Effect of hypodermin A, an enzyme secreted by Hypoderma lineatum (Insect Oestridae), on the bovine immune system

Veterinary Immunology and Immunopathology, 31 (1992) 167-177

167

Elsevier Science Publishers B.V., Amsterdam

Effect of hypodermin A, an enzyme secreted by Hypoderma lineatum (Insect Oestridae), on the bovine immune system N. Chabaudie and C. Boulard INRA, Station de Pathologie Aviaire et de Parasitologie, 37380 Nouzilly, France (Accepted 8 March 1991 )

ABSTRACT Chabaudie, N. and Boulard, C., 1992. Effect ofhypodermin A, enzyme secreted by Hypoderma lineaturn (Insect Oestridac), on the bovine immune system. Vet. Immunol. Immunopathol., 31: 167177. The absence of any inflammatory reaction around the first instar larvae (LI) ofHypoderma sp. in previously uninfested cattle suggested that these larvae may escape the non-specific defence system of the host. Immunosuppression had been noted during an experimental infestation. The aim of this work was to determine more precisely the potential role of hypodcrmin A (HA), an enzyme secreted by the larvae, in this immunosupprcssion. HA was found to have no effect on unstimulatcd lymphocytes from naive cattle but could influencc the response of these cells to mitogens. In calves, injection of HA was accompanied by a decrease in the lymphocyte proliferative response to mitogens. This immunodeprcssionlasted only for the duration of enzyme injections. In cattle, when HA is added, the antigen-dependentproliferative response increased significantly after 1 week of injection and disappeared 2 weeks after the end of the injection period. Finally, the rate of production of anti-HA antibodies increased at the same rate for calves and cows, and achieved a similar level. These results suggest that HA significantly modified the lymphoproliferativc response for naive cattle and, to a lesser extent, immune cattle during the time of administration only.

ABBREVIATIONS

ConA, concanavalinA; HA, hypodcrrnin A; PBL, peripheralblood lymphocytes;PBM, periphcralblood mononuclear cells;PHA, phytohcmagglutinin;P W M , pokcwecd mitogcn. INTRODUCTION

Hypoderma boris and Hypoderma lineatum are parasites of cattle only in the Northern hemisphere. Infestation occurs in summer as the first instar larvae penetrate the skin and then undergo an 8-month migration through the © 1992 Elsevier Science Publishers B.V. All fights reserved 0165-2427/92/$05.00

168

N. CHABAUDIE AND C. BOULARD

connective tissue. No inflammatory reaction accompanies this primary infestation. It has already been demonstrated how hypodermosis results in the disturbance of various mechanisms contributing to inflammation. For example, it has been reported that collagenolytic and fibrinolytic activity of the larvae (Boulard, 1970, 1975) and a complementary depleting effect (Boulard and BenCharif, 1984) mediated via a drastic proteolysis of bovine C3 (Boulard, 1989). This impairment of inflammation is largely caused by two of the secreted larval enzymes, hypodermins A and B. In investigating the effect of hypodermosis on the immune system, attention was initially directed to the humoral response (Boulard, 1970; Robertson, 1980). More recently, cellular immunity has been studied during a primary infestation and after reinfestation (Baron and Weintraub, 1987). During the first instar migration, the authors observed an inhibition of peripheral blood lymphocytes (PBL) reactivity to the crude larval antigen and a decreased responsiveness of PBL to a non-specific lymphocyte mitogen stimulation. Hypodermin A (HA) induces the highest anti-inflammatory effect; therefore it was investigated whether HA might also act on the cellular immune system and participate in immunosuppression. The purpose of this study was therefore to determine the effect of HA on the immune cellular response in vitro and in vivo. Bovine lymphocyte proliferation was stimulated with mitogens phytohemagglutinin (PHA), concanavalin A (ConA) or pokeweed mitogen (PWM), or with HA antigen. In vivo administration of HA was performed with naive and preinfested cattle and the PBL responsiveness to antigen and mitogens evaluated. Simultaneously, the humoral response was assayed by ELISA. MATERIAL AND METHODS

