Opsonization of Anaplasma marginale mediated by bovine antibody against surface protein MSP-1

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37 ( 1993) 343-350

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B.V., Amsterdam

Short Communication

Opsonization of Anaplasma marginale mediated by bovine antibody against surface protein MSP-1 Glenn H. Cantofl.h, Carol H. Pontzer’ and Guy H. Palmer’ “Dcpavtrmwt

(?I‘ I ?tcrinary

Mimhiologv P ~t/h~rl.

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Lrt~iwr.sity.

99/64- 7040. US.4

(Acccptcd 28 October 1992)

ABSTRACT Cantor. G.H.. Pontzer. C.H. and Palmer. G.H., 1993. Opsonization of .-lwp/asr,m nmrgirmlc mediated by bovine antibody against surface protein MSP- I 1>t. I~III,II~~I~/.Il,lr,lrlriol,al/~o/.. 37: 343350. Antibody from cattle immunized with purified major surface protein-I (MSP-I ) was demonstrated to significantly enhance phagocytosis of Florida strain .-lr~aplnsr~ra margitlak by bovine macrophagcs in vitro. Serum immunoglobulin from individual MSP-I immunized, protected cattle varied in ability to promote phagocytosis. however all scra were significantly opsonic as compared with sera from sham immunized control cattle. ABBREVIATIONS BSA. bovine serum albumin; MHC, major histocompatibility tein-l: PBS, phosphate buffered saline.

complex: MSP-I, major surface pro-

INTRODUCTION

Protective immunity against Anaplasma marginale, the rickettsial pathogen responsible for bovine anaplasmosis, develops following recovery from acute infection and can be induced by immunization with killed whole organism vaccines (Montenegro-James et al., 1991), outer membrane fractions (Tebele et al., 199 1 ), or purified surface proteins (Palmer et al., 1986; 1988b, 1989). Induction of immunity, defined as significant reduction in peak rickettsemia and protection from anemia, following outer membrane immunizaCormpotldmcc

Washington

to: G.H. Cantor, Department of Veterinary State University, Pullman, WA 99 164-7040. USA.

0 I993 Elsevier Science Publishers

Microbiology

B.V. All rights reserved 0165-2427/93/$06.00

and Pathology.

344

Ci.H.CANTOK

ETAL.

tion correlates with antibody titer against surface polypeptides (Tebele et al., 1991). Antibody against the whole organism (Palmer and McGuire, 1984) and specific antibody against the major surface protein complex- 1 (MSP- 1) (Palmer et al., 1986) has been shown to neutralize A. marginale infectivity for splenectomized calves. However the mechanism by which antibody contributes to protective immunity is unknown. Postulated mechanisms for antibody mediated immunity include direct antibody and complement mediated killing, antibody blockade of organism attachment and penetration into host erythrocytes, antibody-dependent cellular cytotoxicity by major histocompatibility complex (MHC) non-restricted lymphocytes, and antibody mediated opsonization for phagocytosis. A role for antibody mediated opsonization followed by killing by activated macrophages in immunity to A. marginale is supported by several observations. Phagocytosis occurs within splenic macrophages in vivo and splenomegaly develops during acute infection (Schmidt, 1937; Trueman and Wilson, 1979). Secondly, cattle splenectomized prior to infection develop a higher peak rickettsemia, more severe clinical disease, and have a higher case fatality rate than non-splenectomized cattle (Jones et al., 1968). Thirdly, persistently infected carrier cattle recrudesce and develop microscopically detectable rickettsemia within l-2 weeks following splenectomy or treatment with immunosuppressive drugs (Kuttler and Adams, 1977; Corrier et al., 198 1). Recrudescence within this brief period during which antibody levels would decline only minimally indicates a suppressible cellular function is involved. Finally, a correlation between antibody mediated opsonization/phagocytosis and in vivo protection against Plasmodium suggests this mechanism is involved in immunity against intraerythrocytic pathogens (Michel et al., 1983; Druilhe and KImsmith, 1987). We hypothesize that antibody specific for MSP1 promotes macrophage phagocytosis of A. marginale. In this short communication, we present results of in vitro experiments that test this hypothesis. MATERIALS

