Differences in the surface radioiodinated proteins of skin and uterine microfilariae of Onchocerca gibsoni

Molecular and Biochemical Parasitology, 10 (1984) 217-229 Elsevier

217

MBP 00395

DIFFERENCES IN THE SURFACE R A D I O I O D I N A T E D PROTEINS OF S K I N AND UTERINE M I C R O F I L A R I A E O F O N C H O C E R C A G I B S O N I

KAREN P. FORSYTH t, D. BRUCE COPEMAN 2 and GRAHAM F. MITCHELL l ~lmmunoparasitology Unit, The Walter and Eliza Hall Institute of Medical Research, Melbourne. Victoria 3050, Australia and 2Department of Tropical Veterinary Medicine, James Cook University of North Queensland, Townsville 4811, Australia. (Received 25 April 1983; accepted 23 July 1983)

Surface labeling studies using tWO populations of Onchocerca gibsoni microfilariae revealed important differences in major radioiodinated proteins. Small numbers of microfilariae harvested from the skin of cattle or the uteri of adult worms from skin nodules were purified, radioiodinated, solubilized and the proteins analysed by two dimensional gel electrophoresis and autoradiography. As reported previously, uterine microfilariae showed a complex profile of radioiodinated proteins, none of which appeared to be bovine albumin or immunoglobulin. In contrast, application of the same techniques to skin microfilariae demonstrated only one major labeled protein complex of approximate M r 67 000. This protein complex was immunoprecipitated with an antiserum to bovine serum albumin. Surprisingly, fluorescence techniques failed to show bovine serum albumin on the surface of living microfilariae. Although the evidence is circumstantial at present, acquisition of host albumin (perhaps oriented in a particular way) may be a means whereby skin microfilariae evade immune effector mechanisms and, when living, generally fail to elicit inflammatory reactions in the skin of the host. Key words: Onchocerca gibsoni; Microfilariae; Surface proteins; Carbohydrates and antigens; Albumin; Two dimensional gel electrophoresis; Onchocerciasis

INTRODUCTION L i v i n g m i c r o f i l a r i a e o f O n c h o c e r c a s p p . i n d u c e m i n i m a l i n f l a m m a t o r y r e a c t i o n s in the subcutaneous

t i s s u e s o f i n f e c t e d h o s t s [1,2]. I t is b e l i e v e d t h a t t h e m a j o r c l i n i c a l

manifestations of onchocerciasis are caused by reactions to dead and dying microfilariae. A v i o l e n t h o s t r e s p o n s e t e r m e d t h e M a z z o t t i r e a c t i o n , c a n b e i n d u c e d in p a t i e n t s w i t h o n c h o c e r c i a s i s a f t e r a d m i n i s t r a t i o n o f a m i c r o f i l a r i c i d a l d r u g s u c h as d i e t h y l c a r b a m a z i n e ( D E C ) [3]. D E C a p p e a r s t o e n h a n c e m i c r o f i l a r i a l k i l l i n g in v i v o , in c o n c e r t with the host's immune

s y s t e m [2,4]. O n e cell t y p e o b s e r v e d t o a c c u m u l a t e

and

Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; BSA, bovine serum albumin; BTPBS, bovine tonicity phosphate buffered saline; DEC, diethylcarbamazine; IEF, isoelectric focussing; IFAT, indirect fluorescent antibody test; Mr, relative molecular weight; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate. 0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.

