Induction of specific humoral immunity to soluble proteins in the American cockroach (Periplaneta americana). I. Nature of the primary response

DEVELOPMENTAL AND COPLPARATIVE IM/~UNOLOGY, Vol. 4, pp. 447-458, 1980. 0145-305X/80/030447-12502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.

I N D U C T I O N OF S P E C I F I C H U M O R A L

I M M U N I T Y TO SOLUBLE PROTEINS

IN THE A M E R I C A N C O C K R O A C H I.

(Periplaneta americana). 1 NATURE OF THE PRIMARY RESPONSE.

2 L a w r e n c e A. Rheins, R i c h a r d D. Karp , and A n d r e w Butz D e p a r t m e n t of B i o l o g i c a l Sciences U n i v e r s i t y of C i n c i n n a t i Cincinnati, Ohio 45221

ABSTRACT

A d u l t m a l e ~ a e r i c a n c o c k r o a c h e s (Periplaneta americana) g e n e r a t e d a specific p r o t e c t i v e r e s p o n s e when injected w i t h s o l u b l e p r o t e i n toxins. This r e s p o n s e d e v e l o p e d over a p e r i o d of time, peaking w i t h i n 2 weeks and then g r a d u a l l y s u b s i d i n g by the fifth week. S p e c i f i c i t y of this r e a c t i v i t y was d e m o n s t r a t e d by the fact that in~aunized animals w e r e only p r o t e c t e d against the origi nal i m m u n i z i n g toxin, and not to a h e t e r o l o g o u s toxin. Passive t r a n s f e r studies r e v e a l e d that p r o t e c t i o n could be t r a n s f e r r e d to v i r g i n animals w i t h c e l l - f r e e immune hemolymph. Thus, this a d v a n c e d i n v e r t e b r a t e is capable of g e n e r a t i n g a specific a d a p t i v e humoral immune response to these soluble proteins.

INTRODUCTION

A d i s t i n c t i v e feature of all v e r t e b r a t e immune r e s p o n s e systems s t u d i e d to date is that they are c o m p r i s e d of both c e l l u l a r and h u m o r a l elements (1,2). A l t h o u g h it has now been e s t a b l i s h e d that several classes of i n v e r t e b r a t e s possess a d a p t i v e cellm e d i a t e d i m m u n e r e a c t i v i t y (3-7), the q u e s t i o n of w h e t h e r or not these lower animals are c a p a b l e of specific humoral immunity

i S u p p o r t e d by NIAID r e s e a r c h grant AI15601, and a r e s e a r c h g r a n t from the U n i v e r s i t y of C i n c i n n a t i R e s e a r c h Council. 2To w h o m r e p r i n t requests

should be sent.

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remains unanswered. There have been numerous reports demonstrating the existence of naturally occurring humoral factors reactive for such antigens as vertebrate erythrocytes, bacteria, and foreign proteins in the body fluids of molluscs (9-11), sipunculid worms (12), annelids (13), arthropods (10,14-17), echinoderms (10,18-22), and tunicates (23). In addition, some investigators have reported examples of invertebrate agglutinins, opsonins or lysins that could be induced by the injection of the corresponding antigen (24-28). In a few cases, this reactivity has been transferred passively to virgin animals via the hemolymph from previously injected animals (21,25,27). In general, these responses usually have two things in common: i.) They are of very short duration, peaking within 24-48 hours, and then disappearing; and, 2.) one does not observe an anamnestic response upon repeated injection of the antigen (21,26). The studies reported here demonstrated that an adaptive humoral response was induced in the American cockroach (Periplaneta americana) to the soluble protein complexes Honeybee toxin and Cottonmouth Moccasin venom. These soluble proteins were chosen as antigens for two reasons: I.) Unlike particulate antigens, they would not be quickly cleared by phagocytic mechanisms upon injection, and thus would promote antigen persistence; and, 2.) they provide a natural biologic assay because of their lethality for roaches. The response to these proteins was found not only to be specific, but demonstrated kinetics rather similar to those characteristic of vertebrate reactivity.

