Brugia pahangi: Immunologic evaluation of the differential susceptibility to filarial infection in inbred Lewis rats

EXPERIMENTAL PARASITOLOGY 52, 147- 159 (1981)

Brugia pahangi: Susceptibility

Immunologic Evaluation of the Differential to Filarial Infection in Inbred Lewis Rats

R. D'A. GUSMAO,’ Laboratory

of Parasitic

A. M. STANLEY,

AND

E. A. OTTESEN

Diseases. National Institute of Allergy and Infectious of Health. Bethesda. Maryland 20205. U.S.A. (Accepted

for publication

5 October

Diseases.

National

Institutes

1980)

GUSMAO, R. D.A., STANLEY, A. M., AND OTTESEN, E. A. 1981. Brugia pahangi: Immunologic evaluation of the differential susceptibility to tilarial infection in inbred Lewis rats. Experimental Parasito/og.v 52, 147- 159. After inoculating inbred Lewis rats with infective larvae ofBrugia pahangi, we found consistently that patent infection was established in only about two-thirds of experimental animals. To determine which aspects of the host response were responsible for this differential susceptibility to infection, we evaluated all animals with respect to blood leukocyte levels, antililarial IgG and IgE antibody production, and specific lymphocyte responses to mitogens and tilarial antigens during the first 50 weeks of infection. In the critical “prepatent” period of infection, each of these responses developed in an essentially identical fashion in the infected and resistant groups except for the production of specific IgE antibodies. While those animals destined to resist infection developed specific IgE antibodies between Weeks 5 and 8, none of the animals that became microfilaremic showed a similar IgE response. These findings suggest that the development of specific IgE antibodies played a role in protecting the animals from acquiring tilarial infection. INDEX DESCRIPTORS: Brugia pahangi: Nematode; Filaria; Microfilaria; Rat; Immunity: Eosinophil; Lymphocyte: Antibody, IgE; Protection. INTRODUCTION

Though a number of experimental models exist for studying lymphatic-dwelling Iilariae (Schacher and Sahyoun 1967; Ewert and Singh 1969; Ash and Riley 1970b; Malone and Thompson 1975; Denham and McGreevy 1977), most of these models involve animal hosts such as the jird, hamster, or cat which are relatively difficult to investigate and manipulate immunologically. Recent work by several investigators, however, has attempted to overcome this problem by establishing such infections in strains of either inbred rats (Fox and Schacher 1976; Weller 1978) or inbred mice (Suswill0 et al. 1980), animals for which a large repertoire of immunologic techniques and reagents are readily available. 1 Current address: Tropical Hopkins University School Health, Baltimore, Maryland

Medicine Center, Johns of Hygiene and public 21205, U.S.A.

While it might be anticipated that these inbred animals would respond to a given infection in a relatively uniform mar,‘_::-, it has been of interest to us to note, both in our own work with Brugia pahangi infection in Lewis rats as well as in the observations of others (Fox and Schacher 1976; Weller 1978), that not all such infected animals develop patent filarial infection. Indeed, despite essentially identical experimental conditions, only 55-80% of Lewis rats given equivalent inocula of B. pahangi infective larvae (L3’s) develop patent infection; the remainder appear to resist the establishment of infection. Because similar patterns of infection and apparent resistance to infection are seen in human populations residing in areas endemic for Brugian and Bancroftian filariasis, we felt it important to analyze the immune mechanisms potentially responsible for these observations. Therefore, both cellular and 147 00144894/81/040147-13$02.00/O Copyright0 1981 by Academic Press, All rights of reproduction

Inc. in any form rererved.