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degranulate in the vicinity of dying microfilariae is the eosinophil granulocyte [1,5,6]. Recently, Greene et al. [7] have shown that O. volvulus microfilariae taken from the skin of an infected patient can be killed in vitro in the presence of eosinophils (or neutrophils) and serum taken from that same patient. The above observations raise the question of how established microfilariae evade microfilaricidal responses which, from in vitro experiments, appear to involve at least antibody-dependent cell-mediated cytotoxicity (ADCC). One possible explanation is that the microfilarial surface is masked by a coat of host protein which renders the parasite surface immunologically inert. Some support for an antigen masking mechanism of immune evasion has been gained in the Schistosoma mansoni system (reviewed in [8-12]). Host components have been reported on the surface of microfilariae [13-15], other helminths [16,17] as well as protozoan parasites [18,19]. Lectin binding and protein labeling studies have been performed on microfilariae of the cattle parasite O. gibsoni which is closely related to the parasite of man, O. volvulus. Microfilariae derived from either the uteri of adult worms (in brisket nodules) or the skin of infected cattle were compared since they must differ in exposure to host proteins and possibly in surface antigens. The demonstration of stage-specific surface antigens of nematodes [20-22] indicates that an alteration of surface molecules accompanies larval development. Using a fluoresceinated lectin assay, Furman and Ash [23] have shown differences in carbohydrates on the surface of uterine and blood-borne microfilariae of Brugia pahangi. Further evidence for differences in surface molecules of these two populations ofB. pahangi microfilariae comes from the observation that complement-mediated cell adherence reactions occur with blood-derived microfilariae and not with microfilariae taken from the uteri of adult worms [24]. Wegerhof and Wenk [25] have shown that uterine microfilariae are more potent than blood microfilariae at inducing anti-microfilarial immunity in cotton rats infected with Litomosoides carinii. Consequently, protein a n d / o r carbohydrate antigens on the cuticle of skin and uterine microfilariae of O. gibsoni should differ. A comparison of the surface radioiodinated proteins of the two microfilarial populations was performed in order to determine the extent of differences in surface proteins. In addition, fluoresceinated lectins have been used to examine gross differences in expression of carbohydrates. MATERIALS AND METHODS

Parasites. The brisket regions of cattle infected with O. gibsoni were collected from carcases held overnight in a 4°C chiller at abattoirs in Townsville. Nodules containing adult worms were removed from this tissue and sent to Melbourne at 4°C. Strips of bovine skin were also cut from this tissue, slashed at 5 cm intervals and placed in 250 ml sterile glass bottles containing Medium 199 (Gibco, New York, U.S.A.) supplemented with 200 units of penicillin and 200 lag of streptomycin per ml. Skin samples were incubated at room temperature for 20-24 h during transit to, and storage in,

219 Melbourne. Approximately 103 skin m icrofilariae were collected by centrifugation of the supernatant of each skin sample at 1 000 X g in a refrigerated Heraeus Christ minifuge. Uterine microfilariae and eggs were obtained from fragments of female adult worms dissected from nodules [26]. Both uterine and skin microfilariae were purified by a Ficoll-Paque method as described previously [26]. Each preparation of purified microfilariae contained less than 0.1% contaminating bovine cells.

Antisera. Sera were obtained from 2-3 year old O. gibsoni-infected cattle shown to have high numbers of skin microfilariae by the technique of Beveridge et al. [27]. A calf given multiple injections of living uterine microfilariae subcutaneously, and shown to have no detectable skin microfilariae, was bled for serum. Control bovine sera were obtained from calves maintained in insect-free facilities in Townsville. Antisera to bovine serum albumin (BSA) were raised by injecting SJL / J mice with 10 lag of Fraction V BSA (Armour Pharmaceutical, Eastbourne, England) in Complete Freund's Adjuvant (CFA) (Difco Laboratories, Detroit, MI, U.S.A.) into the footpads. Mice were boosted 1 month later with 100 lag of BSA in saline given intraperitoneally and bled one week after this injection. Specificity of this reagent was checked by immunoprecipitation of ~25I-BSA labeled by the IODO-GEN (Pierce Chemical Co., Rockford, IL, U.S.A.) procedure [27] and subsequent two dimensional gel analysis and autoradiography. Further confirmation of the specificity of these sera has been provided by Handman [19]. Control mouse sera were collected from SJL/J mice which had been immunized with an irrelevant antigen.