Animals For in vitro experimentation Lymphocytes were collected from five naive Holstein female calves aged 68 months, kept in an hypodermosis-free environment. For in vivo experimentation and cattle immunization The control and uninfested groups consisted of four naive Holstein female calves per group aged 6-9 months. Sera were subjected to ELISA testing to ensure that all animals were hypodermosis-free. The infested group was made up of four cattle infested for the second time. These cows were strongly seropositive. Uninfested and infested groups were injected, via the intradermal or subcutaneous route with 250/tg of HA three times weekly for 3 1/2 weeks, i.e. 2.5 mg per animal. The control group received injections of saline solution.

EFFECT OF HYPODERM1N A ON BOVINE IMMUNE SYSTEM

169

For the first 4 weeks, lymphocyte cultures were initiated twice a week and then once a week for the following 4 weeks. Serum samples were collected each week during the first 2 months and twice a month for the following months.

Mitogens ConA and PHA were purchased from Pharmacia and PWM from Sigma.

Hypodermine A antigen HA was purified from the first instar larvae ofHypoderma lineatum as previously described (Lecroisey et al., 1979).

Preparation of bovine PBM Peripheral blood mononuclear cells (PBM) were isolated from blood collected in EDTA solution, by centrifugation on Ficoll-Paque (Pharmacia). PBM isolated from the interface of plasma and FicoU-Paque were washed three times in PBS without Ca-Mg pH 7.2. PBM were resuspended at a concentration of 2 × 10 6 cells ml-~ in RPMI 1640 (Flow Laboratories) supplemented with 10% heat-inactivated foetal bovine serum, glutamine (2 mM), 2 mercaptoethanol (5 × 10-5 M), penicillin (50 IU ml-~ ) and streptomycin (40/tg ml-~ ) and pyruvate ( 1 mM ). The cells were more than 95% viable as tested by Trypan blue dye exclusion.

Lymphocyte proliferation assay In vitro experimentation PBM suspension ( 100/A) was added in triplicate to a microtitre plate (Poly Labs. ). In addition, PBM were cultured in the presence of 50/zl of ConA (20 /tg m l - ~), PHA ( 1 : 25) or PWM (20 #g m l - 1) and in the presence of 50/tl HA at different concentrations ( 15, 30, 60, 120 #g ml-~ ). The optimal concentration of mitogens was determined with reference to previous work (Pearson et al., 1979) and from our preliminary assays. Thus, 20 #g m l - I for ConA and PWM and 1 : 25 for PHA were selected. Negative control cultures received 100 #1 supplemented RPMI 1640 medium in place of the mitogens and antigen. Positive control culture contained mitogens but no antigen.

In vivo experimentation PBM suspension ( 100/A) was added in triplicate to a microtitre plate. In addition, PBM were cultivated in the presence of 50/tl of ConA, PHA or PWM (at the same concentration as above ) or in the presence of 50/tl of HA antigen at three concentrations (60, 120 and 200 #g m l - 1). Control cultures

170

N.CHABAUDIEANDC.BOULARD

received 100 #l supplemented RPMI 1640 m e d i u m in place of the mitogens or antigen.

Culture conditions Microtitre plates were incubated at 37°C in a humidified atmosphere with 6% CO2. All cultures were in triplicate. Plates were incubated either for 3 days (with mitogens or m i t o g e n + a n t i g e n ) or for 6 days (with antigen alone). Then, 1/tCi of [ 3H ]thymidine (CEA) was added to each well. After 18 h, the cultures were stored at - 2 0 °C until the cells were harvested. The plates were harvested using a multiple automated sample harvester ( P H D cell harvester, Cambridge Technology) on glass microfiber filters. Samples were dried, then counted in a liquid scintillation counter (Kontron).