148

GUSM.&O,STANLEY,AND

OTTESEN

humoral responses of such rats were evalu- fractions of the blood from mf-negative ated in detail, with our goal being to discern animals were filtered through Nuclepore those mechanisms responsible for deter- filters to detect even very low levels of mimining which of these inbred animals ac- crofilaremia. quire and which “resist” the experimental Peripherul Blood Leukocyte Evaluation filarial infection. Blood was obtained at weekly intervals MATERIALS AND METHODS from the tail vein of infected animals for Experimental Infections leukocyte enumeration. Total white blood A total of 39 male Lewis rats (Microbio- cell counts (wbc) were performed with a logical Assoc., Rockville, MD, USA) were Coulter Counter (Coulter, Inc., Hialeah, used in three separate experiments. All rats FL, USA). Differential counts of the leukowere 8- 10 weeks of age and in good health cytes were made by counting 200 cells after at the time of infection. They were main- the peripheral blood smears were stained using fast green with a neutral red countained with food and water ad libitum. terstain, a technique designed to facilitate Infective larvae (L,) of Brugia pahangi were obtained from Aedes aegypti mos- distinction of eosinophils from other leukoquitoes infected 12- 14 days previously by cytes (Ottesen and Cohen 1978). Absolute blood meals on infected jirds. Raising of the counts of each cell type were determined mosquitoes and collection of the larvae from the total wbc and the differential were carried out either at NIH (by Dr. counts. Robert Gwadz) or at the University of Georgia (by Dr. John McCall) according to Parusite Antigens standard techniques (Ash and Riley 1970a). Saline extracts of B. pahungi adults, B. Twenty-three rats were infected once each pahungi microfilariae, and Dirofilaria imwith 100L,‘s subcutaneously through a No. mitis adult parasites were used as antigens. 23 needle in the left hind footpad. In the The Brugia adults and mf were collected by other 16 rats, two L,‘s were administered peritoneal lavage of jirds infected inby subcutaneous injection into the left hind traperitoneally 2-3 months previously with footpad twice a week for 25 weeks (total of 400 L,‘s. Male and female adult worms 100 L:,‘s). were frozen after extensive washing and maintained at -20 C. Microfilariae were Detection of Microfilariae separated from jird peritoneal cells by graBlood was taken from either the tail vein dient density centrifugation at room temor the heart and examined for microtilariae perature over a cushion of Hypaque (9.7%, (mf) weekly after the eighth week of infec- Winthrop Laboratories, New York, NY, tion. In all cases, a minimum of 0.5 ml of USA) and Ficoll (6.5%. Pharmacia Fine blood was filtered through 3.0 pm Nucle- Chemicals, Piscataway, NJ, USA). After pore filters (Nuclepore Co., Pleasanton, washing twice with RPMI-1640 medium CA, USA) and the number of trapped mi- (Grand Island Biologicals, Grand Island, crofilariae counted after the filter was NY, USA) containing 124 pg/ml penicillin stained with Wright’s stain. In animals per- and 70 pug/ml streptomycin, the microsistently mf negative, an additional 0.5- 1 filariae were cultured overnight in this ml of blood was examined by Knott’s tech- medium at a concentration of <20,OOO/mlin nique (Knott 1935). Furthermore, when an environment of 37 C and 5% CO,. Mimononuclear cells were separated from crotilariae were harvested the next day, whole blood over gradients for the cellular washed in phosphate-buffered saline (PBS, immune studies (see below), the remaining pH 7.4), and frozen at -20 C.

h-ugia

fXZhUngi:

RAT SUSCEPTIBILITY

D. immitis adult worms (male and female) were collected from the hearts of experimentally or naturally infected dogs, washed extensively, and frozen at -20 C. Saline extracts were prepared from the frozen parasites by first sonicating or pulverizing the worms in a Ten Brock glass homogenizer, and then incubating the material in PBS initially at 37 C for 4 hr and then overnight at 4 C. Following high-speed centrifugation at 20,OOOgfor 30 min, the soluble supernatant material was passed through a 0.45pm filter (M&pore Corp., Bedford, MA, USA) and stored in aliquots at -70 C. The antigens were standardized by Lowry protein determinations (Lowry er al. 1951). Approximately 250 pg of antigen could be obtained from either 100 adult worms or IO5 microlilariae by this technique . Mitogens

Phytohemagglutinin (PHA-P: BurroughsWellcome, Research Triangle Park, NC, USA) was used at concentrations of 1, 2, and 5 pg/ml. Pokeweed mitogen (PWM; GIBCO, Grand Island, NY, USA) was added to cultures at its previously determined optimal stimulatory concentrations of 0.1 and 0.2%.

