Synergistic interaction between immune serum and thoracic duct cells in the adoptive transfer of rapid expulsion of Trichinella spiralis in adult rats

71,9&99(199O)

EXPERIMENTALPARASITOLOGY

Synergistic Interaction Cells in the Adoptive ALI AHMAD, James

CHINGHUAWANG,MASATAKA KORENAGA,R.G. LINCOLN SADAMS

A. Baker

AHMAD,

between Immune Serum and Thoracic Duct Transfer of Rapid Expulsion of Trichinella spiralis in Adult Rats

Institute for Animal Health, New York State College of Veterinary Cornell University, Ithaca, New York 14853, U.S.A. A.,

WANG,

C. H.,

KORENAGA,

M.,

BELL,

R. G.,

AND

ADAMS.

BELL,' AND Medicine,

L. S. 1990.

Synergistic interaction between immune serum and thoracic duct cells in the adoptive transfer of rapid expulsion of Trichinella spiralis in adult rats. Experimental Parasitology 71, 9O-99. Rapid expulsion of Trichinella spiralis could be transferred to naive adult rats with thoracic duct lymphocytes and immune serum. Thoracic duct cells collected from Days 3-5 and immune serum collected on Day 28, respectively, after infection were effective. Both cells and serum were unable to transfer rapid expulsion when given alone, even in large volumes. Recipients of immune serum and cells eliminated a significantly higher number of larvae than control rats by 1 hr after challenge with muscle larvae. Rapid expulsion produced 3O-8O% larval worm rejection but could not be increased by the transfer of more cells or immune serum. Mucus trapping did not appear to play a role in the rejection process. After transfer of 2 x 10’ cells and 4.0 ml immune serum, rapid expulsion persisted for less than 1 week. However, after adoptive transfer of cells alone, the gut remained functionally receptive to the passive transfer of immune serum for 7 weeks. Therefore, the changes effected by transfer of cells were long lived in contrast to the 1 week, or less, of functional persistence by transferred immune serum. The data indicate that two separate processes, one cell mediated and the other immune serum mediated, interact synergistically in the intestine and lead to the expression of rapid expulsion. o 1990AC&~ PUSS, IW. INDEX DESCRIPTORS AND ABBREVIATIONS: Trichinella spiralis; Immune serum; Thoracic duct cells; Rapid expulsion; Adoptive transfer; Rats; Hank’s balanced salt solution (HBSS); Muscle larvae (ML); Thoracic duct lymphocytes (TDL); T helper cells (Th).

intestine within minutes (McCoy 1940; Russell and Castro 1979). This phenomenon has been termed rapid expulsion (Bell et al. 1979). Rapid expulsion is clearly an important immune response in eliminating the parasite from the host and in reducing infection-related pathology. Although not expressed in all species, e.g., swine (Murrell 1985), rapid expulsion nevertheless appears to have a wide distribution in mammals, as is suggested by the occurrence of similar responses in sheep infected with Haemonthus contorfus (Miller et al. 1983) and guinea pigs infected with Trichostrongylus colubriformis (Rothwell and Grifftths 1977). Despite the strong immunity produced by T. spiralis, the passive transfer of immunity

INTRODUCTION

Trichinella spiralis is, overwhelmingly, an intracellular parasite. The adults live within intestinal epithelial cells (Wright 1979), the “intramulticellular niche” (Despommier 1983), and infective muscle larvae live for years within modified striated muscle cells, the nurse cell (Despommier 1983), of the host. In rodents, the adult worm is eliminated from its intestinal niche by a vigorous immune response 1 to 3 weeks after infection. Infected rats are refractory to reinfection and will eliminate 9&95% of a challenge infection from the ’ To whom correspondence

should be addressed.