METHODS

Animals. Adult male American cockroaches weighing approximately 1 g. were purchased from Carolina Biological Supply Co., Burlington, N.C., and Ward's Natural Science Establishment, Rochester, N.Y. Stock animals were housed in 5 gallon plastic drums lined with vaseline to prevent escape. Animals used in experiments were transferred into individual pint size glass bottles covered with a cheese cloth lid. Roaches were fed Purina rat chow and water ad libitum, and maintained at 25°C. Anti@en Preparations. Honeybee toxin (Apis mellifera, United States Biochemical Corp.) and Western Cottonmouth Moccasin venom (A~kistrodon piscivorus, Sigma Chemical Co.) were diluted in Burns-Tracey saline (BTS) (29) to make a stock solution of 1.0% w/v. The LDI00 doses for the toxins were determined by injecting various amounts of the 1% solutions into animals to find the dosage that achieved 100% lethality. This was repeated for all batches of the toxins used during the study. Toxoids were prepared by heating Honeybee toxin (HBT) and Cottonmouth Moccasin venom (CMV) at 60°C for 1 hour and then subjecting them to formalization. Immunization Procedures. Roaches were CO 2, injected with 5 ~i of toxoid, and then times with 7 ~i (LDI00 dose) of the toxin. injections were carried out with a Hamilton

anesthetized with challenged at various The intrahemocoelic i0 ~i syringe, and

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a d m i n i s t e r e d b e t w e e n the fourth and fifth a b d o m i n a l sternites. D u r i n g this procedure, the n e e d l e was inserted h o r i z o n t a l l y and a n t e r i o r l y in a slow fashion. If e x c e s s i v e coelomic fluid leaked out u p o n w i t h d r a w a l of the syringe, the animal was discarded. The d o s a g e s i n j e c t e d w e r e a d j u s t e d to the w e i g h t of each animal. R o a c h e s r e c o v e r e d from the a n e s t h e t i c state w i t h i n 20 m i n u t e s w i t h no ill effects. T r e a t e d animals that were alive 36 hours p o s t - i n j e c t i o n w e r e s c o r e d as positive. H e m o l y m p h and H e m o c y t e Preparation. Immune and normal hemolymph w e r e o b t a i n e d from a n e s t h e t i z e d roaches by severing the a n t e n n a e at their base, and the legs b e t w e e n the t r o c h a n t e r and the femur, and then c o l l e c t i n g the e x p r e s s e d fluid w i t h a 20 ~i E p p e n d o r f pipet. These m a n i p u l a t i o n s w e r e carried out in the cold (0°C) to p r e v e n t c o a g u l a t i o n . The support m e d i u m for cellular m a n i p u l a t i o n s was G r a c e ' s Insect M e d i u m (GIM, G r a n d Island B i o l o g i c a l Co.), pH 7.2, s u p p l e m e n t e d w i t h i0 m l / L antibiotica n t i m y c o t i c s o l u t i o n (GIBCO) and 5.955 g/L HEPES buffer. The h e m o l y m p h was p o o l e d and c e n t r i f u g e d at 200 x g for 5 m i n u t e s in an IEC c l i n i c a l centrifuge. The s u p e r n a t a n t was r e m o v e d as the s o u r c e of hemolymph, and the cell p e l l e t was gently r e s u s p e n d e d in 5 ml of cold GIM. V i a b i l i t y was d e t e r m i n e d by T r y p a n Blue dye exclusion, and the cell s u s p e n s i o n a d j u s t e d to 1 x 106 v i a b l e h e m o c y t e s per ml. D i l u t i o n s of the stock s u s p e n s i o n were made in GIM to o b t a i n the d e s i r e d cell c o n c e n t r a t i o n s for i n j e c t i o n into animals. S t a t i s t i c a l Analysis. Tests for h o m o g e n e i t y among the r e p l i c a t e trials w i t h i n t r e a t m e n t s w e r e m a d e by c a l c u l a t i n g the n o r m a l d e v i a t e z, w h i c h allows a n o r m a l a p p r o x i m a t i o n for binomial p r o p o r t i o n s (30). The s t a n d a r d d e v i a t i o n for the t r e a t m e n t means was c a l c u l a t e d using s = ~ - - / ~ for the p r o p o r t i o n of successes in a binomial distribution. The c o m p a r i s o n b e t w e e n two t r e a t m e n t s was m a d e u s i n g the 2 x 2 h e t e r o g e n e i t y X 2 test, w i t h c o r r e c t i o n for continuity.