149

dicator rats in 0.05-ml volumes. Seventytwo hours later these rats received 4 mg of Dirofilaria sp. adult antigen intravenously along with 0.5 ml of a 3% solution of Evans blue dye (J. T. Baker Chemical Company, Phillipsburg, NJ, USA). Thirty minutes later the animals were sacrificed and the skin reflected to determine the positive bluing reactions. Reactions greater than 2 mm diameter were considered positive, and results are expressed as the reciprocals of the greatest dilution of sera yielding positive reactions. The Dirofiluria sp. antigen was used for these “specific” IgE antibody determinations because of the difficulty in obtaining adequate amounts of the homologous Brugia sp. antigen. (A total of 160 mg of DirojZaria sp. antigen was used in the 40 indicator rats needed for these PCA studies.) No sera from uninfected animals ever showed positive PCA reactivity. Furthermore, sera from animals injected with the fluid collected by processing uninfected mosquitoes in the same manner as infected mosquitoes showed no positive PCA reactivity to DirojTluria sp. antigen over the 15 weeks of observation post injection. Lymphocyte

Cultures

Blood obtained from individual rats by cardiac puncture was heparinized and the Specific IgG antibodies were determined red cells sedimented with 3% gelatin (Type in an enzyme-linked immunosorbant assay P-20, Kind and Knox Gelatin Co., Camden, NJ, USA) used in a ratio of 1 part gelatin to (ELISA) with both adult and microfilarial B. pahangi antigens. The optimal concen- 2 parts whole blood. If the resulting leukotrations of antigen in the test were found by cyte suspension contained microfilariae, “box titration” of serial dilutions of antigen these were removed by passage of the susand antiserum to be 2 pg/ml for each pension over a Hypaque - Ficoll gradient as antigen. Other aspects of the technique described above. The leukocytes recovered have been previously described (Lunde et by either of these techniques were washed three times in Hank’s balanced salt solution al. 1979). before being suspended in RPMI-1640 tisIgE Antibody Determinations sue culture medium supplemented with Antifilarial IgE antibody titers were de- penicillin (124 pug/ml), streptomycin (70 termined in passive cutaneous anaphylaxis pg/ml), glutamine (300 pg/ml), and 5% au(PCA) reactions (Stechschulte et al. 1970). tologous, heparinized plasma or pooled, Test sera were serially diluted with PBS normal heat-inactivated (56 C for 30 min) and injected intradermally into normal, in- Lewis rat serum. IgG Antibody

Determinations

150

GUSM~O,STANLEY,ANDOTTESEN

Cells were cultured at 37 C in 0.2 ml of supplemented medium in wells of round bottom tissue culture plates (Cooke Laboratory Products, Alexandria, VA, USA). The density was 100,000 cells/well; atmosphere, 5% CO, with 95% air;, and temperature, 37 C. Antigens or mitogens were added to the cultures in varying concentrations. On the fourth day (80 hr) of culture (unless specifically noted in certain experiments) 1 &i of [3H]thymidine was added to each well. Cells were harvested 4 hr later onto a glass-fiber filter paper (Reeve Angel No. 943H; Whatman, Inc., Clifton, NJ, USA) with a multiple automated sample harvester (MASH II, Microbiological Associates, Rockville, MD, USA). The activity of the cultures was determined by liquid scintillation spectrometry and expressed as the absolute level of thymidine incorporated by the cells (in disintegrations per minute, dpm) or in terms of the stimulation ratio E/C: average dpm in stimulated cultures average dpm in unstimulated ’ (control) cultures All assays were performed in triplicate (except for the unstimulated, control cultures which were carried out in sextuplicate), and mean values of the responses were used for calculations. RESULTS

