Immunological interactions between Trichinella spiralis and Heligmosomoides polygyrus: Cross reactivity between muscle larvae and antibodies raised to unrelated antigens

Pergamon 0021b7514'97

PII: SOO20-7519@7)00049-0

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crosisl Reactivity Between AmtWxlies Raised to U MICHAEL

ROBINSON.*

THOMAS

R. GUSTAD

and MICHELE

R. ERICKSON

Department of Veterinary and Microbiological Sciences, North Dakota State University. Fargo, ND 58105, U.S.A. (Received

19 August

1996: accepted

6 March

19971

responseby T. spiralis.0 1997 Australian Society for Parasitology. PutrIMed by Ekmier Science Ltd. Key wwds:

Trichinella

spiralis;

Heligmosomoides;

IgGI: affinity chromatography;crossreactivity

INTRODUCTION

A significant feature of many parasites which exist as chronic infections is the ability to manipulate the host immune system (Behnke, 1987), particularly the response to heterologous infections (Ali & Behnke, 1984; Crawford et al., 1989). This mechanism can be demonstrated during concurrent infections in mice with the nematode parasites Trichinella spiralis and Hdigmosomoides polygyrus, where prior exposure to H. polygyrus will reduce the protective immune response to T. spiralis (Behnke et al., 1978), and also increase the “take” of the infection (Bell et al., 1992). Conversely, the inflammation induced by T. spiralis *To whom correspondenceshould be addressed.Fax: 1701) 231-7514; E-mail: [email protected]. 865

can actually act to remove H. pofygyrus infections (Behnke et al.. 1992). Therefore, there is substantial evidence that, although taxonomically unrelated, H. polygyrus and T. spiralis can have considerable influence upon each other. A notable characteristic of both T. spiralis and H. polygyrus infections is the production of high serum levels of IgGl (Chapman et al., 1979a,b; Williams & Behnke, 1983; Denkers et al., 1990). Despite data indicating a host protective role for antibody against both of these parasites (Pritchard et al., 1983; Williams & Behnke, 1983; Bell et al., 1992), there is also an opinion that immunoglobulin induced in some hosts may not have a decisive role in immunity, or may act by blocking other immune responses(Day et al., 1979: Pleass& Bianco, 1994; Venturiello et al., 1996). Therefore, the complex interactions between parasites and

M. Robinson

866

the induced immunoglobulin have not been completely elucidated. As earlier work had suggested that monoclonal antibodies (MAb) to T. spiralis muscle larvae (m.l.) could cross-react with H. polygyrus adult worm homogenate (AWH) (Robinson M. et al., 1991), we decided to investigate whether MAb to H. polygyrus could specifically react with m.1. antigens of T. spiralis. Further, we have recently shown that mouse serum proteins, including antibodies, can bind to specific antigens of H. polygyrus and this feature can be used to affinity purify mouse IgGl, of any specificity (Robinson M. et al., 1997). Thus we have examined whether antibodies of any specificity could bind to T. spiralis in a situation analogous to that which exists for H. pol,vgyrus. MATERIALS Mice. BALB/c were obtained

Department

(H-2d)

AND and

METHODS

outbred

Swiss

Webster

mice

from the Immunology Mouse Colony, of Veterinary and Microbiological Sciences,

North Dakota State University. Mice were 2 months old at the time of experimentation.

a minimum

of

The parasite. The production and maintenance of Heligmosomoidespolygyrus was as described by Jenkins St Behnke (1977). Trichinella spiralis was originally a generous gift from Dr D. L. Wassom, Colorado State University, and was maintained in BALB/c mice. Production of infective muscle larvae was as described by Wassom et al. (1983). Antigen. The production of H. polygyrus AWH and T. spiralis muscle larval homogenate (MLH), was as described previously (Robinson M. et al., 1994). T cell hybridomas. The production, maintenance, of T cell hybridomas in superantigen assays described previously (Robinson M. et al., 1995).