RESULTS

The P r i m a r y Immune R e s p o n s e to HBT. A n i m a l s were injected w i t h a p r i m a r y irmnunizing dose of 5 ~i of 1% H o n e y b e e toxoid (HBTd), and then w e r e r e s t e d for v a r i o u s periods of time b e f o r e b e i n g c h a l l e n g e d w i t h 7 ~i of HBT. The p r i m a r y i m m u n i z i n g dose of H B T d had b e e n d e t e r m i n e d p r e v i o u s l y in pilot experiments. The r e s u l t s (Table i) s h o w e d that by 3 days, a r e s p o n s e was already in evidence, since 43.3% of the animals s u r v i v e d a c h a l l e n g e of the toxin. The r e s p o n s e c o n t i n u e d to gain in s t r e n g t h as reflected by a 62.9% rate of p r o t e c t i o n after 1 week, then peaked at 80% a f t e r 2 w e e k s of rest. The a c t i v i t y then began to decline after 3 weeks, and did not s i g n i f i c a n t l y differ from c o n t r o l s by the f o u r t h week. Less than 10% of the control animals that rec e i v e d BTS i n s t e a d of t o x o i d (with the e x c e p t i o n of the first week) s u r v i v e d the c h a l l e n g e i n j e c t i o n of HBT. Thus, the kinetics of the p r i m a r y r e s p o n s e to HBT d i f f e r s t r i k i n g l y from the shortterm r e a c t i v i t y r e p o r t e d by others.

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TABLE 1

The Primary Im~nune R e s p o n s e of the A m e r i c a n C o c k r o a c h to H o n e y b e e Toxin

Primary Injection

Rest Period (Days)

5 ~i HBTd b

3

5 ~i BTS d

7

5 ~i BTS

14

5 ~i BTS

5 ~i HBTd

21

5 ~i BTS

5 ~i HBTd

28

5 ~i BTS

5 ~i HBTd

7 ~i HBT c 7 ~i HBT

5 ~i HBTd

5 ~i HBTd

Challenge Injection

35

5 ~i BTS

M e a n P e r c e n t S u r v i v o r s + S.D. 36 Hours -Post_Challenge a

43.3 + 9.0

(13/30)

6.7 + 4.6

(2/30)

7 ~l HBT

62.9 ~ 8.2

(22/35)

7 ~i HBT

11.4 + 5.9

(4/35)

7 ~i HBT

80.0 ~ 6.8

(28/35)

7 ~i HBT

5.7 + 3.9

(2/35)

7 ~i HBT

48.6 ~ 8.4

(17/35)

7 ~i HBT

9.1 + 5.0

(3/33)

7 ~i HBT

17.1 ~ 6.4

(6/35)

7 ~i HBT

5.7 + 3.9

(2/35)

7 ~i HBT

0

(0/35)

7 ~i HBT

0

(0/34)

aData from 3 trials w e r e averaged, t i c a l l y homogeneous.

since they w e r e statis-

b H o n e y b e e toxoid. C H o n e y b e e toxin. dBurns-Tracey

saline.

F u r t h e r e x p e r i m e n t s were c o n d u c t e d to e x c l u d e the p o s s i b i l i t y that the toxoid in some f a s h i o n m i g h t be n e u t r a l i z i n g or interfering w i t h the toxin in vivo by n o n - i m m u n o l o g i c a l means. For

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451

TABLE 2

S p e c i f i c i t y of the P r i m a r y Immune R e s p o n s e of the A m e r i c a n C o c k r o a c h To S o l u b l e P r o t e i n Toxins

Rest Period (Weeks)

Secondary Injection

5 ~i HBTd b

2

7 ~i HBT c

84.2 + 8.4

(16/19)

5 ~i HBTd

2

7 ~i CMV d

i0.0 + 6.7

(2/20)

5 ~i BTS e

2

7 ~i HBT

5 ~l CMVd f

2

7 ~l CMV

65.0 + 10.7

(13/20)

5 ~i CMVd

2

7 ~i HBT

5.0 + 4.9

(1/20)

5 ~i BTS

2

7 ~i CMV

Primary Injection

aData from 2 trials w e r e averaged, tically homogeneous. bHoneybee

M e a n P e r c e n t Survivors + S.D. 36 Hours Post C h a l l e n g e a

0

0

(0/i0)

( 0/i0)

since they were statis-

toxoid.