became microfilaremic, the remainder never developing patent (i.e., microfilaremit) infection (Fig. 1). In animals which became mf positive, the prepatent period varied depending on the inoculation schedule, being IO- 16 weeks for the single large inoculum group and 14-20 weeks for the multiple small inocula group (Fig. 1). The degree of microfilaremia which developed varied considerably for rats with both types of infection, ranging from 2 to 796 mu/ml of blood. All positive animals had microfilariae detectable on at least four different bleedings. Amicrofilaremic animals remained negative during all weekly bleedings. Autopsy evaluation of experimental animals for the presence of adult worms met with such limited success that results are not reported here in detail. No adult worms were ever found in mf-negative animals, but even among those animals with patent infections, there were many in which adult worms could not be demonstrated either by direct visualization or after “soaking” the skin and carcass in Hank’s balanced salt solution (Ash and Riley 1970a). Therefore, direct parasitologic evidence for the presence or absence of adult parasites in mf-negative animals is not reliable in these experiments; however, the weight of the immunologic evidence presented below suggests that infection was never successfully established in the mfnegative animals.

Parasitology

Three separate experiments were carried out. In two of these, Experiments I and III, rats were given a single subcutaneous inoculum of 100 infective Brugia pahangi larvae; however, in Experiment II, the protocol was varied so that again a total of 100 L,‘s were given to each rat, but this time in doses of 2 L,‘s twice a week for 25 weeks. The most interesting finding common to all three experiments was the fact that regardless of the schedule used for inoculating infective larvae, only 55-77% of animals

Peripheral

Blood Leukocyte Responses

Detailed evaluation of the peripheral blood leukocyte response to infection was carried out only in Experiment III in which 11 rats were each exposed to a single inoculum of 100 L,‘s. Six of these rats developed patent infections and five did not. The peripheral blood leukocyte responses of these two groups of animals are depicted in Fig. 2. An early lymphocytosis and neutrophilia seen in both groups of animals (Figs. 2B and C) diminished after the first several

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RAT SUSCEPTIBILITY

,

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,

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FIG. 1. Cumulative percentage of animals developing Brugia pahangi microfilaremia. Solid line records data for the 12 animals in Experiment I (single large inoculum group) and dotted line, data for the 16 animals in Experiment II (multiple small inocula group).

weeks of infection, and at no time were there significant differences between the lymphocyte and neutrophil responses of the two groups of animals. Because there was no “control” group of uninfected animals studied concurrently, these early changes

must be interpreted with caution. Eosinophil responses, however, (Fig. 2A) did differ significantly between the animals that developed patent infection and those that did not. Though an equivalent blood eosinophil response developed in both

TABLE I Detection of Antifilarial IgE Serum Antibodies (Experiment III) Rats developing microtilaremia with Brugio pohangi

Rats not developing microtilaremia

No.

Weeks 5-8

Week 17

No.

Weeks 5-8

Week 17

1 2 3 4 5 6

- 0 -

+* + + + + +

7 8 9 10 11

+ + + +

-

fl PCA-negative sera. * PCA-positive sera.

152

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STANLEY,

AND

OTTESEN

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2

,6,&30~

;’

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LYMPHOCYTES

i?ij~

’

L--L-I.!2

4

6

8

10

WEEKS

~I-II 12

14

16

18

: 20

I 22

24

POST INFECTION

FIG. 2. Blood leukocyte responses to Brugicr pcrl~lgi infection. Data were collected from the 11 animals in Experiment III. The six rats which developed patent infection are represented by the solid lines and closed circles while values for the five rats which remained amicrofilaremic are presented as dashed lines and open symbols. Error bars signify &SEM.

groups during the early, “prepatent” phase of infection (peaking in the fifth week), only the animals with patent microfilaremia developed a second phase of eosinophilia beginning after the twelfth week of infection, the time at which those animals became microfilaremic. This second phase of eosinophilia was sustained in these animals throughout the remaining 3 months of observation.