Coupled HA on gel beads This technique was used to determine whether interaction of HA occurred with mitogens or lymphocytes. HA was coupled to CNBr-activated Sepharose 6B (pharmacia) as described by the manufacturers. HA-Sepharose was, then, incubated with the different mitogens for 5 hours in culture plates at the same concentration as that used in the microtitre plates. Controls were set up containing mitogens and sepharose uncoupled with HA. Supernatants containing each of the incubated mitogens were collected into the culture medium. Bovine PBM cultures were then treated and harvested as described above.

Measurement of anti-HA antibodies We used an ELISA test to detect anti-HA antibodies, as described by Boulard ( 1985 ). Each well of the microtitre plate (Flow Labs. ) was coated with 5/zg HA ml-1 of carbonate buffer. Included in each microplate were reference positive and negative sera. Results are expressed as a percentage of the mean value of the sample to these reference pools.

Presentation of results and statistical analys& In vitro experiment Each set of results is the mean value obtained from five animals. Experiments were repeated three times for each mitogen. Data are presented as a percentage of the positive control value (culture in the presence of mitogens and with no antigen). The above parameters were compared by analysis of variance. Significance was ascribed at P < 0.05.

EFFECT OF HYPODERMIN A ON BOVINE IMMUNE SYSTEM

171

In vivo experiment After subtraction of the CPM of parallel non-mitogen-stimulated cultures, data from injected groups were expressed as a percentage of this control value. These results were also analyzed using analysis of variance: values of P < 0.05 were considered to be significant. RESULTS

In vitro HA effect on the mitogenic-induced P B M proliferation Exposure of PBM to HA did not cause cytotoxicity, affect the viability of PBL, or lead to proliferation (not shown). The results of co-incubation of PBM from naive cattle with PHA, ConA or PWM and HA are presented in Fig. 1. A marked dose-dependent inhibition of the PHA-induced PBL response was obtained in the presence of HA. This inhibition was statistically significant ( P < 0.05 ) with HA at 60 and 120/zg m1-1, but not significant at 15 and 30/~g m l - ~. A moderate proliferative hyporesponsiveness, which only reached 20% suppression (Fig. 1 ), was observed with ConA co-incubated with HA. An obvious proliferative response was induced when HA was co-administrated with PWM (Fig. 1 ) reaching an increase of 50% significantly different ( P < 0.05 ) at concentrations of 30, 60 or 120/zg m l - ~of HA. A control assay to test for any suspected interaction of HA with mitogens was also performed. PHA and PWM were incubated with HA coupled to sepharose CN-Br or with activated sepharose CN-Br alone, prior to PBM culture. No modifications of lymphocyte proliferation were observed (not shown), suggesting that HA did not interfere with the PBL response by directly affecting the mitogens. In vivo HA effect on the immune response The kinetics of the proliferative response of PBL from uninfested or previously infested cattle to mitogens PHA, ConA and PWM during and 3 weeks after the course of HA inoculation are shown in Fig. 2. In naive cattle, HA administration resulted after the first injection ( 500 #g per animal) in a significant ( P < 0.05 ) hyporesponsiveness of PBL to PHA stimulation (80% inhibition), ConA (60% inhibition) and PWM (80% inhibition). The PBL proliferation inhibition was also observed for the previously infested cattle; however, suppression was delayed on exposure to PHA and remained less pronounced on exposure to PWM than observed for naive cattle. The preinjection level of mitogenic response of PBL from naive or immune cattle was restored after the HA injections were stopped. The kinetic responses of PBL to homologous antigenic stimulation within

172

N. C H A B A U D I E A N D C. B O U L A R D

60 f 40 E o

"6

-

ConA

20~0

-

t

Eu ~ -20

-40 F -601 0

;