Radioiodination ofmicrofilariae. Approximately 103 purified uterine or skin microfilariae were surface radioiodinated by either the IODO-GEN technique [28] or the Bolton and Hunter method [29]. Surface labeling of microfilariae using IODO-GEN was performed as described previously [30] using 500 laCi of Nal-'5I (Amersham Searle Corp., Arlington Heights, IL, U.S.A.). Autoradiography and electron microscopy studies of transverse sections of uterine microfilariae labeled by the IODO-GEN procedure had previously shown that this technique labeled the outermost layer of the cuticle [30]. Purified microfilariae to be radioiodinated by the Bolton and Hunter method were washed three times in ice cold 0.1 M borate buffer pH 8.5. Parasites were then added in a volume of 100 lal of borate buffer to a glass tube coated with 500 laCi of Bolton and Hunter reagent (Amersham Searle Corp.). The reaction proceeded on ice for 15 min and was terminated by addition of 0.5 ml 0.1 M borate buffer, pH 8.5, containing 2 M glycine for 5 min on ice. The labeled microfilariae were transferred to a clean glass tube and washed by centrifugation at 1000 X g in bovine tonicity phosphate buffered saline (BTPBS). Skin and uterine microfilariae were shown to be viable after both radioiodination procedures by motility observed under microscopic examination. Parasites were stored at -70°C in BTPBS containing 2% (w/v) ovalbumin (Sigma, St. Louis, MO, U.S.A.), 5 mM phenylmethylsulphonyl fluoride (Sigma)and 5 mM EDTA. Extracts of microfilariae labeled by the IODO-GEN procedure were

220 prepared for immunoprecipitation analysis by solubilization of 500-1000 microfilariae in a 0.15 M NaC1 buffer containing 1.5% Triton X-100 (BDH, Poole, England), 2% (w/v) ovalbumin, 5 mM PMSF and 5 mM EDTA. Microfilariae were incubated in this buffer for 30 min on ice, sonicated, maintained on ice for a further 30 min and centrifuged at 12 000 × g for 10 min [30].

Immunoprecipitation of Triton X- 100 extract of ~251-1abeled microfilariae. Supernatants of solubilized ~25I-labeled microfilariae (2 >( 105 trichloroacetic acid (20%)precipitable cpm) were precleared with 100 I.tl o f 10% (v/v) heat killed and formalin fixed Staphylococcus aureus of the Cowan I strain (Commonwealth Serum Laboratories, Parkville, Victoria, Australia). The cleared extract was then added to 10 lal of the appropriate bovine or mouse serum and incubated for 2 h on ice. Immune complexes were isolated by binding to protein A-bearing Staphylococcus aureus as described by Kessler [31]. Precipitated immune complexes were washed 3 times in a 0.05 M Tris-HCl buffer, pH 8, containing 0.5% Triton X-100, 0. !5 M NaCI, 0.05 M E D T A as described by Kessler [31] with a tube change before the last wash, and counted in an autogamma counter. Immunoprecipitates were then analysed by one or two dimensional gel electrophoresis.

Polyacrylamide gel electrophoresis. One-dimensional polyacrylamide gel electrophoresis (PAGE) was carried out in the presence of sodium dodecyl sulfate (SDS) according to the method of Laemmli [32], using 10% acrylamide slab gels. ~25I-labeled microfilariae or immunoprecipitates were prepared for electrophoresis by addition of a Tris-HCl buffer, pH 6.8, containing 3% w/v SDS, 6 M urea, 50% glycerol and 5% v/v I~-mercaptoethanol. Alternatively, samples were electrophoresed in two dimensions by the method developed by O'Farrell et al. [33] using 10% acrylamide slab gels for the second dimension. Prior to two-dimensional gel analysis, immune complexes were incubated for 15 min at room temperature in 25 Ixl of SDS-lysis buffer which was identical to the O'Farrell isoelectric focussing (IEF) buffer [33] except that the Triton X-100 was replaced with 2% SDS (w/v). 100 lal of IEF-lysis buffer was then added and the mixture incubated for 15 min at room temperature. Insoluble material was removed by centrifugation at 10 000 X g using a Beckman microfuge (Fullerton, CA, U.S.A.). The supernatant which was then ready for isoelectric focussing, contained 90-95% of the total immunoprecipitated counts. Gels included molecular weight standards (LMW calibration kit, Pharmacia Fine Chemicals, Uppsala, Sweden): phosphorylase b (M r 94 000), bovine serum albumin (M r 67 000), ovalbumin (M r 43 000), carbonic anhydrase (M r 30 000), soybean trypsin inhibitor (M r 20 000), and a-lactoalbumin (M r 14 400) and stained with 0.01% Coomassie Blue in 50% methanol/10% acetic acid. They were destained in 7% acetic acid, dried and autoradiographed using Kodak X-Omat S film and Dupont Cronex Lighting plus intensifying screens (Du Pont Wilmington, DE, U.S.A.). Exposure times varied between I and25d.