first lo- 15 weeks of infection regardless of the inoculation schedule used. Thereafter in the single-inoculum experiment (Fig. 3A), animals with patent infections demonstrated persistently high or increasing antibody titers while those not developing patency showed a progressive falloff in levels of specific antibody. While titers in patent animals from the multiple inocula group (Fig. 3B) also were generally higher than the nonpatent animals in this group Specific ZgG Antibody Responses after the beginning of patency, the differFor Experiments I and II, IgG antitilarial ences were not so striking as in the singly antibody titers were determined in an inoculated animals, probably because both ELISA assay using cross-reactive Di- of these groups of animals continued to rerojiluria sp. antigen. The results (Fig. 3) ceive injections of L,‘s twice weekly show no appreciable differences in anti- through Week 25 of the experiment. body titers between the microfilaremic To examine the development of IgG and nonmicrofilaremic groups during the antililarial antibodies with more specific re-

BrUgiU @WZgi:

BAT SUSCEPTIBILITY

153

1:160

1:40

I

’ 2

I 6

1 10

I I I I 14 18 22 26 WEEKS OF INFECTION

I 28

I 34

I 38

FIG. 3. Development of antifilarial IgG antibodies during Bvugia pahangi infection. Values are geometric mean titers (?SEM) determined by microplate ELISA techniques using Dirojilaria adult antigen. Panel A depicts data from the single large inoculum group (Experiment I). Solid line follows titers of the 9 animals that developed microtilaremia and the dotted line, titers of the 3 animals remaining negative. Panel B presents data from the multiple small inocula group (Experiment II). Solid line marks titers of the 10 animals that developed microfilaremia and the dotted line, titers of the 6 animals remaining negative.

agents, ELISA determinations in Experiment III were carried out with adult and microfilarial Brugia sp. antigens. When adult Brugia antigen was used, the differences between the groups of animals becoming or not becoming patent in this “single inoculum” experiment were almost identical to the findings in Experiment I in which adult Dirofilaria sp. antigen was used (Fig. 4A); that is, there were similar high titers in both groups until the time of patency when the two curves diverged. With the use of microfilarial Brugia sp. antigen, however, the two groups were seen to be even more clearly distinguished. Following

patency, high titers of microfilaria-specific antibody developed in the group with patent infection though no such antibody was produced by the nonpatent group (Fig. 4B). Filaria-Speci$c

IgE Antibody

Responses

Two distinct patterns emerged when the development of filarial-specific IgE antibodies was examined in all animals inoculated with infective larvae. Twelve animals received a single large inoculum of L,‘s in Experiment I. Of these, all three in which patent infection was never established developed IgE antibodies demonstrable during the early period of infection

154

GUSM/iO,

STANLEY.

FIG. 4. IgG antibody responses to Brrcgirr pahongi antigens in the 11 animals of Experiment III. Panel A records the responses to adult worm antigen and Panel B, the responses to microfilarial antigen. Solid circles and solid lines mark the geometic mean titers of the six rats which developed patent infection and open symbols and dashed lines, the titers of the five amicrotilaremic animals. Error bars signify tSEM.

(2-7 weeks), whereas none of the nine which subsequently became microtilaremic had detectable IgE antibodies during this same period. Once microfilaremia became established in these latter animals, however, antifilarial IgE antibodies were produced, and these persisted at high levels for the duration of the experiment (Fig. 5a). An essentially identical pattern of IgE antibody development was also found in the group of 16 animals inoculated with multiple small doses of infective larvae in Experiment II (Fig. 5b). Here again, only the six animals that never became microfilaremic had detectable IgE antibodies during the early period of infection, and only after the infection became patent did the microfilariapositive animals manifest antifilarial IgE antibodies. In Experiment III, such detailed titration of the specific serum IgE antibody was not