C H o n e y b e e toxin. d c o t t o n m o u t h M o c c a s i n venom. eBurns-Tracey

saline.

f C o t t o n m o u t h M o c c a s i n toxoid.

this purpose, a n i m a l s r e c e i v e d a p r e - m i x e d i n j e c t i o n of 7 ~i of bee t o x o i d plus 7 ~i of HBT. None of the animals so treated survived, showing that the toxin was still lethal in the presence of the toxoid. To ensure that the animals were not just s u c c u m b i n g to the v o l u m e of the injection, controls r e c e i v e d an equal a m o u n t (14 ~i) of BTS. F o u r t e e n of the 15 animals so t r e a t e d survived. Thus, the toxoid was g e n e r a t i n g a p r o t e c t i v e f a c t o r in the p r e v i o u s experiments, rather than d i r e c t l y interfering w i t h the toxin challenge. S p e c i f i c i t y of Western Cottonmouth a n t i g e n in o r d e r to mals w e r e i m m u n i z e d

the Primary Response. An u n r e l a t e d toxin, M o c c a s i n v e n o m (CMV), was used as a second test the s p e c i f i c i t y of this response. Aniw i t h either bee t o x o i d or C o t t o n m o u t h toxoid,

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TABLE 3

Passive T r a n s f e r of P r o t e c t i o n to H o n e y b e e T o x i n W i t h C e l l - F r e e Immune H e m o l y m p h

M e a n P e r c e n t S u r v i v o r s + S.D. 36 Hours -Post-Challenge

Primary Injection

Rest Period

Secondary Injection

7 ~i IH a

1 Hour

7 ~i HBT b

65.7 + 8.0

(23/35) c

7 ~i NH d

1 Hour

7 ~i HBT

20.0 + 8.9

(4/20) e

7 ~i BTS f

1 Hour

7 ~i HBT

2.9 + 2.8

(1/35) c

a C e l l - f r e e irmmune hemolymph. b H o n e y b e e toxin. CData from 4 trials w e r e averaged, tically homogeneous.

since they w e r e statis-

dNormal c e l l - f r e e hemolymph. eData from 2 trials w e r e averaged, tically homogeneous. fBurns-Tracey

since they w e r e statis-

saline.

and then r e s t e d 2 weeks to a l l o w for a m a x i m u m r e s p o n s e to d e v e l op. The i m m u n e animals w e r e f u r t h e r subdivided, and then challenged w i t h LDI00 doses of e i t h e r the h o m o l o g o u s or h e t e r o l o g o u s toxin. The results (Table 2) i n d i c a t e d that the p r o t e c t i o n was indeed specific. A n i m a l s r e c e i v i n g s e c o n d a r y i n j e c t i o n s of h e t e r o l o g o u s toxins did not show good p r o t e c t i o n since only 10% of the r o a c h e s i m m u n e to bee t o x o i d and c h a l l e n g e d w i t h CMV survived, and only 5% of the roaches immune to C o t t o n m o u t h t o x o i d and c h a l l e n g e d w i t h HBT survived. On the o t h e r hand, i m m u n e animals c h a l l e n g e d w i t h h o m o l o g o u s toxins w e r e well p r o t e c t e d as r e f l e c t e d by 85% of the animals s u r v i v i n g in the case of HBT, and 65% in the case of CMV. None of the c o n t r o l animals r e c e i v ing BTS as a p r i m a r y i n j e c t i o n s u r v i v e d c h a l l e n g e s of e i t h e r toxin. Thus, the r e s p o n s e g e n e r a t e d was s p e c i f i c in nature, b e i n g e f f e c t i v e only a g a i n s t the initial i m m u n i z i n g toxoid. P a s s i v e T r a n s f e r of Protection. A series of e x p e r i m e n t s w e r e c a r r i e d out to d e t e r m i n e if the i n d u c e d p r o t e c t i o n to HBT c o u l d be p a s s i v e l y t r a n s f e r r e d via i m m u n e hemolymph, h e m o c y t e s

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TABLE 4

Passive Transfer of Protection Against Honeybee With Hemocytes F r o m Immune Animals

Number V i a b l e Hemocytes Injected a

Toxin

Rest Period (Hours)