AND METHODS

Source of organism

and sera

The Florida strain of A. marginale was used in all experiments. Intact organisms (initial bodies) were obtained from cryopreserved infected erythrocytes by differential centrifugation (Palmer and McGuire, 1984). The purity and infectivity of isolated A. marginale has been previously described (Palmer and McGuire, 1984). Initial bodies were quantitated based on the percentage of infected erythrocytes. Sera for opsonization studies were obtained from cattle immunized with either affinity-purified MSP- 1 (five individuals) or sham immunized on an identical schedule with ovalbumin (five individuals). The sera used for these opsonization studies were collected from the

animals after immunization and before challenge.The immunization of these cattle, determination by immunoblotting of antibody titers to the MSP- la and MSP-1 b polypeptides that form the MSP-1 complex, and challengewith A. marginale have been described (Palmer et al., 1989). All live MSP-1 immunized cattle were completely protected against microscopically detectable rickettsemia upon challengeand did not develop anemia (Palmer et al., 1989). All ovalbumin immunized cattle developed detectablerickettsemia and acute, mild anaplasmosis (Palmer et al., 1989). For in vitro opsonization studies, immunoglobulin was partially purified from each serum by precipitation with saturated ammonium sulfate (Heide and Schwick, 1973). Immunoglobulin fractions were dialyzed against phosphate buffered saline (PBS), pH 7.4, and standardized to the original serum volume. Reactivity of isolated immunoglobulin was confirmed by binding to affinity purified MSP-1 in enzymelinked immunosorbent assay (Palmer et al., 1985). The opsonization studies were performed with antibody which was pooled from the five MSP-1 immunized individuals or with antibody pooled from the live ovalbumin immunized individuals. Additionally, opsonization was determined with antibody from each individual animal. Macrophages

Bovine macrophages were derived from neonatal undifferentiated bone marrow stem cells (Pontzer and Russell, 1989). Briefly, bone marrow cells were passagedto remove plastic adherent libroblasts and cultured in the presence of fibroblast-conditioned medium, as a source of macrophage-colony stimulating factor (Pontzer and Russell, 1989). We have previously defined bovine cells prepared by this technique as macrophages,basedon expression of Fc receptorsfor aggregatedIgG, bovine macrophagedifferentiation markers, and Class I and Class II MHC antigens, in addition to ability to ingest opsonized sheep red blood cells (Pontzer and Russell, 1989). Viability was assessedby Trypan Blue dye exclusion and the proportion of macrophages assessedby examination of Diff-QuickR-stained cytospin slides. Only samples exhibiting > 90% viability and containing > 80% macrophageswere used. Phagocvtosis

assay

Macrophages (5 x 1OScells) were grown in Lab-TekR tissue culture chamber/slides (Miles Scientific, West Haven, CT), which had been previously blocked for 1 h, 37°C with a mixture of growth medium and 10 mg ml-’ bovine serum albumin (fraction V) (BSA). The cells were incubated for 2 h, 37°C to allow macrophagesto adhere to the slides, washed twice, and incubated in 300 ~1of growth medium containing 15% fetal bovine serum for 18 h. A 1:30 dilution of antibody in growth medium with 10 mg ml-’ BSA was

346

G.H.