AND

OTTESEN

FIG. 5. Development of antitilarial IgE antibodies during Bncgia pahdngi infection. Values are geometric mean titers (-cSEM) obtained by PCA reactions. Panel A presents data from Experiment I, the single large inoculum group. Solid line follows titers of the 9 animals that developed microtilaremia and the dotted line, titers of the 3 animals remaining negative. Panel B presents data from Experiment II, the multiple small inocula group. Solid line marks titers of the 10 animals that developed microtilaremia and the dotted line, titers of the 6 animals remaining negative. Dirojilnrirr antigen was used to elicit PCA responses.

carried out. Rather, sera from each of the 11 inoculated rats were “screened” for such antibody by carrying out the PCA test on three dilutions (1:2, 1:4, 1:8) of each serum collected at Weeks 5, 7, and 8 during the “prepatent” period and at Week 17 after patency was (or would have been) established. The detection of specific IgE antitilarial antibodies in these two groups of animals is recorded in Table I. Four of the five animals in which patent infections did not develop showed specific IgE antibody during the early phase of the infection, and none had such antibodies by 17 weeks after inoculation. In contrast, none of the six animals that went on to harbor a patent infection had any “early” IgE antifilarial

Brugia pahangi:

RAT SUSCEPTIBILITY

155

antibodies, but all developed such antiAdult Antigen Microfilmid Antigen 12 bodies after patency was established. t t That the specific reaginic antibodies being detected by PCA were of the IgE class and not the short-acting, heat-stable IgG reagins sometimes found in the rat (Morse et al. 1969) was shown by the fact that heat inactivation of individual sera for 4 hr at 56 C completely destroyed the activity of these reagins. Indeed, though antifilarial IgG reaginic antibodies were specifically sought in the sera by techniques I I I T 5 5 2 3 4 2 3 4 optimal for their detection (Morse et al. DAYS OF LYMPHOCYTE CULTURE 1969), none appeared during the course FIG. 6. Lymphocyte responses to Brugiu pahangi of infection (data not shown). antigens in two rats immunized 3 weeks previously Cell-Mediated

Immune Responses

Extensive studies of the lymphocyte proliferative responses to mitogens and parasite antigens were carried out during all three experiments. Though the culture techniques and antigens used for study clearly detected specific proliferative responses to filarial antigens in rats immunized with these antigens (Fig. 6), consistent patterns of lymphocyte responsiveness to these same antigens during the course of infection were difficult to discern (data not presented in detail). Of particular interest to the present study, however, was the fact that no differences in lymphocyte proliferative responses could be detected between animals which developed patent infection and those that did not. This point is illustrated in Fig. 7 where direct comparisons are made between the patent and “nonpatent” groups of animals with respect to their peak lymphocyte responses to mitogens and parasite antigens both during the prepatent period of infection (Weeks l-8) (Fig. 7A) and during the postpatent period (Weeks 17-84) (Fig. 7B). All animals were studied on multiple occasions and all antigens and mitogens employed over a range of concentrations. The term “peak response” refers to the maximal reaction of each animal to any concentration of antigen or mitogen used at any week

with Dirofihria sp. adult antigen (100 pg) in complete Freund’s adjuvant. Two rats (solid and open symbols) were studied and each antigen was tested~at two or three diierent concentrations; 100(circles), 50 (squares), and 10 &ml (triangles). Cultures were harvested at Days 2, 3, 4, and 5 to assess the kinetics of response.

for either the pre- or postpatent period (see legend to Fig. 7). DISCUSSION

Each of the experimental animal models of human filarial infection has its own particular strengths and weaknesses for study. One of the most intriguing aspects of the Brugia pahangi model in inbred Lewis rats, and the subject around which most of the present investigation has focused, is the fact that despite similar exposures to infective larvae, not all animals in any experimental group acquire patent (i.e., microtilaremic) infection. Instead, a minority of animals in each group remain persistently amicrofilaremic. Since this situation is reminiscent of the epidemiologic findings in human populations from filarial endemic areas, it appeared important to us to better our understanding of these observations by seeking answers to two specific questions. First, had the amicrofilaremic animals in our experimental groups actually resisted the infection successfully or were they in fact infected with adult worms but im-