Challenge Injection

1 x 105

1

7 ~i HBT c

48.0 + 9.9

(12/25)

5 x 104

1

7 ~i HBT

44.0 + 9.9

(11/25)

2.5 x 104

1

7 ~i HBT

28.0 + 8.9

(7/25)

1 x 104

1

7 ~i HBT

8.0 + 5.4

(2/25)

i0 W1 GIM d

1

7 ~i HBT

16.0 + 7.3

(4/25)

aInjected

in a total v o l u m e of l0 ~i of GIM.

bData from 5 trials were averaged, tically homogeneous. CHoneybee dGrace's

Mean Percent Survivors + S.D. 36 Hours b Post-Challenge

since they were statis-

toxin. Insect medium.

from immune animals, or both. Donor animals received 2 injections of 5 ~i of bee toxoid that were spaced 2 weeks apart, and then bled one week after the last injection. The immune hemolymph and cellular fraction were separated from each other as d e s c r i b e d in Methods. U n t r e a t e d animals were similarly processed as a source of normal h e m o l y m p h and cells for control purposes. The first set of experiments were formulated to determine if p r o t e c t i o n to HBT could be t r a n s f e r r e d by the injection of cellfree immune h e m o l y m p h into virgin animals. Animals were injected w i t h 7 ~i of immune hemolymph, rested one hour, and then challenged w i t h 7 ~i of HBT. Table 3 shows that 65.7% of these animals survived a lethal dose of toxin. On the other hand, only 20% of the animals receiving cell-free normal hemolymph survived the HBT challenge. The two treatments differed significantly from each other at the p < .005 level. Only 2.5% of the animals injected with BTS survived the toxin injection. Thus, protection rivalling that g e n e r a t e d by active immunization was passively t r a n s f e r r e d to v i r g i n animals with cell-free immune hemolymph. Next,

experiments w e r e carried out to determine

if the cell-

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TABLE 5

Passive T r a n s f e r of P r o t e c t i o n A g a i n s t H o n e y b e e T o x i n With Hemocytes From Normal Animals

Number Viable Rest Hemocytes Period Injected a (Hours)

Challenge Injection

M e a n P e r c e n t S u r v i v o r s + S.D. 36 Hours b Post-Challenge

1 x 105 4 5 x i0

1

7 ~i HBT c

60.0 + 10.9

(12/20)

1

7 ~i HBT

50.0 + 11.2

(10/20)

2.5 x 104

1

7 ~i HBT

30.0 + 10.2

(6/20)

1 x 104

1

7 ~i HBT

25.0 + 9.7

(5/20)

i0 ~i GIM d

1

7 ~i HBT

15.0 + 7.9

(3/20)

a I n j e c t e d in a total v o l u m e of i0 ~i of GIM. b D a t a from 4 trials w e r e averaged, tically homogeneous.

since they w e r e statis-

C H o n e y b e e toxin. dGrace's

Insect Medium.

ular p o p u l a t i o n from immune animals c o u l d also lend p r o t e c t i o n to v i r g i n animals. A n i m a l s r e c e i v e d i n j e c t i o n s of e i t h e r 1 x 105 , 5 x 104 , 2.5 x i0 ~ or 1 x 104 v i a b l e i m m u n e h e m o c y t e s susp e n d e d in i0 ~i of GIM. F o l l o w i n g one hour rest, t h e s e roaches w e r e then c h a l l e n g e d w i t h 7 ~i of HBT. The r e s u l t s (Table 4) i n d i c a t e d that some p r o t e c t i o n a g a i n s t the toxin was c o n f e r r e d upon v i r g i n animals w i t h the h i g h e r dose levels. However, the data r e s u l t i n g from a n i m a l s i n j e c t e d w i t h a n a l o g o u s n u m b e r s of n o n - i m m u n e h e m o c y t e s (Table 5) m a k e s one q u e s t i o n w h e t h e r there was any specific c e l l u l a r p r o t e c t i o n at all, since s i m i l a r doses of c o n t r o l cells p r o t e c t e d animals from the effects of the toxin as well as the immune cells. This p r o t e c t i v e e f f e c t b e g a n to d i s a p p e a r for both i m m u n e and n o n - i m m u n e cells as the dosages decreased. It w o u l d a p p e a r that the i n j e c t i o n of r e l a t i v e l y large numbers of hemocytes, w h e t h e r immune or n o n - i m m u n e , h e l p e d to fortify the animals a g a i n s t the lethal e f f e c t s of the toxin in a n o n - s p e c i f i c manner.