90 OO14-4894/9O $3.00 CopyrisbtO 19!3Oby AcademicPress.Inc. Au rights of reproduction in any form reserved.

RAPID EXPULSION

OF T. Spidis IN RATS

using serum from infected animals has frequently been unsuccessful (McCoy and Bond 1941; Larsh et al. 1964, 1970; Denham 1969; Bell and McGregor 1980a, b; Bell et al. 1982). However, only the earlier studies from this laboratory specifically examined the transfer of rapid expulsion rather than overall protective immunity (Bell and McGregor 1980a, b). These experiments suggested that successful transfer of rapid expulsion in adult rats required both serum factors and another nonspecific stimulus whose function was restricted to the intestine (Bell and McGregor 198Oa). There has been one recent report of the successful transfer of rapid expulsion with immune serum alone in adult rats (Harari et al. 1987). Immunity is, however, readily transferrable with cells: mesenteric lymph node cells (Love et al. 1976, Grencis et al. 1985, Wakelin and Wilson 1977), thoracic duct lymphocytes (Crum et al. 1977; Bell et al. 1987), and peritoneal exudate cells (Larsh et al. 1964) from immune rats or mice adoptively transfer to naive animals the capacity for accelerated rejection of adult T. spiralis worms. This process is a slow rejection requiring several days to eliminate adult worms from the intestine. The recipients of passively transferred cells usually require at least a week of exposure to worms before rejection commences a few days earlier than in nontransferred controls. The distinguishing features of rapid expulsion are that it is directed against larval worms and operates within minutes to hours of larval contact with the immune intestine (Russel and Castro 1979). The ability of cells from immune rats or mice to specifically transfer rapid expulsion has never been directly explored. In this paper we report that thoracic duct cells and immune serum interact synergistically to successfully transfer rapid expulsion to naive rats. MATERIALSANDMETHODS Experimental

animals. Male or female, adult A0

91

rats (8 weeks or older) were used in this study. The animals were bred and maintained in the J. A. Baker Institute vivarium. The vivarium has a 12-hr light/dark cycle and constant levels of humidity and temperature are maintained. The rats had free access to food and water. Parasite. The strain of T. spiralis used in this study was originally provided by Dr. D. D. Despommier of Columbia University and has been maintained by serial passage in DA, PVG, or A0 rats that had been irradiated with 500 rad prior to infection. The procedures used to isolate infective muscle larvae, infect the rats, and obtain S-day adult worms have been described (Bell and McGregor 1980a). Immunization protocols. All immune serum and thoracic duct cell donor rats were infected with 2000 ML orally. No drug treatment was given to terminate or modify the infection. For thoracic duct cells, rats were cannulated on Day 3 postinfection following the modified technique of Bollman et al. (1948). Protective ceils appear at this time in the thoracic duct (Bell et al. 1987). Procedures for collection, handling, and transfer of thoracic duct cells or thoracic duct T cells into recipient rats have been described (Bell et al. 1987). For immune serum, infected rats were bled from the dorsal aorta on Day 28 postinfection. The serum from a group of 15-30 rats was pooled and stored at - 70°C. The PCA titer of the immune serum was determined following the general method of Ovary (1951). Briefly, a volume of 100 pl of doubling dilutions of immune serum was injected intradermally in rows on the shaved dorsum of exbreeder A0 rats. Anaphylaxis was induced 48 hr later by intravenous injection of 1.5 mg of soluble muscle larvae antigens in 2 ml of 1% Evans blue. Rats were killed and skinned 30 min later for reading PCA titers. Sera with a titer of 11800 or more were used in this study. Standard protocol for cell and serum transfer. Thoracic duct cells were washed twice with HBSS after collection and transferred iv to the recipient rats in RPM1 1640 medium containing 1% normal rat serum. Serum (4 ml/rat) was transferred by ip injection 7 days after cells. This procedure was adopted since rapid expulsion is first identifiable 7 days after infection with T. spiralis (Bell and McGregor 1979). Recipient rats were challenged with 1000 ML 24 hr after serum transfer. This standard protocol is depicted in Fig. 1. Assay procedures. Procedures for determining intestinal worm numbers and rapid expulsion were as previously described (Bell and McGregor 1979b). Mucus trapping and luminal worm counts. In some experiments, rejected worms were examined for mucus trapping. Procedures for counting worms trapped in mucus collected from the lumen of the intestine have been described previously (Bell et al. 1984). Statistical analysis. Worm counts of different groups were compared by using Student’s t test for

92

AHMADETAL.

Day 0

TABLE1

4-B: 4ml immune serum i.p.