156

GUSMAO,

PWM

STANLEY,

B.p Adult

AND

E.pmf

PHA

OTTESEN

PWM

BP. Adult

r3.p.mf

FIG. 7. Lymphocyte transformation responses to Brugiu pcrhangi antigens and to mitogens in infected and “resistant” rats. All animals were studied on multiple occasions both during the prepatent period of infection (Weeks l-8) and during the postpatent period (Weeks 17-84). “Peak responses” of each animal which were used in plotting the figure were defined as the maximal reaction of that animal to any concentration of each antigen or mitogen used at any week during either the pre- or postpatent period. Concentrations of mitogens are given under Materials and Methods while those for both adult and microfilarial antigens were 100, 50, and 5 fig/ml. For the prepatent period, values from 8 animals that remained persistently mf negative are compared with those of 9 animals that became microfilaremic (hatched bars). For the postpatent period (Panel B) values were derived from 7 amicrofilaremic and 16 microfilaremic animals. These data were obtained from lymphocytes cultured in autologous plasma, but findings from cultures using normal rat serum were essentially identical. Error bars are ?SEM.

munologically capable of limiting the expression of microfilaremia, a situation clearly recognized in several other animal and human filariae infections (Wong 1964; Beaver 1970; Bagai and Subrahmanyam 1970; Denham and McGreevy 1977; Haque et al. 1978). Second, if these animals had indeed resisted infection, were there specific immunologic mechanisms which appeared to be involved? The most direct approach to the first question would, of course, have been to examine the amicrofilaremic animals for the presence or absence of adult worms. When this was done both by careful dissection and by soaking techniques, no adult worms ever were found in the amicrofilaremic animals. On the other hand, however, of the nearly 20 microfilaremic animals examined at autopsy, fewer than half harbored adult worms which could be identified. The difficulties in recovering adult worms in rats have been indicated previously (Fox and Schacher 1976; Weller 1978), and we feel that our failure to find worms in the amicrofilaremic animals offers

supportive but not compelling proof for the lack of established infection in those animals. Stronger proof that the amicrofilaremic rats had actually resisted establishment of infection came from immunologic observations on the mf-positive and mf-negative groups of animals. With the exception of specific IgE antibodies (discussed below), responses were generally comparable for both groups during the early period of infection (lo- 12 weeks after inoculation of infective larvae) regardless of whether the L,‘s were administered all at one time or over 25 consecutive weeks. This similarity was seen for peripheral blood leukocyte levels, IgG antibody responses, and specific lymphocyte reactivity. After the prepatent period, however, responses of the mf-positive and mf-negative groups diverged with respect to at least three different parameters. First, a second phase of eosinophilia developed in the microfilaremic animals while eosinophil levels of the amicrofilaremic group fell to normal (Fig. 2A). Second, a similar divergence was

Brugiapahangi:

RAT SUSCEPTIBILITY

seen in the IgG antibody responses to both adult worm (Figs. 3 and 4A) and, even more strikingly, to microfilarial (Fig. 4B) antigens; indeed, though the microfilaremic animals developed high titers of antimicrofilarial antibody after patency was established, at no time during infection did the amicrofilaremic animals produce significant levels of such antibody. Third, the specific IgE antibody responses of these two groups differed markedly in the postpatent period. Those animals with microfilaremia had high titers of specific antifilarial IgE but those that remained amicrofilaremic were uniformly specific-IgE negative at this time (Fig. 5). Furthermore, it was of interest that the absolute level of microfilaremia in each rat did not appear to correlate with the magnitude of any of these immunologic responses, i.e., responses of animals with low microfilaremias were both qualitatively and quantitatively similar to those with high levels of circulating microfilariae and were distinctly different from those animals that remained amicrofilaremic. Previous studies in both experimental models (Wong 1964; Bagai and Subrahmanyam 1970; Denham and McGreevy 1977; Haque et al. 1978; Weiss 1978; Weil et al. 1981)and man (O’Connor 1932; Wong and Guest 1969; Ottesen et al. 1979) have demonstrated clearly that amicrofilaremia may sometimes belie persistent underlying lilarial infection. Interestingly, the amicrofilaremic state in such cases has been regularly associated with (and probably caused by) immunologic hyperresponsiveness to microfilariae, as manifested by high levels of antibodies to microfilariae and other parasite stages, peripheral blood eosinophilia, and increased levels of parasite-specific IgE (Neva and Ottesen 1978). Thus, the observations in our amicrofilaremic rats of normal blood eosinophil levels, falling antibody titers to adult worm antigens, lack of IgG antibodies against microfilariae, and absence of parasite-

