends upon the presence of IgM in the serum and is influenced by the state of activation of the PECs. Although naive cells display limited adherence to larvae, Brugia-primed PECs adhere efficiently, suggesting a requirement for cellular activation to achieve optimal adherence. IgM-depleted wt serum loses its potential to promote adherence of secIgM–/– cells to larvae, whereas the purified IgM fraction restores this effect, confirming the importance of this isotype.

Although IgM is the first isotype produced in response to various infectious agents, class switching and affinity maturation often result in the production of high titers of other isotypes later in infection. These other isotypes play important roles in host protection in other infectious diseases. We and others have shown that there is a robust Ab response to the filarial parasite after exposure. These Abs cover all isotypes (11, 25, 26, 27). Indeed, in the case of some specific somatic Ags, Abs appear to be distributed either to the IgG isotype (for instance, filarial myosin (27)) or IgE (filarial collagen (27)). Other workers have clearly documented the presence of substantial titers of IgG-4 and IgE in human filarial infections (28, 29, 30, 31, 32). Our data, however, would suggest that even though the Ab response, both total and filarial specific, can occur across a broad spectrum of isotypes, the protective Ab may be primarily IgM. The fact that class switching of Ab isotype appears not to take place even in primed mice raises the possibility that this epitope may be nonprotein in nature.

In this regard, one nonprotein epitope that has repeatedly been shown to be immunodominant in filarial infection is phosphorylcholine (33, 34, 35, 36, 37, 38, 39). In preliminary experiments (data not shown), we have found that a mAb of the IgM isotype (PC 140) (40) directed against phosphorylcholine does not facilitate the adherence of activated peritoneal cells to larvae. Nor does the presence of this particular mAb competitively inhibit the ability of primed wt serum to bind to the larvae. These experiments, however, do not conclusively rule out the possibility that phosphorylcholine is a protective Ag. Additional experiments to explore the importance of phosphorylcholine in this regard are currently underway.

Macrophages activated by Brugia infection display high levels of IgM on their surface, in comparison to those from naive mice, suggesting an up-regulation of IgM receptor(s) during the activation process. The candidate receptors for IgM on peritoneal macrophages include the recently documented Fc{alpha}µ and polymeric Ig receptors (41, 42). Experiments to address the role of these receptors in cell adherence and worm elimination are in progress. We hope that these studies will expand our understanding of not only mechanisms involved in host protection from filarial infections but also the hitherto unappreciated role of IgM in cell-mediated immunity.


   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 T.R. and B.R. contributed equally. Back

2 Address correspondence and reprint requests to Dr. T. V. Rajan, Department of Pathology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032. E-mail address: rajan{at}neuron.uchc.edu Back

3 Abbreviations used in this paper: wt, wild type; PEC, peritoneal exudate cell. Back

Received for publication January 20, 2005. Accepted for publication May 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Rajan, T. V., L. Ganley, N. Paciorkowski, L. Spencer, T. R. Klei, L. D. Shultz. 2002. Brugian infections in the peritoneal cavities of laboratory mice: kinetics of infection and cellular responses. Exp. Parasitol. 100: 235-247.[Medline]
  2. Ramalingam, T., B. Rajan, J. Lee, T. V. Rajan. 2003. Kinetics of cellular responses to intraperitoneal Brugia pahangi infections in normal and immunodeficient mice. Infect. Immun. 71: 4361-4367.[Abstract/Free Full Text]
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