Cht4l;gc 1000 ML

Collect intestine count Worms

FIG. 1. Standard protoco1 for transfer of rapid expulsion with thoracic duct lymphocytes and serum. Thoracic duct cells were transferred to the recipient rats iv on Day 0, and immune serum was transferred ip on Day 7. Recipient rats were challenged with 1000 ML orally on Day 8 (24 hr after serum transfer). Rapid expulsion was assayed by intestinal worm counts on Day 9 (20-24 hr after challenge). independent means (Snedecor and Cochran 1980). Probability levels of SO.05 were considered significant.

RESULTS Thoracic Duct Lymphocytes and Serum Induce Rapid Expulsion

Several initial experiments demonstrated that thoracic duct lymphocytes or immune serum alone failed to confer protection when measured in a 24-hr postchallenge assay. Therefore, we determined whether cells and serum in combination could induce rapid expulsion. Immune serum and cells were transferred as outlined in Fig. 1. In a typical experiment, recipient rats were divided at random into four equal groups. Cells and serum were transferred, alone, or in combination and intestinal worms were counted 20 hr after challenge with 1000 ML (Table I). Similar results were obtained in several identical experiments. Protection afforded by cells and serum given in combination usually ranged between 30 and 70% and was influenced by the time of collection of immune serum from donor rats. As a consequence, serum collection was standardized to Day 28 postinfection when PCA antibody titer to muscle larvae reached a peak (data not shown). Znjluence of Large Doses of Cells or Serum Alone

To determine whether the inability of thoracic duct lymphocytes or immune serum transferred alone to protect recipients

Transfer of Rapid Expulsion with Cells and Immune Serum Group”

TDLb

Immune serum’ (4 mllrat ip)

A B c Control

+ + -

+ + -

Worm counts& (mean + 1 SD) 173 537 549 581

k f 2 +

24’ 60 84 62

0 Five rats per group. b Thoracic duct lymphocytes (TDL; 3 x 10s) were injected iv per rat. ’ Immune serum was transferred 7 days after TDL transfer. d All groups were challenged with 1000 muscle larvae orally 24 hr after serum transfer and worm counts were done 20 hr later. * Difference significant (P 5 0.001) from control.

was due simply to their presence in suboptimal amounts, rats were given 6 ml of immune serum and 4 x 10’ thoracic duct T cells per rat. Since TDL contain substantial numbers of B cells (-30-35%) which could theoretically synthesize sufficient antibody to achieve rapid expulsion, B cells were removed by cell affinity chromatography. The serum transferred (6 ml/rat) was equivalent to the recipient’s own serum pool in volume and the number of T cells transferred was equivalent to 6-7 x IO* TDL/rat or the average cell output from the thoracic duct of 1.5-2 donor rats for 24 hr. The results of this experiment, T cells alone: 595 -+ 76; immune serum alone: 545 + 62; cells and serum 229 2 133; control: 535 2 114, demonstrated that even large volumes of immune serum or numbers of thoracic duct cells were unable to confer protection, although they were effective when combined. Specificity of Rapid Expulsion

To determine whether rapid expulsion induced by cells and immune serum was specific for the preadult stage of the parasite, the following experiment was performed. Recipient rats were divided randomly into four equal groups (A-D). Groups A and C

RAPID EXPULSION

received Day 3 TDL and immune serum according to the standard protocol whereas B and D served as controls. A and B were challenged with 1000 muscle larvae whereas 1000 Sday-old T. spiralis adult worms were transferred intraduodenally to each rat in groups C and D. Rats were killed for intestinal worm counts 5 hr after challenge because the immune serum contained anti-adult antibody. As shown in Table II, significant reductions in worm numbers were observed only in rats that had been challenged with muscle larvae. Dose-Responses for Transferred Cells and Serum

Two experiments were conducted to determine the minimum number of cells required to transfer protection and to determine whether protection was dose dependent. In one experiment, different groups of recipient rats received 0, 1.0 x lo*, 1.5 x lo*, or 3.0 x lo* TDL iv followed 1 week later by serum transfer. All groups were challenged with 1000 muscle larvae and harvested 20 hr later. As shown in Table III, significant reductions in intestinal worm numbers were observed in recipients injected with 1.5 x lo* TDL, but no extra effectiveness accrued from a doubling in dose to 3.0 x 10’ TDL/rat. In a similar exTABLE II Rapid Expulsion Induced by Cells and Serum Is Specific for Preadults Group”

TDL’

A B C D

+ + -

Immune serumC + +

Worm count@ (mean * 1 SD) 3442% 645553 237 + 155 393 k 80

93

OF T. SpirUhs IN RATS

P value
n Four rats per group. A and B were challenged with 1000 ML whereas C and D received 1000 5-day adult worms id after laparotomy. b TDL (3.0 x 108)/rat were injected iv. c Immune serum was injected 4 ml/rat ip 7 days after TDL transfer. d Worm counts were done 5 hr after challenge.