' 15

x 30

i

60

120

I 60

J 120

HA # g / m l of c u l t u r e PHA

0 B

u "5

-10 -20 -30

~

40

u

-50

-60

lt5

0

310 HA #g /m[ of c u l t u r e

60

PWM

50 E o

4O

"5

3O

~u 2o ~_ ~o O_~

15 HA

30 ,ug/ml of culture

60

120

Fig. 1. Lymphoproliferation responses of PBM from naive calves exposed to variable concentrations of HA and mitogen, either Con-A, PHA or PWM. Each mean value (five animals) is expressed as a percentage of control values obtained from HA-free cultures. Each assay was repeated three times. ~t', experiment I; A, experiment II; II, experiment III. the course o f HA administration for 3 weeks afterwards is also presented in Fig. 2. Within the first 2 weeks of inoculation o f HA in both groups o f cattle, no specific response was observed. A very low antigen-induced proliferative response (not statistically significant) was observed the following week for the naive cattle. An increased responsiveness which was significant ( P < 0.05 ) was observed for the infested cattle within the next month with a marked peak during week 5. Humoral response Anti-HA antibodies began to increase 1 week after the beginning o f HA injections in the infested group and after 2 weeks in uninfested calves (Fig.

173

EFFECT OF HYPODERMIN A ON BOVINEIMMUNE SYSTEM Con A

100 8O 6O

~

~o

~

2o

v

-!!, 0

~, - 8 0 o. -100

I

'

I

~

1

P

2

~

'

3

I

I

I

I

i

z,

5

6

7

8

I ~

t 5

I 6

i 7

r 8

W e e k s a f t e r first injection

PHA

40

~

zo

u

0

"5 -40 -20 I ~ -60 ~_ -80 -100 0

t

I 1

I

I 2

I

' 3

I

Weeks a f t e r first injection

100 80 60 ! 40 20 u O; '5 -20~ -40 -60 -80 Q. -100 0

PWM

~

~

1

r

r

2

~

I

3

t

I

t

f

r

I

4

5

6

7

8

Weeks a f t e r first injection

Antigen

14 12

u

8

o.

0"1" 0

1

2

3

4

5

6

7

8

Weeks a f t e r first injection

Fig. 2. Kinetics of suppression of ConA, PHA, PWM or antigen-induced PBM blastogenesis as a result of HA administration. HA was injected three times weekly for 3½ weeks. Mean responses of PBM from uninfested (W) or previously infested ( • ) cattle are expressed as a percentage of controls. 0 , P < 0.05 for calves; ~-, P < 0.05 for cattle, (analysis of variance); R , period of injection.

174

N. CHABAUDIE AND C. BOULARD

~,

100

~c o :~

80 60J

"~

40

"6

20 o 1

2

3

4

Weeks o f t e r

5

6

first

injection

7

8

12

1/.

16

Fig. 3. Anti-HA antibody responses in uninfested (11) and previously infested ( • ) cattle after HA administration. Each data point represents the arithmetic mean _+SE of percentage values for the group, m , period of injection.

3 ). At the beginning of the experiment, cattle had antibody levels which were 60% of the reference positive serum, residual from the previous infestation. Anti-HA antibodies reached a maximum value 3 weeks after the first injection and increased from 60 to 100% in previously infested cattle. Similarly, anti-HA antibody levels for calves changed from l 0 to 50%. The level of antibody decreased so that l0 weeks after administration of HA commenced, no detectable antibody was found in the uninfested group, but a level of 15% was found in infested ones. DISCUSSION

The major finding of this study is that the immunosuppression previously described during bovine hypodermosis (Baron and Weintraub, 1987) may be mediated, in part at least, by a molecule secreted by the parasite, hypodermin A, which has also been demonstrated to be a powerful anti-inflammatory factor (Boulard and Bencharif, 1984; Boulard, 1989). This study concerning the effect of HA on mitogen-stimulated lymphocytes provides apparently different results which may be due to the nature of the mitogen used and the immune environment participating in the lymphocyte activation. Co-incubation in vitro of certain concentrations of HA with PBM of naive cattle demonstrated a different effect dependent upon the mitogen used: HA suppressed the PHA-dependent lymphoproliferation response while it increased the PWM one. These observations suggest that HA may impair the T-lymphocyte functions while it may also participate in B-cell activation. The different effect of HA on T-lymphocyte proliferation when co-incubated with various mitogens, substantiates the fact that this parasite product does not interfere with a similar set of signals following each type of mitogen stimula-