CANTOR

ET AL.

mixed with A. marginale initial bodies in BSA-blocked, siliconized glasstubes, and was incubated for 1 h, 37°C. The antibody-initial body mixture was washed with growth medium-BSA and centrifuged for 30 min at 10°C 1100xg. The supematant was discarded and 50 ,~lof the antibody-initial body mixture was added to each well. Initial bodies were added to macrophagesat a ratio of 50:1. Allocation of treatments to wells was determined by a random number table. To ensure contact of the initial bodies with the monolayer of macrophages,the tissue chamber/slides were centrifuged for 10 min, 25“C, at 1OOOxg.Chambers were incubated at 37°C 1 h. Following phagocytosis, the supematant was removed and the macrophageswere washed three times with growth medium. Fluorescentantibody staining Acetone-fixed slides were incubated for 30 min with murine monoclonal anti-MSP 1 antibody (ANA 22Bl), 100 ~1 at 0.1 mg ml-‘, or, as a negative control, murine monoclonal anti-Trypanosoma brucei antibody (TRYP 1E 1) (Palmer et al., 1988a). Afterwards, slides were washed three times in PBS and stained for 30 min with fluorescein isothiocyanate-labelled rabbit antimouse immunoglobulin antibody. This antibody was obtained by immunizing rabbits with mouse immunoglobulin (&A, G,, GZa,GZb,Gj, and M ), followed by purification of rabbit immunoglobulin by diethylaminoethyl ether cellulose chromatography and coupling to fluorescein isothiocyanate (Johnson and Holborow, 1973). P-phenylenediamine-buffered glycerine was used as a mounting medium (Johnson and Nogueria Araujo, 1981). Data analysis Slides were examined without foreknowledge of identity of treatments. From each well 50- 100 macrophages,from at least six areasof the well, were examined, and the number of fluorescent intial bodies within or adherent to eachcell counted. Data were analyzedby the Mann-Whitney two-sample test. RESULTS

Phagocytosisof opsonizedinitial bodies The number of initial bodies ingested by macrophages was significantly greater when initial bodies were pre-incubated with anti-MSP-1 serum antibodies (pooled from 5 MSP-1 immunized cattle), as compared with those pre-incubated with anti-ovalbumin serum antibodies (pooled from five ovalbumin immunized cattle) (PcO.01; Figs. 1(a) and 1(b) ). The mean number of MSP-1 antibody treated initial bodies ingested per macrophageswas

OPSONIZATION

OF .-th’:IPL4SA/.4

347

A~:IHGIIV:ILL

MSP-1 antibody 40 a 35302 0 "a e

25-

i 6

20.

"E z

15-

:

lo507

. 0

2

4

6

8

Number

10 12 14 16 18 20 22

of initial bodies

I 24 26 28

Ifi 30 32

par macrophage

Oval bumin antibody 40

b

Fig. 1. Increased phagocytosis of A. ~~arginale opsonized with MSP-I antibody. Initial bodies incubated with a pool of serum antibodies from (a) five MSP-1 immunized individuals or with a pool from (b) five ovalbumin-immunized individuals were added to macrophages at a ratio of 50: I. Initial bodies in each of 50 macrophages were counted. The experiment was repeated five times with similar results.

348

G.H. CANTOR

ET AL.

10.4, and the median was 16. The percentage of macrophages which had ingested two or more initial bodies was 80%. By contrast, the mean number of ovalbumin antibody-treated initial bodies ingested per macrophages was 0.8, and the median was zero. Only 18% of the macrophages ingested two or more initial bodies.

Phagocytosisqf initial bodiesopsouizedby sewn antibodJl.f,.ornindividual animals Initial bodies were pre-treated with antibody from each of the ten individual animals. Pretreatment with serum antibody from each of the live MSP-1 immunized animals resulted in more phagocytosis than pretreatment with antibody from each of the live ovalbumin-immunized animals (P~0.05) (Table 1 ). There were also statistically significant differences in the ability of antibodies from different MSP- 1 immunized animals to promote phagocytosis (Table 1). Among the individual anti-MSP- 1 sera, there was no significant correlation between the antibody titer to either MSP-la or MSP-1 b and opsonization. Sera from all ovalbumin-immunized cattle were unreactive with A. marginale polypeptides, including MSP-la and MSP-1 b, at the lowest dilution tested, 1:500. TABLE

I

Increased phagocytosis from individual animals

of :l.