TABLE III Minimum Requirement of Cells for Protection

Group” A : D Control

No. of TDL/ratb Wlos) 0 1.0 1.5 3.0 -

Immune serumc (4 ml/rat) + + + +

Worm counts‘+ (mean f 1 SD) P value 538 + 479 + 231 + 255 + 544 +

111 % 71 84 95

N.S. N.S. <0.005 <0.005

a Four rats per group. b TDL were injected iv. ’ Immune serum was injected ip 1 week after TDL. d All groups were challenged with 1000 ML 24 hr after serum transfer and harvested for intestinal worm counts 20 hr later.

periment, it was found that 0.5 or 1.O x IO* TDL/rat did not protect the recipient whereas 2.0 or 4.0 x lo* TDL per rat did. However, no difference was observed in intestinal worm counts between recipients that had 2.0 or 4.0 x lo* TDL per rat (data not shown). This experimental procedure was repeated using different amounts of immune serum and a dose of 3.0 X lo* cells/rat. Rats were given none or 2,4, or 6 ml of immune serum 1 week after TDL transfer. As shown in Table IV, significant reductions were observed in recipients of as little as 2 ml of immune serum but again, no dosedependent increase in protection was observed. In parallel experiments, no gradation in effectiveness was found with immune serum doses of 0.5 or 1.0 ml, which never conferred protection. Similarly, TABLE IV Effect of Different Doses of Serum on Protection

Group” A B C D Control

TDLb + + + + -

Serum transferred’ (ml) 0 2 4 6 -

Worm count@ 513 f 279 2 244 2 285 2 537 2

90 54 57 48 54

P value

N.S.
a Four rats/per group. b TDL (3 x 10’) were injected iv. ’ Serum was injected ip 7 days after TDL transfer. d All groups were challenged with 1000 ML 24 hr after serum transfer and harvested 20 hr later.

94

AHMAD

ET AL.

doses of 2 ml did not always work, suggesting that the threshold dose was around 2 ml. In all cases, when immune serum was effective, rejection was around 40-60%. Longevity of the Inductive Rapid Expulsion

Steps for

The object of these experiments was to determine the period for which rapid expulsion would persist in rats after the transfer of cells and serum. Recipient rats were given 2.0 x 10’ TDL iv and 7 days later 4 ml of serum ip per rat. These rats were divided at random into three groups in each experiment. In one experiment, each group was challenged with 1000 muscle larvae I, 3, or 8 days later and in the other 1, 5, or 7 days after serum transfer. The results are shown in Fig. 2. Worm counts were significantly lower than controls only when recipients of cells and serum were challenged 1 or 3 days after serum transfer. In another series of experiments, the longevity of the intestinal priming effect produced by adoptively transferred TDL was investigated. In these experiments, recipi110

100 90

E

n

Experiment

#l

0

Experiment

#2

ent rats were given 1.5-2.5 x 10’ TDL per rat iv and divided randomly into different groups. The number of cells transferred to the recipient rats varied between experiments but was the same within each experiment. Serum was transferred to the recipient rats at different time points ranging from 24 hr to 84 days after TDL transfer. One day after serum transfer to any individual group, rats were challenged with 1000 muscle larvae orally and intestinal worm counts were performed 20-24 hr later. As shown in Fig. 3, significantly reduced worm counts were observed at each time point up to 7 weeks after cell transfer but not at 12 weeks after cell transfer. Transfer of Serum after or at the Time of Challenge

Our standard protocol involved transfer of immune serum 7 days after TDL transfer, followed by the challenge infection 24

T

T

L -

1

Day of Challenge

3

5

7

8

after Serum Transfer

FIG. 2. Recipient rats were injected with 2 x 10’ Day-3 thoracic duct lymphocytes iv and 4.0 ml of immune serum ip 7 days later, then randomly divided into three groups in both of the two experiments. On Days 1,3, and 8 in Experiment 1 and Days 1,5, and 7 in Experiment 2 after serum transfer, the recipient groups were challenged with 1000 ML, along with controls, and harvested for intestinal worm counts 20-24 hr later. *P < 0.05.