and use has been

Monoclonal antibodies. MAb 9A3, 13B3, HpD (all IgGl), 8B3 (IgM), for H. polygyrus AWH; MAb EA3. FD3 (all IgGl) to mouse thymulin and MAb lA6 (IgM) to KLH, were produced in BALB/c mice at NDSU, according to accepted methodology (Goding, 1986) and their specificity, or otherwise. for AWH has been described previously (Robinson M. et al., 1997). All other MAb were produced from hybridomas purchased from American Type Tissue Culture (ATTC). The hybridoma lB7.11 (IgGl) produces mouse MAb specific for 2,4,6 trinitrophenyl (TNP). Hybridoma BP107.2 produces a mouse IgG3 MAb specific for mouse Ia. All MAb were produced in cell culture and concentrated using 45% saturated ammonium sulfate (SAS), followed by

dialysis. k. polygyrus-specific, hyperimmunized mouse serum (HHIS) was bled from BALB/c mice, immunized during the nroduction of MAb. T. spiralis infection serum (TIS) was obtained from mice bledhuring the production of muscle larvae. Immune T. spiralis serum (THIS) was obtained from mice immunized with MLH, as for the production of HHIS. Normal mouse serum (NMS) was pooled from several naive BALB/c mice, bred in the mouse colony at NDSU. As thymulin is only 9 amino acids long (EAKSQGGSD), a thymulin-BSA conjugate was used as the antigenic stimulant and a thymulin-KLH (keyhole limpet hemocyanin) con-

er (I/. jugate was used to test for the specificity of the monoclonal antibodies produced. The thymulin-specific MAb used in these studies do not bind to KLH in isolation (data not shown). The specificity of the hybridomas was tested by ELISA, with AWH or thymulin-KLH as the target antigen. MAb 13B3 has been demonstrated as being positive for AWH, while MAb BP107.2 and FD3 show no cross reactivity for AWH by ELISA (Robinson M. et al., 1997). The specificity, or otherwise, of MAb 8B3, HpD, EA3, 9A3 and lB7.1 I for AWH is shown in Fig. 1. ELLSA. Evaluation of parasite-specific antibody levels was carried out as described previously (Robinson M. & Gustad. 1996). Protein (Western) blotting. One-hundred micrograms of T. spiralis MLH were electrophoretically separated on a 10% SDS-PAGE gel, then blotted onto a polyvinylidene fluoride (PVDFI membrane using Mini-PROTEAN II electrophoresis and blotting equipment (BIO-RAD), according to the manufacturers’ instructions. The blot was blocked overnight with Tris buffered saline (TBS), containing 3% bovine serum albumin (BSA) with, or without, 1% NMS, then washed 3 times for 10 min each wash, with TBS-0.2% Tween 20. prior to being transferred to a Mini-PROTEAN II Multi Screen apparatus (BIO-RAD) for probing. The blot was incubated with the appropriate monoclonal antibodies, diluted with TBS-1% BSA for 1 h, then washed 3 times, followed by a l-h incubation with AP conjugated goat antimouse IgG and IgM polyclonal antibody (Pierce), diluted 1:5000 with TBS-1% BSA. This was followed by a further 3 washes. The blot was then removed to a plastic container and washed once in TBS for 10 min, followed by the addition of BIO-RAD Immuno-Blot colour development reagent, 5bromo-4-chloro-3-indoyl phosphate (BCIP) and nitroblue tetrazolium (NBT), in dimethylformamide (DMF) for 7 min. The reaction was stopped by washing with distilled water. All procedures were carried out at room temperature. Afjnity chromatography. Twenty-five milligrams of 45% SAS precipitated MAb FD3 was incubated overnight with 3 g of pre-swelled CNBr-activated Sepharose CL-4B (Pharmacia) in counline buffer (0.1 M NaHCO,, 0.5 M NaCl, pH 8.3). The resuhingmixture was then washed repeatedly with blocking buffer (0.2 M glycine, pH 8.0). followed by alternate washes with acetate buffer (0.1 M sodium acetate, 0.5M NaCI, pH 4.0) and coupling buffer, followed by a final wash with PBS. The MAb-Sepharose mixture was poured into a plastic liquid chromatography column and allowed to settle. A solution of T. suiralis MLH (1.5 mg in 2ml). in loading buffer (20mM MbPS, 20mM‘ NaC< pH 7.2) was then allowed to pass through the column, under gravity. The column was washed with loading buffer, followed by the addition of elution buffer (4M NaCl, 1OOmM Tris, pH 8.5) to remove the bound protein. The protein removed from the column was quantified, approximately, by absorption at 260nm and 280nm using the formula given below, then diluted and the excess NaCl removed by centrifugation, using a 10000mw Centriprep concentrator (Amicon). Protein concentrations were calculated using the formula: 1.55 x A ,,,-0.76x AzeO (=mgml-‘).