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Albumin adsorption assay. Ten thousand purified uterine microfilariae were incubated at 37°C for 3 h with 1.0 ml of either a pool of 5 sera from cattle infected with O.

gibsoni or a pool ofsera from calves never exposed to O. gibsoni. As a control, the same number of uterine microfilariae was incubated at 37°C for 3 h in Leibowitz L-15 medium (Gibco, New York, U.S.A.). After this incubation period, the microfilariae were washed 3 times in BTPBS and radioiodinated by the I O D O - G E N procedure. Radioiodinated microfilariae were solubilized in IEF-lysis buffer in the presence of ovalbumin as carrier protein and subsequently analysed by two-dimensional gel electrophoresis and autoradiography.

Indirect fluorescent antibody test. Samples of 50 Ixl BTPBS containing 50 living skin or uterine microfilariae, were added to each of the wells of a round bottom microtiter tray (Linbro McLean, VA, U.S.A.). 50 ~tl samples of appropriate sera, diluted 1:10, were added to the microfilariae and allowed to react for 30 min on ice. Microfilariae were then washed three times with ice cold BTPBS by centrifugation at 250 X g in the microtiter tray followed by addition of 50 ~tl of a 1:25 dilution of fluorescein isothiocyanate-conjugated rabbit anti-bovine Ig or a fluorescein conjugate of rabbit antimouse Ig. Reagent controls were wells containing washed parasites not exposed to mouse or bovine sera. Fluorescein isothiocyanate conjugates were prepared from affinity-purified antibodies according to the method of Goding [34]. Microfilariae were incubated with the fluorescein conjugates for 30 min on ice and subsequently washed three times with ice cold BTPBS. Fluorescence of the microfilariae surface was detected using a Zeiss microscope with vertical illumination and appropriate filters.

Lectin binding assay. Eggs, uterine and skin microfilariae were purified using the Ficoll-Paque procedure [26] and washed in a 0.02 M Tris-HCl buffer, pH 7.4 (Tris buffer). Aliquots containing 100 washed parasites in 100 I.tl of Tris buffer were incubated with 10 lal of fluoresceinated lectins (200 lag ml -~) (E-Y Laboratories, Inc., San Mateo, CA, U.S.A.) for 30 min on ice. The microfilariae or eggs were then washed in Tris buffer and resuspended to a final volume of 50 lal. A 20 lal sample was examined by fluorescence microscopy. RESULTS Skin and uterine O. gibsoni microfilariae were labeled by either the I O D O - G E N or Bolton and Hunter radioiodination methods. Two-dimensional gel analysis and autoradiography of solubilized 125I-labeled microfilariae was performed to compare the spectrum of radioiodinated surface proteins. Fig. 1 (A and C) reveals that a complex pattern of radioiodinated proteins was obtained when uterine microfilariae were 125I-labeled by either of the 2 methods. At least 30 proteins were detected, which varied in molecular weight from M r 20 000 to M r 120 000 and displayed a broad range of charge heterogeneity. Considerable variation between the 125I-labeled protein

222

Fig. 1. Autoradiographs of two dimensional gels (run under reducing conditions) of radioiodinated O.

gibsoni uterine (A,C) or skin microfilariae (B,D) labeled by either the IODO-GEN (A,B) or Bolton and Hunter (C,D) techniques. The most acidic proteins are to the left of each panel. Numbers on the left of each panel represent Mr standards. X-ray films were exposed to gels A and C for 2 days and gels B and D for 7 days. profiles o f uterine microfilariae resulted f r o m the use o f the 2 a b o v e m e n t i o n e d labeling p r o c e d u r e s (Fig. I A a n d C). Whilst s o m e o f the p r o t e i n s l a b e l e d by either o f the 2 m e t h o d s h a d similar m o l e c u l a r weights, the overall charge characteristics o f the l a b e l e d p r o t e i n s were quite different. The d i s p a r i t y in charge o f l a b e l e d p r o t e i n s p r e s u m a b l y results f r o m a greater a l t e r a t i o n o f charge t h r o u g h s u b s t i t u t i o n o f a free a m i n o g r o u p b y an i o d o p h e n o l g r o u p in the case o f the B o l t o n a n d H u n t e r technique, c o m p a r e d with direct i o d i n a t i o n b y the I O D O - G E N reagent [29]. A n analysis o f r a d i o i o d i n a t e d p r o t e i n s o f skin microfilariae labeled by the 2 p r o c e d u r e s is s h o w n in Fig. IB a n d D. O n l y one r a d i o i o d i n a t e d p r o t e i n c o m p l e x o f m o l e c u l a r weight M r 67 000 c o u l d be l a b e l e d by either m e t h o d . This p r o t e i n c o m p l e x was not p r e c i p i t a t e d b y sera f r o m either an O. gibsoni-infected cow o r a calf injected with O. gibsoni uterine microfilariae. T h e M r 67 000 p r o t e i n c o m p l e x was, however, i m m u n o p r e c i p i t a t e d by an a n t i - B S A s e r u m (Fig. 2). In c o n t r a s t , i m m u n o p r e c i p i t a t i o n s t u d i e s using a T r i t o n X-100 detergent extract o f uterine microfilariae labeled by the