1

Elapsed

3

7

12

time (days)

21

35

49

84

after tell transfer

FIG. 3. Results of three separate experiments are depicted here. Recipient rats were injected with Day-3 thoracic duct lymphocytes iv as detailed above. Cell dose per rat varied between experiments (1.5-2.5 x lO’/rat) but was the same for each group within an experiment. After the cell transfer, immune serum was given (4 ml/rat ip) at different time points (l-84 days) as shown on the x-axis. AU recipient rats, along with appropriate controls, were challenged with 800-1000 muscle larvae 24 hr after serum transfer and harvested for intestinal worm counts individually. The intestinal worm burden was significantly (P S 0.05) reduced from Days 1 to 49 when cell and serum recipients were compared to the control rats. On Day 84 there was no difference (P > 0.05) between control and cell and serum recipient intestinal worm counts.

RAPID

EXPULSION

OF

hr later (Fig. 1). These experiments were designed to determine whether immune serum transfer in TDL recipients immediately after or at the time of challenge would be protective. As shown in Table V, significantly reduced worm counts were observed when serum was transferred at the time of challenge and also 6 hr after challenge. Other Considerations

The cell and immune serum transfer system was also used to define the following points with regard to adoptive rapid expulsion. Larvae were rejected within 1 hr of the challenge infection (cell and serum recipients 58 ? 25, control 251 + 31). No time points earlier than 1 hr were examined. High doses, 2000 or more, overcame rapid expulsion in adoptively transferred rats and no rejection was seen (cell and serum recipients, 974 + 292; control, 1114 ? 206; challenge dose, 2000; cell and serum recipients; 1942 -+ 321; control, 2250 + 365; challenge dose, 4000). Mucus trapping was not a critical element in the rejection of worms by adoptive recipients undergoing rapid expulsion (adoptive recipients 29% trapped, control 21%, P > 0.05). DISCUSSION

In the experiments reported here we have demonstrated that thoracic duct lymTABLE

V

Serum Transfer after or at the Time of Challenge

Group”

TDLb

A B C Control

+ + + -

Time of serum transfer in hours after (+) or before ( - ) challenge= -24 0 +6 -

worm countsd (mean f 1 SD) 359 407 436 692

2 + + 2

64 68 61 42

P value
a Four rats/per group. b TDL (2.5 ’ All groups TDL transfer. d Intestines

x 108) were injected per rat. were challenged with 1000 ML 8 days after Each rat received 4 ml of immune serum ip. were harvested 24 hr after challenge.

T. s@ra/is

IN

RATS

95

phocytes and serum collected from rats infected with T. spiralis consistently transfer rapid expulsion to adult rats. The role of cells from immune rats and mice in the transfer of immunity to this parasite in rodents is well documented (Love et al. 1976; Grencis et al. 1985; Wakelin and Wilson 1977; Despommier et al. 1977; Bell et al. 1987) whether measured by the accelerated rejection of adult worms, 7-9 days after the challenge infection, or by reduced muscle larvae burden, determined at least 28 days after challenge. The latter, of course, does not measure any particular immune response but accumulates the effects of several immune responses (Bell et al. 1979). In none of the above reports was the capacity of transferred cells to confer rapid expulsion on adoptive recipients specifically explored. Our experiments have shown that thoracic duct lymphocytes collected 3 days after infection of donor rats with 2000 muscle larvae can transfer rapid expulsion provided immune serum is also transferred. Immune serum alone did not transfer rapid expulsion in over 30 individual experiments. This is in accordance with the tindings of a number of investigations (McCoy and Bond 1941; Denham 1969; Gore ef al. 1970; Larsh et al. 1970), all of whom found that immune serum did not confer immunity of any sort on recipients when adult worms were counted 5 or more days after a challenge infection. These results necessarily also demonstrate the absence of rapid expulsion after serum transfer, as previously reported by us in experiments specifically directed at rapid expulsion (Bell and McGregor 1980a, b; Bell et al. 1982). Although immune serum, alone, failed to transfer rapid expulsion in these studies, there have been reports that immune serum is protective (Culbertson 1942; Hendricks 1953; Mills and Kent 1965; Love er al. 1976). Immunity was determined by adult worm counts in these experiments and rapid expulsion was not specifically investigated. Harari et al. (1987) have, however,