Immunogcn and Animal No.

,\ISl’-

rrrargir~~~lc

Titer

opsonizcd

of antibody

with

MSP-I

antibody:

cffcct

to:

MSP-la

MSP-lb

polypeptide

polypcptide

64 000 64 000 64 000

I’8 000 64 000 I28 000

of strum

antibodies

Number of initial bodies per IO0 macrophagcs

I

B194 B203 B187

I303 754” 8 [email protected]

8208

I28

000

I28

000

460b

Bl96

I28

000

1'8000

258

Oulltwl?lilr

Bl95 B217 B207

‘33’ I 5Yd 175d.c 86d,c 53’

Bl89 B197 Each

of the groups

ovalbumin antibody, indicate no significant

incubated

with

MSP-I

antibody

is statistically

greater

than

those

based on the Mann-Whitney two-sample test (P-cO.05). Identical differences (P=O.O5) based on the Mann-Whitney two-sample

incubated

with

superscripts test.

349 DISCUSSION

The mechanism by which MSP- I immunization induces protection in cattle is unknown. Although antibody titer to surfacepolypeptides correlateswith protective immunity (Tebele et al., 1991) and in vitro incubation with antibodies against MSP-1 neutralizes A. marginale infectivity (Palmer and McGuire, 1984; Palmer et al., 1986), the critical role of the spleen and suppressible cellular functions suggeststhat protective immunity is not strictly humoral. Protective immunity in other non-erythrocytic rickettsial infections has been shown to depend upon phagocytosis and killing by activated macrophages (Nacy and Meltzer, 1979, 1984). In this study, we demonstrated that antibody from MSP-1 immunized cattle promotes phagocytosis of A. marginale initial bodies by bovine macrophages.Demonstration of opsonization by MSP-1 antibody, from cattle which were shown to be protected against challenge, suggeststhat this may be a significant protective mechanism in vivo. Consistent with the prior observations on immunity againstA. marginale, phagocytosisas a key mechanism of immunity would be adversely affected by both splenectomy (Joneset al., 1968) and suppressionof lymphocyte function (Corrier et al., 1981). Optimization of protective immunization may require both stimulation of appropriate opsonizing antibody and also enhancement of macrophage function. ACKNOWLEDGMENTS

The authors thank Dr. Michael Conlon, Division of Biostatistics, J. Hillis, Miller Health Center, University of Florida for statistical advice, and Dr. Kristy Truenbach for technical assistance.This study was funded by Agency for International Development project DAN-4 178-A-00-7056-00and USDABARD project US-l 561-88. REFERENCES Corricr. D.E.. Wagner, G.G. and Adams. L.G., 198 1. Recrudescence of .-I,ru,&zs,?ru mzrgidc induced by immunosuppression with cyclophosphamide. Am. J. Vet. Rcs.. 42: 19-2 1. Druilhc, P. and Khusmith, S., 1987. Epidemiological correlation between levels of antibodies promoting merozoite phagocytosis of Plusr,lodilrl,l./~k.i~u~lrr,rand malaria-immune status. Infect. Immun., 55: 888-89 I. Hcide. K. and Schwick. H.G., 1973. Salt fractionation of immunoglobulins. In: D.M. Weir (Editor), Handbook of Experimental Immunology, 2nd Edn.. Blackwcll. Oxford, UK, pp. 6. I6.1 I. Johnson, G.D. and Holborow, E.J., 1973. Immunofluorcsccnce. In: D.M. Weir (Editor), Handbook of Experimental Immunology, 2nd edn.. Blackwell, Oxford, UK, pp. 18. l-l 8.20. Johnson, G.D. and Nogueira Araujo, G.M.C.. I98 I. A simple method of reducing the fading of immunoiluorescence during microscopy. J. Immunol. Methods.. 43: 349-350.