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DISCUSSION

T h e r e have been n u m e r o u s studies in the past r e p o r t i n g attempts to induce a s p e c i f i c h u m o r a l r e s p o n s e to various p a r t i c u late or s o l u b l e s u b s t a n c e s in invertebrates. Some instances of s h o r t - t e r m responses, d e v e l o p i n g w i t h i n 24-48 hours and then disappearing, have b e e n observed. The studies r e p o r t e d here have shown that a long lasting specific humoral response to soluble p r o t e i n c o m p l e x e s could be g e n e r a t e d in the A m e r i c a n cockroach. The k i n e t i c s of this r e s p o n s e w e r e s u r p r i s i n g l y similar to w h a t one m i g h t e x p e c t for a v e r t e b r a t e , since it took 2 w e e k s for the r e a c t i v i t y to r e a c h a m a x i m u m b e f o r e slowly declining. Control e x p e r i m e n t s e n s u r e d that the toxoid form of the a n t i g e n was not i n t e r f e r i n g w i t h the l e t h a l i t y of the toxin, w h i c h m i g h t have p r o d u c e d m i s l e a d i n g results. The r e s p o n s e also p r o v e d to be s p e c i f i c in that animals w e r e only p r o t e c t e d from the toxin that they w e r e o r i g i n a l l y i m m u n i z e d against, w h e r e a s they s u c c u m b e d to a c h a l l e n g e of a h e t e r o l o g o u s toxin. P a s s i v e t r a n s f e r e x p e r i m e n t s were c o n d u c t e d in o r d e r to d e t e r m i n e if the p r o t e c t i o n g e n e r a t e d was due to a humoral or a c e l l u l a r factor. The results clearly d e m o n s t r a t e d that p r o t e c t i o n was s u c c e s s f u l l y t r a n s f e r r e d w i t h c e l l - f r e e immune hemolymph. In fact, animals p r e - t r e a t e d w i t h immune h e m o l y m p h were able to withstand a lethal dose of HBT as well as animals that had been actively i m m u n i z e d against the toxin. The e x p e r i m e n t s d e a l i n g with c e l l u l a r t r a n s f e r of p r o t e c t i o n w e r e harder to interpret. Hemocytes from n o r m a l donors c o n f e r r e d as good a rate of p r o t e c t i o n upon v i r g i n animals as immune hemocytes. S i n c e it is e s t i m a t e d that A m e r i c a n c o c k r o a c h e s have a total of only 1.2 x 107 hemocytes, the i n j e c t i o n of an a d d i t i o n a l 1 x 105 cells may have p r o t e c t e d them n o n - s p e c i f i c a l l y by i n c r e a s i n g their total cell number enough so that they w e r e b e t t e r able to survive the lethal dose of toxin. In any case, the d i f f e r e n t i a l p r o t e c t i o n o b s e r v e d w i t h immune h e m o l y m p h v e r s u s n o r m a l h e m o l y m p h (Table 3) was not evident in the e x p e r i m e n t s using i~mnune h e m o c y t e s and normal h e m o c y t e s (Tables 4 and 5). Thus, it w o u l d a p p e a r that the p r o t e c t i o n o b s e r v e d was b e i n g s p e c i f i c a l l y m e d i a t e d by a humoral factor. In summary, a specific a d a p t i v e immune r e s p o n s e to soluble p r o t e i n c o m p l e x e s was induced in an a d v a n c e d invertebrate, the A m e r i c a n cockroach. P a s s i v e t r a n s f e r studies w o u l d indicate that this r e s p o n s e is humoral in nature. F u r t h e r study and c h a r a c t e r i z a t i o n of this r e s p o n s e will d e t e r m i n e how analogous it may be to c l a s s i c a l v e r t e b r a t e - t y p e reactivity.

ACKNOWLEDGMENTS

We w o u l d like to thank Dr. Carl A. H u e t h e r for his help w i t h the s t a t i s t i c a l a n a l y s i s of the data.

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S t a t i s t i c a l Methods,

6th