157

specific IgE all support the negative autopsy findings and argue strongly that these amicrofilaremic animals had in fact successfully resisted the establishment of filarial infection at the same time that other similarly inoculated animals in our study acquired it. The means by which this minority of animals resisted infection is not entirely clear. The “trivial” explanations involving damage to the infective larvae or uncontrolled environmental differences appear unlikely because of the reproducibility of the finding from experiment to experiment and the fact that resistence was evident in both the single and multiple inoculum experiments. We therefore turned our attention to potential immunologic differences between the two groups of animals. Of the numerous humoral and cellular immune parameters assessed in these animals (Figs. 2-7), responses of both groups were found to be essentially identical during the critical prepatent period except for the development of specific IgE antibodies. While those animals destined to resist infection developed early specific IgE against the parasite, none of the animals in which patent infection was eventually established showed a similar IgE antibody response (Fig. 5). These findings, though they do not prove it, suggest that IgE antibodies played a role in protecting the animals from acquiring tilarial infection and support the notion that the ability to produce such antibodies may be important in protective immunity to helminths. IgE antibodies generally exert their effects through cellular amplification systems. Most reports have focused on the special relation between IgE and the mast cell or basophil with its numerous inflammatory mediators, but recent evidence has also demonstrated potentially significant interactions of IgE antibodies with both macrophages (Dessaint et al. 1980) and eosinophils (Hubscher 1975). With these cell types, one can envision numerous

158

GUSMAO,

STANLEY,

mechanisms through which IgE might mediate parasite killing, but what is really surprising is the fact that there is as yet very little in vivo data that clearly implicate IgE in the protection of animals from helminth infection. What studies do exist (Rousseaux-Prevost et al. 1977; Musoke et al. 1978) are certainly suggestive, but at this stage only phenomenologic. Therefore, while it is possible that the early IgE response of the protected animals in our study was only a secondary reaction to dying parasites killed by some unrelated mechanism, we believe that it is important to define more fully the nature of this response. Both the precise specificities of this early IgE antibody and the ways in which it might differ from the IgE produced after patency is established in the microfilaremic animals are important. Indeed, we hope that these findings with Brugia pahangi in the rat will offer a valuable experimental system with which to explore both the biologic importance and the functional mechanisms involved in production of the specific IgE antibodies so characteristically associated with the host response to parasitic helminth infections. ACKNOWLEDGMENTS We appreciate the constructive suggestions of Dr. Allen Cheever as well as his help with the autopsies. The assistance of Ms. Frances Ottman in the preparation of the manuscript is also gratefully acknowledged. Certain of the materials used in this study were supplied by Drs. J. MC Call and L. Cowgill of the University of Georgia, Athens, Georgia, U.S.A., under the U.S.-Japan Program contract with the U.S. National Institute of Allergy and Infectious Diseases. REFERENCES ASH, L. R., AND RILEY, J. M. 1970a. Development of Brugia pahangi in the jird, Meriones unguiculatus. with notes on infections in other rodents. Journal oj Parasitology 56, 962-968. ASH, L. R., AND RILEY, J. M. 1970b. Development of subperiodic Brugia malayi in the jird, Meriones unguiculatus. with notes on infections in other rodents. Journal aj” Parasitology 56, 969-973. BAGAI, R. C., AND SUBRAHMANYAM, D. 1970. Natural and acquired resistance to filarial infection in albino rats. Nature (London) 228, 682-683.

AND

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