RESULTS

The initial exercise was carried out to determine if MAb to H. polygyrus could be demonstrated, by

Interaction

between

H. polygyrus

antibodies

and T. spiralis

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Antibody Fig. I. Kesults from an ELlSA to demonstrate the binding of anti-H. polygyrus MAb YA3. HpD, 8B3 to AWH (IO /~gml ’ 1. MAb 167.1 I (anti-TNP) and EA3 (anti-thymulin) are included as control. All MAb ‘were adjusted to 100 ~8 ml ’ before use. Absorption data are for the appropriate MAb. minus the value for NMS.

ELISA, to cross react with T. spiralis MLH. The results of this gave a mixed pattern, with MAb 8B3 (IgM) and 9A3 (IgGl) being positive, while in contrast MAb 13B3 (IgGl) had undetectable cross reactivity using this technique (Fig. 2). A control MAb, IA6 (IgM; anti-KLH) also showed no binding to MLH. To continue this investigation another ELISA was carried out, this time using antisera to either T. spiralis or H. pa/~>g~rus. These results indicated that HHIS,

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raised to H. polygyrus. did bind to the T. spirulis homogenate. As mig,ht be expected, the binditlg of THIS and TIS to MLH exceeded any cross reactivity between antiserum tie H. polygyrus and MLH. All of the antisera binding demonstrated a pronounced prozone effect (Fig. 3). The results so far indicated that antibodies and antiserum specific fos H. poiygwvrus could cross react with T. spiralis MLH, but that this cross reactivity was not universal for all antibodies tested. and neither

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Fig. 2. Results from an ELISA demonstrating the binding of anti-H. polygyrus MAb 13B3,9A3 and 8B3, to T. spiralis MLH (IO~grnl--I). MAb IA6 (anti-KLH) is included as a control.

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M. Robinson et al

868

was it restricted to specific immunoglobulin isotypes. To extend these observations a Western blot was carried out using T. spiralis MLH, where the probes used were various MAb and antisera. The results confirm the data obtained with the ELISA and suggest that 2 MAb, 9A3 and 8B3, identified specific but dissimilar antigens. Two other anti-H. polygyrus MAb, 13B3 (see also Fig. 2) and HpD, failed to bind to any MLH antigen. Control MAb, including lA6 (anti-KLH; see Fig. 2) also failed to bind. Furthermore, antisera to H. polygyrus (HHIS) also cross reacted strongly with specific MLH antigens (Fig. 4a). As we were concerned about the possibility of immunoglobulins nonspecifically binding to MLH, we repeated the Western blot, shown in Fig. 4a, except this time we included 1% NMS in the blocking buffer. The results indicate that in addition to the previously identified antigens, all lanes, including those without antiserum or MAb as probes, showed distinct bands (Fig. 4b). Interestingly, 2 of those bands, of 43 and 50 kDa approximate molecular weight, were also specifically recognized by MAb 9A3 (Lane 14) and H. polygyrus antisera (HHIS; Lane 18), in addition to T. spiralis antisera (Lanes 2, 3, 16 and 17) in Fig. 4a. Also, MAb 8B3 recognized a number of protein bands, many of which appear to correspond to antigens recognized by T. spiralis antisera (Fig. 4a; Lanes 8, 16 and 17). The results shown in Fig. 4b appeared to indicate that some factor contained in NMS was non-specifically binding to MLH, and this in turn was recognized by the 2” antibody used in the Western blot. On the assumption that this factor was mouse immunoglobulin, this non-specific binding could be utilized to extract those antigens which bound in this way to mouse immunoglobulins. Therefore, an affinity column was produced, using MAb FD3 coupled to CNBr-activated Sepharose CL4B. This MAb was chosen becauseit is highly specificfor mouse thymulin and does not cross react with either T. spiralis MLH (Fig. 4) or H. polygyrus (Robinson M. et al., 1997). Using this affinity column, we were able to extract specific proteins from MLH. The chromatogram from the affinity column in shown in Fig. 5 and the SDSPAGE of the extracted protein is shown in Fig. 6. As predicted from the Western blot, the affinity column was able to successfully extract proteins from the MLH with proteins of approximate molecular weight of 25, 33,38 and 43 kDa being noticeable. Conversely the major protein of approximately 68 kDa, seen in both MLH and T. spiralis AWH, was not bound by MAb FD3. DISCUSSION