EFFECT OF HYPODERMIN A ON BOVINE IMMUNE SYSTEM

17 5

tion. It might be speculated that various pathways of interaction of HA in vitro are related to its biological and biochemical properties. HA is a serine protease (Boulard and Garrone, 1977; Lecroisey et al., 1979) which exhibits a high affinity and powerful enzymatic activity on bovine C3 (Boulard, 1989). C3 by-products modulate the immune response even in an in vitro PBM culture (Weiler et al., 1982). HA could potentially impair the immune process at various levels when C3 is involved through the peptides specific for the cell complement receptors such as CRI and CR3 on macrophages or CR1/CR2 on B-cells (Fingeroth et al., 1989). HA may also exert a direct enzymatic effect on accessory cells by activating other serine proteases or interfere with other receptors which participate in cell metabolism (Hameed et al., 1988; Simon et al., 1988 ) and in the delivery of cell signals. Studies are currently in progress to characterize the class and subsets of the cell populations affected by PBM mitogen stimulations in a co-culture with HA. As HA may act directly on cytokines, the interactions of HA with these in the course of PBM activation are also under study. These data are required to determine the molecular mechanisms by which HA affects cellular responses to mitogens as used in this in vitro assay. In contrast, the immunological consequences of HA injections in vivo on peripheral blood cellular and humoral responses of previously uninfested or infested cattle are consistent and comparable with most of the immuno-suppressive phenomena described in the course of parasitic infections. These phenomena resulted in an overall decrease of the lymphocyte responses to Tand B-mitogens and to antigen (Dessaint et al., 1977; Burger et al., 1986). The duration of the immunodepression was strictly dependent on the period of HA injection and was alleviated soon after administration ceased. The kin6tics of the proliferative response of PBL to PWM appeared to be equally affected in both groups in total contrast with the results obtained from in vitro experiments. Differences in the humoral immune system environment in vivo may account for this discrepancy. The anti-inflammatory activity of HA may participate in the decreased responsiveness of the in vitrostimulated B-lymphocytes. The specific proliferative response to the HA antigen was delayed by 2 weeks for the previously infested cattle, and lasted only 4 weeks. These data could be related to the previous observations of defective T-cell activity associated with the presence of HA. This lack of specific cellular responsiveness is frequently mentioned in other parasite infections (Ottesen et al., 1977; Oldham and Williams, 1985 ). This lack of specific response may reflect a delay in the memory process. The fact that the anti-HA antibody responses displayed a similar pattern in uninfested and infested cattle receiving HA injections substantiates this hypothesis. The immunoglobulin class of the antibodies detected is now being investigated to determine whether the switch from IgM to IgG has been affected (Abbas, 1988; Shinomya et al., 1989).

176

N. CHABAUDIE AND C. BOULARD

In c o n c l u s i o n , t h e s e results i n d i c a t e that, as well as h a v i n g a n t i - i n f l a m m a t o r y a c t i v i t y , H A m a y significantly r e d u c e the specific cellular i m m u n e res p o n s e a n d p r e v e n t the i n i t i a t i o n o f the i m m u n e m e m o r y m e c h a n i s m s . T h e b o v i n e t a r g e t cells o f H A a n d the i m p a i r m e n t o f t h e u n d e r l y i n g m o l e c u l a r mechanisms need further investigation. ACKNOWLEDGEMENT T h e excellent t e c h n i c a l a s s i s t a n c e p r o v i d e d b y C. V i l l e j o u b e r t d u r i n g this i n v e s t i g a t i o n is g r a t e f u l l y a c k n o w l e d g e d .