223

Fig. 2. An autoradiograph of a one dimensional gel (run under reducing conditions) of immunoprecipitates of sera reacted with the Triton X-100 detergent extract of O. gibsoni skin microfilariae labeled by the IODO-GEN technique. Labeled proteins specificallyrecognised by sera from control mice,either of 2 mice immunised with BSA and a calf injected with uterine microfilariae are shown in lanes A, B, C and D, respectively. The band immediately above M 43 000 in lanes B, C and D is not an immunoprecipitated labeled protein but it is an .artifact due to an overload of unlabeled immunoglobulin heavy chain in that region of the gel. Numbers to the left of the figure represent M standards, Exposure time for this gel was 25 days. I O D O - G E N procedure a n d a p p r o p r i a t e sera, indicated that host a l b u m i n was n o t present o n the surface o f uterine microfilariae [30]. At least 9 proteins were specifically i m m u n o p r e c i p i t a t e d by serum from a calf i m m u n i s e d with O. gibsoni uterine microfilariae (Fig. 3A) a n d 5 proteins were recognised by serum from a n O. gibsoni infected cow (Fig. 3B). To s u p p o r t the surface labeling studies, an indirect fluorescent a n t i b o d y test ( I F A T ) was p e r f o r m e d using the 2 p o p u l a t i o n s of microfilariae. As expected, the surface of uterine microfilariae reacted specifically with sera from both infected a n d

224

Fig. 3. Autoradiographs of two dimensional gels (under reducing conditions) of the immunoprecipitates of sera reacted with a Triton X-100 detergent extract of O. gibsoni uterine microfilariae labeled by the IODO-GEN technique. Labeled proteins recognised by serum from a calf immunised with O. gibsoni uterine microfilariae or serum from an O. gibsoniinfected cow are shown in A and B, respectively. Numbers to the left of each panel represent M standards. Exposure time for gels was 14 days. m i c r o t i l a r i a e i m m u n i z e d cattle b u t not with a n t i - I g o r a n t i - B S A sera. H o w e v e r , no positive fluorescence was o b s e r v e d with a n y o f the a b o v e m e n t i o n e d sera (including a n t i - B S A serum) when r e a c t e d with s k i n - d e r i v e d microfilariae. The ability o f uterine m i c r o f i l a r i a e to a d s o r b B S A o r o t h e r host s e r u m proteins, was e x a m i n e d by i n c u b a t i n g uterine m i c r o f i l a r i a e at 37°C for 3 h in sera f r o m cattle infected with O. gibsoni o r a n i m a l s not e x p o s e d to this parasite. A c o n t r o l i n c u b a t e d at 37°C with no s e r u m was also included. A f t e r the i n c u b a t i o n p e r i o d , uterine microfilariae were w a s h e d in BTPBS a n d ~25I-labeled using I O D O - G E N . R a d i o i o d i n a t e d m i c r o f i l a r i a e were solubilized a n d s u b s e q u e n t l y a n a l y s e d by 2 - d i m e n s i o n a l gel elect r o p h o r e s i s a n d a u t o r a d i o g r a p h y . T h e typical uterine m i c r o f i l a r i a e r a d i o i o d i n a t e d p r o t e i n profile was o b t a i n e d for p a r a s i t e s i n c u b a t e d with o r w i t h o u t s e r u m (see Fig. I A ) , i.e. no a d s o r b e d a l b u m i n c o u l d be d e t e c t e d using this i n c u b a t i o n a n d labeling procedure. T a b l e I s u m m a r i z e s the results o f a lectin b i n d i n g s t u d y b a s e d on the use o f a p a n e l o f fluoresceinat'ed lectins. N o n e o f 6 lectins b o u n d to either uterine o r skin microfilariae. H o w e v e r , five o f the lectins t e s t e d b o u n d u n i f o r m l y to O. gibsoni eggs indicating t h a t the assay was c a p a b l e o f detecting lectin b i n d i n g a n d t h a t N - a c e t y l g l u c o s a m i n e , N - a c e t y l g a l a c t o s a m i n e , glucose, galactose a n d m a n n o s e residues were present o n the surface o f all uterine eggs. DISCUSSION T h e presence o f host a l b u m i n a n d the a p p a r e n t m a s k i n g o f c u t i c u l a r antigens on the surface o f s k i n - d e r i v e d 0. gibsoni m i c r o f i l a r i a e has been d e m o n s t r a t e d by surface