96

AHMAD

reported passive transfer of rapid expulsion in adult rats with immune serum alone. The role of antibody alone in promoting worm rejection, as defined by passive transfer techniques, has been controversial since the first attempts to achieve this in the 1940s and it remains so. It is important to note that reported failures of protection with transferred immune serum alone in rats and mice outnumber successful passive transfers. The real incidence of failed passive transfer of immune serum is almost certainly higher since negative results are infrequently reported. The failure of experienced experimental scientists over a period of almost 50 years to establish a consistent and reproducible system for passive transfer of protection with immune serum is particularly noteworthy. We have demonstrated that, in the presence of intestinal inlIammatory conditions produced by H. polygyrus, which penetrates and then dies in the gut wall of rats, passively transferred immune serum consistently induces rapid expulsion (Bell and McGregor 1980a, b). Our earlier results suggested that this initial step was antigenically nonspecific and was related to the function of stimulated T helper cells in the gut (see Discussion). However, it also seems entirely feasible that a variety of other pathogens and perhaps nonpathogenic agents might also produce intestinal changes that facilitate the subsequent expression of an antibodymediated response in the gut. These infections, or changes in pathology, may not be readily detectable, would not be anticipated, and could therefore pass unnoticed by investigators. The changes produced by thoracic duct cells are relatively long lived as cell recipients are receptive to transferred antibody for 7 weeks after cell transfer, thus events that precede passive transfer by almost 2 months could exert an effect. The Baker Institute rat colony has none of the common viral, bacterial, or helminth infections of rats with the exception of a ~3% incidence of Killem rat virus (by

ET AL.

serology). Adult rats in this colony have never shown rapid expulsion after the transfer of immune serum alone (even in volumes up to 10 ml) and controls for this have been included in numerous transfer experiments over a 1Zyear period. We hypothesize that inapparent infections or other factors (dietary?) have primed the gut and thus facilitated passive transfer of protection with immune serum alone in some laboratories but not in others. This may have been the uncontrolled, unobserved factor that has resulted in the inability to transfer protection with immune serum in many cases. Differences in the strains of rats used or in the immunization protocols for the collection of immune serum could have contributed but these are likely to be of less significance. Rats of all strains tested so far (R. G. Bell, unpublished) exhibit strong rapid expulsion and we have conducted cell and serum transfers successfully in A0 and PVG strains, suggesting that the strain variation evident in mice is not a factor in rats. Furthermore, in more recent experiments (A. Ahmad and R. G. Bell, in preparation), we demonstrate that the reaction is mediated by antibodies of several different isotypes that are present in rat serum over an extended period (2 weeks-3 months) after a single or multiple infections. Consistent transfer of rapid expulsion with immune serum alone has been reported in suckling rats (Culbertson 1942; Appleton and McGregor 1984, 1985). In these studies, infected dams were also found to transfer rapid expulsion to their suckling pups for at least three breeding cycles. Considering the differences in the gastrointestinal physiology of suckling and adult rats, e.g., antibody transport, it can be argued that suckling rats do not need the “intestinal stimulus” that adult rats need to express rapid expulsion. These as yet unspecified changes may already be present in the immature intestine and therefore antibodies alone, whether fed in colostrum or injected ip, will induce rapid expulsion. Al-