REFERENCES Abbas, A.K., 1988. A reassessment of the mechanisms of antigen-specific T-ceil-dependent Bcell activation. Immunol. Today, 9: 89-94. Baron, R.W. and Weintraub, J., 1987. Lymphocyte responsiveness in cattle previously infested and uninfested with Hypoderrna lineaturn (De Vill) and Hypoderma boris (L) (diptera:oestridae). Vet. Parasitol., 24: 285-296. Bou!ard, C., 1970. Etude pr61iminaire d'une collag6nase brute extraite de la larve de premier stade d'Hypoderma lineaturn (De Villiers). C.R. Acad. Sci. Paris., 270: 1349-1351. Boulard, C., 1975. Modifications histologiques de la sous-muqueuse oesophagienne de bovins parasit6s par Hypoderrna lineatum (De Vill. ) (Diptera-Oestriforme). Ann. Rech. V6t6r., 6" 131-142. Boulard, C., 1985. Avantages de l'immunodiagnostic de l'hypodermose bovine 6tablis par h6magglutination passive et par Elisa,/l partir du s6rum et du lactos6rum, sur la num6ration des varons. Ann. Rech. Vet., 16(4): 335-343. Boulard, C., 1989. Degradation of bovine C 3 serine proteases from parasites Hypoderma lineaturn (diptera,oestridae). Vet. Immunol. Immunopathol., 20: 387-398. Boulard, C. and Bencharif, F., 1984. Changes in the haemolytic activity of bovine serum complement by Hypoderrna lineaturn (insect oestridae) larval proteinases in naive and immune cattle. Parasitol. Immunol., 6: 459-467. Boulard, C. and Garrone, R., 1977. Characterization of a collagenolytic enzyme from larvae of Hypoderrna lineaturn (Insecta: Diperta, Oestriform). Comp. Biochem. Physiol., 59 (B): 251255. Burger, C.J., Rikihisa, Y. and Lin, Y.C., 1986. Taenia taeniaformis: inhibition of mitogen induced proliferation and interleukin 2 production in rat splenocytes by larval in vitro product. Exp. Parasitol., 62: 216-222. Dessaint, J.P., Camus, D., Fisher, E. and Capron, A., 1977. Inhibition of lymphocyte proliferation by factor (s) produced by Schistosoma mansoni. Eur. J. Immunol., 7: 624-629. Fingeroth, J.D., Heath, M.E. and Ambrosino, D.M., 1989. Proliferation of resting B cells is modulated by CR2 and CR1. Immunol. Lett., 21:291-302. Hameed, A., Lowrey, D.M., Lichtenheld, M. and Podack, E.R., 1988. Characterisation of three serine esterases isolated from human IL2 activated killer cells. J. Immunol., 141: 3142-3147. Lecroisey, A., Boulard, C. and Keil, B., 1979. Chemical and enzymatic characterisation of the collagenase from the insect Hypoderma lineatum. Eur. J. Biochem., 101: 385-393. Oldham, G. and Williams, L., 1985. Cell mediated immunity to liver fluke during experimental Fasciola hepatica infection of cattle. Parasitol. Immunol., 7:503-516.

EFFECT OF HYPODERMIN A ON BOVINE IMMUNE SYSTEM

177

Ottesen, E.A., Weller, P.F. and Heck, L., 1977. Specific cellular immune unresponsiveness in human filariasis. Immunology, 33:413-421. Pearson, T.W., Roelants, G.E., Lundin, L.B. and Mayor-Withey, K.S., 1979. The bovine lymphoid system: binding and stimulation of peripheral blood lymphocytes by lectins. J. Immunol. Methods, 26: 271-282. Robertson, R.H., 1980. Antibody production in cattle infected with Hypodermaspp. Can. J. Zool., 58: 245-251. Shinomya, N., Kuratsuji, T. and Yata, J.I., 1989. The role of T cells in immunoglobulin class switching of specific antibody production system in vitro in humans. Cell. Immunol., 118: 239-249. Simon, H.G., Fruth, U., Eckerskorn, C., Lottspeich, F., Kramer, M.D, Nerz, G. and Simon, M.M., 1988. Induction of T cell serine proteinase 1 (TSP-I)-specific mRNA in mouse T lymphocytes. Eur. J. Immunol, 18: 855-861. Weiler, J.M., Ballas, Z.K., Needleman, B.W., Hobbs, M.V. and Feldbush, T.L., 1982. Complement fragments suppress lymphocyte immune responses. Immunol. Today, 3: 238-243.