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TABLE I A comparison of lectin-binding properties of uterine eggs and microfilariae, and skin microfilariae, of

Onchocerca gibsoni Lectina

Presenceof competing sugar

Sugar specificity of lectin

Fluorescence Eggs

Con A

+

D-mannose, D-glucose.

UEA

+

0t-L-fucose

PNA

+

13(l--3)-N-acetyl glucosamine, o-galactose,

-

N-acetyl-D-galactosamine.+

SBA

Uterine microfilariae

Skin microfilariae

+b -

+ -

+ Hp

WGA

a b

-

ct-D-N-acetylgalactosamine, +

+

a-D-galactose.

4-

+

[~(l--4)-N-acetyI-D-glucosamine]~

+ +

Abbreviations: Con A = concanavalin A; UEA = Ulexeuropaeusagglutinin; PNA = peanut agglutinin; SBA = soybean agglutinin; Hp = Helix pomatia agglutinin; WGA = wheat germ agglutinin. 100%of eggs examined were positive in all reactions designated +.

labeling studies using 2 r a d i o i o d i n a t i o n methods. N o evidence for a stable i n t e r a c t i o n between BSA a n d cuticular (surface) antigens was gained by i m m u n o p r e c i p i t a t i o n with sera from calves i m m u n i s e d with uterine microfilariae. This serum recognizes n u m e r o u s microfilarial antigens (Fig. 3A) a n d if BSA is associated with these antigens (protein or n o n protein), then labeled a l b u m i n should have been co-precipitated. This negative result m a y indicate that a l b u m i n masks all epitopes on solubilized antigens such that they are n o t available to a n t i b o d i e s or that a l b u m i n dissociates from 'receptor' molecules at some stage prior to i m m u n o p r e c i p i t a t i o n . D e m o n s t r a t i o n of a l b u m i n o n the surface o f O. gibsoni skin microfilariae as well as L. carinii [ 14] a n d Wuchereria bancrofti b l o o d - d e r i v e d microfilariae ([ 15] a n d Forsyth, K.P., u n p u b l i s h e d observations) indicates that a d s o r p t i o n of host a l b u m i n (-_t: masking of target antigens) o n the surface of microfilariae may be a general p r o p e r t y o f systemic microfilariae. A l b u m i n is k n o w n to interact with a variety of proteins, fatty acids a n d phosphates a n d is also k n o w n to interact with itself a n d other proteins t h r o u g h sulphydryl groups [35]. The presence of collagen molecules with SH groups