The interrelationships between 2 pathogens inside 1 host are complex and in many cases difficult to

predict. However, it is important to understand how these interactions are resolved because in nature, multiple infections with a variety of parasitic organisms are always a consideration (Udonsi et al., 1996). H. polygyrus, for example, has been demonstrated to adversely affect the immune response to Trichuris muris (Jenkins & Behnke, 1977) and Hymenolepis citelli (Alghali et al., 1985) in addition to T. spiralis (Behnke et al., 1978). More recently, it has been shown that even pathogens with non-cross reacting antigens, such as Mycobacterium sp., can influence the course of infection in certain mouse strains (Robinson K. et al., 1996). The data contained here have attempted to rationalize some of these findings and to show that antigenic crossreactivity existsbetween T. spiralis muscle larvae and H. polygyrus AWH. However, the relationship is by no means straightforward, as the cross reactivity is restricted to a few MAb of differing isotypes. This suggeststhat the interaction is not due to just 1 immunodominant, but conserved, epitope produced by both species, but is in fact more complex. This is demonstrated by the fact that the 2 H. polyg-vrusspecific MAb, which cross reacted with T. spiralis MLH recognized differing proteins, which in both casesare also antigens recognized during a T. spiraiis infection (Fig. 4a; Lanes 8, 14 and 16). The ability of MAb 8B3 to bind to so many proteins of T. spiralis (Fig. 4a; Lane 8) suggests the recognition of a conserved epitope, perhaps carbohydrate, found on many proteins of dissimilar size. Such an observation was also noted by Denkers et af. (1991). Further information, which can be gleaned from the Western blots, is that the antigens recognized by the 2 MAb are also recognized by mice which have been infected with T. spiralis (Fig. 4a), and are not simply an artifact of injecting mice with parasite homogenate. Therefore, it is possible that this specific cross reactivity could influence the course of an infection with T. spiralis. We have considered the possibility that there is a bacterial contaminant in both H. polygyrus AWH and T. spiralis MLH, which is the source of the cross reactivity, but comparative Western blots indicate that the protein bands recognized in each speciesare dissimilar (data not shown). Therefore, we must conclude that antibodies which are raised against H. polygyrus can, and do, bind to specific antigens of T. spiralis. This could account for at least part of the complex interactions observed between the 2 parasites (Behnke et al., 1978, 1992; Bell et al., 1992). It is perhaps the second section of data contained here which is most surprising. We have already shown that antibodies bind non-specifically to H. polygyrus (Robinson

M. et al., 1997) and suspected that this

Interaction between H. polygvrus antibodies and T. spimlis I 1

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Fig. 4. Protein blot showing the binding of MAb and antisera to T. spiralis MLH. All MAb were adjusted to 1OOpgmJ -I before use. All antisera were diluted 1:lOO. Blots were blocked with (a) TBS-3% BSA, or (b) TBS-3% BSA-1% N&&S. Key to lanes: 1, cell culture medium; 2, THIS; 3, TIS; 4, NMS; 5, cell culture medium; 6, blank; 7, FD3; 8, 8B3; 9, BBlg7.2; 10. HpD; 11, 1383; 12, EA3; 13. 1A6; 14, 9A3; 15, lB7.11; 16, TIS; 17. THIS; 18. HHIS; 19. NMS; molecular weight marker (kDa).