RAPID

EXPULSION

OF

tematively, there may be more than one mechanism of rapid expulsion, and that operating in suckling rats may be distinct from that of adult rats. The induction of rapid expulsion by thoracic duct cells and immune serum together points toward a synergistic interaction rather than just an additive effect. Earlier studies from this laboratory showed that successful induction of rapid expulsion in adult rats required two discrete stimuli: a serum factor and a nonspecific intestinal stimulus (Bell and McGregor 1980b). These experiments used infection with H. polygyrus and active transfer of a blood-borne factor from parabiosed rats infected with T. spiralis. Infection with H. polygyrus provided the “intestinal stimulus.” In the experiments reported here, thoracic duct lymphocytes from immune rats apparently provide the “intestinal stimulus” needed to induce rapid expulsion with immune serum. This intestinal stimulus may be in the form of inflammatory changes produced in the intestine by the transferred cells. It has been demonstrated in mice that the intestinal mastocytosis and eosinophilia accompanying infection with T. spiralis can be adoptively transferred with immune mesenteric lymph node T cell populations and antigen-specific T cell lines (Alizadeh and Wakelin 1982; Riedlinger et al. 1986). In rats, dividing T cells in thoracic duct lymph home to the gut in large numbers and promote eosinophilia and mastocytosis (C. H. Wang et al., submitted for publication). We hypothesize that the transferred cells promote the growth and maturation of a second cell population and that antibody provides the secondary amplification that actually confers rapid expulsion and its specificity. The absolute requirement for the transfer of immune cells and serum for expulsion of challenge larvae, reported here, is not without precedent. Love (1975) demonstrated that the transfer of both immune cells and serum was essential for rejection of developing Nippostrongylus bra-

T. Spiralis

IN

RATS

97

siliensis larvae from the intestine of recipient rats. When rapid expulsion occurs after a challenge infection in previously infected rats, the rejected worms are often found trapped in mucus in the lumen of the small intestine. This has led to the suggestion that mucus may be an effector of rapid expulsion (Lee and Ogilvie 1982; Miller et al. 1981). In our experiments the same percentage of worms in immune and control rats were trapped in mucus. We also found that immune serum transferred to cell recipients 6 hr after challenge (Table V), when virtually all worms have already been established in the mucosal epithelium, still produced rapid expulsion. Therefore, a role for mucus in excluding worms or preventing their establishment is not a necessary part of rapid expulsion (Bell et al. 1984). Rapid expulsion induced by cells and serum is kinetically similar to that seen after active infections with T. spiralis. McCoy (1941), in his original description of rapid expulsion, demonstrated a reduction in worm numbers in immune rats 1 hr after challenge as we found here. The percentage of worms rejected in our experiments (3O80% of the challenge) as compared to 9& 95% reported in rapid expulsion after infection with muscle larvae (McCoy, 1941) is lower. The results of Harari et al. (1987) were very similar to those reported here, 45-81% rejection, reflecting a reduction of efftciency in adoptive rapid expulsion compared to active immunity. This may be due to additional mechanisms operating in actively immune rats. This view is buttressed by the fact that there was essentially no dose-response increment for increased doses of cells or of immune serum. In addition, Harari et al. (1987) used 6 ml of immune serum but gained no increase in the efficiency of rejection over that seen here. Rapid expulsion induced in cell and immune serum recipients persisted less than 1 week in our hands and less than 2 weeks for Harari e? al. (1987) using 50% more serum.

AHMAD ETAL.

98

After infections resulting in established muscle larvae, rapid expulsion persists for at least 1 year (Bell et al. 1979). This difference is probably due to the short half-life of transferred antibody which, in actively immune rats, is constantly being stimulated by antigen released from implanted muscle larvae (Pritchard 1985). In contrast, the priming effect of transferred cells persists for at least 7 weeks after transfer. These differences between the longevity of the cellular versus the serum effect further support the view that these are quite independent processes. The period for which cellular priming persists is reminiscent of the “short-term” rapid expulsion produced in rats given abbreviated infections. This form of rapid expulsion has a self-limited life of 5-6 weeks in the absence of muscle larvae implantation (Bell and McGregor 1979a, b). The results presented here demonstrate that rapid expulsion can be produced in adult rats by a cell and immune serum transfer procedure that will facilitate analysis of the underlying mechanisms. ACKNOWLEDGMENT This work was supported by FAO exchange visitor program No. G 4 111 and NIH Grant AI 17484. A.A. is a FAO fellow from Pakistan. The authors thank Mrs. Anita Hesser for typing the manuscript and figure compilation.

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APPLETON, J. A., AND MCGREGOR, D. D. 1985. Lifephase specific induction and expression of rapid expulsion in rats suckling Trichinella spiralis infected dams. Immunology 55, 225-232. BELL, R. G., ADAMS, L. S., AND OGDEN, R. W. 1984. Intestinal mucus trapping in the rapid expulsion of Trichinellu spiralis by rats: Induction and expression analyzed by quantitative worm recovery. Infection and Immunity

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