226 in the nematode cuticle [36-38] and polypeptides capable of undergoing disulphide bond dependent aggregation [39], suggests that binding of BSA by SH groups to the microfilarial cuticle is possible. Experiments with reducing agents or alternatively, a comparison of ~25I-labeled skin microfilariae proteins run on reduced or non reduced acrylamide gels, may define the role of SH groups in the interaction between BSA and the cuticle of O. gibsoni microfilariae. The fact that systemic microfilariae can be used in in vitro A D C C reactions with immune sera indicates that some surface antigens are available or that the albumin is readily displaced under some in vitro incubation conditions. Another possible role for adsorbed albumin on microfilariae besides antigen masking may be as a local inhibitor of eosinophil peroxidase mediated oxidative damage. BSA is a known scavenger of hypochlorous acid which is generated by reaction of eosinophil peroxidase with H202 and Cl- and which is capable of killing new born Trichineila spiralis larvae [40]. The inability of uterine microfilariae to adsorb host albumin in vitro could reflect suboptimal incubation conditions. (This also shows that the source of BSA is unlikely to be an artifact of the preparation of microfilariae from skins.) More interestingly, this negative result could be real and indicate that some maturation process must occur for uterine microfilariae to adsorb albumin. The uterine microfilariae used in these experiments were taken randomly from the entire length of the female worm uterus and may therefore have varied in maturity. Only a subpopulation ofmicrofilariae which are released or about to be released, from the adult worm may have the molecular characteristics to adsorb albumin. With their albumin coat, they can migrate freely through host tissues without eliciting an inflammatory response. Other subpopulations of microfilariae lacking this characteristic may be responsible for the stimulation of the progressive inflammation observed over the course of the untreated disease. Alternatively, the pathology observed in the untreated disease may be caused by responses due to the natural death of microfilariae whether they are coated with albumin or not. Data from the I F A T study provided further evidence for the lack of antigenic reactivity of skin microfilariae compared to the surface of uterine microfilariae. However, the negative I F A T result with anti-BSA serum is puzzling. The immunochemical evidence for BSA on skin microfilariae was an early observation but the negative I F A T result has necessitated extensive confirmation of the labeling results. No entirely satisfactory explanation of the I F A T result can be provided. Perhaps albumin is incorporated or organized in the cuticle in such a manner that relevant epitopes (responsible for elicitation of anti-BSA antibodies when injected into mice in adjuvants) are no longer available. A comparable phenomenon has been observed with BSA associated with lymphocytes [41]. From the results of the lectin study there is no evidence for exposed cuticle carbohydrate on either skin or uterine microfilariae whereas eggs appear to have significant amounts of carbohydrate present. Furman and Ash [23] have reported similar findings on carbohydrates of sheathed and exsheathed B. pahangi microfila-

227 riae. It would appear that O. gibsoni eggs express carbohydrates with the same lectin-binding specificities as those on the sheath of B. pahangi microfilariae. This result is not surprising as the sheath is derived from the vitelline layer of the egg shell. The absence of carbohydrate on the surface of the cuticle of uterine microfilariae implies that egg debris is unlikely to have contaminated the surface of these microfilariae and thus contribute to the complexity of the protein profile of iodinated uterine microfilariae. The relevance of adsorption of host albumin to the surface of filarial parasites as an immune evasion mechanism remains conjectural. This also applies to those parasite systems where surface Ig has been implicated as a 'blocking antibody' [17]. More conclusive evidence may be gained by correlating the loss of host albumin to the microfilaricidal action of DEC. The treatment of skin microfilariae with DEC, subsequent surface labeling, and analysis of radioiodinated proteins, may reveal the presence of previously undetected antigens. Experiments to test this possibility using O. gibsoni microfilariae have been inconclusive to date (Forsyth, K.P., unpublished observations). A similar experiment using B. malayi microfilariae in an antibody-dependent cell-mediated adherence system revealed that enhanced antibody-mediated adherence of cells to microfilariae was achieved as a result of prior incubation of the parasites in D E C [4]. Aside from the unknown relevance of an albumin surface coat, the principal observation of this work, i.e. that there is a major difference between the surfaces of uterus- and skin-derived microfilariae, implies that the latter source of microfilariae will be unsuitable for studies of surface protein antigens. In addition, the lack of detectable surface Ig on skin microfilariae indicates that such microfilariae are unlikely to be an important 'sink' for anti-microfilarial antibody leading to artificially low titers in sera from heavily-infected individuals (see [42]). ACKNOWLEDGMENTS This work was supported by the Filariasis component of the U N D P / W o r l d B a n k / W H O Special Programme for Research and Training in Tropical Diseases and the Australian National Health and Medical Research Council. We thank Robyn Neuparth and Paul Verral for technical assistance. REFERENCES 1 2 3

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