scenario might also exist for T. spiralis (Robinson M. et al., 1991) and these results prove that this is so. Once again, the proteins which are bound nonspecifically by antibodies are also antigens recognized by antiserum from a patent infection, and hence there exists the likelihood of there being a functional role for this interaction. The data also indicate that other proteins, including BSA, can bind to the same sites as the antibodies, because these sites can be blocked by a BSA-containing buffer. This would have occurred

in the ELISA (Figs 1-3) and in the Western blot which had been blocked by TBS3% BSA (Fig. 4a). However, in a competitive situation, mouse immunoglobulins appeared to bind pre&entiaIly as indicated in Fig. 4b, and by the results from the affihity column (Fig. 6). We have no data suggesting the nature of these antigens of T. spiralis that are bound by non-spe&c antibodies, and cannot speculate upon their biochemical profiles. Lik:ewise, any functional role for

870

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Sample Number

Fig. 5. Affinity chromatogram of the isolation of T. spiralis MLH antigens, using an affinity column of MAb FD3, coupled to CNBr-activated Sepharose 4B. Unbound protein was contained in samples 621. Bound protein was eluted in samples 3542. these antigens has yet to be determined. However, other groups have shown that affinity purified T. spirulis m.1. antigens are capable of transferring protection to naive animals. Although the MAb used in these purification procedures were specific for T. spiralis (Gamble, 1985; Ortega-Pierres et al., 1989;

Fig. 6. SDS-PAGE bound

of affinity purified protein

fraction;

T. spiralis

2, unbound

et al. Denkers et al.. 1990; Appleton & Usack, 1993) the data presented here suggest that in these situations other, non-specific, antigens might also have been extracted. It remains to be seen whether the proteins that bind non-specifically to antibody have any role to play in the immune response to T. spiralis. Nevertheless, MAb FD3 does extract a protein of 43 kDa which might be comparable to one of similar molecular weight identified as being host protective by several other groups (Silberstein & Despommier, 1984, 1985; Robinson M. et al., 1991; Appleton & Usack, 1993). It has already been suggested that a protective 43 kDa T. spiralis antigen may be masked by carbohydrate antigens, which may serve to divert the immune response (Robinson K. et al., 1995). These data may indicate that the masking of antigens is more extensive than was previously suspected, and could extend to immunoglobulins which are not directed at the organism causing the actual infection. As a final point, the fact that mouse IgGl binds so effectively to T. spiralis could also be important, as this isotype has been identified as being raised during infection (Pritchard et al., 1984; Denkers et al., 1990; Wahid & Behnke, 1993), and by stimulation with soluble parasite antigens (Denkers et al., 1991; Robinson M. & Gustad, 1996), for both H. polygyrus and T. spiralis. In conclusion, we have shown that there is crossreactivity between T. spirafis muscle larval antigens, and both antisera and MAb specific for the unrelated

MLH proteins. Key to lanes: mwm, molecular weight marker (kDa); 1, fraction; 3, unfractionated MLH; 4. T. spiralis AWH.

protein

Interaction nematode

H. pol.vgyrus.

Although

between

the cross

H. pol~vqyrus

reactivity

by MAb is not universal, both IgGl and IgM MAb are able to bind, and recognize dissimilar antigens. .4dditionally. IgGI which does not specifically bind to T. spiralis muscle larvae could be used to extract proteins via affinity chromatography. These nonspecifically bound proteins were found to be also recognized by T. spirulis infected mice, and some were apparently present in T. spiralis AWH. It is postulated that binding of these disparate antibodies may be important in protection against. and/or susceptibility to. T. .spiralis.

A~knowl~d~emrr?ts---This USDA/NRICGP Grant thank Dr D. L. Wassom, generous gift of Trichinrlla

work was supported in part by 94-37208-1036. We would like to Colorado State University, for the spiralis.

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Venturiello S. M., Costantino S. N. & Giambartolomei G. H. 1996. Blocking anti-Trichinella spiralis antibodies in chronically infected rats. Parasitology Research 82: 77-8 1,

Wahid F. N., Behnke J. M. 1993. Immunological relationships during primary infection with Heligmosomoidespolvgyrus (Nematospiroides dubius): parasite specific IgG 1 antibody responses and primary response phenotype. Parasite Immunology 15: 401413. Wassom D. L., Brooks B. O., Babish J. G. & David C. S. 1983. A gene mapping between the S and D regions of the H-2 complex influences resistance to Trichinella spiralis infections of mice. Journal of Immunogenetics 10: 371378.

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