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ET AL.

Jones, E.W., Norman, B.B., KIiewer, 1.0. and Brock, W.E., 1968. Anaplasma marginale infection in splenectomized calves.Am. J. Vet. Res., 29: 523-533. Kuttler, K.L. and Adams, L.G., 1977. Influence of dexamethasone on the recrudescenceofAnaplasma tnarginale in splenectomized calves. Am. J. Vet. Res., 38: 1327-l 330. Michel, J.C., Fandeur, T., Neuilly, G., Roussilhon, C. and Dedet, J.P., 1983. Opsonic activity of ascitic fluids from Plastnodiutnfalciparutn-infected Saimiri monkey: positive correlation with protection in passivetransfer assay.Ann. Immunol., 134D: 373-383. Montenegro-James, S., James, M.A., Toro Benitez, M., Leon, E., Back, B.K. and Guillen, A.T., 1991. Efficacy of purified Anaplustna marginale initial bodies as a vaccine against anaplasmosis. Parasitol. Res., 77: 93- 10 1. Nacy, C.A. and Meltzer, MS., 1979. Macrophages in resistance to rickettsial infections: macrophage activation in vitro for killing of Rickeltsia tsulsugumushi. J. Immunol., 125: 25442549. Nacy, C.A. and Meltzer, M.S., 1984. Macrophages in resistanceto rickettsial infections: protection against lethal Ricketlsia lsutsugatnushi infections by treatment of mice with macrophage activating agents. J. Leuk. Biol., 35: 385-396. Palmer, G.H. and McGuire, T.C., 1984. Immune serum against Anuplasma marginale initial bodies neutralizes infectivity for cattle. J. Immunol., 133: 10 lo- 1015. Palmer, G.H., Kocan, K.M., Barron, S.J., Hair, J.A., Barbet, A.F., Davis, W.C. and McGuire, T.C., 1985. Presenceof common antigens, including major surfaceprotein epitopes, between the cattle (intraerythrocytic) and tick stagesof Anaplasma marginale. Infect. Immun., 50: 88 l-886. Palmer, G.H., Barbet, A.F., Davis, W.C. and McGuire, T.C., 1986. Immunization with an isolate-common surface protein protects cattle against anaplasmosis. Science,23 1: I299- 1302. Palmer, G.H., Barbet, A.F., Musoke, A.J., Rurangirwa, F., Katende, J., Pipano, E., Shkap, V., Davis, W.C. and McGuire, T.C., 1988a. Recognition of conserved surface protein epitopes on Anaplasma centraleand Anaplastna tnarginale isolates from Israel, Kenya and the United States.Int. J. Parasitol., 18: 33-38. Palmer, G.H., Oberle, S.M., Barbet, A.F., Davis, W.C., Goff, W.L. and McGuire, T.C., 1988b. Immunization with a 36-kilodalton surface protein induces protection against homologous and heterologous Anaplasma tnarginale challenge. Infect. Immun., 56: 1526- 153 1. Palmer, G.H., Barbet, A.F., Cantor, G.H. and McGuire, T.C., 1989. Immunization of cattle with the MSP-1 surface protein complex induces protection against a structurally variant Anaplasma tnarginale isolate. Infect. Immun., 57: 3666-3669. Pontzer, C.H. and Russell, S.W., 1989. Culture of macrophages from bovine bone marrow. Vet. Immunol. Immunopathol., 21: 351-362. Schmidt, H., 1937. Anaplasmosis in cattle. J. Am. Vet. Med. Assoc.,90: 723-736. Tebele, N., McGuire, T.C. and Palmer, G.H., 199 1. Induction of protective immunity by using Anaplasma marginale initial body membranes. Infect. Immun., 59: 3 199-3204. Trueman, K.F. and Wilson, A.J., 1979. Observations on the pathology ofAnaplasma marginale infections in cattle. Aust. Adv. Vet. Sci., 5: 75.